biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 23-25 | doi: 10.14421/biomedich.2021.101.23-25 issn 2540-9328 (online) brittle bone brothers: osteogenesis imperfecta conventional serial case marsha ruthy darmawan1, elysanti dwi martadiani2,* 1radiology resident; 2musculoskeletal radiologist, radiology department, faculty of medicine, universitas udayana sanglah hospital denpasar, bali, indonesia. corresponding author* elysantiidm@gmail.com manuscript received: 08 october, 2020. revision accepted: 01 july, 2021. published: 13 july, 2021. abstract osteogenesis imperfecta is a hereditary connective tissue disorder due to col1a1/2 mutation causing gene defect encoding proteins to metabolize collagen. the skeletal manifestation of oi causing bone incompetence, hence the name brittle bone disease. here we report three cases of oi type iv in adults. skeletal conventional x-rays were performed to all patients and all of them has similar results such as bowing deformities of long bones, old union and some non-union fractures with extreme angulation and severe osteoporosis. oi are classified based on skeletal structure, sclera colorization, dentinogenesis, and functional metabolic defect genetically. oi type i and iv can live until adults; also, the same type of oi can be found in siblings. skeletal conventional x-rays can solely make the diagnosis. keywords: osteogenesis imperfect; conventional x-ray; osteoporosis; bone deformity; brittle bone. introduction osteogenesis imperfecta (oi) or “brittle bone disease” is a hereditary form of early osteoporosis in children with an incidence reaching 1: 20.000 births. (hoyer-kuhn, et al., 2015) according to the national institutes of health, 90% of oi genetic mutation occur due to a mutation of col1a1 and col1a2 genes. (blom, et al., 2017) it means disturbance to all connective tissue from collagen type i; therefore, patients can have fractures all their lives. (van dijk, & sillence, 2014) a nomenclature in 2014 by the international nomenclature group of constitutional disorders of the skeleton (incds)classified oi into primary type i-iv and adding type v based on their causative genes and manifestation in deformed bones. (van dijk, & sillence, 2014) here we present three cases of oi type iv in one family of three brothers in their 40s and the only adults with oi in our hospital. case report case 1 a 40-year-old indonesian male came to the hospital with small stature for a medical check-up. he is unusually short for his age; while he was young, he had multiple fractures of left radial bone and right tibia. on physical examination, we found over-bending to all extremities without abnormal sclera colorization or teeth. imaging examination includes conventional x-raysbut none of bone mineral density (bmd). on upper and lower extremities x-ray, there was generalized bowing to the radial shaft, ulna, metacarpal bones, femur, tibia, and fibula. acute-angled long bones with missing bone parts in both humerus caused by non-union fractures. chest x-ray showed bowing of ribcage and left clavicle. there were no ecg done in any of these patients. radiographic images are in figure 1. figure 1. a 40-year-old man with oi presented with small stature. skeletal x-rays showed generalized bowing and angled long bonesand missing bone parts in both humerus caused by non-union fractures. https://doi.org/10.14421/biomedich.2021.101.23-25 24 biology, medicine, & natural product chemistry 10 (1), 2021: 23-25 case 2 a 41-year-old indonesian male came to the hospital with short stature and limitation to self-activity. he had fractured almost all his long bones. physical examination showed generalized bent deformation to his extremities with a normal sclera, yellowbrownopalescent discoloration of anterior teeth, and many missing ones. further examinations were only conventional x-ray, bowing to all long bones in the upper and lower extremities. there were missing bone parts on medial and lateral thirds of right humerus that indicates non-union fracture and enlarged metaphysis of proximal as well as distal upper long bones. chest x-ray showed bowing of the ribcage, old fracture to the left clavicle, and all bones appeared porotic severely. moreover, no cardiopulmonary abnormalities found in this patient. radiographic images are in figure 2. figure 2. a 41-year-old man with oi who presented with short stature. skeletal x-rays showed porotic, bent to all long bones and missing bone parts on medial and lateral thirds of right humerus indicates the non-union fracture. case 3 a 42-year-old indonesian male came to the hospital complaining his stature is getting smaller compared to his brothers. he was in a wheelchair throughout his life, unable to complete physical activities due to pain and fragile bones, which would easily break when exposed to blunt forces. physical examination showed bent extremities with normal sclera and teeth. further x-rays showed, bowing and bent deformities to all long bones, missing bone part on medial third of left humerus and left femur, also enlarged metaphysis on all sites of long bones. chest x-ray showing bent ribs and deformed left clavicle. all bone trabeculation appeared severely porotic, and no cardiopulmonary abnormalities found. radiographic images are in figure 3. figure 3. a 42-year-old man with oi who presented with small stature. skeletal conventional x-rays showed porotic, bending on all long bones, missing bone parts on the medial third of left humerus, and left femur, also kyphoscoliosis. discussion oi is a hereditary connective tissue disorder due to col1a1/2 mutation causing gene defect encoding proteins to metabolize collagen. the skeletal manifestation of oi mainly causing bone incompetence, vulnerable to fractures, deformed, and joint laxity, therefore bones are fragile, hence the name brittle bone disease. (hoyer-kuhn, et al., 2015) (blom, et al., 2017) from the new oi nomenclature 2014 revised nosology, there are five types of oi. type i related to deficiency of normal collagen, type ii is lethal, type iii severe, type iv mutation in collagen structure, and type v is oi with calcification in the interosseus membrane. (van dijk, & sillence, 2014) forlino and marini in 2016 described nineteen types of oi with five categories based on functional metabolic defects genetically, such as defects in collagen synthesis, structure or processing (group atype i-iv, xiii), defects in collagen modification (group b-vii-ix, xiv), defects in collagen folding and cross-linking (group ctype x-xi), defects in bone mineralization (group dtype v-vi), and defects in osteoblast development with collagen insufficiency (group e-type xii, xv-xvi). (forlino & marini, 2016) current therapy for oi is integrative, pain management, muscle rehab for regaining strength and range of movement also regain mobility to increase the quality of life, and a regular check-up for dentition and hearing. bisphosphonate treatment with cyclic intravenous pamidronate given in infancy proved to help increase bone density and reduce fractures. (scheres, et al., 2018) in these cases of the brothers, their ages range from 40-42 years old; they have a small stature, normal sclera, darmawan & martadiani – brittle bone brothers: osteogenesis imperfecta … 25 two patients have normal teeth and one with dentinogenesis imperfecta (di). these brothers generally have the same type of oi which is oi type iv but different subtype, iv a without di and iv b with di. (van dijk, & sillence, 2014) (scheres, et al., 2018) genetically categorized as group a by forlino and marini (forlino & marini, 2016), type iv oi resulted in col1a 1 or 2 mutation and genetic workup must be done to determine which one. unfortunately, genetic workup was not administered to all patients. type iv oi categorized as group a in forlino and marini classification is caused by collagen deficiency creating structural inadequacy. glycine substitutions in the helical domain are the most common problem, which can delay helical folding and prolonged time to modify enzymes. another mutation common for oi is impaired chain of procollagen c-pro-peptide. inadequate collagen structurally can manipulate intracellular metabolism and matrix architecture rather than a deficiency in collagen quantity. (forlino & marini, 2016) general radiographic findings mostly consist of osteopenia, deformities, and fractures. common findings to lower extremities include anterior or lateral bowing of the femur, anterior bowing of tibia, protruded acetabulum, and ‘shepherd’s crook’ deformities of the proximal femur. ‘popcorn’ appearance in the metaphysis, multiple areas, or radiolucent scalloping with thick rims, can be seen in some patients with oi. specific findings of the spine include compressed vertebrae between cartilaginous disc space, called codfish vertebrae. abnormalities were found not only in long bones but also in the skull, which is caused by excessive bone malleability and plasticity. the more common findings to the skull are multiple wormian bones, a physiological finding in the skull, but considered abnormal if there were more than ten found and usually present in patients with severe oi. (blom, et al., 2017) diagnosis of oi in childhood made solely with conventional skeletal x-ray, simple yet effective. an optimal method to determine quantitative osteopenia is by dual-energy x-ray absorptiometry (dexa), and bone mineral density (bmd) score will reveal if there is osteopenia or already in osteoporosis state. another method to diagnose oi in children is with dna analysis with an examination of cultured fibroblast. this method showed a decreased quantity of cultured fibroblast in children with oi than healthy children, also abnormality of type 1 procollagen molecules or mutation to col1a1 or col1a2 genes that encrypt type 1 procollagen chains. (scheres, et al., 2018) bone histomorphometry examined in oi type i-iv (collagen defect group) showed low bone volume and trabecular quantity with high replacement kinetic rates. if more than one family member has this disease, the clinician should explore oi gene panel for better therapy plan. (bishop & walsh, 2014) conclusion oi is a rare inherited abnormality to the skeletal because of mutations to type 1 collagen in connective tissue. oi are classified based on skeletal structure, sclera colorization, dentinogenesis imperfecta, and functional metabolic defect genetically. oi type i and iv can live until adults; also, the same type of oi can be found in siblings. skeletal conventional x-rays can solely make the diagnosis of oi. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references bishop, n. j., & walsh, j. s. (2014). osteogenesis imperfecta in adults. the journal of clinical investigation, 124(2), 476–477. https://doi.org/10.1172/jci74230 blom, a., warwick, d., & whitehouse, m. (eds.). (2017). apley & solomon's system of orthopaedics and trauma (10th ed.). crc press. https://doi.org/10.4324/9781315118192 forlino, a., & marini, j. c. (2016). osteogenesis imperfecta. lancet (london, england), 387(10028), 1657–1671. https://doi.org/10.1016/s0140-6736(15)00728-x hoyer-kuhn, h., netzer, c. & semler, o. (2015). osteogenesis imperfecta: pathophysiology and treatment. wien med wochenschr 165, 278–284. https://doi.org/10.1007/s10354015-0361-x scheres, l., van dijk, f. s., harsevoort, a. j., van dijk, a., dommisse, a. m., janus, g., & franken, a. (2018). adults with osteogenesis imperfecta: clinical characteristics of 151 patients with a focus on bisphosphonate use and bone density measurements. bone reports, 8, 168–172. https://doi.org/10.1016/j.bonr.2018.04.009 van dijk, f. s., & sillence, d. o. (2014). osteogenesis imperfecta: clinical diagnosis, nomenclature and severity assessment. american journal of medical genetics. part a, 164a(6), 1470–1481. https://doi.org/10.1002/ajmg.a.36545 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 5-8 | doi: 10.14421/biomedich.2023.121.5-8 issn 2540-9328 (online) high performance liquid chromatography (hplc) for detection of glucosamine and chondroitin sulfate compounds rakhmiyati1,*, tetri widiyani1,2, agung budiharjo1,2 1department of bioscience, graduate program; 2department of biology, faculty of mathematics and natural sciences, sebelas maret university. jl. ir sutami no. 36a, kentingan, surakarta, 57126, central java, tel./fax. +62-271-663375, indonesia. corresponding author* miarakhmiy@gmail.com manuscript received: 26 march, 2022. revision accepted: 24 august, 2022. published: 14 september, 2022. abstract glucosamine and chondroitin sulfate are compounds found in shark cartilage (carcharhinus sorrah). the two compounds have many health benefits, that is wound healing and helping the process of angiogenesis. this study aims to determine the content of glucosamine and chondroitin sulfate compounds in shark cartilage (sc) extract. the method used was high performance liquid chromatography (hplc) with potassium phosphate buffer solution at ph 3. the results of this research were sc extract contained glucosamine and chondroitin sulfate compounds with a retention time of 1.914 minutes. keywords: hplc; shark; glucosamine; chondroitin sulfate. introduction shark cartilage (sc) has several benefits, including controlling the growth and spread of tumor cells, cancer, helping to reduce bone pain, avoiding rheumatic diseases, strengthening and maintaining bone function, relieving pain and gout, maintaining body health and vitality, and avoid curvature of the spine (sulityowati et al., 2015; dean and summers, 2006). according martelpelletier (2015) sc can also treat osteoporosis and osteoarthritis because it contains chondroitin sulfate. the extraction and purification of chondroitin sulfate was first carried out in 1960 (miller and clegg, 2011). research conducted by sulityowati et al (2015) showed that shark cartilage powder contained 28.36% glucosamine and 6.06% chondroitin. chondroitin sulfate powder is white to cream in color with a ph value between 5.5 to 7.5. this compound is easily soluble in water and is hygroscopic. chondroitin sulfate becomes unstable when exposed to direct light and at high temperatures (marzuki et al., 2014). according garnjanagoonchorn et al. (2007) in xie et al (2014) states that dry sc contains glycosaminoglican 10-40% and collagen type ii 25-55%. glycosaminoglycan (gag) is components of the extracellular matrix of connective tissue. chondroitin sulfate is a type of gag whose components consist of n-acetyl-galactosamine, sulfuric acid and glucuronic acid. chondroitin sulfate can be used in joint ailment therapy, anti-inflammatory, arthritis, atherosclerosis and cancer (siagian, 2014; widyaningsih et al, 2016), and immunostimulant (bargahi and rabbani-chadegani, 2008). according to kerri et al. (2003) and huskisson (2008) the chemical structure of chondroitin sulfate and glucosamine were shown in figures 1 and 2. figure 1. the chemical structure of chondroitin sulfate compound. figure 2. the chemical structure of glucosamine compound. chondroitin sulfate is a polysaccharide anion consisting of the disaccharide unit naacetylgalactosamine 4or 6-sulfuric acid and dhttps://doi.org/10.14421/biomedich.2023.121.5-8 6 biology, medicine, & natural product chemistry 12 (1), 2023: 5-8 glucuronic acid repeated in cartilage tissue. these polysaccharides covalently bind to proteins to form proteoglycans. chondroitin sulfate has a wide range of applications in the pharmaceutical, cosmetic and food industries (nakano et al., 2000). according to siagian (2014), chondroitin is found in hyaline cartilage and can be distinguished structurally by the position of the sulfate ion in the monosaccharide bonds. materials and methods tools and materials the tools used in this research include: styrofoam ice box, measuring tape, sitting scale, digital scale, surgical instrument set, knife, tray, oven, aluminum bowl, blander (miyako), sieve (size mesh 80), plastic bags, plastic clips, gloves, digital analytical scales, petri dishes, aluminum foil, surgical mats, rotary evaporator (rvo 400 sd boeco germany), beakers, vials glass, filtering funnels, hot plates, measuring cups, plastic pot bottles, hplc tools (shimadzu), column c-18 (dimensions 4.6 x 250 mm, size of diameter pore 5µm), rid detector (refraksi index detector), cpu, stainless steel spatula, tweezers, stirring rod. the material used in this research is shark (carcharhinus sorrah) obtained from depok beach, yogyakarta, ice cubes, methanol pa, acetonitrile, potassium dihydrogen phosphate powder (1.36 grams), distilled water, g-nutri, chondroitin sulfate powder, and phosphoric acid (h3po4). shark cartilage powder shark (carcharhinus sorrah) was obtained commercially from the coast of depok, yogyakarta, then all flesh and tissue attached to the cartilage were removed. the cartilage was cut into ±1 cm in size and had been dried using an oven at 500 c for 24 hours. furthermore, it was mashed using a blender, sieved with a sieve of 80 mesh. shark cartilage powder was stored in a cool place prior to extraction (sulityowati et al., 2015; davis, c., 1994). shark cartilage extract 60 grams of shark cartilage powder was dissolved in 1200 ml of methanol as a solvent. the immersion time was 7 days. every day the solution was stirred for 3 hours. then after that filtered using filter paper. the filtrate obtained from the filtering was collected, while the residue was discarded. the filtrate was then processed using a rotary evaporator machine. the temperature used on the hot plate was 400c and the speed of the driving rotor was 2 turns. the result of the rotary evaporator was a thick, milky white liquid which was an extract of shark cartilage (iffah et al., 2018). detection of glucosamine and chondroitin sulfate compounds instrument setup hplc (high performance liquid chromatography) the first step was to turn on the electric power, then turn on the stabilizer. after that turn on the pump on the tool. next, turn on the water column by pressing the polar button. then turn on the detector rid (refractive index detector) [set the wavelength (γ)], then modular and finally turn on cpu (central processing unit) on computer. if the chromatogram shows a flat base line then the instrument can be used. preparing the mobile phase mobile phase was using potassium phosphate buffer solution ph 3. the buffer solution was made by adding 1.36 grams of potassium dihydrogen phosphate powder into 800 ml of distilled water, then adding phosphoric acid (h3po4). the ratio between the potassium phosphate buffer and the acetonitrile was 99.5:0.5. the flow rate is 1 ml/minute. the hplc column used was c18 4.6×250 mm 5µm merck. the temperature in the column used was 280c. the eluent will carry the components of the mixture to the rid (refractive index detectors) detector. (jahangir, et al., 2015; nagarajan, et al., 2013). results and discussion based on the results of high performance liquid chromatography (hplc), it was found that the extract of shark cartilage (carcharhinus sorrah) was proven to contain glucosamine and chondroitin sulfate compounds. shark cartilage extract chromatogram based on hplc method is shown in figure 3. figure 3. hplc chromatogram on shark cartilage extract. the hplc method is a suitable method to determine the presence of glucosamine and chondroitin sulfate compounds in shark cartilage extracts. separation of analytes were using phosphate buffer solution and acetonitrile with a ratio of 99.5: 0.5. the buffer solution ph 3 is a mixture of phosphoric acid solution = 88 ml + 1000 ml aquades + 1.3 grams of potassium dihydrogen phosphate and the flow rate used was 1 ml/minute. in rakhmiyati et al. – high performance liquid chromatography (hplc) for … 7 figure 1 it can be seen that the elution substance has formed a good symmetrical peak. figure 4. hplc chromatogram results on glucosamine (a) and chondroitin sulfate (b) parameters. from the hplc output in the form of a chromatogram (figure 3), it can be seen that the shark cartilage extract sample (carcharhinus sorrah) has a retention time of 1.917. meanwhile, the retention time for glucosamine and chondroitin sulfate parameters were 1.914 and 1.914 (figure 4). this retention time proves that the shark cartilage extract contains glucosamine and chondroitin sulfate compounds. according to research that has been done by sulityowati et al. (2015) stated that in 40 grams of shark cartilage powder it contains 28.36% glucosamine and in 240 grams of shark cartilage powder there is 6.06% chondroitin. glucosamine, 2-amino-2-deoxy-d-glucose (c6h14no5) is a monosaccharide having a molecular weight of 197.2 da (agiba, 2017; huskisson, 2008). this compound is the main component of glycosaminoglycans (gags) in cartilage and synovial fluid (sulityowati et al., 2015). glycosaminoglycans (gags) are heteropolysaccharides that have a negatively charged protein at the edge and function as a binder called mucopolysaccharide (sulityowati et al., 2015). glucosamine is found in almost all connective tissue, but the most abundant content is in cartilage (dahmer and schiller, 2008; sulityowati et al., 2015). chondroitin sulfate (cs) is a heteropolysaccharide with long and unbranched chains called glycosaminoglycans with a molecular weight of 50-100 kda, but after the extraction process the molecular weight becomes 10-40 kda (sulityowati et al., 2015; henrotin et al., 2010). but according to huskisson (2008) the molecular weight of chondroitin is 10,00050,000 da. cs compounds have the same properties as gc which are hydrophilic, can dissolve in water and produce a liquid that resembles sodium hyaluronate. conclusion based on observations that have been made using the high performance liquid chromatography (hplc) method, it can be concluded that the shark cartilage extract (carcharhinus sorrah) contains glucosamine and chondroitin sulfate compounds with a retention time of 1.917 minutes. conflict of interests: authors state that there is no conflict of interest in this research output. references agiba, a.m. (2017). nutraceutical formulations containing glucosamine and chondroitin sulphate in the treatment of osteoarthritis: emphasis on clinical efficacy and formulation challenges. international journal of current pharmaceutical r esearch 9 (2): 1-7. american college of rheumatology subcommittee on osteoarthritis guidelines. 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(2005). pharmacokinetic profile of glucosamine and chondroitin sulfate association in healthy male individuals. acta ortop bras 13(5): 235-237. václavíková, e., and kvasnička, f. isotachophoretic determination of glucosamine and chondroitin sulphate in dietary supplements. czech j. food sci 31 (1): 55-65. vasiliadis, h.s., tsikopoulos, k. (2017). glucosamine and chondroitin for the treatment of osteoarthritis. world journal orthopedics 8 (1): 1-11. widyaningsih td, wd rukmi, e sofia, sd wijayanti, n wijayanti, r ersalia, n rochmawati, d nangin. (2016). extraction of glycosaminoglycans containing glucosamine and chondroitin sulfate from chicken claw cartilage. research journal of life science 3 (3): 181-189. xie, j., hy ye and xf luo. (2014). an effcient preparation of chondroitin sulfate and collagen peptides from shark cartilage. international food research journal 21(3): 1171-1175. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 91-95 | doi: 10.14421/biomedich.2020.92.91-95 issn 2540-9328 (online) a chemical overview of azanza garckeana yilni edward bioltif1,*, naanma bioltif edward2, terry dalyop tyeng1 1department of chemistry, faculty of natural science, plateau state university, bokkos, nigeria. 2department of plant science and biotechnology, faculty of natural science, university of jos, nigeria. corresponding author* edwardyilni405@gmail.com manuscript received: 18 may, 2020. revision accepted: 10 november, 2020. published: 17 november, 2020. abstract azanza garckeana is a popular fruit tree in nigeria, specifically in gombe state, where it is locally called ‘goron tula’ which means ‘kola of tula’. it is also found in part of some african countries. different plant part of this small tree/shrub has recorded different uses by the locals; uses ranging from its fruits being edible and others parts helping to remedy different diseases, especially sexually related diseases. it also records use as booster for sexual performance. the uses of the plant are majorly attributed to the presence of chemicals. its local use initiates the necessity of this review to enhance the research for drug discovery since chemicals are the chief constituencies responsible for its medicinal importance. keywords: azanza garckeana; chemical; compounds; mansonone. introduction the indigenous fruits collected from the wild play a significant role in food and nutrient security of the poor and rural dwellers. some wild fruits have been identified to have better nutritional value than cultivated fruits (musinguzi et al., 2007) as a result, in recent years, a growing interest has emerged to evaluate various wild edible plants for their nutritional features (nkafamiya, 2007; aberoumand and deokule, 2009; nazarudeen, 2010). nutritional value of indigenous fruit bearing tree species indicates that many are rich in sugars, essential vitamins and minerals, while others are high in vegetable oil and protein contents. in addition to fruit production and cash, the extensive list of benefits includes firewood, fodder, building material, shade and medicine especially to rural communities. edible wild leaves and fruits are consumed frequently in northern nigeria especially in rural communities where a variety of edible leaves and fruits abound. some of these are cultivated while others grow in the wild (nkafamiya et al. 2016). azanza garckeana is a member of the malvaceae family. the generic name “azanza” is derived from the word “azania”, a word meaning black and surviving in zanzibar. the specific name “garckeana” is in honour of professor august garcke (1819-1904), a german botanist and plant collector who specialized in pharmacognosy (maroy, 2017). botanical classification of azanza garckeana kingdom : plantae – plants subkingdom : tracheobionta – vascular plants superdivision : spermatophyta – seed plants division : magnoliophyta – flowering plants class : magnoliopsida – dicotyledons subclass : dilleniidae order : malvales family : malvaceae – mallow family genus : azanza specie : azanza garckeana english (common name) tree hibiscus, azanza, snot apple nigeria (hausa): goron tula, bostwana – morojwa south africa thespesia garckeana (mojeremane and tshwenyane, 2004; and ochokwu et al., 2014). description azanza garckeana is a deciduous shrub; the tree can grow to a height of 3-15m high depending on the climate condition stem diameter at breast height of up to 25cm. the tree is multi-stemmed with straight or crooked stem, which is sometimes forking from the base. the bark is rough and greyish-black or brown, fibrous with longitudinal fissures. the twigs are hairy when young but become smooth with age and branches have woody hairs. the leaves are distinctively rounded, 8 by 12cm on long stalks. they are always simple, alternate and roundish. the leaves have 3 to 5 lobes, which are covered in brown star-shaped hairs, and have longitudinal fissures in the midrib. the tip of the leave is usually bluntly pointed or rounded. the base of the https://doi.org/10.14421/biomedich.2020.92.91-95 92 biology, medicine, & natural product chemistry 9 (2), 2020: 91-95 leave is heart-shaped and is 5 to 7 nerved. the young leaves are brown in colour and velvety. the flowers are large up to 6cm long, solitary on long pedicels in the axils of uppermost leaves, yellow with a purple-brown centre; the petals are globose and capsules are up to 4cm long, the thickness is 3cm. the fruit is globose and have woody capsules of up to 3 to 4cm in diameter, it is divided into 5 segments with each segment containing a seed, the remains of the calyx and epicalyx at the base; the seeds are hemispherical, up to 10 mm long, 7 mm thick, with brownish and woolly floss (orwa et al., 2009). a. shoot b. leaves and fruits c. mature fruit figure 1. different parts of azanza garckeana. table 1. uses of azanza garckeana. medicinal use plant part(s) used country practised dietary uses edible fruits fruits botswana, kenya, malawi, nigeria, sudan, tanzania, zambia, zimbabwe food additive fruits sudan, tanzania medicinal uses abscesses fruit poultice applied nigeria anemia ripe fruits sudan antiemetic root infusion taken orally zimbabwe aphrodisiac ripe fruits taken orally nigeria asthma root decoction mixed with sterospermum kunthianum cham. malawi chest pains root infusion taken orally nigeria, zimbabwe cough root infusion taken orally kenya, nigeria, zimbabwe diabetes leaf decoction taken orally drc earache root infusion dropped into ear zimbabwe edema leaf decoction taken orally drc epilepsy leaf decoction taken orally drc fever root decoction taken orally malawi gonorrhoea roots and stem bark taken orally malawi, nigeria induce labour root decoction taken orally tanzania infertility ripe fruits or root decoction taken orally botswana, malawi, nigeria liver problems stem and leaf decoction taken orally kenya, nigeria madness (mental illness) root decoction taken orally zimbabwe malaria eat raw fruit or cook and eat as relish zambia membrane rupture root decoction taken orally drc menstruation root infusion taken orally nigeria, zimbabwe retained placenta root infusion taken orally zimbabwe sexually transmitted diseases root and bark infusion taken orally zambia syphilis root decoction taken orally nigeria maroyi, 2017 bioltif et al. – a chemical overview of azanza garckeana 93 table 2. compounds extracted from azanza garckeana. compounds extract plant parts sesquiterpenoids gossypol, 6, 6-dimethoxygossypol, 6-methoxygossypol ethyl acetate in nhexane; methanol in dichloromethane root phytosterol stigmasterol ethyl acetate in nhexane; methanol in dichloromethane root and stem bark e-docosyl 3-(3, 4dihydroxyphenyl) acrylate ethyl acetate in nhexane; methanol in dichloromethane root and stem bark o-naphthoquinones n-hexane heartwood mansonones e, f, g, h azanzone a, b n-hexane heartwood triterpene betulinic acid ethyl acetate in nhexane; n-hexane; methanol in dichloromethane fruit pulp, root, stem bark dikko et al., 2016 & maroyi, 2017 azanza garckeana is widely distributed in the east, west and southern africa. it generally grows naturally in all types of woodlands from sea level to about 1700m above sea level. it also grows in semi-arid areas. azanza garckeana grows in a variety of soils and is found near termite mounds and deserted areas while in nigeria it grows in open woodland in the north eastern part of the country (ochokwu et al., 2015). azanza garckeana (goron tula) as an edible indigenous fruit in north eastern part of nigeria (particularly gombe state). fao (1983) reported that a. garckeana grows naturally in semi-arid areas receiving annual rain fall that range from 250mm to 1270mm. flowering takes place during the raining season, while fruit ripening occurs during the dry season, hence it takes about six months from flower fertilization to ripening of the fruit. in southern africa, flowering occurs from december to may and fruiting from february to september while in north eastern nigeria flowering occurs from may to october and fruiting/ripening from november to april (ochokwu et al., 2015). oh oh oh oh oh oh ch3 ch3ch3 ch3 ch3 ch3 o ch2 h h ch3 ch3 ch3 ch3 o o ch3 ch3 ch3 ch3 o o ch3 ch3 ch3 ch3 o o o gossypol mansonone c azanzone mansonone e ch3 ch3 ch3 o o o oh ch3 ch3 ch3 ch3 o o oh ch3 ch3ch3 h ch3 ch3 h h h oh ch3 mansonone h mansonone g stigmasterol ch3 h ch3 ch3 h h h oh ch3ch3 ch3 ch3 ch3 h ch3 oh h o oh h ch2 h ch3 ch3 o o phytosterol butulinic acid o-naphthoquinone figure 2. some compounds isolated from azanza garckeana. 94 biology, medicine, & natural product chemistry 9 (2), 2020: 91-95 effects of chemicals extracted from azanza garckeana mansonone e, c, g and h displayed antifungal activity against p. parasitica, with mansonone e showing the highest activity. also suggests potential mansonone e as a new natural pesticide for agricultural plant pathogen management. (mongkol1 and chavasiri, 2016). a computational and experimental study carried out on the anticancer ability of mansonone g by βcyclodextrin-based host-guest complexation revealed that the inclusion complex formation between mansone g and β-cyclodextrin was confirmed by dsc and sem techniques. notably, the mansone g/β-cyclodextrin inclusion complexes exerted significantly higher cytotoxic efect on a549 lung cancer cells than the uncomplexed mansonone g (mahalapbutr, 2019) anticancer activity, anti-hiv activity, antimalarial activity was recorded for betulinic acid, it also shows pronounced antinociceptive properties, antiinflammatory activity (moghaddam, 2012) the effect of the terpenoids gossypol, 6methoxygossypol, 6,60-dimethoxygossypol, gossypolone and apogossypolone on growth of fungal soil pathogens were investigated. gossypol, gossypolone and apogossypolone demonstrated strong growth inhibitory activity (≥90%) against pythium irregulare, pythium ultimum and fusarium oxysporum. these same terpenoids provided good growth inhibition against most rhizoctonia solani isolates. methylated gossypol derivatives generally yielded reduced growth inhibition against the tested fungi compared with gossypol. dose– response effects of gossypol, gossypolone and apogossypolone were determined over a concentration range (mellon, 2014). extracts from plants with high phytosterol (stigmasterol and β-sitosterol) content is used in the treatment of inflammatory conditions and prevention of cancers and cardiovascular diseases (ivanescu et al., 2013). sitosterols have been found to induce apoptosis when added to cultured human prostate, breast and colon cancer cells. therefore, they may play significant roles in the management and prevention of human cancers. beta-sitosterol preparation improved symptoms, increased peak urinary flow, and decreased post-void residual urine volume. however, relatively few controlled studies have examined the efficacy of phytosterol supplements in men with symptomatic bph. (ogbe, 2015). the plant has antifertility/contraceptive, antitumor properties (anticancer properties of gossypol against many types of cancer cell lines), antioxidant properties, antiparasitic properties, antivirus properties, antimicrobial properties, plasma cholesterol reduction properties. (keshmiri-neghab & goliaei, 2014). conclusion the various pharmacological activities of the chemicals extracted from the plant justifies the use of azanza garckeana as plant effective for various diseases and health conditions, especially sexually trelated issues, as exposed by various researches. the use of the plant for further research will help to make even drug discovery. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references dikko, y. j., khan, m. e., tor-anyiin, t. a., anyam, j. v. and linus, u. a. (2016). in vitro antimicrobial activity of fruit pulp extracts of azanza garckeana (f. hoffm.) exell and hillc. and isolation of one of its active principles, betulinic acid. br. j. pharm. res., 14: 1-10 maroy, a. (2017). azanza garckeana fruit tree: phytochemistry, pharmacology, nutritional and primary healthcare applications as herbal medicine: a review. res. j. med. plants, 11 (4): 115-123, 2017. musinguzi, e. l., kikafunda, j. k. and kiremire, b. t. (2007). promoting indigenous wild edible fruits to complement roots and tuber crops in alleviating vitamin a. deficiencies in uganda. proceedings of the 13th istrc symposium.; 763769. nkafamiya, i. i., modibbo, u. u., manji, a. j. and haggai, d. (2007). nutrient content of seeds of some wild plants. afr. j. biotech. 6(14):1665-1669. aberoumand, a. and deokule, s. s. (2009). studies on nutritional values of some wild edible plants from iran and india. pak. j. nutrition. 8(1): 26-31. nazarudeen, a. (2010). nutritional composition of some lesserknown fruits used by ethnic communities and local folks of kerela. ind. j. traditional knowl., 9(2): 398-402. food and agricultural organization (fao) (1983). food and fruit bearing forest species. examples from eastern africa. food and agricultural organization, forestry rome.14-16. nkafamiya, i. i., ardo, b. p., osemeahon s. a. and akinterinwa, a. (2016). evaluation of nutritional, non-nutritional, elemental content and amino acid profile of azanza garckeana (goron tula). british journal of applied science & technology, 12(6): 1-10. keshmiri-neghab, h. and goliaei, b. (2014). therapeutic potential of gossypol: an overview. pharmaceutical biology, 52(1): 124–128. https://doi.org/10.3109/13880209.2013.832776 mellon, j.e., dowd, m.k., beltz, s.b. and moore, g.g. (2014). growth inhibitory effects of gossypol and related compounds on fungal cotton root pathogens. letters in applied microbiology 59, 161-168. ogbe, r. j., ochalefu, d. o., mafulul, s. g. and olaniru, o. b. (2015). a review on dietary phytosterols: their occurrence, metabolism and health benefits. asian j. plant sci. res., 5(4):10-21. mongkol1, r. and chavasiri, w. (2016). antimicrobial, herbicidal and antifeedant activities of mansonone e from the bioltif et al. – a chemical overview of azanza garckeana 95 heartwoods of mansonia gagei drumm. journal of integrative agriculture 15(12): 2795–2802. moghaddam, m. g. ahmad, f. b. h. and samzadeh-kermani, a. (2012). biological activity of betulinic acid: a review. pharmacology & pharmacy, 3, 119-123. mahalapbutr, p., wonganan, p., charoenwongpaiboon, t., prousoontorn, m., chavasiri, w. and t. rungrotmongkol (2019). enhanced solubility and anticancer potential of mansonone g by β-cyclodextrin-based host-guest complexation: a computational and experimental study, biomolecules, 9: 1-17. doi:10.3390/biom9100545 ivanescu, b., vlase, l. and a. corciova (2013). importance of phytosterols and their determination in herbal medicines, the 4th ieee international conference on e-health and bioengineering, ochokwu, i. j., dasuki, a., & oshoke, j. o. (2015). azanza garckeana (goron tula) as an edible indigenous fruit in north eastern part of nigeria. journal of biology, agericulture and healthcare, 5(15), 26-31. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 133-136 | doi: 10.14421/biomedich.2022.112.133-136 issn 2540-9328 (online) the triterpenes of kageneckia oblonga jorge tapia-merino1,*, lily arrue2, josé san martín3, orlando muñoz4 1universidad bernardo o´higgins, facultad de salud, departamento de ciencias químicas y biológicas, general gana 1702, santiago, chile. 2centro de investigación de estudios avanzados del maule (cieam) vicerrectoría de investigación y postgrado, universidad católica del maule, chile. 3instituto de ciencias biológicas, universidad de talca, talca, chile. 4departamento de química, facultad de ciencias, universidad de chile, casilla 653, santiago, chile. corresponding author* jorgeat@uc.cl manuscript received: 27 june, 2022. revision accepted: 22 july, 2022. published: 01 august, 2022. abstract three known triterpenes were isolated from leaf extracts of kageneckia oblonga by conventional chromatographic methods: ursolic acid, benthamic acid and a third called kc-iii. the structure of kc-iii was determined by rmn spectroscopy, ft-it and hr-ms. the compound was identified as fern-7-en-3β-ol (motiol), not previously reported in kageneckia. keywords: kageneckia oblonga; motiol; triterpene derivative; hopane. introduction kageneckia oblonga ruiz & pav (known as “bollén”) is an evergreen tree native to chile. it is found from the northern part of coquimbo to malleco in north-central and central chile, in dry or semi-humid soils below 1800 m elevation in the lower part of the andes range. its wood is hard and resistant, due to which the natives used it to construct tools.gay, 2010) . leaves and roots of k. oblonga are used in ethnomedicine; infusions or powders are used to treat fevers, renal and hepatic lesions and digestive problems. these medicines should be used in moderation, since an excess may cause poisoning. its use as a medicinal plant has decreased due to this property. its seeds are sometimes used to treat the “evil eye” (berguecio, nicolás garcía. pagliotti, 2008; gay, 2010; mélica. muñoz & barrera, 1981). previous studies of k. oblonga have reported triterpenes and cyanogenic glucosides, ursolic acid and benthamic acid (cassels et al., 1973); cucurbitacins and a new cucurbitane triterpene (23,24 dihidrocucurbitacin f). biological and toxicological trials of cucurbitacins isolated from different extracts of the tree showed high toxicity in dichloromethane and methanol extracts. biological activity trials to test anti-inflammatory, antipyretic and analgesic effects showed that the cucurbitacins found are partly responsible for these biological effects (delporte et al., 2002; o. muñoz et al., 2002). a screening of 31 chilean medicinal plants evaluated for anti-trypanosoma cruzi activity found that the methanol extract of showed significant inhibition in the mtt trial: mtt ic50 = 35.7 ± 40 ug/ml. these trials were performed with t. cruzi trypomastigotes (o. muñoz et al., 2013). the compound fern-7-en-3β-ol was previously isolated from several plant species, including ainsliaea yunnanensis, hibiscus cannabinus, rhododendron macrocepalum, rhodendron linearifolium and scorzonera latifolia, among others (acikara et al., 2014; ageta & ageta., 1984; nakamura et al., 1965; seca et al., 2000). the compound kc-iii has some important pharmacological properties. it has been reported that it has anti-cancer agonistic properties against the cell line thp-1 (ic50 of 1.75 µm) used to study leukemia (li et al., 2016). the presence of this triterpene in k. oblonga reinforces the biological effects of this tree. material and methods experimental section samples of k. oblonga were collected in april, 2017 near the las trancas bridge in the quebrada del cepillo sector, road g-546, 8 km from the laguna aculeo, province of maipo, metropolitan region, chile (33°50'02.3"s 71°01'17.2"w). the plants (4.2 kg) were identified by dr. josé san martin (u. of talca). a herbarium specimen was stored in the natural products laboratory of the science faculty of the university of chile, identified as n°0017-017. the leaves (used for extraction) were removed from the branches and dried for three weeks in the shade. then they were ground in an electric grinder with a 1.5 mm grill. the powdered material (2.6 kg) was placed in 5l glass recipients and degreased with hexane (3*4l), after which a sufficient https://doi.org/10.14421/biomedich.2022.112.133-136 134 biology, medicine, & natural product chemistry 11 (2), 2022: 133-136 volume of dcm was added to cover the plant material (3*3 l). the mixture was percolated with sporadic agitation for 72 hours. the extract was then filtered and concentrated by distilling to dryness under reduced pressure. this procedure was repeated three times, obtaining a total of 18g of dcm extract. extraction and isolation the total h-dcm extract was placed on a silica gel 60 chromatographic column (merck). dichloromethane was used as the mobile phase; 35 fractions were obtained. chromatographic analysis of the fractions revealed a mixture of the triterpenes ursolic acid and benthamic acid. these compounds were identified by tlc, using commercial standards and estimating their fusion points. the remaining fractions were concentrated in a rotatory evaporator, dried with anhydrous na2so4, filtered and concentrated to a yellow residue. this fraction was purified by crystallization, dissolving the sample in methanol and then cooling to 0 °c. the result was 0.14g of white kc-iii crystals. analysis of the sample some of the separation and purification steps were performed in the department of phytochemistry and bioactive natural products university of geneva unige(suiza). luhplc was performed on an acquity i-class plus uplc system hyphenated with an acquity photodiode array (pda) detector (waters). the separation was performed in an acquity beh c18 uplc column (50 mm × 2.1 mm i.d.; 1.7 μm, waters), using a generic gradient (mecn and h2o both containing 0.1% formic acid) of 5% to 98% mecn in 4 min, followed by a washing step with 98% mecn for 2 min. after the washing step, the column was equilibrated with 5% mecn for 2 min before the next injection. the flow rate was set to 0.6 ml/min, the temperature to 40 °c, and the injection volume was 1 μl. hrms: hrms spectra were obtained on a q exactive focus hybrid quadripole-orbitrap mass spectrometer (thermo scientific, waltham, ma, usa) using electrospray in positive or negative mode. the spray voltage was set at 3.5 kv or 2.5 kv; the sheath gas flow rate (n2), 50 units; the capillary temperature, 320 °c; the s lens rf level, 50 and the probe heater temperature, 425 °c. spectroscopy and complementary spectroscopic analyses of 1h-rmn, 13c-rmn, cosy, hmqc and hmbc were performed in a bruker avance 400 mhz nuclear magnetic resonance spectrometry at 25 ºc. the kc-iii sample was dissolved in deuterated chloroform. tetramethylsilane was used as internal standard. the spectra were processed using the mestre nova 9.0 program. the ft-ir spectrum was obtained from a bruker ft-ir perkin-elmer 1310 spectrometer in kbr disks recorded from 500-4000cm-1. these last rmn and ft-ir analyses were performed in the instrumentation unit of the pontificia universidad católica de chile. results compound kc-iii was identified as fern7-en-3β-ol (motiol) by spectroscopic comparison and mp: 210-213 °c. (ageta & ageta., 1984; nakamura et al., 1965; seca et al., 2000). figure 1. kc-iii structure, fern7-en-3β-ol (motiol). fern-7-en-3β-ol (motiol): translucent yellow crystals, apparently rectangular in shape. yield: 0.14g, 0.004% of dried leaf. mp: 210-213 °c. ir: 3500 cm-1, 2941 cm1,2852 cm-1,2361 cm-1,1469 cm-1,1386 cm-1. esims: 514.25 m/z, [ m+ch3cn+ hcooh] +; 498.26 m/z, [ m-ch3 + ch3cn + hcooh] +; 404.20 m/z, [ m -oh ch3cn] +; 227.10 m/z y 167.01 m/z [ fragmentation d ring] +. 1h-rmn: δ 5.37 (d, j = 3.6hz, 1h, h-7), 3.23 (1h, h-3), 2.34 (m, 1h, h-9), 2.17 (m, 1h, h-6a), 2.00 (m, 1h, h-6b), 1.82 (m, 1h, h-20), 1.73 (m, 1h,h-16a), 1.69 (m, 4h, h-1,2), 1.58 (m, 1h, h-11a), 1.54 (m, 1h, h-11b), 1.50 (m, 1h, h-16), 1.48 (m, 1h, h-18), 1.46 (m, 1h, h-15), 1.43(m, 1h, h-22), 1.40(m, 1h, h-19a), 1.35 (m, 1h, h-12a), 1.33 (m, 1h, h-5), 1.32 (m, 1h, h12b), 1.25 (m, h, h-19b) 1.24 (m, h, h-20), 1.06 (m, 1h,h-2), 0.99 (s, 3h, h-26), 0.96 (s, 4h, h-24,h-21), 0.91 (s, 3h, h-30), 0.89 (s, 3h, h-27), 0.85 (s, 3h, h23), 0.83 (d, j = 6.7 hz3h, h-29), 0.74 (s, 3h, h-25), 0.73 (s, 3h, h-28).13c-rmn: δ 145.29 (c-8), 116.28 (c-7), 79.41 (c-3), 59.71 (c-21), 54.26 (c-18), 50.89 (c-5), 48.04 (c-9), 42.99 (c-17), 41.66 (c-14), 39.10 (c-4), 36.95 (c-2); 36.41 (c-16), 36.21 (c-13), 35.48 (c-10), 32.47 (c-12), 30.82 (c-22), 30.44 (c-15), 28.37 (c-20), 27.85 (c-1), 27.67 (c-24), 24.29 (c-6), 24.15 (c-26), 23.14 (c-29), 22.26 (c-30), 21.20 (c-27), 20.15 (c-19), 16.22 (c-11), 14.76 (c-23), 14.19 (c-28), 13.01 (c-25). tapia-merino et al. – the triterpenes of kageneckia oblonga 135 discussion the ir spectrum of compound kc-iii allowed the rapid assignment of the signal at 3500 cm-1, assigned to an alcohol. the absorbances between 2350 and 2450 cm-1 were assigned to an alicyclic skeleton, with possible presence of a double bond, and the proton of neighboring hydrogens to oh. complementary analysis with reported spectroscopic data for this kind of structure allowed deducing that kc-iii should have a hopane skeleton. the mass spectrum signals showed that ion 514.25 m/z could be assigned to the sum of the masses of the sample, acetonitrile and formic acid [m+ ch3cn+ hcooh]+. the 498.26 m/z and 496.24 m/z fragments correspond to the mentioned adduct, but with a loss of 15 uma, typical of the loss of a methyl group [mch3+ ch3cn+ hcooh] + for 498.26m/z. by the 18 m/z difference from 514.25m/z it may be inferred that the signal at 496.24 m/z is due to dehydration, [m+ ch3cn+ hcoohh2o] +. ion 404.20 m/z was assigned to the mass of the molecule, with loss of the hydroxyl at c-3 and isopropyl in c-21 forming two new double bonds in their respective carbons that underwent the elimination and addition of acetonitrile [m-oh-58]+. in consequence, compound kc-iii has the molecular formula c30h50o. the c-nmr showed 30 signals, including eight methyl groups, nine methylene groups, seven methine groups and four quaternary carbons, all deduced from the dep 135 experiments. the high-field signals of the h-nmr spectrum showed six singletons, assignable to tertiary methyl groups and two secondary methyl groups, assigned as doublets. the δ 5.37ppm signal (1h, q, j = 3.3 hz) is typical of double bonds, ratifying the ir signal at 3050 cm-1 and 1639 cm-1, while the h-3 axial singleton at δ 3.23 ppm is displaced to the low field due to the presence of the 3 β-oh group in c-3. the analysis of the bi-dimensional spectra and the conclusions of the previous paragraphs allow assigning the double bond to the b ring, and the connectivity shown in figure 2. the interactions of the hydrogens (cosy) show the correlations of the h-22 with the hydrogens of the h-29 and h-30 methyl groups; the interaction of h-22 with h-21. both interactions describe the union of the isopropanol group in the c-21 carbon, which coincides with the fractioning of the ms of 167 m/z, corresponding to a retro diels-alder ion of the e ring after losing a methyl group. the hmbc spectrum (s12) shows evidence of the union of the a-b, b-c, c-d and d-e rings by the interactions of the c-3 and h-5 atoms; h-5 with c-9, h9 with c-12; h-12 with c-18 and h-18 with c-21, respectively. the position of the geminal group formed by c-23 and c-24 in the a ring is inferred by the interactions with c-5 and h-3 with the methyl substituents in c-4. the position of c-24 between the union of the a-b rings is deduced from the interactions of c-5 and c-9. c-26 and c-27 in the union of the c-d rings is interpreted by the interaction of its hydrogens with c-13, c-14, c-15 and c-18. the c-28 methyl is assigned as the substituent in c-17 by the interactions of h-28 with the four carbons that surround c-17. the interaction of c-29 and c-30 and the interaction of its ch3 hydrogens with c-21 reaffirm the isopropyl structure in the molecule. figure 2. hmbc coupling of the kc-iii structure. conclusion the triterpene fern-7-en-3β-ol (motiol) is reported for the first time in the genus kageneckia, and thus in k. oblonga. the presence of motiol in the leaves of k. oblonga along with the presence of other substituted triterpenes and cucurbitacins would explain some of the medicinal properties of this tree. the toxicity of its 136 biology, medicine, & natural product chemistry 11 (2), 2022: 133-136 extracts recommends against its medicinal use. however, this native chilean tree has potential uses as an antiparasite, anti-inflammatory and anti-carcinogen. thus pharmacological in vitro, in vivo, pre-clinical and clinical trials should be continued, to produce the bases for a specific and sure phytopharmaceutical for the population, which self-medicates with crude extracts. acknowledgments: we warmly thank professor dr. philippe christen and dr. sylvian creton for advice and assistance with spectroscopic measurements. competing interests: the authors declare that there are no competing interests. references acikara, b., glu, g. s. c., dall’acqua, s., özbek, h., ka, j. c., žemlič ka, m., & šmejkal, k. (2014). bioassay-guided isolation of the antinociceptive compounds motiol and βsitosterol from scorzonera latifolia root extract. pharmazie, 69(9), 711–714. https://doi.org/10.1691/ph.2014.3920 ageta, h., & ageta., t. (1984). ercaceous constituyents: seventeen triterpenoids isolated from the buds of rhododendron macrocepalum. chem. pharm. bull., 32, 369– 372. https://doi.org/10.1061/͑asce͒1090-02412͑0031͒29:11͑2 ͒ berguecio, nicolás garcía. pagliotti, c. o. (2008). arboles nativos chilenos. enersis s.s. http://fundacionphilippi.cl/sites/default/files/arboles-nativosenersis.pdf cassels, b., urzúa, a., cortez, m., & gabardino, juan. (1973). triterpenoid constituents of kageneckia oblonga. phytochemistry, 12, 3009. delporte, c., muñoz, o., rojas, j., ferrándiz, m., payá, m., erazo, s., negrete, r., maldonado, s., san feliciano, a., & backhouse, n. (2002). pharmaco-toxicological study of kageneckia oblonga, rosaceae. zeitschrift fur naturforschung section c journal of biosciences. https://doi.org/10.1515/znc-2002-1-218 gay, c. (2010). historia física y política de chile. botánica, tomo vii. biblioteca fundamentos de la constrcción de chile. li, j., zhang, b., liu, h., zhang, x., shang, x., & zhao, c. (2016). triterpenoids from ainsliaea yunnanensis franch. and their biological activities. molecules, 21(11), 1–7. https://doi.org/10.3390/molecules21111481 muñoz, mélica., & barrera, elizabeth. (1981). el uso medicinal y alimenticio de plantas nativas y naturalizadas en chile. museo nacional de historia natural. publicacion ocacional, 33, 63. muñoz, o., maya, j., ferreira, j., & christen, p. (2013). medicinal plants of chile: evaluation of their antitrypanosoma cruzi activity. zeitschrift fur naturforschung c, 198–202. https://doi.org/10.5560/znc.2013.68c0198 muñoz, o., ravelo, a., & gonza, a. (2002). cucurbitacin f in seeds of kageneckia angustifolia (rosaceae). zeitschrift fur naturforschung, 24–26. nakamura, s., yamada, t., wada, h., & inoue., y. (1965). the structures of five new triterpenoids obtained from rohodendron linearifolium. tetrahedron letters, 6(24), 2017– 2022. seca, a., silva, a., silvestre, a., cavaleiro, j., domingues, f., & neto, c. (2000). chemical composition of the light petroleum extract of hibiscus cannabinus bark and core. phytochemical analysis, 11(6), 345–350. https://doi.org/10.1002/10991565(200011/12)11:6<345::aid-pca540>3.0.co;2-t biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 1-4 | doi: 10.14421/biomedich.2023.121.1-4 issn 2540-9328 (online) preliminary investigative study on the blood pressure-lowering potential of aqueous leaf extract of simarouba glauca (aesg) on normotensive adult wistar rats sammy davies e. osagie-eweka1,*, noghayin e.j. orhue1, fabian c. amaechina2, eric k.i. omogbai2, emuesiri g. moke3 1department of biochemistry, faculty of life sciences, university of benin, benin city, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, university of benin, benin city, nigeria. 3department of pharmacology, faculty of basic medical sciences, delta state university, abraka, nigeria. corresponding author* davies.osagie-eweka@uniben.edu manuscript received: 26 july, 2022. revision accepted: 31 august, 2022. published: 13 september, 2022. abstract studies have shown that plants possess medicinal properties and compounds are beneficial in managing and treating diseases, including high blood pressure and related cardiovascular conditions. simarouba glauca (sg) has been widely reported to possess antibacterial activity, anti-oxidant, anti-proliferative and hemolytic activity; amongst others. however, there is paucity of data on its effect on blood pressure. hence, the study research aimed at assessing the hypotensive prospect inherent in the aqueous leaf extract of simarouba glauca (aesg) on normotensive male wistar rats. the study was conducted using adult male wistar rats (n = 3), a urethane/thiopental (1205/20 mg/kg) anesthesia and a chart paper attached to ugo basile uni-recorder model 400700 data capsule. under full anesthesia, the rat’s trachea and the carotid artery were cannulated for assisted respiration and blood pressure measurement. at stable variables; following the administration of 0.2 ml normal saline, the aesg was administered intravenously via the caudal vein at 2.5 and 5.0 mg/kg body weight dose respectively. the data was recorded on a chart; indicated the characteristic dose-dependent hypotensive effect of aesg on normotensive rats; at doses of 2.5 mg/kg and 5.0 mg/kg, with marked decreases in the systolic blood pressure (sbp), diastolic blood pressure (dbp) and mean arterial pressure (map) from basal levels of 127.83 ± 1.01 mmhg, 91.00 ± 1.00 mm hg and 103.27 ± 0.99 mm hg respectively. the outcome of the preliminary investigation indicates that the aesg demonstrated a hypotensive effect on the bp of normotensive male wistar rats dependent on varying doses administered; indicative of further evaluation. keywords: cardiovascular; caudal vein; invasive blood pressure; simarouba glauca. abbreviations: sg simarouba glauca; aesg: aqueous leaf extract of simarouba glauca; sbp: systolic blood pressure; dbp: diastolic blood pressure; map: mean arterial pressure; acei: angiotensin converting enzyme inhibitors; ara: angiotensin receptor antagonists. introduction hypertension is a public health condition characterized by chronic cardiovascular disease and end-stage renal diseases (fuchs and whelton, 2020). untreated hypertension predisposes risk factors like stroke, myocardial infarction, arteriosclerosis, cardiac arrest, heart attack, cardiomegaly and amongst others (landazuri et al., 2017). some allopathic agents applied in the management of hypertension include; adrenergic antagonists (alpha and beta receptor blockers) like prazosin and atenolol, centrally acting sympatholytic agents (alphamethyldopa, guanabenz and clonidine), calcium channel blockers (nifedipine and amlodipine), diuretics (hydrochlorothiazides), angiotensin converting enzyme inhibitors – acei (lisinopril and ramipril), angiotensin receptor antogonists – ara (losartan and valsartan) and the aldosterone antagonists (spironolactone and eplerenone) have been extensively reported to elicit adverse effects like dry cough, severe hypotension, depressed libido and sometimes erectile dysfunction (landazuri et al., 2017; olowofela and isah, 2017; moke et al., 2022); as such, the antihypertensive effect achieved with these agents is always short lived. in addition to the arrays of these side effects, virtually all these antihypertensive agents are cost implicative, creating a huge burden in the purse of affected lowincome earners worldwide (lacy et al., 2008; pr et al., 2014). hence, there is the need to adequately evaluate the anti-hypertensive (hypotensive) potential of cheap and available plants with proven medicinal properties as have been the case in the last four decades (tabassum and ahmad, 2011; pr et al., 2014). there are a number of medicinal plants with folk history that have been applied to treat hypertension; a https://doi.org/10.14421/biomedich.2023.121.1-4 2 biology, medicine, & natural product chemistry 12 (1), 2023: 1-4 few investigations have shown their effectiveness, while others have been disproved by scientific findings (tabassum and ahmad, 2011; kamyab et al., 2021). literatures have reported vast findings on the ethnomedicinal benefits of s. glauca (patil and gaikwad, 2011; ramasamy et al., 2022) with no record on the effect on cardiovascular system and blood pressure; hence this study. materials and methods collection of plant material and preparation of aqueous extract fresh leaves of simarouba glauca were procured from cercobela farms®, ubiaja. fresh plant specimen was authenticated and deposited with voucher specimen no. ubhs382 at plant biology and biotechnology department herbarium, university of benin. the plant leaves were properly washed with clean water and then dried at room temperature for twenty-eight (28) days. fine powdery particles were obtained following pulverization of the dried crispy leaves of s. glauca. five hundred grams (500 g) of the leaf powder was macerated in 2.5 l distilled water and stimulated intermittently for forty-eight hours (48 hrs.) to obtain a filtrate. the filtrate was lyophilized with a freeze-drier to obtain the aqueous extract (osagie-eweka et al., 2016). materials for invasive procedure for the invasive procedure, the materials used included the following: an intravenous cannula, eighteen g needles, a surgical table, respiratory tubing (6′′ pediatric ryle's tube may be used). one milliliter tuberculin syringe, 5, 10 ml syringes, small (3′′) and medium (5′′) c, adson dissecting forceps (toothed and non-toothed) (5′′), artery forceps (5′′), small and medium forceps (with teeth, blunt and pointed), a bulldog clamp, a surgical lamp, an insertion needle, a surgical blade, normal saline, a thread, adhesive tape and prepared stock solution of the aqueous extract with appropriate concentrations. distilled water was used in the preparation of the required stock solution of the aqueous leaf extract of s. glauca. pressure transducer calibration procedure calibration is an imperative step in the experiment; it was conducted with a sphygmomanometer at a specific pressure. the pressure cuff was disconnected from the sphygmomanometer; linked to the transducer with a physiograph data acquisition system. inflating to a required specific pressure was performed to check the pressure transducer. the mathematical conversion factor to express the blood pressure was established. the calibration between the voltage (millivolts) and pressure in the data acquisition system was previously performed; results were automatically calculated relative to the system calibrated value (ordodi et al., 2005). animal experimental procedure for cannulation of the caudal vein adult male wistar rats (210 220 g) were procured from the rat housing facility of pharmacology and toxicology department, university of benin. experimental animals were anaesthetized with urethane/thiopentone (1250/20 mg/kg) (amaechina and omogbai, 2007; wang et al, 2013) administered intra-peritoneally. an established protocol for invasive blood pressure assessment (amaechina and omogbai, 2007; wang et al, 2013) was used. the caudal vein of the rat was cannulated with a heparinized saline-filled-23 g scalp-vein needle for the administration of extract intravenously and was fastened to the dissecting table dorsally. the cervical region was shaved and dissected open to reveal both the trachea and carotid artery (plehm et al., 2006). the trachea was isolated, cleared of connective tissues and cannulated with a 2 mm diameter polythene tube for assisted respiration (kramer and remie, 2005). the carotid artery was isolated, cleared of adhesive tissues, and cannulated with a heparinized saline-filled teflon polyethylene tube connected to a pressure transducer for the transmission of blood pressure variations to ugo basile uni-recorder (model: 400700). an angle poised lamp with a 60 watts electric bulb is positioned over the anaesthetized animal, for the purpose of maintaining the temperature within normal range. when all the measurable variables remained stable as confirmed following the administration of normal saline, aesg was administered to experimental rats intravenously at two varying doses of 2.5 and 5.0 mg/kg respectively. this procedure was conducted in triplicate; the effects of the administered doses were recorded on the ugo basile chart recorder. results the data presented in table 1 reveal that aesg elicited significant (p ˂ 0.05) dose-dependent decreases in the sbp, dbp and map at 2.5 mg/kg (122.00 ± 1.15 mmhg, 84.67 ± 2.40 mmhg; 97.10 ± 1.99 mmhg) and 5.0 mg/kg (84.00 ± 2.31 mmhg, 62.67 ± 1.45 mmhg; 69.80 ± 1.73 mmhg) doses respectively; compared to the experimental rat treated with normal saline (127.83 ± 1.01 mmhg, 91.00 ± 1.00 mmhg; 103.27 ± 0.99 mmhg). likewise, the polygraph presented in figure 1 indicates marked dose-dependent decreases in the hemodynamic parameters considered at 2.5 and 5.0 mg/kg body weight respectively, when compared to the normal saline group. osagie-eweka et al. – preliminary investigative study on the … 3 table 1. effect of aesg on hemodynamic parameters of normotensive rats. parameters systolic blood pressure (mmhg) diastolic blood pressure (mmhg) mean arterial pressure (mmhg) normal saline 127.83 ± 1.01 91.00 ± 1.00 103.27 ± 0.99 aesg 2.5 mg/kg 122.00 ± 1.15 84.67 ± 2.40 97.10 ± 1.99 aesg 5.0 mg/kg 84.00 ± 2.31*# 62.67 ± 1.45*# 69.80 ± 1.73*# all values expressed as mean ± sem, where n=3, all data were analyzed by using one-way anova followed by tukey’s post hoc test. *p<0.05 compared to the normal saline control group; #p<0.05 compared to aesg 2.5 mg/kg group. figure 1. the effect of aesg on blood pressure of normotensive rats. discussion the study reveal a 4.6 % decrease in systolic blood pressure (sbp), 7.0 % decrease in diastolic blood pressure (dbp); 6.0 % decrease in mean arterial pressure (map) at 2.5 mg/kg compared to the normal saline group of the experimental rats. furthermore, at 5.0 mg/kg. aesg showed a significant (p < 0.05) decrease in sbp (34.29%), dbp (31.13%) and map (32.41% ) relative to the normal saline group. additionally, the group treated with 5.0 mg/kg showed 31.15%, 25.98%, 28.12% significant (p < 0.05) decreases in sbp, dbp and map respectively relative to the group treated with 2.5 mg/kg. the outcome of the study therefore indicate a better and promising blood pressure lowering effect at 5 mg/kg when administered intravenously. the data presented in figure 1 likewise indicate that there was instananeous recovery of the blood pressure to the basal level which may be obviously not unconnected to the reflex compensatory mehanism aimed at restoring the blood pressure to normal after the adminisration anti-hypertensive (kuogias et al., 2010). in the present study, a similar effect was observed at a dose of 5.0 mg/kg. however, the recovery was not sustained as there was a second phase derease in the blood pressure which was more sustained as observed in figure 1. thus, the outcome and resultant effect on blood pressure suggests that s. glauca may possess some promising active principles capable of eliciting hypotensive effect on the cardiovascular system. in fact, studies have reported the hypotensive and (or) the blood pressure-lowering potentials of several known medicinal plants (anaka et al., 2009; imafidon and amaechina, 2010; amaechina et al., 2017; alawode et al., 2021; kamyab et al., 2021) with less adverse effects. however, pharmacologist must continue to conduct systematic inquiry into the therapeutic benefits of plants with hypotensive potentials in the quest to discover the most effective mechanistic treatment approach for hypertension considering the complexities associated with the condition. accordingly, it is recommended that s. glauca may be subjected to detailed and extensive laboratory investigation to ascertain its pharmacological pertinence. conclusion the outcome of the preliminary investigative study of aesg (aqueous leaf extract of s. glauca) on cardiovascular system indicate a strong blood pressure lowering potential and a promising vaso-relaxant bioactive compound that may be beneficial in managing hypertension related conditions author(s) contributions statement: sdeo, nejo, fca, ekio and egm participated in research design. sdeo and egm conducted the preliminary experiments. sdeo and fca participated in data analyses. sdeo, fca and egm wrote the manuscript. 4 biology, medicine, & natural product chemistry 12 (1), 2023: 1-4 all authors have approved the final version of the manuscript. conflict of interests: authors state that there is no conflict of interest in this research output. ethical approval: the experimental protocols were approved by the faculty of pharmacy, university of benin ethics committee with reference number ec/fp/021/11. funding: the study was self funded at the departments of biochemistry/pharmacology. references alawode d, asiwe j, moke e, okonofua d, sanusi k, adagbada e, yusuf m, fasanmade a (2021). the effect of ethanol leaf extract of cnidosculus aconitifolius on cardiorenal functions in hypertensive and normotensive male wistar rats. international journal of nutrition sciences 6(3): 155160. amaechina fc, omogbai eki (2007). hypotensive effect of aqueous leaves extract of phyllanthus amarus schum and thonn (euphorbiacee). acta poloniae pharmaceutica-drug research 64: 547-52. amaechina fc, uchendu ap, oboh ci, agokei ni, eboka cj (2017). preliminary comparative effect of the aqueous extract of persea americana seeds on the blood pressure of normotensive rabbits and rats. journal of science and practice of pharmacy 4(1):177-181. anaka on, ozolua ri, okpo so (2009). the effect of the aqueous seed extract of persea americana mill (lauraceae) on the blood pressure of sprague dawley rat. african journal of pharmacy and pharmacology 3(10): 485-490. fuchs fd, whelton pk (2020). high blood pressure and cardiovascular disease. hypertension 75(2):285-292. imafidon ke, amaechina fc (2010). effects of aqueous seed extract of persea americana mill. (avocado) on blood pressure and lipid profile in hypertensive rats. advanced biomedical research 4(2): 116-121. kamyab r, namdar h, torbati m, ghojazadeh m, araj-khodaei m, fazljou smb (2021). medicinal plants in the treatment of hypertension: a review. advance pharmaceutical bulletin 11(4):601-617. kougias p, weakley sm, yao q, lin ph, chen c (2010). arterial baroreceptors in the management of systemic hypertension. medica science monitor 16(1): ra1-8. kramer k, remie r (2005). measuring blood pressure in small laboratory animals. methods in molecular medicine 108: 51-62. lacy cf, armstrong ll, goldman mp (2008). drug information handbook. 17th (ed.), hudon, oh: lexi-comp; ahfs drug information. bethesda ed. american society of healthsystem pharmacists. landazuri p, chamorro nl, cortes bp (2017). medicinal plants used in the management of hypertension. journal of analytical and pharmaceutical research 5(2):00134. moke eg, ekuerhare b, enaohwo mt, asiwe jn, ofulue oo, umukoro ek, isibor np (2022). resistant hypertension. journal of drug delivery and therapeutics 12(3-s):230-235 olowofela ao, isah ao (2017). a profile of adverse effects of antihypertensive medicines in a tertiary care clinic in nigeria. annals of african medicine 16(3):114-119. ordodi vl, mic fa, mic aa, toma o, sandesc d, paunescu va (2005). simple device for invasive measurement of arterial blood pressure and ecg in the anesthesized rat. timisoara medical journal 55: 35-37. osagie-eweka sde, orhue nej, ekhaguosa do (2016). comparative phytochemical analyses and in-vitro antioxidant activity of aqueous and ethanol extracts of simarouba glauca (paradise tree). european journal of medicinal plants 13(3): 1-11. patil ms, gaikwad dk (2011). a critical review on medicinally important oil yielding plant laxmitaru (simarouba glauca dc) journal of pharmaceutical sciences and research 3(4): 1195-1213. plehm r, barbosa me, bader m (2006). animal models for hypertension/blood pressure recording. methods in molecular medicine 129: 115-126. pr r, hv a, shivamurthy m (2014). anti-hypertensive prescribing patterns and cost analysis for primary hypertension: a retrospective study. journal of clinical and diagnostic research 8(9): hc19-22. ramasamy sp, rajendran a, pallikondaperumal m, sundararajan p, husain fm, khan a, hakeem mj, alyousef aa, albalawi t, alam p, ali hm, alqasim a (2022). broad-spectrum antimicrobial, antioxidant, and anticancer studies of leaf extract of simarouba glauca dc in vitro. antibiotics (basel) 11(1):59. tabassum n, ahmad f (2011). role of natural herbs in the treatment of hypertension. pharmacognosy reviews 5(9): 30-40. wang y, cong y, li j, li x, li b, qi s (2013). comparison of invasive blood pressure measurements from the caudal ventral artery and the femoral artery in male adult sd and wistar rats. plos one 8(4): e60625. biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 15-19 | doi: 10.14421/biomedich.2014.31.15-19 the effect of water-soluble stem extract “kayu kuning” (arcangelisia flava l. merr) on the growth inhibition of candida albicans atcc 10231 and trichophyton mentagrophytes in vitro rini setyowati, sudarsono* and setyowati e. p faculty of pharmacy ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract “kayu kuning” (arcangelisia flava l.merr) was used when someone has a skin problem caused by candida albicans and trichophyton mentagrophytes. scientific based medicine on this traditional knowledge was necessary be done. stem powderwas extracted by distilled water.the extract was then evaporated. qualitative and quantitative analysis of the active substance e.g., berberin chloride by thin layer chromatography (tlc) the antifungal activity againts candida albicans and trichophyton mentagrophyteswere tested by using agar diffusion and microdilution methods. the absorbance from microdilution were analized by one way anova. the conclusion showed that the extract contained 1.55±0.12% w/walkaloid calculated as berberine chloride. the inhibition zone for candida albicans and trichophyton mentagrophytes were 16.65±4.52 and 6.55±0.05 mm respectively. the mic vallue for both fungi was 10 mg/ml.the mbc value for candida albicans was 40 mg/ml and for trichophyton mentagrophytes was 50 mg/ml. from the analysis with one-way anova, shows that there are significant differences between the positive control group and the test solution with the negative control group with p=0.020 for candida albicans and p=0.028 for trichophyton mentagrophytes (p<0.050). post hoc tukey analysis results showed that both intergroup and between the concentration of the test solution to the control group did not differ significantly positive because the value of p>0.050. keywords: arcangelisia flava l.merr, “kayu kuning”, growth inhibitor, candida albicans, trichophyton mentagrophytes introduction candidiasis caused by c.albicans, water fleas and dermatophytosis caused by t.mentagrophytes. this infection disease is usually treated by azoles derivatives which are ketoconazol, fluconazol, miconazol, and also the poliena classes, such as nistatin, contemporarily for the dermatophyte can be treated by gryseofulvine (jawetz et al., 2001). however, using antibiotic reported often causes microbes resistance (who, 2009). indonesia has occupied on eight ranking out of 27 countries by heavy burden for multi-drugs resistancy/mdr around the world. according to health ministry of republic of indonesia (2011).the use of antibiotics for health service was often not appropriate, so it cause less effective treatment, increasing risk for the patients, widespread resistance and costly treatment. therefore, it needs the proper solution to prevent the problems. one of solution is using traditional herbal medicine, as like conducted by remote communities that are far from goverment health services, so they can also conserve the local knowledge. “kayu kuning” or a. flaval.merr is one of plant used as traditional health care in the suban jeriji village, rambang dangku, muara enim, south sumatra, indonesia.this plant in muna village, southeast sulawesi is used for diarrhea, sore eyes, jaundice, oral ulces and water flea medicine (larisu, 2011). this plant was known by its bright yellow wood’s color, a herb, climbing, annual, wild growth and can be found in rocky beach or in edge of forest (sitepu and sutikno, 2001). a.flava l. contains saponin, flavonoid, polyphenolic substance, glycoside and alkaloid. berberine derivative are the the main group found in this plant. it was reported by singh, et. al. (2010). whereas terpenes are found in this plant e.g., fibrauleusin, fibraurin (siwon, 1982).berberine is alkaloid in form of chloride or sulfate salts, they are existing on menispermaceae plant, the mechanism of action as antimicrobial agent could be changing the arrangement of amino acid chain on dna that rises balances changes of genetics on dna, so that the dna of microbial will be defeat, this causes a core of microbial cell to be defeat and dead (dassonneville et al., 2000). methodology materials stem of a.flava l. was come from suban jeriji village, rambang dangku, muara enim, south sumatra. voucher specimen was found at pharmaceutical biology department, faculty of pharmacy ugm; berberine chloride (b2251-10g, sigma), c.albicans atcc 10231 and t.menta-grophytes, media nutrient broth and agar media, nystatin, mtt (methyl thiazol tetrazolium, sigma), n-butanol, glacial acetic acid, distilled water, 0.9% nacl (merck germany) and tlc(thin layer 16 biology, medicine, & natural product chemistry 3 (1), 2014: 15-19 chromato-graphy) plates silica gel 60 f254 (e.merck germany). apparatus set of reflux, incubator, autoclave, laminair air flow (laf), oven, flat microplate, microplate reader, petri dish, micropipette, white tip, yellow tip and blue tip, a set of tools thin layer chromatography (tlc), ultra violet (uv) light and tlc scanner procedure 20 gram of drug powder was extracted by distilled water 100 ml for 2 hours (reflux). the water extract was was filtered evaporated by reduced vapor. qualitative and quantitative analysis. it was done by tlc; 5 μl test solution and berberine chloride were spotted on tlc plate on silica gel 60 f254. the mobile phase was n-butanol, acetic acid, water (3:1:1 v/v/v). the spots were observed under uv254 nm, uv366 nm and visible light. the spots of test substance and berberine chloride were scanned between 200 700 nm. the standard curve ob berberin chloride was conducted from the series of berberine chloride, which was 100; 50; 25; 12,5; and 6,25 μg/ml. the solution test was made by 5 mg/ml in methanol. it was eluted by the same tlc system. the spot area under curve (auc) that was suspected as alkaloid measured by densitometer. it was made standard curve by regressing content (μg) vs area under curve. the alkaloid content was calculated as berberine chloride. antifungal activities test by agar diffusion method (kirby-bauer) the sterile agar media was diluted, after the lukewarm 10 ml, the media was added by 100 µl of fungi suspension, whipped homo-geneously. the concentration was 5x102– 2,5x103 cfu/ml of fungi in media. mixture was poured into a sterile petri disk, waited until condensing. steril paper diskwas mounted on the surface in order to be drop by sample 20 µl, each 10µl for nistatin and distilled water , then it was incubated on the temperature 37oc for 24 hours. clear zone was measured by using vernier caliper. anti-fungi test was done by microdillution method the test solution were dissolved into distilled water at several concentration 10 %; 8%; 6%; 4%; 2%; 1%; 0,5%; 0,25% and 0,125% b/v. each 50 µl solution test was poured into the well and added by fungi suspension in nb media. the fungi concentration becomes 5x102 cfu/ml. the sample was incubated on 37oc temperature for 24 hours. then, mtt was added to make easy observation. od (optical density) value could be seen through the absorption. it could be computed by formula: the clear pitting should be scratched on solid media for knowing a mec value. bioautographic assay the solution test was spotted for 5 µl on tlc plate and eluted by mobile phase n-butanol, acetic acid, water (3:1:1 v/v/v). it was prepared the media in order to the sterile mixed by standardized fungi suspension, poured into sterile petri and wait until condensing. the tlc plate was eluted, and dried, then it should be placed on the media for 30 minutes. after tlc plate had been taken, incubated the petri disc by 24 hours on 37oc. their clear zone was observed. results and discussion figure 1 showed that the hrf value of berberine chloride was 61 and spot like berberine solution test was 62 at visible light, uv 254 nm and 366 nm. the spot of sampel and standard was yellow fluorescence under the uv 366 nm. the scanning result for the two spots can be seen in figure 2 at λmax 349 nm. the standard equation curve was y = 58596,5419 x + 2512,5 with r value = 0,9956. the berberine chloride solution test were 1,55 ± 0,12% b/b (table 2). the sample had antifungal activities for tested microbial. the complete activities as table 3. to know potential of extract for anti-fungi activities was conducted the potential test using liquid dilution method, which was micro-dilution. parameter of antifungi activity was minimum inhibitory consentration (mic) value and minimum fungicidal concentration (mfc). the smallest concentration that was still showing clearness was mic, see figure 4 and table 4. the standard equation curve was y = 58596,5419 x + 2512,5 with r value = 0,9956. the berberine chloride solution test were 1,55 ± 0,12% b/b (table 2). figure 1. chromatogram of water-soluble stem extract of a.flava (x) and berberine chloride (b) examination by: (a) visible light; (b) uv 366 nm and; (c) uv 254 nm solid phase: silica gel 60 f254; mobile phase system: n-butanol, acetic acid, water (3:1:1 v/v/v). rini setyowati, et al. – the effect of water-soluble stem extract “kayu kuning” … 17 figure 2. the pattern of uv spectrum of berberine chloride and sample test between 200-700 nm. the sample had antifungal activities for tested microbial. the complete activities as table 3. to know potential of extract for anti-fungi activities was conducted the potential test using liquid dilution method, which was micro-dilution. parameter of antifungi activity was minimum inhibitory consentration (mic) value and minimum fungicidal concentration (mfc). the smallest concentration that was still showing clearness was mic, see figure 4 and table 4. table 1. berberine chloride content vsauc. berberinechloride (µg/µl) volume (µl) berberinechloride content (ng) hrf auc 0,00625 5 0,03125 63 2774,9 0,0125 5 0,0625 63 6586,6 0,025 5 0,125 63 10958,5 0,05 5 0,25 63 17700,2 0,1 5 0,5 63 31307,7 table 2. berberinechloridecontent in water-soluble stem extract of “kayu kuning”. sample consentration (mg/ml) volume (µl) sample weight (µg) hrf auc berberinechloride content/spot (ng) content (%b/b) 5 5 25 63 25308,30 389,01 1,56 5 5 25 64 23427,00 356,90 1,43 5 5 25 64 26777,20 414,10 1,66 average 25170,83 386,67 1,55 sd 1679,32 28,67 0,12 table 3. diameter of inhabitation extract is for test microbial. solution diameter inhibition c.albicans (mm) average (mm) diameter inhibition t.mentagrophytes (mm) average (mm) 1 2 3 1 2 3 sample 13,73 21,53 13,68 16,31 ± 4,52 6,52 6,52 6,60 6,55 ± 0,05 nystantin 17,00 16,85 18,10 17,32 ± 0,68 7,50 7,75 8,00 7,75 ± 0,25 aquadest table 4 showed that, the mic value of sample were 1% w/v for c.albicans and t.mentagrophytes. while table 5 showed that the mfc of sample for c.albicans was 4%w/v and for t.mentagrophytes was 5% w/v. result of bioautography assay showed that there was only an inhibition for c.albicans, but on t.mentagrophytes was not appearing the presence of a clear zone. tlc spot which was showing an activity of inhibition for c.albicans growth was the spot with 62 hrf value. it was berberine chloride, as like appeared on figure 5. this evidenced that solution test containing … … berberine chloride was a bio-active compound that was responsible for the presence of anti-fungi activities of c.albicans. while on t.mentagrophytes couldn't be determined if the compound has activity as anti-fungi, this should be caused by synergetic system. 18 biology, medicine, & natural product chemistry 3 (1), 2014: 15-19 figure 3. microdilution for (a) c.albicans and (b) t.mentagrophytes using mtt reagent. table 4. mic of sample for fungi tested using mtt reactant. fungi mic (mg/ml) average(mg/ml) c.albicans 10 10 10 10 t.mentagrophytes 10 10 10 10 table 5. mfc of sample for fungi tested. sample consentration (% w/v) c.albicans t. mentagrophytes 1 2 3 1 2 3 1 + + + + + + 2 + + + + + + 3 + + + + + + 4 -* -* -* + + + 5 -* -* -* nistatin5000 iu control fungi + + + + + + control media control solvent description: (+): there microbial growth (-): there is no microbial growth (*): mfc from result of analysis using one way anova, both c.albicans and t.mentagro-phyteswas exist significant differences between negative control group with all test solution group, this was by p-value = 0,020 for c.albicans, and p=0,028 for t.mentagrophytes (p<0,05). this result showed that giving test solution causing the growth inhibition of microbial significantly. in addition to significant differences was also showed between positive control group and negative control both c.albicans and t.mentagrophytes tests. table 6. percent inhibition of water-soluble extract of a.flava (“kayu kuning”) against microbial test. no sample consentration (% w/v) average of % inhibition (%) c.albicans t.mentagrophytes 1 0,0625 67,16 48,22 2 0,125 70,67 51,78 3 0,25 73,9 65,21 4 0,5 76,25 66,63 5 1 77,13 74,67 6 2* 85,92* 84,23* 7 3 87,98 89,22 8 4 92,38 93,39 9 5 97,07 96,03 nistatin 5000 iu 100,22 100,41 description: (*): mic figure 4. bioautografi assay results on (a) c.albicans (b) t.mentagrophytes. analysis result of post hoc tukey for c.albicans and t.mentagrophytes, correlation between positive control group and test solution group were not showing significant differences, except group number 1 (0,0625% concentra-tion), that was by p>0,05 value. this was showed that the test solution had the same effect with the positive control (antibiotic) for fungi growth. that was rini setyowati, et al. – the effect of water-soluble stem extract “kayu kuning” … 19 inhibiting the growth of fungi. while correlation between concentration groups on all test microbial, were not giving a significant differences, by p > 0,05 value. the data showed that an increasing of test solution concentration from the smallest concentration 0,0625% until 5% w/v were not showing a significant differences for the tested fungi growth inhibition. an activity and potential of sample could inhibited c.albicans and t.mentagro-phytes growths that was caused by the compound contained in the extract, berberine chloride. the activity of berberine chloride was known as antimicrobial (hwang et al., 2003; karou et al., 2006; scazzocchio et al., 2001; swabb et al., 1981; kaneda et al., 1991). conclusion 1. water-soluble extract of “kayu kuning”(a.flava) was containing of 1.55 ± 0.12% w/w alkaloid berberine chloride. 2. water-soluble extract of “kayu kuning” (a.flava) was active as antifungal against c.albicans colony with mic value of 10 mg/ml and mfc of 40 mg/ml while the colony t.mentagrophytes with mic and mbc values of 10 mg/ml and 50 mg/ml. references backer, c.a., and van den brink, jr.r.c.b., 1969, flora of java, vol. i, 153, 157, n.v.p. noordhoff, groningen. chitwood, l. a., 1969, tube dilution antimicrobial sucseptibility testing, applied microbiologi: 707-709. gupte, s., 1990, mikrobiologi dasar, edisi iii, diterjemahkan oleh dr. julius, e.s., 1992, 69-76, 321, binarupaaksara, jakarta. harborne, j. b., 1973, metode fitokimia, penuntun cara modern menganalisis tumbuhan, diterjemahkan oleh dr. kosasih dan dr. iwang soediro, 1984, terbitan kedua, 234-245, itb, bandung. heyne, k. 1927, tumbuhan berguna indonesia. jilid ii, diterjemahkan oleh badan litbang kehutanan, 1987, yayasan sarana wana jaya, jakarta. hwang, b.y., roberts, s.k., chadwick, l.r., wu c.d., and kinghorn, a.d., 2003, anti microbial constituents from goldenseal (the rhizomes of hydrastin canadensis) againt selected oral phatogens, planta med., 69 (7 : 623-627). karou, d., savadogo, a.yomeogo, s., and montesano, c., 2006, antibacterial activity of alkaloids from sida acuta, j. afric.bio.,5 (2): 195-299. jawetz, e., melnick, j.l., and adelberg, e.a., 2001, mikrobiologi kedokteran, diterjemahkan oleh dr. nani widori, 2005, edisi 22, 313-325, 344-346 penerbit salemba medika, jakarta. kaneda, y., torii, m., and tanaka, t., 1991, in vitro effects of berberine sulfate on the growth of entamoeba histolytica, giardia lamblia and trichomonas vaginalis. ann. trop. med.parasitol., 85:417-425 keawpradub, n., dejadisai, s. and yuenyongsawad, s., 2005, antioxidant and cytotoxic activities of thai medicinal plants named khaminkhruea: arcangelisia flava, coscinium blumeanum and fibraurea tinctori, songklanakarin, j. sci. technol., 27 (suppl. 2) : 455-467. mahon, c. r., and manuselis j.r. g., 1995, textbook of diagnosis microbiology, w. b sanders company, philadephia. nakamoto, k., tamamoto, m., and hamada, t., 1995, in vitro study on the effects of trial denture cleansers with berberine hydrochloride. j. prosthet. dent., 73:530-533. ongsangkul. m., jindarat. a., and rajona. c., 2009, antibacterial /effect of crude alcoholic and aqueous of six medical plants against staphylococus aureus and eschericia coli, j health res no. 23 vol. 3 : 153-156 praptiwi, jamal. y., fathoni, a., and agusta. a., 2009, antimicrobial metabolit from the culture of endophytic fungus afk-8 from kayu kuning (archangelisia flava l. merr), research centre of biologi, indonesian institute of science, bogor. scazzocchio, f., cometa, m.f., tomassini, l., and palmery, m., 2001, antibacterial activity of hydrastis canadensis extract and its major isolated alkaloids, planta med., 67 (6): 561-564. schwalbe, r., steele-moore, l., and goodwin, a.c., 2007, antimicrobial susceptibility testing protocols, 186-193, crc press taylor & francis group, london. singh, a., duggal, s., kaur, n., and singh, j., 2010, berberine: alkaloid with wide spectrum of pharmacological activities, j. nat. products, vol.3:64-67. siwon, f., 1982, a pharmacognostical study of some indonesian medicine of the family menispermaceae, disertasi, 10, 43, drukkij j h pasmans b. v’s gravenhage. stahl, e., 1973, analisis obat secara kromatografi dan mikroskopi, diterjemahkan oleh dr. kosasih padmawinata dan dr. iwang soediro, 1985, penerbit itb, bandung. swabb, e.a., tai, y.h., and jordan, l., 1981, reversal of cholera toxin-induced secretion inrat ileum by luminal berberine. j. am. physiol., 241 : 248-252. content_v3n1_2.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 123-127 | doi: 10.14421/biomedich.2021.102.123-127 issn 2540-9328 (online) stability analysis of mathematical modeling of interaction between target cells and covid-19 infected cells sugiyanto1*, mansoor abdul hamid2, alya adianta1, hanny puspha jayanti1, muhammad ja'far luthfi 3 1mathematics department, faculty of science and technology, universitas islam negeri sunan kalijaga, indonesia. 2school of food science & nutrition, universiti malaysia sabah, malaysia. 3department of biological education, faculty of tarbiyah and education, universitas islam negeri sunan kalijaga, indonesia. corresponding author* sugiyanto@uin-suka.ac.id manuscript received: 19 october 2021. revision accepted: 30 october, 2021. published: 02 november, 2021. abstract the stability analysis in this mathematical model was related to the infection of the coronavirus disease 2019 (covid-19). in this mathematical model there were two balance points, namely the point of balance free from covid-19 and the one infected with covid-19. the stability of the equilibrium point was influenced by all parameters, i.e. target cells die during each cycle, number of t arget cells at 𝑡′ = 0, target cells infected during each cycle based on virion unit density, effective surface area of the network, the ratio of the number of virus particles to the number of virions, infected cells die during each cycle, the number of virus particles produced by each infected cell during each cycle, and virus particles die during each cycle. in the simulation model, immunity is divided into high, medium and low immunity. for high, moderate and low immunity, respectively, the highest number of target cells is in high, medium and low immunity, whereas for the number of infected cells and the number of covid-19, it is in the opposite sequence of the number of target cells. keywords: coronavirus disease 2019; equilibrium point stability; target cells and infected cells. introduction coronavirus disease 2019 (covid-19) was first known to infect residents in wuhan city, china, and was notified by the chinese government to who in december 2019 (sugiyanto & abrori, 2020). covid-19 belongs to subfamily orthocoronavirinae, family coronaviridae, and order nidovirales (tan et. al., 2020). about 80% of covid-19 illness show mild symptoms and 20% have severe symptoms. some of the 20% patients who contract covid-19 develop severe pneumonia, sometimes with acute respiratory distress, which can lead to organ failure and death. the stability analysis of mathematical modeling is used to determine the recovery period of covid-19 patients. there are many factors that determine a person would get into mild, severe or severe symptoms. we can classify these symptoms into three things depend on the immunity of the covid-19 patient. in this modeling, categorization were done using the t – i – v model. the target cell subpopulation (t) is cells in several organs, such as the lungs, heart, arteries, intestines and kidneys. the infected cell subpopulation (i) is a cell that is infected through a receptor on the surface called angiotensin converting enzyme 2 (ace2) (diaz, 2020). target cells were epithelial cells in all of these organs. this target cell was ace2. the conversion of angiotensin ii (vasoconstruction peptide) to angiotensin 1-7 (vasodilator) was catalyzed by ace2 (zhang et. al., 2020). 83% of normal lung cells express ace2, namely type ii alveolar epithelial cells (aecii), which make these cells viral reservoirs. the spike protein (shaped like a nail) stuck to the surface of the sars-cov virus (zoufaly et. al., 2020). the ace2 enzyme attaches to the cell membranes of several organs (bourgonje et. al, 2020). stability analysis the mathematical model obtained in system (1) refers to du and yuan's (2020) paper. 𝑑𝑇 𝑑𝑡′ = (𝑑𝜏)𝑇0 − (𝑑𝜏)𝑇 − (𝑘𝜏) 𝐴𝛼 𝑉𝑇 (1a) 𝑑𝐼 𝑑𝑡′ = (𝑘𝜏) 𝐴𝛼 𝑉𝑇 − (𝛿𝜏)𝐼 (1b) 𝑑𝑉 𝑑𝑡′ = (𝑝𝜏)𝐼 − (𝑐𝜏)𝑉 (1c) description of the target cell subpopulation, covid19 infected cells, virus population and parameters are shown in table 1. https://doi.org/10.14421/biomedich.2021.102.123-127 124 biology, medicine, & natural product chemistry 10 (2), 2021: 123-127 table 1. target cell subpopulation, covid-19 infected cells, virus population and parameters. no. symbol explanation unit 1 𝜏 average cycle time for viral replication 𝑑𝑎𝑦 2 𝑡′ = 𝑡/𝜏 number of virus replication cycles 3 𝑇 number of target cells at 𝑡′ 𝑐𝑒𝑙𝑙 4 𝐼 number of infected cells at 𝑡′ 𝑐𝑒𝑙𝑙 5 𝑉 number of virus particles at 𝑡′ 𝑣𝑖𝑟𝑢𝑠 6 (𝑑𝜏) target cells die during each cycle 7 𝑇0 number of target cells at 𝑡 ′ = 0 𝑐𝑒𝑙𝑙 8 (𝑘𝜏) target cells infected during each cycle based on virion unit density 9 𝐴 effective surface area of the network 𝑚𝑚2 10 𝛼 the ratio of the number of virus particles to the number of virions 𝑣𝑖𝑟𝑢𝑠 /𝑚𝑚2 11 (𝛿𝜏) infected cells die during each cycle 12 (𝑝𝜏) the number of virus particles produced by each infected cell during each cycle 13 (𝑐𝜏) virus particles die during each cycle theorem 1. equilibrium point there are two equilibrium points of system (1), namely: free from the covid-19 virus and infected with the covid-19 virus. the covid-19 virus-free equilibrium point is 𝐸𝑃0 = (𝑇, 𝐼, 𝑉) = (𝑇0, 0,0). the equilibrium point for contracting the covid-19 virus is 𝐸𝑃1 = (𝑇, 𝐼, 𝑉) = (𝑎1, 𝑎2, 𝑎3), where 𝑎1 = 𝐴𝛼(𝛿𝜏)(𝑐𝜏) (𝑘𝜏)(𝑝𝜏) , 𝑎2 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝑝𝜏)(𝛿𝜏)(𝑘𝜏) , 𝑎3 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝛿𝜏)(𝑐𝜏)(𝑘𝜏) . proof. from equation (1a) and 𝑑𝑇 𝑑𝑡′ = 0, we get 𝑇 = (𝑑𝜏)𝑇0𝐴𝛼 (𝑑𝜏)𝐴𝛼+(𝑘𝜏)𝑉 (2) from equation (1c) and 𝑑𝑉 𝑑𝑡′ = 0 obtained 𝐼 = (𝑐𝜏) (𝑝𝜏) 𝑉 (3) from 𝑑𝐼 𝑑𝑡′ = 0 and substituting equations (2) and (3) into equation (1), we get 𝑉 = 0 (4) or 𝑉 = (𝑑𝜏)[(𝑘𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)𝐴𝛼] (𝛿𝜏)(𝑐𝜏)(𝑘𝜏) = 𝑎3 (5) from equation (2) and equation (4), we get 𝑇 = 𝑇0. (6) from equation (3) and equation (4), we get 𝐼 = 0. (7) from equations (6), (7) and (4) it is proven that the covid-19 virus-free equilibrium point is 𝐸𝑃0. if equation (5) is substituted into equation (2), then we get 𝑇 = 𝐴𝛼(𝛿𝜏)(𝑐𝜏) (𝑘𝜏)(𝑝𝜏) = 𝑎1 (8) if equation (8) is substituted into equation (3), then we get 𝐼 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝑝𝜏)(𝛿𝜏)(𝑘𝜏) = 𝑎2 (9) from equations (8), (9) and (5) it is proven that the equilibrium point for contracting the covid-19 virus is 𝐸𝑃1. ■ from theorem 1 it can be conveyed, if there is no covid-19 virus then someone will be safe or someone is virus free, and if there is a virus then a person's healing point is influenced by all parameters. virus-free can be achieved if there is no person carrying the virus or complying with health procedures such as wearing a mask, keeping a distance and washing hands as often as possible. when a person gets a virus, only the immune (target cells) can fight the infected cells. theorem 2. existence of the equilibrium point existence 𝐸𝑃0 fulfilled in any non-negative number parameter and existence 𝐸𝑃1 fulfilled if (𝑘𝜏)𝑇0(𝑝𝜏) − (𝛿𝜏)(𝑐𝜏)𝐴𝛼 > 0. proof. from theorem 1, that existence 𝐸𝑃0 and 𝐸𝑃1 proven. ■ from theorem 2 it can be seen that all parameters do not affect the existence of the equilibrium point 𝐸𝑃0. all parameters are target cells die during each cycle, number of target cells at 𝑡′ = 0, target cells infected during each cycle based on virion unit density, effective surface area of the network, the ratio of the number of virus particles to the number of virions, infected cells die during each cycle, the number of virus particles produced by each infected cell during each cycle, and virus particles die during each cycle. this means that if a person is not exposed to the covid-19 virus, the target cells would not affected or the condition of a person is healthy without the virus. for someone who is infected with the virus, all parameters affect the existence of the equilibrium point 𝐸𝑃0. this means that a person's condition will remain sugiyanto et al. – stability analysis of mathematical modeling of interaction … 125 healthy or even die depending on the target cells working well or not. theorem 3. stability of the equilibrium point (1) if √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)), then the equilibrium point 𝐸𝑃0 is locally asymptotically stable. (2) if √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)(𝑎1) 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then the equilibrium point 𝐸𝑃1 is locally asymptotically stable. proof. for example, in system (1) it is written 𝑓1 = 𝑑𝑇 𝑑𝑡′ = (𝑑𝜏)𝑇0 − (𝑑𝜏)𝑇 − (𝑘𝜏) 𝐴𝛼 𝑉𝑇 (10a) 𝑓2 = 𝑑𝐼 𝑑𝑡′ = (𝑘𝜏) 𝐴𝛼 𝑉𝑇 − (𝛿𝜏)𝐼 (10b) 𝑓3 = 𝑑𝑉 𝑑𝑡′ = (𝑝𝜏)𝐼 − (𝑐𝜏)𝑉 (10c) jacobian matrix function 𝑓 from system (10) written can be obtained by first performing the partial derivation of the functions 𝑓1 = (𝑇, 𝐼, 𝑉 ) (11a) 𝑓2 = (𝑇, 𝐼, 𝑉 ) (11b) 𝑓3 = (𝑇, 𝐼, 𝑉 ) (11c) as follows. (i). partial derivative 𝑓1 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓1 𝜕𝑇 = −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 𝑉; 𝜕𝑓1 𝜕𝐼 = 0; 𝜕𝑓1 𝜕𝑉 = 0; (ii). partial derivative 𝑓2 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓2 𝜕𝑇 = (𝑘𝜏) 𝐴𝛼 𝑉; 𝜕𝑓2 𝜕𝐼 = −(𝛿𝜏); 𝜕𝑓2 𝜕𝑉 = (𝑘𝜏) 𝐴𝛼 𝑇; (iii). partial derivative 𝑓3 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓3 𝜕𝑇 = 0; 𝜕𝑓3 𝜕𝐼 = (𝑝𝜏); 𝜕𝑓3 𝜕𝑉 = −(𝑐𝜏); the jacobian matrix is 𝐽(𝑇, 𝐼, 𝑉) = [ −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 𝑉 0 0 (𝑘𝜏) 𝐴𝛼 𝑉 −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 𝑇 0 (𝑝𝜏) −(𝑐𝜏)] (1) for 𝑬𝑷𝟎, we get 𝐽(𝑇0, 0,0) = [ −(𝑑𝜏) 0 0 0 −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 (𝑇0) 0 (𝑝𝜏) −(𝑐𝜏) ] we find the eigenvalues of 𝐽(𝑇0, 0,0) that is 𝜆𝑖, for 𝑖 = 1,2,3, where  0 , 0, 0 0.j t i  we get the eigenvalues of the jacobian matrix which is represented by                             1 2 2 2 3 0 0 1 , , 4 4 2 1 . 2 d k p t c c a k p t c c a                                             we know that ((𝛿𝜏) − (𝑐𝜏)) 2 ≥ 0 and (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0, so that ((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0. since the parameters are greater than zero, we get 1 20, 0,   and because √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then we get 𝜆3 = 1 2 [−((𝛿𝜏) − (𝑐𝜏)) ± √((𝛿𝜏) − (𝑐𝜏)) 2 − 4 ((𝛿𝜏)(𝑐𝜏) − (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 )] < 0. we get all negative eigenvalues, so that 𝐸𝑃0 is locally asymptotically stable. (2) for 𝑬𝑷𝟏, we get 𝐽(𝑎1, 𝑎2, 𝑎3) = [ −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 (𝑎3) 0 0 (𝑘𝜏) 𝐴𝛼 (𝑎3) −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 (𝑎1) 0 (𝑝𝜏) −(𝑐𝜏) ] we find the eigenvalues of 𝐽(𝑎1, 𝑎2, 𝑎3) that is 𝜆𝑖, for 𝑖 = 1,2,3, where  1 2 3, 0.,j a a a i  we get the eigenvalues of the jacobian matrix which is represented by                                   3 1 1 1 2 2 2 3 , 1 4 , 2 1 4 2 k d a a k p c c a k p a a c c a                                                     we know that ((𝛿𝜏) − (𝑐𝜏)) 2 ≥ 0 and (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0, so ((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0. since the parameters are greater than zero, we get 𝜆1 < 0, 𝜆2 < 0, 126 biology, medicine, & natural product chemistry 10 (2), 2021: 123-127 and because √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)(𝑎1) 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then we get              2 3 11 4 0 2 c a ak p c                      . we get all negative eigenvalues, so that 𝐸𝑃1 is locally asymptotically stable.■ from theorem 3 the stability point is affected by all parameters. this means that a person will recover depending on the target cells that work. the better the target cells work, the healthier the person would be and those who have been infected with covid-19 will recover. simulation the parameters in this simulation are taken from du and yuan's (2020) paper. table 2 shows the parameter values. in this simulation, we replace the symbol (𝑝𝜏) with 𝑏. this is because in matlab there is no insert legend that can be written (𝑝𝜏). table 2. parameter values for simulation. no. parameter value 1 𝜏 7 2 𝑑𝜏 2 × 10−4 3 𝑇0 10 8 4 (𝑘𝜏) 𝐴𝛼 𝑇0 0.075 5 𝛿𝜏 0.4 6 𝑐𝜏 0.4 7 𝐼0 10 8 𝑉0 100 figure 1. changes in the number of target cells against the presence of the covid-19 virus. target cells reflect the number of cells in people with three conditions, namely: low, moderate and high immunity conditions. figure 1, figure 2 and figure 3 represent person with high immunity ((𝑝𝜏) = 𝑏 = 50), moderate immunity ((𝑝𝜏) = 𝑏 = 100), and low immunity ((𝑝𝜏) = 𝑏 = 150). person with good immunity shows the target cell from 10,000,000 cells in 13.09 days to 101,800 cells. person with moderate immunity shows the target cell from 10,000,000 cells in 8,514 days to 104,300 cells. person with low immunity shows the target cell from 10,000,000 cells in 2,398 days to 105,000 cells. the order of decline in target cells from the longest to the fastest is good, medium and low immunity. table 3 describes the descending order of the target cells. table 3. target cell decrease. no. immunity initial amount (cell) total ten thousand (cell) time (day) 1 high 10,000,000 101,800 13.09 2 medium 10,000,000 104,300 8.514 3 low 10,000,000 105,000 2.398 figure 2. changes in the number of infected cells against the presence of the covid-19 virus. figure 2 shows the peak number of infected cells differed between individuals with high, moderate and low immunity. a person with low immunity on day 6,155 the number of infected is 7.146 × 107 cell. a person with moderate immunity on day 7,685 the number of infected is 6.65 × 107 cell. a person with high immunity on day 11.46 the number of infected is 5.655 × 107 cell. briefly, this explanation is in table 4. table 4. increase in the number of infected cells. no. immunity highest number of cells (cell) time (day) 1 high 5.655 × 107 11.46 2 medium 6.65 × 107 7.685 3 low 7.146 × 107 6.155 figure 3. changes in the number of virus particles. sugiyanto et al. – stability analysis of mathematical modeling of interaction … 127 figure 3 shows the number of viruses with high, medium and low immunity conditions. for someone with high immunity the maximum virus count on day 13.19 is 4.31 × 108 virus. for someone with moderate immunity the maximum virus count on 9,492 days is 8.946 × 109 virus. for a person with low immunity the maximum viral load on day 8,068 is 1.356 × 1010 virus. table 5 describes the amount of virus in the condition of a person with high, medium and low immunity. table 5. increase in the number of virus particles. no. immunity highest number of viruses (virus) time (day) 1 high 4.31 × 108 13.19 2 medium 8.946 × 109 9.492 3 low 1.356 × 1010 8.068 conclusion the stability of being free of the covid-19 virus and infected with the virus is influenced by all parameters. the number of target cells, virus-infected cells and virus particles is affected by a person's immunity. if a person has high immunity, the number of target cells would decrease slowly. vice versa, if a person has low immunity, then the number of target cells will drop rapidly. in a person having low immunity, the infected cells and viruses will quickly increase in number compared to the one with high immunity. conflicts of interest: mjl is on the editorial board of the biology, medicine, & natural product chemistry, and was recused from this article’s review and decision. the authors declare that there are no conflicts of interest. references bourgonje, a. r., abdulle, a. e., timens, w., hillebrands, j. l., navis, g. j., gordijn, s. j., ... & van goor, h. 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(2020). ace2 and covid-19 and the resulting ards. postgraduate medical journal, 96(1137), 403-407. zoufaly, a., poglitsch, m., aberle, j. h., hoepler, w., seitz, t., traugott, m., ... & penninger, j. m. (2020). human recombinant soluble ace2 in severe covid-19. the lancet respiratory medicine, 8(11), 1154-1158. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 47-52 | doi: 10.14421/biomedich.2014.32.47-52 a discovery and characteristics description of telosma puberula (asclepiadoideae) in mount gedang atas and mount ijo, baturagung mountain yogyakarta widodo faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: wwidodo594@gmail.com abstract population of telosma puberula in community forest bushes was identified at s.07.48'.44.1"; e.110.31.15.8", 411m, mount gedang atas and s 07o 04 '04.1"; e 110o 30 '47.9 ", 415m, mount ijo. telosma puberula was also found in mount parangan, mount mintorogo, mount nglanggeran, and baturagung mountains yogyakarta. the identification was based on herbarium specimens collected by horsfield in 1802 and 1859 from java island, indonesia (k000873052, k000873053). information about telosma puberula is very limited. this article describe photographs of morphological characters of the plant’s stems, leaves, flowers, and pollinia. keywords: telosma puberula, asclepiadoideae, mount gedang atas, baturagung mountains introduction in an exploration, observation and assessment of wild plants in mount gedang atas baturagung mountains in yogyakarta, the author was collecting fruits of vines from family apocynaceae, sub family asclepiadoideae in a community forest at a location of s.07.48'.44.1"; e.110.31.15.8". (widodo, 411m dpl, 26 september 2012). furthermore, a specimen collection process and observation was completed in a flowering season in 2013. the same specimen was also found in mount ijo within a radius of 5 km at a location of s.07.47'.04.4 "; e.110.30'.47.9 ". (widodo, 415m dpl, 31 december 2012). from a thorough identification process using literature and herbarium reviews, it was identified that the specimen collected is telosma sp. telosma is belong to marsdenieae tribe of the subfamily asclepiadoideae (takhtajan, 2009: 522). according to backer and bakhuizen (1965: 273), there are two types of telosma in java: telosma accedens and telosma cordata. meanwhile, hooker (1885: 38) states that in java, there is telosma puberula (a new name for pergularia puberula). telosma cordata (a new name for pergularia odoratissima) is predicted to be a native to china. in java, the plant has been cultivated as floral fragrance (backer and bakhuizen, 1963: 273). meanwhile telosma accedens can be found in central java, east java and madura at an altitude of 10-800 m in woody shrubs, cleared forests, and teak forests. the previous name of pergularia accendens was telosma accedens, blume. backer corrected the name into telosma accedens (bl.) back. (backer, 1949: 63). the name pergularia accedens was firstly published in bijdragen tot de flora van nederlandsch indie (blume, 1826). the herbarium of pergularia accedens was collected by carl l von blume from the java island in 1836 (mnhn p03858919). meanwhile, the herbarium of telosma accedens collection (bl.) back was collected by ca backer from the java island (jember-bondowoso) in 1920 (mnhn p03858922). studies on telosma cordata or tonkin flowers hold are abundant on the internet because this plant is cultivated as floral fragrance plants although there is no identified source from indonesia. based on the writer’s knowledge, there are no further information or study about telosma accedens and telosma puberula in indonesia after backer and bakhuizen (1965: 273), hooker (1885: 38) and blume (1836). according to the plant list (2010), the genus telosma consists of many species: telosma accedens (blume) backer, telosma africana (nebr.) nebr., telosma angustiloba (warb. in perkins) merr., telosma celebica (warb.) ma rahman & wilcock, telosma cordata (burm. f.) merr., telosma filipes (schltr.) ma rahman & wilcock, telosma pallida (roxb.) craib, telosma procumbens (blanco) merr., and telosma puberula (miq.) kerr. based on the information obtained from backer and bakhuizen (1965: 273) and hooker (1885: 38), the telosma species that are available in the java island is telosma accedens, telosma cordata, and telosma puberula. there is very little information about telosma accedens and telosma puberula both in written literature and published pictures on the internet. this paper attempts to present a description of the characteristics, specimen and herbarium photographs of telosma specimens found in mount gedang atas and mount ijo, baturagung mountains. the discovery of the telosma species in the baturagung mountains yogyakarta needs to be disseminated to present the richness of flora in java. wild plants are no longer recognized despite the fact that there were some report in the books of flora and 48 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 herbarium in the past hundreds years by european explorers. a publication of plant species in the nature is highly needed in attempt to complete the flora world data, to re-check and rediscover old flora, to correct description of the characters for further research in the study of plant biology. the study of biology is the basis for conservation of earth plants. the purpose of this study is to present a visual characteristics description of the morphology of leaves, stems, flowers, pollinia, and fruit of telosma puberula found in mount gedang atas and mount ijo, baturagung mountains for identification verification. materials and methods this research employs one kind of research methode that explores and visits as well as collects data information (exploration and collection trip, (singh, 1999)). an early exploration was performed in september 2012 along with an exploration of wild plants. picture taking was completed for the first step of the identification process. specimen sampling for herbarium was also performed with an awareness of the preservation of the population. along with the identification process, monitoring and visitation processes were also kept on being conducted based on a prediction on the flowering season and fruit formation, i.e. december 2012, january 2013, march 2013, april 2013, december 2013, and october 2014. flowers specimen collection was completed using wet preservation technique for further identification. the tools used for observation and collection were: sony nex f3 digital camera, sony cyber-shot dscw180 digital camera, canon dslr digital camera, rulers, micrometers, calipers, small meter roller, plastic for sample collection, scissors, cutter, paper for labeling, gps (global positioning system), dried herbarium collection equipment, flacon bottle, stereo microscope nikon smz 1500 equipped with a camera, nikon eclipse 50 light microscope equipped with nikon dsf1camera. materials for observation and collection comprise: aquadest, alcohol 70%, faa (formalin acetic alcohol) solution. steps of the study include: (1) photograph the specimens under natural conditions on site, (2) photograph the specimens under the dried herbarium process preparation conditions, (3) photograph the details of the flower, (4) making the dried herbarium, (5) photograph the dried herbarium specimens, (6) collecting and observing the structures of the preserved flowers, (7) observing and photographs the pollinia, (8) early identifying of specimens for the member of asclepiadaceae based on the book flora of java vol. 2 (backer and bakhuizen, 1965), (9) further identifying of specimens for other members of asclepiadaceae based on existing literature, (10) checking and matching with the herbarium types. results and discussion in the exploration, observation and assessment of wild plants in mount gedang atas baturagung mountains in yogyakarta, the author collected fruit from the vines family apocynaceae sub-family asclepiadoideae in a community forest located at s.07.48'.44.1” location; e.110.31.15.8" (widodo, 411m dpl, 26 september 2012). further, at the initial period of the rainy season at the same location, it was found the shoots with leaves and flowers as such that facilitated further identification s.07.48'.44.1 "; e.110.31.15.8 ". (widodo, 411m dpl, 31 december 2012). the fruits were initially identified by the author as the fruit of cynancum callialatum. another visit conducted at the beginning of the rainy season 31 december 2012 a set of data was obtained on the habitus and the structure of the specimen because leaves had been grown at the branches and they were flowering. at the same time, an observation at mount ijo which is approximately 5 km from the starting location and the author found the same plants which were also flowering. an identification using the book flora of java, vol. 2 (backer and bakhuizen, 1965: 273) mentioned that the plant identified was telosma. subsequent visitations and repeated observations were then completed on 19 january 2013 and 29 january 2013 to obtain a complete set of on the plant structure (figure 1 a, b, c, d, e, and figure 2). the specimen collection process was carried out in the laboratory for further observation. on 24 march 2013 visit, a young fruit was identified (figure 4). further identification results using the book flora of java, vol. 2 (backer and bakhuizen, 1965: 273) showed that this telosma held the characteristics of telosma accedens (bl.) back. a specimen comparison with the 1917 backer herbarium specimens collection in java (mnhn p03858922) or figure 1 f demonstrated the identified specimen compound flower structure of the specimen to be slightly different with the herbarium. the flowers units on the compound stalks converge or congregate in a way so that the compound flower compositions construct a sphere shape, meanwhile the compound flower at the herbarium exhibited characteristics that shaped like a bunch with accompanying braktea (supporting leaves). another comparison with the telosma procumbens herbarium specimens collection made by ramos 1918 from luzon, the philippines (mnhn p03858921) it was found that the obtained structure of the compound flower to be similar to the specimen of the identified telosma but there was a slight difference in terms of the units of flower. a single flower stalk and crown neck tube of the specimen was found to be shorter than that of the telosma procumbens. the centre of the crown round end when it was still in the form of bud looked much clearer in telosma procumbens. a comparison of the form and size of the fruit of telosma procumbens from the philippines in the united states national herbarium (usnh623771) collection was the same as the identified specimen but widodo – a discovery and characteristics description of telosma puberula … 49 the four sides of the fruit wing lines of the specimen identified were more apparent. from a comparison with the herbarium specimens of telosma puberula from the horsfield collection in 1859 of java (k000873052, k000873053) in figure 1 g, several characteristics were found to be the same in: the flowering type, size of the flower parts and shape as well as size of ovatus-oblong leaves. the sketched figure of pollinia in the herbarium (k000873053) also showed similarities with the pollinia of telosma identified in mount ijo (figure 3 a, b). characteristics of the pollinia of telosma puberula foundwere as shown in figure 3a. the location of the pollinia was upright next to the anther. the shape of the pollinia lobes was ovatus-oblong. the translator were very short. the corpuscullum or caudicle (caudicula) was round egg shaped almost like a short triangle, shiny brown. a comparison with the herbarium specimens of telosma puberula collected by kerr in 1928 from thailand (mnhn p03858930) was shown in figure 1h and another one by petelot in 1941 from tonkin (mnhn p03858929) there were some characteristics found to be the same in: the structure of compound flower, stalk compound flower, the size of the parts of the flower as well as the shape and size of the leaves. from the discussions, it was found that the specimen of telosma from mount gedang atas, mount ijo, mount parangan, mount mintorogo in baturagung mountains was telosma puberulens. figure 1. a. photo stature (habitus) of telosma puberula in the natural habitat found by the author. b. flowering branch of telosma puberula in the natural habitat found by the author. c. flowering branch of telosma puberula under the laboratory conditions. d. herbarium flower twig of young telosma puberula from the author’s collection. e. herbarium of the blooming twig flower telosma puberula from the author’s collection. f. herbarium type of telosma accedens (bl.) back. (mnhn, paris). g. herbarium type of telosma puberula (miq.) kerr. (© copyright of the board of trustees of the royal botanic gardens, kew). h. herbarium type of telosma puberula (miq.) kerr. (mnhn, paris). taxonomic information telosma puberula (miq.) kerr, fl. siam. enum. 3 (1): 32. 1951; pergularia puberula miq, fl. brit. india 4: 38. 1885. type: java, 1802 & 1859, t. horfsfield, k000873053, k000873053 (holotype, k!) description liana, stem 4 m or more, smooth surface, young puberulen twigs/branches. 3-6 cm; round-egg shaped to elongated elliptic leaf blade, 6-13 × 3-8 cm, thin, smooth to the puberulen along the vein, flat base to almost heart shape, tapered tip; lateral veins 4-6 pairs. extra axillary compound flower, umbrella to round shape, comprising a b c d e f g h 50 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 several to many flowers; compound flower stems 4-5 cm; flower stalk unit 1-1.5 cm. round-egg shaped petal lobes unit, 2 × 2.5 mm, slippery until puberulen, slightly hairy edges. bright green or greenish yellow crown, slightly fragrant, crown 1.5 cm long; crown tube 6-8 mm long, the tube length is equal to the length of the crown lobe; oblong crown lobes narrowed at the end, 6-8 mm long, 2-2 mm wide, slightly hairy at the edge tip, yellowish green, rounded ends folded out. rounded corona tip, folded out; additional parts of the corona (corollines corona) are higher than the position of pollinia, covering the anthers, pointed tip, thin, yellowish white color. suboblong pollinia lobe, the half-width long, upright, short triangular corpuscullum, short caudicle. follicle fruit, 12-18 cm long, 2-3 cm wide, almost rectangular cross-sectional sepal, with four wing lines on the long side; flat seed, round-egg shaped, 1-1.5 cm in diameter, 4-5 cm seed hair. flowering season from december to january, fruit formation: july to september. specimens examined baturagung mountains, mount gedang atas, s.07.48'.44.1 "; e.110.31.15.8 ", widodo, 411m dpl, 31/12/2012; baturagung mountains, mount ijo, s. 07 ', 47 ", 04.4"; e. 110, 30 ", 47.9", widodo, 415m dpl, 19/01/2013. figure 2. the structure of telosma puberula flower found by the author (a. compound flower, b. flower buds unit, c. blooming flower unit (side view), unit d. blooming flower unit (top view). figure 3. a. the structure of gynostegium and pollinia of telosma puberula flower found by the author (1. gynostegium, 2. corona and corollines corona, 3, 4. the orientation or layout of pollinia, 5, 6. pollinia unit and its parts. b. sketched figure of parts of gynostegium and pollinia of telosma in literature (1. sketch of corona in the telosma puberula herbarium (rbg kew), 2. sketch of pollinia unit in the telosma puberula herbarium (rbg kew), 3. satan pollinia telosma procumbens in the book of flora of china (1995: 273). a b c d 1 2 3 4 5 6 1 2 3 a b widodo – a discovery and characteristics description of telosma puberula … 51 figure 4. telosma puberula fruit found by the author. (a. young fruit, b. ripen fruit, c. fruit with seeds and hairless seed, d. wall fruit after the seeds release). note telosma puberula was found to be twining vines in cassia fistula and schoutenia ovata along with other vines, namely: dioscorea hispida, dioscorea alata, vitis trifolia, and gliricidia sepium. the initial specimen was found by the author during an exploration of the existence of plants of the genus capparis on site. the author found the telosma puberula was bearing some fruit on 24 march 2013 in mount parangan and mount ijo. on 13 april 2013, on the east side of mountain mintorogo the author also found telosma puberula bearing some fruit. conservation regarding the limited presence of this plant in the location of the discovery and other locations, it is then necessary to study the status of its cultivation range. along with the assessment processes, there is a need for live specimen preservations. conclusion telosma puberula (miq.) kerr was found in mount gedang atas and mount ijo, baturagung mountains, yogyakarta. the morphological characteristics of stature (habitus), leaves, of the telosma puberula flower specimen in mount gedang atas and mount ijo shows similarities with the kew herbarium holotype collected by horsfield in 1802 & 1859 (k000873053, k000873053) from java. the existence of telosma puberula (miq.) kerr in java complete and confirm the descriptions made by bakhuizen and backer (1965: 273) on the genus telosma. acknowledgements the author would like to express his gratitude to the herbarium museum national d'histoire naturelle, paris (mnhn) and the herbarium royal botanic garden of kewensis edinburg (kew) on the types of herbarium photo credit. the author would also thank mr. dr. m. jafar luthfi in the biology laboratory of uin sunan kalijaga yogyakarta for his testimony, discussion and confirmation of the discovery of this plant specimen on site. references backer, c. a. &bakhuizen. 1965. flora of jawa (spermatophytes only).vol i, ii, iii. groningen: n. v. p. noordhoff. backer, c. a. 1949. beknopte flora van java (nood unitgave) 8(a. fam. 173): 63. 1949. http://www.tropicos.org/name/2609608 accessed 7 october 2014. blume, c. l. 1826. bijdragen tot de flora van nederlandsch indië 1056. flora of china editorial committee. 1995. flora of china (gentianaceae through boraginaceae). 16: 1–479. in c. y. wu, p. h. raven & d. y. hong (eds.) fl. china. science press & missouri botanical garden press, beijing & st. louis.http://www.tropicos.org/image/22 934. accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. pergularia accedens. http:// colb .mnhn. fr/catalognumber/mnhn/p/ p03858919., accessed 7 october 2014. herbarium museum national d’histoire naturelle paris (mnhn). 2014. telosma procumbens. http:// colb .mnhn .fr/catalognumber/mnhn/p/ p03858921., accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma accedens. http:// colb .mnhn. fr/catalognumber/mnhn/p/ p03858922., accessed 7 october 2014. a b c d 52 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma puberula. http:// colb. mnhn. fr/catalognumber/mnhn/p/ p03858929., accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma puberula. http:// colb. mnhn. fr/catalognumber/mnhn/p/ p03858930.,accessed 7 october 2014. hooker, j. d. 1885. flora of british india (vol. iv). london: reeve and co. http://specimens.kew.org/herbarium/k0 00873052. diakses 7 oktober 2014 http://www.tropicos.org/name/2602046. accessed 7 october 2014. ipni (international plant name index). 2014. telosma (http:// www. plantsystematics. org, accessed 7 october 2014. kerr, a. f. g. 1951. flora siamensis enumeratio 3(1): 32. http:// www. tropicos. org/name/2609609. accessed 7 october 2014. merill, e.d. 1912. philippine journal of science 7: 243. http:// www. tropicos. org/ name/2607466. accessed 7 october 2014. royal botanic garden, kew. 2014. telosma puberula. singh, g. 1999. plant systematics. new hampshire: science publisher. takhtajan, a. 2009. flowering plant. st petersburg: springer. the plant list (2010). version 1. published on the internet; http:// www.theplantlist .org/ tpl/ search?q=telosma+ . diakses 7 oktober 2014. united states national herbarium. 2014. telosma procumbens. https://www. flickr. com/ photos/ filibot/8045825440. accessed 7 october 2014. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 161-167 | doi: 10.14421/biomedich.2022.112.161-167 issn 2540-9328 (online) chemical compositions and antioxidant activity of volatile oils from morinda citrifolia and beta vulgaris leaves from nigeria adesegun olusimbo onanuga1, ejike onwudiegwu okpala2,* 1organic chemistry unit, department of chemistry, university of ibadan, ibadan, nigeria 2department of chemistry, faculty of science, federal university lokoja, kogi state-nigeria. corresponding author* ejike.okpala@fulokoja.edu.ng; okpalaejike@gmail.com manuscript received: 27 july, 2022. revision accepted: 15 august, 2022. published: 31 august, 2022. abstract morinda citrifolia l. and beta vulgaris l leaves are both ethnomedicinal use for the treatment of arthritis, indigestion and skin infections with no reports on their essential oils compositions. the colourless volatile oils with a percentage yield of 0.6 and 0.4 (w/w) for morinda citrifolia l. and beta vulgaris l respectively were obtained. forty-five compounds representing 94.31 % of the total percentage compositions were identified in the leaf essential oil of m. citrifolia with the most abundant compound as 14-beta-h-pregna(33.13%). forty-eight compounds representing 74.18% of the total oil composition were identified in the leaf oil of b. vulgaris with phytol (24.20%) as the dominant compound. the essential oils showed good free radical scavenging activity when compared to ascorbic acid used as control, with % inhibition varying from 88.74 ± 0.010 to 96.61 ± 0.004 as compared to 95.68 ± 0.010 to 97.31±0.003 of the ascorbic acid at (100 to 6.25 mg/ml) concentrations. the leaves essential oils of morinda citrifolia l. and beta vulgaris l contains chemical compounds that might be responsible for their antioxidant activity. this result validates the traditional usage of these plants in the treatment of arthritis, indigestion and skin infections. keywords: morinda citrifolia l.; beta vulgaris l.; antioxidant; free radical scavenging activity volatile oil. introduction the morinda citrifolia l (figure 1), also known as noni in nigeria, belongs to the rubiaceae family (arunachalam, 2018). it is a plant native to southeast asia and australia that has long been used medicinally. according to reports, m. citrifolia can treat a wide range of medical conditions, including arthritis, heartburn, headaches, wounds, and skin infections. additionally, there are some reports on the antitumor and anticancer activity of this plant and biological activities such as antihypoglycemia have been reported (algenstaedt et al., 2018). beta vulgaris l. (figure 1), popularly known as beetroot, is a member of the chenopodiaceae family (mello et al., 2008). it is a biennial herbaceous plant. it is eaten in nigeria as a vegetable. it has traditionally been believed to treat or prevent conditions like arthritis, colon, prostate, dyspepsia, and skin infections. beta vulgaris leaf consumption lowers the risk of diabetes mellitus, obesity, and cardiovascular illnesses. it is also recognized for its potential to treat cancer (kumar et al., 2016). it is known to have a wide range of biological activities, including properties that are antibacterial, antioxidant, anticancer, antiviral, and anti-diabetic (kavitha et al., 2020). consequently, in continuation of our search for biologically active compounds from plants with ethnomedicinal uses (okpala et al., 2021; okpala et al., 2022; onanuga and oloyede, 2021), we present chemical compositions of the volatile oils and antioxidant activities of leaves of beta vulgaris l. and morinda citrifolia l. the chemical compositions of the essential oils (eos) of the leaves of both plants have not been reported in the literature to the best of our knowledge. beta vulgaris leaves morinda citrifolia leaves figure 1. the image of the studied plants. material and methods plant material the samples of morinda citrifolia l. leaves were obtained fresh from akobo ibadan, oyo state, southhttps://doi.org/10.14421/biomedich.2022.112.161-167 162 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 west, nigeria (7o22' 39’’n; 3o 54’21’’e), on 10th april, 2021 while beta vulgaris l. leaves were purchased from bodija market ibadan, oyo state. the plants were identified by dr. s. k. odewo of forestry research institute of nigeria (frin) ibadan, oyo state. extraction of the essential oil the air dried and pulverized leaves of m. citrifolia (200 g) and b. vulgaris (200 g) were weighed and separately subjected to extraction using hydro-distillation method with clevenger type apparatus for four hours following british pharmacopoeia specifications with modifications (british pharmacpoeiae, 1980). the samples were added into a 2 l round-bottomed flask containing 1.0 l distilled water and heated to boiling. there was the evaporation of the essential oils together with water vapour and these were collected in a condenser. the upper phase that contained the eos was separated from the lower one and anhydrous sodium sulphate was used for drying the oils isolated. oils extracted were preserved in a sealed amber glass vial at 4oc until analyses. the percentage yields (w/w) were determined. gas chromatograph-mass spectrometry (gc-ms) of the oils gc-ms analysis of the oils was performed using gas chromatography 7890 coupled with mass spectrometer 5975 agilent technology. the chemical components were identified by matching their mass spectra with those recorded in the mass spectra library (w11n17 main). the stationary phase was the column of model agilent technologies hp-5 ms of length 30 m, the internal diameter of 0.320 mm and thickness of 0.25 µm while the mobile phase was helium gas. the oven temperature was at 80oc held for 2 mins at 12 degrees per minute to the final temperature of 240oc held for 6 mins. the ion source was set at 240oc and electron ionization at 70 ev. the scan ranges were from 50 to 550 amu and the interface temperature between gc and ms was 250oc. a sample of (1.0 µl) of diluted oils in hexane was manually injected into the gc-ms. antioxidant assay the antioxidant activity of the essential oils was evaluated using the dpph (2, 2-diphenyl-1picrylhydrazyl) free radical scavenging ability method. the concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 µg/ml) of the essential oils were mixed with 100 µm methanoldpph solution (2.0 ml) prepared by dissolving 3.94 mg of dpph in 100 ml of methanol to give a purple solution. the mixture was shaken vigorously and left to incubate for 30 minutes in the dark at room temperature and the absorbance was then measured at 517 nm and recorded as a (sample) using a gs uv-12, uv-vis spectrophotometer. in its radical form, dpph absorbs, but upon reduction by antioxidant species, its absorption reduces. a blank experiment was carried out applying the same procedure without essential oil (dpph + methanol) and the absorbance was recorded as a (control). ascorbic acid was used as a standard antioxidant for comparison. the free radical scavenging activities of each essential oil were then calculated as percentage inhibition according to the following equation: % 𝐼 = acontrol − asample acontrol × 100 results the physical properties and percentage yields of the volatile oils obtained from m. citrifolia and b. vulgaris leaves are presented in table 1. forty five compounds representing 94.31 % of the total percentage compositions were identified in the leaf essential oil of m. citrifolia (figure 2). the most abundant compounds are 14-beta-h-pregna(33.13%), 1-hexacosane (11.11%), heneicosane (7.90%) and tricosane (7.17%). the constituents of the oil were mainly non-terpenoids: hydrocarbon (43.67%), steroids (33.13%), alcohols, esters and fatty acids (9.26%) while the terpenes present are monoterpene (0.40%), diterpene (6.05%) and triterpene (1.28%) table 2. forty eight compounds representing 74.18% of the total oil composition were identified in the leaf oil of b. vulgaris (figure 3). the dominant compounds are phytol (24.20 %), 1, 3-dimethylbenzene (14.84%) and neophytadiene (6.13%). the major class of compounds identified are diterpene alcohol (24.20) and aromatic compounds (18.70%) table 3. some similarities of the ethnomedicinal uses of the two plants could be related to the presence of compounds such as phytol, neophytadiene, mesitylene, 3-hexanol, decane, docosane, tetracosane, eicosane and heneicosane which were identified in the essential oils of both plants. the gc-ms chromatograms of the essential oils are given in figures 2 and 3. the antioxidant activity of the essential oils of the leaves of m. citrifolia and b. vulgaris are presented in tables 4 and 5. the dpph scavenging ability of the essential oils was compared with ascorbic acid a known standard antioxidant. onanuga & okpala – chemical compositions and antioxidant activity of … 163 figure 2. the gc-ms chromatogram of the leaf essential oil of m. citrifolia. figure 3. the gc-ms chromatogram of the leaf essential oil of b. vulgaris. 164 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 table 1. physical properties and yields of essential oils from m. citrifolia and b. vulgaris leaves. plant weight of leaf sample (g) weight of oil obtained (g) % (w/w) yield of the oil obtained physical properties morinda citrifolia l. 200 1.2 0.6 colourless, herbaceous beta vulgaris l. 200 0.8 0.4 colourless; leafy and aromatic odour table 2. chemical compositions of m. citrifolia essential oils. s/n rt (min) chemical constituents % composition class of compound 1 3.260 3-hexanol 0.14 alcohol 2 3.325 6-methyl-2-heptanol 0.25 alcohol 3 3.800 ethyl-cyclohexane 0.15 cycloalkane 4 4.124 2-hexenal 0.24 aldehyde 5 4.189 3-hexen-1-ol (z) 4.66 alcohol 6 4.357 2-hexen-1-ol (e) 0.77 alcohol 7 4.394 1-hexanol 0.74 alcohol 8 4.465 3-methylene-1-vinyl-1-cyclopentene 0.25 cycloalkane 9 4.902 2-heptanol 1.20 alcohol 10 5.858 2-hexanol 0.22 alcohol 11 6.204 1-octen-3-ol 0.35 alcohol 12 6.415 mesitylene 0.13 aromatic 13 6.534 decane 0.11 alkane 14 6.663 3-hexen-1-ol, acetate 0.57 ester 15 7.684 benzofuran 0.94 heterocyclic 16 8.133 3-carene 0.12 monoterpene 17 9.235 cinnamaldehyde (e) 0.30 aldehyde 18 9.872 2-methyl-2-nonen-4-one 0.10 ketone 19 10.050 citronellol 0.28 monoterpene 20 10.520 2-decanal (e) 0.15 aldehyde 21 13.502 4-(2,6,6-trimethyl-1-cyclohexen-1-yl 3-buten-2-one 0.19 ketone 22 17.408 neophytadiene 0.66 diterpene 23 17.473 3-octadecene (3e)0.18 alkene 24 17.840 3-eicosene (e) 0.24 alkene 25 18.013 nonadecane 0.13 alkane 26 18.623 n-hexadecanoic acid 0.34 fatty acid 27 18.985 eicosane 1.05 alkane 28 19.093 14-beta-h-pregna 33.13 steroid 29 19.785 1-nonadecene 0.68 alkene 30 19.920 heneicosane 0.85 alkane 31 20.66 phytol 5.39 diterpene 32 20.207 1-heneicosene 0.36 alkene 33 20.325 1-eicosene 1.20 alkene 34 20.482 1-tetracosene 0.62 alkene 35 20.817 docosane 1.69 alkane 36 21.670 tricosane 7.17 alkane 37 21.750 1-hexacosene 11.11 alkene 38 22.491 tetracosane 3.16 alkane 39 23.291 heneicosane 7.90 alkane 40 24.053 hexacosane 3.65 alkene 41 25.317 9-hexacosene 0.53 alkene 42 25.500 octacosane 0.98 alkane 43 25.733 supraene 1.28 triterpene 44 26.186 nonacosane 0.44 alkane 45 27.499 3-ethyl-2,6,10-trimethyl undecane 0.24 alkane total percentage composition 94.31 onanuga & okpala – chemical compositions and antioxidant activity of … 165 table 3. chemical compositions of b. vulgaris essential oils. s/n rt (min) chemical constituents % composition class of compounds 1 3.125 3-hexanone 0.31 ketone 2 3.152 1-ethyl-3-methyl cyclopentane 0.41 cycloalkane 3 3.179 2-hexanone 0.89 ketone 4 3.260 3-hexanol 1.22 alcohol 5 3.250 2,5-dimethyl-1-hepten-4-ol 1.20 unsaturated alcohol 6 3.384 1, 3-dimethyl-transcyclohexane 0.39 cycloalkane 7 3.730 1,2-dimethylcyclohexane 0.33 cycloalkane 8 3.800 ethyl-cyclohexane 0.82 cycloalkane 9 4.081 1,2,4-trimethyl cyclohexane 0.26 cycloalkane 10 4.124 2-hexanal (e)0.60 aldehyde 11 4.243 ethylbenzene 2.57 aromatic 12 4.308 nonane 0.31 alkane 13 4.470 1,3-dimethylbenzene 14.84 aromatic 14 4.659 cis-1-ethyl-3-methyl-cyclohexane 0.13 cycloalkane 15 4.886 nonane 0.18 alkane 16 5.858 2-methyl-3-propyl-trans oxirane 1.30 cyclo ether 17 6.415 mesitylene 0.56 aromatic 18 6.534 decane 0.61 alkane 19 6.885 1-ethyl-3-methyl benzene 0.12 aromatic 20 7.360 1-methyl-3-propylbenzene 0.13 aromatic 21 7.479 1-ethyl-3,5-dimethyl benzene 0.14 aromatic 22 7.895 1,2,4,5-tetramethyl benzene 0.13 aromatic 23 8.203 nonanal 0.12 aldehyde 24 8.754 3-methyl-6-(1-methyl ethyl ) cyclohexene 0.68 cycloalkane 25 9.251 4-acetyl-1-methyl cyclohexene 0.24 cycloalkane 26 9.872 1-cyclopropyl-2-propanone 0.72 cycloketone 27 9.964 1-carboxaldehde, 2,6,6-trimethyl-1cyclohexene 0.33 cycloalkane 28 11.039 tridecane 0.17 alkane 29 12.363 tetradecane 0.15 alkane 30 13.059 6,10-dimethyl, 5,9-undecadien-2-one 0.18 unsaturated ketone 31 13.173 1-[(e)-3-methylbut-1-enyl]cyclohexene 0.32 cycloalkane 32 13.281 1-(1,1-dimethylethyl)4-ethyl benzene 0.21 aromatic 33 13.502 4-(2,6,6trimethyl-1-cyclohexen-1-yl) 3-buten-2-one 1.83 cycloketone 34 13.967 copaene 0.23 alkene 35 14.707 2-methyl-4-(2,6,6, trimethyl-1-cyclohexen-1-yl) 2 butenal 0.16 cycloaldehyde 36 15.922 heptadecane 0.27 alkane 37 17.354 3,7,11,15 tetramethylhexadec-2-ene 1.78 alkene 38 17.408 neophytadiene 6.13 diterpene 39 17.948 1-nonadecene 0.18 alkene 40 19.780 e-15-heptadecanal 0.34 aldehyde 41 19.839 1-octadecene 0.40 alkene 42 19.920 heneicosane 0.29 alkene 43 20.066 phytol 24.20 diterpene 44 20.325 diallylacetal, palmitaldehyde 3.18 aldehyde 45 20.811 docosane 0.87 alkane 46 21.665 eicosane 1.98 alkane 47 22.491 tetracosane 0.68 alkane 48 23.285 3-pentacosene (e) 1.09 alkene total percentage composition 74.18 166 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 table 4. absorbance values at 517 nm of dpph method of antioxidant assay. conc. (mg/ml) m. citrifolia b. vulgaris asc. acid 100 0.251 ± 0.003 0.102 ± 0.004 0.081 ± 0.003 50 0.270 ± 0.002 0.106 ± 0.007 0.097 ± 0.004 25 0.280 ± 0.013 0.116 ± 0.007 0.105 ± 0.009 12.5 0.312 ± 0.005 0.123 ± 0.005 0.127 ± 0.005 6.25 0.339 ± 0.010 0.133 ± 0.005 0.130 ± 0.010 absorbance values in mean ± standard error; asc.acid = ascorbic acid at 517nm; ± standard deviation for measurement, absorbance of control =3.010±0.005 table 5. percentage inhibition calculated from dpph method of antioxidant assay. conc. (mg/ml) m. citrifolia b. vulgaris asc. acid 100 91.66 96.61 97.31 50 91.02 96.48 96.78 25 90.69 96.15 96.51 12.5 89.63 96.91 95.78 6.25 88.74 95.58 95.68 discussion methyl hexanoate, methyl octanoate, ethyl octanoate and methyl 4 e-decanoate have been reported in the volatile oil of m. citrifolia fruit, they are found to contain flavonoids as the major phytochemicals (pino et al., 2010). 14-b-h-pregna, a steroid considered to be a sex pheromone specific to males, and also a defensive chemical with diabetic retinopathy prevention and treatment effects. the presence of 14-b-h-pregna has been reported in the essential oils from different parts of some plants, including scutellaria plants, urginea indica kunth, allium rotundum, gundelia tournefortii l (farhang et al., 2016). citrus limon (akhila et al., 2015). dehpour et al.2012 reported that 14-b-h-pregna was the major compound in the essential oil of lower allium rotundum which displayed antibacterial activity. neophytadiene is a good analgesic, antimicrobial, antipyretic, antioxidant and anti-inflammatory compound (venkata et al., 2012). phytol, known to exhibit antioxidant and antinociceptive effects is a precursor of synthetic vitamins e and k and is cytotoxic against breast cancer cell lines (mcf7) (casuga et al., 2016; sermakkani and thangapandian, 2012). the measured absorbance values and calculated percentage inhibition show that the antioxidant activity of the two essential oils and standard (ascorbic acid) is concentration dependant. from the results, the % inhibition of the essential oils of both plants exhibited good scavenging ability on dpph radical which were comparable to ascorbic acid at all concentrations (1006.25 mg/ml) investigated. conclusions analysis of the essential oils of m. citrifolia and b. vulgaris leaves showed that they contained different major constituents. the major constituent of m. citrifolia leaves essential oil were steroids: 14-beta-h-pregna (33.13%) and 1-hexacosane (11.11%), while the dominant compounds identified in b. vulgaris were phytol (24.20 %) and 1,3-dimethylbenzene (14.84%. these major constituents of both oils have been reported to possess similar properties such as antibacterial, antioxidant and antinociceptive. most of the chemical compounds identified from the essential oils of m. citrifolia and b. vulgaris were biologically active compounds and the essential oils exhibited good scavenging ability at all concentrations investigated. the plants’ parts could be a source of drug development for oxidative diseases. competing interests: a. o. onanuga and e. o. okpala declare that they have no competing interests. acknowledgements: the authors are grateful to the university of ibadan, nigeria for providing the laboratory space and facilities for the extraction and antioxidant analysis of the essential oils. references akhila s, bindu a, bindu k. (2015). phytochemical and pharmacological evaluation of citrus limon pell. world j. pharm. pharma. sci. 4(3):1128-135. algenstaedt, p, stumpenhagen, a, westendorf, j. (2018). the effect of morinda citrifolia l. fruit juice on the blood sugar level and other serum parameters in patients with diabetes type 2. evi. compl. alt. med. 3565427:10 arunachalam, v. (2018). morinda citrifolia l. (rubiaceae): a multipurpose tree for coastal ecosystems and its variability in konkan region of india. genetic resources and crop evolution, 65(6):1751-1765. british pharmacoepia. (1980). london: h. m., stationary office, pa. pp. 109. casuga, f., a. castillo, a., corpuz, m. (2016). gc–ms analysis of bioactive compounds present in different extracts of an endemic plant broussonetia luzonica (blanco) (moraceae) leaves asian pac. j. trop. biomed. 6, 957-961. dehpour a.a., yousefian m., kelarijani s.j., koshmoo, m., mirzanegad s., mahdavi, v. (2012). antibacterial activity and composition of essential oils of flower allium rotundum. adv. env. bio. 6(3):1020-1025. deng s, west bj, palu ak, jensen cj. (2012). phytochemical, antioxidant and toxicological investigation of morinda citrifolia l. blossoms, anal chem. 2012:1-5. farhang, hr, vahabi, mr, allafchian, ar. (2016). chemical compositions of the essential oil of gundelia tournefortii l.(asteraceae) from central zagros, iran. j. herbal drugs. 6(4):227-233. guldiken b, toydemir g, memis kn, okur s, boyacioglu d, et al. (2016). home-processed red beetroot (beta vulgaris l.) https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ onanuga & okpala – chemical compositions and antioxidant activity of … 167 products: changes in antioxidant properties and bioaccessibility. int. j. mol. sci. 17: 858. okpala, eo, onocha, pa, ali, ms, zikr-ur-rehmen s, lateef, m. (2021). zenkeramide: a new iso-benzofuranone propanamide and urease inhibitory constituents of celtis zenkeri engl stem bark (ulmaceae) nat. prod.res.1-6. doi:10.1080/14786419.2021.19546443. okpala, e., onocha, p., ali, m. (2022). antioxidant activity of phytol dominated stem bark and leaf essential oils of celtis zenkeri engl., trends in phytochemical research, 6(2), 137144. doi:10.30495/tpr.2022.1952985.1246 onanuga ao, oloyede gk. (2021). two new biologically active steroids from costus lucanusianus (costaceae) steroids (175) 108913. pino ja, eliosbel m, clara eq, déborah c. (2010). volatile compounds in noni (morinda citrifolia l.) at two ripening stages. ciênc. tecnol. aliment. 30(1):183-87. sermakkani m., thangapandian, v. (2012). gc-ms analysis of cassia italica leaf methanol extract. asian j. pharm. clin. res., 5, 90-94. venkata, rb, samuel la, pardha sm. (2012). antibacterial, antioxidant activity and gc-ms analysis of eupatorium odoratum. asian j. pharma. clin. res. 5(2) 99-106. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 41-46 | doi: 10.14421/biomedich.2021.101.41-46 issn 2540-9328 (online) computational chemical study of pigment of mangosteen (garcinia mangostana) rind extract as dye compound in dye-sensitized solar cell (dssc) didik krisdiyanto*,1, sudarlin1, hikmah supriyati2 1department of chemistry, faculty of science and technology, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no.1 yogyakarta 55281, tel. + 62-274-540971, fax. + 62-274-519739, indonesia 2post graduate program of biological education department, universitas negeri yogyakarta jalan colombo no. 1 yogyakarta 55281, indonesia corresponding author* didik_kris@yahoo.com manuscript received: 03 march, 2021. revision accepted: 30 june, 2021. published: 19 july, 2021. abstract the electronic transition processes of α-mangostin and β-mangostin as dye compounds in dssc and their ability to transfer electrons to semiconductors have been studied in theory. the research was carried out computationally using the nwchem application. the methods used are density functional theory (dft) for structure optimization and time-dependent dft (td-dft) for electronic transitions. the results showed that the energy of homo β-mangostin was lower that it was more stable or easier to form bonds with semiconductors. likewise, its lumo energy is lower than α-mangostin that it is easier to inject electrons into the semiconductor. the energy difference of homo-lumo β-mangostin is smaller than α-mangostin. this shows that β-mangostin is more easily excited or more efficient in exciting solar energy to convert it into electricity. this is consistent with the molecular spectra where λmaxβ-mangostin is at a higher wavelength. orbital analysis shows electron injection of α-mangostin and β-mangostin into the semiconductor via double o atoms in each molecule. the injection is influenced by the bond length, where the bond length of α-mangostin to tio2 is smaller than that of β-mangostin to tio2. keywords: α-mangostin; β-mangostin; energy homo-lumo; dft; td-dft; mangosteen rind; dye-sensitized solar cell. introduction research on dye compounds for dssc (dye-sensitized solar cell) has been widely carried out, but synthetic dyes' efficiency is still higher than natural dyes, which are only ~ 1%. however, synthetic dyes have drawbacks including difficult preparation, expensive production costs, and a toxic risk to the environment. the use of natural dyes becomes a very attractive alternative to develop because they are easy to obtain, have unlimited sources, have a long absorbance coefficient, high light capture efficiency, low production costs, easy preparation, and are environmentally friendly (luo et al., 2009). however, not all dyes can be used as sensitizers because they must qualify, namely adsorption intensity at visible wavelengths, strong adsorption on the semiconductor surface, has the ability to inject electrons into the semiconductor conduction band, and has groups = o or –h to bind with the tio2 surface which can increase the rate of the electron transfer reaction (ludin et. al., 2014). various studies on dssc using natural dyes from plant extracts have been conducted and these studies have proven that natural dyes can provide a photovoltaic effect. one of the sensitizers used is mangosteen rind pigment extract. chairat et al. (2007) in their research used mangosteen rind pigment extract which was proven to be used as a sensitizer on solar cells of the dssc type. mangosteen is very easy to obtain. this is because indonesia is a tropical country that is very suitable for the growth of mangosteen. the main component in the mangosteen pigment extract which is potential as a dssc sensitizing dye is shown in figure 1. the α-mangostin and β-mangostin compounds are two of the several components in the mangosteen rind pigment extract which has absorption at visible wavelengths as one of the requirements for the dssc dye. further studies on the adsorption of compounds on semiconductor surfaces, electronic energy levels, and electron transfer capabilities need to be done. the study in this research was done with the help of computational chemistry. two basic structures of the main components of mangosteen rind extract, the electro-optical properties of dssc will be studied based on computational calculations to study adsorption on semiconductors, adsorption spectrum, energy levels, and charge transfer. https://doi.org/10.14421/biomedich.2021.101.41-46 42 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 a b figure 1. basic structure of mangosteen rind extract (a) α-mangostin (b) β-mangostin. dye-sensitized solar cell (dssc) is a series of devices that can convert visible light into electricity based on semiconductor band gap sensitization and its bonding with groups on the surface of a thin layer cell (ludin et. al., 2014). the dssc material consists of a pair of glass substrates coated with tco (transparent conducting oxide) material which acts as an electrode and a counter electrode separated by a redox electrolyte which has high transparency and low resistance characteristics, can be indium tin oxide (ito), aluminum zinc oxide (azo), and fluorine tin oxide (fto) mounted opposite each other. fto and ito are most often used as dssc, where the process of sintering the oxide layer on the substrate at a temperature of 450500oc, these materials have good conductivity and do not experience defects or defects in that temperature range (halme, 2002). figure 2. schematic and working principle of dye-sensitized solar cell (dssc). the redox pair that is often used is i/ i3(iodide / triiodide). the solvent used in the electrolyte solution is polyethylene glycol (peg) because it can penetrate the tio2 dye absorption for both small particle size comparisons and nano-scale pore diameters, and can maintain work stability (misbahudin et.al., 2013). at tco, the counter electrode is coated with a catalyst in the form of a carbon layer to accelerate the redox reaction, while the electrode is positioned as a porous tio2 nanocrystalline layer as a photoanode and is sensitized by dye as a photosensitizer which functions to capture photons which then will be absorbed into tio2 nanoparticles. an efficient photosensitizer must qualify, namely dye particles can be adsorbed onto the semiconductor surface, the capacity to capture light in the visible light range, the ability to inject electrons into the conduction band of the semiconductor, and have an = o or –h group to bind to the tio2 surface. the working principle of the dssc is shown in figure 2. basically, the working principle of dssc is a reaction of electron transfer where the first process begins with the excitation of electrons in the dye molecule due to photon absorption. electrons are excited from the ground state (d) to excited state (e). s + photon  s* (absorption) (1) the electrons from the excited state are then directly injected into the conduction band (ecb) of the titania so that the dye molecules are oxidized (s +). with the presence of an electron donor by the electrolyte, the dye molecules return to their ground state and prevent the recapture of the oxidized dye electrons. s* + tio2 e(tio2) + s+ (injection process) (2) the injected electrons are carried from the tio2 nanopore thin layer to the conductive electrode (anode). e(tio2)+the conductive electrodetio2+e(c.e) (3) after reaching the tco electrode, electrons flow towards the electrode counter through the external circuit and approach the cathode (counter electrode). the electrons are transferred to the electrolyte at the electrodes. with a catalyst on the counter electrode, the oxidized dye accepts electrons from the ion (i-), a redox reaction occurs to replace the lost electrons, and the iodide molecule is oxidized to the triiodide ion (i3-). s+ + 3 2 i- s + 1 2 i3 (4) ion (i3-) is used to donate electrons to the oxidized dye permitting an electron transport cycle to be formed, with this cycle a direct conversion from sunlight to electricity. 1 2 i3+ e(c.e)  3 2 i+ c.e (5) (narayan, 2012). sunlight produces 45% of the spectra in the visible and 5% of the uv spectra so that in the use of dyes in the dssc, the absorption of photon energy from visible light is carried out by light-sensitive materials (dyes that function as sensitizers). with the presence of a sensitizer, it is possible to inject/transfer electrons to the tio2 semiconductor material even though the received photon energy is smaller than the band gap of the tio2 semiconductor (this event is called sensitisation). to krisdiyanto, et al. – computational chemical study of pigment of … 43 support this process, the tio2 semiconductor material must be able to entangle as many molecules of the color substance as possible so that more electrons can be accepted. some of the criteria that must qualify by tio2 as a semiconductor in dssc include the particle size being on the nanometer scale. this is necessary because with the particle size that is on the nanometer scale the surface area of the particles as a whole becomes larger so that more dye molecules are possible to absorb. in addition, tio2 particles are also expected to have a porous (mesoporous) morphology, allowing the dye molecules enter between the pores and can be absorbed on the surface of the tio2 particles, which implies that they will increase the amount of light absorbed (zhang et.al., 2008). in addition, the use of semiconductor oxides in photoelectrochemistry is due to its stability against corrosion photo and also its large energy band (3.2-3.8 ev) which in dssc is needed for semiconductor transparency in most of the sunlight spectrum. although tio2 is a material that is often used because the efficiency of dssc using tio2 is still unmatched, several materials can act as semiconductor oxides including zno, cdse, cds, wo3, fe3o3, sno2, nb2o5 and ta2o5 (halme, 2002). the dye in the dssc oxide layer serves to capture photons of light. furthermore, these photons are absorbed into the tio2 nanoparticle. the requirements for dye as a photosensitizer in dssc are absorption in the visible light region or near the infrared region of the sunlight spectrum and bind to the tio2 semiconductor (cherepy et.al., 1997). functional groups are needed to interact with tio2 surfaces such as carboxylates or around other acid groups (galopini, 2004). several functional groups have the possibility to bind with tio2. the best groups are metal oxides such as phosphonic acid followed by carboxylic acids and their derivatives such as chlorides, amides, esters or carboxylic salts (galopini, 2004). figure 3. possible bond models between -cooh groups and tio2 (hug et. al., 2014). plant extracts or pigments used as photosensitizers in visible light areas can be in the form of chlorophyll extract (amoa, 2003). chlorophyll is the main plant pigment which functions to absorb light and convert it into chemical energy needed to reduce carbon dioxide into carbohydrates in the photosynthesis process. figure 3 shows several bonding models between tio2 and the dye molecule with at least one carboxylate group. another requirement for this material to be an active ingredient in solar cells is that the material must be able to become a transfer medium for electric charge carriers as a result of the absorbed photons (supriyanto et. al., 2010). to obtain a suitable dye as a sensitizer requires a long time and resources in research which is computational chemistry can be an alternative. by knowing in more detail the electro-optical properties of a dye, it can be used as a basis for the development of efficient dye alternatives. density fungtional theory (dft) and time dependent dft (td-dft) methods can be used for large molecules with an accurate approach to calculate the uv spectrum of dyes as well as for complex systems between dyes and semiconductors. methods the research was conducted computationally using the nwchem application. the methods used are density functional theory (dft) for structure optimization and time dependent dft (td-dft) for electronic transitions. all calculations use b3lyp functional and 6-31g * base set. data visualization using ecce and chemcraft applications. in the study of dyestuffs for dssc in computational chemistry involving large molecules and complex systems, a suitable approach is needed. results and discussion method selection the calculation results obtained were compared with the results of experiments conducted by madihah et al. (2012). the parameter being compared is the αmangostin excitation energy in units of ev as shown in table 1. other parameters such as bond length cannot be compared because experimental data are not available. the results of the calculations as shown in table 1 show the difference in excitation energy from the calculation of α-mangostin and the experimental results of 0.01ev. the similarity between the calculation results and the experimental results reached 99.71%. these results indicate the selection of methods and basis sets in this study are correct and quite accurate. 44 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 table 1. comparison of the first excitation energy of α-mangostin on experimental and calculated results. no α-mangostin data excitation energy (ev) similarity level 1. experiment 3.522 99,71% 2. calculation 3.532 supporting data that can be compared is the display of α-mangostin spectra on the experimental and the calculation results at wavelengths above 280 nm, which shows the similarities as shown in figure 4. figure 4. the α-mangostin spectra of the experimental results and the calculation results above 280 nm. optimization of homo-lumo molecules and energy optimization is carried out on each molecule using the same method. the optimization results are in the form of a stable structure as shown in figure 5 and the multivariate load value as shown in table 2. a b figure 5. the results of the calculation of α-mangostin (a) and βmangostin (b). based on the optimization results of α-mangostin and β-mangostin in figure 5, each red o atom has a different mullicen charge value as shown in table 2. based on the table, the o-4 atom has a more negative charge so it is more reactive in binding to other atoms. thus, o-4 atoms can be used to bind tio2. the homo and lumo energies of each compound as a result of the optimization can be seen in table 3. based on the homo energy in the table, β-mangostin has a lower homo energy. based on the requirements of the dye compound, this indicates that β-mangostin is more stable so it is easier to form bonds with semiconductors. likewise, the lumo β-mangostin energy is lower than the lumo α-mangostin energy so that β-mangostin is easier to inject electrons into the semiconductor. table 2. mullicen charge values of o α-mangostin and β-mangostin atoms. atom o α-mangostin β-mangostin o-1 -0,135014 -0,135076 o-2 -0,240754 -0,240796 o-3 -0,360894 -0,361235 o-4 -0,377779 -0,380988 o-5 -0,370443 -0,200601 o-6 -0,341725 -0,341406 table 3. the energy difference between homo-lumo α-mangostin and β-mangostin. energy α-mangostin β-mangostin homo (ev) -5.647 -5.677 lumo (ev) -1.547 -1.783 δ (ev) 4.100 3.894 figure 6. the energy bands of α-mangostin and β-mangostin calculated. the difference in energy bands produced is the difference between homo energy and lumo energy. the smaller the value of the energy band difference, indicating that the easier the electrons in the molecule move from a lower energy level to a higher energy level. figure 6 shows that the smaller homo-lumo energy difference is β-mangostin. this shows that β-mangostin is more easily excited. that is, β-mangostin is more efficient in exciting solar energy to convert it into electricity. the shape of the electron orbitals in the homo and lumo states the shape of the homo α-mangostin and β-mangostin orbitals as presented in figure 7 shows the same pattern. td spectrum wavelength, nm 360 350 340 330 320 310 300 290 280 f 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 krisdiyanto, et al. – computational chemical study of pigment of … 45 the figure shows the delocalized homo orbital electrons on the o-4 atom. this corresponds to the high mullicen charge of o-4 atoms, thus strengthening the reason that tio2 semiconductors tend to bond to o-4 atoms, both to α-mangostin and β-mangostin. a b figure 7. form of the calculated α-mangostin (a) and β-mangostin homo orbitals (b). as with the homo orbitals, the lumo α-mangostin and β-mangostin orbitals show the same pattern as shown in figure 8. the figure shows the delocalized molecular electrons on the o-6 atom. this shows the electron injection of the dye compound into the semiconductor via o-6 atoms, both in α-mangostin and β-mangostin. a b figure 8. form of the calculated lumo α-mangostin (a) and βmangostin (b) orbitals. bond length the calculation of the bond length is carried out between ti on tio2 and o-6 atoms in α-mangostin and βmangostin. in the previous section, it was explained that electrons are more easily injected into semiconductors via o-6 atoms based on the electron composition of the lumo orbitals. thus, the distance between ti and o-6 atoms at α and β mangostin will affect the electron transfer rate. the closer distance makes the electron transfer easier. a b figure 9. optimization results for α-mangostin + tio2 (left) and βmangostin + tio2 (right). the calculation results as shown in table 4 show the length of each bond. the bond lengths between ti and o-6 in α-mangostin and β-mangostin are 1.989 ǻ and 1.990 ǻ. when compared with the total radius length between ti and o of 2.36 ǻ, it can be concluded that there is a bond between tio2 and α-mangostin and tio2 and β-mangostin. comparison of the bond length between ti and o-6 in α-mangostin and β-mangostin as in the table shows that α-mangostin tends to transfer electrons more quickly to the tio2 semiconductor. table 4. bond lengths of each compound. spectra the spectra in the figure show that the β-mangostin spectra have undergone a bathochromic shift. this is the effect of the addition of the ch3 group as an electron booster that is able to shift the wavelength to a higher wavelength. the shift in wavelength to vis shows the efficiency of β-mangostin as a dye compound is better than α-mangostin. this is because excitation at visible light (vis) to infrared wavelengths is generally an excitation that involves the transfer of charge. excitation involving charge transfer is an excitation that provides an effective injection of electrons into the semiconductor and thus plays a major role in determining the efficiency of dssc cells. figure 10. the calculated spectra of α-mangostin (black curve) and βmangostin (red curve). in addition, the amount of sunlight reaching the earth's surface is dominated by uv rays at a wavelength of 300-400 nm, which is about 95%. uv light at 200300 wavelengths only reaches 5% of the earth's surface because most of it has been absorbed by airborne molecules such as ozone. compound bond length (ǻ) α-mangostin ti – o(6) 1,989 β-mangostin ti – o(6) 1,990 46 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 conclusion the results showed that the energy of homo βmangostin was lower that it was more stable or easier to form bonds with semiconductors. likewise, lumo energy is lower than α-mangostin so that it is easier to inject electrons into the semiconductor. the energy 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(2008). betalain pigments for dyesensitized solar cells. photochemistry and photobiology. 7280. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 67-71 | doi: 10.14421/biomedich.2021.101.67-71 issn 2540-9328 (online) effect of ethanol extracts of musa paradisiaca fruit pulp and peels on haematological indices and liver enzymes of experimental rats emuesiri goodies moke1,*, emuesiri kohworho umukoro1, evelyn tarela ojugbeli2, theresa ezedom2, tarela melish elias daubry3, iziegbe lisa omorodion1 1department of pharmacology and therapeutics; 2department of medical biochemistry; 3department of physiology, faculty of basic medical sciences, delta state university, abraka, nigeria corresponding author* hiligoodies@gmail.com manuscript received: 12 february, 2021. revision accepted: 19 july, 2021. published: 29 july, 2021. abstract medicinal plants usage in traditional medicine has risen over the years. musa paradisiaca has been reported that it possesses various therapeutic efficacies. this study is aimed at evaluating the effect of parts of the ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. thirty wistar rats were grouped into five groups of six animals each which were administered 200 mg/kg and 400 mg/kg of musa paradisiac fruit (mpf) pulp extract or peel extract. haematological indices and liver enzymes were assayed for at the end of the 14-days experimental period. mpf pulp and mpf peel at 400 mg/kg showed a significant (p<0.05) increase in red cell count and haematocrit level as compared to the control. there was a non-significant (p>0.05) increase in haemoglobin concentration in the treated rats as compared to the control. there was also a non-significant (p>0.05) change in ast, alt, and alp level of the treated rats as compared to the control. fruit pulp and peel of musa paradisiaca improve erythrocytes count and haematocrit level, and they may not be associated with liver toxicity. keywords: anaemia; haematology; liver enzymes; medicinal plants; musa paradisiaca. introduction plants possess the ability to synthesize a variety of chemical compounds with various biological functions. the plant kingdom remains a harvest for many species of plants with key medicinal value which are yet to be discovered. reports show that a large population (80%) of people in developing countries depend primarily on medicinal plants for their primary health care (mahomoodally, 2013; ekor, 2014). these plants and herbs are taken in different ways and forms (whole or in parts) for the alternative management of diseases traditionally (benzie and wachtel-galor, 2011; emudainohwo et al., 2015; moke et al., 2019; okafo et al., 2019). musa paradisiaca belongs to musaceae family and is popularly known as plantain. the plant is widely distributed in the southern part of nigeria, west and east africa, malaysia, cameroun and southern parts of united states (uwaoma, 2003; nayar, 2010). musa paradisiaca (plantain) is an important staple crop that contributes to the calories and substance economic in africa (oyeyinka and afolayan, 2019). abundant medicinal activities of parts of musa parasidiaca have been reported. it has been shown to possess hepatoprotective activity (nirmala et al, 2012; issa et al., 2018) as well as antidiabetic (vilhena et al., 2020), antiulcer (onasanwo et al., 2013; ezekwesili et al., 2014; moke et al., 2017), antimicrobial (fagbemi et al., 2009), wound healing (agarwal et al. 2009), and antioxidant properties (yin et al., 2008). the present study is aimed at evaluating the effect of parts of the ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. material and methods plant collection and preparation musa paradisiaca fruits (unripe) were purchased locally from the market, and were identified and authenticated by a taxonomist with existing specimen deposited at the herbarium of the department of botany, delta state university, abraka, nigeria. the fruits were rinsed with water, and both the fruit pulps and fruit peels were air dried. the dried pulps and peels were grinded separately into pulverized powder using a grinding machine for ease of extraction. the powdered materials of musa paradisiaca fruits (400 g each) were separately extracted exhaustively with ethanol using soxhlet extractor at 25 0c. the filtrates were concentrated using rotary evaporator at 40 0c. the percentage yields were 8.4% (fruit pulp) and 9.73% https://doi.org/10.14421/biomedich.2021.101.67-71 68 biology, medicine, & natural product chemistry 10 (1), 2021: 67-71 (fruit peel). the concentrated ethanol extracts were refrigerated prior to use. animals wistar rats (150 – 180 g) were obtained from the animals’ house facility of the faculty. the animals were acclimatized for 7 days prior to the study, and were fed with rat feed and clean water ad libitum. guidelines followed in the handling of animals were in accordance with the ethical standards of the institutional animals ethics committee (iaec), as adopted by the ethical committee of the faculty of basic medical science, delta state university, abraka, nigeria. experimental design the animals were divided into five (5) groups of six animals each:  group 1 – normal saline (control) 10 ml/kg  group 2 – musa paradisiac fruit pulp extract (mpf pulp) 200 mg/kg  group 3 – musa paradisiac fruit pulp extract (mpf pulp) 400 mg/kg  group 4 – musa paradisiac fruit peel extract (mpf peel) 200 mg/kg  group 5 – musa paradisiac fruit peel extract (mpf peel) 400 mg/kg the experimental animals were administered the extracts orally daily for 14 days according to their body weights. sample collection at the end of the 14-days treatment period, the animals were anesthetized using chloroform. blood samples were collected by cardiac puncture into labeled edta bottles for haematological analysis and liver function tests. determination of haematological indices the method as described by tietz (1976) and baker et al. (1998) were used for determining the red blood cells (rbc) counts, haemoglobin (hb) concentration, and haematocrit level. determination of liver function test alkaline phosphatase (alp), aspartate aminotransferase (ast), and alanine transaminase (alt) in serum were determined according to methods described by reitman and frankel (1957) and roy (1970). statistical analysis results are presented as the mean ± standard error of the mean (sem). data were analysed using one-way analysis of variance (anova) followed by tukey’s post hoc test. p-values < 0.05 were taken as significant. results and discussion effect of ethanol fruit extracts of musa paradisiaca on haematological indices of wistar rats figures 1-3 depict the effect of ethanol fruit extracts (pulp and peel) of musa paradisiaca on the red blood cell count, haematocrit level, and haemoglobin concentration of normal wistar rat. mpf pulp and mpf peel at a dose of 200 mg/kg had a non-significant (p>0.05) increase in red blood cell count when compared to the control, however, at 400 mg/kg, there was a significant (p<0.05) increase in red cell count. high dose (400 mg/kg) of both fruit extracts significantly (p<0.05) increased the haematocrit level as compared to the control. there was a non-significant (p>0.05) increase in haemoglobin concentration in the treated rats as compared to the control. g r o u p s r e d b lo o d c e ll ( r b c ) c o u n t ( × 1 0 1 2 /l ) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 2 4 6 8 * * figure 1. the effect of ethanol fruit extracts of musa paradisiaca on red blood cell count of wistar rats. g r o u p s h a e m a t o c r it ( % ) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 2 0 4 0 6 0 * * figure 2. the effect of ethanol fruit extracts of musa paradisiaca on haematocrit level of wistar rats. moke, et al. – effect of ethanol extracts of musa paradisiaca fruit pulp and … 69 g r o u p s h a e m o g lo b in ( h b ) ( g /d l) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 5 1 0 1 5 2 0 figure 3. the effect of ethanol fruit extracts of musa paradisiaca on heamoglobin concentration of wistar rats. blood is composed of a variety of living cells that circulate through the heart and the blood vessels carrying nutrients, hormones, vitamins, antibodies, heat and oxygen to the body’s tissue. the components of blood include red blood cells, white blood cells and platelets which are suspended in plasma (basu and kulkarni, 2014). red cell contains haemoglobin, a protein that carries oxygen to all the tissues of the body. haematocrit or packed cell volume is a measurement of the proportion of blood that is made up of cells. following centrifugation, it is an estimate of the ratio of the volume of red blood cells to the total volume of blood (mondal and budh, 2020). haematocrit and haemoglobin values are useful for assessing anaemia, polycythemia, and also for estimating response to treatment (northrop-clewes and thurnham, 2013; white, 2018; mondal and budh, 2020). hematopoiesis is the process involved in the formation of blood cells (rieger and schroeder, 2012). this study showed the positive effect of the fruit pulp and peel of musa paradisiaca on hematopoiesis. musa paradisiaca was also revealed to have to be non-toxic effect on liver enzymes. the assessment of haemotological parameters could be used to reveal the deleterious effect of foreign compounds including plant extract on the blood constituent of animals. they can also be used to determine possible alteration in the levels of biomolecules, metabolic products, as well as histomorphology of the organs (magalhães et al., 2008). following the administration of the extracts, there was an increase in red cells count and haematocrit level, which was significant at a higher dose of 400 mg/kg as compared to the control group. mpf pulp had as better increase in red cell count when compared with mpf peel, whereas mpf peel showed a much effect in increasing the haemotocrit level as compared to mpf pulp. the results also revealed an increase in haemoglobin concentration (figures 1-3). these increments indicate that m. paradisiaca contains phytochemicals that stimulate the synthesis of erythrocytes possibly by stimulating erythropoietin formation and secretion. erythropoietin is a glycoprotein hormone which stimulates stem cells in the bone marrow to produce red blood cells (ohlsson and aher, 2009). effect of ethanol fruit extracts of musa paradisiaca on serum liver enzymes of wistar rats figure 4 shows the effect of ethanol fruit extracts (pulp and peel) of musa paradisiaca on the liver enzymes of normal wistar rat. there was a non-significant (p>0.05) change in ast, alt, and alp level of the treated rats as compared to the control. l iv e r e n z y m e s ( iu /l ) a s t a l t a l p 0 2 0 4 0 6 0 c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g figure 4. the effect of ethanol fruit extracts of musa paradisiaca on liver enzymes of wistar rats. an assessment of the effect of ethanol fruit extracts of musa paradisiaca on liver enzymes of wistar rats showed a non-significant change in serum aspartate aminotransferase (ast), alanine aminotransferase (alt) and serum alkaline phosphatase (alp) levels of the treated rats as compared to the control (figure 4). serum ast, alt, and alp, which are cytoplasmic enzymes released into circulation after cellular damage are useful enzymes biomarkers in predicting liver damage (ramaiah 2011; zhao et al., 2018). alt and ast are largely used in the assessment of liver damage by drugs or any other hepatotoxins, while alp is a marker enzyme for the plasma membrane and endoplasmic reticulum (giannini et al., 2005; mcgill, 2016). the observed non-significant differences in the liver enzymes are an indication that both fruit pulp and peels extracts of musa paradisiaca are non-toxic to the hepatic cells, thus, suggesting that musa paradisiaca fruit extracts may not possess hepatotoxic effects, perhaps, a protective effect by stabilization of plasma membrane thereby preserving the structural integrity of the cell (pari and murugen, 2004). this is corroborated by the findings of iweala et al (2011) which reported significantly reduced liver enzymes level with the consumption of a musa paradisiaca-supplemented diet by wistar rats. the hepatoprotective properties of musa 70 biology, medicine, & natural product chemistry 10 (1), 2021: 67-71 paradisiaca against experimentally induced hepatotoxic models have also been reported (nirmala et al., 2012) conclusion this study evaluated the effect of ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. fruit pulp and peel of musa paradisiaca improve erythrocytes count and haematocrit level, and they may not be associated with liver toxicity. fruits of musa paradisiaca can be used as food therapy in raising red cells synthesis in anaemic conditions. conflict of interest: the authors declare that there are no conflicts of interest concerning the publication of this article. references agarwal pk, singh a, gaurav k, goel s, khanna hd, goel rk (2009). evaluation of wound healing activity of extracts of plantain banana (musa sapientum var. paradisiaca) in rats. indian j exp biol 47: 322-40. baker fi, silverton re, pallister cj (1998). baker and silvertons introduction to medical laboratory technology, 7th edition, bounty press ltd, nigeria, 339-373. basu d, kulkarni r (2014). overview of blood components and their preparation. indian j anaesth 58(5): 529-37. benzie iff, wachtel-galor s (2011). herbal medicine: biomolecular and clinical aspects. 2nd edition, crc press/taylor & francis, boca raton (fl). ekor m (2014). the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. front pharmacol 4:177. emudainohwo jot, erhirhie eo, moke eg, edje ke (2015). a comprehensive review on ethno-medicine, phytochemistry and ethnopharmacology of chrysophyllum albidum. journal of advances in medical and pharmaceutical sciences 3(4): 147-154 ezekwesili cn, ghasi s, adindu cs, mefoh nc (2014). evaluation of the anti-ulcer property of aqueous extract of unripe musa paradisiaca linn. peel in wistar rats. african journal of pharmacy and pharmacology 8(39): 1006-1011. fagbemi jf, ugoji e, adenipekun t, adelowotan o (2009). evaluation of the antimicrobial properties of unripe banana (musa sapientum l.), lemon grass (cymbopogon citratus s.) and turmeric (curcuma longa l.) on pathogens afr j biotechnol 8(7): 1176-1182. giannini eg, testa r, savarino v (2005). liver enzyme alteration: a guide for clinicians. cmaj 172(3): 367-79. issa mt, agbon an, balogun su, mahdi o, bobbo ka, ayegbusi fo (2018). hepatoprotective effect of methanol fruit pulp extract of musa paradisiaca on carbon tetrachlorideinduced liver toxicity in wistar rats. j exp clin anat 17: 1-7 iweala eej, obichi ic, omotosho oe (2011). biochemical and histological responses of hepatotoxic rats fed musa paradisiaca l. supplemented diet. int j pharmacol 7(4): 471477. magalhães p, appell h, duarite j (2008). involvement of advanced gyration and production pathogenesis of diabetes complication: the protective role of regular physical activity. eur rev aging phys act 5: 17-29. mahomoodally mf (2013). traditional medicines in africa: an appraisal of ten potent african medicinal plants. evid based complement alternat med, 617459. mcgill mr (2016). the past and present of serum aminotransferases and the future of liver injury biomarkers. excli j 15: 817-828. moke eg, anachuna kk, edje ke, ojezele mo (2019). hepatoprotective effect of methanol seed extract of citrus tangerina on paracetamol-induced hepatotoxicity in wistar rats. niger j nat prod med, 2019, 23: 83-87. moke eg, omorodion li, akpoguma ha, imere p, ahante e (2017). anti-ulcerogenic activity of aqueous extract of unripe fruit of musa sapientum linn in combination with vitamin c on ulcer induced models in experimental rats. eur j med plants 19(2): 1-6 mondal h, budh dp (2020). hematocrit [updated 2020 jul 10]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 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(2018). comparison of the hepatoprotective effects of four endemic cirsium species extracts from taiwan on ccl4-induced acute liver damage in c57bl/6 mice. int j mol sci 19(1329): 1-18. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 105-110 | doi: 10.14421/biomedich.2021.102.105-110 issn 2540-9328 (online) phytochemical screening, antioxidant and antibacterial activities of the root extract of cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm abdulbasit haliru yakubu1,3,*, mohammed mustapha mohammed2, abdulqadir bukar bababe1, hassan yesufu braimah1 1department of pharmaceutical chemistry, pmb 1069, faculty of pharmacy, university of maiduguri, borno state, nigeria. 2department of microbiology, faculty of science, pmb 1069, university of maiduguri, maiduguri, nigeria. 3department of pharmaceutical service, pmp 1414, university of maiduguri teaching hospital, maiduguri, nigeria. corresponding author* pharmahy071@gmail.com manuscript received: 13 september 2021. revision accepted: 10 october, 2021. published: 11 october, 2021. abstract plant secondary metabolites have provided important bioactive principles for developing new lead compounds. within their confinement, they exhibit unique chemical diversity, which influences their diverse biological properties. the vitaceae family is known for its potent antioxidant and antibacterial phytoconstituents, among other biological properties. cyphostemma adenocaule is one of the family members explored for its ethnomedicinal properties. this study undertook the evaluation of the phytochemical, antioxidant, and antibacterial properties of the root extract of cyphostemma adenocaule. preliminary phytochemical screening revealed the presence of flavonoids, alkaloids, carbohydrates & glycoside, saponins, and tannins. the methanol root extract had the highest activity in the dpph assay, providing ic50 (50% inhibition) of 10.87µg/ml, followed by n-hexane (ic50 74.10µg/ml) and chloroform (ic50 74.31µg/ml) extract. in the antibacterial assay, the chloroform extract was active against e. coli (24.00±0.15) and had moderate activity against staph. aureus (12.5±0.18). the n-hexane extract was completely inactive against the test organisms while the methanol extract showed poor activity against the test organisms. the present study adds to the existing literature on cyphostemma adenocaule with scientific evidence into its biological properties. keywords: cyphostemma adenocaule; phytochemical screening; antioxidant and antibacterial activity. abbreviations: ca1 – cyphostemma adenocaule 1; chcl3 – chloroform; cosy – correlation spectroscopy; dept – distortions enhancement by polarization transfer; eta – ethyl acetate; hmbc – heteronuclear multiple bond correlation; hsqc – heteronuclear single quantum correlation; khso4 – potassium bisulphide; meoh – methanol; moa – mechanism of action; nhex – hexane; nmr – nuclear magnetic resonance; tlc – thin layer chromatography; who – world health organization. introduction natural products have been in existence for ages and evolved with unique chemical diversity, which results in their diverse biological activities and drug-like properties. these compounds present as important resources for developing new lead compounds and scaffolds (galm & shen, 2007). morphine from the opium poppy plant is considered the first pharmacologically active compound isolated by friedrich sertürner (hamilton & baskett, 2000; joo, 2014). natural products are important for the development of new drugs, and these products have been in constant use. drugs used as anticancer, antihypertensive, and antimigraine medication, have benefited greatly from natural products (joo, 2014; newman et al., 2003) plants have been part of traditional medicine systems, which have been used for thousands of years (iwu, 2014). these plant-based systems continue to play an essential role in health care, and it has been estimated by the world health organization (who) that approximately 80 % of the world’s inhabitants rely mainly on traditional medicines for their primary health care (who, 2017) cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm is a climbing, scrambling, or trailing herb that belongs to the vitaceae family (bello et al., 2019; wickens & burkill, 1986) and is locally known as yáákùwár fátààkéé (hausa, nigeria) (wickens & burkill, 1986). the plant is a popular, non-cultivated vegetable eaten in many parts of africa i.e., nigeria, ghana, congo, uganda, ethiopia, and eritrea (bello et al., 2019). the plant had been documented for its https://doi.org/10.14421/biomedich.2021.102.105-110 106 biology, medicine, & natural product chemistry 10 (2), 2021: 105-110 ethnomedicinal value, with a comprehensive review given by bello and colleagues (2019). the effectivity of plant bioactive compounds against oxidative stress-related diseases and as an anti-infective had been well explored. this study entails investigating the phytochemical, antioxidant, and antibacterial properties of the root extracts of c. adenocaule by employing standard protocol. the results from the study will justify the ethnomedicinal uses of the plant and underscore its potentials as a source of antioxidants and antimicrobial agents. materials and method plant collection and identification fresh root parts of c. adenocaule were collected aseptically in july 2019 from shuwarin town, dutse lga, jigawa state, nigeria, and identified at the medicinal botany section, of the department of biology, ahmadu bello university, zaria, nigeria. preparation of plant extract and its fractions the preparation of plant material and fractions employed in our previous work on c. adenacaule was adopted with modifications (yakubu et al., 2020). in this study, one and a half kilograms (1.5kg) of the pulverized sample material was extracted with hexane, chloroform, and methanol. preliminary phytochemical screening phytochemical screening was carried out on the crude extracts to detect the presence of plant secondary metabolites; alkaloids, anthraquinones, flavonoids, glycosides, steroids, tannins, terpenoids, and carbohydrates using standard procedures as described in the literature (brain, kr and turner, 1975; evans, 2009; markham, 1982; sofowora, 1996; vishnoi, 2009). biological activity test for antioxidant activity: dpph assay the radical scavenging potential was done using dpph assay (brand-williams et al., 1995). 3 ml of 0.004% dpph working solution (prepared using dpph stock solution and methanol in correct proportions to give 0.899 abs) was added per every 100 µl of different concentrations of the extract and incubated at 37 ℃ for 30 minutes in dark. then absorbance was taken at 517 nm wavelength in a uv spectrophotometer. the negative control contained 100 µl of methanol in place of the sample solution. the percentage antioxidant inhibition (ai) was obtained by the equation: % ai = control (abs) − sample (abs) ÷ control (abs) × 100 ascorbic acid (aa) was used as the positive control. inhibition curves were made and ic50 value per sample was calculated. antimicrobial assay test organisms the organisms employed in this study are; escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, and streptococcus epidermis, clinical isolates got from the microbiology department, university of maiduguri, and were stored at 2-8 ℃ until required. preparation of extract solutions for pathogenic assay a stock solution of the extracts was prepared by dissolving 10 g of extract in 10 ml of distilled water, and a 1000 mg/ml solution was obtained. a two-fold serial dilution was carried out to obtain working solutions of varying concentrations. preparation of test organisms test organisms cultured for 24 hours were suspended in a sterile bottle containing pure broth. normal saline was added gradually to it and the turbidity was observed and compared to that of 0.5 mcfarland standard which corresponds to approximately 108 cells/ml. this was then diluted to produce 106 cells/ml and used in the experiments. the dilution ratio was 1:1000 and 1:1500 for gram-positive and gram-negative organisms respectively (usman et al., 2009). preparation of agar plates nutrient agar was prepared accurately to the manufacturer’s specification (i.e. by dissolving 18.5 g powder in 500 ml of distilled water) and sterilized at 121 ℃ for 15 min. the sterilized agar was allowed to cool to 50 ℃ in a water bath. the test organism (1 ml) (106 cells/ml) was inoculated into pre-labeled petri plates (90 mm diameter), then 19 ml of the molten agar was added to each petri plate, shacked, and allowed to sit at room temperature on a flat surface. antimicrobial susceptibility assay (agar well diffusion method) the antibacterial activity of the crude extracts was determined by following the agar-well diffusion method described by igbinosa and colleagues (igbinosa et al., 2009) with modification. the bacterial isolates were grown for 18 h in a nutrient broth and standardized to 0.5 mcfarland standards (10 6 cfuml-1). two hundred microliter of the standardized cell suspensions were spread on mueller-hinton agar (oxoid) and wells were bored into the agar using a sterile 6 mm diameter cork borer. approximately 100 µl of the crude extract at 100, 75, 50, and 25 mgml-1 were introduced into the wells, allowed to stand at room temperature for about 2 hr, and then incubated at 37 °c. controls were set up in parallel yakubu et al. – phytochemical screening, antioxidant and antibacterial activities … 107 using the solvents that were used to reconstitute the extract. after 24 hr, the plates were observed for the zones of inhibition. the effects were compared with those of ciprofloxacin at a concentration of 5 mg/ml. antibacterial activity was evaluated by measuring the diameters of zones of growth inhibition in triplicates and results were presented as mean±sem. statistical analysis the obtained antioxidant and antibacterial results were expressed in mean ± standard error with observation recorded in triplicates. analysis of variance for individual parameters was performed based on mean values to determine the significance at p < 0.05 using spps v20. regression analysis was deployed to calculate and obtain the ic50 from the regression equation using excel 2016. percentage yield in this experiment, 1.5kg of the resultant size reduced root powder of c. adenocaule was used. the meoh extract showed the highest yield of 44.6g. the percentage yield is given in the table1 table 1. percentage yield of c. adenocaule root extracts. extract weight of extract (g) percentage (%) yield nhex 5.4 0.36 chcl3 12.5 0.83 meoh 44.6 2.97 phytochemical screening of root extract of c. adenocaule. the result for the preliminary phytochemical screening of root extract of c. adenocaule is shown in table 2. table 2. preliminary phytochemical screening of methanolic extract of c. adenocaule. phytoconstituent test result nhexane chloroform methanol alkaloids dragendorff's + + + mayer's + + + anthraquinones freeanthraquinones borntrager's + combine anthraquinones borntrager's carbohydrates general test molisch's + + + monosaccharide barfoed's + + + free reducing sugar fehling's + + combine reducing sugar fehling's + cardiac glycosides steroidal nucleus salkowsi's + + steroidal nucleus liebermann-buchard's + + terpenoids + flavonoids lead acetate + ferric chloride + + shinoda's + + sodium hydroxide + + saponins glycosides frothing + tannins ferric chloride + + lead acetate + + key: + = present: = absent biological activity dpph assay: in-vitro antioxidant activity table 3. in vitro antioxidant activity (dpph assay) of c. adenocaule root extract fractions. concentration (µg/ml) % of dpph scavenging activity nhex chcl3 meoh aa 6.25 22.8 33.5 32.8 46.5 12.5 29.9 35.9 55.2 53.7 25 36.8 39.4 67.2 64.1 50 40.5 44.6 79.3 77.3 100 55.2 50.9 97.2 96.8 ic50 74.1 74.6 10.87 4.51 key: aa: ascorbic acid, chcl3: chloroform extract, nhex: n-hexane extract, meoh: methanol extract results of the in vitro antioxidant assay (in ic50) of the crude root extracts of c. adenocaule are given in table 3 below. the meoh extract showed relatively invitro dpph scavenging activity compared to other extracts. 108 biology, medicine, & natural product chemistry 10 (2), 2021: 105-110 figure 1. dpph assay; ic50 extrapolation graph of nhex, chcl3, and meoh extracts of c. adenocaule. antimicrobial susceptibility assay the chcl3 showed the highest activity against the test organism. the result of the in-vitro antimicrobial susceptibility assay is given in table 4. table 4. in vitro antimicrobial activity of c. adenocaule extract. concentration (mg/ml) test organism extracts hex chcl3 meoh cip(5mg/ml) e. coli 00.00±0.00 24.00±0.15 0.10±0.02 31.00±0.75 p. aeruginosa 00.00±0.00 2.00±0.02 2.00±0.00 27.30±0.41 100 stap. aureus 00.00±0.00 12.50±0.18 5.50±0.13 29.70±0.17 s. epidermis 00.00±0.00 5.50±0.07 1.5.00±0.1 25.02±0.84 e. coli 00.00±0.00 12.00±0.15 0.10±0.00 p. aeruginosa 00.00±0.00 1.00±0.15 2.00±0.32 75 stap. aureus 00.00±0.00 13.50±0.4 2.00±0.01 s. epidermis 00.00±0.00 7.50±0.12 1.0.00±0.00 e. coli 00.00±0.00 4.50±0.2 0.10±0.00 p. aeruginosa 00.00±0.00 0.00±0.00 0.00±000 50 stap. aureus 00.00±0.00 9.10±0.16 2.00±0.10 s. epidermis 00.00±0.00 2.60±0.05 1.70±0.18 e. coli 00.00±0.00 3.00±0.21 0.10±0.00 p. aeruginosa 00.00±0.00 0.00±0.00 0.20±0.00 25 stap. aureus 00.00±0.00 2.50±0.4 1.00±0.10 s. epidermis 00.00±0.00 1.00±0.00 1.50±0.00 key: cip: ciprofloxacin discussion many solvents including hex, chcl3, and meoh had been employed for the extraction of bioactive plant principles. meoh had shown effective as an extraction solvent in many plant drug analyses especially in the isolation of phenolics and flavonoids content (do et al., 2014; truong et al., 2019). the percentage yield result y = 0,6731x r² = -1,351 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay nhex y = 0,6729x 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay chcl3 y = 0,5155x + 47,704 r² = 0,967 0 20 40 60 80 100 120 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay aa y = 0,5861x + 43,629 r² = 0,8424 0 20 40 60 80 100 120 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay meoh yakubu et al. – phytochemical screening, antioxidant and antibacterial activities … 109 from this study shows meoh to be with the highest extraction yield of 2.97%, followed by chcl3 (0.83 %) and nhex (0.36 %) respectively. (table 1). phytochemical screening of c. adenocaule using standard methods revealed the presence of alkaloids, carbohydrates, saponins, and tannins in the nhex extract. flavonoids, carbohydrates and glycoside, alkaloids, saponins, terpenoids, and tannins were present in both the chcl3 and meoh extracts (table 4). similar results were reported by akinwunmi and colleagues on the phytochemical screening of the ethanol root extract of c. adenocaule (akinwunmi et al., 2015). dpph assay remains one of the commonly employed methods for the analysis of the antioxidant activity of plant phytochemicals and employs spectrophotometric application. the ability of a test compound to scavenge dpph radical is determined based on its concentration providing 50% inhibition (ic50), which is the value of the concentration of the sample to cause 50% inhibition and is obtained by the interpolation from the linear regression analysis (figure 1). in this study, different fractions of the extracts were screened for their antiscavanging activity, and it was observed that the meoh extract showed the highest potential with an ic50 10.87 µg/ml scavenging activity, followed by nhex (74.1 7 µg/ml) and chcl3 (74.6 7µg/ml) with the least scavenging activity. though, the results are below that of the standard; ascorbic acid aa with ic50 4.50 µg/ml (table 4). this activity might be due to the presence of phenolics and flavonoids and other secondary metabolites present in the meoh extract that are known potent antioxidants, and taking into consideration, c. adenocaule belong to the vitaceae family which are known for their potent antioxidant principles (murias et al., 2005; piotrowska et al., 2012; rivière et al., 2012). akinwunmi and colleagues, reported a dpph scavenging activity of ic50 38.42 µg/ml for the ethanol root extract of cissus adenocaule with the root total phenolic and flavonoid content to be 182±0.38 mg/g tae (tannic acid equivalent) and 103±0.42 mg/g qe (quercetin equivalent) [akinwunmi et al., 2015]. the use of the plant as an antimicrobial agent in the ethnomedicinal space cannot be overemphasized, as they continued to be used to date. this augments their exploitations for the discovery of lead and novel molecules for antimicrobial drug discovery. they provide starting materials and derivatives that are employed as ligands in the drug discovery and development process. this study explored the antimicrobial activity of the c. adenocaule root extracts (table 4). the n-hexane extract was completely inactive against the test organism. the chcl3 extract was active at 100mg/ml and showed good activity against e. coli (24.00±0.15) and moderate activity against staph. aureus (12.5±0.18). the meoh extract showed poor activity against the test organism. this result is inconsonant with an earlier report by hamil and colleagues on the activity of meoh root extract of c. adenocaule on e. coli. p. aeruginosa and staph. aureus (hamill et al., 2003). conclusion the present study undertook the phytochemical screening, isolation, and characterization of chemical compounds present in the methanol root extract of c. adenocaule, as well as, determination of their antioxidant and antibacterial activity. methanol presents the best extraction solvent in terms of percentage yield. flavonoids, alkaloids, carbohydrates and glycoside, saponins, and tannins were present while anthraquinone were absent. in the chloroform and hexane extracts; anthraquinones and flavonoids were absent, with carbohydrates also absent in the hexane extracts. in the assessment of the biological properties, the antibacterial sensitivity assay showed chloroform to have activity against e. coli and moderate against staph. aureus at 100mg/ml respectively, while poor activity was recorded with the methanol and n-hexane extract. the dpph antioxidant assay revealed the free radical scavenging activity, methanol extract yields the best result with an ic50 of 10.87µg/ml. the results from this study add to the existing literature on c. adenocaule with scientific evidence into its biological properties. acknowledgments: the authors will like to acknowledge mr. namadi sanusi of the department of biological science, ahmadu bello university, zaria, nigeria for the collection and identification of the plant materials. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest: the authors declare no conflict of interest. references akinwunmi, k. f., ajala, v. o., & oyedapo, o. o. 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(2020). a natural triglyceride from the methanol root extract of cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm. https://doi.org/10.26434/chemrxiv.13296539.v1 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 111-115 | doi: 10.14421/biomedich.2021.102.111-115 issn 2540-9328 (online) acute toxicity and hypoglycemic effect of a polyherbal formulation on blood glucose in oral glucose tolerance test (ogtt) and alloxan-induced diabetic rats 1department of pharmacology, therapeutics and toxicology, faculty of basic medical sciences, university of lagos, lagos, nigeria. 2department of pharmacology and therapeutics, faculty of basic medical sciences, delta state university, abraka, nigeria. 3department of pharmacology and toxicology, faculty of pharmaceutical sciences, chukwuemeka odumegwu ojukwu university, awka, nigeria. corresponding author* abedoke@gmail.com manuscript received: 04 october 2021. revision accepted: 10 october, 2021. published: 15 october, 2021. abstract madam f. kayes bitters® is an herbal formulation commonly used in nigeria and some african countries in the management of diabetes mellitus and other diseases conditions. this study evaluated the in-vivo hypoglycaemic activity, as well as acute toxicity of the polyherbal formulation to provide its efficacy and safety. healthy albino mice (20-30 g) and sprague dawley female rats (90-130 g) were used for this study. acute toxicity study (ld50) of the herbal formulation was determined by methods originally described by miller and tainter in 1994. following oral dosing with glucose (2 g/kg) in normal fasted animals, herbal formulation (hf) at various doses was administered and blood glucose levels at 30 minutes, 60 minutes, 90 minutes, and 120 minutes were taken and recorded. diabetes was induced using alloxan 150 mg/kg and diabetic rats were given the hf at doses of 50, 100, and 200 mg/kg with glibenclamide 2.5 mg/kg used as standard drug treatment. blood glucose level was determined on 1st day, 7th day, 14th and 21st day. the ld50 was greater than 5g/kg with oral administration. the oral glucose tolerance test showed that the group that received 100 mg/kg hf showed a significant reduction (p<0.05) in glucose level after 120 minutes when compared to the basal level of glucose recorded. all treated diabetic groups showed a significant decrease in glucose level on the 21st day. the herbal formulation of hydrastis canadesis aloe capensis, echinacea angustifolia and honey exhibited a significant glucose-lowering activity in alloxan-induced diabetic rats. keywords: alloxan monohydrate; diabetes mellitus; herbal formulation; ld50 value. introduction diabetes mellitus (dm, diabetes) is a chronic disease characterized by persistent elevation of blood glucose level of an individual which resulting from defects in insulin secretion, insulin action, or both (ada, 2009). this disease is also marked by altered lipids, carbohydrates and protein metabolism (ozougwu et al., 2013; ezuruike and prieto, 2014). the hyperglycaemic condition takes place due to the inappropriate secretion of insulin hormone or the inappropriate use of insulin hormone by the body itself. insulin hormone is secreted by the islet of langerhans located in the beta cells of pancreas and it helps to maintain the glucose level in the blood (xavier, 2018). besides the storage of glucose, insulin also inhibits the secretion of glucagon and lowers the concentration of serum fatty acids leading to a decline in liver glucose production (asmat et al., 2016). insufficient insulin or resistance to insulin in the body results in reduced tissue uptake of glucose that results in intracellular hypoglycemia and extracellular hyperglycemia. the intracellular hypoglycemia causes glucogenesis and gluconeogenesis that leads to fats breakdown (causing diabetic ketoacidosis) and decreases protein synthesis and gamma globulins (causing cachexia, polyphagia, and impaired wound healing), while the extracellular hyperglycemia leads to hyperglycemic coma and osmotic dieresis (ozougwu et al., 2013). based on the requirements of insulin, diabetes is classified into insulin-dependent diabetes mellitus (type 1), and non-insulin-dependent diabetes mellitus (type 2) (ada, 2009). madam f. kayes bitters® is an herbal formulation commonly used in nigeria and some african countries in the management of diabetes mellitus and other diseases conditions. this formulation is listed to be composed of aloe capensis, hydrastis canadesis, echinacea angustifolia and honey. in spite of significant advancements in the development of conventional drugs, herbal medicines remain useful in the management of diseases, especially in developing countries (ekor, 2014; erhirhie et al., 2015; moke et al., 2021). this may be due to the adverse effects and high cost of therapy abednego okeoghene warri1*, emuesiri goodies moke2, aishat oyinkansola balogun1, kennedy chibogu nzeh1, emuesiri kohworho umukoro2, earnest oghenesuvwe erhirhie3 https://doi.org/10.14421/biomedich.2021.102.111-115 112 biology, medicine, & natural product chemistry 10 (2), 2021: 111-115 associated with conventional drugs (gurib-fakim, 2006; ekor, 2014). medicinal plants are plants whose parts contain substances of therapeutic important which can be constituted into drug and used for treatment of various disease (sofowora et al., 2013). many conventional drugs such as quinine, aspirin, digoxin, amongst others, are of plant origins (vickers et al., 2001; veeresham, 2013). presently, there is an urgent need to develop safer drugs for the management of chronic diseases such as diabetes mellitus. consequently, assessment of various medicinal plants used in traditional systems have grown (lahlou, 2013; sofowora et al., 2013; ekor, 2014, anachuna et al., 2018; okafo et al., 2019; moke et al., 2020). however, high cost of production is a factor, developmental processes for newer drugs is a way of evaluating of efficacy and adverse effects of chemical substances. the present study aimed to evaluate the invivo hypoglycaemic activity, as well as acute toxicity of the polyherbal formula extracts (madam f. kayes bitters®) on experimental animals. material and methods herbal formulation the herbal formulation used was purchased from a local pharmacy. it was stored in a cool dry place during the period of the study. experimental animals healthy albino mice (20-30 g) and sprague dawley female rats (90-130 g) were used for this study. these were procured from the animal house of the college of medicine, university of lagos, nigeria. the animals were kept in polypropylene cages with wire mesh for proper ventilation throughout the study. the animals were given free access to standard diet (lifestock feed plc, ikeja, nigeria) and water ad libitum. the animals were kept in room temperature with a 12:12 day and night cycle and maintained at temperature of 27 ℃. the animals were allowed to acclimatize for two weeks before commencing the study. the maintenance and treatment of the animals was according to the principles of the guide for care and use of laboratory animals in research and teaching prepared by the national academy of science and published by the national institute of health (nih) publication 86-23 revised in 1985. acute toxicity study acute toxicity study of the herbal formulation was determined by methods originally described by miller and tainter of 1994 (erhirhie et al., 2018). mice (20-25 g) were fasted for 12 hours and divided into four groups of 5 mice each. in order to determine the ld50 of the formulation, different doses 1000 mg/kg, 2500 mg/kg and 5000 mg/kg were administered orally to different groups of mice while the control group received 10 ml/kg distilled water (ph = 6.9). all animals were closely observed for symptoms of toxicity and the mortality noted. based on data obtained, the ld50 was determined. oral glucose tolerance test (ogtt) fifteen adult female rats (100-126 g) were fasted overnight. the animals were divided into five different groups with three animals each. blood samples were obtained from each rat by gently nipping the tail with a lancet, and then gently squeeze the tail to let out 2 drops of fresh venous whole blood on the glucometer strip inserted and the readings recorded for the 0 minute. different doses of the herbal formulation were orally administered. glucose (2 g/kg) was orally administered 30 minutes after extract administration to each animal according to their body weight. blood glucose level at 30 minutes, 60 minutes, 90 minutes, and 120 minutes were taken and recorded. alloxan-induced diabetic study eighteen healthy female rats were randomly divided into 6 groups of 3 animals each. the rats were fasted for 24 hours after which group i rats were given 10 ml/kg normal saline while groups ii-vi were made hyperglycemic with a single intraperitoneal injection of 150 mg/kg of alloxan monohydrate (sigma chemicals company, st. louis, mo., u.s.a) (gbolade et al., 2008; moke et al., 2015). the baseline fasting blood glucose was determined prior to the administration of alloxan. after 48 hours, blood was collected from the tail vein of each rat and the fasting blood glucose level determined. a blood glucose ≥ 200 mg/dl was considered diabetic. animals were further tested after 72 hours and animals with stable hyperglycemia were selected for the study. the treatment schedule of the rats is as follows:  group i (control) – normal saline 10 ml/kg  group ii (diabetic and untreated) – normal saline 10 ml/kg  group iii – herbal formulation 50 mg/kg  group iv – herbal formulation 100 mg/kg  group v – herbal formulation 200 mg/kg  group vi – glibenclamide 2.5 mg/kg determination of fasting blood glucose in diabetic rats the blood glucose level was determined using the tail tipping method after 12-16 hours of fast. the tail was gently squeezed to let out 2-3 drops of blood which is placed on the test spot of the glucose strip after which the test strip was gently inserted into the digital glucometer. blood glucose level was determined on 1st day, 7th day, 14th and 21st day. data analysis data were presented as mean ± standard error of mean (sem) using graph pad prism. test of statistical warri et al. – acute toxicity and hypoglycemic effect of a polyherbal … 113 significance was carried out using a one-way anova. p-values lesser than 0.05 (p<0.05) were considered statistically significant. results and discussion acute toxicity test of herbal formulation when the herbal formulation was administered orally, there was no death of experimental animals even at a dose of 5g/kg. hence, for the oral route, the ld50 is greater than 5g/kg. (table 1) table 1. oral acute toxicity of herbal formulation. group/dose (mg/kg) number of mice number of death control 5 nil 1000 5 nil 2500 5 nil 5000 5 nil ld50oral ˃5000mg/kg body weight of animals there was no statistically significant change in body weights of the animals (p>0.05) when compared to control as shown in table 2. table 2. herbal formulation treatment on body weights. treatment day 1 (g) day 7 (g) day 14 (g) day 21 (g) control (ns) 103.70±4.91 105.00±5.29 107.3±3.84 115.30±2.60 untreated (ns) 106.70±4.49 105.00±3.06 99.67±2.60 97.00±3.22 glb 2.5 mg/kg 100.70±6.36 101.00±7.21 99.67±2.60 68.83±28.67 50 mg hf 107.00±6.56 106.00±5.69 103.70±3.48 105.0±2.52 100 mg hf 107.00±6.08 104.30±6.94 105.00±3.61 108.00±2.31 200 mg hf 105.00±5.57 105.70±6.84 105.30±7.42 113.30±16.05 values are expressed as mean ± sem (n=3). p<0.05 statistically significant when day 1 is compared to other respective groups using one way -anova followed by tukey’s post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf =herbal formulation oral glucose tolerance test of herbal formulation. the oral glucose tolerance test showed a sharp decrease in the glucose level after 30 minutes when compared to the level at 0 minute. this was followed by a gradual reduction in glucose level after 60, 90 and 120 minutes. the control group showed a statistically significant reduction in glucose level at the 90th minute (p<0.01) when compared with the basal level recorded (78.33±0.88). glibenclamide showed a significant reduction (p<0.01) in glucose level after 120 minutes (26±13.01). also the group that received 100 mg/kg hf showed a significant reduction (p<0.05) in glucose level after 120 minutes (39.67±2.33) when compared to the basal level of glucose recorded. (table 3) table 3. oral glucose tolerance test (ogtt) of herbal formulation. treatment basal (mg/dl) 0 minute (mg/dl) 30 minutes (mg/dl) 60 minutes (mg/dl) 90 minutes (mg/dl) 120 minutes (mg/dl) control (ns) 85.33±1.86 104.33±6.57 91.67±5.90 77.33±1.86 87.54±0.88 90.67±2.96 glb 2.5 mg/kg 90.33±7.86 106.67±6.77 87.67±12.25 66.00±10.82 43.67±6.64 26.00±13.01** 50 mg hf 83.33±6.23 104.67±4.41 85.33±9.70 52.67±12.03 67.67±6.94 54.00±13.58 100 mg hf 87.00±5.86 114.00±10.60 88.33±7.84 60.00±5.29 61.00±4.58 39.67±2.33* 200 mg hf 82.67±4.48 116.67±8.84 110.33±8.29 74.33±5.36 71.00±1.53 63.67±6.36 values are mean ±sem (n=3): *p<0.05, **p<0.01 statistically significant compared to basal within group with other respective minutes using one wayanova followed by tukey's post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf = herbal formulation blood glucose level of alloxan-induced diabetic animals the 21 days treatment of alloxan-induced diabetic rats with herbal formulation resulted in statistically significant reduction in blood glucose (table 4). there was a statistically significant difference between all treated groups and the untreated diabetic group when compared with the non-diabetic control indicating initial hyperglycemic condition upon induction of diabetes. on the 7th day, there was a statistical significant difference in glucose level in the untreated group (p<0.05) and groups administered glibenclamide (p<0.01) and 200 mg/kg hf (p<0.001) compared to nondiabetic control. on the 14th day, there was no statistically significant difference in all the treatment groups when compared to both diabetic untreated group and non-diabetic control group. however, the untreated control showed a significant difference (p<0.01) in 114 biology, medicine, & natural product chemistry 10 (2), 2021: 111-115 glucose level when compared with non-diabetic control. all treated groups showed a significant decrease in glucose level on the 21st day. the p values were as follows: glibenclamide (p<0.001), 50 mg/kg hf (p<0.001), 100 mg/kg hf (p<0.001) and 200 mg/kg hf (p<0.01). table 4. blood glucose level of alloxan-induced diabetic animals. treatment baseline (mg/dl) day 1 (mg/dl) day 7 (mg/dl) day 14 (mg/dl) day 21 (mg/dl) control (ns) 53.33±7.31 54.67±7.31 59.33±8.29 48.33±1.20 55.67±5.90 untreated (ns) 66.67±11.79 307.70±56.69* 335.30±51.27* 338.30±49.97** 360.00±55.08**** glb 2.5 mg/kg 52.00±1.53 386.70±51.57*** 207.00±67.35** 126.00±62.98 78.67±18.49b 50 mg hf 78.67±7.75 336.00 ±64.29* 289.30±35.97 116.00±52.81 83.33±13.42b 100 mg hf 67.00±16.50 383.30±52.03** 260.00±60.65 124.70±11.05 55.00±4.73b 200 mg hf 72.33±22.72 399.67±25.11** 284.00±44.23** 192.33±39.07 106.70±8.74a values are mean ±sem (n=3): *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus control; ap<0.001, bp<0.0001 versus untreated control. analysis by one way anova followed by tukey’s post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf = herbal formulation over centuries, herbal products of medicinal value have been recruited in traditional medicine for the management and treatment of several ailments including diabetes mellitus (jung et al., 2006). although there have been a significant advancement in development of conventional drugs in recent times, herbal remedies are still used especially in developing countries in the treatment of diseases (ekor, 2014; moke et al., 2021). numerous medicinal plants are widely used in nigeria to manage diabetes mellitus (abo et al., 2008; ezuruike and prieto, 2014; moke et al., 2015; abubakar et al., 2017; okafo et al., 2019). the crude extracts of these plants may be used alone or mixed with extracts from other plants or other sources. madam f. kayes bitters® is a poly-herbal formulation of aloe capensis, hydrastis canadesis and echinacea angustifolia plus honey. diabetes mellitus is characterized by persistent elevations of fasting blood glucose above 200 mg/dl due to insufficient or complete cessation of insulin synthesis or secretion and/or peripheral resistance to insulin action (adeneye and agbaje, 2008; kitabchi et al., 2009). in this study, acute toxicity test revealed that the polyherbal formulation is quite safe for oral consumption. the oral glucose tolerance test (ogtt) showed a gradual reduction in the glucose level from 0 minute to 120 minutes. this decrease was found to be significant for the groups administered the standard treatment (glibenclamide) and hf 100mg/kg which suggests that the herbal formulation has a glucose lowering effect. following 21 days treatment of alloxan-induced diabetic rats with the herbal formulation, there was a significant reduction in blood glucose level in all groups receiving treatment compared to the untreated diabetic control. the reduction in glucose level was gradual in all groups with glucose level brought to below 150 mg/dl on the 14th day with the exception of the group receiving 200mg/kg. the hypoglycemic effect was more profound in the group receiving 100 mg/kg than that of 50 mg/kg and 200 mg/kg which suggests that the hypoglycemic effect is not dose-dependent. the result obtained is consistent with earlier reports in scientific literature demonstrating the hypoglycemic effect of aloe barbadensis/aloe vera (used synonymously with aloe carpensis) in alloxan-induced diabetic rats (nwanjo, 2006; choudhary et al., 2014; pothuraju et al., 2016; hammeso et al., 2019). also honey has been shown to exert a dose-dependent hypoglycemic effect in diabetic rats (erejuwa, 2014). honey is sweet and rich in sugars. it is therefore surprising that it has a hypoglycemic effect. it has been hypothesized that fructose and oligosaccharides present in honey may in some way contribute to its observed hypoglycemic effect (erejuwa et al., 2012). hence, the hypoglycemic effect of the herbal formulation investigated is consisted with scientific reports on the constituents. conclusion the polyherbal formulation of hydrastis canadesis aloe capensis, echinacea angustifolia and honey exhibited a significant glucose-lowering activity in alloxan-induced diabetic rats. this suggests that this formulation may be useful for the management of diabetes mellitus. this study therefore corroborates the claim that this herbal formulation is effective in managing diabetes mellitus. acknowledgements: the work was supported by grant received from federal ministry of health through postgraduate scholarship administered by the federal scholarship board (fsba/fgss/pg/16/212). we wish to acknowledge the contributions and mentorship role of dr. nwaiwu obiyo (of blessed memory). warri et al. – acute toxicity and hypoglycemic effect of a polyherbal … 115 conflict of interest: the authors declare that there are no conflicts of interest concerning the publication of this article. references abo ka, fred-jaiyesimi aa, jaiyesimi ae (2008). ethnobotanical studies of medicinal plants used in the 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se, moke eg, obi cs (2019). formulation and evaluation of anti-diabetic tablets containing aqueous extract of moringa oleifera seeds. jophas 16(5): 3167-3176. ozougwu jc, obimba kc, belonwu cd, unakalamba cb (2013). the pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. j physiol pathophysiol 4(4): 46-57. pothuraju r, sharma rk, onteru sk, singh s, hussain sa (2016). hypoglycemic and hypolipidemic effects of aloe vera extract preparations: a review. phytother res 30(2): 200-7. sofowora a, ogunbodede e, onayade a (2013). the role and place of medicinal plants in the strategies for disease prevention. afr j tradit complement altern med 10(5): 21029. veeresham c (2012). natural products derived from plants as a source of drugs. j adv pharm technol res 3(4): 200-1. vickers a, zollman c, lee r (2001). herbal medicine. west j med 175(2): 125-8. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 55-63 | doi: 10.14421/biomedich.2022.111.55-63 issn 2540-9328 (online) a mathematical model of cervical cancer treatment by radiotherapy followed by chemotherapy murtono1,*, sugiyanto2, mansoor abdul hamid3 1department of education physics, 2department of mathematics; universitas islam negeri sunan kalijaga yogyakarta, indonesia. 3food technology and bioprocess, universiti malaysia sabah, malaysia. corresponding author* hasnamur@yahoo.co.id manuscript received: 15 april, 2022. revision accepted: 10 may, 2022. published: 21 may, 2022. abstract cervical cancer is the second most common type of malignant tumor found in women aged 15-44 years worldwide. radiotherapy is one form of treatment that uses radiation that can eliminate or kill cancer cells and one way to treat cervical cancer is qui te popular. chemotherapy is a cancer treatment using anticancer drugs designed to kill or slow the growth of cancer cells that divide rapidly in the body. the method used is to study mathematical model of cervical cancer with radiotherapy and radiotherapy treatment followed by chemotherapy. the results of this study are for the early stages of cervical cancer with radiotherapy is quite effective, while for the late stages of radiotherapy and chemotherapy is less effective. therefore, the need for other treatments for end -stage cervical cancer in addition to radiotherapy and chemotherapy. keywords: cervical cancer; radiotherapy; chemotherapy. introduction cervical cancer is still the leading cause of cancer deaths among women in developing countries including indonesia (hoffman, et.al, 2008 and woodman et al., 2007). indonesia cancer foundation explained, the mortality rate of cervical cancer most among other types of cancer among women. it is estimated that 52 million indonesian women are at risk for cervical cancer, while 36 percent of all women with cancer are cervical cancer patients. there are 15,000 new cases per year with the death of 8,000 people per year (kompas, 2008). in southeast asia the incidence of cervical cancer becomes number two in women after breast cancer. nevertheless, in globocan (2012) the mortality rate due to cervical cancer is higher than the mortality rate due to breast cancer. radiotherapy is one of treatments that can be done for a patient with cervical cancer. radiotherapy is used as a therapy for cancer patients by using radiation that can eliminate and kill cancer cells, a way to treat cervical cancer which is quite popular. treatment with radiation therapy is usually done with chemotherapy, or accompanied by other therapies and sometimes only with radiotherapy alone depending on the type of cervical cancer suffered and the level of cancer. model formation this model develops from asih, et al. (2015) on the development of cervical cancer, liu and yang (2014) on general cancer treatment models with radiotherapy and pillis et al. (2007) on general cancer treatment models with chemotherapy. figure 1. transfer diagram of cervical cancer treatment with radiotherapy and followed by chemotherapy. caption figure 1:  1 1sl s k ms s   , 2 2( ) il i k mi i   , 3 3( ) pl p k mp p   , https://doi.org/10.14421/biomedich.2022.111.55-63 56 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 4 4 ( ) c l c k mc c  is a function of chemotherapy and radiotherapy. table 1. subpopulations, parameters and units. symbol explanation unit  s t normal cells density cells/mm3  i t infected cells density cells/mm3  p t pre-cancerous cells density cells/mm3  c t cancer cells density cells/mm3  v t free virus density virus/mm3  m t concentrations of chemotherapy drugs mg r intrinsic cell growth rate is normal 1/ day n homeostatic carrying capacity cells/mm3  the rate infection 1/(day x virus) 1 a the rate of proliferation of infected cell 1/day 1 d the rate of apoptosis of infected cell 1/day  the rate of progression, from being infected to pre-cancer 1/day 2 a the rate of proliferation of pre-cancerous cells 1/day 2 d the rate of apoptosis of pre-cancerous cells 1/day  the maximum invasion rate, from precancer to cancer 1/day k half-saturation concentration cells/mm3 3a the rate of proliferation of cancer cells 1/day 3 d the rate of apoptosis of cancer cells 1/day n the average number of viruses produced one infected cell constant 4 d the death rate of free virus 1/day 1  the proportion of radiation to susceptible cells (normal cells) constant 2  the proportion of radiation to infected cells constant 3  the proportion of radiation to pre-cancerous cells constant  strategy of radiotherapy 1/day s k fractional susceptible cells killed by chemotherapy 1/day i k fractional infected cells killed by chemotherapy 1/day p k fractional pre-cancerous cells killed by chemotherapy 1/day c k fractional cancer cells killed by chemotherapy 1/day  the rate of chemotherapy drug decay 1/day m v the rate of chemotherapy drug enter mg/day murtono et al. – a mathematical model of cervical cancer treatment by … 57 the dynamics of cervical cell changes from normal cells to cancer cells are given in the following differential equation systems (1). 1 1 s ds s i rs sv k ms s dt n              (1a) 1 1 2i di sv a i d i i k mi i dt          (1b) 2 2 2 32 2 p dp p i a p d p k mp p dt k p           (1c) 2 3 32 2 c dc p a c d c k mc c dt k p        (1d) 1 4 dv nd i d v dt   (1e) m dm m v dt    (1f) the point of equilibrium the equilibrium point with radiotherapy alone, without chemotherapy system (1) without chemotherapy is 1 1 ds s i rs sv s dt n             (2a) 1 1 2 di sv a i d i i i dt         (2b) 2 2 2 32 2 dp p i a p d p p dt k p          (2c) 2 3 32 2 dc p a c d c c dt k p       (2d) 1 4 . dv nd i d v dt   (2e) the requirement to determine the equilibrium point of system (2) is 0, 0, 0, 0, 0 ds di dp dc dv dt dt dt dt dt      . so the point of equilibrium with radiotherapy in cervical cancer, without chemotherapy, is    * * * * *1 2 3 1 51 4, , , , , , , ,r rep s i p c v p       * * * * *2 2 3 2 52 4, , , , , , , ,r rep s i p c v p    (3)    * * * * *3 2 3 3 53 4, , , , , , , ,r rep s i p c v p    , where 1 1 4 nd d   , 1 2 1 2 1 d a         ,  1 2 3 1 r n r r n          , 4 1 3   , 3 1 2 2 3 a a d         , 2 2 2 2 2 3 1 2 2 3 a k d k k b a d          , 2 3 1 2 2 3 k c a d       , 3 1 1 1 1 1 2 9 27d a a b c   ,     2 3 3 2 1 1 1 1 1 1 1 2 9 27 4 3e a a b c a b     ,    1 3 31 1 1 1 1 1 1 1 1 3 3 2 3 2 r a p d e d e      ,    1 3 32 1 1 1 1 1 3 1 1 3 1 3 6 2 6 2 r a i i p d e d e         ,    1 3 33 1 1 1 1 1 3 1 1 3 1 3 6 2 6 2 r a i i p d e d e         ,    2 5 2 2 3 3 i i i p k p d a        . 58 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 existence of the equilibrium points with radiotherapy alone, without chemotherapy if it satisfies 1. 1 2 1 0d a      , 2.    1 2 1 4 1 1 0 d a d r n r nd                 , 3. 0, 1, 2, 3ip i  and 4. 3 3 0d a   . the equilibrium points with radiotherapy and followed by chemotherapy in this system is the same as system (1). to find the equilibrium point in a way 0 ds dt  , 0 di dt  , 0 dp dt  , 0 dc dt  , 0 dv dt  , 0 dm dt  . so the equilibrium point of radiotherapy followed by chemotherapy in cervical cancer is    * * * * * *1 7 9 8 9 1 101 1 9 6, , , , , , , , , ,rc rc rcep s i p c v m p            * * * * * *2 7 9 8 9 2 102 1 9 6, , , , , , , , , ,rc rc rcep s i p c v m p         (4)    * * * * * *3 7 9 8 9 3 103 1 9 6, , , , , , , , , ,r rc rcep s i p c v m p         where 6 m v    , 1 7 1 n r           , 6 1 8 s nk n n r r        , 1 8 1 1 6 2 9 1 7 i a d k                , 11 2 2 2 6 3 , p a a d k             22 2 6 3 2 2 2 6 3 p p a d k k b a d k              , 2 11 2 2 2 6 3p k c a d k         , 3 2 2 2 2 2 2 9 27d a a b c   ,     2 3 3 2 2 2 2 2 2 2 2 2 9 27 4 3e a a b c a b        2 3 31 2 2 2 2 1 1 1 1 3 3 2 3 2 rc a p d e d e      ,    2 3 32 2 2 2 2 1 3 1 1 3 1 3 6 2 6 2 rc a i i p d e d e         ,    2 3 33 2 2 2 2 1 3 1 1 3 1 3 6 2 6 2 rc a i i p d e d e         ,    2 10 2 2 3 3 6 4 irc irc irc c p k p a d k           . existence of the equilibrium points with radiotherapy and followed by chemotherapy, if it satisfies 1. 6 1 7 9 0s nk n n r r         , 2. 1 8 1 1 6 2 0ia d k            and 3. 0ircp  . stability of the equilibrium point stability of the equilibrium points with radiotherapy alone, without chemotherapy theorem 1. if 3 3 0a d    ,   2 2 3 32 2 2 2 0i i pk a d k p        , 0 i a  , 3 2 9 27 0 i i i i a a b c   , 2 3 0 i i a b  where 3, 4, 5i  then the equilibrium point murtono et al. – a mathematical model of cervical cancer treatment by … 59    * * * * * 2 3 6 5, , , , , , , ,ir i iep s i p c v p    asymptotically stable. proof. from system (2) is obtained characteristic equation in equation (5).            2 1 3 3 2 3 32 2 2 1 3 232 51 1 1 1 2 4 32 51 1 1 1 2 4 2 1 1 2 4 2 1 51 32 51 1 2 2 2 2 p k a d a d k p rr r a d d n n rr r a d d n n r a d d nd n rr r n n                                                                                                     1 1 2 4 2 1 2 51 4 2 1 0 a d d nd r d nd n                                               (5) stability of the equilibrium points with radiotherapy and followed by chemotherapy theorem 2. if 3 3 4 0 m c v a d k            ,   2 2 2 32 2 2 2 0irc m p irc p k v a d k k p               , where 3, 4, 5i  and 6 0a  , 3 6 6 6 6 2 9 27 0a a b c   , 2 6 6 3 0a b  then the equilibrium point    * * * * * * 7 9 8 9 10 1 9 6, , , , , , , , , ,irc irc ircep s i p c v m p         asymptotically stable. proof. from system (1) is obtained characteristic equation in equation (6).            2 2 3 3 2 3 32 2 2 2 3 232 52 1 1 1 2 4 32 52 1 1 1 2 4 2 1 1 2 4 2 1 52 32 52 1 2 2 2 2 p k a d a d k p rr r a d d n n rr r a d d n n r a d d nd n rr r n n                                                                                                     1 1 2 4 2 1 2 52 4 2 1 0 a d d nd r d nd n                                               (6) 60 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 simulation in this section will be discussed about the numerical simulation and medical interpretation of the mathematical model of cervical cancer in the presence of radiotherapy and chemotherapy effects are divided into 2 cases. 1. the influence of radiotherapy 2. the influence of radiotherapy and followed by chemotherapy simulation of radiotherapy effect the parameter values for this case are given in the following table 2. table 2. values of parameters for cases of radiotherapy effects. symbol value reference r 0.0255 asih et al. (2015) n 9900.99 asih et al. (2015)  0.00001 asih et al. (2015) 1  0.004 liu and yang (2014)  0.05 liu and yang (2014) 1 a 100 asih et al. (2015) 1 d 100.01 asih et al. (2015)  0.0082 asih et al. (2015) 2  0.03 liu and yang (2014) 2 a 101 asih et al. (2015) 2 d 100 asih et al. (2015)  20.03 asih et al. (2015) k 2 asih et al. (2015) 3a 0.03 asih et al. (2015) 3 d 101.01 asih et al. (2015) n 1 asih et al. (2015) 4 d 500 asih et al. (2015) figure 2 shows the first 30 days or the first month after radiotherapy is showed a decrease in the number of cells from 13 cell/mm3 to 12.78 cell/mm3. this means that the decrease in cell number is 0.22 cell/mm3. at six months and one year of radiotherapy if it continues to show a successive decrease to 11.8 cell/mm3 and 10.73 cell/mm3. this shows the ill effects of radiotherapy because of healthy cells. for infected cells in the first and third months after radiotherapy successively from 13 cell/mm3 to 4,156 cell/mm3 and 0.4364 cell/mm3. this is good for curing cancer, because the infected cells will eventually become pre-cancerous cells. while the cancer cells on the fourth day after radiotherapy immediately fell rapidly, from 13 cell/mm3 to 0.3526 cell/mm3. for pre-cancerous cells almost the same as cancer cells, that is down very quickly. for the virus goes down quickly, because the infected cell goes down as well. this is because the virus replicates itself from infected cells. murtono et al. – a mathematical model of cervical cancer treatment by … 61 figure 2. trajectory diagram the influence of radiotherapy. simulation of the influence of radiotherapy and followed by chemotherapy the parameter values for this case are given in the following table 3. table 3. value of case parameters the influence of radiotherapy and followed by chemotherapy. symbol value reference r 0.0255 asih et al. (2015) n 9900.99 asih et al. (2015)  0.00001 asih et al. (2015) 1  0.004 liu and yang (2014)  0.05 liu and yang (2014) 1 a 100 asih et al. (2015) 1 d 100.01 asih et al. (2015)  0.0082 asih et al. (2015) 2  0.03 liu and yang (2014) 2 a 101 asih et al. (2015) 2 d 100 asih et al. (2015) symbol value reference  20.03 asih et al. (2015) k 2 asih et al. (2015) 3a 0.03 asih et al. (2015) 3 d 101.01 asih et al. (2015) n 1 asih et al. (2015) 4 d 500 asih et al. (2015) s k 0.01 pillis et al. (2007) i k 0.01 pillis et al. (2007) p k 0.01 pillis et al. (2007) c k 0.01 pillis et al. (2007)  0.1 pillis et al. (2007) m v 0.01 pillis et al. (2007) figure 3 shows a trajectory with the effects of radiotherapy and followed chemotherapy, which is an advanced patient of cervical cancer. normal cells during the first 30 days showed a rapid decline, from 13 cell/mm3 to 3,638 cell/mm3. this makes patients with 62 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 advanced cervical cancer in this condition will get worse. therefore, in this study advised not to use this treatment. cancer cells also drop very rapidly, in the first 4 days of radiotherapy and chemotherapy, from 13 cell/mm3 to 0.2061 cell/mm3. figure 3. trajectory diagram the influence of radiotherapy and followed by chemotherapy. conclusion cervical cancer is one type of cancer and second highest incidence of cancer in women compared to other cancers. treatment of cervical cancer with radiotherapy at an early stage is quite effective. this mathematical model showed that treatment of cervical cancer at an advanced stage with radiotherapy and followed by chemotherapy is not effective. because normal cells die very quickly, and will aggravate the patient's condition of cervical cancer. this research needs to be continued with other more effective treatments, especially for advanced stages of cervical cancer. conflict of interest: the authors declare that there is no conflict of interest. references asih t. s. n., suzanne lenhart, steven wise, lina aryati, f. adikusumo, mardiah s. hardianti, jonathan forde, (2015), the dynamics of hpv infection and cervical cancer cells, bull math biol, doi 10.1007/s11538-015-0124-2. globocan, (2012), download february 5, 2018. http://globocan.iarc.fr/old/factsheets/cancers/cervix-new.asp hoffman b., schorge j., schaffer j., halvorson l., bradshaw k., cunningham f., (2008), williams gynecology, second edition, mc grawhill’s, usa. kompas.com, july 10, (2017). download february 5, 2018. http://lifestyle.kompas.com/read/2017/08/15/071700520/kank er-serviks-bisa-dicegah. liu z. and yang c., (2014), researcharticle: a mathematical model of cancer treatment by radiotherapy, hindawi publishing corporation computational and mathematical methods in medicine volume 2014, article id 172923, 12 pages http://dx.doi.org/10.1155/2014/172923. murtono et al. – a mathematical model of cervical cancer treatment by … 63 pillis l.g. de, w. gua, k.r. fister, t. head, k. maples, a. murugan, t. neal, k. yoshida, (2007), chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls, mathematical biosciences 209 (2007) 292–315. woodman c. b. d., collins s. i., young l. s., (2007), the natural history of cervical hpv infection: unresolved issues, nat rev cancer, vol. 7(1): 11-22. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 33-37 | doi: 10.14421/biomedich.2020.91.33-37 issn 2540-9328 (online) toxicological effects of ethanolic stem bark extract of xylopia aethiopica on testicular oxidative stress markers and histology of male rats elias adikwu1,*, ben enoluomen ehigiator2 1department of pharmacology and toxicology, faculty of pharmacy, niger delta university, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, nigeria corresponding author* adikwuelias@gmail.com manuscript received: 22 april, 2020. revision accepted: 10 may, 2020. published: 11 may, 2020. abstract impairment in testicular function can occur through perturbations in testicular oxidative stress markers and histology. xylopia aethiopica (xe) is used to enhance fertility in males, but with information gap on its effect on testicular oxidative stress markers and histology. the present study assessed the effects of ethanolic stem bark extract of xylopia aethiopica (eexa) on testicular oxidative stress markers and histology of male albino rats. sixty adult male albino rats (200g-250g) were randomly grouped into 4 (a-d) of 15 rats per group. the rats in the control group a (a1-a3) were administered per oral (p.o) with water (0.2 ml/day) for 15, 30 and 60 days respectively. the rats in groups b (b1-b3), c(c1-c3) and d (d1-d3) were administered p.o with eexa (200, 400 and 800 mg/kg/day) for 15, 30 and 60 days respectively. the rats were anesthetized at the termination of eexa administration and were dissected and testes removed. the testes were weighed and evaluated for oxidative stress markers and histology. testicular weights were decreased in a dose and-time dependent fashion in eexa-treated rats. significant decreases in testicular superoxide dismutase, glutathione, catalase, and glutathione peroxidase levels with significant increases in malondialdehyde levels in a dose and time-dependent fashion were observed in rats administered with eexa. testicular histology showed cellular necrosis, degeneration and loss of interstitial tissues in rats administered with eexa. this study observed that eexa perturbed testicular oxidative markers and histology. its use may impair testicular function. keywords: xylopia aethiopica; testis; oxidative stress; histology; rat. introduction infertility is defined as the inability to conceive after 1 year of regular unprotected sexual intercourse (rowe et al., 1993). infertility can be caused by a number of factors including drugs and exposure to chemical substances (slade et al., 2007). one of the proposed mechanisms by which drugs and chemical substances cause infertility is through the induction of oxidative stress via uncontrollable activity of reactive oxygen species (ros) (geng et al., 2015; alahmar, 2019). studies have shown that 40%–88% of infertile patients have high levels of seminal ros (lewis et al., 1995). increased ros can lead to testicular redox imbalance, reduced sperm quality and increased sperm dna damage. spermatozoa are highly vulnerable to the deleterious activities of excess ros because of the presence of unsaturated fatty acids found in their cell membranes. the unsaturated fatty acids undergo oxidation which is detrimental to sperm cell, germ cell membrane, eventually inducing cell death (proudfoot, 2007). excess ros can also damage biomolecules (dna, lipids and proteins) thus, altering the morphology and functions of the testes (uzunhisarcikli et al., 2007; afolabi et al., 2018). in traditional medicine, the use of herbal remedies to treat ailments including infertility is one of the most important therapeutic approaches used by man (kashani et al., 2017). evidence based herbal remedies can serve as effective treatments for infertility among males and females. herbal remedies produced from special plant parts are believed to improve reproductive organ functions, hormonal system, and sex drive (kashani et al., 2017). however, the indiscriminate use of herbal remedies to enhance fertility is becoming worrisome due to possible adverse effects on reproduction function (leke, 2018). studies using animals have shown spermatogenesis arrest, impaired gonadal hormone function, distorted testicular morphology and altered testicular redox status caused by some herbal products (kusemiju et al., 2012). xylopia aethiopica (annonaceae) (xe) is an aromatic tree that grows up to 15-30 m high. it is native to lowland rainforest and moist fringe forest in savannah zones of africa (orwa et al., 2009). xe is commonly used in traditional medicine for the treatment of diseases https://doi.org/10.14421/biomedich.2020.91.33-37 34 biology, medicine, & natural product chemistry 9 (1), 2020: 33-37 (ogbonnia et al., 2008). its bark and leaves are used to treat boils, sores, wounds and cuts. the stem bark is used in combination with other medicinal plants as topical remedy for post-partum breast infections. the decoction of its fruit is used as treatment for bronchitis, rheumatism, asthma, and dysenteric conditions. its fruit and bark extracts are also used as tonic to facilitate or stimulate fertility (burkill, 1985) which has not been proven scientifically. however, we have shown that the ethanolic stem back extract of xe decreased sperm quality and impaired reproductive hormones in male albino rats (ehigiator and adikwu, 2020). this study further examined the toxicological effects of the ethanolic stem bark extract of xylopia aethiopica (eexa) on testicular oxidative stress markers and morphology of male albino rats. this study is imperative due to the fact that experimentally, anti-fertility effects of xenobiotics have been associated with perturbations in testicular oxidative stress markers and histology (agarwal et al., 2005) materials and methods plant material xe stem bark was sourced from imo state, nigeria and was identified at the federal ministry of environment and forestry research institute of nigeria, benin city, edo state. xe stem bark was air dried and powdered using mortar, pestle and manual grinder. thereafter, 900g of the powder was macerated in 200ml of ethanol for 72 hours with intermittent shaking. the resultant extract was filtered at the end of maceration. the filtrate was concentrated using a rotary evaporator and the yield of the extract was used for this study. animals in bred adult male albino rats (200g-250g) were sourced from the department of pharmacology and toxicology, madonna university, nigeria. the rats were housed in clean gauze cages with free access to diet and water and maintained under standard laboratory conditions. the rats were acclimatized for 2 weeks prior to the experiment. the study was approved by the research ethics committee of the department of pharmacology and toxicology, madonna university, nigeria. the rats were handled according to the recommendations of the research ethics committee. animal grouping and treatment  sixty adult male albino rats were randomized into 4 groups (a-d) of 15 rats each. each group was further divided into 3 subgroups of 5 rats each.  group a which served as control was administered per oral (p.o) with water (0.2ml/day) for 15, 30 and 60 days.  group b (b1-b3) was administered p.o with eexa (200mg/kg/day) for 15, 30 and 60 days.  group c (c1-c3) was administered p.o with eexa (400mg/kg/day) for 15, 30 and 60 days.  group d (d1-d3) was administered p.o with eexa (800mg/kg/day) for 15, 30 and 60 days. animal sacrifice, collection of samples and oxidative stress assay the rats were anesthetised at end of extract administration after overnight fast. testes were excised and washed in cold physiological saline. the testes were homogenized in 0.1 m tris-hcl solution buffered (ph 7.4) and centrifuged at 3000 g for 20 min. the supernatants were collected and assessed for oxidative stress markers. testicular total protein was measured according to gonall et al. (1949) whereas malondialdehyde (mda) was assayed as reported by buege and aust, (1978). reduced glutathione (gsh) was analysed according to sedlak and lindsay, (1968) whereas superoxide dismutase (sod) was assayed as reported by sun and zigma, (1978). the method of aebi, (1984) was used to determine catalase (cat) whereas glutathione peroxidase (gpx) was assessed according to rotruck et al. (1973). histological examination of the testes testicular tissues were routinely processed and dehydrated in graded alcohol and embedded in paraffin wax. sections 3-5μm thick were prepared using a rotary microtome and stained with hematoxylin and eosin (h and e) and viewed with the aid of a light microscope for histological changes. statistical analysis graph pad prism 5.03 (graphpad software inc., ca, usa) statistical package was used for the analysis of data. results are expressed as mean ± standard error of mean (sem). results were subjected to two-way analysis of variance (anova) followed by tukey’s multiple comparison test. values at p<0.05; 0.01 and 0.001 were considered significant. results testicular weights were decreased in a dose and time dependent fashion in rats administered with eexa (200, 400 and 800 mg/kg) when compared to control (table 1). the decrease in testicular weight was significant at p<0.05 in rats administered with eexa (200 mg/kg) for 60 days whereas decreases were significant at p<0.01 in rats administered with eexa (400 and 800 mg/kg) for 30 and 60 days when compared to control (table 1). the administration of eexa produced dose and timedependent increases in testicular mda levels when compared to control (table 2). the increases were significant at p<0.05 and p<0.001 in rats administered with eexa (800 and 400 mg/kg) for 15 and 30 days adikwu & ehigiator. – toxicological effects of ethanolic stem bark extract of xylopia aethiopica … 35 respectively when compared to control. however, at p<0.001 increase was significant in rats administered with eexa (800mg/kg) for 60 days when compared to control (table 2). furthermore, the administration of eexa decreased testicular sod, cat, gsh and gpx levels in a dose and timedependent fashion when compared to control (tables 3-6). testicular sod, cat, gsh and gpx levels were significantly decreased at p<0.05 in rats administered with eexa (200 and 400 mg/kg) for 15 and 30 days. on the other hand, significant decreases at p<0.01and p<0.001 were observed in rats administered with eexa (200, 400 and 800mg/kg) for 60 days when compared to control (tables 3-6). h and e stained section of the testis of control rat showed normal histology (figure a). in contrast, the testis of rat administered with eexa (800mg/kg) for 15 days showed showed necrosis, loss of interstitial tissues and cellular degeneration (figure b). the testis of rat administered with eexa (800mg/kg) for 30 days showed enlarged interstitial space, loss of interstitial tissues, and cellular degeneration (figure c). the testis of rat administered with eexa (800mg/kg) for 60 days showed loss of interstitial tissues, cellular degeneration and necrosis (figure d). table 1. effect of ethanolic stem bark extract of xylopia aethiopica on relative testicular weight of albino rats. dose (mg/kg) 15 days 30 days 60 days control 0.71± 0.03 0.72± 0.05 0.70± 0.01 200 0.67± 0.02 0.62± 0.07 0.41± 0.06* 400 0.50± 0.06* 0.40± 0.11** 0.30± 0.03** 800 0.43± 0.04* 0.31± 0.36** 0.25± 0.44** data are expressed as mean ±sem, n=5, * p<0.05 when compared to control ** p<0.01 when compared to control table 2. effect of ethanolic stem bark extract of xylopia aethiopica on testicular malondialdehyde of albino rats. dose (mg/kg) 15 days 30 days 60 days control 0.46 ± 0.09 0.42 ± 0.01 0.45 ± 0.04 200 0.47 ± 0.05 0.48 ± 0.03 0.78 ± 0.05* 400 0.65± 0.03* 0.72± 0.07* 1.00 ± 0.03** 800 0.76 ± 0.08* 0.89± 0.04** 1.45 ± 0.07*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control figure 1. fig a. the testis of control rat showing normal seminiferous tubules (st). fig b: testis of rat treated with eexa(800mg/kg) for 15 days showing necrosis (n) loss of interstitial tissue (it) and cellular degeneration (cd). fig c: testis of rat treated with eexa (800 mg/kg) for 30 days showing enlarged interstitial space (es), loss of interstitial tissues (li), and cellular degeneration (cd). fig d: testis of rat treated with eexa (800mg/kg) for 60 days showing loss of interstitial tissues (lt) cellular degeneration (cd) and necrosis (cn) (h&e)x 200 . fig a fig b fig c fig d st st n cd it es li cd lt cn cd 36 biology, medicine, & natural product chemistry 9 (1), 2020: 33-37 table 3. effect of ethanolic stem bark extract of xylopia aethiopica on testicular superoxide dismutase of albino rats. dose (mg/kg) 15 days 30 days 60 days control 17.5± 2.63 16.9± 1.69 17.9± 2.00 200 13.4 ± 0.19* 10.7 ± 1.11* 7.62 ± 0.47** 400 9.14 ± 0.55* 6.32 ± 0.35* 4.57 ± 0.02** 800 6.26 ± 0.32** 3.68 ± 0.19** 1.17 ± 0.04*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 4. effect of ethanolic stem bark extract of xylopia aethiopica on testicular catalase of albino rats. dose (mg/kg) 15 days 30 days 60 days control 25.3 ± 3.00 26.5± 2.57 26.9 ± 3.90 200 20.1 ± 2.25* 15.8± 1.00* 9.37 ± 0.77** 400 15.6 ± 1.53* 9.96 ± 0.21* 6.44 ± 0.62** 800 9.90 ± 0.61** 6.32 ± 0.51** 2.51± 0.54*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 5. effect of ethanolic stem bark extract of xylopia aethiopica on testicular glutathione of albino rats. dose (mg/kg) 15 days 30 days 60 days control 15.0 ± 0.11 16.7± 1.04 15.9± 0.33 200 10.9 ± 0.62* 7.08 ± 0.39* 5.43± 0.57** 400 7.32± 0.65* 5.61 ± 0.91** 3.24 ± 0.73*** 800 5.14 ± 0.58** 3.65 ± 0.63** 1.22 ± 0.09*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 6. effect of ethanolic stem bark extract of xylopia aethiopica on testicular glutathione peroxidise of albino rats. dose (mg/kg) 15 days 30 days 60 days control 20.2 ± 2.55 20.9 ± 2.43 21.9± 2.78 200 15.7 ± 1.25* 11.7 ± 1.00* 8.9 9± 0.52** 400 11.6 ± 0.11* 9.05 ± 0.61* 5.76 ± 0.28** 800 8.48 ± 0.32** 6.71 ± 0.22** 2.32 ± 0.32*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control discussion one of the primary objectives of the preclinical toxicological assessment of xenobiotics is the identification of target organs which can help clinicians to monitor the adverse profile of xenobiotics during clinical development. experimentally, perturbation in testicular weight is an index for the adverse effect of xenobiotics on testicular function (stevens and gallo,1989). the current study observed dereases in testicular weights in a dose and time-dependent fashion in eexaadministered rats. testis contains antioxidants including sod, cat, gsh, and gpx which prevent free radical-induced damage (quinn and payne, 1984). free radicals especially ros are very important for cell signalling and essential physiological functions in the testis. however, excessive production of free radicals can alter cellular redox balance through oxidative stress thereby disrupting normal biological functions. the male reproductive system especially the testes are susceptible to ros-induced oxidative stress (sabeti et al., 2016; asadi et al., 2017). most health related issues that impaired testicular function have been associated with ros-induced oxidative stress and decreased testicular antioxidant defence (halliwell, 2006). the present study observed dose and time-dependent decreases in testicular sod, cat, gsh and gpx levels in rats administered with eexa. this observation is a sign of testicular oxidative stress caused by ros.the overwhelming activity of ros in the testis might have surpassed the regulatory capacity of antioxidants leading to their depletion. ros production is regulated by antioxidants to prevent oxidative damage, including lipid peroxidation (lpo). lpo is a ros-induced oxidation of polyunsaturated fatty acids. lpo has been related to various health conditions including infertility because of the oxidative products produced that can be detrimental to testicular function (nam, 2011). the present study assayed testicular mda level to ascertain the extent and magnitude of testicular lpo caused by eexa. testicular mda levels were elevated in a dose and timedependent fashion in eexa administered rats. this observation can be attributed to excess ros production which oxidized testicular polyunsaturated fatty acids. furthermore, the present study correlates perturbation in testicular redox status with testicular morphology of eexa-administered rats. the testes of eexa administered rats showed varying degrees of damage including necrosis, loss of interstitial tissues, and cellular degeneration. the present observation can be attributed to eexa-induced testicular oxidative stress causing lpo. extensive lpo in biological membranes can cause loss of fluidity, decrease membrane potential, increased permeability and eventual rupture leading to release of cell and organelle contents. damage to lipids can alter and modify cellular membranes, cellular function and structure (esterbauer et al., 1991). conclusion the findings in this study showed that eexa perturbed testicular oxidative stress markers and morphology in a dose and time-dependent fashion. its use may impair testicular function. acknowledgment: the authors appreciate animal handling by mr eze ihukumere of the department of adikwu & ehigiator. – toxicological effects of ethanolic stem bark extract of xylopia aethiopica … 37 pharmacology and toxicology, faculty of pharmacy, madonna university, nigeria. source of fund: none conflict of interest: authors declare no conflict of interest references aebi h. 1984. catalase in vitro. methods enzymol, 105:1216. afolabi, ok, wusu ad, ugbaja r, and fatoki jo 2018. aluminium phosphide-induced testicular toxicity through oxidative stress in wistar rats: ameliorative role of hesperidin. toxres and appl 2: 1–11 agarwal a, prabakaran, sa. 2005. oxidative stress and antioxidants in male infertility: a difficult balance. iran j reprod med 3; 1-8 alahmar at. 2019. role of oxidative stress in male infertility: an updated review j hum reprod sci. 12(1): 4–18 asadi n, bahmani m, kheradmand a, rafieian-kopaei m 2017. the impact of oxidative stress on testicular function and the role of antioxidants in improving it: a review. j clin diagn res. 11(5):1-5 buege ja, aust sd. 1997. microsomal lipid peroxidation. methods enzymol. 52:302-10. burkill, h. m 1985. entry for xylopia aethiopica dunal a. rich.: family annonceae". the useful plants of west tropical africa, vol 1 (jstor). retrieved 1 january 2013. ehigiator be, adikwu e. 2020. toxicity study of ethanolic stem bark extract of xylopia aethiopica on fertility indexes in male rats: an experimental study. int j reprodbiomed2020; 18: 1– 10 esterbauer h, schaur r, j, zoilner j. 1991. chemistry and biochemistry of 4-hydroxynonenal malondialdehyde and related aidehydes (review]. free radic biol med. 11:82-128. geng x, shao h, zhang z, ng jc, peng c 2015. malathioninduced testicular toxicity is associated with spermatogenic apoptosis and alterations in testicular enzymes and hormone levels in male wistar rats. environ toxicol pharmacol 39(2):659–667 gornall ag, bardawill cj, david mm. 1949. determination of serum proteins by means oof the biureto reaction. j biol chem. 177:751-66. halliwell b. 2006. oxidative stress and neurodegeneration: where are we now? j neurochem. 58:1634–36. kashani l, akhondzadeh s 2017. female infertility and herb. med 16, 61: 1-7 kusemiju to, yama oe, okanlawon ao. 2012. effect of carica papaya bark extract on oxidative stress parameters in testes of male albino rats; int. j. appl. res. nat. prod. 4(4):1-6 leke, r. reproductive health in cameroun. geneva, who. collaborating centre for research in human reproduction. 2008 lewis se, boyle pm, mckinney ka, young is, thompson w. 1995. total antioxidant capacity (tac) of seminal plasma is different in fertile and infertile men. fertil steril. 64:868–70 nam t. 2011. lipid peroxidation and its toxicological implications toxicol res. 27(1): 1–6. ogbonnia, s, adekunle, a.a, bosa, m.k, and enwuru, v.n. 2008. evaluation of acute and sub-acute toxicity of alstonia congensis engler bark and xylopia aethiopca (dunal) a. rich (annonaceae) fruits mixtures used in treatments of diabetes. african j biotechnol. 7: 701-705 orwa, c., mutua, a., kindt, r., jamnadass, r. and simons, a. agroforestree database:a tree reference and selection guide 2009. version 4.0 (http://www.worldagroforestry.org/af/treedb/) proudfoot at. 2009. aluminium and zinc phosphide poisoning. clin toxicol. 47: 89–100. quinn pg, payne ah. 1984. oxygen mediated damage of microsomal cytochrome p-450 enzymes in cultured leydig cells: role in steroid genic desensitization. j biol chem. 259:4130–15 rotruck jt, pope al, ganther he, swanson ab, hafeman dg, hoekstra wg. 1997. selenium: biochemical role as a component of glutathione peroxidase. scie. 179:588-90. rowe p, comhaire fh, hargreave t. who manual for the standardized investigation of the infertile couple. cambridge: cambridge university press; 1993. sabeti p, pourmasumi s, rahiminia t, akyash f and reza a. talebi. 2016. etiologies of sperm oxidative stress int j reprod biomed. 14; 231-240 sedlak j, lindsay rh. 1986. estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with ellman’seeagent. anal biochem. 25:192-205. slade p, o'neill c, simpson aj, lashen h. 2007. the relationship between perceived stigma, disclosure patterns, support and distress in new attendees at an infertility clinic. hum reprod. 22:2309–17 stevens k r, gallo m a: practical considerations in the conduct of chronic toxicity studies. in: principles and methods of toxicology. raven press, new york, edition 4, 1989: 237250. sun m, zigma s. 1978. an improved spectrophotometer assay of superoxide dismutase based on epinephrine antioxidation. anal biochem. 90:81-9. uzunhisarcikli m, kalender y, dirican k, kalender s, ogutcu a, buyukkomurcu f 2007. acute, subacute and subchronic administration of methyl parathioninduced testicular damage in male rats and protective role of vitamins c and e. pestic biochem physiol 87(2):115–122 https://www.ncbi.nlm.nih.gov/pubmed/?term=asadi%20n%5bauthor%5d&cauthor=true&cauthor_uid=28658802 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3834518/ this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 83-87 | doi: 10.14421/biomedich.2022.111.83-87 issn 2540-9328 (online) anti-oxidative effects of butanol seed extract of parkinsonia aculeata on carbon tetrachloride-induced liver damage on wistar rats muhammad bashiru abdulrahman1,*, yusuf gumburawa malami2, sanusi wara hassan3, mansur lawal3, waliu temitope adanlawo4, mansur mohammed birnin kebbi4, kamaldeen olalekan sanusi5 1department of chemical pathology and immunology, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria; 2department of science laboratory technology, umaru ali shinkafi polytechnic, sokoto, nigeria; 3department of biochemistry, faculty of science, usmanu danfodiyo university, sokoto, nigeria; 4department of chemical pathology, usmanu danfodiyo university teaching hospital, sokoto, nigeria; 5department of physiology, faculty of basic medical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria. corresponding author* abfmhzs69@gmail.com manuscript received: 06 may, 2022. revision accepted: 27 may, 2022. published: 30 june, 2022. abstract medicinal plants have protective effect because of the presence of several compounds which have different mechanism of action . this study sought to assess the anti-oxidative effects of butanol seed extract of parkinsonia aculeata on carbon tetrachloride (ccl4)-induced liver damage on wistar rats. the wistar rats were put into five groups, each with six rats: group a received a daily dosage of liquid paraffin (1ml/kg); group b received 1ml/kg body weight of ccl4 (30% in liquid paraffin intraperitoneal); group c, d, and e received the seed extracts at 100, 120, and 160 mg/kg every day for two weeks. induction of ccl4 was three times a week for two weeks simultaneously with the extract to the last day. after sacrificed, the liver was harvested and homogenized, and used for further analyses. there was a significant increase (p<0.05) in the levels of superoxide dismutase and catalase in all extract treated groups compared to positive control, except the catalase levels of group treated with 160mg/kg. similar results was observed in vitamin c, vitamin e and glutathione in rats treated with 100 and 120mg/kg of the extract. the results of this study revealed that butanol s eed extract from p. aculeata has antioxidant properties and can protect wistar rats' livers from the damaging effects of ccl4. keywords: antioxidant; carbon tetrachloride; liver damage; oxidative stress; parkinsonia aculeata. abbreviations: cat – catalase; gsh reduced glutathione; sod superoxide dismutase; ccl4 carbon tetrachloride; mda – malondialdehyde; ros reactive oxygen species; h2o2 hydrogen peroxide. introduction antioxidant activity is an important approach to defend against liver damage. antioxidants are chemicals that inhibit the oxidation of a molecule in the cells when present in extremely low concentrations. it has the capacity to nullify the effects of oxidation (lipid peroxidation) caused by free radicals in the liver of an organisms. the impaired electrons of free radicals are highly reactive and neutralize the harmful reactions of human metabolism (serbinova et al., 1991). protection of the liver against free radicals effects is provided by enzymatic antioxidants such as catalase (cat), reduced glutathione (gsh), and superoxide dismutase (sod) and non-enzymatic antioxidants e.g. vitamin c and vitamin e that play a crucial role in lipid peroxidation process. the rising amount of chemical compounds and pollution in the environment are causing an increase in liver illnesses (wadekar et al., 2008). many of these chemicals cause harm to cells and molecules by producing reactive oxygen species and other free radicals. because of its critical function in the body's metabolism of external chemical compounds, liver is one of the organs that can cause toxic reactions and is a popular subject of toxicological research (beckman & ames, 1998). carbon tetrachloride (ccl4) is frequently used as a chemical inducer of experimental liver cirrhosis. this toxic agent activates liver damage by forming reactive intermediates, such as trichloromethyl free radicals, via cytochrome p450-related functions in the oxidase system (recknagel et al., 1989). the main causes of ccl4-induced hepatic damage is related to lipid peroxidation enzymes, and generation of free radicals caused by this agent (poli, 1993). medicinal plants have protective effect because of the presence of several compounds which have different https://doi.org/10.14421/biomedich.2022.111.83-87 84 biology, medicine, & natural product chemistry 11 (1), 2022: 83-87 mechanism of action. some of the components of this plant are proteins and enzymes of low molecular weight such as vitamin, flavonoids and carotenoids (zhang & wang, 2002). many of these components especially phytochemical constituents exhibit hepatoprotective effects due to their antioxidant property (madrigalsantillán et al., 2014). the leaf, stem bark and the seed extracts of parkinsonia aculeata are used traditionally in the northern nigeria for the treatment of hepatopathy, bacterial diseases, typhoid fever, diabetes, malaria and trypanosomiasis (hassan et al., 2010; leite et al., 2007). leaf extracts of p. aculeata were reported to have hepatoprotective and antioxidant activities (hassan et al., 2005). however, there is paucity of scientific report on the seed extract. this study therefore sought to assess the anti-oxidative effects of butanol seed extract of p. aculeata on carbon tetrachloride-induced liver damage on wistar rats. materials and methods. plant collection, identification and storage parkinsonia aculeata seeds were obtained on the campus of usmanu danfodiyo university in sokoto, nigeria. the plant components were identified and taxonomically validated at usmanu danfodiyo university, sokoto botany unit. in the departmental herbarium of the botany unit, a voucher specimen of the plant (uduh/ans/0038) was deposited for reference. the seeds were opened and air dried for one week in the shade before being pounded into a fine powder with a mortar and pestle and stored at room temperature until use. experimental animal wistar rats of either sex weighing 120-200 g were obtained from animal house, department of biological sciences, usmanu danfodiyo university, sokoto, nigeria. they were kept in wire mesh cages with free access to food and water for one week to acclimatize. they were maintained on standard optimal feeds and clean tap water before and after daily administration of plant extract between 9:30 to 10:30 hours. experiments were performed according to ethical guidelines for investigation of experimental pairs in conscious animals. the standard orogastric cannula was used for oral administration of the seed extract. preparation of plant extracts two hundred grams (200g) of fine powder of the plant seed were extracted with two (2) litres of methanol at room temperature overnight and filtered through whatman no. 1 filter paper. the filtrate was concentrated to dryness using rotary evaporator and percentage yield was calculated (5.6%). the residue was dissolved in distilled water and partitioned with butanol (saturated with water). the butanol fraction was thereafter screened for antioxidant properties. experimental design the animals were divided into 5 groups of six (6) rats. group a: (normal control) received daily dose of liquid paraffin (1ml/kg) body weight per day for 14 days. group b: (positive control) received 1ml/kg body weight of ccl4 (30% in liquid paraffin intraperitoneal) three (3) times a week for two (2) weeks. group c, d and e: received the seed extracts of p. aculeata at 100, 120 and 160 mg/kg per day respectively for two weeks. induction of ccl4 was three times a week for two weeks simultaneously with the extract to the last day. animals were sacrifice 24 hours after the last day. liver was removed and rinsed in ice-cold 1.5% kcl, dried and weighed homogenized in 4x ice-cold isotonic phosphate buffer ph 7.4 and the centrifuged at 9000g for 20 minutes to obtain post mitochondria supernatant at 105,000xg for 60 minutes to obtain the microsomes cytosolic fractions of the supernatant was immediately frozen on dry ice and microsome was suspended in 0.15 sucrose solution. sample preparation for the determination of markers of antioxidant activity the liver was perfused with 0.86% cold saline to completely remove the red blood cells, it was suspended in 10% (w/v) ice-cold. 0.1cm3 phosphate buffer at ph 7.4. the liver was then cut into small pieces, some quality was weighed and homogenized. the homogenate used for the estimation of enzymatic and non-enzymatic antioxidants. estimation of vitamin c vitamin c estimation was carried out using the method of rutkowski et al. (rutkowski et al., 1998). briefly, 1ml of sample was measured into test tubes, 1ml of pr (50nm solution of oxalic acid) was added and mixed thoroughly at room temperature for 30 minutes. it was centrifuged at 700g for 16 minutes, supernatant was collected with pipette and used as a test sample for spectrophotometric measurements. standard sample was prepared using 1ml of standard solution without centrifugation. absorbance of test sample (ax) and standard sample (as) was measured at 700nm against the mixture of pr as the reference sample. concentration cx of vitamin c (µm) in the sample was determined using the formula 𝐶𝑥 = 𝐴𝑥 as . 𝐶𝑠 where; cs = concentration of standard solution abdulrahman et al. – anti-oxidative effects of parkinsonia aculeata 85 estimation of vitamin e vitamin e was determined using the method of rutkowski et al. (rutkowski et al., 2005). briefly, 0.5ml of sample was measured into test tube, 0.5ml of anhydrous ethanol were added and shaken vigorously then plugged for 1 minute. a 3ml of xylene was added again and shaken vigorously for another 1 minute, centrifuged at 1500g for 10 minutes simultaneously. a 0.25ml solution of batophenanthroline was measured into test tube ii. a 15ml of the extract was collected and transferred to test tube ii, the content was mixed. a 0.25ml of fecl3 solution was also added to test tube ii, mixed then 0.25ml of h3po4 solution was also added and mixed again and the absorbance was measured spectrophotometrically. 0.5ml of standard solution prepared using trolox as test sample using α tocopherol. 0.5ml of deionized water was added. absorbance of the test (ax) and standard (as) was measured at 539nm against blank test. concentration of vitamin e was thereafter calculated using the formula: concentration of vitamin e (cx) 𝐶𝑥 = 𝐴𝑥 as . 𝐶𝑠 where; cs = concentration of standard solution. estimation of reduced glutathione and enzymatic assay of superoxide dismutase and catalase the colorimetric assay for catalase (cat), superoxide dismutase (sod), and reduced glutathione (gsh) was carried out using commercial kits (randox lab) according to the manufacturer’s protocol. malondialdehyde (mda) determination tissue supernatant (150µl) was diluted to 500µl with double deionized water. two hundred and fifty (250) µl of 1.34% thiobarbituric acid was added to all the test tubes followed by addition of an equal volume (250µl) of 4% trichloroacetic acid (tca). the resulting mixture was shaken and incubated for 30 minutes in a water bath temperature greater than 900c. the test tubes were allowed to cool at room temperature and the absorbance of the complex formed was read at wave length of 532nm (hartman, 1983). the absorbance was extrapolated from a standard curve generated by using a standard (1,1,3,3-tetraethoxy propane). the result was expressed as nanomoles of mda per cubic centimeter of supernatant. results estimation of non-enzymatic antioxidants. there was a significant increase (p<0.05) in the levels of vitamin c, vitamin e and gsh in rats treated with 100 and 120mg/kg of the extract. however, it was observed that the higher the dose, the lower the values (table 1). table 1. non-enzymatic antioxidant properties of rats administered butanol seed extracts of parkinsonia aculeata and carbon tetrachloride. group vitamin c (umol/l) vitamin e (mg/dl) gsh (mg/ml) a 121.07±24.62 96.88±27.87 22.90±1.24 b 110.46±21.34 α 89.23±7.61α 14.84±5.35α c 156.91±14.94αβ 173.36±5.88αβ 48.15±2.00αβ d 130.88±7.17αβ 107.29±8.79αβ 27.44±1.41αβ e 111.59±25.97α 103.87±3.04β 20.37±1.46β values are expressed as mean ± sem (n = 5) α = significantly (p<0.05) different vs a, β = significantly (p<0.05) different vs b, by using the analysis of variance and lsd multiple comparison test on spss software (ibm corp.usa, 2011). gsh: reduced glutathione. group a: liquid paraffin treated group, group b: 30% ccl4 treated group, group c: administered 100mg/kg of butanol extract + ccl4, group d: administered 120mg/kg of butanol extract + ccl4 group e: administered 160mg/kg of butanol extract + ccl4 estimation of enzymatic antioxidant markers a significant increase (p<0.05) was observed in the levels of antioxidant enzymes such as sod and cat in all treatment groups compared to positive control, except the cat levels of group treated with 160mg/kg of the extract. moreover, the levels of lipid peroxidation marker (mda) was only significantly lower (p<0.05) in the group treated with 160mg/kg of the extract (table 2) compared to positive control. table 2. enzymatic antioxidant properties of rats administered butanol seed extracts of parkinsonia aculeata and carbon tetrachloride. group sod (u/ml) cat (u/ml) mda (umol/l) a 14.30±6.01 288.88±43.09 130.34±32.93 b 8.06±6.38 α 279.14±20.62α 195.94±58.65α c 44.53±21.40αβ 410.06±23.47αβ 217.17±99.33αβ d 89.94±25.44αβ 352.65±21.31αβ 222.22±8.79αβ e 56.93±35.72 αβ 276.44±8.95 α 96.15±5.25 αβ values are expressed as mean ± sem (n = 5) α = significantly (p<0.05) different vs a, β = significantly (p<0.05) different vs b, by using the analysis of variance and lsd multiple comparison test on spss software (ibm corp.usa, 2011). group a: liquid paraffin treated group, group b: 30% ccl4 treated group, group c: administered 100mg/kg of butanol extract + ccl4, group d: administered 120mg/kg of butanol extract + ccl4 group e: administered 160mg/kg of butanol extract + ccl4 mda: malondialdehyde, cat: catalase; sod: superoxide dismutatse. discussion free radicals play a role in the aetiology of a variety of diseases (covacci et al., 2001). they are easily produced in the body through regular metabolic pathways. antioxidants, on the other hand, can neutralise free radicals and thereby prevent disease. natural antioxidants derived from plant sources have been linked to a lower risk of chronic disease due to their ability to stop free radical growth in the biological system (covacci et al., 2001). antioxidant activity, or the prevention of free radical formation, is critical in the 86 biology, medicine, & natural product chemistry 11 (1), 2022: 83-87 prevention of ccl4-induced hepatopathy (venukumar & latha, 2002). to avoid and neutralise free radical-induced damage, the body possesses an excellent defence mechanism. catalase, superoxide dismutase, and glutathione peroxidase are examples of endogenous antioxidant enzymes that help with this. these enzymes form a mutually beneficial defence team against reactive oxygen species (ros) (hewawasam et al., 2003). in ccl4 induced hepatotoxicity, the balance between ros production and antioxidant defences may be lost hence oxidative stress result, which through a series of event regulate cellular functions leading to hepatic necrosis. from the result of this study, rats administered ccl4 showed significant changes (p<0.05) on the liver function and antioxidant parameters when compared with normal control group. the reduced activity of antioxidant enzymes observed point out the hepatic damage as the rats administered with ccl4. the seed extract had indicated reversed changes in liver function and antioxidant parameters when compared with ccl4 treated group. in terms of non-enzymatic antioxidants, gsh is a significant predictor of tissue sensitivity to oxidative damage, and hepatic gsh depletion has been linked to increased toxicity to compounds such as ccl4 (kidd, 1997). in the present study, decrease in hepatic tissue of gsh level was observed in ccl4 treated groups. moreover, the findings showed significant decrease in the levels of vitamin c and vitamin e in ccl4 treated group when compared to the normal control group. this is in agreement with the findings of narasimhanaidu and ponnain, (2006) that indicated that condition of severe oxidative stress may also lead to the depletion of protective physiological moieties such as gsh, vitamin c and vitamin e in rats (kamalakkannan & prince, 2006). glutathione through its significant reducing power contributes to the recycling of other antioxidants such as vitamin c and e that have become oxidized (kidd, 1997). interestingly, the reduced levels of gsh, vitamin c and vitamin e caused by ccl4 was attenuated by the butanol seed extract of p. aculeata. lipid peroxidation is a measure of membrane damage and changes in the structure and function of cellular membranes, and it was found to be higher in the ccl4-treated group. the observed increase in mda levels of ccl4 groups suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defence mechanism to prevent formation of excessive free radicals (achliya et al., 2004). this is also in agreement with findings of moscrella et al. (1994) and patrick-iwuanyanwu et al. (2007). the treatment with 160mg/kg of the extract was however able to reduce the mda levels, which suggests a hepatoprotective effect. similarly, the activities of sod and cat was observed to increase as result of the ccl4 administration. sods are the first line of defence against harm caused by ros. these proteins catalyse the dismutation of superoxide anion free radical (o2-) into molecular oxygen and hydrogen peroxide (h2o2), lowering the level of o2-, which can harm cells at high levels. 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[et al.], 55(9), 359–363. https://doi.org/10.1007/s00011-006-5195-y zhang, h.-y., & wang, l.-f. (2002). theoretical elucidation on structure-antioxidant activity relationships for indolinonic hydroxylamines. bioorganic & medicinal chemistry letters, 12(2), 225–227. https://doi.org/10.1016/s0960894x(01)00724-7 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 81-86 | doi: 10.14421/biomedich.2021.102.81-86 issn 2540-9328 (online) risk assessment of heavy metals in chromolaena odorata collected around gemstone mining site in ijero-ekiti efe sylvanus abiya, foluso akinbode ologundudu*, ekpo wisdom department of biology, federal university of technology akure, nigeria. corresponding author* akinbodefoluso@gmail.com manuscript received: 27 july 2021. revision accepted: 02 august, 2021. published: 01 october, 2021. abstract in nigeria, like many developing nations, the resultant effect of land degradation: aggravated soil erosion, flood disasters, salinization or alkalisation, and the desertification have been a major public health concern for the past decades, however this study highlighted some of the factors that leads to the menace of soil fertility. the study was conducted at a gemstone mining site in ijero ekiti, ekiti state, nigeria. the soil samples were collected at a depth of 0-15 cm top soil and 0-30 cm subsoil. a line transect of 20 cm was drawn and soil sample was collected, all samples were kept in a clean container and labeled accordingly before been transported to the laboratory for analysis. the plant samples were thoroughly washed with distilled water to remove dust and other particles, air dried in a dust free wire meshed cage. all data obtained from this research were subjected to one-way analysis of variance (anova). the result obtained in this study indicated that the levels of heavy metal concentration tested were still within the permissible limit in the root and shoot of chromolaena odorata between the mine and control site. the implication of this is that chromolaena odorata is safe for human and animal consumption. the said plant can readily undergo photosynthetic activity to aid growth by exploiting the presence of these metals either as a macro-nutrient or micro-nutrient as seen from the translocation factor and metal transfer factor. the study concludes that soil at ijeroekiti mine site were slightly acidic soil ph, reduced organic carbon, total nitrogen, available phosphorus, exchangeable cations and averagely elevated heavy metal contents. keywords: chromonlaena odorata; gemstone; permissible limit; photosynthetic ability; transfer factor; heavy metal. introduction major problem of land degradation and pollution arise as a result of exploitation and excavation of the natural environment with an increase in sophisticated tools and methods and also revolution in industrialization in large scale pose a serious threat to the world’s resources and environmental degradation with socio-economic impacts katar (2009). the main causes of this degradation are transformation of fertile cultivated land into wasteland and in some cases pose serious environmental pollution and ecological degradation which can leads to loss of biodiversity (keskin and makineci, 2009). mining is one of the major factors that have pose serious threat and hazards that can jeopardize ecosystems of nations. nigeria has been actively engaged in solid mineral exploitation for more than decades and endowed with deposit of more than 34 solid minerals including coal, tin, gold and many more across the country. adekoya et al., 2003, southwestern part of the country has about 25% of the total land mass consisting of sedimentary rocks and hence mining activities are common. mining operations alter a site’s ecosystem by disrupting the ecological balance, landscapes, agricultural lands, forests, plantations and vegetation as well as the economic food and tree crops. other impacts of mining include alteration of the soil structure, loss and overturning of the fertile top soil, air, soil and water pollution, instability of soil and rock masses, destruction of flora and fauna, casing mass exodus of species of animals (adegboye, 2012). however, the health implications associated to mining as recorded by kitula (2004) in tanzania that the symptoms of heavy metals poisoning such as sensory disturbance, metallic taste and night blindness are common and also the world health organizations reported the prevalence of human diseases during the past decade is rapidly increasing due to effect of heavy metals. methods study area the study was conducted at a gemstone mining site in ijero ekiti, ekiti state, south western region of nigeria. sample collection soil and plants samples were collected at the different spots at the gemstone mining site in ijero ekiti, ekiti state. the soil samples were collected at a depth of 0-15 https://doi.org/10.14421/biomedich.2021.102.81-86 82 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 cm top soil and 0-30 cm subsoil. a line transect of 20 cm was drawn and soil sample was collected, all samples were kept in a clean container and labeled accordingly before been transported to the laboratory for analysis. the plant samples were thoroughly washed with distilled water to remove dust and other particles, then air dried in a dust free wire meshed cage. the analysis was carried out at the sustainable environmental laboratory and crop, soil and pest management in federal university of technology akure. soil physico-chemical properties, ph and conductivity of soil ten gram of the soil sample was weighed and placed into a sample cup; 20 ml of water was measured and poured into it. setup was allowed to stand for 30 minutes. afterwards, the ph meter was inserted into the dissolved soil sample and the reading was taken. at a room temperature of 28oc, soil conductivity was conducted using the auto-conductivity machine and the reading were recorded. organic carbon and organic matter little of the soil sample was pulverized. 1 gram was weighed into a 250ml conical flask. 10ml of potassium dichromate (k2cr2o7) was added. 20ml of sulphuric acid (h2so4) was further added and the flask was swirl vigorously for one minute. afterwards, 100ml of distilled water was added after standing for 30minutes. 3-4 drops of ferroin indicator and titrate with 0.5m iron (ii) ammonium sulphate (fe2nh4so2), takes a greenish cast and then changes to dark green. at this point, ferrous sulphate was added drop by drop until colour change was observed from green to brownish red. metal analysis about 1.0 to 2.0 gram of the sample was weighed into a 250 ml conical flask after dried in an oven for one hour. 20 ml of trioxonitrate acid (hno3) was added. then it was heated in a heater starting with low temperature for about 15 to 20 minutes. the heat was increased to medium temperature for about 30 minutes again and finally at high heating until complete digestion is required. the flask was rotated at intervals until the digest is clear (white fumes) continue heating for few minutes after that to ascertain complete digestion, that is, a clear solution is an evidence of complete a complete digestion. after cooling the sample residue was filtered and use to make up the digest up to 50 or 100ml or as appropriate. after being placed in a sample bottle the concentration using atomic absorption spectrophotometer (aas) or flame photometer was carried out for the elements like (ni, cd, as, cu, zn, pb and cr). most importantly, the machine (aas) must be powered for about 30 to 45 minutes before introducing any sample into it. this is done to increase the efficiency of the machine. statistical analysis all data obtained from this research were subjected to one-way analysis of variance (anova) and means separated with new duncan’s multiple range tests using spss 17.0 version of windows 7 statistical package was used. results table 1. soil textural class of the soil samples. soil samples % clay % silt % sand soil textural class transect site 30.48 15.28 54.2 sandy clay mining site 30.48 15.28 54.28 sandy clay control site 26.48 9.28 64.2 sandy laomy physico-chemical characteristics of soil at the mining and control sites. table 2 shows the physical and chemical (physicochemical) characteristics of soil sampled at the mine and control sites. the ph value at the mining site (6.81 ± 0.01) is slightly elevated compared to the control site (6.51 ± 0.01) and are significantly different from each other (p < 0.05). generally, elevated levels of concentration were observed for conductivity, bulk density, phosphorus, exchangeable cation (mg, k, na, ca), cation exchange capacity, organic carbon and total nitrogen between soils of mine site and control site respectively which are also not significantly different (p < 0.05). table 2. physico-chemical characteristics of soil at the mining and control sites. soil parameters pair mean±s.e n s.d t. cal sig remarks ph m 6.51±0.01 3 0.01 36.742 0.001 s c 6.81±0.01 3 0.01 conductivity m 644.3±0.88 3 1.53 378.976 0.000 ns c 171.6±0.88 3 1.53 porosity m 47.2±0.57 3 1.00 -7.232 0.005 ns c 51.9±0.32 3 0.55 bulk density m 1.32±0.01 3 0.01 12.017 0.001 ns abiya et al. – risk assessment of heavy metals in chromolaena odorata … 83 soil parameters pair mean±s.e n s.d t. cal sig remarks c 1.19±0.01 3 0.02 sodium m 2.52±0.01 3 0.02 -70.835 0.000 ns c 1.77±0.01 3 0.01 phosphorus m 4.66±0.01 3 0.02 -231.715 0.001 ns c 7.55±0.01 3 0.02 potassium m 5.21±0.01 3 0.01 4.000 0.000 ns c 7.46±0.01 3 0.01 calcium m 1.59±0.01 3 0.02 32.571 0.000 ns c 1.25±0.01 3 0.01 magnesium m 1.91±0.01 3 0.01 39.528 0.000 ns c 1.49±0.01 3 0.02 cec m 3.17±0.01 3 0.01 86.963 0.000 ns c 2.25±0.01 3 0.02 org. carbon m 0.94±0.01 3 0.01 -53.889 0.001 ns c 1.38±0.01 3 0.01 nitrogen m 0.85±0.01 3 0.01 -52.664 0.00 ns c 1.28±0.01 3 0.01 note: s – significant; ns – not significant; m – mining; c – control; s.d – standard deviation; t.cal/t-calculated; s.e-standard error; nnumber of replicates physical and chemical characteristics of soil at the line transect and control sites. table 3 shows the physical and chemical (physicochemical) characteristics of soil samples at the line transect site and control sites. the ph value at the line transect site (6.99 ± 0.01) is slightly elevated compared to the control (6.51 ± 0.01) site and are not significantly different to each other (p < 0.05). generally, there are no significant differences observed for the concentration of conductivity, bulk density, organic carbon, phosphorus, exchangeable cation (mg, k, na, ca) and total nitrogen between soils of the line transect site and control site and are also not significantly different (p < 0.05) from each other. table 3. physico-chemical characteristics of soil at the line transect and control sites. soil parameters pair mean±s.e n s.d t.cal sig remarks ph t 6.99±0.01 3 0.01 58.788 0.000 ns c 6.51±0.01 3 0.01 conductivity t 364.0±0.57 3 1.00 182.463 0.000 ns c 171.6±0.88 3 1.52 porosity t 45.5±0.57 3 0.01 -9.697 0.001 s c 51.9±0.32 3 0.55 bulk density t 1.35±0.01 3 0.02 12.829 0.000 ns c 1.19±0.01 3 0.02 sodium t 3.43±0.01 3 0.01 111.452 0.000 ns c 2.52±0.01 3 0.01 phosphorus t 14.6±0.01 3 0.02 570.870 0.000 ns c 7.55±0.01 3 0.02 potassium t 8.82±0.01 3 0.01 166.565 0.001 ns c 7.46±0.01 3 0.01 calcium t 1.49±0.01 3 0.02 23.085 0.000 ns c 1.25±0.01 3 0.01 magnesium t 2.11±0.01 3 0.01 58.502 0.001 ns c 1.49±0.01 3 0.02 cec t 4.12±0.01 3 0.01 177.088 0.001 ns c 2.25±0.01 3 0.02 org. carbon t 1.95±0.01 3 0.02 54.391 0.000 ns c 1.38±0.01 3 0.01 nitrogen t 1.38±0.01 3 0.01 -37.967 0.001 ns c 0.97±0.01 3 0.01 note: s – significant; ns – not significant; t – transect; c – control; s.d – standard deviation; t.cal/t-calculated; s.e-standard error; nnumber of replicates. the nutrient of zinc (zn), copper (cu), cadmium (cd), chromium (cr), nickel (ni) and lead (pb) in the soil at the mine site are slightly higher in abundance compared to the control site. paradoxically, arsenic (as) seems to have a low nutrient concentration in the soils at the mining and control sites. 84 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 table 4 translocation factor of each element at the mining and control site for chromolaena odorata elements mining site control site tf (cu) 0.796 0.456 tf (zn) 0.982 0.853 tf (pb) 0.939 0.876 tf (ni) 0.759 0.704 tf (as) 0.001 0.001 tf (cr) 0.953 0.014 tf (cd) 0.800 0.333 note: tf – translocation factor. translocation factor (tf) = concentration of metals (mg/kg) in the receiving level (shoot) concentration of metals (mg/kg) in the source level (root) translocation factor of each element between the soils at the mining and control sites in tree herb the nutrient of copper (cu), zinc (zn), lead (pb) and cadmium (cd) in the soil at the mine site are considerably higher in abundance compared to the control site. in contrast, nickel (ni), arsenic (as) and chromium (cr) seems to have a low nutrient in soil at the mine and control site. table 5 translocation factor of each element both at the mining and control site for the tree herb. elements mining site control site tf (cu) 1.224 0.589 tf (zn) 1.176 0.902 tf (pb) 1.040 0.759 tf (ni) 0.590 1.516 tf (as) 0.500 0.500 tf (cr) 0.956 0.936 tf (cd) 1.000 0.400 tf –translocation factor. 𝑇𝑟𝑎𝑛𝑠𝑙𝑜𝑐𝑎𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (𝑇𝑓) = concentration of metals (mg/kg) in the receiving level (shoot) concentration of metals (mg/kg) in the source level (root) metal transfer factor of each element between the soils at the mining and control sites in chromolaena odorata the metal transfer factor of copper (cu), zinc (zn), lead (pb) and chromium (cr) seems to have a high transfer factor in the soils at the mine and control site. in contrast, arsenic (as) and nickel (ni) seems to have a low metal transfer at the mine and control site respectively. table 6 indication of the values of the metal transfer factor of each element both at the mining and control site for chromolaena odorata. elements mining site control site mtf (cu) 2.640 1.666 mtf (zn) 3.761 3.130 mtf (pb) 9.818 9.636 mtf (ni) 0.000 0.000 mtf (as) 5.000 3.000 mtf (cr) 13.700 10.200 mtf (cd) 0.000 0.00 mtf – metal transfer factor metal transfer factor = mp = metal content in plant shoot (mg/kg) ms = metal content in soil (mg/kg) metal transfer factor of each element between the soils at the mining and control sites in tree herb table 7 metal transfer factor of each element both at the mining and control site for the tree herb. elements mining site control site mtf (cu) 6.800 2.813 mtf (zn) 4.500 2.488 mtf (pb) 6.818 6.000 mtf (ni) 0.000 0.000 mtf (as) 3.000 1.000 mtf (cr) 11.800 11.000 mtf (cd) 0.000 0.000 mtf – metal transfer factor. metal transfer factor = mp = metal content in plant shoot (mg/kg) ms = metal content in soil (mg/kg) discussion international organizations such as united state environmental protection agency (usepa), world health organization (who) has given some guidelines for the presence of heavy metals in plant and soil (marcovecchio et al; (2007). therefore, heavy metals like copper (cu), zinc (zn), lead (pb), nickel (ni), arsenic (as), chromium (cr) and cadmium (cd) has their respective permissible limit in plant and soil as specified by (who). the maximum permissible level in plant for (cd) is 0.02mg/kg; (zn) is 50mg/kg; (cu) is 10mg/kg; (pb) is 2mg/kg; (ni) is 10mg/kg; (cr) is 0.03mg/kg and arsenic (as) is 0.05mg/kg. the maximum permissible limit in soil for (cd) is 45.00mg/kg; (cu) is 30.00mg/kg; (pb) is 35.00mg/kg; (ni) is 20.00mg/kg; (cr) is 30.00mg/kg and arsenic (as) is 20.00mg/kg. the result for heavy metals in plant in this study indicated that the levels of heavy metal concentration tested were still within the permissible limit in the root and shoot of chromolaena odorata between the mine and control site. the implication of this is that chromolaena odorata is safe for human and animal consumption; the said plant can readily undergo photosynthetic activity to aid growth by exploiting the presence of this metals either as a macro-nutrient or micro-nutrient as seen from the translocation factor (tables 3 and 4) and metal transfer factor (table 5) calculated independently for each of the metals (mysliwa-kurdziel et al; (2004). however, the result also shows that the level of heavy metal concentration is significantly higher at the mine site compared to the control site. this is so because as distance from the point source increases, the concentration or toxicity level abiya et al. – risk assessment of heavy metals in chromolaena odorata … 85 decreases considerably (olafisoye et al; (2013). another distinction observed is that zn, as, and cd are significantly different from each other (both in the root and shoot) of the aforementioned plant. this can also be due to the rate of absorption or uptake experienced between the mine and control site considerably (yang et al., (1998) which characteristically influenced the soil type of the mine site (sandy-clay) and the control site (sandy-loam) (table 1). similar result was also noted for in the root and shoot of the tree herb collected at the same site. the heavy metal concentrations tested were also within permissible limit. heavy metal pollution of soil is regarded as one of the severe environmental challenges in many countries of the world (facchinelli et al., (2001). concentrations of cu, zn, as, cd, ni and pb in the soil are among the heavy metals investigated for in the mine and control site. concentration zinc (zn), copper (cu), lead (pb) were significantly higher at the mining site compared to the control site although it is within the permissible limit. a previous study also showed that mining around kabwe was responsible for heavy metal pollution, especially by pb (tembo et al., (2006). that paper indicated that the heavy metal concentrations decreased with increasing distance from the mine, confirming that mining activities are the main cause of soil contamination. lead (pb) toxicity causes many diseases including hematological, gastrointestinal and neurological dysfunctions, and nephropathy (lockitch,1993). it is reported that children have a greater susceptibility to pb toxicity because intestinal absorption of pb is five times greater in children than in adults. oelofse (2008) also reported elevated concentrations of as, cu, cd and pb at the ijero-ekiti mine soil. cooke and johnson (2002), akcil and koldas (2006) and arogunjo (2007) observed that low ph in mine soils promoted solubility of heavy metals. the slightly acidic soil ph at ijero-ekiti mine site is due to manual technologies adopted which resulted in the overturning of the top soil. while concentrations of heavy metals in the mine soils were slightly elevated, plant nutrient concentrations were lower at the line transect and mining site respectively (table 6 and 7). low levels of conductivity, bulk density, organic carbon, phosphorus, exchangeable cation (mg, k, na, ca) and total nitrogen were reported between soils of the line transect and mine site, and high zn and cu concentration occurred in the mine and transect sites, as supported by martinez and motto (2000) and oelofse (2008), that mining activities negatively affected the mineralization, absorption and uptake of nutrients by the root and shoot of plant. conclusion the primary target for the toxicity of heavy metals is still not clear yet. however, this study has showed the impact of mining activities on the plant and soil. it has provided up-to date empirical data on the current state of soil and plant degradation as a result of exploitation of solid mineral in ijero-ekiti southwestern nigeria. the study concludes that soil at ijero-ekiti mine site were slightly acidic soil ph, reduced organic carbon, total nitrogen, available phosphorus, exchangeable cations and averagely elevated heavy metal contents. since most of the metals analyzed for were more or less within the permissible limit according to the guidelines of world health organization (who), that means the plant is considerably safe for human and animal consumption. the reason for this safety may be due to the manual technologies adopted at the ijero-ekiti mine sites as compared to other sites such as: awo and itagunmodi where mechanized technologies were used, thereby resulting to greater discharge and accumulation of this respective metals causing more damage and degradation to the plants and soil. the study underscores the need for strict mining operation policies in nigeria with quick remediation strategies to restore degraded soil and plant life. consent for publication: all authors are aware of the publication of this manuscript. availability of data and material: the datasets used and/or analysed during the current study are available from the corresponding author on request. competing interest: the authors declare that they have no competing interest. funding: the research was self-funded. authors’ contributions: mr. e. s. abiya designed the experiment, ekpo wisdom carried out the laboratory works. dr. f. a. ologundudu carried out the statistical analysis and interpretation of the results. the author(s) read and approved the final manuscript. acknowledgement: the researchers want to appreciate the technical staff of the department of biology, federal university of technology, akure, nigeria. references adegboye ma (2012) effect of mining on farming in jos south local government area of plateau state. j. soil sci. environ. manage. 3:77–83. doi: 10.5897/jssem11.048 adekoya ja, kehinde-phillips o, odukoya am (2003) geological distribution of mineral resources in southwestern nigeria. in: elueze a.a. (ed.) prospects for investment in mineral resources of southwestern nigeria. nig. mining and geosci. soc. (nmgs), 113. akcil a, koldas s (2006) acid mine drainage (amd) causes, treatment and case studies. journal of cleaner production, 14, 1139-1145. 86 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 arogunjo a (2007) heavy metal composition of some solid minerals in nigeria and their health implications to the environment. journal of biological sciences 10(24):4438-43 doi: 10.3923/jbs.2007.4438.4443· facchinelli, a., scchi, e., & mallen, l. (2001). multivariate statistical and gis-based approach to identify heavy metal sources in soils. environ. pollution. 114, 313–324. katar s., (2009). environmental degradation and measures for its mitigation with special reference to india’s agricultural sector. india. journal of agriculture. econ. 64, 1, jan.-march keskin, k. & makineci, e. 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2department of biological science and biotechnology, college of natural and computational sciences; 3department of food science and post-harvest technology, institute of technology; haramaya university, ethiopia. corresponding author* kelemumuluye@gmail.com manuscript received: 14 december, 2020. revision accepted: 25 june, 2021. published: 02 july, 2021. abstract pectinases are the group of enzymes that degrade pectin. this study was conducted with the aim of isolation of efficient pectinase producing pectinolytic fungi from the decomposing mango peels using extracted mango peels pectin as a growth substrate under submerged fermentation, determining optimum pectinase production conditions with regards to some physicochemical parameters. the organisms were screened for the production of pectinase using pectin agar media, and the two active pectinolytic fungi (p1 and p2) were isolated. pectinase production media was later used for the lab scale production of pectinase by inoculating p1 and p2 and incubating for 7 days. the enzyme was extracted after seven days of fermentation and every day tested for their pectinolytic activity. p2 showed relatively higher pectinolytic activity and was therefore used for further studies. p2 was inoculated into a broth containing mango pectin under submerged fermentation. results indicate that a pectin yield of mango peel 17.75%. different parameters optimization processes were investigated on submerged fermentation namely ph, incubation period, temperature and substrate concentration optima were found 6, 4 days, 35oc and 1.5% respectively. the result suggests that mango peels have high pectin content and can be used for the value-added synthesis of pectinase. keywords: mango peels; pectinase; pectin; submerged fermentation. introduction enzymes are biological catalysts that are prerequisite for various chemical reactions produced by plants, animals and microbes. they are specific for carrying out a task and efficiently speedup the rate of reaction ultimately accelerates all metabolic processes. microbial enzymes are gaining consideration with the current development of enzyme technology (gurung et al., 2013). as nature has vast potential of microbial enzyme resource and enzymes from microbes are of great significance in developing bioprocesses for industries. there is continuous increasing demand for industrial enzymes with the rising need for sustainable solutions (adrio and demain, 2014). pectinases are among the first enzymes to be used at home. their commercial application was first observed in 1930 for the preparation of wines and fruit juice. pectinase are today one of the upcoming enzymes of the commercial sector. it has been reported that microbial pectinase account for 25% of the global food enzymes sales (jayani et al., 2005). pectinase, that break down pectin is a well-known term for commercial enzyme preparation; a polysaccharide substrate, found in the cell wall of plants. this enzyme splits polygalacturonic acid into monogalacturonic acid by opening glycosidic linkages. through this process, it softens the cell wall (khan et al., 2012). plants and microorganisms are two major sources of the pectinase enzyme. but, microbial source of pectinase has become increasingly important from both technical and economic point of view. among microbes, fungi as enzyme producers have many advantages since they are normally gras (generally regarded as safe) microorganisms and their enzymes are extracellular which makes it easy recuperation from fermentation broth (murthy and naidu,2010). maximum yield of pectinase can be achieved using agricultural wastes. the use of waste products is economically viable. furthermore, the production can also be increased after optimization of fermentation conditions and different cultural parameters (bibi et al. 2016). optimization of different physicochemical parameters, including incubation period, temperature, ph, carbon, and nitrogen sources for selected fungal https://doi.org/10.14421/biomedich.2021.101.15-21 16 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 strains, is a key step for the enhanced production of pectinase (neeta et al., 2011). mango peel is one of the major by-products from the mango pulp processing industries. during the processing of mango fruit, peel and stone are generated as waste (40–50% of total fruit mass). waste generated from mango processing constitutes 20–25% peel, which was found to be a good source for the extraction of pectin of good quality, with a high degree of esterification and phenolic compounds (berardini et al., 2005). pectin acts as the inducer for the production of pectinase enzymes by microbial systems, and pectin-rich mango peels are considered to be a good source for pectinase production and ideal substrate for the decomposition of mango peels by microorganisms (kumar et al., 2012). many urban areas in ethiopia have simple fruit/juice processing cottage industries which usually discard mango peels after extracting the flesh for mango juice. these produce a huge amount of waste throughout the year. these waste materials can be constructively used to produce important products including pectin and pectinases as a replacement for dumping them in the environment where they might at times generate pollution. this is a promising way of converting wastes to wealth. therefore, optimization of in-vitro production of pectinase by pectinolytic fungi using pectin obtained from mango peels as growth substrate under submerged fermentation. desired to extract partially purified pectin from powdered mango peels, isolate and screen active pectinolytic fungi from decaying vegetables and fruits as well as from soils containing decomposed mango peels, and optimize the effect of some physicochemical parameters. materials and methods substrate preparation mango (mangifera indica) peels were collected from tsega fruit and juice house of harar city, ethiopia. the peels were washed and cut into small pieces and then treated with 97% ethanol to reduce the microbial load. the ethanol treated peels were washed with distilled water and sun-dried for 7 days. the dried peels were then ground to a powder using a milling machine and stored in a clean container at room temperature until used for extraction. extraction of pectin from mango peels pectin was extracted using the method described by mccready (1970) as cited by ezugwu et al. (2014). one hundred gram ground mango peels were weighed into a 2000 ml beaker containing 800 ml of distilled water and 12 g of sodium hexa-metaphosphate. the ph adjusted with 3n hcl to 2.2±0.1. the mixture was heated in a water bath at 70oc for 1hr and stirred with a stirrer. the extract was filtered through a muslin cloth and the residue washed with 200 ml of distilled water. the washings were added to the filtrate. the filtrate was concentrated by evaporation on a hot plate to 1/5th of the initial volume. the concentrated pectin was cooled to 50oc and poured into a volume of 0.5 m hcl-ethanol (1:3), solution. the mixture was stirred for 30 min and allowed to stand for 1hr. the precipitate was vacuum filtered, washed with hcl-ethanol solution and finally washed with acetone to remove traces of hcl and ethanol. the extract was dried in an oven at 40oc for a few hours to constant weight, finely ground and then used as mango pectin. %𝑌𝑖𝑒𝑙𝑑 𝑜𝑓 𝑝𝑒𝑐𝑡𝑖𝑛 = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑝𝑒𝑐𝑡𝑖𝑛 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 (𝑔) 𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑚𝑎𝑛𝑔𝑜 𝑝𝑒𝑒𝑙 𝑝𝑜𝑤𝑑𝑒𝑟 𝑢𝑠𝑒𝑑 (𝑔) × 100 collection of soil samples containing decomposing mango peels soil samples were collected from dumping sites of decompose waste that contained mango peels at addis ababa, koshe massive garbage dump, ethiopia, using the methods described by martin et al. (2004). the soil samples were collected in a clean dry plastic container and transported to the laboratory. preparation of broth media soil samples (3 g) collected from the site of decomposing/ decaying fruit and vegetable containing mango peels were homogenized in sterile medium containing 1% mango pectin, 0.14% of (nh4)2 so4, 0.2% of k2hpo4, 0.02% of mgso4.7h2o, 0.1% of nutrient solution containing 5 mg/l feso4.7 h2o, 1.6 mg/l mnso4.h2o, 1.4 mg/l znso4.7h2o, 2.0 mg/l cocl2. the mixture was at 30 oc for 24 hours. preparation of solid media the pectin agar medium (pam) contained 1% mango pectin, 0.14% of (nh4)2so4, 0.2% of k2hpo4, 0.02% of mgso4.7h2o, 0.1% of nutrient solution containing; 5 mg/l feso4.7h2o, 1.6 mg/l mnso4.h2o, 1.4 mg/l znso4.7h2o, 2.0 mg/l cocl2 and 3% agar-agar (the gelling agent) (w/v). the medium was autoclaved at 121 oc for 15min. it was allowed to cool to about 45 oc and then poured into sterile petri dishes and allowed to gel. the plates were then incubated in an incubator at 37 oc overnight to check for sterility (ezugwu et al., 2014). mulluye, et al. – production and optimization of pectinase from … 17 inoculation of plates and sub-culturing a loop of homogenized culture from the broth medium was streaked onto the solid medium under the flame of bunsen burner. streaks were made from each side of the plate, marking an initial point, with sterilization of the wire loop after each side has been completed. the plates were thereafter incubated at 35 oc till visible colonies were observed. all morphologically contrasting colonies were purified by repeated streaking and subculturing on separate plates. this process was continued till pure fungal cultures were obtained (ezugwu et al., 2013). storage of pure fungal isolates pure fungal isolates were maintained on potato dextrose agar (pda) slopes or slants as stock cultures. pda media were prepared according to the manufacturer’s specification. in the latter, 3.9 g of pda powder was weighed and added in a small volume of distilled water and made up to 100 ml. this was boiled and simultaneously stirred until a clear solution of molten pda was produced. microscopic and macroscopic features of the isolated fungi and identification three days old pure cultures were examined. the colour, nature of mycelia or spores and growth patterns were also observed. the three days old pure culture was used in preparing microscopic slides. a little bit of the mycelia was dropped on the slide and a drop of lacto phenol blue was added to it. a cover-slip was placed over it and examination was performed under the light microscope at x400 magnification. identification was carried out by relating features and the micrographs to identification of common aspergillus species (klich, 2002). the fermentation broth pectinase was produced by submerged fermentation according to the method of martin et al. (2004). submerged fermentation (smf) technique was employed using a 250 ml erlenmeyer flask containing 100 ml of sterile cultivation medium optimized for pectinase with 0.1% nh4no3, 0.1% nh4h2po4, 0.1% mgs04.7h2o and 1% mango pectin. the flask was covered with aluminium foil and autoclaved at 121 oc for 15 min. inoculation of the broth from the pda slants, fresh plates were prepared and inoculated. three days old cultures were used to inoculate the flasks. in every sterile flask, two discs of the respective fungal isolates were added using a cork borer of diameter 10 mm and then plugged properly. the culture was incubated for 7 days at 30 oc. extracting the crude enzyme at each day of extract, flasks were selected from the respective groups and mycelia biomass separated using filtration technique. the broth was filtered through cheesecloth followed by whatman no. 1 filter paper. each day, the filtrate was analysed for pectinase activity till the 7th day of fermentation. pectinase assay pectinase activity was evaluated by assaying for polygalacturonase (pg) activity of the enzyme. this was achieved by measuring the release of reducing groups from pectin using 3, 5-dinitrosalicylic acid (dns) reagent assay method described by miller (1959) as cited in wang et al. (1997). the reaction mixture containing 0.5 ml of 0.5% pectin in 0.05 m sodium acetate buffer of ph 5.0 and 0.5 ml of enzyme solution was incubated for 1 hr. one ml of dns reagent was added and the reaction was stopped by boiling the mixture in a boiling water bath for 10 min. the volume of the mixture was made up to 4 ml with 1 ml of 1.4 m of rochelle salt (sodium potassium tartrate) solution and 1 ml of distilled water. the reaction mixture was allowed to cool and the absorbance was read at 575 nm. one unit of enzyme activity was defined as the amount of enzyme that catalyses the release of one micromole of galacturonic acid per minute. optimization of physicochemical parameter for pectinase production the isolate was grown under different temperature, ph, and substrate concentration to evaluate the influence of growth conditions on pectinase production. the optimization was carried out according to meena et al. (2015), with slight modification. thus when one parameter (e.g. temperature) was evaluated for its effect on pectinase production the other parameters (e.g. ph, and incubation period) were kept constant at 5 and 96 hours, respectively, and similarly, when the effect of ph was evaluated, the other two parameters (i.e. temperature and incubation period) were kept constant at 30 oc and 96 hours, respectively. effect of temperature on the production of pectinase the isolate was inoculated into a 50 ml production medium kept in 250 ml capacity erlenmeyer flask and was subsequently incubated in an incubator whose temperatures are adjusted to 25, 30, 35, 40, 45, 50 and 55 oc with the aim of determining the optimum temperature required for the production of the pectinase.the liquid medium was prepared by weighing 1 g of the extracted mango pectin and mixing with 0.1 g of nh4no3, 0.1g of nh4h2po4, 0.1 g of mgso4.7h2o, and 100 ml distilled water. the mixture was then autoclaved at 121 oc for 15min. the medium was cooled to room temperature in a 18 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 bio-safety cabinet and inoculated with the three days old pure culture of the fungal isolate. in every sterile flask, disc of the pectinolytic fungi isolates was added using a cork borer of 10 mm diameter and then plugged properly. the culture was incubated for 4 days. after incubation, the culture broth was filtered and assayed (bezawada and raju, 2018). effect of substrate concentration on pectinase production the isolate was inoculated into each of the 50 ml production medium consisting of varying concentrations (i.e. 0.2, 0.5, 1, 1.5, and 2%) of the extracted mango pectin in 250 ml erlenmeyer flask and incubated at 30 oc in an incubator for 4 days. after incubation, each culture broth was filtered and was assayed. all pectin broth media preparations and inoculations were done. effect of ph on the production of pectinase in this experiment, all the procedures followed in the preparation of media and inoculations were similar to those described in effect of temperature on the production of pectinase except that the incubation was done at varying ph (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0) and at fixed temperature and concentration of pectin (30 oc and 1%, respectively). furthermore, assay for pectinase activity was done after 4 days of incubation (ketipally and ram, 2018). data analysis the data were entered into the computer and statistical analysis was done using anova provided by the sas version 9.1 and significant difference examined for different parameters and between pectinolytic fungal isolates at 95% confidence level of significance and at p <0.05 results and discussion yield of extracted mango pectin in this research, pectin was extracted from mango peel with a yield of 17.75% at ph 2.2, the temperature of 70 oc and extraction time of 60 min using the ethanolhcl method described by mccready (1970). rehman et al. (2004) reported mango pectin extraction yields of 13.45%, 21.0% and 15.1% using hydrochloric acid, sulphuric acid, and nitric acid respectively at the of ph 2.5, temperature 80 oc and extraction time of 120 min. the differences in the yields could be as a result of differences in the sources of pectin and other factors such as extraction technique, changes in ph, temperature, extraction time and environmental factors (kertesz 1951; rehman et al., 2004). table 1. experimental observation yield of extracted mango pectin. contents unit value solution of ph –– 2.2±0.1 volume taken for extraction ml 800 amount of mango peel sample added g 100 amount of sodium hexametaphosphate added g 12 extraction temperature oc 70 extraction time min 60 amount of pectin obtained g 17.75 yield of pectin obtained % 17.75 isolation and screening of pectinase producing fungi in total, two isolates were identified from soil samples that contain mango peel. based on the colony characteristics the fungi exhibited on the selective growth media, the two isolates were assigned the codes p1 and p2 for further characterization and identification purpose. figure 1 shows microscopic photographs of the two fungal isolates. the isolates were identified based on their microscopic features. table 2. identification of pectinolytic fungal based on their colony morphology, growth characteristics, microscopic features and growth at temperature of 35 oc. code of the isolate colony characteristics on pectin agar media growth on petri plate (85 x 15mm diameter) microscopic features probable identity p1 moderately growing colonies with carbon black /deep brownish black colour conid i.a e.; reverse-colorless to pale yellow, exudates lacking produced a submerged form of mycelium covered the plate in 8 days conidial heads radiate, vesicles nearly globose, conidiophores up to 3 mm in length, phialides biseriate aspergillus niger p2 rapidly spreading, colonies are colorless to yellow or velvety, dull blue-green, reverse colourless to varying g in shades, produces tufted aerial mycelium up to felted floccose forms covered the plate in 7 days conidial heads columnar, densely crowded, flask-shaped vesicles, conidiophores short in length, and phialides uniseriate. aspergillus fumigates mulluye, et al. – production and optimization of pectinase from … 19 p1 p2 figure 1. micrograph of pectinolytic fungal isolate (40x). production of pectinases by submerged fermentation (smf) pectinase production increase with the increase in time duration up to 96 hr then it decreases (table.3). the maximum production of both isolates occurs on 4th day (96 hr) incubation period. production of pectinase exhibited the highest enzyme activity of p1 and p2 was at 0.0276 ± 0.0015 and 0.0290 ± 0.0107 u/ml respectively. from the two isolates, p2 showed relatively maximum pectinolytic activity; therefore, isolate p2 was used for further studies of pectinase optimization and characterization. table 3. effect of incubation period on enzyme production. the incubation period (days) isolated pectinolytic fungi enzyme activity u/ ml p1 p2 1 0.0144 ± 0.0012d 0.0145 ± 0.0009c 2 0.0170 ± 0.0011dc 0.0209 ± 0.0021abc 3 0.0177 + 0.0014c 0.0219 ± 0.0020abc 4 0.0276 + 0.0015a 0.0290 ± 0.0107a 5 0.0258 ± 0.0006a 0.0259 ± 0.0076a 6 0.0218 ± 0.0029b 0.0259 ± 0.0076ab 7 0.0171 ± 0.0018dc 0.0162 ± 0.0005bc p1 = pectinolytic fungal isolate one p2 = pectinolytic fungal isolate two values are mean ± s.d. of 3 replicates a,b,c,d means within a column with values followed by different superscripts are significantly different at (p < 0.05), values followed by same superscripts are not significantly different at (p < 0.05). determination of optimum temperature required for pectinase production by p2. the experiment was run at various temperatures between 25 and 55 oc (table 4). 35 oc temperatures were the most suitable temperature for the growth and production of pectinase activity. maximum enzyme activity 0.0298 ± 0.00047 u/ml was found at 35 oc and lower activity 0.0020 ± 0.0013 u/ml was showed at 55oc. any increase in temperature above 35 oc affects metabolic activities of the microorganism which results in reduced enzyme production. table 4. effect of temperature on pectinase production. temperature ( oc ) enzyme activity u/ml 25 0.0093±0.00034c 30 0.0292±0.0107a 35 0.0298±0.00047a 40 0.0211±0.0021b 45 0.027±0.0024ab 50 0.0024±0.0010c 55 0.0020±0.0013c determination of optimum concentration of mango pectin required for pectinase production by p2. the effect of substrate concentration for pectinase production p2 was incubated at different concentration of mango pectin as 0.1, 0.2, 0.5, 1.0, 1.5 and 2% (fig.2). enzyme production increased with increase in concentration. a slight decrease in enzyme production was observed until the concentration reached 2%. the maximum 0.013109 u/ml enzyme production was observed at 1.5% of mango pectin. figure 2. pectinase production by p2 versus substrate concentration. determination of optimum ph required for pectinase production by p2. the ph of the growth medium was found to have a marked effect on pectinase production (table 5). the highest enzyme yield was observed at ph 6.0. ph alters enzyme conformation, recognition site, active site, and substrate conformation, and hence determining its optimum value. this is very critical in biochemical characterization of enzymes (palmer, 1995). 20 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 table 5. effect of ph pectinase production. ph enzyme activity u/ml 4 0.0095±0.00091bc 5 0.0100 ±0.00093b 6 0.0123±0.00036a 7 0.0122±0.00037a 8 0.0086±0.00037c 9 0.0096±0.0011bc summary and conclusions an abundant amount of waste materials are produced by agricultural and fruit processing industries, which pose considerable disposal problems and ultimately leads to pollution. vast varieties of microorganisms are present in the environment which can be exploited for the utilization of waste materials. for example in the processing of mango fruits, a large proportion of the produced wastes are in the form of peel. these peels contain substantial amounts of pectin. from these investigations, it is evidenced that the mango peels with 17.75% pectin content were successfully used to encourage the production of pectinase under submerged fermentation process. the pectin extracted from mango peels act as the inducer for the production of pectinase enzyme. microorganisms serve as a major source of enzyme. even the majority of industrial enzymes are of microbial origin. thus an attempt of the present study is to isolate and screen for potential producers of pectinase from microorganisms. total two pectinolytic fungi isolates were identified as the efficient producer between them screening pectinolytic fungi isolate p2 was found as efficient producers. in order to obtain high yields of pectinases, it is essential to optimize the fermentation medium used for pectinase production. isolate p2 was grown for different temperature, ph, time and substrate concentration because it is essential to maximize optimum condition for production. the evidence from this study suggests that, it is highly promising to use pectin extracted from mango peels as a substrate for the production of pectinases in a submerged fermentation system using p2 isolate and a high extent solve the problem of environmental pollution by reducing mango wastes (peels). acknowledgments: we are thanks to haramaya university laboratory management directorate (hulmd) and the school of animal and range sciences for providing all the required laboratory facilities. we are also grateful to mr. yimeslal atnafu, ms. yehararwork abebaw and mr. dilnessa baile for their technical assistance. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references adrio, j. l. and a. l. demain. 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(2010). protease production by aspergillus oryzae in solid-state fermentation utilizing coffee by-products. world applied sciences journal, 8(2): 199-205. mulluye, et al. – production and optimization of pectinase from … 21 neeta, r., anupama, s., anjuvan, s. and giridhar, s. (2011). production of polygalacturonase and pectin methylesterase from agro waste by using various isolation of aspergillus niger. insight microbiology, 1(1):1-7. palmer, t. (1995). understanding enzymes. ellis horwood ltd. england. 104: 352 – 365. rehman, z.u., salariya, a.m., habib, f. and shah, w.h. (2004). utilization of mango peels as a source of pectin. journalchemical society of pakistan, 26:73-76. wang, g., michailides, t.j. and bostock, r.m. (1997). improved detection of polygalacturonase activity due to mucor piriformis with a modified dinitrosalicylic acid re agent. phytopathology, 87(2): 161-163. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 59-63 | doi: 10.14421/biomedich.2014.32.59-63 substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cyt b of indonesian swamp buffalo (bubalus bubalis) akhmad sukri1*, mohamad amin2, aris winaya3 and abdul gofur2 1department of biology, ikip mataram, jl. pemuda no. 59a mataram, nusa tenggara barat, indonesia 2state university of malang, jalan semarang 5 malang 65145, east java, indonesia 3university of muhammadiyah malang, jl. bandung 1 malang, east java, indonesia author correspondency*: sukri_bio04@yahoo.co.id abstract this research aims to investigate the substitution pattern of nucleotide base and haplotype diversity of indonesian swamp buffalo (bubalus bubalis) based on the mitochondrial dna cyt b partial gene sequence. 17 samples were chosen from 7 different regions with each uniquely represents indonesian biogeography which comprise aceh, riau, madiun, blitar, lombok, south borneo and tana toraja. the result of cyt b gene sequence alignment showed the presence of transition and transversion substitutions and the absence of insertion and deletion. the amount of transitions was found to be higher than that of transversions and the amount of substitution in pyrimidine was also higher than that in purine. the highest amount of transitions happened in base tc which is a silent substitution. the result of median joining network analysis showed that indonesian haplotype bubalus bubalis could be classified into 16 haplotypes which form different haplogroups unique to their geographical region. the result of median joining network analysis also indicated that the genetic relationships of swamp buffaloes (bubalus bubalis) in indonesia are highly influenced by their geographical locations. keywords: substitution, haplotype diversity, bubalus bubalis, cyt b gene introduction there are three kinds of genetic marker: morphological, biochemical, and dna marker. dna marker has some characteristics; it has a high rate of polymorphism, is abundant in amount, is uninfluenced by environment and has a rate of heritability of 100 percent. molecular marker is divided into two kinds: nuclear dna, and non-nuclear dna marker. one of the non-nuclear dna markers mostly used for molecular analyses is mitochondrial dna (surahman, 2002). there are two vantage points of mitochondrial dna: the evolution of mitochondrial dna happens through the pair substitution of a singular base (wolstenholme, 1992) and the velocity of mitochondrial dna evolution is 10 times faster than that of nuclear dna (brown et al, 1979). mitochondrial dna contains 13 protein-coding genes and one of them is cyt b protein-coding gene which is seen as a powerful marker for genetic analyses (arif & khan, 2009). due to the fact that cyt b gene is various and experiences evolution quickly, it is then widely used and is suitable for discovering variations on species level (bruford et al, 2003). besides, cyt b is also one of the best samples of mitochondrial dna of mammals (arnason & gullberg, 1996). cyt b gene has been used mostly for studying evolution and genetic relationships of mammals, such as that in the studies of cows and buffaloes (schreiber et al, 1999; kumar et al, 2007; li et al, 2007; qifa et al, 2007; tobe et al, 2010; xuan et al, 2010). in this research, the partial sequence of mitochondrial dna cyt b is used for identifying the substitution pattern and haplotype diversity of indonesian swamp buffalo (bubalus bubalis) as an actualisation for genetic inventory of indonesian swamp buffalo which is also considered as an indirect endeavour to preserve and to improve the genetic quantity and quality of the population of swamp buffaloes in indonesia. materials and method blood sample, dna isolation and pcr blood samples were taken from 17 indonesian swamp buffaloes (bubalus bubalis) from 7 sampling regions, comprising aceh, riau, madiun, blitar, south borneo, tana toraja and lombok. the swamp buffalo blood was taken through jugular vein using a venoject and vacuum tube and was then mixed with an edta material as much as 0.1 gram in order to keep the blood liquid. next, the tube containing the blood sample was labeled to mark the sample number and location of the blood sampling. the blood was then stored in a refrigerator until the dna isolation process was conducted. the process of total dna isolation was carried out using nucleospinr quickpure blood kit and was complied with some established procedures. in order to detect the result of the dna isolation, agarose gel electrophoresis as much as 0.8% was used. after that, pcr (polymerase chain reaction) was conducted using a pair of partial mitochondrial dna cyt b genes with forward primers l14841: 60 biology, medicine, & natural product chemistry 3 (2), 2014: 59-63 aaaaagcttccatccaacatctcagcat gatgaaa, and reverse primers h15149: aaactgcagcccctcagaatgatatttg tcctca (kocher et al, 1989; irwin et al, 1991). the positions of the primers are illustrated in figure 1. the process of dna amplification using pcr consisted of the reaction mixture of 2,5 µl dna template, 2,5 µl forward primers, 2,5 µl reverse primers, 12,5 µl pcr mix, and 5,0 µl dh2o. the pcr process comprised 30 cycles with the following phases: pre-denaturation phase at 930c for 30 seconds, denaturation phase at 930c for 1 minute, annealing phase at 500c for 1 minute, elongation at 720c for 5 minutes and post-elongation phase at 40c. sequencing and data analysis double-stranded dna, which is the product of pcr, was then purified and sequenced using one forward primer: aaaaagcttccat ccaacatctcagcat gatgaaa (kocher et al, 1989) using taq dye deoxy terminator cycle sequencing kit and 373s dna sequencer (perkin elmer, usa). the total of 17 cyt b gene sequences was then edited using a programme called bioedit sequence alignment editor and all the cyt b gene sequences were aligned using clustalx programme (thompson et al, 1997) with african buffaloes (syncerus caffer) as the outgroup. the results of the sequence alignment were then used for discovering the variable and conserved regions, as well as the substitution pattern of the cyt b gene sequences of the indonesian swamp buffaloes (bubalus bubalis) which was assisted by the use of mega 4 programme (kumar et al, 2007). in order to discover the haplotype diversity of the indonesian buffaloes, a median joining network analysis method was conducted using dna sp and network 4.1 programme (bandelt et al, 1999). results fragments of dna which were successfully amplified using a pair of primers l14841 and h15149 in this research were about 307 bp (anderson et al, 1981; kohcer et al, 1989; irwin et al, 1991, and tanaka et al, 1996). the achieved findings were then confirmed using blast (basic local alignment search tool) method to discover whether the gained sequences in the research matched with the cyt b gene sequences of bubalus bubalis. the result of alignments of the gene sequences with query from genbank showed the rate of sequences homologous ≥ 200 bp and the similarity rate of 98%. the high percentage of similarity indicated that the sequences gained in this research were the actual sequences of cyt b gene of bubalus bubalis. the characteristics of a cyt b gene are that it does not have any stop codon (termination codon) and it has the lowest guanine as well as the highest cytosine of all genes (avise, 1994). the translation result of the dna sequences to amino acids found that there was no stop codon found in the whole cyt b gene samples. this showed that the amplified dna sequences were the target gene sequences. the composition of nucleotide and variable region the nucleotide base composition of adenine, thymine, guanine and cytosine is consecutively 29.3%, 26.7%, 27.1% and 16.9%. the total amount of nucleotide a+t = 56% and g+c = 44%. hence, gc<at and this is congruent with what was stated by sueoka (1962), that the comparison of cc<at is relative equal to the content of gc by 40-45% in vertebrate. according to the sample and outgroup sequence alignment result, it was found that the conserved region was bigger than the variable region with the former as much as 80.13% and the latter as much as 19.87%. this finding showed that indonesian bubalus bubalis has quite bigger homologous than african buffaloes, syncerus caffer. this is because african buffaloes or syncerus caffer is within the same family with bubalus bubalis, called bovidae family, and in the group of buffalo of genus of bubalus with two different kinds of species: syncerus caffer and bubalus bubalis. figure 1. the positions of the primers l14841 and h15149 in amplifying mitochondrial dna cyt b gene sequences (source: irwin et al, 1991). this finding was supported by cai et al (2013), who studied the phylogeny among goats, cows and buffaloes species under the family of bovidae based on the sry gene sequence variations. they found that cows are classified in genus bos, goats in genus capra, while buffaloes (both syncerus caffer and bubalus bubalis) in akhmad sukri, et al. – substitution and haplotype diversity analysis on the partial sequence … 61 genus bubalus. a similar finding was also stated by maceachern et al (2009) who conducted species phylogeny reconstruction under the cluster of bovini, who found that species from genus bubalus and syncerus are under the same cluster of buffaloes. transition and transversion substitution cyt b gene has a characteristic of having the amount of transition substitution more than that of transversion (avise, 1994). this could be observed from the amount of nucleotide base which experienced transition substitutions was 17, while that which experienced transversion substitution was 6. (table 1). the nucleotide base which experienced transition substitutions was the base numbered 7, 31, 47, 64, 76, 94, 127, 148, 163, 167, 172, 223, 226, 230, 266, 277, and 288, while the nucleotide base which experienced transversion substitutions was the base numbered 43, 73, 121, 166, 220, and 259. the transition substitutions mostly took place in base tc and consecutively followed by ct, ag and ga, while the transversion substitutions mostly took place in base ac and ca and consecutively followed by base substitution tg and at (table 1). the amount of transition substitutions in pyrimidine base excelled that in purine base. it could be observed either from the substitutions of base t to c or those of base c to t which happened a lot more than the substitutions of base a to g or those of base g to a. a similar finding was reported by tamura and nei (1993) and was also supported by li et al. (2007) who proposed the taxonomy status of gayal (bos frontalis) founded on the partial sequence of cyt b gene which showed the amount of substitutions in pyrimidine base (tc) was more than that in purine base (ag). table 1. the variations of nucleotide base of cyt b gene sequences of the indonesian bubalus bubalis and of the outgroup. haplotype diversity the result of median joining network analysis showed that indonesian bubalus bubalis could be classified into 16 haplotypes according to their own biogeography (figure 2). the haplotypes form a haplogroup in accordance to the biogeography of each sample of indonesian swamp buffalo (bubalus bubalis). based on figure 2, it was known that the variations of nucleotide base between the haplotypes of the buffaloes from the same region were smaller than the variations of nucleotide base between the haplotypes of the buffaloes from different regions. this suggested that the buffaloes from the same region shared a higher rate of genetic relationships than with those from different regions. this phenomenon could be observed from the haplotypes of the buffaloes from aceh region which formed the haplogroup of aceh region (a1, a2 and a3) had relatively smaller variations of nucleotide base between themselves than with those of other distant regions (figure 2). discussions table 1 illustrates the absence of insertion or deletion but it indicates the presence of the nucleotide substitutions in 307 bp of the cyt b gene sequence of indonesian bubalus bubalis which were transition and transversion substitutions. transition substitution is a substitution between purine base (a and g) or between pyrimidine base (c and t), while transversion substitution is a substitution between purine and pyrimidine base (graur and li, 2000). cyt b gene has more transition than transversion substitutions (brown et al, 1982; avise, 1994). the same thing happened in this research as there 7 16 20 22 28 31 43 47 60 64 67 73 76 84 94 10 1 11 5 12 1 12 7 13 4 14 1 14 6 14 8 15 0 15 1 15 7 16 3 16 6 16 7 16 9 17 2 17 7 17 9 18 6 18 8 19 0 19 3 20 6 21 1 21 4 22 0 22 3 22 6 23 0 23 1 23 3 23 7 24 1 25 1 25 3 25 6 25 9 26 6 27 0 27 7 28 0 28 3 28 6 29 5 30 5 30 7 syncerus caffer c t t c c a a t t t c a t c t g a t c a g a g a c c c a a a c t t a a a c c c t c c t t t g c a g a c c a t t g a c t t a madiun (1) t . . . t g c c . c . c c . c a g g t t . t a . . g t t g . t c g c t c g a . c a t c c . . . . . . . a g . c t . t c . . madiun (2) t . . . t g c c . c . c c t c a g g t t . t a . . g t t g . t c g c t c g a . c a t c c . . . . . . . a g . c t . t c . . aceh (3) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g g c a . t g . . blitar (1) t c . . t g c c . c . c c . c a . g t . a . a . . . t t g g t c . . . c . t . g a t c c a . . . . t . a g g c a . t c . . blitar (2) t c . . t g c c . c . c c . c a . g t . . . a . . . t t g g t c . . . c . t . c a t c c a . . . . t . a g . c a . t c . . riau t c . . t g c c . c . c c . c . g g t t a . a . . . t t g . t . . . . . . a . c a t c c . . . g . . . a g . c a . t c . . tator (2) t c g . t g c c g c . c c . c c g g t . a . a . . . t t g . t . . . . . . a . c a t c c . . . . a . . a g . c a . t c . . kalimantan (3) t c . . . g c c . c . c c . c . g g t . a . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . kalimantan (1) t c . . . g c c . c . c c . c . g g t . a . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . kalimantan (2) t c . . . g c c . c . c c . c . g g t . . . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . lombok (3) t c . . . g c c g c . c c . c . g g t . a . a . . . t t g . t . . . . . . a g g a t c c . a a . a . . a g . c a c t c . g lombok (2) t c . . . g c c g c . c c . c a g g t . a . a g . g t t g . t . . . c . . a g g a t c c . a a . a . . a g . c a c t c . g tator (3) t c g . t g c c g c . c c . c c g g t . a . a . . g t t g . t . g . . . . a . c a t c c . a . . a . . a c . c a . t c . . lombok (1) t c . . . g c c g c . c c . c . g g t . a . a g . g t t g . t . . . . . . a g g a t c c . a a . a . . a g . c a c t c . g tator (1) t c g . t g c c g c g c c . c . g g t . . . a . . . t t g . t . . . . c . a . c a t c c . . . . . . . a g . c a . t c . . aceh (1) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g . c a . t g . . aceh (2) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g . c a . t g . . samples nucleotide base variations (base to-) 62 biology, medicine, & natural product chemistry 3 (2), 2014: 59-63 were 17 transition substitutions and 6 transversion substitutions. the fact that the transition substitution is higher than transversion substitution is the result of the saturation happens on the divergence levels in purine base is lower than that in pyrimidine base; this makes the frequency of a and g base to be much different than the frequency of c and t (kocher & carleton, 1997). the amount of transition substitutions which was more than the amount of transversion substitutions identified in this research is supported by winaya (2013) who found that there were 16 transition substitutions of cyt b gene sequence in some kingdoms of cows with the most amount of base substitutions of t to c base, and vice versa, from c to t base. the substitution of t to c base does not necessarily change amino acids since this substitution is silent substitution. this is supported by anderson et al. (1981), who conducted sequencing and organising the human mitochondrial genome, who remarked that the substitution of t to c base is a hidden substitution and does not change amino acids. based on the result of median joining network analysis, there were 16 haplotypes of indonesian bubalus bubalis (figure 2). haplotype is the variation of nucleotide base of a species. the 16 indonesian haplotypes bubalus bubalis identified are then classified into 7 different haplogroups: the haplogroup of borneo (k-1, k-2 and k-3), the haplogroup of aceh (a-1, a-2 and a-3), the haplogroup of madiun (m-1 and m-2), the haplogroup of riau (r-1), the haplogroup of tana toraja (t-1, t-2 and t-3), the haplogroup of lombok l-1, l-2 and l-3) and the haplogroup of blitar (b-1 and b-2). the result of median joining network analysis showed that each individual of swamp buffalo formed a haplogroup according to their own biogeography, comprising aceh, riau, borneo, madiun, blitar, tana toraja and lombok. this result indicates that the samples taken for this research are actually originated from the locations in which the samplings were conducted. however, a different finding was shown by the haplotype t-1 which was separated from the haplotype t-2 and t-3 even though it is still in the same haplogroup with the two others, the haplogroup of tana toraja. this indicates that the sample t-1 is from a different population of t-2 and t-3, although it is still in the same region (figure 2). figure 2. the haplotypes of the indonesian bubalus bubalis and the outgroup based on the cyt b gene sequences. haplotype is illustrated in different circle shapes (big = 2 individuals, small = 1 individual). the red number in the branch point shows the position of various base between haplotypes. l = the buffaloes from lombok, t = the buffaloes from tana toraja, m = the buffaloes from madiun, r = the buffaloes from riau, a = the buffaloes from aceh, k = the buffaloes from borneo (kalimantan), b = the buffaloes from blitar and sc = syncerus caffer. based on figure 2, the amount of variations of nucleotide base of each haplotype will be increased along with the improvement of the distance amount between regions or biogeography. this could be observed from the haplotype of madiun, aceh and riau which have one branch point and are separated by the variations of nucleotide base which are relatively small. nevertheless, the amount of variations of nucleotide base will get increased when the haplotype of aceh is compared with the haplotype of lombok which has a huge amount of akhmad sukri, et al. – substitution and haplotype diversity analysis on the partial sequence … 63 variations of nucleotide base and which biogeographically is very much distant between them. indirectly, the bigger the variations of nucleotide indicates the more distant the genetic relationships, and this is valid conversely. therefore, the geographical location or biogeography influences the genetic relationships between indonesian swamp buffaloes (bubalus bubalis). conclusions based on the analysis result it was found that there were transition and transversion substitutions in cyt b gene sequences of indonesian swamp buffaloes (bubalus bubalis), and there was not any insertion or deletion. also, it was found that the amount of transition was higher than transversion substitution. the result of median joining network analysis showed that there were 16 haplotypes of indonesian swamp buffaloes which formed different haplogroups based on their own region or biogeography. hence, it was concluded that the geographical location influenced the relatives rate or genetic relationships of indonesian swamp buffaloes (bubalus bubalis). acknowledgments the authors would like to thank the fund management institution of education ministry of finance of the republic of indonesia, which has funded this research (no:prj 481/lpdp/2013). references anderson, s.,bankier, a.t.,barrel, b.g.,de bruijn, m.h.l.,coulson, a.r.,drouin, j.,eperon, i.c.,nierlich, d.p.,roe, b.a.,sanger, f.,schrier, p.h.,smith, a.j.h.,staden, r.,& young, i.g. 1981. sequences and organization of the human mitochondrial genome, nature, vol 290: 457-465. arif, i.a & khan, h.a. 2009. molecular markers for biodiversity analysis of wildlife animals: a brief review. anim. biodiv and conserv. vol 32 (1): 9-17. arnason, u & gullberg, a. 1996. cytochrome b nucleotide sequences and the identification of five primary lineage of extant cetaceans. mol biol evol, 13(2): 407-417. avise, j.c. 1994. molecular markers, natural history & evolution. new york: chapman & hall. bandelts, h.j.,forster, p.,& rohl, a. 1999. median joining network for inferring intraspesific phylogenies, molecular biological evolution, 16: 37-48. brown, w.m.,george, jr.m.,& wilson, a.c. 1979. rapid evolution of mitochondrial dna. proceeding of the national academy and science of usa, 76: 1967-1971. brown, w.m.,prager, e.m.,wang, a.,& wilson, a.c. 1982. mitochondrial dna sequences of primates: tempo and mode of evolution, j.mol.evol, vol 18: 225-239. bruford, m.w.,bradley, d.g.,& luikart, g. 2003. dna markers reveal the complexity of livestock domestication. genetics, vol 4: 900-910. cai, x.,zhang, h., & mipam, t.d. 2013. unique variations of sry gene result in distinct patrilineal phylogeny of capra hircus and another domestic bovidae, animal science papers and reports, vol 30(3): 219-227. graur, d. & li, w.h. 2000. fundamentals of molecular evolution. usa: sinauer associates inc. irwin, d.m.,kocher, t.d.,& wilson, a.c. 1991. evolution of the cytochrome b gene of mammals, j mol evol, vol 32: 128-144. kocher, t.d.,thomas, w.k.,meyer, a.,edwards, s.v.,paabo, s.,villablanca, f.x.,& wilson, a.c. 1989. dynamics of mitochondrial dna evolution in animals: amplification and sequencing with conserved primers, proc.natl.acad.sci, vol 86: 6196-6200. kocher, r.d & carleton, k.l. 1997. base substitution in fish mitochondrial dna: pattern and rates. in molecular systematics on fish. kocher, t.d and ca stepien (eds.). academic press: san diego. kumar, s.,nagarajan, m.,sandhu, s.,kumar, n.,behl, v.,& nishanth, g. 2007. mitochondrial dna analysis of indian water buffalo support distinct genetic origin of river and swamp buffalo, animal genetics, 38:227-232. li, s.,chang, h.,ma, g.,chen, h.,ji, d.,& geng, r. 2007. the origin taxonomic status of the gayal based on cytochrome b gene partial sequences, research journal of animal sciences, vol 1(4):114-119. maceachern, s.,mcewan, j, & goodard, m. 2009. phylogenetic reconstruction and the identification of ancient polymorphism in the bovini tribe (bovidae, bovinae), bmc genomics, vol 10: 2-17. qifa, l.,yinxia, l.,xingbo, z.,zhenshan, l.,yefen, x.,dawei, s.,xunguang, q.,ning, l.,& zhuang, x. 2007. study on the origin and taxonomic status of yak (poephagus) using cytochrome b gene of mitochondrial dna, front agric china, vol 1(3): 329-333. schreiber, a.,seibold, i.,notzold, g.,& wink, m. 1999. cytochrome b gene haplotypes characterize chromosomal lineages of anoa, the sulawesi dwarf buffalo (bovidae: bubalus sp), the american genetics association, 90: 165-176. surahman, m. 2002. peta genetika tanaman, prinsip dan aplikasinya. bul.agron, vol 30 (1): 27-30. tamura, k dan nei, m. 1993. estimation of the number of nucleotida substitutions in the control region of mitochondrial dna in humans and chimpanzees, mol. biol. evol, vol 10: 512-526. tanaka, k.,solis, c.d.,masangkay, j.s.,maeda, k.,kawamoto, y.,& namikawa, t. 1996. phylogenetic relationship among all living species of the genus bubalus based on dna sequences of the cytochrome b gene, biochemical genetics, vol 34: 443452. thompson, j.d.,gibson, t.j.,plewniak, f.,jeanmough, f.,& higgins, d.g. 1997. the clustal-x windows interface: flexible stategies for multiple sequences alignment aided by quality analysis tools, nucleic acids research, vol 25: 4876-4882. tobe, s.s.,kitchener, a.c.,& linacre, m.t. 2010. reconstructing mammalian phylogenies: a detailed comparison of the cytochrome b and cytochrome oxidase subunit i mitochondrial genes, mammalian phylogeny best gene, vol 5:1-14. winaya, a. 2010. variasi genetik dan hubungan filogenetik populasi sapi lokal indonesia berdasarkan penciri molekuler dna mikrosatelit kromosom y dan gen cytochrome b. disertasi tidak diterbitkan. bogor: institut pertanian bogor. wolstenholme, d.r. 1992. animal mitochondrial dna: structure and evolution, international review of cytology, 141: 173-215. xuan, t.p.,georgescu, s.e., manea, m.a., hermenean, a.o., & costache, m. 2010. genetic diversity and phylogenetic relationship of romanian cattle breeds inferred from cytochrome b gene partial sequences, romaian biotechnological letters, vol 15(2): 5154-5158. content_v3n2_3.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 135-140 | doi: 10.14421/biomedich.2021.102.135-140 issn 2540-9328 (online) repurposing dihydroartemisinin-piperaquine-doxycycline as an antimalarial drug: a study in plasmodium berghei-infected mice udeme owunari georgewill1,*, elias adikwu2 1department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* udgeorgewill@yahoo.com manuscript received: 06 december 2021. revision accepted: 12 december, 2021. published: 16 december, 2021. abstract artemisinin-based combination (act) therapy is the mainstay for malaria treatment. however, plasmodium parasite with decreased susceptibility to act has emerged. hence, it is imperative to discover new drugs or explore new drug combinations that can decrease plasmodium parasite resistance. this study assessed the antiplasmodial activity of dihydroartemisinin-piperaquinedoxycycline (d-pdx) on mice infected with plasmodium berghei. swiss albino mice (25-30g) of both sexes inoculated with 1x107 plasmodium berghei intraperitoneally were used. the mice were randomly grouped and orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily in curative, suppressive and prophylactic studies. the negative and the positive controls were treated daily with normal saline (0.2ml) and chloroquine (cq) (10mg/kg), respectively. after treatment, blood samples were assessed for percentage parasitemia, hematological and lipid parameters. the mice were observed for mean survival time. d-p, dx, and d-p-dx produced significant decreases in percentage parasitemia at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control. in the curative study, d-p, dx, and d-p-dx produced 64.9%, 71.1%, and 93.6% parasitemia inhibitions when compared to 75.0% inhibition produced by cq. plasmodium berghei -induced alterations in packed cell volume, white blood cells, red blood cells, hemoglobin, high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were significantly restored by dx (p<0.05) and d-p (p<0.01) and d-p-dx (p<0.001) when compared to the negative control. d-p-dx showed significant antiplasmodial activity against plasmodium bergheiinfected mice. it may be clinically useful for the treatment of malaria. keywords; artemisinins; doxycycline; malaria; repurposing; resistance. introduction world health organization (who) estimates that nearly half of the world’s population lived in malaria endemic areas (who, 2016). malaria, a plasmodium parasite infection is one of the greatest health challenges in tropical regions, despite the availability of antimalarial drugs, mosquito repellents and insecticide-treated nets. malaria chemotherapy remains a major focus of research, and new molecules are being discovered prior to the emergence of drug-resistant strains of plasmodium parasite (gaillard et al., 2015). the use of antimalarial drugs is faced with the development of resistance from plasmodium falciparum in primarily endemic areas. other challenges include financial costs, contraindications, and clinical tolerance (gaillard et al., 2015). doxycycline (dx), a broad-spectrum bacteriostatic agent is synthetically obtained from naturally occurring tetracyclines produced by streptomyces sp. (mcevoy et al., 2008). it acts by binding to several proteins in the 30s ribosomal small subunit and to different ribonucleic acids in the 16s ribosomal rna. in addition to its antimicrobial activity, it is a partially efficacious prophylactic drug with activity against liver stage of plasmodium and blood schizontocides. it is highly effective for the prevention of malaria. the u.s. food and drug administration (fda) approved the use of dx for prophylaxis of plasmodium falciparum in short-term travelers to areas with chloroquine or pyrimethaminesulfadoxine-resistant strains (tan et al., 2011). dx can be used for the treatment of malaria in children less than 8 years old and non-pregnant adults in combination with quinine sulfate for uncomplicated and chloroquineresistant plasmodium falciparum. it is also used with primaquine and quinine sulfate for uncomplicated chloroquine-resistant plasmodium vivax and with parenteral quinidine for severe malaria (griffith et al., 2007). artemisinin derivatives are highly potent with fast acting antiplasmodial activity. however, due to short half-life, and plasmodium parasite resistance to artemisinin derivatives and older antimalarial drugs, artemisinin derivatives are often combined with partner drugs with longer half-life for fast clearance of malaria parasites (nosten, 2007). this led to the development of https://doi.org/10.14421/biomedich.2021.102.135-140 136 biology, medicine, & natural product chemistry 10 (2), 2021: 135-140 artemisinin-based combination therapies (acts), which have become the mainstay for the treatment of malaria especially in malaria endemic regions (basco et al., 2017). however, plasmodium parasites with decreased susceptibility to acts’ have emerged due to both decreased susceptibility to artemisinins and partner drugs (leang et al., 2013; sanders et al., 2014). also, dihydroartemisinin-piperaquine (d-p), one of the currently used acts, which is efficacious against malaria (tayler et al., 2020) and is being considered for the prevention of malaria in pregnancy (kakuru et al., 2016; desai et al., 2016) has experienced plasmodium resistance in malaria endemic regions. plasmodium resistance was attributed to decreased susceptibility to both piperaquine and dihyroartemisinin (amaratunga et al., 2016; amato et al., 2017). hence this study aimed to evaluate if the antiplasmodial effect of d-p can be augmented by dx in mice infected with plasmodium berghei. materials and methods animals, drugs and parasites swiss albino mice of both sexes (25-30 g) used for this study were sourced from the animal husbandry of the department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state. the mice were housed in cages at temperature 20oc with cycles of 12 h light/12 h darkness. the mice were acclimated for 2 weeks and fed with food pellets and given water ad libitum. cq was manufactured by alben healthcare ind ltd, d-p was manufactured by bliss gvs pharma ltd india whereas dx was manufactured by ranbaxy laboratories ltd, india. doses were selected based on previous studies: cq (10mg/kg) (somsak et al., 2018), dx (2.2 mg/kg) (gaillard et al., 2015) and d-p (1.71/13.7 mg/kg) (yavo et al., 2011). cq-sensitive plasmodium berghei (p. berghei) (nk65) was used. p. berghei was obtained from nigerian institute of medical research, yaba, lagos. mice used were inoculated intraperitoneally (i.p.) with 0.2 ml of infected red blood cells (rbcs) containing p. berghei (1 × 107) obtained from the donor mice. evaluation of antiplasmodial activity  evaluation of curative activity curative activity was performed as reported by ryley and peters (1970). twenty-five swiss albino mice were inoculated i.p with blood containing 1 × 107. p. berghei and grouped into 5 of 5 mice each. the first two groups, ai (negative control) and a2 (positive control) were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups a3-a5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-p-dx daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice; thin blood films were produced on slides and stained with giemsa stain and viewed with the aid of a microscope. evaluations for percentage parasitemia and inhibitions were performed using the formula below. % parasitemia = number of parasitized red blood cells (rbcs) × 100 total number of rbcs count % inhibition = (%parasitemia of negative control − %parasitemia of treated group) × 100 %parasitemia of negative control  evaluation of prophylactic activity prophylactic test was performed using an established method described by peters (1965). twenty-five swiss albino mice were assigned to 5 groups of 5 mice each. group bi served as the negative control and group b2 served as the positive control and were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups b3-b5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily for 4 days, respectively. on day 5, the mice were inoculated i.p with 0.2 ml of infected blood containing 1x107p. berghei. after, 3 days, tail blood samples were collected and percentage parasitemia and inhibitions were calculated using the formula above.  evaluation of suppressive activity suppressive test was carried out as described by knight and peters (1980). twenty-five mice were selected and inoculated i.p with blood containing 1x107 p. berghei. after 3 h, the mice were randomized into 5 groups of 5 mice each. the first two groups, ci (negative control) and c2 (positive control) were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups c3-c5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily for 4 days, respectively. on day 5, tail blood samples were collected and evaluated for percentage parasitemia and inhibitions using the formula above. evaluation of biochemical parameters blood samples were collected from the mice used for the curative test and evaluated for white blood cells (wbcs), hemoglobin (hb), packed cell volume (pcv), red blood cells (rbcs), triglyceride (tg), total cholesterol (chol), and high-density lipoprotein cholesterol (hdl-c) using an auto analyzer. georgewill & adikwu – repurposing dihydroartemisinin-piperaquine-doxycycline as … 137 statistical analysis results as mean ± s em (standard error of mean). data was analyzed using one-way analysis of variance (anova) and tukey’s test. differences between means were considered significant at p < 0.05, 0.01 and 0.001, respectively. results curative activity treatment with dx, d-p, and d-p-dx significantly decreased percentage parasitemia levels at p<0.05, p<0.01 and p<0.001, respectively when compared to the negative control (table 1). the observed parasitemia inhibitions produced by dx, d-p, and d-p-dx and cq were 64.9%, 71.1%, 93.6%, and 75.0%, respectively (table 2). mst was significantly prolonged in mice treated with dx, d-p, and d-p-dx at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 1). suppressive activity percentage parasitemia levels were significantly decreased in mice treated with dx, d-p and d-p-dx at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 2). treatment with dx, d-p and d-p-dx produced 66.5%, 75.0%, and 95.1% parasitemia inhibitions, respectively whereas cq produced 81.9% parasitemia inhibition (table 2). significant prolongation of mst in dx, d-p and d-pdx-treated mice occurred at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control (table 2). prophylactic activity the prophylactic test showed significant decreases in percentage parasitemia levels in mice treated with dx (p<0.05), d-p (p<0.01) and d-p-dx (p<0.001) when compared to the negative control (table 3). the percentage parasitemia inhibitions produced by dx, d-p and d-p-dx were 65.1%, 86.8%, and 98.9%, respectively while cq produced an inhibition of 85.7% (table 3). mst was prolonged in dx, d-p, and d-pdx-treated mice. this occurred at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 3). determination of mean survival time the mice in the control and the treated groups were observed for mortality and expressed in days. mortality represented as mean survival time (mst) was calculated using the formula bellow. 𝑀𝑆𝑇 = sum of survival time of all mice in a group (days) total number of mice in that group hematological and lipid parameters in p. berghei-infected mice, tg, chol, ldl-c and wbcs increased whereas hb, pcv, rbcs and hdl-c decreased significantly (p<0.001) when compared to the normal control (tables 4 and 5). on the other hand, tg, chol, ldl-c wbcs levels were decreased whereas hb, pcv, rbc and hdl-c levels were increased significantly in mice treated with dx, d-p, and d-p-dx at p<0.05, p<0.01 and p<0.001, respectively when compared to the negative control (tables 4 and 5). table 1. curative antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst (days) pc 35.10±3.00 0.0 9.22±0.18 cq 8.78±1.54a 75.0 27.62±2.39 a. dx 12.30±1.27b 64.9 14.15±1.66b d-p 10.11±1.11a 71.1 25.83±2.65 a d-p-dx 2.25±0.15c 93.6 36.96±3.48 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time. a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. table 2. suppressive antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst(days) pc 17.60±2.54 0.0 9.40±0.33 cq 3.19±0.01a 81.9 31.61±2.72a dx 5.90±0.08b 66.5 20.47±2.67 b d-p 4.40±0.09a 75.0 29.73±3.11 a d-p-dx 0.86±0.07c 95.1 37.12±3,29 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time. a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. 138 biology, medicine, & natural product chemistry 10 (2), 2021: 135-140 table 3. prophylactic antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst (days) pc 15.80±1.47 0.0 9.79±0.22 cq 2.58±0.06a 85.7 33.43±2.53 a dx 5.51±0.07b 65.1 22.75±3.57 b d-p 3.03±0.04a 86.8 30.91±2.89 a d-p-dx 0.17±0.08c 98.9 38.02±3.86 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. table 4. effect of dihydroartemisinin-piperaquine-doxycycline on hematologic parameters of mice infected plasmodium berghei. treatment rbc (x106) wbc (cells/l) pcv (%) hb (g/dl) nc 6.83±0.33 7.55±0.09 60.91±5.91 16.93±1.35 pc 3.21±0.43a 15.71±1.55a 26.72±3.45a 6.01±0.17a cq 5.44±0.28b 9.44±0.19b 44.21±4.33b 13.45±1.38b dx 4.37±0.27c 11.00±0.37c 31.94±3.47c 10.73±0.81c d-p 5.40±0.11b 9.57±0.16b 42.62±3.67b 13.07±1.11b d-p-dx 6.61±0.32d 7.25±0.01d 55.9±4.41d 16.72±1.37d data presented as ± sem, n= 5, nc: normal control, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time, rbcs: red blood cell, wbcs: white blood cell, pcv: packed cell volume, hb: haemoglobin. a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pc, sem: standard error mean. table 5. effect of dihydroartemisinin-piperaquine on lipid parameters of mice infected with plasmodium berghei. group tg (mg/dl) tchol (mg/dl) hdl-c (mg/dl) ldl (mg/dl) nc 80.8±7.03 110.8±11.4 50.4±4.00 44.2±3.11 pc 250.3±18.9a 273.4±18.0 a 22.2±1.41 a 201.1±18. a cq 150.1±5.87 b 181.6±22.4 b 39.7±4.63 b 111.9±11.6 b dx 200.7±3.03 c 220.0±12.0 c 30.6±4.22 c 149.3±15.0 c d-p 159.0±4.87b 170.7±13.5b 38.7±4.00b 100.2±12.5b d-p-dx 97.4±8.88d 127.7±10.6 d 47.9±5.43d 60.3±10.1d data presented as mean ± sem, n= 5, nc: normal control, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisininpiperaquine, tg: tryglyceride, tchol: total cholesterol, hdl: high density lipoproteins, ldl: low density lipoprotein. a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pc, sem: standard error mean. discussion malaria is a major health challenge in developing countries of sub-saharan africa and south east asia. the emergence of widespread resistance of plasmodium species to most antimalarial drugs, the increasing insecticide resistance by mosquitoes, and the lack of vaccines have made the fight against malaria seriously tasking (beeson et al., 2016; joseph et al., 2020). hence there is an urgent need to discover alternative drugs with novel modes of action or a combination of currently existing antimalarial drugs to overcome these challenges. the present study, assessed whether antimalarial activity of d-p can be augmented by dx in mice infected with p. berghei. this study used invivo model, because it takes into cognizance the possible prodrug effect and the involvement of the immune system in eradicating malaria infection. p. berghei has been used in antiplasmodial studies in predicting experimental treatment outcomes and hence was appropriately used for the study (satish et al., 2017). this study used suppressive test which determines the activity of a drug candidate on early infection and curative test, which evaluates the curative activity of a drug candidate on established infection (mekonnen, 2015; hiben et al., 2016). in the present study, in the curative test, d-p-dx decreased percentage parasitemia levels most when compared to individual doses of d-p, dx and cq. the observed parasitemia inhibitions in the curative test were 64.9%, 71.1%, 93.6% in dx, d-p, and d-p-dx-treated mice, respectively. also, in the suppressive and prophylactic tests, best decreases in percentage parasitemia levels occurred in d-p-dxtreated mice in comparison to individual doses of dx, d-p, and cq. in the suppressive test, 66.5%, 75.0%, and 95.1%, parasitemia inhibitions were observed in dx, dp, and d-p-dx treated mice respectively. in view of the antiplasmodial activity of d-p-dx observed in the georgewill & adikwu – repurposing dihydroartemisinin-piperaquine-doxycycline as … 139 present study, the ability of d-p-dx to prolong mst in mice was also evaluated. treatment with d-p-dx prolonged mst in the curative, prophylactic and suppressive tests. the prolongation of mst by d-p-dx was best when compared to individual doses of dx, dp, and cq. hematological abnormalities like anemia caused by erythrocyte destruction are common characteristics of p. berghei-infected mice. rodent malaria causes parasite-induced decrease in pcv, which occurs approximately 48 h post-infection (nardos and makonnen, 2017). in the present study, notable signs of anemia marked by low levels of hb, pcv rbcs and increased wbcs levels were observed in p. bergheiinfected mice. however, p. bergheiinduced anemia was curtailed in d-p-dx treated mice when compared to individual doses of dx, d-p, and cq. studies have reported that changes in serum lipids could be possible features of malaria (visser et al., 2013). the present study observed elevated tchol, tg, and ldl-c and decreased hdl-c levels in p. berghei-infected mice. however, d-p-dx restored serum lipid characterized by decreased tchol, tg, ldl-c and increased hdl-c levels. the observed antiplasmodial effect of d-p-dx may be due to different modes of antiplasmodial activity of the partner drugs. dihydroartemisinin acts through the cleavage of the endoperoxide bridge and the production of free radicals (meshnick, 1994). piperaquine is suggested to have similar mode of action as cq (meshnick, 1994). in parasite food vacuole, concentrated cq binds free hematin forming cqhematin complex. this interferes with enzymatic processes in the parasite causing parasite death (tärning et al., 2007). the antiplasmodial mode of action of dx is not clear, but studies suggested the inhibition of mitochondrial protein, nucleotides and deoxynucleotides syntheses in plasmodium (yeo et al., 1997; 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(2011). multicentric assessment of the efficacy and tolerability of dihydroartemisinin-piperaquine compared to artemether-lumefantrine in the treatment of uncomplicated plasmodium falciparum malaria in sub-saharan africa. malarial journal. 2011; 10:198. yeo ae, edstein md, shanks gd, & rieckmann kh (1997). potentiation of the antimalarial activity of atovaquone by doxycycline against plasmodium falciparum in vitro. parasitology research. 83:489–91. https://www.ncbi.nlm.nih.gov/pubmed/?term=tan%20kr%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=magill%20aj%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=parise%20me%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=arguin%20pm%5bauthor%5d&cauthor=true&cauthor_uid=21460003 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 27-32 | doi: 10.14421/biomedich.2020.91.27-32 issn 2540-9328 (online) the potential of chrysin of oroxylum indicum l. to induce carbonic anhydrase (ca) to improve cattle fertility mohamad amin1,*, muhammad najib fahmi2, muhammad andi ali ridho2, nurul fitri2, umie lestari1, dina maulina3, ihya fakhrurizal amin4 1department of biology, faculty of mathematics and sciences, universitas negeri malang, jl. semarang 5, malang, indonesia 2postgraduate program of biological education, universitas negeri malang, jl. semarang 5, malang, indonesia 3biology education, faculty of teacher training and education, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, lampung, indonesia 4faculty of medicine, university of indonesia, universitas indonesia, jl. salemba raya no.4, central jakarta city, jakarta, indonesia corresponding author* mohamad.amin.fmipa@um.ac.id manuscript received: 11 april, 2020. revision accepted: 15 april, 2020. published: 16 april, 2020. abstract the artificial insemination (ai) aims to develop the potential of the cattle reproduction comprehensively. the success of ai results is highly dependent on the level of livestock fertility. therefore, improv the quality and quantity of semen could be done by ho rmone induction. however, hormone prices are expensive. this problem could be overcome using alternative way with the natural compound containing chrysin which might has the same function. the purpose of the study was analyze the potential of chrysin compound from bungli plant (oroxylum indicum) as fertilizer of males through bioinformatics approach. methods performed through data search by webserver with the following stages: (1) pubchem, (2) swiss (3) target prediction, (4) uniprot, (5) protein data bank and (6) docking software using pyrx, pymol and discovery studio. the results showed that the chrysin compound interact with carbonic anhydrase (ca) expressed from the testes of the cattle, through van der waals and pi-alkyl bonds. chrysin is effective as ca ligand inducers based on affinity bonds (-8.4 kcal/mole) and more negatively than flavonol as a control compound with binding affinity (-6.2 kcal/mol). this suggests that chrysin is an effective compound as a potential drug for improved livestock fertility. keywords: bioinformatic; carbonic anhydrase; chrysin; fertility. bungli plant (oroxylum indicum l.). introduction indonesia has a wealth of natural resources that can be optimized to support the productivity of livestock, including herbs that it mixed in the form of traditional herbal medicine (motaleb et al., 2010; salim & munadi, 2017). herbal plants can be processed into livestock herbs and are useful for increasing the cattle fertility and can reduce the use of hormones (balittro, 2015; affandhy et al., 2017). increasing the productivity of beef cattle through artificial insemination (ai) is one of the achievement programs of beef self-sufficiency (direktorat pangan dan pertanian, 2010). the success of the artificial insemination program (ai) is highly dependent on the fertility of the cattle (foote et al., 1999). the quality and quantity improvement of semen is conducted through hormones function (walker et al, 2005; afriani, et al., 2014). one of the active compounds that play a role to increase the fertility of cattle is chrysin, a herb plant compound found in bungli plant (oroxylum indicum l.). oroxylum indicum l. (lamiales: bignoniaceae) is native to india that used as an ingredient in the manufacture of medicines (deka et al., 2013). this plant is found in primary and secondary forests or in open areas (djarwaningsih, 1992). the distribution of the plant is covering at philippines, indochina, siam, india and indonesia (java, sumatra, borneo, sulawesi, nusa tenggara and maluku). it has many benefits for health, all part of this plant contain useful compounds that can be used for traditional medicine (deb, et al., 2016). the previous research showed that this plant is not toxic when consumed by human and animal even in high doses (lawania et al., 2010). research about the bungli plant based on in vivo and in vitro tests revealed that bungli has benefit as antimicrobial (islam, 2010), antifree radical (kalaivani et al., 2009), antiulcer (khandar et al., 2006), genotoxic (tepsuwan et al., 1992), anti cancer (roy et al., 2007), antioxidants (upaganlawar et al., 2007; tenpe et al., 2009), and many other functions that have yet proven. this plant contains flavonoids and oils almost in all its parts (sankara et al., 1972). flavonoids consist of a large group of polyphenolic compounds that have benzo-γ-piron structures and are ubiquitous in plants. they are synthesized by phenylpropanoid pathways. available reports tend to show that phenolic secondary metabolites including flavonoids are responsible for https://doi.org/10.14421/biomedich.2020.91.27-32 28 biology, medicine, & natural product chemistry 9 (1), 2020: 27-32 various pharmacological activities (mahomoodally et al., 2005; pandey, 2007). chrysin and baicalein are the most commonly found flavonoid compounds in bungli plant (yan et al., 2011). the leaf part contains baicalein, 6 and 7-glucoronide, chrysin, anthraquinone and aloe-emodin. the bark contains flavones of oroxylin, chrysin, baicalein, and biochanin-a. while at the root of this plant contains chrysin, skutelarin-7-rutinsococic, weak acid, alkaloids, cytosterol, galactose, baicalein, and biochanin-a (lawania et al., 2010). chrysin, in the chemical name called 5.7 dihydroxyflavone, is a natural polyphenol compound found in bungli plant, honey and several other plants (morissette, et al., 2017). chrysin is a flavone-derived compound that has biological activity, has various pharmacological effects including anti-inflammatory, anti-cancer, and antioxidant (manoharan et al., 2012). chrysin is a compound of the flavonoid group found in honey, propolis, and plant extracts, which is a powerful antioxidant that has been widely studied because of its mechanism of action to prevent the conversion of testosterone hormones into estrogen through inhibition of the cyp19 enzyme (ommati et al., 2013; akhlaghi et al., 2014). the benefits of these compounds are commonly used by bodybuilding athletes as a testosterone enhancer, but also known as antineoplastic (neuman et al., 2002), anti-inflammatory (eid et al., 2006; rengaraj et al., 20015), anti hypertension (min et al., 2016), antiaging (mantawy et al., 2014). in addition, it can protect against the effects of oxidative and inflammatory injury (mantawy, et al., 2014) along with steroidogenic activity (jana et al., 2008) and antiaromatase (campbell et al., 1993). based on these reasoning, chrysin is an attractive candidate to evaluate its effect on reproduction. several studies using animal models reported that chrysin improved sperm parameters (jana et al., 2008; campbell et al., 1993), raising testosterone level, and preventing oxidative damage caused by exposure to toxic chemicals (dhawan et al., 2002; ciftci et al., 2012). ca (carbonic anhydrase) is a zinc metalloenzyme class that catalyzes reversible hydrogen carbon dioxide. ca has many different physiological functions and seven different isozymes have been characterized in mammals (hewett-emmett et al., 2000). ca is abundant in spermatozoa (parkkila et al., 1991) and its presence may be associated with maintenance of adequate intraspermatozoal bicarbonate concentration required to control sperm motility and acrosome reactions (tajima et al., 1987). the presence of ca in male reproductive organs was first shown about 50 years ago. mawson & fisher (1952) showed that biochemically approach the homogenate dorsolateral mouse prostate contained a large amount of ca activity. many researchers then confirm these findings (fisher et al., 1955; pincus & bialy 1963; leiter 1964; mcintosh 1969). biochemical studies have also shown that human and mice seminal vesicles contain the activity of ca (miyake & pincus, 1959; maren 1967). ca activity in rats' prostate and seminal vesicles was found to be regulated by androgens. the benefits of chrysin compounds in the bangli plants that could increase male fertility by binding to carbonic anhydrase (ca) protein had tested in silico. this study aims to investigated the chrysin compound from bungli as a male cattle fertility drug based on in silico screening. methods retrieval of sample this research used chrysin as spesific compound from oroxylum indicum l. the substance is the most abundant compound in oroxylum indicum l (manoharan et al., 2012; morissette, litim, & paolo, 2017) and it has been reported to have antioxidant activity. therefore, chrysin was selected as a specific sample for analysis. in addition, the compositions of chrysin were obtained from the compounds database of pubchem. ligand preparation the chemical structure of 3d and smiles ligands (chrysin) is taken from pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) with id number: 5281607. target selection input chrysin’s smiles on pharmmapper (http://lilab.ecust.edu.cn) to identify potential target candidates using mapping approaches swiss target predictions (http: //www.swisstargetprediction.c/) and chemical structure associations with molecular 3d. molecular docking process the molecular reverse docking was used to observe the interactions of chrysin with a target protein. it was identified using: collection of 3d natural compounds structure, predictions of target protein, receptor profiling, and clarification of the potential of chrysin compounds based on mode of action using pyrx 0.8 software. the collection of natural compounds structure from pubchem was used to identify the biological activity from small molecule. the pubchem was confirmed by three databases that are connected with ncbi’s information systems, including pubchem substance, pubchem compound, and pubchem bioassay. therefore, 3d structure of the chrysin would be obtained. predictions of target protein was identified using pharmmapper, superpred and swiss target prediction software. they were used to identify the bioactive amin et al. – the potential of chrysin of oroxylum indicum l. to induce … 29 molecule in the animal body. it was performed in order to observe the molecular mechanism that is fundamental as phenotype and their site of biological activity. the next process, receptor profiling process was obtained using two online databases: uniprot and rscb protein data bank. the 3d structures are required to further analyze the receptor proteins. the last stage was performed using the vina wizard feature which is integrated in pyrx 0.8 software. it was minimized to obtain the most suitable conformation before the interaction with the target protein. information obtained from pathway analysis was clarified to identify the action mechanism of chrysin compounds towards the target protein concerned. the natural compound and the control inhibitor compounds (inhibitor compounds or target protein activator) were used as a ligand in this step (maulina, et al., 2018). molecular interaction and visualization finally, the molecular interaction and visualization of the docking results were analyzed using pymol and ligplus+ software. in this stage, a profiling visualization of an interaction between chrysin compounds with the target protein was conducted. all of the bound chrysin and receptor that interact directly can be observed. therefore, the new chrysin receptor was analyzed for their structure and function mechanisms in the animal body metabolism specificly in the cattle (maulina, et al., 2018; amin, et al., 2018). result and discussion reverse docking is a method used to predict the interaction activity between ligand (compound/drug) with receptors (protein/target) (kharkar et al., 2014). based on the docking of ca (carbonate anhydrase) pdb id: 1jd0) with a resolution of 1.95 å, chrysin compound was known as a ligand informing that both have binding affinities (-8.3 kcal/mol). the results of molecular visualization and molecular interaction using pymol software found that chrysin binds to target the protein (ca) in the same binding site with the control compound (flavonol) via binding for van der waals, unfavorable bump and pi-alkyl arg b: 2401, pro b: 240, hoh: b, 2617, thr b: 233, tyr b: 231, hoh b: 2572 glu b: 248 ser b: 239 as shown in figure 1. so it can be suggest that chrysin has the same function as flavonol as ligand ca for male fertility. chrysin as a natural favonoid compound, previously used to increase testosterone (brown, et al., 2001; dhawan, et al., 2002). ciftci, et al., (2011) had studied the steroidogenic effects of flavonoid compounds and found that chrysin significantly increases steroidogenesis in leydig cells primarily by increasing star gene expression. the effects of chrysin compound was confirmed by ciftci, et al., (2012) reporting where the levels of testicular antioxidant enzymes such as sod, cat and gsh-px coincide with gsh increased significantly after chrysin administration. the chrysin-treated male albino mice had more sperm count, a much higher fertility rate when mated to proestrous female mice (dhawan, et al., 2002). higher sperm count and motility along with lower percentage of sperm abnormality were also noted in their study. in addition to the antioxidant effects, both in vivo and in vitro studies have confirmed the potential of chrysin to increase testosterone levels and then male sex drive (jana et al., 2008; ciftci et al., 2012). figure 1. (a) the interaction between target protein (ca) with chrysin and flavonol shows that binding to target proteins on the same site. green (chrysin), purple (flavonol), blue (ca); (b) interaction through van der waals bond, unfavorable bump and pi-alkyl arg b: 2401, pro b: 240, hoh: b, 2617, thr b: 233, tyr b: 231, hoh b: 2572 glu b: 248 ser b: 239. the result of target selection using swiss target prediction found that chrysin interacts with ca expressed on the testes for sperm motility and male fertility. this compound is an interesting object of this research for the development of male cattle fertility drug. as a natural compound in bungli plant, this compound is predicted as an effective substance for male fertility because it is directly linked to ca. in addition, bungli has active compound of flavonoids as aphrodisiac compounds that can increase sexual arousal (uddin et al., 2003). histochemical studies have demonstrated that ca activity acts and work in the testes, the efferent ducts, epididymis, ductus deferens, seminal vesicles, and prostates in some species (waldeyer & häusler, 1959; 30 biology, medicine, & natural product chemistry 9 (1), 2020: 27-32 cohen et al., 1976; goyal et al., 1980). in the testes, ca activity has been found in sertoli and leydig cells and in interstitial tissues in rats. capillary endothelial cells and some larger vessels in the testes have been shown that it contains ca activity in many species, including humans (cohen et al., 1976; ridderstråle et al., 1985; ekstedt et al., 1991). related to this explanation, mezquita et al., (1999) reported that the expression of ca conducted during spermatogenesis in rats and the main structure of testicular transcript for ca isolated from adult and human testes. mezquita, et al. (1999) also suggest that certain transcriptional and posttranscriptional mechanisms regulate the expression of ca during mammalian spermatogenesis. harris's et al., (1984) studied the increasing of total semen and seminal volume of plasma per semen collection related to increasing of the ca activity of testicular and ductus deferens. it was showed that the processes occurring in the testes and ductus deferens are closely involved with the total ejaculatory volume. inhibition of ca activity by acetazolamide showed that it was influenced for decreasing of in semen or seminal plasma volume. this result may be regarded as further evidence of the importance of a bicarbonate buffer system in seminal production. this confirms the linear relationship found in experiments between semen production and ca activity of the testes (ridderstråle et al., 1985). in addition, sperm motility, sperm concentration and serum testosterone levels increased significantly, whereas abnormal sperm levels decreased significantly after chrysin treatment (zhandi, 2017). in conclusion, it was suggested that treatment with chrysin may affect the reproductive system positively in mice, and may be used for the treatment of male infertility (ciftci at al., 2011). findings of the relationship between the volume of semen, spermatozoa or seminal plasma and ca activity in the reproductive system show that ca is involved in some reproductive functions (harris and goto, 1984). conclusion this study proves that chrysin is a potential compound as an inducer of ca based on affinity and intermolecular binding interactions. chrysin is a potential herb for increasing fertility in male cattle, interacts with ca expressed on the testes for sperm motility and male fertility. references affandhy, l., ratnawati, d., & luthfy, m. 2017. herbal combination effect on the quality of semen and libido in ongole cattle cross-breed. trad. med. j. vol. 22(2), p 84-90 issn-p: 1410-5918 issn-e: 2406-9086 afriani, t., 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number 2, october 2020 | pages: 97-103 | doi: 10.14421/biomedich.2020.92.97-103 issn 2540-9328 (online) microbial qualities of nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria osita gabriel appeh1,*, tochukwu frank egwuatu2, chiamaka maryann ogbunta1 1department of microbiology, college of natural science, michael okpara university of agriculture umudike, p.m.b. 7267, umuahia, abia state, nigeria 2department of cell biology and genetics, faculty of science, university of lagos, akoka-yaba lagos state, nigeria corresponding author* ositaappeh@yahoo.com manuscript received: 02 august, 2020. revision accepted: 06 november, 2020. published: 22 november, 2020. abstract water samples from nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria were evaluated for the presence of microbial contaminants. both samples were subjected to physicochemical analysis, total bacterial count and isolation of microbial pathogens, using conventional techniques. physicochemical characteristics of both water samples showed that nkwuaku stream had the least total dissolved solid value of 210mg/l while the highest value of 270mg/l was recorded for ogbaru stream. total heterotrophic bacteria count revealed that ogbaru stream water sample had a higher bacterial population of 5.1x107cfu/ml than nkwuaku stream water sample which had a bacterial population of 4.2x107cfu/ml. a total number of twelve (12) microorganisms were isolated from both water samples analyzed. eight (8) bacteria genera isolated include: escherichia coli, enterococcus faecalis, salmonella spp., klebsiella spp., staphylococcum aureus, pseudomonas aeruginosa, proteus spp. and campylobacter spp. four (4) fungi were isolated and they include: aspergillus niger, aspergillus flavus, aspergillus fumigatus and fusarium oxysporum. escherichia coli, salmonella spp., staphylococcus aureus and proteus spp. had the highest percentage occurrence (16.66%) each while enterococcus faecalis, klebsilla spp., pseudomonas aeruginosa and campylobacter spp. had the least percentage occurrence (8.33%) each. also, aspergillus recorded the highest percentage occurrence of 40% for fungi while the least percentage (20%) was recorded for aspergillus niger, fusarium oxysporium and aspergillus fumigatus each. this study reveals that microbial qualities of these streams render them unfit for human consumption as sources of portable water although they can be used for other household purposes. keywords: microorganisms; contaminants; physicochemical; pathogens; streams. introduction water is the most vital element among the natural resources; it is the most indispensable need for existence of all living things. its decreasing availability in terms of quality and quantity has been a major public health concern in africa, particularly in nigeria (who, 2004; saraveanan and peter, 2009). according to a recent unicef report, about 80 million people in asia and africa are living without access to safe water. consequently, this has caused many people to suffer from various diseases (tanwir et al., 2003). in developing countries such as nigeria, most of the rural community lack access to potable water supply and rely mainly on river and stream sources for their household use and other purposes (banwo, 2006). many water sources in developing countries are unhealthy because they contain harmful physical, chemical and biological agents. unfortunately, many of the available water sources are not potable without some form of treatment which is seldom or not available in most rural settings which expose the rural populace to waterborne diseases (oketola et al., 2006). in some rural areas in nigeria, domestic wastes, sewage and faeces are discharged into streams which also serve as their water sources for daily needs. when the load of organic matter or wastes is too heavy, the self-purification power of the stream are unable to remove these materials added and there will be pollution of these water sources which can be dangerous to human and the environment as a whole (adetokunbo and grilles, 2003). the microbiological quality of drinking water is of a great primary importance, and the monitoring of bacterial indicators such as total coliform. microbial indicators have been used worldwide to indicate if human wastes have contaminated water body. microbes typically utilized are those that are found in elevated concentrated in human faecal coliform, escherichia coli and enterococci (brooks et al., 2006). an additional indicator, clostridium perfringes can be used for monitoring stream water quality (egberongbe et al., 2010). the outbreaks of diarrhea or gastroenteritis in rural communities have all been attributed to the consumption of water of poor microbial https://doi.org/10.14421/biomedich.2020.92.97-103 98 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 quality (ashbolt, 2004). it is therefore not an option but an imperative to critically monitor the quality of water supply in rural areas in order to further highlight their despicable water supply situation and to provide the impetus for sustainable government intervention (gucker et al., 2006). the aim of this study was to evaluate the microbial qualities of nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria. materials and methods study area the study took place in awgu local government area of enugu state, nigeria. the local government is made up of towns such as agbogugu, isu-awa, ituku, ihe, ogbaku, owelli, ogugu, agbudu, amoli, mmaku, ugbo, obeagu, mgbidi, ugwueme, nkwe, ezere, awugu, nenwenta, awgunta and mgbowo. it has clay and stony lands with hilly topography. subsistence farming, petty trading, livestock rearing, palm wine tapping and stone quarrying are the major occupation of the people. there is no notable industry located in this area and its environs. collection of samples water samples for this study were collected from two different streams (nkwuaku and ogbaru) within the study area. the samples were collected in sterile containers at different collection points and analyzed within 24 hours for physicochemical and bacteriological qualities. sterilization of materials all glassware which were used for this study were properly washed and rinsed using distilled water and sterilized in a hot air oven at 250oc for 1 hour. these glassware included glass petri-dishes, test tubes, pipette, bijou bottles and mccartney bottles. determination of physicochemical characteristics of the sample the physicochemical properties which were examined included temperature, ph, taste, turbidity, colour, conductivity, odour, total solids, total dissolved solids, total suspended solids, acidity and alkalinity. determination of ph, temperature and conductivity the ph was determined using a ph meter (hi96107 hanna ph meter), which was standardized with a neutral buffer solution of ph 7.0 in a beaker. a 100ml aliquot of each sample was measured into a beaker and the ph probe was immersed into the sample. this was allowed for some minutes until a stable reading was obtained, and value was recorded. an aliquot of 50ml of each sample was measured into a 100ml beaker and a simple mercury-in-glass thermometer calibrated in degrees centigrade immersed in the water. the reading on the thermometer for each sample was recorded. conductivity of the two samples was determined using a digital conductivity meter (4520jenway, serial no. 01263). the meter was turned on and allowed to warm up for about 15 minutes. it was then standardized with 0.01m kcl solution where a conductivity value of 1413 microsiemen per-centimetre was obtained. the electrode was thoroughly rinsed with distilled water, wiped and then dipped into 100ml of each water sample in the beaker and left for some minutes to obtain a stable reading. the value for each sample was recorded. determination of colour and turbidity colour was determined immediately each sample arrived the laboratory. this was carried out after the two samples had been allowed to rest on a bench to attain room temperature. approximately 25 ml of water from each of the samples was measured into the sample cell for colour analysis. colour instrument (hach dr/890) was zeroed with 25ml distilled water before samples were determined and the readings were recorded. approximately 10ml of distilled water was measured into the sample cell of a turbidity meter (hanna, lp2000), which was used to zero the instrument. later, 10ml each from samples was measured into the same cell and the readings were recorded. determination of total solids (ts) and total dissolved solids by gravimetric method total solids were determined as the residues left after evaporation of the unfiltered samples. approximately 10ml of each of the two unfiltered samples was taken and evaporated in an evaporating dish in an oven at a temperature of 103-105oc for two and half hours. the evaporating dish was then cooled in desiccators and weighed. total dissolved solids were determined as the residues left after evaporation of the filtered samples, approximately 10ml of filtered sample from each of the samples was measured into a pre-weighed evaporating dish. the residues collected were then dried in an oven at temperature of 103-105oc for one hour and final weights were taken after cooling the evaporating dish in a desiccator. total solids were calculated using the formula given below: total solids (mg l⁄ ) = (𝑊1 − 𝑊2) x 1000 𝑉 where: 𝑊1 : initial weight of dried residue + dish in mg 𝑊2 : final weight of the dish in mg 𝑉 : volume of sample used in ml determination of total suspended solids (tss) for total suspended solid (tss), 10ml each of the water samples were filtered through a pre-weighed filtered appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 99 paper. the filtered papers were dried at 103-105oc in oven and tss was determined by the following formula. tss (mg l⁄ ) = filter post weight – filter pre weight x 1000 volume of sample (ml) determination of alkalinity and acidity to 100ml of each of the water samples, 3 drops of phenolphthalein indicator was added. the samples were titrated with 0.02n h2s04 to ph 8.3 and phenolphthalein alkalinity was estimated (phenolphthalein indicator was changed colour from pink to colourless at ph 8.3). finally, the phenolphthalein alkalinity of water was calculated as follows: alkalinity (mg l⁄ ) = 𝐴 x 𝑁 x 50 x 1000 𝑉 where: 𝐴 : volume of h2s04 in ml 𝑁 : normality of h2s04 used to titrate 𝑉 : volume of sample used in ml determination of acidity to 100ml of each of the water samples, 3 drops of methyl orange indicator was added. the samples were titrated with 0.02n standard naoh to ph 8.3. (methyl orange indicator was changed colour from orange-red to yellow at ph 8.3). the acidity of water was calculated as follows: acidity (mg l⁄ ) = 𝐴 x 𝑁 x 50 x 1000 𝑉 where: 𝐴 : volume of naoh titrant used in ml 𝑁 : normality of naoh used 𝑉 : volume of sample used in ml microbiological analysis preparation and inoculation of samples serial dilution of each sample was made in sterile distilled water by inoculating 1ml of each sample into the first test tube containing 9ml of distilled water. the solution was mixed thoroughly and 1ml of it was transferred into the second test tube containing 9ml of distilled water. the procedure was repeated with all the remaining tubes. 0.1ml was pipetted out from 105 of the diluted factor and was inoculated in the plate using spread plate technique. the plates were incubated for 24 hours at 25oc for bacterial isolation and at 20oc for 3 days for fungal isolate using potato dextrose agar. the number of colonies were counted and recorded for both bacteria and fungi. purification of isolates the resulting colonies from the nutrient agar medium, macconkey, potato dextrose agar, eosin methyl blue agar, manitol salt agar, blood agar and salmonella – shigella agar plates were purified by sub-culturing on freshly prepared nutrient agar plates. the plates were incubated at 25oc for 24 hours for bacterial incubation and 20oc for 3 days for fungi. after the appropriate periods of incubation, the resulting discrete colonies were transferred onto agar slants in mccartney bottles and kept in the refrigerator as stock culture for subsequent tests during identification. characterization and identification of bacterial isolates bacterial isolates were analyzed based on morphological features, gram staining and other biochemical characterization which included citrate, indole, catalase, coagulase, motility, oxidase, urease, glucose, lactose, fructose and mannitol tests. confirmation of biochemically characterized bacteria was made using the most probable number (mpn) technique. identification of fungal isolates fungal isolates were identified based on their colonial morphology and cell morphology. a small portion of fungal growth was isolated with a sterile wireloop and placed on a grease free glass slide and teased with a drop of distilled water. a drop of lactophenol cotton blue stain was added and covered with a greased free cover slip. the slide was observed using x10 and x40 objective lenses. lactophenol cotton blue wet mount preparation is the most widely used method of staining and observing fungi. results table 1 shows the various physicochemical characteristics exhibited by the stream water samples. ogbaru stream had higher colour value of 10hu than nkwuaku stream which had a colour value of 6hu. nkwuaku stream sample had no objectionable odour while ogbaru stream sample had an objectionable odour. nkwuaku stream had a lower ph value of 6.7 while ogbaru had a ph value of 8.3. turbidity was higher in ogbaru stream and lower in nkwuaku stream sample. ogbaru stream had a higher temperature of 27oc than nkwuaku (26oc). conductivity was higher in ogbaru stream sample (678 𝜇s/cm) than in nkwuaku stream sample (468 𝜇s/cm). the value for total solids was lower (430 mg/l) in nkwuaku stream and higher (530 mg/l) in ogbaru sample. nkwuaku sample recorded a lower total dissolved solids (210 mg/l) than ogbaru (270 mg/l). total suspended solids was 220 mg/l in nkwuaku stream and 260 mg/l in ogbaru stream sample. ogbaru stream sample’s acidity was 4.0 mg/l and that of ogbaru stream was 6.0 mg/l. alkalinity was recorded to be 5.0 mg/l in ogbaru sample and 6.0 mg/l in nkwuaku sample. 100 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 table 1. physicochemical characteristics of the stream water samples in awugu local government area, enugu state. parameters sample a nkwuaku stream sample b ogbaru stream ph 6.7 8.3 temperature (oc) 26 27 conductivity (𝜇s/cm) 468 678 colour (hu) 6.0 10.0 turbidity (ntu) 8.0 10.0 odour u o total solids(mg/l) 430 530 total dissolved solids(mg/l) 210 270 total suspended solids(mg/l) 220 260 alkalinity(mg/l) 6.0 5.0 acidity(mg/l) 4.0 6.0 key: u = unobjectionable; o = objectionable; hu = hazen unit; ntu = naphelometric turbidity unit. table 2 shows the colonial characteristics of each isolates on nutrient agar, eosin methylene blue agar, macconkey agar, manitol salt agar, salmonella-shigella agar and blood agar. the isolates which were obtained include staphylococcus aureus, enterococcus faecalis, escherichia coli, salmonella species, klebsiella species, pseudomonas aeruginosa, proteus species and campylobacter species. table 3 shows the total bacterial and fungal plate counts of the stream samples. the total bacteria count revealed that ogbaru stream had a higher total bacteria count of 5.1 x 107 cfu/ml than nkwuaku stream which had 1.2 x 107cfu/ml. this indicates that nkwuaku stream sample had the lower bacterial indicators compared with ogbaru stream sample. also, the total fungal count revealed that nkwuaku had a higher total fungi count (4.0 x 107 cfu/ml) than ogbaru stream (1.6 x 107 cfu/ml). table 2. colonial characteristics of isolates from ogbaru and nkwuaku stream water on different growth media. isolate code media plates na emb mac msa ssa ba og1 smooth, golden yellow, convex colonies pink, smooth, convex and opaque colonies yellow, smooth, convex and opaque colonies light golden yellow, smooth, convex and hemolytic colonies og2 thick, grayish white, moist, smooth & opaque colonies green metallic sheen smooth colonies moist, smooth, flat & pink colonies slight growth pink colonies gray, moist & hemolytic colonies og3 low convex, smooth, grayish white translucent colonies low convex, smooth, colourless and transparent colonies black colonies with offensive odour low convex, smooth, grayish white, translucent and non-hemolytic colonies og4 irregular, low convex, smooth, mucoid, greenish yellow opaque colonies metallic sheen and glass appearance colonies low convex, smooth, mucoid, colourless transparent colonies irregular slight growth & nearly colorless colonies flat, smooth, mucoid, grayish white, opaque, hemolytic colonies nk1 whitish, round raised, glistering colonies light pink raised large colonies yellow colonies white nonhemolytic colonies nk2 mucoid, grayish white, raised opaque colonies convex, mucoid, pink translucent colonies convex, mucoid, pink-red opaque colonies smooth slight growth, pink colonies mucoid, grayish white, opaque non hemolytic colonies nk3 smooth, opaque, irregular, glistening grayish white translucent colonies glistening, colourles, transparent colonies low convex, smooth, colourless transparent colonies colorless with black center colonies irregular, glistening, grayish white, opaque, non-hemolytic colonies nk4 whitish droplet colonies smooth, grayish, convex, glistening & non-hemolytic colonies = no growth; na = nutrient agar; emb = eosin methylene blue agar; mac = macconkey agar; msa = manitol salt agar; ssa = salmonella – shigella agar; ba = blood agar; og = ogbaru; nk = nkwuaku. appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 101 table 3. total bacterial and fungal plate counts obtained from the stream water samples. sample source bacteria (cfu/ml) fungi (cfu/ml) na emb mac ssa pda nkwuaku 4.2 x107 2.8 x107 3.9 x 107 2.2 x 107 4.0 x 107 ogbaru 5.1 x107 1.5 x 107 3.5 x 107 1.9 x 107 1.6 x 107 pda = potato dextrose agar; na = nutrient agar; emb = eosin methylene blue agar; mac = macconkey agar; ssa = salmonella – shigella agar table 4 shows various groups of microorganisms isolated and identified from this study. they include; staphylococcus aureus, escherichia coli, enterococcus faecalis, klebsiella pneumonia, proteus spp, pseudomonas aeruginosa, salmonella spp. and campylobacter spp. table 4. morphological and biochemical characteristics of bacteria present in stream water samples in awgu local government area, enugu. is o la te c o d e g r a m s ta in s h a p e c it r a te u ti li z a ti o n t e st in d o le t e st c a ta la se t e st c o a g u la se t e st m o ti li ty t e st o x id a se t e st u r e a se t e st m e th y l r e d t e st g lu c o se f e r m e n ta ti o n f r u c to se f e r m e n ta ti o n l a c to se f e r m e n ta ti o n p r o b a b ly o r g a n is m s og1 + cocci + + + + ag na ag staphylococcus aureus og2 rod + + ag ag ag escherichia coli og3 rod + + ag na na salmonella spp og4 _ rod + + ag ag ag pseudomonas aeruginosa nk1 + cocci + ag ag ag enterococcus faecalis nk2 rod + + + nag ag ag klebsiella spp. nk3 _ rod + + ag ag na proteus spp nk4 spiral + + + + na na na campylobacter spp. + = positive; = negative; ag = acid and gas production; na = no acid production; nag = no acid and gas production; og = ogbaru; nk = nkwuaku. table 5. morphological characteristics of fungal isolates in the stream water samples. macroscopy microscopy organism(s) dark-brown mycelium conidiophores smooth walked and non-septate aspergillus niger yellow pink creamy colonies cylindrical to ovoid conidia, curved septate conidiophores fusarium oxysporum light green and powdery long, erect septate, conidiophores aspergillus flavus gray-green fluggy colonies long, erect, non-septate conidiophores aspergillus fumigatus table 5 shows various fungi isolates and identified from the stream samples by their morphological characteristics. they are; aspergillus niger, fusarium oxysporum, aspergillus flavus and aspergillus fumigatus. table 6 shows the result of the mpn tests for occurrence of the presumptive coliforms (e. coli). the result revealed a gross coliform contamination in the two stream samples. table 6. mpn values per 100ml of the stream water samples. no. of tubes giving a positive result water samples 5 of 10ml 5 of 1ml 5 of 0.1ml mpn (per 100ml) nkwuaku stream 0 1 0 2 1 1 0 4 1 0 1 4 1 1 1 6 1 0 0 2 ogbaru stream 2 0 0 5 2 0 1 7 3 1 0 11 1 1 1 6 0 2 0 4 mpn (per 100ml) = most probable number per 100ml of water sample. 102 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 table 7 shows the percentage occurrence of bacterial isolates from the stream water samples which includes; escherichia coli, 2(16.66%); enterococcus faecalis 1(8.33%); salmonella spp. 2(16.66%); klebsiella spp. 1(8.33%); staphylococcus aureus 2(16.66%); pseudomonas aeruginosa 1(8.33%); proteus spp. 2(16.66%) and campylobacter spp. 1(8.33%). table 7. percentage occurrence of bacterial isolates from stream water samples. s/n isolates frequency occurrence % occurrence 1. escherichia coli 2 16.66% 2. enterococcus faecalis 1 8.33% 3. salmonella spp. 2 16.66% 4. klebsiella spp. 1 8.33% 5. staphylococcus aureus 2 16.66% 6. pseudomonas aeruginosa 1 8.33% 7. proteus spp. 2 16.66% 8. campylobacter spp. 1 8.33% 12 100% table 8 shows the percentage occurrence of fungi isolates from stream water samples. they are; aspergillus niger 1(20%); aspergillus flavus 2(40%); fusarium oxysporum 1(20%) and aspergillus fumigatus 1(20%). table 8. percentage occurrence of fungal isolates from stream water samples. s/n isolates frequency occurrence % occurrence 1. aspergillus niger 1 20.0% 2. aspergillus flavus 2 40.0% 3. fusarium oxysporum 1 20.0% 4. aspergillus fumigatus 1 20.0% 5 100% discussion evaluation of microbial contamination of nkwaku and ogbaru streams showed that the ph of nkwaku stream sample was 6.7 while that of ogbaru sample was 8.3. this is in agreement with ph assigned by the us environmental protection agency (epa) as the standard ph of water which ranges from 6.5 – 8.5 (epa, 2002). the higher ph values measured from the stream samples could be attributed to more rain water deposition which dilutes the acidity of water by raising the ph. thus indicated that the measured ph values of the stream samples were within permissible value which will not cause any harmful effect to the consumers. ogbaru stream sample had a higher temperature value (27oc) than nkwaku sample (26oc) and this could be attributed to the fact the samples were collected early in the morning. this conforms with the findings of okoye and umo (2006) who stated that cool water are generally more potable for drinking, because high water temperature enhances the growth of microorganisms and causes taste, odour, colour and corrosion problems. in the case of turbidity, ogbaru sample recorded higher value of 10 ntu while nkwuaku recorded a lower value of 8 ntu. the obtained values are far greater than the value specified and approved (5ntu) by the world health organization (who) (who, 2004). the higher values obtained could have been caused by rainfall and human activities such as; grazing of ruminants and improper management of solid and liquid wastes within the streams. high turbidity is also associated with high levels of disease causing microorganisms such as bacteria, fungi and other parasites. thus, waters from these streams are not good for drinking purpose. the total dissolved solid (tds) of the samples are in agreement with the epa standard of 500mg/l. ogbaru recorded higher value of 260mg/l while a lower value of 210mg/l was recorded in nkwuaku sample. odour of nkwaku sample was perceived to be unobjectionable whereas the odour of ogbaru sample was objectionable. the objectionable odour of ogbaru sample could be due to the presence of aquatic lives (fungi, bacteria etc) and waste disposal from toilets, industries, household etc. and this makes the water unfit for drinking. the percentage occurrence of bacterial and fungal isolates from the stream samples were determined and the result revealed that escherichia coli, salmonella spp., staphylococcus aureus and proteus spp. recorded the highest percentage occurrence (16.66%) while enterococcus faecalis, klebsiella spp., pseudomonas aeruginosa and campylobacter spp. recorded lower percentage occurrence (8.33%). the high number of salmonella, proteus spp and staphylococcus aureus in the stream samples are not in agreement with epa water standard for recreational use which states that these pathogenic organisms must not be present in water, because they are of public health significance, having been associated with gastrointestinal infections, diarrhoea, typhoid fever and other form of infection (epa, 2005). the total bacteria and fungi counts for the two samples were generally high, exceeding the limit of 1.0 x 102cfu/ml which is the standard limit of heterotrophic count for drinking water (epa, 2002). this indicates the presence of high organic matters and nutrient sources which supported the growth of the microbes. the primary sources of microbial contamination include the surface runoff, sewage treatment facilities and improper management activities of the inhabitants like washing, refuse dumping, open defaecatio and dropping of different materials into water bodies. various groups of microorganisms were isolated and identified during the study. they include escherichia appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 103 coli, staphylococcus aureus, enterococcus faecalis, klebsiella pnueumonia, proteus sp, pseudomonas aeruginosa, salmonella spp. and campylobacter spp. for bacteria. the fungi isolated include aspergillus niger, fusarium oxysporum, aspergillus flavus and aspergillus fumigatus. the presence of some of these organisms signifies contamination of water from domestic sources. the staphylococcus species are known to produce enterotoxins (okonko et al., 2008). proteus species are intestinal flora, but also widely distributed in soils and water (sclegel, 2002). pseudomonas aeruginosa is an example of non-faecal coliforms, while e. coli is a faecal coliform. conclusion from this study it can be observed that from the microbial qualities of these that they are unfit for human consumption though they can be used for other purposes. many of the inhabitants of awgu local government area depend on these two streams for drinking water supplied. in view of this, it is recommended that the government should ban and discourage all indiscriminate disposal of wastes within stream environments. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references adetokunbo, d. and gilles, f. 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(2003). water contamination, health hazards and public awareness: a case of the urban puijab, pakistan. international journal of agriculture and biology. 5: 460-462. who (2004). water sanitation and health programme. managing water in the home: accelerated health gains from improved water sources. flourosis and sanitation 11:7-11. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 27-32 | doi: 10.14421/biomedich.2021.101.27-32 issn 2540-9328 (online) extraction, phenolic content and hydrogen peroxide scavenging capacity of extracts from some honey samples, propolis and bee pollen zohra mohammedi department of biology, faculty of natural sciences and life, university of mustapha stambouli, bp 305 street of mamounia, mascara 29000, tel: 0551724433, algeria. corresponding author mdi3zhr@gmail.com manuscript received: 01 october, 2020. revision accepted: 24 june, 2021. published: 16 july, 2021. abstract honey and propolis is natural food, produced by honey bees (apis mellifera) and largely used by the local population for its medicinal properties. our work aims to extract and evaluate the hydrogen peroxide scavenging capacity of different phenolic extracts from some bee products. phenolic compounds from honey samples, propolis, and bee pollen were extracted by methanol and subjected to radical scavenging activity towards hydrogen peroxide. the results showed the highest values for the total phenolic and total flavonoïd contents in propolis and bee pollen, and a great hydrogen peroxide (h2o2) inhibition (ic50: 0.205 2.210 µg/ml) with honey extracts, while sample “multiflower” is the better antioxidant, more than ascorbic acid used as control. the less scavenging activity was observed with the extract from bee pollen (ic50: 39.383 µg/ml). to combat the harmful effects of free radicals, especially reactive oxygen species including hydrogen peroxide, it is important to use phenolic extracts instead of using honey as it is, and extracts from the honey o f different types are excellent antioxidants compared to other bee products. keywords: apis mellifera; honey; propolis; phenolic compounds; hydrogen peroxide. introduction honey is the nectar collected and processed from different plants by honey bees (apis mellifera) and is known for its high nutritional value (an et al., 2016). it has been used in many cultures for its medicinal properties, as a remedy for burns, cataracts, ulcers and wound healing, because it exerts a soothing effect when initially applied to open wounds. according to its origin, honey can be classified in different categories: blossom honey, honeydew honey, monofloral honey, and multifloral honey that has several botanical sources (alvarez-suarez et al., 2014). honey is composed of approximately 82.4% total carbohydrates (almasaudi et al., 2017). the polyphenols constitute minor components of honey (istasse et al., 2016). honey has been reported to promote numerous nutritional and biological effects, such as antioxidant, antimicrobial, antiviral, antiparasitic, anticancer, antiinflammatory, and immunosuppressive activities (duddukuri et al., 1997; othman, 2012; shahzad and cohrs, 2012; borsato et al., 2014; samarghandian et al., 2017; sowa et al., 2017; sinha et al., 2018). however, the antioxidant properties of honey are well known because it contains a number of compounds with antioxidant properties such as polyphenols, amino acids, ascorbic acid, and some enzymes. the most important classes of antioxidants are the phenolic acids and flavonoids (ayoub et al., 2009). they have a promising effect on the treatment of some chronic diseases (isla et al., 2013). a wide range of phenolic constituents in honey are known such as gallic acid, syringic acid, vanillic acid, benzoic acid, chlorogenic acid, ferulic acid, protocatechuic acid, 3-o-caffeoylquinic acid, phydroxybenzoic acid, 5-o-caffeoylquinic acid, caffeic acid, p-coumaric acid, ellagic acid, dihydroxybenzoic acid methyl ether, benzyl caffeate, protocatechualdehyde, gentisic acid, vanillin, sinapic acid, salicylic acid, dihydrocinnamic acid, 2,2,4trihydroxybenzoic acid, hesperetin, naringin, naringenin, hesperidin, tricetin, myricetin, quercetin, quercitrin, quercetin 3-orhamnoside, kaempferol 3-orhamnoside, kaempferol 7-orhamnoside, luteolin, pinobanksin 5methyl ether, naringenin, apigenin, kaempferol, methoxy-kaempferol, pinobanksin, isorhamnetin, chrysoeriol, rhamnetin, chrysin, sakuranetin, pinocembrin, galangin, kaempferide, acacetin, morin, tangeretin, catechin, genistein, ectochrysin, and other compounds such as caffeic acid phenethyl ester, kojic acid, 5-hydroxymethylfurfural, dehydrovomifoliol, leptosin, glyoxal, methylglyoxal, 3-deoxyglucosulose (an et al., 2016; alvarez-suarez et al., 2014; isla et al., https://doi.org/10.14421/biomedich.2021.101.27-32 28 biology, medicine, & natural product chemistry 10 (1), 2021: 27-32 2013; lachman et al., 2010; cianciosi et al., 2018; cheung et al., 2019; nešovíć et al., 2020). another bee product is propolis, which is a resinous substance collected by honeybees from various plant sources. this natural product has been used for thousands of years in folk medicine for several purposes and contains amino acids, terpenes, tannins, polysaccharides, phenolic acids, phenolic acid esters, cinnamic acid, caffeic acid, phenethyl caffeate, pcoumaric acid and flavonoids such as kaempferol, naringin, chrysin, and galangin. propolis possesses several biological activities such as anti-inflammatory, immunostimulatory, antiviral, and antibacterial. propolis extract and its active components showed strong antioxidant activities, a free radical scavenging effect, a significant inhibition of xanthine oxidase activity, ferric reducing antioxidant power, and an anti-lipoperoxidative capacity (russo et al., 2002; kumazawa et al., 2004; korayem et al., 2012; socha et al., 2015). a large number of studies have estimated the antioxidant activities of honey by a range of methods (dpph free radical scavenging activity, abts radical cation decolorization assay, frap assay, orac assay, etc.), but the step of dilution is a major drawback in antioxidant tests since it generates a toxic oxidant substance (h2o2), the cause of the exclusion by researchers of the antioxidant test by hydrogen peroxide scavenging activity in entire honey. so, the aim of this study was to extract phenolic and flavonoids compounds, then evaluate the antioxidant activity of obtained extracts from different types of honey against hydrogen peroxide, and to compare with two phenolic extracts from propolis and bee pollen. also, we highlight the ability of the phenolic compounds in honey to eliminate hydrogen peroxide, and therefore the step of extraction of polyphenols is important in the context of the use of honey in the fight against oxidative stress. materials and methods bee products honey samples (multiflower and monofloral honey: eucalyptus, thyme, sidr, citrus, and epins honey), propolis and bee pollen are the local products of tlemcen, algeria. the 100% natural honey samples from apis mellifera were obtained directly from the beekeeper in 2019 from different vegetation sources in tlemcen city. these honey samples were chosen for their quality, in terms of low content of water and hydroxymethylfurfural (hmf). extraction of phenolic compounds from honey samples the extraction of phenolic compounds from the honey samples was performed according to the method described by an et al. (2016). the stationary phase of the glass column, amberlite xad-2 resin, was prepared by soaking 100g of amberlite xad-2 resin in methanol. the packed column was washed successively with acidified distilled water followed by neutral distilled water. the honey sample was diluted with acidified water and loaded into the packed glass column. sugars were eliminated with hydrochloric acid solution and water washes. phenolics compounds were desorbed with methanol and then dried at 50°c using a rotavapor. the dried product was protected from light and stored at 5°c until use. extraction of phenolic compounds from propolis and bee pollen for extract phenolic compounds from propolis and bee pollen samples, the hydroalcoholic solutions (methanolwater) were used. for each 5g sample, 100ml of methanol 80% were added, the mixture was covered with aluminum paper and then filtered after 24h. the filtrates were dried at 50°c using a rotavapor. the dried extracts were stored at 5°c until use. total phenolic contents the total phenolic contents of honey samples, propolis, and bee pollen extracts were analyzed by the folinciocalteu method (singleton et al., 1999) using gallic acid as a standard. the methanolic solution of each sample of honey, propolis, bee pollen, and gallic acid was prepared. the test samples and standard solutions of different concentrations were introduced into separate test tubes. folin-ciocalteu reagent, diluted in water (1:1), and na2co3 saturated solution were added. the tubes were vortexed, incubated in the dark at room temperature for 25 minutes and then the absorbance was read at 725nm against the blank using a spectrophotometer. the total phenolic content was determined by comparison with a calibration curve of gallic acid and represented as mg gallic acid equivalents per 100 gram (mggae/100g). total flavonoid content the total flavonoïd content was determined by the alcl3 method (cottica et al., 2011) using rutin as standard. 250µl of alcl3 5% (m/v in methanol) were added to separate test tubes containing the methanolic solution of each honey sample, propolis, bee pollen extracts, and rutin solutions of different concentrations. after 30 min incubation at room temperature, the absorbance was read at 425nm using a spectrophotometer. the total flavonoid content was determined by comparison with the calibration curve of the rutin and represented as mg rutin equivalents per 100gram (mgre/100g). antioxidant activity (hydrogen peroxide scavenging activity) the hydrogen peroxide scavenging activity was measured by the method described by ruch et al. (1989). a volume of 0.6 ml of the solution of hydrogen mohammedi – h2o2 scavenging activity of bee products 29 peroxide (0.089 mm), prepared in phosphate buffer (0.1m, ph 7.4) was added in a series of test tubes, containing 3.4ml phenolic extract solutions (310µg/ml) from honey samples, propolis, bee pollen and standard (ascorbic acid). the percentage of hydrogen peroxide scavenging activity was calculated by the formula: % scavenged [h2o2] = [(ac – at)/ac] x 100 where ac is the absorbance of the control and at is the absorbance in the presence of the phenolic extracts from different samples (tests) and standard. statistical analysis the results of experiments were expressed as (mean ± sd; n=3), and subjected to statistical analysis at 95% confidence level (p0.05) by student's t-test using graphpad prism software. results and discussion the results of phenolic content and flavonoid content were reported in table 1. total phenolic content in six honey samples was ranged from 4.36 mggae/100g to 26.29 mggae/100g honey, with the average value of 12.755 mggae per 100g honey. the multiflower type of honey was the sample containing a high amount of phenolic compounds and the monofloral type "citrus" honey was the sample with the lowest quantity of phenolic compounds compared to all honey samples. the same observation was showed in flavonoid content. the multiflower type remains the richest honey in phenolic compounds compared to other honey. while the lowest content is also observed in the "citrus" type. the flavonoids content obtained was from 1.95 ± 0.352 mgre/100g to 10.65 ± 1.020 mgre/100g, with an average amount of 4.607 mgre per 100g honey. the evaluated results were in close agreement with the results reported by meinen et al. (2014), which have reported that total phenolic content is ranged between 8.114 mg and 109 mg per 100g honey and for the total flavonoid content is ranged between 0.655 mg and 212 mg per 100g honey. this study was perfected in 30 honey samples. boulanouar et al. (2017) have observed higher levels of phenolic compounds in two types of algerian honey, which are 38 ± 0.009 mggae/100g for the type “zizyphus” and 86±0.008 mggae/100g for the type “harmala”, whereas for the flavonoids, they found contents, which belong to the range of values that we recorded, the values of flavonoids were 5 ± 0.003 mgre/100g and 8 ± 0.002 mgre/100g for “zizyphus” and “harmala” types, respectively. also, the same remark is observed with the works of khalil et al. (2012). these authors analyzed four samples of algerian honey and registered mean values of 45.983 ± 0.192 mg/100g for phenolic compounds and values of 5.423 ±0.062 mg/100g for flavonoids. table 1. total phenol content and flavonoids of honey samples, propolis and bee pollen. sample average amount of phenolic average amount of flavonoids (mggae/100g) mgre/100g multiflower honey 26.29 ± 9.764a 10.65 ± 1.020a thyme honey 8.67 ± 2.494b 2.61 ± 0.558b citrus honey 4.36 ± 2.441c 1.95 ± 0.352b eucalyptus honey 8.97 ± 1.033b 2.21 ± 1.068b sidr honey 14.20 ± 3.278d 5.31 ± 0.112c epins honey 14.04 ± 4.260d 4.91 ± 0.232c propolis 9161.0 ± 6.906e 2189.62 ± 1.975d bee pollen 4794.15 ± 6.169f 1267.38 ± 0.912e data are (mean ± sd, n = 3). means within a column sharing the same letter are not significantly different by student’s t-test (p < 0.05). total phenol and flavonoids were also compared to the values reported for the honey from different countries. some authors have found results, approaching our range of values, such as the work of reshma et al. (2016), almasaudi et al. (2017) and krpan et al. (2009). the first authors have shown that the phenolic content in 24 samples of honey was ranged between 20.2 ±1.2 mg/100g to 30.78 ± 2.5 mg/100g, the second authors have observed that the high total phenolic content was 10.399 ± 0.168 mg/100g in “manuka” type of honey and the lowest phenolic content was 9.6 ± 0.002 mg/100g for “sidr” type of honey, while the third authors have ranged the total phenolic content from 3.172 mg/100g to 8.011 mg/100g honey and this later study was performed only in one type of honey, which is the "acacia" type. in contrast, other authors have deferred high and even very high levels, for examples, for chua et al. (2013) the total phenolic content of honey samples was in the narrow range from 110.394 to 196.500 mggae/100g, arahaman et al. (2013) showed that the total phenolic contents for honey samples were ranged from 38.379 to 60.617 mg gae/100g, pontis et al. (2014) ranged the total phenolic content from 25 to 54.8 mg gallic acid per 100g of honey, while for buba et al. (2013), the mean values of total phenolic content are around 65.31 ± 19.50 mg gallic acid equivalent per 100g. in addition, some authors have reported very small quantities of phenolic content in honey, for example, ferreres et al. (1994) reported 0.50 to 2.0 mg per 100g honey. for flavonoids, pontis et al. (2014) have shown less content than obtained in our studies, the total flavonoid content was ranged from 0.9 to 4.86 mg per 100g honey. the results obtained experimentally in table 1 show very high levels of phenolic compounds and flavonoïds in propolis and bee pollen compared to those obtained in honey samples and the amount of phenolic and 30 biology, medicine, & natural product chemistry 10 (1), 2021: 27-32 flavonoids in propolis was higher than bee pollen. the quantities registered in propolis and bee pollen were 9161.0 ± 6.906 mggae/100g, 4794.15 ± 6.169 mggae/100g for total phenolic content and 2189.62 ± 1.975 mgre/100g, 1267.38 ± 0.912 mgre/100g for total flavonoids content, respectively. boulanouar et al. (2017) have also observed that propolis contains more phenolic and flavonoïds than honey, the respective quantities registered were 2385±2.90 mggae/100g and 379±0.54 mg re/100g. čeksterytė et al. (2016) have also observed high levels of phenolic compounds in bee pollen, but are less relative to the quantities we have obtained. the values of total phenolic content found were 2330 mg gae/100g. the antioxidant capacity of honey and bee products is due mainly to the phenolic compounds. the samples exhibited various antioxidant activity measured towards hydrogen peroxide, which is concentration-dependent (figure 1). results show that the scavenging activity values on hydrogen peroxide of phenolic extracts from honey samples were more efficient than other bee products. lower antioxidant activity was observed in extracts from propolis and bee pollen. figure 1. hydrogen peroxide (h2o2) scavenging activity of phenolic extract from honeys, propolis, bee pollen, and ascorbic acid. the antioxidant activity of honey has been reported in many scientific works (krpan et al., 2009; pontis et al., 2014; reshma et al., 2016; chua et al., 2013; arahaman et al., 2013). currently, some very limited research on antioxidant activity has been conducted on phenolic extracts of honey (lianda et al., 2012; bridi et al., 2017; estevinho et al., 2008; halagarda et al., 2020), but no research has assessed the capacity of extracts or phenolic fractions isolated from honey by the hydrogen peroxide scavenging method. hydrogen peroxide is not very reactive, but it can be toxic because it may produce hydroxyl radicals in the cells. thus, removing h2o2 is very important in the cell. the scavenging ability of honey samples, propolis, and bee pollen was examined by comparing to that of the known antioxidants ascorbic acid. the extract of honey type "multiflower" showed powerful activity on hydrogen peroxide and decreases its concentration more than the positive control, ascorbic acid. antioxidant activity, expressed as ic50 (table 2) was 0.206 ± 0.0243 µg/ml, while the standard given an ic50 value of 0.694 ± 0.0238 µg/ml. this parameter ic50 defined the concentration of the sample, which scavenge 50% of hydrogen peroxide. the sample with lower ic50 presents potent antioxidants. the phenolic extracts from extracts of others honey types exhibited also a good antioxidant activity on h2o2. the scavenging ability is in the following order: “multiflower” ˃ ascorbic acid ˃ “citrus” ˃ “epins” ˃ “sidr” ˃ “thyme” ˃ “eucalyptus”. it has been reported that the antioxidant capacity of honey was correlated with the biochemical constituents, in particularly with phenolic compounds and flavonoids (chua et al., 2013; buba et al., 2013). table 2. hydrogen peroxide scavenging capacity of phenolic extract from honey samples, propolis and bee pollen, expressed as ic50 (µg/ml). extract ic50 (µg/ml ) multiflower honey 0.206 ± 0.0243a thyme honey 1.928 ± 0.0209b citrus honey 0.771 ± 0.0239c eucalyptus honey 2.211 ± 0.0216d sidr honey 1.259 ± 0.0224e epins honey 0.951 ± 0.0099f propolis 35.116 ± 0.4049g bee pollen 39.383 ± 0.2622h ascorbic acid 0.694 ± 0.0238i data are (mean ± sd, n = 3). means within a column sharing the same letter are not significantly different by student’s t-test (p < 0.05). poor scavenging activity is observed with extracts from propolis (35.116 µg/ml) and bee pollen (38.383 µg/ml). in similar studies, the h2o2 scavenging assay of phenolic compounds extracted from propolis by various solvents (distilled water, absolute methanol, 50% methanol, and 70% methanol) presented different action on h2o2, which ic50 values were between 11.72 µg/ml and 765.75 µg/ml (farooq wali et al., 2016; ramnath et al., 2016; kızılpınar temizer et al., 2017). although these two bee products are quantitatively rich in phenolic compounds, but their extracts have a low antioxidant capacity compared to honey extracts, which suggests that the antioxidant capacity is not always dependent on the amount of phenolics and flavonoids, honeys are therefore a good source of antioxidant substances. also, the botanical origins of honey greatly influence hydrogen peroxide scavenging capacity. the results of the ic50 obtained in table 2 clearly confirm these observations. mohammedi – h2o2 scavenging activity of bee products 31 conclusion in this present study, it had been established that phenolic extracts from all types of honey, propolis, and bee pollen contain phenolic and flavonoids compounds and possessed antioxidant property toward hydrogen peroxide, and therefore the preventive and medical application of phenolic extracts from honey, and other bee products against oxidative stress are recommended. all honey types after extraction of phenolic compounds are able to scavenge efficiently the hydrogen peroxide, and this capacity is as much greater than that observed in other bee products. antioxidant activity against h2o2 in honey samples is related to botanical type, which is strongly affected by the floral origin, and therefore varies quantitatively and qualitatively with the content of antioxidant phenolic compounds. it is well shown in the results that extract from 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(2017) honey and health: a review of recent clinical research. pharmacognosy res 9: 121-127. shahzad a, cohrs rj (2012) in vitro activity of honey against varicella virus (vzv): a translational medicine study for potential remedy for shingles. transl biomed 3: 1-7. singleton vl, rudolf o, rose lr (1999) analysis of total phenols and other oxidation substrates and antioxidant by means of folin-ciocalteu reagent. methods enzymology 299: 152-178. sinha s, prakash a, sehgal r, medhi b (2018) comparative effect of manuka honey on anaerobic parasitic protozoans with standard drug therapy under in vitro conditions: apreliminary study. indian j pharmacol 50:197-203. sowa p, grabek-lejko d, wesolowska m, swacha s, dzugan m (2017) hydrogen peroxide-dependent antibacterial action of melilotus albus honey. lett appl microbiol 65: 82-89. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 7-13 | doi: 10.14421/biomedich.2020.91.7-13 issn 2540-9328 (online) the effect of cassava peel starch addition to bioplastic biodegradation based on chitosan on soil and river water media mahfud syuhada1, sintia ainus sofa2,*, endaruji sedyadi3 1,3chemistry; 2chemistry education program, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author* sintiashofa@gmail.com manuscript received: 28 november, 2019. revision accepted: 28 february, 2020. published: 13 april, 2020. abstract the study of the relationship between starch addition to biodegradation of bioplastics has been carried out. this study aims to understand the biodegradability of chitosan-based bioplastics with additional cassava peel starch on soil and river water media. this research was conducted through four stages, namely making starch from cassava peel waste, making bioplastics using the blending and castingwet processes method with variations of starch 5, 10, 15 and 20 ml. testing physical mechanical properties including water resistance, thickness, tensile strength, elongation, and modulus young. testing the characteristics of bioplastics functional groups was carried out using ftir (fourier transform infrared) and biodegradation testing of bioplastics carried out on soil and river water media. the results of bioplastics research with variation 5 ml produce good mechanical physical properties. bioplastics produced water resistance value of 45.03%, thickness of 0.0190 mm, tensile strength of 49.93 mpa, elongation of 3.068% and young modulus of 1627.63 mpa. bioplastics biodegradation test was observed by measuring the decrease in sample mass. the biodegradation test results in soil and river water media respectively showed a decrease in bioplastic mass up to 63% and 54%. the biodegradation rate of the calculation results on soil media is −0,1502 and in river water media is −0,0948. keywords: chitosan; cassava peel starch; bioplastic; mechanical properties; biodegradation introduction the annual production of petroleum based plastics (pmb) was recorded more than 300 million tons in 2015 (emadian, 2016). in the fact, p-mb production produced above 34 million tons of water which continues to grow every year and indonesia itself is the second largest producer of p-mb waste in the world after china (wahyuningtyas et al., 2017). as much as 93% of the waste tends to accumulated on the surface of the land and oceans so that it was very detrimental to humans and the environment (pathak et al., 2014). one of the efforts to save the earth from the explosion of p-mb waste is the making biodegradable bioplastics. biodegradable bioplastics are plastic polymers that will undergo decomposition by fungi or microorganisms. decomposition can occur up to one hundred percent if discharged into the environment with the final result in the form of water and carbon dioxide gas without leaving a toxic residue. this bioplastic is ecofriendly, non-toxic and renewable because its constituent compounds come from plats such as starch cellulose, and lignin as well as from animals such as casein, protein and lipids (widyaningsih et al., 2012). one of the raw materials for making biodegradable bioplastics in this research is chitosan. chitosan can be used as a raw material in the production of the biodegradable bioplastics due to its nature which can form clear, strong and flexible thin layers (mackay & tait, 2011) non-toxic (jara, et al., 2018). besides that, other raw materials used in the form of cassava peel starch. cassava peel starch is added as bioplastic material because it is believed to accelerate the biodegradation process. it happens because the ability of starch to absorb high water then becomes the optimum place for the growth of degrading microorganisms (wahyuningtyas et al., 2017). the use of chitosan and cassava peel as an ingredient in the production of biodegradable bioplastics will increase its use value (anita, akbar, & harahap, 2013). the selection of chitosan with cassava peel as a bioplastic base is expected to be an alternative solution for making bioplastics. biodegradability of bioplastics in the environment has been tested through various types of experiments. it was started from using one of type of organism to complex systems such as compost and seawater (emadian, 2016). based on the background above, a study was conducted on the study of biodegradable plastics with raw materials in the form of chitosan with the form of chitosan with the addition of cassava peel starch in surface media and river. https://doi.org/10.14421/biomedich.2020.91.7-13 8 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 materials and methods tools and materials non-electronic devices used include a set of glassware, chopsticks, tweezers, thermometers, magnetic stirrers, bioplastic molds (mica), filter paper soil testers and biodegradation media tubes. electronic device that will be used include blenders, hot plates, ovens, and analytical tools such as analytical balances, ftir spectrophotometers, and a set of tensile and elongation strength gauges. while the ingredients used are chitosan, cassava peel starch, 1% acetic acid and distilled water. procedures  step 1. synthesis of cassava peel starch. cassava peel remove the outer skin, then washed, added distilled water and blended to produce pulp. the resulting pulp is then separated between pulp and starch by filtering. then it was deposited for several hours. the precipitate is then dried in an oven. cassava skin starch was then analyzed using ftir.  step 2. synthesis of bioplastic chitosan solution and cassava peel starch solution were taken with several variations in volume, the comparison is as follows: variation 0. chitosan : cassava peel starch (10:0) = 50 : 0 ml variation 5. chitosan : cassava peel starch (9:1) = 45 : 5 ml variation 10. chitosan : cassava peel starch (8:2) = 40 : 10 ml variation 15. chitosan : cassava peel starch (7:3) = 35 : 15 ml variation 20. chitosan : cassava peel starch (6:4) = 30 : 20 ml both solutions with each of the above comparisons were stirred for 25 minutes using a magnetic stirrer. bioplastic solutions that have been thoroughly mixed are carefully poured into the mold. then the printed solution is dried in an oven, then cooled to room temperature. bioplastic that have been formed are then released for further analysis and testing. data analysis  fourier transform infrared spectroscopy characterization of functional groups carried out by ftir using a kbr pellet method. one mg of sample was mixed with 200 mg of kbr powder. samples were mixed kbr pellets by pressed mixture into a thin transparent using 10 tons pressure (2000 psi). pellet samples were then measured its infrared absorption at wavenumber 4000–400 cm-1 (sedyadi, anggraini, & ekawati, 2016).  mechanical properties test mechanical testing is carried out to determine thickness, tensile strength, elongation and modulus young test. film thickness were measured using micrometer to the nearest 0.001 mm. thickness measurements were taken at five different points for each film sample (top right corner, bottom right corner, middle, upper left corner and the bottom left corner). film thickness value is a average of the results of measurements at five points in units of mm. a set equipment test is prepared and arranged. sample of edible film mounted at the ends of both clamping and wedged firmly. the measurement area is set with loads corresponding to pen recorder off. tensile strength is determined by maximum load, the elongation is determined and calculated at the time the film broken. the modulus young are comparison between tensile strength and elongation.  biodegradation test biodegradation test is carried out on two media, namely surface soil and river water. bioplastic samples were cut 3x3 cm2 in size and then weighed. the samples are stored on media in an open for 14 days. samples are examined once every two days by means of being taken from the media, then cleaned using distilled water, dried in the oven and weighed dry mass. the mass lost from the sample is referred to as percent biodegradation which can be calculated by the equation: % 𝐵𝑖𝑜𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝑎1 − 𝑎2 𝑎1 information a1 = mass before testing (gram) a2 = mass after testing (gram) results and discussion synthesis cassava peel starch cassava peel starch is obtained from extraction process with a smooth and abrasive surface. the total starch obtained was 26 grams from 400 gram of cassava peel waste. the process of getting cassava starch is done by making cassava peel pulp using a blender with the addition of distilled water, then the pulp is filtered and precipitated to obtain starch. the pulp filtering process is carried out in stages to produce purer starch. distilled water are used because the nature of starch granules did not dissolve in distilled water at room temperature, so that starch can be deposited and separated easily(kusnandar, 2010). the results of the starch are dried using an oven to reduce water content, so that it can inhibit the growth of bacteria, yeast or mold and extend the shelf life (jabbar & fatimah, 2017).the dried starch is crushed to be smooth and then filtered to obtain a uniform size. syuhada et al. – the effect of cassava peel starch addition to bioplastic … 9 identification of functional groups for cassava peel starch powders have been produced from a simple extraction process is carried out using ftir spectrophotometer at wavelength 400 – 4000 cm-1. the results of ftir analysis of cassava peel starch performed are presented in the spectra in figure 1. figure shows the widening absorption at the 3387 cm-1 wave number that characterizes the -oh group. absorption in the area of 1026.13 cm-1 indicates the presence of c-o groups, and absorption in the area of 2931.8 cm-1 indicates the presence of c-h. in addition there is a peak in the wave number 1635.54 cm-1 which shows the bond that occurs cyclic or aromatic. figure 1. spectra ftir of cassava peel starch. synthesis of bioplastic bioplastic were made by the blending method, which was a method that combined or mixed two or more raw materials into one. this method has advantage that the material used is relatively small, the time required is shorter and the cost is cheaper (nurseha, 2012). bioplastics could be formed after going through several stages. in this research, a transparent and thin bioplastic was produced with slightly rough surface texture and a bit stiff. chitosan is dissolved on 1% acetic acid to get chitosan solution. the use of acidic solvents was due to the nature of chitosan itself which can only dissolve in organic acids such as formic acid or lactic acid, and can dissolve completely in acidic acid. furthermore, cassava peel starch is dissolved in distilled water and stirred using a magnetic stirrer and heated. the purpose of this warm-up is that the cassava starch and aquades can be homogenous and not separate. after the two solutions are obtained, the volume was measured according to the variations made. the two raw materials were mixed according to their variations and stirred again using magnetic stirrers until it was homogeneous. the thin layer which was referred to as bioplastic was produced thorough a wet process molding method, in which chitosan and cassava peel starch were converted into a form of solution then dried. bioplastic thin layers could form if the cohesive attraction that occurred between molecules is adequate, sufficient diffusion and evaporation of intact water, so that the polymer chains will adjust to form layers (kandasamy, 2005) bioplastic characterization the total content of the components used to prepare bioplastics will affect the physical and mechanical properties produced from bioplastic raw materials. the results of the physical and mechanical properties of the bioplastics carried out are shown in table 1 as follows: table 1. bioplastic characterization test results. v a r ia ti o n w a te r r e si st a n c e ( % ) thickness (mm) tensile strength (mpa) elongation (%) modulus young (mpa) 0 45,268 0,0220 45,7567 2,6549 1723,4811 5 45,034 0,0190 49,9342 3,0679 1627,6345 10 40,360 0,0180 48,4456 2,9836 1623,7297 15 36,154 0,0175 46,7011 2,9902 1561,8052 20 29,749 0,0755 9,6806 3,3622 287,9246 water resistance is an important property for bioplastic that is directly affected by the water absorption of bioplastic-forming polymers. table 1 showed that there was a decrease in water resistance along with the addition of cassava peel starch variations. it occurred because the hydrophilic nature of starch allows for the absorption of water. the more starches were used, the lower the resistance of the bioplastics produced. based on the results of water resistance obtained in this research, bioplastic chitosan cassava peel starch did not meet the standard criteria set by sni. sni set the plastic water resistance value of 99%, while the bioplastics made are less than 99%. the addition of cassava peel starch would initially reduce the thickness but then increase again as the volume of starch solution variation increased. chitosan solution in this study has physical properties that are thicker than cassava peel starch solution(nahir, 2017), high chitosan concentration will increase the total solids in solution. so, as the volume of chitosan decreases, the total solids in the solution will decrease so the bioplastic thickness value decreases to a certain point which increase again. in addition to the volume of material solution, the bioplastic molding process on the printing plate also influences the thickness value. the bioplastic printing process is one of the crucial things that determine how bioplastics will be formed, whether thick or thin. if it is not appropriate in pouring, leveling the surface of the solution, or slope of the laying plate then the bioplastic solution will gather at one point and produce a non-uniform thickness in a single sheet of bioplastics. chitosan bioplastic thickness with the addition of cassava peel starch variations on average under 0.25 mm, so it can be said that some of the standard value set by jis (japanese industrial standard) of < 0.25 mm, with the exception of variations of 20. 10 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 based on table 1, the addition cassava peel starch to chitosan bioplastics could increase the tensile strength of bioplastics. in variation 5, the tensile strength of value is the highest, then in variations 10, 14, and 20 there was a decrease in the value of bioplastics will be high if the composition of the composition was optimum, and the addition of starch could result in a decrease in tensile strength values. the optimum composition occurred in perfect balance between chitosan macromolecules and macromolecules of cassava peel starch (nahir, 2017). if the condition of the mixed composition was only close to a little or far from the optimum condition, then with the addition of starch the value of bioplastic tensile strength would decrease because polymer bonding chain of starch would be between the chitosan polymer bonding mechanism so the interaction that occurred between the chitosan polymer chains was reduced (saputro & ovita, 2017). chitosan had a linear polymer chain so that chitosan tends to form a crystalline phase so that it can provide strength and stiffness in bioplastics(agustin & padmawijaya, 2016). the results if the tensile strength of chitosan bioplastic with cassava peel starch variations have met the plastics standards set by sni (indonesian national standard. in the case of variations in bioplastics 20, with the tensile strength produced by the bioplastics only 9.6806 mpa, they cannot be said to meet the standard. this may be influenced by the inhomogeneity of the distribution of bioplastics molecules which results in a decrease in tensile strength (utami et al., 2014). based on table 1 showed that along with addition of cassava peel starch variations, the elongation valueof bioplastics is increasing. in starch there are two main constituent components, namely amylose and amylopectin chain structure is branched. this branched chain structure had an amorphous phase. when bioplastic was given a tensile load, this amorphous part was the first to experience an elongation, the amorphous phase would be attracted and stretched to form a parallel arrangement. amorphous phase elongation process can be seen in figure 2(agustin & padmawijaya, 2016). amylopectin was a cause of increased elongation of bioplastic chitosan-cassava peel starch. although there was an increase in elongation, the percentage value of elongation obtained by bioplastic chitosan cassava peel starch has not fulfilled sni of 21-220%. figure 2. bioplastic stretch in amorphous phase when given tensile load. the addition of cassava peel starch variations is the cause of young’s modulus obtained by bioplastics which is decreasing. this can occur because with the addition of starch, bioplastics stiffness decreases. bioplastic stiffness decreases due to the presence of starch branched amylopectin chains that tend to form an amorphous phase. as previously known, bioplastic stiffness is due to linear chains which tend to form crystalline phases(agustin & padmawijaya, 2016). chitosan-cassava peel starch bioplastics has two linear chains, each derived from chitosan and amylose starch. when compared with the plastic standards issued by sni of 117.62-137.27 mpa, the young bioplastic modulus value is far more rigid. young modulus values for polymer-based materials range between <10-10.000 mpa (hastomo, 2009). analysis of bioplastic function group the biodegradable bioplastic characterization that has been made was done using ftir to determine the functional groups contained in bioplastics. the spectra of the results of ftir analysis of chitosan and bioplastic raw materials with the best tensile strength could be seen in figure 3. figure 3. ftir spectra of chitosan (a) bioplastic (b) and chitosan bioplastic with cassava peel starch (c). figure 3 showed that chitosan spectra have a sharp absorption with wave number 3425.58 cm-1 indicating the presence of o-h groups overlapping with n-h, absorption at wave number 2877.79 cm-1 indicating the presence of c-h and 1072.42 cm-1 indicating the presence of c-o. after bioplastics were formed, the peak absorption pattern produced is slightly different from the basic material, namely chitosan. there was a widening strong absorption at a wavelength of 3425.58 cm-1 indicates an o-h group overlapping with n-h. ch was indicated by the absorption which was slightly shifted at wave number 2885.51 cm-1, and for c-o it was marked by absorption at wave number 1064.71 cm1. absorption of functional groups with similar wave numbers was also produced by bioplastics chitosan syuhada et al. – the effect of cassava peel starch addition to bioplastic … 11 added with cassava peel starch. in the vicinity of these wavelengths also experience widening uptake, this was due to the increase in the o-h group due to the addition of cassava peel starch. the peak at 3448.72 cm-1 indicates the o-h group overlaps with n-h, the peak at wave number 2877.79 cm-1 indicates the presence of ch and the peak at the wave number 1080.14 cm-1 indicates the presence of c-o. based on the analysis of figure 3, ftir spectra of chitosan, chitosan bioplastics and chitosan bioplastics with addition of cassava peel starch did not show at new functional groups. this means that the bioplastic are formed come from the result of physical mixing(saputro & ovita, 2017). proposed interactions that occurred in bioplastics can be seen in figure 4 (afif, wijayanti, & mursiti, 2018). figure 4. proposed bioplastic interaction. pearson correlation test the pearson correlation test results on mechanical properties presented in table 2 can be interpreted by matching the correlation coefficient value obtained with the correlation coefficient value in table 3. table 2. pearson correlation test. it appears that the addition of cassava peel starch variation had a positive pearson correlation value of 0.669 with a significance value of 0.225. it showed a strong relationship between the additional of cassava peel starch to thickness. meanwhile, the pearson correlation value generated for tensile strength is negative 0.698 with a significance value of 0.190. it showed that the addition of cassava peel starch variation has a strong negative correlation to the value of bioplastic tensile strength produced. the addition of cassava peel starch tends to reduce the value of tensile strength. pearson correlation between variation and elongation is 0.838 and has a significance of 0.076. based on these data it can be concluded that the addition of cassava peel starch variations has a very strong relationship to the value of the resulting bioplastic elongation. through statistical calculations it can also be seen that the addition of cassava peel starch variation has strong relationship to young bioplastic modulus produced with a negative value meaning that the addition of cassava peel starch tends to decrease young’s modulus value. the resulting relationship value using pearson correlation calculation is -0.768 with significance of 0.139. overall it can be said that the addition of cassava peel starch is strongly related to thickness and tensile strength, and is strongly related to young’s elongation and modulus. table 3. level of relationship of two variables (qudratullah, 2014). correlation coefficient relationship level 1 perfect 0,75-0,99 very strong 0,50-0,74 strong 0,25-0,49 weak 0,01-0,24 very weak 0 none biodegradation test  soil media based on the graph in figure 5, it was known that the bioplastics made have biodegradable properties, this is evidenced by the reduction in the bioplastic mass of each variation within the observation period for 14 days. the graph also showed a percent reduction in mass on one day which was equal to the increase in cassava peel starch as a variation the amorphous part of starch polymers was a less organized part compared to chitosan polymers, and enzymes from microorganisms were easier to attack and more biodegradable of less organized parts (rohaeti, 2009). amorphous polymers were easier to biodegrade than crystalline polymers and starch cyristallinity was lower than chitosan, so adding starch to bioplastics will further increase the rate of biodegradation(asiah, 2010). percent decrease in bioplastic mass was further analyzed using analysis of variance (anava) to find out whether or not there were differences resulting from each measurement of biodegradation test on the second, seventh, and fourteenth days. on the second day of the biodegradation test, the results of the analysis of variance did not show any significant difference, meaning that variations in the addition of cassava peel starch didn’t significantly affect the decrease in bioplastic mass on the second day. on the seventh day of the biodegradation test, based on the analysis of variance conducted showed the same results on the second day, no significant differences occurred. this shows that the addition of cassava peel starch variation didn’t produce a significant effect on the decrease in bioplastic mass on the seventh day. on the fourteenth day, the results of the analysis of variance showed a 12 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 significant difference, which means that the addition of cassava peel starch variations resulted in a marked difference in the decrease in bioplastic mass on the fourteenth day. the results of analysis of variance in soil media can be seen that the effect of adding starch was seen to significantly occur on the fourteenth day. figure 5. graph of bioplastic biodegradation of chitosan-cassava peel starch on soil media.  river water media figure 6. graph of bioplastic biodegradation of chitosan-cassava peel starch on river water media. figure 6 shows that chitosan bioplastics with additional variations of cassava peel starch made have biodegradable properties, this is evidenced by the reduction in the bioplastic mass of each variation during the 14 days observation. graph 6 showed a reduction in mass with increasing variations of cassava peel starch. in addition to the hydrophilic nature of starch which causes bioplastics to degrade more quickly, starch polymer are also favored by microorganisms which can result in the formation of large cracks and pore in bioplastic until the reduction of bioplastics mass (alam et al., 2018). percent decrease in bioplastic mass was further analyzed of variance (anava) to find out whether or not there were differences resulting from each measurement of biodegradation test on the second, seventh and fourteenth days. on the second day of the biodegradation test, the result of the analysis of variance showed a significant difference, meaning that variations in the addition of cassava peel starch significantly affected the bioplastic mass reduction on the second day. on the seventh day of the biodegradation test, based on the analysis of variance conducted, there was a significant difference. this shows that the addition of cassava peel starch variation produced a significant effect in the decrease in bioplastic mass on the seventh day. on the fourteenth day, the result of the analysis variance showed a significant difference, which meant that the addition cassava peel starch variation significantly affected the decrease in bioplastic mass in the fourteenth day. data results above it was concluded that the addition cassava peel starch variation significantly affected the bioplastic mass reduction on the second, seventh and fourteenth days of biodegradation test in river water media. comparison of biodegradation test figure 7. graph of bioplastic mass reduction in soil and river water media. figure 7 showed bioplastic mass decreases more rapidly in soil media than in river water media. this is corroborated by the slope value of the biodegradation test linier equation in soil media of -0.1502 and for biodegradation test of river water media of -0.0948. several factors including the availability of water temperature, the amount of oxygen used, minerals, carbon ph and energy sources affect the growth of microorganisms in each test medium (manika et al., 35.00 45.00 55.00 65.00 75.00 85.00 95.00 105.00 0 2 4 7 8 10 11 14 0 2 4 7 8 10 11 14 0 grams 100.00 88.70 86.54 83.99 79.08 76.19 71.36 65.97 5 grams 100.00 94.11 89.59 86.02 83.49 77.81 73.01 69.11 10 grams 100.00 93.45 85.35 82.77 76.39 72.06 64.45 58.42 15 grams 100.00 92.84 84.23 78.24 72.91 65.22 60.78 54.79 20 grams 100.00 90.58 82.50 77.21 74.09 57.55 42.87 37.05 40.00 50.00 60.00 70.00 80.00 90.00 100.00 0 2 4 7 8 10 11 14 0 2 4 7 8 10 11 14 0 grams 100.00 80.08 77.39 70.07 67.75 64.55 61.94 57.97 5 grams 100.00 81.69 79.65 78.03 75.85 69.79 67.35 61.25 10 grams 100.00 81.99 78.05 76.11 67.57 65.80 59.67 56.33 15 grams 100.00 76.33 74.26 66.22 64.11 60.67 56.94 54.49 20 grams 100.00 87.98 81.69 66.70 63.26 57.57 53.03 46.31 -2.2037 -2.1936 -2.9367 -3.3015 -4.5749 -2.6433 -2.3038 -2.8235 -2.8421 -3.8778 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0 5 10 15 20 25 soil water syuhada et al. – the effect of cassava peel starch addition to bioplastic … 13 2015). the possibility of soil media is an optimal place for growth of microorganisms becomes abundant. this causes the bioplastic biodegradation process to occur more quickly in soil media. conclusion based in the results obtained, it was concluded that bioplastics during biodegradability tests on both media from the first day to the fourteenth day experienced a decreases in mass. the increasing amount of starch variation, the faster biodegradation occurs. the fastest mass reduction occurs in variation 20 bioplastics, while the slowest decrease occurs in variation 5 bioplastics. if a comparison of biodegradation rates is taken in both media, the fastest biodegradation process occurs in soil media. acknowledgements the author would like to thank integrated laboratory of uin sunan kalijaga yogyakarta for permission to conduct experiments. references afif, m., wijayanti, n., & mursiti, s. 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(2017). sintesis dan karakteristik bioplastik dari kitosan-pati ganyong (canna edulis). kimia dan pendidikan kimia, 13-21. sedyadi, e., anggraini, d., & ekawati, d. p. (2016). strachglycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surini dumbo catfish (clarias gariepinus) addition on its mechanical properties. biology, medicine & natural product chemistry, 33-40. utami, m. r., latifah, & widiarti, n. (2014). sintesis plastik biodegradable dari kulit pisang dengan penambahan kitoan dan platicizer gliserol. indonesian journal of chemical science, 3(2), 163-167. wahyuningtyas, nanang, e., & suryanto, h. (2017). analysis of biodegradation of bioplastics made of cassava starch. mechanical engineering science and technology, 1(1). widyaningsih, s., kartika, d., & nurhayati, y. t. (2012). pengaruh penambahan sorbitol dan kalsium karbonat terhadap karakteristik dan sifat biodegradasi film dari pati kulit pisang. molekul, 7(1), 69-81. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 73-79 | doi: 10.14421/biomedich.2021.102.73-79 issn 2540-9328 (online) effect of diabetes mellitus and hypertension on osmotic fragility and hemorheological factors in male wistar rats david ehikhuemen okonofua1, jerome ndudi asiwe1,2,*, kenneth kelechi anachuna3, emuesiri goodies moke4, kamaldeen olalekan sanusi1, ebunoluwa oluwabusola adagbada1, mariam onono yusuf1, damilola ifeoluwa alawode1, adesoji adedipe fasanmade1 1cardiorespiratory research unit, department of physiology, college of medicine, university of ibadan, ibadan, nigeria. 2department of physiology, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, faculty of basic medical sciences, delta state university, abraka, nigeria. 4department of pharmacology and therapeutics, faculty of basic medical sciences, delta state university, abraka, nigeria. corresponding author* asiwejerome@yahoo.com; tel: +2348163727468 manuscript received: 20 july 2021. revision accepted: 29 july, 2021. published: 01 october, 2021. abstract diabetes mellitus is a common risk factor for erythrocyte osmotic stress. this study was aimed at exploring the effect of streptozotocin (stz)-induced diabetes mellitus and salt-induced hypertension on osmotic fragility and hemorheological variables in male wistar rats. thirty male rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet 0.3% salt); high salt diet (8% salt) (hsd only); stz induced diabetes and normal salt diet (stz only); stz induced diabetes and high salt diet (stz + hsd). at the end of a 4 weeks period, hematological variables, osmotic fragility, rheology and cardiovascular responses were assessed. there was an increase (p<0.05) in the mean arterial pressure and heart rate of hsd, stz and hsd + stz groups indicating a salt induced hypertension. there was a decrease in the body weight of stz and hsd +stz groups. there was significant increase (p<0.05) in the haematocrit, platelets estimates and fibrinogen concentrations in the experimental groups when compared with the controls. the stz and stz + hsd groups showed a reduced clotting time which corresponded to the increased platelet estimates and fibrinogen concentration. the increase in haematocrit, platelet and plasma protein resulted in the increased blood viscosity and a decreased flow rate. the osmotic fragility test was also observed to be increased (p<0.05) in hsd, stz only and stz + hsd groups. diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte, as well as increase blood viscosity. keywords: diabetes mellitus; hemorheology; high salt diet; hypertension; viscosity. abbreviations: bp – blood pressure; dm – diabetes mellitus; hr – heart rate; hsd – high salt diet; nacl – sodium chloride; rpv – relative plasma viscosity; sbp – systolic blood pressure; stz – streptozotocin; wbv – whole blood viscosity. introduction diabetes mellitus (dm) is a chronic metabolic disorder in the endocrine system marked by abnormalities in insulin secretion and/or insulin action that leads to the progressive deterioration of glucose tolerance, which causes hyperglycemia (granner, 2000). in postmodern times, dm has now become a trending public health problem which calls for serious care and concern (harika et al., 2014). various medical therapies are current being used in management of diabetes (patel et al., 2010; chaudhury et al., 2017; palanisamy et al., 2018; okafo et al., 2019). dm is one of the major risk factors for increased osmotic fragility. this is because hyperglycemia causes structural and functional changes in erythrocytes which can result in osmotic stress (eze et al., 2017). according to the american heart association, high salt consumption is a serious essential/primary hypertension cause and risk factor. go reported that an average american adult consumes about 5400 mg of sodium per day which is about 130% increase more than the recommended 2300 mg of sodium per day (go et al., 2013). this high salt intake per day is a major risk factor for essential hypertension. the rate of erythrocyte osmotic fragility increase in hypertensive subjects is higher than in normotensive subjects (fasanmade, 1999). high salt intake which induces hypertension will cause an increase in the osmotic fragility of red blood corpuscle (baskurt and meiselman, 2003). high salt diets have been greatly linked to be a serious risk factor for cardiovascular diseases relating to high blood pressure, endothelial dysfunction, stroke, ventricular hypertrophy and fibrosis, arterial and ventricular stiffening, myocardial infarction, arrhythmias, and heart failure (mohan and campbell, 2009) while diabetes mellitus is a common risk factor for erythrocyte osmotic stress (eze et al., 2017). this suggests that the https://doi.org/10.14421/biomedich.2021.102.73-79 74 biology, medicine, & natural product chemistry 10 (2), 2021: 73-79 constituents of blood and its flow properties serve as a link between dm and hypertension. hemorheology is the study of the flow properties of blood and its elements (plasma and formed elements, including erythrocytes, white blood cells and platelets) baskurt et al., 2007). hemorheological parameters, such as hematocrit, plasma and whole blood viscosity, plasma proteins, erythrocyte deformability and aggregation are basic characteristics of blood flow (rabai, 2013). there is growing evidence that the flow properties of blood are among the main factors of proper tissue perfusion, and shifts in these properties play significant roles in disease processes (mohan et al., 2001). the viscosity of blood is directly proportional to the hemoconcentration and inversely proportional to the flow rate. this implies that factors which increase blood constituents and decrease plasma will elevate the blood viscosity which will decrease flow rate and alter tissue perfusion (chang et al., 2017; sloop et al., 2020). there are adequate evidences on diabetes that the elevated blood viscosity is a pathogenetic factor of diabetic microangiopathy, altering microcirculation and leading to insufficient tissue nutrition (grigoleit et al., 1973; cho et al., 2008). alterations in tissue perfusion will result in microvascular and macrovascular complications (cade et al., 2008). hence, this study provides an investigation into the rheological properties of blood in diabetic and hypertensive male wistar rats. materials and methods experimental design a total of thirty male wistar rats (120-150 g) were used for the experiment. they were acclimatized for two weeks prior to commencement of the experiment at the central animal house of the college of medicine, university of ibadan, nigeria, and kept under standard laboratory conditions and fed standard rat pellets and water ad libitum. animal handling was done in accordance to established guidelines by the national institute of health for care and use of laboratory animals. ethical approval was given by the college of medicine ethics committee (ui-acurec/19/0140). the rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet 0.3% salt); high salt diet (8% salt) (hsd only); stz induced diabetes and normal salt diet (stz only); stz induced diabetes and high salt diet (stz + hsd). preparation of feed rations three different rations were used for the study. the high salt diet was prepared by mixing 8g of table salt with 92 g of standard rat chow (asiwe et al., 2020). hsd only and stz +hsd groups were given this ration and water ad libitum. the normal salt diet was prepared by mixing 0.30 g of table salt with 99.70 g of standard rat chow which was given to the positive control group and water ad libitum. while the zero-salt diet was given to the negative control group. induction of diabetes mellitus diabetes was induced by single intraperitoneal injection of 60 mg/kg body weight dose of streptozotocin (stz) dissolved in freshly prepared 0.1m cold citrate buffer of ph 4.5 into the animals according to the stz-induced hyperglycemia in rats’ model (eze et al., 2017; asiwe et al., 2020; akbarzadeh et al., 2007). the experimental animals were fasted overnight (18 hours) prior to diabetes induction while allowed access to drinking water. seventytwo hours after streptozotocin injection, a drop of blood was drawn from tail vein of the rats to measure their blood glucose level. animals having fasting blood glucose levels ≥ 200 mg/dl were considered diabetic and used in the study. measurement of blood glucose level in order to ascertain the diabetic state of the animals, the fasting blood sugar level was measured every week after being fasted overnight using the accu-chek active glucometer (model gb06140695). the results obtained in mmol/l were converted to mg/dl by multiplying with 18 (conversion factor). blood samples were collected from tail artery of the rats for evaluation of the blood glucose. body weights of animals were also measured weekly in grams to estimate the effect of the induced diabetes and high salt diet on body composition. the weight was measured with a standard weighing scale. blood pressure and heart rate blood pressure and heart rate were done at the small animal ward of the veterinary clinic university of ibadan, ibadan, nigeria. systolic blood pressure (sbp) was measured indirectly in a conscious and slightly restrained rat using the tail cuff plethysmography method (kent scientific, usa). heart rate (hr) tracings were observed and recorded as obtained during blood pressure (bp) measurement. for these measurements, rats were conditioned to the restraint (cone) and the warming chamber for about 20 minutes before the measurement. sbp and hr measurements were performed in a very quiet environment to avoid sound interference by the same investigator. there were two sensors to measure bp and vascular peripheral resistance. after stabilization in the chamber, an acclimatization run was performed for 5 cycles which was immediately followed by the typical run involving 10 repetitions of the automated inflation-deflation cycle. examination of blood samples blood samples were collected by ocular puncture at the end of the fourth week and stored in heparinized bottles which were used to consider effect of high salt diet and okonofua et al. – effect of diabetes mellitus and hypertension on … 75 diabetes mellitus on hematology, osmotic fragility and rheology. four animals were used for each group for analysis (n=4). rheological analysis whole blood viscosity and relative plasma viscosity were estimated using the method described by reid and ugwu (1987), and the flow rate was calculated. plasma fibrinogen concentration was estimated by clot weight method of ingram (1952). haematocrit was estimated using microhaematocrit reader. statistical analysis data were analyzed with graphpad prism version 7.0 (graphpad software, san diego, ca) and expressed as means ± sem (standard error of mean). one-way anova was used for comparisons, followed by post hoc newman-keuls multiple comparison test. p < 0.05 was considered statistically significant. results and discussion osmotic fragility figure 1 shows the rate of erythrocyte osmotic fragility (%). the percentage erythrocyte osmotic fragility decreased significantly with an increase in sodium chloride (nacl) concentration. there was complete (100%) haemolysis at 0.0% of nacl. and there were no significant changes in erythrocyte osmotic fragility when observed at 0.0% and 0.1% of nacl concentration in all control and experimental groups when compared. however, significant (p < 0.05) changes in percentage erythrocyte fragility were recorded at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations, when the experimental groups were compared with the control groups. *a, *b, *c, and *d p<0.05 at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations. platelet counts figure 2 shows the platelets estimates (mm3) in diabetic and hypertensive male wistar rats. there was significant increase when high salt diet + stz groups were compared with the controls. p ˂ 0.05 is significant when compared with negative control and positive control groups. fibrinogen estimates figure 3 shows the fibrinogen estimates (mm3) in diabetic and hypertensive male wistar rats. there was significant increa se when other groups were compared with the controls. *p ˂ 0.05 is significant when compared with negative control and positive control groups. hematocrit figure 4 shows the hematocrit (%) in diabetic and hypertensive male wistar rats. there was significant increase when other groups were compared with the controls. *p ˂ 0.05 is significant when compared with negative control and positive control groups. whole blood viscosity figure 5 shows the whole blood viscosity (wbv) (mpa.s) in diabetic and hypertensive male wistar rats. from the values expressed there was significant increase between the negative control group and positive control group. there was significant inc rease when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. relative plasma viscosity figure 6 shows the relative plasma viscosity (rpv) (mpa.s) in diabetic and hypertensive male wistar rats. from the values expressed there was significant increase between the negative control group and positive control group. there was significant inc rease when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. plasma flow rate figure 7 shows the plasma flow rate (cm/sec) in diabetic and hypertensive male wistar rats. there was significant increase between the negative control and positive control groups. there was significant incre ase when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. 0 0 .1 0 .3 0 .5 0 .7 0 .9 0 2 0 4 0 6 0 8 0 1 0 0 n a c l c o n c e n t r a t i o n ( m g / m l) p e r c e n t a g e h e m o ly s is ( % ) n e g a tiv e c o n tr o l g r o u p p o s itiv e c o n tr o l g r o u p h s d o n ly s t z o n ly s t z + h s d* * * * * * * * * * * * figure 1. rate of erythrocyte osmotic fragility (%) in diabetic and hypertensive male wistar rats. 76 biology, medicine, & natural product chemistry 10 (2), 2021: 73-79 a b c d e 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 p la t e le t c o u n t ( m m 3 ) * * n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in figure 2. platelet count (mm3) in diabetic and hypertensive male wistar rats. a b c d e 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 f ib r in o g e n ( m m 3 ) * * n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in figure 3. fibrinogen estimates (mm3) in diabetic and hypertensive male wistar rats. a b c d e 0 1 0 2 0 3 0 4 0 5 0 p a c k e d c e ll v o lu m e ( % ) * ** n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in figure 4. hematocrit (%) in diabetic and hypertensive male wistar rats. a b c d e 0 2 4 6 8 w h o le b lo o d v is c o s it y ( m p a .s ) n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in * * * * * figure 5. whole blood viscosity (mpa.s) in diabetic and hypertensive male wistar rats. a b c d e 0 1 2 3 4 5 r e la ti v e p la s m a v is c o s it y ( m p a .s ) n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in * * * * * figure 6. relative plasma viscosity (mpa.s) in diabetic and hypertensive male wistar rats. a b c d e 0 5 1 0 1 5 p la s m a f lo w r a t e ( c m /s e c ) n e g a tiv e c o n tro l p o s itiv e c o n tro l h ig h s a lt d ie t s t r e p to z o to c in h ig h s a lt d ie t + s t r e p to z o to c in * * * * * figure 7. plasma flow rate (cm/sec) in diabetic and hypertensive male wistar rats. in this study, the percentage erythrocyte osmotic fragility decreased significantly with increasing nacl concentration. there was complete (100%) hemolysis at 0.0% and 0.1% of nacl. no significant changes in erythrocyte osmotic fragility were observed at 0.0% and 0.1% of nacl (distilled water) in both controls and experimental groups when compared. however, the results of the osmotic fragility showed a significant increase in the rate of erythrocyte osmotic fragility at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations in hsd only, stz only and hsd +stz groups, when compared with the control groups. this finding was in agreement with the works of other researchers (shin et al., 2007; megha and sehyun, 2009; eze et al, 2017) who showed that erythrocyte osmotic fragility was significantly increased in streptozotocin-induced diabetic animals. the increased rate of erythrocyte osmotic fragility in hypertensive subjects has also been reported by fasanmade (1999). the implication of this finding suggests that stz induced diabetes can alter the integrity of the red blood cell (rbc) membrane. erythrocyte osmotic fragility test gives an in-vitro measure of the tensile strength of erythrocyte membrane and it is an indirect method of evaluating lipid peroxidation in animals (akande et al., 2014). it is also used to measure the tensile strength of erythrocytes and its ability to resist alterations in osmotic gradients and it has been discovered to be increased during oxidative stress (aldrich et al., 2006; adenkola et al., 2010). hence, this suggests that the greater the erythrocyte osmotic fragility, the weaker the tensile strength of erythrocyte membrane (ogundeji et al, 2013) and a resultant hemolysis of erythrocyte is observed. the mechanism for increased fragility of erythrocytes have been reported to be due to increased glycosylation of the erythrocyte membrane protein or/and alteration of the na+/k+ atpase on the erythrocyte membrane (arun, 2013). the viability of an erythrocyte depends greatly on the maintenance of its membrane. studies have shown that dm and hypertension can cause an increase in lipoperoxidative changes in the membranes of erythrocytes (qujeq et al., 2005; ahmed et al., 2006; gauri and vijaya, 2008) which account for the increase in the rate of osmotic fragility. hemorheology is the study of the flow properties of blood and its elements such as plasma with its dissolved okonofua et al. – effect of diabetes mellitus and hypertension on … 77 components and formed elements, which include erythrocytes, leucocytes and platelets (baskurt et al., 2007). the flow properties of blood are among the main determinants of proper tissue perfusion, and alterations in these properties play vital roles in disease processes (lowe et al., 1980). in this study, hemorheological factors were measured with focus on blood viscosity and flow rate. the viscosity of blood is determined by the hematocrit (rabai, 2013; cho et al., 2008), increase in plasma viscosity which is determined by plasma proteins (fibrinogen and globulin) and water (mohan et al., 2001). from this study, there was a significant increase in the blood viscosity and plasma viscosity in the hsd only, stz only and stz+hsd groups when compared with the control groups. the observed increase in viscosity could be due to the significant increase in hematocrit, fibrinogen, and platelet estimates as recorded in stz only and stz+hsd groups. this finding was in accordance with the works of reid and memeh, and others (reid and memeh, 1988; khan et al., 2005; okomafe et al., 2017). increased blood viscosity and the onset of diabetic angiopathy have been related to abnormal hematocrit, plasma viscosity, fibrinogen concentration, erythrocyte deformability and rouleaux formation (winberger and baskurt, 2007). the increase in hematocrit in the experimental groups appears to have contradicted the increased rate of erythrocyte osmotic fragility as reported earlier in this study. a possible mechanism to support this finding is linked to hemoconcentration. as the osmolarity of the blood increases due to increased glucose level, the capillary permeability increases, resulting in a decrease in plasma water and thus increasing hematocrit and subsequently the blood viscosity (meiselman et al., 1967; rizvi and zaid, 2001). however, hsd only group had a significant increase in hematocrit only with a decrease in fibrinogen and platelet estimates when compared with other experimental groups. this increase in hematocrit could be the major reason behind the increase in blood viscosity (macrury et al., 1988). another interesting finding in this study was the significant increase in blood and plasma viscosity when the positive control group with normal salt intake was compared with the salt deficient negative control group. the possible mechanism underlying this claim is yet to be fully understood. the flow rate of blood is determined by three main factors – vessel diameter, perfusion pressure and blood viscosity (lowe et al., 1980). although vessel diameter has been primarily linked with blood flow rate, in pathological conditions, the contributions of perfusion pressure and viscosity becomes greatly enhanced. it has been reported from studies that the viscosity of blood is directly proportional to the hemoconcentration and inversely proportional to the flow rate (sloop et al., 2020). this implies that factors which increase blood constituents and decrease plasma water will elevate the viscosity of blood which will decrease flow rate and alter tissue perfusion (chang et al., 2017; sloop et al., 2020). from this study, it was also observed that there was a corresponding significant decrease in the flow rate of plasma across the experimental groups when compared with the control groups. hence, the increase in blood viscosity could be a factor for the decreased flow rate of plasma. the alterations in the blood flow patterns in dm is produced by a combination of reduced erythrocyte deformability and increased erythrocyte aggregation due to variations in the plasma protein (mcmillian et al., 1978). the changes in plasma proteins are linked with the development of glucose intolerance (mcmillian, 1983). a decrease in flow rate can possibly result in circulatory insufficiency marked with poor tissue perfusion which in a long term can lead to the development of vascular complications as commonly observed in subjects with dm (lowe et al., 1980). there was a significant decrease in plasma flow rate when the positive control group with normal salt intake (with higher blood and plasma viscosity) was compared with the salt deficient negative control group. this also verified the inverse relationship between blood viscosity and plasma flow rate. conclusion from the findings of this study, it can be stated that diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte. there is also an increase in the hematocrit which significantly increases the blood 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u, baskurt ok (2007). comparative hemorheology. baskurt ok, hardeman mr, rampling mw, meiselman hj, editors. handbook of hemorheology and hemodynamics, ios press, amsterdam, 267-285. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 27-33 | doi: 10.14421/biomedich.2022.111.27-33 issn 2540-9328 (online) the bioprospecting of mangrove red snapper cultivation (lutjanus argentimaculatus forsskål, 1775) using floating cages angga puja asiandu1,*, achmad gusti malayudha2 1faculty of biology, universitas gadjah mada, jl. teknika selatan, yogyakarta 55281, indonesia. 2faculty of communication and multimedia, universitas mercu buana yogyakarta, jl. ring road utara, yogyakarta 55281, indonesia corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 24 january, 2022. revision accepted: 14 february, 2022. published: 01 march, 2022. abstract mangrove red snapper is one of the potential and valuable aquaculture products. the high demand for this fish causes high fishing activities. this condition can threaten their existence in the environment. it is necessary to have aquaculture activities to preserve their existence and meet market demand. indonesia as a country with wide marine waters should be able to take advantage of the potential of fish farming by using a floating cage system. thus, this article was written to analyze the bioprospecting of red snapper aquaculture. based on the literature study, the floating net system has the potential to be developed on a large scale because the system has various advantages over fishing directly from nature. things that must be considered in the cultivation of mangrove red snapper using floating nets are the area and quality of the floating nets, the composition of the feed, and the chemical components of the feed given to the fish. based on economic potential, mangrove red snapper cultivation has high prospects which can be used as one of indonesia's leading export products. keywords: aquaculture; feed; floating net cages; snapper. abbreviations: amp (l-ascorbyl-2-monoposfate-mg); fcr (feed conversion ratio), hcg (human chorionic gonadotrophin); pb (puberogen) introduction the high growth of the human population in the world causes the need of increasing food sources, including food sources containing protein. fish is an important source of animal protein for humans. however, fish consumption in indonesia is still relatively low when compared to other country such as japan. it means that fish farming in indonesia has yet fulfill animal protein needs of the indonesian people (langkosono, 2007). red snapper (lutjanus argentimaculatus) is a potential source of fishery culture in indonesia. the high demand for red snapper encourages the cultivation of red snapper which can reduce dependence on wildcaught red snapper supply. some of the advantages of red snapper cultivation are relatively fast growth rate, high tolerance for water turbidity and salinity, low cannibalism, tolerance for high fish density, and good consumption of artificial feed (melianawati and aryati, 2012). red snapper (l. argentimaculatus) is one of the aquatic commodities with high economic value. the price in the international market in 2003 ranged from usd 5,50 to usd 18,10. in indonesia, the price is quite varied. in 2011, in west java province, the price of the fish was rp. 35,000/kg (yasad, 2011; melianawati and aryati, 2012). in lampung, the price of the fish ranged from rp. 40.000 to rp. 50.000. red snapper is a commodity that is in great demand by the public, so the cultivation and production of red snapper is very potential and promising (melianawati and aryati, 2012). red snapper is one of the most popular fish in southeast asia. the high demand for red snapper has not been matched by cultivation at this time, the supply still relies on direct catch from nature which is seasonal, varied, unstable, and less sustainable. in addition, several red snapper cultivation activities are carried out by directly catching the young snapper in the estuary or beach area. this results in a threat to the sustainability and abundance of these fish in nature. thus, it is necessary for cultivation to be carried out so that the red snapper population in nature is maintained (chi and true, 2017). cultivation of red snapper can be done by using floating cages in the sea. the floating cage is a place made of nets tied to a frame, square in shape, with a size that can reach hundreds of square meters. the size of the cage can be adjusted to the size and number of fish to be cultivated. these cages can be placed in bays, lakes, or reservoirs (purba, 1994). however, there are several https://doi.org/10.14421/biomedich.2022.111.27-33 28 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 factors that can affect the success of the red snapper (l. argentimaculatus) cultivation. some of these factors are feed, feed composition (giri et al., 2007), design of floating net cages, as well as various environmental factors such as temperature, salinity, light, and so on (affan, 2011). thus, in this paper, the authors discuss several factors that determine the success of red snapper cultivation in more detail. methods the writing of this paper was carried out by exploring various literature related to red snapper cultivation and other related sources. a literature study is an activity of searching various sources of information or reading thoroughly on a topic to be discussed. materials or sources of information that can be used in library research include books, journals, magazines, and other sources. the steps in the literature study are choosing topics to be discussed, exploring topics and related information, conducting research focus, collecting information sources, managing and presenting data, and compiling reports (sari, 2020). the data used in writing this paper is secondary data. secondary data is data obtained indirectly. secondary data is obtained from various data sources, such as the internet, statistical data, books, journals, and so on (tanujaya, 2017). in writing this paper, secondary data were obtained from journals and theses related to red snapper cultivation. results and discussion mangrove red snapper (lutjanus argentimaculatus) snapper belongs to the lutjanidae family, which consists of 103 species. these fish live in shallow to medium sea areas to a depth of 100-500 m. these fish are classified as predatory fish. snapper is actively looking for prey at night. the natural foods for these fish are crabs, shrimp, small fish, plankton, and squid. in general, snapper reproduces by involving mating between female and male snapper (wwf-indonesia, 2015). one of the snappers that are known among the public is the red snapper. figure 1. the morphology of mangrove red snapper (lutjanus argentimaculatus) (obtained from fishider.org). this fish has a distribution area that includes the indo-pacific waters, the line islands in north africa to the waters of australia, and the ryukyu islands, japan. these fish can be found in bays and beaches, to estuaries (purba, 1994). the classification of red snapper is as follows: kingdom : animalia phylum : chordata class : teleostei order : perciformes family : lutjanidae genus : lutjanus species : lutjanus argentimaculatus forsskål, 1775 the red snapper has a slightly flattened body, the back is higher, the head is pointed, the upper jawbone sinks when the fish opens its mouth. the fish has a forked caudal fin, red on top, and silvery-white on the underside. the dorsal fin has 10 spines, 13 to 15 weak rays, the anal fin has 3 spines and 8 to 19 weak rays, the pectoral fin has 14 to 15 rays weak finger. the lateral line consisted of 45 to 48 pieces. the structure of the gill cover in these fish is grooved (purba, 1994). the existence of red snappers in nature is also influenced by their habitat. red snapper spawning occurs in estuaries. the destruction of the estuary area causes red snapper spawning to decrease. this also has an impact on the red snapper population in nature (chi & true, 2017). red snapper fry is often found in the river estuaries that are in direct contact with mangroves with a salinity level of 10-25 ppt. the tillers are found on rocks and sandy areas. saplings aged 3-10 cm are often found on rocks. tillers measuring less than 3 cm are often found in sandy areas and seaweed. the natural feed for red snapper fry is shrimp, small fish, zoobenthos, and zooplankton (chi & true, 2017). snapper (lutjanus sp.) is one of the demersal fish that has high economic value and is in demand by the public. snapper is not very active, can form small groups, the migration rate is not too far, and the life cycle is stable. this is because this fish lives on the seabed which is relatively more stable than the surface. coastal degradation is one of the threats to the existence of this fish. the distribution area of snapper is not too wide so environmental degradation will directly affect the existence of these fish. in addition, the high fishing activity causes the population to become increasingly threatening in nature (sriati, 2011). reef fish such as snappers play an important role in ecological functions. the presence of these fish in their habitat supports the productivity of the habitat. however, fishing for reef fish that is not environmentally friendly has caused damage to these habitats. until now, about 75% of coral reef habitats in indonesia have been damaged due to destructive fishing by fishermen. in addition to habitat destruction, another asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 29 factor is the over-exploitation of these fish. some fishermen use potassium or a kind of anesthetic poison to catch the fish. this is because the selling price is higher when the fish are still alive (wwf-indonesia, 2015). cultivation of mangrove red snapper (lutjanus argentimaculatus) indonesia is the second-largest producer of cultivated fish in the world after china. in 2012, aquaculture production in indonesia reached 9.675.553 tons. the production of snapper itself ranks fourth with total production reaching 6.198 tons. from 1999 to 2012, the percentage increase in snapper production in indonesia increased by 11.43% (ministry of marine affairs and fisheries, 2013; giri et al., 2007). the cultivation of red snapper attracts attention because of its high growth. red snapper is a euryhaline fish that has good adaptation to salinity concentrations in the waters. this fish is able to live in waters containing high salinity of 35 ppt to freshwater with a salt concentration of 0 ppt. this ability causes the red snapper to have the potential to be cultivated in ponds filled with fresh water. the development of red snapper cultivation in freshwater opens new opportunities for entrepreneurs and the community in providing a supply of red snapper stock in the market (muyot et al., 2021). cultivation of red snapper can be done by giving a stimulant hormone. the hormones used in the spawning induction are hcg (human chorionic gonadotrophin) and pb (puberogen) hormone. induction is done by injecting hormones into the body of the fish through the back of the pectoral fins, in the area between the backs, or on the line of the ribs. the size of the broodstock of red snapper that is ready to spawn is 3 kg to 7 kg. at this weight, the broodstock red snapper is about 4 to 5 years old. in general, red snapper gonad maturation occurs in january and october. the best spawning time for red snapper is 11:00 am to 04:30 pm (purba, 1994). in the cultivation of red snapper, it is necessary to pay attention to feeding. the quality and nutritional content of the feed given must be maintained so that the cultivated snapper can grow well. availability of feed is one of the determining factors for the success of red snapper cultivation. in providing appropriate feed, it is necessary to pay attention to the adequacy of nutrients or macro and micronutrients needed by the fish. however, feeding should be done by having a feed that has economic value so that it can maximize the benefits that will be obtained from the red snapper cultivation process (giri et al., 2007). one of the important sources of nutrition in the process of fish growth is the provision of proteincontaining feed. protein is one of the important nutrients for the growth of red snapper. provision of optimum feed protein levels can stimulate the growth process of fish. however, protein levels in the feed must be considered and adjusted to the needs of the fish. the protein content in the feed must also be adjusted to the content of other nutrients so that the desired growth rate of fish can be achieved so that the yield will be high (giri et al., 2007). cultivation of mangrove red snapper in freshwater is also very potent. the analysis that should be carried out in cultivaing are the growth rate, endurance, feed conversion ratio (fcr), and the profit. cultivation of red snapper in freshwater ponds can be fed in the form of dry pellets. acclimatization that can be done on red snapper to the level of salinity is carried out for 1 to 2 weeks. cultivation of red snapper in freshwater can be done in rivers, ponds, or lakes. the use of cages can maximize the number of fish cultivated, the costs used are also economical, also valuable (muyot et al., 2021). cultivation of mangrove red snapper (l. argentimaculatus) using the floating net cage system fish cultivation in the open ocean can be carried out using a floating net cage system. a floating net cage is a container that is placed in the water and filled with fish that is used to maintain the cultured fish. the cages must have an effective and strong design so that they are durable (tatengkorang, 2020). the success of fish culture in waters is determined by the carrying capacity of the aquatic environment. the carrying capacity of the environment is the ability of an environment to support the life processes of living things in the long term. the carrying capacity of the environment is the ability of the environment to support the welfare of the life of living things that live or exist in the environment. in another sense, the carrying capacity of the environment is closely related to the availability of natural resources and environmental factors in determining the rate of life of living things. the environmental carrying capacity of waters is very important for the success of aquaculture in that environment (anrosana & gemaputri, 2018). the level of depth of water used in fish farming determines the success of aquaculture. the depth from the bottom of the cage to the optimum bottom at low tide is about 4-5 m. the depth of the cage and the waters used to determine whether or not a location is suitable for use as a fishery cultivation area. another factor that also influences is the level of lighting or water brightness. this is related to the level of light penetration into the waters. the level of water clarity is influenced by the presence of suspended particles in the water environment. light is needed by photosynthetic organisms to carry out the process of photosynthesis. this is very necessary for the phytoplankton contained in the cages to carry out the photosynthesis process. the phytoplankton can also be used as natural food for fish cultured in cages. the optimum brightness value for aquaculture in the sea is 70.35% (affan, 2011). another factor that also affects the success of aquaculture is strong currents. the flow rate of water in 30 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 an environment will affect the distribution of suspended particles in the water. in addition, currents are also related to the amount of dissolved oxygen in the waters. the design and construction of cages must be adapted to the flow of water. this is because the strength of the current can reduce the risk of fouling in the cages used. if the current is less than 25 cm/sec, the fouling organisms will be more easily attached to the cages used. the attachment of fouling organisms will reduce and disrupt the circulation of dissolved oxygen in the cage. the minimum current velocity that can still be used in cage cultivation is 5-15 cm/sec (affan, 2011). temperature also plays an important role in the success of aquaculture in waters. temperature affects the metabolic rate of cultured organisms. an increase in temperature can reduce dissolved oxygen levels in the water. thus, at too high temperatures, the respiratory activity of the cultured fish will be disturbed. the optimum temperature in fish farming is 27-37 oc (affan, 2011). dissolved oxygen is one of the key factors in fish farming. lack of dissolved oxygen can reduce the consumption of fish feed, this results in the inhibition of the growth of the fish (riadhi et al., 2017). fish farming using cages placed in saltwater or the sea is also affected by salinity. the optimum salinity in the cultivation is 30-35 ppt. however, the optimum salinity must be adjusted to the type of fish to be cultivated. certain fish have a higher salinity tolerance value than other species. the degree of acidity or ph of the water environment used also affects the success of the cultivation carried out. the ph value of seawater, in general, is in the range of 7.5-8.4. the closer to the river estuary area, the ph will change due to the influence of freshwater flowing into the sea (affan, 2011). one of the factors that cause the failure of fish farming is the presence of parasites that attack the cultured fish. endoparasites are parasites that usually attack the organs and body cavities of cultured fish. some of the endoparasites that attack fish are nematodes, trematodes, cestodes, and several types of protozoa. the parasitic infection causes the growth of fish to be stunted. parasitic infection also causes a decrease in the consumption of fish feed. another thing to worry about is the zoonic nature of the parasite so that it can infect humans who eat the fish (puspitarini et al., 2018). the endoparasites reported infecting red snapper are anisakis physeter and cucullanus heterochorus. anisakis larvae at stage two can be eaten by small fish which are then eaten by red snapper. then it will develop into a third-stage larva in red snapper. one of the factors that cause red snapper infection is a dirty water environment (puspitarini et al., 2018). in indonesia, a lot of fish farming is also done openly in nature. the cultivation is carried out using floating cages placed in reservoirs, rivers, ponds, and so on. aquaculture can be divided into two, namely marine aquaculture and freshwater aquaculture. both types of cultivation have their own characteristics. in fish farming, there are several subsystems that are interconnected with each other. the system includes the procurement of facilities and infrastructure, production systems, post-production systems, and supporting subsystems. in the production subsystem, various cultivation activities are involved, starting from the initial maintenance process to harvesting. the system also involves feeding the cultured fish (deswati & adrison, 2019). some of the advantages of aquaculture using cages are that it can maximize the location of the waters as a location for fish cultivation, the population of cultivated fish is easy to control, avoids predators, is easy to transfer, is more economical, can maximize the water used, maintained water circulation, is easy to harvest, and lower maintenance costs. low (purba, 1994). red snapper cultivation includes several activities. these cultivation activities include parental rearing, larval rearing, and seed maintenance. one of the determining factors for the success of red snapper cultivation is the eggs to be developed. when the eggs have hatched and developed into larvae, the feeding and the amount of feed given should be appropriate. in the larval phase, the food for the red snapper chicks can be in the form of small rotifers that match the larval mouth opening (melianawati & aryati, 2012). marine fish cultivation using floating cages has several impacts on fishermen. in fish farming using floating cages, feed is given to support the growth process of the fish. not all of the feed given will be eaten by the fish in the cage, some of the feed will be released into the environment outside the cage. the positive side of this is the approach of the fish in the cages to the cage area so that it can help fishermen in harvesting fish in the area (deswati & adrison, 2019). meanwhile, incentive feeding can also be misused as a “feed pump”. the feed pump is an excessive feeding of fish that is carried out continuously until the fish that are cultivated are completely full. this practice leads to overfeeding. overfeeding is a bad practice for feeding fish. this excessive feed reduces feed efficiency and increases the amount of feed that is wasted in the environment (deswati & adrison, 2019). excessive feeding in fish farming activities using cages in the ocean is the potential for upwelling. the upwelling that occurs is caused by the buildup or deposition of excessive feed and accumulates below sea level. this triggers a backflow due to changes in water temperature. this backflow will bring the leftover feedback to the surface which can eventually poison the fish in the cage (deswati & adrison, 2019). a study on the cultivation of lutjanus species using floating net cages had been carried out by castillovargasmachuca et al. (2012). it was conducted to observe the growth of pacific red snapper (l. peru). the results of their study stated that the fish had a potential asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 31 growth rate and biomass. the cultivation process using floating net cages also is prosperous to be developed on a commercial scale to meet market demand. another study was conducted by castillo-vargasmachuca et al. (2007) who analyzed the growth of rose snapper (l. guttatus) cultivated in floating cages. they found that the cultivated fish had a high survival rate. subadults have a survival rate of up to 74.7%. however, some aspects need to be considered in cultivating this fish in floating net cages, such as the availability of natural feed. marine cultivation or sea farming in indonesia is highly prosperous as a maritime country with most of its territory consisting of sea water. if this potential can be optimized, the community's economy will also be higher. still, the cultivation process requires strong management effectiveness (sunardi et al. 2020). therefore, in this paper, the authors discussed the bioprospecting of mangrove red snapper cultivation using floating net cages, as well as feed needs and the economic bioprospects. mangrove red snapper (l. argentimaculatus) feed needs the concentration of certain proteins in the feed can affect the rate of feed consumption by farmed fish. improper protein levels will reduce the rate of feed consumption by the fish. a feed with protein concentrations of 32% to 40% did not reduce the rate of fish feed consumption. however, increasing the protein content to 52% reduced the consumption of red snapper feed. as a result, the higher the protein content in the feed, the faster the protein needs to be needed by the fish will be fulfilled so that the consumption of fish feed will decrease. as a result, the rate of feed consumption by sea bass is low when the protein content is too high in the feed (giri et al., 2007). feed efficiency is influenced by the protein content in the feed. feed efficiency is the proportion of the addition of fish biomass to the amount of feed given and consumed by the fish. the higher the feed efficiency value, the better the quality of the fish feed. in red snapper, the highest feed efficiency is in feed containing protein with a concentration of 40% or more than 40%. thus, at this concentration, red snapper were able to utilize their feed efficiently for increasing their biomass (giri et al., 2007). in the red snapper feed, the protein content in the feed was 45.4%, the fat content was 9.3%, the fiber content was 1.7%, the ash content was 13.8%, and the nitrogen-free extract was 29. 8%. in the feed also added other substances such as beta-carotene which will stimulate redfish meat. in addition, some vitamins are added to support the growth process of the snapper (catacutan et al., 2011). vitamin c is very important for the growth of red snapper. the addition of l-ascorbyl-2-monoposfate-mg (amp) affected the growth of the red snapper. the increase in the bodyweight of the fish increased 6 times for 17 weeks. the weight growth decreased on day 60 on media that did not contain amp. fish that were not given amp experienced several abnormalities, such as swelling of the eyes, soft bodies, and abnormalities in the fins. this abnormality is caused by an abnormality in the synthesis of collagen. amp deficiency causes collagen synthesis to be strained so that the process of forming connective tissue in these fish is abnormal (catacutan et al., 2011). protein requirements in the feed are adjusted to the type of fish being farmed. each species of fish requires varying levels of protein. in addition, the protein content of the feed must also be adjusted to the size of the fish, the quality of the protein, the energy content of the feed, the nutritional balance of the feed, and the level of feeding (furnichi, 1988; giri et al., 2007). protein quality is determined by the profile of amino acid content of the protein. the amino acid content in feed determines the adequacy of protein needed by fish (giri et al., 2007). one source of protein feed is fish meal. as for other protein sources, namely vegetable protein derived from nuts, seeds, such as soybeans. however, not all types of fish can utilize these vegetable protein sources properly. the provision of protein in the diet of fish also determines and affects the protein levels of the fish's body. the higher the protein content fed, the higher the protein concentration in the fish's body. in the feed containing 32% protein, the total protein of red snapper was 54.7%. meanwhile, feeding containing 40% protein produced red snapper with 58.7% body protein. meanwhile, feed containing protein with a concentration of 52% resulted in red snapper containing 61.9% protein (giri et al., 2007). feeding done three times a day (morning, afternoon, evening) can increase the amount of feed that is wasted in the environment. in this feeding, as much as 25-30% of the feed given is wasted in the environment. in cultivation with a fast and flowing water environment, an excess feed can be channeled to other areas so that it does not accumulate in that area. however, if cultivation is carried out in a calm aquatic environment, there will be a buildup of feed at the bottom which causes an accumulation of phosphate and nitrate. this will reduce water quality and interfere with the health of the fish (deswati & adrison, 2019). natural feeed found in red snapper (l. argentimaculatus) especially in the juvenile phase is acetes indicus, acetes sp., some members of mysidae, and some zooplankton such as clanoida and melita longidactyla. in addition, they also found feed in the form of members of the luciferidae, alpheidae, and so on (chi & true, 2017). in general, the sufficient protein content in feed ranges from 30% to 55% (nrc, 1993; giri et al., 2007). in carnivorous fish, such as grouper, the protein requirement given in the feed reaches 47% to 60%. red snapper is also a carnivorous fish. thus, the protein 32 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 requirement in a red snapper is also relatively high (giri et al., 2007). research by giri et al., (2007) showed that the protein content in the feed affected the rate of fish weight gain, feed consumption, feed efficiency, and growth of red snapper fry. provision of red snapper feed containing protein with a content of 32% resulted in a low growth rate and mass weight gain of snapper. feeding with a protein content of up to 40% resulted in the maximum growth rate and weight gain of snapper. however, feeding with protein content exceeding 40% did not significantly affect the weight gain and growth of red snapper. thus, in this study, the optimum protein concentration in the feed was 40%. giving excessive levels of protein in the fish feed will cause a negative effect. the negative effect that arises is in the form of slowing the growth rate of fish. excessive feeding results in not having enough energy for fish cells to manage these proteins. this causes an increase in the rate of deamination and excretion of excess amino acids due to the high protein in the feed consumed by fish. in addition, the negative impact of adding feed with too high a protein content is that it can reduce the growth rate of fish (giri et al., 2007). in addition, other nutrient compositions such as vitamin c are also needed. the addition of l-ascorbyl2-monoposfate-mg (amp) affects the growth of red snapper. the increase in the bodyweight of the fish increased 6 times for 17 weeks. the weight growth decreased on day 60 on media that did not contain amp. fish that were not given amp experienced several abnormalities, such as swelling of the eyes, soft bodies, and abnormalities in the fins. this abnormality is caused by an abnormality in the synthesis of collagen. amp deficiency causes collagen synthesis to be tense so the process of forming connective tissue in these fish is abnormal (catacutan et al., 2011). giving amp as much as 60 mg/kg of feed weight is equivalent to giving ascorbic acid as much as 20 mg/kg of feed. this composition is a good composition in overcoming vitamin c deficiency in red snapper. administration of amp in this concentration was effective in supporting the weight growth of red snapper. however, the addition of amp in higher doses had no effect on fish weight gain. addition in too high a dose can be toxic to red snapper cells (catacutan et al., 2011). economic aspects of mangrove red snapper (l. argentimaculatus) cultivation fish is one of the foodstuffs that contain high protein needed by body cells. fish, in general, contain 18-20% protein which is good for the body. thus, fish has become an important commodity in the economy. one of the fish with high economic value is the red snapper (reo, 2013). based on fao, from 2010 to 2013, the average catch of red snapper in indonesia is the highest in the world. the supply of snappers from indonesia reaches 48.3% of the total supply of snappers in the world. this amount is equivalent to more than 120,000 tons. next, followed by the philippines with a total percentage of 8.1%, brazil 7.7%, malaysia 7.3%, mexico 4.9%, nigeria 3.1%, thailand 2.0%, venezuela 1.8%, us 1.8%, and australia 1.6%. meanwhile, according to fao in 2014, the number of import and export markets for snappers has increased significantly. global snapper export activities in 1990 were around 5.000 tons and increased to more than 10.000 tons. meanwhile, global snapper import activity in 1990 amounted to 5.000 tons until in 2010 it has increased to 25.000 tons. meanwhile, the united states is the highest importing country of snapper in the world. the imports come from mexico, brazil, nicaragua, panama, and suriname (amorim & westmeyer, 2016). mangrove red snapper is a high-value aquatic product. market demand for these fishes is also relatively high with relatively high prices. red snapper is the most popular snapper in various restaurants in singapore and hong kong (muyot et al., 2021). based on fao (2018), global red snapper production in 2016 reached 9,815 tonnes, which came directly from fishermen's catch, and 10,240 tonnes came from aquaculture. the highest fish farming comes from southeast asia. many of these fish are cultivated using floating cages placed directly in the ocean and using brackish water ponds (muyot et al., 2021). fish that live in reef areas such as snapper and grouper are a very important resource. from an economic point of view, this fish is one of the leading commodities and is the livelihood of fishermen in indonesia. indonesia's reef fish production reaches 7% of the world's total reef fish production. this amount is one source of foreign exchange for the country (wwfindonesia, 2015). conclusions the factors that influence the success of red snapper (l. argentimaculatus) cultivation using a floating net cage system are the size of the cage, the density of fish in the cage, feeding, and other environmental factors such as temperature, ph, dissolved oxygen, and salinity. analysis of the growth of red snapper (l. argentimaculatus) cultivated using a floating net cage system which includes fish weight gain, absolute growth, specific growth rate, fcr, and survival rate. red snapper is classified as a carnivorous fish so it requires feed with a protein content of 40%. acknowledgements: the first author thanks lembaga pengelola dana pendidikan (lpdp) for the scholarship given to them. also, the second authors thank m. nastain. m.ikom as the research methodology lecturer and didik haryadi santoso s.kom.i.,m.a as the dean asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 33 of the faculty of communication and multimedia, umby, who inspired us to write this article. authors’ contributions: all the authors are involved in idea developing, secondary data collecting, manuscript writing, language editing, and formatting. competing interests: all the authors declare that there is no conflict interest regarding the writing and publishing the article. funding: the authors received no financial funding regarding the data collection, manuscript writing, authorship, and publication of this article. references affan, j. m. 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(2015). better management practices seri panduan perikanan skala kecil. in wwf-indonesia (2nd ed., vol. 2, issue 1/november). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 47-55 | doi: 10.14421/biomedich.2020.91.47-55 issn 2540-9328 (online) prevalence of psychoactive substance use among nigerian male commercial vehicle drivers selected from the three major ethnic groups in nigeria tochukwu frank egwuatu*, onyekachi ogbonnaya iroanya, khalid olajide adekoya department of cell biology and genetics, faculty of science, university of lagos, nigeria corresponding author* tochukwufrankegwuatu@gmail.com manuscript received: 16 june, 2020. revision accepted: 23 july, 2020. published: 26 july, 2020. abstract the use of psychoactive substances for several reasons by commercial vehicle drivers has been documented as a major cause of increased incidences of traffic accidents globally. the study aimed at determining the prevalence of psychoactive substance use among nigerian male commercial vehicle drivers randomly selected from the three major ethnic groups in nigeria (igbo, hausa and yoruba). a structured questionnaire which included data on sociodemographic status and psychoactive drug use history was introduced to all participants. generic multi-drug urine dip card test kit and shimadzu chromatograph system (shimadzu corporation, kyoto, japan) were used for toxicological analyses. out of the 264 nigerian male commercial vehicle drivers who participated in this study, 111(42.0 %) had used illicit drugs in recent times, 55(20.8 %) took drugs for improved energy and 90(34.1 %) felt high after using illicit drugs. marijuana was the most abused substance; 77(29.2 %) followed by benzodiazepine; 34(12.9) across all ethnicities studied. across all ethnicities, hplc revealed that the hausa drivers had the highest concentration of psychoactive substances in their urine samples. these findings, reiterates the need for the nigerian anti-drug agency to develop new strategies in conducting a repressive drug war and extending the same to bus stations. keywords: psychoactive drug; accident; drivers; forensic toxicology; nigeria. introduction forensic toxicology is the science which applies the principles of analytical chemistry and clinical toxicology for medico-legal purpose where the results are likely to be used in court (wyman, 2012; smith and bluth, 2016). forensic toxicology covers human performances such as impaired driving due to drug consumption, use of steroids by athletes and workplace drug testing (jenkins, 2010, wyman, 2012). substance abuse is a patterned use of a drug in which the user consumes the substance in amounts or with methods which are harmful to themselves or others (nutt et al., 2010). psychoactive substances are classified into three main groups: those that alter perception, those that stimulate the brain and those that depress it (vij, 2011). in 2018, the prevalence of any drug use in nigeria is estimated at 14.4 per cent or 14.3 million people aged between 15 and 64 years and the extent of drug use in nigeria is comparatively high when compared with the 2016 global annual prevalence of any drug use of 5.6 per cent among the adult population (unodc, 2018). psychoactive substances are prevalent in biological samples taken from injured drivers at the emergency units of several hospitals. several studies have reported high prevalence rates not only of alcohol but also of medicinal and illicit drugs in such samples (bogstrand et al., 2011; drummer et al., 2012). a lot is known about the magnitude of problems associated with drink-driving and the effectiveness of related countermeasures, but much less is known about driving under the influence of other psychoactive substances. currently, there are no global estimates of deaths resulting from drug-driving, nor is the prevalence of drug-driving known. however, growing recognition of the problem of drug-driving has led to increased focus on this area among road safety policy-makers and researchers (emcdda, 2015). the issues linked with driving under the influence of alcohol and narcotic substances are subject to continuous interest not only for legal practitioners, but also for the police, prosecution, and medico-legal teams (emcdda, 2012). the use of psychoactive substances among commercial vehicle drivers is not only increasing but strangely predisposing the drivers and their passengers to health risks. there is however paucity of empirical data on the factors associated with this increase (yunusa et al., 2017). road traffic injuries claim more than 1.2 million lives each year and have a huge impact on health and development (gopalakrishnan, 2012). road traffic accidents are the leading cause of death among young https://doi.org/10.14421/biomedich.2020.91.47-55 48 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 people aged between 15 and 29 years. the risk of a road traffic death varies significantly by region, and there has been little change in the regional rates of death since 2010. injury and deaths arising from road traffic accidents are a major public health problem in developing countries where more than 85 % of all deaths and 90 % of disability-adjusted life years were lost from road traffic injuries (nantulya and reich, 2002). road traffic accidents have emerged as an important public health issue which needs to be tackled by a multidisciplinary approach (gopalakrishnan, 2012). the highest rates are still in the african region, while the european region has a rate far below the global average (9.3 per 100 000 populations, relative to the global rate of 17.4 (who, 2015). almost half of all deaths on the world’s roads are among those with the least protection motorcyclists, cyclists and pedestrians (who, 2015). motor vehicle accident is a significant cause of mortality and morbidity in nigeria, and driving under the influence of psychoactive substances to improve their performance and keep sleep at bay for as long as possible. the causes of motor vehicle accidents are varied and multi-factorial. however, the use of psychoactive substances plays a major role in the occurrence of motor vehicle accidents. studies in nigeria and other countries have shown a high prevalence of use of psychoactive substances, among various categories of drivers (adekoya et al., 2011). pela (1989) documented the use following drugs in decreasing order of frequency: alcohol, cigarettes, stimulants, cannabis (indian hemp) and sedativehypnotics by adolescents in benin, nigeria. males had been documented to have a higher prevalence of use of all the psychoactive substances (igwe et al., 2009; oshodi et al., 2010). the lifetime use of caffeine, alcohol, and tobacco has been noted to be significantly associated with male gender (akanni and adayonfo, 2015). a significant substance abuse problem in the undergraduate population in botswana with alcohol being the most commonly used substance has also been reported (olashore et al., 2018). the prevalence of psychoactive substances use among middle and high school students in the north center of morocco was comparable to the national prevalence. hence, there is need to initiate psychoactive substance prevention programs among school students which should be based on factors associated with psychoactive substance use (zarrouq et al., 2016). the study aimed at determining the prevalence of psychoactive substance use among nigerian male commercial vehicle drivers randomly selected from the three major ethnic groups in nigeria (igbo, hausa and yoruba). material and method study design, location and population this was a cross sectional study which employed both experimental and quantitative research design. the study adopted the purpose/judgmental sampling technique. the technique is a non-probability sampling method because the participants of interest which were drivers were selected from different motor parks located in anambra, abia (igbo–speaking), kaduna, sokoto (hausa–speaking), oyo and lagos states (yoruba– speaking) ethnic group in nigeria. the target population for the study was the male commercial driver’s population (motorcycle riders, tricycle riders, taxi and bus drivers). ethical approval prior to sample collection, the research details were explained to all the participants after which they willingly consented to participate in the study. questionnaires were completed by all the study participants and informed written consent was obtained. the study protocol was approved by the health research ethics committee (cmulhrec number: cmul/hrec/05/18/349) of the university of lagos, nigeria. permission was also requested and obtained from the national union of road transport workers (nurtw) in all sample states before the study was conducted. sample and data collection approximately 15 ml of urine was collected from each driver who participated in this study using sterile sample bottles. an on-site urinalysis was carried out using 5 ml of each urine sample to determine the general health status of the subjects. afterwards, the remaining urine samples of the subjects were transported to the laboratory for toxicological analysis. each driver in the various motor parks was interviewed until the desired sample size was attained. they responded to a sociodemographics and a semi-structured pro forma, which sought information on the type of substance commonly used, reasons for their use, usage frequency and related accident history. toxicological analysis the urine samples were tested using 10 panel generic multi drug urine dip card test kit. the kit tested for the presence of ten psychoactive drugs (methamphetamine (meth), cocaine (coc), oxycodone (oxy), morphine (mop), amphetamine (amp), methadone (mtd) and barbiturates (bar), marijuana (thc), benzodiazepines (bzo) and phencyclidine (pcp)). the tip of the kit was dipped into the urine sample and allowed to wait for minimum of 10 seconds. the kit had a positive and negative panel for each drug. each sample that tested positive to a particular drug would indicate a red line while negative showed two lines. eqwuatu et al. – prevalence of psychoactive substance use among … 49 quantification of drugs in polysubstance users high performance liquid chromatographic (hplc) analysis was performed with the use of a shimadzu liquid chromatograph equipped with a model lc2010aht gradient pump, cbm-20a system controller and shimadzu class-vp uv-visible detector (shimadzu corporation, kyoto, japan). all chemicals and reference standards (morphine, phencyclidine, benzodiazepine and δ9-tetrahydrocannabinol solution used for the hplc analysis were all of analytical grade. purchased from sigma-aldrich. the chromatographic separations were performed using a sepax br-c18 column (250×4.6 mm; sepax technologies, inc.) with 5µm particle diameter. the column temperature was set at 25°c, and the mobile phase was water (0.1 % formic acid) and acetonitrile (0.1 % formic acid). this was delivered at a flow rate of 0.8 ml/min. the wavelength of the detector was set at 254 nm, and the injection volume of each extract was either 10 µl for assay determination. data analysis all numerical data collected were statistically analysed using statistically package for the social sciences (spss) for windows version 20.0 software. frequency counts and percentage were generated for all variables, and statistical tests of significance were performed with correlation and regression analysis. significance was fixed at p ≤ 0.05 and highly significant if p ≤ 0.01. concentrations of illicit drugs in the urine samples of polysubstance users were deduced from the chromatograms using the formula; concentration of drug (mg/l ) = concentration of standard x area of drug area of standard results out of the 264 nigerian male commercial vehicle drivers who participated in this study, 72 (27.27 %) were hausas, 96 (36.36 %) each were igbos and yorubas respectively. in table 1, the data obtained from the questionnaires revealed that the lowest mean ± se value for age among the three ethnicities studied was 41.47 ± 1.02 years (igbo) while highest was 42.07 ± 1.56 years (hausa). the highest numbers of single (33(34.4 %)) and married (73(76.0 %)) drivers were obtained from yoruba and igbo drivers respectively. also, the lowest numbers of single (13(18.1 %)) and married (59(81.9 %)) drivers were obtained from hausa drivers. table 1. descriptive statistics of sociodemographic parameters in all drivers. parameters variables distribution across ethnicities (%) hausa (n=72) igbo (n=96) yoruba (n=96) age (years) minimum 19.0 21.0 24.0 minimum 72.0 67.0 72.0 mean ± s.e 42.07 ± 1.56 41.47±1.02 41.96 ± 1.21 marital status (%) single 13 (18.1) 23 (24) 33 (34.4) married 59 (81.9) 73 (76) 63(65.6) the study showed that 236 (89.4 %) were bus divers while 15 (5.7 %) and 13 (4.9 %) were car and tricycle drivers respectively. in table 2, igbo drivers had the highest (88 (91.7 %)) number of bus drivers while hausas had the least (63 (87.5 %)). hausa participants had the highest (28 (38.9 %) years) number of experienced divers while those with the highest number of individuals with the lowest (32 (33.3) years) years of experience were igbo drivers. yoruba participants had the highest (59 (61.5 %)) number of drivers who have had accidents followed by the hausa (27 (37.5 %)) and igbo (23 (24.0 %)) drivers respectively. in table 3, hausa drivers had the highest (54 (75.0 %)) number of illicit drug users while the igbo drivers had the lowest (20 (20.8 %)) number. marijuana was indicated to be the most used drug by the drivers compared to other psychoactive drugs across all ethnicities; hausa (36(50.0 %)), igbo (13(13.5 %)) and yoruba (36(37.5 %)). across all ethnic groups, increased energy was observed to the major reason why drivers used illicit drugs. additionally, smoking was indicated as the major mode of administration of illicit drugs among the drivers across all ethnic groups. feeling high after drug use was indicated as the major post administration effect of illicit drug use across all ethnic groups. 50 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 table 2. descriptive statistics of driving experience and accident history parameters in all ethnicities. parameters variables frequency across ethnicities ( %) hausa igbo yoruba vehicle type bus 63 (87.5) 88 (91.7) 85 (88.5) car 7 (9.7) 0 (0) 8 (8.3) tricycle 2 (2.8) 8 (8.3) 3 (3.1) driving experience ˂ 5 years 14 (19.4) 32 (33.3) 22 (22.9) ˂ 10 years 30 (41.7) 38 (39.6) 52 (54.2) ≥ 10 years 28 (38.9) 26 (27.1) 22 (22.9) accident history yes 27 (37.5) 23(24.) 59 (61.5) no 45 (62.5) 73 (76) 37 (38.5) number of times ˂ 5 times 20 (27.8) 20 (20.8) 50 (52.1) ˂ 10 times 5 (6.9) 3 (3.1) 8 (8.3) ≥ 10 times 2 (2.8) 0 (0) 1 (1.0) none 45(62.5) 73 (76) 37(38.5) table 3. descriptive statistics of psychoactive drug use history parameters in all drivers. parameter variable frequency across ethnicities (%) hausa (n=72) igbo (n=96) yoruba (n=96) have you used drugs before? yes 54 (75) 20 (20.8) 37 (38.5) no 18(25) 76 (79.2) 59 (61.5) type of drug maijuana 36 (50) 13 (13.5) 36 (37.5) tablet 18 (25) 6 (6.3) 1 (1) syrup 0(0) 1 (1) 0 (0) none 18(25) 76 (79.2) 59 (61.5) reason for drug use alertness 6 (8.3) 4 (4.2) 10 (10.4) increased energy 26 (36.1) 10 (10.4) 19 (19.8) improve sexual performance 2(2.8) 1 (1.0) 4 (4.2) pleasure 18 (25) 2 (2.1) 1 (1) peer pressure 2(2.8) 3 (3.1) 3 (3.1) none 18(25) 76 (79.2) 59 (61.5) did you use it in the past 90 days? yes 54 (75) 18 (18.8) 36 (37.5) no 0(0) 2 (2.1) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) frequency of use everyday 35 (48.6) 18 (18.8) 32 (33.3) weekends 8 (11.1) 2 (2.1) 4 (4.2) weekly 11(15.3) 0 (0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) last time used last 24 h 33 (45.8) 19 (19.8) 34 (35.4) 1-3 day(s) ago 12 (16.7) 1 (1) 2 (2.1) last week 9 (12.5) 0 (0) o (0) ≥ 1 month 0 (0) 0 (0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) mode of administration smoking 35 (48.6) 14 (14.6) 36 (37.5) ingestion 19(26.4) 6 (6.3) 1 (1.0) none 18 (25) 76 (79.2) 59(61.5) do you feel addicted to it? yes 13 (18.1) 6 (6.3) 15 (15.6) no 41(56.9 14 (14.6) 22 (22.9) none 18 (25) 76 (79.2) 59 (61.5) how do you get it? park sellers 52 (72.2) 14 (14.6) 29 (30.2 private suppliers 2(2.8) 6 (6.3) 8 (8.3) none 18 (25.) 76 (79.2) 59 (61.5) is the drug costly? yes 9 (12.5) 3 (3.1) 3 (3.1) no 45(62.5) 17 (17.7) 34 935.4) none 18 (25) 76 (79.2) 59(61.5) can you do without it? yes 21 (29.2) 12 (12.5) 23 (24) no 33 (45.8) 8 (8.3) 14 (14.5) none 18 (25) 76 (79.2) 59 (61.5) feeling after use high 35.00 (48.6) 19 (19.8) 36 937.5) sleepy 19 (26.4) 1 (1.0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) eqwuatu et al. – prevalence of psychoactive substance use among … 51 table 4 shows the distribution of the participants by psychoactive drug use history. out of the 264 participants across all ethnic groups, 111(42.0 %) had used illicit drugs in recent times while 153(58.0 %) did not, 55(20.8 %) abused illicit drugs for increased energy and 85(32.2 %) used illicit drugs daily. additionally, 90(34.1 %) of the respondents indicated that they felt high after taking illicit drugs. table 4. distribution of the respondents by psychoactive drug use history. parameter variable frequency ( %) have you used drugs before? yes 111 (42) no 153 (58) reason for drug use alertness 20 (7.6) increased energy 55 (20.8) improve sexual performance 7 (2.7) pleasure 21 (8) peer pressure 8 (3) none 153 (58) frequency of use everyday 85 (32.2) weekends 13 (4.9) weekly 13(4.9) none 153 (58) mode of administration smoking 85 (32.2) ingestion 26 (9.8) none 153 (58) do you feel addicted to it? yes 35 (13.3) no 76 (28.8) none 153 (58) how do you get it? park sellers 94 (35.6) private suppliers 17 (6.4) none 153 (58) feeling after use high 90 (34.1) sleepy 21 (8) none 153 (58) n= 264 pearson correlation analysis was carried out to determine the relationship between sociodemographics, driving experience history and psychoactive drug use history. across all ethnic groups (table 5), ethnicity correlated with drug use, drug name, reason for drug use, frequency of drug use, feeling of addiction, acquisition of drugs and cost and post administration effects of drug use at 0.01 level of significance. however, there was no correlation between ethnicity and mode of administration (p≥0.05). accident history correlated with drug use, drug name, reason for drug use, frequency of drug use, mode of administration, feeling of addiction, acquisition of drugs and cost and post administration effects of drug use at 0.01 level of significance. all urine samples were analysed using drug test kits. in table 6, marijuana 77(29.2 %) was the most abused substance followed by benzodiazepine 34(12.9 %) across all ethnicities studied. marijuana had the highest 32(44.4 %) number of users among the hausa drivers, followed by benzodiazepine 28(38.9 %) while the least was morphine 3(4.2 %). for the igbo drivers, the use of marijuana was the highest 14(14.6 %), followed by methadone 10(10.4 %) with the least being morphine and phencyclidine 1(1 %). amongst the yoruba drivers, marijuana was also most abused substance 31(32.3 %) followed by benzodiazepine 4(4.2 %) while the least abused drugs were barbiturate and methadone 1(1 %) each. across all ethnicities, hausa drivers had the highest number of polysubstance users compared to other ethnic groups as shown in table 7. 52 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 table 5. correlation between sociodemographics, driving experience history and psychoactive drug use history in ethnic groups. p a r a m e te r d r u g u se n a m e o f d r u g s r e a so n f o r d r u g u se u se d p a st 9 0 d a y s f r e q u e n c y o f u se l a st t im e u se d m o d e o f a d m in is tr a ti o n f e e li n g o f a d d ic te d h o w d o y o u g e t it is t h e d r u g c o st ly c a n y o u d o w it h o u t it f e e li n g a ft e r u se age 0.02 -0.02 -0.09 -0.02 0.01 -0.01 -0.02 -0.05 -0.00 0.00 0.03 -0.04 ethnicity 0.26** 0.20** 0.18** 0.26** 0.19** 0.22** 0.12 0.18** 0.39** .026** 0.17** 0.19** marital status -0.02 -0.02 -0.07 -0.04 0.02 0.00 -0.03 -0.07 -0.03 -0.02 -0.02 -0.03 vehicle type -0.04 -0.02 .028 0.01 -0.05 -0.05 -0.02 -0.02 0.01 -0.04 -0.08 -0.05 driving experience -0.01 -0.04 -0.09 -0.05 -0.02 -0.02 -0.03 -0.02 -0.03 0.00 -0.00 -0.03 accident history 0.33** 0.37** 0.34** 0.31** 0.34** 0.35** 0.37** 0.36** 0.18** 0.29** 0.31** 0.36** number of times 0.26** 0.30** 0.28** 0.25** 0.26** 0.27** 0.30** 0.30** 0.14* 0.25** 0.24** 0.28** * = correlation is significant at the 0.05 level. ** = correlation is significant at the 0.01 level. table 6. frequency distribution of drugs across ethnic groups rom toxicological testing. ethnicity drug thc (%) mop (%) bar (%) coc (%) oxy (%) amp (%) met (%) pcp (%) bzo (%) mtd (%) hausa (n=72) 32 (44.4) 3 (4.2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (5.6) 28 (38.9) 0 (0) igbo (n=96) 14 (14.6) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (1) 2 (2.10) 10 (10.4) yoruba (n=96) 31 (32.3) 3 (3.1) 1 (1) 0 (0) 0 (0) 0 (0) 2 (2.1) 2 (2.1) 4 (4.2) 1 (1) all ethnicities (n=264) 77 (29.2) 7 (2.7) 1 (0.4) 0 (0) 0 (0) 0 (0) 2 (0.8) 7 (2.7) 34 (12.9) 11 (4.2) key: thc = tetrahydrocabinnol bar = barbiturate pcp = phencyclidine coc = cocaine mop = morphine amp = amphetamine mtd = methadone bzo = benzodiazepines oxy = oxycodone met = methamphetamine table 7. frequency distribution of polysubstance users across all ethnic groups. ethnicity drug bzo+thc (%) thc+pcp (%) thc+mop (%) bzo+thc+pcp (%) bzo+thc+mop (%) pcp+thc +mop (%) bzo+mop+ pcp (%) hausa (n=72) 1 (1.4) 1 (1.4) 0 (0) 3 (4.2) 2 (2.8) 0 (0) 1 (1.4) igbo (n=96) 2 (2.1) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) yoruba (n=96) 1 (1) 0 (0) 2 (2.1) 1 (1) 1 (1) 1 (1) 0 (0) all ethnicities n=264 4 (1.5) 2 (0.8) 2 (0.8) 4 (1.5) 3 (1.1) 1 (0.4) 1 (0.4) key: thc = tetrahydrocabinnol, bzo = benzodiazepine, mop = morphine, pcp = phencyclidine chromatographic analysis was performed using the urine samples of participants who tested positive to two or more drugs. table 8 shows that the polysubstance users used mainly marijuana, benzodiazepine, morphine and phencyclidine. across all ethnicities, the hausa drivers had the highest concentration of psychoactive substances in their urine samples. the chromatographs did not show thc only in samples h14, h17 and ig58. tetrahydrocabinnol and bzo only were not detected by hplc in samples h53 and h66. in samples h16 and y22, bzo only and mop only were not detected by hplc respectively. also, bzo and mop were not detected by hplc in sample h07 but pcp was detected. eqwuatu et al. – prevalence of psychoactive substance use among … 53 table 8. concentrations of different drugs in polysubstance users. ethnicity subject drug concentrations in urine (mg/l) pcp thc bzo mop hausa h07 1321.3 671.7 0 0 h12 0 210.6 15.9 0 h14 0 0 0.02 0 h16 0 0.2 0 0.5 h17 0 0 0.1 93 h40 10.7 8 0. 0 h53 122.9 0 0 0 h66 167.4 0 0 0 igbo ig25 34.7 8.7 0 0 ig36 0 0.9 1.2 0 ig58 0 0 2.3 0 yoruba y03 108.1 25.6 0 1.2 y04 0 19.5 0 32.8 y06 0.02 0.8 1.2 0 y10 0 0.8 1.5 0 y21 0 0.1 0.3 0.2 y22 0 0.1 0 0 standards: pcp = (2.5) thc = (2.4) mop = (3.99) bzo = (2.39) key: mop = morphine pcp = phencyclidine bzo = benzodiazepines thc = tetrahydrocabinnol h = hausa ig = igbo y = yoruba discussion the ages of the commercial vehicle divers who participated in this study ranged from 19 to 72 years old. majority of the drivers studied fell with the age range (15-64 years) given by unodc, (2018) as the age range that has the highest prevalence of drug use. across all ethnic groups, 111 (42.0 %) of the respondents had used illicit drugs in recent times and this value is higher than what was reported in the report of iroanya et al., (2018) who reported that only 5.7 % of their subjects had taken other stimulants aside alcohol in recent times. the study revealed that 55 (20.8 %) abused illicit drugs for increased energy, 20(7.6 %) for alertness, 21(8.0 %) for pleasure and 85 (32.2 %) used illicit drugs daily. additionally, 90 (34.1 %) and 21(8.0 %) of the respondents indicated that they felt high or sleepy respectively after taking illicit drugs. these findings agree with the report of yunusa et al., (2017) who stated that the desires to relax/sleep after a hard days job (84.8 %), work hard (48 %), relieve stress (81 %), relieve anxiety (66.5 %) and pleasure (72 %) were the major factors associated with the abuse of substances by the respondent. this study revealed that 52(72.22 %) of drivers from the hausa ethnicity used one or more psychoactive substance(s) and this agrees with the study of yunusa et al., (2017) who reported that eight out of every ten (81.1 %) of drivers who participated in their study has abused a substance. this study showed that 25(26.04 %) of igbo drivers used one or more drug(s) and this is lower than the findings of aniedu and okonkwo, (2008) who reported the prevalence of psychoactive drug use amongst taxi drivers to be 85.4 %. it was also observed that 36(37.5 %) of the yoruba drivers also used one or more psychotropic substance(s) and this is also higher than the report of iroanya et al, (2018) who reported that 24.6 % of their participants tested positive to at least one psychoactive drug in a study conducted at two bus stations in lagos state, nigeria. additionally, 113(42.8) of the participants across all ethnic groups tested positive to one or more psychoactive substance(s). this is lower than the finding of hamman et al., (2018) who documented that 57.7 % of the subjects tested positive for substance abuse in a study conducted among professional minibus drivers at zagazig city, sharqia governorate, egypt. the study showed that the substances commonly used by the drivers were marijuana (29.2 %), benzodiazepine (12.9 %), methadone (4.2 %), phencyclidine (2.7 %), morphine (2.7 %) and methamphetamine (0.8 %). this is significantly higher than the findings of bogstrand et al., (2012) who reported that most commonly found substances among drivers in normal traffic were cannabis (0.7 %), benzodiazepines (0.8 %), methadone (0.1 %), methamphetamine (0.2 %) and morphine (0.0 %). from this study, marijuana was the most used drugs among the drivers who participated in the study. this agrees with the report of unodc, (2018) which reported that cannabis was the most commonly used drug in the nigerian population. hamman et al., (2018) also reported that the most common abused substance was cannabis which represented 80 % of the positive participants. cannabis is the world’s most commonly used illicit drug which occupies fourth place in worldwide popularity amongst psychoactive drugs, after caffeine, nicotine and alcohol (vij, 2011). it was also observed from this study that the use of benzodiazepine by the subjects was also common after marijuana’s use. this disagrees with the report unodc (2018) which stated that cannabis’ use was followed by opioids. interestingly, the use of methodone was highest among the igbo drivers. this disagrees with the findings of aniedu and okonkwo, (2008) who reported that the commonest drugs used by taxi drivers in enugu were alcohol, tobacco and central nervous system stimulants such as coffee and kolanut. it was observed that none of the drivers across all ethnic groups tested positive to cocaine, oxycodone and amphetamine. this might be due to the unavailability of these drugs within their environments, moreover, they are also very expensive. this however is in disagreement with the findings of dini et al., (2019) who documented that 21.3 % and 2.2 % of the truck drivers who took part in their study consumed amphetamine and cocaine respectively. this study revealed that the hausa drivers had the highest number of polysubstance users. the concentrations of pcp, thc and bzo were generally higher in the hausa 54 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 drivers than other ethnic groups. this agrees with the report of ndlea (2011) regarding the urgent need for coordination and collaboration between tiers of government to tackle the scourge of drug abuse in the northern nigeria. it has been predicted that the use of illicit drugs kill around 0.2 million people each year, shattering families and bringing misery to thousands of other people. illicit drugs undermine economic and social development and contribute to crime, instability, insecurity and the spread of hiv (ndlea, 2011). conclusion and recommendation the prevalence of hypertension among the nigerian male commercial drivers in the three major ethnicities studied is high and of public health importance. the threat of drug abuse is real and enormous. hence, the nigerian government and relevant non-governmental organizations should come up with viable strategies on how to identify areas with high drug addiction prevalence, train specialist staff to handle drug abuse issues, educate drivers and the public on the negative effects of drug abuse. this study has revealed that a sizable proportion of the nigerian male commercial drivers operating in the selected bus stations in anambra, abia, kaduna, sokoto, oyo and lagos states, nigeria use psychoactive substances as diagnosed by the drug kits. it is very crucial for the nigerian anti-drug agency (national drug law enforcement agency (ndlea)) to develop new tactics in conducting a repressive drug war, and make attempts to understand the psychological world of addicts in order to formulate appropriate policies against drug abuse. there can be no better time and appropriate turning point than now. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references akanni, o. o. and 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2018. drug use in nigeria. vienna. 61pp. eqwuatu et al. – prevalence of psychoactive substance use among … 55 vij, k. 2011. forensic medicine and toxicology: principles and practice. 5th edition. elsevier: a division of reed elsevier india private limited. india. 445pp. world health organization (who). 2015. global status report on road safety. geneva. 340pp. wyman, j. 2012. principles and procedures in forensic toxicology. clinics in laboratory medicine. 32:493-507. yunusa, u., bello, u. l., idris, m., haddad, m. m. and adamu, d. determinants of substance abuse among commercial bus drivers in kano metropolis, kano state, nigeria. 2017. american journal of nursing science. 6(2): 125-130. zarrouq, b.,el asri, b. a., achour, s., rammouz, i., aalouane, r., lyoussi, b., khelafa, s., bout, a., berhili, n., hlal, h., najdi, a., nejjari, c. and el rhazi, k. 2016. psychoactive substances use and associated factors among middle and high school students in the north center of morocco: a crosssectional questionnaire survey. bmc public health. 16(468): 1–9. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 69-78 | doi: 10.14421/biomedich.2023.121.69-78 issn 2540-9328 (online) combined anthocleista vogelii and alstonia boonei stem barks extract alleviates hyperlipidaemia and renal malfunctions in benign prostatic hyperplasia-induced rats robert ikechukwu uroko1,*, mercylyn ezinne uche2, paul chukwuemaka nweje-anyalowu3, ikenna obiwuru2, chinedu aguwamba3, chinomso friday aaron2 1department of biochemistry, college of natural sciences, michael okpara university of agriculture, umudike, abia state, nigeria 2department of biochemistry, faculty of biological and physical sciences, abia state university, uturu, nigeria. 3biochemistry unit, department of chemical sciences, faculty of sciences, clifford university, owerrinta, abia state, nigeria. corresponding author* ir.uroko@mouau.edu.ng manuscript received: 28 july, 2022. revision accepted: 19 august, 2022. published: 19 october, 2022. abstract benign prostatic hyperplasia (bph) is a urological disease prevalent among the ageing male population, which impairs the quality of life, including hyperlipidaemia and a decline in renal functions. combining anthocleista vogelii and alstonia boonei stem bark extract has effectively managed bph and its associated complications. this study evaluated the effects of a combined anthocleista vogelii and alstonia boonei stem bark extract (caasbe) on the lipid profile and renal functions of rats induced benign prostatic hyperplasia with testosterone propionate injection. the study comprised five treatment groups, with groups 1 – 5 being the normal control, bph control, standard control, bph+200 mg/kg caasbe, and bph+400 mg/kg caasbe, respectively. bph was induced in the groups 2 – 4 rats by subcutaneous administration of testosterone propionate injection (5 mg/kg) for 28 days, and treatment with finasteride and caasbe were administered orally. the bph control rats exhibited a significant (p < 0.05) increase in the total serum cholesterol, triacylglycerol (tag), low-density lipoprotein cholesterol (ldl-c), urea, creatinine and significant (p < 0.05) decline in the serum high-density lipoprotein cholesterol (hdl-c) compared to the normal control. conversely, treatment of the bph rats with 200 and 400 mg/kg of caasbe significantly (p < 0.05) reversed the altered total serum cholesterol, tag, ldl-c, hdl-c, urea and creatinine to normal levels comparable to that of the normal control and standard control respectively. these findings show that caasbe alleviates hyperlipidaemia and renal malfunctions in the bph rats suggesting it could be effective in managing bph complication. keywords: alstonia boonei; anthocleista vogelii; benign prostatic hyperplasia; hyperlipidaemia; renal functions. abbreviations: bph = benign prostatic hyperplasia; tag = triacylglycerol; hdl-c = high density lipoprotein cholesterol; ldl-c = low-density lipoprotein cholesterol; caasbe = combined anthocleista vogelii and alstonia boonei stem bark; a. vogelii = anthocleista vogelii; a. boonei = alstonia boonei. introduction among all the ailments and medical conditions affecting men's quality of life, benign prostatic hyperplasia (bph) remains the most challenging urological disorder confronting the ageing male population worldwide. the probability of senior male increases as he attains 50 years old based on available autopsy that 42 % and 85 % of men within 51-60 and 80-above years respectively had bph condition (gacci et al. 2017). the increased prostate stromal and epithelial cell multiplication and proliferation in response to the high circulating testosterone and dihydrotestosterone have been implicated in the bph pathogenesis. there is a decline in the quality of life as bph progresses because of complications, including urinary disorder occasioned by bladder outlet obstruction and lower urinary tract symptoms, which are difficult to manage. genetic factors influence bph pathogenesis as some races have an increased risk of developing bph than others, but recently dietary habits, physical inactivity, and unhealthy lifestyle have been shown to increase the risk of bph in ageing men (vignozzi et al. 2014). abnormal lipid profile, obesity, diabetes inflammatory conditions and hypertension are linked to bph development and progression. effective management of these common metabolic disorders in ageing men could be key to staying from bph complications (dahle et al. 2002). aged men with abdominal obesity and abnormal serum lipid profile had a very low amount of high-density lipoprotein cholesterol (hdl-c), along with elevated low-density lipoprotein cholesterol (ldl-c), total cholesterol, and triacylglycerols demonstrated the increased amount of testosterone and oestradiol which https://doi.org/10.14421/biomedich.2023.121.69-78 70 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 increased their risk for bph (adaramoye et al. 2008; ugwu et al. 2019). the role of dyslipidaemia in the bph pathogenesis is believed to be through stimulation of increased synthesis of androgens, including testosterone, dihydrotestosterone and oestradiol hormones that promote the growth of prostate cells. therapeutic agents against bph pathogenesis target and inhibit 5αreductase enzyme activities, thereby preventing the conversion of testosterone to dihydrotestosterone, which retard the multiplication of prostate stromal and epithelial cells and shrink the prostate size. a suitable therapeutic agent against bph pathogenesis must be able to prevent dyslipidaemia in the subject to achieve the desired end. anthocleista vogelii planch belongs to the loganiaceae family utilised for various medicinal purposes in africa. the stem bark has been therapeutically effective against prostate enlargement, male infertility, obesity, hyperlipidaemia, and renal and hepatic disorders (sunday et al. 2016; chukwu et al. 2020; uroko et al. 2021). a phytochemical study of anthocleista vogelii has revealed that it is rich in alkaloids, phenol, flavonoids, saponins, glycosides, terpenoids, steroids and tannins with minerals and vitamins (ikpe et al. 2020). a. vogelii is used as a therapeutic against sleeping sickness in traditional medicine. various experimental studies involving animal models have shown that it has anti-trypanosomal and anti-inflammatory activities (eze et al. 2019). it has wide medicinal properties and has been useful in managing various ailments, including diabetes, jaundice, pains, venereal diseases, malaria, ulcer, and prostate enlargement (okon et al. 2014; chukwu et al. 2020). alstonia boonei de wild is a well-known medicinal plant from the family of apocynaceae that has demonstrated high therapeutic potential against many diseases and medical conditions confronting man. the plant is found mainly in the tropical rainforest across west african countries, but it can also be found in varying proportions worldwide. the plant extracts are pharmacologically against asthma, ulcers, toothache and impotence (akinmoladun et al. 2007). the stem bark of alstonia boonei has been reported to contain substantial amounts of phytochemicals such as flavonoids, phenols, alkaloids, tannins, cardiac glycosides, and saponins, along with high mineral contents and antioxidant vitamins, which are responsible for some of its medicinal properties (akinmoladun et al. 2007; oppongbekoe et al. 2020). traditionally, a. boonei is used for treating menstrual pains, snake bites, the expulsion of residual placental in women after birth, bone remodelling, lactation induction, venereal diseases, jaundice, diabetes, rheumatism, malaria, and bacterial infections (abbiw 1990). it has also been reported to possess anti-anaemic effects, antioxidative stress and hepatoprotective properties (uroko et al. 2020; okpashi et al. 2022). this study evaluated the pharmacological impact of combined anthocleista vogelii and alstonia boonei stem bark extract (caasbe) on lipid profile and renal functions of rats induced benign prostatic hyperplasia (bph) with testosterone propionate injection. materials and methods plant material five hundred (500) g each of the coarsely ground a. vogelii leaves and a. boonei stem barks corresponding to 1000 g of the combined plant samples were weighed into a clean, sterile container, and 2.5 l of absolute ethanol were to macerate for three days under regulation agitation. the macerated combined plant sample was filtered with a whatman no. 1 filter paper, and the filtrate was concentrated with a rotary evaporator. the concentrated caasbe was weighed, and the percentage yield was calculated as 9.78 %, corresponding to 97.8 g of caasbe. experiment animals this study used thirty mature male wistar albino rats within a close body range. chemicals and drugs the finasteride, testosterone propionate injection and pentobarbital were purchased from merck company, usa; health biotech ltd, india; and medica men biotech limited, india. the analytical grade absolute ethanol solvent and randox commercial assay kits were purchased from sigma aldrich, usa, and randox laboratories, uk. study design male wistar albino rats numbering thirty with a close weight range of 160-168 g were purchased from the animal breeding unit of the department of zoology, university of nigeria nsukka, nigeria, and kept in our animal house for seven days. the rats were provided with a standard finisher feed and drinking water and humanely handled following normal regulations and guidelines for the use of animals for an experiment. our study design adopted five groups containing six male wistar albino rats of similar body weight. the study groups are as follows:  normal control: rats without bph induction received only 1 ml/kg of distilled water.  bph control: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection for 28 days consecutively without any treatment.  standard control: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 5 mg/kg finasteride for 28 consecutive days. uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 71  bph+ 200 mg/kg caasbe: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 200 mg/kg caasbe for 28 consecutive days.  bph+400 mg/kg caasbe: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 400 mg/kg caasbe for 28 consecutive days. the rats fasted overnight on the 28th day of the experiment. they were anaesthetised on the 29th by the intraperitoneal injection of 25 mg/kg pentobarbital, followed by blood collection through cardiac puncture biochemical analyses and harvesting of kidney tissues for histopathological evaluation. determination of lipid profile, serum urea and creatinine concentrations the lipid profile parameters, including total serum triacylglycerols (tag), cholesterol, high-density lipoprotein cholesterol (hdl-c), and low-density lipoprotein-cholesterol (ldl-c), were measured following the procedures outlined in the randox commercial assay kit for lipid profile. also, the serum urea and creatinine concentrations were measured using the assay procedures in their respective randox commercial assay kit. histopathological examination of kidney tissues the kidney sections collected from each of the five groups for the histopathological examination were fixed in 10% phosphate-buffered formalin for two days. the tissues were dehydrated in four different alcohol strengths in ascending order. the histopathological examination of the kidney sections was carried out according to the procedures outlined by uroko et al. (2020). ethics approval the study design was reviewed by the ethical board of the department of physiology, biochemistry, and pharmacology, michael okpara university of agriculture, umudike and approved with reference number: mouau/vpp/ec/18/005. statistical analysis all the data generated from the study were compared and analysed for statistically significant differences with the duncan multiple range comparison test and one-way analysis of variance (anova) using statistical products and service solutions version 22. mean with a p-value < 0.05 are considered statistically different, while a mean with a p-value > 0.05 indicated no significant difference. results and discussion results effects of caasbe on the serum tag concentrations of bph-induced rats figure 1 indicated a substantial elevation in the tag concentration of the bph control compared to the normal control. the standard control and bph rats, which received 200 and 400 mg/kg caasbe, showed no significant decline in serum tag concentrations relative to the normal control. conversely, the standard control that received 5 mg/kg finasteride and bph rats that received 200 and 400 mg/kg caasbe showed significantly reduced serum tag concentrations compared with the bph control. figure 1. serum tag concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum hdl-cholesterol concentrations of bph rats the result in figure 2 showed a significant reduction in the serum hdl-cholesterol concentration of the bph control relative to the normal control. the standard control administered finasteride, and bph rats that received 200 and 400 mg/kg caasbe, respectively, demonstrated no significant decline in the serum hdl0,00 0,50 1,00 1,50 2,00 2,50 3,00 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbet s e ru m t a g . c o n c. ( m m o l/ l) treatment groups a b a a a 72 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 cholesterol concentrations compared to the normal control. conversely, the serum hdl-cholesterol concentrations in the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, exhibited a significant increase in the serum hdlcholesterol concentration compared to the bph control. figure 2. serum hdl concentrations in bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the total serum cholesterol concentrations of bph -induced rats figure 3 showed a significantly elevated total serum cholesterol concentration in the bph control compared with the normal control. however, there was no significant variation in the total serum cholesterol concentration in the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, compared with the normal control. conversely, the total serum cholesterol concentrations in the standard control and bhp rats treated with 200 mg/kg caasbe decreased significantly compared to the bph control. figure 3. total serum cholesterol concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum ldl-cholesterol concentrations of bph rats figure 4 demonstrated a significant rise in the serum ldl-cholesterol concentration of the bph control rats compared to the normal control. contrarily, the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, showed no significant increase in the serum ldl-cholesterol concentration relative to the normal control. the level of serum ldl-cholesterol in the standard control and bph rats treated with 200 and 400 mg/kg caasbe were significantly reduced compared to the bph control. figure 4. serum ldl-cholesterol concentrations of bph treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. 0,00 0,50 1,00 1,50 2,00 2,50 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbe s e ru m h d l c o n c. ( m m o l/ l) treatment groups b a b bb 0,00 1,00 2,00 3,00 4,00 5,00 6,00 normal control bph control standard control 200 mg/kg caasbe 400 mg/kgcaasbe t o ta l se ru m c h o e st e ro l (m m o l/ l) treatment groups a b a a a 0,00 0,50 1,00 1,50 2,00 2,50 3,00 normal control bph control standard control 250 mg/kg caasbe 500 mg/kg caasbe m e a n l d l c o n c. ( m m o l/ l) treatment groups a b a a a uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 73 effects of caasbe on the serum urea concentrations of bph rats figure 5 indicated a significant increase in the serum urea concentration of the bph control compared to the normal control. there were mild variations in the serum urea concentrations of the standard control and bph rats treated with 200 and 400 mg/kg caasbe, respectively, compared to the normal control. the serum urea concentrations of the standard control and bph rats administered varying doses of caasbe showed significant reduction compared with the bph control. figure 5. serum urea concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum creatinine concentrations of bph rats the serum creatinine concentration in the bph control was significantly elevated compared to the normal control, standard control and bph rats administered 200 and 400 mg/kg caasbe, respectively (figure 6). in contrast, the standard control and bph rats treated with 200 and 400 mg/kg caasbe showed no significant difference in their serum creatinine concentrations relative to the normal control. figure 6. serum creatinine concentrations in bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the kidney histomorphology of bph rats the kidney section from the normal control rats indicated a normal renal histomorphology for laboratory rodents, containing normal glomeruli (g) in the cortex containing intact renal tubules indicated by the arrow. the renal tubules also contain unaltered outer and inner medulla, as shown in figure 7 a. similarly, the sections of kidneys from bph control rats (fig. 7b), standard control (fig. 7 c), and bph induced rats treated with 200 mg/kg and 400 mg/kg of caasbe in fig. 7 d and fig. 7 e, respectively, showed normal kidney histomorphology of normal rodents. 0 10 20 30 40 50 60 normal control bph control standard control 200 mg/kgcaasbe 400 mg/kg caasbe m e a n u re a c o n c. ( m g /d l) treatment groups a b a a a 0,00 0,50 1,00 1,50 2,00 2,50 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbe s e ru m c re a ti n in e c o n c. ( m g /d l) treatment groups a b a a a 74 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 figures 7. a e. histomorphology of kidney section from normal control rat, bph control rat, standard control, bph rats treated with 200 mg/kg caasbe, and bph rat treated with 400 mg/kg caasbe, respectively. discussion the combined a. vogelii and a. boonei stem bark (caasbe) is a potent therapeutic agent against bph and several diseases in traditional medicine across south-eastern nigeria. it has little scientific data on its effects on other biochemical and physiological functions. the bph is a medical condition common in the ageing male population worldwide but with increased occurrence in males of african origin. it is caused by prostate enlargement occasioned by the rapidly dividing and proliferating prostate stromal and epithelial tissues in response to the stimulatory actions of increased dihydrotestosterone (dht) concentration (gacci et al. 2017). this study evaluated the effects of a combined caasbe on the lipid profile and renal functions in benign prostatic hyperplasia (bph) induced rats. in this study, the bph induction without administration of any therapeutic agent resulted in a significant elevation of the serum tag, cholesterol, ldl, urea and creatinine concentrations along with a significantly reduced hdl concentration in the bph control compared to the normal control. the significantly elevated serum tag concentration in the bph control relative to the control indicates the adverse effect of bph on lipid metabolism, which promoted hyperlipidaemia in the bph rats. the elevated serum tag level in the bph control agrees with ugwu et al. (2019) that high serum tag level is associated with bph pathogenesis. the bph control rats could have experienced impaired carbohydrate catabolism and conversion of excess amount of free fatty acids resulting in increased serum tag concentration due to the decline in the transport of tag to the liver for catabolism because of the inadequate amount of hdl needed for the vehicle. the bph rats could have suffered acute pancreatic inflammation that impaired carbohydrate metabolism and elevated serum tag concentration. the bph induction might have impaired the metabolism of tag ingested from dietary sources that could have contributed to the increased circulating serum tag concentration. the effect of bph on tag indicates that increased serum tag is associated with bph progression, which predisposes the bph patient to the increased risk of arteriosclerosis, cardiovascular dysfunction and paralysis of some parts of the body. this finding agrees with güven and gökhan (2022) report that hyperlipidaemia could accelerate bph progression. the substantially reduced serum tag concentration in all the bph rats administered caasbe relative to the bph control but comparable to the normal control and standard control, respectively, could be attributed to the antihyperlipidemic effects of caasbe. the caasbe might be rich in bioactive phytoconstituents such as flavonoids, alkaloids, terpenoids and vitamins that could be responsible for lowering the serum tag concentrations in the bph rats. the low serum tag concentrations in the caasbetreated bph rats indicated improved carbohydrate uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 75 metabolism and efficient transport of tag to the liver for catabolism which might have contributed to reduced risk of stroke, hypertension and heart diseases which are associated with abnormal serum tag levels in line with güven and gökhan (2022). the hdl cholesterol is a very important type of cholesterol that maintains a healthy balance of lipid profile via its role in moving ldl-cholesterol, potentially unhealthy cholesterol, to the liver for catabolism and elimination from blood circulation. aside from the clearance of ldl-cholesterol from clogging the arterial walls, many researchers have suggested that hdl possesses antioxidant, antiinflammation and anticoagulant properties. the significantly decreased serum hdl-cholesterol concentration in the bph control showed the adverse effects of bph on the serum hdl-cholesterol concentration. the reduction in the hdl level in the bph control suggests that alterations in lipid profile, especially the serum hdl-cholesterol concentration, are associated with the bph pathogenesis, and adequate regulation or restoration of altered lipid profile could play an important role in alleviating the adverse effects of bph on general body functions. the reduced hdlcholesterol level in the bph control is in line with the findings of gacci et al. (2017) that lower hdlcholesterol level is associated with prostate enlargement and that hyperlipidaemia could accelerate bph progression by promoting prostate cell inflammation. the low serum hdl-cholesterol concentration in the bph control has great health consequences as there could be a decline in the transport of ldl-cholesterol and other cholesterol molecules to the liver for metabolism and clearance, which will increase the risk of narrowing the arterial walls and heart diseases as earlier reported by uroko et al. (2021). however, the significantly increased serum hdl-cholesterol concentrations in the bph rats treated with finasteride and caasbe, respectively, compared to the bph control showed the anti-dyslipidaemia effects of the caasbe against bph-associated alterations in the lipid profile. the high serum hdl-cholesterol level in the caasbe-treated bph rats is indicative that there would be effective extraction of ldl-cholesterol from the blood vessels to the liver for breakdown, thereby freeing the arteries from the deposition of ldl-cholesterol, narrowing, increased blood pressure and complications that might result in heart diseases. thus, treating bph rats with caasbe improves serum hdl-cholesterol levels and prevents adverse health effects associated with its low blood level. the increased hdl in the caasbe-treated bph rats agrees with gacci et al. (2017). cholesterol is an important lipid profile component in synthesising bile acids and many steroid hormones, including gonadal and adrenal steroid hormones, and maintaining membrane structure and integrity. still, its high serum concentration could accelerate the risk of developing cardiovascular diseases. the substantially increased serum cholesterol concentration in the bph control compared to the normal control demonstrated that bph progression is linked to the abnormally elevated serum cholesterol concentration. the increased serum cholesterol level in the bph control aligns with previous findings that increased serum cholesterol level, including in the prostate tissues, is associated with the pathogenesis of benign and malignant prostatic diseases (rohrmann et al. 2005; yat-ching et al. 2011). increased circulating cholesterol could induce signalling pathways that enhance bph pathogenesis. the elevated serum cholesterol concentration in the bph control suggests that there is an increased likelihood of the high serum cholesterol being deposited on the blood vessel walls, thereby forming arterial clots and hindering blood flow which might increase the blood pressure and heart diseases that would adversely affect the survival of the bph control rats. in contrast, the substantially reduced serum cholesterol concentration of the bph rats treated with 200 and 400 mg/kg caasbe showed the antihypercholesterolaemia effects of the phytoconstituents present in the caasbe. the anti-hypercholesterolaemia effects demonstrated by caasbe in this study were comparable to the finasteride effect in the standard control, suggesting that caasbe possesses high therapeutic potential against bph and its associated health complications. the drastically reduced cholesterol coupled with the low serum ldl-cholesterol level in the caasbe-treated bph rats showed a low risk of atherosclerosis and cardiovascular diseases in the bph rats treated with caasbe. the substantial reduction in the serum total cholesterol level in the bph rats treated with caasbe aligns with previous findings that inhibiting cholesterol synthesis by therapeutic agents could reduce bph progression and that hypercholesterolaemia aggravates bph progression (rohrmann et al. 2005). the ldl-cholesterol is one of the components of the lipid profile parameters commonly evaluated to monitor one’s lipid profile status. the elevation in the serum ldl-cholesterol indicates impending health consequences, mostly when there is a substantially low serum hdl-cholesterol level (kang et al. 2015). ldlcholesterol is the main lipid deposited on the arteries, which clogs and narrows the arterial area, thereby increasing the blood flow pressure that has a farreaching implication on heart functions and general wellbeing. the significantly elevated serum ldlcholesterol in the bph control showed that bph caused an imbalance in the lipid profile composition of the rats, exposing them to the increased health risk of accumulation of ldl-cholesterol in the arteries, which agrees with the findings of uroko et al. (2021). the decline in the serum hdl-cholesterol needed to remove and transport ldl-cholesterol from the blood vessels to the liver could be responsible for the elevated serum ldl-cholesterol in the bph control. the high serum 76 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 ldl-cholesterol level in the bph control agrees with güven and gökhan (2022) findings that increased ldlcholesterol is linked with bph progression. conversely, the substantial decrease in the serum ldl-cholesterol concentrations in the finasteride and caasbe treated bph rats, respectively, relative to the bph control, showed each therapeutic agent's ability to restore the altered ldl-cholesterol to the normal level. the power of caasbe to lower ldl-cholesterol indicates that it could promote normal biochemical and physiological functions aside from restoring an enlarged prostate to normal size, as uroko et al. (2021) and güven and gökhan (2022). this finding suggests that caasbe could play a key role in preventing and managing high blood ldl-cholesterol, arteriosclerosis and cardiovascular disorders. evaluating the serum urea level is a very reliable indirect method of ascertaining the renal function status and serves as a guide for taking necessary action to restore normal renal functions. urea is an excretable end product protein and nitrogenous base metabolism that is filtered by the glomerular of the kidney and excreted with excess fluid in the body. the high serum urea concentration points to renal dysfunction. the elevated serum urea concentration in the bph control relative to the normal control suggested an impaired renal glomerular filtration and excretion of urea from the circulating blood. the substantially elevated serum urea concentration in the bph control finding agrees with the previous findings that increased serum urea levels in bph rats are associated with impaired renal functions (weinstein et al., 2009; uroko et al., 2021). the persistent rise in the serum urea level could lead to various adverse health complications, including hypertension and cardiac dysfunctions. this finding suggests impaired glomerular filtration rate and increased serum urea level may be associated with bph progression if not effectively managed. in contrast, the significantly reduced serum urea level in the standard control and all the caasbe-treated bph rats showed that treatment with finasteride and caasbe alleviated the adverse effects of bph on renal functions, according to the findings of uroko et al. (2021). the ability of caasbe to drastically restore the elevated serum urea level in the bph rats similar to the normal control could be attributed to the therapeutic effects of the bioactive constituents in the combined extract, which is an indication that caasbe could be used to manage renal disorders. the serum creatinine level is the metabolic waste product of creatine catabolism that is regularly filtered from the blood by the renal glomerular and eliminated from the body via urine. the serum creatinine level, like serum urea level, reflects the glomerular filtration rate and the renal function status. the sensitivity of serum creatinine level as a measure of renal function status is very reliable when coupled with the serum urea level. the high serum creatinine level in the bph control relative to the normal control could be attributed to impaired creatinine filtration and clearance by the renal glomerular in line with weinstein et al. (2009). the high serum creatinine level in the bph control indicated impaired removal of excess wastes in the circulating blood via urinary excretion, which could adversely affect the regulation of biochemical and physiological activities in the body. contrarily, the significantly reduced serum creatinine level in the bph rats treated with caasbe suggests that the combined extract relief the adverse effects of bph induction by testosterone propionate injection and restored the ability of the renal glomerular to filter and remove excess creatinine from the blood in line with the findings of uroko et al. (2021). the very low serum creatinine level showed that caasbe could be used as a therapeutic agent against renal disorders, which agrees with the earlier reports by reshma et al. (2014). the normal renal histomorphology observed in the bph control, standard control, and bph rats treated with 200 and 400 mg/kg caasbe, respectively, compared to the renal histomorphology of the normal control, showed that it caused no renal injury in the rats. these suggest that bph interfered with renal functions and reduced the glomerular filtration rate and clearance of wastes out of the body. the absence of observable alterations in the renal histomorphology is in order because the testosterone propionate injection administered to induce bph is not nephrotoxic. the increased serum urea and creatinine concentrations indicated impaired renal functions due to stress associated with bph pathogenesis and complications rather than renal injury. the absence of histomorphological alterations of the kidney sections of the bph control and the bph rats treated with finasteride and caasbe are consistent with uroko et al. (2021). conclusions the findings of this study showed that treatment of bph rats with caasbe significantly restored the altered lipid profile to a normal level. the caasbe-treated bph rats had considerably reduced serum urea and creatinine concentrations compared to the bph control but no alterations in the renal histomorphology. the findings suggest that treatment with caasbe could prevent hyperlipidaemia and renal malfunctions in bph rats. acknowledgements: the authors wish to express profound gratitude to asogwa edith ogechi, egba simeon ikechukwu and okwor josephat ani for their assistance during the study. uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 77 authors’ contributions: robert ikechukwu uroko, mercylyn ezinne uche, and paul chukwuemaka nwejeanyalowu designed the study; robert ikechukwu uroko, ikenna obiwuru, chinedu aguwamba, and chinomso friday aaron carried out the experimental analysis. robert ikechukwu uroko analysed the data statistically and interpreted the results while mercylyn ezinne uche, paul chukwuemaka nweje-anyalowu, ikenna obiwuru, and chinomso friday aaron discussed the results. robert ikechukwu uroko supervised the study and wrote the manuscript. all the authors read and approved the final manuscript for publication. competing interests: the authors have expressed no conflict of interest. funding: not applicable. references abbiw d. useful plants of ghana: west african uses of wild and cultivated plants. kew, london: intermediate technology publications, royal botanical garden; 1990. adaramoye, o.a., akintayo, o., achem, j. & fafunso, m.a. 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(2011). the role of cholesterol in prostatic diseases. urological science, 22(3): 97-102. https://doi.org/10.1016/j.urols.2011.08.002 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 35-43 | doi: 10.14421/biomedich.2022.111.35-43 issn 2540-9328 (online) liver and renal cell damage following excess bee honey consumption in male wistar rat akpevwoghene agbatutu1, jerome ndudi asiwe*,2,3, olusegun gafar adebayo2,3 1department of botany, university of ibadan, nigeria 2department of physiology, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, college of medicine university of ibadan, nigeria. corresponding author* asiwejerome@yahoo.com; +2348163727468 manuscript received: 14 january, 2022. revision accepted: 11 february, 2022. published: 01 march, 2022. abstract honey is a widely used natural product with several health benefits. however, there is paucity of information on its excessiv e usage. the present study investigated the effect of excess honey consumption on hepato-renal functions in male wistar rats. twenty-eight adult male wistar rats were selected into four groups (n=7) and treated with distilled water (control) and 1ml, 2ml and 3ml of honey res pectively for 5 weeks. thereafter, the animals were euthanised and blood as well as kidney and liver were collected for further studies. there was a significant increase in creatinine, bilirubin, urea ast, alp, total protein as well as a significant decrease in rbc, wbc, ha emoglobin, lymphocyte and pcv. histology of the liver and kidney revealed a significant degeneration and necrosis in a dose dependent manner. this study suggest that excess honey consumption causes liver and renal cellular damage as well as haematological alterations . keywords: honey; excess consumption; liver enzyme; electrolyte; renal function; haematological indices; histopathology. introduction natural honey is a sweet, flavourful liquid with high nutritional value and therapeutic potential (eterafoskouei and najafi, 2013). it is a natural product made by honeybees (apis mellifera; apidae) from nectar collected from flowers (dashora et al., 2011). honey has been consumed by humans since almost five centuries ago for both nutritional and therapeutic purposes (adebolu, 2005; ashrafi et al., 2005). however, it is the only naturally occurring insect-derived substance with nutritional, aesthetic, medicinal, and industrial benefits (bansal et al., 2005). honey has been used as a natural sweetener since ancient times because of its high fructose content and it was reported to be 25% sweeter than tablet sugar (babacan and rand, 2007; pataca et al., 2007). the increasing popularity shown by the usage of honey in beverages is also accounted to its high fructose content (babacan and rand, 2007). as of today, approximately 300 different varieties of honey have been identified and many forms of nectar gathered by honeybees are related to these variations (lay-flurrie, 2008). proximate analysis showed that honey's main components are carbohydrates (95–97% of its dry weight), proteins, vitamins, amino acids, minerals, and organic acids (betts, 2008; helmy and el-soud, 2012). meanwhile, phytochemical screening of pure honey also revealed that it contains flavonoids, polyphenols, reducing compounds, alkaloids, glycosides, cardiac glycosides, and volatile compounds (white, 1980; islam et al., 2012). regardless of its strong phytochemical properties, honey's vitamin content is poor, and it falls far short of the daily requirements but the most abundant is the vitamin c. honey is a good source of antioxidant and it has been reported to exhibit strong anti-oxidative (ahmed and othman, 2013; kasala et al., 2015), and anti-inflammatory (khalil et al., 2012) potential. due to the high polyphenols of honey, various studies reported the pharmacological usage of honey in the treatment and management of pathological conditions like increase blood pressure (ahmed and othman, 2013), diabetes (estevinho et al., 2008), neuropsychiatric symptoms (ghosh and playford, 2003; rahman et al., 2014), bacterial infections (attia et al., 2008; kasala et al., 2015) as well as respiratory and gastrointestinal disorder (abdulrhman et al., 2008; israili, 2014), microbial infection (israili, 2014; saikaly and khachemoune, 2017). the antioxidant properties of honey have been implicated through its reactive oxygen species (ros) scavenging ability and by increasing the intracellular activity of glutathione (gsh), uric acid, beta-carotene, and vitamin c (ahmed et al., 2018). moreover, certain phenolic ingredients of honey were reported to inhibit the activity of nitric oxide synthase, decrease inducible nitric oxide synthase (inos) and cyclooxygenase-2 https://doi.org/10.14421/biomedich.2022.111.35-43 36 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 (cox-2) activities that cause inflammation of cells and tissues in human body (ahmed et al., 2018). also, honey displayed very strong anti-inflammatory effect reported in several studies. reports stated that honey can inhibit or slows down pro-inflammatory cytokine release, nitric oxide synthase (inos) expression, generation of ros, and reduce the level of prostaglandin: which are the major players culminating processes involved in inflammation (al-waili and boni 2003; candiracci et al. 2012). according to candiracci et al., (2012), honey exacerbated nitric oxide release in acute and severe inflammation. however, this mechanism was related to cox-2 and inos inhibition leading to suppression of mediators of pro-inflammation (pge2, no, tnf-α, and il-6) (hussein et al. 2012). another area where honey is adopted is its effect on wound healing. several preclinical and clinical trials have reported the application of honey in both acute and chronic wounds including injuries resulting from burns (moore et al. 2001). this reported have demonstrated honey’s ability to reduce the level of edema, enhance granulation and epithelization during the proliferative stage as well as decreased wound healing time and scarring and decreases contractures in cases with burn wounds with no adverse effect (allergy or toxicity) whatsoever (yaghoobi et al. 2013). meanwhile, the side effect of honey consumption has not been well studied and there are major concerns with respect to excessive intake of honey causing increase in blood sugar level. there seems a very little or no report on the excessive or prolonged consumption of honey which might lead to toxicity in the body system. hence, the goal of this present study is to investigate the high consumption of honey on haematological parameters, liver function indices and kidney function in male wistar rats. materials and methods drugs and chemicals honey was purchased at gembu local market in saurdana local government area of taraba state, nigeria. creatinin and urea kit was obtained from immunometrics limited (uk) and total proteins kit, albumin kit and bilirubin kit were all obtained from sigma aldrich (st. louis, mo, usa). other reagents and solvents were of analytical graded level. experimental animals twenty-eight adult male wistar rats (weighing 180200g) were obtained from the physiology department central animal house (pdcah), university of ibadan, ibadan, oyo state, nigeria and managed under normal laboratory condition according to the university ethical guideline which adheres strictly to the “principle of laboratory animal care” (nih publication no. 85-23). the dosed selected for this study was according to fasanmade and alabi, (2008) with slight modifications. treatment design the animals were arranged into four groups (n =7). group 1 serves as the non-treated animal (control animals) and received distilled water (10 ml/kg) alone. group 2 was administered with 1ml/100g of body weight while group 3 received 2ml/100g of body weight and group 4 received 3ml/100g of body weight. all treatments were daily administered orally using oral gavage and lasted for 35 days. the animals were carefully observed for change in body weight and blood glucose level evaluated weekly and after overnight fasting on the last day of sacrifice (24 hours after the last day of treatment) using a glucometer (accu-check active, roche diagnostic, mannhein germany). preparation of blood samples for biochemical analysis after the conclusion of the treatment, all the rats were subjected to ketamine anaesthesia and then sacrificed through cervical dislodge. cardiac puncture was used for blood collection for haematological variables. further, plasma was also obtained from the collected blood by centrifugation (3000 rpm; 15 minutes) at room temperature using a bench top centrifuge (bosch, uk) for the assessment of electrolytes concentration, renal and liver function markers. estimation of liver function markers alanine transaminase (alt), aspartate aminotransferase (ast) and alkaline phosphatase (alp) activities for liver injury markers in the plasma was determined according to the protocol previously described by reitman and frankel (1957) using randox test kit. estimation of kidney function markers assessment of serum creatinine concentration plasma (50 ml) was taken and mixed to a monoreagent (1000 ll) obtained from the assay kit. the mixture was then incubated for 60 seconds. thereafter the absorbance was read (k 492 nm) twice within the interval of 1 min. furthermore, the concentration of creatinin was calculated using the kit manufacturer method (immunometrics limited uk). assessment of serum urea concentration four parts of monoreagent taken from reagent i and onepart monoreagent taken from reagent ii were mixed together and incubated at 15–25°c for 30 minutes. following incubation, the mixture was kept in amber bottle prior to use. 10 ml of plasma sample and urea standard were further mixed to 1000 ll monoreagent each and then further incubated at 20-25°c for 60 seconds. the absorbance was read at wavelength of 340 agbatutu et al. – excess bee honey consumption induces toxicity 37 nm twice within the interval of 1 minute. finally, the concentration of urea was determined and calculated as proposed by the kit manufacturer (immunometrics limited uk) using the formula: urea concentration = mg/dl change in sample absorbance change in standard absorbance × standard concentration/cal assessment of serum albumin concentration plasma albumin was determined using randox reagent kits as according to the instructions and method in the manual further corroborated by doumas et al. (1971). estimation of plasma electrolyte concentration the plasma electrolytes: sodium, potassium and chlorides were assayed using their respective commercial kits. all assays were done using microplate reader spectramax plus (a molecular device product). estimation of haematological indices the haematological parameters were determined using an automated haematology analyzer (abx micros 60 from horiba abx, france). the red blood cells (rbc), packed cell volume (pcv), haemoglobin and other haematological indices such as, lymphocytes, neutrophils, monocytes, eosinophils and basophils were determined. the mean corpuscular haemoglobin concentration (mchc), mean corpuscular hemoglobin (mch) and mean corpuscular volume (mcv) were calculated from rbc, haemoglobin (hb) and packed cell volume (pcv) values. the white blood cell (wbc) count was also determined. histological assessment the kidney and liver tissues were harvested and fixed in 10% phosphate buffered formalin for histomorphological examination. the kidney and liver tissues were directly fixed through immersion in a 4% paraformaldehyde of 0.1 mol/l phosphate buffers at ph 7.2 for 48 h. all tissues were then further dehydrated and embedded in a paraffin wax and thereafter sliced transversely using the microtome machine into sections (5 μm). after tissue slicing, it was then stained in haematoxylin and eosin solution. finally, all the sections were examined by using optical microscope for cell condensation and death (asiwe et al., 2021). statistical analysis data are presented as mean ± standard error of mean and analysed with one-way analysis of variance (anova) and compared by newman-keuls test using the graphpad prism 7.0 (graphpad software, san diego, ca, usa). p<0.05 was considered statistically significant. results honey consumption (hc) maintains blood glucose level and reduced body weight gain in rats the effect of honey consumption on blood glucose level and body weight was presented in table 1-2. following administration of honey, there was no significant difference in the glucose level across the treatment group comparative to the control (table 1). however, there was a significant (p < 0.05) progressive decrease in body weight from the week 2-5 in hc (2ml/100g and 3ml/100g) when compared with control (table 2). honey consumption (hc) induced electrolytes imbalance in rats table 3 showed the impact of honey consumption on the plasma level of electrolytes following administration in the rats. the plasma concentration of sodium and chlorine ion significantly (p < 0.05; [689 ± 7.98 and 450 ± 3.13 vs 736±3.36] and [29.1 ± 2.61 and 26.1 ± 11.70 vs 31.3 ± 0.92]) decreased in the animals administered hc (1ml/100g and 3ml/100g), but there was significant (p < 0.05; [747 ± 5.29 vs 736±3.36] and [69.8 ± 1.33 vs 31.3 ± 0.92]) increase in the animals administered hc (2ml/100g) relative to the control. the plasma concentration of potassium ion decreased significantly (p < 0.05; [5.88 ± 0.41, 6.08 ± 0.42 and 3.92 ± 0.30 vs 7.58 ± 0.08]) across all the groups administered hc (1ml/100g, 2ml/100g and 3ml/100g) when compared to the control (table 3). honey consumption (hc) exacerbates liver function markers in rats the data for markers of liver toxicity in rats was presented in fig. 1a-c. data showed significant decrease level of alt in the animals treated with hc (2ml/100g and 3ml/100g) compared to the control (fig. 1a). however, the animals treated with higher doses of hc (2ml/100g and 3ml/100g) significantly decreases the level ast and alp comparative to the control (fig. 1bc). honey consumption (hc) initiates renal toxicity via increased inflammatory markers in rats the results obtained from this result indicating renal toxicity following honey consumption is represented in figure 2a-e. there was a significant (p < 0.05) increase in the urea and creatinine concentration in the animals treated with hc (2ml/100g and 3ml/100g) when compared to the control (fig. 2a-b). we also observed significant (p < 0.05) increased total protein and bilirubin level in the animals that were administered hc (1ml/100g, 2ml/100g and 3ml/100g) (fig. 2c-d), while 38 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 a significantly (p < 0.05) decreased albumin level was recorded in them (fig. 2e). the effect of honey consumption on haematological indices in rats table 4 showed the values of the haematological indices in the rats after administration of honey. the results showed that there were significant (p < 0.05; [3.06 ± 0.54 and 2.05 ± 0.31 vs 5.41 ± 0.39]) decrease in wbc in the hc (1ml/100g and 2ml/100g) treated rats and a significant (p < 0.05; [7.59 ± 0.55 vs 5.41 ± 0.39]) increase in the rats treated with hc (3ml/100g) when compared to the control. the lymphocyte counts decrease significantly (p < 0.05; [1. 26 ± 0.17 and 0.42 ± 0.05 vs 2.96 ± 0.22]) in the rats treated with hc (1ml/100g and 2ml/100g) when compared to the control. the concentration of haemoglobin decreases significantly (p < 0.05; [12.5 ± 0.17, 11.4 ± 0.16 and 13.1 ± 0.17 vs 14 ± 0.29]) in the hc (1ml/100g, 2ml/100g and 3ml/100g) treated rats when compared to the control. the value of rbc and pcv decreases significantly (p < 0.05; [6.98 ± 0.16 and 6.42 ± 0.41 vs 8.2 ± 0.18]) and (p < 0.05; [39.5 ± 0.17 and 37.1 ± 2.27 vs 44.9 ± 0.44]) in the hc (1ml/100g and 2ml/100g) treated rats when compared to the control. additionally, we observed that the value of pct decreases significantly (p < 0.05; [0.50 ± 0.14 vs 0.64 ± 0.40]) in the hc (3ml/100g) when compared to the control. however, there were no significant (p > 0.05) change mcv, mch, mchc and platelet levels when compared with group (table 4). honey consumption (hc) induces hepato-renal degeneration in rats figure 3 showed the photomicrograph of the liver and kidney tissues. histomicrograph of the liver in control rats showed normal cellular architecture with no lesion (plate 1). however, the rats administered hc (1ml/100g) showed centrilobular, hepatocellular vacuole change while hc (2ml/100g) showed diffused atrophy of hepatic cords and focal hepatocyte vacuolation. also, those given the higher dose of hc (3ml/100g) showed hepatocellular degeneration and fibroblast reaction as well as diffuse atrophy cords and portal inflammation. the photomicrograph of the kidney showed patchy epithelia degeneration as well as necrosis in hc (1ml/100g and 2ml/100g) while hc (3ml/100g) showed tubular epithelia coagulation as well as necrosis as shown in figure 3. figure 1. (a) alt (b) ast (c) alp. values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control. table 1. effect of bee honey consumption on fasting blood glucose (g/dl). honey consumption weeks control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) week 1 120 ± 2.03 118 ± 2.71 114 ± 2.20 115 ± 2.23 week 2 120 ± 2.03 118 ± 2.71 114 ± 2.20 115 ± 2.23 week 3 112 ± 1.11 116 ± 1.69 122 ± 2.07 120 ± 1.24 week 4 133 ± 1.29 121 ± 2.78 107 ± 2.71 110 ± 2.71 week 5 116 ± 3.69 116 ± 3.67 114 ± 1.82 114 ± 2.74 values are expressed as mean ± standard error of mean, (n=5). hc = honey consumption agbatutu et al. – excess bee honey consumption induces toxicity 39 figure 2. (a) urea (b) creatinine (c) total protein (d) billirubin (e) albumin. values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control. table 2. effect of bee honey consumption on body weight (grams). honey consumption weeks control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) week 1 203 ± 7.69 206 ± 5.13 205 ± 3.96 204 ± 8.22 week 2 226 ± 7.46 219 ± 10.0 210 ± 7.01* 183 ± 20.6* week 3 223 ± 8.62 220 ± 12.1 206 ± 5.85* 198 ± 8.07* week 4 231 ± 11.4 227 ± 18.3 206 ± 7.18* 208 ± 12.7* week 5 249 ± 12.2 223 ± 14.2 212 ± 8.05* 209 ± 12.5* values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control, hc = honey consumption table 3. effect of bee honey consumption on electrolytes after 35 days (5 weeks). honey consumption variables control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) na (g/dl) 736±3.36 689 ± 7.98* 747 ± 5.29* 450 ± 3.13* k (g/dl) 7.58 ± 0.08 5.88 ± 0.41* 6.08 ± 0.42* 3.92 ± 0.30* cl (g/dl) 31.3 ± 0.92 29.1 ± 2.61* 69.8 ± 1.33* 26.1 ± 11.70* values are expressed as mean ± standard error of mean, (n=5), *p< 0.05 is significant when compared with control, hc = honey consumption. ml of honey table 4. effect of bee honey consumption on haematological indices after 35 days (5 weeks). honey consumption variables control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) wbc 5.41 ± 0.39 3.06 ± 0.54* 2.05 ± 0.31* 7.59 ± 0.55* lymphocyte 2.96 ± 0.22 1. 26 ± 0.17* 0.42 ± 0.05* 3.22 ± 0.21 rbc 8.2 ± 0.18 6.98 ± 0.16* 6.42 ± 0.41* 7.66 ± 0.40 hb (g/dl) 14 ± 0.29 12.5 ± 0.17* 11.4 ± 0.16* 13.1 ± 0.17* pcv 44.9 ± 0.44 39.5 ± 0.17* 37.1 ± 2.27* 42 ± 0.45 mcv 55 ± 0.41 56.7 ± 0.47 58 ± 0.41 57.2 ± 1.24 mch 17.1 ± 0.04 17.8 ± 0.17 17.8 ± 0.24 17.1 ± 0.23 mchc 31.3 ± 0.17 31.5 ± 0.24 30.8 ± 0.10 31.2 ± 0.37 platelet 761 ± 56 709 ± 0.71 749 ± 49.10 623 ± 20.50 pct 0.64 ± 0.40 0.56 ± 0.01 0.63 ± 0.07 0.50 ± 0.14* values are expressed as mean ± standard error of mean, (n=5), *p< 0.05 is significant when compared with control, hc = honey consumption. ml of honey 40 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 figure 3. histology of liver and kidney. the arrows showing significant leisions (h&e x400). group 1= normal control, group 2= 1ml of honey, group 3= 2ml of honey and group 4= 3ml of honey discussion the constituents of honey make it one of the widely used natural products for treating many diseases. however, our present study evaluated the effect of excess honey consumption on serum electrolyte, liver and renal function markers as well as haematological variable in male wistar rats. there was a progressive decrease in the body weight of the animals which suggest the antiobesogenic potential of honey as reported by yaghoobi et al., (2008) and bahrami et al., (2009). the weight loss was highest in the group fed with 3 ml of honey which corroborated the studies of chepulis, (2007) that protective effects of honey against obesity and weight gain depend on quantity or duration of exposure. we also observed a decrease in blood glucose level in week 4 and week 5 after an initial increase in week 3. this observation was consistent with the report that administration of honey in stz-induced diabetic rats and non-diabetic rats for 3 days significantly reduces blood glucose levels (özta¸san et al., 2005; jansen et al., 2012). however, the antidiabetic effect of honey can be attributed to its ability to modulate adiponectin secreted by adipose tissue which regulate glucose and lipid metabolism as reported by (li et al., 2009). the adiponectin modulating potential of honey though not within the scope of our present study, could suggest the mechanism of weight loss in a dose depended manner. electrolytes are essential for survival because of the electrical charges they provide. they interact with each other and the cells in the tissues, nerves and muscles. imbalance in this electrolyte can alter the normal physiology of the body. na and k ions are essential contractile tools of excitable tissues such as the heart, muscles and nerves, a decrease in this ions concentration as observed in this study suggest a modulating role of honey in electrolyte balance. the decrease in the levels of haematological components in this study suggests that honey supplementation directly destroy the blood components or disturb the processes involved in their formation. it can also be attributed to loss of blood due to haemorrhage as hematurea was observed during the study. however, this finding was not consistent with the studies of al-waili et al., (2006) and abdelaziz et al., (2012) who reported a protective role of honey against haemorrhagic and anemic conditions. other studies such as bazzoni et al., (2005), al-qayim et al., (2014), ghorbel et al., (2015) also showed conflicting results. liver is an essential organ for food metabolism and detoxification of substances that threatens the normal functions of the body. the biomarkers assayed in this study indicated the state of health of the liver as it was previously reported to indicate toxicity when there is significant increase in their concentration (murugavel and pari, 2007; asiwe et al., 2022). previous studies by al-waili et al. (2006) and halawa et al., (2009) showed that honey attenuates elevated liver enzymes during acute blood loss and food restriction. however, we observed that honey supplemented diet reduces alt and increases ast and alp in a dose dependent manner. most studies reported the preventive effect against liver agbatutu et al. – excess bee honey consumption induces toxicity 41 injury due to its free radical scavenging potentials. however, the present study was not consistent with previous studies and the inconsistency can be attributed to the dose and duration of the study. honey has been reported by rashed and soltan, (2004) to contain several trace elements as well as heavy metals such as cadmium (cd) and lead (pb) as well as aluminium (al) which previously has been confirmed to be toxic to liver and kidney functions. this may contribute to the observed dose and time dependent toxicity of honey. on the other hand, kidney is one of the important organs in the body saddled with the responsibility of excreting waste metabolic product from the body and a dysfunction of the kidney has been reported to be characterised by increased serum concentrations of creatinine and urea (asiwe et al., 2022). halawa et al., (2009) reported ameliorating effect of honey in environmental toxicant induced kidney dysfunction. however, our present findings suggest that honey supplementation could cause renal damage as both creatinine and urea concentrations were significantly increased when compared to the control group. the photomicrograph of the liver showed centrilobular, focal hepatocyte vacuolation, hepatocellular degeneration and fibroblast reaction as well as diffuse atrophy cords and portal inflammation. several trace elements as well as heavy metals such as cadmium (cd) and lead (pb) which was previously confirmed by asiwe et al., (2022) to be toxic to liver and kidney functions by inducing cellular stress, injury or damage in body tissues was reported by rashed and soltan, (2004) to be part of the constituents of honey as contaminants. this could suggest the adverse effect of honey in this study. this observation was consistent with the study of wilson et al., (2011) who reported adverse effect of excess honey intake on histology of the liver. the renal tissue was not exempted from this effect as the photomicrograph of the kidney showed patchy epithelia degeneration, necrosis as well as tubular epithelia coagulation in a dose dependent manner. this could contribute to the increased creatinine and urea concentrations observed in this study. this observation was in contrast with the study of onyije et al., (2011) who reported normal renal architecture with increased urea and creatinine levels. conclusion in conclusion, this study suggest that excess honey consumption causes adverse effects on liver and renal tissues as well as haematological alterations as shown both in biochemical assays and histological examination. it is therefore recommended that proper awareness should be raised to educate the public as well as agencies regulating food and drug intake of these possible adverse effect of excess consumption of honey or honey products. ethics approval and consent to participate: ethical approval was given by the college of medicine ethics committee. animal handling was done in accordance to established guidelines by the national institute of health for care and use of laboratory animals as adopted by the college of medicine, university of ibadan, nigeria. consent for publication: not applicable availability of data and materials: all data produced and analyzed during this study are included in this article. competing interest: the authors declare that they have no competing interests. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors’ contributions: aa and ajn designed the study. ajn, aa and aog performed the experiments, analyzed the data, and wrote the manuscript. all authors read and approved the final manuscript. acknowledgements: not applicable references abdelaziz i, elhabiby mi and ashour aa (2012). toxicity of cadmium and protective effect of bee honey, vitamins c and b complex. human and experimental toxicology 1–9. abdulrhman m, el-hefnawy m, ali r, el-goud aa (2008). honey and type 1 diabetes mellitus. in: liu cp, editor. type diabetes – complications, pathogenesis, and alternative treatments. croatia: in tech; 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al-waili, n ghayour-mobarhan, m. parizadeh, s.m.r. abasalti, z. yaghoobi, z. yaghoobi, f esmaeili, h. kazemi-bajestani, s.m.r. aghasizadeh, r (2008). natural honey and cardiovascular risk factors; effects on blood glucose, cholesterol, triacylglycerole, crp, and body weight compared with sucrose. sci. world j. 8, 463–469. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 45-54 | doi: 10.14421/biomedich.2022.111.45-54 issn 2540-9328 (online) the roles of the fluorescent in situ hybridization (fish) and comparative genomic hybridization (cgh) techniques in the detection of the breast cancer harem othman smail department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f.r. iraq. corresponding author harem.othman@koyauniversity.org manuscript received: 25 february, 2022. revision accepted: 11 march, 2022. published: 24 march, 2022. abstract this paper aimed to understand and compare the two popular cytogenetic techniques of fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) in detecting breast cancer chromosomal abnormality. several chromosomal anomalies play a role in the development of breast cancer, and the two above approaches play an important role in confirming fluorescence in situ hybridization in particular (fish). however, comparative genomic hybridization has developed dna copy number profiles for most of the publicly available breast cancer cell lines for the fish methods rely on the fluorescent probes. chromosomal profiles can be generated for the suspected chromosomal abnormality, copy number changes between the tumour and the dna control can be compared, and the results can be registered. today, modern cytogenetic tools such as fluorescence in situ hybridization (fish) are more commonly used to detect any microdeletion that cannot be detected by conventional cytogenetic karyotypes that involve a high rate of cell division and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and research. usuall y, this is a problem for physicians, and there are still many drawbacks and disadvantages concerning the high benefits, such as false findings. normal chromosome in situ hybridization requires the hybridization of a labelled dna probe into denatured chromosomal dna present in metaphase chromosomes in an air-dried microscope slide preparation. metaphase spreads are used for traditional chromosome fish (metaphase fish). positive and positive signs of hybridization also appear as a double spot, corresponding to the hybridized probe for both sister chromatids. a further extension of chromosome painting is comparative genomic hybridization (cci-i). cch involves simultaneous chromosome painting in two different colours using complete dna from two similar sources as probes, which reveal variations concerning the benefit or loss of sub-chromosomal regions or even entire chromosomes. keywords: fish; cgh; probe; classical cytogenetic; chromosomal regions and hybridization. introduction in terms of histology, clinical response, diffusion trends to distant locations, and patient outcomes, breast cancer is heterogeneous (prat et al., 2011). nevertheless, the most common cause of death from cancer worldwide is breast cancer. rates differ about five-fold across the world, but they are growing in regions with low disease rates until recently (key et al., 2011). furthermore, the availability of cytogenetics and cytogenetics technologies has enhanced the detection and recognition of molecular tumour signatures and the understanding of the initiation and progression of cancer (ribeiro et al., 2019). the fluorescence in situ hybridization (fish) technique that plays a leading role in diagnostic pathology has provided valuable information on genomic variations in malignant cells for its single-cell study (das et al., 2013). fish is a molecular tool in which both metaphase chromosomes and interphase nuclei identify numerical and structural defects. fish is widely used to identify translocations and determine gene deletion and amplification in tumours as a prognostic and diagnostic method (brown et al., 2007). the technique utilizes the inherent capacity to hybridize complementary strands of dna or rna from various sources. the theory of annealing a labelled nucleic acid probe to complementary sequences inside cells or tissue mounted (in situ) on a microscope slide is based on in situ hybridizations (karimi et al., 2020). genomic changes, amplifications, and deletions may reduce the complexity of genomic data observed by comparative genomic hybridization (przybytkowski et al., 2014). by comparing the samples with reference dna, cgh detects changes in the copy number of individual chromosomes or chromosomal regions, such as changes in relative genome size and ploidy levels in test samples. this technique was initially designed to examine the differences between solid tumours and normal tissues in chromosomal supplements (zhang et al., 2015). it is expected that the precise classification of these genomic changes would have an important effect https://doi.org/10.14421/biomedich.2022.111.45-54 46 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 on translational and fundamental research (climen et al., 2007). for example, comparative genomic hybridization has been instrumental in the dissection of distinct molecular pathways to malignancy of the breast and developing a direct relationship between genotype and clinical pathology (reis et al., 2005). characterization types of breast cancer over 100 types of cancers consist of multiple subtypes capable of arising in a single type of organ or tissue. most cancers can be defined at a very general level based on their originating tissue location. in epithelial cells, the most common type of cancer, comprising more than 80 per cent of all cancers, occurs; these cancers are called carcinomas. depending on whether the epithelial cells of origin are part of the protective epithelial layer (squamous cell carcinomas) or have secretory properties (adenocarcinomas), most carcinomas can break into squamous cell carcinomas or adenocarcinomas (smail 2016 and smail 2020). breast cancers are a complex and heterogeneous community of diseases, and patients are treated with clinicopathological features and estrogen receptor status and her2 guidance. however, for the individualization of treatment, these factors are not necessary. microarray-based gene expression profiling has contributed to a paradigm change in the understanding of breast cancer. it has been shown conclusively that breast cancer is not a single disease (weigelt et al.,2010). joint hormone receptor-defined subtypes of breast cancer (h.r.; estrogen receptor [e.r.] and progesterone receptor [p.r.]) and her2 status (howlade et al., 2014). triple-negative (tnbc) breast cancers do not express hormone receptors or over-express her2. as characterized by low five-year survival and high recurrence rates after adjuvant therapy, poor prognosis is associated with these conditions. overall, tnbc has striking similarities with basal-like breast cancers (bbc), but they are found the same in various studies (de et al., 2011). five tumour subtypes (luminal a, luminal b, her2-enriched, basal, and claudin-low) have been identified in breast cancer gene expression studies, each of which has specific biological and prognostic features (prat et al.,2010). four major intrinsic molecular subtypes of breast cancer, known as luminal a, luminal b, her2-enriched [her2e], and basal-like, were identified in global gene expression analysis studies. these molecular entities have shown major differences in terms of occurrence, risk factors, baseline prognosis, age at diagnosis, and response to treatment. because of its high frequency, lack of successful targeted therapies, weak baseline prognosis, and propensity to impact younger women, the basal-like subtype is of particular clinical concern (prat et al., 2013). a promising new diagnostic area for estimating the risk for metastatic relapse and metastatic progression in cancer patients is the circulating tumor cell (ctc) study (lianidou et al., 2011). rna sequencing found that pdx cells developed in the mammary gland were identical to those studied in culture for global gene expression. carboplatin was cytotoxic to whim30 but not whim2 in vitro, while both lines were cytotoxic to bortezomib, dacarbazine, and cyclophosphamide. however, these drugs have proven unsuccessful in treating in vivo both primary and metastatic whim2 tumours. on the other hand, carboplatin and cyclophosphamide have successfully treated whim30 mammary tumours and reduced brain, liver, and lung metastatic burden (turner et al., 2018). the metastatic cascade is a series of biological processes that cause tumour cells to migrate from the primary site to a distant location and create new cancer growth. circular tumour cells (ctcs) play a crucial role in tumour propagation. the role of ctcs in treatment failure and disease progression can be explained by their association with biological processes, including epithelial-tomesenchymal transformation and self-seeding, defined as primary infiltration of the tumour or metastasis formed by more aggressive ctcs. ctcs are a rare and heterogeneous population of cells of proven prognostic and predictive value in some clinical contexts (mego et al., 2010). in addition to the typical hormone receptor-positive and hormone receptor-negative forms, studies of breast cancers using gene expression profiling have identified many major breast cancer subtypes. the luminal a and luminal b classes are the most reproducibly defined molecular subtypes among the hormone receptorpositive cancers. the major molecular subtypes identified in hormone-receptor-negative breast cancers are the her2 and basal-like classes. some studies have also described other molecular subtypes, such as luminal c and regular breast-like classes. still, they are less well characterized than luminal a, luminal b, her2, and basal types (schnitt 2010). several molecular-targeted therapies for breast cancer have been tested since the application of endocrine therapy for estrogen receptor (e.r.)-positive tumour types1. an example of effective gene-targeted therapy is genome alteration-matched treatment of breast cancer to target amplification of human epidermal growth factor receptor 2 genes (erbb2 receptor tyrosine kinase, erbb2 also referred to as her2). gene expression-based molecular subtyping has also been broadly applied to breast cancer to aid treatment decisions (chung et al., 2017). there are many cellular functions for brca1. dna repair, cellcycle regulation, transcriptional regulation, and chromatin remodelling have been implicated. on the contrary, functions assigned to brca2 were specifically limited to dna recombination and repair processes. in the regulation of rad51 activity, brca2 smail – the roles of the fluorescent in situ hybridization (fish) and … 47 has a role. rad51 is a highly conserved dna recombinase involved in double-strand break repair and replication fork arrest (da and lakhani, 2010). chromosomal abnormality in breast cancer the most common cytogenetic anomalies observed in human breast carcinoma are modifications to the long arm of chromosome 1 (bièche and lidereau, 1995). for four ductal breast carcinomas, the cytogenetic analysis showed a net benefit of 1 q in all tumours. the only improvement in the first tumour was that one chromosome 16 was replaced by a chromosome derivative consisting of 16p and 1q (pandis et al., 1992). active x chromosome duplication and xi loss characterized almost half of the cases of sporadic basallike cancers studied (richardson et al., 2006). grade i and tubular breast carcinomas have a limited number of genomic alterations with extremely recurring 16q losses, while grade iii breast carcinomas also have complex genotypes with 11q, 14q, 8p, 13q loss; 17q, 8q, 5p gain; and high-level gains (amplification) on 17q12, 17q2224, 6q22, 8q22, 11q13, and 20q13 (simpson et al.,2005). amplification of chromosome band 11q13 protooncogenes (myc, erbb2) and dna; tp53 mutation; and loss of heterozygosity of chromosome and chromosome arms 1, 3p, 6q, 7q, 8p, 11, 13q,16q, 17, 18q, and 22q are the main forms of genetic defects commonly found in breast tumours (bièche et al.,1995). in breast cancer, abnormalities of chromosome 17, recognized over two decades ago to be important in tumorigenesis, frequently occur. changes in unique chromosome 17 loci, including amplification of erbb2, loss of p53, loss of brca1, and amplification or deletion of top2a, are considered to play an important role in breast cancer pathophysiology (reinholz et al., 2009). a single amplicon spanning several megabases was originally thought to include 11q13 amplification. still, more recent data identified four core regions within 11q13 that can be amplified separately or separately together in various combinations (ormandy, 2003). chromosomal alterations can be tested using gbanding karyotype and multicolour fluorescence in situ hybridization (m-fish) on metaphases (rondón et al.,2014). fluorescence in situ hybridization (fish) allows the number of the gene or chromosome copies in archival tissues to be measured in situ and linked to morphology and clinical outcome (watters et al., 2003). for example, cytokinetic defects are characterized by chromosomal instability in an inherited cancer syndrome and may help explain why brca2-deficient tumours are also aneuploidy-deficient (daniels et al., 2004). the innovation is known as comparative genomic hybridization (cgh), sets out methods for evaluating the relative number of copies of nucleic acid sequences in or in parts of one or more subject genomes (e.g. a tumour cell) as a function of the position of certain sequences in the reference genome (e.g. a regular human genome) (pinkel et al., 2011). initial cgh array applications for breast cancer analysis and the mechanisms by which various types of copy number changes can occur have been described (albertson 2003). how does fluorescence in situ hybridization works? fluorescence in situ hybridization by a fluorescently labelled probe detects nucleic acid sequences that precisely hybridize within the intact cell to its complementary target sequence (moter and göbel, 2000). fluorescence in situ hybridization has been used in interphase nuclei to image complex genomic dna sequences. unreplicated dna parts give singlet hybridization signals in normal diploid cells, whereas replicated loci are characterized by doublets (selig et al., 1992). fish on 3d preserved nuclei, compared to fish on metaphase chromosomes and traditional interphase cytogenetics, needs special requirements concerning the consistency of the probe, fixation, and pretreatment steps of the cells to achieve the two objectives, namely the best possible preservation of the nuclear structure and at the same time, the efficient accessibility of the probe (cremer et al.,2012). the concepts of in situ hybridizations of fluorescence are as follows: (a) dna probe and a target sequence are the basic elements. (b) before hybridization, the dna probe is indirectly marked with a hapten, specifically marked with hapten by adding a fluorophore probe (right panel). (c) the labelled probe and the target dna are denatured to yield single-stranded dna. (d) then they are combined, which allows complementary dna sequences to be annealed. (e) if the probe has been indirectly labelled, the visualization of the non-fluorescent hapten using an enzymatic or immunological detection system involves an additional step. finally, fluorescence microscopy tests the signals. speicher and carte adapted from (speicher et al.,2005 and bishop, 2010). hapten binding is visualized using a subsequently applied fluorochromeconjugated antibody (waminal et al.,2018). the choice of the probe is one of the most significant factors in fish research. it is possible to use a large variety of probes, from whole genomes to tiny cloned probes (1–10 kb). there are three types of probes in general, each with several applications: fullchromosome painting probes, repetitive sequence probes, and locus-specific probes (bishop, 2010). wellprepared chromosome distribution and an effectively labeled probe are prerequisites for a good fish experiment. nick-translation, random priming, and pcr provide multiple enzymatic methods for labelling probes. the most common use of nick-translation is 48 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 (gozzetti and le, 2000). in conducting efficient 3d fish experiments, there are two major considerations. to maintain nuclear morphology as much as possible while keeping dna sufficiently accessible for probe hybridization, the option of cellular treatments, including fixation, pre-and post-hybridization steps (bolland et al., 2013). figure 1. the fish spot detection algorithm (les et al., 2014). figure 2. fluorescence in situ hybridization (fish) applications in genetic diagnostics on ffpe content in solid tumours: a-1p/19q probe: a1 deletion of 1p32 locus, a-2 natural signal pattern (cell on the left) and 19q13 locus deletion (cell on the right), a-3 normal signal pattern (abbott molecular), b-dual fusion probe: col1a1 and pdgfb loci fusion and normal signal pattern (zytovision), c-break apart probe: c-1 alk gene rearrangement, c-2 normal signal pattern (abbott molecular), d-break apart probe: ewsr1 locus rearrangement (abbott molecular), f-break apart probe: f-1 rearrangement (empire genomics), g-locus specific probe: g-1 her2 locus amplification, g-2 normal signal pattern (abbott molecular), e-break apart probe: normal ss18 locus signal pattern, green colour—pdgfb gene locus, yellow colour—the fusion of col1a1pdgfb and pdgfb-col1a1 (chrzanowska et al.,2020). figure 3. detection of di-centric, tri-centric, and tetra-centric ring chromosome 18 using a centromeric probe d18z2 for chromosome 18. the left panel shows regular chromosome 18, the top dicentric ring 18 and the bottom tetracentric ring 18. the right panel shows dicentric ring 18 and fish insets pericentric/tetracentric ring 18. (b) identification of a 2q32/16p13.3 translocation derivative of chromosome 16 by complete chromosome painting probes for chromosome 2 (wcp2) and 16 (wcp2) (wcp16). (c) identification by dual-colour double fusion probes of abl1/bcr gene fusions in interphase and metaphase cells (thin arrows point to the normal signal and thick arrows point to the abnormal fusion signals). (d) diagnostic use of the etv6 and runx1 probes for the identification of two cryptic t(12;21) (p13;q22) fusion signals, loss of etv6 signal and gain of three additional runx1 signals (thin arrows point to the fusion signals and thick arrows to extra runx1 signals). all images are from yale clinical cytogenetics laboratory (cui et al.,2016). smail – the roles of the fluorescent in situ hybridization (fish) and … 49 figure 4. using whole-chromosome-painting probes, fluorescence in situ hybridization (fish). to rule out the presence of an additional copy of chromosome 19 (i.e. trisomy 19) in this metaphase distribution, derived from a glial cell taken from a mouse's brain, chromosome painting probes were used. chromosome 19 is green in colour (mcneil and ried 2000). important comparative genomic hybridization in early detection of breast cancer comparative genomic hybridization (cgh) has emerged to promote the aggregation of high-resolution data of cancer-associated genomic imbalances as a highthroughput genomic technology. high-resolution genomic microarray cgh (li et al., 2020). the rapidly growing cgh publication database already contains around 1500 tumours and is beginning to reveal genetic anomalies characteristic of certain forms of tumour or stages of tumour progression (forozan et al.,1997). comparative genomic hybridization has created dna copy number profiles for most of the publicly available breast cancer cell lines (forozan et al., 2000). highresolution cgh analysis of breast cancer shows nonrandom associations between particular amplicons in many regions where dna copy number is frequently obtained or lost. that specific genetic modification is preserved in breast cancer cell lines despite repeated passage through tissue culture (climent et al., 2007). recent results released and discussed at scientific meetings have suggested higher rates of implantation and pregnancy after microarray testing, resulting in changes expected for quite some time. by using markers spanning much of the genome, it is not only possible to detect aneuploidy in single cells but also translocations. the validation results indicate that the cgh array in single cells has a resolution of 6 mb. therefore, most translocations can be tested as this is also the limit of karyotyping. translocations of smaller exchanged fragments may also classify the translocation, as three out of the four fragments are above 6 mbb fragment ((munné, 2012). over the past decade, genomic microarray technology has greatly matured. the technique offers a locus-by-locus measure of the variance of dna copy-number (cnv) and represents another way to improve mapping resolution. postnatal chromosomal array techniques have far higher diagnostic results (15-20 per cent) than g-banded karyotyping does for genetic testing of people with unexplained developmental delay, intellectual impairment, autism, or other congenital abnormalities, as the first-tier cytogenetic diagnostic test for people with these diseases, the international standards for cytogenomic array consortium recommends it (lee et al.,2012). the genotyping of metastatic samples, primarily focused on array-based comparative genomic hybridization (acgh) and next-generation sequencing, is the growth of targeted therapies and the emergence of personalized medicine (ngs), another solution to resolving acgh's drawbacks, such as the repeat-rich regions, is next-generation sequencing. the genomic analysis should be paired with expression analysis to elucidate individual genes related to breast cancer development and progression. identification of new molecular targets for breast cancer eradication will contribute to the elucidation of the functions of the affected genes (ueno et al., 2012). figure 5. the photomicrograph of fluorescence shows the effects of comparative genome hybridization of invasive ductal carcinoma tissue. (a) tumour tissue extracted dna was labelled green, and (b) natural reference dna was labelled red. (c) the chromosome of the regular metaphase was counterstained blue with dapi. (d) tumour and normal dna have been hybridized to the normal chromosome of the metaphase. there is a 50 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 predominantly green colour in chromosomal regions, which were overrepresented in the tumour, while regions with deletions in the tumour show a predominantly red colour. the overlap reflects the ratio of changes in the copy number between the tumour and the dna control (zhang et al., 2015). comparative classical cytogenetic tools with fluorescence in situ hybridization hybridization of fluorescence in situ (fish) enables cytogenetic analyses of primary tumours without culture (thompson and gray 1993). in the identification and evaluation of human malignancies, one of the greatest impacts has been in the non-dividing interphase nucleus, chromosome translocations, deletions, amplification of particular genes, and chromosome number changes identified using probes ranging from whole chromosome 'paints' to individual gene-specific probes. progress in fish technology has also benefited from gene mapping (price 1993). in comparison to the methods mentioned previously separate denaturation or proteinase k digestion changes are not needed for each sample. this technique allows retrospective studies of large series of tumors and is also useful for the routine diagnostic use of formalin-fixed material to apply fish (hyytinen et al., 1994). the fish technique also helped us determine the degree of amplification and the size of the intrachromosomal amplified regions at metaphase and interphase (bar et al., 1992). fluorescence in situ hybridization (fish) is used in genetic toxicology for the study of chromosome damage with improved efficiency and precision to distinguish certain forms of chromosome aberrations, in addition to classical cytogenetic methods for scoring chromosomal aberrations (hovhannisyan, 2010). the study shows the utility of a combination of classical karyotyping and fish to determine the chromosome origin of doubleminute chromosome amplified dna sequences in cancer cells (giollan et al.,1996). fish identified cells with clonal chromosomal defects with rates of aneuploidy ranging from 6% to 92% (median 59%). in addition, there was a gain of centromeric signals for chromosome 11, most likely corresponding to hyperdiploid; aberrations of chromosome 17 in specimens from 26 patients (87%) were hyperploid as well; however, four cases (13%) showed loss of chromosome 17 centromeres (fiegl et al., 1995). modern high-throughput techniques affect research to classify new genomic regions associated with tumours (liehr et al., 2015). in correlating karyotype abnormalities with diagnosis, prognosis, and response to therapy in haematological neoplasias, classical cytogenetics described by chromosomal banding techniques has been effective. such approaches, however, require a high cell division rate and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and study, which is typically a challenge for doctors (varella, 2003). also, fish is considered safer and has the added benefit of using several fluorochromes to differentiate between different targets simultaneously (bartlett, 2004). these approaches range from dna fluorescence in situ hybridization (fish)based kilobase-level resolution imaging approaches of individual cells to genome-wide sequencing strategies that collect nucleotide-level data from different sample types. in conjunction with the combinatorial use of multiple approaches, technical developments have led to new rearrangement groups and mechanistic insights into processes that drive structural changes in the human genome (hu and ly 2020). fish and traditional cytogenetic experiments often offer a false negative outcome (alayed et al., 2013). figure 6. schematic diagram of karyotyping (qaisar and bhat, 2017). smail – the roles of the fluorescent in situ hybridization (fish) and … 51 figure 7. the concepts of in situ hybridization of fluorescence. (a) a dna probe and a target sequence are the basic elements. (b) before hybridization, the dna probe is indirectly marked with hapten (left panel) or specifically marked with hapten (left panel) by adding a fluorophore probe (right panel). (c) the labelled probe and the target dna are denatured to yield single-stranded dna. (d) they are then mixed, which enables complementary dna sequences to be annealed. (e) if the probe has been indirectly labelled, the visualization of the non-fluorescent hapten using an enzymatic or immunological detection system involves an additional step. finally, fluorescence microscopy tests the signals (shakoori, 2017). limitation of the fluorescence in situ hybridization while direct preparations may be carried out, cell culture is usually needed (1-10 days), complex karyotypes with suboptimal morphology may be encountered, submicroscopic or cryptic rearrangements can result in a false-negative result, normal karyotypes may be observed after therapy-induced tumour necrosis or overgrowth of normal stromal cell support low cell density and the release of cells from the bone matrix are also issues with bone tumours (bridge, 2008). there is still a lack of a mechanistic approach to the whole fish system, and the key limiting steps for hybridization remain uncertain (lima et al., 2020). the rate-limiting phase was still the slow reaction rate of the reagents used in the probe solution. in particular, hybridization was slowed by using formamide, which acts as a doublehelix destabilizing agent (nguyen et al., 2018). however, a fluorescence microscope is required for fish, and the signals are labile and easily fade over time (kim et al., 2011). in recent years, the combination of microfluidic techniques and fish have tackled weaknesses in the consumption of probes and hybridization times, making the experimental process more sustainable and adaptable to high-throughput innovations (huber et al., 2018). as cytogenetic defects have been found in samples that appear normal by morphological and conventional cytogenetic examination, the fish analysis provides enhanced sensitivity in many cases. the combination of cytogenetic, fish and molecular studies offers a powerful method for diagnosing and sub classifying malignant diseases into clinically and biologically important subgroups, selecting effective therapies, and monitoring the effectiveness of therapeutic regimens (gozzetti and le, 2000). it is also recommended that fish and cgh findings be re-evaluated by one another 52 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 to resolve these technological artefacts. however, cgh is of potential benefit in characterizing chromosomal alterations and could help produce tumour-specific sets of fish probes within a few days to obtain genetic information of prognostic value (jacobsen et al., 2000). conclusions from this review article conducted the following points as follows: 1. fluorescence in situ hybridization (fish) more accurate than classical cytogenetic such as karyotyping 2. comparative genomic hybridization (cgh) is very important for the create a wide range of chromosomal profile 3. both fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) have many limitations and disadvantages 4. probes is the key success for both fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) conflict of interest: the author declares no conflicts of interest. references alayed, k., medeiros, l. j., schultz, r. a., cortes, j., lu, g., bueso-ramos, c. e., & konoplev, s. 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(2015). comparative genomic hybridization analysis of invasive ductal breast carcinomas in the chinese population. oncology letters, 10(4), 2100-2106. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 65-75 | doi: 10.14421/biomedich.2020.92.65-75 issn 2540-9328 (online) polyphenols content and antimicrobial, antioxidant and hemolytic activities of essential oils from four selected medicinal plants growing in algeria noureddine halla1,2,*, kebir boucherit2, bankaddour zeragui1, abdelkader djelti1, ziane belkhedim1, rachida hassani1, saada benatallah1, hassiba djellouli1, oumlkheir kacimi1, zahia boucherit-otmani2 1laboratory of biotoxicology, pharmacognosy and biological recovery of plants, department of biology, faculty of sciences, university of saida dr. moulay-tahar, 20000, algeria. 2antibiotics antifungal laboratory, physical chemistry, synthesis and biological activity (lapsab), department of biology, faculty of sciences, university of tlemcen, bp 119, tlemcen, 13000, algeria. corresponding author* halla.nour@yahoo.fr; noureddine.halla@univ-saida.dz manuscript received: 12 july, 2020. revision accepted: 10 november, 2020. published: 13 november, 2020. abstract the saharan and steppe spontaneous plants are very characteristic because of their particular adaptation to the desert and extreme environment. some species have pharmacological properties that give them a medicinal interest. the aim of the present work was to determine the polyphenol contents of essential oils obtained from four endemic plants growing in algeria (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita), and study its biological activity, including antimicrobial, antioxidant, and hemolytic. the antimicrobial activity was evaluated by the microdilution method against twelve strains. the antioxidant activity was carried out by two methods (dpph radical scavenging and reducing power). however, the hemolytic effect has been evaluated against the red blood cells. p. scoparius and m. piperita showed yields of essential oils higher than 1%. all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans treated by r. officinalis essential oils. the most sensitive strain was c. albicans treated by p. scoparius essential oils by mic of 0.0781 mg/ml, it was the same plant that shows the highest polyphenol content (14.78 ± 0.72 g gae/g ds). the antioxidant activity by the dpph method was greater for all essential oils tested by ic50 ranging from 0.69 ± 0.07 (r. officinalis) to 30.67 ± 2.12 mg/ml (m. nivellei). the r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). in reducing iron, it was the r. officinalis essential oils which were found to be the most active with an ec50 concentration of 9.67 ± 1.36 mg/ml. after 120 min incubation, minimal haemolysis (10%) was obtained with essential oils of r. officinalis at a concentration of 0.39 mg/ml. we conclude that p. scoparius essential oils showed the high content of polyphenols and r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). keywords: polyphenols; antimicrobial; antioxidant; essential oils; hemolytic; mentha piperita; myrtus nivellei; pituranthos scoparius; rosmarinus officinalis; sahara. introduction the total algerian flora has about 16,000 plant species, of which more than 1,000 species with medicinal properties and 700 species are endemics. algeria is the source of significant taxonomic, ecosystem and landscape diversity (aps, 2019). the spontaneous plants in sahara area have many uses, traditionally practiced by the local population, in terms of pharmaceuticals, food and domestic use. these plants have the ability to synthesize many compounds called secondary metabolites and thus constitute an immense reservoir of compounds of great chemical diversity, possessing a wide range of biological activities (seca and pinto, 2019). this is the case, for example, of plant essential oils which are widely used in therapeutics. in recent years, studies of biological activities of medicinal plants have increased remarkably because of their potential to be used as sources of drugs, food additives or active ingredients in cosmetics (haddouchi et al., 2016). in this context, the aim of the present work was to determine the polyphenol contents of essential oils obtained from four endemic plants growing in algeria (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita), and study its biological activity, including antimicrobial, antioxidant, and hemolytic. pituranthos scoparius and myrtus nivellei are from tassili n'ajjer (sahara), rosmarinus officinalis and mentha piperita are from el bayadh and tiffrit, respectively (steppe area). https://doi.org/10.14421/biomedich.2020.92.65-75 66 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 material and methods plant material and essential oil extraction for the four plants studied, the flowering aerial parts were used. the plant material was identified by dr. tayeb si tayeb (laboratory of biotoxicology, pharmacognosy and biological recovery of plants, university of moulay-tahar, saida, algeria). a voucher specimen was deposited at the herbarium of the laboratory under the following code numbers lbpbpts03-13. pituranthos scoparius and myrtus nivellei were collected in october 2018 and april 2018, respectively, from tassili n'ajjer in south-east algeria (25°30'0" n and 9°0'0" e). rosmarinus officinalis was collected in october 2018 from el bayadh (33°40′49″ n and 1°01′13″ e). mentha piperita was collected in july 2018 from tiffrit (wilaya of saida) (34°54'0.01" n and 0°24'0" e). using a clevenger-type apparatus, 400 g of dried plant materials were subjected to hydro-distillation in 4,000 ml of distilled water for 4 h. the obtained oil was dried over anhydrous sodium sulfate and then stored in sealed glass vials at 4 °c prior to analysis (el asbahani et al., 2015). total polyphenols content the polyphenols are assayed according to the method described by dewanto et al. (2002). a 100 µl quantity of each essential oil was mixed with 2 ml of a freshly prepared sodium carbonate solution (2%). after five minutes, 100 µl of the folin-ciocalteu reagent (1 n) was added to the mixture, the whole was left for 30 minutes at room temperature and the reading is performed against a blank using a spectrophotometer at 750 nm. a standard range based on gallic acid is also prepared. the total polyphenol contents of the essential oils are then expressed in milligrams gallic acid equivalent per gram of the dried sample (mg eag/g ds) (halla et al., 2019a). antibacterial and antifungal activity the antimicrobial activity of the essential oils was evaluated using different strains; gram-positive bacteria: staphylococcus aureus atcc 25923 and bacillus cereus atcc 11778; gram-negative bacteria: escherichia coli atcc 25933, pseudomonas aeruginosa atcc 27853, pasteurella multocida atcc 43137, salmonella typhimurium atcc 13311, salmonella enterica atcc 13312, campylobacter fetus atcc 27374, klebsiella pneumonia atcc 700603 and enterobacter cloacae atcc 13047; and fungal microorganisms (yeasts): candida albicans atcc 10231 and candida albicans ip 444. bacteria were cultured at 37 °c for 18 h under aerobic conditions in nutrient agar medium (fluka, usa). before experimental use, the cultures from different solid mediums were cultivated in liquid media, incubated, and used as the inoculum for each experiment. muellerhinton broth (bacteria) and rpmi-1640 (yeast) were used for antimicrobial tests (clsi, 2008; clsi, 2012). the minimum inhibitory concentration (mic) was determined based on the methods approved by the national committee for clinical laboratory standards (clsi, 2008; clsi, 2012), with slight modifications (halla et al., 2019b). the microtiter plates were inoculated within different concentration of essential oils and then incubated at 37 °c for bacteria or 35 °c for c. albicans in a moist dark chamber. the mic of each sample was recorded after 16-20 h of incubation for bacteria and 24 h for c. albicans. end points were defined as the lowest concentration of antibacterial agent resulting in total inhibition of visual growth compared to the growth in the control wells containing no essential oil. the minimum bactericidal concentration (mbc) and fungicidal concentration (mfc) were also determined. after determining the mic, 50 μl (bacteria) or 20 µl (c. albicans) samples were withdrawn from each well of the microtiter tray with a 96-pin replicator (boekel scientific, feasterville, pa, usa) and plated onto nutrient agar (bacteria) or sabouraud dextrose agar plates (c. albicans). inoculated plates were incubated at 37°c for 24 h (bacteria) or 35 °c for 48 h (c. albicans) before determining the mbc (or mfc), which was defined as the lowest concentration of essential oil that resulted in total inhibition of visible growth (espinelingroff and cantón, 2007; qaiyumi, 2007). antioxidant activity  dpph radical scavenging activity the experimental protocol followed that of prieto et al. (1999). fifty microliters of essential oil at different concentrations were added to 1950 μl of a methanolic solution of dpph at 6.34 × 10-5 m. a blank (negative) control was prepared by mixing 50 μl of methanol with 1950 μl of the methanolic solution of dpph. after incubation in the dark for 30 min at room temperature, the reduction of dpph was evidenced by the color change of the solution from violet to yellow. the absorbance of this solution was then determined at 515 nm using a spectrophotometer. the positive control used is ascorbic acid. the results were expressed as percent inhibition (pi), and this was calculated based on the reduction of the color intensity of the solution using the formula: pi = (odcontrol odessential oils / odcontrol) x 100 where odcontrol is the absorbance of the negative control and odessential oils is the absorbance with the essential oils. ic50 value is the concentration corresponds to 50% inhibition (zeragui et al., 2019).  reducing power assays the reducing power is determined according to the method described by oyaizu (1986). a volume of 1 ml halla et al. – polyphenols content and antimicrobial, antioxidant and … 67 of each essential oil at different concentrations was mixed with 2.5 ml of 0.2 m phosphate buffer (ph 6.6) and 2.5 ml of 1 % potassium ferricyanide solution. the mixture obtained was incubated for 20 min at 50°c. after this period, 2.5 ml of 10 % trichloroacetic acid was added to stop the reaction. the resulting mixture was centrifuged at 650 × g for 10 min at room temperature, and 2.5 ml of the resulting supernatant was added to 2.5 ml of distilled water and 0.5 ml of 0.1 % (w/v) iron chloride. the absorbance of this solution at 700 nm was compared to that of a blank solution and the activity of the essential oil was compared with that of the positive control, i.e., butylhydroxyanisole (bha). the results allow calculation for effective concentration (ec50), concentration of the corresponding essential oils with an absorbance equal to 0.5 (halla et al., 2019a). hemolytic assay red blood cells were isolated and suspended in pbs (100 mm at ph 7.4) at a rate of 4000 cells/ml. the erythrocyte suspension was incubated at 37 °c under continuous agitation for 120 min, from the addition of essential oil solution with dmso (dimethyl sulfoxide) at different final concentrations. samples of 50 μl from the reaction solution were made at regular intervals to which we added 2 ml of cold washing solution (150 mm of nacl, 2 mm of mgcl2). after centrifugation at 4000 rpm for 5 min, the absorption of the resulting supernatant was determined at 548 nm by photometric monitoring against a blank sample. control samples of 0% lysis (in buffer) and 100% lysis (in double-distilled water) were employed in all experiments (bolard, 1986; halla et al., 2013; silva et al., 2017). statistical analysis the yields obtained, polyphenols content and antioxidant activity tests were carried out in triplicate and the results are expressed in mean values ± standard deviation. all analyzes were performed in the microsoft excel for windows program, 2007. results and discussion extraction yield in this study, we were interested in the study of different essential oils obtained by hydrodistillation of the aerial parts of four algerian plants. the two studied plants, pituranthos scoparius and myrtus nivellei, were collected in the tassili n'ajjer region in the wilaya of tamanrasset (south of algeria). the tassili national park has been listed as a unesco world heritage site since 1982 and has been classified as a man and biosphere reserve since 1986. it is characterized by a contrasted landscape of rugged mountainous terrain and desert plateau of black rocks which form the reg or white sands. the central barrier of 1500–2000 m in altitude extends over 800 km and covers 80,000 km2 (hammiche and maiza, 2006). the two plants rosmarinus officinalis and mentha piperita were harvested in the steppe area (saida and el bayadh). in algeria, the steppe constitutes a vast region which extends between the tellian atlas in the north and the saharan atlas in the south, extending over a land area of around 20 million hectares. the altitude ranges from 400 to 1,200 meters. the steppe is characterized by a strong climatic constraint (insufficient rainfall with an isohyet varying from 100 to 400 mm, strong and sometimes hot winds, etc.) and edaphic (vulnerable soils, thin and poor in organic matter) (khaldi, 2014). the various yields obtained are reported in table 1. p. scoparius and m. piperita showed yields higher than 1%, however, the yields of m. nivellei and r. officinalis were of the order of 0.81 and 0.76 %, respectively. table 1. yields and total polyphenols content of essential oils of pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita. yields (%) total polyphenols content mg gae/g ds* pituranthos scoparius 1.081 ± 0.061 14.78 ± 0.72 myrtus nivellei 0.81 ± 0.02 0.611 ± 0.056 rosmarinus officinalis 0.76 ± 0.42 0.279 ± 0.087 mentha piperita 1.10 ± 0.13 0.104 ± 0.012 * mg gallic acid equivalents per g dried sample pituranthos scoparius essential oil had a specific odour (odour of fennel) and was colorless. the yield of essential oils of pituranthos scoparius was 1.081 ± 0.061%, which is in agreement with that obtained by lograda et al. (2013) from the same plant collected in mechouneche (biskra). however, the same authors found yields of 0.47%, 0.85% and 2.29% in different regions (t'kout (batna), boussâada (m'sila), and elkantra (biskra), respectively) when the plant was harvested in october. gourine et al. (2011) obtained yields ranging from 0.6 up to 2.8%. ksouri et al. (2017) studied the essential oil of pituranthos scoparius harvested in the wilaya of tamanrasset, they found a yield of 0.4% which is different than that obtained in our study. our result was not correlated with that obtained by vérité et al. (2004) (0.77% from the seeds and 0.50% from the stems of the plant harvested in april), kalla et al. (2010) (0.25% and 0.3% of the aerial part of the plant 68 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 harvested in february and april, respectively), abderrazak et al. (2013) (0.25% of the aerial part of the plant harvested in october) and chikhoune et al. (2017) (0.502% of the aerial part of the plant harvested in april). the essential oil of m. nivellei was pleasant with a light yellow colour. the yield was 0.81 ± 0.02%. this result is supported by that obtained by bouzabata et al. (2013) from the hoggar zone, which was not the case with that harvested from tassili n'ajjer where these authors found yields ranging from 0.5 to 0.9%. boukhalfa (2017) obtained that the yield was 1.6% of this plant harvested in the months of september and october in the tagmart region in tamanrasset. unlike the abundant literature relating to the essential oil of myrtus comunis in algeria, studies on the essential oils of m. nivellei are counted on the fingers. pereira et al. (2009) suggested that the highest yields of the essential oils of myrtus comunis were obtained for the leaves in october. as well as, the samples taken in may and october produced very little oil. altogether, september seems to be the month with the best yields for the different parts of the plant. the yield of essential oils of rosmarinus officinalis (0.76 ± 0.42%) is lower than those found by jordán et al. (2013) of the various essential oils of rosmarinus officinalis harvested in thermo mediterranean, upper mesoand supra-mediterranean areas in spain, which are of the order of 1.74 ± 0.38, 2.44 ± 1.26 and 2.58 ± 0.75%, respectively. zaouali et al. (2010) reported that the yields of essential oils of this plant harvested in tunisia from three different zones (sub-humid, upper semi-arid and upper arid zone) are between 1.17 and 2.7%. a study by djeddi et al. (2007) recorded a yield of 0.82%. this result seems very close to our result, may be due to the same origin of the two plants (algeria). the essential oil of mentha piperita was pale yellow with a strong aromatic odour. the essential oil yield of mentha piperita was 1.10 ± 0.13%. work carried out on the same species in algeria (ghardaïa) revealed a yield close (1.325%) to the value obtained by our study on the essential oil of m. piperita (laghouiter et al., 2015). another study in morocco showed a yield around 1.02% (derwich et al., 2010). on the other hand, yield values for essential oil of m. piperita have been recorded by several authors from different countries, and which are higher or lower than that obtained by our study. singh et al. (2015) revealed a yield of about 0.64% from the leaves of m. piperita harvested in libya. a yield of 2.29% was recorded by gavahian et al. (2015) of the aerial part of the same plant harvested in iran. laghouiter et al. (2015) reported that the extraction yield was very low during the cold period (autumn and winter), however the best yields were obtained in summer and spring during the flowering period of the plant. rohloff et al. (2005) studied the effect of the harvest time and the drying method on the essential oil yield of mentha piperita, and they concluded that the harvest of this plant should be carried out at full flowering to obtain the best yield. this difference may be due to the geographical origin of the plant as well as to the harvest period. according to some authors, the harvest season can affect the variation in the yield of essential oils (boukhalfa, 2017). others explain this difference by the harvest period and the drying method (rohloff et al., 2005). other factors are likely to influence the difference in yield. precipitation can particularly affect the yield of essential oils (sangwanet al., 2001). plant ontogenesis can be one of the most important factors that influence the accumulation of essential oils in plants. since many transformations and changes occur inside cells due to physiological processes, the harvest season and the organ used are often considered to be critical parameters that can affect the chemical compositions of essential oils (lee and ding, 2016). total polyphenols content the total phenol content is determined from the equation of the linear regression of the gallic acid calibration curve: y = 0.005 x + 0.185 (r² = 0.996) and the results are expressed in mg gallic acid equivalents per g of dried sample (mg gae/g ds). table 1 summarizes the results for essential oils of four species tested. from the results obtained, we observed a high content of polyphenols in the p. scoparius essential oils (14.78 ± 0.72 mg gae/g ds), however, the essential oils of m. nivellei, r. officinalis and m. piperita contain 0.611 ± 0.056, 0.279 ± 0.087 and 0.104 ± 0.012 mg gae/g ds, respectively. the total phenol contents are variable between the four plants. the total phenol content found by wojdylo et al. (2007), in essential oils of rosmarinus officinalis (1.71 ± 0.02 mg gae/g ds) is greater than our value (0.279 ± 0.087 mg gae/g ds). for the essential oils of m. piperita, the total phenol content is lower than that of the same species previously studied by zheng and wang (2001); it is about 2.26 ±0.16 mg of gae/g of fresh weight. studies on the extract of m. nivellei leaves indicated that the hydromethanolic, ethyl acetate, butanolic and aqueous fraction had the highest values in phenolic content (222.98 ± 4.70, 308.4 ± 7.40, 414.96 ± 2.40 and 128.03 ± 1.87 mg gae/g ds, respectively) (ramdane et al., 2017). in another study on the aqueous extract of the leaves of m. nivellei, the level of total polyphenols was estimated around 242.68 ± 9.79 mg gae/g ds (rached et al., 2010). lograda et al. (2013) reported the variation in the composition of the essential oils of pituranthos scoparius in algeria and they recorded that these oils are very rich in alcoholic compounds such as terpinene-4-ol, p-cymèn-8-ol, β-eudesmol, methyl-eugenol, spathulenol, linalool, γ cadinol and t-muurolol. halla et al. – polyphenols content and antimicrobial, antioxidant and … 69 the value of the polyphenol content of mentha piperita is not in agreement with those obtained by sharafi et al. (2010), from the plant harvested in iran, where the polyphenol level was 89.43 ± 0.58, 40.43 ± 0.58, 15.10 ± 1 and 9.43 ± 0.58 μg gae/mg sample for oil concentrations of the order of 10, 5, 2.5 and 0.2 µg/ml. atanassova et al. (2011) showed that the methanolic extract of mentha piperita contains a polyphenol level of 0.4525 mg gae/g ds where this plant was harvested in bulgaria. the level of total polyphenols was estimated to be around 71.8 ± 5.1 and 55.1 ± 3.6 mg gae/g ds of the methanol/chloroform extract and the aqueous extract from the leaves and stems of mentha piperita harvested in pakistan (akhtar et al., 2018). riachi and de maria (2015) concluded that the composition of mentha piperita oil is very sensitive to environmental variations where the maturity of the leaves plays an important role in the composition of the essential oil. thus, mint plants harvested at the flowering stage usually have a higher concentration of menthol (phenol) than plants in the bud-forming process, which contain a high amount of menthone. another factor, the exposure of the plant mentha piperita to uv-a radiation in addition to white light, during the night, increased the phenol content (maffei et al., 1999). in short, longer days, colder nights, older leaves, plants harvested at the flowering stage, and adequate hydration of plants should provide good quality peppermint essential oil (riachi and de maria, 2015). by comparison with previous studies, it seems that the extraction of phenolic compounds is governed by several factors which directly influence the levels of these molecules among these factors; increasing the extraction temperature, contact time of the plant material with water, and decreasing the particle size to increase the diffusion coefficient of the water. antibacterial and antifungal activity the obtained results of the antibacterial and antifungal activity (minimum inhibitory concentration ‘mic’ and minimum bactericidal (fungicidal) concentration ‘mbc’ -‘mfc’) of the essential oils of the four plants are presented in table 2. all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans against r. officinalis essential oils. all essential oils tested are less active than the positive control (gentamicin or ketoconazole) with the exception of rosmarinus officinalis essential oils against salmonella typhimurium (mic and mbc = 0.625 mg/ml), salmonella enterica (mic = 0.0825 mg/ml and mbc = 0.312 mg/ml) and klebsiella pneumonia (mic = 0.3125 mg/ml and mbc = 1.25 mg/ml), as well as essential oils of mentha piperita against staphylococcus aureus (mic = 0.078 mg/ml). the strains treated by p. scoparius essential oils showed that the most sensitive strain was c. albicans atcc 10231 by mic of 0.0781 mg/ml. on the other hand, all bacteria tested against p. scoparius in this study revealed mics of 5, 10 or higher than 10 mg/ml. e. coli was the most sensitive bacteria tested by m. nivellei essential oils with mic around 2.5 mg/ml, while, the fungal strains showed good mic (0.312 mg/ml) by comparing with those obtained against bacteria. table 2. minimum inhibitory concentrations and minimum bactericidal (fungicidal) concentrations(mg/ml) of essential oils (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita) and controls. test organisms pituranthos scoparius myrtus nivellei rosmarinus officinalis mentha piperita controld mica mbcb (mfcc) mic mbc (mfc) mic mbc (mfc) mic mbc (mfc) mic mbc staphylococcus aureus atcc 25923 5 5 10 10 10 10 0.078 0.156 0.156 0.156 bacillus cereus atcc 11778 10 10 5 5 2.5 10 5 5 0.156 0.156 escherichia coli atcc 25933 10 10 2.5 2.5 1.25 5 0.312 0.625 0.312 0.312 pseudomonas aeruginosa atcc 27853 10 10 5 5 2.5 5 5 5 0.625 1.25 pasteurella multocida atcc 43137 10 10 10 10 2.5 10 0.156 1.25 0.039 0.039 salmonella typhimurium atcc 13311 ˃ 10 ˃10 10 10 0.625 0.625 1.25 2.5 0.625 0.625 salmonella enterica atcc 13312 5 5 10 10 0.0825 0.312 10 10 0.625 0.625 campylobacter fetus atcc 27374 ˃ 10 ˃ 10 10 10 5 10 5 5 0.019 0.039 klebsiella pneumonia atcc 700603 10 10 10 10 0.3125 1.25 2.5 5 5 10 enterobacter cloacae atcc 13047 ˃ 10 ˃ 10 5 5 5 10 5 5 2.5 5 candida albicans atcc 10231 0.0781 0.0781 0.312 0.312 / / 0.625 2.5 0.019 0.019 candida albicans ip 444 0.039 0.039 0.312 1.25 / / 5 5 0.019 0.019 a mic: minimum inhibitory concentrations, bmbc: minimum bactericidal concentrations, cmfc: minimum fungicidal concentrations d control: gentamicin for bacteria and ketoconazole for candida albicans concerning r. officinalis, s. enterica recorded the lowest mic compared to the other tested bacteria (0.0825 mg/ml). by comparison, gram negative bacteria were more sensitive than gram positive against r. officinalis essential oils. indeed, the registered mics of m. piperita essential oils for b. cereus, p. aeruginosa, 70 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 s. enterica, c. fetus, e. cloacae and c. albicans ip 444 (from 5 to 10 mg/ml) were higher than those obtained for s.aureus, e.coli, p. multocida, s. typhimurium, k. pneumonia and c. albicans atcc 10231 (from 0.078 to 2.5 mg/ml). the mics for pituranthos scorparius essential oil were similar to those reported by boutaghane et al. (2004) for enterobacter, klebsiella pneumoniae and salmonella thiphymurium where they found mics of the order of 256, 16 and 128 mg/ml, respectively. on the other hand, the mic results obtained by these authors against escherichia coli (256 mg/ml), pseudomonas aeruginosa (1 mg/ml) and staphylococcus aureus (256 mg/ml) are not in agreement with our results. ksouri et al. (2017) found that no antibacterial activity of pituranthos scorparius essential oil has been revealed against escherichia coli and klebsiella pneumoniae. however, the mics obtained for staphylococcus aureus and candida albicans were of the order of 1 and 0.5 mg/ml. the mic obtained by ksouri et al. (2017) against pseudomonas aeruginosa (greater than or equal to 2 mg/ml) is correlated with that of our result. bouzabata et al. (2013) studied the antifungal activity of the essential oil of myrtus nivellei (same species in algeria), they showed mics and mfcs between 1.25 and 2.5 µl/ml against candida albicans atcc 10231. these can be correlated with the cmf obtained against candida albicans ip 444 (1.25 mg/ml). a work by kabouche et al. (2005) on essential oils of rosmarinus officinalis grown in algeria showed mics greater than 0.128 mg/ml against both strains e. coli and s. aureus. the results of the cmi are in agreement with those obtained by celiktas et al. (2007) from turkey (izmir, çanakkale) of the essential oils of the plant harvested in september against the staphylococcus aureus (10 mg/ml). however, a tunisian study reported that essential oils of this species revealed mics between 1.25-2.5 μl/ml against e. coli and between 1.25-2.5 μl/ml against bacillus cereus (zaouali et al., 2010). it is comparable to our results. according to jordán et al. (2013), these essential oils have an mic of 2-5 μl/ml against e. coli. i̇şcan et al. (2002) tested the essential oil of m. piperita from different origins (turkey and india), they reported mics between 1.25 and 2.5 mg/ml against escherichia coli, between 0.625 and 2.5 mg/ml against staphylococcus aureus, between 1.25 and 2.5 mg/ml against salmonella typhimurium, of 2.5 mg/ml against klebsiella pneumoniae, of 1.25 mg/ml against bacillus cereus and between 0.312 and 0.625 mg/ml against candida albicans. these results are correlated with our results, with the exception of those obtained for escherichia coli and bacillus cereus. the mic values for essential oil of m. piperita against different bacterial strains, reported by mahboubi and kazempour (2014), range from 0.125 to a value greater than 64 μl/ml. the mics are of the order of 1, 0.25, 1, 2, 16, 0.25 and 0.125 μl/ml against s. aureus, b. cereus, e. coli, s. typhimurium, p. aeruginosa, k. pneumioniae and c. albicans, respectively. however, the mbcs are of the order of 2,> 64, 1, 2, 16 0.5 and 0.125 μl/ml against s. aureus, b. cereus, e. coli, s. typhimurium, p. aeruginosa, k. pneumioniae and c. albicans, respectively. mohammadi et al. (2016) revealed percentages of 0.16%, 0.8% and 2% of mic50, mic90 and mbc, respectively, of the essential oil of m. piperita against e. coli. compared with our result, it seems that the oil tested has a strong activity to that tested by mohammadi and al. tyagi and malik (2011) recorded mic and mbc (or mfc) of 1.13 to 2.25 mg/ml (mic) and 2.25-9 mg/ml (mbc) for bacterial strains and 1.13 mg/ml (mic) and 2.25 mg/ml (mfc) for yeasts. according to the study by saharkhiz et al. (2012), the essential oils of m. piperita have an antifungal activity with an mic of 1.5 μl/ml against c. albican strain. according to the study by saharkhiz et al. (2012), the essential oils of m. piperita have an antifungal activity with a mic of 1.5 μl/ml against c. albicans. samber et al. (2015) found that m. piperita essential oil is a bioactive fungicidal compound that has a strong effect on pm-atpase (pm: plasma membrane) in candida species. they suggested these essential oils enter the cell membrane and target the pathway for ergosterol biosynthesis, thereby compromising its biosynthesis. simultaneously, they react with the membrane itself with their reactive hydroxyl moiety, and the extensive lesion on the membrane is a combined effect of the two events. antioxidant activity the evaluation of the antioxidant activity of the essential oils of the four plants was carried out by two conventional methods in order to test these essential oils by various reaction mechanisms involved in these antioxidant tests. the dpph radical is one of the most widely used substrates for the rapid and direct evaluation of antioxidant activity due to its stability in radical form and the simplicity of this analysis. the antioxidant power of the essential oils has been compared to that of the positive control used (ascorbic acid). the values of the concentrations corresponding to the inhibition of 50% of the free radical dpph (ic50) are summarized in table 3. from the results obtained, we observed that the essential oils of the four plants showed an antioxidant activity by scavenging of the free radical dpph. by comparing the antioxidant power of ascorbic acid (positive control) with that of the essential oils of p. scoparius, m. nivellei and m. piperita, we note that these essential oils were less active and showed a weak antioxidant activity relative to ascorbic acid. however, the r. officinalis essential oils (ic50 = 0.69 ± 0.07 halla et al. – polyphenols content and antimicrobial, antioxidant and … 71 mg/ml) reported more antioxidant power than the ascorbic acid (ic50 = 0.81 ± 0.14 mg/ml). from the results obtained, the essential oils of p. scoparius, m. nivellei and m. piperita have ic50 values of 3.26 ± 0.65, 30.67 ± 2.12 and 18.33 ± 1.84 mg/ml, respectively. table 3. ic50 values for reduction of radical dpph and ec50 of reducing power of essential oils of pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita (mg/ml). ic50 dpph ec50 reducing power ascorbic acid 0.81 ± 0.14 / bha / 0.42 ± 0.12 pituranthos scoparius 3.26 ± 0.65 484.40 ± 5.14 myrtus nivellei 30.67 ± 2.12 31.2 ± 2.59 rosmarinus officinalis 0.69 ± 0.07 9.67 ± 1.36 mentha piperita 18.33 ± 1.84 11.07 ± 0.07 the values of the optical densities obtained by reducing power assays allowed to drawing curves for each essential oil. in this test, the increase in absorbance means an increase in the reducing power of the essential oils tested. in order to compare the antioxidant activity of the essential oils tested by this method, we calculated the ec50. the results obtained are illustrated in table 3. we note that the essential oils of the p. scoparius showed a very low reducing power by comparing them with the positive control used (bha) and the essential oils of the other plants (484.40 ± 5.14 mg/ml). the essential oils of r. officinalis reported an ec50 of 9.67 ± 1.36 mg/ml, this time their antioxidant activity have not powerful to those of the positive control bha (0.42 ± 0.12 mg/ml). the ec50 of m. nivellei and m. piperita essential oils are 31.2 ± 2.59 and 11.07 ± 0.07 mg/ml, respectively. the essential oils of pituranthos scoparius have an ic50 value of 3.26 ± 0.65 mg/ml which results in a much higher antioxidant power than the value obtained by ksouri et al. (2017) which is of the order of 11.21 ± 0.26 mg/ml of the same species. to our best knowledge, there is no work done on the antioxidant activity of the essential oils of m. nivellei. touaibia and chaouch (2014) studied some extracts of this plant, they showed that the extracts studied all had a very good reducing activity, especially for the ethanol extract (ec50 equal to 0.59 mg/ml) by the dpph method and methanol extract by the frap test (66.7%). rached et al. (2010) evaluated the antioxidant activity of the fractions obtained from the raw extract of the leaves of m. nivellei. they found that the best activity was reported for the ethyl acetate and n-butanol fractions with ic50 values of 3.08 ± 0.40 μg/ml and 4.40 ± 0.43 μg/ml, respectively. in addition, ramdane et al. (2017) reported ic50 values between 4.97 μg/ml and 16.33 μg/ml, for the different extracts obtained from m. nivellei leaves (hyudromethanol, ethyl acetate, butanolic and aqueous). the antioxidant activity of essential oils of rosmarinus officinalis is greater compared to that found by zaouali et al. (2010) and wojdylo et al. (2007), which revealed a low antioxidant power, by the two dpph and frap methods. for the essential oils of m. piperita, we compared our results with the work carried out by singh et al. (2015), on the same species of libya, which showed a higher antioxidant activity by scavenging the dpph radical, with an ic50 value of 15.2 ± 0.9 μg/ml. according to sharafi et al. (2010), the m. piperita essential oil has shown a percentage inhibition of dpph activity of 63.82 ± 0.05% at the concentration of 10 µg/ml where the ic50 was around 3.9 μg/ml. laghouiter et al. (2015) recorded an ec50 of around 208.495 ± 4.247 μg/ml of m. piperita essential oil. gavahian et al. (2015) extracted the essential oil from m. piperita by four different methods (hydrodistillation; steam entrainment, hydrodistillation assisted by microwaves and hydrodistillation assisted by ohmic heating). they found that the antioxidant activity was almost similar for the essential oil obtained by these different methods whose ec50 values were between 9.6 ± 0.7 and 10.4 ± 0.6 μg/ml. our results, in most cases, are not in agreement with those obtained by previous work. this difference in the results is probably due to the diversity of the chemical composition and according to intrinsic and extrinsic factors, namely the harvest region and the evaluation method used. hemolytic asssay the hemolysis test was evaluated because, even if a plant has potent antioxidant power or good antimicrobial activity, its use in traditional medicine and in pharmacological preparations will be impossible in the presence of their hemolytic effect, which is an indicator of cytotoxicity. when the plasma membrane of red blood cells is altered by the action of essential oil, it follows lysis resulting in the release of hemoglobin in the red blood cell, which is why we assayed the extracellular hemoglobin after the addition of different concentrations of essential oils. final concentrations of essential oils were chosen according to the mic founded in the first part. figure 1 shows the effect of essential oils at different concentrations (0.39, 0.781, 1.56 and 3.125 mg/ml) on the release of hemoglobin from the red blood cells at 37 °c. the results obtained show that the percentages of hemolytic effect are directly proportional to the increase in the concentrations of the essential oils of the four tested plants (fig. 1). after 120 minutes of incubation and for all the concentrations tested, the hemolysis percentages are between 10 and 93%. therefore, the hemolytic effect of the various essential oils tested, at the concentration of 3.125 mg/ml after 120 minutes of contact with human erythrocytes, can be classified as 72 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 follows: p. scoparius (93%) m. piperita (72.88 %) > m. nivellei (72.75%) > r. officinalis (43.26%). after 120 min incubation, minimal haemolysis (10%) is obtained with essential oils of r. officinalis at a concentration of 0.39 mg/ml, so these essential oils may be slightly hemolytic at this concentration after two hours of incubation. on the other hand, the other essential oils have an important hemolytic effect against isolated erythrocytes, with a hemolysis rate that exceeds 41% at a concentration of 0.39 mg/ml. figure 1. release of hemoglobin by erythrocytes induced by different concentrations (mg/ml) of essential oils (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita). our results show that the essential oils of rosmarinus officinalis have a very low toxic effect compared to isolated erythrocytes, with a haemolysis rate not exceeding 45% at a concentration of 3.1225 mg/ml, what characterizes it by the absence of risk of cytotoxicity. for other plants, they may be slightly hemolytic at high concentrations with respect to human erythrocytes. samber and his collaborators have shown that the rate of hemolysis of essential oil of mentha pipireta against human red blood cells does not exceed 6% at a concentration of 2 mg/ml after one hour of incubation (samber et al., 2015). mendanha et al. (2013) report that terpenes can compete with the intermolecular hydrogen bond between lipid molecules and thereby disrupt the network of hydrogen bonds in the lipid bilayer, which weakens the membrane. as well, jain et al. (2002) found that terpenes, which have alcoholic ‘oh’ groups and which act as hydrogen bond donors, can disrupt the hydrogen bond network in the membrane bilayer. according to some authors, the cytotoxicity of essential oils against red blood cells is due to their hydrophobic nature which is accentuated by the synergetic effect between their compounds (sacchetti et al., 2005). silva et al. (2017) suggest that the constituents present in oils can interact with the components of the erythrocyte membrane, leading to destabilization of its structure and to a disordered influx of ions and water which leads to rupture of the membranes. conclusion the plants studied were harvested from areas of specific climate in algeria. p. scoparius and m. piperita showed yields higher than 1%. according to the antimicrobial activity results, all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans against r. officinalis essential oils. the high content of polyphenols was reported in p. scoparius essential oils (14.78 ± 0.72 mg gae/g ds). the antioxidant test shows that the the essential oils of the four plants showed an antioxidant activity by scavenging of the free radical dpph. signally, the r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). the reducing power for all essential oils tested was by ec50 ranging from 9.67 ± 0 20 40 60 80 100 0 5 10 15 30 60 120[ h e m o g lo b in ] re le a se d ( % ) time (min) pituranthos scoparius witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[ h e m o g lo b in ] re le a se d ( % ) time (min) myrtus nivellei witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[ h e m o g lo b in ] re le a se d ( % ) time (min) rosmarinus officinalis witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[ h e m o g lo b in ] re le a se d ( % ) time (min) mentha piperita witness 0,39 0,781 1,56 3,125 halla et al. – polyphenols content and antimicrobial, antioxidant and … 73 1.36 (r. officinalis) to 484.40 ± 5.14mg/ml (p. scoparius). our results show that the essential oils of rosmarinus officinalis had a very low toxic effect compared to isolated erythrocytes (45% at 3.1225 mg/ml). from the results obtained, the geographical origin and period of harvest can influence the yield and the antioxidant activity of the essential oils of the studied plants. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references abderrazak k, messoued r, azzedine z (2013). etude phytochimique et de l’activité antimicrobienne des huiles essentielles de pituranthos scoparius de la region de biskra (sud-est algérien). tunisian journal of medicinal plants and natural products 10(2): xx-xx. akhtar n, ihsan-ul-haq mirza b (2018). phytochemical analysis and comprehensive evaluation of antimicrobial and antioxidant properties of 61 medicinal plant species. arabian journal of chemistry 11 (8): 1223-1235. aps (algeria press service) (2019). environnement: l'observatoire national de la biodiversité inscrit dans la loi de finances 2020 (la ministre de l'environnement et des energies renouvelables, fatima zohra zerouati). algeria press service. http://www.aps.dz/economie/89734environnement-l-observatoire-national-de-la-biodiversiteinscrit-dans-la-lf-2020. atanassova m, georgieva s, ivancheva k (2011). total phenolic and total flavonoid contents, antioxidant capacity and biological contaminants in medicinal herbs. journal of the university of chemical technology & metallurgy 46(1): 8188. bolard j (1986). how do the polyene macrolide antibiotics affect the cellular membrane properties?. biochimica et biophysica acta (bba)-reviews on biomembranes 864(3-4): 258-303. boukhalfa d (2017). contribution à l’étude des plantes aromatiques et médicinales de la région de l’ahaggar (doctoral dissertation, university of algiers, algeria). http://193.194.83.98/jspui/bitstream/1635/14379/1/boukha lfa_djamel.pdf boutaghane n, nacer a, kabouche z, ait-kaki b (2004). comparative antibacterial activities of the essential oils of stems and seeds of pituranthos scoparius from algerian septentrional sahara. chemistry of natural compounds 40(6): 606-607. bouzabata a, bazzali o, cabral c, gonçalves mj, cruz mt, bighelli a, cavaleiro c, casanova j, salgueiro l, tomi f (2013). new compounds, chemical composition, antifungal activity and cytotoxicity of the essential oil from myrtus nivellei batt. & trab., an endemic species of central sahara. journal of ethnopharmacology 149(3): 613-620. celiktas oy, kocabas eh, bedir e, sukan fv, ozek t, baser khc (2007). antimicrobial activities of methanol extracts and essential oils of rosmarinus officinalis, depending on location and seasonal variations. food chemistry 100(2): 553559. chikhoune a, damjan pavleca j, shashkov m, berroua z, chebbi k, bougherra h, zeroual b, aliane k, gagaoua m, boudjellal a, vovk i, krizman m (2017). antioxidant effect induced by the essential oil of pituranthos scoparius in a formulation of a whey spread emulsion. journal of food processing and preservation 41(5): 1-12. clsi (clinical and laboratory standards institute) (2012). methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 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(ssp. sahariensis) flowers as a natural cosmetic preservative: chemical composition, and antioxidant and antibacterial activities. journal of essential oil bearing plants 22(3): 685-694. zheng w, wang sy (2001). antioxidant activity and phenolic compounds in selected herbs. journal of agricultural and food chemistry 49(11): 5165-5170. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 59-65 | doi: 10.14421/biomedich.2021.101.59-65 issn 2540-9328 (online) hibiscus rosa-sinensis l. (red hibiscus) tea, can it be used as a home-remedy to control diabetes and hypercholesterolemia? subhashinie sanadheera, deepanjana subasinghe*, melissa nethmi solangaarachchi, manju suraweera, noshara yushanthi suraweera, nadeesha tharangika rajarata university of sri lanka, mihintale 50300, sri lanka. corresponding author* deepanjana.subasinghe@gmail.com manuscript received: 20 october, 2020. revision accepted: 24 july, 2021. published: 27 july, 2021. abstract according to traditional medicine, hibiscus is used to treat hypertension, heart diseases and many other ailments. commercially available hibiscus tea is prepared with h. sabdariffa and is expensive. h. rosasinensis is the common variety which is abundant in tropical homegardens, however the scientific data are lacking on the effects. the present review focuses on the available scientific data on the effects of h. rosasinensis on controlling diabetes and hypercholesterolemia. the endeavour was to identify whether h. rosasinensis flower petals as a ‘tea’ is effective for diabetic and hypercholesterolemic patients. medical databases such as medline, bmc, bmj, research gate, mendelay literature search database, google scholar and the general engine google were searched from november 2018 to april 2019. search was carried out using keywords such as “hibiscus rosasinensis”, “antidiabetic effects”, “antihyperlipidemic effects”, “toxic effects”, “anti-inflammatory effects”, “phytochemicals in hibiscus” etc. data were critically analyzed to assess whether the effective doses of the research studies on a par with the doses present in h. rosasinensis teas. we found thirty-nine journal articles fulfilled the criteria. the data were categorized and extracted on uses of h. rosasinensis, anti-diabetic effects, anti-inflammatory effects, antihypercholesterolemic effects and its phytochemicals. the review revealed that the dose of h. rosasinensis petals present in a home-made hibiscus tea is theoretically sufficient to elicit anti-hyperglycemic and anti-hyperlipidemic effects. home-made hibiscus tea is effective in controlling diabetes and hypercholesterolemia without causing acute toxicity. keywords: hibiscus rosasinensis; hibiscus sabdariffa; diabetes; hypercholesterolemia. introduction the trend of the use of herbal remedies is rapidly growing globally (ekor & pistelli, 2013). based on 2002 data, almost four billion people in the world relied on herbal remedies for primary health issues(mukherjee, 2002). although the beneficial effects of these herbal remedies on health problems have been observed and experienced by humans for thousands of years, yet minimum scientific data are available on these treatments (ekor & pistelli, 2013). lack of data on the mode of action, potential adverse reactions, contraindications and interactions with existing western medical treatments has restricted these products from being ‘competitive leading herbal supplements’ in the market. hibiscus is a family of flowering plants which the leaves and the red flower petals are used as herbal remedies by most people in the world. yet data are scarce on the effects of different varieties. consumption of tea made of flower petals of hibiscus family is being practiced by a large number of patients in the world to control hypertension and hyperlipidemia (haji faraji & haji tarkhani, 1999; mckay et al, 2010) thus hibiscus tea is commercially available in international markets. to prepare hibiscus tea, a variety of flowers which are belonged to hibiscus family is used. hibiscus sabdariffa is the mostly consuming variety which also has scientifically proven hypotensive and hypolipidemic effects (haji faraji & haji tarkhani, 1999; mckay et al., 2010). however, hibiscus rosasinensis is the most common, easily cultivated plant which is accessible to most people in the world as it is grown massively in tropics and sub-tropics (it can be cultivated in glass chambers in cold weathers as it does not tolerate temperatures below 10oc). aqueous extract of hibiscus rosasinensis red flower petals is a common herbal home-remedy consumed by people in asian countries however, has minimum scientific data to prove the effects on humans. this plant is a glabrous shrub cultivated as an ornamental plant. among the different colors, red flowered variety is mostly used as a herbal remedy (jadhav et al., 2009). https://doi.org/10.14421/biomedich.2021.101.59-65 60 biology, medicine, & natural product chemistry 10 (1), 2021: 59-65 due to universal availability and good palatability, herbal home-made tea could be prepared with the petals of hibiscus rosasinensis with a minimum preparation time. prior a study on the effects of hibiscus rosasinensis tea on humans, the present review mainly focuses on the effects of this flower on diabetic and hypercholesterolemic animals. methods several medical databases such as medline, bmc, bmj, research gate, mendelay literature search database, google scholar and the general engine google were searched from november 2018 to april 2019. some full articles were accessed through hinari gateway. we used categorized search using keywords such as “hibiscus rosasinensis”, “antidiabetic effects”, “antihyperlipidemic effects”, “toxic effects”, “anti-inflammatory effects”, “phytochemicals in hibiscus” etc. we included trials conducted with animals and humans that had examined the effectiveness of hibiscus rosasinensis and safety in the treatment of hyperglycemia and hyperlipidemia. as the review focuses on the effectiveness of hibiscus tea, the initial endeavour was to review the studies carried out only with water extracts of the flower petals and to exclude the studies carried out with other solvent extraction methods. however, due to scarcity of published data, studies conducted with other solvent extraction methods were also included. researches carried out with parts of the hibiscus plant except for petals were excluded. after gathering the relevant articles, we crossreferenced to see the validity and traced back to the original articles to avoid any plagiarism. most commonly discussed most recent research findings in the original articles were included to conclude this review. the articles were categorized to extract data on uses of h. rosasinensis by humans, anti-diabetic effects, antiinflammatory effects, anti-hypercholesterolemic effects and phytochemicals in h. rosasinensis. data were critically analyzed to assess whether the effective doses of the research studies on a par with the doses present in h. rosasinensis teas. description of the plant different names used for this plant in different countries are given in the first volume of the “medicinal plants of the world” by ivan a. ross (ross, 2003). the plant is a shrub with long slender branches of about 6 m tall. the flowers are borne singly at the apexes or ends of the branches, on long stalks. flowers possess colorful overlapping petals (ross, 2003). classification: kingdom : plantae clade : angiosperms clade : eudicots clade : rosids order : malvales family : malvaceae genus : hibiscus species : h. rosa-sinensis evidence for the use of the h. rosasinensis flower by humans to regulate the menstrual cycle, females of bangladesh use a decoction of the flower petals with green beetle nut. hot water extract of flowers is used by chinese as an emmenagogue and a tonic. flower extract is used by east indies to regulate menstrual cycle and to produce abortion. in fiji, hot water extract of flowers and leaves is used orally to ease childbirths and the infusion of dried flower is used to aid digestion. people in haiti use decoction of flowers to cure flu and cough while hawaiians eat flowers to produce lactation. decoction of dried flowers is used by indians for abortion and the hot water extraction of flowers as a contraceptive (ross, 2003). flowers, crushed with sugar, are used in indian folk medicine to control excessive uterine bleeding. ancient indian medicinal literature reveals the use of hibiscus flowers for heart diseases. flowers have cooling and soothing effects, anti-inflammatory effects and emmenagogue effects. petals are used to stimulate hair growth, to prevent premature graying and hair loss and used as a hair conditioner (linn et al, 2009; upadhyay et al, 2011). in sri lankan ayurveda medicine, flowers are used to treat anaemia, constipation and to stimulate hair growth (ayurvedic medicinal plants of sri lanka, n.d.). sanadheera et al. – hibiscus rosa-sinensis l. (red hibiscus) tea … 61 antidiabetic effects a study conducted in india with alloxan induced diabetic wistar rats revealed that the ethanolic extract of h. rosasinensis elicits acute hypoglycemic effects (1, 3, 5 h) and sub-acute (1, 3, 5, 7 days) hypoglycemic effects at doses of 250 mg/kg and 500 mg/kg (venkatesh et al, 2008). thus, to obtain this effect, a human should consume around 40 mg/kg bw of dried ethanolic extract of h. rosasinensis (reagan-shaw et al, 2008). required weight of dried petals to make hibiscus tea to obtain the above effect will be much higher. when streptozotocine induced diabetic female wistar rats were mated, and treated orally with aqueous extract of hibiscus rosa-sinensis during pregnancy, an increased maternal and fetal weights, reduced atherogenic index, coronary artery risk index and reduced pre-implantation loss rate were observed compared to the untreated diabetic group (afiune et al., 2017). a study conducted by bhaskar and vidhya revealed that administration of aqueous extract of h. rosasinensis flowers at a oral dosage of 500 mg/kg to normal rats, lowered the postprandial (2hr) blood glucose level significantly (p<0.001) after an oral glucose load. moreover, administration of the extract at the same dose over 21 days, significantly lowered the fasting blood glucose level (p<0.001), glycosilated hemoglobin level and serum lipid levels of stz induced diabetic wistar rats, compared to the control group, although a significant change in insulin levels were not exhibited (bhaskar & vidhya, 2012). scientific data is scarce on human studies of the effect of h. rosasinensis flowers. however, data on human studies are available for tea made with h. sabdariffa, a similar flower variety to h. rosasinensis. h. sabdariffa is the variety commonly used in manufacturing hibiscus tea. arabic people used to consume karkadeh tea made from h. sabdariffa mainly after meals. in a study, this tea was prepared by boiling 10g of dried petals in 500ml of water for 60 minutes. final volume was 250 ml which was refrigerated at 4oc until consumption. this tea elicited a slow rise in blood glucose level compared to english breakfast tea, after a high glycemic meal. conversely, the meal with karkadeh tea showed a 20% higher area under the curve (auc) compared to the meal with english breakfast tea (harrison et al, 2009) indicating the slow glucose absorption. the only human-study carried out with h. rosasinensis flowers has been conducted for 60 days providing 2g of hibiscus rosasinensis flower powder incorporated in to mathri (an indian rotti variety) to type 2 diabetes patients. this study revealed a significant decrease in mean fasting blood glucose, post prandial blood glucose level, mean glycosylated hb level, mean total cholesterol, triglyceride level, total ldl and total vldl cholesterol level (sharma et al, 2016). however, the study has been carried out with 6 subjects in each test and control groups and the groups have not been crossed over. anti-inflammatory effect type 2 diabetes mellitus is an inflammatory disease (donath & shoelson, 2011) thus the herbs which possess anti-inflammatory effects might be beneficial in controlling diabetes. ethanolic extract of h. rosasinensis showed the significant anti-inflammatory activity at doses of 125, 250 and 500 mg/kg on carrageenin induced paw edema, cotton pellet induced granuloma and xylene induced mice ear edema (birari et al., 2009). ethanolic extract of white hibiscus (hibiscus rosasinensis var alba) elicits more potent anti-inflammatory effects over red hibiscus (hibiscus rosa-sinensis l) with doses of 50 and 100 mg/kg and elicited a significant reduction (p<0.05) in polymorphonuclear leukocytes infiltration (raduan et al., 2013). antihypercholesterolemic effects a significant improvement in dyslipidemia caused due to diabetes mellitus was observed in alloxan induced diabetic wistar rats with oral doses of 50, 100, 200 mg/kg of hydroalcoholic extract (70%) of flowers of hibiscus rosasinensis, administered for 4 weeks. researchers reported a significant reduction in total cholesterol, triglycerides, vldl, ldl and elevation in hdl levels (m pethe & guptha, 2011). a decrease in the triglycerides and alt levels were observed in pregnant diabetic wistar rats fed with h. rosasinensis aqueous extract at a dose of 100 mg/kg from day 0 to 7 of pregnancy, 200 mg/kg from day 8 to 14 and 400 mg/kg from day 15 to 20. however, no effect on glycemia was observed in this study (silva et al., 2015). when alloxan induced diabetes rats were administered with hydroalcoholic extract (70%) of hibiscus rosa-sinensis, at doses of 50,100 and 200 mg/kg bw for 4 weeks, a significant improvement in diabetes induced dyslipidemia was observed by the researchers with a significant decrease in total cholesterol, triglycerides, vldl, ldl and elevation in hdl levels (m pethe & guptha, 2011). further, significant anti-diabetic effects which were comparable to glibenclamide and sulphonylurea were observed with the same dosed administered for 28 days. histopathological studies showed an increase in the size and number of islets, diameter of the islet cells and decrease in the necrosis and atrophy of the islets (pethe et al, 2017). after administrating ethanolic hibiscus flower extract for 21 days, a maximum blood glucose lowering of 41-46% and total cholesterol and serum triglycerides lowering of 22 and 30%, respectively were observed with streptozotocine induced diabetic rats. the effect was comparable to glibenclamide, however with no increase in insulin level (sachdewa & khemani, 2003). 62 biology, medicine, & natural product chemistry 10 (1), 2021: 59-65 furthermore, anthocyanin compounds delphini din-3-o-sambubioside and cyanidin-3-o-sambubioside, extracted from hibiscus rosasinensis petals elicited potent competitive ace inhibitor effects agreeing to the use of h. rosasinensis petal extracts to treat hypertension in folk medicine (ojeda et al., 2010). moreover, khan et al. (2011) revealed that the chronic use of ethanolic and chloroform extracts of h. rosasinensis petals has anti-anxiety effects without producing any cns depression signs in experimental rats (mohammed j et al., 2011). phytochemicals in flower on blood glucose and serum cholesterol scientific data revels that the edible parts of the h. rosasinensis flowers contain 89.8% moisture, 0.064% nitrogen, 0.36% fat, 1.56% crude fibre. moreover, flower extract contains tannins (7.5±0.20 %), phenols (0.678±0.14%) and alkaloids (0.51±0.16 %) while the total phenol content in flowers was 735±46 mg gallic acid equivalent /100g. the four main types of flavanoids in petals are rutin, quercetin, kaempferol and myricetin (purushothaman, meenatchi, s, sundaram, & saravanan, 2018). petals contain various flavones such as quercetin-3-di-0-beta-d-glucoside, quercetin-3-7-di0-beta-d-glucoside, cholesterol, campesterol, quercetin3-0-beta-d-sophorotrioside, ß-sitosterol10, kaempferol3-0-beta-d-xylosyl-glucoside19, catalase13. red and magenta colour flower petals possessed dark-purplish dye, anthocyanin pigment and cyanidin diglucoside (subramanium & nair, 1972; wealth of india, 1997) along with proanthocyanidins (nakamura et al, 1990). another study reveals the presence of terpenoids in flower petals (patel & adhav, 2016). all these compounds elicit antioxidant properties, thus the antioxidant capacity of the petals were significantly high [ascorbic acid equivalent antioxidant capacity (aeac): 640 ± 56 mg ascorbic acid /100g; total anthocyanin content (tac): 284 ± 17 mg cyanidin-3-glucoside equivalent/100g; ferric-reducing power (frp): 4.0 ± 0.3 mg gallic acid equivalent /100g (sk, lim, & chan, 2009)]. in another study, aqueous extract of hibiscus elicited high tannin and anthocyanin contents, and possessed high ferric reducing antioxidant power (mak et al., 2013). further, fresh flowers of hibiscus rosasinensis possess 0.30-0.50 v/w % of essential oils. most of the beneficial effects of this flower variety could be due to the presence of antioxidants, which play a key role in controlling diabetes and hypercholesterolemia. phenolic compounds in tea inhibit α amylase and α glucosidase enzymes, the key enzymes of carbohydrate digestion (hara & honda, 1990). furthermore, polyphenolics in tea inhibit glucose uptake by intestinal caco-2 cells (johnston, 2005). tannic acid, a major compound of tannins and alkaloids, stimulates the translocation of glucose transporters (glut 4) and increase phosphorylation of insulin receptors thus increasing glucose clearance from blood and activating insulin action respectively (li et al., 2005). the whole plant of hibiscus rosasinensis increase insulin secretion from the pancreatic beta cells, probably by the actions exerted by alkaloids, flavanoids and terpenoids (mishra, 2009). owing to the above actions on glut 4 receptors and insulin receptors, polyphenolic compounds might increase deposition of excess glucose in adipose tissues as fat, increase uptake of triglycerides from adipose tissues by activating lipoprotein lipase and inhibit hormone sensitive lipase which involve in adipolysis. summative action will be the decrease of circulatory triglycerides and phospholipids. further, anthocyanins in hibiscus sadariffa inhibit angiotensin converting enzyme (ace) by competing with the substrate of the enzyme, thereby controls hypertension (ojeda et al., 2010). terpenoids are present in many herbal plants which can modulate activities of ligand-dependent transcription factors [peroxisome proliferator-activated receptors (ppars)] and thus can prevent obesity-induced metabolic disorders, such as type 2 diabetes, hyperlipidaemia, insulin resistance, and cardiovascular diseases (goto et al., 2010). quantitative analyses have revealed that the aqueous extract of hibiscus rosasinensis flower petals have high amounts of tannins and anthocyanin thus show high ferric reducing antioxidant power. among the phenols, flavonoids, tannins, flavonol and anthocyanins are the most potent antioxidants in hibiscus (mak et al., 2013) thus it can be hypothesized that the above beneficial health effects also could be obtained by hibiscus tea. however, all above beneficial effects have been observed with isolated polyphenolic compounds. therefore, it is not clear whether a tea made from the herbal plant or flower will exert the same effect. although data on animal studies are available to prove the anti-hyperglycemic and anti-hyperlipidemic effects of hibiscus rosasinensis extracts, human studies to observe the activity of these compounds in human body, are yet to be carried out. does the dose in hibiscus rosasinensis tea is sufficient to cause the antihyperglycemic and antihyperlipidemic effects? the above evidences show that the h. rosasinensis could be beneficial as a tea, after meals in terms of controlling postprandial blood glucose level, which is a key goal to minimize diabetes related macro and microvascular complications. however, in most of the animal studies, the beneficial effects were obtained with the powder of the dried or evaporated solvent extracts of the petals, which the concentration of the active compounds ought to be higher than the amounts present in a tea. as a commercially available tea bag weighs 2 – 2.5g, this amount might not be sufficient to provide the beneficial sanadheera et al. – hibiscus rosa-sinensis l. (red hibiscus) tea … 63 effects as shown in research. yet, the study conducted by sharma et al. in 2016, with diabetic patients, emphasizes the presence of anti-hyperglycemic effect of the petal powder (2 g) when incorporated in to a food (sharma et al., 2016). furthermore, to obtain the similar effects observed in animal studies (studies using rats), only one sixth of the animal dose has to be given to humans. therefore, present review reveals the necessity of further studies on antihyperglycemic and antihyperlipidemic effects of hibiscus rosasinensis tea. toxic effects antifertility effects: a study was conducted to observe the effect of h. rosasinensis on diabetes during pregnancy. dried and powdered flower petals (20 g) were boiled in 1 l of water for 5 minutes, filtered and evaporated in a rotary evaporator to obtain the test samples. stz induced diabetic female rats were administered with an initial dose of 100 mg/kg/day of the h. rosasinensis extract, from day 0 until the 7th day of pregnancy (implantation period). the dose was increased to 200 mg/kg/day from day 8 to 14 of pregnancy (embryonic period), and to 400 mg/kg/day from day 15 to 20 (fetal period). the study revealed beneficial effects of the flower extract on diabetic pregnant rats and their offspring with no effect on non-diabetic pregnant rats. the non-diabetic treated group elicited decreased high density lipoprotein cholesterol, increased atherogenic index (ai) and coronary artery risk index (cri), and increased preimplantation loss rate compared to the non-diabetic untreated group. treatment has not shown toxicity, however, could be deleterious to cardio vascular system and reproductive functions. therefore, the researchers indicate that the indiscriminate intake of h. rosasinensis extract may be harmful to healthy individuals and further emphasize that this extract should be completely avoided during pregnancy (afiune et al., 2017). extractive value of water soluble extractives of h. rosasinensis is 5.3% (al-snafi, 2018). therefore, according to the previous study, from 20 g of dried petals 1.06 g of water soluble extractives could be gained. the highest water soluble extractives dose/ day which caused the deleterious effects on rats, was 400 mg/kg/day. however, the dose for humans which causes same effect will be six times lower than that of rats (11). therefore, the deleterious water soluble extractives dose will be 3.3g/day. this amount is present in 62.9 g of dried petals. a tea bag contains 2-2.5 g of dried petals which is significantly lower than the toxic dose. acute toxic effects: when powder of the methanolic extract of h. rosasinensis was administered to rats, in 100, 200, 400, and 800 doses, no significant changes in behavior, skin effect, breathing, defecation, postural abnormalities, impairment in food intake and water consumption and yellowing or loss of hair were observed. no changes were observed for one week. however, 20% mortality was observed with a 1600 mg/kg dose (meena et al, 2014). ethanol soluble extractive value for h. rosasinensis was 2.6% (al-snafi, 2018). thus by considering the ratio of the dose for rats and humans, to obtain a 20% mortality rate, 13 g of ethanol soluble extractives have to be consumed by a 50 kg human. this amount is present in 500 g of dried petals. a tea bag contains 2-2.5 g of dried petals which is significantly lower than the toxic dose. genotoxic effects: genotoxicity was not observed with the flower extract, therefore hibiscus rosasinensis could be used as a safe pharmaceutical material (meena et al., 2014). cytotoxic effects: when a decoction was prepared with 15 g of dried and milled hibiscus petals and diluted to 5g/l and 10g/l, it showed a reduction in mitotic index for l allium cepa l., significantly. therefore, h. sinensis contains antimitotic constituents which block anywhere of the cell cycle (ali, 2010). these concentrations are similar to 1.25g and 2.5 g of petals in a tea cup of 250 ml respectively. however, the study was carried out with plant materials and may not be applied to the human body. chronic toxic effects of hibiscus rosasinensis flower extract have not been reported yet, although tea and drinks made from hibiscus rosasinensis flower petals have been used globally from ancient times as a home remedy and a beverage. conclusion the dose of h. rosasinensis petals present in hibiscus tea is sufficient to elicit anti-hyperglycemic, antihyperlipidemic, anti-hypertensive, anti-inflammatory, anti-anxiety and anti-oxidant properties without causing acute toxicity. author contribution: study concept, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript: senadheera s. p. a. s. 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(2013). antiinflammatory effects of hibiscus rosa-sinensis l. and hibiscus rosa-sinensis var. alba ethanol extracts. international journal of pharmacy and pharmaceutical sciences. reagan-shaw, s., nihal, m., & ahmad, n. (2008). dose translation from animal to human studies revisited. the faseb journal, 22(3), 659–661. https://doi.org/10.1096/fj.07-9574lsf ross, i. a. (2003). hibiscus rosa-sinensis. in medicinal plants of the world (pp. 253–266). totowa, nj: humana press. https://doi.org/10.1007/978-1-59259-365-1_12 sachdewa, a., & khemani, l. . (2003). effect of hibiscus rosa sinensis linn. ethanol flower extract on blood glucose and lipid profile in streptozotocin induced diabetes in rats. journal of ethnopharmacology, 89(1), 61–66. https://doi.org/10.1016/s0378-8741(03)00230-7 sharma, k., pareek, a., & chauhan, e. s. (2016). evaluation of hyperglycemic and hyperlipidemic mitigating impact of hibiscus rosa sinensis (gudhal) flower in type ii diabetes mellitus subjects, 7(2), 223–229. silva, t. l., afiune, l. a. f., de moraes souza, r. q., de sousa soares, t., carneiro, t. b., américo, m. f., volpato, g. t. (2015). effect of hibiscus rosa sinensis aqueous extract treatment on biochemical parameters in diabetic pregnant rats. diabetology & metabolic syndrome, 7(s1), a77. https://doi.org/10.1186/1758-5996-7-s1-a77 sk, w., lim, y., & chan, e. (2009). antioxidant properties of hibiscus: species variation, altitudinal change, coastal influence and floral colour change. journal of tropical forest science, 21(4), 307–315. subramanium, s., & nair, a. (1972). flavonoids of four malvaceous plants. phytochemistry, 11(4), 1518–1519. upadhyay, s. m., upadhyay, p., ghosh, a. k., singh, v., & dixit, v. k. (2011). effect of ethanolic extract of hibiscus rosa sinensis l., flowers on hair growth in female wistar rats. der pharmacia lettre, 3(4), 258–263. venkatesh, s., thilagavathi, j., & shyam sundar, d. (2008). antidiabetic activity of flowers of hibiscus rosasinensis. fitoterapia. https://doi.org/10.1016/j.fitote.2007.06.015 wealth of india. (1997). raw materials (vol. vi). new delhi. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 105-107 | doi: 10.14421/biomedich.2020.92.105-107 issn 2540-9328 (online) a simple technique for rapid assessment of rat (rattus norvegicus) sperm motility muhammad ja’far luthfi1,*, mahanem mat noor2 1department of biological education, faculty of tarbiyah and education, universitas islam negeri sunan kalijaga yogryakarta, indonesia 2school of bioscience and biotechnology, faculty of science and technology, universiti kebangsaan malaysia, malaysia corresponding author* jafarluthfi@yahoo.com manuscript received: 20 september, 2020. revision accepted: 19 february, 2021. published: 25 february, 2021. abstract assesment of rat sperm motility can be carried out on sperm sample from epididimal source using minimal amount of equipment. this method will aid researcher and practitioner working in the field of sperm quality to determined rat sperm motility rapidly and efficiently. keywords: sperm quality; sperm motility; rat; haemocytometer. introduction sperm motility is one of the most important parameters determining sperm quality (martinez et al., 2000; talarczyk-desole et al., 2017). the results of sperm motility assessment can be used more directly to address problems affecting male reproductive organs (björndahl, 2010). the other parameters are sperm count and sperm morphology (freund & carrol 1964). these three components (sperm motility, sperm count, and sperm morphology) compose of sperm quality (perreault & cancel 2001). the most frequently used techniques to assess sperm qualiy are counting chambers (e.g. haemocytometer and makler chamber). these techniques, however, are subject to a high variability (yang et al., 2019). much effort has been made to speed up the objective measurement of sperm motility. casa (computer aided sperm analysis) and ivos (integrated visual optical system) are automatic methods of sperm quality testing. the techniques, however, need sophisticated equipment not available in many laboratories. they are too technically complex and too expensive for routine use. manual observation is still the most widely used method for testing sperm motility (das 1985; kuster 2005; macpherson, 2001). the aim of this study was to determine the optimal manual technique for assessment of rat sperm motility using arbitrary scale. the technique was based on years of practices in our laboratories. it provides a practical and simple means for assessment of rat sperm motility for the purpose of determining the fertility status the animal. materials and methods equipments and materials the equipment needed in the analysis of rat sperm motility were as follows: a light microscope, micropipette, petri dishes, improved neubauer haemocytometer, co2 incubator, small animal surgical instruments, and hand counter. the materials needed in the analysis of rat sperm motility were as follows: three adult male spraguedawley rats (rattus norvegicus), biggers, whitten, & whittingham (bww) medium, and cover glasses. the study was done in the zoology laboratory of university kebangsaan malaysia and the zoology laboratory of uin sunan kalijaga yogyakarta. preparation of sperm suspension sperm samples were taken from the left cauda epididymis. the cauda epididymis was separated based on the epididymal division by hamilton (1975), then placed in a petri dish, minced and incubated in 15 ml of biggers, whitten & whittingham (bww) media solution (biggers et al. 1971) for 30 minutes in 37°c in an incubator of 5% co2 to let sperm swim in the media (swim-up technique) (luthfi, 2015; luthfi & noor, 2015). procedure for assessing sperm motility sperm motility were determined using improve neubauer haemocytometer as described previously (prasad et al., 1972; who, 1999) with modification. the cover glass was attached firmly to the improved neubauer haemocytometer counting grid area. the interference pattern (> 10 newton's rings/fringes) should be seen between the glass surfaces of the two areas https://doi.org/10.14421/biomedich.2020.92.105-107 https://www.ncbi.nlm.nih.gov/pubmed/?term=bj%26%23x000f6%3brndahl%20l%5bauthor%5d&cauthor=true&cauthor_uid=20111079 106 biology, medicine, & natural product chemistry 9 (2), 2020: 105-107 where the glass cover is attached to the haemocytometer. the line/newton's rings that appear too few indicate that the distance between the cover glass and the haemocytometer is widened, therefore the counting chamber volume becomes larger and the assessment would be incorrect. the center of the haemocytometer counting grid area is different from the other ones because it consists of 25 larger squares bounded by triple lines each, while the ones in the corners consist of 16 larger squares. in addition, the larger squares inside the central counting grid are subdivided into 16 even smaller squares each (figure 1). a total of 10 μl of sperm suspension from the preparation of sperm samples were taken with a micropipette, then inserted into the space between the cover glass and the haemocytometer in one of its counting chamber. the other counting chamber was also filled in the same way. let the sperm settled in the chambers for 5 minutes. each counting chamber must be filled correctly. the suspension was inserted slowly to let the liquid evenly distributed by capillary force. if the filling on to the counting chamber was uneven, wipe the slide and then the process of filling the chamber must be repeat. the removal of excessive volume from the counting chamber must not be done because it will change the density of sperm in the counting chamber. figure 1. haemocytometer. a. side view. b. top view. c. one of the two haemocytometer counting chambers. d. insert of iv. legend: i. cover glass; ii. counting chamber; iii. counting grid area; iv. one of the 25 larger squares. after both counting chambers were filled, assessment of sperm motility was carried out. the sperm motility was observed at 200x or 400x magnification using a light microscope. systematically scan the center of the haemocytometer counting grid area, started with the uppermost left square of the 25 large squares, continued to the next three squares to the right, and the squares at the row below, and so on. assessment were done only for intact sperm (defined as having a head and a tail). approximately 100 sperm in each counting chamber were assigned to minimize deviation. sperm motility is estimated to follow the criteria as defined by who (1999; 2010) and is denoted by the letters a, b, c (from the fastest to the slowest) as follows: a: progressive motility; ≥ 25 μm/s where 25 μm is the same as the length of 2 squares (smallest square) of the haemocytometer (see red rectangle in figure 1d) b: non-progressive motility; <5 μm/s c: no motility result and discussion determination of sperm motility is an important step in the analysis of sperm quality. however, there are various methods of assessing sperm motility in terms of equipment, technicality and practicality. different laboratories use different methods. this study was use haemocytometer to determine sperm motility. the determination of the method is mostly based on the practice and experience of testing rat sperm samples carried out at the zoology laboratory of the faculty of science and technology, uin sunan kalijaga and zoology laboratory of university kebangsaan malaysia (data not shown). the sperm motility of the laboratory animals can be determined from ejaculate or epididymal samples. the determination of epididymal sperm motility is using only sperm from the caudal part (clegg et al., 2001). changes in the progression/rate of sperm motility after substance or drug treatment can provide important clues as to the effect of a substance on sperm fertilizing capacity (working, 1988). motility pattern is one of the most important and simplest variables in determining the fertility potential of sperm because immotile sperm will not be able to penetrate the mucus layer of the cervix to reach the ovum and fertilization to occur. sperm motility patterns is an important factor in fertilization, as a strong swing of the sperm tail is necessary for the sperm head to penetrate the lining of the ovum (partodihardjo, 1992). progressive motility is a normal sperm movement. circular motion and reversion movements are often signs of cold shock or due to abnormal sperm morphology. meanwhile, wavy motion and vibrating motion occur more frequently in old men (atherton, 1977). luthfi & noor – a simple technique for rapid assessment of … 107 sperm motility is observed with either physiological saline or other nutrient medium. sperm were observed under a microscope slide with/without a cover glass or with a capillary tube. casa (computer aided sperm analysis) and ivos (integrated visual optical system) are automatic objective methods for testing sperm motility. however, manual observation is still the most widely used method for testing sperm motility using an arbitrary scale (das 1985; kuster 2005; strader 1996). this study revealed that using this method, 25-45 percent of rat sperm would fall into category of progresssive motility, 45-65 percent were of nonprogressive motility category, and 15-35 were of no motility category. aproximately, those numbers could be used as a reference for normal sperm motility of rat sperm. several things need to be considered in order to achieve consistency in using this method. first, we must ensure that the cutting of cauda epididymis was done consistently in every replication. second, micropipette insertion of samples in each counting chamber must be perfect. third, the room temperature used in determining the sperm motility must be the same for all sperm sample assessed. fourth, the time needed to assess the sperm motility should consistent from slide to slide. all these things affect the consistency of the sperm motility assesment. the procedures that are followed strictly will ensure the accuracy of the assessment. conclusion using this method, researchers can determine the rat sperm motility in a practical way. most of the materials and equipments used in this method are widely available. the increasing importance of sperm motility analysis will be consistent with the adoption and development of this simple and rapid method. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references atherton, r.w. 1977. evaluation of sperm motility in hafez, e.s.e. (ed). techniques of human andrology. 175-179. elsevier. amsterdam. biggers, j.d., whitten, w.k. &whittingham, d. 1971. the culture of mouse embryos in vitro in daniel, j.c. (ed). methods in mammalian embryology. 86-116. freeman, san francisco, ca. björndahl, l. 2010. the usefulness and significance of assessing rapidly progressive spermatozoa. asian j androl. 2010 12(1): 33–35. clegg, e.d., perreault, s.d. & klinefelter, g.r. 2001. assesment of male reproductive toxicity in hayes, a.w. (ed). principles and methods of toxicology. 1264-1292. fourth editions. taylor & francis. philadelphia. das, r. p. 1985. assessment of spermatozoal function. j. biosci. 7 (2): 245–255. freund, m. & carol, b. 1964. factors affecting haemocytometer counts of sperm concentration in human semen. journal of reproduction and fertility 8: 149-155. hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat in hamilton, d.w. & greep, r.o. (eds). handbook of physiology, section vii, endocrinology, vol.5, male reproductive system. 259-301. american physiological society, washington d.c. kuster, c. 2005. sperm concentration determination between hemacytometric and casa systems: why they can be different. theriogenology 64: 614–617. luthfi, m.j. 2015. a simple and practical method for rat sperm epididymal sperm count (rattus norvegicus). biology, medicine & natural product chemistry 4 (1): 1-3. luthfi, m.j., noor, m.m. 2015. analisis kualitas sperma tikus percobaan (jumlah, motilitas, dan morfologi). uns press. surakarta. indonesia. macpherson, m.l. 2001. how to evaluate semen in the field. proceedings of the annual convention of the aaep. volume 47. usa. martínez, c., mar, c., azcárate, m., pascual, p., aritzeta, j.m., lópez-urrutia, a. 2000. sperm motility index: a quick screening parameter from sperm quality analyser-iib to rule out oligoand asthenozoospermia in male fertility study. human reproduction 15 (8): 1727–1733. partodihardjo, s. 1992. ilmu reproduksi hewan. cetakan ke-3. 2537. penerbit mutiara sumber widya. jakarta. perreault, s.d. & cancel, a. m. 2001. significance of incorporating measures of sperm production and function into rat toxicology studies. reproduction 121: 207–216. prasad, m.r.n., chinoy, n. j., kadam. k.m. 1972. changes in succinic dehydrogenase levels in the rat epididymis under normal and altered physiologic conditions. fertility and sterility 23 (3): 186-190. strader, l.f., linder, r. e. & perreault. s.d. 1996. comparison of rat epididymal sperm counts by ivos htm-ident and hemocytometer. reproductive toxicology 10 (6): 529-533. talarczyk-desole, j., anna berger, a., taszarek-hauke1, g., jan hauke, j., pawelczyk, l., jedrzejczak, p. 2017. manual vs. computer-assisted sperm analysis: can casa replace manual assessment of human semen in clinical practice? ginekologia polska 88 (2): 56–60. white, w.j. 2001. the use of laboratory animals in toxicologic research in hayes a.w. (ed). principles and methods of toxicology. fourth edition. 773-775. taylor & francis. philadelphia. who. 1999. laboratory manual for the examination of human semen and semen-cervical mucus interaction. new york: cambridge university press. who. 2010. who laboratory manual for theexamination and processing of human semen. fifth edition. who press. geneva, switzerland. working, p.k. 1988. male reproductive toxicology: comparison of the human to animal models. environmental health perspectives 77: 37-44. yang, y., zhang, y., ding, j. 2019. optimal analysis conditions for sperm motility parameters with a casa system in a passerine bird, passer montanus. avian res 10 (35) https://doi.org/10.1186/s40657-019-0174-5 https://www.ncbi.nlm.nih.gov/pubmed/?term=bj%26%23x000f6%3brndahl%20l%5bauthor%5d&cauthor=true&cauthor_uid=20111079 javascript:; javascript:; javascript:; javascript:; javascript:; javascript:; http://www.sciencedirect.com/science/journal/08906238 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235156%231996%23999899993%2378585%23flp%23&_cdi=5156&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=abaf66d755593badcfa6b6be4a9497c9 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 117-121 | doi: 10.14421/biomedich.2021.102.117-121 issn 2540-9328 (online) antibacterial activity of mangrove root extracts from ngurah rai mangrove forest, denpasar-bali made dharmesti wijaya1*, anak agung gede indraningrat2 1pharmacology and pharmacy department; 2microbiology and parasitology department, faculty of medicine and health sciences, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@gmail.com manuscript received: 08 october 2021. revision accepted: 15 october, 2021. published: 21 october, 2021. abstract the increasing rate of antimicrobial resistance in the past decades has motivated the search for novel antibacterial compounds to overcome infectious diseases. among diverse natural sources, mangrove ecosystems offer untapped sources of biological active compounds for future antibacterial medicine. this research was aimed to evaluate antibacterial activities of crude extracts of four dominant mangrove plants from the ngurah rai mangrove forest namely rhizophora mucronata, avicennia marina, rhizophora apiculata, and sonneratia alba. roots of these four plants were extracted using methanol, chloroform, and n-hexane. these crude extracts were tested against two gram positive bacteria (staphylococcus aureus and streptococcus mutans) and two gram negative bacteria (escherichia coli dan klebsiella pneumoniae) by disc difussion assay. we found that 3 mg/ml of n-hexane crude extracts from r. apiculata yielded the highest zone of inhibition of 8.64 mm against s. aureus. while, 3 mg/ml of chloroform crude extract of r. apiculata yielded the highest inhibition of 19.83 mm against s. mutans. unfortunately, no zone of inhibition was observed when crude extracts were tested against gram negative indicator strains. our results indicate that the root crude extracts of r. apiculata yielded the highest zone of inhibition against gram positive indicator strains compared to root crude extracts of r. mucronata, s. alba, and a. marina. further research is required to determine the antibacterial activities of the mangrove crude extracts against other bacterial indicator strains to determine their spectrum of activities. keywords: mangrove; roots, antibacterial; rhizopora apiculata. introduction antimicrobial substances play a crucial role during prevention and medication of infectious diseases, postoperation infections, infections after chemotherapy or infections for diabetic patients, late stage of kidney diseases and patients with rheumatoid arthritis (ventola, 2015). however, incidence rate of antimicrobial resistance has increased dramatically in the past decades (ventola, 2015), which cause difficulty to treat infectious diseases, increase cost for medication, and lead to higher mortality rate (world health organization, 2020). world health organization reported approximately 700,000 mortality every year until 2014 due to antimicrobial resistance and it is predicted the casualties of antimicrobial resistance reached 10 million in 2050 (departemen kesehatan republik indonesia, 2016). immediate actions are required to overcome the increasing rate of antimicrobial resistance such as a better diagnose and prescription of antibiotics, optimization of therapy regimen, and prevention of infectious diseases (ventola, 2015). in addition, the search for novel antimicrobial compounds are urgently needed since many antibiotics on the market are no longer effective to eradicate bacterial infections (ventola, 2015). among natural resources, mangrove ecosystems are the ideal habitat to search for novel antimicrobial activities. mangrove refers to ecosystem where seawater and fresh water dissolve, which characterize by plants that can tolerate high salinity and limited oxygen (friess, 2016). a number of studies showed that mangrove plants are rich of various of active compounds such as alkaloid, steroid, terpenoid, saponin, tannin, flavonoid dan poliphenol (dahibhate et al., 2018; gouda et al., 2015; patra & mohanta, 2014). furthermore, diversity of these secondary metabolites led to broad biological activities such as antibacterial, antifungal, antioxidant, antiviral, and anticancer, which overall could potentially be developed as the source of new drugs (patra & thatoi, 2011; saranraj, 2015). a study conducted in kalimantan showed antibacterial activities of root of mangrove plant of rhizophora apiculata (usman, 2018). however, a similar study from mangrove plants found in bali have not been conducted. the ngurah rai mangrove forest is the biggest mangrove ecosystem in bali which has more than 16 different mangrove spesies and the four https://doi.org/10.14421/biomedich.2021.102.117-121 118 biology, medicine, & natural product chemistry 10 (2), 2021: 117-121 dominant species are rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba (lugina et al., 2017). to date, antibacterial properties of these four dominant mangrove species are rather unexplored. therefore, we aimed to screen for antibacterial activities of crude extract of the root of these four mangrove species by using three different solvents: methanol, chloroform and n-hexane against gram negative and positive indicator strains. it is expected antibacterial activities of these crude extracts will shed light to explore antibacterial active compounds from root of mangrove plants. materials and methods materials roots of the four dominant mangrove plants: rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba were collected from the ngurah rai mangrove forest (8o43’40.4886” s, 115o11’42.80313” e), bali, indonesia, on august 2019 during the low tide. the species determination was carried out in eka karya botanical garden, bedugul, tabanan, bali, indonesia. the solvents (methanol, chloroform, and n-hexane) were purchased from merck, germany. methods sample extraction for each of mangrove species, 50 g of each dry powder of mangrove root were macerated twice using 250 ml of individual solvent (methanol, chloroform, or n-hexane) at room temperature for 24 hours and filtered using whatmann filter paper no. 1. the extracts were then evaporated using rotary evaporator (ika®, rv8) at 50°c, 90 rpm and stored in closed and air tight vial at 4°c until further used. antibacterial screening from each mangrove root extract, three level of concentrations were prepared: 1 mg/ml, 3 mg/ml and 5 mg/ml. subsequently, each crude extract was exposed against bacterial indicator strains gram positive (staphylococcus aureus dan streptococcus mutans) and gram negative (escherichia coli dan klebsiella pneumoniae) by disc diffusion assay. one hundred microlitre of bacterial indicator strains were spotted on luria bertani agar and spread using a sterile spreader. thirty microlitre of crude extracts were spotted on a 6 mm paper disc and air dried for 30 minutes. paper discs in triplicates containing crude extracts were placed on agar media containing bacterial indicator strains. ampicillin and streptomycin were used as positive controls, while the solvents, methanol, chloroform, and n-hexane were used as negative controls. antibacterial activities were calculated based on average zone of inhibition (zoi) that were formed from triplicate disc. results and discussion the results showed that antibacterial activities were displayed by some extracts against gram positive strains, s. aureus and s. mutans (table 1), but none against gram negative strains, e. coli dan k. pneumoniae. among the four mangrove species, only r. apiculata exhibited antibacterial properties against both gram positive indicator strains. the concentration of 3 mg/ml n-hexane extract showed the highest zoi of 8.64 ± 0.13 mm against s. aureus while 3 mg/ml chloroform extract yielded the highest zoi of 19.83 ± 0.63 mm against s. mutans. a study by seepana and co-workers (2016) also showed that methanol, ethanol, and hexane extract of r. apiculata roots is active against salmonela typhi and s. aureus. their study also showed that methanol, hexane, and ethanol r. apiculata root extracts have no activity against e. coli. however, there are activities against other gram-negative bacteria salmonella typhi, shown by methanol, hexane, and ethanol extracts of r. apiculata root. ethanolic extract of r. apiculata root also displays activity against k. pneumonia (seepana et al. 2016). figure 1. antimicrobial activity of r. apiculata n-hexane extracts in triplicates against s. aureus. the concentration of 3 mg/ml n-hexane extract showed the highest zoi (8.64 ± 0.13 mm). 5 mg/ml 5 mg/ml 5 mg/ml 3 mg/ml 3 mg/ml 1 mg/ml 1 mg/ml 3 mg/ml 1 mg/ml wijaya & indraningrat – antibacterial activity of mangrove root 119 figure 2. antimicrobial activity of r. apiculata chloroform extracts in triplicates against s. mutans. the concentration of 3 mg/ml chloroform extract yielded the highest zoi of 19.83 ± 0.63 mm against s. mutans. table 1. antibacterial activities of mangrove root extracts. bacterial strains samples solvents concentration zoi (mm) staphylococcus aureus rhizopora apiculata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 8.05 ± 0.64 3 mg/ml 8.64 ± 0.13 5 mg/ml 7.84 ± 0.61 rhizopora mucronata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml sonneratia alba methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml avicennia marina methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml positive controls ampicillin 10 μg 19.20 ± 0.98 streptomycin 10 μg 15.54 ± 0.22 negative controls methanol chloroform n-hexane 3 mg/ml 3 mg/ml 3 mg/ml 1 mg/ml 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml 5 mg/ml 120 biology, medicine, & natural product chemistry 10 (2), 2021: 117-121 bacterial strains samples solvents concentration zoi (mm) streptococcus mutans rhizopora apiculata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 13.98 ± 1.49 3 mg/ml 19.83 ± 0.63 5 mg/ml 15.92 ± 0.68 n-hexane 1 mg/ml 3 mg/ml 5 mg/ml rhizopora mucronata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml 7.32 ± 0.43 sonneratia alba methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 7.39 ± 0.21 5 mg/ml 7.96 ± 0.25 avicennia marina methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml positive controls ampicillin 10 μg 31.72 ± 3.03 streptomycin 10 μg 20.76 ± 1.49 negative controls methanol chloroform n-hexane in addition, other rhizopora species namely r. mucronata also displayed antibacterial activity in which 5 mg/ml n-hexane extract showed zoi of 7.32 ± 0.43 mm against s. mutans. rhizopora species including r. apiculata and r. mucronata contain many medically important secondary metabolites such as essential oils, glycosides, alkaloids, phenolic compounds, and tannins (habib et al., 2018). tannins is known to have antimicrobial properties against both gram-positive and gram-negative bacteria. this compound passes the bacterial cell wall and intrudes the cell metabolism, resulted in bacterial death (kaczmarek, 2020). furthermore, n-hexane extract of s. alba root showed activity against s. mutans in which the concentration of 3 mg/ml and 5 mg/ml yielded zoi of 7.39 ± 0.21 and 7.96 ± 0.25 mm respectively. although study on s. alba root extract is still limited, there are a number of studies focusing on leaves and bark of this plant. a study by saad and co-workers (2012) showed that methanolic extract of s. alba leaves antibacterial activities were observed against gram-negative bacteria e. coli (17,5 mm), as well as gram-positive bacteria namely s. aureus (12.5 mm) and bacillus cereus (12.5 mm) (saad et al., 2012). our result indicate that overall, crude extract of mangrove root displayed much lower antibacterial activities compared to positive control ampicillin (10 μg) dan streptomycin (10 μg). however, zone of inhibition r. apiculata crude extracts using n-hexane (3 mg/l) were comparable to that of streptomycin. therefore, r. apiculata could potentially contain novel antibacterial compounds. however, composition of active compounds in the crude extracts need to be confirmed. it is expected that once this active compound wijaya & indraningrat – antibacterial activity of mangrove root 121 has been identified, it can be isolated and further developed as new antibiotics drugs. conclusion in conclusion, crude extracts of root r. apiculata displayed the highest antibacterial activities compared to other crude extracts of different mangrove plants. we found that extraction using n-hexane yielded the highest inhibition zones compared to methanol and chloroform. further research is required to elucidate the active compounds in n-hexane. acknowledgements: we would like to acknowledged financial support from up2m of faculty of medicines and health sciences under grant no. 417/unwar/fkik/up2m/kp-02/vii/2019. conflicts of interest: the authors declare that there are no conflicts of interest. references dahibhate, n., saddhe, a., & kumar, k. (2018). mangrove plants as a source of bioactive compounds: a review. 08. doi:10.2174/2210315508666180910125328 departemen kesehatan republik indonesia. (2016). mari bersama atasi resistensi antimikroba (amr). friess, d. a. (2016). mangrove forests. current biology magazine, 26, r746-r748. gouda, s., das, g., & patra, j. k. (2015). mangroves: a rich source of natural bioactive compounds. in s. k. panda (ed.), recent advances in natural products. usa: studium press llc. habib, m. a., khatun, f., ruma, m. k., chowdhury, a., silve, a. r., rahman, a., & hossain, m. i. (2018). a review on phytochemical constituents of pharmaceutically important mangrove plants, their medicinal uses and pharmacological activities. kaczmarek, b. (2020). tannic acid with antiviral and antibacterial activity as a promising component of biomaterials—a minireview. materials, 13(14), 3224. retrieved from https://www.mdpi.com/1996-1944/13/14/3224 lugina, m., alviya, i., indartik, & pribadi, m. a. (2017). strategi keberlanjutan pengelolaan hutan mangrove di tahura ngurah rai bali (strategy of mangrove management in ngurah rai grand forest park). jurnal analisis kebijakan kehutanan, 14(1), 61-77. patra, j. k., & mohanta, y. k. (2014). antimicrobial compounds from mangrove plants: a pharmaceutical prospective. the chinese journal of integrated traditional and western medicine, 1-10. doi:10.1007/s11655-014-1747-0 patra, j. k., & thatoi, h. n. (2011). metabolic diversity and bioactivity screening of mangrove plants: a review. acta physiologiae plantarum, 33(4), 1051-1061. doi:10.1007/s11738-010-0667-7 saad, s., taher, m., susanti, d., qaralleh, h., & awang, a. f. i. b. (2012). in vitro antimicrobial activity of mangrove plant sonneratia alba. asian pacific journal of tropical biomedicine, 2(6), 427-429. doi:https://doi.org/10.1016/s2221-1691(12)60069-0 saranraj, p. (2015). mangrove medicinal plants: a review (vol. 7). seepana, r., karthick, p., narayana murthy, k., ramesh, c., mohanraju, r., & vijayakumar, a. (2016). evaluation of antimicrobial properties from the mangrove rhizophora apiculata and bruguiera gymnorrhiza of burmanallah coast, south andaman, india. journal of coastal life medicine, 4(6), 475-478. doi:10.12980/jclm.4.2016j6-52 usman. (2018). uji fitokimia dan uji antibakteri dari akar mangrove rhizopora apiculata terhadap bakteri escherichia coli dan staphylococcus aureus. jurnal kimia dan pendidikan kimia, 2(3), 169-177. doi:10.20961/jkpk.v2i3.11850 ventola, c. l. (2015). the antibiotic resistance crisis: part 1: causes and threats. p & t: a peer-reviewed journal for formulary management, 40(4), 277-283. retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25859123 https://www.ncbi.nlm.nih.gov/pmc/pmc4378521/ ventola, c. l. (2015). the antibiotic resistance crisis: part 2: management strategies and new agents. p t, 40(5), 344-352. world health organization. (2020). global antimicrobial resistance surveillance system (glass) report: early implementation 2020. retrieved from geneva: https://apps.who.int/iris/bitstream/handle/10665/332081/9789 240005587-eng.pdf?ua=1 this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 65-67 | doi: 10.14421/biomedich.2014.32.65-67 the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a.1, arief r.h.2* and sudarsono3 1faculty of pharmacy; 2department of pharmacology; 3department of pharmaceutical biology, ugm, indonesia author correspondency*: ar_hakim2000@yahoo.com abstract this study was conducted to examine / determine the antidepressant effects of (arcangelisiaflava l.) on immobility time of the white male mice strain balb-c by the forced swim test method. the method of research using laboratory animals such as 25 micewas divided into 5 groups. as a negative control group was only given distilled water ad libitum. amitryptiline was used as the positive control group; the experiment group was a water-soluble extract of a.flava by multiple doses. the results showed that the best antidepressant effects were 312 mg/kg bw; it had a minimum of immobility time compared with the other groups. keywords: acangelisiaflava, berberine, immobility time, antidepressants introduction depression is usually as a public problem. world health organi-zation (who) in 2001 stated that depression was the fourth most commonbasic problem in the world. symptoms of depression that appears in the form of complaints related to mood (e.g depressed, sad, despair) to make a diagnosis of depression can be easily enforced, but when psychomotor and somatic complaints that appear frequently undiagnosed depression (amir, 2005). therapy for depression people are medications that can improve mood, better known as antidepressant drugs (grollman, 1972).antidepressant are drugs which can improve depressive symptoms. antidepressant can be classified as desipramine type; imipramine type; amytriptylin type. this antidepressant drug has the side effects of these drugs are : due to anticholinergic properties i.e dry mouth; accommodation disorders, constipation (mutschler, 1995), the development of local wisdom e.g. the people of west sulawesi were using hot water soluble extract of archangelisiaflava l. merr for their gastrointestinal problem (diarrhoea). according to their experience in using hot water soluble of a.flavastem, could be work for the treatment of their problem. according to some references, one of the constituent of a.flava stem are a group of berberine derivative alkaloid (siwon, 1982). this alkaloid berberine chloride is as one of the specific active constituent that active against shigella sp. in vitro and in vivo (larisu, 2010). in this research of interest was to examine, that berberine chloride constituent of the hot water soluble extract of a.flava, had an anti-depressant effect in vivo in mice. the active constituent of the a.flavais composed of alkaloid berberine, jatrorrhizin, palmatin and kolumbamin (keawpradub et al., 2005). from the research, it was known that berberinechloride was an isoquinolinealkaloid group found in a.flava. the group of isoquinolin alkaloid had many pharmacological activities, namely as an antihypertensive, anti-inflammatory, antidepressant, anticancer, antimicrobial, hypolipidemic, hepatoprotective and antidiabetic (singh et al., 2010). n a b c d + 1 2 3 4 5 6 8 9 10 11 12 13 r1 r2 r3 r4 r5 r1 r2 r3 r4 r5 berberine o-ch2-o och3 och3 h jatrorrhizine och3 och3 och3 hoh palmatine och3 och3 och3 och3 h figure 1. berberinederivative (siwon, 1982). materials and method plant material a.flava (“kayukuning”) was obtained from sorong, west papua, irian jaya. this experimental plants was identified by mr.jokosantosom.si and voucher specimen was found at the department of pharmaceutical biology faculty of pharmacy ugm. 66 biology, medicine, & natural product chemistry 3 (2), 2014: 65-67 figure 2. i. a.flava stem; ii. a.flava powder. animals male white mice balb-c strain 2-3 months old, weighing 30-45 grams were used in the present study. the minimum number of animals and duration of observations required to obtain consistent the data were employed. amitriptiline tablets was obtained from “kosudgama” drugstore ugm, was used as a positive control. the dose of the water-soluble extract containing berberine chloride of a.flava based on empirical dose by the local people, while the administration of one dose amitriptiline based on usage which was then converted to the test animals dose. qualitative and quantitative analize of plant material qualitative – quantitative analyze of berberine chloride were done by thin layer chromato-graphy 5 μl.extractand reference standard berberine chloride was spotted on silica gel 60 f254 each. mobile phase was a mixture of nbutanol-acetic acid and water (3:1:1 v/v/v). the spot visualization were uv254 and uv366 separated spots was scanned between 200 – 700 nm. the quantification was done by the use basic stock for 5 mg/ml.stock solution of test solution was 5 mg/ml and the several concentration of berberine chloride in ethanol with in 250; 200; 100; 50; dan 25 μg/ml. preparation of water soluble extract the extraction was done by hot water extraction method (infundation) 3,12%. 3,12gram stem powderand 100 ml aquadest for 15 minutes at 90oc.there was 100 ml filtrate. figure 3. preparation of water soluble extract. qualitative and quantitative analyze thin layer chromatography method, was used to analyze the constituents of this extract. quantitative analyze of the alkaloids constiturnts has done by tlc-scanner 5 μlextract was spotted on the tlc plate silica gel 60 f254 ; mobile phase was n-butanol-acetic acid-water (3:1:1 v/v/v) for 8 cm;visible light, uv254 nm, uv366 nm were used for spot detection. scanningwas done by 200-700 nm. pharmacological treatments forced swim test (fst) can be used as a sign of antidepressants effect. mice were divided into 5 groups, with the control group 2 positive control (+) and negative control (-) and 3 treatment groups (p1, p2, and p3). mice were adapted to feeding ad libitum for 1 week.after the mice were created stress by forcible allow to swim as in the forced swim test procedures on the first day up until the 14th day. then from day 15 to day 28, every day is oral, positive control group (+) was given po at a dose of amitriptyline were 3.25 mg / kg, negative control group (-) were given a placebo in the form of distilled water, groups p1, p2 and p3 were each given a stock solution in test p.o. at a dose of 78, 156, and 312. on day 29, all groups treated with the forced swim (forced swim test) for 6 minutes and measured the immobility time. 100 75 67 50 25 0 tiara a., et al. – the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract … 67 im m ob ili ty ti m e (d et ik ) ekstrak (mg/kgbb) statistical analysis all results were expressed as mean ± sd. data were analyzed by one-way anova followed by scheffe's multiple range test. the criterion for statistical significance was p≤ 0.05. all statistical analyses were carried out by using spss for windows (spss inc.). results effect of water-soluble containing berberine chloride extract of a.flava in accordance with the forced swim test results of the testing solution, the results of immobility time in the p3 group was the shortest namely 128.6 ± 44.8 seconds and a negative control with an average immobility time later than the 213.6 ± 43.6 seconds. based on the results of the anova analysis found a significant difference only in the p3 group (water-soluble extract of a.flava stem with a dose of 312 mg/kgbw) compared to the negative control group (distilled water). this indicates that the water-soluble containing berberine chloride extract of a.flavawith a dose of 312 mg/kgbwhad an effect as as an antidepressant. figure 4. influence of water-soluble containing berberin chloride extract vs the immobility time in mice. * = significantly different from the negative control (p ≤ 0.05). conclusion the results showed that the water-soluble extract of a.flava at a dose of 312 mg/kgbw were able to give antidepressants effect on white mice balb-c strain in terms of immobility time in the forced swim test method. as a suggestion research needs to be done the same with the number of animals per group and using comparative more pure and more detailed observation so as to give the best results. references amir, n.a. 2005.depresi: aspek neurobiology diagnosis dan tatalaksana. penerbit fkui. jakarta. grollman, a. 1972.pharmacology and therapy of depression. the american journal of psychiatry.113:950. kametani, t. 1969. the chemistry of the isoquinoline alkaloids. hirokawa publishing company. tokyo. 109-111. larisu, m. a. 2011. kajian ilmiah air rebusan batang katola (arcangelisia flava (l) merr) obat tradisional diare berdarah masyarakat kabupaten muna sulawesi tenggara. tesis. program pascasarjana fakultas farmasi universitas gadjah mada. mutschler e., darendorf h., 1995, drug action, medpharm scientific publisher, stuttgart, p.126-128 peng, w.h., lo, k.l., lee, y.h.,hung, t.h., lin, y.c. 2007. berberine produces antidepressant-like effects in the forced swim test and in the tail suspension test in mice. journal of life sciences. 933-938. porsolt, r.d., bertin, a., jalfre, m., 1977. behavioral despair in mice: a primary screening test for antidepressants. archives internationales de pharmacodynamieet de therapie. 229, 327– 336. porsolt r.d, le pinchon m, jalfre, m. 1977. depression: a new animal model sensitive to antidepressant treatments. nature. perancis.730-732. singh, a., duggal, s., kaur, n., singh, j. 2010.berberin: alkaloid with wide spectrum of pharmacological activities. journal of natural product.3:64-75. siwon, 1982, apharmacognostical study of some indonesian plants of the family menispermaceae, dissertation, p.73., drukkerij j.h., pasmans b.v,’s-gravenhaage world health organization. 2011. depression. http://www.who.int/en/ diakses juni 2013. zomkowski, a.d.e., rosa, a.o., lin, j., santos, a.r.s., calixto, j.b., rodrigues, a.l.s., 2004. evidence for serotonin receptor subtypes involvement in agmatine antidepressant-like effect in the mouse forced swimming test. brain research 1023.253– 263. content_v3n2_4.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 21-25 | doi: 10.14421/biomedich.2020.91.21-25 issn 2540-9328 (online) analysis of body posture using rapid entire body assessment (reba) and rapid upper limb assessment (rula) to improve the posture of sand paper machine operators and reduce the risk of low back pain trio yonathan teja kusuma department of industrial engineering, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author trio.yonathan@gmail.com manuscript received: 26 september, 2019. revision accepted: 14 april, 2020. published: 17 april, 2020. abstract the metal casting industry, nitikan is a micro business whose production process is carried out manually. the equipment used is simple and without considering the user's health. this can be seen in the product finishing process that uses a sanding machine. the equipment is not designed according to the user's posture so the operator bends in doing work movements, of course this is very risky to experience lower back pain injuries. this study aims to analyze the work activities of the finishing process using the rapid entire body assessment (reba) and rapid upper limb assessment (rula) approach and design work tools that can be applied to improve work posture. the results of data processing and analysis obtained a rapid entire body assessment score on a sandpaper machine single head of 5 and a rapid upper limb assessment score of 4, the score indicates that the work position has the potential to experience a risk of low back pain, so there must be an improvement in work posture. work posture improvement is done by designing a tool in the form of a sandpaper machine that takes into account ergonomic dimensions to reduce the risk of lower back injury. the final result after the improvement is obtained rapid entire body assessment score of 3 and rapid upper limb assessment score of 4. this shows that the risk of lower back injuries can be minimized. keywords: rapid eentire body assessment; rapid upper limb assessment; low back pain introduction the metal casting industry, nitikan is a micro business which can be said to be the entire production process carried out by human labor. the equipment used is simple and without considering the user's health. this can be seen in the product finishing process that uses a sanding machine. the equipment is not designed according to the user's body posture so operators bend in doing work movements, of course this is very risky to experience low-back pain injuries. in addition to injuries, the process was fairly long because it took 11 minutes. this process is somewhat longer than the other processes which on average only takes 5 minutes. thus it is necessary to anticipate this, business owners need to pay attention to the comfort of their workers so that work health and safety are guaranteed. this can be done by adjusting workers with work methods, work processes and the work environment. this approach is known as the biomechanical approach which in this context is reba and rula analysis. this analysis serves to analyze the work posture is not good and the risk of low-back pain. the analysis then becomes the basis for providing solutions in the form of work postures in the form of tools to reduce these risks. by implementing this it is expected to minimize the risk of low-back pain injuries and to increase process productivity. materials and methods rapid upper limb assessment (rula) rapid upper limb assessment (rula) is the method used to estimate the risk of injury to the upper body such as the upper arm (forearm), forearm (wrist) and wrist (wrist rotation), wrist (neck), torso. by using rula, fast and systematic results will be obtained in determining postural risk of workers (mcatamney & corlett, 1993). the rula measurement method has several main applications such as: 1. measuring the risk of musculoskeletal (muscle), usually part of a wider increase in ergonomics. 2. comparing muscle loads (muscles) between current and modified work station designs. 3. evaluate results such as productivity or suitability for equipment use. https://doi.org/10.14421/biomedich.2020.91.21-25 22 biology, medicine, & natural product chemistry 9 (1), 2020: 21-25 4. train workers about muscle loads (muscles) caused by differences in work posture. posture with the rula method is divided into two parts, namely group a posture consisting of the upper arm (upper arm), forearm (forearm), wrist (wrist) and wrist rotation (wrist rotation); and group b posture consists of the neck, body and legs. the final results obtained from the two final scores for group a and group b are combined, the combined score results from group a and group b are classified into several categories. the development in the rula method consists of three stages, namely: 1. selection of work posture for assessment or measurement. 2. development of an assessment system. 3. the development of a large score is converted to one of four levels of action. figure 1. rula analysis sheet (source: mc atamey & corlett, 1993). rapid entire body assessment (reba) rapid entire body assessment (reba) is an ergonomic method (hignett & mcatamney, 2000) developed to measure the types of postures that pose a risk to health. reba assesses work positions on body postures such as the neck, back, arms, back of the hands and feet of the operator. reba depends on the coupling factor, external loads supported by the body and worker's activities. determination of reba score manually, discussing the level of risk of work posture, starting with determining the a score for posture of the body, neck and legs plus the load score (load) and the b score for the posture of the upper arm, forearm and the hand is added with a score clutch. both scores are used to determine c scores. reba scores are obtained by adding activity scores to c scores from reba values to determine the level of risk of injury. measurement of body posture using the reba method has six stages including: 1. observation of body posture can be done by taking workers' posture data using video or photo assistance. 2. selection of posture that must be done. 3. assessment of body posture score. 4. the score calculation process. 5. determine the results of the reba assessment for the owned posture. 6. check the results of the reba assessment according to the level of action to make improvements or changes. the reba method can be used when an assessment of ergonomic work posture identifies the need for further analysis. the things that need to be done are: 1. all body parts used. 2. static, dynamic, fast changing or unstable posture. 3. modification of workplaces, equipment, training, and monitoring of worker risk behavior. results and discussion the object of research was carried out in the sandpaper process section. the sanding process is carried out in 2 stages, namely coarse sanding and fine sanding. because it only has one sanding machine, in doing these 2 steps, it has to replace the sanding eye, this causes the processing time to be longer. another problem is the design of the sandpaper machine that does not consider the health of workers, causing workers to have to bend for a long time, causing lower back pain. figure 2. sandpaper machine operator posture. table 1. reba angle dimensions for sanding machine operators. grup dimensions angle a neck 29 back 19 knee kaki 87 load 0 b upper arm 39 lower arm 38 wrist 32 kusuma – analysis of body posture using rapid entire body assessment … 23 table 2. reba score on the sanding machine operator's work posture. group dimensions a n g le s c o r e table a score a score c grade score reba a neck 29o 2 4 4 4 5 back 19o 3 knee 87o 1 load 0 group dimensions a n g le s c o r e table b score b b upper arm 39o 2 3 3 lower arm 38o 2 wrist 32o 2 coupling 0 activity score 1 for the number of scores in table a, a result of 3 is obtained and added to a load score of 0 because the load weight is less than 5 kg. then the a score has a score of 0 because the load weight is less than 5kg. then the score a has a score of 4. from each angle of group b the table b score is generated that is equal to 3 and coupled with a coupling score of 0 because the grip is right and in the middle so that the grip on the load is strong. then the resulting b score is 3. based on the a score and the b score, it can be seen the value of the c score is 4. to get the reba score, by adding up the c score of 4 with an activity score of 1, the result score is obtained 5. based on the risk table it is known that reba score of 5 indicates a moderate level of risk and work position needs to be improved. table 3. rula angle dimensions for sanding machine operators. grup dimensions angle a upper arm 39 forearm 38 wrist 32 twist 0 b neck 29 back 19 feet 87 based on data processing using rula, then the results obtained by 3 and then coupled with muscle and energy scores. muscles have a score of 1 and a score of energy usage of 0 due to occasional loading or energy of less than 20kg and retained. so as to get a c score in group a of 4. while in group b the results obtained in table b are as big as 3 then added with muscle and energy scores. muscles have a score of 1 and a score of energy use of 0 due to occasional loading or less than 20kg of energy and retained. so as to get a d score in group b of 4. based on the scores of c and d scores obtained, the rula value was obtained. for the c score in group a obtained by 4 and the d score in group b obtained a result of 4. by looking at the grand score table based on the c and d scores, a rula score is 4. the score indicates that changes need to be made, figure 2 shows the operator’s body position is less comfortable. table 4. rula score of sanding machine operator posture. group dimensions angle score table a score c score rula a upper arm 39o 2 3 4 4 fore arm 38o 2 wrist 32o 3 twist 1 muscle 1 power 0 group dimensions angle score table b score d b neck 29o 3 3 4 back 19o 2 feet 87o 1 muscle 1 power 0 improvement the use of a sanding machine was done by sitting in a chair. dimension is done only on the front side of the sanding machine, because it is adjusted to the use. the engine dimension data of the ideal chair dimensions were adapted in kusumawati’s research (2011). figure 3. sanding machine design improvements. 24 biology, medicine, & natural product chemistry 9 (1), 2020: 21-25 figure 4. the operator's body position angle. table 5. reba angle dimensions for sanding machine operators. grup dimensions angle a neck 9 back 23 knee 110 load 0 b upper arm 37 forearm 77 wrist 34 table 6. reba score on the sanding machine operator's work posture. group dimensions a n g le s c o r e table a score a score c score reba a neck 9o 1 2 2 2 3 back 23o 3 knee 110o 1 load 0 group dimensions a n g le s c o r e table b score b b upper arm 37o 2 2 2 lower arm 77o 1 wrist 34o 2 coupling 0 activity score 1 table 7. rula angle dimensions for sanding machine operators. group dimensions angle a upper arm 37 lower arm 77 wrist 34 twist 0 b neck 9 back 23 feet 110 based on a and b score, it can be seen the value of the c score is equal to 2. to get the reba score, by summing the results of the c score that is equal to 2 with an activity score of 1, then the results obtained score of 3. table 8. rula score of sanding machine operator posture. group dimensions angle score table a table c score rula a upper arm 37o 2 3 4 4 fore arm 77o 1 wrist 34o 3 twist 1 muscle 1 power 0 group dimensions angle score table b score d b neck 9o 1 3 4 back 23o 3 feet 110o 1 muscle 1 power 0 based on data processing using rula, the c score in group a is 4 and the d score in group b is 4. by looking at the grand score table based on c and d scores, the rula score is 4. conclusion from the analysis and results of the improvements obtained, it can be concluded; the sandpaper machine operator's posture has a reba score of 3 with a level 1 action with a low level of risk, whereas the rula score has a score of 4 with action level 2 with moderate risk level. this result shows that the design of sandpaper machine improvement is effective in reducing the risk of injury to low back pain operators, and can also speed up the processing time in its use without the need to change of the coarse and fine sandpaper eyes. references hignett, s. and mcatamney, l. (2000) rapid entire body assessment (reba). applied ergonomics, 31, 201-205. kusumawati, intan. 2011.usulan perancnagan ulang meja kursi baca berdasarkan aspek fungsi dan kenyamanan sesuai kebutuhan pengguna perpustakaan (studi kasus di kantor arsip dan perpustakaan kabupaten klaten). surakarta: universitas sebelas maret surakarta. lueder, r., ( 1996), a proposed rula for computer users, procceding of the ergonomic summer workshop, san francisco. kusuma – analysis of body posture using rapid entire body assessment … 25 mcatamney, l. and corlett, e.n., (1993), “rula: a survey based method for the investigation of work related upper limb disorders”, applied ergonomics, 24(2).91-99. prasetyo. (2012). fasilitas kerja dengan perbaiakan postur kerja pada aktivitas manual handling menggunakan analisis metode rapid entire body assessment (reba) dan ovako working posture anlysis system(owas) studi kasus perusahaan tempo pedro yogyakarta. yogyakarta: uin sunan kalijaga yogyakarta. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 21-23 | doi: 10.14421/biomedich.2014.31.21-23 larvicidal activity of the mixture of cashew nut shell liquid (cnsl) and aqueous extract of sapindus rarak dc against larvae of culex quinquefasciatus rahmi safarina fauziah1, sudarsono1* and budi mulyaningsih2 1pharmaceutical biology department, faculty of pharmacy; 2parasitology laboratory, faculty of medicine, ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract the aim of this study was to evaluate the larvicidal activity of cashew nut shell liquid (cnsl) against the culex quinque fasciatus in larval stage. the cnsl was diluted in water by addition of aqueous extract of sapindus rarak dc to increase its solubility. larvae were exposed to varying concentrations of that mixture. the larvae mortality was observed after 24 h exposure. lc50 and lc90 value by extrapolation were 20,52 ppm and 55,41 ppm respectively. cnsl were specified by characterizing its physico-chemical properties and anacardic acid as marker compound by high performance chromatography (hplc). the results were the mixture of cashew nut shell liquid (cnsl) and aquous extract of sapindus rarak dc had larvicidal activity against cx. quinque-fasciatus and further investigations were needed to identify the fatty acid derivative as active compound of cnsl which responsible for larvicidal activity. keywords: larvicidal, larvae, culex quinquefasciatus, cashew nut shell liquid, sapindus rarak introduction culex quinquefasciatus is the most common mosquito species found in indonesia. there are the main cause of the mosquito bites in the evenings and night. the main factor supporting this species is probably due to poor sanitation caused by human migration to urban areas. interest in the control of cx.quinquefasciatus lies in the fact that it acts as a vector of filarial disease as a serious public health problem in indonesia and many tropical developing countries. filariasis is an endemic, disabling, and disfiguring disease. the filarial worm, wuchereria bancrofti responsible for human filariasis is carried by cx.quinquefasciatus which is a tropical pest and probably the most abundant and ubiquitous house mosquito in towns and cities, in the tropical countries. one of the strategies of who in combating tropical disease is to destroy their vectors or intermediate hosts. since no effective vaccine is available for filariasis, the only effective approach of minimizing the incidence of this disease is to eradicate and control mosquito vectors mainly by application of organic insecticides to larval habitats. control of mosquito at this stage is efficient because during the immature stages, mosquitoes are immobile. the use of natural products for controlling of insect pests offers an economically viable and eco-friendly approach, besides being harmless to beneficial insects when adopted on a larger scale. chemical pesticides have been used for several decades in controlling pests and vectors of various human diseases as they have a quick knock down effect. however, their indiscriminate use resulted in several problems such as resistance and resurgence of pests, elimination of natural enemies, toxic residues in food, water, air, and soil which affect human health and disrupt the ecosystem, leading to the threat that their continued use may further harm the environ-ment. anacardium occidentale l. (anacardiaceae), a fruit tree grown widely in tropical and sub-tropical areas is cultivated in indonesia for its cashew nuts. tyman & morris in 1989 described the composition of cnsl which found between the seed coat (pericarp) and the nuts. it is not a triglyceride and contains a high portion of phenolic compound, mainly are anacardic acid, cardol, and cardanol. recently, lomonaco and oliveira noted insecticidal action of cnsl on larvae of aedes aegypti (diptera: culicidae). the three cnsl compo-nents demonstrated good larvicidal activity against ae. aegypti. despite the potential to be developed as larvicides, phenolic compounds in the cnsl was toxic because it induced dermatitis. in addition, application of cnsl as larvicides in the medium of water is also cumbersome, so it is necessary to add other materials intended to dilute it. in this study, aqueous extract of sapindus rarak dc was added to increase the solubility of cnsl in water. materials and methods plant materials cashew nuts were obtained from cashew trees (a.occidentale) at wonogiri, central java, indonesia. the fruits of sapindus rarak dc were collected from the areas of “perhutani” located in situbondo, east java, indonesia. the plant material was identified by mr.joko santosa and voucher specimen was deposit at deprtment of pharmaceutical biology faculty of pharmacy, ugm. 22 biology, medicine, & natural product chemistry 3 (1), 2014: 21-23 oil extraction of cnsl the dried cashew nut shell (1,3 kg) were pressed by hydraulic pressor to obtain cashew nut shell liquid (cnsl). preparation of aquous extracts of s.rarak the dried pericarp from s.rarak (10 g) were cut into small pieces then extracted with 1000 ml of distilled water by reflux process at 50°c for 30 minutes. the concentration of aqueous extract of s.rarak fruits was 1% b/v. reagents n-toluene (e.merck), ethyl acetate (e.merck), acetic acid glacial (e.merck), chloroform (brataco), methanol (brataco). iodium p, anisaldehyde-sulphuric acid lp, vanilin-sulphuric acid lp, ethanol 96% (brataco), nhexane (brataco), hydrochloric acid (e.merck). all other chemicals used were analytical grade. larvicidal assay the activity of the test materials were evaluated by the world health organization recommended guidelines. the larvicidal assay consisted of two steps. first was the orientation to determine a certain concentration of aqueous extract of s. rarak dc. (it was the biggest concentration which is not found any died larvae). the chosen concentration then was used for making the mixture of cnsl and aquous extract of s. rarak dc. the next step was dissolving cnsl in various amount so that it was obtained 6 concentration series of the mixture for used in the testing, which were 0; 8; 8,56; 9,16; 9,8; and 10,49 ppm. characterization of cnsl’s physico-chemical properties physico-chemical properties of cashew nut shell oil was characterized through the iodine number, esther number, and acid number. those properties were characterized by pusat antar universitas (pau) ugm. hplc analysis of cnsl constituents the identity of anacardic acid was confirmed by hplc. the analysis was carried out using a shimadzu spd10vp chromatograph, uv-vis detector, which utilized a lichochart rd-c18 analytical column. the mobile phase consisted of methanol-acetic acid 4% (9:1v/v), which was run in the isocratic mode phase (1 ml/min) at a uv wavelength of 280 nm. determination of foaming index of s.rarak source 5 g of dried s.rarak fruits was dissolved in distilled water and then heated 50oc for 5 minutes. 10 ml filtrate was used for foaming test. saponin test was performed by agitation in 10 ml of the filtrate in a closed tube for 10 minutes. incidence foam up interval of 10 minutes (stable foam) showed a saponin. figure.1. chromatogram of csnl. figure.2. chromatogram of anacardic acid. results and discussion one of the marker constituent of cnsl, anacardic acid, was detected in the sample of cnsl in relative concentration of 2,58%. the hplc chromatograms were shown in fig.1 and fig.2. due to its bad solubility, cnsl was diluted in water with s.rarak’s pericarp extract as a co-solvent, in concentration which was determined by the orientation test. concentration of 400 ppm was selected as the optimum concentration to dillute cnsl in water but it had no effect to cause larval mortality. the mixture of cnsl and s.rarak extract used in this study was visually clear. there were also not detected visible oil particles or any breaking phase. rahmi safarina fauziah, et al. – larvicidal activity of the mixture of cashew nut shell liquid … 23 table 1. physico-chemical properties of cnsl used in this study. physico-chemical properties of cnsl value iodium number (g iod/10 g cnsl) 6,3 acid number (mg naoh/g cnsl) 104,1 saponification number 127,1 esther number 45,9 density (g/ml) 0,8232 it had no published data available of effect of cnsl on immature stages of culex mosquitoes. effect of different concentrations of cnsl solution on aquatic stages of cx.quinquefasciatus larvae in the laboratory conditions was shown in table 2. there was significant decreasing mortality percentage of the larvae by increasing cnsl concentration (table 2). as shown in that table, the biggest death rate was noticed in exposure to 10,49 ppm concentration. lc line equation obtained from probit analysis was y = 3.01 x + 1.05, so that the calculation could be known that lc50 and lc90 value were 20.52 ppm and 55.41 ppm, respectively. this value was the result of extrapolation and therefore could not guarantee that these results show the actual value of lc50 and lc90. essential oils or plant extracts with lc50 values below 100 ppm can be considered potentially have larvicidal activity. our present observation reveals that cx.quenquefasciatus larvae were susceptible to 8 ppm to 10,49 ppm cnsl. in indonesia, population of cx.quin-quefasciatus is controlled by using of organophosphate insecticides, such as temephos and malathion. temephos, which is also known as “abate” has been used in areas of water where the cx.quinquefasciatus mosquito breeds in order to reduce the population of this disease-carrying insect. resistance to temephos by cx.quinquefasciatus has been reported in brazilian and malaysia. temephos was very lethal toward cx.quinquefasciatus larvae with a lc50=0,00088 ppm. cnsl consti-tuents were less active than temephos, but could be important models for the further development of new larvicides. it is necessary to investigate the effectiveness of these mixture compounds in a field setting and the toxicity toward other organisms, for example by doing brine shrimp lethality test. conclusions in the present work the mixture of cnsl in aquaeous extract of s.rarak dc could be used as larvicide against. cx.quinquefasciatus. given the continual search for renewable and biodegradable sources of new medicinal products, cnsl from cashew nut processing industries in indonesian agribusiness represents an opportunity to increase the value of this by-product by developing green larvicidal compounds to be used in filariasis control, which is cheap, non-toxic, and biodegradable. table 2. larvicidal activity of cnsl and s.rarak extract mixture against cx.quinque fasciatus larvae. acknowledgement i would like to thanks to faculty of pharmacy and parasitology laboratory ugm for the possibility for doing experiment. references cheng, s.s., chang, h.t., chang, s.t., tsai, k.h. & chen, w.j., 2003, bioactivity of selected plant essential oils against the yellow fever mosquito aedes aegypti larvae, bioresearch technology, 89, 99-102. devine gj, furlong mj. insecticide use: contexts and ecological successions. agric hum values 2007; 24: 281-306. evans, f.j. & schmidt, r.j., 1980, plants and plant products that induce contact dermatitis, planta medica, 38, 289-316. lima, j.b.p., da-cunha, m.p., silva júnior, r.c., galardo, a.k.r., soares, s.s., braga, m.a., ramos, r.p., valle, d., 2003. resistance of aedes aegypti to organophosphates in several municipalities in the state of rio de janeiro and espírito santo, brazil. am. j. trop. med. hyg. 68, 329–333. lomonaco, d., gilvandete, m.p.s., yana, s.f., angela, m.c.a., selma, e.m., mele, g., & vasapollo, g., 2009, study of technical cnsl and its main components as new green larvicides, green chemistry, 1, 31–33. oliveira, m.s.c., de morais, s.m., magalhaes, d.v., batista, w.p., vieira, g.p., & craveiro, a.a., 2011, antioxidant, larvicidal, and antiacetylcholinesterase activities of cashew nut shell liquid constituents, acta tropica, 117, 165-170. samuel t, jayakumar m, william sj. 2007, culex mosquito: an overview. in: william sj. defeating the public enemy, the mosquito: a real challenge. loyola publications: chennai, p. 95-116. shian, l.c., 2007, the effect of sublethal concentration of abate on aedes aegypti (linnaeus) and culex quinquefasciatus (say), tesis, universiti sains malaysia, subang jaya. tennyson, s., ravindran, j.k. & arivoli, s., 2012, screening of twenty five plant extracts for larvicidal activity against culex quinquefasciatus say (diptera: culicidae), asian pacific journal of tropical biomedicine, s1130-s1134. tyman jhp, morris lj. composition of cashew nut shell liquid. j chromatography 1989; 27: 287–8. world health organization (who), 2005, guidelines for laboratory and field testing of mosquito larvicides, geneva. cnsl (ppm) number of larva died (3 replications) total larvae mortality (%) 1 2 3 0 1 1 0 75 2.67 8 2 4 4 75 13.33 8,56 5 3 3 75 14.67 9,16 7 3 3 75 17.33 9,8 6 9 0 75 20 10,49 8 5 3 75 21.33 content_v3n1_3.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 53-57 | doi: 10.14421/biomedich.2014.32.53-57 larvicidal activity of a mixture of cashew nut shell liquid and water-soluble extract of soap nut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati1, sudarsono2 and budi mulyaningsih3 1faculty of pharmacy; 2laboratory of parasitology faculty of medicine; 3department of pharmaceutical biology, ugm, indonesia author correspondency: sudarsono: ugmpsot@yahoo.com; budi mulyaningsih: budimulyaningsih@yahoo.com abstract cashew nut shell liquid (cnsl) which has been known as a waste of processing cashew fruits which is contain phenolic compounds have activity as larvicides. cashew nut shell liquid is not soluble in the water where the larvae grow. cashew nut shell liquid mixed with watersoluble extract of soapnut fruit which serves as a natural surfactant that can emulsify oil in water. the test subjects were larvae of aedes aegypti third instar. test subjects were divided into treatment group and control group. in the treatment group, test subjects were the mixture of cnsl and ethylacetat soluble extract (ese) in tap water. the larvae mortality observations were done 24 hours after the treatment. lc50 and lc90 as final test data were analyzed using probit analysis. extract constituents of cnsl and water soluble extract of soapnut fruit (wseosn) were investigated using thin layer chromatography (tlc) method. the effect of cnsl as larvicides against third instar larvae of ae. aegypti with were lc50 of 14,12 ppm, while the lc90 of 24,85 ppm. keywords: larvacidal activity, thin layer chromatography, sapindus rarak dc introduction aedes aegypti is a mosquito species that can carry and transmit the dengue virus causes dengue hemor rhagic fever (dhf), yellow fever virus and chikungunya virus (who, 2004). how to control the mosquito population that most rapidly break the cycle of transmission is the use of chemical compounds, one of them is the use of synthetic insecticides and larvicides. the use of synthetic compounds can lead to increased mos quito resistance, environmental pollution caused by their residue, even the death of non-target creatures (munif, 1993). the use of plants as a biopesticide is a one of alternative natural way for controlling which is safer for sustainable environment balance. the natural biopesticides were usually easy to apply and not harmful to natural enemies and other beneficial insects (adebowale & adedire, 2006). cnsl is a viscous oil, black color and has a very strong irritant properties on the skin because of the phenolic substances derivatives e.g. cardol, anacardic acid and 2-methyl kardol (kubo et al., 1986); anacardic acid is not soluble in water. this sticky fluid was extracted from cashew nut shell by the use of hydraulic pressure pump at a certain temperature (mulyono and abdul gani, 1996). anacardic acid has several biologic effect such as anti-microbial, anti-acne (kubo et al., 1993; 1994) and as molluscicides (kubo et al., 1986). the main constituent of cnsl i.g. anacardic acid, cardanol, and cardol also reported as larvicidal activity against the larvae of ae. aegypti with lc50 of 12,40 ppm for anacardic acid, 14,45 ppm for cardanol, and 10,22 ppm for cardol (oliveira et al., 2010). soapnut in indonesia called “lerak” (sapindus rarak dc.) is a plant that from southeast asia and can be able grow well in tropical climate such in indonesia (afriastini, 1990). the active compounds from the soapnut pericarp are saponins and sesquiterpenecompounds. the main of non-sugar saponins compound are usually triterpenes and steroids derivatives (schneider, 1980; wina et al., 2005).saponins as well as surfactants that have both hydrophilic and hydrophobic pool which were able to lower the interfacial tension between oil and water that immiscible; so as to form a colloidal solution which forms a foam when shaken (matheson, 1996; gunawan & mulyani, 2004). the purpose of this study was to determine the activity of cashew nut shell oil (cashew nut shell liquid / cnsl) as an alternative natural larvicides. methodology preparation and extraction of plant materials cashew nut shell obtained from jatibedug village, wonogiri and soapnut fruit from perhutani area, situbondo. the identification of plant material was carried out by mr.joko santosa m.si, and voucher specimen was found at the department of pharmaceutical biology faculty of pharmacy, ugm, yogyakarta. cashew nut shell that had been oven-dried at 50°c for 1 hour obtained 1,313 kg net weight then pressed using a hydraulic press by 150-160 kg/cm2 to obtain cashew nut shell liquid (cnsl); whereas 10 g of soapnut fruit chopped and then extracted by heating at 50°c for 30 min in the distilled water ad 1000 ml to obtain a water-soluble extract of soapnut fruit 1% b/v. 54 biology, medicine, & natural product chemistry 3 (2), 2014: 53-57 preparation of water-soluble extract of soapnut fruit which did not kill larvae as a main solution of water-soluble extract of soapnut fruit was 1% b/v diluted by taking a volume of 6, 8, 10, and 12 ml for each dissolved in 100 ml of tap water to obtain test solutions with concentrations of 600, 800, 1000, and 1200 ppm. four plastic cups with a mouth diameter of approximately 10 cm glass used as a test medium. each test cups contain10 larvae of third instar ae. aegypti. each concen-tration of repetitions performed 3 times. larval mortality was observed 24 hours after treatment by calculating the percentage of larval mortality. the biggest concentrations that doesn’t kill any larvae chosen for making the mixture solution with cashew nut shell liquid. bioassay for larvicidal toxicity mixture concentration series was made by mixing watersoluble extract of soapnut fruit at a concentration elected with cashew nut shell liquid. the mixture of cnsl and water soluble extract of soapnut fruit homogenized using a vortex for 5 minutes, then stirred using a magnetic stirrer at medium speed for 5 minutes. the results observed visually mixing at room temperature (25°c). subsequently the mixture was dissolved in tap water to obtain a final volume of 100 ml of solution for each concentration. the cnsl concentration used was 6 ppm, 7,56 ppm, 9,53 ppm, 12,01 ppm, 15,13 ppm, and 19,06 ppm. each test cups contain 25 larvae of third instar ae. aegypti. each concentration of repetitions performed 3 times. larval mortality was observed 24 hours after treatment by calculating the percentage of larval mortality. larvicidal test data were analyzed using probit analysis to obtain lc50 and lc90 (finney, 1971).the concentrations in probit analysis were transformed to logarithm (log concentra-tion) and the lethal concentrations (lc50 and lc90) were calculated manually. specification of test material using thin layer chromatography (tlc) a. cashew nut shell liquid cashew nut shell liquid that dissolved in n-hexane (1:10b/v) and then filtered with silica gel 60 used as test solution. components of cnsl are further separated by thin layer chromatogra phic (tlc) method. chromtography was perfomed using aluminium plates silica gel 60 f254 (merck). three microliters of test solution was applied to the plate with distance of development 8 cm. plate inserted in a chamber that had already saturated by mobile phase ntoluene: ethyl acetate: glacial acetic acid (7:2:1 v/v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with iodine vapor and vanillin sulfuric acid. b. water-soluble extract of the soapnut fruit thirty milliliters of water-soluble extract of soapnut fruit was evaporated and then hydrolyzed by boiling with 30 ml of 2n hcl using reflux distillation method for 30 minutes. samples that had been hydrolyzed were then fractionated with 30 ml of n-hexane. nhexane fraction was then washed with distilled water until water fraction ph was neutral. the results of this fraction was then added anhydrous na2so4 to remove water traces. furthermore, the fraction of n-hexane which has been neutralized partitioned again using 30 ml of ethyl acetate. components of both fractions were then separated by tlc. samples of both fractions was applied to the plate as much as 3 spots using a capillary tube with distance of development 8 cm. plate inserted in a chamber that has been saturated with mobile phase. mobile phase that used was chloroform: methanol (9.5: 0.5 v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with anisaldehyde sulfuric acid. results and discussion larvicidal effect of a mixture of cashew nut shell liquid and water-soluble extract of soapnut fruit probit analysis was used to study the response of larvae to the exposure of test solution in a biological assay (umniyati, 1990). probit analysis is a statistical method used to determine the relationship between dose and response (finney, 1971). percentage larval mortality was calculated after 24 hours. dead larvae are larvae that does not move when touched with a pipette or larvae that sink to the bottom of container that does not respond to stimulation. table 1. test results of water-soluble extract of the soapnut fruit preliminary test. soapnut concentration (ppm) total mortality of larvae total larval test1 2 3 600 (*) 0 0 0 30 800 1 0 1 30 1000 1 2 2 30 1200 4 3 4 30 in table 1, it can be seen that the biggest concentration of water-soluble extract of the soapnut fruit that does not make larvae dead were at a concentration 600 ppm. this preliminary test aim is to minimize the effect of watersoluble extract of the soapnut fruit in the death of larvae so the final assay results that obtained later is the result of larvicidal activity of cashew nut shell liquid. the largest concentration of water-soluble extract of soapnut fruit that does not kill larvae was used for the mixture becausehigher levels of water-soluble extract of soapnut fruit will increase the solubility of cashew nut shell liquid in the water. glory resia raraswati, et al. – larvicidal activity of a mixture of cashew nut shell liquid … 55 in table 2, data percentage mortality test results are used to calculate the lc50 and lc90 values are percent mortality at levels of 7,56 ppm, 9,53 ppm, 12,01 ppm, 15,13 ppm, and 19,06 ppm.the percentage mortality data is plotted in probit tables to obtain theprobit value, then made the graph between log concentration (x) and probit value (y) to obtain the linear regression equation y = a + bx. probit calculation steps performed according to the method of finney (1971). table 2. results of probit analysis of a mixture of cashew nut shell liquid and water-soluble extract of soapnut fruit against ae. aegyptilarvae (total larval test = 75). cnsl concentration (ppm) total mortality of larvae percentage larval mortility (mean) linier equation lc50 (ppm) lc90 (ppm) 0 0 0 y = 5,20921x – 0,9891 14,12 24,85 6 0 0 7,56 8 10,67 9,53 14 18,67 12,01 20 26,57 15,13 41 54,67 19,06 60 80 linear regression equation obtained from the probit analysis is y = 5.20921 x 0.9891 so that the calculation of lc50 and lc90can be obtained.lc50 and lc90 calculated were 14.12 ppmand 24.85 ppm. a material that has a larvicidal test lc50 values below 100 ppm can be said to have larvicidal activity against the larvae test (cheng et al., 2003). lc50 values in this study were at 14.12 ppm so cashewnutshell liquid that is used as the test material can be said to have potential as larvicidal agent against the third instar larvae of ae. aegypti. cashew nut shell oil is known to have 3 main compound content anacardic acid, cardol, and cardanol which showed inhibition of the acetylcholinesterase enzyme activity as well as the mechanism of action of organophosphate and carbamate (oliveira et al., 2010). inhibition of the acetylcholinesterase enzyme causes accumulation of acetylcholine which triggers central nervous system disorders, convulsions, respiratory paralysis, and death in larvae (rosenberryet al., 2008). phytochemical compund of cashew nut shell liquid and water-soluble extract of soapnut fruit in figure 1, elution of cnsl results showed 2 spots on the hrf 63 and 73, that drown under 254 nm uv light. furthermore, the silica plate inserted in a closed chamber that has been filled by iodine vapor and left a few minutes. figure 1. tlc-chromatogram of cashew nut shell liquid. detection: visible light (a), under 254 nm uv light (b), under 366 nm uv light (c), after iodine vaporized (d), and after spraying vanillin sulfuric acid were observed in visible light (e). figure 1.d were a yellow spot; the hrf 63 and 73. it could be the presence of an unsaturated compound; it was perhaps an side chain of the phenolic compounds. iodine vapor was used for the detection of side chain compounds 56 biology, medicine, & natural product chemistry 3 (2), 2014: 53-57 that had a double bond; a positive result is a yellowbrown spots on the visible light (fessenden and fessenden, 1994). the mechanism of iodine to form the color is an addition reaction on double bond in unsaturated fatty acids (simpen, 2008). results of detection by vanillin sulfuric acid reagent (figure 1.e) were observed under visible light were the presence of spots on hrf 63 with red-orange color, while on hrf 73 as pink-brown color spot. based on these results, spots on the hr f63 and 73 suspected as phenolic compounds anacardic acid derivatives. identification of the compounds in water-soluble extract of soapnut fruit was identifying saponin. saponins are triterpenoid glycosides or glycoside steroid with sapogeninaglycone (schneider, 1985; gunawan and mulyani, 2004). the basic science of saponin identification was made for the existence of the sapogenin aglycone. (a) (b) (c) (d) : 1 = n-hexane soluble fraction; 2 = ethyl acetate soluble fraction figure 2. tlc chromatogram profile of n-hexane and ethyl acetate fraction of water-soluble extract of soapnut fruit. detection before spraying anisaldehyde sulfuric acid: under visible light (a), uv 254 nm (b), and uv 366 nm (c) and detection after spraying anisaldehyde sulfuric acid under visible light (d). in figure 2, the chromatogram of n-hexane and ethyl acetate of water-soluble extract of soapnut fruit had no spots after observed under visible light, uv light 254 nm, and 366 nm uv. it was due to saponins had no aromatic ring in the structure. it could be detected by coloring spray reagent (wagner, 1984). if there is purple-red or purple after spraying with anisaldehyde sulfuric acid reagent showed the presence of terpenoids or steroids (wagner & bladt, 1996). after spraying with anisaldehyde sulfuric acid reagent (aas) and it was heated for 5 minutes at a temperature of 105°c, there was 3 purple spots (hrf 49; hrf 67, and hrf 79) in ethyl acetate fraction and the fraction of n-hexane had pink spots on the hrf 82 and 93. the purple color in ethyl acetate fraction was a steroid sapogenin compounds, and the pink spots on n-hexane fraction could be as triterpene sapogenin. steroid compounds are more polar than the triterpenoid compounds so that would be more soluble in more polar solvents as well, in this case is ethyl acetate. based on the above observations, the water-soluble extract of soapnut fruit suspected of containing sapogenin steroid and triterpenoid sapogenin which is the aglycone of saponin glycosides and it can be used as a natural surfactant to emulsify cashew nut shell oil in aqueous media. the larvicidal properties exhibited by cashew nut shell liquid in this study might be related to presence of phenolic derivative components. conclusion the results of this study indicated that a mixture of cashew nut shell liquid and water soluble extracts of soapnut fruit (sapindus rarak dc.) had an effect as larvicides against third instar larvae of ae. aegypti. further research needs to be done about the toxicity effects of a mixture of cashew nut shell liquid and water soluble extract of soapnut fruit against non-target organisms i.g fish, and frog. references adebowale, k.o. & adedire, c.o., 2006, chemical composition and insectisidal properties of the underutilized jatropha curcas seed oil. african j. biotech. 5 (10), 901-906. afriastini, j.j., 1990, daftar jenis nama tanaman, 53-60, penebar swadaya, jakarta. cheng, s.s., chang, h.t., chang, s.t., tsai, k.h., chen, w.j., 2003, bioactivity of selected plant essential oils against the glory resia raraswati, et al. – larvicidal activity of a mixture of cashew nut shell liquid … 57 yellow fever mosquito aedes aegypti larvae. bioresource technology, 89, 99-102. finney, d.j., 1971, probit analysis, 3rd ed., 333-340, cambridge university press, great britain. gunawan, d. & mulyani, s., 2004, ilmu obat alam (farmakognosi), jilid 1, 87-95, penebar swadaya, depok. kubo, i., muroi, h., & kubo, a., 1994. naturally occurring antiacne agents, j nat prod, 57, 9 – 17. kubo, i., muroi, h., himejima, m., yamagiwa, y., mera, h., tokushima, k., ohta, s., & kamikawa, t., 1993, structure antibacterial activity relationships of anacardic acids, j agric food chem, 41, 1016 –1019. kubo, i., komatsu, s., & ochi, m., 1986, molluscicides from cashew (anacardium accidentale) and their largescale isolation. j. agric. food chem, 34, 970-973. matheson, k.l., 1996, surfactant raw materials: classification, synthesis, and uses. in spitz, l. (ed). soap and detergents: a theoretical and practical review, 288-303, aocs press, illinois. mulyono, e., & abdulgani, l.y., 1996, karakterisasi cnsl dan metode ekstraksinya; isolasi kardanol dan karakteristiknya; isolasi pektin dan karakteristiknya, laporan penelitian, balittro, bogor. munif, a., 1993, cendawan patogen pada larva anopheles aconitus yang resisten dpt di daerah endemis malaria banjarnegara, jawa tengah. majalah kesehatan indonesia xxi, 8, 472-6. oliveira, m.s.c., de morais, s.m., magalhães, d.v., batista, w.p., vieira, i.g.p., craveiro, a.a., de manezes, j.e.s.a., carvalho, a.f.u., de lima, g.p.g., 2010, antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents, acta tropica, 117 (2011), 165–170. rosenberry, t.l., sonoda, l.k., dekat, s.e., cusack, b., johnson, j., 2008, monitoring the reaction of carbachol with acetylcholinesterase by thioflavin t fluorescence and acetylthiocholine hydrolysis, chem. biol. interact, 175, 235– 241. schneider g., 1980, pharmazeutisch bio logie, b.i.varlag mannheim p. 230, simpen, i.n., 2008, isolasi cashew nut shell liquid dari kulit biji jambu mete (anacardium occidentale l) dan kajian beberapa sifat fisiko-kimianya. jurnal kimia 2 (2), 71-76. umniyati, s.r., 1990, analisa probit secara aritmatis untuk pengujian toksisitas insektisida terhadap serangga, 1-13, 2736, laboratorium parasitologi fakultas kedokteran universitas gadjah mada, yogyakarta. wagner, h., 1984, plant drug analysis a thin layer chromatography atlas, 164, springer, new york. wagner, h & bladt, s., 1996, plant drug analysis, 2nd ed., 305317, 359, springer, new york. wina, e., muetzel, s., hoffmann, e.m., makkar, h.p.s., & becker, k., 2005, saponin containing methanol extract of sapindus rarak affect microbial fermentation, microbial activity and microbial community structure in vitro, animal feed science technology, 121, 59-174. world health organization (who), 2004, prevention and control of dengue and dengue haemorrhagic fever, terjemahan dari who regional publication searo no. 29, new delhi content_v3n2_2.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 87-91 | doi: 10.14421/biomedich.2021.102.87-91 issn 2540-9328 (online) potential of tithonia diversifolia hemsley a. gray (kembang bulan) leaf extract as anti-cancer agents muflihah rizkawati department of pharmacology, faculty of medicine, universitas islam indonesia, indonesia. corresponding author dr.rizkawati@uii.ac.id manuscript received: 22 september 2021. revision accepted: 04 october, 2021. published: 07 october, 2021. abstract the main objective of this review is to explain the great potential of herbal plants as anticancer agents. cancer is a disease caused by abnormal cell growth in the body. the high number of cancer incidents still become a global concern because of the high mortality rate. the treatments of cancer such as chemotherapy can cause serious side effects by killing the normal cells. this is the reason why it is necessary to develop an alternative treatment of cancer. i discussed a plant that is believed to has health benefit. many studies have showed the positive effect of tithonia diversifolia plant for health. after 2000, the researchers discovered a new potential through its cytotoxicity to neoplastic cells. this plant needs to be developed sustainably. however, in the future this plant might become an effective alternative to treat cancer with lower side effects. keywords: tithonia diversifolia (hemsley) a. gray; kembang bulan; anticancer. introduction cancer is a disease with a multifactorial etiology which is initiated by an abnormal and uncontrolled expression of transcription factors. this can be affected by a gene mutation (babazadeh et al., 2018). cancer is one of the main causes of death in the world and still become a global concern. cancer has the second highest risk of death with a mortality ratio of 1:6 or around 9.6 million deaths in 2018. the most common types of cancer in men are lung, prostate, colorectal, stomach, and liver and the most common types of cancer in women are breast, colorectal, lung, cervical and thyroid cancers (who, 2021). cancer can develop in any part of the body and can spread (metastasize) to other parts of the body. although some types of cancer can be similar in some ways, how they develop, grow, spread, and respond to drugs can be very different (american cancer society, 2021). the research and development data in 2019 showed the incidence of cancer in indonesia reached 136.2/100,000 population and reach 8th place in southeast asia. the highest incidence rate of cancer for men is lung cancer, which is 19.4 per 100,000 population with an average death rate of 10.9 per 100,000 population, while the highest incidence rate of cancer for women is breast cancer, which is 42.1 per 100,000 population with an average 17 deaths per 100,000 population (kemenkes ri, 2019). cancer treatment methods are still being developed. there are several types of local treatment such as surgery and radiation therapy, and systemic treatment such as chemotherapy, immunotherapy, or targeted therapy that can affect not only cancer cells but also the entire body (american cancer society, 2021). understanding the causes, how they are formed, and especially the factors that promote or inhibit their growth required to solve the complex biological and medical problems. currently, there are many successful results from cancer treatment. for example, over the past half century, the survival rate for childhood leukemia has increased from 10% to more than 80%. but the healing method (or extended remission) is difficult, and is only available to those who living in affluent countries. there is nothing like "medicine" for cancer, and much remains for medical researchers to do (haussman, 2019). the cancer treatment ideally can stop the growth of cancer cells with a selective toxic effect on the targeted cancer cells without damaging other normal tissues. however, most of today's anticancer drugs are still potentially toxic to normal tissues. the condition can cause harmful side effects to the body. therefore, the discovery of anti-cancer drugs that have more positive effects with low toxicity is being developed. many potential phytopharmaceuticals have been investigated to find chemical elements in plants that are beneficial for treatment (wahyuningsih et al., 2013). the development of herbal studies using in vitro and in vivo methods has https://doi.org/10.14421/biomedich.2021.102.87-91 88 biology, medicine, & natural product chemistry 10 (2), 2021: 87-91 shown the beneficial effects of medicinal plants and their bioactive compounds. the most common anticancer mechanism is by inducing apoptosis in neoplastic cells. currently, more than 25% of drugs used over the last 20 years are derived directly from plants. according to who, 80 percent of the world's population has benefited from traditional medicine (aryan, 2018). nowadays, the efforts to develop new anti-cancer medicines and particularly novel drug delivery systems should be done. it is important to improve the pharmacodynamics and bioavailability of drugs and to deliver specific drug concentration to cancer cells so it can produce minimum cytotoxic effects on normal cells (babazadeh et al., 2018). tithonia diversifolia characterization the asteraceae family is known for its therapeutic effect such as antihelmintic, anti-inflammatory, astringent, antispasmodics, cholesteric, antioxidant, antihemorrhagic, antimicrobial, diuretics, and analgesics. one type of the species from this family is tithonia diversifolia (farias et al., 2019). t. diversifolia can be described as a flowering shrub-like species, growing up to over 2–3 m high. the flowers are 5–15 cm wide and shaped like a daisy. the leaves (15–30 cm long) can be described as sub-ovale, petiolate, 3to 7-lobed delicately hairy, and alternately to oppositely arranged (tagne at al., 2018). t. diversifolia (hemsl.) a. gray (mexican sunflower) originally from mexico and central america, but is now widely known in various parts of the world as an ornamental plant and a plant used for medicine. there are more than 150 types of secondary metabolites isolated from t. diversifolia, including the sesquiterpene lactones thyrotundin, tagitinin a, and tagitinin c (miranda et al., 2015). t. diversifolia can be found in tropical and subtropical climes. traditionally, all parts of the plant especially the leaves, are used by people to treat wounds, musculoskeletal disorders, abscesses, dermatological conditions, stomach pains, oral treatment for diabetes, malaria, fever, hepatitis and infectious diseases. some studies showed the heterogenous evidence from in vitro and in vivo research that supporting most of the traditional therapeutic claims. t. diversifolia can grow up for all year-round and used fresh or dry as traditional remedies in several cultures. all parts of the plant are useful include the leaves, flowers, stems and roots are mentioned in some ethnobotanical reports (tagne et al., 2018). the studies on phytochemical screening not only showed the presence of metabolites phenolics, flavonoids and tannins but also absence of alkaloids and saponins in t. diversifolia ethanolic flower extract. phenolics are well known for their antioxidants and antimicrobial properties. this plays a role in prevention activities of phenolic against infection and degenerative disease, inflammation and allergies through antioxidants, antimicrobial and protein or enzyme neutralization or modulation mechanism. the examples of phenolic compounds are flavonoids and tannins. flavonoids have many beneficial health effect and physiological properties. flavonoids have antioxidant effects, anti-inflammatory, anti-mutagenic, anticarcinogenic property and as a modulator to the function of cellular enzymes. it is also have a great potensial inhibitors of xanthine oxidase, cyclooxygenase, lipoxygenase and phosphoinositide 3-kinase. tannins has antioxidant, anticarcinogenic and antimutagenic potential. tannins are also known to accelerate blood clotting, lower blood pressure, lower serum lipid levels and modulation of immunoresponse. terpenoids has an antibiotic, chemopreventive and therapeutic effects on cancer. terpenoids have been developed in the prevention, inhibition and therapy of several diseases including cancer, antivirals, antimicrobial, anti-allergic, antispasmodic, antidiabetic, anti-inflammatory, anticancer and immuno-modulatory properties (gaule ang et al., 2019). farias et al, in 2019 also evaluate the composition, antioxidant, cytotoxic and microbiological activity of the essential oil of the leaves of the species t. diversifolia. the main constituent they was found are α-pinene (9.9%), limonene (5.40%), (z)-β-ocimene (4.02%), pcymen-8-ol (3.0%), piperitone (11.72%), (e)-nerolidol (3.78%) and spathulenol (10.8%). the activity of antioxidant from thee essential oil extracted from the leaves of t. diversifolia can be observed from the significant result with p < 0.0001 at the concentration of 5 mgml-1 with %aa 54.6 ± 0.06. the minimum inhibitory concentration (ic50) was 4.30 mgml-1 , with a strong correlation coeffificient (r2) of 0.9965. although t. diversifolia has good antioxidant activity, its ic value still lower than ascorbic acid (farias et al., 2019). anti-cancer potential activity-guided fractionation technique of an ethyl acetate extract from t. diversifolia, using an antiproliferative bioassay against human colon cancer cells (col2), resulted in the isolation of three new sesquiterpenoids, 2r-hydroxytirotundin (1), tithofolinolide (2), and 3r-acetoxydiversifolol. (3), together with eight known sesquiterpene lactones, 3'acetoxy-8'-isobutyryloxyreynosine (4), tagitinin c (5), 1',2repoxytagitinin c (6), 4r,10r-dihydroxy-3-oxo-8'isobutyryloxyguaia -11(13)-en-12,6r-olide (7), 3racetoxy4r-hydroxy-11(13)-eudesmen-12-oic acid methyl ester, 17,20-dihydroxygeranylnerol, tagitinin a, and thyrotundine. measurement of antiproliferative activity in col2 cells and induction of cellular differentiation in human promyelocytic leukemia (hl60) cells were carried out to determine the potential of these isolates as cancer chemopreventive agents. in this rizkawati – potential of tithonia diversifolia as anti-cancer agents 89 study, a culture test was used on rat breast organs that had been induced by 7,12-dimethylbenz[a]anthracene and the selected compound was tested for its ability to inhibit preneoplastic lesions. isolates tagitinin c and 1',2 repoxytagitinin c showed significant antiproliferative activity. isolat tithofolinolide, 3'-acetoxy-8'isobutyryloxyreynosine, and 4r,10r-dihydroxy-3-oxo8'-isobutyryloxyguaia -11(13)-en-12,6r-olide induced cellular differentiation of hl-60. the isolate 3'-acetoxy8'-isobutyryloxyreynosine significantly inhibited (63.0% at 10 μgml-1) lesion formation in rat breast organ culture assays. among these isolates there were several sesquiterpenoids and flavonoids that showed anti-human pro myelocytic leukemia (hl-60) activity and tagitinin c was the main sesquiterpenoid that had been identified (gu et al., 2002). the methanol extract of tithonia diversifolia showed antiproliferative activity against glioblastoma cells u373, with an ic50 value of 59.2 ±3.7 μgml -1, while tagitinin c showed an ic50 value of 6.1 ±0.1 μgml -1. based on flow cytometric analysis and pan-caspase inhibitory activity, the anti-glioblastoma effect was independent of apoptosis and it was concluded that tagitinin c induces u373 cell death through autophagy under certain conditions. autophagy is a transport pathway to all eukaryotic cells. cells degrade part of their own intracellular constituents, including cytoplasm and organelles. the first process for autophagi mechanism is the formation of a double membrane bound vacuole from endoplasmic reticulum. the autophagosomes receive hydrolases to form an autolysosome. this study also found that human glioblatoma u373 cell death induced by tagitinin c was apoptosis-independent and induced autophagosome (lee at al., 2011). another study showed the activity of the extract t. diversifolia in inhibiting the viability of keloid fibroblasts. antifibrotic activity of the ethanolic extract of t. diversifolia on keloid fibroblast cell proliferation and collagen accumulation was expressed by the ic50 value using probit regression analysis. the percentage of inhibition of keloid fibroblast proliferation increases gradually with the given dose extract of t. diversifolia with a very strong correlation (r = 0.838; p = 0.000 in 72 hours; r = 0.924; p = 0.000 in 120 hours of incubation) with the lowest ic50 value of 3.624 μgml -1 at an incubation time of 120 hours (wahyuningsih et al., 2015). furthermore, ranti et al, in 2018 tested the cytotoxic activity of tagitinin c on keloid fibroblast cells with ic50 values of 0.122 μgml -1 (72 hours incubation) and 0.039 μgml-1 (120 hours) which affected the deposition of 53.1% of keloid collagen (72 hours) and 44.3% (120h). the selectivity index of tagitinin c in normal fibroblasts (nf) was 287 (72 hours incubation) and 791 for (120 hours incubation). based on these results, it was concluded that the ability of tagitinin c to inhibit keloid fibrosis viability and reduce keloid collagen deposition was consistent with the concentration and incubation time. the process of collagen synthesis happened when the fibroblast got a stimulus from tgf-β that was bound to tgf-β receptors on the fibroblasts membrane. when an injury occured, the tgf-β will be separated from the binding protein give respond to mechanical trauma system. however, there have not been many studies on the inhibitory activity of tgf-β by t. diversifolia compound. but, another study have found that the sesquiterpene lactones is known to inhibit the keloid fibroblasts proliferation, inhibit tgf-β1, and inhibit collagen synthesis of keloid through the nf-κb (ranti et al., 2018). the next study was carried out by wahyuningsih, et al in 2019 to determine the antifibrocytic mechanism of t. diversifolia extract. the study showed that the ethanolic extract of t. diversifolia at concentrations of 20 gml-1, 10 gml-1, and 5 gml-1 for 24 hours in keloid fibroblast culture showed slower migration activity compared to untreated keloid fibroblast culture (p<0, 05). the study also showed that the expression of tgfβ1 and vegf in the treatment group given the ethanolic extract of t. diversifolia was significantly lower than untreated keloid fibroblasts (p<0.05). from those result, it can be concluded that the ethanolic extract of t. diversifolia can inhibit fibroblast migration activity and reduce the expression of vegf and tgf-β1 in keloid fibroblasts. this mechanism further strengthens that t. diversifolia has great potential to be used as a keloid therapy (wahyuningsih et al., 2019). traditionally, t. diversifolia have been used to treat chronic hepatitis and liver diseases in chinese herbal medicine. further researh must be done to explore whether t. diversifolia can be used to treat hepatoma. (lu et al., 2017). the study of the cytotoxic activity of the methanol extract of the leaves of t. diversifolia and tagitinin c against human hepatoma cells (hep-g2) showed positive results. the test results with the mtt assay showed ic50 values 40.0 ± 2.0 and 2.0 ± 0.1 μgml-1. this compound induces an increase population in the sub-g1, exhibited s phase arrest and results in the activation of caspase 3 and caspase 8 which confirms the data that the antiproliferative effect of this compound is through caspase-dependent apoptotic activity. tagitinin c caused membrane blebbing, cell shrinkage, nuclear fragmentation, and chromatin condensation in hepatoma cells. caspases, have an essential role in the regulation and the execution of apoptotic cell death. caspase 3 well known as a key effector caspase in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase 8). this study show that tagitinin c induced apoptosis done by the activation of the caspase cascade (liao at al., 2012). a follow-up study was conducted by lu in 2017 to showed another cytotoxic effect of t. diversifolia on hep g2 cells. the results of the study showed that the induction of apoptosis such as dna fragmentation could increase the level of apoptosis and apoptosis of the sub-g1 population in the treatment group that given the acetate extract leaves of t. 90 biology, medicine, & natural product chemistry 10 (2), 2021: 87-91 diversifolia. in addition, the acetate extract of the leaves of t. diversifolia was also increase the expression of the proapoptotic protein bax bcl-2. based on this study, it prove the cytotoxicity induced by ethyl acetate extracts of t. diversifolia leaves (td-l-ea) in human hepg2 hepatoma cells that strengthen ethnopharmocologic use of t. diversifolia (lu at., 2017). the cytotoxic effect of t. diversifolia leaf extract on raw264.7 cells and human mononuclear peripheral blood cells (pbmc) by mitochondrial respiration method was also studied. the results of the study showed that the ic50 concentration in pbmc proliferation was 4.42 μgml-1 and in raw264.7 cells the ic50 value was 11.63 μgml -1. the study demonstrated an immunomodulating effect by the leaf extract of t. diversifolia, which was produced the inhibition of phytohemagglutinin-m-induced pbmc proliferation and lps-induced nitric oxide production in raw264.7 macrophages. the hydrogen peroxide are able to induce cell death under stress conditions through apoptosis, but the anti-oxidant compound can protect this condition. the data showed that t. diversifolia aqueous extract showed an anti-oxidative capacity equivalent of (241.04 ± 11.93 mmol troloxg-1) from abts assay and (94.89 ± 2.69 mmol troloxg-1) from dpph assay, respectively. the anti-oxidative capacity of t. diversifolia aqueous extract was equivalent to (32.62 ± 1.87) and (20.99 ± 2.79) mg nacg-1 in the abts and dpph assays, when nac was used as the standard compound. however, the calculated concentration was out of the non-cytotoxic range, which should be lower than 30 mg/ml. thus, this calculated concentration based on the antioxidant capacity could not be used in practice in the hydrogen peroxide-induced cell death model. it could be conclude that the effects of the immunomodulation caused by the aqueous extract from t. diversifolia. the mechanism can desccribe such as anti-pha-m-induced pbmcs proliferation and antilpsinduced nitric oxide production from macrophages in the range of non-cytotoxic concentrations (hiransai et al., 2016). conclusion based on the literature study, it is found that some isolate from the extract of tithonia diversifolia (hemsley) a. gray) have the anticancer activity in several different types of cancer cells. the active substances contained in the leaves of the t. diversifolia. the compound material which has been known to become an anticancer alternative therapy is tagitinin c. the extract of the leaves t. diversifolia has several anticancer mechanisms related to antifibrotic activity and the induction of apoptosis. further research can be develop to find out another potential compound from the t. diversifolia extract. we hope there will be a new chance to find better alternative therapy for cancer. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references aryan, h. 2018. the role of herbal medicine as anti-cancer medicine: from claim to truth. golden medical journal, 7:e1179. doi: 10.22086/gmj.v0i0.1179 american cancer society. 2021.basis cancer. https://www.cancer.org/cancer/cancer-basics.html babazadeh, a., zeinali, m., hamishehkar, h. 2018. nanophytosome: a developing platform for herbal anti-cancer agents in cancer therapy. bentham science. 19: 170-180. 10.2174/1389450118666170508095250 farias, a., rodrigues, a., martins, r., rabelo, e., farias, c., de almeida, s. 2019. chemical characterization, antioxidant, cytotoxic and microbiological activities of the essential oil of leaf of tithonia diversifolia (hemsl) a. gray (asteraceae). pharmaceutical. 12, 34; doi:10.3390/ph12010034 gabule ang, a., enot, m., baltazar, g., alinapon, c., buncales, e., barbosa, g. 2019. antioxidant and cytotoxic activity of the leaf ethanolic extracts of tithonia diversofilia and gliricidia sepium from bukidnon, philippines. asian journal of biological and life science. 8(1): 8-15. doi: 10.5530/ajbls.2019.8.2 gu, j., gills, j., park, e., greenwood, e., hawthorne, m., axelrod, f., chavez, p., fong, h., mehta, r., pezzuto, j., kinghorn, a. 2002. sesquiterpenoids from tithonia diversifolia with potential cancer chemopreventive activity. journal natural products. 65: 532-536. hausman, d. 2019. what is cancer?. perspective in biology and medicine, 6(4): 778-784. doi: https://doi.org/10.1353/pbm.2019.0046. hiransai, p., tangpong, j., kumbuar, c., hoonheang, n., rodpech, o., sangsuk, p., kajklangdon, u., inkaow, w. 2016. anti-nitric oxide production, anti-proliferation 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of tithonia diversifolia (hemsley) a.gray) against hela cells. traditional medicine journal. vol. 18(1), p 2228. wahyuningsih, m., wirohadidjojo, y., hidayat, r., sadid, a. 2015. antifibrotic effect of standardized ethanol extract of tithonia diversifolia (hemsley) a. gray on keloid fibroblasts. international journal of pharmacognosy and phytochemical research. 7(4); 642-647. wahyuningsih, m., nugrahaningsih, d., budiyanto, a.2019. ethanolic extract of tithonia diversifolia (hemsley) a. gray inhibits migration activity and decrease the transforming growth factor-beta1, vegf expression on keloid fibroblasts. asian journal of pharmaceutical and clinical research. 12(1): 342-345. doi: http://dx.doi.org/10.22159/ajpcr.2019.v12i1.29850 world health organization, 2021. cancer. https://www.who.int/health-topics/cancer#tab=tab_1 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 129-133 | doi: 10.14421/biomedich.2021.102.129-133 issn 2540-9328 (online) antibacterial screening of bacterial isolates associated with mangrove soil from the ngurah rai mangrove forest bali anak agung gede indraningrat1, made dharmesti wijaya2*, putu arya suryanditha1, ayu savitri siskayani3, ni made defy janurianti4 1microbiology and parasitology department; 2pharmacology and pharmacy department; 3laboratory of biomedical science faculty of medicine and health sciences, 4faculty of agriculture, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@gmail.com manuscript received: 20 october 2021. revision accepted: 30 october, 2021. published: 02 november, 2021. abstract in this study we reported cultivation of bacteria associated with mangrove soil from the ngurah rai mangrove forest, bali. mangrove soil samples were serially diluted using sterile artificial seawater, spread onto starch casein m agar and incubated at 28oc for 28 days. cultivation of mangrove soil samples yielded 165 bacterial colonies with 68 isolates were selected and purified based on different morphology. of these 68 isolates, 22 isolates displayed antibacterial activities ranging from weak to strong inhibition against at least one of four bacterial indicators namely staphyloccocus aureus, streptococus mutans, escherichia coli and klebsiella pneumoniae using perpendicular streak method. overall, 19 out of 22 bacteria isolates displayed weak antibacterial potential and two isolates exhibited moderate antibacterial activity. the isolate sa4 was the only bacterium with strong antibacterial potential with measured clear distance ≥ 10 mm against the four bacterial isolates. sequence analysis based on 16s rrna gene fragment assigned the isolate sa4 as bacillus subtilis strain bil/bs-168. overall, this study confirmed the untapped potential of antibacterial activities from bacteria associated with mangrove soil. keywords: mangrove soil; bacteria; antibacterial screening. introduction bacterial resistance against antibiotic drugs is currently an emerging global health threat that require immediate actions (world health organization, 2018; centers for disease control and prevention, 2018). antibiotic resistance arises because bacteria develop immune mechanism by mutation, horizontal gene transfer and enzyme inactivation (munita and arias, 2016). as a rough estimation, resistance against antibiotics will reach 10 millions cases in 2050 and is predicted to result in financial loss of 100 trillions usd due to increased cost for hospitalization and loss of productivity. to date, overused and misused antibiotics are the main factors of the increasing rate of antibiotic resistance (ventola, 2015). one of the efforts to overcome the increasing rate of resistance of pathogenic bacteria is to discover new sources of novel antibiotics with much stronger efficacy compared to current drugs that are available on the market (roca et al., 2015; cheng et al., 2016). exploration of bacteria with a potent antibacterial activity has been mainly focused on terrestrial ecosystem (elbendary et al., 2018; singh et al., 2016; mohamed et al., 2017; assis et al., 2014). however, a number of studies indicated the highest de-replication of active compounds that have been previously reported. (debbab et al., 2010). therefore, exploration of marine habitat should also be given more priority as bacteria in this habitat is rather unexplored yet yield (debbab et al., 2010). mangrove forest is characterized with extreme salinity, high-low tide, wind pressure , muddy and low oxygen concentratio (friess, 2016). therefore, microbe in this habitat tend to be able to adapt on harsh environmental conditions (booth, 2018). the group of actinobacteria in mangrove forest synthesize various of secondary metabolites to survive with this extreme condition (van der heul et al., 2018; bentley et al., 2002). bioactive compounds from mangrove origins have been shown to display therapeutics potential and could be developed as new drugs including antibiotics. a number of studies have isolated bacteria from mangrove and these isolates could inhibit wide range of pathogenic bacteria (azman et al., 2015; lee et al., 2014; jiang et al., 2018). the ngurah rai mangrove forest is the biggest mangrove ecosystem in bali with over 19 mangrove plants species inhabited the area and dominated by four main species namely: rhizophora mucronata, avicennia https://doi.org/10.14421/biomedich.2021.102.129-133 130 biology, medicine, & natural product chemistry 10 (2), 2021: 129-133 marina, rhizophora apiculata dan sonneratia alba (balai pemantapan kawasan hutan wilayah viii denpasar, 2018). to date diversity of bacteria in the ngurah rai mangrove forest is rather unexplored, therefore this research aims to isolate and pre-screen bacterial isolates with antibacterial activities. materials and methods mangrove soil sampling mangrove soil samples were collected from the ngurah rai mangrove forest (8o43’40.4886” s, 115o11’42.80313” e), in august 2019 during the low tide. four sediment samples were taken from habitat of the four dominant mangrove plants rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba. from each sampling point, approximately 100-gram sediment was taken from 10 cm depth using a clean masonry trowel and stored individually in a sterile 50 ml falcon tube (figure 1). soil samples were stored in 4oc until further used. figure 1. soil samples collected from the ngurah rai mangrove forest. cultivation of bacterial isolates from mangrove soils ten grams of each mangrove soil samples were pretreated with wet heating in a water bath at 60oc for 15 minutes by combining sterile artificial seawater and distilled water (1:1 v/v) (azman et al., 2015; lee et al., 2014; jiang et al., 2018). subsequently, 1 ml of each of soil sample suspension was serially diluted (10-1 to 10-3) in 9 ml sterile artificial seawater. one hundred µl of each diluted soil sample was spread using sterile cotton swab (onemedia) onto starch m-protein agar (63 gram/l, himedia, india) which was supplemented with 100 μg/ml nalidixic acid and 25 μg/ml nystatin. agar plates were sealed with parafilm and incubated at 28oc for 28 days. observation of colonies grew on agar was performed every two days and the total colonies observed on agar media were counted. bacterial colonies with different morphologies (colour, form, elevation) were picked up from each agar plate and these colonies purified individually by streaked onto isp-2 agar media (4.0 gram/l yeast extract, 10 gram/l malt extract, 4 gram/l dextrose, 20 gram/l bacto agar). each of the purified bacterial isolate was stained using gram staining dan observed under microscope to identify their morphological form. antibacterial prescreening antibacterial activities of each purified isolates were pre-screened using perpendicular streak method (boontanom and chantasari, 2020) with a slight modification against four bacterial indicator strains which represented gram-negative bacteria (escherichia coli dan klebsiella pneumoniae) and gram-positive bacteria (staphylococcus aureus dan streptococcus mutans). in brief, each of pure bacterial indicator strains was streaked as a five cm vertical line and incubated for 48 hours at 37oc until fully grown. bacterial indicator strains were streaked five cm perpendicular from the original line of the bacterial isolate (figure 2). antibacterial potential was determined based on the distance formed between an isolate and a bacterial indicator according to four categories: no activity (a bacterial indicator show no distance with an isolate), weak (1 – 4 mm), moderate (5 – 9 mm) and strong ≥ 10 mm. figure 2. schematic overview of perpendicular streak for antibacterial pre-screening of bacterial isolates from mangrove soil. genetic analysis of bacterial isolates with strong antibacterial activities bacterial isolates with a strong antibacterial activity were genetically identified using polymerase chain reactions followed by sanger sequencing. the genomic dna of selected bacterial isolates was extracted using bacteria dna preparation kit jena bioscience (jena, germany) by following the manufacturer instruction. genomic dna of each the selected isolates were amplified by targeting 16s rrna gene fragment using primer pairs 27f: 5’-agagtttgatcmtggctcag3’ and 1492r 5’-ggttacsttgttacgactt indraningrat et al. – antibacterial activity of mangrove soil bacteria 131 3’(lane, 1991). the 50 µl pcr master mix contained 25 µl my taq hs red mix 2 x, 1 µl (20 µm) of each forward and reverse primer, 22 µl of sterile dna/rna free water and 1 µl of genomic dna. the pcr cycles consisted of 95°c for 5 minutes pre-denaturation, 30 cycles consisted of 95oc for 1 min denaturation, 55oc for 1 minutes annealing, 72oc for 1.5 min extension, and a final extension for 7 minutes. amplified pcr product was analysed using 1% agarose supplemented with sybr safe by gel electrophoresis for 45 minutes (80 volt/200 watt) and visualised using uv light in a gel documentation machine. subsequently, pcr products were sent to pt genetika science (https://ptgenetika.com/) for sanger sequencing. the obtained sequence data were subjected to ncbi blast database (https://blast.ncbi.nlm.nih.gov/blast.cgi) to assign the closely identity of related bacterial sequence. results and discussion bacterial isolates with antibacterial activities a total of 68 bacterial isolates were selected over 165 bacterial colonies observed on agar plates after 28 days of incubation based on morphological observations. of these 68 isolates, only 22 isolates showed potential antibacterial activities against at least one bacterial isolate using perpendicular streak method. these bacterial isolates were grouped as gram positive cell wall with rod morphology (table 1). table 1. list of isolates that displayed inhibition against at least one bacterial indicator strains with their morphological characteristic. an initial code indicated the dominant mangrove plant species where soil sample was collected namely sa (sonneratia alba), am (avicenna marina), ra (rhizopora apiculata), rm (rhizopora mucronata). isolate morphology gram staining distance against bacterial indicator strains (mm) e. coli k. pneumoniae s. aureus s. mutans sa1 rod positive no activity no activity no activity 7 sa3 rod positive 2 2 2 2 sa4 rod positive 15 10 20 15 sa9 rod positive no activity no activity 5 no activity sa11 rod positive no activity no activity no activity 3 sa14 rod positive no activity no activity 5 no activity am1 rod positive no activity 4 no activity no activity am3 rod positive 2 2 2 3 am4 rod positive no activity no activity 2 no activity am8 rod positive no activity 2 no activity no activity am9 rod positive no activity no activity no activity 3 am12 rod positive no activity 4 no activity 4 am13 rod positive no activity 4 no activity no activity am20 rod positive no activity 3 3 2 am23 rod positive no activity 4 3 5 am30 rod positive no activity 3 3 2 ra1 rod positive 2 1 5 no activity rm2 rod positive no activity no activity 5 no activity rm3 rod positive no activity 5 no activity no activity rm9 rod positive no activity no activity no activity 5 rm10 rod positive 6 no activity 8 7 rm18 rod positive 4 5 3 5 the level of antibacterial inhibition varied among the 22 bacterial isolates. in general majority of isolates weakly inhibited at least one indicator bacterium. two isolates (sa1 and rm10) displayed moderate antibacterial activity. sa4, however, is the only bacterial isolates with the strongest antibacterial potential against all the four bacterial indicator (figure 3). perpendicular streak was selected as the prescreening method because the approach is rather straight forward and has been applied effectively to determine isolates with antibacterial activities (boontanom and chantasari, 2020; balouiri et al., 2016). it could be assumed that isolate sa4 could potentially synthesis active antibacterial molecules based on substantial distance formed between the isolate and bacterial indicators. antibacterial substances produced by an active bacterial isolate were diffused on agar media so that growth of other bacteria was inhibited (balouiri et al., 2016). nucleotide sequences comparison of isolate sa4 against ncbi blast database indicated that the top hit was assigned as bacillus subtilis strain bil/bs-168 with 97,36% identity. bacillus subtilis in general has been regarded for its ability to produce antimicrobial compounds and has been applied for food preservation and crop protection (caulier et al., 2019). for example, bacteriocin is one example of an active compound 132 biology, medicine, & natural product chemistry 10 (2), 2021: 129-133 responsible for antimicrobial activities in b. subtilis (sharma et al., 2018). figure 3. perpendicular streak of isolate sa4 against gram positive and negative bacteria. a clear distance of ≥10 mm between each bacterial indicator and the isolate sa4 indicated strong antibacterial properties synthesized by the bacteria. however, at this stage it is still unknown what type of antibacterial substances that could be produced by the sa4 isolate. further research, therefore should be focused to identify type of antibacterial compounds synthesized by the sa4 isolate. in addition, genetic analysis should also be done to analyze metabolic pathways that are responsible to synthesize antibacterial compounds in the sa4 isolate. despite, the remaining 21 bacterial isolates only showed weak to moderate antibacterial activities based on perpendicular streak do not automatically exclude the full potential of these isolates. it could be that each isolate requires different time to accumulate their antibacterial substances. therefore, a liquid fermentation followed by organic extraction of these isolates using different solvents depending on polarity of the expected compounds should be done to fully unravel the true antibacterial potential or other therapeutic capability. conclusion in conclusion, this study obtained 22 bacterial isolates with antibacterial potential from mangrove soil of the ngurah rai mangrove forest. all of these isolates were characterized by rod shape and gram-positive cell wall under microscope observation. isolate sa4 displayed the strongest antibacterial potential based on the perpendicular streak method with clear distance zone above 10 mm. isolate sa4 was closely related to bacillus subtilis strain bil/bs-168with 97.36% sequence identity based on 16s rrna gene sequences. further studies should be focused to extrapolate the antibacterial potential of these 22 isolates especially the isolate sa4 by performing organic extractions and screening against different bacterial pathogen. apart from antibacterial activities, other therapeutic potential of the obtained isolates should also be explored such as antifungal, antioxidant, or anticancer in order to fully unravel the biological capabilities of these isolates. acknowledgements: this research was fully funded by the research and community development unit (unit penelitian dan pengabdian masyarakat) faculty of medicine and health sciences, warmadewa university under grant no. 417/unwar/fkik/up2m/kp02/vii/2019. we would also thank ir. magdalena hehakaya, m.si from the department environment and forestry bali province for her guidance during the sampling trip at the ngurah rai mangrove forest. conflicts of interest: the authors declare that there are no conflicts of interest. references assis, d. a., rezende, r. p., & dias, j. c. (2014). use of metagenomics and isolation of actinobacteria in brazil's atlantic rainforest soil for antimicrobial prospecting. isrn biotechnol, 2014, 909601. doi:10.1155/2014/909601 azman, a. s., othman, i., velu, s. s., chan, k. g., & lee, l. h. (2015). mangrove rare actinobacteria: taxonomy, natural compound, and discovery of bioactivity. front microbiol, 6, 856. doi:10.3389/fmicb.2015.00856 balai pemantapan kawasan hutan wilayah viii denpasar. (2018). potensi wisata taman hutan raya ngurah rai provinsi bali. in. http://bpkh8.menlhk.go.id/pdf/karya_tulis_mandiri/liflet_tahu ra.pdf. balouiri, m., sadiki, m., & ibnsouda, s. k. (2016). methods for in vitro evaluating antimicrobial activity: a review. journal of pharmaceutical analysis, 6(2), 71-79. doi:https://doi.org/10.1016/j.jpha.2015.11.005 bentley, s. d., chater, k. f., cerdeno-tarraga, a. m., challis, g. l., thomson, n. r., james, k. d., . . . hopwood, d. a. 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(2018). diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in beilun estuary national nature reserve of guangxi, china. front microbiol, 9, 868. doi:10.3389/fmicb.2018.00868 lane, d. j. (1991). 16s/23s rrna sequencing (e. stackebrandt & m. goodfellow (eds.); nucleic ac. john wiley and sons, pp. 115–175. lee, l. h., zainal, n., azman, a. s., eng, s. k., goh, b. h., yin, w. f., . . . chan, k. g. (2014). diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in malaysia. scientificworldjournal, 2014, 698178. doi:10.1155/2014/698178 mohamed, h., miloud, b., zohra, f., garcia-arenzana, j. m., veloso, a., & rodriguez-couto, s. (2017). isolation and characterization of actinobacteria from algerian sahara soils with antimicrobial activities. int j mol cell med, 6(2), 109120. doi:10.22088/acadpub.bums.6.2.5 munita, j. m., & arias, c. a. (2016). mechanisms of antibiotic resistance. microbiol spectr, 4(2). doi:10.1128/microbiolspec.vmbf-0016-2015 roca, i., akova, m., baquero, f., carlet, j., cavaleri, m., coenen, s., . . . vila, j. (2015). the global threat of antimicrobial resistance: science for intervention. new microbes new infect, 6, 22-29. doi:10.1016/j.nmni.2015.02.007 sharma, g., dang, s., gupta, s., & gabrani, r. (2018). antibacterial activity, cytotoxicity, and the mechanism of action of bacteriocin from bacillus subtilis gas101. med princ pract, 27(2), 186-192. doi:10.1159/000487306 singh, v., haque, s., singh, h., verma, j., vibha, k., singh, r., . . . tripathi, c. k. (2016). isolation, screening, and identification of novel isolates of actinomycetes from india for antimicrobial applications. front microbiol, 7, 1921. doi:10.3389/fmicb.2016.01921 van der heul, h. u., bilyk, b. l., mcdowall, k. j., seipke, r. f., & van wezel, g. p. (2018). regulation of antibiotic production in actinobacteria: new perspectives from the postgenomic era. nat prod rep, 35(6), 575-604. doi:10.1039/c8np00012c ventola, c. l. (2015). the antibiotic resistance crisis: part 1: causes and threats. p & t : a peer-reviewed journal for formulary management, 40(4), 277-283. retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25859123 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4378521/ world health organization. (2018). antibiotic resistance. retrieved from geneva: https://www.who.int/news-room/factsheets/detail/antibiotic-resistance this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 77-80 | doi: 10.14421/biomedich.2020.92.77-80 issn 2540-9328 (online) isolation and characterization of stigmasterol from fritillaria roylei gunpreet kaur1,*, vikas gupta1, r. g. singhal2, parveen bansal1 1baba farid university of health sciences, faridkot, india 2shobhit university, meerut, india corresponding author* reetjattana21@gmail.com manuscript received: 26 june, 2020. revision accepted: 06 november, 2020. published: 16 november, 2020. abstract fritillaria roylei (kshirakakoli) is the threatened species of “ashtwarga” group suffers lot of confusion for identification & authentification in ayurvedic system of medicine. due to lack of natural sources and insufficient availability of kshirakakoli, chances of adulteration and substitution increases which in turn leads to loss of faith of people in herbal drugs. thus for identification and differentiation, quality standardization and quality assurance of kshirakakoli containing herbal formulations there is a need to isolate chemical marker compound using advanced analytical techniques. the methanol extract of root samples of plant was prepared and phytochemical screening was performed. marker compound was isolated from the extract using column chromatography. single compound having rf value 0.31 was isolated with tlc by using mobile phase n-hexane: ethyl acetate: formic acid (8:2:0.1 v/v/v) and purified by re-crystallization with methanol. isolated compound was further characterized by using melting point and spectral analysis. the methanol extract was dark brown in color and showed the presence of steroids, amino acids and flavonoids. the isolated compound was found to be white crystalline powder with melting point range of 167-169°c. spectral analysis confirmed the presence of stigmasterol. in present study stigmasterol was isolated for the first time and can be used as chemical marker for identification and differentiation of the plant from its substitutes. keywords: isolation; kshirakakoli; marker; standardization; stigmasterol. introduction ayurveda is one of the most distinctive systems of medicine known to man (jaiswal and williams, 2017; sreena et al., 2011). it is regarded as the oldest divine knowledge in the humankind which is based on the principle of maintaining a balance between the interconnected relations within the body and mind. ayurvedic medicines include herbs, herbal materials, herbal preparations, minerals and finished herbal products. ayurveda is blessed with plentiful speculate medicinal plants including ashtawarga group of eight medicinal plants i.e. kakoli (roscoea purpurea), kshirakakoli (fritillaria roylei), jeevaka (microstylis muscifera), rishabbhaka (malaxis acuminatea), meda (polygonatum cirrhifolium), mahameda (polygonatum verticillatum), riddhi (habenaria edgeworthii) and vridhi (habenaria internedia) (balkrishna et al., 2012; saha d et al., 2015). in recent days, herbal medicines are getting more popular with the comprehensive movement of people towards natural therapies. this increasing demand of the population towards herbal medicines results in shortage of authentic raw materials leading to increase in chance of adulteration and substitution because the regulatory authorities lack the strict quality control measures of herbal medicines (shukla and dhanya, 2017). this same situation happens with plants of ashtawarga group. according to international union of conservation of nature (iucn) and conservation assessment and management plan (camp), fritillaria roylei (kshirakakoli) is considered as threatened medicinal plant (saha et al., 2015; iucn 2001; kuniyal et al., 2015). from various studies, it has been found that due to lack of natural sources and insufficient availability to meet the requirements of market for the raw material the department of ayush, govt. of india, permitted the use of available substitutes in place of original plant. the substitution of fritillaria roylei is done with ashwagandha (root) (withania somnifera) or safed musli root (chlorophytum arundinaceum boker) (balkrishan et al., 2012; sagar, 2014). however, literature survey reveals that ayurvedic parameters as well as pharmacological actions of the ashtawarga plants do not match with their substitutes. the substitute of f. roylei that is w. somnifera or c. arundinaceum shows 33% or 16% similarity which ultimately results in reduced efficacy of the drugs along with loss of faith of people towards use of herbal drugs (virk et al., 2015). thus, drug standardization and quality assurance is essential to assess the safety, efficacy and quality of herbal drugs. lack of chemical markers is a major problem for the https://doi.org/10.14421/biomedich.2020.92.77-80 78 biology, medicine, & natural product chemistry 9 (2), 2020: 77-80 regulatory authorities for enforcement of quality regulations. therefore, the study was designed to isolate chemical markers from fritillaria rolei (kshirakakoli) using advanced analytical techniques. materials and methods chemicals in the present study all the reagents and solvents used were of analytical grade. for isolation of marker compound, precoated aluminum-backed tlc plates with 0.2 mm layer of silica gel 60 f254 (20 cm × 10 cm) manufactured by e. merck (germany) were purchased from local authorized dealer. plant material the root samples of fritillaria roylei were procured from cultivar source of himachal pradesh and authenticated by national botanical research institute, lucknow wide authentication letter no. nbri/cif/535/2017 dated 04/01/2017. root samples of the plant were washed, shade dried and stored in air tight container. extract preparation the shade dried roots of fritillaria roylei (500gm) were coarsely powdered and defatted with petroleum ether followed by continuous hot extraction process with methanol. the methanol extract thus obtained was filtered and then evaporated to obtain a concentrated semisolid mass. the final extract thus obtained was then stored in vacuum desiccator for further use. phytochemical screening preliminary phytochemical screening of the extract were performed for the detection of phytoconstituents like alkaloids, flavonoids, steroids, proteins, tannins, saponins, phenolics, carbohydrates and amino acids (banu and cathrine, 2015; kokate et al., 2000; tiwari et al., 2011). isolation of chemical marker about 8.4g of methanol extract was mixed with methanol and then silica gel having pore size 60–120 mesh was added to form slurry which was then dried on water bath to form a free flowing powder. silica gel (675g) suspended in n-hexane was poured into the glass column having dimensions 1000mm x 50 mm to give rise to silica bed. saturated silica bed was allowed to stand overnight for uniform bed packing. after 12hrs, elution was started with non-polar solvent n-hexane followed by an increase in polarity of the solvents and fractions were collected with optimum flow rate of 10ml/min. thin layer chromatography (tlc) of collected fractions were performed using different solvents selected by hit and trial method and on the basis of tlc profile similar fractions were pooled together. elution with the solvent system n-hexane: chloroform: ethyl acetate 60:20:20 yielded a pool of two compounds with rf 0.31 and 0.42 on tlc plates by the use of mobile phase n-hexane: ethyl acetate: formic acid (8:2:0.1 v/v/v) along with mixture of other compounds on tlc plates. single compound was isolated by cutting and pooling of tlc plate of compound having rf value 0.31 and purified by re-crystallization with methanol. the fraction was kept in a refrigerator to get the crystallized compound (virk et al., 2016; cannell, 1998). characterization of isolated compound isolated crystallized compound was characterized by using different chemical test, melting point and spectral analysis (ir, nmr, mass, and uv spectroscopy). figure 1. nmr spectra of isolated compound stigmasterol from fritillaria roylei (kshirakakoli). kaur et al. – stigmasterol from kshirakakoli 79 result and discussion physical evaluation and percentage yield of extract the methanol extract of fritillaria roylei was dark brown in color, and the percentage yield of extract was 7.48%. phytochemical screening of extract preliminary phytochemical analysis confirmed the presence of steroids, amino acids, flavonoids and proteins characterization and identification of isolated compound isolated compound was found as white crystalline powder after crystallization from chcl3-meoh. the compound was shown positive test of steroids. melting point of the compound was found to be 167-169°c (lit. 164-171°c). spectroscopic data ir: the infra-red spectra of isolated compound, showed very intensely broad peak at 3428 cm-1 and moderately intense peak at 1192 and 699 cm-1 were observed for the o-h bond vibrations of hydroxyl group. the unsaturated part of c-h and c=c vibrations was observed at 881 cm1 and 1642 cm-1. the vibrations of -ch3 were observed at 2937 cm-1and at 1465 cm-1 and vibrations of =ch2 were observed at 2852 cm-1 and at 1460 cm-1 respectively. the c-c vibration peak was shown at 1053 cm-1which confirm the structure of stigmasterol. nmr: in the 1h-nmr spectrum of isolated compound showed two olefenic protons appeared downfield at δ 4.57 (m) and δ 4.14 (m) which were identical with the chemical shift of h-22 and h-23, respectively of stigmasterol. six methyl protons at δ 1.23(s, 3h), δ 1.19(s, 3h), δ 1.06(s, 3h), δ 1.00, δ 0.98(s, 3h) and δ 0.91 (s, 3h); [(3h each, s, ch3)] confirms the structure of stigmasterol. mass spectra: mass spectrum of isolated compound showed parent molecular ion [m+] peak at m/z 412 which was being in agreement with the proposed structure of stigmasterol. structure and molecular formula of isolated compound the molecular formula of isolated molecule stigmasterol is c29h48o that is confirmed by ir, mass spectra and nmr data. its iupac name is (3s,8s,9s,10r,13r,14s,17r)-17-[(e,2r,5s)-5-ethyl-6methylhept-3-en-2-yl]-10,13dimethyl,2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro1h-cyclopenta[a] phenanthren-3-ol. as phytosterols are not synthesized by humans and animals so they are needed to be included in the diet. stigmasterol is present in various naturally occurring items of plant origin like vegetables, nuts, seeds and unpasteurized milk. edible oils contain higher amount of stigmasterol than vegetables. it is widely used as food additives in food and beverage industries for production of sausages, yogurt, cold cuts, margarines, bakery products, milk and spreads (aboobucker et al., 2019; jun-hua et al., 2008; siddiqui et al., 1990). stigmasterol is also used as a precursor or intermediate for synthesis of various human steroid hormones like progesterone, androgen corticoids, estrogens etc (sundararaman and djerassi, 1977; hogg, 1992). stigmasterol is used as raw material in pharmaceutical industries for commercial synthesis of vitamin d3 (kametani and furuyama, 1987). in addition to this various studies showed that stigmasterol possesses various pharmacological properties like cytotoxic activity, antioxidant activity, anti-hypercholestrolemic, anti-osteoarthritic activity, anti-inflammatory activity, hypoglycemic activity etc. high therapeutic value and regulatory approval status as generally recognized as safe (gras) in the u.s., followed by an approval from the fda and by the eu scientific committee on food (sfc) increases the demand of phytosterol significantly (ghosh et al., 2011; chandler et al., 1979; batta et al., 2006; gabay et al., 2010; panda et al., 2009; navarro et al., 2001). as the market price of stigmasterol is 32,334/10g (approximately) and it will be difficult for commercial manufacturers to replace fritillaria roylei plant with stigmasterol just to claim the presence of fritillaria roylei (kshirakakoli). presence of stigmasterol has not been ever reported in its substitutes/adulterants. it has been reported first time from root of kshirakakoli plant. thus, for quality standardization of kshirakakoli containing formulations isolated compound i.e. stigmasterol may be used as chemical markers for identification and differentiation of authentic plants from its substitutes and common adulterants. conclusion in the present study, stigmasterol was isolated from roots of fritillaria roylei using column chromatography and tlc. as per the knowledge, this is first report on chromatographic method of isolation of stigmasterol from natural source that is fritillaria roylei plant. this compound can be used as a chemical marker by regulatory authorities for identification of kshirakakoli plant from other substitutes. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. source of funding: the authors gratefully acknowledge authorities of baba farid university of health sciences, faridkot for necessary permission and providing all research facilities. this work was supported by the national medicinal plants board, ministry of ayush, govt. of india, new delhi, india [grant numbers r&d/ch-01/2012]. 80 biology, medicine, & natural product chemistry 9 (2), 2020: 77-80 references aboobucker, s. i., suza, w. p. 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(2015). lack of pharmacological basis of substitution of an endangered plant group ashtawarga–a significant ingredient of polyherbal formulations. am j phytomed clin ther., 2 690712. biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 25-30 | doi: 10.14421/biomedich.2014.31.25-30 identification of migratory birds and their spesific characteristics of habitat in the salt water lake of gili meno, north lombok distric diah purwitasari1, luh gde sri astiti1,2 and supriadi1* 1faculty of veterinary medicine, university of west nusa tenggara, mataram, indonesia 2research institute for agricultural technology west nusa tenggara, indonesia author correspondency*: supriadi@yahoo.com abstract the aim of this research was to identify the species of migratory birds in the ecosystem of salt water lake of gili meno and their specific characteristics of habitat. data collection for birds, mangrove and fish species has been carried out in september and october 2013. in this study, a shannon-wiener diversity index and importance value index (ivi) of mangrove vegetation were calculated to identify carrying capacity of mangrove population in the form of specific habitat in salt water lake of gili meno. this research has identified 17 species of birds which are divided into 5 families: scolopacidae, charadriidae, ardeidae, meropidae and alcedinidae. moreover, 3 species of mangrove were discovered that are a. marina, a. lanata and e. agallocha, as well as one species of fish (mujair fish/o. mossambicus). n. nycticorax and b. striatus were well-known to have higher population than other bird species. the diversity index showed that mangrove vegetation in the ecosystem of salt water lake of gili meno has a low species diversity (0.565). this is due to higher dominance of one species than the others. what is more, the ivi of a. marina demonstrated a fairly significant value compared to that of other species (189,01). mangrove vegetation which surrounds the ecosystem of salt water lake of gili meno has formed a unique habitat and and an ideal stopover site for migratory birds. the ecosystem not only provides shelters from predators but also supplies for various abundant feeding sources. the lake it self is rather shallow and muddy around the shore which gives advantages for the migratory birds to obtain plenty small fish from the lake. keywords: migratory birds, salt water lake, gili meno, characteristics of habitat introduction indonesia is the fifth country after colombia, peru, brazil and ecuador which have the highest megabiodiversity of birds, as many as 1539 species (sudaryanto, 2006). those species are divided into 20 order and 90 families (yamin and jamaluddin, 2002) and some of them are endemic (around 381 spesies). the increasing habitat degradation has led to the fact that nearly 400 species of birds are now threatened with extinction (thinh, 2006). one of bird groups that are interesting to study is migratory birds. this group is a bird group that move from one region to another, either inter-island or intercontinent, and will return to their origin (shackelford et al., 2005). this migration occurs because of seasonal change or the birds feeding habits in a relatively far distance. small islands in indonesia provide habitats for a variety of migratory bird species to stopover. habitat characteristic of various ecosystems in each island possesses its own specific and unique characteristics. often, these migratory birds can attract tourists visiting the islands (aeny, 2004). one of small islands (lombok: small island = gili) that exist around lombok island is gili meno. this small island is one of three islands included in gili matra village. gili meno is well-known for its specific and unique salt water lake (rachman, 2004) and has a pleasing panorama. moreover, its ecosystem also saves a great potential of various exotic animal species. research conducted by setiawan (2008) explained that no less than 11 species of migratory birds use the habitat to stopover in migration season. some of them are phalacocorax melanoleucos, actitis hypoleuco, ardea purpurea and charadrius dubius. to date, several researches have been carried out to identify many migratory bird spesies in the ecosystem of salt water lake of gili meno, one of them is by setiawan (2008). however, in the research, field observation was only performed once and the researcher has not done a study of microhabitat and factors that make the salt water lake as one of preference habitat for migratory birds. therefore, this research was conducted to identify the diversity of migratory bird species in the ecosystem of salt water lake of gili meno and recognize the habitat characteristics that could support the wild birds’ lives in the ecosystem. materials and methods the objects of this research were migratory birds utilizing the lake as their activity place. this research was undertaken in september and october 2013. the first data collection was performed on the 24th-26th of september and the second data collection was completed on the 18th20th of october 2013. data collected were bird species, mangrove vegetation and fish species. data collection of bird species was conducted on field in the morning from 07.00-10.00 and in the afternoon from 15.00-17.00. the observation was performed in 4 26 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 spots which were determined purposively on field. bird species were observed and identified by directly observing morphological features and characteristics of the birds based on a guide to the birds of wallacea (coates and bishop, 2000). morphological features observed included: (i) shape and size of body, beak and feet, (ii) the colours of feather on body, beak, and feet, (iii) visible specific features, as well as (iv) sounds produced. analysis of mangrove vegetation was performed on sampling spots that have been directly marked by a gps device. the vegetation analysis was undertaken by making 10 x 10 m quadrants. obtained vegetation data included vegetation species, trunk diameter, and the individual number of each species. furthermore, fish were caught utilizing a fish net in cooperation with local community. the vegetation data were then analysed by calculating density, frequency, dominance, ivi, and shannon-wiener diversity index (h’). results and discussions a total of 17 species belonging to 5 families have been identified in this research. the five bird families are scolopacidae, charadriidae, ardeidae, meropidae dan alcedinidae. of the 17 identified species, 16 species are migrants (table 1). the non-migrant bird and inhabitants of gili meno is the species of halcyon sancta. black-crowned nirgt-heron (n. nycticorax) is a species with the highest population identified. as stated by coates and bishop (2000), this species is a migratory species that reproduce in their migration sites. on several observation spots in the vicinity of mangrove forest, their active and already inactive nests were discovered. in addition to that, it was observed that striated heron (b, striatus) was also on fairly high population number. both bird species are known to prefer similar habitat which is mangrove area, muddy sites and shallow puddle (zulfan, 2009; setiawan 2008; mustari, 1992). these species difference is in their feeding activity. nycticorax nycticorax was more often spotted perched on tree branches and once in a while went down to catch small fish. meanwhile, b. striatus was more active and often walked around the lake to find preys. this research discovery is supported by previous research conducted by setiawan (2008) who also discovered both bird species in high individual number. it was further stated that mangrove ecosystem in salt water lake of gili meno could highly sustain the existence of the two species population. additionally, food availability in the vicinity of lake and the lake’s proximity to adjacent beach also provided more varied habitat choices for these birds. other fairly numerous migratory bird species discovered during this research were those belong to trinil category i.e: common sandpiper (actitis hypoleucos), wood sandpiper (tringa glareola), marsh sandpiper (tringa stagnatilis) and common redshank (tringa tonatus). detailed observation has successfully identified the four species clearly. detailed observation was necessary because occasionally, these four species were in large group (>10). the observation of these bird species was basically simple to perform due to significant differences in size and features of each species. nevertheless, a. hypoleucos and t. glareola were often found feeding in pairs or individual on the lake shore. figure 1. documented bird species. a. cerek jawa (charadrius javanicus chasen, 1938); b & c. cerek besar (pluvialis squatrola); d. trinil semak (tringa glareola linnaeus, 1758); e. kokokan laut (butoroides striatus); f. kuntul kecil (white) (egretta garzetta), trinil pantai/small brown (actitis hypoleucos). another migratory bird category is cerek including little ringed plover (charadrius dubius), javan plover (c. javanicus) and grey plover (pluvialis squatarola). two of the three species (c. javanicus and pluvialis squatarola ) have not been reported in the study conducted by setiawan (2008), this might be because of different observation time. according to coates and bishop (2000), both species are migratory birds and do not reproduce so that sometimes they are not observed in certain time. it was further explained that the three species prefer shallow and calm mangrove habitat, natural habitat and far from human activity. this result demonstrate that the thre migratory species presence can be utilized as habitat indicator that salt water lake of gili meno can support the lives of wild animals. mulyawati (2007) also reported that this bird group likes muddy mangrove area and shallow water. the next bird category observed was kuntul comprising 3 species: little egret (egretta garzetta), reef egret (e. sacra) and intermediate egret (e. intermedia). the population of e. garzetta was the diah purwitasari, et al. – identification of migratory birds and their spesific characteristics … 27 highest of the three species with the highest individual number observed on field was 16 individuals. this species occupied almost half of the lake’s shore around 08.00-09.00 and 15.00-17.00 when they are actively feeding. on the other hand, e. intermedia was only found 4 individuals at the most during the research. posture, larger body size and typical beak colors (bright yellow) appears to distinguish the two species. unlike e. garzetta and e. scara, e. intermedia often becoming active between 09.00-10.00 and sometimes it was not observed in the afternoon. meanwhile, e. sacra was rarely seen lingering around the lake. this may be due to food competition on the lake shore. the species was mostly spotted on the beach shore at low tide in the afternoon then returned to the mangrove ecosystem afterward. table 1. birds species discovered in salt water lake ecosystem of gili meno. family indonesia english latin scolopacidae trinil pantai common sandpiper actitis hypoleucos trinil semak wood sandpiper tringa glareola trinil kaki merah common redshank tringa tonatus trinil rawa marsh sandpiper tringa stagnatilis gajahan erasia eurasian curlew numenius arquata charadriidae cerek kalung kecil little ringed plover charadrius dubius cerek jawa javan plover charadrius javanicus cerek besar grey plover pluvialis squatarola ardeidae cangak merah purple heron ardea purpurea kuntul perak intermediate egret egretta intermedia kuntul kecil little egret egretta garzetta kuntul karang reef egret egretta sacra kowak malam abu black-crowned nirgt-heron nycticorax nycticorax kowak malam merah rufous nigtt-heron nyicticorax caledonicus kokokan laut striated heron butoroides striatus meropidae kirik-kirik laut blue-tailed bee-eater merops philipinnus alcedinidae cekakak suci sacred kingfisher halcyon sancta other bird species that were observed around the lake were sacred kingfisher (halcyon sancta) and bluetailed bee-eater (merops philippinus). sacred kingfisher often noticed on several spots on the lake shore to catch small fish. decreasing lake water has led to higher salinity causing small fish to surface, so that it was easier for the birds to catch their prey. occasionally, sacred kingfisher produced strong sounds when they flew or met the same species. in contrast, m. philippinus was more aereal. this species make use of mangrove habitat as feeding site which provide abundant insects. mangrove ecosystem is an integral part of the ecosystem of salt water lake of gili meno. this mangrove ecosystem provide excellent microhabitat for many wild animals, especially migratory birds. as stated by elfidasari and junardi (2006), one of many functions of mangrove is as nesting and stopover sites for various bird species. healthy mangrove ecosystem is more preferred and visited by many bird species as it provide huge potential as food sources. mangrove ecosystem in gili meno consist of three mangrove species: avicennia lanata, avicennia marina dan exoecaria agallocha. the calculation of species relative dominance showed that a. marina dominate the habitat in the ecosystem of the salt water lake (69.45%), followed by a. lanata (23.81%) and e. agallocha (6. 74%). moreover, relative species density index also illustrated a fairly dense coverage of a. marina. in contrast, species diversity index (0.565) demonstrated the low species diversity in the mangrove ecosystem because of a. marina dominance. in addition to plant resources, the habitat of salt water lake of gili meno also offer animal resource for migratory bird feeding. the mangrove ecosystem ability to maintain the stability and fluctuation of salinity and temperature causes fresh water species capable to adapt in the saltwater habitats. from the results of fishing using fishing nets and rods it was found that only one species of fish inhabit the whole habitat of the salt water lake. the identified species is mujair fish (oreochromis mossambicus). as indicated by kosztowny et al (2008), this fish species can adapt well in salt water from juvenile. this is becaue of its ability to activate atpase anzyme to maintain cell membrane activity, especially on the gill rackers to keep active despite the high salinity. further research to identify other animal species which is potential as feeding resource for migratory birds is of high importance. moreover, research on the histological and anatomic differences of mujair fish from salt water and fresh water is required. 28 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 figure 2. coverage shape and characteristics of mangrove a. avicennia marina with close coverage along the lake shore b. oval leaf tip shape with white lower leaf surface is the diagnostic character of a. marina. the calculation of importance value index (ivi) showed that the most important species was a. marina. this species was discovered almost in all ecosystem area around the salt water lake. this was supported by the calculated diversity index of shannon-wiener which showed the low diversity of mangrove vegetation in the ecosystem. in other words, the ecosystem is dominated by a. marina. the existence of mangrove vegetation in a habitat will form a spesific ecosystem and provide abundant resources. davies et al., (1996) stated that mangrove ecosystems have important roles in sustaining the lives of animals on coastal line and terestrial. mangrove vegetation in the ecosystem of salt water lake of gili meno also provide abundant feeding resource for migratory birds like small insects and fish. this is also supported by abdurakhman (2002) research who discovered that food availability in a habitat will be more frequently visited by animals (burung). yamin and jamaludin (2002) also explained that excellent vegetation will provide diverse resources to sustain bird lives in a habitat. dominant bird species observed in this study is blackcrowned nirgt-heron (n. nycticorax) and striated heron (b. striatus) which are migratory birds that reproduce in their migration sites (coates and bishop, 2000). both species made many nests on dense mangrove tree canopy. these species have successfully dominated the ecosystem of salt water lake of gili meno may because both species can obtain their suitable life requirements such as food, nesting place and shelter from predators. other migratory birds which belong to trinil category (a. hypoleucos and t. glareola) often spotted fly actively and walked along the shallow lake shore to find food. both species are different from other two species from the same group (t. stagnatilis and t. totanus). t. stagnatilis and t. totanus are relatively not very active and often observed in groups. the high availability of small fish and water insects on the surface of the lake make the lake habitat is mostly preferred by many migratory bird species. the lake ecosystem which almost 90% covered by mangrove vegetation contribute to the ideal condition of the habitat for migratory birds to stopover. the fairly dense mangrove vegetation which surround the salt water lake ecosystem has formed a characteristic and sustainable habitat for many wildlife including migratory birds. habitat with dense canopy and which tend to be wet is more favored by birds (mulyawati, 2007; anonima, 2000). additionally, a relatively shallow, sandy and muddy area is a preferred area for migratory bird species. this research discovered that the migratory birds were more concentrated on the southern area of the lake. this area is relatively shallow, muddy to sandy, has denser mangrove coverage and more importantly, is far from road access (human activity). birds distribution map that has been prepared cleary shows that the main characteristic of preferred habitat of migratory birds is habitat with wet, shallow, sandy to muddy land, as well as dense vegetation canopy and which is far from human activity. table 2. data analyses of mangrove vegetation biodiversity. name sum value indonesia latin rden rf rd ivi h’ api-api putih avicennia marina 23 22,55 42,11 23,81 88,46 0,565api-api avicennia lanata 79 77,45 42,11 69,45 189,01 buta-buta exoecaria agalocha 8 7,84 15,79 6,74 30,37 explanation: rden : relative density rf : relative frequency ivi : importance value index h’ : shannon-wiener diversity index diah purwitasari, et al. – identification of migratory birds and their spesific characteristics … 29 figure 3. distribution map of migrant birds in a salt water lake of gili meno. from this map, it is clear that migratory birds (red spots) are more active in the south of the lake. this is supported by the shallow banks of the, dense mangrove coverage and the far location from human activity. natural and undisturbed habitat is more favored by migratory birds. habitats with high canopy and dense mangrove coverage provide nesting places, protection, nursing site and suitable feeding resources. shallow and sandy to muddy areas can facilitate birds to find food especially small fish. in addition, areas that are far from road access and human activity, are safer and unstressful. these characteristic factors are specific for the habitat of salt water lake of gili meno which in turn make it a favored place to stopover during migratory season. new species observed in this habitat which was undiscovered in previous research was javan plover (charadrius javanicus). this species has been reported to migrate along the shoreline of sumatera, kalimantan, java as well as bali and just reported in 2011 to stopover in several habitats in west nusa tenggara (anonimc, 2013; mckinnon, 1991). this may because of decreasing quality of habitats in western part of lombok island so that the species chose healthier habitat to migrate to. the presence of this migratory bird also indicates that the ecosystem of salt water lake of gili meno is still in a good condition and therefore should be protected. overall, bird species discovered during this research were higher in number than those observed by setiawan (2008) in august 2008. it is assumed that different observation time take part in the different result from this research. however, this research did not discovered pecuk padi belang (phalacocorax melanoleucos) which were present in setiawan’s research. these data should be updated to identify whether habitat change (habitat degradation) or migration season and observation time is the determining factor which affect the absence of several bird species. conclusion there are 17 bird species identified in this research belonging to 5 families i.e. scolopacidae, charadriidae, ardeidae, meropidae and alcedinidae. of the 17 species discovered, 16 species were migratory birds. in addition, this research also uncovered the especially high population of n. nycticorax followed by b. striatus. this study also identified 3 species of mangrove: avicennia marina, a. lanata and exoecaria agallocha as well as one species of fish (mujair fish/oreochromis niloticus). the existence of the fairly good mangrove vegetation and sufficient food availability contributed to the ideal condition of the habitat of saltwater lake of gili meno as migration site for birds during migration seasons. acknowledgment we express our gratitude to the birdwatching group in gili meno who supported us in fieldwork and provided us more information about several mangrove species. we also thank mr. andy setiawan who has provided his assistance in the indentification of birds’ population and fieldwork documentation. all parties who cannot be all mentioned in this article is also acknowledged for their support during field study and data analyses. petadistribusiburungmigran padaekosistem danaugili meno legenda : s n ew insert : ## ## ## ## ## ## ## ## ## ## ## 395640 395640 395820 395820 396000 396000 90 76 86 0 9076860 90 77 04 0 9077040 ; r ; r r r r r r r 1 2 3 ; 4 5 6 7 8# # # # # # # # # # # ## # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # ; r # : belukar : danau : kebun : tegalan : berugaq : lokasi anveg : distribusi burungmigran # # # # # # # # # # # # # # # gilimeno s n ew 0 60 meters 30 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 references abdurakhman. 2002. stratifikasi penggunaan habitat berbagai jenis burung di sekitar danau gili meno-lombok barat. skripsi fkip jurusan biologi universitas mataram. mataram. aeny, w. 2004. keanekaragaman jenis dan pemanfaatan jenis tumbuhan oleh burung liar di kota mataram. skripsi fkip jurusan biologi universitas mataram. mataram. anonima. 1992. ensiklopedia indonesia seri fauna burung. pt. intermasa. jakarta. anonimb. 2006. jenis-jenis burung dilindungi yang sering diperdagangkan. http://www.kehati.or.id/news/data/8,%20)kt2000%20info %20kehati.pdf. downloaded on 19/11/2012. 10.30 wita. anonimc, 2013. burung-burung di kawasan indonesia. www.kutilang.or.id coates, b. j., dan bishop, k. d. 2000. panduan lapangan burungburung di kawasan wallacea. bird life internationalindonesia programme & dove publications pty. ltd. davies, j., g. claridge, dan c.h.e. niranita. 1996. manfaat lahan basah dalam mendukung dan memelihara pembangunan. bogor: direktorat jendral phpa & asian wetland bureau. elfidasari, d dan junardi. 2006. keanekaragaman burung air di kawasan hutan mangrove peniti kabupaten pontianak. biodoversitas. vol.7 no.1, pp: 63-66. kusmana, c. 1997. metode survei vegetasi. pt. institut pertanian bogor. bogor. kosztowny, a.l., t. hirano and e. g. grau. 2008. developmental changes in na+ , k+ -atpase activity in mozambique tilapia (oreochromis mossambicus) embryos and larvae in various salinities. international symposium on tilapia in aquaculture. mackinnon, j. 1991. panduan lapangan pengenalan burungburung di jawa dan bali. gadjah mada university press. yogyakarta. mulyawati, d. 2007. migrasi burung. buletin burung iii/edisi januari 2007. bogor. mustari, a.h., 1992. jenis-jenis burung air di hutan mangrove delta sungai cimanuk indramayu-jawa barat. media konservasi. vol. iv; pp: 39-46. noor, y. r., m. khazali dan i. n. n. suryadiputra. 2006. panduan pengenalan mangrove di indonesia. phka/wi-ip. bogor. rachman, r. 2004. identifikasi potensi wilayah nusa tenggara barat. percetakan sahabat. mataram. setiawan, a. 2008. karakteristik habitat burung di gili meno desa gili indah kecamatan pemanang kabupaten lombok barat. skripsi mipa jurusan biologi universitas mataram. mataram. shackelford, c. e., e. r. rozenburg, w.c. hunter and m.w. lockwood. migration and migratory birds of texas 4th edt. texas parks and wildlife pwd, usa. sudaryanto. 2006. birdwatching. accessed from 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[skripsi]. institut pertanian bogor, bogor. [indonesia]. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 1-5 | doi: 10.14421/biomedich.2021.101.1-5 issn 2540-9328 (online) the effectiveness of giving plant pgpr rhizosphere bamboo on cocoa seeds germination at the nursery level muhammad yusril hardiansyah*, yunus musa, abdul mollah jaya department of agronomy, faculty of agriculture, universitas hasanuddin, jl. perintis kemerdekaan km. 10 tamalanrea indah, makassar, indonesia. corresponding author* yusrilhardiansyah1@gmail.com manuscript received: 27 september, 2020. revision accepted: 26 june, 2021. published: 01 july, 2021. abstract the low productivity of cocoa plantations in indonesia is partly due to the low quality of seeds, which refers to the impeded growth of cultivated cocoa nurseries. seed is the initial growth of plants so the importance of giving special treatment to seeds will refer to better seed growth. provision of plant growth promoting rhizobacteria (pgpr) microbes can produce indoleacetic acid (iaa) in plants to improve the quality of plant growth. this study aims to determine the effectiveness of the provision of plant growth promoting rhizobacteria bamboo rhizosphere against cocoa seed germination. the study was carried out in the farmer group garden, gantarangkeke district, bantaeng. this study was arranged in the form of a two-factor factorial design (f2f) in a randomized block design (rbd). the use of cocoa seed type as the first factor consisted of gtb (gantarangkeke bantaeng) local cocoa seed and mcc 01 cocoa seed and seed immersion treatment at pgpr rhizosphere bamboo concentration as the second factor consisting of 0% (control) concentration, 5%, 10 % and 15%. the results obtained indicate that administration of seeds with bamboo rhizosphere pgpr affects the germination (100.00%), the speed of seed growth (7.14%/etmal), as well as on abnormal seeds (10.00%). so that the provision of bamboo rhizosphere pgpr on cocoa seeds has an effective influence on seed germination and cocoa seedling development. keywords: cocoa seed; plant growth promoting rhizobacteria; seedbed. introduction the soaring increase in world cocoa demand is due to the increasing demand for cocoa beans which is getting higher every year. the annual world demand for cocoa reaches 6.7 million tons and only 2.5 million tons can be met. this means that there are still more than 4 million tons to meet the increasing market needs, so that this can still be an opportunity for indonesia, especially south sulawesi (yusniar, 2013). cocoa (theobroma cacao l.) is a strategic commodity which ideally is able to play a maximum role in increasing farmers' income in indonesia. this can happen because cocoa has always had a good price development in meeting market needs. however, the reality is that even though every year there is an increase in the planting area, it cannot increase production. the average farmer production is only 1/5 of the potential productivity of the cocoa plant, which ranges from 1.2 3 tonnes/ha (junaedi, et al., 2017). of course, these results are still far from expectations because of the many obstacles that occur in the cocoa cultivation process. one of the factors thought to be the main cause of the low productivity of cocoa plantations in indonesia is the low quality of seeds which refers to the inhibition of growth in the vegetative phase of cultivated cocoa (mertade & basri, 2011; sugiharti, 2006). in general, farmers in indonesia cultivate cocoa using seeds derived from beans. although generative seeding using seeds is easy to do and can be produced in large quantities at low cost, seedlings of cocoa using seeds will produce genetically non-uniform plants. this is because cocoa is a cross-pollinating crop (li et al., 1998). this statement indicates the emergence of a problem currently being faced, namely whether to increase cocoa production to meet cocoa needs in the future, while the availability of good quality plant vegetative phase conditions is currently difficult to obtain. cultivation of cocoa starts from the nursery stage or the vegetative phase and from this stage will determine the success of plants in the generative phase. during this nursery period, good quality seeds must be obtained so that they will be used as a benchmark for optimum growth and produce fruit and good quality cocoa beans in the future. of course, in order to get quality seeds, the selection or use of good seeds in germination will be a major factor. one way to get good seeds is by soaking or soaking the seeds. soaking is a hydration activity slowly before the seeds are germinated so that the potential of seed water reaches a balance to activate metabolic activities in the https://doi.org/10.14421/biomedich.2021.101.1-5 2 biology, medicine, & natural product chemistry 10 (1), 2021: 1-5 seeds to improve germination and seed growth (rouhi et al., 2011). the immersion treatment can be combined with the provision of biological agents capable of improving the quality of seed germination, for example with microbes that are capable of producing growth hormones in plants. one of the microbes that can be used is the microbes that come from plant growth promoting rhizobacteria (pgpr) as a hormone for plants that can provide more nutrients to spur plant growth in the vegetative phase of plants. the use of pgpr in this plant is proven from several research results that have been conducted. the results of research by baihaqi, et al. (2018) show that the use of pgpr on cucumber plants can increase plant growth from 68.6 to 77.7% compared to without pgpr. other research on plantation crops, namely the results of research from widyaningrum (2017) shows that the application of pgpr can increase root length, number of roots, number of leaves, plant height, total dry weight, shoot root ratio, seed steadfastness and robusta coffee seed quality index. in the rhizosphere or bamboo roots there are pseudomonas flourenscens bacteria and bacillus polymixa bacteria which can help the decomposition process (decomposer). the bamboo root pgpr bacteria can secrete a liquid that can dissolve minerals so that they become available nutrients, break down and decompose organic matter (decomposition of organic matter) into plant nutrients. in addition, the pseudomonas fluorenscens and bacillus polymixa bacteria can release enzymes and substances that are useful to stimulate plant growth and release antibiotics that can inhibit the growth and development of pathogenic microbes (microbes that cause disease) (efendi, 2011). research on the presence and diversity of microbes in the rhizosphere of bamboo plants has been conducted by several previous researchers. according to baharuddin et al. (2007) found antagonistic bacteria such as pseudomonas flourences, bacillus subtilis and streptomyces in the roots of healthy bamboo plants. meanwhile, research conducted by asniah (2013) showed that the inoculation of fungi from bamboo root soil into the nursery soil had a significant effect on increasing the wet weight of broccoli plants. this study aims to obtain data and information on the effectiveness of giving plant growth promoting rhizobacteria rhizosphere of bamboo against cocoa seed germination. information from the results of this study is expected to be a solution for cocoa farmers in improving their cultivation systems, especially in the nursery phase. methods this research was conducted at the talaka farmer group garden, gantarangkeke district, bantaeng regency. this research took place from august to november 2019. the tools used were 30 liter buckets, hoes, shovels, scissors, knives, machetes, rulers, meters, calipers, ph meters, carts, analytical scales, sprayers, scales, jerry cans, funnels, aqua plastic bottles, hoses, stoves, pans, paranets, cloths, plastic cups, brown envelopes, millimeter paper blocks, cameras, signage, matches, and writing instruments. the materials used were soil samples from the research location, local gtb (gantarangkeke bantaeng) cocoa seeds, mcc 01 clone cocoa seeds, bamboo rhizosphere (bambusa blumeana), molasses, bran, raw shrimp paste, rice water, water, husk charcoal, sawdust, clear plastic, dab of soap, polybags 12 x 17 cm, label paper, dolomite and label paper. the study was conducted using a two-factor factorial design (rf2f) in a randomized block design (rbd) as an environmental design. this research consists of 2 factors. the first factor is the type of cocoa seed (b) which consists of: local cocoa seeds gtb (gantarangkeke bantaeng) (b1) and cocoa seeds clone mcc 01 (b2), while for the second factor is the initial soaking of seeds with pgpr solution for cocoa seeds. with various concentrations of seed priming (k) consisting of: 0% concentration (0 ml of pgpr solution or 1000 ml of water), 5% concentration (50 ml of pgpr + 950 ml water), 10% concentration (100 ml of pgpr + solution 900 ml of water) and a concentration of 15% (150 ml of pgpr solution + 850 ml of water). the flow of this research includes making a plant growth promoting rhizobacteria solution which is carried out by taking bamboo rhizosphere then growing it by fermentation process in a container for 14 days, preparing the seeds by selecting seeds from mother pods of mcc 01 and gtb cocoa then separating the placenta from the seeds. giving of pgpr solution to cocoa seeds (initial soaking of cocoa seeds) simultaneously according to the treatment for 18 hours (ratnawati et al., 2013), preparation of sprouts media using sterile cloth and sawdust, planting in seedbeds (seedbeds), maintenance and observation. observation parameters in this study are as follows: 1. germination capacity (%), measurement based on observing the number of normal germinated seeds marked with cotyledons on the raised seed. calculation of germination that is on day 7 and day 14 (deptisari et al., 2018). sprouts are calculated using the formula: db = ∑ kn observation i + ∑ kn observation ii number of seedlings planted 𝑥 100% information: db : germination capacity (%) ∑ kn observation i : the number of sprouts is normal on the 7th day ∑ kn observation ii : the number of sprouts is normal on the 14th day hardiansyah et al. – the effectiveness of giving plant pgpr rhizosphere bamboo on … 3 2. seed growth speed (%/etmal), measurement based on observing the number of normal germinating seeds every day until the day 14 and expressed on a percent scale (tefa, 2017). seed growth rate is calculated using the formula: 𝐾𝐶𝑇 = (% 𝐾𝑁 𝑒𝑡𝑚𝑎𝑙 ) = ∑ 𝑁 𝑡 𝑡𝑛 0 information: 𝑡 : observation time 𝑁 : percentage of normal sprouts every time of observation 𝑡𝑛 : end time of observation (day 14) 1 etmal : 1 day (24 hours) 3. abnormal seeds (%), measurement based on observing the number of seeds that germinate abnormally. observation of abnormal seeds is carried out on the day 14 after the nursery (deptisari et al., 2018). abnormal seeds are calculated using the formula: abnormal seeds = ∑ abnormal seeds number of seedlings planted 𝑥 100% result and discussion the results showed that the use of two types of cocoa seeds in the initial soaking treatment of seeds with pgpr with seed concentration and the interaction of pgpr application on two types of cocoa seeds had a very significant effect on seed germination, seed growth speed and abnormal seeds in cocoa. the average parameter results are presented in table 1. table 1. germination (%), seed growth speed (% / etmal) and abnormal seed (%) of cocoa on the 14th day of giving pgpr bamboo rhizosphere to seeds. treatment germination capacity (%) seeds growth speed (%/etmal) abnormal seeds (%) gtb local seeds + pgpr concentration 0% 90,00d 6,43b 10,00a gtb local seeds + pgpr concentration 5% 90,00d 6,43b 10,00a gtb local seeds + pgpr concentration 10% 93,33c 6,67b 6,67b gtb local seeds + pgpr concentration 15% 100,00a 7,14a 0,00d mcc 01 seeds + pgpr concentration 0% 96,67b 6,90ab 3,33c mcc 01 seeds + pgpr concentration 5% 100,00a 7,14a 0,00d mcc 01 seeds + pgpr concentration 10% 96,67b 6,90ab 3,33c mcc 01 seeds + pgpr concentration 15% 100,00a 7,14a 0,00d bnj 0,05 0,03 0,45 0,40 information: the numbers followed by the same letter (abcd) in the same row and column mean not significantly different in the bnj 0.05 level test. bnj test results table 1. the interaction between the use of cocoa species and the treatment of bamboo rhizosphere pgpr concentration has a very significant effect on cocoa seed germination. the highest germination parameter was 100.00%, the highest seed growth speed parameter was 7.14% and the highest abnormal seed parameter was 10.00%. use of two types of cocoa seeds in seed germination in the nursery phase, the results showed that the average use of mcc 01 cacao seed type had the highest effect on germination and seed growth speed, while local gtb seeds had the highest effect on abnormal seed conditions. the seeds of mcc 01 had a very significant effect on germination, which was counted twice on the 7th and 14th day with the highest germination rate of 100%, the observed seed growth rate for 14 days with the fastest seed growth percentage of 7.14%/etmal and the percentage of abnormal seeds calculated on the 14th day with the highest abnormal seeds, namely 10%. this is due to the influence of the environment where the seeds grow at the time of the nursery, where the humid conditions and long seeding times lead to optimum germination and seed growth speed. in addition, the genetic factor of the seed itself is also a major influence on germination, because if the genetic of the seed is not strong enough to accept the environmental conditions where it is grown and the initial treatment, it will cause the seeds to not have the potential to germinate normally or in other words, abnormal seeds. this is in accordance with saleh (2004) statement, which states that cocoa has an epigeal germination type, so the time needed to produce radicles greatly affects the speed of procuring ready-to-channel seeds. the process of seed germination is influenced by genetic and environmental factors. the improvement of the external environment will significantly encourage the emergence of radicles as the beginning of the seed 4 biology, medicine, & natural product chemistry 10 (1), 2021: 1-5 germination process. the emergence of sprouts above the soil surface is a factor that reflects the vigor of a seedling. to find out which treatment can increase vigor, the sprouts that can appear above the soil surface are observed from the number of seeds germinated. the average germination rate with the highest effect was the type of mcc 01 seed when compared to the local gtb seed germination capacity. the average seed germination rate in this study had optimum growth because the seeds had been given previous treatment, where the highest germination growth in mcc 01 seeds and local gtb seeds had been treated with pgpr with a concentration of 5% and 15%. giving pgpr treatment with the highest concentration is a major factor in the germination power of seeds. this is in line with the opinion expressed by schmidt (2002), that pre-treatment is carried out with the aim of increasing the power, speed and uniformity of seed germination. the average seed growth speed with the fastest effect was the mcc 01 seed type when compared to the local gtb seed growth rate. the average seed growth speed of mcc 01 seeds and local gtb seeds with pgpr treatment with a concentration of 5% and 15% showed the fastest growing results when compared to other seeds. the growth rate of mcc 01 seeds and local gtb seeds is influenced by the optimum condition of the seeds at the time of the seed nursery and the provision of special treatment for seeds so that the metabolism of seed growth can run well. this is in line with the statement of lesilolo et al. (2013) stated that the speed of seed growth is a process of rapid reactivation of seeds if the surrounding conditions are for optimum growth and the metabolic process is not hampered. abnormal seeds in the use of local gtb seeds without pgpr treatment or control and with pgpr treatment with a concentration of 5% showed the highest yield of abnormal seed conditions when compared to other seeds. abnormal seeds occur due to the lack of pre-treatment of local gtb seeds, besides the physical and genetic characteristics of local gtb seeds which tend to be weaker than mcc 01 seeds. the occurrence of abnormal seeds in the nursery process is due to the deterioration of the seeds so that the quality of the seeds decreases which resulting in inhibition of the germination process. this is in line with the statement of sutopo (2002), that the decline in seeds that occurs indicates a decrease in seed quality or viability which results in low vigor and poor plant growth and production. this is also reinforced by the statement of debtisari et al. (2018), that abnormal seeds are seeds that are capable of germinating but do not show the potential to develop into normal sprouts. the seed is said to grow abnormally if the important structure for the germination process is lost or damaged, the growth of sprouts is weak due to defective or disproportionate sprouts structure, and sprouts with slow growth until the end of the test. when compared to normal seed sprouts, the abnormal seed growth is smaller than normal sprouts. concentration of pgpr rhizosphere bamboo on seeds in the germination of seeds in the nursery phase, the results showed that the treatment of seed concentration using the bamboo rhizosphere pgpr had a very significant effect on germination capacity, germination speed, and the percentage of abnormal seeds. this is because the need for pgpr or initial immersion of seeds can result in a more active metabolism of seeds to support germination, besides that the use of pgpr microorganisms can improve seed quality because pgpr acts as a growth stimulant (biostimulant) by synthesizing and regulating the concentration of various regulatory substances. growing (phytohormones) very well on seeds. this is supported by research from baihaqi et al. (2018) which states that soaking seeds with pgpr is intended so that the bacteria contained in pgpr are able to colonize the seeds as early as possible. the right long soaking treatment can increase plant yield because the bacteria will bind the seedcoat and imbibe it into the seeds. conclusion giving pgpr bamboo rhizosphere from several concentrations gave different responses to cocoa seed germination including the parameters of germination (100.00%), seed growth speed (7.14%/etmal), and abnormal seeds (10.00%). so that seed treatment with bamboo rhizosphere pgpr has an effective effect on cocoa seed germination. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references asniah, widodo, & wiyono s. (2013). potensi cendawan asal tanah perakaran bambu sebagai endofit dan agen biokontrol penyakit akar gada pada tanaman brokoli. j. fitopatol indones. 1 (2): 61-68 baharuddin, nur r., & sayifudin a. (2007). pengembangan usaha perbenihan kentang hasil kultur jaringan. forkom iptekda lipi. makassar: gedung iptek. universitas hasanuddin baihaqi a.f., yamika w.s.d., & aini n. (2018). pengaruh lama perendaman benih dan konsentrasi penyiraman dengan pgpr pada pertumbuhan dan hasil tanaman mentimun (cucumis sativus l.). jurnal produksi tanaman. 6 (5): 899905. deptisari h.e., erawati d.n., & sugiyarto. (2018). pengaruh cara penyimpanan terhadap viabilitas benih kakao (theobroma hardiansyah et al. – the effectiveness of giving plant pgpr rhizosphere bamboo on … 5 cacao l.) klon sulawesi 01. agropross national conference proceedings of agriculture. jember: politeknik negeri jember effendi m.s. (2011). kinetika fermentasi asam asetat (vinegar) oleh bakteri acetobacter aceti dari etanol hasil fermentasi limbah cair pulp kakao, jurnal teknologi dan industri pangan. 8 (2): 125-135. junaedi, thamrin s., & baba b. (2017). kajian penggunaan klon unggul kakao pada perkebunan rakyat di kabupaten bone. j. agroplantae. 6 (1): 46-49. leisolo m.k.j., rirydan e.a., & matatula. (2013). pengujian viabilitas dan vigor benih beberapa jenis tanaman yang beredar dipasaran kota ambon. jurnal agrologia, 2 (1): 1-9. li z., traore a., maximova s., & guiltinan m.j. (1998). somatic embryogenesis and plant regeneration from floral explants of cacao (theobroma cacao l.) using thidiazuron. in vitro cell. dev. biol-plant. 34: 293–299. rouhi h.r., surki a.a., sharif-zadeh f., afshari r.t., aboutalebian m.a., & ahmadvand g. (2011). study of different priming treatments on germination traits of soybean seed lots. notulae sci biol. 3 (1): 101-108. saleh m.s. (2004). pematahan dormansi benih aren secara fisik pada berbagai lama ekstraksi buah. agrosains. 6 (2): 79-83. schmidt l. (2002). guidelines for handling tropical and subtropical forest seed plants. danida forest seed centre. krogerupvej 21. dk-3050. denmark. humlebaek. sugiharti e. (2006). budidaya kakao. bandung: nuansa. sutopo l. (2002). teknologi benih. jakarta: pt raja grafindo persada. tefa a. (2017). uji viabilitas dan vigor benih padi (oryza sativa l.) selama penyimpanan pada tingkat kadar air yang berbeda. jurnal pertanian konservasi lahan kering. 2 (3): 48-50. widyaningrum a. (2017). pengaruh aplikasi pgpr (plant growth promoting rhizobacteria) dan kompos azolla terhadap mutu bibit asal stek kopi robusta. jember: fakultas pertanian universitas jember. yusniar. (2013). membangun kesejahteraan petani lewat nagari model kakao (nmk). padang: dinas perkebunan sumatera barat. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 145-150 | doi: 10.14421/biomedich.2022.112.145-150 issn 2540-9328 (online) co-existent hypertension with diabetes mellitus exacerbates renal dysfunctions eruore amalaka obore1, jerome ndudi asiwe2,3,*, vincent i. iyawe1, andrew e. edo4 1department of physiology, faculty of basic medical sciences, university of benin, benin-city, nigeria. 2department of physiology, faculty of basic medical sciences, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria. 4department of medicine, faculty of clinical sciences, university of benin, benin-city, nigeria. corresponding author* asiwejerome@yahoo.com manuscript received: 26 july, 2022. revision accepted: 04 august, 2022. published: 15 august, 2022. abstract hypertension as well as diabetes mellitus has been reported to be a major risk factor for deteriorating kidney functions. however, there is paucity of reports on renal functions in a co-existence of hypertension and diabetic condition, hence, this study evaluates renal functions in hypertensive and diabetic co-morbidity. participants were categorised into healthy, hypertension, diabetes and hypertension with diabetes group. blood pressure was measured as well as fasting blood glucose and blood samples were collected from each participant to assay renal function indices. the results showed that there was substantial uraemia as well as a significant reduction glomerular filtration rate in co-morbid hypertension and diabetic patients. fasting blood glucose and mean arterial blood pressure were considerably elevated while creatinine concentration was not significantly altered in comorbid hypertension and diabetic patients. this study revealed that coexistence of hypertension and diabetes mellitus exacerbated renal dysfunction comparatively to hypertension and diabetes mellitus. keywords: hypertension; diabetes mellitus; renal functions; co-morbidity; uraemia. introduction as part of the metabolic syndrome, diabetes mellitus (dm) is characterized by hyperglycemia and/or glucose intolerance as a result of inadequate insulin production, improper insulin action, or both (galicia-garcia et al., 2020). an important factor in the pathogenesis of this disease has been reported to be dysregulations in the regulatory systems for storage and mobilization of metabolic fuels including the catabolism and anabolism of carbohydrates, lipids, and proteins resulting from defective insulin secretion, insulin action, or both (gonzalez-gil and elizondo-montemayor 2020). multiple organ failures and increasing metabolic problems such as retinopathy, nephropathy, and/or neuropathy are among the severe symptoms of dm (preguiça et al., 2020). about 171 million individuals worldwide have been diagnosed with dm and its frequency is rising quickly, especially in developed nations (cao et al., 2020). the world health organization (who) predicted that by 2030, the number would have doubled (who, 2006). urbanization and lifestyle changes have been blamed for the rise in diabetes cases in emerging nations, although the processes are still poorly understood (akdis, 2021). it was discovered in a follow-up cross-sectional study of people with diabetes in rivers state (the central part of the niger delta region) that diabetes significantly contributes to the health issue with type 2 diabetics presenting upon diagnosis (maiga et al., 2020). several cardiovascular problems including hypertension, vasculopathy, and congestive heart failure (chf) have been related to diabetes mellitus by a body of research evidence. a chronic medical disorder known as hypertension (htn) is caused by sustained high blood pressure in the arteries (gupta et al., 2021). usually, high blood pressure has no symptoms. however, persistently high blood pressure is a significant risk factor for dementia, atrial fibrillation, peripheral vascular disease, vision loss, chronic renal disease, heart failure and coronary artery disease (panula et al., 2020). systolic and diastolic pressures; the highest and lowest lateral pressures applied to the arterial wall respectively are the two parameters used in clinical blood pressure measurements. most persons' typical resting blood pressure falls within the ranges of 60-80 mmhg diastolic and 100-130 mmhg systolic (balwan and kour, 2021). the majority of persons have high blood pressure if their resting blood pressure is consistently 130/80 or 140/90 mmhg or higher (balwan and kour, 2021). children are subject to different numbers (balwan and kour, 2021). numerous patients have been reported to have both diabetes mellitus and hypertension. nigerians have both diabetes and https://doi.org/10.14421/biomedich.2022.112.145-150 146 biology, medicine, & natural product chemistry 11 (2), 2022: 145-150 hypertension rates of 10 to 15% and 1 to 2% respectively (edeogu et al., 2020). most cases of hypertension in people with type 2 diabetes are caused by essential hypertension (akalu and belsti, 2020). according to studies, patients with diabetes experience hypertension two times more frequently than people without the condition (asiwe et al., 2021a). hypertension is strongly and independently correlated with diabetes incidence (palaiodimos et al., 2020). elevated blood pressure makes diabetic individuals more susceptible to problems such cardiovascular disease, stroke, renal disease, and retinopathy (cardoso et al., 2020). the kidney is a crucial organ for homeostasis and has been linked to both diabetes and hypertension (tinti et al., 2021). chronic kidney disease is frequently detected after an examination of those thought to be at risk for renal problems such as those with high blood pressure and/or diabetes because the symptoms of failing kidney functions are not always clear (asiwe et al 2021b). there are not many researches looking into the connection between diabetes, hypertension, and the onset of renal failure. recent investigations on patients with type 1 and type ii diabetes found a link between dietary salt consumption and diabetic complications (asiwe et al., 2021a). to the best of our knowledge, however, there is a dearth of information in the literature about the kidney's functionality in a condition where diabetes and hypertension are coexisting. as a result, the goal of this current study was to evaluate renal functions in comorbid state of hypertension and diabetes. materials and procedures ethical consideration before starting the study, a formal approval was received from the university of benin teaching hospital's (ubth) ethics and research committee (adm/e22/a/vol.vii/148283) which adhered strictly to the national guideline for research and experimentation (nih publication no. 85-23). criteria for inclusion and exclusion the study excluded participants who are diagnosed within the last three years as well as those who are type i diabetes and also those with other health complications that might add bias to our study study plan a total of 100 willing participants who met the inclusion criteria were recruited for the cross-sectional analytical study from the diabetes and hypertension clinic, outpatient department (opd) of university of benin teaching hospital (ubth), benin, nigeria. they were divided into four groups, each with 25 participants (n=25), consisting of healthy individuals (controls), hypertensive patients (htn), diabetes mellitus patients (dm), and patients with both hypertension and diabetes mellitus (htn/dm). sample collection consented participants were given a set of structured questionnaire in order to get information on their medical history. the blood pressure of the participants was measured using a sphygmomanometer and weight was measure using a weighing scale. the blood samples gotten from the participant were drawn into 5ml edta bottle for fasting blood glucose level check and thereafter centrifuged at 3000 rpm for 5 minutes and the plasma decanted for other assays such as urea and creatinine test. this procedure was carried out early in the morning when the participants had not had their breakfast. measurement of plasma creatinine the alkaline jaffe's picrate assay was used to measure the plasma creatinine level (owen et al., 1954). the jaffe's method for measuring blood creatinine is based on the idea that when creatinine combines with picric acid in an alkaline media, an orange-colored complex with the alkaline picrate is formed. at 490nm, a spectrophotomer measures the complex's absorbance, whose strength is directly proportional to the amount of creatinine present in the sample. plasma urea level determination according to cheesbrough (2005), the plasma urea level was measured using the diacetylmonoxime technique. the diacetylmonoxime method produces a reddish solution whose absorbance is measured at 530nm in a spectrophotomer by reacting urea with diacetylmonoxime at high temperature in an acid medium in the presence of cadmium ions and thiosemicarbazide. calculated creatinine clearance determination using the cockcroft-gault formula, the creatinine clearance, which is nearly identical to the glomerular filtration rate, was calculated (cockcroft and gault, 1976). since plasma creatinine is so strongly influenced by age, sex, and body size, the method takes into account a number of variables that have an impact on the estimation of muscle mass and assumed creatinine generation. creatinine clearance is expressed as follows: creatinine clearance (mol/l) = (140 age) x weight)/ (0.814 x plasma [creatinine]) x (0.85 if patient is female), where creatinine clearance is expressed in milliliters per minute age is expressed in years, weight is expressed in kilograms, and [creatinine] is expressed in micromoles per liter. statistical analysis the results were statistically evaluated using graphpad prism version 8. mean and standard error of the mean obore et al. – co-existent hypertension with diabetes mellitus … 147 were used to express the results. one way analysis of variance (anova) and tukey post hoc test were used to check the data for any significant differences. at p<0.05, mean variation was considered significant. results co-morbidity of hypertension and diabetes mellitus has been reported to be a major risk factor for deteriorating kidney functions. following one-way analysis of variance (anova) and student t-test, we evaluated the renal function indices in patients with hypertension (htn), diabetes mellitus (dm) and co-morbid condition of hypertension and diabetes (htn/dm). there was a significant (p<0.05) elevation of plasma urea [f(3,96)=4.45, p=0.0057] in dm and htn/dm group comparatively to control (fig. 4). also, in htn/dm group, there was a significant (p<0.05) reduction in egfr [f(3,96)=3.08, p=0.0311] comparatively to control (fig. 5). however, plasma concentration of creatinine [f(3,96)=0.537, p=0.6580] was not significantly (p>0.05) altered in any of the groups when compared with control (fig. 3). diabetes mellitus and hypertension was confirmed with a significant (p<0.05) elevation in fasting blood glucose (fbs) [f(3,96)=13.3, p<0.0001] and mean arterial blood pressure (map) [f(3,96)=8.48, p<0.0001] in dm and htn/dm as well as htn, dm and htn/dm respectively when compared to control as shown in figure 1 and figure 2. figure 1. fasting blood glucose. values are represented as mean ±sem, n=25 and *p<0.05 was significant when compared to control while #p<0.05 was significant when compared to hypertension group. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 2. mean arterial blood pressure (map). values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 3. plasma creatinine concentration. values are represented as mean ± sem, n=25 and *p>0.05 was not significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 4. plasma urea concentration. values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. 148 biology, medicine, & natural product chemistry 11 (2), 2022: 145-150 figure 5. estimated glomerular filtration rate (egfr). values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. discussion there are no specific signs or symptoms of deteriorating kidney functions, however they could include feeling generally unwell and losing appetite. diabetes mellitus has long been known to negatively impact blood pressure, cardiovascular risk and survival. people who are known to be at risk of kidney issues, such as those with high blood pressure and diabetes mellitus, are frequently examined in order to diagnose chronic kidney disease (byrne and targher 2020). this study was conducted to assess renal function in patients with diabetes and hypertension as well as a co-morbid condition of hypertension and diabetes. in this study, patients with diabetes as well as hypertension and diabetes had substantial uraemia. additionally, it was found that diabetic and hypertensive (htn/dm) patient’s egfr dramatically decreased as compared to healthy patients (control group). however, none of the patients' creatinine levels were considerably changed. the possibilities of having renal problem and other complications that involves organ dysfunction increases significantly when diabetes mellitus and hypertension are co-diagnosed. this is especially true if the underlying illnesses are left untreated. deteriorating kidney functions are most frequently caused by diabetes in nigeria (okaka et al., 2020; ibitoba et al., 2022). diabetic and hypertensive patients also have increased prevalence of cardiovascular (cv) risk factors, such as dyslipidemia, microalbuminuria, hyperuricemia, a propensity to clot, and left ventricular hypertrophy (mantovani et al., 2020; sanusi et al., 2020; okonofua et al., 2021). we reported an elevated mean arterial blood pressure in diabetic patients (fig.2) which was consistent with previous reports that vascular consequences of diabetes includes renal disease, coronary heart disease, stroke, peripheral vascular disease, lower extremity amputations as well as retinopathy and these are exacerbated by hypertension (asiwe et al., 2021b). furthermore, our research showed that individuals with both diabetes and hypertension have a 5to 6-fold increased risk of developing renal failure compared to individuals with hypertension alone and no sign of diabetes (fig. 5). although renal failure is a significant consequence for people with diabetes and hypertension, however, cardiovascular disease events account for the majority of deaths (copur et al., 2021). renal failure caused by diabetes and hypertension is becoming more common despite improved knowledge and suggestions for preventive measures put out by many public health organizations. in nigeria, the number of people beginning dialysis has steadily increased over the past 15 years mostly due to diabetes. this could be explained in part by a decrease in cv mortality which has led to a rise in the number of people living long enough to experience deteriorating kidney functions. a second, possibly more significant problem is that patients themselves may not be adhering to prescribed drug regimens as well as caregivers not been adequately able to educate the patients. as a result of these, target blood pressure goals are not met which increases the likelihood of developing renal failure. the general population's rates of controlling hypertension have dropped recently, according to data from the third national health and nutrition examination survey (nhanes iii) (zheutlin et al., 2022). the prevalence of diabetes has continued to climb which is a third factor that contributes to the rising incidence of renal failure (cheng et al., 2020). last but not least, the rise in cv and subsequent renal morbidity in recent years has also been influenced by the rising frequency of heart failure; as a result, even while mortality related to hypertension has declined, morbidity has grown. diabetes often results in hypertension, especially when nephropathy is also present. about 85% of persons with type 2 diabetes mellitus that have nephropathy before needing dialysis or a kidney transplant also have hypertension, which is roughly twice as common in those with type 2 diabetes as in those without it (asiwe et al., 2021). renal failure can be a third limb of a tripod-stand disease condition of hypertension and diabetes because of their common path-biological etiologies. however, to prevent or treat one, the others must be taken into consideration to reduce mortality. conclusion the results of our study reveal that diabetes and hypertension are risk factors for developing renal failure and also hypertension and diabetic co-morbidity exacerbated the deteriorating kidney functions by obore et al. – co-existent hypertension with diabetes mellitus … 149 significantly reducing glomerular filtration rate and increasing plasma urea concentration. however, it is therefore recommended that awareness and enlightenment programs should be encouraged to educate diabetic patients as well as hypertensive patients the need to take their medications serious to avoid renal function complications. acknowledgements: the authors appreciate the technical assistance of mr. obi udogwu, dr peter akpotaire and dr pauline akpe during the data collection and laboratory assay. author contributions: conceptualisation of the study was done by e.a obore and v.i iyawe data collection and laboratory assay was done by e.a obore, a. edo and j.n asiwe. funding was done by e.a obore, v. iyawe and a. edo, writing of the manuscript was done by j.n asiwe and e.a obore. all authors approved the final draft of the manuscript. data availability statement: all data associated with this study are included in this manuscript funding: this study was entirely funded by the authors conflict of interest: the authors declare that they have no conflict of interest. ethical consideration: the approval for this study was granted by the university of benin teaching hospital's (ubth) ethics and research committee with approval number adm/e22/a/vol.vii/148283. consent to participate: participants willingly consented to participate in this 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(2006). prevention of blindness from diabetes mellitus: report of a who consultation in geneva, switzerland, 9-11 november 2005. world health organization. zheutlin, a.r., derington, c.g., king, j.b., berchie, r.o., herrick, j.s., dixon, d.l., cohen, j.b., shimbo, d., kronish, i.m., saseen, j.j. and muntner, p., (2022). factors associated with antihypertensive monotherapy among us adults with treated hypertension and uncontrolled blood pressure overall and by race/ethnicity, national health and nutrition examination survey 2013-2018. american heart journal, 248; 150-159. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 137-143 | doi: 10.14421/biomedich.2022.112.137-143 issn 2540-9328 (online) ethnobotanical survey of plants used in the management of peptic ulcer diseases in wukari metropolis akpevwoghene agbatutu1,2, francis nosakhare imade2,*, efosa a. ogie-odia2, mary oromioshemime ifedele3, funmilola modinat abdulrasaq3, daniel eseigbe2 1department of biological sciences, kwararafa university wukari, taraba state, nigeria. 2plant science and biotechnology department, faculty of life sciences, ambrose alli university, ekpoma, edo state, nigeria. 3department of botany, university of ibadan, ibadan, oyo state, nigeria. corresponding author* imadefrancis@gmail.com manuscript received: 26 may, 2022. revision accepted: 28 july, 2022. published: 03 august, 2022. abstract peptic ulcer diseases (pud) are sores formed in soft tissues present in the lining of the digestive tract as a result of excessive stomach acid or inability of the alimentary tract or stomach to protect itself. the increasing surge for plant based drugs in the management of pud has increased scientific investigation of herbs and recipes from traditional medicinal practitioners (tmp) to ascertain their efficacy through pharmacological studies. ethnobotanical survey of plants and recipes from tmp used in the management of pud in wukari metropolis, taraba state, northeastern nigeria were documented. a total of twenty (20) tmp located in ten (10) different area of the town were interviewed using a semi structured questioner. twenty-four (24) plant species from twenty (20) different families and sixteen (16) recipes were documented from the survey. it was observed that plant leaves were mostly used while c. longa (zingberaceae), m. paradisiaca (musaceae) had the highest frequency of occurrence in recipes formulation. therefore, due to the proven efficacy of these herbal recipes as reported from correspondents, there is need for proper documentation, conservation, cultivation and use of these medicinal plants in the management of pud to avoid them being endangered or going into extinction. keywords: peptic ulcer; wukari; plant; taraba. introduction peptic ulcer disease (pud) is a common gastrointestinal disorder which occurs on inflamed break in the skin or mucus membrane lining the alimentary tract. it is the most predominant chronic and recurrent gastrointestinal diseases and a global health problem leading to morbidity and mortality worldwide (sen et al., 2009). pud manifest in different unit of the alimentary tract like the stomach (gastric ulcer), duodenum (duodenal ulcer), esophagus (esophageal ulcer) resulting in different types of peptic ulcer. the specific cause of pud remains unclear over the years though studies report that the lack of equilibrium between the mucosal defensive factors (secretion of bicarbonate, prostaglandins, innate resistance of the mucosal cell) and gastric aggressive factors (acid, pepsin, h. pylori infection) are possible causes of pud (roy et al., 2013; dashputre et al., 2011; roberts, 2003). presently h. pylori, non-steroidal anti-inflammatory drugs (nsaids) and zollinger-ellison syndrome (a dramatic hypersecretion of acid) are three primary factors in peptic ulcer disease though, environmental factors (smoking, excessive alcohol intake), genetic, dietary and physiologic stress also increases gastric acid secretion, weakens mucosal barrier leading to pud (malfertheiner et al., 2009; wu and fassihi, 2005). the pathophysiology of pud injury mechanism in gastric and duodenal ulcer differs distinctively such that duodenal ulcer is essentially an h. pylori related disease while gastric ulcer is commonly associated with nsaid ingestion but in both conditions inflammatory symptoms is observed at the onset leading to an imbalance between protective and aggressive factors (chan and leung, 2002). orthodox drugs used in the treatment of pud are histamine-receptor blockers (ranitidine, cimetidine), proton pump inhibitors (omeprazole, pantoprazole) and antibiotics (metronidazole, amoxycillin) which are sometimes combined to achieve better result. they are often directed at reduction of aggressive factors with some reported side effects such as dizziness, headache, constipation, impotence or breast enlargement in men and the use of antacids leads to stomach distention, belching, constipation (gulmez et al., 2007, sarkar et al., 2008, reilly, 1999, franco and richter, 1998). previous studies reveal that about 5–10% of world population suffer from pud though with a recent decrease in the incidence, rates of hospital admissions and mortality (lanas and chan, 2007, lanas et al., 2011, https://doi.org/10.14421/biomedich.2022.112.137-143 138 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 sonnenberg, 2013). the prevalence of h. pylori, a major cause of pud is higher in developing countries present in africa, central america, central asia and eastern europe where the bacteria is mostly acquired during childhood in an unhygienic environment especially in communities with lower socioeconomic status (kuna et al., 2019, hooi et al., 2017). most people in these developing countries live below poverty level, do not have access to standard healthcare facilities, rely on nsaids and painkiller drugs to subside pains resulting from stress, are unable to afford balanced diet therefore consume food substances which tend to increase gastric acid production and these factors creates an imbalance between aggressive and defensive factors in gastrointestinal tract. these classes of people who constitute about 80% of the world’s population rely on herbs from plants as alternative medication in management of sickness and diseases. traditional healers have used and relied on plants materials as main source of natural therapeutic medicines from time immemorial in the treatment and management of various infectious diseases (akinwumi and sonibare, 2019; beverly and sudarsanam, 2011; dike et al., 2012). scientific studies have established that medicinal plants used by traditional healers have displayed proven pharmacological, antioxidant and therapeutic activity against degenerative diseases. many herbs and plant products have been found to play a role in protecting or helping to heal stomach and peptic ulcers with the presence of important secondary metabolites like flavonoids and tannins reported as the active principle responsible for their anti-ulcer activity (nihar et al., 2017). ethnobotanical studies or survey are recognized as the most viable method of identifying novel medicinal plants or refocusing on those earlier reported for bioactive constituents (alebiosu et al, 2005). there is little or no documentation of medicinal plants used for the treatment of pud in wukari metropolis, northeastern nigeria, taraba state therefore this documentation will serve as reference to scientific researchers in the development of potent drugs and recipes from natural plants in treatment and management of pud with fewer or no side effects. this present study aims to document medicinal plants used in the management of pud in wukari, taraba state. methodology study area this survey was conducted in wukari metropolis the headquarters of wukari local government area of taraba state, nigeria. it has an area of 4,308km2 and a population of 238,283 at the 2006 census which is located in the south of the benue river basin (nbs, 2006). the town is located in the southern taraba with its coordinates between latitude 7o 51’n to 7o 85’n and longitude 9o47’e to 9o78’e which is about 200km from jalingo the state capital. the city has a tropical continental climate characterized by a marked distinctive wet and dry season, an annual rainfall between 1000-1500mm, temperatures range between 2040°c with a lengthy wet season (7 months) throughout the course of the year (oyatayo et al., 2017). wukari is mainly an agrarian town that lies within the savannah zone and has mainly grassland vegetation and scattered trees and shrubs in the southern part of the state. its inhabitants are the jukuns, who are predominantly traditionalists though some of them are christians and muslims. the survey was conducted in sampled areas that are close to farms and garden in the extended city areas of wukari, with the aim to capture both rural and urban populace and locate the elderly people with the knowledge of traditional medicine. figure 1. map of nigeria showing taraba state. agbatutu et al. – ethnobotanical survey of plants used in the management of … 139 figure 2. map of taraba state showing wukari local government area and areas visited. data collection the main data sources consisted of a series of semi– structured and open-ended questionnaires as well as informal interviews administered on local herb sellers, herbalists, aged and other groups of people rich in traditional medicine knowledge. the survey was conducted within the span of eight (8) months from january 2019 to august 2019 with repeated visits to the respondents. questionnaires were administered and hausa language was used to obtained the information from the respondents. such information includes the local plant names, useful plant parts and methods of preparation. local names of plants mentioned were validated using literatures and proper taxa nomenclature was validated in the plant list database at www.plantlist.org. demographic data a total of twenty respondents were interviewed consisting of twelve males (60%) and eight females (40%) in wukari metropolis. the respondents include traditional healers (30%), herbalist (20%), herb sellers (40%) and the aged people (10%) all of which are nigerians from the jukun ethnic group comprising of (40%) christians, (20%) muslims and (40%) practicing traditionalists. their ages were within 31-70 years with the age bracket of 51-60 (45%) with no formal education (60%) being the highest among them (table 1). results a total of twenty-four (24) plant species belonging to twenty (20) families were identified as plants used in treatment of pud in wukari metropolis (table 2). the plant families, local names, part used and frequency of occurrence were documented. the anarcardiaceae, alliaceae, malvaceae and musaceae had two plants each among the twenty-four plant species while the leaves were mostly used in preparation of various herbal recipes. some of the plants were reported to be harvested from the forest while others from home garden and environs. the respondents claimed that their medicinal knowledge was inherited, few trained and some believed to be divine. table 1. source of ethnomedicinal information. demography frequency (n=20) percentage (%) traditional healers 6 30.0 herbalist 4 20.0 herb seller 8 40.0 aged 2 10.0 gender male 12 60.0 female 8 40.0 age range below 30 0 0.0 31-40 2 10.0 41-50 7 35.0 51-60 60 and above 9 2 45.0 10.0 religion christianity 8.0 40.0 islam 4.0 20.0 traditional religion 8.0 40.0 education no formal 12 60.00 primary 4 20.00 secondary 4 20.00 diploma 0 00.00 degree 0 00.00 140 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 table 2. list of plants used in management of pud in wukari, taraba state. s/n plant name family part used common name hausa name frequency of occurrence 1 azadirachta indica (a.) juss. maliaceae leaves neem doogon yaaroo 1 2 psidium guajava linn. myrtaceae leaves guava gweebaa 1 3 anarcardium occidentale linn. anarcardiaceae roots, stems leaves cashew jambe 1 4 carica papaya linn. caricaceae fruits, seeds pawpaw gwandar masar 1 5 ocimum gratissimum linn. lamiaceae leaves scent leaf tagida, daddooya 1 6 musa sapientum linn. musaceae unripe fruits banana ayama 2 7 talinum triangulare (jacq.) wild. portulacaceae leaves water leaf alenyruwa 1 8 curcuma longa linn. zingberaceae rhizome tumeric zabibi 4 9 musa paradisiaca linn. musaceae fruit plantain agada 4 10 aloe vera (l.) burm. f alliaceae gel aloe hantsar giwaa 1 11 mangifera indica linn. anarcardiaceae leaves mango mangoro 1 12 hibiscus sabdariffa linn. malvaceae calyx roselle soboroto 1 13 sida acuta burm.f. malvaceae leaves wire weed tsadar iamarudu 1 14 lantana camara linn. verbenaceae stem, whole plant red sage kimbama halba 1 15 euphorbia hirta linn. euphorbiaceae leaves garden spurge noonon kurciyaa 2 16 ipomoea batatas linn. convolvulaceae tuber sweet potato ba fadamee 1 17 moringa oleifera lam. moringaceae leaves drumstick zogelle 1 18 bryophyllum pinnatum lam. crussalaceae leaves life plant harfifi 1 19 calotropis procera (ait.) ait.f. ascclepidiaceae leaves sodom apple baabaa ambele 1 20 allium cepa linn. alliaceae bulb onion albasa 1 21 datura metel linn. solanaceae leaves devil trumpet zakami 1 22 vitellaria paradoxa gaertn.f. sapotaceae leaves and fruit shea butter kadee 1 23 sorghum bicolor (l.) moench poaceae seed guinea corn mazakuwa 2 24 ampelocissus africanus (lour.) merr. vitaceae leaves simple-leaved wild grape siling siame 1 figure 3. frequency of plant parts used in management of pud in wukari metropolis 0 2 4 6 8 10 12 14 bulb tuber leaves root stem fruits seeds rhizome gel calyx whole plant agbatutu et al. – ethnobotanical survey of plants used in the management of … 141 table 3. pud herbal recipes from tmp in wukari, taraba state. s/n method of preparation 1. dry the half ripe m. paradisiaca and m. sapientum peels and grind into powder, mix one teaspoon of the powder with honey. 2. squeeze the fresh leaves of t. triangulare, e. hirta in clean water, add edible salt (nacl), then sieve the extract, take a glass cup twice daily 3. dried rhizome of c. longa is chewed with leaves of e. hirta in an oily juice (palm oil, olive oil). 4. dried half ripe peels of m. paradisiaca and m. sapientum is powdered, a powdered teaspoon is mixed with honey and taken daily for two months. 5. cut two whole fruits of c. papaya pieces including the peel, soak in six liters of water, sieve to remove the cubes and add 750ml of honey into the extract, and drink a glass cup twice a day. 6. blend four matured leaves of a. vera with 750ml of pure honey and 750ml of clean water respectively, filter the syrup and keep in a container to be taken twice daily. 7. dry leaves of c. procera are crushed and mixed with v. paradoxa and swallowed twice daily. 8. gentle heat is applied to the leaves of b. pinnnatum until it brings out oil which is then crushed to paste and mixed with honey before consumption twice daily. 9. boiled or crushed leaves of m. oleifera in water, is taken twice daily. 10. unripe m. paradisiaca and c. papaya fruits are sliced to cubes, soaked in water for three days and taken daily. 11. powdered leaves and rhizome of a. africanus, c. longa is mixed with s. bicolor paste and consumed morning and evening daily. 12. bulbs of a. cepa and leaves of s. acuta are crushed, mixed with s. bicolor paste and natural honey. 13. decoction of the calyx of h. sabdariffa is mixed with powdered leaves of a. occidentale and p. guajava and taken twice daily. 14. a tea spoon of powdered m. paradisiaca fruit in mixed in a maceration of a. indica leaves and l. camara whole plant for seven days be taken twice daily for two months. 15. paste of boiled c. longa mixed with a pinch powdered leaves of d. metel leaves be eaten with boiled tuber of i. batatas should be taken early morning daily. 16. decoction of fresh leaves of o. gratissimum, m. indica mixed with s. bicolor paste and natural honey figure 4. frequency of plants used in pud herbal recipes in wukari metropolis. discussion the importance of plant based drugs from traditionalists and herb sellers in the management of pud remains vital in the search of novel active compounds with little or no side effects. a total of twenty-four (24) plant species from twenty (20) families with sixteen (16) recipes were recorded from twenty (20) traditional medical practitioners present in wukari metropolis. (figure 2 to 4, tables 1 to 2). the plants c. longa, m. paradisiaca recorded the highest frequency of occurrence while various plant parts such as leaves, stem, fruit bark, rhizome and bulb were frequently used in the prescribed recipes. these formulations were either mixed or prepared with pap, water and honey as major vehicle suggested during preparation, administration and consumption of prepared recipes. there are reports on the scientific investigation on pud and gastro-protective 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 142 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 potentials of some of these plants such as c. longa (savaringal and sanalkumar, 2018), a. vera (subramanian et al., 2007), a. occidentale (ajibola et al., 2010), c. papaya (okewumi and oyeyemi, 2012), o. gratissimum (amadi et al., 2014), m. paradisiaca (rao et al., 2016), m. indica (neelima et al., 2012), t. triangulare (onwurah et al., 2013) and m. sapientum (prabha et al., 2011) have been evaluated using different models on experimental animals. some of these plants have been reported to display strong antimicrobial (a. cepa, c. longa), anti-inflammatory (s. acuta), relaxant (o. gratissimum, m. oleifera) and acid reflux (m. sapientum, a. vera) activity which could confirm their mode of activity or mechanism of action against the reported major causes of pud such as h. pylori a bacteria, nsaids related drugs, zollinger-ellison syndrome and environmental stress (iqbal et al., 2018, gupta et al., 2015, arciniegas et al., 2016, malfertheiner et al., 2009). the therapeutic activity of these herbal recipes as reported from respondents and their customers suggest that these herbal products contain numerous bioactive constituents that are a part of the physiological functions of living flora and hence they are believed to have better compatibility with human body (kamboj, 2000). akinwumi and sonibare (2019) stated that cultivation and proper documentation of medicinal plants from ethnobotanical survey is essential to prevent them from being endangered and going into extinction. this confirms and addresses the worries from respondents who reported the scarce availability of some plants in their natural habitats due to their increasing demands for food and medicine. therefore, the increasing dependence of these plant based herbal drugs mainly in developing countries for primary health care could be properly harnessed and managed only if there are measures to ensure sustainable cultivation that will have a considerable long term effect on the environment, health care and economy. conclusion twenty-four (24) plant species with sixteen (16) recipes from traditional medicinal practitioners and herb sellers were documented and reordered for the treatment and management of pud. the major essence of this survey is to harness and document the rich knowledge of the traditional medicinal practitioners on the use of medicinal plants in the treatment of pud which could pave the way for detailed scientific research in the discovery of novel compounds with better activity, new mechanism of action and less toxic side effects. authors’ contributions: agbatutu designed the study. agbatutu carried out the laboratory work. ifedele and imade analyzed the data. agbatutu and imade wrote the manuscript. abdulrasaq and ogie odia review the manuscript all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references ajibola es, adeleye oe, okediran bs and rahman sa (2010). effect of intragastric administration of crude aqueous leaf extract of anacardium occidentale on gastric acid secretion in rats. nig. j. physiol. sci. 25. 59 – 62. akinwumi ia and sonibare ma (2019). use of medicinal plants for the treatment of gastric ulcer in some parts of southwestern nigeria. african journal of pharmacy and pharmacology vol. 13(15), pp. 223-235 alebiosu co, ugwa om, ugwah-oguejiofor cj and 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negeri sunan kalijaga yogyakarta jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author* sugimath@yahoo.co.id manuscript received: 29 march 2020. revision accepted: 10 april, 2020. published: 15 april, 2020. abstract covid-19 stands for corona (co), virus (vi), disease (d) and year 2019 (19), which is covid-19 first appeared in 2019. mathematical model of covid deployment in indonesia under and without lockdown case uses the sirv model, such as susceptible, infected, recovery, and virus. the results of this model state that under lockdown the spread of covid-19 could be stopped. if it were not under lockdown it can multiply 1,276 times higher over two months. keywords: corona virus novel 2019 (covid-19); mathematics model; lockdown; indonesia introduction novel corona virus can pneumonia (pan et al., 2020). pneumonia is an inflammatory lung disease which is characterized by coughing, chest pain, fever, and difficulty breathing. covid-19 is a very contagious disease and declared by who as a pandemic on march, 12, 2020 (chen et al., 2020). pandemic is an infectious disease that is widely spread and it can reach all countries in the world. covid-19 was first identified in wuhan, hubei province, china in december 2019 (liu et al., 2020). the understanding of lockdown in a pandemic context of covid-19 is an emergency state where someone is not allowed to enter or out of a certain area in a certain period of time. this kind of action is carried out during the emergency situation (ku et al., 2020). mathematical model of covid-19 deployment is using susceptible sub populations, infected, recovery and virus or abbreviated as sirv. this population is divided into four subpopulations. susceptible subpopulation (s) is a subpopulation that is vulnerable to covid-19. infected subpopulation (i) is subpopulation that is infected by covid-19. recovery subpopulation (r) is subpopulation that recover from the covid-19 virus. the population of covid-19 (v) is the population number of covid-19 outside the human body. model formulations the covid-19 deployment model uses several parameters. parameter 𝑑1 is the rate of natural death and other diseases besides covid-19. parameter 𝑎1 is the level of susceptibility to covid-19 disease. parameter 𝑎2 is the level of covid-19 virus infection. parameter 𝑎3 is the cure rate of covid-19 disease. parameter 𝑑2 is the death rate due to covid-19 virus. this models are defined into: a. the susceptible subpopulation are affected by: (1) the increasing in birth rate; (2) the increasing in susceptible individuals from other regions; (3) the increasing number of individuals recovering from the diseases that is caused by covid-19; (4) the reduce susceptibility of covid-19 which infected individuals; (5) the reduce susceptible individuals going to other areas; (6) the loss of individuals due to natural deaths, other than diseases caused by covid-19. b. the infected subpopulation are affected by: (1) the increasing number of individual births from a mother that infected by covid-19; (2) the increasing in individuals due to arrivals from other regions; (3) the increasing in individuals from susceptible and infected by covid-19; (4) the reduce individual recovery from covid-19; (5) the reduction in infected individuals who went to other areas; (6) the https://doi.org/10.14421/biomedich.2020.91.15-19 16 biology, medicine, & natural product chemistry 9 (1), 2020: 15-19 reduction in individuals who die from diseases that caused by covid-19; (7) the loss of individuals due to deaths other than covid-19. c. the recovery subpopulation are affected by: (1) the increase in births at recovery time; (2) the increase in individual recovery from other regions; (3) the increase in infected; (4) the reduce susceptible individuals; (5) the reduction in recovery individuals that went to other areas; (6) the loss of individuals due to deaths other than covid-19. d. the population of covid-19 (population of virus in the environment spread by infected people) are affected by: (1) the covid-19 deployment because individuals are infected; (2) the unsurvive of covid-19 in a free environment due to physical and chemical factors. table 1. the population, subpopulations, and parameters used in the covid-19 spread by mathematics model. no. symbol explanation unit 1. s susceptible subpopulation person/km2 2. i infected subpopulation person/km2 3. r recovery subpopulation person/km2 4. v the population of covid-19 virus/km2 5. 1a birth rate of susceptible subpopulation day -1 6. 2a birth rate of infected subpopulation day -1 7. 3a birth rate of recovery subpopulation day -1 8. 1b arrival rates from other countries susceptible subpopulation day -1 9. 2b arrival rates from other countries infected subpopulation day -1 10. 3b arrival rates from other countries recovery subpopulation day -1 11. c death rates due to disease caused by covid-19. day-1 12. 1d natural death rates are not due to covid-19 susceptible subpopulation. day -1 13. 2d natural death rates are not due to covid-19 infected subpopulation. day-1 14. 3d natural death rates are not due to covid-19 recovery subpopulation. day -1 15. 4d the covid-19 mortality rates in environment. day-1 16. 1e rates of traveling to other countries susceptible subpopulation day -1 17. 2e rates of traveling to other countries infected subpopulation day -1 18. 3e rates of traveling to other countries recovery subpopulation day -1 19. f the degree of covid-19 emergence in environment virus person-1 20. 1k carrying capacity of the susceptible birth subpopulation person 2 21. 2k carrying capacity of the infected birth subpopulation person 2 22. 3k carrying capacity of the recovery birth subpopulation person 2 23. 1l carrying capacity of the susceptible natural death subpopulation person 2 24. 2l carrying capacity of the infected natural death subpopulation person 2 25 3l carrying capacity of the recovery natural death subpopulation person 2 26.  the degree of interaction between susceptible subpopulation become infected subpopulation km2.day-1.virus-1 27.  the degree of interaction betweem infected subpopulation become recovery subpopulation day -1 28.  the degree of interaction between recovery subpopulation become susceptible subpopulation. day-1 in this model it is assumed that: (1) open population, means people are free to enter and exit a country/region; (2) newborn individuals are included in the susceptible subpopulation; individuals who recover from covid are not immune; (3) every individual who infected by covid-19 will become infected; (4) after the incubation period of two weeks are already considered an infected individual; (5) a vaccine for covid-19 has not been found yet, it means that healing is due to good individual immunity, not because of vaccine; (6) people who recovery from covid-19 can be a susceptible subpopulation again; (7) birth rate and natural mortality rate of the infected subpopulation and recovery does not exist. in this model not analyzed local stability of the equilibrium point, and it become our next job. figure 2 sugiyanto & abrori – a mathematical model of the covid-19 cases in … 17 is covid-19 deployment transfer diagram. based on the figure 2, transfer diagram, formed the covid-19 spread mathematics model equation (1a) – (1d). figure 1. covid-19 distribution between individuals. 1 1 1 1 1 1 1 1                    ds s s a s b s r sv e s d s dt k l   (1a) 2 2 2 3 2 2 1 1                     di i i a i b i sv i ci e i d i dt k l   (1b) 3 3 3 3 3 3 1 1                    dr r r a r b r i r e r d r dt k l   (1c) 2 4 2 1         dv i fa i d v dt k (1d) figure 2. transfer diagram of covid-19 deployment model. simulation in this section, we will discuss about numerical simulation and its intepretation by covid-19 deployment model. the initial value starts from the spread of covid-19 in indonesia on march, 12–23, 2020 that was announced by the indonesian goverment. the initial value of the susceptible is taken from the total population of indonesian divided by the population of indonesian, 𝑆0 = 260,000,000 260,000,000 = 1. the infected value was taken from the number of people infected on march 23, 2020 divided by the population of indonesian, 𝐼0 = 579 260,000,000 = 0.0000022269. the recovery values is taken from the number of people recovered on march 23, 2020 divided by the total population of indonesia, 𝑅0 = 30 260,000,000 = 0.0000001. covid-19 value is taken by the estimation, 𝑉0 = 1,000,000 260,000,000 = 0.0038461538. the parameter values are as follows. indonesia area 1.905 x 109 km2. in 2019, the birth rate is = 4.4 x 106 people. in 2019, the mortality rate is = 1.6 x 106 people. there are 365 days in one year. 6 3 1 1 9 4.4 10 6.3 10 1.905 10 365     x a x day x x 6 1 3 1 1 9 1.6 10 2.3 10 1.905 10 365      x d day x day x x from the assumption of natural birth and death that the infected subpopulation does not exist, then the parameter 𝑎2 = 0 and 𝑑2 = 0. from the assumption of natural birth and death recovery subpopulation does not exist, then the parameter 𝑎3 = 0 dan 𝑑3 = 0. because the indonesia country has suspended its arrival visa and the other countries have also applied for a visa suspension, then the parameter 𝑏1 = 𝑏2 = 𝑏3 = 𝑒1 = 𝑒2 = 𝑒3 = 0. from the table 2, we take the highest gradient that is from march 17–18, 2020 are 14 souls per day (zonautara, 2020). it means, the death rates due to the diseases caused by covid-19 is the highest mortality divided by indonesian population or 𝑐 = 14 260,000,000 = 0.0000000538 𝑝𝑒𝑟𝑠𝑜𝑛−2𝑑𝑎𝑦−1. value 260,000,000 are taken from the indonesian population. the value of covid-19 mortality rates in the environment ( d ) be accepted 𝑑4 = 2 260,000,000 = 7.7𝑥10−9 𝑑𝑎𝑦−1 (bnpb, 2020). the estimated value of covid-19 emergence rate in the environment (𝑓) be accepted 𝑓 = 260,000 260,000,000 = 1𝑥10−3 𝑣𝑖𝑟𝑢𝑠 𝑝𝑒𝑟𝑠𝑜𝑛−1𝑑𝑎𝑦−1. the value of the level of interaction between susceptible subpopulation become infected subpopulation (𝛼) taken from the slope of the highest infected divided by indonesian population. the highest slope is on march, 18–19, 2020 or march, 20–23, 2020 there were 81 people died in one day. so, the value of 𝛼 is 81 divided ten times of 579 people were infected (ten fold was infected but its unknown, because it’s not covid-19 tested), 𝛼 = 81 579𝑥10 = 3.6𝑥10−3 𝑑𝑎𝑦−1𝑣𝑖𝑟𝑢𝑠−1. the value of the level of interaction between recovery subpopulation become susceptible 18 biology, medicine, & natural product chemistry 9 (1), 2020: 15-19 subpopulation (𝛽) taken from the highest slope recovery divided by the population of indonesian. the highest slope was on march 21–22, 2020 as many as 9 people died in one day. so, the 𝛽 value is 9 divided by a hundred multiple of 30 people who recover (one hundred multiples are recovery but not known, because it does not test by covid-19), 𝛽 = 9 30𝑥100 = 3𝑥10−3𝑑𝑎𝑦−1. the value of the level of interaction between recovery subpopulation become susceptible subpopulation (𝛾) taken the same as 𝛽. the value of carrying capacity of natural birth and death in the susceptible sub population taken with an estimated number of deaths of 500,000 people divided indonesian population, that is 3 1 2 3 1 2 3 500, 000 1.923 10 . 260, 000, 000        k k k l l l x to summarize the parameter values, we shown in table 3. table 2. the data that died in indonesia because of covid-19 (zonautara, 2020). no. by the date the number of people died the number of people recovered the number of people infected 1. 12 march 2020 1 4 34 2. 13 march 2020 4 4 69 3. 14 march 2020 5 5 96 4. 15 march 2020 5 5 117 5. 16 march 2020 5 8 134 6. 17 march 2020 5 9 172 7. 18 march 2020 19 11 227 8. 19 march 2020 25 15 308 9. 20 march 2020 32 17 369 10. 21 march 2020 38 20 450 11 22 march 2020 48 29 514 12 23 march 2020 49 30 579 table 3 is multiplied by 260,000,000 people; this number is taken from the indonesian population. lockdown case is the level of interaction between susceptible subpopulation become infected subpopulation, the degree of interaction between infected subpopulation becomes recovery subpopulation, and the degree of the interaction between recovery subpopulation being a susceptible subpopulation doesn not exist, it means the value of 0     . table 3. the parameter value without covid-19 deployment lockdown in indonesia. no. symbol value no. symbol value 1. 1a 3 6.3 10  x 13. 2e 0 2. 2a 0 14. 3e 0 3. 3a 0 15. f 3 1 10  x 4. 1b 0 16. 1k 3 1.923 10  x 5. 2b 0 17. 2k 3 1.923 10  x 6. 3b 0 18. 3k 3 1.923 10  x 7. c 85.38 10  x 19. 1l 3 1.923 10  x 8. 1d 3 2.3 10  x 20. 2l 3 1.923 10  x 9. 2d 0 21. 3l 3 1.923 10  x 10. 3d 0 22.  3 3.6 10  x 11. 4d 9 7.7 10  x 23.  3 3 10  x 12. 1e 0 24.  3 3 10  x figure 3. diagram of trajectory subpopulation infected between under lockdown and without lockdown. figure 3 shows the case trajectory diagram under and without lockdown in infected subpopulation. in the case of not lockdown on day-0 showed 579 people infected. on 30th for the case to be 677,600 people infected. on 60th showed 738,900 people infected. on day-0 of lockdown case showed 579 people infected and the 30th day showed 579 people infected. because the incubation period of 14 days, then in the case of lockdown on the 15th day no one has been infected. conclusion covid-19 is a vicious virus, because its spread from modeling result is very fast. from the indonesia government’s data on march 12–23, 2020 up to 579 sugiyanto & abrori – a mathematical model of the covid-19 cases in … 19 people have been infected, while from the model when not in lockdown of 579 people become 738,900 people have been infected. this is 1,276 times in two months. under lockdown, it can minimize casualties because covid-19 does not spread. lockdown is a solution to reduce deaths due to covid-19. references bnpb, 2020 in https://www.covid19.go.id/ketahui-caramengurangi-risiko/ downloaded on march 20, 2020. chen, h., guo, j., wang, c., luo, f., yu, x., zhang, w., ... & liao, j. (2020). clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records. the lancet. ku, c. c., ng, t. c., & lin, h. h. (2020). epidemiological benchmarks of the covid-19 outbreak control in china after wuhan’s lockdown: a modelling study with an empirical approach. available at ssrn 3544127. liu, y., gayle, a. a., wilder-smith, a., & rocklöv, j. (2020). the reproductive number of covid-19 is higher compared to sars coronavirus. journal of travel medicine. pan, f., ye, t., sun, p., gui, s., liang, b., li, l., ... & zheng, c. (2020). time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology, 200370. zonautara, 2020 in https://zonautara.com/2020/03/22/tabelsebaran-virus-corona-per-provinsi-22-maret-2020/ downloaded on march 20, 2020. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 109-115 | doi: 10.14421/biomedich.2020.92.109-115 issn 2540-9328 (online) callus induction of leaves and stems in krisan (chrysanthemum morifolium ramat cv dewi ratih) with alternative foliar fertilizers media ahmad saifun naser*, muhammad wisnu department of biology, faculty of sciences and technology, universitas islam negeri sunan kalijaga yogryakarta, jl. marsda adisucipto, yogyakarta, 55281, indonesia. corresponding author* saifun98@gmail.com manuscript received: 11 october, 2020. revision accepted: 16 november, 2020. published: 18 july, 2021. abstract availability of quality seeds in production of krisan (chrysanthemum morifolium ramat cv dewi ratih) cultivation is still rare, therefore research on seed multiplication through tissue culture is needed. the media used in tissue culture is relatively expensive for home industry. this study aims to determine the respond of leaf and stem explants using foliar fertilizers (growmore, gandasil d and mutiara) as an alternative media for callus inductions. this study used a completely randomized design (crd) consisted of 4 treatments: p0: ½ ms + 0,25 mg/l bap, p1 (growmore + 0,25 mg/l bap), p2 (gandasil d + 0,25 mg/l bap), p3 (mutiara + 0,25 mg/l bap). the variables observed in this study included callus appearance time, callus color and callus texture. the result of this study indicated that the use of bap (6-benzyl amino purine) affected the time of callus formation and callus morphology. callus was formed on leaf explants 13 days after planting while on stem explants 7 days after planting and compact texture. growmore + 0,25 mg/l bap treatment yields the best callus on leaf explant, while gandasil d + 0,25 mg/l bap treatment yields the best callus on stem explant. keywords: bap (6-benzyl amino purine); krisan (chrysanthemum morifolium ramat cv dewi ratih); plant tissue; alternative media (growmore, gandasil d and mutiara). introduction chrysanthemum is an ornamental plant that has a high economic value due to the beauty of the flowers. according to mustakim et al., (2015) the flowers of chrysanthemum containing antioxidants that play a role in detoxifying toxins in the body and improve blood circulation. chrysanthemum plants could be used as a raw material for antibiotic drugs and sharpens eye vision (mani & senthil 2011; lin & herly, 2010). chrysanthemum plant (chrysanthemum morifolium ramat cv dewi ratih) is cultivated in almost all of indonesia. the dominating area includes east java, central java, west java, south sulawesi and north sumatra (badan pusat statistik dan direktorat jendral holtikultura, 2017). c. morifolium plants contained secondary metabolite of flavonoid and volatile compounds. the examples of the compound are quercetin-3-galactoside, luteolin 7-glucoside, quercitrin, myricetin, luteolin, apigenin, and kaempferol (sun et al., 2010). the successful morphogenesis from cultivated plants determined by the explants. according vidyasagar, (2006) explant is part of the plant that is used as a material for the initiation of culture. explant can be cells (cell culture), protoplast (protoplast culture), medulla (tissue culture), apical meristem or lateral (shoot culture) and cut stems or leaves (organ culture) (dwiyani, 2015). the juvenile physiological explants are better for the tissue culture then the old one. the part of juvenile plants have higher regenerations than the mature one (gunawan, 1987). callus is an unorganized and actively dividing tissue that usually produced by injured plants in tissue culture. according to dwiyani, (2015) callus can be obtained by tissue roots, leaves and stems in the plant that occurred through the process of indirect embryogenesis. explant derived from meristematic tissue developed faster than tissue of thin-walled cells that containing lignin (nugroho & sugito, 2001). plant growth regulators (pgr) could be used for additional plant tissue cultured media. according from gunawan (1987) pgr usage affected growth and morphogenesis in cell, tissue and organ culture caused by the interaction between media determined culture development. media are very important in tissue culture methods, even the success of the tissue culture is determined by the media used (gunawan, 1987). the essential elements in the media included inorganic salts, vitamins, energy source and carbon. the essential nutrients are nutrients https://doi.org/10.14421/biomedich.2020.92.109-115 110 biology, medicine, & natural product chemistry 9 (2), 2020: 109-115 that used by plant to completed their life cycle. the functions of these nutrients cannot be replaced by other elements. it is required by the plants in the metabolic process as inorganics components or cofactors in enzyme reactions (orcutt & nilsen, 2000). foliar fertilizers like mutiara and growmore can be alternative substitution for micronutrient and macronutrient ms (murashage and skhoog) media (shintiavira et al., 2012). gandasil d and growmore are compound fertilizers that contain complete macronutrient and micronutrient. sometimes the alternative media usage added by some nutrient like casein hydrolysate (ch), corn oil, coconut water and tomato juice. widiastoety & purbadi (2003) stated that 150 ml/l usage of coconut water that pour into the media could have an effect for increased growth, height, length and width leaves and the number of roots to dendrobium orchid plantlet. this study aims to determine the response of leaf and stem explants to use foliar fertilizers (growmore, gandasil d & mutiara) with added plant growth regulators (pgr) as an alternative media for callus inductions of chrysanthemum morifolium ramat cv dewi ratih. it is hoped that this research can be used as material for consideration of a substitute for ms (murashige and skoog) which is relatively expensive for home-scale tissue culture farmers given the nutrient content in foliar fertilizers meet the needs for optimal plant growth and development. methods this research was conducted from 15th february to 20th september 2019 at the embryology laboratory of uin sunan kalijaga yogyakarta. materials the plant parts used as explants in this study were the leaves and stems of chrysanthemum morifolium ramat cv dewi ratih. the growth media used in this study were ½ ms medium (murasahge and skoog) and compound fertilizer gandasil d, growmore and mutiara. furthermore, the chemicals used were agar, bap powder, 1 m hcl solution, distilled water, 70% alcohol, sterile tissue paper, sterile aluminium foil, filter paper and plastic cling wrap. procedure 1. sterilization of ingredients the explants that had been prepared for sterilization were put in a tea filter, then washed with running water for 5 minutes. remove the explants and soak them in a 10% alcohol solution for 3 minutes while shaking them manually then rinsing with distilled water 3 times (1-3 minutes), then the explants were removed and immersed in 5% chloroc (naocl) solution while shaking it manually for 35 minutes then 10 % chlorine for 3-5 minutes. then rinse with distilled water 3 times (1-3 times). the explants were then let dry in erlenmeyer and covered with sterile tissue in an inverted position. 2. preparation of hormone solution 100 mg of bap powder was dissolved in 2.5 ml of 1 n hcl solution. the material was heated and stirred until dissolved. then, add distilled water slowly until the volume of the solution reaches 100 ml. furthermore, the ph of the solution is adjusted to 5.0. the solution was transferred to the stock container and labelled. 1000 ppm bap stock solution (1 ppm = 1 mg/l) were stored in a refrigerator. 3. media media ms (murashige and skoog) 2.215 grams of instant ½ ms medium diluted in 1000 ml of distilled water. then added the hormone 0.25 mg/l bap. the ph measurement of the solution was carried out in the range of 5.6 5.9. after that, 8 grams of agar powder and 30 grams of sugar are added to the mixture of media and hormones. making ½ ms resulted in a positive control, namely media ½ ms + 0.25 mg/l bap, each consisting of 10 replications. ½ ms solution was heated to a boil, stirring chili sauce until the powder was completely dissolved. after boiling, ½ ms liquid medium was poured into the culture bottle and closed tightly with aluminium foil. the culture bottles that have been filled with the media were then put into an autoclave to be sterilized at a pressure of 17 psi at 121°c for 15 minutes. finally, the culture bottles were stored in the culture room at 12°c. after one to two days, the culture bottles are examined for clots or for contamination. 4. explant inoculation and culture incubation explant was obtained from the leaves of the chrysanthemum. each culture bottle was planted with three leaf explants with a size of 0.5 cm x 0.5 cm. each explant was planted 10 times in a variety of different growth media according to table 1. the culture bottles that have been planted with explants were then stored in the culture room at a temperature of 16°c. table 1. design of research treatment chrysanthemum morifolium ramat cv dewi ratih. treatment a. positive control (p0: ½ ms +0,25 mg/l bap) b. alternative media (p1: growmore + 0,25 mg/l bap) c. alternative media (p2: gandasil d + 0,25 mg/l bap) d. alternative media (p3: mutiara + 0,25 mg/l bap) 5. observation of parameters observations were made for once every two days after the day after planting (dap). until callus formed on all naser & wisnu – callus induction of leaves and stems in krisan … 111 explants. the parameters observed during the study are as follows:  callus morphology and color the morphology observed included crumb and compact callus texture and a white, green or brown callus color  callus appearance time the time of callus formation was determined by observing the explants from the beginning of planting until the first callus appeared. results and discussion leaf and stem explants on chrysanthemum morifolium ramat cv dewi ratih are able to form callus. callus formation on leaf and stem explants had different times. on the stem, callus formed 7 dap (days after planting) while on callus leaves formed 13 dap (days after planting). it can be seen in tables 2 and 3. the callus formed in all treatments was compact type. the compact callus texture in this study was caused by the provision of cytokinins, as the statement by purwaningsih et al., (2016) that compact callus has the effect of giving cytokines to the media that play a role as nutrient transfer. table 2. callus texture and color, as well as other descriptions of leaf explants (c. morifolium ramat cv. dewi ratih). treatment days after planting color callus other information ½ ms + 0,25 mg/l bap 7 dap clear green stems are still fresh, shoots appearead in 13 dap. compact callus. mutiara+0,25 mg/l bap 7 dap clear green callus grows on the scar, stems are still fresh, shoots appearead in 13 dap. compact callus. gandasil+0,25 mg/l bap 7 dap clear green compact callus, stems are still fresh, shoots appearead in 13 dap. growmore+0,25 mg/l bap 7 dap clear green compact callus. stems are still fresh. 13 dap shoots grow in new branches. table 3. callus texture and color, as well as other descriptions of stems explants (c. morifolium ramat cv. dewi ratih). treatment days after planting callus texture other information color callus ½ ms + 0,25 mg/l bap (+) control 13 dap compact the leaves are swollen, the scars form a clear white callus. clear green callus mutiara + 0,25 mg/l bap (-) 13 dap compact there is browning on the scar, getting thicker, on the scar forming a clear white callus. clear green callus gandasil + 0,25 mg/l bap (-) 13 dap compact the leaves are swollen, browning on the wound, forming a clear callus on the wound. clear green callus growmore + 0,25 mg/l bap 13 dap compact the leaves are swollen, callus is found all over the leaves. clear green callus p0 p1 p2 p3 figure 1. callus on leaves explants with the treatment p0, p1, p2, and p3. ( : callus) in the formation before, callus was followed by swelling of the explants. this is in accordance with the statement of hariyanti et al., (2016) stating that callus is formed followed by a whitish or clear swelling around the scar which eventually covers the entire surface of the explants. callus on the leaf (table 3) is formed marked 112 biology, medicine, & natural product chemistry 9 (2), 2020: 109-115 by the swelling of explant tissue on 13 dap, then followed by the formation of clear grains on scar and the uneven explant surface. callus formed in each treatment leaf explants could be seen in figure 1. the callus that formed on the stem (table 2) is at tip and base of the stem because that part is a place of scar on leaves. on the stem explants also formed shoot on 13 dap (table 2). callus and shoots formed on stem explants can be seen in figure 2. p0 p1 p2 p3 : soots, : callus figure 2. callus on stem explants with the treatment p0, p1, p2 and p3. leaves treatment of p1 (figure 1) showed that growmore + 0,25 mg/l bap foliar fertilizers able to become an alternative medium for callus induction, because growmore fertilizer contained sufficient nutrient. callus was formed on 13 dap which is compact texture, fresh green in the entire leaves surfaced and accompanied by swelling explants. the formation of callus is a sign that the explants are competent. according lubis (2016) chrysanthemum morifolium ramat cv merahayani which is cultured by growmore media 2 g/l did not significant with ½ ms media in media ½ ms in variable shoot height, shoot weight and number of roots. treatment p0 (½ ms +0,25 mg/l bap) in leaves explants could be callus inductions. callus was formed at 13 dap by initiating the formation of white grains then growing / forming calluses on the scar. (figure 1: p0). this is different from the growmore treatment where callus grows evenly over the surface of the leaf explants. the color of callus ½ ms is clear green and compact texture. callus color can be used as an indicator of the quality of the callus. callus which is green in color has good quality because it still contains chlorophyll (siregar et al., 2013). there was browning in the ½ ms (p0) explant (figure 1) which was caused by the presence of phenolic compounds which were derived from the role of the polyphenoloxidase enzyme. phenolic compounds are derived from plant secondary metabolites stored in vacuoles. when the explant is sliced, the vacuole breaks, the phenol compound is oxidized and then the compound is oxidized. this will cause the explants to turn brown. when phenol compounds are oxidized on the media, the media must be replaced. if not replaced it will lead to explant poisoning and death. (dwiyani, 2015). the treatment p2 (gandasil d + 0.25 mg/l bap) showed that the explants were able to form callus. the callus formed 13 dpa which compact texture, clear green color that is on the scar and surface of the explant (table.2). the callus that grows on the scar has a grainy texture so that the scar turns white. there is a little browning on scar in leaf explant indicated when cutting, however, the part that experienced browning could still induced callus on the surface of leaf explants (figure 1). the treatment p2 (gandasil + 0.25 mg/l bap) indicated that it could be used as an alternative medium for homescale tissue culture. nutrient that contained in foliar fertilizer gandasil d have contained sufficient nutrient for callus induction. matatula (2003) the addition of coconut water to gandasil d media caused an increase in plant height, number of leaves, number of roots, shoot wet weight and root wet weight in vitro. before the callus formed, leaf explants have some symptoms. on 3 dap the surface of the leaf explants began to become uneven and stretched up to 9 dap then on 13 dap leaf explant begin formed callus until 22 dpa. callus began to appear clearly almost all over the leaf surface. callus on the treatment p3 (mutiara + 0,25 mg/l bap) was compact in the texture and clear green color that grow up in the scar and surface leaf explant. p3 leaf explants on 3 dap have responded to the treatment of the media used this causes the leaf surface to be uneven and there are spots. this condition is stilled same until on 7 dap. the p3 leaf explants before forming the callus were stretched this is due to the role of the hormone cytokinins then in the scar begun to form clear white grains and spread on the leaf surface. some of the p3 leaf explants experienced browning at 9 dap. however, the explants are stilled able to form callus. callus begun to grow on leaf explants after 13 dap. leaf explants treated p3 can be classified as competent because they are able to form callus in response to the media. p3 treatment explant media (mutiara + 0,25 mg/l bap) can be used as an alternative media. treatment of the three leaf explants (figure 1) showed that foliar fertilizer has the potential as a alternative medium to replace ½ ms. the treatment of p1 leaf explant (growmore +0,25 mg/l bap) yielded the best callus then the others treatment. because callus on the p1 leaf explant has a fresh green color and grow entire the surface leaf explant (on surface and inside). naser & wisnu – callus induction of leaves and stems in krisan … 113 stem the callus was formed from stem explant that showed the equal result (table.2). callus on the treatment ½ ms, growmore, gandasil d and mutiara appeared on 7 dap. the four treatments indicated that there is potential of the media in growing callus on stem explants. stem explants which formed callus indicated that organogenesis had occur. the formation of callus on stem explants is due to the presence of endogenous auxin hormones that initiated in the scar. callus is formed at the tip and base of the stem in the explant because that position has been injured (figure 2). shoots appeared on the nodes in all treatments stem explants (½ ms, growmore, mutiara and gandasil d) after 13 dap until 22 dap. shoot formation occurs due to the role of the hormone cytokinin (bap). according dwiyani (2015). if want to form callus on the explants, the explants are planted in callus inducing media like medium + auxin. however, if want to shoot inducing, the explant planted in shoot inducted, like medium + sitokinin (bap). its can be inferred that the stem explant has cytokinin hormone higher than auksin. stem explant in p1 treatment (growmore + 0,25 mg/l bap) (figure 2) formed callus at stem tips scar. the texture callus is compacted and clear green color that appear on 7 dap. callus was formed due to endogenous auxin hormone in stem. shoots grow on the nodes stem after 13 dap in scar. the p1 treatment stem explants were swollen due to the effects of the cytokinin hormone. new leaves grow on shoots after 15 dap and the callus is getting bigger. p2 treatment stem explants (gandasil d + 0.25 mg/l bap) (figure 2) were able to form callus and shoots. browning appear on p2 treatment started 3 dap, although browning, callus can still be induced on the cutting marks. callus was formed at the base of stem while shots are formed in nodus. in 7 dap, callus can be seen with clearly, compact texture and clear green color. meanwhile, shoots can be seen clearly on 13 das. stem explants in p2 treatment (gandasil d + 0,25 mg/l bap) get the best callus than the others treatment. two young leaves clearly appear on the shoot after 15 dap. callus and shoot have pale green color and getting a bigger until 17 dpa. the color of callus turns white and shoot color getting green on 20 dpa. in p0 treatment (½ ms +0,25 mg/l bap) (figure 2), callus appeared on 7 dpa on the cut marked at stem tips. on 9 dpa, browning and callus could be seen clearly. two leaves appear on the shoot at nodus stem on 13 dpa. after that the new leave appeared in shoots on 15 dpa. the stem of p0 treatment explant still looked fresh. the leaves on the shoot begun to enlarge and the callus can still be seen clearly on 20 dpa. browning appeared in the p3 (mutiara+ 0,25 mg/l bap) treatment stem explant getting on 3 dpa. on 6 dpa the stem explant is still green fresh and the callus appeared in stem explant started on 7 dpa at cutting marked stem tips. the callus was compact in texture and had a clear white color, but the callus formed was not as much as in the p0 stem explant treatment. callus was seen more clearly at 9 dap and 2 new leaves appeared on shoots located on the nodus stem on 13 dap. on 15 dap there are addition two new leaves again on the stem. on 17 dpa there are new leaves again on the explant stem shoots. the shoots are still developed and the color of stem still fresh green until 20-22 dpa. the stem of p3 treatment has a characteristic that the shoot bigger than p0 treatment. but the number of leaves formed on the stem explants in treatment p3 was less than in treatment p0. conclusion the foliar fertilizer were able to induce callus in the chrysanthemum morifolium ramat cv dewi ratih in vitro, the p1 treatment (growmore) yielded the best callus in the leaf explants and the p2 treatment (gandasil d) yielded the best callus in the stem explants. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references badan pusat statistik dan direktorat jendral holtikultura (2017) luas panen, produksi dan produktivitas tanaman krisan, 2016-2017. dwiyani, r (2015) kultur jaringan tanaman, pelawa sari percetakan & penerbit., bali. gunawan, l.w (1987) teknik kultur jaringan tumbuhan. pau bioteknologi ipb. bogor. hariyati, m., i. bachtiar, & p. sedijani (2016) induksi kalus tanaman krisan (chrysanthemum morifolium) dengan 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(2012). studi pengaruh substitusi hara makro dan mikro media ms dengan pupuk majemuk dalam kultur in vitro krisan. jurnal hortikultura, 22(4), 334-341. doi: http://dx.doi.org/10.21082/jhort.v22n4.2012.p334-341 siregar, l.h., siregar l.a.m., putri, l. p (2013) pengaruh α benzil aminopurin dan αasam asetat naftalena terhadap pertumbuhan akar boesenbergia flava secara in vitro. jurnal agroekoteknologi 1 (3): 511-522. sun, q.l., s. hua, s., ye, j.h., zheng, x.q., & liang, y.r (2010) flavonoids and volatiles in chrysanthemum morifolium ramat flower from tongxiang county in china. african journal of biotechnology 9(25):3817-3821 vidyasagar, k (2006) national conference on plant biotechnology. china. widiastoety, d., & -, p. (2003). pengaruh bubur ubikayu dan ubijalar terhadap pertumbuhan plantlet anggrek dendrobium. jurnal hortikultura, 13(1), 1-6. doi: http://dx.doi.org/10.21082/jhort.v13n1.2003.p1-6. naser & wisnu – callus induction of leaves and stems in krisan … 115 table s1. observation of daily development on leaf explants. date ms mutiara gandasil d growmore 26-07-2019 3 dpa uneven surface, there are spots uneven surface, there are spots starting to warp, uneven surface. some look flat, uneven surface. 29-07-2019 6 dpa still same the day before still same the day before still same the day before many are curved, uneven surface 30-07-2019 7 dpa still same the day before still same the day before still same the day before still same the day before 1-08-2019 9 dpa more uneven surface more uneven surface, browning in edge still fresh, uneven surface uneven surface, still fresh 5-08-2019 13 dpa enlarged leaves and callus appeared there are already 9 explants with callus appeared callus in 1explant dan 1 browning enlarged leave and callus appeared 7-08-2019 15 dpa browning appeared, 1 explant enlarged callus visible more clearly and enlarged callus visible more clearly and leaf enlarged callus visible more clearly at cutting marks and surface 9-08-2019 17 dpa still same the day before the part of leaf browning callus visible more clearly increased callus and clearly, explant enlarged 12-08-2019 20 dpa some of the leaves have blackened and enlarged leaf still fresh, callus visible 1 explant browning increased callus and exsplan enlarged 14-08-2019 22 dpa there were explants that just appeared callus leaf still fresh, callus visible callus is visible on almost all surfaces 3 explants browning and 1 explant appeared callus table s2. observations of the daily development of observations on the stem. date ms mutiara gandasil d growmore 26-07-2019 3 dpa still green fresh, browning at cutting edges. still green fresh, browning at cutting edges. still green fresh, browning at cutting edges still green fresh browning at cutting edges 29-07-2019 6 dpa still green fresh, browning at cutting edges. still green fresh, there is browning at the base of the cut still green fresh, browning at cutting edges still green fresh, some of explant browning at cutting edges 30-07-2019 7 dpa callus appeared at cutting marks base brown, callus appears but not as much as ms callus appears but not as much as ms callus appeared. 1-08-2019 9 dpa the callus on the cut mark was clearly visible, browning is starting to clear callus is starting to clear, some explant browning callus clearly, the stem begins to swell at the bottom (elongation) callus is starting to clearer, the stem begins to swell 5-08-2019 13 dpa shoots are visible, there are 2 new leaves on nodus, new shoots shoots are visible, callus is still visible on the cut marks. callus still fresh, cut mark browning, new shoots at nodus. new shoots at nodus. callus still fresh. 7-08-2019 15 dpa 3 shoods appeared in nodus 3 new leaves, the leaves are longer and clearer looks new shoots tips, 2 new leaves the callus is getting bigger, young leaves grow, there are no new leaves yet 9-08-2019 17 dpa new shoots and new leaves grow in the nodus shoots tips still fresh, small leaves become large and there are new leaf candidates the callus is getting bigger, there is new leaf, then another candidate for leaves appears again, green clear color new leaf, callus still fresh 12-08-2019 20 dpa the stems are growing and still fresh, leaves start bigger, callus is still clearly visible new shoots develop on the nodes the callus is getting whiter, the leaves are getting green clearly . still same 14-08-2019 22 dpa still bigger explant and shoots are still small. green shoots color, looks bigger than ½ ms but leaves less leaf green clearly, leaves a little but big. there are some that haven't appeared yet. dark green shoot color this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 75-81 | doi: 10.14421/biomedich.2022.111.75-81 issn 2540-9328 (online) phytochemical constituents of f. sagittifolia warburg ex mildbraed & burret leaves with antimicrobial activity olayombo margaret taiwo1,2,*, olaoluwa omosalewa olaoluwa1, olapeju oluyemisi aiyelaagbe1, josphat clement matasyoh2 1department of chemistry, faculty of science, university of ibadan, 200284, ibadan, nigeria 2department of chemistry, faculty of sciences, egerton university, 20115 egerton, kenya. corresponding author* olayombotaiwo@gmail.com manuscript received: 06 april, 2022. revision accepted: 07 june, 2022. published: 30 june, 2022. abstract the leaves and bark of ficus sagittifolia have been used as a cure for stomach and pulmonary disorders, respectively. the bark is edible and is taken against colic. from the leaves of f. sagittifolia, a steroidal glycoside named stigmast-5,22-diene-3-o-β-d-glucopyranoside 1 and three isoflavonoids named 5-hydroxy-3-(4-hydroxyphenyl)-7-methoxy-4h-chromen-4-one 2, 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one 3 and 5-hydroxy-3-(4-hydroxyphemyl)-8,8-dimethylpyrano[3,2-g}-chromen-4(8h)-one 4 were isolated, and this is the first report of the isolation of these compounds from this plant. the structural elucidation of the compounds was based on 1d and 2d nmr, ir and ms data analyses. compounds 1 and 2 inhibited the growth of pseudomonas aeruginosa and aspergillus niger at 6.25 mg/ml, respectively while compounds 2 and 4 were active against helicobacter pylori at 6.25 mg/ml. these findings corroborate the ethno-medicinal use of f. sagittifolia leaves as a treatment for stomach disorders. keywords: antimicrobial activity; f. sagittifolia; isoflavonoids; natural products; steroidal glycoside. abbreviations: fsl – ficus sagittifolia leaves; hex – hexane; etoac – ethyl acetate; tlc – thin layer chromatography; chcl3 – chloroform; meoh – methanol; hplc – high performance liquid chromatography; p –int – p-iodonitrotetrazollium; dmso – dimethylsulphoxide; nmr – nuclear magnetic resonance; 1h nmr – proton nuclear magnetic resonance; 13c nmr – carbon nuclear magnetic resonance; cosycorrelation spectroscopy; hsqcheteronuclear single quantum correlation spectroscopy; hmbc heteronuclear multiple bond correlation spectroscopy; tms – tetramethylsilane; hresims – high-resolution electrospray ionization mass spectrometry; lresims – low-resolution electrospray ionization mass spectrometry; ir – infrared spectroscopy; uv – ultraviolet; na – nutrient agar; tsb – tryptic soy broth; sdb – sabouraud dextrose broth; mic – minimum inhibitory concentration; mmc – minimum microbial concentration; (cd3)2so – deuterated dimethylsulpoxide; (cd3od) – deuterated methanol; multi – multiplet; j – coupling constant; tr – retention time; rf – retention factor. introduction ficus sagittifolia (warburg ex mildbraed & burret) belongs to the family moraceae and a member of the genus ficus. it is an epiphytic shrub, often on oil palms, becoming a tree to 30 feet high. it is native to benin, cameroon, ghana, guinea, guinea-bissau, ivory coast, liberia, nigeria, senegal, sierra leone, and togo (powo, 2022). ficus species have been used locally as stomach herbs, anthelmintics, vermonicides, astrigents, carminatives, hypotensives, and anti-dysentery drugs (salem et al., 2013). the leaves and bark of f. sagittifolia are used as a cure for stomach and pulmonary disorders, respectively. the bark is edible and is taken against colic (burkill, 1997). species of ficus are known to possess antioxidant, cytotoxic, antimicrobial, anti-inflammatory, antidiabetic, antiulcer, and anticonvulsant activities (salehi et al., 2021). phytochemicals such as genistein, protocatechuic acid, stigmatserol, rutin, bergapten, psoralen, alpinumisoflavone, betulinic acid, umbelliferone, apigenin, derrone, β-sistosterol and chlorogenic acid, amongst others, were common compounds reported in this genus (nawaz, et al., 2019; ateba et al., 2019). a recent study on the phytochemical screening of f. sagittifolia leaf and stem bark extracts revealed the presence of phenolics, flavonoids, steroids, tannins, saponins, alkaloids, and terpenoids with good antioxidant activities (taiwo and olaoluwa, 2020). however, there is little or no information on the isolation and characterization of phytochemicals constituents from f. sagittifolia to support its ethnomedicinal use. hence, this paper reports four known compounds from f. sagittifolia leaves and their antimicrobial activity for the first time. https://doi.org/10.14421/biomedich.2022.111.75-81 76 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 materials and methods plant collection and identification leaves of f. sagittifolia were harvested during the dry season in ikire, osun state, nigeria in april 2018. identification and authentication were done at the herbarium unit of the forest research institute of nigeria (frin), ibadan. a voucher specimen of f. sagittifolia (fhi 111988) was documented for reference purposes. plant preparation and extraction f. sagittifolia leaves were air-dried and subjected to pulverization. ground leaves were macerated in ethanol for 72 h and was concentrated under reduced pressure to give ethanol extract. the ethanol extract was partitioned in hexane and subsequently in ethyl acetate to obtain the respective fractions. general experimental procedure tlc was performed on precoated silica gel 60gf24 by merck. column chromatography with a column length – 60 cm; internal diameter of 32.5 mm; external diameter of 36.5 mm and silica gel of 60 – 200 mesh size was used as the stationary phase. eluates were concentrated at reduced pressure at 40°c using the buchi rotavapor r-200 rotary evaporator. compounds were purified using a shimadzu preparative hplc equipped with a uv detector, a reversed phase column (luna®, c18, 5 μm particle size, 10 × 2500 mm, phenomenex), a shimadzu lc – 20ap pump equipped with a dgu – 20a5r degassing unit, a shimadzu spd – m20a detector, and a shimadzu sil – 20acht autosampler, using labsolutions software. nmr experiments for 1h and 13c were performed on the bruker avance 600 and 700 mhz nmr spectrometers for 1h; 125 and 150 mhz nmr spectrometers for 13c. the readings were taken in deuterated methanol and deuterated dimethylsulphoxide with tms serving as reference. 1h and 13c nmr spectra were elucidated using mestrenova software. hresims were performed on a bruker daltonik maxis 4g equipped with ultra-high resolution time-of-flight electrospray ionization in both negative and positive ion modes. lresims were carried out on bruker amazone speed etd ion trap and 8-dionex ultimate 3000 lc in both negative and positive ion modes. in the analysis, thermo xcaliburqual computer software was used in analysis of the mass chromatogram. ir spectra were recorded on the perkinelmer spectrometer instrument. fractionation the ethylacetate fraction (10 g) of f. sagittifolia was subjected to column chromatography and fractionated by the gradient elution method using a solvent mixture of hexane and ethyl acetate as the mobile phase. a total of 102 fractions (100 ml) were collected from the column, and fractions with similar tlc profiles were pooled together to obtain 16 fractions (fsl 1-16). isolation and purification of compounds fraction 15 was eluted by hex/etoac (10:90), as a white precipitate to give compound 1 (92.2 mg) which was soluble in dmso and was confirmed as a spot on the tlc plate (rf = 0.58). fractions 13 (hex/etoac, 30:70), 14 (hex/etoac, 20:80) and 16 (hex/etoac, 0:100) were further purified using preparative hplc analysis. the solvents used were double distilled water with 0.1% formic acid as solvent a and hplc grade meoh as solvent b. gradient elution was carried out with 60% meoh for 7 min and thereafter, isocratic condition at 100% meoh for 5 min. the system returned within 0.5 min to the initial condition of 60% meoh and was equilibrated for 10 min. the eluted fractions were detected by absorbance between 254 370 nm and the flow rate was 3 ml/min to obtain compounds 2 (tr = 15.2 min; 2.1 mg), 3 (tr = 18.3 min; 5.3 mg) and 4 (tr = 18.9 min; 4.4 mg). subsequently, compounds 1 – 4 were subjected to spectroscopic analysis. antimicrobial assay microbial cultures gram negative bacteria: escherichia coli (atcc 11175), pseudomonas aeruginosa (atcc, 27853), salmonella typhi (atcc, 14028), helicobacter pylori, and fungi: aspergillus niger and candida albicans were maintained on na and sda, respectively. a single colony of each organism was inoculated into 5 ml of tsb and sdb for the preparation of bacterial and fungal cultures, respectively. all the microbes were subcultured from the original culture and incubated overnight at 37°c for 24 h and at 25°c for 48 h for bacteria and fungi respectively. the bacteria except h. pylori were obtained from the pharmaceutical microbiology department, university of ibadan, ibadan, nigeria, and the fungi and h. pylori were obtained as clinical isolates from the university college hospital (uch), ibadan. the standard drugs, gentamycin and ketoconazole, were used as the positive control. the negative control used was 1% dmso. minimum inhibitory concentration and minimum microbial concentration the antimicrobial assay was done using the broth microdilution method and 96-well plates were used. compounds 1-4 were dissolved separately in dmso to obtain a stock solution of 50 mg/ml each. this was diluted serially in the microplate wells to obtain a concentration range of 25 to 0.391 mg/ml. tsb was used, and standard drugs were gentamycin and ketoconazole (10 𝜇g/ml) for the anti-bacterial and antifungal assays, respectively. the reference drugs were also diluted to obtain concentration range of 10 – 0.3125 taiwo et al. – phytochemical constituents of f. sagittifolia … 77 𝜇g/ml. each of the microplate wells was inoculated with 10 𝜇l of the test organisms and incubated at 37°c and 25°c for 24 h and 48 h for bacteria and fungi, respectively. the least concentrations that showed no growth or turbidity after hours of incubation were streaked on na and sda for bacteria and fungi, respectively. the concentration with no trace of growth was taken as the mic. also, after incubation, 10 𝜇l of 0.2 mg/ml p –int was added to each well and incubated for another 30 min at 37°c. wells with a color change from yellow to pinkish red were an indication of microbial growth. the least concentration that showed no trace of color change was taken as the mmc (bacteria/fungi). results and discussion phytochemical investigation the ethyl acetate fraction of f. sagittifolia leaves yielded a steroidal glycoside 1, and fractions 13, 14, and 16 that were purified by preparative hplc gave three isoflavonoids, 2 4 respectively. stigmast-5,22-diene-3-o-β-d-glucopyranoside (stigmasterol glycoside) 1 was isolated as a white powder. lresims gave [m h] ion at m/z 573.75 with the molecular formula c27h35o8 (calculated for c27h35o8; 574.843). in the 1hnmr spectrum of 1 (table 1), the presence of six methyl protons was confirmed by signals at δh 0.67 s (h-18), 0.91 s (h-19), 0.96 d (h-21), 0.81 m (h-26), 0.85, 1.39 m (h-27) and 0.82 d (h-29); three olefinic protons at δh 5.38 (h-6, t, j=4.8 hz), 5.13 (h-22, dd, j=9.0 hz, 13.8 hz) and 5.03 (h-23, d, j=8.4) and one anomeric proton at δh 4.18 (h1’, d, j=7.8 hz). the 13cnmr spectrum showed six signals in the methyl region (δc 12.2, 12.3, 19.1, 19.4, 20.2 and 23.1). four methine resonances at δc 73.9, 77.2, 70.6 and 77.1, as well as one methylene resonance at δc 61.6, were due to c-2’, c-3’, c-4’, c-5’, and c-6’, respectively, of the β-d-glucopyranoside. (table 1). the olefinic resonances at δc 121.7, 138.5 and 129.3 corresponded to the c-6, c-22, and c-23 methine carbons. an anomeric carbon signal at δc 101.2 indicated the presence of a sugar d-glucose moiety. the value of j = 7.8 on 1' (anomeric proton) reflected an axial-axial position to the c-2' proton, which confirms that the glucopyranoside moiety binds to the sterol moiety in a β-position (silverstein et al., 1962; bai et al., 2005). the connectivity of protons and carbonprotons was determined from a combination of cosy, hsqc, and hmbc data as shown in table 1. the ir band (cm-1) at 3589, 2850, and 1104 are typical for hydroxyl, carbon-hydrogen, and carbon-oxygen groups, respectively. this explanation was based on a comparison of 1h and 13cnmr spectra with those previously reported in literature (valizadeh et al., 2014; ilango, 2018; olawumi and koma, 2019). 5-hydroxy-3-(4-hydroxyphenyl)-7-methoxy-4hchromen-4-one (prunetin) 2, is pale yellow in color and powdery, with a molecular formula of c16h12o5, m/z of 285.0756 [m+h] + in hresims (calculated for c16h12o5, 284.2634). sixteen signals were displayed from the 13cnmr spectrum of 2 (table 2). seven aromatic carbons were observed at δc 91.8 (c-8), 97.8 (c-6), 114.9 (c-3’, c-5’), 130.0 (c-2’, c-6’) and 153.7 (c-2). four oxygenated aromatic carbons at δc 157.5 (c4’), 158.2 (c-9), 162.3 (c-5) and 165.9 (c-7). one carbonyl carbon at δc 181.1 (c-4). the δc 55.1 (och3, c-7) and δh 3.91 (3h, s, h-7) as well as the hmbc correlations confirmed the presence of a methoxy group at position c-7. the 1h nmr also showed two doublets of four aromatic protons with vicinal coupling constants. the δc 153.7 (c-2) is a characteristic of ring b of an isoflavone (table 2). the cosy correlation of h-6 with h-8 and h2’/3’ with h5’/6’ supported the positioning of these four aromatic protons. the hmbc correlations from the methoxy protons (δh 3.91) to c-7 supported the attachment of the methoxy group to c-7. the ir spectra indicated the presence of hydroxyl, carbonyl, and aromatic groups (3671, 1717, and 1454 cm-1, respectively). this elucidation was in agreement with the literature (máximoa et al., 2002; awouafack et al., 2011). 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one (derrone) 3, is a pale yellow powder with a molecular formula of c20h16o5, m/z of 337.1070 [m+h] + in hresims (calculated for c20h16o5, 336.3). the presence of an isoflavone skeleton was confirmed from the 1h nmr and 13c nmr spectra, with proton and carbon signals at δh 7.43 (h-2 ’), δc 153.4 (c-2), δc 123.6 (c-3) and δc 181.1 (c-4). the the 1h nmr showed two doublets of four aromatic protons with vicinal coupling constants and one proton signal at δ 6.23 s of aromatic proton in ring a. the 2,2-dimethylpyran substituent was confirmed by signals at δh 5.73 (h-3’’) d (j3’’/4’’ = 9.8 hz), δh 6.78 (h-4’’) d (j3/4, 8.4 hz), δh 1.50 (h5’’me,6’’me), δc 127.4 (c-3’’), δc 114.9 (c-4’’) and δc 27.0 (c-5’’/6’’) whose location in ring a was established by hmbc correlation of a methine carbon δc-6 99.4 with a methine carbon δc-5 161.9 and a quaternary carbon δc-9 159.5 (table 3). the ir bands at 3714cm-1 and 1635cm-1 are typical for hydroxyl and a α,-β unsaturated ketone group respectively. this elucidation was also in agreement with those reported in literature (maximoa et al., 2002; ediziri et al., 2012). 5-hydroxy-3-(4-hydroxyphemyl)-8,8dimethylpyrano[3,2-g}-chromen-4(8h)-one (alpinumisoflavone) 4, is pale yellow and powdery, having a m/z of 337.08 [m+h]+ in lresims with a molecular formula of c20h16o5. a pair of doublets δh 6.87 d (j=7.7 hz) and 7.41 d (j=7.0 hz) integrated for two protons and were allocated to the h-3’/h-5, h-2’/h6’ of the parasubstituted aromatic ring. the high chemical shift of δh 6.87 (j=7.7 hz) signified the 78 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 presence of an oxygened substituent (oh) at c-4’of the aromatic nucleus. the ring b of the isoflavone was also confirmed with signals at δh 7.41 (h-2), δc 153.5 (c-2), δc 123.4 (c-3) and δc 181.0 (c-4). the evidence of proton signals at δh 5.75 (h-3’’) d (j3’’/4’’ = 9.8 hz), δh 6.71 (h-4’’) d (j3/4, 10.5 hz), δh 1.48 (5’’me/6’’me) and carbon signals at δc 128.2 (c-3’’), δc 114.7 (c-4’’) and δc 27.1 (5’’/6’’) confirmed the attachment of the 2,2-dimethylpyran substituent on ring b. compounds 3 and 4 have the same molecular formula and mass but different structures due to the position of the 2,2dimethylpyran substituent on ring b. the positon was further confirmed using hsqc and hmbc, which revealed different chemical shifts for δc (c-6) and δc (c8) and different hmbc correlations as shown on table 4. the δc (c-6) is a quaternary carbon in 4, whereas in 3, δc (c-6) is a methine carbon and vice versa for δc (c-8). the ir spectra indicated the presence of hydroxyl, carbonyl, and aromatic groups (3571, 1717, and 1454 cm-1). this spectral data was compared to those previously published for alpinumisoflavone (rahman et al., 2007; hussain et al., 2011 tjahjadarie et al., 2016). table 1. 1h nmr (600 mhz) and 13c nmr (150 mhz), hmbc, cosy assignment of compounds 1 in (cd3)2so. p o si ti o n δc, type δh mult. (j in hz) hmbc (h/c) c o s y 1 37.3, ch2 0.99, m;1.78, m c-14, 4 2 29.7, ch2 1.23, m; 1.81, m 3 77.4, ch 3.46, m 4 42.2, ch2 1.96, m 5 140.9, c 6 121.7, ch 5.38, d (j=4.8) c-8, 10 h-8 7 31.8, ch2 1.39, m 8 31.9, ch 1.53, s c-6,7,13,14 h-6 9 50.1, ch 0.88, m 10 36.7, c 11 21.1, ch2 0.97, m 12 38.5, ch2 2.11, m 13 42.3, c 14 56.6, ch 0.99, m 15 39.6, ch2 1.95, m c-13 16 24.3, ch2 1.54, m c-5,6,8,14 17 55.9, ch 1.10, m 18 12.2, ch3 0.67, s c-4,14 19 19.1, ch3 0.91, s c-17, 20 20 36.0, ch 1.30, m 21 19.4, ch3 0.96, d (j=7.2) h-29 22 138.5, ch 5.13, dd (j=9.0, 13.8) 23 129.3, ch 5.03, dd (j=8.4, 15.0) 24 45.6, ch2 0.92, m 25 29.1, ch 1.63, m 26 19.6, ch 0.81, m 27 20.2, ch3 0.85, m; 1.39, m 28 23.1, ch3 0.76, m 29 12.3, ch3 0.82, d (j=7.2) c-24, 25, 26 h-21 1’ 101.2, ch2 4.18, d (j=7.8) c-3’ 2’ 73.9, ch 2.89, m 3’ 77.2, ch 3.12, m 4’ 70.6, ch 3.01, m c-5’,6’ 5’ 77.1, ch 3.06, m 6’ 61.6, ch2 3.39, m; 3.63, m c-5’ taiwo et al. – phytochemical constituents of f. sagittifolia … 79 table 2. 1h nmr (700 mhz) and 13c nmr (175 mhz) assignment of compounds 2-4 in (cd3od). 2 3 4 position δc, type δh mult. (j in hz) δc, type δh mult. (j in hz) δc, type δh mult. (j in hz) 2 153.7, ch 8.15, s 153.4, ch 8.17, s 153.5, ch 8.10, s 3 121.8, c 121.6, c 121.7, c 4 181.1, c 181.1, c 181.0, c 5 162.3, c 161.9, c 157.5, c 6 97.8, ch 6.40, d (j=2.8) 99.4, ch 6.23, s 105.2, c 7 165.9, c 152.2, c 156.3, c 8 91.8, ch 6.58, d (j=2.1) 105.6, c 94.4, ch 6.38, s 9 158.2, c 159.5, c 159.4, c 10 105.7, c 101.1, c 105.6, c 1’ 123.6, c 123.6, c 123.4, c 2’, 6’ 130.0, ch 7.42, d (j=8.4) 130.0, ch 7.43, d (j=11.9) 130.0, ch 7.41, d (j=7.0) 3’,5’ 114.9, ch 6.88, d (j=9.1) 114.9, ch 114.9, ch 6.87, d (j=7.7) 4’ 157.5,c 157.5, c 157.4, c 2’’ 78.0, c 77.9, c 3’’ 127.4, ch 5.73, d (j=9.8) 128.2, ch 5.75, d (j=9.8) 4’’ 113.9, ch 6.77, d (j=8.4) 114.7, ch 6.71, d (j=10.5) 5’’, 6’’ 2.70, ch3 1.50, s 27.2, ch3 1.48, s och3 55.1, ch3 3.91, s (j=2.8) antimicrobial assay the broth dilution method was used to determine the mic and mmc of compounds 1-4 against different bacteria and fungi strains: escherichia coli (atcc 11175), pseudomonas aeruginosa (atcc, 27853), salmonella typhi (atcc, 14028), helicobacter pylori, aspergillus niger and candida albicans. compounds 14 showed good antimicrobial activity as demonstrated by their mic values (6.25-25 mg/ml), in table 3a. the lowest mic value (6.25 mg/ml) was obtained from compound 1 and 2 against p. aeruginosa and a. niger. at the same concentration, compounds 2 and 4 inhibited the growth of h. pylori, among others. compounds 2, 3, and 4 showed moderate inhibition (mic =12.5 mg/ml) against the growth of e. coli while for compound 1, 25 mg/ml was required. compounds 1, 3 and 4 inhibited the growth of c. albicans at a concentration of 12.5 mg/ml. compound 2 had the lowest mic and mmc values against s. typhi (12.5 mg/ml) and h. pylori (6.25 mg/ml), respectively in table 3a. gentamicin had an mic value ranging from 10 to > 10 µg/ml while ketoconazole had an mic values ranging from 1-0.5 µg/ml. overall, compounds 2 and 4 showed very good antimicrobial activity against all the test organisms among others. flavonoids and isoflavonoids are wellknown natural products with extensive pharmacological activities and extremely low toxicity (wang et al., 2020). more importantly, they possess a wide range of biological activities, such as antibacterial, antifungal, antiviral (dastidar et al., 2004; orhan et al., 2010), antitumour (kopustinskiene et al., 2020), antiinflammatory (sychrová et al., 2020) and antiaging activities (gupta et al., 2014). table 3a. mic and mmc of compound 1-4. organisms compound 1 compound 2 compound 3 compound 4 mica mmca mica mmca mica mmca mica mmca h. pylori 12.5 12.5 6.25 6.25 12.5 25 6.25 6.25 e. coli 25 25 12.5 12.5 12.5 12.5 12.5 12.5 p. aeruginosa 6.25 12.5 25 25 25 25 12.5 12.5 s. typhi 25 25 12.5 12.5 25 25 25 25 a. niger 25 25 6.25 25 12.5 12.5 12.5 12.5 c. albicans 12.5 25 25 25 12.5 12.5 12.5 25 note: a: mg/ml table 3b. mic and mmc of bacteria and fungi standards. organisms gentamycin ketoconazole mica mmca mica mmca h. pylori 10 >10 na na e. coli 10 10 na na p. aeruginosa >10 >10 na na s. typhi 10 10 na na a. niger na na 1 1 c. albicans na na 0.5 0.5 note: a: µg/ml 80 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 conclusions a steroidal glycoside namely, stigmast-5, 22-diene-3-oβ-d-glucopyranoside (stigmasterol glycoside) 1 and three isoflavanoids, namely 5-hydroxy-3-(4hydroxyphenyl)-7-methoxy-4h-chromen-4-one (prunetin) 2, 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one (derrone) 3, and 5-hydroxy-3-(4-hydroxyphemyl)-8,8dimethylpyrano[3,2-g}-chromen-4(8h)-one (alpinumisoflavone) 4, were isolated from the ethylacetate fraction of f. sagittifolia leaves for the first time. compounds 1 and 2 showed inhibition against p. aeruginosa and a. niger; 2 and 4 inhibited the growth of h. pylori. therefore, compounds 1, 2, and 4 could be antimicrobial agents for diseases related to stomach disorders. this study also provided a scientific justification for the ethnomedicinal use of f. sagittifolia leaves for the treatment of stomach disorders, particularly those caused by microbes. acknowledgements: o. m. taiwo is thankful to the department of chemistry, university of ibadan for the equipment rendered for the extraction and ir studies of samples. o. m. taiwo is grateful to african german network of excellence in science (agnes) for a research visit to the department of chemistry, egerton university, kenya which enabled the purification and characterization of the compounds and also to the members of j.c.m. research group. all authors are grateful to the institute of environmental research (infu), tu-dortmund for nmr and ms equipment. dr. s. a. odewo is acknowledged for the identification and authentication of the plant species. competing interests: the authors declare that there are no competing interests. funding: this study was financially supported by the african german network of excellence in science (agnes), through the programme “advocating women in science, technology, engineering, and mathematics’’ with a mobility scholarship to o.m.t. references ateba, s.b., mvondo, m.a., djiogue, s., zingué, s., krenn, l. and njamen, d., 2019. a pharmacological overview of alpinumisoflavone, a natural prenylated isoflavonoid. frontiers in pharmacology, 952. doi: https://doi.org/10.3389/fphar.2019.00952 awouafack, m.d., spiteller, p., lamshoft, m., kusari, s., ivanova, b., tane, p., & spiteller m. 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(2020). naturally occurring flavonoids and isoflavonoids and their microbial transformation: a review. molecules, 25(21), 5112. doi: https://doi.org/10.3390/molecules25215112 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 33-39 | doi: 10.14421/biomedich.2021.101.33-39 issn 2540-9328 (online) ethnobotanical study of edible plant communities on the slopes of mount merapi and merbabu, indonesia lita ayu umartani*, maizer said nahdi** department of biology, faculty of science and technology, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no.1 yogyakarta 55281, tel. + 62-274-540971, fax. + 62-274-519739, indonesia corresponding author ayulita1998@gmail.com*, maizer.nahdi@uin-suka.ac.id** manuscript received: 09 april, 2021. revision accepted: 13 july, 2021. published: 19 july, 2021. abstract ethnobotany is a study of the interaction between local people and their natural environment, especially regarding the use of plants as food and medicinal ingredients. edible plants are a daily basic need whose existence is a necessity for the people on the slopes of mount merapi and merbabu. how to use plants is transferred from generation to generation to form a culture. the research was carried out in march-may 2020 with the aim of digging local knowledge about plants used as food, including staple food, vegetables and medicines by studying the species diversity, including the benefits of plant parts, habit, how to use, process and how to obtain these species. the data were collected by using a combination of qualitative and quantitative methods with in-depth interviews through 40 respondents who were selected by purposive sampling. the results showed that the communities on the slopes of mount merapi and merbabu used 74 plant species from 37 families as food sources. the favorite family is fabaceae, followed by zingiberaceae and solanaceae. the most widely used habitus of plants were herbs (36.49%), followed by bush (28.38%), shrubs (18.92%) and trees (16.22%). plant parts that are widely used are leaves (29.73%), fruit (17.57%), tubers (10.81%), seeds (9.46%), roots, rhizomes and flowers (6.76%), shoots (5.40%), stems (2.70%) and water, skin and heartwood (1.35%). the most common ways to use it are eaten raw as vegetables (29.73%), boiled (16.22%) and drink (12.16%). how to obtain it are through own cultivation (72.97%), and buying at the market (21.62%). the highest use values were adas (foeniculum vulgare mill.) (0.25), chili pepper (capsicum annum l.) (0.20), turmeric (curcuma dosmetica loir) and water spinach (ipomoea aquatica forsk.) (0.17). the highest importance values were rice (oryza sativa l.) (5.23%), and fennel (foeniculum vulgare mill.) (4.57%). keywords: culture; ethnobotany; in-depth interviews; rice (oryza sativa l.); mount merapi; mount merbabu. introduction the existence of plants is a pleasure and a blessing given by allah swt to his creatures in interacting and managing nature. humans are given a mandate from allah, namely first, allah allows humans to take and make the best use of natural products, humans are required to be able to take lessons from natural phenomena and events that occur. humans are required to maintain and maintain environmental sustainability (zuhdi, 2012). ethnobotany is a study of the interaction between local communities and their natural environment, especially regarding the use of plants in their daily life (martin, 1998). these uses can be in the form of plants as food and medicine. the demand for food has undergone a shift from being very dependent on nature, switched to unhealthy fast food (anggraini, 2013). some people nowadays are starting to realize the importance of healthy living by trying to consume foods that have physiological effects to the body to get maximum health (astawan, 2003). based on the food law (uu) number 18 of 2012, food is anything that comes from biological sources intended for human consumption that has certain functions to the body, used for medicinal purposes and as daily food (hariyani, 2013). each region has a specific plant use system and differs from other regions according to the diversity of plants in its environment. this includes the use of food on the slopes of mount merapi and merbabu to support human life. the area is well-known as a center for producing green vegetables with a majority of the population of farmers, besides that the area also has high enough rainfall causing the soil to become fertile and the abundance of plants and has a high variance. edible plants in the area are used by the community as medicinal plants, food as well as traditional ceremonies used in everyday life. how to use plants by the community is obtained and has been carried out from generation to generation to form a culture (gunawan, 2014). advances in science and technology as well as the increase in the level of public education are very influential in the knowledge of the younger generation https://doi.org/10.14421/biomedich.2021.101.33-39 34 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 of the culture that has been formed in society. one such knowledge is the use of the diversity of edible plants, resulting in this knowledge being increasingly marginalized, even though so far little has been stated in writing. based on this, a comprehensive research is needed to answer the problems that arise, namely how the community's knowledge of the use of food plants on the slopes of mount merapi and merbabu, precisely in mliwis village, cepogo, boyolali on plants that are used as food, including use as staple food, vegetables and medicine by specifically studying the diversity of species, including plant parts, habits, their use, processing methods and how to obtain these species. materials and methods research period and location the research was carried out on the slopes of mount merapi and merbabu, mainly in mliwis village, cepogo district, boyolali regency, covering 2 hamlets, namely baksari and ngarsapura (figure 1). the reason for choosing the two locations was because these two hamlets were the centers of agricultural and food production in the boyolali area. the research began in march may 2020. mliwis is located at 110 "22" 110 "50" east longitude and 7 "36" 7 "71" ls. figure 1. research locations on the slopes of mount merapi and merbabu, in the hamlets of baksari and ngarsapura. data collection data collected by all edible plants that have been used by the people on the slopes of mount merapi and merbabu including staple food, vegetables, medicines including species, plant parts, habits and methods of use. this information was obtained with the help of 40 respondents who were selected by purposive sampling with a total of 10% of the number of households (heads of households) in the study location. collecting data on communities consisting of traditional, local, vegetable farmers, vegetable sellers, herbalist sellers and other users through in-depth interviews through open questionnaires to be analyzed descriptively, qualitatively, and quantitatively. plants found were identified using the book flora of java volume i (1963), volume ii (1965), and volume iii (1968) by backer and van der brink jr., and indonesian useful plants by heyne (1987). meanwhile, quantitative data are used for botanical data collection, including inventory and identification of plants used by the community as food plants. the benefit value data uses a modified formula from cotton (1996) and nahdi and ardyan et al., (2019), as follows: 1. use value uvs = information: uvs : total number of use values of a type s uvis : total value of type s given by the informant i is : total number of informants interviewed for the type s use value 2. important value importance value index calculation using a formula iv = x 100 % information: iv : important value ni : number of respondents using type a n : total number of respondents the profile of respondents a b c figure 2. the profile of the respondents in the studied area based on: a. education; b. gender; c. employment. respondents are considered as representatives of the people on the slopes of mount merapi and merbabu, represented by the mliwis community as users of edible plants. they were selected from various profiles of gender, educational level and occupation. the percentage of female respondents are higher than male sd 40% smp 30% sma 30% female 60% male 40% farmer groups 10% vegetable farmers 20% food plant users30% vegetable sellers 17,5% guest sellers 10% elders 12,5% information: ngarsapura baksari umartani & nahdi – ethnobotanical study of edible plant communities on … 35 respondents (60% and 40% respectively). based on the level of education, 40% of them are only elementary school (sd) graduates, 30% junior high school (smp) graduates, and 30% junior high school (sma) graduate. according to occupations include vegetable farmers (20%), vegetable sellers (17.5%), elders (12.5%), farmer groups (10%), guest sellers (10%) and food plant users (30%). meanwhile, according to gender 60% of them are women, and 40% are men (figure 2). results and discussion a b c d figure 3. the summary of information on the use of edible plants based on: a. habitus; b. the part of the plant being used; c. how to use; d. the locations where the plants being collected. the diversity of edible plants. based on the results of the research, the people on the slopes of mount merapi and merbabu use 74 species of food plants from 37 families (table 1) with details, baksari village has 64 plant species as food used by the community and ngarsapura village has 59 species from 40 respondents. the same number of species found in the two hamlets is used by the community either as daily staples, vegetables or medicine. the number of food and medicinal plant species in this study is more than the research conducted by nita (2017) in the arfak tribe, namely 29 species of edible plants and 16 species of medicinal plants. this is because the use of edible and medicinal plants by local communities is different from one region to another. some of the factors that influence it are the efficacy and availability, language, social relations, cultural history, understanding, local beliefs and beliefs (silalahi, et al., 2018). usually, food and medicinal plants that are easily available with sufficient availability will be more used by the community. however, gudro, et al., (2014) stated another reason, where the factors that influence the use of plants by the community is the purpose of their use. the food plants, most widely used by the people on the slopes of mount merapi and merbabu come from the fabaceae family, which is used as a vegetable, followed by the zingiberaceae family as a cooking and medicinal spice. meanwhile, there are fewer types of plants from other families. previous research conducted by zulharman (2015) in the sambori tribe, bima, east nusa tenggara showed the same results, namely the fabaceae family is the family most widely used by the community as food. based on the results of interviews with respondents, the main reason for the higher use of plants of the fabaceae family is that these families are easily found in gardens, yards and in markets for sale. zulharman (2015) adds, the fabaceae family is widely used by the community because it is easy to cultivate and does not really need specific soil. in addition, according to permana et al., (2011) that almost every community has unique local wisdom as an adaptation to the environment and food in the form of fruit, vegetables and medicinal. plant habitus used as food. the results showed that there were four habitus of plants that were used by the community as food ingredients, namely trees, shrubs, shrubs and herbs. herbs are the most widely used habits habitus with a percentage of 36.49% of 74 species, followed by bush (28.38%), shrubs (18.92%) and trees (16.22%). the results of research conducted by nita (2012) in the arfak tribe, manokwari regency, sukmawati, et al., (2013) in toga village, central sulawesi and nahdih, et al., (2016) in the turgo area, yogyakarta showed the same results, namely the majority of herbal habitus are the habitus most widely used by people as raw material for food and medicine with varying percentages. the reason the people of the slopes on mount merapi and merbabu use more of the herb and bush habitus as food plants compared to others, which is easy to obtain and has highly adapted to the environment so that the growth rate and development are very fast and easy to 36. 49 0 5 10 15 20 25 30 35 40 29.73 se e d fr u it le a f tu b e rs ro o t r h iz o m e fl o w e r sh o o ts w a te r st e m sk in h e a rt 29.73 b o il e d p o u n d e d sh re d d e d fr ie d ro a st e d st e a m e d b u rn e d co co n u t m il k v e g e ta b le d ru n k d ir e ct ly … 72, 97 36 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 grow in various locations besides that there is no need for proper cultivation specifically (sukamto, 2007). the part of food plants. based on the results of the study, some people use food plants using plant parts or organs for consumption, which can be grouped into twelve (12) types of organs. leaves occupied the top position with the most percentage (29.73%), followed by fruit (17.57%), tubers (10.81%), seeds (9.46%), roots, rhizomes and flowers, respectively (6.76%), shoots (5.40%), stems (2.70%) and the last position was occupied by water, skin and heart, respectively (1.35%) (figure 3.b). sukmawati, et al., (2013) in the kaili rai tribe also showed the same thing, namely that people use more leaf parts than other parts with a percentage of 47.36% of 46 species. the same thing also happened in the research by nahdi, et al., (2016) that leaves were the highest part that was used by the community with a percentage of 51.39%. based on the results of interviews, favorite leaves are used because they are easy to obtain, there are always many leaves in one plant and easy to produce, so that people use them more than other parts, beside leaves also contain various substances needed by the body. according to payung, et al., (2016) the high utilization of leaves is because the leaves are used almost every day for vegetables and spices. apart from leaves, fruit and tubers are also parts that are widely used, because both are places to store food reserves that contain carbohydrates and other substances that are beneficial to the body (savitri, 2008). the serving method of food plants. based on the way it is used, people use food plants in eleven ways: boiling, pounding, grated, fried, roasted, steamed, roasted, coconut milk, vegetable, drunk and immediately consumed. the most widely used method by the community is by using vegetables with a percentage of 29.73%. the reason for the high processing method using vegetables is because people generally consume it as a complement to rice in their daily lives, it is easy to process, besides that people are also aware of their health and the many substances contained in plants that are very beneficial for the body. according to teo (2001) vegetables are a source of vitamins, minerals and phytochemicals that contain dietary fiber which is good for health. in contrast to research conducted by efremila, et al (2015) by drinking directly is the most common way, because beside being practical, cost-effective, it can also react directly in the body both for food and internal medicine. the source of food plants. the people on the slopes of mount merapi and merbabu obtain food plants by taking plants that come from wild plants, self-cultivating products, and buying at the market with details that the most widely used methods is self-cultivation (72.97%) (figure 3.d) compared to plants illegal (5.4%) and buying in the market (21.62%). this is because the cultivation process is very simple, namely by using the empty land around the house or garden using makeshift tools. generally, land in yards and gardens is used by the community to grow vegetable plants so that it is easy to collect them without having to travel long and cheap distances. in addition, medicinal plants are usually used as spices in cooking. in contrast to zaman's research, et al (2013), the people of sumenep district, east java mostly uses wildly obtained plants because of their economic value without spending money to get them. the way to get the second most widely used food plant by the community is buying at the market (21.62%) because food plants are the main and important need for the local community, and are an easy and practical way without carrying out the planting and harvesting process which requires time quite long and usually carried out by landless communities. meanwhile, how to obtain it by taking wild plants is very little due to the lack of public knowledge of the potential of these plants. value benefits (uvs) and important value (inp). based on the research results, the food plants that had the highest use value were fennel species (foeniculum vulgare mill.) with a value of 0.25, followed by chili pepper (capsicum annum l.) with a value of 0.20, and turmeric (curcuma dosmetica loir) and water spinach (ipomoea aquatica forsk.) with a value of 0.17 (table 1). several types of food plants above, are already well known among the surrounding community because in addition to being used for daily food they also have many benefits to cure various diseases, fennel (foeniculum vulgare mill.) in addition to plants that are unique and favored by the surrounding community, are also a plant that has many medicinal benefits. according to (kurniawan, et al., 2015), fennel can treat diseases such as itching, coughing and motion sickness. the essential oil content in fennel has anethol and estragole compounds as antipyretics (newall et al., 1995). the highest importance value based on the research results was padi (oryza sativa l.) with a value of 5.23%, followed by fennel (foeniculum vulgare mill.) 4.57%, and cassava (3.27%) (table 1). the important value of these two plants is not too far apart, so it can be said that both of them are excellent plants and are typical of the people of the slopes on mount merapi and merbabu. it is said so because at the beginning of the interview with respondents almost all of them mentioned these plants. regardless of the plant's content, the community's use of plants as a source of food and medicine is only based on hereditary knowledge and personal experience without knowing its contents. people make use of plants based on knowledge obtained from their parents or elders. based on the use value (uvs) and important value (inp), it is not always the plants that have the highest use value also have the highest importance value. this is umartani & nahdi – ethnobotanical study of edible plant communities on … 37 because the use value only looks at how many benefits a species has, while the important value is seen not only in terms of benefits, but how much people use these plants, so that even if the benefits are not as many as other species, these plants trusted and idolized by the community. table 1. list of edible plants used by the community of village of baksari and ngarsapura, boyolali. family scientific name local name benefits uvs inp amaranthaceae amaranthus tricolor l. bayem cabut detoxify body toxins 0,12 1,96 anacardiaceae mangifera indica l. pelem cure anemia 0,1 1,31 apiaceae foeniculum vulgare mill. adas overcome menstrual disorders, cure fever 0,25 4,57 daucus carota l. wortel improve eye health, 0,1 2,61 apium graveolens l. ledri anti inflammatory 0,07 1,31 araceae colocasia esculenta l. tales overcoming heart disease 0,07 0,65 xanthosoma sagittifolium l. kimpul overcoming digestive problems, 0,07 0,65 amorphophallus paeoniifolius suweg anti-poison, treat wounds 0,07 2,61 arecaceae cocos nucifera l. kelopo neutralizing body toxins 0,1 1,96 asteraceae tagetes arecta l. ningkir treat ulcer disease 0,1 0,01 lactuca sativa l. selada maintain eye health 0,07 0,01 bombaceae durio zibethinus l. duren fight infection (antibacterial) 0,05 1,96 brassicaceae brassica rapa l. sawi bakso lose weight 0,1 0,65 brassica oleracea l. kobis increase immunity 0,07 0,01 brassica olearacea l. brokoli improve the health of bones and teeth, 0,12 0,65 nasturtium officinale l. cenil prevent constipation 0,12 0,65 cactaceae hylocereus undatus haw. buah naga blood circulation 0,1 0,01 cannaceae canna discolor l. ganyong relieves heartburn symptoms 0,07 0,01 caricaceae carica papayal. kates digestion 0,12 1,96 chenopodiaceae beta vulgaris l. bit lower blood pressure 0,07 2,61 convolvulaceae ipomoea batatas l. telo source of carbohydrates, lose weight, 0,1 0,01 ipomoea aquatica forsk. kangkung against liver damage, 0,17 0,01 cucurbitaceae sechium edule l. jepan mineral source of the body 0,1 0,01 cucurbita moshcata durch. pumpkin improve the immune system, 0,07 0,01 cucumis sativus l. timun antioxidants, smooth digestion 0,07 0,01 momordica charantia l. pare relieves asthma, fights cancer cells, 0,1 0,65 cucumis melo l. melon prevent skin aging 0,07 0,65 dendrocalamaeae dendrocalamus asper l. boung lowering the risk of stroke 0,07 0,65 dioscoreaceae dioscorea alata l. uwi lowering blood sugar and cholesterol levels 0,05 0,65 dioscorea esculenta l. mbili prevent diabetes, 0,07 0,65 ebenaceae diospyros kaki l. tledung stop bleeding 0,07 1,96 euphorbiaceae manihot utillisima pohl. pohung relieves pain 0,07 3,27 euphorbia heterophylla l. patik mas reduces swelling due to snake bites 0,07 1,96 fabaceae clitoria ternatea l. telang contains antioxidants, heal wounds 0,1 0,01 glycine max l. dele lower blood pressure 0,07 0,01 arachis hypogea l. kacang brol prevent cancer 0,07 0,65 vigna cylindrica l. kacang panjang increase immunity 0,1 0,65 sesbania grandiflora l. turi treat coughs, dysentery 0,1 0,01 phaseolus vulgaris l. buncis maintain heart health 0,07 0,01 pisum sativum l. kapri free radical scavenger 0,07 0,65 leucaena leucocephala metir eradicate worms, prevent diabetes 0,07 0,65 gnetaceae gnetum gnemon l. melinjo smooth urine, 0,1 0,65 graminaceae zea mays l. jagung source of carbohydrates (daily staple food) 0,02 0,01 lamiaceae ocimum sanctum l. kemangi prevent fever 0,07 0,65 liliacea allium cepa l. brambang prevent colds, 0,1 0,65 allium sativum l. bawang putih natural antibiotics 0,12 0,65 38 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 family scientific name local name benefits uvs inp aloe vera l. lidah buaya speed up wound healing, 0,07 0,01 malvaceae abelmoschus esculentus l. okra prevent kidney disorders 0,07 0,01 marantaceae maranta arundinacea l. garut overcoming digestive diseases 0,02 0,65 moraceae artocarpus altilis park. sukun treat gout 0,07 0,65 artocarpus heterophyllus lam. gori cure insomnia 0,1 0,65 musaceae musa paradisiaca l. gedang kepok accelerating the digestive system 0,07 0,65 myrtaceae psidium guajava l. jambu klutuk tackle dengue fever 0,1 0,01 syzygium aromaticum l cengkeh treating stomach ulcers, 0,1 0,01 pandanaceae pandanus amaryllifolius roxb. pandan strengthens nerves 0,12 1,96 phyllanthaceae sauropus androgynus l. katu smooth out milk production 0,1 1,96 phyllanthus niruri l. meniran cure liver disease 0,07 0,01 piperaceae piper betle l. suroh antiseptic 0,12 0,01 poaceae oryza sativa l. pari source of carbohydrates 0,02 5,23 cymbopogon citratus l. sere antioxidants, control, regulate muscle function 0,07 0,65 portulaceae portula oleracea l. krokot prevent kidney stones, treat rheumatism 0,07 0,01 rosaceae rosa l. mawar improves immunity 0,07 0,01 fragaria anansa l. stoberi maintain heart health, prevent allergies 0,07 1,96 rutaceae citrus hystrix d.c jeruk purut clean the blood, increase endurance 0,07 1,96 solanaceae solanum lycopersicum l. tomat maintain eye health, treat acne 0,12 0,65 capsicum annum l. lombok streamlining bowel movements, treating thrush, 0,2 1,96 solanum melongena l. terong overcoming stomach acid 0,15 2,61 solanum tuberosum l. kentang maintain heart health, antioxidants, 0,15 0,01 capsicum annum l. paprika prevent anemia, control blood sugar 0,07 0,01 zingiberaceae curcuma dosmetica loir kunir overcoming menstrual problems 0,17 0,65 kaempferia galanga l. kencur treating coughs, treating diarrhea 0,15 0,01 zingiber officinale rosc jahe strengthens immunity, warms the body 0,12 0,01 curcuma zanthorrhiza l. temu lawak pain reliever, increases milk production 0,1 0,65 alpinia galaga l. laos cure asthma 0,07 0,65 conclusion based on the results and discussion, it can be concluded that the people on the slopes of mount merapi and merbabu use 74 species of plants from 37 families. the most commonly used plants are dominated by the fabaceae, zingiberacee and solanaceae families. the most widely used plants organs were leaves (29.73%), fruit (17.57%), and tubers (10.81). based on its use, it has the highest value (29.73%), followed by direct consumption (10.81%) and boiled (16.22%). the method of obtaining food plants by cultivation was in the highest ranking (72.97%), followed by buying in the market (21.62%) and wild plants (5.4%). plants that have a high use value (uvs) of fennel with a value of 0.25, chili pepper 0.20 and turmeric 0, 17. food plants that are excellent with the highest index of importance value are rice 5.23%, fennel 4.57% and cassava 3.27%. all the knowledge they have is knowledge acquired from generation to generation. acknowledgments: the results of this study involved various parties, many thanks to the people on the slopes of merapi merbabu, to be precise, mliwis village, especially the baksari and ngarsapura hamlets who have provided information about local knowledge on food plants and medicines they have, as well as to colleagues who have helped us. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references anggraini f. 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(2015). etnobotany and food plant community sambori district bima regency west nusa tenggara indonesia. natural b, journal of health and environmental sciences 3(2): 198-204. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 151-160 | doi: 10.14421/biomedich.2022.112.151-160 issn 2540-9328 (online) anti-oxidant, anti-inflammatory and anti-atherosclerotic activity of bioactive peptide hpaedr isolated from catla catal muscle on lps induced inflammation on 246.7raw macrophage cells and hcf induced hyperlipidemic zebrafish larvae sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah* sadakathullah appa college, thirunelaveli – 627011, india. corresponding author* janetgnana@gmail.com manuscript received: 23 july, 2022. revision accepted: 13 august, 2022. published: 23 august, 2022. abstract a muscle wasdissected from the catla catlafish and enzyme hydrolysis was done using various digestive enzymes such as pepsisn, protease, papine, trypsin and alcalase at verity of time intervals (0th, 3rd, 6th, 9th and 12th) hour respectively. followed by, the amino acid composition was identified and the confirmative assays such as, the anti-oxidant assays (dpph and hydroxy radical scavenging activity) and anti-inflammatory assays (hrbc and ad) were done for various peptide hydrolysate. the active hr was identified as 9th hr alcalase hydrolysate which was purified through ultrafiltration (>30 kda, 30-10 kda, 10-3 kda and <3 kda). these fractions were again studied for its anti-oxidant and anti-inflammatory activity. based on the results obtained, the active fraction was identified as 10-3kda which was further purified and identified using gel filtration chromatography and lc-ms/ms as hpaedr (723.76 da). further, for in vitro and in vivo studies the peptide derived from ccm was synthetically designed with 98% purity (phtdpeptides co., ltd. zhemgzhou, china). additionally, the physiochemical properties (solubility, emulsifying properties and foaming properties) of these fraction was studied. finally, the purified fraction was tested for in vitro activity through cell viability, cox-2 production, no production and tnf-α production. moreover, the in vivo protective effect is tested on zebrafish larvae. the results suggest that the active purified peptide fraction isolated from catla catla muscle has a strong natural anti-oxidant, anti-inflammatory and cholesterol reduction activity which can be used in functional foods and pharmaceuticals. keywords: anti-inflammatory; antioxidant; peptide; zebrafish larvae; 246.7raw macrophage; inflammation. abbreviations: hcf-high cholesterol food; ad-albumin denaturation assay; hrbchuman red blood cell membrane stabilization assay; ccm-catla catla mussel introduction generally, fishes are considered as easily putrescible food hence, it starts to blemish as soon as it was harvested. to overcome these problems, processing and packaging methods such as drying and smoked fish that are required to increase its shelf life (majidiyan et al. 2022). moreover, the fish waste such as bones, muscles and viscera are not disposed in a proper way (hotel 2001). the fish muscles are rich in proteins and collagen which can be used in development of novel pharmaceutical and nutritious functional food (hadidi et al. 2021; zamorano-apodaca et al. 2020). these collagen and muscle proteins are rich in amino acids such as glycine and proline which can be hydrolyzed using digestive enzymes to develop a bioactive peptide (nurilmala et al. 2020). these enzymatic production of bioactive peptides will have various productive activities such as, anti-inflammatory, anti-oxidant and anti-microbial activities (qian et al. 2020). the peptide isolation via enzyme hydrolysis is a productive and non-toxic method because even when it comes to provide non-synthetic hydrolysed peptide it will be less toxic than chemical or solvent based extraction. mostly, digestive enzymes such as trypsin, pepsin, papine, protamex and alclase are used in enzyme hydrolysis, where alclase is considered as one of the active enzyme with ability to create bioactive peptide especially from fish species (qiu et al. 2019). salmon skin (grundtman 2012) and yellow tuna (ray et al. 2019) are some of the examples from which bioactive peptide is isolated using alclase enzyme. the reactive oxygen species (ros) is one of the major markers which caused verity of oxidative stress related diseases such as, cardiovascular diseases and cancer (huertas‐alonso et al. 2021). fish isolated bioactive peptide are a good source of anti-oxidant molecules which can reduce ros production. moreover, https://doi.org/10.14421/biomedich.2022.112.151-160 152 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 the research shows that the peptides with anti-oxidant activity will posses other important activities such as anti-inflammatory and anti-microbial activities (taheri et al. 2014). carpfish (catla catla) is one of the common fish that lives on fresh water (zamora-sillero et al. 2018). fresh water fish production highly depends on carp fish because it is available globally (franěk et al. 2021). many studies show that fishes can posses a high anti-oxidant activity when enzyme hydrolysis is done (chalamaiah et al. 2015). in this current study, we had investigated the anti-inflammatory and anti-oxidant activity of the bioactive peptide isolated from the carp fish. additionally, we have investigated the gene expression and toxicity studies on macrophage cells and zebrafish larvae. materials and methods sample collection the catla catla was obtained from coastal area thoothukudi (8° 45' 50.9976'' n 78° 8' 5.4024'' e) of tamil nadu, india and was verified from zoological surveyof india (zsi), chennai. chemicals used the chemicals used in this study were as follows: opa, dtt, dpph, pepsin, trypsin, papain, protamex, alcalse sephadex g-25, lps, phosphatase inhibitor (sigma). other chemicals were purchased commercially proximate analysis and amino acid quantification the catla catla muscle (ccm) dissected and the protein percentage is measured (covey et al., 1991). the other parameters of the proximate analysis were also measured using commercially available kits. the ccm was hydrolyzed using hcl at 100 degree celcius for whole day and sent for hplc to quantify the percentage of essential and non-essential amino acids present (waterborg and matthews 1984). enzyme hydrolysis the ccm was hydrolyzed using various digestive enzymes such as, pepsin, trypsin, papin, protamex and alcalase at respective ph and temperature (adler-nissen 1979). the extraction buffer was used to hydrolyze the ccm. the samples were taken for lyophilisation at different time intervals 0th hr, 3rd hr, 6th hr, 9th hr and 12th hr respectively. finally, the various lyophilized samples were used to determine the anti-inflammatory and anti-oxidant activity of the peptide hydrolysate. acid hydrolysis the degree of hydrolysis percentage (dh %) was calculate using opa method where, the various peptide hydrolysate were taken and dissolved in 1mg/ml concentration and 400 μl of sample was mixed with 300 μl of opa after 20 minutes incubation at 37 degree celsius the od was measured at 340nm (chandra et al. 2012). determination of anti-oxidant activity for dpph assay, 50 μl of each peptide hydrolysate (1mg/ml) was mixed with 500 μl of dpph (95% 1:1 v/v ethanol) and kept in 37 degree celcius for 30 minutes and the od was measured at 517nm. the ascorbic acid was used as a positive control (haghani et al. 2021). for hydroxyl radical scavenging assay, 200 μl of each peptide hydrolysate (1mg/ml) was mixed with 900 μl of pbs, 100 μl of edta, 100 μl of ferrous sulphate and 500 μl of deoxyribose followed by, 250 μl of h202 and incubated for 30 minutes at room temperature. finally, the od reading was taken at 532 nm. the ascorbic acid was used as a positive control (ghanbari et al. 2016). determination of anti-inflammatory activity in hrbc assay, the blood sample was collected from volunteries and mixed with equal volume of alsever solution and centrifuged at 1000 rpm for 15 min followed by, the addition of isosaline and incubated for 30 minutes at room temperature and finally, the absorbance is measured at 560 nm. the diclofenac was used as a positive control (mizushima 1966). in albumin denaturation (ad) assay, the sample is mixed with specimen 1% bsa and the mixture is incubated for 30 minutes at room temperature additionally, kept in water bath (60 degree celsius) for 5-6 minutes and the od reading was measured at 340nm. the diclofenac is used as a positive control (rajapakse et al. 2005). separation, purification and identification of peptide sequence ultracentrifugation was done for the active hour (9th) hydrolysate with different cutoff units (3, 10 and 30 kda) to separate peptide hydrolysate into various fractions based on molecular weight. then gel filtration coromatography was performed to purify the active fraction by maintain 1000 μl per minute speed at 280nm. the obtained fractions were again tested for inhibition activity and protein percentage (ranathungaet al., 2006). finally, the amino acid sequence of the purified fraction was identified using lc-ms/ms as hpaedr (723.76 da) and the results obtained were examined using bio tools software. physiochemical properties of peptide to determine the solubility of peptide the peptide was mixed with 1ml of water with a ph of around 3-9 and the mixture was centrifuged (8000 rpm) for 20 minutes. additionally, to determine the protein content, sodium hydroxide was used as solvent (ranathunga et al., 2006). for emulsifying property, 1ml of maize oil was mixed with 30ml of peptide solution followed by sethuramalingam et al. – effect of catla catla muscle derived peptide 153 adjusting the ph between 2-10 respectively and the mixture was centrifuged (20,000) for 1 minutes. then 0.5ml of sds was added and the od was measured at 500nm after 10 minutes incubation (coveyet al.,1991). to examine the foaming property the pure peptide was taken (200 μl) and centrifuge at 16000 rpm for 1 minutes. finally the entire volume of the solution was determined (pearce and kinsella 1978). in vitro assessment peptide activity on 246.7raw macrophage cells raw264.7 macrophage cells were cultured in dmem media along with 10 percent fbs and 1 percent (v/v) antibiotic at 36 °c in a 5 percent co2 incubator. cell viability raw264.7 macrophage cells (1 104 cells/well) are incubated for 3–4 h at 37 °c with 50–1000 g/ml of pure peptide concentrations. after 1x pbs washing, 20 l of mtt reagent was added afterwards. after 3 hours of incubation, 200 l of dmso was added to each well, and the od was measured at 570 nm. triton x-100 served as control substance (mosmann 1983). determination of nitrous oxide production raw264.7 macrophage cells (1 104 cells/well) were incubated for one hr at 37 °c with doses ranging from 60 to 260 g/ml, followed by a 24-hour incubation at 37°c with lps (1 g/ml). later, griess reagent (1:1) was added to the supernatant and maintained for 10 minutes. finally, od was measured at 540 nm was observed (nambiar et al., 2015). determination of pro-inflammatory cytokine synthesis the cells (1 104 cells/well) were treated with peptides (50–250 g/ml) and lps (1 g/ml) at 37 °c and 5% co2 for one day; diclofenac served as the reference. the quantitative analysis was performed by measuring the absorbance at 450 nm. in addition, standard concentrations versus absorbance values were used to produce standard curves. in vivo activity of purified ccm peptide on zebrafish larvae zebrafish maintenance and feeding zebrafish were purchased from local shops and grown and maintained in a cycling zebrafish aquaculture system at 28 °c with a 14:10 light-dark cycle. factors such as ph, ammonia and nitrite concentrations are maintained to ensure water purity. a cholesterol diet (hcd) for zebrafish was prepared from a cholesterol solution in diethyl ether (sigma) to obtain a cholesterol content of 4% (w/w) in artificial brine shrimp after evaporation of the ether. five-week-old zebrafish were fed a diet three times a day. the control group was given regular fish kernels and the experimental group was given purified ccm peptide. cholesterol measurement the control and experimental zebrafish larvae were euthanized using cold pbs and homogenized. by centrifugation at 1500 g at 4° c. for 5 minutes the supernatant was separated from 10 zebrafish larvae in each group. total cholesterol was measured using a commercially available test kit. in vivo microscopy for in vivo microscopy, sedated fish larvae were stored in droplets containing tricaine in a sealed, temperaturecontrolled environment. zebrafish larvae were observed under a microscope. pictures of zebrafish larvae were collected every 200 nm and all images were inspected and processed using software. gene expression and survival percentage the expression of tnf-α and nf-kβ is studied on zebrafish larvae and the survival percentage was calculated during the feeding and peptide treatment statistical analysis all the experiments were done in triplicate and the results were represented in the mean ± sd. the analysis was done using graphpad prism 8. results and discussion proximate analysis of ccm the muscle protein percentage was determined to be 33.67 ± 1.68% which has a similarity with other protein percentage (gökoolu and yerlikaya 2003). the percentage of other parameters of proximate analysis is shown (table 1). other studies reveals that higher protein percentage will eventivually have higher possibility of getting active bioactive peptide. table 1. protein percentage of baf. proximate analysis percentage (%) protein 35.77 ± 0.46 moisture 54.53 ± 0.03 (table 1) protein percentage and moisture of baf (mean ± sd) amino acid composition of ccm the anti-oxidant, anti-inflammatory and physiochemical properties of peptides are related to the amino acid composition of the samples (chen et al. 2017). the amino acid composition of the ccm is shown in the table 2. the results shows that, it has both essential and non-essential amino acids where it has higher percentage of amino acids such as, histidine and glutamine which is highly known amino acids for its anti-oxidant and antiinflammatory property (liu et al. 2010). it also has 154 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 arginine and lysine amino acids which are responsible for high anti-microbial activity (chi et al. 2014; liu et al. 2010). observed amino acids are known to posse’s anti-oxidant activity of peptides (alemán et al. 2011). table 2. amino acid composition of ccm. amino acids ccm (%) asparticacid 15.7 glutamic acid 25.7 serine 19.2 histidine* 7 glycine 0.9 threonine* 3.0 arginine 17.3 alanine 4.2 tyrosine 6.9 methionine* 0.9 valine* 0.9 phenylalanine* 5.2 isoleucine* 6.0 leucine* 9.5 *essential amino acids (table 2) it shows the amino acids composition present in the acid hydrolysis sample of oliva oliva visceral mass percentage of protein hydrolyzed (dh %) the percentage increases in the 9th hr hydrolysate as, 99.69 ± 1.79 %, 89.7 ± 1.54 %, 66.89 ± 0.85 %, 65.99 ± 0.77 % and 60.42 ± 0.39 % for trypsin, alcalase, papine, pepsin and protamex hydrolysate respectively (figure 1). similar studies show that the dh % can be calculate in the same method for marine organisms (ray et al. 2019). other studies shows that the pepsin and alclase has more dh% that other enzymes. in this study, we have used opa method of dh % estimation which is easy to perform and cheaply affordable. 0th 3rd 6th 9th 12th 0 20 40 60 80 100 trypsin alcalase papain pepsin protamex time (hr) d h ( % ) figure 1. it shows the percentage of degree of hydrolysis of oliva oliva visceral mass with five digestive enzymes (mean ± sd, n=3), *p>0.05. anti-inflammatory and anti-oxidant activity of ccm peptide the anti-inflammatory activity is quantified by albumin denaturation assay (ad) and hrbc membrane stabilization assay. current results show that in ad assay the alcalase 6th, 9th and 9th hr hydrolysate has higher inhibitory activity of 67.20 ± 0.88, 76.55 ± 0.37 and 69.84 ± 1.14 percentages respectively. when compared with other hydrolysate inhibitions the 12th hr alcalase shows higher activity (figure 2a). the diclofenac potassium is used as standard drug (95.97 ± 0.27 %). the inflammation may also leads to the lysosomal membrane rupturing hence hrbc membrane is performed because the rbc membrane are similar to lysosomal membrane (babu, pandikumar and ignacimuthu 2011). results shows that the 9th hr alcalase hydrolysate of ccm shows a higher hrbc membrane stabilization assay about 69.03 ± 1.04 % (figure 2b). the diclofenac potassium is used as standard drug (93.77 ± 0.99 %). figure 2. the ad and hrbc assay of different time variables during enzyme hydrolysis are shown in a and b respectively (mean ± sd, n=3), *p>0.005. the dpph is performed since the dpph free electron is paired off (taheri et al. 2014). the antioxidant capacity of ccm peptide shows that the 9th hr hrydrolysate has more activity when compared with sethuramalingam et al. – effect of catla catla muscle derived peptide 155 other hydrolysate (figure 3a). this can be due to the presence of alanine and glycine amnioacid (hajfathalian et al. 2018; taheri et al. 2014). hydroxyl radicals are the major reactive oxygen species (pavithra and vadivukkarasi 2015). the current results show the similar activity with higher anti-oxidant activity at 9th hr hydrolysate (figure 3b). some aminoacids detected also have the ability to chelate pro-oxidative transition metals, thus favoring the reduction and deactivation of oh˙ free radicals (hajfathalian et al. 2018). all these facts could explain the dependency between oh˙ scavenging activity and concentration. figure 3. the dpph and hydroxyl assay of different time variables during enzyme hydrolysis are shown in a and b respectively (mean ± sd, n=3), *p>0.005. purification and sequencing of active fraction the active hr sample was purified using the gel filtration chromatography (figure 4) and the fraction was subjected to amino acid sequencing and molecular weight analysis using lc-ms/ms. the sequence was obtained as hpaedr (723.76 da). further, for in vitro and in vivo studies the peptide derived from ccm was synthetically designed with 98% purity (phtdpeptides co., ltd. zhemgzhou, china) (figure 5). figure 4. the gel filtration chromatogram of the 9th hr alcalase hydrolysate. figure 5. the amino acid sequencing of purified fractions and its molecular weight. physiochemical properties the functional property relies on the structure and amino acid sequences of the protein. the results obtained show that, an increase in solubility was observed between ph (4-10) up to 84.37 ± 1.03% (figure 6). the observed increase in solubility is due to 156 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 amino acid serine which is reported earlier as an efficient amino acid regarding to solubility of protein (trevinoet al., 2007). similarly, the emulsifying ability also relies on the structure and amino acid sequences of the protein. another study shows that the foaming property are also responsible for the ph change and said that the ph (6-10) is steady in foaming property (majidiyan et al. 2022). additionally, the emulsification activity are active at ph 5 and its stability varies based on the ph (ma et al. 2018). figure 6. the functional properties of oliva oliva veseral mass derived peptide (mean ± sd, n=3), *p<0.05 cytotoxisity and pro-inflammatory cytokines expression the cell viability of the drug treated cells are generally evaluated using mtt assay whereas, the current results on mtt assay shows that the cell viability was more than 80% (figure 7) and hence the purified peptides were not toxic to the macrophage raw264.7 cells. similarly, on another study conducted using sargassum polycystum shows higher cell viability. additionally, a anti-inflammatory peptide derived from arca subcrenata shows lower cell toxicity on macrophage raw264.7 cells (chen et al. 2017; li et al. 2014). figure 7. cell viability (a) and anti-inflammatory activity of ccm peptide over (b) tnf-α, (c) il-1 β, (d) il-6, (e) cox2 and (f) no production inlps-induced raw264.7cells, where n and prepresent negative and positive control (mean ± sd) and there is significant difference, p< 0.05 the pro-inflammatory cytokines expression analyzed at a dosage of 50,100 and 200 μg/ml respectively. the results show that increase in the concentration of peptides increases the cox-2 inhibition (figure 7). it is found that comparatively, the 200μg/ml and higher concentration are effective in inhibition than the lower concentration. further, the studies carried out on oyster and yan-hou-qing shows the similar activity on cox-2 inhibition (hwang et al. 2012; ray et al. 2019). additionally, the expression of tnf-α and nf-kb of peptide treated zebrafish larvae is studied (figure 8). sethuramalingam et al. – effect of catla catla muscle derived peptide 157 figure 8. the peptide reduced the weight (a) of zebrafish. it has a considerable amount of mortality (b) throughout the experiment. the c and d shows the gene expression (hadhaa and hadhb) in the zebrafish liver, intestine and muscle of the atherosclerotic zebrafish against fish oil zebrafish group. ccm peptide downregulated the emergence of plaques in atherosclerotic zebrafish figure 9 shows that zebrafish of the ac group showed a huge amount of deposition of lipids near the vascular area, but there was no change in the control group. interestingly, the addition of 1 mg/l and 10 mg/l ccm peptide to the bath significantly reduced lipid deposition in zebrafish larvae compared to the ac group. there were no significant differences between the as (atherosclerotic) group and the 0.1 mg/l peptide group. figure 9. larvae induced with 4% red florescence attached high cholesterol food (hcf) for 10 days where, the dotted box represents the crucial area of accumulated lipids. ccm peptide improved liver metabolism and oxidative stress in atherosclerotic zebrafish compared to the manage organization, the as organization exhibited a enormous growth in lipid degrees, which have been considerably decreased with the aid of using 1mg/l and 10mg/l ccm peptide (figure 10(a)). figures 10(b) and 10(c) exhibit that tc and tg degrees have been additionally considerably reduced in as zebrafish dealt with with 1mg/l and 10mg/l peptide, however now no longer with 0.1mg/l peptide. in conclusion, peptide should successfully lessen lipid degrees in as zebrafish. 158 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 figure 10. nail red staining images of control anpeptide treated (a) at various concentration (exposed for a period of 10 days). total cholesterol (b) and triglycerides (c) of zebrafish. significance was calculated by one-way anova and unpaired t-test using graphpad prizom. ccm peptide protected against lesion in atherosclerotic zebrafish neutrophil is widely regarded to be the principal cell type responsible for tissue damage and acute lesion. to determine the characteristics of the lesion response in the early stages of atherosclerosis, we first created a genetically modified zebrafish line that upregulated the gfp of neutrophils in transgenic mpx: “egfp” zebrafish. we discovered a significant upregulation of green fluorescentlylabeled neutrophils in vascular locations in atherosclerotic zebrafish larvae group (figure 11). in contemplation to determine the benefit of fish oil as a therapeutic medication that inhibits gfpneutrophils transhumance and lesion response, we administered varying dosages of ccm peptide to zebrafish larvae fed an hcd diet. both the 1mg/l and 10mg/l concentrations of ccm peptide resulted in a reduction of neutrophils in the tails of zebrafish larvae, however the 0.1mg/l concentration was unaffected. in conclusion, ccm peptide protected zebrafish from oxidative damage and inflammation. figure 11. anti-inflammatory effect of peptidein hcf induced atherosclerosis on zebrafish (egfp) larvae. the green fluoresce represents the inflammation level of neutrophils. sethuramalingam et al. – effect of catla catla muscle derived peptide 159 conclusions the catla catla muscle(ccm) derived peptide is isolated and purified using ultrafiltration and gel filtration chromatography. by the anti-inflammatory assays (ad and hrbc) the active hour is identified as 9th hr alcalase hydrolysate. additionally, the fractions are purified and the sequence is found using lc/ms-ms as hpaedr with a molecular weight of 723.76 da. further, in vitro studies were carried out using lps induced inflammation on raw264.7 macrophage cells against synthetically designed peptide and found that the peptide is non-toxic and posses ability to suppress a proinflammatory cytokine in increased quantity. additionally, it exhibits reflex effects in hcd-induced hyper-lipidemia, lesion and oxidative stress and inhibits formation of atherosclerotic plaque. hence this study suggests that the ccm derived peptide may be work as a supplementary medicine to treat chronic inflammatory diseases and may posses cholesterol reducing effect. code availability: no code availability data availability: data sharing not applicable to this article as no datasets were generated or analysed during the current study. authors’ contributions: sabarinathan s and revathy lr have contributed in all aspects of research and manuscript preparation under the guidance of janet rani r. competing interests: the authors declare that there are no competing interests. funding: the authors declare that nofunding was received to assist this research references adler-nissen, 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akachukwu1, ndukwe onyeani atuh1 1department of biochemistry, college of natural sciences, michael okpara university of agriculture, umudike, p.m.b. 7267, umuahia, abia state, nigeria. 2department of biochemistry, faculty of biological and physical sciences, abia state university, uturu, nigeria. 3department of public health, gregory university uturu, abia state, nigeria. corresponding author* ir.uroko@mouau.edu.ng manuscript received: 05 june, 2022. revision accepted: 18 july, 2022. published: 31 july, 2022. abstract medically active compounds in plants confer biological effects including antioxidant properties. this study evaluated the phytochemical content and in vitro antioxidant properties of combined funtumia africana leaves and abutilon mauritianum extract (cfae). the 1,1diphenyl-2-picrylhydrazyl (dpph), ferric reducing antioxidant power (frap), thiobarbituric acid reactive substances (tbars), total antioxidant capacity (tac), and nitric oxide radical (no.) scavenging capabilities were used as antioxidant assay models. the results of the phytochemical analysis showed that cfae is rich in alkaloids, flavonoids, tannins, saponins, phenols and cardiac glycosides. the extract contains antioxidant vitamins a, c and e, vitamin e being the most abundant. the cfae showed a dose-dependent tac based on the observed frap, tbars, and dpph scavenging activity which could be attributed to the presence of phenolic compounds and vitamins e. these strongly suggest that cfae is a potential source of phytochemicals and antioxidants which could be exploited in the food and pharmaceutical industries in production of potent nutraceuticals or therapeutically-important products. keywords: abutilon mauritianum; antioxidant activity; antioxidant vitamins; funtumia africana; free radicals; phytochemicals. abbreviations: dpph = 1-diphenyl-2-picrylhydrazyl, frap = ferric reducing antioxidant power, tbars = thiobarbituric acid reactive substances, tac = total antioxidant capacity, no = nitric oxide radical, cfae = combined funtumia africana leaves and abutilon mauritianum extract. introduction medicinal plants are the bio-resource of many drugs and are used in modern medicine as well as food supplements and pharmaceutical intermediates (ahn, 2017). medicinal plants contain a wide range of natural products known as phytochemicals, which are compounds manufactured by plants via major or minor metabolism (molyneux et al., 2007). plant-based natural products have played an important role in drug discovery and were the basis of most early medicines. these phytochemicals including saponins, flavonoids, alkaloids, polyphenols, terpenes, tannins etc. generally possess potent biological activities which play important roles in growth of plants or defence against pathogens or predators (molyneux et al., 2007). also, they serve as sources of therapeutic agents used in the management of several ailments because of their antimicrobial, anticancer and antioxidant effects (riganti et al., 2011). hence, antioxidants play a vital role in the defence system of the body against harmful oxygen species. in addition to the application of natural products such as drug, they also contribute significantly towards nutraceuticals as well as ingredients for food products. in recent times, the search for safer natural products and more potent antioxidants have necessitated the need to harness plant parts which are capable of producing potent secondary metabolites (phytochemicals) to replace synthetic antioxidants. several reports have denoted the antioxidant effects in vitro, of phytoconstituents in biological systems (papuc et al., 2017,). on the other hand, the need for improved therapeutic effects may warrant the combination of plant extracts due to synergistic properties. this concept is found in ayurveda and medicinal systems where more than one herb in a precise proportion could be used in the management of many ailments (aslam et al., 2014). funtumia africana (family = apocynaceae) is a flowering plant native to africa with narrow crown and can grow up to 30 metres tall. the slender, cylindrical stem can be up to 50 cm in diameter (burkil, 2014). funtumia africana is harvested from remote areas for indigenous use as a medication and source of materials. https://doi.org/10.14421/biomedich.2022.112.111-118 112 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 it is used by african natives to treat fever, inflammation, burns, malaria, cancer and urinary incontinence (burkil, 2014). abutilon mauritianum (family = malvacedae) is also a widely used plant in traditional african medicine. the plant is extensively dispersed in the drier regions of african tropics and widely used for its edible leaves and medicinal properties (ruffo et al., 2012). the root, stem, bark and leaves are utilized in the management of several maladies including kidney problems, diarrhoea, dysentery, haemorrhoids, stomach-ache, fire burns, sore throat, cough and cold (ruffo et al., 2012; burkil, 2014). therefore, this research evaluated the phytochemical constituents and in-vitro antioxidant properties of a combined extract of f. africana leaves and a. mauritianum stem bark (cfae). materials and methods collection and identification of plant material fresh samples of f. africana leaves and a. mauritianum stem bark were collected from the botanical gardens at michael okpara university of agriculture, umudike and identified at the herbarium unit of the department of forestry in the same institution with specimen voucher number 2694-5 (acuss 1899) and jones fhi 13749 respectively. preparation of plant materials the samples were washed with clean water and later dried under shade at room temperature for thirteen days. they were ground into fine powder, weighed and then stored in a dry, clean, sterile container for subsequent extraction. extraction of plant materials five hundred gram (800 g) of the finely ground f. africana leaves and a. mauritianum barks (1:1) was soaked in 2 l of ethanol for 72 hours in a sterile vessel. it was filtered, first with a mesh cloth, and later with a whatman no.1 filter paper. the filtrate was concentrated in a water bath (50℃) and allowed to evaporate completely. the evaporated extract was weighed and the percentage yield was calculated. the extraction of 800 g of cfae gave a percentage yield of 22.7% (113.5 g). the obtained combined extract (cfae) was covered with aluminium paper foil and stored in the refrigerator (4℃) for further analyses. qualitative phytochemical analysis the presence of phytochemicals including alkaloids, flavonoids, saponins, tannins, terpenoids, cardiac glycosides, and steroids were screened according to the methods of harborne (1998). quantitative determination of phytochemicals alkaloid and flavonoid contents were determined quantitatively as per the protocols outlined by harborne (1998). phenol, saponin and terpenoid contents were quantified using the methods of harborne (1984), while tannin, cardiac glycoside and steroid contents of the extract were evaluated as stated by the method of pearson (1976). determination of antioxidant vitamins vitamins a (retinol), c (ascorbic acid) and e (αtocopherol) contents were determined utilizing the protocols of pearson (1976). their concentrations were extrapolated from the respective standard curves. determination of 1,1-diphenyl-2-picrylhydrazyl (dpph) radical-scavenging activity scavenging activity on dpph free radicals was assessed according to the method of gyamfi et al. (1999) with slight modifications. exactly 2.0 ml of extract was mixed with 1.0 ml of methanol and 0.3 mm dpph in a dilute 2-fold solution. the mixture was shaken vigorously to prevent it from freezing and left for 25 minutes in the dark. the absorbance of the reaction mixture was measured at 520 nm against each blank. % scavenging activity = 100 − (abssample − absblank) (abs control) 𝑥 100 determination of nitric oxide radical (no) scavenging activity nitric oxide was measured according to the protocol of marcocci et al. (1994). reaction mixtures containing nitroprusside (5 mm) and the plant extract were incubated at 25℃ for up to 180 minutes in front of a polychromatic light source. the no radical generated, interacted with oxygen to yield no2 which was then analysed at 30-minutes intervals by mixing an equivalent volume (1.0 ml) of greiss reagent and incubation mixture. the absorbance was measured at 546 nm. determination of ferric reducing antioxidant power (frap) the frap of the extract was determined according to the protocol of yen and chen (1995) with little alteration. exactly 0.3 ml of plant extract, ascorbic acid and rutin which were prepared in distilled water, was mixed with a reacting mixture [(25 ml of phosphate buffer and 2.5 ml of kfe(cn)]. the resultant mix was incubated at 500c for 20 minutes followed by adding of tca (2.5 ml, 10% w/v) and then incubated at 25℃ for 15 minutes. absorbance was measured at 700nm against the blank. determination of total antioxidant content (tac) the tac was determined using the phosphomolybdate method earlier reported by el-hashash et al. (2010). exactly 1.0 ml aliquot of the extract and vitamin c was mixed with 1 ml of reagent solution (600 mm h2so4, 25 mm na2(po)4 and 4 mm ammonium molybdate – uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 113 1:1:1). the test tubes were incubated at 95℃ for 90 minutes and cooled. absorbance was read at 765 nm against a blank. ascorbic acid served as the reference. 𝑇𝐴𝐶(%) = 𝐴𝑜 − 𝐴𝑠 𝐴𝑜 𝑥 100 𝐴𝑜 = absorbance of control; 𝐴𝑠 = absorbance of extract determination of thiobarbituric acid reactive substances (tbars) activity the tbars activity was determined according to the method of banerjee et al. (2005) where egg yolk homogenates served as a lipid-rich media. exactly 0.5 ml of egg homogenate and 100 ml of sample were added to all test tubes and made up to 1.0 ml with distilled water. thereafter, 50 ml of feso4 (0.075 m) and 20 ml of lascorbic acid (0.1 m) were introduced and incubated for 60 minutes (37℃) to induce lipid peroxidation. afterwards, 0.2 ml of edta and 1.5 ml tba reagent were added to the respective samples and heated for a 15 minutes at 100℃. after cooling, samples were centrifuged at 3000 rpm for 10 minutes. percentage inhibition(%) = 𝐴𝑜 − 𝐴𝑠 𝐴𝑜 𝑥 100 𝐴𝑜 = absorbance of control, 𝐴𝑠 = absorbance of sample. data analysis the data obtained were subjected to one-way analysis of variance (anova) with a statistical products and service solution (spss) version 22 and the statistical significance ascertained at p<0.05. results were presented as mean ± standard deviation (n =3). results and discussion qualitative phytochemical composition of cfae the qualitative phytochemical analysis of cfae (table 1) showed that tannins, phenolics, steroids and terpenoids are the most abundant phytochemicals in the extract. flavonoids were present in moderate amount, while glycosides and saponins were the least present. table 1. qualitative phytochemical composition of cfae. key: + = low concentration; ++ = moderate concentration; +++ = high concentration quantitative phytochemical composition of cfae the result of the quantitative phytochemical analysis of cfae (table 2) showed very high concentrations of alkaloids (3547.46±17.54 mg/100g), total phenolics (1398.42±15.72 mg/100g), and terpenoids (1528.01±0.58 mg/100g). tannins, flavonoids and steroids were present in moderate amounts while glycosides and saponins were present in minute concentrations. table 2. quantitative phytochemical composition of cfae. phytochemicals concentration (mg/100 g) glycoside 3.33 ± 0.77 alkaloids 3547.46 ± 17.54 saponins 5.74 ± 0.01 tannins 128.42 ± 1.79 total phenols 1398.42 ± 15.72 flavonoids 147.25 ± 4.58 steroids 131.13 ± 0.73 terpenoids 1528.01 ± 0.58 values are mean ± standard deviation of triplicate determinations (n = 3). antioxidant vitamin composition of cfae as shown in table 3, cfae contains antioxidant vitamins a, c and e. vitamin e was present in high concentration while vitamins a and c were present in relatively low amounts. table 3. composition of antioxidant vitamins of cfae. vitamins concentration vitamins a (µg/g) 5.52 ± 0.11 ascorbic acid (mg/100 g) 3.76 ± 0.28 vitamin e (mg/100 g) 113.48 ± 3.97 values are mean ± standard deviation of triplicate determinations (n = 3). tbars free radical scavenging activity of cfae the data in figure 1 show tbars free radical scavenging activity of cfae and an antioxidant standard, butylated hydroxytoluene (bht). a dosedependent increase in tbars activity was observed for the cfae. however, the extract showed considerable lower activity at lower concentrations (7.81µg/ml and 15.63µg/ml) when compared with the bht standard. increase in concentration of the extract led to increase in scavenging activity. the half maximal effective concentration (ec50) of the extract for tbars was 73.86±1.22 µg/ml, while that of bht standard was 40.75±0.92 µg/ml. phytochemicals composition glycoside + tannins +++ flavonoids ++ total phenolic +++ steroids +++ terpenoids +++ saponins + alkaloids ++ 114 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 figure 1. tbars radical scavenging activity of cfae. dpph free radical scavenging activity of cfae as shown in figure 2, a dose-dependent increase in dpph inhibition was observed for cfae, whereas the dpph inhibition for ascorbic acid remained constant regardless of concentration. the extract showed considerably lower dpph inhibition at lower concentrations when compared with that of ascorbic acid. increase in concentration of the extract led to increase in mean percentage dpph inhibition. the ec50 of the extract and ascorbic acid for dpph were 49±0.08 and 34.07±0.20 µg/ml respectively. figure 2. dpph radical scavenging activity of cfae. ferric reducing antioxidant power (frap) of cfae figure 3 shows the frap of cfae. a dose-dependent increase in frap was observed. the extract showed lower frap at lower concentrations but reached its peak at 640 µg/ml. 0 10 20 30 40 50 60 70 80 7.81 µg/ml 15.63 µg/ml 31.25 µg/ml 62.5 µg/ml 125 µg/ml 250 µg/ml 500 µg/ml m e a n % i n h ib it io n t b a r s ( % ) concentrations combined extract bht each bar represents mean±standard deviation (n = 3) 0 20 40 60 80 100 120 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m e a n d p p h i n h ih ib it io n ( % ) concentration combined extract ascorbic acid each bar represents mean±standard deviation (n = 3) uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 115 figure 3. ferric reducing antioxidant power (frap) of cfae. total antioxidant capacity (tac) of cfae figure 4 shows the tac of cfae. a dose-dependent decrease in tac was observed. at lower concentrations, the extract showed higher tac which decreased as the concentration increased. figure 4. total antioxidant capacity (tac) of cfae. nitric oxide (no) scavenging activity of cfae the no activity of cfae (figure 5) increased slightly as concentration increased in line with the observed no activity for ascorbic acid. however, the no activity for ascorbic acid begins to decrease at 80 µg/ml concentration and reaches its least activity at 640 µg/ml, whereas that of the extract remained stable. the ec50 of the extract and ascorbic acid for nitric oxide were 54.93±0.28 µg/ml and 23.71±0.56 µg/ml respectively. figure 5. nitric oxide free radical scavenging activity of cfae. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m e a n f r a p ( g a e ) concentrations each bar represents mean ± standard deviation (n = 3) 0,0 0,2 0,4 0,6 0,8 1,0 1,2 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m e a n t a c ( a a e ) concentrations each bar represents mean ± standard deviation (n = 3) 0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0 80,0 90,0 100,0 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/mlm e a n n it ri c o x id e i n h ib it io n ( % ) concentrations combined extract ascorbic acid each bar represents mean ± standard deviation (n = 3) 116 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 discussion this study was undertaken to evaluate the phytochemical compositions and antioxidant properties of combined extract of f. africana leaves and a. mauritianum stem bark (cfae). therapeutic tendency of plants can be evaluated by performing preliminary qualitative screening to ensure the presence of phytochemicals (batool et al., 2019). in this study, bioactive constituents that confer biologically active effects to cfae were screened and the results confirmed the existence of alkaloids, flavonoids, tannins, glycosides, phenols, steroids, terpenoids, saponins and antioxidant vitamins. the high levels of alkaloids, phenols, flavonoids and terpenoids in this study strongly suggest that cfae could be a potential source of natural products, which could serve as effective free radical scavengers and/or inhibitors; hence could be a good plant-based pharmaceutical product for management of several diseases caused by free radicals. this is in line with several reports which highlighted the therapeutic effects of the above-mentioned phytochemicals (adamski et al., 2020; aloko and bello, 2021). steroids found in plants play critical roles in some ailments including prostate cancer (lubik et al., 2016). the moderate concentration of steroids in the sample could justify its wide use for the formulation of various herbal medicines. tannins had long been thought to be anti-nutritional, but their helpful or anti-nutritional characteristics are now understood to be dependent on their chemical structure and dosage (schiavone et al., 2008). however, consumption of tannin-rich food has been implicated in esophageal cancer (kanzaki et al., 2001) suggesting that tannins might be carcinogenic and hence, the consumption of polyherbal extract containing f. africana leaves and a. mauritanum stem barks should be monitored. plant-based vitamins including a, c and e are potent antioxidants present in varying amounts in plants. these vitamins are of great impact to human health. they are essential for metabolism because of their redox chemistry and role as enzymatic cofactors (asensifabado and munne-bosch, 2010). the high level of vitamin e in cfae observed in this study could have contributed to its free radicals scavenging property. a review had shown antioxidant potentials of african natural products (lawal et al., 2017). validating the presence of antioxidant abilities of plants entails the use of established antioxidant protocols with varying principles, mechanisms and experimental conditions. antioxidants act as radical scavenger by electron donating mechanisms or by hydrogen donating mechanisms – hence preventing the deleterious role of free radicals in different diseases. therefore, to evaluate the antioxidant capacity of cfae, several antioxidant assays were carried out. the extract showed considerable lower tbars activity at lower concentrations when compared with the bht standard, but increased slightly as concentration increased. lipid peroxidation of membranes is one of the early events at the onset of inflammatory response by stimulated polymorphonuclear cells; hence, agents that are capable of averting lipid peroxidation of membranes could potentially serve as appropriate candidate in antiinflammatory drug discovery process (anyasor et al., 2014). the potent scavenging activity exhibited by cfae at increased concentrations could be attributed to the high concentration of phenolics, which confer antioxidant effects possibly by targeting the neutralization of reactive radicals and/or quenching of singlet oxygen. plant phenolics can react with active oxygen radicals like hydroxyl, superoxide anion and lipid peroxy radicals to prevent early lipid oxidation (muanya and odukoya, 2008). the effect of antioxidants on dpph is thought to be due to their hydrogen donating ability (adesegun et al., 2017). free radical scavengers are proton donors that help to mop up excess free radicals (adesegun et al., 2017). ascorbic acid is a potent antioxidant as an electron donor showing free radical scavenging ability; hence, it is commonly used in dpph radical scavenging assay as a reference compound for measuring antioxidant activity. the presence of phenolic compounds could have contributed to the scavenging activity observed. this is in agreement with earlier reports (jimoh et al., 2008), which posit that phenolic constituents of the combined extract contributes to its scavenging ability. presence of phenolics and flavonoids impart the scavenging capabilities to plants, because they are significantly extracted in the polar solvents and hence, donate electron or hydrogen to stabilize dpph free radicals. the development of the o-phenanthroline-fe(2+) complex and its destruction in the presence of chelating drugs is the basis of the frap assay. this test is frequently used to determine the antioxidant capacity of polyphenol-containing foods, drinks, and nutritional supplements (benzie and strain, 1996). a dosedependent increase in frap of cfae was observed (figure 5). the extract showed lower frap at lower concentrations but reached its peak at 640ug/ml. earlier report had shown a direct correlation between antioxidant activity and reducing power, which was attributed to the presence of phenolic compounds (ojewunmi et al., 2013). this present study has revealed that the extract certainly has a proton-donating property and could work as a free radical inhibitor or scavenger, possibly acting as a key antioxidant. due to the fact that plants contain several classes and types of antioxidants, it is imperative to ascertain their cumulative antioxidant capacity of scavenging free radicals (pellegrini et al., 2003). this formed the basis for determining the tac of cfae, which measures the amount of free radicals scavenged by a test solution uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 117 (ghiselli et al., 2010) and is used to evaluate the antioxidant capacity of biological samples (pinchuk et al., 2012). in the present study cfae showed high tac values at lower concentrations. however, the antioxidant capacities decreased dose-dependently as the concentration of the extract increased. the high antioxidant capacity shown by the extract (especially at lower concentrations) could be attributed to the high content of phenolics (jimoh et al., 2008), which has demonstrated strong antioxidant activities in different models. a positive correlation between total phenolic content and tac was also observed in a related study using f. africana stem barks (frempong et al., 2021). endothelial cells, macrophages, neurons, and other cells create nitric oxide (no), a chemical mediator involved in the regulation of many physiological processes. on the other side, high no levels have been related to a variety of illnesses. in models of inflammatory bowel disease and diabetes mellitus, no inhibitors have been found to reduce inflammation and tissue alterations (aydin et al., 2010). increasing data suggests that oxidative stress and alterations in nitric oxide production or activity are important factors in the development of diabetes problems (maritim et al., 2003). the extract efficiently inhibited the formation of nitric oxide from sodium nitroprusside in the current investigation by competing with oxygen for the reaction with nitric oxide and therefore inhibiting the generation of nitrite anions. the extract showed a slightly lower nitric oxide scavenging activity at lower concentrations than the standard ascorbic acid. however, at higher concentrations, the radical scavenging activity increased considerably as that of the standard decreased. thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences, not repeat them. concluding sentence should be given at the end of the discussion. conclusions the findings from this study showed that the cfae is a rich source of natural products including; alkaloids, flavonoids, tannins, glycosides, phenols, steroids, terpenoids and saponins, and possesses good antioxidant property which could be attributed to the presence of phenolic compounds and antioxidant vitamins (a, c and e). these findings strongly suggest that their constituents can be used as an easily accessible source of natural antioxidant, which could be exploited in the food and pharmaceutical industries in production of potent nutraceuticals or therapeutically-important products. acknowledgements: the authors expressed profound gratitude to okwor josephat ani for his technical assistance during the conduct of this study. authors’ contributions: uroko ri and ndukwe oa designed the study. uroko ri, akachukwu, aaron cf, umezurike bc, ndukwe oa and chukwu cn carried out the laboratory work. uroko ri analyzed the data. uroko ri and chukwu cn wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references adamski z, blythe ll, milella l, bufo sa. 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(1995). antioxidant activity of various tea extracts in relation to their antimutagenicity. journal of agricultural and food chemistry, 43(1): 27-32 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 7-14 | doi: 10.14421/biomedich.2021.101.7-14 issn 2540-9328 (online) compliance level of textual therapeutic usage of kshirakakoli containing formulations with a serial ethnomedicinal survey and modern system of medicine gunpreet kaur1, vikas gupta1, ravinder sharma2, sanjiv kumar3, rg singhal4, ranjit singh4, parveen bansal1,* 1university centre of excellence in research, baba farid university of health sciences, faridkot, punjab, india 2university institute of pharmaceutical sciences, faridkot, punjab, india 2regional ayurveda research institute, ranikhet, uttarakhand, india 3shobhit university, meerut, uttar pradesh, india 4shobhit university, gangoh, uttar pradesh, india corresponding author* ucer_bfuhs@rediffmail.com, reetjattana21@gmail.com manuscript received: 19 november, 2020. revision accepted: 28 june, 2021. published: 01 july, 2021. abstract fritillaria roylei (kshirakakoli) is a primal plant used in ancient times. but nowadays, due to biotic and abiotic stress the plant has entered in the list of threatened medicinal plant. in ancient texts effective uses of formulations containing kshirakakoli are well mentioned but the information is not written in simple language due to which the therapeutic value of the plant is not well understood by scientific fraternity. so, there is a major need to perform ethno medicinal survey for the formulations containing kshirakakoli and compare their therapeutic uses as mentioned in text with the modern system of medicine. in this study, a field survey was performed in 4 states i.e., uttar pradesh, uttrakhand, punjab and himachal pradesh where the conversation regarding usage of this plant or formulation was done with 24 local medical practitioners, 18 shopkeepers and 4 traditional healers. the information thus obtained were recorded and then compared. results showed that the usage of kshirakakoli containing formulations was highest in himachal pradesh and uttrakhand. only few clinical studies have been done on these formulations. the effectiveness of the formulations against remedies alluded by the trado-medical practitioners claimed was found accurate as per ayurvedic textual literature. hence, the ethno medicinal survey provides a precise guidance to scientists for future research on these kshirakakoli containing formulations that are useful in plethora of disorders. keywords: ethnomedicinal survey; kshirakakoli; formulations; ayurveda; fritillaria roylei. introduction today, plant products, animal products, minerals and metals are used by pharmaceutical industries for production of majority of the ayurvedic as well as allopathic medicines. on the basis of information obtained from the traditional healers nearly 121 pharmaceutical products have been discovered (anesini and perez, 1993; kshiti, 2012). the knowledge of medicinal plants started fading away with the desertion of gurukul system of ancient teaching, as written details of most of the medicinal plants are not available (sharma and balkrishna, 2005). this also happened with eight plants of ashtawarga that are considered as very good rasayana with rejuvenating and health promoting properties that strengthens the immune system. due to high therapeutic properties, these plants are used in various ayurvedic formulations like taila (medicated oil), ghritam (medicated clarified butter), churana (powder) and other formulations of traditional medicinal system including chywanprash and other disease preventive tonics (dhyani et al., 2010). ‘ashtawarga’ a significant constituent of a variety of classical ayurvedic formulations has been assigned a variety of medicinal properties by ancient materia medica dealing with ayurveda and a subject of rigorous botanical research. kshirakakoli known as fritillaria roylei (one out of group of eight ashtwarga plants) come under the category of threatened species (saha et al., 2015). due to limited distribution in their natural habitat, today substitutes of ashtawarga plants are commonly used in ayurvedic formulations to meet the market demand. department of ayush, govt. of india has suggested use of substitutes in formulations in absence of original plants however this option is being exploited by manufacturers rendering such an important and precious plant in ignored condition (sagar, 2014; tewari,1991). hence it becomes important to highlight the therapeutic potentials of this plant in front of scientists so that a justified research shall be carried out on an important but ignored plant. fritillaria roylei contain various https://doi.org/10.14421/biomedich.2021.101.7-14 8 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 active compounds like peimine, peiminine, peimisine, peimiphine, peimidine, peimitidine, propeimin, sterol and these active compounds possess galactogogue, haemostatic, ophthalmic and cytotoxic properties (chi et al., 1940; wu, 1944; chou, 1947; chatterjee et al., 1976; jiang et al., 2006). due to the lack of authentic species in natural habitats, systemized studies/clinical studies have not been carried out on this group of plants. a very few clinical studies related to its therapeutic potentials have been carried out. moreover, the information available is also highly scattered that is not organized in proper manner due to which it cannot be used by scientist groups working on such important plants. this plant is being used in a number of formulations of high therapeutic value as mentioned in ancient texts however a very limited data with scientific evidences is available in modern literature as well as on internet sources. ancient texts claim very potent uses of these formulations but as the information is available in regional languages or in sanskrit, so the real uses of the plant are not well understood by scientific fraternity. hence it becomes important to know the real status of the mentioned therapeutic potentials as well as practiced potentials by trado-medical practitioners (tmps) and put it in front of scientists so as to give a thrust to clinical studies on therapeutic effects as well as pharmacological actions of the plant. material and methods in india, particularly in the rural and remote areas, there are number of trado-medical practitioners (tmps) and various kshirakakoli containing ayurvedic formulations are used by these practitioners as a traditional medicine for the treatment of a variety of ailments. so, a folklore survey was taken in 2019 to recognize the therapeutic importance of available kshirakakoli containing formulations mainly in four states of northern india. a field survey was conducted around and in district headquarters and discussion was done with the 24 local medical practitioners, 18 shopkeepers and 4 traditional healers and the information related to these ayurvedic formulations was recorded. the information procured was validated by comparing the information given by at least four tmps. the medicinal uses of these formulations were recorded from the folklore claims and the standard literature of the indian systems of medicine. an effort has been made to highlight the traditional use of these formulations so as to enable the scientists to explore these formulations for further research studies. result and discussion the folklore survey was done in various districts in himachal pradesh (shimla, dharamshala, kangra, mandi, kullu and manali), punjab (sangrur, barnala, ludhiana, bhatinda, patiala, ferozepur, faridkot), uttrakhand (dehradun, haridwar, pauri garhwal, rudraprayag) and in uttar pradesh (agra, meerut, moradabad, mathura). it was observed that usages of 20 formulations containing kshirakakoli were maximum in himachal pardesh and uttrakhand followed by uttar pradesh and least in punjab. the excerpts of ayurvedic formulations containing kshirakakoli as one of the ingredients are listed in table 1. the compliance level of therapeutic uses of kshirakakoli containing formulations as per ancient literature with trado-medical practitioners (tmps) and pre-clinical/clinical trials/case studies as per modern system of medicine are enumerated as in table 2. these formulations have been indicated in a plethora of reproductive disorders, loss of digestion, insanity, depression, depletion of body tissue, emaciation, phthisis, cures gout arthritis pervading the whole body, heart disease, facial paralysis, diseases of the head/neck and epilepsy. similar type of ethnomedicinal survey was conducted by the authors for kakoli containing formulations (kaur et al., 2019). so, there is a need of hour to explore kakoli and kshirakakoli containing important formulations for further trials. table 1. excerpts of ayurvedic formulations containing kshirakakoli. s. no. formulation shloka’s references 1. jeevaneeya gana तद्यथा – जीवकर्षभकौ मेदा महामेदा काकोली क्षीरकाकोली मुदपर्णीमार्पर्ण्यो जीवन्ती मधुकमममत दशेमामि जीविीयामि भवन्तन्त hebbar, 2015a 2. shukrala / kamdev ghrita शुक्रलैजीविीयैश्च ब ृंहरै्णबषलवधषिैैः | क्षीरसञ्जििैशै्चव पयैः मसद्धृं प थक् प थक्||६|| युक्तृं गोधूमचूरे्णि सघ तक्षौद्र शकष रम्| पयाषयेर्ण प्रयोक्तव्यममच्छता शुक्रमक्षयम्||७|| hebbar, 2015a 3. khuddaka taila/ khuddaka padmaka tailam पद्मकोशीर यष्ट्याह्व रजिी क्वाथ सामधतम्| स्यात् मपषै्ैः सजष ममञ्जष्ठा वीरा काकोमल चन्दिैैः ||११४|| खुड्डाक पद्मकममदृं तैलृं वातास्र दाहिुत्|११५| इमत खुड्डाक पद्मकृं तैलम्| hebbar, 2016a kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 9 4. madhuparnyadi taila मधुकस्य शतृं द्राक्षा खजूषरामर्ण परूर्कम्| मधूकौदिपाक्यौ च प्रस्थृं मुञ्जातकस्य च||९६|| काश्मयाषढकममते्यतच्चतुद्रोरे्ण पचेदपाम्| शेरे्ऽष्भागे पूते च तन्तमृंसै्तलाढकृं पचेत्||९७|| तथाऽऽमलक काश्मयष मवदारीकु्ष रसैैः समैैः | चतुद्रोरे्णि पयसा कल्कृं दत्त्वा पलोन्तितम्||९८|| कदम्बामलकाक्षोट पद्म बीज कशेरुकम्| hebbar, 2016a 5. mahasneham ghrita जीवकर्षभकौ मेदाम ष्यप्रोक्ताृं शतावरीम्| मधुकृं मधुपर्णीं च काकोलीद्वयमेव च||७२|| मुद्ग मार्ाख्यपमर्णषन्यौ दशमूलृं पुििषवाम्| बलाम ता मवदारीश्च साश्वगन्धाश्मभेदकाैः ||७३|| एर्ाृं कर्ाय कल्काभ्ाृं समपषसै्तलृं च साधयेत्| लाभतश्च वसा मज्ज धान्व प्रातुद वैन्तिरम्||७४|| चतुगुषरे्णि पयसा तत् मसद्धृं वात शोमर्णतम्| hebbar, 2016a 6. paste prepared of drugs belonging to jivaniya group, cow-milk and muscle fat/ dhanwantharam thailam समूलाग्रच्छदैरण्डक्वाथे मद्व प्रान्तस्थकृं प थक्| घ तृं तैलृं वसा मज्जा चािूप म ग पमक्षर्णाम्||१४१|| कल्काथे जीविीयामि गव्यृं क्षीरमथाजकम्| हररद्रोत्पल कुषै्ठला शताह्वाश्वहिच्छदाि् ||१४२|| मबल्व मात्राि् प थक् पुष्पृं काकुभृं चामप साधयेत्| मधून्तच्छष् पलान्यष्ौ दद्याच्छीतेऽवताररते||१४३|| hebbar, 2016a 7. sneha parisheka से्नहै मषधुर मसदै्धवाष चतुमभषैः परररे्चयेत्| स्तम्भाके्षपक शूलातं कोषै्णदाषहे तु शीतलैैः ||१२५|| hebbar, 2016a 8. parushaka ghrita त्रायन्तन्तका तामलकी मद्वकाकोली शतावरी| कशेरुका कर्ायेर्ण कलै्करेमभैः पचेद्ध तम्||५८|| दत्त्वा परूर्का द्राक्षा काश्मयेकु्षरसाि् समाि्| प थन्तिदायाषैः स्वरसृं तथा क्षीरृं चतुगुषर्णम्||५९|| एतत् प्रायोमगकृं समपषैः पारूर्कमममत म तम्| वातरके्त क्षते क्षीरे्ण वीसपे पैमिके ज्वरे||६०|| इमत पारूर्कृं घ तम्| hebbar, 2016a 9. bala ghrita/taila बलाममतबलाृं मेदामात्मगुप्ाृं शतावरीम्| काकोलीृं क्षीरकाकोलीृं रास्नाम न्तद्धृं च पेर्येत्||५६|| घ तृं चतुगुषर्ण क्षीरृं तैैः मसद्धृं वातरक्तिुत्| हृत्पाणु्डरोग वीसपष कामला ज्वर िाशिम्||५७|| hebbar, 2016a 10. sukumara taila/ sukumara kashaya मधुकस्य शतृं द्राक्षा खजूषरामर्ण परूर्कम्| मधूकौदिपाक्यौ च प्रस्थृं मुञ्जातकस्य च||९६|| काश्मयाषढकममते्यतच्चतुद्रोरे्ण पचेदपाम्| शेरे्ऽष्भागे पूते च तन्तमृंसै्तलाढकृं पचेत्||९७|| तथामलक काश्मयष मवदारीकु्षरसैैः समैैः | चतुद्रोरे्णि पयसा कल्कृं दत्त्वा पलोन्तितम्||९८|| कदम्बामलकाक्षोट पद्म बीज कशेरुकम्| शृङ्गाटकृं शृङ्गवेरृं लवार्णृं मपप्पलीृं मसताम्||९९|| hebbar, 2016a 11. snehopaga gana/ (adjuvants of snehana/oleation treatment) म द्वीका मधुक मधुपर्णी मेदामवदारी काकोली क्षीरकाकोली जीवक जीवन्ती शालपर्ण्यष इमत दशेमामि से्नहोपगामि भवन्तन्त hebbar, 2016a 12. shukrala shukrajanana/ shukra-shodhana-janana granules जीवकर्षभक काकोली क्षीरकाकोली मुदपर्णी मार्पर्णी मेदाव द्धरूहा जमटला कुमलड्गा इमत दशेमामि शुक्रजििामि भवन्तन्त hebbar, 2016a 13. jivaniya mahakashaya तैलप्रस्थृं घ तप्रस्थृं जीविीयैैः पलोन्तितैैः | क्षीरद्रोरे्ण पचेत् मसद्धमपमारमविाशिम्||२८|| hebbar, 2016a 14. amruta prasha ghruta/ amritaprasha ghrita जीवकर्षभकौ वीराृं जीवन्तीृं िागरृं शटीम्| चतस्रैः पमर्णषिीमेदे काकोल्यौ दे्व मिमदन्तिके||३५|| पुििषवे दे्व मधुकमात्मगुप्ाृं शतावरीम्| ऋन्तद्धृं परूर्कृं भागीं म द्वीकाृं ब हतीृं तथा||३६|| शृङ्गाटकृं तामलकीृं पयस्याृं मपप्पलीृं बलाम्| बदराक्षोट खजूषर वातामामभरु्कार्ण्यमप||३७|| hebbar, 2015b 10 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 15. phalakalyan grita/ taila uttara basti कमर्णषन्य चरर्णाशुियोमि प्राक्चरर्णासु च||१०२|| कफवाते च दातव्यृं तैलमुिर बन्तस्तिा| hebbar, 2016b 16. siva gutika/shiva gritham मेदाृं पयस्याृं जीवन्तीृं मवदारी ृं कण्टकाररकाम्| श्वदृंष््ाृं क्षीररकाृं मार्ाि् गोधूमाञ्छामलर्मष्काि्||८|| पयस्यधोदके पक्त्वा कामर्षकािाढकोन्तिते| मववजषयेत् पयैः शेर्ृं तत् पूतृं क्षौद्रसमपषर्ा||९|| युक्तृं सशकष रृं पीत्वा व द्धैः सप्मतकोऽमप वा| मवपुलृं लभतेऽपत्यृं युवेव च स हृष्यमत||१०|| hebbar, 2016b 17. vrishya pooplika फलािाृं जीविीयािो न्तििािाृं रूमचकाररर्णाम् । कुडवशू्चमर्णषतािाृं स्यात् स्वयड्गुमाफलस्य च ॥ १५ ॥ कुडवशै्चव मार्ार्णाृं द्वौ च मतलमुद्र्यो :। गोघूमशामलचूर्णाषिाृं कुडव: कुडवो भवेत्॥ १६ ॥ hebbar, 1998 18. upatyakari shashtikadi gutika आमसक्त क्षीरमापूर्णषमशुिृं शुद्ध र्मष्कम्| उदूखले समापोथ्य पीडयेत् क्षीरममदषतम्||३|| ग हीत्वा तृं रसृं पूतृं गवे्यि पयसा सह| बीजािामात्मगुप्ाया धान्य मार् रसेि च||४|| hebbar, 2016c 19. mahamayura ghrita/ mahanarayan tailm एतेिैव कर्ायेर्ण घ त प्रस्थृं मवपाचयेत्| चतुगुषरे्णि पयसा कलै्करेमभश्च कामर्षकैैः ||१६६|| जीवन्ती मत्रफला मेदा म द्वीकमधष परूर्कैैः | समङ्गा चमवका भागी काश्मरी सुरदारुमभैः ||१६७|| आत्मगुप्ा महामेदा ताल खजूषर मस्तकैैः | hebbar, 2015c 20. jivantyadi ghrita एवमेव क्षीर समपष जीविीयोपसामधतम्||६९|| गभषदृं मपिलािाृं च योिीिाृं स्यान्तिर्न्तितम्|७०| hebbar, 2015c table 2. compliance level of therapeutic uses of kshirakakoli containing formulations as per ancient literature with trado-medical practitioners (tmps) and pre-clinical/clinical trials/case studies as per modern system of medicine. sr. no. formulations therapeutic uses as per texts therapeutic uses as mentioned by trado-medical practitioners (tmps) pre-clinical/clinical trials/case studies as per modern system of medicine 1. amruta prasha ghruta/ amritaprasha ghrita 1. cough 2. hiccup 3. fever 4. asthma 5. burning sensation 6. morbid thirst 7. an ailment characterized by bleeding from different parts of the body 8. vomiting 9. fainting 10. diseases of heart 11. female genetial tract 12. urinary tract 13. procreation of male child 14. joint problems and other diseases caused by unbalanced vata & pitta doshas. yes yes yes yes yes yes yes yes yes no yes no yes yes no no no no no no no no no no no no no yes improves the nonspecific immunity against rheumatoid arthritis (lekurwalel et al., 2010). 2. jeevaneeya gana 1. enlivening 2. anti-aging 3. antioxidant yes yes yes no no no 3. jivantyadi ghrita 1. cure female infertility 2. cure cataract and glaucoma yes yes no yes effective in treatment and controlling of computer vision syndrome and helpful in curing myopia (vinaik et al., 2013; shukla et al., 2011). kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 11 4. khuddaka taila/ khuddaka padmaka tailam 1. gout 2. burning sensation yes yes no no 5. madhuparnyadi taila 1. cures vatarakta 2. pain in limbs 3. affliction of the whole body 4. it also promotes strength and complexion. yes yes yes yes no no no no 6. mahamayura ghrita/ mahanarayan tailm 1. neuro-muscular disorders 2. cough 3. asthma 4. inflammation 5. diseases of the female genital tract 6. menstrual disorders yes yes yes yes yes yes no no yes yes used to treat inflammation, pain and arthritis, useful in treatment of chronic back pain, useful in treatment of female infertility, provide strength to the local soft tissues, thus play significant role in the management of cervical spondylosis or osteoarthritis of cervical spine (pawar et al., 2011; panda and debnath, 2011; kaushik et al., 2017; kunjibettu et al., 2017). 7. mahasneham ghrita 1. gout 2. serious diseases caused by the aggravated vata 3. arthritis yes yes no no 8. paste prepared of drugs belonging to jivaniya group, cow-milk and muscle fat/ dhanwantharam thailam 1. cures pain 2. gout 3. kyphosis (spinal disorder) no yes yes no no no 9. phalakalyan grita/ taila uttara basti 1. uterine tonic 2. gynecological disorders yes yes no yes useful in treatment of female infertility (kunjibettu et al., 2017). 10. shukrala/ kamdev ghrita 1. improves semen and sperm production yes no 11. siva gutika/shiva gritham 1. diabetes mellitus 2. bronchitis 3. anemia 4. cardiac diseases 5. epilepsy 6. psychotic diseases 7. skin diseases 8. infertility in women/men 9. liver diseases no no yes yes yes yes no yes yes no no no no no no no no yes used in treatment of chronic liver and highly effective in treatment of hiv/aids and acute deep vein thrombosis (wasedar et al., 2017; rathod et al., 2013; pusuluri et al., 2017). 12. sneha parisheka 1. stiffness 2. convulsion 3. gout 4. burning sensation yes yes yes yes no no no no 12 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 13. sukumara taila/ sukumara kashaya 1. lower abdominal pain in women 2. menstrual pain 3. inflammation 4. constipation 5. promotes robustness in body and positive health yes yes yes yes yes yes yes yes yes yes helps in improving the quality of the ovum by regularizing the menstrual cycle which enhanced rate of conception and also used in treatment of complete rectal prolapse (vanishree and kumari, 2017; shripathi et al., 2017). 14. upatyakari shashtikadi gutika 1. male fertility yes no 15. vrishya pooplika 1. aphrodisiac yes no 16. bala ghrita/taila 1. gout 2. enemia 3. jaundice 4. uterine diseases 5. provides strength to children no yes relieves constipation in pregnant women (sharma et al., 2017). no yes helps in improving females reproductive disorders (patel, 2018). yes message contributes positive effects to health of new born child (sharma et al., 2017). 17. parushaka ghrita 1. menstrual pain 2. menorrhagia 3. anemia 4. respiratory disorder 5. jaundice yes yes act as good uterine tonic by providing strength to uterus (vartak and mehere, 2019) no no no 18. shukrala shukrajanana/ shukrashodhanajanana granules 1. improves quality and quantity of semen and sperm yes provides strength and increases sperm count (girish, 2017). 19. snehopaga gana/ (adjuvants of snehana/oleation treatment) 1. dry skin conditions 2. neuromuscular disorders 3. muscular dystrophy 4. wasting disorders no no no no 20. jivaniya mahakashaya 1. provides strength 2. immunity booster 3. improves quality of semen 4. aphrodisiac 5. hormonal disturbances in women 6. nervousness dizziness no no no no no no conclusion the analysis of literature reveals that fritillaria roylei is a wonder plant used by saints/rishies since ages however due to a number of reasons; plant has been ignored for its therapeutic uses. this survey clearly indicates that there is a need of deciphering the textual references given in regional languages and use them for new drug development process. as clearly indicated in textual as well as in survey, most of the kshirakakoli kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 13 containing formulations have been recommended for number of disorders in men and women. however, there is lack of such patent formulations as well systemized clinical trials that could prove to be useful in highlighting the real therapeutic potentials of kshirakakoli containing formulations. hence this survey provides a template for scientists for further screening and research on these formulations that are useful in plethora of disorders. financial support: none acknowledgment: the authors are thankful to the tradomedical practitioners (tmps) for sharing their precious traditional knowledge. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references anesini, c., perez, c. 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(1944). the constituents of fritillaria roylei. j. am. chem. soc., 66(10) 1778-1780. doi: https://doi.org/10.1021/ja01238a048. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 1-6 | doi: 10.14421/biomedich.2022.111.1-6 issn 2540-9328 (online) potential antimalarial activity of artemether/lumefantrine/doxycycline: a study in mice infected with plasmodium berghei udeme owunari georgewill1,*, elias adikwu2 1department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* udgeorgewill@yahoo.com, tel+234 8036662936 manuscript received: 08 december 2021. revision accepted: 11 january, 2022. published: 09 february, 2022. abstract antimalarial drug resistance is one of the greatest challenges towards eradicating malaria. exploring new combination therapies can overcome resistance challenges. the present study examined the antiplasmodial effect of artemether/lumefantrine/doxycycline (a/l/d) on a mouse model infected with plasmodium berghei. adult swiss albino mice (22-30g) intraperitoneally infected with blood containing 1x107 plasmodium berghei were randomly grouped and orally treated daily with d (2.2 mg/kg), a/l (1.71/13.7 mg/kg) and a/l/d. the negative control was treated daily with normal saline (0.2ml) whereas the positive control was treated daily with chloroquine (cq) (10mg/kg). after treatment, blood samples were assessed for percentage parasitemia and biochemical parameters. mice were observed for mean survival time (mst). d, a/l and a/l/d produced significant decreases in percentage parasitemia levels at p<0.05; p<0.01 and p<0.001, respectively when compared to negative control. in the curative test, d, a/l and a/l/d produced 60.4%, 70.3%, and 90.0% parasitemia inhibitions, respectively whereas cq produced 76.0% parasitemia inhibition. d, a/l, a/l/d and cq produced 63.2 %, 80.1%, 92.3% and 83.6% parasitemia inhibitions, respectively in the suppressive test. d, a/l, and a/l/d prevented plasmodium bergheiinduced alterations in biochemical parameters by increasing packed cell volume, red blood cells, hemoglobin, and high-density lipoprotein and decreasing white blood cells, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels significantly at p<0.05 and p<0.01 and p<0.001, respectively when compared to the negative control. a/l/d produced significant antiplasmodial activity therefore, it may be used clinically for the treatment of malaria. keywords; antiplasmodial; artemether; tetracycline; antimalarial; lumefantrine; plasmodium berghei. introduction antimalarial drug resistance has been acknowledged to be one of the greatest challenges to the roll back malaria programme. the situation is highly precarious due to the rising incidence of plasmodium resistance to currently available antimalarial drugs (yeung et al., 2004). chloroquine (cq) resistant plasmodium falciparum now predominates in southeast asia, south america and africa. resistance to sulphadoxinepyrimethamine is widespread in asia and south america and is spreading in africa and even quinine has become less effective over time (khan et al., 2004). the use of combination therapy with artemisinins and partner drugs has been a rational approach to combating drug resistance. drugs with different mechanisms of action may enhance their respective efficacies and extend their therapeutic life spans (white & olliaro, 1996). despite, the combination therapy approach, plasmodium resistance is still a serious challenge (yeung et al., 2004). doxycycline (d) is one of the most active antibiotics against plasmodium parasites. it belongs to the tetracycline family. the tetracyclines, which have a very wide spectrum of activity are bacteriostatic and inhibit bacterial protein synthesis (gaillard et al., 2015). among the tetracyclines, d is widely used for malaria prophylaxis and is highly acceptable for long-term therapy, except in pregnant women and children (who, 2005). in-vitro and in-vivo investigations have shown that d may be an effective antimalarial drug against drug-resistant plasmodium strains (basco & bras, 1993). it is used in combination with quinine as an effective standby emergency treatment of malaria associated with plasmodium falciparum (who, 2005). recently, it has shown antimalarial potential against plasmodium bergheiinduced cerebral malaria in experimental models by inhibiting brain inflammation, tumour necrosis factor and chemokines expressions (schmidt et al., 2018). artemether/lumefantrine (a/l) is an artemisininbased antimalarial drug approved by the us food and https://doi.org/10.14421/biomedich.2022.111.1-6 2 biology, medicine, & natural product chemistry 11 (1), 2022: 1-6 drug administration in 2009 for the treatment of plasmodium falciparum malaria. the dual mechanisms of action of a/l provide fast and sustained plasmodium clearance (stover et al., 2012). it is the most widely used antimalarial drug combination in endemic regions. in 2017, a/l accounted for almost 75% of all purchased and clinically used artemisinin based combination therapies (acts) (nsanzabana, 2019). artemisinin derivatives rapidly clear parasites through a number of proposed mechanisms such as interference with plasmodial transport proteins, interference with plasmodial mitochondrial electron transport, and the production of free radicals (stover et al., 2012). the precise mechanism for the antiplasmodial activity of lumefantrine is not well defined, but it is proposed to inhibit β-hematin formation, which is an important detoxification pathway for plasmodium parasites (stover et al., 2012). despite the success achieved with the use of a/l in combating malaria scourge, the emergence of plasmodium parasite resistance in some endemic countries has become a significant drawback for the fight against malaria (nsanzabana, 2019). due to the challenge posed by plasmodium parasite resistance, there is a strong advocacy for the rational use of antimalarial drugs with antibiotics. this will afford the synergistic or additive killing of plasmodium parasites, and thus prevent or reduce drug resistance (miller et al., 2006; alecrim et al., 2006). this study, therefore assessed the antiplasmodial effect of a/l/d on a mouse model infected with plasmodium berghei. materials and methods drugs and dose selection artemether/lumefantrine (a/l) (ipac laboratory, india), chloroquine (cq) (evans medical nigeria plc), and doxycycline (d) (ranbaxy laboratories ltd, india) were used. the following doses were used: a/l (2.3/13.7 mg/kg) (sirima et al., 2016), cq (10mg/kg) (somsak et al., 2018), and d (2.2 mg/kg) (gaillard et al., 2015) animals adult swiss albino mice (22–30 g) were used. the mice were obtained from the animal unit of the department of pharmacology, faculty of basic clinical sciences, college of health sciences, university of port harcourt, rivers state. the mice were housed and fed throughout the study period as per recommended standards. the mice were allowed for 2 weeks to acclimatize to working environment and were handled according to the international animal care and welfare guidelines (ilar, 2011). parasites inoculation cq-sensitive plasmodium berghei (p. berghei) (nk65 strain) was used for malaria induction in the experimental mice. p. berghei was obtained from malaria research laboratory, centre for malaria research and phytomedicine, university of portharcourt, rivers state, nigeria. mice previously infected with p. berghei were used as donor mice and parasites were kept alive by continuous intraperitoneal (i.p) passage of blood from donor mice to uninfected mice weekly. percentage parasitemia was determined using the formula below. protocol for antiplasmodial test  protocol for curative test the curative test was performed as described by ryley and peters (1970). thirty mice randomly grouped into 6 (a1-a6) of 5 mice each were used. group a1 served as the normal control while a2-a6, which served as the experimental groups were inoculated with 1 × 107 p. berghei-infected blood (i.p). three days later, the mice were treated orally as follows: group a1 (normal control) was treated with normal saline (0.2ml) daily for 4 days. group a2 (negative control) and group a3 (positive control) were treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups a4-a6 were treated with d (2.2mg/kg), a/l (2.3/13.7 mg/kg) and a/l/d daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice and thin smears were prepared on slides and stained with 10% giemsa stain. the stained slides were examined microscopically with an oil immersion objective of 100× magnification power. the percentage parasitemia and inhibitions were calculated using the formula shown below. % 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑎𝑒𝑚𝑖𝑎 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑧𝑒𝑑 𝑟𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙𝑠 (𝑅𝐵𝐶𝑠) total number of rbcs count × 100% % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (% parasitemia of negative control − % parasitemia of treated group) × 100 % parasitemia of negative control  protocol for suppressive test the suppressive test was performed as described by knight and peters (1980). twenty-five adult swiss albino mice were inoculated with blood containing 1 × 107p. berghei and randomized into 5 groups (b1-b5) of 5 mice each. the mice were treated after 3 hours of georgewill & adikwu – potential antimalarial activity of artemether/lumefantrine/doxycycline: … 3 inoculation as follows: group b1 (negative control) and group b2 (positive control) were orally treated daily with normal saline (0.2ml) and cq (10mg/kg), for 4 days, respectively. groups b3-b5 were orally treated with d (2.2mg/kg), a/l (2.3/13.7 mg/kg) and a/l/d daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice and thin smears were prepared on slides and stained with 10% giemsa stain. the stained slides were examined microscopically with an oil immersion objective of 100×magnification power. the percentage parasitemia and inhibitions were calculated as explained above.  protocol for prophylactic test the prophylactic test was performed as described by peters (1965). twenty-five adult swiss albino mice randomized into 5 groups (c1-c5) of 5 mice/group were orally treated as follows: group c1 (negative control) and group c2 (positive control) were treated with normal saline (0.2 ml) and cq (10 mg/kg) daily for 4 days, respectively. groups c3 – c5 were treated with d (2.2 mg/kg), a/l (2.3/13.7 mg/kg), and a/l/d daily for 4 days, respectively. on day 5, the mice were inoculated with blood containing 1 × 107 p. berghei. after 2 days, tail blood samples were collected and percentage parasitemia and inhibitions were determined as explained above. determination of mean survival time from the time of inoculation with p. berghei until death, mortality of each mouse was monitored and recorded. mean survival time (mst) was determined using the formula below. 𝑀𝑆𝑇 = 𝑆𝑢𝑚 𝑜𝑓 𝑠𝑢𝑟𝑣𝑖𝑣𝑎𝑙 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑎𝑙𝑙 𝑚𝑖𝑐𝑒 𝑖𝑛 𝑎 𝑔𝑟𝑜𝑢𝑝 (𝐷𝑎𝑦𝑠) total number of mice in that group evaluation of biochemical parameters in the curative study, blood samples were collected from the mice and evaluated for red blood cells (rbcs), hemoglobin (hb), packed cell volume (pcv), white blood cells (wbcs), total cholesterol (chol), triglyceride (tg), low-density lipoprotein cholesterol and (ldl-c) and high-density lipoprotein cholesterol (hdl-c) using an auto analyzer. statistical analysis values were expressed as mean ± sem (standard error of mean) of n=5. values were analyzed using one-way anova, followed by tukey’s post hoc test. p values less than 0.05, 0.01 and 0.001 were considered significant. results curative antiplasmodial test treatment with d, a/l, and a/l/d produced significant decreases in percentage parasitemia at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control (pu) (table 1). d, a/l and a/l/d produced parasitemia inhibitions of 60.4%, 70.3%, and 90.0%, respectively whereas cq produced 76.0 % parasitemia inhibition. mst was significantly prolonged in mice treated with d, a/l, and a/l/d at p<0.05, p<0.01, and p<0.001, respectively when compared to pu (table 1). suppressive antiplasmodial test significant decreases in percentage parasitemia levels at p<0.5, p<0.01 and p<0.001 were produced by d, a/l, and a/l/d, respectively when compared to pu (table 2). parasitemia inhibitions, which represent 63.2 %, 80.1%, 92. 3% and 83. 6% were produced by d, a/l, a/l/d and cq, respectively (table 2). treatment with d, a/l and a/l/d significantly prolonged mst at p<0.5, p<0.01, and p<0.001, respectively when compared to pu (table 2). prophylactic antiplasmodial test percentage parasitemia levels were significantly decreased in mice treated with d (p<0.05), a/l (p<0.01) and a/l/d (p<0.001) when compared to pu (table 3). d, a/l and a/l/d produced parasitemia inhibitions of 65.1%, 82.8%, and 93.9%, respectively whereas cq produced 86.7% parasitemia inhibition. significant prolongations of mst occurred in mice treated with d, a/l, and a/l/d at p<0.5, p<0.01 and p<0.001, respectively when compared to pu (table 3). hematological and lipid profile p. berghei infected mice showed significant (p<0.001) increases in serum tg, chol, ldl-c and wbcs levels with significant (p<0.001) decreases in serum hb, pcv, rbcs and hdl-c levels when compared to control (tables 4 and 5). on the other hand, treatment with d, a/l, and a/l/d significantly decreased serum tg, chol, ldl-c, wbcs levels and significantly increased serum hb, pcv, rbcs and hdl-c levels at p<0.05, p<0.01and p<0.001, respectively when compared to pu (tables 4 and 5). 4 biology, medicine, & natural product chemistry 11 (1), 2022: 1-6 table 1. curative effect of artemether/lumefantrine/doxycycline on plasmodium berghei infected mice. data as mean ± sem (standard error of mean) n= 5, pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 2. suppressive effect of artemether/lumefantrine/doxycycline on plasmodium berghei infected mice. treatment % parasitemia % inhibition mst (days) pu 20.30±1.03 0.00 9.66±0.45 cq 3.33±0.32a 83.6 30.41±3.71a d 7.47±0.11b 63.2 22.73±2.78b a/l 4.04±0.05a 80.1 31.02±3.48a a/l/d 1.56±0.02c 92.3 37.62±3.67c data as mean ± sem (standard error of mean) n= 5, pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 3. prophylactic effect of artemether/lumefantrine/doxycycline on plasmodium berghei-infected mice treatment % parasitemia % inhibition mst (days) pu 18.50±1.57 0.00 9.66±0.61 cq 2.46±0.01a 86.7 32.03±3.71a d 6.46±0.35b 65.1 25.52±2.60b a/l 2.44±0.17a 82.8 32.60±3.38a a/l/d 1.29±0.03c 93.9 39.21±4.61c data as mean ± sem (standard error of mean) n= 5. pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 4. effect of artemether/lumefantrine/doxycycline on hematologic parameters of plasmodium berghei-infected mice. treatment rbc (x106) wbc (cells/l) pcv (%) hb (g/dl) nc 5.11±0.24 6.61±0.11 54.63±5.00 16.31±0.08 pu 2.41±0.29a 13.91±1.06a 20.72±3.17a 6.63±0.37a cq 4.00±0.11b 9.11±0.09b 38.80±3.54b 11.93±0.15b d 3.10±0.17c 10.27±0.17c 26.54±3.76c 9.65±0.45c a/l 3.99±0.12b 9.42±0.09b 38.43±4.49b 10.04±0.80b a/l/d 4.91±0.09d 6.33±0.21d 50.25±4.21d 13.95±0.05d data as mean ± sem (standard error of mean) n= 5, nc: normal control pu: negative control cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time, rbcs: red blood count, wbcs: white blood count, pcv: packed cell volume hb: haemoglobin, a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pu. table 5. effect of artemether/lumefantrine/doxycycline on lipid parameters of plasmodium berghei-infected mice. treatment tg mg/dl chol mg/dl hdl-c mg/dl ldl mg/dl nc 76.8±8.03 100.4±14.0 55.9±5.66 29.1±3.11 pu 266.3±15.0a 298.4±14.1 a 24.6±2.32 a 220.1±18.1a cq 169.1±10.6 b 190.8±16.4 b 39.1±5.72 b 117.8±11.6b d 210.7±11.6 c 247.5±13.7 c 30.0±3.19 c 175.4±15.0c a/l 179.0±14.0b 199.6±14.3b 37.3±3.67b 126.5±12.5b a/l/d 80.9±7.49d 117.8±12.5 d 49.5±4.39d 41.3±10.1d data as mean ± sem (standard error of mean) n= 5, nc: normal control pu: negative control cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, tg: tryglyceride, chol:total cholesterol hdl: high density lipoproteins, ldl: low density lipoprotein, vldl: very low density lipoprotein. a p<0.001 when compared to nc, b p<0.01, c p<0.05, d p<0.001 when compared to pu. discussion artemisinin based combination therapies (acts) are used as treatment for uncomplicated malaria (who, 2015). unfortunately, the emergence of plasmodium parasite resistant to acts has been reported in endemic regions (cui et al., 2015; who, 2016). antimalarial drug resistance poses one of the greatest threats to malaria control. in africa, the efficacy of affordable antimalarial drugs is rapidly declining, and efficacious treatment %parasitemia %inhibition mst (days) pu 38.10±4.95 0.0 9.02±0.11 cq 9.14 ±0.20a 76.0 25.30±2.37a d 13.72±0.67b 60.4 16.21±1.57b a/l 11.30±0.13a 70.3 24.82±3.48a a/l/d 3.43±0.08c 90.0 31.63±3.21c georgewill & adikwu – potential antimalarial activity of artemether/lumefantrine/doxycycline: … 5 antimalarial drugs tend to be too expensive. costeffective methods are needed to fight against antimalarial drug resistance. one of the primary solutions to challenges associated with plasmodium parasites resistance to antimalarial drugs is to explore new combination therapies. in combination therapy, the possibility of the plasmodium parasites developing resistance simultaneously to two or more combined drugs with different mechanisms of action is extremely low (who, 2001). the current study examined the antiplasmodial activity of a/l/d on p. berghei-infected mice. p. berghei is used in predicting treatment outcomes of antimalarial drug candidates, due to its sensitivity, making it an important parasite for antiplasmodial studies (unekwuojo et al., 2011). studies have shown that suppressive and curative tests are effective in the antiplasmodial evaluation of candidate drugs on early and established infections, respectively. importantly, suppressive and curative tests give vital information on percentage parasitemia, and parasitemia inhibitions (bobasa et al., 2018). in the current study, treatment with a/l/d produced the best curative and suppressive antiplasmodial effects in relation to individual doses of a/l, d and cq. in the curative study, treatment with d, a/l, and a/l/d produced 60.4%, 70.3%, and 90.0% inhibitions, respectively. in the suppressive study, treatment with d, a/l and a/l/d produced 63.2 %, 80.1% and 92. 3% inhibitions, respectively. the prophylactic study showed best decrease in parasitemia level in a/l/d-treated mice when compared to individual doses of a/l, d and cq. the observed parasitemia inhibitions in the prophylactic study were 65.1%, 82.8%, and 93.9%, and 86.7% in mice treated with d, a/l, a/l/d and cq, respectively. in addition to percentage parasitemia and inhibition, this study determined the mst of the mice used for the curative, suppressive and prophylactic tests (georgewill et al., 2021) to further buttress the antiplasmodial activity of a/l/d. studies have shown that candidate drugs that can appreciably prolong the mst of parasitized animals may be active against malaria (oliveira et al., 2009). in the current study, curative, suppressive and prophylactic tests showed best prolongation of mst in mice treated with a/l/d than d, a/l, and cq alone. in the curative study, d, a/l and a/l/d prolonged mst to 16.21±1.57, 24.82±3.48 and 31.63±3.21 days, respectively. studies have shown that plasmodium depend on host hemoglobin as a nutrientsource for growth and multiplication. it consumes more than 75% of haemoglobin during its intra-erythrocytic phase and metabolizes heme into hemozoin (inbaneson and sundaram 2012). p. berghei-infected mice are prone to anemia due to erythrocyte destruction, as a consequence of parasite multiplication or by spleen reticuloendotelial cell action causing the production of phagocytes by the spleen due to abnormal erythrocytes (nardos and makonnen, 2017). in the current study, anemia was conspicuous in p. berghei-infected mice characterized by decreased pcv, hb, and rbcs with increased wbcs levels. however, p. berghei–induced anemia was vividly reduced in mice treated with a/l/d which was characterized by increased pcv, hb, and rbcs levels and decreased wbcs levels. interestingly, the anti-anemic activity of a/l/d was best when compared to individual doses of a/l, d and cq. changes in serum lipid profile related to malaria infection have been reported in some studies. the underlying biological mechanisms for malaria related lipid changes remain unclear, but may be host related, parasite-related or a combination of the two factors (visser et al., 2013). the present study observed significant alterations in lipid profile in parasitized mice marked by increased chol, tg, ldl-c and decreased hdl-c levels. the altered lipid parameters were best restored in a/l/d-treated mice marked by elevated hdl-c and decreased chol, tg, ldl-c levels. the observed antiplasmodial effect of a/l/d may be due to the independent mechanisms of action of the constituent drugs. the antiplasmodial mechanisms of d are not well defined, but d can inhibit mitochondrial protein synthesis and also decrease the activity of mitochondrial enzyme (dihydroorotate dehydrogenase) involved in pyrimidine synthesis (prapunwattana et al., 1998). also, d can inhibit the syntheses of nucleotides and deoxynucleotides in plasmodium (yeo et al., 1997). the artemisinins are speculated to interfere with plasmodial mitochondrial electron transport, transport proteins, and the production of free radicals (stover et al., 2012). lumefantrine is thought to inhibit β-hematin formation, an important detoxification pathway in plasmodium parasites (stover et al., 2012). conclusion results of the study concluded that a/l/d significantly decreased percentage parasitemia, increased percentage inhibition and prolonged mst in p. berghei-infected mice. the results of this study therefore showed prospect for the use of a/l/d for malaria treatment. conflict of interest: the authors declare no conflicts of interest. references alecrim mg, lacerda mv, mourao mp, 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(2004). antimalarial drug resistance, artemisinin based combination therapy, and the contribution of modelling to elucidating policy choices. american journal of tropical medicine and hygiene. 71(2): 179-86 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 81-89 | doi: 10.14421/biomedich.2020.92.81-89 issn 2540-9328 (online) non-alkaloidal compounds from khat (catha edulis) leaves tsegu kiros department of chemistry, college of natural sciences, haramaya university, dire dawa, p.o.box 138, ethiopia. corresponding author kirosorg@gmail.com manuscript received: 23 may, 2020. revision accepted: 01 october, 2020. published: 17 november, 2020. abstract khat belongs to the family celastraceae, genus catha, and species edulis. more than 200 compounds have previously been identified in khat leaves, including: 40 alkaloids, terpenoids and sterol, flavonoids, glycosides, tannins, amino acids, vitamins and minerals. researchers have spent their effort and time merely on study of the alkaloidal components (mainly the stimulant agents, cathinone and cathine) of khat both qualitatively and quantitatively. the two principal khat stimulant compounds, cathinone and cathine, by now are well established. but, on the contrary, previous studies on the non-alkaloidal constituents of the plant were limited. the objective of this work was therefore to isolate and characterize compounds from non-alkaloidal fractions of the plant’s leaves. in this work, two nonalkaloidal compounds (kna-1 and kna-2) were isolated and characterized from the acid-etoac extract of fresh and oven-dried leaves of khat (catha edulis). from the present study, it is possible to conclude that investing more effort and time on searching additional nonalkaloidal principles from the leaves of khat is so necessary. and further works could be done in the future to isolate extra non-alkaloidal compounds from the leaves and other parts of khat and evaluate their biological activity. keywords: khat; catha edulis; non-alkaloid; kna-1; kna-2. introduction khat (catha edulis) belongs to the family of celastraceae and genus of catha. it is an evergreen shrub growing to a bush or a large tree. the first scientific description and the name catha edulis was given by the swedish botanist peter forskal. the plant is known by a variety of names, including: abyssinian tea, african salad, chat, jaad, mirra, qat, etc. the name khat is commonly used for c. edulis (lamina, 2010). khat is used commonly for mastication and its sympathomimetic actions. consumption of khat leaves is common in yemen, madagascar, saudi arabia and east african countries (kenya, ethiopia, djibouti, somalia, uganda, and tanzania). regardless of sociodemographic characters, the number of khat chewers in ethiopia is increasing from time to time (belwal and teshome, 2011; mathewson et al.,2013) beyond its deep rooted sociocultural tradition, khat is also used to some extent as medicine. processed leaves and roots of khat, for example, are used to treat influenza, cough, gonorrhea, headache, asthma and other chest problems (numan, 2003). and the leaves have been used for the treatment of depression, gastric ulcers, hunger, obesity, and tiredness (mathewson et al., 2013; numan, 2003; lemessa, 2001). moreover, khat plays an important role in the economy of ethiopia. it has become the source of livelihood for millions of people and is sometimes considered as a rival of coffee for income generation to ethiopia (ambaye, 2012). regarding the chemistry aspect, there are more than 200 compounds that have been identified in khat leaves (szendrei, 2004), including: 40 alkaloids, terpenoids and sterol, flavonoids, glycosides, tannins, amino acids, vitamins and minerals (lamina, 2010). the chemical constitute of khat have been studied since the late 19th century, in which an alkaloidal fraction was found in this plant by fluckiger and gerok and called it “katin”. this was followed by the isolation of many other substances and it was not until the year 1975 that the most important component of khat was isolated and named cathinone (s (-)-alpha-aminopropiophenone)) at the united nations laboratories (dhaifalah and`šntavy, 2004). the compounds reported so far from khat can generally be classified into five main compound classes, namely, flavonoids, terpenoids and sterol, alkaloids, amino acids, and vitamins. some of the representative compounds reported from khat plant so far are: dihydromyricetin (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), dihydromyricetin-3-orhamnoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), kaempferol (szendrei, 2004; almeshal et al., 1985; ermias et al., 1984), myricetin (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984; bredholt, 2010), myricetin-3-o-²̭d-galactoside (szendrei, 2004; almeshal et al., 1985; ermias et al., 1984), myricetin-3-ohttps://doi.org/10.14421/biomedich.2020.92.81-89 82 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 rhamnoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), quercetin (szendrei, 2004; almeshal et al., 1985; bredholt, 2010), quercetin-3-o-²̭dgalactoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), celastrol (bredholt, 2010; brossi, 1990); baxter et al., 1979), iguesterin (brossi, 1990; baxter et al., 1979), pristimerin (bredholt, 2010; brossi, 1990; baxter et al., 1979),tingenin a (bredholt, 2010; brossi, 1990); baxter et al., 1979), tingenin b ( bredholt, 2010; brossi, 1990; baxter et al., 1979), friedeline (brossi, 1990; baxter et al., 1979), sitosterol (bredholt, 2010; brossi, 1990; baxter et al., 1979), (+)cathine (brossi, 1990;wabe and mohammed, 2012; feyissa and kelly , 2008), (-)-cathinone (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly, 2008), 3,6-dimethyl-2,5-diphenyl pyrazine (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly, 2008), merucathine (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly , 2008), merucathinone (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly , 2008), cathedulins e2e6 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k1 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k2 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k6 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k12 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k15 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), alanine (feyissa et al., 2008; halbach, 1972), ±̭aminobutyric acid (feyissa et al., 2008; halbach, 1972), arginine (feyissa et al., 2008; halbach, 1972), asparaginic acid , (feyissa et al., 2008; halbach, 1972), choline (feyissa et al., 2008; halbach, 1972), glutamic acid (feyissa et al., 2008; halbach, 1972), glycine (feyissa et al., 2008; halbach, 1972), histidine (feyissa et al., 2008; halbach, 1972), isoleucine (feyissa et al., 2008; halbach, 1972), leucine (feyissa et al., 2008; halbach, 1972), ornithine (feyissa et al., 2008; halbach, 1972), ascorbic acid (feyissa et al., 2008; halbach, 1972), niacin (feyissa et al., 2008; halbach, 1972), riboflavin (feyissa et al., 2008; halbach, 1972) and thiamine (feyissa et al., 2008; halbach, 1972). although the above mentioned classes of compounds were reported from the stimulant green plant, khat, researchers have spent their effort and time merely on study of the alkaloidal components (mainly the stimulant agents, cathinone and cathine) both qualitatively and quantitatively. as a result, the two principal khat stimulant compounds, cathinnoe and cathine, are now well established. but, on the contrary, previous studies on the non-alkaloidal constituents of the plant were limited. it therefore became apparent that it is necessary to undertake a phytochemical study in order to give an attention to the chemical constituents of khat other than the most studied alkaloidal one. the objective of this work was therefore to isolate and characterize compounds from non-alkaloidal fractions of the plant’s leaf. materials and methods plant material khat plant (c. edulis) was collected from the province known as sebeta, 20 km far from the capital city, addis ababa, ethiopia. two types of leaves are easily recognized in khat plant; the chewable and “nonchewable”. the chewable parts are usually shiny tender greenish young leaves found on the tip of a branch. the “non-chewable” leaves known locally as “geraba” are found on the lower part of a branch and are much older, harder and deep green in color. the fresh chewable leaves were used for immediate extraction and the “nonchewable” leaves were oven-dried (50 oc). chemicals, apparatuses and instruments chemicals such as visualizing agent (vanillin/meoh/ conc.h2so4), hcl (37%), etoac, meoh and acetone were used. besides, apparatuses and instruments including tlc plate (pre-coated aluminum sheet silica gel 60 f254), chamber, capillary tube, shaker 3020, nmr (bruker avance 400 mhz spectrometer), vacuum oven (sv40), rotavapor (r-114), sonicator (3210 branson), uv lamp (cc-8), uv/vis spectrophotometer (t60) and digital melting point (electrothermal ia 9200) were employed in the present study. isolation of non-alkaloidal compounds from khat leaves the fresh chewable khat leaves (60 g) were extracted with 0.1n hcl (300 ml) and filtered by suction filtration. the filtrate was extracted with etoac (100 ml) and separated the organic phase by separatory funnel, concentrated by rotavapor affording 140 mg. the plant extract (140 mg) was dissolved in meoh (10 ml) and adsorbed on 1 g silica gel (230 400 mesh size). the adsorbed sample was chromatographed by applying on top of column chromatography packed with silica gel (12 g). elution was carried out using etoac/ acetone with increasing polarity and five fractions were collected (table 1). table 1. collected fractions of acid-etoac extract of chewable leaves. fraction no. solvent system volume (ml) amount (mg) 1 etoac (100%) 5 4 2 “ 10 15 3 “ 20 15 4 etoac/ acetone (2:1) “ 20 5 “ “ 14 kiros – non-alkaloidal compounds from khat (catha edulis) leaves 83 as shown from the tlc profile (figure 1) of the above fractions (table 1), fraction 4 (kna-1) was seen as a better spot; it was then concentrated (20 mg) and obtained as yellow solid; mp 190-192oc (literature value 192-195oc, dictionary of natural product); rf in tlc: 0.6 (etoac/ meoh/ acoh; 4.5: 0.5: 1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5); uv (meoh) λmax 293 nm. 1h nmr (400mhz, meod): δh 5.01 (1h, br s, h-2), 4.56 (1h, d, h-3), 5.93 (1h, br s, h-6), 5.92 (1h, br s, h-8), 6.54 (2h, s, h-2’, 6’), 4.10 (1h, br s, h-1”), 3.61 (1h, br s, h-2”), 3.70 (1h, d, h-3”), 3.33 (1h, br s, h-4”), 4.28 (1h, m, h-5”), 1.22 (3h, d, h-6”). 13c nmr (400mhz, meod): δc 82.68 (c-2), 77.06 (c3), 95.99 (c-6), 94.89 (c-8), 106.25 (c-2’, 6’), 100.69 (c-1”), 70.36 (c-2”), 70.74 (c-3”), 72.43 (c-4”), 69.11 (c-5”), 16.51 (c-6”). dept-135: δc (194.53 (c-4), 164.06 (c-5), 167.17 (c-7), 162.65 (c-9), 101.05 (c10), 126.99 (c-1’), 145.63 (c-3’, 5’), 133.68 (c-4’). same extraction method as above was applied for the vacuum oven dried powder “non-chewable” (25 g) khat leaves and afforded 150 mg of etoac extract for the acid extract. the plant extract (150 mg) was dissolved in meoh (10 ml) and adsorbed on 1 g silica gel. the adsorbed sample was chromatographed by applying on top of a column chromatography packed with silica gel (13 g) and eluted using etoac/ meoh with increasing polarity. six fractions were collected (table 2). table 2. collected fractions of acid-etoac extract of “non-chewable” leaves. fraction no. solvent system volume (ml) amount (mg) 1 etoac (100%) 10 10 2 “ 15 15 3 “ 20 40 4 “ 10 20 5 “ 30 15 6 etoac/ meoh(4:1) 25 20 as shown from the tlc profile (figure 2) of the above fractions (table 2), different spots were observed in the chromatogram after it was subjected to uv lamp at 254 nm. among the fractions, fraction 1(kna-2) was concentrated (10 mg) and obtained as an orange solid; mp 165-168oc; uv (meoh) λmax 292 nm; rf in tlc: 0.8 (etoac/ meoh/ acoh; 4.5: 0.5: 0.1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5). 1h nmr (400 mhz, meod): δh 4.86 (1h, d, h-2), 4.49 (1h, d, h-3), 5.90 (1h, br s, h-6), 5.93 (1h, br s, h-8), 6.54 (2h, br s, h-2’, 6’). 13c nmr (400 mhz, meod): δc 83.90 (c-2), 72.28 (c-3), 94.85 (c-6), 95.87 (c-8), 106.61 (c-2’, 6’). dept-135: δc 196.93 (c-4), 163.91 (c-5), 167.30 (c7), 163.05 (c-9), 100.41 (c-10), 133.51 (c-1’), 145.47, 145.47 (c-3’, 5’), 127.66 (c-4’). results and discussion two types of leaves are easily recognized in khat plant; the chewable and “non-chewable”. the chewable parts are usually shiny tender greenish young leaves found on the tip of a branch. the “non-chewable” leaves known locally as “geraba” are found on the lower part of a branch and are much older, harder and deep green in color. in the present work, these leaves were analyzed to isolate non-alkaloidal components. in order to isolate non-alkaloidal components, the khat leaves were first extracted with 0.1n hcl, filtered, and extracted with etoac. this etoac extract was fractionated to isolate the non-alkaloidal compounds as described in the experimental section. characterization of two khat non-alkaloidal (kna-1 and kna-2) compounds the etoac extract of fresh chewable khat leaves (see section 2.3) was chromatographed by applying on top of column chromatography packed with silica gel. elution was carried out using etoac/ acetone with increasing polarity and five fractions were collected (table 1). similarly, the etoac extract of vacuum oven dried “non-chewable” khat leaves (see section 2.3) was chromatographed on column silica gel using etoac/ meoh with increasing polarity and six fractions were collected (table 2). thin layer chromatographic (tlc) analysis using etoac/ meoh/ acoh (4.5: 0.5: 0.1) as developing solvent, the fractions (table 1) were spotted and chromatogramed on a tlc plate. the tlc plate was then sprayed with vanillin/ meoh/ conc. h2so4 (1: 95: 5). as shown from the tlc profile (figure 1), fraction 4 (kna-1) was seen as a better spot, concentrated (20 mg) and characterized. figure 1. tlc profile of fractions of acid-etoac extract of chewable leaves. kna-1 84 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 the same solvent system with same ratio and spraying reagent as above were applied here for the fractions stated in table 2. as shown from the tlc profile (figure 2), fraction 1(kna-2) was observed as a better spot. figure 2. tlc profile of fractions of acid-etoac extract of “nonchewable” leaves. characterization of compound kna-1: it was obtained as yellow solid; mp 190192 oc (literature value 192-195oc, dictionary of natural product); rf in tlc: 0.6 (etoac/ meoh/ acoh; 4.5: 0.5: 1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5); uv (meoh) λmax 293 nm. the 1h nmr (400 mhz, meod) spectrum showed a doublet at δ 4.56 (1h, d, j = 10.8 hz, h-3) and a broad singlet at δ 5.01 (h-2) which were due to the two protons at c-3 and c-2 of flavanol skeleton, respectively. the two broad singlets observed at δ 5.92 (h-6) and 5.93 (h-8) were due to the aromatic protons of a-ring of a flavonoid skeleton at c-6 and c8, respectively. the broad singlet at δ 6.54 (h-2’ & -6’) integrated to two protons was due to the overlapping of signals for the symmetric aromatic protons of b-ring at c-2’ and c-6’ of flavonoid skeleton. the oxymethinic proton signals between δh 3.33 4.28, together with the methyl proton signal at δh 1.22 (3h, d, j = 6.4 hz, h6”), and the oxymethinic carbon signals between δc 69.11-100.69, including the methyl carbon signal at δc 16.51 indicated that a rhamnoside sugar was attached. the doublet signal at δh 1.22 (3h, d, j = 6.4 hz, h-6”) was a characteristic for the rhamnoside moiety. the three broad singlets at δh 3.33 (h-4”), 3.61 (h-2”), and 4.10 (h-1”) were due to the rhamnoside protons positioned at c-4”, c-2”, and c-1”, respectively. the methine proton at c-3” of the sugar moiety was observed as a doublet at δh 3.70 (1h, d, j = 9.6 hz, h3”), and the muliplet signal at δh 4.28 (h-5”) was characteristic signal for the rhamnoside proton attached at c-5” which contains a substituted methyl group. the 13c nmr spectrum showed the presence of nineteen signals attributed to twenty-one different carbons. the spectral region between δc 94.89-167.17 was characteristic of aromatic carbons of aand b-rings of flavonoid skeleton with the exception of the signal at δc 100.69 (c-1”) which was due to the anomeric carbon of the rhamnoside moiety. the remaining carbon atoms of the sugar moiety were observed in the spectral region at δc 69.11-72.43 including the methyl carbon appeared at δc 16.51 (c-6”). and the signal at δc 194.53 (c-4) was characteristic of carbonyl carbon of ketone functional groups (table 3). signals at δc 77.06 (c-3) and 82.68 (c-2) were due to α, β carbons of c-ring (pyranone), and were compatible with a dihydroflavanol structure which were comparable with a literature value [18] medeiros aan, medeiros fa, queiroz tm, tavares jf, silva ms, medeiros ia (2010). from the dept-135 spectrum, there were nine quaternary carbons one to a carbonyl carbon at δc 194.53 (c-4), and eight to aromatic carbons of aand b-rings. the eleven signals appeared in the positive direction were due to the methine (-ch) carbons containing two symmetric carbons overlapped with each other at δc 106.25 (c-2’, -6’), and a methyl carbon at δc 16.51 (c-6”). the above 1d nmr data is summarized as follows (table 3). table 3. 1h, 13c and dept nmr data (δppm) of compound kna-1. isolated compound (kna-1) literature value (medeiros et al., 2010) (2, 3dihydromyricetin3-o-rhamnoside) c/h 1h 13c dept-135 1h 13c 2 5.01, br s 82.68 -ch4.86, d 83.20 3 4.56, d 77.06 ch-o4.61, d 76.80 4 194.53 c=o (q) 194.30 5 164.06 q 165.40 6 5.93, br s 95.99 =ch 5.90, d 96.00 7 167.17 q 166.90 8 5.92, br s 94.89 =ch 5.87, d 95.00 9 162.65 q 162.10 10 101.05 q 101.00 1’ 126.99 q 126.80 2’ 6.54, s 106.25 =ch 6.50, s 108.00 3’ 145.63 q 145.80 4’ 133.68 q 135.20 5’ 145.63 q 145.80 6’ 6.54, s 106.25 =ch 6.50, s 108.00 1” 4.10, br s 100.69 ch-o4.07, s 100.00 2” 3.61, br s 70.36 ch-oh 3.36, br s 70.10 3” 3.70, d 70.74 ch-oh 3.41, dd 70.40 4” 3.33, br s 72.43 ch-oh 3.20, dd 71.60 5” 4.28, m 69.11 ch-o3.88, m 68.90 6” 1.22, d 16.51 -ch3 0.92, d 17.60 the extensive 2d nmr experiments involving 1h1h cosy, hmqc, hmbc spectra supported the 1d nmr data above for the proposed structure of kna-1. kna-2 kiros – non-alkaloidal compounds from khat (catha edulis) leaves 85 the cosy spectrum of kna-1 is used to determine 1h1h correlations. as stated in table 4, except the protons at δh 5.93 (h-6), 5.92 (h-8), and 6.54 (h-2’, 6’) which showed a correlation only with themselves, all the rest protons of the rhamnoside moiety and the pyranone (cring) made a correlation more than one bond. the two protons at δh 5.01 (h-2) and 4.56 (h-3) are coupled through vicinal (3j) coupling with each other. the anomeric proton of the rhamnoside moiety at δh 4.10 (h-1”) is correlated with the proton at δh 3.61(h-2”). the methyl group of the rhamnoside at δh 1.22 (me-6”) showed a correlation with a proton at δh 4.28 (h-5”). the hmqc spectrum of kna-1 is utilized to determine direct 1h-13c correlations (table 4), while the hmbc correlations described the long range 1h-13c connectivities. the h-2 proton of the c-ring at δh 5.01 displayed hmbc correlations with c-2’, 6’ (δc 106.25) and c-1’ (δc 126.99) of the b-ring. the h-3 proton at δh 4.56 showed a correlation with c-1” (δc 100.69), which indicated the rhamnoside moiety is attached at c3 position. the symmetric protons of the b-ring at δh 6.54 (h-2’, 6’) showed a correlation with c-2 of the pyranone (c-ring). table 4. cosy, hmqc and hmbc data of compound kna-1. proton no. cosy hmqc hmbc 2 h-2 h-3 h-2 c-2 h-2 c-1’, c-2’, c-6’ 3 h-3 h-2 h-3 c-3 h-3 c-1” 6 h-6 c-6 h-6 c-8, c-10 8 h-8 c-8 h-8 c-6, c-9 2’ h-2’ c-2’ h-2’ c-2 6’ h-6’ c-6’ h-6’ c-2 1” h-1” h-2” h-1” c-1” h-1” c-3 2” h-2” h-1”, h-3” h-2” c-2” h-2” c-3”, c-4” 3” h-3” h-2”, h-4” h-3” c-3” h-3” c-2”, c-4” 4” h-4” h-3”, h-5” h-4” c-4” h-4” c-2”, c-6” 5” h-5” h-4”, h-6” h-5” c-5” h-5” c-2”, c-6” 6” h-6” h-5” h-6” c-6” h-6” c-2”, c-5” o ooh h ho h h h oh oh oh o h h o oh oh oh h h h h h h h h o o o oh h h h h h h oh ho h oh oh oh h h h h oh h ho h o h cosy correlation selected hmbc correlation the structure of kna-1 was deduced as 2, 3dihydromyricetin-3-o-rhamnoside (figure 3) in comparison of the data with the literature and then confirmed by the 2d nmr data. this compound belongs to a group of secondary metabolites called flavonoids. characterization of compound kna-2: it was obtained as an orange solid; mp 165-168 oc; uv (meoh) λmax 292 nm; rf in tlc: 0.8 (etoac/ meoh/ acoh; 4.5: 0.5: 0.1) sprayed with vanillin/ meoh/ con. h2so4; 0.3: 95: 5. oho oh o o oh oh oh o oh oh oh h3c 1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 1'' 2'' 3''4'' 5'' 6'' figure 3. suggested chemical structure of compound kna-1 86 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 the 1h, 13c, and dept nmr spectral values of kna-2 are tabulated and compared with literature value (jr et al., 2009) as follows (table 5). table 5. 1h, 13c and dept nmr data (δppm) of compound kna-2. isolated compound (kna-2) literature value (jr et al., 2009) (2, 3dihydromyricetin) c/h 1h 13c dept-135 1h 13c 2 4.86, d 83.90 -ch4.96, d 84.40 3 4.49, d 72.28 -ch-oh 4.57, d 72.90 4 196.93 c=o (q) 197.9 5 163.91 q 164.00 6 5.90, br s 94.85 =ch 5.94, s 95.70 7 167.30 q 167.60 8 5.93, br s 95.87 =ch 5.98, s 96.80 9 163.05 q 164.8 10 100.41 q 101.40 1’ 133.51 q 134.00 2’ 6.54, br s 106.61 =ch 6.62, s 107.90 3’ 145.47 q 146.10 4’ 127.66 q 128.90 5’ 145.47 q 146.10 6’ 6.54, br s 106.61 =ch 6.62, s 107.90 depending on the above nmr data along with the literature value, the structure of kna-2 was suggested as 2, 3-dihydromyricetin (figure 4) which belongs to a group of secondary metabolites called aglycones flavonoid. this compound was first reported in khat leaves in 1980 (szendrei, 2004). it was also isolated from a plant known as elderberry (sambucus nigra l.) in 2009 and has biological activities as anti-virus and antiinfluenza (jr et al., 2009). oho oh o oh oh oh oh 1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' figure 4. suggested chemical structure of compound kna-2 conclusion from the present study, it is possible to conclude that investing more effort and time on searching additional non-alkaloidal principles from the leaf part of khat is so necessary. and further works could be done in the future to isolate extra non-alkaloidal compounds from the leaf and other parts of khat and evaluate their biological activity. acknowledgements: the author would like to thank the ethiopian ministry of education and addis ababa university for their financial support. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references al-meshal ia, hifnawy, ms, asir m (1985) myricetin, dihydromyricetin, and quercetin glycosides from catha edulis. j. nat. prod. 49(1): 172. ambaye gg (2012) production and consumption trends of khat in ethiopia: a big business or a big worry. advances in agriculture, sciences and engineering research 2(10): 414 427. baxter rl, crombie l, simmonds dj, whiting da (1979) alkaloids of catha edulis (khat). part i, isolation and characterization of eleven new alkaloids with sesquiterpene cores (cathedulins); identification of the quinone methide root pigments. j.c.s. perkin i 2965 2971. baxter rl, crombie wml, crombie l, simmonds dj, whiting da (1979) alkaloids of catha edulis. part 4, structures of cathedulins e3, e4, e5, e6, and k12. novel sesquiterpene alkaloids with monoand bismacrolide bridges. j.c.s. perkin i 2982 2989. belwal r, teshome h (2011) khat exports and the ethiopian economy: opportunities, dilemmas and constraints. african journal of business management 5(9): 3635 3648. bredholt t (2010) delineating cellular and molecular mechanisms of toxicity of an extract of khat (catha edulis forsk.) in leukemia and normal peripheral blood cells. [dissertation]. brossi a (1990) the alkaloids: chemistry and pharmacology. academic press, inc. dhaifalah i, `šntavy j (2004) khat habit and its health effect, a natural amphetamine. biomed. papers 148(1): 1115. ermias d, sebsebe d, kalman s, brenneisen r, peter k, amha m, zein az (1984) international symposium on khat: chemical and ethnopharmacological aspects of khat. proceedings, addis ababa university, addis ababa, 1984. [ethiopia]. feyissa am, kelly pj (2008) a review of the neuropharmacological properties of khat. progress in neuropsychopharmacology and biological psychiatry 32: 11471166. halbach h (1972) medical aspects of the chewing of khat leaves. bull. org. mond. sante 47: 2129. jr br, fink rc, mcmichael md, li d, alberte rs. (2009) elderberry flavonoids bind to and prevent h1n1 infection in vitro. phytochemistry 70: 1255 -1261. lamina s (2010) khat (catha edulis): the herb with officio-legal, sociocultural and economic uncertainty. s. afr. j. sci. 106(3/4): 14. lemessa d (2001) khat (catha edulis): botany, distribution, cultivation, usage and economics in ethiopia. unemergencies unit for ethiopia, 114. mathewson h, james k, schifano f, sumnall h, wing a, anderson d (2013) khat: a review of its potential harms to kiros – non-alkaloidal compounds from khat (catha edulis) leaves 87 the individual and communities in the uk. advisory council on the misuse of drugs. 196. medeiros aan, medeiros fa, queiroz tm, tavares jf, silva ms, medeiros ia (2010) effects of extract, fractions and 2,3dihydromyricetin-3-oα-l-rhamnoside from pradosia huberi (ducke) ducke on rat isolated mesenteric arteries. brazilian journal of pharmacognosy 20(4): 542 548. numan nmd (2003) the green leaf concept of khat chewing in yemen: social, cultural, psychological and medical aspects for khat use. [dissertation]. szendrei k (2004) the chemistry of khat. unodc-bulletin on narcotics 535. wabe nt, mohammed ma (2012) what science says about khat (catha edulis forsk)? overview of chemistry, toxicology and pharmacology. journal of experimental and integrative medicine 2(1): 29 37. 88 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 figure s1. uv (meoh) spectra of kna-1 and kna-2. figure s2. 1h nmr (400 mhz, meod) spectrum of kna-1. figure s3. 13c nmr (400 mhz, meod) spectrum of kna-1. figure s4. dept-135 spectrum of kna-1. figure s5. cosy spectrum of kna-1. figure s6. hmqc spectrum of kna-1. kna-1, λmax = 293 nm kna-2, λmax = 292 nm kiros – non-alkaloidal compounds from khat (catha edulis) leaves 89 figure s7. hmbc spectrum of kna-1. figure s8. 1h nmr (400 mhz, meod) spectrum of kna-2. figure s9. 13c nmr (400 mhz, meod) spectrum of kna-2. figure s10. dept-135 spectrum of kna-2. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 39-46 | doi: 10.14421/biomedich.2020.91.39-46 issn 2540-9328 (online) isolation and characterization of sesquiterpenes from stem bark of warburgia ugandensis sprague teshome gonfa hordofa department of chemistry, college of natural and computational sciences, haramaya university, ethiopia p. o. box 138, dire dawa corresponding author tashe2002@gmail.com, teshome.gonfa@haramya.edu.et manuscript received: 06 may, 2020. revision accepted: 12 may, 2020. published: 17 may, 2020. abstract warburgia ugandensis sprague is one of the medicinal plants traditionally used to treat a number of diseases like asthma, cough, diarrhea, common cold, stomachache and toothache in ethiopia. however, there is still insufficient information on the isolation and evaluation of bioactive compounds from this plant species. extraction, purification and isolation of the stem bark of this plant by dichloromethane and chloroform as solvents afforded two sesquiterpenes; namely, wu-1 (ugandensidial) and wu-2 (cinnamolide-3β-acetate) respectively. the structural elucidations of these bioactive compounds were accomplished by using a variety of spectroscopic methods (ir, uv and nmr). the spectroscopic results compared with the reported data in the literature. keywords: medicinal plants; natural products; sesquiterpenes; warburgia ugandensis sprague. introduction natural products extracted from medicinal plants are the major sources of modern drugs that have been used for treatment of human pathogens (sasidharan et al., 2011). the studies of these medicinal plants were the basis of earliest medicines. plant derived medicines constitute a substantial component of present day human healthcare systems (gómez et al., 2007, butle 2004). according to world health organization (who), more than 60% of the world’s population and over 80% of the people in low incoming/developing countries depend upon traditional medicine for their primary health care needs (sukirtha et al., 2012). in recent years, medicinal plants as natural products have attracted attention due to their important bioactive content such as terpens, alkaloids, anthraquinones, steroids, tannins, glycosides, saponins, volatile oils, resins, phenols and flavonoids that are deposited in their different parts (saiprasanna et al., 2012). the medicinal value of these plants also depends on bioactive phytochemical constituents that produce a definite physiologic action on the human body (mohammed, 2018). the genus warburgia is one of the flowering plants belongs to canellaceae family (rabe et al., 2000). the four species of warburgia namely, warburgia elongate verdc, warburgia salutaris (bertol. f.) chiov., warburgia stuhlmannii engl. and warburgia ugandensis sprague are distributed restrictly in the eastern and southern africa (maroyi, 2013; muchugi et al., 2008). warburgia ugandensis sprague is highly aromatic evergreen trees that has restricted distributions in the evergreen forests (rabe et al., 2000; drage et al., 2014) and widely used as traditional medicinal plant in many parts of east africa (kenya, uganda and ethiopia) (drage et al., 2014, rajab et al., 2000). the plant is known with different names: east african green wood, east african green heart, east african pepper bark tree, kenya green heart, green heart, pepper-bark tree (english) (maundu et al., 2005; wube et al., 2005), zogdom (wube et al., 2005) or kenaffa (amharic) (dagne et al., 2009) and befti (oromo language). the stem bark and roots have been used for the treatment of various diseases such as diarrhea, cough, common cold, general muscular pains, internal wounds, loss of appetite, malaria, syphilis, gonorrhea, stomachache, toothache, colds, throat and chest infections, fever, weak joints and general body pains (rabe et al., 2000, dharan et al., 2008). the compounds isolated from this plant also possess antifeedant, antibacterial, antifungal, anti-mycobacterial, cytotoxic (rabe et al., 2000, rajab et al., 2000) anti-plasmodial, anti-trypanosomal (wube et al., 2010) anti-asthmatic (karani et al., 2013) and antileishmanial activities (ngure et al., 2009). however, the leaves of plant has weaker property compared to the stem bark and roots (mbwambo et al., 2009). in ethiopia, particularly in bale zone, stem bark of this plant is traditionally used https://doi.org/10.14421/biomedich.2020.91.39-46 40 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 for treatment of a wide range of diseases, such as stomachache, toothache, malaria, tuberculosis, gonorrhea, asthma (wube et al., 2010, wube et al., 2005), cough and rabies (geyid et al., 2005). it is also used to remove tapeworm from human body. many drimane and coloratane sesquiterpenes was isolated from the stem barks of warburgia ugandensis sprague. some of these include waburganal (1), polygodial (2) (kioy et al., 1990; xu et al., 2009), mukaadial (3), warburgin (4) warburgiadione (5) (wube et al., 2005; kioy et al., 1990; xu et al., 2009), pereniporin b (6) (rajab et al., 2000), cinnamolide (7), cinnamolide-3β-acetate (8), ugandensolide (9) (wube et al., 2005; xu et al., 2009), dendocarbin a (10), ugandenial a (11), 9α,11α-dihydroxy, 6β-acetylcinnamolide (12), dendocarbin l (13), dendocarbin m (14), 9α-hydroxycinnamolide (15) (xu et al., 2009), muzigadial (16), muzigadiolide (17) (wube et al., 2005; kioy et al., 1990; rukutt et al., 2005), 4(13),7coloratadien-12,11-olide (18), 6α,9α-dihydroxy-4(13),7coloratadien-11,12-dial (19)and 7β-hydroxy-4(13),8coloratadien-11,12-olide (20), 7α-hydroxy-8-drimen11,12-olide (21) (wube et al., 2005), cinnamolide-3β-ol (22), deacetylugandensolide (23) (xu et al., 2009), 11α– hydroxymuzigadiolide (24) (rajab et al., 2000; wube et al., 2005), ugandensidial (25) (rajab et al., 2000; wube et al., 2005; kioy et al.,1990). 1 2 3 4 5 oh cho cho h h cho cho h oh cho cho h h oh o o o o o o h h o h oh o ho o o h h 6 7 8 9 10 o h o o h o h h h o ho o oh o h h ho o 11 12 13 14 15 h o ho o oh h o h o oh h o ho o o o oh o h o oh ho o h ho o oh 16 17 18 19 20 h o h o h h h h oh h h o h o oh cho cho h h cho cho ohh oh o o h h h oh 21 22 23 24 25 o ho o h h o h oh oh o o o h oh h h h o ho o oh h oh o cho cho oh figure 1. drimane and colorotane sesquiterpenes isolated from stem bark of warburgia ugandensis sprague. many of sesquiterpenes have been extracted and isolated from this plant species. despite of its medicinal uses, but still, extraction, isolation then characterization of active compounds from this medicinal plant is not sufficient. these, therefore, give emphasis on the choice of appropriate solvents and selection of the plant part (stem bark) to isolate the phytochemical constituents and characterize with very visible spectroscopic data. materials and methods experimental materials freshly stem bark of warburgia ugandensis sprague (canellaceae) was collected from barbere wareda, bale zone, south eastern part of ethiopia. the plant was then shade-dried at room temperature for one month. the dried stem barks were crushed to fine powder using an electrical grinder. chloroform, dichloromethane, etoh, petroleum ether, n-hexane, deuterated chloroform (cdcl3) were used. analytical tlc was performed using 20 x 20 cm, 0.20 mm thick silica gel 60 with fluorescent indicator uv254 to determine the number of components in a mixture and the purity of compounds. kieselgel 40*, particle size of 0.063-0.200 mm and 70-230 mesh astm silica gel was used for column chromatography. compounds on tlc were first detected by ultraviolet lamp, multiband 254/366 nm and then sprayed with 1% vanillin in sulfuric acid. heidolph rotary evaporator was used for removal of solvent under reduced pressure. melting points were determined by using stuart smp3 melting point apparatus. uv/vis spectra were recorded on genesy’s 2pc uv-vis scanning spectrometer (200-800 nm) in ch3cn. the 1d ( 1h at 400.13 and 13c at 100.6 mhz) were recorded on a bruker advance 400 spectrometer in cdcl3. the residual proton signal of the solvent is used as reference. ir spectra were recorded with kbr pellets on perkin elmer bx infrared spectrometer in the range 4004000 cm-1. optical rotations were measured on autopol iv automatic polarimeterat 20° in chcl3. experimental procedures the air-dried and finely powdered stem bark (100 g) was soaked and extracted successively with 600 ml of n-hexane, dichloromethane and chloroform for 48hours. each extract was filtered and the solvent was removed by rotary evaporator under reduced pressure. the crude dichloromethane extract (5.5 g) was subjected to column chromatography on a silica gel using n-hexane (100%) as eluent. out of 25 fractions collected, fractions 11 and 12 were combined, concentrated and further fractionated over a silica gel column using n-hexane/etoac 85:15 as eluent. fractions 6 and 7 which showed a single spot with same rf value were combined and concentrated to give 21 mg of white solid. the compound was coded as wu-1. the crude chloroform extract (1.93 g) was applied on a column silica gel using 100% hexane as mobile phase. fractions 11-15 were combined, concentrated and further purified on silica gel column using nhexane/etoac 70:30. fractions 10 and 11 showed a gonfa – isolation and characterization of sesquiterpenes from stem bark … 41 single spot with the same rf value and were combined and concentrated to afford 44mg of white solid. the compound was labeled as wu-2. results and discussions characterization of compound wu-1 wu-1 was isolated as a white crystalline solid with melting point of 131-134°c and rf value 0.34 using nhexane: ethyl acetate (3:2) as a solvent system. wu-1 was reacted with 2,4-dinitrophenylhydrazine (dnp) in ethanol and h2so4 gave yellow precipitate indicating the presence of carbonyl groups in the compound. the optical rotation ([α]d 20, chcl3) of wu-1 measured to be -197o indicating that the compound is optically active. the uv(ch3cn) spectrum (figure 2) showed maximum absorption (λmax) at 220nm indicating the presence of unsaturated group in the compound. figure 2. uv-visible spectrum of wu-1. the ir spectrum (figure 3) displayed strong absorption band at 3431 cm-1 due to vibrational stretching of o-hgroup. absorption band at 2926 cm1indicated the presence of c-h stretching of saturated group. ir spectrum demonstrated, in addition to strong absorption bands at 1744 and 1721 cm-1 due toester carbonyl and aldehyde, the presence of olefinic group at 1693 cm-1. figure 3. ir spectrum of wu-1. the 1h nmr spectrum (figure 4 and table 1) demonstrated a doublet at δ 9.78 and a singlet at δ 9.5 each integrating for one proton due to two aldehyde protons. the doublet at δ 7.03 integrating for one proton corresponded to anolefinic proton. the triplet at δ 5.92 which integrated for one proton is due to a proton on a methine carbon attached to an electronegative atom whereas the peak at δ 4.10 indicated the presence of oh group. the spectrum also showed a singlet at δ 2.16 due to an acetyl methyl group and three methyl groups at δ 1.35, 1.18 and 1.04 attached to quaternary carbon atoms. figure 4.1h nmr spectrum of wu-1. the 13c nmr spectrum (figure 5 of wu-1 showed well-resolved resonances of the 17 carbon atoms. the 13c nmr and the dept135 (figure 6 and table 2) spectra displayed four quaternary carbons, one ester carbonyl carbon (δ 170.09), three methine carbons, two aldehyde carbonyl carbons (δ 201.17 and 193.08), three methylene carbons and four methyl carbons. figure 5. 13c nmr spectrum of wu-1. 200 250 300 350 400 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 220 a b so rb an ce wave length, nm 4000 3500 3000 2500 2000 1500 1000 500 0 60 62 64 66 68 70 72 74 76 78 80 3431 2926 1744 1371 1234 1043 802 605 1693 1721 % t ra n sm it a n c e wave number cm -1 ppm (t1) 1.02.03.04.05.06.07.08.09.010.0 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 1 .0 7 1 .0 0 1 .0 3 1 .1 0 1 .0 7 2 .8 2 1 .0 4 3 .0 1 1 .1 6 3 .0 4 1 .9 3 2 .9 7 1 .2 3 1 .1 8 1 .1 4 ppm (t1) 7.02507.03007.0350 ppm (t1) 5.9005.9105.9205.930 ppm (t1) 4.10504.1100 ppm (t1) 2.05502.06002.0650 ppm (t1) 1.7501.8001.850 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 1.3901.4001.4101.4201.430 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 1.5501.6001.650 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 0102030405060708090100110120130140150160170180190200210 2 0 1 .1 6 6 1 9 3 .0 8 2 1 7 0 .0 9 2 1 4 8 .7 0 0 1 4 0 .9 1 7 7 7 .3 6 7 7 7 .0 4 9 7 6 .7 3 2 6 6 .0 5 0 4 4 .9 3 8 4 4 .0 0 0 4 1 .6 5 4 3 4 .0 1 9 3 2 .5 9 5 3 1 .8 1 4 2 4 .7 7 5 2 1 .4 9 8 1 9 .9 6 7 1 7 .6 8 3 42 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 figure 6. dept 135 spectrum of wu-1. table 1. proton decoupled 13c nmr and dept 135 (100.6 mhz, cdcl3) spectroscopic data of wu-1. carbons no. 13c nmr δ (ppm) dept 135 δ (ppm) remark c-1 31.81 31.81 ch2 c-2 17.68 17.68 ch2 c-3 44.00 44.00 ch2 c-4 34.02 c (quaternary carbon) c-5 44.94 44.94 ch c-6 66.05 66.05 ch c-7 148.70 148.70 ch c-8 140.92 c (quaternary carbon) c-9 c-10 41.65 c (quaternary carbon) c-11 201.17 201.17 ch c-12 193.08 193.08 ch c-13 32.60 32.60 ch3 c-14 24.77 24.77 ch3 c-15 19.97 19.97 ch3 ch3co 21.50 21.50 ch3 ch3co 170.09 c (quaternary carbon) the 1h and 13c nmr results obtained for wu-1 were comparable with 1h nmr (kioy et al., 1990) and 13c nmr (rukutt et al., 2005) spectral data of ugandesidial (25) (table 2). table 2. comparison of 1h (400.13, mhz, cdcl3) and 13c nmr (100.6 mhz, cdcl3) spectroscopic data of wu-1 and ugandensidial (25). position of carbons wu-1 ugandensidial δ 13cnmr 1hnmr δ (ppm) (multiplicity, integration) δ 13cnmr 1hnmrδ(ppm) (multiplicity, integration) c-1 31.81 1.83-1.76 (2h, m ) 32.02 c-2 17.68 1.65-1.55 (2h, m) 17.88 c-3 44.00 1.43-1.40 (1h, m) 1.33 (1h, m) 44.21 c-4 34.02 34.21 c-5 44.94 2.09(1h, d, j= 4.8hz) 45.16 2.04 (1h, d, j=4.7 hz c-6 66.05 5.92 (1h, t, j= 4.8hz) 66.26 5.89 (1h, t, j=4.7hz) c-7 148.70 7.03 (1h, d, j= 4.8hz ) 148.86 7.00 (1h, d , j= 4.7hz c-8 140.92 141.14 c-9 4.10 (1h, d, j= 1.2 hz) 4.10(1h,d, j=1.4hz) = 1.4 hz, 9-oh)= 1.4 hz, 9-oh) c-10 41.65 41.85 c-11 201.17 9.78 (1h, d, j= 1.2 hz) 201.32 9.76 (1h, d, j= 1.4 hz c-12 193.08 9.5(1h, s) 193.24 9.48(1h, s) c-13 32.60 1.04 (3h, s) 32.79 1.03 (3h, s) c-14 24.77 1.18 (3h, s) 24.96 1.17 (3h, s c-15 19.97 1.35(3h, s) 20.15 1.34 (3h, s) ch3co 21.50 2.16 (3h, s) 21.67 2.14 (3h, s) ch3co 170.09 170.27 in addition, the melting point of wu-1 (131-134°c) was in good agreement with the reported melting point of ugandensidial (138-140°c) (xu et al., 2009). based on the above data and in comparison with the literature (kioy et al., 1990), wu-1 (figure 7) is most probably ugandensidial. hp-67 ugandensidial (24) cho cho o ch3 oh o cho cho o ch3 oh o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 h h16 figure 7. the structure of wu-1 and ugandensidial (24). ppm (t1) 0102030405060708090100110120130140150160170180190200 2 0 1 .1 6 6 1 9 3 .0 8 0 1 4 8 .7 0 0 6 6 .0 4 8 4 4 .9 3 5 4 3 .9 9 7 3 2 .5 9 4 3 1 .8 1 2 2 4 .7 7 4 2 1 .4 9 8 1 9 .9 6 7 1 7 .6 8 2 gonfa – isolation and characterization of sesquiterpenes from stem bark … 37 characterization of compound wu-2 wu-2 was obtained as a white crystalline solid (melting point 155–157oc) with rf value 0.41 using n-hexane: ethyl acetate (3:2) as solvent systems. it is optically active with optical rotation ([α]d 20, (chcl3) +16. an absorption maximum (λmax) at 221 nm in the uv (ch3cn) spectrum (figure 8) was characteristic of a molecule possessing an unsaturated lactone structure. figure 8. uv-visible spectrum of wu-2. the ir(kbr) spectrum (figure 9) displayed an absorption band at 3439 cm-1 due to the stretching vibration of o-h group whereas absorption bands at 2969 cm-1 and 2914 cm-1 demonstrated c-h stretching vibration of alkane. strong absorption bands at 1760 cm1 and 1680 cm-1 showed the presence of an α,βunsaturated carbonyl group while absorption band at 1731 cm-1 indicated the presence of ester carbonyl group. the strong absorption bands at 1246 cm-1 and 1197 cm-1 demonstrated c-o stretching. figure 9. ir spectrum of wu-2. the quartet at δ 6.9 observed in the 1h nmr spectrum (figure 10 and table 4) of wu-2 integrating for one proton corresponded to an olefinic proton attached to c-7. the tripletsat δ 4.41 and 4.05 integrated for one protoneach due to oxymethyleneprotons on c11. the doublet of doubletat δ 4.57 which integrated for one proton indicated oxymethine proton attached to c-3. the 1h-nmr spectrum also showed three methyl protons signals at δ 1.00(3h, s), 0.93 (3h, s) and 0.84 (3h, s) whereas the acetate methyl protons appeared at δ 2.08. figure 10.1h nmr spectrum of wu-2. the 13c nmr (figure 11) and dept135 experiments (figure 12 and table 3) displayed 17 carbon atom resonances comprising of two carbonyl carbons (δ 169.81 and 170.79), three quaternary carbons (δ 34.01, 37.63 and 127.16), four methine (δ49.28, 50.57, oxymethine δ80.13 and olefinic methine δ 135.82), four methylene (δ23.52, 24.65, 36.67 and oxygenated methylene δ 66.95) four methyl carbons (δ13.49,15.99, 21.23 and 27.80). figure 11.13c nmr spectrum of wu-2. 200 250 300 350 400 0.0 0.2 0.4 0.6 0.8 1.0 221 a b so rb a n c e wave length, nm 4000 3500 3000 2500 2000 1500 1000 500 0 10 20 30 40 50 60 70 80 3439 2914 1760 1376 1246 1009 743 624 1680 1731 % t ra n sm it a n c e wave number cm -1 ppm (f1) 1.02.03.04.05.06.07.0 6 .9 0 3 6 .8 9 5 6 .8 8 7 6 .8 7 8 4 .5 7 3 4 .5 6 2 4 .5 4 5 4 .5 3 4 4 .4 2 9 4 .4 0 6 4 .3 8 3 4 .0 7 3 4 .0 5 0 4 .0 2 7 2 .8 2 2 2 .8 1 1 2 .4 2 3 2 .4 1 1 2 .0 8 1 1 .7 3 8 1 .7 2 8 1 .6 4 0 1 .4 6 7 1 .4 5 4 1 .0 0 0 0 .9 3 5 0 .8 4 0 1 .0 0 0 .9 8 1 .0 2 0 .9 9 3 .1 0 3 .0 2 3 .0 0 2 .0 4 2 .9 8 0 .9 8 0 .9 0 1 .5 1 1 .7 9 ppm (f1) 4.4004.4504.5004.550 ppm (f1) 4.050 ppm (f1) 2.8002.850 ppm (f1) 2.4002.450 ppm (f1) 1.4001.450 ppm (f1) 6.8806.8906.900 ppm (f1) 102030405060708090100110120130140150160170 1 7 0 .7 9 2 1 6 9 .8 0 7 1 3 5 .8 2 3 1 2 7 .1 6 4 8 0 .1 3 2 7 7 .3 7 9 7 7 .0 6 1 7 6 .7 4 3 6 6 .9 5 5 5 0 .5 6 5 4 9 .2 8 4 3 7 .6 3 4 3 6 .8 6 8 3 4 .0 1 4 2 7 .7 9 9 2 4 .6 4 8 2 3 .5 2 4 2 1 .2 3 3 1 5 .9 8 7 1 3 .4 9 4 38 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 figure 12. dept 135 spectrum of wu-2. table 3. proton decoupled 13c nmr and dept 135 (100.6 mhz, cdcl3) data of wu-2. carbons no. 13c nmr δ (ppm) dept 135 δ (ppm) remark c-1 36.67 36.67 ch2 c-2 23.52 23.52 ch2 c-3 80.13 80.13 ch c-4 37.63 c (quaternary carbon) c-5 49.28 49.28 ch c-6 24.65 24.65 ch2 c-7 135.82 135.82 ch c-8 127.16 c (quaternary carbon) c-9 50.57 50.57 ch c-10 34.01 c (quaternary carbon) c-11 66.95 66.95 ch2 c-12 169.81 c (quaternary carbon ) c-13 27.80 27.80 ch3 c-14 15.99 15.99 ch3 c-15 13.49 13.49 ch3 c-16 21.23 21.23 ch3 c-17 170.79 c (quaternary carbon ) the 1h nmr and 13c nmr data obtained for wu-2 are comparable with data obtained for cinnamolide-3β-acetate (8) (isolated and identified from the same plant) in the literature (wube et al., 2005). moreover, the melting point of wu-2 (155–157oc) and cinnamolide-3β-acetate (153–157°c) (kioy et al., 1990) are comparable. table 4. comparison of 1h(400.13, mhz, cdcl3) and 13c-nmr(100.6 mhz, cdcl3) spectral data of wu-2 with those of cinnamolide-3β-acetate (8). carbons no. observed nmr data of hp-81(ppm) cinnamolide-3β-acetate ( ppm) δ 13c nmr δ 1hnmr(ppm) (multiplicity,integration) δ 13c nmr δ 1hnmr(ppm) (multiplicity, integration) 1 36.67 1.42(1h, ddd, j= 13.6, 4hz) 1.65 (1h, td, j= 11.6, 5.2 hz) 36.9 1.42 (1h, ddd,j= 13.5, 4.0 hz, α) 1.64 (1h, dt, j= 13.5, 3.5hz, β) 2 23.52 1.73(2h, m) 23.5 1.68 (1h, ddd, j= 12.5, 1.5 hz, β) 1.74 (1h, ddd, j= 13, 4 hz, α) 3 80.13 4.57 (1h, dd, j= 11.2, 4.4hz) 80.2 4.55 (1h, dd, j= 11.5, 4.5 hz, α) 4 37.63 37.7 5 49.28 1.48 (1h, dd, j= 11.6, 5.2 hz) 49.4 1.48 (1h, dd, j= 10.5, 4.5 hz) 6 24.65 2.22(1h, td, j= 11.6, 4 hz) 2.45 (1h, qd, 20, 4, 8.8 hz) 24.7 2.22 (1h, ddq, j= 12.0,3.5, 1.5 hz, β) 2.44 (1h, dq, j= 20, 5, 4.0 hz, α) 7 135.82 6.9 (1h,q, j= 3.2 hz) 135.7 6.89 (1h, q, j= 3.5 hz) 8 127.16 127.2 9 50.57 2.82 (1h, m) 50.6 2.82 (1h,m) 10 34.01 34.1 11 66.95 4.05 (1h, t, j= 9.2 hz), 4.41 (1h, t, j= 9.2hz) 66.9 4.05 (1h, t, j= 9.0 hz, β), 4.41 (1h, t, j= 9.0 hz, α) 12 169.81 169.7 13 27.80 0.93 (3h, s) (3h, s, h-13) 27.6 0.94 (3h, s) (3h, s, h-13) 14 15.99 1.00 (3h, s) 15.9 1.00 (3h, s) 15 13.49 0.84 (3h, s ) 13.5 0.84 (3h, s ) 16 21.23 2.08 (3h, s) 21.2 2.07 (3h, s) 17 170.79 170.7 ppm (f1) 102030405060708090100110120130140 1 3 5 .8 2 8 8 0 .1 3 2 6 6 .9 5 9 5 0 .5 6 6 4 9 .2 8 4 3 6 .8 6 9 2 7 .8 0 0 2 4 .6 4 9 2 3 .5 2 6 2 1 .2 3 8 1 5 .9 8 8 1 3 .4 9 4 gonfa – isolation and characterization of sesquiterpenes from stem bark … 45 based on the above spectroscopic data and in comparison with literature data (wube et al., 2005), wu-2 is proposed to be (+) cinnamolide-3β-acetate (8). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 hp81 (+)cinnamolide-3-acetate (8) 16 o o o h3c o o o o h3c o h h h figure 13. the structure of wu-2 and of cinnamolide-3β-acetate (8). conclusion phytochemical investigation of the stem bark of warburgia ugandensis sprague (canellaceae) afforded two bioactive sesquiterpenes namely wu-1 (ugandensidial) and wu-2 (cinnamolide-3β-acetate) isolated from dichloromethane and chloroform extracts respectively. identification of these compounds was based o the melting point, array of spectroscopic data (uv/visible and nmr) and comparison of their spectroscopic data with reported literature values. the traditional medicinal use of this plant may be attributed to its high content of these bioactive constituents. acknowledgement: the author would to 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(2009). ugandenial a, a new drimane-type sesquiterpenoid from warburgia ugandensis. molecules, 14(10) 3844-3850. doi: 10.3390/molecules14103844 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 47-57 | doi: 10.14421/biomedich.2021.101.47-57 issn 2540-9328 (online) the effect of balimo (zanthoxylum nitidum) immersion water on the hematological profile of white rats (rattus norvegicus) that given liquor (ciu) panca buana wijaya*, tyas rini saraswati, silvana tana, sunarno, erma prihastanti department of biologi, faculty of science and mathematics, universitas diponegoro, jl. prof soedarto, sh, tembalang. semarang, indonesia. corresponding author* pancabuanawijaya@gmail.com manuscript received: 21 december, 2020. revision accepted: 11 june, 2021. published: 23 july, 2021. abstract consumption of liquor such as ciu in excessive doses can cause a decrease in hematological status. balimo stem is an alternative treatment to improve hematological status due to excessive alcohol consumption because it contains alkaloids, flavonoids, and other secondary metabolic compounds, that have functions as antioxidant effects. this study aims to examine and analyze the effect of balimo immersion water on the hematological status of mice with the observed variables, namely the erythrocytes count, hemoglobin levels, hematocrit value, and total count of leukocytes in rats that had been given ciu. the study used 20 rattus norvegicus male rats which were divided into 4 groups. the data were analyzed using one-way anova. the results showed no significant differences (p <0.05) on the balimo immersion water treatment, but if it was seen from the difference in the mean data of each variable, it could still be seen the difference from each treatment. in this study, it can be concluded that balimo immersion water was able to improve the hematological status of rats that had been given ciu liquor with a 0,2 ml dose. keywords: antioxidant; balimo; zanthoxylum nitidu; hematological status; rattus norvegicus. introduction ciu is a traditional liquor of banyumas and bekonang regions, sukoharjo. ciu is made from fermented cassava or sticky rice, which produces ethanol and carbon dioxide with ± 30% ethanol (bpom, 2014). the world health organization (who) also states that consuming liquor regularly can cause long-term diseases including hepatitis, tuberculosis, heart disease, cancer, cirrhosis of the liver, an increase in infectious diseases such as hiv / aids, and can even cause death (hammer et al., 2018). laboratory examinations of 179 respondents who have the habit of drinking liquor found that the hemoglobin levels, erythrocyte count, and the hematocrits value showed lower values compared to normal conditions, besides the data showed that the total count of leukocytes was higher than normal (riany & rahmayanti, 2013). drunk liquor will undergo an absorption process in the digestive system, the absorption process occurs in the oral mucosa, stomach, and small intestine. alcohol is absorbed through the walls of the stomach and intestines, then carried by the blood circulation to all organs in the body, especially the liver. alcohol that has reached the liver then undergoes a series of metabolism (katzung & trevor, 2015). ethanol metabolism in the liver produces acetaldehyde (wilson & matschinsky, 2020). too much acetaldehyde makes acetaldehyde more toxic. the acetaldehyde in mitochondria will be synthesized again into triglycerides or fatty acids through an oxidation process (king, 2019). the increase in triglycerides that occurs causes a buildup of fat in the liver, it triggers fatty liver or steatosis and eventually becomes alcoholic liver cirrhosis (shah, 2015). patients with alcoholic liver cirrhosis have abnormal hematological parameters, which lead to a tendency to bleeding, anemia, leukopenia, and thrombocytopenia (lindseth, 2013). the main factors causing complications of bleeding that occur are reduced clotting factors due to liver cirrhosis and the destruction of blood cells resulting in abnormalities in the number of blood cells (armitage, 2011). the progressive decrease in hemoglobin level in liver cirrhosis patients. hematological abnormalities are often found in acute alcohol drinkers in the study (jain et al., 2016). the direct effect of excessive alcohol consumption is the toxic effect on bone marrow, blood cell precursors, erythrocyte mature cells, and leukocytes. the indirect effect of alcohol consumption is nutritional deficiencies that can interfere with blood cell production (hematopoiesis) and the function of various blood cells, https://doi.org/10.14421/biomedich.2021.101.47-57 48 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 causing an increase in blood pressure or hypertension (ifeannyi et al., 2014). the balimo plant (zanthoxylum nitidum) is used by the kanayatn dayak tribe in tapakng village as a medicinal plant for traditional medicine (raja and hartana, 2017). balimo plants are used to treat alcohol poisoning after drinking wine (andasputra & julipin, 2011). the balimo plant is also used as a medicine for a cough with phlegm and hemoptysis treatment (sepsamli & jumari, 2019). balimo plant (zanthoxylum nitidum) is used to improve blood circulation, relieving blood stasis in traditional chinese medicine. phytochemical investigations showed the presence of alkaloids, flavonoids, and true amino acids in the bark of zanthoxylum nitidum (bhattacharya & zaman, 2009). these compounds have benefits as anti-viral, antiinflammatory, antioxidant, and anti-cancer activities (van nguyen et al., 2019). balimo stem is expected to be an alternative to improve hematological status due to consuming liquor because it contains flavonoids, alkaloids that have antiinflammatory and antioxidant effects that function to improve hematological status (aiba et al., 2016). antioxidants are compounds that can inhibit oxidation reactions by binding to free radicals and highly reactive molecules (suryani et al., 2013). the mechanism of action of flavonoid compounds as antioxidants is to maintain erythrocytes, by inhibiting peroxidation of lipid peroxidation caused by h2o2 and preventing protein degradation and hemolysis (yousif et al., 2012). flavonoids play an important role in regulating blood vessel health, namely maintaining maximum cardiac function (catherine et al., 2015). based on the background above, to determine the effect of compounds in plants balimo (zanthoxylum nitidum), it will do the research and observation of the hematological status of male rats (rattus norvegicus) who had been treated with alcohol and then given balimo (zanthoxylum nitidum) immersion water. materials and methods research ethics this study has received ethical clearance from the ethics commission for health and medical research, faculty of medicine, diponegoro university semarang no. 164 / ec / h / kepk / fk-undip / xii / 2019. research design and research locations research on the effect of balimo (zanthoxylum nitidum) stem immersion water on the hematological status of rats that have been given liquor (ciu) was carried out in animal cages, and animal structure and function laboratory, department of biology, fsm-undip. this study used 20 male rats (rattus norvegicus) which were divided into 4 treatment groups, including: a0: represents rattus norvegicus control, a1: represents cirrhosis rattus norvegicus that given ciu for 2 weeks at a dose of 0.2 ml, a2: represents cirrhosis rattus norvegicus by giving balimo immersion water (ciu for 2 weeks and 50% balimo immersion water for 2 weeks with a dose of 0.2 ml each), a3: represents cirrhosis rattus norvegicus with balimo immersion water (ciu for 4 weeks along with 50% balimo immersion water for 2 weeks with a dose of 0.2 ml each). each treatment was repeated 5 times. treatment for 2 weeks of acclimation and 4 weeks of treatment. tools and materials the tools used in this study include 35 sets of mouse cages, wire caps, food and drink containers, gloves, feeding bowls, sprayers, syringes, sonde, measuring cups, analytical scales, paraffin tubs, surgical instruments, edta tubes, dropper pipettes, hemometer sahli, sahli pipette, aspirator, erythrocyte pipette, leukocyte pipette, microscope, object-glass, object glass cover, counters, cameras and counting booths improved neubauer, centrifuge, hematocrit tube, microhematocrit reader. the materials used in this study included 35 male rats (rattus norvegicus) 60 days old, standard organic feed, rice husks, drinking water, balimo immersion water, detergent, distilled water, 39% liquor (ciu), 70% alcohol, 0.1 ml hcl, hemotoxin-eosin dye, hayem's solution, turk solution, chloroform, glass objects, and blood samples. making the balimo immersion water making balimo immersion water is done by cutting the balimo stem with a size of 0.5 cm, then weighing it using 1 g of analytical scales and put in 10 ml of 36oc warm water stored in a tightly closed glass bottle and left for 3 days. the dosage levels is determined based on according to the ethical commission which has been converted based on the dose to humans and the determination of the concentration is determined based on the results of preliminary tests that are previously recognized. blood data collection and observation hematological status data were obtained, then observed appropriately based on variables, such as: erythrocyte count observation of the erythrocytes count was carried out using a microscope, improved neubauer hemocytometer counting chamber, erythrocyte pipette and aspirators, counters, hayem solution, aquades. observations were carried out by observing and counting erythrocyte cells using a microscope, improved neubauer counting chamber using the 5-square wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 49 technique in the large square in the middle of the counting chamber, then the count of all erythrocyte cells in the five squares multiplied by 5000 (oktiyani et al., 2017). hemoglobin levels hemoglobin levels are observed with hemometer sahli method, by equating blood color on the color indicator (block comparator) in hemometer sahli, then calculated the existing high blood fluid in the tube hemometer sahli, aquades supplemented by the addition dropwise (a'tourrohman, 2020) hematocrit value observation of the hematocrit value is carried out on blood in a centrifuge then the blood is fed into the microhematocrit reader or using the hematocrit value formula (nurrahman & mariyam, 2019). total count of leukocytes observation of the total count of leucocytes was carried out using a microscope, counting booth improved neubauer, leucocyte pipette and aspirator, counter, turk solution, and distilled water. observations were made by observing and counting leukocyte cells using a microscope, an improved neubauer counting chamber in the five boxes located diagonally in 4 large squares in the corner of the counting room, the results were x 50 cells / mm3 of blood (bakhri, 2018). data analysis hematological status data that had been observed were then analyzed using analysis of variance (anova) using a completely randomized design with 4 treatments and five replications (gomez, 2005). results and discussion the results of the statistical analysis of the effect of balimo (zanthoxylum nitidum) stem immersion water on the hematological status of rats (rattus norvegicus) given liquor (ciu). based on the statistical analysis test using the one-way analysis of variance (anova) test with a confidence level of 95%. based on the results of data analysis using anova, the effect of balimo stem immersion water (zanthoxylum nitidum) on the hematological status includes the erythrocytes count, hemoglobin levels, hematocrit values, and the total count leukocytes of white rat (rattus norvegicus) which have been given ciu shows no different results. real or (p> 0.05). hematological status includes the erythrocytes count, hemoglobin levels, hematocrit values, and the total count leukocytes of white rat (rattus norvegicus) which are not different due to several factors, including the length of time given treatment in all groups is still too short, which causes the treatment effect has not reached an acute point. also, the body's immune system can maintain a state of hematological status, especially in mice when they are 6 weeks old, so that the mice can restore the state of hematological status. guyton & hall (2014) states that the body has a defense system that can maintain and improve the condition of the body that is experiencing disorders. erythrocyte count the results of statistical tests based on the mean erythrocytes count of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the number of erythrocytes still change based on the group and can be seen in the graph (figure 1). figure 1. erythrocyte count. the average number of erythrocytes when compared with the a0 treatment group, the erythrocytes count in group a1 decreased due to the toxicity effect of the alcohol content in ciu drinks, resulting in damage to erythrocyte blood cells and suppression of erythrocyte formation (erythropoiesis) which occurs due to ethanol compounds scattered in the blood vessels and spinal cord. the erythrocytes count in group a2 increase because treatment of a2 was able to inhibit free radicals that interfere with the formation of blood cells, and flavonoid and alkaloid compounds can also stimulate the central nerve, which stimulates the spinal cord so that the formation of red blood cells increases. the formation of red blood cells is not disturbed and will continue to form blood cells in the bone marrow, so the erythrocytes count can increase and tends to increase the hematological status of the a2 treatment group mice. the erythrocytes count in group a3 has decreased and then increased because a3 approaches a0, this is because the erythrocytes count in group a3 has increased again and tends to maintain the erythrocytes count by protecting and repairing damage to blood cells from free radicals due to the presence of flavonoids and alkaloids that function as antioxidants in balimo immersion water. flavonoid and alkaloid compounds 5,1 4,97 5,47 5,07 4,6 4,8 5 5,2 5,4 5,6 a0 a1 a2 a3 erythrocyte count (×106/µl) 50 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 can also stimulate the central nervous system, which stimulates the spinal cord so that the formation of red blood cells increases. hartono et al., (2019), states that alcoholic drinks can have a toxic effect on bone marrow which causes suppression of blood production and abnormal blood structure so that the erythrocytes count is reduced. stanley, et al. (2014) explained that alkaloid and flavonoid compounds act as antioxidants which can play a role in preventing oxidative stress due to exposure to free radicals. hemoglobin levels the results of statistical tests based on the mean hemoglobin levels of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of hemoglobin levels continued to change based on groups and can be seen in graphs (figure 2). figure 2. hemoglobin levels. the average hemoglobin level when compared with the a0 treatment group, the hemoglobin level of group a1 decreased because it was caused by the toxic effect of ethanol compounds that spread in the body, both in the bone marrow and blood vessels due to giving ciu for 2 weeks, without water treatment. balimo immersion water. this causes damage to erythrocyte blood cells and reduces or destroys the ability of erythrocyte blood cells to synthesize hemoglobin so that the hemoglobin level in the blood is also reduced. the level of hemoglobin in the a2 group has increased due to the effect of giving balimo immersion water which functions as a therapy for the toxic effects of the ciu liquor, and balimo immersion water has flavonoids which are useful as antioxidant compounds, and as a source of protein that can help form hemoglobin in blood cells. the hemoglobin level of the a3 group which was expected to increase, showed a decrease in hemoglobin levels. this is due to the prolonged administration of the ciu liquor, which causes more damage to blood cells. this also affects the formation of hemoglobin in blood cells and bone marrow, which becomes inhibited and disturbed. this is explained by schalm (2010) that the hemoglobin synthesis that occurs in blood cells in the bone marrow will be disrupted and damaged due to the toxic effects caused by ethanol compounds in alcoholic drinks. aiba et al., (2016) explain that balimo stems have flavonoid compounds and alkaloids that can be used for antioxidants to repair cells damaged by free radicals. hematocrit value the results of statistical tests based on the mean hematocrit value of mice in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the hematocrit value continue to change based on the group and can be seen in graphs (figure 3). figure 3. hematocrit value. the mean hematocrit value when compared to the a0 treatment group, the hematocrit value of group a1 decreased due to the toxic effect of ciu alcoholic drink which resulted in a decrease in the number of erythrocytes and hemoglobin levels, so the hematocrit value also decreased because the hematocrit value was proportional to the erythrocytes count and hemoglobin levels. the hematocrit value of group a2 increased due to the effect of balimo immersion water treatment for 2 weeks which functions as an antioxidant against the toxic effects of ciu for the previous 2 weeks, this resulted in a return to the condition of the hematological status and was able to increase the erythrocytes count and hemoglobin levels. hematocrit value in group a3 showed a decrease in the hematocrit value with the same results as in group a1 because the ciu was longer than the other groups so that the treatment ability of balimo immersion water was not able to increase the hemoglobin level, because according to the hemoglobin a3 chart, it was less versus a1, and shows the same hematocrit value as a1. this is 15,33 14,97 16,23 14,83 14 14,5 15 15,5 16 16,5 a0 a1 a2 a3 hemoglobin levels (g/dl) 46 44,67 49,33 44,67 42 43 44 45 46 47 48 49 50 a0 a1 a2 a3 hematocrit value (%) wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 51 parallel with rosita, et al. (2015) which states that normal hematocrit values are proportional to the number of erythrocytes and hemoglobin levels. if the erythrocytes count and hemoglobin levels decreased, the percentage of the hematocrit value also decreased. total count of leukocytes the results of statistical tests based on the mean total count leukocytes of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the total number of leukocytes still change based on the group and can be seen in graphs (figure 4). figure 4. total count of leukocytes. the mean total count of leukocytes when compared with the treatment group a0, the total count of leukocytes in groups a1, a2, a3 increased. the increase in the total count of leukocytes in group a1 occurs due to the presence of free radical compounds caused by liquor so that the defense system reacts by increasing the count of leukocytes so that foreign compounds including free radicals in the body decrease. the increase in the total count of leukocytes in group a2 was due to the provision of balimo immersion water for 2 weeks after ciu treatment for 2 weeks. balimo immersion water intake after consuming liquor initially provides a therapeutic effect and eliminates the acid toxicity effect of alcohol to improve the hematological status. balimo immersion water intake can cause an increase in the total count of leukocytes in the body because, there are anti-inflammatory alkaloid compounds, and can also be active toxin if given excessively so that the leukocyte defense system considers these compounds to be harmful foreign compounds. the increase in the total count of leukocytes in group a3, which should have shown a result of the total count of leukocytes that is close to a0, but has not shown a decrease that is close to a0. this is because in the a3 treatment group there was a reaction caused by the ciu toxic compound given for 4 weeks and the compound in the balimo immersion water given for the last 2 weeks. compounds in liquor for 4 weeks caused a toxic effect on the hematological status of the rats, resulting in a significant increase in the total count of leukocytes. after that there was a decrease in the total count of leucocytes due to the provision of balimo immersion water, however, because the provision of ciu continued in the last 2 weeks, the function of the balimo immersed water compound was inhibited, due to the reaction of ethanol compounds from ciu, and caused the total number of leukocytes not showed signs of a significant decline approaching group a0. this is in line with the statement of fitria, (2014) which states that leukocytes are closely related to the body's defense system. leukocytes in normal conditions are very few, and leukocytes will be increased by pathological conditions. this is following the statement of aziyah et al. (2014), and dumeva et al. (2016) which states that alkaloid compounds have active toxic properties when consumed without recommendations. alkaloids are composed of carbon, hydrogen, and nitrogen which can damage the nervous system, interfere with breathing, and interfere reproductive abilities. stanley, et al, (2014) also explained that flavonoid compounds can bind and neutralize alcohol so that the total number of leukocytes can decrease. lenny, (2006) also explains that flavonoid and alkaloid compounds that can bind ethanol compounds reduce the ability to repair cells so that leukocytes help repair and protect the body, especially in areas affected by inflammation. conclusion based on the results of the study, it was shown that balimo stem soaking water intake affected the number of erythrocytes, hemoglobin levels, hematocrit value, and the total number of leukocytes in rats that had been given ciu. intake of balimo immersion water can improve the hematological status of rats that have been given ciu. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references aiba, s., manalu, w., suprayogi, a., & maheswari, h. 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(2012). structure-activity relationships regarding the antioxidant effects of the flavonoids on human erythrocytes. natural science 4(94): 740-747. wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 53 table s1. result of the normality test. tests of normality variabel kolmogorov-smirnova shapiro-wilk statistic df sig. statistic df sig. erythrocytes number ,215 12 ,131 ,935 12 ,442 hemoglobin level ,156 12 ,200 ,953 12 ,676 hematocrit value ,136 12 ,200 ,970 12 ,908 total leukocytes ,207 12 ,166 ,909 12 ,206 *. this is a lower bound of the true significance. a. lilliefors significance correction table s2. result of the homogeneity test. test of homogeneity of variances variabel levene statistic df1 df2 sig. erythrocytes number 2,270 3 8 ,157 hemoglobin level 1,964 3 8 ,198 hematocrit value 2,013 3 8 ,191 total leukocytes 2,729 3 8 ,114 table s3. anova test anova variabel sum of squares df mean square f sig. erythrocytes between groups number within groups total ,430 3 ,143 ,808 ,524 1,420 8 ,178 1,850 11 hemoglobin between groups level within groups total 3,583 3 1,194 1,031 ,429 9,267 8 1,158 12,849 11 hematocrit between groups value within groups total 43,667 3 14,556 1,456 ,298 80,000 8 10,000 123,667 11 total between groups leukocytes within groups total 269,576 3 89,859 ,789 ,533 911,573 8 113,947 1181,149 11 54 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 table s4. results of the descriptive data analysis of the treatment. descriptives variabel n mean std. deviation std. error 95% confidence interval for mean minimum maximum lower bound upper bound erythrocytes a0 number a1 a3 a3 total 3 5,10000 ,400000 ,230940 4,10634 6,09366 4,700 5,500 3 4,96667 ,057735 ,033333 4,82324 5,11009 4,900 5,000 3 5,46667 ,472582 ,272845 4,29271 6,64062 5,100 6,000 3 5,06667 ,568624 ,328295 3,65413 6,47921 4,600 5,700 12 5,15000 ,410100 ,118386 4,88944 5,41056 4,600 6,000 hemoglobin a0 level a1 a3 a3 total 3 15,33333 1,159023 ,669162 12,45416 18,21251 14,100 16,400 3 14,96667 ,251661 ,145297 14,34151 15,59183 14,700 15,200 3 16,23333 1,365040 ,788106 12,84239 19,62428 15,300 17,800 3 14,83333 1,167619 ,674125 11,93281 17,73386 13,800 16,100 12 15,34167 1,080790 ,311997 14,65497 16,02837 13,800 17,800 hematocrit a0 value a1 a3 a3 total 3 46,00000 3,605551 2,081666 37,04331 54,95669 42,000 49,000 3 44,66667 ,577350 ,333333 43,23245 46,10088 44,000 45,000 3 49,33333 3,214550 1,855921 41,34795 57,31872 47,000 53,000 3 44,66667 4,041452 2,333333 34,62714 54,70619 41,000 49,000 12 46,16667 3,352972 ,967920 44,03629 48,29704 41,000 53,000 total a0 leukocytes a1 a3 a3 total 3 11,23333 1,803700 1,041367 6,75269 15,71397 9,500 13,100 3 19,20000 10,400000 6,004443 -6,63503 45,03503 12,800 31,200 3 24,43333 18,123557 10,463641 -20,58808 69,45475 5,800 42,000 3 19,70000 3,988734 2,302897 9,79144 29,60856 17,200 24,300 12 18,64167 10,362297 2,991337 12,05778 25,22556 5,800 42,000 table s5. tukey test. multiple comparisons (i) (j) dependent variable perlakuan perlakuan mean difference (i-j) std. error sig. 95% confidence interval lower bound upper bound erythrocytes tukey number hsd a0 a1 a3 a1 a3 a3 ,133333 ,343996 ,979 -,96826 1,23493 -,366667 ,343996 ,718 -1,46826 ,73493 ,033333 ,343996 1,000 -1,06826 1,13493 a0 a3 a3 -,133333 ,343996 ,979 -1,23493 ,96826 -,500000 ,343996 ,504 -1,60160 ,60160 -,100000 ,343996 ,991 -1,20160 1,00160 a0 ,366667 ,343996 ,718 -,73493 1,46826 wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 55 a3 a1 a3 ,500000 ,343996 ,504 -,60160 1,60160 ,400000 ,343996 ,664 -,70160 1,50160 a0 a1 a3 -,033333 ,343996 1,000 -1,13493 1,06826 ,100000 ,343996 ,991 -1,00160 1,20160 -,400000 ,343996 ,664 -1,50160 ,70160 hemoglobin tukey level hsd a0 a1 a3 a3 a0 a1 a3 a3 a0 a1 a3 a3 a1 a3 a3 ,366667 ,878762 ,974 -2,44744 3,18077 -,900000 ,878762 ,741 -3,71410 1,91410 ,500000 ,878762 ,939 -2,31410 3,31410 a0 a3 a3 -,366667 ,878762 ,974 -3,18077 2,44744 -1,266667 ,878762 ,511 -4,08077 1,54744 ,133333 ,878762 ,999 -2,68077 2,94744 a0 a1 a3 ,900000 ,878762 ,741 -1,91410 3,71410 1,266667 ,878762 ,511 -1,54744 4,08077 1,400000 ,878762 ,433 -1,41410 4,21410 a0 a1 a3 -,500000 ,878762 ,939 -3,31410 2,31410 -,133333 ,878762 ,999 -2,94744 2,68077 -1,400000 ,878762 ,433 -4,21410 1,41410 hematocrit tukey value hsd a1 a3 a3 1,333333 2,581989 ,953 -6,93510 9,60177 -3,333333 2,581989 ,593 -11,60177 4,93510 1,333333 2,581989 ,953 -6,93510 9,60177 a0 a3 a3 -1,333333 2,581989 ,953 -9,60177 6,93510 -4,666667 2,581989 ,337 -12,93510 3,60177 ,000000 2,581989 1,000 -8,26844 8,26844 a0 a1 a3 3,333333 2,581989 ,593 -4,93510 11,60177 4,666667 2,581989 ,337 -3,60177 12,93510 4,666667 2,581989 ,337 -3,60177 12,93510 a0 a1 a3 -1,333333 2,581989 ,953 -9,60177 6,93510 ,000000 2,581989 1,000 -8,26844 8,26844 -4,666667 2,581989 ,337 -12,93510 3,60177 total tukey leukocytes hsd a1 a3 a3 -7,966667 8,715758 ,798 -35,87759 19,94426 -13,200000 8,715758 ,473 -41,11093 14,71093 -8,466667 8,715758 ,769 -36,37759 19,44426 a0 a3 a3 7,966667 8,715758 ,798 -19,94426 35,87759 -5,233333 8,715758 ,929 -33,14426 22,67759 -,500000 8,715758 1,000 -28,41093 27,41093 a0 a1 a3 13,200000 8,715758 ,473 -14,71093 41,11093 5,233333 8,715758 ,929 -22,67759 33,14426 4,733333 8,715758 ,946 -23,17759 32,64426 a0 a1 a3 8,466667 8,715758 ,769 -19,44426 36,37759 ,500000 8,715758 1,000 -27,41093 28,41093 -4,733333 8,715758 ,946 -32,64426 23,17759 56 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 table s6. result of the tukey & duncan test. homogeneous subsets total leukocytes treatment n subset for alpha = 0.05 1 tukey hsda a0 a1 a3 a3 sig. 3 11,23333 3 19,20000 3 19,70000 3 24,43333 ,473 duncana a0 a1 a3 a3 sig. 3 11,23333 3 19,20000 3 19,70000 3 24,43333 ,192 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. hemoglobin level treatment n subset for alpha = 0.05 1 tukey hsda a3 a1 a0 a3 sig. 3 14,83333 3 14,96667 3 15,33333 3 16,23333 ,433 duncana a3 a1 a0 a3 sig. 3 14,83333 3 14,96667 3 15,33333 3 16,23333 ,172 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. hematocrit value treatment n subset for alpha = 0.05 1 tukey hsda a1 a3 a0 a3 sig. 3 44,66667 3 44,66667 3 46,00000 3 49,33333 ,337 duncana a1 a3 a0 a3 sig. 3 44,66667 3 44,66667 3 46,00000 3 49,33333 ,128 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. erythrocytes number treatment n subset for alpha = 0.05 1 tukey hsda a1 a3 a0 a3 sig. 3 4,96667 3 5,06667 3 5,10000 3 5,46667 ,504 duncana a1 a3 a0 a3 sig. 3 4,96667 3 5,06667 3 5,10000 3 5,46667 ,209 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 57 figure s1. research photographs (a. cage; b. male rats; c. weighing of test animals; d. batang balimo; e. ciu; f. water immersion of balimo stems; g. feed for rats; h. process of treatment; i. 10% bnf fixative solution; j. anesthesia process of rats; k. surgical process; l. topography of internal organs from test animals) a b c d e f g h i j k l this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 17-26 | doi: 10.14421/biomedich.2022.111.17-26 issn 2540-9328 (online) kidney evaluation in hyperuricemia rats treated with green tea leaves (camellia sinensis l.) extract putranty widha nugraheni1,*, chanif mahdi2 1department of environmental engineering, faculty of engineering, universitas tanjungpura, jl. prof. dr. h. hadari nawawi, kota pontianak, kalimantan barat 78124 tel. +62-561-739630, indonesia. 2department of chemistry, faculty of mathematics and natural science, universitas brawijaya, jl. veteran, kota malang, jawa timur 65145 tel. +62-341-551611, fax. +62-341-575777 indonesia. corresponding author* putranty@teknik.untan.ac.id manuscript received: 03 february, 2022. revision accepted: 07 february, 2022. published: 23 february, 2022. abstract uric acid is an oxidation product of the xanthine oxidase enzyme found in extracellular fluid, and when it exceeds, uric acid will build up and cause hyperuricemia. tnf-α is released by epithelial cells and mesangial cells when inflammation occurs and causes apoptosis in epithelial cells, causing damage to kidney structures and initiating acute kidney poisoning. green tea extract (camellia sinensis l.) contains many antioxidants, especially flavonoids with potent antioxidant properties such as lipid peroxidase and free radical absorbers, inhibiting xanthine oxidase. this study expresses the potential of green tea extract on kidney repair caused by hua. twenty-four male albino rats (175-225 g) of wistar strain being fed a high purine diet in 60 consecutive days and divided into six groups randomly, i: negative control, ii: positive control, iii: allopurinol, iv: green tea extract 150mg of body weight, v: green tea extract 300mg of body weight, and vi: green tea extract 600mg of body weight. treatment was done for 14 days and measured by total creatinine levels, malondialdehyde levels, and kidney histopathology. the statistical analysis using one way anova and post hoc tukey analysis by spss 23.0 proved that green tea extract with a dose of 600 mg/kg of body weight green could lower levels malondialdehyde of the kidney as much as 58.85% (p<0.01), decreased creatinine level by 24.5% (p>0.05), and improved kidney histopathology. this study proved that green tea extract is a promising alternative for hyperuricemia while improving kidney tissues and lowering malondialdehyde and creatinine levels. keywords: camellia sinensis l.; hyperuricemia; kidney creatinine; kidney histopathology; kidney malondialdehyde. abbreviations: ua, uric acid; hua, hyperuricemia; ros, radical oxygen species; nfκb, nuclear factor kappa-light-chain-enhancer of activated b cells; gte, green tea extract; alp, allopurinol; mda, malondialdehyde; bw, body weight; xod, xanthine oxidase; urat1, urate transporter 1; oat1, organic anion transporter 1; oat3, organic anion transporter 3; pbs, phosphate buffered sucrose; pfa, paraformaldehyde introduction uric acid (ua) comes from endogenous sources and exogenous sources. endogenous sources come from protein and nucleoprotein breakdown products, while exogenous sources come from purine foods. the body's average ua levels are 3.4-7.0 mg/dl in men and 2.4-6.0 mg/dl in women, and 75% of the urate will be excreted in the urine (lippi et al., 2008). ua that is ionized to urate will dominate the extracellular plasma and synovial fluid as monosodium urate at ph 7.4 so that the plasma will experience saturation. when ua levels in the body exceed normal limits, ua will build up and cause hyperuricemia (hua). if ignored, hua can impair kidney function, such as nephrolithiasis and urate nephropathy, leading to gouty arthritis and cardiovascular disease (cirillo et al., 2006). kidneys play an essential role in filtering blood so that urine is excreted by the body, including maintaining the homeostatic system of ua because the kidneys carry out 70% of ua expenditure. therefore, hua and gout are mainly due to a decrease in the relative kidney excretion of ua (lipkowitz, 2012). when there is an increase in ua levels in the urine, ua will experience saturation and form crystals that eventually form urinary tract stones, which can inhibit and injure the kidneys' tissues and functions and lead to uricosuria. previous studies (angielski, 1992) demonstrated heterogeneity of several enzymes for metabolizing adenosine centered on the rat kidney. the highest activity of ada, xanthine oxidase, and nucleoside phosphorylase was found in the cytosol in the glomerulus, where the glomerulus is a complex of three different cell types: epithelial, endothelium, and mesangial cells. increased excessive activity triggers https://doi.org/10.14421/biomedich.2022.111.17-26 18 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 tissue damage in the kidneys. several studies have shown that nfκb causes multiple organ damage, such as kidneys, liver, and pancreas (tugcu et al., 2006). activated nfκb plays an essential role in expressing pro-inflammatory cytokines such as tnf- and other mediators involved in the acute inflammatory response and different conditions associated with ros generation (arjumand et al., 2011). several studies have shown this to cause apoptosis and epithelial cells in tubular structures, causing damage to kidney structures and initiating acute kidney poisoning (abraham, 2003). hua is increasing significantly globally because it affects more than 2 billion people (kramer & curhan, 2002). in indonesia, the prevalence of this disease is the second highest, which is 24.7%, and possibly caused mainly by unhealthy eating habits. allopurinol (alp) is the most widely used drug because it is considered the most effective in inhibiting the formation of ua by inhibiting the activity of xanthine oxidase (boudiaf et al., 2010). however, its use can cause serious side effects, such as nephropathy, allergic reactions, and digestive disorders (feig et al., 2008). the side effects caused by drugs cause some people to choose to use medicinal plants that are considered capable of healing with minimal side effects. several medicinal plants have been tested for their effectiveness, such as indian nettle, rosella (astuti, 2011), and coffee (lelyana, 2008). green tea extract (gte) (camellia sinensis l.) contains many polyphenol antioxidants, especially flavonoids. the types of flavonoids in gte are (+)-catechin (c), (‒)-epicatechin (ec), (‒)-gallo catechin gallate (gcg), (‒)epigallocatechin (egc), (‒)-epicatechin gallate (ecg), and (‒)-epigallocatechin gallate (egcg) (jatuworapruk et al., 2014). gte has strong antioxidant properties by performing several mechanisms, such as anti-lipid peroxidase, free radical scavenger, metal binding, and inhibition of several enzymes, including xanthine oxidase (xod). previous studies have shown that chinese gte at the right dose can reduce ua content, decrease xanthine oxidase activity (xod) and urat1 expression, and increase oat1 and oat3 term in the kidneys of mice with hua (chen et al., 2015; fraga et al., 2010). gte also contains 8-30 mg of caffeine per cup (240 ml) (bolignano et al., 2007). caffeine is a type of alkaloid with the chemical formula 1,3,7trimethylxanthine (c8h10o2n4), is a diuretic. although several studies have stated that caffeine consumption can trigger excessive ua formation, low intake of caffeine can provide several benefits, such as treating headaches, increasing alertness, and relaxing muscles (paganini-hill et al., 2007; zhen et al., 2007). in gte, caffeine activity is inhibited because of the amino acid l-theanine. to provide a calming effect on the brain (rogers et al., 2008), l-theanine is also a caffeine antagonist to balance the activity of caffeine in the body (kakuda et al., 2000). thus, in this study, gte decaffeination did not perform. materials and methods procedures gte leaves extraction dry gte leaves (50 g) were crushed by blending and then sifted by a sieve of 80 mesh. gte powder was brewed using boiled water (± 95 ºc) with a ratio of 1:10. then, the result of maceration was cooled to room temperature, filtered, and separated between the filtrate and the dregs. the brewing process was repeated up to 3 times and concentrated using a vacuum rotary evaporator at a temperature of 85 °c and 110 rpm. the gte's extraction yield was 24.94% (w/w) and kept in a 20 ºc freezer. acclimatization of experimental animals the experimental animals used in the study were 24 male albino rats (rattus norvegicus) wistar strain aged 2-2.5 months with an average body weight of 175-225 g, which were purchased from the provider of laboratory animals in bandung, indonesia. all the ethical animal procedures have been approved by ub's research commission (approval code 690-kep-ub). the rats were placed in a polyethylene cage filled with wood husks inside with a dimension of 45 x 35 x 20 cm with wire enclosures at the animal laboratory of biosains of universitas brawijaya, malang, indonesia. the room temperature was 23±2 ºc. the rats were acclimated for a week before the experiments with a standard feed and water ad libitum. induction of high purine diet and drugs treatment twenty-four rats were divided into six groups, which each group had four rats: (1) negative control group, (2) positive control group, (3) alp therapy of 5mg/kg of body weight, (4) gte therapy of 150 mg/kg of body weight, (5) gte therapy of 300 mg/kg of body weight, and (6) gte therapy of 600 mg/kg of body weight. each rat was fed a high purine diet, except the (1) group. based on a previous study (rahmawati et al., 2018), we used a high-purine diet consisting of cow's liver, cow's spleen, fried melinjo (gnemon gnetum), and fried peanuts blended and dissolved into the water. the filtrate was introduced was 3 ml/rats, twice a day (at 8:00 and 14:00) daily for 60 consecutive days. the ua level was examined every five days. when ua levels were more than 7 mg/dl, rats were ready to be treated. after ua levels of five groups reached 7 mg/dl, rats in (3) group were administered using 3 ml alp with a dose of 5 mg/kg of body weight. rats in (4), (5), and (6) groups were treated with 3 ml of gte with various doses. all rats were sacrificed for kidney and blood collection on the same day. nugraheni & mahdi – kidney of hyperuricemia rats 19 blood and organ collections blood was drawn from the rats every five days to check the ua levels. measurements were carried out using a digital tool, "easy touch gcu." after calibrated, the test strip was inserted into the detector slit on the device until the screen displayed a "blood drop" image that indicated the device was ready for use. the tail of the rat disinfected with 70% ethanol was massaged, then the tip of the rat's tail was pierced with a sterile blood lancet. the first drop of rat blood was discarded, then the next drip was absorbed into the test strip. within 20 seconds, the ua level will appear on the device screen in mg/dl. the test was performed on each rat in all groups every day until sacrificing day. the rats were dislocated in the neck, and the rats were arranged in a supine position ventral above on a surgical board and dissected by splitting the inguinal area. then both kidneys were taken and washed with 0.9% nacl. the first kidney was immersed in pbs, while the other was immersed in 4% pfa. after rats sacrificing, about 2 hours after blood collection, blood was centrifuged at 2000 rpm for 10 min to collect blood serum. serum was moved into the eppendorf tube and centrifuged at 1000 rpm for 10 minutes. serum samples were transferred into a new eppendorf tube and stored in a -20 ºc freezer. jaffe test's measurement of creatinine levels serum creatinine levels were determined by enzymatic reactions using the "point scientific creatinine kit.”. 1000 l of creatinine reagent was taken and put into a microtube, then incubated at 37 ºc for 5 minutes. furthermore, 50 l of the protein-free filtrate was added, transferred into a cuvette, and the stopwatch was turned on for 60 seconds. the absorbance was measured (a1) using a spectrophotometer with a wavelength of 510 nm, then waited again for 60 seconds, the absorbance was measured again (a2). the absorbance value used is a2-a1. measurement of mda levels half gram of kidney were crushed with quartz sand in a mortar placed on ice gel. grounded kidneys were added by 1 ml of 0.9% nacl solution and mixed until homogeneous. the homogenate was put into microtubes, sonicated for 10 minutes, centrifuged at 8000 rpm for 20 minutes, then the supernatant formed was taken carefully using a micropipette. furthermore, 100 l of kidney supernatant was taken from each microtube, wrapped in aluminum foil, and added 550 l of distilled water. each tube was added with 100 l of 10% tca, 100 l of 1% na-thio, and 250 l of 1m hcl and each tube was homogenized with a vortex. each tube was incubated in a water bath at 100 ºc for 30 minutes and centrifuged at 500 rpm for 10 minutes. then the centrifuged supernatant was transferred to a new eppendorf tube. after heating, the supernatant was cooled at room temperature until a pink complex was formed. the supernatant was then measured for absorbance with a uv-vis spectrometer at a wavelength of 530 nm. then the absorbance results were plotted on a curve to calculate the mda levels. kidney's histological evaluation embedding kidney organs using the bancroft method kidneys were soaked into a fixative solution of paraformaldehyde, then into 70% ethanol for at least 24 hours, followed by immersion in 80% ethanol for 2 hours. the kidneys were washed with 90% and 95% ethanol, respectively, for 30 minutes. then, the kidneys were immersed into absolute ethanol three times for 30 minutes, then xylol two times for 30 minutes each in an incubator at 60-63 ºc for 30 minutes. the kidneys were immersed in xylol and paraffin three times each. the embedding process was continued by dipping the kidneys into liquid paraffin poured into a container. kidney slide set making the kidney on the embedding paraffin block was inserted into the microtome block holder, and the cutting surface was aligned with the microtome blade. cutting begins by adjusting the thickness of the slices above 10 m. kidneys were sliced to a size of 5 m. slices were taken with a brush and immersed in water at room temperature to reveal possible creases in the slides. the slices were transferred with a brush into warm water at 38-40 ºc to straighten the wrinkles then placed on to object-glass. the pieces were dried and placed on a hot plate at 38-40 ºc until the slides dry. furthermore, the slides were stored in an incubator at a temperature of 3840 ºc for 24 hours. hematoxylin eosin staining procedures deparaffinization was carried out by putting the slides into xylol three times for 5 minutes each. then, the rehydration stages were done by putting slides into graded ethanol, starting with absolute ethanol, 95%, 90%, 80%, and 70% ethanol, and distilled water for 5 minutes. the staining stages were carried out by putting the slides into a hematoxylin dye container for 10 minutes. after completed, the slides were washed with running water for 30 minutes, then rinsed with distilled water. the slides were then immersed in eosin alcohol dye for 5 minutes. for the dehydration process, the slides are added to graded ethanol from 80%, 90%, 95%, and absolute ethanol concentrations. the slides are put into xylol two times, dried, and mounted. statistical analysis the data obtained were analyzed using a data normality test using shapiro-wilk statistic and homogeneity to determine the normality of data distribution. effects of treatment on parameters were analyzed using anova, which was completed by tukey test with 99% 20 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 confidence level to know the difference between treatments. statistical analysis was performed using statistical package for social sciences 23.0 software. results were significant when p < 0.01. results and discussion uhplcms/ms gte analysis the gte used was tested qualitatively using uhplcms/ms. the result of the qualitative analysis of gte was presented in figure 1, figure 2, and table 1. table 1. parameters in qualitative analysis using uhplcms/ms of gte flavonoid. rt peak fragment ions (m/z) standard fragment ions (m/z) molecular ions [m]+ (m/z) prediction of flavonoid compounds 3.72 136.5-137.5 137 305 (‒)-gallo catechin (gc), (‒)-epigallocatechin (egc) 4.29 168.5-169.5 169 457 (‒)-epigallo catechin gallate (ecgc), (‒)-gallo catechin gallate (gcg) 4.44 204.5-205.5 205 289 (+)-catechin (c), (‒)-epicatechin (ec) 4.94 168.5-169.5 169 441 (‒)-epicatechin gallate (ecg), (‒)-catechin gallate (cg) based on figure 1, the gte was analyzed qualitatively some peaks appeared with its retention time. the highest chromatogram peak from uhplcms/ms spectra formed at the retention time of 4.29 min with molecular ions [m+] weight of 457 m/z. based on the test analysis, the most abundant components in gte are epigallocatechin gallate (ecgc) or gallo catechin gallate (gcg) and epicatechin gallate (ecg) or catechin gallate (cg) with the molecular ions [m+] weight of 441 m/z and standard fragment ion of 169 m/z. this peak was identified as epigallo catechin gallate (egcg) or its epimer, gallo catechin gallate (gcg), with a molecular formula of c22h18o11. figure 1. uhpmcms/ms chromatogram of gte compounds and epigallo catechin gallate (egcg) or gallo catechin gallate (gcg) molecular structure. gte effects on mda levels in kidneys the test was carried out with the thiobarbituric acid test based on the color changes when mda and tba reacted. mda levels in the kidneys indicate how much cell damage occurs due to lipid peroxidation due to free radicals formed due to xod activity. the pink color created was calculated for absorbance at a wavelength of 532 nm. figure 2. the reaction of mda and thiobarbituric acid (nair et al., 1981). nugraheni & mahdi – kidney of hyperuricemia rats 21 the statistical test results using one way anova analysis showed that the administration of gte with three different dosage variations could reduce mda levels in the kidneys with a very significant difference between treatments (p < 0.05). figure 3. graph of mda levels in the kidneys. $p < 0.01 compared to (1). #p < 0.05 compared to positive control. the (1) group had the lowest kidney mda level of 0.47 ± 0.14 g/dl. this group was a group of rats that were not given any treatment, only given standard food and drink, so they are a group of healthy rats because their kidney mda levels are still below the normal range, at 1.04 ± 0.43 g/dl. the (2) group was the group that was given a highpurine diet so that the mda level reached 2.84 ± 0.59 mg/dl. this level is high compared to the (1) group because of the increase of 507.07% compared with the (1) group. in the (3) group, the levels of mda formed in the kidneys were 1.91 ± 0.36 g/dl, with a decrease in levels of 32.67%. in the (4), (5), and (6) groups, the kidney mda levels were 2.54 ± 0.61 g/dl, 2.22 ± 0.61 g/dl, and 1.31 ± 0.26 g/dl with decreased levels of 10.30 %, 21.56%, and 53.85% respectively. gte effects on creatinine levels in serum determination of creatinine levels in blood serum was determined by the jaffé reaction colorimetric method, with the principle of measuring the absorbance of the picrate reaction in alkaline conditions at a wavelength of 540 nm and creating a complex with a reddish color. figure 4. the reaction mechanism for the formation of the janovsky complex (toora & rajagopaj, 2002). the results of the statistical test using one-way analysis of variation (one way anova) showed that giving gte with three different dosage variations could reduce creatinine levels in the kidneys, but not significant (p > 0.05). 22 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 figure 5. graph of creatinine levels in serum. the lowest serum creatinine level was in the (1) group, 0.59 ± 0.08 mg/dl. the positive control group was the group that was given a high-purine diet so that the creatinine level reached 1.44 ± 0.19 mg/dl. in this study, alp showed its ability to significantly lower creatinine levels than the (1) groups at 1.18 ± 0.13 mg/dl, with a reduced rate of 22.41%. in this study, there was a significant change and relationship between the decrease in ua levels between positive controls and alp therapy (p = 0.05). in the (4), (5), and (6) group, the creatinine levels were 1.35 ± 0.18 mg/dl, 1.26 ± 0.19 g/dl, and respectively. 1.01 ± 0.10 g/dl with decreased levels of 6.11%, 12.21%, and 24.50% respectively. gte effects on kidneys histopathology kidneys of rats were examined in histopathology to investigate the level of damage caused by hua and the improvement that occurred due to alp therapy and gte. figure 6. kidney histopathology with he staining (400x magnification); (a) negative control; (b) positive control; (c) alp therapy; (d) gte therapy of 150 mg/kgbw; (e) gte therapy of 300 mg/kgbw; (f) gte therapy of 600 mg/kgbw; (g) glomerulus; (bc) bowman's capsule; (pl) parietal layer; (vl) visceral layer; (v) vacuolization/fat accumulation; (p) pyknosis; (kr) karyorrhexis; (kl) karyolysis discussion the structure of egcg and gcg has eight glycosylate groups that can interact with the active site of the xod enzyme so that it can inhibit the formation of ua. this fact is in line with previous studies (chen et al., 2015; jatuworapruk et al., 2014; nugraheni et al., 2017) which reported that gte could reduce ua levels by inhibiting xod enzyme activity and modulating urate transporters in the kidneys, thereby increasing the excretion of ua through the kidneys. in this study, mda levels were also determined to determine whether there was an effect of induction of a nugraheni & mahdi – kidney of hyperuricemia rats 23 high-purine diet on the lipid peroxidation process and the impact of gte on decreasing mda levels after a high-purine diet was induced. the (1) group had the lowest kidney mda level. this group was used to compare the increase or decrease in kidney mda levels due to treatment, induction of a high-purine diet, and gte therapy. the (2) group was the group that was given a high-purine diet so that the mda level reached 507.07% compared with the negative controls. this high level of mda is caused by excessive xod activity and produces ros in the form of h2o2 and o2 -• as a form of innate immunity (boumerfeg et al., 2012). oxidative stress is generally defined as an excess of ros generation that can damage essential biomolecules such as dna and lipids massively and cause the formation of chronic diseases such as atherosclerosis, cancer, diabetes, aging, and others, including hua (doehner et al., 2016). excessive xod activity causes the formation of ros, which can cause damage to living tissue in the body due to lipid peroxidation. (d.-h. kang et al., 2002). in the (3) group, the levels of mda formed in the kidneys were 1.91 ± 0.36 g/dl, with a decrease in levels of 32.67%. alp can reduce mda levels well and prevent kidney damage, consistent with previous studies (asghar et al., 2007; chang et al., 2021; noeman et al., 2011). in the (4), (5), and (6) groups, the kidney mda levels were decreased 10.30 %, 21.56%, and 53.85%, respectively. this fact is in line with previous studies (asghar et al., 2007; noeman et al., 2011), which reported that gte could reduce mda levels in the kidneys by inhibiting xod enzyme activity. the antioxidant capacity of a phenolic antioxidant is directly associated with the ability to donate hydrogen radicals from the phenol group and the presence of unpaired electrons in the aromatic ring. figure 7. the structure and parts of flavonoids that have potential antiinflammatory activity (unsaturated c chain, number and position of hydroxyl groups on ring b, carbonyl groups on c-4 carbon, and nonglycosylation) (lago et al., 2014). the antioxidant activity of non-glycosylated flavonoids is related to the number and position of the hydroxyl groups present in the molecule. the hydroxyl group in the aromatic core has a low antioxidant capacity (d. kang et al., 2005; soobrattee et al., 2005). this result is in line with previous studies (cao et al., 2010), (forester & lambert, 2011) which reported that flavonoid compounds such as c, ec, and egc could be ros absorbers due to 3'-oh and 4'oh hydroxyl groups at ortho positions that increase its antioxidant capacity. the three most important structures are (a) the orthodihydroxy catechol at ring b, (b) a double-chain c sigma bond combined with a carbonyl bond at c-4 in ring c, and (c) a hydroxyl group at the c-3 and c-5 position. the egc structure that fulfills the three requirements above is concluded as the primary agent in the process of reducing ros activity and reducing lipid peroxidation. the decrease in kidney mda levels in alp therapy was lower than gte's decrease in mda levels. this result indicates that alp can reduce oxidative stress levels by lowering ua levels and improving kidney function. still, the flavonoids in gte are more effective in acting as nephroprotection because of their antioxidant activity and hypouricemic effect. several species of flavonoid antioxidants, especially catechins, are the best ros absorbers (boudiaf et al., 2010) by inhibiting the mechanism of ros generation in tissues and inhibiting the formation of hydroxyl radicals (lin et al., 2000). creatinine was measured as a marker of kidney damage. creatinine levels above normal limits usually characterize the diagnosis of kidney failure due to low creatinine clearance by the kidneys. the (2) group was the group that was given a high-purine diet so that the creatinine level increased by 145.07% compared to the (1) group. however, these levels are still within the normal range, so this shows that a high-purine diet for 60 days does not cause significant damage to the kidneys. hua can induce high blood pressure, increased hydrostatic pressure, and kidney injury (goicoechea et al., 2010). high ua levels are associated with the onset of kidney-related disease in the late phase. creatinine is a toxic substance produced when the body usually breaks down creatinine phosphate in muscles and at a constant rate depending on muscle mass. a previous study (d. kang et al., 2005) found that hua rats showed higher proteinuria, blood pressure, and serum creatinine levels than the control group treated with alp as the most effective ua lowering agent. in this study, alp showed its ability to significantly lower creatinine levels than the (1) group with a reduced rate of 22.41%. by reducing and excreting excess levels of ua in blood serum, alp can indirectly act as a glomerular hydrostatic pressure-lowering agent and thereby relieve kidney damage. in this study, there was a significant change and relationship between the decrease in ua levels between (2) group and (3) group (p = 0.05). thus, the effect of alp in reducing the progression of kidney disease may be related to its ability to lower serum ua levels. 24 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 in the (4), (5), and (6) groups, the creatinine levels decreased 6.11%, 12.21%, and 24.50%, respectively. based on statistical analysis using the post hoc tukey test, the decrease in creatinine levels due to alp therapy and the three doses of gte therapy did not show a significant average difference. so, it can be concluded that the ability of creatinine-lowering agents from gte is as good as alp. the formation of creatinine begins with the transamination process from arginine to glycine to form glycocyamine or guanidoacetic acid (gaa). this reaction generally occurs in the kidneys, but the response also appears in the mucosa of the small intestine and pancreas. the creatinine concentration tends to be constant because it is evenly distributed throughout the body. under normal conditions, creatinine is excreted through the kidneys, but its production increases with age due to decreased muscle mass. with a small molecular weight of about 113 daltons, creatinine is easily filtered by the glomerulus and is not reabsorbed or affects the urinary rate (hosten, 1982). the presence of flavonoids that can inhibit the activity of the xod enzyme can reduce the production of free radicals and reduce the level of oxidative damage (dahal & mulukuri, 2015). by decreasing the level of oxidative damage, creatinine can be re-filtered by the glomerulus and excreted adequately by the kidneys. the (1) group showed normal rat kidney condition, characterized by no vacuolization or fat accumulation, standard glomerular shape, and no signs of necrosis, either pyknosis, karyolysis, or karyorrhexis. this condition is because group a was not given a highpurine diet and was only fed standard water, so there was no significant tissue damage. meanwhile, (2) group were given a high-purine diet until the rats experienced hua. shriveled glomerulus and blackened nucleus indicate pyknosis caused by damage to membranes, mitochondria, and the golgi apparatus. the high xod activity increased the production of ros and increased the occurrence of lipid peroxidation, which caused tissue damage. vacuolization also occurs in more significant numbers, caused by cell degeneration due to lipid metabolism disorders that lead to excessive triglyceride accumulation in the cytoplasm with the formation of vacuoles. the cells will experience hypoxia and metabolic disorders. if this happens continuously, it will cause more severe tissue damage, such as karyorrhexis (nuclear rupture and scattering of chromatin fragments scattered around the cell) and karyolysis (the loss of the nucleus due to its inability to be stained), so that the cell pales and disappears. the resulting cell death or necrosis elicits an inflammatory response in living tissues (asghar et al., 2007). kidney histopathology in (3) group showed some improvements. although vacuolization and pyknosis still happen, the condition of the glomerulus has improved, and no karyorrhexis and karyolysis has occurred. this condition is due to the ability of alp to inhibit xod enzyme activity and inhibit the formation of ros. the ultrafiltration of urine causes glomerular damage through the glomerulus, which causes urate crystals not to penetrate the filtration membrane and cause hypertension. the difference in pressure between the glomerular capillary blood pressure and the colloid osmotic pressure in bowman's capsule can cause glomerular damage. decreased glomerular filtration is evidenced by increased blood creatinine levels and decreased creatinine clearance so that this condition can be associated with nephrotic syndrome (dahal & mulukuri, 2015). histopathological appearance of kidneys treated with gte therapy groups showed some tissue improvement. there was a reduction in vacuolization, pyknosis, and karyorrhexis. this condition shows that green tea can improve the histopathological picture of the kidneys because of its antioxidant activity. it can protect the kidneys from oxidative stress and lipid peroxidation because of its ability to reduce ros. overall, a diet high in purines can cause damage to kidney cell tissue, especially the glomerulus. this fact is shown by creatinine levels that are still in the normal range. however, if a high-purine diet is continued for a long time, glomerular damage can become more severe and lead to further complications of hyperuricemia. conclusions it can be concluded from this study that gte significantly inhibited the activity of the xanthine oxidase enzyme, can lower blood creatinine levels, and reduce malondialdehyde levels in the kidneys. gte also can improve the kidney histopathological picture of rats with hua due to a high-purine diet. this study proved that gte is a promising alternative treatment for hua. for the following research, it is necessary to conduct further studies on the mechanism of reducing ua with other flavonoid products, such as quercetin, silybin, and luteolin, to compare their effectiveness and determine the effective dose of gte therapy in lowering ua levels. acknowledgements: financial support for this work from the fund grant of the indonesia endowment fund for education ministry of finance republik indonesia (lpdp kementerian keuangan ri) is gratefully acknowledged. authors’ contributions: putranty widha nugraheni and chanif mahdi designed the study and analyzed the results. putranty widha nugraheni performed the material preparation and data collection. chanif mahdi supervised the laboratory work. putranty widha nugraheni & mahdi – kidney of hyperuricemia rats 25 nugraheni wrote the first draft of the manuscript. chanif mahdi provided the critical reading and insightful recommendations of the manuscript. all the authors have read the final 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abstract this study investigates the spermicidal and contraceptive effect of azadirachta indica seed aqueous extract in male and female wistar rats. the animals were divided into four groups (n=6), graded doses (2.5, 5, and 10 mg/kg) of the extract. they were exposed to female rats in a ratio of 2:1 after 24 hours, 7, and 14 days of administration. the spermicidal properties were evaluated. the female contraceptive study involved 14 days of pre-coital and post-coital administration of the neem seed extract, and contraceptive indexes were investigated. the results obtained from the spermicidal activity showed a significant decrease in male hormonal levels at 2.5, 5, and 10 mg/kg of a. indica (0.73, 0.50, and 1.08 ng/ml); and the sperm cells (102.5, 111.5, and 97 counts) after 24 hours. administration and mating, compared to the control. also, the histopathology of the testes showed normal testicles. the contraceptive study elicited a significant decrease in estrogen (1.46 ng/ml), luteinizing hormone (0.094 mg/dl) µand progesterone (1.82 ng/ml), at 5 mg/kg in day 14 post-coital study compared with the control. the histopathology of the treated uterus had no deteriorating effect compared with the control. the extract elicited spermicidal and contraceptive potential at a reduced dose, validating its folklore claim. keywords: spermicidal; contraceptive; azadirachta indica; wistar rats. introduction azadirachta indica a. juss. is a prevalent and highly wonderful tree (alzohairy, 2016). it was known as neem a distinct tree associated closely with the socio-cultural and holistic aspects of the life of indians since ancient times. it is a fast-growing tree with edible fruits and aromatic leaves. a mature tree can produce three hundred and fifty kilograms of leaves a year (kelli, 2010). it can grow to a height of fifteen-twenty meters and seldom thirty-five meters, located in the subtropical and tropical world. neem is in the mahogany family, meliaceae, native to the indian subcontinent. neem oil is usually produced from its fruits and seeds, the leaves and oil have antifertility effects (ruchi et al., 2014). indra the king of the gods, was on his way back to his kingdom with the pot of amirithan (the divine nectar) which he had gotten from the devils after a tough-fought battle. probably, he was fagged out, maybe careless or probably he did it intentionally, for little drops of the pot content poured over a neem tree, from that day henceforth, the neem tree had good qualities for healing ailments, of course, this is a folk tale. meanwhile, the neem is a superb tree for millions of people and its parts have several uses in healthcare, medicines, and agriculture, and this is no folklore (ajayi, 2002). neem is called indian village pharmacy in the west, it is also called arishta in the sanskrit language which means relieving sickness. africans usually call it muarubaini which means forty uses or forty cures. meanwhile, the persians have given the most perfect name azaddirakht-hind which means a free tree of india, and perhaps from this its latinized botanical name a. indica (asiif, 2013). almost all the parts of the wonderful tree have been used medicinally since ancient times. this is a rampant tree found in different countries including; india, pakistan, srilanka, burma, malaya, indonesia, japan, australia (tropical region) even in the middle east. it is amongst the trees tagged holy by hindus all over the world. contraception is defined as the inhibition of conception, but generally, it is taken to mean the prevention of pregnancy (bansal et al., 2010). family planning has been encouraged through numerous means of contraception, but these contraceptives have several side effects produced by their steroid content (chandramouli et al., 2014), such as contraceptive pills, intra uterine devices, tubectomy, condoms, diaphragm, and https://doi.org/10.14421/biomedich.2023.121.79-88 80 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 coitus interruptus. these methods are also typically female-oriented. contraceptive pills are generally female sex hormones such as estrogen, progesterone, or their derivatives either single or together. the concept of sterilization by female sex hormone is very old and it was initiated at the beginning of the twentieth century. novid was the first "pill" permitted by the fda for usage as a birth control agent (chaudhuri, 2007). however, these pills lead to the development of some unwanted effects such as obesity, dysmenorrhea, vomiting, cardiovascular disorders, and carcinoma of the breast and uterus. these side effects from the use of the pills make them unsafe for long-term use. countless measures have been taken to decrease the side effects of these pills but there is little success (dash et al., 2014). as a result of the serious adverse effects caused by synthetic steroidal contraceptives, the focus has been shifted to local plants for possible contraceptive effects. although contraceptives comprising estrogen and progesterone are effective and common, the risks accompanying the drugs have prompted the need to synthesize newer molecules from medicinal plants. this study evaluates the antifertility properties of azadirachta indica in male and female wistar rats. materials and methods collection of plant material fresh seeds of the plants were collected from kaduna state, in the northern part of nigeria. it was identified and authenticated by dr. timothy odaro in the herbarium unit of the department of plant biology and biotechnology, with the voucher number ubt-b184. it was further authenticated using theplantlist.org. fresh seeds were harvested and sun-dried for two weeks. the dried seeds were ground to powder using a mechanical grinder. preparation of plant material two thousand (2000) grams of the sample, weighed using a scale was measured into a glass jar. 2l of nhexane was poured into the glass jar containing the sample. the mixture was stirred, covered, and left for 36 hours. the mixture was then strained after 36 hours and a dark brown oily semi-solid extract was obtained. the extract was concentrated using a water bath at about 45oc. it was then kept in a sterile container in the refrigerator until needed for use. experimental animals sixty-eight (68) healthy whisker (albino) rats (male and female) weighed 180-250 g. the animals were acquired from animal and environmental biology, university of benin animal house. they were housed in wellventilated woody cages in a normal laboratory state (12 hours light/dark cycle: 23 ± 2°c) and fed using a standard diet. food and water were administered at free choice (ad libitum) to the animals designed for experiments. the animals were properly handled using the ethics of laboratory animals’ approval from the ethical committee of the faculty of life sciences with the ethical number ls20619. experimental design this study involved two experimental protocols; male spermicidal activities and female contraceptive properties. the male wistar rats were randomly divided into 4 groups (n=9). three of the groups were administered with graded doses (2.5, 5, and 10 mg/kg) of a. indica seed aqueous extract, the last group serves as the control group (ogbuewu, 2011). they were administered with graded doses of the extract orally for 14 days. after 24 hours, 3 rats across the groups (making a total of 12 rats) were exposed to female rats using a ratio of 2 male to 1 female and then sacrificed. the same process was repeated for 7 and 14 days. sperm, blood, and reproductive organs (testes, and epididymis) were collected from the sacrificed rats and were analyzed (khillare and shrivastava, 2003; koresriem, 2013). the female contraceptive study was carried out in two (2) phases; the pre-coital and post-coital treatment with neem seed aqueous extract. phase 1: the pre-coital treatment involved 4 groups (n=4); the control group, graded doses (2.5, 5, and 10 mg/kg) of neem seed aqueous extract for 14 days, on the 14th day all the female animals were paired with male wistar rats during their heat period for another 7 days using ratio 1:1, and the female rats were checked at every interval for possible signs of vaginal sperm plug as an indicator for mating (gbotolorun et al., 2008; dreweke, 2014). after 7 days of pre-exposure to the male animals, the female rats were observed for traces of pregnancy for another 23 days, which is the gestational period of rats. the weight of the rats was obtained and sacrificed in mild anesthetics, and blood and female reproductive organs were isolated. phase 2: the post-coital treatment involved 4 groups (n=4); the control group, graded doses (2.5, 5, and 10 mg/kg) of neem seed aqueous extract (harris, 2018; hatcher et al., 2007). before the administration of the extract, the female rats were paired with male animals' using a ratio of 1:1 for 48 hours, the female rats were checked at every interval for possible signs of vaginal sperm plug as an indicator for mating (nripendra et al., 2019; prakash et al., 1988). the female animals were administered with graded doses of a. indica seed aqueous extract for 7 days of oral administration, the female rats were observed for traces of pregnancy for another 23 days, which is the gestational period of rats. the weight of the rats was obtained and sacrificed in mild anesthetics, and the number of resorption, number of embryos, number of corpora, pre-implantation, and post-implantation mortality, and total prenatal mortality idu et al. – anti-fertility property of azadirachta indica 81 were evaluated. blood and female reproductive organs were isolated for analysis. sperm cell procedure sperm cells were collected from the vas deferens and placed in a sterile petri dish. to the petri dish, 6 µl of normal saline already adjusted to 37°c was added. a drop of the sperm cell suspension was taken from the petri dish and dispensed on a clean grease-free slide, further covered with a transparent cover slip. the slide was placed on the microscope and viewed with the 20x and 40x objective magnification lens. the motility was scored in percentage according to their nature of motility as, progressive, nonprogressive, and immotile sperm cells (sethi et al., 2017; suryawanshi 2011; ibeh et al., 2018). one volume of semen (a drop) was milted into two volumes of eosin solution (1% diluted water). after 30 seconds three volumes of nigrosine solution (10% nigrosine) were added and the sample was homogenized. a thin smear was then made immediately and air dried. the stained slide using an improved eosin and leishman stain (umadevi et al., 2013; world health organization, 2010), was examined under the oil immersion objective lens (100x). live spermatozoa were unstained (white) and the dead ones were red. the slide was viewed with at least 30 magnification fields, and the normal and abnormal sperm cells were spotted and scored in percentage (suryawanshi 2011). determination of serum testosterone concentration serum testosterone level of the tested animals’ plasma was investigated using an established protocol from the manufacturer’s manual. this was established on the standard competitive requisite between testosterone in the test plasma specimen (serum). this assay method involved the dispensing of 10 μl of testosterone reference standards at (0, 0.1, 0.5, 2.0, 6.0, and 18.0 ng/ml), serum (diluted ×5), and the controls of testosterone 1 and 2 into a goat anti-rabbit igg-coated microtitre wells (96 wells), 100 μl of testosterone-hrp conjugate reagent (blue color) and 50 μl of rabbit antitestosterone reagent were distinctly distributed into each well. the resultant solution was systematically mixed for 30 seconds and allowed for incubation at 37oc for 90 minutes. this was left to a stable quantity of hrplabelled testosterone to contend with endogenous testosterone of the standard, sample, or quality control serum for a constant number of bindery spots of a detailed testosterone antibody (since the quantity of testosterone peroxidase conjugate with the immunological bound of the well gradually decreases as the testosterone concentration in the specimen increases) (aversa et al., 2000). the microwells were washed and skimmed 5 times in distilled water (to eliminate unbound testosterone peroxidase conjugate) previously dispensing 100 μl of tmb reagent into the well. the resultant solution was properly mixed mildly for 5 seconds. this was later incubated at room temperature for about 20 minutes to achieve blue coloration. the color change was ceased with an additional 100 μl of stop solution (1n hcl) to each well and when slightly mixed, the color altered from blue to yellow. the absorbance was recorded within 15 mins at 450 nm in a microtitre well reader. the greatness of the color made was comparative to the quantity of enzyme extant and was inversely relative to the total of unmarked testosterone in the sample (gauthaman and adaikan 2008). the testosterone level of the serum in the animals was considered from the calibration curve (plotting the concentration of the standard against the absorbance) using the expression: 𝑇𝑒𝑠𝑡𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑛𝑔/𝑚𝐿 = 𝐶𝑠 × 𝐹, (1) where cs is the corresponding testosterone concentration from the calibration curve and f is the dilution factor progesterone assay protocol all reagents should be allowed to reach room temperature (18-25oc) before use. pipette 50 µl of standards (ready to use) and diluted samples into appropriate wells within 5 minutes. add 100 ul of progesterone enzyme conjugate solution to each well (except those set for blanks). mix well for 30 second. and incubate for 60 minutes at 37°c. you may use par film to cover the wells or use an appropriate zip-lock bag to store the plate during the incubation. discard the contents of the wells and wash the plate 5 times with wash solution (250-300 µl) per well. invert the plate, and tap firmly against absorbent paper to remove any residual moisture. add 100 µl (tmb) substrate solutions to all wells. remember to follow the pipetting order. incubate the plate at room temperature (18-28°c) for 10 minutes without shaking. stop reaction by adding 50 µl of stopping solution to wells in the same sequence that the substrate solution was added and gently mixed. read the absorbance at 450 nm with a microwell reader. luteinizing hormonal assay secure the desired number of coated wells in the holder. dispense 50 µl of standards, specimens, and controls into appropriate wells. dispense 100 µl of enzyme conjugate into each well. mix for 30 seconds. it is very important to have completed mixing at this step. incubate at room temperature (37oc) for 2 hours. remove the incubation mixture by flicking the plate contents into a waste container. rinse and flick the microtiter wells five (5) times with wash buffer. strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets. dispense 100 µl of tmb solution into each well. gently mix for 10 seconds. incubate at room temperature for 20 minutes, in the dark. stop reaction by adding 50 µl (one drop) of 2n hcl to each well. gently mix for 30 seconds. it is 82 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 important to observe a color change from blue to yellow. read optical density at 450 nm with a microtiter well reader. follicle-stimulating hormone bring all reagents and samples to room temperature before use. centrifuge the sample again after thawing before the assay. it is recommended that all samples and standards be assayed in duplicate. prepare all reagents and samples as directed in the previous sections. determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°c. set a blank well without any solution. add 50 μl of standard or sample per well. standard need test in duplicate. add 50 μl of hrp-conjugate to each well (not to blank well), then 50 μl antibody to each well. mix well and then incubate for 60 minutes at 37°c. aspirate each well and wash, repeating the process two times for a total of three washes. wash by filling each well with wash buffer (200 μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or auto-washer, and let it stand for 10 seconds, complete removal of the liquid at each step is essential to good performance. after the last wash, remove any remaining wash buffer by aspirating or decanting. invert the plate and blot it against clean paper towels (neychev and mitev 2016). add 50 μl of substrate a and 50 μl of substrate b to each well, and mix well. incubate for 15 minutes at 37°c. keeping the plate away from drafts and other temperature fluctuations in the dark. add 50 μl of stop solution to each well, and gently tap the plate to ensure thorough mixing. determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm. histopathological analysis testes and uterus were fixed in neutral bouin’s fluid. affixed organs were utterly dehydrated with 99.9 % ethanol along with 70 % ethanol, and 96 % ethanol and washed using distilled water. 4 µm sections were prepared and stained in hematoxylin-eosin dye. stained tissues were subjected to optical photomicroscope (leica mc170 hd, leica biosystems, germany) and viewed at 400x magnification (drury and wallinton, 2013). statistical analysis results obtained are presented as mean ± sem. data were analyzed via one-way analysis of variance (anova) and means were compared using the dunnete test. significant differences were measured at p< 0.05. graph pad prism (version 7, usa) was the statistical tool used. results and discussion spermicidal property the body and organs weight of the treatment groups elicited a slight significant increase when compared with the control at 24 hours. 7 and 14 days of the study (tables 1 and 2). sperm cells in the graded doses of the extract (2.5, 5.0, and 10 mg) at 24 hrs. 7 and 14 days elicited a significant decrease in sperm count, progressive motility with an increase in non-progress motility, and immobility when compared with the untreated control as shown in figure 1. the testosterone level, follicle-stimulating hormone, luteinizing hormone, and progesterone had a significant reduction in the treatment groups when compared with the control, eliciting spermicidal and antifertility properties of the extract as shown in figure 2. the abnormal weight loss or gain serves as an indication of toxicity possibly due to structural protein breakdown or muscle wastage (kumar and mishra, 2010). this study present showed that a. indica seed aqueous extract at graded doses maintains the level of a significant increase in the body weight with the spermicidal effect for 24 hours. 7 and 14 days (table 1-3). this result consented with singh et al. (2018) report on grapefruit components to ameliorate reproductive toxicological organs' weight loss. similarly, the visceral organs such as the testes, cowpea gland, and epididymis, elicited a slight significant increase with no damaging effect across the treatment groups compared with the control at 24 hours, 7, and 14 days. table 1. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 24 hours. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 193.50±0.50 185.50±5.50 1.20±0.00 0.40±0.04 1.05±0.15 a. indica 5.0 207.50±1.50 187.00±2.00 1.25±0.05 0.60±0.10 0.80±0.05 a. indica 10 182.00±1..80 178.00±1..70 1.25±0.05 0.35±0.05 0.90±0.05 control 0.5 ml/kg 204.00±3.40 204.50±3.40 1.35±0.15 0.30±0.00 0.70±0.04 the results were expressed in mean±sem, p>0.05. idu et al. – anti-fertility property of azadirachta indica 83 table 2. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 7 days. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 212.50±1.70 223.50±1.50 1.50±0.10 0.60±0.02 0.85±0.05 a. indica 5.0 182.00±1.50 185.00±3.00 0.75±0.07 0.40±0.01 0.55±0.05 a. indica 10 186.50±1.50 191.50±2.50 1.25±0.15 0.55±0.02 0.55±0.05 control 0.5 ml/kg 205.00±3.60 207.50±3.50 0.60±0.05 0.20±0.01 0.65±0.05 the results were expressed in mean±sem, p>0.05. table 3. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 14 days. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 177.50±1.60 199.00±1.00 1.15±0.05 0.35±0.02 0.45±0.05 a. indica 5.0 174.50±1.20 199.00±1.30 1.10±0.00 0.50±0.03 0.45±0.05 a. indica 10 184.50±6.50 199.50±2.50 1.15±0.05 0.15±0.00 0.50±0.02 control 0.5 ml/kg 179.50±4.50 187.00±4.00 0.90±0.00 0.55±0.05 0.55±0.05 the results were expressed in mean±sem, p>0.05. the semen analysis serves as the onset of selection for fertility evaluation and is frequently utilized in the definition of semen quality (morphology, viability, and sperm motility) and quantity (sperm count) (khan, 2008). the findings from this study elicited a significant decrease in sperm count, progressive motility, and a significant increase in non-progressive, and immotile sperm cells across 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract displaying the spermicidal effect of the extract when compared with the control in 24 hrs., 7 and 14 days of the exposure as shown in figure 1. the semen analysis serves as an onset target for spermicidal activities utilized in the definition of an effective sperm killer. this agreed with the report of asiif (2013) on a review of spermicidal activities on azadirachta indica. studies had shown that certain constituents in several plant materials possess spermicidal properties, which aid in controlling overpopulation (umadevi et al., 2013; suryawanshi, 2011). a. indica seed aqueous extract serves as a potent spermicidal. figure 1. spermicidal effect of azadirachta indica seed aqueous extract on the sperm analysis after 14 days. keywords: total sperm cell count (tsc), progressive motility (pm), nonprogressive motility (npm), immotile (im). the extract promotes a significant decrease in the level of testosterone which further triggers reduced libido activity as shown in figure 2. this is in line with the report of khan, m. a. (2008). a. indica seed aqueous extract, also are linked with the androgen inhibitory function, which could be responsible for the detraction of male sexual performance as shown in this study (deshpande et al., 1980). certain phytoconstituent stimulate sexual urges with spermicidal properties (chauhan, 2008). enhancement in the spermicidal effect 0 50 100 150 200 250 300 350 400 450 control (dw) 2.5 mg/kg a. indica 5.0 mg/kg a. indica 10 mg/kg a. indica s p e r m a n a ly s is groups/doses 24 hr. tsc 7 days tsc 14 days tsc 24 hr. pm 7 days pm 14 days pm 24 hr. npm 7 days npm 14 days npm 24 hr. im 7 days im 14 days im 84 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 in this research study could be a result of the active constituents in a. indica seed aqueous extract. an increase in the level of androgen enhanced leydig cells to stimulate luteinizing hormone resulting in an increased in spermatogenesis and increase epididymal sperm, but for the associated mechanism of action of a. indica seed aqueous extract the reverse is the case due to its spermicidal property. findings by chaudhuri, (2007) practice of fertility control in weight, size and secretory role of the epididymis, testes and auxiliary organs. modifications in the circulating level of androgen may be distorted by a. indica seed aqueous extract leading to interference during spermatogenesis (jensen, 2002). variation in the reproductive organ can be used as a marker to improve androgen levels in reproductive glands, in the case of this study, the extract possibly impedes the feedback action involved in sperm formation. meanwhile, the androgenic property is associated with serum testosterone concentration (harris, 2018), the extract interferes with the function of testosterone discharge consenting by blocking the secretion of the hormone in gonads as displayed in figure 2. a contradictory study by gbotolorun et al. (2008) reported on mondia whitei hexane extract on male sexual reproductive in rats. testosterone is the main androgenic hormone responsible for the sexual application to display a vital role in spermatogenesis. findings showed that sexual zeal is triggered and maintained by the penis tissues that facilitated erection (aversa et al., 2000). figure 2. spermicidal effect of azadirachta indica seed aqueous extract on the hormonal level after 14 days. keywords: follicle stimulating hormone (fsh), testosterone (tt), luteinizing hormone (lh). studies from gauthaman and adaikan (2008) showed that male sexual dysfunction is related to several factors such as; androgen insufficiency. testosterone agents (hormonal replace therapy) are exhibited to develop sexual roles and libido. some plant extracts such as a. indica seed aqueous extract inhibit the synthesis or secretion of testosterone and serves as potent spermicidal or antifertility properties (bansal et al., 2010). a significant decrease in the level of testosterone is effective at a reduced dose as shown in a. indica seed aqueous extract when compared with the control (figure 2), also a decrease in the folliclestimulating hormone, luteinizing hormone, and progesterone inhibits the release of testosterone thereby eliciting antifertility effect of the extract (figure 2). the histopathological study of the testes exposed the standard architectural framework when compared with the control as shown in plate 1. the treated organs at 2.5, 5.0, and 10 mg/kg of the a. indica seed aqueous extract showed an enhanced architecture structure of the testes. however, across the treated groups, an absence of spermatogenic action in the lumen of the seminiferous tubule was observed. this stimulated cellular activity occurrence from the cellar membrane all through to the lumen in seminiferous tubules found in the testes inhibits the secretion of the primary spermatogonia as proof for this study. it is agreed with gbotolorun et al. (2008) reported results. 0 1 2 3 4 5 6 7 8 9 control (dw) 2.5 mg/kg a. indica 5.0 mg/kg a. indica 10 mg/kg a. indica h o r m o n a l a s s a y groups/doses 24 hr. fsh (mg/dl) 7 days fsh (mg/dl) 14 days fsh (mg/dl) 24 hr. tt (ng/dl) 7 days tt (ng/dl) 14 days tt (ng/dl) 24 hr. lh (mg/dl) 7 days lh (mg/dl) 14 days lh (mg/dl) idu et al. – anti-fertility property of azadirachta indica 85 plate 1. effect of azadirachta indica seed aqueous extract on the histopathology of testes after 14 days. (a. control testis: seminiferous tubules with spermatocytes, interstitial space, and sertoli cells. b. 2.5 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation of spermatocytes. c. 5.0 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation of spermatocytes. d. 10 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation and mild spermatogenic arrest (h&e x 1).) contraceptive study oestrogen is secreted during the menstrual cycle. the serum fuels of estrogen are usually low during every follicular phase rising gradually until about 1 day before ovulation. this present study in pre-coital treatment as shown in table 3 elicited no significant increase in estrogen level, with a slight increase in folliclestimulating and luteinizing hormone concentration across the treatment groups (2.5, 5.0, and 10 mg/kg a. indica seed aqueous extract) when compared with untreated control. while progesterone controls ovulation and investigated luteal functions at 0.2-0.8mg/ml (follicular phase) and 4.0-20.0mg/ml (luteal phase), it is the hormone responsible for pregnancy. this study showed a significant decrease in the progesterone level across graded doses of the extract when compared with an increase elicited contraceptive property of the extract tables 4 and 5. since antiquity, traditional medicines and several formulations from the herbal plant are known to be effective as a natural contraceptives (umadevi et al., 2013). it has been established that a decrease in serum estrogen, showed the defect of the extracts against fertility property with the magnitude response which stimulates antifertility known as a contraceptive (sedgh et al., 2014). in this present study, the female contraceptive activities in female animals treated groups with graded doses of 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract, elicited no significant difference in serum estrogen level in the pre-coital treatment but a slight significant increase in the post-coital phase of the contraceptive study when compared with the control. this concurred with sharma et al. (2013) work on antifertility. this proposed product cannot stimulate estrogen levels serving to be a potent contraceptive agent either by decreasing its production or via metabolic impairment as shown in tables 4 and 5 (rajandeep et al., 2011). table 4. contraceptive effect of azadirachta indica seed aqueous extract on pre-coital treatment after 14 days in female animals. treatment dose (mg/kg) lh (mg/dl) pg (ng/dl) est (ng/dl) fsh (mg/dl) control (dw) 0.5 ml/kg 0.68±0.01a 0.24±0.01a 0.21±0.02a 3.82±0.07a a. indica 2.5 0.75±0.01b 0.19±0.01a 0.23±0.02a 4.05±0.17b a. indica 5.0 0.74±0.02b 0.31±0.03b 0.22±0.04a 4.28±0.17b a. indica 10.0 0.71±0.04a 0.34±0.03b 0.25±0.02a 4.41±0.17b the results were expressed in mean±sem, p>0.05; keywords: follicle stimulating hormone (fsh), progesterone (pg), luteinizing hormone (lh), and estrogen (est). a b c d 86 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 table 5. contraceptive effect of azadirachta indica seed aqueous extract on post-coital treatment after 14 days in female animals. treatment dose (mg/kg) lh (mg/dl) pg (ng/dl) est (ng/dl) fsh (mg/dl) control (dw) 0.5 ml/kg 0.64±0.01a 0.44±0.11a 0.17±0.02a 4.84±0.09a a. indica 2.5 0.79±0.01b 0.77±0.04b 0.32±0.01b 4.84±0.08a a. indica 5.0 0.82±0.01b 0.77±0.08b 0.39±0.03b 4.99±0.12a a. indica 10 0.88±0.01b 0.82±0.08b 0.33±0.01b 5.70±0.02b the results were expressed in mean±sem, p>0.05; keywords: follicle stimulating hormone (fsh), progesterone (pg), luteinizing hormone (lh), and estrogen (est). in regards to a. indica seed aqueous extract intake, an increase in the luteinizing hormone and folliclestimulating hormone in female rats was recorded. this finding is similar to previous studies reported by pathak et al. (2005) to clarify the potential effect of female sex hormones with a decrease in luteinizing hormone and follicle-stimulating hormone impairment due to higher doses of toxic substances, affecting the production and secretion of this hormones. hence, this study showed that luteinizing and follicle-stimulating hormones showed a slight significant increase in the treated groups when compared with the control (tables 4 and 5). also, prolonged intake of a. indica seed aqueous extract rather reduced plasma progesterone concentration in females (shaikh et al., 2009). the results from this study showed a significant decrease in serum progesterone level across a graded dose of 2.5, 5.0, and 10 mg/kg of the treatment when compared with the control in the pre-coital phase with a slight increase in the post-coital phase (table 4 and 5). a decrease in progesterone concentration as recorded in this study enhances the contraceptive effect in a dose-dependent manner (neychev and mitev, 2016). plate 2. contraceptive effect of azadirachta indica seed aqueous extract on the uterus in rats (a. control. rat uterus: ecto cervical epithelium, and subepithelial stroma. b. 2.5 mg/kg a. indica seed aqueous extract: normal endometrial lining, stromal infiltrates of inflammatory cells and endometrial glands. c. 5.0 mg/kg a. indica seed aqueous extract: stromal congestion, infiltrates of inflammatory cells, and glandular epitheliosis. d. 10 mg/kg a. indica seed aqueous extract: stromal infiltrates of inflammatory cells (h&e x 100).) histopathological examination of the uterus administered with graded doses of 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract reveals a normal structure of the uterus muscles when compared with the untreated control (drury and wallinton 2013) (plate 2). this suggested that the extract possesses a a b c d idu et al. – anti-fertility property of azadirachta indica 87 protective effect on the uterus by inhibiting the stroma and other cells from secreting progesterone responsible for the occurrence of pregnancy. this concurred with the report of dosaa et al. 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(2010). laboratory manual for the examination and processing of human semen 5th edition.pp 1-260 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 65-73 | doi: 10.14421/biomedich.2022.111.65-73 issn 2540-9328 (online) ethnomedicinal, phytochemicals, and pharmacological aspects of sentul (sandoricum koetjape) made dharmesti wijaya pharmacology and pharmacy department, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, indonesia. tel. +62 361 240727. corresponding author dharmestiwijaya@gmail.com manuscript received: 09 march, 2022. revision accepted: 05 april, 2022. published: 18 april, 2022. abstract sentul (sandoricum koetjape) is a tropical plant that has been used as traditional medicine in some asian countries for decades. research on phytochemicals and pharmacological activities of this plant extracts has been conducted and shows promising medicinal properties. this review aims to integrate knowledge about s. koetjape focusing on three main aspects namely ethnomedicinal, phytochemicals, and pharmacological, in order to encourage further research on this plant for future drug development. traditionally, all plant parts of s. koetjape have been used for treating various health problems and diseases such as diarrhea, fever, colic, and leucorrhoea. more than 30 chemicals have been identified from s. koetjape, which the most important compounds are ring-a secotriterpene, oleane-type triterpene, secomultiflorane-type triterpene, hydroxymultiflorane triterpene, and limonoids. in vitro studies showed pharmacological potential of the extracts and phytochemicals constituents of s. koetjape including antibacterial, antifungal, antitumoral, anticancer, insecticide, and antioxidant. keywords: ethnomedicine; pharmacology; phytochemicals; sandoricum koetjape; sentul. introduction since prehistoric times, human have been utilized medicinal plants for their health benefits, as evidently shown by fossil records from the middle paleolithic age, approximately 60,000 years ago (solecki, 1975; fabricant and farnsworth, 2001). the oldest written evidence of the utilization of the medicinal plants for drugs preparation has been verified by the 5000 years old sumerian clay slab which described 12 drug synthesis recipes by referring to more than 250 plants (srivastava, 2018). in addition, traditional medicines such as ayurveda, traditional chinese medicine, unani, traditional korean medicine, and kampo have applied medicinal plants to alleviate and treat wide range of diseases such as malaria, diarrhoea, and microbial infections (yuan et al. 2016). currently, it is estimated that about 80% of the world’s population still depend on herbs and herbal products for primary health care (subramani et al. 2017). despite the fact that traditional medicinal plants have been established through empirical practices and evidences, their importance have often been undervalued and disregarded (kaliyaperumal et al. 2013). it is estimated that only less than 10% of the world’s medicinal plant biodiversity has been scientifically studied for their potential pharmacological activities (dias et al. 2012). the awareness of medicinal plants and their ethnopharmacological relevance is crucial to discover novel plant-based drugs and medicines, as well as in disease prevention (sofowora et al. 2013; jamshidi-kia et al. 2018). plants with ethnopharmacological practices have become the main sources of pharmaceuticals in early drug discovery. therefore, efforts must be directed towards measures that will improve the efficacy, effectiveness, and rational use of medicinal plants (veeresham, 2012; sofowora et al. 2013). among the traditional medicinal plants, members of mahogany or meliaceae family have been regarded for their medicinal properties. this family consists of approximately 1400 species belonging to more than 50 genera. modified triterpene compounds called limonoids are abundantly found in meliaceae and are chemically more diverse in this family compared to any other plant families (paritala et al. 2015). a large quantity of other triterpenoid derivatives as well as other compounds such as flavonoids, alkaloids, phenols, coumarins, lignans, and chromones are also present in different meliaceae genera (paritala et al. 2015; yadav et al. 2015). these very diverse compounds are responsible for the wide range of medicinal properties of meliaceae such as antiviral, antiparasitic, and antimicrobial activities, as well as cytotoxic, anticancer, and anti-inflammatory properties (paul et al. 2011; https://doi.org/10.14421/biomedich.2022.111.65-73 mailto:dharmestiwijaya@gmail.com 66 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 xavier-jr et al. 2015; yadav et al. 2015; mubeen et al. 2018). the members of this family have been traditionally used for treating a wide range of diseases (sujarwo et al. 2016; agyare et al. 2018). among the meliaceae family, only a few members have edible fruits, in which sandoricum koetjape (burm.f.) merr. is one of the most popular species (yadav et al. 2015). the species s. koetjape belongs to genus sandoricum cav, that is distributed mainly in tropical areas in asian countries and among plant that have been utilized for traditional medicines (ismail et al. 2003a). the root, bark, leave, and the whole plant of s. koetjape are used in folk medicine for generations in some asian countries such as india, malaysia, indonesia, and thailand for treating various diseases such as diarrhea, leucorrhea, colic, or drunk as a tonic after childbirth (perry and metzger, 1980; kaneda et al. 1992; ismail et al. 2003a). a number of phytochemicals and bioactive compounds from s. koetjape have been identified and tested in vitro which showed large therapeutic potentials (ismail et al. 2003a; aisha et al. 2009a, b; chudzik et al. 2015;). this review aims to comprehensively integrate current knowledge on s. koetjape by focusing into three main aspects namely ethnomedicinal, phytochemistry, and pharmacology. moreover, we discussed toxicological aspects that have not been studied from s. koetjape and some issues to translate the therapeutic capacity of this plant into consumable product. botany and ethnomedicinal aspects of sandoricum koetjape s. koetjape is possibly indigenous plants of indochina and peninsular malaysia, and later the plant has been introduced and naturalized in the philippines, borneo, indonesia, india, the andaman islands, mauritius, australia, taiwan, china, and also into a few other locations in southern florida and central america (lim, 2012). s. koetjape is locally called kechapi, lolly fruit, santol, sentol, wild mangosteen (english), faux mangostan, and sandorique mangousteiner savage (french). this plant is classified into the genus sandoricum, of the meliaceae family, order sapindales, and division tracheophyta (barstow, 2018). other synonymies for s. koetjape are melia koetjape burm.f., sandoricum indicum cav., sandoricum maingayi hiern, sandoricum nervosum blume, sandoricum nervosum (vahl) m.j. roem., sandoricum vidalii merr., trichilia nervosa vahl (lim, 2012; mabberley, 1985). distribution of s. koetjape is mainly in primary and secondary rain forests below 1000 m which are characterized by deciduous, small to large tree, up to 50 m tall with a straight trunk, flaky or fissured, lenticillate, greyish to pale pinkish-brown bark (orwa et al. 2009; lim, 2012). the tree bole morphology of s. koetjape is straight but often crooked or fluted, branchless for up to 18–21 m and with a trunk diameter up to 100 cm (lim, 2012). bark surface smooth or sometimes flaky or fissured, lenticillate, greyish to pale pinkish-brown, inner bark pale brown or red-brown to pink, exuding a milky latex (orwa et al. 2009). it produces fleshy fruits that are round or flattened ball-shaped, yellow or brownish, and 5-8 cm across, with arils part range from sour to sweet (figure 1) (chen et al. 2015). the fruit has 3 5 brown, ovate to ellipsoid seeds, 2–3.5 by 1.2–2.1 by 0.9–1.6 cm, which are usually tightly associated to the pulp (chen et al. 2015). figure 1. sandoricum koetjape fruits. traditionally, s. koetjape has been applied as medications to treat a number of diseases in thailand, malaysia, philippines, and indonesia. in thailand, this plant is locally known as krathon or sathon and its bark decoction is traditionally used to treat diarrhea (kaneda et al. 1992). meanwhile, the aqueous extract of s. koetjape or santol bark in malaysia is consumed after childbirth as a tonic (nassar et al. 2010). moreover, the stem bark of s. koetjape or locally called sentul or kecapi in indonesia is used by local people for colic, leucorrhoea, and stomach ache treatment (kosela et al. 1995; novaryatiin and indah, 2019). moreover, the pounded bark is applied for treating ringworm (perry and metzger, 1980). beside the bark, other plant parts of s. koetjape such as leaves and roots are also conventionally used for several diseases treatments. decoction of the leaves of s. koetjape is being used to treat diarrhea and water from the pounded leaves is used as intermittent fever medication (perry and metzger, 1980). in the philippines, fresh leaves of s. koetjape are put on the body to induce sweating while santol herbal tea is used wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 67 to bath to bring down fever (lim, 2012). the leaves are also used for inflammation or swelling poultice by applying directly to the affected area (agapin, 2020). the root is used to cure leucorrhoea, dysentry, and as general tonic (perry and metzger, 1980). the decoction or infusion of s. koetjape roots is also used for diarrhea and spasm treatment (cabi, 2008). a record of balinese traditional healing therapies written in palm leaves called taru pramana stated that “loloh” or traditional herbal drink of s. koetjape roots and leaves is employed to treat diarrhea, while the bark can be chewed and then sprayed into the stomach (pulasari, 2013). the bark and leaves of s. koetjape are also used in treating diarrhea by the baduy ethnic in indonesia (khastini et al. 2021). phytochemicals of sandoricum koetjape the phytochemicals constituent of s. koetjape has been examined since 1960. to date, more than 30 compounds have been isolated from different parts of this plant, in which triterpenes are the ubiquitous plant components. various group of triterpenes were identified in this plant such as ring-a secotriterpene, oleane-type triterpene, secomultiflorane-type triterpene, hydroxy-multiflorane triterpene, and limonoids. besides, sesquiterpenes and polyalcohols were also detected in stem and fruit hulls of s. koetjape. polyphenols such as quercetin (flavonoid) and tannin were also observed in the plant extracts of s. koetjape (table 1). table 1. compounds identified from the sandoricum koetjape extracts. class compounds plant parts ref triterpenoind acids katononic acid (3-oxo-olean-12-en-29-oic acid) stem (kaneda et al. 1992) katonic acid (3α-hydroxyolean-12-en-29-oic acid) heartwood (king and morgan, 1960) indicic acid heartwood (king and morgan, 1960) bryonolic acid fruit hulls (sim and lee, 1972) ring-a secotriterpene koetjapic acid stem (kaneda et al. 1992) sentulic acid bark (efdi et al. 2012) oleane-type triterpenoid 3-oxo-olean-12-en-27-oic acid bark (efdi et al. 2012) 20-epikoetjapic acid (3,4-seco-olean-4(23),12-diene3,29-dioic acid) stem bark (tanaka et al. 2001) 3-epikatonic acid stem bark (tanaka et al. 2001) briononic acid stem bark (tukiran et al. 2010) secomultiflorane-type triterpene bryononic acid fruit hulls, stem bark (sim and lee, 1972; kosela et al. 1995) secobryononic acid stem bark (kosela et al. 1995) secoisobryononic acid stem bark (kosela et al. 1995) 12β-hydroxymultiflorane triterpenoid acid sandorinic acid a (12β,18-dihydroxy-3oxomultiflora-8-en-29-oic acid) stem bark (tanaka et al. 2001) sandorinic acid b (12β,18-dihydroxy-3oxomultiflora-7-en-29-oic acid) stem bark (tanaka et al. 2001) sandorinic acid c (12β-hydroxy-3-oxomultiflora-8en-29-oic acid) stem bark (tanaka et al. 2001) limonoids/ tetranorterpenoid [2α-(2-methylbutanoyl)oxy]sandoricin leaves (pancharoen et al. 2005) [2α-(2-methylpropanoyl)oxy]sandoricin leaves (pancharoen et al. 2005) sanjecumins a and b leaves (nagakura et al. 2013) trijugin-class limonoids sandrapins a-c leaves (ismail et al. 2003b) sandrapins d-e leaves (ismail et al. 2005) koetjapin d seed (bumi et al. 2019) andirobin-class limonoids sandoripin a and b leaves (pancharoen et al. 2009; nagakura et al. 2013) sandoricin seed (powell et al. 1991) 6-hydroxysandoricin seed (powell et al. 1991) koetjapins a-c seed (bumi et al. 2019) sesquiterpenes (–)-alloaromadendrene (–)-caryophyllene oxide (+)-spathulenol stem (kaneda et al. 1992) polyalcohols mesoinositol fruit hulls (sim and lee, 1972) dimethyl mucate fruit hulls (sim and lee, 1972) flavonoid quercetin n/d (kaewkod et al. 2021) tannin n/d (kaewkod et al. 2021) note: n/d = not determined 68 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 pharmacological activities of sandoricum koetjape antibacterial activity new antibiotic agents are urgently needed due to high antibiotics resistance incidence worldwide that threaten our action to combat serious bacterial infections (mayor, 2018). plants produce a wide range of phytochemicals and secondary metabolites that have therapeutic properties and therefore they are one of the most crucial sources of antimicrobial agents. a study by subramani and co-workers reported that a total of 60 plant extracts and 110 purified compounds were acquired from 112 plants against multi-drug resistant (mdr) pathogens including mdr-mycobacterium tuberculosis, methicillin resistant staphylococcus aureus (mrsa), and plasmodium spp. between 2005 and 2015 (subramani et al. 2017). plants belong to meliaceae family have been shown to have antibacterial activity, including s. koetjape. a number of antibacterial activities have been documented from this plant (table 2). methanol extract of s. koetjape seed seems promising as it shows strong antibacterial activity with minimum inhibitory concentration (mic) at 0.25, 0.50, and 0.50 µg/ml against bacillus subtilis, pseudomonas aeruginosa, and staphylococcus aureus respectively (azziz et al. 2013). meanwhile, the aqueous extract of the seed exhibit weak antibacterial property with mic at 250 mg ml-1 against escherichia coli, s. aureus, candida albicans, and streptococcus pneumoniae (elijah et al. 2016). the observed antibacterial activities in s. koetjape could be related to arrays of secondary metabolites such as alkaloids, flavonoids, and phenolic compounds found in this plant. flavonoid is known by its ability to interact with bacterial membrane proteins, causing the increase in membrane permeability and lead to membrane disruption (gupta and birdi, 2017). terpenoids that ubiquitously present in this plant have been reported to display antibacterial in nature (gupta and birdi, 2017). table 2. antibacterial activity of sandoricum koetjape extracts. solvents suggested constituents property target ref mic zoi seed methanol alkaloid, flavonoid 0.25 µg ml-1 b. subtilis (azziz et al. 2013) 0.50 µg ml-1 p. aeroginosa 0.50 µg ml-1 s. aureus aqueous phenols 250 µg ml-1 e. coli, s. aureus, c. albicans, and s. pneumoniae (elijah et al. 2016) leaf aqueous phenols 100 µg ml-1 e. coli and s. aureus (elijah et al. 2016) fruit juice phenols 8.3±0.6 mm e. faecalis (toobpeng at al. 2017) 13.0±1.0 mm p. aeruginosa mdr1 14.0±1.0 mm e. coli p174 esbl 15.0±0.6 mm e. coli esbl 15.0±1.5 mm p. aeruginosa mdr2 15.0±1.0 mm a.baumannii mdr2 15.0±1.5 mm a.baumannii mdr1 16.0±1.5 mm s. aureus mrsa1&2 fruit hulls bryononic acid (triterpenoid) 6 µg ml-1 salmonella enterica (heliawati et al. 2019) root aqueous n/a 500 µg ml-1 11 mm s. pyogenes nprc 101 (limsuwan and voravuthikunchai, 2013) ethanol n/a >1000 µg ml-1 15 mm s. pyogenes nprc 101 plant extracts aqueous tannin, quercetin (flavonoid) 16 mg ml-1 10.3±0.6 mm e. coli atcc 25922 (kaewkod et al. 2021) 63 mg ml-1 10.3±0.6 mm e. coli k-12 note: n/a = not available; mic = minimum inhibitory concentration; zoi = zone of inhibition anticancer/antitumor activity s. koetjape is known to be rich in triterpene compounds. in recent years, triterpenoids-related studies show that these compounds have potential roles for tumor or cancer prevention and treatments (gill et al. 2016; patlolla and rao, 2012). although many triterpene compounds have been proven to give effective results in treating cancers, only some of them have passed the wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 69 clinical trial (e.g. 12-dioxooleana-1,9(11)-dien-28-oic acid or cddo) (gill et al. 2016). a number of triterpenoids isolated from s.koetjape such as katonic acid, sandorinic acid a, sentulic acid, and koetjapic acid have been noted to have cytotoxic activity against leukemia, colon, and breast cancer cell lines (table 3) (kaneda et al. 1992; tanaka et al. 2001; efdi et al. 2012; nassar et al. 2012a). koetjapic acid has also shown to have cancer chemopreventive and antitumor properties, as well as antimetastatic and antiinflamation activities (ismail et al. 2003a; rasadah et al. 2004; nassar et al. 2012b). table 3. anticancer and antitumor activity of sandoricum koetjape extracts. type of extracts suggested constituents dosage/results ref seed methanol koetjapin d cytotoxic activity against murine leukemia p-388 cell lines with ic50 of 16.8 ± 1.8 µg/ml (bumi et al. 2019) stem diethyl ether 1) 3-oxo-olean-12-en29-oic acid 2) katonic acid cytotoxic activity against p-388 leukemia cells (ed50 0.61 µg/ml (1) and 0.11 µg/ml (2)) (kaneda et al. 1992) bark purified compounds 1) sentulic acid 2) 3-oxo-olean-12-en27-oic acid cytotoxic activity against human promyelocytic leukemia hl-60 cell line by inducing apoptosis (efdi et al. 2012) hexane koetjapic acid cancer chemopreventive. significantly delayed tumor promotion in two-stage mouse skin carcinogenesis (ismail et al. 2003a) hexane 1) koetjapic acid 2) 3-oxo-olean-12-en29-oic acid 3) katonic acid anti-tumor promoting agents. inhibit epstein-barr virus early antigen (ebv-ea) activation (ismail et al. 2003a) stem bark purified compound sandorinic acid a cytotoxic activity against human leukemia hl-60 cells (ic50 15 µg/ml) (tanaka et al. 2001) n-hexane n/a ic50 values of 23, 14, 50, and 52 µg/ml against human umbilical vein endothelial cell (huvec), human colon cancer cells hct-116 and ht-29, and normal cell line ccd-18co (aisha et al. 2009b) n-hexane n/a 50 µg/ml extract showed potent apoptotic cell death induction on hct-116 colon cancer cell by inducing caspases 3 and 7 activity (aisha et al. 2009b) purified compound koetjapic acid cytotoxic activity with ic50 value of 18.88 µg/ml against hct-116 colon cancer cells by inducing caspase-3/7, -8, and -9, inducing morphological changes and nuclear condensation, causing dna fragmentation, disrupting mitochondrial membrane potential, downregulating wnt, hif-1α, map/erk/ jnk, and myc/mac signalling pathways, up-regulating nf-κb signalling pathway (nassar et al. 2012a) synthetic potassium koetjapate (salt form of koetjapic acid) enhanced cytotoxicity against hct-116 cells compared to koetjapic acid (jafari et al. 2014) n-hexane n/a ic50 values between 44 – 48 µg/ml against breast cancer cells mcf7, mda-mb-231, and t47d, and normal cell line mcf-10a (aisha et al. 2009a) n-hexane n/a 100 µg/ml extract showed apoptotic cell death induction on mcf-7 breast cancer cell by inducing caspases 3 and 7 activity (aisha et al. 2009a) methanol koetjapin d cytotoxic activity against murine leukemia p-388 cell lines with ic50 of 16.8 ± 1.8 µg ml-1 (bumi et al. 2019) purified compound koetjapic acid cytotoxic activity with ic50 value of 68.88 µg/ml against mcf-7 breast cancer cells; significantly inhibit cell migration and invasion at 15 µg/ml (sub-toxic dose); significantly inhibit the colony formation properties of mcf-7 (nassar et al. 2012b) other activities triterpenes such as koetjapic acid, katonic acid, and 3oxo-olean-12-en-29-oic acid are found to inhibit dna polymerase β (sun et al. 1999; hu et al. 2004). the mentioned compounds and sentulic acid are also known to have antiinflammation properties (rasadah et al. 2004; itoh et al. 2018;). limonoids compounds isolated from s. koetjape leaves namely sandoripins a and b exhibit antioxidant activity by inhibiting no production in j774.1 cell line 70 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 (nagakura et al. 2013). in addition, phenolic content and flavonoid from s. koetjape fruit extract show antioxidant activity by decreasing ros production and increasing antioxidant enzyme gpx-1 (anantachoke et al. 2016). tannins extracted from methanolic extract of the stem bark also display radical scavenging activity (cavin et al. 1999). beside aforementioned pharmacological activities, s. koetjape extracts also show antiangiogenic, antifungal, antifeedant, ichthyotoxic, and insecticidal against lepidopteran larvae (tabel 4) (mikolajczak and reed, 1987; powell et al. 1991; cavin et al. 1999; ismail et al. 2003a; leatemia and isman, 2004;). table 4. other activities of sandoricum koetjape extracts. bioactivities extract/ constituents dosage/results ref seed antifeedant ethanol, hexane ethanol and hexane extracts strongly inhibited feeding and resulted in high mortality (feeding ratio 0.05 and 0.21; mortality 90 and 100% respectively) of fall armyworm, s. frugiperda (mikolajczak and reed, 1987) sandoricin and 6hydroxysandoricin 100% effective against larvae of spodoptera frugiperda and ostrina nubilalis at 200 ppm or above. (powell et al. 1991) insecticidal ethanol ineffective (49-97% larval growth – relative to control) (leatemia and isman, 2004) leaves antioxidant 1) sandoripins a 2) sandoripins b inhibit no production with ic50 of 16.4 µm (1) and 30.4 µm (2) in mouse macrophage-like j774.1 cells stimulated by lps (nagakura et al. 2013) fruits antioxidant phenolic content, flavonoid 1 mg/ml extract shows dpph scavenging activity of 84.73% (ic50 of 415.8 µg/ml); supressed ros production that induced by h2o2; significantly increase the protein level of an antioxidant enzyme gpx-1 in human embryonic kidney hek-293 cell line (anantachoke et al. 2016) bark ichthyotoxic 1) koetjapic acid 2) 3-oxo-olean-12en-29-oic acid ichthyotoxic activity with tlm of 1.8 and 1.9 ppm respectively for compounds 1 and 2 (ismail et al. 2003a) stem antiinflammation 1) 3-oxo-olean-12en-29-oic acid; 2) katonic acid 3) koetjapic acid oedema inhibition of 94% (crude methanolic extract); 100% (dichloromethane fraction); 90% (hexane fraction); 77% (methanol fraction); 64% (ethyl-acetate fraction); 14% (water fraction); 94% (1); 81% (2); 13% (3) (rasadah et al. 2004) stem bark antiinflammation sentulic acid (purified compound) reduced the production of nitric oxide after co-stimulation with lps/ifnγ in raw264.7 cell line by inhibiting the binding of lps to tlr4f (itoh et al. 2018) antioxidant tannins show radical scavenging activity against dpph radical (cavin et al. 1999) antifungal 1) dichloromethane extract 2) methanol extract active against candida albicans (1) and cladosporium cucumerinum (2) (cavin et al. 1999) antiangiogenic n-hexane and methanol extract n-hexane and methanol extracts showed 97% and 90% blood vessel outgrowth inhibition using rat aortic ring assay (aisha et al. 2009c) n-hexane extract 100 µg/ml extract showed 94±5.5% blood vessel outgrowth inhibition using rat aortic ring assay; and ic50 values of 23, 14, 50, and 52 µg/ml against human umbilical vein endothelial cell (huvec), human colon cancer cells hct-116 and ht-29, and normal cell line ccd-18co (aisha et al. 2009b) koetjapic acid (purified compound) 20 µg/ml and 40 µg/ml koetjapic acid in ethanol showed 50% and 100% vascularitation inhibition using rat aortic ring assay; noncytotoxic against huvecs (ic50 40.97 ± 0.37 µg/ml) (nassar et al. 2011) potassium koetjapate (salt form of koetjapic acid) supressed angiogenesis by inhibiting endothelial functions and expression of angiogenic cytokine vegf (jafari et al. 2020) stem bark and wood dna polymerase β inhibitor 1) 3-oxo-olean-12en-29-oic acid 2) katonic acid 3) koetjapic acid ic50 values of 22, 36, and 20 µm for dna polymerase β inhibitors compounds 1-3 respectively (hu et al. 2004; sun et al. 1999) wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 71 drawbacks and future direction of research many studies on antibacterial activity screening of s. koetjape were mostly carried out using crude extracts. the results seem promising, but nevertheless has limited impact on further drug development since crude extracts contain many types of compounds with different activities, side effects, as well as toxic effects. researches related to antitumor and anticancer activity of s. koetjape are generally more focused on a specific compound which would be more convenient to be developed into further step in drug development. a milestone in developing a new therapeutic agent from s. koetjape has been reached by jafari and co-workers (2020). their research chemically modifies the poorly soluble koetjapic acid into more soluble form namely potassium koetjapate, and thus enhanced its antiangiogenesis efficacy in rats. in the future, research about discovering medicinal properties of s. koetjape should be focus more on a single compound, rather than the whole extract. further preclinical and clinical research are also needed to develop the promising compounds contained in s. koetjape as new therapeutic agents against a wide range of diseases. conclusions in conclusion, phytochemicals of s. koetjape have been reported to have promising bioactivities that can potentially be used for therapeutic applications. however, more knowledge is required regarding to mode of actions, biosynthetic pathways, and toxicological aspects of these identified compounds. importantly, many of reported bioactivities were conducted in vitro, while compelling evidence of the application of such compounds from in vivo studies are rather limited. toxicological aspects are especially the utmost important particularly to determine the limit concentrations that are safe to be applied to treating such diseases. acknowledgements: we would like to thank the faculty of medicine and health sciences, warmadewa university, bali-indonesia for providing financial support for this study. competing interests: the author declares that there are no competing interests. references agapin, j. s. 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(2016). the traditional medicine and modern medicine from natural products. molecules, 21(5). doi:10.3390/molecules21050559 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 7-16 | doi: 10.14421/biomedich.2022.111.7-16 issn 2540-9328 (online) peptide fractions from chymotrypsin-hydrolyzed moringa oleifera seed proteins inhibit α-amylase and α-glucosidase in vitro oluwafemi emmanuel ekun*, augustine olusegun olusola, joseph adaviruku sanni, feyisayo ishola department of biochemistry, faculty of science, adekunle ajasin university, akungba akoko, ondo state, nigeria. corresponding author* oluwafemi.ekun@aaua.edu.ng manuscript received: 14 january, 2022. revision accepted: 03 february, 2022. published: 23 february, 2022. abstract this study attempted to investigate the activities of chymotrypsin-digested moringa oleifera seed proteins and their peptide fractions on carbohydrate-hydrolyzing enzymes. proteins from m. oleifera seeds were isolated using isoelectric point precipitation and hydrolyzed using chymotrypsin. the hydrolysates obtained were fractionated into peptide fractions of <1 kd, 1-3 kd and 3-5 kd ranges by means of gel-filtration chromatography. the inhibitory effects of the hydrolysates and their fractions on -amylase and -glucosidase were evaluated, and kinetics of inhibition were also determined. using starch and p-nitrophenyl glucopyranoside as substrates, the hydrolysate and fractions demonstrated concentration-dependent inhibition of -amylase and -glucosidase respectively (ic50 of 0.172 ± 0.005 mg ml-1 to 1.312 ± 0.267 mg ml-1, for -amylase inhibition and ic50 of 0.463 ± 0.008 mg ml-1 to 0.696 ± 0.051 mg ml-1 for -glucosidase inhibition). kinetic analysis revealed that selected hydrolysate fractions competitively inhibited -amylase while displaying a mixed mode of inhibition of -glucosidase. this study suggests that subjecting m. oleifera seed proteins to proteolysis could yield therapeutic peptide products having immense potentials that could be harnessed to develop novel anti-diabetic agents and additives to food, which could serve as cost effective alternatives to current therapies. keywords: moringa oleifera; hydrolysate; peptide; chymotrypsin; α-amylase; α-glucosidase. introduction bioactive peptides and protein hydrolysate preparations obtained either by enzymatic hydrolysis or microbial fermentation of plant and animal proteins have been investigated for their therapeutic capacities and other health promoting benefits (lopez-barrios et al., 2014, ulagesan et al., 2018). peptide products have been reported to demonstrate numerous bioactivities against hypertension (yamada et al., 2013; majumder and wu 2015), cancer (vileghe et al., 2010; thundimadathil 2012) oxidative stress (olusola et al., 2018), pathogenic microorganisms (ulagesan et al., 2018) and recently, diabetes mellitus (olusola and ekun 2019, famuwagun et al., 2020), in various in vitro assays and/or animal models. diabetes mellitus is a metabolic disorder that is caused by an absolute or relative insulin deficiency (rhoades and bell 2013), and it is the fourth leading cause of health problem globally (idf 2020). it is characterized by chronic hyperglycemia associated with derangements in the regulation of carbohydrate, fat and protein metabolism (olusola and ekun 2019). this results in the appearance of metabolites such as ketone bodies and advanced glycated end products in the blood, and these in turn cause oxidative tissue and organ damage, ketoacidosis among other complications at later stages of the disease (arise et al., 2016). carbohydrate – hydrolyzing enzymes (such as α-amylase and αglucosidase) and incretin degrading enzymes (such as dipeptidyl peptidase iv) have been key pharmacologic targets for many hypoglycemic drugs (arise et al., 2019) and as such have been used in the management of diabetes mellitus, in addition to lifestyle changes. however, owing to certain adverse effects caused by several of these drugs (yu et al., 2012), in addition to high cost of procurement especially in third world countries, attention has turned to alternatives from natural sources, and this has included peptide products from proteins in seeds and leaves of plants. moringa oleifera is a fast growing, drought – resistant and perennial plant, belonging to the genus moringaceae (anwar et al., 2007). it is widely known as the “horseradish” family and native to india, especially in the himalayan regions. it is now known to be cultivated in tropical and subtropical areas such as in tropical africa and in south west asia, where its young seed pods and leaves are used as vegetables as well as in herbal medicine (leone et al., 2015; abd-rani et al., 2018). m. oleifera is a plant whose leaves and seeds have been excellent sources of essential oils and other nutrients (texeira et al., 2014). texeira et al. (2014) https://doi.org/10.14421/biomedich.2022.111.7-16 mailto:oluwafemi.ekun@aaua.edu.ng 8 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 found that whole moringa leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ash, 44.4% carbohydrates, in addition to 3.0 mg/100g of calcium and 103.1 mg/100g iron. also, another study reported that proximate analysis of its seeds indicated that the percentage nutrient composition of its protein, lipid, ash, fiber, and carbohydrate were about 35.5, 29.3, 4.6, 11.5, and 19.6% respectively (kwaambwa et al., 2015; munemune et al., 2016). proteins in m. oleifera leaves and seeds consist of albumin, prolamins, globulins and glutelins (mune-mune et al., 2016) which is mostly the case with many oil seeds (wani et al., 2011). its relatively high protein content makes it an excellent source of potential biologically active peptides (olusola et al., 2018). furthermore, freire et al., (2015) and mune-mune et al., (2016) reported that m. oleifera leaves and seeds are especially rich in glycine, isoleucine, glutamate, aspartate, leucine, arginine, proline, threonine among other amino acids. parts of the plant (roots, leaves, stem bark and seeds) have been used for nutritional purposes and as traditional medicine (leone et al., 2015; abd-rami et al., 2018). various parts of m. oleifera have been demonstrated to possess a myriad of bioactivities such as purgative, antimicrobial as well as normoglycemic effects (siddhuraju and beck, 2003; divi et al., 2012). its stem bark has been determined to have anti-proliferative, anti-ulcerative, as well as anti-inflammatory properties (mahajan et al., 2009). the presence of certain polyphenols as well as other bioactive secondary metabolites in m. oleifera leaf extracts were reported to exert antihypertensive (ndong et al., 2007), hypolipidemic and hypoglycemic effects (anwar et al., 2007). recently, crude enzymatic hydrolysates of m. oleifera seed proteins have been reported to possess enzyme-inhibitory activities in vitro (olusola et al., 2018, olusola and ekun 2019). however, hydrolysate fractionation is essential in reducing peptide aggregation, liberating peptides in solution, which in turn leads to increased peptide bioactivities in the process (awosika and aluko 2019). therefore, this study aims to examine the carbohydrase – inhibitory activities of peptide fractions obtained from chymoytrpsin – digested m. oleifera seed proteins in order to harness them as possible sources of novel antidiabetic peptides, and to further justify the value added uses of m. oleifera seed proteins. materials and methods materials collection of m. oleifera seeds m. oleifera seeds were collected from farms in akungba akoko, ondo state and identified, after which voucher samples were deposited at the department of plant science and biotechnology, adekunle ajasin university, akungba akoko. chemicals and reagents chymotrypsin (from bovine pancreas), and α-amylase (fungal), α-glucosidase (human) were products of sigma-aldrich laboratories, co-artrim, united kingdom. all other chemicals and reagents used were of analytical grade, and were also products of sigmaaldrich laboratories, co-artrim, united kingdom. methods isolation of m. oleifera seed proteins the seeds were dried and pulverized before being kept in an air-tight container at 4oc. this was subsequently defatted using n-hexane as was previously described by arise et al., (2016) with slight modifications. the meal was extracted three times with n-hexane using a meal/solvent ratio of 1:10 (w/v). the meal was then dried at 40oc in a vacuum oven and ground again to obtain a fine powder, termed defatted seed meal, which was stored at -20oc. the protein component of the defatted meal was extracted using the method described by alashi et al., (2014). defatted seed meal was suspended in 0.5 m naoh ph 12.0 at a ratio of 1:10, and stirred for one hour to facilitate alkaline solubilization. this was centrifuged at 18°c and 3000 g for 10 min. two additional extractions of the residue from the centrifugation process were performed with the same volume of 0.1 m naoh and the supernatants were then pooled. the ph of the supernatant was adjusted to 4.0 to facilitate acid-induced protein precipitation using 0.1 m hcl solution; the precipitate formed was recovered by centrifugation. the precipitate was washed with distilled water, adjusted to ph 7.0 using 0.1 m naoh, freeze-dried and the protein isolate was then stored at -20°c until required for further analysis. preparation of m. oleifera seed protein hydrolysates the protein isolate was hydrolysed using the methods described by onuh et al., (2015) and olusola and ekun (2019b) with slight modifications. the conditions for hydrolysis was tailored for each enzyme in order to optimize its activity. hydrolysis was carried out using chymotrypsin (ph 8.0, 37ºc). the protein isolate was dissolved in 0.2m phosphate buffer, ph 8.0. the enzyme was added to the slurry at an enzyme-substrate ratio (e:s) of 2:100. digestion was performed at the specified conditions for 8 hours with continuous stirring. the enzyme was then inactivated by boiling in water bath (95–100oc) for 15 minutes and undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh followed by centrifugation at 7000 g for 30 minutes. the supernatant containing target peptides were then collected. protein content of samples were determined using biuret assay method with bovine serum albumin (bsa) as standard. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 9 fractionation of m. oleifera seed protein hydrolysates the moringa oleifera seed protein hydrolysates were separated into molecular weight fractions using gel filtration chromatography as described by boyer (2012) and tounkara et al., (2014) with some modifications. briefly, 5 ml of the clear supernatant resulting from protein hydrolysis, at a protein concentration of 10 mg/ml was filtered, suspended in 50 mm phosphate buffer ph 7 and passed into a sephadex g25 chromatographic column of dimensions 30 cm x 4 cm which had earlier been equilibrated with the buffer. the same phosphate buffer was used to elute the separating fractions, and the elution peaks were monitored at 400 nm according to prasad et al. (2017). the separating fractions eluted under the same elution peak were collected, pooled and their molecular weights were determined by comparison with the graph of the logarithm of molecular weights against elution volumes of known standards. the eluates, according to their molecular weights, were then sorted into <1 kd, 1-3 kd and 3-5 kd ranges. peptide fractions of molecular weights higher than 5 kda were removed and discarded. the collected peptide fractions were stored at -20°c for further analysis. determination of degree of hydrolysis degree of hydrolysis (dh) was determined by calculating the percentage of soluble protein in 10% trichloroacetic acid (tca) in relation to total protein content of the protein isolate according to the method described by olusola et al. (2018). one ml of protein hydrolysate was added to 1 ml of 20% tca to produce 10% tca soluble material. the mixtures was left to stand for 30 minutes to allow for precipitation, followed by centrifugation at 4000 g for 20 minutes. the supernatants were then analyzed for protein content using biuret assay method with bovine serum albumin (bsa) as standard. the degree of hydrolysis (dh) was then calculated as the ratio of soluble peptide in 10% trichloroacetic acid (in milligrams) to the total protein content of isolate (in milligrams), expressed in percentage. determination of peptide yield the percentage peptide yield was determined using the method described by girgih et al. (2011). the peptide yields (%) of moringa oleifera seed protein hydrolysates and fractions, were calculated as the ratio of peptide content of lyophilized hydrolysate/fraction to the protein content of unhydrolysed protein isolate. determination of α-amylase inhibition an α-amylase-inhibitory assay was performed according to the method reported by oboh et al. (2011). briefly, 125 µl of hydrolysate (0.2 to 1.0 mg ml-1) was placed in test tubes and 125 µl of 20 mm sodium phosphate buffer (ph 6.9, with 6mm nacl) containing α-amylase solution (0.5 mg ml-1) added. the content of each tube was pre-incubated at 25°c for 10 min, after which 125 µl of 1% starch solution in 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) was added at timed intervals. the reaction mixtures were incubated at 25°c for 10 min. the reaction was terminated by adding 250 µl of dinitrosalicylic acid (dns) colour reagent and further incubated in boiling water for 5 min and cooled to room temperature. the content of each test tube was diluted with 2.5 ml distilled water and the absorbance measured at 540 nm. a control was also prepared using the same procedure except that the hydrolysate was replaced with distilled water. the α-amylase-inhibitory activity was calculated as shown: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙– 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) / 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 the concentration of hydrolysate resulting in 50% inhibition of enzyme activity (ic50) was determined from a plot of percentage inhibition against hydrolysate concentrations using graphpad prism version 6.0 (graphpad software, san diego, ca, usa). determination of kinetic parameters of α-amylase inhibition the kinetic study of α-amylase inhibition was conducted according to the method described by olusola and ekun (2019a). 125 µl of the hydrolysate was pre-incubated with 125 µl of α-amylase solution for 10 min at 25°c in a set of tubes. in another set of tubes, 250 µl of phosphate buffer (ph 6.9) was also pre-incubated with 125 µl of α-amylase solution. starch solution (125 µl) of increasing concentrations (1.0 to 8.0 mg ml–1) were added to both sets of reaction mixtures to initiate the reaction. the mixture were then incubated for 10 min at 25 °c, and then boiled for 5 min after the addition of 250 µl of dinitrosalicylic acid (dns) reagent to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically from a maltose standard curve and converted to reaction velocities as shown below: 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (µ𝑚𝑜𝑙 𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛– 1) 𝑚𝑖𝑛– 1) = 𝑀𝑎𝑙𝑡𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 / 𝐼𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 × 𝑀𝐸. where: maltose concentration is in µmol ml–1; incubation time: 10 min; me: amount of enzyme (in mg) in reaction mixture a double reciprocal plot (1/v versus 1/[s]), where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition and the kinetic parameters of α-amylase inhibition by hydrolysates were determined by analysis of the double reciprocal plot. the inhibition constant (ki) was determined using a secondary plot known as the dixon plot (palmer and bonner, 2007), by plotting a graph of inverse of initial 10 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 velocities on the x-axis against inhibitor concentrations on the x-axis, at fixed concentration of substrate. determination of α-glucosidase inhibition the effect of the hydrolysates on 𝛼-glucosidase activity was determined according to the method described by kim et al., (2005) using 𝛼-glucosidase from saccharomyces cerevisiae. the substrate solution pnitrophenyl glucopyranoside (pnpg) was prepared in 20 mm phosphate buffer, and ph 6.9. 100𝜇l of 𝛼 glucosidase (1.0 u/ml) was pre-incubated with 50𝜇l of the different concentrations of the hydrolysates for 10 min. then 50𝜇l of 3.0 mm (pnpg) as a substrate dissolved in 20 mm phosphate buffer (ph 6.9) was added to start the reaction. the reaction mixture was incubated at 37∘c for 20 min and stopped by adding 2ml of 0.1 m na2co3 solution. the 𝛼-glucosidase activity was determined by measuring the yellowcolored para-nitrophenol released from p-npg at 405 nm. the results were expressed as percentage of the blank control. percentage inhibition was calculated as: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙– 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) / 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetic parameters of α-glucosidase inhibition the kinetic parameters of 𝛼-glucosidase by the hydrolysates was determined according to the modified method described by ali et al. (2006). briefly, 50 𝜇l of the (5 mg ml-1) hydrolysate was pre-incubated with 100 𝜇l of 𝛼-glucosidase solution for 10 min at 25°c in one set of tubes. in another set of tubes, 𝛼-glucosidase was pre-incubated with 50𝜇l of phosphate buffer (ph 6.9). 50 𝜇l of pnpg at increasing concentrations (0.5–2.0 mg ml-1) was added to both sets of reaction mixtures to start the reaction. the mixture was then incubated for 10 min at 25°c, and 500 𝜇l of na2co3 solution was added to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically at 405nm using a paranitrophenol standard curve and converted to reaction velocities. a double reciprocal plot (1/v versus 1/[s]) where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition of the hydrolysates on 𝛼-glucosidase activity was determined by analysis of the double reciprocal (lineweaver-burk) plot using michaelis menten kinetics. the inhibition constant (ki) was also determined using the dixon plot (palmer and bonner, 2007), by plotting a graph of inverse of initial velocities on the x-axis against inhibitor concentrations on the xaxis, at fixed concentration of substrate. statistical analysis results were expressed as mean of triplicate observations ± standard deviation. the data were statistically analyzed using one way analysis of variance (anova) and duncan’s multiple range tests. differences were considered statistically significant at p<0.05 using graphpad prism version 6.0 (graphpad software, san diego, ca, usa). results peptide yield and degree of hydrolysis the enzyme chymotrypsin was used to hydrolyze the protein isolate and the degree of hydrolysis was found to be 51.180 ± 2.461 %. the peptide yields of the hydrolysate and its fractions are displayed in table 1. table 1. peptide yield of m. oleifera seed proteins hydrolyzed by chymotrypsin and fractionated using gel filtration chromatography. sample peptide yield unfractionated hydrolysate 87.571±2.342a fraction 1 (<1 kd) 12.791±0.535c fraction 2 (1-3 kd) 13.548±1.070bc fraction 3 (3-5 kd) 13.656±0.076b -amylase inhibitory activity the percentage -amylase inhibitory activity of the hydrolysate and its fractions in relation to the control, acarbose, was illustrated in figure 1 and their ic50 values were displayed in figure 2. this result showed that, at lower concentrations, the hydrolysate fractions demonstrated -amylase inhibitory effects comparable to the control. however, the unfractionated chymotrypsin hydrolysate demonstrated a concentrationdependent reduction in activity, attaining a value of 15.487 ± 1.125 % at a final concentration of 1.0 mg ml1, which is significantly lower (p<0.05) than those of the hydrolysate fractions. chymotrypsin hydrolysate fractions displayed percentage inhibitions above 50% at all study concentrations, such that the fractions f1, f2 and f3 reached 61.859 ± 2.490 %, 76.440 ± 1.220% and 67.195 ± 0.528 % inhibition respectively at a final concentration of 1.00 mg ml-1. among the chymotrypsin hydrolysate fractions, fraction f2, had the highest (p<0.05) inhibitory activity. the hydrolysate and its peptide fractions f1 – f3, inhibited -amylase to a 50% extent at concentrations of 1.312 ± 0.267 mg ml-1, 0.172 ± 0.005 mg ml-1, 0.892 ± 0.038 mg ml-1 and 0.526 ± 0.093 mg ml-1 respectively. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 11 figure 1. percentage -amylase inhibition by moringa oleifera seed protein hydrolysates and their fractions obtained by chymotrypsin digestion. bars are expressed as means ± standard error of mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). figure 2. values of 50% inhibition of -amylase activity by chymotrypsin-derived m. oleifera seed protein hydrolysate and its fractions. bars are expressed as means ± standard error of means of triplicate determinations (n=3). values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another. kinetics of -amylase inhibition the effect of a selected peptide fraction (fraction 2) on the catalytic activity of -amylase in converting starch to maltose was presented in figure 3. kinetic parameters determined from lineweaver-burk plots in the absence and presence of two different concentrations of the hydrolysate fraction were summarized in table 2. in the absence of the hydrolysate fractions, the michaelis constant, km, of -amylase for its substrate was found to be 0.552 mg ml-1of starch while maximal velocity, vmax was 3.890 mm mg-1 min-1. inhibition of -amylase activity increased with increasing concentrations of the peptide fraction, such that the km of the enzyme was increased while vmax and catalytic efficiency, ce, of amylase were reduced in the presence of the inhibitory peptide fractions. the peptide fraction demonstrated competitive inhibition at both 0.5 mg ml-1 and 1.0 mg ml-1. figure 3. lineweaver-burk plot of -amylase inhibition by moringa oleifera seed protein hydrolysate fraction 2 (1-3 kd) obtained from chymotrypsin hydrolysis 12 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 table 2. kinetics of -amylase-catalyzed reactions in the presence and absence of m.oleifera seed protein hydrolysate fraction 2. kinetic parameters no inhibitor chymotrypsin hydrolysate fraction 2 (mg ml-1) 0.5 1.0 km or kˈm (mg ml-1) 0.552 0.610 0.659 vmax or vˈmax (mm mg-1 min-1) 3.890 3.632 3.750 ce (mmol ml-1 min-1) 7.053 5.953 5.717 ki (mg/ml) 0.735 km or kˈm: michaelis constant in the absence or presence of the inhibitory peptide fraction; vmax or vˈmax: maximum velocity in the absence or presence of the inhibitory peptide fraction; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. -glucosidase inhibitory activity the inhibitory activities of the m. oleifera seed protein hydrolysate and its fractions on α-glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside at varying concentrations in comparison to acarbose (control) are presented in figure 4. their ic50 values were also depicted in figure 5. m. oleifera seed protein hydrolysate and its fractions, as digested by chymotrypsin, also displayed increased inhibition of glucosidase with increasing concentration (figure 4). however, these were significantly lower (p<0.05) when compared to control at all study concentrations. the unfractionated hydrolysate and its gel-filtration fractions f1, f2 and f3 attained maximal inhibitory activities of 38.723 ± 1.508 %, 73.077 ± 1.110 %, 83.474 ± 2.691% and 85.282 ± 1.295% respectively at a final concentration of 1.0 mg/ml. chymotryptic hydrolysate fraction f2 had significantly higher (p<0.05) activity than other fractions at 0.4 mg ml-1, 0.6 mg ml-1 and 0.8 mg ml-1, but there was no significant (p<0.05) difference in its activity when compared to fraction f3 at 1.0 mg ml-1. in addition, chymotrypsin hydrolysate and its fractions f1, f2 and f3 inhibited -glucosidase to a 50% extent at concentrations of 0.509 ± 0.025 mg ml-1, 0.651 ± 0.025 mg ml-1, 0.463 ± 0.008 mg ml-1, and 0.696 ± 0.051 mg ml-1 respectively. these ic50 values were higher (p<0.05) than those of acarbose. chymotrypsin hydrolysate fraction 2 had the lowest (p<0.05) ic50 value among the hydrolysates and fractions derived from chymotrypsin proteolysis. figure 4. percentage -glucosidase inhibition by moringa oleifera seed protein hydrolysates and their fractions obtained by chymotrypsin digestion. bars are expressed as means ± standard error of mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). figure 5. ic50 values of -glucosidase activity by chymotrypsin-derived m. oleifera seed protein hydrolysate and its fractions. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 13 bars are expressed as means ± standard error of means of triplicate determinations (n=3). values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another. kinetics of α-glucosidase inhibition the effects of a selected m. oleifera seed protein hydrolysate fraction (fraction 2) on the kinetics of α glucosidase–catalyzed hydrolysis of p-nitrophenyl glucopyranoside, p-npg, to p-nitrophenol were illustrated in figure 6, while the kinetic parameters from the resulting line-weaver burk plot were summarized in table 3. in the absence of inhibitory hydrolysates, the michaelis constant, km of αglucosidase for its substrate was determined to be 0.297 mg ml-1 p-npg, while maximum velocity, vmax, was 270.27 mm mg -1 min-1. all hydrolysate fractions caused decreases in the vmax and catalytic efficiency, ce of the enzyme. figure 6. lineweaver-burk plot of -glucosidase inhibition by moringa oleifera seed protein hydrolysate fraction 2 (1-3 kd) obtained from chymotrypsin digestion. table 3. kinetic parameters of -glucosidase inhibition by m. oleifera seed protein hydrolysate fractions. kinetic parameters no inhibitor chymotrypsin hydrolysate fraction 2 (mg ml-1) 0.5 1.0 km or kˈm (mg ml-1) 0.297 0.456 0.220 vmax or vˈmax (mm mg-1 min-1) 270.270 147.059 100.000 ce (mmol ml-1 min-1) 910.001 322.498 454.545 ki (mg ml-1) 0.136 km or kˈm: michaelis constant in the absence or presence of inhibitory peptide fraction; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory peptide fraction; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. discussion degree of hydrolysis and peptide yield the degree of hydrolysis (dh) estimates the amounts of cleaved peptide bonds in a protein hydrolysate. dh can affect the molecular sizes and amino acid compositions of the peptides and consequently influence the biological activities of the peptides formed during hydrolysis (olusola et al., 2018). the degree of chymotrypsin hydrolysis of m. oleifera seed proteins was 51.59 ± 3.81 % which was higher than 38.66% gotten for obtained for whey protein hydrolysates at an e/s ratio of 1:100 but slightly lesser than 57.34% obtained for the same whey hydrolysate at an e/s ratio of 2:100 (galvao et al., 2001). also, it was higher than the dh value of 11.2 ± 1.4% determined for chymotrypsin digests of buffalo casein at an e/s ratio of 1:100 (shanmugam et al. 2015). the observed differences could be due to different protein sources, which invariably leads to varying number of sites susceptible to chymotrypsin hydrolysis, as the enzyme is known to preferentially hydrolyze the c-terminal peptide bonds of aromatic aminoacyl residues (voet et al., 2016). in addition, it appears that an increased enzyme/substrate ratio is required to improve the degree of hydrolysis as regards proteolysis by chymotrypsin. other factors such as hydrolysis time and ph of the buffer medium could affect enzymatic activity and by extension, the degree of hydrolysis. peptide yield measures the amount, in percentage, of peptides generated relative to the whole protein subjected to enzymatic proteolysis; thus representing an important index in determining the efficiency of the overall process (alashi et al., 2014), as these enzymes degrade the proteins into several peptides of varying lengths and sizes (girgih et al., 2011). the peptide yield obtained by chymotrypsin digests was higher than the 48.18 ± 0.89% reported for chymotrypsin hydrolysates of yellow field proteins (awosika and aluko, 2019). this could be as a result of a relative abundance of 14 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 aromatic aminoacyl residues in m. oleifera seed proteins (mune-mune et al., 2016) which provides more cleavage points for enzymatic hydrolysis. the peptide yield of the unfractionated hydrolysates was higher than those of their corresponding fractions put together, and this could be due to peptide loss during the process of chromatographic sepaprtion and the removal of peptides whose molecular weights were higher than 5 kda. this is consistent with the reports of awosika and aluko (2019) in their work with yellowfield pea protein hydrolysates and peptide fractions. α-amylase inhibitory activity and kinetics the enzyme α-amylase, a digestive carbohydrase, catalyzes the hydrolysis of complex polysaccharides in the mammalian gut. thus, the inhibition of α-amylase activity is a key pharmacologic intervention in the management of type 2 diabetes mellitus (olusola and ekun, 2019a). in this study the unfractionated hydrolysates showed lower α-amylase inhibitory activities when compared to acarbose, and this is because acarbose is a synthetic inhibitor of α-amylase. in addition, the peptide fractions demonstrated better inhibitory activities than the unfractionated hydrolysate, and this is in consonance with the reports of awosika and aluko (2019) that hydrolysate fractionation improves peptide bioactivity, such that more peptides are able to access substrate binding sites on the enzyme. in the same vein, malomo and aluko, (2016) reported that unfractionated hydrolysates contained high molecular weight peptides which could act antagonistically to smaller peptides, thus reducing inhibitory activity. among the peptide fractions, the chymotrypsin hydrolysate fraction f2 (1-3 kd) demonstrated the highest inhibitory activities at a maximum concentration of 1.0 mg ml-1. this could be a direct result of the nature of the peptides released by these enzymes during proteolysis. it is known that chymotrypsin as an endoprotease hydrolyzes peptide linkages from c-terminal residues of phenylalanine, tyrosine and tryptophan. reports from previous studies stated that phenylalanine, leucine, proline and glycine residues are required for the inhibition of α-amylase (yu et al. 2012, garza et al. 2017). in the same vein, arise et al. (2016) also suggested that α-amylase binds to peptides containing aromatic residues such as phe, tyr and trp. thus, these peptides obtained from enzymatic proteolysis could contain these specific amino acid residues that locks into sites on the enzyme, inhibiting its activity in the process. the lineweaver-burk plot was used to determine the mode of α-amylase inhibition by varying two concentrations of a selected peptide fraction, f2. also, the kinetic parameters determined from the double-reciprocal plots were summarized in table 2; suggesting that the michaelis constant, km of αamylase (from saccharomyces cerevisiae) in the absence of inhibitory hydrolysates is 0.552 mg ml-1 of starch, which is lower than 1.4 mg ml-1 (acharya et al., 2014) for α-amylases obtained from aspergillus oryzae. the selected peptide fraction in this study exhibited a competitive type of inhibition at both study concentrations. this suggests that these peptides are capable of binding α-amylase in its free form, at the same substrate-binding site for starch, creating a deadend complex as a result. this is in contrast to the findings of arise et al. (2016) that reported a mixed type of inhibition of α-amylase for peptic, tyrptic and alcalase hydrolysates of citrullus lanatus seed protein hydrolysates, but literature has been scarce for the αamylase – inhibitory activity of peptides obtained by chymotrypsin hydrolysis. however, the ki value obtained suggest that these peptides bind α-amylase with lesser affinity when compared to enzyme – substrate binding. α-glucosidase inhibitory activity and kinetics the enzyme α-glucosidase occurs mostly as a membrane-bound enzyme on the brush border membranes of the ileum, and digests carbohydrates by hydrolyzing glucose residues from a number of oligosaccharides. thus, modulating α-glucosidase activity represents another important strategies in the control of blood glucose levels in the management of diabetes mellitus (qaisar et al. 2014). chymotrypsin hydrolysate fractions displayed a concentrationdependent increase in α-glucosidase inhibitory activities, such that fractions f2 and f3 showed better inhibitory effects. this is consistent with the reports of awosika and aluko (2019) who reported that chymotrypsin hydrolysate fractions of yellow field pea protein hydrolysates inhibited α-glucosidase. it therefore follows that the process of fractionation may have helped liberate these bioactive peptides so that they could bind to and inhibit α-glucosidase. in addition, since chymotrypsin cleaves peptides after aromatic residues (voet et al. 2016) and that a tyrosinyl residue is a likely requirement for α-glucosidase inhibitory activity (ibrahim et al., 2018), it is therefore suggested that the presence of a one or more well positioned aromatic side chains may have contributed positively to the inhibition of α-glucosidase activity. the kinetic parameters obtained from the double reciprocal plots of αglucosidase inhibition by selected peptide fractions of m. oleifera seed proteins in figure 6 were summarized in table 3. the michaelis constant, km, of α-glucosidase for p-nitrophenyl glucopyranoside in the absence of inhibitor was determined to be 0.297 mg ml-1 p-npg in this study. this is slightly higher than 0.211 mg ml-1 (0.7mm) p-npg obtained by awosika and aluko (2019) but lower than 6.31 mg ml-1 reported by arise et al. (2019). vmax, in the absence of inhibitory hydrolysates was 270.27mm mg-1ml-1. chymotrypsin hydrolysate fraction f2 demonstrated a mixed mode of inhibition at 0.5 mg ml-1 while at 1.0 mg ml-1 the inhibition ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 15 mechanism shifted to an uncompetitive subtype of mixed inhibition. this is in contrast to what was obtained with the <1 kd fraction of chymotryptic digests of yellowfield pea protein hydrolysate (awosika and aluko 2019) as they inhibited α-glucosidase via a noncompetitive inhibition mechanism. this suggests that peptide concentration could have significant effects on mechanism of enzyme inhibition and this could be evaluated in further studies. the enzyme-inhibitor binding constant, ki, revealed that the peptide fraction possessed stronger binding affinity for α-glucosidase than for α-amylase. ibrahim and others (2018) stated that aminoacyl residues such as tyrosine are needed at terminal ends of a peptide for α-glucosidase inhibitory activity. chymotrypsin is a protease that could cleave and release peptides with aromatic c-terminal ends (voet et al., 2016), and this might explain the αglucosidase inhibitory activities observed by these peptide products. conclusion this study concludes that peptide fractions obtained from chymotrypsin digests of m. oleifera seed proteins elicited antidiabetic potentials by inhibiting carbohydrate hydrolyzing enzymes – α-amylase and αglucosidase – by differing mechanisms in vitro. in addition, fraction 2 containing peptide sizes of 1-3 kd recorded the highest activity against the carbohydrases. further studies such as structural identification, modifications, and chemical synthesis of peptides responsible for the observed bioactivities are being embarked upon, and are currently in progress. conflict of interest: the authors declare no conflicts of interest. references abd-rani nz, husain k. kumolosasi e (2018) moringa genus: a review of 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10.14421/biomedich.2020.92.57-64 issn 2540-9328 (online) the role of gene therapy in the treatments of type 1 diabetes mellitus: a review harem othman smail department of biology, faculty of science and health, koya university koya koy45, kurdistan region f. r. iraq corresponding author harem.othman@koyauniversity.org manuscript received: 22 july, 2020. revision accepted: 06 november, 2020. published: 10 november, 2020. abstract the main aims of this review were to understand the roles of gene therapy in the treatment and prevention of type 1 diabetes mellitus and i will discuss a brief history, approaches, vector types with the future of diabetes following clinical use. type 1 diabetes mellitus is a metabolic condition that is identified by insufficient insulin development due to pancreatic damage to beta cells. control, long life, and diagnosis of these metabolic disorders have become vital sources for many scientists and researchers. after 2000, the latest approaches to molecular medicine were introduced as one of the possible therapeutic options for diabetes type 1 diagnosis. many genes have been reported as a clinical trial so that damaged genes can be treated and three main approaches shown about 50 years ago are islet transplantation, β cell regeneration, and insulin gene therapy to cure and prevent diabetes type. treating diabetes through gene therapy can promise children and adolescents, but more clinical applications are needed to recognize it as a permanent route. keywords: type 1 diabetes mellitus; gene therapy; β cells; virus vectors and islet. introduction diabetes is one of the metabolic disorders characterized by hyperglycemia due to insulin secretion deficits, insulin action, or both (smail et al., 2019). type 1 diabetes mellitus is caused by extreme insulin deficiency secondary to pancreatic beta cell autoimmune damage (kolodka et al., 1995). the middle east and north africa region have the highest average prevalence of diabetes in adults (10.9%), while the western pacific region has the largest number of adults diagnosed with diabetes and the highest prevalence of diabetes in countries (37.5%). in terms of diagnostic criteria, etiology, and genetics, various classes of diabetes mellitus, namely type 1, type 2, gestational diabetes, and other types of diabetes mellitus are compared (kharroubi et al., 2015). gene therapy is a new form of molecular medicine that will have a significant impact on human health in the next century (verma et al., 2000). visionary scientists had hypothesized almost five decades ago that genetic modification by exogenous dna could be an effective treatment for inherited human diseases. this strategy of “gene therapy” offered the theoretical advantage that one single treatment would achieve a lasting and possibly curative clinical benefit (dunbar et al., 2018). recent gene therapy clinical trials have shown considerable therapeutic benefits and an excellent safety record. they show the long-sought-after potential of gene therapy to provide 'cures' after certain potentially terminal or seriously disabled disorders (naldini, 2015). to date, approximately 2600 clinical trials on gene therapy have been completed, are underway, or licensed worldwide (ginn et al., 2018). gene therapy is intended to inject genetic material into patient cells to compensate for damaged genes or to include therapeutic transgenes. gene therapy has advanced from the initial human gene transfer trials to approved clinical therapies over the past three decades (wang et al., 2020). modern therapeutic strategies seek to restore the endogenous insulin production rather than traditional insulin injection treatment (memon and abdelalim 2020). differentiation of insulin-producing pancreatic β cells from induced pluripotent stem cells (ipscs) originating from diabetes patients aims to provide autologous cells for cell replacement therapy for diabetes. present methods, however, generate patient ipsc-derived β (sc-β) cells with low in vitro and in vivo function (maxwell et al., 2020). in several ways, cell replacement therapy for type 1 diabetes (t1d) is a perfect first drug model to follow in the emerging field of regenerative medicine (brandon et al., 2020). history of gene therapy the ideas of gene therapy emerged in the 1960s and early 1970s, while in 1968 rogers & pfuderer demonstrated a proof of concept for virus-mediated gene transfer (wirth et al., 2013), development of genetically https://doi.org/10.14421/biomedich.2020.92.57-64 58 biology, medicine, & natural product chemistry 9 (2), 2020: 57-64 marked cell lines and explanation of the polyoma and sv40 papovavirus mechanisms of cell transformation (friedmann, 1992). modern transfection methods were combined with cultured cell selection systems and recombinant dna technology in the late 1970s.the possibility of successful gene transfer into mammalian cells for gene therapy purposes was generally recognized with the creation of retroviral vectors in the early 1980s (wolff et al., 1994). however, clinical efficacy in a small-scale clinical trial curing an otherwise fatal immunodeficiency disorder in children could not be demonstrated until the start of the new century (escors et al., 2010). pancreatic transplantation, first performed in 1966, exists in the new millennium as a radical treatment for particularly intractable type i diabetes with advanced complications. gene therapy with molecules like leptin and insulin, still in experimental mode, may one day become a reality (lakhtakia, 2013). clinically active islet-free implants began in 1989 which require immunosuppression for long life. for more than 30 years, several encapsulated islet approaches have been ongoing without the definition of a clinically relevant product (scharp et al., 2014). approaches for gene therapy in type 1 of diabetes a cure for type 1 (insulin-dependent) diabetes could be found to replace β cells in the generation of surrogate insulin-producing cells (samson and chan, 2006). several different approaches have long been used to seek a cure for diabetes, including islet transplantation, β cell regeneration, and insulin gene therapy (lee et al., 2000). islet transplantation the idea of t1d cell replacement therapy remained dormant for 80 years until 1972 when ballinger and lacy reversed chemical diabetes by islet transplantation in rats (shapiro, 2012). following the success of steroidfree immunosuppression transplantation of fresh human islets (froud et al., 2005). the inexorable decline in insulin independence following islet transplantation alone (ita) has raised concerns regarding its clinical utility (bellin et al., 2012). allotransplantation of islets into patients with autoimmune type 1 diabetes is a reexposure to autoantigen (bosi et al., 2001). although immunosuppression protocols have improved islet transplant outcomes, more research is required to maximize islet availability for transplantation and improve the viability of islets once transplanted (zarinsefat and stock, 2020). islet transplantation in patients with type 1 diabetes can reduce or eliminate the insulin requirement. exenatide is a long-acting glucagon-like peptide-1 (glp-1) analog that increases glucose-induced insulin secretion and may increase the mass of β cells (al et al., 2007). glucagon responses of islet transplant recipients to hypoglycemia were significantly lower than those observed in control subjects (incremental glucagon [mean ± se]: −12 ± 12 vs. 64 ± 22 pg/ml, respectively; p < 0,05), and not significantly different from those of non-transplant type 1 diabetic subjects (−17 ± 10 pg/ml) (paty et al., 2002). a glucose-potentiated arginine test, conducted only in insulin-independent transplant subjects (n = 5), showed significant impairments in the glucose-potential slope (p < 0.05) and the maximum response to arginine (armax; p < 0.05), a measure of βcell secretory capacity. because armax provides an estimate of the functional β-cell mass, these results suggest that the functional defects observed after islet transplantation may account for a low engrafted β-cell mass (rickels et al., 2005). moreover, approximately 70 percent of transplanted type 1 diabetic patients achieved insulin independence (matsumoto, 2010). by contrast, a clinical review at the bmj in 2001 anticipated that transplantation of langerhans islets would be the treatment of choice for most type 1 patients diabetes by 2010. islet transplantation is currently an option for a specific group of patients with type 1 diabetes only — those with severe glycaemic lability, recurrent hypoglycemia, and unawareness of hypoglycemia (de et al., 2011). iak was associated with better glucose control and a slower decrease in egfr than standard insulin therapy in patients with type 1 diabetes and a functioning kidney transplant (maanaoui et al., 2020). in this first doubleblind randomized trial, the islet transplant evidence obtained with reparixin does not support the role of cxcr1/2 inhibition in preventing inflammationmediated damage to the islet (maffi et al., 2020). regeneration of β cells one goal of regenerative medicine is the instructive incorporation of adult cells into other forms of cells for tissue repair and regeneration (zhou et al., 2008). two major efforts are underway to address the β-cell diabetes deficit: one would produce ex vivo β-cells that are appropriate for transplantation, and the other would promote β-cell regeneration in the pancreas (bonner et al., 2005). the regeneration of pancreatic β cells which produce insulin is a key therapeutic strategy for diabetes (ackeifi et al., 2020). through adulthood, β cell mass increasing the ability to suit peripheral requirements. insufficient insulin secretion by β cells leads to mellitus diabetes (teta et al., 2017). pancreatic insulin-producing β-cells have a long lifespan, so they replicate very little during a lifetime in healthy conditions (thorel et al., 2010). regeneration of pancreatic β-cell mass following either toxinor autoimmune-mediated destruction in young rodents is probable, but the degree of recovery declines with age and is incomplete in adult life. similarly, in children and adolescents with type 1 diabetes, there is histological evidence of islet cell smail – the role of gene therapy in the treatments of … 59 neogenesis and regenerative response (thyssen et al., 2006). single-cell rna sequencing (scrna-seq) of islets identified β-cell dedifferentiation and dysfunction markers and pathways (sachs et al., 2020). three regeneration modes were established according to the various cellular regeneration origins. in the first mode, the undifferentiated progenitor cells proliferate and differentiate as a self-renewing source in response to injury to repair the lost cell population. the second mode, de-differentiate, and then proliferate the highly defined remaining cells (wang et al., 2020). regeneration of lost or dysfunctional islet β cells in vivo can fulfill the promise of improved treatment for patients with diabetes. numerous mitogenic factors have been attempted to achieve this, including gammaaminobutyric acid (gaba) (yi et al., 2020). results indicate that curcumin has anti-diabetic properties because of its superior immunomodulatory behavior on t-associated cytokines and immunosuppressive activity on pro-inflammatory cytokines may enhance damage to pancreatic β cells (badr et al., 2020). proliferation is the primary mechanism of b-cell expansion during the early postnatal period to produce sufficient b-cell mass in an organism. however, the proliferation of b cells declines rapidly in early life, and the rate of division of b cells in adults is very low (guney et al., 2020). however, there is still a great need for new treatments, as tid remains an incurable illness. natural products, primarily phytochemicals, are a tremendous source of drug lead discovery for diabetes (apaya et al., 2020). nevertheless, conventional cell-based research approaches have weaknesses in identifying the precise processes of β-cell differentiation and transdifferentiation, as well as the related regulatory mechanisms (yu and xu, 2020). data cumulated over the past few years suggest that increased β-cell dedifferentiation plays a crucial role in diabetes progression, shedding new light on potential targets for β-cell replacement therapy (zhang et al., 2020). based on a new understanding of structural and functional dynamics of the bone marrow, a conditioning-free bone marrow transplantation (bmt) with reduced adverse effects opens the possibility of evaluating β cell regeneration and euglycemic restoration by inducing allogeneic chimerism in t1dm patients, as shown in a mouse model (black and zorina, 2020). a new study now shows that a pharmacological combination of insulin and glp-1–estrogen conjugate not only decreases the daily requirements for insulin but also improves β-cell function (de et al., 2020). lif receptor expression is limited to a subset of transcriptionally isolated human β cells with the enhanced proliferative ability (rosado et al., 2020). residual β-cells play a major role in the design of clinical trials: they may not only respond to combination therapies that involve metabolic function stimulants but are also the possible source of new β-cells (akirav et al., 2018). insulin gene therapy insulin treatment options for type 1 and type 2 diabetics have increased since the launch of the insulin analogs in 1996. insulin therapies are now able to mimic the physiological insulin secretion more closely and thus achieve better glycemic control in diabetes patients (donner et al., 2019). gene therapy is one of the diabetes mellitus treatment methods (ramezani et al., 2019). the two key issues are efficacy and safety in developing insulin-replacement gene therapy as a possible treatment for type 1 diabetes. in addition to using a safe and efficient gene transfer vector, the physiological regulation of the expression of the insulin gene is important (chen et al., 2011). insulin gene therapy refers to the targeted expression of insulin in non-β cells, the primary therapeutic target being hepatocytes (handorf et al., 2015). the human insulin gene wrapped in chitosan nanoparticles can be successfully transfected through the gastrointestinal tract into rats (niu et al., 2008). differentiation between adsc and tissue-specific promoters will improve the therapeutic gene expression. the use of microcarriers can facilitate posttransplantation cell survival and hold potential for longterm cell therapy (fang et al., 2019). insulin-producing cell replacement through transplantation shows significant promise but is limited in application due to supply constraints (cadaver-based) and chronic immunosuppression. important progress has been made over the past decade in tackling these obstacles to broad cell therapy adoption (latres et al., 2019). the possibility that the risk variants of bach2 and clec16a could contribute to the development of insulin-triggered type 1 diabetes cannot be excluded in addition to the type 1 diabetes high-risk human leukocyte antigen class ii and the class i allele of the insulin gene variable number tandem repeat genotype (onuma et al., 2019). therapeutic studies assess the efficacy of antigenspecific and antigen-nonspecific immune therapies, which also include reconstruction of the damaged betacell mass via islet transplantation, neogenesis, and regeneration, and their combinations (van et al., 2011). cells that express insulin and have molecular characteristics that closely resemble bona fide insulinsecreting cells have been produced by the most effective protocols to date; however, these cells are often nonresponsive to glucose, a feature that should be addressed in future protocols (aguayo et al., 2010). such beneficial effects of leptin are not underpinned by pancreatic β-cell regeneration, since circulating insulin levels were undetectable at basal and glucose overload levels (fujikawa et al., 2010). however, contrary to insulin monotherapy, leptin reduces both lipogenic and cholesterologenic transcription factors and enzymes, and reduces lipids from plasma and tissue (wang et al., 2010). 60 biology, medicine, & natural product chemistry 9 (2), 2020: 57-64 prevent of type 1 of diabetes by gene therapy since no existing targeted immunotherapies are currently unable to replace the normal insulin delivery, it is important to have a deep understanding of t1d pathophysiology to prevent t1d development (kaur, 2020). the study shows that aat gene therapy decreases cell-mediated autoimmunity, changes the repertoire of t-cell receptors and effectively prevents type 1 diabetes in the nod mouse model. these findings strongly suggest that aat gene therapy mediated by raav1 may be useful as a novel approach to preventing type 1 diabetes (lu et al., 2006). the data indicate that expression of defensive mhc class ii alleles in bone marrow-derived cells provides robust self-tolerance to islet autoantigens and is adequate to prevent recurrence of autoimmune diabetes following islet transplantation (tian et al., 2007). dual activity of heparanase inhibitors / hs replacers as a novel therapeutic class to prevent t1d progression and potentially to mitigate secondary vascular disease developed with long-term t1d (simeonovic et al., 2020). an independent study showed that the optimal rise of gcs in beta-cells was a compensatory mechanism that prevented high-fat diet-induced betacell failure (cobo et al., 2020). the role of combination therapy in the prevention of diabetic cardiomyopathy, without forgetting the major contribution of insulin, the preferred medication for the treatment of t1dm (cieluch et al., 2020). in several chronic diseases, including t1d, a large number of proinflammatory ligands that can signal via rage have been implicated. it is therefore unbelievable that rage has become a possible therapeutic target for disease treatment and prevention (le et al., 2020). as an antigen-specific monotherapy, adi-100 is highly e cacious in reversing elevated hyperglycemia to prevent diabetes, where a promising feature of immune tolerance is the increased apoptosis-inducing bax content (alleva et al. ,2020). it has been shown that the formulated autoantigenic islet peptides (gad65206-220, gad65536-550, insulin b923, and c17-a1) used as a tolerogenic vaccine prevented t1d from developing in prediabetic nod mice (zhou et al., 2020). the coxsackievirus b association with the onset of t1dm has been observed. accurate trigger detection will contribute to the creation of suitable preventive steps. it can become a foundation for advanced clinical studies to prevent t1dm (desai et al., 2020). recognizing that t1d is primarily an autoimmune betacell disorder that only in its final stage progresses to a metabolic syndrome will expand therapeutic options for earlier intervention and improve the prospects for prevention (desai n et al., 2020). gene expression with immuno-regulatory capacity may potentially decrease allograft rejection. recent studies have shown that viral interleukin (il)-10 can decrease immune response during allotransplantation and is one of the most promising methods for preventing rejection (jeong et al., 2020). vectors for gene therapy in diabetic virus vectors were used as gene transfer vehicles for various applications in preclinical and clinical gene therapy and with the approval of glybera (alipogene tiparvovec) as the first gene therapy product as standard medical treatment (goins et al., 2020). the use of viruses as carriers of genetic products requires a detailed and thorough understanding of viral vectors from viral preparation to clinical use (afzal et al., 2020). adenoviral vectors as one of the most common groups used in gene therapy have a high human capacity. they 're not actually integrated into the host genome (arjmand et al., 2020). in a cerebellar granule cell culture (cgc), treatment with gad cdna-containing hsv vectors increases gad65 and gad67 expression and gaba release evoked by stimulation (kanao et al., 2020). the definition of ad, aav and lv (shirley et al., 2020) is given on the other hand to competent (rc) viral vectors (de et al., 2020). the advantage of adenoviral vectors over retroviral vectors is that they can transduce both dividing and non-dividing cells and can be prepared in high titers. adenoviruses can infect insulin-secreting cells, and it has been shown that they can transduce rodent islets (borkar et al., 2020). co-precipitation of calcium phosphate is a simple and cheap method for the genetically modified pancreatic cells being non-viral vectors (wong et al., 2010). nearly all of the non-viral vectors used to date are plasmids of expression that were engineered for high expression when transmitted to striated muscle or other cells (prud et al., 2007). non-viral hvj liposomemediated human hgf gene transfer has potential for safe and successful diabetic sensorimotor neuropathy treatment (kato et al., 2005). future gene therapy in diabetes: in the future, therapeutic strategies should be personalized, given the high variance in genetics and islet autoimmunity among patients (liu et al., 2020). the recent increase in global stem cell research funding is largely based on the promise to translate scientific understanding of stem cells into regenerative medicine (wainwright et al., 2006). as the approaches to immunotherapy have remained unsuccessful, transplantation of donor-derived pancreas or islets is the only cure for t1d (aghazadeh et al., 2017). the urgent need for a much anticipated insulin-secreting substitute for β-cells led researchers to focus on stem cells (scs) to produce insulin-secreting β-cells. sc-based methods have opened up the new horizons to treat t1dm for being more precise and focused therapeutic approaches (farooq et al., 2019). replacing pancreatic islets with corpse-derived islets has proven to be a successful functional cure for some t1d patients which allows smail – the role of gene therapy in the treatments of … 61 them to be independent of exogenous insulin (sluch et al., 2019). there was an explosion of interest in developing methods for transplantation to replace the islets lost during natural diabetes development (emerich, 2002). clinically appropriate and healthy, the procedure is associated with a low risk of adverse effects (ahrén, 2011). clinical trials to improve the engraving, the availability of insulin-producing cell sources and alternative transplant sites are currently under investigation to expand treatment (bruni et al., 2014). glucagon-like peptide 1(glp-1) analogs and alternative insulin pathways, particularly oral (entericgastrointestinal, inhaled) pathways, are now most promising and attractive (takei et al., 2004). conclusions 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cyclosporin a induces ptreg and prevents type 1 diabetes in murine model. human vaccines & immunotherapeutics, 16(2), pp.240-250. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 89-109 | doi: 10.14421/biomedich.2022.112.89-109 issn 2540-9328 (online) the antiviral potential of iranian herbal pharmacopoeia (ihp) on herpes simplex viruses (hsv): a review article shahin gavanji young researchers and elite club, isfahan (khorasgan) branch, islamic azad university, isfahan, iran university blvd, arqavanieh, jey street, p.o. box:81595-158, isfahan, iran. corresponding author gavanji.shahin2@gmail.com manuscript received: 18 june, 2022. revision accepted: 19 july, 2022. published: 30 july, 2022. abstract herpes simplex viruses (hsv) viruses are highly contagious that commonly cause dermatitis, encephalitis, meningitis and genitourinary infections and also can lead to cervical cancer. for treatment of hsv infections, several physical methods and antiviral drugs are introduced, antiviral medications can also prevent or reduce outbreaks. the use of herbal medicine with antiviral effects attracted worldwide attentions. the aim of this review article is to introduce the iranian herbal pharmacopoeia (ihp) with antiviral potential against two hsv serotypes and help to evaluate and develop the new drugs from natural sources. to provide this review, relevant articles in some authentic databases including pubmed, web of science, science direct, scopus, google scholar and sid (scientific information database), from 1967 to april 2022 were collected, and selected botanicals from (ihp) with their scientific names and classifications and their outcome (cc50 and ic50) were introduced. in this review, scientific data regarding anti-herpetic activities of iranian herbal pharmacopoeia (ihp) showed that 34 herbs from 17 families have antiviral potential to inhibit two hsv serotypes. according to families, lamiaceae family has the highest percentage (29.41 %) of plants with antiviral activity against two hsv serotypes, also review of recent data showed that salvia officinalis, melissa officinalis, securigera securidaca, hyssopus officinalis, quercus brantii, artemisia aucheri and curcuma longa have remarkable antiviral activity against two hsv serotypes. results of this review suggest that further research to identify and purify the bioactive compounds to determine the molecular mechanisms of action is needed. keywords: herpes simplex; herbal medicine; pharmacopoeia; antiviral; drugs. introduction herpes simplex virus (hsv) belongs to the herpesviridae family and alphaherpesvirinae subfamily (álvarez et al. 2020), is a global public health issue that can cause serious infection and classified into 2 serotypes: hsv-1 and hsv-2 (gavanji et al. 2015; pebody et al. 2004). the hsv genome consists of linear double-strand dna, that causes primary and recurrent lesions (reuven et al. 2003) hsv viruses are highly contagious that commonly cause dermatitis, encephalitis, meningitis and genitourinary infections and also can lead to cervical cancer (kłysik et al. 2020; elad et al. 2010). hsv-1 or oral herpes mainly occurs on the face, chin, lips and mouth area which infection with this serotype causes small blisters or cold sores and inflammation of oral and eye cells (asai and nakashima 2018; vaghela et al. 2021), ranging from mild to severe, that may develop and increase the risk of serious illness (farooq and shukla 2012; anderson et al. 2014). this type of virus usually transmitted via oral secretions or direct contact with cold sores and it can cause more severe complications such as conjunctivitis (koujah et al.2019), herpetic stromal keratitis (hsk) (stuart and keadle 2012), gingivostomatitis (george and anil 2014) and hsv-1 encephalitis (hse) (feola et al. 2018). hsv-2, infection is usually transmitted through sexual contacts (zhang et al. 2022) and affects the genital area and increased risk of human immunodeficiency virus (hiv) transmission (crisci et al. 2019). for treatment of hsv infections, several physical methods and antiviral drugs such as valacyclovir, acyclovir, penciclovir, cidofovirare, and famciclovir are introduced (sadowski et al. 2021). the antiherpetic agents target and inactive viral dna polymerase enzyme and inhibit the proliferation and viral replication (li et al 2019). acyclovir (acv) is an antiviral agent which widely used to control and treatment of hsv infections (wei et al. 2021). in the process of drug action, the acv converts to acyclovir triphosphate (acyclo-gtp) and inhibits viral dna polymerase (fyfe et al. 1978). nowadays, due to the resistance mutations and tolerant enzyme production, especially in people with immunodeficiency disorders, urgent need to investigate the new antiviral drugs has been highlighted (roy et al. 2022; majewska and mlynarczyk-bonikowska 2022; chuerduangphui et al. 2022). numerous research studies have been done to investigate the novel antiviral agents https://doi.org/10.14421/biomedich.2022.112.89-109 90 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 (jassim and naji 2003), and many anti-herpetic drugs have been approved (sadowski et al. 2021), also researchers have been contributing to develop and produce a new vaccine against herpes infection but, no preventive hsv vaccines have been approved (krishnan and stuart 2021). the use of herbal medicine with antiviral effects attracted worldwide attentions (gavanji et al. 2014). scientific research has shown that natural bioactive compounds like flavonoids, therpens, phenols and alkaloids have anti-hsv properties (pesola and coen 2008; tolo 2006). among the compounds mentioned, phenolic classes are the most commonly used for their anti-hsv properties (treml et al. 2020) (table1). this article is a review which provide various information about the potential antiviral activity of iranian herbal pharmacopoeia (ihp) on herpes simplex viruses (hsv), and help to evaluate and develop the new drugs from natural sources. methods to provide this review, all reported herbal medicine with antiviral effects against herpes simplex viruses (hsv) were collected through iranian herbal pharmacopoeia (ihp) (ghasemi dehkordi et al. 2003) and the most relevant articles in some authentic databases including pubmed, web of science, science direct, scopus, google scholar and sid (scientific information database), from 1967 to april 2022 were searched. the search with different combinations of keywords were herbal medicine, herpes simplex viruses, antiviral agents, simplex virus, plants, iran, herb. in this review, the selected articles and books were used to sets of the general and specific criteria to examine and present the medicinal plants with antiviral activities. selected botanicals with their scientific names and classifications and their outcome (cc50 and ic50) are presented in the article. antiviral activity of phytochemicals against hsvs todays, various compounds with antiviral activity are contributing in the most of the medicinal process for the cure of human viral infections (singh et al. 2021; mukhtar et al. 2008). many studies have shown that naturally produced compounds with antiviral activity against two serotypes of hsv are polysaccharides (jin et al. 2015), flavonoids (flores et al. 2016), terpenes (soares et al. 2007), steroids (da rosa guimarães et al. 2013), saponins (ogawa et al. 2021), tannins (lin et al. 2011), phenolic (hassan et al. 2011), alkaloids (chen et al 2015), lignans (chen et al 2015), miscellaneous (treml et al. 2020), proanthocyanidin (terlizzi et al. 2016), quinones (caruso et al. 2020) and thiosulfinates (rouf et al 2020). several antiviral mechanisms of these phytochemicals have been identified, which most of them were verified against rna and dna viruses (jang et al. 2021; zrig 2022), such as herpes simplex virus type 1 and type 2 (dna viruses) (liu et al. 2019). these compounds inhibit the specific processes in the viral replication cycle (hassan et al. 2018), and viral entry to the target cells (rouf et al 2020), also they inhibit the viral gene and protein expression and suppress the nfkb activity (hutterer et al. 2017) that can help to prevent the spread of viruses. several studies have shown the antiviral mechanisms of these natural compounds against two hsv serotypes which are summarized in table 1. antiviral potential of iranian herbal pharmacopoeia (ihp) the present review focuses on selected iranian herbal pharmacopoeia (ihp) with antiviral potential against two hsv serotypes. in this review, the articles were read and 34 herbs from 17 families were selected based on anti-herpetic activity and percentages of each families were calculated. according to families in ihp, lamiaceae has the highest percentage (29.41 %) of plants with antiviral activity against two serotypes of hsv (figure 1). figure 1. the percentages of plant families against two serotypes of hsv. gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 91 table 1. anti-herpetic activity of natural compounds. references serotype mechanisms compositions chemical groups no (kutluay et al. 2008; zalilawati et al. 2015; zandi et al. 2010; flores et al. 2016) hsv-1/hsv-2 affecting the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication, inhibition of hsv-1 and hsv-2 replication and adsorption curcumin flavonoid 1 (lyu et al. 2005) hsv-1/hsv-2 inhibition of viral adsorption galangin flavonoid 2 (lee et al 2017) hsv-1 inhibiting expression of glycoprotein d (gd) and infected cell protein 0 (icp0), and suppresses the tlr-3 quercetin flavonoid 3 (li et al 2017) hsv-1 blocking membrane fusion houttuynoid flavonoid 4 (de oliveira et al. 2013; isaacs et al. 2008; subramanian and geraghty 2007) hsv-1 inhibition of viral adsorption, binding to hsv glycoproteins epicatechin gallate flavonoid 5 (hung et al. 2015) inhibition of nf-κb activation isoquercitrin flavonoid 6 (hutterer et al. 2017; chen et al 2015) hsv-1/hsv-2 inhibition of viral replication and gene expression, reduction the nf-κb activation, and iκb-α degradation harmine alkaloid 7 (hassan et al. 2011) hsv-1 inhibition of hsv-1 dna polymerase psoromic acid phenolic 8 (li et al 2005; hao 2019) hsv-1 inhibition of viral replication protocatechuyl aldehyde phenolic 9 (ma et al. 2016) hsv-1 inhibition of hsv-1 adsorption and reduction of (ie) gene expression and viral dna synthesis kuwanon x phenolic 10 (lin et al. 2011) hsv-1 blocking the interactions of virus and target cells chebulagic acid (chla) tannins 11 (lin et al. 2011) hsv-1 blocking the interactions of virus and target cells punicalagin (pug) tannins 12 (kesharwani et al. 2017) hsv-2 inhibition of viral attachment to the target cells chebulinic acid ( tannins 13 (kuo et al. 2002) hsv-1 inhibition of viral replication samarangenin tannins 14 (soares et al. 2007) hsv-1 maybe target the hsv-1 replication epitaondiol diterpenes 15 (álvarez et al. 2015) hsv-1 inhibition of viral gene expression (e)-2-(2,4-hexadiynyliden) 1,6-dioxaspiro[4.5]dec-3-ene miscellaneous 16 (jin et al. 2015) hsv-1 blocking the viral entry to the target cells by inactivating the viral particles, inhibition of viral intracellular biosynthesis eucheuma gelatinae polysaccharide (egp) polysaccharide 17 (zhu et al. 2006) hsv-1 inhibition of hsv-1 replication and viral entry to the target cells sulfated polysaccharide polysaccharide 18 (bouhlal et al. 2011) hsv-1 inhibition of virus adsorption polysaccharide boergeseniella thuyoides polysaccharide 19 (saha et al. 2012) hsv-1 inhibition of viral attachment to the target cells by interaction with virus particles alginic acids and sulfated polysaccharide 20 (zheng and lu 1990) hsv-1 inhibition of viral replication mangiferin polyphenol 21 (astani et al 2014; chen et al 2017) hsv-1/hsv-2 inhibition of viral replication and viral attachment to the target cells rosmarinic acid polyphenol 22 (kuo et al 2006) hsv-1 inhibiting expression of infected-cell polypeptide 4 (icp4) and infected cell protein 0 (icp0), and viral dna synthesis yatein polyphenol 23 (hassan et al. 2018) hsv-2 inhibition of viral replication geraniol monoterpenoid 24 (wiart et al. 2005) hsv-1 inhibiting expression of glycoprotein d andrographolide diterpenoid 25 (da rosa guimarães et al. 2013) hsv-1 inhibition of viral attachment to the target cells halistanol sulfate steroids 26 (rouf et al 2020) inhibition of viral entry to the target cells allicin organosulfur 27 (ikeda et al. 2005) hsv-1 inhibition of viral replication glycyrrhizic acid methyl ester triterpenoid 28 (tshilanda et al. 2020; bag et al. 2012) hsv-1/hsv-2 inhibition of viral replication [78,79] ursolic acid triterpenoid 29 92 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 apiaceae apiaceae family which is called umbelliferae, includes a large number of medicinal plants with various therapeutic properties has a significant role in pharmaceutical development (amiri and joharchi 2016; ekiert 2000). cuminum cyminum belonging to family apiaceae is an aromatic plant that can be used as medicinal herbs to treat various diseases (sowbhagya 2013; mnif and aifa 2015; gavanji et al. 2015) and flavoring agents to improve the flavor of foods (johri 2011). researchers have reported that methanol extract of c. cyminum showed the antiviral effects against hsv1 (table 2). based on this study, the cc50, ic50 values were 0.45 and 0.18 mg/ml, respectively (motamedifar et al. 2010). the molecular mechanism of c. cyminum against herpes simplex virus has not yet been characterized, but the polyphenolic compounds in the extract may inhibit the hsv virus (ani et al. 2006). asteraceae the asteraceae family, or sunflower family, including several thousand plants, has a long history of use in traditional herbal medicine (thm), for the treatment of diseases (amiri and joharchi 2016; ekiert 2000). artemisia aucheri belonging to family asteraceae is an aromatic and endemic plant in iran, which is widely used for treatment of various diseases (gavanji et al. 2014). this plant has been reported for its antiviral activities (kshirsagar and rao 2021), and karamoddini et al assessed the antiviral properties of artemisia species against hsv-1 which artemisia annua inhibited this serotype at different concentrations (karamoddini et al. 2011). another study indicated that aqueous extract of a. aucheri had anti-herpetic property on hsv-1 and reduced the expression of ul46 and us6 genes, and the ic50 of this extract, 24.7 μg/ml was determined (zamanian et al. 2021). in the other study the antiherpetic activity of aqueous extract of a. aucheri against hsv-1 was evaluated that viral infection significantly reduced at 50 and 75 μg/ml (zamanian et al. 2021). research in 2015 showed that phenolic compounds in artemisia has antiherpetic activity which can cause some abnormalities in the function and structure of herpes simplex virus (gavanji et al. 2015). arctium lappa is another member of asteraceae family which possesses various therapeutic potential to treat infectious diseases (bai et al. 2016), in addition a. lappa exhibited, anti-inflammatory (pirvu et al. 2017), anticancer (leonard et al. 2006) and antiviral effects against two serotypes of hsv (chan et al. 201; dias et al. 2017). a study has been reported, that 400 mg/ml of hydroalcoholic extract of a. lappa inhibited the hsv-1 in in vitro condition (dias et al. 2017) (table 2). echinacea purpurea a species of asteraceae family is widely used for the treatment of diseases (shemluck 1982), such as chest & lung conditions, colds, coughs, candidiasis and influenza (hudson et al. 2005). furthermore, e. purpurea exhibited significant antiviral activity against hsv-1 (thompson 1998). also, garcia et al. assessed the antiherpetic activity of e. purpurea against hsv-1 which the ic50 and cc50 values, were determined to be 500 and 900 μg/ml, respectively (farahani 2013). according to a study, chicoric acid exhibited antiviral properties against hsv-1 (binns et al. 2002). another in vivo study showed that e. purpurea polysaccharide (ep), antiviral effects on the development of hsv by promoting the immune response (ghaemi et al. 2009). another important species of asteraceae family is tanacetum parthenium which has been traditionally used to treat various diseases (ghaemi et al. 2009). benassi-zanqueta et al. assessed the antiviral efficacy of chlorogenic acids and parthenolide which derived from t. parthenium against hsv-1 that results showed that chlorogenic acids was effective against hsv-1 (benassi-zanqueta et al. 2019). base on this research, the hydroethanolic extract inhibited viral replication and the ec50 value was18.1 mg/ml. avicenniaceae avicenna marina is a member of avicenniaceae family, which traditionally used for treatment of small pox, rheumatism and respiratory problems (afzal et al. 2011; namazi et al 2013). and has strong antiviral activity against hsv-1 (chiang et al. 2003; namazi et al, 2013; behbahani et al. 2013). a study showed that glycerin extract of a. marina, inhibited the hsv-1 and the ic50 values before and after virus attachment and cc50 were determined to be 87.1, 41.9 and 5750.96 μg/ml, respectively (zandi et al. 2009). based on the previous studies, a. marina, contains many phytocompounds with antiviral potentials to inhibit herpes simplex viruses (hsv). several studies have shown that flavonoids in the a. marina extract plays a crucial role in antiviral activities against hsv-1 (chiang et al. 2003; namazi et al, 2013; behbahani et al. 2013). (table 2). gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 93 table 2. antiviral mechanism of iranian herbal pharmacopoeia (ihp) against herpes simplex viruses (hsv-1 and -2) no plant name family type of study (in vitro or in vivo) type of virus compounds and mechanisms references 1 aloe vera xanthorrhoeacea e in vitro hsv-1/ hsv-2 aloe-emodin inhibits the replication of viral enveloped (lin et al. 2008; zandi et al. 2007; rezazadeh et al. 2016) 2 artemisia aucheri asteraceae in vitro hsv-1 artemisinin inhibits the central regulatory processes and blocks the metabolic requirements of replication, reduces the ul46 and us6 genes (kshirsagar and rao 2021; karamoddini et al. 2011; zamanian et al. 2021a; zamanian et al. 2021b). 3 arctium lappa asteraceae in vitro hsv-1 arctigenin inhibits the viral replication, phenolic constituents such as caffeic acid and chlorogenic acid inhibit the viral multiplication (dias et al. 2017; wang et al. 2019; yang et al 2005; lal et al 2020; chiang et al. 2002; chan et al. 2011) 4 avicenna marina avicenniaceae in vitro hsv-1 luteolin inhibits the viral replication (chiang et al. 2003; namazi et al. 2013) 5 camellia sinensis theaceae in vitro hsv-1 epicatechin gallate inhibits the viral adsorption, binding to hsv glycoproteins (de oliveira et al. 2013; isaacs et al. 2008; subramanian and geraghty 2007) 6 cuminum cyminum apiaceae. in vitro hsv-1 ehp [1-(2-ethyl, 6-heptyl) phenol] effects on the percentage of plaque (mohamadein et al. 2015; motamedifar et al. 2010) 7 curcuma longa zingiberaceae in vitro hsv-1/ hsv-2 affecting the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication, inhibition of hsv-1 and hsv-2 replication and adsorption (hutterer et al. 2017; kutluay et al. 2008; zalilawati et al. 2015; flores et al. 2016) 8 echium amoenum boraginaceae in vitro hsv-1 rosmarinic acid inhibits the viral attachment to host cells (flores et al. 2016; chen et al. 2017) 9 echinacea purpurea asteraceae in vitro/in vivo hsv-1 cichoric acid interact and inhibit the virus activities (binns et al. 2002; pluymers et al. 2000; burlou-nagy et al. 2022; zhang et al. 2014) 10 euphorbia spinidens euphorbiaceae in vitro hsv-1 betulin and (3β,23e)-cycloarta-23-ene-3,25-diol, the extract inhibits the viral replication and adsorption (shamsabadipour et al. 2013; karimi et al. 2016) 11 eucalyptus caesia myrtaceae in vitro hsv-1 unknown (schnitzler et al. 2001; brezáni et al. 2018; mierescastro et al. 2021) 12 eucalyptus globulus myrtaceae in vitro hsv-1 grandinol, sideroxylin, and tereticornate inhibit the viral replication (schnitzler et al. 2001; brezáni et al. 2018; mierescastro et al. 2021; ma and yao 2020; mohan et al. 2020) 13 glycyrrhiza glabra leguminosae in vitro hsv-1 glycyrrhizic acid (glycyrrhizin) inhibits the viral replication, suppress the growth (ghannad et al. 2014; fukuchi et al. 2016; van rossum et al. 1998; cohen 2005; huan et al. 2021) 14 hypericum perforatum hypericaceae in vitro/in vivo hsv-1/ hsv-2 hypericin inhibits the viral adsorption (huan et al. 2021; weber et al. 1994; mohamed et al. 2022; fritz et al. 2007; westh et al. 2004; béjaoui et al. 2017) 15 hyssopus officinalis lamiaceae in vitro hsv-1/ hsv-2 unknown (akram et al. 2018; behbahani 2009; schnitzler et al. 2019) 16 melissa officinalis lamiaceae in vitro hsv-1/ hsv-2 rosmarinic acid inhibits the viral attachment to host cells (astani et al 2014a; astani et al. 2012b; mazzanti et al. 2008) 17 mentha piperita lamiaceae in vitro hsv-1/ hsv-2 piperitenone oxide (peo) and menthol inhibit the viral replication and adsorption (wolbling et al. 1994; koytchev et al. 1999; civitelli et al. 2014; herrmann et al. 1967) 18 myrtus communis myrtaceae in vitro hsv-1 unknown (moradi et al. 2011; alipour et al. 2014; isaacs et al. 2008) 94 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 no plant name family type of study (in vitro or in vivo) type of virus compounds and mechanisms references 19 olea europaea oleaceae in vitro hsv-1 phenolic compounds such as caffeic acid inhibits the viral multiplication (motamedifar et al. 2015; ben-amor et al. 2021; ikeda et al. 2011) 20 ocimum basilicum lamiaceae in vitro hsv-1/ hsv-2 ursolic acid and apigenin inhibit the viral replication (chiang et al. 2005; bag et al. 2012; lin et al. 2008) 21 plantago major plantaginaceae in vitro hsv-1/ hsv-2 caffeic acid inhibits the viral multiplication (chiang et al. 2002; ben-amor et al. 2021; ikeda et al. 2011) 22 quercus persica fagaceae in vitro hsv-1 tannic acid inhibits viral replication (chiang et al. 2005; wu et al. 2022; karimi et al. 2013; kaczmarek 2020; nance and shearer 2013) 23 ricinus communis euphorbiaceae in vitro hsv-1 the alkaloid and phenolic compound have antiherpetic activity (elkousy et al. 2021; abdul et al. 1996) 24 rheum palmatum polygonaceae in vitro/in vivo hsv-1 unknown (abdul et al. 1996; kurokawa et al. 1993; chang et al. 2014; shen et al. 2019) 25 rosmarinus officinalis lamiaceae in vitro hsv-1/ hsv-2 rosmarinic acid inhibit of viral replication and viral attachment to the target cells (al-megrin et al. 2020; mancini et al. 2009; hitl et al. 2021; chen et al. 2017; astani et al 2014) 26 salvia officinalis lamiaceae in vitro/in vivo hsv-1/ hsv-2 thujone, β-caryophyllene linalyl acetate, alpha terpinyl acetate, and germacrene d have anti-herpetic activity (shen et al. 2019; al-megrin et al. 2020; mancini et al. 2009; hitl et al. 2009; schnitzler et al. 2008; rajbhandari et al. 2001; santoyo et al. 2014; ezema et al. 2022) 27 satureja hotensis lamiaceae in vitro hsv-1 unknown (hamidpour et al. 2014; khalil et al. 2020) 28 securigera securidaca leguminosae in vitro hsv-1 kaempferol and kaempferol-7-o-glucoside inhibit the hsv infection but the mechanism is unknown (behbahani et al. 2014; behbahani et al. 2013) 29 solanum paniculatum solanaceae in vitro hsv-1 unknown (valadares et al. 2009; kaunda and zhang 2019) 30 tanacetum parthenium asteraceae in vitro/in vivo hsv-1 parthenolide did not directly act against hsv, and it can able to handle the defense mechanisms in host cells against viral particles (benassi-zanqueta et al. 2019; benassi-zanqueta et al. 2018) 31 thymus vulgaris lamiaceae in vitro hsv-1/ hsv-2 thymol reduces the viral transmission (gavanji et al. 2015; catella et al. 2021; nolkemper et al. 2006; sharifi-rad et al. 2017; lai et al. 2012) 32 thymus kotschyanus lamiaceae in vitro hsv-1/ hsv-2 borneol, and isoborneol that inhibits the viral replication (farahani 2017; yang et al. 2020) 33 zataria multiflora lamiaceae in vitro hsv-1 rosmarinic acid inhibits the viral attachment to host cells (arabzadeh et al. 2020; ben-shabat et al. 2020; mardani et al.. 2012; astani et al 2014; chen et al 2017) 34 zingiber officinale zingiberaceae in vitro/in vivo hsv-1 unknown (schnitzler et al. 2012; camero et al. 2019; koch et al. 2008; hayati et al. 2021) gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 95 boraginaceae echium amoenum, commonly known as borage, belongs to the family boraginaceae (zannou et al. 2021), which has a long history in iranian traditional medicine (itm) for treatment of influenza and infectious diseases (ranjbar et al. 2006). this plant has antiviral properties against hsv-1, and the ic50 and cc50 values were determined to be 500 and 1000 μg/ml, respectively, also the results of this research showed that virus replication was inhibited at the lower concentration than 400 μg/ml (abolhassani 2010; farahani 2013). euphorbiaceae euphorbia spinidens, a member of euphorbiaceae family, is traditionally used as antiasthmatic, relieving stomachache, laxative and emetic (ghanadian et al. 2016). this plant contains several types of bioactive compounds which exhibited significant antimicrobial activities (li et al. 2009). furthermore, a research demonstrated that hexan fraction of e. spinidens showed the inhibitory activity against hsv-1, and the ic50 value of 9.92±0.072 mg/ml was determined (li et al. 2009). another study stated that the methanolic extract of e. spinidens was active against hsv-1 with ec50 and si values, of 0.34mg/ml and 14.9 respectively, and this research showed that e. spinidens can inhibit the viral replication and adsorption (karimi et al. 2016). ricinus communis is another member of euphorbiaceae family (abdul et al. 2018), which contains many phytochemicals and can be used to treat a wide range of diseases (scarpa and guerci 1982; ohishi et al. 2014). a study showed that ethanolic extracts of r. communis has potent antiviral activity against hsv-1 and inhibited this virus at concentration of 0.1 mg/ml (abdul. et al. 1996). fagaceae quercus persica, commonly known as oak, belonging to the family fagaceae, has been used in iranian traditional medicine(itm) for the treatment of various disease (karimian et al. 2020). a study demonstrated that hydroalchoholic extract of q. persica has anti-hsv activity. in this research the authors conclude that q. persica extract has inhibited the hsv‑1, and the ic50 values, before and after attachment to bhk, were determined to be 1.02 and 0.257 μg/ml, respectively (karimi et al. 2013). another study by karimi et al. stated that quercus brantii extract showed the inhibitory activity against hsv-1, and the ic50 and si values of 4.3 μg/ml and 48.4 were determined, respectively (karimi et al. 2017). hypericaceae hypericum perforatum, a member of hypericaceae family, is a medicinal plant which has been widely used as antidepressants, anti-cancer and psychotic disorders (klemow et al. 2011). a number of studies demonstrated that h. perforatum, has strong antiviral activity against numerous viruses, including radiationleukaemia virus (radlv), friend virus (fv), human immunodeficiency virus type 1 (hiv-1) and hsv-1 (weber et al.1994). also a scientific research showed that the complex of h. perforatum and lysine hydrochloride, remarkably inhibited the hsv‑1 and the ic50 value was from 6.8 to 9.7 mg/ml (hu et al. 2004). lamiaceae hyssopus officinalis belongs to the family lamiaceae, which is traditionally used for treatment of chest & lung conditions, coughs, colds and infectious diseases (vlase et al. 2014), also several studies demonstrated that h. officinalis extract has antifungal and antiviral activity (fathiazad et al. 2011). a study by behbahani, showed that methanolic extract of h. officinalis significantly inhibited two hsv serotypes, which the ec50 and cc50 values, against hsv‑1, were determined to be 4.1±0.40 and 960 μg/ml, respectively, and for hsv‑2, the ec50 and cc50 values were > 5.0 and 100 μg/ml, respectively (behbahani 2009). another study stated that essential oil of h. officinalis was active against hsv-1, and ec50 and cc50 values, were determined to be 0.0001±0.00001 and 0.0075±0.002 % respectively (schnitzler et al. 2007). another important species of lamiaceae family is melissa officinalis, which is a well-known and it has been used in traditional medicine for treatment of various diseases (miraj et al. 2017). furthermore, a research showed that m. officinalis has inhibitory activity against hsv‑1 and inhibited the viral attachment to the host cells, and the ic50 and si values of 0.4 μg/ml and 350 were determined, respectively (astani et al 2014). mentha piperita is antother important species of lamiaceae family which has therapeutic potential to treat different diseases (zaker et al. 2014; alves et al. 2012). schuhmacher et al., demonstrated that the essential oil of m. piperita, can able to inhibit the herpes simplex virus type 1, and reduce the plaque formation by up to 82% for hsv-1 (schuhmacher et al. 2003). another research stated that the m. piperita extract was effective against hsv-1 and inhibited the viral replication cycle, which ed50 and ti values were determined to be 62.70 mg/ml and 1.79, respectively (omidian et al. 2014). several studies exhibited that many species of lamiaceae family has potential to inhibit the two hsv serotypes. ocimum basilicum or great basil, a traditional medicinal plant, belongs to the family lamiaceae which is widely used for treatment of different diseases including, headaches, diabetes, nerve pain and anxiety (bora et al. 2011). a study revealed that water and ethanolic extracts of o. basilicum have inhibited two hsv serotypes, the result of this research showed that the ec50 and si values of water extracts against hsv-1, were determined to be 90.9 mg/ml and 16.2, also for hsv-2 were 51.4 mg/ml and 28.6 respectively. ethanolic extract showed inhibitory activity against hsv-1, and ec50 and si values of 108.3μg/ml and 6.3 were determined, respectively (chiang et al. 2005). another important 96 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 species of lamiaceae family is rosmarinus officinalis, which is widely used in treatment of rheumatic pain, headache, hysteria, stomachache, depression and infectious diseases (ghasemzadeh rahbardar et al. 2020). a study reported that extract of r. officinalis, exhibited potential antiviral activity against hsv-1 and hsv-2, which this extract at the concentration of 30 μg/ml inhibited the 55% of hsv-1 and at 40μg/ml inhibited the 65% of hsv-2 plaques and extract of r. officinalis showed the significant inhibitory effect at the 50 μg/ml concentration two hsv serotypes (al-megrin et al. 2020). another important species of lamiaceae family is salvia officinalis, that is used in traditional medicine to treat different kinds of diseases, such as rheumatism, diarrhea, ulcers, inflammation and paralysis. this plant contains several types of phytochemicals which exhibited significant antibacterial, antifungal and antiviral activities (ghorbani and esmaeilizadeh 2017). a study demonstrated that two diterpenoids compounds (safficinolide and sage one) which is isolated od aerial parts, have antiviral activity (smidling et al. 2008). a number of research studies demonstrated that s. officinalis has antiviral activity against two serotypes of hsv. the extract of s. officinalis was used in the study, against hsv-1, which the ic50 value of this extract, 199.0 μg/ml was determined (smidling et al. 2008), and in another studies ic50 value was 1.41–1.88 μg/ml and inhibited the plaque formation (santoyo et al. 2014). schnitzler et al. studied the antiviral activity of s. officinalis extract against two serotypes of hsv which ic50 values for hsv-1 and 2 were 0.18 and 0.04 μg/ml respectively (schnitzler et al. 2008). satureja hotensis is another important species of lamiaceae family which can use in treatment of many ailments and diseases. in this plant, various types of phytochemicals such as flavonoids steroids, triterpenoids, and and sesquiterpenoids, have been identified (gursoy et al. 2009; golestannejad et al. 2015), which are used in pharmaceutical industries (tepe and cilkiz 2016). a study showed that this plant has potential antiviral activity against hsv-1, which ic50 and cc50 values were determined to be 0.008% and 0.245%, respectively (gavanji et al. 2015). one of the important species of lamiaceae family is thymus vulgaris commonly known as thyme and is the rich sources of phytochemicals which are widely used for the treatment of inflammation, cancers, and infectious diseases (gavanji and larki 2017). a study demonstrated that the essential oil of t. vulgaris was effective against two serotypes of hsv and reduce the viral infectivity, which ic50 for 1,8-cineole, 1200 μg/ml was determined (gavanji and larki 2017). furthermore, a study revealed that aqueous extract of t. vulgaris has inhibitory effects against two serotypes of hsv, which ic50 and si values for hsv-1, were 0.065 mg/ml and 954, also for hsv-2, 0.077 mg/ml and 805 were determined, respectively (nolkemper et al. 2006). another member of lamiaceae family is thymus kotschyanus which has anti-viral activity and inhibited hsv-1 at the higher concentration of 400 μg/ml (farahani 2017). based on a study, t. kotschyanus contains many bioactive compounds such as borneol, has impressive antiviral potentials to inhibit herpes simplex viruses (armaka et al. 1999) (table 2). zataria multiflora is one of the most important species of lamiaceae family which is used to relieve some of illnesses such as fever, bone pain, flatulence, cough, cold and infectious diseases (ghorani et al. 2022; dadashi et al. 2016). a research study has been documented, reporting that methanolic extract of z. multiflora at the 1000 mg/ml concentration, remarkably reduce the plaque formation of hsv‑1 (arabzadeh et al. 2013). moreover, essential oil of z. multiflora exhibited antiviral potential against hsv‑1, in which ic50 and si values were determined to be 0.0059% and 11.7, respectively (mardani et al. 2012). another study revealed that essential oil of z. multiflora contains rosmarinic acid which is an antiviral compound and inhibited the viral attachment to host cells. this study demonstrated that z. multiflora oil, has strong antiviral activity which ic50 and cc50 values were determined to be 0.003% and 0.166%, respectively (gavanji et al. 2015). zingiber officinale or ginger is another important species of lamiaceae family which is widely used for treatment different diseases in traditional medicine (grzanna et al. 2005). z. officinale has broad-spectrum antiviral potential on human respiratory syncytial virus (hrsv) (chang et al. 2013), hepatitis c virus (hcv), influenza a (h1n1) (sahoo et al. 2016), and two serotypes of hsv [123,180]. the study has demonstrated that z. officinale inhibited the hsv-2 with ic50 and si values of 0.0001% and 40 respectively (allahverdiyev et al. 2013). another study by koch et al stated that z. officinale possess antiviral activity, and the ic50 value of 0.001% was determined (koch et al. 2008) (table 2). leguminosae securigera securidaca, a species of leguminosae family, is widely used as herbal for the treatment of several diseases in iranian traditional medicine (itm). s. securidaca is a rich source of flavonoids having significant antibacterial, antifungal and antiviral activities (raesi vanani et al. 2019). also the result of a research study showed that, two major compounds, including kaempferol and kaempferol-7-o-glucoside which is isolated from s. securidaca, can inhibit the hsv infection (behbahani et al. 2013). another study showed that methanolic extract of s. securidaca has inhibited the hsv‑2 with ic50 and cc50 values of 1.6 and 130 μg/ml, respectively (sayedipour et al. 2012). furthermore, s. securidaca exhibited significant antiviral activity against hsv-1 which ic50 and cc50 gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 97 values of 2 and 500 μg/ml, were determined, respectively (behbahani et al. 2013). glycyrrhiza glabra is another medicinal plant from leguminosae family which has been reported for healing gastroesophageal reflux disease, liver diseases, tuberculosis and infectious diseases (wahab et al. 2021). numerous antiviral phytochemicals, such as glycyrrhetinic acid, and glycyrrhizin were isolated from g. glabra which have antiviral activity against hsv-1 (ming and yin 2013; huan et al. 2021). based on the results of a research, g. glabra extract has inhibited the hsv‑1 with ic50 and cc50 values of 500 and 800 μg/ml, respectively (monavari et al. 2008). another study by fukuchi et al. stated that water extract of g. glabra with ec50 and si values of 650 to 740 mg/ml, and 2.0 to >4.6, respectively, showed a strong inhibitory activity against hsv-1, compared to alkaline extracts of g. glabra with ec50 and si values of 600 to >3000 mg/ml, and to 3.2, respectively (fukuchi et al. 2016). myrtaceae eucalyptus caesia and eucalyptus globulus are the two most common species of myrtaceae family which have numerous phytopharmacological potential to treat different kinds of diseases, such as asthma, pulmonary, cold, bronchitis, and infectious diseases (mierescastroet al. 2021). both of these species exhibited antiviral properties against hsv-1 and hsv-2 (mierescastroet al. 2021). a study demonstrated that essential oil of e. globulus inhibited two hsv serotypes which ic50 and si values for hsv-1, were determined to be 0.009% 3.3, respectively and for hsv-2, were 0.008% and 3.75, respectively (schnitzler et al. 2001). furthermore, a research demonstrated that 1,8-cineole reduced the hsv infection under in vivo condition (behbahani et al. 2013). another study researchers compared the effect of e. globulus oil and individual monoterpenes against hsv-1, which result of this study showed that e. globulus 1,8-cineole, α-pinene, γterpinene, p-cymene, α-terpineol and terpinen-4-ol, have antiviral activity against hsv-1 with ic50 of 55, 1.20, 4.5, 7.0, 16.0, 22.0, and 60.0 μg/ml, respectively (astani et al. 2010). another study revealed that essential oil of e. caesia inhibited two hsv-1 which ic50 and cc50 values, were determined to be 0.004% and 0.287%, respectively (gavanji et al. 2015). myrtus communis is another important member of myrtaceae family, which has been used in traditional medicine to treat many diseases such as hemorrhoid, recurrent aphthous stomatitis, diarrhea, and infectious diseases (mahboubi 2016; alipour et al. 2016). based on a study, the hydroalcoholic extract of m. communis, has impressive antiviral potentials to inhibit hsv-1, which ic50 and cc50 values, were determined to be 3100 and 4960 μg/ml, respectively (moradi et al. 2011). this herb showed anti-herpetic activity, under clinical trial condition, which the result of this study demonstrated that myrtle oil, reduced the signs and symptoms of disease in the treated group, in comparison to other control groups (zolfaghari et al. 1997). oleaceae olea europaea, a member of oleaceae family, can be used to treat a wide range of diseases in traditional medicine, such as diarrhea, hemorrhoids, infectious diseases, inflammation and rheumatism (alipour et al. 2016). o. europaea has antiviral activities against many types of viruses, including canine parvovirus (cpv), hepatitis virus, bovine rhinovirus (brav), herpes virus (hsv) and feline leukemia virus (felv) (ben-amor et al. 2021). a study demonstrated that hydroalcoholic extract of o. europaea var. sylvestris exhibited anti hsv-1 activity that ec50 and cc50 values, for preinfection assay, were determined to be 0.12 and 0.2 mg/ml, respectively and for post-infection assay were 0.15 and 0.2 mg/ml, respectively (ben-amor et al. 2021).another study by motamedifar et al. stated that hydroalcoholic extract of o. europaea with ic50 and cc50 values of 660 and 1750 μg/ml, respectively, showed a strong inhibitory activity against hsv-1 (motamedifar et al. 2015). plantaginaceae plantago major belongs to the family plantaginaceae, which is commonly known as greater plantain, and it has been traditionally used to treat many diseases such as fever, constipation and wounds and bleeding. furthermore, p. major has been reported to contain caffeic acid, that demonstrated the strongest antiviral activity against two serotypes of hsv (najafian et al. 2018). a study showed that aqueous extract of p. major has weak antiviral activity against hsv-1 and hsv-2, in extracts, pure compounds such has caffeic acid demonstrated the strongest antiviral activity against hsv-1. moreover, this study stated that aqueous extract of p. major inhibited hsv-2 with ec50 and si values, of 843 mg/ml and 2.2, respectively and caffeic acid isolated from p. major exhibited the antiviral activity against hsv-1 with ec50 and si values, of 15.3 μg/ml and 671, and hsv-2 ec50 and si values, were determined to be 87.3 μg/ml and 118, respectively (chiang et al. 2002; chiang 2003). polygonaceae rheum palmatum a flowering plant of polygonaceae family, which can be used to treat a wide range of diseases such as gasteroenteritic, herpes and kidney disease (chang et al. 2014). r. palmatum contains several types of natural compounds such as emodin, chrysophanol and aloe-emodin which exhibited significant anti-viral activities (li et al. 2007). a study has been documented that aloe-emodin isolated from r. palmatum has antiviral activity against hsv (types 1 and 2), influenza virus (sydiskis et al. 1991), human cytomegalovirus (hcmv) (barnard et al. 1992), and polio viruses (semple et al. 2001), also emodin and 98 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 chrysophanol have antiviral property against hepatitis b virus (hbv) (shuangsuo et al. 2006), hepatitis c virus (hcv) and human immunodeficiency viruses (hiv) (kubin et al. 2005). a study demonstrated that r. tanguticum nanoparticles suppressed the hsv-1, which ec50 and cc50 values, were determined to be 194.1 and 415.3 μg/ml, respectively (shen et al. 2019). solanaceae solanum paniculatum or jurubeba, a member of solanaceae family, which is widely used to treat several diseases including hypertension, anemia, inflammation and tuberculosis (tenório et al. 2016). a research study by valadares et al., showed that ethanolic extract of s. paniculatum exhibited antiviral property against hsv-1, which ec50 and si values, were determined to be 298 mg/ml and 1.4, respectively (valadares et al. 2009). theaceae camellia sinensis or green tea, is a medicinal plant of theaceae family which is used as the most consumed and favorable drink in the world. c. sinensis has many medicinal properties (singhal et al. 2017). based on a study, c. sinensis contains many bioactive compounds that has impressive antiviral potentials to inhibit herpes simplex viruses tape 1, with ic50 and si values, of 20 mg/ml and 50, respectively (farahani et al. 2014). another study, stated that aqueous extract of c. sinensis exhibited the antiviral activity against hsv-1, which ic50, cc50 and si values, were determined to be 50, 1000 μg/ml and 20, respectively (farahani et al. 2013) another scientific research reported that methanolic extracts of c. sinensis completely inhibited two hsv serotypes at 12 μg/ml concertation (deepika et al. 2014). xanthorrhoeaceae aloe vera, commonly known as aloe barbadensis, is a member of xanthorrhoeaceae family which has been traditionally used to treat various diseases such as abdominal pains, malaria, arthritis, fever, and skin diseases (adams et al. 2014). a. vera produces a wide range of phytochemicals, such as emodin and aloeemodin which exhibit potential strong antiviral activity against two serotypes of hsv, immunodeficiency virus (hiv), and influenza virus (brahimi et al. 2021). another an anthraquinone compound isolated from a. vera, is aloe-emodin which inhibits the viral replication of hsv serotypes and the ic50 value, of 1.5–6.0 μg/ml was determined (sydiskis et al. 1991). a study demonstrated that topical a. vera gel at 0.2 to 5% concentrations can inhibit hsv-i growth (rezazadeh et al. 2016). another study by ebrahimi et al. revealed that a. vera extract inhibited the hsv-1 with ic50, cc50 and si values, were determined to be 10000 ± 55, 20000 ± 94 μg/ml, and 2.0 respectively (brahimi et al. 2021). also the result of a study showed that hot glycerine extract of aloe vera can able to inhibit the hsv-2, which the ic50, cc50 and si values, were determined to be 428, 3238 μg/ml and 7.56, respectively (zandi et al. 2007). zingiberaceae curcuma longa belongs to the family zingiberaceae, which is used in iranian traditional medicine(itm) for healing rheumatism, anorexia, wounds, cough and respiratory diseases (trujillo et al. 2013). a research study showed that, 2 mg/ml of c. longa extract reduced the plaque formation of hsv-1 (fani et al. 2015). furthermore, a research demonstrated that c. longa has inhibitory effect against hsv-1, and the ic50, cc50 and si values were 33.0, 484.2 μg/ml and 14.6, respectively (lyu et al. 2005). another study revealed that c. longa extract contains polyphenolic compounds such as curcumin which affects the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication. this study stated that curcumin has significant has antiherpetic activity and inhibited hsv-2 with ed50 value, of 0.32 mg/ml (kutluay et al. 2008). zingiber officinale or ginger is another antiviral species of zingiberaceae family which exhibited antiviral activity against numerous viruses (wang et al. 2020). based on a study, the essential oil of z. officinale, has impressive antiviral potentials to inhibit hsv-1, whit ic50 value, of 0.001% (koch et al. 2008). conclusions since time immemorial, a human being has sought medications to relieve pain and remedy for various diseases. weighty evidence demonstrates the use of medicinal plants for therapeutic purposes and numerous research studies have been done to investigate the novel antiviral agents. in this review, all reported data of herbal medicines (34 herbs from 17 families) with antiviral activity against hsv through iranian herbal pharmacopoeia (ihp) were collected. in some cases, screening test of medicinal plants for anti-herpetic activity was done under in vitro condition and only a few of them were done under clinical trial condition. additionally, in many research studies, bioactive phytochemicals and mechanisms of actions, were not identified. results of this review suggest that further research to identify and purify the bioactive compounds to determine the molecular mechanisms of action are needed. acknowledgements: i would like to thank dr. forough mortezaienejad for guidance on this project. competing interests: the authors declare that there are no competing interests. gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 99 references abdul, a. m., mackeen, m. m., ei-sharkawy, s. h., abdul, h., junainah, n. h., ismail, n. h., ahmad, f., & lajis, m. 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(2020). advances in pharmacological activities of terpenoids. natural product communications, 15, 1934578x20903555. https://doi.org/10.1177%2f1934578x20903555 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 93-103 | doi: 10.14421/biomedich.2021.102.93-103 issn 2540-9328 (online) a physical chemistry study of black powder materials by solution combustion synthesis method fitria hidayanti1,*, kiki r. lestari1, nano sujani1, jarot raharjo2 1engineering physics department, universitas nasional, jakarta selatan 12520, indonesia. 2center for materials technology, agency for the assessment and application of technology puspiptek building no. 224, south tangerang, banten 15314, indonesia. corresponding author* fitriahidayanti@gmail.com manuscript received: 14 august 2021. revision accepted: 30 september, 2021. published: 08 october, 2021. abstract a study on the synthesis of black powder (la2nio4) material using the solution combustion synthesis method at a variation of synthesis temperature of 60, 70, and 80°c was carried out. it produces a mass of black powder of 2 grams by four times of synthesis process. then, material characterization was performed on the black powder samples obtained by using x-ray diffraction (xrd) to determine the phases formed, scanning electron microscopy energy dispersive x-ray spectroscopy (sem-eds) to determine the morphology and analyze the composition elemental on the microscale and fourier transform infra-red (ftir) to determine chemical bonds. from the whole black powder sample, xrd analysis showed the phases of dilantanum nickel tetraoxide (la2nio4), nickel oxide (nio), lanthanum oxide (la2o3), and lanthanum oxide ht x-form (la2o3 ht (x-form)). in addition, it was seen from the visible compositions of the phases that the nio phase looks more dominant and the variation of the synthesis temperature shows that the la2o3 phase was increasing. this was supported by the eds analysis, which showed that the eds spectrum contains elements la, ni, and o where the element o indicates that oxidation occurs in the elements ni and la. on the other hand, the sem analysis results confirm that the black powder sample contains the elements la and ni, based on the high and low electron images contained in the morphology of the black powder sample. in addition, it was also known that the particles in the black powder sample were micron size and had porous morphology. this occurs due to rapid thermal decomposition events and excessive gas development. in addition, ftir analysis showed that the o-h bond had been reduced and there are still c-o and c-h bonds indicating the presence of organic elements possessed by glycine. keywords: la2nio4, solution combustion synthesis; battery nimh; hydrogen storage alloy. introduction the development of electronic technology and electric cars can not be separated from the role of energy sources as a power to use these technologies. one source of energy booster is the battery. a battery is a device that has a working principle to convert chemical energy into electrical energy through electrochemical processes. one fairly rapid development occurred in the type of rechargeable batteries. a rechargeable battery is a type of battery that can be recharged again with the charge & discharge system. similar to the type of primary battery, batteries generally consist of 3 main components, namely the anode, cathode, and electrolyte. there are very rapid development in nickel metal hydride (nimh) batteries (chang et al., 2016, young et al., 2017). highpower international inc., which is a clean energy supplier company integrated with research and development, manufacturing and sales of nimh and li-ion rechargeable batteries, as well as energy storage and battery recycling systems, explains that the majority of the 74% nimh battery consumer market share in china, followed by japan as 14% and europe as 10%, and 2% are the others. in addition, european countries still have a large market level for the consumption of nimh batteries. for example, in 2013, germany, the netherlands, belgium, and the uk accounted for 8.3% of nimh battery needs from china, and in february 2014, north america received 51.76% of nimh battery needs, followed by europe receiving 32.7%, and 13.73% (chang et al., 2016). the data shows that the market share of nimh batteries is still growing, and indonesia may still be able to develop nimh batteries given the abundant natural resources for raw materials for nimh batteries. indonesia is a country that has the second-largest wealth of nickel in the world. nickel ore is contained in nickel oxides or laterites, which are spread in eastern indonesia (prasetiyo, 2008). based on data from the geological resource center in 2016, indonesia has nickel resource potential of 5,756,362,683 (ore) and 79,172,702 (metals) and nickel reserves of 3,197,178,940 (ores) and 50,872,304 (metals) with elemental content nickel average 1.20-3.25%. these https://doi.org/10.14421/biomedich.2021.102.93-103 94 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 nickel resources and reserves in 2011-2015 continued to increase due to the discovery of new nickel resources (haryadi, 2017). in contrast, the potential of soil metals is rarely found in monazite and xenotime minerals with reserves of more than 951,000 tons (inatadon et al., 2015). some of indonesia's rare earth metal minerals are contained in an associated tin in bangka belitung and gold in kalimantan (virdhian and afrilinda, 2014). both of these minerals are raw materials from nimh batteries. nimh batteries are secondary batteries in which metal hydride acts as a negative electrode and hydrogen-absorbing alloy acts as an active substance in the negative anode. the development of nimh batteries received considerable attention from researchers, especially their use in hybrid electrical vehicles (hev) technology. in addition, the unique characteristics of the working principle of the battery, especially in the hydrogen storage alloy as the anode of the battery, is an interesting topic for writers and has been developed by several researchers, such as research conducted by several institutions in china, japan, europe, and america (chang et al., 2016, young et al., 2017, ouchi et al., 2016, chang, et al., 2017). the research mainly focuses on nimh battery anode components, where there is a hydrogen storage alloy material. the role of hydrogen storage alloy material in this case are greatly affects to nimh battery power density. furthermore, its role is very important in the absorption/adsorption process of h2 at negative electrodes. nickel metal hydride (nimh) batteries nimh batteries are rechargeable batteries by utilizing electrochemical reactions found in active materials or electrodes. like most batteries, the main components in nimh batteries are the same as batteries in general, consisting of negative and positive electrodes and electrolyte solutions. nimh battery's active material used on the positive electrode (cathode) is ni(oh)2. at the same time, the hydrogen storage alloy material is used as active material on negative electrodes. in addition, the electrolyte solution that is usually used in nimh batteries is potassium hydroxide (koh). in nimh battery cathode an electrochemical reaction (equation (1)) is occur as follows: 𝑁𝑖(𝑂𝐻)2 + 𝑂𝐻 − → 𝑁𝑖𝑂𝑂𝐻 + 𝐻2𝑂 + 𝑒 − (1) in the charging process, atoms in nickel hydroxide (ni(oh)2) compounds will dissociate to form nickel oxide hydroxide (niooh) compounds and water and electrons. then the hydrogen atom moves through the diaphragm (electrolyte) to the nimh battery anode in combination with the metal hydride material. meanwhile, electrons flow to the anode through an external circuit, then the reaction occurs (equation (2)) in the anode as follows: 𝑀 + 𝐻2𝑂 + 𝑒 − → 𝑀𝐻 + 𝑂𝐻− (2) then the charging process at the cathode occurs where an hsa material and a hydrogen atom dissociate at the cathode and an electron to form a metal hydride material and a hydroxide ion. briefly, the process of charging and discharging is shown in figure 1. figure 1. charge and discharge mechanism on nickel metal hydride batteries (chang, s., young, k. h., & lien, y. l., 2017). solution combustion synthesis method solution combustion synthesis (scs) is an effective method for the synthesis of nanoscale materials, versatile and efficient in the use of energy that has been used in the production of various types of ceramic powders for several types of advanced applications (li et al., 2016, khina, 2010). the scs method is a simple method but an important technique in synthesising and processing nanostructures of metal, oxide, spinel, alloy, and intermetallic (xanthopoulou et al., 2018). this method is based on an exothermic reaction between nitrates (oxidizing agents) and organic substances (reducing agents) by directly producing nanostructured powders (xanthopoulou et al., 2018, deganello et al., 2018). utilization of the exothermic reaction as a source to drive the reaction itself so that no external source is needed and only requires ignition temperature to start the process (gonzález-cortés et al., 2013). crucial advantages of the scs method are (i) mixing of the initial reaction that occurs in a liquid state, thereby facilitating control of the composition, structure, homogeneity, and stoichiometry of the reaction product; (ii) the possibility of incorporating impurity/dopant ions in the form of oxides, to prepare materials that are suitable for industrial needs; (iii) the very fast scs method allows the formation of metastable phases (xanthopoulou et al., 2018, aruna, 2017, manukyan, 2017). in addition, the main thing that is important from the use of the scs method is that it can be used with various precursors, both soluble and non-dissolved (deganello et al., 2018). hidayanti et al. – a physical chemistry study of black powder materials … 95 material characterization material characterization is an activity carried out to determine the physical properties, mechanical properties, and chemical properties through various tests. among the tests are thermogravimetry analysis (tga), differential thermal analysis (dta), dsc, fourier transform infra-red (ftir), x-ray diffraction (xrd), x-ray fluorescence (xrf), scanning electron microscopy (sem), energy dispersive x-ray spectroscopy (eds), uv-vis, and many other tests. this study, it was conducted characteristic tests on three main types of tests, including (1) x-ray diffraction spectroscopy (xrd), (2) fourier transform infrared spectroscopy (ftir), and (3) scanning electron microscope (sem). therefore, this research aimed to obtain hydrogen storage alloy material which is still in the scope of research in black powder synthesis with variations in temperature of synthesis/reaction using the solution combustion synthesis (scs) method. then the material characterization analysis was carried out, including crystal size, chemical bonds, functional groups, and morphology on the influence of temperature. research methods black powder material synthesis black powder synthesis was carried out by dissolving the three precursors as presented in the synthesis process flow in figure 2. the precursors used in this synthesis process include 0.433 grams (1 mmol) lanthanum nitrate hexahydride (la (no3)3.6h2o) p.a. which produced by merck, 1.454 grams (5 mmol) nickel nitrate hexahydride (ni (no3)2.6h2o) p.a. produced by merck, and 0.976 grams (7.2 mmol) glycine (c2h5no2) is produced by merck. then, it is dissolved at 10 ml distilled water which is equivalent to 10 grams (adziimaa et al., 2013). after that, simultaneously stirring using magnetic stirring with a total mass of 12.863 grams. the temperature variations carried out in the stirring process were at temperatures 60, 70 and 80 °c (xanthopoulou et al., 2018, liu et al., 2016, patil et al., 1997, xanthopoulou et al., 2017). when the stirring process reached the desired temperature, the temperature of the solution was stabilized at a set point at 60, 70, and 80 °c. after that, the transparent green solution that had been carried out in the stirring process on the beaker glass is transferred into the cup for further drying process which aims to remove the formed water element. this drying process was carried out for 3 hours 20 minutes until the gel was formed (liu et al., 2016). the purpose of forming the gel from the solution between the fuel and the oxidant was tightly connected in the gel network (deganello et al., 2018). figure 2. the flow of black powder material synthesis. the gel obtained from the drying process was then calcined by heating the powder at high temperatures but still under the melting point (latif et al., 2014). in addition, this process aimed to eliminate volatile elements, heat decomposition and form new phases of the three precursors used. the condition of this gel calcination with a temperature of 500 °c and a heating rate of 10°c/min is then held for 2 hours (liu et al., 2016). then the gel would reach ignition temperature to undergo combustion and change into a sponge shape with volume development. the expanded sample was then mashed using a mortar to obtain a sample in the form of powder. x-ray diffraction this process was carried out on the x-ray diffraction characterization at the lipi physics research center with the xrd smartlab, rigaku. xrd characterization was carried out under test conditions with a voltage of 40 kv and a current of 30 ma. the target element used to radiate the sample is cu kα with wavelength specifications 0.1541862 nm. besides the step mode scan with a scan speed of 0.25 and a scan range of 10 90 deg for each sample. then an analysis of the x-ray diffraction pattern of the black powder sample was conducted using the rietveld method using panalytical high score plus software. sem-eds the scanning electron microscopy characterization has been carried out at the bppt technology incubator center using the scanning electron microscope (sem) type quanta 650 to test three samples with magnifications of 500, 1000, 1500 and 10000 and the energy dispersive x-ray spectroscopy (eds) of oxford instruments. it was calcined on 500 oc for 2 hours with rate 10 oc/min it was dryed on 100 oc it was stirred on 60, 70 and 80 oc it was dissolved in 10 ml of distilled water 96 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 fourier transform infrared (ftir) fourier transform infrared (ftir) characterization has been conducted at the material technology center bppt. the ftir test was carried out using the nocolet is50 thermo scientific type ftir under the conditions of the spectra scan speed test per second 65 (at 16 cm-1) and 95 (at 32 cm-1). with a scan range of 400-4000 cm-1 for each black powder sample. result and discussion x-ray diffraction the diffraction pattern of the sample for black powder with a variation of synthesis temperature at 60 ºc, 70 ºc and 80 ºc is shown in figure 3. it is known that in the angular range of 10 to 90 degrees, there is an identical diffraction peak pattern of the four black powder samples, the identical peak pattern in the 2theta angles, including 37°, 43°, 62°, 75° and 79° which are identified as phases of nickel oxide. figure 3. x-ray diffraction pattern for each sample back powder. meanwhile, figure 4 was a magnification of figure 3 with an angle range of 2 theta 20º to 40º, where there were differences in the peak pattern in the angle range of 2 theta. in the sample, black powder room temperature can be indicated at magnification with a range of angles that the peak at an angle of 2 theta 26°, 29°, 30°, and 31° is the peak of the lanthanum oxide (la2o3) phase. while at an angle of 2 theta 23° and 31° was the phase of the lanthanum nickel tetraoxide (la2nio4). in addition, it was also observed at the diffractogram peaks for black, 60 °c, 70 °c, and 80 °c black powder samples. an identical peak was seen at an angle of 2 theta were 25° and 30° which is the phase of lanthanum oxide. while the peak at an angle of 2 theta was 28°, it experiences shrinkage as the temperature of the synthesis rises, which indicates that the angle represents the lanthanum oxide ht (x-form) phase. on the other hand, in figure 5 there was also the formation of peaks in the range of 30° to 60°, at an angle of 39° and 44° observed that there is a paw that shows the lanthanum oxide phase, as the temperature rises, the formation of the peak shows more clearly that the lanthanum phase is formed oxide. similarly, around the angle of 2 theta 40° and 45° also formed a diffractogram peak which showed the development of the phase of lanthanum nickel trioxide at room temperature, but along with the increase in temperature appeared lanthanum oxide phase at the peak at 53° and 55°. figure 4. magnification of diffractogram black powder sample on 2: 30 60°. figure 5. magnification of diffractogram black powder sample on 2: 20 40°. judging from the suitability criteria for the profile fitting process based on the literature, the good refinement match is determined by the weighted rprofile (rwp) and goodness of fit (gof) parameters. rwp is a weight comparison pattern of the difference between observation patterns with xrd calculations (speakman, 2012). the ideal value of rwp is <10% 10 20 30 40 50 60 70 80 90 (011) (010) (222) (113) (022) (002) bp 80 bp 70 bp 60 in te n si ty ( a .u ) 2 theta (degree) bp rt (111) 30 40 50 60 (013) (021) (112)(020)(110)(002) (012) (110) (013) (112) (017)(011)(012) in te n s it y ( a .u ) 2 theta (degree) bp rt bp 60 bp 70 bp 80 20 25 30 35 40 (011) (011) (011) (010) (011) (011) (010) (011) (010) (002) (010) (011) in te n s it y ( a .u ) 2 theta (degree) (013) bp rt bp 60 bp 70 bp 80 hidayanti et al. – a physical chemistry study of black powder materials … 97 (speakman, 2012, sarwanto and adi, 2018). while rexpected is a statistical evaluation of noise data on diffractograms. in addition, the parameter of goodness of fit (gof) or commonly known as chi-squared is a ratio of xrd observation patterns that are comparable with expected, with an ideal value of no more than 4% (speakman, 2012). the goodness of fit and rwp factors, as well as the size of the crystals that have been calculated using high score plus for each black powder sample, are known respectively in table 1. table 1. match parameters and crystal size of each black powder sample. sample uniform parameter crystal size rexpected rwp gof black powder room temperature 9.1453 7.9019 1.1902 2.0737 black powder 60°c 9.0551 8.1407 1.2738 5.0808 black powder 70°c 9.1597 8.1181 1.2420 4.3323 black powder 80°c 8.9618 7.6367 1.1468 3.4879 from table 1 it can be concluded that the level of fitting for each black powder sample can be categorized very well according to the literature mentioned earlier. in addition, the results of the analysis of the composition of compounds contained in each black powder sample are listed in table 2. table 2 showed that in the black powder room temperature sample, there is a la2nio4 phase with a composition of 27.4%. table 2. phase composition contained in the black powder sample. compound composition of compounds (%) r.t 60 °c 70 °c 80 °c la2nio4 27.4 nio 71.3 84.6 82.0 80.7 la2o3 1.3 15.3 17.8 19.3 la2o3 – ht (x-form) 0.1 0.2 total 100% 100% 100% 100% in each black powder sample 60-80 °c, the la2nio4 phase was not formed and tended to show a very dominant nio phase and it was observed that with increasing synthesis temperature, la2o3 phase composition tends to increase. scanning electron microscopy (sem) analysis the analysis was carried out with three magnifications, namely 500x, 1000x and 10000x. the sample chamber is vacuum up to 3.54x10-4 pa for 500x magnification and 19.4x10-2 pa for 1000x and 10000x magnification, respectively, to ensure that the sem column is free of air molecules. the sem was operated with a standard high voltage operation parameter of 10 kv with a spot size of 2.5 and a work distance (wd) of 9.6 mm for a 60°c black powder sample. sem results for the microstructure test of a 60°c black powder sample are shown in figure 6 taken with a se detector. figure 6. the microstructure of black powder 60°c (a) with a magnification of 500x (b) with a magnification of 1000x (c) the measurement of molecular samples of black powder and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. at 500x magnification (figure 6a), black powder particles are scattered to form small fragments and some are porous chunks. to clarify the observed porous profile, a 1000x magnification was performed. the analysis results with higher magnifications observed that there were agglomerates in the porous lump (figure 6b). the average particle size observed at 1000x magnification is in the micron range with an average particle size of 11,8093 µm (figure 6c), but it has not provided information about the porosity of the black sample particles the powder. on that basis, the analysis continued with 10000x magnification. in micrographs with a magnification of 10000x, it was able to show important information, which was very clearly visible agglomerates contained in the porous lump (figure 6d) and the size of the pores in the micron order with an average pore diameter of 22.8361 µm (figure 6e). then the analysis for the 70 °c black powder sample is presented in figure 7. with the sem operation specifications for the high voltage operation parameter of 10 kv with spot size 2.5 and work distance (wd) of 10 mm for the 70°c black powder sample. the sample chamber is vacuum up to 1.30x10-4 pa for 500x magnification and 1.41x10-4 pa for 1000x magnification and 1.52x10-4 for 10000x magnification. in micrographs with a magnification of 500x, it was seen that in the 70°c black powder sample, the particles in the sample 98 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 were scattered with mostly dominated by lumps (figure 7a). then, it was clear that the lump had pores on each of the lumps (figure 7b) with an average size of the lumps of 20,89 µm (figure 16c). in addition, at 10,000x magnification (figure 7d), it was observed that there were agglomerates with an average of 0,5632 µm and an average pore diameter of 0,7508 µm (figure 7e). figure 7. the microstructure of black powder 70 °c (a) with a magnification of 500x (b) with a magnification of 1000x (c) the measurement of molecular samples of black powder and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. figure 8. the microstructure of black powder 80 ° c (a) with a magnification of 500x (b) with a magnification of 1000x (c) molecular measurements of the black powder sample and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. after that, the 80 °c black powder micrograph sample was shown in figure 8. the micrograph is obtained with a high voltage operation parameter of 10 kv with a spot size of 2.5 and a work distance (wd) of 10 mm. the sample chamber is vacuum up to 1.07x10-4 pa for 500x magnification and 1.16x10-4 pa for 1000x and 10000x magnification, respectively. in figure 8, it appears that the 80 °c black powder sample does not look too many lumps that appear and most are shaped granules. then a magnification of 1,000x was carried out so that it was observed that the particle structure of the black powder sample tended to have pores that were slightly unlike the micrographs of the black powder samples 60 °c and 70 °c. in addition, information was obtained in the form of particle size with an average of 6.7447 µm (figure 8c). in micrographs with 10,000x magnification (figure 8d and 8e), it is known that in the black powder sample 80 °c, the shaft/pores of the particles shrink and tend to be more solid with an average shaft/pores of 0.4676 µm tend to be smaller compared to the black powder samples in figure 6 and figure 7. the morphology of black, 60 °c, 70 °c, and 80 °c samples in the figure 6d, figure 7d, and figure 8d with dark colours that appear to be more dominant in each sample is a constituent element with a low atomic number. in contrast, the bright colours seen in agglomerates are constituent elements that have high atomic numbers (tutu et al., 2015). so it can be concluded that the dark side of the sample contains ni atoms and agglomerates that appear to be representations of la atoms. from the three micrographs, it appears that the high surface area was affected by the type of glycine fuel which causes rapid thermal decomposition and excessive gas development, resulting in greater porosity and crystal size (deganello and tyagi, 2018; fathi et al., 2017). it is also known that the influence of temperature variations in the synthesis can affect the porosity and size of the crystal. energy dispersive x-ray spectroscopy (eds) analysis eds analysis on each black powder sample obtained from the solution combustion synthesis (scs) synthesis method was carried out to determine the composition of the elements in the sample. the results of the eds analysis for each black powder sample are presented in figures 9, figure 10 and figure 11. based on the results of the eds analysis in figure 9, it was known that the microstructure for black powder samples with a temperature variation of 60ºc contains the composition of the elements o, ni, and la at each observation point with the relative percent mass of the element at the first observation point (figure 9a) for ni of 57.8%, element la is 26.6%, and element o is 15.6%. then at the second observation point (figure 9b) shows the relative mass of each element, each for ni is 51.4%, element la is 27.8%, and element o is 20.9%. similarly, the relative mass at the third observation point (figure hidayanti et al. – a physical chemistry study of black powder materials … 99 9c) for each element is as follows for ni of 54.1%, element la for 26.3%, and element o for 19.6%. figure 9. the eds spectrum and composition of the 60 °c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. in addition, figure 10 shows the eds spectrum for black powder samples with a temperature variation of 70 ºc containing the composition of the elements ni, la and o. with each relative mass of the element at the first observation point (figure 10a), namely ni of 56.3%, element la is 24.8%, and element o is 18.9%. furthermore, at the second observation point (figure 10b), it was found that the relative mass of each element, including ni was 55.2%, la was 27.9% and o was 16.9%. then the observation at the third point (figure 10c) obtained the relative mass composition of each element, including ni of 61.2%, la of 27.1% and o of 11.7%. figure 10. the eds spectrum and the composition of the 70 ° c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. on the temperature of 80 ºc, the black powder sample presented in figure 11 showing three observation points on the surface of the black powder sample, the results show that there are elemental compositions as follows ni, la, and o with the relative mass content of each element at the first observation point (figure 11a) ni at 57.1%, element la at 25.1%, and element o at 17.8%. whereas at the second observation point (figure 11b), each element's relative mass composition as follows on the ni element is 56.4%, la element is 26.9%, and element o is 16.7%. at the last observation point (figure 11c), the relative mass composition of each element is 57.2% for the ni element, 25.2% for the la element, and 17.6% for the o element. figure 11. the eds spectrum and the composition of the 80 ° c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. the following is summarized in table 3 results of the analysis of energy dispersive x-ray spectroscopy (eds). table 3. eds test analysis results for each black powder sample. observation element (%) ni la o total black powder 60 point 1 57.8 26.6 15.6 100 point 2 51.4 27.8 20.9 100 point 3 54.1 26.3 19.6 100 black powder 70 point 1 56.3 24.8 18.9 100 point 2 51.4 27.8 20.9 100 point 3 61.2 27.1 11.7 100 black powder 80 point 1 57.1 25.1 17.8 100 point 2 56.4 26.9 16.7 100 point 3 57.2 25.2 17.6 100 it appears that there was three percent by weight of the dominant atom contained in each black powder sample. in addition, it was confirmed that the nickel element has a composition of atomic weight that is greater and most dominant for each black powder 100 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 sample and the element la is the lowest element. then in the results, it can be concluded that the eds test is eds-oxide seen in the presence of element o. so that due to the presence of element o, oxidation occurs in the elements of ni and la and the compound element is formed which is ni into nio (nickel oxide), and the element it becomes la2o3 (setiadi, 2018). therefore, this result is in accordance with the compounds' composition results where the phases formed are nickel oxide and lanthanum oxide and the nickel oxide compound phase has a very dominant compound composition. this data supports data from xrd where no other peaks are found as impurities outside the reactants used. fourier transform infrared (ftir) analysis the fourier transform infrared spectrum for the three black powder samples synthesized using the solution combustion synthesis (scs) method with temperature variations (room temperature, 60, 70, and 80°c) and the forming material is shown in figure 12. figure 12. ftir spectrum of each black powder sample with its raw material. the fourier transform infrared spectrum (ftir) of the sample is then compared with each of its constituent materials. so it is known that each chemical bonding group is formed and disappears in the black powder sample. based on the analysis that has been done, there are several peaks in the spectrum of lanthanum nitrate hexahydrate (la (no3)3.6h2o) which have been identified in table 4. in the ftir spectrum of the material forming the lanthanum nitrate hexahydrate, it is known that in the forming material, several chemical bonding groups appear, which is found in the wavenumber 3200-3550 cm-1, which indicates the existence of o-h stretching vibrations with strong and wide spectrum characteristics. the o-h bonding group shows that the sample contains water molecules (nedumkallel et al., 2014). table 4. ftir spectrum of lanthanum nitrate hexahydrate, nickel nitrate hexahydrate, and glycine. wavenumber (cm-1) assignment of vibrations la(no3)3.6h2o 3200–3550 o-h bending ~2.980 c–h bending, medium 1293–1434 no3 asymmetric raw material 1368–1464 no3 bonding asymmetric sample black powder. 1649, 1646, and 1640 n=o bonding on black powder samples 60, 70, and 80 ºc 415–423 la-o group ni(no2)3.6h2o 3550–3200 o–h bonding 1619 hydroxyl group 1110 carbonate group 2980 c-h bonding 633 ni-o-h bonding 447–552 ni–o bonding glycine 607, 1389, and 1570 carboxylate group coo 1389 carboxylate group symmetrical coo 1125, 1489, 2600 ammonium group nh3+ 2600 and 3090 ammonium group nh3+ 889 and 1044 ccn asymmetric and simetric bonding 926 ch2 rocking 1322 ch2 twisting 1435 ch2 bonding on tabel 4, peaks around ~ 2980 cm-1 were observed due to vibration stretching of the c-h bond with the characteristic of stretching with medium intensity (kumari et al., 2015). in addition, it was observed that there was no3 stretching asimeter which appeared at 1293-1434 cm-1 in the forming material and at 13681464 cm-1 in each black powder sample. this shows the change in the no3 stretching wave number due to the possibility of constant changes in the force and dipole moment of the no3 group vibrations (trivedi et al., 2015). the characteristic vibration peaks for stretching n = o also appear at 1649, 1646, and 1640 cm-1 respectively in black powder samples 60, 70, and 80 ºc which were previously shifted at wave number 1634 cm1 in the forming material (lanthanum nitrate hexahydrate). whereas la-o chemical bonding group based on literature shows the wavenumber 415-423 cm-1 (radev et al., 2008). however, figure 12 only covers the range of 500-4000 cm-1. then in the infrared spectrum of ni nickel nitrate hexahydrate (no3)2.6h2o material, it was seen that several peaks were formed, including the wave number hidayanti et al. – a physical chemistry study of black powder materials … 101 3550-3200 cm-1 which was an o-h bond and around the wave number 1619 cm-1 associated with the group hydroxyl (rahdar et al., 2015). however, stretching vibration modes of h-o-h around the wave number 1658 cm-1 does not appear in the spectrum pattern (segawa et al., 2015). when compared with the infrared spectrum in the black powder sample, it was observed that the stretching of the o-h bond was reduced, which was caused by the drying and calcination treatment process carried out on the black powder sample. the absorption of the wavenumber 1110 cm-1 shows the presence of carbonates and bonds at around 2980 cm-1 according to the c-h stretch (rahdar et al., 2015). the ni-o-h stretching bond was observed at wave number 633 cm-1, but the absorption of the wavenumber at 447 552 cm-1 associated with the ni-o bond stretching was not observed, due to the range of wave numbers which only ranged from 4000 to 500 cm-1 (nedumkallel et al., 2014, rahdar et al., 2015). figure 13. the infrared spectrum of a black powder sample has been tested with ftir. in the last infrared spectrum, which is glycine material, it was known that in this comparison, there was a free glycine carboxylic group (coo) which is usually observed at 607, 1414 and 1605 cm-1. as in the case of glycine in this forming material, the peak shifts to 607, 1388, and 1570 cm-1 (ahamed et al., 2013, sankar et al., 2010). observed at the peak of 1389 cm-1, there was a stretching of the coosymmetric carboxylate group according to the reported value of literature (azhagan & ganesan, 2013). in addition, peaks in the wavenumbers 1125, 1489, 2600 cm-1 are shifted from the values presented in the literature at wavenumbers 1133, 1507, and 2614 cm-1 of the ammonium group (nh3+) (ahamed et al., 2013). whereas the absorption peak with wavenumbers of 2600 and 3090 cm-1 was indicated as stretching nh3+ (peter & ramasamy, 2010). the peaks were observed at wavenumbers 889 and 1044 cm-1 which are asymmetric and symmetric ccn stretches. whereas the peaks observed at wavenumbers 926, 1322, and 1435 cm-1 are respectively ch2 rocking, ch2 twisting, and ch2 bending (azhagan & ganesan, 2013). so from the analysis of the three row materials, it was found that there were several new absorption peaks that appeared, and some absorptive peaks were lost as shown in figure 13. from the peak of the absorption that appeared based on merck infrared spectrum table and chart, among them, there were around wavenumbers 1464 and 1462 cm-1 in each black powder sample showing the presence of c-h bending bonds with medium intensity and indicated as a class of alkane compounds. then, there is also the absorption peak at the wavenumbers 1390 and 1391 cm-1 in the black powder samples 60 ºc and 70 ºc which were indicated as c-h bending bonds with medium intensity and were observed as an aldehyde class. a strong c-o stretching bond was also observed at the absorption peak around the wave number 1087 cm-1 with indications belonging to the secondary alcohol class. these are summarized in table 5. table 5. ftir spectrum of black powder sample. wavenumber (cm-1) assignment of vibrations black powder 60 1464 c–h bending, medium, alkane 1390 c–h bending, medium, aldehyde 1368 1087 c–o stretching, strong, secondary alcohol 854 649 black powder 70 1462 c–h bending, medium, alkane 1391 c–h bending, medium, aldehyde 1087 c–o stretching, strong, secondary alcohol 854 656 591 black powder 80 1462 c–h bending, medium, alkane 1087 c–o stretching, strong, secondary alcohol 854 649 579 it was observed that all of the three black powder samples as a whole o-h stretching bond groups were reduced. this can be influenced by the drying process (evaporate process). so the o-h stretching bonds that indicate the element h2o evaporates at their boiling temperature. while the c-o and c-h bonds in the infrared spectrum indicate that there are still organic elements owned by glycine, which acts as fuel, so it can be concluded that the temperature during the calcination 102 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 process was still not optimal. not yet the optimal formation of this phase refers to the calcination process in accordance with its purpose, so that the optimal temperature needed in the calcination process to get the phase in accordance with the reaction equation from the reference in this study. conclusion based on the results of the research that had been carried out, some conclusions can be taken as follows: 1. in this study, black powder (la2nio4) material with a variation of synthesis temperature of 60, 70, and 80 °c with the solution combustion synthesis method produces a sample weight of 0.5 grams 4 times the synthesis process so that each sample is obtained by 2 grams. in addition, it is known that there is heavy evaporation of 96.11% of the total weight of the synthesized precursors. 2. x-ray diffraction analysis results from each sample indicated that there were several phases contained in the black powder, namely nickel oxide, lanthanum oxide, and lanthanum oxide ht (x-form). by varying the synthesis temperature, the composition of the lanthanum nickel oxide (la2nio4) phase disappears and makes a tendency for chemical reactions to help the two dominant phases, namely the nickel and the lanthanum oxide phase. this is supported by the results of scanning electron microscopy with micrographs that appear dominant with a dark colour that shows a low atomic number indicating the element ni and light colours on agglomerates that show a high atomic number which indicates the element la. in addition, the phase formation previously carried out by xrd analysis is strengthened through eds analysis where the results show that the graph is eds-oxide, seen in the presence of element o. so because of the presence of element o, oxidation occurs in the elements ni and la and a compound element occurs formed namely ni to nio (nickel oxide) and the element la to la2o3 (lanthanum oxide). in addition, fourier transform infrared analysis shows that there are still c-o and c-h bonds which are bonds of glycine surfactants, thus indicating that the process of phase formation in accordance with the reference reaction is not optimal. then we need an optimal calcination temperature. acknowledgments: we are thanks to the wp team rare earth metal prototypes at the center for material technology-agency for the assessment and application of technology for providing all the required laboratory facilities. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references adziimaa a. f., risanti d. d., and mawarni j. (2013). synthesis of sodium silicate from lapindo mud as corrosion inhibitor. j. tek. its, 2(2), 384–389. ahamed, s. z. a., dillip, g. r., raghavaiah, p., mallikarjuna, k., & raju, b. d. p. (2013). spectroscopic and thermal studies of γ-glycine crystal grown from potassium bromide for optoelectronic applications. arabian journal of chemistry, 6(4), 429-433. aruna, s. t. (2017). solution combustion synthesis. in concise encyclopedia of self-propagating high-temperature synthesis (pp. 344-346). elsevier. azhagan, s. a. c., & ganesan, s. (2013). crystal growth, structural, optical, thermal and nlo studies of γ-glycine single crystals. optik, 124(23), 6456-6460. chang, s., young, k. h., nei, j., & fierro, c. (2016). reviews on the us patents regarding nickel/metal hydride batteries. batteries, 2(2), 10. chang, s., young, k. h., & lien, y. l. (2017). reviews of european patents on nickel/metal hydride batteries. batteries, 3(3), 25. deganello, f., & tyagi, a. k. (2018). solution combustion synthesis, energy and environment: best parameters for better materials. progress in crystal growth and characterization of materials, 64(2), 23-61. fathi, h., masoudpanah, s. m., alamolhoda, s., & parnianfar, h. (2017). effect of fuel type on the microstructure and magnetic properties of solution combusted fe3o4 powders. ceramics international, 43(10), 7448-7453. gonzález-cortés, s. l., & imbert, f. e. (2013). fundamentals, properties and applications of solid catalysts prepared by solution combustion synthesis (scs). applied catalysis a: general, 452, 117-131. haryadi, h. (2017). analysis of indonesia's iron sand and nickel ore resources balance. journal mineral technology dan batubara. 13 (2), 153–169. inatadon, n. f., abdurrachman, m., and aziz, m. (2015). geology and study of rare earth metals botor peanut area and surrounding areas, badau district, belitung regency, province of bangka belitung islands. proceeding, national seminar on earth ke-8. 744–753. khina, b. b. (2010). combustion synthesis of advanced materials. new york: nova science publishers, inc. kumari, a., soni, a. k., dey, r., and rai, v. k. (2015). white light emission and optical heating in er – tm – yb codoped la2o3 phosphor. j. disp. technol., 12(1), 99–105. latif, c., triwikantoro, and munasir. (2014). effect of calcination temperature variation on silica structure. j. sains dan seni its, 3(1). 4–7. li, m. m., yang, c. c., wang, c. c., wen, z., zhu, y. f., zhao, m., ... & jiang, q. (2016). design of hydrogen storage alloys/nanoporous metals hybrid electrodes for nickel-metal hydride batteries. scientific reports, 6(1), 1-10. hidayanti et al. – a physical chemistry study of black powder materials … 103 liu, w., & aguey-zinsou, k. f. 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(2017). reviews on chinese patents regarding the nickel/metal hydride battery. batteries, 3(3), 24. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 1-3 | doi: 10.14421/biomedich.2015.41.1-3 a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja’far luthfi biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract sperm sample from epididymal source can be determined its number using minimal amount of equipment. these method will aid researcher and practitioner in sperm quality analysis to determined sperm number rapidly and practically. keywords: sperm quality, sperm number, sperm count, haemocytometer introduction rat is the most widely used testing animal in reproductive biology for several reasons. the rat is an animal model that resembles human physiology. this animal has long been used as test animals in screening a compound to determine the pharmacological effects include mechanisms, distribution, and toxicity (briggs & oehme 1980). the extensive use of the rat in research has led to a relatively complete biological data (golden 2002; white 2001). several methods have been developed for the assessment of male reproductive function. one of the most widely used methods is sperm quality analysis. analysis of sperm quality can give us information about the fertility status of the male genital organ. the purpose of the analysis is to assess the sperm descriptive parameters. usually, the parameters used to predict male fertility are the sperm number/sperm count, sperm morphology and sperm motility. the analysis may show an increase or decrease in the fertility of the animal testing. methodology equipments equipment required in rat sperm motility analysis is as follows:  light microscope  improved neubauer haemocytometer  micropipette  petri dish  surgical scissors  incubator  hand counter sperm sample preparation sperm samples were taken from the cauda epididymis. cauda epididymis were separated as determined by hamilton (1975), then placed in a petri dish, minced and incubated in 15 ml of media biggers, whitten, and whittingham (bww) (biggers et al. 1971) for 30 minutes at 37 ° c in 5% co2 incubator to allow the sperm to swim in the medium bww (swim-up technique). sperm count procedure mount the cover glass on neubauer improved neubauer haemocytometer. interference pattern (> 10 newton's rings/fringes or iridescence lines) should be seen between the glass surface of the area where the glass cover attached to the haemocytometer. too little line/newton's rings shows that the distance between the cover glass and haemocytometer widened, therefore counting chamber volume becomes larger and the counting will be inaccurate. a total of 10 ml sperm suspension were taken from sperm sample preparation, and then inserted into the space between the cover glass and haemocytometer. other counting chamber also filled in the same way. each chamber sould be completely filled. suspension inserted slowly to let the liquid evenly by capillary forces. if a chamber is overfill, it must be discarded and fill a new chamber. removal of the superfluous chamber must not be done since this will change the sperm concentration in the chamber. let haemocytometer 10-15 minutes to allow the sperm settled on counting grid. counting were done with 200x magnification using a light microscope. at counting chamber central area there are 25 large square. each "large squares" is bounded on all sides by a triple line (figure 1). counting performed on all of 25 large squares at each counting chamber. for sperm located on the borderline, only sperm lies on the upper or left line (triple 2 biology, medicine, & natural product chemistry 4 (1), 2015: 1-3 line) should be count as “belonging” to that square. plot is calculated as the property of the plot. therefore, do not count the sperm that is located on the bottom line or the right line. figure 1. haemacytometer. a. side view (cover glass shown by 1). b. top view. c. one of the counting chambers (one of the 25 large squares shown by 2). approximately 200 sperm in each chamber should be count to minimize standard deviation. if the number of sperm obtained from the count at one counting chamber is less than 150, made of new sperm suspension samples by reducing the volume of bww medium. calculation of cauda epididymal sperm number firstly, the sperm number from both chamber were averaged. secondly, the mean of counted sperm divided by volume within which they were count (volume of 25 large square= 100 nl). the sperm concentration obtained is the number of sperma per nl, whic equals millions of sperm/ml (sperm /10-9 l= sperm x 106/10-3 l). thirdly, to obtain the number of sperm per cauda epididymis, sperm concentration obtained multiplied by the number of bww medium volume used in swim up. example of sperm number calculation 15 ml solution of bww media was added to epididymal sperm sample (swim-up method). counting on one chamber produces 320, whereas in the other counting chamber is obtained 280. both these results are summed and divided by two to get the average, obtained number 300. to obtain the concentration of sperm per nl, average of sperm number of both chamber was divided by 100, to obtain the numbers 3 x 106 sperm per ml of sperm suspension. to obtain the number of sperm per cauda epididymis, sperm concentration (3 x 106 sperm per ml) was multiplied by the volume of bww used to swim up (15 ml) yields: 30 x 106 sperm per cauda epididymis of rat. discussion determination of sperm number is one of the important aspects in the analysis of sperm quality. however, there are so many variations on the method in the aspects of equipment and technical details. various different laboratories using different methods. in this study we use improved neubauer haemocytometer to count sperm number. the method mostly based on the practice and experience of testing of 300 rat samples. the study were done at uin sunan kalijaga yogyakarta and laboratory of zoology faculty of science and technology universiti kebangsaan malaysia (data not shown). sperm count or sperm concentration of the testing species can be determined from a sample of ejaculate, epididymis, or the testes. determination of the caudal epydidimal sperm number usually only use sperm from the cauda (clegg et al., 2001). difference in the sperm number after treatment of materials or drugs may give important clues about the effect of a substance on sperm production (working, 1988). epididymal sperm counts are generally used in toxicology studies to assess the damage to the male reproductive system, or vice versa, to determine the therapeutic effect of a substance. epididymal sperm count reduction indicates a reduction in daily sperm production by the testes, the transport obstacles from the testes to the muhammad ja’far luthfi. – a simple and practical method for rat epididymal sperm count … 3 epididymis, or changes in the epididymal sperm transit time. in general, the determination of epididymal sperm count was performed using haemocytometer (strader et al., 1996). determination of sperm concentration with haemocytometer is the main basis for determining whether a sample categorized as normal, and to predict whether an individual is fertile. counting with haemocytometer has become a standard method for measuring the concentration of sperm (freud & carol, 1964). compared with other methods, haemocytometer is still a better method for determining the sperm number (kuster, 2005). in this study, the amount of bww volume used in a sperm sample dilution is 15 ul. however, this number can be increased or decreased to obtain suitable sperm density. in some cases, it is necessary to add 20 ml bww solution for dilution in order to obtain the ideal sperm density to be calculated (200-400 sperm in a haemocytometer counting chamber). three factors need to be considered to achieve accuracy uisng this method. firstly, we must ensure that the incision of the cauda cauda epididymis was consistent. secondly, the insertion of the sample with a micropipette on each space calculation must be completely filled. thirdly, the room temperature in the determination of sperm numbers should be the same for the entire counting sperm samples. these factors affect the consistency of counting of the sperm number. the strict procedure will ensure the accuracy sperm count. references biggers, j.d., whitten, w.k. &whittingham, d. 1971. the culture of mouse embryos in vitro dlm daniel, j.c. (ed). methods in mammalian embryology. page 86-116. freeman san francisco, ca. briggs, g.b. & oehme, f.w. 1980. toxicology dlm baker, h.j. et al. (ed). the laboratory rat. volume ii. researhapplication. page 104-118. academic press. new york. golden, a.l. 2002. biomarkers of male reproductive health dlm wilson, s.h. & suk, w.a. (ed). biomarkers ofenvironmentally associated disease. technologies, concepts, and perspectives. page 387-410. lewis publisher. new york. hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat dlm hamilton, d.w. & greep, r.o. (ed). handbook of physiology, section vii, endocrinology, vol.5, male reproductive system. page 259-301. american physiological society, washington d.c. white, w.j. 2001. the use of laboratory animals in toxicologic research dlm hayes a.w. (ed). principlesand methods of toxicology. fourth edition. page 773-775. taylor & francis. philadelphia. content_v4n1_1.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 11-15 | doi: 10.14421/biomedich.2015.41.11-15 the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi* and maratun janah faculty of veterinary medicine, university of west nusa tenggara, mataram jl. tawak-tawak karang sukun, mataram, tel. +62-370-636875 author correspondency*: supriadi@yahoo.co.id abstract the application of intensive keramba in rivers could affect the presence of parasite organisms throughout the river downstream. the aims of this research are to find out the diversity of parasite species and the effect of intensive aquaculture method developed by the community on the presence of various parasitic organisms, particularly in the downstream area. a total of 65 tilapia fish samples (o. niloticus) that was collected from 3 areas (15 samples from upstream, 25 samples in keramba and 25 samples from downstream areas) have been examined in the laboratory of faculty of mathematic and natural science, university of mataram. methods employed to identify parasites that infected fish samples are native method and flotation method. this research has identified 7 species of parasites which were divided into 2 groups: ectoparasites (trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp., trematode) and endoparasites (entamoeba sp. dan camallanus sp.). diversity index calculation indicated that parasite organisms in upstream area were lower in number than that in the downstream and intensive karamba area (h’= (0,825; 1,596 dan 1.324 respectively). these data has showed there was a difference in species diversity and evenness index of parasite organisms in the upstream, downstream and intensive keramba area. in conclusion, there was significant influence of the application of intensive keramba on the appearance of various parasite organisms that could affect the sustainability of fish aquaculture. keywords: intensive keramba, parasites organism and diversity index introduction fishery products have become the highest animal protein sources demanded in indonesia with the consumption rate of these products has reached 30.4 kg/capita/year or 72% of animal protein consumption/capita/year (anonima, 2013). although the prospect of fishery sector development in indonesia is high, especially in freshwater fishery sector, there has been insignificant production growth in the last several years. this may be due to various factors such as fish farmers’ low interest in the cultivation, capital shortage, lack of knowledge, stream water pollution and various diseases (parasites, bacteria and viruses) which result in considerable loss. one of many factors that can lead to a decrease in the quality and quantity of fishery products is parasite infections (akbar, 2011). the impact of the infection can be a decrease in fish population for consumption, reduction in fish weight, morphological changes and even in some cases; this can cause illness in humans (zoonoses parasite infection) (herman and chiodini, 2009; mccarthy and moore, 2000; punyagupta et al., 1990). declining fish population triggered by the degraded water quality in the cultivation sites is a direct aspect and unsettling condition for fish farmers (bell and burt, 1991). the presence of parasite organisms such as digenea worms, cestodes, nematodes, acantochepals and several ectoparasites from copepode group greatly affects aquaculture activities (kennedy, 1990; lawton, 1989). according to salgado-maladona et al., 2005, the presence of parasites in freshwater fish populations are often linked to the quality of the aquatic environment. moreover, environmental degradation is also often associated with a greater diversity of parasitic infections (chubb, 1963). dominance and density of infection of pathogenic organisms is often directly proportional to the environmental degradation (quiroz-martı´nez and salgado-maldonado, 2013). one method of fish farming that is directly cultivated in the river is intensive keramba method. this method is very simple and easy, even though its feasibility has not been tested ecologically. to anticipate the possibility of eutrophication impact in the downstream and the risk of pathogenic organism growth, it is necessary to conduct this research to study the diversity of parasitic organism caused by the application of intensive keramba method as well as the method’s impact on the presence of parasitic organisms particularly in the downstream area. research methods this research was conducted from july to august 2014. sampling was carried out in the river which became the center of cultivation, located in lingsar sub-district, west lombok regency. fish sample examination was performed in the laboratory of biology, faculty of mathemathics and natural science, university of mataram. parameters to be measured and examined in this research were the species and the diversity of parasitic organisms, species eveness index and the frequency of 12 biology, medicine, & natural product chemistry 4 (1), 2015: 11-15 parasitic organisms’ presence in wild fish as well as in keramba-farmed fish. fish collection was performed in sampling spots which were purpospreively determined and refered to the actual condition on field. the fish were collected utilizing fishnets and fishing rods. the fish net was thrown over 35 times to obtain as a minimum 10 fish on the certain spot. farmed fish samples were acquired from the fish farmers on field. parasite specimens collected on field were preserved in 70% alcohol for further analysis in the laboratory of the faculty of mathematics and natural science, university of mataram. fish organs, such as gill, digestive organs, and flesh (muscle around abdominal cavity), were examined for the presence of parasitic organisms. the obtained organisms were then transferred a physiological saline solution and were identified using a light microscope. staining on the parasitic specimen was necessary in order that the parasite organisms could be identified acurately. infestation rate of the organisms from various samples was calculated by identifying the species discovered from the host tissue (gill, digestive track, flesh) and then counting the individual numbers. the species identification refered to identification books by noble & noble (1989) & taylor et al., (2007). endoparasite examination was performed utilizing saturated sugar method. prevalence = x100% diversity index = log/ln similarity index = ( ) x100% (choi et al., 2011; kusmana, 1997) data obtained from this research were analysed descriptively by linking up the data and facts found on fields; whilst data interpretations were presented in the form of tables, figures and graphs. subsequently, the conclusion was drawn deductively by describing the issues from general to specific points results and discussion results there were 25 fish samples that were collected randomly from several intensive keramba in the river of lingsar village. the examination results showed that there were various parasite infections. parasites identified from this research were devided into ectoparasite and endoparasite group. the species that were included in ectoparasite group were trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp and 1 species of trematodes; while the endoparasites were camallanus sp. dan entamoeba sp (figure 1). figure 1. parasites found in tilapia fish from the river of lingsar village. a. amylodinium sp., b. trichodina sp., c. oogonium sp., d. entamoeba sp., e. dactylogirus sp., f. trematoda dan g. camallanus sp. supriadi and maratun janah. – the effect of intensive keramba on the presence of parasite organisms … 13 the shanon-wiener diversity index of parasites in tilapia fish (oreochromis niloticus) from three sampling location (upstream, middle/keramba and downstream) were 0.8247, 1.3244 and 1.5962 respectively. these numbers demonstrated that the species diversity of parasites in the upstream, middle and downstream area were moderate. however, there were differences in the numbers and infection intensity in the upstream to downstream area; where the frequency of infection in the upstream was very low in contrast to the very high infection frequency in the middle and downstream area. table 1. comparison of diversity index value (h’), species eveness (e) and frequency (f) of parasite organisms presence in three sampling location. no. sampling location h’ e f (%) 1 river upstream 0.8247 0.4238 46.7 2 intensive keramba 1.3244 0.6806 96.0 3 river downstream 1.5962 0.8203 92.0 . the calculation results of the presence frequency of parasites organisms illustrate that the infection frequency tend to get higher in the downstream area. in the river upstream, the infection frequency was 46.7%, in the intensive keramba was 96.0% and in the downstream was 92.0%. according to the graph in figure 2, a number of 7 parasite species infected fish were collected in the river downstream and in the intensive keramba owned by the community. on the other hand, only 3 species of ectoparasite were discovered infecting the fish collected from the upstream. further, it can be explained that the dominant species infecting the fish was trichodina sp. this species was found in all sample groups. what is more, the species of amylodinium sp and oodinium sp. were also had the same presence frequency as trichodina sp. however, the intensity (density) of infection of the two species were lower compared to that of trichodina sp (figure 2). figure 2. comparison of the infection frequency in tilapia fish from 3 three sampling location. data of parasite species diversity index from three location show that there was a significant difference. in the upstream area, the parasite species diversity index is low which counted at 0.8247. meanwhile, the parasite diversity index in the intensive keramba area and downstream area were counted at 1.3244 and 1.5962 respectively. based on these values of shannon-wiener diversity index, it can be stated that the upstream area has lower parasite species diversity, while the keramba area and downstream area have moderate diversity. evenness index values of the parasite species show increasing trends toward the downstream area. in the upstream area, only 3 species of parasites were discovered, whereas in keramba site and downstream area, 7 species of parasites were identified. however, there was similar data pattern i.e all parasite species found in the upstream were also found in tilapia fish collected from keramba site and the downstream. figure 3. comparation graph of shannon-wiener species diversity index and species evennes index for parasite organisms on the 3 (three) sampling location. the number of parasite species identified in each sampling location illustrates the drastic increase from upstream (3 species) toward downstream area (7 species). the main difference is the level of parasites density. even though there were only 7 species discovered in keramba site and downstream area, the parasites individual number than infects the keramba-cultured fish was higher (figure 3). figure 4. graph for difference in species number of parasites that infect tilapia fish in three different sampling location. 14 biology, medicine, & natural product chemistry 4 (1), 2015: 11-15 discussion based on the analysis and laboratory examination results, as many as 7 species of parasites infected tilapia fish caught in the upstream, keramba site and the downstream. the species include ectoparasite trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp, 1 species of trematode, and 2 endoparasites (camallanus sp. and entamoeba sp (figure 1)). dominant species found in this study is trichodina sp. which was discovered in all sampling locations. according to eronimo et al., (2012), these parasite species are common ectoparasites that infect tilapia fish farmed in freshwater. further explained is that this parasite infection mostly happen in gill and skin area, which reduce the immunity system of the infected fish (martin, 2012). amylodinium sp. and gymnodinium sp. were two other ectoparasites that were discovered in all sampling locations, although with lower individual density. this may be due to the two species competition with trichodina sp. other than protozoa, this research also found trematode worm (ectoparasite) on the fish gill and was identified as dactylogyrus sp. this species is mostly discovered in the fish collected from the downstream and keramba sites. this trematode worm is pathogenic and often become the cause of fish mortality in several fish farming sites. by infesting the gill of freshwater fish (tilapia), this parasite infection would escalate the heart rate to pump up the blood to the gill and increase mucus production, causing the difficulty in taking in oxygen from water (eva, 2007). beside dactylogyrus sp., there was also another unidentified treamatod worm. however, this species is rarely found infecting the sampled tilapia fish. two endoparasites species identified in this study, i.e camallanus sp. and entamoeba sp., are nematode worms that were mostly found in fish intestine. kabata (1985) also explained that these worms often infect the intestine of freshwater fish. furthermore, these parasite species are often discovered infecting freshwater fish in aquariums. these nematodes infection is easily spreading as the worms do not require intermediate hosts in their life cycles (untergasser, 1989). besides, the worms infection is very damaging to their hosts as the worm can wound the host’s intestine by biting the intestine wall using the worm’s bucal capsules. this research identified camallanus sp. with the length of 10-15 cm. this discovery is supported by research results of buchmann & bresciani (2001) and arie (2008) who found that the female worms could reach the length of 22 cm while the male ones could have the length of 11cm. analysis results of species diversity showed that there was significant difference in species diversity index from the upstream to downstream. species diversity index values for upstream, keramba, and downstream area are 0.8247, 1.3244 and1.5962 respectively. these data display a pattern that the number and density of parasite infection were changing from the upstream to downstream. in the upstream, parasite infection in tilapia fish was very low with only a total of 3 parasite infection and low parasite density. increasing individual number and species of parasites infecting the fish from the upstream to downstream can be triggered by various biotic and abiotic factors. biotic factors include organisms carrying parasites (hosts) and abiotic factors include aquatic environmental pollution as a result of human activities which produce wastes or change the condition of aquatic environment. as stated by quiroz-martı´nez and salgado-maldonado (2013), intensive keramba culture would increase the risk of change in various environmental condition as a result of feeding residue pollution. it can be further explained that feeding remnants from the intensive keramba farming can pollute the aquatic environment in the form of changing water ph, eutrophication and decreasing water clarity. however, these factors are not the only factors that pollute the aquatic environment. species evenness index on the three sampling locations showed that there was also a significant difference. the values of species evenness index for upstream area, keramba sites, and downstream area are 0.4238, 0.6806 and 0.8203 respectively. the values illustrated that parasites presence in the upstream was rare in contrast to the higher species and individual number of parasites in the intensive keramba site and downstream area. this means that fish farming which employing intensive keramba method in the river has a significant effect on the presence of various parasite species. the high parasite infection on keramba-cultured fish is a result of high density of the fish farmed in the intensive keramba. in contrast, fish density outside keramba was very low. the dense environment inside keramba has made it possible for the parasite to easily transmit from one individual fish to another. this is also supported by the parasite density data observed in downstream area. although the species number of parasite identified in the downstream was the same as those found in keramba, the density of the individual parasite found in fish caught from the downstream was lower than those infecting keramba-fish. furthermore, the sparse distribution of fish in the downstream contributed to the lower possibility of parasite organism to transmit between individual or species of fish. data from this research has shown the effect of intensive keramba culture on the presence of parasite organisms in farmed-fish and wild fish. however, it is also important to conduct a research in different season and perform measurement of other factors such as the effect of habitation in the vicinity of river and water pollutant from the surrounding rice field. therefore, recommendation for the application feasibility of intensive keramba system in rivers can be comprehensively established. conclusion from this research, it can be concluded that parasite species diversity of fish in downstream area and intensive supriadi and maratun janah. – the effect of intensive keramba on the presence of parasite organisms … 15 keramba sites were higher than that in the upstream; with the values of species diversity index was 0.8247 in the upstream, 1.3244 in keramba sites and 1.5962 in the downstream. in other words, fish culture employing intensive keramba system has a significant effect on the presence of various parasite organism on fish in the downstream area. references akbar, j. 2011. identifikasi parasit pada ikan betok (anabas testudieus). bioscientiae. volume 8, nomor 2, juli 2011, page 36-45. anonima, 2013. bisnis ikan di indonesia timur: tantangan dan potensinya. http://nyataindonesiaku.com/bisnis-ikan-diindonesia-timur-tantangan-dan-potensinya./akses 29/11/2013. 21.30 wita. arie, u. 2001. pembenihan dan pembesaran nila gift. penebar swadaya. jakarta. bell, g., and a. burt. 1991. the comparative biology of parasite species diversity: internal helminths of freshwater fish. journal of animal ecology. (1991), 60, 1047-1063. buchmann k & bresciani j. 2001. an introduction to parasitic diseases of freshwater trout. denmark: dsr publisher. choi, s. h., j. kim, j. okjo, min k. cho, h. s. yu, h. j. cha and m. s. ock. 2011. anisakis simplex larvae: infection status in marine fish and cephalopods purchased from the cooperative fish market in busan, korea. korean j parasitol. vol. 49: no. 1: 39-44. chubb, j.c. 1963. on the characterization of the parasite fauna of the fish of llyn tegid. proc zool soc lond. 141:609–621. eva.g. 2007. ectoparasites and endoparasites among oreochormis niloticus (tilapia). liceo journal of higher education research. herman, j. s and p. l, chiodini. 2009. gnathostomiasis, another emerging imported disease. clin microbiol rev. 22: 484–492. irianto. a. 2005. patologi ikan teleostei. gadjah mada university press. yogyakarta. jeronimo, g.t., n. da c. marchiori1, s. b. de pádua, j. d. neto, f. pilarski, m. m. ishikawa, and m. l martins. 2012. trichodina colisae (ciliophora: trichodinidae): new parasite records for two freshwater fish species farmed in brazil. rev. bras. parasitol. vet., jaboticabal, v. 21, n. 4, p. 366-371. kabata, z. 1985. parasites and diseases of fish cultured in the tropics. taylor and francis. london. 318 pp. kennedy, c.r. 1990. helminth communities in freshwater fish: structured communities or stochastic assemblages? parasite communities: patterns and processes (ed. by g.w. esch, a.o. bush & j.m. aho), pp. 131-156. chapman and hall, london. martin, ml, marchiori, n., roumbedakis, k. and lami, f. trichodina nobilis chen, 1963 and trichodina reticulata hirschmann et partsch, 1955 from ornamental freshwater fishes in brazil. braz. j. biol. vol. 72, no. 2, p. 281-286 mccarthy, j and t. a. moore. 2000. emerging helminth zoonoses. int j parasitol. 30: 1351–1360. noble, e.r & noble g.a. 1989. parasitologi biologi parasit hewan. edisi ke-5.wardiarto, penerjemah; soeripto n. editor. yogyakarta. gadjah mada university press. translation from: parasitology: the biology of animal parasites 5th edition. punyagupta, s., t. bunnag and p. juttijudata. 1990. eosinophilic meningitis in thailand. clinical and epidemiological characteristics of 162 patients with myeloencephalitis probably caused by gnathostoma spinigerum. j neurol sci. 96: 241–256. purbomartono. c, isnaetin. m dan suwarsito 2010. ektoparasit pada benih ikan gurami (osphronemus gouramy, lac) di unit pembenihan rakyat (upr) beji dan sidabowa, kabupaten banyumas. sains aqutic journal. quiroz-martı´nez, b., and g. salgado-maldonado. 2013. patterns of distribution of the helminth parasites of freshwater fishes of mexico. plos one 8(1): sukardi, m.f, 2002. peningkatan teknologi budidaya perikanan. jurnal lktiologi indonesia vol.2, no. 2. taylor, m. a., r. l. coop, and r. l. wall. 2007. veterinary parasitology 3th edition. blackwell publishing; victoria australia. utergasser, d. 1989. handbook and diseases. t.f.h publication inc. translated by howard h. hirschron. neptune city. united states. 159 h. content_v4n1_3.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 17-24 | doi: 10.14421/biomedich.2015.41.17-24 krokot extract (portulaca oleracea. l) as natural light-harvesting pigments for dye-sensitized solar cells (dsscs): influence of dye acidity cici nurfaizah, didik krisdiyanto*, khamidinal and sudarlin department of chemistry, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency*: didik_kris@yahoo.com abstract dye-sensitized solar cells (dsscs) was fabricated using natural dyes extracted from krokot (portulaca oleracea. l). the effect of dye acidity was investigated on natural ph extract, 5.00, 4.00 and 3.00 of ph. the efficiency and stability dsscs as a function of the dye acidity was studied. the result of the uv-vis shows that the absorption of wave-length from dye extract of krokot is located in the visible region with the absorbance peak in 410.5 nm and 664.5 nm which are the peak of chlorophyll. the efficiency of extract krokot dye sensitized solar cells was decreasing 6.88 x 10-3 % to 0.42 x 10-3 % when ph of the dye was adjusted from 6.27 to 3.00. dsscs stability was also decreased look for efficiency loss from 5.27% to 97.49% in the same conditions. keywords: dye sensitized solar cell (dsscs), chlorophyll, efficiency introduction portulaca oleracea l. is a warm-climate, herbaceous succulent annual plant with a cosmopolitan distribution belonging to the portulacaceae family (xi zhou et al., 2015). many constituents of portulaca oleracea have been isolated, including alkaloids, beta-carotene, betasitosterol, caffeic acid, catechol, chlorophyll, coumarins, dha, epa, ferulic acid, flavanoids, saponins and tannin (esiyok et al., 2004), (palaniswamy et al., 2001), (mohamed and hussein, 1994). the green pigmented chlorophyll has the desirable photovoltaic properties that are utilized in today’s organic photosynthetic solar cells. figure 1. the portulaca oleracea l plants. chlorophyll is a light harvesting pigment that absorbs light in the visible spectrum of solar radiation which promotes electron transfer. carotenoids are also an important part of the photosynthetic process. they aid in energy transfer to the chlorophyll molecule,and serve to supplement the light gathering properties of chlorophyll (diarra et al., 1986). appropriate plant species for use in photosynthetic solar cell applications are those with high concentrations of chlorophyll alpha and chlorophyll beta. krokot or purslane (portulaca oleracea) have been found to contain high concentrations of chlorophyll alpha and beta (griffin et al., 2004). the idea of using the reactions of photosynthesis to convert light into electrical power appeared already melvin calvin before 1974 (hug et al., 2013). sun light excited the electrons of pigment into higher energy level then transfered to the conduction band of the wide band gap semiconductor. the model based on a synthetic membrane where carotenoids were used as a wire inside the membrane. after absorption of a light by a sensitizer molecule at one side of the membrane, the electron is transfered to a carotenoid and then diffuse through the membrane the the other side where it is captured by ellectron acceptor. dye-sensitized solar cells (dsscs) are belonging to the third generation photovoltaics concept where used natural dyes as light harvesting pigment (kalyanasundaram and graetzel, 2010; hagfeldt et al., 2010). they are also called graetzel cells (o’regan and graetzel, 1991) the advantages of natural dyes as photosensitizer are large adsorption coefficients, high light-harvesting efficiency, no resource limitations, low cost, easy prepared and no harm to the environment (luo et al., 2009). chlorophyll is the principal pigments in natural photosynthetic system. it is a green pigment found in the 18 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 leaves of most green plant, algae and cyanobacteria. six different types of chlorophyll pigment exist and the most occuring types is chlorophyll α. the molecular structure include a chlorine ring with mg center, along with different side chains and a hydrocarbon trail depending on the chlorophyll type (ludin et al., 2014). their function include harvesting sunlight, converting solar energy to chemical enery and tranferring electrons. chlorohyll and their derivatives are inserted into dsscs as dye sensitizer because of their beneficial light absorption tendency modes. chlorophyll has an absorption maximum at 670 nm because of an attractive compound that acts as an photosensitizer the visible light range (wang et al. 2010) a dssc consists of a pair of coated glass tco substrate (transparent conducting oxide) as the electrode and the counter electrode, the redox electrolyte that contains iodide and triiodide ion (i /i ) carbon layer as the catalyst, porous tio2 nanocrystal as fotoanoda, and a dye photosensitizer. all components are arranged in front of the sandwich structure where the top layer is the working electrode as the initial layer in receiving photons and the lower layer is the counter electrode and the middle is electrolyte to regenerate electron. dye criteria that can be used as a dye sensitizer is adsorption intensity at visible wavelengths, strong adsorption on the surface of the semiconductor, has the ability to inject electrons to the band conduction of the semiconductor, and has a group = o or -h to bind to the surface of tio2 which can increase the reaction rate of the electron transfer. figure 2. dssc sceme (khuzaifah et al., 2015). therefore, this study will utilize the potential of the natural dye that derived from extracts of krokot which are expected to fulfill the requirement as a natural sensitizer. optical and electrical test are done in order to determine the compliance of the requirement and can be used in dssc system. to further understand the effect of dye acidity was used to investigate the electron transport characteristics of the fabricated cells. this study shows the correlation between efficiency and stability as a function of the dye acidity. experiment chemicals and instrumentation chemicals used for research are krokot, indium transparent oxide (ito), tio2 (degusa), ki, i2, polyethylene glycol, ethanol 96%, polyvinyl alcohol (pva), aquades, graphite pencil 8b and detergent. instrumentation applied for research are sonikator, ultrasonic cleaner, hotplate, glassware, aluminium foil, paper clips, scothlite, screen proyektor (gasket) and cutter. spektrofotometer uv-vis single beam, uv-vis spekular reflektansi uv 1700 pharmaspec, fourier transformation infra-red (ft-ir) shimadzu and i-v meter keithley 2400 source meter. preparation of dye-sensitizer ten grams of krokot powder are macerated with 120 ml of 96% ethanol for 24 hours. then, it is filtered by using vacuum filtration and before it is used for further processing, it should be analyzed first using uv-vis spectrophotometer in the wavelength range 400-700 nm. the effect of ph of dye solution was studied by adjusting cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 19 ph from the original ph of 6.27 using 0.1 m hcl solution to three different phs (3.0, 4.0 and 5.0). preparation of electrodes tio2 powder was weighed as much as 1.5 grams and then inserted into erlenmeyer and added with 3 ml of aquades. then, it is stirred with a magnetite stirring spoon and sonicated with 20 khz frequency for 2 hours. the next solution was then added with polyvinyl alcohol solution which previously has been made from 0.5 grams pva added with 6 ml of aquades by heating at a temperature of 150oc until all of pva are dissolved. the mixing is followed by stirring for 10 minutes until it is formed a homogeneous paste.. then, it is performed tio2 paste deposition on surfaces glass of transparent indium oxide (ito) with doctor blanding technique. but before it, ito should be washed with detergent and followed by aquades using ultrasonic cleaner for 10 minutes and rinsed with ethanol. before tio2 paste is dropped on ito glass, the conductive part should be found and then each of it is given a scotchlite restraint and it is made a rectangular pattern by leaving a 1.8 x 1.3 cm room. furthermore, in above of that fields, the tio2 paste is distributed evenly with a glass rod and then dried in the air and the scotchlite is opened, then it is heated at 80 ° c for 1 hour. tio2 film is inserted into the krokot extract, the container is covered with aluminum foil and then it is saved for an hour. the film which has been soaked then removed and rinsed with ethanol to clean the edge of the layer. then,it is dried at room temperature and analyzed by uv-vis reflectance spectrometer and ft-ir. the graphite of a 8b pencil is spread into the surface of ito on the conductive layer with the shading manner to average carbon layer. then, it is heated at temperature of 300oc for 1 hour. preparation of electrolytes potassium iodide (ki) is weighed as much as 0.815 grams and then dissolved in 10 ml of polyethylene glycol (peg) 400 and stirred until dissolved then added with 0.128 grams of i2 and stirred again until completely mixed. the finished electrolyte solution then stored in the dark bottles and also sealed. assembly of dye-sensitized solar cells (dsscs) dssc fabrication which is used is a sandwich construction with a composition such as: glass-ito working electrode (tio2 layer) that has been coated with dye-screen projector-counter electrode (carbon layer) – ito glass. the use of screen proyector is intended to prevent the short on dssc system. at the ends of the glass that does not stick together is spilled with the electrolyte solution and allowed to seep between the two layers after it is clamped with paperclip (binder clips) on two opposite sides are not coated. measurements dssc prototype was tested by measuring the i-v characteristic curve using a digital multimeter keithley 2400, in the light of a xenon lamp at an intensity of 1000 w / m2.the result of the i-v characteristic curves test were then analyzed voc, isc, fill factor, and the efficiency of solar cells [6] by the equation:= .. (1) = ( ). ( ).. ( ) (2) result and discussion the effect of dye ph on the absorption spectra the result of characterization of krokot extract color absorption spectrum in figure. 3 shows the krokot extract absorbs the blue spectrum (400-450 nm) and red (650700 nm) with peak absorbance is absorbed at λ = 410.5 with a absorbance value of 0.91 abs, λ = 536.5 with a absorbance value of 0.567 abs, λ = 608 with a absorbance value of 0.473 abs and λ = 664.5 with absorbance values of 0.30 abs so that from the great absorbance at a wavelength of 410.5 nm and 664.5 nm can be known that the more dominant krokot extract contains the pigment chlorophyll. figure 3. absorbance of krokot extract. the effect of ph was also investigated in absorbance of krokot extract as a dye sensitizer. as shown in table 1, the ph extract solution has not a significant effect on the absorbance. the absorbance was found the chlorophyll pigment in different ph extract solution of krokot dye. the variation of ph extract did not effect the absorption peak, two maximum peak were detected in 410.5 nm and 664.5 nm. 20 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 table 1. absorbance of dye sensitizer. ph λ maximum (nm) absorbance (a.u.) 6.27 664.50 0.30410.50 0.91 5.00 664.00 0.29410.50 0.90 4.00 664.00 0.30410.50 0.95 3.00 663.50 0.29412.00 1.02 figure. 4 showed the absorbance graphic of plot toward different ph of krokot extract as tio2-dye system. the absorbance indicated that tio2-dye system of extract krokot dye had a wide electronic absorbance in visible light region. extract absorbance was shifted into higher wave length or red shift that indicated dye was absorb into tio2 surfaces than the tio2-dye had lower exited state energy. the red shift showed an electron injection from dyes into semiconductor surfaces. figure 4. the graph of absorbance to wavelength of thin layer tio2 – dye in different ph extract dye. table 2. absorbance shift of extract dye. ph λ dye λ tio2-dye shift 6.27 664.50 670.00 5.50 5.00 664.00 669.00 5.00 4.00 664.00 666.00 2.00 3.00 663.50 684.00 20.50 figure 5. the graph of reflectance to wavelength of thin layer tio2 – dye in different ph extract dye absorbance is used to calculate the band gap energy (eg) in the tio2-dye film by tauc plot method. figure 6 showed that dyes acidity increased from 3.28, 3.61, 3.42 and 3.38 ev when the ph of the dyes was adjusted from 6.24, 5.00, 4.00 and 3.00. the band gap was influence the semiconductor performance when apllied into dye sensitized solar cells (dsscs) system. the wide band gap (more than 3.00 ev) absorb high foton energy from sunlight. figure 6. energy gap thin layer tio2-dye. the effect of dye ph on the ftir spectrum infrared absorption spectrum of a material has a distinctive pattern so that it possible to identify the material and also shows the existence of the major functional groups in the identified structure. the bond can be estimated if the ir spectra of tio2-dye system shows the appearance of a significant new peak or functional groups shift if it is compared with the spectra of dye and spectra of tio2 film. krokot extract used in this study contains carboxyl and carbonyl because in the analysis that uses uv-vis is identified to contain chlorophyll so it is possible there is efficient sensitization through the formation of a bond between the dye and tio2. cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 21 figure 7. infrared spectra (a) thin layer tio2, (b) thin layer tio2-dye ph 6.27 (c) thin layer tio2-dye ph 5.00 (d) thin layer tio2-dye ph 4.00 and (e) thin layer tio2-dye ph 3.00. the results of ftir analysis for tio2 film, and tio2dye film of krokot extract is shown in the figure. 4 which shows the existence of the carbonyl absorption at wave number 1635.64 cm-1 and there is absorption at 3410.15 cm-1 region which is the absorption area of hydroxyl groups. carbonyl and hydroxyl group that is owned by krokot extract that can bind to the group of ti (iv) on tio2 (kuzaifah et al 2015). table 3. infrared spectra thin layer tio2-dye. wavenumber (cm-1) groups tio2 tio2-dye ph 6.27 ph 5.00 ph 4.00 ph 3.00 3425.58 3425.58 3448.72 3425.50 3410.00 -oh 2931.80 2931.80 2939.53 2931.80 2932.00 c-h 1635.64 1635.64 1635.64 1635.60 1636.00 c=o 1427.23 1427.57 1381.03 1381.00 1396.00 c-c 1095.57 1095.57 1095.57 1095.60 1096.00 c-o 678.94 678.94 678.94 666.66 678.90 ti-o from ftir analysis is not seen the significant difference from treatment with variation of extract solution acidity. it is only seen the carbonyl absorption shift of in tio2 film is at a wavelength of 1635.64 cm-1 for natural ph shifted to a wave number 1636.00 cm-1 for 3.00 ph of extract krokot dye. this shows that krokot extract does not bind yet optimally with a film of tio2 or the interaction possibility that occurs only physical interaction. the effect of dye ph on the dsscs efficiency a dsscs efficiency can be characterized with an ivdiagram where the corresponding current (i) at rising voltage (v) is plotted. at a bias of 0 v the short circuit current (isc) is measured and when the current 0 v the open circuit voltage (voc) is defined. the maximum power output (pmax) generated by dsscs is reached when the product of the current and the voltage is maximal. how efficient a solar cell can convert the power of the incident light into electricity described by electricity conversion efficiency (η) (hug et al., 2013). an iv-diagram of dsscs with different dye solution ph was showed in figure 8. 22 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 figure 8. current-voltage curve for krokot extract dye sensitized solar cell in ph 6.27 (a), 5.00 (b), 4.00 (c) and 3.00 (d). table 4 present the performance of dsscs in the term of short-circuit photocurrent (isc) , open circuit voltage (voc), fill factor (ff) and energy convertion efficency (η) (wongcharee et al 2007). the efficiency was found to decrease with decreased ph and had maximum at the natural solution ph. the photoanode made from the krokot extract can absorb more light from chlorophyll pigment. as another pigment, chlorophyll was easy to degradation with heat, light, oxidator and ph (gross et al., 1991). pheophytin, a degradation product of chlorophyll, which represents chlorophyll that has lost the central mg ion replace with h+ in acidic solution that was colorless breakdown products. table 4. photoelectrochemical parameter of the cells sensitized by krokot extract dye. ph isc(m.a.cm-2) . 10-5 voc (mv) ff η (%) . 10-3 6.27 31.00 0.28 0.32 6.88 5.00 12.50 0.36 0.33 3.62 4.00 11.50 0.44 0.33 4.08 3.00 35.00 0.14 0.34 0.42 in this study, the little dssc efficiency which is produced can be caused by the performance of the natural dye used is still low due to the effect of the extract used still contains a lot of pigment with the long structure r which lead steric hindrance of the pigment to the surface band structure so as to prevent oxidation of tio2 molecules bind with tio2 in effective to cause the transfer of electrons from the conduction band to the dye molecule is reduced (khuzaifah et al., 2015). the effect of dye ph on the dsscs stability dye sensitized solar cells with krokot extract stability was also decrease by adjusting the ph of the extract from 6.27 to 3.00. as can be seen from figure 10 efficiency loss of dye sensitized solar cells increased with decreasing ph from 5.27% to 97.49% after irradiated for 2 hours. cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 23 figure 9. current-voltage curve for krokot extract dye sensitized solar cell in ph 6.27 (a), 5.00 (b), 4.00 (c) and 3.00 (d) after irradiated. table 5. photoelectrochemical parameter of the cells sensitized by krokot extract dye after irradiated. ph isc(m.a.cm-2) . 10-5 voc (mv) ff η (%) . 10-3 6.27 28.00 0.26 0.37 6.52 5.00 8.90 0.31 0.45 3.02 4.00 11.00 0.31 0.29 2.41 3.00 0.09 0.06 0.70 0.01 a reason for the worse stability is that, at natural krokot extract acidity the chlorophyll was existed. the pigment are labile and can transformed into the colorless coumpound with decreasing ph. chlorophyll that has lost the central mg ion replace with h+ in acidic solution was degradation into pheophytin. figure 10. stability cells sensitized krokot extract. 0 2 4 6 8 6.27 5 4 3 initial irradiated for 2 hours 24 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 conclusion the results shows that the absorbance spectrum of the krokot extract dye is stretched in the range of visible light to the maximum absorbance peak at a wavelength of 410.5 nm and 664.5 nm so that can be known that krokot extract dye contains chlorophyll. the efficiency of extract krokot dye sensitized solar cells was decreasing 6.88 x 10-3 % to 0.42 x 10-3 % when ph of the dye was adjusted from 6.27 to 3.00. dsscs stability was also decreased look for efficiency loss from 5.27% to 97.49% in the same conditions. acknowledgment department of chemistry, faculty of science and technology uin sunan kalijaga yogyakarta for academic supported. references esiyok, d. ötles, s. and akcicek e., 2004 “herbs as a food source in turkey,” asian pacific journal of cancer prevention, vol. 5, no. 3, pp. 334–339, yan-xi zhou, hai-liang xin,khalid rahman, su-juan wang, cheng peng, and hong zhang, 2015.,portulaca oleracea l.: a review of phytochemistry and pharmacological effects, biomed research international, u. r. palaniswamy, r. j. mcavoy, and b. b. bible, “stage of harvest and polyunsaturated essential fatty acid concentrations in purslane (portulaca oleraceae) leaves,” journal of agricultural and food chemistry, vol. 49, no. 7, pp. 3490– 3493, 2001 a. i. mohamed and a. s. hussein, “chemical composition of purslane (portulaca oleracea),” plant foods for human nutrition, vol. 45, no. 1, pp. 1–9, 1994 diarra, a. , hotchandan i, s., max j j. and leblanc r. (1986) photovoltaic properties of mixed monolayers of chlorophyll a and carotenoids canthaxanthin, j. cher n . so c. , faraday tran . 2, 8 2 , 2217 2231. griffin, w., quach, h. and steepe r, r. (2004) extraction and thin layer chromatography of chlorophyll a and b from spinach, chern . e d. , 81, 385 – 387 hug, h., bader, m., mair, p., and glatzel, t., 2014, biophotovoltaics : natural pigments in dye-sensitized solar cells., applied energy., 115., 216-225 kalyanasundaram, k., and graetzel, m., 2010, artificial photosynthesis : biomometric approaches to solar energy conversion and storege., curr. opin.biotechnol, 21: 298-310 hagfeldt, a.,boschloo, g., sun, i., and kloo, p., 2010., dyesensitized solar cells., chem. rev.,110, 6 : 595-663 o’regan, b and graetzel, m ., 1991., a low cost high efficiency solar cells based on dye sensitized colloidal tio2 films., nature, 353: 737-740 ludin, n.a.; narosikin, a.a.m.; mahmoud, a.a.; muhammad, a. a.; kadhum a.a.h..; sopian, k.; nor karim, s.a. review on the development of nature dye photosensitizer for dye sensitized solar cell. renewable and suinable energy. 2014, 386-396. wang, x.f., ossamu, k.,eiji, h., haoshen, z.,shinichi, s., hitoshi, t., 2010. tio2 and zno-based solar cells using chlorophyll a derivative sensitized for light-harvesting and energi conversion. journal of photochemistry and photobiology, 145-152 biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 25-29 | doi: 10.14421/biomedich.2015.42.25-29 krokot (portulaca oleracea. l) as a natural sensitizer for tio2 dye-sensitized solar cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi and didik krisdiyanto* department of chemistry, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency*: didik_kris@yahoo.com abstract the solar cell is formed by a sandwich structure, in which two electrodes flank the primary electrolyte that is containing redox i-/i based on peg (polyethylene glycol). the working-electrode which is tio2 layer on an ito glass substrate is sensitized with krokot dye as the electron donor. the counter electrode is a layer of carbon. the fabrication cell is immersed with the krokot dye with 40°c, 50°c, and 60°c extract temperature. the result of the uv-vis shows that the absorption of wave-length from dye extract of krokot is located in the visible region with the absorbance peak in 420.5 nm and 665.5 nm which are the peak of chlorophyll. for the uv-vis solid system, there are the highest band gap in 50°c extract temperature that make the capability of absorption toward uv spectrum is large. furthermore, in the functional group analysed by ft-ir, there are shiften-carbonil and hydroxyl group after they are sensitized. from the current and voltage test with i-v meter keithley 2400 is resulted that on the 50°c extract temperature produces the highest efficiency of reaches which is 2.63 x 10-3 %. keywords: dye sensitized solar cell (dssc), krokot dye, chlorophyll, tio2 introduction the solar cell is one way to harness solar energy in which the device is able to convert the sunlight energy into electrical energy. in principle, the solar cell work is similar to the photosynthesis work in plants. light energy is used to produce free electrons. solar cell uses free electron to generate electrical energy while the plant uses the free electron to produce chemical energy (yuwono. et al. 2011). the development of solar cells based on dye sensitized began in 1991 when for the first time grätzel and o'regan designed the basic forms of solar cell based on film of titania semiconductor that is known as dyesensitized solar cell (dssc) in which these systems can convert solar energy into electrical energy (o'regan & gratzel, 1991). this mechanism shows the optical absorption and charge separation processes through the association of a sensitizer as a light absorber with a nanocrystal semiconductor that has a wide bandgap (gratzel, 2003). a dssc consists of a pair of coated glass tco substrate (transparent conducting oxide) as the electrode and the counter electrode, the redox electrolyte that contains iodide and triiodide ion (i /i ) carbon layer as the catalyst, porous tio2 nanocrystal as fotoanoda, and a dye photosensitizer (o'regan & gratzel, 1991). all components are arranged in front of the sandwich structure where the top layer is the working electrode as the initial layer in receiving photons and the lower layer is the counter electrode and the middle is electrolyte to regenerate electron. dye criteria that can be used as a dye sensitizer is adsorption intensity at visible wavelengths, strong adsorption on the surface of the semiconductor, has the ability to inject electrons to the band conduction of the semiconductor, and has a group = o or -h to bind to the surface of tio2 which can increase the reaction rate of the electron transfer (ludin, et al, 2014). therefore, this study will utilize the potential of the natural dye that derived from extracts of krokot which are expected to fulfill the requirement as a natural sensitizer. optical and electrical test are done in order to determine the compliance of the requirement and can be used in dssc system. materials and methods materials krokot, indium transparent oxide (ito), tio2 (degusa), ki, i2, polyethylene glycol, ethanol 96%, polyvinyl alcohol (pva), aquades, graphite pencil 8b and detergent methods sonikator, ultrasonic cleaner, hotplate, glassware, aluminium foil, paper clips, scothlite, screen proyektor (gasket) and cutter. spektrofotometer uv-vis single beam, uv-vis spekular reflektansi uv 1700 pharmaspec, fourier transformation infra-red (ft-ir) shimadzu and i-v meter keithley 2400 source meter procedure krokot extraction (portulaca oleracea l.) ten grams of krokot powder are macerated with 120 ml of 96% ethanol for 24 hours at 40°c, 50°c, 60°c and 26 biology, medicine, & natural product chemistry 4 (2), 2015: 25-29 normally solution temperature. then, it is filtered by using vacuum filtration and before it is used for further processing, it should be analyzed first using uv-vis spectrophotometer in the wavelength range 400-700 nm. preparation of working electrode tio2 powder was weighed as much as 1.5 grams and then inserted into erlenmeyer and added with 3 ml of aquades. then, it is stirred with a magnetite stirring spoon and sonicated with 20 khz frequency for 2 hours. the next solution was then added with polyvinyl alcohol solution which previously has been made from 0.5 grams pva added with 6 ml of aquades by heating at a temperature of 150oc until all of pva are dissolved. the mixing is followed by stirring for 10 minutes until it is formed a homogeneous paste. then, it is performed tio2 paste deposition on surfaces glass of transparent indium oxide (ito) with doctor blanding technique. but before it, ito should be washed with detergent and followed by aquades using ultrasonic cleaner for 10 minutes and rinsed with ethanol. before tio2 paste is dropped on ito glass, the conductive part should be found and then each of it is given a scotchlite restraint and it is made a rectangular pattern by leaving a 1.8 x 1.3 cm room. furthermore, in above of that fields, the tio2 paste is distributed evenly with a glass rod and then dried in the air and the scotchlite is opened, then it is heated at 80 ° c for 1 hour. preparation of elektrode tio2/ dye tio2 film is inserted into the krokot extract, the container is covered with aluminum foil and then it is saved for 1, 8, 18 and 26 hours. the film which has been soaked then removed and rinsed with ethanol to clean the edge of the layer. then,it is dried at room temperature and analyzed by uv-vis reflectance spectrometer and ft-ir. gap energy calculation using the following equation: ∞= ( )( ) (1) ( ∞) ′∞ ′∞ (2) the calculation is performed on each sample by using the kubelka munk method in which the gap energy is obtained from the graph of hv (ev) vs. (f (r'∞) hv) 1/2 (mikrajuddin & khairurijal, 2010). preparation of the counter electrode the graphite of a 8b pencil is spread into the surface of ito on the conductive layer with the shading manner to average carbon layer. then, it is heated at temperature of 300oc for 1 hour. preparation of electrolytes potassium iodide (ki) is weighed as much as 0.815 grams and then dissolved in 10 ml of polyethylene glycol (peg) 400 and stirred until dissolved then added with 0.128 grams of i2 and stirred again until completely mixed. the finished electrolyte solution then stored in the dark bottles and also sealed. fabrication of dye-sensitized solar cell (dssc) dssc fabrication which is used is a sandwich construction with a composition such as: glass-ito working electrode (tio2 layer) that has been coated with dye-screen projector-counter electrode (carbon layer) – ito glass. the use of screen proyector is intended to prevent the short on dssc system. at the ends of the glass that does not stick together is spilled with the electrolyte solution and allowed to seep between the two layers after it is clamped with paperclip (binder clips) on two opposite sides are not coated. characterization of currents and voltage dssc dssc prototype was tested by measuring the i-v characteristic curve using a digital multimeter keithley 2400, in the light of a xenon lamp at an intensity of 1000 w / m2.the result of the i-v characteristic curves test were then analyzed voc, isc, fill factor, and the efficiency of solar cells (lee & misook, 2010) by the equation: = .. (3) = ( ). ( ).. ( ) (4) results and discussion characterization of optical properties krokot dye at diffrerent temperature extract the result of characterization of krokot dye absorption spectrum in different extract temperature was showed at figure 1. figure 1. the graph of krokot dye (a) normally solution temperature extract (b) 40°c temperature extract (c) 50°c temperature extract (d) 60°c temperature extract. reyza anni mufidah, et.al. – krokot (portulaca oleracea. l) as a natural sensitizer for tio2 … 27 figure 1 shows the krokot extract absorbs the blue spectrum (400-450 nm) and red (650-700 nm) with peak absorbance is absorbed at λ = 420.5 with a absorbance value of 2.678 abs, λ = 536.5 with a absorbance value of 0.567 abs, λ = 608 with a absorbance value of 0.473 abs and λ = 665.5 with absorbance values of 1.753 abs so that from the great absorbance at a wavelength of 420.5 nm and 665.5 nm can be known that the more dominant krokot extract contains the pigment chlorophyll. this is because the wavelength is included in the wavelength range of visible light. so that chlorophyll can be used as a dye in dssc because it has characteristic to absorb the visible light that produced by sunlight. temperature extract was influence the absorbance intensity that in hight temperature was decrease the adsorbance intensity. it was indicated that the pigment chlorophyll decresed in dye with hight temperature extract. the pigment chlorophyll was convert into pheophytin pigment in heat treatment (putri et al 2013, gross, 1991). characterization of thin layer electronic properties tio2-dye figure. 2 shows the absorbance graphic of plot toward thin film wavelength of tio2 that has been soaked in dye with variation of temperature extract. figure 2. energy gap (a) thin layer tio2, (b) thin layer tio2-dye solution temperature extract (c) thin layer tio2-dye 40°c temperature extract (d) thin layer tio2-dye 50°c temperature extract and (e) thin layer tio2-dye 60°c temperature extract. absorbance is used to calculate the band gap energy (eg) in the film of tio2 and tio2-dye film with temperature extract variation by tauc plot method. figure. 2 shows that the form of the tio2 band gap is 3.3 ev. this value fullfills the major requirements of semiconductor material that will be used as a dssc where its band gap energy should more than 3.0 ev so that be able to absorb the energy of the photon in the most of the spectrum of sunlight. at the variation temperature extract of film tio2 immersion in the dye for 1 hour there was an eg increasing. this is as expected that the higher temperature extract capable to increase eg on tio2 so that allowing the electrons injection of dye sensitized into the conduction band of nanoparticles tio2 becomes easier because it takes a lower photon for excitation mechanism or in other words the deposited tio2 has been more active in the area of low energy or visible light region. characterization with fourier transform infra-red (ftir) infrared absorption spectrum of a material has a distinctive pattern so that it possible to identify the material and also shows the existence of the major functional groups in the identified structure. the bond can be estimated if the ir spectra of tio2-dye system shows the appearance of a significant new peak or functional groups shift if it is compared with the spectra of dye and spectra of tio2film. krokot extract used in this study contains carboxyl and carbonyl because in the analysis that uses uv-vis is identified to contain chlorophyll so it is possible there is efficient sensitization through the formation of a bond between the dye and tio2. figure 3. infrared spectra (a) thin layer tio2, (b) thin layer tio2-dye solution temperature extract (c) thin layer tio2-dye 40°c temperature extract (d) thin layer tio2-dye 50°c temperature extract and (e) thin layer tio2-dye 60°c temperature extract. 28 biology, medicine, & natural product chemistry 4 (2), 2015: 25-29 the results of ftir analysis for tio2 film, and tio2dye film of krokot extract is shown in the fig. 4 which shows the existence of the carbonyl absorption at wave number 1627.92 cm-1 and there is absorption at 3425.58 cm-1 region which is the absorption area of hydroxyl groups. carbonyl and hydroxyl group that is owned by krokot extract that can bind to the group of ti (iv) on tio2. from ftir analysis is not seen the significant difference from treatment with variation of temperature extract. it is only seen the carbonyl absorption shift of in tio2 film is at a wavelength of 1635.64 cm-1 shifted to a wave number 1627.92 cm-1. this shift occurs when a film of tio2 is coated with krokot extract. current and voltage measurement systems solar cells figure 4. iv meter grafic (a) thin layer tio2-dye solution temperature extract (b) thin layer tio2-dye 40°c temperature extract (c) thin layer tio2-dye 50°c temperature extract and (d) thin layer tio2-dye 60°c temperature extract in table. 1 the efficiency obtained by determining the amount of voltage and the maximum current shown in the fig. 5. from that figure, it can be seen that the current legible shows the dye on tio2 layers undergo the electron transfer from the excited state of the dye to the conduction band of tio2. the highest efficiency found in a layer of tio2-dye with a 40°c temperature extract with an efficiency of 2.63 x 10-3%. table. 1. the parameters of solar cells. ekstrak krokot isc( ) voc(mv) ff (%) ekstrak krokot a 0.092 0.32 24.4565217x10-2 1.44x10-3 ekstrak krokot b 0.165 0.31 25.7576x10-2 2.635x10-3 ekstrak krokot c 0.08 0.135 29.1667x10-2 6.3x10-4 ekstrak krokot d 0.034 0.09 34.3137x10-2 2.1x10-4 in this study, the little dssc efficiency which is produced can be caused by the performance of the natural dye used is still low due to the effect of the extract used still contains a lot of pigment with the long structure r which lead steric hindrance of the pigment to the surface band structure so as to prevent oxidation of tio2 molecules bind with tio2 in effective to cause the transfer of electrons from the conduction band to the dye molecule is reduced. figure 5. iv meter grafic after 2 hours irradiated (a) thin layer tio2dye solution temperature extract (b) thin layer tio2-dye 40°c temperature extract (c) thin layer tio2-dye 50°c temperature extract and (d) thin layer tio2-dye 60°c temperature extract. table. 2. the parameters of solar cells after irradiated. ekstrak krokot isc ( ) voc (mv) ff % ∆ (%) ekstrak krokot a 0.092 0.32 24.4565217 x 10-2 1.44 x 10-3 40.27778 0.09 0.17 28.104575 x 10-2 8.6 x 10-4 ekstrak krokot b 0.165 0.31 25.7576 x 10-2 2.635 x 10-3 86.33776 0.045 0.1 0.4 3.6 x 10-4 ekstrak krokot c 0.08 0.135 29.1667 x 10-2 6.3 x 10-4 -11.1111 0.135 0.145 17.8799 x 10-2 7 x 10-4 ekstrak krokot d 0.034 0.09 34.3137 x 10-2 2.1 x 10-4 16 0.035 0.092 27.3913 x 10-2 1.76 x 10-4 reyza anni mufidah, et.al. – krokot (portulaca oleracea. l) as a natural sensitizer for tio2 … 29 conclusion the result of the uv-vis shows that the absorption of wave-length from chlorophyll pigment. for the uv-vis solid system, there are the highest band gap in 50°c extract temperature. the functional group analysed by ft-ir, there are shiften-carbonil and hydroxyl group after they are sensitized. from the current and voltage test with i-v meter is resulted that on the 50°c extract temperature produces the highest efficiency of reaches which is 2.63 x 10-3 %. acknowledgment department of chemistry, faculty of science and technology uin sunan kalijaga yogyakarta. references yuwono, a. h., dhaneswara, d., ferdiansyah, a., rahman, a. 2011. sel surya tersensitasi zat pewarna berbasis nanopartikel tio2 hasil proses sol gel dan perlakuan pasca hidrotermal. jurnal material dan energi indonesia vol 01 no. 03. 127-140. o'regan, gratzel. 1991. a low cost, high-eficiency solar cell based on dye-sensitized coloidal tio2 film. nature 737-740. gratzel, m. 2003. dye-sensitized solar cell. journal of photochemistry and photobiology 145-153. ludin, n.a., narosikin, a.a.m., mahmoud, a.a., muhammad, a. a., kadhum a.a.h., sopian, k., nor karim, s.a. 2014. review on the development of nature dye photosensitizer for dye sensitized solar cell. renewable and suinable energy 386396. mikrajuddin, a., khairurijal. 2010. karakterisasi nanomaterial teori, penerapan, dan pengolahan data. cv. rezeki putera bandung: bandung. lee, yeji., misook, k. 2010. the optical of nanoporous structured titanium dioxide and the photovoltaic efficiency on dssc. material chemistry and physics. 284-289. content_v4n2_1.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 49-51 | doi: 10.14421/biomedich.2015.42.49-51 tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik faculty of languages and science, university of wijaya kusuma surabaya, indonesia author correspondency: marni.kelik@yahoo.com abstract tapak liman (elephantopus scaber l) is one of the plants that have medicinal properties and has been used for maintenance and improvement of health and disease treatment. the purpose of this study was to determine the effect of extracts of tapak liman (elephantopus scaber l) as immunostimulant to the development of lymphocytes in mice balb / c. the procedure of this study was to test aqueous extracts in vivo with various treatments (control, treatment of 0.5 g / kg, 1.0 g/ kg, 2.0 g / kg) in healthy mice balb / c for 2 weeks. after the treatment carried out analysis of the percentage and number of cells that express cd4+, cd8+ and cd4+ cd8 + in thymus organ, using flowcytometry. analysis of data using one-way anova followed tukey's test with spss. from the analysis showed that the extract of tapak liman at various doses showed no significant effect on the percentage expression of cd4 + cd8 + and cd4 + cd8 + in thymus organs. while the analysis of the number of cells, extracts of tapak liman show its effect on the number of cells that express cd4+, cd8 + and cd4 + cd8 + in thymus organs. concentration of 1.0g / kg of mice showed a good effect on the increase in t helper cells (cd4 +), cytotoxic t cells (cd8 +) and prothymosit cells (cd4 + cd8 +). keywords: tapak liman, immunostimulant, lymphocyte introduction elephantopus scaber linn, is a small herb, which grows in the wild throughout the tropical regions of the world. the major phytochemical constituents of the plant are elephantopin, triterpenes, stigmasterol, epofriedelinol and lupeol (rastogi and mehrotra, 1990; kritikar and basu, 1991). the plant has been used in the indian system of medicine as analgesic, diuretic, astringent and antiemetic. the leaves of the plant were known to be used for bronchitis, small pox and diarrhea and as a brain tonic (sankar et al., 2001). recently, it has been shown to possess anti-inflammatory and anti tumour activity in animal models (reico, 1989) and also found to have antibacterial activity against a few standard bacterial strains (avani and neeta, 2005). the genus elephantopus consists of approximately 30 species distributed in the neotropicsand the old world, and its lectotype species, e.scaber, occurs in all tropical regions (cabrera and klein, 1980; chen, 1985; cao and but, 1999). in southern china, hong kong and taiwan, the whole plant of e. scaber, a perennial herb, is well known as a folk medicine widely used in the treatment of nephritis, edema, dampness, chest pain, fever, pneumonia, scabies, and arthralgia due to wounding (peer and metzger, 1980; hsu, 1986; tsai andlin, 1999). in brazil, the infusion and the decoction of the whole plant are used to stimulate diuresis, reduce fever, and eliminate bladder stones (cabrera and klein, 1980; poli et al., 1992). it has also been popular as a medicinal herb in many countries of southeast asia, latin america and africa for a long time (hammer and johns, 1993; cao et al., 1997). since the 1970’s, a number of chemical constituents and pharmacological evaluations of e. scaber have been reported. for example, kurokawa et al. (1970) and govindachari et al. (1972) reported elephantopin, deoxyelephantopin, and isodeoxyelephantopin in this species; de silva et al. (1982) found that both alcoholic and chloroformic extracts of e. scaber contain cytotoxic germacranolide-type sesquiterpene lactones; poli et al. (1992) tested the aqueous and hydroalcoholic extracts of whole plants for acute toxicity, analgesic, antipyretic, anti-inflammatory, cardiovascular, diuretic, and constipating activities; hammer and johns (1993) reported that the plant extract of e. scaber was subjected to bioassays; lin et al. (1995) and tsai and lin (1999) evaluated the hepatoprotective and anti-inflammatory effects of the taiwanese folk medicine “teng-khia-u”, derived from three plant species including e. scaber, and but et al. (1997) described the isolation and structure elucidation of three germacranolide sesquiterpene lactones from e. scaber. the e. scaber roots and leaves aqueous extracts showed excellent hypoglycemic effect in diabetic rats by lowering the blood glucose level and serum insulin level. a decrease in the elevated levels of glycosylated hemoglobin, liver glycogen, triglycerides and cholesterol serum in alloxan-induced hyperglycemic rats was also reported by daisy et al. (2007). besides hypoglycemic activity in mice models, aqueous extract of e. scaber also showed significant antiinflammatory effect in both experimental acute and chronic arthritis rat models. the aqueous extract from the whole plant significantly inhibited the development of pad swelling in the acute experimental arthritis rats at a dose of 300 mg/kg while higher concentration of the 50 biology, medicine, & natural product chemistry 4 (2), 2015: 49-51 extract (500 mg/kg) was required to inhibit the development of chronic joint swelling in the chronic inflammatory model (tsai and lin, 1999). during thymocyte development, immature thymocytes that express both cd4 and cd8 genes must choose either a helper cd4+ or cytotoxic cd8+ t-cell fate. over the past two years, there have been some important advances regarding t-cell lineage choice, including the identification of transcription factors required for cd4 gene silencing by cd8-lineage cells (runx3) or for cd4+ t-cell differentiation (gata3), and a better understanding of how t-cell receptor (tcr) signalling correlates cd4/cd8-lineage differentiation to mhc specificity. this review summarizes these recent advances and highlights potential links between tcr signals and nuclear effectors of lineage differentiation (bosselut r, 2004) the purpose of this study was to determine the effect of extracts of tapak liman (elephantopus scaber l) as imunostimulant to the development of lymphocytes in mice balb/c. materials and methods this experiment used 4 groups, 1 group control and 3 treatment groups, with simple randomization. the samples taken at random (random) of the population reached the inclusion criteria as follows: murinestra in balb/c female, age 6 weeks, and healthy. 12 mice balb/c as are divided into 4 groups, each group consists of three mice. each group of mice is given the same standard food and drink ad libitum. four groups of mice are given: control (k): aquades without extract of tapak liman leaf treatment1 (p1): tapak liman leaf extract 0.5 grams/kg body weight/day treatment2 (p2): tapak liman leaf extract 1.0 g/kg body weight/day treatment3 (p3): tapak liman leaf extract 2.0g/kg body weight/day. the effect of extracts on the development of lymphocytes in thymus can be determined by measuring the percentage and number of lymphocytes expressing the express cd4 +, cd8 +and cd4+ cd8 + in thymus organ using flowcytometry on the 16th day, after the treatments of tapak liman extract at various doses. the data obtained in the form of percentage and number of cells that express the cd will be analyzed statistically by calculating the standard deviation in each treatment and one-way anova analysis to determine the effect of treatment on the development of lymphocytes. if there are significant differences in each treatment, tukey test would be done. analysis were done using spss with a p value<0.05. results and discussion thymus is the primary lymphoid organs, bone marrow as a producer of t cell precursors are derivatives of progenitor cells after differentiation in the thymus to form a functional t cells, and after 4 stages of maturation involves a variety of protein expression and t cell receptor (tcr) as an end to the circulation cell t peripherals (keer, 1998). transitional stages of thymocyte maturation can be characterized on phenotip cells with the tcr-cd3 complex, the presence of cd4 and cd8 coreceptor (kuper et al., 2002). based on the results of flowcytometry analysis (figure 1) note that the percentage of cd4 + expression on the control has an average value of 13:04%, in the treatment of 0.5 g / kg had a mean value 10.67%, in the treatment of 1.0 g / kg had an average of 9.85%. while on treatment of 2.0 g / kg had an average of 10.81%. based on the results of statistical analysis using one-way anova (appendix 8) found that the expression of cd4 +, with a significance value of 0864 (> 0.05). for the expression of cd8 + with a significance value 0676 (> 0.05), whereas for the expression of cd4 + cd8 + has a significance value 0.496 (> 0.05). it could be argued that there is no real influence liman extract of tread on all expressions of concentration versus percentage of cd4 +, cd8 +, cd4 + cd8 + in thymus organs. figure 1. cell profile cd4+, cd8+ and cd4+ cd8+: prosentage cell exspression cd4+, cd8+, cd4+ cd8+ on thymus. control (k), (p1:0.5 gr/kgbw), (p2:1.0 gr/kg bw), (p3:2.0 gr/kg bw). total cells (figure. 2) shows that the concentration of 1.0 g/kg increased the number of cd4+ cells (1615946) when compared to the control and the concentration of 0.5g/kg (976 610). concentration of 2.0 g/kg would reduce the number of cells that express cd4+ (1438173) but still better than in the control and treatment of 0.5 g/kg. likewise, the cd8+ cells that express at a concentration of 1.0 g/kg caused the number of cd8+cells at high (764 786) when compared to controls, 0.5 g/ kg, 2.0g/ kg. concentration of 2.0 shows thedecline inthe number of cytotoxic cells but still higher when compared with the concentration of 0.5g/kg and control. so it can be said that the concentration of 2.0g/kg gave the best effect on the number of cytotoxic cells in mouse thymus organs. total cells that express cd4+ cd8+ (prothymocyte) showed that consentration1.0g/kg showed the best effect when compared with control (7395966) and the treatment of 0.5g/kg (7,455,115), and marmi kelik – tapak liman (elephantopus scaber l) as immunostimulant … 51 2.0 g treatment/kg. showed that the concentration of 1.0g/kg increased the number of cells prothymocyte. figure 2. total cell cd4+, cd8+,cd4+cd8+ on thymus at control, 0.5 gr/kg bw, 1.0 gr/kg bw, and 2.0 gr/kg bw. lymphocyte progenitor cells derived from bone marrow, some of which are relatively non-lymphocyte differentiation have migrated to the thymus and reproduce themselves, here is to obtain properties of lymphocytes t lymphocytes at first thymosit not express cd4 and cd8, cells then develop into double positive (cd4 + cd8+) and eventually mature into single positive (cd4 + or cd8+) can then log back into the blood stream, back into the bone marrow or lymphoid organs and peripheral can live several months or years (schwarz and bandola, 2008). conclusion extract of tapak liman at various doses showed no significant effect on the percentage expression of cd4 + cd8 + and cd4 + cd8 + in thymus organs and cd4 + cd8+ in thymus organs. while the analysis of the number of cells, extracts of tapak liman show its effect on the number of cells that express cd4 +, cd8 + and cd4 + cd8 + in thymus organs. concentration of 1.0g / kg of mice showed a good effect on the increase in t helper cells (cd4 +), cytotoxic t cells (cd8 +) and prothymocyte cells (cd4 + cd8 +). references avani, k. and s. neeta, 2005. a study of the antimicrobial activity of elephantopus scaber. indian j. pharmacol., 37: 126-127. bosselutr, 2004. cd4/cd8-lineage differentiation in the thymus: from nuclear effectors to membrane signals, nature reviews immunology 4, 529-540 (july 2004) | doi: 10.1038/nri1392 but p.-p.-h., hon p.-m., cao h., chan t.-w.-d., wu b.kurokawa t., nakanishi k., wu w., hsu h.-y., maruy-m., mak t.-c.-w., and che c.-t. (1997), sesquiterama m., and kupchan s.-m. (1970), deoxyelephanpenelactones from elephantopus scaber. phytochemtopin and its interrelation with elephantopin. tetraheistry 44, 113d116. dron lett. 33, 2863d2866. cabrera a.-l. and klein r.-m. (1980), flora ilustrada lin c.-c., tsai c.-c., and yen m.-h. (1995), the eva-catarinense: compositae. 3. tribo: vernoniae, vi. luation of hepatoprotective effects of taiwan folk genero: elephantopus. itajaı´, brasil, pp. 397d402. medicine “teng-khia-u”. j. ethnopharm. 45, 113d daisy p, rayan na, rajathi d (2007). hypoglycemic and other related effects of elephantopus scaber extracts on alloxan induced diabetic rats. j. biol. sci. 7: 433-437. hammer m.-l.-a. and johns e.-a. (1993), tapping an ered in la gomera, canary islands, spain. j. chro-amazonian plethora: four medicinal plants of marajo mat. a 1011, 241d244. island, para (brazilourna). j. ethnopharm. 40, 53d75. kritikar, k.r. and b.d. basu, 1991. indian medicinal plants. 2nd edn, allahabad publisher, lalit mohan basu,.livermore, d.m., t.g. winstanley and k.p. shannon, 2001. interpretative reading; recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. j. antimicrob. chemother, 48: 87-102. rastogi, r.p. and b.n. mehrotra, 1990. compendium of indian medicinal plants. vol. 1, central drug research, lucknow and national institute of science communication, new delhi. sankar, v., r. kalirajan, f. sweetlin vivian sales and s. raghuraman, 2001. antiinflammatory activity of elephantopus scaber in albino rats. indian j. pharm. sci., 63: 523-525. schwarz, b. a., bandoola, a., 2006. trafficking from bone marrow to the thymus: a prerequisite for thymopoesis. immunol rev.209: 47-54. tsai cc, lin cc (1999). anti-inflammatory effects of taiwan folk medicine ‘teng-khia-u’ on carrageenan-and adjuvant-induced paw edema in rats. j. ethnopharmacol. 64: 85-89. wang, l., jian, s., nan, p., liu, j., zhong, y., 2005. chemotypical variability of leaf oils in elephantopus scaber from 12 locations in china. chem. nat. compd.41: 491-493. waters, w.r., 2003. expression ofl-selectin (cd62l), cd44, and cd25 on activated bovine t cells .infect immun. 71(1): 317– 326. content_v4n2_5.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 61-67 | doi: 10.14421/biomedich.2023.121.61-67 issn 2540-9328 (online) synthesis, characterization and activity test of natural zirconium zeolite (zr-za) catalyst in the esterification reaction of glycerol with acetic acid anhydride didik krisdiyanto1,*, tutik farihah2, hikmah supriyati3 1chemistry department, faculty of science and technology, uin sunan kalijaga yogyakarta, indonesia. 2department of industrial engineering, faculty of science and technology, uin sunan kalijaga yogyakarta, indonesia. 3postgraduate biology education program, yogyakarta state university, indonesia. corresponding author* didik_kris@yahoo.com manuscript received: 26 september, 2022. revision accepted: 05 october, 2022. published: 17 october, 2022. abstract synthesis of zr-za catalyst used as catalyst for esterification reaction of glycerol with acetic acid anhydrous has been done. catalysts are characterized using an infrared spectrophotometer, x-ray diffraction, and measurement of surface acidity, while reaction products are characterized using a gas chromatography-mass spectrometer. effect of catalyst acidity, reaction time and re-addition of acetic acid anhydrous studied by glycerol conversion and selectivity value of triacetin were yielded. infra-red spectrophotometer and x-ray diffraction analysis of the catalyst showed that the synthesized catalyst was zirconium zeolite (zr-za), while esterification analysis using a gas chromatography-mass spectrometer showed a triacetin product. keywords: glycerol; triacetin; acetic acid anhydrous. introduction for muslims, the halal food is absolute, even if it contains only a trace of additives it would not be allowed. as a result, the production of halal-certified additives is critical, both in terms of sources and production processes; currently, many additives are obtained from non-halal sources and processes. for example, some monoacetyl glycerol (mag), diacetyl glycerol (dag), and tri acetyl glycerol (tag)/triacetin are derived from the esterification of non-halal animal glycerol, such as pork (gültekin, et al., 2020: alzeer and hadeed 2021). triacetin has many uses in both food and non-food applications. triacetin can be used as an aroma ingredient in candy (confectionery), milk drinks, soft drinks, and chewing gum. as for non-food ingredients, triacetin can be used as a solvent in perfumes, printing inks, solvents for aromas, plasticizers for cellulose resins, polymers, co-polymers, and can even be used as a fuel additive to reduce knocking in car engines. in addition to triacetyl glycerol (tag) and triacetin, monoacetyl glycerol (mag) and diacetyl glycerol (dag) are formed during the esterification of glycerol with acetic acid (nuryoto et al., 2010; gama et al., 2019; yanti et al., 2019) glycerol derivatives such as monoacetyl glycerol (mag), diacetyl glycerol (dag), and triacetyl glycerol (tag)/triacetin can be used as alternative food additives (additives). food additives are ingredients that are intentionally added to food to improve the appearance, texture, taste, and shelf life of the food. food additives such as monoacetyl glycerol (mag) and diacetyl glycerol (dag) are used as thickening agents or emulsifiers. meanwhile, triacetil glicerol (tag)/triacetin is a solvent for other additives such as flavorings and dyes (ogawa., et al.,1992; fiume 2003; jiang et al 2010) glycerol is a by-product of biodiesel from the transesterification process to obtain methyl esters. in 2010, it is estimated that glycerol production was around 1.2 million tons, of which more than half came from biodiesel production (appleby, 2003). as a by-product of the biodiesel industry, glycerol has not been processed much, so its selling value is still low. therefore, it is necessary to process glycerol so that it can become a product with higher selling value and more benefits. one of them is by making glycerol derivatives through the esterification process. one of the glycerol esterification products is triacetin. (ghoreishi., 2013; setyaningsih et al., 2020; reddy, 2010) the glycerol esterification reaction usually uses acetic acid. silva et al. (2010) have conducted a study to compare the glycerol esterification reaction using acetic acid and anhydrous acetic acid. in the study described, under the same conditions, esterification of glycerol https://doi.org/10.14421/biomedich.2023.121.61-67 62 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 using anhydrous acetic acid gave high selectivity to triacetin (100% at 80 minutes) compared to acetic acid (7% at 120 minutes). glycerol derivatives are made from the esterification process between glycerol and acetic acid with the help of a catalyst. in previous research using a homogeneous catalyst as done by widayat et al. (2013), they used a sulfuric acid catalyst because it is superior in its hygroscopic properties, which can absorb water, for the esterification reaction goes towards the product. however, the use of homogeneous catalysts has disadvantage that the catalyst is difficult to separate from the product after the reaction because it is in one phase (dewajani., et al., 2019) the use of heterogeneous catalysts is an alternative in the glycerol esterification reaction. zirconium is widely used in catalytic processes as a support and a promoter in catalysts (tanabe and yamaguci., 1994; teterycz et al., 2003). zirconium is also an important metal because of the possibility of strong bond polarization between sio2 and zrd+ (beck et al., 1992). in addition, zirconium has a low surface area (usually 50 m2 g-1) (nawrocki, j. et al., 1993). for this reason, this research would focus on the synthesis of glycerol derivatives, namely triacetin, using heterogeneous zirconium catalysts immersed in natural zeolites. then proceed with the optimization of reaction conditions and the analysis of halal results through food critical point tests. methods natural zirconium zeolite catalyst synthesis zeolite pretreatment the first stage was natural zeolite obtained from klaten, central java, was pulverized to form a powder with a size of 100 mesh, then washed with distilled water and heated at a temperature of 120°c for 1 hour. activation of natural zeolite 80 grams of natural zeolite were soaked with 100 ml of 1% hf solution in a plastic container, then washed and dried in an oven for 1 hour at 120 °c. then soaked again in 100 ml of hcl 2m for 30 minutes and washed again with distilled water until the cl ions disappear. then dried again in the oven. making natural zirconium zeolite catalyst by the impregnation method the natural zeolite obtained was impregnated with zirconium metal with concentrations of zr 0%, 1%, 2%, 3%, 5% and immersed in zrocl2.8h2o solution while stirring with a magnetic stirrer, then washed with distilled water and calcined for 4 hours at 400°c. synthesis of glycerol derivatives this step was carried out by heating glycerol and anhydrous acetic acid separately until it reaches 80 °c. both are reacted in a three-neck flask after reaching 80 °c, and a zirconium zeolite catalyst was added. the combined solution was then heated to 115 °c and stirred with a magnetic stirrer at 1000 rpm for 4 hours. discussion catalyst characterization characterization using ft-ir characterization using ft-ir is shown in figure 1. infrared analysis was carried out to determine the active group in the zeolite solid. hendayana (1994) stated that infrared spectroscopy is used to determine the structure, which is important information about the functional groups of a molecule. infrared spectra analysis is grouped into 2 parts, namely wave numbers 4000–1250 cm-1 and 1250-350 cm-1. observations with an infrared spectrophotometer showed specific peaks. generally, there is no significant difference in absorption in the infrared spectrum of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). in the spectrum, it can be seen that there are peaks at the same wave number, namely 3448.72 cm-1 which indicates oh absorption. at absorption wave numbers 2360.87cm-1 and 2368.59 cm-1, it shows the stretching vibration of sioh. the absorption is sharply widening in the wavenumber region. internal and external zeolites interwoven are shown at an absorption of about 1000 – 1250 (cm-1) figure 1. infrared spectrophotometer results of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). krisdiyanto et al. – synthesis, characterization and activity test of … 63 which indicates the presence of o-si-o and o-al-o asymmetric stretching vibrations of the alumina silicate framework. the absorption area 770-803 (cm-1) is the fingerprint region of the zeolite, which shows the presence of o-si-o and o-al-o vibrations. the spectra show the absorption of o-si-o and o-al-o asymmetric stretching vibrations in the 1087.85cm-1 and 1080.14 cm-1 regions and the o-si-o and o-al-o symmetric stretching vibrations in the 794 regions. 67 cm-1. the sio and al-o buckling vibrational regions are shown in the absorption regions of 447.49 cm-1 and 462.92 cm-1 in the wave number table of 1660 cm-1 to 1620 cm-1, there is a reduction in intensity from 1651.07 cm-1 to 1635.64 cm-1 which indicates that there is a release of water molecules bound to the zeolite physically. the loss of absorption proves that the zeolite is cleaner than impurities, so the pores are getting bigger. the increase in intensity at wave numbers 500 cm-1 420 cm-1 indicates that there are more silanol groups in the zeolite framework. this shows that there is a reduction in al-o bonds in the zeolite framework when hcl is added. the width of the peak indicates the increasing number of silanol groups until the crystallinity decreases. where the si-o bond is stronger than the al-o bond, resulting in a higher wave number used to vibrate. this is also supported by the increase in intensity at wave numbers 2385 cm-12363 cm-1 which indicates the presence of more silanol groups in the zeolite framework. table 1. zeolite ftir spectrum interpretation. spectral interval (cm-1) wave number (cm-1) functional group interpretation natural zeolite active zeolite zeolite-zr 5% 3620 3420 3448,72 3448,72 stretching vibration –oh 2385 2363 2360,87 2368,59 stretching vibration si-oh 1660 – 1620 1651,07 1635,64 bending vibration h2o 1213 – 1000 1087,85 1080,14 asymmetric stretching vibration o-si-o and o-al-o 803 – 770 794,67 794,67 symmetric stretching vibration o-si-o and o-al-o 500 – 420 447,49 462,92 bending vibration si-o and al-o characterization using xrd the results of the crystallinity test using xrd can be seen in figure 2. the crystallinity characterization of zeolite was carried out qualitatively using xrd. the xray diffractograms of the za/zr-0 and za/zr-5 samples provide information about the type of mineral and the degree of crystallinity of the structural components that make up the sample. the type of mineral that composes the sample is indicated by the area of peak appearance (2θ), while the level of crystallinity of the component structure is indicated by the high and low peak intensity. the mineral diffractogram from the xrd results is matched its 2θ value with jcpds data (joint committee on powder diffraction standards) so that the type of mineral in the sample will be known. the diffractogram of the zeolite test results using xrd can be seen in figure 2. the diffractogram pattern of za/zr-0 and za/zr-5 in figure 2 looks almost the same. there are certain peaks that experience a change in intensity and a shift in the value of 2θ. changes in intensity there is an increase and there is a decrease depending on the crystal structure, the position of atoms in the unit cell, and thermal vibrations. however, the crystal structure of za did not change much because it was stable when activated. it is as shown in the table. based on table 2 in the za/zr-0 and za/zr-5 diffractograms there are many peaks of interpretation of mordenite character zeolite, this proves that the za/zr catalyst has been dealuminated after activation using 2m hcl. with the decrease in the composition of al cations, the si/al ratio in natural zeolite changed from previously clinoptilolite with the molecular formula [na1.84k1.76mg0.2ca1.24(h2o)21.36] [si29,84al6.16o72] to mordenite with the molecular formula na8(h2o)24] [si40al8o96] here the si/al ratio increases. the results of the xrd diffractogram show that zr metal has been successfully distributed in active natural zeolite, which is indicated by the presence of peaks at 2θ = 22.09 and at 2θ = 35.55. 64 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 figure 2. diffractogram data of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). table 2. interpretation of diffraction peaks at za/zr-0 and za/zr-5. 2θ jcpds 2θ za/zr-0 interpretation 9,77 9,78 mordenite 13,41 13,39 mordenite 19,58 19,58 mordenite 21,24 22,238 mordenite 25,62 25,603 mordenite 26,25 26,25 clinoptilolite 27,60 27,64 clinoptilolite 30,068 30,069 clinoptilolite 30,828 30,83 mordenite 35,65 35,66 mordenite 48,21 48,34 mordenite 2θ jcpds 2θ za/zr-5 interpretation 13,41 13,33 mordenite 19,36 19,47 clinoptilolite 21,79 21,84 mordenite 22,05 22,09 zr 25,56 25,51 mordenite 26,10 26,13 mordenite 27,54 27,54 mordenite 35,53 35,55 zr acidity test for zirconium-impregnated natural zeolite (za-zr) catalyst zeolite acidity can be measured gravimetrically using the ammonia adsorption method. zr metal impregnation treatment on zeolite is expected to increase the acidity of the zeolite. this increase in acidity is due to the exchange of the zr4+ group with the h+ group found in the active zeolite. table 3. catalyst acidity. catalyst acidity (𝒎𝒎𝒐𝒍/𝒈𝒓𝒂𝒎) za/zr-0 4,52 za/zr-5 5,88 za/zr-10 6,79 from the results of the ammonia adsorption analysis, it was found that the total acidity contained in the zeolite increased with the increase in the amount of zr metal impregnation into the active zeolite (table 3). this is due to the interaction between the nh3 base and the acid in the zeolite. h+ is a bronsted acid, which will form nh4+ ions when it interacts with nh3. the presence of zr metal is possible to cause an increase in the acidity of the catalyst because zr metal has a (d) orbital that is not fully filled so it effectively accepts electron pairs from the adsorbate base. the contribution of the number of acid sites of zr metal is a lewis acid site (comelli et al., 2006). the presence of a large number of active sites,increases the adsorption power of the reactants. analysis of glycerol acetylation results the product of glycerol esterification reaction with acetic acid anhydride using zirconium-impregnated natural zeolite (za-zr) as catalyst was analyzed qualitatively using an infrared spectrophotometer. this analysis is intended to determine the presence of esters (triacetin or mono acetin and diacetin) in the esterification reaction product. the results of the infrared spectra can be seen in figure 3. figure 3. infrared spectra of glycerol esterification reaction with acetic acid anhydride using zirconium-impregnated natural zeolite (za-zr) as catalyst. 2-theta (deg) in te n s it y ( c p s ) 20 40 60 0.0e+000 2.0e+003 4.0e+003 6.0e+003 8.0e+003 1.0e+004 2-theta (deg) in te n s it y ( c p s ) 20 40 60 0 1000 2000 3000 4000 5000 krisdiyanto et al. – synthesis, characterization and activity test of … 65 the infrared spectra show a strong absorption at a wave number of 1728.22 cm-1, a strong absorption at a wave number of 1226.73 cm-1, and a medium absorption at 1373.32 cm-1, where the absorptions successively indicate the presence of vibrations from groups c=o, c-o (from the ester), and ch3. the presence of these three functional groups corresponds to the product resulting from the glycerol esterification reaction, which is in the form of an ester. with a reaction like figure 4 (silva et al., 2010). figure 4. reaction of glycerol esterification with acetic acid anhydride using acid catalyst and with the proposed reaction mechanism, namely: figure 5 the mechanism of the esterification reaction for the formation of monoacetate/monoacetin. the reaction mechanism in the figure continues until all the hydroxy groups in glycerol are replaced by acetate groups and triacetin is formed. for one triacetin compound, it takes three compounds of acetic acid or acetic acid anhydride to react with glycerol. the spectrum of the hydroxy group (–oh) also appears in the infrared spectra of the product, which is in the form of a wide absorption at wave numbers of 2962.66 cm-1 to 3600 cm-1, which indicates the presence of vibrations from the hydroxy group (o-h) (sastrohamidjojo, 2007). the hydroxy group can come from acetic acid as a by-product, or from glycerol and (excess) acetic acid anhydride that have not reacted. the results of the complete interpretation of infrared spectra data are presented in table 4 below: table 4. interpretation results of infrared spectral data of glycerol esterification products. wavenumber (cm-1) functional group research result reference specta (sastrohamidjojo, 2007) 1728,22 (strong absorption) 1600-1820 (strong absorption) c=o 1226,73 (strong absorption) 1000-1300 (strong absorption) c-o (of ester) 1373,32 (medium absorption) ±1375 (medium absorption) -ch3 2962,66-3600 (widen) 2400-3400 (widen) -oh from the ft-ir data, it can be seen that glycerol has undergone an esterification reaction and has been converted into an ester, namely monoacetin, diacetin, and triacetin. the ft-ir test performed on the product was not sufficient to detect the presence of triacetin. therefore, it is necessary to carry out further tests using a gas chromatography instrument (gas chromatography/gc) which is connected to a mass spectrometer (mass spechtrometer/ms). with this instrument triacetate compounds can be identified and the percentage can be calculated in the resulting product. the results of the analysis with gc-ms in the form of 66 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 two data, namely the chromatrogram derived from the results of the gc analysis and the mass spectra from the results of the ms analysis. chromatogram analysis was based on the level of similarity or similiarity index (si) between the retention time (tr) obtained and the retention time (tr) from the library search report (wiley7nist05.l). the chromatogram results of the esterification reaction products are presented in figure 6. (a) (b) figure 6. chromatogram of esterification reaction products (a), mass spectrometry triacetin spectra (b). in the chromatogram, the triacetin peak is located at the retention time (tr) 7.863 minutes, according to the library search report (wiley7nist05.l) on gc. the complete chromatogram analysis is presented in table 4. table 5. results of chromatogram analysis and results of glycerol esterification reactions. no retention time (minutes) compound si (%) (wiley7nist05.l) 1 2,075 acetic acid 91 2 2,930 acetic acid anhydride 64 3 6,527 diacetin 83 4 7,863 triacetin 90 the results of the chromatogram regarding the presence of triacerin can be seen in (figure 8 (a) ), while the mass spectrometer data can be seen in (figure 8 (b)). from these data we can know the relative molecular mass of a compound and the fragmentation of the compound. the mass spectrometer data of the triacetin compound present at the retention time of 7.866 is presented in figure 7. the figure shows that the molecular mass/charge (m/e) of the compound is 219.1 m/e. (widayat et al, 2013). figure 7. fragmentation of triacetin. krisdiyanto et al. – synthesis, characterization and activity test of … 67 conclusion zirconium-impregnated natural zeolite (za-zr) catalyst has been successfully synthesized were indicated by difractogram of x ray diffraction analysis. zr metal impregnation treatment on zeolite was increase the acidity of the zeolite into 6,79 mmol/gram for za/zr-10 catalyst. the results of the analysis of the esterification product base on infrared spectra and mass spectrometry chromatogram showed the presence of triacetin products. acknowledgments: lppm uin sunan kalijaga for funding assistance for this research in the 2017 beginner research program. competing interests: there was no conflict of interest in this study. references alzeer, j., hadeed, k.a. (2021). halal certification of food, nutraceuticals, and pharmaceuticals in the arab world. in: laher, i. (eds) handbook of healthcare in the arab world. springer, cham. appleby, d. (2003). the impact of biodiesel production on the glycerine market. oral precentation of procter & gamble at american oil chemist society, champain, illinois. beck, j.s. vartuli, j.c. roth, w.j. leonowicz, m.e. kresge, c.t. schmitt, k. d. chu, c.t.w olson, d.h. sheppard e. w., mccullen, s.b. higgins, j.b. schlenker j.l. a new family of mesoporous molecular sieves prepared with liquid crystal templates j. am. chem. soc., 114 (1992), pp. 10834-10843 comelli, n.a., e.n. ponzi, m.i. ponzi. (2006). pinene isomerization to camphene effect of thermal treatment on sulfated zirconia. chemical engineering journal, 117 (2): 93–99 dewajani h, zamrudy w, saroso h, paramarta s, mulya w (2019), conversion of crude glycerol from by-product biodiesel into bio-additive of fuel through acetylation reaction based on modified zeolite catalyst, alchemy volume 7 no 2 page 46-52 fiume mz, (2003)., cosmetic ingredients review expert panel. final report on the safety assessment of triacetin. international journal of toxicology.; 22 suppl 2:1-10. gama, n., santos, r., godinho, b. 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(2020). triacetin production by selective esterification of glycerol over activated zeolite and lewatite as catalyst. materials science forum, 988, 122–127. silva. leonardo n, valter l.c, goncalves, claudio j.a. mota., (2010). catalytic acetylation of glycerol with acetic anhydride. catalysis communications catal commun. 11. 1036-1039. 10.1016/j.catcom.2010.05.007. sastrohamidjojo. h (2007). spektroskopi. yogyakarta: liberty. tanabe k., yamaguchi t., (1994) acid-base bifunctional catalysis by zro2 and its mixed oxides, catalysis today, volume 20, issue 2, pages 185-197, teterycz, h., klimkiewicz, r., laniecki, m.. (2003). the role of lewis acidic centers in stabilized zirconium dioxide. applied catalysis a: general. 249. 313-326. widayat, satriadi h, abdullah, ika windrianto k. handono. (2013). proses produksi triasetat dari gliserol dengan katalis asam sulfat. jurnal teknik kimia indonesia. 12. 192. 10.5614/jtki.2013.12.1.3. yanti, n.r., heryani, h., putra, m.d., nugroho, a., (2019). triacetin production from glycerol using heterogeneous catalysts prepared from peat clay. international journal of technology. volume 10(5), pp. 970-978 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 175-180 | doi: 10.14421/biomedich.2022.112.175-180 issn 2540-9328 (online) inhibitory effect of mammea africana on alpha-amylase and alpha-glucosidase enzymes of rats nwakaego omonigho ebong1,*, jude efiom okokon1,2, jesse idakwoji1 1department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. corresponding author* nwakaebong@gmail.com manuscript received: 10 august, 2022. revision accepted: 23 august, 2022. published: 12 september, 2022. abstract mammea africana sabine (guttiferae), a medicinal plant used traditionally in the treatment of diseases including diabetes was evaluated for effect on alpha-amylase and alpha-glucosidase enzymes in vivo. the stembark extract (30, 60 and 90 mg/kg) of m. africana were investigated in vivo for inhibitory effect on alpha-amylase and alpha-glucosidase enzymes using starch, sucrose and maltose as substrates. acarbose was used as reference drug. the stembark extract caused significant (p<0.05) reduction in blood glucose levels of treated rats with the various substrates used. the results suggest that the stembark extract of m. africana have the potentials to inhibit alpha-amylase and alpha-glucosidase in rats. keywords: alpha-amylase; alpha-glucosidase; hypoglycaemia; mammea africana. abbreviations: blood glucose level (bgl). introduction mammea africana sabine (guttiferae) (syn. ochrocarpus africana oliv.) (m. africana) is a large forest tree of 50 to 100 feet high with bark often yellow with pale scales and resinous yellow sap (hutchison and daziel, 1958). the plant is widely distributed in tropical africa. traditionally, the stem bark of the plant is used by the ibibios, of the niger delta region of nigeria, in the treatment of malaria related fever, diabetes, microbial infections and mental disorders. the stembark is also traditionally used to treat stomach pains, rheumatism pains, scabies, cough and hypertension (raponda-walter and sillans, 1961; adjanohoun et al. 1996). the stembark extract has been reported to possess cytotoxic activity, in vitro (chapuis et al. 1988; okokon et al. 2012). ouahouo et al. (2004) reported cytotoxic coumarins with anti-microbial activity against staphylococcus aureus from the plant stembark. the stembark has been reported to have anti-plasmodial (okokon et al. 2006), cardioprotective (okokon and antia,2007), anti-diabetic, hypolipidaemic (okokon et al. 2007), vasorelaxant (dongmo et al. 2007), antihypertensive (nguelefack-mbuyo et al. 2008), antiinflammatory, analgesic (okokon et al. 2009), antioxidant (nguelefack-mbuyo et al. 2010), antidiarrheal, anti-ulcer (okokon et al. 2010), immunomodulatory, anti-lesihmanial (okokon et al. 2012), depressant and anti-convulsant (okokon and davis, 2014) as well as nephroprotective (okokon and bawo, 2014) and hepatoprotective (okokon et al. 2016) activities. the stembark has been reported to have 5,-7dihydroxy-8-(12-methyl-butryl)–4–n-pentylcoumarins (carpenter et al. 1970, 1971; crichton and waterman, 1978), 4-phenyl and 4alkylcoumarins (games, 1972), mesuxanthone b (carpenter et al. 1971). alkaloids have been reported to be absent in the entire plant parts (gartlands et al. 1980). we report in this study the effect of leaf extract and fractions of the plant on alphaamylase and alpha-glucosidase of rats. materials and methods plants collection the plant material mammea africana (stembark) were collected in anwa forest in uruan area, akwa ibom state, nigeria in january 2022. the plant was identified and authenticated by dr. margaret bassey, at the department of botany and ecological studies, university of uyo, uyo, nigeria with voucher number fphuu 381. extraction the stembarks were washed and shade-dried for two weeks. the dried plants’ materials were further chopped https://doi.org/10.14421/biomedich.2022.112.175-180 176 biology, medicine, & natural product chemistry 11 (2), 2022: 175-180 into small pieces and reduced to powder using electric grinder. the powdered material (1.5 kg) was macerated for 72 h in 50% ethanol. this was thereafter filtered and the liquid filtrate was concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract was stored in a refrigerator at -4˚c, until used for the proposed experiments. animals albino wistar rats (120 -135 g) of either sex were used for these experiments. the animals were housed in standard cages and were maintained on a standard pelleted feed (guinea feed) and water ad libitum. in vivo alpha-amylase and alpha-glucosidase inhibition study alpha-amylase inhibitory study thirty-five wistar rats were divided into 6 groups of 5 rats each. the rats in all groups were fasted for 18 hours and fasting blood glucose concentration was first taken at 0 minutes before administration. group i, as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. groups iv, v and vi were administered simultaneously, starch (2 g/kg) and mammea africana stembark extract at 30, 60 and 90 mg/kg respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 90, 120 and 180 minutes (gidado et al. 2019). alpha-glucosidase inhibitory study the procedure as described above was used for this study but with sucrose and maltose used as substrates (gidado et al. 2019). blood glucose determination drops of blood from tip of rats’ tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (accu-chek, indiana). the glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). the reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. light from a light emitting diode strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. an electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the resulting current is directly proportional to the amount of glucose in the sample (who, 2011). statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% level of significance i.e. p≤ 0.05. results and discussion in vivo alpha-amylase and alpha-glucosidase inhibition assay administration of starch (2g/kg) to fasted rats caused varying percentages of increase in blood glucose concentrations of the treated animals after 30 min. the percentages were starch (63.18%), extract-treated groups (6.95-42.73%) and acarbose-treated group (17.97%). these increases were reduced after 60 min to 0%, 15.29% and 20.51% in animal groups treated with 60, 90 and 30 mg/kg of the extract respectively. all the extract-treated groups had their blood glucose level (bgl) reduced to normal without any further increase from 90 to 180 minutes. also, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (table 1). there was 60.78% increase in blood glucose concentration 30 minutes following maltose administration in the control group. however, 17.0321.05 % increases were observed in the extract-treated groups. at 60 minutes, the bgl of the extract-treated groups were significantly reduced with groups treated with 30, 60, and 90 mg/kg having percentage increments of 6.11, 8.63 and 13.15% in bgl respectively. there reductions were sustained and significant throughout the duration of the study (180 minutes) with no increment in bgl recorded in any group (table 2). administration of sucrose (2 g/kg) produced a 46.01% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group and 20.95-56.03% increases in blood glucose concentration of extract-treated groups. the blood glucose concentrations were significantly reduced after 60 minutes post-administration of sucrose in all the extract-treated groups and sustained for 180 minutes with the group treated with the lowest dose of the extract (30 mg/kg) having no increment in bgl followed by groups treated with 60 and 90 mg/kg of the extract with 1.16 and 13.35 % increases respectively (table 3). ebong et al. – inhibitory effect of mammea africana … 177 table 1. effect of ethanol leaf extract of mammea africana on blood glucose level of rat after oral administration of starch load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 73.66±6.17 91.0±7.50(5.81) 80.00±6.02 starch 2000 73.33±8.25 119.66±5.45a(63.18) 115.66±1.33a (57.72) 104.66±2.60a (42.72) 95.66±3.75a(30.45) 92.0±6.35(25.46) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 76.33±3.48(5.53) 74.0±1.00(2.30) 72.33±8.68(0) extract 30 78.0±4.35 111.33±4.97(42.73) 94.0±3.51(20.51) 78.0±1.73() 72.33±0.88() 66.66±0.88() 60 86.33±3.75 92.33±6.33(6.95) 80.0±2.64() 74.00±2.30() 70.00±1.15() 68.66±2.72() 90 82.10±5.85 100.0±13.15(21.80) 94.66±10.72(15.29) 82.0±4.61() 76.66±3.84() 71.0±1.73() data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 2. effect of ethanol leaf extract of mammea africana on blood glucose level of rat after oral administration of maltose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25 90.33±2.33 89.0±4.35 87.33±3.84 maltose 2000 92.0±4.04 134.33±2.90b(46.01) 128.66±5.45a(39.84) 117.33±4.66a(27.53) 97.66±0.66(6.15) 104.16±2.48(13.21) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 79.33±2.96 71.66±3.75 78.0±3.78 extract 30 73.33±1.45 86.0±1.73(17.27) 79.66±0.33(8.63) 74.66±0.33() 72.33±1.45 68.33±5.16 60 76.33±0.66 92.0±3.78(17.03) 81.00±2.51(6.11) 76.0±0.57() 70.6±0.33() 68.66±0.33() 90 76.0±2.51 92.0±5.03(21.05) 86.0±5.50(13.15) 81.0±5.03(6.57) 74.0±3.05() 70.33±2.96() data is expressed as mean ± sem. significant at ap<0.05, bp< 0.01, when compared to control (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 3. effect of ethanol leaf extract of mammea africana on blood glucose level of rsat after oral administration of sucrose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 90.33±2.33(3.62) 89.0±4.35(1.55) 87.33±3.84(3.98) sucrose 2000 82.30±2.14 132.33±1.90b(60.78) 130.22±2.45(58.22) 120.66±3.22a(46.60) 115.0±2.46(39.73) 106.22±4.24(29.06) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20c(0.77) 85.33±2.15c() 84.26±1.14a() 82.28±2.26a() extract 30 84.6±2.60 102.33±8.64(20.95) 94.33±1.20a(11.50) 91.66±2.40b(8.34) 86.66±2.90b(2.43) 81.33±5.69() 60 85.66±5.36 111.6±6.93b(30.28) 98.0±5.29(14.40) 92.0±4.35(7.40) 90.66±4.91a(5.83) 86.66±3.38(1.16) 90 77.33±1.85 120.66±2.60(56.03) 112.0±3.05b(44.83) 98.0±1.52b (26.72) 93.66±1.85b(21.11) 87.66±1.45c(13.35) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion mammea africana stembark is used in ibibio traditional medicine in the treatment of diseases such as diabetes among others. this work investigated the effect of m. africana stembark on alpha-amylase and alphaglucosidase activities in rats. the extract was found to inhibit increases in blood glucose concentration following starch administration though non-dosedependently. complete digestion of dietary polysaccharides like starch is achieved by the combined action of alpha-amylases and alpha-glucosidase enzymes. the alpha-amylase enzyme digests alphabonds of the alpha-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound alpha-glucosidase enzymes (alongi and anese, 2018; kalra, 2014). inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for 178 biology, medicine, & natural product chemistry 11 (2), 2022: 175-180 managing type 2 diabetes mellitus, many medicinal plants have been reported to possess alpha-amylase and alpha-glucosidase inhibitory potential (esimone et al. 2001; ibrahim et al. 2014). similarly, the stembark extract significantly and non dose-dependently inhibited blood glucose rise when coadministered with maltose and sucrose. acarbose, the standard drug used in this study significantly inhibited blood glucose rise when co-administered with starch, maltose and sucrose. the results of this study corroborate earlier reported antidiabetic activity of the stembark extract of m. africana in rats (okokon et al. 2007; tchamadeu et al. 2010). this further suggest that inhibition of alpha-glucosidase and alpha-amylase activities may be one of the antidiabetic modes of action of the extract. the inhibitory activities of plant extract are linked to their phytochemical constituents especially polyphenols. the stembark extract has been reported to contain 5,-7-dihydroxy-8(12-methyl-butryl)-4-npentylcoumarin (carpenter et al. ;1970;1971; crichton and waterman, 1978), mesuxanthone b (carpenter et al. 1971), 4-n-propylcoumarins and 4-phenyl coumarins (ouahouo et al. 2004) have also been isolated from the stembark. polyphenolic compounds have been variously reported to inhibit alpha-glucosidase and alpha-amylase activities (proenca et al. 2017; su and tang, 2019; proenca et al. 2017). coumarins in particular have been reported to inhibit alpha-glucosidase and alpha-amylase activities (zhao et al. 2015; karakaya et al. 2018). also, xanthones are reported to possess the potentials to inhibit alpha-amylases and alpha-glucosidase enzymes (malik et al. 2020). the presence of these compounds in the stembark extract could have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of the stembark of m. africana. alpha-amylase and alpha-glucosidase inhibitions by plants extracts have been reported severally (ishnava and metisariya, 2018; shirwaikar et al. 2005). phytochemicals implicated as anti-diabetic agents, do so possibly through alpha-amylase and alpha-glucosidase inhibition. the phytochemicals implicated include; flavonoids, saponins, tannins and terpenoids (ishnava and metisariya, 2018; ortiz-andrade et al. 2007; yoshikawa et al. 1998). also, polyphenolic compounds from plants are known to cause several effects on the biological systems which include enzymes inhibitions (funke and melzig, 2005; kalita et al. 2018). the phenolic compounds are known to be strong metal ion chelators and protein precipitation agents forming insoluble complexes with proteins as well as acting as biological oxidants (ishnava and metisariya, 2018). the presence of the polyphenolic compounds in the stembark extract in addition to the xanthones may suggests that their inhibitory potential on alpha-amylase and the membrane-bound intestinal alpha-glucosidase enzymes. conclusions the results of this study suggest that inhibition of alpha amylase and alpha glucosidase enzymes maybe one of the modes of antidiabetic activity of the stembark extract of mammea africana which can be attributed to the activities of its phytochemical constituents. acknowledgements: the authors greatly acknowledges mr nsikan malachy of pharmacology and toxicology department for his technical support. authors’ contributions: noe, jea and ji conceived and designed this study. jea and ji carried out the experiments and drafted the manuscript. noe performed the statistical analysis, edited and reviewed the manuscript. jea, noe and ji read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. references adjanohoun je, aboubakar n, dramane k, ebot me, ekpere ja, enoworock eg, foncho d, gbile zo, kamanyi a, kamoukom, et al (1996). traditional medicine and pharmacopeiacontribution to ethnobotanical and floristic studies in cameroon. porto-novo, benin: cnpms. pp 15. alongi m, anese m (2018). effect of coffee roasting on in vitro α-glucosidase activity: inhibition and mechanism of action. food research international 111: 480-487. carpenter i, mc garry ej, scheimann f (1971). extractives from guttiferae. part xxi. the isolation and structure of nine coumarins from the bark of mammea africana g. don. journal of the chemical society 22: 3783-3789. carpenter i, mc garry ej, scheimann f (1970). the neoflavonoids and 4-alkylcoumarins from mammea africana g. don. tetrahedron letters 46: 3983-3986. chapius jc, sordat b, hostettman k (1988). screening for cytotoxic activities of plants used in traditional medicine. journal of ethnopharmacology 23: 273-284. crichton eg, waterman pg (1978). dihydromammea c/ob: a new coumarin from the seed of mammea africana. phytochemistry 17: 1783-1786. dongmo ab, azebaze agb, nguelefack tb. 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reticulata. chemical and pharmaceutical bulletin 46(8): 1339-1340. 180 biology, medicine, & natural product chemistry 11 (2), 2022: 175-180 zhao dg, zhou ay, du z, zhang y, zhang k, ma yy (2015). coumarins with α-glucosidase and α-amylase inhibitory activities from the flower of edgeworthia gardneri. fitoterapia 107: 122-127. biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 31-33 | doi: 10.14421/biomedich.2015.42.31-33 effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja’far luthfi biology department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract sanrego (lunasia amara), has been used in the folk medicine to increase and/or to treat male fertility. however there is no scientific evidence to confirm the positive effect of the plant on an improvement of male fertility. the objective of this research was to study the effects of the plant (on adult sprague-dawley male rats) at the doses of 30 mg/kg, 60 mg/kg, and 90 mg/kg on the sperm count, motility, and testicular histology. administration were given by force-feeding between 10.00 am and 12.00 pm daily for a period of 42 days followed by sperm quality analysis and testicular histology evaluation. the sperm analysis showed that the sanrego increased the sperm count and sperm motility. the testicular histology also revealed positive effect of the plant on spermatogenesis. overall the present study showed the sanrego is potential plant to increase male fertility. keywords: sanrego, male fertility, testis histology, sperm quality, sperm number, sperm motility introduction male infertility is an increasingly problem in the modern world. inspite of the many effort that have been employed over the years, medical treatment of male infertility has been a failure (hamadeh, 2001; kohn, 2001). in majority of cases of male infertility, the cause is low sperm quality (shen and ong, 2000; clouatre, 2005). one of the important factor that influence fertility are environment. unlike genetic causes of male factor subfertility, environmental causes of subfertility are of particular interest, because of the possibility of its curative or preventive properties (ebisch et al., 2006). a significant environmental factor is nutrition. it is in this area that natural remedies could shine. animal studies have demonstrated the importance of the effects of nutrition on spermatogenesis (mackay 2004; sinclair, 2000). plant have been used by human since time of unmemorable. however, most claims that plant could enhance virility, increase fertility, or arouse sex libido are root in folklore with very little scientific evidence to support them (anonym, 2005; nickel, 2001). despite gap between science and traditional medicine, there are historical evidence to the betterment of the sexual function by medicinal plant used by many culture (waddell, 1980). sanrego (lunasia amara blanco) has been used by local people in some area of sabah (goh et al., 1997), philipina and indonesia (perry, 1980) as folk medicine to cure many ailment. the plant use to treat snake bite, skin diseases, swollen limb, also as a remedy for stomach trouble (perry, 1980). in the recent years, however, there is an extention of the plant’s initial use. based on observation on the goodness of the sexual function of horse graze on sanrego, sulawesian people popularized sanrego to treat male sexual problem. some studies were conducted to determine the effect of sanrego on male reproductive system of rat (darise, 1999; sutanto, 2002). however the result was far from complete. a systematic approach to provide scientific evidence to confirm the claims of the traditional used is greatly needed. the present study was designed to determine the effect of an aqueous extract of lunasia amara blanco stem on the sperm quality and testicular histology of male rats. materials and methods animals fifteen weeks male sprague-dawley rats from the animal house of the university kebangsaan malaysia were used. animals were housed under standarized conditions. rats were divided at random into 4 groups including control. six rats per group received 0, 30, 60, or 90 mg/kg aqueous extract of sanrego each day for 42 days. preparation of aqueous extract of sanrego sanrego was obtained from south sulawesi, indonesia. the plant was identified by djoko santoso, a botanist of the departement of pharmaceutical biology, gadjah mada university. the voucher number 53/biofar/020307 was deposited at the department. aqueous extract was prepared according to a traditional method (gonzales, et. al., 2004; gonzales et al., 2006). in brief, 500 g of the dry powder of sanrego stem were placed in a container with 1500 ml of distilled water and boiled for 30 minutes. the extract was left to cool and 32 biology, medicine, & natural product chemistry 4 (2), 2015: 31-33 was then filtered. the filtrate then freeze-dried and kept in 4° c refrigerator until use. sperm analysis cauda epididymis were separated according to the hamilton (1975). sperm counts were determined using improve neubauer hemocytometer as described previously (prasad et al., 1972; nafa & eshre-siga, 2002) with some modification. in brief, cauda epididimis was minced in 15 ml bww medium (biggers et al., 1971) and incubated with 5 % co2 for 30 minutes at 370c. data were expressed as number of sperm per cauda epididymis. progressive sperm motility was assesed subjectively based on who laboratory manual (1999). treatment to administer sanrego or vehicle, an appropriate oral needle were used to give, once a day, 2 ml aqueous sanrego extract or distilled water. one day after cessation of treatment, the rats were sacrificed. the left cauda epididymides were removed and cleared off the attached fat and connective tissue. the cauda epididymides were prepared for sperm analysis. the testis were prepared for histological preparation. testicular histology after fixation in bouin’s solution, testes were transferred to 70% ethanol, embedded in paraffin and cut at 5 µm. sections were stained with hematoxilin and eosin and digital images of sections were captured using an olympus bx51 microscope and software analysis pro. morphometrical evaluation was done using coreldraw x3 version 13. the diameter of seminiferous tubules was measured in 15 round tubular sections per animal (n = 3 rats/group) at 100×. statistical analysis data are expressed as mean + s.d. and analysed for statistical significance by using one-way analysis of variance (anova). when f test was found significant (p < 0.05), the data were further subjected to the tukey test. data of seminiferous tubule measurement were analyzed using t-test. data were analyzed using software spss 14.0. results and discussion the average sperm number of rats consuming sanrego at dose 30 mg/kg (43.4375 ± 5.22280 x 106), dose 60 mg/kg (49.3000 ± 2.43475 x 106), and dose 90 mg/kg (47.000 ± 1.59992 x 106) were significantly increased compared to the average number of sperms from control groups (35.3375 ± 4.74091 x 106) (table 1). the sperm number from rats consuming sanrego at dose 60 mg/kg was higher than sperm number from rats consuming sanrego at dose 90 mg/kg. however, this were not significantly different. the increase of sperm number indicates the beneficial effect of sanrego on testicular spermatogenesis. however, histological testicle analysis are needed to verify this observation. table 1. sperm count and motility grade of rats after 42 days treatment with water extract of sanrego. treatment dose sperm count(x 106) motility grade control distilled water 35.3375 ± 4.74091 b sanrego 30 mg/kg 43.4375 ± 5.22280 b 60 mg/kg 49.3000 ± 2.43475 a 90 mg/kg 47.000 ± 1.59992 a note for the grade of motility who (1999) (a): rapid progressive motility ; ≥ 25 μm/s, (b): slow or sluggish progressive motility, (c): non progressive motility ; < 5 μm/s, (d): immotile the effect of sanrego on the sperm motility were showed in table 1. the treatment groups have the betterment of sperms motility compared to the control groups. treatment of sanrego with dose 60 mg/kg and dose 90 mg/kg showed ‘a’ grade movement, while control group showed ‘b’ grade movement. sperm motility has been considered as one of the most important predictors of fertility. several reports have demonstrated the correlation of motion parameters with fertilization rates (liu et al., 1991). further studies are required to confirm the mechanisms of action of sanrego on sperm motility. table 2. average of seminiferous tubules diameter of control rats group compare to treatment group. group tubules seminiferuos diameter average + sd control 44.587 ± 3.096 dose 60 mg/kg 48.969 ± 3.504 treatment group provided significant increases in the diameter of seminiferous tubules (48.969 ± 3.504) when compared to the control group (44.587 ± 3.096) (p < 0.005) (table 2). this result was coincide with the sperm number and sperm motility analysis. in general, the present study revealed that aqueous extract of sanrego enhanced the sperm motility, and seminiferous tubules diameter in rats. this biological activity may be due to one or more of the compound/phytochemicals present in the extract. at least ten alkaloids and quinolines are among the constituent of sanrego (goodwin, 1959). this could directly or indirectly lead to an increase in the potential of fertility in male rats, mainly by acting directly on the pituitary gland and influencing its secretion and/or the pituitary gland– spermatogenic axis. however, there is still unknown which particular metabolite of the sanrego has effect on muhammad ja’far luthfi – effect of lunasia amara blanco on sperm number, sperm motility … 33 the variables studied. further studies are required and are in progress to isolate and identify the active principle(s) that acts upon this axis, influencing and increasing this process and the precise mode of its action. conclusion in conclusion, this study support the extention of the traditional use of this herb to treat male fertility problem. the sperm count analysis showed that the sanrego increased the sperm count, progressive motility, and seminiferous tubules diameter. overall the present study showed that sanrego is potential herb to increase male fertility. references anonym, 2005. aphodisiac galore? total health 27 (1): 7 adimoelja, a. 2000. phytochemicals and the breakthrough of traditional herbs in the management of sexual dysfunctions. int. j. androl. 23, suppl.2: 82-84 biggers, j.d., whitten, w.k. and whittingham, d. 1971. the culture of mouse embryos in vitro. in methods in mammalian embryology pp86-116. ed. jc daniel. freeman, san francisco, ca. clouatre, d. 2005. new help male fertility. total health 26/4: 2627 darise, m. 1999. obat kuat: berkuda dengan sanrego. gatra 23: 13. ebisch, i.m.w., f.h. p. pierik, f.h. de jong, c.m.g. thomas, r.p.m. steegers-theunissen. 2006. does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in man? int. j. andr. 29(2): 339-345 foster, s., j.a. duke. 2000. a field guide to medicinal plants and herbs. second edition. houghton mifflin company. boston. goh, s.h., k.h. lee, c.h. chuah. 1997. phytochemical study of borneo: selected plants from sabah lowland forest. j. herbs spices med. pl. 5: 29-52 goodwin, s., a. f. smith, a. a. velasquez, e. c. horning. 1959. alkaloids of lunasia umara blanco. isolation studies. j. am. chem. soc. 81: 6209-6213 hamadeh, m. e., t. zeginiadov, p. rosenbaum. 2001. predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. archives of andrology 46: 99-104 hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat. in: handbook of physiology, section vii, endocrinology, vol.5, male reproductive system (eds d.w. hamilton and r.o. greep). pp. 259-301. american physiological society, washington d.c. humason, g.l. 1979. animal tissue techniques. freeman and company. san fransisco. kohn, f.m. 2001. nonmedical and naturopathic approaches to treatment of male fertility. in proceedings of the 7th andrology symposium. treatment of male infertility viewpoints, controversies, perspectives. giessen, germany. p: 337 liu, d. y., clarke, g. n., & baker, h. w. g. (1991). relationship between sperm motility assessed with the hamilton-thorn motility analyzer and fertilization rates in vitro. journal of andrology, 12, 231-239. mackay, d. 2004. nutrients and botanicals for erectile dysfunction: examining the evidence. alter. med. rev. 9 (1): 4 -16 nafa, eshre-siga. 2002. manual on basic semen analysis. pei, j., e. strehler, u. noss, m. abt, p. piomboni, b. baccetti, k. sterzik. 2005. quantittive evaluation of spermatozoa ultrastructure after acupunture treatment for idiopathic male infertility. fertility and sterility 84(1) : 141-147 perry l. m. 1980. medicinal plants of east and southeast asia: attribute, properties and uses. the m.i.t press. massachusetts. prasad, m. r. n., n.j. chinoy, k.m. kadam. 1972. changes in succinic dehydrogenase levels in the rat epididymis under normal and altered physiologic conditions. fertility and sterility 23 (3): 186-190. shen, h., c. ong. 2000. detection of oxidative dna damage in human sperm and its association with sperm function and male infertility. free radical biology and medicine 28(4): 529-536 sinclair, s. 2000. male infertility: nutritional and environmental considerations. alternative medicine review 5 (1): 28-38. sutanto, h. 2002. uji efek androgenik ekstrak kayu sanrego (lunasia amara blanco) pada tikus jantan.. tesis sarjana. universitas katholik widya mandala surabaya. unpublished. nickel, l.n. 2001. nature’s aphrodisiacs. crossing press. usa waddell, t.g., h. jones, a. l. keith. 1980. legendary chemical aphrodisiacs. journal of chemical education 57(5): 341-342. who. 1999. laboratory manual for the examination of human semen and semen-cervical mucus interaction. new york: cambridge university press. content_v4n2_2.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 35-39 | doi: 10.14421/biomedich.2015.42.35-39 antioxidant potential of black, greenand oolong tea methanol extracts wahyu widowati1*, tati herlina2, hana ratnawati1, gabriella constantia1, i dewa gde sathya deva3 and maesaroh maesaroh3 1medical research center, faculty of medicine, maranatha christian university bandung, jl. prof. drg. suria sumantri mph no 65, bandung 40164, west java, indonesia 2chemist program, faculty of mathematics and natural sciences, university of padjadjaran, bandung. west java, indonesia 3aretha medika utama biomolecular and biomedical research center, bandung jl. babakan jeruk 2 no 9, bandung 40163, west java, indonesia author correspondency*: wahyu_w60@yahoo.com abstract degenerative diseases and chronic diseases are often caused by oxidative stress. oxidative stress caused by free radicals. antixodant as inhibitor are needed to prevent it which is one of antioxidant sources is tea. tea processing generally produce various kinds of teas such as black, green and oolong tea. tea processing affect the content of phenolic compounds. the aim of the research is to evaluate phytochemical content, total phenolic content of black tea, green tea and oolong tea extracts using catechin, quercetin, kaempferol, myricetin as standard, and to evaluate the antioxidative potency of black tea, green tea and oolong tea extracts compared to catechin, quercetin, kaempferol, myricetin. phytochemical assay using modified farnsworth method, the antioxidant activity were measured by by its 1,1-diphenyl-2-picrylhydrazyl (dpph) scavenging activity. green tea extract contained highest phenolic and flavonoid. the highest antioxidant activity was green tea extract with ic50=0,487 μg/ml. green tea extract content phenol and flavonoid are higher compared to the other extracts, green tea extract has the highest antioxidant activity. keywords: antioxidant, black tea extract, green tea extract, oolong tea extract, total phenolic content introduction various degenerative and chronic diseases are often caused by oxidative stress. oxidative stress is caused by free radicals. it highly reactive and unstable because of its unpaired electron in the outer atomic orbital. free radical can react with the cells molecules by binding to it. it can oxidize the nucleic acid, proteins, fats and even the cells dna and initiate the degenerative disease (halliwell & gutteridge, 2007). free radicals are derived either from normal essential metabolic process in the human body or from external exposure (bagchi & puri, 1998). the antioxidants as oxidation inhibitor are needed to overcome the negative effect of the free radicals. antioxidant inhibits the oxidation by reacting to reactive free radicals to form reactive substances that relatively stable. endogenous antioxidant already present in the human body. however the exogenous antioxidant still needed if the free radicals present in copious amounts (johnson, 2002). a balance between free radicals and antioxidant is necessary for proper physiological function. antioxidant may exert their effect on biological systems by different mechanism, including electron donation, metal ion chelation, co-antioxidants, or by gene expression regulation (krinsky, 2000; lobo et al., 2010). there are two kinds of antioxidant based on its source, natural antioxidant and synhetic antioxidant. synthetic antioxidants are carcinogenic when it consumed for long term. the needs of natural antioxidant that have fewer side effect and less toxic continues to rise.natural antioxidants can protect the body against the damage caused by reactive oxygene species (ros), inhibit the degenerative disease and inhibit the lipid peroxidase activity (wiseman et al., 1997). tea-one of the most popular beveragecomponents possess antioxidant activity.most commercially prepared tea is obtained form the leaf of the plant camelia sinensis (satoh et al., 2005). among tea, 69% of consumption is black tea, 28% of consumption is green tea and 3 % others is oolong tea (cabrera et al., 2008). teas of c.sinensis undergo different manufacturing processes. green tea is produced by steaming (japan) or panning (china) to prevent cathechin oxidation by polyhenol oxidase. oolong tea is semi-fermented while black tea is fully fermented (eric et al., 2011). many studies have shown that green, black and oolong tea has antioxidant properties (xie et al., 1993; wiseman, 1997; mckay & blumberg, 2002; zhu et al., 2002; higdon & frei, 2003; satoh et al., 2005). tea processing will affect the phenol content and ultimately will affect the antioxidant activity of it (higdon & frei, 2003). therefore, it become important to investigate the antioxidant activity of the extract of black tea, green tea, and oolong tea and test the phenol content based on the cathechin, quercetin, kaempferol and myricetin standard. methodology plant material preparation, extraction and phyrochemical content assay dried leaves green tea and black tea were obtained from cisaruni plantation, ptpn viii, west java, indonesia 36 biology, medicine, & natural product chemistry 4 (2), 2015: 35-39 and dried oolong tea was obtained from tea plantation, east java. extraction was performed based on maceration method using methanol 96% as the solvent (widowati et al., 2011a; widowati et al., 2013a; widowati et al., 2014a; widowati et al., 2014b). the extraction yielded 20.139 % of black tea, 22.11 % of green tea, and 17.39 % of oolong tea. the green tea, black tea, and oolong tea extract were tested by phytochemical assay using modified fransworth method including terpenoid, phenol, steroid, triterpenoid, flavonoid, tannin, alkaloid, and saponin assay (fransworth et al., 1966; widowati et al., 2010; bera et al., 2014). total phenol assay briefly 25 µl standard solution in 10 concentration level (500; 250; 125; 62,5; 31,25; 15,625; 7,81; 3,95; 1,98; and 0,98 µg/ml) of catechin, quercetin, kaempferol, and myricetin and sample (extract from green tea, black tea, and oolong tea) in concentration of 500 µg/ml were prepared for total phenol assay. each standard and sampled was mixed with 125 µl follin 10% and 100 µl of na2co3 7,5 % in microplate. the reaction then incubated at 45°-50°c for 10 minutes. the absorbance was measured in 760nm of wavelength using microplate reader. the linear regression equation (y= + ) was created based on the standard (cathechin, quercetin, kaempferol, myricetin) absorbance value. the analysis of phenol content of sample was performed based on the each of standard linear regression equation (ivanova et al., 2005; widowati et al., 2011a; widowati et al., 2015). dpph scavenging activity assay dpph (2,2-diphenyl-1-picrylhydrayl) free radical scavenging activity were analyzed based on the linear regression equation continued by median inhibitory concentration (ic50)value determination. ten concentration levels (500; 250; 125; 62,5; 31,25; 15,625; 7,8; 3,9; 1,9; and 0,9 µg/ml) of black tea, green tea,and oolong tea extract as working solution was prepared for this assays. briefly, 50µl samples (working solution) were added to a microplate followed by 200µl dpph (sigma-aldrich) solution (0,077 mmol/l in methanol). the reaction was shaken vigorously and kept in the dark for 30 minutes at room temperatures. dpph scavenging activity was determined by microplate reader at 517 nm. metanhol absolute was used as blanko. the ic50 value then determined. the radical scavenging activity of each sample was measured according to equation 1 (han et al., 2004; widowati et al., 2013b; widowati et al., 2013c; widowati et al., 2015).%=( − )/( ×100) (1) description as = sample absorbance ac = negative control absorbance (without sample) results phytochemical content of tea extracts table 1 shows the different phytochemical content, including terpenoid, phenol, steroid, triterpenoid, flavonoid, tannin, alkaloid, and saponin of green tea, black tea, and oolong tea methanol extract. all of phytochemical tested was found in the green tea extract. high level of phenol also found in green tea and oolong tea extract. green tea also containts the highest level of alkaloid compared to the other tea extract. otherwise, steroid and alkaloid did not observed in the black tea extract. triterpenoid and tannin also not found in the oolong tea extract. less content of saponin also found in all of the extract. table 1. phytochemical content assay of black tea, green tea, and oolong tea methanol extract. sample phytochemical assay terpenoid phenol steroid triterpenoid flavonoid tannin alkaloid saponin black tea ++ ++ + ++ + + green tea + ++++ + + +++ + +++ + oolong tea + ++++ ++ +++ + + description: ++++ : very high content, +++: high content, ++: moderate content, +: low content, : undetected phenolic compound of tea extracts statistical assay based on linear regression equation was performed to analyze the total phenol of black tea, green tea, and oolong tea based on the standard solution (cathechin, quercetin, kaempferol and myricetin). the highest level of total phenol was showed by green tea extract (table 2). the degree of oxidation affected by the polyphenols profile of the tea. (balentine et al., 1997). wahyu widowati, et.al. – antioxidant potential of black, greenand oolong tea methanol extracts 37 table 2. the average of total phenol level concentration of black tea, green tea, and oolong tea (μg/mg). sample catechin equivalent(ce) quercetin equivalent (qe) kaempferol equivalent (ke) myricetin equivalent (me) black tea extract 14,33 4,50 4,33 4,17 green tea extract 23,33 9,71 4,33 6,17 oolong tea extract 16,08 5,09 3,33 4,67 dpph scavenging activity the most active extract in dpph scavenging activity showed by green tea extract indicated by the lowest ic50 value compared to the other extract and standard (table 3). dpph is a stable free radical because of the unpaired electron. the unpaired electron of the radical becomes paired in the presence of hydrogen donor, decreasing the absorption in 517 nm of wavelength. the dpph scavenging activity has been widely used to test the compound ability as a free radical scavenger and antioxidant activity in food or plat extract (satoh et al., 2005). previous study already obtained that the black tea has the antioxidant activity through dpph free radical scavenging activity with ic50= 5,405 µg/ml (widowati et al., 2011a), catechin (c) diluted in methanol with ic50 =8.11 µm (evacuasiany et al., 2014) and catechin diluted in dmso with ic50 =7.02 µg/ml (budiman et al., 2014). table 3. inhibitory concentration (ic50) value of antioxidant dpph scavenging activities of tea extract and standard. samples replication linear equation r2 ic50(µg/ml) average ic50 green tea extract 1 y= 4,299x + 48,70 0,767 0,30 0,487 ± 0,2582 y= 4,539x + 46,45 0,735 0,78 3 y= 4,342x + 48,37 0,751 0,38 oolong te extract 1 y= 7,363x + 12,93 0,911 5,03 5,005 ± 0,060 2 y= 7,225x + 13,55 0,918 5,04 3 y= 7,286x + 14,03 0,911 4,94 2 y= 6,592x + 3,575 0,989 7,04 3 y= 6,666x + 3,671 0,987 6,95 quercetin 1 y= 6,153x + 23,65 0,901 4,28 4,279 ± 0,0652 y= 6,095x + 24,32 0,893 4,21 3 y= 6,128x + 23,39 0,878 4,34 kaempferol 1 y = 8,731x 11,662 0,823 7.06 7.154±0.1342 y = 8,063x 8,924 0,887 7.31 3 y = 9,039x 14,107 0,827 7.09 myricetin 1 y = 12,803x 6,749 0,829 4,43 2 y = 11,588x 4,270 0,809 4,69 4,496±0,058 3 y = 10,89x + 2,464 0,719 4,37 discussion tea is one of the most widely consumed beverages in the world which is grouped into three main groups, including green, black, and oolong tea, according to the fermentation process, fermentation of tea can affect the phytochemical content of different tea extract confirmed by the result that highest phenol and flavonoid content was observed in green tea (non fermented tea) and oolong tea (semi fermented tea) compared to black tea (fully fermented tea). in the fermentation process, the catechin is oxidized resulting thearubigin, theaflavin including theaflavin (tf1), theaflavin-3-gallate (tf2a), theaflavin3’-gallate(tf2b), and theaflavin-3,3’-digallate (tf3b) which is the key component of the black tea flavour (leung et al., 2001;. yang & landau, 2002; usda, 2003). phytochemical assay of green tea extract catechins are a group of natural polyphenols found in green tea. previous study of green tea exract using ehanol 70% resulted phenols (+++), triterpenoids (++), steroid (), terpenoids (+++), saponins (+), alkaloids (++), flavonoids (++), tannins (+++) (fanny et al., 2015). different solvent concentration resulted different compounds, yield and bioacivities (tiwari et al., 2011) the different solvent would result the different compound and bioactivity (pujimulyani et al., 2004; widowati et al., 2011b)., validated by previous study that water extract of forsythia koreana flowers exhibited a higher phenolic content than ethanol extract (yang and kang, 2011). otherwise, theaflavins are another group of polyphenol 38 biology, medicine, & natural product chemistry 4 (2), 2015: 35-39 pigment found in both black and oolong teas.oolong tea that partially oxidized and retains a considerable amount of the original catechin (nor & mohd, 2003). many studies have demonstrated that both catechin and theaflavin have strong free-radical-scavenging activity both in vivo and in vitro (lai et al., 2001). the chemical composition of tea includes proteins, polysaccharides, polyphenols (cathecins or flavan-3-ols, theaflavins, thearubigins, and proanthocyanidins), chlorophyll, minerals, and trace elements volatile compounds, amino and organic acids, lignins and alkaloid (chen et al., 2009). tea is wellknown for it health benefits because of its poliphenol bioactive content, especially chatechin which have antioxidants activity that play a role in reducing the free radical effect (vecchia et al., 1992; bravo et al., 1998; nagao et al., 2005). catechin belong to flavonoid that posses the high antioxidant activity in biological system. (gramza et al., 2005; evacuasiany et al., 2014) according to the table 2. the most active extract in dpph scavenging activity was the green tea extract showed by the lower of ic50 values compared to the catechin, quercetin, kaempferol, and myricetin. the green tea extracts have the higher antioxidant activity through dpph scavenging activity and ic50 value compared to the black tea extracts (widowati et al., 2011a). that result is in accordance to the phytochemical assay (table 1) and total phenol assay (table 2) showed that the green tea extract containts the higher total phenol content compared to black tea and oolong tea extracts and the higher flavonoid compared to the black tea extracts. high correlations were observed between antioxidants activities and polyphenol phytochemicals content. the antioxidants activities most probably might be contributed by polyphenols contents in the plant extracts (bakar et al., 2009; ling et al., 2010; nor & mohd, 2013). the result of this study also confirmed that the green tea extract posses the higher antioxidant activity compared to the catechin, quercetin, kaempferol and myrietin. table 2 also confirmed that teas extract contain catechin and various compound including quercetin, kaempferol and myricetin (cabrera et al., 2006). some research also investigated that black tea, oolong tea and green tea posses the antioxidant activity (xie et al., 1993; zhu et al., 2002). this study also in line with the research that polyphenol compound have antioxidant activity (higdon & frei, 2003; widowati et al., 2011a). green tea is the good source of polyphenol especially for flavanol and flavonol compounds which is found in 30% of tea leaf dry mass (wolfram et al., 2008). the antioxidant activity of green tea extracts was higher than oolong tea and black tea extracts (gramza et al., 2005). satoh et al (2005) also found that the green tea has the highest percent dpph scavenging activity followed by roasted tea, oolong tea, and black tea 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online available zhu q, hackman r, ensunsa j, holt r, keen c (2002) antioxidative activities of oolong tea. j agric food chem 50: 6929-6934. content_v4n2_3.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 15-18 | doi: 10.14421/biomedich.2016.51.15-18 determination of leisure levels of village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural green village and environmentally friendly supriatna1, thaqibul fikri niyartama2, and iwan kuswidi2 1faculty of sharia and law, 2faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: soepriatna@yahoo.co.id abstract this study took place in the village of patronage uin sunan kalidjaga yogyakarta that consist of 13 hamlets (klidon, banjarsari, wonosalam, dongkelsari, puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong and surirejo), sukoharjo village, district ngaglik, diy sleman regency. data are collect and analysed in order to obtain results in a level of comfort. the analysis was performed by using a formula based on the comfort level temperature humidity index (thi). the results showed that hamlet klidon, banjarsari, wonosalam, and dongkelsari shows not comfortable, whereas hamlet puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong, surirejo and mujen show strongly comfortable mainly on the clock 10:00 to 15:00. keywords: patronage village, comfort level, temperature humidity index (thi). introduction the village is basically an ideal residential location. this is because the cool environment filled with trees as a source of oxygen and water supplies are abundant. however, there is a negative impact on rural development which cannot be avoided that include changes in environmental quality. the change of the environmental quality of course have an impact on people and life. one of the changes is a decrease in the environmental quality of air quality including the change in climate parameters, especially temperature. the impact of a decrease in air quality influenced the quality of the rural environment. this is because the human and natural environment cannot be separated, they interact and influence each other, including influenced the surrounding neighborhoods. therefore, one of the factors to consider in choosing a dwelling is a neighborhood that has a good quality of aspects of climate and air temperature. climate is one of the biophysical components are taken into account in the framework of human adaptation to the natural environment, one of them in terms of choosing a dwelling. so it can be assumed that the climatic conditions both macro and micro affect the quality of neighborhoods. changes in the amount of rainfall, air temperature, wind speed and humidity affect the quality criteria of a good village environment. this needs to be considered in the choice of residential location in order to achieve comfort in an activity for the entire population. so far, studies have been conducted regarding the level of comfort (humidity index) in relation to the climatological factors, but most of these studies are not spatially or research areas include a point / block large areas. this is understandable, because the studies conducted terrestrial, which of course takes time and labor costs are great. therefore, research in areas that are quite specific coverage is rarely done. actually, the development of remote sensing technology capable of overcoming the obstacles encountered by research conducted using terrestrial methods. the use of remote sensing data can be used to obtain information that is directly or indirectly related to comfort, then the information can be used to measure the comfort level of a region. in this study, in order to determine the level of comfort in the village of patronage universitas islam negeri (uin) sunan kalijaga necessary information relating to changes in the climate elements such as air temperature, relative humidity, and wind speed. finally, this study is expected to provide information about the quality of the environment in the village of patronage uin sunan kalidjaga as a first step to designing governance of the village towards the village green and environmentally friendly. tools the tools used in this study are: 1. global positioning system (gps) is used to determine the location of the measurement and facilitate plotting the data on a map. 2. thermo hygrometer used to measure air temperature and relative humidity. 16 biology, medicine, & natural product chemistry 5 (1), 2016: 15-18 research data the following data is taken during this study, the village: sukoharjo, district: ngaglik, district: sleman, yogyakarta. table 1. data of air temperature and humidity. no village time position air temperature humidity 1 klidon 10.29 -7.7041547, 110.4261053 33.6 46% 2 banjarsari 10.40 -7.6975199, 110.4247859 32.8 49% 3 wonosalam 10.46 -7.6937960, 110.4280415 33.9 46% 4 dongkelsari 10.58 -7.6836879, 110.4307653 33.6 45% 5 puntuk 11.10 -7.6886566, 110.4313688 38.1 36% 6 tanjung sari 11.23 -7.6968149, 110.4332366 35.6 41% 7 karang lo 11.36 -7.7006707, 110.4361444 39.4 35% 8 purworejo 11.52 -7.7055966, 110.4323726 40.1 32% 9 tanjung 12.42 -7.7050605, 110.4396199 40.4 33% 10 banturejo 12.52 -7.7089683, 110.4313996 40.8 31% 11 nglengkong 13.00 -7.7085144, 110.4207388 40.8 31% 12 surirejo 13.12 -7.7189814, 110.4244178 42.1 30% 13 mujen 13.25 -7.7189814, 110.4210858 40.4 32% analysis thi value of each criterion comfort level residential areas in table 2. table 2. criteria temperature humidity index (thi). no criteria thi comfort level 1 < 29 comfortable 2 29 – 30.5 uncomfortable 3 > 30.5 very uncomfortable from table 2 can be classified village built which area is comfortable, uncomfortable, and very uncomfortable. to facilitate the reading can be seen in table 3 below. table 3. level of leisure village patronage uin sunan kalijaga. no village thi comfort level 1 klidon 29.9712 uncomfortable 2 banjarsari 29.4544 uncomfortable 3 wonosalam 30.2388 uncomfortable 4 dongkelsari 29.904 uncomfortable 5 puntuk 33.2232 very uncomfortable 6 tanjung sari 31.3992 very uncomfortable 7 karang lo 34.278 very uncomfortable 8 purworejo 34.6464 very uncomfortable 9 tanjung 34.9864 very uncomfortable 10 banturejo 35.1696 very uncomfortable 11 nglengkong 35.1696 very uncomfortable 12 surirejo 36.206 very uncomfortable 13 mujen 34.9056 very uncomfortable supriatna, et.al. – determination of leisure levels of village patronage uin sunan kalijaga yogyakarta … 17 description: horizontal axis : temperature humidity index (thi) : uncomfortable : very uncomfortable figure 1. level of leisure village patronage uin sunan kalijaga. figure 2. temperature humidity index (thi) village patronage uin sunan kalijaga. 0 5 10 15 20 25 30 35 40 klidon banjarsari wonosalam dongkelsari puntuk tanjung sari karang lo purworejo tanjung banturejo nglengkong surirejo mujen 18 biology, medicine, & natural product chemistry 5 (1), 2016: 15-18 in hamlet klidon, banjarasri, wonosalam, and dongkelsari the hours shown in table 3 shows not comfortable, whereas hamlet puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong, surirejo and mujen the hours shown in table 3 shows strongly comfortable. this is because the vegetation is not considered ole surrounding population. conclusion assuming that the temperature humidity index (thi) is a real comfort level, it is necessary to increase the patronage of vegetation in the village in an effort to increase the level of comfort in residential areas. this is seen in the results of the analysis. so the local community can feel comfort and also can improve the welfare of residents of the results of such vegetation. references bintarto, r., s. hadisumarmo, 1978, metode analisis geografi, yogyakarta: lp3es. dewi, anggraini., 1996, penggunaan foto udara dan sistem informasi geografis (sig) untuk mengkaji kualitas lingkungan permukiman dalam hubungan dengan pola penggunaan air minum (air domestik), thesis-s1, faculty of geography, universitas gadjah mada, yogyakarta. ediyono, setijati h., dkk, 1999, prinsip-prinsip lingkungan dalam pembangunan berkelanjutan, direktorat jenderal pendidikan tinggi, departemen pendidikan dan kebudayaan, jakarta. indarto, eddy, 1993, pengaruh suhu udara dan kelembaban udara terhadap tingkat ketidaknyamanan fisiologis penghuni rumah tinggal di perumnas banyumanik semarang, thesis-s2, program pasca sarjana, universitas gadjah mada, yogyakarta. lillesand & kiefer, 1993, penginderaan jauh dan interpretasi citra, yogyakarta: gadjah mada university press. mather, j.r., 1974, climatology: fundamentals and aplications, mcgraw-hill: new york. murdiyarso, d., s. heny, 1992, peranan hutan kota dalam pengendalian iklim kota, seminar sehari iklim perkotaan, jakarta. pudjiastuti, l., r. septa, r.s. happy, 1998, kualitas udara dalam ruamg, direktorat jenderal pendidikan tinggi, departemen pendidikan dan kebudayaan. sulistyaningsih, astin., 1995, distribusi suhu udara dan faktorfaktor lingkungan yang mempengaruhinya serta pengaruhnya terhadap curah hujan di kotamadya surakarta, thesis-s1, faculty of geography, ugm, yogyakarta. sutanto, 1986, penginderaan jauh jilid 1, yogyakarta: gadjah mada university press. sutanto, 1994, penginderaan jauh jilid 2, yogyakarta: gadjah mada university press. suwarsih, kristianan, 1996, evaluasi kesehatan lingkungan permukiman kota berdasarkan foto udara dan sistem informasi geografis (sig) kasus di kecamatan pasar kliwon, kotamadya surakarta, thesis-s1, faculty of geography, ugm, yogyakarta. widyatmanti, wirastuti, 1998, pengaruh perubahan liputan lahan terhadap variasi spasio temporal suhu perkotaan skala mikro dengan bantuan penginderaan jauh dan sistem informasi geografis , kasus studi di daerah semarang utara, thesis-s1, faculty of geography, ugm, yogyakarta. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 171-176 | doi: 10.14421/biomedich.2023.121.171-176 issn 2540-9328 (online) lupenone isolated from diospyros melanoxylon bark non-competitively inhibits -amylase activity mohan krishna durgam1, praveen kumar vemuri2, vijaya lakshmi bodiga3, sreedhar bodiga1,* 1laboratory of biochemistry, department of basic sciences, forest college and research institute hyderabad, mulugu, telangana, india 2department of biotechnology, koneru lakshmaiah university, green fields, vaddeswaram, guntur, andhra pradesh, india 3department of biochemistry and molecular biology, institute of genetics & hospital for genetic diseases, osmania university, begumpet, hyderabad, telangana, india. corresponding author* sbodiga@gmail.com abstract diabetes mellitus is a chronic disease that poses a serious global health problem, due to its associated effects on obesity a nd aging. therapeutic strategies for targeting diabetes include the downregulation and/or inhibition of enzymes such as -amylase and glucosidase, hydrolyzing the dietary carbohydrates in intestine. there is increasing interest for -amylase inhibitors from natural sources. our objective was to undertake the phytochemical screening of bark extracts of diospyros melanoxylon for potential -amylase inhibitory activity and further identification of the active principle and the underlying mechanisms of inhibition. enzyme-assay guided fractionation of the diospyros melanoxylon bark extract led to the isolation of a triterpene, lupenone as a potential inhibitor of -amylase, with a noncompetitive inhibition and inhibitor constant = 30 m. lupenone-mediated inhibition of -amylase responsible for the breakdown of dietary sugar may be effective in preventing postprandial hyperglycemia in the diabetic subjects. keywords: diospyros melanoxylon; ebenaceae; triterpenoid ketone; pancreatic amylase; non-competitive inhibition. introduction diabetes is a chronic disease of multiple etiologies characterized by chronic hyperglycemia with alterations in carbohydrate, fat and protein metabolism. this disorder affects various organs, including eyes, kidneys, nerves, heart and blood vessels. the prevalence of diabetes in india is exploding and 2017 estimates by the international diabetes foundation indicate that 72.9 million people are affected by diabetes in india. type 2 diabetes population in india has a high burden (76.6%) of poor glycemic control(borgharkar & das, 2019). inhibition of intestinal absorption and digestion of carbohydrates targeting the carbohydrate-hydrolyzing enzymes such as -amylase and -glucosidase is an attractive strategy (matsui, ogunwande, abesundara, & matsumoto, 2006; tundis, loizzo, & menichini, 2010). research efforts directed towards screening and developing natural compounds with potential antidiabetic properties is on the rise. -amylase recognizes a consecutive glucose chain as a substrate using its subsite (brayer et al., 2000). acarbose, a typical -amylase inhibitor, has a strong affinity for the enzyme due to pseudo-tetrasaccharide structure (c. li et al., 2005). most of the small molecule inhibitors from natural sources against -amylase include polyphenols with low enzyme specificity (kim, kwon, & son, 2000; h. li, tanaka, zhang, yang, & kouno, 2007; lo piparo et al., 2008; mcdougall et al., 2005). a triterpene glycoside is reported as a specific inhibitor (tarling et al., 2008), and a simple low-polar ketone, chalcone was also found to exhibit -amylase inhibitory activity (najafian et al., 2011). natural triterpenoids have wide spectrum of biological activities (siddique & saleem, 2011). most triterpenes consist of 30 carbon atoms and can be regarded as a compound of 6 isoprene structural units and distributed in plants with both monocotyledons and dicotyledons. two main classes containing either tetracyclic or pentacyclic triterpenes exist. lupene is the most basic structure of lupane-type pentacyclic triterpienoid. it contains a, b, c and d 6-carbon rings and a pentacyclic e ring with an isopropryl group at the 19th position. lupenone is a typical polar lupine type triterpenoid, and it has a ketone group at position 3 in the nuclear ring (meng, huang, & liu, 2008). lupenone is a secondary metabolite in many plants (lee & lee, 1999; na, kim, osada, & ahn, 2009) and possess anti-inflammatory, anti-diabetic, and anti-tumor activity(wang, liu, wang, & zhong, 2012; xu et al., 2014). in this paper, we present the results of a study on amylase inhibition and identification of the active manuscript received: 30 november, 2022. revision accepted: 17 january, 2023. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.171-176 172 biology, medicine, & natural product chemistry 12 (1), 2023: 171-176 principle from the extract of leaves of d. melanoxylon. disopyros melanoxylon roxb (family: ebenaceae), the coromandel ebony or east indian ebony (tendu in hindi), is a middle sized deciduous tree or shrub native to india and srilanka. this plant is considered to be a minor forest produce (mfp) in india, as its leaves are used for making bidi, a traditional cigarette. the leaves are arranged opposite, mostly subopposite or alternate, coriaceous, up to 35 cm long with most of them between 6 and 15 cm; tomentose on both sides when young but when full grown glabrous above, tomentose or pubescent beneath, parrot green; petiolate, with petiole up to 1 cm long; exstipulate, simple and entire; secondary nerve 6-10 pairs, often irregular and branching; tertiary nerves reticulate and raised on upper side; shape variable in the same plant but predominantly of one type out of the four basic forms, namely ellipsoidally lanceolate, ovate, elliptic and orbicular (sometimes cuneate). the bark is pelican in colour, exfoliating in rectangular scales. the wood is also utilized for making boxes, combs, ploughs and beams. the fruits are eaten and sold commercially. the bark is burnt by tribals to “cure” small-pox. the seeds are prescribed as cure for mental disorders, palpitation of heart and nervous breakdown (j. rathore, 1972). recent studies have reported anti-adipogenic, anti-diabetic and hypolipidemic pharmacological activities of d.melanoxylon leaf extracts (k. rathore et al., 2014). however, the phytochemicals responsible for the observed effects have not been elucidated. materials and methods materials porcine pancreatic -amylase (ppa) was obtained from sigma aldrich, usa. ethyl acetate, methanol, benzene, n-hexane were obtained from merck chemicals ltd. acarbose was from bayer pharmaceuticals pvt ltd. soluble starch (extra pure) was obtained from himedia laboratories. all the chemicals and reagents procured were of ar grade. extraction and isolation of inhibitor from the bark of d. melanoxylon the barks of d. melanoxylon were collected from bhadrachalam district of telangana. the plant was authenticated by prof. m. mamatha from the forest college and research institute, mulugu. a digital specimen voucher was deposited in the herbarium of fcri. the bark was shade dried, cut into small pieces, and powdered in a pulverizer. the powdered bark (1.5 kg) of d. melanoxylon was extracted with ethanol (3 x 18 l) at room temperature for 24 h. the extract was filtered and concentrated in vacuo, suitably diluted with water, then partitioned with dichloromethane (3 x 1.5 l), ethyl acetate (3 x 1.5 l) and n-butanol (3 x 1.5 l), successively. column chromatography of the dichloromethane-soluble layer (20 g) over silica gel using n-hexane-acetone mixture with increasing polarity yielded 15 fractions. fraction 2 (2.0 g) that exhibited ppa-inhibition was further applied to a flash column chromatography with rp-18 using methanol/water (30:70 to 80:20) yielded four fractions. ppa-inhibitor was isolated form fraction 4 by silica gel column chromatography eluting with dichloromethane-acetone gradient (1:0, 50:1, 20:1, 10:1, 5:1, acetone). 1hand 13c-nmr spectra were recorded on a bruker advance iii 500 mhz nmr spectrometer using cdcl3 as a solvent. mass spectra were obtained using a waters q-tof micro mass spectrometer. spectral analysis indicated that the isolated compound is lupenone. -amylase inhibitory activity -amylase inhibition assay was carried out according to the method of sudha et al. (2011) based on the starchiodine test with little modification (sudha, zinjarde, bhargava, & kumar, 2011). 75 l of different fractions (100-500 mg/ml) were added to 150 l 0.02 m sodium phosphate buffer (ph 6.9 containing 6 mm sodium chloride) and 75 l -amylase solution, and incubated at 37c for 15 min. subsequently, 100 l from each sample reaction solution was taken and added to 750 l soluble starch (1%, w/v). 500 l phosphate buffer solution was added and incubated at 37 c for 45 min. 25 l of the above mixture was taken and added to 2.5 ml of iodine reagent (5 mm i2 and 5 mm ki) and mixed thoroughly. the color change was observed and the absorbance was taken at 565 nm. control sample was without any inhibitor representing 100% enzymatic activity. to eliminate the absorbance produced by plant fractions, appropriate fraction controls without the enzyme were also included. the standard drug acarbose (-amylase inhibitor) was used as a positive control. it is observed the dark-blue color which indicates the presence of starch, a brownish color indicates partially degraded starch and a yellow color indicates the absence of starch in the reaction mixture. in the presence of inhibitors from the fraction the starch added to the enzyme assay mixture is not degraded and gives a dark blue color complex whereas no color complex is developed in the absence of the inhibitor, indicating that starch is completely hydrolyzed by -amylase. the percentage inhibition of -amylase was calculated using the following formula: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡) 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100 statistical analysis: data were expressed as mean ± standard deviation (sd). statistical analysis was performed using two-way analysis of variance (anova). durgam, et al. – lupenone isolated from diospyros melanoxylon bark non-competitively … 173 results and discussion bioassay-guided fractionation yielded a potential inhibitor against porcine pancreatic -amylase, which was obtained as a white amorphous powder, with a melting point of 168-170c. figure 1. ei-ms spectrum of the isolated inhibitor. the mass spectrum of the isolated inhibitor as shown in figure 1exhibited a molecular ion peak [m]+ at m/z 424, corresponding to a molecular formula c30h48o. fragments at m/z 203 and 218, characteristic of pentacyclic triterpenes were also present in the mass spectrum of compound. the mass spectrometry data also suggested the presence of a carbonyl group (fragment at m/z 205) and the accurate mass of this fragment indicated that it was attached to rings a or b of the pentacyclic ring. the fragment at 409 corresponding to [m-ch3]+ was also observed. the 1h nmr data of the isolated inhibitor represented in figure 2 showed signals for six tertiary methyl groups, which were observed as singlets at 0.79 ppm, 0.97 ppm, 1.04 ppm, 0.84 ppm, 0.74 ppm and 0.92 ppm. the same spectrum also showed resonances for olefinic methylene protons at 4.74 ppm and 4.60 ppm and a vinyl methyl singlet at 1.68 ppm which was shown to be coupled to one of two vinylic protons (h-29a, 4.74 ppm), thus indicating the presence of an isopropenyl group as well as a lupane skeleton. figure 2. 1h nmr spectrum of the isolated inhibitor. the 13c nmr data of the isolated inhibitor shown in figure 3 were in agreement with the mass spectral data, as it revealed the presence of 30 carbon atoms. 13c nmr data of compound was also in agreement with the existence of an isopropenyl group, evidenced by the characteristic vinylic carbon atom resonances at 151.6 ppm and 109.6 ppm, corresponding to carbon atoms 20 and 29 respectively. the existence of an isopropenyl 174 biology, medicine, & natural product chemistry 12 (1), 2023: 171-176 group was also supported by the olefinic methylene protons seen as singlets at 4.74 ppm and 4.60 ppm in the 1h nmr spectrum of compound. the carbon resonance at 218.0 ppm, in the 13c nmr spectrum was assigned to a carbonyl carbon and a resonance at 1.55 ppm, in the 1h nmr spectrum, to two alpha protons of the carbonyl group. figure 3. 13c nmr spectrum of the isolated inhibitor. the chemical structure of the inhibitor ascertained from the chemical shifts of nmr data and mass spectra is presented in figure 4. figure 4. structure of lupenone. -amylase catalyzes the hydrolysis of internal (14) glucosidic bonds. the porcine pancreatic amylase, a 469amino acid containing protein exhibits (/)8 barrel along with a c-terminal -stranded domain with an -crystalline topology (buisson, duee, haser, & payan, 1987; m. qian, haser, & payan, 1993). lupenone showed a dose-dependent decrease in amylase activity with 50% inhibition at 30 m, 85% inhibition at 50 m and 89% inhibition at 100 m. figure 5. effect of different concentrations of lupenone on amylase activity. figure 6. non-competitive inhibition of amylase by lupenone as demonstrated by lineweaver-burk plot. durgam, et al. – lupenone isolated from diospyros melanoxylon bark non-competitively … 175 figure 7. dixon plot for calculation of inhibitor constant. the positive control, acarbose exhibited 50% inhibition at 21.28 ± 1.42 μm. enzyme kinetics data as shown in figure 5 shows a dose-dependent decrease in amylase activity with increasing concentrations of lupenone. further kinetic analysis using lineweaverburk plot shown in figure 6 indicated that vmax decreased, without change in km (2.4 mg/ml) with increasing concentrations of lupenone. these data suggest that lupenone is a non-competitive inhibitor of ppa. further, dixon plot of 1/v vs. [lupenone] shown in figure 7 with increasing concentrations of starch as substrate indicated the inhibitor constant to be 30 m. considering the characteristics of noncompetitive inhibition, lupenone likely binds at an allosteric site separate from the active site of substrate binding, regardless of the presence of a bound substrate. lupenone thus may have the same affinity for both the enzyme alone and the enzyme-substrate complex. binding of lupenone to the enzyme or enzyme-substrate complex inhibits the enzyme, disallowing the production of its end product. inhibition of the enzyme decreases the maximum rate of the reaction (vmax), defined as the rate of the reaction at a substrate concentration that fully saturates all active sites of the specific enzyme. the affinity of the enzyme for its substrate (km) remained unchanged as the active site is not competed for by the inhibitor. inhibition of intestinal enzyme such as amylase responsible for the breakdown of dietary sugar by lupenone may be effective in preventing postprandial hyperglycemia in the diabetic subjects. a large number of in vitro and in vivo studies have shown that lupenonecontaining plants and lupenone have significant anti-diabetic activity. according to the reports, the plants and herbs containing lupenone have good anti-diabetic activity, including rhizoma musae (the root of musa basjoo sied. et zucc.), banana peel (musa nana lour.), thespesia populnea bark and leaf, acanthus ilicifolius l., adenophora triphylla var. japonica, pueraria lobata root, etc. (ahmad et al., 2015; ahn & oh, 2013; callies et al., 2015; parthasarathy, ilavarasan, & karrunakaran, 2009; seong, roy, jung, jung, & choi, 2016). the ethyl acetate and petroleum ether fraction of rhizoma musae could inhibit the activity of α-glucosidase in vitro, and the inhibitory activity of rhizoma musae petroleum ether fraction is higher (zhang, chang, & kang, 2010). the rhizoma musae fraction with anti-diabetic activity could improve the glucose metabolism and increase the oral glucose load in alloxan-induced diabetic mice (h. qian, hao, & wang, 2012). the chemical constituents of euonymus alatus (thunb.) sied. have good inhibitory effects against the protein tyrosine phosphatases 1b (ptp1b) and α-glucosidase enzyme activity, and the lupenone isolated from euonymus alatus (thunb.) sied. and sorbus commixta also could inhibit the ptp1b enzyme activity, while lupenone could not inhibit the αglucosidase enzyme activity (jeong et al., 2015; na et al., 2009). the lupenone from abrus precatorius leaves has α-amylase-inhibitory activity (yonemoto, shimada, gunawan-puteri, kato, & kawabata, 2014). in another study, the ethyl acetate and petroleum ether fraction of banana peel (musa nana lour.) show the antihyperglycemic activity. furthermore, the lupenone isolated from banana peel show promising antihyperglycemic activity (wu, xu, hao, yang, & wang, 2015). our study provides mechanistic evidence that lupenone isolated from d. melanoxylon exhibits potent inhibitory activity against -amylase and therefore could be tested further in animal models of diabetes. conclusion α-amylase enzyme assay guided fractionation of the diospyros melanoxylon bark extract yielded a triterpene, lupenone that exhibited non -competitive inhibition. thus, lupenone-based natural inhibitors of α-amylase may aid in limiting the breakdown of dietary sugar and also may be effective in preventing postprandial hyperglycemia in the diabetic subjects. conflict of interest: the authors declare that there is no conflict of interest. references ahmad, s., sukari, m. a., ismail, n., ismail, i. s., abdul, a. b., bakar, m. f. a., . . . ee, g. c. 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(2010). study on αglucosidase inhibitory activity of extracts from musa basjoo sieb. et zucc. sci tech food ind, 31(2), 125-130. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 289-293 | doi: 10.14421/biomedich.2023.121.289-293 issn 2540-9328 (online) glut4 as a protein target for t2dm therapy with natural compounds vivi hendra sutandar1,*, mgs. irsan saleh2, ziske maritska3 1biomedical science master program; 2department of pharmacology; 3department of biology medicine, faculty of medicine, universitas sriwijaya jl. masjid al gazali, palembang, sumatera selatan 30128, tel. +62-11-58069, fax. +62-11-580664, indonesia. corresponding author* vivihendrasutandar@gmail.com manuscript received: 01 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract who reported 1.9 million death cases of diabetes patients in 2019. diabetes is caused by damage in the pancreas which resulted in a lack of insulin or insulin resistance. medication for t2dm mainly focuses on lowering blood glucose and treating affected organs. current medications are still lacking, thus research is needed in finding novel medications to accommodate t2dm. this paper aims to pres ent the current research on potential plant extract in increasing glut4 translocation in diabetes conditions. insulin resistance st ate affecting glut4 translocation which is important in affecting glucose uptake. some research shows that plant extract proved to be potential in increasing the translocation of glut4 and helping lowering blood glucose levels. keywords: diabetes mellitus; t2dm; glut4; extract. introduction diabetes is a chronic disease either caused by damage to the pancreas which resulted in a decreased production of insulin or the body's unable to use available insulin. diabetes is a public health problem1. there is an increase in mortality rate by 3% caused by diabetes between 2000 and 2019, while in lower-middle-income countries the number increased to 13% mortality rate (who, 2022). type 2 diabetes (t2dm) is a condition characterized by the presence of insulin resistance and damage to the cell  pancreas. insulin resistance is caused by several factors such as high lipid, increased inflammation signal, and activation of stress pathway in reticulum endoplasma. damage in cells  pancreas prompt a decrease in pancreas function that caused dysfunction causing insulin deficiency (muoio and newgard, 2008). several oral medicines are regularly used by t2dm especially sulfonylurea and metformin, alongside some drugs which targeted the affected organ of diabetes. sulfonylurea and thiazolidinedione each have roles in increasing insulin secretion and the latter in increasing insulin sensitivity (perkeni, 2021). nowadays, t2dm medication focuses on the use of metformin and a healthier lifestyle. the use of second-line and third-line drugs helps improve the condition of t2dm patients. however, current medications are still lacking because of the presence of side effects. metformin in a contraindicative individual could increase the risk of lactic acidosis including liver disease, kidney disorder, and lung disease. thus, there is room for improvement in the research in finding novel medications for t2dm to overcome the shortcomings of current drugs (he et al., 2015). until recently, society use plants as a traditional medicine to help treat illness (rahayu et al., 2006). glut (glucose transporter) is a transporter that plays roles in molecules exchanges through membrane cells with (quistgaard et al., 2016) each type has a unique and specific affinity towards its substrates, distribution, location, mechanisms, and physiological function (navale and paranjape, 2016). glut4 is typically found in skeletal muscle and adipose tissue. insulin-induced glucose transport within fat and muscle tissue by stimulating glut4 increased glucose uptake (pessin et al., 1999; saltiel and kahn, 2001). in the insulin resistance state, insulin is unable to increase the translocation of glut4 to the membrane and caused accumulation of glut4 in the membrane compartment. thus, insulin resistance involves a defect in glut4 traffic (garvey et al., 1998). glut4 may show potential as a protein target for t2dm medication due to their roles in regulating glucose uptake by depending on insulin which is present in t2dm patients. traditional medication may prove to be potential in targeting glut4 due to their nature of natural products thus safe to use. several natural compounds derived from plants show their potential in targeting the activation of glut4. this paper's objective https://doi.org/10.14421/biomedich.2023.121.289-293 290 biology, medicine, & natural product chemistry 12 (1), 2023: 289-293 is to present the current research focusing on the potential of plant extract in increasing the activity of glut4 translocation to the membrane with the condition of diabetes. glut4 glucose act as a source of energy for the most organism. glucose characteristics are polar and have large molecule sizes so it cannot pass cell membrane lipids through diffusion. glucose molecules pass the membrane cell with the help of a structural transport protein family known as glucose transporter. two main kinds of glucose transporter have already been identified which are sodium-glucose linked transporters (sglts) and facilitated diffusion glucose transporters (gluts). glut protein consists of 12 membrane-spanning regions with amino acid chains and carboxyl. based on its amino acid sequences and several studies of its sequence alignment found three subclasses of facilitative transporter have been discovered (navale and paranjape, 2016). glut4 fall in class 1 glucose transporter which is insulin-responsive transporter, found in the heart, skeletal muscle, adipose tissue, and brain. glut4 can be found in the vesicle inside the cytoplasm and would be translocated to the membrane after insulin binds to the insulin receptor. after binding, glut4 activation increased 10-20 folds (bryant et al., 2002). glut4 structure is built with a unique sequence of n-terminal and cooh-terminal that play roles in responsibility towards insulin signal and membrane traffic. glucose transfer along the membrane occurs via the glut4 mechanism through facilitated diffusion atp-independent. after glucose influx into the cell, glucose would then be metabolized as energy or lipid synthesis or stored as glycogen (huang and czech, 2007). the translocation of glut4 from the intracellular domain to the plasma membrane would occur after stimulation. during the unavailability of insulin or exercise, 90% of glut4 would be stored inside intracellular. if there is the availability of insulin or exercise hence the vesicle storing glut4 undergoes exocytosis toward the plasma membrane, sarcolemma, and t-tubules of skeletal muscle and plays its role in transporting glucose (shepherd and kahn, 1999). the increase of glut4 molecules on the surface of the membrane would accelerate the rate of glucose transport inside the cell. however, in the condition without stimulation from insulin, glut4 would undergo endocytosis back inside the cell through the process of budding of vesicles on the plasma membrane which contained clathrin, and be stored back inside intracellular vesicles (bryant et al., 2002; shepherd and kahn, 1999). activation of glut4 on skeletal muscle begins with insulin binding to the surface receptor which is a  subunit insulin receptor consisting of a tyrosine kinase domain that would autophosphorylate. phosphoryla  subunit then triggers irs and phosphorylates irs. after irs is phosphorylated then it would bind and activate pi3k and transfer toward the plasma membrane and convert pip2 become pip3. then, after pip3-dependent protein kinase has been activated, it would phosphorylate and activate akt (pkb). akt activation leads to vesicle fusion which involves the translocation of vesicles containing glut4 from the intracellular compartment to the plasma membrane. glut4 elevation on the plasma membrane causes an increase in glucose uptake into cell 15. potential plant bioactive compounds recent researches initiated investigations on plant extract in enhancing glut4 translocation in dm conditions. the table below presents the summary of each article. table 1. summary of study in glut4 activation with various plant extract. study plant extract contents length of follow up outcome yamashita et al., 2012 japan cacao liquor procyanidin extract epicatechin, catechin, and other procyanidins l6 myoblast icr and c57bl/6 male mice (4 weeks old) 7 days of treatment methods: western blot (wb) increased glucose uptake and promote glut4 translocation in l6 myotubes. suppressed hyperglycaemic response after carbohydrate ingestion and enhanced glut4 in mice skeletal muscle. lv et al., 2020 china lipophilic extract from flowers of wisteria sinensis na l6 irap-morange and male c57bl/6j mice (8 weeks old) 4 weeks of treatment methods: wb strongly increased glucose uptake to 2.0 fold compared to normal control and increased glut4 expression while stimulating akt in l6 myotubes in mice, treatment ameliorated hyperglycemia, insulin resistance, and dyslipidemia. also, increased glut4 expression by akt activation in white adipose tissue and skeletal muscle. sutandar et al. – glut4 as a protein target for t2dm therapy … 291 table 1. cont. study plant extract contents length of follow up outcome zhao et al., 2018 china dandelion chloroform extract (dce) na l6 irap-morange methods: wb and realtime pcr dce increased glut4 translocation and expression through the ampk pathway, glut4 fusion to the plasma membrane (pm) independent of ca2+. feng et al., 2022 taiwan anthocyanin-rich extract from black rice (oryza sativa l.) anthocyanin c2c12 myotubes methods: wb hualien and changhua black rice (hbre and cbre) promote glucose uptake. cbre did not affect irs-1 downstream but enhanced pampk/ampk. hbre target pi3k/akt and p38 mapk/erk which both promote glut4 glucose uptake. naowaboot et al., 2012 thailand mulberry (morus alba l.) leaf extract 1deoxynojirimycin (dnj), gallic acid, quercetin. male sprague-dawley rats 6 weeks of treatment. methods: wb increased glucose uptake, and enhanced glut4 translocation while mediated via pi3k signalling pathway. park et al., 2018 korea portulaca oleracea l. extract (poe) flavonoids and triterpenoids (data not shown) mouse 3t3-l1 preadipocyte cells methods: wb poe enhances glucose uptake, by increasing glut4 on pm. increase of pm-glut4 associated with irs-1 phosphorylation, pi3k activation, and akt phosphorylation. pi3k inhibitor and ampk inhibitor inhibit glucose uptake on poe. pujimulyani et al., 2020 indonesia white saffron rhizomes curcuma mangga val. ethanol extract (eecm) curcumin, gallic acid, catechin, epicatechin, epigallocatechin, epigallocatechin gallate, and gallocatechin gallate. 3t3-l1 fibroblastsderived adipocyte cells methods: rt-qpcr eecm increased glucose uptake and glut4 mrna expression. wulansari et al., 2017 indonesia papaya seed extract (carica papaya linn.) na white wistar strain rats 2 weeks of treatment methods: ihc fasting blood glucose (fbg) decrease and increase of glut4 expression based on skeletal muscle staining with ihc after extract treatment zhang et al., 2018 china semen cassia (cassia obtusifolia l.) extract (sce) anthraquinones male sprague-dawley rats 1.30 days of treatment (sce on normal rats) 2.5 weeks of treatment (sce on diabetic rats) methods: wb fbg and serum lipids decrease, while oral glucose tolerance (ogt), and fasting serum insulin (fsi) increase after sce. sce restored the low expression of glut4 translocation in diabetic rats. sce increased thr642, ser473 and pi3k in skeletal muscle lipophilic extract from flowers of wisteria sinensis is the potential as an antidiabetic compound, due to its activity in stimulating glucose uptake by activating protein akt (protein kinase b) and increasing glut4 translocation on l6 cells in vitro. in vivo, the extract increases glut4 expression and helps ameliorate t2dm symptoms in t2dm mice from hyperglycemia to pancreatic islet destruction (lv et al., 2020). cacao liquor procyanidin (clpr) extract is the potential to increase glut4 translocation and glucose uptake in skeletal muscle, due to their content such as epicatechin, catechin, and other procyanidins. clpr help suppressed hyperglycemia response in mice after carbohydrate ingestion while enhancing glut4 translocation in skeletal muscle, due to their beneficial potential clpr help improves glucose tolerance (yamashita et al., 2012). chloroform extract from the dandelion (dce) plant may be another potential extract to help increase glut4 translocation. the research was performed in vitro using l6 cells and the protein was measured by western blot. the result shows that dce upregulated glut4 expression which leads to an increase in glut4 translocation to the membrane, hence is instrumental to the enhancement of glucose uptake to the cells (zhao et al., 2018). anthocyanin-rich extract from black rice (oryza sativa l.) extracted with ethanol from hualien (hbre) and changhua (cbre) region help promotes glucose uptake in c2c12 myotubes by increasing glut4 292 biology, medicine, & natural product chemistry 12 (1), 2023: 289-293 expression and phosphorylation of irs-1. cbre increased p-ampk/ampk while hbre involves in pi3k/akt and mapk/erk (feng et al., 2022). phenolic compounds from mulberry (morus alba l.) leaf extract such as gallic acid help improves glucose uptake and enhanced glut4 translocation thus may be potential as antihyperglycemic. mulberry leaf extract help enhances glut4 translocation by mediating through the pi3k pathway due to glucose uptake getting inhibited by pi3k inhibitor (in this paper used wortmannin). (naowaboot et al., 2012). portulaca oleracea extract (poe) helps enhance glucose uptake by increasing glut4 expression. while enhancing glut4 translocation, poe helps the activation of irs-1, pi3k, and akt phosphorylation. although poe did not involve in protein kinase c phosphorylation, poe involves in ampk pathway activation (park et al., 2018). curcuma manga val. rhizomes ethanol extract (eecm) can increase glucose uptake in lipid-laden 3t3l1 cells, and increase glut4 mrna expression (pujimulyani et al., 2020). papaya seed extract (carica papaya linn.) had a significant influence on glut4 expression in the skeletal muscle of diabetic mice, measured by immunoreactive score and immunohistochemical staining (wulansari et al., 2017). total anthraquinone from semen cassia (cassia obtusifolia l.) extract (sce) was evaluated for its action in improving glucose metabolism in diabetic rats. the result shows that sce enhances glut4 translocation while increasing the expression of phosphorylatedas160 (thr642), phosphorylated-akt (ser473), and pi3k in skeletal muscle. sce help promotes glut4 translocation due to the involvement in pi3k-akt-as160 signaling pathway activation (zhang et al., 2018). conclusion glut4 plays a role in glucose uptake and affects blood glucose levels. t2dm patients' condition is mostly caused by insulin resistance, with the condition of hyperglycemia. glut4 may be a potential protein target to help cope with the symptom of hyperglycemia. various beneficial plants show their ability in increasing glut4 expression on pm through activation either with pi3k or ampk signaling pathway, which contributed to the increase of glucose uptake in vivo or in vitro. based on the research mentioned above, research in finding precise extract and investigation to its exact mechanism pathway in promoting glut4 translocation is needed to discover the best potential plants to target the activation of glut4 in t2dm conditions. competing interests: the authors declare that there are no competing interests. references bryant, n. j., govers, r., & james, d. e. (2002). regulated transport of the glucose transporter glut4. nature reviews molecular cell biology, 3(4), 267–277. https://doi.org/10.1038/nrm782 feng, s. y., wu, s. j., chang, y. c., ng, l. t., & chang, s. j. (2022). stimulation of glut4 glucose uptake by anthocyanin-rich extract from black rice (oryza sativa l.) via pi3k/akt and ampk/p38 mapk signaling in c2c12 cells. metabolites, 12(9). https://doi.org/10.3390/metabo12090856 garvey, w. t., maianu, l., zhu, j. h., brechtel-hook, g., wallace, p., & baron, a. d. (1998). evidence for defects in the trafficking and translocation of glut4 glucose transporters in skeletal muscle as a cause of human insulin resistance. the journal of clinical investigation, 101(11), 2377–2386. https://doi.org/10.1172/jci1557 he, z.-x., zhou, z.-w., yang, y., yang, t., pan, s.-y., qiu, j.-x., & zhou, s.-f. 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(2017). effect of papaya seed extract (carica papaya linn.) on glucose transporter 4 (glut 4) expression of skeletal muscle tissue in diabetic mice induced by high fructose diet. traditional medicine journal, 22(2), 131–137. yamashita, y., okabe, m., natsume, m., & ashida, h. (2012). cacao liquor procyanidin extract improves glucose tolerance by enhancing glut4 translocation and glucose uptake in skeletal muscle. journal of nutritional science, 1, e2. https://doi.org/doi: 10.1017/jns.2012.2 zhang, m., li, x., liang, h., cai, h., hu, x., bian, y., dong, l., ding, l., wang, l., yu, b., zhang, y., & zhang, y. (2018). semen cassiae extract improves glucose metabolism by promoting glut4 translocation in the skeletal muscle of diabetic rats. frontiers in pharmacology, 9. https://www.frontiersin.org/articles/10.3389/fphar.2018.00235 zhao, p., ming, q., xiong, m., song, g., tan, l., tian, d., liu, j., huang, z., ma, j., shen, j., liu, q. h., & yang, x. (2018). dandelion chloroform extract promotes glucose uptake via the ampk/glut4 pathway in l6 cells. evidence-based complementary and alternative medicine, 2018. https://doi.org/10.1155/2018/1709587 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 363-369 | doi: 10.14421/biomedich.2023.121.363-369 issn 2540-9328 (online) fiber concentration on fermentation of cleome gynandra l based on storage time and solvent change lily restusari1,*, ayu komala dewi2, alinea dwi elisanti3 1poltekkes kemenkes riau, pekanbaru, jl melur no 103, pekanbaru 28122, indonesia 2langsat public health center, pekanbaru, jl. langsat no.1, jadirejo, kec. sukajadi, kota pekanbaru, riau 28121, indonesia 3clinical nutrition, health departemen politeknik negeri jember, jl. mastrip, krajan timur, sumbersari, kec. sumbersari, kabupaten jember, jawa timur 68121 indonesia. corresponding author* lily.restusari@pkr.ac.id manuscript received: 11 october, 2022. revision accepted: 11 march, 2023. published: 21 june, 2023. abstract cleome gynandra l (cgl) or maman plant, is the basic ingredient of malay food in riau province, indonesia. the young leaves and stems of cgl are processed into fermented food (joruk maman). it contains crude fiber and is useful for lowering blood cholesterol levels. however, effective storage of this cgl has not been carried out. this study wants to see the effect of storage time and solvent change on the fiber content of joruk maman. an experimental study applied a completely randomized design (crd) using 5 groups and 2 repetitions. this sample of cgl leaves was taken from one seller in the rokan hilir market of riau province. the primary outcome was a difference to the number of fibers in joruk maman without solvent change (p =0.001) and with solvent change (p = 0.001) based on the day group and there was no difference base on the temperature group. secondary outcome was the difference in duration time to produce the highest fiber content at room temperature with the solvent change and not. the highest fiber content occurred at 5 days of storage at room temperature without solvent changing. meanwhile, by changing the solvent, the fiber content would be optimal for 1-day of storage. keywords: cleoma gynandra l; joruk maman; fiber; storage; solvent change. abbreviations: cleoma gynandra l (cgl) introduction the world health organization (who) and the food and agriculture organization (fao) have recommended high consumption of fruits and vegetables to prevent various chronic diseases such as hypertension, coronary heart disease, and stroke. however, who and fao emphasize consuming fresh fruits and vegetables to get high nutritional content (endrizzi et al., 2009) one of the examples is cleoma which belongs to the capparaceae family. the people of rokan hilir, riau, indonesia recognize the maman plant which originated from south africa. throughout africa, the tender leave or young shoots, and often the flower are boiled and consumed as a potherb, delicious dish, stew, or side dish. the fresh leaves are used as an ingredient in other mashed foods, and the dried leaves are ground and put into weaning foods. however, boiling the leaves can reduce the vitamin c content by up to 81%, while drying the leaves reduces the vitamin content by up to 95% (heever & venter, 2007). this maman plant is used as a traditional antidiabetic, anti-agingg, anti-cancer, and cardiovascular disease prevention (mishra et al., 2011). this plant is often found as a wild plant and it grows anywhere. people in this village usually consume this plant as a fermented vegetable. this fermented product of the maman plant (cleome gynandra l) is processed from young leaves and stems which are mixed with salt, rice, and warm water and then it is left for 2-3 days before the people consume it. the villagers of bunga tanjung rohul and tanah putih rohil usually call this food by the name of cgl fermentation or joruk maman. fermented products of the maman plant are commonly consumed by the public, and fermented products can be consumed with or without rice (saida, e, 2014). fermented products of maman plant or cgl fermentation contain crude fiber and lactic acid bacteria (lab) which function as probiotics. dietary fiber has a very important function for health maintenance and prevention of various degenerative diseases such as diabetes, hypercholesterolemia, stroke, coronary heart disease, and obesity as well as digestive disorders such as constipation, hemorrhoids, and colon cancer (winarti, 2010). based on a review (institute of medicine, 2005) the adequacy of total dietary fiber in adolescents and adults https://doi.org/10.14421/biomedich.2023.121.363-369 364 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 is 14 g/1000 kcal. the highest crude fiber content was obtained in cgl fermentation with the addition of 2% salt and 15% rice, which was 0.43 g per 100 g of fermentation. maman has crude fiber content which can reduce cholesterol levels and also contains lactic acid bacteria. the content of lactic acid bacteria in maman's fermentation with the addition of 5% salt and 10% rice had the highest result of 2.40 x 108 cfu/g (lily restusari, muharni, 2019). people can make joruk maman, or they can purchase at traditional markets in rokan hulu and rokan hilir, riau indonesia. traditionally, local people will replace the fermented soaked water with boiled water if cgl fermentation is stored for a long time and the sensory taste change. moreover, after they change the water, they leave it overnight and cgl fermentationis ready to be consumed again with the same taste as the new cgl fermentation produced. materials and methods study area the determination of maman in sekeladi village, rokan hilir regency and determined at the andalas university herbarium (figure 1). figure 1. cleome gynandra l (maman’s plant) by the author on sekeladi village, rokan hilir regency. this research type is an experimental study conducted to determine the storage time and solvent change to fiber content in cgl fermentation by applying a completely randomized design using 1 treatment and 2 repetitions. maman fermentation was stored at room temperature and then was taken aseptically every day on days 1, 2, 3, 4, and 5. on day 5, the solvent was replaced by boiled water with the same amount of water replaced. then, it was continued the inspection of dietary fiber on days 1, 2, 3, 4, and 5 after the solvent was replaced. the research tools used were analytical balance, niemeyer, measuring cup, condenser, funnel, vacuum pump, oven, desiccator, heater, and watch glass. the materials used were maman (cleome gynandra l) leaves, 0.85% nacl diluent solution, table salt (pn salt) and local rice-pandan wangi varieties, sulfuric acid, naoh, whatman filter paper, alcohol, micropipette tips, and distilled water procedures this research was carried out by weighing 1-2 grams of fermented maman samples and put into a 500 ml erlenmeyer, and then it was added 50 ml of hot 1.25% h2so4 and refluxed for 30 minutes. after that, it was added 50 ml of 3.25% naoh and refluxed for 30 minutes. the heated sample was then filtered in hot condition with whatman 42 filter paper whose weight was known. after being filtered, the sample was washed with 50 ml of 1.25% h2so4 and 50 ml of 36% alcohol, then the precipitate was dried in an oven at 105°c and weighed to a constant weight. this research was conducted at the laboratory of agricultural polytechnic of andalas university in payakumbuh and the microbiology laboratory of the health polytechnic of the ministry of health riau. the research time was approximately 6 months from july to november 2020. this research involved 1 alumnus as a field survey officer and 1 laboratory analyst. data analysis the survey primarily collected the sample at the rokan hilir market on the same day, with direct contact with the seller. passing the fermentation process, data was collected at the laboratory. data analysis of dietary fiber results was carried out using spss 25 as the statistic program. all variables were tested for normality, then if the variables were normally distributed, anova block design was applied. to see the variation difference among treatments, it is followed by a posthoc test, namely lsd and duncan to find out the real difference between treatments. results and discussion results explain fiber content in cgl fermentation (joruk maman) base on storage time and solvent change and difference fiber concentration (figure 2-3; table 1-9). fiber content of joruk maman table 1. description of fiber content of joruk maman base on storage time. storage time average of fiber content (gr/100gr) refrigerator temperature room temperature day 1 0.46 0.51 day 2 0.56 0.58 day 3 1.69 1.41 day 4 1.62 1.69 day 5 1.65 1.74 note: primary data sources restusari et al. – fiber concentration of joruk maman 365 the graph of fiber content in cgl fermentation which was stored at refrigerator temperature and room temperature (figure 2) figure 2. fiber content of joruk maman against temperature and storage time. the normality test for the effect of temperature and storage time on fiber content in cgl fermentation are normally distributed. so it is continued with the anova block design test. the results of the anova block design test were shown at table 2. table 2. the analysis of anova block design, temperature effect, and storage time on fiber content of joruk maman. treatment group average + sd p-value storage time 0.891 refrigerator temperature 1.19 + 0.48 room temperature 1.18 + 0.48 storage time 0.001* day 1 0.48+0.77 day 2 0.57+0.77 day 3 1.55+0.77 day 4 1.65+0.77 day 5 1.69+ 0.77 note: * significantly different in the anova block design test (α<0.05) the significance value of the block (day 1,2,3,4,5) was 0.001<0.05. there was a difference between the day group on the number of fibers; the posthoc test could be continued. the results showed in table 3-4. table 3. post-hoc lsd test, temperature effect, and storage time on fiber content in joruk maman. post-hoc lsd test average difference (gr/100gr) p day 1 day 2 -0.0850 0.476 day 3 -1.0650* 0.001 day 4 -1.1700* 0.000 day 5 -1.2100* 0.000 day 2 day 1 0.0850 0.476 day 3 -0.9800* 0.001 day 4 -1.0850* 0.001 day 5 -1.1250* 0.000 day 3 day 1 1.0650* 0.001 day 2 0.9800* 0.001 day 4 -0.1050 0.387 day 5 -0.1450 0.252 day 4 day 1 1.1700* 0.000 day 2 1.0850* 0.001 day 3 0.1050 0.387 day 5 -0.0400 0.731 day 5 day 1 1.2100* 0.000 day 2 1.1250* 0.000 day 3 0.1450 0.252 day 4 0.0400 0.731 note: * significantly different in post-hoc lsd test (p<0.05). 366 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 based on the post-hoc lsd test, the different mean pairs are on days 1, 2, 3, 4, and 5 pairs. the number of differences in the average fiber is highest on day 5 and day 1 which is 1.21. table 4. temperature effect, and storage time on fiber content in joruk maman. treatment average fiber content (gr/100gr) storage time day 1 0.48a day 2 0.57a day 3 1.55b day 4 1.65b day 5 1.69b note: numbers followed by the same lowercase letters in one column show no significant difference in (p < 0.05). the fiber content in joruk maman with a solvent change towards temperature and storage time table 5. description of fiber content in cgl fermentation with solvent change towards temperature and storage time. storage time average fiber content (gr/100gr) refrigerator temperature room temperature day 1 1.79 1.89 day 2 1.65 1.57 day 3 1.24 1.11 day 4 1.11 1.08 day 5 1.1 1.04 note: primary data sources. the fiber content in cgl fermentation which was stored at refrigerator temperature and at room temperature with solvent change also describe of figure 3. figure 3. fiber content in cgl fermentation with changes in solvents against temperature and storage time. the normality test for the temperature effect and storage time on fiber content in cgl fermentation with solvent change were normally distributed, so it was continued with the anova block design test. the results are in table 6. table 6. description of anova block design, temperature effect, and storage time on fiber content in cgl fermentation with solvent change. treatment group average + sd p-value storage temperature 0.359 refrigerator temperature 1.37 + 0.27 room temperature 1.33 + 0.27 storage time day 1 1.84 + 0.43 0.001* day 2 1.61 + 0.43 day 3 1.17 + 0.43 day 4 1.09 + 0.43 day 5 1.07 + 0.43 note: *significantly different in the anova block design test (α<0.05) the anova block design test showed the significant value of the block (day 1,2,3,4,5) was 0.001 < 0.05. there was a difference between the day group with the fibers content; the lsd post hoc test was shown in table 7 and table 8. table 7. description of the lsd test, temperature effect, and storage time on fiber content in joruk maman. post-hoc lsd test average difference (gr/100gr) p day 1 day 2 0.2300* 0.020 day 3 0.6650* 0.000 day 4 0.7450* 0.000 day 5 0.7700* 0.000 day 2 day 1 -0.2300* 0.020 day 3 0.4350* 0.002 day 4 0.5150* 0.001 day 5 0.5400* 0.001 day 3 day 1 -0.6650* 0.000 day 2 -0.4350* 0.002 day 4 0.0800 0.260 day 5 0.1050 0.160 day 4 day 1 -0.7450* 0.000 day 2 -0.5150* 0.001 day 3 -0.0800 0.260 day 5 0.0250 0.703 day 5 day 1 -0.7700* 0.000 day 2 -0.5400* 0.001 day 3 -0.1050 0.160 day 4 -0.0250 0.703 note: *significantly different in the lsd post-hoc test (p<0.05) based on table 7, we know the pairs of average differences are on the 1st, 2nd, 3rd, 4th, and 5th -day pairs. thus, the number of differences in the average fiber is highest on day 1 and day 5 which is 0.77 restusari et al. – fiber concentration of joruk maman 367 table 8. description of the duncan's test analysis of temperature effect and storage time on fiber content in cgl fermentation with solvent change. treatment average fiber content (gr/100gr) storage time day 1 1.84c day 2 1.61b day 3 1.17a day 4 1.09a day 5 1.07a note: numbers followed by the same lowercase letters in one column show no significant difference in (p < 0.05) the difference in fiber content in cgl fermentation before and after dissolving, the paired t-test can be seen in table 9. table 9. paired t-test of fiber concentration in cgl fermentation before and after dissolving. solvent change mean ± std. deviation p-value before 1.19 ± 0.57 0.572 after 1.35 ± 0.33 note: there is a significant difference in the paired sample t-test (∝<0.05) there is no difference in fiber concentration of cgl fermentation before and after solvent changes discussion the fiber content in cgl fermentation at room temperature ranged from 0.51 to 1.74 g/100 g, and fiber content in cgl fermentation at refrigerator temperature ranged from 0.46-1.69 g/100g. both at room temperature and refrigerator temperature, the lowest fiber content was obtained on the first day, namely 0.51 g/100 g and 0.46 g/100g respectively; and the highest fiber content was obtained on the fifth day, namely 1.69 g/100 g and 1.74 g/100g. during the fermentation process, there were bacteria that played an important role, namely acetobacter xylinum which has the ability to convert ethanol into vinegar. however, these bacteria required oxygen in the process. crude fiber it self was the result of sugar reform in the fermentation medium by the activity of acetobacter xylinum (saleh, 2011) (putriana & siti aminah, 2013). there are not many research results that report fiber content related to temperature in this fermented food of the malay tribe, named joruk maman. however, there are similar studies that examine the crude fiber content of the leaves and seeds of the rubber tree (hevea brasiliense). it is known that the crude fiber content of leaves and seeds of rubber trees (hevea brasiliense) after a 2-day fermentation period is much lower than the 5-day and 8-day fermentation periods. however, at 34 and 44 degrees celsius, the crude fiber content is higher than the 2 and 5-day fermentation period. this result occurs because at high temperature, enzyme activity is reduced due to enzyme damage, and the water content of the substrate becomes much reduced. as a result, the raw fiber is converted into sugar. on the first day of the fermentation period, the addition of incubation time will reduce the crude fiber content of the leaves and seeds of the rubber plant (hevea brasiliense). the decrease in crude fiber from a temperature of 24-34 degrees celsius is higher than the decrease in crude fiber from a temperature of 34-44. similar results are also obtained at the 5 and 8-day fermentation periods. these changes are closely related to the growth of molds and cellulase enzyme activity. with increasing incubation time, some cellulase enzymes can break down higher crude fiber; so that, the fiber content decreases (syahruddin et al., 2016). the research that we have done has obtained that the fiber content in cgl fermentation stored at refrigerator temperature compared to room temperature without any changes had the same graphic pattern. on day 3 of storing cgl fermentation at both temperatures, the fiber content increased which was quite high. the block significance value (day 1,2,3,4,5) was different among day groups on the number of fibers. while the different mean pairs were on day 1, 2, 3, 4, and 5 pairs, where the highest number of fiber average differences occurs on day 5 and day 1, namely 1.21. storage time on day 1 (0.48 gr/100gr) had the smallest significant difference compared to other treatments, and storage on day 5 (1.69 gr/100gr) was significantly different from other treatments. the final results showed that the fiber content of cgl fermentation with a combination of 5-day fermentation treatment at room temperature was the best treatment because it had the highest fiber content. during the fermentation process, the ability of lactic acid bacteria (lab) continued to increase in order to produce sufficient lactic acid to loosen the bonds of lignocellulose and lignohemicellulose which are components of crude fiber. thus, the increase of crude fiber was in line with the fermentation time. the ph condition that was not optimal in lab could not produce lactic acid in sufficient quantities to loosen lignocellulosic and lignohemicellulose bonds; so lab had not caused changes in the crude fiber content of the silage. therefore, at the beginning of storage, the amount of lab in cgl fermentation was less; so that, the lactic acid produced was also less to produce crude fiber. another finding of fiber content in cgl fermentation based on temperature and storage time on solvent change was a decrease in fiber content of cgl fermentation from the first day to the fifth day; the decrease reached 0.85 g/100 g. meanwhile, at refrigerator temperature, the fiber content of cgl fermentationday 1 to day 5 decreased to 0.69 g/100g. based on the graph, it can be seen that on day 3 the fiber content decreased quite high. there was a difference between the day groups in the number of fibers. the different mean pairs were on the 368 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 1st, 2nd, 3rd, 4th, 5th day pairs. the highest number of fiber average differences on day 1 and day 5 was 0.77. this finding is in line with olive pomace fermentation with simultaneous production of gallic acid, where the fermentation changes the chemical composition of olive pomace so that crude fiber decreases by 8.56% (fathy et al., 2018). the crude fiber content on the first day continued to decrease significantly to the fifth day until it reached the lowest fiber content. this is inversely proportional to the fiber content of cgl fermentation without solvent change. this decrease in fiber content can occur because it is closely related to the constituent components of the fiber, especially lignin. high lignin will make it difficult for microorganisms (bacteria) to degrade the material, so fiber content is decreased to low. the fiber content of cgl fermentation with the combination of 1 day fermentation period treatment at room temperature was the best treatment because of the highest fiber content in this treatment. the research results are different from other studies related to fiber in nata de cassava which states that the length of nata de cassava fermentation causes acetobacter xylinum bacteria to work depending on differences in the number of nutrients to meet their needs. if the number of nutrients is sufficient, then the amount of cellulose formed is also big, whereas if the number of nutrients is not sufficient, the growth of acetobacter xylinum bacteria is inhibited as a result, a small amount of cellulose is produced (putriana & siti aminah, 2013). the average value of the highest fiber content was obtained in the nata de cassava product on the 7th day of fermentation, which was 94.31 mg. the duration of data de cassava fermentation did not affect the fiber content of the data formed. this was because on day 7 of fermentation, the acetobacter xylinum bacteria were in an exponential phase because the acetobacter xylinum bacteria concealed as much extracellular polymerase enzymes to arrange glucose polymers into cellulose; so that, more data matrix was produced in this phase. the 9th and 11th fermentation times decreased because acetobacter xylinum bacteria were in a slow growth phase because the availability of nutrients had decreased. the 13th fermentation time increased because more data matrix was produced in this phase. there is no explanation regarding the solvent change in the nata fermentation, this is certainly different from the conditions of the cgl fermentationfermentation that we did. in the cgl fermentationfermentation process, we used two groups, namely without solvent change and using solvent change. it turned out that the fermentation of cgl fermentationwhich was replaced by a solvent (using solvent change) was not significantly different. this could be triggered by the previous initial conditions, where the fiber content of cgl fermentation before and after being given dissolving did not have a much different numerical difference. meanwhile, the highest cgl fermentationfiber content occurred on day 5. this could be due to a significant increase in the size of the cellulose fraction compared to the raw material, which was caused by the loosening of the cell walls and release of cellulose, and a reduction in the size of the hemicellulose and pectin fractions, such as the research on sauerkraut. one of the more important functional properties of dietary fiber is its water-holding capacity which reflects the fiber's ability to swell. the results obtained that the ability of cabbage fiber to bind water depended on the cultivar and the length of storage time of fermented cabbage. the rehydration capacity of dried sauerkraut increased with storage time. the increase, compared to fresh cabbage, was an average of 22% after 10 days from the end of fermentation; after 30 and 90 days were at the same level with an average value of 35%. this difference in water absorption probably arises from changes in the fractional composition of the fiber, namely from the increase in the size of the cellulose fraction observed during the storage of sauerkraut (elkner, krystyna, kosson, 2009). conclusions there was a different the number of fibers in cgl fermentation without solvent change and with solvent change based on the day group. the highest fiber content production has 5 days duration, without solvent change and at room temperature. meanwhile with solvent change, the optimal fiber content at 1 day and at room temperature. regarding the length of storage in cgl fermentation, it has the potential to become a probiotic product. acknowledgments: the authors express their gratitude to the health polytechnic of the ministry of health of riau, the laboratory of agricultural polytechnic of andalas university have given permission and examined the samples in this research. hence, it can be carried out well. there is no potential conflicts of interest are noted authors’ contributions: lily restusari designed the study. ayu komalasari carried out the laboratory work. alinea dwi elisanti analyzed the data. lily restusari and alinea dwi elisanti wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: this research supporting from dipa funds poltekkes kemenkes riau 2020 restusari et al. – fiber concentration of joruk maman 369 references elkner, krystyna, kosson, r. (2009). dietary fibre content and its fractional composition in cabbage as affected by cultivar earliness and sauerkraut storage period. journal of fruit and ornamental plant research, 69(1), 165–175. doi: https://doi.org/https://doi.org/10.2478/v10032-008-0031-2 endrizzi, i., pirretti, g., calò, d. g., & gasperi, f. (2009). a consumer study of fresh juices containing berry fruits. journal of food and agriculture, 89(7), 1227–1235. doi: https://doi.org/https://doi.org/10.1002/jsfa.3580 fathy, s. a., mahmoud, a. e., rashad, m. m., ezz, m. k., & mohammed, a. t. (2018). improving the nutritive value of olive pomace by solid state fermentation of kluyveromyces marxianus with simultaneous production of gallic acid. international journal of recycling of organic waste in agriculture, 7(2), 135–141. doi: https://doi.org/10.1007/s40093-018-0199-5 heever, e. van den, & venter, s. l. (2007). nutritional and medicinal properties of cleome gynandra. international conference on indigenous vegetables and legumes. prospectus for fighting poverty, hunger and malnutrition, 752. doi: https://doi.org/10.17660/actahortic.2007.752.17 institute of medicine. (2005). dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). in dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). institute of medicine of the national academies. doi: https://doi.org/10.17226/10490 lily restusari, muharni, f. (2019). activities of fermented maman (activities of fermented maman (cleome gynandra l) on blood sugar level in hyperglycemic white rats. doi: https://doi.org/10.31227/osf.io/4j8q6 mishra, s., moharana, s., & dash, m. (2011). review on cleome gynandra. 1(3), 681–689. putriana, i., & siti aminah. (2013). mutu fisik, kadar serat dan sifat organoleptik nata de cassava berdasarkan lama fermentasi physical quality, dietary fiber and organoleptic characteristic from nata de cassava based time of fermentation. 04(07). saida, e, m. (2014). identifikasi bakteri patogen pada produk fermentasi cleome gynandra. politekkes kemenkes riau saleh, d. (2011). sintesis dan karakterisasi selulosa mikrobial dari whey serta pengaruh iodium pada sifat mekanik, listrik dan absorbsi terhadap limbah mgcl2, mg(oh)2, dan hcl disertasi djonaedi saleh syahruddin, e., herawaty, r., & azhar. (2016). improving the quality of the leaves and seeds of rubber trees (hevea brasilliensis) for poultry feed through various types of microbial biotechnology. pakistan journal of nutrition, 15(11), 963–968. doi: https://doi.org/10.3923/pjn.2016.963.968 winarti, s. (2010). makanan fungsional. graha ilmu https://doi.org/https:/doi.org/10.2478/v10032-008-0031-2 https://doi.org/https:/doi.org/10.1002/jsfa.3580 https://doi.org/10.1007/s40093-018-0199-5 https://doi.org/10.17660/actahortic.2007.752.17 https://doi.org/10.17226/10490 https://doi.org/10.31227/osf.io/4j8q6 https://doi.org/10.3923/pjn.2016.963.968 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 9-16 | doi: 10.14421/biomedich.2023.121.9-16 issn 2540-9328 (online) proximate composition, levels of some essential mineral elements and anti-nutritional components of some yam species found in minna, niger state eneogwe okechukwu godfrey1,*, ibrahim izihyi esther2, obuye faith1 1department of chemistry; 2department of industrial chemistry, federal university lokoja, p.m. b. 1154, lokoja, nigeria. corresponding author* godfrey.eneogwe@fulokoja.edu.ng manuscript received: 18 august, 2022. revision accepted: 06 september, 2022. published: 27 september, 2022. abstract samples of dioscorea dumenturom, dioscorea rotundata and dioscorea cayenensis were investigated for their proximate composition, anti-nutritional and mineral contents using standard analytical methods. these varieties of dioscorea analysed showed a significant difference (p≤0.05) amongst them. however, from the results, it was observed that dioscorea rotundata had the highest ash (8.05±0.05 %) and crude fibre content (13.11±0.10 %) which indicates that it contains more mineral stuffing and is best for softening of stool. dioscorea cayenensis had the highest fat content (16.31±0.30 %), indicating that it is a better source of calories than other yam species analysed. dioscorea dumenturom had the lowest moisture content (3.51±0.01 %) as well as the highest crude protein (12.29±0.01 %) and carbohydrates (69.04±0.10 %) than other yam species analysed, indicating its longer shelf-life, high bodybuilding capacity and better source of energy than other yam species analysed. the anti-nutritional constituent of alkaloid and tannin were lowest in dioscorea cayenensis while dioscorea rotundata had the least cyanide, phytate and oxalate content. this implies that these particular yams are safer for consumption. the elemental analysis in mg/100g indicated that the yam species contained appreciable levels of essential minerals, with dioscorea dumenturom having the highest sodium, calcium, iron, potassium, phosphorous and magnesium concentration of 32.05±0.07 mg/100g, 190.57±0.01 mg/100g, 5.98±0.03 mg/100g, 80.12±0.17 mg/100g, 237.10±0.48 mg/100g and 100.22±0.03 mg/100g respectively. all these mineral concentrations exist within the permissible limit of who and hence indicate that the yam species can serve as a good source of minerals. keywords: proximate composition; anti-nutritional; mineral content; dioscorea dumenturom; dioscorea rotundata; dioscorea cayenensis. introduction yam is a staple cuisine in many tropical and subtropical areas throughout the world. nigeria in particular has the world’s largest annual output and consumption per capita (ukom et al., 2014). there are over 600 dioscorea species, with more than 10 species farmed for food and 6 species used in pharmaceuticals (okigbo et al., 2015). however, white yam (dioscorea rotundata), water yam (dioscorea alata), aerial yam (dioscorea bulbifera), yellow yam (dioscorea cayenensis), trifoliate yam (dioscorea dumentorum) and chinese yam (dioscorea esculenta) are the six most economically important species. yam cultivation not only helps rural farmers survive but also provides a significant number of calories, essential micro-nutrients and phytochemical compounds such as iron, zinc, ascorbic acid and flavonoids (ukom et al., 2014). in many regions of west africa particularly nigeria, yam is processed in a variety of culinary forms, such as pounded yam, fried yam, roasted yam, boiled yam, yam balls, mashed yam, yam chips and flakes (orkwor et al., 1997), which are typically served with protein-rich soups and sources. the species has historically influenced how yams are processed. west africa is the world’s most important yamproducing region, with nigeria as the leading producer, accounting for more than half of global production (modu et al., 2015). despite these facts regarding yam, it continues to be overlooked in west african national food policy programs. this has resulted in limited dioscorea species research and development on the continent (sanoussi et al., 2016). this study compares the proximate composition, mineral content and anti-nutritional constituents of several yam varieties which includes dioscorea rotundata, dioscorea dumenturom and dioscorea cayenensis obtained from minna, niger state. https://doi.org/10.14421/biomedich.2023.121.9-16 10 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 materials and method sample collection matured accessions of the three cultivated yam species were harvested randomly from rural farms in the chanchaga, mekunkele and gunu areas of niger state. the samples include cultivars of yellow yam (dioscorea cayenensis), a variety of white yams (dioscorea rotundata) and trifoliate yam (dioscorea dumentorum). the samples were cleaned by brushing off soil particles and transported at tropical ambient temperature to the laboratory for analysis. dioscorea dumenturom dioscorea rotundata dioscorea cayenensis figure 1. the images of the studied plants. sample pre-treatment the yam samples were washed thoroughly with water, peeled and cut using a knife. these yam species were ground separately using a laboratory mortar and pestle and then sieved using a 250 μm mesh size sieve. the three samples were stored in airtight properly labeled polythene bags and kept in a cool and dry place before analysis. determination of proximate composition the proximate analysis of samples for moisture, crude fat, crude fiber and ash was determined using the method described by aoac (2006). the protein content was determined using the micro kjeldahl method (n x 6.25) and the carbohydrate was calculated by difference. mineral analysis the digestion of samples for mineral analysis was carried out according to the method described by aoac (2006). a 250 cm3 beaker was filled with 1.00 g of the pulverized sample. the beaker was filled with an acid mixture (15.00 cm3 concentrated hno3 and 5.00 cm 3 concentrated perchloric acid). the mixture was agitated thoroughly to ensure adequate mixing, then heated on a hot plate until a clear digest appear. the digest was allowed to cool and filtered quantitatively into a 100 cm3 volumetric flask. the filtrate was made up to the 100 cm3 mark, transferred to a plastic bottle and aspirated into the machine for trace metal analysis. determination of anti-nutrients determination of total alkaloids this was accomplished utilizing the method described by aoac (2005). 0.50 g of the sample was dissolved in 5.00 cm3 of 96% ethanol and 5.00 cm3 of 20% h2so4 (1:1) and the resulting solution was filtered. 1.00 cm3 of the filtrate was added to 5.00 cm3 of 60% h2so4 and allowed to stand for 5 minutes. after that, 5.00cm3 of 0.5% formaldehyde was added and allowed to stand for 3 hours. the reading was taken at an absorbance length of 565 nm using an ultra-violet (uv) spectrophotometer. the extinction coefficient of vincristine (e296, ethanol {etoh} = 15136 m¹־cm¹־) was chosen as a reference alkaloid (aoac, 2005). determination of saponins this was accomplished utilizing the method described by krishnaiah et al. (2009). 0.50 g of the sample was boiled for 4 hours in a 20.00 cm3 of 1 mol/dm-3 hcl solution. after cooling, 50.00 cm3 petroleum ether was added to the filtrate for the ether layer, which was then evaporated to dryness. 5.00 cm3 of acetone ethanol was added to the residue and 0.40 cm3 of each was divided among three test tubes. they were filled with 6.00 cm3 of ferrous sulfate reagent followed by 2.00 cm3 of concentrated h2so4. the absorbance was measured at 490 nm after 10 minutes of complete mixing. the calibration curve was established using standard saponin. determination of tannin jaffe (2003) described that 1.00 g of each sample a and b were dissolved in 10.00 cm3 distilled water and agitated, left to stand for 30 minutes at room temperature. the extract was obtained from each sample godfrey et al. – proximate composition, levels of some essential mineral elements … 11 after centrifugation. in a 50 cm3 volumetric flask, 2.50 cm3 of the supernatant was dispersed. similarly, in a separate 50.00 cm3 flask, 2.50 cm3 of the standard tannic acid solution was dispersed. in each flask, a 1.00 cm3 folin dennis reagent was added, followed by 2.50 cm3 of saturated na2co3 solution. the mixture was then diluted to 50.00 cm3 and incubated for 90 minutes at room temperature. the sample’s absorbance was measured at 250 nm with the reagent blank at zero. the % tannin was calculated using the formula: 𝑇𝑎𝑛𝑛𝑖𝑛 (𝑚𝑔 𝑔) = (𝐴𝑠−𝐴𝑏 )−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 𝑆𝑙𝑜𝑝𝑒×𝑑×𝑊 × 10⁄ (eqt. 1) where 𝐴𝑠 is the sample absorbance, 𝐴𝑏 is the blank absorbance, 𝑑 is the density of the solution (0.791g/ml), 𝑊 is the weight of the sample in grams and 10 is the aliquot. determination of phytic acids markkar et al. (1993) described the process for determining phytic acid. in a 250 cm3 conical flask, 2.00 g of each sample (a and b) were weighed. each sample was soaked in 100.00 cm3 of 2% concentrated hcl acid for 3 hours in the conical flask before being filtered through a double layer of hardened filter papers. 50.00 cm3 of each filtrate was placed in a 250 cm3 beaker and 100 cm3 of distilled water was added to each to give proper acidity. 10.00 cm3 of 0.3% ammonium thiocyanate solution was added to each solution as an indicator. each solution was titrated with standard iron chloride solution, which contains 0.00195 g iron per cm3. the endpoint color was slightly brownish-yellow which persisted for 5 min. the percentage of phytic acid was calculated using the formula: % 𝑃ℎ𝑦𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 = 𝑦 × 1.19 × 1 (eqt. 2) where 𝑦 is the titre value × 0.00195 determination of cyanides cyanide content was determined by the alkaline picrate method as described by onwuka (2005). here 5.00 g of powdered sample was dissolved in 50.00 cm3 of distilled water in a corked conical flask and the extraction was allowed to stand overnight and filtered. in a corked test tube, 1.00 cm3 of the filtered sample was mixed with 4.00 cm3 alkaline picrate and incubated in a water bath for 5 minutes. the absorbance of the blank containing 1.00 cm3 distilled water and 4 cm3 alkaline picrate solution was also measured at 490 nm after colour development (reddish-brown colour). the cyanide content was extrapolated from a cyanide standard curve prepared from a different concentration of kcn solution containing 5-50 μg cyanide in a 500 cm3 conical flask followed by 25.00 cm3 of 1 mol/dm-3 hcl. it was calculated as: 𝐶𝑦𝑎𝑛𝑖𝑑𝑒 (𝑚𝑔 100𝑔⁄ ) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒×𝐺𝐹×𝐷𝐹 𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (eqt. 3) where 𝐺𝐹 is the gradient factor and 𝐷𝐹 is the dilution factor. determination of oxalates the oxalate content of the samples was determined using the titration method described by munro and bassiro (2000). in a 250 cm3 volumetric flask suspended in 190.00 cm3 distilled water, 2.00 g of each sample a and b was inserted. each sample received a 10 cm3 of 6 mol/dm-3 hcl solution, which was digested at 100ºc for 1hour. the samples were then cooled and made up to the 250 cm3 mark of the flask. the samples were filtered and a duplicate portion of 125.00 cm3 of the filtrate was measured into a beaker and 4 drops of methyl red indicator were added, followed by the addition of concentrated nh4oh solution (dropwise) until the solution changes from pink to yellow colour. each portion was then heated to 90ºc, cooled and filtered to remove the precipitate containing ferrous ion. each of the filtrates was again heated to 90ºc and 10.00 cm3 of 5% cacl2 solution was added to each of the samples with consistent stirring. after cooling, the samples were left overnight. the solutions were then centrifuged for 5 minutes at 2500 rpm. the supernatant was decanted and the precipitates completely dissolved in 10.00 cm3 20% h2so4. the total filtrate resulting from the digestion of 2.00 g of each of the samples was made up to 200 cm3. the filtrate was heated to near boiling points in aliquots of 125.00 cm3 and then titrated against 0.05 mol/dm-3 standardized kmno4 solution to a pink colour which persisted for 30 seconds. each sample of the oxalate contents was then calculated using the formula: 𝑂𝑥𝑎𝑙𝑎𝑡𝑒 = 𝑇 ×(𝑉𝑚𝑒)(𝐷𝑓)×105 (𝑀𝐸)×𝑀𝑓 (eqt. 4) where 𝑇 is the titre value of kmno4, 𝑉𝑚𝑒 is the volume-mass equivalent, 𝐷𝑓 is the dilution factor, 𝑀𝐸 is the molar equivalent of kmno4 in oxalate and 𝑀𝑓 is the mass of the sample. statistical analysis the obtained results were subjected to statistical analysis using mean standard deviation and analysis of variance (anova) as described by duncan’s multiple range test to determine the level of significance between different samples and significance was set at p ≤ 0.05. 12 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 results and discussion table 1. proximate composition of selected yam species (%). yam species ash content moisture content crude fat crude fibre crude protein carbohydrate d. cayenensis 3.09±0.19a 4.51±0.07d 16.31±0.30j 8.30±0.40b 10.14±0.40h 57.65±0.10b d. dumenturom 5.06±0.80c 3.51±0.01c 3.56±0.03a 6.54±0.01a 12.29±0.01f 69.04±0.10g d. rotundata 8.05±0.05f 5.60±0.06f 12.46±0.10h 13.11±0.10e 2.15±0.03b 58.63±0.08d values are means ± standard deviation of triplicate analysis. moisture content the moisture content of the various yam species ranged from 3.51±0.01c % for dioscorea dumenturom to 5.60±0.06f % for dioscorea rotundata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the highest moisture content. however, these values are comparable to literature values as reported by oko and famurewa (2014) that ranged from 2.1% to 9.2% for dioscorea dumenturom and dioscorea alata respectively and lower than the research carried out by anthony et al. (2014) which reported its moisture content within the range of 30.51±0.06d % to 37.90±0.08a % for xanthosoma maff and dioscorea cayenensis respectively. as such it could be said that dioscorea dumenturom has a higher resistance to deterioration and longer shelf life than any of the selected yam species. crude fibre content the crude fibre content ranged from 6.54±0.01a % for dioscorea dumenturom to 13.11±0.10d % for dioscorea rotundata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the highest crude fibre content. however, these results can be compared with reports by afiukwa et al. (2013) that ranged from 6.01±0.04b % to 13.03±0.80a % for varieties of dioscorea dumenturom and is different from reports by oko and famurewa (2014) that ranged from 3.31% to 3.53% for their dioscorea alata species. studies have shown that an increase in fiber consumption in foods reduces the incidence of obesity, cardiovascular disease, diabetes and digestive disorders (turner, 2014). ash content ash contents of the yam varieties ranged from 3.09±0.19a % for dioscorea cayenesis to 8.05±0.05f % for dioscorea rotundata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea rotundata showing the highest ash content. however, these results were different from reports by sorh et al. (2015) that ranged from 1.64±0.03a % to 1.78±0.03a % for varieties of dioscorea alata. the ash content is an indication of the extent of mineral stuffing in the dioscorea species (akonor et al., 2017). as such, dioscorea rotundata is stuffed with more minerals than the other yam tubers analysed. crude protein content the crude protein content showed a significant difference (p≤0.05) between the yam varieties. it ranged from 2.15±0.03b % for dioscorea rotundata to 12.29±0.01f % for dioscorea dumenturom. however, dioscorea dumenturom proved to be the tuber variety with the highest protein content. nevertheless, the result can be compared with reports by ojinnaka et al. (2017) that showed 2.43±0.11b % for dioscorea bubilfera and in contrast with the report by ukom et al. (2014) that showed crude protein of dioscorea dumenturom to be 69.15±4.49b %. as such, this shows that dioscorea dumenturom is the richest in protein among the analysed dioscorea species. crude fat content the fat content in these analysed varieties of yam tubers ranged from 3.56±0.03a % for dioscorea dumenturom to 16.31±0.30j % for dioscorea cayennesis. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea cayenensis showing the highest fat content. however, these results contradict ukom which showed 4.4±1.91a % for dioscorea cayenensis. that dietary fat supplies most of the energy required by man suggests that dioscorea cayenensis is a better source of calories than other dioscorea species analysed. carbohydrate content the carbohydrate content of the analysed yam varieties was significantly different (p≤0.05) and ranged from 57.65±0.10b % for dioscorea cayenensis to 69.04±0.10g % for dioscorea dumenturom. despite the huge carbohydrate content contained by the dioscorea species, dioscorea dumenturom appeared to be more than dioscorea cayenensis and dioscorea rotundata. however, these values are comparable to literature by ukpabi and akobundu (2014), which had 78.32±0.29 % for dioscorea dumenturom and in contrast with frank and kingsley (2014) that ranged from 24.25±0.62b % for dioscorea alata to 32.03±0.89c % dioscorea rotundata. carbohydrates are considered as the primary source of energy for all organisms, playing a nutritional as well as structural role (ojinnaka et al., 2017). godfrey et al. – proximate composition, levels of some essential mineral elements … 13 table 2. mineral concentration of selected yam species (mg/100g). yam samples ca fe mg na k p d. cayenensis 67.12±0.11b 3.09±0.01a 74.38±0.03d 24.10±0.14b 50.06±0.09d 131.51±0.05b d. dumenturom 190.57±0.01i 7.37±0.04i 100.22±0.03i 32.05±0.07e 72.23±0.37g 193.11±0.01b d. rotundata 60.60±0.17a 3.18±0.03a 60.85±0.21b 31.10±0.14g 42.08±0.11b 117.20±0.01b values are means ± standard deviation of triplicate analysis. iron concentration minerals are an important component of diet because of their physiological and metabolic function in the body. table 2 shows iron concentrations that ranged from 3.09±0.01a mg/100g for dioscorea cayenensis to 7.37±0.04i mg/100g for dioscorea dumenturom samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest iron concentration. however, this result was low when compared to results reported by mergedus et al. (2015) which ranged from 10.10±0.01a mg/100g to 11.60±0.01a mg/100g for cultivars of colocasia esculenta (cocoyam). iron is a major component of hemoglobin, a type of protein in red blood cells that carries oxygen from the lungs to all parts of the body (mergedus et al., 2015). the recommended dietary allowance for iron is 13.7–15.1 mg/day for children and 17.0–18.9 mg/day for adults (who, 2014). sodium concentration the sodium concentration in this study ranged from 24.10±0.14b mg/100g for dioscorea cayenensis to 32.05±0.07e mg/100g for dioscorea dumenturom. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea dumenturom showing the highest sodium concentration. however, the sodium concentration was comparable to the report by oko and famurewa (2015) which had 24.84±0.37a mg/100g and 21.06±0.77b mg/100g for dioscorea vilgaris and dioscorea villosa respectively. sodium as a macronutrient plays an important role in various metabolic processes including excitation and transmission of nerve impulses during action (olajumoke et al., 2014). the recommended daily dietary intake for sodium is 10 mg/day for adult males and below 15 mg/day for females (who, 2014). potassium concentration potassium is a macro-nutrient required by both plants and animals and is involved in various metabolisms. in this study, the concentration of potassium ranged from 42.08±0.11b mg/100g for dioscorea rotundata to 72.23±0.37g mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest potassium concentration. however, these values were lower when compared with reports by ellong et al. (2014) that ranged from 338.00±59.29 mg/100g to 407.04±168.36 mg/100g for varieties of sweet potato (ipomoea batatas). potassium is important in the regulation of heartbeat, neurotransmission, signal and immune response and water balance in the body (olajumoke et al., 2014). the recommended dietary intake of potassium is 2000 mg/day for adults and 1000 mg/day for children (who, 2014). calcium concentration the calcium content in this study ranged from 60.60±0.17a mg/100g for dioscorea rotundata to 190.57±0.01i mg/100g for dioscorea dumenturom. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea dumenturom showing the highest calcium concentration. however, this concentration was low when compared to reports by sorh et al. (2015) which ranged from 150±14.50ab mg/100g to 185±18.14d mg/100g for varieties of dioscorea alata. calcium is necessary for blood clotting, muscle contraction, neurological function, bone and teeth formation (trailokya et al., 2017). the recommended dietary intake of calcium is 500–800 mg/day for children and 1000 mg for adults (who, 2014). magnesium concentration magnesium was also present in small quantities in the range of 60.85±0.21b mg/100g for dioscorea rotundata to 100.22±0.03i mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest magnesium concentration. however, its results were comparable to reports by cyrile et al. (2014) that showed 53.70±0.32b mg/100g for its dioscorea dumenturom species. magnesium is involved in muscle degeneration, growth retardation, alopecia, dermatitis, immunologic dysfunction, poor spermato-genesis, congenital abnormalities and bleeding disorders among other things (mergedus et al., 2015). the recommended dietary intake of magnesium is 80–320 mg/day (who, 2014). phosphorous concentration phosphorus, together with calcium, helps to strengthen bones and teeth, particularly in children and breastfeeding mothers. the phosphorus content of the samples analysed ranged from 117.20±0.01b mg/100g for dioscorea rotundata to 193.11±0.01c mg/100g for 14 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest phosphorous concentration. however, these values are low when compared to those of 410 mg/100g of sweet potato reported by sorh et al. (2015). furthermore, phosphorous appeared to be the most abundant mineral in all the yam samples analysed. the recommended dietary allowance for both children and adults is 800 mg/day (who, 2014). table 3. anti-nutritional contents of the selected yam species (mg/100g). yam species tannin phytate cyanide alkaloids oxalate d. cayenensis 30.06±0.30d 49.44±0.30h 1.70±0.06d 8.35±0.10b 4.86±0.08d d. dumenturom 72.99±0.50j 30.00±0.20f 3.38±0.06e 24.17±2.70g 3.67±0.30c d. rotundata 62.00±0.50h 15.06±0.30d 1.54±0.06c 11.58±0.20d 3.19±0.90bc values are means ± standard deviation of triplicate analysis. tannin content tannins have been reported to form complexes with proteins and impair their digestibility and palatability. however, cooking is known to diminish the contents in foods (lewu et al., 2010). tannin concentration in the yam samples studied ranged from 30.06±0.30d mg/100g for dioscorea cayenensis to 72.99±0.50j mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea cayenensis having the least tannin concentration. however, these values are relatively lower than those of 20-255 mg/100g reported on various under-utilized dioscorea tubers (arinathan et al., 2009) and higher than reports by polycarp et al. (2012) that ranged from 4.56±0.01a to 19.23±0.03b mg/100g for different dioscorea species. tannin has been shown to decrease the activity of several enzymes including trypsin, amylase and lipase as well as interfere with the absorption of dietary iron (rao and desothe, 2008). the total acceptable tannin intake for a man is 560 mg/kg (stephene, 2004). as such the tannin contents in these yam species are low and within permissible limits and thus, cannot be harmful to consumers (stephene, 2004). phytate content the phytate contents of the yam tuber ranged from 15.06±0.30d mg/100g for dioscorea rotundata to 49.44±0.30h mg/100g for dioscorea cayenensis samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least phytate concentration. however, these values were higher when compared to the report by otoo et al. (2008) that showed 2.60±0.20a mg/100g for its dioscorea rotundata cultivars and lower than sweet potato which contained 119.98±0.01a mg/100g (akaninwor, 2004). the issue with phytate in diets is that it can bind some essential mineral nutrients in the digestive tract, leading to mineral deficiencies. there is no particular recommended daily allowance for phytic acid as it differs from country to country (bello et al., 2008). oxalate content comparatively, the oxalate content of the various yam species analyzed ranged from 3.19±0.90bc mg/100g for dioscorea rotundata to 4.86±0.08d mg/100g for dioscorea cayenensis samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least oxalate concentration. however, these values are lower than reports according to princewill and ibeji (2012) that had 12.60a mg/100g for their dioscorea bubilfera specie and higher than 0.48±0.01a, 0.50±0.03a mg/100g for dioscorea rotundata and dioscorea alata respectively according to reports by afoakwa et al. (2012). oxalic acid and oxalate are found naturally in plants but they have little or no benefit for human health. though high levels in diet irritate tissues and the digestive system notably the stomach and kidney (ogbuagu, 2008). soluble oxalate is known to be poisonous at high concentrations, particularly above 3mg/kg (norwood and fox, 1994). the oxalates levels found in this study indicate that while the analysed yam tubers were slightly above the maximum permitted limit, they lose most of their toxicity when treated (boiled) and so cannot be consumed raw. cyanide content the cyanide contents ranged from 1.54±0.06c mg/100g for dioscorea rotundata to 3.97±0.06e mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least cyanide concentration. however, these values are low when compared to reports by afiukwa et al. (2013) that showed 26.687±0.081a and 21.827±0.058b mg/100g for dioscorea villosa species (okpura and ighobe) respectively. it is also higher than reports by umoh (2013) that had 0.22±0.02a to 0.53±0.01b mg/100g for raw and processed false yam flour. the permissible limits for cyanide are 0.5-3.5 mg/kg which indicates that the level of the cyanide in the samples is above the acceptable range for human consumption and as such must not be consumed in their raw state (mohammed et al., 2013). godfrey et al. – proximate composition, levels of some essential mineral elements … 15 alkaloid content the observed values for the alkaloid samples ranged between 8.35±0.10b mg/100g for dioscorea cayenensis to 24.17±2.70g mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea cayenensis having the least alkaloid concentration. however, the values obtained for the alkaloids contents of these yam species were higher when compared to some varieties of dioscorea cayenensis that ranged from 0.38±0.12 mg/100g to 0.68±0.02 mg/100g and 1.68±0.01 mg/100g for dioscorea rotundata (okwu and ndu, 2006) and lower than reports by ogbuagu (2008) that contained 30.62±0.03a and 32.46±0.01b mg/100g for different species of dioscorea dumenturom. because of their effect on the nervous system, electrochemical transmission and disruption of the cell membrane in the gastrointestinal tract, alkaloids are considered to be antinutrients (friedman, 2001). however, human lethal dosages range between 3-6 mg/kg body weight and a dose above 3 mg/kg is usually considered toxic (habtamu and ratta, 2014). the good news is that it loses most of its toxicity when treated or processed (cooked or boiled). conclusions this study provided vital information on the proximate composition, mineral content and anti-nutritional constituents of the selected yam tuber species (dioscorea dumenturom, dioscorea rotundata, dioscorea cayenensis). this research observed that these analysed dioscorea species were rich in protein, carbohydrate, fibre, and fat and had a high shelf-life. however, the anti-nutritional constituents of these yam species, if properly treated or processed (cooked or boiled), tend to lose more than 95% of their toxicity and can be exploited as good food sources. furthermore, all the species of yam analysed contained an appreciable amount of macro and micro-nutrients which are essential to human nutrition. specifically, dioscorea dumenturom had the highest concentrations of na, ca, mg, fe, k, and p. competing interests: the authors declare that there are no competing interests. references afiukwa, j.n., okereke, c.o., & odo, m.o. 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(2014). children fund and clinical management of acute diarrhea. who/ unicef joint statement. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 197-203 | doi: 10.14421/biomedich.2023.121.197-203 issn 2540-9328 (online) comparative cough suppression of chitosan crab extract of uca tangeri and dihydrocodeine joshua charles isirima1, precious ojo uahomo2,* 1department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state, nigeria; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com abstract cough is an inmate primitive reflex and acts as a part of the body's immune system to protect against foreign materials from the respiratory tract. this study was done to investigate the cough suppression potential of uca tangeri. a day before the test, guinea pigs were placed individually in a transparent chamber (60 × 36 × 60 cm) for 5 minutes before cough was induced by exposure to 15% citric acid, delivered using an omron compressor nebulizer (rate of 0.4 ml/minutes and particle size 5μm) for 10 minutes. the animals were then monitored visually within this exposure time for cough; the latency and counts, of which, were taken as the basal values. the animals exhibiting 10 20 bouts of cough were selected for the study and fasted overnight but with access to water. the selected animals were randomly allotted to 5 groups (n=5 per group). the animals were treated orally thus: group 1 was the control group and received 2 ml/kg of normal saline; group 2 received 25 mg/kg dihydrocodeine; group 3 received 150 mg/kg extract; group 4 received 300 mg/kg extract and group 5 received 600 mg/kg of the extract. an hour after administration, they were re-exposed to citric acid aerosol (as earlier described) and the latency of cough and cough count were recorded. the procedure was repeated at hours 2 and 3 after treatment. antitussive activity was then evaluated in each guinea-pig as the percentage reduction in the number of coughs also known as percentage suppression of cough and percentage increase in latency of cough. the results revealed that uca tangeri exhibited a dose dependent percentage increase in cough latency period as well as percentage increase in suppression of cough which was inferior to dihydrocodeine, but significantly greater than normal saline and basal levels. keywords: cough; suppression; uca tangeri; chitosan crab; dihydrocodeine, comparative. introduction the body's immune system uses the involuntary primitive reflex of coughing to defend against foreign substances that invade the respiratory tract (sharma et al., 2020). to eliminate debris, excessive mucus, irritants, microbes, or other substances from the respiratory tract, one may cough either voluntarily or involuntarily. coughing is either voluntary or involuntary (chung and pavord, 2008). coughing could be classified as acute, subacute, or chronic depending on the duration (vally and ihuma, 2016). acute cough has the shortest duration. it is considered to be a cough with duration of less than two weeks. sub-acute cough has a duration between three-eight weeks while chronic cough lasts for more than eight weeks (four weeks for children) (vally and ihuma, 2016). adults who have persistent coughing are increasingly being linked to pertussis (irwin et al., 2006). the prevalence of chronic cough is 9.6% worldwide, with oceania having the highest incidence (18.1%), and africa having the lowest prevalence (2.3%), according to song et al. (2015). coughing is a symptom of several diseases and health conditions, including the common cold, acute bronchitis, pneumonia, pertussis, flu, smoking, as well as asthma, tuberculosis, and lung cancer. coughing can cause chest pain, congestion, and an irritated throat. repeated coughing causes irritation and discomfort, which in turn leads to additional coughing (irwin et al., 2006). cough is also brought on by a variety of microbes, including bacteria and viruses, which aids in the spread of the illness to new hosts. regular coughing is typically brought on by a respiratory tract infection, but it can also be brought on by choking, smoking, air pollution, asthma, gastroesophageal reflux disease (gerd), postnasal drip, chronic bronchitis, lung tumors, heart failure, and drugs like ace inhibitors (dicpinigaitis et al., 2009). crabs of various species have been employed in the treatment of various illnesses. cardisoma guanhumi has been used to treat wounds, boils and bronchitis in latin america, according to alves and alves (2011). goniopsis cruentata is used to treat epilepsy and genital problems; plagusia depressa serves as a complementary medicine for epilepsy; emerita portoricensis on earaches' therapeutic applications; ocypode quadrata in treating manuscript received: 17 december, 2022. revision accepted: 11 february, 2023. published: 14 february, 2023. https://doi.org/10.14421/biomedich.2023.121.197-203 198 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 asthma, hemorrhage in women, the flu, and to lessen the effects of naquin poison intoxication (pisces, batrachoididae); ucides cordatus in treating hemorrhage in women, urinary incontinence, osteoporosis, cough, asthma, tuberculosis, womb disorders, arthrosis, and bronchitis; and uca maracoani in curing asthma, whooping cough. dev roy (2014) gathered 22 species of brachyuran crabs from various regions of the world, including india, nepal, and brazil. these crabs are primarily used to treat conditions like whooping cough, bronchitis, pneumonia, asthma, osteoporosis, wounds, boils, womb disorders, tuberculosis, earache, burns, and epilepsy, while hermit crabs are used to treat earache, urethritis; malaria. this research examines uca tangeri and dihydrocodeine’s capability to suppress coughing. materials and methods animals 25 adult guinea pigs of either sex weighing 460-600g were obtained from an animal facility in ogoni, rivers state, and brought to the animal house of department of pharmacology, faculty of basic medical sciences, college of health sciences, university of port harcourt, nigeria. all animals were allowed two weeks acclimatization in the animal facility of the department of pharmacology, university of port harcourt. they were all allowed free access food and tap water and were exposed to natural light-dark cycle and room temperature. all animals were handled according to standard protocols for the use of laboratory animals (national institute of health, 2002). site of collection of crab samples the samples were collected at sivibilagbara along the dor nwezor channel of bodo creek and buguma creek. the buguma creek is a tributary of the bonny river which is located southeast of the niger delta between longitude 6º51'e and 49.8'e and latitude 4º43'n and 47.8'n in asari-toru local government area of rivers state. the creek system consists of the main creek channel with other associated interconnecting creeks which are interconnected and surround buguma and ido communities. the creek serves as a source of tidal water for nigerian institute for oceanography and marine research/buguma brackish water experimental fish farm, which was constructed between 1963 and 1966. the new calabar river brings the salty ocean water as tidal flows diurnally to the fish ponds (dublin-green and ojanuga, 1999). sample collection and identification samples were collected from the creeks. uca tangeri were collected at low tide in the mangrove shores by hand picking. the samples collected were transferred into perforated plastic containers to allow for air during transportation and was transported to the pharmacognosy research laboratory, department of pharmacognosy, university of port harcourt. the samples were identified using food and agriculture organization species identification sheets for fresh water and marine crab species. figure 1. uca tangeri. method of extraction according to shahidi and synowiecki (1991), 60 of the freshly collected crabs (u. tangeri) were sacrificed and the shell separated from the meat and washed with tap water to remove all impurities. the crab shells and meat were then transferred to the oven and dried at 70oc until they were completely dry. using a laboratory mortar and pestle, the dried crab shells and meat were ground and sieved into the size of 500µm. carotenoids extraction four gram of the sieved crab shell was measured using want precision electric weighing balance into a beaker and 200ml of cod liver oil was added and stirred with magnetic stirrer until it was completely mixed for 20minutes. the beaker was then transferred into a water bath at a temperature of 60oc and allowed for 30 minutes. the mixture was then filtered with a white handkerchief to drain off the oil and the residue transferred into a beaker. deproteinization the residue from the carotenoids extract was treated with 2% potassium hydroxide (koh) at a ratio of 1:20 w/v and was stirred continuously for 2 hours at a temperature of 90oc to remove protein from the crab. the sample was filtered and the residues were continuously washed with distilled water until the ph became neutral i.e., ph=7. this was done to ensure that all the salt had been removed after removing the protein. the deproteinized crab was transferred into an oven and dried at 60oc until it was completely dry (shahidi and synowiecki, 1991). isirima & uahomo – comparative cough suppression of chitosan crab extract … 199 demineralization 2.5% w/v of hydrochloric acid was used at room temperature (23oc) for 6 hours to remove the mineral content of the deproteinized crab materials at a ratio of 1:20 w/v. the samples were filtered and washed with tap water until the ph was neutral. the demineralized crab material was then transferred to the oven and dried at a temperature of 60oc until completely dried. (shahidi and synowiecki, 1991). decolouration and dewatering the demineralized crab material was treated with 300ml acetone for 10minutes and dried for 2 hours at an ambient temperature and the residues were removed to achieve decolourization. the decolourized sample was washed in running water, filtered and dried at 60oc until it was completely dried to obtain crab chitin (shahidi and synowiecki, 1991). deacetylation of chitin deacetylation of chitin was carried out using the method of yen et al. (2009). the obtained chitin was treated with 40% w/v aqueous sodium hydroxide in the ratio of chitin to the solution 1:15 w/v at 105oc in a water bath for 2 hours. thereafter, the chitin was filtered with filter pump and washed with deionized water until ph was neutral to obtain chitosan. the obtained chitosan was then dried at 60oc for 2 hours in the oven. the dried chitosan was preserved in a well labelled bottle and kept for the experiment. extract concentration preparation the extract solution for the study was prepared by dissolving 0.5g of the extract in 1ml of di-methylsulfoxide (dmso) solvent to have a stock concentration of 500mg/ml. oral toxicity testing to determine ld50 the bruce method of 1985 was used to determine the ld50 in this study. following this method, swiss mice were dosed one at a time beginning from 1000mg/kg of the extract from the crab because since the extract was from an edible source, there might not be low toxic doses. this was increased by a factor of 1.3 thus after this dose which produced no death, higher doses used were 1300mg/kg, 1690mg/kg, 2197mg/kg, 2856mg/kg, 3713mg/kg, 4827mg/kg and 6275mg/kg. with these doses there was no observed death of the mice and it was concluded that the extract is safe for the study based on pharmacology rule. experimental procedure this was based on the guinea pig cough model of nadig (2005) with minor alterations. a day before the test, guinea pigs were placed individually in a transparent chamber (60 × 36 × 60 cm) for 5 minutes before cough was induced by exposure to 15% citric acid, delivered using an omron (omron health care ltd, japan) compressor nebulizer (rate of 0.4 ml/min and particle size 5μm) for 10 minutes. the animals were then monitored visually within this exposure time for cough; the latency and counts, of which, were taken as the basal values. the animals exhibiting 10 20 bouts of cough were selected for the study and fasted overnight but with access to water. the selected animals were randomly allotted to 5 groups (n=5 per group). the animals were treated orally thus: group 1 was the control group and received 2 ml/kg distilled; group 2 received 25 mg/kg dihydrocodeine; group 3 received 150 mg/kg extract; group 4 received 300 mg/kg extract and group 6 received 600 mg/kg of the extract. an hour after administration, they were re-exposed to citric acid aerosol (as earlier described) and the latency of cough and cough count were recorded. the procedure was repeated at hours 2 and 3 after treatment. antitussive activity was then evaluated in each guinea-pig as the percentage reduction in the number of coughs also known as percentage suppression of cough and percentage increase in latency of cough in comparison with the previously established control basal value, calculated as below: 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 𝑐𝑜𝑢𝑔ℎ 𝑐𝑜𝑢𝑛𝑡 = [1 − ( 𝐶2 𝐶1 )] 𝑥 100 (1) where; c1 is basal values and c2 is the total number of coughs after treatment. 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑖𝑛 𝑙𝑎𝑡𝑒𝑛𝑐𝑦 𝑜𝑓 𝑐𝑜𝑢𝑔ℎ = [1 − ( 𝐿2 𝐿1 )] 𝑥 100 (2) where; l1 is basal values, and l2 is the latency of coughs after treatment. data analysis all results are expressed as means ± sem. an analysis of variance was performed on the different treatment groups to determine significant effects of the treatments. posthoc analysis between the different groups was performed with a dunnett's t-test. a value of p≤0.05 was accepted as the level of statistical significance. 200 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 results and discussion bronchoconstriction is significant in cough induction since the process stimulates intrapulmonary rapidly adapting receptor (rar), a type of cough receptor to cause or enhance the sensitivity of the cough (pavord, 2004). rar activation initiates bronchospasm and mucus secretion via parasympathetic reflexes. this study investigated antitussive properties of the uca tangeri in guinea pigs. guinea pigs were used in the antitussive investigation because their airways possess the needed afferent nerves and can produce cough, just like in humans (agrawal et al., 1991). cough was detected with a characteristic sound and by stretching of limbs accompanied by inspiration and then expiration similar to that described by morice and co-workers (morice et al., 2007). these criteria were adopted so as to distinguish it from other respiratory reflexes like sneezing and expiratory reflex. as a tussigenic agent when inhaled, citric acid is known to stimulate transient receptor potential vanilloid1on the c-fibers. this then causes the release of tachykinnins to mediate bronchoconstriction and mucus secretion, which in turn stimulates rar (bonham et al., 1996 and canning et al., 2001), a widely studied cough receptor. the impulse is then conveyed through the vagus nerve to the cns and then back to respiratory muscle through the efferent pathway to cause cough. dihydrocodeine was used as the positive control because it is the second most specific antitussive of the commonly used opioids (eddy et al., 1969). dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). number of cough in guinea pigs pre-treatment with dihydrocodeine, and uca tangeri and thereafter exposure to citric acid table 1. effect of uca tangeri on latency period (in seconds) in animals treated with acetic acid in a tussive protocol. group blp (seconds) olp (seconds) tlp (seconds) thlp (seconds) normal saline (ns) 43.60±0.25 44.60±0.25 44.80±0.37 45.40±0.40 dihdrocodeine (dh) 43.60±0.25 59.80±0.20 60.00±0.01 60.60±0.25 low dose (uca tangeri) (ldut) 43.60±0.25 48.00±0.01 48.20±0.20 48.60±0.24 medium dose (uca tangeri) (mdut) 43.60±0.24 51.80±0.20 52.00±0.01 52.60±0.24 high dose (uca tangeri) (hdut) 43.60±0.24 55.40±0.24 56.20±0.20 56.60±0.24 blp = basal latency period; olp = one hour latency period; tlp = two hours latency period; thlp = three hour latency period; low dose (uca tangeri) (ldut) = 150mg/g; medium dose (uca tangeri) (mdut) = 300mg/kg; high dose (uca tangeri) (hdut) = 600mg/kg table 1 presents the results of the number of cough bouts produced by the guinea pigs before and after pretreatment with normal saline, 25mg/kg of dihydrocodeine, 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. although, there were no significant differences, observed between the basal i.e., pre-treatment cough bouts (17.60±0.24) and those of normal saline for the various time frames (14.60±0.24, 13.80±0.37 and 13.40±0.24), there were significant differences between the basal and those of 25mg/kg of dihydrocodeine, 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. anova comparison of number of cough bouts between normal saline (14.60±0.24, 13.80±0.37 and 13.40±0.24), and dihydrocodeine (4.00±0.32, 3.20±0.20 and 3.00±0.01) respectively also showed significant differences. also, anova comparison of the number of cough bouts between normal saline (14.60±0.24, 13.80±0.37 and 13.40±0.24) and low dose of uca tangeri (8.20±0.37, 8.40±0.24 and 8.00±0.32 revealed a significant difference. this was also true for both medium dose and high dose. these results implies that normal saline does not reduce the number of cough bouts induced with citric acid, since its administration did not produce any significant difference from animals induced with citric acid without treatment. thus, it could be stated that normal saline does not have any effect on the intrapulmonary rapidly adapting receptor (rar). it neither activates nor inhibits it ability to cause or enhance the sensitivity of the cough. on the contrary, dihydrocodeine and all the different doses of the extract significantly reduced the number of cough bouts in the different time frames, implying that both the standard drug and the extract exerted inhibitory effect on the stimulatory process on the intrapulmonary rapidly adapting receptor (rar). therefore, this inhibitory effect also prevents the frequency of bronchospasm and mucus secretion caused by citric acid. these, antitussive properties were greater for dihydrocodeine, followed by high dose of the extract, medium dose of the extract and low dose of the extract. also, it is known that dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996), hence, this reduction in the number of cough bouts is associated with a stimulation of μ opioid receptors in the cns. in a similar manner the effect produced by the uca tangeri extract, isirima & uahomo – comparative cough suppression of chitosan crab extract … 201 could be associated with cns stimulation of μ opioid receptors. finally, it was noted that the effect of uca tangeri extract on the number of cough bouts was dose dependent, implying that the number of cough bouts decreased as the dose of the extract increased. latency period of cough in guinea pigs pre-treatment with dihydrocodeine and uca tangeri and exposure to citric acid table 2. effect of uca tangeri on number of cough bouts in animals treated with acetic acid in a tussive protocol. group pcb ocb tcb thcb normal saline (ns) 17.60±0.24 14.60±0.24 13.80±0.37 13.40±0.24 dihdrocodeine (dh) 17.60±0.24 4.00±0.32 3.20±0.20 3.00±0.01 low dose (uca tangeri) (ldut) 17.60±0.24 8.20±0.37 8.40±0.24 8.00±0.32 medium dose (uca tangeri) (mdut) 17.60±0.24 7.00±0.32 6.60±0.24 6.40±0.24 high dose (uca tangeri) (hdut) 17.60±0.24 5.80±0.37 5.40±0.24 5.20±0.20 pcb = pre-treatment cough bouts; ocb= cough bouts after one hour of drug administration; tcb = cough bouts after hour of drug administration; thcb = cough bouts after 3 hours of drug administration table 2 presents the results of the latency period caused by uca tangeri and dihydrocodeine in comparison to normal saline. it was observed that the basal latency period (43.60±0.25) was not significantly lower than that produced by normal saline, after one hour (44.60±0.25), two hours (44.80±0.37) and three hours (45.40±0.40) pre-treatment, but was significantly lower than those produced by dihydrocodeine (25mg/kg) after one hour, two hours and three hours (59.80±0.20, 60.00±0.01 and 60.60±0.25) pre-treatment respectively. this was also true for 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. it was also observed that the latency periods of normal saline were significantly lower than those for 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri for the different time frames of one, two and three hours. anova comparison revealed significant differences. this implies that there was no cough suppression associated with normal saline, while on the contrary; significant cough suppression is associated with dihydrocodeine and the extract from uca tangeri. this could be associated with activation of μ opioid receptors, since dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). the effect produced by uca tangeri extract was similar to that of dihydrocodeine, hence a similar assumption could be made for the uca tangeri extract. it is also worth stating here that uca tangeri extract produce a dose dependent increase in the cough latency period, i.e., the cough latency period increasing as the dose increased. also, since cough induction is associated with significant stimulation of the cough receptor ‘intrapulmonary rapidly adapting receptor (rar)’, it can be deduced that both dihydrocodeine and the extract from uca tangeri significantly inhibited the cough receptor ‘intrapulmonary rapidly adapting receptor (rar)’and could be use in acute or chronic conditions of bronchial constriction, since significant bronchioconstriction is associated with cough induction. effect of dihydrocodeine and uca tangeri on percentage reduction in cough counts in animals treated with acetic acid in a tussive protocol table 3. effect of uca tangeri on percentage reduction in cough counts in animals treated with acetic acid in a tussive protocol. group oprcc tprcc thprcc normal saline (ns) 17.06±0.24 21.63±1.28 23.86±1.02 dihdrocodeine (dh) 77.19±2.03 81.83±1.04 82.94±0.24 low dose (uca tangeri) (ldut) 51.24±2.34 52.29±1.05 54.51±1.87 medium dose (uca tangeri) (mdut) 60.19±1.85 62.48±1.41 63.66±1.10 high dose (uca tangeri) (hdut) 66.93±2.52 69.28±1.53 70.46±1.02 oprcc = percentage reduction in cough count after one hour of drug administration; tprcc = percentage reduction in cough count after two hours of drug administration; thprcc = percentage reduction in cough count after three hours of drug administration table 3 presents the results of the percentage reduction in cough counts produced by normal saline, 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. it was 202 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 observed that there was significant difference in the percentage reduction of number of coughs produced by 25mg/kg of dihydrocodeine (77.19±2.03, 81.83±1.04 and 82.94±0.24), as compared to normal saline (17.06±0.24, 21.63±1.28 and 23.86±1.02) in the respective time frames of one hour, two hours and three hours. similar significant differences were observed between normal saline and the different doses (150mg/kg, 300mg/kg and 600mg/kg) of uca tangeri in the various time frames as shown in table 3. thus, normal saline clearly does not decrease the percentage number of cough, as observed in the study, but this is contrary to the effect observed with 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri, which implies that both the standard drug and the test extract actually suppressed the stimulatory process of the intrapulmonary rapidly adapting receptor (rar), which are responsible for the cough or enhancement of the sensitivity of the cough. the fact that dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996) obviously implies that, the decrease in the percentage number of cough is related to the stimulation of μ opioid receptors in the cns. a similar deduction could be made for the uca tangeri extract, since similar and dose dependent pattern was observed with the extract. meaning the extract could possess some cns effect similar to dihydrocodeine. effect of dihydrocodeine and uca tangeri on percentage increase in cough latency in animals treated with acetic acid in a tussive protocol table 4. effect of uca tangeri on percentage increase in cough latency in animals treated with acetic acid in a tussive protocol. group opicl tpicl thpicl normal saline (ns) 2.29±0.01 2.75±0.45 4.13±0.45 dihdrocodeine (dh) 37.13±0.58 37.56±0.73 38.92±0.17 low dose (uca tangeri) (ldut) 10.61±0.62 10.61±0.62 11.92±0.43 medium dose (uca tangeri) (mdut) 18.83±0.95 19.28±0.67 20.65±0.81 high dose (uca tangeri) (hdut) 27.07±0.54 28.91±0.68 29.83±0.85 opicl = percentage increase in cough latency after one hour of drug administration; tpicl = percentage increase in cough latency after two hours of drug administration; thpicl = percentage increase in cough latency after three hours of drug administration table 4 presents the results of percentage increase in cough latency. anova comparison revealed that there were significant differences between normal saline (2.29±0.01, 2.75±0.45 and 4.13±0.45) and 25mg/kg of dihydrocodeine (37.13±0.58, 37.56±0.73 and 38.92±0.17) respectively, for the various time frames of one hour, two hours and three hours. similar significant differences were observed between normal saline and the different doses (150mg/kg, 300mg/kg and 600mg/kg) of uca tangeri for the various time frames. this implies that both 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri cause inhibition to the cough reflex or the stimulation of the intrapulmonary rapidly adapting receptor (rar), leading to a reduction of in the urge for cough, thereby causing a significant increase in the latency period of the cough. dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). thus, the suppression of the cough leading to an increase in latency period is related to μ opioid receptors. it was also noted that the increase in cough latency produced by uca tangeri was dose dependent, meaning a greater suppression was observed with the highest dose, while the smallest suppression was produced by the lowest dose. also, since this suppression was similar to that caused by dihydrocodeine, it could be deduced that extract possesses central nervous system effect similar to that of dihydrocodeine. conclusion dihydrocodeine and uca tangeri were found to be effective antitussive agents. dihydrocodeine was used as the positive control because it is the second most specific antitussive of the commonly used opioids (eddy et al., 1969). dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). uca tangeri also exhibited a dose-dependent antitussive effect reducing both the cough count and latency of cough, similar to dihydrocodeine. thus, it is possible that the extract possesses central nervous system effect similar to that of dihydrocodeine on cough. acknowledgements: the researchers acknowledge all laboratory staffs that assisted in ensuring this research is a success. authors’ contributions: isirima jc designed the study and analyzed the data and uahomo po carried out the laboratory work. isirima jc wrote the manuscript. all authors read and approved the final version of the manuscript. isirima & uahomo – comparative cough suppression of chitosan crab extract … 203 competing interests: the authors declare that there are no competing interests. references agrawal d.k., bergren d.r., byorth p.j., townley r.g. 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(2009). physicochemical characterization of chitin and chitosan from crab shells. carbohydr polym., 75:15–21. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 391-397 | doi: 10.14421/biomedich.2023.121.391-397 issn 2540-9328 (online) analysis of biological activities of two novel metal (ii) complexes of andrographis paniculata crude extract mary adelaide oladipo1, ayodele oluwabunmi ojo2, kayode taiwo ishola3,* 1department of pure & applied chemistry, ladoke akintola university of technology, ogbomoso, oyo state, nigeria. 2department of science laboratory technology, oyo state college of agriculture and technology, igboora, oyo state, nigeria 3department of chemistry, federal college of education (special), oyo, oyo state, nigeria. corresponding author* isholatk@gmail.com manuscript received: 13 february, 2023. revision accepted: 13 march, 2023. published: 24 june, 2023. abstract many diseases in nature have led to the death of many young and old. many bacteria have developed resistance to the available antibiotics on the market. and many drugs employed in treating many diseases such as diabetes mellitus are expensive and are not locally available. therefore, in order to search for more effective, inexpensive, and locally available drugs, this study synthesized and investigated the biological activities of andrographis paniculata crude extract and its co (ii) and ni (ii) complexes. the crude extract and synthesized complexes were characterized using a solubility test, infrared, and ultraviolet-visible spectroscopic analysis. their antibacterial potentials were investigated against two gram-negative bacteria (escherichia coli,staphylococcus aureus) and three gram-negative bacteria (proteus, klebsiella, pseudomonas) while their antidiabetic activities were examined against α-amylase and α-glucosidase enzymes. acarbose was employed as a standard drug. the crude extract and its metal complexes showed different degrees of solubility in the employed solvents. infrared analysis suggested coordination of the crude extract to the metal ions through the oxygen donor atom while the formation of the complexes was affirmed through the occurrence of d-d transitions in the visible region of the metal complexes. the metal complexes were found to display more antibacterial activity than the crude extract. co (ii) and ni (ii) complexes of the crude extract were found to exhibit better activities against α-amylase and α-glucosidase enzymes, respectively than the crude extract and acarbose. it is concluded that the metal complexes could be considered potential antibacterial and antidiabetic agents. keywords: anti-diabetes; anti-bacteria; medicinal plant; metal complexes. introduction high mortality in nature has been attributed to different diseases caused by bacteria, fungi, and viruses. diabetes mellitus described by high blood glucose levels due to insulin deficiency and/or ineffective insulin action is also one of the major deadly diseases that require serious attention. many drugs have been synthesized to combat the diseases. however, many pathogenic organisms have been reported to develop resistance to many of the available drugs while many of the drugs are not locally available. many drugs such as metformin, sitagliptin glimepiride, and acarbose have been widely applied in treating diabetes mellitus. however, these drugs are discovered to possess negative side effects on the patient’s health while some of them are expensive. therefore, a search for more effective and inexpensive antibiotics and antidiabetic drugs without side effects becomes imperative. medicinal plants have become a promising source of natural effective antimicrobial agents. these plants have been observed to possess many bioactive compounds capable of fighting different types of disease. medicinal plants have been widely applied for medical treatment as a result of their ability to exhibit known pharmacological actions for humans and animals. medicinal plants have been the basis of the basic treatment of diseases in many developing countries like africa. andrographis paniculata has been reported to be of great importance in the management of disease and infection. andrographis paniculata (figure 1) also known as ‘king of bitters” in yoruba is known as ‘mejemeje’ is one of the medicinal plants used in treating many diseases such as cancer,ulcer, malaria, and urinary tract infections (karmegam et al. 2015; sachin and kailasam, 2016). the plant has been reported to contain many active organic compounds such as kaempferol, andrographolide, 14-deoxy andrographolide 14deoxy11,12-didehydroandrographolide, quercetin, and other secondary metabolites (subramanian et al. 2008). andrographolide (figure 2) is found to be the main active component of the plant and its medical importance in treating different ailments has been examined by many researchers (flores et al., 2014; daneman & prat, 2015; tao et al. 2018; owoade et al. 2021) https://doi.org/10.14421/biomedich.2023.121.391-397 392 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 the antioxidant and anti-diabetic activity of andrographis paniculate investigated by reddy et al. (2022). the plant was found to possess good antioxidant and anti-diabetic activity. hartini et al. (2021) studied the inhibitory activity of aqueous extract and ethanolic extract of sambiloto andrographis paniculata against the α-amylase enzyme. the ethanol extract of the plant leaf demonstrated higher activity than that of the aqueous extract. the α-amylase enzyme inhibitory activity of andrographis paniculata in aqueous methanol, crude methanol extract, and n-hexane fraction was investigated by ajayi et al. (2021). the plant extract demonstrated more activity than the standard drug employed for the study. the crude methanol extract was observed to demonstrate the highest activity. the contribution of andrographis paniculata in the treatment of metastatic esophageal cancer was investigated by lin et al. (2017). the anti-migratory and suppressive effects on metastasisrelated factors of the absorbed andrographis paniculate were verified. the diterpenes and flavonoid components of the plant component were observed to display esophageal anticancer activity. advancement has been made in the field of medicinal inorganic chemistry in developing different novel organic therapeutic agents as pharmacological and pharmacy technical behaviors of many organic therapeutic agents are observed to increase upon coordination with transition metal ions (farrer and sadler, 2013; newman and cragg, 2016). therefore, there is an apparent need for the study of new metal complex of organic compounds endowed with antimicrobial activities which could be applied in combating multi drug-resistant microorganisms and other diseases. andrographis paniculata has been reported to be of great importance in the management of diseases. however, there is no report on biological activity of metal complex of the plant. therefore, this study synthesized metal (ii) complexes of andrographis paniculata crude extract and evaluated biological activity of the crude extract and its metal complexes against some bacteria and enzymes. figure 1. image of andrograhis paniculata plant. ch3 h ch2 o ch o ho ch2oh h3c ho ch2 figure 2. structure of andrographolide. materials and methods all chemicals used were of analytical grade and they include cobalt acetate, nickel acetate, sodium hydroxide, petroleum ether, hexane, ethanol, methanol, acetone, chloroform, tetra-chloro-methane, distilled water, positive gram strains (escherichia coli, staphylococcus aureus) and negative gram strains (pseudomonas, klebsiella, and proteus). collection and preparation of the plant crude extract the mature leaves of andrograhis paniculata were collected from oyo state college of agriculture, igboora, oyo state. the plant was identified by a botanist in the department of botany the university of ibadan, ibadan with the voucher number uih-23122. the collected leaves were rinsed twice under running water and then put in distilled water to get rid of dirt particles. the plant was air-dried at room temperature. it was then crushed into small particles and then ground into powder. the crude extract of the dried leaves was obtained in n-hexane at 60-80oc where oils, fats, waxes, and terpenes were removed. the extract was then subjected to soxhlet extraction with ethanol (95%) and the solution was concentrated using a rotary evaporator (mousumi et al. 2014). preparation of metal complexes solutions of 10 g of the crude extract and 5 g of cobalt salt in ethanol were mixed together and refluxed for about 4 hr. the mixture was then heated at 80 oc until a precipitate was formed. the precipitate was filtered and dehydrated under a vacuum. the same procedure was repeated for the nickel complex (mousumi et al. 2014). characterization of the metal complexes the metal complexes were characterized by solubility test solvents in water, ethanol, methanol, chloroform, acetone, and diethyl ether. ftir and uv spectroscopic analysis was carried out using a uv-visible spectrophotometer (ce 2021, cecil) and an ftir oladipo et al. – analysis of biological activities of two novel metal (ii) … 393 spectrophotometer (530m, buck) within the range of 600-4000cm-1. antibacterial assay whatman no 1 filter paper was used to prepare a 6 mm diameter disc. the 6 mm diameter discs were sterilized inside an autoclave at 121oc. the moisture discs were dried in a hot air oven at 50 oc. the crude extract and its metal complexes disc and control were prepared. the tests were carried out using the original technique of bauer et al (1996). muller-hinton agar was prepared and autoclaved at 15 bs pressure for 20 mins and cooled. the media was then poured into the sterilized petri dishes and allowed to solidify. the petri plate with the poured media was then seeded, after which the microbial was suspended with the aid of a sterile swab. the plant crude extract and its metal complexes were then placed on each of the plates as well as the control. the plates were then incubated at 37 oc for 24 hr. thereafter, the inhibition zone was measured and expressed in mm. effect of extracts on αamylase activity the extracts (100 μl) and 500 μl of 20 mm sodium phosphate buffer (ph 6.9 with 6 mm nacl) containing pancreatic α-amylase (ec 3.2.1.1) (0.5 mg/ml) were incubated at 25 °c for 10 min. then, 500 μl of 1% starch solution in the same phosphate buffer was added and incubated for another 10 min. 1.0 ml of dinitrosalicylic acid (dnsa) was added, boiled for 5 min, and cooled to room temperature. the reaction mixture was then diluted by adding distilled water (10 ml), and the absorbance of each sample was measured at 540 nm. a complete reaction mixture without acarbose or extract was used as the control. the α-amylase inhibitory activity is expressed as percentage inhibition and calculated using the formula; % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑐 − 𝐴𝑠 𝐴𝑐 𝑋100 ac : absorbance of control (containing all reagents except extracts or acarbose) as : absorbance of the sample (extract or acarbose). four diluted solutions of the crude extract and its metal complexes (40 100 mg/l) were collected for the calculation of the ic50 values. the concentration of the extract required to inhibit the activity of the enzyme by 50% (ic50) was evaluated by ic50 aat calculator (www.aatbio.com). effects of extract on α – glucosidase activity in-vitro the effect of the extract on α – glucosidase activity was determined according to the procedure described by apostolid et al. (2007), using acarbose as a reference. the crude extract and its metal complexes (50 μl) and 100 μl of α-glucosidase solution were incubated at 25°c for 10 min. thereafter, 50 μl of 5 m p-nitrophenyl-α-dglucopyranoside solutions in 0.1 m phosphate buffer (ph 6.9) was added and incubated at 25 °c for 5 min. the absorbance was then read at 405 nm. the α-glucosidase inhibitory activity is expressed as percentage inhibition and calculated as shown below: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑐 − 𝐴𝑠 𝐴𝑐 𝑋100 ac : absorbance of control (containing all reagents except extracts or acarbose) as : absorbance of the extract or acarbose at least four serially diluted solutions of the crude extract and its metal complexes (40 100 mg/l) were taken for calculation of the ic50 values. the concentration of the extract required to inhibit the activity of the enzyme by 50% (ic50) was calculated using the aat calculator (www.aatbio.com). result and discussion the physical properties of andrograhis paniculata crude extract and its metal (ii) complex are shown in table 1 while tables 2 and 3 depict important ir and uv-visible bands of the crude extract and its metal (ii) complexes. antibacterial activities of the plant crude extract and its metal complexes were tested against two-gram-positive and three gram-negative bacteria. the zone of inhibitions of the plant crude extract and its metal complexes against the bacteria in mm are measured as shown in figure 3. figures 4 and 5 depict the compound’s antibacterial activities against the bacteria. table 1. physical characteristics of the plant crude extract and its meeta complexes. compound color crude extract green nickel (ll)complexes green cobalt (ll) complexes pink table 2. solubility property of the pant crude extract and its metal complexes. solvents crude extract cobalt (ii) complex nickel (ii) complex water in in in methanol vs vs ss ethanol vs vs vs chloroform ss ss ss acetone vs vs vs diethyl ether ss ss ss 394 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 table 3. important infra-red data of the plant crude extract and its metal (ii) complexes. compound ʋc-h ʋc-o ʋc=o ʋ-oh plant crude extract 2935 s 1157 s 1717 s 3406 b co (ii) complex 2935 s 1144 s 1702 s 3447 ni (ii) complex 2929 s 1143 s 1698 s 3440 b table 4. electronic data of the plant crude extract and its metal complexes. compound band (cm-1) transition plant crude extract 28169 42553 n→⫪* ⫪→⫪* co (ii) complex 17241 14925 d-d ni (ii) complex 19685 15408 d-d figure 3. measurement of the inhibitory zone of the plant crude extract and its metal complexes. figure 4. histogram representation of antimicrobial activities of the crude extract and its metal complexes against gram-positive bacteria. figure 5. histogram representation of antimicrobial activities of the crude extract and its metal complexes against gram-negative bacteria. table 5. α-amylase and α-glucosidase inhibitory activities of the plant crude extract and its metal complexes. compound ic50 (mg/l) α –amylase α –glucosidase acarbose 55.49 102.66 plant crude extract 85.65 77.98 co (ii) complex 80.52 74.96 ni (ii) complex 42.03 100.79 oladipo et al. – analysis of biological activities of two novel metal (ii) … 395 figure 6. percentage inhibitory effect of andrograhis paniculata crude extract, its metal complexes and standard drug on α-amylase. figure 7. percentage inhibitory effect of andrograhis paniculata crude extract, its metal complexes and standard drug on α˗glucosidase. discussion the various colors of the plant crude extract and its metal complexes are shown in table 1. the plant crude extract and its ni (ii) complex are green in color while the co (ii) of the extract shows is dark pink in color. the plant crude extract and its metal complexes are all very soluble in ethanol, methanol, and acetone, insoluble in distilled water, and slightly soluble in chloroform and diethyl ether as shown in table 2. infrared and uv-visible spectra the relevant infrared spectra data for the crude extract (ligand) and its metal complexes are shown in table 3. the spectrum of the crude extract shows four important bands at 2935 cm-1, 1157 cm-1, 1717 cm-1, and 3406 cm-1 which are ascribed to ν(c – h), ν(c–o), ν(c = o) and ν(c–oh) stretching vibrations, respectively. the bands of ν(c–o) and ν(c = o) observed in the crude extract are observed, upon coordination with co (ii) and ni (ii), to undergo a hypsochromic shift to 1144 cm-1, 1702 cm-1, and 1143 cm-1, 1698 cm-1, respectively. the band shifts and appearance of the new band in the region 400-800 cm-1 in the complexes confirmed the coordination of the ligands to the metal ions through m-o (talavara et al. 2016; teleb et al. 2019) as shown in figure 8. ch3 h ch2 o ch o ho ch2oh h3c ho ch2 ch3 h ch2 o ch o ho ch2oh h3c ho ch2 ch3 h ch2 o ch o ho ch2oh h3c ho ch2 m figure 8. proposed structure for m (ii) complex of mangifera indica leaf crude extract (m= co (ii) and ni (ii)) bands at the visible region of 2817 cm-1 and 4255 cm1 are in the spectrum of the crude extract and the bands are attributed to n– π* and π – π* electronic transitions, respectively as shown in table 4. in the spectra of ni(ii) and co(ii) complexes, bands at 1724 cm-1, 1494 cm-1 and 1969 cm-1, and 1541 cm-1 are observed and are ascribed to d-d electronic transition (table 4) (rasool et al. 2014). antibacterial activities antibacterial activities of the plant crude extract and its metal complexes in ethanol were tested against two gram-negative and three positive bacteria at concentrations of 40 mg/l, 60 mg/l, 80 mg/l, and 1000 mg/l, and the zones of inhibition of the complexes against the bacteria are measured over the disc plates as shown in figure 3. the antibacterial activities of the extract and the complexes at different concentrations as measured are represented in histograms (figures 4 and 5). the solvent employed showed no antibacterial activity at the concentrations. ni (ii) complex was found to show more activity against escherichia coli at the concentrations of co (ii) while the complexes were more active than the plant crude extract. also, only ni (ii) complex was observed to be effective against staphylococcus. the metal complexes exhibited more activity against pseudomonas, proteus, and klebsiella than the extract. co (ii) complex exhibited the highest activity against the pseudomonas and proteus while the plant crude extract was inactive against pseudomonas at all the concentrations. however, ni (ii) complex was observed to display the most activity against klebsiella as shown in figure 5. the antibacterial activities against both gram-negative and positive bacteria increased with an increase in concentration. antidiabetic activities the percentage inhibitory effect of the standard drug (acarbose), andrograhis paniculata crude extracts against α-amylase and α-glucosidase enzymes at different concentrations (40, 60, 80, and 100 mg/l) were represented in figures 6 and 7, respectively. at a 396 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 concentration of 40 mg/l, ni (ii) complex showed more inhibitory effect against α-amylase even than the standard drug while the same activity was exhibited by co (ii) complex and acarbose at a concentration of 60 mg/l. however, the most activity was displayed by acarbose at 80 and 100 mg/l concentrations as shown in figure 6. it is shown in figure 7 that at a concentration of 40 mg/l, ni (ii) and acarbose displayed almost the same activity against α-glucosidase while acarbose exhibited the highest activity at a concentration of 60 mg/l. co (ii) complex and acarbose showed the highest activity at concentrations of 80 and 100 mg/l, respectively. α-amylase and α-glucosidase inhibitory activities of the standard drug (acarbose), andrograhis paniculata crude extract and its metal complexes were calculated and reported as half-maximal inhibitory concentration (ic50) as shown in table 5. ni (ii) displayed the highest inhibitory strength against α -amylas enzyme with an ic50 value of 42.03 while the lowest inhibitory potency against the enzyme was observed to be exhibited by the plant crude extract with an ic50 value of 85.65. the calculated a-amylase inhibitory ic50 values of the crude extracts, its metal complexes, and acarbose in descending order were ni (ii) complex > acarbose > co (ii) complex > andrograhis paniculata crude extracts. conversely, co (ii) showed the highest inhibition against α -glucosidase with an ic50 value of 74.96 mg/l while the lowest activity was displayed by acarbose. the inhibitory effects against α –glucosidase enzyme was found in the order of co (ii) complex > andrograhis paniculata crude extracts > co (ii) complex > acarbose. co (ii) and ni (ii) complexes of the crude extract were found to exhibit better activities than the positive control, acarbose against α-amylase and α-glucosidase enzymes, respectively. the more antidiabetic activities displayed by the metal complexes than the crude extract and acarbose could be ascribed to the capacity of the metal ions to transform the bioavailability and pharmacological behavior of the andrograhis paniculate crude extract. conclusion and recommendation the potential inhibitory properties of co (ii) and ni (ii) complexes of andrograhis paniculata crude extract against some bacteria, α-amylase and α-glucosidase have been examined. the complexes showed more pronounced antibacterial and antidiabetic activities than the crude extract. the metal complexes could be considered potential antibacterial and antidiabetic agents. further characterization analysis should be performed to ascertain the main structures of the complexes. pharmacological and toxicology studies on the metal complexes should be conducted to establish their feasibility as antibacterial antidiabetic agents. conflict of interest: no conflict of interest. references owoade ao, alausa ao, adetutu a, olorunnisola o s, owoade aw (2021). phytochemical characterization and antioxidant bioactivity of andrographis paniculata (nees). pan african journal of life sciences 5(2): 246-256 ajayi os, balogun os, olawuni ij, october n, adigun r, akinlade ig. (2021). alpha amylase inhibition and antioxidant activities of bicyclic diterpenoid lactones from andrographis paniculata. trop j nat prod res.5(6):11101117. doi.org/10.26538/tjnpr/v5i6.22 aposolids e, kwon y i i, shetty k (2007). inhibitory potential of herbs, fruits and fungal enriched cheese against key enzymes linked to type 2-diabetes and hypertension innovative. food science and emerging technologie 8:46-54. bauer a, kirby w, sherris j, turck m (1996). antibiotic susceptibility testing by a standardized single disk method. journal of clinical pathology 45:493-496. reddy css, ramalingam g d, selvaraj j, priya j (2022). in vitro antioxidant and anti-diabetic analysis of andrographis echioides and andrographis paniculata ethanol extract. bioinformation 18(4): 337-342 daneman r, prat a (2015). the blood-brain barrier. cold spring harbor perspective in biology; 7: a020412. farrer nj, sadler pj 2011 medicinal inorganic chemistry: state of the art, new trends, and a vision of the future. in: alessio e (ed) bioinorganic medicinal chemistry. wiley-vch verlag gmbh & co. kgaa, weinheim flores jj, zhang y, klebe dw, lekic t, fu w, zhang jh (2014). small molecule inhibitors in the treatment of cerebral ischemia, expert opin, pharmacother 15: 659–80. hartini ys, setyaningsih d, chang mjv, iglesia mc, nugrahanti a (2021). sambiloto (andrographispaniculatanees.) leaf extract activity as anα amylase enzyme inhibitor. pharmacy education 21(2): 305 – 308 karmegam n, nagaraj r, karuppusamy s, prakash m (2015). biological and pharmacological activities of andrographis spp. (acanthaceae) distributed in southern eastern ghats, india. international journal of current research in biosciences and plant biology 2 :140. lin l, grace g, julia k, eric c, kwok-pui f, jun y, clara b, philip w (2017). the adjuvant value of andrographis paniculata in metastatic esophageal cancer treatment from preclinical perspectives. phytotherapy research 32(7): 13881396 mousumi d, arnab k, garima j, vinod r, aindrila c, tridib d, debajit b, debasish b (2014). andrographolide, one of the major components of andrographis paniculata protects against copper-ascorbate induced oxidative damages to goat cardiac mitochondria in vitro. international journal of pharmaceutical science review and research 28: 237-247. newman dj, cragg gm (2016). natural products as sources of new drugs from 1981 to 2014. j. nat. prod. 3: 629-661. rasool r, hasnain s, nishat n (2014). metal-based schif base polymers: preparation, spectral, thermal and their in-vitro biological investigation. designed monomers and polymers 17: 217-226. sachin a, kailasam k (2016). review of pharmacological investigation of kariyat. journal of pharamaceutical research 6 (1) 270-286. subramanian r, asmawi zm, sadikun a (2008). in vitro α glucosidase and α-amylase enzyme inhibitory effects of oladipo et al. – analysis of biological activities of two novel metal (ii) … 397 andrographis paniculata extract and andrographolide. acta biochimica polonica 55(2): 391–8. talavara v, yadav db, kenchappa ri, sandeep t (2016). synthesis, antimicrobial and antioxidant activity of chalcone derivatives containing thiobarbitone nucleus. medicinal chemistry 6: 440-448. tao l, zhang l, gao r, jiang f, cao j, liu h (2008). andrographolide alleviates acute brain injury in a rat model of traumatic brain injury: possible involvement of inflammatory signaling, front. neurosci. 12: 657. teleb s m, muhammad ea, el-kalyoubi s a, gaballa a s (2019). synthesis, characterization and antimicrobial activities of some 5-bromouracilmetal ion complexes. bulletin of the chemical society of ethiopia 33: 255-268. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 25-32 | doi: 10.14421/biomedich.2023.121.25-32 issn 2540-9328 (online) antioxidative stress and hepatoprotective activities of leaf extract and fractions of setaria megaphylla in plasmodium berghei infected mice ndanti bartholomew william1, augustine lawrence bassey2, john akpan udobang2, jude efiom okokon1,* 1pharmacology and toxicology, faculty of pharmacy; 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 07 september, 2022. revision accepted: 20 september, 2022. published: 04 october, 2022. abstract setaria megaphylla (steud) dur & schinz (poaceae), a perennial grass used traditionally in the treatment of various diseases such as malaria was, investigated for antioxidative stress activity in plasmodium berghei-infected mice. the leaf extract (200-600 mg/kg) and fractions (hexane, dichloromethane, ethyl acetate and methanol; 400 mg/kg) of s. megaphylla were investigated for antioxidative stress and hepatoprotective activities in plasmodium berghei-infected mice using a modified suppressive test model. antioxidative stress and hepatoprotective potentials were assessed by determining oxidative stress markers levels, liver function indices and histopathology of liver. the extract/fractions progressively reduced parasitaemia induced by chloroquine-sensitive p. berghei infection with the methanol fraction exerting the highest activity. the leaf extract and fractions caused significant (p<0.05 – 0.001) increases in the levels of oxidative stress markers enzymes and molecules (sod, cat, gpx, gsh) and also reduced mda level significantly (p<0.05) in the livers of the treated-infected mice. the extract/fractions treatment caused reduction in liver enzymes (alt, ast and alp), total and conjugated bilirubin. histology of livers revealed absence or significant reductions in pathological features in the treated infected mice compared to untreated infected mice. the leaf of s. megaphylla may possess antioxidative stress and hepatoprotective effects which may in part be mediated through the chemical constituents of the plant. keywords: antioxidative stress; setaria megaphylla; antimalarial; antioxidative stress; malaria; plasmodium berghei. introduction global malaria cases in 2019 were estimated to be about 229 million occurring in 87 malaria endemic countries (who, 2020). contrary to significant reduction in malaria related mortality, nigeria recorded the highest mortality rate of all malaria deaths globally in 2019. this portrays a serious threat to life in most countries of africa especially nigeria. oxidative stress associated with malaria infection has been implicated in the pathogenesis and development of systemic complications caused by malaria (guha et al., 2006; ojezele et al., 2017). malaria complications such as anemia, jaundice and pre-eclampsia have been linked to oxidative stress damage caused by the parasite (fabbri et al., 2013; sarr et al., 2017). medicinal plants, therefore, serve as a great reservoir for antimalarial remedies having the advantage of being safer and providing many therapeutic effects. setaria megaphylla (steud) dur & schinz (poaceae), a perennial grass found in tropical and subtropical areas of world (van oudtshoorn, 1999), is traditionally used for the treatment of malaria and diabetes among others (okokon et al., 2007). preliminary reports of antiplasmodial activity on the leaves have been published (okokon et al., 2007; okokon et al., 2017). the leaf extract also possesses antidiabetic and hypoglycaemic (okokon and antia, 2007; okokon et al., 2022), anti-inflammatory, analgesic (okokon et al., 2006), cytotoxic, immunomodulatory and antileishmanial (okokon et al., 2013), antidepressant (okokon et al., 2016), inhibitory effect on α-amylase and α-glucosidase (okokon et al., 2021) activities. phytochemical analysis of the leaf extract shows that it contains phytochemical compounds such as flavonoid, carbohydrate, terpenes, saponins, tannins, anthraquinones, cardiac glycosides (z,z,z)-8,11,14eicosatrienoic acid, phthalic acid, diisooctyl ester, vitamin e, ᵞ-elemene, urs-12-ene, bicyclogermacrene, αmuurolene, germacrene-a, and guaiol have been reported (okokon et al., 2006; okokon et al., 2013). 1triacontanal, 1-triacontanol, 1-dotriacontanol, 1triacontyl cerotate, and stigmasterol have also been isolated from the plant leaves (okokon et al., 2022). we report in this study the antioxidative stress and hepatoprotective potentials of leaf extract and fractions of setaria megaphylla in plasmodium berghei-infected mice. https://doi.org/10.14421/biomedich.2023.121.25-32 26 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 materials and methods plants collection the leaves of setaria megaphylla were collected from farms in the uruan area of akwa ibom state, nigeria. the plant was identified by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. a voucher specimen (uuph. 221 d) of the plant was deposited in the department of pharmacognosy and natural medicine herbarium at the university of uyo. extraction the leaves were washed and shade dried for two weeks. the dried leaves were cut into smaller pieces and pulverized to powder. the powdered leaves was divided into two parts. one part was macerated in ethanol for 72 hours, while the remaining part was successively and gradiently macerated for 72 hours in each of, n-hexane, dichloromethane, ethyl acetate and methanol respectively, which is along their polarities to give the corresponding gradient fraction for each solvent. the liquid filtrate of each extract and fraction were concentrated and evaporated to dryness in vacuo 400c using a rotary evaporator. the various yields were calculated and the extract and fractions were stored in a refrigerator at -4oc, until used for the proposed experiments. microorganism (parasite) chloroquine-sensitive strain of plasmodium berghei anka strain was obtained from the national institute of medical research (nimer), yaba lagos, nigeria and maintained by subpassage of blood from infected to healthy mouse once every 7-8 days. parasite inoculation each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of infected blood containing about 1 x 107p. berghei parasitized erythrocytes collected from an infected mouse with 2030% parasitaemia. the inoculum consisted of 5 x 107 p. berghei infected erythrocytes per milliliter prepared by determining both the percentage parasitemia and the erythrocytes count of the donor mouse and diluting the blood with isotonic saline in proportions indicated by both determinations (atanu et al., 2021). parasitemia was monitored by standard methods; thin blood smears were made on glass slides, fixed using methanol, and stained using giemsa stain parasitema was counted using a microscope and was calculated as a percentage of infected red blood cells (rbcs) relative to the total number of cells in a microscopic field at ×100 magnification according to the formula of peters and robinson (1992) as given below: 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 (%) = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑠𝑒𝑑 𝑅𝐵𝐶𝑠 total number of rbcs × 100 experimental animals male and female swiss albino mice, each weighing 2132 g, were obtained from the university of uyo’s animal house. they were kept in standard cages and acclimatized for a period of 10 days before use in the experiments. the mice were fed on standard pelleted diet and water ad libitum. all animals were kept at room temperature in cross ventilated rooms. the care and use of animals was conducted in accordance with the national institute of health guide for the care and use of laboratory animals (nih publication, 1996). approval for the study was obtained from the university of uyo’s animal ethics committee. drug administration the extract, fractions, chloroquine and pyrimethamine that were used in the antimalarial study, were administered orally with the aid of a stainless metallic feeding cannula. evaluation of antioxidative and liver protective activities of the leaf extract and fractions of saccharum officinarum using 4-day test a modified early infection test model was used to assess the antioxidative stress and hepatoprotective potentials of the leaf extract and fractions as well as chloroquine in p. berghei infected mice. this was done using the method described by knight and peters (1980). fortyfive mice were randomly divided into nine groups of five (5) mice each based on body weights on the first day (d0). they were further infected with the parasite and treated with the leaf extract, fractions, chloroquine and distilled water. based on previously determined median lethal dose (ld50) of 2.4 ± 0.5 g/kg, (okokon et al.,2006), the mice in groups 1-3 were given 200 mg/kg, 400 mg/kg and 600 mg/kg of crude extract respectively, while groups 4, 5, 6, 7 were administered 400 mg/kg of n-hexane, dichloromethane, ethyl acetate, and methanol fractions respectively, group 8 was given 5 mg/kg of chloroquine (positive control) and group 9 was given 10 ml/kg of distilled water (negative control) for four consecutive days (d0-d3) between 8am to 9am. on the fifth day (d4), thin films were made from the tail blood. the films were stained with giemsa stain to reveal parasitized erythrocytes out of 500 in a random field of the microscope. on the tenth day, the mice from various group were sacrificed under diethyl ether vapour. blood samples were collected into edta bottles and plain centrifuge tubes. the blood samples in the edta bottles were used for hematological analysis, while those in centrifuge tubes were centrifuged immediately at 2500 rpm for 15 mins to separate the serum at room temperature to avoid haemolysis and used for biochemical assays such as determination of liver function indices. liver from each mouse was surgically removed, weighed and divided into two parts. one part was fixed in 10% formaldehyde for histological process william et al. – biological activities of setaria megaphylla 27 and the other part stored in ice cold normal saline. the average suppression of parasitemia was calculated according to the formula of peters and robinson (1992) as follows: (average % parasitemia positive control – average % parasitemia negative control) / (average % parasitemia negative control). effect of the leaf extract and fractions on biochemical parameters and histology of livers of p. berghei infected mice serum was separated from the blood of each mouse sacrificed and these sera were stored at -20oc until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase(ast), alanine aminotransferase (alt), alkaline phosphotase(alp), conjugated and total bilirubin to assess the liver functions. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (tietz, 1976). the livers of the sacrificed animals were surgically removed, weighed and a part of each fixed in 10% formaldehyde for histological processes, while the other part was washed with ice cold 0.9% nacl and homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c and the supernatants were used for the assays of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence and burk,1976), and reduced glutathione (gsh) (ellman,1959). malondialdehyde (mda) and glutathione-s-transferase (gst) were determined using commercial diagnostic kits (aldrich-sigma, usa) using standard procedures of the manufacturer's protocol. statistical analysis data collected were analyzed using one-way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem and significance relative to control were considered at p˂0.05. results and discussion effect of leaf extract and fractions on parasitaemia the extract exerted a dose-dependent and significant (p<0.001) reduction in parasitaemia levels of the treated mice at the different doses employed when compared to the control group in the study. the leaf fractions (nhexane, dcm, ethyl acetate and methanol; 400 mg/kg), exerted prominent reductions in parasitaemia levels of the treated mice with chemosuppression of 11.12, 23.96, 20.81 and 41.00% for n-hexane, dichloromethane, ethyl acetate and methanol respectively. however, methanol fraction exerted the highest activity (figure 1). effect of leaf extract and fractions on liver function indices of p. berghei-infected mice. the levels of liver function indices (ast, alt, alp, total protein, albumin, total and conjugated bilirubin) were found to be elevated in untreated p. berghei infected mice. however, treatment of p. berghei infected mice with leaf extract and fractions of s. megaphylla caused non dose-dependent and significant (p<0.01-0.001) reductions in the activities of ast and alt especially at higher doses (400 and 600 mg/kg) with methanol fraction followed by ethyl acetate fraction treated group having the most significant (p<0.001) reduction when compared to control though not comparable to that of chloroquine (table 1). alp levels of the treated groups of infected mice were non dose dependently and significantly (p<0.01-0.001) reduced when compared to control untreated mice with the methanol fraction followed by n-hexane fraction treated group having the highest and most significant (p<0.001) reductions. the total protein levels of the treated mice groups were similarly reduced non dose-dependently and significantly by the extract/ fractions treatments with the n-hexane fraction treated group having the highest significant effect (p<0.01). treatment of infected mice further produced non dose-dependent and significant (p<0.05-0.01) reductions in albumin levels of the treated mice when compared to control. although the fractions-treated groups had reduced albumin levels, these were not significant (p>0.05) when compared to control. the extract/fractions treatment caused non dosedependent reduction of total bilirubin level of the treated infected mice though not significant (p>0.05) when compared to control. however, the methanol fraction significantly (p<0.05) reduced the total bilirubin level of the mice when compared to control (table 5). the leaf extract and fractions of s. megaphylla also caused non dose dependent reductions in the levels of conjugated bilirubin of the treated mice which was only significant (p<0.05) at the middle dose (400 mg/kg) when compared to control. methanol fraction also produced significant (p<0.05) reduction of conjugated bilirubin when compared to control (table 1). effect of leaf extract and fraction on liver antioxidant enzymes of p. berghei-infected mice. the levels of enzymatic and non-enzymatic endogenous antioxidants (gst, sod, cat, gpx and gsh) in the infected mice were found to be lowered by malaria parasite infection, while higher levels of mda were also observed (table 2). treatment of plasmodium bergheiinfected mice with leaf extract and fractions of s. megaphylla caused significant (p<0.05-0.001) dosedependent elevation in the levels of sod with the dcm fraction treated group having the most significant level (p<0.001) followed by ethyl acetate and methanol 28 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 fractions treated groups. treatment of infected mice with leaf extract/fractions of s. megaphylla also caused dose-dependent and significant (p<0.001) increases in the activity of gst especially at the higher doses of the extract (400 and 600 mg/kg) when compared with the control untreated infected mice. methanol fraction followed by n-hexane and dcm fractions produced the most significant effect (p<0.001) when compared to control. cat activity was increased non dosedependently but significant (p<0.05-0.001) by the leaf extract/fractions treatment of the infected mice when compared to control with the n-hexane fraction treated group having the highest activity. leaf extract/fractions treatment caused non dose-dependent elevation of gpx activity in the treated infected mice which was only significant (p<0.001) in the group treated with n-hexane fraction when compared to control. similarly, gsh levels in the treated infected mice were significantly (p<0.001) and non dose-dependently elevated following treatment with the leaf extract and fractions when compared to control. n-hexane fraction followed by ethyl acetate and methanol fractions exerted the highest significant (p<0.001) activity when compared to control. however, there were significant (p<0.01-0.001) and non dose-dependent reductions in the levels of mda of the treated mice with dcm and ethyl acetate fractions having the most significant effects (p<0.01-0.001) when compared to control (table 2). effect of extract and fractions on the histology of liver of p. berghei-infected mice. histologic sections of livers of untreated infected mice showing distorted livers with congested central vein, hepatocytes, sinosoids containing inflammatory cells, necrotic tissues. histologic sections of livers of infected mice treated with 200 mg/kg of leaf extract revealed distorted livers with hepatocytes characterised by fatty degenerations, steatosis (microvasicular, macrovasicular, steatosis). livers of infected mice treated with 400 mg/kg of extract had liver sections revealing normal/borderline liver hepatocytes with mild steatosis, patent central vein and sinosoids. histologic sections of livers of p. berghei-infected mice treated with 600 mg/kg showed distorted liver with congested central vein, hepatocytes, necrotic tissues, and sinosoids. n-hexane fraction treated infected mice showed liver sections with mildly distorted livers/border line liver with hepatocytes, sinosoids and necrotic tissues. infected mice treated with dcm fraction had livers histologic sections showing distorted liver with hepatocytes, sinosoids containing kupffer cells, generalised distribution of necrotic tissue and patent central vein (figure 2). group of infected mice treated with ethyl acetate fraction showed livers histologic sections with normal liver with intact hepatocytes, sinosoids and patent central vein (figure 2). also, infected mice treated with methanol fraction had normal livers sections with intact hepatocytes, sinosoids containing kupffer cells and patent central vein (figure 2). p. berghei-infected mice treated with chloroquine had histologic sections of livers showing normal liver with intact hepatocytes, sinosoids containing kupfers cells and patent central vein (figure 2). figure 1. suppressive activities of leaf extract and fractions of s. megaphylla during early plasmodium berghei infection in mice. values are expressed as mean ± sem. significant relative to control. *p<0.05; **p<0.01; ***p<0.001. n = 6. william et al. – biological activities of setaria megaphylla 29 figure 2. histologic liver sections of plasmodium berghei-infected mice untreated (a), treated with leaf extract of s. megaphylla, 200 mg/kg (b), 400 mg/kg (c), 600 mg/kg (d), n-hexane fraction(e), dcm fraction (f), ethyl acetate fraction (g), methanol fraction (h) and chloroquine, 5 mg/kg (i) at magnification x400. keys: hepatocytes (hep), sinosoids (sin), necrotic tissues (nt), steatosis (st), inflammatory cells (inf). table 1. effect of leaf extract and fractions of setaria megaphylla on liver function parameters of mice infected with plasmodium berghei during established infection. treatment dose (mg/kg) liver function parameters ast (iu/l) alt(iu/l) alp(iu/l) total protein (g/l) albumin (g/l) total bilirubin (µmol/ml) conjugate d bilirubin (µmol/ml) control 61.33±1.84 27.46 ±1.35 31.6 ±1.06 69.6 ±3.38 48.0 ±2.08 13.53±0.63 8.20±0.55 extract 200 54.66±1.17 21.6±1.76 17.33±1.85b 57.0±0.57b 33.0±0.57b 10.93±1.83 7.0±1.65 400 40.0±3.21c 14.66±1.79c 17.16±1.20b 59.0±2.64b 37.3±1.85a 8.16±1.33 4.66±0.79a 600 51.0±1.35b 19.66±1.80a 11.66±1.20c 54.66±2.18c 34.6±2.72b 10.13±0.86 6.06±0.98 n-hexane 400 44.33±3.92b 22.66 ±2.09 19.66±1.85a 57.33±1.20b 39.0 ±3.51 8.70±1.04 5.33±0.68 dichloromethane 400 46.66±3.66b 20.66± 2.17 24.33±2.72 58.0 ±0.57b 38.0 ±1.52 9.16±1.67 5.10±1.20 ethyl acetate 400 41.33±2.45c 16.33± 1.36c 26.0± 3.21 58.33 ±1.20b 40.0 ±1.15 8.93±1.78 6.10±1.60 methanol 400 37.0±3.21c 16.33± 1.10c 17.0± 2.51b 58.66 ±2.33b 39.0 ±2.64 7.43±0.66a 3.80±0.17a chloroquine 5 25.66 ±2.90c 15.36 ±0.85c 18.16 ±4.41b 52.0±1.73c 35.66±1.85a 5.30±0.55b 3.73±0.57a values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. (n = 6). a b c d e f g h i 30 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 table 2. effect of leaf extract and fractions of setaria megaphylla on liver antioxidant enzymes of mice infected with plasmodium berghei during established infection. treatment dose (mg/kg) antioxidant parameters gsh (µg/ml) sod (µg/ml) cat (µg/ml) gpx (µm/ml) gst (µg/ml) mda (µmol/ml) liver weight (g) control 1.16±0.06 0.17 ±0.02 1.03 ±0.13 0.050 ±0.003 0.074 ±0.002 0.57±0.01 2.76±0.16 extract 200 1.30±0.05c 0.25±0.02 3.59±1.27c 0.054±0.002 0.107±0.007 0.46±0.03 2.69±0.19 400 1.56±0.03c 0.34±0.04b 1.78±0.50b 0.049±0.004 0.177±0.004c 0.36±0.02c 2.28±0.16 600 1.50±0.03c 0.39±0.09c 3.73±1.23c 0.054±0.005 0.211±0.006c 0.39±0.03b 2.86±0.18 n-hexane 400 1.95±0.05c 0.31 ±0.01a 6.35±0.08c 0.660±0.003c 0.116 ±0.005c 0.48±0.03 2.41±0.16 dichloromethane 400 1.44±0.02c 0.39± 0.01c 1.99±0.10c 0.060 ±0.007 0.116 ±0.009c 0.35±0.03c 2.24±0.18 ethyl acetate 400 1.70±0.03c 0.33±0.01b 1.67± 0.12b 0.058 ±0.006 0.100 ±0.005 0.41±0.02b 2.39±0.13 methanol 400 1.51±0.04c 0.32±0.03b 2.19± 0.29c 0.062 ±0.001 0.380 ±0.01c 0.47±0.04 2.16±0.10 chloroquine 5 1.18 ±0.04 0.20 ±0.05 2.73 ±0.39c 0.057±0.008 0.155±0.01c 0.56±0.04 2.22±0.18 values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. (n = 6). discussion the leaves of s. megaphylla are used in ibibio traditional medicine as malaria remedy and this work was designed to investigate its antioxidative stress and hepatoprotective potentials in plasmodium bergheiinfected mice. the leaf extract and fractions of s. megaphylla were investigated for antimalarial activity against rodent malaria parasite, p. berghei infection in mice using early infection test model. it was found that the extract and fractions significantly reduced the parasitaemia with methanol fraction exhibiting the highest suppressive activity, confirming the antimalarial potential of these extract and fractions. these activities could have resulted from plasmodicidal or plasmodistatic activity of the extract and fractions. the results of this study corroborate earlier reports of antimalarial and antiplasmodial activities of the leaf extract and fractions (okokon et al., 2007; 2017). this observed activity may have resulted from the activities of the phytochemical constituents of this plant earlier reported (okokon et al., 2006; 2013; 2021). in the present study, higher levels of transaminases and hyperbilirubinemia were observed in untreated infected animals. temporary hepatic dysfunction is associated with malaria infection characterized by the increase of relative liver weight and liver enzymes activities. the distortions in liver may result from alteration in blood flow through the organ as parasitized rbc adhere to endothelial cells, blocking the sinusoids and obstructing the intrahepatic blood flow. similarly, liver damage could be also due to the leakage of some hepatic cells which were killed or injured by the immune response progress and/or by abnormal cell activation induced by the parasites (guthrow et al.,1979). free radicals have been implicated in liver impairment which results in leakage of some enzymes. hyperbilirubinemia results from the impairment of drainage capacity of the liver due to endothelial blockage and distrubance of hepatocytes (onyesom and onyemakonor, 2011). administration of leaf extract and fractions of s. megaphylla to p. berghei-infected mice was found to decreased the elevated total protein, albumin, ast, alt, alp, conjugated and total bilirubin. the increases recorded in serum total protein, albumin, alt, ast, alp, conjugated and total bilirubin in the parasitized non-treated mice was suggested be due to response to hyper-parasitemia (orhue et al., 2005). malaria parasite infections are accompanied by cellular mobilization of t-cells and its complements with a resultant synthesis and secretion of antibody molecules leading to elevated protein levels in parasitized non-treated mice (orhue et al., 2005). the increased activities of serum ast, alt, and alp in the liver and blood of p. berghei infected mice may be due to hepatic dysfunction (george et al 2011; guthrow et al., 2007) or hepatic damage as could be confirmed in the liver histology. the observed increases in activities of markers enzymes of hepatic damage is in agreement with the report of uzuegbu and emeka, (2011). however, administration of s. megaphylla reduced and restored normal levels of total protein, albumin, direct and total protein and the activities of ast, alt and alp in the serum of infected treated mice. the results are indicative of hepatoprotective activity of the leaf extract and fractions. besides, the histological analyses of livers from malaria infected mice showed a significant pathological signs. this reticuloendothelial hyperplasia expressed by general architectural disorganization of liver with inflammatory sites, hepatocyte necrosis, vascular congestion, malarial pigment presence in the kupffer cells and steanosis is suggestive of inflammatory reaction in the tissue. these were reduced or absent following extract/fractions treatments which further confirm the hepatoprotective activity of the leaf extract due to the antioxidant activities of its phytochemical constituents as previously reported (okokon et al., 2013, okokon et al., 2017; okokon et al., 2021). oxidative stress results from an imbalance between the generation of reactive oxygen species and william et al. – biological activities of setaria megaphylla 31 endogenous antioxidant systems (chanda and dave, 2009). the tissue hypoxia cause by malaria infection induces an activation of the natural host defence that generates large amounts of reactive oxygen species, causing an imbalance between the formation of oxidizing species and the activity of antioxidants which can lead to the death of the parasites (becker et al., 2004; percario et al., 2012). studies suggest that the generation of reactive oxygen species and reactive nitrogen species (ros and rns) associated with oxidative stress is implicated in the pathogenesis and development of systemic complications caused by malaria (guha et al., 2006; ojezele et al., 2017). malaria complications including anemia, jaundice and preeclampsia have been linked to oxidative stress damage caused by the parasite (fabbri et al., 2013; sarr et al., 2017). malarial infection has been found to decrease the levels of antioxidant enzymes and other non-enzymatic anti-oxidants such as catalase (cat), glutathione (gsh) peroxidase, super oxide dismutase (sod), albumin, glutathione, ascorbate and plasma tocopherol. malaria severity has also been found to be directly proportional to the level of lipid peroxidation an indication of membrane damage which is associated with increased malondialdehyde levels (asagba et al., 2010; adil et al., 2013). the oxidative stress induction was also described as electrons produced during the oxidation of fe2+ into fe3+ following the hb degradation by the parasites (kumar and bandyopadhyay, 2005). moreover, antioxidant enzymes (catalase and sod), and molecules such as proteins significantly decreased while lipid peroxidation (mda) increased during malaria infection (luersen et al., 2000). these indices have been used to measure the severity of malaria infection. in this study, the activities of sod, cat, gpx and gsh level were found to decrease significantly, while mda level was significantly increased in the untreated infected group. these findings corroborate earlier reports on the rise in mda level as indicative of lipid peroxidation in the liver of p. berghei-infected mice and depletion in host’s sod and cat activities being important features of malaria infections (becker et al., 2004; gora et al., 2006). the plant extract and fractions exerted protective effect by increasing the activity of antioxidant enzymes and molecules as well as reducing the level of mda significantly, thus lipid peroxidation. this activity can be explained by the presence of phenols, flavonoids and tannins that are able to trap free radicals (rice-evans et al., 1995). these activities may have resulted from the antioxidant activities of its phytochemical constituents as previously reported (okokon et al., 2013, okokon et al., 2017; okokon et al., 2021). conclusion the results of this study show that the leaf extract and fractions of setaria megaphylla possess antimalarial, antioxidative stress and liver protective potentials which may be attributed to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to mr. nsikan malachy of pharmacology and toxicology department and mr adewale wole of anatomy department for providing technical assistance. authors’ contributions: jeo, nuw, ail, research concept and design; 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(1999). guide to grasses of south africa. briza publications, cape town,1999. world health organisation (2020). world malaria reports.www. who. int/publications /m/item/who-htm-gmp-2020.08 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 259-266 | doi: 10.14421/biomedich.2023.121.259-266 issn 2540-9328 (online) combination interactive effects of gongronema latifolium leaves and picralima nitida seeds extracts on glucose tolerance maureen ifeyinwa egbuniwe1, harrison anezichukwu ozoani1, amara anwuchaepe ajaghaku2, ikechukwu sonne mbagwu3, uchechukwu harrison orji3, daniel lotanna ajaghaku1,* 1department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu, nigeria 2department of pharmacognosy, faculty of pharmaceutical science, chukwuemeka odumegwu ojukwu university, igbariam, anambra, nigeria. 3department of pharmacology, faculty of pharmaceutical sciences, nnamdi azikiwe university awka, anambra state, nigeria. corresponding author* daniel.ajaghaku@esut.edu.ng manuscript received: 13 december, 2022. revision accepted: 13 march, 2023. published: 18 march, 2023. abstract this study evaluated the combination interactive effects of g. latifolium leaves and p. nitida seed extracts using a metabolic glucose tolerance test. the plant samples were extracted separately using cold maceration and their acute toxicities were determined. doseresponse glucose tolerant tests of both plants were done using a 2 g/kg glucose load monitored over 0 – 1h. a 41% effect isobologram was used to determine the needed dose combinations according to the principle of loewe’s additivity model. the glucose tolera nt tests of dose pairs of the combined extracts were evaluated and their combination indices were calculated to determine the nature of their interaction. the ed41 of g. latifolium (gl) and p. nitida (pn) were 180 mg/kg and 254 mg/kg respectively. the percentage reductions of gl:pn (50:160); gl:pn (100:90); and gl:pn (150:30) dose pairs were 48.23, 50.76 and 42.99 % respectively. their combination i ndex were calculated to be 0.91, 0.91 and 0.95 respectively an indication of synergistic interaction. findings from this study validate the combined use of g. latifolium leaves and p. nitida seeds in folkloric medicine. however, combining the extracts of g. latifolium: p. nitida in the dose ratios of 50:160, 100:90 and 150:30 mg/kg gave the best dose pairs with synergistic outcome. keywords: combination studies; gongronema latifolium; glucose tolerance; picralima nitida. introduction plants have evolved over millennia to address the multifactorial nature of disease pathogenesis by targeting multiple pathways associated with disease initiation, progression and complications through the combined action of structurally and functionally diverse constituents (zaynab et al., 2018). this diversity in plants is better harnessed when they are used in crude unfractionated forms and in combination (che et al., 2013). it is however too simplistic to assume a positive synergy from any two or more herbs combined in traditional practice as some may lead to an unexpected decrease in activity due to competition for the same site of action or through interfering with the pharmacokinetics of each other (sun et al., 2019). similarly, two plants when combined can exhibit both antagonism and synergism depending on the dose pair that is used (foucquier and guedj, 2015). the benefit of combination therapy is not simply attributed to the properties of the drugs but could also depend on the dose ratio. as the cells do not make the difference between a single dose and a combination, two drugs combined at a given dose ratio could be considered as a third agent with its own dose-effect relation (chou, 2010). the establishment of optimal pair of dose levels for each plant extract intended to be used in combination therapy is therefore indispensable in maximizing the treatment efficacy of combination therapy. the use of optimal dose pairs in combination therapies exploits the chances for improved efficacy and decreased adverse effects (podolsky & greene, 2011). thus, owing to these advantages, combination therapies have become a standard for the treatment of several diseases (raskin, 2008). as such, they continue to represent a promising approach in indications of unmet medical needs. glucose intolerance is a general term for a group of metabolic conditions that result from hyperglycemia. it is a dysglycemic state that comprises both intermediate hyperglycemia (formerly called prediabetes) and diabetes. the major categories of glucose intolerance are as follows: impaired fasting glucose (ifg), impaired glucose tolerance (igt), and diabetes mellitus (dm). ifg and igt are intermediate conditions in the transition between normality (normal glucose tolerance, ngt) and diabetes (who, 2021). the development of glucose intolerance is usually initiated by insulin resistance and https://doi.org/10.14421/biomedich.2023.121.259-266 260 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 worsened by compensatory hyperinsulinemia (wang et al., 2019). pancreatic beta-cell dysfunction and/or hepatic and muscle insulin resistance are primary defects responsible for the development and progression of type2 diabetes. impaired glucose tolerance represents an early stage in the development of type-2 diabetes. although it can present as the asymptomatic condition with subtle changes in fasting and/or postmeal serum glucose concentration, it is an important indicator in identifying patients at high risk of developing type-2 diabetes (alok et al., 2018). several studies have demonstrated the medicinal and beneficial effects of gongronema latifolium and picralima nitida plants. both plants are members of the apocynaceae family which have been used in traditional medicines for the treatment and management of malaria, abscesses, hepatitis, pneumonia, diabetes, hypertension, etc. (decampos et al., 2020). the leaves of g. latifolium and seeds of p. nitida have been specifically reported to possess glucose lowering potential both in diabetes and normoglycemic in-vivo experimental models (mohammed et al., 2014). however, none of these reports have considered a combined effect of both plants, a common practice in traditional medicine. this study evaluated the combination interactive effects of g. latifolium leaves and p. nitida seed extracts using a metabolic glucose tolerance test (mgtt) in an animal model. materials and methods collection of plant materials the seeds of picralima nitida and leaves of gongronema latifolium were obtained from nsukka, enugu state, nigeria. the plant samples were identified by mr felix nwafor, a taxonomist of the department of pharmacognosy and environmental medicine, university of nigeria nsukka (unn), enugu state, nigeria. animals wistar albino mice (20 – 25 g) were obtained from the animal house of the department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu state, nigeria. the animals were housed in standard laboratory conditions of 12 h light, room temperature, 40-60% relative humidity and fed with rodent feed (guinea feeds nigeria ltd). they were allowed free access to food and water. all animal experiments were conducted in compliance with the nih guide for the care and use of laboratory animals (national institute of health (nih) (2011) pub no: 85-23). preparation of plant extracts the pods of picralima nitida were opened and the seeds obtained were air-dried under a shade for 30 days. similarly, the fresh plant sample of gongronema latifolium was collected from the wild fields and the leaves were detached from the unwanted parts of the plant. the leaves were air dried under a shade at room temperature for 15 days. after air-drying under a shade, the seeds and leaves were pulverized to a coarse powder using an electrical blender. pulverized samples of picralima nitida (1kg) and gongronema latifolium (1.6 kg) were macerated separately in 1.5 l each of methanol at room temperature for 72 hours with intermittent shaking. the extracts were first filtered, using a cotton wool clogged funnel and then filtered severally through whatman no. 1 filter papers. the extracts were concentrated by evaporating the methanol to dryness under reduced temperature and pressure (below 40oc) using a rotary evaporator. the percentage yields of the extracts were determined and then transferred into air-tight containers and stored in a refrigerator until required for experimentation. qualitative phytochemical analysis the qualitative phytochemical analysis of the extracts was carried out using standard methods as described by odoh et al., 2019. acute toxicity study the acute toxicities of both samples were done using lorke’s method (lorke, 1983). this method has two phases which are phases 1 and 2 respectively. phase 1: in this phase, nine animals were randomly selected to represent the g. latifolium group and another nine were selected to represent the p. nitida group. for each group, the nine animals were divided into three subgroups containing three animals per sub-group. members of each of the three sub-groups were administered 10 mg/kg, 100 mg/kg and 1000 mg/kg of each of the plant samples respectively. the animals were then placed separately under 24-hour observation to monitor for any sign of behavioral changes as well as mortality that may suggest toxicity. phase 2: this phase involved the use of three animals per plant sample. these animals were distributed into sample groups of one animal per group. the animals were then administered higher doses (1600, 2000 and 5000 mg/kg) for g. latifolium and 300, 500 and 800 mg/kg for p. nitida. they were observed for 24h post treatment for signs of toxicity as well as mortality. then the ld50 was determined using the following equation: 𝐿𝐷 = √(𝐷0 ∗ 𝐷100) (1) d0 : highest dose that gave no mortality, d100 : lowest dose that produced mortality. dose response glucose tolerance effect of extracts of g. latifolium leaves and p. nitida seeds the animals were fasted overnight before the test and their fasting blood glucose levels were measured (fbg). egbuniwe et al. – glucose tolerance herbal combination interaction 261 1hr after the extracts were given, the animals were administered 2 g/kg d-glucose in distilled water. blood samples were taken by tail milking at 15, 30, 45 and 60 minutes after glucose administration and the glucose concentrations were determined. in comparison to the control group, the area under the curve (auc) of the plot of blood glucose against time was used to measure metabolic glucose tolerance. for dose-response effect of methanol extract of g. latifolium leaves, the animals were grouped into 5 groups of 5 mice each. they fasted for 12 h, but water was provided ad libitum before the experiment. the fasting blood glucose of each animal was determined before extracts were administered. groups 1-5 were treated with 12.5, 25, 50, 100, and 200 mg/kg, respectively. change in blood glucose levels was accessed for each animal at 15, 30, 45 and 60 minutes post-treatment. the percent change in blood glucose for each animal was calculated and the average for each group was determined. for p. nitida, the animals were grouped into 4 groups of 5 mice each. they fasted for 12 h, but water was provided ad libitum before the experiment. the fasting blood glucose of each animal was determined before extracts were administered. groups 1-4 were treated with 100, 200, 400, and 800 mg/kg, respectively. change in blood glucose levels was accessed for each animal at 15, 30, 45 and 60 minutes post-treatment. the percent change in blood glucose for each animal was calculated and the average for each group determined. the animals for the control groups were grouped into 2 groups of 5 mice each. group 1 received 5 ml/kg distilled water and served as a negative control, and group 2 received 250 mg/ kg metformin and served as the positive control. the same procedure was carried out for the control groups. combination effect of methanol extract of g. latifolium leaves and p. nitida seeds twenty –five (25) mice grouped into 5 groups of 5 animals per group were used for the combination interaction study. they fasted for 12 h before the experiment. the fasting blood glucose of each animal was determined before extract and drug administration. they were treated with combination doses of g. latifolium: p. nitida as follows: group 1 (91:126.5 mg/kg), group 2 (110:100 mg/kg), group 3 (50:160 mg/kg), group 4 (100:90 mg/kg) and group 5 (150:30 mg/kg). effects of these treatments on blood glucose were evaluated at 15, 30, 45 and 60 minutes for each animal and the average for each group was determined. evaluation of the nature of the interaction of the combination therapy using a dose-effect-based strategy, the interactions of various ratio combinations of p.nitida and g.latifolium extracts were determined. the percentage effect value of the extracts at different concentrations and when combined in different ratios were used in the formula to calculate their combination index. various ratio combination interactions between p. nitida and g. latifolium extracts were determined using a dose-effectbased strategy (loewe additivity). the percentage effect value of the extracts at different concentrations separately and when combined in different ratios were used for the calculation of their combination index using the formula. ci = a a + b b (2) where: a = dose of g. latifolium in the combination and a = effective dose of g. latifolium; while b =dose of p. nitida in the combination and b = effective dose of p. nitida, ci= combination index. when ci= 1 (additive interaction), ci > 1 (antagonistic interaction) and ci < 1 (synergistic interaction). statistical analysis all values were expressed as the mean + standard error of the mean (sem) of five animals per group. data were analyzed using one-way anova. p values less than 0.05 was considered statistically significant. graphical plots were done using microsoft excel 2010. results extraction, yield and phytochemical analysis methanol extracts of g. latifolium produced a higher yield compared with p. nitida (table 1) saponins, alkaloids, tannins, steroids, and flavonoids were present in high amounts in both samples. quinones were not detected in the sample of picralima nitida but were present in high amounts in the sample of gongronema latifolium. glycosides were present in high amounts in the methanol extract of g. latifolium but undetected in the sample of p. nitida. lastly, anthraquinones were not detected in both samples. table 1. extraction yields and phytochemical content of the extracts. phytochemical constituents gongronema latifolium picralima nitida saponins +++ +++ flavonoids +++ +++ steroids ++ ++ alkaloids +++ +++ glycosides +++ quinones +++ anthraquinones tannins ++ ++ yield% 24.69% 15.59% key: +++ = high amount of phytoconstituents present;++ = moderate amount of phytoconstituents present;+ = low amount of phytoconstituents present;– = no phytoconstituents detected 262 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 acute toxicity study in the gongronema latifolium study group, there was no mortality or any signs of behavioral changes or toxicity observed in the mice after oral administration of gongronema latifolium up to the dose of 5000 mg/kg body weight. as such, the ld50 can be said to be greater than 5000 mg/kg body weight. in the picralima nitida study group 100% mortality was observed at 1000 mg/kg dose group in the first phase of the study. using lower dose levels of 300 mg/kg, 500 mg/kg and 800 mg/kg in the second phase, no mortality or any sign of toxicity was observed. hence the lethal dose was above 800 mg/kg. furthermore, using lorke’s method (1983) to calculate the ld50, the ld50 was observed to be approximately 894.4 mg/kg glucose tolerance individual effects of g. latifolium and p. nitida peak hyperglycemia was observed 15 minutes post glucose load and declined continuously thereafter (figures 1a and 2a). at 15 minutes post glucose administration, p. nitida at 200, 400 and 800 mg/kg produced a significant (p < 0.05) reduction in blood glucose levels (bgl) compared to the negative control group just as was recorded for the positive control (p = 0.001). at 30 minutes post-administration, significant differences in bgls were only observed with 400 mg/kg (p = 0.02) and 800 mg/kg (p <0.001) of p. nitida as well as the positive control (p <0.001). at 45 minutes postadministration, a weak significant difference in bgls was observed at 400 mg/kg (p = 0.05) and 800 mg/kg (p = 0.039) of p. nitida; as well as at 200 mg/kg of g. latifolium (p = 0.045). a strong significance difference in bgl was observed for the positive control (p < 0.01). at 60 minutes of post-administration, only the bgl of the positive control was significantly different (p = 0.042) from the bgls of the negative control group. when comparing the area under curve (auc) of the negative control group with the different treatment groups, there was a significant difference in auc of g. latifolium at 100 mg/kg (p = 0.044) and 200 mg/kg (p = 0.038) doses, as well as the positive control (p <0.001). there was no significant difference in the auc of g. latifolium at 12.5, 25 and 50 mg/kg (p >0.05) (figure 1b). there was also a significant difference in auc between the negative control group and p. nitida at doses of 200 mg/kg (p = 0.033), 400 mg/kg (p <0.001) and 800 mg/kg (p < 0.01) but no significant difference at 100 mg/kg (p >0.05) (figure 2b). the percentage glucose reduction in auc of both treatments were presented in figures 1c and 2c. figure 1. effect of g. latifolium on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). 0 50 100 150 200 250 300 350 0 10 20 30 40 50 60 70 b lo o d g lu co se c o n ce n tr a ti o n ( m g /d l) time (min) 12.5 mg/kg 25 mg/kg 50 mg/kg) 100 mg/kg 200 mg/kg 5 ml/kg distilled water 250 mg/kg metformine 0 2000 4000 6000 8000 10000 12000 14000 m e a n a u c 0 10 20 30 40 50 60 70 80 90 100 m e a n % r e d u ct io n egbuniwe et al. – glucose tolerance herbal combination interaction 263 figure 2. effect of p. nitida on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). glucose tolerance effect of the extracts in combination using the loewe additivity combination strategy (equation 2) complemented with isobologram analysis we defined all the pairs of doses of g. latifolium and p. nitida that could lead to the combination effect eab to form additivity and these were drawn as the line of additivity of negative slope on a graph where the x and yaxis represent the dose of g. latifolium and p. nitida (figure 3a). this representation makes clear that when g. latifolium is present at the selected effective dose the quantity of p. nitida needed to reach the specified level is 0, and that the presence of p. nitida reduced the need for g. latifolium in a quantity predicted by the model. all experimental points below the line that could give the desired effect correspond to a combination index (ci) < 1 and indicate synergism. peak hyperglycemia was also observed at 15 minutes just like in the dose response test of the individual extracts (figure 3b). all the combination doses exhibited significant reduction (p<0.05) in blood glucose compared with the negative control group except gl:pn (91:126.5) dose pair (figure 3c). gl:pn (91:126.5) and gl:pn (110:100) combination dose pairs gave the lowest percentage reductions of 29.57 ± 5.4 and 36.59 ± 5.93% respectively while gl:pn (50:160); gl:pn (100:90); and gl:pn (150:30) combination dose pairs were 48.23 ± 3.28; 50.76 ± 4.77 and 42.99 ± 6.88% respectively. the first two combination doses were expected to give an additivity effect based on the model. however, they could not produce the set percentage effective reduction in blood glucose auc which was 41%. since the effects produced from these combinations were below the effective value, these combinations were considered suboptimal. contrary to the behavior of the first two dose pairs, the last three dose pairs produced effects above the set value (41%) and their combination index were calculated to be 0.91, 0.91 and 0.95 respectively an indication of synergistic interaction. 0 50 100 150 200 250 300 350 0 10 20 30 40 50 60 70 b lo o d g lu co se c o n ce n tr a ti o n ( m g /d l) time (min) 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg 5 ml/kg distilled water 250 mg/kg metformin 0 2000 4000 6000 8000 10000 12000 14000 m e a n a u c 0 10 20 30 40 50 60 70 80 90 100 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg 250 mg/kg metformin m e a n % r e d u ct io n 264 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 figure 3. effect of combined doses of p. nitida and g. latifolium on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). discussion control of postprandial hyperglycermia is an essential component of diabetes management. the postprandial pattern of glucose metabolism after glucose load and solid mixed meal containing carbohydrate, fat and protein have been found to be virtually the same and this validates studies like ours employing glucose loads to pertain to those observed after mixed meals (dimitridia et al, 2021). the liver sees the largest change in blood glucose and insulin after a meal and its role is to restrain their acute rise in the bloodstream. the spillover of glucose to the peripheral circulation requires a substantial increase in systemic insulin level and insulin secretion. in the long term, this mechanism provides insight into how a high glycemic index/load of carbohydrate diet producing sustained high insulin secretion could contribute to the development of insulin resistance – the negative consequences of hyperinsulinemia (shanik et al., 2008). under hyperglycermic conditions, the liver uses several mechanisms to bring about glucose homeostasis. these include increased glucose uptake in the hepatocytes, increased activity of glucose utilizing pathways like glycolysis, pentose monophosphate shunt and glucose storage in form of glycogen by glycogenesis. also, acetyl-coa generated from the glycolysis-driven pyruvate pathway is used for fatty acids production which can be transported to adipose tissue for storage (han et al., 2016). agents enhancing these liver mediated activities have been shown to lower post-prandial high blood glucose and are useful in the management of diabetes (rines et al., 2016). g. latifloium extract has been shown to exhibit insulin like activities in the liver by increasing the flux of glucose into the glycolytic pathway and pentose monophosphate shunt in the liver of hyperglycemic animal model (ugochukwu and babady, 2003). these activities were reported to be mediated through increased activities of hexokinase the first enzyme of glycolysis and phosphofructokinase, the rate-determining enzyme of glycolysis. also the activities of glucose-6-phosphate 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 p ic r a li m a n it id a gongrenema latifolium 41% effect isobologram additivity (ci =1) synergy (ci˂1) antagonism (ci >1) a * 0 50 100 150 200 250 300 350 0 20 40 60 80 b lo o d g lu co se co n ce n tr a ti o n ( m g /d l) time (min) gl:pn(91:126.5) gl:pn(110:100) gl:pn(50:160 gl:pn(100:90) gl:pn(150:30) 5 ml/kg distilled water 250 mg/kg metformin 0 2000 4000 6000 8000 10000 12000 14000 m e a n a u c 0 10 20 30 40 50 60 70 80 90 100 m e a n % r e d u ct io n egbuniwe et al. – glucose tolerance herbal combination interaction 265 dehydrogenase (g6pd) a key enzyme in the pentose phosphate pathway has been shown to be increased by g. latifolium (ugochukwu and babady, 2003) in addition to its reported ability to increase liver glycogen synthesis (ajiboye et al., 2019). similarly, p. nitida extract has been shown to promote glucose uptake in the liver through the activation of glucokinase enzyme that plays a critical catalytic role in promoting the addition of a phosphate to glucose, thereby enhancing its uptake into the liver (guzman and gurrola-diaz, 2019). glucose transport is an important step in glucose disposal by peripheral tissues because it controls the rate of glucose utilization in these tissues. in skeletal muscle and adipose tissues, the glucose transporters isoforms expressed are glut1, glut3 and glut4 (shepherd et al., 1999). in the postprandial state, insulin increases the rate of glucose transport mainly by stimulating the translocation of the glut4 isoform from the intracellular pool to the cell membrane. improvement in the sensitivity of tissues to this insulin action avoids marked hyperglycemia and hyperinsulinemia under postprandial conditions. g. latifolium extract was reported to increase muscle glucose uptake significantly in the presence of insulin, indicating an insulinsensitizing potential as well as increased expression and translocation of glut4 glucose transporter (al-hindi et al., 2014; ajiboye et al., 2019). indole alkaloids were the first set of alkaloids isolated from p. nitida: akuamine, pseudoakuamine, akuammidine, akuammicine, akuammigine, pseudoakuammigine, akuammiline, akuamminine and pseudoakuammicine (mawout et al., 2020). most of the effects of p. nitida including modulation of glucose metabolism have been attributed to its rich alkaloid content. stimulation of glucose uptake is one of the known mechanisms of alkaloids containing plant’s hypoglycermic effects (mawout et al., 2020). several alkaloids are allosteric activators of adenosine monophosphate protein kinase – a cellular fuel sensor enzyme and glucose transporter regulator (kumar et al., 2019; song et al., 2021). similarly, alkaloids have been shown to increase the translocation of glut4 and enhancement of glucose-stimulated insulin secretion (kumar et al., 2019). an increase in insulin sensitivity of multiple tissues like the liver, adipose tissue and skeletal muscles by alkaloids has been reported to be mediated through the inhibition of protein tyrosine phosphatase-1b – an enzyme involved in the negative regulation of insulin signal transduction pathways (adhikari, 2021). akuammicine, an indole alkaloid isolated from p. nitida has also been shown to stimulate glucose uptake in adipocytes (shittu et al., 2010). the improved glucose tolerance effect of the individual plant extracts may be attributed to their phytocompounds and through hypoglycemic mechanisms already reported. the multiple targets of regulation of glucose homeostasis by both plants may be responsible for 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(2018). role of secondary metabolites in plant defense against pathogens. microb. pathog.124:198–202. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 159-169 | doi: 10.14421/biomedich.2023.121.159-169 issn 2540-9328 (online) immunoprotective effect of cocos nucifera oil on sheep red blood cell-induced immunocompromised rats macdonald idu1, igori debby1, benjamin ogunma gabriel1,2,* 1department of plant biology and biotechnology, phytomedicine unit; 2science laboratory technology, university of benin, pmb 1154, benin city, edo state. nigeria. corresponding author* alexthemain076@gmail.com abstract the aim of this study is to investigate the immunoprotective effects of cold-pressed coconut oil in immunocompromised rat. standard procedure was used to perform this research work with a modified method. the effect of immunomodulatory properties of coconut oil was evaluated after challenging the animals with 0.3 ml sheep red blood cells (srbcs) intraperitoneally and further treated with graded doses (0.25, 0.5 and 1.0 ml/kg i.p) of the oil extract for 21 days. the hematological, cd4, cd8 biochemical and histopathological analysis were evaluated. result obtained from this study showed a significant increase in hematological indexes (wbc: 11.70; lym: 9.9 gran: 8.0 and hgb: 15.57) across the treated groups, but majorly at lowest dose (0.25 ml/kg). more so a significant increase in cd 4 and cd8 count specifically at 0.25 ml/kg (7.763 and 7.830). anti-oxidant property of the oil extract at 0.25 ml/kg had a significant reduction in malondialdehyde with an increased in antioxidant enzymes (sod, cat and gpx) when compared with untreated control. the body weight of the animals in the treated groups showed a significant increase at 0.25 ml/kg of the oil extract when compared wit h the untreated control. liver function test (alt, ast and alp) showed no significant increase in the treatment groups when compare d with the controls. the histopathological result reveals a normal physiological structure of the heart, lungs, spleen, liver and kidney in lowest dose of the oil extract. in conclusion, this study validated the ethnomedicinal property of the plant. keywords: immunoprotective; cocos nucifera; sheep red blood cell; immunocompromised. introduction immune system is a versatile defense system that protect humans against invading pathogens and inhibit diseases, which can regenerate into variety of cells and molecules proficient in recognizing and eradicating foreign invaders (richard et al., 2003). immunomodulation occurs when host defense mechanism is stimulated under impaired immune response conditions or when immunosuppressant is associated with such state includes; autoimmune disorders. medicinal plants are the current known aspect in phytomedicine, involved in a wider range of herbal materials formulation into supplements. immunomodulatory therapy with the help of medicinal plants serves as an alternative approach to conventional medicine against diseases. hence, host defend mechanism is triggered under impaired immune feedback. immune system is implicated in the investigation and pathological mechanisms of several diseases, with tremendous increased yearning for effective drugs for an improved immune system. to overwhelm the interval of synthetic immunomodulators, there is the need for the development of herbal immunomodulators (z-ziza et al., 2008). immunomodulator is biological or synthetic substances, which stimulate and modulate the components responsible for immune system booster including innate and adaptive immune response (agarwal and singh, 1999; shivaprasad et al., 2006). modulation of immune system using herbal remedy as a therapeutic measure has brought in the need for scientific investigations. coconut (cocos nucifera l.) belongs to the family ‘arecaceae’ and subfamily ‘cocoidae’. it is a tropical tree species extending over large uniform zones. it is usually known as the "tree of life" because of it medicinal usefulness (assa et al., 2010). oil from cocos nucifera is the most well-known food product gotten from dried coconut pulp, called the copra (shankar et al. 2013). law et al. (2014) analyzed the significance of coconut oil for patients with breast cancer. using coconut oil supplement has improved the general wellbeing of patients and reduce possible adverse effect associated with chemotherapy. coconut oil is useful for weight loss due to the biological component of short and medium-chain fatty acids that help in eliminating obesity. it eases digestion and help in healthy functioning of thyroid and endocrine gland. it is known to increases body metabolic rate via pancreatic stress removal; thereby burning more energy and promoting and prevent various stomach and digestion-related manuscript received: 02 october, 2022. revision accepted: 05 december, 2022. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.159-169 160 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 problems include; irritable bowel syndrome (yousefi et al., 2013). the saturated fats present in coconut oil have antimicrobial properties and help in dealing with various bacteria, fungi and parasites that can cause indigestion (shilling et al., 2013). due to the presence of lauric acid, it plays a vital role in preventing diverse cardiovascular diseases (vala et al., 2014). c. nucifera oil has been reported to be an effective as a wound healing, immune booster and other therapeutic agents. (nevin et al., 2010). materials and method collection and preparation of plant materials fresh coconut seeds were procured from egor local government area of benin city, edo state, nigeria. it was identified and authenticated by dr. h. akinnibosun in the departmental herbarium unit of plant biology and biotechnology, with a voucher number (uwh-x219). cold extraction method was involved in the isolation of the coconut milk, allowed to stand for 20-24 hrs. under favorable conditions (35-40˚c, 75% relative humidity), the oil gets separated from the water and the protein. the airborne lactic acid bacteria, which can break the protein bonds, act on the coconut milk mixture for proper separation. the operation conditions and sanitary precautions were strictly followed. the top layer was floating curd. the curd also contained a considerable amount of trapped oil. the separated oil contained some adhering particles and was filtered using a sterilized filter paper. experimental animals thirty (30) wistar male rats weighed between 150-315 g were obtained from the animal house, of the department of biochemistry, university of benin, benin city. they were housed under disinfected and wellventilated plastic cages in a standard laboratory condition (12 hours light/dark cycle: 23 ± 2°c) and fed using bendel meal pelleted grower mesh. the food and water were given at ad libitum. the animals were handled in accordance to the ethical laboratory animals’ guide’ from the approval of the life sciences ethical committee university of benin, issued a voucher number ls21507. preparation of sheep red blood cells (srbc) fresh blood was collected from a healthy sheep sacrificed in the local slaughterhouse in a sterile bottle containing elsevier’s solution (1:1). then it centrifuged at 500 g for 15 min at 4 c in cooling centrifugation apparatus to separate the red blood cells (rbcs). the sheep red blood cells (srbc) were washed three times in normal saline. they were then kept under refrigeration for the immunization. experimental protocol the animals were numbered, weighed and then divided into six groups with five animals in each: all groups will be immunized with 0.3 ml/kg srbc for 7 and 14 days of the study. group a serves as the normal control, group b was administered 0.3 ml/kg srbc alone intraperitoneally (negative control), group c received 0.3 ml/kg srbc and 5 mg/kg azathioprine (standard drug p.o) (positive control), group d, e and f were treated with 0.3 ml/kg srbc pus graded doses (0.25, 0.5 and 1.0 ml/kg of coconut oil p.o). after 14 days, the animals were sacrificed under mild chloroform anesthesia. blood was collected via cardiac puncture and organ samples were isolated. the serum was separated for various biochemical tests include; liver function tests (alkaline aminotransferase (alt), aspartate aminotransferase (alp), alkanine phosphatase (alp), gama glutamine transferase (ggt), total bilirubin (tot. bil), conjugated bilirubin (cong. bil), unconjugated bilirubin (ub) and total protein (tp)); anti-oxidant test. the estimation of superoxide dismutase (sod), catalase (cat), malondialdehyde (mda), and glutathione peroxidase (gpx). hematological indexes the estimation of various (20) parameters was carried out. the white blood cell (wbc), hemoglobin (hgb), red blood cell (rbc), platelet count (plt) and lymphocytes. cd4 and cd8 count were carried out. biochemical assay the blood sample for biochemical assays also collected via cardiac puncture were placed in lithium heparin sample bottles and centrifuged at 3000 revolutions per minute (rpm), the plasma was separated using pasteur pipettes into clear labeled bottles. the samples were stored in deep freezer at -20oc until analyses were carried out (bagul et al., 2005). the blood plasma was used for the evaluation of the following biochemical parameters incudes aspartate aminotransferase (ast), albumin (alb), total protein (tp), total bilirubin (t bil), direct bilirubin (d bil), alanine aminotransferase (alt) alkaline phosphate (alp), malondialdehyde (mda), catalase, superoxide dismutase (sod) and glutathione peroxidase (gpx). the biochemical parameters were evaluated using commercial kits obtained from randox laboratories, uk. histopathological analysis l.iver, kidney, heart, spleen and lungs were fixed in formal saline fluid. affixed organs were utterly dehydrated with 99.9 % ethanol along with 70 % ethanol, and 96 % ethanol and washed using distilled water. 4 µm sections were prepared and stained in hematoxylin-eosin dye. stained tissues were optical photomicroscope (leica mc170 hd, leica biosystems, idu, et al. – immunoprotective effect of cocos nucifera oil in rats 161 germany) viewed at x 400 magnification (drury and wallinton, 2013). statistical analysis the data were calculated as mean ± standard error of mean. significance differences of the mean value with respect to the control group was analyzed by one way anova using graph pad prism 8 statistical software. p<0.05 considered being significant. results and discussion results obtained in table 1, 2 and 3 showed a significant increase (p < 0.05) at lowest dose of 0.25 ml of the treatment groups when compared with untreated control in the hematological parameters. the immune response is a biological process which solely depends on external protection and is differ for all members of vaccinated population (okonkwo et al., 2004; yakubu et al., 2007). the immune response is influenced by many genetic and environmental factors. hematological parameters as one of the indexes in immune system understudied the white blood cell, red blood cell, lymphocyte, hemoglobin, platelet and other. result from table 1 showed a significant increase in the value of wbc counts across the treated groups but more on 0.5 ml/kg when compare with untreated control. bone marrow being a flexible tissue is majorly responsible for blood cells regenerations to improve immunity. lymphocyte and other key cells of the immune system are known to activate the production of several antibody polymorph with nuclear granulocyte to destroy antigen (prescott, 1999). lymphocyte indexes being one of the immune booster that protect the body against invaders showed a slight increase in the treated groups that received 0.25 and 0.5 ml/kg of coconut oil (9.90 and 8.20) when compared with untreated control. a significant increase in the level of granulocyte across the treated groups specifically at 0.25 ml/kg (8.0) when compared with the controls. there is a significant increase in the level of hemoglobin, hematocrit and platelet in the treated groups when compared with the control groups as shown in tables 2-3. this present study agreed with the work of oduola et al. (2005) that reported the leaf extract of j. gossypiifolia on the biochemical and hematological analyses has no damaging effects. table 1. effect of coconut oil on white blood and differentials in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil wbc 103/µl 11.73±1.51b 10.10±0.17a 9.30±0.45a 11.70±1.25b 9.90±0.76a 10.33±1.56a lym % 83.07±3.53a 84.43±0.59a 84.47±1.68a 84.03±2.36a 83.10±1.10a 85.17±1.82a mid % 9.533±1.82b 8.67±0.24a 8.20±0.60a 8.83±0.93a 8.90±0.47a 8.10±1.00a gran % 7.40±1.70a 6.90±0.50a 7.33±1.13a 7.13±1.42a 8.0±0.67b 6.73±0.83a lym 103/µl 9.77±1.39b 8.53±0.18a 7.87±0.41a 9.90±1.34b 8.20±0.50a 8.80±1.36a mid 103/µl 1.10±0.26b 0.87±0.03a 0.77±0.07a 1.00±0.00b 0.90±0.10a 0.83±0.14a gran 103/µl 0.87±0.23b 0.70±0.00a 0.67±0.12a 0.80±0.10b 0.80±0.15b 0.70±0.11a the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. table 2. effect of coconut oil red blood cell and components in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil rbc 106/µl 6.36±0.33a 6.48±0.44a 6.0±0.15a 6.85±0.25a 6.71±0.38a 6.37±0.27a hgb g/dl 13.60±0.53a 14.63±0.89a 13.83±0.50a 15.57±0.67b 15.33±0.70b 14.23±0.62a hct % 37.50±1.47a 40.83±2.24a 38.73±1.31a 42.37±1.48b 41.40±1.68a 38.70±1.11a mcv µm3 59.13±0.84a 63.20±1.60a 64.77±0.64a 61.93±0.09a 62.57±0.37a 60.97±1.70a mch pg 21.37±0.34a 22.57±0.48a 23.03±0.35a 22.67±0.14a 22.83±0.35a 22.30±0.10a mchc g/dl 36.20±0.55a 35.77±0.23a 35.67±0.60a 36.67±0.29a 37.00±0.44b 36.73±1.17a rdws µm3 30.93±1.23a 32.20±0.60a 33.43±1.07a 31.57±1.07a 31.60±0.00a 32.83±3.26a rdwc % 16.07±0.42a 15.80±0.00a 16.13±0.43a 15.70±0.52a 15.73±0.24a 16.50±1.27a the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. 162 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 table 3. effect of coconut oil platelet and factors in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil plt 103/µl 740.00±72.76a 765.30±45.33a 949.70±322.00b 660.30±80.8a 909.00±132.3b 996.30±225.9b mpv µm3 7.43±0.12a 8.03±0.26a 8.47±0.09a 7.77±0.09a 8.07±0.13a 8.20±0.30a pdw % 9.37±0.33a 10.47±0.70a 11.17±0.44a 9.57±0.07a 10.40±0.59a 10.80±0.67a pct % 0.54±0.047a 0.61±0.02a 0.80±0.28b 0.51±0.06a 0.73±0.11a 0.82±0.21b p-lcr % 3.83±1.93a 8.30±3.30a 12.80±1.12b 5.03±0.27a 8.03±2.43a 12.10±3.16b the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. figure 2 showed an increase in the level of cd4 count across the graded doses of the treated groups 0.25ml, 1.0ml extract when compared with untreated control. figure 1 showed an increase in the level of cd8 count across the graded doses of the treated groups 0.25ml, 1.0ml extract when compared with untreated control. cd4 and cd8 cell count provides an improved immune status against opportunistic infections, also it is regarded as a diagnostic decision-making, test particularly with patients suffering from immunosuppressant and hiv disorders. result from this study at graded doses of the extract-treated animals, significantly increase the level of cd4 and cd8 count specifically at 0.25 ml/kg (7.763 and 7.830) when compared with untreated group (figure 1 a and b). this concurred with the work of oduola et al. (2005) on the effect of voacanga africana leaves extract on serum lipid profile and hematological parameters. a b c d e f 0 2 4 6 8 10 treatment group m e a n a n ti b o d y v a lu e a b c d e f 0 2 4 6 8 10 treatment group m e a n a n ti b o d y v a lu e key: group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; gr oup c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. figure 1. (a and b) effect of coconut oil extract on cd8 and cd4 count. figure 2a showed the anti-oxidant test carried out to estimate the in vivo scavenging power of malondialdehyde (mda) of graded doses of coconut oil in wistar rat. the anti-oxidant test carried out to estimate the in vivo scavenging power of catalase response on coconut oil extract in wistar rat as shown in figure 2b. figure 2c elicited the anti-oxidant potential carried out to investigate the in vivo scavenging power of superoxide dismutase responses on coconut oil extract in wistar rat. the results in figure 2d exhibited anti-oxidant effect investigated in the in vivo scavenging power of glutathione peroxidase responses. various antioxidant markers showed the scavenging effect caused by oxidation of lipid peroxidation. hence, the level of malondialdehyde (mda) in the blood was measured (uchiyama et al., 1978). in vivo mda antioxidant level elicited a significantly reduction at lowest dose (0.25 ml/kg) in cocos nucifera oil extract (34.53) when compared with untreated control as shown in figure 3. antioxidant enzymes serves as delicate part associated with cellular defense against responsive oxygen species (ros) with eventual oxidative stress (nevin et al., 2006). the determination of oxidative stress using the balance involved ros generation includes; antioxidant protective systems like superoxide dismutase (sod) and superoxide anion. antioxidants enzymes are implicated in the removal of reactive oxygen species such as gsh, sod and cat. antioxidant enzymes such as catalyst scavenging power significantly increased (p < 0.05) it scavenging capacity at 0.25 ml/kg (241.0) when compared with the control, however, in the activity of sod, there was no significant a b idu, et al. – immunoprotective effect of cocos nucifera oil in rats 163 increase across the treated animals when compared to the control. glutathione peroxidase activity, showed a significantly increased in its scavenging power at (129.0) of 0.25 ml/kg of the oil extract when compared with the control groups. similarly, this study has exhibited its biological effects on cocos nucifera oil established to scavenge oxidative stress via boosting its antioxidant capacity, thereby scavenging free radicals and decreasing lipid peroxidation (nevin et al., 2006 and van et al., 2004). a b c d c f 0 10 20 30 40 50 group c o n c e n tr a ti o n ( m m /m l) a b c d e f 0 100 200 300 400 treatment group c o n c e n tr a ti o n ( m m /m l) a b c d e f 0.0 0.5 1.0 1.5 2.0 2.5 treatment group c o n c e n tr a ti o n ( m m /m l) key: group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; gr oup c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. figure 2. (a, b, c and d) effect of cocos nucifera on enzymatic (malondialdehyde) and non-enzymatic (catalase, superoxide dismutase and glutathione peroxidase) effect on immune response. figure 7 showed the level of liver function tests (alt, ast, alp, ggt, total bilirubin, conjugated bilirubin, unconjugated, bilirubin and total protein) on the extract activity (adeoye and oyedepo, 2004). the eight liver function tests were found to be within normal range in all treated groups. excessive secretion of alanine aminotransferase (alt) and aspartate aminotransferase (ast) are raised in serum when malfunction of the liver occurs. the liver function test results elicited no significant increase in the graded doses of the oil extract (alt, ast and alp) specifically at 1 ml/kg of the treated groups at (15.28) when compared with the control. this is in line with the results showed in the findings of adeosun et al. (2014) that reveal stem latex of j. gossypifolia as a protein precipitant for biochemical investigation with potential defensive measures. no significant difference at (p < 0.05) on the conjugated bilirubin value when compared with the control. 0.25 ml/kg of the oil extract showed a significant increase in the level of ggt, total bilirubin and total protein antibody level at 0.833, 0.053 and 13.57 when compared with the control groups. this agreed with the report of igbe et al. (2013) and magili and bwatanglang (2018). body weight of the animals in the treated groups showed significant increase specifically at 0.25 ml/kg of oil extract, when compared with the control groups (kumar et al., 2008). a sight significant increase in the body weight of the rats was revealed at weeks 1, 2, 3 and 4. afterward treated with c. nucifera oi extract across graded doses of 0.25, .0.5 and 1.0 m/kg had a slightly significant difference in the body weight observed in dose dependent manner (figure 3). reduction in the body weight aids as an indicator and sensitive marker to mark toxicity. this is adhering to the work of witthawaskul et al. (2003). a b c d e f 0 50 100 150 200 treatment group c o n c e n tr a ti o n ( m m /m l) a b c d 164 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 a b c d e f 0 10 20 30 40 concentration (ml/kg) a la n in e a m in o tr a n s fe ra s e r e s p . a b c d e f 0 20 40 60 concentration (ml/kg) a s p a rt a te a m in o tr a n s fe ra s e r e s p . a b c d e f 0.00 0.01 0.02 0.03 0.04 concentration (ml/kg) c o n ju g a te d b il ir u b in r e s p . a b c d e f 0 5 10 15 20 concentration (ml/kg) a lk a li n e p h o s p h a ta s e r e s p . a b c d e f 0.0 0.5 1.0 1.5 concentration (ml/kg) g a m a g lu ta m y lt ra n s fe ra s e r e s p . a b c d e f 0.00 0.02 0.04 0.06 0.08 concentration (ml/kg) t o ta l b il ir u b in r e s p . a b c d e f 0 5 10 15 20 concentration (ml/kg) t o ta l p ro te in r e s p figure 3. (a, b, c, d, e) dose responses on the extract in various concentration of liver function test. group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. a b c d e f 0 100 200 300 treatment group week1 week 2 week 3 week 4 w e e k l y w e ig h t g a in figure 4. dose responses on the extract in various concentration of the body weight. group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group a b c d e f g idu, et al. – immunoprotective effect of cocos nucifera oil in rats 165 receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. the histopathological structure of the liver, kidney, heart, spleen and lungs (plate 1, 2, 3, 4 and 5) at graded doses (0.25ml, 0.5ml and 1.0 ml/kg) specifically 0.25 ml/kg of the extract revealed a distinct centriole and dilated sinusoidal, with no inflammatory cells and steatosis when compared with untreated control that showed mild steatosis in hepatic cells this is in line with the findings of eleazu et al. (2013) and magili and bwatanglang (2018) that worked on the toxicological profile of the aqueous leaves extract of j. gossypiifolia.. the structure of renal cells reveals no necrosis in lowest doses (0.25 ml/kg) when compare with the untreated control with pronounced necrosis. figure 5. effect of cocos nucifera oil extract on histology of the liver. a= normal control received water alone liver: reveals visible centriole (long arrow) with the hepatocytes revealing pyknotic nucleus (short arrow). b= 0.3 ml/kg srbc alone liver: reveals centriole surrounded by mild inflammatory cells (long arrow) with the hepatocytes revealing mild steatosis (short arrow). c= 5 mg/kg standard drug (azathioprine) liver: reveals centriole (long arrow) surrounded by hepatocytes that appear not so distinct. (short arrow). d= 0.25 ml/kg of coconut oil liver: reveals distinct centriole (long arrow) with the hepatocytes and dilated sinusoidal (short arrow). e= 0.5 ml/kg of coconut oil liver: reveals centriole surrounded by focal inflammatory cells (long arrow) with the hepatocytes revealing mild steatosis (short arrow). f= 1 ml/kg of coconut oil liver: reveals centriole (long arrow) surrounded by hepatocytes with hydropic fatty changes and revealing mild steatosis (short arrow). figure 6. effect of cocos nucifera oil extract on histology of the kidney. a b c a d b c e f d e f 166 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 a= normal control received water alone kidney: reveals prominent renal corpuscle (long arrow) and interstitial space and tubules (short arrow). b= 0.3 ml/kg srbc alone kidney: reveals atrophied renal corpuscle (long arrow) and interstitial space with mononuclear cells (short arrow) and mild tubular necrosis. c= 5 mg/kg standard drug (azathioprine) kidney: reveals renal corpuscle appearing not so distinct with granulated nucleus (long arrow) and interstitial (short arrow) and tubular necrosis. d= 0.25 ml/kg of coconut oil kidney: reveals visible renal corpuscle (long arrow) and interstitial space (short arrow) and tubules. e= 0.5 ml/kg of coconut oil kidney: reveals atrophied renal corpuscle (long arrow) and interstitial space with mononuclear cells (short arrow) and tubular necrosis. f= 1 ml/kg of coconut oil kidney: reveals renal corpuscle appearing not so distinct (long arrow) and interstitial (short arrow) and tubular necrosis. figure 7. effect of cocos nucifera oil extract on histology of the heart. a= normal control received water alone heart composed of bundles of myocardial fibres (short arrow), interstitial space and prominent coronary artery (long arrow). b= 0.3 ml/kg srbc alone heart composed of bundles of myocardial fibres (short arrow), interstitial space and atrophied coronary artery (long arrow). c= 5 mg/kg standard drug (azathioprine) heart composed of bundles of myocardial fibers (short arrow), interstitial space and congested coronary artery (long arrow). d= 0.25 ml/kg of coconut oil heart composed of bundles of myocardial fibers (short arrow), interstitial space and large prominent coronary artery (long arrow). e= 0.5 ml/kg of coconut oil heart composed of tight bundles of myocardial fibers (short arrow), interstitial space and coronary artery (long arrow). f= 1 ml/kg of coconut oil heart composed of bundles of myocardial fibers (short arrow), interstitial space and coronary artery (long arrow). figure 8. effect of cocos nucifera oil extract on histology of the spleen. a b c e f a d b e c f d idu, et al. – immunoprotective effect of cocos nucifera oil in rats 167 a= normal control received water alone: spleen shows lymphoid follicles (short arrow) with centrally to eccentrically located large blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which. the red pulps appear distinct. b= 0.3 ml/kg srbc alone: spleen shows lymphoid follicles (short arrow) with centrally to eccentrically located large blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which. the red pulps appear coarse. c= 5 mg/kg standard drug (azathioprine): spleen shows lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which appear activated. the red pulps appear coarse. d= 0.25 ml/kg of coconut oil: spleen shows prominent lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes. the red pulps are prominent. e= 0.5 ml/kg of coconut oil: spleen shows lymphoid follicles (short arrow) with not so prominent eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes. the red pulps appeared coarse. f= 1 ml/kg of coconut oil: spleen shows lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which appear activated. the red pulps are appeared coarse. figure 9. effect of cocos nucifera oil extract on histology of the lung. a= normal control received water alone: lung reviewed prominent blood vessel (long arrow) and visible alveolar ring. the alveolus appears distinct (short arrow). b= 0.3 ml/kg srbc alone: lung reviewed prominent bronchiole (long arrow) and visible alveolar ring. the alveoli appears slightly congested (short arrow). c= 5 mg/kg standard drug (azathioprine): lung reviewed prominent blood vessels (long arrow) and visible alveolar ring. the alveoli appear slightly congested (short arrow). d = 0.25 ml/kg of coconut oil: lung reviewed prominent alveolar sac (long arrow) and alveolar ring. the alveoli appear slightly congested. e= 0.5 ml/kg of coconut oil: lung reviewed prominent alveolar sac (long arrow) and alveolar ring. the alveoli appear slightly congested. f= 1 ml/kg of coconut oil: lung revealed prominent bronchiole (long arrow) and visible alveolar ring. the alveoli appear slightly congested (short arrow). differences between the hearts occurred in coronary artery which stated a large prominent coronary artery in the lowest dose when compared with untreated control that exhibited atrophied with congested coronary artery (magili and bwatanglang, 2018). the alveolus in the lungs showed a distinct structure in 0.25 ml/kg when compared with untreated control that showed alveolus congestion. the extract feedback response triggered by cellular and humoral immune response. hence, this study validated the ethnomedicinal property as immune booster. conclusion this study elicited a feedback response from the coconut extract, triggered by cellular and humoral immune response, and thereby boosted the immune physiological state of the body. this 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(2014). tokoferole i tokotrienole jako witamina e. chemik. 68(7): 585-591. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 225-232 | doi: 10.14421/biomedich.2023.121.225-232 issn 2540-9328 (online) chemical profile and in-silico docking studies on bioactives from essential oil of cymbopogan pendulus targeting penicillin binding proteins (pbps) in bacteria arun dev sharma*, inderjeet kaur pg department of biotechnology, lyallpur khalsa college jalandhar, india. corresponding author* arundevsharma47@gmail.com manuscript received: 25 january, 2023. revision accepted: 15 february, 2023. published: 17 february, 2023. abstract antibiotic resistance in bacteria is the major concern worldwide. pbp (penicillin binding proteins) have been cited as an appropriate target for therapeutic drug design. in the present study molecular docking followed by wet lab validation was designed to estimate the effect of potent bioactive molecules from cymbopogan pendulus essential oil against pbp5 protein. gc-fid (gas chromatography with flame-ionization detection) based composition profile, and in-silico docking study was conducted by using cb-dock 2 analysis followed by 2d and 3d interactions. gc-fid revealed limonene, neral, geranial, linalool, myrcene as major and minor compounds in cymbopogan pendulus essential oil. the docking score indicated effective binding of ligands to pbp5. interactions results indicated that, pbp5/ligand complexes form hydrogen and hydrophobic interactions. wet lab study validated the anti-bacterial potential of oil against gram-positive and gram-negative bacteria. therefore, essential oil from cymbopogan pendulus essential oil may represent potential herbal treatment to mitigate bacterial infections. keywords: bacteria; docking; lemon grass oil; geranial; herbal drug. introduction bacterial peptidoglycan, which is repeating disaccharide unit of n-acetylglucosamine (nag) and nacetylmuramic acid (nam), with nam bearing a peptide stem, provide resistance to bacteria not only by providing capability to resist against internal intracellular pressure but also helps to maintain well-defined cellshape (brockhurst et al., 2019). the final steps involved in cell wall bio synthesis and remodeling are mediated by penicillin-binding proteins (pbps), which comprise highmolecular-mass (hmm) and low-molecular-mass (lmm) pbp subgroups. the term ‘pbp’ has been cited in manuscripts to refer to any enzyme that identifies and/or metabolizes β-lactams, independently of its function in the cell (fair and tor, 2014). pbps are implicated in catalysis such as: in trans-glycosylation (polymerization of the glycan strands) and transpeptidation (the cross-linking between glycan chains), dd-carboxy-peptidation (hydrolyze the last d-alanine of stem pentapeptides) and endo-peptidation (hydrolyze the peptide bond connecting two glycan strands) (simoes et al., 2017). pbps represents excellent targets for β-lactam antibiotics such as penicillin’s, thus inhibiting penultimate physiological role of enzymes involved in bacterial cell wall synthesis and leading to cell death. however, worldwide uncontrolled use of β-lactam antibiotics has led to bacterial resistance to antibiotics which is a swiftly growing apprehension. this problem emerged due to the emergence spread, and persistence of multidrug-resistant (mdr) bacteria, which are frequently isolated in hospitals collectively known as “eskape”, which constitutes gram-positive and gram-negative species (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter spp.), also known as “superbugs”. “eskape” are also resistant to traditional and conventional treatments involved in majority of nosocomial infections (brockhurst et al., 2019). bacteria possess a variable number of pbps and among all pbp5, an lmm trans-peptidase, is the most abundant and a vital enzyme involved in peptidoglycan synthesis in cell wall of bacteria (welsh et al., 2017). in the cell wall, the major function of pbp5 is a ddcarboxypeptidase reaction, that regulates the degree of cross-linking by hydrolytically shortening the peptide stem of the nascent peptidoglycan. it was reported that blocking of either the carboxypeptidation, transpeptidation or reactions by β-lactams antibiotics, worsens the peptidoglycan and may cause cell death (chan et al., 2016). this process has widened the use of antibiotics and its analogs maximally but has been challenged by the transmission of drug-resistant strains, https://doi.org/10.14421/biomedich.2023.121.225-232 226 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 highlighting the inevitability for novel natural antibiotic therapies. in this regard, inhibition of the glycosyltransferase reaction by the natural product moenomycin also has been reported that weakens the peptidoglycan and kills bacterial cells (masters et al., 2020). pbp5, topology constitutes: a trans-membrane anchor, a cytoplasmic tail, and two domains joined by a beta-rich linker located on the outer surface of the cytoplasmic membrane where cell wall peptidoglycan synthesis takes place (lu et al., 2020). the antibacterial activity of β-lactams is arbitrated by covalent binding to pbps, thus inhibiting the transpeptidase (tpase) activity of pbp-mediated bacterial cell wall synthesis (da costa et al., 2018). since bacterial resistance to multiple drugs, including b-lactam antibiotics, is a main therapeutic problem thus development of new chemical entities as antibacterial agents are urgently needed (da costa et al., 2018). it was argued that gram-positive and gramnegative bacteria have mostly established resistance to all the available antibiotics and pose a grave problem not only in hospitals but also for the general population (contreras-martel et al. 2017). therefore, by virtue of its key role, pbps are considered as an appropriate objective for developing bacterial inhibitor. inhibition of pbps protein activity would block replication of bacteria. since in humans, not at all any pbps with comparable cleavage specific are recognized, so inhibitors are improbable to be considered as toxic. lemon grass essential oil (lgo) from cymbopogon species, also known as lemon grass, encompasses a number of bioactives. due to complex nature of essential oil, their anti-fungal mechanism of action is still not completely understood (bhatnagar, 2008, mancianti et al., 2020)]. lgo has long history to be used as complementary and traditional medicine in ancient times. in addition, various potent biological activities like antiamoebic, anti-inflammatory, anti-filarial, anti-diarrheal, anti-malarial, anti-fungal and anti-bacterial agent, antihiv have been attributed to lgo, hence playing a major role as therapeutics in the scientific community (oladeji et al., 2019). this study postulated that due to richness of geranial, essential oil from cymbopogon pendulus plants have potential to inhibit bacterial infections. hence as an objective this study was designed to study molecular docking of geranial and wet lab validation of antibacterial potential of lemon grass oil in relation with pbp5. the present study outcomes would offer scientists and doctors with prospects to identify the key antibacterial drugs to combat mdr. experimental gc-fid analysis lgo was extracted from fresh leaves of cymbopogan pendulus growing naturally at nearby areas of lyallpur khalsa college, jalandhar. the cymbopogan pendulus was authenticated by dr upma from botany dept and voucher with number bt103 was deposited in dept of biotechnology. hydro-distillation method was used for extraction of essential oil by using clevenger-type apparatus (borosil, india) (sharma and kaur 2021). to identify bioactive compounds in eo, gc-fid study was carried out (gc-fid, chemtron 2045). the specifications of column was: 2 m long, stainless steel having 10% ov-17 on 80-100% mesh chromosorb w (hp). nitrogen was used as carrier gas at flow rate of 35 ml/min. 0.2 µl leo sample was used. the temperatures for detector and injector were: 220 °c and 270 °c. oven ramping conditions were: 100°c (firstly maintained) ramped to 2100 °c at 3 °c/min. bioactive constituents in leo were identified by comparing relative retention times (rt) of gc-fid spectra of leo with authentic standards and literature data. ligand preparation for bacterial receptors (pbp5, pdb id: 3mzf), various bioactive compounds such as: limonene, neral, geraniaol, linalool, myrcene which are present in lemon grass essential oil as major and minor amounts were used as ligands for structures. to build 3d structure of ligand, smiles of ligands was recovered from ncbi-pubchem database. the structure was built by using ucsfchimera. molecular docking crystal structures of pbp5 bacterial penicillin binding protein recovered from pdb (https://www.rcsb.org/). before docking analysis, all target enzymes were cleaned from selected h2o molecules, cofactors, co-crystallized ligand, and energy minimized. then all protein target structures were prepared by means of the dock prep set up in ucsf-chimera. it is the process under optimization that bond length, charges anomalies and corrects atomic structure. for docking, cb-dock 2 tool was used for docking of ligands over pbp5 (https://cadd.labshare.cn/cb-dock2/php/index.php). to execute docking, both receptors and ligand molecules as “pdb files” were uploaded to the cb-dock 2 and docking was performed. for 2d and 3d interactions in docked complexes, biovia 2020, ucsf chimera and plip tools were used. active sites prediction in fungal receptors, identification and dimension of cavities on 3d active sites were computed by using castp web tool. for this all structures in “pdb” format were uploaded to server and prediction was executed with probe radius value of 1.4 angstroms. in-vitro anti-bacterial activity the in-vitro antimicrobial activity of lgo was determined through agar disc diffusion method against four test organisms, gram-negative escherichia coli (mtcc 40), pseudomonas aeruginosa (mtcc 424), and sharma & kaur – chemical profile and in-silico docking studies on … 227 gram-positive staphylococcus aureus (mtcc 3160) and bacillus subtilis (mtcc 121). pathogens were purchased from institute of microbial technology, chandigarh. sterile paper discs (10 mm in diameter) were impregnated with 100 µl lgo. 12-h cultures were used inoculums and od of suspensions was adjusted to 0.6. a swab of bacteria suspension was spread on to lb-agar plates and allowed to dry for 30 min. the discs with essential oil were then applied and plates were left for 20 min at room temperature to allow to diffusion of oil followed by incubation at 37oc for 24 hours. vancomycin antibiotic, (10mg) was taken as positive control. zone of inhibition was measured. result and discussion gc-fid analysis of bioactive molecules in lgo the gcfid chromatogram obtained was depicted in figure 3. the peaks observed and their respective retention time was also displayed. the gcfid analysis of lemon grass oil obtained from cymbopogon pendulus revealed 26 compounds for the total of 100%. in the present study, all identified compounds were micrene, limonene, linalool, geranial, neral, undececanone and geranial acetate. gc-fid chromatogram contained three major peaks along with many small peaks indicating the presence of minor compounds. the major constituents were geranial (27%), neral (31%), myrcene (6.7%), limonene (4.9%) and linalool (3.6%). the small peaks may be ascribed to the disintegrated major compounds bioactive compounds or present in small quantities. the literature studies also showed the presence and identification of mycrene, geranial and neral in lemongrass oil obtained from c. flexuous, (oladeji et al., 2019). during the course of time, use of lgo has become a major area of healthand medical-related research due to richness of bioactives. lgo also has been used as therapeutic agent in pharmaceutical preparations as anti-oxidative, antibacterial, antiviral, anti-diabetic, anti-tumor, antifungal, anti-obesity, antihypertensive, anti-histaminic, anti-cancer, anti-hiv and hepatoprotective agent (oladeji et al., 2019). in this study 2 major and 3 minor bioactive compounds as cited above were selected for 3d docking. peak no. retention time (min) biactive compound conc. 1 0.498 micrene 6.7075 2 5.582 limonene 4.9152 3 6.915 linalool 3.6378 4 18.998 geranial 27.0440 5 21.248 neral 31.5752 6 23.998 undececanone 1.5138 7 27.165 geranial acetate 1.3755 figure 1. gc-fid analysis of lemongrass essential oil (lgo). molecular docking structure-based drug design (sbdd) is most widely used in-silico technique in making drugs which is based on 3-d structures (srimai et al., 2013). in-silico docking has simplified investigators to screen conformations and affinities of an assembly of bioactive components against receptors (barcellos et al., 2019). present study aimed at docking of limonene, neral, geranial, linalool, and myrcene bioactive molecules from lgo as key antibacterial inhibitor candidates against pbp5. from docking analysis, it was apparent that ligands efficiently docked with pbp5 bacterial enzyme. 3d docking results illustrated that pbp5 depicted strong binding with ligand limonene (table 1) as apparent from its docking score of -5.3. based on docking score, the order to docking of ligand with pbp5 receptor was: limonene>, neral>geranial>linalool> myrcene. the best pose displaying 3d model of pbp5-ligand based on docking 228 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 score are displayed in figure 2. 2d/3d interaction of ligands with pbp5 is displayed in figure 3. with pbp5, it was revealed that ligands docked with penicillin binding domain of pbp5. the c-terminal module is responsible for the transpeptidase activity of pbps catalyzing peptide cross-linking between two adjacent glycan chains catalyzing peptide cross-linking between two adjacent glycan chains in peptidoglycan cell wall synthesis (contreras-martel et al., 2017). it was cited that blocking of either the transpeptidation or carboxypeptidation reactions by b-lactam antibiotics or therapeutic inhibitors, weakens the peptidoglycan and may engender cell death (straume et al., 2020). once a pbp is acylated by a by therapeutic inhibitors, it is unable to catalyze hydrolysis of the covalent acylenzyme intermediate and is inactivated; peptidoglycan transpeptidation cannot occur, and the cell wall is weakened (masters et al., 2020, moon et al., 2018). based on analysis, it was highlighted that lgo can be used as effective source of anti-bacterial compounds. table 1. docking score of ligands with pbp5. ligand vina score (kcal/mol) cavity volume (å3) center (x, y, z) docking size (x, y, z) interacting residues (4 a˚) h-bond interactions hydrophobic interactions limonene -5.3 1721 39, 4, 27 26, 17, 17 214athr,222atyr,222atyr,224aleu 245aphe, 248aarg, 248aarg neral -5.1 1721 39, 4, 27 26, 19, 19 216a his 198aarg,214athr,216ahis,222atyr, 222atyr,224aleu,248aarg, 248aarg,249aglu geraniaol -4.9 1721 39, 4, 27 26, 19, 19 216a his 198arg, 214athr 216ahis, 222atyr 222atyr, 224aleu 248aarg,249a,glu linalool -4.7 1721 39, 4, 27 26, 18, 18 216ahis 198aarg,214athr,222atyr,248aarg, 248aarg,249aglu myrcene -4.7 1721 39, 4, 27 26, 18, 18 214athr,216ahis,222a tyr,222atyr,222atyr, 248aarg,248aarg,249aglu figure 2. pictorial view of 3d model of pbp-ligand interactions. a: geranial; b: limonene; c: linalool; d: myrcene; e: neral; f: general pictorial view of pbp1 exhibiting domains. through 3d docking, with site residues of receptors, ligand could form h-bonds or hydrophobic bonds which designate affinity of ligand with receptor (lima et al., 2019). hence, docking interactions of limonene, neral, geranial, linalool, myrcene with pbp5 was further evaluated. it was observed that most effective ligand limonene forms hydrophobic interactions with pbp. with pbp5 receptors, hydrophobic interactions were detected a b c d fe sharma & kaur – chemical profile and in-silico docking studies on … 229 via 214thr, 222tyr, 224leu, 245phe, and 248arg active site residues at 3.38, 3.84 3.69, 3.68 and 3.56 å (figure 2). it was noted in addition to hydrophobic interactions that ligands neral, geranial, linalool exhibited h-bond interactions also by 216his. castp active sites prediction quantified interacting residues in the active site cavities of pbp1 receptors (data not shown). in pbp5 enzymes, a main pocket was documented with volume (sa) of 1721(å3) and area (sa) of 376 (å3) main pocket contained active site residues such as ser44 arg198 thr214 gly215 his216 tyr222 asn223 leu224 arg248 glu249. meanwhile, as all ligands shown good affinity to pbp5 enzyme via active site residues so it was conjectured that upon binding with ligand pbp5 becomes closed thus in-turn persuades change in conformation of bacterial enzymes and inhibit biosynthetic pathway involved in cell wall synthesis. earlier studies also documented that β-lactam antibiotics irreversibly acylate the active-site serine of pbps, which deprives bacteria of their biosynthetic functions and results in bacterial death (o’daniel et al., 2014). all these events halts bacterial viability thus mitigate infectivity of bacteria into the host cell. similar in-silico results citing antibacterial potential of polypharmacological natural agents like flavonoids, phenolics, steroids, and terpenoids, which have the ability to inhibit and kill bacteria strains have been, stated (soviati and widyarman, 2020, apriyanti and kurnia, 2020, kurnia and apriyanti, 2019, gartika and pramesti, 2018). anti-bacterial activity in the present study the in-vitro anti-bacterial activity of lgo was quantitatively assessed against drug resistant microbial strains of escherichia coli (mtcc-40), bacillus subtilis (mtcc-121), pseudomonas aeruginosa (mtcc-424) and staphylococcus aureus (mtcc-3160), the results of which are depicted in table-2 and figure 4. the present study shows that lgo exhibits substantial antimicrobial activity against gram negative escherichia coli (mtcc-40) while total inhibition was seen for gram positive bacillus subtilis (mtcc-121), pseudomonas aeruginosa (mtcc-424) and staphylococcus aureus (mtcc-3160) as indicated in figure 3. the variance action of lgo might be owing to the incidence of single target or multiple targets for their activity. the antimicrobial activity of lgo may arise due to the presence of major and minor bioactive component that affected hydrolytic enzyme inhibition (proteases) or inhibited partners like: cell wall envelop proteins, microbial adhesions, and non-specific interactions with carbohydrates (silva et al., 2003, siramon et al, 2007). earlier studies also have cited that anti-microbial activity was not always related to the high content of one major chemical compound, rather than to synergic effects between major and minor components (silva et al., 2003). siramon et al, (2007) also cited incidence of potent bioactive molecules like flavonoids, and terpenoids behind the antimicrobial activity. same authors cited that bioactive molecules have tendency to cross across the cell membranes and to induce biological reactions, thus upsetting electron flow, the proton motive force, active transport and coagulation of the cellular contents. lgo also exhibits high antifungal, insecticidal and bactericidal activity (chen et al, 2016). in present study high antimicrobial toxicity of lgo toward gram negative bacteria was observed which is a noteworthy observation as most studies suggests that the gram negative bacteria are more resistant than gram positive bacteria due to thick peptidoglycan layer, lipopolysaccharides, phospholipids, of cell wall that permit gram negative bacteria to be added resistant to most of the hydrophobic antibiotics and toxic drugs (su et al, 2006). table 2. antimicrobial analysis of lemon grass essential oil. strain strain type zone of inhibition (cm) c eo pseudomonas aeruginosa gram negative 3.0 fi escherichia coli gram negative 2.5 6.5 bacillus subtilis gram positive 2.6 fi staphylococcus aureus gram positive 2.5 fi here: c= positive control (vancomycin antibiotic, 10mg), eo= essential oil, fi: 100% inhibition, values are expressed as mean±sd (n=3), 230 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 figure 3. 3d interaction’s of pbp5 with ligands. a: geranial; b: limonene; c: linalool; d: myrcene; e: neral. figure 4. anti-bacterial activity of lgo against mtcc-121, mtcc-40, mtcc-424 and mtcc-3160. codenc, pc and lgo are for negative control (blank), positive control and lemongrass oil and codelgo-a, lgo-b, lgo-c and lgo-d represents mtcc-40, mtcc-121, mtcc-424, and mtcc3160 respectively. conclusions currently, antibiotic resistance against gram-positive and gram-negative bacteria has emerged in the human population, and is a potential threat to global health, worldwide. currently, the main target for bacterial infections is primarily pbps. the aim of this study was to examine bioactive molecules from lemon grass essential oil that may be used to inhibit the bacterial infection pathway. compositional analysis revealed the presence a b c d e nc-a pc-a lgo-a nc-b pc-b lgo-b nc-c pc-c lgo-c nc-d pc-d lgo-d sharma & kaur – chemical profile and in-silico docking studies on … 231 of bioactive compounds in lemon grass oil. in-silico docking depicted effective docking of all bioactive compounds. wet lab validation documented antibacterial role of lgo. therefore, we suggested that bioactives compounds from lemon grass essential oil may represent potential treatment options, and found in medicinal plants that may act as potential inhibitors of bacterial pbps. however, further 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(2017). identification of a functionally unique family of penicillin-binding proteins. j am chem soc. 13:17727-17730 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 133-141 | doi: 10.14421/biomedich.2023.121.133-141 issn 2540-9328 (online) ethnobotanical survey of aromatic and medicinal plants used in traditional medicine and agri-food in the fez-meknes region hamza el finou*, nadia salhi, asma halmoune, lhoussaine el rhaffari department of biology laboratory of bioactives, health and environment, faculty of sciences, moulay ismail university meknes. morocco. corresponding author* h.elfinou@edu.umi.ac.ma manuscript received: 11 november, 2022. revision accepted: 29 december, 2022. published: 14 january, 2023. abstract in order to identify the medicinal plants used in agri-food and traditional medicine by the population of fez-meknes region (morocco), a floristic and ethnobotanical study was carried out in 4 provinces of this region (fez, meknes, azrou, taza). questionnaire forms were used to survey the usual users of the plants and herbalists and to collect as much information as possible on the therapeutic and dietary use of aromatic and medicinal plants in the region. for plants used for food, our survey identified 29 species divided into 26 genera and 16 families, including herbaceous plants (70%), trees (16.60%), shrubs (10%) and bushes (3.30%). the study of the medicinal flor a used in traditional medicine also allowed the inventory 81 species belonging to 47 families. leaves are the most commonly used part and the majority of remedies were prepared in the form of infusion (47%) and decoction (26%). among all the diseases treated, digestive diseases are the most cited (25%), followed by dermatological diseases (21%). the present study allowed us to evaluate some traditional practices used by the fez-meknes region population. in this context, it is essential to carry out similar investigations in other regions of the kingdom, in order to safeguard this precious natural heritage by means of a monograph that is as complete as possible and to validat e the remedies and preparations identified using rigorous scientific protocols. keywords: agri-food; ethnobotany; medicinal plants; monograph; traditional medicine. introduction according to the who (world health organization), nearly 6377 species of plants are used in africa, of which more than 400 are medicinal plants that constitute 90% of traditional medicine. in 2004, nearly 75% of the african population used plants to treat themselves and do not have access to modern medicines, whose pharmaceutical industry still relies heavily on the diversity of secondary plant metabolites to find new molecules with novel biological properties (mikou et al. 2016) the use of plants in therapy has been known for a long time. due to the richness and original diversity of its flora, morocco constitutes a real phytogenetic reservoir with approximately 4500 species and subspecies of vascular plants. the diversity of the relief and the most varied bio climates associated with them have given rise to a large number of endemic species. the rate of endemism is about 20% of the total number of species (pousset 1989). however, while there is no denying the curative virtues of a large number of plants, a good knowledge of them (toxicity, form of administration, dose and method of preparation) is essential to select in the mass of actions attributed to plants. in this sense, floristic and ethnobotanical studies have been carried out in different regions of morocco and have shown a return of the populations to the traditional use of medicinal plants (hseini and kahouadji (2007), lahsissene and kahouadji (2010) and salhi et al. 2010). the multiplication of these ethnobotanical studies on a national scale will make it possible to gather more information on moroccan medicinal plants, to enhance them and to preserve some of the knowledge acquired by the local population (mikou et al. 2016). these ethnobotanical studies are the most approach for discovering new medical plants or focused on those previously identified for their bioactive ingredients (celestina et al. 2012). in this sense, and in order to highlight the virtues and traditional uses of aromatic and medicinal plants, the present ethnobotanical study was conducted among the population of the fez-meknes region. materials and methods study area the present ethnobotanical study was carried out in the form of a survey using a pre-established questionnaire with various specific questions about the informant, the vernacular identity of the plant, as well as the part used, https://doi.org/10.14421/biomedich.2023.121.134-141 136 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 the modes of preparation, the therapeutic and traditional uses. study zone located in the north centre of morocco, the fezmeknes region is composed of 2 prefectures (fez and meknes) and 7 provinces (taounate, taza, sefrou, el hajeb, boulemane, my yacoub and ifrane) (figure 1). the region has three types of climates; a continental climate in the northern part, very hot and dry in summer and cold and wet in winter. a cold and humid climate in mountainous zones, very cold and snowy in winter, temperate in summer and a semi-arid climate in the high hills of boulemane, where the average rainfall does not exceed 250 mm. the winter is very cold and snowy. figure 1. the fez-meknes region on the national map. survey procedure in order to ensure a high degree of objectivity of the data obtained from our study, the survey is conducted using a survey form or questionnaire based on four axes:  information about the informant's profile (age, gender, level of education, etc.)  choice between the two medicines (modern and traditional)  information on the nature and pharmaceutical techniques of the plants used (local name, part used, method of preparation, dose)  information on the use of medicinal plants for diseases treatment. data analysis for data analysis, ibm spss statistics-21 software was used. the graph-pad prism 8 software was also used to create graphs. the frequency of citation was determined as follows: 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛 = ([ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛𝑠 𝑥 100 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛𝑠 ]) results socio-demographic data for the use of aromatic and medicinal plants, the results obtained showed a dominance of women gender with 87.3% against 12.7% for men, and the age group of 40 to 60 years with 51%. for education level, those not attending school come first at 41%. analysis of medicinal plants catalogue inventoried floristic aspect the study of the medicinal flora has allowed the inventory of 81 species belonging to 47 families. among the 47 families, the most used are the lamiaceae with 14 species followed by the asteraceae (7), apiaceae (7), fabaceae (5) and myrtaceae (3). table 1 presents a complete analysis of the plants. plant parts used and preparation modes the plant parts used are ranked in decreasing order of importance: leaves (42%), seeds (27%), fruits (10%), stem (9%), and bark (9%). other parts were presented by 5% (figure 2.a). to facilitate the use of medicinal plants, several preparation methods were used, such as decoction, maceration and infusion. in this sense, we obtained that infusion (47%), decoction (26%), whole plant (16%) and cataplasms (11%) were the most used preparation methods (figure 2.b). le av es s te m s s ee ds b ar k fr ui t o th er 0 15 30 45 60 42% 9% 27% 9% 10% 5% p e rc e n ta g e % a in fu si on d ec oc tio n c at ap la sm r aw p la nt 0 15 30 45 60 47% 26% 11% 16% p e rc e n ta g e % b figure 2. percentage of parts used (a) and preparation methods (b). el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 135 why do you use traditional medicine in order to find out the reasons for using traditional medicine, the interviewees were asked this question. the efficacy criteria came first with 48%, followed by the availability of plants and the lower cost with 8% and 7% respectively. while 38% of the respondents stated all three criteria at the same time. the use of medicinal plants constitutes a risk to human health, hence the need to know their toxicity status. for this reason, the question of toxicity was asked to the herbalists and phytotherapists, 89.2% of whom declared the nontoxicity of the plants most often used in the region. this percentage correlates with the good expertise of these professionals. the survey revealed that 53.9% of these professionals had 10 to 20 years of experience in traditional herbal medicine field, and 21% had more than 20 years (figure 3). 0 15 30 45 60 under 10 years between 10 and 20 years more than 20 years 25% 53% 20% pourcentage % figure 3. years of experience of herbalists and practitioners interviewed. symptoms treated with aromatic and medicinal plants of all the diseases treated, digestive diseases are the most cited (25%), followed by dermatological diseases (21%) and cardiovascular diseases (15%) (figure 4). 0 5 10 15 20 25 30 neurological-neueropsychic other osteoarticular respiratory cardiovascular dermatological digestive 5% 7% 9% 13% 15% 21% 25% percentage % figure 4. symptoms treated with aromatic and medicinal plants. table 1. list of plants used for food purposes. common name scienific name family biological form preparation method part used nutritional interest number of citations le bugle ajuga iva lamiaceae herbaceae fresh leaves rich in calcium, potassium, and antioxidants. use for the urinary system diseases. 3 malva, la mouve malva sylvestris malvaceae herbaceae cooked leaves, stem rich in vitamins c and e, unsaturated fatty acids with metabolic and digestive benefits. 5 le palmier nain chamaerops humilis arecaceae shrub fresh fruit, root antioxidants, omega 3, 6 and 9 and vitamins a, b, c, e. interest in blood pressure regulation. 1 l’arbousier arbutus unedo ericaceae shrub fresh fruit rich in vitamin c 2 136 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 common name scienific name family biological form preparation method part used nutritional interest number of citations -antioxidants regulation of cholesterol synthesis. chardon d’espagne scolymus hispanicus astéraceae herbaceae cooked and fresh leaves, stem composed of inulin (simple sugar) with metabolic interest in passing kidney stones. 6 la ronce rubus fruticosus rosaceae shrub fresh fruit rich in vitamin c and all forms of vitamin b (except b12). 2 cardon cyanara cardunculus astéraceae herbaceae cooked leaves, stem high mineral and vitamin b content. 3 rhus pentaphylla anacardiace ae shrub fresh fruit -1 l’oléastre olea europea oleaceae tree oil fruit rich in fatty acids: omega 3, 6, 9 with digestive and metabolic benefits. 2 romarin rosmarinus officinalis lamiaceae herbaceae cooked leaves metabolic interest and use in the digestive system. 2 fenouil foeniculum vulgare apiaceae herbaceae cooked stem, bulb rich in vitamins b, c, d, e. metabolic interest and use for digestive system. 2 le jujubier zizyphus lotus rhamnaceae tree fresh fruit use for the digestive and genito-urinary system. 5 le thym thymus bleicherianthus lamiaceae herbaceae cooked leaves and whole plants use for the treatment of digestive tract. 4 herbaceae cooked leaves and stem metabolic interest. 4 pourpier potage portulaca oleracea portulacacea e herbaceae cooked leaves and stem rich in omega 3. 4 chêne verte quercus ilex fagaceae tree fresh seeds potassium, calcium, phosphorus with interest in metabolic, regulation of glycemic and digestive tract disorders. 5 menthe à feuille ronde mentha rotundifolia lamiaceae herbaceae cooked leaves and stem digestive tract and respiratory system. 5 le myrte myrtus commuins myrtaceae tree fresh fruit use for fatigue and digestive tract. 4 petite férule elaeoselinum asclepium apiaceae herbaceae fresh and cooked leaves and stem metabolic interest. 1 la férule ferula communise apiaceae shrub cooked flowers anticoagulation. 2 lavande lavandula multifidal lamiaceae shrub cooked leaves treatment of digestive tract and respiratory system. 3 grande oseille rumex acetosa polygonacée ae herbaceae fresh and cooked leaves rich in vitamin c with metabolic interest. 4 ache aquatique apium nodiflorum apiaceae herbaceae cooked leaves high content of calcium, vitamin e, b9, and antioxidants. 4 la bette beta vulgaris amaranthace aeou chenopodiac eae herbaceae cooked leaves vitamin c, potassium and iron. 3 origan compact origanum compactum bentham. lamiaceae herbaceae cooked leaves and whole plants treatment of gastric pains. 4 moutard des champs sinapis arvensis brassicaceae herbaceae cooked leaves metabolic interest. 3 lavande papillon lavandula stoechas lamiaceae herbaceae cooked leaves and whole plants use for the treatment of digestive tract and respiratory system. 4 el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 137 common name scienific name family biological form preparation method part used nutritional interest number of citations caroubier ceratonia siliqua fabaceae tree flour fruit rich in carbohydrates, vitamin a, amino acids and fatty acids. 6 table 2. list of plants for medical use. common name scientific name family therapeutic use frequency of citation percentage of citation globulaire globularia alypum l. plantaginaceae eczema, gout, digestive problems, diabetes, sports perspiration and wound healing. 10 9.25% thym serpolet thymus serpyllum lamiaceae treatment of cough, digestive disorders, colic. 81 80 .14 % la rue d’alep ruta chalepensis.l rutaceae hair care, dermatological, respiratory, oral and digestive diseases. 6 5.55% caroubier ceratonia siliqua l leguminosae (ceasalpiniaceae) regulation of intestinal transit, hypercholesterolemia. 8 7.40% ricin ricinus communis euphorbiaceae hair care. 1 0.92% aubépine crataegus monogyna rosaceae cardiovascular system, stress and sleep disorders. 6 5.70% chénopode chenopodium ambrosioides amaranthaceae treatment of fever, asthma and other ailments. 8 7.25% marrube blanc marrubium vulgare lamiaceae gastrointestinal diseases, cough and bronchitis. 3 2.77% carvi carum carvi l. apiaceae digestion, expectoration, colic, sometimes for rheumatic problems. 14 12.81% laurier rose nerium oleander.l apocynaceae dermatological problems, dental infections, fever. 20 18.51% verveine odorante aloysa citriodora verbenaceae nervousness, sleep disorders. 7 6.48% lavande lavandula angustifolia p.mill lamiaceae genito-urinary problems, digestive tract, hair and skin care. 11 10.18% khella ammi visnaga (l.) lam apiaceae (umbelliferae) oral and dental infections, respiratory disorders. 78 72.22% romarin rosmarinus officinalis l. lamiaceae asthma and colds, digestive tract, hair care. 63 58.33% girofle syzygium aromaticum myrtaceae antiseptic, urinary tract infections. 6 5.55% fenugrec trigonella foenum graecum fabaceae treatment of gastralgia, appetite, digestion, hypercholesterolemia. 32 29.62% harmel peganum harmala zygophyllaceae rheumatism, healing and burns. 4 3.70% souchet long cyperus longus cyperaceae inflammation and pain, anticonvulsant properties. 1 0.92% jujubier ziziphus lotus rhamnaceae soothing; anxiety, regulation of intestinal transit. 6 5.55% roseau à massette typha latifolia l. typhaceae analgesic, carminative. 2 1.85% thuya de barbarie tetraclinis articulata cupressaceae treatment of intestinal infections, fever, hair and skin care. 3 2.77% herniaire herniaria hirsuta caryophyllaceae renal calculi, urinary tract. 1 0.92% achillée des marais achillea odorata l. subsp astéracées rheumatism, tonic, stimulant. 1 0.92% myrte myrtus communis myrtaceae treatment of arthralgia.. 7 6.48% henné lawsonia inermis lythraceae hair colouring, dermatological problems. 12 11.11% cresson alénois lepidium sativum brassicaceae anti-rheumatic, lung diseases, gastric problems, and to boost immunity. 9 8.33% haricots noirs phaseolus vulgaris fabaceae anti-inflammatory properties. 2 1.85% vigne à vin vitis vinifera l. vitaceae against abscesses and constipation. 1 0.92% 138 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 common name scientific name family therapeutic use frequency of citation percentage of citation le cubèbe piper cubeba l.f. piperaceae tonic, stimulant, culinary, antiashmatic. 1 0.92% menthe odorante mentha suaveolens lamiacées tonic, stomachic and antispasmodic effects. 5 4.62% ortie urtica dioica urticaceae antirheumatic, treatment of inflammation, diuretic, antidiabetic. 1 0.92% souci des champs calendula arvensis asteraceae gastric inflammations. 1 0.92% sauge à feuilles verveine salvia verbenaca lamiaceaa regulation of perspiration; menstrual cycle. 6 5.55% rosier rosa centifolia rosaceae wellness, anxiety. 3 2.77% pin maritime pinus pinaster ait pinaceae treatment of respiratory diseases, pain relief for rheumatic diseases. 1 0.92% laurier laurus nobilis lauraceae antibacterial, antiviral, antiseptic, fungicide, decongestant, nervous system regulator, digestion and anxiety. 8 7.40% inule visqueuse dittrichia viscosa asteraceae constipation, gastrointestinal diseases. 3 2.77% marjolaine origanum majorana lamiacées digestive disorders, flatulence, nausea, intestinal spasms, diarrhea; nervous disorders, migraines, insomnia. 9 8.33% oseille de guinée hibiscus sabdariffa malvaceae treatment of respiratory tract inflammations, high blood pressure, cholesterol, fever and stomach pains. 1 0.92% ail allium sativum amaryllidaceae prevention of cardiovascular disease, cancer. 3 2.77% oursins echinops spinosus asteraceae dermatological diseases. 12 11.11% armoise artemisia vulgaris asteraceae treatment of digestive disorders, joint and muscle pain and insomnia. 11 10.18% magydaris magydaris panacifolia apiaceae hair care and dermatological diseases. 1 0.92% garou daphne gnidium thymelaeaceae hair care and dermatological diseases. 8 7.40% cumin velu ammodaucus leucotrichus apiaceae treatment of respiratory diseases and intestinal disorders. 8 7.40% melissa melissa officinalis lamiaceae calming (nervous disorders, stress, anxiety, anguish), antispasmodic (stomach, intestine), heart problems (tachycardia). 2 1.85% arganier argania spinosa sapotaceae cosmetic application (face and body care). 1 0.92% nerprun alaterne rhamnus alaternus l. rhamnacées anxiety, digestion, constipation. 3 2.77% sauge salvia officinalis lamiacées digestion, stress, anxiety, cough. 5 4.62% eucalyptus eucalyptus gunnii myrtaceae anti-inflammatory and analgesic, treatment of respiratory problems. 9 8.33% jasmin jasminum polyanthum oleaceae anxiety. 1 0.92% lupinus lupinus luteus fabaceae prevention of cardiovascular and skin diseases. 2 1.85% noyer juglans regia juglandaceae natural toothbrush, natural antifungal. 3 2.77% figuier opuntia ficus indica cactaceae diuretic activity. 1 0.92% bergamote citrus bergamia rutaceae digestive disorders, stress, mouth and skin infections. 1 0.92% coriandre coriandrum sativum.l apiaceae digestive disorders and diarrhea, and to induce sleep. 8 7.41% aristoloche aristilochia paucinervis aristolochiaceae digestive disorders, analgesic 1 0.92% origan, thym commun thymus vulgaris lamiaceae tonic, digestive diseases. 29 26.85% https://fr.wikipedia.org/wiki/lauraceae https://www.facebook.com/hashtag/marjolaine?__eep__=6&__cft__%5b0%5d=azxtmc8dyjurivnaleqpm7prq3dusapay0y7x2vfmnx5a-whmrqzjjfkea5tk-zr_gglsyeydi8-bfr8scy9x5ghdvvlbadsvlfzolrmrk_669jegvp2-sxu_lav9yqjojnv2scvukn49aop3pf6tpai&__tn__=*nk-r https://fr.wikipedia.org/wiki/malvaceae https://www.google.fr/search?bih=657&biw=1366&hl=fr&q=amaryllidaceae&stick=h4siaaaaaaaaaongvuluz9u3mcozljdzxmrnmjtyvjmtk5msmjyamaoa1j0dwb4aaaa&sa=x&ved=2ahukewiqypiax_zwahvgzrokhu25bk4qmxmoatalegqijhad https://fr.wikipedia.org/wiki/asteraceae https://fr.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/apiaceae https://fr.wikipedia.org/wiki/lamiaceae https://fr.wikipedia.org/wiki/lamiaceae https://fr.wikipedia.org/wiki/sapotaceae https://fr.wikipedia.org/wiki/myrtaceae https://fr.wikipedia.org/wiki/fabaceae https://fr.wikipedia.org/wiki/salvadora_persica https://fr.wikipedia.org/wiki/salvadoraceae el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 139 common name scientific name family therapeutic use frequency of citation percentage of citation coquelicot papaver rhoeas papaveraceae insomnia, respiratory diseases. 16 14.81% coloquinte citrurllus colocynthis cucurbitacées diuretic activity. 2 1.85% valériane valeriana officinalis valerianaceae sleep disturbances. 2 1.85% garance rubia tinctorum.l rubiacées stomach diseases, anti diarrhea.. 1 0.92% dauphinelle staphisaigre delphinium staphisagria ranunculaceae hair care. 1 0.92% euphorbe euphorbia resiniphera euphorbiaceae wound healing, ophthalmic diseases. 17 15.74% millepertuis hypericum perforatum hypericaceae anxiety, sleep problems. 14 12.96% colchique d’automne, safran des près conopodium majus koch apiaceae weight gain. 1 0.92% séné senna alexandrina caesalpiniaceae laxative, constipation. 1 0.92% anis étoilé illicium verum illiciaceae anti-inflammatory, intestine. 2 1.85% lin linum usitatissium linaceae inflammation, gastritis, hair care. 3 2.77% fenouil, aneth doux foeniculum vulgare apiaceae anxiety, insomnia, vomiting, gastrointestinal and respiratory disorders. 7 6.48% carline atractylis gummifera asteraceae appetite, stomach. 3 2.77% gingembre zingiber officinale zingibéraceae muscle pain, digestion. 1 0.92% géranium geranium robertianum géraniaceae stress and anxiety. 1 0.92% camommile romaine chamomilla nobilis astéraceae anxiety, insomnia. 2 1.88% discussion at the end of this survey, 29 species of plants for food use were identified, which were divided into 26 genera and 16 families, including herbaceous plants (70%), trees (16.60%), shrubs (10%) and bushes (3.30%). the study of the medicinal flora used in traditional medicine has also allowed the inventory of 81 species belonging to 47 families. for the socio-demographic data, the results obtained showed a dominance of female gender with 87.3% against 12.7% for men, and the age group of 40 to 60 years with 51%. for education level, those not attending school come first at 41%. in this sense, several ethnobotanical studies carried out on a national scale have also confirmed these results (ziyyat et al. 1997), hmamouchi (1999), (jouad et al. 2001), (eddouks et al. 2002), (tahraoui et al. 2007), (mehdioui and kahouadji 2007), (salhi et al. 2010) and (benkhnigue et al. 2012). for the used part, we obtained that the leaves are the most used (42%). then, seeds (27%), fruits (10%), stem (9%), bark (9%), ad other parts were presented by 5%. several studies also confirmed these results (el rhaffari and zaid (2008), bammou et al. 2015) and umartani and nahdi (2021). although the use of leaves is represented by an important percentage, it was noted during the survey, that some users proceed to pull the whole plant instead of being interested only in the desired part. on the other hand, there is a clear relationship between the used part of the plant and the effects of this exploitation on its existence (cunningham 1996), this mode of collection seriously compromises the sustainability of medicinal species, especially bulbous ones. knowing that the leaves are the seat of photosynthesis and sometimes of the storage of secondary metabolites responsible for the biological properties of the plant (bigendako-polygenis and lejoly 1990), the ease and speed of harvesting (bitsindou 1986) may be the cause of the high rate of use of foliage by the region population. the use of medicinal plants is done by several modes of preparation. in this study it was obtained that the majority of the remedies are prepared by infusion (47%) and decoction (26%). as the respondents do not have ideas about the precise quantities and measures in the preparation and dosage of phytomedicines. precision is lacking on several plants such as the quantities of plant organs to be prepared, the solvent or vehicle used, the time needed to prepare the solutions (decoction, infusion, powder, fumigation, poultice, maceration and daubing) and the precise dose to be prescribed. however, the infusion (47%) and decoction (26%) remain the most commonly used methods of preparation. this percentage shows that the local population grows to the decoction mode and finds it adequate to warm the body and disinfect the plant (lahsissene and kahouadji 2010). on the other hand, decoction allows the collection of the most active principles and attenuates or cancels the toxic effect of certain recipes. the results obtained showed that most medicinal plants are widely used in the care of the digestive system 140 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 (25%). our results are also confirmed by those of hmamouchi and agoumi (1993) who conducted a study in the mechraâ bel ksiri region, hseini and kahouadji (2007) in the region of rabat, mehdioui and kahouadji (2007) in the province of essaouira, salhi et al. (2010) in kenitra city and lahsissene and kahouadji (2010) in the zaër region. other plants are also used to treat dermatological diseases (21%) and cardiovascular diseases (15%). according to respondents and their clients, these herbal recipes have therapeutic action, which suggests that they include many bioactive ingredients that are essential to the living flora's physiological processes. as a result, they are thought to be more compatible with human bodies (agbatutu et al., 2022). conclusions traditional phytotherapy was and still, currently in demand by people who have confidence in trusting in the popular uses of plants and not having resources and access to modern medicine. the present work was carried out with the aim of making as complete an inventory as possible of the medicinal plants used in the fez-meknes region (morocco) and to gather information concerning the practical therapeutic uses in this region. the results obtained showed that of the 47 families inventoried, the lamiaceaes is the most represented with 14 species (29.78%). on the ethnobotanical and pharmacological side, the leaves are the most commonly used part. decoction and infusion are the most commonly used method. similarly, of all the diseases treated, digestive diseases are the most cited. in addition, our results showed an important diversity of wild edible species in the fez-meknes region, which represents a significant asset for food and nutritional diversification. the present study allowed us to evaluate some traditional practices used by the population of fezmeknes region. a wealth of knowledge and expertise has been shown. in this context, it is essential to carry out similar investigations in other regions of the kingdom in order to safeguard this precious natural heritage by means of a monograph that is as complete as possible, and to validate the remedies and preparations identified by means of rigorous scientific protocols. acknowledgements: the authors would like to thank all the herbalists and citizens of the fez-meknes region who agreed to participate in this survey. authors’ contributions: nadia salhi & asma halmoune and hamza el finou designed the study, collection of data. hamza el finou analyzed the data and wrote the manuscript. lhoussaine el rhaffari: validation and supervision. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references agbatutu, a., ogie-odia, e. a., ifedele, m. o., abdulrasaq, f. m., eseigbe, d., & imade, f. n. 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(1997). phytotherapy of hypertension and diabetes in oriental morocco. journal of ethnopharmacology, 58(1). https://doi.org/10.1016/s03788741(97)00077-9 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 97-108 | doi: 10.14421/biomedich.2023.121.97-108 issn 2540-9328 (online) potential inhibition of ace2 membrane protein by flavone glycosides for blocking entrance of sarscov-2 into the cells; a computational study ahsan ibrahim*, ehtisham ul haq shifa tameer-e-millat university, islamabad-44000, tel. +9251-8438061 fax. +9251-8438059, pakistan. corresponding author* ahsan.scps@stmu.edu.pk manuscript received: 07 october, 2022. revision accepted: 07 december, 2022. published: 10 january, 2023. abstract severe acute respiratory syndrome coronavirus 2 (sarscov-2), since its emergence in wuhan city of china in late 2019, had been a dilemma for the global healthcare system. humongous efforts have been put in ascertaining the effective treatments for attenuation of the spread of corona virus disease (covid-19) pandemic. the aim of this research study is to probe the potential inhibition of angiotensin converting enzyme 2 (ace2) membrane protein by well-known flavone glycosides, hence preventing the binding of spike proteins with ace2 and subsequent prevention of entry of sarscov-2 inside the cells. the molecular docking analysis, for total ten flavone glycosides was carried out, that laid out propitious results in terms of binding energies towards the active residues of ace2 protein with a range of -9.3 to -7.1 kcal/mol. the molecular dynamics simulation also yielded promising outcomes. the in-silico toxicity analysis of all the potential drug candidates was carried out that revealed that all the compounds were non-toxic and safe. studies may be required for optimum formulation development using these compounds as a part of drug discovery and development phenomenon. this study may play a vital part in exploration of natural compounds in pharmacotherapy of covid-19. keywords: ace2 membrane protein; covid-19; flavone glycosides; molecular docking analysis; sarscov-2; spike proteins. abbreviations: sarscov-2 : severe acute respiratory syndrome coronavirus 2 covid-19 : corona virus disease ace2 : angiotensin converting enzyme 2 ssrna : single stranded ribonucleic acid prrar : furin cleavage site in spike protein tmprss2 : transmembrane serine protease 2 s protein : spike protein s cleavage : spike cleavage rbd : receptor binding domain pdb : protein data bank admet : absorption, distribution, metabolism, elimination and toxicity profile introduction the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) emerged as the most common clinical presentation of severe covid-19 (brosnahan et al. 2020). coronavirus disease started in wuhan, china in late 2019, spreading worldwide, affecting 612 million people and total number of deaths exceeds 6.53 million as of september 2022 as per statistics of world health organization (who) (“who coronavirus (covid-19) dashboard | who coronavirus (covid-19) dashboard with vaccination data” n.d.). the advent of covid-19 created serious crisis in health sector, leaving behind huge gaps in the field of disease control and prevention, that could be bridged through new research and investigations. the genome of covid-19 had been the most noteworthy in the ongoing research on sarscov-2. it is a positive sense single-stranded rna (+ssrna) and hence tends to replicate in a swift way within the host cell. the genetic material of the virus encodes for many nonstructural proteins (nsps) that implement enormous functions for virus such as taking the control the host biosynthetic machinery, viral replication, viral protein processing etc. the function of some nsps is still unknown. the structural genes of this virus encode for the structural proteins, which include spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, having a wide spectrum of functions in https://doi.org/10.14421/biomedich.2023.121.97-108 98 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 characterizing the entry and pathogenicity of the viral particle in the host cell. the homotrimers of s proteins make up the spikes on the surface of viral particle and they are involved in the attachment to the host cell receptors. spike proteins hold prime importance in relation to the ace2 proteins to which they bind and provoke the infective cascade of covid-19 (chen, liu, and guo 2020). in host cells, spike protein (s protein) is cleaved at the furin cleavage site i.e. prrar motif by a protease named furin into s1 and s2 to form a heterodimer of s1/s2, which is finally assembled in the form of the trimeric spike protein complex. the s2 subunit of this complex is cleaved by host proteases like transmembrane protease serine 2 (tmprss2) to uncover the fusion peptide for membrane fusion of the host cell and virus (wu et al. 2022). s1 is for binding to the host cell receptor angiotensin-converting enzyme 2 (ace2). it is v-shaped and the second one (s2 subunit) for fusion of the viral and host cell membranes (sternberg and naujokat 2020). ace2 and tmprss2 are abundantly present in the airways, arteries, nasal and oral mucosa, kidneys, the intestine, most profusely expressed on epithelial cells of the respiratory tract i.e. type ii pneumocytes that are the cells responsible for secreting surfactant to lessen the surface tension and prevent lung collapse (sternberg and naujokat 2020; akkız 2022). receptor-binding domain (rbd) contains a receptor binding motif, which is the necessary for spike protein (s1) that binds to the outer surface of ace2 protein. tmprss2 cleaves a part of spike proteins and promote the adhesion of sars-cov-2 s protein to get fuse with host cell membrane as the extended spike fosters the fusion of viral and host cell membrane. subsequently, many hydrophobic amino acids are also generated that get concealed in nearby cell membrane. the virus enters into the cell and have a first encounter with host cell biosynthetic machinery for translation of viral genetic material into viral proteins leading to prompt replication. cells that overexpress ace2 and tmprss2 are more susceptible to sarscov-2 entry (akkız 2022; ahmad et al. 2021). ace2 is a membrane-bound carboxydipeptidase protein. ace2 degrades angiotensin ii to generate angiotensin 1-7, which downregulates regulates a variety of angiotensin ii actions mediated by counteracting effects against the excessively activated ace/angiotensin ii/at1r axis, angiotensin ii type 1 receptor, as seen in hypertension, cardiac hypertrophy, heart failure, and some cardiovascular disorders. on the other hand, human ace2 is a known receptor by which sars-cov-2 particle enters host cells, by mutual binding of the spike protein of sar-cov-2 and ace2 membrane protein. this makes it a critical site for stopping the sarscov-2 to enter the cells using various ligands. so, it is pivotal to inhibit ace2, prevent viral genetic material entry into the cell (kai and kai 2020). the figure 1 summarizes the mechanism of the whole process. flavonoids are the polyphenolic compounds, very popular for the extensive pharmacological effects they provide such as anti-cancer, anti-inflammatory, antioxidant etc. (panche, diwan, and chandra 2016). flavones are one of the subclasses of flavonoids. the glycosidic linkages at positions 3 or 7 of flavones leads to formation of flavone glycosides (kumar and pandey 2013). besides various therapeutic effects such as antimicrobial, antidiabetic, hepatoprotective, antitumor and immunomodulatory effects (xiao et al. 2016), flavone glycosides have also been studied and reported for having antiviral effects against herpes simplex virus (hsv) 1 and 2 (yarmolinsky et al. 2012). keeping in view that fact that ace2 is a potential target, this study is going to use molecular docking as a tool to inquire regarding the activity of flavone glycosides for inhibiting ace2 and blocking the entry of sarscov-2 into the cell and arresting the viral replication process. figure 1. mechanism of entry of sarscov-2 into the cells through ace2 membrane protein materials and methods macromolecule preparation the 3d structure of the ace2 protein was retrieved from rcsb-protein data bank database in pdb format (pdb id: 1r42) (towler et al. 2004a). x-ray crystallography method was used for the determination of the structure of the macromolecule. the macromolecule was prepared by removing the unwanted water molecules, heteroatoms and ligands from the structure of protein on software biovia discovery studio visualizer v17.2.0.16349. the cleaning of protein helps to purely observe the interaction between ligand and the target protein residues, without any influence of these unnecessary entities. ligand preparation the ligands selected were the flavone glycosides and their structures were acquired from pubchem database. ibrahim & ul haq– flavone glycosides attenuate covid-19 99 perkinelmer chem 3d 16.0 was used to convert the 3d structure of the ligands into pdb format. luteolin 3'glucoside (l1), isovitexin (l2), baicalin (l3), scutellarin (l4), diosmin (l5), apigenin 7-o-glucoside (l6), isoorientin (l7), lonicerin (l8), tricin 7-oglucouronide (l9) and swertisin (l10) were the ligands used for studying their potential for inhibition of ace2 protein receptor for preventing the entry of sars-cov2 into the cells containing ace2 membrane protein, as shown in table 1. the two-dimensional structures of the ligands l1 to l10 are shown in figure 2. table 1. the table shows the list of flavone glycoside ligands (l1 to l10) in this study, their chemical structure, botanical source and iupac names. serial number ligands pubchem id structure botanical source literature source l1 luteolin 3'glucoside 12309350 podocarpus nivalis pubchem l2 isovitexin 162350 carex fraseriana pubchem l3 baicalin 64982 scutellaria amoena pubchem l4 scutellarin 185617 scoparia dulcis pubchem l5 diosmin 5281613 asyneuma argutum pubchem l6 apigenin 7o-glucoside 44257792 lonicera japonica pubchem 100 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 serial number ligands pubchem id structure botanical source literature source l7 isoorientin 114776 carex fraseriana pubchem l8 lonicerin 5282152 lonicera japonica pubchem l9 tricin 7-oglucouronide 101939793 scutellaria discolor pubchem l10 swertisin 124034 gentiana orbicularis pubchem ibrahim & ul haq– flavone glycosides attenuate covid-19 101 figure 2. the figure shows the chemical structures of the ligands l1 to l10, along with their iupac names obtained from pubchem. results and discussion molecular docking analysis & results a molecular docking technique has been very handy in drug discovery by pinpointing the drug leads having auspicious therapeutic activity (ferreira et al. 2015). the molecular docking was performed using pyrx virtual screening tool that is highly reliable as it works on the configuration of autodock vina (ekins, mestres, and testa 2007). for the visualization of protein-ligand interactions, biovia discovery studio visualizer software v17.2.0.16349 was utilized. it provided distinct display of the 2d and 3d interactions along with the bond lengths, aromatic, hydrophobic interactions etc. promising results were witnessed after the molecular docking analysis of human ace2 protein receptor with ligands l1 to l10, as shown in table 2. 102 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 table 2. the table shows the findings of the molecular docking and scoring analysis performed for ligands, l1 to l10. ligand-protein interaction binding energy (kcal/mol) bonding residues type of bond bond length (å) other residues l1 -7.5 his 378 pi-pi 5.61 ala 348, asp 350, tyr 385 his 401 pi-pi 4.43 glu 402 pianion 3.49, 3.75 l2 -7.1 his 378 pi-pi 4.93 asp 382, asn 394 his 401 pi-pi 5.71 glu 402 pianion 4.20 l3 -8.1 his 401 h bond 2.53 ser 44, asp 350, asp 382 l4 -8.2 his 401 h bond 2.50 ser 44, trp 349, asp 350, asp 382, tyr 385 l5 -9.3 his 378 h bond 3.31 phe 40, asp 350, asp 382, asn 394 his 401 pialkyl 5.12 l6 -7.9 his 401 pidonor h bond 2.58 ser 44, ala 348, trp 349, asp 350 glu 402 h bond 2.38 l7 -7.4 his 378 pi-pi 4.93 ala 348, glu 375 his 401 pi-pi 5.72 l8 -8.4 his 378 pi-pi 4.94 ser 44, ala 348, trp 349, tyr 385, tyr 515 glu 402 pi-anion 3.57 l9 -8.0 glu 402 h bond 2.11 trp 349, asp 350, arg 514 l10 -7.3 his 378 h bond, pi-pi 3.11, 4.95 pro 346, asp 382, arg 393 his 401 h bond, pi-pi 2.53, 5.77 all the ligands l1 to l10, exhibited their firm interactions with the binding site residues of human ace2 protein receptor i.e. his 378, his 401 and his 402 (towler et al. 2004b), with a binding affinity ranging from -9.3 to -7.1 kcal/mol. the best binding energy of 9.3 kcal/mol was expressed by diosmin (l5), while the binding energies exhibited by other compounds in this study were -8.4 kcal/mol for lonicerin (l8), -8.2 kcal/mol for scutellarin (l4), -8.1 kcal/mol for baicalin (l3), -8.0 kcal/mol for tricin 7-o-glucouronide (l9), 7.9 kcal/mol for apigenin 7-o-glucoside (l6), -7.5 kcal/mol for luteolin 3'-glucoside (l1), -7.4 kcal/mol for isoorientin (l7), -7.3 kcal/mol for swertisin (l10) and -7.1 kcal/mol for isovitexin (l2). all the ligands showed absolute interactions with the active site residues of human ace2 enzyme. luteolin 3'-glucoside (l1) and isovitexin (l2) were bound to his 378, his 401 and glu 402 with considerable binding energies. ligands, baicalin (l3) and scutellarin (l4) manifested binding with his 401 residues with shorter bond lengths. diosmin (l5) and apigenin 7-o-glucoside (l6) showed their interactions with his 378, his 401 and his 401, glu 402 residues, respectively with reasonable bond lengths. ligands, isoorientin (l7), lonicerin (l8), tricin 7-o-glucouronide (l9) and swertisin (l10), all demonstrated their robust binding affinities with active residues his 378, his 401 and glu 402 with substantial bond lengths, with pi-pi and hydrogen bonds. strong interactions and short bond lengths also appeared with some other residues i.e. ser 44, ala 248, trp 349 and asp 350 may also be the active residues of ace2 protein. the interactions of the best docked ligand diosmin (l5) are shown in figure 3. while figure 4 demonstrates the interactions of top three ligands with highest binding energies (l5, l8 and l4) with ace2 binding site. ibrahim & ul haq– flavone glycosides attenuate covid-19 103 figure 3. the best docked ligand, diosmin (l5) blocking the binding site of ace2 (a) and 2d diagram of interactions of l5 with active site residues (b). figure 4. 3d and 2d interactions of top three ligands with respect to binding energies (l5, l8 and l4) respectively, with the active site residues of ace2 protein. 104 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 admet analysis admet profile of a specific ligand serves as a surrogate for its fate to be considered as a potential drug candidate. swiss adme was employed for analyzing the adme profile of the potential drug candidates in this study (daina, michielin, and zoete 2017). toxicity profile was established through datawarrior v5.5.0 (sander et al. 2015). the orisis property explorer was used for the prediction of druglikeness score of all potential drug candidates. the molecular weights of ligands from l1 to l10 are 448.38, 432.38, 446.36, 462.36, 608.54, 432.38, 448.38, 594.52, 506.41 and 446.40 grams per mole (g/mol), respectively. the highest score for bioavailability was expressed by l2 and l10 i.e. 0.55. while l1, l5, l7, l8 conveyed their bioavailability score as 0.17 and a bioavailability score of 0.11 was predicted for l3, l4 and l9. a further invitro and in-vivo studies are recommended for improvement and tailoring of the pharmacokinetic profile of these potential drug candidates, so that these could be formulated in such a way that they may act as potent inhibitors of ace2 in attenuating sars-cov-2. the toxicity profile of these ligands, studied on datawarrior v5.5.0, was unremarkable for all the ligands, l1 to l10. the admet profile of ligands l1 to l10 is shown in table 3. the bioavailability radar of diosmin (l5) is provided in the figure 5. figure 5. bioavailability radar of the best docked ligand, diosmin (l5), obtained through swissadme. table 3. the table represents the in-silico profiling of admet, physicochemical properties, druglikeness and toxicity of the ligands (l1 to l10) drug candidates l1 l2 l3 l4 l5 l6 l7 l8 l9 l10 physicochemical properties mol.w (g/mol) 448.38 432.38 446.36 462.36 608.54 432.38 448.38 594.52 506.41 446.40 h-acc 11 10 11 12 15 10 11 15 13 10 h-don 7 7 6 7 8 6 8 9 6 6 n.r.b 4 3 4 4 7 4 3 6 6 4 tpsa (å²) 190.28 181.05 187.12 207.35 238.20 170.05 201.28 249.20 205.58 170.05 log p -0.06 0.05 0.22 -0.20 -0.44 0.55 -0.24 -1.03 0.30 0.35 b.sc 0.17 0.55 0.11 0.11 0.17 0.55 0.17 0.17 0.11 0.55 drug-likeliness d.l.s -3.4 -1.2 0.8 1.04 3.85 -2.3 -0.7 2.4 2.0 -1.1 n.l.v 2 1 2 2 3 1 2 3 3 1 metabolism cyp 1a2 no no no no no no no no no no cyp 2c19 no no no no no no no no no no cyp 2c9 no no no no no no no no no no cyp 2d6 no no no no no no no no no no cyp 3a4 no no no no no no no no no no p-glycoprotein no no yes yes yes yes no yes yes no toxicity mutagenicity no no no no no no no no no no tumorgenicity no no no no no no no no no no irritant no no no no no no no no no no reproductive effects no no no no no no no no no no mol. w (g/mol) molecular weight, h-acc hydrogen bond acceptors, h-don hydrogen bond donors, n.r.b number of rotable bonds, tpsa (å²) topological polar surface area, log p prediction of octanol/water partition coefficient, b.sc bioavailability score, d.l.s druglikeness score, n.l.v number of lipinski’s rule violations, cyp cytochrome p-450 enzyme. ibrahim & ul haq– flavone glycosides attenuate covid-19 105 molecular dynamics simulation the molecular dynamics simulation is performed to assess the physical dynamics or motions of a molecule, virtually on a computer which imparts a lot of details and facts about motions and gestures of a given docked macromolecule complex. the molecular dynamics simulation for the top docked complex of diosmin (l5) was conducted using imods server (lopéz-blanco, garzón, and chacón 2011). the molecular dynamics simulation was carried upon normal mode analysis (nma). the results for b-factor or mobility were imparted. this is an important factor of the protein crystallography, which specifies the residues with in the complex that deform themselves to interact with other residues, exposing the flexibility of a molecule. the arrow field around the complex shows the orientation of our complexed macromolecule virtually, as shown in the figure 6. figure 6. the figure shows the diagrammatic results of the molecular dynamics simulation done with the best docked complex, diosmin (l5) with ace2 protein. image (a) shows the orientation of the docked complex with the arrow field. image (b) shows the deformability graph and graphical representation of the b-factor. eigenvalue was also yielded through this server that denotes the energy required for deforming a residue in a dynamic protein for interaction with other residues. the less is the eigenvalue, the more is the ease for deformation of the residues. the eigenvalue of the studied complex was 5.6245e-05. covariance map casts a scenario of interactions between the residues of the protein and ligand in the complex while the elastic network deliver a complete picture of the stiffness within the target molecule, hence giving an estimate of deforming in the residues of the target protein, as provided in figure 7. figure 7. image (a) demonstrates the covariance graph of the best docked complex diosmin (l5) and ace2 protein, image (b) represents th e elastic network map of the docked ligand while image (c) represents the eigenvalue of the docked ligand. 106 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 discussion molecular docking is a worthwhile tool in the drug discovery and repurposing phenomenon in the arena of computer aided drug designing (hughes et al. 2011). the interaction of a specific compound with a specific target molecule is predicted computationally, without utilizing the wet lab (meng et al. 2011). in this study, molecular docking was used to recognize the potential inhibition of ace2 enzyme by flavone glycosides. in this study, it was observed that flavone glycosides had an appreciable range of binding affinities with the active site residues of ace2 enzyme i.e. -9.3 to -7.1 kcal/mol. the potential drug candidates exhibited their powerful interactions with the active residues of ace2 i.e. his 378, his 401 and glu 402. luteolin 3'-glucoside (l1) showed binding energy of -7.5 kcal/mol, that is highly encouraging for inhibition of ace2. luteolin 3'glucoside (l1), isolated from podocarpus nivalis (“luteolin 3’-glucoside | c21h20o11 pubchem” n.d.) has also been identified as hepatoprotective and immunoregulatory agent in some studies (park and song 2019). isovitexin (l2), acquired from carex fraseriana (“isovitexin | c21h20o10 pubchem” n.d.), also spotted for its anti-inflammatory and antidiabetic potential (abdulai et al. 2021), has provided very optimistic results regarding binding and potential inhibition of ace2. a reasonable binding energy of -7.1 kcal/mol and linkages with active residues can make a considerable molecule having potential inhibitory activity for ace2. baicalin (l3) is a flavone glycoside derived from scutellaria amoena (“baicalin | c21h18o11 pubchem” n.d.), has anti-inflammatory effects in cardiovascular, neurodegenerative and infectious ailments (hu et al. 2022), while scutellarin (l4) gained from scoparia dulcis (“scutellarin | c21h18o12 pubchem” n.d.), has been reported as to be an antioxidant, antitumor, antiplatelet, cardioprotective and immunomodulatory (wang and ma 2018). both have showed an excellent affinity for ace2 active residue his 401, with binding energies -8.1 and 8.2 kcal/mol with considerably shorter bond lengths of 2.53 and 2.50 angstrom, respectively. diosmin (l5) was the potential drug candidate that delivered the most decent results in terms of binding energy, that was -9.3 kcal/mol and was bound to his 378 and his 401. it has been reported that diosmin (l5), obtained from asyneuma argutum (“diosmin | c28h32o15 pubchem” n.d.), works as an antioxidant as well as relieves venous insufficiencies (feldo et al. 2018). some studies have revealed that apigenin 7-o-glucoside (l6) from plant lonicera japonica (“apigenin 7-o-beta-dglucoside | c21h20o10 pubchem” n.d.), was found to be antimicrobial and cytotoxic (nie et al. 2018) and this study has detected l6 as a potential inhibitor of ace2 with a binding energy of -7.9 kcal/mol with active residues and shorter bond lengths. isoorientin (l7), derived from carex fraseriana (“isoorientin | c21h20o11 pubchem” n.d.), bound to the target with energies of -7.4 kcal/mol and showed potent affinity for active site. a study has outlined the role of isoorientin (l7) in lowering inflammatory mediators, relieving algesia and combating free radicals as well (yuan et al. 2016). lonicerin (l8), a chemical constituent of lonicera japonica (“lonicerin | c27h30o15 pubchem” n.d.), has also been described for its potent antipseudomonal pharmacology (xu et al. 2019). in this study, the binding energy of -8.4 kcal/mol of lonicerin (l8) and its connection with his 378 and glu 402 tells the possible inhibitory activity for ace2. tricin 7-o-glucouronide (l9), isolated from scutellaria discolor (“tricin 7-o-glucuronide | c23h22o13 pubchem” n.d.), is known for its inhibition for cervical cancer cell growth through upheld expression of bax and caspases (laishram et al. 2015), seems to have a significant potential to bind ace2 in human, as per this study, with optimistic energy of -8.0 kcal/mol, with bond length of 2.11 angstrom with glu 401. swertisin (l10), a priniciple component of gentiana orbicularis (“swertisin | c22h22o10 pubchem” n.d.), well known for its antioxidant and inflammation fighting tendencies (“swertisin (chebi:131838)” n.d.), came up with a binding energy value of -7.3 kcal/mol and interactions with his 378 and his 401. hence, all the potential drug candidates have furnished hopeful results in terms of potential inhibition of ace2 in human that can be further studied and validated through in-vitro and in-vivo studies. the molecular dynamics simulation study clues that the studied complex had a substantial degree of deformability. the low eigenvalue, the covariance map and the elastic network of the studied complex are the evidences of the promising deformability and admissible dynamics of the molecule. conclusion in this study, it is concluded that the ligands l1l10 have shown a potent activity for ace2 protein receptor for potentially averting the entry of sarscov-2 into the cells. the ligands have manifested pertinent interactions with ace2 active residues, showing highly encouraging binding energies. the molecular dynamics simulation studies also provided hopeful results. however, in-vitro and in-vivo studies are required to confirm these findings. furthermore, pharmaceutical studies are also recommended for these compounds in formulating them as a suitable drug delivery system for maximal therapeutic efficacy. covid-19 had been very grim challenge for the health system around the globe. the focus of the ongoing research in the field of drug discovery revolves around the drug development for covid-19. many concerns have developed in terms of efficacy as well as safety for many drugs to be used against this virus. on ibrahim & ul haq– flavone glycosides attenuate covid-19 107 the basis of findings of this study, an inference could be drawn that these flavone glycosides could be utilized as potential drug molecules for restricting the entry of sarscov-2 entry into the cells they infect by blocking ace2 receptor protein, especially in lungs. this study will assist the researchers for preclinical and clinical investigation of these potential drug molecules. acknowledgement: the study is carried out entirely by the authors, so there is no need for acknowledgement. authors’ contributions: example: ahsan ibrahim designed the study. ahsan ibrahim carried out the computational work. ahsan ibrahim & ehtisham ul haq wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: there exist no competing interests. references abdulai, ibrahim luru, samuel kojo kwofie, winfred seth gbewonyo, daniel boison, joshua buer puplampu, and michael buenor adinortey. 2021. “multitargeted effects of vitexin and isovitexin on diabetes mellitus and its complications.” the scientific world journal 2021. 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81 (july): 356–62. https://doi.org/10.1016/j.biopha.2016.04.025. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 281-288 | doi: 10.14421/biomedich.2023.121.281-288 issn 2540-9328 (online) phytochemical analysis and antibacterial activity of methanol and ethyl acetate extracts of detarium microcarpum guill. & perr. muhammad mubarak dahiru*, abdulazeez mumsiri abaka, susan pwakangdi artimas department of science laboratory technology, school of science and technology, adamawa state polytechnic, yola, jimeta, 640101, tel. 555-555-1212, adamawa state, nigeria. corresponding author* mubaraq93@gmail.com manuscript received: 30 january, 2023. revision accepted: 11 march, 2023. published: 27 march, 2023. abstract this research aimed to investigate the phytoconstituents and antibacterial effects of methanol and ethyl acetate stem bark extracts of detarium microcarpum (dm). the phytochemicals were detected and quantified while the antibacterial activity against escherichia coli, staphylococcus aureus, and salmonella typhi was established determining the zone of inhibition (zi), minimum inhibitory concentration (mic), and minimum bactericidal concentration (mbc). phytochemical screening showed alkaloids (16.33% ±0.88) were present in the methanol extract only while saponins and flavonoids were detected in concentrations of 31.00% ±2.31 and 21.01% ±2.33 respectively for the methanol extract and 21.67% ±1.76 and 38.01% ±1.16, for the ethyl acetate. the methanol extract exhibited the highest zi on s. aureus (21.3 mm ±1.11) with its least inhibition observed on e. coli (6.5 mm ±0.77) while the ethyl acetate extract demonstrated the highest zi on s. typhi (19.1 mm ±2.01). s. typhi exhibited more sensitivity to dm extracts at the least concentrations of 12.5 mg/ml (methanol) and 25 mg/ml (ethyl acetate) while the mbc results showed that the 12.5 mg/ml and 25 mg/ml were the effective respective concentration for methanol and ethyl acetate extracts against s. typhi. conclusively, dm exhibited an antibacterial effect against the test organisms with notable inhibitory and bactericidal effects. keywords: antibacterial activity; antimicrobial activity; antibacterial resistance; detarium microcarpum; phytochemical analysis. abbreviations: dm (detarium microcarpum), esbe (ethyl acetate stem bark extract), msbe (methanol stem bark extract), mic (minimum inhibitory concentration), mbc (minimum bactericidal concentration), and zi (zone of inhibition). introduction medicinal plants have long been reported to be sources of therapeutics and have been utilized in the management of several ailments in traditional and folkloric practices. different research reported the pharmacological effects of several plants employed to manage ailments including diabetes, cancer, inflammation, parasite, and microbial infections (prasathkumar et al., 2021). in traditional practice, medicinal plant parts such as roots, leaves, and stem bark are prepared in different forms such as decoction and infusions administered by oral or inhalation as an alternative to modern medicines to achieve therapeutic purposes (ullah et al., 2020). there are several reasons for the inclination toward the use of medicinal plants as it offers certain advantages such as being cheap, and available with minimized side effects compared to modern drugs (ahad et al., 2021; rahayu et al., 2020). in other cases, poverty, poor health facilities, and access may contribute to the recognition of drugs from plant sources as alternatives. the therapeutic use of plants as sources of drugs in disease and infection management has been attributed to their phytochemical components extracted via the preparation method which are associated with several biological effects (george et al., 2021; hossen et al., 2022; zheng et al., 2022). bacterial infections are attributed as the cause of millions of deaths and morbidities worldwide and have so far evolved into a major health concern all over the world (ji et al., 2016). although different antibiotics of varying efficacy are employed in the treatment of infections, these antibiotics are losing their efficacy because of antimicrobial resistance which is a major challenge in achieving treatment goals (maillard et al., 2020). antimicrobial resistance forms a major problem in treatment of infectious diseases especially in rural communities and developing countries even though this resistance continues to occur every day during treatment with an estimated projected death of up to 4 million by 2050 (akinde & taiwo, 2017; elton et al., 2020; tong et al., 2019). there is an increase in antimicrobial resistance, especially in africa, however, phytochemicals from plants serve as long-time sources of therapeutics with proven results in treatments of infectious diseases https://doi.org/10.14421/biomedich.2023.121.281-288 282 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 (bouyahya et al., 2017). phytochemicals from plants offer an alternative as utilized in traditional practices as infectious diseases are a worldwide menace and create a burden on an individual and governments. phytochemicals extracted from different plant preparations exert different pharmacological effects individually or synergistically observed in the folkloric use of plant drugs. alkaloids are associated with antidiabetic (adhikari, 2021), anticancer (song et al., 2022), and antimicrobial effects (fan et al., 2022; jafaar et al., 2021). flavonoids exert biological and pharmacological activities against oxidative stress, cancer, inflammation, and tumor through different modes of action (jucá et al., 2020). flavonoids also exert an antimicrobial effect on antibiotic-resistant microbes (biharee et al., 2020; gupta et al., 2022). glycosides exhibit pharmacological effects including anti-diabetic (yang et al., 2022) and antimicrobial activities (tm et al., 2022). terpenoids exert pharmacological effects against a tumor, cancer, inflammation, malaria, diabetes, and microbial infections (wu et al., 2020; yang et al., 2020). saponins exhibit pharmacological effects against cancer, oxidative stress, inflammation, and microbial infections (fang et al., 2020). several phytochemicals exert different pharmacological activities working either individually or in synergy with each other. thus, this research aimed to establish the phytochemical composition and antibacterial effects of methanol and ethylacetate extracts of dm. materials and methods study area detarium microcarpum (dm) sample collection was carried out in the girei local government area of adamawa state, nigeria. the authentication of the plant sample was done by a forest technologist from the department of forestry, adamawa state polytechnic yola, where a voucher specimen (asp/ft/0212) was deposited. the stem bark was dried and ground into powder using a blender. chemicals and reagents all the chemicals and reagents used for the study were of anarlar. procedures extract preparation stem bark powder (300 g) of dm was macerated in 1 l of 70% v/v methanol and ethyl acetate for 48 hours, followed by filtration and concentrated to dryness over reduced pressure (evans, 2009). qualitative phytochemical analysis the detection of phytochemicals in methanol (msbe) and ethyl acetate (esbe) stem bark extracts of dm was carried out following the methods previously described (evans, 2009). quantitative phytochemical analysis  alkaloids alkaloids were quantified as previously described (harborne, 1998).  saponins saponins were estimated by the method previously described (obadoni & ochuko, 2002).  flavonoid quantitation of flavonoids was carried out by the method previously (harborne, 1998). antibacterial activity  test organisms the bacteria isolates (e. coli, s. aureus, and s typhi) were collected from modibbo adama university teaching hospital microbiology laboratory, yola, nigeria. the characterization of the test organisms was done through observation of their cultural growth characteristics according to a previously described method (idris abubakar & abubakar usman, 2016). confirmation of clinical isolates’ identity was carried out via biochemical tests following the standard method previously described (cheesbrough, 2002; talaiekhozani, 2013). the pure cultures were streaked onto a nutrient agar slant followed by 24 h incubation at 37 °c and subsequent storage at 4 °c until needed for use.  mcfarland standard preparation exactly 9.95 ml of 1% h2so4 and 0.05 ml of 1.17% bacl were mixed to form a precipitated suspension acting as 0.5 mcfarland standard set as the turbidity for the test organisms (cheesbrough, 2002).  inoculum standardization the confirmed isolates were subjected to further culturing on nutrient agar containing petri-dishes followed by overnight incubation at 37 °c to form colonies which were transferred into test-tubes / containing 5 ml of 0.9% normal saline adjusted to the turbidity of previously prepared mcfarland's standard (andrews, 2005). determination of antibacterial activity a slightly modified agar well diffusion technique was adopted to establish the antibacterial effects of dm (biradar et al., 2008). the bacterial inoculation was carried out with a sterile swab onto a prepared solidified mueller-hinton agar, and allowed to stand for 15 min, followed by the addition of 0.2 ml of the extracts of varied concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 mg/ml) to five wells bored with cork borer with the addition of ciprofloxacin to the other well as a positive control and incubated overnight at 37°c. the diameter of the zones of dahiru et al. – phytochemical analysis and antibacterial activity of … 283 inhibition was used to determine the antibacterial effects of the extracts. determination of minimum inhibitory concentration (mic) the previously described method was adopted to ascertain the mic of dm following the procedures described by national committee for clinical laboratory standards (nccls) (lar et al., 2011). briefly, one ml of concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, and 6.25 mg/ml) of the extract was introduced into seven tubes with 5 ml of muller-hinton broth and thoroughly mixed, then 0.1 ml of broth cultures of the test organism and overnight incubation at 37 °c for bacterial growth to be observed. the minimum extract concentration to inhibit bacterial growth was defined as the mic of the extracts. determination of minimum bactericidal concentration (mbc) the evaluation of the mbc was carried out by further culturing the test-tubes with no visible growth in the mic test on mueller-hinton agar by spreading 0.1 ml of the inoculum with a sterile loop, followed by overnight incubation at 37 °c. the minimum concentration with no visible growth was defined as the mcb (de & ifeoma, 2002). data analysis data obtained were expressed as mean ± standard error of triplicate determinations' mean (± sem) and evaluated with statistical package for the social sciences (spss) version 22 software. results and discussion the phytochemicals detected in the msbe and esbe of dm are presented in table 1. alkaloids were detected in only the msbe while saponins and flavonoids were detected in both the msbe and esbe. however, steroids, glycosides, and terpenoids were absent in both the msbe and esbe. table 1. qualitative phytochemical test of msbe and esbe of dm. phytochemicals inference msbe esbe alkaloids + saponins + + steroids glycosides terpenoids flavonoids + + note: + = present, = absent. the phytochemicals quantified in the msbe and esbe are shown in table 2. alkaloids were quantified up to 16.33 % ±0.88 in the msbe while saponins quantified up to 31.00 % ±2.31 and 21.67 % ±1.76 for msbe and esbe respectively. the concentration of flavonoids in the msbe was 21.01 % ±2.33 while that of the esbe was 38.01 % ±1.16. table 2. quantitative phytochemical composition of msbe and esbe of dm. phytochemicals concentration (%) msbe esbe alkaloids 16.33 ±0.88 saponins 31.00 ±2.31 21.67 ±1.76 steroids glycosides terpenoids flavonoids 21.01 ±2.33 38.01 ±1.16 note: values are in triplicate determinations ± sem the antibacterial activities of msbe and esbe showed varying degrees of inhibition (figure 1-3). the msbe exhibited a maximum mean zone of inhibition against s. aureus (21.3 mm ±1.11 at 100 mg/ml) with its least inhibitory effect observed against e. coli (6.5 mm ±0.77) at 25 mg/ml. the extracts were ineffective against all the isolates at 6.25 mg/ml (figure 1-3). the esbe demonstrated a maximum mean zone of inhibition against s. typhi (19.1 mm ±2.01 at 100 mg/ml concentrations) with no effect at 12.5 mg/ml and 6.25 mg/ml against e. coli and 6.25 mg/ml against both the s. aureus and s. typhi. 284 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 figure 1. zi of s. aureus. figure 2. zi of s. typhi. figure 3. zi of e. coli. the inhibitory effects of msbe and esbe of dm are displayed in table 3. mic values for the msbe and esbe are between 100 mg/ml to 3.125mg/ml for s. aureus, s. typhi, and e. coli. s. typhi was the most sensitive to the msbe and esbe at 12.5 mg/ml and 25 mg/ml respectively. 21,3 17 12,5 9,5 18,8 16,4 12,6 8,5 40 0 5 10 15 20 25 30 35 40 45 100 50 25 12,5 6,25 z o n e o f in h ib it io n ( m m ) concentrations (mg/ml) m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin 19,8 14,5 9,5 7 19,1 15 11,4 7,4 35 0 5 10 15 20 25 30 35 40 100 50 25 12,5 6,25 z o n e o f in h ib it io n ( m m ) concentrations (mg/ml) m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin 12,1 9,5 6,5 13,2 10,4 7,2 25 0 5 10 15 20 25 30 100 50 25 12,5 6,25 z o n e o f in h ib it io n ( m m ) concentrations mg/ml m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin dahiru et al. – phytochemical analysis and antibacterial activity of … 285 table 3. mic of msbe and esbe. concentrations (mg/ml) s. aureus s. typhi e. coli m.e e.e m.e e.e m.e e.e 100 50 -* -* 25 + -* -* + 12.5 -* + -* + + + 6.25 + + + + + + 3.125 + + + + + + note: m.e= methanol extract, e.e= ethylacetate extract, * = mic value, + = turbidity , = no turbidity table 4 displays the results of the mbc which reveal the minimum extract concentration needed to fully kill the bacterial isolates. a concentration of 12.5 mg/ml was required to kill s. typhi for the msbe while 25 mg/ml was effective for the esbe. table 4. mic of msbe and esbe. test organism mbc (mg/ml) m.e e.e s. aureus 12.0 ±1.34 25.0 ±2.33 s. typhi 12.5 ±1.12 25.0 ±1.89 e. coli 25.0 ±2.21 50.0 ±2.62 note: values are in triplicate determinations ± sem, m.e= methanol extract, e.e= ethylacetate extract discussion the result indicated a variation in the presence of concentrations of phytochemicals in the msbe and esbe of dm which might be the difference in the polarity of the two solvents as the extraction process depends on the ability of the solvents to penetrate the extracts and solubilize the phytochemicals. thus, different solvents extract different phytochemicals with variable concentrations (aboshora et al., 2014). this might also account for the detection of alkaloids in the msbe as they are more polar. flavonoids were previously reported in the ethyl acetate fraction of stem bark of dm with the absence of alkaloids and saponins which were all absent in the methanol extract including flavonoids (ibrahim et al., 2021). our report agrees with a previous report (abdullahi et al., 2021; mu'azu et al., 2022) for alkaloids, saponins, and flavonoids detection in msbe of dm. the phytochemicals detected in our study were previously reported to exert antibacterial effects (akinpelu et al., 2014; donadio et al., 2021; liu et al., 2020; yan et al., 2021). alkaloids exert their antibacterial activities through the inhibition of bacterial cell walls, protein, nucleic acid, metabolic pathways, and by changing cellular membrane permeability (yan et al., 2021). another mechanism of action for flavonoids (indole) was reported to be by inhibiting efflux pumps, biofilm formation, filamentous temperature-sensitive protein z, and pyruvate kinase (liu et al., 2020). the positions of the nitrogen and methylenedioxy of isoquinoline were previously implicated in the antibacterial activity of the alkaloids (qing et al., 2017). saponin fractions of erythrophleum suaveolens exhibited antibacterial effects against some gram-positive cocci and gram-negative organisms indicating a good source of antibiotics (akinpelu et al., 2014). flavonoids demonstrated antibacterial activity by inhibiting biofilm formation and attachment, nucleic acid synthesis, energy metabolism, membrane properties, and function, reducing pathogenicity (xie et al., 2015). additionally, flavonoids further exhibit antibacterial effects by binding to microbial proteins required for basic cellular functions (donadio et al., 2021). the antibacterial effects of the msbe and esbe were measured at 100, 50, 25, 12.5, and 6.25 mg/ml (figure 1-3). both of the extracts demonstrated an antibacterial effect against the test organism with the highest mean zi of 21.3 mm ±1.11 observed against s. aureus at 100 mg/ml by the msbe. a similar result showed the superior effectiveness of the msbe against s. aureus against the esbe (tiwari et al., 2011). the zi difference might be attributed to the solvent polarity which influences the types and concentrations of the phytochemical extracted, thus influencing the antibacterial effects of the extracts (aboshora et al., 2014; gomashe et al., 2014). the antibacterial effects demonstrated by dm might be attributed to growthinhibiting phytochemicals such as flavonoids which were reported to possess antibacterial potentials (sanusi et al., 2022). although in the traditional and folkloric practice the primary solvent used for extraction is water for preparation, extraction with organic solvents exhibits superior antimicrobial effects compared to aqueous extracts (gomashe et al., 2014). the best practice for the extraction of broad-spectrum antimicrobial compounds employs alcohol as an extraction solvent as seen in the present study (gomashe et al., 2014). the effective concentration of the esbe was against the s. typhi as presented in table 1. the nature of the gram-negative bacteria cell wall might contribute to the resistance of the organism to the extract by limiting the penetration of the extract to affect its action due to the thin lipopolysaccharide exterior membrane (biswas et al., 2013). however, the mesh-like peptidoglycan layer of gram-positive bacteria allows for permeability and access to the extract (malanovic & lohner, 2016). the mic describes the minimum concentration of antimicrobial agent needed to inhibit microbial growth though not applicable in clinical practice for the determination of antibiotic dosage for a patient. however, it is applicable in the determination of the effective target antidiabetic with a lower chance of developing resistance (wiegand et al., 2008). the inhibitory effect of methanolic and ethyl acetate extracts dm was observed to be between 3.125mg/ml to 100 mg/ml for s. aureus, s. typhi, and e. coli with s. typhi 286 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 being the most sensitive. this variation in mbc values might be attributed to the phytoconstituents of the extract which vary due to solvent difference, thus, exhibiting inferior different inhibitory and bactericidal effects to the standard (aboshora et al., 2014; i. abubakar & a. usman, 2016; gomashe et al., 2014; sanusi et al., 2022). similar results to the present study were reported on the phytoconstituents and antibacterial effect of stem bark extracts of dm (sanusi et al., 2022). conclusion the study investigated the phytoconstituents and antibacterial effects of d. microcarpum which exhibited antibacterial effects 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(2022). potential roles and molecular mechanisms of phytochemicals against cancer. food & function, 13(18), 9208-9225. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 109-117 | doi: 10.14421/biomedich.2023.121.109-117 issn 2540-9328 (online) antioxidant and antibacterial activity of pomegranate extract (punica granatum l.) in lip balm formulation maria grasela kase, aniek prasetyaningsih, dwi aditiyarini* biology study program, faculty of biotechnology, duta wacana christian university jl. dr. wahidin sudirohusodo no. 5-25 yogyakarta, 55224, indonesia. corresponding author* dwi.aditiyarini@staff.ukdw.ac.id manuscript received: 31 october, 2022. revision accepted: 06 december, 2022. published: 11 january, 2023. abstract lips are a part of the face that does not have hair follicles making them easily dry and crack. lipbalm is a beauty product that could be used to solve this problem by increasing lip moisture. however, the synthetic ingredient in commercial lip balm can have side effects on the body in the long term. pomegranate fruit is one of the natural ingredients containing anthocyanin which can be used as a natural dye, antioxidant, and antibacterial. therefore, this study was performed to study the potency of pomegranates as natural dyes, antioxidants, and antibacterial in lip balm. pure pomegranate juice was obtained through squeeze step. lip balm was prepared in several concentrations of pomegranate juice which were 0%, 12.5%, 18.75%, and 25%. phytochemical screening shows the content of anthocyanin, saponin, tannin, and flavonoid. in this study, pomegranate juice has low antioxidant activity with ic50 449 ppm. lip balm formula with 18.75% and 25% of pomegranate juice can inhibit the growth of the bacteria staphylococcus aureus. keywords: antibacterial; antioxidants; lip balm; lips; synthetic ingredients. introduction one part of the face that most influences the aesthetic perception of the face is the lips. according to mulyawan and suriana (2016) in nazliniwaty et al., (2019) the most sensitive and unprotected part of the face is the lips. lips often look dry and cracked when it is exposed to cold air or excessive heat so that they are unsightly. this is because the lip does not have sweat glands and hair follicles that can protect it in the environment (trookman et al., 2009, yayang et al., 2019). one of the cosmetics products that can overcome this problem is lip balm. lip balm is a preparation that is applied to the lips and functions as a moisturizer by forming an immiscible oil layer on the surface of the lips. the layer formed by the lip balm is a protective layer of the lips from the influence of the external environment. in addition to lipstick, lip cosmetics that are often used by women are lip balms. the purpose of lip balm is to increase moisture in the lips. the main components in lip balms are waxes, oils, and fats which aim to prevent dry lips from occurring by keeping the lips moist and protecting the lips from environments that are too hot and too cold (agustiana et al., 2019). lip balm also contains antioxidants, preservatives, and coloring compounds. antioxidants in lip balm function to prevent free radicals that are harmful to the skin. preservatives are used to prevent irritation and prevent bacterial growth, while the dye in lip balm is added only to make the product (lip balm) look attractive (rini, 2012). antioxidants that are often used in the manufacture of lip balms are bht (butylated hydroxytoluene) and other preservatives. pomegranate is a tree that has a height of 2 to 5 meters. the pomegranate tree has a single leaf opposite each other (scattered). the large pomegranate flowers have red and white colors. the pomegranate flower is a pansy flower, and separated. the flower axis is hollow with a conical shape, 5 to 7 corollas in irregular buds, and many stamens, free stamens, and ovary sink. while for the pomegranate itself has a size 5 inches with red and dark red skin and is shaped like a grenade. the taste of the pomegranate itself varies depending on the level of maturity of the pomegranate (sharrif and hamed, 2012). pomegranate fruit, also known as pomegranate fruit, has several active compounds, namely alkaloids, flavonoids, saponins, tannins, and triterpenoids. pomegranate contains two types of polyphenol components, namely flavonoids (anthocyanins) and hydrolyzed tannins which have antioxidant activity. the antioxidant activity of pomegranate is higher than the antioxidant activity of red wine and green tea. in addition, the antioxidants in pomegranate also have the potential to reduce lipid peroxidation (roswiem et al., 2014). https://doi.org/10.14421/biomedich.2023.121.109-117 110 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 therefore, in this research, researchers would make a lip balm made from natural ingredients. one of the natural ingredients used in this research is pomegranate. the use of natural ingredients aims to reduce and replace synthetic ingredients that exist in lip balm preparations. the compounds in the pomegranate (punica granatum l.) to be tested are anthocyanins, saponins, tannins, and flavonoids, each of which has a role to replace or reduce synthetic ingredients commonly used in the manufacture of lip balms, so that in the manufacture of lip balms from natural ingredients is expected to bring out the natural color of the pomegranate, as well as antioxidants and antibacterial from the natural ingredients used. materials and methods this study uses petri dishes, beakers (pyrex), object glassed, measuring cups, oven (memmert), microscopes, test tubes, laf (laminar air flow), micropipettes, stoves, uv-vis spectrophotometers (genesys 10) as an antioxidant test and testing the total phenolic content, filter cloth, ph meter, oven, knife, and lip balm container, while for the used materials is a pomegranate from a fruit shop in yogyakarta, olive oil, beeswax, oleum cacao (kimia jaya labora), glycerin, aquadest, cetyl alcohol, dpph solution, methanol pa, hcl 2n solution, fecl3 solution, folin ciocalteau reagents, na2co3 solution, naoh solution, and staphylococcus aureus bacteria culture. in sample preparation, the used fresh pomegranate is ± 1 week after harvest that has red color. three kilograms of pomegranate fruit is separated between the skin and flesh, then the flesh is washed until clean and weighed (yuska et al., 2018). procedures extraction the extraction process was carried out by squeezing the pomegranate and filtering the pomegranate to get pure juice using a white filter cloth. the filtering process was carried out by squeezing the pomegranate which is still together with the seeds then placed on a filter cloth and then the juice was put into a 1000 ml sterilized beaker glass. yield is calculated using this formula: 𝑦𝑖𝑒𝑙𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑗𝑢𝑖𝑐𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑖𝑚𝑝𝑙𝑖𝑐𝑖𝑎 × 100% phytochemical screening of pomegranate juice anthocyanins the color test of anthocyanin compounds was carried out according to harbone (1987). red pomegranate juice 0.5 g was added to 2m hcl in 2 drops and then heated at 100°c for 5 minutes. a positive result is indicated with a red color. the several dropwise of 2m naoh was added while observing the color changes. positive result is indicated by the changes of red color to a blue-green color which then fades slowly (putri ni ketut et al., 2015). saponin the red pomegranate juice was weighed as much as 0.3 g and then put into a test tube. then 5 ml of distilled water was added and shaken for 30 seconds. the positive result is marked by the formation of foam which is stable for 30 seconds on the surface of the solution. tannin the red pomegranate extract 1 g was added to 10 ml of distilled water and then boiled. after cooling, 5 ml of 1% fecl3 was added. if the color changes to dark blue, then the sample contains tannins (novilia, 2014). flavonoid the extracted sample 1 g was put into a test tube. then 3 drops of concentrated hcl were added and then heated for 15 minutes in a water bath. if red or yellow color is formed, it means that the juice contains flavonoids (flavones chalcones and aurons) (muthmainnah, 2017). determination of total phenol content a total of 2 mg of pomegranate juice was dissolved in 2 ml of methanol and homogenized. then 0.2 ml of extract was put into a test tube and added 1.8 ml of distilled water and 0.1 ml of folin ciocalteu reagent. the solution was homogenized, vortex and incubated for 5 minutes. next, 1 ml of 5% na2co3 was added and homogenized. distilled water 1.9 ml was added to obtain final volume of 5 ml. then solution was homogenized and incubated for 5 minutes. the absorbance was measured at a wavelength of 760 nm. gallic acid was used as a standard curve with a concentration of 12,5; 25; 50;100 and 200 ppm. a linear equation is obtained by plotting a linear graph between the gallic acid concentration (x) and the absorbance value (y) (yismairai et al., 2019). calculation of total phenolic content is performed using the formula below: 𝑇𝑃𝐶 = 𝐶 × 𝑉 × 𝑓𝑝 𝑚 annotation: c : fenolic concentration (x score) v : volume of extract used (ml) fp : dilution factor g : weight of sample used (g) tpc : total phenolic content (mg gae/ g sample) antioxidant activity test antioxidant activity was determined by the dpph free radical method according to santosa et al. (1998). in this test, the sample of pomegranate juice were made in several concentrations which are 200,400, 600, 800, and 1000 ppm. the concentration of dpph is 10 ppm. the kase et al.– antioxidant and antibacterial activity of pomegranate extract … 111 stock solution was 1000 ppm. the dpph solution was put into each tube with various concentrations, namely 2 ml of dpph 10 ppm + 1 ml of sample solution, while for the control, 1 ml of dpph 10 ppm was made, then incubated for 30 minutes in the dark room temperature, then the absorbance was measured at a wavelength of 517 nm. furthermore, the value of inhibition concentration 50% (ic50) was determined, namely the concentration of the sample that can reduce dpph free radicals by 50% by using the equation y= ax+b, thus obtaining ic50 in units of g/ml. (abdullah et al., 2014). extract dosage formulation in this study, four lip balm formulations were made, consist of 0, 12.5, 18.7, and 25% of pomegranate extract with a weight of 12.8 g each container. the composition of the pomegranate extract lip balm is presented in table 1. table 1. formulation of natural lip balm. ingredient red pomegranate extract formulation (%) f0 f1 f2 f3 red pomegranate extract 0 12.5 18.75 25 olive oil 10 10 10 10 beeswax 25 25 25 25 glycerin 10 10 10 10 cetyl alcohol 50 50 50 50 oleum cacao ad 100 ad 100 ad 100 ad 100 annotations: f0 = lip balm formula as a base f1 = lip balm formula with concentration extract 12.5% f2 = lip balm formula with concentration extract 18.75% f3 = lip balm formula with concentration extract 25% in the process of making lip balm, the first step was to put ethyl alcohol in a beaker glass, heated and added pomegranate juice slowly. the solution was stirred until evenly mixed between ethyl alcohol and extract. after that, beeswax and cacao oleum was added and melted a temperature of 65°c. next, olive oil was added, heated, and stirred until homogeneous. lastly, the solution was taken out from water bath and poured into a container lip balm mold evenly and cooled until it hardens at room temperature (dirjen pom, 1985). organoleptic test an organoleptic test was carried out using the five senses. the parameters include odor, color, and texture of lip balm (yulyuswarni, 2018). evaluation of the preparation was carried out for 3 weeks with inspections carried out every week (days 1, 5, 10 and so on). homogeneity test this test was carried out by testing the homogeneity of the preparation of each lip balm preparation by taking the lip balm preparation and placing it on the glass slide and then visually observing whether there were coarse grains or not (keithler, 1956). lip balm weight test this test was carried out by weighing the lip balm preparation including the container. this weighing was carried out every 7 days for 3 weeks at a closed room temperature of 27°c. this test was carried out to determine the increase or decrease in weight at the beginning of manufacture and during storage. melting point test this test was done by melting 1 g of the lip balm preparation in a beaker. the melting temperature was measured at the initial melting (dian, 2019). ph test ph value was measured using a ph meter where 1 g of the sample was inserted and then dissolved in 10 ml of distilled water, after that electrode is inserted or dipped in the lip balm solution, then the numbers show on the ph meter are seen (risnawati et al., 2012). irritation test this test was carried out to see and evaluate whether the product can cause irritation or not. the technique used in this irritation test is an open patch test on the forearms of 30 panelists. an open patch test was carried out by applying the prepared preparation at the location of the attachment, leaving it open and, observing what happens. reaction was observed. positive irritation reactions are characterized by redness, itching, or swelling (risnawati et al, 2012). favorite test this test was conducted to determine the level of panelist preference. the age of 30 female panelists was around 18-22 year old. the panelist was not having sensitive or allergic skin. each other panelist was asked to apply lip balm of various concentrations of red pomegranate to the back of the hand. then they were asked to fill out the provided questionnaire. the duration of trial was ± 15 minutes and after trying the lip balm, the panelists are expected to wash their hands. antibacterial test antibacterial activity test was done using pour plate method. bacterial incubation was carried out for 24 hours using the pour plate method at 37c. then diameter of the inhibition zone was measured for each sample. the list of samples is presented in table 2. 112 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 table 2. list of samples. no code sample 1 e pomegranate extract with concentration 12.5%, 18.75% dan 25% 2 b olive oil, beeswax, oleum cacao, glycerin dan cetyl alcohol 3 f1 lipbalm formula with concentration extract 12.5% 4 f2 lipbalm formula with concentration extract 18.75% 5 f3 lipbalm formula with concentration extract 25% 6 k commercial lipbalm antibacterial test was performed to determine the ability of antibacterial activity against staphylococcus aureus. this first step is to culture the bacteria on nutrient agar (na) and nutrient broth (nb) medium. nutrient agar (na) is a medium for rejuvenating staphylococcus aureus which is in solid form, while nutrient broth (nb) has same function as (na) for growing bacteria but in liquid form. then, 100 μl nutrient broth (nb) containing bacteria was poured aseptically to each petri disk. mha media 20 ml was added into each petri disk. this step can also be performed by formerly mixing the bacteria and mha media, then small holes are made after the media hardens to put the sample. plates were divided into 3 to 4 zones to facilitate the distribution of treatments. after samples had been put into all petris, each petri was wrapped and incubated at 37°c for 24 hours under aerobic conditions for staphylococcus aureus. the ability of antibacterial activity was shown through the inhibition zone. the diameter of the finhibition zone was measured using a ruler. results and discussion secondary metabolites of pomegranate juice the yield of pomegranate juice is 76.80% (1.565 kg of pomegranate juice from 2.038 kg of peeled pomegranate) by squeezing the pomegranate. dian (2019) also carried out this method on dragon fruit with the similar yield as this study. phytochemical test results from pomegranate juice is presented in table 3. table 3. phytochemical screening data of pomegranate juice. compound parameter analysis result description anthocyanin blue green + green saponin stable foam + presence of foam tannin dark blue + dark blue flavonoids red/yellow + red annotation: (+) there are chemical compounds, (-) no chemical compounds in this study, pomegranate extract showed a positive result for the anthocyanin test, namely a change in color to green. this pigment belongs to the flavonoid compound and is responsible for the appearance of red, orange, blue and purple colors in some leaves, fruits, and flowers. anthocyanins have potential natural dyes to replace synthetic dyes (gross, 1987). saponin is also detected in the pomegranate juice. saponin compounds are included in triterpenoids, usually found in higher plants. saponins are abundant in leaves and roots but are rarely found in fruit (cseke et al, 2006). saponin compounds are indicated by the presence of stable foam or foam. the next compound identified was tannin. tannin compounds have positive results by showing a dark blue color. tannins are a class of polyphenolic compounds that are also commonly found in plants. tannins can be defined as polyphenolic compounds with very large molecular weights, which are more than 1000 g/mol, and can form complex compounds with proteins, besides that, tannins have a major biological role because of their function as protein precipitants and metal chelators. it also acts as a biological antioxidant. (noer et al, 2018) the last test of flavonoid compounds in this research showed positive results where the color produced was red. flavonoids are secondary metabolites of polyphenols, which are found widely in plants and food and have various bioactive effects including anti-viral and anti-inflammatory (qinghu wang et al, 2016). flavonoids are also one of the natural compounds that are commonly found in plants and food to treat various diseases such as cancer, antioxidants, bacterial pathogens, inflammation, cardiovascular malfunction, and have antioxidant abilities in preventing injury caused by free radicals (arifin et al, 2018) total phenolic content in pomegranate extract this research used gallic acid as a standard according to singleton et al. (1999). the standard curve of gallic acid is presented in figure 1. gallic acid has been used as a standard of comparison which is stated as milligrams of gallic acid equivalent per gram or milliliters of extract between samples (tapera et al., 2019). the gallic acid standard is the relationship between gallic acid concentration (x) and absorbance value (y), which this relationship will produce a linear regression equation y = 0.0026x – 0.0116 and r2 value is 0.9903. from the equation in figure 1, it was carried out to calculate the total phenol in the pomegranate extract. total phenol levels are expressed in units of mg gallic acid equivalent/g sample, % gallic acid equivalent (gae) (mg gae/g) (hala et al., 2020). these results indicate that the total phenol content in the pomegranate extract is 0.012 mg/mg gae. kase et al.– antioxidant and antibacterial activity of pomegranate extract … 113 figure 1. gallic acid standard curve. pomegranate extract antioxidant activity the result of antioxidant measurements on pomegranate extract can be seen in figure 2. based on the data in figure 2, the results of linear regression are obtained, namely y = 0.0649x + 20.805 with a relation coefficient r2 = 0.924 so that the ic50 can be determined. the ic50 value is the concentration of a test sample solution that provides 50% dpph reduction (molyneux, 2004). from the equation data, it was found that ic50 in pomegranate extract had a value of 449 ppm. figure 2. antioxidant activity of pomegranate extract. according to molyneux (2004), the antioxidant activity in a compound is said to be very strong if the ic50 value is <50ppm, strong if it has an ic50 value between 50-100 ppm, moderate if ic50 value around 100-150 ppm and is said to be weak if the ic50 value 151-200 ppm. the ic50 value obtained from this research is 449 ppm, which is a weak ic50 value. in wulandari et al. (2017), ic50 of pomegranate peel extract with the extraction process, namely maceration, got a value of 2.39 ppm, which means the ic50 is very strong. the low ic50 of pomegranate extract in this study is probably caused by the difference of extraction process. preparation of pomegranate extract lip balm lip balm preparation from pomegranate extract is pink. the concentrations used were 0%, 12.5%, 18.75%, and 25%. figure 3 shows that the higher concentration, the more concentrated the color will be. the result of the lip balm preparation can be seen in figure 3. f0 (0%) fi (12.5%) f2 (18.75%) f3 (25%) figure 3. lip balm from pomegranate extract in several concentrations. organoleptic test results lip balm preparations from various extract concentrations 0%, 12.5%, 18.75%, 25%, had different characteristics of color, smell, and texture. the organoleptic test aims to determine the stability conditions of each concentration of lip balm tested for 3 weeks. it is included color, odor, and texture of each concentration of lip balm. based on organoleptic data for 3 weeks, changes in texture, aroma and color began to occur in the second week. the organoleptic test is conducted by dian (2019) also showed changes in the second week. according to dian (2019), physical differences (color and texture) are caused by a high room temperature factor which cause faster water evaporation so the particles in the lip balm clump together and cause the formation of crystals. according to pertiwi et al., (2020), the higher the concentration of extract used in the lip balm formulation, the stronger the color intensity. homogeneity test results based on homogeneity data, at concentrations of 0%, 12.5%, 18.75%, and 25% of the lip balm were homogeneous, while for a concentration of 12.5%, the results were not homogeneous. the homogeneity data showed that all lip balm formulations did not show any coarse granules, while the concentration of 12.5% was not mixed evenly for color distribution. therefore, it can be said that the preparations are 0%, 18.75%, and 25% and have a homogeneous composition in which the color distribution is even and there are no coarse grains on the glass object. this is in line with research conducted by (yusuf, 2019) ph test results testing the ph of the pomegranate extract lip balm in this study was using a ph meter. the purpose of ph testing is to determine and obtain a ph value that is close to the physiological ph of the skin, namely 4.56.5. the results of the ph test can be seen in table 4. y = 0,0026x 0,0116 r² = 0,9903 0 0,1 0,2 0,3 0,4 0,5 0,6 0 50 100 150 200 250 a b so rb a ti o n concentration (ppm) y = 0,0649x + 20,805 r² = 0,9424 0 20 40 60 80 100 0 200 400 600 800 1000 1200 % in h ib it io n concentration (ppm) 114 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 table 4. ph lip balm from pomegranate juice. concentration ph f0 (0%) 5.41 f1 (12.5%) 4.61 f2 (18.75%) 4.24 f3 (25%) 4.01 according to table 4, ph value of the lip balm without extract (f0) is 5.41, the concentration of f1 (12.5%) containing pomegranate extract is 4.61, then for the concentration of lip balm using pomegranate extract f2 (18.75%) was 4.24 and the concentration of f3 (25%) had a ph of 4.01. the difference in ph was caused by differences in the concentration of the extract. at concentrations of 0% (f0) and 12.5% (f1) it is already in the vulnerable ph of the lip skin (tranggono and latifah, 2007) which says the physiological ph of the lip skin is between 4.5-6.5, which means lip balm from extracts. pomegranate is safe to use, while the concentrations of 18.75% and 25% do not meet the physiological ph requirements of the lip skin. this is because the amount of pomegranate extract that is included in the lip balm preparation during the manufacturing process, so that the more concentrations used in the preparations, the more acidic it will be because the ph of the pomegranate is acidic, which 3.51. melting point test results in this research, the melting point test was also carried out. a melting point test is done to find out at what temperature the lip balm can melt completely. the result data are presented in table 5. table 5. lip balm melting point from pomegranate extract. concentration melting point (°c) f0 (0%) 55 f1 (12,5%) 53 f2 (18,75%) 52 f3 (25%) 52 from the results of the melting point test on the lip balm preparation, f0 (0%) has a melting point of 55ºc, f1 (12.5%) has a melting point of 53°c, then for f2 (18.75%) is 52°c and the last one for f3 (25%) which is 52°c. all melting point results have been included in the standard based on sni 16-5769-1998 in ratih (2014) which is in the range of 50-70°c. it can be said that the melting point of the prepared lip balm has good melting point and is up to standard. according to (agustiana et al., 2019) the difference in melting point in each preparation is due to the difference in the amount of cacao oleum included in each lip balm, the higher the concentration, the less cacao oleum is added so that the resulting melting point decreases. data shows that f0 (0%) has the highest melting point which is caused by the amount of cacao oleum that is the most compared to other concentrations is 5,2 g, while the least amount of cacao oleum is in the concentration of f3 (25%) which is 2 g, so it has a lower melting point. lip balm weight test result the result of the observed lip balm weight for 3 weeks is presented in table 6. table 6. lip balm weight test data. week weight (g) f0 (0%) f1 (12.5%) f2 (18.75%) f3 (25%) 0 average 6.207 6.695 6.871 6.697 1 average 6.207 6.693 6.868 6.695 2 average 6.208 6.692 6.861 6.655 3 average 6.210 6.692 6.859 6.644 lip balm weight test data for all concentrations were included in the paired samples test using spss to determine whether there was a difference in lip balm weight. tests using paired sample test are seen based on the comparison of week 0 and week 1, week 1 and week 2, week 2 and week 3, and the last week 0 and week 3. based on the results of the paired samples test, there was no significant difference in the weight of the lip balm which was weighed every week. if the sig value >0.05 then there is no significant difference in the lip balm weight test which is carried out every week. favorite test results in this research, the preference test was carried out by applying the four preparations of pomegranate extract lip balm (f0, f1, f2, f3) to 30 panelists, them the panelists were asked to fill out a questionnaire to find out which lip balm preparation was the most attractive according to the panelists. based on the test result, it was found that those who liked the lip balm preparation for the texture were 63.3% for the 25% formulation, the panelists who liked the lip balm preparation for the aroma were 76.19% for the 25% formulation, while the largest color was 77.4% for the concentration of 25% and overall including texture, aroma and color is 67.7% for a concentration of 25% as well. based on the lip balm preference test with aroma texture and color parameters, the lip balm most preferred by the panelists was lip balm with a concentration of 25%. the 25% concentration is the lip balm concentration with the most pomegranate extract compared to other concentrations. kase et al.– antioxidant and antibacterial activity of pomegranate extract … 115 irritation test result the result of the irritation test is shown in table 7. table 7. irritation test result data on pomegranate lip balm. irritation test f0 (0%) f1 (12.5%) f2 (18.75%) f3 (25%) number of respondent itchy rash 0 0 0 0 0 redness 0 0 0 0 0 bump 0 0 0 0 0 other irritation 0 0 0 0 0 based on the results of the tests carried out, the panelists gave negative results or there were no signs indicating irritation such as the absence of red skin, itching, or the presence of swelling and other allergic reactions. this shows that the ingredients contained in the lip balm from pomegranate extract are not irritating. this is in accordance with research (nazliniwaty et al., 2019) that lip balm from pomegranate extract does not cause irritation. the conclusion that can be drawn is that all the lip balm concentrations tested did not cause irritation. antibacterial test results in this research, antibacterial testing was carried out to determine the level of antibacterial in the sample to be tested against staphylococcus aureus bacteria which is a gram-positive bacterium that grows at an optimum temperature of 37°c, this bacterium that is usually present on the skin, digestive tract, and intestines. human respiratory tract can be found on the lips of humans (kusuma, 2009). this antibacterial test used the pour plate method which was repeated 3 times for each sample tested to determine the inhibition zone formed in the sample tested using staphylococcus aureus bacteria. the result of antibacterial testing can be seen in table 8. table 8. inhibition zone measurement data. sample average diameter of inhibition zone (mm) extract 12.5% 13.33a,b,c extract 18.75% 14.66 b,c extract 25% 22.66 c lipbalm 12.5% 0 a lipbalm 18.75% 5.33 a,b lipbalm 25% 6 a,b control + (ciprofloxacin) 40.94 d control – (aquadest) 0a description: a: mean are included in subset a and have no significant difference b: mean are included in subset b and have no significant difference c: mean are included in subset c and have no significant difference d: mean are included in subset d and have no significant difference the presence of an inhibitory zone in the pomegranate extract was due to the pomegranate extract containing antibacterial compounds (saponins) so that it could inhibit the staphylococcus aureus bacteria. in this research, the three extracts (12.5%, 18.75% and 25%) had an inhibitory zone with a vulnerability of 11-20 mm which means strong, while for positive control it had an inhibition zone of 21 so it was very strong (yunus et al., 2018). the inhibition zones of the pomegranate extract lip balm formed were 18.75% and 25% concentrations, only in the first repetition. the lip balm inhibition zones with concentrations of 18.75% and 25% had a strong inhibitory power with a range of 21 (yunus et al., 2018). one of the constituent materials formed by the inhibition zone is glycerin, where glycerin has a very strong inhibitory zone strength 21 as well as a positive control (yunus et al., 2018). the inhibitory zone data for the antibacterial test in this research were then entered into the anova test using spss. the anova test was used to determine whether or not there was a difference in the sample with an inhibitory zone. the anova results showed a significant difference in the inhibition zone formed in the pomegranate extract and lip balm preparations from the pomegranate extract because 000<0.05 so h0 was rejected. and h1 is accepted. the results of the subset table are then entered into table 8. table 8 column 1 (a) shows that f1, f2, f3 lip balm, and 12.5% extract have similarities and there are no differences. table subset 2 (b) shows that lip balm f2, f3 extract 12.5% and 18.75% there is no difference, different from the table for subset 1 (a) earlier, next is table subset 3 (c) where extract 12.5%, 18.75%, and 25% have no significant difference, in contrast to table subset 2 (b), that table subset 2 has similarities with lip balm f2 and f3. the last table subset 4 (d) is the positive control which has differences with pomegranate extract and lip balm preparations because it has the greatest value among the others. from the results of the antibacterial test in table 8 and the test using spss the largest diameter of the inhibition zone was pomegranate extract with a concentration of 25% and the lowest was lip balm with a concentration of 12.5%. conclusions the ability of pomegranate extract as an antioxidant is 449 ppm, which in pomegranate extract contains 116 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 antioxidants, but based on ic50 it is very low. the ability of pomegranate extract as an antibacterial in inhibiting the growth of staphylococcus aureus bacteria was found in the three extract concentrations (12.5%, 18.75% and 25%) while in lip balm preparations there were concentrations of 18.75% (f2) and 25% (f3), while the ability of pomegranate extract as a colorant in lip balm preparations can last for 3 weeks at room temperature and the lip balm which produces the brightest color is a concentration of 25% (f3). this concentration is also the most preferred by the panelists. acknowledgements: the authors would like to thank all staff in laboratory biotechnology, faculty of biotechnology, duta wacana christian university, for helping and supporting this research. competing interests: authors state that there is no conflict of interest in this research output. references abdullah, w., & runtuwene, m. r. j. 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(2019). total phenolic content (tpc) determination and phytochemical screening of plecranthus esculentus tubers of rusape, zimbabwe. international journal of advance study and research work, 2(2), 1-6. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 41-47 | doi: 10.14421/biomedich.2015.42.41-47 medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja’far luthfi1*, mahanem mat noor2 and jalifah latip3 1biology department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 2the study center of bioscience and biotechnology, 3the study center of chemical science and food technology, faculty of science and technology, ukm 43600 bangi, selangor, malaysia author correspondency*: jafarluthfi@yahoo.com abstract medicinal plants have been a revolutionary breakthrough in the treatment of male sexual dysfunction. traditional medicine based on a holistic philosophy is quite different with the practice of “western” medicine. phytochemical substances focus their mechanisms of healing to the root of cause, i.e. the inability of controlling the proper function of the whole body system. hence, medicinal plants manage sexual dysfunction and male fertility in the frame of sexual dysfunction as a whole entity. some previous researches prove that the use of medicinal plants have a good impact in the treatment of a variety of male sexual problems. this paper will discuss several important aspects of aphrodisiac plants and preliminary study regarding them in indonesia. difficulties inherent to activity guided isolation and the specific requirements of bioassays are also discussed. keywords: medicinal plants, male sexual problem, traditional medicine introduction male sexual dysfunction is a very serious problem. (mackay, 2004; clourate, 2005). approximately 10% men are infetile, and 50% infertile couple are from male factors. (pei et al., 2005). the medical treatment for male sexual problem has not succeeded yet although many efforts have been done (hamadeh, 2001; kohn, 2001). the existed modern methods, like assisted reproductive technology (art), need high cost while the result is not consistent (orgebin-crist, 1998), even the methods have side effects (levie’vre et al., 2007). people have used plants as medicine since the beginning of human civilization, even probably have been used before they evolved to be man, as shown in the practice of using medicinal plants by non-human primates (newton, 1991). one of the use of medicinal plants is to overcome sexual dysfunction. indonesia is one of the mega-biodiversity countries. there are many potential plant as aphrodisiac agent. however there is little scientific evidence supporting the effectiveness of using aphrodisiac plants. besides that, there is still less studies of the action mechanism and its active components. the profound research of the effect of plants towards male reproductive system will support the use and the effectiveness of the medicinal plants. the paper will discuss about several important issues of medicinal plants to cure male sexual problem covering the characteristics, the research, and the development of the medicinal plants. those plants include aphrodisiac plants and those which function to improve the fertility. it also discusses about the research of several plants used by local people. the research of drug from plant sources plants is a main source of new drug discovery (balandrin et al., 1985), approximately 40% modern drugs are from botanical substances (lafrance jr. et al., 2000). however, recently many pharmaceutical industries have reduced plant drugs studies. compared with synthetic drugs, plant drugs are not suitable for high-throughput screening. the study and evaluation of plant drug are very complicated, time-consuming, and more expensive compared to conventional drugs (cordell,. 2000; etkin, 2000; balunas & kinghorn, 2005; cordell & colvard, 2005; who, 2005). it is believed that combinatorial chemistry will be a the main source of synthetical medicine in the future (simmonds & grayer, 1999) . in fact, however, combinatorial chemistry fails to provide a model for drug structure in several medical treatment (simmonds, 2003; butler, 2004), whereas the plants still provide more various structures than combinatorial chemistry (muller, 2004). it is assumed that plants, as chemical factories, have continuously evolved their biosynthetic programs for more than 400,000 million years. as long as evolutionary process, these plants synthesize compounds in which the structural variety is far beyond the imagination of synthetical chemistry experts. it is really possible that the evolutionary pathway of secondary metabolite produces compounds enabling to cure diseases that cannot be healed by conventional medication. (simmonds & grayer, 1999). plants as a treatment for male sexual problem environment is one of crucial factors affecting human fertility. contrast to infertility caused by genetical 42 biology, medicine, & natural product chemistry 4 (2), 2015: 41-47 factors, infertility caused by environmental factor have possibility to cure and prevent (quallich, 2006). one of the most important environment factors is nutrition (ebisch et al., 2005). in this prespective, a traditional medicine using medicinal plant is very promising. some research has shown a positive effect of nutrition in spermatogenesis. recent studies show medicinal plant potency in improving male fertility and aphrodisiac on animal (sinclair, 2000; mackay, 2004). the improvement of sexual function has been proven through the use of medicinal plants, like ginseng, yohimbe, tribulus terestis, and maca (burkill, 1965; waddell et.al., 1980; lewis & elvin-lewis, 2003). the traditional medication that is orally done by drinking the steeped water from the parts of the plant has been practiced over hundred years ago. in a medical treatment, the oral medication of sexual problem is a new thing. viagra, the first pill to heal impotence, had just been launched in 1998. this medicine is a revolution in medical treatment, in which the previous medication was treated only by injection and operation. (eardley, 1998; mulhall, 2000; elferink, 2000; morales, 2001). however, the development of the plant research is very slow. it happens because many researches conducted by pharmacy industries depend on the patent to get the profit. plants cannot be patented therefore the industries are reluctant to invest their money to prove that certain plants are safe and effective (foster & duke, 2000). there are no plants or botanical derivation compound that are legally certified by food and drug association or other similar institutions to be used as a medication of sexual dysfunction (sinclair, 2000; nickell, 2001; mackay, 2004). the difference between traditional medicine and modern medicine in the medical world in america and other developed countries, there is just a single chemical compound that is recognized as a medicine, not in the form of mixed chemical components existed in the plants. this recognition is preferably caused by the drug regulations and laws, not by the scientific consideration (foster & duke, 2000) or philosophical belief that medicine works as ‘one-target-one-disease’ (adimoelja, 2000; wermuth, 2004). a dominant belief indirectly puts minor beliefs aside, consequently it reduces the supports towards scientific fields based on different philosophies (kuhn, 1996). modern medicine relies on the view that a disease is caused by a very specific pathogen invaded to the body therefore the healing must be done by eradicating the source of the disease (adimoelja, 2000). the medication using medicinal plants uses holistic approach by investigating the disease up to the main origin causing the unbalancing and deficiency in the body function (cracker & giblette, 2002). the whole body functions caused by the synergy and the existence of individual actions from body parts are the characteristics of living creatures therefore the reductionist view of the disease concept, that just blames an organ or a certain isolated mechanism, is an incomplete approach (rangel, 2005). the model of medicinal plant study nowadays, in vitro test for screening pharmaceutical agent is a main choice for many pharmacy companies but the success of the effort is limited on the beginning of the drug discovery program. this test has not yet fully replaced the use of animals in many toxicology studies and pharmacology experiments (white, 2001). the understanding on molecular level cannot be automatically applicated to show that the medical strategy is effective. in vitro physiological reaction of a pharmaceutical can have an inaccurate prediction of in vivo physiological reaction. many pharmaceutical agents show off different activities in various cell population. that ails in predicting the whole effect of giving an agent in the body (walsh, 1998; barton & andersen, 1998; jobe et al., 1994). in vitro data is indirectly equivalent with in vivo data although in vitro test can provide the foundation to determine the study objective. the studies are continuously conducted to ensure how far in vitro data can replace in vivo data (rodrigues, 1997). the drug discovery program is basically based on the use of the experimental animals to determine the pharmacological effect and the compound/chemical metabolism (briggs & oehme, 1980). an experimental model is important from a clinical point of view because there are many aspects of human physiological and biological reproduction that cannot be studied directly (plant & marshall, 2001). a conventional test of medicinal plants using rodensia as an experimental animal model is really needed before pre-clinical and clinical tests can be conducted (farnsworth, 1992). the result is often the only reference to determine the drug development from the pre-clinical test (greaves, 1990). a rodent as an experimental animal has an important role in screening chemical agents to study the pharmacological effect covering the distribution, mechanism, and toxicity (briggs & oehme, 1980). evaluation of the effect of an agent towards a male reproduction system generally uses rodents as an animal model. the wide use of mouse species in the research has produced complete biology data. the research on fertility and male reproduction is conducted using various methods taken from toxicology, medical, ecology, and epidemiology (golden, 2002). bioassays for bioactive compound evaluation the combination of chemical and biological screening is the quickest method to get an active compound of the plant. to get this, the bioassays provision or simple pharmacological test is very important to focus on certain activities from the plants or various plant fraction as a guidance to obtain pure active component(s). this bioassays must be very sensitive because the active substance in the plants may be in a very low quantity. this muhammad ja’far luthfi, et.al. – medicinal plants: a prospect in developing male … 43 bioassays must also be specific towards the desired target. the main target for biology test can be divided into six groups, namely:  low organisms: microorganism  invertebrata: insects, crustacean, mollusca  isolated sub-celullar system: enzyme, receptor  animal and human cell culture  isolated organ  intact animal most bioassays are used in in vitro test in cells or subcells and the low animal test. in vivo test in complete animals is less conducted and less interested because of ethical issue and it is time-consuming (hawcroft et al., 1987; hamburger & hostettmann. 1991; hostettmann & marston, 2002; preusch, 2004). determining a simple and quick bioaassay to evaluate plant for improving sexual function is very difficult. reproduction system is a very complicated system having existed complex interactions in the level of organs, cells and sub-cells. moreover, there are many aspects of reproduction system that have not been studied and not certainly known (kierzenbaum, 1994; de kretser & baker, 1999; liska, 2003; huggins, 2003; lopez-gatius, 2006). because of that, it is quite difficult to find simple and quick in vitro bioassays in those fields – those are not always provided. the only bioassay, that are reliable to test plants in improving sexual function, is in vivo test in intact animal although the test is time-consuming and expensive. bioassays offer an huge advantage in standardization and quality control of plant based product. the products are heterogen because there are a mixture of bio-active components from both a plant and mixture of several plants. the physical analysis method, like chromatography, cannot be used for this purpose because it is not sensitive towards the chemical complexity existed in plant crude extracts. it often happens that the desired biological activities are not evoked by single plant component but a mixture of various plant components therefore just relying on physical and chemical analyses towards a single component on a mixture is not really satisfying (mclaughlin, 1998). unfortunately the objectives of many phytochemist is just isolate, characterize and publish various new compounds without testing their bio-activities. to attain practical uses, natural chemical substances must combine bioassays inside those substances. the extracts must be screened for biological activities, active extracts must be chosen, fraction must be directed to bioassays, bioactive compounds are finally identified and exploited (mclaughlin, 1998). experiment on fertility and male sexual function the researches conducted by luthfi et al (2008) and mat noor & luthfi (2006) on tongkat ali (eurycoma longifolia), sanrego (lunasia amara), ginger (alpinia galanga), dan clove (syzygium aromaticum) show the potency of aphrodisiac and sanrego to improve man fertility. this study uses rat as tested animals. rats are divided into two groups for different plant extracts, namely every group is given plant extracts in dose 3.33 mg/ml and 333 mg/ml each and control group is given distilled water. plant extracts or distilled water are given through force feeding once a day at 11.00 a.m. for 50 days. after having given plants extraxts for 50 days, the rat are sacrificed using chloroform and the epididymis were dissected out. cauda epididymis were separated according to the hamilton (1975). sperm counts were determined using improve neubauer hemocytometer as described previously (prasad et al., 1972; nafa & eshre-siga, 2002) with some modification. in brief, cauda epididimis was minced in 15 ml bww medium (biggers et al., 1971) and incubated with 5 % co2 for 30 minutes at 370c. data were expressed as number of sperm per cauda epididymis. progressive sperm motility was assesed subjectively based on who laboratory manual (1999). a study of sperm morphology is conducted by providing three slides of sperm smear for each rat. after fixated in methanol, the slide were stain with giemsa. the dry slide is observed in a light microscope. one hundred sperms are counted randomly from every slide. the percentage of morphology of normal/abnormal sperms is determined referring to criteria stated by wyobek and bruce (1975). table 1 shows the number of each rat sperm which has been given aphrodisiac, sanrego, ginger and clove for 50 days. the average number of mouse sperms treated by aphrodisiac extracts at 333 mg/ml dosage (46.23±1.77) and sanrego extracts at 333 mg/ml dosage (47.30 x 106 + 3.47) show that there is a significant improvement (p < 0.05) compared with the average number of sperms given ginger, clove extracts and control (distilled water). however, the intake of tongkat ali and sanrego extracts at 3.33 mg/ml dosage does not show any significant different in sperm number. a statistical analysis does not show any significant difference between groups treated with ginger extract, clove extracts, and control. the morphology analysis result of rat sperm shows that the percentage of sperm with normal morphology in treated rat group and control group exceeds 95%. a statistical analysis using turkey test shows that there is no significant difference (p > 0.05) between control group and a treated group from the percentage of formal morphology sperm. table 1. sperm number (x106) and percentage of normal morphology sperm a of rat group given aphrodisiac, sanrego, ginger and clove extracts each at 3.33 mg/ml and 333 mg/ml dosage compared to control group. treatment dosage sperm number (x 106) % normal morphology sperm tongkat ali 333 mg/ml 46.23±1.77 96.24±1.16 3.33 mg/ml 34.16±3.37 96.8±0.70 44 biology, medicine, & natural product chemistry 4 (2), 2015: 41-47 sanrego 333 mg/ml 47.30±3.47 96.66±0.80 3.33 mg/ml 38.43±3.09 96.04±1.10 ginger 333 mg/ml 27.21±2.76 95.70±1.39 3.33 mg/ml 29.21±4.46 96.83±0.21 clove 333 mg/ml 31.67±1.91 96.48±1.35 3.33 mg/ml 32.43±2.76 96.71±0.47 control distilled water 33.17±4.25 96.67±0.63 the effect of aphrodisiac, sanrego, ginger and clove on sperm motility is shown in table 2. a sperm motility analysis shows that tongkat ali and sanrego have the best effect in improving sperm movements compared with groups given clove, ginger extracts and control group (distilled water). the treatment of aphrodisiac and sanrego at high dosage (333 mg/ml) shows that 80% of sperm movements are in the a grade while control group shows only 40% of sperms movements are in the a grade. a group treated using ginger extracts at 333 mg/ml dosage on the contrary shows the lowest level of sperm motility, namely 80% in the level c. sperm motility has been considered as one of the most important predictors of fertility. several reports have demonstrated the correlation of motion parameters with fertilization rates (liu et al., 1991). further studies are required to confirm the mechanisms of action of tongkat ali and sanrego on sperm motility. table 2. sperm motility grade in the treated group given aphrodisiac, sanrego, ginger and clove extracts each at 3.33 mg/ml and 333 mg/ml dosage compared with a control group. motility grade (a-d) treatment dosage individual rat 1 2 3 4 5 tongkat ali 333 mg/ml a a b a a 33.3 mg/ml a a a b a sanrego 333 mg/ml a a a b a 33.3 mg/ml a a b b a ginger 333 mg/ml c c c b c 33.3 mg/ml b c c b b clove 333 mg/ml b b b b b 33.3 mg/ml b b b b c control distilled water b a b b a note for the grade of motility who (1999) (a): rapid progressive motility ; ≥ 25 μm/s, (b): slow or sluggish progressive motility, (c): non progressive motility ; < 5 μm/s, (d): immotile. this study shows that cloves and gingers do not improve the parameter of male fertility, namely the numbers of sperms, the movements of sperms, and the normal morphology of sperms. both plants on the contrary decrease the number of sperms although it is not significant (p> 0.05). the report written by jaganath and ng (2000) shows that gingers can improve sperm number and sperm motility is incompatible with the result of this study. this result is difficult to compare because there are differences of tested animals and there may be differences in providing extracts that are not explicitly stated in detail in the report. a study conducted by luthfi and mat noor (2007) also shows that sanrego increase sexual behavior of male rat. table 3 shows the aphrodisiac effect in male rat after they are given sanrego at a certain dosage (the treatment group) and distilled water (a control group). the 60 mg/kg dosage shows that the number of mounting is increase significantly compared with other groups. the numbers of mounting in the group using 60 and 90 mg/kg dosages do not show any significant differences. the decrease of mounting number at 90 mg/kg dosage is difficult to explain with the data from the result of the study. it may be caused by the limited number of tested rat (sample). hayes (2001) stated that the more number of mice, the more consistent the result. the research is still conducted to isolate active compounds from sanrego that can improve aphrodisiac or male fertility. table 3. the average mounting number of male rat after treating sanrego extract using various dosages. treatment dosage the average of mountingnumber + se kontrol 1.33 + 1.54 30 mg/kg 2.33 + 0.57 60 mg/kg 3.33 + 0.57 90 mg/kg 3.00 + 0.00 the early research on purwoceng (pimpinella pruatjan) also shows its potency as aphrodisiac herb (tambunan, 2005). the root of purwoceng can be used as aphrodisiac, diuretic and tonic herbs. at this moment, the information about bioactive content in purwoceng has not been known yet. some bioactive compounds that have been identified are stigmaterol and sitosterol but no one has reported the use of the single compounds. muhammad ja’far luthfi, et.al. – medicinal plants: a prospect in developing male … 45 the direction of the medicinal plant development in the future most research synthetic organic and natural compounds are conducted by pharmaceutical company and industries. the research is focused to look for new and more powerful compounds. in the university, a study must not always be directed to find new compounds but the study can also look for substances showing certain biological activities that may help in understanding on physiological effects. the study of medicinal plants to treat sexual dysfunction is ideally conducted using an experimental approach that is wellknown reliable to find bioactive compounds, that is bioassay-guided isolation. plants are chosen based on ethnopharmacology criteria, then the plants are extracted. extracts produced are tested using relevant bioassays system. after biological activities are determined, active extracts are fractionated and their biological activities are monitored for each level of fraction. active isolat elucidation is finally conducted. there are several possibility if extraxts and fractions show bioactivity but each compound is not active. the first possible reason is that there is a synergy effect in those compounds. bioactivity emerges because there is a synergy among those compounds (williamson, 2001) but there is another possible reason that there are some components that have not been isolated. it occurs because a single plant extract can contain hundreds components from various different groups, either primary and secondary metabolit. those group are isoprenoid, fenol, lipid, cabohydrate and its derivations, amino acid and mineral. all those subtances have possibilities to be bioactive (wildman, 2001). in this case, in a research of plant having a potency to improve fertility, it has a slight possibility to get active components because in vivo testing needs more numbers of components to test. in that case, a pharmacological testing to test extracts that produces standardized extraxts can be conducted. after a study of toxicity and safety of the extract are conducted, the standardized extracts are formulated. then, a product can be developed. this product can be a first step for an innovative local pharmaceutical industry and it can compete with western pharmaceutical industries, not only for common diseases but also for severe diseases (pieters and vlietinck, 2005). conclusion a number of medicinal plants show the promising potency in treating male sexual problems. medicinal plants have their own characteristics that cannot fully fulfilled the research methods of conventional medicines. the results of the research show that some practices using medicinal plants can validly healing male sexual dysfunction and it can be improved in order to have a cheap effective safe therapy of the disease. it is hoped that the wide spread of the botanical medicine interest and knowledge will reduce obstacles in using and developing the medicinal plants. references adimoelja, a. 2000. phytochemicals and the breakthrough of traditional herbs in the management of sexual dysfunctions. int. j. androl. 23, suppl.2: 82-84 aminudin, n. 2004. eurycoma longifolia jack aqueous extract: bioactive compound and its effects toward hormone production and spermatogenesis. doctorate thesis. institute of biology science, faculty of 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antioxidant activities of schefflera elliptica leaves i gede yoga ayuning kirtanayasa1, anak agung gede indraningrat2,*, i putu candra1 1food science and technology study program, faculty of agriculture, warmadewa university 2departement of microbiology and parasitology, faculty of medicine and health sciences, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* indraningrat@warmadewa.ac.id abstract schefflera elliptica or locally called kayu tulak, is one of the balinese herbal plants that have traditionally be used to reject (tulak) negative influences that exist in the human body. althoug, s. elliptica has been routinely used as a part of a ritual in bali, only a limited study has been reported on its bioactivity. this study was designed to analyze the phytochemical content, antibacterial activity, and antioxidant activity of s. elliptica. in this study, simplisia of s. elliptica was extracted using n-hexane and ethyl acetate solvents, then the viscous extracts of the two solvents were carried out for phytochemical tests, antibacterial activity tests with the kirby-bauer method and antioxidant activity tests with based on dpph method. phytochemical screening showed that the n-hexane extract contains active compounds in the form of phenols and steroids while ethyl acetate contains active compounds in the form of phenols, tannins, and steroids. antibacterial screening showed ethyl acetate extract of s. elliptica displayed a diameter zone of inhibition of 10.72±0.71 mm against staphylococcus aureus, 12.17±2.80 mm against streptococcus mutans, 12.40±1.65 mm against escherichia coli and 15.20±2.44 mm against klebsiella pneumoniae. the dpph analysis showed percentages of 61.17% and 67.42% from n-hexane and ethyl acetate extracts respectively, which indicated the antioxidant properties of s. elliptica. overall, this research provides a preliminary report on the bioactivity potential of s. elliptica mainly in term of antibacterial and antioxidant properties which open up possibilities for future drug development. keywords: antibacterial; antioxidant; phytochemical; schefflera elliptica. introduction indonesia is one of the countries with high plant diversity. the biological diversity of flora in indonesia, especially seeded plant species reaches 30,000–40,000 types, or equivalent to 15.5% of the total number of plants in the world (widjaja et al., 2014). furthermore, more than 2,039 plant species are categorized as medicinal plants (zuhud, 2009). among many islands in indonesia, bali is one of the islands with rich biological diversity and balinese people have applied herbal plants for traditional medicine (sutomo and iryadi, 2019). in general, information about traditional balinese medicine has been recorded in a manuscript called lontar usada bali, which explains the function of each herbal function and the procedure to use it (oktavia et al., 2017). schefflera elliptica or locally called kayu tulak is one of the types of local balinese plants listed in usada tiwang (arsana et al., 2020). this plant is commonly found in the traditional balinese offerings called banten byakala has a function to reject (tulak) any impurities or negative influences that exist in the human body (puspa et al., 2019). more specifically, the leaf part of kayu tulak is used as a repellent for disasters or bad luck in human body (hanum, 2011). schefflera elliptica leaves can also traditionally be used as a remedy for skin diseases and fractures (sivaperuman et al., 2018). methanol and ethyl acetate extracts of s. elliptica leaves have also been reported to have antibacterial activity against staphylococcus aureus (purwantoro et al., 2009). however, apart from antibacterial against s. aureus, no other studies have been published on the antibacterial activities of s. elliptica extracts against other gram-positive and gramnegative bacterial species. in addition, rather limited information is available on other aspects of the bioactivities of s. elliptica e.g. phytochemicals and antioxidant activities. this present study aimed to assess the bioactivities s. elliptica leaves focusing on three main aspects namely phytochemical, antibacterial, and antioxidant bioactivities. the obtained information is expected to give further knowledge on the bioactivities of s. elliptica which could be the basis to develop the ethnomedicine and drug development purposes of the plant. manuscript received: 04 march, 2023. revision accepted: 24 march, 2023. published: 01 april, 2023. https://doi.org/10.14421/biomedich.2023.121.329-334 330 biology, medicine, & natural product chemistry 12 (1), 2023: 329-334 methods sample collection and determination leaves samples of s. elliptica plant were obtained from gerih village, bali, indonesia on february 2022 (figure 1). samples were selected by taking 3-5 leaves of s. elliptica which were calculated from the leaflets, provided that it was in full bloom, fresh, without hollow, and free of insect infections. plant determination was performed by sending fresh and dried vouchers to the characterization laboratory of the botanical garden "eka karya" bali – national research and innovation agency (brin), candikuning, tabanan, bali. the purpose of plant determination is to obtain a clear identity of the plant under study and avoid errors in collecting the main research material. sample preparation and extraction two kilograms of s. elliptica leaves were washed in running tap water to remove debris. subsequently, leaves samples were drained, and dried using an oven at 40oc. the dried simplisia was dried and were sorted by separating foreign objects that occurred during drying. the dried simplisia was turned into powder using a blender and was sieved with a 60-mesh sieve. finally, the powder was stored in a clean glass jar to prevent contamination and other impurities before extraction. two types of crude extracts were prepared using two different chemical solvents namely ethyl acetate (smartlab) dan n-hexane (merck). for each of solvent, 100 gram of dry powder leaves of s. elliptica was macerated with a ratio of 1:5 (w/v) (wijaya and indraningrat, 2021). maceration was carried out for 24 hours and each mixture was stirred every five minutes with a time span of six hours. for each of the solvent, remaceration was carried out after 24 hours. subsequently, after maceration and remaceration were completed, each macerate was separated from the residue using a vacuum filter, followed by evaporation in a rotary evaporator (ika rv 10, germany) at a speed of 100 rpm at a temperature of 40ºc. figure 1. s. elliptica plant (left), and s. elliptica leaves (right). phytochemical screening each the ethyl acetate and n-hexane s. elliptica crude extracts was tested to detect the presence of a group of compounds based on the following methods. phenol one ml of each s. elliptica extract was transferred into a test tube followed by the addition of 2 – 3 drops of iron (iii) chloride (fecl3) 5%. the presence of phenols was indicated by a blue-black color (friany et al., 2017). flavonoids one ml of each s. elliptica extract was transferred into a test tube. subsequently, 2 mg of magnesium powder and 3 drops of concentrated hcl were added to the tube. the mixture was shaken and the formation of a red, yellow, or orange color on the solution indicated the presence of flavonoids (purwati et al., 2017). tannins one ml of each s. elliptica extract was mixed with a few drops of 10% iron (iii) chloride (fecl3) solution. a dark blue or greenish-black color indicates the presence of tannins in a solution (baud et al., 2014). alkaloids two ml of each s. elliptica extract was mixed with 3 drops of concentrated hcl and 5 drops of mayer reagent. a white precipitate indicates that a sample contains alkaloids (ergina and pursitasari, 2014). steroid/terpenoid two ml of each s. elliptica extract were mixed with liebermann burchard reagent, a mixture of concentrated hcl and concentrated h2so4. positive results were indicated by the presence of a red-orange color for triterpenoids and blue for steroid (sangi et al., 2008; ergina and pursitasari, 2014). saponin two ml of each s. elliptica extract were mixed with 10 ml of aqua dest. the mixture was shaken for 1 minute and subsequently, two drops of hcl 1 n was added. the presence of a stable foam for approximately 7 minutes indicated that the mixture contains saponins (mondong et al., 2015). antibacterial activity screening for each ethyl acetate and n-hexane extract, paper discs with a diameter of 6 mm were prepared. each paper disc was soaked into a viscous extract and allowed to dry for 15 minutes until the extract was evenly absorbed. paper discs containing extracts were transferred into luria bertani agar which already contained one of the following test bacteria namely staphylococcus aureus atcc 25923, streptococcus mutans fncc 0405, escherichia coli atcc 25922 and klebsiella pneumoniae atcc 700603. triplicate paper discs of kirtanayasa et al. – phytochemical, antibacterial and antioxidant activities of … 331 each extract were used for each of the tested bacteria. nalidixic acid (oxoid) paper discs were used as a positive control, while ethyl acetate and n-hexane were used as negative controls. antibacterial activities were calculated based on the triplicate average zone of inhibition (zoi) that were formed on each of the lawn bacterial species. antioxidant assay the radical inhibition activity of the sample was carried out based on its inhibition against free radicals 1, 1diphenyl-2-picrylhydrazyl (dpph) (pangestuty, 2016). sample absorbance was measured with a uv-vis spectrophotometer. the magnitude of antioxidant activity was measured by the following formula: antioxidant activity (aa%) = 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100 result and discussion phytochemical screening results phytochemical screening is a preliminary test and qualitative analysis that can be used as initial information about a group of compounds present in a plant. in this research, phytochemical screening of n-hexane and ethyl acetate extracts of s. elliptica were summarized in table 1. in general, there were discrepancies in the presence of specific groups of compounds by comparing the phytochemical constituents of each extract. alkaloids tests showed clean deposits were formed in both extracts. this result indicated the absence of alkaloids in ethyl acetate and n-hexane. a positive result was indicated by the presence of white deposit (ergina and pursitasari, 2014) table 1. phytochemical composition of s. elliptica extracts. test extract n-hexane ethyl acetate alkaloid flavonoid tannin + phenol + + saponin steroid + + terpenoid the presence of flavonoids in n-hexane and ethyl acetate extracts was tested using two methods, namely by adding concentrated magnesium (mg) + hydrochloric acid (hcl) and using h2so4. positive signs were indicated by a change of color in the mixture to red or orange. in this study, n-hexane and ethyl acetate samples did not change color to red or orange, so it was concluded that flavonoids were absent in both extracts (friany et al., 2017; purwati et al., 2017). a positive test of tannin was indicated by the formation of blackish-green color (baud et al., 2014). the occurrence of this green color change is due to the reaction between fe metals and tannins to form complex compounds due to the presence of coordination covalent bonds between metal ions or atoms and non-metallic atoms (effendy, 2007). our result indicated that tannin was present in ethyl acetate extract based on the color change that was observed. meanwhile, no color change was observed from the n-hexane extract. this could happen mainly because n-hexane is a solvent that has non-polar properties while tannins are polar compounds and tend to dissolve in polar or semi polar solvents (muthmainnah, 2017). the presence of phenol was screened by adding 10% of fecl3 reagent and positive signs were indicated by a color change to blackish-green (friany et al., 2017). color changes to green-black were observed from both s. elliptica extracts to indicate both extracts contained phenol compounds. the discoloration of n-hexane and ethyl acetate extracts of s. elliptica was because phenol compounds can dissolve in polar solvents as well as nonpolar solvents (wongso, 2014). in the saponin test, n-hexane and ethyl acetate extracts of s. elliptica were heated followed by addition of 10 ml of aquadest. the mixture was shaken strongly and a positive sign was indicated by the presence of a stable foam of 1-10 cm high which was stable after the addition of 1 drop of hcl 1n (mondong et al., 2015). the appearance of foam was present because saponin compounds have physical properties that are easily hydrolyzed in water so that saponin compounds will cause foam when shaken. in both n-hexane and ethyl acetate extracts of s. elliptica the saponin test did not show positive results because the foam formed after shaking only lasted for a few seconds. the presence of steroids and terpenoids were screened by the addition of liebermann-burchard reagents. the positive result for steroid compounds was indicated by the color change to blue or purple while for triterpenoid compounds was indicated by a brownish-red color (sangi et al., 2008; ergina and pursitasari, 2014). the test results showed that n-hexane and ethyl acetate of s. elliptica extracts underwent a blue color change so that they were positive for containing steroid compounds. antibacterial activities screening the antibacterial screening showed that the crude extractof n-hexane displayed lower antibacterial activity against test bacteria compared to the crude extract of ethyl acetate (figure 2, table 2). ethyl acetate extract has an average zoi of 10.72±0.71 mm against s. aureus. ethyl acetate extract had an average zoi of 12.17±2.80 mm against s. mutans. meanwhile, in the ethyl acetate extract has an average zoi of 12.40±1.65 mm against e. coli and an average zoi of 15.20±2.44 mm against k. pneumoniae. 332 biology, medicine, & natural product chemistry 12 (1), 2023: 329-334 figure 2. antibacterial activities of n-hexane and ethyl acetate extract againts bacteria. annotations: h = n-hexane, e= ethyl acetate; sa = staphylococcus aureus atcc 25923; sm = streptococcus mutans fncc 0405; ec= escherichia coli atcc 25922; kp=klebsiella pneumoniae atcc 700603 table 2. antibacterial activities of s. elliptica extract against testing bacteria. diameter zone of inhibition of each treatment (ethyl acetate, nhexane, positive and negative controls) were calculated from triplicate samples. bacterial strains samples solvents zoi (mm) s. aureus s. elliptica n-hexane ethyl acetate 10,72±0,71 + control nalidixic acid 15,34±0,03 control n-hexane ethyl acetate s. mutans s. elliptica n-hexane ethyl acetate 12,17±2,80 + control nalidixic acid 16,29±0,12 control n-hexane ethyl acetate e. coli s. elliptica n-hexane ethyl acetate 12,40±1,65 + control nalidixic acid 16,58±0,04 control n-hexane ethyl acetate k. pneumoniae s. elliptica n-hexane ethyl acetate 15,20±2,44 + control nalidixic acid 15,50±0,13 control n-hexane ethyl acetate when compared to the results of the study of purwantoro et al, (2009) which stated that the ethyl acetate extract of s. elliptica at a concentration of 50 μg/ml, 100 μg/ml, and 200 μg/ml had a zoi with an average of 7.5 mm against s. aureus and e. coli bacteria, the crude extract of ethyl acetate with a concentration of 100% had a bigger zoi against s. aureus and e. coli bacteria. the difference in inhibitory power can occur due to the difference in the concentration of the extract where the higher the concentration of the extract, the higher the inhibitory power will be. the same thing is also stated by (zuhud et al., 2001) who mentioned that the higher the concentration of the extract, the more the amount of antimicrobial compounds released will be, thus facilitating the penetration of compounds into cells. davis and stout, (1971) classified the diameter zone of inhibition into four categories, namely weak, medium, strong, and very strong. zones of inhibition with a diameter of ≤5 mm are categorized as weak, 6-10 mm are categorized as medium, 11-20 mm are categorized as strong, and above 20 mm are categorized as very strong. based on these categories, the zoi formed by ethyl acetate extracts of s. elliptica could be considered as a strong activity. however, the observed antibacterial activity was still lower compared to positive control nalidixic acids. nevertheless, the observed antibacterial activity from ethyl acetate extracts provides a valuable insight on bioactive compounds that present in s. elliptica leaves. the presence of antibacterial activities in the ethyl acetate extract of s. elliptica may occur due to the content of its secondary metabolites. the mode of action of phenol compounds is in general by denaturing cell proteins. hydrogen bonds formed between phenol compounds could damage protein layers in cell structures (bontjura et al., 2015) meanwhile, tannin was also reported to display have antibacterial activity against gram-positive and gram-negative bacteria by entering the cell wall (kaczmarek, 2020). tannin forms hydrogen bonds with bacterial cell’s proteins and subsequently hydrogen bonds formed between tannins and proteins will disrupt bacterial cell walls (mailoa et al., 2014). antibacterial screening showed that the zone of inhibition of ethyl acetate extract of s. elliptica against gram-positive bacteria (s. aureus and s. mutans) was smaller compared to gram-negative bacteria (e. coli and k. pneumoniae). such discrepancies could probably happen because the cell wall of gram-negative bacteria is thinner compared to gram-positive bacteria so the protein structure in the cell wall of gram-negative bacteria was damaged by the presence of tannin compounds (mailoa et al., 2014). furthermore, grampositive bacteria have a thick and rigid peptidoglycan layer while gram-negative bacteria have a thinner peptidoglycan layer (sudarmi et al., 2017). the absence of zoi observed in n-hexane extracts even though qualitatively the extract contained phenol compounds could be influenced by differences of the polarity of solvents in extraction which affecting the total content of bioactive compounds in the extract (santoso et al., 2012). the total content of phenol compounds according to hidayah et al., (2017) has an influence on antibacterial activity as the higher the levels of oxidized phenol compounds, the stronger the antibacterial activity kirtanayasa et al. – phytochemical, antibacterial and antioxidant activities of … 333 will be. the extract of n-hexane s. elliptica did not display a zone of inhibition power even though it qualitatively contained steroid compounds. such a condition could happen because lipids have large sizes of molecules which interfere with the diffusion process and consequently, n-hexane extracts are unable to inhibit bacterial growth (naufalin et al., 2005). antioxidant activities test results antioxidant activities were analyzed based on the 2,2diphenyl-1picrilhydrazyl (dpph) method. in theory, the higher the concentration of antioxidant activity added to the dpph solution, the more the absorbance value will decrease (sapri et al., 2013). the absorbance value of nhexane and ethyl acetate s.elliptica samples (table 3) showed that the antioxidant activities (aa) of n-hexane extract was 61.17% while for ethyl acetate extract was 67.42%. according to rahmawati (2004) in (parwata et al., 2009) the inhibition value of 0% means that an extract has no antioxidant activity and the inhibition value of 100% means total dampening. an extract could be classified as an active antioxidant when its inhibition percentage is more than or equal to 50%. therefore, based on this criteria, crude extracts of n-hexane and ethyl acetate of s. elliptica are classified as active antioxidants. table 3. antioxidant percentages of s. elliptica crude extracts. sample crude extracts concentrations aa% s. elliptica n-heksan 1000 ppm 61,17 etil asetat 1000 ppm 67,42 factors that affect the absorbance are the type of solvent, the ph of the solution, the temperature, the high concentration of the solution, and the presence of a disruptive substance. the amount of antioxidant activity of n-hexane and ethyl acetate extracts from s. elliptica is due to the content of secondary metabolites such as phenols, steroids, and tannins in the extract. tannins are compounds composed of polyphenols that have free radical capture activity. the more tannin content contained in the extract, the more antioxidant activity (malangngi et al., 2012). according to (amarowicz, 2007) tannins not only function as primary antioxidants but also function as secondary antioxidants. phenolic compounds have the ability to contribute hydrogen atoms or electrons to free radicals. this process converts phenols into phenoxyl radicals that can stabilize themselves so that there is no radical formation reaction (pangestuty, 2016; diniyah and lee, 2020). conclusions in conclusion, this study confirmed antibacterial and antioxidant activities from s. elliptica crude extracts. in addition, s. elliptica leaves contain phytochemicals in the form of phenol compounds, tannins, and steroids. the selection of solvents seems to play an important role in extracting bioactive compounds from s. elliptica leaves. in terms of antibacterial activity, ethyl acetate which is a semipolar solvent seems more suitable to extract the active antibacterial substances compared to n-hexane which is a non-polar solvent. further studies should be focused to explore the ideal solvent for chemical extraction. comparison of polar, semipolar, and nonpolar solvents for extraction of s. elliptica should also be done to provide an ideal comparison of bioactivities. in addition, antibacterial screening should also be expanded against multi-drug resistance bacteria and fungi. further studies should also explore other aspects of bioactivity screenings such as cytotoxicity, anti-larvicide, and anticancer tests of s. elliptica to elucidate possible untapped bioactivities of the plant. acknowledgments: the author would like to thank the research unit of the faculty of medicine and health sciences at the university of warmadewa denpasar-bali for providing a research grant to perform this research under grant number: 227/unwar/fkik/unitpenelitian/pd-13/ix/2022. competing interests: the authors declare that there are no competing interests. references amarowicz, r. 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(2001) ‘aktifitas antimikroba ekstrak kedawung (parkia roxburghii g. don) terhadap bakteri patogen’, jurnal teknologi dan industri pangan, pp. 6–12 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 295-303 | doi: 10.14421/biomedich.2023.121.295-303 issn 2540-9328 (online) effect of quarry activities on some morphological parameters of two maize varieties (swan 1 and sammaz 52) bridget odiyi, olubukola maku, foluso akinbode ologundudu*, sylvanus efetobor abiya department of biology, school of life sciences, federal university of technology, akure, nigeria. corresponding author* akinbodefoluso@gmail.com manuscript received: 11 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract the effect of quarry activities on some morphological parameters of two maize varieties (swan 1 and sammaz 52) was investigat ed with the aim of determining the impact of quarry activities on some growth parameters of the maize varieties under study. the seeds were collected from the seed bank department of the ondo state ministry of agriculture, akure, ondo state. they were authenticated at the herbarium unit of the federal university of technology, akure, and the voucher was deposited. soil sampl es were collected at 50m, 100m, 150m, 200m, and 250m from the quarry site and transferred to the laboratory for analysis. a screen house experiment was set up to house the pots. seeds of swan 1 and sammaz 52 were sown into perforated plastic pots (30 cm diameter and 33 cm depth) filled with 10 kg of quarry soil. the following morphological parameters were determined; shoot height, leaf area, plant dry weight, shoot dry weight, root dry weight, root-shoot ratio, leaf area, and determination of photosynthetic parameters especially chlorophylls a and b. the result revealed that at 50 meters from the quarry site, sammaz 52, one of the maize varieties grown in soil taken from the site, had the highest shoot height (94 cm). which showed that plants growing in higher concentrations of dust pollution respond to nutrient stress by devoting more of their available carbon to shoot growth, resulting in elongated stems, were consistent with the observed high er shoot height in sammaz 52, daily variations in photosynthetic activity and the rate of nitrogen uptake are to blame for these alterations in plant behavior. the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust. the observed higher biomass (3.84g) under sammaz 52's management regime can be attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. keywords: dust pollution; quarry, maize; morphological. introduction despite its importance as a major food in many parts of the world, corn is inferior to other cereals in nutritional value. its protein is of poor quality, and it is deficient in niacin. diets in which it predominates often result in pellagra (niacin-deficiency disease). corn is high in dietary fiber and rich in antioxidants. corn oil can be converted into margarine by hydrogenation, a process in which the oil is combined with hydrogen at high temperature and pressure in the presence of a catalyst. corn is also used to produce ethanol (ethyl alcohol), a first-generation liquid biofuel (upadhay et al., 2015). the response of the plant to dust accumulation may vary according to different species, as dust deposition fluctuates with plant species due to leaf orientation, leaf surface canopy, phyllotaxy, epidermal and cuticular features, leaf pubescence, height and canopy of roadside plants (wang et. al.,2019; yun et. al., 2017) with the accumulation of dust, the roadside plant may exhibit adaptive response by changing morphological and physiological attributes. air pollution stress leads to stomatal closure, which reduces co2 availability in leaves and inhibits carbon fixation. the net photosynthetic rate is a commonly used indicator of the impact of increased air pollutants on tree growth (cao et al., 2015). plants that are constantly exposed to environmental pollutants absorb, accumulate and integrate these pollutants into their systems. the relationship between traffic density and photosynthetic activity, stomatal conductance, total chlorophyll content, and leaf senescence has been reported (silva et al., 2016). one of the most common impacts of air pollution is the gradual disappearance of chlorophyll and concomitant yellowing of leaves, which may be associated with a consequent decrease in the capacity for photosynthesis (gupta et al., 2015). methods seed collection/authentication seeds of two maize varieties swan 1 and sammaz 52 were utilized in the experiment. the seeds were collected from the seed https://doi.org/10.14421/biomedich.2023.121.295-303 296 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 bank department of the ondo state ministry of agriculture, akure, ondo state. they were authenticated at the herbarium unit of the federal university of technology, akure, and the voucher was deposited. study site the study was conducted in a screen house behind the department of biology, federal university of technology, akure. soil sample collection soil samples were collected at 50m, 100m, 150m, 200m, and 250m from the quarry site. the soil samples were taken at a plowing depth of 0 10 cm using a calibrated soil auger. the samples from each site were bucked together to obtain a composite sample and the replicate samples were packed in polyethylene bags, labeled appropriately, and transported to the laboratory for analysis. the topsoil taken from the garden served as the control. a completely randomized design (crd) was set up which comprises two maize varieties, six plowing depths, and 3 replicates. experimental setup a screen house experiment was set up to house the pots. this was necessary to protect the plants from rainfall contaminations and to avoid being destroyed by rodents as the plants develop. soil analysis soil analysis was carried out at the department of crop, soil and pest management, school of agriculture and agricultural technology, federal university of technology, akure. the following physicochemical properties of the quarry soil were determined; dissolved oxygen, conductivity, nitrogen, phosphorus, copper, iron, organic matter, and organic carbon. planting procedure seeds of swan 1 and sammaz 52 were sown into perforated plastic pots (30 cm diameter and 33 cm depth) filled with 10 kg of quarry soil. the seedlings were allowed to establish for 14 days before data were taken. weight analysis on the zero-day i.e. the day when feeding with appropriate nutrient solution commenced and at intervals of seven days, weight analysis was carried out on five seedlings harvested at random. the plants were carefully uprooted, blotted dry, weighed fresh, and then placed inside a labeled envelope and kept in a gallenkamp drying oven set at 80oc to dry to constant weight. measurement of growth parameters a meter rule was used to measure the following; leaf length, leaf width, and shoot height from soil level to the terminal end, and the number of leaves per plant were noted. the fresh weight was taken after which the plants were dried at 80oc in a gallenkamp oven until a constant weight was achieved. after cooling, the dry weight was determined. the dried samples were separated 19 into leaves, shoots, and roots and their different dry weights were determined. these were kept for further analysis. growth parameters: the following plant growth parameters were determined from the data obtained from the physical parameters: leaf area (la) = the unit of la is cm2 l and w are leaf length and width respectively while 2.75 is the correction factor for maize respectively. la = l x w x 2.75 (maize) anderson et al (2005) 3.4.5.2. root shoot ratio (rsr) rsr = w3/w2 root shoot ratio defines the method of assimilate partitioning, w2, and w3 are shoot and root dry weights respectively, the unit is g-1 3.5. photosynthetic pigment analysis chlorophyll extraction: 5g each of the leaves of the seedlings were grounded in 20 ml of 80% (%) acetone using a mortar and pestle. the brew was filtered through a whatman’s no 1 filter paper. the pigment quantities in the acetone extract were determined on a ce 373 (visible) linear readout spectrophotometer at a wavelength of 664nm and 647nm, chlorophyll “a” and “b” and the total chlorophyll quantities were determined using the formula. chlorophyll ‘a’ (µm) = 13.19a664 – 2.37a647 chlorophyll ‘b’ (µm) = 22.10a647 – 5.26a664 total chlorophyll (µm) = 7.93a664 + 19.53a67 20 a664 is the absorbance at 664nm a647 is the absorbance at 647nm (coombs et al; 1993). result and discussion particulate emissions from a wide range of industrial operations may hinder plant growth and development. at 50 meters from the quarry site, sammaz 52, one of the maize varieties grown in soil taken from the site, had the highest shoot height (94 cm). given that stem extension and apical dominance were more pronounced in the plants than in swan 1, it can be said that they devoted more of their nutrients to these processes. the ability to measure changes in the general growth habit of soybeans caused by the environment using apical dominance has been found to be valuable (thomas and raper, 2013). the results of bouma et al. (2010) and bonifas et al. (2015), show that plants growing in higher concentrations of dust pollution respond to nutrient stress by devoting more of their available carbon to shoot growth, resulting in elongated stems, were consistent with the observed higher shoot height in sammaz 52. according to reynolds et al. (2016), daily variations in photosynthetic activity and the rate of nitrogen uptake are to blame for these alterations in plant behavior. the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust (ralphs et al; 2013). the observed odiyi et al. – effect of quarry activities on some morphological parameters of … 297 higher biomass (3.84g) under sammaz 52's management regime can be attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. the findings of peace and grubb (2018) and tischer et al (2010) that higher dry weight was due to ideal leaf expansion rates were supported by an increase in the generation of dry matter under optimal conditions. plants swan 1 and sammaz 52 accumulated less biomass, as nutritional effects on photosynthetic rate per unit leaf area (greenwood, 2016). the low shoot biomass (0.04g) under swan 1 was because more carbon was diverted for greater root growth than in sammaz 52 plants and this carbon may be from the stem tissues (peace and grubb, 2018). when the nutrient supply was high, in the control regime (normal soil), sammaz 52 plants had more stem components than swan 1 plants because the soil had an adequate nutrient supply. the lowering of the shoot biomass under a low nutrient supply may not be unconnected with the reduction in the production of photosynthates as more carbon was diverted to root growth from both stem and leaf tissues (morgan and smith, 2011). plants reduce root growth relative to leaf area as an adaptation to dust pollution (chung et al; 2013). the reduced root and shoot biomasses in swan 1 at 250 m and 200 m from the quarry site, respectively, were explained by the aforementioned. according to thompson et al. (2008), low carbon content caused root growth to be reduced at low nutrient delivery levels. richer soil encourages better shoot development with roots that are less compact and branches out less (oke, 1985). nutrient-stressed plants expanded their roots quickly in search of nutrition. there was an increase in vegetative growth with maximum leaf production in sammaz 52 at the control regime compared to swan 1. lindquist et al (2016) reported that in velvetleaf, a reduction in nitrogen content increased leaf number but decreased leaf area. in phaseolus vulgaris, bridges (2012) found leaf number to increase with increased nitrogen application. the production of more leaves under the control regime may be a mechanism evolved by maize plants to increase the total surface area for photosynthesis due to reduced leaf area (morgan et al, 2018). in swan 1, at 200m from the quarry site, there was an increase in leaf abscission and a reduction in the number of leaves produced due to the fact that the extra carbon needed for greater root growth as a result of low nutrient supply was taken from their non-assimilatory tissue as well as leaf tissue (peace and grubb, 2012). roots are very important not only for absorbing water and nutrient but also for optimizing plant growth (renalto et al; 2017). the ability of a plant to compete for soil nitrogen is dependent upon root morphological characteristics such as root radius, root length, and root surface area (hilbert, 2010). previous research has shown that excessive dust pollution can inhibit root growth in corn (wang et al, 2018). the above is in agreement with the low root biomass accumulation recorded in swan 1 at 250m. plants respond to limiting soil nutrients by increasing the amount of biomass allocated to roots (bonifas et al; 2015). this substantiates the observation recorded in swan 1 plants. plants may modify their root morphology to further aid their capacity to take in nutrients (clemens, 2018). the relationship between nitrogen uptake and root growth was demonstrated in the result observed in the root-shoot biomass ratio. according to bonifas et al (2000), velvetleaf had greater nitrate uptake efficiency than corn, suggesting that velvetleaf may have more roots with a smaller radius and/or greater specific root length which would increase the surface area available for uptake. in the swan 1, there was a reduction in leaf length, leaf width, and consequently leaf area. this can be attributed to the transfer of photosynthates (assimilates) for stem elongation and the production of new leaves. according to cracker, 2013, one of the most important factors affecting leaf development is a nutrient. according to junk et al; 2012, the size of the leaf is determined by nutrient and carbon supply. since swan 1 plants had a lower assimilation rate, they were expected to have lower leaf areas. according to cardwell et al; 2016, nitrogenstressed plants adapt by producing leaves with longer internodes and an increase in leaf surface area at the expense of food allocation to the roots. leaf area may be decreased by nitrogen deficiency depending on the severity (bonifas et al; 2005). nutrient stress reduces crop photosynthesis by reducing leaf area development and leaf photosynthesis rate and by accelerating leaf senescence (pandy et al; 2000). the trend in root shoot ratio showed that the sammaz 52 higher leaf area ratio than the swan 1 plants as a consequence of nutrient addition; more leaves led to a higher surface area for photosynthesis and so a proportionate high dry matter in plant tissues. the lower leaf area ratio observed in the swan 1 plants may be due to reduced carbon allocation to the leaf tissues for leaf development (morgan and smith, 2018). there was more or less a direct correlation between the root shoot ratio (rsr) and shoot dry weight. in the study conducted, the proportionate decrease in the root shoot ratio noticed in swan 1 plants compared to the sammaz 52 plants could be attributed to lower biomass partitioned to the root than to the shoot. hong et al (2000) while working with cucumber plants found dust particles to affect root shoot ratio. according to summerfield et al (2016), the root shoot ratio declines with an increase in nutrient application in cowpea because there is a decline in the symbionts and therefore a reduced carbon allocation to the roots. the relationship between photosynthetic capacity and dust pollution had been documented by evans and seemann, 2009. also, alt et al; 2010, reported a correlation between photosynthetic capacity dust emissions from industries in vernonia herbacea. nitrogen deficiency leads to disruption of the fine structure of chlorophyll and instability of the pigment 298 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 protein complex (reddy et al; 2017). reddy et al (2017) also reported a positive correlation between leaf nitrogen, net photosynthetic rate, stomata conductance, and ion transport in cotton. hence, this was in agreement with the result obtained under swan 1 (10.54µm) at 250m from the quarry site plants which showed a lower accumulation of chlorophylls a and b. about 75% of leaf nitrogen is allocated to chloroplasts (hak, 2013), most of which is being used for the synthesis of components of photosynthetic apparatus, in particular, “rubisco”, the most representative leaf protein, playing a key role in carbon assimilation (evans and terashima, 2017). the above accounted for the chlorosis, necrosis, abscission, and senescence of older leaves noticed in the latter part of the experiment in swan 1 plants. the chlorosis spreads from older to younger leaves because of the mobility of nitrogen from older to younger leaves. (mengel et al; 2017). the accumulation of chlorophylls “a” and “b” showed consistent patterns. this was in agreement with the findings of ashraf and rehman (2019) that with adequate nutrients, chlorophylls “a” and “b” are increased, hence these substantiated the highest chlorophylls accumulation (“a” and “b”) recorded in sammaz 52 plants compared to swan 1. the lowest values of photosynthetic apparatus in swan 1 plants could be a result of low stomata conductance and protein contents, affecting rubisco activity and electron transport (evans, 2013). the higher accumulation of calcium in sammaz 52 plants at 100m from the quarry site substantiates the earlier findings of ashraf et al (2019) while working on sorghum. a comparison of calcium ion and magnesium ion concentrations in both regimes reveals that the two ions show antagonism in uptake (ashraf et al; 2000). the calcium and magnesium contents in the two varieties followed inconsistent patterns. this confirms the result of the experiment indicating the highest magnesium accumulation in swan 1 plants while calcium accumulation was observed to be relatively low. there is a correlation between nitrogen and magnesium accumulation as magnesium ion plays a vital role in chlorophyll biosynthesis (walker et al; 2011), protein synthesis, and photosynthesis (marschner et al; 2017). analysis of potassium ions showed different patterns of accumulation in sammaz 52 and swan 1. the percentage of potassium content which decreases in the latter part of the distance from the quarry site can be explained in view of the argument that a high amount of potassium is needed to maintain nitrogen metabolism when large amounts of nitrogen are supplied in the external medium (leigh et al, 2014). however, these results were at variance with the observation of ashraf et al, (2000) who found a lower accumulation of potassium in nutrient-stressed plants grown under normal conditions. magnesium limitation resulted in a reduction in shoot growth and photosynthetic capacity in maize (foyer et al; 2010). supraoptimal levels of magnesium have been reported to increase in corn (diao et al; 2018, tsialtas et al; 2018). this can therefore be interpreted to mean greater metabolic activities, utilization and uptake noticeable in both sammaz 52 and swan 1 plants. however, this was at variance with the observation of tischer et al (2000) that an increase in magnesium supply not only delays senescence and stimulates growth but also changes plant morphology in a typical manner, particularly if the magnesium availability is high in the rooting medium during the early growth, shoot elongation is enhanced and root elongation inhibited, a shift which is unfavorable for nutrient acquisition and water uptake in later stages. the efficient translocation of photosynthate from source to sink organs is the key factor driving plant growth and increased crop yield (dakora, 2003; cornu, 2007). photosynthesis was reported to be affected by sink strength as an increased photosynthate supply is required to meet the increasing demand for photoassimilates during early vegetative growth and seed development in zea mays (richards, 2000). strong positive correlations have been found between the photosynthetic capacity of leaves and their magnesium content, most of which is used for the synthesis of components of the photosynthetic apparatus (gastal et al, 2012). conclusion the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust. the observed higher biomass (3.84g) under sammaz 52's management regime was attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. this can be attributed to the transfer of photosynthates (assimilates) for stem elongation and the production of new leaves. there were correlations between nitrogen and magnesium accumulation as magnesium ion plays a vital role in chlorophyll biosynthesis, protein synthesis, and photosynthesis. there were clear inconsistent patterns in the accumulation of the chlorophylls a and b. abbreviations: not applicable. ethics approval and consent to participate: not applicable. consent for publication: all authors are aware of the publication of this manuscript. odiyi et al. – effect of quarry activities on some morphological parameters of … 299 availability of data and material: the datasets used and/or analyzed during the current study are available from the corresponding author on request. competing interest: the authors declare that they have no competing interest. funding: the research was self-funded. authors’ contributions: prof mrs b. o. odiyi designed the experiment, maku olubukola carried out the laboratory works. dr. f. a. ologundudu carried out the statistical analysis and interpretation of the results. the author(s) read and approved the final manuscript. acknowledgement: the researchers want to appreciate the technical staff of the department of biology, federal university of technology, akure, nigeria. references ahmad, s.d., m. liebman and a.s.davis (2012). integration of soil, crop and weed management in low external input farming systems. weed res.40:27-47. alt, a.p., f.makini and n.kidula (2010). effect of intercropping legume with maize on soil fertility and maize yield. plant and soil.54:109-115. anderson m.k. and h. ambus (2012). reduction of pest attack on sorghum and cowpea by intercropping. entomology experimental application70:179-184. bonifas, k.d., d. t walters,.and j.l. lindquist (2005). nitrogen supply affects root: shoot ratio in corn and velvetleaf. weed science 54, pp.133-137. bonifas, k.d. and j.l. lindquist (2015). predicting biomass partition to root versus shoot in corn and velvetleaf. weed science 53 .670-675. brook, s.f., b.b. singh and d.l. smith (2007). evaluation of yield stability of cowpea under sole and intercrop management in nigeria. euphytica, 61:193-201. boston, r.s., p.v. viitanen and e. vierling (1996). molecular chaperon and protein folding in plants. plant mol.biol.32:191222. bouma, t. j .and k.l. nielsen (2010). sample preparation and scanning protocol for computerized analysis of root length and diameter. plant and soil 218, 185-196. cao, s.d. and mc nelly (2015). the effects of growing beans together with maize on incidence of bean diseases and pests. neth. j. plant pathol.78:12-18. darley m, amaliotis, d. (2016). effect of nitrogen fertilization on growth, leaf nutrient concentration and photosynthesis in three peach cultivars. international symposium on irrigation of horticultural crops.ishs acta horticulture.449:36-42. gupta r.k, and p jensen (2016). biomass production, symbiotic nitrogen fixation and inorganic n use in dual and tricomponent annual intercrops. plant and soil 266:273-287. kan, a.f.and m.a.m. carvalho (2009). fructan metabolizing enzymes in rhizosphores of vernonia herbacea upon excision of aerial organs. plant physiology and biochemistry 42:313319. layett, m. p., g.s. aharon and w.a. snedden (2009). salt tolerance conferred by over expression of vacuolar na+/h+ antiport in arabidopsis. science285:125-258. reddy s. t.w. kuyper (2017). farmers agronomic and social evaluation of productivity, yield and n2 fixation in different cowpea varieties and their subsequent residual n effects on a succeeding maize crop. nutrient cycling in agroecosystems 80:199-209. seyenejad, t.j. and a.p.rehman (2019). use of velvet bean to improve soil fertility and weed control in corn production in northern belize. commun. soil sci. plant anal. 27(9-10). siyuan, s.a.and m. silberbush (2018). plant root morphology and nutrient uptake, roots, nutrient and water influx and plant growth: american society of agronomy, 65-878. upadhay, j.r., w. robbins and j. w.shive (2015). nutrition studies with corn iii. a statistical interpretation of the relation between nutrient ion concentration, carbohydrate and nitrogenous content of the tissue. soil sci.49:211-238. . 300 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 table s1. shoot height of zea mays at different distances from the quarry site. varieties days distance (m) control 50 100 150 200 250 var 1 28 43.67±1.86a 39.33±1.33a 37.33±1.33a 36.00±5.29a 39.00±4.58a 33.00±5.19a 35 51.33±1.76b 45.33±1.76b 43.33±2.40ab 38.67±4.81a 44.00±3.06ab 37.33±4.67a 42 58.00±1.16c 56.00±1.16c 48.67±2.33ab 42.67±5.18a 49.33±0.67ab 46.00±2.31a 49 61.33±0.67b 58.67±0.67b 50.33±2.03a 45.67±5.67a 50.67±1.59a 49.33±0.67a 56 63.67±0.88b 62.00±1.16b 52.33±1.45a 46.67±5.69a 54.00±1.16a 51.33±0.67a 63 70.67±0.67b 67.67±1.45b 57.67±1.45a 52.67±4.67a 59.33±0.67a 57.00±0.58a 70 77.67±2.19c 72.33±1.45bc 63.67±0.88a 57.33±5.46a 64.67±0.67ab 63.67±0.33a var 2 28 49.67±3.28b 48.00±1.16b 38.33±2.03a 33.33±1.76a 38.67±1.20a 39.33±2.96a 35 57.00±1.53c 56.00±1.16c 46.33±0.88b 37.33v0.67a 44.33±2.33b 45.00±2.52b 42 66.00±1.00c 67.33±1.76d 54.00±1.16b 40.67±0.67a 51.00±2.08b 49.00±2.08b 49 72.67±1.76d 73.67±2.03e 60.00±0.00c 46.67±0.67a 55.33±1.76bc 53.33±1.76b 56 80.00±1.16d 80.00±1.16e 64.00±1.16c 52.33±1.45a 58.00±1.16b 57.00±1.53b 63 86.00±1.16d 86.33±0.88f 69.33±0.67c 58.67±0.67a 63.33±2.40b 61.67±1.67ab 70 92.67±1.76c 94.00±2.08g 76.00±0.58b 65.00±0.00a 68.67±2.40a 68.00±2.31a mean follow by the same alphabet in a row are not significantly different (p>0.05) from one another, using duncan new multiple range test, var 1 = swan 1, var 2 = sammaz 52 shoot height of zea mays at different distances from the quarry site ranged between 33cm and 94cm respectively relative to the control. the highest shoot height (94cm) was observed in sammaz 52 in the 7 th at 50m from the quarry site. the lowest shoot height was however noticeable in swan 1 at 250m. there is a significant difference in the shoot height of swan 1 at 50m from the quarry site between the first and the third week. also, significant differences were also observed in sammaz 52 at 50m from the quarry site and from the beginning till the end of the experimental period. table s2. plant dry weight (pdw) of maize at different distances from the quarry site. varieties distance dry weight (g)/days after planting (dap) 28 35 42 49 56 63 70 swan 1 0 0.06±0.02a 0.36±0.00ab 0.16±0.00a 0.80±0.00b 0.65±0.00a 0.48±0.00abc 0.52±0.07bc 50 0.04±0.01a 0.40±0.04a 0.21±0.04a 1.01±0.11abc 0.62±0.12a 0.40±0.04ab 0.55±0.04c 100 0.05±0.02a 0.48±0.02c 0.21±0.02a 1.19±0.14abcd 0.69±0.42a 0.32±0.07a 0.54±0.06bc 150 0.04±0.01a 0.46±0.05bc 0.28±0.02ab 1.21±0.10abcd 0.70±0.29a 0.31±0.11a 0.31±0.04a 200 0.09±0.03a 0.51±0.02c 0.16±0.04a 1.51±0.08bcd 1.11±0.30a 0.48±0.10abc 0.32±0.07a 250 0.04±0.00a 0.42±0.04abc 0.23±0.04a 2.06±0.43cd 1.40±0.49a 0.71±0.27abc 0.38±0.06ab sammaz 52 0 0.07±0.01a 0.45±0.02abc 0.18±0.04a 3.84±0.23e 3.29±0.54b 0.98±0.31c 0.47±0.07abc 50 0.07±0.01a 0.34±0.04a 0.21±0.03a 3.35±0.23e 2.84±0.61b 0.89±0.34bc 0.46±0.03abc 100 0.12±0.11a 0.46±0.06bc 0.28±0.14ab 2.19±0.98d 1.57±0.72a 0.22±0.05a 0.54±0.03bc 150 0.28±0.07b 0.47±0.02bc 0.29±0.08ab 0.26±0.05a 0.38±0.09a 0.28±0.04a 0.52±0.03bc 200 0.28±0.04b 0.51±0.03c 0.28±0.03ab 0.27±0.03a 0.36±0.15a 0.33±0.07a 0.56±0.04c 250 0.33±0.07b 0.52±0.04c 0.45±0.05b 0.26±0.04a 0.33±0.09a 0.38±0.05ab 0.46±0.04abc mean follow by the same alphabet in row are not significantly different (p>0.05) from one another, using duncan new multiple range test biomass accumulation in the two maize varieties ranged between 0.04g and 3.84g. the highest biomass was recorded in sammaz 52(3.84g) in the 4th week while the least was recorded in swan 1(0.04g) at the beginning of the experimental period. there was no significant difference in the pdw of the varieties relative to the control. odiyi et al. – effect of quarry activities on some morphological parameters of … 301 table s3. root dry weight (rdw) of the varieties at different distances from the quarry site. v a rie tie s d a y s distance (m) control 50 100 150 200 250 v1 28 0.033±0.003c 0.027±0.003bc 0.023±0.003b 0.010±0.000a 0.063±0.003d 0.030±0.000bc 35 0.117±0.072a 0.193±0.013ab 0.270±0.034b 0.273±0.013b 0.307±0.024b 0.227±0.052ab 42 0.217±0.087a 0.467±0.012b 0.480±0.047b 0.483±0.063b 0.500±0.020b 0.500±0.030b 49 0.050±0.012a 0.063±0.012a 0.037±0.003a 0.050±0.027a 0.050±0.012a 0.067±0.003a 56 0.391±0.015a 0.391±0.028a 0.452±0.054a 0.421±0.022a 0.437±0.032a 0.456±0.019a 63 0.113±0.002a 0.111±0.002a 0.134±0.021a 0.119±0.008a 0.116±0.008a 0.123±0.006a 70 0.550±0.072bc 0.580±0.042c 0.560±0.050bc 0.317±0.047a 0.377±0.068ab 0.407±0.056abc v2 28 0.073±0.003d 0.040±0.000c 0.017±0.003a 0.010±0.000a 0.033±0.003bc 0.027±0.003b 35 0.217±0.038a 0.277±0.012a 0.240±0.055a 0.553±0.209b 0.560±0.021b 0.563±0.013b 42 0.510±0.25b 0.289±0.008a 0.277±0.004a 0.351±0.038a 0.305±0.015a 0.312±0.023a 49 0.317±0.020a 0.347±0.026ab 0.447±0.015c 0.380±0.017ab 0.373±0.013ab 0.387±0.022b 56 0.193±0.003a 0.233±0.039a 0.230±0.015a 0.290±0.023a 0.223±0.038a 0.257±0.044a 63 0.112±0.002a 0.144±0.025ab 0.164±0.015b 0.141±0.001ab 0.146±0.008ab 0.132±0.002ab 70 0.540±0.070a 0.500±0.025a 0.550±0.032a 0.523±0.034a 0.593±0.043a 0.490±0.035a mean follow by the same alphabet in a row are not significantly different (p>0.05) from one another, using duncan new multiple range test. var 1 = swan 1, var 2 = sammaz 52 root dry weight of the varieties at different distances from the quarry site ranged from 0.030g at 250m (swan 1) from the quarry site and 0.593g at 200m (sammaz 52) from the quarry site. there were significant differences in rdw at 50m in swan 1 and sammaz 52 respectively between the first and the second week. however, there were no significant differences between the varieties at 100-250m from the quarry site. table s4. shoot dry weight (sdw) of the maize varieties at different distances from the quarry site. v a rie tie s d a y s distance (m) control 50 100 150 200 250 v1 28 0.030±0.006c 0.025±0.000b 0.015±0.001a 0.008±0.000a 0.037±0.001c 0.029±0.001b 35 0.173±0.001a 0.170±0.015a 0.187±0.009a 0.190±0.025a 0.197±0.013a 0.170±0.006a 42 0.064±0.005d 0.051±0.001c 0.038±0.004b 0.016±0.001a 0.099±0.003e 0.059±0.002cd 49 0.079±0.003ab 0.073±0.003a 0.082±0.003ab 0.089±0.006b 0.08±0.003b 0.082±0.004ab 56 0.088±0.003d 0.080±0.002bc 0.069±0.000a 0.074±0.003ab 0.09±0.005d 0.092±0.004d 63 0.080±0.003b 0.073±0.002a 0.082±0.004b 0.092±0.002c 0.115±0.005d 0.067±0.004a 70 0.750±0.067d 0.559±0.034c 0.399±0.009b 0.178±0.018a 0.71±0.014d 0.602±0.018c v2 28 0.066±0.003c 0.049±0.005b 0.035±0.002a 0.028±0.004a 0.046±0.002b 0.035±0.001a 35 0.023±0.005a 0.026±0.003a 0.033±0.003a 0.033±0.004a 0.029±0.002a 0.032±0.002a 42 0.138±0.004e 0.088±0.006d 0.050±0.003b 0.037±0.003a 0.079±0.017d 0.062±0.004c 49 0.061±0.011a 0.081±0.010a 0.108±0.07b 0.078±0.005a 0.076±0.001a 0.068±0.005a 56 0.065±0.009a 0.087±0.005b 0.125±0.002c 0.930±0.003b 0.095±0.003b 0.084±0.003b 63 0.056±0.008a 0.086±0.009bc 0.099±0.004bc 0.079±0.005b 0.103±0.004c 0.095±0.006bc 70 0.095±0.009d 0.783±0.034c 0.512±0.016b 0.258±0.013a 0.733±0.048c 0.588±0.057b mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test. var 1 = swan 1, var 2 = sammaz 52 there were significant differences in the shoot biomass of swan 1 at 50m and at 200m between the first and the 5th week relative to the control. similarly, significant differences were also observed in sammaz 52 between the 28 th and the 42nd day at 50m relative to the control. the highest shoot dry weight (0.71g) was observed in swan 1 at 200m while the least sdw was noticeable at 150m. 302 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 table s5. mineral nutrient composition of maize varieties at different distances from the quarry site. varieties distance nutrients na k mg ca swan 1 0 0.31±0.05b 0.57±0.03b 0.23±0.05ab 0.23±0.05ab 50 0.33±0.04bc 0.47±0.25b 0.26±0.03abc 0.26±0.03ab 100 0.21±0.02a 0.48±0.02b 0.34±0.04bc 0.33±0.03b 150 0.18±0.01a 0.56±0.04b 0.36±0.04c 0.33±0.04b 200 0.33±0.01bc 0.59±0.06b 0.55±0.03d 0.29±0.02ab 250 0.42±0.05cd 0.50±0.06b 0.17±0.02a 0.32±0.02b sammaz 52 0 0.38±0.01bc 0.27±0.09a 0.28±0.01abc 0.34±0.05b 50 0.36±0.02bc 0.51±0.07b 0.34±0.04bc 0.29±0.03ab 100 0.50±0.03d 0.75±0.04c 0.25±0.04ab 0.33±0.04b 150 0.43±0.04cd 0.50±0.06b 0.29±0.02bc 0.27±0.02ab 200 0.40±0.03bc 0.55±0.03b 0.48±0.01d 0.21±0.02a 250 0.40±0.02bc 0.44±0.03b 0.51±0.06d 0.24±0.04ab table s6. leaf area (cm2) of maize varieties at different distances from the quarry site leaf area (cm2) distance (m) swan 1 sammaz 52 50 32.50±0.07d 33.06±0.03d 100 18.50±0.01b 22.18±0.04b 150 29.00±0.07c 28.37±0.02c 200 17.50±0.04b 19.32±0.01a 250 12.45±0.12a 15. 24±0.07a control 35.50±0.08d 35.24±0.06d the leaf area of both varieties increased gradually for a greater part of the experimental period. similar growth patterns were recorded throughout the experimental period. gradual increases were observed at 200m and at 250m from the quarry site. the highest leaf area was recorded in the control regime followed by 50m while 250m was observed to be the least. results of the anova showed that there were significant differences (p<0.05) in the varieties grown on the soils from the quarry site. chlorophyll ‘’a’’ accumulation of maize varieties at various distances from the quarry site there were gradual decreases in a linear fashion in chlorophyll ‘a’ accumulation for a greater part of the experimental period in the two maize varieties. both maize varieties had approximately equal chlorophyll accumulation at 150m from soil collected from the quarry site. these were followed by a gradual increase between 50 and 100m from the quarry site. the highest chlorophyll ‘a’ accumulation was recorded in control, while 250m was ob served to be the lowest (swan 1). results of the anova showed that there were significant differences (p<0.05) at 50 and 250m respectively relative to the control. table s7. chlorophyll ‘’a’’ accumulation of maize varieties at various distances from the quarry site. chlorophyll a (µm) distances (m) swan 1 sammaz 52 50 8.57±0.07a 24.55±0.02d 100 10.27±0.05b 12.50±0.04a 150 10.78±0.01b 10.45±0.03a 200 8.23±0.02a 16.99±0.02c 250 7.52±0.02a 17.72±0.01c control 24.52±0.04c 35.76±0.02d mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test odiyi et al. – effect of quarry activities on some morphological parameters of … 303 table s8. chlorophyll ‘’b’’ accumulation of maize varieties at various distances from the quarry site. chlorophyll b (µm) distance(m) swan 1 sammaz 52 50 11.64±0.02a 25.43±0.01c 100 16.24±0.02b 23.23±0.02c 150 14.58±0.01b 18.76±0.06b 200 12.58±0.01a 15.57±0.05a 250 10.54±0.03a 12.78±0.05a control 18.76±0.04c 38.56±0.08d mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test in the same pattern like chlorophyll ‘a’ contents, the chlorophyll ‘b’ contents in maize followed similar pattern except that initial contents of chlorophyll ‘a’ were lower than those of chlorophyll ‘b’ also, both varieties decreased gradually between 100 and 200m in the soil collected from the quarry site. sammaz 52 had the highest accumulation under the control regime while the least was observed in swan 1 at 250m. results of the anova showed that there was no significant difference (p>0.05) in the two maize varieties. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 305-313 | doi: 10.14421/biomedich.2023.121.305-313 issn 2540-9328 (online) antidiarrhoeal activities of lime (citrus aurantiifolia) extract in experimentally-induced diarrhoea model joshua charles isirima1, precious ojo uahomo2,* 1department of pharmacology, faculty of basic clinical sciences; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com manuscript received: 20 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract this study investigated the effects of lime on diarrhoea in wistar rats. a total of 60 wistar rats were procured and randomly divided into 3 groups of 20 animals each for each of the three t-test models. the twenty healthy wistar rats for each diarrhoea model were fasted for 6 hours prior to the experiment but allowed free access to water. the twenty animals were randomly divided into 5 groups of 4 animals each for each experiment. established antidiarrhea models were followed. the test groups received various doses (97.65mg/kg, 195.3mg/kg, and 390.6mg/kg) of citrus aurantiifolia juice extract; whereas positive controls received loperamide (2.5mg/kg) and negative controls received distilled water (1ml/kg). the administration was done once daily for 15 days, and the faeces of each animal was collected on the 5th, 10th and 15th day. the result of this study showed that medium and high dose citrus aurantiifolia has an anti-diarrhoeal effect on castor oil-induced diarrhoea over repeated administration for a minimum of 15 days as it prolonged the onset of diarrhoea, decreased the frequency of defecation and gastrointestinal transit time in wistar rats. this study shows that citrus aurantiifolia demonstrates significant anti-diarrhoeal activity and can be used as an anti-diarrhoea agent. keywords: diarrhoea; citrus aurantiifolia; castor oil; lime juice extract; gastrointestinal tract; enteropooling; diarrhoea model. introduction limes (citrus aurantifolia) are a small citrus fruit either sour or sweet. sour limes possess a greater sugar and citric acid content than lemons and feature an acidic and tart taste. lime contains unique flavonoid compounds that have antioxidant and anticancer properties. while these flavonoids have been shown to stop cell division in many cancer lines, they are perhaps most interesting for their antibiotic effects (bina et al., 2010). citric acid the major organic acid in these juices has been found to be responsible for inhibiting the growth of vibrio (gram-negative bacteria of the family vibronaceae). the natural biocidal activity of lime juice was studied in order to explore its possible use as a disinfectant and inhibitor of vibrio cholera in drinking water for areas lacking water treatment plants (d'aquino et al., 1994). owing to the support of the national and international organizations for the studies on the treatment and prevention of these diseases based on traditional practices, medicinal plants are becoming a hopeful source of antidiarrhoeal drugs (lin et al., 2002). according to ekwawati and darmanto (2019), medicinal plants are one of the natural resources that can be explored by humans. various secondary metabolites from plants can be used as medicines, agrochemicals, flavours, fragrances, dyes, biopesticides and food addictive. citrus fruits contain nutrients and phytochemicals that are beneficial to health. citrus juice contains various substances including carbohydrates, fibre, vitamin c, potassium, folate, calcium, thiamine, niacin, vitamin b6, vitamin a, phosphorus, magnesium, copper, riboflavin, pantothenic acid and various phytochemicals. these substances are needed by the body. some compounds in citrus fruit can provide additional protection against chronic disease and basic nutrition. citrus fruits also contain lots of phytochemicals, essential oils, alkaloids, flavonoids, psoralens, carotenoids, limonoids, tannins, and terpenoids. previous pharmacological studies revealed that citrus fruits have antimicrobial, anthelminthic, antioxidants, anticancer and many other pharmacological effects (prastiwi and ferdiansyah, 2013). diarrhoea is an alteration in the normal bowel movement characterized by an increase in the volume, fluidity, frequency, and passage of loose or watery stool at least three times a day (unicef/who, 2009). it is a common symptom of gastrointestinal infection due to the ingestion of many bacteria, viruses, or parasites that may be transmitted by water, food, utensils, hands, and flies (unicef/who, 2009). it is the mechanism by which https://doi.org/10.14421/biomedich.2023.121.305-313 306 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 the body rids itself of pathogenic organisms, with excessive stimulation of intestinal motility, leaving insufficient time for the absorption of intestinal fluid (keusch et al., 2006). diarrhoea is one of the most important health problems in developing countries affecting people of all ages (snyder and merson, 1982) and results in electrolyte loss, dehydration, shock, and sometimes death (unicef/who, 2009). diarrhoeal diseases account for 1 in 9 child deaths worldwide, making diarrhoea the second leading cause of death among children under the age of 5 (hutton et al., 2007), while lakshminarayana et al. noted that diarrhoea is a leading cause of mortality and morbidity in children under 5 years old, and it accounts for 5–8 million deaths worldwide each year (lakshminarayana et al., 2011; world health organization, 2006). a report in 2015 revealed that diarrhoea is one of the main killers of children that accounts for 9% of all deaths among kids below the age of 5 years worldwide (unicef, 2019). according to this report, sub-saharan africa and southern asia were recorded as the regions that experienced the highest child death toll because of diarrhoea (sow et al., 2016). in nigeria, the prevalence of diarrhoeal infection is as high as 18.8%, above the average of 16%, making it one of the worst in sub-saharan africa (olawuyi et al., 2004). it accounts for an annual estimated 300,000 deaths mainly amongst children under five in nigeria (olawuyi et al., 2004), while 7-to-12-month-old babies continue to be the most susceptible (audu et al., 2000) caused mainly by poor sanitation and hygiene practices. the disease may be caused by a wide array of agents such as enteropathogenic microorganisms (shigella flexneri, staphylococcus aureus, escherichia coli, salmonella typhi, and candida albicans) (jouret-mourin and geboes, 2002), alcohol, irritable bowel syndrome, bile salts and hormones (teke et al., 2007), secretory tumours, and intoxication (brijesh et al., 2011), whereas susan & mays said the causes of diarrhoea include infectious agents, gastrointestinal disorders such as inflammatory or dysmotility problems, and substances that increase gastrointestinal tract secretions (susan and mays, 2005). despite different pathophysiological changes in different types of diarrhoeas, there are four major mechanisms responsible for pathophysiology in electrolyte and water transport that is, increased luminal osmolarity, increased electrolyte secretion, decreased electrolyte absorption and accelerated intestinal motility causing decreased transit time (lutterodt, 1992). management of diarrhoea comprises both nonpharmacological and pharmacological interventions. in general, the treatment is aimed at reducing the discomfort and inconvenience of frequent bowel mobility and frequency of faecal passage (suleiman et al., 2008). several options employed in the management of the diseases include oral agents such as metronidazole, antibiotics, and oral rehydration therapy (ort) (hardman et al., 2001; sastry and burgard, 2005). with over a decade of the practice and promotion of ort, diarrhoea is still the second the cause of child death (grant, 1993). in addition, these management options which often fail during high stool output state are also associated with undesirable side effects such as headache, convulsion, stomach cramps, vomiting, constipation, and hallucination. consequently, attention is now being shifted to alternatives in medicinal plants for the management of the disease. medicinal plants represent a promising source for the discovery of new antidiarrhoeal agents. these plants are cheaper and more easily available than conventional medicines. even the world health organization has encouraged studies for the treatment and prevention of diarrhoeal disease using traditional medicinal practices (atta et al., 2004). various plants are being investigated for their possible antidiarrhoeal activities to provide safe and inexpensive alternatives to standard drug therapies. materials and methods animals procurement animals were procured from the department of pharmacology, faculty of basic clinical sciences, university of port harcourt and were acclimatized for a period of two weeks with their weights checked constantly during this period. all the animals (160±0.02g) were housed in clean plastic cages that were placed in a well-ventilated house (temperature: 22 ± 3oc; photoperiod: 12 hours; humidity: 45–50%). the animals were fed on rat pellets (premier feed mill co. ltd., ibadan, nigeria) and clean tap water, except when fasting was required during the experiment. the cages and the animal house were cleaned on daily basis. plant collection fresh lime fruits were purchased from choba market and were identified and authenticated at the department of plant science and biotechnology, university of port harcourt, herbarium. extraction of plant the extraction method by amita and shalini (2014) with slight modifications was used. the lime was sliced and the juice was squeezed out into a beaker and after which the extract was filtered. the filtrate was then centrifuged for clarification of the extract (das et al., 2010) and stored in the refrigerator for use. drugs and chemicals loperamide hydrochloride was products of laborate pharmaceuticals (india) and richy gold international limited (nigeria), respectively. castor oil was a product isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 307 of bills sons and co. (druggist) limited, southport, england. experimental protocols castor oil-induced diarrhoea in rats twenty (20) healthy wistar rats were fasted for 6 hours prior to the experiment but allowed free access to water. the experimental rats were completely randomized into five groups of four animals each. the procedure described by bajad et al. (2001) was adopted with slight modification. animals in group a which received 1.0ml of distilled water served as a negative control, while those in groups b (positive control), c, d, and e (test groups) received 1.0ml each corresponding to 2.5mg/kg body weight of loperamide (a reference drug), 0.25, 0.5, and 1.0ml of the extract, respectively. thirty minutes after administration, all the animals were orally administered 1ml of castor oil and thereafter placed in cages lined with a pre-weighed transparent paper. during the 6-hour observation period, the time of onset of diarrhoea, the total number of faeces, diarrhoeal faeces, total weight of faeces, and percentage inhibition of diarrhoeal defecation in each group were computed. the weight of the faeces was obtained from the difference in the pre-weighed transparent paper and the fresh weight of the stool. the dry weight of the faeces was obtained by drying the fresh faeces in the laboratory oven (uniscope laboratory oven, sm9053, surgifriend medicals, england) at 1000c until a constant weight was obtained. the difference in the fresh weight of the faeces and the dry weight of the faeces gives the water content of the faeces. at the end of the 6-hour exposure period, the animals were sacrificed under the effect of diethyl ether anaesthesia and the small intestine of the animals was dissected and removed. thereafter, the contents of the small intestines were squeezed out. castor oil-induced enteropooling the procedure described by chitme et al. (2004) was adopted for the castor oil-induced enteropooling study. the animals were fasted without food for 6h prior to the experiment but were allowed free access to water. four animals were randomly selected for each group and placed in their respective cages. animals in the negative control group (group a) received 1.0ml of distilled water, while those in the positive control group (group b) received 1.0ml of loperamide (2.5mg/kg body weight). rats in groups c, d, and e (test groups) were orally administered 0.25, 0.50, and 1.0ml of the sap, respectively. immediately afterwards, 1.0ml of castor oil was administered orally to each of the rats in all the groups. after 30 minutes, each rat was sacrificed according to the method described by akanji and yakubu (2000) and the ends of the pylorus and caecum of the small intestine were tied. the small intestine was dissected and its intestinal content was squeezed into a measuring cylinder. the volumes and the masses of the intestinal contents were noted and used to compute the percentage of inhibition of intestinal content. gastrointestinal motility the method described by teke et al. (2007) was adopted for the evaluation of the effect of the sap on gastrointestinal transit in rats. twenty, healthy wistar rats were fasted for 6 hours prior to the experiment but were allowed free access to water. the experimental rats were completely randomized into five groups of four animals each. the negative control group (group a) received 1.0ml of distilled water while the positive control group (group b) received 1.0ml of loperamide (0.6mg/ml) intramuscularly. animals in groups c, d, and e (test groups) were orally administered 0.25, 0.50, and 1.0ml of the sap, respectively. charcoal meal (10% charcoal suspension in 5% agarose agar, prepared by weighing 10 g of charcoal powder and 5 g of agarose agar into 100ml distilled water and mixed thoroughly) was administered orally, 30 minutes after the administration of atropine sulphate and the citrus aurantiifolia. the animals were then sacrificed after 45 minutes of charcoal administration, using the diethyl ether as anaesthesia as described by akanji and yakubu (2000). the small intestine was removed very carefully and the length of the intestine as well as the distance travelled by the charcoal meal through the intestine was measured. the percentage of gastrointestinal motility was computed as the ratio of the distance moved by the charcoal meal to the length of the small intestine. methods of data analysis the experimental results were analyzed using the statistical package for the social sciences (ibm spss), version 25.0. results were expressed as a mean ± standard error mean (sem), and statistical analyses were carried out by employing a one-way analysis of variance (anova), followed by tukey’s post hoc test to compare the results of controls and the groups. in all cases, statistical significance was set at p<0.05. results analysis of the effect of the extract on castor oilinduced diarrhoea in wistar rats below is a table showing the observed effect of citrus aurantiifolia on castor oil-induced diarrhoea and enteropooling respectively in wistar rats. the low dose, medium dose and high dose of the extract were administered and the effect of these substances on the wistar rats was observed and analysed alongside that of the well-known drug and the control substance. 308 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 table 1. effects of citrus aurantiifolia (lime juice extract) on castor oil-induced diarrhoea in wistar rats. group mot mnd mnws mnds mfws mwcs control 63.25±0.47 6.00±0.41 2.50±0.29 2.00±0.41 4.75±0.25 2.20±0.41 loperamide 121.75±1.37 2.50±0.29 1.25±0.25 1.25±0.25 0.83±0.025 0.48±0.025 low dose 93.50±1.55 2.75±0.48 1.50±0.29 1.50±0.29 0.93±0.03 0.55±0.03 medium dose 108.00±0.91 2.50±0.29 1.00±0.41 1.50±0.29 0.78±0.03 0.45±0.03 high dose 140.00±1.08 1.75±0.48 0.75±0.25 1.70±0.25 0.65±0.00 0.30±0.41 mot = mean onset time (min); mnd = mean number of defecations; mnws = mean number of wet stool; mnds = mean number of dry stool; mfws = mean fresh weight of stool; mwcs = mean water content of stool table 2. effects of citrus aurantiifolia (lime juice extract) on castor oilinduced enteropooling in wistar rats. group mwic mvic control 2.88±0.14 2.60±0.09 loperamide 1.63±0.09 1.60±0.07 low dose 2.15±0.10 1.90±0.27 medium dose 1.65±0.25 1.33±0.11 high dose 0.98±0.17 1.10±0.04 mwic = mean weight of intestinal content; mvic = mean volume of intestinal content table 3. effects of citrus aurantiifolia (lime juice extract) on castor oilinduced transit time in wistar rats. group mdtcm control 77.25±0.63 loperamide 35.50±0.29 low dose 40.25±0.25 medium dose 31.50±0.64 high dose 27.75±1.11 mdtcm = mean distance travelled by charcoal meal table 4. effects of citrus aurantiifolia (lime juice extract) on the onset of diarrhoea in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mot 63.25±0.47 121.75±0.53 93.50±1.55 108.00±0.91 140.00±1.08 dfreq 92.49±0.09 47.83±1.08 70.75±0.24 121.34±0.64 𝐷𝑓𝑟𝑒𝑞 = 𝑀𝑂𝐷𝑇𝐺−𝑀𝑂𝐷𝑁𝐺 𝑀𝑂𝐷𝑁𝐺 𝑥 100 (1) where, mot = mean onset time, dfreq= percentage inhibition in terms of control of the onset of diarrhoeal stool, modtg = mean onset of diarrhoea in the treated group and modng = mean onset of diarrhoea in the negative control (schuster, 2001). table 5. effects of citrus aurantiifolia (lime juice extract) on the mean number of defecations in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mnd 6.00±0.41 2.50±0.29 2.75±0.48 2.50±0.27 1.75±0.48 pid 58.33±0.41 54.17±0.18 58.33±0.43 70.83±0.07 𝑃𝐼𝐷 = 𝑀𝑁𝐷𝐶𝐶−𝑀𝑁𝐷𝐶𝐷/𝐸 𝑀𝑁𝐷𝐶𝐶 𝑥 100 (2) where, mnd = mean number of defecations, pid = percentage inhibition of defecation, mndcc = mean number of defecations caused by castor oil and mndcd/e = mean number of defecations caused by drug/extract (kola-mustapha et al., 2019). table 6. effects of citrus aurantiifolia (lime juice extract) on mean number of wet faeces in castor oil-induced diarrhoea in wistar rats. parameter /dose (ml) distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mnws 2.50±0.29 1.25±0.25 1.50±0.17 1.00±0.41 0.75±0.25 pfreq 50.00±0.03 40.00±0.12 60.00±0.14 70.00±0.04 isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 309 𝑃𝑓𝑟𝑒𝑞 = 𝑀𝑁𝑊𝑆𝐶−𝑀𝑁𝑊𝑆𝑇 𝑀𝑁𝑊𝑆𝐶 𝑥 100 (3) where, mnws = mean number of wet stools, pfreq = percentage inhibition in terms of the purging frequency (number of wet stools), mnwsc = mean number of wet stool in control group and mnwst =mean number of wet stool in treated group. table 7. effects of citrus aurantiifolia (lime juice extract) on number of dry faeces, fresh weight of faeces and water content of faeces in castor oilinduced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mndf 2.00±0.41 1.25±0.25 1.50±0.29 1.50±0.29 1.70±0.25 mfwf 4.75±0.25 0.83±0.025 0.93±0.03 0.78±0.03 0.65±0.01 mwcf 2.20±0.41 0.48±0.025 0.55±0.03 0.45±0.03 0.30±0.41 where, mndf = mean number of dry faeces, mfwf = mean fresh weight of faeces and mwcf = mean water content of faeces table 8. effects of citrus aurantiifolia (lime juice extract) on weight of intestinal content in castor oil-induced diarrhoea in wistar rats. paramete distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mwic 2.88±0.14 1.63±0.09 2.15±0.10 1.65±0.25 0.98±0.17 piic 43.40±0.24 25.35±0.04 42.71±0.29 65.97±0.64 𝑃𝐼𝐼𝐶 = ( 𝑀𝑊𝐼𝐶𝐶−𝑀𝑊𝐼𝐶𝑇 𝑀𝑊𝐼𝐶𝐶 ) 𝑥 100 (4) where, mwic = mean weight of the intestinal content, piic = percentage inhibition of intestinal content, mwicc = mean weight of the intestinal content of the control group and mwict = mean weight of the intestinal content of the test group (giday et al., 2010). table 9. effects of citrus aurantiifolia (lime juice extract) on volume of intestinal content in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mvic 2.60±0.09 1.60±0.07 1.90±0.27 1.33±0.11 1.10±0.04 piifv 38.46±0.02 26.92±0.21 48.85±0.17 57.69±0.05 𝑃𝐼𝐼𝐹𝑉 = ( 𝑀𝑉𝐼𝐶𝐶−𝑀𝑉𝐼𝐶𝑇 𝑀𝑉𝐼𝐶𝐶 ) 𝑥 100 (5) where, mvic = mean volume of the intestinal content, piifv = percentage inhibition of intestinal fluid volume, mvicc = mean volume of the intestinal content of the control group and mvict = mean volume of the intestinal content of the test group (ekor, 2014). table 10. effects of citrus aurantiifolia (lime juice extract) on charcoal meal distance travelled in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mdtcm (cm) 77.25±0.63 35.50±0.29 40.25±0.25 31.50±0.64 27.75±1.11 pi 68.97±0.13 31.70±0.21 33.04±0.11 28.13±0.12 24.78±0.45 gmeq 54.04±0.13 42.72±0.12 59.21±0.02 64.07±0.31 mean length of small intestine (mlsi) = 112±0.56cm 𝑃𝐼 = 𝑀𝐷𝑇𝐶𝑀 𝑀𝐿𝑆𝐼 𝑥 100 (6) where, pi = peristaltic index, mdtcm = mean distance travelled by charcoal meal and mlsi = mean length of the small intestine (agegnehu et al., 2019) 𝑃𝐼𝑇 = 𝑀𝐷𝑇𝐶𝑀 𝑀𝐿𝑆𝐼 𝑥 100 (7) where, pit = percentage intestinal transit, mdtcm = mean distance travelled by charcoal meal, and mlsi = 310 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 mean length of small intestine (kola-mustapha et al., 2019). 𝑃𝐼 (𝐺𝑚𝑒𝑔) = 𝑃𝐼𝐶−𝑃𝐼𝑇 𝑃𝐼𝐶 𝑥 100 (8) where, gmeq = percentage inhibition and pic = peristaltic index of control and pit = peristaltic index of test group (bern et al., 1992). or 𝐺𝑚𝑒𝑞 = 𝑃𝐼𝐶−𝑃𝐼𝑇 𝑀𝑃𝐼𝐶𝐺 𝑥 100 (9) where gmeq = percentage inhibition in terms of control in the gut travelled distance, mpicg = mean peristaltic index of control group and mpitg = mean peristaltic index of treated group. table 11. the in vivo anti-diarrhoeal index (adi in vivo) (hussain et al., 2009; than et al., 1989). parameter loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 2.5 97.65 195.3 390.6 dfreq 92.49±0.09 47.83±1.08 70.75±0.24 121.34±0.64 gmeq 54.04±0.13 42.72±0.12 59.21±0.02 64.07±0.31 pfreq 50.00±0.03 40.00±0.12 60.00±0.14 70.00±0.04 adi in vivo 62.99±0.07 43.40±0.25 63.11±0.09 81.64±0.2 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √𝐷𝑓𝑟𝑒𝑞 𝑥 𝐺𝑚𝑒𝑞 𝑥 𝑃𝑓𝑒𝑞 3 (10) adi in vivo for loperamide 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(92.49𝑥54.04𝑥50.00) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √249907.98 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 62.99 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.09𝑥0.13𝑥0.03) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.000351 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.07 adi in vivo for low dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(47.83𝑥42.72𝑥40.00) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √81731.904 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 43.40 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(1.08𝑥0.12𝑥0.12) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.015552 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.25 adi in vivo for medium dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(`70.75𝑥59.21𝑥60.00) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √251346.45 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 63.11 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.24𝑥0.02𝑥0.14) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.000672 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.09 adi in vivo for high dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(121.34𝑥64.07𝑥70.00) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √544197.766 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 81.64 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.64𝑥0.31𝑥0.04) 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.007936 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.2 discussion effect of citrus aurantiifolia (lime juice extract) on castor oil-induced diarrhoea in wistar rats. the result of the effect of citrus aurantiifolia on castor oil-induced diarrhoea in wistar rats is presented in table 1. it was observed that the animals had diarrhoea after treatment with castor oil. however, when loperamide (standard drug) was administered there was an observed significant reversal reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) compared to the mean onset time (mot) and the normal control group, this means that loperamide is effective in the treatment of diarrhoea. this reversal is not shocking because loperamide is a known antidiarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, it is expected that loperamide will reverse the isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 311 process. furthermore, when the low dose of citrus aurantiifolia was administered there was also an observed significant reversal and reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. also, when the medium dose of citrus aurantiifolia was administered to the wistar rats, there was an observed significant reversal and reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. since the medium dose of citrus aurantiifolia caused a reversal of the diarrhoea caused by the castor oil, it could mean that it works with the same mechanism with loperamide. therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, it is expected that loperamide will reverse the process. furthermore, when the high dose of citrus aurantiifolia was administered to the wistar rats, there was an observed significant reversal reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. generally, the citrus aurantiifolia is observed to be effective in the treatment of diarrhoea just like loperamide, with the high dose seen to be more effective than other doses. effect of citrus aurantiifolia (lime juice extract) on castor oil-induced enteropooling in wistar rats. the result of the effects of citrus aurantiifolia on castor oil-induced enteropooling in wistar rats is presented in table 2. there was an increase in the enteropooling level of the wistar rats when exposed to citrus aurantiifolia, but when the standard anti-diarrhoeal was administered, there was an observed reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that loperamide can reverse reduction in accumulation of fluids in the small intestine. this reversal is possible because loperamide is a known anti-diarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowel. however, when the standard anti-diarrhoeal was administered, there was an observed reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that loperamide can reverse reduction in accumulation of fluids in the small intestine. furthermore, when the medium dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that the medium dose of citrus aurantiifolia the ability to cause a reversal reduction in accumulation of fluids in the small intestine. since the medium dose of citrus aurantiifolia caused a significant reduction in the enteropooling level of the wistar rats. also, when the high dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. it indicates that the high dose of citrus aurantiifolia could cause a reversal reduction in accumulation of fluids in the small intestine. hence, the loperamide is effective for the enteropooling reversal and citrus aurantiifolia is also effective for enteropooling reversal with the high dose seen to be more significant followed by the medium dose and then the low dose. effects of citrus aurantiifolia (lime juice extract) on castor oil-induced transit time in wistar rats the result of the effect of citrus aurantiifolia on castor oil-induced transit time in wistar rats is presented in table 3. during the study, it was observed that there was an increase in the transit time of the wistar rats when exposed to castor oil but when loperamide was administered, there was an observed reversal reduction in the mean distance travelled by charcoal (mdtcm) meal when compared with the normal control group. this means that loperamide is effective in the reversal of the inability of the ingested food to travel on time. loperamide is a known anti-diarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). and castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, therefore loperamide could reverse the process. furthermore, when the low dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean distance travelled by charcoal meal (mdtcm) when compared with the normal control group. when the medium dose of citrus aurantiifolia was administered there was a significant reduction in the mean distance travelled by charcoal meal (mdtcm) when compared with the normal control group. when the high dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean distance 312 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 travelled by charcoal meal (mdtcm) when compared with the normal control group. the high dose of citrus aurantiifolia caused a significant reduction in the time it takes for ingested food to travel through the gut of the wistar rats, hence, loperamide is effective in the treatment of delay in transit time and citrus aurantiifolia is effective with high dose seen to be more effective followed by medium dose and then the low dose. this finding is similar to a previous study on the antidiarrheal properties of aqueous leaf extract of cyathula prostrata by uahomo and isirima (2022). in conclusion, the results of this study have shown that citrus aurantiifolia demonstrates significant antidiarrhoeal activity and may be working through antisecretory and anti-motility mechanisms or through inhibition of prostaglandin activities and/or synthesis. acknowledgements: the researchers acknowledge all laboratory staffs that assisted in ensuring this research is a success. authors’ contributions: isirima jc designed the study and analyzed 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https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 17-23 | doi: 10.14421/biomedich.2023.121.17-23 issn 2540-9328 (online) the effect of pumpkin fruit ripeness (cucurbita moschata. d) on total flavonoid levels and antioxidant activity linda astutik, eka fitri yanti* departement pharmacy, harapan bangsa jember school health jl. teuku umar no. 67 jember 68133, tel./fax.0331-5102836, indonesia. corresponding author* rofi3k4@gmail.com manuscript received: 01 august, 2022. revision accepted: 31 august, 2022. published: 03 october, 2022. abstract pumpkin fruit (cucurbita moschata. d) belongs the cucurbitaceae family which is a functional vegetable widely distributed in indonesia because it has nutritional value and health benefits. this study aims to determine the value of total flavonoid content and antioxidant activity in the ethanol extract of unripe, mature and ripened pumpkin. simplicia powder was extracted by maceration method using 96% solvent. testing the total flavonoid content with the addition of alcl3 at a wavelength of 425 nm and the antioxidant activity test was carried out using dpph (1-1-diphenyl-2-picrylhydrazyl) as a free radical with a maximum wavelength of 515 nm using microplate reader. the results of the study concluded that the ethanolic extract of pumpkin flesh had a total flavonoid content of 0,146 mgqe/100g in unripe fruit, 0,221 mgqe/100g in mature pumpkin, 0,191 mgqe/100g in ripened pumpkin. the antioxidant activity of the ethanol extract of unripe, mature, and ripened pumpkin fruit obtained was not active or did not completely reduce free radicals. keywords: pumpkin fruit (cucurbita moschata d); total flavonoid levels; antioxidant activity. introduction pumpkin is an annual plant that has been known to the indonesian people for a long time and is widely used in traditional food preparations. pumpkin is considered a functional vegetable that is widespread in indonesia and is very adaptable to various environmental conditions. the availability of pumpkin in indonesia is relatively large. however, the high production of pumpkin in indonesia is not matched by the use of pumpkin (purwanto et al., 2013). the use of yellow pumpkin are still limited to the household scale, which is processed into cooked vegetable from unripe unripe fruits, or dodol, cakes, compotes, and pastries from ripe fruits (hamdi et al., 2017). sharma et al., (2020) suggested that this plant has a broad spectrum for the treatment of diseases associated with its constituent compounds. the skin, flesh, and seeds of pumpkin have high nutritional value because they contain a lot of total phenols, total carotenoids, flavonoids and a large number of macro and micro nutrients (hussain et al., 2021). flavonoids are well-known active substances from plants that act as drugs in the human body. pumpkin seeds and flesh have higher total flavonoid content due to higher metabolism in plant parts, resulting in more metabolites. although the total flavonoid content in pumpkin is less than the total phenol content, the total flavonoid content at low concentrations also has strong antioxidant potential (asif et al., 2017). gumolung et al., (2013) suggested that ethanol extract of pumpkin flesh produced an antidote to free radicals of 62,16%. the ethanolic extract of pumpkin fruit has phenolic compounds, especially flavonoids as antioxidant activity in the moderate category with an ic50 (inhibitory concentration) value of 175,672 μg/ml and as a comparison is trolox with an ic50 value of 33,177 μg/ml (lukita, 2021). this flavonoid compound can release hydrogen radicals contained in the hydroxyl group (-oh) to attach to dpph radicals, so that dpph radicals become stable (sabarudin et al., 2021). in addition, flavonoids have the ability to scavenge free radicals and inhibit lipid oxidation (zuraida et al., 2017). as antioxidants, flavonoids are able to inhibit degenerative and chronic diseases. in addition to antioxidants, flavonoids are said to have hepatoprotector, antithrombotic, anti-inflammatory, and antiviral properties (dewi et al., 2018). the effects of flavonoid compounds are very diverse and very beneficial especially in relation to traditional medicine (sopan et al., 2014). previous studies by mokhtar et al., (2021) conducted an analysis of the phenolic content, flavonoids of (cucurbita moschata d) at various stages of ripening (unripe, mature, ripened) and determine antioxidant activity. the content of polyphenolic and flavonoids https://doi.org/10.14421/biomedich.2023.121.17-23 18 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 compound in riped pumpkin was 97.4 mggae/gram and 28.6 mgqe/gram. ripe pumpkin showed high antioxidant activity against dpph radicals, which was 0.065 ± 0.010 mol te/gram. based on the description above, it is necessary to conduct research to identify total flavonoid compounds and antioxidant activity of yellow pumpkin fruits during the ripening stage (unripe, mature, ripened). so that this research aims to study the changes in the profile of total flavonoid levels during ripening, and the development of antioxidant activity. samples had been taken from tegal rejo village which is part of tegalsari district, banyuwangi regency, east java. the livelihood of the people is 100% in agriculture, one of the cultivated commodities is yellow pumpkin fruit (fauzi dan purnomo, 2016). three fruit samples from each stage of development were selected based on their morphological attributes. materials and methods material unripe, mature, ripened pumpkin fruit obtained from tegal rejo village, tegalsari district, banyuwangi regency, east java, ethanol solvent 96%, methanol, sodium nitrite (nano2) 5%, sodium hydroxide (naoh) 1 m, quercetin, dpph (1,1-diphenyl-2pikrihildrazil), aluminum chloride alcl3 10%, and distilled water. table 1. identification of pumpkin fruits (cucurbita moschata d) of different degrees of maturity. identification description picture unripe pumpkin fruit light green, the shape of the fruit is flat-rounded. the grooves of the fruit are indistinct, fruit patches are present, the color of the flesh is yellowish-green. mature pumpkin fruit orange green, the shape of the fruit is flat-rounded, the grooves of the fruit are not clear, the color of the flesh is orange. ripened pumpkin fruit brown, flat round fruit shape, fruit spots are present, fruit groove is clear, flesh color is dark orange. procedures sample and extract preparation unripe, mature, ripened pumpkins were separated from the seeds and skin, then washed with running water and sliced thinly. then dried using the oven at a temperature of 50o-70o c and put in a blender into powder. furthermore, 100 grams of simplicia powder were weighed at various levels of fruit maturity and extracted with 500 ml of 96% ethanol for ± 3-5 days and stored at room temperature, after which it was filtered. the result obtained is called the filtrate. the filtrate was then concentrated with rotary evaporatoruntil a thick extract is obtained. determination of total flavonoid level contents in pumpkin extracts the method used for total flavonoid contents determination in pumpkin powders was alcl3 colorimetric assay as described by dona et al., (2020) with slight modification. a sample of 5 mg was dissolved in 5 ml of ethanol, so that the mother liquor with a solution concentration of 1000 ppm was obtained. then 100 µl was pipetted, and put into a microwell plate with three replications. then added 50 µl nano2 5% and 50 µl alcl3 10%, and incubated for 5 minutes in a dark room at room temperature, and added 50 µl naoh 1 m, then let stand for 30 minutes in a dark place. then the absorbance was measured using a microplate reader at a wavelength of 425 nm. determination of antioxidant activity contents in pumpkin extracts determination of antioxidant activity contents in pumpkin extracts was performed through a method by nasution dan ardhiyati, 2019). as much of 5 mg of the sample was dissolved in 5 ml of methanol so that the sample concentration was 1000 μg/ml. as much was added 50 μl (plate consists of rows a-h with each astutik & yanti – the effect of pumpkin fruit ripeness (cucurbita moschata. d) … 19 totaling 12 holes). as much as 50 μl was put into each well of rows f, e, d, c, b. furthermore, samples with a concentration of 1000 μg/ml as much as 50 μl were inserted into rows g and f. then diluted rows f, e, d, c, b. row f 50 μl pippeted into row e, row e pipetted 50 μl into row d, row d 50 μl pipetted into row c, row c 50 μl pipetted into row b. row b 50 μl pipetted then discarded, so that the concentration of the test solution is 32 ppm (μg/ml), 63 ppm (μg/ml), 125 ppm (μg/ml), 250 ppm (μg/ml), 500 ppm (μg/ml), dan 1000 ppm (μg/ml). next row g, f, e, d, c, b dan a added 80 μl dpph with a concentration 80 ppm. then incubated for 30 minutes at room temperature to protect from light. the absorbance of the sample was measured using microplate reader at the maximum wavelength 515 nm. then the calculation of the value of % inhibition and calculation of ic50 were performed. % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100 % description: 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 : absorbance of dpph 𝐴 𝑠𝑎𝑚𝑝𝑒𝑙 : absorbance of the dpph solution containing the sample after calculating the % inhibition, the ic50 value was calculated from the linear regression equation, 𝑦 = 𝑎𝑥 + 𝑏 description: 𝑥 : as sample concentration 𝑦 : % antioxidant activity data analysis we analysis of the total flavonoid compounds obtained from the absorbance of the comparison quercetin solution, presented a calibration curve and obtained a linear regression. the average absorbance was fitted in the standard curve equation as the y value, where the x value obtained was the concentration in mgqe/100g. analysis of antioxidant activity was obtained from the value of % inhibition and calculation of ic50. results and discussion yield of pumpkin fruit ethanol extract yield of 96% ethanol extract of unripe pumpkin is 34,2%, ripe pumpkin is 33,1% and mature pumpkin is 19,7% (table 2). the yield shows the active component that was successfully extracted (zuraida et al., 2017). yield results can be influenced by several extraction factors, including time, temperature, type of solvent, ratio of material and solvent, particle size and processing of the plant (chairunnisa et al, 2019). table 2. yield of pumpkin fruit ethanol extract. sample simplicia weight extract weight % yield unripe pumpkin 100 gr 34,2 34,2% mature pumpkin 100 gr 19,7 19,7% ripened pumpkin 100 gr 33,1 33,1% total flavonoid content this analysis aims to determine the total flavonoid content in the extract obtained from the standard curve equation. the choice of quercetin as a standard solution is due to its compounds that are widely distributed in plants (hasanah & novian, 2020). the following absorbance data on the concentration of quercetin produces a standard curve line y= 0,0013x + 0,0783 with an value r2 = 0,945 (figure 1). from this equation, the total flavonoid content obtained is 0,146 mgqe/100g extract of unripe pumpkin, 0,221 mgqe/100g extract of mature pumpkin and 0,191 mgqe/100g extract of ripened pumpkin as shown in table 3. figure 1. the standard curve of quercetin. table 3. data in the total flavonoid content of the ethanol extract of unripe, mature, ripened pumpkin. sample absorbance measurement average ktf (mgqe/100g) sd ktf ± sd (mgqe/100g) 1 2 3 unripe pumpkin 0,111 0,117 0,130 0,146 0,0079 0,146 ± 0,0079 mature pumpkin 0,164 0,176 0,182 0,221 0,0075 0,221 ± 0,0075 ripened pumpkin 0,149 0,151 0,153 0,191 0,0016 0,191 ± 0,0016 antioxidant activity in the antioxidant activity test, that ripe, mature and unripe pumpkins did not completely reduce the antioxidant activity (table 4). y = 0,0013x + 0,0783 r² = 0,945 0,00 0,05 0,10 0,15 0,20 0,25 0 50 100 150 a b so rb a n c e concentration (µ/ml) quercetin 20 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 table 4. the results of the measurement if % inhibition and ic50 value from ethanol extract of unripe, mature, ripened pumpkin. sample konsentrasi (µg/ml) % inhibition ic50 (µg/ml) rata-rata ic50 ± sd (µg/ml) ic50 (µg/ml) raw pumpkin 63 125 250 500 1000 1,1765 5,8824 5,8824 24,7059 35,2941 1337 1443,8 ± 102,37 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 63 125 250 500 1000 1,1765 7,0588 9,4118 15,2941 35,2941 1454 63 125 250 500 1000 3,5294 7,0588 12,9412 15,2941 34,1176 1541 mature pumpkin 32 63 125 250 500 1000 2,198 5,495 7,692 10,989 16,484 31,868 1637 1751 ± 242,05 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 32 63 125 250 500 1000 3,297 5,495 8,791 12,088 17,582 32,967 1587 32 63 125 250 500 1000 4,396 5,495 7,692 10,989 16,484 26,374 2029 ripened pumpkin 32 63 125 250 500 1000 2,198 4,396 5,495 5,495 12,088 20,879 2563 2283,6 ± 319,04 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 32 63 125 250 500 1000 2,198 4,396 6,593 7,692 12,088 23,077 2352 32 63 125 250 500 1000 4,396 3,297 6,593 6,593 14,286 27,473 1936 discussion pumpkin has many health benefits, traditionally used to treat skin diseases, measles, jaundice, insomnia, cancer and can help increase endurance due to its antioxidant activity (sabarudin et al., 2021). different plant parts can have different phytochemical compounds, which can cause different pharmacological effects (sembiring et al., 2018). the extraction method used is maceration. astutik & yanti – the effect of pumpkin fruit ripeness (cucurbita moschata. d) … 21 the maceration method is used because the tools and methods are simple, and it does not use high temperatures which are at risk of damaging the chemical components of materials that are not resistant to high temperatures (prasetya et al., 2020). 96% ethanol is used as a solvent which is polar, so it is good to be used as an extract solvent to extract polyphenol compounds and a solvent that is safe for drugs (dai & mumper., 2010). analysis of the total flavonoid content test was carried out using the instrument microplate reader elisa (λ) 425 nm, because it is simple, easy to operate, faster, can use many samples at once in measurement and can use smaller volumes such as 200-500µl for microplate 96 -well (berg et al., 2016). the measurement of total flavonoid levels using the colorimetric method is based on the formation of a complex reaction between flavonoids and aluminum chloride (alcl3) (zuraida et al., 2017). reagent alcl3 added after nano2 will form a stable complex with a c4 keto group and a c3 or c5 hydroxyl group on flavones and flavonols (syafitri et al., 2014), resulting in a shift in wavelength towards the visible which is indicated by the solution producing a more yellow color. the results of the total flavonoid content obtained are in the unripe pumpkin of 0,146 mgqe/100g extract, mature pumpkin of 0,221 mgqe/100g extract, and ripened pumpkin of 0,191 mgqe/100g extract can be seen in table 2. the values obtained in this study were not much different from those of hasanah dan novian (2020) of 0,00288 mg/g. flavonoids will experience a decrease due to the influence of temperature during the drying process because these compounds are sensitive to light and heat. according to zainol et al (2009) the degradation of flavonoids occurs due to the termination of the molecular chain and the occurrence of an oxidation reaction that causes the oxidation of the hydroxyl group and will form other volatile compounds quickly. testing the antioxidant activity of the ethanolic extract of pumpkin fruit by reducing the free radical 1.1diphenyl-2 pikhrylhydrazil (dpph) using elisa microplate reader (λ) 515 nm. the results can be seen in table 4. that unripe, mature, and ripened pumpkins did not completely reduce the antioxidant activity with ic50. the results obtained in unripe pumpkin of 1432,37 µg/ml, mature pumpkin of 1751 µg/ml, ripened pumpkin of 2283,67 µg/ml which could be categorized as very weak or inactive antioxidant activity (waode rustiah et al., 2018). the difference in activity obtained in each extract is probably due to differences in the content and number of active compounds contained in the extract, so that the antioxidant activity obtained is also different (purwanto et al. 2017). in general, the smaller value ic50 obtained means the higher the antioxidant activity (dona et al., 2020). this is possible that in pumpkin which has flavonoid levels in addition to antioxidant activity and also acts as antifungal, diuretic, antihistamine, antihypertensive, insecticide, antiparasitic, anthelmintic, and antiviral (hasanah dan novian., 2020). another factor that causes weak antioxidant activity is that the compound is still not pure, so it is necessary to do fractionation and purification in the hope that the value will be obtained ic50 of specific compounds that have stronger antioxidant activity. the presence of secondary metabolites other than flavonoids may not provide a synergistic response so that the antioxidant activity produced does not completely reduce (inactive) free radicals (mz et al., 2017). in addition, the cause of the ethanol extract of unripe, unripe, and ripe pumpkin fruit does not completely reduce free radicals due to the phytochemical test results which show a small total flavonoid content value. this is supported by research from gumolung et al., (2013) who stated that the part of the pumpkin that contained high free radical scavenging activity specifically was in the skin, stump and seeds compared to the pumpkin flesh. sopan et al., (2014) also suggested that the flesh of pumpkin (cucurbita moschata d) has large phenolic compounds which are very strong chain-breaking antioxidants and it has been reported that phenolic compounds are associated with antioxidant activity. this phenolic compound has many hydroxyl groups including o-hydroxy groups which have very strong antioxidant potential. according to supriatna et al., (2019), the level of age and maturity of a plant affects the maximum active content of secondary metabolites in the plant. reinforced by the statement metusalach (2007) suggests that the growth of a plant is influenced by external and internal factors. external factors such as habitat, season, water temperature, types of food available and other environmental factors, while internal factors, namely age, size and other biological factors determine antioxidant properties. one of the supporting factors of antioxidant activity is the presence of phenolic compounds and flavonoids that can reduce free radicals, as stated by nuret et al. (2019) which states that there is a correlation between phenolic and flavonoid content on antioxidant activity. the content of phenolic compounds and flavonoids in the extract will act as hydrogen donors, reducing agents and unpaired oxidant absorbers. however, another factor that is no less important to the high antioxidant activity is the other bioactive compounds present in the sample, such as tannins and quinones which also have potential as antioxidants whose levels have not been studied in this study. as for the possibility of other compounds in pumpkin fruit that will have potential as antioxidants when extracted with semi-polar and non-polar solvents. there is necessity to aim of taking non-polar fractions in pumpkin which are thought to have potential as antioxidant compounds. 22 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 conclusions pumpkin (cucurbita moschata d) unripe has a total flavonoid content of 0,146 mgqe/100g, mature pumpkin of 0,221 mgqe/100g, ripened pumpkin of 0,191 mgqe/100g. antioxidant activity in pumpkin fruit (cucurbita moschata d) unripe, mature, and ripened are classified as very weak or inactive because they do not completely scavenge free radicals. conflict of interests: authors state that there is no conflict of interest in this research output. references asif, m., raza naqvi, s. a., sherazi, t. a., ahmad, m., zahoor, a. f., shahzad, s. a., hussain, z., & mahmood, h. & mahmood, n. 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(2017). phenols, flavonoids, and antioxidant activity on the bark extract of pulai stem (alstonia scholaris r. br) (phenolics, flavonoidas, ands antioxidant activity of alstonia scholaris r. br stem bark extract. journal of forest products research. vol 35 no.3: 211-219 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 215-224 | doi: 10.14421/biomedich.2023.121.215-224 issn 2540-9328 (online) effects of ouabain in ehrlich tumor development in vitro and in vivo amanda costa ayres salmeron1, maria beatriz calado2, mateus da silva matias antunes3,*, gabriel rodrigues da silva4, deyse cristina madruga carvalho4, beatriz fernandes de souza4, márcia regina piuvezam5, sandra rodrigues mascarenhas4 1edmond and lily safra international institute of neuroscience, santos dumont institute. macaíba, rio grande do norte, brazil; 2laboratory of immunopathology keizo asami, federal university of pernambuco, recife pernambuco, brazil; 3school of pharmaceutical sciences, university of são paulo. ribeirão preto, são paulo, brazil; 4biotechnology center, federal university of paraíba, joão pessoa, paraíba, brazil; 5health sciences center, federal university of paraíba, joão pessoa, paraíba, brazil. corresponding author* mateusmatias08@gmail.com abstract ouabain (oua) is a cardiotonic steroid with an immunomodulatory and anti-inflammatory role in different experimental models. currently, the potential antineoplastic effect of oua has been studied, however, research is needed to better understand oua role during tumor development. therefore, our aim was to investigate the oua effects on ehrlich tumor (et) development in vitro and in vi vo. to evaluate the cytotoxic effects of oua on et in vitro the cells were incubated with different concentrations of oua during 24h and 48h and our results showed that only the [1000 μm] decreased the number and viability of et cells in the two analyzed times. to s tudy the oua effects on et in vivo, swiss mice were pretreated with 0.56 mg/kg of oua intraperitoneally (i.p.) for three consecutive days. to develop et in the solid form, one hour after the last day of pretreatment, et cells were inoculated subcutaneously into the footpad and the animals were monitored for 13 days. to develop the ascitic form, et cells were inoculated (i.p.) and the animals were monitor ed for 3 days. oua was able to reduce the thickness and weight of the tumor paw, in addition to reduce the weight of the popliteal lymph node. in the ascitic tumor, oua reduced the number of neutrophils and macrophages and increased the lymphocytes in the peritoneum. thus, we demonstrated that oua affects et development both in vitro and in vivo. our results suggest a new perspective in the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain and brings new concepts about the pathophysiological role of this substance. keywords: ehrlich tumor; inflammatory microenvironment; ouabain; solid tumor; tumor development. introduction cancer is characterized by disordered cell growth with the ability to metastasize and interfere with organs function (lópez-lázaro, 2018). the tumor development follows several complex steps, in which the cells gradually acquire a neoplastic phenotype (arneth, 2019). many tumors can alter and control the microenvironment, as a survival strategy, to interfere with the host immune response. one of the cancer hallmarks is the inflammatory microenvironment induced by tumor development (hanahan & weinberg, 2011). a better understanding of the inflammation role during tumor evolution and looking for new substances that can alter inflammatory parameters, such as cell migration and number of viable cells, can be a way to delay tumor development, allowing a longer time window of treatment and resulting in a better prognosis. (kimizgebologlu et al., 2018; wellenstein et al., 2019). ehrlich's transplantable tumor originated from a spontaneous mammary gland carcinoma in female mice and has been used widely for several studies on experimental oncology (havelek et al., 2017; safwat et al., 2020). like other transplantable tumors, this model is very useful since it is possible to determine and adjust the concentration of neoplastic cells to be inoculated and to quantify the growth and regression of the tumor. ehrlich tumor cells develop ascetically when the cells are injected intraperitoneally (fernandes et al., 2015) or solidly when the cells are injected subcutaneously or in the footpad (bahr et al., 2015). this tumor corresponds to an aggressive carcinoma with fast growth, which induces myelosuppression and affects the inflammatory response (brozovic et al., 2009), being a good experimental model for studying the interaction of different substances and the immune system in the tumor development (da mota et al., 2018; machado et al., 2017; moura et al., 2018). to search for new substances that can modulate inflammation and the immune system, and possibly delay the tumor development, ouabain (oua) has shown broader effects (cavalcante-silva et al., 2017; galvão et manuscript received: 19 january, 2023. revision accepted: 12 february, 2023. published: 16 february, 2023. https://doi.org/10.14421/biomedich.2023.121.215-224 216 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 al., 2017; jacob et al., 2013; leite et al., 2015; takada et al., 2009). different in vitro and in vivo models have demonstrated the modulation of cytokines by oua, establishing this molecule as a striking immunomodulator. however, the ability to interfere in immunologic functions is not restricted to the expression of cytokines, because oua also modulates the migration of inflammatory cells, inhibits the pathway of inflammatory mediators, and decreases edema (cavalcante-silva et al., 2017; rodrigues-mascarenhas et al., 2011). in addition, oua has been described as a substance capable of inhibiting neuroinflammation. moreover, in vitro studies have shown the antineoplastic potential of oua in different human tumor cell lines as lung a549 cancer cells, breast mcf7 cancer cells, prostate du 145 cancer cells, osteosarcoma u-2 os cells, melanoma a375 and sk-mel-28 cells, and glioma u‑87mg cells (chang et al., 2019; chou et al., 2018; wang et al., 2021; xiao et al., 2017; yang et al., 2018). modulating the immune system has proven to be one of the most important strategies to fight against tumors. while oua exerts a meaningful role on inflammation, these effects during tumor development have not been properly explored. therefore, to gain more insights about new compounds to control tumor growth, we investigate the effects of oua on ehrlich tumor cells in vitro and on animal experimental models of solid and ascitic tumors. materials and methods animals female swiss mice aged between six and eight weeks, weighing between 25 and 30 g, were used. the animals were kept in polypropylene cages at a temperature of 25 ± 1ºc. they were subjected to light/dark cycles of 12 hours and with free access to water and food throughout the experimentation period. the experimental protocols were submitted to the animal research ethics committee of the pharmaceuticals and medicines research institute/ufpb and were approved under number 093/2016. euthanasia of the animals in all experiments was done with ketamine/xylazine solution (ketamine 100 mg/kg and xylazine 10 mg/kg) injected intramuscularly (i.m.) followed by cervical dislocation. ehrlich tumor cells and in vitro culture cryopreserved samples of ehrlich tumor cells diluted in 1 ml of fetal bovine serum (fbs) + dimethyl sulfoxide 10% (dmso) were thawed and grown in 14 ml dulbecco's modified eagle's medium high glucose (dmem; sigma d1152), supplemented 2,0 g/l sodium bicarbonate, 1 mm/l sodium pyruvate (0,11g/l), 1% penicillin-streptomycin (gibco, usa) + 10% fbs. for experiments a concentration of 5x105 viable cells/ml were used. cell viability was determined by neubauer chamber counting using the trypan blue 0.4% (gibco, usa) exclusion method and an optical microscope (200x increase). after thawing, the cells were maintained in culture for 7 days and subcultures were performed every 2 days, in a 1:3 ratio. after the initial 7 days of cell maintenance and stabilization, the contents of the culture flask was centrifuged at 235 xg at 37°c for 4 minutes (hermle z 326 k centrifuge), the supernatant was discarded and the cells resuspended in 1 ml of dmem + 10% fbs. after, ehrlich tumor cells were seeded in 96-well plate at a concentration of 5x104 viable cells/well together with oua at the concentrations: 1 nm, 10 nm, 100 nm, 1 μm, 10 μm, 100 μm and 1000μm based on studies that describe the concentrations at which digitalis are capable of inhibiting the na+/k+ atpase pump (aizman et al., 2001; bortner et al., 1997; fontana et al., 2013; goto et al, 1992). for the control group phosphate-buffered saline solution (pbs) was used. the plates were incubated in a humid incubator at 37°c and 5% co2, protected from light. all tests were performed in triplicate and two different times were analyzed: 24 hours and 48 hours. procedures  cell viability evaluation and basal cytokine production after oua incubation, cell viability was evaluated by neubauer chamber counting using the trypan blue exclusion method and mtt assay. to perform mtt assay, the 96-well plates were centrifuged at 100 xg at 4°c for 6 minutes (hermle z 326 k centrifuge). the supernatant was discarded and 100 μl of dmem supplemented with 10% fbs + 10% mtt reagent (pbs + 0.5 g/ml mtt) was added to the wells. then the plate was incubated for 4 hours in a humid incubator at 37°c and 5% co2, protected from light. after the incubation time, 100 μl of sds 10% was added to the wells. the plates were under agitation, and protected from light, overnight. subsequently, the plates were analyzed on a spectrophotometer at a wavelength of 570 nm and the absorbance values of each sample were obtained. to measure the production of pro-inflammatory cytokines in vitro, the plates were centrifuged at 100 xg at 4°c for 6 minutes (hermle z 326 k centrifuge). the supernatant was collected, and the cytokine production was evaluated by sandwich enzyme-linked immunosorbent assay (elisa). the cytokines il-6, tnf-α and il-1β were quantified using the kit and protocol indicated by the manufacturer (bioscience, usa). the analysis was performed on a spectrophotometer, at a wavelength of 450 nm, where the absorbance values of each sample were obtained.  induction of ehrlich’s tumor in vivo cryopreserved samples of ehrlich tumor cells were thawed, diluted in pbs and inoculated intraperitoneal salmeron et al. – ouabain in ehrlich tumor development 217 (i.p.) at the concentration of 1x106 cells per animal in a volume of 300 µl, this process is called zero passage. after five days the animals were euthanized and collected the peritoneal lavage and performed the first passage, as described previously. after two passages in vivo, the cells were inoculated into the experimental animals. for experiments, 1x105 cells in a volume of 300 µl was inoculated via i.p. in mice for ascitic tumor model and 1x105 cells in a volume of 50 μl was inoculated subcutaneously in the footpad of the left paw for solid tumor model. in the solid ehrlich tumor model, the contralateral paw was used as a control for the group itself.  animal pretreatment protocol all the animals were pre-treated with pbs or oua at concentration 0.56 mg/kg i.p. for three consecutive days (jacob et al., 2013; leite et al., 2015; rodriguesmascarenhas et al., 2006). this dose of oua was chosen because this concentration corresponds to the levels found physiologically in the human organism (blaustein, 1993). moreover, for solid ehrlich tumor model, on the third day of pretreatment, 1 hour after the last pretreatment, 50 μl of pbs or ehrlich tumor cells were inoculated in the footpad of the left paw and 50 μl of pbs in the right paw. for ascitic ehrlich tumor model, 1 hour after the last pretreatment, 300 μl of pbs or ehrlich tumor cells were inoculated in the animal peritoneum.  solid ehrlich tumor model mice were randomly separated into three different groups: g1, g2 and g3. g1 group (n = 24) was the positive control and was pre-treated with 200 µl of pbs and ehrlich tumor cells were inoculated in the footpad of the left paw. g2 group (n = 24) was the experimental group pre-treated with 200 µl of oua and ehrlich tumor cells were inoculated in the footpad of the left paw. at least, g3 group (n = 20) was the negative control pre-treated with 200 µl of oua and pbs were inoculated in the footpad of the left paw. tumor mass thickness was measured using a digital micrometer (digimess, são paulo, brazil) one hour after inoculation, 24 hours later and thereafter every four days until the 13th day, when the animals were euthanized. after euthanasia, the popliteal lymph nodes of the right and left paw were removed using surgical forceps and the organs were weighed. the right and left foot pads were removed by dislocating the feet at the tibio-tarsal junction and were also weighed. subsequently, the lymph nodes were macerated in 6 ml of pbs. this suspension was centrifuged (200 xg, 4°c, 5 min), the supernatant discarded, and the pellet formed resuspended in 1 ml of pbs solution and cell count was performed using trypan blue exclusion method. for differential counting, 50 μl of cell suspension was centrifuged in a cytospin centrifuge serocite® fanem mod2400 crosshead ref.: 248.059.600 (1500 rpm for 10 min). then, the slides were stained with a panotic kit. after 24 hours of drying, the slides were analyzed using an optical microscope using immersion oil (1,000x increase). the slides were run in the tail region until the count of 100 cells.  ascitic ehrlich tumor model mice were randomly separated into three different groups: g1, g2 and g3. g1 group (n = 6) was the negative control and was pre-treated with 200 µl of saline and 1 hour after the last pretreatment, saline was inoculated. g2 group (n = 6) was the positive control group pre-treated with 200 µl of saline and 1 hour after the last pretreatment, ehrlich tumor cells were inoculated. at least, g3 group (n = 14) was the experimental group pre-treated with 200 µl of oua and 1 hour after the last pretreatment, ehrlich tumor cells were inoculated. the animals were daily weighed before, during the pretreatment, and after the inoculation of ehrlich cells. after 72 hours of tumor cells inoculation, all animals were euthanized, and the abdominal circumference was measured and calculated the tumor volume. after, using a 5 ml syringe, 3 ml of cold pbs was introduced into the peritoneum and the volume of ascitic fluid was aspirated. the samples were centrifuged at 200 xg 4°c for 5 minutes (hermle z 326 k centrifuge), the supernatant was discarded, and the cell pellet formed was resuspended in 1 ml of pbs and used for the perform total cell count and differential leukocyte count. differential cell counting was performed as described previously. data analysis statistical analyses were performed using prism® 6.0 software (graphpad, usa). agostino-pearson and shapiro wilk tests were performed to verify the gaussian distribution. when necessary, the data were normalized using the formula: y = [(y * 100 / 1.647)]. all results were analyzed using the one-way anova method for non-parametric data with tukey's post-test. values of p <0.05 were considered statistically significant. results and discussion oua decreases the number and viability of ehrlich tumor cells after 24 and 48 hours at 1000 μm to understand whether the oua has an influence on ehrlich's tumor cells growth, in vitro assays were performed. at 1000 μm of oua, ehrlich tumor cells reduced cell viability in 39.6% and 49.3% at both 24 and 48 hours, respectively, when assessed by the mtt test (figure 1a-b). moreover, by trypan blue exclusion method, oua has also been shown to be able to reduce the number of total cells in culture (figure 1c-d) and cell viability of at 1000 μm concentration after 24 and 48 hours, reducing viability in 15.8% and 28.1%, respectively (figure 1e-f). 218 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 figure 1. effect of ouabain on number and viability of ehrlich tumor cells in vitro. cell viability was tested by mtt after (a) 24 hours and (b) 48 hours of treatment with oua. cell viability was tested by trypan blue exclusion after (c) 24 hours and (d) 48 hours of treatment with oua. the total cell numbers were analyzed by trypan blue after (e) 24 hours and (f) 48 hours of treatment with oua. although, oua did not alter the basal production of tnf-α (g) and il-1β (h). the graphs a and b were normalized and plotted as the mean ± sem. the other graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. ** p < 0.01 and **** p < 0.0001. oua does not alter basal cytokine production in ehrlich cells our results showed that ehrlich tumor cells produces a low level of basal cytokines tnf-α and il-1β. although, oua does not alter the basal production of the evaluated cytokines (figure 1g-h). moreover, our results did not detect the presence of il-6 in any group (data not shown). salmeron et al. – ouabain in ehrlich tumor development 219 oua delay solid tumor development in mice footpad on the fifth day, it was a significant increase in paw thickness between g1 and g3. on the 9th and 13th day, g2 group showed a reduction in the thickness of the paw with the solid tumor in relation to g1 (figure 2a). moreover, in g2 group, oua reduced the weight of the tumor paw (figure 2b) and increased the weight of the left popliteal lymph node (lymph node closest to the tumor site) in relation to g1 group (figure 2c). regarding the total cell count in the left popliteal lymph node, it was possible to observe that there was no significant difference between groups g1 and g2 (figure 2d). furthermore, the differential cell count indicated that was no significant difference in the pattern of cell migration between the groups analyzed (data not shown). figure 2. effect of ouabain pretreatment on tumor development of the solid ehrlich tumor in the footpad of mice. paw thickness with the tumor was measured on the day of ehrlich cell inoculation and 3, 5, 9 and 13 days after tumor inoculation (a). on the 13th day the animals were euthanized. the (b) paws and (c) popliteal lymph nodes on the right and left side were weighed. the total cell counting of lymph node maceration was also performed (d). the graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. * p < 0.05, ** p < 0.01 and **** p < 0.0001. oua decreases neutrophils and macrophages migration to the tumor microenvironment and increases the migration of lymphocytes our results showed that there was no significant difference between g2 and g3 in relation to the gain or reduction in animal weight (figure 3a), abdominal circumference (figure 3b) and the ascitic fluid volume (figure 3c) of the animals. regarding the total cell count, there was no significant difference between g1, g2 and g3 (figure 3d), however, the cell profile of the peritoneal lavage was different between the groups. the g2 showed a 42.72% increase in the number of neutrophils, a 40.9% reduction in the number of lymphocytes and a 23.3% increase in the number of macrophages when compared to g1. it was observed that oua pretreatment reduced cell migration to the tumor microenvironment. the g3 had a reduction of 81% in the number of neutrophils and 55% in the number of macrophages when compared to the g2. on the other hand, the oua increased the quantity of lymphocytes by 182.17% also in relation to the g2 (figure 3e). 220 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 figure 3. effect of ouabain pretreatment on tumor development of the ascitic ehrlich tumor. (a) the animals were monitored and weighed during the 3 pretreatments (-48, -24 and -1h) and 72 hours after the inoculation of ehrlich tumor cells intraperitoneally. after 3 days of tumor growth, the animals were euthanized. (b) the abdominal circumference was measured and the peritoneal lavage (c) was analyzed by tumor volume, (d) cell viability and (e) differential leukocyte count. the graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. * p < 0.05, ** p < 0.01 and **** p < 0.0001. discussion the development of malignant tumors and the inflammatory process are closely linked events. previous studies have shown that chronic inflammation can make individuals more susceptible to the progression of different tumors (diakos et al., 2014; korniluk et al., 2017). moreover, the tumor development interferes with the innate immune response altering production and secretion of signaling molecules, such as cytokines and chemokines, capable of promoting alterations in tissue structure, formation of new vessels or other events that assist in tumor progression (misra et al., 2018; taniguchi & karin, 2018; velloso et al., 2019). there are different strategies to try eliminating or delay tumor growth, thus, one of the areas of experimental oncology has been looking for, is substances that can control the inflammatory microenvironment during tumor development such as oua. first, to analyze the effects of oua treatment on ehrlich tumor cells, we evaluated cytotoxicity and proinflammatory cytokine basal production in vitro. the results of cell viability tests showed that only the highest concentration tested (1000 μm) was able to decrease cell viability of ehrlich tumor cells in both times analyzed, suggesting a dose-dependent response. this dosedependent response was also observed by other researchers who evaluated the cytotoxicity of five glycosides, including ouabain, in several human tumor cell lines (johansson et al., 2001). furthermore, at both times tested, the viable cell concentration was significantly lower in the 1000 μm concentration suggesting that oua has a cytotoxic effect on cells only at this concentration. regarding the mechanism of action that triggered the reduction of cell viability and concentration of cells in the 1000 μm group, we hypothesize two possibilities: the salmeron et al. – ouabain in ehrlich tumor development 221 first involves classic inhibition of the na+/k+ atpase pump, which can results in a reduction in the concentrations of k+ in the cytoplasm concomitant with higher ca2+ concentrations in the cell cytoplasm, triggering cell death by apoptosis, through the activation of caspases-3 (diakos et al., 2014); and the second possibility involves inhibiting the na+/k+ atpase pump, or altering its kinetics, increasing na+ concentrations along with a reduction in k+ concentration in the cell cytoplasm, favoring the polarization of the cytoplasmic membrane of the tumor cell. thus, cells with lower levels of glutathione, commonly seen in tumor cells, inhibit the depolarization of the cytoplasm membrane causing an increase of tyrosine kinase expression and ras protein, in addition to stimulating the production of superoxide ions, inducing the cell to apoptosis (valente et al., 2003). second, to assess the effects in vivo of oua pretreatment on the development of the ehrlich’s solid tumor, our data showed a reduction in the thickness of the paw with the solid tumor in oua pretreated animals. we observed a weight loss of the tumor paw and the left popliteal lymph node. other studies have already shown that oua can reduce vascular permeability and edema (leite et al., 2015; rodrigues-mascarenhas et al., 2009, 2011). thus, to investigate whether these observed reductions could be associated with alterations in the lymphatic organ drainage, we evaluated the total and differential cell count of the popliteal lymph node. it is interesting to note that although a reduction in the weight of the left lymph node is observed, whose paw received ehrlich cells together with the pretreatment with oua, there was no statistically significant change in the cell count and cell profile, although the graph shows a trend. the increase in the volume of lymph nodes is known as lymphedema and occurs when there is a functional overload of the lymphatic system, either due to hereditary causes or acquired through obstruction or injury caused by infectious processes, diseases, surgery, obesity or as a consequence of malignancies (grada & phillips, 2017; petrek et al., 2000). therefore, it is possible that the reduction in the lymph node weight of the animal pretreated with oua occurred due to the reduction of fluid leakage in the inflamed site, where the ehrlich cells were inoculated, and not due to the reduction in the number of cells in the popliteal lymph node. this is a hypothesis supported by previous data from our group that report the oua ability to reduce plasma exudate leakage in an experimental model of edema in mice (rodrigues-mascarenhas et al., 2011). regarding the ascitic tumor model, our study showed that pretreatment with the oua reduced the migration of cells from the innate immune system to the tumor microenvironment and it is one of the oua antiinflammatory mechanisms of the action. our group has already demonstrated the reduction of eosinophils migration in the experimental model of allergic pulmonary inflammation (galvão et al., 2017) and neutrophils in the experimental model of peritonitis (leite et al., 2015). in addition, the reduction of inflammatory cell migration may indirectly, delaying tumor progression (tsutsui et al., 2005). moreover, a study carried out by our group already demonstrated that oua can inhibit the activation of the nf-κb and p-38 proteins (mascarenhas et al., 2014), both playing an important role in the carcinogenic process, especially in the early stages (agrawal et al., 2001). furthermore, the immune system has a dual role in tumor development may help in the process of carcinogenesis, through the release of proinflammatory cytokines and stimulation of angiogenesis that favors metastases, but it may also be involved in the process to eliminate the tumor with the expression of tumorassociated antigens (de visser et al., 2006). the level of lymphocytes can be a prognostic marker of tumor aggressiveness. it was observed that lower levels of lymphocytes were associated with a greater degree of tumor development and metastasis formation (coffelt et al., 2015). the m1 macrophages are associated with the secretion of pro-inflammatory cytokines, such as interleukin-1β (il-1β) and tnf-α; and m2 have an antiinflammatory profile, with greater secretion of interleukin-10 (il-10) (najafi et al., 2019). additionally, n1 neutrophils can express more immune-activating cytokines and chemokines, lower levels of arginase and greater ability to kill tumor cells. the n2 phenotype is related to the depletion of polymorphs in experimental models to reduce the number of metastases, without the aid of the primary neoplastic focus (giese et al., 2019; park et al., 2016; sagiv et al., 2015). although the oua reduced the tumor volume in the ehrlich experimental models in vivo, this reduction is probably not associated with the cytotoxicity of the glycoside in ehrlich cells. mathematically, the oua concentration used in our in vivo experiments approaches 96 μm/dose and, at that concentration, the oua did not demonstrate cytotoxicity to ehrlich cells in the tests conducted in vitro. most likely, the oua has negatively modulated the development of the tumor due to its antiinflammatory activity. this does not exclude the fact that the oua has previously reported antitumor activity. however, the oua antitumor activity has been demonstrated only in human tumor cell lines as a549 cells, mcf7 cells, du 145 cells, u-2 os cells, a375 cells, sk-mel-28 cells and u‑87mg cells (chang et al., 2019; chou et al., 2018; wang et al., 2021; xiao et al., 2017; yang et al., 2018). here we evaluated the effect of oua on murine cells that are about 1000 times more resistant to the cytotoxic effects of oua compared to human cells due to a difference in the isoform of the α1 subunit of the na+/k+ atpase pump that makes murine cells less sensitive to the oua (akimova et al., 2015). the pathophysiological role of ouabain on tumor development is still little explored, however, here we demonstrate that oua can play an immunomodulatory role in both ascitic and solid ehrlich tumors and delay 222 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 tumor development. it is known that at a concentration of 1000 μm oua acts by inhibiting na+/k+ atpase, consequently reversing the kinetics of the na+/ca2+ exchanger, resulting in an increase in intracellular ca2+ that triggers apoptosis via activation of caspase-3. however, in the in vivo experiments the doses administered to the animals (96 μm/dose) was much lower, precisely to understand the role of the oua in hormonal levels. therefore, our hypothesis is that ouabain in hormonal concentrations acts by downregulating inflammation and thereby interfering with the migration of inflammatory cells to the ascitic tumor and decreasing the extravasation of plasma exudate reducing the tumor mass in mice with solid tumor (rodrigues-mascarenhas et al., 2011). this could be related to the capacity of ouabain to inhibit the nuclear factor nf-kb in low concentrations (leite et al., 2015). this work presents new perspectives on the antiinflammatory potential of oua and brings new ideas about the pathophysiological role of this hormone. conclusions oua in higher doses can decrease the number and viability of ehrlich tumor cells in vitro. in addition, animals that were pretreated with the physiological concentration of ouabain showed a delay in the development of solid and ascitic tumors. pretreatment with ouabain reduced the paw thickness and weight in animals with a solid tumor, in addition to reducing the weight of the lymph node closest to the paw with tumor. in animals with ascitic tumor, pretreatment with ouabain altered the migration profile of lymphocytes, neutrophils and macrophages to the microenvironment of tumor development. taken together, our work demonstrates that ouabain can delay tumor development both in vitro and in vivo, this expands the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain. acknowledgements: the group thanks éssia lima, juliane frança, josé marreiro, josé guilherme, luiz agra, vivian rumjanek for technical assistance. funding was supported by brazilian federal agency for support and evaluation of graduate education (capes), national council for scientific and technological development (cnpq) and federal university of paraiba. authors’ contributions: sandra rodrigues mascarenhas & deyse cristina madruga carvalho designed the study. amanda costa ayres salmeron, maria beatriz calado & mateus da silva matias antunes carried out the laboratory work, collected and analyzed the data, and wrote the manuscript. gabriel rodrigues da silva & beatriz fernandes de souza helped in the treatment of animals. márcia regina piuvezam kindly provided the place where the experiments were carried out. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: supported by the national council for scientific and technological development – cnpq, brazilian federal agency for support and evaluation of graduate education – capes and federal university of paraíba – ufpb. references agrawal, a., choudhary, d., upreti, m., rath, p. c., & kale, r. k. 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(2018). ouabain suppresses the growth and migration abilities of glioma u-87mg cells through inhibiting the akt/mtor signaling pathway and downregulating the expression of hif-1α. molecular medicine reports, 17(4), 5595–5600. https://doi.org/10.3892/mmr.2018.8587 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 169-173 | doi: 10.14421/biomedich.2022.112.169-173 issn 2540-9328 (online) the weight performance stability of mice on modeling obesity-associated hyperglycemia induced by dextrose monohydrate deksa yudha syach putra1, setiyo budi santoso1,2,*, heni lutfiyati1,2 1department of pharmacy, faculty of health sciences; 2consortium for pharmacology and clinical pharmacy studies, universitas muhammadiyah magelang, indonesia. corresponding author* sb@unimma.ac.id manuscript received: 11 august, 2022. revision accepted: 26 august, 2022. published: 06 september, 2022. abstract previously, streptozotocin and alloxan were employed to imitate hyperglycemia in mice. high doses of sucrose were also induced as an alternative. due to body mass index has been associated with hyperglycemia, the evidence of weight body index in various induction alternate kinds, however, have not been fully reported. here-in, we report on the weight performance stability of mice body weight induced by dextrose, streptozotocin, and alloxan. to begin, all mice were divided into six groups of five, with one reserve in each. following seven days of acclimatization, the mice were induced for nine days of hyperglycemia modeling; alloxan (groups a and d), streptozotocin (group b and e), dextrose monohydrate (groups c and f). on preclinical research animals modelling related to obesityassociated hyperglycemia in mice, dextrose monohydrate induction was most successful than streptozocotin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). overall, dextrose monohydrate is most suitable to be used for modeling type 2 diabetes mellitus test animals rather than alloxan and streptozocotin. keywords: alloxan; preclinical research animals; streptozotocin; type 2 diabetes mellitus. abbreviations: type 1 diabetes mellitus (t1dm), type 1 diabetes mellitus (t2dm), glucose transporter 2 (glut2), body weight (bw) introduction diabetic mice are commonly employed in the development of anti-diabetes agent. alloxan and streptozotocin are commonly used to induce diabetic in the mice (al-awar et al., 2016; king & austin, 2017; kottaisamy et al., 2021). in a single induction, alloxan elevated the glycemic index to 127 mg/dl (irdalisa et al., 2015), whereas three days or more raised it to 156 270 mg/dl (dewi et al., 2021; hamdani & nurman, 2020; lolok et al., 2019). streptozotocin had a comparable impact, increasing glucose levels to 136 mg/dl after three days (apriani et al., 2011) and 220244 mg/dl after five days (ocktarini et al., 2011; suwanto & rahmawati, 2019). as an alternative to alloxan and streptozotocin, glucose induction groups have been induced. fructose intake for 12 weeks hold glycemic levels at 81 mg/dl (tillman et al., 2014). dextrose outperformed fructose after seven days of induction, while by 10%concentration raised glycemic at 148 mg/dl (pramushinta et al., 2019) and by 40% at 154 mg/dl (santoso & suryanto, 2017). diabesity is a new term for diabetes that comes alongside obesity (serván, 2013). obese are roughly three times more vulnerable to diabetic (prasetyani & sodikin, 2017). diabetes, most especially type 2 diabetes mellitus (t2dm), is correlated with weight gained (droz et al., 2017) or obesity (fang et al., 2019; finkelstein et al., 2012). weight gained and body mass are two factors that contribute to rising glycemic levels (algoblan et al., 2014), so obesity and t2dm called the twin pandemic (scheithauer et al., 2016). previously, streptozotocin and alloxan were employed to imitate hyperglycemia in mice. high doses of sucrose were also induced as an alternative. due to body mass index has been associated with hyperglycemia, the evidence of weight body index in various induction alternate kinds, however, have not been fully reported. here-in, we report on the weight performance stability of mice body weight caused by dextrose, streptozotocin, and alloxan. https://doi.org/10.14421/biomedich.2022.112.169-173 https://www.google.com/search?client=firefox-b-d&q=glycemic&spell=1&sa=x&ved=2ahukewjxwktujmt5ahvvh7cahrgec14qbsgaegqiaraz https://www.google.com/search?client=firefox-b-d&q=glycemic&spell=1&sa=x&ved=2ahukewjxwktujmt5ahvvh7cahrgec14qbsgaegqiaraz 170 biology, medicine, & natural product chemistry 11 (2), 2022: 169-173 materials and methods ethical clearence the medical and health research ethics committee ethics of the faculty of medicine, universitas gadjah mada, has accepted the whole series of research procedures under registration number ke/fk/0328/ec/2022. animals male balb/c mice were around 2-3 months that bred by pharmacy laboratory of universitas gadjah mada (yogyakarta, indonesia) and treated in accordance with current norms. materials alloxan monohydrate (sigma-aldrich), streptozotocin (sigma-aldrich), and dextrose monohydrate 40% (otsuka) were used to induce mice. metformin (dexa medica) and natrium carboxy methylcellulose (daiichi kogyo seiyaku) were prepared to assess the stability of hyperglycemia treatment. citrate buffer ph 4.5 is solved for intraperitoneal preparation. supplies for glycemic testing, such as strip and glucometer (easytouch). among the other consumables are a handscoon, syringe for 1ml and 5 ml, blood lancet, and aquadest. as support equipment, analytical scales, beakers, mouse cages, markers, mortars and stampers, and oral probes were used. procedures all mice were divided into six groups of five, with one reserve in each. following seven days of acclimatization, the mice were induced for nine days of hyperglycemia modeling and five days of metformin intervention.  induction period to get baseline data, we assessed the weight of the mice following the acclimatization. for 9 days, groups a and d were induced alloxan 0.12 mg/gram body weight (bw) intraperitoneally, group b and e were induced streptozotocin 0.05 mg/gram bw intraperitoneally, and groups c and f were induced dextrose monohydrate 6 mg/gram bw orally. at the end of the induction, we weighed the mice again.  intervention period following the induction period, groups a, b, and c were treated metformin orally, whereas groups d, e, and f as negative controls and were treated na cmc orally. dextrose monohydrate continued to activate groups c and f throughout the intervention. on day 14, the body weight was measured. results and discussion performance of alloxan induction mice in groups a and d gained 35.86 grams and 37.98 grams of body weight after nine days of alloxan induction, respectively. mice on group a grew to 37.50 grams after a five-day metformin intervention, whereas control mice (group d) stayed constant at 37.98 grams. (figure 1). figure 1. the body weight gain due to alloxan induction in the intervention group (a) and the control group (d). the solid line represents the induction period, the dashed line represents the intervention period. performance of streptozotocin induction mice in groups b and e gained 35.80 grams and 36.60 grams of body weight after nine days of streptozotocin induction, respectively. mice on group b grew to 39.10 grams after a five-day metformin intervention, whereas control mice (group d) stayed constant at 36.60 grams. (figure 2). figure 2. the body weight gain due to streptozotocin induction in the intervention group (b) and the control group (e). the solid line represents the induction period, the dashed line represents the intervention period. performance of dextrose monohydrate induction mice in groups c and f gained 40.33 grams and 36.08 grams of body weight after nine days of dextrose monohydrate induction, respectively. both groups thereafter continued to rise to 41.90 grams and 39.36 putra et al. – the weight performance stability of mice on … 171 grams respectively, after receiving metformin intervention for five days (figure 3). figure 3. the body weight gain due to dextrose monohydrate induction in the intervention group (c) and the control group (f). the solid line represents the induction period, the dashed line represents the intervention period. induction variant performance in mice treated with metformin was compared. after the 9-day induction period, dextrose-induced mice gained the most weight (40.33 grams), followed by alloxan-induced mice (35.86 grams), and streptozotocininduced mice (35.80 grams). after 5 days of metformin treatment, dextrose-induced mice had the best body weight performance (41.90 grams), followed by streptozotocin-induced (39.10 grams) and alloxaninduced (37,50 gram) figure 4. the body weight gain in mice following 9 days of induction (solid line) to alloxan (a), streptozotocin (b), and dextrose monohydrate (c), and persistence rate following 5 days of metformin intervention (dash line) after obesity (day 14th). dextrose monohydrate induction in mice was most successful than streptozocotin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). table 1. percentage of body weight gain in mice following 9 days of induction to alloxan (a), streptozotocin (b), and dextrose monohydrate (c), and persistence rate following 5 days of metformin intervention after obesity (day 14th). group label induction treatment body weight (percentage increase) baseline 9th day treatment 14th day treatment a alloxan 31.341.27 (-) 35.863.58 (14%) 37.504.54 (20%) b streptozotocin 31.862.69 (-) 35.803.58 (12%) 39.103.01 (23%) c dekstrose monohydrate 30.882.35 (-) 40.332.66 (31%) 41.903.28 (36%) discussion on preclinical research animals, we simulate obesityassociated hyperglycemia in mice using dextrose monohydrate induction. the bulk previous studies employed streptozotocin and alloxan to imitate hyperglycemia. however, as one of the important criteria in hyperglycemia etiology, the investigation of body weight issues was disregarded. due to the importance of publishing our findings, we would show the evidence of steady rates of dextrose monohydrateinduced body weight during the experiment. according to the availability of test animals in preclinical research on the developing of anti-diabetic agents, there are two options: genetic modification, or chemical induction (kottaisamy et al., 2021). in the unavailability of genetically diabetic test animals, alloxan and streptozotocin are widely used to cause diabetes in mice (al-awar et al., 2016; kottaisamy et al., 2021). streptozotocin and alloxan, on the other hand, act on beta cells via the glucose transporter 2 (glut2) and cause complete ablation of beta cells inside the islets, leading in severe insulin insufficiency, hyperglycemia, and weight loss, they are more suited for modeling type 1 diabetes (king & austin, 2017). mice given alloxan or streptozotocin lost weight because they were unable to use the available glucose for energy. to meet the body's energy requirements, excessive catabolism of protein and fat in muscle and adipose tissue was performed (malik et al., 2015; sinata & arifin, 2016; suwanto & rahmawati, 2019). since the great majority of t2dm are overweight (finkelstein et al., 2012; wilding, 2014), body mass index has long been linked to hyperglycemia and t2dm 172 biology, medicine, & natural product chemistry 11 (2), 2022: 169-173 (algoblan et al., 2014; jung & choi, 2014), with the potential of obesity to promote insulin resistance being the primary explanation (jung & choi, 2014). obesity was proclaimed in mice as test animals when they weighed more than 20% of their age-matched mates. (patonah et al., 2018). common sugar exacerbates obesity and increases adiposity in mice (kleinert et al., 2018). adipocyte hypertrophy and hyperplasia lead to excessive lipid accumulation (lipotoxicity), which activate insulin resistance (lee et al., 2020; longo et al., 2019). indeed, the diets manipulated of mice with high sucrose feeding generate a compensatory response from some beta cells, (king & austin, 2017). additionally, the weight gain was also linked to the disfunction of leptin (anorexigenic hormone) due to the circulating of glucose which led the test animals to consume more feed (tillman et al., 2014). throughout the investigation, the induction performance of dextrose monohydrate has been shown to increase glycemia. dextrose monohydrate is more suitable to be used for modeling type 2 diabetes mellitus test animals due to the aspect of increasing body weight, rather than the two toxic compounds that actually suppress body weight, and they are more suitable for modeling mice with diabetes mellitus type 1 as well, aside from having a higher level of glucose elevation than alloxan and streptozotocin. conclusions on preclinical research animals modelling related to obesity-associated hyperglycemia in mice, dextrose monohydrate induction was most successful than streptozotocin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). throughout the investigation, dextrose monohydrate is most suitable to be used for modeling type 2 diabetes mellitus test animals rather than alloxan and streptozotocin. acknowledgements: this study is part of the acceleration research scheme for institutional vision revitalization research output (refference number: 012/kontrak/prvi-pa/2022) in the subject of exploring the biological and pharmacological effects of employing plants, fungi, animals, microbes, and minerals. authors’ contributions: conceptualization, sbs; methodology, sbs and hl; data collection, dysp; writing-original draft preparation, sbs and dysp writing-review & editing, dysp, sbs. and hl. all authors read and approved the final version of the manuscript. competing interests: the authors claim to have no conflicts of interest. the funders had no say in the design of the investigation, data collection, analysis, or interpretation, article writing, or decision to publish the findings. the authors declare that there are no competing interests. funding: the authors are thankful to the research, development, and community service department of universitas muhammadiyah magelang for financial supporting. references al-awar, a., kupai, k., veszelka, m., szűcs, g., attieh, z., murlasits, z., török, s., pósa, a., & varga, c. 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(2014). the importance of weight management in type 2 diabetes mellitus. international journal of clinical practice, 68(6), 682–691. https://doi.org/10.1111/ijcp.12384 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 55-60 | doi: 10.14421/biomedich.2023.121.55-60 issn 2540-9328 (online) acute toxicity ld50 fraction ethyl acetate aquilaria malaccensis, ficus benjamina, mikania micrantha, and fraction water cinnamomum burmanii in mus musculus rizky yulion1,*, santi perawati1, barmi hartesi1, lia anggresani2, lili andriani1, lesra indriani1, lara syahila1, suci ramadani1, nadia monika1 1program studi farmasi, stikes harapan ibu jambi jl. kol. tarmizi kodir no.71, pakuan baru, kec. jambi sel., kota jambi, jambi 36122, indonesia. 2sekolah tinggi ilmu kesehatan syedza saintika padang jl. prof. dr. hamka no.228, air tawar tim., kec. padang utara, kota padang, sumatera barat 25132, indonesia. corresponding author* rizkyyulionputra10@gmail.com manuscript received: 28 september, 2022. revision accepted: 05 october, 2022. published: 17 october, 2022. abstract research on the acute toxicity of ld50 had been carried out on the ethyl acetate fraction of gaharu leaves (aquilaria malaccensis), beringin leaves (ficus benjamina), sembung rambat leaves (mikania micrantha), and kayu manis cortex (cinnamomum burmanii) water fraction the base of use as traditional treatment by the suku anak dalam (sad) empirically obtained from ancestors. this research method was experimental and it used male and female mice. the control group was given a 1% nacmc doses of 625 mg/kg body weight, 1,250 mg/kg body weight, 2,500 mg/kg body weight, and 5,000 mg/kg body weight. the test preparation was administered orally once per day. the thomson-weil method was used for the measurement of ld50 values. the weight ratio of organs was applied for heart, liver, lungs, kidneys, and stomach. the results showed that the ld50 value for male mice was 2454mg/kg body weight; 2454mg/kg body weight; 1546 mg/kg body weight and 2065mg/kg body weight respectfully. the ld50 value for female mice was a pseudo ld50. the value of the organ weight ratio in each sample showed p<0.05 values that were obtained in gaharu leaves in the liver and kidney organs (male); heart in females. beringin leaves value p<0.05 lung organs (male). sembung rambat leaves p<0.05 values of the heart, liver, and stomach organs (male); heart and stomach (female). cinnamomum cortex value p<0.05 liver and stomach organs (male). the conclusions showed that the ld50 value of the ethyl acetate fraction of gaharu leaf, sembung rambat leaf, beringin leaf, and kayu manis cortex in male mice was slightly toxic, while in female mice it could not be counted. keywords: beringin leaf (ficus benjamina l.); gaharu leaf (aquilaria malaccensis); kayu manis cortex (cinnamomum burmanii); sembung rambat leaf (mikania micrantha kunth); toxicity acute ld50. introduction suku anak dalam (sad) is part of a minority group on the island of sumatra, precisely in the interior area of jambi province, indonesia (setiyadi et al., 2020). at the end of the 18th century, the jungle people (suku anak dalam) encountered foreigners who brought in the infectious disease, smallpox, and reached epidemic and severe levels. this encouraged people to seek healing from plants in the forest and in addition to the knowledge of traditional medicine obtained from ancestors (ayuningtyas et al., 2020; mustika & dastina, 2020; najib, 2020). some plants that can be used in traditional medicine are gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. gaharu leaves are used to overcome insomnia, beringin leaves are usually used as influenza medicine, sembung rambat leaves are used as wound medicine and kayu manis cortex can be used as a solution for diabetes, this is used empirically. several studies have been reported related to the plant that gaharu leaf contains secondary metabolites, namely flavonoids, steroids, tannins, triterpenoids, saponins, alkaloids, and sesquiterpene metabolites (δcadinene, gurjunene) (fitriani & erlyn, 2019; wong et al., 2015). beringin leaves contain kaempferol, chlorogenic acid, alkaloids, flavonoids, saponins, steroids, and polyphenols (aslamiah & haryadi, 2013; corrêa et al., 2012). sembung rambat leaf contains lignin (lim et al., 2020), isoledene, δ-cadinene, debromofiliformin, trans-caryophyllene, β-bisabolene, germacrene-d, zingiberene (saikia et al., 2020), protein, phenolics, and proline (jali et al., 2021). the kayu manis cortex contains polyphenols (rozi et al., 2022) alkaloids, tannins, phenolics, flavonoids, quinones, and triterpenoids (hananti et al., 2012). https://doi.org/10.14421/biomedich.2023.121.55-60 56 biology, medicine, & natural product chemistry 12 (1), 2023: 55-60 the acute toxicity test is a pre-clinical trial aimed at measuring the degree of the toxic effect of a compound within 24 hours after a single dose. the purpose of the acute toxicity test is to determine the potential for acute toxicity (ld50) (khumaidi et al., 2018; oecd, 2022; ugwah-oguejiofor et al., 2019). based on literature studies that have been carried out, no research has been found using the ethyl acetate fraction of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex water fractions to test for acute toxicity of ld50. we interested to know the acute toxicity test of ld50 in mice as a research experimental animal so that the use of active doses is more guaranteed. materials and methods the study area quantitative experimental studies have been conducted to trace the acute toxicity value of ld50 in four types of medicinal plants in jambi province. the results of a fraction of ethyl acetate aquilaria malaccensis, ficus benjamina, mikania micrantha, and fraction water cinnamomum burmanii were used as samples on albino mice (mus musculus). furthermore, tracing the impact of sampling on the liver, kidney, heart, lung and stomach organs was carried out. tools and materials the tools used in this study were a set of vacuum rotary evaporators, analytical scales, balance, lumps and stampers, glass bottles, stirring rods, filter paper, funnels, measuring cups, aluminum foil, erlenmeyer, measuring flask, separator funnels, spatulas, vaporizing cups, drip pipettes, masks, hand scoops, tissue, napkins, syringe, needles, surgical tools mice, styrofoam, and squeak basins. the ingredients used were mice (25 males and 25 females), gaharu leaf extract, beringin leaf extract, sembung rambat leaf extract, kayu manis cortex extract, 70% ethanol, n-hexane, ethyl acetate, distilled water, hgcl2, ki, i2, subnittric bismuth, glacial acetic acid (ch3cooh), hcl, ammonia (nh3), chloroform (chcl3), magnesium (mg), water, iron (iii) chloride solution (fecl3), ether, sulfuric acid (h2so4), and acetic acid anhydride (c4h6o3). procedure plant determination determination of plants is was carried out to avoid mistakes in the identification of plants. determination of gaharu leaf plants, beringin leaf, sembung rambat leaf, and kayu manis cortex at the biota identification and determination laboratory, padjadjaran university (unpad) indonesia. ethical clearance ethical clearance is a written statement given by the research ethics commission for research involving living things and which states that a research proposal is feasible to be carried out according to 7 who standards. ethical clearance was carried out at the ethics committee of the health service poltekkes of the ministry of health, jambi. ethically feasible information that has been carried out consecutively for acute toxicity tests ld50 ethyl acetate fraction of gaharu leaf, beringin leaf, sembung rambat leaf and kayu manis cortex water fractions in white mice no. lb. 02.06/2/ 104/2021; no. lb.02.06/2/090/2021; no. lb. 02.06/2/122/2021. sample and material processed samples of gaharu leaf were used as much as 5 kg, beringin leaf as much as 1 kg, sembung rambat leaves as much as 5 kg, and kayu manis cortex as much as 1 kg. the sample was washed thoroughly using running water first to separate the dirt, the next step was drying it by aeration. samples that had been dried then chopped for maceration (hermes et al., 2021). extraction in the extraction process of samples that had been cleaned, dried, and pureed, the next step was to extract by the maceration method using 70% ethanol (1: 10 b/v) in a closed dark bottle allowed to stand for 24 hours. further filtered and squeezed, the pulp is added again 70% ethanol until submerged, soaked, and filtration is carried out for 3 days with 3 changes of solvent or carried out until the color of the solvent was clear. after all, the resulting filtrates were then mixed, then the next step was concentration using a rotary evaporator at a temperature of 500c until finally the results of the viscous extract were obtained (dechayont et al., 2021; khan & islam, 2012). the working principle of a rotary evaporator was to evaporate the extraction solvent and leave only the distraction resulting compound called extract. fractionation extracts of gaharu leaf, beringin leaf, spliced leaves of propagation, and kayu manis cortex are concentrated and then fractionated by liquid-liquid extraction (ecc) using a solvent of 1: 10 based on the degree of polarity. the first thing to do was that the sample extract was stirred well-used water (polar), after mixed was put in the separator funnel then n-hexane (non-polar) is added in a ratio of 1: 1, close the separator funnel put under the hand then shake several times interspersed with opened the faucet of the separator funnel to remove the gas, so as not to cause too strong pressure to occur. then after the sound of the gas does not come out again, it was allowed to stand for a few minutes so that there was a separation of 2 phases, namely the n-hexane phase and yulion et al. – acute toxicity ld50 fraction ethyl 57 the water phase, separate the n-hexane phase to the limit of solubility to the container. furthermore, the ethyl acetate (semi-polar) fraction with the same workmanship was used, after all the fractionation of the n-hexane solvent, ethyl acetate and the remained water obtained, were separated first based on the solvent. then re-concentrated using a rotary evaporator until it gets a viscous fraction result (liu et al., 2013; rachmawaty et al., 2019; shah & gilani, 2012). selection and setup of test animals the test animals used were healthy male and female mice, aged 2-3 months with a body weight of mice of 20-30 grams. prepared 50 heads of mice. mice are divided into 5 groups and each group consists of 5 male mice and 5 female mice. mice are acclimatized for 7 days and before treatment were fasted for 18 hours. the doses used were the control group given na cmc 1%, the treatment group was given a dose of 625 mg/kgbb, 1,250 mg/kgbb, 2,500 mg/kgbb and 5,000 mg/kgbb (oecd, 2022; vogel, 2002). organ weight ratio all groups of test animals performed surgery and observations on the organs of the liver, heart, lungs, stomach, and heart. the organ was then weighed and compared with body weight to obtain the organ index (rajeh et al., 2012). data analysis used the spss (statistical product and service solutions) program to see the difference in the ratio of squeak organ weights between sexes and dose groups after administration of the test preparation, if the data was not distributed normally, the kruskal-wallis test is continued. the significance of the obtained results was judged at the 5% level results and discussion results plant determination plants were determined at the biota identification and determination laboratory, padjadjaran university (unpad). the determination results obtained that the sample used was aqularia malaccensis lam, ficus benjamina l., mikania micrantha kunth, and cinnamomum burmanii. extraction results the extraction method used was the maceration method. the sample was soaked with 70% ethanol solvent and let stand in a sealed dark vial for 24 hours. soaked and filtration are carried out for 3 days with 3 solvent changes. after all, filtrates were obtained and then evaporated using a rotary evaporator (dillasamola et al., 2021; halim et al., 2020). a total of 1,400 g, 1,000 g, 1,000 g, and 1,000 g of gaharu leaf powder extracted, beringin leaf extracted, sembung rambat leaf extracted, and kayu manis cortex extracted respectfully were successively obtained condensed extracts of 85.41 g, 77.72 g, 70.29 g, 81.6 g with a yield of 6.10%, 6.4%, 7.02%, 8.16%. fractionation results fractionation aims to separate the components of the active compound from the extract that has been produced. the results of the viscous extract of the sample are further fractionated by separation based on their polarity. fractionation in this study used non-polar solvents, namely n-hexane, and semi-polar solvents, namely ethyl acetate (liu et al., 2013; rachmawaty et al., 2019; shah & gilani, 2012). the results of the yield of the fractionation of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex successively accorded to the polarity of the solvent obtained an n-hexane fraction of 13.43%; 10,8%; 12,73%; 0,06%. the ethyl acetate fraction was obtained 7.62%; 6,09%; 11,50%; 2.16% and the residual fraction of water obtained 23.00%; 22%; 35,86%; 30,00%. table 1. total number of male mice deaths on gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. normal 625mg/kgbb 1250mg/kgbb 2500mg/kgbb 5000mg/kgbb total survive dead survive dead survive dead survive dead survive dead survive dead gaharu leaf 5 0 4 1 5 0 2 3 2 3 18 7 beringin leaf 5 0 3 2 4 1 2 3 3 2 17 8 sembung rambat leaf 5 0 5 0 3 2 2 3 2 3 17 8 kayu manis cortex 5 0 5 0 4 1 3 2 1 4 18 7 58 biology, medicine, & natural product chemistry 12 (1), 2023: 55-60 table 2. total number of female mice deaths on gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. na-cmc 1% 625mg/kgbb 1250mg/kgbb 2500mg/kgbb 5000mg/kgbb total survive dead survive dead survive dead survive dead survive dead survive dead gaharu leaf 5 0 5 0 4 1 3 2 3 2 20 5 beringin leaf 5 0 4 1 5 0 5 0 2 3 21 4 sembung rambat leaf 5 0 5 0 4 1 4 1 3 2 21 4 kayu manis cortex 5 0 5 0 4 1 3 2 3 2 20 5 table 3. the data value of the influence of the ratio of organ weights on each sample to the organ of mice (p>0,05). heart liver lungs kidney stomach male female male female male female male female male female gaharu leaf no yes yes yes no no yes yes no no beringin leaf no no no no yes yes no no no no sembung rambat leaf yes yes yes yes no no no no yes yes kayu manis cortex no no yes yes no no no no yes no discussion ld50 in this study, the fifty mice used were divided into 5 groups, each group consists of 5 male mice and 5 female mice. before treatment, all mice were acclimatized for 7 days with the aim that the test animals could adapt to the laboratory atmosphere (tested environment). before being given the treatment of mice, they were fasted for 18 hours but were still given a drink. mice are satisfied so that when given the sample treatment were expected to directly interact with the digestive system and not be disturbed by the presence of food contained in the digestion of mice. mice were administered sample extracts orally (vogel, 2002). in male mice with gaharu leaf samples, the results of this study showed that the value of ld50 ethyl acetate fraction of gaharu leaves obtained a result of 2,454 mg/kg body weight are included in the category of slightly toxic. the toxicity ethyl acetate fraction of beringin leaves obtained a result of 2,454 mg/kg body weight belongs to the category of slightly toxic. the toxicity ethyl acetate fraction of sembung rambat leaf obtained a result of 1546 mg/kgbb, belonging to the category of slightly toxic. the toxicity water fraction of the cinnamomum cortex obtained a result of 2065 mg/kg body weight belongs to the category of slightly toxic. in female mice, no meaningful value of ld50 founded after being given extracts of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. it can be said that this is included in the pseudo ld50. this is possible due to the influence of hormonal cycles that can cause estrogen hormone levels to increase. if the level of the hormone estrogen increases then the immune system also increases or was stronger than the immune system of the male mice, so that the male mice can experience death. in female mice, ld50 calculations could not be carried out because they did not meet the mortality criteria of the thompson-weil method. organ weight ratio in the statistical test of the organ weight ratio after administration of the ethyl acetate fraction of gaharu leaf, there was a significant difference in female mice in the heart organs. while in male mice there are significant differences in the liver organs and kidney organs. in the ethyl acetate fraction of the leaves of the splice of the sembung rambat leaf, there were significant differences in female mice in the organs of the heart and stomach. while in male mice there were significant differences in the organs of the liver, heart, and stomach. this was likely due to the presence of content of secondary metabolite compounds. secondary metabolites are chemical compounds that were formed in plants. secondary metabolites in addition to having physiological effects that can nourish or cure diseases can also cause undesirable side effects on the human body or certain animals. in plants, each compound works together to influence the other to increase its activity or effectiveness. as for compounds that can damage liver cells, including alkaloids and saponins, both compounds can damage cells and tissues and even cause death in experimental animals. for data on the ratio of organ weights after administration of the ethyl acetate extract fraction of beringin leaf to the heart, liver, kidney, and stomach organs of male and female mice, data were obtained with p>0.05 meant that there was no noticeable difference in the weight of the heart, liver, kidney and stomach organs at various doses, while data on the weight ratio of male lung organs were obtained data marked p <0.05 which means that there was a difference yulion et al. – acute toxicity ld50 fraction ethyl 59 in the weight of the male lung organs in various doses. in statistical tests of kayu manis cortex fraction on the weight of mice organs, there were significant differences in the lung organs of male mice and the gastric organs of male and female mice. the ethyl acetate fraction of beringin leaf and the water fraction of kayu manis cortex may contain flavonoid compounds, which is known if flavonoids at excess levels in the cells can cause a halt in active transport which causes an uncontrolled intake of ions in the cells so that it can cause cell death (necrosis) in the lung organs (tanduwinata et al., 2015). in the lungs, there is an alveolus that functions as a place for gas exchange, where each wall is coated by cells. the alveolus is declared damaged when dilation of the alveolus is founded and this can trigger the onset of inflammatory infiltrates. there are significant differences in the gastric organs of male and female mice, it can be concluded that there is gastric damage in male white mice and female white mice. kayu manis cortex may contain flavonoid compounds that are thought to have an inhibitory effect against lipoxygenase and cyclooxygenase. where one of the product pathways of cyclooxygenase, namely prostaglandins, is thought to have a protective effect on the stomach. a deficiency of prostaglandins is suspected to lead to a decrease in the production of mucus, phospholipids, hco3 secretions, the proliferation of mucosal cells, and microvascular flow of the stomach that is suspected to cause discontinuities in the epithelium of the gastric mucosa known as peptic ulcers. the mechanism is suspected to be the cause of swelling of the gastric organs in male white mice and female white mice after administration of the kayu manis cortex fraction (astri et al., 2012). conclusions based on the studies that have been carried out, it can be concluded that the ld50 values of gaharu leaf, geringin leaf, sembung rambat leaf, and kayu manis cortex in male mice are categorized as slightly toxic and in female mice cannot be calculated or show pseudo-ld50 values. the value of the organ weight ratio in each sample shows p<0.05 values were obtained in gaharu leaves in the liver and kidney organs (male); heart in females. beringin leaves value p<0.05 lung organs (male). sembung rambat leaves p<0.05 values of the heart, liver, and stomach organs (male); heart and stomach (female). cinnamomum cortex value p<0.05 liver and stomach organs (male). acknowledgements: thanks are given to all colleagues who have helped in the process of this research. authors’ contributions: rizky yulion designed the study. rizky yulion, lia anggresani and lara syahila carried out the laboratory work for gaharu leaves (aquilaria malaccensis). rizky yulion, lili andriani and lesra indriani carried out the laboratory work for beringin leaves (ficus benjamina). rizky yulion, santi perawati and suci ramadani carried out the laboratory work for sembung rambat leaves (mikania micrantha). rizky yulion, barmi hartesi and nadia monika carried out the laboratory work for kayu manis cortex (cinnamomum burmanii). rizky yulion analyzed the data. rizky yulion wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: there was no conflict of interest in this study. funding: there is no cost support from any party for this research. the study was carried out at the own expense of the researchers. references aslamiah, s., & haryadi, h. 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(2015). evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant-fungus interaction in aquilaria malaccensis. journal of chromatography a, 1387, 104–115. https://doi.org/10.1016/j.chroma.2015.01.096 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 119-131 | doi: 10.14421/biomedich.2022.112.119-131 issn 2540-9328 (online) detection of the atherosclerotic pcsk9 gene inhibitors through in silico method to improve targeted therapy sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah* sadakathullah appa college, thirunelaveli – 627011, india corresponding author* sethuraji90@gmail.com manuscript received: 27 june, 2022. revision accepted: 19 july, 2022. published: 31 july, 2022. abstract the pcsk9 is one of the most important marks for the evolution of therapeutic agents for atherosclerosis because its interaction with lowdensity lipoprotein receptors causes atherosclerosis. protein-ligand interactions help us to understand the true mechanism of pharmacological action. this study seeks to identify the most powerful suppression options for pcsk9. initially, the reported ace inhibitors were included in pharmacophore modeling using pharmagist. next, zincpharmer was used to screen the selected model against a zinc database to identify putative drug candidates docked to the target protein to understand the interactions. the 10 best pharmacological candidates for pcsk9 with a binding energy of 9.8-8.2 kcal mol-1 were identified by molecular docking and their pharmacokinetic properties and oral bioavailability were evaluated. the (s) severalplant obtained chemicals have been discovered, including anti-hypersensitive drugs such as “canadine, hesperetin, and labetalol”. according to biochemistry, these compounds formed a stable “protein-ligand” complex. the (s) canadine pcsk9 complex had the lowest rmsd and was the most stable. future in vitro studies could identify (s) canadin as a promising atherosclerosis inhibitor for the evolution of novel pcsk9 inhibitors. keywords: pcsk9; therapeutics; ace inhibitors; protein – ligand; docking. introduction highly elevated ldl cholesterol known as “familial hypercholesterolemia” (fh) is recognized as a hereditable disease. it can lead to an increase of low density lipoprotein (ldl) cholesterol in the blood. this can increase ldl quantity by more than 4 mmol / l (> 180 mg/dl) (zhao et al., 2019), increasing the threat of cardiovascular diseases (cvds) such as atherosclerosis, heart failure and myocardial infarction, etc. patients with genetic modification in the familial hypercholesterolemia gene, such as low density lipoprotein receptor (ldlr), proprotein convertase subtilisin/kexin type 9 (pcsk9), and apob, are at increased risk of atherosclerosis (ascvd) than those without the mutation but with comparable cholesterol levels (khera et al., 2016). mutant fh-related genes can cause an increase in low-density lipoprotein cholesterol (ldlc) (semenova et al., 2020), cause vascular endothelial dysfunction. ascvd, including cad, is a well-known disease characterized by endothelial dysfunction. endothelial dysfunction is the first step in the development of atheroma. hypercholesterolemia is one of the most common causes of endothelial dysfunction that causes stenosis of the aorta of the heart (zhang et al., 2020). cholesterol is required for some physiological activities. cell membranes are important organelles that require large amounts of cholesterol. in addition, cholesterol is essential for the production of bile acid and steroid hormones. an increase in cholesterol, particularly under oxidizing circumstances, may culminate in atherosclerosis, causing carotid, peripheral artery, and coronary heart disease (andreadou et al., 2017). one of the leading causes of stroke and heart attack is atherosclerosis. cholesterol buildup may result in the formation of arterial plaques, leading to atherosclerosis. elevations in ldlc and apolipoprotein b100 (apob100) are precisely connected to cardiovascular events such as atherosclerosis. decreased ldlc levels may reduce the risk of cardiovascular events, as reduction of apob on the arterial wall can cause lesion and lead to the formation of atherosclerosis (mahley, 2016). ldlc deposits can result in fatty streaks accumulation and reduced flow of blood to many organs as a result of inflammatory changes in the arterial wall. pcsk9 interacts with the ldl receptor (ldlr) and causes liposomal degradation that promotes atherosclerosis. therefore, pcsk9 inhibition is essential for risk halting of cvds (latimer et al., 2016). pcsk9 is a protein with 692 amino acids which in mainly expressed in liver and intestine (horton et al., 2007). pcsk9 is synthesized in the endoplasmic reticulum (er), and its 73 kda precursor peptide is zymogen. this zymogen makes many changes before it stick out the https://doi.org/10.14421/biomedich.2022.112.119-131 mailto:sethuraji90@gmail.com 120 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 cell surface. there are auto-catalytic activity in-between 152nd and 153rd amino acids. the n-terminal pro-domain stays firmly linked with the catalytic domain in the secretory route(hyock et al., 2008). pcsk9 can break ldlr with its proteolysis activity when in contact with the extra-cellular sites of ldlr. pcsk9 has a subtilisin-like catalytic domain that can bind to the ldlr-egfa domain. this domain contains asp374, and changing to tyr increases pcsk9's affinity for ldlr (hyock et al., 2008). binding of low density lipoprotein to low density lipoprotein receptor is important for reducing lipid sedimentation and the threat of cvds. when pcsk9 binds to ldlr, it protects ldl from attaching to low density lipoprotein (ldlr) and causes lipid sedimentation. to avoid cad, you should block the association between pcsk9 and its ldlr (horton et al., 2009). natural treatments using mab and short molecule drug therapies are the two main approaches used in pcsk9 targeted therapies. unfortunately, certain advances in small molecule drug therapy for pcsk9 inhibition are hampered by the fact that the molecular structure of pcsk9 required for small molecule binding to reduce the activity of this protein has not been identified (joseph & robinson, 2015). computational methods based on bioinformatics and chemoinformatics help identify new binding sites for inhibiting proteins. bioinformatics and chemoinformatics deal with the creation and manipulation of databases and statistical methods for the management and analysis of biological and chemical data. these devices help to find and study about targets, chemical structures, and mobile sites. these devices can also discover potential medicinal molecules for specific therapeutic targets and quantify their medicinal similarity via molecular docking (wishart, 2005). medicinal candidates are evaluated for their binding affinity and their properties are subject to change as needed. the concept of computationally-based drug discovery is called structural design. therefore, the in silico approach can provide a broad and favorable approach for identifying new target proteins and predicting their biological activity (ramharack & soliman, 2018). the drug components such as, “captopril, zophonopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, sirazapril, moexipril, trandolapril, alicin, teprotide” were used in this incilico study (attique et al., 2019). using computational approaches such as the pharmacophore design of existing drugs, new small inhibitors of pcsk9 have been identified. molecular docking was performed to study the interaction of pcsk9 target proteins with computer-identified new drugs. this study is essential for the discovery of new drugs targeting pcsk9 that can be administered to cure or reduce heart disease and address the increasing challenges associated with excess lipids for the reduction and treatment of atherosclerosis or its symptoms. materials and methods 3d structure of the protein of interest (pcsk9) the 3d structure of the target protein was obtained from the pdb library (https://www.rcsb.org/search) (burley et al., 2021). the interaction between the egfa domain of pcsk9 and ldlr occurs at the target site. this interaction (du et al., 2011) between the pro-domain and c-terminal of pcsk9 increases blood ldlc quantity and thus increases susceptibility to cvd. the pdb id 6u26 (petrilli et al., 2020) was determined based on its quality (1.49) after analyzing all known structures in the pdb database, including domains with one active site. pcsk9 active site characterization the active site of pcsk9 was found in-between a unique pocket, catalytic and c-terminal domain(petrilli et al., 2020). lig plot is used to plot this pocket (figure 1). ligplot provides data on hydrogen bonds and hydrophobic contacts (ra & mb, 2011). figure 1. ligplot for 6u26: (figure 1) it shows data on hydrogen bonds and hydrophobic contacts. preparation of the protein target and ligand an autodock tool(hanwell et al., 2012) was used to purify target protein to remove heteroatom, polar hydrogen, and colman charges. the 3d structure of the pcsk9 target protein was generated by converting to the pdbqt format. angiotensin-converting enzyme sethuramalingam et al. – identification of pcsk9 inhibitors 121 inhibitor (acei) with atherosclerosis activity was found in ligand synthesis (table s1). those 2 dimensional structures were taken from the pubchem database(kim et al., 2021) using individual pubchem ids and energy was reduced using the chemaxon marvin suite. we used raccoon software (hanwell et al., 2012) to convert the energy-optimized structure to pdbqt format. table 1. the ei and pi of the molecules. molecule name pubchem id binding energy residues ei pi captopril/ 44093 -7.0 asp 651 and arg 545 0.60 0.98 zofenopri 92400 -9.3 arg 458 and trp 461 0.24 0.89 enalapril 5388962 -8.8 arg 458 0.28 0.80 ramipril/ 5362129 -9.4 arg 458 0.33 0.88 quinapril 54892 -9.9 arg 458 0.24 0.60 perindopril 107807 -8.2 arg 458 0.30 0.93 lisinopril 5362119 -8.8 arg 458 0.49 0.92 benazepri 5362124 -8.3 arg 458 and arg 476 0.51 2.03 fosinopri 55891 -8.7 arg 357 0.08 0.74 cilazapril 56330 -9.6 arg 458 0.09 0.46 moexipril 91270 -9.4 arg 458 0.27 0.86 trandolapril 5484727 -9.1 arg 458 2.52 2.40 allicin 65036 -9.9 arg 458 2.35 2.68 teprotide 443376 -9.7 arg 458, arg 357, asn 298, trp 461, cys 323 and arg 458 2.77 2.67 (table 1) it shows the ei and pi of the molecules with its binding energy. molecular docking of recognised ace inhibitors with psck9 the energy-reducing structure (pdbqt format) was docked to the target protein using autodock vina (ukuku et al., 2012). all known molecules with above average binding energies (moexipril, trandolapril, allicin, teprotide, zophenapril, lamapril, quinapril) were selected for the pharmacophore design. inhibitor selection was based on intracting energy and structural comparison (table 1). pharmacophore designing/modeling the pharmacophore was designed using pharmagist (laskowski et al., 2018), and the pharmacophore was selected predicted on its high range and the number of molecules placed in the design. pharmacophores are a three-dimensional sequence of properties (chemical bond donors, chemical bond acceptors, anions/cations, aromatic rings, hydro-phobic groups) required for a ligand to communicate with a particular target (schneidman-duhovny et al., 2008). screening virtually for the pharmacophore prior to wet-lab investigations, one of the fundamental levels in drug improvement is digital screening. this manner consists of estimating the binding affinity of a capability medicine candidate to a goal protein. virtual screening is likewise utilised to evaluate capability binding modalities of the medicinal candidate and different drug-like short molecules throughout interplay with the goal protein. using hpc “high-performance computing” configuration technologies (jaghoori et al., 2016), the maximum distinguished medicine applicants displaying capability binding affinity in the direction of the goal protein can be eliminated. through the virtual screening manner, numerous bioactive compounds that could have interaction with the goal protein can be found (langer & hoffmann, 2008). the pharmacophore become absolutely screened towards the zinc drug database the use of zincpharmer (koes & camacho, 2012), and 1033 hits/ compounds have been identified. molecular docking for the last molecules screened sdf-formatted structures (each molecule in a single file) were obtained and used to dock the terminal of screened 1033 compounds (using the desired pharmacophore). i used a python script to separate and reduce the energy of these structures. the resulting structure was changed to pdbqt format using vina to dock to the required protein. all compounds were docked to autodock vina (guidi et al., 2006) and the top results (contingent on interaction and affinity energy) were examined using autodock tools (hanwell et al., 2012). the docking grids coordinates were 40.516000 (x), 24.375625 (y), and 25.098375 (z), and each grid had dimensions of 25. there were 20 modes in all. table 2 shows the 10 most important zinc molecules selected as potential treatment candidates. 122 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 table 2. amino acid interactions of molecules. molecule name binding energy number of interactions amino acid interaction apomorphine -10.8 7 arg 458, pro 438, arg 357, trp 461 and val 460 oxyphencyclimine -10 8 cys 358, pro 331, ala 478, val 460, arg 458, val 359, thu 459 and pro 438 canadine -10 7 arg 357, asp 360, pro 331, ala 478, val 460 and pro 438 naltrexone -9.8 10 arg 476, arg 458, val 460, pro 331, ala 330, thr 335, ala 328 and cys 358 oxolinic -9.6 7 leu 440, pro 438, trp 461, val 650, thr 437 and thr 459 norfloxacin -9.6 9 trp 461, leu 436, ile 416, ala 649, pro 438, arg 458, arg 357 and arg 412 canadine -9.6 7 arg 458, asp 360, pro 438, ile 416, leu 440, thr 459, val 460 and arg 357 hesperetin -9.4 9 arg 412, ala 330, arg 357, cys 358, pro 331, trp 461, thr 459 and val 460 labetalol -9.3 9 trp 461, cys 358, thr 437, pro 438, val 650 and asp 651 benorilate -9.2 6 arg 458, val 460, arg 357, pro 438 and leu 436 (table 2) the binding energy of top ten molecules are shown with their amino acid interactions. final medication candidates' pharmacokinetic characteristics we used openbabel (http://openbabel.org/wiki/main page) to create a simplified molecular input system & # 40; smiles & # 41; autodockvina for the top 10 compounds identified during virtual screening. the resulting smiles code is zinc (sterling & irwin, 2015), molinspiration cheminformatics' free online service (,pubchem. analyzed using a comparison website. , swiss adme (daina et al., 2017) and admet sar (yang et al., 2019). swissadme was used to calculate the oral bioavailability of each drug (table 3). for bioactivity prediction and drug similarity from lipinski's rule 5, use the predict bioactivity tool available to calculate molecular properties and bioactivity scores. was evaluated by. the le and lelp parameters were found from the specified results. table 3. bioavailability of drugs. molecule formula mw ali log s bioavailability score synthetic accessibility apomorphine c17h18no2 268.33 -3.88 0.65 4.29 oxyphencyclimine c20h28n2o3 344.45 -4.89 0.65 5.08 (s)-canadine) c20h22no4 340.39 -4.65 0.65 4.68 naltrexone c20h24no4 342.41 -3.69 0.65 5.76 oxolinic acid c13h11no5 261.23 -3.16 0.66 3.26 norfloxacin c16h19fn3o3 320.34 -1.14 0.65 1.5 (r)-canadine c20h22no4 340.39 -4.65 0.65 4.68 hesperetin c16h14o6 302.28 -5.27 0.65 4.22 labetalol c19h25n2o3 329.41 -5.86 0.65 4.04 benorilate c17h15no5 313.3 -4.5 0.65 3.16 (table: 3) the molecular weight and the bioavailability value of various molecules are given with its synthetic accessibility. molecular dynamics of complexes of protein and ligand the molecular dynamics of the protein-ligand complex was prepared and calculated using gromacs (abraham et al., 2015) version 2018.4 on a desktop computer with a 2.80 ghz and nvidia graphics geforce gtx 1060. we did it with a 6gb card. the gromacs source code was taken from the website http://www.gromacs.org/ and built using opencl version 1.2 of microsoft visual studio community msvc19.16.27025.1 and nvidia gpu computing toolkit cuda v10.0 for windows 10. the pdbqt file of the protein-ligand complex obtained by virtual screening at autodock vina is used as the first coordinate source for both psck9 and the screened virtual inhibitor. using open babel, the coordinates were converted to in pdbqt format, pdb for proteins and mol2 for ligands. the generated pdbqt file was reconstructed in swissmodel (waterhouse et al., 2018) using the entire human pcsk9 amino acid sethuramalingam et al. – identification of pcsk9 inhibitors 123 sequence (bateman et al., 2015; leinonen et al., 2004) and lacked the original 3d structure. i filled in the loop that is. new pdb coordinates were retrieved and utilised by gmx pdb2gmx with the charmm36 allatom force field to generate the gro protein topology (july 2020). in the avogadro programme (http:// avogadro.cc/) (hanwell et al., 2012), mol2 files were updated with all the missing hydrogen atoms. the updated mol2 files were visually verified and modified for problems. using sort mol2 bonds, a perl script created by justin a. lemkul and provided under the gpl3.0 licence, the bonds in the coordinate files were separated in ascending order. such processed mol2 data was sent to swissparam (https://swissparam.ch/) to build a ligand topology for retrieving “itp and pdb” files. the received pdb file was converted to gro using gmxeditconf and the itp file was correctly placed in the respective topol.top file for each ligand. next, we defined the unit cell and filled it with water. gmxeditconf generated a dodecahedron cell from the proteinligandgro file and the correct topol.top file. sodium and chloride ions were added to achieve a concentration of 0.155 m with a neutral charge while energy was minimised. the ligands and protein were individually restrained by constructing position restraint topologies, and twophase 100 ps, 310 k, and 1 bar equilibrations were carried out to create first nvt and then npt outputs. terminal molecular dynamics production was set for “10 nanoseconds” of simulation at 320 kelvin and 1 bar of pressure, and a single simulation of a proteinligandcombination on the previously reported computer equipment lasted around 24 hours. for both energy minimization and equilibration, the similar temperature and pressure conditions were employed. visual molecular dynamics (vmd) (yamada et al., 2017) was used to visualise the results, and gromacs gmx rms was used to determine the rootmeansquare deviation (rmsd). results and discussion results active site 3d structure of the target protein (pcsk9) figure 1 shows the ligplot of the structure expressing the residues participated in the interaction. the dashed line shows the hydrogen bond interaction between the amino acids and legends of the target protein. figure 2 uses discovery studio to contrast this interaction and distinguish the active area residues from the rest of the structure. figure 2. active cite pocket. (figure 2) ball sticks representation of active site pocket utilizing molecular docking research to identify known inhibitors for pharmacophore design. to identify the optimal structure of pcsk9 and its active sites and recognized ace inhibitors such as, “captopril, zophenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, silazapril, moexipril, trandolapril, alicin”, etc. we reviewed the literature in (s1 table). all known acei were docked to the target protein pcsk9 and the top results were evaluated based on the interaction between binding energy and the active site. molecules evaluated on the basis of pharmacophore in this study, pharmacophore was adopted as a drug discovery strategy and a novel pcsk9 inhibitor was used with established inhibitors such as, “moexipril, trandolapril, alicin, teprotide, zophenopril, ramipril, quinapril” shown in table s1. the pharmacophore model created by pharmagist was evaluated and the optimal pharmacophore was selected based on a score of 18.102 and 5 attributes (1 hydrophobicity, 1 negative ion, and 3 hydrogen bond acceptors). rice field. in zincpharmer, pharmacophores were screened along the entire zink drug database and 1033 hits / molecule were identified. molecular docking to identify the top 10 most promising medicinal candidates after evaluating all 1033 molecular docking complexes with the required protein, their affinity energies and interactions were saved. the docking results of the top 10 zinc molecules were sorted based on the interaction of the active sites assessed for lower binding energies calculated by autodock (table 2 and figure 3a–3j). lower energies exhibit stronger affinities and sustained interactions. therefore, the top compounds may be more effective in exerting the expected activity of the pcsk9 and are therefore excellent therapeutic candidates. 124 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 final 10 drug candidates the drugs such as, “apomorphine, oxyphencyclimine, (s) canadine, naltrexone, oxolinic acid, norfloxacin, (r) canadine, hesperetin, labetalol, and benolylate” were the top 10 choices. table s4 shows their pubchemcid, name, structural formula, putative characteristics, and drug similarity according to lipinski's rule of five. according to pubchem, apomorphine (zinc00009073) is the first molecule with the least change in docking energy, a non-selective dopamine used to treat parkinson's disease. oxyfencyclimin (zinc00020260) is an oral muscarinic receptor antagonist used to treat gastric ulcers and gastrointestinal problems. the third is (s) canadine which is also called as (s) tetrahydroberberine and xanthopsin (zinc00033518), which is a benzylisoquinoline alkaloid (bia) in the protoberberine structural subgroup (xu et al., 2019) and is found in some poppy and corydalis plants. the next substance (zinc00001773) is naltrexone, which is commonly used to treat alcohol or opioid addiction. oxolinicacid, zinc00001875, is a synthetic antibiotic used for veterinary medicine. the antibiotic zinc00003742 is norfloxacin, which has broadspectrum antibacterial activity over the gram-negative and gram-positive bacteria. (r) canadine is an enantiomer of (s) canadine. zinc00039092 is a secondary plant metabolic product, hesperetin, that acts as an antioxidant and antitumor agent. labetalol is the chemical name for 2 hydroxy 5 [(1s) 1 hydroxy 2 [[(2r) 4 phenylbutane 2 yl] amino] ethyl] benzamide, also known as zinc00000416. it is a third-generation vasodilator, an antihypertensive drug selective α1 adrenergic blocker and non-selective β-adrenergic blocker. the final product (zinc00001003) is benorilate, an esterified codrug of aspirin and acetaminophen. it is used as an anti-inflammatory and antipyretic drug(bass, 1973). figure 3. the 10 drug candidates. (figure 3) it shows the final drugs table 4. pubchem ids and names of molecules. molecule name cids le lelp alogp milogp drug similarity apomorphine 6931310 0.5 8.7 2.43 4.16 pass oxyphencyclimine 667690 0.5 9.9 3.73 4.57 pass (s)-canadine 21171 0.5 9.3 2.67 3.99 pass naltrexone 5360515 0.5 4.9 1.11 2.37 pass oxolinicacid 4628 0.6 2.5 2.42 1.68 pass norfloxacin 4539 0.5 -2.8 1.24 -1.69 pass (r)-canadine 443422 0.4 9.7 2.67 3.99 pass hesperetin 72281 0.5 6.1 3.52 2.94 pass benzamide labetalol 134045 0.4 9.2 2.11 3.85 pass benorilate 21102 0.5 8.3 3.79 3.61 pass (table 4) the pubchem ids and the drug similarity report based on the “lipinski rule” is given in detail sethuramalingam et al. – identification of pcsk9 inhibitors 125 the pharmacokinetic characteristics and bioavailability of the medication will be evaluated. using tools, “molinspiration, knime, swissadme, and admetsar, computational structural analysis, druglikeness, adme characteristics, oral bioavailability, and toxicity profiling” have been conducted (3 and 4 tables). using smiles codes, the pinnacle molecules have been re-searched towards the zinc database, molinspiration, and pubchem, and that they have been all recognized as acknowledged compounds, a number of plant beginning such as (s)-canadine and hesperetin. an in addition antihypertensive agent, labetalol, turned into found (4 table). depending at the diploma of nitrogen protonation, numerous chemical substances containing nitrogen atoms may also exist in lots of paperwork. these compounds incorporate numerous pubchem codes, distinct smiles, however are stated as "discern compounds" on this database. consequently, it is able to be concluded that they are the equal chemical compounds that tackle numerous paperwork in accordance at the ph. figure 4 depicts the availability radars of the pinnacle ten nominated compounds. figure 4. molecules radar chart. (figure 4) – this shows the molecular radar chart evaluation of the stability of the suggested proteinligand complexes upon visual inspection with vmd, (s) canadin, hesperetin, and labetalol produced a strong proteinligand complex throughout molecular dynamics and remained at the docking targets for at least 10 ns. the rmsd value of the heavy atom of the ligand in the 10ns lasting simulation of the three compounds was 0.007. 0.13 0.024; 0.13 and 0.035 (mean sd) (figure 5), respectively, suggesting that the position of the ligand changes only slightly. the value of (s) canadine is 0.1, indicating the excellent stability of this compound. this molecule produced the most stable (s) canadine complex in terms of duration and molecular dynamics, with the lower rmsd (fig. 5). this compound can be further researched for the discovery of new pcsk9 inhibitors. 126 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 figure 5. rmsd stimulation. (figure 5) – it shows the rmad stimulation discussion the manufacturing of arterial plaques is one of the main reasons of atherosclerosis, which in the end outcomes in arterial blockage and coronary heart attack. moreover, better stages of apob-a hundred and ldl cholesterol are without delay connected to atherosclerosis-associated cvds. the lipid cycle commonly begins with the liver`s launch of im-mature vldl. in addition, apob-a hundred, cholesteryl esters, triglycerides, and ldl cholesterol are present. vldl broaden and end up a supply of electricity in “adipose tissues, skeletal and cardiac muscular tissues” all through movement withinside the blood (chaudhary et al., 2017). one of the jobs of vldl is the trade and elimination of triglycerides, which ends up withinside the conversion of vldl into intermediate-density lipoproteins (idl) (shelness & sellers, 2001). some of those idl can be removed through the liver all through endocytosis, whilst idl with a more ldl cholesterol content material are transformed to ldl, which additionally consists of apob-a hundred. this apob-a hundred attaches to ldlr at its n-terminal area after which passes via the acidic endosome via endocytosis. ldlr in the end breaks from ldl and is reintroduced into the mobileular membrane (ding et al., 2015). at an acidic ph, structural changes in ldlr might also additionally decorate pcsk9's affinity for ldlr relative to ldl(horton et al., 2009). although mutations might also additionally dispose of the catalytic hobby of pcsk9, they do now no longer have an effect on its cappotential to bind to ldlr. a stoppage withinside the protein's manufacturing might also additionally bring about blockading its binding to ldlr. pcsk9 mrna degradation might also additionally in the end hinder its reference to ldlr, or a tiny drug meant to save you this courting also can do so (mcnutt et al., 2007). atherosclerosis is as a result of the interplay among pcsk9 and ldlr (egf-a area or epidermal boom factor-like repeats), which inhibits the endocytosis of ldl. the connection among pcsk9 and ldlr can be blocked to halt the buildup of ldl. pcsk9 is produced withinside the er and transferred to the plasma membrane to be able to have interaction with ldlr. protein-protein interactions govern a extensive quantity of organic activities. the law of those interactions is a famous and potential drug improvement pathway. to save you the egf area of ldlr from interacting with pcsk9, it's far important to perceive the interplay's goal location. it is acknowledged that antihypertensive medications, specifically ace inhibitors, have a essential position in atherosclerosis (pitt, 1995). acei are putative plaque formation inhibitors (ferrari et al., 2010). experimental, epidemiological, and scientific research have proven that ace inhibitors decorate arterial endothelial feature and, thus, lower the improvement of atherosclerosis (lonn, 2001). it has been proven that they lower erythropoietin (epo) stages and hematocrit. acei might also additionally modulate a whole lot of bioactive peptides, along with angiotensin 1–7 and substance p. in hematopoiesis, numerous sethuramalingam et al. – identification of pcsk9 inhibitors 127 peptides had been located to have a position. reducing the quantity of angiotensin ii might also additionally restrict erythropoiesis. acei additionally have an effect on the renin-angiotensin system (younas et al., 2017). several ace inhibitors, along with “captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, cilazapril, moexipril, trandolapril, allicin, and teprotide”, had been used on this study. captopril decreases ldl oxidation, which is understood to make contributions to the improvement of atherosclerosis (k.r. et al., 2014). zofenopril and enala prilare acei which can lessen vascular harm and enhance endothelial progenitor mobileular movement. they inhibit endothelial harm and the improvement of atherosclerosis (pines & fisman, 2003). arterial plaque formation is one of the leading causes of atherosclerosis and ultimately causes arterial occlusion and heart attack. in addition, high levels of apob100 and ldl cholesterol are directly associated with cardiovascular disease associated with atherosclerosis. the lipid cycle usually begins with the release of immature vldl by the liver. it also contains apob100, cholesteryl ester, triglycerides and cholesterol. vldl develops in adipose tissue, skeletal muscle, and myocardium during circulation in the blood and becomes an energy source (chaudhary et al., 2017). one of the roles of vldl is the exchange and removal of triglycerides, converting vldl to intermediate density lipoprotein (idl) (shelness & sellers, 2001). some of these idls can be eliminated from the liver during endocytosis, but idls with high cholesterol content are converted to ldl, which also contains apob100. this apob100 binds to ldlr at its n-terminal domain and passes through acidic endosomes by endocytosis. ldlr is eventually cleaved from ldl and reintroduced into the cell membrane (ding et al., 2015). at acidic ph, changes in ldlr conformation may increase the affinity of pcsk9 for ldlr compared to ldl [10]. mutations can eliminate the catalytic activity of pcsk9, but do not impair its ability to bind to ldlr. interfering with protein production, can block binding to the ldlr. degradation of pcsk9 mrna can ultimately prevent its association with ldlr. alternatively, the same can be done with small medicines designed to prevent this relationship (mcnutt et al., 2007). atherosclerosis is caused by the interaction of pcsk9 with ldlr (egfa domain or epidermal growth factor-like repeat) and inhibits ldl endocytosis. you can block the connection between pcsk9 and ldlr to stop ldl accumulation. pcsk9 is produced in the er and transcribed into the plasma membrane to interact with the ldlr. proteinprotein interactions control a significant number of biological activities. ace inhibitors (table 1) have been shown to play an active role in the prevention of atherosclerosis. therefore, pharmacophore modeling and screening using the zinc database has been introduced into the drug development process. the binding affinity of the newly identified chemical (1033) from the zinc database was assessed using the autodock tool. the program calculated several factors, including reduced binding energy, interaction with the active site, and the number of hydrogen bonds. comparing these criteria yielded a list of the 10 most promising drug candidates for atherosclerosis (table 2). some of the molecules virtually screened from the zinc database showed good interactions, while others showed no or weak interactions. the results show that the top 10 compounds with a binding energy of 9.8-8.2 kcal / mol (table 2) are similar to the reference compounds (table 1) and are therefore desirable and viable drug candidates. candidate target for pcsk9 in atherosclerosis. in other words, in this study, the topselected compounds showed the highest binding affinity for the pcsk9 target protein for atherosclerosis. in addition, molinspiration, computational structural analysis of these compounds using swiss adme, drug similarity, adme properties, and oral bioavailability, based on these results, (s) canadin, hesperetin, and labetalol contain heavy atoms and have le and lelp values, which is why further research and possible design of new drugs based on pharmacophore structure is the most. we can conclude that it is a promising candidate. same range as known ace inhibitors (tables 1 and 4). the results of autodockvina led to the selection for further molecular dynamics analysis of the complex with pcsk9. during molecular dynamics, all three compounds appeared to form a stable pcsk9ligand complex, staying at the docking site for at least 10 ns, and their heavy atom rmsd values were minimal. a notable advantage of (s) canadine, also known as (s) tetrahydroberberine, is a significant reduction in rmsd (figure 5), which promotes the formation of a highly stable complex with pcsk9. this is an interesting finding, as studies suggest that berberine inhibits psck9 (xu et al., 2019). although the direct inhibitory effect of such compounds is unknown, this study provides insights into (s) canadin and berberine with very similar pharmacophore structures that differ only in the degree of hydrogen atom saturation. increase. berberine is an aromatic chemical, but (s) canadine is not. experimental studies in mice have found that berberine delays the onset of atherosclerosis in cholesterol-fed apoe / knockout mice (wan et al., 2018). in addition, a study of the coptis chinensis decoction network path, a traditional chinese healing method, revealed: based on these results, (s) canadin, hesperetin, and labetalol are the most promising candidates for further research, and (seidah et al., 2017) the potential for new drug development based on the study is (s) canadin and berberine. we can conclude that it indicates that. of the most probable drug for atherosclerosis. in vivo studies conducted with hesperetin to assess its role in ldlr expression showed that hesperetin dose (200m) stimulated ldlr gene expression, increased mrna levels of transcription factors srebp1 and srebp2, 128 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 and plasma levels. the effect of ldlc on reducing the risk of cardiovascular disease has been suggested to reduce (bawazeer et al., 2016). in addition, studies suggest that activation of srebp2 causes co-expression of pcsk9 and ldlr, leading to ldlr depletion and increased ldlc circulation (maxwell et al., 2003; urban et al., 2013). as a result, the effects of hesperetin can be counteracted. studies show that saturation increases with increasing fsp3 levels (lovering et al., 2009; wei et al., 2020). the relationship between fsp3 and the solubility and melting point attributes of 1000 compounds in the dataset showed a positive correlation between fsp3 and solubility and a negative correlation with melting point (lovering et al., 2009). compounds with a lower melting point show better absorption than compounds with a higher melting point (chu & yalkowsky, 2009). therefore, low chemical saturation (fsp3) can lead to high melting points, limited drug absorption, and reduced oral bioavailability. therefore, it is assumed that low levels of hesperetin saturation (table 3) reduce oral bioavailability. although various studies have shown that hesperetin has low oral bioavailability (kanaze et al., 2007; liu & chen, 2008), it is possible to increase oral bioavailability by specific chemical approaches [69]. (s) and (r) canadins have unique bioavailability properties due to their ability to efficiently cross the blood-brain barrier (bbb) and reach their targets (table 3). their high gastrointestinal absorption and ability to bind to pgp make these molecules excellent candidates for orally administrable drugs (laskowski et al., 2018). based on current research, the pharmacophore structure of (s) canadin may function as an inhibitor of the pcsk9 protein molecule. using existing ace inhibitors, pharmacophore was used as a drug discovery method to identify new pcsk9 inhibitors. a literature search was conducted to identify the optimal structure of pcsk9 and the active site of known ace inhibitors. the results of molecular docking, incorporating a computational and unbiased approach, highlighted the relevance of drug development in atherosclerosis. in this study, the binding affinities were analyzed and the molecules with the highest affinities for the target of atherosclerosis were zinc000073, zinc00020260, zinc00033518, zinc00001875, zinc00003742, zinc0003517, zinc000390, zinc000390, zinc0003900, zinc0000390, zinc000390, zinc000390, zinc000390, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900 lipinski's rule 5 was applied and passed for these major drug candidates. several molecules from virtual screening and drug database searches were found as herbal chemicals such as (s) canadine, hesperetin, or the antihypertensive drug labetalol. the formation of stable protein-ligand complexes by these chemicals has been demonstrated using molecular dynamics. for (s) canadine, the complex prepared with this chemical is the most stable in molecular dynamics and has the lowest rmsd. conclusions the pcsk9 inhibitors are found through the computational methods, these inhibitors can be used as a vital target for the novel drug screening and discovery for atherosclerosis in future. the computational findings should be conformed in future using invitro and invivo method for further understanding the inhibitors role in atherosclerosis. hence a depth wet lab research need to be done with is inhibitors as a target for treating atherosclerosis conflict of interest: the author has no relevant financial or non-financial interests to disclose. code availability: no code availability data availability: data sharing not applicable to this article as no datasets were generated or analysed during the current study. authors’ contribution: first author s sabarinathan and second author revathy l r have contributed in all aspects for the prepration of manuscript and generation of idea under the guidance of dr r janet rani (corresponding author) competing interests: the author declares there is no competing interest funding: no funding was received to assist with the preparation of this manuscript references abraham, m. j., murtola, t., schulz, r., páll, s., smith, j. c., hess, b., & lindah, e. 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(2019). genetic determinants of myocardial infarction risk in familial hypercholesterolemia. cjc open, 1(5), 225–230. https://doi.org/10.1016/j.cjco.2019.06.001 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 315-321 | doi: 10.14421/biomedich.2023.121.315-321 issn 2540-9328 (online) synthesis, spectroscopic analysis and antidiabetic properties of copper (ii) complex of mangifera indica leaf crude extract mary adelaide oladipo1, folasade omobolanle ajao2, adewusi john adepoju1,*, kayode taiwo ishola3, olalekan jamiu ajeigbe1 1department of pure & applied chemistry; 2department of physiology, ladoke akintola university of technology, p.m.b. 4000, ogbomoso, oyo state, nigeria. 3department of chemistry, federal college of education (special), p.m. b. 1089, oyo, oyo state, nigeria. corresponding author* ajadepoju@lautech.edu.ng manuscript received: 23 january, 2023. revision accepted: 13 march, 2023. published: 30 march, 2023. abstract many applied conventional drugs in treating diabetes have been reported to possess some drawbacks which necessitate a search for alternative therapies. in order to search for a more active antidiabetic agent, this study synthesized and evaluated antidiabetic properties of mangifera indica crude extract and its cu (ii) complex in alloxan-induced diabetic albino rats. the leaf crude extract and its metal complex were characterized using percentage metal analysis and ir spectroscopic data. experimental animals were induced by a single intraperitoneal injection of alloxan monohydrate at a single dose of 140 mg/kg body weight and animals with fasting blood glucose level (bgl) > 200 mg/dl were considered diabetic. metformin was used as a standard drug. fasting blood glucose level and body weight were used to assess the antidiabetic activity. one-way anova was used to determine the level of statistically significant at p< 0.05. the crude extract was found to coordinate with the metal ion through o donor atom of c=o and o-h of phenol and ketone respectively. the cu (ii) complex of the crude extracts at tested dose of 600mg/kg demonstrated more antidiabetic activity without weight gain than the standard drug. it is concluded that the cu (ii) complex could be a potential material in the development of more active and negative-side-effectfree antidiabetic drug. keywords: albino rats; body weight; blood glucose; metal complex. introduction the increase in mortality caused by diabetes has become one of the biggest health challenges of the 21st century. according to (who, 2016), diabetes is responsible for about 0.02% of mortality worldwide in 2015 and about 8.8% of adults were evaluated by international diabetes federation (idf) to possess diabetes. diabetes has become rampant and steadily increasing worldwide. however, the widespread of this disease can harshly impact the finances of individuals and their families, and the economies of nations. many individuals with type 2 diabetes which is typified by hyperglycemia and abnormal carbohydrate metabolism, who are dependent on insulin for survival, suffer due to a dearth of affordable insulin (who 2016; upadhyay et al., 2018). the number of mortality caused by diabetes is envisaged to reach about 10.4% unless effective prevention is accessible (patarakijavanic eta al., 2019). many synthetic drugs have been applied to combat diabetes and these drugs are reported to act in different ways by lowering the level of blood glucose, for example, increased insulin secretion (sulfonylureas and meglitinides), decreased insulin resistance (biguanides and thiazolidinediones), increased prandial insulin secretion (dpp-4 inhibitors), reduced carbohydrate absorption (αglucosidase inhibitors), and inhibiting glucose reabsorption in the proximal renal tubule, resulting in increased renal glucose excretion and lower blood glucose levels (sglt2 inhibitors) (chao and henry, 2010). many of the present antihyperglycemic agents have been reported to be less effective, expensive and many drawbacks such as hypoglycemic episodes, gastrointestinal disturbances, skin reactions, lactic acidosis, fluid retention, and weight gain are associated with them (krentz and bailey, 2005). since many orthodox drugs and other conventional therapies are less effective or ineffective with several shortcomings in treating diabetes, there is an increase in the study of medicinal plants with hypoglycemic properties to replace the conventional drugs. recently, there is a rapid evolution of studies in the area of herbal medicine and an upsurge in the use of medicinal plants in treating many ailments both in developing and developed countries. their use is attributed to their natural origin, https://doi.org/10.14421/biomedich.2023.121.315-321 316 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 fewer side effects, accessibility, effectiveness, and affordability (bandaranayake, 2006). the leaves of mangifera indica linn. commonly called mango have been applied for the treatment of fever, diarrhea, fainting, abnormality of lymph nodes, diabetes, and many ailments (aderibigbe et al., 2001; dineshkumar et al., 2010; andrew et al., 2013; garridosuarez et al., 2014; ganogpichayagrai et al., 2017). the bioactivities of the leaf are attributed to the presence of secondary metabolites which include flavonoids, tannins, alkaloids, terpenoids, anthraquinones, saponins, cardiac glycosides, and steroids. mangiferin, c19h18o11 (figure 1), a glucosyl xanthone (1, 3, 6, 7-tetrahydroxyxanthonec2-β-d-glucoside) has been reported to be a prominent polyphenolic constituent found in mongo and it is mainly found in mango leaves, barks, and fruit peels (patarakijavanic et al. 2019). o o oh h oh oh oh oho ho ho figure 1:mangiferin figure 1. mangiferin. the discovery of secondary metabolites from plants for clinical applications has provided a model for drug synthesis. in the last few years, there is a developing interdisciplinary field in the development of metal-based drugs using natural products. transition metals show different oxidation states and can coordinate with molecules containing donor atoms or anions. these properties of transition metals are responsible for the development of metal-based drugs. the pharmacological properties and bioavailability of natural products can be modified by metal coordination (jurca et al. 2017). the dearth of access to affordable insulin and synthetic drugs has become a fundamental barrier to the successful treatment of diabetes and results in unnecessary problems and premature deaths in many developing and underdeveloped countries. therefore, in order to overcome the spread of diabetes through the use of inexpensive and readily available drugs, this work investigated the antidiabetic activity of mangifera indica leaf crude extract and its metal complex. experimental reagents and equipment all the chemicals and solvents employed are of analytical grade and they are used without further purification. they include n-hexane, ethyl acetate, methanol, distilled water, normal saline, alloxan monohydrate, calcium chloride, copper (ii) acetate, sawdust, pelletized rat feed, and metformin hydrochloride. the infrared spectra of the leaves crude extract and its metal complexes were recorded on agilent ftir spectrophotometer while percentage metal analysis was carried out using atomic absorption spectrometer model pg990. collection and identification of plant leaves mangifera indica leaves were collected from the surroundings of the department of pure and applied chemistry, ladoke akintola university of technology, nigeria. the plant specimen was properly identified and authenticated at the herbarium of the department of pure and applied biology, ladoke akintola university of technology (lautech). the collected leaves were washed, shade dried at room temperature, and powdered in a grinder mill. extract preparation about 1.2 kg of the washed, dried, and powdered leaves of mangifera indica were extracted by cold maceration method using 96 % n-hexane and ethyl acetate solvents for 72 h (pepato et al. 2005). after 72 h of maceration, the ethyl acetate soluble portion was cautiously decanted and concentrated using a rotary evaporator. the residual solvent in the crude extract obtained after concentration was allowed to evaporate at room temperature to avoid the decomposition of the natural metabolites. the dark green colored mangifera indica leaves crude extract obtained was kept in an air-tight desiccator over calcium chloride. the crude extract was weighed and a fresh stock for treating the diabetic animals was daily prepared (wadood et al. 2013; mohammed et al. 2015) preparation of copper (ii) acetate complex of the crude extract a solution of 0.5 g (2.75 × 10-3 mol) of copper (ii) acetate was dissolved in distilled water. the resulting metal solution was added drop wisely to a solution of 1g mango crude extract in methanol. the mixture was stirred on a magnetic stirrer for 1 hr at room temperature. the metal complex formed was filtered in a vacuum system, washed with water, and dried in a desiccator over calcium chloride (cacl2). a pure green solid was obtained and weighed. experimental animals male albino rats weighing 120-150 g body were used and acclimatized at a closely maintained temperature of 25 ± 2 ∘c with a standard relative humidity under photoperiodicity of 12 light:12 dark cycles for 4 weeks with free access to standard rodent pellet diet and water ad oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 317 libitum. adult male albino rats weighing around 180-220 g were selected for the study. induction of experimental diabetes the male albino rats were allowed to fast overnight with their blood glucose levels and body weight recorded prior to the induction of the alloxan prepared in normal saline. the animals were chemically induced by intraperitoneal injection of freshly prepared alloxan monohydrate in saline at a dose of 160 mg/kg b.wt. the animals were kept under observation and after 48 hr of alloxan, the blood glucose was confirmed using an accuchek glucometer. all animals with plasma glucose levels > 200mg/dl were separated and considered diabetic for the study and the rats having a blood glucose lesser than 200mg/dl glucose level were rejected (sabu and subburaj, 2002; mohammed et al. 2015). administration of the leaf extract and its metal complex the diabetic rats were divided into seven groups for the experimental study with four rats in each group as shown below: a) normal (non induced) rats b) diabetic control (untreated rats) c) diabetic rats treated with metformin hydrochloride (500mg/kgb.wt) d) diabetic rats treated with aqueous mango leaf extract (400mg/kgb.wt), e) diabetic rats treated with aqueous mango leaf extract (600mg/kgb.wt) f) diabetic rats treated with an aqueous complex of mango leaf extract with copper (ii) (400mg/kgb.wt), g) diabetic rats treated with an aqueous complex of mango leaf extract with copper (ii) (600mg/kgb.wt) the blood glucose and body weight of rats in each group were measured and evaluated on day 0, day 5, day 10, and day 15 using accuchek glucometer with disposable test strips and digital weighing balance respectively monitoring of blood glucose concentration and animal weight the accuchek glucometer with disposable test strips was used to determine the blood glucose level in rats. blood samples were collected through the tail of the animals. the tail in each case was first wiped with ethanol and then nibbled with a set of new blades. a test strip was fully inserted into the glucometer before applying a drop of blood to fully cover the test area inside the grey target (sharma et al. 2003). after the collection of blood, the nibbled side of the tail was rubbed with cotton wool soaked in ethanol in order to protect the animal from infection and to arrest further bleeding. blood glucose levels and weight of each animal were monitored and taken three times in three weeks using the accuchek and digital weighing balance respectively (shastr, 1980). statistical analysis the results from the number of experiments were expressed in mean and standard deviation, and subjected to the analysis of variance (one-way anova) to determine the significant levels difference between the groups using a t-test. the values with p < 0.05 were considered significant. results and discussion the mangifera indica leaves crude extract and its metal complex were characterized using infrared spectroscopic and aas analyses. the physical and chemical properties of the mango leaf crude extract, its metal complex, ir, and effects of the leaf extract, its metal complex, and the standard drug on diabetics are presented in tables 1, 2, 3, and 4 respectively. the anti-diabetic property of the mango leaves crude extract and its metal (ii) complex was evaluated through the body weight and blood glucose level. the body weight and blood glucose level before and after inducing diabetes were compared to obtain the anti-diabetic efficacy of the complex. the induction of alloxan monohydrate produced hyperglycemia in albino rats. the mango leaf extract and its metal complex caused a significant (p < 0.05) decrease in the fasting blood glucose of treated rats (table 3) when compared with the diabetic control and the metformin-treated group. this decrease was comparable with that of the normal control on certain days. also, extract-treated groups showed statistically significant (p < 0.05) increases in weight gain at the end of 15 days when compared with diabetic control (table 5). table 1. the physical and chemical properties of the mangifera indica leaf crude extract and its cu (ii) complex. compound color (%wt) cu (ii) mangifera indica leaf crude extract dark green 0.0052 [cu(ii) crude extract] dark green 0.0071 table 2. important ir spectra of the mangifera indica leaf crude extract and its cu (ii) complex. compounds oh (cm-1) c=o (cm-1) c-o (cm-1) m-o (cm-1) mangifera indica leaf crude extract 3308 b 1701 s 1194 s [crude extract cu] 3276 b 1640 m 1190 m 800-1000 m b-broad, s-strong, m-medium 318 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 table 3. effects of mangifera indica leaf crude extract and its cu (ii) complex on blood glucose level of diabetic albino. group pre-treatment(mg/dl) post treatment(mg/dl) day 0 day 5 day 10 day 15 normal control 76.4 + 8.76 73.25 + 7.63 78 + 24.52 82.3 + 22.60 diabetic control 225.7 + 67.52 244.1+ 120.59 304.2 + 45.80 269.9 + 11.81 metformin hcl (500mg/kgb.wt) 337.2 + 51.33 289.3 + 99.78 244 + 82.65 197.9 + 7.69 leaf crude extract (400mg/kgb.wt) 333.5 + 40.99 278 + 106.02 263 + 23.39 206.1 + 4.00 leaf crude extract (600mg/kgb.wt) 301.4 + 129.06 103.7+ 107.19 93 + 90.93** 77.2 + 5.68 ** [crude extract cu] (400mg/kgb.wt) 269.2 + 28.18 296.2+ 115.59 187.4+98.19 102.3 + 9.55 [mango extract cu] (600mg/kgb.wt) 305 + 123.89 264.2+ 130.47 128 + 53.82** 80.4 + 5.25** the values are expressed as means ± sem; n = 4. values are statistically significant at **p < 0.05 compare to diabetic control group (anova). figure 2. histogram representation of effects of mangifera indica leaf crude extract and its metal complex on blood glucose level. table 4. effect of standard rug, mangifera indica leaf crude extract and its cu (ii) complex on body weight of diabetic albino rats. group(n=4) average body weight (g) ±sem day 0 day 5 day 10 day 15 normal control 186.5 + 47.96 185.71 + 60.58 186.25 + 46.95 190.2 + 45.96 diabetic control 225.86 + 17.61 214.85 + 18.95 201 + 11.18 181.32 + 18.20 metformin hcl (500 mg/kgb.wt) 202.71 + 11.31 186.83 + 21.38 197 + 19.98 212.5 + 7.64 leaf crude extract (400 mg/kgb.wt) 189.86 + 14.36 184 + 28.35 205 + 30.35 213.31 + 27.19** leaf crude extract (600 mg/kgb.wt) 209.71 + 26.94 217 + 41.17 217.29 + 25.33 227 + 11.43 ** [crude extract cu] (400 mg/kgb.wt) 204.6 + 15.85 211.4 + 36.10 226.14 + 26.37 247.56 + 1.57 ** [crude extract cu] (600 mg/kgb.wt) 176.86 + 24.71 186.2 + 33.62 169.6 + 17.77 188.21 + 2.88** values are expressed as mean±sem. **p<0.05 as compared to diabetic control group; n= number of animals (one-way anova) treatment l) d g / (m l e v e l s e c o u g l o o d b l normal control diabetic control metformin (500mg/kg) mangifera indica leaves extract (400mg/kg) mangifera indica leaves extract (600mg/kg) [mangifera indica extract cu](400mg/kg) [mangifera indica extract cu] (600mg/kg) 350 300 250 200 150 100 50 day 0 day 5 day 10 day 15 0 oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 319 day 0 day 5 day 10 day 15 0 20 40 60 80 100 120 140 160 180 200 220 240 260 normal control diabetic control metformin (500mg/kg) mangifera indica leaves extract (400mg/kg) mangifera indica leaves extract (600mg/kg) [mangifera indica extract cu](400mg/kg) [mangifera indica extract cu] (600mg/kg) a v e ra g e b o d y w e ig h t (g ) treatment figure 3. histogram representation of effects of standard drug, mangifera indica leaf crude extract and its cu (ii) complex on body weight of diabetic albino rats. discussion the mangifera indica leaves crude extract and its metal complex after complexation reaction were dark green in color and the percentage of cu (ii) presents in the crude extract and its metal complex are 0.0052 and 0.0071 respectively as shown in table 1. the important ir bands were shown in table 2. the spectrum of mango leaves crude extract showed two bands at 3308 cm-1 and1701 cm-1. the bands were assigned to o-h of phenol and c=o of ketone respectively (coate, 2001). these were found to shift to 3276 cm-1 and 1640 cm-1 upon coordination with the metal ion. the shift of the bands to the lower frequency and appearance of bands between 1000-800 cm-1 confirmed the formation of the complex and binding of the extract to cu (ii) through oh and c=o of ketone, respectively as shown in figure 4. o o ho h ho oh ho ho o ho ho o o oh h oh oh oh oho ho ho cu figure 4. proposed structure for cu (ii) complex of mangifera indica leaf crude extract. the rate of change in the blood glucose levels in the animals was shown in table 3 and was represented by a histogram (figure 2). no significant variation was observed in the blood glucose level of normal control within 15 days while a significant increase in the glucose level of blood was observed in the diabetic group during the study period compared to the normal control group. the standard drug, the crude extract, and cu (ii) complex of the extract when administered to an alloxan-induced diabetic rat produced a significant reduction in the blood glucose level on the 10th and 15th days of the treatment as compared to control rats. the standard drug at a dose of 500 mg/kgb.wt and mangifera indica leaf crude extract and its cu (ii) complex were administered orally at doses of 400 mg/kgb.wt and 600 mg/kgb.wt for 15 days. on the 15th day, the blood glucose levels of the rats treated with the crude extract and its metal complex at doses of 400 mg/kgb.wt and 600 mg/kgb.wt were observed to significantly decrease (p < 0.05) when compared with the diabetic group. the decrease was found to be more significant in rats treated with the cu (ii) complex of the crude extract at a dose of 400 mg/kgb.wt while the crude extract was more effective at 600 mg/kgb.wt. a change in body weight was observed in the treated rats as shown in table 4 and represented by figure 3. no significant change in the body weight of normal animals from the 0th to the 15th day of treatment was observed. however, the body weight levels of the diabetic control group decreased gradually when compared to the normal non-diabetic rats. the weight levels of the rats treated with the crude extract at doses of 400 mg/kgb.wt and 600 320 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 mg/kgb.wt groups increased significantly at (p < 0.05) when compared to the diabetic control group on day 15th. also, there was a significant increase in the body weight levels of the rats treated with cu (ii) complex at doses of 400 mg/kgb.wt and 600 mg/kgb.wt groups at (p < 0.05) when compared with the diabetic control group on day 15th. however, cu (ii) complex at a dose of 600 mg/kgb.wt produced a normal weight gain in the treated rat as compared to the normal control group. the standard drug was observed to produce abnormal weight gain which could be ascribed to the negative side effect associated with the drug. therefore, the problem of the negative side effect was overcome with the administration of cu (ii) complex at a dose of 600 mg/kgb.wt. in this study, the cu (ii) of the crude extract was found to be more effective than the leaf crude extract. the more increase in body weight and decrease in blood glucose level exhibited by the metal complex of the crude extract could be attributed to the ability of the metal ion to modify the bioavailability and pharmacological properties of the mangifera indica leaf crude extract. the weight gain which was comparable with that of the normal control on the 15th day of administration of cu (ii) complex could be attributed to improved glycaemic control (atangwho et al. 2007; adeoye et al. 2017). conclusion and recommendation in this study, the cu (ii) complex of mangifera indica leaf crude extract showed more diabetic activity than the standard drug. our results provide information on the antidiabetic activity of the metal complex and therefore the metal complex could be considered a potential antidiabetic drug without abnormal-weight-gain side effects. it is recommended that further studies such as nuclear magnetic resonance spectroscopy, magnetic moment susceptibility, and x-ray should be conducted to establish the real structure of the complex. also, the toxicity of the extract and its metal complex should be examined in order to establish its ideality as a potential antidiabetic drug. acknowledgment: the authors are grateful to prof. a.t.j ogunkunle of the department of pure and applied biology, ladoke akintola university of technology (lautech), ogbomoso, oyo state, nigeria for authenticating the plant material. ethical considerations: all experimental procedures were in agreement with the ladoke akintola university of technology ethics committee on research in animals and internationally approved principles for laboratory animal upkeep and use. conflict of interest: no conflict of interest. references adeoye a t, oyagbemi a a, adedapo a d, omobowale t o, ayodele a e, adedapo a a (2017). antidiabetic and antioxidant activities of the methanol leaf extract of vernonia amygdalina in alloxan-induced diabetes in wistar rats’j. med. plant. econ. dev. 1(1): 30-45 http://doi.org/10.4102/jomped.v1i1.30 aderibigbe a o, emudianughe t s, lawal b a s (2001). evaluation of the antidiabetic action of mangifera indica in 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(2006). quality control, screening, toxicity, and regulation of herbal drugs, in: i. ahmad, f. aqil, and m. owais (eds), modern phytomedicine turning. medicinal plants into drugs. wiley-vch gmbh & co. kgaa, weinheim chao ec, henry r r (2010). sglt2 inhibition–a novel strategy for diabetes treatment.nat. rev. drug discov. 9: 551–559. coates j (2001). interpretation of infrared spectra, a practical approach, encyclopedia o of analytical chemistry in r.a. meyers (ed.), john wiley & sons ltd, chichester. dineshkumar b, mitra a, manjunatha m (2010). studies on the antidiabetic and hypolipidemic potentials of mangiferin (xanthone glucoside) in stz-induced type 1 and type 2 diabetic rat models. int j adv pharm sci. 1: 75–85. ganogpichayagrai a, palanuvej c, ruangrungsi n (2017) antidiabetic and anticancer activities of mangifera indica cv. okrong leaves. j adv pharm technol res., 8:19-24 garrido-suarez b b, garrido g, castro-labrada m, merino n, valdes o, rodeiro i, delgado hernandez r (2014). antihypernociceptive effect of mangiferin in persistent and neuropathic pain models in rats. pharmacol. biochem. behav. 124, 311–319. 10.1016/j.pbb.2014.06.019 jurca t, marian e, vicaş l g, mureşan m e (2017). metal complexes of pharmaceutical substances, spectroscopic analyses developments and applications, in: e. sharmin editor and f. zafar editor ed(s)., intechopen, london. krentz aj, bailey cj (2005) oral antidiabetic agents: current role in type 2 diabetes mellitus. drugs 65: 385–411 https://doi.org/10.2165/00003495-200565030-00005 mohammed a, kumar d, rizvi s i (2015). antidiabetic potential of some less commonly used plants in traditional medicinal systems of india and nigeria. j intercult ethnopharmacol, 4(1): 78-85. doi: 10.5455/jice.20141030015241. patarakijavanic p, sato v h, kongkiatpaiboon s, chewchinda, s. (2019) a review of the antidiabetic potential of mangifera indica leaf extract. songklanakarin j. sci. technol. 41 (4): 942 950 pepato m t, mori d m, baviera a m, harami j b, vendramini r c, brunetti i l (2005). fruit of the jambolan tree (eugenia http://doi.org/10.4102/jomped.v1i1.30 https://doi.org/10.4314/gjpas.v13i1.16677 oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 321 jambolana lam.) and experimental diabetes j. ethnopharmacol 96:43-48 sabu m c, subburaj t (2002). effect of cassia auriculata linn. on serum glucose level, glucose utilization by isolated rat hemidiaphragm, j ethnopharmacol 85: 201-206 sharma s, nasir a, prabhu k m, murthy p s, dev g (2003). hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of eugenia jambolana in alloxan-induced diabetic rabbits. j ethnopharmacol 85: 201-206 https://doi.org/10.1016/s0378-8741(02)00366-5 shastr k (1980). comments on charaka samhita. chanukah bharati, varanasi, india. upadhyay j., polyzos s.a., perakakis n, thakkar b, paschou s a, katsiki n (2018) pharmacotherapy of type 2 diabetes: an update. metabolism 78:13-42. wadood a, ghufran m, jamal s b, naeem m, khan a, ghaffar r (2013). phytochemical analysis of medicinal plants occurring in local area of mardan, biochem anal biochem 2: 144. doi: 10.4172/2161-1009. world health organization (who)m. (2016). global report on diabetes. retrieved from https://apps.who.int/iris/bitstream/handle/10665/204871/97892 41565257_eng.pdf;jsessionid=f e60bb6c97974a6bcd4d02f64f018ea?sequence=1 https://doi.org/10.1016/s0378-8741(02)00366-5 https://apps.who.int/iris/bitstream/handle/10665/204871/9789241565257_eng.pdf;jsessionid=f https://apps.who.int/iris/bitstream/handle/10665/204871/9789241565257_eng.pdf;jsessionid=f this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 127-132 | doi: 10.14421/biomedich.2023.121.127-132 issn 2540-9328 (online) in-vivo alpha-amylase and alpha-glucosidase inhibitory activities of solanum anomalum leaf extract and fractions jude efiom okokon1, idongesit charles etuk1, john akpan udobang2, nwakaego omonigho ebong3,* 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, nigeria 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. 3department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, nigeria. corresponding author* nwakaebong@gmail.com manuscript received: 14 october, 2022. revision accepted: 05 december, 2022. published: 13 january, 2023. abstract solanum anomalum thonn. ex schumach. (family solanaceae), an edible shrub whose fruits and leaves are used medicinally to treat diseases including diabetes was evaluated for effect on alpha amylase and alpha glucosidase enzymes in vivo. the leaf extract (70-210 mg/kg) and fractions (hexane, dichloromethane, ethyl acetate, methanol, 140 mg/kg) of s. anomalum were evaluated in vivo for inhibitory effect on alpha amylase and alpha glucosidase enzymes using starch, sucrose and maltose as substrates. acarbose was used as reference drug. the leaf extract especially middle dose (140 mg/kg) and fractions (ethyl acetate and hexane) caused significant (p<0.05) reduction in blood glucose levels of animals treated with the various substrates used. ethyl acetate fraction exerted the highest inhibitory effect when starch and maltose were used as substrates followed by n-hexane and methanol. n-hexane was the most active fraction followed by ethyl acetate when sucrose was used as substrate. the results suggest that the leaf extract and fractions of s. anomalum have the potentials to inhibit alpha amylase and glucosidase in rats. keywords: alpha amylase; alpha glucosidase; hypoglycemia; solanum anomalum. introduction solanum anomalum thonn. ex schumach, a plant whose fruits and leaves are used medicinally and nutritionally is commonly found growing in west and east africa sub-regions. its parts are utilised locally to treat diabetes, gastrointestinal disorders, infections, inflammation and pains (burkill, 2000; bukenya and hall, 1988; offor and ubengama, 2015). hypoglycemic and antidiabetic activities of the fruits and leaves have been reported (offor and ubengama, 2015; okokon et al., 2022). moreso, in vivo and in vitro antiplasmodial (okokon et al., 2016; okokon et al., 2017a), antioedema (okokon et al., 2017b), antioxidant and antiulcer (okokon et al., 2019a), anticonvulsant and depressant (okokon et al., 2019b), analgesic (okokon et al., 2020) and antidiarrhoeal (udobang et al., 2022) properties of the leaf extract have also been reported. phytochemical constituents such as alkaloids, flavonoids, saponins, tanins, diosgenin, a diosgenin glycoside (25(r)-diosgenin-3-o-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside, uracil, 5-methyluracil, 1octacosanol, and octacosane have been reported on the leaves of the plant (okokon et al., 2016; okokon et al., 2022). we report in this study the effect of leaf extract and fractions of the plant on alpha amylase and alpha glucosidase of rats. materials and methods plants collection fresh leaves of solanum anomalum were collected in compounds in uruan area, akwa ibom state, nigeria in august, 2020. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. hebarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo (uuh.75a). extraction fresh leaves of s. anomalum were washed, cut into smaller pieces and dried under shade for two weeks. the leaves were further pulverized to powder using electric grinder. the powdered leaves material was divided into two parts; one part (1.5 kg) was macerated in 50% ethanol (7.5 l) for 72 hours at room temperature (28 ±2 ˚c). while the other part, (1.5 kg) was successively and gradiently macerated for 72 h in each of these solvents (2 x 5l), n-hexane, dichloromethane, ethyl-acetate and methanol to give corresponding fractions of these solvents. these were thereafter filtered and the liquid filtrates were concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract and fractions were stored in a https://doi.org/10.14421/biomedich.2023.121.127-132 128 biology, medicine, & natural product chemistry 12 (1), 2023: 127-132 refrigerator at -4˚c, until used for the proposed experiments. animals wistar rats (138-150 g) of either sex were obtained from the university of uyo animal house. they were maintained on standard animal pellets and water ad libitum. permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, university of uyo. in vivo alpha-amylase and glucosidase inhibition study  alpha-amylase inhibitory study fifty wistar rats were divided into 10 groups of 5 rats each. the rats in all groups were fasted for 18 hours and fasting blood glucose concentration was first taken at 0 minute before administration. based on predetermined ld50 value of 724.56 mg/kg (okokon et al., 2016), the animals were treated as follows; group i, which served as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. groups iv, v and vi were administered simultaneously, starch (2 g/kg) and s. anomalum leaf extract at 70, 140 and 210 mg/kg respectively while groups vii, viii, ix and x rats were administered starch (2 g/kg) and fractions (n-hexane, dichloromethane, ethyl acetate and methanol) at 140 mg/kg respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 90, 120 and 180 minutes (gidado et al., 2019).  glucosidase inhibitory study the procedure as described above was used for this study but with sucrose and maltose used as substrates (gidado et al., 2019). blood glucose determination drops of blood from tip of rats’ tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (accu-chek, indiana). the glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). the reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. light from a light emitting diode strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. an electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the resulting current is directly proportional to the amount of glucose in the sample (who, 2011). statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% and 0.1% level of significance ie p≤ 0.05 and 0.001. results and discusssion in vivo alpha amylase and glucosidase inhibition assay administration of starch (2g/kg) caused varying percentages of increase in blood glucose concentrations after 30 minutes. the percentages were starch (63.18%), extract/fractions treated groups (10.97-32.69%) and acarbose-treated group (17.97%). these increases were reduced after 60 minutes with animals treated with the ethyl acetate fraction (10.53%). these decreases were significant and sustained for 180 minutes in ethyl acetate-treated group, followed by middle dose group, 140 mg/kg, (9.22%) and methanol-treated group (12.15%). however, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (table 1). okokon et al. – in-vivo alpha amylase and alpha glucosidase inhibitory 129 table 1. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of starch load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 73.66±6.17 91.0±7.50(5.81) 80.00±6.02 starch 2000 73.33±8.25 119.66±5.45a(63.18) 115.66±1.33a (57.72) 104.66±2.60a (42.72) 95.66±3.75a (30.45) 92.0±6.35(25.46) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 76.33±3.48(5.53) 74.0±1.00(2.30) 72.33±8.68(0) crude extract 70 70.12±2.00 89.33±1.45(23.11) 100.33±4.05(42.12) 99.66±2.90(42.12) 89.0±2.08(26.92) 80.0±4.16(14.09) 140 72.33±8.87 89.33±8.68(23.50) 94.66±2.90(30.87) 94.0±0.57(29.95) 84.66±4.41(17.04) 79.0±3.21(9.22) 210 62.33±1.45 74.33±7.83(19.25) 86.0±10.14(37.97) 96.0±2.64(54.01) 83.0±2.08(33.16) 82.20±2.30(31.87) n -hexane fraction 140 66.66±1.33 90.33±1.85(35.50) 93.0±3.21(39.52) 108.0±3.60(62.01) 97.00±3.00(45.51) 85.66±4.48(28.50) dichloromethane fraction 140 69.33±4.41 92.0±2.51(32.69) 104.3±14.71(34.97) 108.6±12.81(56.64) 107.33±11.78\(54.81) 96.66±7.53(39.42) ethyl acetate fraction 140 82.00±4.04 91.0±3.21(10.97) 91.66±5.60(10.53) 93.33±1.20(13.81) 91.0±2.30(10.97) 81.66±3.75() methanol fraction 140 71.33±1.20 90.00±4.35(26.17) 93.33±5.78(30.84) 92.0±5.19(28.97) 85.33±3.18(19.62) 80.0±4.54(12.15) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. administration of sucrose (2 g/kg) produced a 46.01% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group and 25.95-70.73% increases in blood glucose concentration of extract/fractions-treated groups. the blood glucose concentrations were significantly reduced in hexane-treated group (13.02%) after 60 minutes postadministration of sucrose. however, n-hexane fraction had the highest effect (0.84%) throughout the duration of the study (180 minutes) followed by middle dose, 140 mg/kg, group (6.47%) (table 2). table 2. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of sucrose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25 90.33±2.33 89.0±4.35 87.33±3.84 sucrose 2000 92.0±4.04 134.33±2.90b(46.01) 128.66±5.45a (39.84) 117.33±4.66a(27.53) 97.66±0.66(6.15) 104.16±2.48(13.21) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 79.33±2.96 71.66±3.75 78.0±3.78 crude extract 70 70.66±5.92 89.0±7.55b(25.95) 92.33±8.83(30.66) 78.33±9.13(10.85) 76.33±2.18(8.02) 77.66±0.66(9.90) 140 82.33±3.75 117.33±4.80b(42.51) 102.0±2.08(23.89) 88.66±5.45(7.68) 85.66±5.23(4.04) 87.66±1.33(6.47) 210 72.0±2.64 114.66±6.38(59.25) 113.0±11.24(56.94) 91.33±7.68(26.84) 93.22±9.33(29.47) 83.00±2.08(15.27) n -hexane fraction 140 79.33±1.45 101.0±3.05(27.31) 89.66±1.45(13.02) 86.0±3.05(8.40) 81.66±1.45(2.93) 80.0±1.00(0.84) dichloromethane fraction 140 73.66±7.53 120.33±4.37(55.66) 106.33±7.35(44.35) 113.66±9.38(54.30) 95.00±5.68\(28.97) 90.33±2.90(22.63) ethyl acetate fraction 140 68.33±3.18 116.66±11.72(70.73) 117.66±6.88(72.19) 110.33±8.53(61.46) 89.66±2.72(31.21) 86.66±1.85(26.82) methanol fraction 140 79.33±6.06 106.33±7.86(34.03) 96.66±2.66(21.84) 108.0±2.51(36.14) 91.33±3.33(15.12) 96.66±9.52(21.84) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. there was 60.78% increase in blood glucose concentration 30 minutes following maltose administration in the control group. however, 27.0857.80% increases were observed in the extract/fractionstreated groups. at 60 and 90mins, the ethyl acetate fraction group had blood glucose level of 18.44 and 0.61% respectively. at 180 minutes, n-hexane fraction and middle dose (140 mg/kg)-treated group had blood glucose level of 2.02% and 2.52% respectively (table 3). 130 biology, medicine, & natural product chemistry 12 (1), 2023: 127-132 table 3. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of maltose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 90.33±2.33(3.62) 89.0±4.35(1.55) 87.33±3.84(3.98) maltose 2000 82.30±2.14 132.33±1.90b(60.78) 130.22±2.45(58.22) 120.66±3.22a(46.60) 115.0±2.46(39.73) 106.22±4.24(29.06) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20c(0.77) 85.33±2.15c() 84.26±1.14a() 82.28±2.26a() crude extract 70 83.3±1.20 131.45±3.28(57.80) 125.17±4.39b(50.26) 115.33±1.66a(38.45) 104.35±1.12(25.27) 95.42±2.28(14.54) 140 81.28±5.28 128.14±1.98(56.05) 105.23±1.52a(29.46) 95.56±3.88(17.56) 90.44±1.86(11.26) 83.33±3.99(2.52) 210 85.24±7.34 125.19±7.22(46.86) 90.50±4.86a(6.17) 87.25±8.65b(2.35) 81.45±5.60a() 77.0±5.21 b () n -hexane fraction 140 80.55±4.89 118.24±5.28(46.79) 104.28±2.37a(29.45) 92.22±3.34 a(14.48) 87.28±2.28 b (8.35) 82.18±3.28(2.02) dichloromethan e fraction 140 83.82±5.49 122.98±4.29b(46.71) 110.37±3.29(31.67) 102.19±2.19(21.91) 98.28±6.18a(17.25) 91.88±1.83(9.61) ethyl acetate fraction 140 82.36±6.54 132.46±7.38(59.61) 97.55±7.44 b(18.44) 82.87±9.28b (0.61) 81.65±2.27 b 74.33±8.44c methanol fraction 140 90.10±10.16 114.50±12.18(27.08) 108.53±5.68a(20.45) 94.66±8.93a(5.06) 82.63±5.68 b 73.26±6.79c data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion various parts of s. anomalum, a medicinal plant, is used in ibibio traditional medicine in the treatment of diseases such as diabetes among others. this work focused on the evaluation of s. anomalum leaf extract and fractions for in vivo inhibitory effect on alpha amylase and alpha glucosidase activities in rats, as well as isolation and characterization of the active antidiabetic principles from this plant. the extract was found to non dose-dependently inhibit increases in blood glucose concentration following starch administration with the middle dose, 140 mg/kg, and ethyl acetate fraction exerting the most inhibition. complete digestion of dietary polysaccharides like starch is achieved by the combined action of alpha-amylases and alpha-glucosidase enzymes. the alpha-amylase enzyme digests alphabonds of the alpha-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound alpha-glucosidase enzymes (kalra, 2014; alongi and anese, 2018). inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for managing type 2 diabetes mellitus, many medicinal plants have been reported to possess alpha-amylase and alpha-glucosidase inhibitory potential (esimone et al., 2001; ibrahim et al., 2014). similarly, the leaf extract and fraction significantly inhibited blood glucose rise when co-administered with maltose and sucrose with n-hexane and ethyl acetate fractions exerting the highest inhibition. acarbose, the standard drug used in this study significantly inhibited blood glucose rise when co-administered with starch, maltose and sucrose. the results of this study corroborate that reported on other species of solanum such as s. nigrum, s. melongena depressum, s. gilo, s. melogena, s. melongena l., s. macrocarpon and s. diphyllum (hossain et al., 2009; nwanna et al., 2013; dasgupta et al., 2016; nwanna et al., 2019), which significant inhibition of alpha –amylase and alphaglucosidase activities were observed. diosgenin present in this plant is implicated in antidiabetic activities of plants (pari et al., 2012; saravanan et al., 2014). diosgenin has been reported to exert its activity through various mechanisms such as inhibiting alpha-amylase and alpha-glucosidase (gosh et al., 2014) to reduce intestinal glucose absorption, inhibiting the sodiumglucose cotransporter-1 (sglt-1) and reducing intestinal na+-k+atpase activity (gan et al., 2020). also, diosgenin glycosides and other steroidal saponins commonly present in the solanum species (kaunda and zhang, 2019) are reported to exert hypoglycaemic activities (wang et al., 2010; wang et al., 2012; elekofehinti, 2015). similarly, β-sitosterol present in the leaf extract and fraction of this plant has been reported to possess inhibitory potentials on alpha-glucosidase and alpha-amylase enzymes (kumar et al., 2013). these could have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of this extract. alpha-amylase and alpha-glucosidase inhibitions by plants extracts have been reported severally (shirwaikar et al., 2005; ishnava and metisariya, 2018). phytochemicals implicated as anti-diabetic agents, do so possibly through alpha-amylase and alpha-glucosidase inhibition. the phytochemicals in question include flavonoids, saponins, tannins and terpenoids (ortizandrade et al., 2007; ishnava and metisariya, 2018). also, polyphenolic compounds from plants are known to cause enzymes inhibitions on the biological systems (kaita et al., 2018). the phenolic compounds which are biological oxidants, strong metal ion chelators and okokon et al. – in-vivo alpha amylase and alpha glucosidase inhibitory 131 protein precipitation agents form insoluble complexes with proteins (ishnava and metisariya, 2018). the presence of the polyphenolic compounds in the leaf extract and fractions in addition to diosgenin, diosgenin glycosides and β-sitosterol may suggests that their inhibitory potential on alpha-amylase and the membrane-bound intestinal alpha-glucosidase enzymes. conclusion the results of this study suggest that inhibition of alphaamylase and alpha-glucosidase enzymes maybe one of the modes of antidiabetic activity of the leaf extract and fractions of solanum anomalum which can be attributed to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to mr nsikan malachy of pharmacology and toxicology department, university of uyo for providing technical assistance. authors’ contributions: joe was involved in the conceptualization, design, execution, supervision, sourcing of materials, statistical analysis, draft and review of the manuscript; ice participated in the sourcing of material, design, execution, statistical analysis, drafting and review of manuscript; while jau and noe were involved in the design, execution, statistical analysis, review and editing of the manuscript. all authors read and approved the final manuscript. competing interests: the authors declare that there are no conflicts of interest related to this article. references alongi m, anese m. 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(online) comparative assessment of the proximate composition, functional properties and amino acid profile of dioscorea bulbifera, dioscorea alata and dioscorea rotundata found in minna, niger state eneogwe okechukwu godfrey1,*, obuye faith1, ibrahim izihyi esther2 1department of chemistry; 2department of industrial chemistry, federal university lokoja, p.m. b. 1154, lokoja, nigeria. corresponding author* godfrey.eneogwe@fulokoja.edu.ng abstract the proximate composition, functional properties and amino acid profile of samples of dioscorea alata, dioscorea rotundata and dioscorea bulbifera were investigated using standard analytical methods. the results showed that dioscorea alata had the highest ash (5.59±0.06 %) and crude fiber content (12.12±0.20 %), indicating that it has more mineral stuffing and is best to reduce the risk of obesity. dioscorea rotundata had the highest fat content (11.63±0.04 %) as well as the lowest moisture content (7.04±0.06 %), indicating that it is a better source of calories and has a longer shelf-life than other yam species analysed. dioscorea bulbifera also had the highest crude protein (8.64±0.03 %) and carbohydrates (77.51±0.08 %) than other yam species analysed, indicating high bodybuilding capacity and a better source of energy than other yam samples analysed. dioscorea alata showed the highest bulk density (0.87±0.02 g/cm3) and swelling capacity (15.25±0.03 g/g). it is indicating its usefulness in the reduction of paste thickness and water-holding capacity of starch granules respectively while dioscorea rotundata, showed the highest water absorption capacity (164.02±0.02 %), oil absorption capacity (149.76±0.02 %) and dispersibility (72.17±0.01 %). this indicates its importance in the consistency and bulking of products, flavour retaining in food and reconstitution of flour samples in water to give a fine consistent paste during mixing. the yam species were also rich in amino acids which are building blocks of protein. however, dioscorea rotundata was the richest in amino acid content, as it had 36.32±0.16 g/100g and 36.49±0.16 g/100g, for essential and non-essential amino acids respectively. keywords: amino acid; functional properties; proximate composition; dioscorea alata; dioscorea bulbifera. introduction roots and tubers allude to any developing plant that stores food in the subterranean roots, corm and tuber. the nutritional value of roots and tubers lies in their capacity to provide one of the cheapest sources of dietary energy in the form of carbohydrates in impoverished climes (ugwu, 2009). roots and tubers crops are an important source of food, nutrition and financial revenue for many impoverished farmers and food-insecure individuals in developing countries (oluwamukomi & akinsola, 2015). tubers are also used for the treatment of purgative, deflatulent, aphrodisiac, hemorrhoids, scrofula and polyureic (dutta 2015). furthermore, dietary plant estrogens of dioscorea provide various health benefits including defence against cancers, osteoporosis, cardiovascular disease, and asthma, as well as being utilized in the preparation of contraceptives and the treatment of numerous genetic abnormalities (sheikh et al., 2013). yam is a popularly consumed tuber in the tropics with several varieties such as dioscorea rotundata (white yam), dioscorea esculenta (chinese yam), dioscorea alata (water yam), dioscorea bulbifera (aerial yam), and dioscorea dumenterum (trifoliate yam) among the economically important species (ike and inoni, 2006). however, this study focuses on aerial yam (dioscorea bulbifera), white yam (dioscorea rotundata) and water yam (dioscorea alata). according to faostat (2006), nigeria is the world’s largest producer of yam, accounting for 67 percent of global production and 72 percent of west african production in 2005. yam is an important staple meal in west africa and a vital source of carbohydrates for over 300 million people worldwide (ettien et al., 2009). despite these well-known facts regarding yam, it continues to be overlooked in west african national food policy plans. this has resulted in limited dioscorea species research and development on the continent (sanoussi et al., 2016). this study investigates the proximate composition, functional properties and amino acid profile of dioscorea rotundata, dioscorea alata and dioscorea bulbifera obtained from minna, niger state. manuscript received: 15 october, 2022. revision accepted: 22 january, 2023. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.177-185 178 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 materials and method sample collection matured accessions of the three cultivated yam species were harvested randomly from rural farms in chanchaga, mekunkele and gunu areas of niger state. the samples include cultivars of water yam (dioscorea alata), a variety of white yam (dioscorea rotundata) and aerial yam (dioscorea bulbifera). the samples were cleaned by brushing off soil particles and transported at tropical ambient temperature to the laboratory for analysis. dioscorea bulbifera dioscorea rotundata dioscorea alata figure 1. pictures of the studied dioscorea varieties. sample pre-treatment the yam samples were washed thoroughly with water, peeled and cut using a knife. these yam species were ground separately using a laboratory mortar and pestle and then sieved using a 250 μm mesh size sieve. the three samples were stored in airtight properly labeled polythene bags and kept in a cool and dry place before analysis. determination of proximate composition standard analytical procedures for food analysis were adopted for the determination of moisture content, crude protein, crude fibre, percentage fat, carbohydrate and ash content. moisture content two grams of the sample were placed in the crucibles and were then dried overnight at 105°c in the oven. after cooling in a desiccator for 30 minutes, the dry sample was weighed to a constant weight. on a dry weight basis, the percentage of weight loss was reported as a percentage of moisture content (aoac, 2006). to acquire triplicate values, this was done three times. the moisture content was calculated as: % 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑟𝑦𝑖𝑛𝑔 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑟𝑦𝑖𝑛𝑔 × 100 ash content two grams of the dried and pulverized sample were taken in triplicates, put in pre-weighed crucibles and ashed for three hours at 600°c in a muffle furnace. after cooling in a desiccator, the hot crucibles were weighed. the percentage residual weight was expressed as ash content (aoac, 2006). the ash content was calculated as: % 𝐴𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛 × 100 crude fat content the percentage fat content of the samples was obtained using the method according to aoac (2006). five grams of the sample was weighed into a pre-weighed fat-free extraction thimble which was plugged tightly with cotton wool. on a heating mantle, the thimble was placed in the soxhlet extractor fitted up with reflux condenser connected to a boiling flask containing 200 ml of petroleum ether (boiling point 60oc). as the flask and petroleum ether were heated, the solvent evaporated and condensed into the thimble extracting oil from the sample and refluxed into the boiling flask with the extracted oil. this was done for 4 hours. at the end of extraction, the solvent (petroleum ether) was evaporated by heating at 70oc on a hot plate leaving the lipid extract in the flask. the flask together with the sample was placed in an oven and dried at 110oc for 1 hour, cooled in a desiccator and re-weighed. the percentage crude fat content was calculated using the formula: % 𝐶𝑟𝑢𝑑𝑒 𝑓𝑎𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑜𝑖𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 crude protein determination the kjeldahl method was used to determine the total protein. a kjeldahl flask containing 0.5 g of the sample was filled with 8–10 cm3 of concentrated h2so4 after godfrey et al. – comparative assessment of the proximate composition, … 179 being weighed in triplicate and the solution was digested in a fume cupboard until it became colourless. a 10 % naoh solution with a 40 % concentration was used for distillation. the boric acid solution glowed green after the condenser tip was dipped into a conical flask containing 5 cm3 of 4 % boric acid in a mixed indicator. hcl of 0.01m was titrated in the receiver flask until the solution turned red (aoac, 2006). the crude protein content was calculated as: % 𝑁 = (𝑎 − 𝑏) × 0.01 × 14 × 𝑣 𝑊 × 𝐶 × 100 where 𝑎 is the titre value of the digested sample, 𝑏 is the titre value of the blank sample, 𝑣 is the volume after dilution, 𝑊 is the weight of the dried sample, 𝐶 is the aliquot of sample used and 14 is the atomic weight of nitrogen. 𝐶𝑟𝑢𝑑𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 6.25 × % 𝑁 crude fibre content using 20 % h2so4 and 20 % naoh solutions, 2.0 g of the pounded sample was utilized in triplicates to estimate the crude fibre by acid and alkaline digestion techniques (aoac, 2006). the crude fibre content was calculated as: % 𝐶𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑟𝑒 = 𝐿𝑜𝑠𝑠 𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑛 𝑖𝑔𝑛𝑖𝑡𝑖𝑜𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 carbohydrate determination the carbohydrate content was calculated by difference using the following formula: 𝐴𝑣𝑎𝑖𝑙𝑎𝑏𝑙𝑒 𝑐𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒 (%) = 100 − [𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (%) + 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 (%) + 𝐴𝑠ℎ (%) + 𝐹𝑖𝑏𝑟𝑒 (%) + 𝐶𝑟𝑢𝑑𝑒 𝑓𝑎𝑡 (%)] determination of functional properties dispersibility kulkani et al. (1991), described a method for determining flour dispersibility. ten grams of flour was weighed into a 100 cm3 measuring cylinder, followed by 100 cm3 of distilled water. for 1 minute, the setup was vigorously agitated. after a regular time-step of 30 minutes, the volume of the settled particles was measured. the volume of settled particles was subtracted from 100. the difference was expressed as a percentage of dispersion. bulk density the bulk density was determined using the method published by oladele and ainaby (2007). in a 100 cm3 measuring cylinder, 50 g of samples were placed. the measuring cylinder was then tapped repeatedly on a laboratory table until it reached a fixed volume. the bulk density was calculated using the formula: 𝐵𝐷 (𝑔 𝑐𝑚3⁄ ) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑡𝑎𝑝𝑖𝑛𝑔 water absorption capacity (wac) phillips et al. (1988) and anderson et al. (1969) techniques were used to determine the water absorption capacity and solubility index of flours from the sample. one gram of flour samples (mo) was weighed in a centrifuge tube and 10 cm3 distilled water was added. in a ks 10 agitator, the content of the centrifuge tube was shaken for 30 minutes. the mixture was centrifuged at 5000 rpm for 15 minutes after being maintained in a water bath (memmert) at 37°c for 30 minutes. the resulting sediment (m2) was weighed and dried to a consistent weight at 105°c (m1). after that wac was calculated using the formula: 𝑊𝐴𝐶 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑤𝑎𝑡𝑒𝑟 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑤𝑎𝑡𝑒𝑟 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑙𝑜𝑢𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 oil absorption capacity (oac) eke and akobundu (1993) techniques were used to assess the oil capacity of the sample. in a weighed 20 cm3 centrifuge tube, 1 g of sample (mo) was mixed with 10 cm3 of oil. the slurry was stirred for 2 minutes in a vortex mixer, then kept at 28°c for 30 minutes before being centrifuged at 4500 rpm for 30 minutes. the clear supernatant was decanted and discarded. the adhering drops of oil were removed and the tube was weighed (m1). the oac was determined as follows: 𝑂𝐴𝐶 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑖𝑙 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑖𝑙 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑓𝑙𝑜𝑢𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 180 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 swelling capacity kaushal et al. (2012) described the method that was used. one gram of flour sample was weighed into a graduated cylinder measuring 10 cm3. the volume occupied by the sample was measured after 5 cm3 of distilled water was added. the sample was left standing in water for 1 hour without being disturbed. the volume occupied after swelling was recorded and calculated as: 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑐𝑐𝑢𝑝𝑖𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑐𝑐𝑢𝑝𝑖𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 determination of amino acid profiles this was evaluated by extracting 3.00 g of the sample using a soxhlet extractor for six hours with petroleum ether (40–60°c) (copper, 2000). in a glass ampoule, 30.00 mg of the defatted samples were weighed and 7.00 cm3 of 6.00 mol/dm3 hydrochloric acid was added. by injecting nitrogen into the ampoule, oxygen was expelled (to avoid possible oxidation of some amino acids during hydrolysis). the ampoule was sealed with bunsen flame and put in an oven preset at 105°c for 22 hours, after which it was allowed to cool, broken at the tip and the content filtered. in a rotary evaporator, the filtrate was evaporated to dryness at 40°c under a vacuum. the residue was dissolved with 5.00 cm3 of acetate buffer (ph 2.0), then stored in a plastic bottle for 24 hours in the deep freezer. the technicon sequential multi-sample (tsm) amino acid analyser was loaded with five to ten microliters of the hydrolysate. this was dispensed into the cartridge of the analyser and the analysis lasted for 76 minutes. statistical analysis the obtained results were subjected to statistical analysis using mean standard deviation and analysis of variance (anova) as described by duncan’s multiple range test to determine the level of significance between different samples and significance was set at p ≤ 0.05. results and discussion table 1. proximate composition of selected yam species (%). yam species ash content moisture content crude fat crude fibre crude protein carbohydrate d.alata 5.59±0.06a 9.54±0.02b 7.52±0.03d 12.12±0.20c 7.85±0.02a 57.38±0.20d d.rotundata 3.05±0.06f 7.04±0.06h 11.63±0.04e 7.59±0.10a 2.19±0.01c 68.50±0.20ab d.bulbifera 2.34±0.02a 8.63±0.45b 1.60±0.01a 1.28±0.10e 8.64±0.03f 77.51±0.08f values are means ± standard deviation of triplicate analysis. proximate composition of selected yam species moisture content the moisture content of the various yam species ranged from 7.04±0.06h % for dioscorea rotundata to 9.54±0.02b % for dioscorea alata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea alata showing the highest moisture content. however, according to oko and famurewa (2014), these values are comparable to its literature values that ranged from 2.1 % to 9.2 % for dioscorea dumenturom and dioscorea alata respectively. this means that dioscorea rotundata, when compared to the other yam species chosen for analysis, has a stronger resistance to deterioration and a longer shelf life. crude fiber content the crude fiber content ranged from 1.28±0.10e % for dioscorea bulbifera to 12.12±0.20c % for dioscorea alata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea alata having the highest crude fibre content. however, this result can be compared with reports by afiukwa et al. (2013) that ranged from 6.01±0.04b % to 13.03±0.80a % for species of dioscorea dumenturom and differs from reports by oko and famurewa (2014) that ranged from 3.31% to 3.53% for their dioscorea alata species. according to studies, fiber intake reduces the risk of obesity, cardiovascular disease, diabetes and softening stools (turner, 2014). ash content ash contents of the yam varieties ranged from 2.34±0.02a % for dioscorea bulbifera to 5.59±0.06a % for dioscorea alata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea alata having the highest ash content. however, these were different from reports by sorh et al. (2015) that ranged from 1.64±0.03 % to 1.78±0.03 % for dioscorea alata species. ash content reveals how heavily the yam cultivars are stuffed with minerals (akonor et al., 2017). as a result, compared to the other yam tubers examined, dioscorea alata has a higher mineral stuffing. crude protein content the crude protein content of the yam varieties ranged from 2.19±0.03c % for dioscorea rotundata to 8.64±0.03f % for dioscorea bulbifera. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea bulbifera having the highest crude protein content. however, the result can be godfrey et al. – comparative assessment of the proximate composition, … 181 compared with reports by ojinnaka et al. (2016) that showed 2.43±0.11b % for dioscorea bubilfera and in contrast with the report by ukom et al. (2014) that showed crude protein of dioscorea dumenturom to be 69.15±4.49b %. this demonstrates that of the dioscorea species examined, dioscorea bulbifera is the greatest source of protein. crude fat content the fat content in these analysed yam varieties ranged from 1.60±0.01a % for dioscorea bulbifera to 11.63±0.04e % for dioscorea rotundata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea rotundata showing the highest fat content. these findings, however, differ from those reported by ukom et al. (2014), who found that dioscorea cayenensis had an abundance of 41.91%. dioscorea bulbifera may be a superior source of calories than the other dioscorea species examined because dietary fat provides the majority of the energy needed by humans. carbohydrate content carbohydrate content of the dioscorea varieties were quite high and ranged from 57.38±0.20d % for dioscorea alata to 77.51±0.08f % for dioscorea bulbifera. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea bulbifera having the highest carbohydrate content. however, these values are comparable to literature values by ukpabi and akobundu (2014) that had 78.32±0.29 % for dioscorea dumenturom and in contrast with frank and kingsley (2014) that ranged from 24.25±0.62b % for dioscorea alata to 32.03±0.89c % for dioscorea rotundata. carbohydrates are considered the primary source of energy for all organisms (ojinnaka et al., 2016). table 2. functional properties of selected yam species. yam species bulk density (g/cm3) wac (%) oac (%) dispersibility (%) swelling capacity (g/g) d.alata 0.87±0.02a 155.34±0.02b 140.71±0.01d 62.85±0.02e 15.25±0.03b d.rotundata 0.85±0.02d 164.02±0.02f 149.76±0.02h 73.16±0.01c 14.29±0.01g d.bulbifera 0.57±0.03a 148.73±0.03a 133.85±0.01g 60.54±0.04a 8.44±0.03c values are means±standard deviation of triplicate analysis, wac: water absorption capacity, oac: oil absorption capacity. functional properties of selected yam species bulk density bulk density is a measure of the heaviness of a flour sample (oladele and aina, 2007). the bulk density of the dioscorea varieties ranged from 0.57±0.03a g/cm3 for dioscorea bulbifera to 0.87±0.02a g/cm3 for dioscorea alata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea bulbifera having the least bulk density while dioscorea alata had the highest. however, this result is comparable to that obtained from different cultivars of aerial yam which showed 0.810±0.01 g/cm3 for purple cultivars and 0.573±0.01 g/cm3 for white cultivars (ojinnaka et al., 2016). bulk density indicates the relative volume of packaging material required, as well as material handling and application in wet processing in food industries (tapti et al., 2018). dioscorea alata having the highest bulk density amongst the analysed yam species is often better for dispersibility and pastes thickness reduction, which is vital in convalescent child feeding (udensi and eke, 2000) while dioscorea bulbifera having the least bulk density is most useful in the formulation of infants weaning foods (akpata and akubur, 1999). water absorption capacity the ability of flour or starch to hold water against gravity is referred to as water absorption capacity (moure et al., 2006). the water absorption capacity of the dioscorea varieties ranged from 148.73±0.03 % for dioscorea bulbifera to 164.02±0.02 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest water absorption capacity. however, this result is comparable to that obtained from oluwamukomi and akinsola (2015) which showed 174.60±9.3 g/cm3 for dioscorea rotundata and 167.00±0.17 g/cm3 for dioscorea bulbifera by tapti et al. (2018). the water absorption capacity is an important parameter since it shows if the flours may be used in aqueous food formulations, product bulking and uniformity, as well as in several baking applications (iwe et al., 2016). consequently, dioscorea rotundata having the highest water absorption capacity than other analysed yam species is best suited for this. oil absorption capacity oil absorption capacity has been attributed to the physical entrapment of oil. the oil absorption capacity showed a significant difference (p<0.05) between the yam varieties. the oil absorption capacity of the dioscorea species ranged from 133.85±0.01 % for dioscorea bulbifera to 149.76±0.02 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest oil absorption capacity. however, this result is higher than the results obtained by kimbonguila et al. (2019) which ranged from 83.33% to 100% for different cultivars of dioscorea alata analysed. oil absorption capacity is an 182 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 indication of the rate at which the protein binds to fat in food formulations. it is important for flavour retention and boosting the mouthfeel of food. consequently, dioscorea rotundata is best suited for this, since it has the highest oil absorption capacity than the other yam species analysed (abu et al., 2005). swelling capacity the swelling capacity the dioscorea varieties ranged from 8.44±0.03 % for dioscorea bulbifera to 15.25±0.03 % for dioscorea alata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea alata having the highest swelling capacity. however, this result is comparable to that obtained from eke-ejiofor and owuno (2012) which showed 10.81±0.01 % for dioscorea dumenturom and slightly higher than reports by ojinnaka et al. (2016) that showed 7.58±0.01 % for dioscorea bulbifera sample. swelling power is a measure of starch hydration and is used to show associative binding force within starch granules (bello and ekeh, 2014). basically, it shows how much water starch granules can store (soison et al., 2015). as such, dioscorea alata is the best flour sample amongst the other yam species analysed for the formulation of infant weaning foods, since it has the highest swelling capacity (ojinnaka et al., 2016). dispersibility the percentage dispersibility gives an indication of water absorption capacity. the dispersibility of the yam species ranged from 60.54±0.04 % for dioscorea bulbifera to 72.17±0.01 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest dispersibility. however, this result is comparable to that obtained from bashirat et al. (2015) which showed 72.17±0.01 % for dioscorea rotundata and 62.85±0.01 % for dioscorea alata. a dispersibility of fifty percent or more is considered high, implying that the higher the dispersibility, the better the flour’s capacity to reconstitute in water to form a fine and consistent paste when mixing (adebowale et al., 2005). table 3. amino acid profile of selected yam species (g/100g). amino acids dioscorea alata dioscorea rotundata dioscorea bulbifera leucine 6.29±0.06a 8.46±0.04a 7.18±0.02e lysine 4.37±0.05a 4.04±0.02d 4.82±0.01f isoleucine 3.59±0.16b 3.66±0.02c 3.54±0.04i phenylalanine 6.65±0.04e 5.68±0.12e 4.44±0.03a tryptophan 0.95±0.01c bdl 0.89±0.01b valine 4.36±0.02f 5.78±0.03a 3.97±0.03d methionine 1.76±0.03a 2.37±0.02b 2.07±0.06c histidine 1.98±0.02d 1.83±0.02c 1.78±0.02a threonine 3.46±0.03i 4.49±0.01h 3.18±0.02b *proline 1.98±0.04a 1.64±0.02a 2.03±0.03e *arginine 8.94±0.03b 6.97±0.03d 8.27±0.03f *tyrosine 3.10±0.02b 4.14±0.02e 2.76±0.03a *cystine 1.09±0.01e 2.78±0.03a 1.22±0.01b *alanine 3.27±0.03c 2.99±0.03c 2.99±0.03d *glutamic acid 5.76±0.02f 6.96±0.01h 8.55±0.01c *glycine 2.14±0.02d 2.04±0.02b 1.68±0.02a *serine 2.22±0.02i 3.00±0.01c 2.73±0.02h *aspartic acid 5.49±0.02h 5.96±0.02a 6.22±0.02f teaa 33.42±0.41 (49.61 %) 36.32±0.16 (49.88 %) 31.89±0.24 (46.60 %) tneaa 33.95±0.18 (50.39 %) 36.49±0.16 (50.12 %) 36.45±0.19 (53.34 %) values are means ± standard deviation of triplicate analysis, teaa: total essential amino acid, tneaa: total non-essential amino acid, bdl: beyond detection limit, *non-essential amino acids. amino acid profile table 3 depicts the results of the amino acids profile. amino acids are the building blocks of proteins and they serve an important role in the body. the findings imply that dioscorea species are rich in amino acids such as essential and non-essential amino acids. the essential amino acids include lysine, phenylalanine, valine, threonine, isoleucine, methionine, histidine and leucine. while the non-essential amino acids are alanine, arginine, aspartic acid, serine, tyrosine, proline, cysteine and glutamic acid. however, the results obtained showed that the non-essential amino acid was more than the essential amino acids in the yam species. the total nonessential amino acid values were 33.95±0.18 g/100g, 36.49±0.16 g/100g and 36.45±0.19 g/100g representing 50.39 %, 50.12 % and 53.34 % for dioscorea alata, dioscorea rotundata and dioscorea bulbifera respectively. while the total essential amino acid contents were 33.42±0.41 g/100g, 36.32±0.16 g/100g and 31.89±0.24 g/100g representing 49.61 %, 49.88 % godfrey et al. – comparative assessment of the proximate composition, … 183 and 46.60 % for dioscorea alata, dioscorea rotundata and dioscorea bulbifera respectively. this result can be compared to reports by alozie et al. (2009). dioscorea alata had the least non-essential amino acid content with 33.95±0.18 g/100g while dioscorea rotundata had the highest with 36.49±0.16 g/100g. also, for the essential amino acid, dioscorea bulbifera had the least content with 31.89±0.24 g/100g, while dioscorea rotundata had the highest with 36.32±0.16 g/100g. as such from the research it can be seen that dioscorea rotundata had the highest total amino acid content. furthermore, the percentage ratio of the essential amino acid (eaa) to the total amino acid (taa) in the samples ranged from 46.66 % to 49.88 %. these values are well above the 39% considered adequate for ideal protein food for infants, 26% for children and 11% for adults. glutamic acid appeared to be the most abundant amino acid in dioscorea bulbifera at 8.55±0.01c g/100g. while leucine was the highest in dioscorea rotundata at 8.46±0.04a g/100g. like valine and isoleucine, leucine is a branched-chain amino acid (bcaa) that is essential for protein synthesis and muscle repair. it also aids in blood sugar regulation, wound healing and the production of growth hormones (shimomura et al., 2004). arginine which can also be considered a conditionally essential amino acid just like glycine was the most abundant amino acid in dioscorea alata at 8.94±0.03b g/100g. the high level of arginine in dioscorea alata indicates its usefulness as a supplement during pregnancy, trauma and illness (cancer). tryptophan which is a sole precursor to serotonin, a neurotransmitter that regulates appetite, sleep and mood, was quite low in all the samples but was relatively absent in dioscorea rotundata (slominski et al., 2002). histidine levels were highest in dioscorea alata at 1.98±0.02d g/100g. this shows its likeliness to produce more histamine which is a neurotransmitter that is vital to immune response, digestion, sexual function, maintaining levels of hemoglobin and sleep-wake cycles. it is also required for the growth and repair of tissues, red blood cell production and protecting tissues from damage from radiation and heavy metals. it is especially very important for the formation of myelin sheaths, which are layers surrounding nerves that enables faster transmission of signals to the brain (ncbi, 2022). the minimum amino acid intake of 1.5 g/kg/day is reported to be necessary in preventing negative nitrogen balance while 2.5 g/kg/day is not advisable (espghan, 1997). conclusions this study provided vital information on the proximate composition, amino acid profile and functional properties of the selected yam tuber species (dioscorea alata, dioscorea rotundata and dioscorea bulbifera) analysed. the generally high carbohydrate content indicates that these species are reliable sources of energy. they can also be considered to be rich in fibre, mineral stuffing and have a high shelf life due to their generally high crude fibre and ash content as well as low moisture content respectively. the amino acid profile of these studied species, suggests that they are rich in protein. however, dioscorea rotundata was the richest in amino acid content, as it had 36.32±0.16 g/100g and 36.49±0.16 g/100g, for essential and non-essential amino acids respectively. furthermore, the wide variation observed in the functional properties of the flour samples serves as a database for the selection and improvement of the yam species for specific food applications to stimulate their industrial processing and utilization. competing interests: the authors declare that there are no competing interests. references abu, j. o., muller, k., duodu, k. g., minnaar, a. 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(2014). production and quality evaluation of pre-gelatinized fermented breakfast food produced from edible trifoliate yam (dioscorea dumenturom) using intermediate technologies in nigeria. american journal of food science and nutrition, 1(4), 60-7. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 143-150 | doi: 10.14421/biomedich.2023.121.143-150 issn 2540-9328 (online) phytochemical, antioxidant screening, antinociceptive, and anti-inflammatory activities of boswellia dalzielii hutch (burseraceae) root ethanol extract using animal model macdonald idu1, anthonia omoregbee1, benjamin ogunma gabriel1,2,* 1phytomedicine unit, department of plant biology and biotechnology; 2science laboratory technology, university of benin, pmb 1154, benin city, edo state. nigeria. corresponding author* alexthemain076@gmail.com manuscript received: 28 september, 2022. revision accepted: 05 december, 2022. published: 14 january, 2023. abstract this study investigated the biological activities and phytochemical screening of boswellia dalzielii root ethanol extract. standard procures were used to evaluate the phytochemicals and antioxidant capacity, antipyretic activity in baker’s yeast-induced pyrexia in mice, analgesic property (hotplate and acetic acid-induced in mice), acute anti–inflammation (carrageenan-induce in rats) and chronic arthritis (formalin– induced in rats) on boswellia dalzielii root ethanol extract. the phytochemical results revealed the presence of phenol, ascorbic acid, flavonoids, alkaloids, cardiac glycoside, tannin, saponin. the extract had a significant reduction in the body temperature in graded doses and 100 mg/kg paracetamol at 60 minutes when compared with the control, but 400 mg/kg was more effective (p<0.01). morphine a nd plant extract showed a slight significant analgesic property at 0 and 30 minute compared to the control. the ext ract at 100 mg/kg elicited a significant increase at 60 and 90 minutes compared with the control, and it is comparable to 5 mg/kg morphine. the plant extract (100, 200, and 400 mg/kg) and aspirin (100mg/kg) shows significant analgesic properties compared to control (p<0.01) but 200 mg/kg of extract revealed highest percentage inhibition. the extract produced no significant reduction on carrageenan induced inflamma tory at all dose level compared to control (p>0.05). the plant extract (100, 200, 400 mg/kg) and indomethacin (1 mg/kg) reduced paw volume across the doses from day 4 compared to the control (p<0.01). the boswellia dalzielii root extract is a promising anti–inflammatory agent, it also possesses antipyretics, and analgesics effect validating the folklore claim. keywords: phytochemicals, ethanol; boswellia dalzielii; pyrexia; anti-inflammatory; mice. introduction phytochemicals with medicinal properties are also known as secondary metabolism. it ranges from cell wall substances through photosynthesis pigments, terpenes and terpenoids, the alkaloids, plants phenolics to plant hormones, plant non-proteins, amino acids and cyanogenic glycosides (harborne, 1973). many phytochemicals have been shown to be bioactive, that is they exhibit pronounced biological activity in other living organisms (harborne, 1973). many of the bioactive phytochemicals have found usefulness as chemotherapeutic agents, pesticides, food additives, and other biological (tripathi and tripathi, 2003; odesanmi et al., 2009). modern medicine recognizes herbal medicine as a form of alternative medicine, as the practice of herbal medicine is not strictly based on evidence gathered using the scientific method. modern medicine, however, make use of many plant-derived compounds as the basis for evidence-tested pharmaceutical drugs, and phytotherapy works to apply modern standards of effectiveness testing to herbs and medicines that are derived from natural sources. boswellia dalzielii hutch (burseraceae) commonly known as the frankincense tree. the bini called it ebanarukhoe, fulani called it januhi, while the hausa called it ararrabi, basamu and hanu. the stem bark secretes a fragrant white gum that is burnt to fumigate cloth and to drive out flies, mosquitoes, antiseptic, fever, rheumatism, gastrointestinal, antidotes for arrow poison, giddiness, palpation, cancers/fibrosis, inflammation, snake bite, arthritis, asthma, microbes, ulcer, syphilis and diarrhea (nwinyi et al., 2004; moses et al., 2005; abubakar et al., 2007; bello et al., 2013). bello et al., 2013 reported that the phytochemicals detected in ethanol and aqueous stem bark extracts include; flavonoids, terpenoids, alkaloids, tannins, saponins, quinones, and cardiac glycosides. this study investigated the phytochemical screening, antipyretic, analgesic, and anti-inflammatory activity of boswellia dalzielii ethanol extract. https://doi.org/10.14421/biomedich.2023.121.143-150 https://en.wikipedia.org/wiki/alternative_medicine https://en.wikipedia.org/wiki/evidence https://en.wikipedia.org/wiki/scientific_method https://en.wikipedia.org/wiki/pharmaceutical_drug https://en.wikipedia.org/wiki/phytotherapy 144 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 materials and methods collection of plant material the fresh root of boswellia dalzielii was collected from makaya forest, kibiya local government area, kano state in the mouth of may, 2015. the plant root was identified and authenticated by dr. timothy odaro in the herbarium unit of the department of plant biology and biotechnology, with the voucher number ughk257. the roots were chopped, air dried for four weeks and ground to course powder. the dried seeds were ground to powder using a mechanical grinder. preparation of plant extract the dried powder was weighed (100 g) and then macerated in 2.5 liters of ethanol for 72 hours. the solution of the root was decanted into another flask. the solution was filtered and concentrated at 40oc by means of a rotary evaporator. the evaporated extract was transferred into an oven at 40oc and evaporated to dryness for 24 hours. the concentrated extract was transferred into a sample bottle of known weight. the weight of the ethanol root extract was 111.5 g giving a yield of 15 %. phytochemical screening the preliminary qualitative and quantitative phytochemical test was screening and identification of bioactive chemical constituents in the ethanol root extract of boswellia dazielii under study that was carried out in extract. specimens using follows the standard procedures as described by sofowora (1993) and tiwari et al. (2011); naredra devanboyina et al. (2013). experimental animal adult swiss mice and wistar rats of male and female sexes weighed between (20 – 30 g) and (180-220 g) were obtained from the animal house, department of pharmacology, faculty of pharmacy, university of benin, benin city. they were acclimatization for 14 days. they were housed in a well-ventilated woody cages in a normal laboratory state (12 hours light/dark cycle: 23 ± 2°c) and fed using a standard diet. food and water were administered at free choice (ad libitum) to the animals designed for experiments. the animals were properly handled using the ethics of laboratory animals’ approval from the ethical committee of the faculty of life sciences with the ethical number ls21592. experimental design the animals were transferred into the laboratory 24 hours before the commencement of the experiment and were randomly grouped into five (n=5). this was done based on the design protocol of the study incudes: the groups for antipyretic study, rectal temperature were taken in order to accustom handling and the environment. the animals were exposed to natural lighting conditions and a room temperature of between 22-26oc and has free access to water and laboratory diet. the animals were carefully handled and the ethical committee of the faculty of life sciences issued an ethical number ls20619. antipyretics activity the animals had their rectal temperature measured, using a sfda lubricated probe digital thermometer (kft–04). a stock solution of 30% (w/v) of baker’s yeast in 0.9% nacl solution was prepared. the intended doses were 10ml/kg and the doses to be administered to individual mouse were calculated with respect to their weights. the different doses were administered intraperitoneally and the time of administration was noted. the different groups were observed for 6 – 7 hours (bafor et al., 2010). at the 7th hour, the rectal temperature was taken for the different groups and a calculated dose of 0.9% nacl solution (control), extract and paracetamol (standard). the control group received 10 ml/kg of nacl orally while the extract groups received 100, 200, 400 mg/kg orally and the standard group received 100 mg/kg of paracetamol intraperitoneally. the different groups were observed for change and sign and their rectal temperature were recorded for 2 hours at 30 minutes hour interval (bafor et al., 2010). analgesic property hot plate method the paws of mice are very sensitive to heat at temperature which is not damaging the skin. the responses are jumping, withdrawal of the paws and licking of the paws. the hot plate (ugo basile hot/cold plate 35100) consist of electrically heated surface, the temperature is controlled to 55.5oc. the animals were placed on the hot plate for 30 minutes before the experiment in order to accustom the environment, after which each animal was placed on the hotplate and the time until either licking or jumping is recorded (shanmugasundaram and venkataraman, 2005). the control group received 10 ml/kg 0.9% nacl orally, while extract groups received 100, 200, 400 mg/kg orally and the standard group received 5 mg/kg morphine intraperitoneally. after 30 minute the animals were placed on the hot plate and the observation were recorded and the time interval of 60, 90 and 120 minutes for all groups. the result of the hot plate method was tabulated. acetic acid induced writhing method the writhing model represents a chemical nociceptive test based on the induction of peritonitis like condition in animals by injecting irritant substances intraperitoneally. the control group received 10ml/kg 0.9% nacl orally, while extract groups received 100, 200, 400 mg/kg and the standard control received 100 mg/kg of aspirin orally and 30minute later all groups idu, et al. – phytochemistry, biological activity of boswellia dalzielii hutch (burseraceae) 145 received 10mg/ml of 1% acetic acid solution intraperitoneally (vogel, 2002). the mice were placed individually into glass beakers and immediately were observed for a period of 20 minutes and the numbers of writhes were recorded in each animal. for scoring purpose, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb percentage inhibition was calculated using the following formula: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = {(𝑊𝑐 − 𝑊𝑡) × 100} 𝑊𝑐 where, 𝑊𝑐 : no. of writhes in control group, 𝑊𝑡 : no. of writhes in test group. carrageenan-paw induced edema in evaluating carrageenan-induce paw edema, 25 wistar rats were randomly divided into 5 groups (n=5). the control group was treated with distilled water, 10 ml/kg, the test groups and graded doses (100, 200 and 400 mg/kg) of the treatment groups, while the standard group received indomethacin (10 mg/kg) orally. after one hour, 0.1 ml/kg of 1% carrageenan was injected into the subplantar tissue of the right hind paw (vogel, 2002). paw edema was measured using a vernier caliper at 30, 60, 120, 180, and 240. formaldehyde induced arthritis arthritis was induced by injecting 0.1 ml of 1% of formaldehyde into the hind paw of albino rat using the model described by fatima and fatima (2016). twentyfive (25) wistar rats weighing 180-220 mg/kg were randomly divided into 5 groups (n=5). group 1 received the vehicle (distilled water) (1 ml/kg) without arthritis (normal control). group 2 received the vehicle (distilled water) with arthritis. group 3 received the standard drug dexamethasone (2 mg/kg). group 4, 5, 6 and 7 received the extract (100, 200, and 400 mg/kg). before administration of the standard and test drug, the rat was induced arthritis by injecting 0.1 ml of formaldehyde into the hind paw of the animal. at the third day, formaldehyde was re-injected again into the hind paw followed by treatment with the test agent. the thickness of the paw was measured at days 2, 3, 4, 5, 6, 7, 8, 9 and 10. statistical analysis data were expressed as the mean ± sem. the data were analyzed using one-way analysis of variance (anova) followed by tukey's post hoc test. statistical analysis was performed using graph pad prism version .6.01. p < 0.05 was considered significant results and discussion the qualitative and quantitative phytochemistry, antioxidant, identification and in vitro antioxidant property of boswellia dalzielii root ethanol extract. table 1 showed the presence of alkaloid, flavonoid and cardiac glycoside in abundantly while carbohydrate, saponin and tannin were moderately present. the total antioxidant capacity was shown in figure 1, which indicates that the total phenolic content was high compared to ascorbic acid that was very low. the phytochemicals screening of boswellia dalzielii root ethanol extract revealed the presence of alkaloid, flavonoid, saponins, tannins, cardiac glycoside and carbohydrate. alkaloids are naturally occurring chemical compounds with nitrogen atoms (andreas luch, 2009), and it is use as a starting points for drug discovery in traditional or modern medicine used for analgesic and anti-bacterial activities. the findings of raymond et al. (2010); cushnie et al. (2014), adhere to the analgesic and anti–inflammatory activities of this present study. study of raymond et al. (2010) showed the effect of flavonoids as a potent antioxidant, antiinflammatory, vasculopropertor, anti-hepatotoxic, antiallergic and antitumor properties (singh and gambhir, 1998). the presence of phenolic and ascorbic acid antioxidant contents of the extract may have contributed to the analgesic and anti–inflammatory properties. table 1. the qualitative and quantitative phytochemical screening of boswellia dalzielii root methanol extract. phytochemicals constituents observations concentration(mg/kg) mean ± sem alkaloid +++ 127 ± 0.01 flavonoid +++ 77.84 ±0.00 carbohydrate ++ – cardiac glycoside +++ – saponin ++ 124 ± 0.00 tannin ++ 145 ± 032 key: +++ = abundantly present, ++ – = moderately present n u m b e r o f r e p l i c a t e a n t i o x i d a n t v a l u e 0 1 2 3 4 0 5 0 1 0 0 1 5 0 t a c ( m g v c e ) t p c ( m g g a e 1 0 0 -1 ) t a c ( m g /k g ) figure 1. total antioxidant capacity, phenolic content and ascorbic content of the ethanolic extract of boswellia dalzielii root. https://en.wikipedia.org/wiki/natural_product https://en.wikipedia.org/wiki/chemical_compound https://en.wikipedia.org/wiki/nitrogen https://en.wikipedia.org/wiki/drug_discovery https://en.wikipedia.org/wiki/drug_discovery https://en.wikipedia.org/wiki/traditional_medicine https://en.wikipedia.org/wiki/pharmaceutical_drug 146 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 the extract produced significant reduction (p < 0.05) in body temperature at all dose level and standard (100 mg/kg paracetamol) when compared with control. the antipyretics activities of the extract was in dose dependent and also comparable to the standard. the results also show that the antipyretics effect of the extract increase with time, in table 2. the root extract of boswellia dalzielii displayed a significant decrease in baker’s yeast-induced pyrexia with an increase in grade time, starting from 60 minutes and progresses a through the study when compared with the control, but at highest dose of 400 mg/kg, the extract showed more effect. antipyretic effect of the extract was comparable to that of the standard drug (paracetamol), with an underlying mechanism of action unknown. yeast is known to be an exogenic pyrogen responsible for an increased in the body temperature via the formation and synthesis of cytokines called as endogenic pyrogens, hence this endogenic pyrogen centrally promote thermosensitive neutrons in the preoptic area of the hypothalamus, increasing heat and decreasing heat loss. the body temperature increases to a set point that leads to fever (kelly et al., 2003). certain phytochemicals including tannins, flavonoids, and cardiac glycosides of the root extract as a potent antipyretic action observed. flavonoids have been reported in previous study carried out by germain et al. (2011) to inhibit prostaglandins and arachidonic acid peroxidation, which surmount to the lowering of the prostaglandin thus causes a reduction in fever occurrence (houghton and raman, 1998; germain et al., 2011). the main mechanism of action through which acetaminophen act is by the inhibition of cyclooxygenase (cox), or highly selective cox–2 receptors (hendrickson et al., 2006). paracetamol reduces the oxidized form of the cox enzyme, thereby reducing the release of prostaglandin e2 in the central nervous system to lower its binding from the hypothalamus set point of thermoregulatory center (hendrickson et al., 2006) which may be consider also as mechanism of action of the extract. table 2. the effect of boswellia dalzielii root ethanol extract on barker’s yeast-induced pyrexis in mice. groups dose mg/kg base line 0c 6 hrs 7 hrs control (nacl) 10 ml/kg 39.43±0.10a 39.65±0.17a 39.95±0.15a boswellia dalzielii 100 39.90±0.18a 37.15±0.16b 37.05±0.17b boswellia dalzielii 200 39.00±0.09a 37.28±0.16a 37.13±0.13b boswellia dalzielii 400 39.88±0.79a 37.30±0.15b 37.18±0.18b aspirin 100 39.08±0.14a 37.22±0.14b 37.20±0.19b the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05 boswellia dalzielii root ethanol extract exhibited a significant decrease in hot plate-induced analgesia when compared to the control. the analgesic effect of the extract was comparable to the morphine standard analgesic, at the lowest concentration. the presence of certain phytochemicals like alkaloids displayed a major role in the analgesic action observed from the extract. this agreed with sinatra et al. (2010) study on analgesic. the significant increase in pain threshold synthesized by hot plate model suggested the connection with the central pain pathways. pain is centrally modulated via series of multifaceted processes on the opiate, dopaminergic descending noradrenergic and serotonergic systems (headley and shaughnessy, 1985; wigdor and wilcox, 1987; yekkirala et al., 2012). the analgesic potential of the treatment groups act with central mechanisms of action connecting the receptor and the inhibitory effect of prostaglandins, leukotrenes, and other endogenous neurotransmitters that triggered pain. morphine being a phenanthrene opioid receptor agonist elicited its main effect is an requisite and activation of μ–opioid receptors in the central nervous system. the μ–δ–opioid receptor heteromer (yekkirala et al., 2010). the μ–binding sites are discretely spread across human brain, with a high densities in the posterior amygdala, hypothalamus, thalamus, nucleus caudatus, putamen, and certain cortical areas. they are also present in the terminal axons of the primary afferents within the laminae i and ii (substantia gelatinosa) of the spinal cord and in the spinal nucleus of the trigeminal nerve. morphine exerts its main action on the central nervous system and gastrointestinal tract to excite possible analgesic action. it also serve as an κ– opioid and δ–opioid receptor agonist, associated with spinal analgesia, meiosis (pinpoint pupils) and psychotomimetic effects. δ–opioid is thought to play a role in analgesia (chien and pasternak, 1995). https://en.wikipedia.org/wiki/phenanthrene https://en.wikipedia.org/wiki/opioid_receptor https://en.wikipedia.org/wiki/receptor_agonist https://en.wikipedia.org/wiki/mu_opioid_receptor https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/gpcr_oligomer https://en.wikipedia.org/wiki/human_brain https://en.wikipedia.org/wiki/amygdala https://en.wikipedia.org/wiki/hypothalamus https://en.wikipedia.org/wiki/thalamus https://en.wikipedia.org/wiki/nucleus_caudatus https://en.wikipedia.org/wiki/nucleus_caudatus https://en.wikipedia.org/wiki/putamen https://en.wikipedia.org/wiki/chemical_synapse https://en.wikipedia.org/wiki/posteromarginal_nucleus https://en.wikipedia.org/wiki/substantia_gelatinosa_of_rolando https://en.wikipedia.org/wiki/substantia_gelatinosa_of_rolando https://en.wikipedia.org/wiki/trigeminal_nerve https://en.wikipedia.org/wiki/human_gastrointestinal_tract https://en.wikipedia.org/wiki/kappa_opioid_receptor https://en.wikipedia.org/wiki/kappa_opioid_receptor https://en.wikipedia.org/wiki/delta_opioid_receptor https://en.wikipedia.org/wiki/miosis https://en.wikipedia.org/wiki/psychotomimetic idu, et al. – phytochemistry, biological activity of boswellia dalzielii hutch (burseraceae) 147 g r o u p s r e a c t in g t im e ( s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g ) m o r p h in (5 m g /k g ) 0 2 4 6 8 g r o u p s r e a c t in g t im e ( s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g ) m o r p h in (5 m g /k g ) 0 2 4 6 8 g r o u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g `2 0 0 m g /k g 4 0 0 m g /k g ) m o r p h in (5 m g /k g 0 5 1 0 1 5 ** ** * g r o u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g m o r p h in (5 m g /k g ) 0 5 1 0 1 5 ** * * ** figure 2. (a, b, c, and d) effect of boswellia dalzielii root ethanol extract on hotplate-induced pain. plant extract at 100mg/kg, 400mg/kg and morphine increase the reaction time to pain induced by hotplate at 0, 30, 60 and 90 minutes exposure compared to control (**p < 0.01, *p < 0.05). the plant extract 100, 200, 400mg/kg and aspirin (100mg/kg) reduced the number of writhing compared to control (p<0.01), in table 2. the root ethanol extract of boswellia dalzielii exhibit a significant decrease in acetic acid induced analgesia when compared with the control. the analgesic effect of this present study was comparable to the standard analgesic, aspirin. abdominal constriction reaction induced by acetic acid serves as a sensitive procedure for the evaluation of peripherally pain acting analgesics (gene et al., 1989). acetic acid is a peripheral pain triggering factor capable of liberating the endogenous substances (histamine, prostaglandins (pgs), bradykinins and substance p, endings). the local peritoneal receptors are proposed to be complicated in the abdominal with a constrictive response (bentley et al., 1983). prostanoids is a general stimulatory factor that increases the levels of pge2 and pgf2α in the peritoneal fluids as well as lipoxygenase products (derardt et al., 1980). the analgesic effect instigated by the treatment via peripheral mechanisms could be involved in prostaglandins, leukotrenes, and other endogenous substances with inhibitory effect. the presence of certain phytochemicals (alkaloids) in the facilitated analgesic action observed from this study (sinatra et al., 2010). aspirin as a pure compound suppresses the production of prostaglandins and thromboxanes to trigger irretrievable inactivation of the cyclooxygenase (cox; known as prostaglandin– endoperoxide synthase, ptgs) enzyme needed for prostaglandin and thromboxane production. it also acts via acetylating agent where acetyl group is covalently bond to serine residue in the active site of ptgs enzyme. this makes aspirin different from other nsaids, which are reversible inhibitors (burke et al., 2006). table 3. the effect of boswellia dalzielii root ethanol extract on acetic acid-induced writhing in mice. group doses mg/kg numbers of writhing % inhibition control (nacl) 10 ml/kg 91.75±10.48a boswellia dalzielii 100 45.00±10.34b 50.95 boswellia dalzielii 200 34.00±7.22b 62.94 boswellia dalzielii 400 36.50±5.33b 60.22 aspirin 100 42.00±5.95b 54.22 the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05. the ethanol root extract of boswellia dalzielii exhibit a significant decrease in carrageenan-induced inflammatory when compared with inflammatory control. the report of cushnie et al. (2014) concurred to this present ant-inflammatory potential. various components leads to inflammatory response stimulating edema formation, leukocyte infiltration and granuloma formation inked with the components of inflammation (mitchell and cotron, 2010). edema formation in the paw is as a result of the synergistic action between inflammatory mediators that triggered an increase in the vascular permeability of blood flow (lalenti et al., 1995). the inhibitory effect of carrageenan-induced inflammation in rats was an established model for the evaluation of anti–inflammatory ingredient, with the frequency to evaluate the anti–inflammatory potential on natural remedies. the development of carrageenaninduced edema is consist of two phase system such as first phase (within one hour of carrageenan inflammation) and is usually intermediate with the release of cytoplasmic enzymes serotonin, discharged from mast cells. the second phase is (mediated release of prostaglandins) in most inflammatory area and linger between the two phases provided by kinins (goel et al., 2001). a b c d https://en.wikipedia.org/wiki/cyclooxygenase https://en.wikipedia.org/wiki/serine 148 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 table 4. the effect of boswellia dalzielii root ethanol extract on carrageenan induced inflammation in rats. paw volume (mm) treatment doses mg/kg t30 min t60min t120min t180min t240min control (nacl) 10 ml/kg 1.28±0.51a 1.46±0.55a 1.62±0.59a 1.08 ±0.29a 0.74±0.25a boswellia dalzielii 100 0.83±0.l14b 1.08 ±0.16b 2.06±0.27a 1.56±0.44a 1.12±0.47a boswellia dalzielii 200 0.69±0.62b 0.63±0.35b 0.72±0.37b 0.77±0.39b 0.49±0.37b boswellia dalzielii 400 0.99±0.80b 0.91±0.17b 1.70±0.44a 0.73±0.25b 0.56±0.20b indomethacin 10 0.79±0.34b 0.78±0.35b 0.18±0.17b 0.32±0.21b 0.09±0.05b the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05. the effect of ethanolic extract of boswellia dalzielii root on formalin–induced inflammation (arthritis) in rats (table 4). the plant extract 100, 200, 400 mg/kg and indomethacin (1mg/kg) reduced paw volume starting from day 4 and progresses through the study compared to control (p < 0.01). boswellia dalzielii root ethanol extract exhibit a significant decrease in formalin– induced arthritis across the graded doses when compared with the arthritic control. the anti-arthritic effect of the extract had a significant reduction when compared with indomethacin. the possible underlying mechanism of the extract has not be established rather it triggered an inhibitory effect of edema induced by formalin in rats, which is a most suitable test procedures to screen for anti–arthritic and anti–inflammatory agents (mitchell and cotron, 2010). table 5. the effect of boswellia dalzielii root ethanol extract on formalin–induced arthritis in rats. treatment doses mg/kg day 2 day3 day4 day5 day6 day7 day8 day9 day10 control (nacl) 10 ml/kg 3.18±0. 49a 1.54±0.38a 2.69±0.04a 2.36±0.64a 2.45±0.20a 2.40±0.26a 2.54±0.10a 3.28±0.20a 3.79±0.38a boswellia dalzielii 100 3.15±0.16a 1.06±0.23b 1.36±0.13b 0.72±0.19b 0.78±0.19b 0.66±0.19b 0.46±0.15b 0.56±0.15b 0.77±0.12b boswellia dalzielii 200 2.10±0.294b 0.79±0.10b 0.99±0.12b 0.79±0.09b 0.37±0.10b 0.51±0.16b 0.38±0.12b 0.56±0.15b 0.32±0.16b boswellia dalzielii 400 2.20±22.63b 0.55±0.18b 1.60±0.38b 0.99±0.33b 0.67±0.37b 0.49±0.27b 0.40±0.60b 0.39±0.14b 0.44±0.12b indomethacin 1 2.18±0.22b 0. 80±0.61b 0.80±0.30b 0.90±0.20b 1.10±1.00b 0.11±0.90b 1.13±0.10b 1.12±1.15b 0.64±.10b the results gotten from the formalin-induced tests is associated with cell and tissue damages instigated arthritic conditions. this is as a result of the changes in connective tissue metabolism, which is one of the major biochemical cascade events leading to inflammation. these changes occurred in the alteration of relative composition of the various constituents in the connective tissue incudes; mucopolysaccharides, glycoprotein, hexosamine and hydroxy proline, sialic acid (houck and jacob, 1969). hence the concentration of hexosamine and hydroxyproline had a significant increase in formalin-induced rats. boswellia dalzielii extract pretreatment elicited an inhibitory effect with the accumulation of hydroxyl proline and hexosamine in the edematous tissue of the formalin–induced rats. indomethacin is a nonselective inhibitor of cyclooxygenase (cox) i and ii, enzymes that triggered prostaglandin synthesis from arachidonic acid. prostaglandins are hormone–like molecules present in the body, with wide variety of effects, some of which it excite pain, fever, and inflammation (brayfield, 2014) conclusion the result from this investigation shows that the ethanolic extract of boswellia dalzieliiroot is a potent antipyretic analgesic and anti-inflammatory agent, as further study should be done to improve on it. conflict of interest: the authors declare that there is no conflict of interest. references abubakar, m. s., musa a. m., ahmed a. and hussaini, i. m. 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(2010). standard opioid agonists activate heteromeric opioid receptors: evidence for morphine and [d–ala(2)–mephe(4)– glyol(5)]enkephalin as selective μ–δ agonists. acs chemical and neuroscience, 1(2): 146–154. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 407-412 | doi: 10.14421/biomedich.2023.121.407-412 issn 2540-9328 (online) the anticancer properties of guava leaves (psidium guajava l.) and turmeric rhizome (curcuma longa l.) against breast cancer: a literature study angga puja asiandu1,2,*, widya sari3,4, septi widiya sari5, alif syahrul abdul majid6 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia. 2laboratory of biology, sma it harapan mulia, palembang 30113, indonesia. 3laboratory of physics, sma it harapan mulia, palembang 30113, indonesia. 4department of physics, faculty of math and natural science, universitas gadjah mada, yogyakarta 55281, indonesia. 5department of sociology, university of bengkulu, bengkulu 38731, indonesia. 6faculty of medicine, public health and nursing, universitas gadjah mada,yogyakarta 55281, indonesia. corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 30 november, 2022. revision accepted: 22 july, 2023. published: 03 august, 2023. abstract breast cancer, one of the most deadly diseases occurring in women, is caused by factors. in the healing process, the sufferer needs treatment, such as radiation techniques, surgery, and chemotherapy. but, these techniques have avoidable weaknesses that damage healthy cells. to date, natural sources can be utilized in medicine field. guava (psidium guajava l.) and turmeric (curcuma longa l.) are two common plants obtaining compounds that inhibit the growth of cancer cells based on phytochemical properties. this study was written to review the potential both of plants as anticancer agents. through the literature, guava leaf extract consists of flavonoids, tannins, alkaloids, saponins, terpenoids that inhibit the proliferation of cancer cells. besides, turmeric also has tannins, alkaloids, terpenoids, flavonoids, glycosides, sterols, and curcumin. the presence of curcumin reduces histamine production which induce inflammation and decrease toxin. because of curcumin, breast cancer cells have dehydration before apoptosis. keywords: apoptosis; curcumin; natural sources; phytochemical. introduction various deadly diseases always lurk in human welfare at any time worldwide. one of those diseases suffered in many countries is cancer. cancer is the second highest cause of death after cardiovascular disease. based on who data, the worldwide mortality rate due to cancer reached 13%. every year, there were 12 million people in the world suffering from cancer and as many as 7.6 million of them died. the number of cancer patients will continue to increase. in 2030, it is estimated that the number will reach 26 million people with death is expected about 17 million (ministry of health 2015). based on basic health research data in 2007, the national rate of cancer patients in indonesia was 4.3 per 1000 population, with higher cancer rates occurring in women than men. in women, 5.7 per 1000 people in indonesia suffered from cancer. meanwhile, 2.9 per 1000 male population experienced cancer (ministry of health of indonesia 2013; dewi and hendrati 2015). the common type of cancer attacking women is breast cancer which causes death in women. the american cancer society (2008) reported that as many as 1.3 million women worldwide were diagnosed with breast cancer. every year, approximately 465,000 women died from breast cancer. within 25 years, the number of patients had increased by around 30% in developed countries (wahyuni 2015). the primary cause of breast cancer is not yet fully known, but it is believed to be multifactorial or caused by many factors. there are several factors of breast cancer, such as genetic disorders facilitating the emergence of cancer cells, chronic irritation and inflammation, radiation, exposure to certain chemical compounds, carcinogenic foods, and others (suryaningsih and sukaca 2009; dewi and hendrati 2015). treatments of breast cancer are usually done with radiation techniques, surgery, and chemotherapy. however, these techniques have side effects because they sometimes enhance cancer cells to spread to other parts of the body, damage healthy cells, and also trigger cancer cells to mutate. thus, the discovery of new drugs that are safer with fewer side effects is needed in overcoming this problem (muhartono and subeki 2015). it may be overcome by utilizing plants as herbal medicines as some plants produce bioactive compounds https://doi.org/10.14421/biomedich.2023.121.407-412 408 biology, medicine, & natural product chemistry 12 (1), 2023: 407-412 potentially used as anticancer. guava (psidium guajava l.) and turmeric (curcuma longa l.) are two common plants producing secondary metabolites such as tannins, flavonoids, alkaloids, and saponins (correa et al. 2016). turmeric (curcuma longa l.) rhizome contains curcumin which also has anticancer properties which can inhibit carcinogenesis (meiyanto 1999; nurrochmad 2004). thus, the combination of guava (psidium guajava l.) and turmeric (curcuma longa l..) can be used as an alternative to prevent breast cancer without or with fewer side effects. methods this review paper was done with a literature study. a literature study was carried out to support ideas that are based on a strong theoretical basis on several sources consisting of several leading journals. the secondary data were obtained to support the idea (asiandu and malayudha, 2022) and used in analyzing the usefulness of the idea to conclude the final conclusion through some stages (sari et al. 2020). furthermore, the benefits to be achieved from writing this scientific work are to provide information about the dangers of breast cancer and to socialize these two materials as the formation of pharmaceutical drugs to reduce the number of breast cancer patients safely. results and discussion cancer cell cancer cells are normal cells that have undergone a genetic mutation causing their growth to be uncontrolled and uncoordinated with other body cells. the process of forming cancer cells is known as carcinogenesis, a somatic event, and is caused by an accumulation of genetic and epigenetic changes leading to changes in the normal regulation of molecular control of cell growth (figure 1). these genetic changes can be in the form of activation of proto-oncogenes and/or inactivation of tumor suppressor genes which can trigger tumor formation (kondo 1993; nurhayati and lusiyanti 2006). figure 1. cancer cell invasion (taken from gupta and massague, 2006). generally, there are two types of cancer cells, benign, and malignant cancer. benign cancer cells have a low growth rate and do not spread to other parts. meanwhile, malignant cells have a rapid growth rate, invade and damage surrounding tissue, and spread to other organs. invasion of cancer cells allows these cells to move into the blood vessels to be transported to other organs emerging new cancer in other organs (lumongga 2008). the general characteristic of cancer cells is the ability to grow rapidly and reduce the body's normal control mechanisms. abnormal genes in cancer are called oncogenes (guyton and hall 1997; putri et al. 2012). cancer cells can not maintain when to stop cell division. they divide uncontrollably which will compete with normal cells using oxygen and nutrients from the body. these cells will replace normal cells and cause pain that ends in death (nurhayati and lusiyanti 2006). breast cancer breast cancer is one of the most common types of cancer in women, although this cancer can also occur in men. the risk of breast cancer cases increases at the age of 40 years, which attacks more on the left breast and the upper part of the breast, close to the arm (wijayakusuma 2008; rahmatari 2014). according to the indonesian ministry of health in 2013, breast cancer contributed 30% and was the most dominant type of cancer in indonesia (dewi and hendrati 2015). breast cancer is a carcinoma originating from the ducts or lobules of the breast which is an international women's health issue. breast cancer is the most common problem in developed countries and is the number two problem in developing countries after cervical cancer. overall, breast cancer is the second leading cause of death after lung cancer (suyatno 2010; sumiatin, 2013). a common symptom of breast cancer is a lump in the breast that can be felt and will get harder, irregular, and cause pain. other symptoms are changes in size and shape, wrinkling of the breast skin that resembles an orange peel, presence of pus, blood, watery fluids, and discharge of milk in a woman who is not pregnant or breastfeeding. breast cancer is also characterized by swelling in one breast, itchy and painful nipples, bone pain, swelling of the arms, and weight loss (suryaningsih and sukaca 2009; dewi and hendrati 2015). breast cancer can be caused by several factors. these factors are combined into hormonal factors which include age at menarche, age at first pregnancy, parity, history of breastfeeding, infertility, and long-term use of hormonal contraception. early menarche or first menstruation at the age of below 12 years will increase the likelihood of breast cancer. the risk of breast cancer increases with the increasing age of women (priyatin et al. 2013). breastfeeding duration affects the risk of breast cancer. women who breastfeed in the 4-6 month range have a greater risk of breast cancer than 7-24 months. it asiandu et al. – anticancer of guava and turmeric 409 shows that the longer the breastfeeding time, the lower the risk of breast cancer. meanwhile, the consumption of fatty foods also affects the high risk of breast cancer in women. in addition, passive smoking, alcohol consumption, and low physical activity also affect the occurrence of breast cancer (yulianti et al. 2016). side effects of breast cancer treatment there are several methods used to treat breast cancer. methods of treating breast cancer include radiation, surgery, and chemotherapy. however, these methods have side effects, such as the spread of cancer cells to other organs and the mutation of cancer cells (muhartono and subeki 2015). side effects caused by chemotherapy can be in the form of physical and physiological disorders. effects that may appear include nausea, vomiting, alopecia, and so on. patients who feel the side effects of chemotherapy treatment, try to overcome these effects by resting or even using anti-emetic or analgesic drugs (wahyuni 2015). severe side effects also often occur in postchemotherapy patients, and often these side effects cannot be tolerated by patients which can even cause death. in addition, the side effect of post-chemotherapy patients may feel is anxiety (setiawan 2015). meanwhile, radiation therapy for cancer patients also causes side effects, especially in children. those effects can appear faster or slower and can occur locally or systemically, from small to large depending on age, location of irradiation, area of irradiation, vital organs around the tumor, and also the dose (yunus 2008). therefore, the use of natural herbal ingredients is very important in preventing the growth and spread of breast cancer cells. these herbal ingredients can be derived from guava leaves (psidium guajava l.) which contain secondary metabolites such as tannins, flavonoids, alkaloids, and saponins that have anticancer properties inhibiting the growth and spread of cancer cells (correa et al. 2016). meanwhile, turmeric rhizome (curcuma longa l.) contains curcumin which has anticancer properties which can inhibit the process of carcinogenesis at the stage of initiation and promotion of cancer cell progression. guava leaves (psidium guajava l.) as anticancer guava or psidium guajava linn is a tropical plant rich in vitamins a, and c, and secondary metabolites which are beneficial for health. guava leaves contain chemical compounds such as flavonoids, quercetin, tannins, saponins, essential oils, and alkaloids used as medicines for various diseases such as coughs and diarrhea. flavonoids are secondary metabolites of the phenol groups, efficacious as antioxidants, anti-inflammatories, and anticancer (aziz and djamil 2013). water extract from guava leaves had been reported to be able to inhibit the spread of prostate gland cancer cells. the compounds found in the extract were polyphenolic and flavonoid (chen et al. 2007; lee and park 2010). meanwhile, secondary metabolites such as tannins, alkaloids, and saponins contained in guava leaves also inhibited the growth and spread of cancer cells by inhibiting the proliferation of cancer cells (correa et al. 2016). table 1. phytochemical properties of guava leaves (psidium guajava l.). phytochemicals result flavonoids tannins alkaloids terpenoids + + + + source: kariawasam et al. 2017. guava leaf extract was reported to have considerable anticancer activity. based on dwitiyanti (2015), 70% ethanol extract from guava leaves had cytotoxic properties against t47d breast cancer cells with a concentration of 130.62 µg ml-1 capable of killing breast cancer cells by 88.52% which were incubated for 24h. meanwhile, the concentration of 5 µg ml-1 was able to kill 12.49% of t47d breast cancer cells. identification of the chemical groups contained in the 70% ethanol extract of guava leaves indicated the presence of alkaloids, flavonoids, saponins, tannins, and triterpenoids. also, kaileh et al (2007), cited in fathilah et al. (2010), stated that guava extract was effective in inhibiting the growth of mcf7 breast cancer cells within 24h with an ic50 value of 55 µg ml−1 ml-1 capable of killing cancer cells by 50%. moreover, the extract also had immunomodulatory activity in the form of nfkb (transcription factor protein) regulated the expression of genes involved in apoptosis. the mechanism of flavonoids in inhibiting and even killing cancer cells can be done in several ways. the first way is to act as an antioxidant which will inactivate free radicals. then, flavonoids will also bind to electrophilic compounds, induce the work of protective enzymes conjugate activity, increase the rate of apoptosis, inhibit cell proliferation, and inhibit lipid peroxidation. furthermore, flavonoids will also inhibit angiogenesis and inhibit dna oxidation (rahayu and roosmarinto 2017). additionally, alkaloids have properties as antineoplastic agents that can inhibit and even kill cancer cells by inhibiting dna synthesis and inhibiting mitosis at the metaphase and anaphase stages of cancer cells (purwaningsih et al. 2015). steroids function as topoisomerase ii inhibitors which can prevent and inhibit the cancer cell cycle (afandi 2006; sahid et al. 2013). saponin compounds also play a role in inhibiting cancer cells because they are antiproliferative, antimetastatic, and also antiangiogenesis. saponins induce apoptosis and cell differentiation (xu et al. 2016). angiogenesis by tumor cells is shown in figure 2. 410 biology, medicine, & natural product chemistry 12 (1), 2023: 407-412 figure 2. angiogenesis by tumor cells (taken from hejmadi, 2010). turmeric rhizome (curcuma longa linn.) as anticancer turmeric (curcuma longa l.) is a plant growing in tropical and subtropical regions. the main ingredient in turmeric is curcumin. it is a substance with antioxidant, anti-inflammatory, anti-proliferative, and cytotoxic effects that can induce apoptosis in malignant blood cells, breast, colon, liver, and ovaries (kurniawan et al. 2016). curcumin is a yellow polyphenol-derived pigment found in the rhizome of the turmeric plant (figure 3). the curcumin found in turmeric can act as an antioxidant, antiviral, antifungal, and anti-inflammatory by inhibiting several important molecules triggering inflammation. curcumin reduces the production of histamine (a substance inducing inflammation), increases the production of cortisol which has an antiinflammatory effect and removes toxins from the body (hutomo et al. 2016). figure 3. structure of curcumin (nurrochmad, 2004). turmeric rhizome extract suppressed the growth of mcf-7 and mda-mb-231 breast cancer cells. curcumin contained in turmeric rhizome extract triggered apoptosis. the rate of apoptosis of mcf-7 and mdamb-231 breast cancer cells correlated with the curcumin concentration. it induced the bax protein used in cell apoptosis and inhibited bcl-2 protein expression as an antiapoptotic protein (lv et al. 2014). table 2. phytochemicals properties of turmeric water-extract (curcuma longa l.). phytochemicals result alkaloid glycosides flavonoid tannin sterol terpenoid + + + source: deb et al., 2013 turmeric n-hexane extract inhibited the growth of qu-db and t47d breast cancer cells. data analysis on the anticancer activity of turmeric rhizome extract showed that the ic50 value for qu-db cells was 74 µg ml-1 and for t47d cells the ic50 value was 57 µg ml-1 in the 48-hour mtt assay. the concentration of 68 µg ml-1 of turmeric extract was able to reduce the rta (relative telomerase activity) rate in qu-db cells by 76.4% (ranjbari et al. 2014). the curcumin induced cell cycle arrest and apoptosis through the cell-signaling pathway (kurniawan et al. 2016). curcumin damages cancer cells by referring to the process of apoptosis. in the event of apoptosis, cells will experience dehydration. loss of intracellular fluid causes the cytoplasm to condense and change shape with smaller and flattened cell sizes. cells undergoing apoptosis can be characterized by the presence of pyknosis due to chromatin condensation (hutomo et al. 2016). further mechanisms of curcumin's anticancer effects may include suppression of nf-kb activity through inhibition of ikkb activity resulting in suppression of genes inducing tumorigenesis which include tnf, cox2, cyclin d1, c-myc, mmp-9, and interleukins. curcumin also controls the cell cycle and stimulates apoptosis by regulating p16 and p53. curcumin also acts as an autophagy modulator which is a tumor angiogenesis and metastatic inhibitor through the suppression of various growth factors including vegf, cox-2, mmps, and icams (wilken 2011; kurniawan et al. 2016). apart from curcumin, turmeric (c.longa l.) rhizome also contains several compounds that play a role in inhibiting or even killing cancer cells such as alkaloids, flavonoids, and tannins with certain mechanisms. they enhance the anticancer potential of turmeric. conclusions guava leaves (psidium guajava linn.) contain several compounds that act as anticancer consisting of alkaloids, flavonoids, saponins, and steroids. turmeric (curcuma longa linn.) rhizome contains curcumin and several other compounds such as alkaloids, flavonoids, and tannins which have anticancer properties. the asiandu et al. – anticancer of guava and turmeric 411 mechanisms of these compounds as anticancer include inhibiting the proliferation of cancer cells and inducing apoptosis of breast cancer cells. further research is needed to determine the perfect combination of both ingredients as herbal medicine in preventing breast cancer. acknowledgments: the authors thank all the authors cited in this article for their valuable works. authors’ contributions: all the authors involved in this review contributed to developing ideas, collecting secondary data, writing the manuscript, editing language and formatting. competing interests: the authors declare that there are no competing interests. funding: the authors received no financial funding from any institution regarding the data collection, manuscript writing, authorship, and this published article. references asiandu, a. p., & malayudha, a. g. 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(2016). risk factors for breast cancer (case study at ken saran hospital, semarang) (faktor-faktor risiko kanker payudara (studi kasus pada rumah sakit ken saran semarang)), jurnal kesehatan masyarakat, 4(4), 401-409. yunus, b. (2008). side effects of radiation therapy in head and neck cancer patients on the salivary glands (efek samping terapi radiasi penderita kanker kepala dan leher pada kelenjar saliva), jurnal dentofasial, 7(1), 57-62. biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 9-14 | doi: 10.14421/biomedich.2016.51.9-14 local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini lecturer of study program mathematics education, stkip pgri tulungagung jl. mayor sujadi timur no. 7 tulungagung, 66221 tel/fax: (0355) 321426 author correspondency: anggreini_004@yahoo.com abstract mathematical model has many benefits in life, especially the development of science and application to other fields. the mathematical model seeks to represent real-life problems formulated mathematically to get the right solution. this research is the application of mathematical models in the field of biology that examines the interaction of the two populations that host populations and parasitoid populations. this study differs from previous studies that examine the interaction of two more species that prey and predators where predators kill prey quickly. in this study the parasitoid population slowly killing the host population by living aboard and take food from the host population it occupies. in this study of differential equations are used to construct a mathematical model was particularly focused on the stability of the local mathematical model of interaction of two differential equations that host and parasitoid populations. stability discussed in this study are stable equilibrium points are obtained from the characteristic equation systems of differential equations host and parasitoid interactions. type the stability of the equilibrium point is determined on the eigenvalues of the jacobian matrix. analysis of stability is obtained by determining the eigenvalues of the jacobian matrix around equilibrium points. having obtained the stable equilibrium points are then given in the form of charts and portraits simulation phase to determine the behavior of the system in the future. keywords: mathematical model, the interaction of two populations model, stability introduction mathematics has many benefits in the areas of science and everyday life, especially in the field of biology. according widowati and sutimin (2007:1). the mathematical model is a mathematical field that seeks to represent and describe physical systems or a problem in the real world in a mathematical statement, in order to obtain an understanding of these real world problems to be more precise. representation of this process is known as a mathematical model. construction analysis and use of mathematical models is seen as one of the most important applications of mathematics. have been many studies that discuss the interaction of two species or two populations such as predator-prey or predator – prey. unlike the predator prey models that describe the interaction where a predator as prey and prey as prey, predators will kill its prey directly in the process of natural selection. while in this study established the relationship between host and parasitoid where the host does not kill quickly, but slowly with the way of life of a ride or take in nutrients from the host they occupy. this research is exciting to do because there are still few studies that discuss mathematical models of host and parasitoid interactions in particular discuss the stability of the system. in this study, given how differential equations can be used to construct a mathematical model was particularly focused on the stability of differential equations mathematical model of the interaction of the two populations that host and parasitoid. stability discussed in this study are stable equilibrium points are obtained from the characteristic equation systems of differential equations host and parasitoid interactions. having obtained the stable equilibrium points are then given a simulation to determine the type of stable equilibrium points using matlab program. type the stability of the equilibrium point is determined on the eigenvalues of the jacobian matrix. some types of stability based on the roots of the characteristic equation is as follows. if the value of the roots of the characteristic equation real, distinct, and equally a sign of the system asymptotically stable equilibrium point. if the value of the roots of the characteristic equation complex conjugate purely imaginary but not the asymptotically stable equilibrium point. if the value of the roots of the characteristic equation real, distinct, and not as a sign of the equilibrium point of the system is unstable. if the value of the roots of the characteristic equation purely imaginary ekuilibrim point system is stable but not asymptotically stable. based on the interaction of host and parasitoid, a mathematical model in the form of differential equations and systems of differential equations is a suitable way to describe the interaction between host and parasitoid. parasitoid is an organism that spends most of his life history to rely on a single host organism which eventually kills (and often took food) in the process. parasitoids laid eggs in the body of the target animal after hatching larvae then sucking the bodily fluids of the target animal to death when the host-parasitoid find a place to live, it must be appropriate host physiology 10 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 and nutrition for the successful development of the parasitoid. the goal in conducting this study was to examine how the stability of differential equations of mathematical models the interaction of two populations (host and parasitoids) and examines how the stability of mathematical models simulating the interaction of the two populations of differential equations (host and parasitoid) with matlab program. this research is the development of a mathematical model, especially the model of differential equations formed from the interaction between host and parasitoid. applications using matlab help to find graph and phase portraits of systems of differential equations stability so they can know the behavior of the system in the future. as study materials for researchers who like or are interested in the development of mathematical models especially differential equations. the interaction of two populations models (predatorprey) the basic model predator-prey or predator prey interaction of the two populations is given by the equation lotka-volterra (1926) *dh mh a hp dt   *dp qp ra hp dt   in this equation ℎ is the prey populations and is a predator population, with t is time. the equation states the prey population changes over time and the equation stated predator population changes over time. so the equation: * b sn a ht (1) with captured prey populations of prey each unit time.   * 1 a h f h bh   (2) with b states the time to deal with a host, f(h) stating the number of hosts attacked parasitoid per unit time or the number of hosts diparasit, a stated figures hosted by parasitoid attack . equation (2) is holling type ii functional response to the host parasitoid interactions. dynamic systems in general, the dynamic system is defined as a real problem that is modeled mathematically using differential equations in the equation which contains parameters that are interconnected , as well as changes in the parameters of the equation will lead to changes in the stability of the equilibrium point . the equilibrium point is one of the key concepts in a dynamic system. more general system can be expressed in the following form.̇1 = 1( 1, 2,…, , )2̇ = 2( 1, 2,…, , )⋮̇ = ( 1, 2,…, , ) (3) with =( , ,…, , ), =1,2,…, n is a general function of , =1,2,…, and time is t. the system can be further simplified into a system that is not dependent on the function of time (autonomous system) as followṡ1 = 1( 1, 2,…, )2̇ = 2( 1, 2,…, )⋮̇ = ( 1, 2,…, ) (3) with =( , ,…, , ), =1,2,…, n is a function that does not depend explicitly on the time t . the definition of equilibrium point (perko, 1991) point =( , ,…, ) ∈ called the equilibrium point of (3) if ( ̅)̇ = 0, =1,2,…, stability of equilibrium point definition of local stability the equilibrium point ∈ on the system (3) said: 1) local stable if for every å>0 there ä>0 such that for every solution ( ) that satisfies ‖ ( )− ̅‖<ä apply ‖ ( )− ̅‖< å for each ≥ . 2) local asymptotically stable if the equilibrium point∈ stable and there >0 such that for every solution ( ) that satisfies x ‖ ( )− ̅‖< applylim→ ( )= ̅. 3) do not be stable if the equilibrium point ∈ does not meet 1. below is given a definition of linear and nonlinear systems granted system(3), with ⊆ and : → continuous function on e. system (3) is said to be linear if , ,…, each linear with respect , ,…, . so the system (3) can be written in the form ̇1 = 11 1+ 12 2+⋯+ 1 ,̇2 = 21 1+ 22 2+⋯+ 2 ,̇ = 1 1+ 2 2+⋯+ , (4) dewi aggreini. – local stability analysis of a mathematical model of the interaction of … 11 with ̇ = , continuous on ≤ ≤ , , ,=1,2,…, . then the system (3) can be expressed in the form ̇ =̇ , with ∈ , ̇ = ( ̇ , ̇ ,…, ̇ ) and a matrix size nxn . granted system (3) with ⊆ and : → continuous function on e. system (3) is said to be nonlinear if there is i such that nonlinear. the nature of the solution around the equilibrium point of nonlinear systems (3) can be determined through linearization around the equilibrium point of the system. definition 2.1 jacobian matrix if the function belongs =( , ,…, ) on the system(3) with ∈ ( ), =1,2,3,…, . matriks ̅ ̅ … ̅̅ ̅ … ̅⋮ ⋮ ⋱ ⋮̅ ̅ … ̅ named jacobian matrix of f in point ̅. (kocak, 1991). by using jacobian matrix ( ̅), point stability properties ekuilibrim ̅ it can be seen as long as the point is hyperbolic eigenvalues and eigenvectors formally the definition of eigenvalues and eigenvectors is as follows. definitions (anton and rorres, 2014) let a nxn matrix and ∈ , ≠0. vector x is called an eigenvector / characteristic vector of a if=ë for a ë∈ . numbers ë that satisfies the above equation is called the eigenvalues / value characteristics. vector x is called an eigenvector corresponding to ë. stability analysis of fixed point (boyce & diprima, 2001) consider the linear system = with a, b , c and d constants (5). linear systems (5) commonly called "data model species" in population dynamics. if the matrix a = and such λ are the eigenvalues of matrix a, then: a =a ↔ −ë −ë = 00 phase portrait and linear systems (boyce & diprima, 2001) belongs to the following linear systeṁ = (6) suppose det (a) ≠ 0, so x are the eigenvalues of matrix a , ie ë is the root of the characteristic equationë − =0 (7) images phase and the system (6) is almost entirely dependent on its eigenvaluesfigure. 2 shows the absorbance graphic of plot toward thin film wavelength of tio2 that has been soaked in dye with variation of temperature extract. 1. if the eigenvalues different real negative is called a node, all trajectories toward zero, which means no points of zero is stable. 2. if the eigenvalues of positive reals different then called nodal source, all trajectories out of the points become unstable 3. if the eigenvalues of different real opposite in sign, with this so-called saddle point, all trajectories will be away to infinity along the eigenvectors, this resulted critical point will always be unstable. 4. if the eigenvalues of different real opposite in sign, with this so-called saddle point, all trajectories will be away to infinity along the eigenvectors, this resulted critical point will always be unstable. 5. if the eigenvalues equal, with two linearly independent eigenvectors, you will get what is called a star point or propernode, when λ < 0 then the point of criticism would be stable and unstable for λ > 0. 6. if the eigenvalues equal to the eigenvectors will be obtained by so-called improper node, when λ < 0 then the point of criticism would be stable and directions trajectory will go to zero. whereas for λ > 0 directions trajectory going out leaving the zero point and the point of criticism would be unstable. if the eigenvalues are complex numbers ë± = ± with <0, it will produce a stable behavior called spiral, 7. if the eigenvalues are complex numbers ë± = ± dengan >0, it will generate a behavior called unstable spiral, all trajectories going out leaving the zero point and the point of criticism would be stable. 12 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 method this research is a study of library studies, which is used in literature studies of relevant literature sources used to gather the necessary information in the study. a literature study by collecting literature sources which may include books, texts, papers and so on. after the collected literature sources ollowed by a review of the literature sources. at the end of the library resources as a basis for analyzing the problem. mathematical modeling the interaction of two populations of host and parasitoid in this research is needed to determine how the shape of the equation, and then to look for form completion hosted model and the parasitoid. steps to resolve the problem that is used to analyze the shape and model of the study are as follows: a. lowering the mathematical model the interaction of two populations of host and parasitoid with holling response function of type ii. b. analysis of the models begins with finding the equilibrium point of the model systems of differential equations. c. determining the characteristic equation and the eigenvalues of the jacobian matrix system which is calculated at each point of equilibrium. d. then check the stability of the equilibrium point. e. creating a numerical simulation of the results of the analysis of mathematical models the interaction of two populations of host and parasitoid with holling response function of type ii. research result in the discussion this time will be discussed on a mathematical model of interaction between host and parasitoid host. in this model there are two species that interact namely host and parasitoid. the population of the host is assumed to have enough food. prior to their predators, the intrinsic growth rate (birth rate minus death rate) host proportional to its population. in this case the host becomes the main food for parasitoid so before their host, the parasitoid population growth will decline due to the mortality rate of the parasitoid. parasitoid attack based on the functional response of the host. in contrast with these interactions, the parasitoid population will be converted to its growth. the ratio of parasitoid immature to adult parasitoids is constant and the population has the maximum growth rate. the parameters given to model the interaction between the host and parasitoid is ℎ( ) which states the host population at time t, ( ) states parasitoid population at the time, (ℎ) stated functional response, stating the effect of oviposition on adult parasitoid population, r states the intrinsic growth rate of the host ,d declared the death rate of the parasitoid . all of these parameters is positive. functional response (ℎ) assumed to be a function : → , , (ℎ) function with a continuous rise (0)=0, and (ℎ) diferensiabel kontinu. based on the assumptions above were obtained host and parasitoid interaction model as follows: phfrh dt dh )( ( ( ) ) . dp af h d p dt   (8) with (ℎ)= ∗ a holling type ii functional response to the interaction of host and parasitoid. here is a system of differential equations after the interaction of the two populations substituted into the equation (8), namely: ℎ= ℎ− ∗ℎ1+ ℎ= ∗ℎ1+ ℎ− (9) from the point equilibrium model in this section we will look for a solution to the 2r , because the population must not be negative and the solutions are beyond 2r cannot be interpreted biologically. equilibrium point equation (9) is obtained if: 0 dt dh (10) 0. dp dt  (11) from equation (10) and (11) was obtained 0 1 *         bh pa rh (12) * 0. 1 a h a d p bh       (13) from equation (12) and (13) was obtained 3 obtained equilibrium point           dbaa ar ph *11 ,0,           0,, *22 dbaa d ph           dbaa ar dbaa d ph **33 ,, . dewi aggreini. – local stability analysis of a mathematical model of the interaction of … 13 on the condition that the equilibrium point ∗ −>0 or ∗ > based on these results it can be concluded that: if∗ > then between host and parasitoid populations can coexis. ∗ > means that the effects of oviposition on adult parasitoid population multiplied by the time available for the parasitoids to chase and finds a host is greater than the mortality rate of parasitoid multiplied by time to handle one host . if ∗ > then between host and parasitoid populations cannot coexist. stability of equilibrium point one way to determine the stability of equilibrium points of the system (9) is to use linearization. based systems (9), for example ℎ= ( , ) and = ( , ) with,   p bh ha rhyxg         1 , * 1 and  yxg ,2 pdbh ha a        1 * . obtainable       * * 1 1 2 * * 2 2 2 11 , . 11 a p a hg g r bhbhh p j h p g g a ap a ah d h p bhbh                           local stability analysis in the following theorems will discuss the local stability of equilibrium points:(ℎ , )= ∗ − , ∗ −(ℎ , )= 0, ∗ and (ℎ , )= ∗ ,0 theorem if ∗ > , ∗ > ( + ), ∗∗ > − ,∗∗ > and ∗∗ + − < ∗∗ − then the equilibrium point (ℎ , )= 0, ∗ local asymptotically stable. theorem if ∗ > , ∗ > ( + ) and( ∗ )∗ − < ( ∗ )∗ , and ( ∗ )∗ > then the equilibrium point           dbaa ar dbaa d ph **33 ,, local asymptotically stable. theorem if 0 d d r a         and 2 4 d rd d r rd a a                then the equilibrium point  2 2 *, , 0 d h p a a db      unstable. conclusion the following will be given some conclusions related to this research. 1. from the system of differential equations interaction of two species , namely: = ℎ− ∗ and = ∗ − obtained equilibrium points(ℎ , )= 0, ∗ , (ℎ , )= ∗ , ∗ and (ℎ , )= ∗ ,0 . 2. if ∗ > , ∗ > ( + ), ∗∗ > − ,∗∗ > and ∗∗ + − < 4 ∗∗ , , then the equilibrium point (ℎ , )= 0, ∗ local asymptotically stable. 3. if ∗∗ + − <4 ∗∗ , ,∗∗ > − , and ∗∗ > thenë , = ∗∗ ± ∗∗ ∗∗ have a negative real part . so based on the equilibrium point theorem (ℎ , )= 0, ∗ local asymptotically stable. 4. by theorem if: ∗ > , ∗ > ( + ),∗∗ > − , ∗∗ > and ∗∗ + −< 4 ∗∗ − , then at the beginning of time very small number of host population , while close to the parasitoid population ∗ . so for a long time, the parasitoid population will approach ∗ while the host population will become extinct. 14 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 5. if ∗ > , ∗ > ( + ), and ( ∗ )∗ −< ( ∗ )∗ , and ( ∗ )∗ > then the equilibrium point (ℎ , )= ∗ , ∗ local asymptotically stable. because (ℎ , )= ∗ , ∗ have negative real part, the equilibrium point (ℎ , )= ∗ , ∗ local asymptotically stable. by theorem if: ( ∗ )∗ − − ( ∗ )∗ , ∗> , ∗ > ( + ), and ( ∗ )∗ > , then at the beginning of time close to the host population ∗ while close to the parasitoid population∗ . so for a long time, will host population approaching ∗ and the parasitoid population will approach ∗ . if + + >0 and+ + >4 + then the equilibrium point (ℎ , )= ∗ ,0 unstable. references anton, h. and rorres, c., 2014, elementary linear algebra, john wiley & sons, inc., new york. boyce, w.e. & diprima r.c. 2001. elementary differential equations and boundary value problems. seventh edition, john wiley & sons: new york. edelstein, l., keshet, 2005, mathematical models in biology, siam, new york. haberman, r. 1977. mathematical models in mechanical vibrations, population dynamics, and traffic flow. prenticehall: new jersey. hanh, w., 1967, stability of motion, springer-verlag, new york. hale, j. k. & h. kocak. 1991. dynamics and bifurcations. new york: springer verlag. kuznetsov, y.a., 1998, elements of applied bifurcation theory, springer-verlag, new york. ni'mah, khoirun, 2015, analysis of model s-i-p interaction of two species predator-prey with response function holling type ii, semarang state university. perko, l., 2001. differential equations and dynamical systems, springer-verlag, new york. sulistyowati, e. et al., 2001, functional response parasitoid cephalonomia stephanoderis betr against coffee fruit powder, hypothenemus hampei ferr, pest plant science program, university of gadjah mada. tarumingkeng, r.c., population dynamics (quantitative ecological assessment), 1994, pustaka sinar harapan and activities of discourse christian university, jakarta. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 151-157 | doi: 10.14421/biomedich.2023.121.151-157 issn 2540-9328 (online) a new ent-kaurene diterpenoid isolated from leaves of espeletia semiglobulata cuatrec. and its potential antimicrobial activity andrés márquez1,*, alida pérez1, †luis rojas1, rosa aparicio1, freddy ramos2, ysbelia obregón1, alfredo usubillaga1 1research institute, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela. 2department of chemistry, faculty of science, national university of colombia, bogotá d.c. 111321, colombia. corresponding author* andresmqch@gmail.com manuscript received: 03 december, 2022. revision accepted: 16 january, 2023. published: 24 january, 2023. abstract the fraction of the neutral extraction of the leaves of espeletia semiglobulata cuatrec. was subjected to chromatographic separation, it yielded four ent-kaurene type diterpenoids: three known [ent-kaur-16-en-19-al (i), ent-kaur-18-nor-16-en-4-ol (iii), ent-kaur-16-en-19-ol (iv)] and a new one elucidated as ent-kaur-3-acetoxy-15-ene (ii), based on the physicochemical and spectroscopic data of ftir, gc-ms, and 1d and 2d nmr. these compounds were subjected to antimicrobial bioassay studies. this new ent-kaurene showed a significant inhibition potential against the growth of gram negative bacterial strains [escherichia coli (atcc 25922): 8 mm, klebsiella pneumoniae (atcc 23357): 10 mm, pseudomonas aeruginosa (atcc 27853): 8 mm], it also showed inhibition against the growth of fungal strain (candida krusei: 8 mm), at a 2 mg/ml concentration. the compounds (i), (iii) and (iv) failed to show any significant results in the antimicrobial screening against five bacterial strains [staphylococcus aureus (atcc 25923), enterococcus faecalis (atcc 29212), klebsiella pneumoniae (atcc 23357), escherichia coli (atcc 25922) y pseudomonas aeruginosa (atcc 27853)] and one fungal strain [candida krusei (atcc 6558)]. these results reveal a remarkable natural structure-activity relationship of the ent-kaurene core regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen bond acceptor or donor group, improves the antimicrobial activity. keywords: diterpene; ent-kaurene; asteraceae; natural products; antimicrobial activity. abbreviations: nmr – nuclear magnetic resonance; 1h nmr – proton nuclear magnetic resonance; 13c nmr – carbon nuclear magnetic resonance; 13c-apt – attached proton test; cosycorrelation spectroscopy; hsqc – heteronuclear single quantum correlation spectroscopy; hmbc – heteronuclear multiple bond correlation spectroscopy; ir – infrared spectroscopy; gc-ms – gas chromatography–mass spectrometry; mp – melting point; mh – müeller-hinton; dmso – dimethyl sulfoxide; etoac – ethyl acetate. introduction bioactive natural products have played an important role in treating and preventing human diseases. these are important sources for new drugs and also good lead compounds suitable for further modification during drug development (atanasov et al., 2021). the terpenoids are among the most important natural compounds known for their medicinal value (kamran et al., 2022; proshkina et al., 2020); within them, ent-kauranes represent an important group of tetracyclic diterpenes that possesses a wide spectrum of biological and pharmaceutical activities such as antimicrobial, antiparasitic, insect antifeedant, cytotoxic, antitumour, anti-hiv, steroidogenic, antifertility, hypotensive, antiinflamatory, among others (ghisalberti, 1997). this type of diterpene is extensively found in different species of espeletiinae (asteraceae), a family of resinous plants, popularly known as “frailejon”, that grow at an altitude of 2500 m in the andes of northern south america. these plants are used by the inhabitants of the high moors to treat asthma and rheumatic conditions (usubillaga et al., 2003; aparicio et al., 2013). ent-kaurenes have been identified in some studies about the espeletia semiglobulata cuatrec. species. it grows above an altitude of 3900 m at the “piedras blancas” moor, which is part of sierra la culata, located near the city of mérida, venezuela. ent-kaur-16-en-19-oic acid has been identified on the fraction of acid extraction of the leaves of this plant as the most abundant component and known for its diverse biological properties (sosa-sequera et al., 1996; cavalcanti et al., 2005; kim et al., 2016; zhang et al., 2017; cotoras et al., 2004; mongelli et al., 2002), and ent-kauran-19-oic acid, ent-kaur-9(11)-16-dien-19oic acid, ent-kaur-15α-isovaleroxy-16-en-19-oic acid, ent-kaur-15α-hidroxy-16-en-19-oic acid, ent-kauran16α-hidroxy-19-oic acid, and ent-kaur-15-en-19-oic acid have been identified as the less abundant components. on the other hand, ent-kaur-16-en-19-al and ent-kaurhttps://doi.org/10.14421/biomedich.2023.121.151-157 152 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 16-en-19-ol have been identified as majority components in the fraction of neutral extraction, and entkaur-16α-hidroxy-16-en-19-al, ent-kaur-18-nor-16-en-4ol, ent-kaur-16-en-19-ol acetate have been identified as minority components without any biological activity reported on any of them (usubillaga et al., 1988; aparicio et al., 2013). in this study, we are reporting the isolation, structural identification, and antimicrobial activity of a new ent-kaurene-type terpenoid natural product (ii) from neutral fraction of espeletia semiglobulata cuatrec. leaves, also, the evaluation of antimicrobial activity of some known ent-kaurenes coming from this neutral extraction is being reported for the first time. materials and methods general methods melting points were determined on a fisher-johns melting point apparatus. ir spectra were measured on a perkin elmer spectrum two, 10.03.06 version, as kbr disks. nmr spectra was recorded with a brukerultrashieldtm 400 mhz instrument for solutions in cdcl3. gc-ms were performed on a hewlett-packard msd 5973 instrument fitted with a 5 % phenylmethyl polysiloxane fused-silica column (hp-5ms, 30 m, 0.25 mm, film thickness 0.25 μm). the initial analysis temperature was 250 °c, which was increased at 5 °c/min. to a final temperature of 300 °c. analytical thin-layer chromatography was performed on e. merck aluminum-backed silica gel foils (f254). flash chromatography was performed on silica gel e. merck grade 60, 63-200 mesh, by gradient elution with hexane and hexane-etoac mixtures. plant collection leaves of espeletia semiglobulata cuatrec. were collected at 3900 m of altitude in “piedras blancas” moor and along the road to piñango, about 13 km from the mountain’s peak “pico del aguila”. voucher specimens (au30 and au21) were deposited at the faculty of pharmacy and bioanalysis herbarium, university of los andes (merf herbarium). extraction and neutral fraction obtainment espeletia semiglobulata cuatrec. leaves were dried at 40 °c during 48 hours and they were grinded. the grinded material (200 g) was extracted at room temperature with a mixture of hexane/diethyl ether (3:1, v/v) for a period of two days. the solvents were removed under reduced pressure and the solid residues were dissolved in hexane/etoac, and shaken with 5 % aqueous naoh. the aqueous phase was taken to ph 3 by careful addition of concentrated hcl and shaken with hexane to obtain the acid fraction. the original hexane-diethyl ether solution, left over after the alkaline treatment surplus, was mixed with active charcoal and boiled for 10 minutes. a neutral fraction of 25 g was obtained after an open column chromatography treatment. isolation of kaurene diterpenes most of the leaves neutral fraction was submitted to flash chromatography over silica gel. the column (a) was eluted with hexane and hexane/acoet mixtures and 100 ml fractions were collected, which were constantly motorized by tlc. fractions 1-35 eluted with hexane yielded 840 mg of a mixture that contained plant waxes and (i). fractions 36-39 eluted with hexane rendered 500 mg of pure (i) as a white solid, mp 113-116 ºc, identical to an authentic sample of ent-kaur-16-en-19-al (mp,1h-nmr, 13c-nmr) obtained from espeletia semiglobulata (usubillaga et al., 1988; aparicio et al., 2013). the elution continued with hexane (40-45 fractions), it yielded 150 mg of a mixture of (i) and (ii). the elution of the 46-61 fractions with hexane yielded 1000 mg of pure (ii), which was crystallized from hexane/et2o as white crystalline solid. this compound has a very similar polarity to (i), however, they are not the same (mp, 1h-nmr, 13c-nmr). the elution of the 82-94 fractions with hexane/etoac 5 % yielded 300 mg of pure (iii), mp 146-148 °c, identical to an authentic sample of ent-kaur-18-nor-16-en-4-ol (mp,1h-nmr, 13c-nmr) obtained from espeletia nana (peña et al., 2012). the elution of the 101-106 fractions with hexane/etoac 10 % yielded 200 mg of pure (iv), mp 142-144 °c, identical to an authentic sample of ent-kaur16-en-19-ol (mp,1h-nmr, 13c-nmr) obtained from espeletia semiglobulata cuatrec. (usubillaga et al., 1988; aparicio et al., 2013). finally, the elution using hexane/etoac 20 % yielded a complex mixture (21.4 g). antimicrobial assay  antibacterial activity the antibacterial activity was determined by the agar diffusion method with discs. the following microorganisms were tested: staphylococcus aureus (atcc 25923), enterococcus faecalis (atcc 29212), klebsiella pneumoniae (atcc 23357), escherichia coli (atcc 25922) y pseudomonas aeruginosa (atcc 27853). an 18 hours culture of each microorganism was used in 2.5 ml müeller-hinton (mh) broth at 37 °c. the bacterial inoculum was adjusted with saline physiological solution to the mc farland turbidity standard n° 0.5 (106-8 cfu/ml). each inoculum was spread with a swab on the surface of a plate containing müeller-hinton agar and then a disc of filter paper (6 mm diameter) previously impregnated with 10 μl of the compound dissolved in dmso was placed on the surface at a 2 mg/ml concentration for each one [(i), (ii), (iii), (iv)]. a disc impregnated with dmso was included as a negative control. in addition, the standard disc of the reference antibiotic was placed as a positive control for each of the microorganism (oxacillin 1 μg márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 153 for staphylococcus aureus; vancomycin 30 μg for enterococcus faecalis; tobramycin 10 μg for escherichia coli; aztreonam 30 μg for klebsiella pneumoniae; cefepime 30 μg for pseudomonas aeruginosa). after having placed the discs on the petri plates, they were refrigerated at 4 °c for 24 h (velasco et al., 2007). the reading of the inhibition halos was performed at 24 h and it was measured (mm) around the disc. all the antibacterial activity trials were done twice. the measurement of the inhibition halos of the antibacterial activity was done thus: c= a-b (c= size of the inhibition halo; a= size of the halo plus the disk of filter paper; b= size of the filter paper disk, 6 mm).  antifungal activity the antifungal activity was determined by the agar well diffusion method. the microorganism tested was candida krusei (atcc 6558), using fluconazole 100 μg as positive control, and dmso as negative control. the strain repacked 12 hours before, coming from a savory dextrose agar, it was used to prepare the inoculum which was compared using the mc farland turbidity standard n°0.5 (106-8 cfu/ml). each inoculum was spreaded with a swab on the surface of a plate containing müeller-hinton agar and then 6 mm diameter holes were opened in it, where 20 μl of the compounds dissolved in dmso were placed at a 2 mg/ml concentration for each one [(i), (ii), (iii), (iv)]. one well was included with the dmso as a negative control and another well with the reference antibiotic as positive control. then the petri plates were refrigerated at 4 °c for 24 h (velasco et al., 2007). the reading of the inhibition halos was performed at 24 h and was measured (mm) around the well. all the antifungal activity trials were done twice. results and discussion phytochemical analysis and structure elucidation the structures of the ent-kaurene diterpenoids isolated from neutral fraction of e. semiglobulata cuatrec., leaves are presented on figure 1. figure 1. molecular structure of ent-kaurenes from neutral fraction of aerial parts of espeletia semiglobulata cuatrec. compound (i) was isolated as a white powder, mp 113-116 ºc. gc-ms gave [m]+ molecular ion at m/z: 286.1 (calcd. for c20h30o; m/z: 286). ft-ir, vmax (cm 1), functional group: 2729, v(c-h, aldehyde); 1711, v(c=o); 1655, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 9.73 (h1-19, s, cho), 4.74 (h1-17a, s, =ch), 4.79 (h117b, s, =ch), 2.64 (h1-13, s, c-h), 2.05 (h2-15, m, ch2), 0.98 (h3-18, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 205.94 (c-19, cho), 103.24 (c-17, =ch2), 43.75 (c-13, ch), 49.05 (c-15, ch2), 24.29 (c-18, ch3). this data completely matches with the spectral data reported in the bibliographic references (usubillaga et al., 1988; aparicio et al., 2013), this leads us to the conclusion, the compound (i) is an ent-kaurene-type diterpene called ent-kaur-16-en-19-al, commonly known as kaurenal. this compound was first isolated and characterized as a natural product of espeletia grandiflora humb. et bompl. (piozzi et al., 1971). it is particularly abundant in species of espeletia genus (morales et al., 1973; bohlmann et al., 1980; usubillaga et al., 1988), and in fact, it has already been isolated and reported from e. semiglobulata cuatrec. leaves (usubillaga et al., 1988; aparicio et al., 2013). compound (ii) was obtained as a crystalline white powder. the analysis of its 13c nmr and gc-ms data (m/z 330 [m]+) deduced its molecular formula c22h34o2, which suggested 6 degrees of unsaturation. the ir spectrum showed the absorption bands of ester group [1735 cm-1 (o−c=o) and 1243 cm-1 (o−c)] and typical bands of alkene group [3040 cm-1 (=c−h) and 1661 cm1 (c=c)]. the 1h nmr (400 mhz, cdcl3) and 13c nmr (cdcl3, 100 mhz) data (table 1) demonstrated the signals of thirty-four protons and twenty-two carbons sorted by attached proton test (13c-apt) and heteronuclear single quantum coherence (hsqc) spectra, which can be classified as: a terminal ester group [δh/δc: 1.98 (h3-22, s, ch3) / 21.47 (c-22, ch3); 171.17 (c-21, >c=o)], a trisubstituted alkene group [δh/δc: 5.06 (h1-15, s, =ch) / 124.47 (c-15, =ch); 139.78 (c-16, >c<)], four methyl groups [δh/δc: 0.81 (h3-18, s, ch3) / 28.22 (c-18, ch3); 0.80 (h3-19, s, ch3) / 22.70 (c-19, ch3); 0.73 (h3-20, s, ch3) / 22.70 (c-20, ch3)] including an olefinic methyl [δh/δc: 1.80 (h3-17, s, ch3) / 23.37 (c-17, ch3)], a methinic proton bonded to a carbon bearing heteroatom (deshielded proton) [δh/δc: 4.50 (h1-3, t, o−ch) / 80.84 (c-3, o−ch)] seven sp3 methylene [δh/δc: 1.55 (h2-1, m, ch2) / 33.01 (c-1, ch2); 1.57 (h2-2, t, ch2) / 23.52 (c2, ch2); 1.49 (h2-6, m, ch2) / 21.07 (c-6, ch2); 1.30 (h2-7, m, ch2) / 36.69 (c-7, ch2); 1.48 (h2-11, m, ch2) / 18.39 (c-11, ch2); 1.54 (h2-12, m, ch2) / 26.75 (c-12, ch2); 1.35 (h2-14, t, ch2) / 41.49 (c-14, ch2)], three sp3 methines [δh/δc: 1.51 (h1-5, m, ch) / 52.51 (c-5, ch); 0.79 (h1-9, m, ch) / 55.41 (c-9, ch); 1.86 (h1-13, m, ch) / 47.79 (c-13, ch)], and three sp3 quaternary carbon [δc: 37.86 (c-4); 40.17 (c-8); 39.68 (c-10)]. detailed analysis of these nmr data suggested the presence of a typical ent-kaurene-type diterpenic 154 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 skeleton [perhydrophenanthrene unit (a, b and c rings) fused to a cyclopentane unit (d ring) formed by a twocarbon bridge between c-8 and c-13 (figure 1)] (batista et al., 2005). this partial structure was further confirmed by correlations spectroscopy (cosy) and heteronuclear multiple bond correlation (hmbc), which has been shown in figure 2. by analysis of the cosy correlations, it was possible to identify six structural areas (a f) of this compound. the fact that the terminal ester group [c-21 (δc 171.17, o-c=o)/c-22 (δc 21.47, ch3); h3-22(δh 1.98, s, ch3)]; where the terminal arrangement was confirmed by the hmbc correlation between the methylic protons h3-22 and carbonyl group c-21 (δc 171.17, c=o)] was on c-3 (δc 142.7) was confirmed by the hmbc correlation from methinic proton h1-3 (δh 4.50, t, ch) to ester carbonyl group c21 (δc 171.17, >c=o), as well as, the correlation of h1-3 with methylene c-2 (δc 23.52, ch2) and quaternary carbon c-4 (δc 37.86, >c<). the presence of two methyls on c-4 was confirmed by its hmbc correlation with h3-18 (δh 0.81, s, ch3) and h3-19 (δh 0.80, s, ch3), in addition to the hmbc correlation of these methylic prontons with methine c-3 (δc 80.84, o-ch), as well as, by the correlations: h3-19 (δh 0.80, s, ch3) / c-5 (δc 52.51, ch) and h1-5 (δh 1.51, m, ch) / c-4 (δc 37.86, >c<). the mutual correlations of both methyls, both by hmbc [h3-18 (δh 0.81, s, ch3) / c-19 (δc 22.70, ch3); h3-19 (δh 0.80, s, ch3) / c-18 (δc 28.22, ch3)] and by cosy [h3-18 (δh 0.81, s, ch3) / h3-19 (δh 0.80, s, ch3)] also confirmed the vicinal arrangement of them on c-4. the existence of one methyl on c-10 was verified by the hmbc correlation from methylic protons h3-20 (δh 0.73, s, ch3) to quaternary carbon c-10 (δc 39.68, >c<) and methylene c-1 (δc 33.01, ch2). on the other hand, the presence of an endocyclic c-c double bond between c-15 and c-16, and the presence of an olefinic methyl on c-16 (d ring) were confirmed by hmbc correlations between methylic prontons h3-17 (δh 1.80, s, ch3) with olefinic methine c-15 (δc 124.47, =ch) and methine c-13 (δc 47.79, ch), and at the same time, the methinic proton h1-13 (δh 1.86, m, ch) correlates with quaternary carbon c-16 (δc 139.78, =c<) and methylene c-12 (δc 26.75, ch2). this partial structure has also been confirmed by cosy correlation from olefinic methyl protons h3-17 (δh 1.80, s, ch3) to olefinic methine proton h1-15 (δh 5.06, s, =ch), and vice versa, as well as, the mutual correlation between olefinic methyl protons h3-17 with methinic proton h1-13 (δh 1.86, m, ch), and this last, mutual correlated with methylenic ptotons h2-14 (δh 1.35, t, ch2) and h2-12 (δh 1.54, m, ch2). finally, the central ring of ent-kaurene core (b ring) was confirmed by the hmbc correlations between methinic proton h1-9 (δh 0.79, m, ch) with quaternary carbon c-10 (δc 39.68, >c<), c-8 (δc 40.17, >c<) and methylene c-11(δc 18.39, ch2), as well as, this ring was confirmed by cosy correlations between methinic proton h1-9 with h1-5 (δh 1.51, m, ch), h2-7 (δh 1.30, m, ch2) and h2-11 (δh 1.48, m, ch2). based on the spectroscopic analysis performed, the isolation of an ent-isokaurene-type diterpene is confirmed, especially ent-kaur-3-acetoxy-15-ene, which have also be called 3acetoxy-isokaurene, this being a natural product reported for the first time. compound (iii) was isolated as a white powder, mp 146-148 °c. gc-ms analysis shown [m]+ molecular ion at m/z: 274.3 (calcd. for c19h30o; m/z: 274). ft-ir, vmax (cm-1), functional group: 3500, v(o-h, alcohol); 1600, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 4.76 (h1-17a, s, =ch), 4.82 (h1-17b, s, =ch), 3.65 (h1-18, s, oh), 2.66 (h1-13, t, c-h), 2.16 (h2-15, m, ch2), 1.20 (h3-18, s, ch3), 1.11 (h3-20, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 79.31 (c-4, >coh), 44.20 (c-13, ch), 49.40 (c-15, ch2), 102.93 (c-17, =ch2), 21.52 (c-18, ch3), 17.74 (c-20, ch3). this data completely matches with the spectral data reported in the bibliographic references (bohlmann et al., 1980; peña et al., 2012), this leads us to the conclusion that the compound iii) is an ent-kaurene-type diterpene called ent-kaur-18-nor-16-en-4-ol, commonly known as ruilopeziol. this compound has only been isolated and reported as a natural product of ruilopezia linden, coespeletia lutescens, and espeletia nana cuatrec. (bohlmann et al., 1980; peña et al., 2012), therefore, its isolation and characterization from e. semiglobulata cuatrec. leaves is a new discovery. márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 155 table 1. 1h-nmr (cdcl3, 400 mhz), 13c-nmr (cdcl3, 100 mhz), cosy ( 1h↔1h) (cdcl3, 600 mhz) and hmbc [ 1h (600 mhz) → 13c (100 mhz), cdcl3] of compound (ii) (ent-kaur-3-acetoxy-15-ene). position δ 1h (ppm), mult. δ 13c (ppm), type cosy ( 1h↔1h) hmbc ( 1h→13c) 1 1.55, m 33.01, (>ch2) h2-1 ↔ h2-2 - 2 1.57, t 23.52,(>ch2) h2-2 ↔ h2-1 h2-2 → c-3 3 4.50, t 80.84, (o-ch-) h1-3 ↔ h2-2 h1-3 → c-2, c-4, c-21 4 -37.86, (>c<) -- 5 1.51, m 52.51, (>ch-) h1-5 ↔ h3-19 h1-5 → c-4, c-19, c-6 6 1.49, m 21.07, (>ch2) -h2-6 → c-5 7 1.30, m 36.69, (>ch2) -h2-7 → c-8 8 -40.17, (>c<) -- 9 0.79, m 55.41, (>ch-) h1-9 → h2-7, h1-5, h2-11 h1-9 → c-10, c-11, c-8 10 -39.68, (>c<) -- 11 1.48, m 18.39, (>ch2) -h2-11 → c-12 12 1.54, m 26.75, (>ch2) h2-12 ↔ h1-13 h2-12 → c-13, c-11 13 1.86, m 47.79, (>ch-) h1-13 ↔ h2-12, h3-17 h1-13 → c-17, c-16, c-12 14 1.35, t 41.49, (>ch2) h2-14 ↔ h1-13 - 15 5.06, s 124.47, (=ch) h1-15 ↔ h3-17 h1-15 → c-17 16 -139.78, (=c<) -- 17 1.80, s 23.37, (-ch3) h3-17 ↔ h1-15, h1-13 h3-17 → c-13, c-15, c-16 18 0.81, s 28.22, (-ch3) h3-18 ↔ h3-19 h3-18 → c-3, c-4, c-19 19 0.80, s 22.70, (-ch3) h3-19 ↔ h3-18, h1-5 h3-19 → c-3, c-4, c-5, c-18 20 0.73, s 17.66, (-ch3) -h3-20 → c-1, c-10 21 -171.17, (>c=o) -- 22 1.98, s 21.47, (-ch3) -h3-22 → c-21 figure 2. selective 1h↔1h cosy (▬) and 1h→13c hmbc (▬) correlations of (ii). compound (iv) was isolated as a white powder, mp 142-144 °c. gc-ms analysis shown [m]+ molecular ion at m/z: 288.3 (calcd. for c20h32o; m/z: 288). ft-ir, vmax (cm-1), functional group: 3406, v(o-h, alcohol); 1026, v(c-o); 1600, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 4.73 (h1-17a, s, =ch), 4.79 (h1-17b, s, =ch), 3.44 (h1-19a, dd, ch2), 3.74 (h1-19b, dd, ch2), 2.63 (h1-13, t, c-h), 2.06 (h2-15, m, ch2), 1.01 (h3-18, s, ch3), 0.96 (h3-20, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 65.60 (c-19, ch2oh), 155.90 (c-16, >c=), 103.00 (c-17, =ch2), 27.10 (c-18, ch3), 18.21 (c-20, ch3), 44.20 (c-13, ch), 49.10 (c-15, ch2). by comparing this experimental data with the spectroscopic data reported in the bibliography (bohlmann et al., 1980; piozzi et al., 1971), it is evident that this compound is an ent-kaurene-type diterpene called entkaur-16-en-19-ol, commonly known as kaurenol. currently, this diterpene is considered a widely distributed product in the asteraceae family, since it has been isolated in different species, including the species of the espeletia genus (seaman et al., 1990), like espeletia semiglobulata cuatrec. (usubillaga et al., 1988; aparicio et al., 2013). antimicrobial activity the isolated compounds, ent-kaur-16-en-19-al (i), entkaur-3-acetoxy-15-ene (ii), ent-kaur-18-nor-16-en-4-ol (iii) and ent-kaur-16-en-19-ol (iv) were evaluated for in vitro growth inhibitory potential against different bacterial [staphylococcus aureus (atcc 25923), 156 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 enterococcus faecalis (atcc 29212), escherichia coli (atcc 25922), klebsiella pneumoniae (atcc 23357), pseudomonas aeruginosa (atcc 27853)] and fungal [candida krusei (atcc 6558)] strains, according to the previously described procedure to agar diffusion method with discs (strain of bacteria), and agar well diffusion method, (strain of fungi). the results in table 2, shows that all the compounds are inactive againt the bacterial and fungal strains tested, except compound (ii), which showed growth inhibition against gram-negative bacterial strains (escherichia coli: 8 mm, klebsiella pneumoniae: 10 mm, pseudomonas aeruginosa: 8 mm), as well as it showed growth inhibition against the fungal strain (candida krusei: 8 mm), at a concentration of 2 mg/ml. these results reveal a remarkable natural structureactivity relationship of the ent-kaurene core with regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or structural modification apparently, improves the antimicrobial activity. similar results have been found synthetically, by preparing derivatives of hydroxylated ent-kaurenes at c-3, improving their biological activity (ohkoshi et al., 2004). moreover, the evaluation of the relationship between structure and antimicrobial activity of some diterpenes suggested that the presence of a substituted decalin skeleton (essential hydrophobic portion for crossing membranes), and a hydrophilic region bearing a donor or acceptor group able to interact with hydrogen-bond acceptor/donor groups on the membrane were necessary for their insertion in a phospholipid bilayer model. this feature enables the diterpenes to cross the bacterial cell membrane causing cell damage (urzúa et al., 2008). in this context, the ent-kaurenes tested have the structural characteristics described above: a substituted decalinic system that confers a lipophilic character, and a hydrophilic region bearing a hydrogen-bond donor or acceptor group. however, the compounds (i), (iii) and (iv), which possess these polar groups in the c-4 position, they are sterically limited to form hydrogenbond with acceptor/donor groups on the microorganism membrane, and therefore cannot cause damage to it. on the contrary, compound (ii) has this moderately hydrophilic group (ester) in c-3 position, whose steric hindrance is low and this confers the advantage to interact and establish hydrogen bonds with acceptor/donor groups on the microorganism membrane, being able to cross and cause damage to it. the position and steric hindrance of this hydrogen-bond donor or acceptor group on decalinic system are important characteristics for the biological activity of ent-kaurenes (urzúa et al., 2008). table 2. antimicrobial activity of natural compounds (i)-(iv) expressed as diameters of the inhibition zone (mm). microorganism compound positive control (i) (ii) (iii) (iv) ox va to az cf fl staphylococcus aureus (atcc 25923) ia ia ia ia *23 enterococcus faecalis (atcc 29212) ia ia ia ia *19 escherichia coli (atcc 25922) ia 8 ia ia *26 klebsiella pneumoniae (atcc 23357) ia 10 ia ia *34 pseudomonas aeruginosa (atcc 27853) ia 8 ia ia *32 candida krusei (atcc 6558) ia 8 ia ia *15 ia: inactive; ox: oxacillin®; va: vancomycin®; to: tobramycin®; az: aztreonam®; cf: cefepime®; fl: fluconazole®; *: inhibition halo in mm, for control groups (clsi, 2020). conclusions ent-kaurene-type diterpenes, ent-kaur-16-en-19-al (i), ent-kaur-3-acetoxy-15-ene (ii), ent-kaur-18-nor-16-en4-ol (iii), ent-kaur-16-en-19-ol (iv), were isolated from the neutral fraction of espeletia semiglobulata cuatrec. leaves, being ent-kaur-3-acetoxy-15-ene (ii) a natural product reported for the first time. compound (ii) was the only one that showed antimicrobial activity, but only against gram-negative bacterial strains (escherichia coli: 8 mm, klebsiella pneumoniae: 10 mm, pseudomonas aeruginosa: 8 mm) and fungal strain (candida krusei: 8 mm), at of 2 mg/ml concentration. these results reveal a remarkable natural structure-activity relationship of the ent-kaurene core with regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen-bond acceptor or donor group, improves the antimicrobial activity. márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 157 acknowledgements: all authors are grateful to the research institute “dr. alfredo nicolás usubillaga del hierro”, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela for the support. the research institute authors are grateful to the prof. freddy ramos from the department of chemistry, faculty of science, national university of colombia, bogotá d.c. 111321, colombia, for nmr and ms equipment. besides, all authors are grateful to the msc. ana deixy flores from the “instituto clínico santa filomena”, microbiology laboratory, mérida 5101, venezuela, for the antimicrobial assays. competing interests: the authors declare that there are no competing interests. funding this research was supported by research institute “dr. alfredo nicolás usubillaga del hierro”, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela. references aparicio, r., villasmil, t., peña, a., rojas, j., & usubillaga, a. 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2540-9328 (online) interleukin-1 as a predictor cytokine sars-cov: article review lisa savitri*, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 14 december, 2022. revision accepted: 07 february, 2023. published: 11 february, 2023. abstract severe acute respiratory syndrome coronavirus (sars-cov) is an etiologic agent of respiratory disease that has a mortality rate of 10%. il-1 actively participates in the inflammatory response to infection. sars-cov-2 appears to act on the activation and maturation of il1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf-. therefore, il-1β is part of the cytokine storm generated by coronavirus infection. elevated levels of the il-1 receptor antagonist (il-1ra) in severe cases of covid-19, and this marker have been associated with increased viral load, loss of lung function, lung damage, and risk of death. in addition, there is an increase in il-1α levels in patients with severe covid-19, and this is strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers coronavirus. keywords: cytokines; interleukin-1; severe acute respiratory syndrome coronavirus (sars-cov). abbreviations: severe acute respiratory syndrome coronavirus (sars-cov), il-1 receptor antagonist (il-1ra), interleukin-1 (il-1), sars-cov lacking the e gene (sars-cov-δe), open reading frame 3a (orf3a), tnf receptor-associated factor 3 (traf3), recruitment caspase domain (asc) interleukin-1 and infection the interleukin-1 (il-1) family of ligands and receptors plays a central role in the formation and regulation of inflammation as part of the immune response. the il-1 molecule is best known as a member of a family of proinflammatory proteins called cytokines. they have been shown to play a role, not only during inflammation, but also in influencing additional physiological and pathological functions such as autoimmune diseases, malignancies, and many others. in addition, the biological balance between proinflammatory and inhibitory activity is critical, as any change in the expression of the il-1 family has the potential to cause disease (etti, et al., 2018). inflammation that occurs as a result of exposure of tissues and organs to noxious stimuli such as microbial pathogens, irritants, or toxic cellular components. the main physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function in the affected area. this process involves the main cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, t cells, and b cells. however, examination of various inflammatory lesions reveals the presence of specific leukocytes in certain lesions. that is, the inflammatory process is regulated in such a way as to ensure that the right leukocytes are recruited. these events are in turn controlled by a number of extracellular molecular regulators, including members of the cytokine and chemokine family that mediate immune cell recruitment and the complex intracellular signaling control mechanisms that characterize inflammation. this review will focus on the role of major cytokines, chemokines, and their receptors in the pathophysiology of autoinflammatory disorders, pro-inflammatory disorders, and neurologic disorders involving inflammation. il-1 actively participates in the inflammatory response to infection (turner, et al., 2014), and its main source is activated monocytes and macrophages. interleukin-1 as a predictor cytokine sars-cov severe acute respiratory syndrome coronavirus (sarscov) is the etiologic agent of respiratory disease that has a mortality rate of 10%. previous studies have shown that sars-cov lacking the e gene (sars-cov-δe) is attenuated in several animal model systems. this https://doi.org/10.14421/biomedich.2023.121.187-190 188 biology, medicine, & natural product chemistry 12 (1), 2023: 187-190 suggests that the absence of protein e resulted in reduced expression of proinflammatory cytokines, decreased number of neutrophils in the lung infiltrates, reduced lung pathology, and increased viability of mice, suggesting that lung inflammation contributes to sarscov virulence. furthermore, sars-cov-δe infection resulted in decreased nf-b activation compared to levels of wild-type virus. most importantly, treatment with drugs that inhibit nf-kb activation led to a reduction in inflammation and lung pathology in sars-cov-infected cells and mice and significantly improved mouse survival after sars-cov infection. these data suggest that activation of the nf-b signaling pathway represents a major contribution to inflammation induced after sarscov infection and that nf-b inhibitors hold antiviral promise in infections caused by sars-cov and other potentially pathogenic human coronaviruses (dediego, et al., 2014). deletion of the sars-cov envelope (e) gene attenuates the virus. gene e encodes a small multifunctional protein that has ion channel (ic) activity, an important function in virus-host interactions. to examine the contribution of the ic activity of protein e in viral pathogenesis, two mouse-adapted sars-cov, each containing a single amino acid mutation that suppresses ion conductivity, were engineered. after serial infection, mutant viruses, in general, introduce compensatory mutations in the e gene that create active ion channels. furthermore, ic activity provided better fitness in competition assays, suggesting that ionic conductivity is an advantage for viruses. interestingly, virus-infected mice exhibiting ic protein e activity, either with a wild-type protein e sequence or with a revertant that restores ion transport, rapidly lost weight and died. in contrast, mice infected with a mutant lacking ic activity, which did not introduce the mutation in the e gene during the experiment, recovered from the disease and mostly survived. levels of inflammasome-activated il-1β were reduced in the lung airways of virus-infected animals lacking ic protein e activity, suggesting that ic protein e function is required for inflammasome activation. the reduction in il-1β is accompanied by a decrease in the number of tnf and il-6 in the absence of ion conductivity protein e. all of these key cytokines promote the development of lung damage and ards pathology. in conclusion, the ic activity of protein e is a novel determinant for the virulence of sars-cov (nieto-torres, et al., 2014). siu et al. (2019) demonstrated that the open reading frame 3a (orf3a) accessory protein of sars-cov activates the nlrp3 inflammasome by promoting tnf receptor-associated factor 3 (traf3)-mediated ubiquitination of an apoptosis-associated spot-like protein containing a recruitment caspase domain (asc). sars-cov and its orf3a protein were found to be potent activators of pro-il-1β gene transcription and protein maturation, 2 signals required for nlrp3 inflammasome activation. orf3a induces pro-il-1β transcription via nf-b activation, which is mediated by ubiquitination and traf3-dependent p105 processing. orf3a-induced increase in il-1β secretion is independent of its ion channel activity or is absent in melanoma 2 but requires nlrp3, asc, and traf3. orf3a interacts with traf3 and asc, localizes with them in discrete mottled structures in the cytoplasm, and facilitates the formation of asc specks. traf3dependent ubiquitination of k63-associated ascs was more prominent in cells infected with sars-cov or when orf3a was expressed. overall, the findings of siu, et al. (2019) revealed a novel mechanism by which the sars-cov protein orf3a activates nf-b and the nlrp3 inflammasome by promoting p105 and asc.siu-dependent ubiquitination of traf3. the severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent asc ubiquitination (siu, et al., 2019). sars-cov-2 appears to act on the activation and maturation of il-1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf (dediego, et al., 2014; nieto-torres, et al., 2014; siu, et al., 2019). sars-cov-infected dcs show low expression of antiviral cytokines (interferon [ifn-α], ifn-β, ifn-γ, and interleukin 12p40 [il-12p40]), upregulation of proinflammatory cytokines (tumor necrosis). factors [tnf-α] and il-6) but significant upregulation of inflammatory chemokines (macrophage inflammatory protein 1α [mip-1α], upregulated on normal-expressed and secreted t cell activation [rantes]), interferon-inducible proteins from 10 kda [ip-10], and monocyte chemoattractant protein 1 [mcp1]). therefore, il-1β is part of the cytokine storm generated by coronavirus infection (law, et al., 2005; chu, et al., 2016; hui, et al., 2019; conti, et al., 2020; mehta, et al., 2020; wan, et al., 2020) yang, et al. (2020) detected elevated levels of the il1 receptor antagonist (il-1ra) in 14 severe cases of covid-19, and this marker has been associated with increased viral load, loss of lung function, lung damage, and risk of death. liu, et al. (2020) also found elevated levels of il-1α in patients with severe covid-19, and this was strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers. covid-19 patients with severe symptoms have elevated levels of il-1β, which has been associated with sars, hypercoagulation, and disseminated intravascular coagulation (zhang, et al., 2020). for this reason, several therapeutic strategies have used il-1 inhibition in an attempt to avoid cytokine storms (tanaka, et al., 2016; kritas, et al., 2020). in this way, mesenchymal stem cells (mscs) have been used to savitri et al. – interleukin-1 as a predictor cytokine sars-cov: article review 189 inhibit proinflammatory cytokines such as il-1α and tnf (chen, et al., 2020). conclusions the conclusions of the study may be presented in here.il-1 actively participates in the inflammatory response to infection. sars-cov-2 appears to act on the activation and maturation of il-1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf-. therefore, il-1β is part of the cytokine storm generated by coronavirus infection. elevated levels of the il-1 receptor antagonist (il-1ra) in severe cases of covid-19, and this marker have been associated with increased viral load, loss of lung function, lung damage, and risk of death. in addition, there is an increase in il1α levels in patients with severe covid-19, and this is strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers coronavirus. acknowledgements: acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted.the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study. lisa savitri and elfred rinaldo kasimo analyzed the articles. lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors stated there was no funding. references chen c., zhang x.r., ju z.y., he w.f. 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(2020) the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china. clin. immunol. 2020;214 doi: 10.1016/j.clim.2020.108393. biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 5-9 | doi: 10.14421/biomedich.2015.41.5-9 the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani biology education, mathematics and science faculty, ikip pgri madiun jl. setiabudi 85 madiun, tel. +62-351-462986, fax. +62-351-459400 author correspondency: primianibiomipa@yahoo.co.id abstract the use of estrogen hormone by public has significantly been improved either as prevention or treatment of disease. menopausal issues in women are often treated using hormone replacement therapy. in regard to this, yam bean is found to contain genistein and daidzein compounds with a chemical structure that resembles estrogen hormone, therefore yam bean is categorized in the phytoestrogen group. the purpose of this study was to identify the potential of yam bean on ovarian and uterine histology of mice. this research employed a completely randomized design of experimental research approach of one factor namely yam bean in three different dosage treatment: 0.3 g/kg, 0.6 g/kg, and 0.9 g/kg of yam bean for 24 days. the surgery and organ harvesting of ovary and uterus were conducted on day 25 along with the making of histological preparat using paraffin method and hematoxylin-eosin (he) staining. the data was then analyzed descriptively. this research found that there were both secondary and tertiary follicle proliferation as the antrum contains some estrogen level. meanwhile, the endometrial tissue of the uterus experienced uterine glandular proliferation. to conclude, yam bean was found to be a natural estrogen source. keywords: yam bean, isoflavone, phytoestrogen, ovarium, uterus, menopause introduction hormone replacement therapy (hrt) is an action which is conducted through giving estrogen hormone especially to women who experience some reduction in the estrogen hormone. hrt is usually undergone by women experiencing menopause. in particular, estrogen replacement therapy (ert) can also be used for conducting cardiovascular disease therapy (stampfer et al., 1991 and sourander et al., 1998), reducing the risk of osteoporosis (grady et al., 1992 and nurochmad et al., 2010), and reducing the symptoms of menopause (barrett, 1998). the use of synthetic estrogen compound in a long term often gives negative impacts so that a natural alternative is needed to replace estrogene hormone. yam bean plant (pachyrhizus erosus) as a tuberlegume crop, is categorised as a legume with the dissemination area of sumatera, java, bali, sulawesi and east nusa tenggara (karuniawan & wicaksana, 2006). people usually prefer to consume fresh yam bean either in the form of salad or rujak (indonesian traditional fruit an vegetable salad dish). yam bean extract is used in the cosmetics industry in whitening, compact powder, and moisturizer products. yam bean contains isoflavone compounds with estrogen-like chemical structure (wanibuchi, 2003; abid, 2005; and lukitaningsih, 2009). the chemical isoflavones structure resembles 17βestradiol and has the efficacy like estrogene hormone (delmonte and rader, 2006; barlow et al., 2007) so that yam bean is also included in phytoestrogen group (urasopon et al., 2008). the largest components of isoflavone are genistein and daidzein which are often found in fabaceae family, including pachyrhizus erosus (kang et al., 2006). pachyrhizus erosus at least contain isoflavones such as, daidzein and genistein (primiani, 2013), the analysis of hplc daidzein and genistein of yam bean is 110,454 mg/100 g and 165,530 mg/100 g respectively. the chemical structure of geninstein and daidzein can bind to estrogen receptor and compete with endogenous estrogen such that they may provide both estrogenic and anti-estrogenic effects (adlercreutz, 1990; griffiths et al., 1996; adlercreutz and mazur, 1997). utilization of yam bean as phytoestrogen is not conducted much, a study by nurrochmad et al., (2010) showed that providing yam bean extract of 400 mg/kg and 800 mg/kg dosage for 4 weeks to mice through ovariectomy could prevent bone fragility; phytoestrogen was then proven to be able to improve uterine mass (ford et al., 2006). meanwhile, genistein affects on the increasing of weight of uterus by stimulating uterine endometrial thickening (santell, 1997). genistein dose 26,6 mg/day eqiuvalen with human dose 0,625 mg/day within 6 months on monkey cause vaginal maturation (marquez et al., 2012). research methods research design the research was conducted using experimental research approach with a completely randomized design, the treatment was completed through providing grated yam bean in three different dosage: 0.3 g/kg, 0.6 g/kg, 0.9 g/kg. the observation is focused on the changes of ovarian and uterine tissue structure. 6 biology, medicine, & natural product chemistry 4 (1), 2015: 5-9 tools and materials the research tools used in this study were: a gavage tube, plastic mice cages (50 x 30 x 20cm size), drinking bottles for mice, a digital scale (hm-200 brand), 1ml syringes with 3ml disposable needles (g23), an incubator, optical miscroscope, digital optilab camera microscope, a triple beam balance ohaus 700 series scale, analytical balance type hm-200 with the capacity of 210 grams and accuracy level of 0.1 mg, pr-50 microtome, surgical equipment, a surgical board, object glasses and lenses, glass beakers, spiritus lamp, a cube made of calendar paper, blender, flour sifter, a nife, a pipette, and a couple. the research materials were yam bean obtained from takeran village madiun indonesia, ovarian and uterine tissue, milk a granule for the mice food produced by pt. charoen pokphand indonesia, husk, cotton, tissue paper, aqua destillata, water tap, paraffin, 0.9% physiological saline, bouin fixative solution, 50%, 70%, 90% and absolute alcohol, pure xylol, xylol-alcohol mixture, with xylol and acohol comparison consecutively 1:3, 2:2, and 3:1, li2co3 solution, 1% hcl, 3% formalin, and haupt adhesive. animal experiments the experimental animals used were mice (mus musculus) strain balb/c female, in a good health condition, aged 12 months, 24 mice in total. every mouse had an initial body weight ranging from 20-25 grams before treatment, the mice were kept in a cage located in the biology education ikip pgri madiun, indonesia maintenance of mice and manufacture of test materials the mice were placed in the mice cages, given food and drink ad libitum and acclimatized for 14 days prior to induction treatment. the mice were maintained at the room temperature of (± 270c), relative humidity between 50-60% and 12 hour lighting cycle. every day the mice were weighed as the basis for determining the provision of grated yam bean. the manufacture of grated yam bean as a research material was conducted through grating the yam bean using a grater. the amount of the yam bean was then weighed and complied with the amount given to the mice. the provision of yam bean and manufacture of preparat of ovarian and uterine histology the provision of yam bean was coducted through direct induction to the stomach using a gavage tube once a day for 24 days. the mice were then dislocated on the 25th day and dissected. the organ harvesting of their ovaries and uterus were then performed. the manufacture of the preparat of ovarian and uterine histology was completed using paraffin method in attempt to determine any ovarian and uterine tissue structure changes. data analysis the ovarian and uterine tissue structure changes were analyzed descriptively based on the changes that happened in the ovarian follicle, myometrium layer, endometrium and uterine mucosa. results and discussions the results of observation on the ovarian tissue showed that there were changes in the ovarian follicles (figure 1). figure 1. the ovaries of mice (mus musculus); he stained sections; 400x description: a. control (p1); b. the dose of 0.3 g/kg (p2); c. the dose of 0.6 g/kg; d. the dose of 0.9 g/kg (p4). a. ovary with the control treatment, there are some corpus albikans. b. ovary with mature follicles and granulosa layer. c. secondary follicular proliferation, tertiary follicles begin to develop. d. tertiary follicles with follicular fluid. a b c d cicilia novi primiani – the phytoestrogenic potential of yam bean (pachyrhizus erosus) … 7 the results of observation on the uterine tissue showed that there were changes in the uterus endometrium tissue (figure 2). figure 2. the uterus of mice (mus musculus); he stained; 400x description: a. control (p1); b. the dose of 0.3 g/kg (p2); c. the dose of 0.6 g/kgd. the dose of 0.9 g/kg (p4). a. uterine wall consists of tunica serosa, tunica muscularis and tunica mucosa. b. proliferation of tunica mucosa (uterus endometrium layer). c. proliferation of uterine glands in uterine endometrium layer. d. proliferation of endometrium and myometrium layers, proliferation of uterine glands. the observation results of ovarian histology using hematoxylin-eosin (he) staining are depicted in figure 1. the control treatment (p1) shows follicles that experienced atresia (corpus albikans) and there is no indication of any primary follicle proliferation. the atresia folikuli phase shows histological features that represent disintegration in the form of fat drops and coarse granules in the ova (figure 1a). in the ovary that is given grated yam bean of 0.3 g/kg dose, some follicles with a thick layer under the tunica albuginea occur and they have a unique characteristic that the ova contained do not have any vitelina membrane (figure 1b). the ova are surrounded by layers of follicular cells which form granula layers on the more mature follicles afterward. in the provision of yam bean of 0.6 g/kg dose, secondary follicular growth takes place, the follicles are more eggshaped (oval) and has been moving away from the cortex approaching the medulla ovary and forming a room that is filled with fluid (antrum) around the ova and the granulosa cells layer are coating it (1c). liquid called liquor folliculi makes the follicles developed into tertiary follicles (apparent from the picture of ovary with 0.9 g/kg dose yam bean treament) (1d). changes in the uterine tisue structure of the mice after being given grated yam bean are depicted in figure 2. the results of observation on uterine histology with hematoxylin-eosin staining (he) show that the uterine wall of the control treatment (p1) is relatively thick and formed in 3 layers: (1) tunica serosa or outer perimetrium in the form of connecting tissue that consists of a layer of mesothelium supported by a thin connective tissue, (2) tunica muscularis or myometrium is the thickest layer which is composed of smooth muscle tissue. myometrium is the thickest tunica that consists of bundles of smooth muscle fibers which are separated by connective tissue. the endometrium consists of epithelial siliata and non siliata as well as lamina propria or stroma endometrialis containing simple tubular glands (uterine glands) that are branched on the inside (near the endometrium) (figure 2a). the provision of grated yam bean of 0.3 g/kg (p2) causes endometrium and myometrum proliferation of the uterus, uterine luminal has been narrowing due to the epithelialization process. the proliferation of uterine glands of the uterus begin to exist despite the decrease of premeneopausal period proliferation (figure 2b). the grated yam bean provided to the mice at a dose of 0.6 g/kg (p3) dan 0,9 g/kg (p4) apparently causes proliferation of the uterine endometrium and myometrium layers and increases the number of uterine glands (figure 2c and 2d). genistein and daidzein are categorised in isoflavone group with the chemical structure that is similar to estrogen hormone and able to work as estrogen. the similarity of the structure of genistein and daidzein demonstrate the ability to bind to estrogen receptors (setchell & cassidy, 1999). the decrease of ovarian follicles proliferation during the premenopause causes delays of tertiary follicle growth such that the secretion of estrogen hormone in the tertiary follicle antrum does not occur. the provision of grated yam bean can optimize the level of estrogen so that it is able to stimulate the development of primary follicles (primordial), secondary follicles, and tertiary follicles. the provision of phytoestrogen during the premenopause period improves the secretion of estrogen hormone which later causes an increase in the endometrium proliferation and epithelial cells replacement to cover the surface of mucosa. phytoestrogens have a similar structure to that of estradiol a b c d 8 biology, medicine, & natural product chemistry 4 (1), 2015: 5-9 which may also occupy estrogen receptors and lead to estrogen-like effects such as endogenous itself (harrison et al., 1999). myometrium may develop due to the phytoestrogens yam bean effect. genistein and daidzein in yam bean have an effect on uterine epithelium resulting in proliferation and cornification of epithelial cells as well as in the optimization of the secretion of estrogen. decreased estrogen level in premenopausal and postmenopausal period, resulting in an excess amount of estrogen receptors that are not bound. genistein and daidzein ability to bind to estrogen receptors called sex hormone binding globulin (hgb) serves to increase the production of steroid hormones and is responsible for binding estrogen and circulating it through the blood vessels (setchell et al., 1998). genistein as one of the isoflavone compounds has the effect of increasing the weight of the uterus by stimulating uterine endometrial thickening (santell et al., 1997). decreased levels of endogenous estrogen in premenopausal period can cause the estrus phase to not occur, thus giving yam bean to premenopausal mice can stimulate the estrus phase. genistein and daidzein as a group of phytoestrogens bind to estrogen receptors in the ovaries and uterus. the resulting response when estrogen levels are low is that the receptor and phytoestrogens binding will help in balancing estrogen levels. the function of estrogen in relation to reproduction is causing proliferation to occur and tissue in the reproductive organs to grow. the use of natural materials as hormone replacement therapy in postmenopausal women is one of the alternatives that can be done to overcome the side effects. component compounds contained in natural materials are very complex and often interact each other to provide a physiological effect (ioannides, 2002 and zhou et al., 2003). multi component contained in herbal medicine is a compounds complexity that provides optimal effect (lan and jia, 2010). conclusions yam bean can be used as natural estrogen hormone source which can increase the proliferation and maturation of ovarian follicles and proliferation of uterine endometrial glands of the uterus in premenopausal period. following up the need for estrogen therapy for premenopausal and postmenopausal women, it is necessary to study the effectiveness and safety ranging from experimentin on animals to humans, so its use as hormone therapy can be trustworthy. references abid, m. 2005. pharmacological evaluation of pachyrhizus erosus (l) seeds for cns activity. disertasi, bangalore: rajiv gandhi university of health science. adlercreutz, h. 1990. western diet and western diseases: some hormonal biochemical mechanism and associations. scandinavian j. clin. lab. invest. 50s 201:3-23. adlercreutz, h., mazur, w. 1997. phyto-oestrogens and western diseases. j. anayls of med. 29:95-120. barlow, j., johnson, j.a., scofield, l. 2007. fact sheet on the 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2department of biological education, faculty of tarbiyah and education, uin raden intan lampung jl. let.kol endro suratmin, sukarame, bandar lampung, 35131, indonesia. corresponding author* aulia@radenintan.ac.id manuscript received: 30 january, 2023. revision accepted: 13 march, 2023. published: 30 march, 2023. abstract crystal guava (psidium guajava var. crystal) is one type of guava plant that has high economic value due to its thick flesh and few seeds. the crystal guava plant is believed to have antioxidant activity, which is a substance that can prevent the formation of free radicals in the body. this research aims to determine the level of antioxidants in chloroform and methanol extracts of the skin and flesh of crystal guava fruit using the dpph method. samples were taken through a stepwise maceration process and solvents of chloroform and methanol, then analyzed using probit analysis and spss 25 software. the results of the study showed that both chloroform and methanol extracts of the skin and flesh of crystal guava fruit have antioxidant activity. the results showed that the ic50 value of the chloroform extract of crystal guava fruit skin is 218.88 ppm and is classified as moderate, the methanol extract of crystal guava fruit skin is 89.78 ppm and is classified as strong, the chloroform extract of crystal guava fruit flesh is 270.56 ppm and is classified as weak, and the methanol extract of crystal guava fruit flesh is 185.72 ppm and is classified as moderate. keywords: antioxidant; dpph; chloroform; crystal guava; fruit flesh; methanol; peel. introduction one horticultural product that is widely found and used in tropical countries is the crystal guava. this guava plant is widely cultivated by farmers and bears fruit throughout the year. in addition to its fruit, almost every part of the guava plant can be utilized. guava fruit is commonly consumed to help improve digestion and lower blood sugar levels. guava leaves can even be used as medicine to treat diarrhea, vomiting, and sore throat. guava is also easily found in the market at affordable prices (rustani and susanto, 2019). crystal guava, which is rich in vitamin c and has a high economic value, is very suitable for agro-tourism development programs (haidawati et al., 2015). vitamin c is one of the natural antioxidants found in fruit, in addition to vitamin e, polyphenols, carotenoids, and flavonoids (febrianti et al., 2016). it can not only be consumed directly but crystal guava can also be used for other purposes and has its own advantages compared to other types of guava. crystal guava is believed to have a lot of phytochemicals such as flavonoids, polyphenols, isoflavonoids, and tannins in its leaves and fruit, providing natural antioxidants. flavonoids are the main bioactive compounds found in guava leaves, while the majority of vitamin c content is found in the fruit skin (jamieson et al., 2021). guava leaves also contain quercetin, which has antioxidant properties and is considered the most active and strong antioxidant in guava seed leaves (sharma and borah, 2021). the presence of natural antioxidants plays an important role in managing various types of degenerative diseases caused by free radicals. free radicals are small molecules with unpaired electrons, making them highly reactive (hartati et al., 2020). their reactive nature causes free radicals to seek out electrons in other compounds, forming new chains of radicals and damaging the structure of certain compounds (setiabudi et.al., 2020). free radicals can also continuously form in the body, so antioxidants are needed to balance them. antioxidants can reduce the negative impact of many free radicals. free radicals can be neutralized by antioxidants through electron donors, making free radicals more stable and less reactive. the presence of antioxidant compounds prevents free radicals from bonding with electrons in other compounds, as their electrons are already bonded with electrons from the antioxidants (handayani et al., 2020). therefore, antioxidant compounds play an important role in the human body. https://doi.org/10.14421/biomedich.2023.121.323-328 324 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 the testing of antioxidant levels in crystal guava is widely conducted. typically, the antioxidant levels test uses ethanol as the solvent in extraction and the maceration method. in this research, extraction was conducted using chloroform and methanol solvents. the use of chloroform and methanol as solvents is based on their different polarities. chloroform is non-polar, while methanol is polar. the principle of solubility says that polar solvents will dissolve polar compounds, and nonpolar solvents will dissolve non-polar compounds. with the use of different solvents, it is hoped that the observed components will be separated according to their level of polarity (taroreh et al., 2015). the compounds that have the potential as antioxidants are flavonoids and phenols, which are included in the polar fraction (yusriyani and syarifuddin, 2021). plants also contain non-polar compounds such as waxes, lipids, and proteins (dewitasari, 2020). non-polar solvents such as chloroform are needed to cleanse polar antioxidant compounds to be more optimally absorbed by polar solvents such as methanol. if non-polar compounds such as proteins and lipids are not cleaned by non-polar solvents, it will interfere with the process of capturing free radicals by flavonoid compounds (rahmadani and nasution, 2021). the maceration method used is a stepwise maceration, so this is one of the innovations in this research. this study aims to determine the antioxidant level in chloroform and methanol extracts of crystal guava skin and flesh using the dpph method. materials and methods materials the equipment used in this research includes an oven, electric balance, filter paper, hot plate, blender, reaction tube, dropper, maceration bottle, knife, distillator, rotary evaporator, uv-vis spectrophotometer (shimadzu), vortex, reaction tube rack, and plastic bags. the materials used in this research include the skin and flesh of crystal guava, methanol, p.a ether, water, ethyl acetate, 1,1diphenyl-2-picrylhydrazyl (dpph), ascorbic acid, and chloroform. the guava fruit used in this study was those with a green-yellow and green maturity level. this is because the fruit with green-yellow maturity has the highest vitamin c content compared to fruits that are green and bright green. (dewi et al., 2017) figure 1. crystal guava (psidium guajava var. crystal) (source: personal documentation). procedures preparation of simplicia and extraction 20 kg of crystal guava fruits is peeled and separated between the skin and flesh. the skin and flesh are washed and drained. then, they are cut and dried in an oven at 450°c until dry. in the next step, the dried samples are blended into a fine powder and sieved using a 25-mesh sieve. the prepared crystal guava skin and flesh simplicial will be extracted using a sequential maceration method. the solvents used for sequential maceration are chloroform and methanol. both solvents are used with a ratio of each solvent to a simplicial of 1:2. extraction is performed by weighing 125 grams of guava skin and flesh simplicial and placing them into a container. then, it is poured with 250 ml of chloroform solvent, covered and allowed to stand for 24 hours in a light-protected condition, and stirred every 8 hours. afterward, the macerate is collected and concentrated using a rotary evaporator to form a concentrated extract. to make a methanol extract, the dried chloroform extract residue is processed in the same way as the chloroform extraction, but using methanol solvent. the extract is then concentrated again using a rotary evaporator to make a concentrated methanol extract. the yield of the extract was calculated using the formula (edison et al. 2020): % yield = weight of extract (𝑔) weight of sample (𝑔) x 100% antioxidant testing 25 mg of each dried sample was weighed and dissolved in p.a methanol to make the volume 50 ml (500 ppm). then, a series of concentrations of the three samples ulmillah et al. – uncovering the antioxidant power: investigating the skin and … 325 were created, namely 50 ppm, 100 ppm, 150 ppm, 200 ppm, and 250 ppm. the testing process was carried out by adding 0.5 ml of the solution from the two sample extracts with various concentrations. then, each was added with 3.5 ml of 50 ppm dpph. the mixture was then vortexed and incubated for 30 minutes in a dark environment and at 37°c. its absorbance was then measured at a wavelength (λ) of 517 nm. an anti-oxidant control test was used using ascorbic acid at 10 mg weighed and dissolved in p.a methanol to have a concentration of 100 ppm. then, a series of ascorbic acid concentrations were made with concentrations of 5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm. the test was then carried out by pipetting 0.5 ml of the sample solution from different concentration series. each solution was then added with 3.5 ml of 50 ppm dpph. the mixture was then vortexed and incubated for 30 minutes at 37°c and in a dark room. after that, its absorbance was measured using a spectrophotometer at a wavelength of 517 nm. the anti-oxidant activity was calculated using the following formula (febrianti and ariani, 2020): inhibition (%) = absorbance of blank − absorbance of sample absorbance of blank 𝑋 100 % the ic50 value was obtained from linear regression analysis by substituting the value of y as 50 from the equation y = a + bx. the smaller the ic50 value, the greater the antioxidant activity of a substance (martiningsih et al., 2016). data analysis the uv-vis spectrophotometry measurement data was were analyzed using the linear regression analysis method (anova) with the help of spss 25 software at a 5% level to decide on the hypothesis proposed. a graph was made using the probit analysis between the log concentration of the sample as the x-axis and the percentage of antioxidant activity as the y-axis, to find the linear regression equation. thus, the ic50 can be found in the methanol and chloroform extracts. results and discussion extraction results the extraction conducted in the research produced four types of concentrated extracts, namely chloroform extract of the fruit skin, chloroform extract of the fruit flesh, methanol extract of the fruit skin, and methanol extract of the fruit flesh. the obtained concentrated extracts were then calculated for yield values as shown in table 1. the highest yield value was in the sample of chloroform extract of the fruit skin (5.92%), followed by chloroform extract of the fruit flesh (5.8%), methanol extract of the fruit skin (2.08%), and methanol extract of the crystal fruit flesh (1.68%). this is not directly proportional to the weight of the obtained concentrated extract. the result of the extraction from each 125 gram of the plant material showed that the highest extract was obtained from the methanol extract of the fruit skin (7.4 g), followed by the methanol extract of the fruit flesh (5.8 g), chloroform extract of the fruit skin (2.6 g), and chloroform extract of the fruit flesh (2.1 g). this result indicates that the extract produced from chloroform solvent resulted in a higher yield value compared to the yield value obtained from the extraction using methanol solvent. chloroform is nonpolar, while methanol is polar and a compound is separated based on its polarity (taroreh et al., 2015). therefore, it is possible that the group of compounds in both samples that were well absorbed in chloroform solvent were nonpolar compounds. table 1. extraction results using chloroform and methanol on skin and flesh of crystal guava fruit. no sample name weight of chloroform extract yield (%) methanol extract yield (%) simplicia (grams) chloroform extract (grams) methanol extract (grams) 1 skin fruit 125 2,6 7,4 5,92 2,08 2 flesh fruit 125 2,1 5,8 4,64 1,68 antioxidant test results the ic50 value was obtained by first conducting a probit analysis. the results obtained from the r value show that the probit data from the chloroform and methanol extracts from the skin and flesh of crystal guava are very good, as they approach the value of 1 (fadiyah et al., 2019). a positive value of +1 indicates that the higher the extract concentration, the higher the antioxidant activity. antioxidant activity is usually expressed in percentage of dpph reduction or with the ic50 value. the line equation obtained was used to calculate the ic50 value. after the calculation, the ic50 value for the skin of the crystal guava fruit is 218.88 ppm for the chloroform extract and 89.78 ppm for the methanol extract. 326 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 meanwhile, for the flesh of the crystal guava fruit, the ic50 value is 270.56 ppm for the chloroform extract and 185.72 ppm for the methanol extract, and 18.09 ppm for ascorbic acid. the results of the antioxidant test can be seen in table 2. table 2. ic50 values of antioxidants in chloroform and methanol extracts from skin and flesh of crystal guava fruit. sample concentration % inhibition log concentration probit ic50 value (ppm) skin fruit extract (c) 50 26,82 1,6990 4,36 218,88 100 27,78 2,0000 4,39 150 29,94 2,1761 4,45 200 30,23 2,3010 4,48 250 31,62 2,3979 4,50 skin fruit extract (m) 50 30,91 1,6990 4,48 89,78 100 33,95 2,0000 4,56 150 37,90 2,1761 4,67 200 39,46 2,3010 4,72 250 45,57 2,3979 4,87 flesh fruit extract (c) 50 31,20 1,6990 4,50 270,56 100 31,79 2,0000 4,50 150 33,75 2,1761 4,56 200 34,23 2,3010 4,59 250 35,18 2,3979 4,61 flesh fruit extract (m) 50 32,96 1,6990 4,53 185,72 100 33,83 2,0000 4,56 150 34,50 2,1761 4,59 200 38,08 2,3010 4,69 250 38,99 2,3979 4,69 ascorbic acid 5 44,19 0,6990 4,85 18,09 10 60,78 1,0000 5,25 15 77,28 1,1761 5,74 20 88,98 1,3010 6,18 25 96,39 1,3979 6,75 *(c) = chloroform, (m) = methanol the ic50 value shown in table 2 indicates that ascorbic acid as a comparison has the strongest antioxidant activity, this is due to ascorbic acid being a pure antioxidant compound. methanol extract from the skin and flesh of crystal guava fruit has a higher ic50 value compared to the chloroform extract from the skin and flesh of crystal guava fruit, as shown in figure 2. figure 2. the relationship between the concentration of chloroform and methanol extracts from the skin and flesh of the crystal guava fruit (psidium guajava var. crystal) and antioxidant activity. note skin 1= chloroform extract from the skin of the fruit; skin 2= methanol extract from the skin of the fruit; flesh 1= chloroform extract from the flesh of the fruit; flesh 2: methanol extract from the flesh of the fruit. discussion in the drying process, both samples were dried using an oven at a temperature of 45 degrees celsius for 150 hours until the dried simplicia, seen from the brownish color and easy to break. then, these dried samples were blended and filtered with the aim of breaking down the cells and expanding the sample surface so that it is easier to extract. the size of the fine powder produced makes it easier for the solvent to extract the contents directly because the larger the surface area and the more effective the interaction with the solvent (pertiwi et al., 2022). in this study, extraction was carried out using a multistep maceration method. maceration means soaking and is a method for isolating active substances using a solvent and stirring at room temperature several times over a certain period. maceration is a cold method that can protect secondary metabolites such as flavonoids from the effects of heating. the solvents used in the extraction are 75% chloroform and 75% methanol. the ulmillah et al. – uncovering the antioxidant power: investigating the skin and … 327 extraction was carried out through a multistep maceration method, which is slightly different from the ordinary maceration method. in this multi-step method, solvents with different polarity levels are used. the first extraction was carried out using a non-polar solvent, chloroform, and then the residue from the chloroform maceration was processed again using methanol as the solvent. the use of the multistep maceration method aims to extract active substances (antioxidants) more optimally. in addition, the use of solvents with different polarity levels also aims to extract active substances with different polarities, so that the extraction results can be better (kuspradini et al., 2016). antioxidants have polar properties, so the use of non-polar chloroform solvent in the initial extraction is intended to pull non-polar substances from the sample, so the sample will be free of non-polar substances. the second extraction using methanol will optimally pull polar substances, so when the dpph test is performed, non-polar substances will not interfere and affect the results that are not maximal. the extraction yields a concentrated extract which is then calculated for its yield value. the yield in this study was measured by comparing the dry extract mass (grams) with the initial mass of the material before the extraction process (grams). the dry extract was obtained after the sample was dried in an oven until it had a constant weight. this calculation was performed to determine the percentage of material that remained after the extraction process and to determine the level of effectiveness of the process produced (senduk et al., 2022). the yield value can also indicate the number of secondary metabolite compounds present in the sample. therefore, this calculation can serve as a reference in antioxidant testing or as a comparison of antioxidant testing results. the second antioxidant test of the two sample types used chloroform and methanol solvents and produced positive results. this is indicated by the color change produced during testing with dpph. in the antioxidant activity study, the addition of dpph solution to the sample was marked by a change in color from purple to yellow. this indicates the occurrence of free radical capture. the change in intensity of the purple color is due to the free radical scavenging produced by the reaction of the dpph molecule with hydrogen atoms released by the sample molecule. this causes the fading of the dpph color from purple to yellow. dpph, which has unpaired electrons, will give a purple color, and the color will change to yellow when the electrons have been paired (konda et al., 2020). the test was performed using a uvvis spectrophotometer with a wavelength of 517 nm, which is the maximum wavelength of dpph. this wavelength will provide the best absorption results from the test solution and provide high sensitivity, thus it is expected to obtain optimal absorbance values for the sample (konda et al., 2020) the samples tested for antioxidant activity include chloroform and methanol extracts of the skin and flesh of crystal guava fruit and ascorbic acid (vitamin c) as a reference. vitamin c was used because it has an antioxidant function, capturing free radicals and preventing chain reactions. vitamin c has free hydroxyl groups that function as free radical captures, and if it has polyhydroxy groups it will increase antioxidant activity. the ic50 values for the skin and flesh of the crystal guava fruit show that the antioxidant activity in the skin of the crystal guava fruit is stronger than in the flesh of the crystal guava fruit. this is due to the higher vitamin c content in the skin of the fruit (jamieson et al., 2021). a smaller ic50 value indicates stronger antioxidant activity, while a larger ic50 value indicates weaker antioxidant activity (andriani and murtisiwi, 2020). research has been conducted on the health benefits of guava leaves, which are attributed to the various phytochemicals they contain such as quercetin avicularia, apigenin, guaijaverin, kaempferol, hyper in, myricetin, gallic acid, catechin, epicatechin, chlorogenic acid, epigallocatechin gallate, and caffeic acid. the extracts from guava leaves (gls) have been investigated for their biological properties such as anticancer, antidiabetic, antioxidant, antidiarrheal, antimicrobial, lipid-lowering, and liver protection activities (kumar et al., 2021). various factors can affect the antioxidant content in fruits, such as variety, species, cultivar, harvesting conditions, ambient temperature, photosynthesis process, relative humidity, oxidative stress, and exposure to sunlight. pollution also plays a role in antioxidant content variation. the ripeness of the fruit also affects the vitamin c content. the riper the fruit, the higher the ascorbic acid content, but overheating during the washing and cooking process can cause a loss of antioxidants due to their easily soluble and easily damaged nature with heating (febrianti et al., 2016). conclusions the results of the study showed that both chloroform and methanol extracts of the skin and flesh of crystal guava fruit have antioxidant activity. the results showed that the ic50 value of the chloroform extract of crystal guava fruit skin is 218.88 ppm and is classified as moderate, the methanol extract of crystal guava fruit skin is 89.78 ppm and is classified as strong, the chloroform extract of crystal guava fruit flesh is 270.56 ppm and is classified as weak, and the methanol extract of crystal guava fruit flesh is 185.72 ppm and is classified as moderate. authors’ contributions: all authors are major contributors to this research. aum, aa, and nw collaborated to design the research and write the initial research draft. aa: executed the experiment, aum: 328 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 wrote the article draft and revised the draft, nw: contributed suggestions for article improvement competing interests: the authors declare that there is no potential for conflicting interests in this research study. references andriani, disa & murtisiwi, lusia. 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(2015). ekstraksi daun gedi (abelmoschus manihot l) secara sekuensial dan aktivitas antioksidannya. agritech, 35(3), 280287. https://doi.org/10.22146/agritech.9338 yusriyani, y., & syarifuddin ka (2021). uji aktivitas antioksidan fraksi polar ekstrak kulit buah naga merah menggunakan metode dpph (1, 1 diphenyl-2-picryl hydrazil). jurnal kesehatan yamasi makassar, 5(2), 59-67. pissn:2548-8279. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 33-44 | doi: 10.14421/biomedich.2023.121.33-44 issn 2540-9328 (online) solanum anomalum leaf extract and fractions attenuate oxidative stress and liver injuries in alloxan-induced diabetic rats idongesit charles etuk1, john akpan udobang2, nwakaego omonigho ebong3, jude efiom okokon1,* 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. 3department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele campus. rivers state, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 03 september, 2022. revision accepted: 20 september, 2022. published: 04 october, 2022. abstract the leaf of solanum anomalum used in ethnomedicine for the treatment of various ailments such as diabetes was evaluated for antioxidative stress and hepatoprotective potentials against hepatic injuries in alloxan-induced diabetic rats. antioxidative stress and hepatoprotective activities of leaf extract and fractions (70-210 mg/kg) were assessed by determining oxidative stress markers levels, liver function indices and histopathological study of livers of treated rats. the leaf extract and fractions caused significant (p<0.05 – 0.001) increases in the levels of oxidative stress markers (sod, cat, gpx, gsh) in the livers of the treated diabetic rats. the extract/fractions treatment caused reduction in liver enzymes (alt, ast and alp), total and direct bilirubin. histology of the livers revealed absence or significant reductions in pathological features in the treated diabetic rats compared to untreated diabetic rats. the results show that the leaf extract and fractions of s. anomalum has antioxidative stress and hepatoprotective potentials which may be due to the antioxidant activities of their phytochemical constituents. keywords: solanum anomalum; medicinal plant; liver protective; antioxidant; antioxidative stress. abbreviations: gas chromatography mass spectrometry (gcms) introduction diabetes mellitus is a chronic metabolic disorder which is associated with increased generation of free radicals especially reactive oxygen species (ros) (okutana et al., 2005). alloxan biotransformation to dialuric acid in the body is accompanied by generation of h2o2, •oh and superoxide radicals via iron catalyst which attack organs like kidney, liver and pancreas etc (mathews and leiter, 1999) and cause oxidative stress which is implicated in the pathogenesis of diabetes complications in animals and humans (baynes and thorpe,1999). ros produce cellular and tissue injury through covalent binding, dna strand breaking, lipid peroxidation and augment fibrosis which is also implicated in other disease conditions (paradies et al., 2011). medicinal plants which are used traditionally in the management of diabetes, therefore serve as a great repository for antidiabetic remedies having the advantage of being safer and providing many therapeutic effects. solanum anomalum thonn. ex schumach, a plant whose fruits and leaves are used medicinally and nutritionally, is commonly found growing in west and east africa sub-regions. its parts are utilised locally to treat diabetes, gastrointestinal disorders, infections, inflammation and pains (burkill, 2000; bukenya and hall, 1988; offor and ubengama, 2015). hypoglycemic and antidiabetic activities of the fruits and leaves have been reported (offor and ubengama ,2015; okokon et al., 2022). more so, in vivo and in vitro antiplasmodial (okokon et al., 2016; okokon et al., 2017a), antioedema (okokon et al., 2017b), antioxidant and antiulcer (okokon et al., 2019a), anticonvulsant and depressant (okokon et al., 2019b), analgesic (okokon et al., 2020) and antidiarrhoeal (udobang et al., 2022) properties of the leaf extract have also been reported. phytochemical constituents such as alkaloids, flavonoids, saponins, tanins, diosgenin, a diosgenin glycoside (25(r)-diosgenin-3-o-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside, uracil, 5-methyluracil, 1octacosanol, and octacosane have been reported on the leaves of the plant (okokon et al., 2016; okokon et al., 2022). we report in this study the effect of the leaf extract and fractions s. anomalum on oxidative stress markers, liver function indices and histology of alloxaninduced diabetic rats. https://doi.org/10.14421/biomedich.2023.121.33-44 34 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 materials and methods plants collection fresh leaves of solanum anomalum were collected in compounds in uruan area, akwa ibom state, nigeria in august, 2020. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. hebarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo (uuh.75a). extraction fresh leaves of s. anomalum were washed, cut into smaller pieces and dried under shade for two weeks. the leaves were further pulverized to powder using electric grinder. the powdered leaves material was divided into two parts; one part (1.5 kg) was macerated in 50% ethanol (7.5 l) for 72 hours at room temperature (28 ±2 °c). while the other part, (1.5 kg) was successively and gradiently macerated for 72 hours in each of these solvents (2 x 5l), n-hexane, dichloromethane, ethylacetate and methanol to give corresponding fractions of these solvents. these were thereafter filtered and the liquid filtrates were concentrated and evaporated to dryness in vacuo 40°c using a rotary evaporator (buchilab, switzerland). the extract and fractions were stored in a refrigerator at -4°c, until used for the proposed experiments. animals wistar rats (138-150 g) of either sex were obtained from the university of uyo animal house. they were maintained on standard animal pellets and water ad libitum. permission and approval for animal studies were obtained from the faculty of pharmacy animal ethics committee, university of uyo. induction of experimental diabetes using alloxan monohydrate sixty (60) healthy albino wistar rats (male and female) of known weights were fasted for 24 hours, they were reweighed before the induction by a single intra peritoneal injection of freshly prepared solution of alloxan monohydrate (150 mg/kg) in ice cold 0.9% saline (nacl solution). according to the method of pari and saravanan, (2002), the animals were given 2 ml of 5% dextrose solution using orogastric tube immediately after induction to overcome the drug induced hypoglycaemia. a rest period of 72 hours was allowed during which the rats were allowed access to food and water and the diabetes to be fully developed during these 72 hours. after the rest period, rats with moderate diabetes, having persistent glycosuria, and hyperglycaemia (i.e with blood glucose levels 200 mg/dl and above), (lenzen, 2008) were considered diabetic and selected for the experiments. the diabetic animals were randomised and divided into 9 (nine) treatment groups of 6 rats each. based on the value of previously determined median lethal dose (ld50) (okokon et al., 2022), suitable dose regimens were selected and the rats were treated as follows. group 1 was given 10 ml/kg/day of normal saline orally for 14 days, group 2 was administered with 5 mg/kg/day of glibenclamide orally for 14 days, group 3 was given 70 mg/kg/day of s. anomalum leaf extract orally for 14 days and group 4 rats were given 140 mg/kg/day of s. anomalum leaf extract orally for 14 days, group 5 was administered with 210 mg/kg/day of s. anomalum leaf extract orally for 14 days. groups 6 -9 were respectively administered with 140 mg/kg/day of n-hexane, dichloromethane, ethyl acetate and methanol fractions of s. anomalum leaves orally for 14 days. effect of administration of leaf extract and fractions of s. anomlum on fasting blood glucose of alloxan-induced diabetic rats. the fasting blood glucose (fbg) of all the rats was measured after 14 days of administration of the leaf extract and fractions. the method that was employed was “the tail-tipping method”. the blood obtained from the tail vein of the rats was dropped on the dextrostix reagent pad and the pad inserted into a microprocessor digital blood glucometer and the readings were recorded (who, 1980). all the treatments were administered between 7.008.00 am daily throughout the experimental period and food was withdrawn from the experimental animals 12 hours before measurement of fbg to create the necessary fasting period for measurement of the fasting blood glucose concentrations. determination of the body weights changes of the treated diabetic rats throughout the experimental period the body weights of the experimental animals were monitored and recorded at the following points; just before the fasting in preparation for induction of the diabetes, after induction, on stabilization of diabetes and after the prolonged study collection of blood samples and organs after 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. blood samples were collected by cardiac puncture and used immediately. blood were collected into plain centrifuge tubes and edta bottles. the blood in the centrifuge tubes were centrifuged immediately at 1500 rpm for 15 min to separate of serum at room temperature to avoid haemolysis and used for biochemical assays. blood that was collected into edta bottles were taken for haematological analysis. the livers and kidneys of the diabetic rats were surgically removed, weighed and fixed in 10% formaldehyde for histological process. etuk et al. – biological activities of solanum anomalum 35 hematological study after the animals were sacrificed under diethyl ether anesthesia, blood samples were collected from each rat by cardiac puncture using 21 gauge (21 g) needles mounted on a 5 ml syringe into ethylene diamine tetraacetic acid (edta) – coated sample bottles for analyzed. hematological parameters such as red blood cell count (rbc), hemoglobin, (hb), packed cell volume (pcv), platelet concentration (plc) and total and differential white blood cell count (wbc). these parameters were analyzed using automatic hematological system (sysmex hematology – coagulation system, model mo-1000 i, trans asia, japan). liver function test the following parameters were determined; aspartate transaminase (ast), alanine aminotransferase (alt), total cholesterol, alkaline phosphatase (alp), total and direct bilirubin. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (tietz, 1976) at the chemical pathology department of university of uyo teaching hospital. evaluation of the protective effect of the leaf extract and fractions on biochemical parameters and histology of livers of alloxan-induced diabetic rats serum was separated from the blood of each animal sacrificed and the sera were stored at -20oc until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphotase (alp), direct and total bilirubin. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (reitman and frankel, 1957; tietz, 1976)). the livers of the animals were surgically removed, weighed and a part of each fixed in 10% formaldehyde for histological processes, while the other part was washed with ice cold 0.9% nacl and homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c and the supernatants were used for the assays of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence and burk,1976), and reduced glutathione (gsh) (ellman,1959). malondialdehyde (mda) and gutathione-s-transferase (gst) were measured using commercial diagnostic kits (sigmaaldrich, usa) according to standard manufacturer's protocols. gas chromatography-mass spectrometry (gcms) analysis gc-ms analyses of hexane and dichloromethane fractions were performed using an agilent 7890a gas chromatograph connected with an 5975c msd detector (agilent technologies, usa). 1-2 µl of each fraction was injected to a hp5-ms column (5 % phenylmethylpolysiloxane, 30 m × 0.25 mm × 0.25 µm) and eluted with helium gas under a pressure of 10 psi. the oven temperature gradient was: keeping at 150 °c for 3 min, increasing to 300 °c at 10 °c/min, and keeping at 300 °c for 5 min. electron ionization mode of gc-ms at 70 ev was applied (okokon et al., 2022). the compounds in the hexane and dichloromethane fractions were determined by comparison of spectral data in the nist 2011 database. statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% level of significance i.e. p≤ 0.05. results and discussion effect of leaf extract and fractions on body weights of rats the body weights of the treated and untreated alloxaninduced diabetic rats were observed to have changed considerably within the period of study (table 1). treatment of the diabetic rats with the leaf extract and fractions produced a non – dose dependent increases in the body weight of the diabetic rats with the middle dose (140 mg/kg) having the highest weight increase. these increases which were significant (p<0.05-0.001) when compared to control were highest in the middle dose treated group (10.02%) followed by methanol fractiontreated group (8.77 %). (table 1). effect of extract and fractions on weights of organs treatment of alloxan–induced diabetic rats with leaf extract and fractions of s. anomalum caused prominent decreases in the liver weights of the treated rats though non dose-dependently (table 1). these decreases which were pronounced in groups treated with higher doses of the extract (140 and 210 mg/kg) and n-hexane fraction were only significant (p<0.001) in the group treated with methanol fraction (table 1). antidiabetic activity of the leaf extract and fractions during prolonged treatment the leaf extract produced significant (p<0.05-0.001) reductions in fbg levels of the diabetic rats following repeated treatment. these reductions which were non 36 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 dose dependent were sustained throughout the duration of the study. these effects were comparable to that the standard drug, glibenclamide. on day 14, the effects were 64.08%, 63.43%, 65.43% and 64.54% for 70, 140, 210 mg/kg and glibenclamide respectively (table 1). the various fractions produced sustained significant (p<0.05-0.001) reductions in fbg of the diabetic rats. the effects of the fractions on day 14 were 67.99, 54.66, 66.08 and 66.77% respectively for n-hexane, dichloromethane, ethyl acetate and methanol fractions. the effects exerted by n-hexane, ethyl acetate and methanol were better than that of standard drug, glibenclamide (table 1). effect of leaf extract and fractions on liver function test parameters of diabetic rats. treatment of the diabetic rats with leaf extract and fractions of s. anomalum caused significant (p<0.050.001) reductions in the levels of total bilirubin, alt, alp and ast when compared to control. direct bilirubin level was not affected by treatment with the extract and fractions. although reductions were observed in ast levels of the treated diabetic rats, it was only significant (p< 0.01-0.001) at the highest dose (210 mg/kg) and methanol fraction treated group (table 2). effect of leaf extract and fraction on liver antioxidant enzymes. treatment of alloxan-induced diabetic rats with leaf extract and fractions of s. anomalum caused significant (p<0.05-0.001) dosedependent elevation in the levels of the antioxidant enzymes (sod, cat, gpx) when compared to control. similarly, gsh level was significantly (p<0.001) elevated following treatment with the extract and fractions when compared to control. n-hexane fraction followed by methanol fraction exerted the highest activity. similarly, there were significant (p<0.05-0.01) reductions in the level of mda of the treated rats with ethyl acetate and dcm fractions having the most significant effects. also, significant (p<0.050.01) increases in enzymes and gsh levels as well as significant (p<0.01) reductions in mda level were observed with the standard drug, glibenclamide (table 3). effect of extract and fractions on the histology of livers of diabetic rats histologic sections of livers of untreated diabetic rats revealed numerous hepatocytes with pyknotic nucleus, cellular and vascular degeneration, vascular congestion, hepatocytes hyperplasia and periportal inflammation. livers of diabetic rats treated with glibenclamide (10 mg/kg) revealed reduced pathological signs with normochromic pyknotic nucleus. (figure 1). livers of rats treated with leaf extract (70 – 210 mg/ kg) revealed hyperchromic cellular profile with vascular congestion and numerous pyknotic nucleus. livers of diabetic rats treated with the middle dose of the extract (140 mg/kg) showed no visible cellular abnormality (figure 1). livers of diabetic rats treated with n-hexane, dichloromethane, ethyl acetate and n-butanol fractions (140 mg/kg) revealed normochromic cellular profile containing periportal inflammation and pyknotic nuclei. there was no obvious cellular abnormality in the livers of diabetic rats treated with ethyl acetate fraction of the leaf. it could be considered slightly affected. (figure 1). effect of leaf extract and fractions on haematological indices of diabetic rats administration of the leaf extract and fractions of s. anomalum caused significant (p< 0.05-0.001) increases in the levels of white blood cells, lymphocytes, and eosinophils, which was significant at the middle dose (140 mg/kg) when compared to control. neutrophils, monocytes, basophils and platelets were reduced, while rbc, hb concentration, platelets and pack cell volume were not affected significantly (p>0.05) by the administration of extract and fraction (table 4). gas chromatography-mass spectroscopy (gcms) analysis the phytochemical analysis of the most active fractions (n-hexane and dichloromethane) of s. anomalum revealed the presence of major compounds. the compounds in n-hexane fraction were bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alpha-, squalene and beta.-sitosterol trimethylsilyl ether (table 5). while 3,7,11,15-tetramethyl-2-hexadecen-1ol, squalene and heptacosane were revealed to be present in dcm fraction (table 6). etuk et al. – biological activities of solanum anomalum 37 table 1. effect of leaf extract and fractions of solanum anomalum on body and liver weights of alloxaninduced diabetic rats. treatment dose mg/kg body weight (g) weights of liver (g) fasting blood glucose (mg/dl) day 0 day 15 % increase 0 hr 14th day control normal saline 132.6 ± 18.34 129.3± 20.43 -2.48 6.72±0.98 266.0±17.16 249.0±14.01 glibenclamide 10 132.0 ± 9.45 141.3± 12.33 7.04 6.10±0.15 233.0±12.00 82.6±8.19b extract 70 152.8 ± 15.56 163.0± 9.29 6.67 6.28±0.23 279.3±14.74 100.3±16.69b 140 143.6 ± 12.55 158.0 ± 3.15 10.02 5.80±0.32 260.6±8.32 95.3±11.29 a 210 140.8 ± 13.29 152.6 ± 7.22 8.38 5.82±0.67 257.0±3.71 88.3±8.83b nhexane fraction 140 142.9 ± 8.54 153.6 ± 6.48 7.48 5.82±0.65 264.3±14.50 84.6±40.05c dichloromethane fraction 140 138.4 ± 6.26 144.6± 13.71 4.68 6.14±0.35 236.0±37.60 107.0±14.52b ethyl acetate fraction 140 144.31 ± 8.50 156.6± 9.88 8.51 7.07±0.22 271.3±13.04 92.0±14.29a methanol fraction 140 145.8 ± 7.45 158.6± 10.55 8.77 0.98±0.07c 245.6±12.33 81.6±18.49b data are expressed as mean ± sem, significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). table 2. effect of s. anomalum leaf extract and fractions on the liver function parameters of alloxan-induced diabetic rats. treatment dose (mg/ kg) alt (iu/l) alp (iu/l) ast (iu/l) total bilirubin (µmol/l ) direct bilirubin (µmol/l) control 10 mg/ml 97.00± 4.35 109.53±8.95 172.63±10.68 2.16±0.14 1.60±0.10 crude extract 70 77.90±9.49 92.11± 2.67 162.42± 1.45 2.06± 0.29 1.16±0.03 140 75.10±12.61 70.73±8.67 149.69±15.94 1.80± 0.05 1.10±0.15 210 53.23±2.25c 61.92±8.67b 128.07±18.57a 1.50± 0.05 1.10±0.00 nhexane fraction 140 63.73±3.91b 100.23±9.68 130.48±8.54 1.60±0.00 1.10±0.10 dichloromethane fraction 140 68.86±3.33a 62.92±6.76b 155.42±18.27 1.43±0.20a 1.10±0.05 ethyl acetate fraction 140 70.40±4.19 67.43±5.44b 145.13±15.21 1.86 ± 0.31 1.10±0.11 methanol fraction 140 58.2±5.51c 53.33±6.09c 106.98±0.75c 1.36± 0.08a 1.10±0.10 glibenclamide 10 61.12±10.94b 60.40±6.54b 122.00±4.00b 1.66± 0.12 1.30±0.10 data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). table 3. effect of s. anomalum leaf extract on liver antioxidative stress markers in alloxan-induced diabetic in rats. parameters/ treatment dose mg/kg sod (µg/ml) cat (iu/l) gst (µg/ml) gsh (µg/ml) gpx (µg/ml) mda (µmol/ml) diabetic control 10 ml/kg 0.18±0.03 3.68± 0.28 0.16±0.01 0.71±0.02 0.039±0.001 0.54±0.02 crude extract 70 0.24±0.04a 4.11±0.23 0.21±0.02 0.72±0.03 0.055±0.002 0.52±0.02 140 0.20±0.02 2.95±0.28 0.23±0.01a 0.81±0.02 a 0.056±0.002c 0.48±0.03 210 0.30± 0.02c 3.53±0.17 0.31±0.01c 1.01±0.03c 0.059±0.002c 0.41±0.02c n-hexane fraction 140 0.30±0.02c 5.86± 0.48c 0.23±0.01a 0.98±0.02c 0.057±0.002c 0.44±0.0b dichloromethane fraction 140 0.20 ±0.04 2.87±0.18 0.31±0.01c 0.82±0.01a 0.058±0.002c 0.38±0.01c ethyl acetate fraction 140 0.33 0.02c 4.640.26 0.26±0.01 c 0.92± 0.02c 0.048± 0.002a 0.31± 0.02c methanol fraction 140 0.37 0.03c 4.330.56 0.32±0.01 c 0.87± 0.02c 0.049± 0.002a 0.43± 0.02b glibenclamide 10 0.29±0.02c 5.69±0.18 b 0.25±0.02 b 0.85±0.01c 0.051±0.003b 0.40±0.02c data were expressed as mean  sem. significant at ap< 0.05, bp< 0.01, cp< 0.001 when compared to diabetic control. (n = 6). table 4. effect of solanum anomalum leaf extract and fractions on hematological parameters of alloxan-induced diabetic rats. treatment dose mg/kg wbc (l) neut. (%) lym (%) mono (%) eosino (%) baso (%) rbc (l) hgb (g/dl) pcv (%) platelets. (l) control normal saline 10 9.27± 1.95 59.00±1.60 35.66±1.65 4.66± 0.28 0.26± 0.21 1.00± 0.10 6.00± 0.28 12.13± 0.78 43.76±3.22 940.0± 35.76 crude extract 70 11.60±1.56 51.86± 3.16 45.86±2.06 a 2.96± 0.24 a 0.16 ±0.06c 0.90± 0.05 6.72 ± 0.53 12.63± 0.56 43.20±2.36 709.3± 54.14a 140 15.15±3.34 a 50.40±0.64 45.30±0.88 a 2.63± 0.29 a 0.76 ± 0.12c 1.10± 0.20 7.83± 0.78 14.10± 1.25a 53.76±7.61a 684.6± 92.13a 210 9.86±1.66 57.93 ± 0.60 36.53±4.27 3.76± 0.38 0.66± 0.12c 0.50± 0.00 7.17± 0.18 11.10± 0.68 40.80±1.55 925.6± 61.50 nhexane fraction 140 9.90±1.51 59.50 ± 0.60 34.90±1.44 4.96± 0.03 0.43 ± 0.17b 0.60± 0.26 6.08± 0.69 12.46± 0.08 44.50±0.40 768.0± 82.92 dichloromethane fraction 140 9.32±2.31 48.10±14.13 65.30±5.71c 3.40± 0.58 0.36 ± 0.51 0.90 ± 0.05 6.65 ± 0.90 12.56± 1.07 45.90±7.50 723.0± 35.80 ethyl acetate fraction 140 10.31±1.64 50.66±0.82 43.46±0.73 3.60 ± 1.01 0.30± 0.51 0.50± 0.17 7.28± 0.17 12.13± 1.21 43.90±4.43 775.0±59.53 methanolfraction 140 14.55 ± 0.98a 47.60±6.45a 46.93±6.59 a 4.16± 0.72 0.40± 0.15c 0.53 ± 0.03 6.70 ± 0.63 13.33 ± 0.74 48.90±2.82 867.3±74.71 glibenclamide 10 12.03±3.76 33.16±0.79b 61.56±1.66 c 2.23± 0.28 a 0.06± 0.03c 0.55± 0.03 7.56 ± 0.31 12.66±1.18 47.90±2.82 888.0±47.05 data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control. (n=6). 3 8 b io lo g y , m ed ic in e, & n a tu r a l p r o d u c t c h em istr y 1 2 (1 ), 2 0 2 3 : 3 3 -4 4 etuk et al. – biological activities of solanum anomalum 39 table 5. gcms analysis of column hexane fraction of s. anomalum. peak rt compound name formula mol. mass 1. 10.044 bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alphac19h38o5si4 458.18 2. 18.315 squalene c30h50 410.39 5. 18.326 .beta.-sitosterol trimethylsilyl ether c32h58osi 486.43 table 6. gcms analysis of column dcm fraction of s. anomalum. peak rt (min) compound name formula mol. mass 1. 10.044 cis-pinane c10h18 138.25 2. 10.524 3,7,11,15-tetramethyl-2-hexadecen-1-ol c20h40o 296.1 3. 10.547 1,4-eicosadiene c20h38 278.5 4 18.315 squalene c30h50 410.39 5. 22.836 heptacosane c27h56 380.44 keys: vascular congestion (vc), vascular degeneration (vd), cellular degeneration (cd), periportal inflammation (pi), pyknotic nucleus (pn), red blood cell (rbc), hepatocytes (h), hepatocytes hyperplasia(h). hepatic vein (hv), bile duct (bd), and pyknotic nucleus (pn) figure 1: histological sections of livers of alloxan-induced diabetic rats treated with normal saline 10 ml/kg (1), glibenclamide 10 mg/kg bw (2), leaf extract 70 mg/kg bw (3), leaf extract 140 mg/kg bw (4), leaf extract 210 mg/kg bw (5), n-hexane fraction 140 mg/kg (6) dichloromethane fraction 140 mg/kg (7), ethyl acetate 140 mg/kg (8), methanol fraction 140 m/kg (9), at magnification a(x400), stained with h&e method. discussion the leaf of s. anomalum is use in ethnomedicine for the treatment of various ailments such as malaria, gastrointestinal disorders and diabetes (okokon et al., 2016). this work was designed to evaluate antioxidative and hepatoprotective potentials of leaf extract and fractions of s. anomalum in alloxan-induced diabetic rats. the body weights of diabetic rats were found to increase significantly following treatment with the leaf extract and fractions. diabetes is associated with a severe loss in body weight due to loss or degradation of structural proteins (rajkumar and govindarajulu, 1991). treatment with the leaf extract and fractions remedied this situation perhaps due to the alleviation of hyperglycemic state and stimulation of protein synthesis. 987 6 321 54 40 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 the leaf extract and fractions treatments were observed in this study to cause significant increases in wbc, lymphocytes and eosinophils, while neutrophils, monocytes and basophils were significantly reduced. other parameters were not affected. the increases maybe due to immunological responses by the body defence mechanism to heal or repair the injuries caused by alloxan (soetan et al., 2013) which can be attributed to the immunostimulatory effect of diosgenin (nimbalkar et al., 2018) and squalene. metabolism of alloxan to dialuric acid causes the production of h2o2, •oh and superoxide radical (mathews and leiter, 1999)., which destroys β-cell necrosis and induces diabetes by partial destruction of pancreatic β-cells of islet of langerhans (cakici et al., 1994; abdel-barry et al., 1997). this reduces insulin levels, raise the blood glucose level thereby leading to type 2 diabetes mellitus, but with residual pancreatic βcells with potentials to secrete insulin (sheela and augusti, 1992; subramoniam et al., 1996). in this study, s. anomalum leaf extract/fractions were observed to demonstrate sustained significant antidiabetic activities during prolonged study with the methanol, n-hexane and ethyl acetate fractions exerting prominent activities. the fasting blood glucose (fbg) levels of the treated diabetic rats were significantly reduced when compared to those of untreated diabetic rats (control). the antidiabetic results observed in this study corroborate that of other species of solanum such as s. nigrum (sengottaiyan et al., 2012; umamageswari et al.,2017), s. trilobatum (doss et al., 2009), s. xanthocarpum (selvi and yogananth, 2016) and solanum villosum (nyaga et al., 2019) thus, confirming strongly the antidiabetic potentials of this plant in ethnomedicine. the leaf extract and fractions were observed in this study to cause significant decrease in weight of liver. generally, internal organs weights are considered as important indicator to injury and toxicities (farah et al., 2013). hypertrophy of organs often indicates toxicity and damaged to organ (ping et al., 2013). this often results from oedema due to inflammation of the organs. free radicals generated during alloxan metabolism cause destruction of hepatic, pancreatic and kidney cells and tissues (mathews and leiter, 1999). the decrease in weights of liver by the extract/fractions especially dcm and methanol fractions-treated group, is as a result of protective effect of the fractions against the effect of free radicals generated by alloxan and diabetic condition perhaps due to its hypoglycemic and antioxidant activities of the phytoconstituents (okokon et al., 2019a) such as diosgenin (kanchan et al., 2016), 1-octacosanol and octacosane (leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018 ), squalene (gunes, 2013; micera et al., 2020), β-sitosterol (gupta et al., 2011; baskar et al., 2012) and phenolic compounds in the leaf extract. metabolism of alloxan leads to the generation of free radicals which attack organs and cause oxidative stress which contributes to diabetes complications in animals or humans (baynes and thorpe, 1999). reactive oxygen species (ros) produce cellular and tissue injury through covalent binding, dna strand breaking, lipid peroxidation (lpo) and augment fibrosis which is also implicated in other disease conditions (sirnivasan et al., 2007; paradies et al., 2011). the results of this study show that oxidative stress was duly induced by alloxan in the diabetic rats as reflected in the marked reductions in the levels of sod, cat, gsh and gpx in hepatic tissues as well as significant increase in mda levels in the liver of the diabetic rats. this finding agrees with earlier findings that the activities of these antioxidants are known to reduce during diabetes (parmar and kar,2008; kostolanska et al., 2009; dixit and kar, 2010). the oxidative stress status of the diabetic rats is further supported by marked elevations in the serum levels of ast, alt, alp, and total bilirubin levels of the diabetic rats. reduction in liver levels of these oxidative markers following treatments with the leaf extract and fractions strongly suggest the great potentials of leaf extract and fractions in attenuating oxidative stress associated with type ii diabetes mellitus which was probably mediated via free radical scavenging activities and improving glutathione status in the tissues by its phytochemical constituents such as diosgenin, 1octacosanol, octacosane and β-sitosterol (gupta et al., 2011; baskar et al., 2012;kanchan et al., 2016;leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018). disease conditions such as diabetes affect the liver and changes the hepatic function as shown by increases in the levels of ast, alt, alp, total and direct bilirubin indicates liver damage which are also detected in human diabetes (takaike et al., 2004). damage to liver cells often results in leakage of enzymes such as aspartate aminotransferase (ast), and alanine aminotransferase (alt) into the blood, while blood levels of alkaline phosphatase (alp) rise when the bile flow is slow or blocked (prat and kaplan, 2000). in this study, treatment of the diabetic rats with the leaf extract and fractions was found to cause significant reductions in the levels of alt, alp and total bilirubin. the integrity of hepatocytes is assessed by levels of the serum liver enzyme markers such as ast, alt and alp (moss and henderson, 1999). serum aminotransferases levels (alt and ast) are two of the most useful measures of liver parenchymal cell injury. ast is raised in acute liver damage, but is also present in rbcs, kidney, testis, cardiac and skeletal muscles, so it is not specific to the liver, while alt is almost exclusively found in the liver (nyeblom et al., 2006). elevated levels of ast and alt indicate liver damage but are not good measures of liver function since they do not reliably reflect the synthetic ability of the liver and may come from tissues other than the liver such as muscles. the leaf extract and fractions produced etuk et al. – biological activities of solanum anomalum 41 significant reduced the elevated levels of these enzymes. this further corroborates histologic findings. the increased activities of transaminases, which are active in the absence of insulin due to the availability of amino acids in the blood of diabetics and are also responsible for the increased gluconeogenesis and ketogenesis (udayakumar et al., 2009). diabetes and hyperlipidaemia also cause cell damage by altering the cell membrane architecture, which results in enhanced activities of alp in diabetic rats (udayakumar et al., 2009). this suggests that alloxan-induced diabetes caused lipid peroxide mediated tissue damage in the liver. the increase in the levels of these enzymes in diabetes may be as a result of the leaking out from the tissues and then migrating into the blood stream. the decrease in ast, alt and alp levels in leaf extract and fractions treated groups indicates the protective effect on the liver. total bilirubin level which was increased in the untreated diabetic rats was observed in this study to be significantly reduced in groups treated with the husk extract and fractions. bilirubin, is a metabolic product of hemoglobin which undergoes conjucation with glucuronic acid in hepatocytes to increase its water solubility. determination of bilirubin represent an index for the assessment of hepatic function, severity of necrosis, conjugation and excretory capacity of hepatocytes and any abnormal increase in the level of bilirubin in the serum indicate hepatobililiary disease and severe disturbance of hepatocellular function (martin and friedman, 1992). decrease in serum bilirubin level of the diabetic rats after treatment with the leaf extract and fractions indicated extract/fractions effectiveness to restore normal functional status of the liver. the above activities of the extract/fraction show that the leaves extract and fractions posses hepatoprotective potential against alloxan-induced hepatic injury. these results corroborate histologic findings which show significant hepatoprotective potentials of the extract/fractions. this effect could be due to the antioxidant /free radical scavenging activities of the extract and fractions (okokon et al., 2019a), squalene, β-sitosterol (ivorra et al., 1988; gupta et al., 2011; baskar et al., 2012; micera et al., 2020) as well as the isolated compounds; diosgenin, 1-octacosanol and octacosane (kanchan et al., 2016; leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018). reports have been indicated that persistent hyperglycemia causes increased production of oxidative stress in alloxan-induced diabetes (bonnefont et al., 2000). hence, excessive ros produced leads to oxidative damage and increased lipid peroxidation (lpo). the results of this study showed a significant increase in lpo levels resulting in high level of mda in alloxan-induced diabetic rats liver with accompanying reduction in the levels of oxidative stress markers (sod, cat, gpx and gsh) in the liver. this reduction in the levels of antioxidant enzymes in this study corroborates those of earlier reports (parmar and kar, 2008; kostolanska et al., 2009; dixit and kar, 2010). in in vivo experimental models, tissue oxidative stress markers such as sod, cat and gsh are useful and reliable markers of antioxidant status while mda is a sensitive and reliable marker for lipid peroxidation (kumar et al., 2010; feillet-coudray et al., 1999). sod plays an important role in oxygen defense metabolism by intercepting and reducing superoxide to hydrogen peroxide, which in mammals is readily reduced to water principally by cat and gpx. gpx plays a pivotal role in minimizing the oxidative stress. gpx and gst work together with gsh and decompose h2o2 and other organic hydroperoxides to non-toxic products. glutathione, reduced form gsh is ubiquitous tripeptide thiol, is a vital intra/extra-cellular protective antioxidant against oxidative/nitrosative stress. glutathione reductase is an enzyme responsible for its conversion back to the reduced state (kayali and tarhan, 2006; okutana et al., 2005). the antioxidant enzymes levels were found to be reduced in untreated alloxan-induced diabetic rats. hence, the changes of these biomarkers is in accordance with the decrease in antioxidant state in the body as other reports (ei-missiry and ei-gindy, 2000; meral et al., 2001). in the present study, administration of the extract and fractions significantly counteract the changes of oxidative stress biomarkers in alloxan-induced rats thus preventing the accumulation of excessive oxidative stress with corresponding increases in the levels of antioxidant enzymes/oxidative stress markers. this activity is due to the antioxidant activities of the phytochemicals such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane and other phenolic compounds found to be present in the leaf extract. similarly, β-sitosterol and diosgenin present in the extract/fractions have been reported to cause reduction in hepatic lipid peroxidation and elevation in the activities of catalase, superoxide dismutase and glutathione (gupta et al., 2011b; baskar et al., 2012; kanchan et al., 2016). the liver of the extract/fractions treated diabetic rats were found to be significantly protected from the effects of free radicals generated by alloxan. this protection was visible as the livers of the treated rats lacked or had reduced pathological signs such as vascular degeneration and congestion as well as periportal inflammation observed in the untreated diabetic rats. these findings corroborated that of chemical pathology and therefore suggest hepatoprotective activity against oxidative stress induced by alloxan. the protection is due to the free radical scavenging potentials of the phytoconstituents of the extract and fractions such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane as well as the antioxidant activity of other phenolic compounds present in the extract (okokon et al., 2019a). the hepatoprotective effect of solanum anomalum in this study corroborate that previously 42 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 reported on other species of solanum such as s. nigrum, s. macrocapon and s. xanthocarpum (lin et al., 2008; 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(2022). analysis of ethanol extract of solanum anomalum leaves for antidiarhoeal activity. journal of current biomedical research.2(2):145-157. who expert committee on diabetes mellitus. tech. rep. series no.646.world health organisation, geneva, 1980. biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 1-8 | doi: 10.14421/biomedich.2016.51.1-8 new record marsdenia tenacissima (asclepiadoideae, apocynaceae) in gunung ijo baturagung yogyakarta widodo1 and muhammad ja’far luthfi2 1biology education program, 2biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: wwidodo594@gmail.com abstract marsdenia tenacissima population were found among wild bushes at s 07 o 47’ 03.4”; e 110o 30’ 48.0” about 415 meter above sea level in gunung ijo baturagung yogyakarta. identification was based on literature and herbarium specimen. the research was conduct using exploration methods, morphoanatomical observation, and specimen collection. marsdenia tenacissima in jawa was not reported in flora of java. marsdenia tenacissima habitus was liana. the specific character for its identification was pollinia’s structure. this paper presented other important character namely leaf, stem, flower, pollinia, and fruit of marsdenia tenacissima in gunung ijo baturagung yogyakarta. accurate description and examination of any plant species were needed for its conservation and awareness of public to local biodiversity. keywords: marsdenia tenacissima, asclepiadoideae, gunung ijo, baturagung introduction exploration, observation and assessment of wild plants on gunung ijo baturagung yogyakarta had found lianas (widodo, 415 m, december 31, 2012) among wild bushes (s 07 ° 47 '03.4 "; e 110o 30 '48.0 ". after collection and re-observation on flower, analysis of fruit development and the thoroughly identification, it is conclude that the plant species are marsdenia tenacissima. marsdenia belongs to tribe marsdenieae of the subfamily asclepiadoideae (takhtajan, 2009). according to backer and bakhuizen (1965) there are three marsdenia in java, namely marsdenia tinctoria or pergularia parviflora, marsdenia crocea, and marsdenia stenocentra or marsdenia villosa. hooker (1885) states that the distribution of marsdenia tenacissima include western himalayas, north oudh, bengal, mountains of rajmahal, chittagong, ceylon, also found in the east. it is estimated that this species in java was identified as pergularia crocea, zipp. from the description and bakhuizen backer (1965) above, the existence of marsdenia tenacissima in java was unexplained but hooker (1885: 36) insist that this species is very similar to pergularia crocea, zipp. the plant list (2010) states that marsdenia genus comprises about 447 species. the members of this taxon in this genus often unclear and led to misidentification. observation on the species characters are necessary to provide valid identification of marsdenia species. borlage (1895) listed the marsdenia species in indonesia, namely: (1) marsdenia tinctoria r. br or m. parviflora decaisne or pergularia parviflora bl. or pergularia tinctoria spreng. the plants are distributed all over java, kalimantan, sulawesi, and sumatra, (2) marsdenia tenacissima wight. et arn. or asclepias tenacissima roxb. or gymnema tenacissima spreng. its distribution includes timor and sulawesi, (3) marsdenia villosa bl. or pergularia villosa bl, which has distribution in java, (4) marsdenia teysmanni or tetragonocarpus teysmanni hassk. the plants are distributed in bali and java, (4) marsdenia celebica or chlorochlamys celebica miq, which has distribution in sulawesi, 5) marsdenia crocea hook f. or pergularia crocea zipp. or pergularia tomentosa span, which found in java and timor. backer and bakhuizen (1965), borlage (1895), hooker (1885) conclude that marsdenia species in java is marsdenia tinctoria or pergularia parviflora, marsdenia crocea or pergularia crocea or pergularia tomentosa, marsdenia stenocentra or marsdenia villosa, marsdenia teysmanni or tetragonocarpus teysmanni, and also marsdenia tenacissima or marsdenia celebica. few information about marsdenia tenacissima are available in the literature and internet. this paper describes the characteristics, specimen photos, and clarify the identification of specimens marsdenia tenacissima found in mount ijo baturagung mountains through literature. the re-discovery of the marsdenia tenacissima in batur agung yogyakarta needs to be disseminated to confirm biodiversity richness in java. many wild plants is no longer recognized both the name or its specimen despite information documented in the books of flora and herbarium hundreds of years ago by european explorers. publication of plants species in nature are needed to check and re-check the rediscovery of flora, and to complete the data of world flora. the study would support as raw material for the basis of any applied fields. the research objective is to present a description of the morphological characteristics of leaves, stems, flowers, pollinia, and fruit of marsdenia tenacissima of 2 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 candi ijo mountains baturagung as for identification verification. materials and methods equipments and materials equipment for observation and collection comprises: digital camera sony nex f3, digital cameras sony cyber-shot dsc-w180 digital camera canon dslr, rulers, micrometers, calipers, plastic container, scissors, cutter, label paper , gps (global positioning system), dried herbarium collection equipment, bottles, stereo microscope nikon smz 1500 equipped with a camera, nikon light microscope equipped with nikon eclipse 50 dsf1. materials for observation and collection comprises: aquadest, alcohol 70%, faa solution (formalin acetic alchohol). procedures 1. observe and take macro and microphotograph of specimen under natural conditions at the site, herbarium, and flower detail. 2. preparation of dried herbarium 3. early identification of specimens for members asclepiadaceae based on the book flora of java vol. 2 (backer and bakhuizen, 1965). 4. identify asclepiadaceae specimen based on existing literature, including checking and matching with herbarium type. results and discussion on december 16 exploration, author found gummy vine with fine hair on dorsal surface of the leaves. the authors predict that this plant belong to family apocynaceae and sub family asclepiadoideae. on further observation at the end of december, aditional data on bud and flower were obtained. the location of the plant was on the shrubs at the site of s 07 ° 47 '03.4 "; e 110o 30 '48.0 "at an altitude of 415 m dpl (widodo, 415 m dpl, december 31th 2012). identification using flora of java, vol. 2 (backer and bakhuizen, 1965) concluded that this plant is marsdenia. subequent observations on january 4, january 19, and january 29, 2013 obtained data about the twigs and flowers structure (figure 1 a, b, c, d, e; figure 2a, b, c, d), specimen collection process is carried out in the laboratory for continued observation. on march 21, 2013 author found the young fruit (figure 3a). figure 1. marsdenia tenacissima found by author on gunung ijo. a. habitus in nature. b. flowering twigs. c. flowering buds. d. inflorescences. a b c d widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 3 figure 2. marsdenia tenacissima flower. a. arrangement of inflorescences. b. flower unit, top view. c. parts of fower unit, lateral view. d. blooming flower. e. flower completely bloom. this marsdenia shows characteristics of marsdenia crocea (zipp. ex span.) hook. f. ex boerl as described flora of java, vol. 2 (backer and bakhuizen, 1965) with little difference in the length of the petiole and the color when the crown of flowers in full bloom. long petiole (petiolus) specimens ranging from 3-7 cm, while backer and bakhuizen (1965) describes the length of the petiole is only 3 cm. corolla has yellowish color to the reddishbrown yellow flowers in full bloom after folding along the crown out towards the end of basalt. backer and bakhuizen (1965) described marsdenia crocea as shown in table 1. identification using the book flora of british india (hooker, 1885) and flora of china (1995) showed that the characteristics of stem, leaf, and flower of marsdenia from gunung ijo match to marsdenia tenacissima. description of hooker (1885) and flora of china (1995) on marsdenia tenacissima shown in table 2. the characteristics of marsdenia tenacissima flowers specimens from gunung ijo match with the description of flora of china (1995). table 1. description of backer & bakhuizen (1965) on marsdenia crocea. plants parts description flower, pollinia fre part of corona-scales narrowly ovate, obtuse, reaching to about half the height of the stigma; back of connate part ridge shaped, opened towards the base; translators several times shortter than pollinia; panicles broadly pyramidal, c. 5 cm long; pedicels 5-7 mm; calyx c. 3 mm; segments ovate-elliptic, obtuse; corolla c. 6 mm, yellowish white, outside more densely hairy in the upper half than the lower; tube on the side with basal tufts on long hairs, c. 3,5 mm long; segments glabrous inside, with a thickened rim at the base (in the throat), c. 2,5 mm long; gynostegium subequalling the corolla-tube; stigma broadly conical, obtuse. leaves leaves cordate, shortly acuminate, thinly grey-pubescent above, more densely so above, palmatinerved, with distint lateral nerves, finely reticulate-veined, 5-10 cm by c. 6 cm; petiole c. 3 cm. information very long ago collected in east java a corolla tube spreading lobes calyx pedicel b d e c 4 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 table 2. flora of china (1995) and hooker description (1885) on marsdenia tenacissima. flora of china (1995) plants part description habitus flower pollinia information lianas robust, densely pilose to tomentose throughout except for interior of corolla. petiole 5–6 cm, slender; leaf blade ovate, 8–10 × 6–6.5 cm, base deeply cordate with rounded sinus, apex acuminate; basal veins 5–7, lateral veins 2 or 3 pairs. inflorescences much branched, broader than long, to 8 × 12 cm, many flowered; peduncle to 2 cm, shorter than first internode. pedicel 6–8 mm. sepals elliptic-lanceolate, ca. 3 × 1–1.3 mm, tip rounded. corolla “yellow,” campanulate, with spreading lobes, very densely pilose outside; tube ca. 3.5 × 2.5 mm, interior retrorsely pilose toward base; lobes oblong, ca. 4 × 2.2 mm, apex rounded, minutely velvety. corona lobes exserted from corolla tube, oblong, apex truncate-emarginate with corners produced into short horns, sometimes toothed between these. anther appendages oblong, slightly longer than corona lobes; pollinia curved cylindric. stigma head broadly cylindric, concealed by anther appendages. cliffs; 1500 m. yunnan (szemoo) [cambodia, india, laos, myanmar, nepal, sri lanka, thailand, vietnam]. the stems yield very strong fibers, reputedly among the strongest produced by any plant, that are used for making cords and strings. hooker (1885) stem leaves flower fruit stem very stout. leaves 4-7 by 3-5 in., often valvaty above; petiole 2-3 in. cymes much corymbosely branched. corrolla 0,25 in. diam., lobus oblong, ciliate. stigma beetwen conical and dome shaped. follicles 5-6 in. long by 1,5-2 in. diam., lanceolate; pericarp very thick, longitudinally wrinkled, finely pubescent. seed ovate-oblong. 0,5 in long. pergularia crocea, zipp. figures 3a, b, c, de, show marsdenia tenacissima fruits of gunung ijo. a collection of young fruit and ripe fruit were made by the author on march 21, 2013 and august 26, 2015. figure 3e, f, is sketch of marsdenia tenacissima fruit (flora of china, 1995). the characters of fruit and seed of marsdenia tenacissima of gunung ijo match with the illustration marsdenia tenacissima in flora of china (1995), especially young fruit structure and seeds form. figure 3. fruit and seed of marsdenia tenacissima from gunung ijo (a, b, c, d), and illustration on flora of china (1995) (e, f). a. young fruit. b. mature fruit. c. mature fruit and broken fruit. d. seed. e. fruit illustration. f. seed illustration. a b c d fe widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 5 figure 4a shows a comparison of the inflorescence herbarium from gunung ijo and from hooker (figure 4d) in 1863 which is collected from bengal (mnhn p03522167), and 4e herbarium kew (k001129150). figure 4b, 4c is a vegetative branches and fruit herbarium from gunung ijo, while figure 4f fruiting twig herbarium of kew herbarium (k001129153). figure 4. marsdenia tenacissima herbarium from gunung ijo compared to kew (a, b, c) & mnhn herbarium collection (d, e, f). comparison of herbarium specimens to the herbarium collections and skew mnhn in figure 4 shows the similarity in the characteristic of twigs, leaves, inflorescence structure and the size of the fruit. similarity of characteristic leaf are as follows: the heart-shaped, rounded and deep sinus at the base of the heart shape, softer hairy of adaxial surface, brighter color of the abaxial surface. the similarity of the structure of a inflorescence is cymes much corymbosely branched. the similarity of the size of the fruit is the size range of about 5-7 cm long with a width of 0.8 to 1.5 cm. based upon the data in the field, tenacissima marsdenia leaf size varies with length of 5-12 cm and 5-9 cm wide in a single individual. it should be noted that the qualitative characteristics of the leaves become an important marker in the identification of this species. the similarity of the structure of the leaves, inflorescence, fruit size specimens of gunung ijo (figure 4a, b, c) can be compared with marsdenia tenacissima in herbarium collections bogor (bo1646031, bo1646032, bo1646038, bo? ex herb. koordersianum no. 16214) in figure 5. structure of gunung ijo spesimen is similar to figure 5d. this herbarium was from sulawesi and was identified as marsdenia velutina. a b c d e f 6 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 figure 5. marsdenia tenacissima in herbarium bogoriennse. there is a significant difference between fruit size of specimen from gunung ijo (range 5-6 cm long, 0.8 to 1.3 cm wide) with size of the fruit marsdenia tenacissima from hooker (1885), which is 5-6 inches long and 1-1.5 inches wide. i suspect that this is an error in writing the unit. identification of species asclepiadaceae (asclepiadoideae) on the basis of morphological characteristics vegetative structures still confusing because the variation between species in the subfamily is quite high, as well as reproductive structures (flowers and fruit). plant identification is very difficult to do in a group of plants with a number of cultivars and hybrids are large and unknown (simpson, 2006). therefore, it is necessary to rely the identification on the traits that have a strong character. shape or structure of the pollinia is an important character to distinguish species in asclepiadoideae (sreenath, et al., 2012; sinha & mondal 2011). characteristic of pollinia of asclepiadoidea species had been described by experts since hundreds years ago, but visualization using pictures were very rare. structure of gynostegium (unification pistil with the anther) are shown in figure 6a, 6b, and the pollinia marsdenia tenacissima of gunung ijo are shown in figure 6d, 6e. the structure pollinia of marsdenia tenacissima of gunung ijo are shown in thats figure. the pollinia match to the description of marsdenia crocera pollinia (backer & bakhuizan (1965) and a description of the flora of china (1995) (table 2) as well as illustrations of marsdenia tenacissima gynostegium and pollinia (flora of china, 1995) in figure 6c, 6f. figure 6. gynostegium and pollinia of marsdenia tenacissima. from the description and analysis above, it is conclude that the specimen of marsdenia from gunung ijo is marsdenia tenacissima. description hooker (1885) about the existence of marsdenia tenacissima (identified by miquel as pergularia crocea) in java can be justified. marsdenia crocea described by backer and bakhuizen (1965) as the correction for pergularia crocea is basically marsdenia tenacissima. herbarium bo1646038 marsdenia tenacissima support the distribution of this species in java. backer bo1646038 herbarium is a backer’s collection from kangean islands in 1919 and identified by hatusima in 1945 as marsdenia tenacissima. the similarity of character between specimen from gunung ijo and bo1646031, bo1646032, bo? ex herb. koordersianum no. 16214 shows that marsdenia tenacissima also found in sulawesi, although initially identified as marsdenia velutina. in addition to using herbarium, species descriptions and determination keys, identification of plant species need an alternative method by utilizing the digital color image files. baskauf and kirchof (2008) has developed a standard photo living plants as digital plant specimens. error, kucuker and sik (2009) have developed an illustrations technique combined with a virtual observation and herbarium specimens for species identification and determination. improvement and completeness of illustration data in the document and books of flora are critical to improve the works in current and future taxonomy. colored digital image can be used to check and re-check identification. a b c d a b c d e f widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 7 taxonomic treatment marsdenia tenacissima (roxburgh) moon, cat. pl. ceylon 21. 1824; marsdenia tenacissima wight & arn., fl. brithis ind. iv: 35; marsdenia crocea (zipp. ex span.) hook. f. ex boerl.; type: benggala, 1863, w. hooker (mnhn p03522167) lianas robust, stem very stout densely pilose to tomentose. petiole 3–6 cm, slender; leaf blade ovatecordate, 4–10 × 6–6.5 cm, base deeply cordate with rounded sinus, apex shortly acuminate; thinly greypubescent above, more densely so above, palmatinerved, with distint lateral nerves, finely reticulate-veined, basal veins 5–7, lateral veins 2 or 3 pairs. inflorescences much branched, cymes much corymbosely branched broader than long, to 8 × 12 cm, many flowered; peduncle to 2 cm, shorter than first internode. pedicel 6–8 mm. sepals elliptic-lanceolate, ovate, ca. 3 × 1–1.3 mm, tip rounded, corolla “yellow,” campanulate, with spreading lobes, very densely pilose outside, 8 mm diam., lobus oblong ; tube ca. 3.5 × 2.5 mm, interior retrorsely pilose toward base; lobes oblong, ca. 4 × 2.2 mm, apex rounded, minutely velvety. corona lobes exserted from corolla tube, oblong, apex truncate-emarginate with corners produced into short horns, sometimes toothed between these. anther appendages oblong, slightly longer than corona lobes; pollinia curved cylindric, translators several times shortter than pollinia. gynostegium subequalling the corolla-tube stigma head broadly cylindric, beetwen conical and dome shaped, concealed by anther appendages. follicles 5-6 cm. long by 1,5-2 cm. diam., lanceolate; pericarp very thick, longitudinally wrinkled, finely pubescent. seed ovate-oblong. 0,5-0,7 mm long. specimens examined: baturagung, gunung ijo, s 07 o 47’ 03.4”; e 110o 30’ 48.0”, widodo, 415 m dpl, 31.12.2012 note: marsdenia tenacissima found at the site of observation twining other plant namely annona muricata, clausena indica, zizipus oenoplia, lantana camara alongside with other lianas namely telosma puberula asclepiadoideae, and gymnema sylvestris. m. tenacissima was found by author while observe telosma puberula and gymnema sylvestris. m. tenacissima is not mount parangan forest about 5 km southeast of gunung ijo. marsdenia tenacissima known as a traditional treatment for swelling, asthma and cancer in society of southwestern chinese (li et al., 2006). tripathi, et al. (2014) analyzed farmakognosi of marsdenia tenacissima include of character macroscopic, microscopic characters, physicochemical analysis, preliminary analysis of phytochemical content, and hptlc profile. root of marsdenia tenacissima used for traditional medicine: antiscorbutic, urinary diseases, arthritis, heart disease, skin disease, thirst, pruritus, vomiting, intermittent fever and fever. marsdenia tenacissima in kerala india called murva by local people. murva root used as medicinal plant in the form of a single herb or mixed with other materials. concervation because the limited number of this plant at the site of the discovery and other locations, it is necessary to study their distribution status. simultaneously herbarium collection need to be carried on preserve the specimen. conclusion marsdenia tenacissima (roxburgh) moon found on gunung ijo mountains baturagung yogyakarta. morphological characteristics stature (habitus), leaves, and flowers specimen marsdenia tenacissima in gunung ijo show compliance with the kew herbarium collection hooker in 1863 (mnhn p03522167) came from bengal and herbarium collections bogoriense backer 1919 (bo1646038) derived from kangean islands. the existence of marsdenia tenacissima (roxburgh) moon in java correct and acomplish description of backer and bakhuizen in the flora of java book on marsdenia genus. acknowledgements the author would like to thank to the herbarium museum national d 'histoire naturelle, paris (mnhn) and kewensis herbarium royal botanic garden edinburgh (kew) on herbarium type photo credit. thanks also to mr. deden girmansyah and mr. arif hidayat in the herbarium bogoriense for the opportunity to checking herbarium specimen. references backer, c. a. & bakhuizen. 1965. flora of jawa (spermatophytes only) (vol ii). groningen: n. v. p. noordhoff. baskauf s j, kirchoff bk. 2008. digital plant image as speciments: toward standards for photographing living plants. vulpia, 7:16-30. borlage, j. g. 1899. handeiding flora van nederlandsch indie. leiden, e.j brill. dwari, s., mondal, a. k. 2011. systematic studies (morphology, anatomy and palynology) of economically viable grass brachiaria mutica (forsskil) stapf in eastern india. african journal of plant science, 5 (5): 296-304. eror o, kucuker o, sik l. 2009. application of a new illustration technique in plant systematics: composite images of two autumn flowering crocus l. 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(2006). acetonyltenacissoside f acetone solvate: a new 8 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 polyoxypregnane glycoside from the stems of marsdenia tenacissima. acta cryst, 62, 5255–5256. royal botanic garden, kew. 2015. marsdenia tenacissima. http://specimens.kew.org/herbarium/k001129150. diakses 10 oktober 2015 http://specimens.kew.org/herbarium/k001129153. diakses 10 oktober 2015 simpson, m.g. 2006. plant systematics. amsterdam: elsevier academic press. sreenath, k. p., ramakrishna, t.m., babu, t. p. 2012. prespective on pollinial apparatus and carriers of asclepiadaceae sensu lato. global journal of bio-science and biotechnology, 1 (1): 45-53. takhtajan, a. (2009). flowering plant. st petersburg: springer. the plant list (2010). version 1. published on the internet; http://www.theplantlist.org/tpl/search?q=marsdenia+. diakses 2 oktober 2015. tripathi m, shivhare d, tiwari a, ahirwar p and pathak s. 2014. pharmacognostical evaluation of marsdenia tenacissima wight. & arn. root. int. j. rec. biotech., 2 (3): 18-23. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 267-271 | doi: 10.14421/biomedich.2023.121.267-271 issn 2540-9328 (online) effect of female age on crossing over frequency in drosophila melanogaster crosses n♂><bcl♀ and n♂><ym♀ and their reciprocals lisa savitri*, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract crossing over is the occurrence of disconnection and reconnection followed by a reciprocal exchange between the two chromatids in a bivalent form. the crossing event will produce parental type and recombinant type. in the event of crossing over, various factors can influence it. these factors can be due to internal and external. recently, various factors have been reported that influence the incidence of crossing over. these factors include age, temperature, radiation, and changes in chromosome structure. this research is a type of experimental research that uses a randomized block design. randomized block design by crossing d. melanogaster strains ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals. from the results of this cross (f1) then cross again ♀n with the recessive male (from stock) then observe the phenotype of the offspring (f2) and calculate the results of the offspring. the f2 ♀n crosses were treated with age variations, namely 0, 3, 6, 9, 12, 15, and 18 days. based on the results of these crosses, the derived strains that appeared in the f2 cr osses showed the phenomenon of crossing over with the influence of the age of the female and the type of strain on crossing events. the frequency or value of crossing over (formation of recombinants) decreased with the increasing age of the female. if the age of the female affects the frequency of crossing over, then the older the female, the more likely the frequency of crossing over will decrease. however, beca use the data obtained were incomplete, it was not possible to know the effect of female age on the frequency of crossing over of d. melanogaster crosses ♀n>< ♂ bcl and ♀n >< ♂ym and their reciprocals. the condition for crossing over is the formation of a synaptonemal complex. age of d. melanogaster females has an effect on the frequency of crossing over in crosses n♂ >< bcl♀, n♂ >< ym♀, and their reciprocals. the older drosophila melanogaster is, the lower the frequency of crossing over will occur. based on this, it was necessary to cross d. melanogaster with strains n, bcl, and ym. a cross consists of ♂n ><♀bcl and ♂n ><♀ym and their reciprocals. by crossing ♀n with a recessive male from the stock, then observing the f2 phenotype, it is hoped that crossing over will occur. so that you can better understand by doing the practice directly. in this case, the effect of crossing over is seen from the age of the female and the type of strain. keywords: crossing over frequency; drosophila melanogaster; female age; strain ♂n>< ♀bcl; strain ♂n>< ♀ym. abbreviations: adenosine triphosphate (atp), transverse filaments (tfs) introduction in life, all organisms try to maintain the continuity of their generations and to maintain the continuity of these generations, a reproduction process is needed. there are two types of reproduction, namely sexual and asexual reproduction. in sexual reproduction, after the meeting and fusion between the egg and sperm, a zygote is formed. in the process of development or what is called the gametogenesis process, cell division processes occur, initially one cell divides into two, then four, and so on. the process of cell division involves two events namely meiosis and mitosis. meiosis is a cell division event that produces gametes or sex cells and has half the number of chromosomes of each of the parents. meanwhile, mitosis is the division of a somatic cell into two cells containing an identical number of chromosomes. meiosis events are divided into 2, namely meiosis 1 and meiosis 2. in meiosis 1 prophase i, paired chromosomes often show a crossed configuration. as a result, crossing over can occur and produce offspring that are different from their parents. crossing over is the occurrence of disconnection and reconnection followed by a reciprocal exchange between the two chromatids in a bivalent form (corebima, 1997). crossing over was first proposed by t. h. morgan to explain the occurrence of recombination of factors which were concluded to be mutually linked based on genetic data (gardner, et al. 1984 in corebima 1997). the crossing event will produce parental type and recombinant type (corebima, 1997). in the event of manuscript received: 10 february, 2023. revision accepted: 11 march, 2023. published: 21 march, 2023. https://doi.org/10.14421/biomedich.2023.121.267-271 268 biology, medicine, & natural product chemistry 12 (1), 2023: 267-271 crossing over, various factors can influence it. these factors can be due to internal and external. recently, various factors have been reported that influence the incidence of crossing over. rothwell (1983) in corebima (1997) mentions these factors include age, temperature, radiation, and changes in chromosome structure. in addition, suryo (1996) stated that one of the influential factors in crossing over was age. where the older the age of the female, the less crossing that occurs. based on this, it was necessary to cross d. melanogaster with strains n, bcl, and ym. a cross consists of ♂n ><♀bcl and ♂n ><♀ym and their reciprocals. by crossing ♀n with a recessive male from the stock, then observing the f2 phenotype, it is hoped that crossing over will occur. so that you can better understand by doing the practice directly. in this case, the effect of crossing over is seen from the age of the female and the type of strain. materials and methods study area this research is a type of experimental research that uses a randomized block design. randomized block design by crossing d. melanogaster strains ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals. from the results of this cross (f1) then cross again ♀n with the recessive male (from stock) then observe the phenotype of the offspring (f2) and calculate the results of the offspring. the f2 ♀n crosses were treated with age variations, namely 0, 3, 6, 9, 12, 15, and 18 days. based on the results of these crosses, the derived strains that appeared in the f2 crosses showed the phenomenon of crossing over with the influence of the age of the female and the type of strain on crossing events. procedures medium creation 1. consider the medium ingredients, namely rajamala banana, cassava tape, and brown sugar with a ratio of 7:2:1. 2. cut the rajamala banana into several parts and put it in the blender along with the cassava tape and add enough water. 3. carry out the blending process until the tape and bananas become smooth/soft and mixed. 4. heat the brown sugar by adding a little water until the brown sugar dissolves in the water. 5. cook mashed tape and bananas using a saucepan for 45 minutes over medium heat by adding water so that the medium is not too thick. 6. when it starts to boil, put the brown sugar water that has been boiled into the pot and keep stirring it so it doesn't burn. 7. evaporate the jam jar and cork over a medium saucepan, and fill the jam jar with the medium until about a quarter of the jar. 8. close the bottle using a sponge that has been cut according to the diameter of the bottle. 9. waiting for the bottle to cool by immersing the bottom of the bottle in cold water. 10. after the medium is cold, add approximately 4 yeast grains into the bottle containing the medium and put the pupation paper into the bottle, and close it again. stock renewal 1. prepare a bottle of jam containing the medium that has just been heated, and after it's cold, add about 4 grains of yeast and put the pupation paper in it. 2. transferring at least 3 pairs of flies from each strain taken from the old medium and then transferring them to the new medium that has been prepared. 3. label according to the type of strain and date of rejuvenation. 4. observe its development and if there are already blackened pupae, immediately collect them stock collection 1. take carefully the black pupa in the bottle using a brush so that the pupa is not damaged 2. insert the pupa into a short hose (± 6-7 cm) which has been given a banana slice in the middle of the hose. 3. closing the hose by using a small piece of sponge at each end 4. waiting for the pupae to hatch (usually a day or two after being pulled) 5. after hatching, cross ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocal (f1 cross) 6. for recessive males to be used in f2 crosses, the maximum age of male flies that can be crossed is 2 days. f1 crosses (♂n>< ♀bcl and ♂n>< ♀ym, and their reciprocals) 1. collect black pupae of n, bcl, and ym strains, and then after hatching cross ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals 2. after 2 days of crossing, remove the male and let the female remain in the bottle until larvae appear. 3. after the larvae appear, move the female at least 3 times (bottles a-d) 4. gathers a black pupa from the cross for its f2 cross. 5. look for tillers with strain ♀n from each cross (♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals) and give them age treatments. preparing female parent stock 1. transferring strain n female flies from each cross that has been ampoule into a bottle containing medium. 2. each strain is put in a different bottle to make it easier for the practitioner to cross. 3. label each ampoule bottle with the date and strain. 4. waiting for the females of each strain until they are 0 days, 3 days, 6 days, 9 days, 12 days, 15 days, and 18 days. savitri et al. – effect of female age on crossing over frequency 269 f2 cross 1. collect black pupae from each stock strain and wait for them to hatch (for recessive males) 2. crossing ♀n from stock ♀n that has been prepared according to age treatment with recessive male p1 3. after two days, the male is released and the female is left in the bottle 4. after the larvae appear, move the female to the second bottle. minimum of 3 female transfers (bottles a-d) 5. observing the phenotype and counting the number of tillers emerging from each bottle for 7 days. data analysis the data obtained were analyzed in the following way: 1. reconstruct the body's chromosomes 2. make an observation table f2 3. calculating the percentage of crossing over with the formula: frequency of recombinant-type derivatives (%) ∑𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡 ∑𝑝𝑎𝑟𝑒𝑛𝑡𝑎𝑙 + ∑𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡 × 100% results and discussion result because the observational data obtained did not allow statistical calculations to be carried out, the data analysis was carried out using descriptive analysis in the form of calculating the frequency of crossing over at each cross. cross d. melanogaster strain ♀ n (from f1 bcl ♀ >< n ♂) >< bcl ♂ recessive 1. age ♀ 0 days, cross over frequency = 22,69% 2. age ♀ 6 days, cross over frequency = 13,43% 3. age ♀ 9 days, cross over frequency = 11,11% cross d. melanogaster strain ♀ n (from f1 n ♂ >< bcl ♀) >< bcl ♂ recessive 1. age ♀ 0 days, cross over frequency = 25,51% 2. age ♀ 3 days, cross over frequency = 24,93% 3. age ♀ 6 days, cross over frequency = 24,93% 4. age ♀ 9 days, cross over frequency = 15,45% cross d. melanogaster strain ♀ n (from f1 n ♂ >< ym ♀) >< ym ♂ recessive 1. age ♀ 6 days, cross over frequency = 20,18% discussion according to suryo (1996), the possibility of crossing over is influenced by several factors, one of which is the age factor, where the older the female, the lower the frequency of crossing over. whittinghill and hinton (1950) also stated the same thing that the frequency or value of crossing over (formation of recombinants) decreased with the increasing age of the female. if the age of the female affects the frequency of crossing over, then the older the female, the more likely the frequency of crossing over will decrease. however, because the data obtained were incomplete, it was not possible to know the effect of female age on the frequency of crossing over of d. melanogaster crosses ♀n>< ♂ bcl and ♀n >< ♂ym and their reciprocals. the condition for crossing over is the formation of a synaptonemal complex. the synaptonemal complex is a protein needed to carry out the correct pairing of homologous chromosomes (campbell, 2000). the synaptonemal complex is a protein complex that appears to mediate synapses during the zygotene stage and then disintegrates. this complex brings together homologous chromosomes during the prophase of meiosis. according to gardner (1991) crossing over is an enzymatic reaction like other enzymatic reactions. the older the fly, the body's metabolism will decrease. the decrease in body metabolism affects the formation of adenosine triphosphate (atp) in the body which is a source of cellular energy that plays a role in cell activities including protein synthesis. if the process of protein synthesis decreases, the amount of protein including the synaptonemal complex and enzymes that play a role in crossing over decreases so that crossing events decrease. according to ashburner (1989) under normal conditions, d. melanogaster can go through its entire life cycle for 30-32 days, and the time it takes to become an adult is ± 10 days from the time the eggs are released. so that the life span of imago can be known, which is around 20-22 days. the life span of the imago can be grouped into three ranges, namely young, adult, and old. the ages of d. melanogaster, namely 1 to 6 days are classified in the young age range, 7 to 14 days old are classified in the adult age range, and ages 15 to 20 are classified in the old age range. in the observations that have been made, crossing over is characterized by the emergence of non-parental or recombinant strains in the crosses. based on the results of the data obtained, namely in crosses bcl♂ >< n♀ in the treatment of females aged 0 days, the percentage of crossing over was obtained, namely 22.69%, in the treatment of females aged 6 days, the percentage of crossing over was obtained by 13.44%, and in the treatment of females aged 9 days, the percentage of crossover frequency was 11.11%. the results of the cross n♂ >< bcl♀ in the treatment of females aged 0 days obtained the percentage of crossing over frequency which was 25.52%, in the treatment of females aged 3 days the percentage of crossing over was obtained in the amount of 24.93%, and in the treatment of females aged 9 days, the percentage of crossover frequency was 15.45%. the decreasing percentage of crossing over frequency of females aged 0 days, 3 days, 6 days, and 9 days indicates that there is a concordance between the practicum results and theory, that is, the older the females are, the lower the frequency of crossing over 270 biology, medicine, & natural product chemistry 12 (1), 2023: 267-271 occurs. the results of crosses ym♂ >< n♀ in the treatment of 6 days old females obtained the percentage of crossing over frequency which was 20.18%. however, the results obtained in this practicum were inaccurate because the data obtained were still incomplete, namely in crosses bcl♂ >< n♀ treated females aged 3 days, 12 days, 15 days, and 18 days, whereas n♂ >< bcl♀ treatments females aged 6 days, 15 days, and 18 days, and cross data ym♂ >< n♀ for the treatment of females aged 0 days, 3 days, 9 days, 12 days, 15 days, and 18 days. crossing over occurs during the synapsis of homologous chromosomes in the pachytene stage of prophase i of meiosis. paired chromosomes at meiosis often display a crossed configuration. when the chromosomes are about to separate, the crossed chromatids attach and break off at the chiasma, then each piece is attached to the adjacent chromatid (its homolog). when the first homologous chromosomes appear as pairs during prophase i, a set of proteins called synapses joins the chromosomes together so that they are tight to one another. apart from the synaptonemal complex, another structure responsible for crossing over is the recombination nodule. recombination nodules are temporary structures that exist only in the middle of the pachytene stage associated with the synaptonemal complex, thus crossing over is expected to occur at that time limit (carpenter, 1975). genes c(3)g and c(2)m are components of the synaptonemal complex. the c(3)g gene encodes the formation of transverse filaments (tfs) (figure 1). according to page and hawley (2001), tfs are filaments that make up the synaptonemal complex in the form of coils that are in the middle of the synaptinema complex formation. the synthesis of tf will trigger the formation of a synaptinema complex between two homologous chromosomes. figure 1. a simplified schematic of the synaptonemal complex. before the synaptonemal complex is formed, the recombinant complex is composed of double-stranded dna that unravels and helps catalyze crossing-over between non-sister chromatids from opposite sides of the complex. source: albert, et al. (2008). it is well known that the existence of a synaptonemal complex is needed in crossing over. in addition to these genes, according to mckim and hayashi-hagihara (2003), the mei-w68 gene is needed in the initiation of meiotic recombination. the mei-w68 gene encodes the mei-w68 protein which is a type of topoisomerase ii protein. this protein is required in the event of double helix breaking during meiosis. based on this theory, it can be concluded that there are certain enzymes that play an important role in the process of recombination. these enzymes have a role in the process of exchanging chromosome segments on homologous chromosomes that have formed pairs or are called synapses at the prophase stage which are assisted by complex proteins and synaptonemal. this complex holds homologous chromosomes together during the prophase of meiosis. so that the age of the female can be said to affect the frequency of crossing over with respect to the decrease in the working process of enzymes and body metabolism along with the increasing age of d. melanogaster. as it gets older, the body's metabolism in d. melanogaster will also decrease, so that the production of atp as a source of energy in the body will also decrease which also results in a decrease in the work function of enzymes that play a role in the recombination process and the amount of its production, resulting in a decrease in the frequency of crossing over. conversely, if the age of the female does not affect the frequency of crossing over, this is possible because the increasing age of d. melanogaster does not affect the process of atp formation in the body, so there is no decrease in the production of enzymes that play a role in the process of crossing over. finally, the recombination process still occurs and there is no difference in the frequency of crossing over in each treatment of the difference in the age of the females. in addition, another possible reason for the age of females not affecting the frequency of crossing over is the presence of a dna ligase enzyme whose job is to repair the damage that is formed so that there is no exchange of genetic material which will result in crossing over, and the number of these genes varies in each individual. gene repair also affects the magnitude of the frequency of crossing over, namely the genes that undergo recombination have the ability to repair themselves so that the exchange of genetic material does not occur. these things resulted in no significant differences in the frequency of crossing over in various treatments of female age. according to subramanian and bickel (2008), there is another mechanism, namely through the chiasmata pathway so that segregation still occurs properly (figure 2). the chiasmata pathway in drosophila oocytes ensures accurate segregation of recombinant chromosomes that have lost chiasmata as a result of the weakening of the cohesive power of the chromosomal arms during the aging process. cohesin protein has an important role in maintaining the heterochromatin pair of achiasmata savitri et al. – effect of female age on crossing over frequency 271 homolog pairs in drosophila oocytes and as a "glue" to hold sister chromatids together. figure 2. the achiasmata segregation system (shown by green stars between homologous chromosomes) allows for accurate segregation of recombinants that previously lost their chiasmata during aging (top figure). when the achiasmata pathway is dysfunctional (indicated by loss of green stars), recombinants that have lost chiasmata will experience inaccurate segregation (figure below). source: subramanian and bickel (2008). conclusions age of d. melanogaster females has an effect on the frequency of crossing over in crosses n♂ >< bcl♀, n♂ >< ym♀, and their reciprocals. the older d. melanogaster is, the lower the frequency of crossing over will occur. acknowledgments: acknowledgments are expressed in a brief; all sources of institutional, private, and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study. lisa savitri and elfred rinaldo kasimo analyzed the data. lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references ashburner, michael. (1989). drosophila, a laboratory handbook. usa : coldspring harbor laboratory press. borror. (1992). pengenalan serangga. edisi keenam. jogjakarta: ugm press. campbell, neil a. 2000. biologi edisi kelima edisi i. jakarta: erlangga borror. carpenter, adelaide t. c. 1975. synaptonemal complex and recombination nodules in wild type drosophila melanogaster females, (online). (http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1213973/, diakses tanggal 15 november 2014) corebima, a.d. 1997. genetika mendel. surabaya: airlangga university press. gardner, eldon j. 1991. principles of genetics. new york : john wiley & sons inc. mckim k.s dan manheim, e.a. 2003. the synaptonemal complex component c(2)m regulates meiotic crossing over in drosophila. elsevier science ltd. elsevier science ltd. subramanian, v.v. dan bickel, s.e. 2008. aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit smc1 is reduced. plos genet 4(11):e1000263 suryo. 1996. genetika. departemen pendidikan dan kebudayaan direktorat jenderal pendidikan tinggi proyek pendidikan profesi guru. whittinghill, m dan hinton, claude w. 1950. the nature of linkage variation with age in inversion heterozygotes of drosophila melanogaster (online).(www.ncbi.nlm.nih.gov/pmc/articles/pmc1063240/pd f/pnas01559-0034.pdf, diakses 20 november 2011). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 335-341 | doi: 10.14421/biomedich.2023.121.335-341 issn 2540-9328 (online) el hadji gorgui diouf1,*, doudou diop2, abdoulaye gueye1, mbaye diouf1, talibouya ndior1, mamadou latyr ndour1, adama faye1, mamadou kébé1 1laboratory of natural products, department of chemistry, faculty of science and technology; cheikh anta diop university of dakar, senegal. 2botany laboratory, fundamental institute of black africa, cheikh anta diop university of dakar, senegal. corresponding author* goorgui82@yahoo.fr manuscript received: 22 march, 2023. revision accepted: 28 march, 2023. published: 02 april, 2023. abstract crop losses in vegetable crops due to nematodes are a concern in a country like senegal where food demand is increasingly high. the use of chemical pesticides has made great progress in increasing harvests in senegal. but these pesticides have negative eff ects on the environment and on human health. crops are often contaminated as well as groundwater. the use of natural products to remedy t his phenomenon is very important, especially in areas such as the niayes zone where the climate and soil are very favorable for market gardening and where water contamination is very sensitive. the aim of our study is to identify the plants used to control nematodes in the niayes area of thiès. to this end, we conducted an ethnobotanical survey of 100 people of different ages and sexes in this area. the results showed 17 plant species used to control nematodes in 12 families. among these species, the most cited were azadirachta indica a. juss. juss, calotropis procera (ait.) f. and cassia occidentalis l. leaves (71%) are mostly used. the sample is often dried and ground (45%) or fresh (case of hydrodistillation) (53%) before preparation. water (92%) is the most used solvent for extraction. mac eration (85%) and infusion are the most common preparation methods. the application of these preparations by systemic action (94%) is more adopted than fumigation. local people find these preparations rather effective (78%), which is very encouraging. the present study constitutes a database for further studies in the field of senegalese pharmacopeia. in addition, scientific research on bionematicidal substances may be conducted in the future to evaluate the effectiveness of these plants identified in the niayes area of thiè s for the protection of vegetable crops against nematodes. keywords: ethnobotanical survey; senegalese pharmacopoeia; nematicidal plants; market gardening; niayes area; niayes area of thiès. abbreviations: ucad: université cheikh anta diop de dakar; ifan: institut fondamental d'afrique noire. introduction agriculture is a major industry that contributes to socioeconomic development and employs nearly 40% of the world's working population (momagri, 2016). in west africa, agriculture contributes about 35% of the countries’ gross domestic product (gdp). it remains the main provider of employment (60% of the active population) but also contributes to more than 80% of the population's food needs (blein r. et al, 2008). the economic development of these countries can only be based on agriculture. in senegal, agriculture in the niayes area plays an important role in supplying the population with agricultural products. it provides more than 80% of the national vegetable production (dps, 2003). very fertile and favorable for market gardening, the niayes zone is characterized by a shallow groundwater table (0.5 to 5 meters deep) and is made up of dunes and depressions (ansd, 2015). the niayes soils are of the tropical ferruginous type, weakly leached on sand (dior), with a sandy texture and 90 to 95% total sand. they have very low levels of carbon (0.2% on average), total nitrogen (0.15 to 1%), and exchangeable bases (1.77 meq/100 g). the ph is slightly acidic at 5.5 to 6.5 (adejumo, 2013). despite all the advantages of this soil, yields are below expectations because the crops are often attacked by predators that cause huge crop losses. nematodes are the main pests of vegetable crops. they attack crops such as aubergines, tomatoes, cabbage, potatoes, etc. thus, the population often resorts to chemical pesticides (or synthetic pesticides) which are certainly effective against these parasites, but at the same time disrupt biodiversity by eliminating insects that are not necessarily harmful and pollute the soil and contaminate the crops. according to the fao, nearly 750,000 people contract chronic illnesses, including cancers, each year as a result of exposure to pesticides. in addition, several pathologies are directly associated with them in the long term, such as congenital malformations, ethnobotanical survey on the plants used in the control of nematodes in the zone of es niayes de thies/senegal https://doi.org/10.14421/biomedich.2023.121.335-341 336 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 mental deficiencies, neurological and reproductive disorders, endocrine disruptions, weakening of the immune system, and some cases of cancer (dewailly e, 2000; petrelli g, 2001; eriksson m, 2008). yet in senegal, chemical nematicides, although classified as extremely toxic, are sold by dealers (diarra a, 2017) in an uncontrolled manner and with ignorance of the consequences. it is therefore urgent to find solutions against this phenomenon of the spread of chemical pesticides. the idea is to find pesticides that are not toxic to humans and do not pollute the environment. so what better way than to use biopesticides. since the discovery of active ingredients of therapeutic interest from plant species selected according to criteria based on medical ethnobotany and ethnopharmacology is more frequent than in a simple random screening of plants (svetaz l et al, 2010) and given that there are no or almost no nematological studies in the niayes area of thiès, we conducted an ethnobotanical survey among local farmers in this area in order to identify the plants used in the fight against nematodes as well as the methods of preparation of extracts from these plants. the present work is also part of the global framework of the valorization of the senegalese flora. materials and methods selection of the ethnobotanical survey site the niayes zone extends from dakar to saint louis, in a strip 180 km long and 5 to 30 km wide (fall as, 2001). it constitutes a rather original environment characterized by dunes and depressions that are kept wet or flooded for a good part of the year and by a sub-surface water table (see figure 1) (cissé i, 2008). its soils are very favorable for market gardening. administratively, it covers part of the regions of saint-louis, louga, dakar, and part of the region of thiès, namely the departments of tivaouane and thiès. our study area concerns these two departments, more precisely the rural communes/communities of kayar (located in the northwest of the department of thiès), notto gouye diama, and darou khoudoss (located in the west of the department of tivaouane) (see figure 2). very few studies have been carried out on potential nematicidal plants used for the protection of vegetable crops in this area. figure 1. level of fluctuation of the water table according to the seasons (cissé i, 2008). figure 2: location of the study area. (source: inspired by (https://www.au-senegal.com/), realisation: authors) method of data collection the area was divided into different sites and visits were made to each site in july and october 2021. the ethnobotanical study was carried out through a series of surveys using a pre-designed questionnaire divided into two parts. the first question concerned the sociodemographic situation of the respondent and the second question concerned the names of the species used for http://www.au-senegal.com/) diouf et al. – ethnobotanical survey on the plants used in the control of … 337 nematode control, the parts of the plant used, the times of harvest, the methods of preparation of the extracts, etc. the identification of the scientific names of the plants was carried out in the botanical laboratory of ifan/ucad. the scientific name of each species was transcribed into the local language using the lexicon of vernacular names of j. g. adam (adam j.g, 1970). data processing the data recorded on the survey forms were processed and entered into excel. the data were analyzed using simple descriptive statistical methods. quantitative variables are described using the mean and qualitative variables are described using percentages. results in this study, all the respondents were farmers. the population is made up of 100 informants, 43 of whom are from notto gouye diama, 10 from darou khoudoss, and 47 from kayar, i.e. 53 in the department of tivaouane and 47 in the department of thiès. description of the study population by age figure 1 shows the age distribution of the sample studied. we note that the age extremes of the informants vary between 18 and 78 years with an average age of 48 years. the majority (71%) were in the 30-60 age group, while the under-30 age group had the lowest rate of nematicide users (6%). distribution by gender figure 2 shows the gender distribution of the study sample. in this study, the majority of informants were male (90%) and 10% were female, with a sex ratio (male/female) of 9. description of the study population by family status regarding the family situation, 94% of the informants have children, 3% say they have no children and 3% gave no answer (see figure 3). figure 1. distribution of respondents by age group. figure 2. distribution of respondents by gender. figure 3. distribution of the study population by family status. distribution of plants by frequency of citation the surveys carried out in the niayes area of thiès enabled us to identify different plant species used in the control of nematodes. the list of the different plant species selected and their frequency of use are presented in table 1. the ethnobotanical survey identified seventeen (17) plant species of which azadirachta indica a. juss. juss. represents the most used plant with a percentage of 45%, followed by calotropis procera (ait.) f. (20%) and cassia occidentalis l. with a percentage of 9%. table 1. list of plants used for nematode control in the study area, ranked by frequency of citation. family scientific name of the plant local name frequency of citation meliaceae azadirachta indica a. juss. nim 45% asclepiadaceae calotropis procera (ait.) f. paftan 20% cesalpiniaceae cassia occidentalis l. bèntamaré 9% fabaceae (= papilionaceae) arachis hypogaea l. gèrté, tiga. 7% fabaceae (= papilionaceae) capsicum frutescens l. kani 3% caricaceae carica papaya l. papaya 2% liliaceae allium sativum l. garlic 2% meliaceae khaya senegalensis (desv.) a. juss khaye 2% labiatae ocimum basilicum basil ngungun 2% 338 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 table 1. cont. family scientific name of the plant local name frequency of citation combrétacées guiera senegalensis j. f. gmel ngèr (in wolof) 1% solanaceae datura metel l. katidiâdabé 1% fabaceae (= papilionaceae) indigofera suffruticosa mill. ngâdj, ngâdièn 1% euphorbiaceae ricinus communis l. khekhem 1% moringaceae moringa oleifera lam. nevèrday, sap sap 1% euphorbiaceae euphorbia balsamifera ait. salan 1% euphorbiaceae jatropha curcas l. tabanani 1% poaceae (= grasses) zea mays l. mbokha 1% the species listed are divided into twelve (12) families. the most commonly used plants are from the meliaceae (47%) and asclepiadaceae (20%) families. the species used in the medium range belong to the fabaceae (11%) and cesalpiniaceae (9%) families (see table 2). table 2. list of plant families used for nematode control in the study area, arranged in alphabetical order. family scientific name of the plant frequency of citation by family asclepiadaceae calotropis procera (ait.) f. 20% caricaceae carica papaya l. 2% cesalpiniaceae cassia occidentalis l. 9% combrétacées guiera senegalensis j. f. gmel 1% euphorbiaceae ricinus communis l. euphorbia balsamifera ait. jatropha curcas l. 3% fabaceae (= papilionaceae) arachis hypogaea l. capsicum frutescens l. indigofera suffruticosa mill. 11% labiatae ocimum basilicum basil 2% liliaceae allium sativum l. 2% meliaceae azadirachta indica a. juss. khaya senegalensis (desv.) a. juss 47% moringaceae moringa oleifera lam. 1% poaceae (= grasses) zea mays l. 1% solanaceae datura metel l. 1% the most commonly used plant parts the distribution of the study sample according to their use of plant parts for nematode control is shown in figure 4. the most used parts are leaves (71%) and seeds (27%). harvest times the plants are harvested during the day, throughout the year, and especially in august. the physical state of the plant before the preparation of the extracts plant samples are often freshly harvested and then extracted (53%) or first ground and then dried before extracts are prepared (45%). very rarely is plant material dried without being ground (2%) (figure 5). the solvent used for preparation water is the most used solvent (92%) while oils are very little used (8%). figure 4 shows the distribution of solvents used for the preparation of extracts (figure 6). method of preparation of extracts maceration is widely used (85%), followed by infusion (7%). hydrodistillation (4%) and the direct use of plant juice (4%) are not widely practiced (figure 7). how to apply the extracts figure 9 shows the distribution of application modes of plant extracts. the rate of application by systemic action was 94%, while only 6% of the study sample used fumigation (figure 8). diouf et al. – ethnobotanical survey on the plants used in the control of … 339 evaluation of the effectiveness of the plants by the study population in the course of our survey, we asked the respondents to give their assessments of the effectiveness of the plant preparations they had to apply to their plantations. thus, 78% of people said that they were moderately effective, 20% said they were very effective and only 2% said they were not very effective (figure 9). figure 4. distribution of plant organs by use. figure 5. physical state of the plant before preparation of the extracts. figure 6. solvents used. figure 7. methods of preparing extracts. figure 8. the modes of application of extracts. figure 9. effectiveness of plants according to respondents. discussion the african population is attached to traditional phytopharmacy. in senegalese households, men traditionally have the role of doing large-scale fieldwork and any other activity that requires muscular effort. women are only involved in small-scale cultivation. several ethnobotanical surveys conducted in senegal have shown that in the field of traditional phototherapy and everything related to agriculture, men are much more represented than women. thus, men are more likely to have knowledge of nematicidal plants than women. in this sense, our results are similar to those of barry (male: 91%, female: 9%) (barry, 20015) and diop (diop, 2015) in which men are forty (43) times more represented than women. individuals in the 30 to 60 age group are more likely to acquire more knowledge, especially about nematicidal plants, in order to ensure good yields to better feed their families, unlike the very young who do not have many responsibilities (no children to feed). the over-60s are well represented because traditional knowledge is kept by the elderly, who then pass it on to responsible young adults. the massive use of the species azadirachta indica a. juss. and the species calotropis procera (ait.) f. is probably due to their insecticidal powers already observed. the bitter taste, and the insecticidal properties towards mosquitoes (pousset j.l, 1989) of azadirachta indica a. juss. have probably led local people to believe that it is also a plant that can be used against nematodes. the great abundance of this plant in senegal makes it easy to obtain and it is by far the most used plant. azadirachta indica a. does have nematicidal effects (yarou b.b). calotropis procera (ait.) f. is also widespread in senegal. it grows well in the streets of senegalese 340 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 villages. the plant has been reported to have nematicidal properties (rao et al., 1996; ahmed et al., 1996). these plants (azadirachta indica a. juss. and calotropis procera (ait.) f.) are available throughout the year. however, for the rare plants, it is in august that the harvesting is done because it corresponds to the wintering period. we note a great diversity of plant families used in the fight against nematodes. we note that the meliaceae, asclepiadaceae, fabaceae, and caesalpiniaceae are the most cited plant families, but in terms of the number of species represented, we have 3 species among the euphorbiaceae and 3 species among the fabaceae, and 2 species for the meliaceae. the latter may have a common trait of being rich in nematicidal compounds which are found in these species. the majority use of plant leaves can be explained by the fact that leaves are rich in active ingredients and are considered the most accessible part of the plant. leaves are the storage place for secondary metabolites that are responsible for the biological properties of the plant. a study by diatta et al (diatta c.d, 2013) (46% for leaves) showed that leaves were the most used as drugs in the preparation of traditional medicinal recipes. water is a widely used solvent (92%) compared to oils. this says that most extracted compounds are polar. these are probably polyphenols, among which there are nematicidal molecules. as for preparation methods, maceration and infusion are the most commonly used methods. maceration is the easiest extraction to implement and does not require any volume of solvent to be boiled beforehand, unlike other extractions. infusion with a polar solvent allows good extraction of watersoluble active ingredients and even those that are weakly soluble in their pure state (benlamdini a, 2014). the fumigation method of the application represents only 6%. it is a difficult method to implement compared to systemic action (94%). fumigation is only more effective if it is carried out in an enclosed area so that the gas has a much greater effect on the pests. but the means of the local population in thiès, as for most local african populations, are generally modest to manage hectares of field land with fumigation. many informants found the plants they tested to be moderately or very effective. it may be that these plants contain very good amounts of nematicidal secondary metabolites. this is very encouraging and needs to be verified by biochemical studies. conclusion ten (17) species of nematicidal plants were selected for this study. the study revealed that men are more knowledgeable about nematicidal plants than women. individuals in the age group 30-60 years, who most often have children, are more numerous than other individuals. our survey shows that the foliage is the most used part. the plants mentioned are available in the study area and people mostly use the species azadirachta indica a. juss. and calotropis indica a. juss. juss. and calotropis procera (ait.) f. for nematode control. they can harvest these plants throughout the year. before preparation, the plant sample is often dried and crushed or is fresh (case for hydrodistillation). maceration and infusion are the most common methods of preparing extracts. the application of these preparations by systemic action is more widely used than fumigation. local people find these preparations rather effective, which is really encouraging. furthermore, these results can be seen as a source of information for scientific research on biopesticides that may one day permanently replace dangerous chemical pesticides. the plants mentioned in this survey may offer broad answers to nematode-related problems. acknowledgments: acknowledgments are expressed in a brief; all sources of institutional, private, and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. authors’ contributions: el hadji gorgui diouf designed the study. mbaye diouf carried out the fieldwork. doudou diop carried out the botanical identification of the species surveyed. el hadji gorgui diouf and abdoulaye gueye analyzed the data and wrote the article. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references adam j.g. noms vernaculaires de plantes du sénégal (fin). in: journal d'agriculture tropicale et de botanique appliquée, vol. 17, no. 10-11, october-november 1970. pp. 402-460. adejumo, b. a., okundare, r. o., afolayan, o. i. and balogun s. a., quality abattributes of ignam flour (elubo) as affected by blanching water temperature and soakinf time. intl. j. engr. sci (ijes), 2013, 2 (1): 213-221. ahmed et al, effect of soil amendment with calotropis procera for the control of meloidogyne javanica infection on egg plant. pakistan journal of nematology. 1996, 14 (1): 55-59. ansd, situation economique et sociale régionale-2013, 2015. barry, m.; bassene, e.; badji, k.d., contrubition à l'étude des plantes antipaludiques: enquête ethnobotanique dans la région de matam. 20015, thése de doctorat, ucad. benlamdini a., elhafian m., rochdi a., and zidane l., étude floristique et ethnobotanique de la flore médicinale du haute moulouya, maroc, journal.of applied biosciences, 2014. blein r. et al, agricultural potential in west africa (ecowas). farm, 2008. diouf et al. – ethnobotanical survey on the plants used in the control of … 341 cissé i., badiane b., ngom s., diop y. mb., & séne m., usage des pesticides et risques sanitaires sur la production horticole de la zone des niayes au sénégal, rev. sn. res. agric, 2008, vol. 1 (3), 19-26. dewailly e., ayotte p., bruneau s., gingras s., belles-isles m., & roy r., susceptibility to infections and immune status in inuit infant exposed to organochlorines, environ health persp, 2000, vol. 108 (3), 205211. diarra, a.; diallo. b.; implementation of regional pesticide policies: report of the case study in senegal. september 2017. diatta c.d., gueye m., akpo l.e., medicinal plants used against dermatoses in the baïnounk pharmacopoeia of djibonker, senegal. journal of applied biosciences, 2013, (70): 55995607. diop, el.h.m., survey on knowledge, attitudes and practices related to the use of pesticides in the niayes area. thesis of pharmacy, dakar, 2015, n°212. dps (direction de la prévention de la statistique), situation économique et sociale du sénégal. ed. dps. 2003,197p. eriksson m., hardell l., calberg m. & akerman m., pesticide exposure as risk factor of non-hodgkin lymphoma including histopathological subgroup analysis, int. j. canc, 2008, vol. 123 (7), 1657-1663. fall as, fall st, cisse i, badiane an, diao mb, fall ca. characterization of the niayes zone. in cites horticoles en sursis? l'agriculture urbaine les grandes niayes au sénégal. idrc. 2001, http//idrc.ca/en/ev27906-201-1. momagri, key figures on agriculture. 2016. petrelli g. & figa-talamanca i., reduction in fertility in male greenhouse workers exposed to pesticides, eur. j. epidemiol, 2001, vol. 17 (7), 675-677. pousset, j.l., plantes medicinales africaines (pharmacognosie), tome 1, masson, paris, france. 1989, p 24, 156p. rao et al, effect of integration of calotropis procera and glomus fasciculatum on the management of meloidogyne incognita infesting tomato. nematologia mediterranea, 1996, 24 (1): 5961. svetaz l., et al. value of the ethnomedical information for the discovery of plants with antifungal properties. a survey among seven latin american countries. j ethnopharmacol. 2010;127:137-158. yarou b.b., bioefficiency of ocimum spp. (lamiaceae) for integrated pest management in vegetable crops. (phd thesis). gembloux agro-bio tech, liège university, belgium. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 119-126 | doi: 10.14421/biomedich.2023.121.119-126 issn 2540-9328 (online) phytochemical, antimicrobial and cytotoxic activities of strophanthus sarmentosus dc julius leke abiola*, olapeju oluyemisi aiyelaagbe department of chemistry, faculty of science, university of ibadan. ibadan-200284, nigeria. corresponding author* abiolajulius005@gmail.com manuscript received: 22 november, 2022. revision accepted: 09 december, 2022. published: 12 january, 2023. abstract strophanthus sarmentosus dc is used traditionally in the management of snake-bite, arthritis, eye infection, rheumatism, emetic and venereal diseases. freshly collected mature strophanthus sarmentosus plant parts were air-dried at room temperature. each of the plant parts (leaf, stem and roots) was successively extracted by cold extraction method using hexane, ethyl acetate and methanol respectively. the crude extracts were subjected to phytochemical, antimicrobial and cytotoxicity analysis by employing chemical tests, agar diffusion and brine shrimps methods. the phytochemical screening showed the presence of tannins, saponins, glycosides, flavonoids, phenols, steroids, terpenoids and carbohydrates in all the extracts. the extracts demonstrated broad spectrum activities against both grampositive and gram-negative bacteria and the fungi tested. the mic and mmc of ethyl acetate and methanol extracts of the s. sarmentosus (stem) is between 0.3 and 5.0 mg/ml. the cytotoxic activity (lc50) of the s. sarmentosus extracts (leaf, stem and root) ranged between 117 µg/ml and 270 µg/ml, showing that the extracts are within the medium toxic level according to clarkson’s toxicity index. keywords: antimicrobial activity; cytotoxic activity; strophanthus sarmentos dc; apocyanaceae; phytochemicals. abbreviations: met; methanol, ea; ethyl-acetate, hex; hexane, dzi; diammeter of zone of inhibition, s. a. ; staphylococcus aureus, s. t.; staphylococcus typhimonium, b. s. ; bacillus subtilis, e. c. ; escherichia coli, k. p. ; klebsiella pneumonia, p. a. ; pseudomonas aeruginosa, a. n. ; aspergillus niger, c. a. ; candida albicans, g; gentamycin, k; ketoconazole, shlh; strophanthus hispidus leaves hexane, shle; strophanthus hispidus leaves ethyl acetate, shlm; strophanthus hispidus leaves methanol, shsh; strophanthus hispidus stem hexane, shse; strophanthus hispidus stem ethyl acetate, shsm; strophanthus hispidus stem methanol, lc; lethal concentration, cyclo p; cyclophosphamide. introduction plant extracts and essential oils are considered to be the potential sources of compounds with anticancer, antimicrobial and antioxidant properties. several medicinal plants and their bioactive components have been reported to be very important in the management of health through the regulation of biological processes (rahmani & aly, 2015). compounds like tannins, steroids, flavonoids and phenols possess strong biological properties and have found use in many fields, including medicine, pharmacy, food production, beauty care and agriculture (behidj-benyounes et al., 2014). infectious diseases are the world’s leading human and animal agents of death. the situation is further complicated by the rapid development of multi-drug resistance to available antimicrobial agents (alalor et al., 2012), thus, plants still remain the most effective and cheapest alternative sources of drugs for management of diseases. strophanthus sarmentosus dc (apocynaceae), is commonly found in tropical africa. many species of strophanthus possess anti-venom, anti-arthritis, antipyretic, emetics, anti-rheumatism and diuretic properties (onotu et al., 2014). one of the active constituents of strophanthus species is strophanthin which is used as a cardiac stimulant and is comparable to and recommended as a therapeutic substitute of digitalis (agbaje and ajidahun, 2011). bioassay-guided fractionation of an ethanol extract of strophanthus boivinii afforded six cardenolide glycosides-boivinides, as well as four known cardenolide glycosides, digitoxigenin 3-o-[ß-dglucopyrananosyl-(1,4)-α-l-acofriopyranoside], corotoxigenin 3-o-ß-d-boivinoside, 17 α-corotoxigenin 3-o-ß-d-sarmentoside, and uzarigenin 3-o-α-l rhamnoside. the structures of these compounds were elucidated by various 1d and 2d nmr techniques. all the compounds showed significant antiproliferative activity against the a2780 human ovarian cancer cell line, with boivinide a being the most active at ic50 of 0.17 mm (karkare et al., 2007). there is limited information in the literature on the biological activities and chemical constituents of https://doi.org/10.14421/biomedich.2023.121.119-126 120 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 strophanthus sarmentosus, hence, this study was aimed at investigating the phytochemical constituents, antimicrobial and cytotoxic activity of the leaf, stem and root extracts of the plant against some pathogenic bacteria and fungi as well as determine its cytotoxic level. material and methods plant collection and identification strophanthus sarmentosus (leaf, stem and root) was collected from the premises of forestry research institute, ibadan, oyo state, south-west, nigeria (7o39’11”n, 3o85’82”e). the plant samples were identified and authenticated by mr. d. p. o. esimekhuei at the herbarium of the botany department, university of ibadan and voucher specimen (uih 23178) of the plant was deposited at the herbarium of the department for further reference. plant preparation and extraction the plant samples were air-dried for two weeks and ground. the powdered plant materials were weighed and then soaked in the solvents for at least 72 hours. the extractions were carried out successively with n-hexane, ethyl acetate and methanol and the extracts were recovered by filtering the solvents and concentrating on a rotator evaporator at 40oc. the concentrated extracts were kept in the desiccators for further drying. phytochemical analysis the freshly prepared extracts were subjected to standard phytochemical tests to determine the chemical constituents such as tannins, alkaloids, flavonoids, glycosides, saponins and phenols (hetty manurung et al., 2019, labiad et al., 2017). test for glycosides: to 1 ml of the extract was added 2ml of acetic acid and then cooled in an ice bath at 40oc. to this mixture 1 ml of concentrated tetraoxosulphate (vi) acid (h2so4) was added dropwise. the formation of an oil layer on top of solution indicated the presence of glycosides. test for alkaloides: to 3 ml of the extract was added 1 ml of 1% hcl. this resulting mixture was then treated with few drops of meyer’s reagent. the appearance of a creamy white precipitate confirmed the presence of alkaloids. test for saponins: five drops of olive oil was added to 2ml of the plant extract and the mixture shaken vigorously. the formation of a stable emulsion indicated the presence of saponins (trease and evans, 2009) test for tannins: two drops of 5% fecl3was added to 1 ml of the plant extract. the appearance of a dirtygreen precipitate indicated the presence of tannins (trease and evans, 2009) test for flavonoids: to 1 ml of the extract was added 3 drops of ammonia solution (nh3) followed by 0.5 ml of concentrated hcl. the resultant pale brown colouration of the entire mixture indicated the presence of flavonoids. test for steroids and terpenoids: to 1 ml of the plant extract was added 1 ml of concentrated tetraoxosulphate (vi) acid (h2so4). a red coloration confirmed the presence of steroids (trease and evans, 2009). test organisms for antimicrobial assay the microorganisms used for the assay consists of three grampositive bacteria: staphylococcus aureus atcc 29213, staphylococcus typhimonium atcc 14028 and bacillus subtilis atcc 23775: three gramnegative bacteria: escherichia coli atcc 11175, klebsiella pneumonia atcc 700303, pseudomonas aeruginosa atcc 27853 and two fungi: aspergillus niger and candida albicans. they were obtained from the department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, nigeria. determination of antimicrobial activity antimicrobial activity of the plant extracts was evaluated by the cup plate agar diffusion method. bacterial cultures were adjusted to 0.5 mcfarland’s turbidity standard and inoculated onto mha (mueller hinton agar) plates (diameter 15 cm). cultures of candida albicans were suspended in sterile solution of 0.9% normal saline and the spores of the other filamentous fungi were suspended in tanguay buffer and then inoculated onto pda (potato dextrose agar) plates. a sterile cork borer was used to make wells of 6 mm diameter on the mha and pda. in each of the wells in the culture plates previously seeded with the test organisms, 100μl aliquots of extract dilutions reconstituted in minimum amount of solvent at concentrations of 50 and 100 mg/ml were applied. methanol (50%) was used as a negative control. wells containing 20μl aliquots of gentamicin (10 μg/ml), ketoconazole (1%) served as positive controls. bacterial cultures were incubated at 37oc for 24 hr while the filamentous fungal cultures were incubated at 30oc for 36 hr. after incubation, antimicrobial activity was determined by measurement of the diameter of the zones of inhibition. for all the extracts, the tests were carried out in triplicates (frempong et al., 2021). determination of minimum inhibitory concentration (mics) & minimum microbicidal concentration (mmcs) the minimum inhibitory concentration (mic) of the methanol, ethyl acetate and hexane extracts were determined for each of the test organisms in triplicate at concentrations of 0.3125, 0.625, 1.25, 2.5, and 5 mg/ml. to obtain these concentrations, 2ml of each of the dilution from 6.25, 12.5, 25, 50, 100 mg/ml of extracts were seeded into 18 ml of molten mha to achieve abiola & aiyelaagbe – biological activities of s. sarmentosus 121 concentrations of 0.3125, 0.625, 1.25, 2.5, and 5 mg/ml and poured into petri dishes. after the solidification of the aliquot (diluted extracts) and mha, different test micro-organisms (isolates) were streaked on the plates of different concentration. the procedure was repeated using the standards for the control. a petri dish containing nutrient broth only was seeded with the test organisms to serve as a negative control. petri dishes containing bacterial cultures were then incubated at 37oc for 24 hr while petri dishes containing fungal spore cultures were incubated at 30oc for 36 hr. after incubation the petri dishes were examined for microbial growth by observing for turbidity. to determine the minimum microbicidal concentration (mmc), which includes minimum bactericidal (mbc) and minimum fungicidal concentrations (mfc), a loopful of broth was collected from those petri dishes which did not show any growth in the mic determination and inoculated on sterile nutrient agar (na) for bacteria and sabouraud dextrose agar (sda) for fungi by streaking. to serve as a control, na and sda only were streaked with the respective test organisms. plates inoculated with bacteria were then incubated at 37oc for 24 hr, while those inoculated with fungi were incubated at 30oc for 36 hr. after incubation, the concentration at which no visible growth was seen was recorded as the mbc and mfc respectively, (taiwo et al., 2022). cytotoxicity assay brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of methanol, ethyl acetate and hexane extracts of strophanthus sarmentosus. brine shrimps (artemia salina) were hatched using brine eggs in a vessel filled with simulated sterile artificial sea water (brine solution) made up of sea salt (38g) in 1000ml of distilled water with the ph adjusted to 8.5 using 1 n naoh under constant aeration for 48hr. the active shrimps were collected and used for the assay (krishnaraju et al., 2005, osamudiamen et al., 2020). brine solution (4.5 ml) was taken into each test tube. suitable dilution of the extracts was made to give concentration from 1000, 500, 250, 125 and 62.5 µg/ml. the 0.5 ml of diluted test solution was added to each of the test tubes. ten shrimps were added into each test tube by drawing them with glass capillary tube. the surviving shrimps were counted after 24 hr and lethality concentration lc50 was assessed. the mortality endpoint of this bioassay is defined as the absence of controlled forward motion during 30 seconds of observation. the percentage lethality of the nauplii for each concentration and control was calculated (apu et al., 2012). % 𝐷𝑒𝑎𝑡ℎ = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓𝑑𝑒𝑎𝑑 𝑁𝑎𝑢𝑝𝑙𝑖𝑖 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 + 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 × 100 toxicity testing criteria the toxicity of plant extracts of medicinal values expressed as lc50 values is commonly valorized either by comparison to meyer’s or to clarkson’s toxicity index. according to meyer’s toxicity index, substance with lc50<1000 µg/ml are considered as toxic, while substance with lc50>1000 µg/ml are considered as non-toxic (meyer et al., 1982). while clarkson’s toxicity criterion for the toxicity assessment of plant extracts are as follows, extract withlc50 above 1000 µg/ml are non-toxic, extract with lc50between 5001000 µg/ml are low toxic, extract with lc50between100-500 µg/ml are medium toxic, and extract with lc50between0-100 µg/ml are highly toxic (clarkson et al., 2004). figure 1. research summary. result and discussion phytochemical composition of the extracts the yield extracts for all the three plant parts (leaf, stem and root) are presented in table 1. the highest (142 g) and lowest (12.4 g) yields were recorded for leaf methanol and stem hexane extracts respectively. table 1. yield of extracts of strophanthus sarmentosus. leaves (1000 g) stem (1600 g) root (1600 g) extracts hex ea met hex ea met hex ea met weight (g) 15.46 36.36 142.45 12.43 32.46 47.71 20.84 20.91 72.62 yield (%) 1.546 3.636 14.245 0.777 2.029 2.982 1.303 1.307 4.539 122 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 the results for the phytochemical screening are presented in table 2. the extracts showed the presence of tannins, carbohydrates, saponins, steroids, glycosides, terpenoids, flavonoids and phenols. bioactive compounds like tannins, steroids, flavonoids and phenols have been reported to be used by plants for protection against bacterial and fungal infections and other pests, hence they may also be responsible for antimicrobial activity (falodun et al., 2006). table 2. phytochemical screening of strophanthus sarmentosus extracts. leaves stem root sslh ssle sslm sssh ssse sssm ssrh ssre ssrm flavonoids + - -+ - + + -+ saponins - -+ -- ++ + phenolics + + --+ + -+ ++ tannins + + --+ + -+ ++ carbohydrates + + + + + + + + + glycosides -+ + -+ + - + alkaloids + + + -+ + + ++ ++ steroids + + + + + + + + + terpenoids + + -+ + + + + - legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, +: mildly present, ++: highly present. antimicrobial activities the in-vitro antimicrobial activity of the strophanthus sarmentosus extracts (leaf, stem and root) are presented in table 3. the highest susceptible pathogenic microorganisms against the extracts were candida albicans, staphylococcus typhimonium, staphylococcus aureus and pseudomonas aeruginosa while the least susceptible were klebsiella pneumonia and bacillus subtillis. all the extracts demonstrated considerable activity against both gram negative and gram positive bacteria and the fungi tested. the mic and mmc of the extracts ranged between 0.3 and >5 mg/ml as shown in table 3. the stem methanol (sssm) and root ethyl acetate (ssre) extracts were the most active of all the extracts and their activity are comparable to the standard antimicrobial agents used (table 3). secondary metabolites present in plants have been reported to be responsible for their therapeutic activity (fabry et al.,1998). flavonoids and other phytochemical constituents of the plant also have antimicrobial properties and this is in agreement with singh and bhat, (2003), which reported that flavonoids are responsible for the antimicrobial activity associated with some ethnomedicinal plants. the results highlight the fact that the methanol and ethyl acetate extracts exhibited greater antimicrobial activity because the antimicrobial principles were either polar or moderately-polar compounds. this observation agrees with the report in the literature that organic solvents are more suitable for extraction of phytochemicals (singh and singh, 2000). low mic is an indication of high efficacy of the plant extract while high mic may indicate low efficacy or possible development of resistance by the microorganisms (shanmugam et al., 2008). the presence of glycosides and alkaloids in the plant extracts may be attributed to their use by traditional medicine practitioners in healthcare systems in the treatment of some bacterial infections such as, venereal diseases, and other diseases (onotu et al., 2014: agbaje and ajidahun, 2011). despite the significant progress made in the development of antimicrobial drugs and the control of microorganisms, high level of epidermics due to drug resistant microorganisms still pose an enormous threat to public health. thus, the use of medicinal plants for antimicrobial activities needs to be given more attention. abiola & aiyelaagbe – biological activities of s. sarmentosus 123 table 3. antimicrobial activities of extracts (dzi, mm) of strophanthus sarmentosus extracts. test pathogens / concentration (mg/ml) / zone of inhibition (mm) extracts s. a s. t b. s e. c k. p p a a.n c. a 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 sslh 12 10 14 12 16 12 ssle 12 10 12 10 14 12 16 14 14 12 16 14 sslm 10 10 12 10 14 10 10 10 18 16 14 10 18 16 sssh 14 10 14 12 10 16 12 10 10 14 10 18 12 16 14 ssse 16 12 12 10 12 10 14 10 12 10 14 12 14 12 14 12 sssm 14 12 18 14 16 14 18 16 14 12 20 14 20 14 18 16 ssrh 10 10 10 12 10 14 12 ssre 12 10 16 10 14 10 12 10 12 10 14 12 18 12 20 16 ssrm 12 14 12 14 10 12 10 14 10 16 12 met (50%) standards gent. 18 18 20 16 22 14 na na ket. na na na na na na 16 14 legend: dzi: diameter of zone of inhibition, sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, -ve control (met 50%): methanol 50%, +ve control = gent.: gentamycin (10 µg/ml), ket.: ketoconazole (1%), na: not applicable, -= no zone of inhibition. table 4. minimum inhibitory concentration (mic) (mg/ml) of strophanthus sarmentosus extracts. extracts test pathogens s. a s. t b. s e. c k. p p. a a. n c. a sslh 0.625 >5.0 >5.0 1.3125 2.50 0.625 5.0 0.625 ssle 2.50 2.50 2.50 0.3125 2.5 2.5 2.5 2.5 sslm 2.50 2.50 5.0 0.3125 5.0 5.0 1.3 5.0 sssh 2.50 5.0 5.0 2.50 1.25 1.25 5.0 5.0 ssse 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 sssm 0.3125 0.625 0.3125 0.6125 <0.3125 <0.3125 0.3125 0.3125 ssrh 2.50 5.0 >5.0 1.25 1.25 0.3125 >5.0 2.5 ssre 1.25 2.50 5.0 1.25 1.25 1.25 5.0 0.3125 ssrm 2.50 5.0 >5.0 2.50 2.50 2.50 5.0 5.0 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, table 5. minimum microbicidal concentration (mmc) (mg/ml) of strophanthus sarmentosus extracts. extracts test pathogens s. a s. t b. s e. c k. p p. a a. n c. a sslh 0.625 >5.0 >5.0 1.25 2.50 2.50 >5.0 >5.0 ssle 5.0 5.0 5.0 5.0 5.0 2.50 5.0 5.0 sslm 5.0 5.0 5.0 1.25 5.0 5.0 5.0 5.0 sssh 5.0 5.0 5.0 2.50 1.25 5.0 5.0 5.0 ssse 2.50 2.50 2.50 2.50 2.50 5.0 5.0 2.50 sssm 0.3125 5.0 0.3125 2.50 0.3125 0.625 0.625 1.25 ssrh 2.50 5.0 >5.0 5.0 2.50 1.25 >5.0 2.50 ssre 5.0 2.50 5.0 1.25 1.25 1.25 5.0 0.625 ssrm 5.0 5.0 >5.0 5.0 2.50 5.0 5.0 5.0 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosusleaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, 124 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 cytotoxic activities the cytotoxic activities of the plant extracts are presented in table 6. this study determined that the extent of lethality was proportional to the concentration of the extract. after 24 hr all the shrimps survived in the control and maximum mortalities were recorded at a concentration of 1000 µg/ml for all the extracts while at 62.5 µg/ml, zero percent death are recorded in the extracts except for stem hexane extract (sssh) and root ethyl acetate extract of (ssre) which were 24% and 27% respectively. however, it was observed that in higher concentrations of the extracts the shrimps were dying after 10 hr and most of all the shrimps died after 24 hr. also, the lc50is presented in table 7. all the extracts had lc50 values between 117 µg/ml and 270 µg/ml, which show that the lc50 of all the extracts are within the medium toxic level according to clarkson’s toxicity index (clarkson et al., 2004). the presence of alkaloids, tannins and flavonoids could be responsible for their cytotoxic properties (osamudiamen et al., 2020). however, the standard drug, cylophosphamide used for the treatment of cancer diseases, had a lc50 value of 64 µg/ml. thus, the plant extracts demonstrated moderate cytotoxic activities. table 6. cytotoxic activity of strophanthus sarmentosus against nauplii. plant extracts concentrat ion (µg/ml) number of surviving nauplii (after 24 hr) t1 t2 t3 total number of nauplii survivors % mortality lc50 (µg/ml) sslh 62.5 125 250 500 1000 9 8 5 3 1 10 7 6 4 0 9 7 6 5 2 28 22 17 12 3 0±0.333 31.58±0.33 73.68±0.89 84.21±0.58 100±0.58 176.1±0.54 ssle 62.5 125 250 500 1000 9 8 4 4 2 10 9 5 3 2 9 7 6 5 2 28 24 15 12 6 0±0.333 19.05±0.58 61.91±0.58 76.19±0.58 100±0.58 226.9±0.48 sslm 62.5 125 250 500 1000 7 5 3 1 0 7 6 3 2 1 8 6 3 1 1 22 17 9 4 2 0±0.333 23.81±0.33 66.67±0.33 90.48±0.00 100±0.33 195.7±0.27 sssh 62.5 125 250 500 1000 8 7 5 4 0 9 6 4 3 1 8 6 5 4 1 25 19 14 11 2 23.81±0.33 40±0.33 52.38±0.33 66.67±0.33 100±0.33 261.6±0.33 ssse 62.5 125 250 500 1000 10 5 3 1 0 9 5 3 1 0 9 6 4 2 1 28 16 10 4 1 0±0.33 40±0.33 68±0.33 88±0.33 100±0.33 170.9±0.27 sssm 62.5 125 250 500 1000 6 4 2 2 0 8 3 2 2 1 8 3 1 1 0 22 10 5 5 1 0±0.33 30.77±0.33 69.23±0.33 69.23±0.33 100±0.33 202.8±0.33 ssrh 62.5 125 250 500 1000 8 5 3 3 0 9 9 4 2 1 9 5 4 3 0 27 24 11 8 1 0±0.33 27.27±0.33 63.64±0.33 77.27±0.33 100±0.33 117.4±0.33 ssre 62.5 125 250 500 1000 7 6 4 3 0 8 5 2 2 1 9 7 4 2 1 24 18 10 7 2 27.27±0.58 42.11±0.58 63.64±0.67 77.27±0.33 100±0.33 209.5±0.50 abiola & aiyelaagbe – biological activities of s. sarmentosus 125 ssrm 62.5 125 250 500 1000 7 4 2 0 0 5 4 2 0 0 7 3 1 0 0 17 11 5 0 0 0±0.33 42.11±0.58 73.68±0.33 100±0.33 100±0.33 153.5±0.27 standard cyclo p 62.5 125 250 500 1000 6 4 3 2 2 6 6 3 2 2 7 5 3 3 1 19 15 9 7 5 40±0.02 60±0.02 70.68±0.02 75±0.05 80±0.01 63.82±0.33 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosusleaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, cyclo: cyclophosphamide. table 7. cytotoxic activity (lc50) of strophanthus sarmentosus extracts. samples lc50 sslh 176.1±0.54 ssle 226.9±0.479 sslm 195.7±0.266 sssh 261.6±0.333 ssse 170.9±0.266 sssm 202.8±0.333 ssrh 117.4±0.333 ssre 209.5±0.497 ssrm 153.5±0.267 cyclo p 63.82±0.02 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, lc: lethal concentration, cyclop: cyclophosphamide conclusion the inhabitants of sub-sahara africa have been using numerous herbs for therapeutic purpose since time immemorial to cure all forms of diseases like diarrhea, jaundice, rheumatism, dyspepsia, asthma, diabetes, dysentery, gonorrhoea and skin infections. the current investigation evaluated for the first time antimicrobial and cytotoxic potentials of strophanthus sarmentosus. the plant extracts showed potent antimicrobial and moderate cytotoxic activities with ethyl acetate and methanol extracts of the stem and root of the plant displaying higher activities than the other extracts. these results authenticate the traditional use of strophanthus sarmentosus for the treatment of various diseases (onotu et al., 2014: agbaje and ajidahun, 2011). further studies to characterize the bioactive constituents of strophanthus sarmentosus responsible for its biological activities are in progress. conflict of interest: the authors declare that there is no conflict of interest. references agbaje, e.o., & ajidahun, o.a. 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(2009). trease and evans pharmacognosy (16th ed.). edinburgh; new york: saunders/elsevier. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 463-466 | doi: 10.14421/biomedich.2023.122.463-466 issn 2540-9328 (online) identification of medicinal plants and their utilization by community in kendal village, kendal sub-district aghnia rahmi hanum*, erna wijayanti biology education program, faculty of science and technology, uin walisongo semarang, jl. walisongo no.3-5, tambakaji, kec. ngaliyan, kota semarang 50185, tel. +62-247-604554 fax. +62-274-519739, fax 024-7601293, indonesia. corresponding author* aghnia_rahmi_hanum_2008086066@walisongo.ac.id abstract traditional medicine is a cultural heritage from ancestors deeply rooted in the nation's heritage; therefore, its use is still based on spoken and written experience from generation to generation. even though most people have turned to modern medicine, some still use these medicinal plants as ingredients for medicine, food, and other processed consumption. this study aims to identify the types of medicinal plants found in kendal village and how to use them, obtain them, and then transform them into effective medications. this study employs qualitative descriptive methodologies. observation and interviews are employed to collect data. based on the results of community interviews, it was determined that many kendal village residents continue to use plants to treat various diseases. the plant parts utilized are rhizomes, leaves, and stems. this medicinal plant has various uses, ranging from direct consumption to boiling. keywords: medicinal plants; traditional medicine; utilization. introduction the indonesian archipelago is renowned for its rich biodiversity and diverse agricultural output, which includes medicinal plants. this, supported by fertile soil conditions and a favorable climate, makes indonesia a potential producer of natural medicinal products (simbala et al., 2016). traditional medicine is a cultural inheritance from ancestors deeply rooted in the nation's heritage; therefore, its use is still based on experiences passed down orally and in writing from generation to generation (takarasel, 2010; mabel et al., 2016). ethnographically, indonesian society consists of numerous tribes with distinct cultures. this cultural diversity is distinct from one another, particularly concerning the utilization of medicinal plants. this diversity is evident in the types of plants used, the treatment methods, and the diseases that can be cured. according to world health organization (who) data, eighty percent of the world's population uses more than 20,000 species of medicinal plants (who, 2005). until 2001, the plant conservation laboratory, faculty of forestry, ipb, had documented from various research reports and published materials that no less than 2039 species of medicinal plants originated in indonesian forests (zuhud 2009; mulyani, et al., 2020). the community's use of medicinal plants ranges from flavoring ingredients to raw materials for the pharmaceutical and cosmetics industries. in the community health care system, however, the indonesian people have recognized and utilized medicinal plants to prevent health problems. some types of medicinal plants found in indonesia have been patented and massproduced in other nations to generate substantial profits for the country (abdullah, 2010; rizal et al., 2019). the knowledge of medicinal plants and their use has been passed down from generation to generation until the benefits of these natural medicines have been demonstrated, even though they are not widely acknowledged. several communities, including the community in kendal village, have utilized these medicinal plants, illustrating this point. although the majority of people have switched to modern medicine, there are still those who use these medicinal plants as ingredients for medicines, as well as for food and other processed consumption. this study aims to identify the types of medicinal plants found in kendal village, as well as how to utilize them, obtain them, and transform them into effective drugs. methods this study employed qualitative descriptive methodologies. this quantitative study collected data and analyzed the results to obtain conclusive information. manuscript received: 09 june, 2023. revision accepted: 09 august, 2023. published: 15 august, 2023. https://doi.org/10.14421/biomedich.2023.122.463-466 464 biology, medicine, & natural product chemistry 12 (2), 2023: 463-466 the data was collected through observation and interviews. observation involves making direct observations in the field alongside respondents to determine the types of plants commonly used for medicinal purposes. several individuals who knew how to use plants as traditional medicine were interviewed. the interview questions include the plant's local name, the part used, the benefits, and the method of application. the method of data analysis consists of describing the data collected through observations and interviews with ten kendal village residents based on two criteria: the use of medicinal plants and the planting of medicinal plants around the residence. the existing data is then presented as a table with local names, scientific names, parts of plants utilized, benefits, and instructions on how to use them. results and discussion medicinal plant species based on research conducted in kendal village, kendal subdistrict, eight medicinal plant species were discovered to be utilized. these medicinal plant species are typically found in the gardens of simple-minded residents. this is because, based on hereditary knowledge from ancestors, intelligent people, or books on medicinal plants, making people aware of the benefits of these plants and cultivating them by simply planting them in their yards, where the maintenance method does not require special maintenance and includes plants that are easy to overgrow, is feasible (mabel, 2016). the results indicated that residents of kendal village continue to use medicinal plants to treat various diseases in various ways (table 1). table 1. types of medicinal plants utilized by community in kendal village, kendal sub-district. no. local name scientific name plant part type of disease how to use picture 1. kunyit curcuma longa rhizome stomach pain and immunity turmeric rhizome as much as 2 fingers are pounded and then squeezed until water comes out, then the water is drunk 2. daun sirih piper betle leaves period pain, cholesterol betel leaves are boiled with water until it boils then the water is drunk 3. temulawak curcuma zanthorriza rhizome increased appetite, indigestion curcuma is pounded and then squeezed until water comes out, then the water is drunk 4. jahe merah zingiber officinale rhizome diabetes, uric acid ginger is pounded and then squeezed until water comes out, then the water is drunk hanum & wijayanti – identification of medicinal plants and their utilization by … 465 table 1. cont. no. local name scientific name plant part type of disease how to use picture 5. daun salam syzygium polyanthum leaves uric acid, cholesterol, diabetes bay leaves are boiled with water until it boils then the water is drunk 6. daun kelor moringa oleifera leaves eye disease moringa leaves as much as 1 handful are boiled with water until boiling then the water is drunk 7. lengkuas alpinia galanga rhizome diabetes bring 2 thumb-sized galangal rhizomes to a boil with 2 cups of water. 8. serai cymbopogon citratus stem cholesterol, immunity, uric acid lemongrass is soaked in hot water and the water is drunk. 9. lidah buaya aloe vera leaves (gel) skin disease the outer skin of aloe vera is peeled and then washed and the gel is taken from the aloe vera and then applied to the wound. 0. kencur kaempferia galanga rhizome pain and inflammation boil 2 thumb-sized galanga rhizomes with 2 cups of water until boiling and then drink the water. the utilized plant parts include rhizomes, leaves and stems. the plant parts that are most frequently used as medicinal plants are rhizomes (up to 5 species), followed by leaves (up to 4 species), and the stem (up to 1 species). the most widely utilized plant part is the rhizome, as several rhizome plants have anti-microbial, anti-inflammatory, and antibiotic properties that increase endurance (edy & ajo, 2020; setiono, et al., 2022). planting rhizome plants is simple and practical because they can be grown in pots and polybags on a 466 biology, medicine, & natural product chemistry 12 (2), 2023: 463-466 narrow land. the growing conditions for rhizome plants must account for climate, soil, altitude, sunlight requirements, and air temperature (tarigan, 2020; setiono, et al., 2022). another reason rhizome (herbaceous) plants are common in residential gardens is that their maintenance requirements are relatively low, whereas shrubs and trees undergo a lengthy growth process. in addition, shrubs are regarded as hedge plants because their medicinal applications are poorly understood, so they are rarely used in treatment (lestari, et al., 2021). type of disease based on interviews with ten kendal village residents, it was determined that many villagers continue to use plants to treat various diseases. the community learns how to use medicinal plants based on inherited knowledge from ancestors, intelligent people, and neighbors who know how to use them. the use of medicinal plants is also highly varied, ranging from direct consumption to boiling and drinking boiled water. in general, there are two types of treatment administered by the community: treatment for internal diseases and treatment of external illnesses. external treatment includes skin diseases, toothaches, eye conditions, and wounds. in contrast, internal diseases are treated by ingesting or drinking a portion of a medicinal plant (hidayat, et al. 2010; mulyani, et al. 2020). conclusion based on the research conducted, it can be concluded that the use of medicinal plants by the kendal village community has been passed down through the generations. in general, these medicinal plants are cultivated in backyards. turmeric, galanga, curcuma, betel leaves, bay leaves, aloe vera, lemongrass, moringa leaves, and red ginger are the plant species found. the use of these medicinal plants is also quite diverse, ranging from direct consumption to the decoction and maceration of the plant parts. competing interests: the authors declared that they have no competing interests. references lestari, d., koneri, r., maabuat, p. v. (2021). keanekaragaman dan pemanfaatan tanaman obat pada pekarangan di dumoga utara, kabupaten bolaang mongondow, sulawesi utara. jurnal bios logos. 11(2): 82-93. mabel, y., simbala, h., koneri, r. (2016). identifikasi dan pemanfaatan tumbuhan obat suku dani di kabupaten jayawijaya papua. jurnal mipa unsrat. 5(2): 103-107. mulyani, y., sumarna, r., patonah. (2020). kajian etnofarmakologi pemanfaatan tanaman obat oleh masyarakat di kecamatan dawuan kabupaten subang provinsi jawa barat. jurnal farmasi galenika (galenika journal of pharmacy). 6(1): 37-54. rizal, s., sustriana. (2019). inventarisasi dan identifikasi tanaman berkhasiat obat di kabupaten musi banyuasin. jurnal indobiosains. 1(2): 50-62. setiono, novriansyah, y., fitri, d., asman, m., & isman. (2022). pelatihan pembuatan minuman herbal rimpang dan pembudidayaannya masa pandemi covid-19. jurnal masyarakat mandiri (jmm). 6(4): 3331-3340. takarasel, r. (2010). inverntarisasi tumbuhan obat tradisional di kecamatan manganitu, tamako, tabukan selatan dan kendahe kabupaten sangihe. manado: skripsi. fmipa unsrat. who. (2005). review of traditional medicine in the south-east asia region. in who biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 399-405 | doi: 10.14421/biomedich.2023.121.399-405 issn 2540-9328 (online) development of an eco-shampoo formulation using local environmental plant extracts for healthy hair as an effort to increase the potential of environmental resources dwi atmanto*, neneng siti silfi ambarwati department of cosmetology, faculty of engineering, state university of jakarta, jakarta 13220, indonesia. corresponding author* dwiatmanto64@gmail.com abstract this research aims to develop an eco-friendly shampoo formulation (eco-shampoo) by adding local environmental plant extracts for hair health. this research method is a quasi-experimental development of existing shampoo formulations (base formulations) by adding local plant extracts such as aloe vera (aloe vera l.) and lemongrass oil (cymbopogon citratus) in three formulations. the resulting shampoo products were then carried out two evaluations, namely the stability test (organoleptic test), foam height observation, viscosity test, ph test, and preference test (hedonic test) by consumers to determine the feasibility and effectiveness of using shampoo. after a storage period of 2 days showed a stability test, slightly changed color, constant odor, and texture did not change and the ph value was between 5.5 6.5. the smear test on the back of the hand and the skin behind the ear did not show skin irritation, whereas the hedonic test, 10 consumers stated that the color was like, the smell was stated like, and the texture was stated quietly. aloe vera fraction and lemongrass oil can be formulated as a shampoo preparation to maintain hair fertility as well as an anti-dandruff shampoo. keywords: eco-shampoo; formulation development; health hair; local environment; plant extracts. introduction indonesia is a fertile country and has various types of plants. the soil is fertile and the rainfall is sufficient for various types of plants to grow and develop (arifin 2006). plants are biological natural resources that can be managed to become a source for the establishment of industries such as the pharmaceutical industry, the food industry, the metal industry, the animal feed industry, the plastic industry, and the cosmetics industry. several plants have been developed to treat hair problems, one of which is aloe vera and lemongrass leaves. empirically, aloe vera leaves are used by the community to nourish their hair by using the inner gel of the leaf flesh (ariyani et al. 2009). likewise, lemongrass leaves are taken through extracts used by people for anti-dandruff hair treatment. hair contains an important role for every human being. this is because hair can affect a person's appearance (filbert 2014). the number of hairs on the human head is about 100,000 strands (hotmauli 2010). even though hair has natural hair loss, for some people, hair loss is still a worrying thing. hair loss can occur due to several factors such as age, hormonal disorders, pregnancy, drug use, continuous sun exposure, and lifestyle (doughari 2006). to overcome hair problems, intensive care is needed, such as using shampoo, hair tonic, hair mask, hair oil, vitamins, and others. environmental factors that have been polluted in urban areas can disrupt hair health. air or water impurities that hit the hair of the head will cause dandruff. dandruff is a dry form of seborrheic capitis known as seborea sika (dry), which are dry, fragile, easily detached scales that stick to cover the epidermis of the scalp (arifin 2006). one of the causes of dandruff is fungus on the scalp, which is dirty due to sweat, sebum (oil) glands, and dust. the fungus that develops on the scalp is called pityrosporum ovale (ariyani et al. 2009). symptoms of dandruff mainly include itching, flaking, and redness of the scalp. this fungus is naturally found on the scalp and can attack humans of all ages. many anti-dandruff shampoos contain antifungal compounds such as sulfur, salicylic acid, selenium sulfide, and zinc pyrithione which have the effect of damaging the scalp and causing hair loss (trueb 2007). therefore, there need to be other alternatives, especially natural ingredients that can be used as anti-dandruff. lemongrass (cymbopogon citratus) is one of the essential oil-producing plants. in indonesia, this species is usually used as a mixture of herbs and spices because it has a distinctive aroma like lemon (boonme et al. 2011). lemongrass oil is one of the most important types manuscript received: 08 february, 2023. revision accepted: 28 may, 2023. published: 20 july, 2023. https://doi.org/10.14421/biomedich.2023.121.399-405 400 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 of essential oil. this essential oil is used to produce citral which is the main constituent of lemongrass oil. lemongrass oil is a pale yellow liquid that has a strong lemon smell due to its high levels of citral (65% to 85%) making it the most important ingredient in the pharmaceutical and cosmetic industries (saputro 2009). the main constituents of lemongrass essential oil are citral (3,7-dimethyl2,6octadienal), a mixture of geranial (trans-citral a), and neutral (cis-citral b) with small amounts of geranium, geranyl acetate, and monoterpene olefin ((pooja et al. 2009). among the plants that can be used as hair, growers are aloe vera. aloe vera is very effective for hair care because it has a composition similar to keratin, an important hair protein, and complex amino acids identical to hair follicles so that it can rejuvenate hair with the same nutrients, especially the content of the amino acid l-lysine which can help in growth hair (limbani et al. 2009). according to daisy (2011), the main elements of aloe vera liquid are aloin, emodin, resin, gum, and other elements such as essential oils. in terms of nutritional content, aloe vera leaf gel or mucus contains several minerals such as zn, k, fe and vitamins such as vitamins a, b1, b2, b12, c, e, inositol, folic acid, and choline (potruli et al. 2011). aloe vera is also a natural remedy that helps regulate the rich blood supply to the root hair follicles on the scalp, thereby helping to strengthen hair (pooja et al. 2009). treatment using traditional medicine today is very popular and is increasingly favored by the community. this is because affordable prices are easy to obtain and also have relatively few side effects (naitullah et al. 2004; pan et al. 2009). in addition, indonesia is also the second mega biodiversity country after brazil, where it is estimated that there are 30000 species of living plants in the indonesian archipelago and at least 9600 species are known to have medicinal properties (robinson 1995). the cosmetic industry in indonesia developed around the 1950s. previously, traditional cosmetics had developed among kings in java (mahataranti et al. 2012). the indonesian people at that time developed a complex of local plant ingredients that were used as powder, shampoo, and herbal medicine. the use of traditional cosmetics at that time was still limited among the royal family alone. along with the development of chemistry, pharmaceutical science, and medical science, cosmetics and industrial sciences have also developed (mitsui 1997). now the cosmetics industry is growing rapidly because of the demands of modern society. the cosmetic industry is more developed using chemicals such as paraffin, surfactants, dyes, and preservatives. as public awareness of environmental issues has increased, such as green products, environmentally friendly products, and herbal products, the cosmetics industry has begun to compete with industrial expansion related to environmentally friendly cosmetic preparations (deeksha et al. 2014). however, not all the basic ingredients in the manufacture of the cosmetics industry have been replaced by natural ingredients, such as foam, thickeners, and preservatives. more active ingredients can be substituted by natural ingredients such as protein, dyes, and other nutrients (aghel et al. 2007). many cosmetic companies in indonesia produce shampoo, such as the wahdah industry. therefore, the formulation of the research problem arises: (1) can the manufacture of shampoo be developed with local plant materials such as aloe vera and lemongrass oil? (2) how to evaluate shampoo preparations made from aloe vera and lemongrass oil? the objectives of this study were (1) to determine the formulation of shampoo that was given a mixture of aloe vera and lemongrass oil, and (2) to determine the evaluation results of shampoo preparations mixed with local plants such as aloe vera and lemongrass oil. material and methods the tools used in this research are measuring cup (pyrex), beaker glass (pyrex), erlenmeyer (pyrex), test tube (pyrex), 65 mesh sieves, spatula, stirring rod, glass funnel (pyrex), separating funnel (pyrex). petri dishes (pyrex), analytical scales, blenders, hot plates, porcelain cups, shampoo containers, clamps and statives, pipettes, ph meters, filter paper, sterile gauze, and bunsen lamps. the materials used in this research are aloe vera, lemongrass oil, sodium lauryl sulfate, cocamide diethanolamine (cocamide dea), carboxymethyl cellulose (cmc), menthol, citric acid, methylparaben, aquadest, sodium chloride (nacl). aloe vera samples are purchased at supermarkets, as is slow mass oil. the initial stage is to collect fresh aloe vera leaves. then the fresh aloe vera leaves are washed under running water to remove the dirt. the contents of the aloe vera gel are taken, then crushed then filtered with apoach to get a smooth and non-clumping gel. this research method is an experiment, which is carried out in the chemistry laboratory of the faculty of engineering, state university of jakarta. the ingredients used are sodium lauryl sulfate, aloe vera, lemongrass oil, and aquadest. this research method is an experimental design with a one-shot study. where there is one group of independent variables is given treatment, then observations are made (sugiyono 2008). the object research is existing shampoo formulations (base formulations) by adding local plant extracts such as aloe vera (aloe vera) and lemongrass oil (cymbopogon citratus) in three formulations (see table 1) (al-badi and khan 2014). atmanto & ambarwati – development of an eco-shampoo 401 table 1. shampoo formulation for lemongrass and aloe vera oil fractions. ingredients shampoo formulations with various fractions of lemongrass and aloe vera oil f1 f2 f3 lemongrass oil 2% 7% 12% aloe vera 3% 3% 3% sodium lauric sulphate 10% 10% 10% cocamide dea 4% 4% 4% cmc 3% 3% 3% citric acid q.s. q.s. qs. menthol 0.5% 0.5% 0.5% methyl paraben 0.15% 0.15% 0.15% aquadest ad.30 ml ad.30 ml ad.30 ml the formulation results were tested for homogeneity, ph, viscosity, foam density and foam stability, wetting time, and cleaning power test. homogeneity testing using a glass object. anti-dandruff shampoo gel is made of a 10% solution and measured the ph using a ph meter. viscosity check is done using a viscometer. the foam power test was carried out by making a 10% shampoo gel solution, shaken 10 times, and recorded the volume of foam formed (munro 1954; fazlolahzadeh & masoudi 2015). the foam stability test is done by recording the volume of foam reduction that occurs in the foam power test in 1–4 minutes intervals (deeksha et al. 2014). the wetting time test was carried out by making a 1% shampoo gel solution then put it in a measuring cup and the canvas cloth was dropped into the solution. measure the time it takes for the canvas to sink. weigh all the ingredients used according to the formulation. cmc is developed with hot water in a mortar. diluted methylparaben with a few drops of ethanol until dissolved. part of the aquadest is heated on a hot plate at 60°c and added with sodium lauryl sulfate, stirring until homogeneous. cocamide dea was added to the mixture while continuing to stir until it was homogeneous (mitsui 1997). the general process of making shampoo with local plant mixtures is presented in figure 1. figure 1. diagram of the process of making shampoo with local plant mixtures. results and discussion the results of organoleptic observations of the antidandruff shampoo for aloe vera leaf and lemongrass oil fraction with various concentrations showed that the higher the concentration of aloe vera leaf aquadest fraction contained in the anti-dandruff shampoo preparation, the stronger the distinctive odor of aloe vera leaves and lemongrass oil so that it covers the odor of the menthol fragrance used, and the darker brown in the shampoo preparation because of the red brick color of the citronella leaf aquadest fraction (see table 2). table 2. result of organoleptic observations for mixed aloe vera shampoo and lemongrass oil. shampoo preparation formulations observation result shape colour smell f1 liquid, nothing settles light brown menthol, and lemongrass f2 liquid, nothing settles brown menthol, and lemongrass f3 liquid, nothing settles dark brown menthol, and lemongrass table 3. ph scale measurement. shampoo preparation formulations ph f1 6 f2 5.5 f3 5.5 the ph test aims to determine the safety of the preparation at the time of use. shampoo ph that is too acidic or too alkaline will irritate the scalp. the result of ph measurements is presented in table 3. the result of the foam height test is presented in table 4. the result of 402 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 the value of the water content test is presented in table 5. the process of making shampoo from aloe vera and lemongrass oil and shampoo preparation is presented in figure 2. table 4. foaming hight measurement. shampoo preparation formulations foaming hight f1 6.2 f2 7.3 f3 8.1 table 5. results of measurement of water level content for shampoo preparations. shampoo preparation formulations water level (%) f1 89.83 f2 86.71 f3 82.36 figure 2. the process of making shampoo from aloe vera and lemongrass oil and shampoo preparation. based on the results obtained, the distilled water fraction has the greatest fungal inhibiting activity so that anti-dandruff shampoo preparations are made using active ingredients, namely the distilled water fraction of aloe vera leaves and lemongrass oil with a concentration of 5% 10%, and 15%, surfactants, and additives. the surfactants chosen in the manufacture of this shampoo are the surfactants that are widely used in shampoo preparations on the market, namely sodium lauryl sulfate as the primary surfactant and cocamide dea as a secondary surfactant. by using primary and secondary surfactants, dandruff shampoo preparations can clean and form foam better. additional ingredients used are cmc as a thickener, methylparaben as a preservative, citric acid as a buffer, and menthol as a fragrance. the anti-dandruff shampoo for the aloe vera leaf fraction was evaluated to determine its quality and safety. then proceed with testing the antifungal activity of the anti-dandruff shampoo preparation. the foam height test aims to show the surfactant's ability to form foam. the lather from the shampoo is very important. this is because the foam keeps the shampoo in the hair, makes hair easy to wash, and prevents the hair sticks from sticking together, causing tangles (mitsui 1997). the foam height resulting from the three shampoo formulations has increased foam power. this increase was caused by an increase in the distilled water fraction in the shampoo preparation because the distilled water fraction of aloe vera leaves and lemongrass contained saponins. according to harbone (1998), saponins are soap. the foam height test results of the three shampoo formulations met the foam height requirements according to wilkinson and moore (wilkinson 1982), namely 1.3 22 cm. based on the results of ph measurements using universal ph indicator paper, the addition of aloe vera and lemongrass distilled fraction causes a decrease in ph due to the influence of active substances in aloe vera distilled water fraction which has an acidic ph. although the ph of the shampoo decreased, the ph value of the three anti-dandruff shampoo formulations still met the requirements set forth in sni no. 06-2692-1992 which is around 5.0 9.0. testing the value of water content is very important to do in a shampoo product because the moisture content is related to the physical shampoo and affects the shelf life of a shampoo product. from the measurement of water content, anti-dandruff shampoo with various concentrations of aloe vera and lemongrass distilled water still meet the requirements of sni no. 06-26921992 which is a maximum of 95%. based on the results obtained, the water content produced, the greater the concentration of the fraction added, the smaller the percentage of water content obtained. this higher water content comes from hygroscopic materials (the ability of a substance to attract water molecules from its environment), such as cmc. the shampoo preparations made in this study used active ingredients which are obtained from the water pacar leaves. water pacar leaves that are taken, cleaned and washed under running water to remove dirt. the leaves are already aerated and put in the oven to reduce moisture content in the leaves. the dried leaves of aloe vera are then blended until smooth and sieved to obtain a homogeneous powder with a surface area large so that it facilitates the release of active substances in the extraction process. the results of aloe vera leaf extract were obtained from the extraction process using the maceration method and 96% ethanol solvent. this maceration method also has the advantage of being able to maintain the compound content in the sample which is not heat resistant, undamaged and the sample can be atmanto & ambarwati – development of an eco-shampoo 403 extracted directly in large numbers (doughari 2006). this sample extraction using 96% ethanol solvent because ethanol solvent covers almost the entire content simplicia, both non-polar, semi-polar, and polar (iswanti 2009). the concentrated extract that has been obtained is then fractionated by the method partition using n-hexane, ethyl acetate, and distilled water. the use of solvents aims to attract compounds in the extract based on the level of polarity. based on the yield of the extract fractionation, it can be seen that the distilled water solvent fraction had the highest yield. this shows that it is deep. based on the results obtained, the distilled water fraction has activity inhibits the most fungus so that antidandruff shampoo preparations are made with using the active ingredient, namely the water fraction of aloe vera leaves and lemongrass oil with concentration 5% 10%, and 15%, surfactants, and additives. surfactants selected in this shampoo is a surfactant that is widely used in shampoo preparations on the market, namely sodium lauryl sulfate as primary surfactant and cocamide dea as a secondary surfactant. by using primary surfactants and secondary, anti-dandruff shampoo preparations can clean and form more foam well (deeksha et al. 2014). additional ingredients used are cmc as a thickener, methylparaben as a preservative, citric acid as a buffer and menthol as a fragrance. shampoo anti-dandruff water fraction of aloe vera leaves that has been finished evaluated to know the quality and safety. then proceed with testing the antifungal activity of the anti-dandruff shampoo preparation. the results of organoleptic observations of antidandruff shampoo for water fraction of pacar leaf with various concentrations show that the higher concentration of water fraction of pacar leaf contained in the shampoo preparation anti-dandruff, the stronger the distinctive smell of aloe vera leaves so that it covers the smell of the menthol fragrance used, as well as the darker brown in the preparation shampoo because the color of the distilled water of aloe vera leaves is brick red. the ph test aims to determine the safety of the preparation at the time of use. shampoo ph that is too acidic or too alkaline will irritate the scalp. based on the results of ph measurements using universal ph indicator paper, the addition of the distilled water fraction aloe vera causes a decrease in its ph due to the influence of the active substance in the water fraction of pacar leaves which has an acidic ph. although the ph of the shampoo has decreased, the ph value of the three shampoo formulations anti-dandruff still fulfills requirements stipulated in sni no. 06-26921992 which is around 5.0 9.0. the foam height test aims to show the surfactant's ability to form foam. the lather from the shampoo is very important. this matters because the lather keeps the shampoo in the hair, making hair easily washed, and prevents the hair sticks from sticking together to cause tangled (mitsui, 1997). high foam resulting from the three shampoo formulations experienced increased foaming. this increase was caused by the increase in the distilled water fraction in the shampoo preparation was due to the distilled water fraction of pacar leaves containing saponins. according to harbone, saponins are soap. the foam height test results of the three shampoo dosage formulations met the high requirements; foam according to wilkinson and moore (1982) is 1.3 22cm. test the value of water content is very important to do in a product shampoo because the water content is related to the physical shampoo and affects the shelf life of a shampoo product. from the measurement of water content, anti-dandruff shampoo with various concentrations of water fraction of pacar leaf still meets the requirements of sni no. 06-2692-1992 which is a maximum of 95%. based on the results obtained from the resulting water content, the greater the concentration of the fraction added, the smaller the percentage of water content obtained. water content higher comes from hygroscopic materials (the ability of a substance to attract water molecules from the environment) such as cmc. anti-dandruff shampoo preparations with various concentrations tested its activity against the growth of the fungus candida albicans by using media, method, fungus, and positive control were the same as those used on antifungal activity testing of the previous extracts and fractions. negative control anti-dandruff shampoo formula is used without the water fraction of aloe vera leaves (base shampoo). the negative control is used to determine whether there is a basis effect shampoo against the growth of the test fungi, so it can be seen that the activity shown by the shampoo with various concentrations of leaf distilled water. aloe vera is the substance contained in the distilled water fraction shampoo preparations and not derived from the shampoo base used (putluri et al. 2011). the results of testing the antifungal activity of antidandruff shampoo, distilled water fraction. aloe vera and lemongrass oil with concentrations of f1 (5%), f2 (10%), f3 (15%), shampoo formula anti-dandruff without distilled water fraction of aloe vera leave as negative control and shampoo (june 2016). ketoconazole 2% as a positive control in each treatment indicates a zone of inhibition that has formed around the well. obstacles zone the largest indicated by the anti-dandruff shampoo with a concentration of 5% distilled water, followed by anti-dandruff shampoo with the concentration of 10% distilled water and zone fraction. the smallest inhibition was shown by antidandruff shampoo with a fraction concentration of 15% distilled water with an average size of the inhibition zone in a row, namely 13.83 mm, 9 mm, and 7.83 mm. the difference in inhibition is influenced by the addition of distilled water fraction which affects the release of active substances for inhibited fungus (aghel et al. 2007). the higher the concentration of distilled water, the thicker it is anti-dandruff shampoo preparation, the greater the 404 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 viscosity of the shampoo preparation. viscosity is a statement about the resistance of a liquid to flow, the higher the viscosity, the greater the resistance (al-badi and khan 2014). this is what makes it so that it blocks the release of the active substance which results in inhibition of the fungus candida albicans. utilization of organic local environmental resources made from plants like aloe vera, mangrove, and spice plants for a variety of products that help the economy and as natural treatments, particularly cosmetics (daisy r. 2011). phenolic compounds are found in the majority of indonesian plants, including aloe vera and mangrove leaves. phenolic compounds are one of the dynamic mixtures found in aloe vera gel. aloin, aloesin, aloemodin, and tannins are the primary components of these phenolic compounds (iswanti da. 2009). in the past, the community did not know how to make cosmetics like organic shampoos and masks made from local environmental plants, which offered huge opportunities for business. plants that contain nutrients and active substances that function as fundamental ingredients for skin and hair care are the strategy for utilizing the potential of local environmental resources in this instance (deeksha d, malviya r, sharma pk. 2014). after getting to know one another and introducing the community to products that grow in the area, the community now knows how to make organic shampoos, soaps, and masks from local plants. these products are easy to make and can be used again and again, and the materials used are ones that can be found easily (fazlolahzadeh o, masoudi a. 2015). according to pooja a, arun n, and maninder k. (2009), the production of organic shampoos and masks made from aloe vera and other plants could also be a solution to the issue of environmental pollution caused by chemical waste, which is difficult to convert into materials that are friendly to the environment. because there are so many "dysfunction" chemicalbased cosmetics on the market, the usage of plants as cosmetic ingredients will continue to expand in the future. as a result, both the central government and the regional governments must continue to stimulate the exploration of the potential of local environmental resources, both types and benefits, through experiments and research. the cultivation of cosmetic ingredient plants can later become a source of revenue for the affected area (naitullah n, jamin f, frengki, dewi m. 2014). in this way it is important to go to preservation lengths for regions that have neighborhood natural assets through local guidelines so the stock of materials is ensured. on the off chance that the plant is unmistakable for its development climate, it is better assuming development is completed around there to increment plant material (robinson t. 1995). involving the local community in the production process as well as in the cultivation of these plants is an equally crucial step. for instance, for each family, they are responsible for planting ten sticks or ten plants. we will eventually discover a "cosmetic village" (saputro ad: 2006). in addition, it inspires researchers at universities, the private sector, and national research agencies like brin. conclusion based on the results of research on the physical stability test of mixed aloe vera and lemongrass oil shampoo with varying concentrations, namely 5%, 10%, and 15%, it can be concluded that aloe vera contains active compounds, fats, carbohydrates, and proteins. aloe vera fraction and lemongrass oil can be formulated as a shampoo preparation to maintain hair fertility as well as an anti-dandruff shampoo that meets requirements such as organoleptic, ph, high foam, and moisture content. evaluation of shampoos for quality assurance is a physical appearance test that is attractive, homogeneous, does not break, and can form foam. the ph value ranges from 5 6. the test for the ability and stability of the foam of the shampoo is carried out using the cylinder shake method at 6-8 cm, where the formulation of aloe vera is 3% and lemongrass oil is 12%. authors’ contributions: dwi atmanto & neneng siti silfi ambarwati designed the study. dwi atmanto & neneng siti silfi ambarwati carried out the laboratory work. dwi atmanto & neneng siti silfi ambarwati analyzed the data. dwi atmanto & neneng siti silfi ambarwati wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research receives no external funding. acknowledgment: the author would like to express gratitude to the dean of the faculty of engineering at jakarta state university for partnering with blu's asset management on the exploration. the authors also express their gratitude to the students and professionals who assisted with the research center's cycle of item testing and exploration. references aghel n, moghimipour b, dana r.a. 2007. formulation of a herbal shampoo using total saponins of acanthophyllum squarrosum. iranian journal of pharmaceutical research. 6(3), pp. 167-172. doi: 10.22037/ijpr.2010.717 al-badi k, khan sa. 2014. formulation, evaluation, and comparison of the herbal shampoo with the commercial shampoos. beni-suef university journal of basic and applied sciences. 3(4), pp. 301-305. atmanto & ambarwati – development of an eco-shampoo 405 arifin z. 2006. kajian mikoriza vesikula arbuskula (mva) dalam menekan perkembangan penyakit bercak ungu (alternaria porri) pada bawang putih [study of arbuscular vesicle mycorrhizal (mva) in suppressing the development of purple spot disease (alternaria 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2006. potensi antifungi isolat bakteri rizosfer rumput pangola (d. decumbens) terhadap jamur c. albicans [antifungal potential of pangola grass rhizosphere bacterial isolate (d. decumbens) against the fungus c. albicans] [barchelor’s thesis]. faculty of teacher training and education, muhammadiyah university surakarta, surakarta. sugiyono. 2008. metode penelitian pendidikan: pendekatan kuantitatif, kualitatif dan r&d [educational research methods: quantitative, qualitative and r&d approaches], bandung: penerbit alafabeta. trüeb rm. 2007. shampoos: ingredients, efficacy and adverse effects. journal der deutschen dermatologischen gesellschaft. 5(5), pp. 356-365. doi: 10.1111/j.16100387.2007.06304.x. wilkinson jb. moore rj. 1982. harry's cosmeticology, 7 eds. new york: chemical publishing company http://www.mindbodygreen.com/0-2794/why-you-should-use-natural-shampoo.html http://www.mindbodygreen.com/0-2794/why-you-should-use-natural-shampoo.html biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 241-250 | doi: 10.14421/biomedich.2023.121.241-250 issn 2540-9328 (online) synthesis and characterization of cinnamon loaded bsa microparticles with antidiabetic properties department of chemistry, university of kelaniya, sri lanka. corresponding author* bimali@kln.ac.lk manuscript received: 03 february, 2023. revision accepted: 01 march, 2023. published: 03 march, 2023. abstract conventional medicine that is being used to treat diabetes exert adverse side effects and therefore scientists have focused on natural hypoglycemic agents. “sri wijaya” (ccsw) is an accession of cinnamomum zeylanicum, which shows higher hypoglycemic activity. pressured water extract of its dried quills can be used as an antidiabetic nutraceutical. higher stability, ease of storage and transportation, make powder form nutraceuticals more preferred. the objective of this study was to develop cinnamon encapsulated microparticles as a powder form nutraceutical with higher hypoglycemic activity. four different products were synthesized. two of them were synthesized using bovine serum albumin (bsa) (8.8 % (w/v), ph=5) in the presence of citric acid and ascorbic acid as cross-linking agents separately. the other products were synthesized using bsa (20 mg/ ml, ph=9) in the presence of same cross-linking agents. antidiabetic activity of the products was determined using alpha-amylase and alpha glucosidase inhibition assays and compared with that of crude cinnamon extract and positive control acarbose. since the product synthesized using bsa (20 mg/ ml, ph=9) and citric acid showed the highest alpha amylase inhibition activity, solubility, cinnamon loading percentage and cinnamon entrapment efficiency those conditions were concluded as the optimum conditions required to synthesize microparticles with higher hypoglycemic activity. particle size, polydispersity index, and zeta potential of that product were 1.281 ± (0.004) µm, 0.460 ± (0.018) and -1.09 ± (0.03) mv respectively. according to the sem image, microparticles have a spherical morphology. the uv-visible spectrum and the ft-ir spectrum confirm the entrapment of cinnamon compounds. keywords: cinnamon; cross-linking agent; diabetes; microparticles; nutraceutical. abbreviations: ccsw: cinnamomum zeylanicum “sri wijaya”; bsa: bovine serum albumin; clmp: cinnamon loaded microparticles. introduction diabetes mellitus, simply referred to as diabetes is the most common endocrine illness (hudaib et al., 2018). in 2008, world health organization has estimated that approximately 2.9 million deaths occur due to diabetes mellitus every year (desoky et al., 2012). the major types of diabetes mellitus are type 1 diabetes and type 2 diabetes (njagi et al., 2014). type 1 diabetes is an autoimmune disease, and it typically develops in children and young adults. the immune system of the individuals who suffer from this disease, mistakenly destroys the pancreatic beta cells which make insulin. type 1 diabetes patients should regularly administer insulin medication. the most common kind of diabetes mellitus is the type 2 diabetes (riaz, 2009). this is a disorder which can be characterized by insulin resistance and beta cell dysfunction (desoky et al., 2012). since current conventional diabetes medicines exert adverse side effects, scientists have focused more on hypoglycemic agents of natural sources, which have been used in traditional medicine systems such as ayurveda (desoky et al., 2012). nearly 72.8 % of the world diabetic population rely on herbal medicine for the treatment of their diabetes mellitus (hudaib et al., 2018). in traditional medicine systems of china, korea and russia, cinnamon has been used as a medicine for diabetes mellitus (hudaib et al., 2018). various chemical compounds in cinnamon can contribute for the antidiabetic property in different ways. methyl hydroxychalcone polymer of cinnamon has an ability to stimulate glucose oxidation, thus helps to decrease the glucose content. some compounds in cinnamon such as polyphenol type-a polymers act as insulin (gan and rao, 2014). cinnamon can reduce the intestinal glucose absorption by inhibiting the enzymes like α-amylase and α-glucosidase which involve for the carbohydrate metabolism. a proanthocyanidin called cinnamtannin b1 is responsible for this activity. the same compound can further contribute to decrease the blood glucose level by b. s. wanniarachchi, h. m. w. k. sathsarani, b. m. jayawardena*, h. g. n. dewangani https://doi.org/10.14421/biomedich.2023.121.241-250 242 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 stimulating cellular glucose uptake by membrane translocation of glut-4, stimulating glycogen synthesis, inhibiting gluconeogenesis, and stimulating insulin release. cinnamon can also act against diabetic retinopathy and neuropathy (constantine et al., 2013). there are approximately 250 species of cinnamon all over the world (gan and rao, 2014). among them ceylon cinnamon (cinnamomum zeylanicum) can be consumed in higher doses for longer durations without toxic effects due to the very low coumarin content. ccsw is one of the accessions of cinnamomum zeylanicum that is a newly developed accession of cinnamon, and it shows higher anti-diabetic activity (wariyapperuma et al., 2020). therefore, ccsw accession of ceylon cinnamon can serve as a promising candidate to develop antidiabetic nutraceuticals. in developing effective antidiabetic nutraceuticals from ceylon cinnamon, the active compounds should be extracted. according to the previous studies, pressured water extraction is the most effective method to prepare an aqueous extract of cinnamon with antidiabetic activity. it is desirable to have aqueous plant extracts of plants in the powder form to enhance the durability, stability and the ease of storage and transportation. plant extracts can be encapsulated in microparticles to convert them into more stable form (amritham et al., 2016). microencapsulation is an advanced technology using which one or more active compounds can be encapsulated within an inert material, making a tiny sphere of diameter ranging from 1 µm to several 100 µm. microencapsulation is performed for protecting the sensitive bioactive compounds as well as for their safe delivery. nowadays bsa is used as a common encapsulant since the microparticles produced using this protein are less toxic, nonantigenic, biocompatible and biodegradable (choudhury et al., 2021). microparticulate drug delivery systems proffer a number of significant advantages including effective protection of the encapsulated active agent against enzymatic degradation, enhancement of peptide stability, sitespecific and controlled drug release (wong et al., 2018; singh et al., 2010) the objective of the study was to synthesize and characterize cinnamon encapsulated bsa microparticles with antidiabetic properties. materials and methods raw materials cinnamomum zeylanicum quills (accession: sri wijaya) chemicals bovine serum albumin (bsa), ethanol, citric acid, ascorbic acid, sodium hydroxide, hydrochloric acid, acarbose, alpha amylase, starch, dinitrosallicylic acid reagent (dns), sodium hydrogen phosphate, sodium dihydrogen phosphate, alpha glucosidase, paranitrophenyl-d-glucopyranoside (pnpg), dimethyl sulfoxide (dmso) and anhydrous kbr instruments electric grinder (sumeet, india), analytical balance (kern alj 120-4 germany), pressure cooker (prestige india), centrifuge machine, magnetic stirrer, ph meter, thermometer, electric oven, microplate reader (spectra max m5, molecular devices, ca, usa), malvern zetasizer nano zs apparatus (malvern instruments ltd., malvern, uk), field emission scanning electron microscope (hitachi su6600 fe-sem), uv-vis spectrophotometer (agilent technologies, germany. cary 60), ft-ir (fourier transform infrared) spectrometer (perkinelmer, l 1600300 spectrum two lita, liantrisant, uk). preparation of cinnamon quills dried quills of cinnamomum zeylanicum (which were obtained from cinnamon research institute, thihagoda, matara) were ground into fine powder and stored at 10◦c. preparation of the extract cinnamon powder (10.00 g) was mixed with distilled water (200.0 ml) and digested under pressure for 10 minutes using a pressure cooker. the extract was filtered through a muslin cloth. the filtrate was centrifuged at 3000 rpm for 15 minutes and then the supernatant was used. preparation of cinnamon loaded microparticles (clmp) based on previous research studies, two different bsa solutions and two different crosslinking agents were used to prepare four products as mentioned in the table 01. ph values of bsa solutions were set to the required value by using naoh (1 m) and hcl (1 m) solutions. table 1. bsa solutions and crosslinking agents which were used to prepare microparticles. product bsa solution crosslinking agent concentration ph a 20.00 mg/ ml 9 citric acid b 20.00 mg/ ml 9 ascorbic acid c 8.85 % w/v 5 citric acid d 8.85 % w/v 5 ascorbic acid for the synthesis of cinnamon loaded bsa microparticles, the aqueous cinnamon extract (4.00 ml) was mixed with ethanol (16.00 ml). the mixture was added to a bsa solution (4.00 ml) at 1.00 ml/ minute addition rate while constantly stirring the bsa solution at 600 rpm. during the addition process the temperature of the system was maintained at around 4 ◦c. then the crosslinking agent (8 %w/v, 230 µl) was added. the wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 243 reaction mixture was stored for twenty-four hours at 4 ◦c. the microparticle bearing solution was centrifuged at 3000 rpm for 30 minutes and the microparticles were collected. the particles were washed with distilled water and dried at 50 ◦c until a constant weight was observed. the particles were stored at 4 ◦c for further analyses. determining the yield of clmp weight of the product was measured. total concentration of all compounds in cinnamon extract was determined by evaporating cinnamon extract (1.00 ml) and weighing the remainder. the yield was calculated according to the following equation. yield = weight of the product (g) weight of used bsa + weight of cinnamon extract × 100 determining antidiabetic activity of clmp antidiabetic activity of the pressured water extract of cinnamon quills, positive control acarbose and the clmp were determined by carrying out in-vitro alpha amylase inhibition assay and alpha glucosidase inhibition assay. the antidiabetic activity of the products was compared with the antidiabetic activity of the acarbose and the crude extract. alpha amylase inhibition assay alpha amylase inhibition activity of clmp, pressured water extract of cinnamon and acarbose were determined by following the method described by aiyegoro et al. (2017) with slight modifications. (aiyegoro et al., 2017) alpha amylase enzyme (0.05 mg ml-1, 250 µl) in sodium phosphate buffer (0.02 m, ph 6.9) was added to the sample (500 µl). the mixtures were incubated at room temperature for 15 minutes. then starch solution (1 %, 250 µl) in sodium phosphate buffer (0.02 m, ph 6.9) was added. the reaction mixtures were incubated at room temperature for another 15 minutes. dns reagent (250 µl) was added and boiled for 5 minutes, and absorbance was measured at 540 nm by microplate reader. the same procedure was repeated by adding sodium phosphate buffer (0.02 m, ph 6.9, 250 µl) instead of alpha amylase enzyme. in addition to that, the above procedure was repeated for two other reaction mixtures. one amongst them was prepared by adding sodium phosphate buffer (0.02 m, ph 6.9, 500 µl) instead of plant extract and the other was prepared by adding sodium phosphate buffer (0.02 m, ph 6.9, 750 µl) instead of both plant extract and the enzyme. the percentage inhibition of alpha amylase enzyme was calculated using the following formula. ic50 values on alpha amylase enzyme were determined by using graphpad prism 8 software, based on inhibitory percentage values. inhibition (%) = (p-q) (r-s) (p-q) × 100 p : absorbance at 540 nm without inhibitor and with enzyme q : absorbance at 540 nm without inhibitor and enzyme r : absorbance at 540 nm with inhibitor and enzyme s : absorbance at 540 nm with inhibitor and without enzyme alpha glucosidase inhibition assay alpha glucosidase inhibition activity of the products, pressured water extract of cinnamon and acarbose were determined by following the method described by jayawardena et al. (2018) with slight modifications. [14] alpha glucosidase enzyme (0.5 u ml-1, 25 µl) in sodium phosphate buffer (0.10 m, ph 6.8) was added to the sample (50 µl) and incubated at 37 ◦c for 10 minutes. p-npg (1.25 mm, 25 µl) in sodium phosphate buffer (0.10 m, ph 6.8) was added and incubated at 37 ◦c for 10 minutes and the absorbance was measured at 405 nm by a microplate reader. the same procedure was repeated by adding sodium phosphate buffer (0.10 m, ph 6.8, 25 µl) instead of the enzyme, sodium phosphate buffer (0.10 m, ph 6.8, 50 µl) instead of cinnamon extract and sodium phosphate buffer (0.10 m, ph 6.8, 75 µl) instead of both cinnamon extract and enzyme. the percentage inhibition of the alpha glucosidase enzyme was calculated using the following formula. ic50 values on alpha glucosidase enzyme were determined by using graphpad prism 8 software, based on inhibitory percentage values. inhibition (%) = (p-q) (r-s) (p-q) × 100 p : absorbance at 405 nm without inhibitor and with enzyme q : absorbance at 405 nm without inhibitor and enzyme r : absorbance at 405 nm with inhibitor and enzyme s : absorbance at 405 nm with inhibitor and without enzyme characterization of microparticles solubility clmp (5.0 mg) was added to 5.00 ml of distilled water and stirred for 30 minutes at 600 rpm at room temperature. after that the mixture was centrifuged at 10 000 rpm for 15 minutes. pellet was dried at 100 ◦c in an oven until a constant weight was observed. the procedure was triplicated. the solubility was calculated according to the following formula. 244 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 solubility (%) = weight of the product added (g) weight of the pellet (g) weight of the product added (g) × 100 cinnamon loading and entrapment efficiency a concentration series from the pressured water extract of cinnamon quills was prepared. absorbance of the concentration series was measured at 310 nm. a graph of absorbance at 310 nm vs concentration of cinnamon extract was plotted (akkawi et al., 2015). absorbance of the supernatants which remained after the separation of the product containing pellets, were measured at 310 nm. by comparing the absorbance value of each supernatant with the standard curve, the concentration of remaining cinnamon compounds in the supernatants was found. the procedure was triplicated. cinnamon loading and entrapment efficiencies were determined by using following formulae. weight of cinnamon loaded = weight of cinnamon added weight of cinnamon in supernatant cinnamon loading (%) = weight of cinnamon loaded total weight of the product × 100 cinnamon entrapment efficiency (%) = weight of cinnamon loaded weight of cinnamon added × 100 determination of particle size and zeta potential the particle size, polydispersity index (pdi) and zeta potential of the product were determined with the malvern zetasizer nano zs apparatus. an aqueous suspension of clmp was diluted 1:100 with ultrapure water and the solution was placed in a disposable polystyrene cuvette and the particle size measurement was obtained. the solution was also placed in a folded capillary zeta cell and the zeta potential measurement was obtained. both procedures were triplicated. morphological observations field emission scanning electron microscope was used to visualize the morphology and shape of the oven-dried product. sample was mounted onto the sample stub using carbon tapes and the images were taken after gold sputter coating for 15 seconds. uv-visible absorbance spectra the uv-visible absorption spectra were analyzed using a uv-visible spectrophotometer from 200 to 500 nm within a 1 cm quartz cell. a 0.8 mg/ml synthesized nanoparticle sample was prepared after dissolving it in dmso and its absorbance spectrum was obtained. it was then compared with the absorbance spectra of 1 mg/ml pure bsa, 0.07 mg/ml aqueous cinnamon extract and 0.8% w/v citric acid. the absorbance spectrum of distilled water was subtracted from all sample spectra. ft-ir spectroscopy the molecular characteristics of the product were examined and compared with that of pure bsa, oven dried aqueous cinnamon extract and pure cross-linking agent, citric acid using an ft-ir spectrometer. each sample was mixed with anhydrous kbr in a 1:10 ratio and ground using a motor and pestle until a fine powder was obtained. a small portion of the powder was placed in the pellet forming mold and pressed under pressure. then the pellet was placed in the ft-ir spectrometer and scanned in the wavenumber range of 750-4000 cm-1. statistical analysis the obtained data were statistically analysed by one-way analysis of variance (anova) using minitab software package. the results were expressed in the form of mean ± standard deviation of triplicate determinants. the level of significance was taken at 5% confidence interval (p < 0.05). results and discussion physical appearance of the products table 2. yield and morphology of the products. product yield (%) morphology a 71.50 brown colour powder b 75.36 brown colour powder c 69.62 brown colour gel d 67.99 brown colour gel wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 245 antidiabetic activity table 3. ic50 values on alpha amylase and alpha glucosidase enzymes. sample ic50 value on alpha amylase enzyme (µg/ml) ic50 value on alpha glucosidase enzyme (µg/ml) pressured water extract 131.27 (±1.64) e 141.25 (±0.21) b acarbose 140.37 (±1.17) c 224.45 (±0.21) a a 117.60 (±1.73) f 119.25 (±0.07) e b 134.97 (±0.32) d 112.40 (±0.57) f c 151.00 (±0.76) a 137.75 (±0.21) c d 146.23 (±0.56) b 122.70 (±0.28) d means that do not share a letter are significantly different from each other. (p < 0.05) figure 1. the graph of percentage inhibition of α amylase enzyme (%) vs concentration (µg/ ml). figure 2. the graph of percentage inhibition of α glucosidase enzyme (%) vs concentration (µg/ ml). characterization of the products table 4. solubility, cinnamon loading percentage and cinnamon entrapment percentage of the products. product solubility in water (%) cinnamon loading percentage (%) cinnamon entrapment efficiency (%) a 53.00 (±1.00) a 3.69 (±0.01) a 77.97 (±0.03) a b 49.00 (±1.00) a 3.33 (±0.10) b 74.11 (±0.02) b c 30.33 (±1.53) b 0.76 (±0.00) c 67.51 (±0.02) c d 22.33 (±2.52) c 0.72 (±0.01) c 62.03 (±0.05) d means that do not share a letter are significantly different from each other. (p < 0.05) table 5. particle size, pdi and zeta potential of the product. parameter mean ± standard deviation particle size 1.281 ± (0.004) µm pdi 0.460 (± 0.018) zeta potential -1.09 (±0.03) mv figure 3. sem image of clmp. 246 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 figure 4. uv-visible absorbance spectra of pure bsa, aqueous cinnamon extract, citric acid, and clmp. figure 5. ft-ir spectra of pure bsa, aqueous cinnamon extract, pure citric acid, and clmp. discussion micro formulations are an exciting novel method that could be used to improve the stability, bioavailability, poor solubility and absorption of natural products (singh et al., 2010). even though cinnamon has many therapeutic properties, the desired effect could be lost in processing cinnamon products. encapsulating natural products into various matrices is a common technique used to overcome the loss of the active ingredients in natural products during processing. in the current study, 4 different products (product a, b, c and d) were formulated by encapsulating cinnamon into bsa, as mentioned in table 1. products a and b were in powder form while products c and d were gel like. for the preparation of products c and d, the ph value of the bsa solution was set to 5. the isoelectric point of bsa is around 4.7 (amritham et al., 2016). hence ph value of the used bsa solution was very close to the isoelectric point of bsa. therefore, at ph 5, the bsa molecules show less tendency to become ionized. hence the aggregation of bsa molecules with each other occur rapidly in order to minimize the contact with highly polar aqueous medium. this may be the reason for the formation of gel like product instead of a powder form product. for the preparation of products a and b, the ph of the bsa solution was set to 9. at that ph value the protein molecules are ionized. therefore, they tend to repulse each other. this results the separation of the protein particles from each other as much as possible. hence the resulted particles become smaller and smaller (constantine et al., 2013; amritham et al., 2016). this can result in a powder like fine particle in microscale. according to table 2, the highest yield has been obtained for the product b which was followed by products a, c and d. this suggests that the highest yield 230 280 215 280 225 215 235 285 -0,1 0,4 0,9 1,4 1,9 2,4 2,9 3,4 200 250 300 350 400 450 500 a b so rb a n ce wavelength (nm) absorbance of pure bsa absorbance of cinnamon extract absorbance of citric acid absorbance of cinnamon microparticles 0 20 40 60 80 100 7501250175022502750325037504250 t ra n sm it ta n ce ( % ) wavenumber (cm-1) % transmittance of pure bsa % transmittance of citric acid % transmittance of cinnamon extract % transmittance of cinnamon microparticles wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 247 for clmp can be obtained by using ascorbic acid as the cross-linking agent if the ph value of the used bsa solution is around ph 9. the antidiabetic activity of natural products can be measured using the inhibitory potential of alpha-amylase and alpha-glucosidase enzymes. product a has shown the lowest ic50 value [117.60 (±1.73) µg/ml] (table 3). that means the product a has the highest inhibitory activity on alpha amylase enzyme. both products a and b have shown higher inhibitory activity than the crude cinnamon extract and the positive control acarbose. this confirms that the activity of cinnamon extract become enhanced when encapsulated. since products c and d are not powder form products, they may not be in microscale. that may be a reason for their low alpha amylase inhibitory activity. the graph of percentage inhibition on alpha amylase enzyme vs. concentration is also shown in figure 1. cinnamon loading percentage and the cinnamon entrapment efficiencies of the products also support the obtained results for the alpha amylase inhibitory assay (table 4). product a has shown the highest cinnamon loading percentage and cinnamon entrapment efficiency. that may be the reason for its higher activity than the other products. according to table 3, all the products have shown higher inhibitory activity on the alpha glucosidase enzyme in comparison with the crude cinnamon extract and acarbose. however, the inhibitory activity of products c and d is lower than that of products a and b. the higher activity of products a and b may be due to their smaller size and higher surface area. the lowest ic50 value [112.40 (±0.57) µg/ml] has been recorded for product b. therefore, the highest inhibitory activity on alpha glucosidase enzyme has been shown by product b even though the cinnamon loading percentage and cinnamon entrapment efficiency of product a is greater than that of product b. but the ic50 values for products a and b [119.25 (±0.07) µg/ml and 112.40 (±0.57) µg/ml respectively] are somewhat closer to each other. the graph of percentage inhibition on alpha glucosidase enzyme vs. concentration is shown in figure 2. the solubility of the products in water is also given in table 4. the water solubility of products a and b is greater than that of products c and d. as explained by amirtham et al. (2016), higher solubility of the product a and b may be due to their smaller size. smaller the size greater is the surface area. therefore, the solubility becomes greater (amritham et al., 2016; amighi et al., 2020). table 4 shows the cinnamon loading percentage of the products. the percentage values obtained for products c and d do not show statistically significant difference, but they show statistically significant difference to the results obtained for other products. the cinnamon loading percentage of products a and b is significantly greater than that of products c and d. the concentration of the bsa solutions which were used for the synthesis of products c and d were 8.8 % (w/v) while the concentration of the bsa solutions which were used for the synthesis of products a and b were 20 mg/ml. when the concentration of the initially used bsa solution becomes higher, the ratio between the loaded cinnamon compounds and bsa decrease. that is the reason for the observed results. the cinnamon loaded percentage of product a [3.69 (±0.01) %] is greater than that of product b [3.33 (±0.10) %]. according to amighi et al. (2020) the particles synthesized using citric acid as the cross-linking agent is greater in size compared to the particles synthesized using ascorbic acid as the cross-linking agent. when the size of the particle is greater, the particle can accommodate more bioactive compounds (amritham et al., 2016). that may be the reason for the slight increment of the cinnamon loading percentage of product a than product b. table 4 shows the cinnamon entrapment efficiencies of the synthesized products. the data obtained for all the products have shown statistically significant difference from each other. the highest cinnamon entrapment efficiency has been reported for product a, followed by product b, c and d. since products c and d were synthesized at ph 5, bsa molecules tend to aggregate with each other due to their less tendency to become ionized at ph values which are very close to their isoelectric point. the aggregated bsa molecules precipitated and formed the gel like product rapidly with the addition of the desolvating agent. the amount of time that the bsa particles spent in the solution while keeping contact with the dissolved cinnamon compounds was very little. most of the aggregated bsa particles precipitated even before the complete addition of the recommended volume of ethanol/ cinnamon extract mixture in previous studies [amirtham et al, (2016)] and the cross-linker. therefore, the probability to entrap more cinnamon compounds is comparatively less for products c and d. that may be the reason for the observed low cinnamon entrapment percentage for products c and d. products a and b were synthesized at ph 9 which is far away from the isoelectric point of bsa (4.7). hence bsa molecules were ionized and well separated from each other. at that ph value bsa particles do not move out from the solution phase rapidly. therefore, most of the bsa particles get the opportunity to stay in the solution phase until the complete addition of the ethanol/ cinnamon extract mixture and the cross-linker. this enhances the probability to entrap more cinnamon compounds during the formation of microparticles. hence the cinnamon entrapment efficiency of products a and b is greater than that of other products. however, the cinnamon entrapment efficiency of product a is little bit greater than that of product b. this may be again due to the greater size of the microparticles 248 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 which were synthesized using citric acid as the crosslinking agent. since the alpha amylase inhibitory activity, solubility, cinnamon loading percentage and cinnamon entrapment efficiency of product a is greater than other products, product a was selected for further studies. hence characterization using parameters such as particle size, surface charge, morphology, particle structure using ftir and cinnamon entrapment using uv-visible spectrophotometer were carried out only for product a. product a showed a mean particle diameter of 1.281 ± (0.004) µm (table 5). according to amighi et al. (2020), the particle size that is obtained under the conditions that were used in the synthesis of product a with citric acid as the cross-linking agent is 1.201 (±0.058) µm (amighi et al., 2020). as the mean particle size obtained for product a is very close to this value, it is clear that they have been formed effectively. according to danaei et al. (2018), the pdi of a sample with effective particle size distribution should be between 0.05-0.7. as per table 5, the pdi value of the product a falls within this range and therefore it can be stated that a homogenous population of microparticles have been synthesized which can act as safe, stable, and efficient microcarriers of cinnamon. the tendency of these bsa microparticles to accumulate in the target tissue, which depends on the particle size distribution, will be minimal (danaei et al., 2018). microparticles with a zeta potential between -10 and +10 mv are considered neutral, while microparticles with zeta potentials greater than +30 mv are considered strongly cationic and those with zeta potentials less than 30 mv are considered strongly anionic. since most cell membranes are negatively charged, zeta potential can strongly affect a microparticle’s tendency to penetrate membranes, with cationic particles displaying toxicity due to cell membrane disruption (clogston and patri, 2011). according to table 5, as the zeta potential of the synthesized microparticles is -1.09 mv, they are neutral and will easily penetrate the cell membranes without causing any toxicological effects. product a was also studied for morphology by sem. morphological analysis of the microparticles were carried out with fe-sem, and the obtained image is shown in figure 3. the sem micrograph revealed morphological aspects of microparticles with a spherical shape and uniform size. the uv-absorbance spectra of the cinnamon extract and clmp (figure 4) showed a peak at 215 nm, proving that the active compounds of the cinnamon extract have been successfully loaded into the microparticles. a peak corresponding to citric acid cannot be observed as it has been completely washed away during the washing step. the pure bsa spectrum showed two characteristic peaks at 230 nm and 280 nm. however, these two peaks were slightly shifted to 235 nm and 285 nm respectively, in the absorbance spectrum of clmp. this may have resulted due to the protein unfolding that may have occurred when the microparticle was dissolved in dmso, an organic solvent. this agrees with the findings of liu et al. (2009), which states that the spectrum shifts to shorter wavelengths as the polarity of the solvent decreases with the tryptophan residues buried in hydrophobic domains exhibiting a spectral shift of 5 to 20 nm. (liu et al., 2009). ft-ir spectroscopy was performed to the product in order to find out if any chemical bond formation has occurred between citric acid and bsa as well as between the active compounds of cinnamon and bsa during the preparation of microparticles, resulting in any conformational changes in the protein structure of microparticles. furthermore, it was performed to further verify if the cinnamon compounds have been successfully entrapped within the synthesized microparticles. the ft-ir spectrum of microparticles showed characteristic peaks that were also observed in the spectrum of the aqueous cinnamon extract (figure 5). peaks at 2750 cm-1 and 2850 cm-1 that may be due to the cho stretch of cinnamaldehyde, a peak at 1056 cm-1 and a peak at 3000 cm-1 that may be due to the c-o and oh stretches respectively, of cinnamyl alcohol, benzoic acid, benzyl alcohol and methoxyeugenol were observed in the spectra of both aqueous cinnamon extract and clmp. according to wariyapperuma et al. (2020), these are the major compounds that are found in the ccsw aqueous extract, and therefore this further proves that the active compounds present in ccsw accession have been successfully extracted and encapsulated within the synthesized microparticles. (wariyapperuma et al., 2020 ; jayawardena et al., 2018). according to xu et al. (2015), one to two carboxyl groups of one citric acid molecule can react with bsa, resulting in an increase in the total amount of carboxyl groups and a decrease in the total amount of amine groups in the cross-linked microparticles (xu et al., 2015). this explains the presence of a peak corresponding to the oh stretch and decrease in intensity of the peak at 3400 cm-1 corresponding to the n-h stretch in the microparticle spectrum. this means that interactions between bsa and citric acid have caused conformational changes in the protein structure of the synthesized microparticles. a similar observation has been made by amighi et al. (2020) during the synthesis of bsa microparticles using citric acid as the cross-linking agent (amighi et al., 2020) additionally, peaks at 1665 cm-1 and 1537 cm-1 observed in the pure bsa spectrum corresponding to the amide i and amide ii stretches respectively, were absent in the microparticle spectrum. hence, loading of active compounds of cinnamon have also induced conformational changes in the structure of the bsa protein. this means that the active compounds of cinnamon have interacted with the protein matrix of the synthesized microparticles via covalent bonds. this wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 249 result is similar to the results obtained by rani (2016) (rani, 2016). conclusions this study has proven that the antidiabetic activity of the pressured water extract of the quills of cinnamomum zeylanicum can be enhanced by micro-encapsulating the active compounds of the extract. for the microencapsulation of the bioactive compounds, bsa microparticles can be used as the microcarrier system. size of the microparticle, cinnamon loading percentage and cinnamon entrapment efficiency have a direct effect on the antidiabetic activity of the synthesized microparticles. if the size of the particle is in the microscale and the cinnamon loading percentage and cinnamon entrapment efficiency are higher, the activity of the particle becomes higher. the cross-linking agent, ph, and the concentration of the bsa solution which is used to prepare bsa microparticles can affect the abovementioned parameters and the solubility of the product in a significant manner. since the product ‘a’ has shown the highest alpha amylase inhibitory activity, cinnamon loading percentage, cinnamon entrapment efficiency and water solubility, it can be concluded that the clmp with higher antidiabetic activity and desired properties can be synthesized at ph 9, if the concentration of the bsa solution is 20 mg/ ml and the cross-linking agent is citric acid. characterization of product a revealed to have a spherical morphology, uniform size with effective particle size distribution and a neutral surface charge. the uv-visible absorbance spectrum of the product showed the successful entrapment of the cinnamon compounds within the microparticles and the ft-ir spectrum showed that the cross-linking agent, citric acid had caused conformational changes in the protein structure of bsa. it also further proved that the active compounds were successfully loaded into the synthesized microparticles which interacted with the protein matrix via covalent bonds. therefore, according to these characterization data of product a, it can be concluded that the clmp have been synthesized effectively, which can be used as a powder form antidiabetic nutraceutical. authors’ contributions: b.s. wanniarachchi: designed and performed the experiments, analyzed the data, prepared figures and/or tables and authored the article. h.m.w.k sathsarani: designed and performed the experiments, analyzed the data, prepared figures and/or tables and authored the article. b.m. jayawardena: planned and designed the experiments and gave final approval of the article. h.g.n. dewangani: designed the experiments and analyzed the data. competing interests: the authors declare that there are no competing interests. funding: this study was funded by the university grant rp/03/02/06/01/2021 which is gratefully acknowledged. references aiyegoro oa, coopoosamy rm, ijeh ii, ohanyerem pe, oyedemi bo, oyedemi so (2017) alpha-amylase inhibition and antioxidative capacity of some antidiabetic plants used by the traditional healers in southeastern nigeria. scientific world journal. akkawi m, attieh ha, jaber s, lafi sa, lutgen p, remeleh qa (2015) cinnamon bark water-infusion as an in-vitro inhibitor of β-hematin formation. journal of medicinal plant research 9(38). amighi f, djomeh ze, shahi mlm (2020) effect of different cross-linking agents on the preparation of bovine serum albumin nanoparticles. journal of the iranian chemical society 17:1223-1235. amirtham d, aniesrani ds, thangavel k (2016) preparation of curcumin loaded egg albumin nanoparticles using acetone and optimization of desolvation process. the protein journal 35:124-135. choudhury n, meghwal m, das k (2021) microencapsulation: an overview on concepts, methods, properties and applications in foods. food front 2(4):426–42. clogston jd, patri ak (2011) zeta potential measurement. in characterization of nanoparticles intended for drug delivery; mcneil, s. e., ed.; methods in molecular biology; humana press: totowa, nj 697:63–70. constantine gr, galappaththy p, katulanda p, pigera s, premakumara gs, ranasinghe p (2013) medicinal properties of ‘true’ cinnamon (cinnamomum zeylanicum): a systematic review. complementary and alternative medicine 13:275. danaei m, dehghankhold m, ataei s, hasanzadeh davarani f, javanmard r, dokhani a, khorasani s, mozafari mr (2018) impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. pharmaceutics 10 (2):e57. desoky e, gaber e, khalid s, mourad am, numair a, soud a (2012) antidiabetic and hypolipidemic effects of ceylon cinnamon (cinnamomum verum) in alloxan-diabetic rats. journal of medicinal plants research 6 (9):1685-1691. gan sh, rao pv (2014) cinnamon: a multifaceted medicinal plant. evidence-based complementary and alternative medicine. https://doi.org/10.1155/2014/642942 hudaib m, mamoori fa, samydai aa, shehadeh m (2018) anti– diabetic activity of cinnamon: a review. international research journal of pharmacy and medical sciences (irjpms) 1 (5):43-45. jayawardena bm, kannangara sdp, subramanium ss, wariyapperuma wa nm, wijayasinghe ys (2018) pressured water extraction and solvent extraction of cinnamomum zeylanicum (l.) bark and evaluation of anti-diabetic properties. journal of the faculty of graduate studies, university of kelaniya, sri lanka 6. liu pf, avramova lv, park c (2009) revisiting absorbance at 230nm as a protein unfolding probe. anal. biochem 389(2):165–170. njagi jm, nzaro gm, piero mn (2014) diabetes mellitus – a devastating metabolic disorder. asian journal of biomedical and pharmaceutical sciences 4 (40):1-7 250 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 rani k (2016) fourier transform infrared spectroscopy (ftir) spectral analysis of bsa nanoparticles (bsa nps) and egg albumin nanoparticles (ea nps). res. j. chem. sci 6:29–36. riaz s (2009) diabetes mellitus: a review. scientific research and essay 4 (5):367-373 singh mn, hemant ksy, ram m, shivakumar hg (2010) microencapsulation: a promising technique for controlled drug delivery. res pharm sci 5(2):65–77. wariyapperuma wanm, kannangara s, wijayasinghe ys, subramanium s, jayawardena b (2020) in vitro anti-diabetic effects and phytochemical profiling of novel varieties of cinnamomum zeylanicum (l.) extracts. peerj. https://doi.org/10.7717/peerj.10070. wong cy, al-salami h, dass cr (2018) microparticles, microcapsules and microspheres: a review of recent developments and prospects for oral delivery of insulin. int j pharm 537(1):223–44. xu h, shen l, xu l, yang y (2015) controlled delivery of hollow corn protein nanoparticles via non-toxic crosslinking: in vivo and drug loading study. biomed. microdevices 17(1):8. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 89-96 | doi: 10.14421/biomedich.2023.121.89-96 issn 2540-9328 (online) cardiotoxicity effects of herbal medicine, a review article shahin gavanji department of biotechnology, faculty of advanced sciences and technologies, university of isfahan, iran. corresponding author gavanji.shahin2@gmail.com manuscript received: 04 october, 2022. revision accepted: 06 december, 2022. published: 13 december, 2022. abstract with the development of human's modern society more and more people tend to use complementary and alternative medicine (cam). toxicological studies indicated that many herbal medicines have direct toxic effects on the circulatory system or cardiovascular system and cause harmful effects on the body. cardiotoxicity or heart damage is a serious issue defined as heart electrophysiology d ysfunction, affecting the cardiac structure, and muscle damage that arises from the drug or chemical poisoning agents, that may lead to heart failure. the aim of this review article is to provide various information about the potential adverse effects of h erbal medicine on the cardiovascular system and introduce herbs that induced cardiac toxicity. to provide this review, all reported cases of cardia c toxicity induced by herbal medicines and natural products were collected through research articles and documents, and the most relevant articles, and books in various authentic search engines including scopus, pubmed, sid (scientific information database), science direct and google scholar, from 1984 to april 2022 were searched, and selected herbs with therapeutic properties which induce toxic effects on the cardiovascular system are introduced. in this review, scientific data regarding cardiotoxicity showed that 16 herbs from 11 families may increase cardiac toxicity. therefore, it's important to use herbal medicines and natural products under the guidance of medical professionals. keywords: cardiotoxicity; heart damage; herbal medicine; toxicity; adverse effects. introduction throughout history, using of herbal medicines has been playing an increasingly important role to treat various health challenges (chaachouay et al. 2022; liu et al. 2022). based on research, 80% of people all over the world, use complementary and alternative medicine (cam) and traditional medicine to treat different diseases (zhang et al. 2015; ardalan and rafieiankopaei 2013). furthermore, several studies demonstrated that about 20,000 herbal medicines are available in different markets all around the world (bent 2008), and a significant number of people rely on medicinal plants and use herbal remedies along with routine medication to treat various diseases, which increased the risk of herb–drug interactions (brantley et al. 2014; de smet 2007; gavanji and larki 2017). researchers have reported that inappropriate use of natural products can induce various damages including kidney injury (yang et al. 2018), nervous system injury (wattanathorn et al. 2008), liver injury (lin et al. 2019), skin injury (ernst 2000) and heart damages (sheth et al. 2015). toxicological studies indicated that many herbal medicines have direct toxic effects on the circulatory system or cardiovascular system and cause harmful effects on the body (fatima and nayeem 2016; valli and giardina 2002). cardiotoxicity or heart damage is a serious issue defined as heart electrophysiology dysfunction, affecting the cardiac structure and muscle damage that arises from the drug or chemical poisoning agents, that may lead to heart failure (sishi et al. 2013; chung et al. 2018). nowadays there is a growing concern over the cardiovascular adverse effects of herbal supplements and natural products and heart toxicity due to herbal medicines has recently become a source of interest (ernst 2003). several types of research demonstrated that several factors including inappropriate use of herbal remedies, using of herbal medicines for a long time, high-dose therapy and herb-herb or herb-drug interaction (cupp 1999; sun et al. 2021) may induce adverse reactions and side effects on the cardiovascular system, including arrhythmias, sympathomimetic activity, and hypertension and cause the irreversible damages which can lead to death. many people with a lack of knowledge, take herbal medicines based on their false beliefs and information to treat various diseases and increase the rate of heart adverse effects (alonso-castro et al. 2021). several studies have shown that various herbal medicines can interact with cardiac medications and increase the risk of bleeding or inactivate the antiplatelet drugs. also, inappropriate use of herbal medicine can cause problems cardiac cycle and pumping of blood throughout the body and may lead to various health problems. there has been https://doi.org/10.14421/biomedich.2023.121.89-96 90 biology, medicine, & natural product chemistry 12 (1), 2023: 89-96 an overwhelming weight of obsession on monitoring the cardiac patients who use herbal medicine and natural supplement without prescriptions, while they are taking heart medications, and scientific research studies indicated that many common medications including anticoagulants like aspirin (acetylsalicylic acid) (abebe 2002), warfarin (coumadin) (leite et al. 2016), and phenprocoumon (falithrom, marcoumar) (fasinu et al. 2012), also antiplatelet medications (prasugrel and clopidogrel) which have been used for cardiovascular diseases can interact with specific herbs (ma et al. 2018) and should be used under professional supervision. it is very important to increase the knowledge of different societies about the potential health risks of selfmedication and change our attitudes toward the inappropriate use of herbal medicines to reduce the risk of cardiotoxicity (alghadir et al. 2022; eichhorn et al. 2011; consolini, and ragone 2010). this review article provides various information about the potential adverse effects of herbal medicine on the cardiovascular system, and introduces herbs that induced cardiac toxicity. methods to provide this review, all reported cases of cardiac toxicity induced by herbal medicines and natural products were collected through research articles and documents, and the most relevant articles, and books in various authentic search engines including scopus, pubmed, sid (scientific information database), science direct and google scholar, from 1984 to april 2022 were searched. the search was organized with several combinations of search strategies with different keywords including cardiotoxicity, heart damage, herbal medicine, toxicity, and adverse effects. in this review article, the selected herbs with therapeutic properties which induce toxic effects on the cardiovascular system were introduced. cardiotoxic plants ranunculaceae ranunculaceae family which is called buttercup or crowfoot includes over 2,200 flowering plants, and has been widely used in traditional medicine (tm) for cancer treatment (christenhusz and byng 2016; hao et al. 2017). researchers have reported that three species of ranunculaceae family including aconitum napellus, aconitum kusnezoffii and aconitum carmichaeli showed the cardiotoxicity effects and people should be cautious to use these plants (table 1). a. kusnezoffii is a plant which has been traditionally used in the clinical practice as an antimicrobial and pain killer (su et al. 2021). based on a research study, 15 patients who consumed a. kusnezoffii for treatment of rheumatism and respiratory tract infection, encountered tachyarrhythmias, and ventricular tachycardia and fibrillation (tai et al. 1992). another species of this family is a. napellus which is used for muscle pain in folklore medicine (shoaib et al. 2020). a case report study has reported, that ingestion of a. napellus roots induced cardiotoxicity and a 54‐year‐old chinese man underwent ventricular tachycardia or arrhythmia and cardiac arrest (bonanno et al. 2020). the other member of ranunculaceae family is a. carmichaeli which shows wound healing and antiinflammatory properties (xia et al. 2019). according to a study, a 60-year-old man who consumed a. carmichaeli to treat his headache encountered aconitine poisoning which lead to myocardial necrosis (lin et al. 2011). zong and coworkers in 2019 demonstrated that a. carmichaeli contains diterpenoid alkaloids which have cardiotoxic potential (zong et al. 2019). another research study stated that aconitum plants have toxic effects on the cardiovascular system and two alkaloids including aconitine (ac) and mesaconitine (ma) play a crucial role in cardiotoxicity and create some serious problems (liu et al. 2019). dioscoreaceae dioscorea bulbifera is a medicinal plant of dioscoreaceae or yam family which has been traditionally used to treat wounds, rectal carcinoma, sore throat, and stomach or gastric cancer (chaniad et al., 2020). the result of a research study showed that extracting the rhizome of d. bulbifera, induced cardiotoxicity by pirarubicin accumulation which lead to necrosis, and muscle fiber damage, also indicated that cardiotoxicity effects of d. bulbifera is dose-dependent (sun et al. 2021). another study indicated that d. bulbifera can interact with doxorubicin (dox) in cancer treatment and cause dox accumulation which leads to cardiovascular injury including necrosis and breakage in cardiac muscle fibers (qu et al. 2019). ginkgoaceae ginkgo biloba also called ginkgo, belonging to the family ginkgoaceae, has been widely used in traditional medicine(tm) for the treatment of various diseases including asthma, cognitive problems, circulatory disorders, and vertigo (brondino et al. 2013). based on research, g. biloba has mild adverse effects and can cause heart palpitations (nguyen and alzahrani 2022). a study by bent and coworkers in 2005 showed that many people consume ginkgo to decrease platelet aggregation but according to various research ginkgo may interact with warfarin and increase the risk of bleeding (bent et al. 2005). however, a randomized controlled trials study, demonstrated that ginkgo may not interact with warfarin (engelsen et al. 2002; köhler et al. 2004). also, another study stated that concurrent use of gingko and warfarin will increase bleeding adverse effects and patients should be cautious to use of herbs and drugs simultaneously (stoddard et al. 2004). gavanji – cardiotoxicity effects of herbal medicine, a review article 91 table 1. cardiotoxicity effects of herbal medicine. no plant name family potential therapeutic application (traditional medicine) type of adverse effect references 1 atropa belladonna solanaceae treat of asthma, cough, and hay fever flushed and tachycardic (chadwick et al. 2015) 2 aconitum napellus ranunculaceae treat of high fever aconitum napellus (bonanno et al. 2020) 3 aconitum kusnezoffii ranunculaceae treat of sore throat, gout and rheumatism tachyarrhythmias, ventricular tachycardia and fibrillation (tai et al. 1992) 4 aconitum carmichaeli ranunculaceae pain relief cardiotoxicity (tai et al. 1992) 5 datura stramonium solanaceae treat of wounds, inflammation and rheumatism tachycardia (khoshnam et al. 2022; arefi et al. 2016) 6 dioscorea bulbifera dioscoreaceae treat of syphilis, ulcers, cough, leprosy and diabetes accumulation of thp in heart tissue (sun et al. 2021; qu et al. 2019) 7 digitalis purpurea plantaginaceae treat of asthma, epilepsy and tuberculosis heart failure (gerakaris et al. 2022) 8 ephedra distachya ephedraceae weight loss and obesity side effects on the cardiovascular system and cerebrovascular effects (gonzález-juárez et al. 2020; andraws et al. 2005) 9 ephedra sinica ephedraceae treat of asthma, bronchitis, and hay fever serious cardiovascular adverse effects (ekor 2014; nyska et al. 2005; dunnick et al. 2007) 10 ginkgo biloba ginkgoaceae treat of cognitive disorders and dementia increasing the bleeding tendency, heart palpitations (nguyen and alzahrani 2022; stoddard et al. 2004; bent et al. 2005) 11 glycyrrhiza glabra fabaceae treat of respiratory disorders, hyperdipsia and fever cardiac arrhythmias, pulmonary edema (deutch et al. 2019) 12 hypericum perforatum hypericaceae treat of anxiety and depression arrhythmia, hypertension (rubini et al. 2019; guru et al. 2021) 13 juniperus oxycedrus cupressaceae anti-inflammatory and antimicrobial effects tachycardia (achour et al. 2011) 14 mandragora officinarum solanaceae treat of asthma and hay fever serious supraventricular tachycardia (tsiligianni et al. 2009) 15 nerium oleander apocynaceae treat of cancer, painful menstrual periods arrhythmia (guru et al. 2021) 16 piper methysticum piperaceae treat of fever and respiratory disorders cardiovascular abnormalities, arrhythmia (toohey et al. 2013) solanaceae atropa belladonna, mandragora officinarum, and datura stramonium are three species of solanaceae family, which contain various alkaloids with different negative effects on the body. atropa belladonna is listed as a poisonous plant and called deadly nightshade. a. belladonna is traditionally used to treat bradycardia (slow heart rate) (shah et al. 2019). but according to a study, demonstrated that the consumption of a. belladonna in a 49-year-old woman can lead to anticholinergic toxic syndrome and heart rhythm disturbances (demirhan et al. 2013). also, the results of a study showed that ingestion of a. belladonna may cause abnormal heart rate (tachycardia) and other problems (chadwick et al. 2015). m. officinarum is another member of solanaceae family (al-maharik et al. 2022), which contains several phytochemicals including withanolides, tropane alkaloids (scopolamine and hyoscyamine), and coumarins (schlesinger et al. 2019; hanus et al. 2005). the root of this plant has been used in traditional medicine (tm) for the treatment of various diseases (monadi et al. 2021). a case study research indicated that two patients who consumed m. officinarum admitted to hospital with supraventricular tachycardia (svt) (tsiligianni et al. 2009). another important species of solanaceae family is d. stramonium which has been traditionally used to treat many diseases including fever, ulcers, wounds, toothache, rheumatism, and bronchitis (sharma et al. 2021). a research study indicated that alkaloids of d. stramonium can increased heart beat in mice (benouadah et al. 2016), furthermore, researchers have reported that the flower, leave and seed d. stramonium can induce the tachycardia (arefi et al. 2016; amini et al. 2012; disel et al. 2015). 92 biology, medicine, & natural product chemistry 12 (1), 2023: 89-96 ephedraceae ephedra distachya is commonly known as sea-grape belongs to the family ephedraceae (gonzález-juárez et al. 2020), which has a long history in folk medicine to treat bronchitis, asthma, low blood and colds (ghavam, and soleimaninejad, 2020). a study showed that ephedra alkaloids of e. distachya have negative effects on the cardiovascular system and adverse cerebrovascular effects (andraws et al. 2005). another important species of ephedraceae family is ephedra sinica, which is traditionally used to treat cold, asthma, and lung diseases (mei et al. 2021). dunnick and coworkers in 2007 demonstrated that a combination of e. sinica with caffeine can induce cardiotoxicity (dunnick et al. 2007). also, another research study stated that using ephedrine and caffeine affects the cardiovascular system and causes cardiotoxicity (howden et al. 2005; nyska et al. 2005). plantaginaceae digitalis purpurea is a species of plantaginaceae family, which has been traditionally used for the treatment of respiratory problems and heart failure (hf) (gerakaris et al. 2022; aswal et al. 2019; smith et al. 1984). so many people used d. purpurea to enhance their general health and well-being, but a study by gerakaris indicated that people without heart failure should not consume digoxin, and patients can take herbal medicinal products (hmps) with a doctor's supervision (gerakaris et al. 2022; lei et al. 2018). fabaceae glycyrrhiza glabra belonging to the family fabaceae is an important medicinal plant that has been widely used for healing respiratory disorders, gastroesophageal reflux disease, fever, liver diseases, hyperdipsia, rheumatism, tuberculosis, sexual debility, and infectious diseases (wahab et al. 2021; gavanji 2022). a study demonstrated that using of licorice (g. glabra) candies can induce arrhythmias (böcker and breithardt 1991), also another research by konik and coworkers in 2012 indicated that licorice can induce the coronary artery spasm (konik et al. 2012). licorice intake may have harmful effects on the cardiovascular system including cardiac arrhythmias and pulmonary edema, therefore it is essential to be cautious in using licorice (deutch et al. 2019). hypericaceae hypericum perforatum, a species of hypericaceae family, with various therapeutic potential, has been traditionally used to treat infectious diseases, anxiety, and burns, also several studies demonstrated that h. perforatum has anti-cancer activities (gavanji 2022; klemow et al. 2011). various types of research indicated that using h. perforatum may cause adverse events, and induce cardiotoxicity and herb-drug interactions. researchers have reported that using h. perforatum, may also lead to arrhythmia, and hypertension (cohen and ernst 2010; ernst 1999). cupressaceae juniperus oxycedrus is a member of cupressaceae family, which is traditionally used for the treatment of respiratory problems and different infectious diseases (tavares and seca 2018; karaman et al. 2003). a number of studies reported that extract of j. oxycedrus, has toxic effects on the body and may cause fever, tachypnea, and tachycardia (koruk et al. 2005; achour et al. 2011). apocynaceae one of the important ornamental and landscaping herbs is nerium oleander or nerium, which belongs to the family apocynaceae, and is widely cultivated in different areas. this plant has been traditionally used to treat eczema, opthalmia, gastrointestinal disturbances, and ringworm infections (akhtar et al. 2014). several types of research demonstrated that ingesting n.oleander cause different side effects including abnormal heart rhythm (bradycardia) and nervous system disorders (rubini et al. 2019). another study showed that oleander poisoning can lead to arrhythmia and other negative effects (guru et al. 2021). piperaceae piper methysticum, commonly known as kava, belonging to the family piperaceae, has been used for the treatment of various diseases including anxiety, restlessness, and psychological disorders (ernst 2007; boon and wong 2003). based on a study taking p. methysticum and psychotropic medications can increase the risk of herb-drug interaction and will be lifethreatening, also it has negative effects on the heart which lead to cardiovascular abnormalities, and arrhythmia (toohey et al. 2013). conclusions since the dawn of humanity, people have tried to seek new medications to treat various diseases. an issue of concern turning into an obsession for people all over the world is self-medication with herbal medicine which causes several harmful effects. in this review, all reported herbal medicines which induce cardiotoxicity were collected and introduced. the results of this review suggest that herbal medicine should be taken with the guidance of medical professionals. acknowledgements: i would like to thank dr. forough mortezaienejad for guidance on this project. gavanji – cardiotoxicity effects of herbal medicine, a review article 93 competing interests: the authors declare that there are no competing interests. references achour, s., abourazzak, s., mokhtari, a., soulaymani, a., soulaymani, r., & hida, m. 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immunogen; vaccines. introduction the creation of efficient vaccines is the ultimate objective in the fight against the spread and deadly nature of many of these infectious diseases. any vaccination that is secure, stable, and able to trigger a robust immune response with a minimal number of doses is desirable. some alternative vaccine types have demonstrated promising outcomes in their immunogenic profiles, despite the fact that many of the frequently used vaccinations attenuate or kill complete organisms. some of the top options for innovative vaccinations include subunit vaccines, often known as second-generation vaccines, and third-generation vaccines, which can be rnaor dna-based (doria-rose et al.,2021; braz gomes et al., 2021). numerous kinds of nanoparticles have innate physical characteristics that can cause an immunological reaction. it has been discovered that cytokine and antibody responses can be induced by gold, carbon, dendrimers, polymers, and liposome nanoparticles. due to these distinctive properties, nanoparticles can now be used as adjuvants to boost the immunogenicity of potential vaccines rather than just as vaccine delivery systems. the nanoparticles utilized for this purpose are commonly between 20 and 100 nanometers in size and are also known as nano-immune stimulators or activators (nm). inorganic nps like iron and silica, polymeric nps like chitosan and poly(lacticco-glycolic) acid (plga), cholesterol and lipid liposomes, and vlps are a few examples of well-known nano-immune stimulators (bachmann et al., 2010; bachmann et al., 1993; baranov et al., 2020). nps as drug delivery mechanisms, including antivirals, can inhibit viral reproduction in host cells by releasing antivirals from nps that block target cell receptors and releasing antivirals from absorbed nps that are released in a target cell. these main viral replication stages include transcription, replication of phage dna, synthesis of protein, and assembly. because of their potential to create prolonged antigen discharge after vaccination injection and to elicit an immune response, manuscript received: 16 march, 2023. revision accepted: 28 march, 2023. published: 21 june, 2023. https://doi.org/10.14421/biomedich.2023.121.343-361 344 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 nps based on organic and inorganic compositions have received extensive research as innovative vaccine approaches. nps can also create an antigen discharge that is controlled and low speed, creating a depot at the injection site that offers the option of antigen preservation over antigen degradation (de souza et al.,2021; abo-zeid et al., 2020; chen and 2020; chen et al., 2013). when compared to conventional vaccine techniques, nano carrier-based delivery systems have a number of benefits, including as greater adjuvant characteristics, superior stability, and increased protection against premature degradation. nanocarriers can shield the immunogen from early proteolytic degradation, allowing researchers to investigate alternative delivery methods when utilized to encapsulate or coat the surface of an antigen. nanocarriers can enhance the specificity of antigen delivery to apcs and lengthen the time for antigen presentation to these cells and other crucial immune cells essential for long-term immunity in addition to their protective properties. many different types of nanoparticles, including inorganic and polymeric nanoparticles, virus-like particles (vlps), liposomes, and self-assembled protein nanoparticles, have been investigated as potential antigen carriers for vaccines. viral antigens have been effectively delivered using biocompatible inorganic nanoparticles like gold, carbon, and silica (choi et al., 2021; chen et al., 2016). figure 1. schematic representation of different types of nanoparticles (nps) (heinz et al., 2017). materials and methods researchers conduct an examination of articles that are in accordance with the issue to be studied. determination of literature search keywords (search string based on pi (e) cot framework (p=patient/problem; i/e=exposure/implementation; c= control/comparison intervention, o=outcome, t=time) because a good question will help determine the scope of the review and help the strategy of finding the article. articles used in the literature review are obtained through the database of international journal providers through pubmed, from 2021-2023, clinical trials only. the author opens www.pubmed.com. researchers wrote keywords according to mesh (medical subject heading) namely “nanoparticle vaccine” and selected full text. 1. inclusion criteria population or sample is (nanoparticle-based vaccines). 2. exclusion criteria population or sample other (nanoparticle-based vaccines). results and discussion table 1. (alimehmeti, 2021), found that severe covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. moderate, transient reactogenicity after vaccination occurred more frequently in the mrna1273 group. serious adverse events were rare, and the incidence was similar in the two groups. thomas et al., 2021, found that vaccine efficacy of 86 to 100% was seen across countries and in populations with diverse ages, sexes, races, or ethnic groups, and risk factors for covid-19 among participants without evidence of previous infection with sars-cov-2. vaccine efficacy against the severe disease was 96.7% (95% ci, 80.3 to hussein et al. – nanotechnology-based vaccines 345 99.9). in south africa, where the sars-cov-2 variant of concern b.1.351 (or beta) was predominant, a vaccine efficacy of 100% (95% ci, 53.5 to 100) was observed. heath et al., 2021, found that a post hoc analysis showed an efficacy of 86.3% (95% ci, 71.3 to 93.5) against the b.1.1.7 (or alpha) variant and 96.4% (95% ci, 73.8 to 99.5) against non-b.1.1.7 variants. reactogenicity was generally mild and transient. the incidence of serious adverse events was low and similar in the two groups. houser et al., 2022, found that exploratory analyses identified neutralizing antibody responses elicited by the h2ha-ferritin vaccine in both h2-naive and h2exposed populations. furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal h1 and avian h5 subtypes, were induced in the h2-naive population by targeting the ha stem. this ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development. shinde et al., 2021, found that of 41 sequenced isolates, 38 (92.7%) were the b.1.351 variant. post hoc vaccine efficacy against b.1.351 was 51.0% (95% ci, -0.6 to 76.2) among the hiv-negative participants. preliminary local and systemic reactogenicity events were more common in the vaccine group; serious adverse events were rare in both groups. dunkle et al., 2022, found that ten moderate and 4 severe cases occurred, all in placebo recipients, yielding vaccine efficacy against the moderate-to-severe disease of 100% (95% ci, 87.0 to 100). most sequenced viral genomes (48 of 61, 79%) were variants of concern or interest largely b.1.1.7 (alpha) (31 of the 35 genomes for variants of concern, 89%). vaccine efficacy against any variant of concern or interest was 92.6% (95% ci, 83.6 to 96.7). reactogenicity was mostly mild to moderate and transient but was more frequent among nvx-cov2373 recipients than among placebo recipients and was more frequent after the second dose than after the first dose. wei et al., 2022, found that, with a combined endpoint of hbeag seroconversion, alanine aminotransferase normalization and hbv dna < 2,000 iu/ml, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. in stage 2, none (0 of 20) of 900 µg εpa-44-treated patients experienced a serologic relapse. the safety profile of εpa-44 was comparable to that of the placebo. sahin et al., 2021, found that most participants had a strong response of ifnγ+ or il-2+ cd8+ and cd4+ t helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. using peptide-mhc multimer technology, we identified several bnt162b2-induced epitopes that were presented by frequent mhc alleles and conserved in mutant strains. one week after the boost, epitope-specific cd8+ t cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating cd8+ t cells and were detectable (0.01-0.28%) eight weeks later. in summary, bnt162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses. datoo et al., 2021, found that participants vaccinated with r21/mm showed high titers of malaria-specific anti-asn-ala-asn-pro (nanp) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. titres waned but were boosted to levels similar to peak titers after the primary series of vaccinations after a fourth dose administered 1 year later. toback et al., 2022, found that no episodes of anaphylaxis or deaths were reported within the substudy. co-administration resulted in no change to the influenza vaccine immune response although a reduction in antibody responses to the nvx-cov2373 vaccine was noted. nvx-cov2373 vaccine efficacy in the substudy (ie, participants aged 18 to <65 years) was 87·5% (95% ci -0·2 to 98·4), and in the main study was 89·8% (95% ci 79·7-95·5). maruggi et al., 2022, found that high frequencies of spike-specific germinal center b, th0/th1 cd4, and cd8 t cell responses were observed in mice. local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. in hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following the sars-cov-2 challenge. therefore, the sars-cov-2 sam (lnp) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple sars-cov-2 variants, and has been advanced to phase 1 clinical evaluation. madhi et al., 2022, found that, of the serious adverse events that occurred among hiv-negative people (of whom, two [0·1%] were baseline sars-cov-2-negative and four [0·6%] were baseline sars-cov-2-positive) and people living with hiv-1 (for whom there were no serious adverse events) in the nvx-cov2373 group, none were assessed as related to the vaccine. among participants who were baseline sars-cov-2-negative in the nvxcov2373 group, the anti-spike igg geometric mean titers (gmts) and seroconversion rates (scrs) were lower in people living with hiv-1 (n=62) than in hivnegative people (n=1234) following the first vaccination (gmt: 508·6 vs 1195·3 elisa units [eu]/ml; scr: 51·6% vs 81·3%); and similarly so 14 days after the second vaccination for gmts (14 420·5 vs 31 631·8 eu/ml), whereas the scr was similar at this point (100·0% vs 99·3%). in the nvx-cov2373 group, antispike igg gmts 14 days after the second vaccination were substantially higher in those who were baseline sars-cov-2-positive than in those who were baseline sars-cov-2-seronegative for hiv-negative participants (100 666·1 vs 31 631·8 eu/ml) and for people living with hiv-1 (98 399·5 vs 14 420·5 eu/ml). this was also the case for angiotensin-converting enzyme 2 receptor-binding antibody and neutralizing antibody 346 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 titers. stuart et al., 2022, found that, between april 19 and may 14, 2021, 1072 participants were enrolled at a median of 9·4 weeks after receipt of a single dose of chad (n=540, 47% female) or bnt (n=532, 40% female). in chad-primed participants, geometric mean concentration (gmc) 28 days after a boost of sarscov-2 anti-spike igg in recipients of chad/m1273 (20 114 elisa laboratory units [elu]/ml [95% ci 18 160 to 22 279]) and chad/nvx (5597 elu/ml [4756 to 6586]) was non-inferior to that of chad/chad recipients (1971 elu/ml [1718 to 2262]) with a gmr of 10·2 (one-sided 98·75% ci 8·4 to ∞) for chad/m1273 and 2·8 (2·2 to ∞) for chad/nvx, compared with chad/chad. in bnt-primed participants, noninferiority was shown for bnt/m1273 (gmc 22 978 elu/ml [95% ci 20 597 to 25 636]) but not for bnt/nvx (8874 elu/ml [7391 to 10 654]), compared with bnt/bnt (16 929 elu/ml [15 025 to 19 075]) with a gmr of 1·3 (one-sided 98·75% ci 1·1 to ∞) for bnt/m1273 and 0·5 (0·4 to ∞) for bnt/nvx, compared with bnt/bnt; however, nvx still induced an 18-fold rise in gmc 28 days after vaccination. there were 15 serious adverse events, none considered related to immunization. aldrich et al., 2021, found that, as initially tested doses of 5 μg cv7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 μg doses which were better tolerated. no vaccine-related serious adverse events or withdrawals occurred. low, dose-dependent vnt responses were detectable from day 15, and by day 29%, 31%, and 22% of 1, 2, and 5 μg groups, respectively, had vnts ≥ 0·5 iu/ml, considered an adequate response by the who. after two 1 or 2 μg doses all recipients had titers ≥ 0.5 iu/ml by day 43. day 57 gmts were not significantly lower than those with rabipur, which elicited adequate responses in all vaccinees after two doses. cv7202elicited vnt was significantly correlated with rabv-gspecific igg antibodies (r2 = 0.8319, p < 0.0001). shinde et al., 2022, found that more solicited adverse events were reported by participants in the qniv group (551 [41·3%] of 1333) than in the iiv4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qniv group vs 212 [16·1%] in the iiv4 group). formica et al., 2021, found that, neutralizing antibody responses exceeded those seen in a panel of convalescent sera for both age groups. study limitations include the relatively short duration of safety follow-up to date and the current lack of immune persistence data beyond the primary vaccination regimen time point assessments, but these data will accumulate over time. khobragade et al., 2021, found that the socio-demographic characteristics of the two arms were comparable. about 9.9% of subjects in the recombinant rabies g protein vaccine arm and 17.2% of subjects in the reference arm reported adverse events. the sero-protection on day 14 was found to be 99.24% and 97.72% in the recombinant rabies g protein vaccine arm and reference vaccine arm respectively and the difference was statistically nonsignificant. august et al., 2021, found that further evaluation of the potential therapeutic use of mrna-1944 in clinical trials for the treatment of chikv infection is warranted. shinde et al., 2021, found that, overall, similar frequencies of solicited and unsolicited adverse events were reported in all treatment groups. kremsner et al., 2021, found that responses to 12 μg were comparable to those observed in convalescent sera from known covid-19 patients. mallory et al., 2022, found that 1610 participants were screened from aug 24, 2020, to sept 25, 2020. 1282 participants were enrolled, of whom 173 were assigned again to placebo (group a), 106 were re-randomized to nvx-cov2373-placebo (group b1), and 104 were rerandomized to nvx-cov2373-nvx-cov2373 (group b2); after accounting for exclusions and incorrect administration, 172 participants in group a, 102 in group b1, and 105 in group b2 were analysed for safety. following the active booster, the proportion of participants with available data reporting local (80 [82%] of 97 participants had any adverse event; 13 [13%] had a grade ≥3 events) and systemic (75 [77%] of 98 participants had any adverse event; 15 [15%] had a grade ≥3 events) reactions was higher than after primary vaccination (175 [70%] of 250 participants had any local adverse event, 13 [5%] had a grade ≥3 events; 132 [53%] of 250 had any systemic adverse event, 14 [6%] had a grade ≥3 events). local and systemic events were transient in nature (median duration 1·0-2·5 days). in the per-protocol immunogenicity population at day 217 (167 participants in group a, 101 participants in group b1, 101 participants in group b2), igg geometric mean titers (gmt) had increased by 4·7-fold and mn50 gmt by 4·1-fold for the ancestral sars-cov-2 strain compared with the day 35 titers.heath et al., 2023, found that the incidence of serious adverse events and adverse events of special interest were similar between groups. lovell et al., 2022, found that, neutralizing antibodies levels of the low-dose and high-dose ecv19 groups had frnt50 geometric mean values of 129 and 316, respectively. boosting responses and dose responses were observed. antibodies against the rbd correlated with antibodies against the spike and with virus neutralization. gatechompol et al., 2022, found that, an mrna vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic. masuda et al., 2022, found that, between 12 february 2021 and 17 march 2021, 326 subjects were screened, and 200 participants enrolled and randomized: nvx-cov2373, n = 150; placebo, n = 50. solicited adverse events (aes) through 7 days after each injection occurred in 121/150 (80.7%) and 11/50 (22.0%) participants in the nvx-cov2373 and placebo arms, respectively. in the nvx-cov2373 arm, tenderness and injection site pain were the most frequently reported solicited aes after each vaccination, irrespective of age. robust immune responses occurred with nvx-cov2373 hussein et al. – nanotechnology-based vaccines 347 (n = 150) by day 36: igg geometric mean fold rise (95% confidence interval) 259 (219, 306); seroconversion rate 100% (97.6, 100). no such response occurred with a placebo (n = 49). ishikawa et al., 2021, found that the most common adverse event (ae) was injection site skin reaction (86.7%). no grade 3 or higher drug-related aes were observed. no tumor responses were observed, and three patients (30%) had stable disease. the immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. comparing chp-ny-eso-1 alone to the poly-iclc combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. in a mouse model, adding an anti-pd-1 antibody to the combination of chp-ny-eso-1/poly-iclc suppressed the growth of ny-eso-1-expressing tumors. combining the vaccine with pd-1 blockade holds promise in human trials. li et al., 2022, found that, in younger participants, neutralizing antibody (nab) geometric mean titers (gmts) for the 10 and 30 µg dose levels declined from 233 and 254 (21 days after dose 2) to 55 and 87 at month 3, respectively, and to 16 and 27 at month 6, respectively. in older participants, nab gmts declined from 80 and 160 (21 days after dose 2) to 10 and 21 at month 6. overall, higher antibody titers were observed in younger participants, and the 30 µg dose induced higher levels of nab, which declined more slowly by month 6. no serious adverse events were reported in the vaccine group. wei et al., 2021, found that epnp, carrying the neutralizing epitope hla121-138, is a good candidate for a vaccine against sa. table 1. clinical studies using nanotechnology-based vaccines. authors title date and type of the study method results alimehmeti efficacy and safety of the mrna-1273 sars-cov-2 vaccine clinical trial 2021 this phase 3 randomized, observer-blinded, placebocontrolled trial was conducted at 99 centers across the united states. persons at high risk for sars-cov-2 infection or its complications were randomly assigned in a 1:1 ratio to receive two intramuscular injections of mrna-1273 (100 μg) or a placebo 28 days apart. the primary endpoint was the prevention of covid-19 illness with onset at least 14 days after the second injection in participants who had not previously been infected with sars-cov-2. severe covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. moderate, transient reactogenicity after vaccination occurred more frequently in the mrna-1273 group. serious adverse events were rare, and the incidence was similar in the two groups. thomas et al. safety and efficacy of the bnt162b2 mrna covid-19 vaccine through 6 months randomized controlled trial 2021 in an ongoing, placebocontrolled, observer-blinded, multinational, pivotal efficacy trial, we randomly assigned 44,165 participants 16 years of age or older and 2264 participants 12 to 15 years of age to receive two 30-μg doses, at 21 days apart, of bnt162b2 or placebo. the trial endpoints were vaccine efficacy against laboratory-confirmed covid-19 and safety, which were both evaluated 6 months after vaccination. vaccine efficacy of 86 to 100% was seen across countries and in populations with diverse ages, sexes, races, or ethnic groups, and risk factors for covid-19 among participants without evidence of previous infection with sars-cov-2. vaccine efficacy against the severe disease was 96.7% (95% ci, 80.3 to 99.9). in south africa, where the sars-cov-2 variant of concern b.1.351 (or beta) was predominant, a vaccine efficacy of 100% (95% ci, 53.5 to 100) was observed. 348 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 table 1. cont. authors title date and type of the study method results heath et al. safety and efficacy of nvx-cov2373 covid-19 vaccine clinical trial 2021 in this phase 3, randomized, observer-blinded, placebocontrolled trial conducted at 33 sites in the united kingdom, we assigned adults between the ages of 18 and 84 years in a 1:1 ratio to receive two intramuscular 5μg doses of nvx-cov2373 or placebo administered 21 days apart. the primary efficacy endpoint was virologically confirmed mild, moderate, or severe sars-cov-2 infection with an onset at least 7 days after the second injection in participants who were serologically negative at baseline. a post hoc analysis showed an efficacy of 86.3% (95% ci, 71.3 to 93.5) against the b.1.1.7 (or alpha) variant and 96.4% (95% ci, 73.8 to 99.5) against non-b.1.1.7 variants. reactogenicity was generally mild and transient. the incidence of serious adverse events was low and similar in the two groups. houser et al. safety and immunogenicity of a ferritin nanoparticle h2 influenza vaccine in healthy adults: a phase 1 trial clinical trial 2022 conducted a first-in-human, randomized, open-label, phase 1 clinical trial (nct03186781) to evaluate a novel ferritin (h2haferritin) nanoparticle influenza vaccine platform. the h2 subtype has not circulated in humans since 1968. adults born after 1968 have been exposed to only the h1 subtype of group 1 influenza viruses, which shares a conserved stem with h2. including both h2-naive and h2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant ha head domain. exploratory analyses identified neutralizing antibody responses elicited by the h2ha-ferritin vaccine in both h2-naive and h2-exposed populations. furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal h1 and avian h5 subtypes, were induced in the h2naive population by targeting the ha stem. this ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development. shinde et al. efficacy of nvxcov2373 covid-19 vaccine against the b.1.351 variant clinical trial 2021 in this phase 2a-b trial in south africa, we randomly assigned human immunodeficiency virus (hiv)-negative adults between the ages of 18 and 84 years or medically stable hiv-positive participants between the ages of 18 and 64 years in a 1:1 ratio to receive two doses of either the nvx-cov2373 vaccine (5 μg of recombinant spike protein with 50 μg of matrix-m1 adjuvant) or placebo. the primary endpoints were safety and vaccine efficacy against laboratory-confirmed symptomatic covid-19 at 7 days or more after the second dose among participants without previous sars-cov-2 infection. of 41 sequenced isolates, 38 (92.7%) were the b.1.351 variant. post hoc vaccine efficacy against b.1.351 was 51.0% (95% ci, -0.6 to 76.2) among the hiv-negative participants. preliminary local and systemic reactogenicity events were more common in the vaccine group; serious adverse events were rare in both groups. dunkle et al. efficacy and safety of nvx-cov2373 in adults in the united clinical trial 2022 conducted a phase 3, randomized, observer-blinded, placebo-controlled trial in the ten moderate and 4 severe cases occurred, all in placebo recipients, yielding vaccine efficacy against the hussein et al. – nanotechnology-based vaccines 349 authors title date and type of the study method results states and mexico united states and mexico during the first half of 2021 to evaluate the efficacy and safety of nvxcov2373 in adults (≥18 years of age) who had not had severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. participants were randomly assigned in a 2:1 ratio to receive two doses of nvxcov2373 or placebo 21 days apart. the primary objective was to determine vaccine efficacy against reverse-transcriptasepolymerase-chain-reactionconfirmed covid-19 occurring at least 7 days after the second dose. vaccine efficacy against moderate-to-severe disease and against different variants was also assessed. moderate-to-severe disease of 100% (95% ci, 87.0 to 100). most sequenced viral genomes (48 of 61, 79%) were variants of concern or interest largely b.1.1.7 (alpha) (31 of the 35 genomes for variants of concern, 89%). vaccine efficacy against any variant of concern or interest was 92.6% (95% ci, 83.6 to 96.7). reactogenicity was mostly mild to moderate and transient but was more frequent among nvx-cov2373 recipients than among placebo recipients and was more frequent after the second dose than after the first dose. wei et al. efficacy and safety of a nanoparticle therapeutic vaccine in patients with chronic hepatitis b: a randomized clinical trial clinical trial 2022 a two-stage phase 2 trial, which included a 76-week, randomized, double-blind, placebo-controlled trial (stage 1) and a 68-week open-label extension (stage 2), was conducted in 15 centers across china. with a combined endpoint of hbeag seroconversion, alanine aminotransferase normalization and hbv dna < 2,000 iu/ml, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. in stage 2, none (0 of 20) of 900 µg εpa44-treated patients experienced a serologic relapse. the safety profile of εpa-44 was comparable to that of the placebo. sahin et al. bnt162b2 vaccine induces neutralizing antibodies and polyspecific t cells in humans clinical trial 2021 extend a previous phase-i/ii trial report2 by presenting data on the immune response induced by bnt162b2 prime-boost vaccination from an additional phase-i/ii trial in healthy adults (18-55 years old). most participants had a strong response of ifnγ+ or il-2+ cd8+ and cd4+ t helper type 1 cell, which was detectable throughout the full observation period of nine weeks following the boost. using peptidemhc multimer technology, we identified several bnt162b2-induced epitopes that were presented by frequent mhc alleles and conserved in mutant strains. one week after the boost, epitope-specific cd8+ t cells of the early-differentiated effectormemory phenotype comprised 0.022.92% of total circulating cd8+ t cells and were detectable (0.01-0.28%) eight weeks later. in summary, bnt162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses. datoo et al. efficacy of a lowdose candidate malaria vaccine, r21 in adjuvant matrixm, with seasonal administration to children in burkina faso: a randomised clinical trial 2021 in this double-blind, randomized, controlled, phase 2b trial, the low-dose circumsporozoite protein-based vaccine r21, with two different doses of adjuvant matrix-m (mm), was given to children aged 5-17 months in nanoro, participants vaccinated with r21/mm showed high titres of malaria-specific anti-asn-ala-asn-pro (nanp) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. titres waned but were boosted to levels similar to peak titres after the 350 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results controlled trial burkina faso-a highly seasonal malaria transmission setting. three vaccinations were administered at 4-week intervals before the malaria season, with a fourth dose 1 year later. all vaccines were administered intramuscularly into the thigh. group 1 received 5 μg r21 plus 25 μg mm, group 2 received 5 μg r21 plus 50 μg mm, and group 3, the control group, received rabies vaccinations. children were randomly assigned (1:1:1) to groups 1-3. an independent statistician generated a random allocation list, using block randomisation with variable block sizes, which was used to assign participants. participants, their families, and the local study team were all masked to group allocation. only the pharmacists preparing the vaccine were unmasked to group allocation. vaccine safety, immunogenicity, and efficacy were evaluated over 1 year. the primary objective assessed the protective efficacy of r21 plus mm (r21/mm) from 14 days after the third vaccination to 6 months. primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for the inclusion of participants who received the wrong vaccine at any time point. primary series of vaccinations after a fourth dose administered 1 year later. toback et al. safety, immunogenicity, and efficacy of a covid19 vaccine (nvxcov2373) coadministered with seasonal influenza vaccines: an exploratory substudy of a randomized, observer-blinded, placebo-controlled, phase 3 trial clinical trial 2022 a planned exploratory substudy as part of the randomized, observer-blinded, placebocontrolled, phase 3 trial of the safety and efficacy of the covid-19 vaccine (nvxcov2373) by co-administrating the influenza vaccine at four study hospitals in the uk. approximately, the first 400 participants meeting the main study entry criteria-with no contraindications to influenza vaccination-were invited to join the substudy. participants of the main study were randomly assigned (1:1) to receive two intramuscular injections of either nvx-cov2373 (5 μg) or placebo (normal saline) 21 days apart; participants enrolled into the substudy were co-vaccinated with a single (0·5 ml) no episodes of anaphylaxis or deaths were reported within the substudy. coadministration resulted in no change to influenza vaccine immune response although a reduction in antibody responses to the nvx-cov2373 vaccine was noted. nvx-cov2373 vaccine efficacy in the substudy (ie, participants aged 18 to <65 years) was 87·5% (95% ci -0·2 to 98·4) and in the main study was 89·8% (95% ci 79·7-95·5). hussein et al. – nanotechnology-based vaccines 351 authors title date and type of the study method results intramuscular, age-appropriate (quadrivalent influenza cellbased vaccine [flucelvax quadrivalent; seqirus uk, maidenhead] for those aged 1864 years and adjuvanted trivalent influenza vaccine [fluad; seqirus uk, maidenhead] for those ≥65 years), licensed, influenza vaccine on the opposite deltoid to that of the first study vaccine dose or placebo. the influenza vaccine was administered in an open-label manner and at the same time as the first study injection. reactogenicity was evaluated via an electronic diary for 7 days after vaccination in addition to monitoring for unsolicited adverse events, medically attended adverse events and serious adverse events. immunogenicity was assessed with influenza haemagglutination inhibition and sars-cov-2 anti-spike protein igg assays. vaccine efficacy against pcr-confirmed, symptomatic covid-19 was assessed in participants who were seronegative at baseline, received both doses of the study vaccine or placebo, had no major protocol deviations affecting the primary endpoint, and had no confirmed cases of symptomatic covid-19 from the first dose until 6 days after the second dose (per-protocol efficacy population). immunogenicity was assessed in participants who received scheduled two doses of the study vaccine, had a baseline sample and at least one post-vaccination sample, and had no major protocol violations before unmasking (per-protocol immunogenicity population). reactogenicity was analyzed in all participants who received at least one dose of nvxcov2373 or placebo and had data collected for reactogenicity events. safety was analyzed in all participants who received at least one dose of nvxcov2373 or placebo. comparisons were made between participants of the substudy and the main study (who were not co-vaccinated for influenza). 352 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results maruggi et al. a self-amplifying mrna sars-cov-2 vaccine candidate induces safe and robust protective immunity in preclinical models clinical trial 2022 assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger rna (sam) vaccine, encoding a prefusion-stabilized spike antigen of sars-cov-2 wuhan-hu-1 strain and delivered by lipid nanoparticles (lnps). in mice, one immunization with the sam vaccine elicited a robust spikespecific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched sars-cov-2 wuhan strain as well as b.1.1.7 (alpha), b.1.351 (beta) and b.1.617.2 (delta) variants. high frequencies of spike-specific germinal center b, th0/th1 cd4, and cd8 t cell responses were observed in mice. local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. in hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following the sarscov-2 challenge. therefore, the sars-cov-2 sam (lnp) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple sars-cov-2 variants, and has been advanced to phase 1 clinical evaluation madhi et al. immunogenicity and safety of a sarscov-2 recombinant spike protein nanoparticle vaccine in people living with and without hiv-1 infection: a randomized, controlled, phase 2a/2b trial clinical trial 2022 in this randomized, observerblinded, multicentre, placebocontrolled phase 2a/b trial in south africa, participants aged 18-84 years, with and without underlying hiv-1, were enrolled from 16 sites and randomly assigned (1:1) to receive two intramuscular injections of nvx-cov2373 or placebo, 21 days apart. people living with hiv-1 were on stable antiretroviral therapy and had an hiv-1 viral load of few than 1000 copies per ml. vacc ine dosage was 5 μg sars-cov-2 recombinant spike protein with 50 μg matrix-m adjuvant, whereas 0·9% saline was used as a placebo injection (volume 0·5 ml each). all study staff and participants remained masked to study group assignment. we previously reported an interim analysis on the efficacy and safety of the nvx-cov2373 vaccine (coprimary endpoints). in this article, we present an expanded safety analysis for the full cohort of participants and report on the secondary objective of vaccine immunogenicity in the full cohort of people living with hiv-1 and in hiv-negative individuals overall and stratified by baseline sars-cov-2 serostatus. of the serious adverse events that occurred among hiv-negative people (of whom, two [0·1%] were baseline sars-cov-2-negative and four [0·6%] were baseline sars-cov-2positive) and people living with hiv-1 (for whom there were no serious adverse events) in the nvx-cov2373 group, none were assessed as related to the vaccine. among participants who were baseline sars-cov-2-negative in the nvx-cov2373 group, the antispike igg geometric mean titres (gmts) and seroconversion rates (scrs) were lower in people living with hiv-1 (n=62) than in hivnegative people (n=1234) following the first vaccination (gmt: 508·6 vs 1195·3 elisa units [eu]/ml; scr: 51·6% vs 81·3%); and similarly so 14 days after the second vaccination for gmts (14 420·5 vs 31 631·8 eu/ml), whereas the scr was similar at this point (100·0% vs 99·3%). in the nvxcov2373 group, anti-spike igg gmts 14 days after the second vaccination were substantially higher in those who were baseline sars-cov-2-positive than in those who were baseline sars-cov-2-seronegative for hivnegative participants (100 666·1 vs 31 631·8 eu/ml) and for people living with hiv-1 (98 399·5 vs 14 420·5 eu/ml). this was also the case for angiotensin-converting enzyme 2 receptor-binding antibodies and neutralizing antibody.titers. stuart et al. immunogenicity, safety, and reactogenicity of heterologous covid-19 primary clinical trial 2022 com-cov2 is a single-blind, randomized, non-inferiority trial in which adults aged 50 years and older, previously immunised with a single dose of chad or between april 19 and may 14, 2021, 1072 participants were enrolled at a median of 9·4 weeks after receipt of a single dose of chad (n=540, 47% female) or bnt (n=532, 40% female). hussein et al. – nanotechnology-based vaccines 353 authors title date and type of the study method results vaccination incorporating mrna, viral-vector, and protein-adjuvant vaccines in the uk (com-cov2): a single-blind, randomised, phase 2, non-inferiority trial bnt in the community, were randomly assigned (in random blocks of three and six) within these cohorts in a 1:1:1 ratio to receive a second dose intramuscularly (8-12 weeks after the first dose) with the homologous vaccine, m1273, or nvx. the primary endpoint was the geometric mean ratio (gmr) of serum sars-cov-2 antispike igg concentrations measured by elisa in heterologous versus homologous schedules at 28 days after the second dose, with a noninferiority criterion of the gmr above 0·63 for the one-sided 98·75% ci. the primary analysis was on the per-protocol population, who were seronegative at baseline. safety analyses were done for all participants who received a dose of the study vaccine. in chad-primed participants, geometric mean concentration (gmc) 28 days after a boost of sars-cov-2 anti-spike igg in recipients of chad/m1273 (20 114 elisa laboratory units [elu]/ml [95% ci 18 160 to 22 279]) and chad/nvx (5597 elu/ml [4756 to 6586]) was noninferior to that of chad/chad recipients (1971 elu/ml [1718 to 2262]) with a gmr of 10·2 (one-sided 98·75% ci 8·4 to ∞) for chad/m1273 and 2·8 (2·2 to ∞) for chad/nvx, compared with chad/chad. in bntprimed participants, non-inferiority was shown for bnt/m1273 (gmc 22 978 elu/ml [95% ci 20 597 to 25 636]) but not for bnt/nvx (8874 elu/ml [7391 to 10 654]), compared with bnt/bnt (16 929 elu/ml [15 025 to 19 075]) with a gmr of 1·3 (one-sided 98·75% ci 1·1 to ∞) for bnt/m1273 and 0·5 (0·4 to ∞) for bnt/nvx, compared with bnt/bnt; however, nvx still induced an 18-fold rise in gmc 28 days after vaccination. there were 15 serious adverse events, none considered related to immunization. aldrich et al. proof-of-concept of a low-dose unmodified mrna-based rabies vaccine formulated with lipid nanoparticles in human volunteers: a phase 1 trial clinical trial 2021 in this phase 1, multi-center, controlled study in belgium and germany we enrolled 55 healthy 18-40-year-olds to receive intramuscular injections of 5 μg (n = 10), 1 μg (n = 16), or 2 μg (n = 16) cv7202 on day 1; subsets (n = 8) of 1 μg an nd 2 μg groups received second doses on day 29. controls (n = 10) received the rabies vaccine, rabipur, on days 1, 8, and 29. safety and reactogenicity were assessed up to 28 days postvaccination using diary cards; immunogenicity was measured as rabv-g-specific neutralizing titers (vnt) by rffit and igg by elisa. as initially tested doses of 5 μg cv7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 μg doses which were better tolerated. no vaccine-related serious adverse events or withdrawals occurred. low, dose-dependent vnt responses were detectable from day 15, and by day 29%, 31%, and 22% of 1, 2 and 5 μg groups, respectively, had vnts ≥ 0·5 iu/ml, considered an adequate response by the who. after two 1 or 2 μg doses all recipients had titers ≥ 0.5 iu/ml by day 43. day 57 gmts were not significantly lower than those with rabipur, which elicited adequate responses in all vaccinees after two doses. cv7202-elicited vnt was significantly correlated with rabv-g-specific igg antibodies (r2 = 0.8319, p < 0.0001). shinde et al. comparison of the safety and immunogenicity of a novel matrix-madjuvanted nanoparticle influenza vaccine with a quadrivalent seasonal influenza vaccine in older adults: a phase 3 randomized controlled trial clinical trial 2022 this was phase 3 randomized, observer-blinded, activecomparator controlled trial done across 19 us community-based clinical research sites during the 2019-20 influenza season. participants were clinically stable and community-dwelling, aged at least 65 years and were randomisedin a 1:1 ratio using an interactive web response system to receive a single intramuscular dose of qniv or iiv4. the primary objective was more solicited adverse events were reported by participants in the qniv group (551 [41·3%] of 1333) than in the iiv4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qniv group vs 212 [16·1%] in the iiv4 group). 354 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results to describe the safety and show that qniv was immunologically non-inferior to iiv4. the primary outcomes were adverse events by treatment group and comparative haemagglutinationinhibiting antibody responses (assayed with egg-propagated virus) on day 28, summarised in terms of the ratio of geometric mean titres (gmtrqniv/iiv4) and seroconversion rate (scr) difference between participants receiving qniv or iiv4 for all four vaccine homologous influenza strains. the immunogenicity outcome was measured in the per-protocol population. non-inferiority was shown if the lower bound of the two-sided 95% ci on the gmtrqniv/iiv4 was at least 0·67 and the lower bound of the two-sided 95% ci on the scr difference -was at least -10%. formica et al. different dose regimens of a sarscov-2 recombinant spike protein vaccine (nvx-cov2373) in younger and older adults: a phase 2 randomized placebocontrolled trial clinical trial 2021 the phase 2 component of our randomized, placebo-controlled, phase 1 to 2 trial was designed to identify which dosing regimen of nvx-cov2373 should move forward into latephase studies and was based on immunogenicity and safety data through day 35 (14 days after the second dose). the trial was conducted at 9 sites in australia and 8 sites in the united states. participants in 2 age groups (aged 18 to 59 and 60 to 84 years) were randomly assigned to receive either 1 or 2 intramuscular doses of 5-μg or 25-μg nvx-cov2373 or placebo, 21 days apart. primary endpoints were immunoglobulin g (igg) anti-spike protein response, 7-day solicited reactogenicity and unsolicited adverse events. a key secondary endpoint was a wild-type virusneutralizing antibody response. after enrollment, 1,288 participants were randomly assigned to 1 of 4 vaccine groups or placebo, with 1,283 participants administered at least 1 study treatment. neutralizing antibody responses exceeded those seen in a panel of convalescent sera for both age groups. study limitations include the relatively short duration of safety follow-up to date and the current lack of immune persistence data beyond the primary vaccination regimen time point assessments, but these data will accumulate over time. khobragade et al. safety and immunogenicity of a novel three-dose recombinant nanoparticle rabies g protein vaccine administered as clinical trial 2021 a multi-centric, open-label, assessor-blind, center-specific block randomized, parallel design, phase iii clinical study was conducted among 800 subjects. the eligible subjects were randomized in a 2:1 ratio the socio-demographic characteristics of the two arms were comparable. about 9.9% of subjects in the recombinant rabies g protein vaccine arm and 17.2% of subjects in the reference arm reported adverse events. the seroprotection on day 14 was hussein et al. – nanotechnology-based vaccines 355 authors title date and type of the study method results simulated postexposure immunization: a randomized, comparatorcontrolled, multicenter, phase iii clinical study for the recombinant rabies g protein vaccine and the reference vaccine. subjects in the recombinant rabies g protein vaccine arm received three doses of vaccine on days 0, 3, and 7, while subjects in the reference vaccine arm received five doses of who-prequalified vaccine on days 0, 3, 7, 14, and 28. found to be 99.24% and 97.72% in the recombinant rabies g protein vaccine arm and reference vaccine arm respectively and the difference was statistically nonsignificant. august et al. a phase 1 trial of lipid-encapsulated mrna encoding a monoclonal antibody with neutralizing activity against chikungunya virus clinical trial 2021 this phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from january 2019 to june 2020 evaluated the safety and pharmacology of mrna-1944, a lipid nanoparticle-encapsulated messenger rna encoding the heavy and light chains of a chikv-specific monoclonal neutralizing antibody, chkv-24 further evaluation of the potential therapeutic use of mrna-1944 in clinical trials for the treatment of chikv infection is warranted. shinde et al. induction of crossreactive hemagglutination inhibiting antibody and polyfunctional cd4+ t-cell responses by a recombinant matrixm-adjuvanted hemagglutinin nanoparticle influenza vaccine randomized controlled trial 2021 a randomized, observer-blind, comparator-controlled (trivalent high-dose inactivated influenza vaccine [iiv3-hd] or quadrivalent recombinant influenza vaccine [riv4]), safety and immunogenicity trial of qniv (5 doses/formulations) in healthy adults ≥65 years. vaccine immunogenicity was measured by hemagglutinationinhibition assays using reagents that express wild-type hemagglutination inhibition (wthai) sequences and cellmediated immune responses. overall, similar frequencies of solicited and unsolicited adverse events were reported in all treatment groups. kremsner et al. safety and immunogenicity of an mrna-lipid nanoparticle vaccine candidate against sars-cov-2 : a phase 1 randomized clinical trial clinical trial 2021 this is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in hannover, munich, and tübingen, germany, and ghent, belgium. after giving 2 intramuscular doses of cvncov or placebo 28 days apart we assessed solicited local and systemic adverse events (ae) for 7 days and unsolicited aes for 28 days after each vaccination. immunogenicity was measured as enzyme-linked immunosorbent assay (elisa) igg antibodies to sars-cov‑2 s‑protein and receptor binding domain (rbd), and sarscov‑2 neutralizing titers (mn50). responses to 12 μg were comparable to those observed in convalescent sera from known covid-19 patients. mallory et al. safety and immunogenicity following a homologous booster dose of a sarscov-2 recombinant clinical trial 2022 this secondary analysis of phase 2, randomised study assessed a single booster dose of a sarscov-2 recombinant spike protein vaccine with matrix-m adjuvant (nvx-cov2373) in 1610 participants were screened from aug 24, 2020, to sept 25, 2020. 1282 participants were enrolled, of whom 173 were assigned again to placebo (group a), 106 were re-randomized to nvx-cov2373-placebo (group b1), 356 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results spike protein vaccine (nvx-cov2373): a secondary analysis of a randomised, placebo-controlled, phase 2 trial healthy adults aged 18-84 years, recruited from 17 clinical centres in the usa and australia. and 104 were re-randomized to nvxcov2373-nvx-cov2373 (group b2); after accounting for exclusions and incorrect administration, 172 participants in group a, 102 in group b1, and 105 in group b2 were analyzed for safety. following the active booster, the proportion of participants with available data reporting local (80 [82%] of 97 participants had any adverse event; 13 [13%] had a grade ≥3 events) and systemic (75 [77%] of 98 participants had any adverse event; 15 [15%] had a grade ≥3 events) reactions was higher than after primary vaccination (175 [70%] of 250 participants had any local adverse event, 13 [5%] had a grade ≥3 events; 132 [53%] of 250 had any systemic adverse event, 14 [6%] had a grade ≥3events). local and systemic events were transient in nature (median duration 1·0-2·5 days). in the per-protocol immunogenicity population at day 217 (167 participants in group a, 101 participants in group b1, 101 participants in group b2), igg geometric mean titers (gmt) had increased by 4·7-fold and mn50 gmt by 4·1-fold for the ancestral sarscov-2 strain compared with the day 35 titers. heath et al. safety and efficacy of the nvxcov2373 coronavirus disease 2019 vaccine at completion of the placebo-controlled phase of a randomized controlled trial clinical trial 2023 adults aged 18-84 years received 2 doses of nvxcov2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe covid-19 (onset from 7 days after second vaccination). participants who developed immunoglobulin g (igg) against nucleocapsid protein but did not show symptomatic covid-19 were considered asymptomatic. secondary outcomes included anti-spike (s) igg responses, wild-type virus neutralization, and t-cell responses. incidence of serious adverse events and adverse events of special interest were similar between groups. lovell et al. interim analysis from a phase 2 randomized trial of eucorvac19: a recombinant protein sars-cov-2 rbd nanoliposome vaccine clinical trial 2022 an initial study of phase 2 randomized, observer-blind, placebo-controlled trial to assess the immunogenicity, safety, and tolerance of ecv19 was carried out between july and october 2021. two hundred twenty-nine participants were enrolled at 5 hospital sites in south korea. healthy adults aged 19-75 without prior known exposure to covid-19 were vaccinated intramuscularly on day 0 and day 21. of the participants who received two vaccine doses according to protocol, 100 neutralizing antibody levels of the low-dose and high-dose ecv19 groups had frnt50 geometric mean values of 129 and 316, respectively. boosting responses and dose responses were observed. antibodies against the rbd correlated with antibodies against the spike and with virus neutralization. hussein et al. – nanotechnology-based vaccines 357 authors title date and type of the study method results received high-dose ecv19 (20 μg rbd), 96 received low-dose ecv19 (10 μg rbd), and 27 received a placebo. local and systemic adverse events were monitored. serum was assessed on days 0, 21, and 42 for immunogenicity analysis by elisa and neutralizing antibody response by focus reduction neutralization test (frnt). gatechompol et al. safety and immunogenicity of a prefusion nonstabilized spike protein mrna covid-19 vaccine: a phase i trial clinical trial 2022 seventy-two eligible volunteers, 36 of whom were aged 18-55 (adults) and 36 aged 56-75 (elderly), were enrolled. two doses of vaccine were administered 21 d apart at 10, 25 or 50 μg per dose (12 per group). the primary outcome was safety, and the secondary outcome was immunogenicity. all three dosages of chulacov19 were well tolerated and elicited robust dosedependent and age-dependent b and t-cell responses mrna vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic. masuda et al. safety and immunogenicity of nvx-cov2373 (tak-019) vaccine in healthy japanese adults: interim report of a phase i/ii randomized controlled trial clinical trial 2022 this phase 1/2, randomized, observer-blind, placebocontrolled trial conducted in japan (two sites), enrolled healthy japanese adults aged ≥ 20 years with no history/risk of sars-cov-2 infection and no prior exposure to other approved/investigational sarscov-2 vaccines or treatments. participants were stratified by age (< 65 or ≥ 65 years) and randomized to receive two doses of either nvx-cov2373 (5 μg sars-cov-2 rs; 50 μg matrixm1) or placebo, 21 days apart. primary outcomes were safety and immunogenicity assessed by serum igg antibody levels against sars-cov-2 rs protein on day 36. herein, we report the primary data analysis at 4 weeks after the second dose, ahead of the 12-month follow-up completion (data cut-off: 8 may 2021). between 12 february 2021 and 17 march 2021, 326 subjects were screened, and 200 participants were enrolled and randomized: nvxcov2373, n = 150; placebo, n = 50. solicited adverse events (aes) through 7 days after each injection occurred in 121/150 (80.7%) and 11/50 (22.0%) participants in the nvx-cov2373 and placebo arms, respectively. in the nvx-cov2373 arm, tenderness and injection site pain were the most frequently reported solicited aes after each vaccination, irrespective of age. robust immune responses occurred with nvx-cov2373 (n = 150) by day 36: igg geometric mean fold rise (95% confidence interval) 259 (219, 306); seroconversion rate 100% (97.6, 100). no such response occurred with a placebo (n = 49). ishikawa et al. safety and antibody immune response of chp-ny-eso-1 vaccine combined with poly-iclc in advanced or recurrent esophageal cancer patients clinical trial 2021 conducted a phase 1 clinical trial of chp-ny-eso-1 with poly-iclc in patients with advanced or recurrent esophageal cancer. chp-nyeso-1/poly-iclc (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. the primary endpoints were the most common adverse event (ae) was injection site skin reaction (86.7%). no grade 3 or higher drugrelated aes were observed. no tumor responses were observed, and three patients (30%) had stable disease. the immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. comparing chp-ny 358 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results safety and immune response. the secondary endpoint was tumor response. in total, 16 patients were enrolled, and six patients in each cohort completed the trial. eso-1 alone to the poly-iclc combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. in a mouse model, adding an anti-pd-1 antibody to the combination of chp-ny-eso-1/polyiclc suppressed the growth of nyeso-1-expressing tumors. combining the vaccine with pd-1 blockade holds promise in human trials. li et al. immune persistence and safety after sars-cov-2 bnt162b1 mrna vaccination in chinese adults: a randomized, placebo-controlled, double-blind phase 1 trial clinical trial 2022 immune persistence was determined at month 3 in 72 younger participants (aged 1855 years) and at month 6 in 70 younger and 69 older participants (aged 65-85 years). in younger participants, neutralizing antibody (nab) geometric mean titers (gmts) for the 10 and 30 µg dose levels declined from 233 and 254 (21 days after dose 2) to 55 and 87 at month 3, respectively, and to 16 and 27 at month 6, respectively. in older participants, nab gmts declined from 80 and 160 (21 days after dose 2) to 10 and 21 at month 6. overall, higher antibody titers were observed in younger participants, and the 30 µg dose induced higher levels of nab, which declined more slowly by month 6. no serious adverse events were reported in the vaccine group. wei et al. identification and application of a neutralizing epitope within alphahemolysin using human serum antibodies elicited by vaccination clinical trial 2021 collected sera from volunteers in a phase 1b clinical trial of a novel recombinant five-antigen sa vaccine (nct03966040). using a luminex-based assay, we characterized the human serologic response against hla, and identified hla121-138 as a neutralizing epitope. in addition, we successfully produced ferritin nanoparticles carrying the neutralizing hla121-138 epitope (epnp) in e. coli. epnp presented as homogenous nanoparticles in an aqueous solution. immunization with epnp elicited potent hemolysisneutralizing antibodies and conferred significant protection in a mouse model of sa skin infection. data suggest that epnp, carrying the neutralizing epitope hla121-138, is a good candidate for a vaccine against sa. conclusions vaccination has had a major impact on the control of infectious diseases. older vaccines, including those that successfully prevent measles and polio, work by stimulating the body's immune system and antibody production using a dead or weakened (attenuated) pathogen. however, in someone with a compromised immune system, an attenuated pathogen may still cause the disease, and a dead pathogen may not result in a strong enough or long-lasting immune response. many of these vaccines have been shown to elicit protective immunity against many diseases, but they are associated with certain challenges that limit their effectiveness in a clinical setting. dna and rna vaccines, for example, are cost-effective and associated with minimal infection risks but can be easily degraded as a result of delivery challenges to target sites. nanoscale size (<1000 nm) materials such as virus-like particles, liposomes, iscoms, polymeric, and non-degradable nanospheres hussein et al. – nanotechnology-based vaccines 359 have received attention as potential delivery vehicles for vaccines. the vaccine antigen is either encapsulated within or decorated onto the surface of the np. by encapsulating antigenic material, nps provide a method for delivering antigens that may otherwise degrade rapidly upon injection or induce a short-lived, localized immune response. in this review, we assess the potential of these delivery systems for the creation of new vaccines against a variety of infections and compare the usefulness of various np systems for the delivery of subunit vaccines. the effectiveness of vaccines is determined in large part by a number of physicochemical characteristics of nanoparticles. clinical trial studies have shown that nanocarriers can function as effective middlemen in the creation of vaccinations against a variety of diseases. development of np formulations that can deliver immunogens to apcs, particularly dcs, is crucial in this situation to promote efficient antigenspecific t-cell responses. via b-cell receptors, b cells are able to identify and react to microbial surface antigens. for the creation of vaccines against various diseases, artificial nps have been used to activate and clonally expand antigen-specific b-cells. comparing the delivery of exposed vaccine molecules to the encapsulation of antigens in virus-like particles (vlps), studies found that the vlps were able to elicit robust and long-lasting humoral responses. several research clearly proved the effectiveness of non-invasively given vaccines. concerns about particle toxicity, production challenges, and delivering antigens in their 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(2021). identification and application of a neutralizing epitope within alpha-hemolysin using human serum antibodies elicited by vaccination. molecular immunology, 135, 45–52. doi:10.1016/j.molimm.2021.03.028 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 273-280 | doi: 10.14421/biomedich.2023.121.273-280 issn 2540-9328 (online) phytochemical and antioxidant activity of blumea balsamifera and cordyline fruticosa based on ethnopharmacology knowledge of muara tae tribe, east kalimantan nur maulida sari1, farida aryani2,*, wartomo1, muhammad fikri hernandi1, erna rositah3, joko prayitno1 1department of forest product processing; 2department of plantation products technology; 3department of forest management, samarinda state agriculture polytechnic, kampus gunung panjang jalan samratulangi, samarinda 75131, east kalimantan, indonesia. corresponding author* faridaaryani@politanisamarinda.ac.id abstract plant use as traditional medicine is still widely practiced in indonesia. muara tae tribe people, west kutai regency are one of the regions that still rely on blumea balsamifera and cordyline fruticosa plants as traditional medicine. this study aims to determine the potential of blumea balsamifera and cordyline fruticosa leaves as medicinal plants with phytochemicals and antioxidants. phytochemical analysis was tested using harborne and kokate methods. antioxidant activity was evaluated b y dpph radical scavenging assay with slight modification. the results of the phytochemical analysis showed that the extracts of n-hexane, ethyl acetate, and ethanol from the leaves of blumea balsamifera and cordyline fruticosa contained alkaloids, tannins, and triterpenoids. antioxidant activity of blumea balsamifera leaves extract showed that the n-hexane extract display an ability to inhibit dpph free radical by 50% at 100 ppm concentration, while ethyl acetate and ethanol extracts display an ability by 77% and 81% at 50 ppm concentration. ic 50 value of ethyl acetate and ethanol extracts of blumea balsamifera leaves sequentially were 23.68 µg/ml and 17.59 µg/ml. antioxidant activity of cordyline fruticosa leaves extract showed that the n-hexane and ethyl acetate extract display an ability to inhibit dpph free radical by 45% and 56% at 100 ppm concentration, while ethanol extracts display an ability by 76% at 50 ppm concentration. ic50 value of ethyl acetate and ethanol extracts of cordyline fruticosa leaves sequentially were 73.72 µg/ml and 20.17 µg/ml. based on the results, blumea balsamifera and cordyline fruticosa leaves extracts had the potential to develop as natural antioxidants. keywords: blumea balsamifera; cordyline fruticosa; dpph; ethnopharmacology; phytochemical. introduction indonesia has unique flora and fauna that complement its, cultural and ethnic diversity. these ethnic groups occupy certain areas of the country (sreekeesoon & mahomoodally, 2014). each ethnic group has its own lifestyle and traditions, including foods, herbs, and spices. it is also very capable of using and preserving organic and ecological diversity. medicinal plants are used by the local community to treat illnesses, mainly due to health restrictions. the original knowledge of the use of medicinal herbs was probably passed down from generation to generation. this approach has so far succeeded in keeping knowledge alive (yaseen et al., 2015). therefore, local knowledge of medicinal plants has always been a source of research in testing the effects of plants and developing new therapeutic means (bolson et al., 2015). indonesia has the second highest biodiversity in the world after the amazon forests and a population of, people from more than 300 nationalities (elfahmi et al., 2014). most of the studies focused on the prevention of diseases or specific medicinal herbs in indonesia (silalahi et al., 2014). although indonesian researchers have studied traditional knowledge about the use of plants as medicine, most studies have not been published in international journals. about 30,000 medicinal plants grow and develop in indonesia, which covers 90% of medicinal plants in the asian region (syamsiah et al., 2016). the use of traditional medicine is increasing with population growth, healthy lifestyles, and degenerative diseases (triratnawati, 2016). also up to 21,4% of the indonesian population used traditional medicine to self-treat health problems. herbal medicine and massage are the most common treatments offered by traditional healers in indonesia (peltzer & pengpid, 2019). the consumption of traditional medicines in indonesia increased by 5,4% per year. efficiency, lower cost, easy availability, and dissatisfaction with traditional treatment methods are some of the reasons for choosing traditional treatment (riptanti et al., 2018). several studies in recent years manuscript received: 15 january, 2023. revision accepted: 14 march, 2023. published: 23 march, 2023. https://doi.org/10.14421/biomedich.2023.121.273-280 274 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 about medicinal plants use, informed that plants were the best sources of natural antioxidants such as phenolics, alkaloids, and flavonoids compound (zhao et al., 2018). an antioxidant agent from the plants was a huge resource to scavenging free radicals naturally. medicinal plants also prevent oxidative stress, maintain health, and disease and delay aging processes (nguyen et al., 2018). oxidative stress of human lipids is known to have various human health problems such as cardiovascular disease, cancer, diabetes, and others (truong et al., 2019). generally, antioxidants are divided into 2 types natural and synthetic antioxidants. natural antioxidants are widely used as free radical inhibitors, while synthetic antioxidants had a negative effect with long-term used (stoia & oancea, 2022). muara tae was local tribe exist in west kutai, east kalimantan, indonesia. the muara tae community known as a sub-tribe of dayak used medicinal plants as alternative drugs for healthcare treatment and diseases such as fever, diabetes, external wound, stomachache, and others. several studies about potential plant use by dayak people informed the plant used as natural antifungal, antioxidant, and antibacterial agents by bentian tribe (kusuma et al., 2016). blumea balsamifera and cordyline fruticose were the plant’s belief as medicinal plants by muara tae’s people. these plants had only limited attempts to explore the biological properties of plants regards their uses as medicine by the local people. the present study aims to explore the potential of blumea balsamifera and cordyline fruticosa leaves extract for its antioxidant activity from the muara tae tribe in indonesia (figure 1). materials and methods plant collection the leaves of blumea balsamifera and cordyline fruticosa were collected from muara tae village, west kutai, east kalimantan, indonesia. the samples were washed thoroughly with water to remove the extemporaneous and dried for about 3 days in the laboratory with air conditioning (a.c.) set for 20-25ºc. the samples were kept in a.c. room to keep the moisture content stable and milled with a blender. the powdered samples were prepared for further analysis. figure 1. morphology of blumea balsamifera and cordyline fruticosa plants (photo source: personal doc.). procedures maceration about 30 gr powdered samples of blumea balsamifera and cordyline fruticosa were extracts with n-hexane, ethyl acetate, and ethanol solvent at room temperature with continuous shaking on a shaker for 48 hours. following filtration of the suspension through whatman paper no.2 (maidstone, uk), the crude extracts of blumea balsamifera and cordyline fruticosa were evaporated in a rotary evaporator at 38-40 ºc and put in a vacuum over near dryness to yield the plant extract. preliminary phytochemical analysis the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were subjected to preliminary screening of phytochemical such as alkaloids, flavonoids, tannin, steroids, triterpenoids, carbohydrate, and saponin using some following standard procedures (harborne, 1998; kokate, 2001). alkaloids determination: 5 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera sari et al. – phytochemical and antioxidant activity of … 275 and cordyline fruticosa leaves were added to 2 ml hydrochloride acid, then 1 ml of dragendorff solution was added. the color changes in the solution indicated the presence of alkaloids (kokate, 2001). flavonoids determination: about 1 ml of the nhexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were drops of 1% sodium hydroxide. the presence of yellow color at extracts solution and were colorless after the addition of 1 % hydrochloride acid indicated the presence of flavonoids (kokate, 2001). tannin determination: 10 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were added 1 % lead ii acetate. the yellow precipitate reaction in the solution indicated the presence of tannin (kokate, 2001). steroids determination: 1 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves dropped about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the green or blue color changes in the solution indicated the presence of steroids (harborne, 1998). triterpenoids determination: 1 ml of the nhexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves dropped about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the red or purple color changes in the solution indicated the presence of steroids (harborne, 1998). carbohydrate determination: about 1 drop of molisch solution were added to 1 ml of the n-hexane, ethyl acetate and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves. then 1 ml of sulfuric acid was added thourgh the tube glass wall, slowly. the formation of purple ring between 2 layers indicated the presence of carbohydrates (harborne, 1998). saponins determination: 10 ml of hot distilled water was added to 1 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves. the solution was then cooled and shaken vigorously (10 seconds). a stable froth upon standing for 10 minutes after adding 1 drops of hydrochloride acid 2n indicated the presence of saponins (harborne, 1998). antioxidant assay test of antioxidants using 5 concentration samples was grouped into 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, and 6.25 ppm times of dilution, respectively. further, 3 mg of ascorbic acid was weighed, then dissolved in 1000 µl of ethanol solvent and regarded as a positive control. while the ethanol solvent was used as a negative control. about 33 µl sample was mixed in a glass tube with 467 µl of ethanol added, and 500 µl of 2,2-diphenyl-1picryhydrazyl (dpph) radical scavenging activity (shimizu et al., 2001). the mixing of the sample was stopped while the volume reached 1000 µl (1 ml). samples were incubated for 20 minutes with minimum light and a.c. set for 27-30 ºc. the antioxidant activity was determined by decolorization of dpph with a wavelength of 517 nm using a spectrophotometer. measurement was performed in the triplicate examination. the percentage of dpph free radical was calculated using the following equation: % dpph radical scavenging activity = ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙− ∆𝑠𝑎𝑚𝑝𝑙𝑒 ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 x 100 (1) the actual decrease in absorbance caused by the test was compared with positive controls. the values of ic50 (concentration giving 50% of inhibition) were calculated by a dose-inhibition curve over a linear range of by plotting the extract concentration and the corresponding washout effect (tuldjanah et al., 2021). results and discussion plant extracts blumea balsamifera and cordyline fruticosa leave were extracted using 3 different solvents n-hexane, ethyl acetate, and ethanol. maceration is done with yielded 2.18-4.60% extracts on the basis of sample dry weight from blumea balsamifera leaves, while cordyline fruticosa leaves yielded 1.09-5.20% extracts (table 1). table 1. yield of blumea balsamifera and cordyline fruticosa leaves extract. plants solvent extract yield (%) blumea balsamifera n-hexane 2.18 ethyl acetate 3.18 ethanol 4.60 cordyline fruticosa n-hexane 1.09 ethyl acetate 3.03 ethanol 5.20 the results showed the ethanol extracts of blumea balsamifera and cordyline fruticosa produce an extractive content more concentrated than the n-hexane and ethyl acetate extracts. phytochemical screening the n-hexane, ethyl acetate, and ethanol leaf extracts of blumea balsamifera and cordyline fruticosa were analyzed as the secondary metabolites. plants known to contain multiple phytochemical molecules such as terpenoids, lignins, phenolics, vitamins, tannins, and others metabolites as antioxidant agents. several studies showed many phytocompounds possess antidiabetic, antimicrobial, and anti-inflammatory activities (campbell-tofte et al., 2012). the pharmacological activities of secondary plant metabolites are known as large compounds as a source of medicinal agents (muthukrishnan & manogaran, 2018). phytochemical analysis of the n-hexane, ethyl acetate, and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa is presented in table 2. 276 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 table 2. phytochemical analysis of blumea balsamifera and cordyline fruticosa leaves extract. compounds blumea balsamifera cordyline fruticosa n-hexane ethyl acetate ethanol n-hexane ethyl acetate ethanol alkaloids + + + + flavonoids tannins + + + steroids triterpenoids + + carbohydrate saponins the phytochemical screening of blumea balsamifera revealed the presence of alkaloids and triterpenoids in the n-hexane extract, and tannins in the ethyl acetate extract, while the ethanol extract contained alkaloids and tannins. the n-hexane extract of cordyline fruticosa revealed the presence of alkaloids and triterpenoids, while the ethanol extract contained alkaloids and tannins. the bioactivity potency of plants was indicated based on the secondary metabolites compounds. the presence compounds of tannins, flavonoids, and alkaloids are known as antitumor, antibacterial, antivirus also had antioxidant activity, antimicrobial and anticancer (taşkın & taşkın, 2017). phenolic known as the largest compounds were found in plants and it’s an important compound for free radical scavenging, also contains the hydroxyl group compound. flavonoid, phenol, and tannin compound are known as the largest group of phenolic compounds and also had the abilities as antioxidant agents (sen et al., 2013). antioxidant activity free radical dpph scavenging of the n-hexane, ethyl acetate, and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa was determined by its hydrogen donating abilities. the inhibition of blumea balsamifera n-hexane leaves extracts displayed the ability to inhibit free radical dpph formation by 50% at 100 ppm concentration, while the ethyl acetate and ethanol extracts by 77% and 81% at 50 ppm concentration, respectively. the antioxidant activity of the n-hexane, ethyl acetate, and ethanol leaf extracts of blumea balsamifera is presented in figure 2. figure 2. antioxidant activity of blumea balsamifera leaves extracts against dpph. several studies about blumea balsamifera plants informed the plant extracts contained steroids, alkaloids, phenolic and saponins compounds used as traditional medicine for eczema, rheumatic, menstrual pain relief, flu, fever, asthma, diabetes cough, and diarrhea (pang et al., 2014). its also has biological activity as an antiinflammatory, anticancer, and antioxidant and also functions as an antimicrobial (nessa et al., 2010). the inhibition of cordyline fruticosa n-hexane and ethyl acetate leaf extract displayed the ability to inhibit sari et al. – phytochemical and antioxidant activity of … 277 free radical dpph formation by 45% and 56% at 100 ppm concentration, while the ethanol extracts by 76% at 50 ppm concentration. antioxidant activity of the nhexane, ethyl acetate, and ethanol leaves extracts of cordyline fruticosa is presented in figure 3. figure 3. antioxidant activity of cordyline fruticosa leaves extracts against dpph. several studies about cordyline fruticosa leaves plants, informed the plant contained saponins, tannins, flavonoids, polyphenol, and alkaloids compound which has biological activity as antioxidant, anti-inflammatory, antibacterial, anti-allergic, anticancer, and antivirus (dyary et al., 2014). this plant is also known to accelerate the wound healing process, the function of tannins compound as astringents uses shrink skin pores and bleeding minor stopped. the antioxidant activity of blumea balsamifera and cordyline fruticosa leaves extracts indicated plants had a source to inhibit free radical dpph. refers to the results, the ic50 of the ethyl acetate and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa extracts were determined as shown in table 3. table 3. the ic50 of blumea balsamifera and cordyline fruticosa leaves extract. blumea balsamifera cordyline fruticosa ethyl acetate ethanol ethyl acetate ethanol 23.68±19.69 17.59±18.71 73.72±14.42 20.17±17.59 the antioxidant activity of ic50 had a classification of the antioxidant activities compound based on the acquisition of the ic50 value as shown as table 4 (analianasari et al., 2022). table 4. the antioxidant activity of ic50 classification. inhibition value (ppm) category <50 extreme 50-100 strong 100-150 moderate 150-200 weak >200 fragile 278 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 figure 4. antioxidant activity of cordyline fruticosa leaves extracts against dpph. based on figure 4, the linear equation value for blumea balsamifera ethyl acetate extract y = 1.0237x+25.762, the calculation of the ic50 value for blumea balsamifera ethyl acetate extract obtained the following values: y = 1.0237x+25.762 (for y = 50), then the x value is 23.68 ppm. furthermore, based on the calculation of the regression curve in the ethanol extract of blumea balsamifera, the regression equation y = 0.964x + 33.039 (assuming the value of y = 50) is obtained, and the x value is 17.59 ppm. the ic50 value of the blumea balsamifera ethyl acetate and ethanol extracts had extreme ic50 antioxidant activity (<50 ppm). several studies about blumea balsamifera, especially the leaves part informed fresh and dry leaves had antioxidant activity with boiling water method, antidiabetic activity with hydro-ethanol extract, antitumor activity with essential oil extract and also anticancer activity of ethyl acetate extract fraction (widhiantara & jawi, 2021). the linear equation value for cordyline fruticosa ethyl acetate extract y = 0.3628x + 23.253 (assuming the value of y = 50), then the x value is 73.72 ppm. the ic50 value of cordyline fruticosa ethyl acetate extract is strong (50-100 ppm). while the regression of cordyline fruticosa ethanol extract equation y = 0.902x + 31.804 (with the value of y = 50) is obtained, the x value is 20.17 ppm. the ic50 value of cordyline fruticosa ethanol extract had extreme ic50 antioxidant activity (<50 ppm). the bioactive compounds of cordyline fruticosa methanol leaf extract have been isolated. the active compound had antitumor activity, antibacterial activity, and cytotoxic activity (assyifa et al., 2022). conclusions an ethnobotanically-selected medicinal plant, blumea balsamifera, and cordyline fruticosa has been investigated for their antioxidant activities. the results informed that the leaves extract of ethyl acetate and ethanol of the plant showed good antioxidant properties by the extreme-good category of ic50. further investigation is needed to find the responsible compound in the blumea balsamifera and cordyline fruticosa plants and explored the possibility of bioproduction for the active compounds, also developed for healthcare in the future. acknowledgments: the research was funded by the ministry of education, culture, research, and technology through penelitian dosen pemula scheme (junior lecturer research grants) 2022 under contract number 012/pl21.g/pg/2022. acknowledgments were conveyed to the muara tae village, west kutai, east kalimantan, indonesia who have supported the research by sharing ethnopharmacological information and plant specimens. authors’ contributions: in this research, nur maulida sari designed the research, supervised all processes, and wrote the manuscript. wartomo was observing and taking research samples. muhammad fikri hernandi controlled the samples and preparation of materials. joko prayitno 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(2018). effect of drying processes on prenylflavonoid content and antioxidant activity of epimedium koreanum nakai. journal of food and drug analysis, 26(2), 796–806. https://doi.org/10.1016/j.jfda.2017.05.011 https://doi.org/10.1016/j.jep.2014.12.053 https://doi.org/10.1016/j.jfda.2017.05.011 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 413-422 | doi: 10.14421/biomedich.2023.121.413-422 issn 2540-9328 (online) peptide fractions from pepsin-digested moringa oleifera seed proteins inhibit hemoglobin glycation and carbohydrate-hydrolyzing enzymes oluwafemi emmanuel ekun department of biochemistry, faculty of science, adekunle ajasin university, akungba akoko, ondo state nigeria, nigeria. corresponding author oluwafemi.ekun@aaua.edu.ng manuscript received: 29 december, 2022. revision accepted: 28 july, 2023. published: 04 august, 2023. abstract the multidirectional abilities of peptide digests and fractions obtained from the hydrolysis of food-based proteins have been investigated in recent times. this study aims to evaluate the effects of pepsin-derived moringa oleifera seed protein hydrolysates and fractions on hemoglobin glycation and the carbohydrases α-amylase and α-glucosidase. proteins were extracted from m. oleifera seeds and consequently digested using pepsin. the hydrolysates obtained were separated into fractions of <1 kd, 1-3 kd, and 3-5 kd ranges using size-exclusion chromatography and comparison with elution volumes of known standards. the activities of the hydrolysates and peptide fractions against both the non-enzymatic glycation of hemoglobin and the carbohydrases were determined in vitro. results revealed that the hydrolysate and its peptide fractions demonstrated varying abilities against the glycation of hemoglobin, with the unfractionated hydrolysate showing better activities (78.230 ± 0.774 % at a maximum concentration of 1.0 mg/ml) than its peptide fractions. also, the hydrolysates and fractions demonstrated higher inhibitory effects on -amylase (with all fractions displaying above 50% inhibition at a final concentration of 1.0 mg/ml) than against -glucosidase. kinetic analysis of a selected fraction showed that it inhibited α-amylase via a mixed mechanism (ki = 0.029 mg/ml) but displayed an uncompetitive mode for α-glucosidase inhibition (ki = 0.333 mg/ml). therefore, it is inferred that m. oleifera seed proteins encode potentially therapeutic peptide sequences that could be further processed to formulate potential antidiabetic agents. keywords: moringa oleifera; peptide; pepsin; hemoglobin; -amylase; α-glucosidase. introduction diabetes mellitus is an endocrine and metabolic disease, which is characterized by elevated plasma and urinary glucose levels as a direct result of inadequate insulin production and/or action (desmukh and jain, 2015). in its various forms, diabetes mellitus causes a plethora of derangements in the metabolisms of carbohydrates, proteins, and lipids (olusola and ekun 2019a). these cause a wide range of complications such as diabetic ketoacidosis, neuropathy, nephropathy, and cardiovascular and multiple organ damage at later stages of the disease (piero et al., 2014; olusola et al., 2018). one particular aspect in the pathogenesis of diabetes mellitus is the fact that persistent hyperglycemia leads to the formation of advanced glycated end products, (ages), as glucose complexes with proteins, and to a relatively lesser extent, lipids in a non-enzymatic, covalent fashion (ramasamy et al., 2005). proteins such as hemoglobin and albumin are frequently glycated (singh et al., 2014). these glycated end products are known to potentiate certain pro-inflammatory pathways via the production of reactive oxygen species. these ages bind to, and activate the receptor for advanced glycated end products, (rage), which in turn activate downstream signaling cascades which play important roles in the progression of diabetic nephropathy, neuropathy, and retinopathy (singh et al., 2014; perrone et al., 2020). current strategies employed in the management of diabetes mellitus have been aimed at controlling postprandial glucose levels (arise et al., 2016). these include insulin preparations (for type 1 diabetes mellitus) and a combination of lifestyle changes and/or the use of oral hypoglycemic drugs (in the case of type 2 diabetes mellitus), which act by modulating the activities of key proteins and enzymes involved in carbohydrate hydrolysis and transport (olusola and ekun, 2019a). enzymes such as α-amylase, αglucosidase, and dipeptidyl peptidase (iv) have been key pharmacologic targets of oral antidiabetic agents (rosenstock and zimmerman, 2007, arise et al., 2016, olusola and ekun, 2019b). however, owing to certain deleterious effects and cost of procurement on the part of these synthetic drugs, attention has since turned to products from natural sources with the purpose of exploring them as possible alternatives in the management of diabetes mellitus (oboh et al., 2011, parveen et al., 2017). in recent times, peptide products https://doi.org/10.14421/biomedich.2023.121.413-422 414 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 and protein hydrolysate preparations from enzymatic hydrolysis of plant and animal proteins are increasingly exploited and investigated for their numerous bioactivities (lopez-barrios et al., 2014; majumder and wu, 2015; siow and gan, 2016). peptides differing in lengths, sizes, and derivation have been widely used for a number of therapeutic purposes (kumar et al., 2015, vilcacundo et al., 2019). one plant whose proteins contain potentially biologically active peptides is moringa oleifera. moringa oleifera, is a plant that belongs to the family moringaceae. it is originally endemic to the himalayan regions of india but is also abundant in the middle east and cultivated in many regions of sub-saharan africa (madubuike et al., 2015). it is fast-growing, and drought resistant and this may have accounted for its wide geographical spread (madubuike et al., 2015, leone et al., 2016, abd-rami et al., 2018). various parts of the plant have been utilized for numerous purposes such as water treatment, as a food source and supplements, and also in traditional medicine (anwar et al., 2007, abdrani et al., 2018). its seeds and leaves are rich sources of nutrients and essential oils (kwaambwa et al., 2015), and proteins (mune-mune et al., 2016). its proteins consist of albumins, globulins, prolamins, and glutelins (kwaambwa et al., 2015) which could yield potentially bioactive peptide products on enzymatic hydrolysis. in addition, its seeds are abundant in certain amino acids such as glutamate, aspartate, arginine, proline, and threonine, but limited in sulfur-containing amino acids (freire et al., 2015; mune-mune et al., 2016). various extracts of its leaves and seeds have been reported to possess potent pharmacologic effects (abd-rani et al., 2018). solvent extracts of its leaves demonstrated antioxidant properties, anticancer potentials (monera et al., 2008; al-asmari et al., 2015), and antimicrobial activities (walter et al., 2011; abd-rani et al., 2018). in addition, m.oleifera leaf and seed extracts (aqueous and ethanolic) were found to demonstrate hypoglycemic, and hypolipidemic potentials in rat models, by reducing insulin resistance and increasing insulin sensitivity (tuorkey, 2016); as well as inhibiting the formation of advanced glycated end products (nunthanawanich et al., 2016). crude enzymatic digests of m. oleifera seed proteins demonstrated enzyme-inhibitory activities in earlier studies (olusola et al., 2018, olusola and ekun, 2019b). however, hydrolysate fractionation is essential to further characterize bioactive peptides (awosika and aluko, 2019). as a result, this study aims to evaluate the activities of fractionated peptides obtained from pepsinassisted hydrolysis of m. oleifera seed protein on hemoglobin glycation and carbohydrate hydrolyzing enzymes, in a bid to identify novel peptides with antidiabetic potentials. materials and methods materials collection of seeds m. oleifera seeds were obtained from farms in akungba akoko, ondo state and they were identified, and voucher samples were deposited at the department of plant science and biotechnology, adekunle ajasin university, akungba akoko. chemicals and reagents pepsin (from porcine gastric mucosa), and α-amylase (fungal), α-glucosidase (human) were products of sigmaaldrich laboratories, co-artrim, united kingdom. all other chemicals and reagents used were of analytical grade and were also products of sigma-aldrich laboratories, united kingdom. methods isolation of m. oleifera seed proteins the seeds were dried and pulverized before being kept in an air-tight container at 4oc. this was subsequently defatted using n-hexane as was previously described by arise et al., (2016a) with slight modifications. the meal was extracted three times with n-hexane using a meal/solvent ratio of 1:10 (w/v). the meal was then dried at 40oc in a vacuum oven and ground again to obtain a fine powder, termed defatted seed meal, which was stored at -20oc. the protein component of the defatted meal was extracted using the method described by alashi et al. (2014). defatted watermelon seed meal was suspended in 0.1 m naoh ph 12.0 at a ratio of 1:10, and stirred for one hour to facilitate alkaline solubilization. this was centrifuged at 18°c and 3000 g for 10 min. two additional extractions of the residue from the centrifugation process were performed with the same volume of 0.1 m naoh and the supernatants were then pooled. the ph of the supernatant was adjusted to 4.0 to facilitate acid-induced protein precipitation using 0.1 m hcl solution; the precipitate formed was recovered by centrifugation. the precipitate was washed with distilled water, adjusted to ph 7.0 using 0.1 m naoh, freezedried, and the protein isolate was then stored at -20°c prior to further analysis. preparation of m. oleifera seed protein hydrolysates the protein isolate was hydrolyzed using the methods described by olusola and ekun, (2019b) with slight modifications. hydrolysis was carried out using pepsin (ph 2.2, 37ºc). the protein isolate (5% w/v, based on the protein content of the isolate) was dissolved in glycine buffer at ph 2.2. the enzyme was added to the slurry at an enzyme-substrate ratio (e:s) of 2:100. digestion was performed at the specified conditions for 6 hours with continuous stirring. the enzyme was then inactivated by boiling in a water bath (95–100oc) for 15 min and undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh followed by ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 415 centrifugation at 6000 g for 30 min. the supernatant containing target peptides was then collected. protein content of samples was determined using the biuret assay method with bovine serum albumin (bsa) as standard. fractionation of m. oleifera seed protein hydrolysates the moringa oleifera seed protein hydrolysates were separated into molecular weight fractions using gel filtration chromatography according to the method described by ekun et al., (2022). summarily, 5 ml of the clear supernatant resulting from protein hydrolysis, at a protein concentration of 10 mg/ml was filtered, suspended in 50 mm phosphate buffer ph 7, and passed into a sephadex g25 chromatographic column of dimensions 30 cm x 4 cm which had earlier been equilibrated with the buffer. the same phosphate buffer was used to elute the separating fractions, and the elution peaks were monitored at 400 nm. the separating fractions eluted under the same elution peak were collected, pooled and their molecular weights were determined by comparison with the graph of the logarithm of molecular weights against elution volumes of known standards (vitamin b12, tryptophan, aspartame, glycine, and bovine serum albumin). the eluates, according to their molecular weights, were then sorted into <1 kd, 1-3 kd, and 3-5 kd ranges. peptide fractions of molecular weights higher than 5 kda were removed and discarded. the collected peptide fractions were stored at -200c for further analysis. determination of peptide yield the percentage peptide yield was determined using the method described by girgih et al. (2011). the peptide yields (%) of moringa oleifera seed protein hydrolysates and fractions, were calculated as the ratio of peptide content of lyophilized hydrolysate/fraction to the protein content of unhydrolysed protein isolate. inhibition of hemoglobin glycation this was investigated by estimating the degree of nonenzymatic hemoglobin glycation according to the method described by venu et al., (2016) with modifications. glucose solution (2%), 0.06% hemoglobin, and gentamycin (0.02%) solution were prepared in phosphate buffer 0.1 m, ph 7.4. 1 ml of each of the above solutions was mixed. 0.2 mg/ml 1.0 mg/ml of hydrolysate was added to the above mixture. gallic acid was used as standard. the mixture was kept in the dark at room temperature for incubation for 72 hours. at 520 nm, hemoglobin glycation was measured with a spectrophotometer, and % inhibition was calculated thus: % ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑔𝑙𝑦𝑐𝑎𝑡𝑖𝑜𝑛 = 𝐴𝑏𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒) − 𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 𝐴𝑏𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒) 𝑥100% determination of α-amylase inhibition an α-amylase-inhibitory assay was performed according to the method reported by oboh et al. (2011). briefly, 125 µl of hydrolysate (0.5 to 2.0 mg ml–1) was placed in test tubes and 125 µl of 20 mm sodium phosphate buffer (ph 6.9, with 6mm nacl) containing α-amylase solution (0.5 mg/ml) added. the content of each tube was pre-incubated at 25 °c for 10 min, after which 125 µl of 1% starch solution in 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) was added at regular intervals. the reaction mixtures were incubated at 25 °c for 10 min. the reaction was terminated by adding 250 µl of dinitrosalicylic acid (dns) colour reagent and further incubated in boiling water for 5 min and cooled to room temperature. the content of each test tube was diluted with 2.5 ml distilled water and the absorbance was measured at 540 nm. a control was also prepared using the same procedure except that the hydrolysate was replaced with distilled water. the α-amylase-inhibitory activity was calculated as shown: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 – 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetic parameters of α-amylase inhibition the kinetic study of α-amylase inhibition was conducted according to the method described by olusola and ekun (2019a). 125 µl of the hydrolysate was pre-incubated with 125 µl of α-amylase solution for 10 min at 25°c in a set of tubes. in another set of tubes, 250 µl of phosphate buffer (ph 6.9) was also pre-incubated with 125 µl of α-amylase solution. starch solution (125 µl) of increasing concentrations (1.0 to 8.0 mg/ml–1) was added to both sets of reaction mixtures to initiate the reaction. the mixture was then incubated for 10 min at 25 °c, and then boiled for 5 min after the addition of 250 µl of dinitrosalicylic acid (dns) reagent to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically from a maltose standard curve and converted to reaction velocities as shown below: 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ((µ𝑚𝑜𝑙 𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛– 1) 𝑚𝑖𝑛– 1) = 𝑀𝑎𝑙𝑡𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 / 𝐼𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 × 𝑀𝐸 where maltose concentration is in µmol/ml–1; incubation time = 10 min; me= amount of enzyme (in mg) in the reaction mixture a double reciprocal plot (1/v versus 1/[s]), where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition and the kinetic parameters (km, k΄m, vmax, v΄max, ce, and ce΄) of αamylase inhibition by hydrolysates were determined by analysis of the double reciprocal plot. the inhibition constant (ki) was determined using a secondary plot known as the dixon plot (palmer, 2007), by plotting a graph of the inverse of initial velocities on the x-axis 416 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 against inhibitor concentrations on the x-axis, at a fixed concentration of substrate. determination of α-glucosidase inhibition the effect of the hydrolysates on 𝛼-glucosidase activity was determined according to the method described by kim et al., (2005) using 𝛼-glucosidase from saccharomyces cerevisiae. the substrate solution pnitrophenyl glucopyranoside (pnpg) was prepared in 20 mm phosphate buffer, and ph 6.9. 100𝜇l of 𝛼 glucosidase (1.0 u/ml) was pre-incubated with 50𝜇l of the different concentrations of the hydrolysates for 10 min. then 50𝜇l of 3.0 mm (pnpg) as a substrate dissolved in 20 mm phosphate buffer (ph 6.9) was added to start the reaction. the reaction mixture was incubated at 37∘c for 20 min and stopped by adding 2ml of 0.1 m na2co3 solution. the 𝛼-glucosidase activity was determined by measuring the yellow-colored paranitrophenol released from p-npg at 405 nm. the results were expressed as a percentage of the blank control. percentage inhibition was calculated as: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 – 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetics of α-glucosidase inhibition the kinetic parameters of 𝛼-glucosidase by the hydrolysates were determined according to the method reported by ali et al., (2006). briefly, 50 𝜇l of the (5 mg/ml) hydrolysate was pre-incubated with 100 𝜇lof 𝛼glucosidase solution for 10 min at 25∘c in one set of tubes. in another set of tubes, 𝛼-glucosidase was preincubated with 50𝜇l of phosphate buffer (ph 6.9). 50 𝜇l of pnpg at increasing concentrations (0.63–2.0 mg/ml) was added to both sets of reaction mixtures to start the reaction. the mixture was then incubated for 10 min at 25∘c, and 500 𝜇lof na2co3 solution was added to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically at 405nm using a para nitrophenol standard curve and converted to reaction velocities. a double reciprocal plot (1/v versus 1/[s]) where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition of the hydrolysates on 𝛼-glucosidase activity was determined by analysis of the double reciprocal (lineweaver-burk) plot using michaelis menten kinetics. the inhibition constant (ki) was also determined using the dixon plot (palmer, 2007), by plotting a graph of the inverse of initial velocities on the x-axis against inhibitor concentrations on the x-axis, at a fixed concentration of substrate. results peptide yield the peptide yields of the hydrolysate and its fractions are illustrated in table 1. the unfractionated hydrolysate had the highest yield. however, among the fractions, the 1-3 kd fraction had significantly higher yields (p < 0.05) when compared to the other peptide fractions. table 1. peptide yields of m. oleifera seed protein hydrolysate and its fractions. values are presented as means ± standard error of the mean of triplicate determinations. yields carrying different symbols are significantly different (p<0.05). yields carrying the same letter or symbol are not significantly different (p<0.05). inhibition of hemoglobin glycation the effects of moringa oleifera seed protein hydrolysates and fractions on non-enzymatic hemoglobin glycation, in comparison to gallic acid, at a concentration range of 0.2mg/ml to 1.00mg/ml is illustrated in figure 1. the hydrolysate and fractions demonstrated significantly lower (p<0.05) inhibitory effects when compared to gallic acid (control), especially at higher concentrations. the unfractionated peptic hydrolysate had higher (p<0.05) inhibitory activities than its fractions, attaining a maximal value of 78.230 ± 0.774 % at 1.0 mg/ml. considering the peptic hydrolysate fractions, fraction f1 had a maximum inhibition of 66.667 ± 2.138 % at 0.8 mg/ml which was not significantly different (p<0.05) from that of fraction f2, with activity of 62.667 ± 3.079 at a concentration of 0.2 mg/ml, but was higher (p<0.05) than fraction f3 which had 58.730 ± 1.833% inhibition at a final concentration of 1.0mg/ml. sample yield unfractionated hydrolysate 80.545 ± 4.949a fraction 1 (<1 kd) 19.709 ± 1.096c fraction 2 (1-3 kd) 25.835 ± 2.166b fraction 3 (3-5 kd) 14.485±0.968d ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 417 figure 1. percentage inhibition of hemoglobin glycation by peptic moringa oleifera seed protein hydrolysate and its fractions. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). inhibition of -amylase activity the percentage -amylase inhibitory activity of moringa oleifera seed protein hydrolysate and their fractions in relation to arabinose are illustrated in figure 2. the inhibitory activities of peptic hydrolysates and fractions at all study concentrations were significantly (p<0.05) lower than those of arabinose. all hydrolysate fractions demonstrated higher (p<0.05) inhibitory activity than the unfractionated hydrolysate. also, the fractions demonstrated percentage inhibitory activity above 50% at concentrations of 0.40 mg/ml to 1.0 mg/ml. fractions 1 and 2 with 66.8% and 64.59% inhibition respectively at a maximum concentration of 1.0 mg/ml had higher (p<0.05) effects than the unfractionated hydrolysate and fraction 3. figure 2. percentage -amylase inhibitory activity of peptic moringa oleifera seed protein hydrolysates. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). kinetics of -amylase inhibition the effect of a selected peptide fraction – fractions f1, (<1 kd), – on the catalytic activity of -amylase in converting starch to maltose is presented in figure 3. kinetic parameters determined from the lineweaverburk plot in the absence and presence of two different concentrations of the peptide fraction were summarized in table 2. in the absence of the hydrolysate fraction, the michaelis constant, km, of -amylase for its substrate was found to be 0.552 mg/ml of starch while maximal velocity, vmax was 3.890 mm/mg/min. inhibition of amylase activity increased with increasing concentrations of the peptide fraction, such that the km of the enzyme was increased while vmax and catalytic efficiency, ce, of -amylase were reduced in the presence of the inhibitory fraction. the enzyme-inhibitor dissociation constant, ki, 418 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 of -amylase inhibition by peptic hydrolysate fraction f1 was determined to be 0.029 mg/ml. figure 3. lineweaver-burk plot of -amylase inhibition by moringa oleifera seed protein hydrolysate fraction 1 (<1kd) obtained from peptic proteolysis. table 2. kinetics of -amylase-catalyzed reactions in the presence and absence of m. oleifera seed protein hydrolysate fraction 1. kinetic parameters no inhibitor peptic hydrolysate fraction 1 (mg/ml) 0.5 1.0 km or kˈm (mg/ml) 0.552 0.768 0.701 vmax or vˈmax (mm/mg/min) 3.890 3.833 1.789 ce (mmol/ml/min) 7.053 4.993 2.551 ki (mg/ml) 0.029 km or kˈm: michaelis constant in the absence or presence of inhibitory hydrolysate fractions; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory hydrolysate fractions; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. -glucosidase inhibitory activity the inhibitory activities of the m. oleifera seed protein hydrolysates and their fractions on α-glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside at varying concentrations in comparison to arabinose (control) are presented in figure 5. peptic hydrolysate and its fractions displayed lower (p<0.05) inhibitory activities when compared to the standard, arcabose. however, peptic hydrolysate fraction 1 (<1 kd) attained maximum inhibitory activity of 46.09 ± 3.331 % at a concentration of 0.6mg/ml, while fraction 2, pf2, attained a maximum inhibitory extent of 59.449 ± 2.342 % at a concentration of 0.8 mg/ml. fraction 3 had significantly lower (p<0.05) -glucosidase inhibitory activity when compared to the unfractionated peptic hydrolysate and the other low molecular weight fractions obtained from peptic digestion. figure 4. percentage -glucosidase inhibitory activity of peptic moringa oleifera seed protein hydrolysates. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 419 kinetics of α-glucosidase inhibition the effect of a selected m. oleifera seed protein hydrolysate fraction (fraction f1, (<1 kd) on the kinetics of α glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside, p-npg, to p-nitrophenol are illustrated in figure 5. the kinetic parameters from the resulting lineweaver burk plots are summarized in table 3. in the absence of an inhibitor, the michaelis constant, km of α glucosidase for its substrate was determined to be 0.297 mg/ml p-npg, while maximum velocity, vmax, was 270.27 mm/mg/min. the hydrolysate fraction also caused reductions in the vmax and catalytic efficiency, ce of the enzyme at concentrations of 0.5 mg/ml and 1.0 mg/ml. figure 5. lineweaver-burk plot of -glucosidase inhibition by moringa oleifera seed protein hydrolysate fraction 1 (< 1kd) obtained from peptic proteolysis. table 3. kinetic parameters of -glucosidase inhibition by m. oleifera seed protein hydrolysate fraction 1. kinetic parameters no inhibitor peptic hydrolysate fraction 1 (mg/ml) 0.5 1.0 km or kˈm (mg/ml) 0.297 0.262 0.293 vmax or vˈmax (mm/mg/min) 270.270 163.934 172.414 ce (mmol/ml/min) 910.001 625.702 588.443 ki (mg/ml) 0.333 km or kˈm: michaelis constant in the absence or presence of inhibitory hydrolysate fractions; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory hydrolysate fractions; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. discussion peptide yield peptide yield gives an estimate of the amount, in percentage, of peptides generated relative to the whole protein subjected to enzymatic proteolysis; thus it represents an important index in determining the efficiency of the overall process (alashi et al., 2014), as these enzymes degrade the proteins into several peptides of varying lengths and sizes. it, therefore, follows that a high peptide yield is indicative of increased proteolysis and resultant peptide release (girgih et al., 2011). the peptide yield of the unfractionated hydrolysates was higher than those of their corresponding fractions put together, and this could be due to peptide loss during the process of chromatography and the removal of peptides whose molecular weights were higher than 5 kda. this is consistent with the reports of awosika and aluko (2019) in their work with yellowfield pea protein digests and peptide fractions. the yield of peptic hydrolysates obtained in this study was higher than 32.33 ± 1.046% and 68.90 ± 1.00% determined for peptic digests of arachis hypogea (olusola and ekun, 2018) and watermelon seed proteins (arise et al., 2016b) respectively. this implies that m.oleifera seed proteins also had a lot of hydrophobic amino acid residues, making them susceptible to cleavage by pepsin, as this enzyme, though relatively non-specific but has a preference for hydrolyzing peptide linkages at the cterminal ends of hydrophobic aminoacyl residues (munemune et al., 2016; voet et al., 2016). inhibition of hemoglobin glycation the formation of advanced glycation end products (ages) as a result of poorly controlled hyperglycemia in diabetes mellitus leads to a plethora of complications such as retinopathy, renal dysfunction atherosclerosis, among other devastating conditions (ramasamy et al., 2005, singh et al., 2014). these ages cause deleterious effects by promoting the generation of reactive oxygen species which activate a cascade of signaling pathways, leading to an increase in the production of pro 420 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 inflammatory mediators, invariably causing other complications such as the formation of atherosclerotic plaques and culminating in cardiovascular disease in diabetic patients (han et al., 2014). certain plant extracts have been reported to inhibit hemoglobin glycation in vitro (hosseini et al., 2015; venu et al., 2016), but information has been scarce on the abilities of peptides and protein hydrolysates to inhibit hemoglobin glycation. m. oleifera seed protein digests and their fractions exhibited lower inhibitory effects on hemoglobin glycation than gallic acid. however, the unfractionated hydrolysates had better inhibitory activities than their fractions at all study concentrations, and this indicates that fractionation, in this case, may have reduced the inhibitory activities of the peptide fractions to prevent non-enzymatic glycation of hemoglobin. this result suggests that there could be synergistic effects among these peptides, which make them more effective as a mixture than being fractionated. among the fractions, peptic fractions 1 and 2, exhibited inhibitory effects above 60%. pepsin is relatively non-specific in its cleavage specificities (naik, 2012), releasing peptides that have hydrophobic and aromatic side chains that could significantly slow down the glycation of hemoglobin. this is consistent with the reports of han et al., (2014), that asn-trp dipeptides inhibited the formation of ages in mice models. however, more investigation is required in further studies to determine the nature of other aminoacyl residues that may be involved in preventing glycation of proteins in both in vitro and in vivo models, thus charting novel courses in the search for new, peptide-based food additives in the management of diabetes mellitus. α-amylase inhibitory activity and kinetics of inhibition the enzyme α-amylase in mammals is an important component of both saliva and pancreatic juice, and it catalyzes the hydrolysis of α-(1-4) glycosidic bonds of polysaccharides, releasing glucose and maltose in the process (voet et al., 2016). in recent times, protein hydrolysate preparations and peptides from some plant and animal sources have been demonstrated to inhibit αamylase activity, with potential implications for alternate therapies to the management of diabetes mellitus (arise et al., 2016b, olusola and ekun, 2018, awosika and aluko, 2019). in this study, all hydrolysates and peptide fractions demonstrated lower α-amylase inhibitory activities when compared to arabinose, and this is not unexpected because arabinose is a synthetic inhibitor of α-amylase. also, all peptide fractions exhibited better αamylase inhibition than the unfractionated hydrolysates, and this could be that the fractionation process improves bioactivity, by allowing for more bioactive peptides to gain access to the enzyme active site, causing inhibition of the enzyme. this is also consistent with the reports of malomo and aluko, (2016) that unfractionated hydrolysates do contain large molecular weight peptides that possess antagonistic effects to enzyme inhibition. the lineweaver-burk plot was used to determine the mode of α-amylase inhibition by varying concentrations of selected peptide fractions in this study. in addition, the kinetic parameters determined from the double-reciprocal plots were summarized in table 2, and it showed that the michaelis constant, km of α-amylase (from saccharomyces cerevisiae) in the absence of inhibitory hydrolysates is 0.552 mg/ml of starch, which is lower than 1.4 mg/ml (acharya et al., 2014) for α-amylase obtained from aspergillus oryzae. the presence of increasing amounts of the hydrolysate fractions increased the apparent km of the enzyme for its substrate, while also reducing both of maximal velocity, vmax, and catalytic efficiency, ce, of α-amylase. the peptide fraction exhibited a mixed type of inhibition and this suggests that these peptides are capable of binding αamylase in both its free form and in its starch-bound forms, creating dead-end complexes on both occasions. arise et al., (2016b) reported a mixed type of inhibition of α-amylase for peptic, tryptic, and alcalde hydrolysates of citrullus lanatus seed protein hydrolysates. inhibition of α-glucosidase activity and kinetics of inhibition the enzyme α-glucosidase commonly resides on the brush border membranes of the intestinal mucosa and it is involved in carbohydrate digestion by hydrolyzing glucose residues from oligosaccharides (voet et al., 2016). thus, the modulation of the activity of this enzyme represents one of the key strategies in the control of blood glucose levels in the management of diabetes mellitus (qaisar et al., 2014). the hydrolysates and their fractions demonstrated lower α-glucosidase inhibitory activities than the control (acarbose). this is because acarbose happens to be a dual inhibitor of α-amylase and α-glucosidase, with a higher binding affinity for αglucosidase (katzung et al., 2012). the fractionation of peptic digests proved to improve the bioactivities of its fractions, with fractions f1 and f2 also demonstrating inhibitory effects, with maximal activity obtained at 0.6 mg/ml and 0.8 mg/ml respectively for the fractions, but these were higher than those of fraction 3. this also suggests that lower molecular weight peptides demonstrate better α-glucosidase inhibitory activities. however, at higher concentrations, these peptides could have antagonistic effects on one another, which could in turn, result in reduced inhibition of α-glucosidase activity (awosika and aluko, 2019). in contrast to the kinetics of α-amylase inhibition, there is limited data available in the literature regarding the kinetic analysis of α-glucosidase inhibition by protein hydrolysate fractions. the kinetic parameters obtained from the double-reciprocal plots of α-glucosidase inhibition by selected peptide fractions of m. oleifera seed proteins in figure 5 were summarized in table 3. the michaelis constant, km, of α-glucosidase for ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 421 p-nitrophenyl glucopyranoside in the absence of inhibitor determined to be 0.297 mg/ml p-npg in this study was is slightly higher than 0.211 mg/ml (0.7mm) p-npg obtained by awosika and aluko (2019) but lower than 6.31mg/ml reported by arise et al. (2019). vmax, in the absence of inhibitory hydrolysates, was 270.27 mm/mg/ml. peptic hydrolysate fraction 1 comprising peptides of molecular weight less than 1 kd demonstrated an uncompetitive inhibition mechanism, and this indicates that these peptides largely bind to other sites different from the substrate binding site on αglucosidase. the enzyme-inhibitor binding constant, ki value of 0.333 mg/ml obtained for peptic hydrolysate fraction 1 in this study was slightly higher than 0.305 mg/ml determined for unfractionated peptic hydrolysates of m. oleifera seed protein in another study (olusola and ekun, 2019). in addition, fraction 1 demonstrated a lesser binding affinity for α-glucosidase when compared with its binding to α-amylase. this could indicate that these peptides could perform better as αamylase inhibitors. conclusion the proteolysis of m. oleifera seed proteins using pepsin and their subsequent fractionation yielded peptide fractions that slowed the process of hemoglobin glycation and also inhibited carbohydrate–hydrolyzing enzymes in vitro. fraction f1 containing the smallestsized peptides demonstrated the strongest inhibitory activity against hemoglobin glycation and carbohydrates. thus, peptide products from m. oleifera seed proteins possess bioactivities that could be harnessed for the development of novel anti-diabetic agents. competing interests: the author declare that there are no competing interests. references acharya, d.k., shah. i.j., gami, p.n, shukla, r.m. 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(2011). antibacterial activity of moringa oleifera and moringa stenopetala methanol and n-hexane seed extracts on bacteria implicated in water borne diseases. afr. j. microbiol. res. 5, 153–157. doi: 10.5897/ajmr10.457 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 233-240 | doi: 10.14421/biomedich.2023.121.233-240 issn 2540-9328 (online) the effects of frequent therapeutic administration of artesunate-amodiaquine and artemether-lumefantrine on haematological markers in balb/c mice david audu1,*, olufunmilayo a idowu1, vinood b patel2, musa f mshelbwala3, adewumi b idowu1 1department of pure and applied zoology, college of biosciences; 3department of veterinary pathology, college of veterinary medicine, federal university of agriculture abeokuta, ogun state, nigeria 2school of life sciences, university of westminster, 115 new cavendish street, london w1w 6uw, united kingdom. corresponding author* audud@funaab.edu.ng abstract artemisinin combination therapy (act) is readily available in malaria-endemic nations, leading to repeated drug usage by undiagnosed persons. repeated use of act therapy by non-infected individuals may affect blood cells. this study explored how repeated artesunateamodiaquine (a/a) and artemether-lumefantrine (a/l) treatment in non-infected mice affected haematological markers. 100 male balb/c mice were randomly divided into 5 groups: non-infected and plasmodium berghei nk65 infected treated with a/l and a/a 1x, 2x, 3x, 4x, 5x, and 6x, and the control group. packed cell volume (pcv), haemoglobin (hb), and red blood cell (rbc) were redu ced (p>0.05) non-significantly in the non-infected group treated with a/l or a/a six times compared to the control and infected groups. wbc rose in infected and non-infected mice treated with a/l or a/a 1x, 2x, 3x, and 6x, with a substantial rise in non-infected mice treated with a/l (p < 0.01) and a/a (p < 0.001) three times. wbc mainly rose due to lymphocytes, although neutro phils decreased. repeated therapeutic use of a/l and a/a without infection may cause a haematological change. continuous efforts are needed to educate the public about screening for malaria parasites before using drugs. keywords: malaria; artemether lumefantrine; artesunate amodiaquine; hematological parameters; anemia; neutropenia. introduction malaria is caused by plasmodium parasites transmitted by female anopheles mosquito bites (w.h.o, 2022). african countries account for 95% of worldwide malaria. effective malaria case management in children and adults consists of early diagnosis and fast, effective treatment with artemisinin-based combination therapy (koko et al., 2022; w.h.o, 2022). the first-line act medicines for malaria in africa are artemetherlumefantrine (a/l) and artesunate-amodiaquine (a/a), which show modest effectiveness (zongo et al., 2020; audu et al., 2023). malaria should be screened before taking acts to guarantee effective therapy, but this is challenging in africa. malaria patients in africa who lack parasitological confirmation of infection are routinely provided with these medications, leading to the use of act without being infected (mbonye et al., 2010; yeung et al., 2011; cohen et al., 2012; mbonye et al., 2013; rusk et al., 2013; idowu et al., 2015; nwokolo et al., 2018 ). multiple antimalaria dosages are typical in nigeria due to self-prescription and free access to the drug over the counter (owumi et al., 2015). blood is where most antimalarial medication activities occur (madukaku et al., 2015); long-term a/l and a/a use by non-infected people may harm blood cells. animal tests showed that a/l at indicated doses for three days did not influence haematological parameters in non-infected rats. however, after seven days of a/l treatment, red blood cells (rbc), hemoglobin (hb), and packed cell volume (pcv) dropped (ofem et al., 2013). in comparison, the study by (adeleye et al., 2012) showed that a single dose of artesunate and a/l affected white blood cell, neutrophil, and lymphocyte counts. long-term administration of either a/l or a/a to noninfected individuals has also been reported to have harmful effects on blood cells (ijeomah et al., 2016). due to the current misuse of act in malaria-endemic regions, the goal of this experiment was to investigate the impact of repeated therapeutic administration of either a/l or a/a on the blood as further investigation is required to determine whether this substance is safe to use repeatedly. we employed a mouse model to simulate the frequent use of a/l or a/a in infected and noninfected mice. secondly, the haematological parameters of mice were examined after one week of treatment, as manuscript received: 04 november, 2022. revision accepted: 16 february, 2023. published: 02 march, 2023. https://doi.org/10.14421/biomedich.2023.121.233-240 234 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 little is known about delayed or late-appearing anaemia in relation to the use of act for therapeutic purposes (sowunmi et al., 2017). methods procurement of animals and management: 100 male adult balb/c mice with a mean weight of 24.46± 0.07g of 8 weeks of age were used in this study. they were obtained from the university college hospital's (uch) institute for advanced medical research and training in ibadan, nigeria. the mice were not used for any experiments before and are pathogen free. the mice were housed in plastic cages containing beddings of dried wood shavings and were fed with standard feed produced by ladokun feed limited, ibadan, oyo state, nigeria. mice were given constant access to food and water and were kept on a 12-hour light/12hour dark cycle. research design: 100 mice were randomly allocated into 5 groups of 20 using computer-generated numbers (johnson and besselsen, 2002). each group was split randomly into four replicates, each with five mice in a cage. the sample size was calculated using the equation formula (charan and kantharia, 2013). group 1 consisted of mice neither infected nor treated but given distilled water 1x, 2x, 3x, 4x, 5x and 6x times, respectively. groups 2 are non-infected mice treated with artemether lumefantrine (a/l) for 1x, 2x, 3x, 4x, 5x and 6x times, respectively. while group 3 are noninfected mice treated with artesunate amodiaquine (a/a) for 1x, 2x, 3x, 4x, 5x and 6x times, respectively, while group 4 consists of mice infected and treated with a/l for 1x, 2x, 3x, 4x. 5x and 6x times, respectively, and group 5 mice were infected and treated with a/a for 1x, 2x, 3x,4x, 5x and 6x times, respectively. mice were given a week to recover after treatment or infection plus treatment before subsequent exposure. blood was collected from 3 mice for haematological analysis one week after 1x, 2x, 3x and 6x exposure periods, in other to compare the effect of 13 usage with up 6 times usage of the drug on mice. the experiment was conducted following the national institutes of health's guide for the care and use of laboratory animals (nih publication no. 85–23, revised in 1996). the college of veterinary medicine research ethics committee at the federal university of agriculture, abeokuta, approved the experimental protocol (funaab/colvet/crec/2019/07/01). antimalaria drug: artemether plus lumefantrine (a/l) (lumartem anti-malarial tablet, 20 mg/120 mg) were obtained from cipla pharmaceuticals limited in mumbai, india, and artesunate with amodiaquine (a/a) (camosunate; 100 mg/300 mg) from geneith pharmaceuticals limited in lagos, nigeria. treatment was carried out in all treatment groups at therapeutic doses calculated based on the manufacturer's recommendation for a man's assumed weight of 35kg. artemether lumefantrine was administered with a therapeutic dose of 14/6.84 mg/kg/d in six dosages at 0, 8, 24, 36, 48, and 60 hours, respectively. at the same time, artesunate/ amodiaquine was administered 2.86/8.58 mg/kg/d once daily for three days a week (otuechere et al., 2012)(daikwo et al., 2018). in infected groups, seven days after infection, mice were treated. oral dosing was used to administer clinical doses using an intragastric feeding needle. nk65 strain of plasmodium berghei: plasmodium berghei nk65 utilised in this study was obtained from the chemotherapy research laboratory at the university college hospital (uch) in ibadan, nigeria. microscopic slides were made from blood from the donor mice's tails to determine the parasitemia level. after the thinly diffused blood, it was air-dried, fixed, and stained with giemsa. the parasite's presence was seen using an x100 oil immersion objective lens. we multiplied the number of parasites by the number of red blood cells (rbcs) in at least four random fields to calculate parasitemia as a percentage. next, malaria parasite inoculums were created using blood samples from a donor mouse. experimental mice were inoculated intraperitoneally with a low inoculum of 104 parasites using 0.1 ml of the donor mouse's blood. finally, blood smears were taken, stained, and inspected under a microscope to check the establishment and monitoring of infection every other day. at the same time, parasitemia levels were fully counted on day 7th post-infection and post-treatment. haematological studies the animals were treated humanely, and blood samples were collected via the retro-orbital plexus into edta bottles for haematological analysis. packed cell volume (pcv): the pcv was determined by centrifuging the well-mixed anticoagulated blood sample in capillary tubes for 10 minutes at 200 revolutions per minute using a micro haematocrit centrifuge to ensure minimal cell packing. the packed cell volume was then calculated by measuring the height of the red cells with a pcv metre. the percentages of blood volume were used to express the values (everds, 2007)(cheesbrough, 2006). haemoglobin measurement: the cyanmethemoglobin technique was used to determine the haemoglobin concentrations in the blood samples. the blood was diluted in a buffered solution of potassium ferricyanide and potassium cyanide to get cyanmethemoglobin. we created 1 in 25l dilutions by washing 20µl of blood in 5.0ml of modified drabkin's fluid. three minutes were given for complete conversion to cyanmethemoglobin. the absorbance was then measured at 540nm compared to distilled water using a spectrophotometer (everds, 2007)(cheesbrough, 2006). red blood cell counts: we made a dilution of 1 in 20 l of the anticoagulant blood sample, 0.02 ml of the blood was mixed with 4 ml turk's solution. 0.01 ml of audu et al. – the effects of frequent therapeutic administration of … 235 the resulting mixture was inserted into the counting chamber. the red cell present in the four corners and central 1 mm2 was counted, recorded, and the total rbc counts were calculated using formula (everds, 2007)(cheesbrough, 2006) red cell indices: the mean cell volume (mcv), mean cell haemoglobin (mch), and mean cell haemoglobin concentration (mchc) were estimated using the formula described by (everds, 2007)(cheesbrough, 2006) mean cell haemoglobin (mch): this is the quantity of haemoglobin (in pictogram form) contained in an average red cell. the value was derived using the haemoglobin concentration and red blood cell numbers formula. mch = (hb×10) / (total rbc) (pg or 10-12/g) mean cell volume (mcv): this is the average volume of red blood cells measured in femtoliters. the mcv was calculated from the pcv and red cell using the formula. mcv = (pcv x 10) /total rbc (fl or 10-15/l) mean cell haemoglobin concentration (mchc): this is the quantity of haemoglobin in 100ml of packed red blood cells instead of the amount of haemoglobin in whole blood. the mchc was computed from the haemoglobin and pcv represented as g/dl, mchc = (hb x 100) / pcv (g/dl) white blood cell counts: the blood sample was diluted by washing 50µl of blood into 950µl of the diluting fluid to give a final dilution of 1 in 20. the dilution was then mixed and loaded into the counting chamber. the white cells present in the four corners 1 mm2 areas were counted. the final white cell count for the whole blood sample was calculated using (everds, 2007)(cheesbrough, 2006). white blood cell differentials: leishmann staining method was used to determine the percentage of each type of wbc, prepared slides were viewed under the microscope x100 oil immersion objective, and the cells were counted and recorded (everds, 2007)(cheesbrough, 2006). statistical analysis: raw data from the laboratory were analysed, and graphs were plotted using graphpad prism 8.0 computer program. to determine the significant difference between the various treated group and the control, a one-way analysis of variance (anova) was used. the tables provided the findings as mean ± standard error mean (sem) (n =3). shapiro-wilk tests were first used to determine whether the data's distribution was normal. the significance of mean values *, ** and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to the respective control groups. results mean parasitemia levels at day seven post-infection were less than 5% in mice infected in each consecutive time of 1x, 2x, 3x, 4x, 5x and 6x, respectively. day 7 posttreatment with a/l or a/a for 1x, 2x, 3x, 4x, 5x and 6x, respectively, after the corresponding infection recorded 0% parasitemia. effects of repeated treatment of non-infected and infected mice with either a/l or a/a on pcv, hb, rbc and wbc. to ascertain the level of haematological damage of noninfected and infected groups treated repeatedly with either al or aa, the blood pcv, hb, rbc, wbc, rbc indices, and differential wbc count were analysed. non-infected and infected mice exposed to a/l and a/a 1x, 2x, 3x and 6x didn't significantly alter (p>0.05) the level of blood pcv (fig. 1a), hb (fig. 1b), and rbc (fig.2a) respectively when compared with their controls. although there was a non-significant (p>0.05) decrease in pcv, hb, and rbc levels in the non-infected group treated with a/l only once compared to the control 1x and the rest group 1x, this reduction was not later observed after treatment for 2x and 3x period. also, mice infected and treated with a/l 3x recorded non-significant (p>0.05) reduced pcv, hb and rbc compared to the control group 3x, and the rest group treated 3x; this reduction was not later seen after 6x treatment (fig. 1 a, b and 2a). the non-infected groups treated with a/l and a/a for 6x non-significantly (p>0.05) reduced the pcv, hb and rbc levels compared to the control group (fig. 1a,1b and 2a). the mcv, mch and mchc of non-infected and infected mice treated with either a/l or a/a for 1x, 2x and 3x were not altered compared to the control group. the noninfected group treated with a/l for 6x significantly (p < 0.05) reduced the mcv value compared to the control, the infected treated with a/l or a/a 6x and the noninfected treated with a/a 6x (table 1). the wbc counts were lower in control compared to the non-infected and infected groups treated with either a/l or a/a 1x, 2x, 3x, and 6x, except group (inf+aa), treated 6x (fig. 2b). however, wbc counts increased in the non-infected groups treated with either a/l or a/a 1x and 3x following an increasing trend with a significant increase in group al (p < 0.01) and aa (p < 0.001) for 3x but dropped after treatment for 6x (fig. 2b). exposure of either a/l or a/a 1x, 2x, 3x and 6x to non-infected and infected groups reduced the neutrophil level and increased the lymphocyte level compared to the control (table 2). the eosinophils level was significantly higher in non-infected mice treated with either a/l for 236 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 2x and 3x compared with the control, following an increasing trend with a significant highest increase in 2x (p < 0.05) and 3x (p < 0.001), after which it drops in treatment for 6x. infected mice treated with either al or aa 6x recorded higher eosinophil levels than the noninfected and control groups (table 2). basophil level was higher in non-infected and infected groups treated with either a/l or a/a for 2x, 3x, and 6x compared to the control, with a significant (p < 0.001) increase in the non-infected group treated with a/a for 1x and 6x and infected group treated with a/a for 1x, 3x and 6x times. exposure of the non-infected group to either a/l or a/a 1x, 2x and 3x times and only a/a for 6x increased the monocyte level compared to the control group. in contrast, the infected group treated with either a/l or a/a 1x and 2x, and only a/a for 3x and 6x increased mono level compared to the control group (table 2). figure 1. (a)pcv level (b)hb level: of infected and non-infected mice treated with either a/l or a/a regime 1x, 2x, 3x and 6x times. pcv: packed cell volume; hb: haemoglobin al; treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. figure 2. (a) rbc level (b) wbc level: of infected and non-infected mice treated with either a/l or a/a regime for 1x,2x, 3x and 6x times. al: rbc: red blood cell; wbc: white blood cell. treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. 1x 2x 3x 6x 0 20 40 60 80 number of times p c v % 1x 2x 3x 6x 0 5 10 15 20 25 number of times h b ( g /d l) ctl al aa inf+al inf+aa ctl al aa inf+al inf+aa a b 1x 2x 3x 6x 0 5 10 15 number of times r b c × 1 0 1 2 /l 1x 2x 3x 6x 0 5 10 15 20 nunber of times w b c ( × 1 0 9 /l ) ✱✱✱ ✱✱ ✱ ctl al aa inf+al inf+aa ctl al aa inf+al inf+aa a b audu et al. – the effects of frequent therapeutic administration of … 237 table 1. effect of repeated usage of al and aa on the level of mcv, mch and mchc blood parameters in mice. no. of times of infection and/or treatment infection and/or treatment red blood cell indices mcv (fl) mch (pg) mchc (g/dl) 1x ctl 60.35±0.33 20.24±0.00 33.54±0.19 al 59.95±0.14 20.00±0.06 33.35±0.03 aa 59.70±0.17 20.20±0.00 33.85±0.09 inf+al 60.05±0.14 20.05±0.03 33.45±0.03 inf+aa 60.00±0.12 20.15±0.14 33.55±0.14 2x ctl 58.60±0.91 20.42±0.82 34.82±0.94 al 58.15±0.95 19.70±0.46 33.85±0.20 aa 58.85±0.66 19.95±0.14 33.95±0.14 inf+al 60.10±0.06 20.30±0.12 33.75±0.20 inf+aa 59.85±0.20 20.05±0.03 33.50±0.17 3x ctl 61.14±2.03 20.53±1.11 33.54±0.79 al 59.25±0.03 20.05±0.03 33.85±0.03 aa 58.70±0.29 19.90±0.17 33.90±0.23 inf+al 60.00±0.12 20.15±0.14 33.55±0.14 inf+aa 59.30±0.69 20.30±0.17 34.30±0.23 6x ctl 61.84±1.18 20.50±0.61 32.37±0.92 al 58.30±0.06* 19.40±0.40 33.30±0.23 aa 60.00±0.17 20.15±0.14 33.55±0.14 inf+al 59.45±0.20 19.60±0.12 33.00±0.06 inf+aa 60.25±0.03 20.25±0.09 33.60±0.12 mcv: mean cell volume; mch: mean cell haemoglobin; mchc: mean cell haemoglobin; al: treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one times; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. table 2. effect of repeated usage of al and aa on wbc differentials in mice granulocytes. no. of times of infection and/or treatment infection and treatment white blood differentials neutrophils (%) lymphocytes (%) eosinophils (%) basophils (%) monocyte s (%) 1x ctl 36.00±1.73 62.33±2.03 1.00±0.00 0.00±0.00 0.67±0.33 al 26.00±0.58*** 71.00± 0.58** 1.50±0.29 0.00±0.00 1.00±0.00* aa 25.50±0.29*** 71.00± 0.57** 0.50±0.29 1.50±0.29*** 1.50±0.29 inf+al 32.00±2.31 64.00± 2.31 2.00±0.00* 1.00±0.00 1.00±0.00 inf+aa 29.00±0.58** 67.00± 1.16 1.50±0.29 1.50±0.29*** 1.00±0.00 2x ctl 36.67±0.33 61.00±0.58 1.00±0.00 0.33±0.33 0.00±0.00 al 28.50±0.87*** 67.50±0.29* 2.00±0.58* 1.00±0.00 1.00±0.00** aa 28.00±0.58*** 69.50±0.29** 1.00±0.00 0.50±0.29 1.00±0.00** inf+al 27.50±0.29*** 69.50± 0.29** 0.50±0.29 0.50±0.29 1.00±0.00** inf+aa 26.50±0.29*** 70.50±0.29*** 1.00±0.00 0.50±0.29 1.50±0.29*** 3x ctl 38.67±0.67 60.00±0.58 1.00±0.00 0.00±0.00 0.33±0.33 al 28.00±0.58*** 66.50± 0.88* 3.50±0.29*** 1.00±0.00 1.00±0.00 aa 32.00±0.58* 63.00± 1.15 2.00±0.00* 1.00±0.00 2.00±0.00*** inf+al 29.00±2.31*** 69.50± 2.02*** 1.00±0.00 0.50±0.29 0.00±0.00 inf+aa 27.00±0.58*** 67.00± 1.73* 3.00±0.00*** 2.00±0.00*** 1.00±0.00 6x ctl 36.67±2.33 61.00±1.73 1.00±0.00 0.00±0.00 1.00±0.00 al 27.00±0.58*** 69.00±0.58** 1.00±0.00 1.00±0.00 1.00±0.00 aa 29.50±2.60** 66.00± 3.46 1.00±0.00 1.50±0.29*** 1.50±0.29* inf+al 30.00±1.73* 66.50±2.02 1.50±0.29 0.50±0.29 1.00±0.00 inf+aa 30.50±0.29* 64.00± 0.58 2.00±0.00* 2.00±0.58*** 1.67±0.33** al: treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. 238 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 discussion in this study, a/l and a/a were very effective against the malaria parasite. both drugs are seen to clear the parasite after 7-day post-treatment during each repeated infection and treatment. reports have shown that both a/l and a/a have high malaria treatment success (davlantes et al., 2018; marwa et al., 2022). although, several reports have shown that malaria cause anaemia and changes in other haematological variables (osaro et al., 2014; omarine nlinwe and nange, 2020; audu et al., 2021), and recurring malaria attacks can lead to lifethreatening anaemia (bakhubaira, 2013; dhangadamajhi et al., 2019). however, this present study was designed to determine if repeated therapeutic exposure to a/l and a/a when not infected could affect blood cell counts and predispose animals to anaemia, as very little was found in the literature on this question. this investigation shows that the pcv, hb and rbc were not significantly altered by the therapeutic use of a/l and a/a for 1x, 2x, 3x and 6x in non-infected mice compared to the control groups. this outcome is contrary to the previous study, which has shown acute haemolytic anaemia following the use of artemisinin (rehman et al., 2014). furthermore, a study by (geerligs et al., 2003) corroborates that malaria chemoprophylaxis improves mean haemoglobin levels. while the term "haematocrit conservation" was spawned because it was seen that there is little to no drop in haematocrit following acts, even when parasitaemia are heavy (gbotosho et al., 2014; sowunmi et al., 2017). these findings suggest that taking this drug for 1x, 2x and 3x, and 6x therapeutically when not infected would not significantly alter the pcv, hb and rbc after one week of each repeated treatment. prolonged repeated treatment of non-infected mice with either a/l or aa for up to six consecutive times reduced the levels of pvc, hb, and rbc insignificantly compared to the control and infected groups. it indicated that the prolonged use of this treatment in the absence of infection could potentially modify the haematological parameter. evidence suggests that exposure to either al or aa over an extended period can have a detrimental effect on haematological markers (ofem et al., 2013; ijeomah et al., 2016). although the use of a/l 1x when non-infected and when infected 3x insignificantly also reduced the pcv, hb and rbc value, although there was a recovery after subsequent treatment, this shows that the use of a/l over a/a could potentially also alter the pcv, hb and rbc parameters when used. (white, 2018) reported that being infected and treated with artemisinin is seen to cause haematolytic anaemia after 1-3 weeks of drug usage. what is surprising in this experiment is that repeated infecting mice and treatment with either a/l or a/l 1x, 2x, 3x and 6x didn't alter the pcv, hb and rbc. these results reflect those of (sowunmi et al., 2017), who also found that both a/a and a/l have been observed to dramatically lower the prevalence of anaemia in younger and older children who have malaria after therapy (sowunmi et al., 2017). this result supports that repeated malaria infection and treatment with therapeutic doses of either a/l or a/a would improve the haematological parameters. mcv, mch and mchc are indices of erythrocyte shape, size, and haemoglobin content changes. studies have shown that artesunate does not affect the total rbc, mch, and mchc levels (bigoniya et al., 2015)(ijeomah et al., 2016). the current study found that the mcv, mch, and mchc values obtained after treatment of non-infected and infected groups with either a/l or a/a for 1x, 2x and 3x were not significantly different compared to the control group. implies that the red blood cells are normal in size and concentration after treatment of infected and noninfected with the drugs for 1x, 2x and 3x. although after repeated treatment with therapeutic dosages of a/l for 6x to non-infected mice, the mcv value was significantly reduced; this indicates that using a/l repeatedly without being infected could decrease the average size of the red blood cells in mice. white blood cells in the body provide a unique defence system against infections and hazardous substances. in this study, apart from the group repeatedly infected and treated with a/a 6x, there was an increase in wbc count in both infected and non-infected mice treated with either al or aa therapeutic doses for 1x, 2x, 3x and 6x times. the increase was due to a rise in lymphocytes, eosinophils, basophils, and monocytes, but a decrease in neutrophil was observed in the treated groups. the increase in wbc and lymphocyte counts suggests an immunological response induced by the drug as they are mobile components of the body's defence systems. as (adeleye et al., 2012) found that a/l can raise total wbc counts and lymphocyte counts while decreasing neutrophil counts, which they ascribed to the immunological response caused by the medication. in our study, we noticed either a/l or a/a usage once or repeatedly resulted in a significant increase in the wbc of non-infected groups. however, a study by (ijeomah et al., 2016) found a substantial drop in wbc after longterm a/l and a/a usage; also, in this present study, non-infected mice treated with al and aa for three consecutive times had the highest increase in wbc count but dropped after six consecutive times. this result has shown that usage of al and aa could increase the wbc count when used repeatedly but could drop after prolonged usage. in malaria-infected individuals, counts of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, and eosinophils were considerably reduced (kotepui et al., 2014). in this study, repeated usage of al and aa in infected and non-infected mice for 1,2,3 and 6 consecutive times had a significant reduction in neutrophil, with a substantial decrease in non-infected audu et al. – the effects of frequent therapeutic administration of … 239 mice treated with either a/l or a/a for 1 and 6 times compared to the control and infected group. this result implies that treatment with either a/l or a/a without infection could lead to neutropenia. low neutrophil has been associated with intermittent (weekly) doses of amodiaquine for malaria prevention. (zwang et al., 2012). this study reported a significant increase in eosinophil, basophil, and monocyte in groups treated with al and aa; this increase may be due to responses of the antimalaria drugs (adeleye et al., 2012; bigoniya et al., 2015). the limitation of this study lies in the fact that the study did not include taking blood immediately after treatment as a subgroup for comparison. notwithstanding these limitations, the study still shows us the extent of haematological alteration caused when these drugs are taken repeatedly. conclusions the result of this study shows that the use of a/l and a/a therapeutically in a repeated manner without being infected could result in haematological alteration. this study lays the groundwork for future clinical and further animal work on this subject. further research could address more longer-time effects of taking this drug and check the haematological parameter immediately after each treatment. in addition, more significant efforts are needed to enlighten the public on the need to repeatedly screen for malaria parasites before every repeated use of antimalarial drugs. competing interest: the authors report having no competing interests. funding: the research animal and laboratory work was funded by the nigeria federal government tertiary education trust fund (tetfund) authors’ contributions: ad came up with the experiment's idea. ad, iba, ioa, and mfm designed the research methodology and carried out the experiments. pvb provided technical assistance. ad worked on the draft manuscript; the final version was read, edited, and approved by all authors. acknowledgements: firstly, i want to thank the nigeria federal government tertiary education trust fund (tetfund) for the grant support of this work. secondly, i want to thank the research laboratory at the university college hospital (uch) in ibadan, nigeria, for providing us with p. berghei for this study. references adeleye, g. s., nneli, r., nwozor, c. m., and emesiana, m. c. 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(2012) comparing changes in haematologic parameters occurring in patients included in randomized controlled trials of artesunate-amodiaquine vs single and combination treatments of uncomplicated falciparum in sub-saharan africa. malaria journal 11(1): 1– 11. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 251-258 | doi: 10.14421/biomedich.2023.121.251-258 issn 2540-9328 (online) chemical composition and evaluation of anti-tyrosinase and antioxidative effects of topical cream formulation from acacia sieberiana, vitellaria paradoxa and beeswax alfred ngenge tamfu1,3,4,*, alain yaya koudoro2, selcuk kucukaydin3,4, theophile olaye2, pascal dossa cokou agbangnan2, dominique codjo koko sohounhloue2, felicien avlessi2 2laboratory of study and research in applied chemistry, university of abomey-calavi, polytechnic school of abomey-calavi, 2009 abomey-calavi, benin. 3department of medical services and techniques, koycegiz vocational school of health services, mugla sitki kocman university, 48800 mugla, turkey. 4department of chemistry, faculty of science, mugla sitki kocman university, 48000 mugla, turkey. corresponding author* macntamfu@yahoo.co.uk manuscript received: 30 january, 2023. revision accepted: 12 march, 2023. published: 13 march, 2023. abstract skin diseases can get natural therapies from medicinal plant-based products. in this study, a topical cream was formulated from ethanol extract of acacia sieberiana, beeswax and vitellaria paradoxa (shea) butter. gc-ms characterization with co-injection of the topical cream revealed stearic acid (31.43%), palmitic acid (23.15%), oleic acid (21.44%) and linoleic acid (16.20%) as the major components. seven phenolic conpounds were identified and quantified by hplcdad and ferulic acid (12.81±0.26 mg/g) was the most abundant. the cream showed good antioxidant properties evaluated through β-carotene-linoleic acid assay, dpph• radical scavenging, abts•+ assay, cuprac assay, and metal chelating assay. the cream had higher activity in the dpph• assay (ic50 = 32.10±0.84 µg/ml), abts•+ assay (ic50 = 22.49±0.62 µg/ml) and cuprac assay (ic50 = 49.27±0.79 µg/ml) than α-tocopherol. the antioxidant effects are an indication that the cream can reduce oxidative stress on the skin including aging, carcinogenesis and inflammation. at 100 µg/ ml, the topical cream showed tyrosinase inhibition of 48.23±0.87% regarded as relatively good compared to the standard tyrosinase inhibitor kojic acid, which showed 79.50±0.32% inhibition at the same concentration. the cosmetic cream was able to inhibit the melanin production rate-limiting enzyme, tyrosinase, indicating that it can control hyperpigmentation and skin spots. keywords: acacia sieberiana; topical cream; gc-ms; phenolic composition; skin diseases; antioxidant; tyrosinase inhibition. introduction skin conditions constitute a real public health problem in the world especially in tropical countries where they represent nearly 30% of consultations (basset et al., 1999). skin diseases are considered as health priorities, which can cause significant mortalities, and are generally include fungal infection, stretch marks, eczema, acne, oxidative stress, skin burns, hypopigmentation, hyperpigmentation, melasma and dermatitis caused by cosmetic bleaching agents (hay et al., 2006). human skin colour is determined by pigments such as hemoglobin, hemosiderin, carotene and melanins that play protective roles as well as determination of human skin and hair colour (yamaguchi and hearing, 2014). melanin is primarily responsible for skin pigmentation which protects the skin from adverse effects of ultraviolet radiation, skin burn and skin cancer but an over-production or loss of melanin can result in serious skin problems (zolghadri et al., 2019; tamfu et al., 2022b). although melanin has desired effects, an overproduction of melanin caused by the action of tyrosinase is undesirable. the production of melanin involves the enzymatic action of tyrosinase and other tyrosinaserelated proteins which play a critical role in the process. tyrosinase, which is a multifunctional copper-containing metalloenzyme is the major rate-limiting enzyme in melanogenesis (garcia-jimenez et al., 2017; alfred ngenge et al 2021). tyrosinase causes undesired browning of vegetables and fruits, overproduction of melanin and skin patches and spots (tamfu et al., 2020b, masum et al., 2019). inhibitors of tyrosine are therefore finding applications as attractive cosmetic and medicinal products for skin whitening and depigmentation as well as in agricultural and food industries as antibrowning agents (zolghadri et al., 2019; tamfu et al., 2020c). therefore, tyrosinase inhibition provides solution to perishing of vegetables and fruits as well as therapeutic potential in skin cancers, pigmentation and 1department of chemical engineering, school of chemical engineering and mineral industries, university of ngaoundere, 454 ngaoundere, cameroon. https://doi.org/10.14421/biomedich.2023.121.251-258 mailto:macntamfu@yahoo.co.uk 252 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 neurodegenerative disorders (masum et al., 2019; mughal et al., 2022). occurrence of reactive oxygen species (ros) and reactive nitrogen species are at the origin of many human ailments and it is necessary to inhibit their excesses (tamfu et al., 2021a). substances which can inhibit the destructive effects of ros, rns and free radicals and referred to as antioxidants. oxidative stress occurs in the situation of an imbalance between ros and rns production with respect to antioxidants and the oxidative damage that results from this can spread over most cells and tissues (talla et al., 2017; alain et al., 2022; sawalda et al., 2022). most antioxidant substances from natural and synthetic sources exist and they are capable of of suppressing ros and rns and protect the living systems against oxidative stress (tamfu et al., 2020d). synthetic antioxidants like butylated hydroxy toluene (bht) and butylated hydroxy anisole (bha) are usually insoluble and associated with side effects, thereby giving way for rising interest in natural antioxidants (talla et al., 2014; soni and loonker, 2022; tamfu et al., 2021b; lópezpedrouso et al., 2022). being constantly exposed to external aggressions, the skin represents a privileged target of oxidative stress, which leads to multiple skin damage (yapi et al 2019; awadji, 2021). oxidative stress is one of the main aggravating causes of these conditions, and is one of the potential factors of skin disorders that occur especially with age as well as the early aging of the body defenses. the skin regulates the excretion of waste products, controls body temperature, and protects the organism from environmental, chemical and physical stress factors. the skin is exposed to effects of sun rays, ros and rns which can damage the antioxidant defense capacity of the skin causing skin diseases (kruk and duchnik, 2014). many cases of skin problems are observed in the health care systems worldwide, but some (over 70% of individuals) of the patients with chronic transmissible skin diseases especially in tropical areas do not seek treatment but recourse to medicinal plants and traditional medicine as solution to their skin conditions (almoshari, 2022). acacia siberiana is one of those plants that pills in northern benin where it is strongly used in traditional medicine to treat skin conditions. a. sieberiana, is a plant of the fabaceae family, is an essentially thorny ligneous plant. it is native to the savannahs of africa. this plant can reach 25 m in height whose leaves are 10 to 15 cm long with straight white spines at their base (orwa et al., 2009). in order to derive a real benefit from the use of this plant, it becomes important to make a formulation for body use based on its extract with judiciously chosen excipients. the production of home-made medicinal skin ointments from vitellaria paradoxa is popular in most communities in tropical areas of africa. this work investigates the chemical composition by gc-ms and hplc-dad of a topical cream from vitellaria paradoxa, acacia siberiana and beeswax, and evaluation of its antioxidant and anti-tyrosinase activities. materials and methods plant material and extraction acacia sieberiana leaves were collected during the month of january 2021 from north of benin (malanville). the plants were identified and voucher specimens prepared by the botanist professor hounnankpon yedomonhan of the benin national herbarium where they were deposited under the specimen numbers yh 684/hnb, for acacia sieberiana. the extraction was carried out using ultrasound-assisted extraction with ethanol as solvent. briefly, 100 g of powdered plant material biomass from each plant were mixed with 1000 ml solvent (h2o2/etoh, 1/1) and sonicated for two hours at 50 °c with bandelin (sonorex digitech device). further, all the extracts were filtered through whatman no.1 filter paper and concentrated under vacuum on a rotavapor (buchi r215) at 50°c. the solid paste extract obtained was stored in the darkness at 4 °c to avoid the degradations until use. preparation of topical cream for the preparation of ointment, the hydroethanol extract of acacia sieberiana was used as the active ingredient. given the sticky aspect of this extract, 30 g of extract was first dissolved in 100 ml of extraction solvent in order to facilitate the preparation. 25 g of beeswax were melted and added unto the extract. the mixture was blended into a uniform paste at 70oc. 30 g of shea (vitellaria paradoxa) butter were added into the mixture and stirred for further 30 minutes. 5 g of glycerin and 5 g of vaseline were then added while mixing continuously to give a homogeneous pasty ointment. in order to have a perfectly homogeneous mixture, the temperature was maintained at 70°c for 10 min. the ointment was transferred into vials and allowed to cool and solidify before storage. table 1 shows the components of the topical cream with the percentage composition by mass of each component. table 1. components of the topical cream formulation. ingredients % composition by mass vitellaria paradoxa (shea) butter 30 vaseline 5 beeswax 30 glycerine 5 ethanol extract of acacia sieberiana 30 gc-ms characterization of the topical cream the extract (10 mg) was mixed with 150 μl of pyridine and 100 μl of bis(trimethylsilyl) trifluoroacetamide (bstfa) and heated at 80 °c for 20 min and then the tamfu et al. – chemical composition and bioactivities of hand-made cream 253 final supernatant was analyzed by gc-ms (mercan et al., 2006). an ion trap ms spectrometer and a rxi-5sil ms (restek) fused silica non-polar capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) were used for the gc–ms analyses. carrier gas was helium with 1.4 ml/min flow rate. the injector and ms transfer line temperatures were 220 and 290ºc, respectively. the ion source temperature was at 200 ºc. the injection volume was 0.2 µl with a split ratio of 20:1. ei–ms measurements were taken at 70 ev ionization energy. mass range was from m/z 28 to 650 amu. scan time was 0.5 s with 0.1 s interscan delays. for analysis, the oven temperature was held at 100ºc for 10 min, then increased up to 280ºc with 4ºc/min increments and kept at this temperature for 10 min. identification of components of the sample was based on gc retention indices and computer matching with the wiley, nist-2008 and trlib libraries, as well as by comparison of the fragmentation patterns of the mass spectra which reported in the literature and, whenever possible, by coinjection with authentic compounds. determination of phenolic profile of the cream selected phenolic constituents in the extracts were identified and quantified through reversed-phase highperformance liquid chromatography (rp-hplc) linked to diode array detector (dad) as described elsewhere (küçükaydın et al., 2021; tamfu et al., 2022c). summarily, 5 g of each extract were dissolved in water:methanol (80:20), filtered through sterile 0.20 μm filter disk. an intertsil ods-3 reverse phase c18 column was used for the separation with a 1.0 ml/min solvent flow rate and 20 μl injection volume. two mobile phases a (0.5% acetic acid h2o) and b (0.5% acetic acid in ch3oh). a gradient elution was applied as follows: 0–10% b (0–0.01 min); 10–20% b (0.01–5 min); 20– 30% b (5–15 min); 30–50% b (15–25 min); 50–65% b (25–30 min); 65–75% b (30–40 min); 75–90% b (40–50 min) 90-10% b (50–55 min). a photodiode array detector set at 280 nm wavelength was employed in the detection and the uv data together with retention times were compared with authentic standards. each analysis was performed three times. a calibration plot established through the elution of known concentrations (range of 0.0~1.0 ppm) of authentic compounds was used in the identification and quantification of the constituent phenolic compounds. twenty-six phenolic standards (gallic, p-hydroxy benzoic, protocatechuic, ellagic, chlorogenic, trans-cinnamic, 3-hydroxy benzoic, vanillic, syringic, p-coumaric, rosmarinic and ferulic acids; catechin, kaempferol, hesperetin, pyrocatechol vanillin, 6,7-dihydroxy coumarin, coumarin, rutin, myricetin, chrysin, luteolin, apigenin taxifolin and quercetin) were used. the results were expressed as μg per g dry weight of extract. antioxidant activity of the cream β-carotene-linoleic acid method, with a few minor modifications, was used to assess the ability to inhibit lipid peroxidation (tamfu et al., 2020a) the spectrophotometric evaluation of the dpph• and abts•+ radical scavenging activities was conducted as described earlier (tamfu et al., 2020b) the cupric reducing antioxidant capacity (cuprac) was measured using the method as previously described (apak et al., 2004). bha (butylated hydroxyanisole) and α-tocopherol were employed as reference compounds for comparison of the β-carotene-linoleic acid, dpph•, abts•+ and cuprac assays. the metal chelating activity of the extracts for fe2+ was determined spectrophotometrically (decker and welch, 1990). edta was used as the reference compound to compare metal chelating activity. antioxidant activity results were given as 50% inhibition concentration (ic50). anti-tyrosinase activity of the cream the inhibition of tyrosinase enzyme (mushroom source) was determined through the method published elsewhere (masuda et al., 2005) using l-dopa as the substrate. in a 96-well microplate, 10 μl solution of sample or kojic acid (reference) were added to 150 μl of sodium phosphate buffer (ph 6.8, 100 mm) followed by 20 μl of tyrosinase enzyme and the resulting mixture incubated at 37ºc for 10 minutes. 20 μl of l-dopa were added after incubation and the absorbances of the resulting solutions were taken at 475 nm and the results expressed as concentration at which 50% inhibition was observed (ic50). statistical analyses descriptive statistics were applied on the data obtained. each experiment was done in triplicate and the means of three parallel measurements were deduced. the values given are means±sem (standard error of the mean) for three measurements. one-way anova (analysis of variance) was used to compare differences amongst the means and were considered statistically significant p < 0.05. results and discussion natural products and medicinal plant extracts are being used as alternative therapies for skin care purposes and are becoming more prevalent in formulations, due to consumers’ concerns about synthetic ingredients/chemical substances. the main benefits reported for plant extracts, used in skin care, include antioxidant and tyrosinase inhibition effects amongst others. due to their uses in skin care products, it is necessary to investigate their chemical composition to guarantee their safety. 254 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 gc-ms chemical composition of the topical cream the volatile constituents of the topical cream were determined by gc-ms and reported on table 1. a total of thirteen volatile compounds were identified, out of which twelve were fatty acids and one fatty alcohol. amongst the identified constituents, 61.09% were saturated fatty acids while 37.64% were unsaturated fatty acids together with 1.27% of other compounds. gc-ms characterization with co-injection of the topical cream revealed that, stearic acid (31.43%), palmitic acid (23.15%), oleic acid (21.44%) and linoleic acid (16.20%) were the major compounds. table 3. chemical profile of topical cream by gc-ms. no ria compounds cosmetic product (%)b identification methods 1 1152 2-nonen-1-ol 0.52 co-gc, ms, ri 2 1160 benzoic acid 0.75 co-gc, ms, ri 3 1170 octanoic acid 1.73 co-gc, ms, ri 4 1265 nonanoic acid 0.94 co-gc, ms, ri 5 1373 decanoic acid 1.15 co-gc, ms, ri 6 1492 suberic acid 0.31 ms, ri 7 1566 lauric acid 0.77 ms, ri 8 1625 azelaic acid 0.40 ms, ri 9 1760 myristic acid 1.21 co-gc, ms, ri 10 1957 palmitic acid 23.15 co-gc, ms, ri 11 2190 linoleic acid 16.20 co-gc, ms, ri 12 2195 oleic acid 21.44 co-gc, ms, ri 13 2237 stearic acid 31.43 co-gc, ms, ri saturated fatty acids 61.09 unsaturated fatty acids 37.64 other compounds 1.27 total 100 a: kovats index on rxi-5sil ms fused silica column, b: percentage concentration, co-gc: co-injection with authentic compounds; ms: based on comparison with wiley, adams and nist 08 ms databases ri: retention index literature comparison hplc-dad phenolic composition of the topical cream the phenolic profile of the topical cream was evaluated by hplc-dad with twenty-six standard phenolic compounds, and seven of them were identified and quantified in milligrams per gram of extract (mg/g) and recorded on table 1. the phenolic compounds gallic acid (2.70±0.10 mg/g), chlorogenic acid (3.93±0.07 mg/g), ferulic acid (12.81±0.26 mg/g), coumarin (4.10±0.18 mg/g), rutin (0.39±0.04 mg/g), ellagic acid (0.17±0.02 mg/g) and rosmarinic acid (8.48±0.21 mg/g) were identified and quatified. the most abundant consituent was ferulic acid followed by rosmarinic acid and then coumarin and chlorogenic acid. antioxidant activity of the topical cream the antioxidant capacity of the topical cream was evaluated through five complementary assays namely βcarotene-linoleic acid assay, dpph• assay, abts•+ assay, cuprac assay and metal chelating assay and the results presented on table 3. the cream had higher activity in the dpph• assay (ic50 = 32.10±0.84 µg/ml), abts•+ assay (ic50 = 22.49±0.62 µg/ml) and cuprac assay (ic50 = 49.27±0.79 µg/ml) than α-tocopherol which was used as one of the standards, while its activity were relatively close to that of standard bha as well. the antioxidant activity of the cream was however moderate in the β-carotene-linoleic and metal chelating assays. the antioxidant effects is an indication that the cream can reduce oxidative stress and the negative effects of excess reactive oxygen species (ros) and reactive nitrogen species (rns) on the skin including aging, carcinogenesis and inflammation. anti-tyrosinase activity of the topical cream tyrosinase is necessary but excessive tyrosinase is unwanted. inhibitors of excessive tyrosinase are used in treating skin pigmentation problems and the tyrosinase inhibition of the topical cream was evaluated and the results presented on table 3. at a test concentration of 100 µg/ml, the topical cream showed tyrosinase inhibition of 48.23±0.87% and this activity can be regarded good when compared to the standard tyrosinase inhibitor kojic acid, which showed 79.50±0.32% inhibition at the same concentration. the cosmetic cream was able to inhibit the melanin production rate-limiting enzyme, tyrosinase, indicating that it can control melanogenesis, skin spots and skin whitening. tamfu et al. – chemical composition and bioactivities of hand-made cream 255 table 2. phenolic composition of topical cream by hplc-dad (mg/g)a. no phenolic compounds rt (min) cosmetic product 1 gallic acid 5.70 2.70±0.10 2 protocatechuic acid 8.75 3 catechin 10.68 4 pyrocatechol 11.04 5 chlorogenic acid 12.35 3.93±0.07 6 p-hydroxy benzoic acid 12.77 7 6.7-dihydroxy coumarin 14.10 8 caffeic acid 15.09 9 3hydroxy benzoic acid 15.98 10 syringic acid 16.56 11 vanillin 17.78 12 p-coumaric acid 20.56 13 taxifolin 21.26 14 ferulic acid 22.14 12.81±0.26 15 coumarin 24.49 4.10±0.18 16 rutin 25.30 0.39±0.04 17 ellagic acid 26.11 0.17±0.02 18 rosmarinic acid 26.77 8.48±0.21 19 myricetin 27.35 20 quercetin 30.83 21 trans-cinnamic acid 31.33 22 luteolin 31.70 23 hesperetin 32.14 24 kaempferol 33.21 25 apigenin 33.77 26 chrysin 38.40 avalues expressed are means ± s.e.m. of three parallel measurements (p < 0.05). b -: not detected table 4. antioxidant and anti-tyrosinase activities of topical cream. antioxidant activity anti-tyrosinase activity sample β-carotene-linoleic acid assay dpph• assay abts•+ assay cuprac assay metal chelating assay tyrosinase inhibition ic50 (µg/ml) ic50 (µg/ml) ic50 (µg/ml) a0.50 (µg/ml) ic50 (µg/ml) % inhibition at 100 µg/ml topical cream 18.31±0.53 32.10±0.84 22.49±0.62 49.27±0.79 57.60±0.94 48.23±0.87 α-tocopherol 2.10±0.07 38.15±0.50 35.50±0.56 60.25±0.55 nt nt bha 1.45±0.03 19.75±0.33 12.80±0.08 25.40±0.40 nt nt edta nt nt nt nt 5.52±0.35 nt kojic acid nt nt nt nt nt 79.50±0.32 values represent the means ± sem of three parallel sample measurements (p < 0.05). nt: not tested. discussion many fatty acids were identified in the topical cream. gc-ms is suitable for the identification of volatile and fatty components of natural extracts and plant samples including fatty acids (eve et al., 2020; carol et al., 2017; koudoro et al., 2022). it could be suggested that most of the fatty acids are coming from the beeswax and shea butter which were used as important matrix components for the production of the cream. bee products such as beeswax used as component in the production of the skin ointment can suitably be characterized by gc-ms since they are made of volatile constituents (ngenge et al., 2016). important skin protective fatty acids such as palmitic acid, linoleic acid, oleic acid and stearic acids were identified as major constituents. fatty acids and their derivatives are important ingredients in cosmetic 256 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 development which act like brighteners, emulsifiers or softeners especially lauric acid, palmitic acid, myristic acid and stearic acid (yang et al., 2020). fatty acids act as skin barrier and reduce water loss on skin, prevent entry of harmful substances and can play antimicrobial and anti-inflammatory roles (cochran et al., 2015). this indicates that the rich fatty acid content of the cream can improve its protective effects on the skin and also avoid entry of bacteria and fungi as well as other objects. it can also control water loss and uv damage. phenolic extracts and compounds from plants possess anti-inflammatory and antioxidant effects that reduce the damage caused by oxidative species and prevent skin diseases and aging (boo, 2019). phenolic compounds are suitably extracted by using ultrasonic means and identified by hplc-dad method and their major interest is due to their antioxidant effects amongst other beneficial activities (tamfu et al., 2022a). important bioactive phenolic compounds have been identified in the topical cream and they could be from a. sieberiana, a fabaceae plant which is rich in phenolic compounds and possesses antioxidant effects (alain et al., 2022; zongo et al., 2023). the presence of phenolic compounds in the cream could confer oxidative protection and most cosmetologists and dermatologists are searching for safer and more effective natural antioxidants to prevent or treat premature aging and skin diseases. human skin is exposed to oxidative stress resulting from conditions like sunlight, radiations, pollutants, stress and oxidants which results to aging, wrinkles, excess skin pigmentation and skin tone unevenness (chen et al., 2021). the antioxidant effect of the cream was evaluated in five different and complementary assays so as to give a global indication of its antioxidant capacity in the different models (alfred et al., 2020). the phenolic compounds and fatty acids identified in the cream can act as natural antioxidant, antiinflammatory, antimicrobial and anti-aging compounds and also as a skin barrier against other biotic and abiotic factors. one important skin problem is irregular spots and pigmentation disorders. tyrosinase is a copper metallo-enzyme which plays an important role in melanogenesis, over-pigmentation and enzymatic browning and therefore, its inhibitors are used in solving skin disorders, whitening and hyperpigmentation (panzella and napolitano, 2021). the topical cream was able to inhibit tyrosinase which shows that it can be used for skin care and skin whitening purposes. pigmentation determines the color of the skin and uv radiation protection of skin but hyperpigmentation results to spots and melasma, which could be suitably solved by tyrosinase inhibitors. skin diseases such as dermatitis, acne vulgaris, psoriasis, urticaria, melanoma, keratinocyte carcinoma, scabies and fungal skin diseases can get natural therapies from medicinal plant-based products (karimkhani et al., 2017; panzella and napolitano, 2021; chen et al., 2021). skin disorders have significant impacts on the affected person’s quality of life especially oxidative damage and skin spots. skin lightening can be achieved by using natural or synthetic products to improve skin tone, give even skin complexion and reduces melanin production thereby helping patients to treat their skin problems including age spots, acne scars, freckles, skin burns and uneven coloration (salsberg et al., 2016; masum et al., 2019). conclusions the skin is exposed to environmental stress and physical, chemical and biological factors can affect it. also, in its functions of defense and homeostasis, the skin can encounter negative effects which need to be solved. many artificial cosmetic and dermatological treatments are available for skin problems but they are expensive and unaffordable to many and also have side effects. for these reasons, many local solutions from medicinal plants and other natural products are available and are used with positive results. in this study, a topical local cream prepared from a. sieberiana, beeswax and other ingredients was evaluated for its chemical compostion by gc-ms, hplc-dad as well as its antioxidant and antityrosinase activities. important fatty acids were identified most of which are relevant in cosmetic science and play a protective role on the skin. bioactive phenolic compounds were also identified. the good antioxidant and tyrosinase inhibition of the topical crean is attributable to the fatty acids and phenolic compounds whch it contains. the scientifc evidence of the beneficial utility of the skin cream is suggested based on the results and ascertains its use by local populations for various skin disorders as well as wounds and burns. acknowledgements: the authors are grateful to mugla sitki kocman university, the university of ngaoundere as well as university of abomey-calavi. authors’ contributions: alain yaya koudoro, theophile olaye, pascal dossa cokou agbangnan, dominique codjo koko sohounhloue and felicien avlessi obtained materials, prepared samples, designed the study and carried part of experiments. alfred ngenge tamfu and selcuk kucukaydin did the experiments, designed methodologies, analyses, data curation and wrote the first draft. all authors contributed materials, read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references alain ky, tamfu an, kucukaydin s, ceylan o, pascal ad, félicien a, dominique sc, duru me, 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inhibitors. j enzyme inhib med chem. 2019 dec;34(1):279-309. doi: 10.1080/14756366.2018.1545767. zongo, e.; busuioc, a.; meda, r.n.-t.; botezatu, a.v.; mihaila, m.d.; mocanu, a.-m.; avramescu, s.m.; koama, b.k.; kam, s.e.; belem, h.; et al. exploration of the antioxidant and anti-inflammatory potential of cassia sieberiana dc and piliostigma thonningii (schumach.) milne-redh, traditionally used in the treatment of hepatitis in the hauts-bassins region of burkina faso. pharmaceuticals 2023, 16, 133. https://doi.org/10.3390/ph16010133 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 45-53 | doi: 10.14421/biomedich.2023.121.45-53 issn 2540-9328 (online) the association between some endocrine conditions and covid-19: a review harem othman smail department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f.r. iraq. corresponding author harem.othman@koyauniversity.org manuscript received: 22 september, 2022. revision accepted: 03 october, 2022. published: 11 october, 2022. abstract the review aimed to understand and explain the association between some major endocrine conditions and covid-19. since march 2019, covid-19 has been identified as a pandemic by the world health organization (who) and has infected millions of individuals worldwide. according to the literature review, endocrine disorders include diabetes, obesity, hypertension, and thyroid. the development and progress of covid-19 patients could be affected but not yet approved by all the studies. in diabetes mellitus, covid-19 may affect cytokines and increase releases of il-1, il-6, and complications caused by diabetes. in obese patients, there was an increased risk of progressing to severe covid-19. because of the international spread of severe acute respiratory syndrome coronavirus, clinicians should pay special attention to obese patients who should be monitored closely with timely and aggressive care. covid-19 pathophysiology and risk in a high incidence of hypertension has been found in patients with covid-19 and can be studied in many kinds of studies, such as in china. however, hypertension is considered one of the most significant risk factors for covid-19. in addition to the above data on associations between covid-19 and endocrine disorders, data on thyroid function or thyroid disease in covid-19 is not yet available and cannot be commonly reported. keywords: covid-19; diabetes mellitus; bidirectional; obesity; pathophysiology; blood pressure; hypothyroidism. introduction coronavirus disease has affected millions of individuals worldwide (covid-199) (mathew et al., 2020; rostam et al., 2020). this latest coronavirus, which also includes middle east respiratory syndrome (mers)-cov and sars-cov-1, is a human β-coronavirus. these viruses are predominantly connected to respiratory illnesses, such as pneumonia, ards, and pulmonary oedema (di et al.,2020). endocrine disorders are no exception, and some endocrine organs are at risk of direct or indirect covid-19 damage. although there is still no proof of a greater predisposition in patients with diabetes and obesity to contract the infection, the coexistence of both conditions leads to a worse prognosis since both conditions impart an impaired immune system (marazuela et al., 2020). evidence is growing to indicate that patients with endocrinopathies such as diabetes mellitus (dm), hypertension (htn), obesity, and cardiovascular disease are at greater risk of complications associated with covid-19. in covid19 non-survivors and serious cases, studies from the uk and us suggested a high prevalence of dm and obesity. htn (49.7 %), obesity (48.3 %), dm (28.3 %), and cardiovascular disease (27.8 %) are the most widely identified cardiometabolic comorbidities related to covid-19 in the us (shekhar et al., 2020). metabolic syndrome is a constellation of cardiovascular risk factors, including abdominal obesity, high blood pressure, dysglycemia, atherogenic dyslipidemia, and pro-thrombotic and pro-inflammatory states. metabolic syndrome is clinically characterized as the occurrence of three or more of the following factors: increased waist circumference (population and countryspecific cutoff), hypertriglyceridemia (>150 mg/dl or hypertriglyceridemia treatment), elevated blood pressure (systolic 130 and diastolic 85 mm hg or hypertension treatment history), elevated blood pressure (systolic 130 and diastolic 85 mm hg or hypertension treatment history), high-density lipoprotein cholesterol reduction (<40 mg/dl in males; <50 mg/dl in females) and dysglycemia (<100 mg/dl or hyperglycemia treatment)( bonora et al., 2018; bansal et al., 2020). the severity of covid-19 can be impaired by hypertension, diabetes, and coronary heart disease. in addition, it may be associated with an angiotensin-converting enzyme 2 (ace2) deficiency, and a glucolipid metabolic disorder (glmd) mediated cytokine storm (chen et al., 2020). several research studies have targeted the epidemiological and clinical features of patients infected with covid-19, but the risk factors for severity and mortality have not been thoroughly investigated. identifying major risk factors and taking effective https://doi.org/10.14421/biomedich.2023.121.45-53 46 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 clinical steps will greatly contribute to saving lives (zhou et al., 2020; zaki et al., 2020). covid-19 and diabetes mellitus diabetes mellitus (dm) is chronic with catastrophic multi-systemic complications and may be associated with extreme coronavirus disease 2019 (covid-19) (huang et al., 2020) larger studies from china gave more consistent prevalence rates. diabetes was present in 7.4 per cent of 1099 (median age of 47 years) (guan et al.,2020). and 8.2 per cent of 1 590 (median age 48.9 years) hospitalized individuals in two multicenter national studies. also, of 7337 people admitted to nineteen hospitals in the province of hubei (median age 54 years), 952 (13.0%) had type 2 diabetes, while a survey by the chinese center for disease control and prevention (china cdc), which also included nonhospitalized people, showed a lower prevalence of diabetes (5.3%) among 44,672 reported cases of covid-19 through february (pugliese et al.,2020). notably, guo et al. data indicate that an initial milder appearance of sars-cov-2 infection may mask the seriousness of covid-19 in diabetes, with fewer patients reporting fever, chill, chest tightness, and shortness of breath (maddaloni et al., 2020). in addition, the fundamental and clinical science of the possible interrelationships between diabetes mellitus and covid-19 was investigated (drucker 2020). however, knowledge in this area is quickly emerging, with numerous publications appearing frequently. this review summarizes recent developments in diabetes mellitus and covid-19 and emphasizes clinical guidelines for patients at risk of or affected by covid19 with diabetes mellitus. unfortunately, most accessible research does not differentiate between diabetes mellitus and, due to its high prevalence, focuses mostly on t2dm (kumar et al., 2020) compared to non-diabetics, diabetes in patients with covid-19 is associated with a two-fold rise in mortality and covid-19 severity. more research is needed on pathogenic pathways and therapeutic effects (kumar et al., 2020). several data sets from china, italy, and the usa have consistently recorded that in patients with advanced age (>70 years of age) and pre-existing comorbidities, primarily diabetes mellitus (dm), hypertension, and cardiovascular disease, the clinical course of covid-19 is more serious (yang 2020). the dm and covid-19 relationship are bidirectional. on the one hand, dm will increase the risk of sars-cov2 contraction and further complicate the clinical path of covid-19, resulting in increased severity and mortality (onder 2020). certain clinical and biological features determine high-risk phenotypes within the dm population, and such prognostic markers need to be characterized in future studies (smail et al., 2022). in the sense of patient-tailored precision medicine, which emerges as an urgent priority in the era of covid-19, more research is required to explore which subgroups of dm patients are anticipated to benefit most from particular antiviral, immunomodulatory and other treatment strategies (koliaki et al., 2020). given the high risk, people with dm should take special precautions during the covid19 pandemic. the norm should be strict social distancing and proper hanging hygiene. good glycemic regulation should be of utmost significance as it has been shown to improve the innate immune system. although it would be prudent to adhere to or intensify the ongoing treatment, doctors may consider reviewing the prescription (pal et al., 2020). compromised innate immunity, pro-inflammatory cytokine environment, decreased ace2 expression, and the use of antagonists of the renin-angiotensin-aldosterone system in people with diabetes mellitus lead to a weak covid-19 prognosis. direct β-cell injury, cytokine-induced insulin resistance, hypokalemia, and drugs used to treat covid-19 (such as corticosteroids, lopinavir/ritonavir) can, on the other hand, lead to a worsening of glucose control in individuals with diabetes mellitus (pal et al., 2020). several studies have attempted to understand the potentially increased vulnerability of diabetes patients to sars-cov-2 infection. however, no data have shown that these patients are at higher risk of contracting covid-19. via the ace2 receptor, sars-cov-2 reaches the host cell. while consensus on the role of ace2 in the crosstalk between diabetes and covid-19 has not yet been achieved, some argue that diabetes patients have elevated ace2 expression, thus facilitating viral entry and subsequent replication. others show that patients with diabetes have low levels of ace2 and that other causes, such as treatment with ace/arbs, hypoglycemic agents, and statins, are responsible for the observed rise in ace2 (azar et al., 2020). the degree to which clinical and demographic variables moderate this relationship is uncertain, although signs link higher bmi and higher hba1c to worse outcomes in people with covid-19 diabetes. covid-19 also risks leading to worse diabetes outcomes due to disturbances caused by the pandemic, including stress and changes in routine treatment, diet, and physical activity, and posing immediate risks to people with diabetes (hartmann et al., 2020) dysregulated post-infection immune response is characterized by delayed and reduced recruitment in lung tissue of cd4+ t cells, inflammatory monocytes, and macrophages. in addition to the reduced overall cd4+ t cell response, infected diabetic mice also showed a more prominent th17 response with increased il-17a levels, implying that a shift in cytokine profiles could be partially responsible for the severity of the disease (angelidi et al., 2020) an independent risk factor for the prognosis of covid-19 is diabetes. smail – the association between some endocrine conditions and covid-19: a review 47 therefore, the prevention and treatment of diabetic patients, especially those needing insulin therapy, should be given greater attention (shang et al., 2020). from 43 reports, the meta-analysis was based on 23007 patients. the pooled prevalence of diabetes in patients infected with covid-19 was 15% (95% ci: 12%–18%), p = <0.0001. furthermore, in covid-19 patients with diabetes, the risk of mortality was found to be substantially higher relative to covid-19 patients without diabetes, with a pooled risk ratio of 1.61 (95% ci: 1.16–2.25%), p = 0.005 (hussain et al., 2020). the pandemic of covid-19 has questioned both institutional and diabetes self-management. continuing social distancing and lockdowns have adversely influenced access to care and self-management (mukona et al., 2020) sars-cov-2 infected t2dm patients reported decreased body mass index (bmi), lymphocytes, ua, albumin levels, and increased crp levels. oxidative stress response and nutritional intake may be associated with reduced bmi, ua, and albumin levels. in addition, the infection may be associated with reduced lymphocyte counts and elevated crp levels (liang et al., 2020). there are major concerns about worsening glycemic regulation, inaccessibility of suitable drugs, inaccessibility of health care or infection with sars-cov-2, and worse results during the covid-19 pandemic. although there are some recommendations for diabetes treatment and related complications during the covid-19 pandemic, very few discuss the psychological problems of people with diabetes (singhai et al., 2020). several specialist bodies have temporarily updated gestational diabetes mellitus (gdm) testing guidelines during the covid-19 pandemic to minimize person-toperson contact. the current temporary australian guidelines indicate that no glucose tolerance test (gtt) is needed if the fasting glucose is around 4.6 mmol/l (van et al., 2020). a common medical condition in pregnancy is gestational diabetes mellitus (gdm). the australasian pregnancy diabetes society (adips) suggests monitoring all women in each pregnancy, preferably using a two-hour glucose tolerance test (gtt). the diagnostic criteria derive from world health organization (who) guidelines based on the findings of the hyperglycaemia and pregnancy outcomes (hapo) report (metzger et al., 2008). in patients with diabetes, there is evidence of an increased incidence and severity of covid-19. the pathophysiology of diabetes could be influenced by covid-19. therefore, it is critical for patients infected with covid-19 to regulate blood glucose and those without the disease. innovations such as telemedicine effectively treat diabetes patients today (singh et al., 2020). in patients infected with various viruses, including the 2009 pandemic influenza a (h1n1), sars-cov, and mers-cov, diabetes and uncontrolled glycemia have been identified as important predictors of severity and deaths. some studies have not found a strong link between diabetes and serious disease in the emerging sars-cov-2 pandemic. however, other studies from china and italy have shown that elderly patients with chronic diseases, including diabetes, are at higher risk of significant covid-19 and mortality, respectively (hussain et al., 2020). the mortality rate was 9.9 % in a meta-analysis of hospitalized patients in china with a diagnosis of covid19. a higher incidence of diabetes mellitus was separately associated with a worse prognosis. the independent impact of covid-19 mortality on diabetes mellitus should be seen as hypothesis-generating and needs further research (miller et al., 2020). figure 1. covid-19, cytokine storm, diabetes mellitus: a vicious cycle (azar et al.,2020). covid-19 and obesity according to estimates from the world health organization, obesity is a global epidemic, with at least 2.8 million people dying each year due to becoming overweight or obese (hussain et al., 2020). obesity is a condition characterized by the assistance of extra adiposity tissue that provides substantial morbidity and mortality due to its complications linked to several weights. therefore, the diagnostic comparison should consist of an anthropometric test that indicates elevated fat mass and the extent to which the well-being of individual patients is adversely affected by excess adiposity (smail 2019 and smail et al., 2021). although the effects of covid-19 on obese patients have not yet been well established, the experience of h1n1 influenza can serve as a warning for treating obese patients, particularly patients with extreme obesity. from 2009 to 2010, adult obesity and extreme obesity rates rose between 2017 and 2018 and now stand at 42% and 9%, respectively (hales et al., 2020). the findings indicate that relative to the h1n1 experience, the proportion of patients with obesity, extreme obesity, and covid-19 infections will increase, and the disease will likely have a more severe 48 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 path in such patients. these results further highlight the need for improved attention, diagnosis and testing priority, and aggressive care for obesity patients and covid-19 infections (dietz et al., 2020). although the data on the effect of sars-cov-2 in people with obesity are minimal, and their relationship has not yet been fully established, it has been found that people with excessive body weight may experience a more severe covid-19 infection (puig et al., 2020). as stated earlier, it is especially noticeable in people affected by other risk factors associated with a more serious disease course. this correlation was also noted during the 2009 influenza a pandemic, which identified obesity as an independent risk factor for complications. obesity patients suffered more than 40 per cent longer after a high-fat diet than people without excessive body weight (kassir 2020). therefore, obesity during the covid-19 outbreak seems to require more attention, especially in western countries where obesity prevalence is high (rychter et al., 2020). there was an increased risk of progressing to extreme covid-19 in obese patients. since severe acute respiratory syndrome coronavirus two will continue to spread internationally, clinicians should pay particular attention to obese patients who should be handled closely with prompt and aggressive treatment (cai et al., 2020). for variables associated with covid-19 risk, severity, and their potential for decreased therapeutic and prophylactic treatments among these individuals, mechanistic mechanisms for obesity are presented in detail. the substantial main changes in morbidity and mortality from covid-19 are correlated with individuals with obesity. several mechanisms explain this impact jointly. a major concern is that vaccinations for obesity may be less successful (popkin et al., 2020). in theory, early intubation benefits identified in patients with covid-19 can indicate osa alleviation in some patients. similarly, concerns of contamination from using nasal pap in some patients with obstructive sleep apnea could lead to deterioration. mechanistic research is encouraged, given the possible connection between osa, obesity, and covid-19 (mcsharry et al., 2020). in young patients, obesity is a significant predictor of covid-19 severity. the key mechanism is connected to liver and kidney damage (deng et al., 2020) study shows that patients with overweight and obese who have covid-19 are at higher risk for mortality and intubation than those with typical bmi. these results support the hypothesis that obesity is a risk factor for complications of covid-19 and should be considered for covid-19 management (nakeshbandi et al., 2020). in addition, there was an increased risk of progressing to extreme covid-19 in obese patients. since severe acute respiratory syndrome coronavirus two will continue to spread internationally, clinicians should pay particular attention to obese patients who should be handled closely with prompt and aggressive treatment (cai et al., 2020). obesity, especially in male and younger populations, plays a significant role in the risk of death from covid19. the lack of impact of racial/ethnic and socioeconomic inequalities on mortality can be explained by the capitated system with more equalized access to health care. the data highlight the leading role of extreme obesity over correlated risk factors, which offers an early intervention target (tartof et al., 2020) the position of obesity, particularly given its high prevalence worldwide, is of great relevance. however, it is important to bear in mind that the assessment of obesity through bmi is highly arguable, particularly in older people. first, since its numerator (i.e. bodyweight) comes from the fat and fat-free mass amount, it should be noted that bmi does not strictly reflect adiposity. second, the cut points that categorize overweight and obese are subjective and cohort-based for young and middle-aged people and insufficient for older people (sattar et al., 2020). third, with age, body fat accumulates with a decrease in muscle mass. obesity is often underestimated in older people, who may have excess adiposity within the normal/overweight body size (so-called sarcopenic obesity). finally, ethnic differences can determine significant variability in body fat distribution, especially regarding ectopic and visceral fat (azzolino et al., 2020). this relative inefficiency reveals a reduced ventilatory reserve and a risk for respiratory failure, even in mild systemic or pulmonary conditions. taken together, any respiratory insults will seriously compromise the already attenuated respiratory system of obese subjects, ensuring that these patients will fail to recover if they have acquired any serious diseases that can deleteriously impact respiratory function, such as covid-19 (albashir et al., 2020) the results indicate an interplay and dynamic pathophysiology between covid-19 and diabetes. for clinical practice and public health, it is a significant priority to understand how diabetes can lead to extreme covid-19 and how covid-19 can worsen diabetes pathophysiology or its effects. to counter this, a global registry (covid) was introduced to promote the study of covid-19-related diabetes manifestations, their effects, and best care (rubino et al., 2020). a wealth of data has emerged on this subject over the short period of the covid-19 pandemic, but important questions still need to be addressed. here, the current literature on the relationship between covid-19 and diabetes is reviewed, and future studies' priorities are considered (vas et al., 2020). the role of cytokines, rapamycin mammalian target (mtor), and altered polarization of natural killer cells in the hazardous relation between covid-19 and obesity. these pathways encourage and accelerate the deleterious downstream cellular effects of sars-cov2. also, it is well known that obesity is associated with smail – the association between some endocrine conditions and covid-19: a review 49 decreased lung capacity and poor response to mechanical ventilation, putting these individuals at high risk of serious covid-19 disease and mortality. also, obesity can lead to other complications, such as renal failure, cardiovascular disease, hypertension, and vascular injury, further speeding up the negative clinical outcomes of covid-19. obese individuals should be protected from possible viral exposure to sars-cov-2 with compulsory safety devices and social distancing considerations (caci et al., 2020). it is stated that people with obesity or overweight are less involved. moreover, community health centres, gyms, swimming pools, and parks have been closed by law in many countries as part of their quarantine policy during the covid-19 pandemic. such diet and social shifts may have led to a rise in body weight in individuals with obesity and the general population (lim et al., 2020). many variables could be involved in the worsening of covid-19 obesity. obesity can increase the expression in the bronchus and blood of ace2 and cd147-related genes, while the latter are sars-cov-2 receptors that invade (radzikowska et al., 2020). this would make obese subjects more vulnerable to infection and could, to some degree, explain the higher positive sars-cov-2 test rate. obesity and concomitant metabolic syndrome can cause possible harm to the function of the organ, making it more vulnerable to failure in the lungs, kidneys, and other organs (yang et al., 2020). however, in some acute diseases, including acute respiratory distress syndrome, where patients with obesity may have better outcomes, some have proposed an obesity paradox; it is uncertain if this phenomenon exists in patients with covid-19 (goyal et al., 2020). on the one hand, emerging evidence indicates that obesity is a risk factor in adults for a more extreme and complicated course of covid-19. on the other hand, the health emergency triggered by the epidemic diverts attention to infectious diseases from preventing and treating non-communicable chronic diseases (dicker et al., 2020). most covid-19 patients die due to covid-19 pneumonia after requiring artificial ventilation for hypoxemic respiratory failure. emerging covid-19 lung post-mortem histopathology provides insights into the underlying pathophysiology. in short, there is evidence of diffuse alveolar injury, as in other types of viral pneumonia, but sometimes this is patchy (lockhart et al., 2020). therefore, it is important to collect anthropometric information for patients with covid-19 because of the potentially critical role of body weight or adiposity in determining the incidence and severity of pneumonia (and probably other complications) (stefan et al., 2020). therefore, special medical care and effective intervention should be undertaken during hospitalization and later clinical follow-up in obesity patients with covid-19, especially those with additional comorbidities (kang et al., 2020). it is unclear if this is driven by these populations' higher prevalence and risks of obesity (driving diabetes and hypertension). regarding progression to more extreme severity requiring acute treatment or death, we do not yet have data on results for those with obesity and less severe covid-19 disease. such data would be necessary to fully understand the risk of mortality for those with obesity, particularly with the understanding that survival of critical care may be greater for those with moderate levels of obesity (fine et al., 2020). absolute and central obesity, in short, are risk factors for hospital admission to covid-19. even with a moderate weight gain, the high risk was evident. impaired glucose and lipid metabolism may be involved in the mechanisms (hamer et al., 2020). figure 2. potential pathways that increase the risk of serious covid-19 illness and death in individuals with obesity. there are a variety of possible mechanisms by which obesity can impact adverse covid-19 outcomes. these include chronic inflammation, respiratory function deficiency, pulmonary perfusion, practical concerns in critical care environments when treating obese patients, immune dysregulation, obesity, metabolic and vascular complications, and relative decreases in main hormones. tnf-alpha, tumour necrosis factor-alpha; frc, residual functional potential (kwok et al.,2020). hypertesion and covid-19 pathophysiology and risk in patients with covid-19, a high incidence of hypertension have been observed, with htn likely predisposing them to an increased risk of more serious illness. the danger may stem from several factors. first, hypertension is mainly associated with immune dysregulation, which occurs as higher levels of il-17, the irregular activity of natural killer cells, and cytotoxic defects of t cells partially reversible by mineralocorticoid receptor antagonists (shekhar et al., 2020). therefore, the initially reported correlation between hypertension and hospitalization rates for covid-19 in china is not surprising.3 indeed, the 50 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 proportion of self-reported hypertension was 12.6 % in a wide database of 20,982 patients with diagnosed covid-19 infections and information on underlying diseases (kreutz et al., 2020). it is unknown whether or not uncontrolled blood pressure is a risk factor for covid-19 acquisition or whether or not controlled blood pressure is less of a risk factor for hypertension patients. however, some organizations have also emphasized that blood pressure regulation remains a significant factor in minimizing the disease burden, even though it does not impact the vulnerability to sars-cov-2 viral infection (schiffrin et al., 2020). hypertension adversely impacts the health status of patients with covid-19. however, broad prevalence studies showing the effects of comorbid diabetes and hypertension are urgently needed (parveen et al., 2020). however, the outbreak of covid-19 may also be a specific moment when specialists in pulmonary hypertension have to balance the costs and benefits of diagnostic work-up, including possible exposure to covid-19 versus the initiation of targeted pulmonary arterial hypertension therapy in a select high-risk, high probability world symposium pulmonary hypertension group 1 patient with pulmonary arterial hypertension (rayan et al., 2020). a severe and possibly fatal manifestation of covid-19 is cardiac involvement and sars-cov-2-associated myocarditis. hypertension treatment with a drug that can minimize inflammation in viral myocarditis and does not pose a theoretical risk of encouraging the proliferation of covid-19 would appear to be a sensible strategy for improving patient outcomes (antwi-amoabeng et al., 2020). recent studies have shown that among patients with covid-19, arterial hypertension, diabetes, cardiovascular disorders, and chronic obstructive pulmonary disease are prevalent. research on the outcome of these patients is scarce, and there is very little evidence. nevertheless, hypertension is one of covid-19's most important risk factors. the relationship between hypertension and negative outcomes is still questionable (tadic et al., 2020). renin-angiotensin system (ras) dysfunction has been identified in patients with coronavirus infection (covid-19). still, it remains unclear whether ras inhibitors, such as angiotensin-converting enzyme inhibitors (aceis) and type one receptor blockers (arbs) of angiotensin ii, are associated with clinical outcomes. covid-19 hypertensive patients have been enrolled to test the impact of ras inhibitors (meng et al., 2020). protein for ace2-expressing cells to join. ace-2 is a part of activating the renin-angiotensin (ras) system that plays an important role in hypertension. this connection between ace2 and sars-cov-2 sparked interest in investigating the relationship between inhibitors of ras and infection with covid-19 (salah et al., 2021). hypertension was associated with increased poor composite outcomes in patients with covid-19, including mortality, extreme covid-19, ards, need for icu treatment, and disease progression (pranata et al., 2020) thyroid and covid-19 the immune system, especially cellular immunity, is modulated by thyroid hormones. the dysregulation of the immune system in hypothyroidism might increase the risk of infection. however, the immune system recovers its normal function after proper hormone replacement therapy (mitrou et al., 2011). the influence of pre-existing hypothyroidism on the effects of covid-19 is unclear (van gerwen et al., 2020). effects of covid-19 on the hypothalamic-pituitary-thyroid axis: it is known that systemic disorders are associated with low-t3 or non-thyroidal syndrome. severe covid-19 is expected to cause such a disease, especially when the infection is associated with fever and lower respiratory tract involvement. in addition, sars-cov-2 infection has been reported to influence the nervous system, typically affecting smell and taste with the involvement of cranial nerves (boelaert et al., 2020). follicular cell death would manifest as low t3 and t4; damage to parafollicular cells would potentially result in low serum calcitonin levels. this has been suggested as a possible femoral head osteonecrosis mechanism in recovered sars patients; calcitonin deficiency contributes to osteoclast disinhibition leading to osteonecrosis (kaiser et al., 2020). data on thyroid function or thyroid pathology in covid-19 is not yet available. a consensus statement concerning problems related to thyroid dysfunction during the covid-19 pandemic has been released by the british thyroid association and the society for endocrinology (bta/sfe). patients with underlying hypothyroidism or hyperthyroidism are recommended to continue their prescription drugs as normal (pal et al.,2020). hyperthyroidism is an endocrine condition characterized by the thyroid gland's inappropriately high thyroid hormone output. it is usually caused by thyroid gland autoimmune dysfunction and has a population prevalence of 1%-15% (kaur et al., 2020). in addition to clear ultrasonographic evidence indicating subacute thyroiditis, the patient presented with tachycardia, anterior neck pain, and thyroid function tests showing hyperthyroidism. corticosteroid therapy resulted in a fast clinical resolution. the case shows that virus-related subacute thyroiditis such as sars-cov-2 should be regarded as a complication of covid-19 and treated as a differential diagnosis in infected patients with tachycardia without evidence of developing covid-19 disease (mattar et al., 2020). smail – the association between some endocrine 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(2020). association of hypertension, diabetes, stroke, cancer, kidney disease, and high-cholesterol with covid-19 disease severity and fatality: a systematic review. diabetes & metabolic syndrome: clinical research & reviews, 14(5), 1133-1142. zhou, f., yu, t., du, r., fan, g., liu, y., liu, z., ... & guan, l. (2020). clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 371-389 | doi: 10.14421/biomedich.2023.121.371-389 issn 2540-9328 (online) medicinal biospecificity of ginger and its efficacious bioactive compounds in the context of its biological activities against predominant health issues: current study and new avenues haseeba shahzad1,4,*, shaifa saleem1, waqas hanif2, zareena ali3, muhammad zeeshan ahmed4, haq nawaz4, zelle humma3,5, laraib rana6, muhammad qamar farid7, hajira kalsoom1, samavia jaan1 1school of biochemistry and biotechnology, university of the punjab, quaid-i-azam campus, lahore 54590, pakistan. 2department of pathology, combined military hospital, bannu, pakistan. 3department of biochemistry and biotechnology, the women university multan, pakistan. 4department of biochemistry, bahauddin zakariya university, multan 60800, pakistan. 5department of experimental medicine, university of genoa, italy. 6department of biochemistry, faculty of biological sciences quaid-i-azam university, islamabad 45320, pakistan. 7department of chemistry, faculty of science, chulalongkorn university, bangkok 10330, thailand. corresponding author* haseebashahzad388@gmail.com abstract there is a multitude of life-threatening and widespread health issues worldwide, regarding weak immunity, severe inflammation, viral infections, bacterial infections as well as antimicrobial resistance (amr), high free radicals generation, and cancer. ginger, a perennial plant of the zingiberaceae family with several authentic nutritional and medicinal values used in many countries as traditional medicine. that is why, the study was designed to highlight recent studies about medicinally most efficacious bio-active compounds of ginger along their biological significance related to immuno-stimulatory, anti-inflammatory, anti-viral, anti-bacterial, anti-oxidant, and anti-cancer effects. our study also recognized future gaps in research. the study included professional research data under duration from 2001-2022 appearing in books and scholarly journals, collected from scientific database platforms via pubmed, web of science, google scholar, springer nature, science direct and scopus. the present study includes the medicinal effects of almost 44 most influential ginger compounds like phenolics, terpenoids, flavonoids, and vinyllyl ketonic compounds etc. our results revealed the strong alleviating effects of gingerols, shogaols, paradols, and polyphenols. moreover, the ginger essential oil has proven to be very effective both for antiviral and antibacterial activity. however, no data is available in previous literature for components of ginger involved in immuno-stimulatory, effects. there is also a need to explore components for antibacterial activity. however, research has been conducted on ginger for only a few viruses despite its strong alleviating effects. besides this, more study is needed to comprehend the comprehensive mechanism of action (especially at the molecular level) regarding the anti-bacterial and anti-viral activity of ginger and its constituents. keywords: anti-bacterial; anti-cancer; antioxidant; antiviral; immunostimulatory; anti-inflammatory; ginger bioactive compounds; new avenues; mechanism of action. introduction herbal medicines’ use is increasing as compared to the use of chemicals day by day, therefore, more research has been performed on phytochemicals to make them more effective for the intervention of various ailments (ishiguro et al., 2007). but it is very troublesome to explore the effectiveness of herbal treatment because of the presence of many compounds in various forms (raynor et al., 2011). ginger (zingiber officinale) is an angiospermae and belongs to the family zingiberaceae. its rhizomes are widely used as a spice and traditional medicine in many countries like the subcontinent and the united states (shahrajabian et al., 2019). due to the diverse phytochemistry of ginger, the components of ginger are classified as volatiles and non-volatiles. volatile components of ginger include sesquiterpene and monoterpenoid hydrocarbons which provide a distinct taste and aroma to ginger while the non-volatile pungent components include shogaols, paradols and zingerone (jolad et al., 2005). ginger has diversified nutritional and medicinal importance. the general nutritional and nonnutritional components of ginger and their quantities have been represented in table 1. ginger contains more than 400 different compounds. the major compounds present in ginger are terpenes, phenolic compounds, carbohydrates, proteins, lipids, gingerols, and shogaols. there are many phytochemicals present in ginger that play an effective medicinal role. the major classification of phytochemicals has been represented in figure 1 and manuscript received: 04 january, 2023. revision accepted: 19 april, 2023. published: 24 june, 2023. https://doi.org/10.14421/biomedich.2023.121.371-389 mailto:haseebashahzad388@gmail.com 372 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 the structures of major bioactive compounds of ginger have been depicted in figure 2. moreover, the oil extract of ginger has been observed to contain e-citral, z-citral, ocimene, and camphene (munda et al., 2018). ginger shows a wide range of prophylactic and curative functions. it suppresses the level of cholesterol in the blood, prevents excessive clotting, is used to treat dyspepsia, and improves appetite (palatty et al., 2013). other properties of ginger include fat loss, prevention of cardiovascular diseases, anti-cancer, and antiinflammatory action (elshater et al., 2009). gingerols play an important role in the alleviation of arthritis and pain. ginger also possesses antioxidant, antimicrobial, and anti-allergic properties because of the presence of gingerols and shogaols (semwal et al., 2015). ginger is consumed as a painkiller for chest pain, menstrual pain, and back pain. it is utilized to cure cough, bronchitis, and upper respiratory tract infections (shukla & singh, 2007). besides this, in china and many other countries, ginger is used for the treatment of rheumatism, nervous disorders, toothache, stroke, asthma, constipation, catarrh, gingivitis, diabetes, and migraine. due to its antiviral properties and warming effect, it is also used for relieving flu and colds (qidwai et al., 2003). moreover, it is also used as a stimulant, diuretic, and carminative in asian medicines (shadmani et al., 2004). ginger essential oil has proved to be very effective, particularly concerning antibacterial and antiviral activity (koch et al., 2008; mostafa et al., 2018). the constituents present in ginger essential oil and their percentage have been represented in table 2. besides this, the strong effects of ginger oleoresin, particularly, regarding antioxidant activity have been observed (ji et al., 2017). in previous literature, there is abundant data available regarding various effective and authentic medicinal approaches of ginger for various ailments but there is no such review available, in which data has been analyzed with a special focus on recent studies (2001-2022) regarding specificity of ginger bioactive compounds with respect to its biological activities to demonstrate the immuno-stimulatory, anti-inflammatory, anti-viral, antibacterial, antioxidant and anticancer effects. besides this, no data is available in past in which after the most recent study (up to 2022), a future research gap got identified. this review has been planned to keep in view the most common and serious health problems and their intervention issues of the day. our review will provide valuable data for drug manufacturers to isolate the bioactive components of ginger for manufacturing herbal drugs against particular disorders. besides this, it will also provide new pathways for the researchers to fulfill the future gap to remove the hot issues of the day easily. table 1. general composition of ginger (agriculture, 2018; nawaz et al., 2018). components value per 100g water 78.89g carbohydrates 17.77g total lipids 0.75g proteins 1.82g ash 0.77g total dietary fiber 2.0g total phenolic acids 0.63g total tannins content 0.28g total flavonoid content 3.93g ca 16mg fe 0.60mg mg 43mg p 34mg k 415mg na 13mg zn 0.34mg vitamin c 5.0mg choline 28.8mg figure 1. classification of major phytochemicals present in ginger (ghasemzadeh et al., 2010; shukla & singh, 2007). shahzad et al. – medicinal importance of ginger 373 figure 2. structures of major phytochemicals present in ginger (drawn by chem draw). table 2. representation of percentage composition of constituents of ginger essential oil. compounds % reference β-bisobeolene 12.5 (varoni et al., 2018) α-zingiberene 25 β-sesquiphellandrene 18 β-phellandrene 8 (ferreira et al., 2018) α-zingiber 24 geraniale 15 farnesene 7.6 (singh et al., 2008) geraniale 26 α-zingiberene 9.5 neral 7.4 1,8-cinerol 10 (snuossi et al., 2016) camphene 12 α-zingiberene 7 β-phellandrene 11 camphene 5 (mesomo et al., 2013) ar-curcumene 11.3 eucalypto 3 geraniale 11 β-cedrene 8.6 (moreira da silva et al., 2018) geraniale 16 geranyl acetate 8.4 z-citral 9.2 zingiberene 14 (borah et al., 2017) β-sesquiphellandrene 27 α-farnesene 10.5 caryophyllene 15.3 β-bisabalene 11 (wang et al., 2006) α-zingiberene 20 β-sesquiphellandrene 13 ar-curcumene 15 citral 7.5 (nogueira de melo et al., 2011) ar-curamene 59 α-zingiberene 7.5 1,8-cinerol 8 farnesene 7.6 (chmit et al., 2014) geraniale 26 neral 7.4 α-zingiberene 9.5 table 3. representing the percentage composition of constituents of ginger oleoresin by different methods (supardan et al., 2012). components composition (%) method nortrachelogenin 6.74 ultrasonicassisted extraction ar-curcumene 8.9 β-sesquiphellandrene 5.02 zingerone 13.85 shogaol 7.92 farnesene 6.29 soxhlet zingiberene 5.13 zingerone 14.47 β-sesquiphellandrene 5.44 shogaol 7.14 β-sesquiphellandrene 6.54 co2 supercritical zingiberene 16.77 geraniol 9.01 gingerol + shogaol 3.48 β-phellandrene 12.86 ginger as an immuno-stimulant immunostimulant is an agent that aids in increasing the body’s immune-response. immunity is defined as the body’s ability to resist infection or toxins by the action of sensitized white blood cells and specific antibodies (divangahi et al., 2021). immune-deficiency leads to various abnormalities and disorders including digeorge syndrome, chronic mucocutaneous candidiasis, interleukin-12 receptor deficiency, hyperimmunoglobulin m syndrome, severe combined immunodeficiency disease, leukocyte adhesion deficiency syndrome, chronic granulomatous disease, and wiskott-aldrich syndrome, etc (vaillant & qurie, 2021). ginger exhibits immuno-stimulatory and antiinfection properties against pathogenic bacteria, worms, and viruses (sanderson et al., 2002). it also shows its immunomodulatory effects in animals, like fish (nya & austin, 2009). white blood cells (wbc) are considered to be primary defenders of the body. in an experiment, when the rainbow trout were fed with ginger, an increased number of wbc, neutrophils, and other blood cells was observed (nya & austin, 2009). it has been noticed that herbal plants boost immunity by elevating the number of blood cells (sahu et al., 2007). like humans, fishes also have specific and nonspecific defense systems to protect themselves against microbes. in fishes, mucus and skin are the primary non-specific defenses. when any pathogen enters the body both humoral and cellular defenses are activated and phagocytosis is part of non-specific immunity. the increased phagocytotic activity of wbcs was observed in fishes fed with ginger (haghighi & rohani, 2013). the phagocytotic activity was compared in two groups of fish. in a group of fishes, with a 0.1% ginger diet, the mean phagocytosis index was 2.21±0.082 while it was greater, 2.37±0.263 in fishes fed with a 1% ginger diet 374 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 (dugenci et al., 2003). besides this, plasma proteins that play a role in humoral defense were also found to increase. it was concluded that both humoral and cellular immunity were enhanced by using ginger extract in the case when any pathogen entered the body through injury (dügenci et al., 2003). when plasma protein level was compared between two groups of fishes, it was observed that in a group with a 0.1% ginger diet the level of protein in fishes was 2.26±0.2541(g/dl) while it was significantly greater, 3.84±0.13(g/dl) in fishes fed with 1% ginger diet (dugenci et al., 2003). ginger shows immunomodulatory effects due to the presence of gingerols, shogaols, zingerone, and paradols (rasmussen, 2011). it has been observed that 6-gingerol suppressed the expression of tnf-α and inos by blocking pkc and nf-кb signaling pathways in macrophages of lipopolysaccharide-stimulated mice (lee et al., 2009). it has been reported that pkc stimulates nf-кb which in turn regulates the inos expression at the transcriptional level (simon et al., 2015). similarly in another study, 6gingerol was observed inhibiting the inos expression and no production in activated macrophages of j774.1 mice (ippoushi et al., 2003). in another study, 6-gingerol inhibited the formation of il-12, tnf-α, and il-1β in macrophages as well (tripathi et al., 2007). the other mechanistic actions of ginger as an immunostimulant are represented in figure 3. figure 3. mechanistic action of immune-stimulatory effects of ginger (shokr & mohamed, 2019). (upward arrow-indicating the enhancing activity, downward arrow-indicating the reducing activity) ginger as an anti-inflammatory when inflammation happens, certain chemicals from the white blood cells of the body enter tissues to protect them from invaders. this increases the blood flow to the site of damage or infection and thus causes redness. besides this, the leakage of fluid in tissues by certain chemicals results in swelling as well (varela et al., 2018). sometimes, acute inflammation (uncontrolled) may become chronic and leads to a variety of inflammatory disorders including bowel and cardiovascular diseases, cancer, and arthritis (zhou et al., 2016). ginger has proved to be very beneficial for the intervention of inflammatory disorders. ginger shows strong anti-inflammatory activity due to the presence of vinyllyl ketonic compounds. besides this, 6-paradol, 6shogaol, and 1-dehydro-6-gingerol are the constituents of ginger that have been observed to cause an antiinflammatory effect (ezzat et al., 2018). the antiinflammatory mechanism of the actions of ginger connecting its constituents has been illustrated in table 4. many enzymes are involved in causing inflammation like lipoxygenase shows its action by forming inflammatory lipid mediators such as hepoxilins, hydroxy fatty acid derivatives, lipoxins, and leukotrienes (kuhn et al., 2007). cyclooxygenase is involved in the formation of prostaglandins and thromboxane. ginger shows its action by decreasing the effect of cyclooxygenase, lipoxygenase, and arachidonic acid (lantz et al., 2007). in the experimental model of rheumatoid arthritis, more anti-inflammatory action has been noticed by combining both gingerols and ginger extract oil as compared to gingerols alone (funk et al., 2009). in inflammatory bowel diseases (ibd) which include ulcerative colitis and crohn's disease, an elevated level of tnf and pge2 has been observed (nakamura et al., 2006) because both diseases are responsible for inflammation (kawahara et al., 2015). ginger shows a protective effect against ibd by decreasing the level of both tnf and pge2 (el-abhar et al., 2008). in one study, tnf-α expression was compared in two groups of rats. in one group, liver cancer was induced by a choline-deficient diet and in the other group, a choline-deficient diet was given along with ginger extract. it was observed that the expression of tnf-α in rats with only a choline-deficient diet was 83.3 ± 4.52% while it was reduced in the group, that was also given the ginger diet along with choline-deficient diet where it was 7.94 ± 1.32% (p< 0.05). similarly the expression of nfκb was also observed in both these groups and it was observed that it was 88.3 ± 1.83% in group with only choline-deficient diet and it was significantly reduced in group that was also given ginger diet along with choline-deficient diet where it was 32.35 ± 1.34% (p<0.05) (habib et al., 2008). besides this, ginger has been observed to reduce the symptoms of gout (grzanna et al., 2005) and also relieve osteoarthritis of the knee (altman & marcussen, 2001). monocyte chemoattractant protein-1 (mcp-1) is a protein that is involved in inflammation. inflammation is also associated with the increased level of rantes production which activates the t cells (appay & rowland-jones, 2001) due to which the formation of il2 and ifn-ϒ also increases. in an experiment, when ginger was given to rats with high rantes levels, it was observed that ginger not only reduced inflammation and restricted t-cell activation but also suppressed the level of rantes and mcp-1( macrophage shahzad et al. – medicinal importance of ginger 375 inflammatory protein) (ezzat et al., 2018). besides this, the anti-inflammatory role of ginger oil has been noted by inhibition of il-1α (zhou et al., 2006). 6-gingerol decreases no production by inhibiting cytokines (ifn-γ and tnf-α) that stimulate the production of no (amri & touil-boukoffa, 2016). gingerol and shogaol inhibit the activity of prostaglandin synthetase enzymes and thus suppress the formation of prostaglandin. they also inhibit the action of cytokines i.e il-1 and il-8 that are involved in inflammation (verma et al., 2004). the comparison of factors involved in inflammation and counter effects of ginger has been shown in figure 4 in a nutshell. figure 4. comparison of factors involved in causing inflammation and therapeutic effects of ginger in a nutshell (ezzat et al., 2018; verma et al., 2004; zhou et al., 2006). abbreviations: tnf: tumor necrosis factor; nfkb: nuclear factor kappa-light-chain-enhancer of activated b cells; il: interleukin; ifn: interferon; pge-2: prostaglandin e-2; mcp-1: monocyte chemo-attractant protein-1; rantes: regulated on activation, normal t cell expressed and secreted. (upward arrow-indicating increasing activity, downward arrow-indicating decreasing activity) table 4. representing the anti-inflammatory mechanisms of ginger connecting its constituents. abbreviations: no, nitric oxide; tnf-α, tumor necrosis factor α; pge2, prostaglandin e2; gdnps, ginger-derived nanoparticles. sr. no. ginger/constituents dose research subjects mechanism of action reference 1 6-gingerol-rich fraction 50 & 100 mg/kg female specie wistar rats levels of no, tnf-α and myeloperoxidase enzyme, enhance (abolaji et al., 2017) 2 extract of ginger and zinger-one 0.1,1, 10&100 mg/kg female specie (balb/c mice) reduces the level of il-1β and inhibits the stimulation of necrotic factor-κb (hsiang et al., 2013) 3 6-shogaol 100 μm human intestinal epithelial cells (ht29/b6, caco-2) ceased the pi3k/akt, nf-κb signaling pathways (luettig et al., 2016) 4 gdnps 0.3 mg female mice (c57bl/6 fvb/nj) by using gdnps 2, levels of il-10 and il-22 and level of tnf-α, il-6, and il-1β decrease. (zhang et al., 2016) 5 extract of ginger 50 mg/ml mice (c57bl6/j) ginger extract prevents the production of tnf-α and activates the akt & nf-κb. (ueno et al., 2014) 6 6-gingerol, 6dehydroshogaol, 6shogaol 2.5 and 5,10 μm macrophage cells of the mouse (raw 264.7) no, pge2 production is stopped. (zhang et al., 2013) ginger as an antiviral the world health organization (who) and other public health agencies have warned about the emergence of infectious diseases at alarming rates that have not been previously noted (organization, 2021). the centers for disease control and prevention (cdc) elaborates on emerging infectious diseases. among all categories of infectious diseases, viral agents are the most serious threat to global populations (prevention., 2021). the 21st century is marked by major pandemics and epidemics due to novel viral agents that have been the source of mortality and morbidity around the world such as ebola virus, zika virus, middle-eastern respiratory syndrome 376 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 (mers), influenza a (h1n1) pdm/09, human immunodeficiency virus (hiv), middle-eastern respiratory syndrome (mers), west nile virus, chikungunya virus and currently severe acute respiratory syndrome virus 2 (sars-cov-2) (ong et al., 2020). besides this, the hepatitis c virus (hcv) is a critical illness. hcv may lead to acute or chronic hepatitis and principally causes liver cancer. universally, approximately 58 million human beings have chronic hcv infection. according to an approximation of who, almost 290 000 human beings expired from hepatitis c in 2019. at present, no efficient vaccine exists against hepatitis c (who, 2022) and pakistan is ranked second after china which is suffering from hepatitis at an alarming rate. in 2018, a very alarming prevalence rate of hepatitis c virus was observed in small towns in pakistan (ahmed et al., 2020). in china, fresh ginger is used as a folk medicine for the treatment of airway infections. ginger shows antiviral activity against various types of viruses like human respiratory syncytial virus (hrsv) and it has been observed that the effects of dried ginger are different from fresh ginger against hrsv because both dried and fresh ginger contain different constituents. fresh ginger shows better results against the virus as compared to dried ginger (jolad et al., 2005). hrsv causes lethality and death by inducing bronchiolitis and pneumonia (san chang et al., 2013). ginger reduces viral invasion by preventing the attachment and internalization of the virus into the cells. at high concentrations, fresh ginger stimulates the low respiratory tract mucosal cells to secrete ifn-b, the cytokine which inhibits the replication of viruses (ali et al., 2008). experimental studies have shown that an aqueous extract of ginger also reduces the infectivity of feline calicivirus (fcv). antiviral activity occurs due to a chemical interaction between phytochemicals like proanthocyanins, polysaccharides, polyphenols, and some other compounds present in natural extracts and the virus (aboubakr et al., 2016). the aqueous extract of ginger contains 1,2-propanediol, 1,2-benzene dicarboxylic acid, and 2,3-butanediol which does not affect the receptors of the cell for fcv but inhibits the attachment of viruses with cells by causing alterations in viral capsid. fcv is used as a surrogate for norovirus in experiments because it is very difficult to grow noroviruses in cell culture (aboubakr et al., 2016). norovirus is an enteric virus and is considered to be a major contributor to foodborne illnesses. aqueous extract of ginger has proved to be very beneficial in the prevention of foodborne diseases. we can also use its extract to wash vegetables and fruits in combination with water to prevent them from viral contamination (aboubakr et al., 2016). several terpenes in the alcoholic extract of ginger have been observed with anti-rhinoviral activity (adom et al., 2019). besides this, gingerenone has been observed in causing the inhibition of many types of influenza a viruses including h9n2, h1n1, and h5n1 (onyiba, 2022). propanediol in an aqueous extract of ginger has revealed strong antichikungunya activity (kaushik et al., 2020; wang et al., 2020). ginger essential oil (composition shown in table 2) has depicted antiviral activity with high accuracy. the inactivation of caprine alphaherpesvirus-1 (up to 100%) by disruption of the virus envelope and other structures needed for virus attachment to host cells has been observed by ginger essential oil (camero et al., 2019). more than 90.0% reduced activity of hsv-2 by preincubation with ginger oil also indicated its effective antiviral influence (koch et al., 2008). moreover, in invitro experimentation, direct inactivation of tulane and hepatitis a viruses have been observed by gingerol (patwardhan et al., 2020) while zerumbone has been observed with reduced activity of epstein–barr virus (onyiba, 2022). in hepatitis c-infected patients, ginger administration revealed the reduction in viral load by suppression of aspartate aminotransferase, α-fetoprotein and alanine aminotransferase (abdel-moneim et al., 2013). however therapeutic effects of ginger have also been observed against sars-cov-2. in coronavirus disease, the cleavage of polyprotein a/b (pp a/b) takes place via papain-like protease (plpro) at different sites, producing several proteins necessity for viral replication and survival (goswami et al., 2020). molecular docking has disclosed the potential inhibition of plpro by 10gingerol, 6-gingerol and 8-gingerol (shin et al., 2020). the binding potential of shogaol, gingerol, zingiberene, zingerone, zingiberenol and geraniol with catalytic domain of mpro has also been observed in docking studies. meanwhile, interference with s protein-ace2 binding has also been remarked by shogaol, zingiberenol, geraniol, zingiberene, gingerol and zingiberene (ahkam et al., 2020). s protein inhibition of corona virus has been examined by 10-shogaol, 10paradol, 10-gingerol, 8-paradol, 8-gingerol, 10-gingerol and sesquiphellandrene (terpene) (joshi et al., 2020). in a study carried out in saudi arabia, a decrease in hospitalization (28.0%) has been observed in covid-19 patients using the ginger diet as compared to nonusers (38.0%) (aldwihi et al., 2021). similarly in another study, in covid patients treated with ginger extract, low serum level of tnf-α, il-1, and il-6 was observed along with short-time mechanical ventilation as compared to the control (shariatpanahi et al., 2013) ginger as an antibacterial bacterial infections are the most common infections everywhere. not only this but with time antibiotic stewardship issue has become the most serious health threat worldwide. the problem can be resolved by replacing allopathic medicines with herbal drugs. antimicrobial resistance (amr) is a threat worldwide to development and health. it necessitates quick and crucial multisectoral action to attain sustainable development https://www.sciencedirect.com/topics/medicine-and-dentistry/propylene-glycol https://www.sciencedirect.com/topics/medicine-and-dentistry/alanine-aminotransferase https://www.sciencedirect.com/topics/medicine-and-dentistry/polyprotein https://www.sciencedirect.com/topics/medicine-and-dentistry/proteinase https://www.sciencedirect.com/topics/medicine-and-dentistry/shogaol https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gingerol https://www.sciencedirect.com/topics/medicine-and-dentistry/zingiberene https://www.sciencedirect.com/topics/medicine-and-dentistry/zingerone https://www.sciencedirect.com/topics/medicine-and-dentistry/geraniol https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gingerol shahzad et al. – medicinal importance of ginger 377 goals. who has stated that worldwide, antimicrobial resistance is among the 10 topmost health threats for human beings (who, 2021). globally, one of the most common reasons for mortality is antimicrobial resistance (amr), especially in settings where health care organizations do not get the least possible standards set by the world health organization (who) or some other quasi-governmental organization. according to analytical statistical models, in 2019, the bacterial amr led to approximately 4·95 million demises. owing to amr, 6 main pathogens caused 929 000 (660 000–1 270 000) demises, these pathogens include escherichia coli, streptococcus pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, and acinetobacter baumannii (murray et al., 2022). ginger shows its action against both gram-positive and gram-negative bacteria (abdulzahra & mohammed, 2014). the oil extract of dried ginger has proven to be very efficacious against various types of pathogens involved in food-borne diseases like e.coli, vibrio cholera, salmonella spp, and klebsiella (islam et al., 2014). in an experiment when v.harveyi infected lates calcarifer which is an asian sea bass and was given a ginger diet, it was observed that ginger controlled the infection caused by v.harveyi (talpur et al., 2013). v.harveyi is a gram-negative bacteria that causes death in asian sea bass (austin & zhang, 2006). ginger is found to increase the lysozyme activity (talpur et al., 2013). lysozymes are the enzymes that carry the hydrolysis of peptidoglycans present in the cell wall of bacteria. in this way, ginger inhibits the penetration of detrimental bacteria (nile & park, 2015). besides this, the antibactericidal action of serum (which plays an important role in removing pathogens) also increases by ginger (ellis, 2001). ginger increases anti-protease activity and thus suppresses the attacking ability of bacteria. the increased respiratory burst action of neutrophils by ginger has also been reported (talpur et al., 2013). a respiratory burst is a reaction in phagocytes that causes the degradation of bacteria. in fishes with a 0.1% ginger diet, 1.05±0.050 (nmol o2 /10 5 leucocyte) respiratory burst activity was observed while it was significantly greater, 1.26±0.230 (p<0.001) in fishes fed with a 1% ginger diet (dugenci et al., 2003). the ginger essential oil has been observed with strong anti-microbial properties against many bacteria. the percentage composition of ginger essential oil is represented in table 2. after the extraction of ginger essential oil by microwave-assisted hydrodistillation method and its chemical composition analysis by ftir spectrometer, citral was found to be the most abundant (89.05%) chemical compound in oil (kalhoro et al., 2022). it was observed in the study that ginger oil obtained through hydrodistillation showed more sensitivity against l.monocytogenes as compared to other bacteria and depicted a big inhibition zone (37mm). it was also observed to be active against the strain v.alginolyticus, despite its high mic value (minimum inhibitory concentration) (0.05-0.2mg/ml)(mostafa et al., 2018). the moderate activity of ginger oil with mic value 0.16-0.63 mg/ml against gram-positive bacteria demonstrated the high resistance of gram-negative bacteria against ginger oil as compared to gram-positive bacteria (snuossi et al., 2016). ginger essential oil has been shown to inhibit activity against e.coli and shigella due to the presence of bioactive constituents like gingerol, endo borneol, and zingiberene (sivasothy et al., 2011). similarly in another research conducted in brazil, zerumbone isolation from ginger oil depicted its efficacy against s.mutans with 500 µg/ml mbc (minimum bactericidal concentration) and 250 µg/ml mic. the efficacy of oil against biofilm formation and growth activity of s.pyrogenes resulted in 1mg/ml mbc and mic(wijesundara & rupasinghe, 2018). the more sensitivity of gram-positive strains suggested that the thick peptidoglycan surrounding the membrane of the cytoplasm would be the bacterial target of essential oil (cox & markham, 2007). however, the possibility of another target cannot be proscribed. the better effect of oil against gram-negative bacteria suggested other microbial targets like plasma membranes as bioactive components of essential oil possess lipophilic properties which interact with membranes via influencing their permeability and fluidity (rouseff & perez-cacho, 2007). the effective doses of ginger (halo, mbc, and mic) against different bacteria connecting different countries have been represented in table 5. 378 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 table 5. representing the effective doses of ginger against different bacteria connecting different countries. abbreviations: mbc, minimum bactericidal concentration; mic, minimum inhibitory concentration. bacteria halo (mm) mbc (μl/ml) mic country reference e. coli o157:h7 18.7 9.4 microliter/ml brazil (da silva et al., 2018) p. aeruginosa 4.7 2.3 microliter/ml s. typhimurium 18.7 9.4 microliter/ml s. aureus 4.7 2.3 microliter/ml l. monoctogenes 9.4 4.7 microliter/ml v. alginolyticus >25 tunisia (snuossi et al., 2016) shigella 119.79% saudi arabia (ashraf et al., 2017) e. coli 106.02% e. faecalis 61.94% p. aeruginosa 21.65% b. spizizenii 0.24 milligram/ml negeri sembilan (sivasothy et al., 2011) e. coli 0.31 milligram/ml p. stutzeri 0.63 milligram/ml b. licheniformis 0.16 milligram/ml k. pneumoniae 0.47 milligram/ml l. plantarum 7.0 brazil (ambrosio et al., 2017) s. enteritidis 8.8 e. faecalis 1.0 milligram/ml mexico (lópez et al., 2017) s. aureus 0.25 milligram/ml s. epidemidis 0.5 milligram/ml s. mutans 500 μg/ml 250 μg/ml brazil (moreira da silva et al., 2018) s. aureus 8.90 india (bag & chattopadhyay, 2015) m. l nkluteus 6.86 l. monocytogenes 9.00 s. typhimurium 6.61 b. cereus 9.11 e. coli 8.00 b. cereus 8.3 saudi arabia (mostafa et al., 2018) s. typhi 0.0 s. aureus 15.8 p. aeruginosa 11.2 e. coli 0.0 k. pneumoniae 20.5 india (singh et al., 2008) p. vulgaris 18.4 s. pyogenes >1000 μg/ml >1000 μg/ml canada (wijesundara & rupasinghe, 2018) ginger as an antioxidant the oxidative stress develops when in the organs and tissue, the development of extremely reactive species, for example, reactive nitrogen species (rns), reactive sulfur species (rss), and reactive oxygen species (ros), prevail over the endogenous antioxidant defense system competence, brings about the dysfunctions and cellular damage, and ultimately gives rise to a broad range of diseases. free radicals produce oxidative stress in the living system which is the major cause of many chronic diseases like cataracts, cancer, aging, rheumatoid arthritis, and autoimmune diseases (pham-huy et al., 2008). the inequality between vulnerable defense systems and too many reactive species causes harm to various cell structures as well as to many molecules like dna, lipids, and proteins, and finally leads to a broad range of diseases (janssen-heininger et al., 2008). medicinal plants contain many phytochemicals which are proved to be effective in alleviating the toxic effects of free radicals. ginger also contains many phytochemicals like 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol which possess antioxidant properties due to their solubilizing side chains and hydroxyl groups which are further classified into two major groups diarylheptanoids and gingerol-related compounds (si et al., 2018). results on hplc-ms/ms of both extracts confirmed that eight different types of phenolic acids are present in ginger that proved the effective antioxidant activity and cause a delay in the diseases which are caused by oxidative stress (tohma et al., 2017). shahzad et al. – medicinal importance of ginger 379 ginger is very useful for chemotherapeutic patients because patients produce many reactive oxygen species which are responsible for oxidative stress followed by more lipid peroxidation products like nitric oxide (amin et al., 2012; gupta et al., 2010; srivastava et al., 2010). when the ginger extract was given to such patients, it showed its efficacy by enhancing the level of antioxidant enzymes and suppressing the level of lipid peroxidation products (danwilai et al., 2017). 6-gingerol is the most abundant compound in ginger that shows antioxidant activity (baliga et al., 2011). the comprehensive mechanism of action of 6-shogaol for anti-oxidant effects has been illustrated in figure 5. newly identified cancer sufferers taking strong beneficial chemotherapy have been randomized to take 20 mg of 6-gingerol, a ginger extract per day which enhances the antioxidant enzymes (danwilai et al., 2017). in one study, the level of antioxidant enzymes was compared between two groups of rats. in one group of rats, there were diabetic rats as control and in the other group, diabetic rats were fed with a ginger diet. a very low level of superoxide dismutase enzyme was observed in the control group which was 90.40±3.10 (u/ml) but it was significantly higher in ginger fed group where it was 109.30±3.70(u/ml) (p<0.05). similarly, the level of glutathione peroxidase (u/ml) was 22.90±0.89 in the control group while it was significantly higher, 26.31±2.10 in the ginger-fed group. the overall antioxidant capacity (mm) of the control group was 0.99±0.17mm while in the ginger treated group it was 1.28±0.29 (p<0.05) (abdullah, 2012). ginger prevents the formation of hydroxyl radicals due to the presence of polyphenols because they form a chelate with fe[3+] (stoilova et al., 2007). polyphenol compounds show high antioxidant activity because they are reducing agents and free radical scavengers (juntachote et al., 2006). anthocyanins and ascorbic acid are two phenols that act as free radical scavengers (pantelidis et al., 2007). phenolics also improve the quality of food by inhibiting the degradation of lipids which is why their use in the food industry is increasing day by day (wojdyło et al., 2007). it has been observed that gingerol plays a dominant role in the inhibition of phospholipid peroxidation as well as inhibits the xanthine oxidase, the enzyme that is involved in the formation of reactive oxygen species (nile & park, 2015). the other mechanisms of different constituents of ginger for antioxidant effects have been shown in table 6. figure 5. the comprehensive mechanism of action of 6-shogaol for antioxidant activity (chen et al., 2014): 6-shogaol causes the translocation of nrf2 in the nucleus and enhances the expression of genes that target nrf2 by modification of keap1 and protects nrf2 from proteasomal degradation. in this way the gsh level increases and the ros level suppresses. abbreviations: keap1, kelch-like ech-associated protein 1; nrf2, nuclear factor erythroid related factor 2; are, antioxidant response element; ftl, ferritin light chain; ho-1, heme oxygenase-1; trx1, thioredoxin 1; nqo1, nicotinamide adenine dinucleotide phosphate (nadph) quinone dehydrogenase 1; trxr1, thioredoxin reductase 1; gclc, glutamate-cysteine ligase catalytic subunit; ggtla4, γglutamyltransferase-like activity 4; gclm, glutamate-cysteine ligase modifier subunit; akr1b10, aldo-keto reductase family 1 member b10; gsh, glutathione; ros, reactive oxygen species. table 6. representing the antioxidant mechanism of action of different components of ginger. abbreviations: mda, malondialdehyde; gssg, glutathione disulfide; glutathione s-transferase p1; mt1, metallothionein. sr. no ginger/constituents dose research subjects mechanism of action reference 1 6-shogaol 20 μm human colon carcinoma cells hct-116 enhancing the activation of mt1, ftl, akr1b10, ho-1,ggtla4, mt1,gclm, and gclc genes; decreasing the level of ros; enhancing the intracellular gsh/gssg ratio; reducing the level of ros (chen et al., 2014) 100 mg/kg mice with wild-type and nrf2/c57bl/6j mutations upregulation of ho-1, gclc, and mt1 expression 2 ginger oleoresin (composition shown in table 2) 100 μg/ml mesenchymal, stem cells from humans stimulating ho-1, nqo1 gene expression; reducing ros generation; stimulating nrf2 translocation to the cell nucleus (ji et al., 2017) 3 ginger phenylpropanoids 40 μg/ml bj fibroblasts from the foreskin upregulation of nrf2 activity and gstp1 level (schadich et al., 2016) 380 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 table 6. cont. sr. no ginger/constituents dose research subjects mechanism of action reference 4 6-gingerol rich fraction 50, 100 mg/kg wistar rats, females h2o2 and mda levels are being reduced, while antioxidant enzyme activity and gsh levels are being increased. (abolaji et al., 2017) 5 ginger extract 100 mg/kg wistar albino rats, males lowering the mda level; preventing catalase activity & gsh content from being depleted (saiah et al., 2018) 200, 400 μg/ml human fibrosarcoma cells, strain ht1080 reducing the production of reactive oxygen species (ros) (romero et al., 2018) 5, 25 μg/ml human chondrocyte cells c28i2 increasing antioxidant enzyme gene expression and lowering ros as well as lipid peroxidation levels (hosseinzadeh et al., 2017) 78 to 313 μg/ml homogenates of rat hearts mda levels decreased (akinyemi et al., 2013) ginger as an anticancer cancer is a disease in which abnormal cells grow and spread uncontrollably throughout the body and damage normal body tissues (fearon et al., 2011). cancer is the second leading cause of death in the world. according to who's (2022) cancer report, 10 million deaths cases were observed in 2020 with nearly one in six deaths. how-ever in lower-middle-income countries, 30% of cancer cases were observed (organization, 2022). globally, approximately 10.0 million cancer-related mortalities (9.9 million without non-melanoma skin cancer) and almost 19.3 million new patients of cancer (18.1 million without non-melanoma skin cancer) arose in the year 2020. breast cancer in females has exceeded lung cancer as the most frequently detected cancer, with almost 2.3 million new patients (11.7%), after that lung, colorectal, prostate, and stomach cancers were 11.4%, 10.0 %, 7.3%, and 5.6% respectively. the highest cause of cancer-related death is lung cancer, with approximately 1.8 million mortalities (18%), afterward here comes colorectal, liver, stomach, and female breast cancers in the range of 9.4%, 8.3%, 7.7%, and 6.9% respectively. on the whole, the frequency was 2 to 3 fold greater in transitioned countries when compared with transitioning countries (sung et al., 2021). studies suggest that ginger causes the cell death of various types of cancerous cells including ovarian, colon, brain, liver, cervical, skin, prostate, renal, pancreatic, gastric, and breast cancer (srinivasan, 2014). phytochemicals like gingerols, shogaols, paradols, and polyphenolics are the compounds that are part of ginger and are involved in the anticancer activity and reduce its risk by targeting at different stages (cheng et al., 2011). the therapeutic mechanisms of different constituents of ginger against various types of cancer are mentioned in table 7. the potential mechanisms of 6-gingerol for suppressing proliferation and induction of apoptosis in cancer (figure 6) (liu et al., 2017; tahir et al., 2015). figure 6. schematic diagram depicting the various signaling pathways of 6-gingerol for anti-cancer activity. abbreviations: pi3k: phosphoinositide 3kinase; ampk: 5 adenosine monophosphate-activated protein kinase; bcl-2: b-cell lymphoma 2; bax: bcl-2-associated x protein; cdk: cyclin-dependent kinase; mtor: mammalian target of rapamycin; akt: protein kinase b. (upward arrow-indicating increasing activity, downward arrow-indicating decreasing activity) shahzad et al. – medicinal importance of ginger 381 table 7. representing the therapeutic mechanism of action of different constituents of ginger against different types of cancer. abbreviations: nf-κb, nuclear factor kappa light chain-enhancer of activated b cells; ampk, 5 adenosine monophosphate-activated protein kinase; gdnp, ginger derived nanoparticles; erk, extracellular signal-regulated kinase; bcl-xl, b-cell lymphoma-xl protein; kras, kirsten rat sarcoma viral oncogene; mtor, mammalian target of rapamycin; stat3, signal transducer and activator of transcription 3; c-myc, cellular myelocytomatosis; bcl-2, b-cell leukemia/lymphoma 2; gstπ, glutathione-s-transferase; mrp1, multidrug resistance associated protein 1. sr. no. ginger/constituents dose research subjects mechanism of action reference 1 10-gingerol 50-100 and 200 μm carcinoma cells from mouse breasts and human inhibit cell growth, reduce the cell division and responsible for s phase cell cycle apoptosis (bernard et al., 2017) 2 gdnps -2 0.3 mg female, mice (c57bl/6) by using gdnps-2 the expression of cyclin d1 is restrained and intestinal epithelial cell multiplication is also restricted. (zhang et al., 2016) 3 extract of ginger based on fluorescent carbon nano-dots 1.11 mg/ml carcinoma cells (hepatocellular, hepg2 human boost up the apoptosis, enhance the ros level, and dominate the expression of p53. (li et al., 2014) 4 ginger extract 100 mg/kg female mice (swiss albino) ginger extract activates the ampk, and also by using it level of nf-κb is reduced, p53 expression is enhanced and the level of nf-κb, d1 is reduced. (el-ashmawy et al., 2018) 2–10 mg/ml human colorectal, adenocarcino-ma (ht29) stimulate the cell apoptosis, and functions of the caspase-9 gene and downcast the level of erk, bcl-xl, kras, etc (tahir et al., 2015) 5 6-gingerol 60,100,140 μm hela human (cervical adenocarcinoma cells) it is responsible for the cell cycle, arrest in the resting phase of the cell (g0/g1-phase), and also it is responsible for the downregulation of cyclins levels (cyclins a, d1, and e1) and it enhances the caspase gene expression and stops the mtor signaling pathway (zhang et al., 2017) 6 ginger extract with alginate beads 50 mg/kg male specie (wistar rats) responsible for enzymatic activity i.e nadh dehydrogenase and succinate dehydrogenase activity (deol & kaur, 2013) 7 6-shogaol 10, 20, 40 μm lncap, du145,pc3, human prostate (cancer cells) responsible for the activation and deactivation of different mechanisms in signaling pathways i.e it stops stat3, nf-κb signaling pathway, and it downregulates the expression of d1, c-myc, bcl2, surviving, and it is also responsible for cell apoptosis. (saha et al., 2014) 8 gingerol-6, shogaol6, gingerol-10, shogaol-10. 10,100 μm pc-3, human prostate cancerous cells. stops the multiplication of prostate cancer cell and suppress mrp1, gstπ expression. (liu et al., 2017) gastric cancer (stomach cancer) another health-related problem is gastric cancer which is also known as stomach cancer. this cancer is the 5th most prevalent cancer and the 3rd most popular basis for cancer-related mortality worldwide. the affliction of gastric cancer is incredibly more in asia, central and eastern europe, and latin america while in most western european countries and north america, it is not a more widespread cancer (ferro et al., 2014). studies have shown that 6-gingerol and 6-shogaol, the active components of ginger, are involved in an anticancer activity that shows a different mode of action against gastrointestinal cancer (ishiguro et al., 2007; prasad & tyagi, 2015). tumor necrosis factor-related apoptosis-inducing ligand (trial) is a cytokine that plays a role in apoptosis and thus is involved in the suppression of metastasis in the host (thorburn, 2007). 6-gingerol upregulates the trial-induced apoptosis by enhancing the trial-induced caspase-3/7 activation. caspase 3/7 are proteases that are involved in cell death machinery (ishiguro et al., 2007). 6-gingerol suppresses the activity of inhibitors of caspase-3/7 like ciap1 and 382 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 thus promotes cell death (prasad & tyagi, 2015). on the other hand, 6-shogaol disturbs the intracellular level of tubulin, a protein in microtubules that performs many cellular functions including mitosis. this disturbing level of tubulin induces mitotic arrest and thus causes apoptosis of cancerous cells (mollinedo & gajate, 2003). pancreatic cancer medically, the malignant tumor that develops in epithelial cells of glandular structures in the pancreatic ductal cells is called pancreatic cancer (aier et al., 2019). now, the frequency and deaths related to pancreatic cancer are rising every year universally, no matter in japan, europe, the us, or china (hu et al., 2021). it has been observed that 6-shogaol and 6-gingerol are the components of ginger that induce apoptosis and antiproliferative activity in tumor cells (li et al., 2012; lu et al., 2014). 6-gingerol inhibits the growth of pancreatic cancer cells by hindering the cell cycle at the g1 phase. retinoblastoma is a protein that regulates the cell cycle. the phosphorylated retinoblastoma is mainly involved in controlling the cell cycle at g1 to s phase. it has been observed that 6-gingerol inhibits the phosphorylation of retinoblastoma after suppressing the expression of cyclin a and cyclin-dependent kinase and thus blocks the cell cycle of cancerous cells by not allowing it to enter in s phase (park et al., 2006; stone et al., 2011). it has also been noticed after experiments that normal cells like human umbilical vein endothelial cells (huvec) are unaffected by ginger as compared to panic-1 cells. features of apoptosis-like an increase in subg1 cells, fragmentation of nuclei, and caspase-3 activation have been noticed by the ginger extract in pancreatic cancer cells. however, treatment of cells with ginger for a long time increases the formation of reactive oxygen species which causes autoptic cell death (akimoto et al., 2015). prostate cancer one of the most prevalent types of malignant cancer (after skin cancer) in males is prostate cancer, which is linked to the reproductive system. in 2012, world statistics stated that 15% of male cancers are prostate cancers as well as it is the 2nd major cause (after lung cancer) of deaths in males related to cancers (khazaei et al., 2019). the cell cycle is maintained by cyclin-dependent kinases (cdks). in prostate cancer cells, ginger exhibits its activity by decreasing the level of cyclin d1 and cdk4 levels. it also reduces the cyclin e levels which control the cell cycle through s-phase (karna et al., 2012). besides this, ginger stalls the cell cycle by increasing the level of p21 which is a cdks inhibitor (besson et al., 2008). ginger also activates caspase-3 which cleaves many cellular proteins such as parp (karna et al., 2012). parp play role in many cellular activities including repairing of dna. the inhibition of parp causes an increase in apoptosis due to a reduction in dna repairing capacity (morales et al., 2014). it concludes that ginger inhibits the growth of prostate cancer cells by apoptosis and by causing derangements in the cell cycle (karna et al., 2012). limitations in addition to the medicinal effects of ginger, some side effects of ginger have also been observed. more bleeding has been observed during surgery in patients, who take regular doses of ginger (ghorbanian p., 2017). it means ginger is also unsafe for patients who have a bleeding disorder. taking ginger more than 5 grams a day enhances the risk of side effects. its high consumption causes skin rashes, mouth irritation, upset stomach, heartburn, and gas. in some cases, the consumption of ginger in combination with medicines also shows harmful effects (line, 2018; mohan, 2017). it is even important to consult with a doctor before taking ginger while pregnant. ginger is more treated as food rather than medicine therefore like drug manufacturers, the supplier of ginger does not show whether it is safe or not before selling in the market (heitmann k1, 2013). conclusion ginger is broadly used in ethnomedicines, having several bioactive compounds with apprehensible pharmacological actions. this review demonstrates the recent studies (under duration from 2001-2022) about the pharmacological actions of ginger and it's almost 44 bioactive compounds. in this study, ginger has unveiled immunostimulatory, anti-inflammatory, anti-viral, antibacterial, anti-oxidant, and anti-cancer effects by stimulating and inhibiting various mechanisms at the cellular, molecular and enzymatic levels. our study provides a comprehensive overview of botanical authentication, experimental studies (in-vitro, in-vivo, and computational), pharmacological properties, and phytoconstituents profile of ginger along with their biological activities. not only this, but this review has also recognized research deficiencies after analyzing the data (up to the year 2022) and has provided new pathways for future research. the present review is beneficial for the development of ginger-based ethnomedicines, for the treatment of the most common and serious health issues of the day with minimum cost and side effectiveness. the major components of ginger and their mechanism of action regarding immunostimulatory, anti-inflammatory, anti-viral, antibacterial, anti-oxidant, and anti-cancerous effects have been concluded in figure 7. shahzad et al. – medicinal importance of ginger 383 figure 7. showing the immunostimulatory, anti-inflammatory, anti-viral, anti-bacterial, anti-oxidant, and anti-cancerous effects of different components of ginger along their mechanism of action in a nutshell. (upward arrow-indicating the increasing level, downward arrow-indicating the decreasing level) recommendations we should consider any confounding factors when assessing the medicinal beneficial effects of ginger against chronic diseases. increasing verification has depicted that different constituents of ginger have different molecular targets, metabolic pathways and bioactivities suggesting the significance of identifying the bioactive compounds and their biological mechanisms for a specific disease. gingerols and shogaols have been observed to be very potent bioactive compounds of ginger regarding its biological significance. it has been noticed that a few rodent and invitro studies directly made comparisons between the bioactivities of shogaols and gingerols in limited disease models. it is suggested to compare the effectiveness of gingerols and shogaols in more models and thus develop unusual ginger formulations for particular diseases. to determine the optimum oral ginger composition will also enforce the study of the additive and synergistic effects of gingerols, shogaols and other bioactive compounds of ginger which will assist researchers to develop authentic standardization techniques to monitor the quality and composition of ginger formulations for specific disease. once the unique ginger formulation is developed against a particular disease, the effective dose frequency and dose range should be established. conflict of interest statement: no conflict of interest is to be declared by any author. authors contribution: haseeba shahzad: conceptualization, design and directed the project, writing-review, editing, investigation, validation, data curation, data analysis, results interpretation, writing original draft and final approval. shaifa saleem: writing-review, data analysis, critical revision and investigation. waqas hanif: design, validation, critical revision, review editing, investigation, manuscript review and final approval. zareena ali: writing-review, data analysis, and investigation. muhammad zeeshan ahmed: writing-review, validation, and critical revision. haq nawaz: critical revision, validation and investigation. zelle humma: review editing and critical revision. laraib rana: writing-review and investigation. laraib rana contributed equally to this work with muhammad qamar farid, hajira kalsoom, and samavia jaan. references abdel-moneim, a., morsy, b. m., mahmoud, a. m., abo-seif, m. a., & zanaty, m. i. 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(2016). triptolide attenuates inflammatory response in membranous glomerulo-nephritis rat via downregulation of nf-κb signaling pathway. kidney and blood pressure research, 41(6), 901-910. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 205-213 | doi: 10.14421/biomedich.2023.121.205-213 issn 2540-9328 (online) bioactive compound in solanum torvum and its potential as functional food and drink: a review nina nurazizah purnomo putri*, rista anggriani, sukardi food technology department, faculty of agriculture and animal husbandry, university of muhammadiyah malang, jl. raya tlogomas no 246 malang 65144, tel. +6282399028714, malang, jawa timur, indonesia. corresponding author* ninaputri171@gmail.com abstract solanum torvum is a fruit used as food and has medical properties. this study aims to provide an updated understanding of solanum torvum’s health benefits as a functional food through a study literature review. the research utilizes electronic databases (pubmed, science direct, scopus, nature, clinical key, and springer) from july-october 2022. solanum torvum contains various bioactive components, vitamins, mineral, nutritions. it shows that solanum torvum has potential sources as functional food. however, studies about solanum torvum consumption as a functional food and the clinical trial of its health benefits in humans are still limited. further in vivo and in vitro studies are necessary to present the effect of solanum torvum consumption on health. keywords: solanum torvum; antioxidant; bioactive compound; nutrition; functional food and drink. introduction the body's immune system uses the involuntary primitive reflex of coughing to defend against foreign substances that invade the respiratory tract (sharma et al., 2020). to eliminate debris, excessive mucus, irritants, microbes, or other substances from the respiratory tract, one may cough either voluntarily or involuntarily. coughing is either voluntary or involuntary (chung and pavord, 2008). solanum torvum has various local names in indonesia, it has known as pokak (east java), cepoka (central java), takokak (west java), rimbang (sumatra and melayu), terong pipit, and many more (zuhud, 2003). it is also called turkey berry, devil’s fig, cherry eggplant, water nightshade, or wild eggplant (lim, 2013). in 1964, lawrence classified solanum torvum into taxonomical classification. kingdom : plantae division : spermatophyta sub division : angiospermae class : dicotyledonae order : solanales family : solanaceae genus : solanum species : solanum torvum swartz distribution of solanum torvum to growth could reach tropical and subtropical territories throughout the world (west and central africa, indian subcontinent, southeast asia, china, japan, australia) and many pacific islands; in the western hemisphere such as mexico, central america, greater and the lesser antilles, south america (brazil, ecuador, french guiana, guyana, venezuela), the united states to the caribbean (scher et al., 2015). the good habitat for species to grow in moist, fertile soil and will tolerate drought; wet thickets, dry brushy plains, woodland clearings, rocky hillsides; weeds in pastures, open native vegetation, swamps, roadsides, and waste places (scher et al., 2015). on the other hand, 1-3 kilogram fruits could be produced from a single tree of solanum torvum. this plant can produce fruit in 5-8 months from the start of the plantation (pratiwi, 2012). this plant is a nutritious food resource in africa that significantly contributes to massive societal nutrition because it has abundant minerals (okoto, 2015). in traditional folkloric medicine, solanum torvum has been used as an antipyretic, anti-rheumatic, anti-infectious, anti-inflammation, diuretic, and analgesic (burkill, 1966; burkill, 2000; jain & borthakur, 1986; stuart, 2022). in modern pharmacological studies, solanum torvum shows antioxidant activity (waghulde, 2011; gyamfi, 1999), antifungal activity (karuppusamy, 2009; beg et al., 2002), antibacterial activity (satish, 1999; lalitha, 2010), antiulcer activity (telesphore, 2008; antonio, 2004), antihypertensive and metabolic correction activity (nguelefack, 2008; jaiswal, 2012; lim, 1992), nephroprotective activity (waghulde, 2011; vaclavikova, 2008; mohan, 2010), cardioprotective activity (mohan, manuscript received: 11 october, 2022. revision accepted: 12 february, 2023. published: 15 february, 2023. https://doi.org/10.14421/biomedich.2023.121.205-213 206 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 2009), antidiabetic activity (gandhi, 2011; keisuke, 2010), analgesic and anti-inflammatory activity (ndebia, 2007; atta, 1997), immunomodulatory and erythropoietic activity (george, 2011; israf, 2004). not only used for medical purposes but solanum torvum also could be used as a food or drink product to get the essential benefit for human health (jaiswal, 2012). recently studies revealed that solanum torvum contains a very potent antioxidant and other useful phytochemistry components (helilusiatiningsi, 2021). the currently popular idea of creating food to get a healthy purpose is called functional food. functional foods are foods that provide health benefits beyond its nutritional value. this paper aims to provide an updated review of the phytochemicals compounds on solanum torvum by promoting its opportunity as a functional food to improve human health. parts and nutritional value of solanum torvum figure 1. solanum torvum. solanum torvum trees could grow up to 2 to 4 meters in height with an ideal environment to grow under full sunlight or partially in shades (pratiwi, 2012). its habitat is commonly found on roadsides and in disturbed soils, with expected growth as an individual plant. commonly found up to an altitude of 1600 m and suitable in moist soil conditions. young and immature fruits are consumed raw, cooked as a vegetable or used as an ingredient in curry sauce. in indonesia, solanum torvum is considered one of the best side dishes to go along with rice as a vegetable called lalapan (lim, 2013). each part of this fruit contains a lot of benefits. plant parts description and nutritional values fruits and seeds the fruit is a spherical round shape berry and has diameter about 1.0 to 1.15 cm. each fruit has about 300 to 400 flat, brown dark seeds inside. berry seeds of medium size (around 1,5 mm long) are hard to distinguish from one another. the fruit color may be affected by aging and the length of the lifetime spent (sirait, 2009). the fully ripped berry has yellow skin, and the youngberry has green with glossy color (scher et al., 2015). the flowers are bisexual. the plant flowers throughout the year. heavy rainfall discourages fruit growth and set (singapore government agency, 2022). extract of the fruit revealed the number of alkaloids, flavonoids, and certain fatty acids such as palmitic and oleic tested analysis by the gc-ms instrument (jaabir, 2015). extraction from seeds of solanum torvum showed a significant amount of phenolic and flavonoid contents which could be the source of natural antioxidants (waghulde, 2011). in pharmacology, alkaloids and solasodina compounds that contain in the fruit can be used as substrates for the production of the important steroid (perez-amador et al. 2007). in addition, the fruit also contains many solosin, chlorogenin, sisalagenone, tervogenin, protein, fat, calcium, phosphorus, iron, vitamins a, b1 and c (zuhud et al. 2003). leaves solanum torvum leaves have a large and green shape with a leaf length range of about 10-15 cm and a width of 810 cm. (jaiswal, 2012). the leaves are simple and ovate to elliptical in shape and lobed. the surface is pubescent. phytochemical analysis revealed the presence of 32 chemical constituents, mainly phenolic compounds, terpenoids, palmitic acid, palmitic acid ester, linoleic acid, linolenic alcohol, linolenic acid ester, and stearic acid found in solanum torvum leaves (naimon et al. 2015). the leaves can be dried, mixed, and added to hot water to putri et al. – bioactive compound in solanum torvum and its potential as … 207 plant parts description and nutritional values make a cold or cough medicine (yousaf et al., 2013). roots and stems solanum torvum is a prickly shrub or small tree, up to 5 m tall. it is cultivated in the tropics for its sharp-tasting, immature fruits (scher et al., 2015). in malaysia, the seeds are smoked to treat toothaches, while the roots are applied as a poultice to treat foot cracks. in china, the roots are believed to disperse blood that has leaked into the surrounding tissue and relieve pain. in india, extracts of the plant are used as an antidote to insect stings, and the fruit is eaten to relieve stomach aches). root powder of the fruit can be used as a remedy for leg fractures and headache relief and can be used as asthma treatment and liver therapy (yousaf et al., 2013). it is reported that solanum torvum roots contain secondary metabolites (sivapriya, 2011). these are potential sources of antimicrobials such as steroid alkaloids, saponins, flavonoids, glucosides, and tannins (bari et al., 2010). the phytochemical compounds and antioxidant of solanum torvum native to and cultivated in africa and the west indies is solanum torvum sw. (solanaceae), sometimes known as turkey berry (adjanohoun, 1996). due to their antioxidant characteristics, fruits and leaves are commonly employed in traditional cameroonian medicine. numerous substances found in solanum torvum have the potential to have pharmacological effects, including isoflavonoid sulfate, steroidal glycosides, chlorogenic and neochlorogenone, triacontane derivatives, 22--o-spirostanol oligoglycosides, 26-o-glucosidase (arthan, et. al., 2006). this plant has secondary metabolites in its leaves, stems, and roots, including steroidal alkaloids, saponins, flavonoids, glucosides, and tannins, which make it a robust and potentially antibacterial source (bari at al., 2010; sivapriya et al., 2011). pérez amador et al. (2017) measured the overall alkaloid content (0.12%), total glycoalkaloids (0.038%), and the glycosylated compounds produced from solasodine, namely solasonine (0.0043%) and solamargine (0.0028%). the amounts of polyphenolic substances (phenols, flavonoids, and tannins) were measured to be 160.30, 104.36, and 65.91 mg/g, respectively (kusirisin et al., 2009). in ethanolic extracts of immature and mature fruits, alkaloids, total saponins, total flavonoids, and vitamin c contents were compared (koomson et al. 2018), and only alkaloids were found to be significantly different (respectively, 16.94 2.3 mg/g in the immature fruits, and 6.32 0.12 mg/g in mature fruits). a large amount of reactive oxygen or as known as free radicals, that the human body produces and receive creates an urgency to develop and have large sources of antioxidant to tackle with. syntethycally antioxidants (for example, butylated hydroxyl anisole, gallic acid ester, butylated hydroxyl toluene, etc.) are associated with the side effect on human health (gao, 1999). synthetic antioxidants show a small level of solubility and a medium level of antioxidant activity (barlow, 1990) the substitution of synthetic antioxidants could be produced by utilizing natural resources from plants (waghulde, 2011). antioxidant activity from plants has a correlation with phenolic compounds (cook, 1996). flavonoids are a group of polyphenol compounds with known properties, including free radical scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory activity (frankel, 1995). wahluge et al, 2011 analyze the antioxidant activity, phenol, and flavonoid contents of seeds of punica granatum and solanum torvum. the result showed that the highest radical scavenging activity was observed with solanum torvum extract, with a significant linear correlation with phenolic and flavonoid contents. antioxidants are compounds that protect compounds or tissues from the damaging effects of oxygen or the effects of oxidation. according to kumalaningsih (2006), antioxidants are compounds with molecular structures that can donate electrons to free radical molecules and cut free radical chain reactions. in other words, antioxidants are able to work as inhibitors that try to inhibit oxidation by reacting with reactive free radicals to form relatively stable non-reactive free radicals. however, when combined with free radicals that can cause disease, antioxidants are defined as compounds that protect the body from the harmful effects of reactive oxygen free radicals (sofia, 2016). saponins is a glycoside group of compounds can be found in plants naturally and has unique properties, such as soap that can produce foam when shaken in the air. classified as a polar compound, this compound is soluble in air and is able to work as an antioxidant component to reduce free radicals in the body, so it can be used as a compound to prevent oxidative stress in liver cells (mardiningsih et al, 2010). tanins is classified as a complex polyphenolic compound, tannins can bind proteins well. the molecular weight is about 500-3000 da. these compounds can also be classified as hydrolysable tannins and condensed tannins. the molecular structure of hydrolyzable tannins is the hydroxyl of esterified phenolics such as gallic acid. tannins also have an anti-oxidant effect (ismarani, 2012). solanum torvum extraction using aquades as a solvent showed the content of alkaloids and tannins (alfarabi et al, 2018). phenolics has high antioxidant capacity due to ability to pass hydrogen on to extremely reactive radicals, therefore preventing further radical 208 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 formation (lapornik et al. 2005; xu et al. 2007). flavonoid are natural active phenolic compounds with a c5-c3-c6 chemical structure. this compound is often found in green plants and is useful as a source of antioxidants that can reduce free radicals or oxidation in the body. flavonoids are also inflammatory agents (rompas et al., 2012). flavonoids have a major role in analgesic activity (acharyya et al., 2018). elements to analyze amount benefit ref macronutrients content (g/100g) total carbohydrates 71.42 ± 0.52  combined with dietary fiber are generally recommended to prevent atherosclerosis, constipation and diseases in the intestine such as appendicitis and colon cancer (melila, et.al, 2021) (melila, et.al, 2021 protein 16.49 ± 0.47  can use in human food can contribute to the fight against proteinenergy malnutrition fat 7.71 ± 0.19  anti-hypertensive diets ash 4.65 ± 0.46  mineral components water content (%/100g) 79.28 ± 0.06 energy (kcal/100g) 421 ± 2,01 minerals (mg/100g) sodium (na) 155.2 ± 44.37  involved in the transmission of nerve impulses and in the water balance of the body (fao, 2004) (melila, et.al, 2021; kouadio et al., 2020) potasium (k) 1307 ± 433.7  involved in muscle contraction (fao, 2004) phosporus (p) 78.70 ± 20.10  necessary for the formation of the skeleton.  essential in the processes of energy storage in the body in the form of atp (kemi et al., 2006) calcium (ca) 38.45 ± 8.54  necessary for the formation of the skeleton.  involved in muscle contraction (fao, 2004)  can be used in diets aimed at skeletal formation (kemi et al., 2006)  balancing the body's ph by neutralizing excess acids (kemi et al., 2006) magnesium (mg) 38.45 ± 8.58  trace element that stimulates the immune system, protects against cell aging and maintains fatty tissue (kala, 2005)  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014)  involved in muscle contraction (fao, 2004)  essential cofactor of the enzymes of carbohydrate metabolism (fiorentini, 2021) iron (fe) 21.04 ± 0.68  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014) zinc (za) 14.65 ± 2.69  trace element that stimulates the immune system, protects against cell aging and maintains fatty tissue (kala, 2005)  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014) vitamin (mg/100g) vit c 2.44 ± 0.36  defense of the body against virus and bacterial infections,  the protection of the blood vessel wall,  the assimilation of iron and has an important antioxidant activity(carr, 2019) (melila, et.al, 2021; vit a 0,078  helpbody's natural defence against illness and infection (the immune system) work properly.  helping vision in dim light (duester, 2000) (okoto, 2015) phytochemical contents (mg/100g) polyphenols 356.70±0.02  anti-inflammatory, urinary antiseptic, anti-free radical, hepaticprotective, immune stimulant, anti-thrombotic and anti-carcinogenic effects (kouadio et al., 2020; vauzour, 2010; acho, 2014) phenol 160.30+3.00  oral analgesic, antioxidant, anti-inflammatory, anti-allergic, anticarcinogenic, antihypertensive, cardioprotective, anti-arthritic and antimicrobial activities (bhuyan, et al., 2017) tannins 685.83±0,01  anti-nutritional compounds that have deleterious effects on digestibility flavonoids 104.36+3.00  regulate cellular activity and fight off free radicals that cause oxidative stress on human body (kusirisin, 2009) (kusirisin, 2009) antioxidant activity 360.53+5.06  protection against damage caused by free radicals (kusirisin, 2009) putri et al. – bioactive compound in solanum torvum and its potential as … 209 value added as functional foods and drinks from solanum torvum the wide traditional medicinal use of s. torvum fruits is nowadays explained by the production of many phytochemicals by this species. alkaloids, flavonoids, tannins, saponins, and glycosides are present in s. torvum extract in sufficient concentrations for protecting the body against oxidative stresses. s. torvum can therefore be considered a promising natural source of phytochemicals displaying a range of medicinal properties, ranging from cardio protection & treatment of heart related diseases, nephro-protection, to analgesic, anti-inflammatory, anti-ulcer, and anti-microbial activities. solanum torvum is having a huge potential resource for developing food products. it’s containing a number of nutritional values, bioactive components, and other physicochemical properties. the fruit's nutritional content could be used as based ingredients or substitute materials to increase the nutrition in food (helilusiatiningsi, 2021). if the turkey berry fruit is used optimally by the community, then the fruit will be an alternative treatment that is cheap, easy, and relatively safe. it's easy because the plant grows and can be found around the community and people can cultivate it themselves at home. the cost of processing the fruit will be much cheaper than treatment with chemical drugs. in addition, traditional medicines that tend to have no side effects are relatively safe for people to use (pratiwi, 2012). flour (dried powder) the most common commercially used product, dried fruit powders are found to be an emerging functional ingredient. it has a long shelf life because of the water content and odorless materials (phan, 2021). to make it efficient to combine with other food products, it is necessary to create solanum torvum powder. helilusiatiningsih, 2021 identifies the phytochemistry component of solanum torvum flour by doing 3 different methods of drying.the types of methods are sunlight drying, vacuum dryer, and try dryer. the result explains phytochemistry compounds and nutritional values. the vacuum drying method showed a higher dpph of 92.81% compared with the try drying method, which has 85.15%, and the sunlight drying method, which has 88.10%. the antioxidant activity of phenolic compounds was attributed to their ability to scavenge free radicals, giving hydrogen atoms, electrons, or chelate metal cations (afanas’ev, 1989). phenol compound is higher found in vacuum drying method (92.8 mg/g) than in sunlight method (37.32mg/g) and the try drying (22.91 mg/g). tannins are phenolic compounds of high molecular weight ranging from 500 da to more than 3000 da they are found in plants, and their parts are located in the tissues, specifically in the vacuoles (hassanpour, 2011). higher tannin content for the flour was found in the vacuum drying method (1.24 mg/g), followed by the sunlight method (1.03 mg/g), and try drying (0.63 mg/g). different from dpph, phenol, and tannins, a higher amount of flavonoids was found in the sunlight method (5.11 mg/g), followed by the vacuum drying method (2.75 mg/g), and try drying method (2.43 mg/g). proximate analysis shows quantitative analysis of macromolecules in food. to compare the nutritional values of the flour, the results were summarized in table 1. table 1. nutritional values of the solanum torvum flour with different drying methods. proximate analysis parameter drying method sunlight drying vacuum drying try drying water content (%) 6.92 7.62 6.89 ash content (%) 3.66 2,82 3,29 protein (%) 11, 51 22.66 21.28 fat (%) 5.38 3.25 7.04 carbohydrates (%) 72.53 65.51 61.5 vitamin c (%) 0.21 2.44 0.39 source: helilusiatiningsih, n. (2021) tea teye, (2017) developed functional drink combination from roselle (hibiscus sabdarffa), ginger (zingiber officinale), and turkey berry (solanum torvum). among several samples, 50% roselle + 25% ginger + 25% turkey berry shows optimum protein content (4.14%), ca (1.80%), fe (0.96 µg/g) and cu (1.18 µg/g). it also showed the best performance for sensory evaluation in terms of color, aroma, flavor, aftertaste, and overall acceptability. furthermore, helilusiatiningsih (2021) developed solanum torvum fruit extract as a potential healthy drink with a combination of black tea and green tea. according to the result, most panelists preferred black tea solanum torvum to green tea solanum torvum by organoleptic test on aroma, taste, and color. a qualitative test shows that black tea solanum torvum contains bioactive compounds, such as saponins, glucosides, steroids, and alkaloids. sample also shows amount of carbohydrates (63,96±0,08%), fat (9,67±0,05%), protein (24,76%), ash content (3,08 ±0,05%), moisture content (6,39 ± 0,12%), vitamin c (6,20±0,05%), and minerals (ca (38,34 ppm), mg (76,3 ppm), fe(4,51 ppm). snack bar novilia, 2017 investigated the food processing of solanum torvum in the snack bar. the investigation focused on the production of solanum torvum flour making, formulation and production of the snack bar, organoleptic testing, analysis of nutritional content, fiber, total phenolic compound, and antioxidant activity on the product. among several samples, 10% extract of solanum torvum showed the best organoleptic test and nutrition content, such as moister content (10.30%), ash 210 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 content (1.35%), protein (9.70%), fat (19,57%), carbohydrates (36.8%), fiber (22.28%), total phenolic (0.47%) and antioxidant activity equivalent vitamin c (704.14 mg/100 g). thus, the analysis of the contribution of nutrients in 1 serving size (37 g), solanum torvum bar contributes energy (5.95%), protein (6.78%), fat (12.21%), carbohydrates (6.30%), dietary fiber (36.54%) and total phenolic (174 mg/37 g) for general nutrition advice. noodle atika et al. (2021) found that solanum torvum extract could be used as an additional ingredient in noodles. adding solanum torvum extract in noodles improves the product with vitamin a. in plants, vitamin a has been found as provitamin a; the most active form of this vitamin is β-carotene. this component prevents vitamin a deficiency, where cells and tissues could be damaged in the human body. furthermore, the researchers discovered that adding solanum torvum with 85% of flour, 75% of solanum torvum, and 40% of water could develop the β-carotene component to 671.2548 g/100g. the control sample with 100% flour and 100% water without solanum torvum fruit extract could only reach 140.8604 g/100g. β-carotene content of 671.2548 g/100g is equivalent to 111.8758 re when associated with the nutritional adequacy rate for vitamin a. juice in ghana, the fruits of solanum torvum are traditional healers for anemia and ailments. the fruit is processed into a drinkable juice. with a high concentration of iron, it is possible to be utilized as a healing treatment for anemia and iron deficiency. iron is a mineral needed for human growth (al-jameil et al., 2014). solanum torvum aqueous extract has the ability as immunomodulatory and haematinic properties where it could be used as a hematinic and food for patients with immunity problems (koffuor, 2011). a study by okoto (2015) investigated the components of solanum torvum juice. the analysis shows that fruit juice has an abundance of water content (86,23%), carbohydrates 7.033%, proteins 2.322%, fats 0.278%, ash 0.143%, and crude fiber 3.993%. the analysis for important minerals content was conducted. according to the obtained results, calcium was the highest amount of mineral (221.583 mg/kg), followed by iron (76.869mg/kg), zinc (21.460mg/kg), manganese (19.466 mg/kg), and copper (2.642mg/kg). besides nutritional and mineral compounds, vitamin was found in the juice. the 100 grams of the sample contain 2.686 mg of vitamin c and 0.078 of vitamin a. conclusions solanum torvum contains various bioactive components, vitamins, mineral, and nutritions. it shows that solanum torvum has potential sources as functional food. however, studies about solanum torvum consumption as a functional food and the clinical trial of its health benefits in humans are still limited. further in vivo and in vitro studies are necessary to present the effect of solanum torvum consumption on health. competing interests: the authors declare that there are no competing interests. references abbaspour n., hurrell r., kelishadj r. 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(2013). phytochemistry and pharmacological studies on solanum torvum swartz. journal of applied pharmaceutical science, 3(4), 152-160. zuhud eam, siswoyo, sandra e, hikmat a, adhiyanto e. 2003. buku acuan umum tumbuhan obat indonesia. jakarta: yayasan sarana wanajaya. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 467-475 | doi: 10.14421/biomedich.2023.122.467-475 issn 2540-9328 (online) identification of primary and secondary metabolites of apis cerana honey using ftir-atr diamond spectroscopy and their botanical origin tiffany hanik lestari, ratna susandarini* faculty of biology, universitas gadjah mada jl. teknika selatan, sekip utara, sleman 55281, indonesia; telp/fax: +62-274-580839. corresponding author* ratna-susandarini@ugm.ac.id manuscript received: 13 april, 2023. revision accepted: 16 august, 2023. published: 21 august, 2023. abstract apis cerana fab. is one of the popular honeybees species among beekeepers in indonesia. this species is easy to care for and produces valuable honey products. honey from a. cerana is abundantly available in traditional and modern markets in indonesia. the purpose of this study was to identify the primary and secondary metabolites in the honey produced by a. cerana using ftir-atr diamond spectroscopy. twelve samples of honey from three provinces in java island were used in this study. in general, all honey samples contained protein, carbohydrate, water, alcohol, cellulose, alkaloid, tannin, and flavonoid. variation on primary and secondary metabolites in honey samples was strongly affected by the botanical origin, geographical origin, and the local condition around beekeeping areas where the honeycombs were placed. keywords: apis cerana; biochemical characterization; honey; spectroscopy analysis. introduction honey is one of the foodstuffs containing many nutrients, such as carbohydrates, proteins, fats, and vitamins (kasprzyk et al., 2018). honey has been produced, marketed, and sold as healthy food and for traditional medicine around the world (formosa et al., 2020). honey is nutritious food safe for consumption by various age groups, ranging from one year old babies to the elderly. in addition, the antioxidant and antibacterial properties of honey make it a great food supplement for human health. honey also contains vitamins, enzyme, and secondary metabolites such as flavonoid, phenolic, and tannin (de almeida-muradian, 2014). in fact, these chemical components affect its medicinal properties as well as the quality, granulation, texture, and shelf life of honey (adeniyi et al., 2014; joshi, 2008). the chemical composition of honey as source of natural products is determined by various variables, such as the floral origin, the climate, environmental and seasonal conditions during the period of beekeeping, and the processing methods from the time of harvesting until its storage (silva et al., 2009). indeed, honey produced by various honeybees species differs not only in their chemical properties, physical properties, and the taste, but also in their biological activities (cimpoiu et al., 2012). honey that has many health benefits is produced by bees who take the best natural ingredients in the form of nectar and pollen from various flowers. honey found on the market in indonesia comes from various species of bees, including apis cerana, a. mellifera, a. dorsata, and trigona spp.. some of these honey bees species are kept on farms, but some live in the wild and their honey is harvested from the forest. in this work, we focused our study on the honey produced by apis cerana. a. cerana is one of the most widely bred honey bees species in indonesia because it is suitable for living in humid rain forests and dry grasslands in the tropics (koetz 2013; radloff et al., 2010). a. cerana does not require a large area for its maintenance and does not need to be fed outside the nest like a. mellifera. this species is also known as a non-aggressive bee species compared to a. mellifera and a. dorsata. although the honey produced by a. cerana is not as much as the other two apis species, this species has good potential to be developed in smallholder beekeeping and large scale farms (theisen-jones and bienefeld, 2016). the criteria to assess the quality of pure honey determined by the indonesian standardization agency are mainly based on physical characteristics. the agency does not include nutritional content as standards for honey marketed in indonesia. considering that honey is generally consumed as a health supplement, its https://doi.org/10.14421/biomedich.2023.122.467-475 468 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 biochemical characterization including the content of primary and secondary metabolites is important to study. currently a more detailed analysis of honey quality has been carried out by researchers around the world (kasprzyk et al., 2018). the analysis includes biological testing, such as identifying the type and amount of pollen contained in honey which become important factor in determining the quality of honey. this is because pollen contains more complex nutrients than nectar as the major plant-based substances to produce honey. currently the testing for honey quality is gaining attention by the community. for example, in poland, honey quality testing was carried out by calculating the amount of pollen and comparing the chemical compounds of the original pollen with the pollen present in honey using the fourier transform infra-red (ftir) analysis using attenuated total reflection (atr) diamond method (kasprzyk et al., 2018). a similar method has also been used in croatia, where the quality of honey is determined from the comparison of chemical compounds of honey with chemical compounds of pollen and nectar using ftir-atr (svečnjak et al., 2017). the ftir-atr diamond is a type of spectroscopy method that uses infrared waves to identify a compound or molecule present in living or dead samples (barth 2007). this type of spectroscopy is very sensitive to the chemical composition and arrangement of a molecule, both proteins and molecular structures that are larger than proteins. the current use of ftir-atr diamond includes the analysis of the chemical structure of living things and the compounds contained in them. one of the application of this method is the qualitative and quantitative analysis of chemical components in honey for determination of botanical or geographical origin, and detection of adulteration (saksangawong et al., 2021). this method is a new, rapid, effective, non-destructive, and cost-effective for analysis honey components (riswahyuli et al., 2020), and therefore suitable to be applied for quality assessment of indonesian honey. this study aims to determine the primary and secondary metabolites in a. cerana honey samples from java island, indonesia. results of this study provide important information on the biochemical content of a. cerana honey in indonesian market, and thus bring beneficial impact for the community as well as for marketing purposes. materials and methods fresh honey samples were collected from 12 a.cerana farming locations covering nine districts in three provinces of java island (figure 1) namely, kulonprogo and gunung kidul (yogyakarta special province); cilacap, solo, magelang, pati, boyolali, and karanganyar (central java province); and depok (west java province). during the visit for collecting honey samples, supporting information from the farmers was noted including the beekeeping management, methods of honey harvesting, and the blooming flowers during the recent seasons. honey samples were stored in a refrigerator at 4oc to maintain their quality prior to ftir-atr analysis. the primary and secondary metabolites content of honey samples was analyzed using ftir-atr diamond (alpha bruker). for the analysis, 1 ml of honey was put in the sample holder. the screening was carried out from the wave number of 4000 to 400 cm–1. the results of the analysis are peak graphs which were then validated according to the provisions as presented in table 1. figure 1. map of honey sampling locations (source: google earth). table 1. interpretation of the results of ftir-atr diamond analysis (kasprzyk et al., 2018). wave number (cm-1) vibrations 1000-1200 c–o and c–c stretching vibrations 1200-1350 n–h deformation and c–n stretching vibrations from amide iii 1370-1420 c–h deformation vibrations of lipids and cellulose 1540 n–h deformation and c–n stretching vibrations from amide ii 1650 c=o stretching vibrations from amide i 1740 c=o stretching vibrations from lipids 2850-3000 c–h stretching vibrations from cellulose and lipids 3250 o–h stretching vibrations from water 3300-3500 n–h stretching vibrations from protein results and discussion visual observations on honey samples and vegetation surrounding a. cerana nests honey samples from nine districts in three provinces in java island were all classified as multifloral honey based on the composition of pollen types identified using melissopalynological analysis (lestari, 2019). honey lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 469 samples showed variation in color as shown in table 2. differences in the color might be attributed to the differences in pollen and nectar sources. table 2. honey samples from a. cerana used in this study. sample code color honey category origin of sample ah-01 yellow multifloral pati, central java ah-02 orange-brown multifloral boyolali, central java ah-03 orange multifloral karanganyar, central java ah-04 orange multifloral kulonprogo, special region of yogyakarta ah-05 yellow multifloral karanganyar, central java ah-06 yellow multifloral gunungkidul, special region of yogyakarta ah-07 yellow multifloral magelang, central java ah-08 brown-red multifloral solo, central java ah-09 dark brown multifloral cilacap, central java ah-10 light orange multifloral solo, central java ah-11 yellow multifloral magelang, central java ah-12 orange-brown multifloral depok, west java general features of vegetation in each location from which honey samples were collected is shortly described as follows. sample ah-01 was labelled as randu tanjung sari honey from pati, central java. the location of the a. cerana nests were in the field near the beekeeper’s house with a white silk-cotton tree (ceiba pentandra) grows nearby. sample ah-02 was known as dasa dharma honey from boyolali, central java. based on direct observation, the honeybee nests were placed in a garden area of about 1 hectare next to the house. various plants species were found in the garden such as longan tree (dimocarpus longan), mango tree (mangifera indica), and bitter bean tree (parkia speciosa). in addition, around the farmer's house there is an area of maize (zea mays) and rice (oryza sativa) fields as well as residential areas with small garden commonly found in front of the houses. sample ah-03 was collected from matesih, karanganyar, central java. the honeybee nests were placed in the house yard, where the farmer's house is bordered by residential areas and plantations of woody plants such as chinese albizia (albizia chinensis) and teak (tectona grandis). sample ah-04 was originated from jatimulyo village, kulonprogo, yogyakarta. the honey was produced by honey bees whose nests were placed in the farmer's house yard adjacent to the community forest with various plants species such as albizzia procera and dioscorea japonica. meanwhile, sample ah-05 was called as kaliandra honey obtained from jumapolo, karanganyar, central java. honeybee nests were placed in the house yard surrounded by residential areas (villages), rice fields, gardens, and cemeteries. sample ah-06 was commercially labelled as sari alami honey collected from kedung poh village, one of the ecotourism villages in gunungkidul, yogyakarta. this village is located adjacent to the community forest, and is a well-known beekeeping ecotourism area for educational purposes in yogyakarta. honeybee nests were placed around the residents' yards. major plant species found in the community forest are acer serrulatum, t. grandis, and c. pentandra. sample ah-07 labelled as kaliandra tanjung sari honey was collected from magelang, central java. the honeybees producing this honey were reared and fed on calliandra plantation in menoreh hill. sample ah-08 was collected from jebres, solo, central java. the honeybee’s nests were placed in farmer's house yard, which is in a densely populated residential area near jebres railway station where various flowering plants in grow in the area, including frangipani (plumeria alba), guava (psidium guajava), acacia glomerosa, and jamaica cherry tree (muntingia calabura). meanwhile, the honey sample ah-09 was obtained from cilacap, central java. the honeybee’s nests were placed in the garden of the farmer’s house which is close to urban area. the plants found around the nest were desmodium paniculatum, panicum grande, dan cassia obtusifolia. sample ah-10 is honey marketed with a label of sekarpace honey from solo, central java. the nests of a. cerana were placed in the house yard where m. calabura, mango (m. indica) and queen’s jewels vine (antigonon leptopus) were planted. the beekeeping area is densely populated settlements in the city and close to public cemetery where frangipani (p. alba) dominated the area. based on information from the farmers, various flowering and fruit plants such as jamaica cherry (m. calabura) and common ornamental vines such as queen’s jewels (a. leptopus) are deliberately planted to improve the quality of honey. sample ah-11 from magelang, central java was named as insulin honey. based on observations during honey collection period, the honeybees producing insulin honey were reared in nests located in farmer's house yard close to menoreh hill, magelang. the location of this bee farming is in a rural area close to community forests, rice fields, and vegetable gardens. in this area, mexican sunflower 470 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 (tithonia diversifolia) and red calliandra (calliandra calothyrsus) are abundant. the honey sample ah-12 is marketed under the name of organic honey from depok, central java. the location of honeybee’s nests in this farm is in the gardens close to residential areas. residents' gardens are planted with various plants such as cottonwood, bananas, and some woody plants such as acacia sp., parasol leaf tree (macaranga tanarius), and african tulip tree (spathodea campanulata). the ftir-atr profiles of a. cerana honey the results of honey analysis with ftir-atr diamond in the form of infrared absorbance spectrum are presented in figure 2 and summarized in table 3 and 4. in the spectrum, the wavenumber indicates the number of infrared waves (the inverse of frequency) emitted to the sample. a chemical bond is detected as a band at a certain position that can be used to identify the type of chemical compound content, especially primary metabolites in honey. the intensity of the band represents the amount of absorbance that occurs after infrared passes through the sample. band intensities greater than or equal to 0.1 will be read and represent a chemical compound content as listed in table 1. in addition, the band intensities also show the quantity of chemical compounds in each honey sample. figure 2. results of ftir-atr diamond analysis. lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 471 figure 2. cont. tabel 3. summary of results of the ftir-atr diamond analysis for primary metabolites. wavenumber (cm-1) honey samples ah-01 ah-02 ah03 ah04 ah05 ah06 ah-07 ah08 ah09 ah-10 ah-11 ah-12 1000-1200 125.93 132.81 127.77 133.37 15.36 129.32 128.81 175.27 14.31 129.51 127.49 132.37 1200-1350 48.03 56.06 47.04 53.51 41.07 53.13 50.71 65.50 41.28 53.41 50.89 57.56 1370-1420 11.66 12.67 10.97 12.42 6.48 12.35 12.01 18.81 6.28 12.46 12.06 13.51 1540 4.81 5.22 4.57 5.16 3.73 5.15 4.95 7.16 3.86 5.11 5.01 5.56 1650 × × × × × × × × × × × × 1740 0.10 0.08 0.11 0.09 0.09 0.10 0.10 0.13 0.09 0.09 0.10 0.08 2850-3000 × × × × × × × × × × × × 3250 10.92 11.85 10.86 11.82 12.94 11.65 11.35 14.14 13.43 11.48 11.43 12.07 3300-3500 0.47 0.47 0.47 0.47 0.48 0.47 0.48 0.49 0.48 0.48 0.46 0.47 notes: the × sign: there is no ftir-atr diamond spectra in this wave. 472 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 tabel 4. secondary metabolites on honey samples detected by ftir-atr diamond and their comparison to results from previous studies. secondary metabolites honey samples references ah01 ah02 ah03 ah04 ah05 ah06 ah07 ah08 ah09 ah10 ah11 ah12 alkaloid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ kavanagh et. al., 2019 flavonoid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ li et al., 2018 tannin ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ malacarne et al., 2018 terpenoid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ mashwani et al., 2016 the ftir-atr diamond has been widely used in the analysis of chemical compounds in honey. several studies showed that ftir-atr was able to analyze secondary metabolite compounds in food products, such as honey (tahir et al. 2017; malacarne et al. 2018). secondary metabolite compounds such as phenolics, flavonoids, tannins, alkaloids, and terpenoids can also be identified by representing the bands that appear on certain wavenumbers. bands at 1000–1200 cm-1 indicate that the honey sample contains c–o and c–c bonds. in infrared (ir) based analysis, the c–o bond is a representation of the alcohol group, so it is likely that in result of honey analysis the band at 1000–1200 cm-1 indicated the presence of alcohol group. the alcohol groups can cause bitter taste in honey with different levels of bitterness. in honey, the alcohol group most likely comes from phenolic compounds which have antibacterial properties (kavanagh et. al., 2019). the band at 1200-1350 cm-1 contains deformed n–h bonds and c–n bonds of amide iii compounds. according to singh et al. (1993), amide iii compounds detected using ftir were used to determine the presence of secondary protein structures. the band at 1370-1420 cm-1 represents the presence of deformed c–h bonds originating from lipids and cellulose. in honey, lipid commonly found at very low level, and this might be because honey contains antioxidants that has beneficial effect of lowering cholesterol level (münstedt et al., 2009). in addition, it is most likely the band at 1370-1420 cm-1 also comes from cellulose as the main component of plant cell walls such as pollen or any plant tissue accidentally carried by the bees during their foraging activity. the presence of cellulose in honey samples might indicate the presence of pollen and thus indicates the authenticity of the honey. the band at 1540 cm-1 contains deformed n–h bonds and c–n bonds of amide ii. in ir analysis, amide ii compounds are used to determine whether a protein structure is unfolding. the unfolding condition of the protein is caused by heating. in this study, honey samples were not heated, so amide ii compounds were not detected in the ftir-atr diamond for all honey samples. the band at 1650 cm-1 represents the c=o bond of amide i. according to singh et al. (1993), amide i compounds in ftir analysis were used to determine the presence of secondary protein structures. however, amide i compounds are more complex than amide iii, so the determination of secondary protein structure is more complicated. figure 2.a shows that the absorbance value of amide i is lower than that of amide iii, and these two compounds indicate the presence of secondary proteins in honey. the band at 1740 cm-1 indicates the presence of c=o bonds derived from lipids. high lipid levels in any food ingredients can cause health problems (majid, et al., 2013). interestingly, the results of the ftir-atr analysis of diamonds did not detect any lipid content or only a small amount of lipid content. this result indicated that all honey samples are of good quality and safe for health. the band at 2850-3000 cm-1 is a representation of the stretching c–h bonds derived from lipids and cellulose. this band is the same as the band at 1370-1420 cm-1 which indicates the presence of cellulose. cellulose is a polysaccharide that has an asymmetrical and symmetrical structure (dassanayake, et al., 2018). these two cellulose structures can be detected by ftir-atr diamond but based on the absorbance value, the band at 2850-3000 cm-1 is lower than the band at 1370-1420 cm-1. the band at 3250 cm-1 indicates the presence of o–h bonds from water. moisture content is one of the indicator to determine honey quality because it is related to honey's resistance to decay, viscosity, and increased microbial contaminants (prica, et al., 2014). in terms of microbial contamination, the presence of polluting microbes most likely comes from the equipment used for harvesting the honey. the microbial contamination is known to affect the hygiene of honey. the water content in honey might indicate the level of rainfall during honey production, because each harvest season may have different rainfall. low water content generally makes honey become semi-solid or has relatively medium viscosity. meanwhile, the band at 3300-3500 cm-1 represents the presence of n–h bonds of the protein. in this case, protein is known to be a primary metabolite that is important for the growth and development of body organs. compared to other studies on metabolite analysis of honey, the presence of band at 750-1800 cm-1 was reported by devi et al. (2018), anjos et al. (2015), and se et al. (2018) which represent the presence of carbohydrates. the band at this spectrum could be used to identify different groups of carbohydrates, such as glucose, fructose, and sucrose. according to se et al. lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 473 (2018), the band at 800-1500 cm-1 indicates the presence of monosaccharides and disaccharides. this is due to the presence of c–o and c–c bonds which are characteristic of carbohydrate structures (bands at 950-1200 cm-1). moreover, se et al. (2018) noted that the bands at 1054 (c–oh: carbohydrate structure), 867, 822, and 779 cm-1 represent the presence of fructose; the bands at 1022, 991, and 898 cm-1 represent the presence of glucose; and the bands at 991 and 921 cm-1 represent the presence of glucose. based on the research of tahir et al. (2017), the bands at 2800-3000 cm-1 represent c–h, o–h, and nh3 bonds indicating carbohydrates, carboxylic acids, free amino acids, and phenolics; the band at 1600-1700 cm-1 contains stretching bands of carbonyl groups, namely c=o and c=c which represent phenolic molecules; while the band at 1175-1540 cm-1 contains deformation bonds of o–h, c–o, c–h, and c=c representing flavonoids and phenolics; and the band at 940-1175 cm-1 contains c–oh groups, c–c and c–o represent carbohydrate structures, and c–o represents phenolics. tannin compounds were also reported to be detectable in the band at 926–2955 cm-1 (malacarne et al., 2018). alkaloid compounds were detected by ftir-atr in the bands at 2300-3040 and 500-1750 cm-1 (monfreda et al., 2015). terpenoid compounds can also be detected with the presence of bands at 1591, 1390, and 1116 cm-1 which represent c―c and c―o bonds as functional bonds of terpenoids (mashwani et al., 2016). flavonoid compounds were detected by ftir-atr in the bands at 1734, 1627, 1522, 1440, 1410, 1367, 1315, and 1255 cm−1 (li et al., 2018). overall, based on the results of the analysis using ftir-atr diamond in this study, all honey samples contain chemical compounds that have bands at wavenumbers of 1000–1200, 1200–1350, 1370– 1420, 1650, 2850–3000, 3250, and 3300–3500 cm-1. concerning on differences of metabolites content in honey samples, the ah-08 honey has the highest alcohol group (band at 1000-1200 cm-1) compared to other honey samples (figure 2.h), while ah-01 (figure 2.a); ah-03 (figure 2.c); and ah-11 (figure 2.k) had the lowest alcohol groups. this might be because the botanical source of ah-8 honey is dominated by poaceae at the proportion 45% as showed from the pollen analysis reported by lestari (2019). this indication was supported by the fact that location of the honey bee nests were close to the place where poaceae plants grow. the presence of pollen of particular plant species might affect the content of chemical compounds in honey such as alcohol groups (kasprzyk et al., 2018). the honey samples ah-01 (figure 2.a) and ah-08 (figure 2.h) had the largest absorbance values in the bands at 1200-1350, 1370-1420, and 1650 cm-1 compared to other samples. this indicates that in both samples the content of amide iii, cellulose, and amide i was higher than the other samples. meanwhile, the amide ii content in the band at 1540 cm-1 was not detected in most of honey samples. in ah-01 (figure 2.a) and ah08 (figure 2.h), the peak appears with an absorbance value of 0.02, but this data did not indicate heating at the time of honey harvesting process. instead, this is most likely due to the effect of light during storage. honey sample ah-01 was stored in the front window of the house, so that it was exposed to sunlight, while honey sample ah-10 was stored inside the house in a densely populated residential area with open spaces that allowing unexpected heating occurred from direct sunlight. the absence of amide ii in all honey samples indicated that there was no heating process to reduce water content in honey. this result showed that all honey samples are of good quality because after the harvesting process the honey is immediately packaged in bottles, making it more natural and healthier. the absence of amide ii compounds in honey is known to be affected by the water content in honey (barth, 2007). environmental changes, such as irregular rainfall intensity might cause the quality of honey to decline. this is due to the high water content, especially during high rainfall in particular times of honey harvesting seasons. the problem of high water content can be modified using unacceptable practices in honey processing by heating the newly harvested honey to reduce the water content. heating process can damage the protein content in honey which affects the enzymes, and thus affect the quality of honey. the lipid content as indicated by the band at 1740 cm-1 was not detected in most honey samples except for ah01(figure 2.a) and ah-10 (figure 2.h). in these honey samples, the band was detected with a fairly small intensity, namely 0.01. this result indicated that all honey samples have no or only a very small amount of lipid content. based on the results of this study and comparison to the honey analysis reported by tahir et al. (2017), all honey samples contain phenolic compounds due to the appearance of bands at 2800-3000, 1600-1700, 11751540, 940-1175 cm-1. in addition, all honey samples also contain tannin compounds, as indicated by the band at 926–2955 cm-1 which was in accordance to malacarne et al., (2018). the presence of alkaloid compounds detected by the bands at 2300-3040 and 500-1750 cm-1 was in line with the study of monfreda et al., (2015), while the presence of terpenoid compounds in the bands at 1591, 1390, and 1116 cm-1 was reported by mashwani et al., (2016). the occurrence of flavonoid compounds as indicated by the bands at 1734, 1627, 1522, 1440, 1410, 1367, 1315, and 1255 cm−1 was in agreement with the study of li et al. (2018). results of ftir-atr diamond form honey samples in this study showed similar patterns to the analysis of pollen and honey using the same method as reported by kasprzyk et al. (2018). therefore, ftir-atr can be used as standard method in determining the quality of a. cerana honey. results of this study showed that all samples are pure honey without any additional ingredients, and showed good practices of honey production process since there are no indications of adding sugar in honey or feeding 474 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 the bees with corn flour. there re also no indication of heating the honey to produce a nice color and adding the water content to increase honey volume. the results of ftir-atr diamond analysis clearly showed that this spectroscopy method is useful in identifying primary and secondary metabolites as one of the quality indicators for a. cerana honey from indonesia. the primary and secondary metabolites analysis using the ftir-atr diamond method is thus can be proposed as standard analysis in determining the quality of honey in indonesia because this method is easy, cheap, and fast to get the results. conclusions honey produced by a. cerana contains various primary and secondary metabolites which include carbohydrates, proteins, cellulose, alcohol, alkaloid, tannin, flavonoid, and terpenoid. the results of ftir-atr diamond analysis indicated that 12 honey samples in this study showed variation in the quantitatively the amount of primary metabolites. finally, given that the ftir-atr diamond is a simple method to practically evaluate the quality of the honey, it is suggested that the improvement of the ftir-atr diamond library of honey is important for quality assessment of honey. acknowledgement: the authors would like to thank the laboratory facilities and the staffs of physics department, faculty of mathematics and sciences, institut teknologi bandung. authors’ contribution: tiffany hanik lestari collected honey samples, carried out laboratory work, and wrote the manuscript. ratna susandarini designed the study, interpreted results of ftir-atr spectroscopy, and wrote the 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10.14421/biomedich.2016.52.49-53 issn 2540-9328 (online) histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati1, muhammad jafar luthfi2 1postgraduate program, universitas sebelas maret jalan ir. sutami 36 a, surakarta, 57126, tel. +62271-646994, fax. +62271-646655, indonesia 2biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: miarakhmiy@gmail.com1 abstract common house gecko (hemidactylus frenatus) belongs to suborder lacertilia that has capacity to shed its tail (autotomy) as a self-defense mechanism. after autotomy, tail regeneration occurs. axiale skeleton of an original tail is composed of bony vertebrae, whereas the the regenerate one is comprised of cartilaginous tube. the purpose of this study was to determine the histological difference between axial skeleton of the original tail and the regenerate one of the common house gecko. twenty four individuals consist of twelve common house gecko with original tail and twelve with regenerate tail were used. microanatomical observations were carried on histological slide of original and regenerated tail stained with hematoxylin-eosin and mallory acid fuchsin. the results showed that the original tail comprised of bony vertebrae whereas regenerated tail supported by cartilaginous tube. keywords: common house gecko (hemidactylus frenatus); autotomy; tail histology; cartilaginous tube of regenerated tail. introduction common house gecko belongs to the family lacertidae that has the ability to autotomized its tail as defense mechanism. caudal autotomy is the ability of an animal to shed off the tail to escape from predator (lutfi, 2002; soesilo, 1992). after autotomy, regeneration occurs (kimball, 1983; soesilo, 1992). the regeneration processes consist of three stages, namely the wound healing stage, blastema formation, differentiation and tail growth (soesilo, 1992). the original tail of lacertid is composed of vertebrae caudales, the spinal cord located within the vertebral canal, perivertebral fat tissue, muscle layers, blood vessels, nerve, skin and scales (white, 1925 in soesilo, 1999). the regenerated tail differs from the original one. according to soesilo (1999), the tail differs mainly in the structure of the vertebrae caudales and spinal cord. instead of vertebrae, regenerated tail is supported by elongated cartilaginous tube, whereas the spinal chord is replaced by ependymal cells, glia cells and nerve fibers without nerve cell body (soesilo, 1999). the muscles of a regenerated tail also have a difference with the original tail muscle. lacertid muscles arranged segmentally, consisted of 8 longitudinal bundles and each is limited by a septum composed by connective tissue. seosilo (1999), stated that tail regeneration can occur when there is ependymal layer on the remaining spinal cord. this study aimed to desribe histology of axial skeleton and muscle of regenerated tail of common house gecko (hemidactylus frenatus). materials and methods twenty four common house geckos (body length ranges from 6.5 to 7.5 cm, with intact tail) were used in this study. histological slides were made using paraffin method. histological observation were done using light microscopy on histological slides stained with hematoxylin-eosin (he) and mallory acid fuchsin. results and discussion from observation of original and regenerated tail of common house gecko it was found that there are difference in histological structure. http://dx.doi.org/10.14421/biomedich.2016.52.49-53 50 biology, medicine, & natural product chemistry 5 (2), 2016: 49-53 figure 1. longitudinal section of original tail (magnification 4x10). hematoxylin eosin (he). 1. skin; 2. muscles; 3. autotomy septum. figure 2. longitudinal section of original (magnification 4x10). mallory acid fuchsin. 1. epidermis; 2. perivertebral fat tissue; 3. muscle segment; 4. scales; 5. muscles; 6. scale joint. autotomy plane is lied transversal (figure 1). autotomy plane serve to facilitate tail sheding for minimalized excessive blooding. muscles pink colored due to eosin staining. the skin on the tail of common house gecko is thin. in the original tail, skin has scales and joints of the scales with thin epidermis (figure 2). muscles are segmental. the vertebral fracture is seen in figure 1. figure 3. cross section of original tail (10x10 magnification). hematoxylin eosin (he). 1. centrum vertebrae; 2. spinal cord; 3. transverse process; 4. muscles. figure 4. cross section of original tail (10x10 magnification). mallory acid fuchsin.1. muscles; 2. spinal chord; 3. centrum vertebrae; 4. caudal artery; 5. perivertebral fat tissue. the muscle in the original tail is divided into 8 bundles and between bundles separated by septum (figure 3). the transverses process is located at both side of vertebra. the original tail has a caudal artery. centrum vertebrae are lied between the caudal artery and the spinal cord (figure 4). the regenerated tail has a cartilaginous tube as a substitute for bony vertebrae and rakhmiyati & luthfi – histological study of common house gecko (hemidactylus frenatus) … 51 also has ependymocyti (figure 5). the regenerated tail does not have a tranverse process like the original one. the figure shows that the cartilaginous tube has chondrocytes. figure 5. longitudinal section of regenerated tail (20x10 magnification). hematoxylin eosin (he). 1. muscles; 2. perivertebral fat tissue; 3. cartilaginous tube; 4. meninx; 5. ependymal cell. figure 6. cross section of lizard tail that has undergone autotomy (4x10 magnification). coloring: mallory acid fuchsin. 1. skin; 2. muscles; 3. perivertebral fat tissue; 4. muscle segment; 5. cartilago pipe. figure 7. cross section regenerated tail (10x10 magnification). hematoxylin eosin (he). 1. perivertebral fat tissue; 2. cartilaginous tube; 3. nerves; 4. septum; 5. meninx; 6. ependymocyti; 7. muscles; 8. myotomes; 9. myoseptum; 10. myotube. figure 8. cross section of regenerated tail (10x10 magnification). mallory acid fuchsin. 1. perivertebral fat tissue; 2. nerves; 3. cartilago pipe; 4. meninx; 5. ependymocyti; 6. septum; 7. muscles; 8. myotomes; 9. myoseptum; 10. myotube. the cartilaginous tube is surrounded by perivertebral fat tissue in which there are nerve cells (figure 7). figure 8 shows that perivertebral fat tissue surrounds the cartilaginous tube and in the fatty tissue there are nerve cells. there are ependymocyti in regenerated tail which play a role in cartilage formation. observation of cross and longitudinal sections on the original tail of the common house gecko (hemidactylus frenatus) observation of cross and longitudinal sections on the original tail of the common house gecko (hemidactylus frenatus) stained with mallory acid fuchsin and hematoxylin-eosin showed autotomy plane and transverse process on vertebrae caudales. autotomy plane is located along the tail from the base of the tail to the tip of the tail. the autotomy pane lies transversely and divides the vertebra and fat tissue into two parts: the anterior and posterior. vertebrae caudales of the original tail is surrounded by muscles. the muscle on the tail is a striated muscle. according to irianto (2005), striated muscle is a muscle attached to the skeleton that can be moved. this muscle contracts quickly and strongly. in addition, striated muscles are also very sensitive to stimuli. the muscle of the regenerated tail is composed of multiple myotubes that merge into a myotomes. myotomes are separated by myoseptum. the muscles of a regenerated tail are divided into 16 bundles. according to research conducted by rachman & hadi (2008), the posterior part of original tail muscle of a lizard (mabouya multifasciata kuhl) has its origo on the chevron bone, spina neuralis, and transverse process, whereas the anterior part of the muscle inserted in the septum. muscle of original tail is divided into 8 bundles. 52 biology, medicine, & natural product chemistry 5 (2), 2016: 49-53 observation of cross and longitudinal sections on common house gecko regenerated tail (hemidactylus frenatus). the observation revealed that the regenerated tail is not supported by the bony vertebrae but cartilage or segmental cartilage (figures 5, 6, 7, and 8). inside the cartilaginous tube there is a layer of ependyma that is located parallel to the cartilage tube. according to soesilo (1999), ependymal layer is very important in the process of tail regeneration and in the formation of cartilaginous tube. rachman & luthfi (2004) stated that calcification in cartilaginous tube of regenerated tail of lizard (mabouya multifasciata) starting at 12 weeks of regeneration. this calcification process starts from the inner side and the outer side of the cartilaginous tube. cartilaginous tube of regenerated tail does not have an autotomy plane so the ability of autotomy in the regenerated tail is lower. autotomy capability on the original tail is higher because it has autotomy plane. conclusion based on the results of the study, we drew conclusion as follows: 1. the original tail is composed of bony vertebrae, while the regenerated tail is composed by cartilaginous tube. 2. the regenerated muscle tail is divided into 16 bundles, while the original tail muscle is divided into 8 bundles. references alibardi, l. 1994. muscle differentiation and morphogenesis in the regenerating tail of lizards. journal of anatomy. 186: 143-151. andayani, m.m.l. 2007. mikroanatomi ekor kadal (mabouya multifasciata kuhl) fase penyembuhan luka setelah autotomi dan irradiasi sinar gamma. thesis universitas gadjah mada (unpublished). anonim. 1985. biology of the reptilia (vol.15). new york: a wiley-interscience publication. bateman, p.w & fleming, p. a. 2009. to cut a long tail short: a reviewof lizard caudal autotomy studies carried out over the last 20 years. 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ekor dan regenerat ekor kadal (mabouya multifasciata kuhl). berkala ilmiah biologi. 6 (2): 81-86. yogyakarta: universitas gadjah mada. irianto, k. 2005. struktur dan fungsi tubuh manusia untuk paramedis. bandung: cv. yrama widya. kardong, k. v. 2006. vertebrates: comparative anatomy, function, evolution. washington state university: mc graw hill higher education. kimball, j. w. 1983. biologi. fifth edition. jakarta: erlangga. luthfi, m. j. 2002. kalsifikasi skeleton aksial dan kemampuan autotomi regenerat ekor kadal (mabouya multifasciata kuhl). tesis. universitas gadjah mada (unpublished). luthfi, m. j, soesilo, n.p, & sagi, m. 2003. kalsifikasi skeleton aksial pada regenerat ekor kadal (mabouya multifasciata kuhl). jurnal berkala ilmiah biologi, 3 (1): 1-8. yogyakarta: universitas gadjah mada. leeson, c.r, leeson, t.s, and paparo, a. a. 1996. buku ajar histologi. translated by yan tambayong dkk. penerbit buku kedokteran egc, jakarta. maria, b. 1998. struktur vertebrae caudales pada 5 species anggota sub ordo lacertilia. skripsi. universitas gadjah mada (unpublished). nahdi, m. s. & solihah, j. 2007. buku ajar: biologi umum. uin sunan kalijaga yogyakarta. pratiwi, i.r. 2009. struktur organ reproduksi dan seksual dimorfisme pada hemidactylus frenatus dumeril & bibron, 1836; cosymbotus platyurus (schneider, 1792); & gekko gecko linnaeus, 1758. thesis universitas gadjah mada (unpublished). pratt, c.w.m. 1946. the plane of fracture of the caudal vertebrae of certain lacertilians. journal of anatomy. 80: 184-188. russell, a.p., bergmann, p.j. & barbadillo, l.j. 2001. maximal caudal autotomy in podarcis hispanica (lacertidae): the caudofemoralis muscle is not sundered. american society of ichthyologists and herpetologists. 1: 154-163. rachman, a & luthfi,m.j. 2004. studi histokimia kalsifikasi skeleton regenerat ekor cicak (hemidactylus sp), jurnal berkala ilmiah biologi. 3 (4): 223-230. yogyakarta: universitas gadjah mada. suntoro, s. h. 1983. metode pewarnaan (histologi dan histokimia). jakarta: bhratara karya aksara. soesilo, n. p. 2002. pengaruh regenerat ekor kadal (mabouya multifasciata kuhl) terhadap angiogenesis. biologi. 2 (14): 833-844. yogyakarta: universitas gadjah mada. soesilo, n. p. 1982. regenerasi ekor kadal (mabuya multifasciata kuhl) setelah mengalami autotomi. thesis. universitas gadjah mada (unpublished). soesilo, n. p. 1992. proses regenerasi ekor kadal (mabouya multifasciata kuhl). biologi. 1 (4): 169-175. soesilo, n.p. 1999. peranan lapisan ependima dalam regenerasi ekor kadal (mabouya multifasciata kuhl). biologi. 2 (8): 419-450. rakhmiyati & luthfi – histological study of common house gecko (hemidactylus frenatus) … 53 takahashi, h. 2009. preferensi pakan cicak rumah (hemidactylus frenatus, gray 1825) dan tokek (gekko gecko, linnaeus 1758) di gamping, sleman, daerah istimewa yogyakarta. thesis. universitas gadjah mada (unpublished). yatim, w. 1996. biologi modern: histologi. bandung: tarsito. zug, g. r. 1993. herpetology an introductory biology of amphibian and reptiles. academic press. london. histological study of common house gecko (hemidactylus frenatus) regenerated tail biology, medicine, & natural product chemistry volume 6 – number 2 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini 37 45 clustering of 18 local black rice base on total anthocyanin kristamtini, endang wisnu wiranti 47 51 vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari, kristamtini, endang wisnu wiranti 53 57 alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati, ahya zhilalikbar amin, mohamad amin 59 62 on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi, riyanto 63 68 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 485-498 | doi: 10.14421/biomedich.2023.122.485-498 issn 2540-9328 (online) determination of oil quality and antifungal effect of selected citronella accessions (cymbopogon nardus, cymbopogon winterianus) to formulate an anti-dandruff shampoo rathnayaka mudiyanselage nipuni wijerathna1, achini anuradha wijeweera2, anushi madushani wijethunga1, mapa mudiyanselage sumudu tharangani mapa1,* 1department of biosystems technology, faculty of technology, university of sri jayewardenepura, sri lanka. 2national cinnamon research and training center, sri lanka. corresponding author* sumudu.mapa@sjp.ac.lk abstract citronella is an aromatic grass of the family poaceae which can be classified into two categories ceylon citronella (cymbopogon nardus) and java citronella (cymbopogon winterianus). the citronella oil was extracted from five selected ceylon citronella (hp t1, hp t2 and hp t3) and java citronella (mp t1 and mp t2) accessions using steam distillation and hydrodistillation methods. citronella oil quantity extracted by hydrodistillation with xylene from ceylon citronella was higher (2.45-2.67 ml/100 g) than the java citronella (1.57-1.64 ml/100 g). the oil quantity of ceylon citronella (hp t1-5.52 %, hp t21.40 %, hp t31.05 %) and the quantity of java citronella (mp t11.25%, mp t21.79%) extracted by hydrodistillation showed a significant difference (p<0.0001) and there was no significant difference (p=0.7055) between the oil quantity of ceylon (hp t11.07%, hp t21.18 %, hp t31.19%) and java (mp t11.16%, mp t21.23%) oils extracted by the steam distillation. both java and ceylon citronella oils showed organoleptic properties with pale yellow to pale brownish yellow colour and a strong citrusy aroma which meets the iso 3848 and iso 3849 standards. the oil of ceylon citronella accessions showed refractive index (1.465-1.487), relative density (0.893-0.910), and ethanol solubility (1:2 ml) within the ranges specified in sls 170 standards. java citronella oil exhibited the refractive index (1.4660-1.4730), relative density (0.880-0.892), ethanol solubility (1:2 ml), and optical rotation (-5˚ to 0˚) which meets the specifications of iso 3848 standards. geraniol, citronellol, and citronellal were identified as the major constituents using the gas chromatography-mass spectrometry (gc-ms) where java citronella oil showed high geraniol content (48.60-49.17%) than ceylon citronella oil (16.93-26.49%). all types of tested citronella oil showed inhibition against candida albicans where hp t3 (1.9 cm) and mp t1(2.0 cm) oils showed the highest promising antifungal activity among ceylon oils and java oils respectively. therefore, these two oils were selected for the antidandruff shampoo formulation. the two antidandruff shampoo samples were formulated with 2% v/v concentrations of hp t3 and mp t1 citronella oil which were determined as mic for the inhibition of c. albicans. antidandruff shampoo tested against c. albicans showed greater antifungal activity (hp t3 2.5±0.05 cm; mp t1 2.5±0.05 cm) than the crude citronella oil (hp t31.9±0.11 cm; mp t1-2.0±0.1 cm), also attained the organoleptic and physical properties such as ph (4.0-8.0), foam height (>100 ml), dirt dispersion (no ink in foam), viscosity, low wetting time and solid content (hp t3-14.75±0.12%; mp t2-12.33±0.19%) in acceptable specification range. this study exhibits that ceylon citronella oil hp t1 has the highest oil quantity from all selected accessions. hydrodistillation can be used to extract high oil quantity than the steam distillation method from both java and ceylon citronella types. compared to ceylon citronella oil, java oil has significant potential industrial applications with high geraniol content and with the highest antifungal activity against c. albicans. also, the tested citronella oil of all selected accessions of both java and ceylon types meet the organoleptic and physiochemical requirements specified by the iso and sls quality standards with excellent antifungal activity against c. albicans, which provides prospective to use citronella oil as a natural, safe, and eco-friendly fungicide in future product formulations. keywords: antidandruff shampoo; antifungal activity; citronella oil; cymbopogon nardus; cymbopogon winterianus. abbreviations: international standard organization (iso); sri lanka standards (sls); minimal inhibitory concentration (mic); saburound dextrose agar (sda); potato dextrose agar (pda); dimethyl sulfoxide (dmso). introduction citronella is an important type of aromatic grass belonging to the family poaceae (devi et al., 2016). citronella can be classified into two categories as ceylon citronella (cymbopogon nardus) and java citronella (cymbopogon winterianus). the essential oil produced from citronella, enhance its economic value as an herb. a number of studies have found that citronella oil has therapeutic value due to the major components geraniol, citronellol, and citronellal (ameliana, almawadah and wulandari, 2019) and shows certain anti-fungal manuscript received: 28 march, 2023. revision accepted: 21 august, 2023. published: 23 august, 2023. https://doi.org/10.14421/biomedich.2023.122.485-498 486 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 properties, which can weaken or destroy certain fungi (li et al., 2012). it also contains many possible bioactive components with great pharmaceutical and medicinal consequences which can act as anti-inflammatory, anticancer, antioxidant, and anticonvulsant agents (devi et al., 2016). these properties of citronella oil enhance its value and the applications in many fields such as cosmetics, food, medicinal, ayurvedic, and herbal products. therefore, there is a huge potential to use citronella oil in herbal cosmetic products to cure common fungal diseases like dandruff. dandruff is a major dermatological problem for many people, and it is observed as white scales, which cause scalp itching and hair loss. excessive sweat gland secretion and the appearance of microorganisms on the scalp can also lead to the formation of dandruff. the most common causes of dandruff are the fungi such as candida albicans (ameliana, almawadah and wulandari, 2019) and malassezia furfur (pingili, vanga and raparla, 2016). antidandruff shampoos and other treatment options contains various anti-dandruff substances, including pyrithione, salicylic acid, imidazole derivatives, selenium sulfide, tar derivatives, ketoconazole (pingili, vanga and raparla, 2016) zinc, menthol, and thymol (ameliana, almawadah and wulandari, 2019), etc. as key ingredients. however, the use of these chemicals can cause hair loss and adverse skin reactions such as rashes, itching, and dermatitis. these cannot prevent the recurrence of dandruff and the side effects caused by these chemicals cannot be ignored. therefore, there is a demand for new antimicrobial agents and alternative therapies with natural ingredients. citronella oil will be a highly effective instinctive remedy for this purpose. introducing the citronella oil from the selected five citronella accessions belonging to cymbopogon nardus and cymbopogon winterianus which are grown in sri lanka, will be a new approach to use citronella oil in the herbal cosmetics industry. direct application of citronella oil on the skin is an impractical and less effective method. therefore, a special formulation incorporated with citronella oil should be prepared to treat candida albicans. suitable topical preparation for fighting dandruff is an anti-dandruff shampoo which is a liquid, gel, lotion, or aerosol that contains surfactants to generate foam and cleanse the scalp and hair. this study focused to determine the antifungal effect of citronella oil (cymbopogon nardus, cymbopogon winterianus) against dandruff causing fungi candida albicans to formulate an anti-dandruff shampoo. the quality of citronella oil, extraction methods, and shampoo preparations were determined according to the to criteria of sls and iso standards. the minimum inhibitory concentration (mic) of citronella oil was determined by antifungal assay of agar well diffusion method. materials and methods study area the plants of cymbopogon nardus (ceylon citronella) and cymbopogon winterianus (java citronella) were selected from the farm of the national cinnamon research and training center of sri lanka, located in palolpitiya, matara, sri lanka (6° 2' 2.94'' n latitude, 80° 33' 38.448'' e longitude). figure 1. a cymbopogon nardus (ceylon citronella), bcymbopogon winterianus (java citronella). citronella oil extraction ▪ sample collection and processing the matured citronella leaves from selected accessions cymbopogon nardus (hp t1, hp t2, hp t3) and cymbopogon winterianus (mp t1, mp t2) were harvested, dried under shade for 3-5 days, and cut into small pieces. ▪ determination of moisture content the entrainment method (sls 86 part 5) was used to measure the moisture content of citronella samples by a b wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 487 using dean and stark apparatus (witeg germany). the moisture content of approximately 10 g of the citronella leaves was measured with 60 ml of saturated toluene for about one and a half hours and refluxing was continued for two intervals of 15 minutes revealing no change. then, the whole unit was left to cool for about 15 minutes, and the readings of moisture amount were taken from the bottom of the dean and stark arm. the percentage moisture content was calculated by the following equation. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 (%) = (𝐴𝑟𝑚 𝑅𝑒𝑎𝑑𝑖𝑛𝑔) (𝑆𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡) × 100 ▪ hydro distillation of citronella oil with xylene and without xylene a modified clevenger method with hydro distillation was used to determine the oil quantity of citronella samples (asta method no 5, fourth edition, 1997). approximately 50 g of citronella leaves with 500 ml of clean water were added to the flask and connected with the clevenger arm where the graduated tube was filled with clean water up to the level of 5 ml and the unit was heated by a heating mantle (whm 12393, wisd laboratory instruments, german) while condensing a cooling system for 5 hours. after distillation, the system was allowed to cool, and the extracted citronella oil was collected into a cleaned and oven-dried glass bottle by removing the water layer of the clevenger arm. then samples were kept for about 24 hours to settle. the minute water particles suspended in oil were removed by a cleaned glass syringe. the purified oil samples were subjected to gc-ms analysis. the oil percentage in 100 g of sample in dry weight basis was calculated by following equations. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 = (𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 %) 100 × 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝐵𝑜𝑡𝑡𝑙𝑒 𝑤𝑖𝑡ℎ 𝑜𝑖𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐸𝑚𝑝𝑡𝑦 𝑏𝑜𝑡𝑡𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑂𝑖𝑙 % (𝑔 𝑖𝑛 100 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 the hydrodistillation with xylene was carried out by adding 1 ml of xylene to the clevenger arm. the dry weight of the samples and the oil percentage in 100 g of sample (dry weight) by hydrodistillation with xylene were calculated by following equations. 𝑂𝑖𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝐴𝑟𝑚 𝑅𝑒𝑎𝑑𝑖𝑛𝑔 − 𝑋𝑦𝑙𝑒𝑛𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑂𝑖𝑙 % (𝑚𝐿 𝑖𝑛 100𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑣𝑜𝑙𝑢𝑚𝑒) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 ▪ steam distillation of citronella oil a steam boiler setup was used for the steam distillation of citronella samples. the distillation was run for 5 hours and the oil with moisture was collected and allowed to cool. samples were subjected to settle for overnight and the oil was separated from the water using a separatory funnel. the remaining minute water particles suspended with oil were removed by adding anhydrous sodium sulfate. oil percentage in 100 g of sample (dry weight) was calculated by following equations. 𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝐵𝑜𝑡𝑡𝑙𝑒 𝑤𝑖𝑡ℎ 𝑜𝑖𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐸𝑚𝑝𝑡𝑦 𝑏𝑜𝑡𝑡𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑂𝑖𝑙 % (𝑔 𝑖𝑛 100 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 determination of citronella oil quality the quality of extracted citronella oil from selected accessions was evaluated in terms of organoleptic properties, relative density, refractive index, optical rotation, and ethanol solubility (iso 3848, iso 3849, sls 170). the colour, odor, and texture of the oil samples were observed under the organoleptic properties and the relative density of oil at 25 ℃ was measured using a standardized 10 ml pycnometer. the refractive index of the citronella oils at 20 ℃ was measured by a j-57wr 488 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 refractometer and p-8000 digital polarimeter (germany) was used to measure the optical rotation at 20 ℃. the 80% ethanol was used to determine the ethanol solubility of citronella oil samples at 20 ℃. the burette was filled with 50 ml alcohol and 1 ml of the oil sample was added to the erlenmeyer flask. then the oil was titrated with 80% alcohol and the burette reading of the endpoint at which the turbidity of the solution disappeared was recorded. the alcohol solubility of the 1 ml oil sample was determined by the following equation. 𝐴𝑙𝑐𝑜ℎ𝑜𝑙 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐹𝑖𝑛𝑎𝑙 𝑏𝑢𝑟𝑒𝑡𝑡𝑒 𝑟𝑒𝑎𝑑𝑖𝑛𝑔 − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑏𝑢𝑟𝑒𝑡𝑡𝑒 𝑟𝑒𝑎𝑑𝑖𝑛𝑔 determination of citronella oil composition the gc-ms analysis to determine the composition of citronella oil was carried out trace 1300 isq qd gas chromatography mass spectrometer (thermo fisher scientific, usa) equipped with tgwaxms column (30 m long × 0.25 mm inner diameter, 0.25 μm film thickness). the sample was prepared by diluting 50 μl of the citronella oil with 950 μl methanol in a vial and 0.5 μl from the dilution was injected into the column through the syringe. the instrument was programmed with a linearly automated oven temperature from 50 ℃ to 220 ℃ in the rate of 3 ℃ min-1 and helium was used as the carrier gas with a carrier flow rate of 1 ml / min. and the pressure was set as 5 bar. the mass spectra were acquired in e1 mode (70ev), in a mass range of 50 to 500 amu and the transfer line and the ion source temperatures were maintained at 220 ℃ and 200 ℃ respectively. the individual oil components were identified by comparing the retention time with standard substances and a computer search by matching mass spectrum data with the ms library (nist ms search 2.0). determination of antifungal effect and minimum inhibitory concentration (mic) of citronella oil the antifungal effect of citronella oil from selected accessions was tested using the agar well diffusion method (balouiri, sadiki and ibnsouda, 2016). saburound dextrose agar (sda) was used as the media for testing and 6.5 g of sda was suspended in distilled water in an erlenmeyer flask and warmed until homogenized. the media, petri plates, and distilled water were autoclaved at 121 ℃ for 15 minutes for sterilization. the sterilized liquid sda was poured into petri plates and left until solidified (ameliana, almawadah and wulandari, 2019). a suspension of candida albicans was made by adding an inoculum of candida albicans to saline water (silva, guterres, weisheimer and schapoval, 2008) and the mixture was vortexed, and the turbidity was checked with 0.5 mcfarland standard (oliveira et al., 2011). then, 100 μl of fungal suspension was spread over the solidified sda medium. after, a hole of 4 mm diameter was punched in the middle of the sda medium aseptically using a cork borer and 20 μl (balouiri, sadiki and ibnsouda, 2016) of citronella oil was pipetted and added into the well. the plates were sealed and incubated at 37 ℃ for 24 72 hours (oliveira et al., 2011). the inhibitory zone diameters of each oil type were recorded. the oil types with the highest inhibitory zone diameters were selected to determine the mic. the mic was tested following agar well diffusion for citronella oil with the highest antifungal activity in several oil concentrations (1%, 2%, 3%, 4%, and 5% v/v) diluted with dimethyl sulfoxide (dmso) solution. the same procedure also was followed using potato dextrose agar (pda) as the growth media. the dmso and captan (n-trichloromethylthio-4cyclohexane-1,2dicarboximide) were assessed as the negative and positive controls, respectively. the lowest concentration shows an inhibitory zone with no visible growth of fungus during the incubation was considered as the mic value of the citronella oil (ameliana, almawadah and wulandari, 2019). formulation of citronella oil antidandruff shampoo the citronella oil shampoo was made with the modified formula shown in table 1. the sodium lauryl sulfoacetate (slsa), sorbic acid, and sodium chloride were measured and added to a 500 ml beaker and the cocamidopropyl betaine, glycerin, tween 80, and the oil were pipetted and added to the beaker and topped up with the distilled water. the mixture was stirred with a magnetic stirrer at 1500 rpm until homogenous. a shampoo sample without citronella oil was formulated as the negative control. the ph of formulated shampoo samples was measured. table 1. citronella oil antidandruff shampoo formulation. material function concentration in 100 ml (%) sodium lauryl sulfoacetate (slsa) primary surfactant 10 cocamidopropyl betaine co surfactant 5 sorbic acid preservative 0.3 sodium chloride (nacl) viscosity adjuster 1 glycerin humectant 3 polysorbate 80 (tween 80) oil solvent 0.5 citronella oil active ingredient (antidandruff agent) 2 distilled water solvent 89.5 total volume 100 ml wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 489 determination of antifungal effect of citronella antidandruff shampoo the antifungal effect of formulated shampoo against candida albicans was examined by following the previous method of agar well diffusion. the well of the medium was filled with 20 μl of citronella oil shampoo. the citronella oil and captan were tested as the positive control and the shampoo without citronella oil was tested as the negative control. evaluation of citronella antidandruff shampoo formulated shampoo samples were tested for quality under different types of organoleptic and physiological parameters. the organoleptic properties were evaluated based on their clarity, colour, odor, and texture (alquadeib, eltahir, banafa and al-hadhairi, 2018). ▪ foaming ability and foam stability the forming ability of the shampoo was evaluated by the cylinder shake method (al badi and khan, 2014). a 50 ml of 1% v/v shampoo solution was added to a 250 ml graduated cylinder and the cylinder was covered by hand and shaken for 10 times. the total volume of that foam that appeared after 1 min shaking was measured and recorded. the foam stability was tested by measuring the foam volume in 1min intervals for 4 min. ▪ determination of ph the ph of the formulated shampoo samples and 10% v/v diluted shampoo samples were directly measured using calibrated cyberscan pc 650 ph meter (eutech instrumets, thermo scientific, singapore) and a strip of ph paper at room temperature 27±2 ℃ (sls 1346:2018) (al badi and khan, 2014). ▪ rheological evaluations (viscosity) the viscosity of the shampoo samples was evaluated by using bdv-1s digital viscometer (biobase meihua, china) with the spindle l1 at speed of 60 rpm. the temperature and the size of the container were kept constant during the study (alquadeib, eltahir, banafa and al-hadhairi, 2018). ▪ dirt dispersion a drop of india ink was added to a large test tube with 2 drops of shampoo in 10 ml of distilled water. then, the test tube was stoppered and shaken 10 times. the amount of ink in foam was evaluated as none, light, moderate or heavy (al badi and khan, 2014). ▪ wetting time drave’s test was used to determine the wetting time of the formulated shampoo samples (alquadeib, eltahir, banafa and al-hadhairi, 2018). a canvas fabric was cut into 1.0 inch diameter disc with an average weight of 0.44 g. the smooth surface of the disc was placed on the surface of 1% v/v shampoo solution and the time required for the disc to begin sinking was measured by a stopwatch as the wetting time. ▪ percentage solid content a cleaned and dried dish was weighed, and 4 g of shampoo was added to the dish the dish with shampoo was weighed and the exact initial weight of shampoo was calculated. the dish was placed on a hot plate until the liquid portion of the shampoo was evaporated. after the remaining solid content was weighed and the percentage (%) of solid content was calculated (al badi and khan, 2014). data analysis data were subjected to one-way anova and two-way analysis of variance according to the glm (general linear mode) at a 0.05 significance level using sas ondemand for academics online version. the mean value, standard deviation, calculations, and figures with graphical representations of mean values were performed by microsoft excel for microsoft 365 mso (version 2205 build 16.0.15225.20028). results and discussion quantity and quality of citronella oil organoleptic properties (colour, odor and texture) of the java and ceylon citronella oil obtained from the selected accessions were determined. the colour measurements were carried out by direct eye observation at a distance of 30 cm (fitri et al., 2022) and the observed colours of each oil type ranged between pale yellow to pale brownish yellow as specified in the iso standard (iso 3849:2003). the characteristic citronella oil aroma (strong citrusy) of each 5 types of oil was sensed at a distance of 5 cm (fitri et al., 2022) as the odor and the oil from java citronella (mp t1 and mp t2) and ceylon citronella hp t3 oils have a stronger aroma than hp t1 and hp t2 due to their high citronellal content (table 3). according to the physical appearance and texture, each type of oil is an oily and viscous liquid that is volatile in the open atmosphere. the moisture contents of the selected citronella accessions shown in table 2 are significantly different (p < 0.0001). the mp t2 java citronella accession showed the highest significant moisture content whereas the hp t3 ceylon citronella accession showed the least significant moisture content. both java citronella accessions showed high amounts of moisture content above 50% compared to the moisture content of ceylon citronella accessions. according to the results of table 2, the % oil content obtained by hydrodistillation with xylene from each accession showed a significant difference (p = 0.0001). the oil quantity (ml) per 100 g of citronella sample from ceylon citronella is significantly higher than the java citronella. the ceylon citronella accession hp t3 490 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 showed the highest oil quantity of 2.67 ml/100 g whereas oil from mp t1 java citronella accession shows the least oil quantity of 1.57 ml/100 g. extracted citronella oil quantities from each accession by hydro distillation without xylene showed a significant difference (p<0.0001) where the hp t1 accession showed the highest oil quantity of 5.52 % where oil from hp t3 accession shows the least oil quantity of 1.05% (table 2). the % oil contents of selected citronella accessions extracted by steam distillation does not show a significant difference (p = 0.7055) and ranged from 1% to 1.25% (table 2). table 2. citronella oil content and quality under different parameters. quality parameter accession of citronella probability cv% hp t1 hp t2 hp t3 mp t1 mp t2 moisture (%) 46.16 c 42.89 d 28.46 e 52.71 b 56.40 a <0.0001 2.23 hydro distillation with xylene % oil v/w (ml/100g) 2.67 a 2.45 a 2.53 a 1.57 b 1.64 b 0.0001 13.67 hydro distillation % oil w/w (g /100 g) 5.52 a 1.40 bc 1.05 c 1.25 bc 1.79 b <0.0001 14.14 steam distillation% oil w/w (g /100 g) 1.07 a 1.18 a 1.19 a 1.16 a 1.23 a 0.7055 9.43 refractive index 1.47 c 1.49 a 1.48 b 1.47 c 1.47 c <0.0001 0.06 optical rotation -2.87˚a -9.89˚b 0.72˚ a -0.82˚a -0.75˚a 0.0003 -94.40 relative density 0.90 b 0.91 a 0.90 b 0.88 c 0.88c <0.0001 0.55 ethanol solubility (ml) 1.25a 1.25a 1.25a 1.25a 1.25a 1.0000 4.61 note: probability<0.05 have a significant difference within mean values. mean within each column followed by the same letter are not significantly different. mean values from highest significant difference have been denoted with “a” the relative density resulted for the oils of selected citronella accessions shows a significant different (p < 0.0001). ceylon citronella oil from accessions hp t1, hp t2 and hp t3 shows a significantly high relative density compared to the java citronella oils of mp t1 and mp t2. according to the relative density of ceylon citronella oil samples, hp t1, hp t2 and hp t3 have been resulted as 0.90, 0.91 and 0.90 respectively, with highest significant difference in hp t2, and in the range of 0.893-0.910, the relative density for ceylon citronella essential oil in 25 ℃ specified in the sls standards (sls 170). also, the relative density values resulted for java citronella oil samples mp t1 and mp t2 are same (0.88) and within the range of 0.880-0.892 specified for the relative density of java citronella essential oil in 25 ℃ (table 2). the measured refractive index values of citronella oil from selected citronella accessions were significantly different (p < 0.0001). the citronella oil of accession hp t2 resulted with the highest significant refractive index, while hp t1, mp t1 and mp t2 shows the lowest significant refractive index (table 2). according to the sls quality standards, the refractive index of ceylon citronella should be in the range of 1.465-1.487. therefore, only the oil from accession hp t2 shows a slightly higher refractive index out of the given range where the oil from hp t1 and hp t3 rely on the given refractive index range in the sls standards (sls 170). although there is no specified refractive index range for the java citronella in sls standards (sls 170), the iso standard have been specified the refractive index for java citronella in the range of 1.4660-1.4730 (iso 3848). the refractive index values of both selected java citronella accessions mp t1 and mp t2 resulted as 1.47 (table 2), which is in the range specified in the iso standard. the optical rotation of the oil samples from selected accessions of java and ceylon citronella has resulted in a significant difference (p < 0.0001). the sample of hp t3 oil has the highest significance positive value (+0.72˚) which indicates that the oil a has dextrorotary polarized light plane. the hp t1 and hp t2 oils are in negative values -2.87˚ and -9.89˚ respectively indicating levorotary of the light plane. however, the oil samples from selected ceylon citronella accessions are not lie down within the iso specified range of -22 ˚ to -12 ˚ (iso 3849:2003) showing an odd optical rotation which might be a result of irregular distribution of oil composition (st-gelais, 2021). optical rotation results of java citronella oil samples mp t1 and mp t2 are -0.82˚ and -0.75˚ respectively are negative values that indicates a levorotary light plane and are not significantly different to each other (table 2). both iso and sls standards have specified that the alcohol solubility of java or ceylon citronella should be determined by using 80% (v/v) ethanol in 20 ℃ and high-quality citronella oil should have 1:2 ethanol solubility (iso 3848, iso 3849, sls 170). all the extracted oil samples were tested for ethanol solubility have been resulted the same value 1.25 ml of ethanol for the solubility of 1 ml of oil and does not show a significant difference (table 2). this illustrate that the oil of selected citronella accessions has their ethanol solubility at an acceptable level by international and local standards. wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 491 chemical composition of citronella oil the citronellal, geranyl acetate, citronellol, geraniol, and geranyl iso butyrate are the main compounds identified from the oil samples of selected citronella accessions according to the gc-ms results (table 3). the citronellal contents available in each oil type are significantly different. the oil from accession hp t3 has the highest significant citronellal content (20.29%), whereas the oil of hp t1 has the least significant citronellal content (2.43%). the oil of java citronella accessions mp t1 and mp t2 have a citronellal content of around 6-7% and which is lesser than the citronellal content of hp t3 oil. the percentage availability of the geranyl acetate has a significant difference among the five oil types of selected accessions where the oil of hp t1 has the highest significant geranyl acetate content (13.48%) and in contrast, the oil hp t2 has the least geranyl acetate content of 0.63% (table 3). citronellol contents identified by the gc-ms showed a significant difference among the tested oil samples. the java citronella oil samples mp t1 and mp t2 have high citronellol content compared to ceylon citronella oil where mp t1 oil has the highest significant citronellol content of 11.41%. the ceylon citronella oil hp t1 has the least significant citronellol content of 2.06% and the high citronellol content of hp t3 (9.63%) compared to the other ceylon citronella oil types of hp t1 and hp t2, is exceptional as it does not have a significant difference to the citronellol content of java citronella oil mp t2. the available proportions of the compound geraniol show a significant difference in tested oil samples. java citronella has very high geraniol content around 50% of its total composition compared to ceylon citronella where java oil mp t1 and mp t2 have the highest significant geraniol contents 48.60% and 49.17% respectively with no significant difference to each other. the ceylon citronella oil hp t1 and hp t3 have geraniol contents 2.49% and 26.12% respectively with no significant difference to each other and ceylon oil hp t2 has the least significant geraniol content of 16.93% (table 3). geranyl iso butyrate is another type of the main compound identified in citronella essential oil in minor quantities. the percentage content of geranyl iso butyrate available in the tested samples has resulted in a significant difference. however, the ceylon oil hp t2 has significantly higher geranyl iso butyrate compared to other types of oil samples (table 3). table 3. chemical composition of the citronella oil. chemical compound accession of citronella probability cv% hp t1 hp t2 hp t3 mp t1 mp t2 citronellal (%) rt-14.73 2.43e 3.46d 20.29a 6.57c 7.39b <.0001 4.83 geranyl acetate (%) rt-24.84 13.48 a 0.63 d 3.48 c 4.95 bc 5.54 b <.0001 18.59 citronellol (%) rt-25.24 2.06 d 3.16 c 9.63 b 11.41 a 10.11b <.0001 8.80 geraniol (%) rt-28.23 26.49 b 16.93 c 26.12 b 48.60 a 49.17 a <.0001 7.09 geranyl iso butyrate (%) rt-29.39 0.99 b 2.75 a 0.44 c 0.23 c 0.21 c <.0001 17.84 note:rtretention time, probability<0.05 have a significant difference within the mean values.mean within each column followed by the same letter are not significantly different. mean values from highest significant difference have been denoted with “a” antifungal effect of citronella oil figure 2. inhibition zones of citronella oil against candida albicans on sda. all five types of non-diluted ceylon citronella (hp t1, hp t2, hp t3) and java citronella (mp t1, mp t2) oil samples that tested positive for the inhibition of candida albicans with evidently visible inhibition zones compare to the negative control (figure 1). the diameters of the inhibition zones that appeared on the 492 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 plates with citronella oil (figure 1) showed that the antifungal activity of the tested oil samples is different from each other. the citronella oil of mp t1 accession of java citronella showed the highest inhibition zone diameter whereas oils of accessions hp t1 and hp t2 showed the least inhibition zone diameter. table 4. inhibition zone diameters (mean) of citronella oil against candida albicans. accession of the oil sample inhibition zone diameter (cm) probability cv% hp t1 1.8 0.1556 5.31 hp t2 1.8 hp t3 1.9 mp t1 2.0 mp t2 1.9 minimum inhibitory concentration of citronella oil table 5. inhibition zone diameters of hp t3 and mp t1 oil concentrations (1%-5% v/v) against candida albicans on pda and sda media. factor level diameter of inhibition zone (cm) pda sda factor 1 – citronella accession hp t3 1.267a 0.722a mp t1 1.361a 0.672b probability 0.0988 <0.0001 factor 2 – concentration (%) 1 -0.00d -0.00d 2 1.30bc -0.00d 3 1.25c 1.00c 4 1.46b 1.03b 5 1.40bc 1.15a positive control 2.47a 1.00c probability <0.0001 <0.0001 interactions – accession * concentration (%) hp t3*1 -0.00d -0.00d hp t3*2 1.43bc -0.00d hp t3*3 1.23c 1.00b hp t3*4 1.27c 1.03b hp t3*5 1.20c 1.30a positive control 2.47a 1.00b mp t1*1 0.00d -0.00d mp t1*2 1.17c -0.00d mp t1*3 1.27c 1.00b mp t1*4 1.67b 1.03b mp t1*5 1.60b 1.00b positive control 2.47a 1.00b probability 0.0122 <0.0001 cv% 12.55 3.38 note: probability<0.05 has a significant difference in mean values. the mean within each column followed by the same letter is not significantly different. mean values from the highest significant difference have been denoted with “a” the 1% v/v concentration of hp t3 and mp t1 oil samples were not showed clearly visible zones against the fungi candida albicans on pda media (table 5). the clearly visible inhibition zones were observed starting from the 2% v/v concentration corresponding to the commercial antifungal agent, captan which was used as the positive control. the results of the same antifungal assay carried out on sda show that there were no inhibition zones for both 1% and 2% v/v concentration and the inhibition zones were observed afterward at the 3% v/v concentration. the positive control also indicated smaller inhibition zones on sda than the inhibition zones for the positive control in pda. wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 493 formulation of citronella oil antidandruff shampoo figure 3. formulated antidandruff shampoos with citronella essential oil hp t3 and mp t1. antifungal activity of citronella oil antidandruff shampoo citronella oil shampoo samples with mp t1 java citronella and hp t3 ceylon citronella showed a high antifungal activity than shampoo without citronella oil (table 6). however, shampoo samples without citronella oil also showed substantial antifungal activity corresponding to the commercial fungicide used as the positive control and the crude citronella oil. table 6. inhibition zone diameters (mean± sd, n=3) of hp t3 and mp t1 oil and shampoo with 2% v/v concentration of oil against candida albicans selected accession inhibition zone diameter (cm) antidandruff shampoo shampoo without oil crude citronella oil distilled water positive control hp t3 2.5±0.05 2.1±0.1 1.9±0.11 0 1.9±0.1 mp t1 2.5±0.05 2.0±0.2 2.0±0.1 0 2.1±0.1 quality of citronella oil antidandruff shampoo the ph of the shampoo formulations with citronella oil of hp t3 and mp t1 accessions resulted within 4-5 while the ph of negative control was higher than 5. the diluted shampoo samples formulated by incorporating citronella oil of hp t3 and mp t1 accessions showed ph values of more than 5 while the diluted negative control also showed a high ph value than the initial ph (table 7). the shampoo formulations with citronella oil of hp t3 and mp t1 and the negative control shampoo samples showed viscosity within 30-50 mpa.s which are significantly different from each other (table 7). although all samples resulted in low viscosity values in high rpm like 60 rpm, the shampoo solution with mp t1 citronella oil shows significantly low viscosity compared to shampoo solution with citronella oil hp t3 and negative control. all shampoo samples with citronella oil hp t3 and mp t1 and negative control samples showed similar results for dirt dispersion indicating no remain of ink in the foam (table 7) the wetting time of the shampoo solutions with citronella oil hp t3 and mp t1 and negative control resulted in very low values of 6.59±0.14 seconds, 5.91±0.19 seconds, and 5.59±0.42 seconds and the wetting time of hp t3 shampoo was higher compared to the other tested samples (table 7). the percentage solid content of the formulated shampoo with citronella oil hp t3 and negative control were lower than the acceptable range of 20-30% and, they were not significantly different from each other (table 7). table 7. physiological properties (mean ±sd, n=3) of citronella oil antidandruff shampoo. parameter hp t3 mp t1 shampoo without oil (negative control) ph 4.53±0.03 4.49±0.03 5.20±0.54 ph of diluted shampoo solutions 5.51±0.02 5.48±0.03 5.53±0.03 viscosity (mpa.s) 46.4±1.08 30.23±0.56 41.99±0.17 dirt dispersion (ink in foam) none none none wetting time (s) 6.59±0.14 5.91±0.34 5.59±0.42 % solid content 14.75±0.12 12.33±0.19 13.47±0.23 the shampoo with mp t1 citronella oil produced significantly high foam volume (170 ml) than foam volumes of shampoo with hp t3 citronella oil (141 ml) and shampoo without citronella oil (negative control). 494 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 as the graph illustrates, the foam volume of each shampoo sample reduced within 3 minutes time and remained constant at certain levels above 100 ml (figure 3). figure 4. the foam volumes of the shampoo samples with time. discussion quantity and quality of citronella oil the citronella plant leaves were harvested before the flowering period to obtain a high oil yield, as flowering causes the aging of plants (blank et al., 2007). shade drying was used to dry the plant leaves without leading to a reduction of oil quality and quantity. direct drying in hot sunlight should be prevented as far as avoiding chemical transformations of this volatile essential oil. drying of citronella leaves aimed to reduce the moisture content with minimum quality reduction. the moisture content should be reduced literally to be at an equilibrium level that is defined for certain relative air humidity and temperature with a minimum quality reduction in terms of active ingredients, colour, flavor and aroma (özgüven, gülseren and müller, 2019). therefore, citronella leaves should be spread out under a shade for drying. in the process of drying, the citronella leaves under the shade should be frequently turned to avoid the formation of molds and the fermentation of leaves which will lead to the oil quality reduction. after the completion of the drying period, the overdried parts and the foreign matters should be removed. the citronella leaves were cut into small pieces, to enhance the even distribution in the still which prevent steam channeling as well as for the easy insertion of plant leaves to the sample container (still) as well as to the round bottom flask of clevenger distillation apparatus. the moisture content of the harvested citronella leaves was measured after shade drying, to calculate the dry weight of the sample. citronella leaves were distilled with toluene which is less concentrated than water and immiscible with water. method of hydro distillation with xylene was performed to determine the oil content that could be obtained from each type of citronella sample and hydro distillation without xylene was used to extract the citronella oil for the gc-ms analysis. the steam distillation was carried out to obtain the citronella oil samples where the higher oil yields can be obtained for further analysis such as determination of refractive index, alcohol solubility, optical rotation, and relative density. the oil yield of citronella plants depends on several factors, including the presence of climatic differences, soil fertility, plant age, and the separation of citronella oil (ameliana, almawadah and wulandari, 2019). however, the study was carried out in the same location and same time period, and the above differences in oil content is due to the genetic variations of each accession. hydrodistillation with the organic compound xylene showed the highest efficiency in citronella oil extraction among the three extraction methods used and hydro distillation with or without an organic compound resulted with high oil quantity than steam distillation in most oil accessions (table 2). however, steam distillation is one of the most common and main methods used for the extraction of citronella oil in bulk quantities for industrial and commercial purposes. all the oil samples from selected java and ceylon citronella accessions that were tested are showing required quality standards for relative density, refractive index, ethanol solubility and can be considered as highquality samples (table 2). essential oils are composed of many different compounds, most of which are optically active. their different compositions allow them to be distinguished by measuring their optical rotation. these measurements also provide comprehension of the quality and purity of essential oil, as any change in its optimal composition can affect the optical rotation (nandapure et al., 2016). although the optical rotation of ceylon 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 f o a m h e ig h t (m l ) time (min) hp t3 mp t1 control shampoo wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 495 citronella oil samples was not in the iso-specified range, java citronella oil samples resulted within the iso-specified range (-5˚ to 0˚) for optical rotation (iso 3848). therefore, the results prove that the oil samples of selected java citronella are within the acceptable range (table 2). chemical composition of citronella oil the composition of the oil samples extracted from the selected citronella accessions was determined by subjecting to the gc-ms and the constituents available in each oil type were identified by comparison of retention time with the library search of mass spectra for fragmentation pattern of reference standard compounds. the proportions of each compound present in five tested oil types were calculated from the gc peak areas using the normalization method. the main constituents identified from the oil samples of selected citronella accessions were geraniol, citronellol, and citronellal. geraniol is the main compound that has been identified in the highest proportions from each oil sample. citronellal and citronellol are the other two types of constituents identified in moderately high amounts in the composition of tested citronella oil. therefore, the gc-ms results of the tested citronella oil samples comparatively correspond to the literature that indicates citronella oil (cymbopogon nardus) contains different types of chemical components, including citronellal, citronellol, and geraniol, which have antifungal and antibacterial activities (ameliana, almawadah and wulandari, 2019). according to aguiar et al., 2014, the high citronellal content of c. nardus (ceylon citronella) oil extremely performs as a potent inhibitor of various fungi at ambient temperatures. therefore, the citronella essential oil from hp t3 accession shows a potential to inhibit the fungi candida albicans with its significantly very high amount of citronellal content (table 3) and also comparatively high amounts of geraniol and citronellol contents. along with this, the oil of selected java citronella accessions mp t1 and mp t2 are also considered to have a high potential antifungal activity due to the availability of high citronellal, citronellol, and geraniol contents in their composition. according to the gc-ms results, there are differences in the chemical composition and the percentage content of the constituents in the citronella oil samples of selected accessions. these changes in the essential oil composition may possibly occur due to several environmental (climatic, seasonal, or geographic) factors and genetic differences (aguiar et al., 2014). antifungal effect of citronella oil the oil of hp t3 resulted the highest inhibition zone among the tested ceylon citronella samples and mp t1 oil showed the highest inhibitory action among tested java citronella samples (table 4). in addition, hp t3 oil showed unique high proportions of the main antifungal constituents citronellal, citronellol, and geraniol (table 3) in its composition (ameliana, almawadah and wulandari, 2019). therefore, the oils of accessions hp t3 and mp t1 were selected as the active ingredients with antifungal properties for the formulation of antidandruff shampoo. and further, the selected oil samples were subjected to antifungal analysis for the determination of minimum inhibitory concentration against candida albicans. identification of the volume of citronella oil that should be incorporated into the shampoo formulation to perform antidandruff function was significant. therefore, the mic of selected citronella essential oils for the inhibition of candida albicans was identified across five concentrations (1%, 2%, 3%, 4%, and 5% v/v) by performing agar well diffusion assay (ameliana, almawadah and wulandari, 2019). the oil samples were evaluated in both sda and pda media to compare the inhibitory activity of selected citronella oil samples in different media (table 5). the positive control also indicated smaller inhibition zones on sda than the inhibition zones for the positive control in pda. therefore, the results of the mic antifungal assay on pda media were considered to be more accurate and precise. table 5 demonstrates that there is no significant difference between the antifungal activity of the citronella oils of hp t3 and mp t2 accessions on pda against candida albicans. in contrast, the antifungal activity of the two oil types shows a significant difference in sda against candida albicans. in the pda media, the positive controls show the highest significant inhibition whereas the 1% v/v concentration of both hp t3 and mp t1 oils show the least significance with no inhibition against the tested fungi. the second least significant inhibition (1.17 cm) of mp t1 oil in pda shows in the concentration of 2% v/v. therefore, the 2% v/v concentration of mp t1 citronella oil is the mic that performs antifungal activity against candida albicans. also, the least concentration of hp t3 oil that shows an inhibition zone diameter is 2% v/v (table 5). therefore, the mic values of both oil samples mp t1 and hp t3 were assumed to be more than 1%v/v concentration and less or equal to 2%v/v concentration (1% < mic ≤ 2% v/v). consequently, the 2% v/v concentration was determined as the mic of both oils hp t3 and mp t1 against candida albicans and referred to as the concentration of citronella oil to be incorporated in the antidandruff shampoo formulation. formulation of citronella oil antidandruff shampoo the citronella antidandruff shampoos formulated with selected citronella oil hp t3 and mp t1 with the highest antifungal activity are shown in figure 2. the sodium lauryl sulfoacetate (slsa; c14h27nao5s) has been registered in drug bank (accession number db13157) as a surfactant which also functions as a wetting agent. according to the final report on the safety assessment 496 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 of slsa by the american college of toxicology, it is a hydrophilic, skin-friendly, mild surfactant derived from coconut and palm plants which use in cleansing products for both skin and hair to remove surface oil, dirt, and bacteria effectively. the hydrophilic nature of the slsa was beneficial for the liquid shampoos as well as it improves the rinsing ability of the product. slsa is a bulky molecule that penetrates the skin and is free from sulfate ions causing less irritation to the skin. this can be effectively used in cosmetic products with ph 5-8.5, which will give better performance for the liquid shampoo prepared for ph 4.0-8.0 (sls 1346:2018). further study of the final report on the safety assessment of slsa by the american college of toxicology (1987), it is a cosmetic ingredient approved by u.s. food and drug administration and used primarily for bath preparations in 5-50%, but the actual concentration contacts with the skin surface are much lesser due to product dilution with water. therefore, it was considered a much safer and more efficient ingredient for the citronella antidandruff shampoo formulation. a commonly used cosmetic ingredient, cocamidopropyl betaine was used as a co-surfactant to increase the function of the slsa (hunter and fowler, 1998), and sorbic acid with low toxicity was used as the natural preservative (dorko et al., 2000). sodium chloride was effectively used as a salt work as a thickening agent (penfield, 2005) whereas polysorbate 80 was used as an emulsifier for the citronella essential oils which are non-soluble in water (ariff, jai, jamaludin and ibrahim, 2022). the distilled water was used as the most abundant and common universal solvent in the preparation of shampoo samples, continuous stirring was performed to obtain a homogenized mixture of both the aqueous and the oil phases. two shampoo samples for mp t1 oil and hp t3 were prepared by adding 2 ml of each oil type separately to obtain a 2% v/v oil concentration in a 100 ml shampoo formulation. antifungal activity of citronella oil antidandruff shampoo according to the inhibition zone diameters tabulated in table 6, both shampoo samples with citronella oil show higher antifungal activity than the crude citronella essential oils used in their formulations, commercial fungicide, and the shampoo without citronella oil. however, in this study shampoo formulated as a negative control without citronella oil also showed inhibitory action against candida albicans. this can be justified as the preservative used in shampoo formulations has increased the antimicrobial activity of the products (sofos and busta, 1981). quality of citronella oil antidandruff shampoo the attractiveness of physical appearance and organoleptic properties are vital factors to be considered in a cosmetic formulation that will enhance consumer preference (chavan et al., 2019). the physical appearance of the formulated shampoo samples by incorporating citronella oil of hp t3 and mp t1 accessions and the shampoo without citronella oil was identified by the visual inspection. according to that, all shampoo mixtures were opaque, and white in colour as shown in figure 2 due to the absence of any synthetic or natural colouring agent. the natural fragrance of the citronella oil blended in both formulations mpt1 and hp t3 had given a citrusy aroma in to the shampoo samples which expresses the availability of citronella oil as the active ingredient. in terms of texture, all shampoo samples were in a viscous liquid form similar to the shampoo formulations in the market and acceptable to the general requirements for the shampoo in sls 1346:2018 standard. according to the resulting organoleptic properties except for negative control shampoo, both citronella antidandruff shampoo samples have no significant difference from each other with good characteristics corresponding to the target market preference. the ph level of a shampoo is accountable for improving and enhancing the hair quality with minimum eye irritation and stabilizing a balanced ecology on the scalp. generally, most of the shampoo formulations are neutral or slightly acidic in ph. the cuticle (outer layer) of the hair is shrink away and lie flatter on the hair shaft with acidic blends (alquadeib, eltahir, banafa and alhadhairi, 2018). also, the mild acidity prevents the swelling and promotes strengthening of the scales, which leads to a shine and smoothness while basic solutions make hair triggers the hair to be frizzier. therefore, the promoting the shampoo formulations with low ph to minimize the hair damage is an emerging trend in the industry (krunali et al., 2013). the acidity of citronella oil has been affected for the low ph of shampoos blended with citronella oil and the formulations were not required to be acid balanced after initial formulation as both shampoo formulations were ranged in sls specified ph levels 4.0-8.0 for the shampoo (sls 1346:2018). the ph of diluted shampoo samples formulated by incorporating citronella oil of hp t3 and mp t1 accessions were in the ph range of 5.5-5.9 near to the skin ph (krunali et al., 2013). also, both shampoo formulations show no significant difference in ph (table 7). the viscosity can be defined as the thickness or the stickiness of a liquid (alquadeib, eltahir, banafa and alhadhairi, 2018). viscosity plays a vital role on a shampoo product in defining and controlling characteristics such as shelf-life stability and product aesthetics such as clarity, ease of flow in removal from packing, ease spreading in application to hair and product reliability in the package (chavan et al., 2019). shampoos are non-newtonian products with plastic or pseudoplastic flow behaviors which shows high viscosity wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 497 in low rpm values and low viscosity in increased shear rates. dirt dispersion is a key criterion for evaluating the cleaning action of shampoo formulations (al badi and khan, 2014). determination of the amount of ink present in the foam phase of the column after shaking the shampoo solution and estimate the dirt dispersion of the shampoo formula (chavan et al., 2019). the shampoos which cause the ink to be concentrated in the foam phase are considered to be poor-quality formulations as the ink used to represent the dirt that remains in the foam is difficult to rinse away and also will be redeposited on the hair. therefore, the ink or the dirt should be always remained in the water phase to accomplish a better performance in cleansing (krunali et al.2013). the results of dirt dispersion ensured that the formulations achieved the cleaning ability and effectiveness in market satisfactory level (al badi and khan, 2014). the wetting time of a surfactant is a function of its concentration which is used to determine the efficacy of the surfactant (krunali et al., 2013). the performance of wetting action is dependent on various factors such as diffusion, surface tension, the concentration of the wetting agent, and the nature of the surface being wetted. an acceptable wetting agent should have the ability to reduce surface tension (alquadeib, eltahir, banafa and al-hadhairi, 2018). a low wetting time indicates a greater wetting efficiency with a maximum concentration of detergents (al badi and khan, 2014). all shampoo samples formulated in this study can be categorized as shampoos with acceptable wetting times due to the maximum concentration of surfactants with high efficacy (alquadeib, eltahir, banafa and al-hadhairi, 2018). the percentage solid content of a shampoo formulation is an important criterion to be considered in terms of quality and performance. too many solids in a shampoo, makes it difficult to work on hair or to wash out where, the absence of enough solids, leads to be too watery and will wash away rapidly. therefore, the solid content of a good shampoo should be in the range of 2030 % (alquadeib, eltahir, banafa and al-hadhairi, 2018). although, the solid contents of both shampoo formulations are slightly lower than the specified range, are expected to perform cleaning and wash out easily (table 7). as a promising fungal inhibitor, citronella oil can be widely used as a strong fungicide in different industrial applications such as in medical field, food industry agricultural industry and etc. the significant antifungal activity of citronella oil against c. albicans shows that citronella oil can be used as a remedy for the infections of candida on human skin such as candidiasis which may cause symptoms including rashes, scaling, itching, and swelling. therefore, citronella oil has the potential of used as an active ingredient in herbal cosmeceutical formulations such as soap, creams, lotions, and ointments etc. conclusions the oil extracted from the selected citronella accessions were in high quality under the tested physiochemical parameters and meet the organoleptic quality requirements with acceptable colour, aroma, and texture. the ceylon oil hp t1 has the highest oil content (ml/100 g) and the oil quantity of ceylon citronella extracted by hydrodistillation with xylene is higher than the oil content of java citronella. compared to steam distillation, hydro distillation can be used to extract high oil quantities from both java and ceylon types. the results of the quality tests performed for the citronella oil samples in terms of refractive index, relative density, optical rotation, and ethanol solubility followed the acceptable ranges under the iso and sls standards. the significant differences in oil quantity and quality of tested citronella accessions show that these accessions should be genetically different from each other. however, clearly, the oil extracted from all the selected citronella accessions are of high quality under the physiochemical parameters. results of gc-ms reveal that the main constituents of the oil samples of selected citronella accessions are citronellal, geranyl acetate, citronellol, geraniol, and geranyl iso butyrate. the oil hp t3 has high contents of the main constituents geraniol, citronellal, and citronellol. the highest geraniol contents are available in mp t1 and mp t2 oil samples. the oil of the hp t3 accession has a unique chemical composition compared to the other tested ceylon citronella accessions. all the oil samples have antifungal activity against candida albicans and mp t1 and hp t3 oils have the highest inhibitory action with a minimum inhibitory concentration (mic) of 2% v/v. antidandruff shampoos formulated blending mp t1 and hp t3 oils show antifungal activity against candida albicans with evidence that both tested oils can be effectively used as an antidandruff agent in hair shampoo. also, both shampoo samples are aligned with the quality standards of the organoleptic properties, and physiological properties such as foaming, ph, viscosity, wetting time, dirt dispersion, and solid content (%) which conclude that the use of citronella oil as an ingredient does not affect to change the acceptable levels of physiochemical parameters of shampoo. acknowledgements: this work was supported by national cinnamon research and training center of sri lanka and department of biosystems technology, faculty of technology, university of sri jayewardenepura, sri lanka. the authors wish to thank ms. w.u.m. pramodani and laboratory staff of biotechnology laboratory of faculty of technology, university of sri jayewardenepura for their support. 498 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 authors’ contributions: r.m.n.wijerathna, a.a. wijeweera & m.m.s.t. mapa designed the study. r.m.n.wijerathna, a.a. wijeweera, & a.m. wijethunga carried out the laboratory work. r.m.n.wijerathna, a.a. wijeweera & m.m.s.t. mapa analyzed the data. r.m.n.wijerathna & m.m.s.t. mapa wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that the research was funded by national cinnamon research institute and training center of sri lanka. references al badi, k., & khan, s. a. 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(1981). antimicrobial activity of sorbate. journal of food protection, 44(8), 614–622. https://doi.org/10.4315/0362-028x-44.8.614 biology, medicine, & natural product chemistry volume 5 – number 1 – 2016 issn 2089-6514 contents new record marsdenia tenacissima (asclepiadoideae, apocynaceae) in gunung ijo baturagung yogyakarta widodo, muhammad jafar luthfi 1 8 local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini 9 14 determination of levels leisure village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural village green and environmentally friendly supriatna, thaqibul fikri niyartama, iwan kuswidi 15 18 modified alizarin red s-alcian blue staining for reptilian skeleton muhammad jafar luthfi 19 22 checklist of macroalgae in waisai coast, raja ampat retno suryandari, widodo 23 32 biology, medicine, & natural product chemistry volume 6 – number 1 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) contents antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate dyah ayu widyastuti, praptining rahayu 1 4 analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan 5 8 comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani, muhammad jafar luthfi 9 12 anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro, muhammad jafar luthfi 13 17 checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo, muhammad jafar luthfi 19 36 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 431-435 | doi: 10.14421/biomedich.2023.122.431-435 issn 2540-9328 (online) assessment of sensorimotor behaviour in konzo-induced rats using the irvine, beattie bresnahan forelimb scale 1department of anatomy, faculty of basic medical sciences; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com; phone: +2347035313368 manuscript received: 24 june, 2023. revision accepted: 26 july, 2023. published: 12 august, 2023. abstract konzo is a neurological disorder of selective upper motor neurons. it is an irreversible paralytic disease associated with prolonged consumption of cassava. it contains cyanogenic glycosides metabolized to hydrogen cyanide, which has been shown by studies to affect the motor neurons of the central nervous system. the irvine, beattie bresnahan (ibb) scale is a recently developed forelimb scale for the assessment of fine control of the forelimb and digits after cervical spinal cord injury such as konzo. 20 adult male wistar rats were assigned to 4 experimental groups (i) control n=5, (ii) konzo-induced group n=5, (iii) induced + complan n=5 (iv) induced + bambara nut (okpa). the bitter cassava foods were taken by oral ingestion for a period of 4 weeks. the assessment of the forelimb and digits were done using the irvine, beattie bresnahan (ibb) with specific parameters such as predominant elbow joint movement, contact volar support, and grasping method. the body weight of the animals was also recorded every week. the data obtained were analyzed using anova. the result obtained showed that there was a significant difference (p<0.05) between the body weight of the animals induced with konzo and rehabilitated with complan milk and bambara nut when compared to the unrehabilitated konzo-induced group. there were differences in the results of the parameters being tested for the irvine, beattie bresnahan (ibb) scaling. the ibb scale confirmed that there was a high level of cyanide content in the cassava which affected the behavioral attributes of the induced group and it also confirmed that the induced group can be ameliorated with the use of complan and bambara nut (okpa) which was shown in the parameters being tested such as predominant elbow joint movement, contact volar support, and grasping method. it was concluded that insufficiently processed bitter cassava is toxic and has neurotoxicity effects on the spinal cord especially on the upper motor neurons and ibb scale is capable of measuring gradual improvements in motor forelimb functions in this model and may be a new and effective assessment tool for peripheral nerve injury. keywords: bambara nut; cyanogenic; neurotoxicity; konzo; bitter cassava; irvine, beattie bresnahan forelimb scale. introduction cassava toxicity has been incriminated in the pathogenesis of tropical myeloneuropathies, such as tropical ataxic neuropathy (tan) and konzo. tan is a progressive myeloneuropathy that was first described in nigeria and is characterized by a progressive onset of ataxia. on the other hand, konzo is a permanent and clinically distinct upper motor neuron disease of abrupt onset first described in the democratic republic of congo (drc). the word “konzo” means tied legs and originated from the yaka tribe in drc to designate a fetish used by hunters to weaken legs and catch wild animals (trolli, 1938; van der beken, 1993). konzo occurs mainly in children and young women of childbearing age. the paralysis occurs quite suddenly, does not progress over time, and is irreversible. it is associated with the consumption of a monotonous diet of high cyanide (bitter) cassava, by poor rural people in africa, many of whom suffer from malnutrition. specifically, konzo is associated with a high cyanide diet of bitter cassava consumed over a period of several weeks combined with a low intake of protein, particularly a shortfall of essential s-containing amino acids that are needed to detoxify cyanide to thiocyanate in the body (howlet et al, 1990). the exact pathogenetic mechanisms of the disease remain unknown. epidemiological studies consistently show an association between outbreaks of the disease and chronic dietary reliance on insufficiently processed cyanogenic cassava (manioc or tapioca). biochemical and toxicological studies suggest that the metabolites of linamarin (α-hydroxyisobutyronitrile β-dglucopyranoside, the main cassava cyanogen), notably cyanide (mitochondrial toxin), thiocyanate (ampa chaotropic agent), and cyanate (protein carbamoylating agent) may play an important role in the pathogenesis of konzo. experimental data suggest that thiol-redox and protein-folding mechanisms may also be perturbed. factors of susceptibility including genetics, poor lekpa kingdom david1, precious ojo uahomo2,*, victor hogan idung2, rachael data dakoru2 https://doi.org/10.14421/biomedich.2023.122.431-435 432 biology, medicine, & natural product chemistry 12 (2), 2023: 431-435 nutrition, poverty, and dietary cyanogen exposure, or their interactions have been suggested. the irvine, beattie bresnahan (ibb) scale is a recently developed forelimb scale for the assessment of fine control of the forelimb and digits after cervical spinal cord injury (sci). several experimental models of cervical spinal cord injury (sci) have been developed recently to assess the consequences of damage to this level of the spinal cord (pearse et al., 2005; gensel et al., 2006; anderson et al., 2009), as the majority of human sci occur here. behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. this is the basis for the study; hence, this study aims to assess the sensorimotor behaviour in a konzo-induced rat using the irvine, beattie bresnahan forelimb scale. methodology animal procurement twenty (20) wister rats weighing between 150-200g were acquired from the animal house of the department of pharmacology, faculty of basic clinical sciences, university of port harcourt for this research work. the animals had free access to water and were fed ad libitum with standard feed (broiler finisher – guinean feed). two weeks before the start of the test, the animals were acclimated. plant collection and identification the cassava plants were gotten at the ministry of agricultural development program, rumuodomaya, port harcourt, rivers state, and were identified by the agriculture department of the university of port harcourt, choba, rivers state. processing of bitter cassava root fresh cassava roots were uprooted from the farm, and immediately after harvesting the outer layer (skin or cortex) was removed with a knife to expose the white inner layer. then the roots were sliced into smaller sizes like chips and allowed to sundry for 3 consecutive days. the dry cassava pieces were manually grinded into powder form using a grinding machine and served to the experimental animal as cassava chow. induction of konzo disease in rats after two weeks of acclimatization, 15 rats were fed with the processed bitter cassava flour constantly to induce the konzo disease and its effects. the oral feeding method used represents the real scenario of food being consumed by ingesting through the mouth, passing through the esophagus into the stomach following the alimentary canal, contacting the necessary visceral organs along with their associated fluids such as saliva in the mouth and gastric juices including any contributions provided via sublingual or buccal absorption during digestion. experimental design the experimental method as described by david et al. (2022) was used in this study. twenty (20) albino wistar rats were used for this study. out of the 20 rats, 15 were induced with konzo disease for two weeks and then randomly selected into three (3) groups of five (5) animals each (groups 2, 3, and 4), while the remaining 5 rats without konzo disease served as control (group 1). rehabilitation group: after the period of konzo disease induction, group 3 and group 4 were completely stopped from consuming the bitter cassava and replaced with rat feed and complan milk for group 3 and bambara nut (okpa) for group 4. the mode of feeding was by oral ingestion. table 1. experimental design. group identity n o . o f r a ts treatment group 1 control 5 2ml normal saline group 2 induced control 5 bitter cassava only group 3 complan 5 bitter cassava + complan milk group 4 bambara nut 5 bitter cassava + bambara nut determination of body weight the animals were weighed daily with an electric weighing scale (sf-400c) and the body weight of the rats was recorded. sensorimotor behaviour assessment the forelimb function was assessed using a special method, which is ibb (irvine, beattis and bresnahan test). the procedure is as explained; rats were given pieces of cereal in their home cage twice daily beginning as soon as they entered the lab. forelimb function was assessed while rats were eating cereal as described previously (irvine et al., 2010). briefly, rats were individually placed in a plexiglas cylinder (diameter = 20 cm; height = 46 cm) or in their home cage and they were given donut-shaped pieces of cereal that were of a consistent size and shape prior to the initiation of eating. rats were not scored when eating cereal pieces that were broken prior to the initiation of testing. each trial was recorded to allow slow-motion hd playback and evaluation of forelimb use. videos of animals eating the cereal were evaluated using a standardized scoring sheet to record observations of forelimb behaviors, including joint position, object support, wrist and digit movement, and grasping method used while consuming both cereal shapes. david et al. – assessment of sensorimotor behaviour in konzo-induced rats … 433 cyanide analysis the samples for analysis of the cyanide in the bitter cassava were prepared as follows: ▪ 10g of the sample was mixed with 50ml of water in a corked conical flask. ▪ allowed to stand for 24hrs to extract the residual cyanoglucosides in the samples. ▪ the mixture was subsequently filtered to obtain the soluble extract containing cyanoglucosides ▪ the same procedure as with standard kcn solutions was followed to determine the free cyanide concentration (as hcn equivalent) in the sample filtrate. ▪ the absorbance of the sample solution was equally measured at 510nm wavelength against a blank devoid of kcn solution. ▪ cyanide levels of the test samples were evaluated from the standard calibration curve by extrapolation. ethical clearance the experimental animals were acquired from the animal house of the department of pharmacology in the faculty of basic medical sciences. all procedures carried out during this research were done in accordance with the guiding principles of research involving animals as recommended by the research ethics committee of the university of port harcourt. animals were kept in standard metal cages and at room temperature. statistical analysis data were analyzed using statistical package for the social sciences (spss ibm version 23.0) and microsoft excel 2019 edition. values were expressed as mean ± sd in descriptive statistics. one-way analysis of variance (anova) was used to analyze the difference between the groups followed by the least significant difference (lsd) post-hoc test. the confidence interval was set at 95%, and therefore p<0.05 was considered significant. results table 2. effect of konzo disease, complan milk and bambara nut (okpa) on the body weight (grams) of wistar rats. group baseline weight week 1 week 2 week 3 week 4 mean weight group 1 263.01±5.13 263.00±5.87 265.02±6.10 260.10±6.32 273.05±6.75 265.25±2.78b group 2 242.30±5.17 175.70±5.16 181.10±4.80 171.80±5.15 172.40±5.90 175.25±2.13a group 3 175.25±4.87 194.10±4.21 192.50±4.11 223.10±8.23 230.10±8.40 209.95±9.72a,b group 4 175.25±5.10 188.30±5.87 190.05±7.02 200.60±7.50 208.90±7.11 196.95±4.82a,b the results are expressed as mean ± sem. level of significance values are ap<0.05 when compared with group 1 (control), bp<0.05 when compared with group 2 (konzo induced). figure 1. comparison of baseline body weight with final mean body weight of wistar rats. table 3. irvine, beatties and bresnahan forelimb scale. group pejp cvs gm group 1 (control) flexed none normal group 2 (induced) partially flexed nearly always abnormal group 3 (induced + complan) flexed some normal group 4 (induced + bambara nut) flexed some normal keys: pejp = predominant elbow joint position; cvs = contact volar support; gm= grasping method 0 50 100 150 200 250 300 baseline body weight mean body weight b o d y w e ig h t (g ) duration (weeks) body weight (g) control konzo induced konzo + complan konzo + bambara nut (okpa) 334 biology, medicine, & natural product chemistry 12 (2), 2023: 431-435 figure 2. contact volar support: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut figure 3. predominant elbow joint movement: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut figure 4. grasping method: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut discussion the effect of bitter cassava has been studied extensively and its neurotoxicity was being studied with the use of experimental animal models to evaluate the effects of diverse substances on motor function and their relationship with cns disorders. (gerhart et al., 1982; kulig et al., 1985; fowler et al., 1990). the weight of the rats was taken and measured accurately from the beginning of the week till the end of the experiment at a significant value of p<0.05. it was observed that there was a significant difference between the mean body weight of the control group 265.25±2.78g and the mean body weight of the induced group (175.25±2.13g). the difference between the weight of the induced was different from that of the induced as a result of the intake of the bitter cassava which has caused a neurotoxic effect on the experimental models. this agrees with the findings of david et al. (2022) and enefa et al. (2020). some nutritional approaches were used to ameliorate the effect of the bitter cassava for a period of 4 weeks after the initial four weeks of being induced with konzo disease. the nutritional approach used was complan and bambara nut (okpa). there was a significant difference between the mean body weight of the ameliorated group and that of the induced. the complan group had a mean body weight of 209.95±9.72g while the bambara nut group has a mean body weight of 196.95±4.82g. this shows that the effect of the bitter cassava on the experimental models was ameliorated as there was a significant increase in the weight of the induced group after being fed with complan and bambara nut (okpa). this is also similar to the findings of david et al. (2022) and enefa et al. (2020). the ibb forelimb scale provided a sensitive measurement scale for detecting recovery of both proximal and distal forelimb function, including digit movements, during a naturally occurring behavior. this behavioral test was developed and tested in rats with spinal cord injury by irvine et al. (2010). this method evaluated the overall integrity of rodent forelimb functionality during a food intake task, measuring how rats grasp a cereal piece. the ibb scale was used to test just 3 parameters which include predominant elbow joint movement, contact volar support, and grasping method. during the assessment of contact volar support, the scale was rated as ‘none’ ‘some’, and ‘almost always’. the rat was assessed for its ability to use the volar (palmar) surface of the impaired forepaw to stabilize the cereal and, in doing so, maintain a position to aid eating. in figure 2, group a being the control group, no volar support by the forelimb during eating (<5% of the time). but in group 2, being the induced group that was affected by the cyanide content of the bitter cassava, the volar support of the object occurred nearly always or always during eating (>95% of the time). this is because the spinal cord had been affected which in turn relates to david et al. – assessment of sensorimotor behaviour in konzo-induced rats … 435 how the rats had to use their volar for support. the ameliorated group which is group 3 (complan) and group 4 (bambara nut) had the support of the volar but did not occur always. this shows that the konzo disease was ameliorated which helped to reduce the use of volar for support in the case of the induced group. the original scale rated the predominant elbow joint position as “extended, partially flexed, or fully flexed.” discrimination between partially and fully flexed appeared to be problematic, and perhaps irrelevant in more recovered animals. looking at figure 3, the rat models were assessed for the most common position (more than 50% of the time). the elbow joint movement for group a was found to be flexed. group b was partially flexed as a result of the cyanide content of the bitter cassava affecting the spinal cord. after the induced were ameliorated, the elbow joint movement was flexed fully. the grasping method was rated to be ‘normal’ and ‘abnormal’ according to figure 4, the experimental rat models were assessed for the most common (more than 50% of the time) grasping technique used during the eating phase. the rat models of group 1 were found to be normal with consistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase. group 2 being induced was abnormal because they consistently used an alternative method of grasping to the method used prior to the injury to support and control the cereal piece during the eating phase. they were able to support their grasping with their volar. this is a result of the spinal cord injury that occurred as a result of the intake of bitter cassava which had a high percentage of cyanide content. the ameliorated group was found to be normal in their grasping method. according to speck et al. (2014), the ibb scale is capable of measuring gradual improvements in motor forelimb functions in this model and may be a new and effective assessment tool for peripheral nerve injury. this was seen in this study as the induced group was being ameliorated with complan and bambara nut and there was gradual improvement in the motor forelimb function using the three parameters, predominant elbow joint movement, contact volar support, grasping method. conclusion this study has shown that insufficiently processed bitter cassava is toxic and has neurotoxicity effects on the spinal cord especially on the upper motor neurons. a balanced diet with sufficient nutrients such as complan and bambara nut (okpa), can ameliorate the effects of konzo disease as there was no effect on motor neurons of the spinal cord of the animals rehabilitated with complan and bambara nut (okpa). competing interests: the authors declare that there are no competing interests. references anderson k., sharp k., hofstadter m., irvine k., murray m., steward o. (2009). forelimb locomotor assessment scale (flas): novel assessment of forelimb dysfunction after cervical spinal cord injury. exp. neurol., 220:23–33. enefa s., chikwuogwo w.p, lekpa k.d. (2020). model of konzo disease: reviewing the effect of bitter cassava neurotoxicity on the motor neurons of cassava-induced konzo disease on wistar rats. saudi journal of medicine; 5(11):336-348. fowler s.c., liao r.m., skjoldager p. (1990). a new rodent model for neuroleptic-induced pseudo-parkinsonism: low doses of haloperidol increase forelimb tremor in the rat. behav. neurosci., 104:449–456. gensel, j.c., tovar, c.a., hamers, f.p.t., deibert, r.j., beattie, m.s., bresnahan, j.c. (2006). behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. j neurotrauma, 23:36–54 gerhart j.m., hong j.s., uphouse l.l., tilson h.a. (1982). chlordecone-induced tremor: quantification and pharmacological analysis. toxicol. appl. pharmacol., 66:234– 243. howlett, w. p, brubaker, g. r., mlingi, n., rosling, h. (1990). konzo, an epidemic upper motor neuron disease studied in tanzania. brain, 113:223–235. kulig b.m., vanwersch r.a.p., wolthuis o.l. (1985). the automated analysis of coordinated hindlimb movement in rats during acute and prolonged exposure to toxic agents. toxicol. appl. pharmacol., 80:1–10. david l.k, idung v.h and uahomo p.o (2022). neurobehavioral and ameliorative effect of complan milk and bambara nut on rats fed with bitter cassava – a nutritional approach. international neuropsychiatric disease journal, 17(1): 7-17 pearse, d., lo t jr, cho, k., lynch, m., garg, m., marcillo, a et al. (2005). histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat. j neurotrauma, 22:680–702. speck a. e., jocemar i., caroline c. s., aguiar a. s. jr., santos a. r., swarowsky a., (2014). the ibb forelimb scale as a tool to assess functional recovery after peripheral nerve injury in mice. journal of neuroscience methods, 226:66–72. trolli, g, (1938). paraplégie spastique épidémique, “konzo” des indigènes du kwango. in: trolli g (ed.), résumé des observations réunies, au kwango, au sujet de deux affections d´órigine indéterminée fonds reine elisabeth, brussels, pp. 1– 36. van der beken, a., (1993). in: karthala (ed 1.), proverbes yaka du zaire harmattan, paris this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 1-4 | doi: 10.14421/biomedich.2017.61.1-4 issn 2540-9328 (online) antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate dyah ayu widyastuti1, praptining rahayu2 1,2biological education, fpmipati universitas pgri semarang, jl. lontar no. 1 phone (024) 8316377 semarang 50125 author correspondency: wid.dyah@gmail.com1; ningbiologi@gmail.com2 abstract annona muricata linn. (soursop) is one of tropical plants which have relatively complete chemical compounds. it has flavonoid, tannin, phytosterol, alkaloid, etc. the high antioxidant compound in soursop is believed as cancer prevention so the cancer threat in the world can be minimized. the antioxidant compound in soursop can be found not only in its fruit, but also in other parts like leaves, seeds, etc. based on that potency, this study aimed to compare antioxidant capacity of soursop leaves and seeds, also to study about the utilization of soursop parts which is usually not used. this research began with maceration to extract leaves and seeds with 96% ethanol. ethanolic extract of soursop leaves and seeds were then tested for antioxidant capacity with dpph (1,1-diphenyl-2-picrylhydrazyl) method. the result showed that antioxidant capacity of soursop leaves and seeds are 85,66875% and 39,0166, respectively. the antioxidant capacity of leaves is higher than seeds due to seed’s extraction difficulty so its antioxidant compound could not be extracted optimally. however, either leaves or seeds extract in this study are potential as antioxidant resources because there are no significant differences between antioxidant capacity of both extract. keywords: annona muricata linn.; antioxidant capacity; dpph; extraction. introduction cancer is the most dangerous killer in the world nowadays. lung cancer cause 1,3 millions of death a year, stomach cancer take the second place with 803 thousands of death a year, then colorectal cancer with 639 thousands of death a year, and breast cancer with 519 thousands of death a year. more than 70% death number were happen in developing countries with low health facilities (who, 2009). cancer can be triggered by accumulation of free radicals compounds. actually, our body needs free radicals in normal amount to prevent inflammation, kill pathogen microbes, and restrain blood vessel’s smooth muscle. the problem is free radicals will be carsinogen in large amount so it can trigger the growth of cancer cells. due to its importance, we need antioxidant to regulate the existence of free radicals inside our body, so its amount can be arranged (limbono, 2013). antioxidant can play a role to supply hydrogen radicals and as free radicals acceptor so its postpone initiation of free radicals forming (puspitasari et al., 2016). antioxidant can be obtained either natural or artificial. synthetic antioxidant are highly effective but does not safe enough to be consumed. based on that fact, the synthetic antioxidant utilizing has to be checked. natural antioxidant has better effect than artificial one. these antioxidant can naturally obtained by consuming fruits and vegetables because both of them contain natural antioxidative effect due to vitamin c, e, betacaroten, and other polyphenolic substances (nihlati et al., 2014). based on research by puspitasari et al. (2016), one of the most valuable fruits which regularly extracted as natural supplement is soursop (annona muricata linn.). it has known that soursop has flavonoid, tannin, phytosterol, and alkaloid which can be play a role as an antioxidant. regularly, soursop consumption limited on its fruit. people only consume the water extract of soursop as cancer prevention. on the other hand, the utilization of leaves and seeds extract of soursop has not been studied yet. both of them has to be compared to determine which one has higher antioxidant capacity. the objective of this research is to find either leaves or seeds of sousop (a. muricata linn.) which has higher antioxidant capacity when extracted with ethanol. so, the utilization of soursop (a. muricata linn.) will be more appropriate. materials and methods this research needs soursop (a. muricata linn.) leaves and seeds, 96% ethanol, hcl, dpph (1,1-diphenil-2http://dx.doi.org/10.14421/biomedich.2017.61.1-4 2 biology, medicine, & natural product chemistry 6 (1), 2017: 1-4 picrylhydrazyl), aquades, h2so4, naoh, mortar and pestle, grinder, rotary evaporator, glassware, analytical balance, sentrifuge, and uv-vis spectrophotometer. figure 1. soursop (a. muricata linn.) leaves. figure 2. soursop (a. muricata linn.) seeds. the dried leaves (figure 1) and seeds (figure 2) were extracted with 96% ethanol by maceration of each 800 gr of leaves and seeds. the whole filtrate was concentrated with rotary evaporator until 1/3 volume remain. the ethanol residues was then be evaporated until concentrated extract was formed with constant weight. the antioxidant capacity of leaves and seeds extract were then measured by dpph test. samples were divide into two groups, leaves extract (l) and seeds extract (s). twenty five mg leaves (l) and seeds (s) extract was measure then dissolved to 25 ml ethanol (stock solution). both l and s stock solution were pipetted 0,1 ml; 0,2 ml; 0,3 ml; and 0,4 ml. so the concentration was 4 ppm; 8 ppm; 12 ppm; and 16 ppm, respectively. five ml of 0,5 mm dpph was added to each solution then its volume was adjusted. the standard solution was made by added 25 ml ethanol to 0,5 mm dpph. the dpph absorbance was measure with uv-vis spectrophotometer in 515 nm wavelength. antioxidant capacity was measured as decreasing of dpph absorbance due to sample addition. the mean of both leaves (l) and seeds (s) antioxidant capacity were then calculated. analysis using non-parametric of two independent samples of mann-whitney test with α 0,05 were done to know the antioxidant capacity of each extract. results and discussion the result of this research showed that antioxidant capacity of soursop (a. muricata linn.) leaves (l) and seeds (s) of ethanolic extract by dpph test have different level. leaves (l) extract showed higher antioxidant capacity than seeds (s) extract (table 1). based on the table 1, the antioxidant capacity of soursop leaves (l) ethanolic extract is 85,67 %. on the other hand, the antioxidant capacity of soursop seeds (s) ethanolic extract is only 39.01 %. the antioxidant capacity play an important role as health protector. this capacity will prevent free radicals so its side effect can be minimalized (shekhar and anju, 2014). due to the advantage, the higher antioxidant capacity will improve the samples potency to be used as protector for some common free radical threat. those result showed that soursop leaves (l) of ethanolic extract has higher potential to obstruct side effect of free radicals as one of cancer growth trigger compared with seeds (s) extract. antioxidant capacity of soursop leaves (l) and seeds (s) ethanolic extract were proved that both of them have ability to decrease the risk of chronic diseases caused by free radicals, include cancer and heart attack. table 1. antioxidant capacity of soursop leaves (l) and seeds (s) ethanolic extract with dpph test. no. samples antioxidant capacity (%) 1. l1 85,99 2. l2 85,35 leaves extract 85.67 3. s1 39,18 4. s2 38,85 seeds extract 39.01 the common antioxidant sources are vitamin c, vitamin e, carotene, and phenolic acid which can be find inside seeds, fruits, and vegetables (shekhar and anju, 2014). those facts strengthen the result of this study widyastuti & rahayu – antioxidant capacity comparison of ethanolic extract of soursop … 3 which said that either soursop leaves (l) or seeds (s) ethanolic extract have potential factor to be an antioxidant sources naturally. soursop (a. muricata linn.) has high vitamin c and polyphenolic compound. phenol and flavonoid which contained in soursop also play a significant role as antioxidant due to molecular structure which give its electron to free radicals molecules. the higher level of phenol and flavonoid compound, the higher antioxidant capacity it have (prasetyorini et al., 2014). reactive oxygen species (ros) and reactive nitrogen species (nos) are free radicals which are formed by either metabolic pathway or external resources. actually, free radical is important substance for energy sources, detoxification, chemical signal, and immunity function. but, the excessive production of free radicals cause biomolecule damage inside cell (such as dna, lipid, protein, carbohydrate), instead of protect them (akomolafe & ajayi, 2015). free radical is also frequency associated with oxidative reaction inside the food and biological system which cause oxidative damage and trigger some human disease, such as neural degeneration, diabetes, and some type of cancer. the danger of free radical discussed above trigger higher research of antioxidant resources which can be an alternative prevention of oxidative damage. chew et al. (2011) said that there are many plant species with bioactive compound which potential as antioxidant resources, including soursop (a. muricata linn.). almost all parts of soursop plant have potency as antioxidant sources although each part has different capacity level. this days, antioxidant capacity of soursop plant often reported to be used as potential anticancer compound. nowadays, usage of soursop plant still limited on its leaves. whereas, antioxidant compound is also find in other parts of soursop plant, such as seeds which not exploited yet. to know about its antioxidant capacity, it can be measured by dpph (diphenylpycryl-hydrazyl) test. extraction process has to be done before dpph test proceed. in pharmaceutical field, extraction use to prepare natural medicine by separate active compound from plant tissues use selective solution and persistent standard procedures. ethanol was used in this research to extract soursop leaves (l) and seeds (s). ethanol is common solvent use at extraction due to its safety. ethanol has characteristic as polar solution so it can solve with water. in this research, maceration was used as extraction method. maceration is extraction process use solution with repeated mixture at room temperature. maceration was used due to its simple process and not time consuming. ninety six persen ethanol was used, so the evaporation of ethanol residue can be faster. after maceration process, the leaves and seeds extract was then measured with dpph test to know about its antioxidant capacity. dpph (1,1-diphenil-2-picrylhydrazyl) is a method that quick, simple, and sensitive to measure antioxidant capacity. it only needs a little sample so we do not have to prepare sample in a large amount. principal characteristic of this test is to measured antioxidant capacity by analyze the changes of dpph color intensity. dpph as free radical have free electron which visualize violet gradation colour. the violet colour change to yellow when the electron paired. decreasing of free radical charges will change the intensity of violet colour. the changes caused by reaction between dpph crystal molecules with hydrogen which release from soursop leaves and seeds ethanolic extract chemical compound. that bond form 2,2-dyphenyl-1picrylhydrazine and cause colour changes from violet to yellow (fathurrachman, 2014). based on the result of this study, leaves (l) extract has higher antioxidant capacity than seeds extract, that is 85,67% and 39,01%, respectively. the leaves (l) extract antioxidant capacity affirm the result of another research by putri (2012) which conclude that 500 μg/ml soursop leaves ethanolic extract showed antioxidant capacity to free radical such as 1,1-diphenyl-2-picrylhydrazyl in the amount of 88,77%. high level of antioxidant capacity which showed by soursop leaves (l) ethanolic extract may potentially be used as natural product for cancer prevention. lower antioxidant capacity on soursop seeds (s) ethanolic extract was caused by its difficult extraction compare with leaves extraction. soursop seeds contain alkaloid compound that is acetogenin and annonaine (idrus et al., 2012). those compounds mention before have potential character as antioxidant. yet, the seeds antioxidant capacity lower than leaves due to its difficult handling and extraction. the extraction difficulties was caused by its hard and rough structure with unbreakable outer layer. acetogenin of soursop seeds (annonaceous acetogenin) has to be examined and proved its characteristic as anticancer, antiparasite, insecticide, antiworm, antibiotic, and antivirus (arifianti et al., 2014). that acetogenin play a role as barrier and killer to cancer cells selectively cause it can detect and discriminate between normal and cancer cells due to atp (adenosine triphosphate) necessity of the cell. cancerous cells grow rapidly so it need bigger amount of atp than normal cells. antioxidant compound of leaves or seeds of soursop (a. muricata linn.) can be used to avoid free radicals so the cancer cell growth can also be prevented. however, the antioxidant capacity in leaves (l) ethanolic extract relatively higher than seeds (s). nonparametric mann-whitney analysis use to know if there is significant differences between antioxidant capacity of leaves (l) and seeds (s). the analysis result showed at table 2. 4 biology, medicine, & natural product chemistry 6 (1), 2017: 1-4 table 2. mann-whitney analysis. test statisticb antioxidant capacity mann-whitney u .000 wilcoxon w 3.000 z -1.549 asymp. sig. (2-tailed) .121 exact sig. [2*(1-tailed sig.)] .333a a. not corrected for ties. b. grouping variable: extract table 2 showed that asymp. sig. (2-tailed) value is 0,121. this value determine if there is significant differences between two samples. if asymp. sig. (2tailed) value is higher than significancy value (α), so there are no significant differences between two samples. on the other hand, if asimp. sig. (2-tailed) value is lower than significant value (α), so there are significant differences between two samples (trihendradi, 2007). asymp. sig. (2-tailed) value in this research is 0,121 (table 2). this number showed there are no significant differences between antioxidant capacity of leaves (l) and seeds (s) ethanolic extract. the values indicate that either soursop leaves (l) or seeds (s) ethanolic extract can be resources of natural antioxidant compound which potential to be an anticancer candidate compound. to improve that potency, it needs simple, quick, and low cost extraction method so that both leaves (l) and seeds (s) can be extracted well. the extraction method between soursop leaves (l) and seeds (s) might be different due to those different shape and texture. conclusion ethanolic extract of soursop (a. muricata linn.) leaves has higher antioxidant capacity than seeds that is 85,67% and 39,01%, respectively. however, there are no significant differences of the antioxidant capacity between those two samples. the higher antioxidant capacity of soursop leaves affirm its potency to be a natural anticancer candidate compound. the lower antioxidant capacity of soursop seeds may be caused by its extraction difficulties so the compound cannot be fully extracted. acknowledgement the authors would like to thank all the participants and partner of this research including biology laboratory technician of universitas pgri semarang and others whom cannot mention each. references akomolafe s. f. & o. b. ajayi. 2015. a comparative study on antioxidant properties, proximate and mineral compositions of the peel and pulp of ripe annona muricata (l.) fruit. international food research journal 22 (6): 2381-2388. arifianti, l., sukardiman, h. studiawan, rakhmawati, & l. megawati. 2014. soursop (annona muricata l.) seeds extract activity test to mammalian cancer cells in vitro. jurnal farmasi dan ilmu kefarmasian indonesia. 1 (2): 63-66. chew, k. k., s. y. ng, y. y. thoo, m. z. khoo, and w. m. a. wan. 2011. effect of ethanol concentration, extraction time and extraction temperature on the recovery of phenolic compounds and antioxidant capacity of centella asiatica extracts. international food research journal 18: 571-578. fathurrachman, d. a. 2014. the effect of solvent concentration to antioxidant capacity of soursop (annona muricata linn.) leaves ethanolic extract with dpph method. medical faculty. uin syarif hidayatullah. jakarta. idrus, r. b., n. bialangi, l. alio. 2012. isolation and characterization of alkaloid compound from soursop (annona muricata linn.) seeds. chemistry education science faculty. universitas negeri gorontalo. limbono, s. 2013. antioxidant capacity of canarium nut (canarium indicum l.) seeds ethanolic extract with dpph (1,1-diphenyl-2-picrylhydrazyl) method. jurnal ilmiah mahasiswa universitas surabaya 2 (2). nihlati, i., a. rohman, & t. hertiani. 2014. antioxidant capacity of ethanolic extract of boesenbergia pandurata (roxb.) schlecth. with dpph (1,1-difenil-2-pikrilhidrazil) method. pharmacy faculty. universitas gadjah mada. yogyakarta. prasetyorini, moerfiah, s. wardatun, & z. rusli. 2014. antioxidant potency of some parts of soursop annona muricata linn.). penel. gizi makan 37 (2): 137-144. puspitasari, m. l., t. v. wulansari, t. d. widyaningsih, j. m. maligan, dan n. i. p. nugrahini. 2016. antioxidant capacity of soursop (annona muricata l.) leaves supplement and mangosteen (garcinia mangostana l.) fruit skin: references study. jurnal pangan dan agroindustri (4) 1: 283-290. shekhar, t. c. & g. anju. 2014. antioxidant activity by dpph radical scavenging method of ageratum conyzoides linn. leaves. american journal of ethnomedicine. 1 (4): 244-249. trihendradi, c. 2007. an easy way to use spss 15 for descriptive, parametric, and nonparametric statistic. penerbit andi. yogyakarta, pp: 127-129. who. 2009. cancers: the problem. http://www.who.int/nmh/publications/fact_sheet_cancers_en. pdf. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 477-484 | doi: 10.14421/biomedich.2023.122.477-484 issn 2540-9328 (online) nephroprotective activities of ethanol root extract and fractions of hippocratea africana against doxorubicin-induced kidney toxicity kufre u. noah1, john a. udobang2, jude e. okokon1,*, martin o. anagboso3, nwakaego omonigho ebong4 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. 3department o microbiology, madonna university, elele, nigeria. 4department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, nigeria. corresponding author* judeefiom@yahoo.com, tel.no. +234-8023453678 abstract hippocratea africana root used locally in the treatment of poisoning was investigated to confirm its antidotal potential in rats. the root extract (200-600 mg/kg) and fractions; dichloromethane (dcm) and aqueous, 400 mg/kg) were evaluated for nephroprotective activity against doxorubicin-induced kidney injury in rats. kidney function parameters, kidney oxidative stress markers and kidney histology were used to assess the kidney protective effect of the extract. the root extract and fractions (200-600 mg/kg) significantly (p<0.05-0.01) reduced the levels of creatinine, urea and electrolytes that were elevated by doxorubicin. also, the mda level elevated by doxorubicin was reduced by the extract and fractions co-administration, while the levels of gsh, gst, sod, gpx, and cat that were decreased by doxorubicin were significantly (p<0.01) elevated by the root extract/fractions. histology of the kidney sections of extract/fractions treated animals showed reductions in the pathological features compared to the organotoxic-treated animals. the chemical pathological changes were consistent with histopathological observations suggesting marked nephroprotective potential. the anti-toxic effect of this plant may in part be mediated through the chemical constituents of the plant. the plant, hippocratea africana possesses anti-toxicant properties which can be exploited in the treatment of doxorubicin related toxicities. keywords: renoprotective; hippocratea africana; doxorubicin; oxidative stress. abbreviations: dichloromethane (dcm), superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), reduced glutathione (gsh), malondialdehyde (mda), haematotoxylin and eosin (h&e), reactive oxygen species (ros), reduced nicotinamide adenine dinucleotide phosphate (nadph). introduction hippocratea africana (willd.) loes. ex engl. (celastraceae) known in english as ‘african paddle-pod’ and ‘eba enang enang’ in ibibio language in nigeria, is a climber perennial plant distributed widely in tropical africa (hutchison and dalziel, 1973). traditionally, the plant root has been variously utilized in herbal preparations to treat diseases like malaria and diabetes (okokon et al., 2006), as well as liver diseases (ajibesin et al., 2008). previous reports showed that the root extract possess antimalarial (okokon et al., 2006; okokon et al., 2021), antioedema and antinociceptive (okokon et al., 2008), antidiabetic and hypolipidemic (okokon et al., 2010; 2022), antidiarrhoeal and antiulcer (okokon et al., 2011), hepatoprotective (okokon et al., 2013a), antileishmanial, cytotoxicity and cellular antioxidant (okokon et al., 2013b), antibacterial, anticonvulsant and depressant (okokon et al., 2014). also, earlier studies had reported the presence of ᵟ-3carene and α-terpineol (okokon et al., 2017), isolation of 1,3,7-trihydroxy-6-methoxyxanthone [isoathyriol] and 1,3,6,7-tetrahydroxyxanthone [norathyriol] (umoh et al., 2021) from ethyl acetate fraction. monoterpenes and sesquiterpenes have been identified in the n-hexane fraction (okokon et al., 2013a). we report nephroprotective and antioxidative stress effects of the root extract and fractions of h. africana against doxorubicin-induced nephrotoxicity in rats. materials and methods plants collection fresh roots of hippocratea africana were collected in bushes in uruan area, akwa ibom state, nigeria in november, 2021. the plant was identified and authenticated by a taxonomist in the department of manuscript received: 28 june, 2023. revision accepted: 17 august, 2023. published: 23 august, 2023. https://doi.org/10.14421/biomedich.2023.122.477-484 478 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 botany and ecological studies, university of uyo, uyo, nigeria. herbarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo. preparation of extract and fractions fresh root of h. africana were washed, cut into smaller pieces and dried under shade for two weeks. they were powdered using electric grinder. the pulverised root of h. africana (hae) was soaked in ethanol (50%) for 72 hours. the liquid filtrate obtained was concentrated in a rotary evaporator at 40˚c. the crude extract (20 g) was dissolved in 500 ml of distilled water and partitioned with equal volume of dichloromethane (dcm, 5 x 500 ml) till no colour change was observed, to obtain dcm and aqueous fractions. the extract and fractions were stored at 4˚c in a refrigerator until used for the experiment. animals in this study, male albino wistar rats were used. the animals were sourced from university of uyo animal house and sheltered in plastic cages. the rats were fed with pelleted standard feed (guinea feed) and given unlimited access to water. the study was approved by college of health sciences animal ethics committee, university of uyo. experimental design in this study, repeated dose model earlier described by raskovi et al. (2011) and olorundare et al., (2020), which lasted for 14 days was used. groups i rats which served as the untreated control were orally pretreated with 10 ml/kg/day of distilled water. group 2 rats were given normal saline (10 ml/kg/day) but equally treated on alternate days with 1.66 mg/kg of doxorubicin hydrochloride dissolved in 0.9% normal saline administered on alternate days for 14 days. groups’ 3-5 rats were orally pretreated with 200 mg/kg/day, 400 mg/kg/day, and 600 mg/kg/day of hippocratea africana dissolved in distilled water 2 hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days, respectively. groups 6 and 7 were pretreated with 400 mg/kg of dcm and aqueous fractions respectively. group 8 rats which served as the positive control group were equally pretreated with 100 mg/kg/day of silymarin two hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days. collection of blood samples and organs after 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. blood samples were collected by cardiac puncture and used immediately. blood samples were collected into plain centrifuge tubes. the blood in the centrifuge tubes were centrifuged at 1500 rpm for 15 minutes to separate of serum at room temperature used for biochemical assays. the rats’ kidneys were identified, harvested, and weighed. kidney function test the following biochemical parameters such as levels of electrolytes (na, k, cl, and hco3), creatinine and blood urea were assayed as markers of kidney function using diagnostic kits at the chemical pathology department of university of uyo teaching hospital. oxidative stress markers the antioxidant enzymes assays were performed on kidney homogenates of rats that were used in this study. these oxidative stress markers were used to assess antioxidative stress potentials of the extract. preparation of renal homogenate in each rat, the kidneys were removed and one kidney was fixed in 10% formaldehyde for histological processes, while the other kidney was dissected free from the surrounding fat and connective tissue and used for assays of oxidative markers. the kidneys were longitudinally sectioned, and renal cortex was separated and kept at -8°c. subsequently, renal cortex was homogenized in cold potassium phosphate buffer (0.05m, ph 7.4). the renal cortical homogenates were centrifuged at 5000 rpm for 10 min at 4°c. the resulting supernatant was used for the determination of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha, 1972), glutathione peroxidase (gpx) (lawrence and burk, 1976), reduced glutathione (gsh) (ellman, 1959) and malondialdehyde (mda) content (esterbauer and cheeseman, 1990). histopathological studies the kidneys of the animals that were surgically removed and fixed in 10% formaldehyde were processed and stained with haematotoxylin and eosin (h&e) (drury and wallington, 1980), according to standard procedures at department of chemical pathology, university of port harcourt teaching hospital, port harcourt. morphological changes observed and recorded in the excised organs of the sacrificed animals. histologic pictures were taken as micrographs. statistical analysis and data evaluation data obtained from this work were analysed statistically using anova (one –way) followed by a post test (tukey-kramer multiple comparison test). differences between means were considered significant at 5% level of significance ie p≤ 0.05. noah et al. – nephroprotective activities of ethanol root extract and fractions … 479 results and discussion effect of root extract and fractions of h. africana on body and organs weights of rats with doxorubicininduced toxicity administration of h. africana root extract and fractions to rats with doxorubicin-induced organs toxicities caused considerable improvement of the body weights compared to the organotoxic group. the crude extract caused a pronounced dose-dependent effect (7.14 -8.09%) when compared to the organotoxic group with the dichloromethane fraction treated group exerting the highest effect (9.30%). silymarin also improved the weight of the treated animals considerably (7.99%). the weights of kidneys of the group treated with doxorubicin only were found to be reduced when compared to those of the normal control group though not statistically significant (p>0.05). however, treatment of rats with doxorubicin-induced toxicities with the root extract and fractions of h. africana improved the organs weights though insignificantly (p>0.05) except in the group treated with aqueous fraction (table 1). evaluation of effect of root extract and fractions of h.africana on kidney function parameters of doxorubicin-induced kidney injury in rats. table 2 shows the effect of root extract/fractions of h. africana on kidney function parameters of rats. administration of doxorubicin (1.66 mg/kg) to the rats caused significant (p<0.05-0.001) elevation of serum urea, creatinine and electrolytes (k+, na+ and hco-3) except clwhen compared to normal control. these increased levels of serum urea, creatinine and electrolytes (k+, na+ and hco-3) were significantly (p<0.05 0.001) reduced when compared to organotoxic group following pretreatment of the rats with silymarin and root extract/fractions (200 – 600 mg/kg), with the dcm fraction having the highest effect in most cases. these reductions were non dose-dependent with the lower doses (200 and 400 mg/kg) exerting more significant effects. however, the cllevel was not affected significantly (p>0.05) with the root extract/fraction treatment (table 2). effect of root extract and fraction on kidney oxidative stress markers of doxorubicin-induced kidney toxicity in rats. the effect of h. africana root extract/fractions on kidney oxidative stress markers of the rats is as shown in table 3. administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.050.001) decreases of kidney antioxidant enzymes activities (sod, gpx, gst, cat) and gsh levels when compared to control. the mda level was also elevated by doxorubicin treatment significantly (p<0.001) when compared to control. however, repeated administration of root extract/fractions of h. africana (200600 mg/kg) concomitantly with doxorubicin for 14 days caused non dose-dependent elevations of the enzymatic and nonenzymatic endogenous antioxidants in the treated rats groups which were mostly significant (p<0.05-0.001) in the higher doses (400 and 600 mg/kg) of the root extract when compared to the organotoxic groups with dcm fraction being the most active. dose-dependent and significant (p<0.05-0.001) decreases in mda levels of the extract/fractions treated groups were recorded when compared to control with aqueous fraction as the most active fraction. similar decreases were also observed in the silymarin-treated group when compared to organotoxic control (table 3). effect of root extract and fractions of h. africana on histology of rat kidney in doxorubicin-induced nephrotoxicity histological sections of kidneys of rats receiving various treatments at magnification (x400) stained with h&e method revealed that group 1 (a) treated with distilled water (10 ml/kg) showed normal renal tubules and glomeruli. no evidence of pathology was seen. the organotoxic group (group 2, b) treated with doxorubicin (1.66 mg/kg) showed focal non-specific inflammation, normal renal tubules and glomeruli (gm) and few congested blood vessels were seen. rats in group 3 (c ) treated with 200 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 4 (d) treated with 400 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 6 (f) treated with 400 mg/kg of aqueous fraction of h. africana root and doxorubicin (1.66 mg/kg), group 7 (g) treated with 400 mg/kg of dichloromethane fraction of h. africana root and doxorubicin (1.66 mg/kg) and group 8 (h) treated with 100 mg/kg of silymarin of h. africana root and doxorubicin (1.66 mg/kg) had kidney sections showing normal renal tubules and glomeruli with no evidence of pathology seen. group 5 (e) rat treated with 600 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg) showed distorted parenchyma with both normal and dilated renal tubules. the dilated tubular epithelium appears flattened. there were also multifocal interstitial inflammatory infiltrates (figures a-h). 480 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 table 1. effect of h. africana root extract on body and kidney weights of rats with doxorubicin-induced toxicity. parameters/ treatment dose mg/kg kidney body weight before after % increase in body weight normal control 1.24±0.10 135.6±18.34 151.0± 12.33 11.35 doxorubicin 1.66 0.94±0.10 130.0 ± 9.45 126.3± 10.43 -2.84 silymarin+dox 100 1.23±0.17 132.6± 14.55 143.2 ± 3.15 7.99 extract+dox 200 1.20±0.05 142.8± 10.56 153.0± 5.29 7.14 400 1.14±0.02 140.3± 7.36 151.6 ± 6.22 8.05 600 1.11±0.06 138.4 ± 8.54 149.6 ± 8.48 8.09 aqueous fraction 400 1.05±0.11 138.4 ± 6.26 144.6± 10.22 4.47 dcm fraction 400 1.17±0.09 134.3± 8.50 146.8± 13.20 9.30 data were expressed as mean ±sem. significant at dp<0.001 when compared to normal control; ap< 0.05, bp< 0.01, cp< 0.001 when compared to organotoxic control. (n = 6). table 2. effect of h.africana root extract and fractions on kidney function parameters of rats with doxorubicin-induced toxicity. treatment dose mg/kg urea (mmol/l) creatinine (µmol/l) chloride (mmol/l) potassium (mmol/l) sodium (mmol/l) bicarbonate (mmol/l) control 10 4.50± 0.30 94.5±5.86 45.75±1.93 3.47± 0.19 111.0± 5.11 22.00± 0.81 doxorubicin 1.66 10.50± 0.40c 171.6±2.02c 46.66±0.88 6.53± 0.08c 165.0± 1.73c 30.66± 0.66a crude extract 200 7.13±0.31e 143.0± 6.35a 57.0±2.51 4.13± 0.78f 125.0±16.37d 22.66± 1.66d 400 5.50±0.30f 112.3±11.34e 48.33±0.88 4.63± 0.28f 138.6± 6.76 25.0± 1.00 600 6.16±0.97f 125.6± 16.33d 48.66±1.20 3.40± 0.05f 107.3±1.45f 24.0± 2.00 aqueous fraction 400 4.80±0.50f 108.5±2.72f 49.50±1.70 4.17± 0.55b 118.7± 6.27b 22.33± 1.33d dcm fraction 400 5.00±0.40f 101.0±8.62f 44.0±1.15 3.10± 0.15f 104.6± 1.45f 25.66± 2.33 silymarin 100 3.60±0.05f 75.6±2.33f 44.60±1.45 3.13± 0.17f 105.0± 2.51f 22.33± 2.60d data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). table 3. effect of h.africana root extract and fractions on kidney oxidative stress markers of rats with doxorubicin-induced toxicity. treatment dose mg/kg sod (u/ml) cat (u/g of protein) gpx (µg/ml) gsh (µg/ml) gst mda (µmol/ml) control 10 0.61±0.02 1.51±0.06 0.090±0.002 1.18±0.02 0.40±0.01 0.22±0.02 doxorubicin 1.66 0.17±0.01c 0.46±0.01a 0.040±0.00a 0.31±0.01b 0.22±0.01c 0.63±0.01c crude extract 200 0.30±0.01b 1.04± 0.15c 0.051±0.001 0.85± 0.15 0.25±0.01b 0.50±0.01b 400 0.39±0.01a,d 1.11±0.12c 0.067±0.001e 1.16±0.01d 0.28±0.01b 0.46±0.03b 600 0.51±0.02e 1.26± 0.05c,e 0.064±0.004 1.12± 0.08 0.34±0.01e 0.31±0.02f aqueous fraction 400 0.23±0.06c 1.15±0.02f 0.045±0.003a 1.02± 0.07 0.31±0.001d 0.39± 0.01e dcm fraction 400 0.48±0.04a,d 1.36±0.02c,d 0.069±0.002e 1.15± 0.14 0.33±0.01e 0.30± 0.01f silymarin 100 0.52±0.02f 1.15±0.06c 0.076±0.002e 1.13±0.01d 0.36±0.01f 0.34±0.02a,d data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). figure a. photomicrograph of kidney section of rat treated with distilled water (10ml/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure b. photomicrograph of kidney section of rat treated with doxorubicin (1.66mg/kg) showing focal non-specific inflammation (red arrowhead) with lower magnification, normal renal tubules (rt), and glomeruli (gm). there are few congested blood vessels (v). h&e stain x400 noah et al. – nephroprotective activities of ethanol root extract and fractions … 481 figure c. photomicrograph of kidney section of rat treated with 200 mg/kg of h. africana root extract and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure d. photomicrograph of kidney section of rat treated with 400 mg/kg of h. africana root extract and doxorubicin (1.66mg/kg) showing normal parenchymal with renal tubules (rt) and glomeruli (gm). no lesion seen. h&e stain, x400 magnification figure e. photomicrograph of kidney section of rat treated with 600 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg) showing distorted parenchyma with both normal (rt) and dilated renal tubules (). the dilated tubular epithelium appears flattened. there are also multifocal interstitial inflammatory infiltrates (red arrowhead). h&e stain, x400 magnification figure f. photomicrograph of kidney section of rat treated with 400 mg/kg of aqueous fraction of h. africana root and doxorubicin (1.66 mg/kg) showing normal parenchyma with renal tubules (rt), glomeruli (gm) and a focal congested blood vessel (v). no degenerative changes seen. h&e stain, x400 magnification figure g. photomicrograph of kidney section of rat treated with 400 mg/kg of dichloromethane fraction of h. africana root and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure h. photomicrograph of kidney section of rat treated with 100 mg/kg of silymarin and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification discussion this work was designed to investigate the effect of root extract and fractions of hippocratea africana on doxorubicin-induced kidney toxicity in rats in a bid to confirm the folkloric claim of its antidotal activity. doxorubicin is an anthracycline glycoside antibiotic that 482 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 possesses a potent and broad spectrum antitumour activity against a variety of human solid tumours and haematological malignancies (calabresi and chamber, 1990). however its use in chemotherapy has been limited largely due to its diverse toxicities, including cardiac, hepatic, hematological and testicular toxicity (yilmaz et al., 2006). the semiquinone form of doxorubicin is a toxic short-lived metabolite which interacts with molecular oxygen and initiates a cascade of reactions, producing reactive oxygen species (ros). ros generation, inflammatory processes and lipid peroxidation have been suggested to be responsible for doxorubicin-induced cardio-, hepatoand nephrotoxicity (injac et al., 2009; kalender et al., 2005). it has been proposed that dox-semiquinone, an unstable metabolite of dox, reacts with o2, producing h2o2 and o2 − (superoxide). in addition, dox enhances the activity of extramitochondrial oxidative enzymes such as xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (nadph) oxidase and also interferes with mitochondrial iron export, resulting in formation of ros (reactive oxygen species) (bachur et al., 1979). in this study, doxorubicin administration was found to have caused elevation of serum urea, creatinine and electrolytes (k+, na+, cland hco-3) levels when compared to normal control, which is an indication of a serious injury to the kidney. this finding is consistent with earlier report of rajasekaran (2019), which similar elevations were reported. it is well documented that kidney injury are indicated by increase in serum level of creatinine and urea (laskshmi and sudhakar, 2010) as well as increase serum levels of na, k, cl and bicarbonate (james and mitchel, 2006). however, these increases were reduced significantly by the coadministration of root extract and fractions of h. africana. doxorubicin is reported to cause nephrotoxicity via oxidative stress as free radicals formed caused tubular atrophy and increased glomerular capillary permeability. nephrotoxicity by doxorubicin can also result from lipid peroxidation and biological macromolecules damage by iron-dependent oxidative damage (mohan et al., 2010). degenerative changes in kidney depend on cumulative dose and duration of treatment as doxorubicin metabolites are partly excreted from the kidney. another mechanism for renal injury is the conversion of dox to semiquinone free radical by nadph-cytochrome p-450 which generates hydroxyl radical and superoxide anion which causes lipid peroxidation (rashid et al., 2013). the reduction of the levels of urea, creatinine and electrolytes by the root extract and fractions in this study is as a result of their free radical scavenging potentials, thereby protecting the kidney against oxidative stress by free radicals generated by doxorubicin. the antioxidative burst and antioxidant activities of the root extract and fractions of h. africana had previously been reported (okokon et al., 2013a; 2022; umoh et al., 2021). moreso, the antioxidative stress activities of the root extract and fractions observed in this study further support the antioxidant potentials of the plant. these activities may have contributed to the observed protective effects in this study. the findings of this study show that administration of doxorubicin (2.5 mg/kg, i.p) on alternate days for 14 days to rats caused significant decreases (p<0.05) in levels of enzymatic and non-enzymatic endogenous antioxidants (gsh,sod, cat, gpx and gsh) when compared to control, while the mda level was elevated, lipid peroxidation is a marker of oxidative stress and elevations in the amount of mda, a lipid peroxidation product, have been reported following doxorubicin treatment (rashid et al., 2013, rehman et al., 2014, khames et al., 2019). this trend was observed in this study. concomitant administration of root extract h.africana (200-600 mg/kg) with doxorubicin caused significant (p<0.05-0.001) non dosedependent elevation in the levels of the antioxidant enzymes (sod, cat, gpx) when compared to control. similarly, gsh level was significantly (p<0.001) elevated following treatment with the extract when compared to control. also, there were significant (p<0.05-0.01) reductions in the level of mda of the extract-treated rats. it has been documented that dox inhibits the activities of endogenous enzymatic and non-enzymatic antioxidants as was the case in this study. so, an imbalance between ros generation and neutralization leads to oxidative stress and injury to the kidney (abushouk et al., 2017; abdel-daim et al., 2017; aboushouk et al., 2019). the reduced mda level caused by the administration of the root extract and fractions may have resulted from reduction in lipid peroxidation and generation of free radicals which might have been scavenged by the phytoconstituents present in the root extract and fractions, revealing the antioxidative stress potentials of the root extract and hence the protective effect on the kidney as was observed. histological findings in this study revealed that kidneys of rats treated with doxorubicin (2.5 mg/kg) alone showed pathological signs of injury which were seen as degenerated microvesicles in the tubular lining cells among others. however, co-administration of h. africana root extract/fractions and doxorubicin reduced the toxic effects of the doxorubicin as normal glomeruli which were devoid of pathological signs were seen in the kidney sections of the extract-treated rats examined. this further confirms the nephroprotective potential of the root extract which may have been exerted through the antioxidant and antioxidative stress activities of its phytochemical constituents. conclusions the findings of this study show that the root extract and fractions of hippocratea africana have the potential to noah et al. – nephroprotective activities of ethanol root extract and fractions … 483 counteract the injurious effect of doxorubicin on the kidney. this activity can be attributed to the antioxidant and antioxidative stress activities of their phytochemical constituents. thus, the root extract can be used to alleviate and/or prevent doxorubicin-induced renotoxicity. acknowledgements: the technical support from mr nsikan malachy of pharmacology and toxicology department was tremendously acknowledged. authors’ contributions: kn, jau and jeo initiated and designed this study. jeo, kn and jau carried out the experiments and drafted the manuscript. noe and moa performed the statistical analysis, edited and reviewed the manuscript. kn, jau, moa, jeo and noe read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. references abdel-daim mm, kilany oe, khalifa ha, ahmed aam. 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(2006) protective effect of lycopene on adriamycininduced nephrotoxicity and nephrotoxicity. toxicology 218: 164-171. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 33-40 | doi: 10.14421/biomedich.2016.52.33-40 issn 2540-9328 (online) starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi1, syafiana khusna aini2, dewi anggraini3, dian prihatiningtias ekawati4 1,2,3,4chemistry department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: endaruji@yahoo.com1 abstract effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on starch-based edible film-glycerol mechanical properties has been done. the purpose of this study is to create an active environment-friendly packaging material. surimi additions are intended to improve the mechanical properties of bioplastics and additions of rosella extract intended as a bio-indicator of acidity. the method used was solvent casting. an amount of surimi and rosella extract varied to obtain the best mechanical properties. the results shows that the addition of surimi and rosella flower extract significantly effect the elongation of edible films produced up to 27%. keywords: edible film; rosella extract; surimi dumbo catfish; mechanical properties. introduction the use of synthetic polymers made from petroleum may have a negative impact on our environment and on human health. synthetic polymers made from petroleum take a long time to degrade (awwaly et al., 2010). the awareness to this problem would then increasing interest to organic materials, which can be destroyed biologically, eco-friendly, and easily obtained in nature as well as suitable for packed food products (embuscado et al., 2009). the ideal solution is the development of natural ingredients for the manufacture of packaging materials which can be decomposed faster naturally, maintain the integrity of the food product to be packed and edible (gennadios et al., 2002). edible film is a thin layer made of edible ingredients and used as food coating. coating of food components can be done by dipping, spraying or wrapping (akbar et al., 2013). the advantage of using edible film is it can be directly consume with the products that are packaged together, eco-friendly, improve the organoleptic properties of the packaged product, serves as a nutritional supplement, as a carrier of flavor, coloring, antimicrobial agents, and antioxidants (murdianto et al., 2005). hydrocolloid such as proteins, polysaccharides, and a mixture of both has been studied intensively as an ingredient in edible film. isnaini (2010) explains that the edible film as bio-indicators can be made by using natural pigments from the plant, one of them is rosella (hibiscus sabdariffa l). the main ingredients of rosella is an anthocyanin. setiono (2013) stated that anthocyanins are dissolved in water to create colored solutions, red under acidic conditions and blue under alkaline conditions. the compound is classified as pigments and determine the plant color which is affected by the ph of its environment. rosella flowers can be used as bio-indicators, which are sensitive to ph. edible films can detect food spoilage by change in ph of the food (akbar et al., 2013). edible films based on protein has been developed, such as the filming of collagen, gelatin, protein maize (corn zein), wheat protein (wheat gluten), soy protein (soy protein), casein and whey protein. based protein film are usually obtained from casting and drying (valenzuela et al., 2013). an alternative materials of edible film made from a protein is surimi. park (2004), explains that the surimi is a myofibril fish protein which has been stabilized and is manufactured through a continuous process stage which involves the removal of the head and spine, meat processing, washing, removal of water and freezing. surimi has the functional ability in gel form and bind water (amaliya et al., 2014). in this research, catfish surimi protein is added to improve the mechanical properties and lowering the water vapor transmission rate so its improve the shelf life of the product that is coated. rosella extract in this study is used as bio-indicators sensitive to ph changes that occur in food to be packaged (amaliya et al., 2014). edible film is expected to detect food spoilage by changes in ph that occur in food, have good mechanical properties and low wvtr. http://dx.doi.org/10.14421/biomedich.2016.52.33-40 34 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 materials and methods materials materials used in this research are rosella flower petals from wonosobo, dumbo catfish obtained from candipitu karangmojo, gunungkidul yogyakarta, commercial tapioca rosebrand, glycerol, sodium hydroxide pellets p.a merck, silica gel obtained from bratachem, 96% ethanol, and distilled water. preparation of rosella’s extract the extraction method in this research was maceration (marwati et al., 2012). a hundred grams of rosella were added to 300 ml 96% of ethanol at room temperature for 24 hours. solution then filtered and evaporate to give a sticky extract of rosella. preparation of dumbo catfish’s surimi surimi preparation of dumbo catfish in this research has done using heruwati & jav method (1995) methods in santoso et al. (2013). one kilogram of catfish was weeded by removing the head and entrails then washed with water. catfish meat are crushed and washed twice with cold water. the mixture was then stirred for 10 minutes. stirring was stopped to precipitate the pulverized meat, while dirt and grease is dumped. the precipitate is then dried by pressing it in a hydraulic press to remove water (bourtoom et al., 2006) preparation of edible film tapioca-surimi edible film edible film preparation was modified from santoso et al. (2011). the procedure of making this edible film are as follows. edible surimi weighed as much as 1%; 1.5%; 2%; 2.5% and 3% (w/v) of the total distilled water used as solvent. a 100 ml of distilled water then added with 1 m naoh and adjust to ph 11. mixture then stired and heated for 30 minutes at 50 c. the mixture reheated to 60 c for 30 minutes. addition of 2.5 gram tapioca and 1.5 ml of glycerol has done when the mixture were homogen. the suspension then heated and degassing for another 25 minutes, respectively. the suspension is then poured over the glass framed and dried at 50 c for 12 hours. tapioca-surimi-rosella edible film edible film preparation method using in this research is similar to the previous method but added rosella’s extract as much as 0%; 1%; 2%; and 3%, respectively. the mixture was heated for 25 minutes at 30 c and degassed at 75 kpa for 20 minutes. mixture is then poured over the glass framed and dried at 50 ° c for 16 hours. characterizations characterization of materials was conducted on the wvtr, mechanical and functional group testing using ft-ir (fourier-transform infrared spectroscopy). color test preliminary test conducted to identify anthocyanin is a color test as conducted by supriyanti et al. (2013). this test were done with the addition of hcl and naoh. hayati et al. (2012) stated that in the acid environment, an anthocyanin will remain red and under alkaline conditions anthocyanin change to bluish-green. thin layer chromatography (tlc) test further testing is to determine the presence of anthocyanin compounds using tlc methods using hcl and baa (butanol: acetic acid: water) as eluent. water vapor transmission rate (wvtr) test measurements were made using the gravimetric method based on astm. e. 96-99. moisture absorbent materials (desiccant) were put in a can, then the sample were placed at the top of the can such that the sample covers cans. wax paper used to cover part of the container with the sample so that no air enters between the boundary and the sample container (bourtoom et al., 2006). cans are weighed to the nearest 1 mg, and then placed in a desiccator at a fixed room temperature. cans are weighed every day at the same hour and determined the increasing of its weight as a consequence of desiccant material that had absorb water. the relationship between weight gain and time were graphed. wvtr value is calculated using the formula: wvtr = slope / sample area x 100 x 100 m2 = g / m2 / days (92% rh, 38oc) mechanic properties test edible film mechanic properties (thickness, tensile strength and elongation) test has done based on astm.d. 882-02. edible films are conditioned at room temperature (30 c), 68% humidity (rh) for 24 hours prior to measurement. film thickness were measured using a micrometer to the nearest 0.001 mm. thickness measurements were taken at five different points for each film sample (top right corner, bottom right corner, middle, upper left corner and the bottom left corner). film thickness value is an average of the results of measurements at five points in units of mm (national standardization agency, 2013). a set equipment test is prepared and arranged. samples of edible film mounted at the ends of both clamping and wedged firmly. the measurement area is set with loads corresponding to pen recorder off. tensile strength is determined by the maximum load, while the elongation is determined and calculated at the time the film broke. fourier transform infrared spectroscopy characterization of functional groups carried out by ftir using a kbr pellet method. one mg of sample was mixed with 200 mg of kbr powder. samples were mixed in kbr pellets by pressed mixture into a thin sedyadi et al. – starch-glycerol based edible film and effect of rosella … 35 transparent using 10 tons pressure (2000 psi). pellet samples were then measured its infrared absorption at wavenumber 4000-400 cm-1. results and discussion rosella extraction rosella (hibiscus sabdariffa l.) was extracted using maceration method to retrieve the active substance in rosella’s petals (hayati et al., 2012). extraction method is done by depositing the active compounds contained in the rosella petals using 96% of ethanol with a ratio of rosella’s powder: ethanol = 1: 5 for 24 hours. the yield obtained is about 26.16% of red extract yield. this thick red rosella extract obtained is not much different from those obtained a yield of 17.7% in the study of suzery et al. (2010). color test one of the active compound in rosella flower petals is anthocyanin. this anthocyanin are red to the blue pigment found in many fruits and flowers (robinson, 1991). this test is performed with the addition hcl and naoh. the results obtained are rosella flower petals extract supplemented with hcl will remain red, whereas one with with naoh will turn green and faded to yellow. this is in accordance with hayati et al. (2012), which states that under acidic conditions the anthocyanin color will remain red and bluish green in alkaline conditions. besides affected by ph, color changes in anthocyanin compounds are also affected by the concentration of pigments, their mixtures with other compounds as well as the number of methyl and hydroxyl groups. hydroxyl groups changes the colors tend to be blue and unstable, while methoxyl group changes of color will tend to be red and stable (warsiki, 2012). thin layer chromatography (tlc) test hcl mobile phase generating rf value of 0.37. this is consistent with the report by hayati et al. (2012) that the value of rf for anthocyanin is eluted with hcl low to mid. research conducted by suzery (2010) about the mangosteen fruit anthocyanins also generate rf value of 0.58. while the mobile phases baa generate rf value of 0.48. rf value for anthocyanin eluted with baa is 0.1-0.4, it is not much different from research conducted by warsiki et al. (2012) which also ranges from 0.25. comparison of anthocyanin of rosella flower petals with harbone (1987) is shown by table 1. based on those preliminary tests, it can be concluded that the extract obtained from rosella contains anthocyanins. these results were then confirmed by ftir characterization to determine the functional groups contained in the extract. ir spectrum of rosella flower extract is presented in figure 9. table 1. comparison antocyanin test. test result this research hayati, 2012 using hcl 2m red red using naoh 2 m turn to green then fade to yellow turn to green then fade to yellow tlc with hcl 1% eluent rf value = 0,371 low to middle rf r tlc with baa eluent rf value = 0,25 middle rf (0.100,40) surimi surimi is a myofibril protein concentrate from successive leaching process to remove unwanted compounds, resulting in a semi-pure protein fraction containing high myofibrils protein. the making of catfish’s surimi is done by separating the meat from the head and the stomach contents of fish with several stages of the rinsing, suppression, the addition of salt (nacl), and the addition of cryoprotectant. rinsing is an important step because it can support the ability of gelation (ashi) and inhibit protein denaturation due to freezing (santoso, 2007). 100 grams of fresh meat catfish yielded 64.86% solid white surimi. the surimi obtained are relatively small due to repeated washings during the washing process. according to santoso (2004), during the washing process many materials are dissolve by water. identification of functional groups on surimi has done using ft-ir at 4000-400 cm-1. ft-ir spectrum of the surimi is presented in figure 10. tapioca-surimi edible film the principle of production edible film manufacture with variations of surimi was prepared by dissolving surimi under alkaline conditions. surimi containing protein myofibril that insoluble in water but soluble in an alkaline. in addition myofibril protein denaturation is essential to form a wider structure for the film formation. cause denaturation of proteins into long parallel chains that can bind with other polymers such as starch. proteins can form edible film because of the ability of the side chain to form intermolecular crosslinking (bourtoom et al., 2006). mechanical properties test of edible film thickness edible film thickness against amount variation of catfish surimi is presented in figure 1 which shows that the surimi edible film thickness ranged from 0.09 to 0.12 mm. krochta (1994) explains that the film thickness correlated to the number of three-dimensional network that is formed so that the film is getting thicker. the concentration of dissolved material in the film-forming 36 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 solution and casting plate size also affects the thickness of the film. figure 1. graph of thickness vs surimi variation. the results showed that the addition of surimi does not significantly affect edible film thickness. tensile strength tensile strength is the maximum achievable pull of a film before it breaking. the tensile strength test with a variety of surimi edible film is presented in figure 2 which shows that the tensile strength ranging between 0.83 to 1.09 n. the best tensile strength value is 1.09 n with surimi concentration as much as 3 g. edible films with surimi addition showed a trend of increasing tensile strength values, though not very significant. figure 2. graph of tensile strength vs surimi variation. this is consistent with chinabhark et al. report (2007) which showed that a protein in a high amount of polymer allows the intermolecular interaction getting bigger and stronger. this causes an increase in the value of tensile strength edible film. elongation elongation is the maximum length changes of edible film during a stretch off. flexibility properties of edible film are very important thing in the coating process food. edible films with a elastic and flexible texture are easier to set up, so that the packing of the food is good (cahyadi, 2009). figure 3. graph of elongation vs surimi variation. elongation measurement is the maximum film length changes when obtaining tensile force to break up the film compared to the initial length (hambali et al., 2007). the greater the elongation of edible film, edible film has more flexibility of being able to do a maximum extension. elongation measurement of edible films in variation of surimi are presented in figure 3. figure 3 shows that the edible film elongation of surimi variations tends to decrease with increasing concentration of surimi. maximum elongation of 21.47% is achieved at a concentration of 2.5% surimi. lowest elongation of 17.99% was obtained at a concentration of 3% surimi. according to sinaga et al. (2013) an edible film elongation value are low, due to the denaturation of myofibril surimi protein, and therefore contributes to a decrease in elongation. water vapor transmission rates (wvtr) water vapor transmission rate is an important parameter in the edible film. water vapor transmission rate indicates the speed of the water vapor to penetrate (per gram per second) per unit area of edible film or the film's ability to inhibit the transmission. the permeability to water vapor should be low (garnidaet al., 2007). the chemical composition of the food surface can be changed due to metabolism of food, microbial respiration, gas solubility, and film permeability to water vapor. edible films rely on its barrier properties against water vapor and gas transfer. edible film with good barrier properties can extend the shelf life of the coated products (skurtys et al., 2009). the wvtr measurement of amount surimi variations edible films are presented in figure 4. it appears that the wvtr values of edible film ranged from 3.59 to 4.01 g / m2hours. the results showed that the addition of surimi concentration tends to decrease the wvtr values. this is because the surimi protein undergo denaturation. protein denaturation cause proteins undergo changes in which the hydrophilic side on the side inside and outside the hydrophobic side will turn into a hydrophobic side out so the water vapor transmission rate to be down (santoso, 2013). 0.0975 0.1002 0.1019 0.0951 0.1196 0 0.05 0.1 0.15 1 1.5 2 2.5 3 0.8349 0.9257 0.9871 0.7021 1.0903 0 0.25 0.5 0.75 1 1.25 1 1.5 2 2.5 3 20.6989 19.8819 19.6328 21.4732 17.9857 0 5 10 15 20 25 1 1.5 2 2.5 3 sedyadi et al. – starch-glycerol based edible film and effect of rosella … 37 figure 4. graph of wvtr vs surimi variation. based on the mechanical test it can be concluded that the addition of surimi is not significantly affect mechanical properties of the resulting edible film. nevertheless it can be seen that the best edible film in this study are edible film with the addition of surimi variation of 2.5% because it has the highest elongation. edible films with this comparison will be used further to add a rosella extract. tapioca-surimi-rosella extract edible film the maximum data obtained will be used for variety addition of rosella flower extract as bio-indicators of the edible film. the production is the same as edible film mentioned above. the suspension of the obtained printed and dried at 40 °c for 24 hours. films were then tested the mechanical properties which include thickness, tensile strength, and elongation, wvtr and ft-ir analysis (cahyadi, 2009). thickness the results of the thickness measurements are presented in figure 5. it appears that the edible film thickness value surimi-starch-glycerol-rosella extract produced ranged from 0.085 to 0.095 mm. results of edible film thickness with 2.5 grams of surimi composition and the addition of 0.75 g roselle extract is greater than the addition of 0.5 grams of roselle extract is equal to 0.09 mm. the thickness of the edible film that is formed is influenced by the film-forming solution concentration and the size of the plate. the results showed that the addition of rosella extract also does not significantly affect the resulting film thickness. the more the concentration of rosella extract is added, the thickness of the resulting film tends to decrease, but not significantly (dewi et al., 2010). leerahawong et al. (2011) explains that the edible film thickness due to differences in the concentration of film-makers, while the volume of film of the solution are poured into each of the same plate. this resulted in the total solids in the film after drying increases and polymers making up the matrix movies more and more. the thickness between the various types of film can cause by the composition formula, of different films. figure 5. graph of thickness vs rosella extract variation. tensile strength the results of the measurement of tensile strength are presented in figure 6. it appears that the value of the tensile strength composite edible film surimi-starchglycerol-rosella extract produced ranged from 0.39 to 0.70 n. the addition of 0.5 gram rosella extracts initially lower the value of the tensile strength of the edible film to 0.39 n. the addition of 0.75 grams rosella extract can increase the value of the tensile strength of the edible film becomes 0.52 n, but still relatively lower than edible film without the addition of extract of rosella (dewi et al., 2010). figure 6. graph of tensile strength vs rosella extract variation. the data obtained show that the more rosella extract is added, and then it tends to lower the value of tensile strength of the resulting edible film. the addition of 0.75 g rosella extract slightly increases tensile strength relative value of the addition of 0.5 grams rosella extract. this is because more film added can increase the density of the resulting film that is not easily torn. figure 6 also shows that the trend of edible film tensile strength value is almost equal to the thickness of the edible film, so it cannot be used as a standard of determining the quality of the food was good coating. elongation elongation measurement results showed in figure 7. result shows that the elongation value of the composite 4.0123 3.9012 3.5925 3.8889 3.7284 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 0.0951 0.0852 0.0915 0 0.05 0.1 0.15 0 0.5 0.75 0.7021 0.3962 0.5213 0 0.2 0.4 0.6 0.8 1 0 0.5 0.75 38 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 edible film of surimi-starch-glycerol-rosella extract ranged 21,47-24.95%. best elongation value contained in the addition of 0.5 grams of rosella extract. figure 7. graph of elongation vs rosella extract variation. elongation measurement showed that the addition of rosella extract tends to increase the value of the elongation of edible film. an edible film is said to be used as food coatings if they have a high value percent elongation and water vapor transmission rate is relatively low. this is because of a growing number of its constituent components, the edible film more tightly. percent elongation produced is inversely proportional to the thickness and tensile strength. water vapor transmission rates (wvtr) the wvtr measurement of edible film composite are shown in figure 8. figure 8. graph of wvtr vs rosella extract variation. based on the figure 8, it showed that the greater the amount of rosella added, the greater the wvtr. a better edible film is a film that has a high elongation and low wvtr values. charaterization of fourier transform infrared spectroscopy rosella petals extracts were analyzed using ft-ir spectrophotometer to determine the character of the resulting functional groups. the spectrum is presented in figure 9. the identification of functional groups on the extract rosella flower petals carried on the wave number 4000-400 cm-1. ft-ir spectrum at rosella flower petals extracts showed some characteristic absorption band in the absorption peaks. figure 9. ft-ir spectrum of rosella extract. based on the ft-ir spectrum in figure 9, it appears the wide absorption at wave number 3356.14 cm-1 indicating the oh absorption band. wavenumber 2947.23 cm-1 indicating their c-h groups are strengthened by their absorption at wavenumber 1411.89 cm-1. both of these absorptions indicate methyl group in edible film cluster. absorption around wavenumber 1728.22 cm-1 indicates the group of carbonyl. while c = c group indicated by adsorption at wave number 1643.35 cm-1. moulana (2012), stated that the absorption at wavenumber 3348.25 cm-1 indicating their alcohol groups are supported by the appearance of absorption at wavenumber 1044.35 cm-1 to c-o bond alcohol. absorption at wavenumber 1706.30 cm-1 indicate the presence of c = o carbonyl group. the presence of the c = c double bonds of aromatic demonstrated by a sharp absorption at wave number 1634.95 cm-1 which is supported also by the appearance of absorption at wave number 709.80 cm-1 to c-h bond. based on the ft-ir spectra interpretation rosella extract, it can be concluded that the rosella extract containing anthocyanin compounds shown by their group c = o, c-o and c = c which is the structure of anthocyanin. figure 10. ft-ir spectrum of surimi. ft-ir spectrum of surimi is presented in figure 10. it showed their wide absorption at 3425.58 cm-1 due to 21.4732 24.9561 22.8954 19 20 21 22 23 24 25 26 0 0.5 0.75 3.8889 5.5555 5.3086 0 2 4 6 8 10 0 0.5 0.75 sedyadi et al. – starch-glycerol based edible film and effect of rosella … 39 the strain nh associated with hydrogen bonds and the oh group. uptake of n-h is not visible as it is covered by the oh absorption. absorption at wavenumber 2924.09 cm-1 indicates the presence of an amine group stretching. a bending amine group showed at wavenumber 2854.65 cm-1 with a sharp peak. this indicates that the amide binds to the ch2 strain. in addition there is absorption at wavenumber 3873.06 cm1 which indicate the presence of free nh groups. absorption peaks at 1643.35 wave numbers indicate a carbonyl (c = o). according to poedjiadi (2009) absorption at wave number 1636-1661 cm-1 showed absorption of amide group. this amide catchment area showed strain c = o and oh groups are paired with a carboxyl group. next on the wave number 1442.75 cm-1 indicates ch2 bending and deformation nh bond in the protein. based on the surimi’s ft-ir spectrum interpretation above, it can be concluded that the surimi protein shown their amide groups (nh). edible films produced spectra identification of cluster compounds using ftir spectrophotometer. the resulting spectra are presented in figure 11. figure 11. ft-ir spectra of edible film starch-glyserol (a), starchglyserol-surimi (b), and starch-glyserol-surimirosella extract (c). starch-glycerol spectrum is shown in figure 11.a, starch-glycerol-surimi in figure 11.b and starchglycerol-surimi rosella extract in figure 11.c. the presence of oh groups on edible film showed by absorption at wavenumber 3448.72 cm-1. the absorption shifted to 3425.58 cm-1 in the edible film with the addition of surimi. the existence of the ch (stretching) groups of edible film showed in wavenumber 2931.80. addition of surimi edible film shown in wave numbers 2924.09 cm-1. new absorption appears on edible film with the addition of surimi. the uptake appears at wave number 2862.36 cm-1 which indicates the presence of an amine group that binds to the stretching of ch2 groups. new absorption also appears on edible film with the addition of surimi. the uptake appears at wave number 2299.15 cm-1. group c = o (carbonyl) on edible film tapioca and edible film with the addition of surimi shown in wave number 1635.64 cm-1. the shift of wave numbers show that the polymerization reaction time of the blending of the edible film and shows the interaction between polymers occurs. figure 11.c of ft-ir surimi edible film-starchglycerol-rosella extract spectra showed some shift wave numbers compared to edible starch-glycerol composite film that is at 2901.60 cm-1 to 2924.09 cm-1 and 1331 cm-1 to 1334.74 cm-1. some of the absorption band is also changing the intensity and emerging new absorption band. ft-ir spectra showed a broad absorption on say 3400-3500 cm-1 wave. khopkar (2008), explains that the absorption band at wave number 3600 cm-1 indicate the presence of oh groups on edible film oh. the absorption of tapioca with the addition of surimi and rosella is located at wavenumber 3448.72 cm-1. ch (stretching) between edible films edible film tapioca with the addition of surimi and rosella extract is shown in wave number 2924.09 and 2854.65 cm-1. the new spectra appear on edible film with the addition of surimi and rosella extract. the uptake appears at wave number 2862.36 cm-1 which indicates the presence of an amine group that binds to a stretch stretching ch2. in addition, the new spectra also appear on edible film with the addition of surimi and rosella extract. the uptake appears at wave number 2299.15 cm-1. group c = o (carbonyl) on tapioca ediblefilm and edible film with the addition of surimi and rosella extract is shown in wave number 1635.64 cm-1. the shift wave numbers show that the polymerization reaction time of the blending of the edible film and shows the interaction between polymers occurs. ft-ir spectra in figure 10 shows that the addition of surimi and rosella extract does not change the pattern of uptake significantly edible film. it is common because the main ingredients of the edible film are tapioca and glycerol so that the pattern of ir absorption of three quite similar edible film. differences absorption occurs only in some shift in absorption of a wave shift. the emergences of new peaks indicate the presences of a bond between the materials were mixed. differences in intensity indicate the bond between the materials grows denser and more complex. conclusion the research showed that the addition of surimi and rosella flower extract significantly increase up to 27% of the elongation of edible film. references akbar, f., anita, z. harahap, h. 2013. pengaruh waktu simpan film plastik biodegradasi dari pati kulit singkong terhadap sifat mekanikalnya. jurnal teknik kimia usu. 2 (2). 40 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 amaliya, r.r., putri w.d.r. 2014. karakterisasi edible film dari pati 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quinoa proteinchitosan-sunflower oil edible film: mechanical, barrier and structural properties. food science and technology. 50: 531537. warsiki, e., putri, c. d. w. 2012. pembuatan label/film indikator warna dengan pewarna alami dan sintesis. ejurnal agroindustri indonesia. 1 (2): 82-87. starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 53-57 | doi: 10.14421/biomedich.2017.62.53-57 issn 2540-9328 (online) vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari1, kristamtini2, endang wisnu wiranti3 1,2,3assessment institute for agricultural technology (aiat) yogyakarta jln. stadion maguwoharjo no. 22, wedomartani, ngemplak, sleman, yogyakarta, indonesia author correspondency: dewindrasari@yahoo.com1 abstract banana is one of the tropical fruits that people like because it tastes good and contains good nutritional value that beneficial for health. the content of vitamin c and total sugar are an important character to complete the morphological characterization of banana accession that can be utilized by breeders in choosing accession as parent. the study aimed to determine the content of vitamin c and total sugar on 31 accessions of banana collection of banana germplasm plants of yogyakarta. the research was conducted at banana germplasm plantation and laboratory of agricultural technology production of gadjah mada university, yogyakarta from january to december 2016. the results indicated that 31 banana accessions showed their susceptibility to vitamin c content 60.42 + 39.22 mg / 100 g and total sugar 22.06 + 16.01%. high standard deviation values indicate the large diversity of banana accessions that were characterized, indicating that the accessions of each characterized banana were separate accessions of one another. keywords: characterization; banana; vitamin c; total sugar. introduction banana is one of the tropical fruits that people like because it tastes good and contains good nutritional value that beneficial for health. in addition to the relatively cheap price, banana was also one of the plants that have bright prospects because everyone in the whole world almost likes to consume bananas (komaryati & adi, 2012). indonesia is one of the centers of banana plant genes (sukartini, 2007). therefore, in 1988, the goverment of yogyakarta special region develop the banana germplasm garden and has been carry out the collection, maintenance and dissemination of various collections of bananas. with a land area of 19,525 square meters, this garden already has 346 varieties of bananas originating from indonesia and from abroad (kristamtini et al., 2016). the banana germplasm garden of yogyakarta was called as the most complete collection of banana conservation garden in indonesia by the ministry of agriculture of indonesia. indonesia has a high diversity of bananas, this condition was very helpful for breeding program in producing superior varieties. the superior varieties of bananas were expected to have high productivity, good quality, early age, resistant to certain pests and tolerance to environmental stress. on the other hand, in the banana diversity was not yet widely known for its characteristics. to support the assembly of superior varieties of bananas, it was necessary to characterize and evaluate existing germplasm. the information obtained from characterization and evaluation, can be used as a material for improving the character through a plant breeding program. banana plants have diverse morphological characters. each banana plant has its advantages and disadvantages based on its morphological appearance (wardiyati et al., 2004). as the first step of breeding program to get information about morphological character of a plant was needed characterization activity (subandiyah, 2002). as simmonds & shepherd (1955) have pointed out, one of the first steps to recognize the magnitude of such biodiversity was to identify genetic diversity. one approach to identifying a banana plant is to use morphological characters that include leaf, stem, fruit, and flower characters (wardiyati et al., 1997) plant breeders can utilize the plant after knowing the character thoroughly through exploration, collection, characterization, evaluation and selection (wardiyati et al., 1997). morphological characterization is the preliminary information required in seeking superior characters and the diversity that was still in need (santos et. al., 2003). morphological characters were considered still not enough to find a clear position so that other methods as complement to evaluate kinship. one method was to do chemical characterization by analyzing the content of vitamin c and total sugar. the content of vitamin c and http://dx.doi.org/10.14421/biomedich.2017.62.53-57 54 biology, medicine, & natural product chemistry 6 (2), 2017: 53-57 total sugar becomes the character that differentiates between accessions, because bananas contain high enough nutrients and high of vitamin c. in addition the content of vitamin c and total sugar was the required character in the description of banana plants the aim of this research was to know and get the diversity of 31 accessions of banana collection of banana germplasm garden of yogyakarta based on vitamin c and total sugar content so that the relation of various bananas based on vitamin c and total sugar content are known. materials and methods the experiment was conducted at banana germplasm plantation and agricultural production technology laboratory of gadjah mada university, yogyakarta from january to december 2016. thirty-one accessions of banana as research material are presented in table 1. banana tree ripe fruit was analyzed for vitamin c and total sugar content. the analysis of sugar content using nelson somogyi method, while analysis of vitamin c content using iodometric titration method. table 1. the banana accessions and its origin. no name origin 1 ambon jaran bantul 2 ambon sepet gunung kidul 3 ambon warangan kulon progo 4 anjasmoro sleman 5 australi kbh dongkelan 6 gajih putih kulon progo 7 kepok brentel bantul 8 kepok bung kulon progo 9 kepok hijau sleman 10 kepok kuning kbh dongkelan 11 kidang hijau sleman 12 kluthuk susu yogyakarta 13 koja pretel gunung kidul 14 koja sirimentak gunung kidul 15 lempereng kulon progo 16 mangsan sleman 17 mas raja sleman 18 mas sloka kulon progo 19 mas tropong sleman 20 morosebo yogyakarta 21 potho merah bantul 22 raja bagus yogyakarta 23 raja bandung bantul 24 raja lini gunung kidul 25 raja pulut yogyakarta 26 raja sabrang kulon progo 27 raja sawi sleman 28 raja sewu yogyakarta 29 rejang sleman 30 serawak kulon progo 31 triolin bantul the data of vitamin c content and total sugar were analyzed by cluster method using hierarchical cluster analysis to know the kinship relationship with the help of sas ver software 9.2. hierarchical cluster analysis was a common way to grouping objects in groups that share the same resemblance to each other. results and discussion the content of vitamin c and total sugar in 31 accessions of bananas is presented in table 2. the content of vitamin c and total sugar from 31 banana accessions varies according to their genetic potential, with an average vitamin c content of 60.42 + 39.22 mg/100 g, average of total sugar 22.06 + 16.01%. the highest vitamin c content is owned by the mangsan accessions (149.34 mg/100g) and the lowest is owned by the mas raja accessions (12.79 mg/100g). the content of vitamin c in 31 accessions of bananas is a hint of accession of bananas to be selected for consumption as fresh fruit that is beneficial to health. table 2. the content of vitamin c and total sugar of 31 bananas accessions. bananas accessions vit c (mg/100 g) total sugar (%) 1. ambon jaran 20.41 20.41 2. ambon sepet 128.20 18.54 3. ambon warangan 52.44 15.19 4. anjasmoro 34.38 17.01 5. australi 53.14 2.42 6. ceni 36.50 21.70 7. gajih putih 76.14 10.66 8. kapok brentel 30.85 20.31 9. kepok bung 135.69 17.25 10. kepok hijau 23.34 23.34 11. kepok kuning 93.48 18.99 12. kidang hijau 82.93 17.90 13. klutuk susu 61.70 21.36 14. koja pretel 27.39 22.73 15. koja sirimentak 50.38 13.20 16. lempereng 20.52 20.07 17. mangsan 149.34 14.36 18. mas jambe 75.53 16.38 19. mas raja 12.79 44.85 20. mas sloka 92.15 18.99 21. mas tropong 98.84 16.96 22. morosebo 85.78 14.31 23. potho merah 19.40 19.40 24. raja bandung 57.93 15.66 25. raja lini 17.79 22.97 26. raja pulut 122.29 20.94 27. raja sabrang 54.43 15.64 28. raja sewu 31.08 22.31 29. rejang 104.37 10.88 30. serawak 17.38 81.80 31. triolin 16.62 73.72 average + sd min max 60.92+39.22 12.79-149.34 22.06+16.01 2.42-81.80 indrasari et al. – vitamin c and total sugar content characterization on 31 accessions of banana … 55 vitamin c or ascorbic acid is needed for health. in human body vitamin c serves as an antioxidant that can protect the body, especially cellular dna from damage due to oxidation of free radicals. antioxidants prevent oxidative stress and play an important role in the development of chronic and degenerative diseases such as cancer, arthritis, autoimmune disease, cardiovascular and neurodegenerative diseases (lian et al., 2008 in harifah et al., 2017). daily requirement for vitamin c vary for each individual, in general experts estimate that 60 mg per day is enough but in certain conditions the body requires more than that (sitohang, 2006). based on widya karya nasional pangan dan gizi v (1993), the requirement of vitamin c per day for baby (7-12 months) is 35 mg, childrem (7-9 year) is 45 mg and women and men (20 59 year) is 60 mg. it appears in table 2 that accessions of bananas that have a high vitamin c content followed by a low total sugar content. this means that there is a negative correlation between vitamin c content and total sugar on 31 accessions of bananas observed. correlation analysis result showed negative correlation equal to 0.41. furthermore, from the chemical characteristic data of vitamin c content and total sugar was used for cluster analysis with the result of dendogram in figure 1. information: 1. ambon jaran : aj 12. kidang hijau : kih 22. morosebo : mro 2. ambon sepet : as 13. klutuk susu : ks 23. potho merah : pm 3. ambon warangan : aw 14. koja pretel : kp 24. raja bandung : rb 4. anjasmoro : am 15. koja sirimentak : ksi 25. raja lini : rl 5. australi : au 16. lempereng : lp 26. raja pulut : rp 6. ceni : ce 17. mangsan : ma 27. raja sabrang : rs 7. gajih putih : gp 18. mas jambe : mj 28. raja sewu : rse 8. kapok brentel : kb 19. mas raja : mr 29. rejang : rj 9. kepok bung : kbu 20. mas sloka : ms 30. serawak : sr 10. kepok hijau : kh 21. mas tropong : mt 31. triolin : to 11. kepok kuning : kk figure 1. dendogram of 31 banana accession of banana germplasm garden collection of based on vitamin c and total sugar content. dendogram of figure 1 shows the kinship relations of 31 banana accessions based on vitamin c and total sugar content. there appears to be some accessions that have very close kinship i.e. accession aj (ambon jaran) with accession lp (lempereng); and accession kk (kepok kuning) with ms accession (mas sloka). but among these accessions there is no equal accession to other accessions, meaning there is no duplication of names between the accessions. and each accession is a separate accession with one accession to another. this result is supported by the morphological character of the fruit, presented in figure 2. name of observation or cluster rj mt ms kk mro kih mj gp ma kbu rp as to sr au ks rb ksi rs aw mr kp rse kb ce am kh rl pm lp aj average distance between clusters 0 10 20 30 40 50 60 70 80 56 biology, medicine, & natural product chemistry 6 (2), 2017: 53-57 dendogram of figure 1 shows that 31 banana accessions based on vitamin c content and total sugar were divided into 5 groups. group i consists of 11 accessions of bananas ie: aj (ambon jaran); lp (lempereng); pm (potho merah); rl (raja lini); kh (kepok hijau); am (anjasmoro); ce (ceni); kb (kepok brentel); rse (raja sewu); kp (kojo pretel) and accession of mr (mas raja). group ii consists of 7 accessions of bananas ie: aw (ambon warangan); rs (raja sabrang); ksi (kojo sirimentak); rb (raja bandung); ks (kluthuk susu); au (australi);. group iii consists of 2 accessions of bananas ie: sr (serawak); and to (triolin). group iv consists of 4 accessions of bananas ie: as (ambon sepet); rp (raja pulut); kbu (kepok bung); and ma (mangsan); and group v consists of 8 accessions of bananas ie: gp (gajih putih); mj (mas jambe); kih (kidang hijau); mro (morosebo); kk (kepok kuning); ms (mas sloka); mt (mas tropong); and rj (rejang). figure 2. bananas fruit of 31 bananas accessions. (a). ambon jaran; (b). ambon sepet; (c). ambon warangan; (d). anjasmoro; (e). australi; (f). ceni; (g). gajah putih; (h). kepok brentel; (i). kepok bung; (j). kepok hijau; (k). kepok kuning; (l). kidang hijau; (m). kluthuk susu; (n). koja pretel; (o). koja sirimentak; (p). lempereng; (q). mangsan; (r). mas jambe; (s). mas raja; (t). mas sloka; (u). mas tropong; (v). morosebo; (w). potho merah; (x). raja bandung; (y). raja lini; (z). raja pulut; (aa). raja sabrang; (ab). raja sewu; (ac). rejang; (ad). serawak; (ae). triolin. indrasari et al. – vitamin c and total sugar content characterization on 31 accessions of banana … 57 conclusion from the result we draw conclusion as follows: 1. the content of vitamin c and total sugar from 31 accessions of bananas vary according to their genetic potential, with an average vitamin c content of 60.42 + 39.22 mg/ 100 g and average total sugar 22.06 + 16.01%. 2. each banana accession from 31 accession was a separate accession one with another accession and is divided into 5 groups based on vitamin c content and total sugar. references anonim. 1993. daftar angka kecukupan gizi yang dianjurkan. widya karya nasional pangan gizi ke v. jakarta komaryati & adi, s. 2012. analisis faktor-faktor yang mempengaruhi tingkat adopsi teknologi budidaya pisang kepok (musa paradisiaca) di desa sungai kunyit laut. kecamatan sungai. kabupaten pontianak. j. tiprekas: 53-61. kristamtini, wiranti, ew., indrasari, sd., wirasti, ch.a., & sutarno. 2016. deskripsi aksesi pisang koleksi kebun plasma nutfah pisang yogyakarta tahun 2016. bptp yogyakarta. harifah, i., mustofa, a., suhartatik, n., 2017. aktivitas antioksidan infuse water dengan variasi jenis jeruk (nipis, lemon, dan baby) dan buah tambahan (strawberry, anggur hitam, dan kiwi). ejurnal.unisri.ac.id/index.php/jtpr/article/download/1517/1335 . retrieved at 23 august 2017. santos, e.a., souza, m.m., viana, a.p., almeida, aaf., freitas, jco., & lawinsky, pr.. 2011. multivariate analysis of morphological charateristics of two species of passion flower with ornamental potential and of hybrids between them. gen. mol. res. 10 (4): 2457-2471. simmonds, n.w. and shepherd, k. 1955. the taxonomy and origins of the cultivated bananas. lennean soc bot. 55: 302312. sitohang, a. pengaruh konsentrasi gula dan suhu pengering terhadap mutu pada pembuatan sirup markisa. media unika tahun 26 no. 87. edisi 1. subandiyah. s, sumardiyono, christanti, rumahlewang & wilhelmina, w. 2002. pengimbasan ketahanan pisang terhadap penyakit layu bakteri (ralstonia solana cearum) dengan pseudomonas cepacia jurnal agrosains 15 (1): 9-16. sukartini. 2007. pengelompokan aksesi pisang menggunakan karakter morfologi ipgri. j. hort 17 (1). wardiyati. t, retnowati. a, & widaryanto, e. 1997. ketahanan empat kultivar pisang terhadap kekeringan. jurnal agrivita 20 (3): 167-170. wardiyati. t, sugianto. a, nugroho. a, lamadji. s, & mugiono. 2004. perbaikan sifat pisang kepok melalui mutasi buatan sinar gamma; keragaman fase genetik. jurnal ilmu hayati 16 (2): 90-98. http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 this page intentionally left blank vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 59-62 | doi: 10.14421/biomedich.2017.62.59-62 issn 2540-9328 (online) alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati1, ahya zhilalikbar amin2, mohamad amin3 1postgraduate, univesitas negeri malang, jl. semarang 5, malang, indonesia 2student of sma negeri 3 malang, sultan agung utara nomor 7 kota malang, indonesia 3department of biology, faculty of mathematics and sciences, universitas negeri malang, jl. semarang 5, malang, indonesia author correspondency: mohamad.amin.fmipa@um.ac.id3 abstract atherosclerosis is a chronic inflammatory disease in the arterial wall mediated by proinflammatory factor. atherosclerosis cause heart disease and stroke is generally believed to be preventable. the single bulb garlic is one of the local plants known as the medicinal plant in indonesia. garlic contains alliin compounds as antiatherosclerotic agent. this study aimed to determine the potential of active compound of single bulb garlic as an antioxidant that improve endothelial function by increasing nitric oxide (no) to increase vasodilation. the bioinformatics webserver used in this study are: pubchem, pharmmapper, swiss target prediction, superpred, uniprot, and protein data bank. docking software using pyrx, pymol and discovery studio. based on these steps, it was found that alliin interacts with nitric oxide synthase (nos) through hydrogen and alkyl bonds. alliin is effective as an inhibitor of nos based on binding afinity (-5.4 kcal / mol) although no more negative than an aspirin inhibitor based on binding afinity (-6.5 kcal / mol). based on the docking results, it is found that alliin is effective as a potential drug to decreased atherosclerosis. keywords: alliin; bulb garlic; nitric oxide; reverse docking; atherosclerosis. introduction indonesia has many medicinal plants containing therapeutic properties and have beneficial pharmacological effects on animal and human body (motalib et al., 2010). garlic is remarkable for the number of compounds it contains, including seventeen amino acids, at least 33 sulphur compounds, 17 amino acids (josling, 2010; febyan et al., 2015), eight minerals (germanium, calcium, copper, iron, potassium, magnesium, selenium and zinc) and the vitamins a, b and c (josling, 2010). in 1944 an italian chemist, c. j. cavallito, as an inventor the isolated an unstable, odorous sulphurcontaining compound with antibacterial properties from extracts of fresh garlic. the name of the substance is allicin, based on the generic name for the plant allium sativum. four years later researchers stoll and seebeck, also working with garlic, discovered an odourless sulphur-containing compound called alliin (josling, 2010). they found that this compound would be converted by a second garlic constituent, an enzyme called allinase, to form allicin. the researchers made an additional, remarkable discovery: when they studied the cloves in cross-section they found that alliin and allinase were stored in different compartments. in an undamaged clove they remained completely separate, but once its structure was rupturedtypically by cutting-the two substances came into contact and formed allicin. atherosclerosis is a chronic inflammatory disease of the arterial wall, especially oxidation of lipoproteins that stimulate the innate immune response and adaptive immune response (tedgui & mallat, 2006; ait-oufella et al., 2011). atherosclerosis cause stroke, hypertension, and coronary heart (tanuwijaya, 2003). atherosclerotic cause by free radicals (sumarno et al., 2012) and unhealthy lifestyle (rahman, 2012). the high free radicals in the body cause degenerative diseases (winarsi, 2007). unhealthy lifestyle like high fat diet cause endothelial disfunction. endothelial disfunction causes decrease production of vasodilation compounds such as nitric oxide (no) (oparil et al., 2003; devlin, 2002). no is the smallest signal molecule produced by three isoforms of nitric oxide synthase (nos) (forstermann & sessa, 2011). the no formation enzymatic of l-arginine is catalyzed by nitric oxide synthase (nos) (schrijvers et al., 2004). therapy used to reducing inflammation and improving endothelial function or rupture of plaque is done by aspirin. drug use to reduce inflammation has side effects. aspirin cause gastrointestinal irritation and bleeding (majid, 2008), therefore safe therapies are required without harmful side effect in patients with atherosclerotic. safe therapy using herbal drug that http://dx.doi.org/10.14421/biomedich.2017.62.59-62 60 biology, medicine, & natural product chemistry 6 (2), 2017: 59-62 contain biochemical compounds, one of them is garlic in indonesia such as single bulb garlic. alliin compounds are potential as antioxidant (febyan et al., 2015) and antiatheroslerotic (hernawan & setyawan, 2003). endothelial disfunction causes a decrease in enos activation resulting in reduced no production (danuyanti et al., 2014). alliin contained in allium sativum lowers blood pressure through complex pathways resulting in vasodilation so that decreasing of plaque atherosclerotic. no is synthesized by nitric oxide synthase (nos) from l-arginine. after synthesis, no diffuses from endothelial cells to smooth muscle cells of the blood vessels and causes an increase in intracellular cyclic guanosine monophosphate (cgmp) to relaxation of smooth muscle of blood vessels so that vasodilation occurs (mckeever, 2009). objective this study aimed to discover alliin compound from single bulb garlic for antiatherosclerotic agent based on insilico screening. methodology ligand preparation the chemical structure of 3d and smiles ligands (alliin) is taken from pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) with id number: 87310. target selection input alliin’s smiles on pharmmapper (http://lilab.ecust.edu.cn) to identify potential target candidates using mapping approaches swiss target predictions (http: //www.swisstargetprediction.c/) and chemical structure associations with molecular 3d (gong et al., 2013). molecular docking molecular docking alliin, target protein, and known inhibitors of target protein used pyrx 0,8 software. visualization of molecule and small molecule interaction the interactions between alliin, target protein, and inhibitors were analyzed using pymol. results and discussion the results of target selection using pharmmapper (job id: job id: 170.228.034.654) dan swiss target prediction, found that alliin interact with nitric oxide synthase (nos) in human vascular endothelium cells. no accumulation induces the formation of a strong oxidizing agent, peroxinitrite (schwartz et al., 2002). nos became an interesting object of this study for the development of drugs as inhibitors of atherosclerotic plaque reduction. alliin as a drug effectively inhibit atherosclerotic disease (zhang et al., 2001; borek, 2001; seo et al., 2012). garlic extract can lowering cholesterol levels significantly, reducing the thickening of the aortic wall and reducing fat accumulation in macrophage cultures. the mechanism is related to the garlic on cholesterol metabolism (campbell et al., 2001). figure 1. (a) the interaction between target protein (nos) with alliin and aspirin inhibitor suggests that binding target protein on the same site. green (alliin), red (aspirin), yellow (nos); (b) interactions through hydrogen and alkyl bonds of ile b: 161, asn b: 157, lys b: 160, and thr a: 146. ilmawati et al. – alliin as a natural bioactive from single bulb garlic (allium sativum) … 61 reverse docking is a method used to predict the interaction activity between ligand (compound / drug) with receptor (protein/target) (kharkar et al., 2014). based on a reverse docking of nos (gdp id: 5unr) with a resolution of 1.95 å, aliin is known as an inhibitor of nos informing that alliin has binding affinity (-5.5 kcal/ mol). the result of molecular visualization and molecular interaction using pymol software found that alliin bind to target protein (nos) at the same site as aspirin inhibitor via hydrogen and alkyl bond of ile b: 161, asn b: 157, lys b: 160, and thr a : 146 as shown in figure 1. so it can be said that alliin has the same function as aspirin as nos inhibitor in atherosclerotic. decreased levels of no cause vasoconstriction of blood vessels and endothelial becomes more proaterogenic and proinflammation (kuzkaya et al., 2003). no plays an important role in blood vessels such as vasodilation, inhibits smooth muscle cell proliferation, platelet aggregation, monocyte and platelet adhesion, low density lipoprotein oxidation (ldl), expression of molecular adhesion and endothelin production (behrendt & ganz, 2002; yetik-anacak & catravas, 2006). alliin as inhibitors of atherosclerotic plaque reduction with improves component of vasodilation. no is synthesized by nitric oxide synthase (nos) from larginine. this arginine will be changed by the enzyme nitrite oxidase becomes no. no stimulate guanylate cyclase to change gtp (guanosine triphosphate) becomes cyclic-gmp to activates protein kinase g which causes the re-taking ca2+ and opening potassium channel which is activated by calcium. decrease of ca2+ concentrations myosin light-chain kinase (mlck) cannot phosphorylates myosin longer, thus stopping the cycle cross bridge and cause relaxation smooth muscle cells of the blood vessels so that vasodilation occurs (ishimura et al., 1998 & mckeever, 2009). allium sativum with water will break down into diallyl sulfide, diallyl disulfide and diallyl trisulfide which then merge into the organic polysulfide that causes red blood cells to produce h2s (hydrogen sulfide). h2s will bind and activate channel katp, so the concentration of ca2+ cell will decrease and hyperpolarization occur vascular smooth muscle cell that cause vasodilation of blood vessel (mckeever, 2009). the result of reverse docking show that alliin interact with nos that can increasing no product, so it can be seen that nos acts as an atherosclerotic inhibitor. conclusion this study proved that alliin has potential as an nos inhibitor based on binding affinity and intermolecular interaction. alliin 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in atherosclerosis from theory to clinical practice. semarang: universitas diponegoro. tedgui, a. & mallat, z. 2006. cytokines in atherosclerosis: pathogenic and regulatory pathways. physiol rev. 86: 515– 581. winarsi, h. 2007. antioksidan alami dan radikal bebas. cetakan ke-2. yogyakarta: kanisnus. p: 11-121. yetik-anacak, g. and catravas, j. 2006. nitric oxide and the endothelium: history and impact on cardiovascular disease. vascul. pharmacol. 45 (5): 268–76. zhang, x., lowe, d., giles, p., fell, s., connock, m., and maslin, d. 2001. gender may affect the action of garlic oil on plasma cholesterol and glucose levels of normal subjects. journal of nutrition. 131: 1471-1478. alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 441-450 | doi: 10.14421/biomedich.2023.122.441-450 issn 2540-9328 (online) alkaloids lead to potential inhibition of the acyl carrier protein reductase to attenuate tuberculosis; an in-silico analysis pernia kamran, ahsan ibrahim* shifa college of pharmaceutical sciences, shifa tameer-e-millat university, islamabad-44000, tel. +9251-8438061, pakistan. corresponding author* ahsan.scps@stmu.edu.pk abstract tuberculosis (tb) is a contagious infection that mostly affects the lungs. mycobacterium tuberculosis causes tuberculosis infection, leading to granulomatous lesions in affected lung tissue. it is one of the most prevalent and deadly infectious diseases among the under developed countries. this study aims to investigate the possible inhibition of the acyl carrier protein reductase for preventing tuberculosis by well-known alkaloids, thereby reducing mycobacterium tuberculosis growth in the lungs and thereby reducing the incidence of latent and active tb. about five natural alkaloids were subjected to the molecular docking analysis, which produced favorable findings in terms of best pose and binding energies of these compounds towards the active residues of mycobacterial acp reductase, with values ranging from -10 kcal/mol to -9.1 kcal/mol. the molecular dynamics simulation produced similar encouraging results. all of the prospective alkaloid compounds were subjected to an in-silico toxicity investigation, which determined that every compound was safe and non-toxic. further studies may be necessary for effective formulation development employing these compounds as part of the process of drug discovery and development. the findings from this study may be helpful in the development of the novel nanoformulations using natural products for pharmacotherapy of tuberculosis infection. keywords: tuberculosis; alkaloids; acp reductase; molecular docking analysis; mycobacterium tuberculosis. abbreviations: mtb: mycobacterium tuberculosis, covid-19: corona virus disease 19, hiv: human immunodeficiency viruses, aids: acquired immunodeficiency syndrome (aids), acp: acyl carrier protein, pdb: protein data bank. introduction tuberculosis (tb) is an infectious disease caused by the bacteria mycobacterium tuberculosis. globally, tuberculosis is the 13th leading cause of death after covid-19 (ahead of hiv and aids) and the second infectious disease killer. in 2021, approximately more than 10 million people worldwide were infected with tuberculosis. this infection affects the people of all age groups in many countries (who, 2023). mycobacterium tuberculosis (mtb) has a unique cell envelope that is linked to its pathogenicity. the presence of trehalose dimycolate (tdm), a free glycolipid that accumulates in a cord-like pattern on the surface of the cells and protects the tubercle bacillus from harmful agents and the host's immune system, is another feature of the cell envelope that is very noticeable. tdm is covalently linked to the cell wall peptidoglycan via an inner leaflet of the outer membrane by a phosphodiester bond. the primary components of this protective layer are mycolic acids. more precisely, the cyclopropane rings in mycolic acid layer help maintain the complex's structural integrity and shield the bacillus from oxidative stress (takayama et al, 2005). the development of an immune response is necessary for the control of mtb infection in individuals who are resistant to the infection. this type of response entails the involvement of resident alveolar macrophages, dendritic cells, t lymphocytes (tcd4+, tcd8+ cells) and the release of proinflammatory cytokines such as interferon-gamma (ifn γ), interleukin-2 (il-2), il-12, il-18, and tumor necrosis factor –alpha (tnfα). all of these are crucial for attracting more immune cells to the infection site to create a granuloma that traps and destroys tuberculosis bacilli while also providing the long-term niche required for latent tuberculosis infection (druszczyńska, kowalewicz-kulbat, fol, włodarczyk, & rudnicka, 2012). if the ongoing bacterial reproduction is not stopped, the bacilli and expanding tubercle may invade the nearby draining lymph nodes. as a result, lymphadenopathy develops, which is a typical sign of primary tb. if the lesion caused by the tubercle's expansion progresses to the lymph node and lung parenchyma, a ghon complex may form. the ghon focus, which describes the main tb infection, is often seen in the center of the lungs and is a hallmark of tb manuscript received: 22 july, 2023. revision accepted: 08 august, 2023. published: 14 august, 2023. https://doi.org/10.14421/biomedich.2023.122.441-450 442 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 infection. latent tb reactivates into active tuberculosis in the presence of immunosuppression in the host (ruiru & isamu, 2013). changes in the host's physiological and immunological condition brought on by aging, malnutrition, diabetes or the emergence of other illnesses, including hiv/aids, as well as latent infections, might trigger their activation. the use of many antibiotics given over the course of six months is required for chemotherapy for active tb caused by drugsensitive bacteria. patient compliance is typically low as a result of this difficult and potentially hazardous treatment regimen. as a result, antibiotic-resistant bacteria have developed, necessitating lengthier treatment regimens, the use of less effective and more toxic medications, and increasing failure rates (reddy et al., 2008). the aim of this study is to analyze the anti-tb effects of several natural alkaloids and their potential to weaken the pathogenesis of tuberculosis using in-silico tools. in order predict the potential activity of alkaloids against the active tb infection, this study utilized molecular docking and molecular dynamics simulation as methodological techniques. the pathophysiology of tuberculosis infection is depicted in figure 1. figure 1. the figure illustrates the pathogenesis of tuberculosis infection caused by mycobacterium tuberculosis. material and methods macromolecule preparation rcsb (protein data bank) was used to retrieve the 3d structure of mycobacterial acp (acyl carrier protein) reductase enzyme (pdb id: 4r9s) in protein data bank (pdb) format. pdb contains the structure elucidation data of proteins obtained by x-ray crystallography and nuclear magnetic resonance (nmr) spectrometry submitted by biologists and biochemists from all over the world. macromolecule was prepared by removing water molecules, heteroatoms, ligands, extra protein chains, and metal ions that could interact with compounds during visualization of docking between compounds and target proteins using biovia discovery studio visualizer v17.2.0.16349 (sharma et al, 2021). ligand preparation the compounds used as ligands for the antimicrobial activity were from the alkaloidal class of phytochemicals. the structures of these compounds were downloaded from the pubchem database and chem3d 16.0 was used to obtain the pdb format of these 3dstructured compounds. c1 (protopine), c2 (apropine), c3 (avicine), c4 (penduline), and c5 (chelerythrine) were used to study the potential inhibition of mycobacterial acyl carrier protein reductase for preventing tuberculosis infection. these five compounds were used against the target protein to check the interaction of the target protein with these compounds (c1c5). figure 2 shows the graphical representation of the complete methodology of the research study. kamran & ibrahim – alkaloids lead to potential inhibition of the … 443 the chemical structures and botanical sources of compounds (c1 c5) are provided in table 1 while figure 3 depicts the iupac names of the compounds c1 – c5. figure 2. the figure shows the step by step methodology of the research study. table 1. the table shows the names of alkaloids employed in this study, their pubchem id, chemical structures and botanical sources. compound compound name pubchem id structure botanical source literature source c1 protopine 4970 corydalis heterocarpa var. japonica pubchem c2 aporphine 114911 antizoma angustifolia pubchem c3 avicine 356657 zanthoxylum asiaticum pubchem c4 penduline 179472 leptopus cordifolius pubchem c5 chelerythrine 2703 zanthoxylum fagara pubchem 444 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 figure 3. the figure demonstrates the chemical structures of compounds c1 to c5, along with their iupac names obtained from pubchem. results and discussion molecular docking analysis & results in structural molecular biology, medicinal chemistry and computer-assisted drug development, molecular docking is a useful technique. predicting the prevailing binding modes and poses of a compound that interacts with a protein having a known three-dimensional structure is the major aim of compound-protein docking. successful docking methods employ a scoring system that accurately ranks the drug candidate dockings and efficiently explores binding affinities and best poses. lead optimization greatly benefits from the use of docking to do virtual screening to provide insights for how the ligands interfere with the target (fan, fu, & zhang, 2019). pyrx was utilized for this experiment. pyrx includes a docking wizard with an easy-to-use user interface, which makes it a valuable tool for computeraided drug design (cadd). pyrx also includes chemical spreadsheet-like functionality that helps in novel drug molecule designing (dallakyan & olson, 2015). software version 17.2.0.16349 of biovia discovery studio visualizer was used. it offered a clear depiction of the 2d and 3d interactions as well as bond lengths, aromatic, hydrophobic, and other interactions. results from the molecular docking studies of the mycobacterial acp protein with compounds c1–c5 were valuable, as shown in table 2. table 2. the table shows the findings and results of the molecular docking analysis done using compounds c1 to c5. compound-protein interaction binding energy (kcal/mol) bonding residues type of bond bond length (å) other residues c1 -10 phe 41 pi pi 4.55 ile 15 ile 95 pi sigma 3.41 thr 39 carbon hydrogen 3.64 c2 -9.8 phe 41 pi pi stacked 3.74 val 65, ile 122. ile 95 pi alkyl 5.23 c3 -9.7 phe 41 pi pi stacked 4.26 ile 122, ile 16 ile 95 pi sigma 3.86 asp 64 carbon hydrogen 3.27 ser 20 conventional hydrogen bond 2.87 c4 -9.6 phe 97 pi pi t shaped 5.92 leu 207, met 103, ile 21, leu 207 ile 16 pi sigma 4.00 c5 -9.1 phe 41 pi pi stacked 4.30 gly 14, ile 122 , phe 97 ile 16 pi sigma 3.78 asp 64 carbon hydrogen bond 3.44 ser 20 conventional hydrogen bond 3.26 kamran & ibrahim – alkaloids lead to potential inhibition of the … 445 all the compounds c1 to c5, exhibited their firm interactions with the binding site residues of mycobacterial acp reductase enzyme receptor i.e., phe 41, ile 95, ile 16 and ser 20 with a binding pose ranging from – 10 to -9.1 kcal/mol. the best binding pose with binding energy of – 10 kcal/mol was expressed by protopine (c1), while the binding energies exhibited by other compounds in this study were -9.8 kcal/mol for aporphine (c2), -9.7 kcal/mol for avicine (c3),-9.6 kcal/mol for penduline (c4) and -9.1 kcal/mol for chelerythrine (c5).all the compounds showed absolute interactions with the active site residues of mycobacterial acp reductase enzyme. protopine (c1), aporphine (c2), and avicine (c3) were bound to phe 41 and ile 95 residues with lower binding energies. penduline (c4) and chelerythrine (c5) manifested binding with phe 97, ile 16, phe 41, ile 16, asp 64, ser 20 residues with reasonable bond lengths. compounds protopine (c1) and aporphine (c2) also exhibited firm interactions with other residues including ile 15, val 65, and ile 122, that may also be the active site amino acid residues of mycobacterial acp reductase enzyme. avicine (c3), penduline(c4) and chelerythrine (c5) demonstrated robust binding affinities with other active residues ile 122, ile 16, leu 207, met 103, ile 21, leu 207 and gly 14, ile 122, phe 97 with substantially shorter bond lengths. figure 4 demonstrates the interactions of all compounds with mycobacterial acp reductase enzyme. admet analysis absorption, distribution, metabolism, elimination and toxicity (admet) analysis is essential for navigating the pharmacodynamics and pharmacokinetics of a potential drug candidate. in-silico adme parameters were studied using swiss adme. the druglikeness score and toxicity profile of the potential drug candidates was probed using datawarrior v5.5.0. table 3 shows the pharmacokinetic and toxicity parameters of the potential drug candidates from c1 to c5. figure 5 manifests the bioavailability radar of best docked ligand, compound c1, obtained from swissadme tool. figure 4. the figure displays the 3d and 2d images of the interactions between active amino acid residues of mycobacterial acyl carrier protein reductase (pdb id: 4r9s) and compounds (c1, c2, c3, c4 and c5). 446 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 table 3. the table provides the pharmacokinetic and toxicity profile of compounds (c1 – c5) using swissadme and datawarrior tools. admet profiling of the potential drug candidates (c1 – c5) potential drug candidates c1 c2 c3 c4 c5 physicochemical properties molecular weight (g/mol) 353.37 g/mol 235.32 g/mol 332.33 g/mol 608.72 g/mol 348.37 g/mol log p 2.67 3.23 2.84 5.17 3.02 h-acceptor 6 1 4 8 4 h-donor 0 0 0 1 0 n.r.b 0 0 0 3 2 tpsa (å²) 57.23 å² 3.24 å² 40.80 å² 72.86 å² 40.80 å² bioavailability score 0.55 0.55 0.55 0.55 0.55 drug-likeness parameters lipinski’s rule violations 0 violations 0 violations 0 violations 1 violation 0 violation drug-likeness score 4.7237 4.9313 2.4707 4.6261 2.1842 metabolic profiling p-glycoprotein substrate yes yes yes no yes gi absorption high high high high high cyp 3a4 yes no no no yes cyp 2d6 yes yes no no no cyp 1a2 yes no yes no yes cyp 2c9 yes no no no yes cyp 2c19 no no yes no yes toxicity profiling tumorgenicity no no no no no mutagenesis no no no no no irritant no no no no no reproductive effects no no no no no mol. w (g/mol) molecular weight, log p prediction of octanol/water partition coefficient, n.r.b number of rotable bonds, tpsa (å²) topological polar surface area, cyp cytochrome p-450 enzyme. figure 5. the figure the bioavailability radar of ligand c1 (having the most favorable binding energies as per the findings of molecular docking). molecular dynamic simulation molecular dynamic (md) simulations depict the dynamics of a macromolecule bound by a ligand, interactions between the amino acid residues and navigating the configurations of a protein molecule (y. wang, ribeiro, & tiwary, 2020). md simulations unveil the dynamics of a protein and assist in discovering the cryptic sites in a protein macromolecule that may have affinity towards a ligand, thereby facilitating its binding to the hydrophobic pocket (kuzmanic, bowman, juarezjimenez, michel, & gervasio, 2020). md simulations were done using the online md simulation tool, imods server (lópez-blanco, aliaga, quintana-ortí, & chacón, 2014; lopéz-blanco, garzón, & chacón, 2011). the ligand and macromolecule docked complex c1 (protopine) was loaded on imods server and md simulation was performed on normal mode analysis (nma). the b-factor yielded information regarding the flexible nature of the macromolecule. it illustrates the deforming tendency of the protein residues in order to interact with protopine molecule. the arrow field shows the orientation of the macromolecule in the virtual experiment. the figures 6,7 and 8 provide the graphical presentation of the finding of molecular dynamic simulation analysis. kamran & ibrahim – alkaloids lead to potential inhibition of the … 447 figure 6. the figure illustrates the orientation of the docked complex (c1 and macromolecule) obtained through molecular dynamic simulations using imods. the eigenvalue for the complex in this virtual experiment was measured as 5.340225e-04. the eigenvalue manifests the energy required to deform a particular amino acid residue while interacting with other residues in a reallife environment created virtually. variance is inversely proportional to eigenvalue and the comparison between the two is shown in the figure. figure 7. the figure shows the findings of molecular dynamic simulation i.e. image (a) shows the deformability of the docked complex. image (b) provides the graphical view of the bfactor. image (c) provides the eigenvalue for the complex while the image (d) demonstrates the percentage variance. the covariance map displays the interactions between the residues of the dynamic protein molecule, with red areas showing the correlating or interacting residues, white areas demonstrating the residues with no interactions, and blue areas showing residues with repulsive behaviour within the protein molecule. the elastic network provides an estimate regarding the ease of deformation of the target amino acid residues within the macromolecule. the dark greyish areas show residues with a higher degree of stiffness, while the lighter areas depict ease of deformation of the target residues. 448 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 figure 8. the image (a) displays the covariance graph of the complex (c1 and macromolecule) while image (b) represents elastic network map of the complex. discussion in this study, five alkaloids were screened computationally for their potential to inhibit the mycobacterial acyl carrier protein reductase and attenuate tuberculosis. molecular docking and scoring is a reasonable method to analyse various drug candidates for their potential for particular biological activity before entering the wet lab (torres, sodero, & jofily, 2019). the results of the molecular docking analysis illustrated optimistic outcomes. all five compounds exhibited strong interactions with the target protein. the binding energies of the best poses of these potential drug candidates were in a range of -10 to -9.1 kcal/mol. protopine (c1) provided most suitable binding energies and poses. strong bonds with catalytic amino acid residues having considerably shorter bond lengths were observed with all five compounds. the compounds showed firm interactions with phe 41, ile 95, and ser 20 residues. the compounds (c1– c5) were alkaloids with multiple botanical sources and a vast range of biological activities. protopine (c1) is obtained from papaver somniferum (pubchem, 2023). the compound has also been tested for its potential biological activity against cancer, alzheimer’s disease, inflammation and other ailments (son et al., 2019; sreenivasmurthy et al., 2022; zhang et al., 2019). in this study, the protopine has been tested in-silico for its potential anti-mycobacterial effect and auspicious results have been furnished in this regard. the second drug candidate, aporphine (c2) has been widely tested for its anti-inflammatory, analgesic, antiparasitic and anti-oxidant effects in various in-silico, invitro and in-vivo models (pieper, mchugh, amaral, tempone, & anderson, 2020; b. wang et al., 2019; r. wang, zhou, shi, liu, & yu, 2020). the exploration of anti-tuberculosis activity was done in this study and promising outcomes were witnessed. the biological activity of avicine (c3) in alzheimer’s disease is well established through animal models (cahlíková et al., 2021; plazas, 2020). avicine has also displayed hopeful anti-mycobacterial activity predictions in this study. penduline (c4) has been studied for its anti-cancer and anti-inflammatory activities while chelerythrine (c5), a potent inhibitor of protein kinase c, has revealed to be active against cancer, bacterial infections and covid 19 (gong et al., 2019; grabarska et al., 2021; lin et al., 2017; valipour et al. 2021). both of these alkaloids have furnished significant results in the molecular docking analysis in this study. the molecular dynamics simulations have further explored the dynamic behavior of the targeted protein. isoniazid is a well-known inhibitor of mycobacterial enoyl-acp reductase (unissa et al, 2016). isoniazid inhibits the synthesis of mycolic acid in mycobacteria and halts the progression of cell wall synthesis, thereby providing bactericidal effects (vilchèze & jacobs, 2019). isoniazid is one of the anti-tubercular drugs that have indicated notable efficacy through the decades. (charan et al., 2018; huang et al., 2018; pease et al., 2017). keeping in view these facts, severe toxicities and adverse drug reactions are also associated with the use of isoniazid in the treatment regimen for tuberculosis (bliven-sizemore et al., 2015). the hepatotoxicity and peripheral neuritis are the most significant toxicities associated with isoniazid (lamb & mauermann, 2021; mafukidze, calnan, & furin, 2016; m russom et al., 2019; mulugeta russom et al., 2018). these considerable toxicities demand a safer treatment option with similar efficacy. in this study, potential drug candidates, i.e., natural compounds (alkaloids) with no reported toxicity and strong affinity for target proteins, could attenuate tuberculosis infection after the development of a stable formulation. this study recommends the formulation scientists to employ novel drug formulation techniques for formulating natural products and secondary metabolites such as alkaloids as nanocarriers in order to achieve their delivery to the targeted sites, maintain efficacy and avoid any undesirable or noxious effect. natural products and phytochemicals have caught the attention of formulation scientists and pharmacologists to a greater extent. the coupling of natural products and kamran & ibrahim – alkaloids lead to potential inhibition of the … 449 nanomedicine can make major breakthroughs in the field of therapeutics. this study also puts emphasis on this idea. conclusion tuberculosis is a public health concern that claims millions of lives annually. the current therapy for tuberculosis has many challenges, with toxicity at the top of the list. natural products have been used traditionally owing to their minimal toxicities and decent efficacy. many studies have established that phytochemicals can attenuate bacterial infections such as tuberculosis. through in-silico analysis, this study has concluded that the alkaloids such as protopine, aporphine, avicine etc. have potential inhibitory activity for the mycobacterial acyl carrier protein reductase enzyme, hence disrupting mycobacterial survival and pathogenesis. the molecular docking and molecular dynamics simulation provided optimistic results regarding the interactions of these alkaloids with the active amino acid residues of mycobacterial acyl carrier protein reductase. this study recommends the conduct of in vitro and in vivo studies in order to validate the findings of this research study and the development of novel formulations for better delivery of these alkaloids for attenuating tuberculosis. acknowledgements: the study is carried out entirely by the authors, so there is no need for acknowledgement. authors’ contributions: ahsan ibrahim designed the study. pernia kamran and ahsan ibrahim carried out the computational work. pernia kamran & ahsan ibrahim wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references bliven-sizemore, e. e., sterling, t., shang, n., benator, d., schwartzman, k., reves, r., . . . consortium, t. t. 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(2019). protopine protects mice against lps-induced acute kidney injury by inhibiting apoptosis and inflammation via the tlr4 signaling pathway. molecules, 25(1), 15. untitled-1 biology, medicine, & natural product chemistry volume 5 – number 2 – 2016 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi, syafiana khusna aini, dewi anggraini, dian prihatiningtias ekawati optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati, muhammad jafar luthfi effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti, endaruji sedyadi, susy yunita prabawati a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto, fajar adi kusumo, lina aryati, mardiah suci hardianti 33-40 41-47 49-53 55-59 61-64 volume 5 number 2 2016 published twice a year printed in indonesia front cover: hoya sp. (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 423-430 | doi: 10.14421/biomedich.2023.121.423-430 issn 2540-9328 (online) weed community structure in patia village rice fields patia sub-district, pandeglang regency ai widiyawati, hadi susilo, mu'jijah, suyamto*, nurullah asep abdilah biology study program, faculty of health pharmacy, mathla'ul anwar university banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 07 may, 2022. revision accepted: 27 july, 2023. published: 10 august, 2023. abstract rice is a very important type of food crop because it is a staple food source. crop losses at the farmer level due to competition with weeds reach 10-1.5%. this study aims to determine the structure of rice weed communities in rice fields in patia village, patia district, and pandeglang regency. the research method uses an observation method with the sampling technique used as a random method. the plot used was 2 m x 2 m. weed sampling was carried out by recording the types of weed types and counting the number of individuals of each species. measurements of environmental factors such as temperature, humidity, ph, and light intensity were measured. the data parameters measured include the shannon-wiener diversity index (h'), evenness index (j'), dominance index (d), wealth index (s), and abundance of rice weed species. the results of the study were that the types of rice weeds found in the rice fields of patia village, district, and patia, pandeglang regency, namely ludwigia octovalvis (jacq) raven, commanded cylindrical. beauv, spigelia anthelmia, ageratum conyzoides, spihemoclea zeylanica, altemanthera sessillis, eclipta prostate, cypress deformed l., digitaria sp. and mimosa chaste l. the structure of the weed community in the rice fields of patia village, patia district, pandeglang regency is the shannonwiener diversity index (h') of 1.94; evenness index (j') of 0.884; dominance index (d) of 0.15. keywords: weeds; competition; rice; productivity; community structure. introduction rice (oryza sativa l.) is an important crop commodity because it is the staple food of the population in indonesia and plays an important role in economic and social stability in indonesia. the population of indonesia reached 270.20 million people with rice production in 2020 reaching 31.33 million tons (bps, 2021). efforts to support the rice self-sufficiency program and meet rice needs need to maintain the stability of rice production. rice production in indonesia can decline due to several factors, including drought, land narrowing, pest disturbance and the influence of weeds (windari et al., 2021). weeds in rice plants are one of the problems that greatly affect productivity (miranda et al 2011). the presence of weeds on rice plants will cause a decrease in production if weeds are not controlled effectively because they can affect growth and reduce food crop production. planting paddy rice using irrigated irrigation will cause weed competition with rice so that it can reduce yields by 10-40% depending on the species and density of weeds, soil type, water supply and climatic state. rice yield losses at the farmer level due to the presence of rivals with weeds reach 10-15% (farmanta et al., 2016). the presence of weeds on cultivated plants will reduce crop yields. losses caused by weeds are caused by competition with cultivated plants in terms of nutrient extraction, water, sunlight and growing space. in addition, weeds can secrete allelopathic compounds and can be hosts for pests and pathogens of cultivated plants (utami &; purdyaningrum, 2012). one of the ways of weed control is by identifying the diversity and dominance of weeds. the results of dahlianah's research (2017) stated that there are 4 families, 5 genera, and 7 species of weeds in tidal rice fields in manggaraya village, tanjung lago district, banyuasin regency, south sumatra province. the structure of rice weeds in tidal fields with the highest inp value is the weed type echinochloa chicken leg (41.16%). the results of agustina & yursida's research (2015) showed that weeds were dominant in rice plantations in tidal land, banyu urip village, tanjung lago district, with an average ratio the highest number of successive predominations were: ludwigia octovalvis, fymbristilis littoralis, alternanthera philoxeroides and cyperus chicken. weed control is effective if we know the types of weeds in rice fields. according to kastanja (2011) the success of weed control must be based on sufficient and correct knowledge of the biological nature of weeds https://doi.org/10.14421/biomedich.2023.121.423-430 424 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 through identification, this method is a step first to explore the possibility of appropriate control. efforts made to determine the type and population of weeds are 1) exploring a rice field, which aims to collect data on weed populations growing in the planting area rice, 2) identification which is an effort to recognize something that observes the characteristics of plants that are analyzed simply. local rice plants often find various types of weeds, but there is still little information or research that explains the types of weeds found in rice fields in the province banten, especially in patia village, patia district, pandeglang regency. research methods the type of research to be carried out is nonexperimental research carried out with exploration methods, namely identifying rice weeds in rice fields in patia village, patia district, pandeglang regency. research tools and materials equipment used in the study includes machetes, boxes, shovels, uls, tape meters, notebooks, stationery, rapia ropes, smartphone cameras, rulers, pegs, google earth, label paper, soil tester, newspaper, cardboard, cardboard and hand sprayer or spray. the material used in this study was rice weeds in rice fields in patia village, patia district, pandeglang regency. procedure ▪ research site survey the survey was conducted by exploring villages covering 5 rws randomly. the survey was also conducted through interviews with local farmers. the existence of rice cultivation, the age of rice, the agricultural system and the presence of rice weeds obtained are then recorded in the research notebook. photo documentation is also carried out during the survey. ▪ determination of research blocks purposive determination of rice blocks based on criteria that are rice plantations using conventional monoculture agricultural systems with non-intensive application of pesticides and rice age 3 weeks after planting and not being carried out weed cleaning. the research block was made as many as 5 blocks in 5 rws because patia village consists of 5 rws. the determination of blocks 1, 2, 3, 4 and 5 is carried out sequentially according to the rw sequence number. in each research block, the measurement of coordinate points was carried out using google earth. ▪ plot creation in each research block, a plot with a size of 2x2 m was made in a square shape using 4 pegs around the sides given raffia rope and the distance between the plot and the ripen was 1 m. the placement of plots in research blocks is carried out randomly. ▪ measurement of environmental factors microclimate components that affect growth are air and soil humidity, air and soil temperature so it is very necessary to measure environmental factors such as temperature, humidity and soil ph. measurement of environmental factors was carried out in the morning at 08.00 wib when sampling rice weeds in the research plot. measurement of environmental factors in the form of ph, humidity and mpatur using a soil tester. ▪ rice weed sampling weed sampling was carried out at 08.00 wib. weed sampling was carried out on each research plot. weeds taken are weeds that have complete organs. picking is not carried out on weeds that are still in the sprouting stage. weeds are taken whole (leaves, stems, and roots) with a small shovel then the roots are carefully separated from the ground then put into a plastic bag to take to the laboratory. documentation is carried out during the weed picking process. the number of weeds grown is obtained by counting the number of weeds growing on each plot plot, then counting all the number of individuals in the weed plot plot. speed esimen was later identified. ▪ weed identification identification of rice weeds was carried out by observing external morphological characters such as leaves, stems and roots and adjusted based on description and identification with reference to the book flora steenis (2008). ▪ data collection weeds that have been identified and species determined, then the data are entered into a table and include the number of individuals in each type. the table of types, types and quantities of weeds is then used in data analysis. data analysis test parameter data used in this study including shannon wiener diversity index (h'), evenness index (j'), dominance index (d), wealth index (s) and abundance of weed species with the formula dombois & ellenberg (mazidaturohmah et al., 2018) widiyawati et al. – rice weed community structure in patia village rice fields … 425 results and discussion types of weeds in rice fields table 1. types and numbers of rice weed individuals in patia village, patia district, pandeglang regency. species region name number of individuals per research block plot 1 plot 2 plot 3 plot 4 plot 5 cecabean ludwigia octavalvis (jacq) raven 14 16 18 8 5 for sake cylindrical orders l. beauv 11 3 14 9 17 meniran spigelia anthelmia 2 0 1 0 3 friendship ageratum conyzoides 5 1 6 7 5 gunda spihemoclea zeylanica 1 2 1 0 4 crema altemanthera sessillis 4 4 2 2 3 how-to eclipse the prostate 2 0 5 4 7 puzzle cypress is deformed l 7 8 10 9 8 jampang grass digitaria sp 4 7 7 6 8 princess malu mimosa chaste l 2 1 0 3 1 the results showed that the types of weeds in the rice fields of patia village, patia district, pandeglang regency include ludwigia octovalvis (jacq), raven, cylindrical ordersl. beauv, spigelia anthelmia, ageratum conyzoides, sphenoclea zeylanica, altemanthera seated, eclipta prostate, cypress deformed l., digitaria sp. and mimosa pudica l. vegetation in rice fields is always damaged periodically due to tillage, or due to control, but there is weed vegetation that always appears in the rice field area. the emergence of weeds in rice fields can be categorized as secondary succession; the community can change in a progressive or retrogressive manner (purnomo, 2011). in the agricultural sector, weeds are plants that have a negative impact on cultivated plants either directly or indirectly. weeds that interfere with productive plants during the growth and development of plant life are one of the important problems that can reduce crop production. the percentage of decline in production of each type of cultivated crop is influenced by the weed community in the agricultural area (suryatini, 201: 8). weeds as plant nuisance organisms (opt) are among the important obstacles that must be overcome in increasing rice production in indonesia. the decline in rice productivity is caused by, among others, competition between weeds and rice plants. the decrease in rice yield due to weeds ranges from 6-87% of weeds have a very competitive nature because they have an efficient breeding mechanism that is able to multiply sexually by producing many seeds and vegetatively, so that it greatly reduces the results of cultivated plants. the decline in crop yield varies greatly depending on various factors, including the ability of plants to compete, types of weeds, plant age and weed age, techniques, their cultivation and duration of competition (utami &; purdyaningrum, 2012). weeds reduce crop yields in competition for light, oxygen and co2, as well as food. the decrease in crop yield is caused by weeds that can reduce growth activities, including stunted plant growth, chlorosis, nutrient deficiencies, and the occurrence of reduction in the number and size of plant organs. symptoms of nutrient deficiency in rice plants can result in complete failure of seedling plants, very stunted plants, symptoms on leaves that are distinctive, and abnormalities – abnormalities arising in plant tissues (antralina, 2012). weeds do not always harm plants, at the population level or certain periods have no effect or little effect on plants so that weeds that grow h in that period are unnecessary controlled. therefore the way of cultivation, the type and density of weeds will largely determine when weeding weeds. the composition of weeds will vary on plants that have different heights. soil type, planting pattern and type of cultivated plants affect the number and diversity of weed species. weed species are also affected by plant density, soil fertility, cultivation and tillage patterns. weed management can be carried out better if the level of weed dominance in rice plants is first known (rohimat et al., 2017). the large number of types of weeds that grow in these rice fields can be caused by tillage and fertilizer inputs. the hoeing process during soil processing can cause the lifting of weed seeds to the soil surface. the storage of weed seeds in the soil (seed bank) can germinate into individual weeds at any time if supported by environmental factors (dahlianah, 2017). ludwigia octavalvis (jacq) raven, weed plants are broadleaf weed plants and are found in many rice farms have fibrous roots, pinnate leaves face to face only leaf picking and yellow flowers (stenis 2008). leaves scattered, opposite, pinnateb-rayed; flowers are plucked leaves or as bunches, numbered 2-6; the leaves of the corolla are yellow, regularly numbered 2-4 at the end of the tubular flower axis. petals clinging. crown leaves are free, seated or hoofed (syaifudin &; nofa, 2020). cylindrical ordersl. beauv is a weed that often appears in tropical and subtropical regions. weed 426 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 commanded cylindrical. beauv is easily spread due to sexual and asexual reproduction. it is assumed that seeds per bunch of flowers are about 500-1.000 seeds, and per m2 about 10-20 bunches of flowers are obtained. seeds of imperata cylindrical. beauv are very easily carried away by the wind, so it is able to spread far. likewise, the possession of allelopathy, makes the reeds very solid in a new place. besides seeds, the weed cylindrical ordersl. beauv produces rhizoma, then when the rhizoma is cut, then each cut (several nodes) will form a new plant bud (juarsah, 2015). spigelia anthelmia is a weed that has taproots. spigelia anthelmia has a cylindrical stem, a smooth or waxy surface, upright growth. the leaves of spigelia anthelmia are oval, green with flat edges. sitting face to face and the number of leaves is compound, it has pinnate leaf bones with tapered leaf tips while the base of the leaves is rounded. flowers are located at the base of the leaves, spherical in shape and yellow in color (kastanja, 2011). ageratum conyzoides belong to flowering plants, members of the family asteraceae originally from tropical america, growing in the tropics. in indonesia, ageratum conyzoides is one of the nuisance plants/weeds that have the potential to inhibit rice growth through rice roots so that it can lack nutrients and ageratumplants conyzoides can live in fields, yards, gardens, banks and waterfronts (astriani, 2010). spihemoclea zeylanica is an annual plant belonging to the broadleaf type. spenochlea zeylanica weed is found in many rice fields. the weed spihemoclea zeylanica has cord-shaped roots, hollow stems, and white grain-shaped flowers. spihemoclea zeylanica multiplies through seeds. altemanthera sessilis is a type of broadleaf weed that has a physical state of creeping growth or growing upwards until scattered, plant height reaches 1 m. in addition, the flowers on alternanthera sessilis berestms white or pink, very small (lestari et al., 2012). the eclipse prostate can be annual (annual weed) and annual (perennial weed), often branched sideways or upright with stands 10-80 cm high, has a taproot, and has the potential to grow roots on each internode. the trunk and branches are green to purplish, and long hairy. the eclipta prostate is able to adapt to changing environments, especially in poorly drained places, wet areas around rivers, ditches, or swamps, but rich in sunlight. eclipta prostate weed resists living on saline soils to a height of 2000 m. high breeding ability, flowering in one year, eclipta prostate is able to produce 17,000 seeds per individual plant (mazidaturohmah et al., 2018). cypress is deformed l. it is a weed that does not shade rice plants, but can compete for water and nutrients. according to steenis (2008) cypress is deformed l. the cyperaceae family has exceptional resistance to mechanical control because it has stem tubers in the soil that can last for months. digitaria sp. is a group of grass weeds (poaceae) which have an important role in competing with staple crops both space, absorption of nutrients and water and development of very fast weed digitaria sp. mimosa chaste l. has a taproot shape, woody stem shape and there are thorns and compound leaf shape. plant mimosa chaste l. included in closed seed plants (angiosperms) and found in the group of two-piece plants. plant mimosa chaste l. these pinnate compound leaves and flat-edged leaves have facing leaves and belong to the legume tribe. leaves of mimosa pudica l. small in size arranged compound, oblong shape with a pointed tip, green color (some are reddish). when leaves mimosa chaste l. touched will be thetup (sensitive plant) menu. the roots are strong taproots, spherical flowers like balls, pink color, stemmed (purnomo, 201). important value index of rice weeds in rice fields figure 1. important value index of weeds (inp) of rice weeds in patia village rice fields patia subdistrict, pandeglang regency. the important value index (inp) of rice weeds in rice fields in patia village, patia district, pandeglang regency is the highest, namely ludwigia octovalvis (jacq) raven at 0.457 and the lowest is spigelia anthelmia at 0.045. the high relative frequency value of a type is an indication that the type is widespread, while a high relative density value indicates that species. ludwigia octavalvis (jacq) raven has the highest number of individuals of any other species. referring to windari et al (2021) stated that inp is divided into 3 (three) classes, namely high class for inp > 20%, medium class for 10 < inp < 20%, and low class for inp <10%. plants belonging to the high class are ludwigia octavalvis (jacq) raven; cylindrical orders l. beauv; cypress is deformed l., digitaria sp., plants belonging to the medium class, namely ageratum conyzoides; altemanthera sessilis; and plants that belong to the lower class, namely spihemoclea zeylanica and mimosa chaste l. the variety of inp values in each species, indicates the influence of the environment where it grows such as humidity, suhu, and competition between species. the highest important value index (inp), namely ludwigia octovalvis (jacq) raven of 0.457 or 45.7%, means that the weed shows density and dominance that is able to dominate the environment grow and have the widiyawati et al. – rice weed community structure in patia village rice fields … 427 ability to compete with growing factors such as nutrients, water, light and space that are getting stronger in rice. this is in accordance with the opinion of saitun et al., (2020) which states that the higher the inp value of a weed in one agricultural area, the higher the occurrence competition for growing factors between weeds and staple crops. this shows that the weed species is able to adapt to different environmental factors. puzzle weeds are included in wild plants that are difficult to eradicate because they produce tubers that make these plants regenerate quickly. an important factor in weeds is the production of tubers and rhizomes. tubers have an asexual reproduction mechanism and are the main dispersal unit that can withstand extreme conditions. tubers make plants difficult to control because only translocation herbicides have the potential to be effective in eradicating weed plants (windari et al., 2021). shannon-wiener diversity index (h') of rice weeds in rice fields table 2. shannon-wiener diversity index (h') of rice weeds in rice fields patia village patia district patia district. no. plot shannon-wiener diversity index (h') 1 1 2.024 2 2 1.718 3 3 1.881 4 4 1.98 5 5 2.09 average 1,94 the diversity index (h') can be calculated using the shannon weaver index. knowing the value of h' aims to determine the percentage of diversity of an organism in an ecosystem. h' aims to determine the number of species that exist at a time in a particular community (yuliana &; ami, 2021). the calculation of the shannon-wiener diversity index (h') of weeds in the rice fields of patia village, patia district, pandeglang regency is 1.94 (moderate diversity). the diversity index describes the level of diversity in a community, the high diversity in a community indicates the more stable or stable the ecosystem. the higher the value of species diversity, the greater the level of diversity, while a low vegetation diversity index value indicates that there has been pressure on vegetation (oktaviani et al., 2015). pressure on rice field vegetation can be in the form of natural factors such as drought disasters or human intervention factors such as intensive tillage measures and weed control with herbicides. ecosystem stability is closely related to diversity, if the ecosystem tends to be stable, then ecosystem diversity is relatively high. if the condition of the ecosystem is low, the environment has diversity disturbances, and if species diversity tends to be low, it means that the ecosystem environment is polluted (windari et al., 2021). weeds tend to exist around cultivated plants in any field under any conditions and multiply rapidly and are difficult to control mechanically. species diversity can be used to express commodity structure, i.e. the ability of a commodity to protect itself to remain stable despite interference with its component components (saitun et al., 2020). the shannon-wiener diversity index (h') value will increase as the number of species in the community increases and the distribution is more even. species diversity is closely related to root rhizomes and can also play a role in dispersal, especially in intensively mechanized agriculture. cutting and removal of rhizome roots will often be done when planting with a cultivated plant. weeds will be able to grow widely if the mechanical work cannot be done properly. furthermore, it was also revealed that the spread of seeds/parts of weeds eaten by the animal and through the digestive tract, can still grow (cyperaceae group, gramineae, and shrubs). then spread through water, usually this happens for aquatic weeds, either in the direction of water flow factors, seeds/parts of weeds that float, or the nature of seeds or weeds that are tolerant of water absorption (suryatini, 2018). abundance of rice weed species in rice fields table 3. abundance of weed species in rice fields, patia village, patia district, pandeglang regency. no species species abundance 1 ludwigia octavalvis (jacq) raven 22.85% 2 cylindrical orders l. beauv 20.22% 3 spigelia anthelmia 2.25% 4 ageratum conyzoides 8.99% 5 spihemoclea zeylanica 3.00% 6 altemanthera sessillis 5.62% 7 eclipse the prostate 6.74% 8 cypress is deformed l 15.73% 9 digitaria sp. 11.99% 10 mimosa chaste l 2.62% the results of the calculation of the abundance of weed species in the rice fields of patia village, district patia of pandeglang regency, namely ludwigia octovalvis (jacq) raven at 22.85%; cylindrical orders l. beauv by 20.22%; spigelia anthelmia by 2.25%; ageratum conyzoides by 8.99%; spihemoclea zeylanica by 3%; altemanthera sessilis by 5.62%; eclipta prostate by 6.74%; cypress is deformed l. by 15.73%; digitaria sp. by 11.99% and mimosa chaste l. by 2.62% these results show that the weed ludwigia octavalvis (jacq) raven has the ability to utilize infrastructure to grow higher than other weeds. differences in weed types are caused by differences in plant management, including water regulation and fertilization as well as differences in morphology and character of constituent plants that can change the microclimate so that it causes different responses in weed types (imaniasita et al., 2020). 428 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 the difference in the species composition of plantain weeds in rice fields in each research plot is influenced by tillage and how weeds spread, there are several kinds of weed distribution, namely dispersal by animals and dispersal through water. dominance index (d) of rice weeds in rice fields table 4. dominance index (d) of rice weeds in rice fields, patia village, patia district, pandeglang regency. no. weed species abundance pi (pi) 2 1 ludwigia octavalvis (jacq) raven 61 0.228 0.052 2 cylindrical orders l. beauv 54 0.202 0.041 3 spigelia anthelmia 6 0.022 0.001 4 ageratum conyzoides 24 0.090 0.008 5 spihemoclea zeylanica 8 0.030 0.001 6 altemanthera sessillis 15 0.056 0.003 7 eclipse the prostate 18 0.067 0.005 8 cypress is deformed l. 42 0.157 0.025 9 digitaria sp. 32 0.120 0.014 10 mimosa chaste l. 7 0.026 0.001 dominance index (d) 0.150 the calculation of the dominance index (d) of weeds in the rice fields of patia village, patia district, pandeglang regency is an average of 0.15. the third highest dominance index in the weed ludwigia octavalvis (jacq) raven is 0.052 or 5.2%, cylindrical orders l. beauv by 0.041 or 4.1% and cypress is deformed l. which is 0.025 or 4.5%. dominance is the ability of a weed to survive in a particular agroecosystem by competing with other weeds. dominance is the proportion between the area covered by plant species and the total area of habitat. bad for the ecosystem can be caused because of the dominance of species in a place. dominance is closely related to invasion and a trait that illustrates the ability of dominant plants and endangered ecosystems, habitats, and other plants found in a place is the meaning of invasion (windari et al., 2021). generally, weeds produce large quantities of seeds, which are in the soil and serve as seed banks in the next growing season. such annual weeds should be controlled in a timely manner to prevent a reduction in crop yield. and it must be controlled before flowering and fruiting weeds. this is to reduce seed production which will become a seed bank (yurlisa, 2021). compared to the dominant weeds in rice crops, there are several different types of dominant weeds. this difference in dominant weed species is thought to occur due to changes in land conditions or cultivation treatment of each type of cultivated plant. in addition, it is also thought to be influenced by the ability of a species to adapt to environmental changes, and the ability of competition between weed species. the dominance of weeds in each experimental plot will affect the control recommendations applied. this situation has implications for different ways of controlling (putra et al., 2018). weed control carried out by farmers at the research site is manual control, but this method requires more time and cost. manual weed control by pulling is effective for controlling annuals or two-season weeds. for annual weeds, removal control will lead to cutting off plant parts (rhizomes, stolons and root tubers) that are dead in the soil. the rest of the plant is an effective part of growing and developing again (farmanta et al., 2016). conditions of environmental factors table 5. measurement of environmental conditions in rice fields of patia village, patia district, pandeglang regency. parameters measurement results on the plot 1 2 3 4 5 average temperature (oc) 26 27 27.5 26 27 26.7 moisture 5 1,5 5 5 1.5 3.6 ph 7,5 7,5 7 7.5 7.5 7.4 light intensity (lux) 700 400 200 700 400 480 measurement of environmental conditions in the rice fields of patia village, patia district, pandeglang regency obtained an average temperature value (oc) of 26.7 oc, humidity 3.6 rh; ph of 7.4 and intensity light widiyawati et al. – rice weed community structure in patia village rice fields … 429 of 480 lux. the community of a plant can be affected by several factors. one of these factors is the difference in location or height of a place. because this can affect light intensity, temperature, and humidity which are climatic factors (suryatini, 2018). the difference in the number of individual weeds obtained between one place and another is also influenced by environmental factors where they grow such as temperature, humidity, soil, space growth and light (yussa et al 2015). light is a factor that affects the number of species living in a community, where light greatly affects the type and number of individuals that can grow in a community. the place. in addition to light, the climate received in a place will also affect differences in the type and number of individual weeds. the existence of differences and changes in the environment can affect the composition of weed communities that occupy an area (farmanta et al., 2016). chairul & rahmatul (2013) stated that weed survival is influenced by several factors, one of which is soil ph. a ph close to neutral (6.5-7.5) is best for growth and availability nutrient content. soil ph determines whether or not nutrients can easily be absorbed by plants. in general, nutrients are easily absorbed by plant roots if the soil ph is around neutral, because at that ph (suryatini, 2018). conclusions based on the results of research and discussion, it can be concluded as follows: ▪ the types of weeds found in the rice fields of patia village, patia district, pandeglang regency are ludwigia octavalvis (jacq) raven, cylindrical ordersl. beauv, spigelia anthelmia, ageratum conyzoides, spihemoclea zeylanica, altemanthera sessillis, eclipta prostate, cypress deformed l., digitaria sp and mimosa chaste l. ▪ the structure of the weed community in the rice fields of patia village, patia district, pandeglang regency is the shannon-wiener diversity index (h') of 1.94; index evenness (j') of 0.884; dominance index (d) of 0.15. suggestion based on the results of research and conclusions, it can be suggested, namely: ▪ weed control should be done as soon as possible to suppress the growth of weeds. control can be done mechanically by using t-style agricultural tools such as hoes, as well as chemically by using 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stricta. 50th annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs (2000) biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. sebelas maret university, surakarta, 17-20 july 2000. 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[indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the 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carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am (2007) seed production and dispersal of rhazya stricta. 50th annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs (2000) biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. sebelas maret university, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto (2004) soil macro-invertebrates diversity and inter-cropping plants productivity in ag roforestry system based on sengon.[dissertation]. brawijaya university, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. offprint or reprint is only available with special request. note: author(s) agree to transfer copy right of published paper to biology, medicine, & natural product chemistry. author shall no longer be allowed to publish manuscript completely without publisher permission. authors or others allowed multiply article in this journal as long as not for commercial purposes. for the new invention, authors suggested to manage its patent before publishing in this journal. notification: all communications are strongly recommended to be undertaken through email. a discovery and characteristics description of telosma puberula (asclepiadoideae) in mount gedang atas and mount ijo, baturagung mountain yogyakarta widodo larvicidal activity of a mixtureof cashew nut shell liquid and water-soluble extract of soapnut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati, sudarsono, budi mulyaningsih substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cytbofindonesian swamp buffalo (bubalus bubalis) akhmad sukri, mohamad.amin, aris winaya, abdul gofur the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a., arief r.h., sudarsono 47-52 53-57 59-63 65-67 volume 3 number 2 2014 published twice a year printed in indonesia cetak cover jurnal biomenaprochy vol 3 num 2 2014 dpn.pdf (p.1) editorial board 2014 v3 n2.pdf (p.2) 4 guidance.pdf (p.3) cetak cover jurnal biomenaprochy vol 3 num 2 2014 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 5-8 | doi: 10.14421/biomedich.2017.61.5-8 issn 2540-9328 (online) analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan institute of islamic studies sunan giri (insuri) ponorogo jl. batoro katong no.32, kertosari, babadan, kabupaten ponorogo, jawa timur 63411 author correspondency: fuadfitriawan@gmail.com abstract sperm abnormalities can occur by various causes. abnormality of sperm is usually characterized by abnormal sperm motility and viability. this was caused by the inability of mitochondria on atp-ase in producing atp and ecto-enzyme cik role in keeping the movement so that movement of sperm motility declines. research that leads to total abnormal sperm dna analysis is still rare. the purpose of this research was to get the results of the molecular characteristic picture of the overall characteristics of dna loci that have abnormalities in bull sperm and get a picture of differences in overall dna loci of abnormal and normal sperm. this research was conducted in october-december 2016. with the results of group i consists of d, e with a percent similarity of 92.308%, group ii consists of c and group i with a percentage similarity of 50.125%, group ii consists of a and b with a percent similarity of 100%, group ii and group iii with a percentage similarity of 0%. based on the above data it can be concluded that the treatment a and b is not suspected to cause dna damage compared to treatment c, d and e. keywords: abnormality; spermatozoa; cow; pcr-rapd; cadmium accumulation. introduction every family usually want to has offspring and feels happiness by the presence of baby in their family. but not all family get the dream to have offspring. one of the things that led to the inability to continue its offspring is infertility that occurs on the husband and/or wife. according to agritubella (2009) infertility is the inability of a couple to have children after intercourse regularly 2-3 times per week, without contraception for 1 year. in women, it is usually caused by an infection of the vagina, cervix disorders, abnormalities of the fallopian tubes and uterus according muchtaromah (1999) besides female cause, the other 50% couple infertility contributed by male due to sperm abnormalities. previous research carried out by fitriawan (2006) showed that sperm infertility largely caused by the sperm abnormalities that can occur due to wrong diet as well as abnormalities in male reproductive organs. the sperm abnormalities are usually marked with the limited ability of sperm in its motility and viability. fitriawan[7] and susilowati (2005) stated that it was caused by the inability of mitochondria on atpase to producing atp and enzyme ecto-cik in keeping the movement according to guven et al. (1998), sperm is said to be abnormal if it had more than 70% morphological abnormalities. abnormality caused sperm mitochondrial deficiency that leads to failure in acrosome and postacrosomal reaction so that sperm is unable to fertilize an egg. mature sperm has a length of approximately 50 micron, morphologically and functionally, sperm is divided into two parts, namely the head and tail (destiany. m. 2007). according to guyton and hall (1997), the head of human sperm is oval shaped with a compact nucleus and few cytoplasm bit, salisbury added that the head of the sperm chromatin contain materials that carry the dna code to be transferred through pellucida zone of the ovum. according to fitriawan (2006) and curry & watson (1995), biochemically, sperm abnormalities caused by the inability at the mitochondrial atpase in producing atp and enzyme ecto-cik in keeping the movement so that the movement of sperm will decrease. research on the causes of infertility of sperm abnormalities in biochemical and physiological processes have been done, but a review of genetic and molecular studies have not been conducted. one method to determine molecular genetic picture of an organism is by using pcr-rapd method, which is a molecular technique to determine the overall picture of the characteristics of nuclear dna and mitochondrial dna which is made possible by the insertion, deletion, or substitution at restriction sites. according to sutarno (2008), the advantage of pcr-rapd method is that we can know the map of particular gene in an organism, and the state of these genes can be duplicated so that it seen more clearly. http://dx.doi.org/10.14421/biomedich.2017.61.5-8 6 biology, medicine, & natural product chemistry 6 (1), 2017: 5-8 materials and methods this research was conducted by taking a sample of bull sperm from the bali cattle center for artificial insemination singosari malang, which then will be treated for abnormality and pcr-rapd analysis in the laboratory of biochemistry and molecular biology department, university of brawijaya. the study was conducted from november to december 2016. the equipments used in this study include a set of tools to handle the semen that consists of one unit of an artificial vagina, semen storage tube (test tube scale), a thermos of hot water and thermometer (for measuring temperature in a thermos). a set of tools to check the quality of the semen including test tubes, test tube rack, centrifuges, ose needles, waterbath, paper labels and a set of tools for observing sperm including a light microscope, object glass, cover glass, co2 incubator, hand counter, a set of haemocytometer and tissue and a set of electrophoresis equipment. the materials used for this study were as follows: bali cattle semen samples, a set of semen material extraction, lysis buffer, protenase k, potassium acetate, phenol, nacl, cdcl, vaseline, distilled water, chloroform isoamyl alcohol and isopropanol. negrosineosin staining materials consist of a mixture of 0.3 g eosin, negrosin 0.5 g, 0.1 g na citrate, 10 ml of distilled water and then in the stirrer with a temperature of 100 °c. 500 ml pbs consists of a mixture of 4 g nacl, 0.1 g kcl, 1.05 g na hpo (h o) and 0.1 g of kh po. the research of characteristic of dna in abnormal sperm are reviewed by pcr-rapd method which is a qualitative descriptive study of data images from pcrrflp to be read in accordance with standard pcrrapd analysis. the technique of collecting data from this study was by random sampling, which is a technique that is done by taking samples directly on the ground which will then be treated to obtain abnormal sperm which will then be analyzed its abnormality using eosin staining and for further dna examination using pcr-rapd (sutarno, 2008; sarkar s, hanry jb. 1996). data were analyzed using descriptive and qualitative analysis (moleong, l. j. 1997) of sperm under light microscope and determined its quality, if 70% sperm are not normal then it is classified as abnormal sperm. then, for further analysis, pcr-rapd was performed to determine the polymorphism characteristics of dna. results and discussion results of total dna electrophoresis on agarose 1% from the research, dna eletroforesis of bali bull sperm are shown in figure 1. qualitatively, there were differences between the control and treatment of dna banding pattern. thickness and thinness of the banding pattern will be the effect of treatment on the stability of sperm. in the sample a (control), almost 'total pattern of dna bands appear not so visible but in treatment (b, c, d, and e) visible banding pattern shown very clearly. a b c d e explanation: a : control b : treatment of cadmium chloride 2 hours c : treatment of cadmium chloride 4 hours d : treatment of cadmium chloride 8 hours e : treatment of cadmium chloride over night figure 1. results of total dna electrophoresis on agarose 1%. according to singer and berg in setyono, et al. (2008) a form of gene expression in organisms are protein and polypeptides which is formed through a series of processes of transcription and translation. badel and tatum in setyono, et al. (2008) put forward a hypothesis about the relationship between genes and enzymes, that one gene-one enzyme which then evolved into one gene-one polypeptide. the assumption of the above changes that can lead to environmental change experiments in the process of transcription of different genes that can cause modifications even to cause mutations in the translation process, and can lead to the expression of different morphological structures. mutations or changes at the gene level basically occur due to changes in the environment, so that the dynamics of different patterns of different dna invaluable in detecting mutations in a timely fashioned (toelihere. 1993). then from the qualitative data above can be seen in quantitative results as follows: results of quantitative measurements of total dna sample of bali bull sperm there is a less significant effect between treatments 0 hours to 2 hours, this is because the treatment process that may not have been so long that transcription of the dna of spermatozoa has not shown any signs of abnormality. fitriawan – analysis of bull sperm dna abnormalities due to cadmium accumulation 7 table 1. results of dna quantitative measurement of total sapi bali. samples a260 a280 levels (µg/ml) purity 0 hour 0.004 0.002 100 2.0 2 hour 0.005 0.003 125 1.67 4 hour 0.005 0.003 125 1.67 8 hour 0.012 0.015 300 0.8 over night 0.006 0.004 150 1.5 the results of rapd sperm with premiere opa2 bali bull, 4 opa and opa 6 m a b c d e a b c d e a b c d e explanation: a : control b : treatment of cadmium chloride 2 hours c : treatment of cadmium chloride 4 hours d : treatment of cadmium chloride 8 hours e : treatment of cadmium chloride over night figure 2. visualization of dna produced by pcr-rapd bull sperm on 1.5% agarose gel. then from the visualization of total dna amplification process is carried out and replication that appear on the visualization of pcr-rapd in figure 2. from the image above, after analyzed by dendogram translation (figure 3) method can be seen as follows: dendogram rapd analysis results using mvsp 3. program 1 (multivariate statistics package 3.1) figure 3. dendogram of pcr-rapd bull sperm with premiere opa2, opa4 and opa6. based on the results of bali bull sperm dna amplification with a variation of the old treatment using a primer opa2 cadmium, opa4, and opa6 showed that the highest percentage of sperm dna damage treated with cadmium is composed of three groups: a) the first group consists of d, e with a percent similarity of 92.308% b) group ii consists of c and group i with a percent similarity of 50.125% c) group ii consists of a and b with the percent similarity of 100% d) group ii and group iii with a percent similarity of 0% if observed further from primer that has been used, it can be seen that the appearance of banding pattern in wells a and b almost non-existent, it is possible not to the effect of dna damage by metal cadmium that has been given that at the time of 0-2 hours, but after the appearance of differences, there is already indications of changes in the physical and molecular of sperm. according destiany (2007) and currt & watson (1995) that cd in the cell is a non-essential subtance (a metal which is not needed by the body), if it settles in a cell, it will form cd2 + that causes toxicity to the cell so that metabolism in the cells is inhibited which causes the generation of atp as mitochondria will undergo changes drastically and immediately this is what causes the loci of genes on dna changes. biochemically, according to fitriawan (2006) sperm abnormalities caused by the inability to precisely at the mitochondrial atpase in producing atp and enzyme ecto-cik role in keeping the movement so that the movement of sperm motility will decrease.the sperm dna abnormalities can be seen physiologically using eosin 2% as follows: description: abnormalities of spermatozoa. figure 4. sperm abnormalities conditions dcl treatment of 300 mg/ml 24h. opa 2 opa 4 opa 6 8 biology, medicine, & natural product chemistry 6 (1), 2017: 5-8 sperm abnormalities were divided into 2 groups: primary and secondary abnormalities. primary abnormality is an abnormality that occurs during spermatogenesis. secondary abnormality is an abnormality that occurs due to environmental factors. the existence of sperm abnormalities in a certain amount (under 5%) is a normal condition as a form of primary abnormality. a toxicant toxicity values against an organism can be expressed by several parameters, including the value of lethal concentration 50 (lc-50), a value of 50% of organisms causing death. from lc-50 we can assigned sub-lethal toxicity that resulted in damaging organ or disruption in growth, reproductive ability and behavior. likewise, chronic toxicity, the effect is shown in a long time. in toxicant heavy metal, the last two things is very important, because the presence of heavy metals in water generally in a relatively low level but in the long term. abnormalities that occur in figure 4 above in the form of abnormalities in sperm coiled tail, broken and bent, allegedly due to exposure to heavy metals cause the solution becomes hypertonic. if sperm are stored in a hypertonic solution will result in cytoplasmic vacuoles open and tail membranes become more permeable, so that the curled tail, until broken. the effect of this condition for fertility is a sperm motility will decreased, so the sperm cannot fertilized. based on the above data it can be concluded that treatment a and b allegedly did not cause dna damage compared to treatment c, d and e. conclusion from the research that has been done can be concluded that: 1. the first group consisting of d, e with a percent similarity of 92.308%, group ii consists of c and group i with a percent similarity of 50.125%, group ii consists of a and b with the percent similarity of 100%, group ii and group iii the percent similarity of 0%. 2. treatment a and b (control) as a treatment for 2 hours allegedly did not cause dna damage compared to treatment c, d and e. references agritubella, m. s. 2009. infertilitas. artikel kesehatan reproduksi http://situs.kesrepro.info/kb/referensi2.htm. retrieved at 29 march 2010. coffee, c. j. 1998. metabolism. 1st ed. fence creek publising. madison: xiii + 454 p. curry mr, watson pf. 1995. sperm structure and function in: graud zinkas jg, yovich jl, (eds). gametes-the spermatozoon. great britain: cambridge university press. pages 45-67. curry, m. r. & watson, p. f. 1995. sperm structure and function. great britain. cambridge university press. pages 45-67. destiany. m. 2007. pengaruh pemberian merkuri klorida terhadap struktur mikroanatomi hati ikan mas. thesis. department of biology universitas negeri semarang fitriawan, f. 2006. kemampuan anti mps dalam menghambat motilitas dan viabilitas spermatozoa kambing, domba, dan sapi. thesis department of biology faculty of science and technology uin malang. ginzburg, sa, 1972. fertilization in fishes and the problem of polyspermy. wiener bindery. jerussalem. guven, m. c., can, b., saran, y. 1998. ultrastructural changes in the spermatozoa of infertil man. journals from dept. histology-embriology, medical school of ankara university turkey. guyton ac, hall je. 1997. buku ajar fisiologi kedokteran. diterjemahkan oleh irwati s, lma ken ariata t, alek s. jakarta: penerbit buku kedokteran egc. pages: 309-314. hafez, e.s.e. 1977. techniques for improving reproduction efficiency, pp.455-479. the cv mosby company: st. louis. mason, cf. 1981. biology of freshwater pollution.longman lnc, new york. moleong, l. j. 1997. metodologi penelitian kualitatif. bandung, p.t. remaja rosda karya. muchtaromah, b. 1999. pengaruh pemberian metoklopramid terhadap gambaran histologis testis tikus putih (rattus norwegicus). thesis postgraduate program department of bioreproduksi unair surabaya. nelson, rw. 2003. morphology of normal and abnormal canine spermatozoa. couto cg. journal of small animal internal medicine. mosby partodiharjo, soebadi, 1992. ilmu reproduksi hewan. mutiara sumber widya. jakarta pusat. cetakan ke iii pages 523-531. rijnders pm. 1996. the spermatozoon-theory. in: brass jw, et al. editors. ivf lab-laboratory as pects of in vitro vertilization. nv organon. pages 23-39. salisbury, g. w. 1985. inseminasi buatan pada sapi. gadjah mada university press yogyakarta. sarkar s, hanry jb. 1996. andrology laboratory and fertility assessment, in: henry jb, editors. clinical diagnosis and management by laboratory metods. philadelphia. setyono, p., soetarto, e. s., 2008. biomonitoring degradasi ekosistem akibat limbah cpo muara sungai mentaya kalimantan tengah dengan metode elektromorf isozim esterase. jurnal biodiversitas vol 9. nomor 3. universitas sebelas maret surakarta. pages 232-236. sutarno, 2008. diktat materi bioteknologi dan seluler. faculty of mathematics and natural sciences universitas sebelas maret surakarta. pages. 35-40. susilowati, t. 2005. fisiologi spermatozoa: kapasitasi, reaksi akrosom dan fertilisasi. faculty of animal husbandry universitas brawijaya malang. toelihere. 1985. fisiologi reproduksi pada ternak. publisher angkasa bandung. pages 93 – 115. toelihere. 1993. analisis kualitas semen pada ternak. publisher angkasa bandung. analysis of bull sperm dna abnormalities due to cadmium accumulation biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 1-14 | doi: 10.14421/biomedich.2014.31.1-14 ecopharmacognosy: exploring the chemical and biological potential of nature for human health geoffrey a. cordell natural products inc., evanston, illinois 60203, usa author correspondency: pharmacog@gmail.com abstract “why didn’t they develop natural product drugs in a sustainable manner at the beginning of this century?” in 2035, when about 10.0 billion will inhabit earth, will this be our legacy as the world contemplates the costs and availability of synthetic and gene-based products for primary health care? acknowledging the recent history of the relationship between humankind and the earth, it is essential that the health care issues being left for our descendants be considered in terms of resources. for most people in the world, there are two vast health care “gaps”, access to quality drugs and the development of drugs for major global and local diseases. consequently for all of these people, plants, in their various forms, remain a primary source of health care. in the developed countries, natural products derived from plants assume a relatively minor role in health care, as prescription and over-the-counter products, even with the widespread use of phytotherapeutical preparations. significantly, pharmaceutical companies have retrenched substantially in their disease areas of focus. these research areas do not include the prevalent diseases of the middleand lower-income countries, and important diseases of the developed world, such as drug resistance. what then is the vision for natural product research to maintain the choices of drug discovery and pharmaceutical development for future generations? in this discussion some facets of how natural products must be involved globally, in a sustainable manner, for improving health care will be examined within the framework of the new term “ecopharmacognosy”, which invokes sustainability as the basis for research on biologically active natural products. access to the biome, the acquisition, analysis and dissemination of plant knowledge, natural product structure diversification, biotechnology development, strategies for natural product drug discovery, and aspects of multitarget therapy and synergy research will be discussed. options for the future will be presented which may be significant as countries decide how to develop approaches to relieve their own disease burden, and the needs of their population for improved access to medicinal agents. keywords: natural products, ecopharmacognosy, sustainable medicines, biotechnology, structure diversification, rain forest resources, strategic implications introduction in lewis carroll’s famous book “alice in wonderland; through the looking glass”, it is the mad hatter who has the perspicacity to say to alice “we are all mad here”. in many ways, given the recent and ongoing relationship between humankind and the resources of the earth, it is we who are living in a world of madness, where resources are squandered now, for instant benefit, with little consideration given for the resource needs of the generations to follow. and it we who, in spite of numerous warnings and international meetings and declarations, still cannot turn and say clearly that what we are doing to the planet and its resources is indeed “madness”, and must change if the human race is to survive and thrive. to borrow a phrase, “we are killing the planet that heals us”. the core madness in the scenario is this: a dramatically increasing population, mostly in the middleand low-income countries, an alarming decrease in the acreage of tropical rain forests, and an expanding dependence on those resources and others for food, shelter, and health care. when, as we have seen, most of the world relies on medicinal plants for their health care, and up to 85% of those plants are harvested in a nonrenewable manner, it is clear to see that new strategies are needed to assure access to medicinal agents in the future (1-12). the fourth factor, oil, remains a mysterious unknown, an enigma with respect to long-term accessibility. optimistic estimates of the current oil resources, although accurate data are basically nonavailable for financial and security reasons, would suggest that they may be viable for the next 40-50 years. however, population is increasing at a faster rate than oil production. so what will be the status of the earth’s resources at that time, say in 2060, when renewable, moderate cost, effective, alternative sources for synthetic medicinal agents will be needed? enzymes, isolated or in intact systems, must be deployed as an essential component of industrial drug synthesis to reduce the dependency on non-renewable resources. the july, 2011 issue of chemical reviews was devoted to a series of eleven reviews of different types of enzymes deployed for selective synthetic transformations, several related to drug processes. the use of renewable, multifunctional enzyme systems which can transpose a compound through to a final product with minimal “chemical” reagent involvement may be an important facet of the production of synthetic drugs within ten years. considerations of the use of inexpensive renewable reagents may also drive the selection of both production processes and compounds evaluated for their drug potential and eventual marketing. also to be developed further is the potential for the use of 2 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 vegetables as chemical reagents for selected enantioselective processes, where intact materials, not isolated enzymes are utilized (13). however, the need for natural products, as quality traditional medicines, and as sources for new medicinal agents will remain. this brief discussion is about some of those opportunities. as natural product scientists, one of our obligations is to initiate the discussion of how to investigate natural resources for health care, and encourage others to see different perspectives of the overall picture. our role is also to examine, define, and articulate the niche of natural products in the future of the population of earth, and particularly the anticipated role of natural products in global health care. it is also important that we, as the content experts, have a scientific role in developing those future plans. the global population is now at 7.31 billion (april 2013), and is projected to rise to 10 billion by about 2035. the causes and the implications for such a dramatic population explosion are philosophical and religious, which are considerations beyond the scope of this short article, but upon which the fate of human existence hinges (14, 15). as one small, yet significant aspect of this huge discussion, some of the many integrated aspects of natural products in health care, we will review some possible relationships between natural products and drug discovery, and look one again at the important question: “what is the future for natural products in global health?” the author has written numerous articles on this area in the past 25 years (1-6,16-24), with several of the more recent articles focusing on two major issues, the concept of medicines as a sustainable commodity (4-12), and enhancing the quality control of traditional medicines in global health (6-12,23-26). on a daily basis, we often forget to acknowledge with gratefulness the vast contributions that plants, their extracts, and the various products derived from them, make to human health and well-being: i) as foodstuffs, ii) as flavoring agents and spices, iii) as perfumes and cosmetics, and iv) as pharmaceutical and biological agents. these categories are not mutually exclusive, for it is apparent that there is significant overlap between foods, spices, cosmetics, and biological (medicinal) agents. it was estimated over 20 years ago that there are more than 120 compounds from over 90 different plant materials which are used on a global basis as prescription products (27), and this number may well have risen since then (28-32). butler, for example, lists 34 natural product based drugs introduced in the period 1998-2007 (31), and harvey lists 108 plant-derived compounds in various development phases (32). from a commercial perspective, world-wide sales for plant-based pharmaceutical agents in 2002 were estimated at us $30 billion (33). in addition, there are also thousands of products, compounds, plant extracts and plant materials, some sanctioned as prescription products, others bought over-the-counter, or in a market place, or through a local medicine man or woman, which are recommended for patient healing in various parts of the world. for the vast majority of the world, approximately 4.5 billion people, these plant resources are their primary source of health care (34). there are many “gaps” in global health care, and per capita government investment in health care in a particular country (see table 1 for some examples), is one of the more staggering examples between the highincome and the low-income countries. that aspect of health care concerned with medicinal agents for patient treatment, pharmacy, is no exception. for example, there is a major regulatory “gap” between the prescription products of the north and the products sold in a marketplace as part of a traditional medicine system in a middleor low-income country. one is highly regulated, costing perhaps $1.3 billion to develop a product and support continuing post-marketing surveillance (35). whereas the traditional medicine is almost completely unregulated; in many countries, a plant material appears in the street market directly from the field, just as it did 4000 years ago, and there are many variations of regulation in this continuum of extremes. the un charter of 1948 clearly indicates that all humans have the universal right to health care. but what is being done by the organizations of the un to achieve this? what does that universal right mean in terms of the quality control of traditional medicines or for drug discovery for global and local diseases? in this regard, we must not be limited to thinking that drug discovery involves only single compounds. in an era of systems biology and network pharmacology, it is clear that the period for thinking of drug discoveries as “magic bullets” is long gone. table 1. government expenditures on health per capita (2009) in us $. country us $ united states 7960 norway 5383 australia 3484 united kingdom 3438 republic of korea 1879 costa rica 1155 turkey 957 south africa 930 brazil 921 iran 728 peru 466 china 347 vietnam 204 philippines 133 india 124 nigeria 136 indonesia 100 myanmar 36 source:http://en.wikipedia.org/wiki/list_of_countries_by_total_health_ex penditure _(ppp)_per_capita in terms of plant-based health care, research is drastically under-funded at the global and national levels in terms of personnel, programs, products, and outcomes. geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 3 the truth is that, as a moral and ethical issue, it is a shameful international embarrassment, and requires a complete reassessment of strategies, programs, and outcomes from the very top of the un through all of the various agencies involved. one group, médecins sans frontieres, justifiably, has called for a complete overall of the whole drug discovery, development and access system for the middle and lower-income areas of the world given the history of drug innovation for the south in the past thirty years (35). patients world-wide who acquire plant-based products, either through prescription or as a street medicine, in their various forms, are rightfully demanding the same assurances of safety, efficacy, and consistency as a patient taking a drug from a pharmaceutical company. the “gap”, of course, is who is standing behind those assurances? is it a government, a pharmaceutical company, a traditional medicine provider? if no-one is making (or can make) those fundamental assurances, based on science, what does that mean for the overall health care of a nation? as the wise man once said, if you want the answer…. “follow the money.” although very basic, this is merely one of many health care “gaps” that exist in the world at this time; another gaggle of “gaps” concern the question of access. in this sense, the term is applied in four ways. the first facet is what are the prevalent diseases in a country for which drugs are not available at all? the second is what are the diseases for which drugs are available globally, but which are either too costly, or not available, locally? the third facet is what are the diseases for which there is resistance to existing treatments? the final facet of access relates to the source of medicinal plant materials, and the long term stability of a disappearing forest as a sustainable source of medicinal agents. these facets, individually or collectively, can form the basis for rationalizing the need for targeted new drug discovery programs, and for placing that rationalization in a conservation and sustainable development framework. access to health care, and to medications in particular, is consequently a global concern for all, with the exception of the very wealthy. another of the medicine-related “gaps” in health care relates to two questions: what are global health care needs? and what is contemporary drug discovery in the pharmaceutical industry targeting? the “gap” between these two responses is vast and expanding. the global health needs for over 1 billion people on earth are treatments for: malaria, hiv-aids, tuberculosis, hepatitis c, diarrheal diseases, ascariasis, leishmaniasis, schistosomiasis, trypanosomiasis, dengue fever, leprosy, rabies, yaws, and necatoriasis. contemporary drug discovery areas in the major pharmaceutical companies have been winnowed to: antivirals, oncology, metabolism, central nervous system ailments, and inflammatory diseases (36). more recently, one major company, astrazeneca, has winnowed its research portfolio to three areas: inflammatory and autoimmune diseases, cardiovascular and metabolic diseases, and oncology (37). the logic behind these strategies is that drug companies will make money when they produce drugs which: i) will not encounter a resistance issue, as occurs with antibiotics, ii) are taken on a chronic basis, and iii) are palliative and not curative. the global health disease burden is not their concern. it is really that simple. for individual countries, and for regional associations of countries, such as asean, many questions must be asked in terms of the present and the future health care needs. there are limited precedents available, but recall that it may take at least fifteen years to move from bench to marketplace, if the strategy involves the development of single agent drugs. there are alternatives which also should be considered, and which, depending on the particular country situation, can serve either as interim steps for enhancing health care, or as longer term solutions. clearly though, not every country will have the human capacity, the infrastructure, and the financial resources to muster a drug discovery effort based on the study of their natural resources. even those countries who do embark on that endeavor will need to clarify whether all of the needed expertise is available in country, or whether other regional or global resources need to be accessed. from a discovery perspective, countries have two main natural resources at their disposal, their natural flora and the indigenous knowledge, which may be within one or several traditional systems of medicine. these resources offer a unique basis with which to develop a strategy or a series of strategies for drug discovery. in this instance, the emphasis here is on a government ministry, or most likely a group of ministries (science and technology, health, education, agriculture, commerce), and local (not the major) pharmaceutical companies, coming together to serve as a catalyst to address this aspect of health care. initiatives such as the discovery of drugs or the enhancement of traditional medicines to meet societal health needs cannot begin at the local level, but must come “top down”, where government is both “calling the shots” through establishing consultative groups, and at the same time providing the highly targeted resources for addressing long-term needs. all parties, government regulators and funding sources, botanical, chemical and biological scientists, agronomists, industrialists, and academics need to come together to address these basic health care issues as a coordinated and cooperative venture for the health of their population. as we consider how to potentiate the available natural resources for health care, it is also important to examine the historical background to the interest in the tropical forests. it is not adequate to state that because plants have yielded drugs in the past they will do so in the future. neither is it appropriate to conclude that because a particular plant has a long history of use as a traditional medicine that it should be approved as a drug. many other considerations come into play, including the need to place such assertions in science; the evidence-based approach. so what is the goal, and what are the strategies, of exploring the rain forest for medicinal plants? are there 4 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 clear and rational reasons which can adequately justify the investment? if so, what is the nature (breadth, depth, duration) of such an interest? what if the discovery of either an effective plant extract or an effective compound is made? what are the issues with respect to next stage development, and how will further development, if warranted, be supported financially? where and how does the patenting of inventions impact the development process? what are the local implications in terms of agricultural or infrastructure development? potentially, unraveling the human genome provides an opportunity for the development of assays related to numerous disease-related, new drug targets, once those correlations have been determined. because of the ethnomedical reputation of tens of thousands (perhaps 810%) of terrestrial plants (4), once a prioritized acquisition and screening schedule is achieved, very specific opportunities for the selective evaluation of the effectiveness and potential for further development of traditional medicines may be achieved. strategically though, for the wider evaluation of plant materials, and for more rapid and effective quality control assessment of traditional medicines an important paradigm needs to be reversed (9-12). as a modality for conducting preliminary screening programs for the biological activity of plant extracts, bringing plant materials from the collection site to the laboratory for extraction and bioassay is an exceptionally inefficient approach. depending where an “active” line is drawn, but probably around the 2-4% level for most programs, the extracts of 96-98% of the collected plant materials are “inactive” in any particular biological assay. while those materials (and their parent plants) can be retained and stored as an asset to the program, and subsequently reassessed against new target assays in the future, the fact remains that there is significant effort in collection, drying, grinding, and extraction which is wasted. consequently, there is an urgent need for the development of bioassay systems, preferably genomebased, which can be performed, at collection site locations, directly on plant extracts prepared in the field using micro-extraction technology to remove unwanted plant constituents. further local plant collection would then focus on those plant extracts which have a demonstrated biological activity. at the same time, the frequent failure of plant extracts to reconfirm biological activity on recollection will be reduced. who is potentially interested in exploring the rain forest biodiversity for bioactive products? several types of companies have a financial interest in plant-based natural products and extracts. in spite of the success of plant-based pharmaceuticals, most of the large pharmaceutical companies have terminated those aspects of their drug discovery programs which are based on plant-derived natural products. the prime reasons for this have been discussed elsewhere (1,3,4,9-12,20). as complex matrices, plant extracts provide a significant challenge for the bioactivity-directed isolation of the active principle(s). in addition, this deconvolution step may yield an undesirable known, rather than a novel, active constituent. the second consideration is that the recollection of a plant may be a time-consuming process, require extensive (re-)negotiations related to access, and may not be biologically active. yet another consideration is the ability to provide an adequate supply of a compound for additional biological evaluation in a timely and reproducible manner. for some biotechnology companies, the use of tissue culture systems in order to examine the ability of cell-free plant systems to produce metabolites which are not present in field-grown plants has proved important, and has provided an opportunity to study plant secondary metabolic enzyme systems and obtain new compounds for evaluation. the botanical supplement companies are finding new products from various parts of the world to market, with only a modicum of concern regarding the sustainability of their sourcing, the consistency of their constituents, and the limited scientific background justifying their use. the nutraceutical companies are looking to exploit the academic discoveries of compounds, such as cancer chemopreventive or cholesterol-lowering agents, which can be added to highvolume foods, or they are seeking plants which offer new and diverse life-style marketing opportunities (noni fruit and acai berry are recent examples). investment in research to justify the safety, use, and quality control of these products is minimal. with this summative background concerning the past and present practices of natural products in the discovery of new medicinal agents, it is opportune to consider the appropriate role of natural products in health care systems globally for the next twenty years. future aspects of natural products in drug discovery for future generations to thrive, be productive, and lead meaningful lives in their respective societies, there are two dominant factors to be considered, the health of the planet and the health of the people. these are not to be separated, as we are all part of one large, fully interacting, biological organism, earth. the goal must be to maintain the health of that whole organism in a cost-effective manner. failure to do so will leave a terrible legacy for our descendants. consideration of all chemical processes, including those for the synthesis of medicinal agents, and the accessibility of drugs to those in need are critical. factoring in the anticipated cost to the patient becomes an important consideration as potential drug candidates in any form, synthetic or natural, make the transition from discovery to clinical development. already there are numerous situations where the latest drugs are becoming (or have become) too costly for health care systems, including insurance companies, and their patients in the high-income world. when it is a health ministry in a country which decides which drugs are to be imported, and at what cost, for their health care systems, very significant compromises are typically made in terms of access to optimal health care. in the course of time, these differences in access are clearly reflected in geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 5 life-expectancy, and become another global pharmaceutical care “gap” between the north and the south. at the same time, it is also recognized that enhancing the quality of traditional medicines will have an impact on product costs. this is contained within the concept of accessibility, the combination of sustainability and affordability. almost lost now in the mists of time, it should be remembered, that before 1899, when the semi-synthetic drug aspirin, based on the natural pain reliever salicin, was introduced, all drugs were derived from natural sources. since then, a vast pharmaceutical industry, with many companies tracing their history to the original sellers of natural products in the latter part of the 19th century, have evolved to develop new drugs for a myriad of diseases. some of the compounds introduced during that period from 1900-2010 have been natural products, and the more recent discovery efforts have been reviewed by newman and cragg (28,29). most of the drugs that have been introduced in the past 110 years are totally synthetic. the primary exceptions are several groups of antibiotics, and the steroid and alkaloid derivatives which are semi-synthetic. it is time now to introduce the new term “ecopharmacognosy” (38). as we look to the future for the practice of using synthetic medicinal agents, one aspect of concern which requires deliberation is the sustainability of the production of those drugs at reasonable cost. both the chemicals and the chemical reagents used in those processes are typically a nonrenewable resource, and their contemporary use depletes the future resources for synthetic drugs. consequently, a fundamental precept for all drug discovery programs, be they synthetic or natural, must now be the concept of sustainability (4-12,38), as an extending consideration of the “green chemistry” movement. one aspect of this topic was discussed earlier in terms of the development of alternative, renewable sources for chemical reagents (enzyme catalysts, vegetables, etc.) (13). selected, valuable traditional medicines have been, or are being, depleted in their natural environment, without alternative resourcing being developed. ecopharmacognosy, defined as the “study of sustainable, biologically active natural resources”, must therefore become the fundamental basis for all natural product research. studying plants, or any natural organism, for their use as a global medicine, must take into account the potential long-term sourcing. we have already seen situations, taxol is an example, where the drug requirements for a clinical trial, came up against serious sourcing concerns. consideration of the enzymes to be used for the large scale synthesis of drugs, must also consider the sustainability of the resource. using the enzymes in plants that are already commercial entities, such as vegetables, assures their long-term accessibility, and reduces cost compared with rare enzyme preparations derived from microbial sources. ecopharmacognosy therefore embraces both the development of biologically active natural products as single agents or as components of traditional medicines, and the resources for the chemical synthesis of single agent drugs. the same considerations must also be applied as marine resources are explored for the development of new biological agents. except for some biological agents, such as vaccines, which will not be discussed here, there are usually three classes of single agent drugs recognized: i) totally synthetic drugs, which are produced from non-renewable resources, such as coal and oil; ii) semisynthetic drugs, such as the steroid hormones which are derived from a chiral natural product core produced in the field, or, in the case of antibiotics, through large scale fermentation. this is followed by structural modification using nonrenewable resources, unless microbial transformations are included in the synthetic strategy; and iii) natural products, such as vincristine or morphine, which are derived from a renewable natural resource. in this instance, the non-renewable aspects involve the materials used for processing the plant material in order to isolate the desired alkaloid. of perhaps thirty different aspects which could be discussed regarding the future for natural products in drug discovery, six will be briefly presented: i) access to the biome, ii) acquisition and analysis of traditional knowledge and on-going research, iii) biotechnology development for secondary metabolites, iv) dereplication studies, v) strategies in natural product drug discovery, and vi) multitarget therapy and synergy research. access to the biome the natural materials to be used, and any ethnomedical knowledge not already in the public domain, are the property of the country in which they are extant historically. the convention on biological diversity (cbd), approved for signature at the earth summit in rio de janeiro in 1992, established those parameters for those signatories who ratify the treaty. as of april 2013, 193 countries were parties to the convention. only three countries in the world had not ratified the convention: andorra, the holy see, and the united states. at the same time, there is a natural, unresolved tension with the trips (trade-related aspects of intellectual property rights) agreement of 1994 in the area of the development of local resources by third parties. in the trips agreement, the country of origin of the genetic material has no sovereign rights over their biological resources or their indigenous knowledge once there is an invention. it is the inventor who can claim the intellectual property rights, with the originating source of the material, whether it is derived from a plant, animal, or fungus in a particular country, having no claim for compensatory loss. the united states was the promulgator and the first signatory country to this agreement. this area, and the whole topic of the impact of the cbd on natural products research, has been reviewed (39), and the discussion will not be repeated here. some selected aspects of the cbd that relate to natural product drug discovery will be mentioned briefly. 6 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 article 15.2 of the cbd indicates that signatory countries should facilitate access to their biome in exchange for present and future considerations which are to be negotiated (39,40). articles 15.5 and 15.6 indicate that all of the collections of biological material which are made within a country should occur with prior informed consent, and that collection of the materials should occur with the accompaniment of local scientists. article 16 is concerned primarily with issues related to access and the transfer of technologies relevant to the conservation and sustainable use of genetic resources under fair and favorable terms. article 17 relates to the information concerned with the conservation and sustainable use of biological diversity and the results from scientific and socio-economic research are promoted. article 18 promotes international technical and scientific cooperation, and the development of joint ventures. following the cbd, there were numerous developments related to the establishment of protocols and systems within countries for the approval and collection of biological materials, and the acquisition of indigenous knowledge (39). indigenous groups, sometimes collaborating across national boundaries, have also sought to establish parameters and protocols to receive authority to provide access. while this has had the beneficial effect of reducing the number of scientists from various countries taking unauthorized plant materials or other biological resources out of a country without local knowledge (sometimes referred to as “biopiracy”), there has been a clear tendency towards the over-protection of access to resources, and/or excessive bureaucracy associated with the approval process (39). it is this outcome of the cbd, doubtless not anticipated by the drafters or the signatory parties, which has led to the overall deleterious impact of the cbd on natural products research on a global basis. this is because the value placed on the access to those resources by an individual country was often too great. the expectation that pharmaceutical companies would line up to seek access to areas of intense biodiversity, never materialized. as a result, if the approval processes, or the restrictions, or the time and the financing required to negotiate the associated considerations, were deemed too onerous, academic and/or industrial laboratories declined to invest, in either the people or the places. particularly, this occurred if more amenable choices for sample acquisition (based on bureaucracy, cost, and time), which might well have the same plant materials, were available. alternatively, many pharmaceutical companies simply chose to eliminate natural product extracts (marine, fungal and plant) from their primary screening programs, and, at the time, turned to other sources, such as combinatorial chemistry, for the guaranteed expansion of their chemical libraries. this resulted in two scenarios. firstly, the biodiversity-rich countries were unable to find collaborations (either academic or industrial) for programs designed to evaluate their biome for a health care impact, and were unable to enhance the development of much-needed taxonomic, chemical, and biological capabilities in-country. in some instances (e.g. the philippines), local scientists were also severely impacted by strict government requirements, and their research was essentially halted for extended periods (34). secondly, local pharmaceutical development, deemed critical for the type of initiatives being discussed to improve local health care, was inhibited. as a direct result, the reliance on externally acquired pharmaceutical and medicinal agents was maintained, benefitting the major drug manufacturers. without access to biological materials, the discovery of new biologically active natural products is diminished and advances in health care are impeded in that country and elsewhere. it is important that prior negotiated agreements are seen as an essential aspect of plant collection programs for drug discovery, and there is a sense of pragmatism required. very, very few compounds, or their semisynthetic derivatives, will ever become a profitable invention. an alternative pathway is to negotiate a graduated, two-tier approval process for the collection of biological materials which distinguishes between the “discovery” phase and the “development” phase. thus, one agreement would cover initial collection and academic/pre-toxicological research on limited size samples, and the second agreement would apply only when the acquired sample size had to be increased for more advanced pharmacology and clinical studies. (39,41). on october 29, 2010, the “nagoya protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization to the convention on biological diversity” was adopted under the auspices of the cbd (42). it is an instrument for the implementation of the access and benefit-sharing provisions of the cbd, and was opened for signatures on february 1, 2011. it was developed after six years of negotiation, and overall content supports and compliments the cbd. only countries which have signed and ratified the cbd are eligible to sign or ratify the nagoya protocol, but a country can also have signed the cbd and choose not to sign the nagoya protocol. when fifty countries had signed it entered into force and countries began implementation. as of april, 2013, 92 countries had signed the nagoya protocol, and 16 had ratified the protocol (43). the primary focus of the protocol is on the equitable sharing of benefits, and the requirements of signatory nations to develop procedures for implementation and regulation of the cbd, with a specific requirement for the issuance of permits with respect to permission granted for access to either genetic resources or indigenous knowledge. the protocol establishes an international clearing house under the cbd secretariat to assist countries with respect to developing various aspects of the implementation process, and requires countries to deposit appropriate records and information from their country with the clearing house for common availability. specific issues relating to trans-boundary situations must be discussed in instances where indigenous groups overseeing knowledge or resources are not located in a geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 7 single country, as may occur in parts of the andean region, in the middle east, and in southeast asia. in response to significant criticism about bureaucratic process and openness, ratifying countries are now required to assure legal certainty, clarity and transparency, both legislatively and in the implementation of regulatory requirements, such as applications for prior informed consent. countries must also provide effective communication systems during periods of application evaluation. permits which are internationally recognized will be issued by the recognized national authority for approved programs in a country based on prior informed consent and mutually agreed terms. this information will be provided to the newly-established cbd access and benefit-sharing clearing house. one can imagine that the permits will be needed both internally at various points in the process, at the collection site, at the exportation site for genetic material, and probably by major international journals in the field to assure compliance with international standards for published research articles. somewhat surprisingly, the contentious issues related to patents are not discussed in the nagoya protocol, even though it is an essential consideration for many groups seeking access to genetic resources and traditional knowledge, and an anticipated constituent aspect of any prior negotiated agreement between parties. consequently, the obvious tensions with the trips agreement remain unresolved. directly related to the concerns regarding patents and the non-obviousness of inventions is the issue of “derivatives”, which the protocol defines as “a naturally occurring biochemical compound resulting from the genetic expression or metabolism of biological or genetic resources.” this is not a robust definition of a derivative, and it is easy to imagine how a corporate entity could develop derivatives, be in accordance with mandates of the nagoya protocol, and still be the sole recipient of patent rights. another significant omission from the nagoya protocol is the absence of mandatory checkpoints or benchmarks which a certificate holder should be required to reach during the application and experimental processes. individual countries are however, permitted to include benchmarks of performance and reporting as they deem necessary. acquisition and analysis of traditional knowledge and on-going research it has been said that the countries which will be successful in science and technology in the future will be those who use the globally available knowledge most creatively to generate new knowledge and inventions. this “smart” technology is desperately needed as strategies and considerations for the exploration of the tropical forest are initiated. information, ethnomedical, botanical, chemical, and biological, is burgeoning. new technologies which may impact drug discovery programs are being developed at an incredible pace. almost every country, and within them most scientific research laboratories, when they are connected to the internet, have essentially unlimited access to this information, most frequently at no or minimal cost. with respect to traditional knowledge, the ease of access to information has its benefits and hazards. the benefit is that more information on the use of a plant can be compared than merely relying on one or two local compendia of information. the hazard is whether all of the available information has been acquired legally under local regulations, particularly knowledge of the use of indigenous plants acquired since the cbd. another consideration is that the vast amount of published ethnomedical information, collected by ethnobotanists and medical anthropologists, on the use of plants for medicinal purposes, is extremely scattered, and is therefore very difficult to acquire in toto. undoubtedly, there is always more literature to be unearthed on the use of a particular plant, if only the resources were available. as stated on several occasions (3,4,6-12), there is a dire and urgent need for an international agency, possibly in collaboration with a global foundation, to fund the development of a central repository of indigenous knowledge, a sort of “wikiethnomed” . but the data base repository should not end there. as well as ethnomedical information, data on the biological evaluation of plant extracts and their constituents, the chemistry of the natural plant sources, and the clinical evaluation of plant extracts, needs to be acquired and collated. data on the safety and possible or observed adverse events associated with traditional medicines are also required to be collated for open access, as a health care consideration for practitioners and patients. at this stage in the 21st century, it should be possible, where ever one is in the world, to indicate a plant name and then find its barcode (vide infra), ethnomedical information, chemical constituents, biological activities, and clinical evaluation data. it is estimated that such an on-line, global health resource, once established, and depending on physical location(s), would cost about $5 to 7 million to operate each year; a veritable bargain given the health care benefits. for the development of rational, sustainable drug discovery from plant sources the first step is a critical evaluation of all of the available information on a plant, or on the plants to be evaluated for a particular health benefit, such as antihyperglycemic activity. this is needed in order to prioritize plant acquisition plans, avoid the unnecessary duplication of research effort, and optimize the consumption of precious (financial, personnel, and oil-based) resources. there are also important ecopharmacognosy considerations of sustainability which enter into these strategies as well. biotechnology development for secondary metabolites when one acquires, dries, and then extracts a plant material, the observed chemical profile is the phytochemical equivalent of a “kodak moment” (10-12). for over 180 years, phytochemistry, and by inference chemotaxonomy, tacitly accepted that this “moment” represented the biosynthetic capacity of that plant. now 8 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 it is well-established, through the use of tissue culture, cell-free systems, and elicitor molecules such as methyl jasmonate, that this is an inappropriate paradigm, and represents only a partial view of genetic capacity for secondary metabolite formation. indeed, it is more like a “still shot” from the dynamic secondary metabolite profile “movie” of the plant. for no single plant on earth has the full metabolic profile yet been determined through analysis of the genes of secondary metabolism and then demonstrated through expression. a more complete understanding of what a plant is in terms of a chemical factory for making secondary metabolites of biological or clinical interest, may be very important to achieve on a selective basis in the future. thus, a single extract of a plant, taken at a single point in time, cannot reflect the constituent range of that plant, ignoring as it does numerous intrinsic and extrinsic factors, including dormant biosynthetic genes which will alter the chemical profile, and thus the biological outcome. the ability to modulate these genes, and thus the enzymes they code for, in a controllable manner, will be a critical aspect to enhancing plant secondary metabolic diversity for biological screening, and in establishing reproducible production levels for needed metabolites, especially for the constituents of traditional medicines. fungal genetics has progressed remarkably from the perspective of understanding secondary metabolic profiles and production in recent years, and inferences with respect to the evolutionary aspects of fungal organisms are developing rapidly (44). because of challenges with the locations of metabolic genes, plant genetics has lagged somewhat. however, as these details of the plant genes responsible for secondary metabolite biosynthesis are recognized, it becomes possible to express the genes in other, faster-growing formats (e. coli, yeast, insect cultures, etc.), and produce either a new range of metabolites previously unknown from that plant, or a desired enzyme, compound or series of compounds with a significantly higher degree of control (45,46). from a biological screening perspective, it will be important to identify the regulatory genes responsible for secondary metabolite production control, and thereby modulate the secondary metabolite profile, so that rather than being a snapshot, it can indeed become a movie. both the short and long-term implications of this are clear. maintaining the genetic capacity of the tropical forests, which has taken billions of years to evolve, through conservation, acquisition and botanic garden development, or sampling and storing germplasm, is a critical aspect of a diversified and well-considered longterm medicinal agent discovery program for a nation. it may be that locked in the dna of a plant are the genes to produce crucial drugs in the future, once it is possible to more completely understand the contortions of their metabolic formation. certainly, it is unconscionable to destroy the forests without first sampling and preserving the breadth and depth of the diversity of genetic resources that are within. there are numerous other aspects of biotechnology which impinge on natural product drug discovery and development, including bioassay design and implementation, drug delivery systems, and enhancement of natural product structural diversity (vide infra). some aspects of these technologies have been discussed elsewhere (5-7,9-12). dereplication studies plants are chemical factories which produce a wide range of metabolites of various structural types. many plants across taxonomic borders have evolved the genes to produce similar or identical metabolites. the wide spread occurrence of common flavonoids, such as quercetin and related derivatives, is an example. once an extract is declared “active”, prioritization along with other “active” extracts for bioactivity-directed fractionation is needed. frequently, there are more active plants than capacity (human) to fractionate. at this point, there are three research options: i) eventually fractionate all the extracts; ii) introduce a secondary bioassay to discern a more selective priority list, or iii) apply a dereplication strategy. dereplication is a process to delineate, in an active extract, the probability that the active principle is likely to be novel. such a determination can re-prioritize active extracts, and/or eliminate extracts for further examination, and it can improve the efficiency of isolating the active principle. several years ago we described an hplc/esms/bioassay/database dereplication system for active natural products in biologically active matrices (47), and went on to describe how it was used on active extracts for the identification of both new and known natural products (48). refinements in dereplication systems include: i) development of software which can rapidly correlate mass and biological data to a chemical and biological database so that searches and conclusions can be achieved on-line, ii) introduction of continuous flow nmr to support postulated structures of active masses, and iii) recognition of carbon-13 nmr to provide an even higher level of certainty regarding the possible skeletal structure and functional group disposition of a molecule within the matrix. over time, the ability to characterize the majority of the known major active constituents in an extract without isolation will become a standard practice. a further use for such hplc/esms/nmr/bioassay system technology, other than drug discovery, will be for the chemical and biological standardization, as well as metabolomic studies of traditional medicine samples, on a batch to batch basis, once the appropriate active principle(s) have been identified. strategies in natural product drug discovery it is an important question to ask whether natural products are drug molecules. several years ago (21), it was shown that those alkaloids which are presently available as pharmaceutical agents are a good fit (with some outlying molecules) to lipinski’s “rule of five”. when an analysis geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 9 of 120,000 compounds in the dictionary of natural products was conducted, 65% had no violations of these rules, and this led to the development of a natural product library of over 500 compounds for drug screening (49). other studies have supported these conclusions (50,51). thus existing natural products can form the basis of a drug discovery program complementing the development of potential new sources of bioactive molecules. strategic considerations for the development of natural product drug discovery programs abound (17,19,52-54). how the plants will be collected (random, phytochemical, ethnomedical), how plants will be extracted (water, non-polar, and polar solvents), which biological assay to use (animal, whole cell, organ tissue, enzyme, receptor), and how to establish levels for “activity” are strategic decisions which must be taken prior to initiating the program and then reviewed regularly during the program. it is also important to recognize that a plant is not a single organism. there are numerous fungi, bacteria, and, in some cases, algae, symbiotically associated with the plant. these organisms are also biosynthetic factories, and the study of pathogenic and parasitic organisms may offer the potential to enhance the range of natural product structures in a sustainable, albeit rather unpredictable, manner. accessibility to resources, reproducibility of activity in recollections, and the time to yield an active isolate are important concerns in the involvement of plant extracts in drug discovery (1-3,10-12). there are several other issues which can also affect the overall discovery process. we have seen that isolation of known compounds with a known bioactivity is a significant issue in some biological areas (such as cancer), and there is also the patent concern of known compounds which may afford new biological activities. patenting inventions has become an important, though not essential (vincristine, taxol, and camptothecin were never patented), function for both industrial and academic discovery programs, and the strongest drug patents are those which claim a new chemical entity with a new biological activity. sample repositories are an essential aspect of any plant-based drug discovery programs. typically, as a program evolves, there are three types of repository that are needed: i) for the plants acquired, ii) for the extracts made, and iii) for the compounds isolated. the first may, in part, be the local, internationally recognized, herbarium where a formal, classically prepared specimen is stored for posterity. also needed is a dry, airconditioned, moderate temperature (0-10˚c) facility for storing the remainder of collected, but unused samples, appropriately labeled and catalogued in a database. the second repository is for residual samples of all extracts and fractions that are prepared, usually held at -20ºc or below, and again systematically catalogued electronically and organized in individual, barcoded vials for easy access. frequently, sample specimens of extracts are also stored in 96-well plates for screening purposes, depending on strategic demands based on the available and projected biological assays. finally, purified and isolated compounds must be catalogued and stored, both in vials, and in 96-well plates for further screening. long term, the drug discovery goal should be to screen every isolate against every relevant bioassay. electronic cataloguing and analysis of this information is important, as is recognizing that data base access should be available to all collaborators. of course a fourth database, not experimental in nature based on the current studies, should constitute an inventory of information on the previously reported studies on all of the plants actively under study. the first step in deciding whether to actively work on a “hit” extract is to become fully aware of all of the prior literature on that plant and related species. it is another example of “information first” as a philosophical precept. identification of the plant materials to be used for discovery purposes is very important as a basis for the whole program. but how is the plant to be identified? preliminary identification may come from morphological examination and herbarium specimen comparison. for several years, dna techniques, such as random amplified polymorphic dna (rapd), amplified fragment length polymorphism (aflp), and inter-simple sequence repeat (issr) analysis were deployed as an adjunct for plant identification, and for studying the genetic variation in medicinal plants (55). now the focus is on the development and application of dna barcoding (56), as one facet of the consortium for the barcoding of life (cbol), and some background details pertaining to medicinal plant identification have been discussed (9-12). the dna fragments which are considered appropriate to use for species identification at this time are focused on the its2, matk, and trnh-pasba genes. reliability rates, the ability to make positive medicinal plant identification based on specific genes at the genus and species level, are typically very high (76-96%). chen and co-workers (57), using seven dna barcodes (psba-trnh, matk, rbcl, rpoc1, ycf5, its2 and its), studied over 6600 plant samples from 753 genera and 4800 species. they found that the its2 barcode gave a 92.7% successful identification rate, and thus could potentially used for the identification of medicinal plants. how this relates to secondary metabolite production, and therefore to a reproducible biological activity, remains an area for future exploration. another significant future development is the incorporation of barcoding technology into highly automated, hand-held devices which could provide extremely rapid, in-field identification of plants (58). principal component analysis (pca) of the low molecular weight (ca. 200-500 daltons) compounds in a plant as a part of metabolomic studies is also changing how a plant is defined (59). it will become an important component of drug discovery programs to assure adequate compound sourcing in recollected materials, either for expanded biological evaluation or possibly to meet production demands. the combination of these techniques means that the identification of plants through gross morphology or macroscopic examination is rapidly being supplanted. 10 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 the gene-based and chemical analysis techniques now demonstrate that within a recognized plant “species” there are many “forms”, “races”, or “chemotypes”. these plants will have different functional genetic profiles under different circumstances, different biosynthetic capacities based on those gene profiles, and therefore variable and complex chemical matrices. this begins to explain why reproducible plant recollection is so challenging and frustrating. within the next few years, dna barcoding, and probably pca, will become essential and integral aspects of all medicinal plant identification, whether for drug discovery programs, or for standardization of traditional medicines and phytotherapeuticals. the result may be that the concept of a latin binomial name for a plant, romantic though that is, will be replaced, or added to, by a series of identifiers which will define both the integrity and the quality of the sample. in the biological evaluation of plant extracts tannins are a frequent confounding factor and must be removed when either enzyme or receptor-based assays are involved (60). this has led to the development of detannification and partial fractionation techniques to form peak libraries of dominant compounds in an extract which can then be screened. while minor bioactive compounds may be missed, this strategy does begin to address the issue of adequate subsequent supply when bioactivity is found, a core consideration of ecopharmacognosy (38). various data mining strategies may also be used to construct libraries of plant extracts or individual compounds, once adequate, pre-existing literature has been compiled. chemical space matches and pharmacophoric models also provide an in silico approach to the identification of compounds of potential interest which can then be isolated or further derivatives sought. rapid access to plant materials as potential resources, based on existing isolation or chemotaxonomic information to identify plants for collection, is then an essential component of the discovery strategy. multitarget therapy and synergy in considering both the quality control and the drug discovery perspectives of traditional medicines, two aspects are significantly undervalued in terms of therapeutic outcome, multitarget therapy and synergy. rather than the classical “magic bullet” approach, therapeutic outcomes have improved significantly for two of the most important disease states, cancer and aids, as a result of strategically assembled multicomponent regimens derived from considering a diverse mechanistic targeting system (61). ethnomedicine has already established that model of therapy. the chemical factory of a plant, and even a hot water extract, will contain a multitude of constituents, and multicomponent plant regimens, in which five, ten, or even twenty plants are used in a prescription, provide an unprecedented range of highly diverse chemical constituents which are affecting multiple sites and acting by diverse mechanistic pathways. an important question is whether there is clear evidence of rational use for the role of each individual component plant in a multicomponent traditional medicine? as evidence-based traditional medicines are explored for drug discovery and validation this will become an important experimental target, and eventually a consideration in the sustainability of that product. a recent discussion on network pharmacology provides an important framework for these deliberations, and for the potential design and discovery of new agents with unsuspected biological activity (62). the biological effects observed in vivo or clinically in an extract may be due to one or more active compounds acting at different sites. alternatively, two, or more, components in the mixture could be acting in a synergistic or in an antagonistic manner. in addition, the effects when a traditional medicine is taken with a single agent drug are unknown, resulting in a possible adverse drug reaction (adr) which may potentiate or inhibit the actions of the single agent drug. when combinations of medicinal plants are used, as in many drug systems around the world, the situation becomes significantly more complex. williamson (63) and wagner (64,65) have stimulated discussion in this area. a significant issue in studying synergy and antagonism in multicomponent traditional medicines has been technique and definition (65). berenbaum (66) used a mathematical definition based on an isobole to represent these biological outcomes, so that the effects of a combination of agents are independent of the mechanism of action, and can be presented graphically. a powerful demonstration of a synergistic interaction between two natural products occurs with mixtures of ginkgolides a and b examining platelet aggregation (67). potentiation of the effects of kava-kava and a passiflora extract as a sedative, and of a complex preparation of nine plants for dyspepsia whose constituent plants demonstrate effects on a range of motility-related disorders have also shown synergistic results (64,65). the protocols developed for these studies may have an important influence on the evolving strategies for the evaluation of the quality, safety, and effectiveness of an individual traditional medicine, and on the discovery and development of new, more effective combinations of medicinal plants (“designer traditional medicines”). in addition, they may offer strategies to increase the sustainability of particular medicinal plants, if the synergistic effects can be quantified and reliably reproduced. critically, such experiments require that the extract is well standardized and the biological mechanisms are well clarified prior to synergy experiments being initiated. challenges and strategies for the future a vision for natural products derived from the tropical forest is based on the concept that sustainable considerations and strategic thinking are needed now. in addition, those resources will be needed even more in the future, in order to provide medicinal agents as fossil geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 11 based resources become depleted during a period when the global population is increasing rapidly. such a vision involves the creation of a new term, ecopharmacognosy, to describe the philosophical shift, and requires new paradigms for the conduct of the natural product sciences on a global basis, including new balances of program development for drug discovery and traditional medicine quality control, new strategies, a new emphasis on evidence-based research, new alliances between various collaborators in academia, industry and government, and new values for what is meaningful, natural product research for health care enhancement (8-12,38). it should be readily apparent that humankind cannot survive another century as destructive of the earths’ resources as the 20th century. twenty-one years after the rio summit of 1992 we are as “irresponsible” as ever with respect to our biodiversity. at the end of the last century one-eighth of all plant species were threatened, 50% of bird species are likely to become extinct by 2050, and affordable oil resources are estimated to last until 2060. the abiding concern which the world must grapple with now is whether, as a human race, we can see these issues as being our destruction and own our responsibility in any attempted restoration of a balance within gaia (1). it is critical that we are mindful of the balance between the conservation of the existing rain forests, and the destruction of these fragile and deeply interwoven ecosystems and their deforestation for crop and grazing lands. our concerns are for ecological, climactic, and geological reasons, and as a way to maintain the bio-, and therefore chemo-, diversity within those forests. as described within ecopharmacognosy, plans for the development of new medicinal plants in any form to fill a market niche must be sustainable. an appropriate balance is needed between intellectual property rights and the burgeoning technology of drug discovery. a balance is also needed between all those who are stake holders for biodiversity and indigenous knowledge and those who have the capacity to potentiate (create value) in that biodiversity for health and economic benefit. this is the balance that is needed between the cbd/nagoya protocols and the trips agreement (39). numerous new alliances, both internal and external will be needed to effect the rational development of tropical forests for new medicinal and biological agents. some may already be in place; they need to be strengthened. other alliances will be new; they will need to be carefully nurtured. diversity of expertise and experience indicates that the alliances will be both local and global in nature. they will involve individuals and groups who have the capacity for high level collaboration and low level ego involvement. several examples of such collaborative programs have been described (1,52-54). the strongest programs, rather than being solely based in academia, will have industrial and government partners working together for a common goal, enhanced health care for the patient. countries which are engaged in medicinal agent discovery programs based on local natural resources are recommended to examine the structure and functioning of these programs as a potential model for collaborative investment to promote the development of local medicinal plants and natural products. in a sound and comprehensive health care program, such alliances are an integral aspect (6-12,38). in order to accomplish these goals though we must create value; value in places, in people, and in plants. the tropical forest is a huge vast ecosystem which is comprised mostly of “weeds”, those “plants whose virtues have yet to be discovered”, as ralph waldo emerson suggested in the late 19th century. an important responsibility for natural product scientists to the generations following is either to demonstrate the value of biodiversity through new discoveries of plant-derived medicinal agents, or to leave the resource alone and protected. solid linkages which unite the interests of environmental preservation, medicinal plant research and drug discovery, and the development of the agroindustrial enterprise are therefore crucial. examining import levels of finished pharmaceuticals and natural products (essential oils, and flavor and fragrance materials) in the context of developing local industrial capacity is important. structured and well-monitored natural product development programs based on tropical forest resources could lead to reduced imports and increased exports. a vision of the natural product sciences contributing to global health care offers very significant challenges to many countries. a primary challenge is to catalog and preserve the bioand chemo-diversity of the rainforests (and the oceans) for the benefit of future generations. a secondary challenge is the need to catalog the ecoand ethno-information on plants, and the chemistry and biology of their products, so that the information can be collated, analyzed, and accessed globally in real time. exploration aimed at potentiation of the biota of the world from any of several aspects, including drug discovery, necessitates systems being available locally which can offer equitable access to the biome and substantial assurances with respect to intellectual property rights and investment commitments. many medicinal plants, some of them threatened or endangered, are presently in commerce in various parts of the world, either for the production of single agent drugs, or in traditional medicine preparations. medicinal plant germplasm banks in selected locations throughout the world are needed to preserve these crucial resources, in the same way that seed crop banks are used for important food crops. at the same time, contemporary techniques for plant identification, including dna barcoding, must become integral aspects of what is considered plant identification, whether these plants are being investigated or used as medicinal plants, or whether they are being pursued for possible drug discovery of standardized preparations or single agents. the sustainable development of plants and their extracts or fractions which are marketed for health care purposes is critical. a “sustainability index” probably needs to be developed, so that patients can clearly see from a label how the product was harvested and prepared. a product derived directly from the forest and extracted 12 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 in a manner where an organic solvent was not fully recycled would get a rating of “0”, whereas one harvested organically in a sustainable manner with full recycling of any solvents, might get a rating of “5”. individual compounds and plant extracts must be evaluated using contemporary procedures and targets for both quality control and drug discovery. strategies should be developed for genomics-based, in-field bioassays to evaluate plant extracts in the field, to avoid bringing dried (or fresh) plant materials to the laboratory for extraction and bioassay. the number of natural products presented to test systems for biological evaluation could be enhanced through chemistry, combinatorial synthesis, combinatorial biosynthesis, and other strategies. drug discovery programs must be introduced, strategically developed, and enhanced based on local plants and for local diseases. most importantly, integrated global alliances are needed both in-country, and between developed and developing countries, for medicinal plant product development, in order to optimize the utilization of facilities, infrastructure, and personnel who are needed to conduct the above programs. how can these goals and the associated programs be initiated? first of all it must be said that plans of the scale described in this article and elsewhere (4-12,38) begin with small scale initiatives, reasonable benchmarks, and an appropriate timetable. they are not instantaneous “fixes” of the situation, nor will they yield rapid results. strategic planning at the highest levels is required, combining government agencies, industrial enterprises, international agencies and foundations, academic institutions, and private consultants with natural product drug discovery and development experience. planning and proposal development may take 1-2 years of meetings, discussions, and consultations to evolve. over time, countries, or consortia of countries, will require an infrastructure to foster the development of their own sustainable medicinal agents from natural sources based on the quality of their natural product sciences. programs will be needed to assist countries to potentiate their infrastructure and resources, including their facilities and their scientists, in order to evaluate and standardize natural product-based medicinal agents on a sustainable basis for their health care systems, and that may take 510 years to evolve. ideally, as proposed originally in 2002 (3), what is drastically needed is a global alliance for natural product development and health care. such an alliance would be composed of international agencies (who, unido, undp, nato, eu, wipo, etc.), government agencies (nih, nsf, nie, src, daad, etc.), global and local pharmaceutical companies, academic institutions, non-government organizations (wwf, wri, cyted, tramil, ifs, twas, etc.), scientific societies (iupac, rsc, asp, pse, ga, jsps, etc.), and major foundations (ford, gates, macarthur, rockefeller, nippon, etc.). a mechanism to bring representatives together to discuss the global issues and implications in new terms, with a new set of goals, with a new agenda, but most importantly with a new vigor, is vital for the global development of natural product based drugs for health care. conclusion globally, natural products, in the form of purified active principles and plant extracts, are the cornerstone of primary health and for the amelioration of life-style conditions, and will remain so for decades to come. with a burgeoning population, the challenges for health care in the future remain significant. for most of the major killer and chronic diseases in the world there are no truly effective drug treatments. drug resistance to existing chemotherapeutic regimens for fungal and bacterial infections, for aids, for cancer, and for malaria, is increasing in an unabated and alarming manner. many killer and debilitating diseases in the middle and lowincome areas of the world exist without any effective drug treatment, or even a drug discovery program, in place. yet, overall health care is slowly improving, life expectancy is rising in most countries, and more children are surviving beyond their first five years. these are contributing factors to the dramatic rise in the global population. however, without a new strategic approach, this population will not have adequate resources to provide basic health care. there is no choice in this matter, medicinal plants must be an essential, sustainable, and fully 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c. gyllenhaal, g.a. cordell, n.r. farnsworth, a.d. kinghorn, and j.m. pezzuto. 2002. the evolution of the university of illinois= policy of benefit-sharing in research on natural products. in: j.r. stepp, f.s. wundham, and r. zarger, eds. ethnobiology and biocultural diversity. university of georgia press, athens, ga, p. 21-30. the text is available at http://www.cbd.int/abs/text/ accessed on august 15, 2011. information at: http://www.cbd.int/abs/nagoyaprotocol/signatories/default.shtml. accessed on april 13, 2013. soejarto, d.d. and n.r. farnsworth. 1989. tropical rain forests – potential source of new drugs? persp. biol. med. 32, 244-256. s. li and b. zhang. 2013. traditional chinese medicine network pharmacology: theory, methodology and application. chin. j. nat. med. 11, 110-120. wagner, h. 2006. multitarget therapy the future of treatment for more than just functional dyspepsia. phytomedicine 13, 122129. wagner, h. and g. ulrich-merzenich. 2009. synergy research: approaching a new generation of phytopharmaceuticals. phytomedicine 16, 97-110. wagner, h. and b. steinke. 2005. natural products chemistry and phytomedicine in the 21st century: new developments and challenges. pure appl. chem. 77, 1-6. wall, m.e., m.c. wani, d.m. brown, f. fullas, j.b. oswald, f.f. josephson, n.m. thornton, j.m. pezzuto, c.w.w. beecher, n.r. farnsworth, g.a. cordell, and a.d. kinghorn. 1996. effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for their removal. phytomedicine 3, 281-285. wetzel, s., a. schuffenhauer, s. rogo, p. ertl, and h. waldmann. 2007. chemoinformatic analysis of natural products and their chemical space. chimia 61, 355-360. williamson, e.m. 2001. synergy and other interactions in phytomedicines. phytomedicine 8, 401-409. volume 4 number 2 2015 i s s n 2 0 8 9 6 5 1 4 biology, medicine, & natural product chemistry volume 4 – number 2 – 2015 issn 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia maizer said nahdi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty 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[indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. offprint or reprint is only available with special request. note: author(s) agree to transfer copy right of published paper to biology, medicine, & natural product chemistry. author shall no longer be allowed to publish manuscript completely without publisher permission. authors or others allowed multiply article in this journal as long as not for commercial purposes. for the new invention, authors suggested to manage its patent before publishing in this journal. notification: all communications are strongly recommended to be undertaken through email. volume 4 number 2 2015 published twice a year printed in indonesia front cover: fagraea ceilanica loganiaceae (photo: widodo) issn: 2089-6514 krokot (portulaca oleracea. l) as a natural sensitizer for tio dye-sensitized solar 2 cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi, didik krisdiyanto effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja'far luthfi antioxidant potential of black, green and oolong tea methanol extracts wahyu widowati, tati herlina, hana ratnawati, gabriella constantia, i dewa gde sathya deva, maesaroh maesaroh medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja'far luthfi, mahanem mat noor, jalifah latip tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik 25-29 31-33 35-39 41-47 49-51 issn 2089-6514 cover jurnal biomenaprochy vol 4 num 2 2015 dpn.pdf (p.1) editorial board 2015 v4 n2.pdf (p.2) guidance_v4n2.pdf (p.3) cover jurnal biomenaprochy vol 4 num 2 2015 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 57-59 | doi: 10.14421/biomedich.2018.72.57-59 issn 2540-9328 (online) alizarin red s-alcian blue staining for regenerated tail of common house gecko (hemidactylus frenatus) rakhmiyati1,*, muhammad ja’far luthfi2 1postgraduate program, universitas sebelas maret jalan ir. sutami 36 a, surakarta, 57126, tel. +62271-646994, fax. +62271-646655, indonesia 2department of biology education, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: miarakhmiy@gmail.com abstract common house gecko (hemidactylus frenatus) is one of reptiles that have ability to autotomy their tails. tail autotomy is a mechanism to protect it self from predators. after the tail broke, there will be wound healing on the tail which is then followed by a tail regeneration event. original tail and regenerate tail is very different morphologically and anatomically. the original tail is composed of bones while the tail of the regenerate is composed of cartilage. histochemical staining using alizarin red-s alcian blue was done to differentiate bone and cartilage. this method will stained bones red while the cartilage will stained blue. keywords: common house gecko (hemidactylus frenatus); autotomy; alizarin reds alcian blue; cartilage; bone (osteon) introduction the tail of the common house gecko (hemidactylus frenatus) is flat and tapered at the tip. in the dorsal area there are fine scales and no spots (takahashi, 2009). these scales are brownish yellow (pratiwi, 2009). the tail comprised of procoel vertebrae (maria, 1998). autotomy is a capacity breaking of the tail, either part or whole of the tail when the animal is chased or captured (lin & lin, 2017; luthfi, 2002; soesilo, 1992). in general, autotomy can be said to be a structural adaptation to minimize tissue damage and ease the wound healing process (gilbert et al, 2013). tail autotomy is a self-protection mechanism that can be carried out by lacertilian (londono et al, 2017). according to pratt (1946) if the tail is held it will cause uneven pressure of muscle contraction across all segments and excessive pressure will occur on the tail held resulting breaking. tail function in lacertilian are as a regulator of balance and movement, storing energy, sexual and reproductive function (jagnandan et al, 2014). the next step is regeneration, which is the ability of living things to replace body parts lost due to injury, tear, damage or autotomy (kimball, 1983; soesilo, 1992). regeneration process occurs through several stages, namely the wound healing stage, cell proliferation, angiogenesis, formation of extracellular matrix, blastema formation stage and stage of tail differentiation and growth (soesilo, 1992; alibardi, 2009; gilbert et al, 2013; lozito & tuan, 2016; jacyniak et al, 2017). mesenchymal cells and several cells accumulate below the wound epidermis form blastema (alibardi, 2017). in order to observe the structure of regenerated tail of common house gecko (hemidactylus frenatus) it is necessary to make the original tail and tail regenerate preparations using alizarin red s-alcian blue staining. materials and methods common house gecko were sacrified using chloroform, then the tails were cut at its base. the tail were then immersed in 96% alcohol solution for 4 days, skinned, and immersed in 96% alcohol solution for 2 days. the tails were soaked in acetone for 3 days, then soaked in a dye solution for 4 days (1 volume 0.3% alcian blue in 70% alcohol + 1 volume 0.1% alizarin red s in 95% alcohol + 1 volume of glacial acetic acid + 17 alcohols 75% volume) and then washed with distilled water. after that the tails were immersed in 1% koh for 3 days. the tails were soaked in a mixture of glycerin and 1% koh with a ratio of 20%: 80%; 50%: 50%; 80%: 20% successively, each for 2 days and after that the preparations were keep in pure glycerin to be observed. results and discussion data obtained from observations on the original tail and the regenerate tail of the wall lizard stained with alizarin red s-alcian blue showed a difference between the original tail bone and the regenerated one. the original https://doi.org/10.14421/biomedich.2018.72.57-59 58 biology, medicine, & natural product chemistry 7 (2), 2018: 57-59 tail is composed of bones (osteon) where as of regenerate is of cartilage. figure 1. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (dorsal view). magnification 3x. (a). processus dorsal; (b). processus transversal; (c). processus ventral. the vertebral type of common house gecko (hemidactylus frenatus) is procoelous. its centrum is concave or curves anteriorly and is convex posteriorly. the original tail has procesus transversus as a posterior attachment of the muscle, while the anterior part of the muscle is attached to the myoseptum (figure 1). viewed from the ventral side, there are prezygapophysis, postzygapophysis, centrum and cevron bone. figure 2. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (ventral view). magnification 3x. (a). postzygapophysis; (b). prezygapophysis; (c). centrum; (d). cevron bone. seen from the ventral part of the tail, there is a joint of prezygapophysis found in the anterior vertebrae and postzygapophysis joints found in the posterior vertebra (figure 2). these two joints are only located in the dorsal part. the original tail stained red along the vertebrae caudales because it is composed of bone tissue starting from the base of the tail to the tip of the tail (figure 3). the processus vertebrae are found along the vertebrae caudales, but its size reduced posteriorly. figure 3. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (transversal view). magnification 3x. (a). autotomy plane; (b). processus dorsal; (c). processus transversal; (d). processus ventral. figure 4. whole preparation of immature regenerate tails of common house gecko (hemidactylus frenatus) of alizarin red s-alcian blue (magnification 7x). (a). bone of original tail; (b). border (crack area) between the original tail and the regenerate tail; (c). cartilagineus tube of immature regenerate tail. the dark red color in the original tail indicates that the tail has many calcium ions which bind to the alizarin red s dye, where as the immature one will stained blue because the it does not have calcium ions. this is consistent with the statement expressed by lozito & tuan (2016). rakhmiyati & luthfi – alizarin red s-alcian blue staining for regenerated tail … 59 figure 5. whole preparations of old regenerate tails (hemidactylus frenatus). alizarin red salcian blue (magnification 11,25x). (a). cracks; (b). cartilago tube of old regenerate tail. in the mature regenerate tail, cartilaginous tube looks red because calcification has occurred. along the mature regenerate tail there are cracks that can break the tail (figure 5). viewed from any side (dorsal, ventral, and transverse), the regnerate tail will look the same color. conclusion from the results of this study it can be concluded that the original tail is composed of bones (osteon) and has complex parts, namely the transverse processes, dorsal processes, ventral processes, prezygapophysis, postzygapophysis, centrum, cevron bone, vertebral cracks (autotomic plane). where as the regenerate tail is composed of cartilage which is tube like shaped and will calcified in mature regenerate and developed crack in a very matured one. references alibardi. 2009. orphological and cellular aspects of tail and limb regeneration in lizards. 1 st edition, pp: 1-49. alibardi. 2017. review: biological and molecular differences between tail regeneration and limb scarring in lizard: an inspiring model addressing limb regeneration in amniotes. journal of ekperimental zoology (molecular and developmental evolution). 00b: 1–22. gilbert et al, 2013. the anatomy and histology of caudal autotomy and regeneration in lizards. physiological and biochemical zoology. vol. 86(6):631–644. jagnandan et al, 2014. tail autotomy and subsequent regeneration alter the mechanics of locomotion in lizards. the journal of experimental biology 217: 3891-3897. jacyniak et al, 2017. tail regeneration and other phenomena of wound healing and tissue restoration in lizards. journal of experimental biology. vol. 220, pp: 2858-2869. kimball, 1983. biologi. edisi kelima. jakarta: erlangga. lin & lin, 2017. tail regeneration after autotomy revives survival: a case from a long-term monitored lizard population under avian predation. the royal society publishing. pp: 1-9. londono et al, 2017. cartilage and muscle cell fate and origins during lizard tail regeneration. frontiers in bioengineering and biotechnology. vol. 5, no. 70, pp: 1-9. lozito & tuan, 2016. lizard tail skeletal regeneration combines aspects of fracture healing and blastema-based regeneration. development. vol. 143, pp: 2946-2957. luthfi, m. j. 2002. kalsifikasi skeleton aksial dan kemampuan autotomi regenerat ekor kadal (mabouya multifasciata kuhl). tesis. universitas gadjah mada (unpublished). maria, b. 1998. struktur vertebrae caudales pada 5 species anggota sub ordo lacertilia. thesis. universitas gadjah mada (tidak dipublikasikan). pratiwi, 2009. struktur organ reproduksi dan seksual dimorfisme in hemidactylus frenatus dumeril & bibron, 1836; cosymbotus platyurus (schneider, 1792); & gekko gecko linnaeus, 1758. thesis universitas gadjah mada (unpublished). soesilo, n. p. 1992. proses regenerasi ekor kadal (mabouya multifasciata kuhl). biologi, vol. 1, no. 4: 169-175. takahashi, 2009. preferensi pakan cicak rumah (hemidactylus frenatus, gray 1825) dan tokek (gekko gecko, linnaeus 1758) di gamping, sleman, daerah istimewa yogyakarta. thesis. universitas gadjah mada (unpublished) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 19-36 | doi: 10.14421/biomedich.2017.61.19-36 issn 2540-9328 (online) checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo1, muhammad jafar luthfi2 1,2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: wwidodo594@gmail.com1 abstract this study aimed to collect data on the species plants in mount nglanggeran, to confirm and update the existence of these plants from flora of java book by backer & bakhuizen, and to showing the visual data of plant species in nglanggeran mountain. this research used survey method through in-situ visit and specimen collection. monitoring and visits were conducted based on prediction of flowering period and fruit-bearing season. eighty plant families of flowering plants were found in the mount nglanggeran. based on the phylogenetic arrangement of flowering plants it was found that all the main groups (clades) of flowering plants were found at this location. keywords: checklist; magnoliophyta; update plant list; mount nglanggeran; local plant. introduction non-cultivated plants in java, especially in yogyakarta tends to be neglected by young generations. the plants left as ornamental plants, wild plant on waste lands or protected places such as in protected forests, cemeteries, etc. more often unknown plant identified only by its scientific name and preserved in herbarium centers in europe and discussed in old books in the past but the current distribution is almost no longer updated. mount nglanggeran area is now an attractive tourist destination. mount nglanggeran is the remnant of ancient volcano with almost parallel magmatic rock area. this area is uninhabited and is a protected area. the status of such area cause the protection of different types of local and wild plants that are no longer in residential areas. in previous research, the author has found plant species that can only be traced from old herbaria and old manuscript sources. publications and data of such plants are very rare. flora of java (backer & bakhuizen, 1962-70) and flora of british india (hooker, 1885) are important manuscripts as guidance for plant identification in java, indonesia, and southeast asia in general. not all local plants can be identified and listed in this book, but through comparison based on the description of the family and genus levels can lead identification to other books and herbaria-herbarium that have been published. the greatest disadvantage of these books is that they are filled with verbal descriptions with no illustrations, making it very difficult to identify even though the plant data is relatively complete. the identification process of plants in mount nglanggeran is relatively difficult and requires a lot of data. often the identity of plant species is known for many years after the observation done. preliminary data from author showed that the mount nglanggeran plant represents nearly 230 flowering plant families in the flora of java (1962-70) from about 560 flowering plant families in the world (takhtajan, 2008) . based on the above description, it is important to document the nglanggeran plants and systematically arranged the hierarchy of plant families. this systematic visual documentation is useful to help the use of flora of java book, to explore and introducing the richness of plants in mount nglanggeran, and revealing the vegetation data that is no longer recognized by people. this paper aimed to show the data of families of plant species in mount nglanggeran, to confirm the existence of the plants in flora of java by backer & bakhuizen, and showing visual photograph data of plant species in mount nglanggeran mountain. materials and methods this researchs were field and literature research. field research was survey method (singh, 2010. collection was done by sampling specimen for herbaria and photograping herbarium sampling with attention on the sustainability of plant population. monitoring and visits are conducted with consideration of the prediction of the period inflorescence and fruit formation. http://dx.doi.org/10.14421/biomedich.2017.61.19-36 20 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 equipments and materials equipments for observation and collection consist of: sony nex f3 digital camera, sony cyber-shot dscw180 digital camera, canon dslr digital camera, glass slide, micrometer, slide length, small roll meter, plastic collection, scissors, cutter, gps (global positioning system), dry herbarium collection kit, flakon bottle, nikon smz 1500 stereo microscope equipped with camera, nikon eclipse 50 light microscope equipped with nikon dsf1 camera. materials for observation and collection consist of: aquadest, alcohol 70%, faa solution (formalin acetic alchohol). work procedure the working procedures were as follows: photograping and observing of specimen in situ, herbarium, and flower/fruit. the data were compared to flora of java (backer & bakhuizen, 1963-70) and other existing literature, checking and matching with herbarium types and illustrations/drawings in the literature to identify the specimen. results and discussion flora of java book by backer & bakhuizen (1963-1968) describes spermatophyta found in java. description is an explanation of important character of plants or groups of plants in the category of family, genus, and species. the pattern of explanation of the characteristics of the plant begins with the description of the characteristics of flowers followed by vegetative characteristics. flora of java book by backer & bakhuizen (19631968) consists of volumes 1, 2, 3. volumes 1 and 2 contain descriptions of dicotyle plants consisting of 190 families. volume 3 contains descriptions of monocotyle containing 48 families. so the total family of plants in flora of java is 238 families. this book contains 2885 genera on volume 1, 2199 genera on volume 2 and 2018 genera on volume 3, or a total of 7112 genera. this book is very useful for the identification of plants found in yards, gardens, fields, and forests. the main obstacles to the use and limitations of this book are the absence of visual illustrations and requirement of flower as main character for identification discovery of interest and its characteristics in the identification process. wild and unknown vegetation in forest areas which is rarely blooming at observation, could be very difficult to identify without the appearance of flowers and fruit. from this study it is found that the diversity of plant species in the mount nglanggeran are 80 families of seed plants from 238 families in the book flora of java (backer and bakhuizen, 1963-1968) or around 33, 19%. details of the percentage of family are 32 out of 110 families in the book volume 1 (29.09%); 33 of the 80 families in the book volume 2 (41.25%); 15 families of 48 families in the book volume 3 (31.25%). the existence of flowering plants genera in mount nglanggeran are 265 genera of 7112 genera in flora of java (backer and bakhuizen, 1963-1965) or 3.73% range. detail of genera percentage are 108 genera of 2885 genera volume 1 (3.74%); 105 of 2199 genera in volume 2 (4, 74%) and 51 of 2018 genera in volume 3 (2.51%). flowering plant is a group of plants that dominate the earth today. seed plants in old taxonomic terms are manifestations of the spermatophyta class, consisting of subclass gymnospermae and angiospermae (flowering plant). in the book backer & bakhuizen (1963-1968), gymnospermae consist of 7 families whereas flowering plants consist of 231 families. table 1. percentage of representation of each category of plant classification in mount nglanggeran. no. clade number of orders order representation in mount nglanggeran number of family family representation in mount nglanggeran 1 unrank 4 0 7 0 2 magnoliids 4 3 or 75 % 20 4 or 20 % 3 monocot commeliniids 5 4 or 80 % 31 7 or 22,58% 4 monocot non commeliniids 7 5 or 71,42 % 46 9 or 19,56 % 5 eudicot unrank 6 1 or 16,67 % 15 2 or 13,33 % 6 core eudicot rosids fabids 8 7 or 87,5 % 71 14 or 19,71 % 7 core eudicot rosids malvids 6 4 or 66,67 % 59 13 or 22,03 % 8 core eudicot unrank 7 6 or 85,71 % 87 15 or 13,21 % 9 core eudicot asterids lamids 5 4 or 80 % 40 14 or 35 % 10 core eudicot asterids campanulids 7 2 or 28,57 % 27 4 or 14,81 % widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 21 according to singh (2010), gymnospermae plants consist of 11 families covering 80 genera, whereas flowering plants consist of 485 families covering 13,372 genera including 253,000 species (10,760 genera, 196,990 species dikotil, 2,612 genera, 56,310 monocots species). currently the reform of the categories of angiospermae plant classification (flowering plants) is carried out by the apg association (angiospermae phylogeny group) (apg iii, 2009). flowering plants comprised about 62 orders that included about 410 families. table 2 shows the checklist of the existence of plant species in mount nglanggeran following the family order based on apg iii (2009). table 1 shows the proportions of each category. from table 1 it is found that all the major flowering plant clusters are in nglanggeran mountain, except for basal groups of mangnoliids which are present only in certain regions of the world. based on the percentage of representation of the order, it is found that the plants in nglanggeran mountain were mainly clade core eudicot rosiid fabids group followed by core eudicot unrank, core eudicot asterids lamids, monocot commeliniids. based on the percentage of representation of existing families, it is found that the composition of plants in mount nglanggeran is mainly from cie eudicot unrank clade, core eudicot rosiid fabids, eudicot asterids lamiids, core eudicot rosids malvids. the representation of all the major clusters of flowering plants in mount nglanggeran shows that this location is an important site for continuous researchs. visual data in the form of specimens, photographs, or living plants in situ can be utilized to introduce more easily to the people about the diversity of flowering plants. detailed photographs or plant images need to be prepared for this step. description of plant character of flora of java should be supported by visual data and real specimens in relation to the importance of identifying species for various purposes. the visual data of plants in mount nglanggeran obtained in this study should be arranged systematically to complement and facilitate the use of the book. preparation of hierarchy of clade, order, family of flowering plants according to apg iii is done to update how to study plant diversity and its identification. conclusion it was found 80 families of flowering plants in the mount nglanggeran. based on the phylogenetic arrangement of flowering plants it was found that all the main groups (clades) of flowering plants were found at this location. acknowledgements the authors wish to thank the society for research and community service uin sunan kalijaga who funded this research, sugeng handoko as the manager of ecotourism area nglanggeran gunungkidul mountain yogyakarta, bayu setya aji, rendi yuntara and didik zulfahmi akbar. references backer, c. a. & bakhuizen. 1963. flora of jawa (spermatophytes only). vol i, ii, iii. groningen: n. v. p. noordhoff. birgitta bremer, kåre bremer, mark w. chase, michael f. fay, james l. reveal, douglas e. soltis, pamela s. soltis and peter f. stevens. 2009. an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg iii. botanical journal of the linnean society, 2009, 161, 105–121. cbd (convention on biological diversity). 2002. strategi global konservasi tumbuhan (translated). bogor: botanic garden conservation international. heim e. 2015. flora and vegetation of bali indonesia. herstellung und verlag, norderstedt. hooker, j. d. 1885. flora of british india (vol. i, ii, iii, iv). london: reeve and co. keng h. 1990. the consist flora of singapore. singapore university press, singapore. knowles, b. 2008. systematics and taxonomy response to the house of lord science and technology committee. united kingdom: bioscience federation. ng, f. s. p. 1972-73. tree flora of malaya vol. 3. syarikat seng teik, selangor. quattrocchi, u. 2012. crc word dictionary of medicinal and poisonous plants. comon names, scientific names, eponyms, synonims and etymology. crc press, london randler, c. 2008. teaching species identification, a prerequisite for learning biodiversity and understanding ecology. eurasia journal of mathematics, science & technology education, 4 (3):223-231. randler, c., and bogner, f. x. 2006. cognitive achievement in identification skills. journal of biology education, 40 (4): 161-165. rustaman, n. y. 2006. arah pembelajaran taksonomi tumbuhan dan assesmennya di lptk dan sekolah (online), http:/www.file.upi.edu), retrieved at 23 june 2012. simpson, m.g. 2006. plant systematics. amsterdam: elsevier academic press. singh, g. 2010. plant systematics. new hampshire: science publisher. takhtajan, a. 2009. flowering plant. st petersburg: springer. 22 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 table family plant list (backer & bakhuizen, 1962) and list of seed plants in mount nglanggeran. no. family availability in location estimation of number species name availability in location estimation of number species name 1 magnoliaceae available 1 magnolia alba 2 schisandraceae 3 annonaceae available 6 uvaria rufa anomianthus dulcis meiogyne sp polyaulax sp annona muricata annona squamosa available 2 stelechocarpus burahol polyaltia sp 4 lauraceae available 2 litsea chinensis available 1 persea americana 5 hernandiaceae 6 myristicaceae available 1 myristica sp 7 ranunculaceae available 1 clematis javana 8 ceratophyllaceae 9 nymphaeaceae 10 berberidacaceae 11 menispermaceae available 6 pycnarhena montana arcangelisia sp tinospora coriacea stephania hernandifolia cissampelos sp pericampylus glaucus cyclea barbata available 1 tinospora crispa 12 aristolochiaceae available 1 aristolochia indica 13 raflesiaceae 14 nepenthaceae 15 piperaceae available 7 piper bettle piper retrofractum piper nigrum piper aduncum peperomia pelucida peperomia sp piper sp 24 saururaceae 25 chloranthaceae 26 papaveraceae 27 fumariaceae 28 turneraceae piriqueta racemosa turnera ulmifolia 29 loasaceae 30 capparaceae available 5 capparis micracantha capparis pyrifolia capparis sepiaria cleome rutidosperma gynandropsis gynandara cleome sp gynandropsis sp 31 moringaceae moringa oleifera 32 brassicaceae 33 violaceae 34 resedaceae 35 polygalaceae 2 polygala paniculata polygala glomerata 36 crassulaceae kalanchoe pinnata 37 saxifragaceae 38 droseraceae 39 podostemataceae 40 elatinaceae 41 caryophyllaceae 42 molluginaceae 1 mollugo pentaphylla glinus lotoides (bawukan berbulu) glinus oppositifolius (bawukan licin) 43 ficoidaceae trianthema portulacastrum 44 portulacaceae 1 portulaca oleracea widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 23 talinum paniculatum talinum fruticosum famili baru: talinaceae 45 polygonaceae polygonum orientale polygonum chinense polygonum barbatum antigonon leptopus muehlenbeckia platyclada 46 phytolaccaceae rivinia humilis 47 chenopodiaceae 48 amaranthaceae celosia argentea amaranthus hybridus amaranthus gracilis amaranthus spinosus cyathula prostrata aerva sanguinolenta achyranthea aspera alternanthera sessilis alternanthera phyloxeroides gomphrena celosioides 49 basellaceae basella rubra anredera cordifolia 50 linaceae 51 zygophyllaceae 52 geraniaceae 53 oxalidaceae 3 oxalis barrelieri oxalis corniculata biophytum reinwardtii averrhoa carambola averrhoa bilimbi 54 tropaeolaceae 55 balsaminaceae 1 impatien platypetala 56 lythraceae 1 lawsonia inermis lagerstroemia indica lagerstroemia speciosa cuphea hyssopifolia 57 crypteroniaceae 58 sonneratiaceae 59 punicaceae punica granatum (famili lythraceae) 60 onagraceae 61 trapaceae 62 haloragaceae 63 callitrichaceae 64 thymelaeaceae 1 phaleria octandra phaleria macrocarpa 65 nyctaginaceae 3 mirabilis jalapa boerhavia erecta bougainvillea spectabili 66 proteaceae 67 dilleniaceae 1 tetracera scandens 68 pittosporaceae 69 bixaceae bixa orellana 70 cochlospermaceae 71 flacourtiaceae (salicaceae) 1 flacourtia indica 72 tamaricaceae 73 passifloraceae 2 passiflora foetida passiflora edulis passiflora suberosa passiflora vitifolia passiflora quadrangularis 74 cucurbitaceae 3 momordica charantia trichosanthes villosa trichosanthes tricuspidata citrulus vulgaris cucumis sativus luffa acutangula cucurbita moschata coccinia grandis sechium edule benincasa hispida 75 begoniaceae 76 datiscaceae 77 caricaceae carica papaya 78 cactaceae pereskia sp nopalea sp 24 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 opuntia sp cereus sp hylocereus sp (buah naga) epyphyllum sp (wijayakusuma) 79 theaceae 80 actinidaceaea 81 saurauiaceae 82 ocnaceae 83 dipterocarpaceae 84 myrtaceae 4 psidium guajava zyzygium cumini zyzigium polyanthum zyzigium javanicum zyzygium aromaticum zyzigium malaccensis melaleuca leucadendron callistemon sp 85 lecythidaceae 1 barringtonia asiatica barringtonia racemosa barringtonia asiatica 86 melastomataceae 3 melastoma malabathricum osbeckia chinensis memecylon caeruleum 87 combretaceae 1 terminalia catappa quisqualis indikca (srigading) 88 rhizophoraceae 89 hyericaceae 90 clusiaceae 2 calophyllum inophyllum garcinia mangostana 91 tiliaceae triumfetta indica schoutenia ovata mutingia calabura corchorus acutangulus 92 elaeocarpaceae 1 mutingia calabura 93 gonystylaceae 94 sterculiaceae 1 helicteres hirsuta helicteres isora 95 bombacaceae ceiba petandra durio zibethinus bombax ceiba 96 malvaceae 8 sida cordata triumfetta indica abutilon crispum abutilon hirtum wissadula periplocifolia sida rhombifolia sida acuta hisbiscus surattensis abelmoschus moschatus corchorus acutangulus melochia corchorifolia waltheria americana hisbiscus rosa-sinensia hisbiscus tiliaceus malvaviscus arboreus 97 malpighiaceae 98 erythroxylaceae 99 euphorbiaceae 20 glochidion eriocarpum glochidion puberum glochidion rubrum breynia oblongifolia phyllanthus muriculatus phyllanthus reticulatus phyllanthus emblica phyllanthus niruri sauropus androgynus bridelia micrantha bridelia stipularis croton hyrtus acalypha indica acalypha boehmerioides jatropha gossypifolia jatropha multifida euphorbia hirta euphorbia prostrata euphorbia heterophylla manihot esculenta manihot glaziovii hevea brasiliensis croton variegatus acalypha wilkesiana ricinus communis jatropha curcas codiaeum variegatum pedilanthus variegatus 100 daphniphyllaceae 101 cunoniaceae 102 escalloniaceae 103 hydrangeaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 25 104 rosaceae available 1 rubus moluccanus 105 dichapetalaceae 106 caesalpiniaceae available 3 cassia siamea cassia occidentalis cassia obtusifolia 107 mimosaceae available 8 albizia montana albizia lebbeck albizia procera leucaena glauca mimosa pdica mimosa invisa acacia auriculiformis parkia speciosa 108 papilionaceae available 15 crotalaria usaramoensis crotalaria striata indiofera sumatrana desmodium pulchellum desmodium gangeticum desmodium triflorum alysicarpus nummularifolius uraria crinita uraria logopoides abrus precatorius centrosema pubescens mucuna pruriens flemingia strobilifera alysicarpus sp gliricidia sepium 109 hamamelidaceae 110 buxaceae 111 salicaceae flacuortia indica 112 myricaceae 113 betulaceae 114 fagaceae 115 casuarinaceae casuarina junghuhnia casuarina equisetifolia 116 ulmaceae 117 moraceae available 16 fatoua sp morus sp malaisa scandens streblus asper streblus taxoides maclura cochinchinensis ficus benyamina ficus septica ficus montana artocarpus integra poikilospermum suaveolens 118 urticaceaea available laportea sp fleurya sp pilea microphyla pouzolzia zeylanica boehmeria sp 119 cannabaceae 120 aquifoliaceae 121 celastraceae available 1 celastrus scandens 122 hippocrateaceae 123 icacinaceae 124 salvadoraceae 125 olacaceae available 1 olax scandes 126 opiliaceae 127 loranthaceae available 1 elythranthe sp 128 santalaceae available 1 santalum album 129 balanophoraceae 130 rhamnaceaea available 2 zizyphus oenoplia 131 elaeagnaceae 132 vitaceae available 7 vitis discolor tetrastigma 26 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 leucostaphylum cissus repens cayratia trifolia leea aequata leea rubra 133 rutaceae 5 glycosmis petaphylla murraya paniculata clausena excavata aegle marmelos zanthoxylum sp 134 simarubaceae available 1 brucea javanica 135 burseraceae 136 meliaceae available 2 swietenia mahagoni chisocheton sp lansium domesticum melia azedarach dysoxylum sp. 137 sapindaceae available 3 cardiospermum halicacabum allophylus cobbe erioglossum rubiginosum shcleicera oleosa euphoria longana nephelium lappaceum pometia pinnata filicium decipiens 138 aceraceae 139 sabiaceae 140 staphyleaceaea 141 anacardiaceae available 4 anacardium occidentale mangifera indica mangifera odorata gluta renghas spondias dulcis lannea coromandeca 142 connaraceae 143 juglandaceae 144 cornaceae 145 alangiaceae 146 nyssaceae 147 araliaceae available 1 schefflera sp nothopanax scutellarium polyscias sp arthrophyllum sp 148 apiaceae available 2 centela asiatica eringium foetidum hydrocotyle sp 149 clethraceae 150 ericaceaea 151 vacciniaceae 152 epacridaceae 153 ebenaceae available 1 diospyros truncata 154 sapotaceae crysophyllum cainito mimusops elingi manilkara kauki manilkara achras 155 myrisinaceae available 2 ardisia humilis ardisia crenata 156 styracaceae 157 symplocaceae 158 loganiaceae available 2 spigelia althemia fagraea ceilanica 159 oleaceae available 1 jasminum pubescens 160 apocynaceae available 8 alstonia sholaris alstonia angustiloba rauvolfia verticilata anodendron paniculatum chonemorpha fragran ichnocarpus frutescens tabernaemontana macrocarpa wrightia pubescens 161 asclepiadaceae available 6 cryptolepis sinensis calotropis gigantea hoya sp marsdenia brunoniana telosma puberula cosmostigma racemosum cynanchum sp marsdenia tenacissima gymnema sylvestris asterostemma repandum 162 rubiaceae available 8 hedyotis corymbosa widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 27 ophiorrhiza mungos nauclea orientalis musaenda frondosa pavetta indica psychotria sp paederia scandens vangueria spinosa-meyna grisea 163 capriofoliaceae 164 valerianaceae 165 dipsaceae 166 asteraceae available 14 vernonia cinerea elephantopus scaber pseudoelephantopus spicatus ageratum conyzoides euphatorium inulifolium erigeron sumatrensis eclipta prostrata wedelia montana wedelia biflora synedrella nodiflora bidens biternata tridax procumbens emilia sonchifolia 167 gentianaceae available 1 isotoma longiflora 168 primulaceae 169 plumbaginaceae available 1 plumbago zeylanica 170 plantaginaceae 171 campanulaceae 172 sphenocleaceae 173 lobeliaceae 174 goodeniaceae 175 stylidiaceae 176 polemoniaceae 177 hydrophyllaceae 178 boraginaceae available 2 ehretia microphylla heliotropium indicum 179 solanaceae 4 physalis minima solanum torvum solanum comitis solanum nigrum 180 convolvulaceae available 5 merremia hastata argyreia mollis 181 scrophulariaceae available 3 lingdernia crustacea scoparia dulcis 182 orobanchaceae 183 lentibulariaceaea 184 gesneriaceae available 1 epithema horsfieldii 185 bignoniaceae available 2 oroxylum indicum crescentia cujete 186 pedaliaceae 187 acanthaceae available 6 thunbergia fragrans andrographis paniculata ruelia napifera strobilanthes crispus asystasia gangetica 188 myoporaceae 189 verbenaceae available 9 tectona grandis lantana camara stachytarpeta jamaicensis vitex sp cleroderdrum serratum clerodendrum inerme vitex sp duranta erecta premna odorata 190 lamiaceae available 4 leucas lavandulifolia salvia riparia 28 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 hyptis rhomboides hyptis suaveolens 191 butomaceae 192 hydrocharitaceae 193 alismataceaea 194 triuridaceae 195 aponogetonaceae 196 potamogetonaceae 197 ruppiaceae 198 zannichelliaceae 199 najadaceae 200 commelinaceae 201 flagellariaceae available 1 fragellaria indica 201 xyridaceae 203 eriocaulaceae 204 bromelliaceae 205 musaceae 206 strelitziaceae 207 zingiberaceae available 5 zingiber cassumunar zingiber zerumber costus speciosus curcuma sp 208 cannaceae 209 marantaceae 210 liliaceae available 1 gloriosa superba 211 tecophilaeacea 212 pontederiaceae 213 smilacaceae available 1 smilax sp 214 philesiaceae 215 araceae available 7 pothos scandens amorphophalullus variabilis alocasia crassifolia typhonium trilobatum 216 lemnaceae 217 typhaceae 218 amaryllidaceae 219 iridaceae 220 roxburghiaceae 221 dioscoreaceae available 6 dioscorea alata dioscorea bulbifera dioscorea aculeata dioscorea pentaphylla dioscorea oppositifolia dioscorea hispida 222 xanthorrhoeaceae 223 agavaceae available 1 agave cantala 224 arecaceae available 1 arenga pinnata 225 pandanaceae available 1 pandanus houlletii 226 cyclanthaceae 227 haemodoraceae 228 hypoxidaceae available 2 curculigo latifolia hypoxis aurea 229 velloziaceae 230 apostasiaceae 331 taccaceae available 1 tacca palmata 332 philydraceae 233 . burmaniaceae 234 thismiaceae 235 orchidaceae available 6 pecteilis susannae liparis sp 236 juncaceaea 237 cyperaceae available 4 scleria laevis widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 29 238 poaceae available 14 imperata cylindrica (alang-alang) pollinia ciliata polytrias amaura (rumput lamuran) pogonatherum paniceum (rumput wesen) andropogon aciculatus (rumput jarum) themeda arguens (rumput merak) oplesmenus compositus setaria sp axonopphus compressus anastrophus compressus table checklist of exist plant species in mount nglanggeran by clade, order, family as suggested by apg iii (2009). clade order family exsistence in mount nglanggeran amborellales amborellaceae nymphaeales cabombaceae hydatellaceae nymphaeaceae austrobaileyales austrobaileyaceae schisandraceae+illiciaceae trimeniaceae cloranthales chloranthaceae magnoliids piperales aristolochiaceae v hydnoraceae lactoridaceae piperaceae v saururaceae canellales canellaceae winteraceae magnoliales annonaceae v deneriaceae eupomatiaceae himantandraceae magnoliaceae mytisticaceae laurales atherospermataceae calycanthaceae gomortegaceae hernandiaceae lauraceae v monimiaceae siparunaceae monocot commelinids commelinales commelinaceae v haemodoraceae hanguanaceae philydraceae pontederiaceae zingiberales cannaceae costaceae v heliconiaceae lowiaceae marantaceae musaceae 30 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 sterlitziaceae zingiberaceae v poales anarthriaceae bromeliaceae centrolepidaceae cyperaceae v ecdeiocaulaceae eriocaulaceae flagellariaceae v joinvilleaceae juncaceae mayacaceae poaceae v rapateaceae restionaceae thurniaceae typhaceae+sparganiaceae xyridaceae arecales arecaceae v dasypogonaceae dasypogonaceae asparagales amaryllidaceae++alliaceae asparagaceae+agaveaceae v asteliaceae blandfordiaceae boryaceae doryanthaceae hypoxidaceae v iridaceae ixioliriaceae lanariaceae orchidaceae v tecophilaeaceae xanthorrhoeaceae xeronemataceae liliales alstroemeriaceae campynemataceae colchicaceae v corsiaceae liliaceae v melanthiaceae petermanniaceae philesiaceae ripogonaceae smilacaceae v pandanales cyclanthaceae pandanaceae v stemonaceae triuridaceae velloziaceae dioscoreales burmanniaceae dioscoreaceae v nartheciaceae petrosaviales petrosaviaceae alismataless alismataceae+limnocharitaceae aponogetonaceae araceae v butomaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 31 cymodoceaceae hydrocharitaceae jncaginaceae posidoniaceae potamogetonaceae ruppiaceae scheuchzeriaceae tofieldiaceae zosteraceae acorales acoraceae eudicot ceratophyllales ceratophyllaceae ranunculales berberidaceae circaeasteraceae eupteleaceae lazirdabalaceae menispermaceae v papaveraceae+fumariaceae ranunculaceae v sabiales sabiaceae proteales nelumbonaceae platanaceae proteaceae buxales buxaceae haptanthaceae trochodendrales trochodendraceae core eudicot gunnerales guneraceae myrothamnaceae rodids fabids cucurbitales anisophyllaceae begoniaceae coriariaceae corynocarpaceae cucurbitaceae v datiscaceae tetramelaceae fagales betulaceae casuarinaceae fagceae juglandaceae myricaceae nothofagaceae ticodendraceae rosales berbeyaceae cannabaceae dirachmaceae elaeagnaceae moraceae v rhamnaceae v rosaceae v ulmaceae urticaceae v fabales fabaceae v polygalaceae v quillajaceae surianaceae celastrales ceastraceae v lepidobotriaceae oxaidales brunelliaceae 32 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 cephalotaceae conaraceae cunoniaceae elaeocarpaceae huaceae oxalidaceae v malpighiales achariaceae balanoporaceae bonnetiaceae calophyllaceae caryocaraceae centroplacaceae chrysobalanaceae clusiaceae ctenolophonaceae dichapetalaceae erytrocylaceae euphorbiaceae v goupiaceae humiriaceae hypericaceae irvingiaceae ixonanthaceae lacistemataceae linaceae lophopixidaceae malphigiaceae ochnaceae+medusaginaceae pandaceae passifloraceae+turneraceae v phyllantaceae v picrodendraceae podostemaceae putranjvaceae rafflesiaceae rhiosporaceae salicaceae v trigoniaceae violaceae zygophyllales krameriaceae zygophyllaceae v malvids malvales bixaceae cistaceae cytinaceae dipterocarpaceae malvaceae v mutingiaceae v neuradaceae sarcolaenaceae thymelaeaceae v sphaerosepalaceae brassicales akanaceae bataceae brassicaceae capparaceae v caricaceae cleomaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 33 emblingiaceae gyrostemonaceae koeberliniaceae limnanthaceae moringaceae pentadiplandraceae resedaceae salvadoraceae setchelanthaceae tovariaceae tropaeolaceae huerteales dipentodontaceae gerrardinaceae tapiscaceae sapindales anacardiaceae v bierbersteiniaceae burseraceae kirkiaceae meliaceae v nitrariaceae rutaceae v sapindaceae v simaroubaceae v picramniales paramniaceae crossosomatales aphloiaceae crossomataceae geissolomataceae guamatelataceae stachyuracaceae staphyleaceae strasburgeriaceae myrtales alzateaceae combretaceae v crypteroniaceae lythraceae v melastomataceae v myrtaceae v penaeaceae vochysiaceae onagraceae geraniales geraniaceae melianthaceae vivianiaceae vitales vitaceae v saxifragales altingiaceae aphanopetalaceae cercidiphyllaceae crassulaceae v daphniphyllaceae cercidiphyllaceae haloragaceae hamamelidaceae iteaceae pterostemonaceae paeoniaceae penthoraceae peridiscaceae 34 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 saxifragaceae tetracarpaeaceae dilleniaceae dilleniaceae v berberidopsidales aextoxicaceae berberidopsidaceae santalales balanophoraceae loranthaceae v misodendraceae santalaceae v olacaceae v opiliaceae shcoepfiaceae caryophyllales achatocarpaceae aizoaceae amaranthaceae v anacampserotaceae ancistrocladaceae asteropeiaceae barbeuiaceae basellaceae cactaceae caryophyllaceae didiereaceae dioncophyllaceae droseraceae drossophyllaceae frankeniaceae gisekiaceae halophytaceae limeaceae lophiocarpaceae molluginaceae v montiaceae nepenthaceae nyctagynaceae v physenaceae phytolaccaceae plumbaginaceae v polygonaceae portulacaceae rhabdodendraceae sarcobataceae simondsiaceae stegnospermataceae talinaceae v tamaricaceae cornales cornaceae curtisiaceae grubbiaceae hydrangeaceae hydrostachyaceae loasaceae ericales actinidiaceae balsaminaceae v clethraceae cyrillaceae diapensiaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 35 ebenaceae v ericaceae fouquieriaceae lecythidaceae v maregravuaceae mitrastemnaceae pentaphylacaceae polemoniaceae primulaceae roridulaceae sapotaceae v sarraceniaceae sladeniaceae styracaceae symplocaceae tetrameristaceae theaceae asterids lamiids garryales eucommiaceae garryaceae gentianales apocynaceae v gelsemiaceae gentianaceae v loganiaceae v rubiaceae v lamiales acanthaceae v bignoiaceae v byblidaceae calceolariaceae carlemanniaceae gesneriaceae v lamiaceae v linderniaceae lentibulariaceae martyniaceae oleaceae v orobanhaceae paulowniaceae pedaliaceae phrymaceae plantaginaceae plocospermataceae schlegeliaceae scrophulariaceae v stilbaceae tetrachondraceae thomandersiaceae verbenaceae v solanales convolvulaceae v hydroleaceae montiniaceae solanaceae v sphenocleaceae boraginaceae boraginaceae v vahliaceae icacinaceae metteniusaceae oncotheaceae 36 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 campanulids aquifoliales aquifoliaceae cardiopteridaceae helwingiaceae phyllonomaceae stemonuraceae escalloniales escalloniaceae aterales alseuosmiaceae argophyllaceae asteraceae v calyceraceae campanulaceae+lobeliaceae v goodeniaceae menyanthaceae pentaphragmaceae phellinaceae rousseaceae stylidiaceae dipsacales adoxaceae capriofoliaceae+dipsacaceae+linnaeaceae+morinaceae+valerianaceae paracryphiales paracryphiaceae+quintiniaceae+sphenostemonaceae apiales apiaceae v araliaceae v griselniaceae myodocarpaceae pennatiaceae pittosporaceae torricelliaceae+aralidiaceae+melanophyllaceae bruniales bruniaceae checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 31-33 | doi: 10.14421/biomedich.2014.31.31-33 larvicidal effect of vinca fruit extract (vinca rosea) against aedes aegypti larvae and secondary metabolites profile by thin layer chromatography rahmawati ekaputri1, sudarsono1* and budi mulyaningsih2 1department of pharmaceutical biology, faculty of pharmacy; 2parasitology laboratory, faculty of medicine, ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract background: vinca rosea is known contain alkaloids, it was usually used to treat various diseases. alkaloids from vinca leaves are also already known have larvicidal activity. based on this toxicological activity, the fruit of vinca rosea was selected to investigation its larvicidal activity against the 3rd instar larvae of the mosquito vector of dengue haemorrhagic fever (dhf) aedes aegypti. five concentrations of vinca fruit extract were tested against the 3rd instar aedes aegypti larvae. the different larval mortality percentages were recorded after 24 hours. lethal concentration (lc50 anf lc90) of vinca fruit extract were calculated using probit analysis. phytochemical compounds of ethanolic extract also investigated using thin layer chromatography (tlc). lc50 and lc90 values of fruit extract were 2.987 mg/ml and 32.861 mg/ml. alkaloids were detected in extract. keywords: larvicidal activity, vinca rosea, chromatography, secondary metabolites, aedes aegypti introduction aedes aegypti transmit a number of diseases, such as filariasis, chikungunya, and dengue haemorrhagic fever (dhf). dhf causing millions of deaths every year. the only way of reducing the incidence of this disease is by vector control using synthetic insecticide. the synthetic insecticide are more hazardous to handle and not biodegradable. substances alternative of chemical pesticides, which pollute and threaten future, can be discovered. more than 300 plant species have been reported to have an activity against mosquitoes included vinca leaves extract (remia and logaswany, 2011). vinca rosea (fam.apocynaceae) is plants grow everywhere in indonesia (fig. 1). vinca rosea has more than 120 indol alkaloids and another alkaloids in the whole plant (van der heijden et al., 2004). the present study was carried out to determine the larvicidal activity of vinca rosea fruit extract against aedes aegypti larvae. methodology preparation and extraction of plant materials the fruits of vinca rosea were collected as wild plants at “north sekip” at yogyakarta. indonesia. fruits were identified at department of pharmaceutical biology faculty of pharmacy, ugm yogyakarta. voucher specimen was found at this department. fruits were washed and dried at 40oc for 12 hours. 370 g dried fruit powder was macerated with 2.5 l of ethanol (96%) for 48 hours. the ethanol was then evaporated to sticky residues. the extract residue was 6.95 % and kept in the refrigerator. figure 1. (a) vinca rosea (b) fruit of vinca rosea. 32 biology, medicine, & natural product chemistry 3 (1), 2014: 31-33 bioassay for larvicidal toxicity different concentrations of extract (0.4 mg/ml, 0.8 mg/ml, 1.6 mg/ml, 3.2 mg/ml, dan 6.4 mg/ml) were prepared using boiled distilled water. bioassay were performed according to standard method (who, 2005). twenty five 3rd larvae of aedes aegypti were introduced in different test concentration with a set of control containing distilled water and water without test solution. the glass with the larvae, were kept at room temperature. after 24 hours of exposure, the number of dead larvae were counted to obtained mortility rate. three replications were maintained for each concentration. larval mortality were counted after 24 hours. the lc50 and lc90 were calculated according to probit methods of finney (finney, 1971). the concentrations in probit analysis were transformed to logarithm (log concentration) and the lethal concentrations (lc50 and lc90) were calculated manually. thin layer chromatography secondary metabolite compounds of vinca fruit extract was analyzed by thin layer chromatography (tlc) and the spot visualitasion was by the spray reagents. chromatography was perfomed using aluminium plates precoated with silica gel 60 f254 (e.merck). extract solution was prepared by dissolving it in ethanol pro analysis. one microliters of the extract solution was applied to the plate. plates were developed face-down, to a distance of 8 cm, in a glass chamber after conditioning until saturated with mobile phase vapor. mobile phase that used was ethyl acetate: methanol (9:1 v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with dragendorff's and ninhidrin reagent. table 1. results of probit analysis of vinca fruit extracts against larvae of ae. aegypti. extract concentration (mg/ml) percentage larval mortility (mean) probit value the linear regression equation lc50 (mg/ml) lc90 (mg/ml) 0,4 21% 4,19 y = 1,229x + 4,416 with r = 0,910 2,987 32,861 0,8 21% 4,19 1,6 27% 4,39 3,2 44% 4,85 6,4 76% 5,17 results and discussions larvicidal effect of vinca fruit extract the 24h bioassay is a major tool for evaluating the toxical effect of vinca fruit extract against aedes aegypti larvae. the mosquito larvae exposed under plant extract showed significant behavior changes. the larvae showed restlessness, loss of equilibrium, and led to death. dead larvae are those that cannot be induced to move when they are probed with a needle in the siphon or the cervical region (who, 2005). in table 1 the 5 concentrations of vinca fruit extract caused larval mortality of 21%, 21%, 27, 44, and 76%. zero percent of mortality was noted in control. lc50 and lc90 calculated were 2.987 mg/ml and 32.861 mg/ml. the larvicidal activity shown by vinca rosea is probably due to the presence of the alkaloid which are toxic substances. phenolic and non-phenolic alkaloids isolated from vinca rosea leaves are known to have toxic effects (waskito, 1999). phytochemical compound of vinca fruit extract zones with rf 0.33-0.39 gave positive results after being sprayed with dragendorff’s reagent and gave negative results after being sprayed with ninhydrin reagent (rf 0.33) that was mean no amino acid spot detected (fig. 2). positive results with dragendorff reagent will give orange-red spots or brown with orange background yellow (wagner and bladt, 1996). a positive result with ninhydrin reagent appeared after heating 95 -1200 c about 10 minutes (jork et al., 1990). samples containing amino acids will give a positive spots that appear blue-violet. tryptophan is the precursor indole alkaloid biosynthesis is a major component of plant alkaloids in vinca rosea (tikhomiroff and jolicoeur, 2002). spraying with ninhydrin is used to ensure that the zones rf 0.330.39 are alkaloids. after isolated brown zones showed the presence of alkaloid compounds in the fruit extract vinca rosea (rf 0.33). the result of vinca fruit extract was also proved that they had larvicidal properties against aedes aegypti larvae. nelson et al., (2006) reported that the whole v. rosea plant is poisonous. whole plant vinca rosea also has a toxic effect on the larvae of artemia salina (brine shrimp) (gadir, 2012 dan rahmatullah, 2010). the larvicidal properties exhibited by vinca extract in this study might be related to presence of vinca alkaloids toxins in the plant. conclusion the present study has demonstrated that an ethanolic extract of vinca fruit had capability as larvicides against rahmawati ekaputri, et al. – larvicidal effect of vinca fruit extract (vinca rosea) … 33 aedes aegypti larvae. further studies are needed to determine the marker compound for producing standard operating procedure (sop) and to study their impacts on human health and non-target organisms in mosquito feeding habitats. figure 2. chromatogram of vinca fruit ethanolic extract. solvent systems: ethyl acetate-methanol (9:1 v/v). detection (a) uv-254nm, (b) uv-365nm, (c) dragendorff’s reagent, (d) ninhydrin reagent, (e) reference for positive result with ninhydrin reagent: yolk. (1b) alkaloids and another compounds with nitrogen shows fluorescence in uv-365 and (1c) react as brown zones with dragendorff’s reagent (rf ~ 0.33-0.39). (2) brown zones indicated negative result with ninhydrin reagent (rf ~ 0.33). (3) amino acids react as blue-violet zones with ninhydrin reagent. acknowledgment i would like to thank to faculty of pharmacy ugm for the permission for doing this experiment. references gadir, a. (2012). assement of bioactivity of some sudanese medicinal plants using brine shrimp (artemia salina) lethality assay. journal of chemical and pharmaceutical research 4 (12): 5145-5148. jork, h., funk, w., fischer, w. & wimmer, h. (1990). thin-layer chromato-graphy: reagents and detection methods volume 1a. vch. new york. nelson, l. s.; r. d. shih and m. j. balick. (2006). handbook of poisonous and injurious plants 2nd edition. springer: 340. rahmatullah, m., sadeak, i., bachar, c., hossain, t., abdullah, montaha, jahan, n., chowdhury, m., jahan, r., nasrin, d., rahman, m. & rahman, s. (2010). brine shrimp toxicity study of different bangladeshi medicinal plants, advances in natural and applied sciences 4: 163-173. remia, k.m. & logaswany, s. (2009). larvicidal efficacy of leaf extract of two botanicals against the mosquito vector aedes aegypti (diptera: culicidae). indian journal of natural products and resources 1(2): 208-212. tikhomiroff, c. & jolicoeur, m. (2002). quantification of indole alkaloids and iridoid precursors in catharanthus roseus hairy roots by high-performance liquid chromatography. journal of chromatography a 955: 87-93. van der heijden, r., jacobs, d.i., snoeijer, w., hallard, d. & verpoorte, r. (2004). the catharanthus alkaloids: pharmacognosy and biotechnology, current medicinal chemistry 11(5): 607-628. waskito, t. (1999). isolasi dan uji aktivitas alkaloid dari daun tapak dara (catharanthus roseus) var albus., tesis, universitas diponegoro. semarang. who. (2005). guidelines for laboratory and field testing of mosquito larvicides, who communicable disease control, prevention and eradication. who pesticide evaluation scheme. geneva. content_v3n1_5.pdf (p.1-3) blank_kosong.pdf (p.4) volume 4 number 1 2015 i s s n 2 0 8 9 6 5 1 4 biology, medicine, & natural product chemistry volume 4 – number 1 – 2015 issn 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia maizer said nahdi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: riyanto & m. iqbal a. t. publisher: state islamic university sunan kalijaga yogyakarta and the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@ yahoo.com guidance for authors biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. scientific feedback (short communicatiom) is only received for manuscript, which criticize published article before. manuscripts will be reviewed by managing editor and invited peer review according to their disciplines. the only articles written in english (u.s. english) are accepted for publication. this journal periodically publishes in may and november. in order to support reduction of global warming as a consequence of transportation vehicles emission and forest degradation for paper manufacturing, management of the journal prefer receiving manuscripts via e-mail rather than in hard copy. manuscripts and its communications can only be addressed to the managing editor; 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[indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. offprint or reprint is only available with special request. note: author(s) agree to transfer copy right of published paper to biology, medicine, & natural product chemistry. author shall no longer be allowed to publish manuscript completely without publisher permission. authors or others allowed multiply article in this journal as long as not for commercial purposes. for the new invention, authors suggested to manage its patent before publishing in this journal. notification: all communications are strongly recommended to be undertaken through email. front cover: pavetta indica (photo: widodo) issn: 2089-6514 volume 4 number 1 2015 published twice a year printed in indonesia 1-3 5-9 11-15 17-24 issn 2089-6514 a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja'far luthfi the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi, maratun janah krokot extract (portulaca oleracea. l) as natural light-harvesting pigments for dyesensitized solar cells (dsscs): influence of dye acidity cici nurfaizah, didik krisdiyanto, khamidinal, sudarlin cover jurnal biomenaprochy vol 4 num 1 2015 dpn.pdf (p.1) editorial board 2015 v4 n1.pdf (p.2) guidance for authors 2015 v4 n1.pdf (p.3) cover jurnal biomenaprochy vol 4 num 1 2015 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 451-455 | doi: 10.14421/biomedich.2023.122.451-455 issn 2540-9328 (online) bio-larvicidal potential of betel leaves (piper betle l) ethanolic extract in addition of peg 400 diluent on aedes aegypti larvae listiana masyita dewi*, hilda zaniba ariffah, riandini aisyah, nurhayani medical faculty, universitas muhammadiyah surakarta jl. garuda mas, kampus 4 ums, gonilan, kartasura, sukoharjo, 57169, telp. (0271) 716844, fax (0271) 724883, jawa tengah, indonesia. corresponding author* lmd123@ums.ac.id manuscript received: 26 march, 2023. revision accepted: 14 august, 2023. published: 15 august, 2023. abstract dengue hemorrhagic fever (dhf) is a kind of vector transmitted disease, by aedes aegypti. it is one of major public health problem around the world, including indonesia, because it may lead to epidemics and death in a short time. the use of plant extracts as biolarvicidal is thought to be a promising solution, and one of them is the betel leaves (piper betle l). the addition of polyethylene glycol (peg) as a diluent is thought may increase the dispersity of plant extract in the water which is larval medium of growth. objectives: to determine the bio-larvicidal potential of 96% ethanolic extract of betel leaves (eebl) in addition of peg 400 diluent on the aedes aegypti larval mortality. material and method: betel leaves were extracted by maceration using 96% of ethanol. there are two kinds of eebl concentration used, 0.2% dan 0.4%. peg 400 was also added as diluent. the samples in this study were aedes aegypti larvae at instar iii-iv, with a total of 400 larvae. evaluation was performed at 6, 12, 18, and 24 hours. the data obtained was then analyzed by kruskall wallis test and post-hoc mann whitney test. result: in all of treatment groups, larval mortality was reached 100% at 24 hours. from the kruskall wallis test, p-value obtained was <0.05. from post-hoc mann whitney test, the p-value obtained in the comparation between treatment groups and positive control group was >0.05, and the p-value obtained in the comparation between treatment groups and negative control group was <0.05. conclusion: eebl in addition of peg 400 diluent is potential as bio-larvicidal on aedes aegypti larvae. it is also known that eebl at concentration of 0.2% and 0.4% in addition of peg 400 are as effective as temephos as larvicides on aedes aegypti larvae. keywords: betel leaves; peg, bio-larvicidal; aedes aegypti. abbreviations: dhf: dengue hemorrhagic fever; peg: polyethylene glycol; eebl: 96% ethanolic extract of betel leaves. introduction dengue hemorrhagic fever (dhf) is one of vector transmitted disease, that mostly known around tropics area. dhf is transmitted by aedes aegypti mosquito and it is still a major public health problem around the world, including in indonesia. this disease may cause epidemic and lead to death in a short time (karyanti and hadinegoro, 2016). data reported in the indonesian health profile in 2021, the number of dhf cases has exceeded 100,000 cases, with 0.7% of them ending in death. the spread of dhf in indonesia is mainly in sumatra and java island, especially in urban areas with a high level of mobility and also in densely populated residential areas (kementerian kesehatan republik indonesia, 2022). aedes aegypti is the main vector of dengue hemorrhagic fever (dhf) and chikunguya. aedes aegypti is a member of the order diptera, brassicaceae. the use of synthetic larvicide in controlling disease vectors, including aedes aegypti, has several negative impacts which may lead to resistance and environmental pollution (waskito and cahyati, 2018). alternative medicine to overcome those problems are being highly investigated. who also recommends proven safe and effective traditional medicinal plants, to be incorporated into the national health system (who, 2018). one of indonesia's native plants that are highly observed was betel leaves (piper betle l). however, betel leaves are evergreen, that are commonly used as flavoring in chewing areca nut (betel nut chewing) (patra, et al., 2022). betel leaves contain several active compounds such as alkaloids, flavonoids, phenolics, saponins, tannins, and also essential oils (maryanti, manalu and lesmana, 2022). these chemical compounds have biolarvicides effect and not necessarily owned by other plants (noshirma, 2019). water is the growth medium for larvae. but, when we applied plant extract on it, sometimes they are hard to dissolves well. one of polyether compounds that is often added in chemical formulation as a dispersant agent, is polyethylene glycol (peg). the addition of peg, https://doi.org/10.14421/biomedich.2023.122.451-455 452 biology, medicine, & natural product chemistry 12 (2), 2023: 451-455 especially peg 400 as a diluent is thought may increase the dispersity of plant extract in the water (hutanu, et al., 2014). previous study performed by sembiring and hasan (2020) stated that the minimum concentration of eebl resulting larval mortality is 2.5%, but it’s only reached 60% at first 6 hours of observation. it needs longer time to reached 100% of larval mortality. it is thought that by adding a dispersant agent, such as peg 400, may increase the extract solubility in the water, so that active substance will be easily contact to the larva and caused death. by considering those conditions, this study aimed to determine the bio-larvicidal potential of 96% ethanolic extract of betel leaves (eebl) in addition of peg 400 diluent on the aedes aegypti larval mortality. materials and methods ethanolic extract of betel leaves (eebl) processing betel leaves collected were washed thoroughly, and then dried under the sun for about a week. when they were completely dry, it is continued by chopped them up using blender so that they turn into powder, and the simplicial were obtained. those simplicial were then weighed and added by 96% of ethanol. this blend was stirred in a few minutes and then left to stand all day, repeated every day, for a week. this process resulting a macerate production, which was need to be concentrated by rotatory evaporator and water bath. hereby, a thick extract was formed. this thick extract was prepared in two variation concentration, 0.2% and 0.4%. this step was performed in pharmacology laboratory, universitas muhammadiyah surakarta. figure 1. dried process of betel leaves under the sun. figure 2. concentrating the macerate using rotatory evaporator. sedimentation test we prepared two container for two variation concentration of eebl. the first container was for 0.2% of eebl, and the other one was for 0.4% of eebl. each of the container were filled with eebl which had been obtained from the extraction process, as much as 0.02 ml and 0.04 ml, respectively. at next, every container was added with 0.3 ml of peg 400 and then added with distilled water until the volume reached 10 ml. the mixture was stirred until homogeneous and then observed for 24 hours to see whether a precipitate formed. this step was also performed in pharmacology laboratory, universitas muhammadiyah surakarta. aedes aegypti larval preparation aedes aegypti’s eggs that had been prepared on filter paper sheets were used the filter paper sheets were placed to a container which has been filled by distilled water before. prepare fish feed and blender a little. mix the fish feed that has been blended slightly, to the container prepared before. wait for 2 to 3 days, so that the eggs will drip. this step was performed in parasitology laboratory, universitas muhammadiyah surakarta. larvicidal test for this study, we adopt the guidelines for laboratory and field testing of mosquito larvicides by who (2005). there were 4 study groups designed. they are the negative control group, the positive control group, the treatment group with 0.2% of eebl, and the treatment group with 0.4% of eebl. a 3% of peg 400 was used as negative control, while 1% of temephos was used as positive control. repetition was done for 4 times, so that we prepared 4 containers for each study group. total number or larva involved in this study was 400, with 25 larvae prepared in each of container. in negative control group, each of container were added with 3 ml of peg 400 and distilled water until the volume reached 100 ml, while in positive control group, each of container were added with temephos and distilled water until the volume reached 100 ml. in the treatment group with 0.2% of eebl were added with 0.2 ml of eebl, 3 ml of peg 400 and distilled water until the volume reached 100 ml. dewi et al. – bio-larvicidal potential of betel leaves (piper betle l) … 453 and in the treatment group with 0.4% of eebl were added with 0.4 ml of eebl, 3 ml of peg 400 and distilled water until the volume reached 100 ml. larval mortality was observed every 6 hours, for 24 hours. the larvae are considered to be dead when they sink or appear unresponsive when touched using a stick. all of this process was performed in parasitology laboratory, universitas muhammadiyah surakarta. data analysis the number of larval-death in each study group and in each observation-period were recorded, and analyzed using kruskall wallis test and post-hoc mann whitney test. results and discussion sedimentation test from both containers prepared for this test, it was obtained that no precipitate formed, directly nor in 24 hours of observation. it means that the addition of peg 400 may increase the dispersion of eebl in water, so that active substance kept soluble in it. larvicidal test in larvicidal test, we count the number of larval mortalities in each study group and in each repetition performed. the data obtained presented in the following table. table 1. larval mortalities. study group larval mortalities (mean ± standard deviation) percentage of larval mortalities in 24 hours 6 hours 12 hours 18 hours 24 hours negative control group 0±0 0±0 0±0 0±0 0% positive control group 25±0 25±0 25±0 25±0 100% 0.2% of eebl 25±0 25±0 25±0 25±0 100% 0.4% of eebl 25±0 25±0 25±0 25±0 100% discussion in first study group, the negative control group, the percentage of larval mortality was 0%. there was no single larva were found to be dead in this study group. here, we could say that peg 400 has no larvicidal effect, especially on aedes aegypti larva. zhong, et al. (2013) stated that peg 400 is a kind of hydrophilic polymer which is inexpensive and non-toxic. peg 400 also has high solubility in water and any other solvent, such as alcohol and acetone. this addition of peg, or we call it by pegylation, aimed to increase the solubility of plant extract in water, so that the active substance will be evenly distributed, and may increasing its exposure to the larva. but, in other side, it’s non-toxic effect will not affect the larval death, so that the larval mortality in this study was purely caused by active substance of the plant extract, not by peg. therefore, the usage of peg as negative control in this study was appropriate. the second study group, that is the positive control group, which using 1% of temephos as larvicidal agent, it was found that the percentage of larval mortality was 100%. all of aedes aegypti larva in this group were considered to be dead since first-6 hour of observation. temephos was neural toxic for larva. there is acetylcholine accumulation in larval tissue because cholinesterase inhibition by temephos. this results in hyperexcitation, tremor, and convulsion of larva, resulting fatigue and lead to death. those mechanism was proven by the determination of temephos as part of aedes aegypti eradication program in indonesia (pambudi, et al., 2018). hence, the determination of temephos as positive control in this study was appropriate. in the treatment groups, 0.2% and 0.4% of eebl, the percentage of larval mortality was 100%. the same condition to the positive control group. it means that eebl was proven to contain active substances that are have larvicidal effect on aedes aegypti larva. various literatures state that betel leaves contain a number of secondary metabolites that are larvicidal, such as alkaloids, flavonoids, saponins, tannins, essential oils, and many more. most of them are mainly act by interference the central nervous system via cutaneous or respiratory absorption. in this type of intoxication, there is acetylcholinesterase (ache) inhibition which may lead to death. and as we know, this mechanism was similar to temephos. some other mechanisms of action were involving gaba system, leading to seizures and inhibition of mitochondrial activity (de souza wuillda, et al., 2019; sembiring and hasan, 2020; and kumara, et al., 2021). another active substance that is thought to have a strong larvicidal effect from betel leaves is caryophyllene. this substance causes a damage on cellular function because it binds strongly to the ns3 protease in the nucleus (prabhu, et al., 2022). the data in table 1 was then statistically analyzed. to see the normality of data distribution, the saphiro-wilk test was used, and the p-value obtained was <0.05. meanwhile, to see the homogeneity of data distribution, the homogeneity of variance test was used, and the pvalue obtained was <0.05. based on these two results, it can be said that the requirements for using the anova test were not met, so an alternative test with krusskal 454 biology, medicine, & natural product chemistry 12 (2), 2023: 451-455 wallis test was used, and the p-value obtained was <0.05. here, we conclude that there is at least one group of data that is significantly different, so the analysis continued with post hoc mann whitney test. comparison of data in the negative control group and the treatment groups, both 0.2% and 0.4% of eebl, obtained the p-value of <0.05. it means that the data in both of study group was significantly different. furthermore, it can be interpreted that eebl with a concentration of 0.2% and 0.4% with addition of peg diluent, was a potential bio-larvicides to aedes aegypti larva. different condition appears when compared to study performed by rosyadi and swastika (2020), where eebl was also used but without the addition of peg, it was seen that there was a difference in the larval mortality within 24 hours. in rosyadi and swastika's study (2020), in eebl with a concentration of 0.2% and 0.4%, larval mortality at 24 hours was 44% and 65%, respectively. whereas in our study, with the same concentration and observation time, but with the addition of peg, larval mortality reached 100%. this result reinforces the previous findings that eebl with a concentration of 0.2% and 0.4% with addition of peg diluent, was a potential bio-larvicides to aedes aegypti larva. the addition of peg 400 as a diluent effectively increases the solubility of eebl in the water, so that it’s active substances evenly distributed and exposed to the larva. we also compared our data in the positive control group and both of treatment groups, where the resulting p-value was >0.05. it means that the data in all of study group was similar. in other words, we could say that eebl in concentration of 0.2% and 0.4% with addition of peg have similar effectivity as larvicide on aedes aegypti larva. conclusions ethanolic extract of betel leaves (piper betle l) in addition of peg 400 diluent is potential as bio-larvicidal on aedes aegypti larvae. it is also known that ethanolic extract of betel leaves at concentration of 0.2% and 0.4% in addition of peg 400 are as effective as temephos as larvicides on aedes aegypti larvae authors’ contributions: listiana masyita dewi designed the study. hilda zaniba ariffah carried out the laboratory work and analyzed the data. listiana masyita dewi and hilda zaniba ariffah wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: authors state that there is no competing interests. funding: the study was funded by universitas muhammadiyah surakarta. references de souza wuillda, a. c. j., martins, r. c. c., & costa, f. d. n. 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(2018). efektivitas granul daun salam (eugenia polyantha wight) sebagai larvasida nyamuk aedes aegypti, spirakel, 10(1), pp. 12–20. available at https://doi.org/10.22435/spirakel.v10i1.603. who. (2005). guidelines for laboratory and field testing of mosquito larvicides. world health organization communicable disease control, prevention and eradication who pesticide evaluation scheme. https://www.who.int/publications/i/item/who-cdswhopes-gcdpp-2005.13 who. (2018). antimicrobial resistance and primary health care: technical series on primary health care. world health organization.https://apps.who.int/iris/handle/10665/326454 zhong, x., dou, g., wang, d. (2013). polyethylene glycol (peg400): an efficient and recyclable reaction medium for the synthesis of pyrazolo[3,4-b]pyridin-6(7h)-one derivatives. molecules. 18, 13139-13147. https://www.researchgate.net/publication/258957692_polyethy lene_glycol_peg400_an_efficient_and_recyclable_reaction_medium_for_th e_synthesis_of_pyrazolo34-bpyridin-67h-one_derivatives this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 61-64 | doi: 10.14421/biomedich.2016.52.61-64 issn 2540-9328 (online) a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto1, fajar adi kusumo2, lina aryati3, mardiah suci hardianti4 1doctoral student of mathematics department; 2,3mathematics department; 4faculty of medicine, universitas gadjah mada, bulaksumur, caturtunggal, kec. depok, kabupaten sleman, daerah istimewa yogyakarta 55281 indonesia 1mathematics department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: sugimath@yahoo.co.id1 abstract this paper discussed the stability of “tumorigenesis models” to link between ebv and carcinoma of the nasopharyngeal from normal cell to invasive carcinoma. the review on this case accomplished the previous theorem of equilibrium point on “tumorigenesis models”. keywords: nasopharyngeal carcinoma; equilibrium point; stability; mathematical model. pacs: 87.19.xj. introduction nasopharyngeal carcinoma (npc) is a malignancy which is derived from epithelial or mucosa and kripta that coat the surface of nasopharynx. according to munir (2006) in indonesia nasopharyngeal carcinoma is the most excessively founded in the ear, nose, throat domain and the most patient are at age 40 or older. soetjipto (1989) said that prevalence of nasopharyngeal carcinoma in indonesia is 4,7/100.000 inhabitant per year. whereas nasopharyngeal carcinoma cases in sardjito hospital in 2011 are comprised of 31 men and 11 women (ratnawati, 2012). in the previous paper we published a mathematical modeling of “tumorigenesis models” for ebvassosiated nasopharyngeal carcinoma and analyzed the equilibrium point of the cell development from normal cell to invansive carcinoma (lo et al., 2012). this paper will described the stability mathematical model from normal cell to invansive carcinoma. mathematical modeling as discussed in previous paper (sugiyanto et al., 2016), with refer to process “tumorigenesis models” for ebvassosiated nasopharyngeal carcinoma (lo et al., 2012), then we can construct the diagram as follows: figure 1. diagram of nasopharyngeal carcinoma infection process. from the diagram above we obtain system of differentional equations that describes “tumorigenesis models” for ebv-assosiated nasopharyngeal carcinoma as follows (lo et al., 2012). http://dx.doi.org/10.14421/biomedich.2016.52.61-64 62 biology, medicine, & natural product chemistry 5 (2), 2016: 61-64 1 1 dn a bn d n dt    (1) 2 1 2 dl a l bn cl i l d l dt      (2) 3 2 3 l l l l dd a d cl i d d d dt     (3) 4 2 4l di a i i d ei l i d i dt      (4) 5 5 h h h h dd a d ei md d d dt     (5) 6 6h dc a c md d c dt    (6) where ( )n t : the density of nasopharynx normal epithelium cell ( )l t : the density of lesions epithelium cell ( ) l d t : the density of low grade dysplastic lesions cell ( )i t : the density of ebv latent infection cell ( ) h d t : the density of high grade dysplastic lesions cell ( )c t : the density of invansive carcinoma. the equilibrium point of system differential equations (1) to (6) is, 1 1 * a n b d   * 1 2 2 * ( ) b n l c i d a     3 2 3 * * ( ) l c l d d i a    2 4 4 * * ( ) i d i e l d a     5 5 * * ( ) h e i d m d a    6 6 * * ( ) h md c d a   where 1 2 2 0c i d a    3 2 3 0d i a   4 4 0e l d a    5 5 0m d a   6 6 0d a  stability equilibrium point the jacobian matrix of system differential equations (1) is, ( , , , , , ) l h j n l d i d c = ( )j e where e is the equilibrium condition. the characteristic equation is, 1 2 2 ( )( ) i b d a c i d        3 2 3 4 4 ( )( )a i d a e l d        5 5 6 6 ( )( ) 0a m d a d       after some calculations we get, 1 1 ( )b d    . 2 2 2i a c i d     . 3 3 2 3 a i d    . 4 4 4 a e l d     . 6 5 5 a m d    . 7 6 6 a d   . lemma 1. the equilibrium point ( *, *, *, *, *, *) l h e n l d i d c is asymptotic stable if, 2 2i a c i d   , 3 2 3 a i d  , 4 4 a e l d   , 5 5 a m d  , and 6 6 a d . in a medical point of view, in order to avoid the cancer cells then the abnormal cell should be dead quickly until the amount of proliferation cells smaller then apoptosis, so a stability will be formed, which mean that the cancer do not develop. killing abnormal cells need a highly immunogenic (janeway et al., 2001). in the stage of nasopharynx carcinoma, killing cancer cells will be more difficult, because cancer cels have characteristic of resisting cell death (hanahan & weinberg, 2011). simulation the selected parameters are given in tabular form below. case i : not infected with ebv. case ii : ebv infected but do not develop nasopharyngeal carcinoma. case iii : infected ebv and nasopharyngeal carcinoma develops. for case i: in the first case of this sub-population: cells infected, high dysplastic lesion cells, and invansive carcinoma cells is zero.   0i t    0hd t    0c t  system differential equations (1) to (6) became system differential equations (1) to (3). sugiyanto et al. – a stability mathematical model of nasopharyngeal carcinoma on cellular level 63 table 1. the values parameter for first case, not infected by ebv. no. parameter value unit 1. 1 a 100 1/day 2. 2 a 1 1/day 3. 3 a 1 1/day 4. 1 d 0.08 1/day 5. 2 d 20 1/day 6. 3 d 2 1/day 7. b 1 1/day 8. c 0.5 1/day 9. 1 i 0.1 1/day 10. 2 i 0.5 1/day figure 2. the process of cell development in the case which is not infected with ebv. in figure 2, ebv is not detected in the body. in the first case, the normal cell decreased, while there was an increase lesion cell. then the cell becomes low dysplastic lesion cell because p16 is inactive. because there is no detection of ebv then the high dysplastic cells did not happen and does not became to invasive carcinoma. this is consistent with description from figure 2. for this case ii: in the second case of this sub-population: high dysplastic lesion cells and invansive carcinoma cells is zero.   0hd t    0c t  system differential equations (1) to (6) became system differential equations (1) to (4). table 2. the values parameter for case infected by ebv, but did not develop nasopharyngeal carcinoma. no. parameter value unit 1. 1 a 100 1/day 2. 2 a 1 1/day 3. 3 a 1 1/day 4. 4 a 1 1/day 5. 1 d 0.2 1/day 6. 2 d 8 1/day 7. 3 d 2 1/day 8. 4 d 2 1/day 9. b 1 1/day 10. c 4 1/day 11. 1 i 0.1 1/day 12. 2 i 2 1/day 13. e 0.1 1/day 14. l 1 1/day figure 3. the process of case development of ebv-infected cells, but did not develop nasopharyngel carcinoma. in figure 3, ebv is detected in the body. in the second case, the normal cells decrease very rapidly in the early years and higher lesions cells which is very fast. when p16 is inactive, the lesion cells becomes low dysplastic cells. presence of ebv lytic cause abnormal cells, low dysplastic cells, can easily be infected by ebv lytic and makes infected cells. the ebv lytic there is an increase, although not significant. for this case iii: system differential equations same with system differential equations (1) to (6). 64 biology, medicine, & natural product chemistry 5 (2), 2016: 61-64 table 3. the values parameter for case infected by ebv and developing nasopharyngeal carcinoma. no. parameter value unit 1. 1 a 100 1/day 2. 2 a 2 1/day 3. 3 a 1 1/day 4. 4 a 1 1/day 5. 5 a 2 1/day 6. 6 a 2 1/day 7. 7 a 2 1/day 8. 1 d 1 1/day 9. 2 d 2 1/day 10. 3 d 2 1/day 11. 4 d 2 1/day 12. 5 d 3 1/day 13. 6 d 3 1/day 14. 7 d 1 1/day 15.  5 1/day 16.  1 1/day 17.  1 1/day 18.  5 1/day 19.  2 1/day 20.  1 1/day 21.  2 1/day 22.  1 1/day figure 4. the process of development case of ebv-infected and developing nasopharyngeal carcinoma. in figure 4, ebv is detected in the body. in the third case, because low immune system, lytic viruses evolve very rapidly. thus making normal cell down very quickly, otherwise latent ebv-infected cells increased. this coincided with increase high dysplastic cells. the high growth fueled high dysplastic cells growth invasive carcinoma very quickly. these cases need special attention. we need for follow-up studies, so there is prevention in anticipation of invasive carcinoma. it should be a particular concern because it studied the beginning of nasopharyngeal carcinoma. conclusion this paper have completed the equilibrium point system in (1). for the asymptotic stability on equilibrium point will be fulfilled if 2 2i a c i d   , 3 2 3 a i d  , 4 4 a e l d   , 5 5 a m d  , and 6 6 a d . it mean that to aim the stability it must fulfilled that the proliferation of lesion cells, low dyplastic cells, invection cells, hight dyplastic cells, and invansive carcinoma cells are smaller than the rate cell that go in the next direction compartment and died cells. acknowledgments thank to mr. m. wakhid musthofa, mr. ario wiraya, miss. dewajani purnomosari, miss. dewi kartikawati paramita, miss. jajah fachiroh, mr. muhammad ghufron over discussion and all their support. references hanahan d., and weinberg r. a., 2011, hallmarks of cancer: the next generation, cell 144, march 4, 2011, elsevier inc. janeway c. a jr., travers p., walport m., et al.immunobiology: the imune system in health and disease. 5th edition, nnew york: garland science: 2001using the immune response to attack tumors. available from: https://www.ncbi.nlm.nih.gov/books/nbk27104/ lo, k.w., chung, g.t., and to k.f., 2012. deciphering the molecular genetic basis of npc through molecular, cytogenetic, and epigenetic approaches, seminars in cancer biology, 22 (2): 79 – 86. munir d., 2006. beberapa aspek karsinoma nasofaring pada suku batak di medan dan sekitarnya, the journal of medicine school university of sumatera utara, 39 (3): 223226. ratnawati h., 2012. major histocompability complex class-i related chain a (mica) sebagai kandidat immunological tumor marker pada penderita karsinoma nasofaring, dissertation: doctoral programs, medicine and health, faculty of gadjah mada university, yogyakarta. soetjipto, d, 1989. karsinoma nasofaring. dalam: tumor telinga, hidung dan tenggorokan. diagnosis dan penatalaksanaan, balai penerbit fk ui jakarta, p. 71-84. sugiyanto, fajar a. k., lina a., mardiah s. h., 2013, mathematical modeling of nasopharynx carcinoma on cell level, aip proceeding, http://scitation.aip.org/content/aip/proceeding/aipcp/10.1063/1 .4866540;jsessionid=2gqg1j7afdq12.x-aip-live-02. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 37-45 | doi: 10.14421/biomedich.2017.62.37-45 issn 2540-9328 (online) the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini mathematics education department stkip pgri tulungagung, jl. mayor sujadi east street, no.7, tulungagung, 66221, indonesia author correspondency: anggreini_004@yahoo.com abstract this research aims to determine the number of female residents in trenggalek regency in 2021 based on data on birth rate and life expectancy. the use of eigenvalues and eigenvectors aims to determine the dividing age distribution by leslie matrix model. the eigenvectors are used to determine the number of female populations of each age interval, while the eigenvalues are used to determine population growth rates. the research method used is to determine the subject of research. the next stage is to collect research data, then analyze the data and last draw conclusions. the research data is obtained from bps kabupaten trenggalek and bps east java province that is data of woman population from year 2010-2015. the result of this research using leslie matrix model for female population in trenggalek regency that is discrete model. the discrete model is divided into fourteen age intervals constructed using the birthrate and life expectancy. the conclusions of the study showed that the number of female population in trenggalek regency tended to increase with positive eigen value greater than one. in other words, the growth rate of female population in trenggalek regency tends to be positive. the success of leslie's matrix model is the application of case studies in predicting the number of female populations in trenggalek district by 2021 using the maple 16 program. keywords: eigen value; eigenvector; leslie matrix. introduction trenggalek regency is a regency in east java province which is located in the southern part of the province of east java with an area of 1,261.40 km². trenggalek regency largely made up of mountainous land with an area of 2/3 covers part of the area. while the rest of his (1/3 part) is the land of the lowlands. trenggalek regency is subdivided into 14 subdistricts da 157 villages. only about 4 subdistricts which are the majority of the village of plains, which are: pogalan, trenggalek subdistrict, district and sub-district durenan monument. while the majority of other sub 10 village mountains. a large number of trenggalek regency year 2015 as much 689,200 inhabitants. number of changes in the populations affected by the internal state of the population is birth, death, and survival. the existence of a number of changes of a population called the growth of population. population growth can provide information as to whether changes in population numbers for next year is always increasing, decreasing or constant. (pratama, prihandono and kusumastuti, 2013, p. 163). population projection is not a forecast but a population of scientific calculations based on the assumption of the components of population growth rate, i.e., birth, death, and displacement. the third component is what determines the magnitude of the age structure of the population and the population in the future. to determine each assumption needed data that illustrates the trend in the past to the present, factors that affect the relationship between a component and a component with another as well as the expected target is reached on the future come. science many rapidly developing lately, including mathematical modeling. according to iswanto (2012, p. 19) in its development of mathematical models can be represented on the problems that occur in everyday life. so there is a strong relationship between applied science and mathematics are represented by mathematical models that are designed and implemented with the help of computer science, to facilitate the simulation of realworld systems. population growth is one of the examples of the application of the linear algebra in field biology and ecology specifically quantitatively. population growth in ecology is often referred to as the "population dynamics". ecology is usually defined as the relationship between living beings with their environment (tarumingkeng, 1994, p. 6). many models that can be used to explain the growth of the population. one of the models used by demographers is the model leslie. where the model using a mathematical approach, i.e. the matrix. in the model leslie that birth and death processes depending on the age and become an important part in the growth of the population. in general, the growth of a living http://dx.doi.org/10.14421/biomedich.2017.62.37-45 38 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 creature is a process of continuous or ongoing process taking place. however, the study population should be also approached from a discrete time review. the use of discrete pattern based also upon the observations of the population that is generally done interval-the interval period such as a day, a week, and the units of time according to a draft of the researchers concerned. based on these considerations, in addition to growth models based on the continuous solutions need to be evaluated more thoroughly with the solution of the discrete basis. (tarumingkeng, 1994, p. 27). in addition, the existence of significant size have yet to know the growth of female population in trenggalek based on birth rate and life expectancy for the coming year as well as the limiting distribution of unknown age by using model the leslie matrix. there is some research on matrix leslie i.e. research corazon, nurul, h. and yusienta, m., (2016, p. 6) which uses a matrix of leslie to predict the number and pace of growth in the province of riau in the year 2017. so the obtained results of the total population of women in riau province tend to experience increased. in addition the results of research conducted by pratama, prihandon. from the description it is necessary to determine the abundance of female population and knowing the limiting age distribution of the female population in trenggalek based on birth rate and life expectancy using the eigen values and eigen vectors matrix of leslie. materials and methods this research is research with quantitative approach with the types of descriptive research. this research is done by taking a secondary data in some of the central bureau of statistics in east java province and trenggalek regency bps. the sample in this study is a population of women of the years 2010 to 2015, by comparison with the number of child birth from the year 2010 to the year 2015. from the results of the data then diplikasikan by applying the matrix model leslie to look for population, the value of the eigenvalues and eigen vectors. procedure research the research methods used in this research are: the first stage is to determine the subject of the research, as for the subject of his research is the female population on the birth rate and life expectancy in east java province and the second stage is to (1) collect research data, with regard to the collection of research data obtained from secondary data in the central bureau of statistics of east java province and trenggalek regency bps (2) data analysis and the last is an interesting conclusion. observed variables observed variables in this study was the value of eigen and eigen vectors of the matrix leslie. eigen values of l are the roots of polynomials of its characteristics. the observed variables in this study was the value of eigen and eigen vectors of the matrix leslie. eigen values of l are the roots of polynomials of its characteristics. polynomial matrix characteristics of leslie are: 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 𝐱 = [ 𝑥1 𝑥2 𝑥3 ⋮ 𝑥𝑛] a vector of eigenvalues of a leslie matrix l related 𝜆1 if and only if x is a non-trivial solution of (𝜆𝐼 − 𝐿)𝐱 = 0. data analysis technique in doing data analysis techniques after the data is finished being processed which do first is find the values of ai (a fertility figure female population) obtained from the quotient between the average number of births born son a mom on 2010-2015 year divided by the number of female population in the year 2010 and bi (life expectancy female population) are obtained from the results of the women's division of the population the year 2015 with total population of woman of the year 2010. second, the mengkontruksi model matrix of leslie on the growth of the population. third after matric leslie was formed later enter the data the number of female population in 2015 to generate predictions of the total population of women in the year 2021. third, find the value of a positive eigenvalues in the matrix. fifth, determine the value of the eigen vectors eigen were positive. sixth, using equation approach to determine the limiting age distribution. the seventh note the abundance of female population for a long period to come. the eighth research report compiled and processed data through the application of maple 16. matrix according to kariadinata (2013, p.11) matrix is the arrangement of a group of numbers in a rectangular shaped ranks arranged based on row and column, and placed between two brackets. meanwhile, according to anton, h. & rorres, c. (2004, p. 23) a matrix with n rows and n columns is called an n order square matrix (in square matrices of order n), and the entries 𝑎11, 𝑎22, … , 𝑎𝑛𝑛 on the main diagonal is said to be from a. when a is the matrix has n rows and n column (type n x n), then a can be written as: [ 𝑎11 𝑎21 ⋮ 𝑎𝑛1 𝑎12 𝑎22 ⋮ 𝑎𝑛2 ⋯ ⋯ ⋯ 𝑎1𝑛 𝑎2𝑛 ⋮ 𝑎𝑛𝑛 ] anggreini – the female population growth projection year 2021 in trenggalek … 39 inverse of a matrix according to adiwijaya (2014, p. 10) suppose a and b are square matrices of the same size are and i is the identity matrix. if a.b=i then b is called the inverse of matrix a (on the contrary, a is the inverse of matrix b). notation that the inverse of matrix b is a is b= a-1 and vice versa a= b-1 matriks elementer according to anton, h & rorres, c (2004, p.56) an n x n matrix is called elementary matrix if the matrix can be derived from in n x n identity matrix by performing a single elementary row operation. each elementary matrix can be reversed, and its inverse is also an elementary matrix. if a is an n x n, matrix, then the following statements are equivalent,they are all true or all wrong: a) a is invertible, b) a x = 0 has only a trivial solution, c) a row equivalent to in or reduced rowechelon form a is in.. determinants according to anton, h & rorres, c (2004, p.92) an elementary product of a matrix a, a, nn x , is a product of entries of, none of which come from the same row or column. eigenvalues and eigenvectors according to kariadinata (2013, p.209) if a is an n x n size matrix, then the nonzero vector x in rn is called an eigenvector of a if a x is a scalar multiple of x; ie, 𝐴𝒙 = 𝜆𝒙 for any scalar λ. the scalar λ is called the eigenvalue of a and x is said to be the eigenvector corresponding to 𝜆. to find the eigenvalue of the matrix a of nxn size then 𝐴𝒙 = 𝜆𝒙 can be rewritten as 𝐴𝒙 = 𝜆𝐼𝒙 or equivalent to (𝜆𝐼 − 𝐴)𝐱 = 0. to be an eigenvalue, there must be a nonzero solution of the equation (𝜆𝐼 − 𝐴)𝐱 = 0, obtained if and only if det (𝜆𝐼 − 𝐴)𝐱 = 0. the equation of det (𝜆𝐼 − 𝐴)𝐱 = 0 is called the characteristic equation of a; the scalar that satisfies this equation is the eigenvalue of a. when expanded, then det (λi-a) is a polynomial p in a variable called characteristic polynomial of a. the characteristic polynomial 𝑝(𝑥) of an nxn matrix has the form: 𝑝(𝜆) = det(𝜆𝐼 − 𝐴) = 𝜆𝑛 + 𝐶1𝜆 𝑛−1 + ⋯ + 𝐶𝑛 diagonalization of the matrix according to anton, h & rorres, c (2004, p.74) the diagonal matrix of a square matrix whose entry does not lie in the main diagonal is zero is called the diagonal matrix. a common diagonal matrix d, n x n can be written [ 𝑑1 0 ⋮ 0 0 𝑑2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝑑𝑛 ] a diagonal matrix can be reversed, if and only if all entries in the main diagonal position are non-zero; in this case the inverse of d is 𝐷−1 = [ 1 𝑑1⁄ 0 ⋮ 0 0 1 𝑑2⁄ ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 1 𝑑𝑛⁄ ] so, 𝐷𝐷−1 = 𝐷−1𝐷 = 𝐼. results and discussion leslie matrix model in population growth in leslie's model, women or females are divided over age groups that are timed together. specifically, uppose that the maximum age reached by any woman or female in the population is m years (or expressed in other units of time) and then the population is divided over n the age group. so the time period in each group m/n years. the age group will be explained by the following table: table 1. age group of leslie matrix model. age group age interval 1 [0,m/n] 2 [m/n,2m/n] 3 [2m/n,3m/n] : : : : (n-1) [(n-2)2m/n,(n-1)m/n] n [(n-1)m/n,m] for example known to the number of women or females in each group of to-n group at time t = 0. specifically, for example, there are x1 (0) women or females in the first group, x2 (0) woman or female in second group, x3 (0) woman or female in the third group, and so on. with the number ke-n will be formed a column vector x(0) that is: 𝐱(0) = [ 𝑥1 (0) 𝑥2 (0) ⋮ 𝑥(0) ] this vector is called the age distribution vector of the origin (initial age distribution vector). over time, the number of women or females in each group of to – n the group will change due to three biological processes, namely: birth, death and aging. by explaining these three processes quantitatively, it can be seen how to project the vector of early age distribution into the future. the easiest way to learn about the aging process is to observe the population at discrete times, say 𝑡0, 𝑡1, . . . , 𝑡𝑘, . ... leslie's model requires that the time span between two consecutive observation times is the 40 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 same as the time period of the hose (interval) age, and is written as follows: 𝑡0 = 0, 𝑡1 = 𝑀 𝑛⁄ , 𝑡2 = 2𝑀 𝑛⁄ , to 𝑡𝑘 = 𝑘𝑀 𝑛⁄ with this assumption, all women or females in the group-(𝑖 + 1) at 𝑡𝑘+1 time have been in group to-i at time 𝑡𝑘. the process of birth and death processes between two consecutive observation times can be explained using demographic parameters. as follows: table 2. parameters in leslie matrix model. parameter model information 𝑎𝑖 𝑖 = 1,2, … , 𝑛 average number of girls who was born by a woman during her time in the age group i. 𝑏𝑖 𝑖 = 1,2, … , 𝑛 the number of women in the i-age group who can be expected is still alive and up to age group i. by defition, it will be obtained that (i) 𝑎𝑖 ≥ 0 for 𝑖 = 1,2, . . . , 𝑛 and (ii) 0 < 𝑏𝑖 ≤ 1 for 𝑖 = 1,2, . . . , 𝑛 − 1.can be seen that, should not allow for 𝑏𝑖 which is equal to zero, because if this happens then there will be no female or female alive after the age group to-i. and also considered that at least there is one 𝑎𝑖 positive that will occur birth. each age group in which the corresponding value 𝑎𝑖 is positive is called the fertile age group (fertile age class). next we will define the age distribution vector 𝑥(𝑘) at the time 𝑡𝑘 with: 𝐱(𝑘) = [ 𝑥1 (𝑘) 𝑥2 (𝑘) ⋮ 𝑥(𝑘) ] where 𝑥𝑖 (𝑘) is the number of women or females in the i-age group at the time 𝑡𝑘. at times, women in the first age group are daughters of women born between time 𝑡𝑘−1 and time 𝑡𝑘. so, it can be written or mathematically, 𝑥1 (𝑘) = 𝑎1𝑥1 (𝑘−1) + 𝑎2𝑥2 (𝑘−1) + ⋯ + 𝑎𝑛𝑥𝑛 (𝑘−1) (1) number of women in the age group to −(𝑖 + 1) ( 𝑖 = 1, 2, . . . , 𝑛 − 1) at the time 𝑡𝑘 are women in groups to −𝑖 at the time 𝑡𝑘−1 who are still alive at the time 𝑡𝑘.or mathematically 𝑥𝑖+1 (𝑘) = 𝑏𝑖𝑥𝑖 (𝑘−1), 𝑖 = 1,2, … , 𝑛 − 1 (2) by using matrix notation, equations (1) and (2) can be written in the form [ 𝑥1 (𝑘) 𝑥2 (𝑘) 𝑥3 (𝑘) ⋮ 𝑥𝑛 (𝑘)] = [ 𝑎1 𝑏1 0 ⋮ 0 𝑎2 0 𝑏2 ⋮ 0 𝑎3 0 0 ⋮ 0 ⋯ ⋯ ⋯ ⋮ 0 𝑎𝑛−1 0 0 ⋮ 𝑏𝑛−1 𝑎𝑛 0 0 ⋮ 0 ] [ 𝑥1 (𝑘−1) 𝑥2 (𝑘−1) 𝑥3 (𝑘−1) ⋮ 𝑥𝑛 (𝑘−1)] or more succinctly 𝐱(𝑘) = 𝐿𝐱(𝑘−1), with 𝑘 = 1,2, … (3) where l is leslie matrix 𝐿 = [ 𝑎1 𝑏1 0 ⋮ 0 𝑎2 0 𝑏2 ⋮ 0 𝑎3 0 0 ⋮ 0 ⋯ ⋯ ⋯ ⋮ 0 𝑎𝑛−1 0 0 ⋮ 𝑏𝑛−1 𝑎𝑛 0 0 ⋮ 0 ] (4) from the equation (3) is found that 𝐱(1) = 𝐿𝐱(1) 𝐱(2) = 𝐿𝐱(1) = 𝐿(2)𝐱(0) 𝐱(3) = 𝐿𝐱(2) = 𝐿(3)𝐱(0) ⋮ 𝐱(𝑘) = 𝐿𝐱(𝑘−1) = 𝐿(𝑘)𝐱(0) (5) thus, if we know the start age distribution 𝐱(0) and leslie matrix l, then we can determine the age distribution of women or females at any later time. limited age distribution although the equation (5) gives the age distribution of the population at any given time, it does not immediately provide an overview of the dynamics of the anggreini – the female population growth projection year 2021 in trenggalek … 41 growth process. it is necessary to investigate the eigenvalues and eigenvectors of leslie's matrix. the eigenvalues of l are the roots of the characteristic polynomial. the characteristic polynomial of leslie's matrix is 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 will be given the theorems related to leslie's matrix, as follows: theorem 1 a leslie matrix l has a unique positive eigen value 𝜆1. this eigenvalue has a multiplicity of 1 and has an eigenvector 𝐱1 that all entries are positive. evidence: (i) the leslie matrix l has a positive eigenvalue 𝜆1. the eigenvalue is the root of the characteristic equation, that is 𝑝(𝜆) = |𝜆𝐼 − 𝐿| = 0 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 if 𝑝(𝜆) = 0, so 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 = 0 (6) because one of the roots of the equation (6) must be the dividing factor of the coefficient 𝑎𝑛𝑏1𝑏2⋯𝑏𝑛−1, i.e ±𝑎𝑛, ±𝑏1, ±𝑏2, ⋯ , ±𝑏𝑛−1 suppose that the solution of one of the divider factors, for example, is taken 𝑏1 > 0 as a solution or 𝑏1 as its root, so the equation (6) becomes 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 = 0 𝜆𝑛 = 𝑎1𝜆 𝑛−1 + 𝑎2𝑏1𝜆 𝑛−2 + 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ + 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 (𝜆 − 𝑏1)(𝑎1𝜆 𝑛−1 + 𝑎2𝑏1𝜆 𝑛−2 + 𝑎3𝑏1𝑏2𝜆 𝑛−3 + ⋯ +𝑎𝑛𝑏2 ⋯ 𝑏𝑛−1) = 𝜆 𝑛 means obtained a positive eigen value 𝜆1, that is 𝜆1 = 𝑏1. it will then be proven that leslie matrix l has a 𝜆1 single eigenvalue that single, that is 𝜆1 = 𝑏1. it will be assumed that leslie's matrix has another positive eigenvalue. so to prove it used a way of contradiction. an example 𝜆2 is another positive eigenvalue assuming 𝜆1 ≠ 𝜆2 or 𝜆2 ≠ 𝑏1. is known 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 if 𝑝(𝜆) = 0, then, 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯ − 𝑎𝑛𝑏1𝑏2 ⋯ 𝑏𝑛−1 = 0 the two segments are divided by 𝜆𝑛, be 1 = 𝑎1 𝜆 + 𝑎2𝑏1 𝜆2 + 𝑎3𝑏1𝑏2 𝜆3 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑛 so 𝑞(𝜆) = 1, for 𝜆 ≠ 0 or 𝑞(𝜆) = 𝑎1 𝜆 + 𝑎2𝑏1 𝜆2 + 𝑎3𝑏1𝑏2 𝜆3 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑛 (7) proven that 𝜆1 and 𝜆2 is the solution of the system of equations 𝑞(𝜆) = 1. so get it 𝑞(𝜆1) = 1 and 𝑞(𝜆2) = 1 or 𝑞(𝜆1) = 𝑞(𝜆2). then by using the equation (7), and if 𝑞(𝜆1) = 𝑞(𝜆2) 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + 𝑎3𝑏1𝑏2 𝜆1 3 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 = 𝑎1 𝜆2 + 𝑎2𝑏1 𝜆2 2 + 𝑎3𝑏1𝑏2 𝜆2 3 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆2 𝑛 if 𝑞(𝜆1) deducted by 𝑞(𝜆2) then 𝑎1 ( 1 𝜆1 − 1 𝜆2 ) + 𝑎2𝑏1 ( 1 𝜆1 2 − 1 𝜆2 2) + 𝑎2𝑏1𝑏2 ( 1 𝜆1 3 − 1 𝜆2 3) + … + 𝑎𝑛𝑏1𝑏2 … 𝑏𝑛−1 ( 1 𝜆1 𝑛 − 1 𝜆2 𝑛) = 0 be 𝑎1 ( 𝜆2−𝜆1 𝜆1𝜆2 ) + 𝑎2𝑏1 ( 𝜆2 2 −𝜆1 2 𝜆1 2 𝜆2 2 ) + 𝑎2𝑏1𝑏2 ( 𝜆2 3 −𝜆1 3 𝜆1 3 𝜆2 3 ) + … + 𝑎𝑛𝑏1𝑏2 … 𝑏𝑛−1 ( 𝜆2 𝑛 −𝜆1 𝑛 𝜆1 𝑛 𝜆2 𝑛 ) = 0 or 𝑎1 𝜆1𝜆2 (𝜆2 − 𝜆1) + 𝑎2𝑏1 𝜆1 2 𝜆2 2 (𝜆2 2 − 𝜆1 2 ) + 𝑎2𝑏1𝑏2 𝜆1 3 𝜆2 3 (𝜆2 3 − 𝜆1 3 ) + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 𝜆2 𝑛 (𝜆2 𝑛 − 𝜆1 𝑛) = 0 (8) the equation (8) will be valid if, 𝜆1 = 𝜆2, or contradictory to, the assumption stating. 𝜆1 ≠ 𝜆2. so the true is the positive eigenvalue of leslie's matrix is singular, that is 𝜆1 = 𝜆2 = 𝑏1. (ii) positive eigenvalues have a multiplicity of 1 𝜆1𝑥4 = 𝑏3𝑥3 𝑥4 = 𝑏3 𝜆1 𝑥3 → 𝑥4 = 𝑏3 𝜆1 ( 𝑏1𝑏2 𝜆1 2 𝑥1) −𝑏𝑛−1𝑥𝑛−1 + 𝜆1𝑥𝑛 = 0 42 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 𝜆1𝑥𝑛 = 𝑏𝑛−1𝑥𝑛−1 𝑥𝑛 = 𝑏𝑛−1 𝜆1 𝑥𝑛−1 𝑥𝑛 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑥𝑛−1 so obtained 𝑥2 = 𝑏1 𝜆1 𝑥1 𝑥3 = 𝑏2 𝜆1 𝑥2 = 𝑏1𝑏2 𝜆1 2 𝑥1 𝑥4 = 𝑏3 𝜆1 𝑥3 = 𝑏1𝑏2𝑏3 𝜆1 3 𝑥1 ⋮ 𝑥𝑛 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑥𝑛−1 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛−1 𝑥1 if for example taken 𝑥1 = 1, then eigenvector 𝐱 corresponds to 𝜆1 that is 𝐱1 = [ 𝑥1 𝑥2 𝑥3 𝑥4 ⋮ 𝑥𝑛] = [ 1 𝑏1 𝜆1⁄ 𝑏1𝑏2 𝜆1 2⁄ 𝑏1𝑏2𝑏3 𝜆1 3⁄ ⋮ 𝑏1𝑏2 … 𝜆1 𝑛−1⁄ ] it is proven that leslie's matrix eigenvect or of all elements is positive. theorem 2 if 𝜆1 is the unique positive eigenvalue of a leslie matrix l and if 𝜆𝑖 is any real eigenvalues or complex of 𝐿, then |𝜆𝑘| ≤ 𝜆1. evidence: it has been proved that 𝜆1 is a single positive eigenvalue, then 𝜆1 is an eigenvalue that can be a real or complex number. if for example 𝜆𝑘 = 0 it proves that |𝜆𝑘| ≤ 𝜆1.then taken any 𝜆𝑘 = 𝑟𝑒 𝑖𝜃 with, 𝑖 = √−1, to prove the same as showing |𝑟| ≤ 𝜆1. the necessary conditions and sufficient conditions 𝜆𝑘 to be an eigenvalue, 𝜆𝑘 must be the solution of the system of equations 𝑞(𝜆) = 𝑎1 𝜆𝑘 + 𝑎2𝑏1 𝜆𝑘 2 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑘 𝑛 = 1, 𝑞(𝜆) = 1 for 𝜆 ≠ 0 𝑞(𝜆) = 𝑎1 𝑟𝑒𝑖𝜃 + 𝑎2𝑏1 (𝑟𝑖𝜃) 2 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 (𝑟𝑖𝜃) 𝑛 = 1 𝑎1 𝑟 (𝑒−𝑖𝜃) + 𝑎2𝑏1 𝑟2 (𝑒−2𝑖𝜃) + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (𝑒−𝑛𝑖𝜃) = 1 𝑎1 𝑟 (cos(−𝜃) + 𝑖 sin(−𝜃) ) + 𝑎2𝑏1 𝑟2 (cos(−2𝜃) (… + 𝑖 sin(−2𝜃))−2 + ⋯ … + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos(−𝑛𝜃) + 𝑖 sin(−𝑛𝜃) ) = 1 𝑎1 𝑟 (cos 𝜃 − 𝑖 sin 𝜃 ) + 𝑎2𝑏1 𝑟2 (cos 2𝜃 − 𝑖 sin 2𝜃) + + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos 𝑛𝜃 − 𝑖 sin 𝑛𝜃 ) = 1 it means 𝑎1 𝑟 (cos 𝜃 ) + 𝑎2𝑏1 𝑟2 (cos 2𝜃) + ⋯ … + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos 𝑛𝜃 ) = 1 and 𝑎1 𝑟 (sin 𝜃 ) + 𝑎2𝑏1 𝑟2 (sin 2𝜃) + ⋯ … + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (sin 𝜃 ) = 0 if taken only the real part is 𝑎1 𝑟 (cos 𝜃 ) + 𝑎2𝑏1 𝑟2 (cos 2𝜃) + ⋯ … + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos 𝑛𝜃 ) = 1 because 𝜆1 is an eigenvalue so 𝜆1 satisfies the equation 𝑞(𝜆) = 1 so 𝑞(𝜆) = 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 = 1 if 𝑎1 𝑟 (𝑐𝑜𝑠𝜃) + 𝑎2𝑏1 𝑟2 (𝑐𝑜𝑠2𝜃) + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (𝑐𝑜𝑠𝑛𝜃) = 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + ⋯ + 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 (9) this equation is subtracted into 𝑎1 ( cos 𝜃 𝑟 − 1 𝜆1 ) + 𝑎2𝑏1 ( cos 2𝜃 𝑟2 − 1 𝜆1 2 ) + ⋯ … + 𝑎𝑛𝑏1𝑏2 … 𝑏𝑛−1 ( cos 𝑛𝜃 𝑟𝑛 − 1 𝜆1 𝑛) = 0 𝑎1 ( 𝜆1 cos 𝜃−𝑟 𝑟𝜆1 ) + 𝑎2𝑏1 ( 𝜆1 2 cos 2𝜃−𝑟2 𝑟2𝜆1 2 ) + ⋯ … + 𝑎𝑛𝑏1𝑏2 … 𝑏𝑛−1 ( 𝜆1 𝑛 cos 𝑛𝜃𝑟𝑛 𝑟𝑛𝜆1 𝑛 ) = 0 equations (9) apply if 𝜆1 cos 𝜃 − 𝑟 = 0 obtained 𝑟 = 𝜆1 cos 𝜃. because −1 ≤ cos 𝜃 ≤ 1 → −𝜆1 ≤ 𝑟 ≤ 𝜆1, it means |𝑟| ≤ 𝜆1. if 𝜆1 meet |𝜆𝑘| < 𝜆1 is said that 𝜆1 is a dominant eigen value of 𝐿. therefore the condition of theorem 2 is not strong enough to prove that the eigenvalues of leslie's matrix are dominant. theorem 3 if two consecutive entries 𝑎𝑖 and 𝑎𝑖+1 in the first row of a leslie matrix 𝐿 are not equal to zero then the positive eigenvalue of 𝐿 is dominant. it is called the dominant eigenvalue j. |𝜆𝑘| < 𝜆1 should not |𝜆𝑘| = 𝜆1 or indicated |𝜆𝑘| ≠ 𝜆1. by definition 𝑎𝑖 > 0 and 𝑎𝑖+1 > 0. with 𝑖 = 1,2, … , 𝑛, without prejudice generaly theorem be understood 𝑖 = 1. so 𝑎1 > 0 and 𝑎2 > 0, so it will be shown |𝜆𝑘| ≠ 𝜆1 with 𝑘 = 2,3, … , 𝑛, where 𝜆1 is positif. to prove it is done way of contradiction. that is, it will be assumed that 𝜆𝑘 = 𝜆1. if 𝜆𝑘 = 𝜆1 then 𝑟𝑒 𝑖𝜃 = 𝜆1, so 𝑟(𝑐𝑜𝑠𝜃 + 𝑠𝑖𝑛𝜃) = 𝜆1. 𝑟𝑐𝑜𝑠𝜃 + 𝑖𝑟𝑠𝑖𝑛𝜃 = 𝜆1 + 𝑖0. obtained 𝑟𝑐𝑜𝑠𝜃 = 𝜆1 and 𝑟𝑠𝑖𝑛𝜃 = 0. anggreini – the female population growth projection year 2021 in trenggalek … 43 if the equation 𝑟𝑐𝑜𝑠𝜃 = 𝜆1 multiplied by 𝑐𝑜𝑠𝜃 be 𝑟𝑐𝑜𝑠2𝜃 = 𝜆1𝑐𝑜𝑠𝜃. it means 𝜆1𝑐𝑜𝑠𝜃 ≠ 𝑟 or 𝜆1𝑐𝑜𝑠𝜃 − 𝑟 ≠ 0 based on the equation (9) that is 𝑎1 ( 𝜆1𝑐𝑜𝑠𝜃−𝑟 𝑟𝜆1 ) + 𝑎2𝑏1 ( 𝜆1 2 𝑐𝑜𝑠2𝜃−𝑟2 𝑟2𝜆1 2 ) + ⋯ ⋯ + 𝑎𝑛𝑏1𝑏2 … 𝑏𝑛−1 ( 𝜆1 𝑛 𝑐𝑜𝑠𝑛𝜃−𝑟𝑛 𝑟𝑛𝜆1 𝑛 ) = 0 earned value 𝑎1 = 0 and 𝑎2 = 0. the statement is contrary to the assumption that states 𝑎1 > 0 and 𝑎2 > 0. meaning the wrong supposition |𝜆𝑘| ≠ 𝜆1. in the next section it will always be assumed that the requirements of theorem 3 are met. it will be assumed that l can be diagonalized. it is not necessary for the conclusions to be taken, but this will simplify the argument. in this case, l has n eigenvalues, 𝜆1, 𝜆2, ..., 𝜆𝑛, which need not be different from each other, and n linearly independent eigenvectors 𝐱1, 𝐱2, ..., 𝐱𝑛 , which corresponds to the eigenvalue. in this list will be placed eigenvalues 𝜆1 the dominant first. a matrix p will be formed whose columns are eigenvectors of l. 𝑃 = [𝐱1|𝐱2|𝐱3| ⋯ |𝐱𝑛] 𝐿𝑃 = 𝐿[𝐱1|𝐱2|𝐱3| ⋯ |𝐱𝑛] = [𝐿𝐱1|𝐿𝐱2|𝐿𝐱3| ⋯ |𝐿𝐱𝑛] = [𝜆1𝐱1|𝜆2𝐱2|𝜆3𝐱3| ⋯ |𝜆𝑛𝐱𝑛] = [𝐱1|𝐱2|𝐱3| ⋯ |𝐱𝑛] [ 𝜆1 0 ⋮ 0 0 𝜆2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 ] = 𝑃𝐷 because column vectors of the linear matrices p are linearly independent, and p is reversible so that the matrix l can be diagonalized. the diagonalization of l will be given by the equation 𝐿 = 𝑃𝐷𝑃−1 𝐿 = 𝑃 [ 𝜆1 0 ⋮ 0 0 𝜆2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 ] 𝑃−1 from this equation is obtained 𝐿𝑘 = 𝑃 [ 𝜆1 𝑘 0 ⋮ 0 0 𝜆2 𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 𝑘 ] 𝑃−1 for 𝑘 = 1,2, … for any vector of age distribution it will first 𝐱(0) be obtained 𝐿𝑘𝐱(0) = 𝑃 [ 𝜆1 𝑘 0 ⋮ 0 0 𝜆2 𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 𝑘 ] 𝑃−1𝐱(0) for 𝑘 = 1,2, … by dividing two sections of this equation by 𝜆1 𝑘 and using the fact that 𝐱(𝑘) = 𝐿(𝑘)𝐱(0), it will be obtained 1 𝜆1 𝑘 𝐱 (𝑘) = 𝑃 [ 1 0 ⋮ 0 0 (𝜆2 𝜆1⁄ ) 𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ (𝜆𝑛 𝜆1⁄ ) 𝑘 ] 𝑃−1𝐱(0) (10) because 𝜆1 is the dominant eigen value that is |𝜆1| > |𝜆𝑖|, then |𝜆𝑖 𝜆1⁄ | < 1 for 𝑖 = 2,3, … , 𝑛. it is clear that (𝜆𝑖 𝜆1⁄ ) 𝑘 → 0 if 𝑘 → ∞ for 𝑖 = 2,3, … , 𝑛. using this fact, it can take limits from both sides of (10) to get lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱 (𝑘)} = 𝑝 [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] 𝑃−1𝐱(𝟎) (11) it will then be declared the first entry of the column vector 𝑃−1𝐱(𝟎) with the constants 𝑐, then 𝑃−1𝐱(𝟎) = [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ]. this result is entered into the equation (11) lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱 (𝑘)} = 𝑝 [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ] = [𝐱1|𝐱2|𝐱3| ⋯ |𝐱𝑛] [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ] = [𝐱1|𝐱2|𝐱3| ⋯ |𝐱𝑛] [ 𝑐 0 ⋮ 0 ] = 𝑐𝐱1 so that the right-hand side of (11) can be written as 𝑐𝐱𝟏, where 𝑐 is a positive controw that depends only on the vector of age distribution first 𝐱(0).so (11) be lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱 (𝑘)} = 𝑐𝐱𝟏 (12) the equation (12) gives an approximation 𝐱(𝑘)~𝑐𝜆1 𝑘𝐱𝟏 (13) for large k values. from (13) also obtain 𝐱(𝑘−1) ≃ 𝑐𝜆1 𝑘−1𝐱𝟏 (14) by comparing equations (13) and equations (14) obtained 44 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) (15) for large 𝑘 values. this means that for large time values each age distribution vector is a scalar multiple of the previous age distribution vector, and the scalar is the positive eigenvalue of leslie's matrix. consequently, the proportion of females in each group of age groups will be constant.then we will review the equations (13) that give the age distribution vector of the population for a long time: )( k x ~ k c 1  1x (16) three cases will appear in accordance with eigenvalues 𝜆1: (i) the total population will eventually tend to increase/increase if 𝜆1 > 1, (ii) the total population will eventually tend to decrease/decrease if 𝜆1 < 1, (iii) population will tend to be stable/fixed if 𝜆1 = 1. if the population tends to decline then it can be said also that the population growth rate is negative, where as if the population number increases it can be said also that the population growth rate is positive. data on population of women and number of children born in 2010 and year 2015 in trenggalek regency the following data on the number of female population from 2010-2015 in trenggalek regency that has been studied in the research. because only a few women over 45 years old who gave birth to a child, it will be limited from the population of women aged between 15 and 49 years this is due to the age of female fertility interval of 15-49 years. this data is for the age group with the period of 5 years, so the total number is 14 age groups. from table 3 data will be analyzed by looking for parameter 𝑎𝑖 (fertility number) and 𝑏𝑖 parameter (life expectancy). leslie matrix model can be used to determine the number of female population the next 6 years. using leslie's matrix, according to table 4. the female population is divided into several age-class intervals, with the interval of female fertility 15-49 years. to calculate the number of female population by matrix method leslie influenced by fertility rate (𝑎𝑖) life expectancy (𝑏𝑖). here is a settlement step to predict the number and growth rate in trenggalek regency in 2021. table 3. number of female population year 2010-2015. age class number of female 2010 number of female 2015 ai bi 0-4 24,284 22,860 0 0.978257 5-9 24,529 23,756 0 1.019365 10-14 26,129 25,004 0 0.933752 15-19 24,704 24,398 0.0099 0.903538 20-24 22,592 22,321 0.2599 1.018900 25-29 24,790 23,019 0.2688 0.943687 30-34 24,627 23,394 0.2698 1.055955 35-39 26,632 26,005 0.2599 1.052418 40-44 28,185 28,028 0.0795 0.969700 45-49 25,671 27,331 0.0374 0.994313 50-54 21,950 25,525 0 0.987198 55-59 17,401 21,669 0 0.925694 60-64 13,651 16,108 0 2.738627 65+ 35,320 37,385 0 0 total 340,465 346,803 next will be analyzed using the maple 16 program to determine the dynamics of the growth process based on the eigenvalues and eigenvectors of leslie matrix. then using the equation approach for the age limited distribution 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) ~ will find the rate growth of female population in trenggalek regency. having formed leslie's matrix, based on equation (3), that is 𝐱(𝑘) = 𝐿𝐱(𝑘−1) with 𝑘 = 1,2, … to predict the number of women in 2021. based on table. 3 we obtain the following leslie matrix 𝐱(𝑘) = 𝐿𝐱(𝑘−1) =                                                                                                  343.39 058,20 198,25 175,27 178.27 368,27 702,24 722,21 742,22 044,22 347,23 216,24 361,22 553,28 37,385 16,108 21,669 25,525 27,331 28,028 26,005 23,394 23,019 22,321 24,398 25,004 23,756 22,860 02.7386000000000000 000.925600000000000 0000.98710000000000 00000.9943000000000 000000.969700000000 0000001.05240000000 00000001.0559000000 000000000.943600000 0000000001.01890000 00000000000.9035000 000000000000.933700 0000000000001.01930 00000000000000.9782 00000.03740.07950.25990.26980.26880.25990.0099000 anggreini – the female population growth projection year 2021 in trenggalek … 45 then by using the equation approach for limited age distribution: 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) so 𝐱(𝑘) ≃ 1,000391𝐱(𝑘−1). from the eigen value obtained 𝜆1 > 1 so that every five years of female population in trenggalek regency will tend to increase or in other words growth rate of woman population in trenggalek regency tends to be positive value. table 4. number of women year 2010 and 2015 & women's prediction year 2021. age class number of female 2010 number of female 2015 number of female 2021 0-4 24,284 22,860 28,553 5-9 24,529 23,756 22,361 10-14 26,129 25,004 24,216 15-19 24,704 24,398 23,347 20-24 22,592 22,321 22,044 25-29 24,790 23,019 22,742 30-34 24,627 23,394 21,722 35-39 26,632 26,005 24,702 40-44 28,185 28,028 27,368 45-49 25,671 27,331 27,178 50-54 21,950 25,525 27,175 55-59 17,401 21,669 25,198 60-64 13,651 16,108 20,058 65+ 35,320 37,385 39,343 total 340,465 346,803 356,007 conclusion based on the data analysis of leslie matrix in female population in trenggalek regency, it can be concluded that eigen value for the total population of women in trenggalek regency in year 2021 amounted to 356.007. distribution age limit for female population in trengggalek regency is 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) with positive eigen value 𝜆1 > 1 is greater than one that is equal to 1,000391 so that the number of female population in trenggalek regency in year 2021 tend to increase or can be said growth rate of population tends to be positive value. references adiwijaya. 2014. application of matrices and vector spaces. yogyakarta: graha ilmu. anton, h. & rorres, c. 1988. application of linear algebra. jakarta: erland. anton, h & rorres, c. 2004. linear elementary algebra (app version). jakarta: erland. central bureau of statistics. 2013. projection of indonesian population (indonesian population projection) 2010-2035, jakarta. corazon, nurul, h and yusianta, m, 2016. leslie's matrix application to predict the number and growth rate of women in riau province by 2017. journal of mathematical and statistical science (jsms), 2 (3), 1-11, ejournal.uinsuska.ac.id/index.php/jsms/article / download / 3098 / iswanto, r.j. 2012. applied and applied mathematical modeling. yogyakarta: graha ilmu. kariadinata, r. 2013. algebra of elementary matrices. bandung: cv pustaka setia. pratama, prihandono and kusumastuti 2013. leslie's matrix application to predict the number and rate of growth of a population. bimaster: math scientific bulletin. stat. and applied, 2 (3), 163-172. http://jurnal.untan.ac.id/index.php/jbmstr/article/view/3859. tarumingkeng, r.c. 1994, population dynamics (quantitative ecological studies), pustaka sinar harapan, jakarta this page intentionally left blank the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 19-22 | doi: 10.14421/biomedich.2016.51.19-22 modified alizarin red s-alcian blue staining for reptilian skeleton muhammad ja’far luthfi biological department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract skeletal staining is an important method in anatomical study. the aim of the research was to develop staining and clearing method of reptilian skeleton using alizarin red s-alcian blue. the specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. this modified double-staining has successfully stain bone and cartilage of reptilian. keywords: skeleton, alizarin red s, alcian blue, reptilia, double-staining introduction skeleton are supporting system for vertebrate body. the skeleton support body, provides for attachment muscles, houses for brain and nerve, and serve for locomotion (kardong, 2002). staining of animal skeleton is important in anatomical study. a lot of papers report methods for skeleton staining. clearing and staining of skeletal system are aimed for demonstrate of animal bone and cartilage. this is a routine method in teratological study and has been widely used for study in fetal and young animal, especially rodent (erdogan et al., 1995). alizarin red s-alcian blue is one of the most successful double staining methods for fetal and young rodent skeleton. application of this method in mature reptile, however, is still scarce (saralamoli et al., 2015). this paper will explain modified alizarin red s-alcian blue for successful staining in mature reptilian skeleton. materials and methods animals used in this study are ahaetulla prasina, gecko gecko, trachemys scripta elegans, and draco volans. equipments used in this study are the surgical scissors, scalpels, and a glass container (jar). materials used in this study are alizarin red s-alcian blue, ethanol, distilled water, glacial acetic acid, glycerin and koh. the method described in this paper is a modification from inouye (1976) that has been used on small vertebrates. inouye was the first researcher who successfully use staining methods for bone and cartilage at once with satisfactory result. inouye (1976) use a double staining method for bone and cartilage in mice for teratological study. using this method cartilage stained blue, bone stained red, whereas muscles and other tissues are transparent. appication of this method for mature and larger vertebrate required a modification of flaking skin and muscles into thinner fragments, aditional and prolonged fixation, fat removal, prolonged staining, and purification. these are because adult tissue and larger vertebrates have massive muscle, thicker skin and dense connective tissue, thus staining and clearing process are more difficult. staining and clearing reptilian specimens includes several stages. the first step is exfoliation. after the scales removed, the specimens were fixed in glass tubes containing 95% alcohol for 3 days. after the skin and muscle in the specimen removed by scissors and a scalpel, the specimen immersed again in the new 95% ethanol for 3 days. to remove fat, specimens are put in tubes containing acetone for 4 days with new acetone replacement on the second day. after that the specimens were kept for 5 days in a solution of the dye at a temperature of 37 ° c (1 volume of 0.3% alcian blue in 70% ethanol + 1 volume of 0.1% alizarin red s in 95% ethanol + 1 volume of glacial acetic acid + 17 volume 70% ethanol). the specimen is washed in water and then soaked in a solution of 1% koh for 5 days (until transparent) and then shaked on a shaker in a mixture of glycerin and koh 1% with a ratio of 20%: 80%; 50%: 50%; 80%: 20%. then the specimens are preserved in pure glycerine. result and discussion this research has succeeded in developing a method for staining and clearing reptile skeleton specimen skeleton (bone and cartilage) with alizarin red s alcian blue (figure 1, figure 2, figure 3, figure 4 and figure 5). figure 1 and figure 2 shows the skeleton of a snake ahaetulla prasina. the preparation showed the bones of the skull and vertebrae. the snake specimen shows a special adaptation of vertebrate skeleton, the vertebrae are huge numbers (+ 300 vertebra vertebrae) suitable for terrestrial life without limbs. figure 3 shows tail skeleton of gecko gecko. the preparation shows the 20 biology, medicine, & natural product chemistry 5 (1), 2016: 19-22 structure of the tail vertebrae in autotomous animals that can spontaneously casting off their tail. figure 4 shows the tail skeleton of turtles (trachemys scripta elegans). figure 5 shows the tail skeleton of draco volans. the turtle and draco volans shows the structure of nonautotomous tail vertebrae. figure 1. structure of snake ahaetulla prasina. note skull and spine with ribs. figure 2. high magnification of skeletal structure of snake ahaetulla prasina. muhammad ja’far luthfi. – modified alizarin red s-alcian blue staining for reptilian skeleton 21 figure 3. high magnification of tail skeleton of gecko gecko. note the presence of autotomous split in the plain where the tail broke in autotomy. figure 4. high magnification of tail skeleton of turtles. turtles cannot autotomized their tail therefore no autotomous split in this vertebrae. figure 5. high magnification of tail skeleton of draco volans. 22 biology, medicine, & natural product chemistry 5 (1), 2016: 19-22 the results show that all specimen are clearly visible. bones stained red whereas cartilage stained blue. muscle and connective tissue are transparent/clear by a clearing process with koh. from the specimens we can observe the vertebrae and its processus, intervertebral discs, ribs, autotomus split, and other details. in this method, the most important aspect for successful staining is exfoliation of skin and muscle, as well as fixation stages. remove the skin and muscle as much as possible. but it should be noted that exfoliation skin and muscles are not cut the skeleton itself. vertebrae have many processes. it is often that the disposal of the skin and muscle also cutting processus. fixation and the removal of fat must be prolonged compared to inouye method for fixative solution penetrate the tick tissue. different specimen takes different times for fixation period, the removal of fat, staining and clearing. it depends on the species, maturity (embryonic or adult), body size, and organ/body part made preparation. the more ticker and mature the tissue, the more times are needed. in general, larger animals and more mature require fixation longer period, longer removal of fat, longer staining and and longer clearing. duration time of fixation, removal of fat, staining and clearing in this study can be used as a guidance in application of this method to other animals. whole body staining and clearing method have certain advantages over methods in which the carcass is macerated and the bones are separated and dried. among these advantages are: (1) there is no chance of losing the small bones, (2) all bones are retained in their original position, (3) there is no chance of wrongly identifying similar bones, (4) in the finished preparations the bones, after identification, may be disarticulated and examined from all angles, equally as well as in dried preparations, and (5) many animals may be processed together without danger of mixing their bones, a great saving in time and effort (green, 1952). conclusion this research has successfully modified alizarin red salcian blue to applied on larger and mature animal (reptilia) with satisfied result. references erdogan, d., kadioglu, d., peker, t. 1995. visualization of the fetal skeletal system by double staining with alizarin red and alcian blue. gazi medical journal 6: 55-58. salaramoli, j., f. sadeghi, h.gilanpour, m. azarnia, t. aliesfehani. 2015. modified double skeletal staining protocols with alizarin red and alcian blue in laboratory animals annals of military & health sciences research. 13 (2):76-81. green, m. c. 1952. a rapid method for clearing and staining specimens for the demonstration of bone. the ohio journal of science 52 (1): 31-33. inouye, m. 1976. differential staining of cartilage and bone in mouse skeleton by alcian blue and alizarin red s. congenital anomalies 16: 171-173. kardong, k.v. 2002. vertebrates comparative anatomy, function, and evolution. international edition. third edition. mcgraw-hill higher education. new york. usa. hal: 233236. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 7-10 | doi: 10.14421/biomedich.2019.81.7-10 issn 2540-9328 (online) statistical analysis of habbatussauda’s benefits for health (blood pressure, glucose and uric acid) sugiyanto1,*, luqyana khalda’ aesa2, meksianis z. ndii3 1,2departement of mathematics, universitas islam negeri sunan kalijaga, yogyakarta, indonesia. 3department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia. author correspondency*: sugimath@yahoo.co.id abstract habbatussauda is one of the traditional medicine existed since a long time ago and this is one of the drugs that recommended by the prophet muhammad. habbatussauda’s benefits have been studied extensively in the healthcare. habbatussauda has also been widely used to cure various diseases. this study revealed the benefits of habbatussauda in lowering blood pressure, glucose and uric acid levels in 20 respondents that given habbatussauda for 2 weeks. the blood pressure, glucose and uric acid levels were measured before and after consuming habbatussauda. blood pressure, glucose, and uric acid levels in the body will decrease after consuming habbatussauda that shown in the statistical analysis of the obtained data. keywords: blood pressure; glucose; habbatussauda; herbal medicine; statistical analysis; uric acid introduction based on data from who (world health organization) in 2010, the population of indonesia is 249.866.000, makes indonesia to be one of the most populated countries. increasing number of population will certainly make many problems, one of them is a health problem. some health problems that frequently occur in indonesia are increased glucose level, increased blood pressure, obesity, and many more, whereas the highest rate of disease caused death are: stroke, coronary heart disease, diabetes mellitus, respiratory infections, tuberculosis, liver cirrhosis, lung disease, and hypertension. it makes a lot of indonesians searching for alternative medicines or just preventive medicines of various diseases. one of them is the herbal treatments, which is currently in great demand, one of them is nigella sativa, or commonly known as black cumin, black seed or habbatussauda. habatussauda is one of the traditional medicines used by people to cure various diseases. prophet muhammad said that habatussauda can cure all diseases except death (osman et al., 2014). contemporary naturopathic medicine all over the world have been using habbatussauda which has thymoquinone as a major component (salahshoor et al., 2018). thymoquinone contained in the habbatussauda has a protection function to counter nephrotoxicity and hepatotoxicity. it also has pharmacological effects which include antihelmintic, anticestoda, and antischistosoma, antibacterials, antifungi, antiviral, antioxidant, antiinflammatory, and can improve the immune response of cells that are mediated t (abdulelah & abidin, 2007). habbatussauda can be used for antihypertensive treatment, antitumor, antibacterial, antioxidant, antidiabetic and antidyslipidemia (hussain & hussain, 2016). other benefit of habbatussauda is antihyperuricemia (suhendi & sutrisna, 2011). habbatussauda is also used as a diuretic (aboul-enein & abou-basha, 1995). the study of habbatussauda’s benefits are quite a lot and still being developed. mathematics has an important role in knowing the benefits of habbatussauda, by using statistics. statistics used to process the primary data the obtained habbatussauda’s benefit data will be processed by statistic methods. figure 1. habbatussauda powder. materials and methods in this study, respondents from ages between 19 – 23 years old with the sample size of 20 respondents (11 https://doi.org/10.14421/biomedich.2019.81.7-10 8 biology, medicine, & natural product chemistry 8 (1), 2019: 7-10 female, 9 male). taken respondents do not have history of serious illness, disease history owned by the respondents include stomach ucler, typhoid, and dengue fever. in this study, respondents did not consume other medicine besides habbatussauda that given by the researchers. respondents were students of uin sunan kalijaga who had a history of stomach ucler, typhoid, and dengue fever. before starting the study, first thing to to check is a blood test to determine the initial value of blood pressure, glucose and uric acid before giving each respondent the habbatussauda. furthermore, respondents who take a blood test will be given 42 habbatussauda capsule for two weeks consumption, the respondent consumes 3 habbatussauda capsules daily, each capsule contains 600 mg of pure habbatussauda. habbatussauda used is the type of powder brand kurma ajwa containing 120 capsules. then the blood test were carry on after two weeks to take final value of respondent’s blood pressure, glucose and uric acid after consuming habbatussauda to know whether it decrease or increase. during the two weeks habbatussauda consumption, respondents were contacted by phone to remind them to consume habbatussauda and ask them if there are any complaints or perceived side effects while taking the habbatussauda. taking blood samples to check the measure of glucose and uric acid is done by taking a blood sample from the respondent, then checked it by using the glucose and uric acid tool check, whereas for checking blood pressure was checked by using sphygmomanometer. the data before and after consumption of habbatussauda taken from respondents were collected, then statistical analysis was perform using several kinds of tests, using statistical package for social science (spss) version 15.0. the first test used to test for normality of 95% significance level to determine whether the obtained data is normally distributed or not. then after the normality test, t test was perform with 95% significance level to determine whether there is a significant difference in the average before and after consumption of habbatussauda. at this t test if the value of sig is more than 0.05 then the two data do not have significant differences, but if sig is less than 0.05 then the data is a significant difference. results the average research results that have been obtained from 20 respondents are as follows (table 1). according table 1. average of the systole before consuming habbatussauda is 119.1 mmhg and average of the diastole before consuming habbatussauda is a 81.6 mmhg. while the average of the systole after consuming habbatussauda is 111.35 mmhg and average diastole after consuming habbatussauda is 75.9 mmhg. table 1. blood pressure average before and after consuming habbatussauda. average blood pressure systole (mmhg) diastole (mmhg) before consuming habbatussauda 119.1 81.6 after consuming habbatussauda 111.35 75.9 table 2. glucose average before and after consuming habbatussauda. average glucose (mg/dl) before consuming habbatussauda 90.3 after consuming habbatussauda 84.6 according table 2, average of the glucose level before consuming habbatussauda is a 90.3 mg/dl, while the average of the glucose after consuming habbatussauda is a 84.6 mg/dl. table 3. uric acid average before and after consuming habbatussauda. average uric acid (mg/dl) before consuming habbatussauda 5.065 after consuming habbatussauda 4.555 according table 3, average of the uric acid before consuming habbatussauda is a 5.065, while the average of the uric acid after consuming habbatussauda is 4.555 mg/dl. furthermore, the test for normality using the shapiro-wilk test described below in the following table. table 4. test result shapiro-wilk blood pressure. shapiro-wilk statistic (mmhg) df p systole before consuming habbatussauda 0.9191 20 0.096 diastole before consuming habbatussauda 0.972 20 0.793 systole after consuming habbatussauda 0.958 20 0.505 diastole after consuming habbatussauda 0.921 20 0.102 according table 4, results of normality test using the shapiro-wilk test showed that probability value (p) of systole before (0.096), diastole before is 0.793, systole sugiyanto et al. – statistical analysis of habbatussauda’s benefits for health … 9 after is 0.505, diastole after is 0.102 is more than 0.05 so it was concluded that the data are normally distributed. table 5. test result shapiro wilk glucose. shapiro-wilk statistic (mg/dl) df p before consuming habbatussauda 0.969 20 0.730 after consuming habbatussauda 0.946 20 0.309 according table 5, results of normality the test results for glucose showed that probability value (p) of before consuming habbatussauda is 0.730 and after consuming habbatussauda is 0.309 is more than 0.05 so it was concluded that the data are normally distributed. according table 6, normality test for uric acid showed that probability value before consumption habbatussauda is 0.113 and probability value after consumption of habbatussauda is 0.883 is more than 0.05 so it was concluded that the data are normally distributed. table 6. test result shapiro wilk uric acid. shapiro-wilk statistic (mg/dl) df p before consuming habbatussauda 0.923 20 0.113 after consuming habbatussauda 0.977 20 0.883 from the results above, it is known that the data are normally distributed with a probability value (sig.) more than 0.05 (p > 0.05). furthermore, the data will be tested average differences using the t test with the following results. table 7 shows decreasing in the average systolic and diastolic blood pressure before and after consuming habbatussauda, a decrease of 7.73 mmhg in systolic and a decrease of 5.7 mmhg in diastole with a probability value (sig.) for systole (0.019) and diastole (0.012) is less than 0.05 it can be seen that there are differences in blood pressure values were significantly before and after consuming habbatussauda. table 7. t test result blood pressure. no variable average standard deviaton t test p systole (mmhg) diastole (mmhg) systole diastole systole diastole systole diastole 1. before 119.01 81.6 16.56852 9.13870 2.556 2.778 0.019 0.012 2. after 11.37067 10.90099 75.9 111.35 table 8. t test result glucose. no variable average (mg/dl) standard deviation t test p 1. before 5.065 1.26419 2.338 0.030 2. after 4.555 0.96381 table 9. t test result uric acid. no variable average (mg/dl) standard deviation t test p 1. before 90.30 12.60368 2.181 0.042 2. after 84.60 13.23631 table 8 shows decreasing the average content of glucose before and after consuming habbatussauda, a decrease of 5.7 mg/dl with a probability value (sig.) 0.042 less than 0.05 so that it can be seen that there is a difference in the value of the content of glucose in the body significant before and after consuming habbatussauda. table 9 shows decreasing the average content of uric acid before and after consuming habbatussauda, a decrease of 0.51 mg/dl with a probability value (sig.) 0.030 less than 0.05 so it can be seen that there are differences in the content value of uric acid in the body significant before and after consuming habbatussauda. 10 biology, medicine, & natural product chemistry 8 (1), 2019: 7-10 conclusions this study shows the results of statistical analysis habbatussauda‘s benefits on blood pressure, glucose and uric acid that showed significant differences between the values before and after consuming habbatussauda. it is visible from a probability value obtained is less than 0.05. from these results and from previous studies show that habbatussauda can lower blood pressure values, glucose and uric acid contained in the body, this research confirm previous studies that have examined the habbatussauda’s benefits both medically and other research. references abdulelah, h. a. a., & zainal-abidin, b. a. h. (2007). in vivo anti-malarial tests of nigella sativa (black seed) different extracts. am j pharmacol toxicol, 2(2), 46-50. aboul-enein, h. y., & abou-basha, l. i. (1995). simple hplc method for the determination of thymoquinone in black seed oil (nigella sativa linn). journal of liquid chromatography & related technologies, 18(5), 895-902. hussain, d. a., & hussain, m. m. (2016). nigella sativa (black seed) is an effective herbal remedy for every disease except death-a prophetic statement which modern scientists confirm unanimously: a review. adv med plant res, 4(2), 27-57. osman, m. t., hamza, a. j. a., omar, e., & adnan, a. (2014). the new miracle of habbatus sauda: its major component thymoquinone can be used in the management of autoimmune diseases. procedia-social and behavioral sciences, 121, 304314. salahshoor, m. r., haghjoo, m., roshankhah, s., makalani, f., & jalili, c. (2018). effect of thymoquinone on reproductive parameter in morphine-treated male mice. advanced biomedical research, 7. suhendi, a., & sutrisna, e. m. (2011). antihyperurisemia activity of water extract of black seed (coleus ambonicuslour) in balb-c mice and its standardization. indonesian journal of pharmacy, 77-84. world health organization. (2010). world health statistics 2010. world health organization. biology, medicine, & natural product chemistry volume 5 – number 2 – 2016 issn 2089-6514 (paper) | issn 2540-9328 (online) contents starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi, syafiana khusna aini, dewi anggraini, dian prihatiningtias ekawati 33 40 optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono 41 47 histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati, muhammad jafar luthfi 49 53 effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti, endaruji sedyadi, susy yunita prabawati 55 59 a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto, fajar adi kusumo, lina aryati, mardiah suci hardianti 61 64 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 51-55 | doi: 10.14421/biomedich.2018.72.51-55 issn 2540-9328 (online) link of nasopharyngeal carcinoma and epstein-barr virus sugiyanto1,2,*, lina aryati1, fajar adi kusumo1, mardiah suci hardianti3 1department of mathematics, universitas gadjah mada, 55281, yogyakarta, indonesia. 2department of mathematics, universitas islam negeri sunan kalijaga, 55281, yogyakarta, indonesia. 3faculty of medicine, universitas gadjah mada, 55281, yogyakarta, indonesia. author correspondency*: sugimath@yahoo.co.id abstract nasopharyngeal carcinoma (npc) is a cancer that occurs in nasopharynx which is associated with epstein-barr virus (ebv). mutation agents in nasopharyngeal neoplasms occur because of ebv infection. transformation of b-cells due to ebv causes hormone imbalance in lymphoid cells or nasopharyngeal epithelial tissue. rates of ebv infection have been shown to be prognostic to npc. the basic level of ebv dna can be used for stratification prognosis, with higher titers showing greater disease severity and worse outcomes. with mathematical models, there is a correlation between the increase in epstein-barr virus and the increase in invasive carcinoma cells or increase in nasopharyngeal carcinoma cells. keywords: nasopharyngeal carcinoma (npc); epstein-barr virus (ebv); mathematical model; invasive carcinoma cells introduction the body consists of trillions of living cells. the normal cells of the body grow, divide, and die regularly. during the early years of a person's life, normal cells divide faster to allow people to grow. once a person becomes an adult, most cells divide only to replace obsolete or dead cells to repair the lesion. (shah et al., 2012) cancer begins when cells in the body part grow out of control. there are many types of cancer, but they all start because of the absence of abnormal cell growth control. (mimi and yuan et al., 2002) nasopharyngeal carcinoma (npc) is a malignancy derived from the epithelium or mucosa and crypts that line the surface of the nasopharynx (morrison, 2004); (kong et al., 2010); (lin et al., 2003). in indonesia, nasopharyngeal carcinoma are most commonly found among malignant tumors in head and neck and age most who suffer is age 40 years and above (adham et al., 2012); (fachiroh et al, 2004); (stevens, 2005). prevalence of nasopharyngeal carcinoma in indonesia is 4.7 / 100.000 population per year (nurhantari et al., 2003). the study was first written old et al. (1966) on the link between epstein-barr virus (ebv) and npc using metallic assays hybridization dan anticomplement immunoflourescent (acid). about 90% of the adult population has undifferentiated nasopharyngeal carcinomas (unpc) are positive of ebv with a blood test (zeng and zeng, 2010). in wu et al. (2003) person with positive ebv, the cancer will be faster than ebv negative. many studies indicate that unpc is always positive ebv (lo and huang, 2004). ebv dna can be identified nasopharynx that the patient has npc disease (sheen et al., 1998). in other words, ebv is one cause of the development of nasopharyngeal cancer. researchers in the medical field have created categories for the development of abnormal cells. categorization of nasopharyngeal cancer is introduced by the world health organization (who) (zeng and zeng, 2010). several methods of cancer treatment have also been developed. the development of normal nasopharynx epithelium to nasopharyngeal cancer is also known as tumorigenesis model for ebv-associated nasopharyngeal carcinoma at the cellular level. on the other hand, mathematically the development of cells from normal cells, then infected with the ebv, until then into cancer cells can be modeled as a model of dynamics. the modeling will be able to see the dynamics of the development of normal cells to become cancer cells and estimated how fast the cancer cells spread. although the development of nasopharyngeal cancer is not only affected by ebv infection, but because ebv infection is more dominant than other factors, factors other than temporary ebv infection may be neglected in mathematical modeling (aryati et al., 2016). nasopharyngeal carcinoma nasopharyngeal carcinoma is a very dangerous abnormality nasopharynx epithelium. the main one of these cancers is endemic and some areas are very low incidence of this cancer (lu et al., 2010); (wang et al., 2011). the hypothesis about the cause of nasopharyngeal cancer began in the early 20th century, first proposed by https://doi.org/10.14421/biomedich.2018.72.51-55 52 biology, medicine, & natural product chemistry 7 (2), 2018: 51-55 jackson in 1901, which proposed the hypothesis that dust irritation in cork workers would damage airway epithelia. since then the pathogenesis of nasopharyngeal cancer has been intensively studied, in particular aimed at geographical features and racial variations. in recent years many environmental and biological factors have shown a link between the risk of nasopharyngeal cancer and recent research results suggest the role of genetic and viral factors in the progression of this disease (mcdermott et al., 2001). there are three types of npc, these are based on cancer cells under a microscope: keratinizing squamous cell carcinoma, non-keratinizing differentiated carcinoma and undifferentiated carcinoma. each type is different from one country to another. in south china, npc are generally found to be type undifferentiated. different cancers are caused by different factors. the factors that cause nasopharyngeal cancer differ from everyone. some of the factors can be changed, such as smoking, on the contrary the factors that cause due to age, family history of cancer, cannot be changed. however, these factors do not tell much about nasopharyngeal cancer. many people have this disease but do not know the cause. epstein-barr virus (ebv) infection combined with frequent exposure to environmental carcinogenic co-factors is suggested to cause npc development (adham et al., 2012). ebstein-bar virus (ebv) ebstein-barr virus (ebv) or other name human herpesvirus 4 (hhv-4), belongs to the family: herpes virus subfamily: gammaherpesvirinae (ebv), genus: lymphocryptovirus (ebv) (hendrix, 2013). the adult ebv particle has a diameter of 120-180 nm and wraps the lipid bilayer. the primary target of cells for ebv infection is b lymphocytes (b-cells) (hendrix, 2013). incubation period of virus 7 to 14 days for children and adolescents is about 30 to 50 days for adults (bale, 1999). the particles caused by this virus are transmitted through saliva and are often incubated in 15 years during their life without symptoms. seroepidemiologic studies indicate that more than 90% of people in the world are infected with ebv (hendrix, 2013). characteristics and mechanism of nasopharyngeal carcinoma dna is a chemical in every human cell that makes genes, a clue to how cells function. dna usually looks like a parent because they are the source of our dna. dna affects more than how people see it. some genes contain instructions to control when cells grow and divide into new cells. viruses such as ebv also contain dna (chua et al., 2016). when cells are infected with viruses, viral dna may be mixed with normal human dna. ebv dna can instruct nasopharyngeal cells to divide and grow in an abnormal way (chong et al., 1996). ebv-infected cells express several specific viral antigens for each period of infection (lin et al., 1997). ebv latent infection is characterized by expression of epstein-barr virus nuclear antigen-1 (ebna-1) and ebna-2, latent protein (lmp) membrane, and epstein barr virus encoded small rnas (eber) (zheng et al., 2007). these proteins can interact or have homology with various body proteins such as antiapoptosis proteins, cytokines and signal transduction. viral proteins play a role in maintaining the ebv genome in b-cells. the ebv is detected in all periods of latent infection (liebowitz, 1994). all nasopharyngeal epithelial cells are considered to have the same receptors so latent ebv can become active in ebv and infect any nasopharyngeal cells, without preference. in other words, it is assumed to be a random contact between the nasopharyngeal epithelial cells and the ebv virus. the population of nasopharyngeal cells is divided into six sub-populations, normal cells, dysplasia cells, ebv-infected cells, high dysplastic cells, invasive carcinoma cells. normal cells are injured, biased due to food preservatives or tobacco. wounded nasopharyngeal cells may progress to lowplated cells, either due to a weak immune system, resulting in no improvement in genes or apoptosis. the ebv-infected cells are infected dysplasia cells, causing the cell to become infected. the ebv-infected that do not occur apoptosis may progress into high dysplastic cells. the high dysplastic cells may progress to invasive carcinoma cells. invasive carcinoma cells can not heal, until it ends with the death of the patient, if no treatment. this process can be explained in figure 1. figure 1. tumorigenesis nasopharyngeal carcinoma. sugiyanto et al. – link of nasopharyngeal carcinoma and epstein-barr virus 53 from figure 1, system of a mathematical model (aryati et al., 2016) in equations (1) (6) as follows: 1 1 dn a n d n dt    (1) 2 2 dd a d n dv d d dt      (2) 3 3 di a i dv i d i dt      (3) 4 4 h h h h dd a d i d d d dt      (4) 5 5h dc a c d d c dt    (5) 3 6 dv ea i d v dt   (6) where, n : subpopulation of normal cells d : subpopulation of dysplasia cells i : subpopulation of ebv infected cells hd : subpopulation of high dysplastic cells or carcinoma in situ (cis) c : subpopulation of invasive carcinoma cells v : subpopulation of viruses 1a : the rate of proliferation of normal cells 2a : the rate of proliferation of dysplasia cells 3a : the rate of proliferation of ebv infected cells 4a : the rate of proliferation of high dysplastic cells 5a : the rate of proliferation of invasive carcinoma cells 1d : the rate of apoptosis of normal cells 2d : the rate of apoptosis of dysplasia cells 3d : the rate of apoptosis of ebv infected cells 4d : the rate of apoptosis of high dysplastic cells 5d : the rate of proliferation of invasive carcinoma cells 6d : the rate of apoptosis of ebv  : the rate of interaction between normal cells that become dysplasia cells  : the rate of interaction between dysplasia cells that become ebv-infected cells  : the rate of interaction between ebv-infected cells that become high dysplastic cells  the rate of interaction between high dysplastic cells that become invasive carcinoma cells e : the rate of ebv increase due to the proliferation of ebv-infected cells. theorem 1. the relationship between the nasopharyngeal carcinoma and epstein-barr virus in equilibrium point is in the following equation: c v where    6 3 5 5 4 4 d ea d a d a        . proof. if 0 dv dt  and from equation (6), then it is obtained 6 3 d v i ea  (7) if 0h dd dt  and from equation (4), then we get 4 4 h i d d a      . (8) if 0 dc dt  and from equation (5), then we gain 5 5 hdc d a    . (9) if equation (8) is substituted for equation (9), then we have   5 5 4 4 i c d a d a       . (10) if equation (7) is substituted for equation (10), then it is acquired    6 3 5 5 4 4 d v c ea d a d a       . ■ (11) from the theorem 1 that the relationship between nasopharyngeal carcinoma and epstein-barr virus is affected by parameters: the rate of interaction between high dysplastic cells, the rate of interaction between ebv-infected cells that become high dysplastic cells, the rate of apoptosis of ebv, the rate of proliferation of ebv infected cells, the rate of proliferation of ebv infected cells, the rate of proliferation of invasive carcinoma cells, the rate of proliferation of invasive carcinoma cells, the rate of apoptosis of high dysplastic cells, and the rate of proliferation of high dysplastic cells. if table 1 is included in equation 11 obtained 3.3c v . simulation in this section, we will discuss numerical simulations and medical interpretations of mathematical models of nasopharyngeal carcinoma. figure 2 shows a trajectory diagram for variables v and c. within 100 days, variables v and c occur at the 54 biology, medicine, & natural product chemistry 7 (2), 2018: 51-55 beginning of the occurrence of npc. within the first 100 days the epstein-barr virus value was 4.92 virus / mm2 and the invasive carcinoma cells value was 3.807 cell/mm2. at the stage of the formation of invasive carcinoma cells are things that need attention, because remember this cancer is a type that is malignant. the earlier the cancer is very related to the treatment process and is related to the survival of npc sufferers. figure 3 shows a portrait diagram of the phase between epstein-barr virus and invasive carcinoma cells. there is a correlation between the increase in epstein-barr virus and invasive carcinoma cells. this has an interpretation that npc occurs because of an increase in ebv (hu, 1996). table 1. the parameter values of the nasopharyngeal carcinoma mathematical model. no. parameter value unit reference 1. 1 a 13 cell mm–3 day–1 (huynh, 2010) 2. 2 a 0,001 day –1 estimation 3. 3 a 0.0138 day –1 (huynh, 2010) 4. 4 a 0.04 day –1 (aryati et al., 2016) 5. 5 a 0.0138 day –1 (aryati et al., 2016) 6. 1 d 1.0412 day –1 (aryati et al., 2016) 7. 2 d 0.02 day –1 (aryati et al., 2016) 8. 3 d 0.0288 day –1 (huynh, 2010) 9. 4 d 0.1152 day –1 (aryati et al., 2016) 10. 5 d 0.0188 day –1 (aryati et al., 2016) 11 6 d 0.1152 day –1 (huynh, 2010) 12. e 2 day–1 (aryati et al., 2016) 13.  0.05 day–1 (aryati et al., 2016) 14.  1 day –1 (aryati et al., 2016) 15.  0.0082 day–1 (aryati et al., 2016) 16.  0.07 day–1 (aryati et al., 2016) figure 2. trajectory diagram of epstein-barr virus and invasive carcinoma cells in the first 100 days of npc development. figure 3. portrait projection of epstein-barr virus and invasive carcinoma cells in the first 100 days of npc development. conclusion the relationship between the nasopharyngeal carcinoma and epstein-barr virus is directly proportional and linear. the increase in epstein-barr virus makes an increase in invasive carcinoma cells by 3.3. in the first 100 days of development of the nasopharyngeal carcinoma increasing of epstein-barr virus are followed invasive carcinoma cells. references adham, m., kurniawan, a. n., muhtadi, a. i., roezin, a., hermani, b., gondhowiardjo, s., ... & middeldorp, j. m. 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(2010). pathogenesis and etiology of nasopharyngeal carcinoma. in nasopharyngeal cancer (pp. 925). springer, berlin, heidelberg. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 33-38 | doi: 10.14421/biomedich.2018.72.33-38 issn 2540-9328 (online) degradation study of biodegradable plastic using nata de coco as a filler tiara nur elfiana1, anisa nur izza fitria2,*, endaruji sedyadi1, susy yunita prabawati1, irwan nugraha1 1chemistry department, 2chemistry education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: anisanurizza17@yahoo.com abstract starch is known as a biodegradable raw material that can be degraded by bacteria and microorganisms in the soil. starch has cellulose which is kind of plant cellulose. this study shows the biodegradation rates of plastic made from ganyong canna (canna edulis kerr) as a cellulose source which is added with nata de coco as a filler. the biodegradable plastic functional group was confirmed by using fitr. the results show that the o-h group of ganyong canna (canna edulis kerr) biodegradable plastic is located at wave number 3298.03 cm1 and shifted to 3290.32 cm-1 after addition of nata de coco. the c-h bonds functional groups in canna biodegradable plastics and nata de coco plastics are at wave numbers 2920.01 cm-1 and 2916.16 cm-1. while the c-o bonds functional groups in biodegradable starch plastics and nata de coco is shown at wave numbers 995.05 cm-1. the mechanical properties of biodegradable plastics testing are thickness, tensile strength, and elongation based on the astm method. the thickness is about 0.1005 mm, the tensile strength of biodegradable plastic is 4,3244 mpa and the elongation value range about 13.9639% while the wvtr range about 14.20 g/m² hours. the results show that the increase of the plastic degradation made from nata de coco occurs between 5% 38% per days. it is faster than the plastic made from pure ganyong canna (canna edulis kerr) starch. these results indicate that nata de coco could be added in biodegradable plastic on packaging materials for better degradation. keywords: biodegradable; cellulose; ganyong canna; nata de coco introduction biodegradable plastic has been predicted being an alternative for environmental problem in the last few decades. biodegradable plastics have the ability to degrade faster than synthetic plastic which is approximately degraded in 50 years for a foam plastic cup (roohi, 2017). the polymer in plastics changes into co2 and h2o in combination of microorganism in the soil (kumar satish and thakur, 2017). the co2 would be used by plants in photosynthesis while the water would dissolve in the soil pores so that biodegradable material would not be harmful in environment. the main constituents of biodegradable plastic are cellulose. the cellulose sources are mostly from plants, such as wood and agricultural waste (mostafa et al., 2018). however, plant cellulose has other components such as lignin, so it needs separation process to get pure cellulose. the purifications has been carried out by vu ngo dinh et al. (2017) using an ultrasound-assisted alkaline methods which required high technology and costs. biodegradable is known as rigid therefore plasticizers are needed to produce the plastic which is more elastic, flexible and resistant to water. one plasticizer that is widely used in manufacture of biodegradable plastics is glycerol. biodegradable plastic from hydrocolloids such as nata de coco and ganyong canna (canna edulis kerr) has a high polarity and it is hydrophilic which produces high water vapor permeability and low oxygen permeability. this is due to the presence of hydrogen bonds in the molecular structure. therefore, it is necessary to add lipids such fatty acids that have low polarity in order to reduce water vapor permeability. saturated fatty acids have hydroxyl groups that are hydrophobic so they can reduce hydrophilic properties. one type of saturated fatty acid that can be used is palmitic acid (krocta, 1994). according to lay and huey (1997), the addition of palmitic acid into the film aims to protect the product against evaporation of moisture. this is related to the ability of palmitic acid to improve the hydrophobic nature of a solution. nata de coco is a food product with high cellulose in the coconut water with excellent minerals such as k, cl, s, ca, na, mg, etc. the sugars content, sucrose, sorbitol, glucose and fructose, are the needs of acetobacter axylum in fermentation process (alexia, 2012). these bacteria produced a million of biomass grown by appearing solid white to transparent in the number of mass about 250 g/l wet weight of cellulose (gayathri, 2015). nowadays, nata de coco is just served as a food desert without any utilizing improvement even thought it has a high tensile strength and biodegradable properties. https://doi.org/10.14421/biomedich.2018.72.33-38 mailto:anisanurizza17@yahoo.com 34 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 in order to meet the need of biodegradable plastic, this paper shows the study of nata de coco as a filler in combination with ganyong canna (canna edulis kerr) as a starch sources including an analyzing of the physical and mechanical properties, thickness, tensile strength, elongation, water vapor permeability and molecular interactions between nata de coco and starch using ft-ir instrument. materials and methods the materials used in this study was ganyong canna (canna edulis kerr) which were the basic ingredients for biodegradable plastics. this canna obtained from regedeg village, gunung kidul regency. other ingredients used was nata de coco, aquades, a soil for degradation media, sodium hydroxide, glycerol, acetic acid. preparations of nata de coco pulp nata de coco was heated with 1% naoh for 60 minutes. this suspension then washed with water and followed by boiling for the second time in water. the nata de coco soaked in water for one night. pulping was done by smoothing nata de coco using a blender and used for the manufacture of biodegradable plastics. afer that, the pulp dried using an oven at 60 °c. preparations of ganyong canna (canna edulis kerr) starch ganyong canna starch was produced by adding water to dried ganyong canna (canna edulis kerr) and mashed into a pulp. the dregs and the starch were separated by filtering with a filter cloth. the filtration results were left for one night until the starch settles. then the resulting precipitate was dried using an oven at 60 °c. preparations of biodegradable plastics first of all, 1.5 gram ganyong canna starch was dissolved with distilled water. the solution was added with 0.75 ml of glycerol and 1.5 ml of acetic acid. then, the solution was added to a volume of 100 ml of distilled water and followed by heating at a temperature of 80-90 °c until homogeneous for 25 minutes. the mixtures were cooled. the air bubbles and impurities were removed. the mixtures were added to nata de coco pulp in 0.5; 1.0; 1.5; and 3.0 grams and poured into a solution casting size of 30 x 20 cm² and dried in an oven at 60 °c for 2 hours. the mold was removed and left at room temperature for 24 hours. data analysis biodegradable plastic was analyzed its functional group using ftir. samples in the form of plastic were placed into a set holder, then the plastic was scanned at a wavelength of 4000-400 cm-1. the mechanical properties of biodegradable plastics were tested including thickness, tensile strength, and elongation based on the astm method. d. 882-02. thickness was measured using a micrometer (accuracy of 0.001 mm) by placing a sample between the micrometer jaw. for each plastic sample to be tested, measurements were made at 3 different points, then the mean value was calculated. the tensile strength and elongation of biodegradable plastic samples were tested using universal testing machine (utm). the plastic was placed at room temperature (30 °c) for 24 hours before measuring. biodegradable plastic samples was placed at the second end of the clamp. the measurement area was set with the appropriate load and set to zero. the drive button and clamp was turned on and the sample test was observed until it broke. tensile strength was determined based on maximum load, while elongation was determined when biodegradable plastic broke. the water vapor transmission rate test was carried out using the gravimetric method (astm 1983). the biodegradable plastic that would be tested was placed in the mouth of a cup with a diameter of 8 cm which contained silica gel 10 g. the edges of the cup and plastic were covered with rubber. the cup was put into a jar containing nacl solution. the water vapor diffused through biodegradable plastic would be absorbed by silica gel and would increase the mass of the silica gel. then it was weighed every 1 hour for 6 hours (starting from 0 to 6 hours). changing in the weight rate indicated the speed of water vapor diffusion through the biodegradable plastic. the data obtained was made by linear regression equation and wvtr value could be determined by the equation: 𝑊𝑉𝑇𝑅 = cup increase slope ( 𝑔𝑟𝑎𝑚 ℎ𝑜𝑢𝑟 ) plastic surface area (𝑚2) biodegradation test was carried out to determine how long plastic can be broken down by microorganisms (bacteria and fungi). the method used was soil burial test, which was planting and burying samples in the soil for 12 days. the soil was placed in a jar with a diameter of 13 cm and a height of 17 cm. plastic samples measuring 3x3 cm² from ganyong canna (canna edulis kerr) starch and variations of nata de coco plastic buried in the soil with a depth of 5 cm. before being buried, biodegradable plastic was left in the desiccator for 24 hours then weighed (w1). every 2 days, plastic was taken, washed with alcohol, dried in an oven and weighed for the second time (w2). the step was repeated for 12 days and the degradation mass was determined. elfiana et al. – degradation study of biodegradable plastic … 35 results and discussion biodegradable plastic can be decomposed naturally by microorganism’s activities in the soil to harmless compounds when discharged into the environment. in this research, biodegradable plastic is made from renewable sources, ganyong canna (canna edulis kerr) as a base material, glycerol as plasticizer, acetic acid as terminator in polymerization reaction, distilled water as a solvent and modified by nata de coco. the film characteristic of nata de coco indicates that the nata main component is a cellulose polymer. it can be seen on the peak absorption of nata de coco cellulose which is shown the o-h groups in wave numbers at 3344.32 cm-1 and the c-o bonds uptake at around 1029.21 cm-1, the c-h absorption is at 2896.87 cm-1 and the c-c is around at 1643.23 cm-1. figure 1. ftir spectrum of ganyong canna (canna edulis kerr). the identification of functional groups is carried out at wave numbers 400-4000 cm-1. the spectrum of ganyong canna (canna edulis kerr) starch shows a widened absorption at wavenumber 3355.89 cm-1 which indicate the presence of o-h stretching groups. the absorption of c-h is at wave number 2927.73 cm-1. this is reinforced of the absorption band around 1357.79 cm-1 which indicates the presence of ch2 groups. the other functional group is ether (c-o) which is shown at 999.05 cm-1. this result indicates that the functional groups are in accordance with the structure of the constituent components of starch, hydroxyl, methyl, and ether. analyzing ftir to find out the functions in biodegradable plastic. in figure 2, biodegradable plastic spectra is seen the ftir spectra of biodegradable plastics, ganyong canna and nata de coco. figure 2. spectra ftir biodegradable plastic (a) canna starch (b) ganyong starch-nata de coco 1.5 g. 36 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 it can be seen that the oh group is located at wave number 3298.03 and shifted to 3290 after the addition of nata de coco. the wave number of c-h bond in ganyong canna biodegradable plastic and nata de coco is shown at 2920, 01 and 2916.16. while the c-o bond in both components is found at wave number 995.05 (sedyadi et al., 2016). starch-based biodegradable plastic is a type of condensation polymerization because it involves a hydroxyl group from one of the monosaccharides with hydroxyl groups from other monosaccharides through glycosidic. this biodegradable plastic has a rigid and easily broken nature because the hydrogen between polymers bonds are very strong, so it is necessary to add plasticizers (glycerol) to reduce hydrogen polymers to produce more elastic and flexible plastics (wirawan et al., 2012). when glycerol was added some structural modifications occurred in the starch. the starch matrix became looser so the polymer chain moved more easily. it caused glycerol were being bridged between starch molecules by cross-linking between starch molecules with another molecules due to the interaction with the oh groups in the amylose and amylopectin structures (kusnandar, 2010). the interaction between starch and glycerol could be seen in (figure 3). figure 3. strach and glycerol reaction. the plastic is used for packaging products mostly. furthermore, in this application, the important thing is a thickness which is affected by the packaging of products. thickness will affect gas permeability and other physical properties such as tensile strength and elongation (sinaga et al., 2010). the thicker the plastic produced, the transmission of water vapor value was lower because the ability to hold water vapor would be greater. figure 4. thickness nata de coco plastic. in figure 4, the thickness shows the irregular value of nata de coco plastics. the plastic thickness on the 0.5 g and 1 g of nata de coco pulp decreases while the 1.5 g and 3 g of nata de coco pulp increase. these results caused the solution do not measure the same on printing process. the thicker the plastic produced, the lower the value of the transmission of water vapor because the greater the ability to hold water vapor. in this study, the thickness of biodegradable plastic is known from variations of nata de coco ranging from 0.0665 mm to 0.1035 mm. the measurement of plastic tensile strength is used to determine the amount of plastic receiving to achieve maximum pull (stretch) in each unit of plastic area. testing of mechanical properties including tensile strength and elongation is very important to determine the strength in resisting the weight and elasticity of plastic. figure 5. the tensile strength and elongation nata de coco plastic. y = -0.0668x3 + 0.3344x2 0.4507x + 0.2496 r² = 1 0 0.05 0.1 0.15 0.2 0 1 2 3 4nata de coco mass (g) th ic kn e ss (m m ) y = -5,6162x3 + 26,876x2 34,259x + 23,836 r² = 1 y = -1,7052x3 + 7,911x2 9,3282x + 6,272 r² = 1 0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 nata de coco mass (g) te n si le s tr e n g h t (m p a ) a n d e lo n g a ti o n (% ) elfiana et al. – degradation study of biodegradable plastic … 37 nata de coco which contained cellulose add tensile strength caused the bonding between hydroxyl (o-h) groups in starch with hydroxyl (o-h) and carboxyl (cooh) groups in cellulose. in this study, the resulting tensile strength of biodegradable plastic is 4,3244 mpa. the resulting value is higher than the research conducted by mery apriani (2014) which made biodegradable plastic using cassava starch which was given aloe vera with a tensile strength of 1.53 mpa. measurement of tensile strength is also followed by measurement of elongation, namely the maximum changing in length before the plastic cut off. these properties is very important to indicate the ability of plastic to withstand a number of loads before the plastic is broken. based on the results of the study shows that the elongation value ranged about 13.9639%. the tensile strength analysis has irregularities. this irregularity occurs because the solution is less homogeneous. there is inter-polysaccharide bonds broken up by glycerol occur unevenly in variations of nata de coco so that intermolecular interactions become reduced and affect the value of the tensile strength produced. this interaction occurs because glycerol inserts and removes the hydrogen bonds between polysaccharide molecules so the bonds between plastic molecules is weak (bourtoom, 2008). other factors due to incomplete nata de coco mixing in plastic. the 0.5 g and 1.0 g of nata de coco mixing have low level of homogeneity so the increasing concentration of nata de coco does not produce strong increasing pull. the graph produced in addition to the mass of nata de coco is seen to be far away which proves that nata de coco in the solution does not give good homogeneity. while the addition of 1.5 g and 3.0 g of nata de coco has a graph that is mutually approaching which proves that the level of homogeneity in the mass of nata de coco is very good. nata de coco which contains cellulose will add tensile strength because cellulose is a reinforcing component in the composite material can increase its mechanical strength. the increase in tensile strength due to the addition of cellulose is caused by an increase in the interaction of the tensile forces between the molecules making up the thin layer. this condition is related to hydroxyl groups that form inter-intramolecular hydrogen bonds to form a thin layer consisting of fibers that are mutually reinforcing (indriyati et al, 2006). the rate of water vapor transmission is the amount of losing water vapor per unit time which divided by the area of plastic. plastic that has the lowest wvtr value is probably good plastic. based on research shows that nata de coco plastic has high value of wvtr. nata is a cellulose compound that is hydrophilic so the water is easily absorbed and migrated. the results show that the resulting wvtr value ranged from 14.20 g / m² hours. this wvtr value is better than one italian company which produced biodegradable plastics named bi mater. it made from starch polymer with wvtr values of 250-1000 g / m² hours. figure 6. water vapor transmission nata de coco plastic. biodegradation test was conducted to find out how fast biodegradable plastic could be degraded by microorganisms when discharged into the environment. in this study, biodegradation test was carried out using soil media because many microorganisms in the soil which could support the plastic degradation process. according to sari (2007), soil moisture is very influential on the degradation process because it will facilitate microbes in consuming plastic as a food source. another factor are ph, temperature, carbon/ nitrogen sources, presence of contaminants, presence availability of nutrients (roohi, 2017). the biodegradation process was carried out by observing the plastics. observations was done by looking at the changing of plastic visually and by weighing the decrease in plastic mass. observations were made for 12 days, where every 2 days the plastic was taken from the soil and then washed and weighed. the results of the degradation of biodegradable plastic mass could be seen in (figure 7). figure 7. biodegradable plastic mass degradation. based on the data, the plastic which was contained nata de coco as filler has more mass degradation than plastic in pure ganyong canna. the percentage mass degradation was between 5%-38% depending on how long the plastic in the soil. in the early days, degradation process was tended faster than another day. it caused there was more cellulose contained in it. nata de coco has ability to absorb water so that the degradation was being faster. when the water contact in 14 14.2 14.4 14.6 14.8 15 15.2 0 1 2 3 4 nata de coco mass (g) w v tr ( g /m ² ja m ) 0 38.57 43.73 59.81 86.34 100 0 13.85 25.02 49.52 79.23 100 0 20 40 60 80 100 120 0 3 6 9 10 12 p e rc e n ta g e o f m a ss d e g ra d a ti o n day of degradation 38 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 the cellulose, there is attacking by microbes which digest the starch (cellulose). the microbes digest this component, leaving behind a porous, sponge-like structure with a high interfacial area, and low structural strength (kumar ashwin, et. al, 2011). it changes the properties such plastic shape, tensile strange and plastic color (roohi, et.al. 2017). conclusion mechanical properties of biodegradable plastic variations of nata de coco have no significant effect on plastic tensile strength. the highest tensile strength value was 4.3244 mpa and the highest elongation was 13.9639% with the highest transmission rate of 14.20 g / m² hours. the addition of nata de coco has an effect on the biodegradability test in the soil. references alexia. 2012. coconut water uses, composition and properties: a review prades. fruits. 67 (2): 87-107, doi: 10.1051/fruits/2012002 apriyani mery and endaruji sedyadi. 2014. sintesis dan karakterisasi plastik biodegradable dari pati onggok singkong dan ekstrak lidah buaya (aloe vera) dengan plasticizer gliserol. j. sains dasar 2015 4 (2) 145 – 152. bourtoom, t. 2008. edible films coatings: characteristics and propeties. international food research journal. 15 (3) gayathry. 2015. production of nata de coco a natural dietary fibre product from mature coconut water using gluconacetobacter xylinum (sju-1). intl. j. food. ferment. technol. 5(2): 231-235, doi: 10.5958/22779396.2016.00006.4. indriyati, l., indrarti., & rahimi, e. 2006. pengaruh carboxymethyil cellulose (cmc) dan gliserol terhadap sifat mekanik lapisan tipis komposit bakterial selulosa. jurnal sains materi indonesia, 8 (1): 40-44. krochta, j., & e. a. baldwin, m.-c. (1994). edible coatings and films to improve food quality. usa: technomic publising co. inc. kumar satish and thakur.2017. bioplastics classification, production and their potential food applications. journal of hill agriculture. 8(2): 118-129. doi: 10.5958/22307338.2017.00024.6. kumar ashwin. 2011. properties of biodegradable polymers and degradation for sustainable development. international journal of chemical engineering and applications. 2 (3). kusnandar, f.2010. kimia pangan: komponen makro. jakarta: dyan rakyat. lay and huey. 1997. properties of microstructures of zain sheets flastiazed with palmitic acid and stearic acid. j. chereal chemistry. 74 (1): 83-90. mostafa. 2018. production of biodegradable plastic from agricultural wastes. arabian journal of chemistry. 11, 546– 553. roohi. 2017. microbial enzymatic degradation of biodegradable plastics. current pharmaceutical biotechnology. 18 (5), doi: 10.2174/1389201018666170523165742. sari, e. 2007. studi biodegradasi poli hidroksi butirat pada medium cair dan padat. tesis. yogyakarta: program pasca sarjana, universitas gadjah mada. sedyadi, e., aini, s. k., anggraini, d., ekawati, d. p. 2016. starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties. biology, medicine, & natural product chemistry. 5(2): 33-40 sinaga, l. l., s. melisa s. r., & sinaga, m. s. (2013). karakteristik edible film dari ekstrak kedelai dengan penambahan tepung tapioka dengan gliserol sebagai bahan pengemas makanan. j. teknik kimia usu. 2 (4): 12-16. sukara endang and meliawati ruth. 2014. potential values of bacterial cellulose for industrial applications. jurnal selulosa. 4 (1): 7 16 vu ngo dinh, et.al. 2017. lignin and cellulose extraction from vietnam’s rice straw using ultrasound-assisted alkaline treatment method. international journal of polymer science, doi: 10.1155/2017/1063695. wirawan, s., prasetya, a., & ernie. (2012). pengaruh plasticizer pada karakteristik edible film dari pektin. journal reaktor. 14: 61-67. biology, medicine, & natural product chemistry volume 3 – number 1 – 2014 issn 2089-6514 contents ecopharmacognosy: exploring the chemical and biological potential of nature for human health geoffrey a. cordell 1 14 the effect of water-soluble stem extract “kayu kuning“ (arcangelisia flava l.merr) on the growth inhibition of candida albicans atcc 10231 and trichophyton mentagrophytes in vitro rini setyowati, sudarsono, setyowati e.p 15 19 larvicidal activity of the mixture of cashew nut shell liquid (cnsl) and aqueous extract of sapindus rarak dc against larvae of culex quinquefasciatus rahmi safarina fauziah, sudarsono, budi mulyaningsih 21 23 identification of migratory birds and their spesific characteristics of habitat in the salt water lake of gili meno, north lombok distric diah purwitasari, luh gde sri astiti, supriadi 25 30 larvicidal effect of vinca fruit extract (vinca rosea) against aedes aegypti larvae and secondary metabolites profile by thin layer chromatography rahmawati ekaputri, sudarsono, budi mulyaningsih 31 33 morpho-anatomical analysis of cosmostigma racemosum (asclepiadoideae) flowers widodo, mohamad amin, mimien henie irawati al-muhdar, muhammad ja’far luthfi 35 46 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 63-68 | doi: 10.14421/biomedich.2017.62.63-68 issn 2540-9328 (online) on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi1, riyanto2 1biological education department, 2integrated laboratory; faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: jafarluthfi@yahoo.com1; justofeel@gmail.com2 abstract this research is an integration between fields of biology, photography, design, and informatics engineering. the study aimed to build an interactive online atlas of reptile anatomy to improve the accessibility and data sharing (free access) of reptile anatomy. website was developed using sdlc (system development life cycle) which consist of five steps as follows: website’s strategic planning, determine the scope of website, website’s requirements analysis, design and implementations of website, and testing. based on the results of testing and system implementation, it can be concluded that online interactive atlas (atlasanatomy.org) had been successfully built as anatomical educational media of reptile. keywords: atlasanatomy.org; online anatomy atlas; vertebrates; reptile; interactive atlas. introduction advances in information technology are increasingly made information exchange easier. facility from information technology becomes important for supporting research and scientific activities. one of the advances in information technology is the rapid development of the internet. data show that the number of internet users has increased from year to year, either globally or in indonesia (apjii, 2014). the data are shown in figure 1. figure 1. (a) world internet users (source: apjii); (b) internet users in indonesia (source: statista.com). from the development of information technology, internet, and growing number of domains (detik.com), a new perspective begins in the development and distribution of science. in 2011, research on digital atlas has been done by jones, stone & karten, entitled "high-resolution digital brain atlases: a hubble telescope for the brain". this research explains the application method for digitalize microscopic part of the brain network containing normal and experimental data and to make the content easily accessible online. the results of this study can be accessed at url www.brainmaps.org. the predecessor http://dx.doi.org/10.14421/biomedich.2017.62.63-68 64 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 study related to brainmaps.org was in 2008 entitled "brainmaps.org interactive high-resolution digital brain atlases and virtual microscopy" (mikula s, et al, 2008). other online atlases avalaible are anatomyatlases.org, innerbody.com, instantanatomy.net, and biodigital.com. all the atlases are human anatomy. this study used reptile as objects in the preparation of interactive online atlas as a reference for learning source. materials and methods vertebrates include all animals that have spines. some of the characteristics possessed by vertebrate animals are having a closed circulatory system, having a backbone in their body, having a complete digestive system, and having a bilateral symmetry body. vertebrates consist of 5 (five) classes, namely pisces, amphibians, reptiles, aves, and mammals. we used mabouya multifasciata as a representation of reptile class. animal 3 lizards (mabouya multifasciata) were used in this study. the lizards were sacrificed, dissected and photographed using canon eos 60d camera; online atlas design for design online atlas we used computer with intel @ 3.40ghz processor i7-4770 cpu (c) i7-4770 equipped with microsoft windows 8.1 operating system software, php 5.3.0 programming language, apache local web server version 2.0, mysql client database server version 5.1.37, mozilla firefox web browser version 56.0.2, text editor sublime text 3, adobe photoshop cs4 extented, coreldraw x6; html, php, javascript, and image map programming languages; servers for data processing and storage; atlasanatomy.org domain name. this research was conducted at integrated laboratory of uin sunan kalijaga yogyakarta. development and manufacture of interactive online atlas system using insourcing method (mulyanto, 2008) sdlc (system development life cycle) covering stages of planning, needs analysis, design, manufacture, and testing. working procedures used in this study include literature studies, data collection & processing, website design, system testing, and system implementation analysis. figure 2. atlasanatomy.org drafting scheme. luthfi & riyanto – on designing interactive online atlas of reptile anatomy … 65 the steps of this research were:  the first step was literature study; namely the study of theories related to animal anatomy, photography techniques, as well as the theory of website making and programming languages.  the second step was data collection & processing; i.e, taking pictures/images on the dissected reptile object and processing the image using software to get the image data. organs were identified and named.  the third step was website making by way of insoursing; there are 5 (five) stages that need to be done namely planning, needs analysis, design, manufacture, and testing.  the fourth step was system testing; there are 2 (two) tests conducted, namely alpha testing conducted directly by the research team about the functional website and beta testing conducted by the general public with a focus on the functionality and interface website. the results of this test are used as material improvement and website development.  the fifth step was system implementation; namely implementation of the website system to obtained evidence or comprehensive facts about animal anatomy website. results and discussion determination of mapping area objects mabouya multifasciata morphology consisted of external morphology, anterior extremities, posterior extremities, organ topography, respiratory system, male reproductive system, female reproductive system, and digestorium system. table 1, figure 3-figure 7 show the segmentation of each section to create image map. table 1. division of mabouya multifasciata image map area. no anatomy system area mapping objects 1 external morphology nares anteriores, thympanic membrane, trucus, rima oris, caput, squama, orgamon visus, cervix, cauda, front limb, hind limb 2 anterior extremities bracium, digit, antebracium, manus, falcuna 3 posterior extremities femur, digit, crus, pes, falcuna 4 organ topography trachea, hepar, hepar, vesica fellea, pulmo, duadenoum, abdominal fat 5 respiration system lingua bifida, rima glottides, trachea, pulmo 6 male reproductive system hemipenis, epidydimis, ren, vas deferent, testis 7 female reproductive system ovarium, oviduct, embrio 8 digestorium hepar, vesica fellea, lien, small intestine, rectum preparation and writing of syntax to create mapping in the truncus section were as follows:  image dimensions: 950 x 364 pixels  syntax determination of truncus part coordinate region: <area name="truncus" alt="" title="" href="#" shape="poly" coords="268,57,274,57,279,57,287,58,297,58,308,59,322,59,329,59,336,59,344,59,356,59,367,59,375,59,389,59,4 04,60,416,60,430,61,446,61,462,63,478,64,492,66,508,69,521,69,535,71,549,73,562,74,578,77,590,79,602,83,611 ,85,612,89,611,96,611,100,611,110,608,118,608,125,608,133,607,140,608,146,608,152,599,151,592,151,581,151, 579,145,583,142,584,136,584,131,582,125,576,120,570,117,564,116,559,114,554,114,548,114,542,115,534,119,5 29,130,530,139,535,148,536,152,522,150,501,150,479,150,457,150,429,151,410,150,389,151,373,151,353,152,34 8,141,342,130,335,119,329,112,320,112,311,112,298,115,285,121,276,126,274,132,276,138,281,144,287,147,294 ,149,299,149,306,146,314,144,324,154,313,157,302,156,291,156,281,156,274,154,267,154,255,151,248,150,242, 149,240,147,245,135,249,126,253,116,257,107,262,98,265,87,268,77" /> figure 3. original images external morphology mabouya multifasciata on the website system. 66 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 figure 4. original images organ mabouya multifasciata on website system. figure 5. output images with image map on external morphology mabouya multifasciata on the website system. (a). caput; (b). truncus; (c). cervix; (d). cauda; (e). digiti; (f). antebracium; (g). manus; (h). bracium; (i). crus; (j). femur; (k). falcuna; (l). pes. figure 6. output images with image map on mabouya multifasciata on the website system. (a). duodenoum; (b). abdominal fat; (c). hepar; (d). pulmo; (e). lingua bifida; (f). pulmo. luthfi & riyanto – on designing interactive online atlas of reptile anatomy … 67 figure 7. output images with image map on mabouya multifasciata on the website system. (a). trachea; (b). rima glottidis; (c). rectum; (d). small intestine; (e). lien; (f). hepar; (g). hemipenis; (h). testis; (i). epidydimis; (j). vas deferent; (k). oviduct; (l). ovarium. the anatomical photographs of reptile in this study had successfully arranged for online access. goubran & vinjamury (2007) stated that online atlas is an effective way for student learning. conclusion based on the results of the study we concluded that this research has succeeded in establishing an interactive anatomy of reptile anatomy available online (atlasanatomy.org). acknowledgements this research is supported and funded by institute for research and community service (lp2m) uin sunan kalijaga fiscal year 2017. references arfian, gilang. 2009. dasar-dasar pemrograman web. elexmedia. jakarta: komputindo. asosiasi penyelenggara jasa internet indonesia. 2015. profil pengguna internet indonesia 2014. jakarta: puskakom ui. asosiasi penyelenggara jasa internet indonesia. infografis penetrasi & perilaku pengguna internet indonesia survey 2016. retrieved from https://apjii.or.id/survei2016 at 7 march 2017, time 13.33 wib bunafit, nugroho. 2004. cascading style sheets (css). yogyakarta: andi. bunafit, nugroho.. database relational dengan mysql. yogyakarta: andi. detik. pengguna internet 2.4 miliar, jumlah situs tembus 634 juta. retrieved from http://inet.detik.com/read/2013/01/21/081040/2147888/398/pe ngguna-internet-24-miliar-jumlah-situs-tembus-634-juta at 27 november 2015, time 18.27 wib flanagan, david. 2011. javascript: the definitive guide (6th ed.). o'reilly & associates goubran, e.z., s. p. vinjamury. 2007. interactive atlas of histology a tool for self-directed learning, practice, and self-assessment. the journal of chiropractic education 21 (1): 13-18. hariyanto, bambang. 2004. sistem manajemen basis data. bandung: informatika. janner. 2009. pengantar sistem dan teknologi informasi. amus. yogyakarta. jones, e. g., stone, j. m. and karten, h. j. (2011), highresolution digital brain atlases: a hubble telescope for the brain. annals of the new york academy of sciences, 1225: e147–e159. doi: 10.1111/j.1749-6632.2011.06009.x kadir, abdul. 2005. pengenalaan teknologai informasi. yogyakarta: andi mikula s, stone jm, jones eg (2008). brainmaps.org interactive high-resolution digital brain atlases and virtual microscopy. in: lorenz s, egelhaaf m (eds): interactive educational media for the neural and cognitive sciences. brains, minds & media, vol. 3, bmm1426. (urn:nbn:de:00093-14269) 68 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 mulyanto, a., 2008. sistem informasi konsep dan aplikasi. yogyakarta: pustaka pelajar. peranginangin, kasiman. 2006. aplikasi web dengan php dan mysql. yogyakarta: andi. ramadhan, a. 2008. pemrograman web dengan html, css dan javascript. jakarta: elexmedia komputindo. sidik, betha. 2009. .pemrograman web dengan html. bandung: informatika bandung. statista. number of worldwide internet users from 2000 to 2015. retrieved from http://www.statista.com/statistics/273018/number-of-internetusers-worldwide/ at 19 november 2015, time 14.33 wib. suntoro, susilo handari, dkk. 1994. anatomi hewan. jakarta: universitas terbuka. sunyoto, andi. 2007. ajax membangun web dengan teknologi asynchronouse javascript & xml, yogyakarta: andi. www.w3school.com www.php.net biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 1-6 | doi: 10.14421/biomedich.2019.81.1-6 issn 2540-9328 (online) novel approaches for detection fluorescent-labeled by cellvizio lab system on hippocampal ca1 region asma ulhusna shaimi1, wan raihana wan aasim2, hasmah abdullah3, wan amir nizam wan ahmad3, tan soo choon4, ang chee wei4, zalina ismail1,* 1brainetwork centre for neurocognitive science, school of health sciences, universiti sains malaysia, 16150 kubang kerian, kelantan, malaysia. 2malaysian technology development corporation sdn. bhd., level 8-9, menara yayasan tun razak, malaysia. 3school of health sciences, universiti sains malaysia, 16150 kubang kerian, kelantan, malaysia. 4institute for research in molecular medicine (informm), universiti sains malaysia, 11800 pulau pinang, malaysia. author correspondency*: drzalina@gmail.com abstract neurosteroids have been identified in the 1981. dehydroepiandrosterone sulphate (dheas) is one of the vital neurosteroids that de novo synthesized in the nervous system from cholesterol precursor (baulieu & robel, 1998). the aim of the study is to develop a method for fluorescence labelling. alexa fluor 488 dye with dheas antibody can binds the dheas antibody in the rat brain monitored by cellvizio lab system. dheas antibody (igg isotype antibodies) was fluorescently conjugated by an amine-reactive compound, alexa fluor 5sdp ester 488 dye. the resultant alexa fluor 488-conjugated antibodies were collected and analyzed by uv-vis spectrophotometer instrument. the absorbance of the protein-dye conjugate at 280 nm and 494 nm were measured. then, the degree of labeling (dol) was calculated to achieve the desired results. fluorescence labelling were carried out into the ca1 region of hippocampus sprague-dawley rat. we reported that the conjugation was successful. optimal labeling depending on degree of labeling (dol) needs some necessity to achieve and effective binding to the target neurosteroid, dheas. cellvizio lab system connected with fiber fluorescence microscopy (ffm) probe is presented as a new approach in real-time imaging of dheas. in conclusion, we have developed a new method of dheas-alexa fluor fluorescence labelling to visualize and evaluate the changes of dheas fluorescence level in the rat hippocampus. this novel approach as a diagnostic tool and can be used to better understand the mechanisms and functions of dheas and other neurosteroids in future research. keywords: dheas; alexa fluor 488 dye; fluorescence labelling; cellvizio lab system introduction dehydroepiandrosterone sulphate (dheas) is a steroid which naturally synthesized in the brain. the steroids that are synthesized in the nervous system have been termed ‘neurosteroids’ (baulieu, 1981). baulieu & robel (1998), reported that concentrations of dheas are much higher in the brain than in plasma. the dheas can enhanced short term and long term memory performances in a variety of learning tasks. treatment aged animals with dheas enhance learning and memory in the t-maze footshock avoidance apparatus (farr et al., 2004). some studies have demonstrated that dheas enhancing memory performances in aged animals (michael et al., 2001; vallée et al., 2001). behavior experiments that use the morris water maze, y maze, and radial arm water maze in animal models of learning and memory showed that dheas supplementation significantly affect behavioural performance (bodensteiner et al., 2008). another study in ovariectomized rats, i.c.v. implants by dheas may decrease escape latencies and distances to platform in the morris water maze (frye & sturgis, 1995). in a study that assessed of learning and memory, fluorescence labelling was develop to conjugate between dheas monoclonal antibodies and alexa fluor 5-sdp ester 488 dye. thus, a sulphodichlorophenyl ester (sdp) functional group is attached to a fluorophore core, alexa fluor 488 dye and bind specificity with monoclonal antibodies directly to form fluorophore-antibody conjugates. moreover, sdp ester indicated hydrolytically stable form of the alexa fluor 488 dye for amine-conjugations (molecular probes, 2007). vira and co-reseachers (2010) reported that the steric hindrance and the absence of additional reactive sites on the fluorophore may affect the conjugation reaction. conjugation between alexa fluor dye and antibodies were given exceptionally bright and most photostable (panchuk-voloshina et al., 1999). in fluorescence labelling, achieveing an optimal degree of labeling (dol) and minimizing fluorescence quenching are the keys for conjugation (berlier et al., 2003). conjugation at high molar ratios of the fluorophore toward antibodies may cause dols become high. therefore, it can be principal to the fluorescence quenching and assume to dye-dye interactions (randolph & waggoner, 1997). the molar coefficient (ɛdye) of the alexa fluor 488 dye https://doi.org/10.14421/biomedich.2019.81.1-6 2 biology, medicine, & natural product chemistry 8 (1), 2019: 1-6 is 71,000 and indicated dols between ranges 4 to 9. further, at high dols, the alexa fluor 488 dye undergo more resistant to fluorescence quenching. the stability to ph of alexa fluor dye was monitored by measuring their absorption spectra in buffers at ph 4-9. the most stable conjugates were performed at ph 8.3. for labelling, alexa fluor 488 dye can be retained the intensity in conjugation with antibodies. in principle, any fluorescent dye can be chosen to conjugate protein for live cell imaging, and many new dyes with improved photochemical properties have been developed (panchuk-voloshina et al. 1999). to complement a develop method of fluorescence labelling, cellvizio lab system connected with ffm probe will be used as an imaging approach to visualize and quantify the fluorescence intensity in vivo and real time imaging. the cellvizio lab system provides to visualize deep brain through fluorescence imaging, high resolution, rapid, in real time image, with minimally invasive, and one animal at one time. besides, stereotaxis apparatus as a platform to position the fiber fluorescence microscopy (ffm) probe to the exact coordinate of target location (visualsonics, 2009). the probe, an approach that designed for cannula implantation on anaesthetized animals, consisting of a diameter tip 0.30 mm, lateral resolution 3.3 µm, optical sectioning 15 µm, and maximum field of view 600x500 µm (vincent et al., 2006). therefore, to understand how the brain activity of rats achieving in behavioral tasks, the cellvizio lab system with flexible ffm probe used to record in vivo imaging attached with dheas-alexa fluor fluorescence labelling at the region of interest. in this article, we report the successful of dheasalexa fluor 488 was expressed in the ca1 region of hippocampus rat brain. here, our goal was to highlight a new method of dheas-alexa fluor 488 as a fluorescence labelling of dheas and expressed the signal of dheas by ffm connected with cellvizio lab system in visualizing real time imaged, hence as a new imaging approach based on fluorescence. materials and methods antibody and reagents preparation dheas monoclonal antibodies (3 mg/ml) were bought from calbioreagents. alexa fluor sdp ester labeling dye (thermo scientific) was used to conjugate with antibodies. the corresponding fluorophore: protein (f:p) ratio, based on a280 and a494 absorption readings, was calculated according to the labeling instructions including the recommended correction factors for the absorbance of the dye at 280 nm. evaluation of dye delivery approaches in ca1 region of hippocampus all procedures were approved by the animal ethics committee universiti sains malaysia and followed the guidelines of the animal research and service centre. one group (n=7) male sprague-dawley (sd) rats (250 300 g) anaesthetised with isoflurane. sd rats were housed in groups of two per cage with twelve-hour light/dark cycle and food and water was provided ad libitum. all surgeries were performed under isoflurane anaesthesia. anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up 4% in the induction phase, at 2.5% during surgical procedures and at 1.0-1.5% during prolonged experimental observations. dheas, neurosteroid in the brain were imaged by using a beveled tip ffm probe through a hole in the skull. the probe was positioned by using the holder of motorized stereotaxis device. one microliter of dheas-alexa fluor 488 dye was carried out into the ca1 region using 10 µl hamilton syringe, set to eject 1.00 µl over about 10 min. injection coordinates were (in mm): for the ca1 hippocampus, ap: -3.00 mm, ml: -1.7 mm and dv: 3.00 mm. optical recording started 1 h later. preparation of alexa fluor 5-sdp ester 488 dye labelled monoclonal antibodies dehydroepiandrosterone sulphate (dheas) was fluorescently conjugated to an amine-reactive compound, alexa fluor 5-sdp ester 488 dye (invitrogen). dheas solution (3.0 mg/ml) was exchanged to sodium bicarbonate by dialysis with against 0.1 m sodium bicarbonate solution (ph 8-9) for 2 hours at room temperature. then, alexa fluor 488 dye were prepared in dmf solution at concentration 10 mg/ml. therefore, 1.2 µl reactive dye was added into the 100 µg dheas solution and incubate the solution for 1 hour at room temperature. the resultant solution was transferred into the membrane dialysis tube and dialyzed against pbs (ph 7.4) an overnight in 4oc with continuous stirring. the resultant dheas-alexa fluor 488 was collected and sterilized via 200 nm polymer membrane filter and was stored at 4oc and -20oc for long storage. the concentration of dheas antibodies and alexa fluor dye were calculated from the absorbance at 280 and 488 nm, measured using an ultraviolet (uv-vis) spectrophotometer, using the following formula: the concentration of protein in the sample: protein concentration (m) = [a280 – (adye x cf280)] x dilution factor 203,000 shaimi et al. – novel approaches for detection fluorescent-labeled … 3 the degree of labeling: moles dye per mole protein = adye x dilution factor ɛdye x protein concentration (m) (molecular probe, 2011) real time imaging by fiber fluorescence microscopy (ffm) probe connected with cellvizio lab system after labelling, rats were anaesthetised and positioned on a stereotaxis apparatus. the ffm probe with a holder was placed into the mount of the stereotaxis device. the probe was fixed into position on top of the cannula implantation and set the coordinate as bregma point (0,0,0). next, the ffm probe connected with cellvizio lab system (le goualher et al., 2004; davenne et al., 2005) and a frame rate was at 12 hz. excitation of cells at 488 nm laser light and emission was measured at 494 nm. the cellvizio lab system have a single-pixel avalanche photodiode detector (apd) for resolution and sensitivity, respectively. results development of dheas-alexa fluor 488 fluorescence labelling we conjugated dheas antibody with reactive dye, excitation and emission wavelength at 488 nm and 494 nm, respectively. for this purpose, we determined by measuring the optimal degree of labeling with different concentration of protein, wherein the buffer ph was constant. table 1 show the absorbance of dheas antibody and reactive dye based on a280 and a494 absorption reading, the concentration of protein, dilution factor, degree of labeling, and molar coefficient of specific dye and correction factor for the fluorophore contribution as constant variables. based the result, the absorption reading for antibody and dye increase, the degree of labeling of fluorescence intensity was increased. therefore, according to obtained result, concentration of antibody, 0.91 m and degree of labeling, 4.63 was chosen for this present study as a new method. table 1. the protein concentration and degree of labeling in different protocols. b a tc h a280 a494 [p] d o l dl ɛdye cf280 λmax em 1 0.82836 0.57950 0.57 2.17 1 71,000 0.11 494 519 1 1.64540 0.92632 1.14 1.70 1 2 1.18320 1.44100 1.51 4.02 2 *constant variables 3 1.45050 1.99340 0.91 4.63 1 * a280 and a494 is an absorption reading by uv-vis spectroscopy. * [p] is protein concentration in mg/ml. * dol is degree of labeling. * dl is dilution factor to modify the calculation. * ɛdye is molar extinction coefficient at λmax in cm -1 m-1. * cf280 is correction factor for absorption readings at 280 nm. * λmax and em are the fluorescence absorbance and emission maxima, in nm, conjugated to an igg antibody. conjugation of dheas-alexa fluor 488 as fluorescence labelling a standardized protocol was implemented in this experiment, and yield less than 4 of dols of conjugation dheas-alexa fluor 488. the experimental workflow has made some modification and introduced two principals in a new method. first, the time incubation need to be longer for conjugation. the conjugates were incubated overnight to increase chemically binding of the dheas-alexa fluor conjugation. to achieve effective labelling, marks et al. (2004) reported that it required the incubation of 1 µm 5’-fluorescein-slf’ for 16 hours. the second modification was increased the volume of the dye from stock solution against antibody to enhance the expression signal of dheas-alexa fluor fluorescence intensity. the standard protocol of dye was 1.2 µl dye in 100 µg antibody (10 mg/ml). therefore, in this study the method was modified, 2.4 µl dye in 100 µg antibody. the uv spectra for batch 2 and 3 has shown in figure 1. figure 1. the uv spectra showing the absorption dheas antibody and alexa fluor dye for batch 1, batch 2 and batch 3. the absorption peaks at 288 nm and 494 nm correspond to the absorption for antibody and excitation of alexa fluor 488 dye, respectively. expression of dheas-alexa fluor fluorescence labelling by ffm imaging ffm probe attached to cellvizio lab system has been used to monitor dheas fluorescence levels in the hippocampal ca1 region. the characteristics of 4 biology, medicine, & natural product chemistry 8 (1), 2019: 1-6 microprobe s300b was shown in table 2. spraguedawley rat was positioned and placement on the motorized stereotaxis apparatus, then dheas-alexa fluor 488 fluorescence labelling was carried out into the ca1 region of hippocampus of anaesthetized animals. one hour after labelling, ffm imaging was visualized and imaged the signal of dheas fluorescence expression. the changes of dheas fluorescence expression was quantified and recorded the images at region of interest (roi)s in real time imaged and minimally invasiveness. table 2. properties of the microprobe s-300b used in this study. probe s-300b diameter (mm) 0.30 maximum field of view (µm x µm) 600 x 500 axial resolution (µm) 15 lateral resolution (µm) 3.3 * values provided by cellvizio lab system, mauna kea technologies, france figure 2. the signal of dheas fluorescence expression. (a) the images visualized indicated the dheas fluorescence was expressed the signal in hippocampal ca1 region. (b) the region of interest (roi) of dheas fluorescence signal were selected. discussion the excitation and emission spectra of alexa fluor 488 dye are illustrated in figure 3. figure 1 in this present study demonstrated the uv-vis spectra of alexa fluor 5-sdp ester 488 dye conjugated with dheas antibodies. the uv-vis spectrophotometer is a useful instrument in most laboratories (shahal et al., 2016). the aim of this study was to develop method of dheas-alexa fluor fluorescence labelling and evaluate the successful conjugation to be used as a tagging for dheas in the ca1 region of hippocampus by imaging approach, ffm connected with cellvizio lab system. this finding indicated that the antibody conjugation was effectively and efficiently to be used as fluorescence labelling to image and quantify dheas in the hippocampus rat brain by ffm imaging. figure 3. the illustration of excitation and emission spectra of alexa fluor 488 dye. coons and co-researchers (1950) showed that antibody conjugation widely uses nowadays in the biological sciences and medicine. in conjunction, labelling method is a direct technique which is the antibodies chemically conjugated to a fluorescent dye. the result of this study indicated that conjugation between alexa fluor 488 dye and dheas antibodies was succeed. the result of the present study also suggest that based on calculation degree of labeling, the f:p ratio the conjugation should be in range 4 to 9 (table 1). this finding indicated that fluorescence labelling can be effective used and given brighter expression in animal studies (hayashi-takanaka et al., 2014; vira et al., 2010). in order to obtain better labelling, the parameters that can affected the fluorescence should be considered. firstly, the antibodies and reactive dye should be in the same buffer. in this case, 0.1 m sodium bicarbonate was used to change the buffer of antibodies. it is necessary to achieve strongly binding in the covalent bond of conjugation. furthermore, longer incubation time required for fluorescence labelling protocols to determine the optimal dol of dheas-alexa fluor 488 conjugates. this suggested that the conjugation leaves for an overnight or at least 16 hours to increase the fluorescence intensity. the absorption data from uv-vis spectra was increased corresponding to the time of incubation. additionally, we modified the approach of shaimi et al. – novel approaches for detection fluorescent-labeled … 5 the dye from standard labeling protocols to increase the efficiency of labeling. initial experiment was conducted with preparation dye in standard protocol (1.2 µl dye in 100 µg antibody). we altered the dye which is increase the volume of dye from stock solution into twice time, 2.4 µl dye in 100 µg antibody. the results in this present study demonstrated that increased the dol of conjugation. there are have several requirements were highlighted in order to achieve the better labelling. this could be due these findings accordance with the previous studies and clearly demonstrated of brightness, photostability, photobleaching, binding affinity, size of fluorophore, fluorescent lifetime and molar coefficient (hayashi-takanaka et al., 2014; shaner et al., 2005; toseland, 2013). additionally, better expression of fluorescent labelling influence by ph sensitivity (shaner et al., 2005). thus, ph conditions performed as an indicator of fluorescent lifetime in conjugation of imaging living cells (lin et al., 2003). the ffm probe was inserted into the brain to recorded and quantified the changes of dheas fluorescence intensity level in the ca1 region of hippocampus. surprisingly, this present study is the first report to evaluate the expression of dheas fluorescence in the target area, ca1 area of hippocampus by ffm connected with cellvizio lab system. according to vincent et al. (2006) demonstrated that cellvizio lab system allowed researchers to visualize calcium signals in deep brain structures. these results are consistent with ffm connected with cellvizio lab system that allowed the direct recording in a short time frame, same animals can be used repeatedly, avoiding shrinkage of cells and tissues, and flexible probes (frykman et al. 1988). thus, ffm connected with cellvizio lab system was essential for in vivo imaging associated with behavioral task in order to time saving and does not require sample preparation or staining to prove the presence of fluorescence labelling. in this present study showed the signal of dheas fluorescence expressed at the region of interest (figure 2). this finding indicate that the signal of expression dheas fluorescence can be used as a potential in vivo imaging to evaluate and quantify the changes of dheas level in the rat brain due to physiology, pathology and pharmacology process (vincent et al., 2006; visualsonics, 2009). accordingly, the results in this study presumed that the changes of dheas fluorescence expression associated with cognition. we have developed new method of fluorescence labelling in conjunction with fluorescence imaging approach, ffm connected with cellvizio lab system that precise and accurate visualization of fluorescence signals in living brains as well as non-invasive method for cellular level imaging in vivo (davenne et al., 2005; vincent et al., 2006; makhlouf et al., 2008). conclusion in conclusion, dheas-alexa fluor 488 fluorescence labelling was developed to monitor and evaluate the changes of dheas fluorescence level that expressed in the ca1 region of hippocampus. beyond imaging, the dheas-alexa fluor fluorescence labelling is one step approach to monitor dynamic behavioral task and better understand the mechanisms of dheas. moreover, this approach can be used to discover the beneficial functions of dheas and other neurosteroids in the hippocampus at preclinical level to encourage in future research. acknowledgement this research was supported by the frgs grant (203/ppsk/6171153) and universiti sains malaysia. references baulieu, e. e. 1981. steroid hormone regulation of the brain, eds. fuxe, k. & gustafsson, j. a. 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dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. brain research reviews, 37: 301-312. vincent, p., maskos, u., charvet, i., bourgeais, l., stoppini, l., leresche, n., changeux, j., lambert, r., meda, p. &paupardin-tritsch, d. 2006. live imaging of neural structure and function by fibred fluorescence microscopy. european molecular biology organization reports, 7(11):1154-1161. vira, s., elena, m., glen, h. & paul, s. b. 2010. fluorescent labeled antibodies-balancing functionality and degree of labeling. anal biochem., 402(2): 146-150. visualsonics. 2009. application brief: cellvizio lab fibered fluorescence microscopy and microdialysis research. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 21-26 | doi: 10.14421/biomedich.2018.71.21-26 issn 2540-9328 (online) physiological response of 'segreng' rice plant (oryza sativa l.) to biogas sludge at wukirsari village, cangkringan, sleman dwi umi siswanti*, sudjino, nindy senissia asri, mifta arlinda, arianda poetri shofia rochman, akrima syahidah faculty of biology, universitas gadjah mada, jl. bulaksumur, yogyakarta, 55281, indonesia tel. +62-274-6492599, fax. +62-274-565223 author correspondency*: dwiumi@ugm.ac.id abstract wukirsari village, cangkringan district is belong to merapi mountain’s slopes which located between the gendol river and yellow river. nowadays, we faced the problem of anorganic fertilizer overused such as urea, za, tsp/sp-36 and kcl in agriculture land. the effort to return the soil organic compound can be done by added some organic compounds or microbial bio -organic fertilizer. sludge is fermented biodigester yield and it has lost its gas. the aim of this research was to understand the physiological response and optimum dose of biogas as planting medium to ‘segreng’ rice planted in the rice field of wukirsari village, cangkringan district, sleman regency. this research was done on greenhouse scale and rice field scale. the treatment given on 0; 1; 1,5; 2 and 2,5 liters per 100 m2 of rice field areas, and given on 0; 4; 8; 12; and 24 ml per 5 kg soil on polybags. data were taken in three repetitions. the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, roots, filled grains, empty grains, and total weight and number of filled grains, empty rains, and the number of panicles. the result were tested with one way anova (analysis of variance) with spss version 19 for windows and followed by duncan's multiple range test with 95% significance level (α = 0.05). generally, the result showed that biogas sludge can increase the vegetative and generative growth of rice plant ‘segreng’ on polybag scale and rice field scale. the rice plant on polybag with 4 ml biogas sludge was significantly different on the vegetative growth and chlorophyll content, while the rice plant on polybag with 8 ml biogas sludge was significantly different on the generative growth and nra levels. keywords: physiological response; sludge; biogas; ‘segreng’; wukirsari introduction rice (oryza sativa l.) is an indonesian main food and strategic commodity. however, rice production in indonesia has not been able to meet the population demands (alavan et al., 2015). in yogyakarta, the rice demand continues to increase because the growth of population is not balanced with agricultural area expansion and rice quality improvement. this in turn led to a decrease in rice production. wukirsari village, cangkringan district belongs to sleman regency which is the supplier of rice in the special region of yogyakarta. the area of this village is 1,456 ha. topographically, the village is located at an altitude of 450 to 600 m above sea level, with an average rainfall of 22 mm/year. the average temperature per year is 21-31oc. wukirsari village is a suitable area for agriculture. wukirsari village is located between the gendol river in the east and the yellow river to the west (rpjm desa wukirsari, 2015; siswanti, 2015). rice production in yogyakarta has decreased from 721,674 tons in 2013 to 713,800 tons in 2014 (bps daerah istimewa yogyakarta, 2015). the productivity decrease becomes a serious obstacle faced by the agricultural sector today. one of the impacts is the use of inorganic fertilizers such as urea, za, tsp / sp-36 and kcl (redono, 2016). the abundance of inorganic materials in the soil reduced the regeneration ability of rice plants (sudadi & widada, 2001 in redono, 2016). according to siswanti (2015), the agricultural land in sleman district, including in wukirsari village contains high chemicals due to excessive use of chemical fertilizers (500 kg/ha). bio organic fertilizers can improve the physical, chemical, and biological properties of the soil that make them quite profitable. one kind of bio organic fertilizers that is expected to contain many nutrients is biogas sludge. biogas produces liquid solid waste in the form of livestock manure that has lost its gas and is rich in the elements needed by plants such as protein, cellulose, and lignin which cannot be replaced by chemical fertilizers (ginting, 2007). until now there has been no research on red rice plant using biogas sludge as fertilizer, therefore, it was not yet known about the effect of biogas sludge as planting medium to vegetative and generative growth, chlorophyll content and nra content of segreng rice https://doi.org/10.14421/biomedich.2018.71.21-26 22 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 planted in the rice field of wukirsari village, cangkringan district, sleman regency. the aim of this research is to understand the effect of biogas sludge as planting medium on the vegetative growth, generative growth, chlorophyll content and the nitrate reductase analysis (nra) value of ‘segreng’ rice, and to find out the optimum concentration of biogas sludge on the growth of 'segreng' rice. materials and methods tools and materials the tools used in this study were plows, bamboo, 1 liter glass cylinder, thermometer, soil ph tester, luxmeter, tube, spectrophotometer, analytical scale, micropipet, pipette, pump pippet, porcelain pallet, whatman no.1 filter paper, aluminum foil, cuvette, scissors, oven, 100 ml measuring flask, and dark flakon. materials used in this study were segreng rice seedlings, paddy soil, biogas sludge, polybag, water, 80% acetone, distilled water, salt, phosphate buffer ph 7.5 0.1 m, nano3 5 m, 0.02% ned, 1% sa in 3 n hcl, aquadest, nano2 0.6 m, tissue paper, and aluminum foil. data collection rice field with an area of 500 m2 was divided into five parts/plots so that each plot size was 100 m2. plot 1 (control) was not added with sludge, plot 2 was added with 1 liter of sludge, plot 3 was added with 1.5 liter sludge, plot 4 was added with 2 liter sludge, and plot 5 was added with 2.5 liter sludge. meanwhile, on polybag media treatments, preparation of rice field soil was done by measuring the weight until 5 kg, mixed with sludge, then put them in polybags with the diameter of 21 cm. the treatment given on p1 (control) was not added with sludge, p2 was added with 4 ml sludge, p3 was added with 8 ml sludge, p4 was added with 12 ml sludge, and p5 was added with 24 ml sludge. parameters measured on the growth of rice plants include the number of leaves, number of seedlings, plant height, and analysis of chlorophyll content by spectrophotometric method (aoac, 1995). while the parameters measured on the productivity of rice crops include dry weight, number of panicles, the weight of filled and empty grains, the number of filled and empty grains, and nra value analysis using modified assay method (hartiko, 1983). the data of vegetative parameter, generative parameter, chlorophyll content and nra level of the 5 treatments were tested with one way anova (analysis of variance) with spss version 19 for windows and followed by duncan's multiple range test with 95% significance level (α = 0.05) observation and measurement of vegetative parameters were done every week for 9 times of observation, followed by observation and measurement of generative parameters done every week for 5 times of observation. parameters observed included plant height, number of leaves, number of seedlings, total chlorophyll content, dry biomass weight, crown weight, root weight, filled grains weight, empty grains weight, number of filled grains, number of empty grains, number of panicles, and nra levels. the steps taken in this research were land preparation, seed preparation, rice seed planting, parameters measurement and rice plant maintenance. preparation of land included the process of jacking rice fields with a depth of 20-25 cm and repeated 2 times with 1 week time difference. this was aimed to ensure the soil conditions become poor of nutrients, so that the effect of biogas sludge becomes significant. in the meantime of waiting the readiness of rice field, preparation of segreng rice seedlings was done. segreng variety rice was chosen for this study because it has several benefits including: 1. high yield, i.e. 3-4 ton/ha, 2. the husks contain β-carotene of 488.65 micro g/100 g, 3. high rice selling value, which is 30% more expensive than ordinary rice, 4. tolerant to water stress, 5. high protein content of about 7.3%, completed with 4.3% iron and 0.34% vitamin b1 (kristamtini & prajitno, 2009). rice seeds were first soaked in 3% nacl solution for 24 hours. this treatment served to separate good quality rice of seeds with the bad ones. good quality rice seeds would sink, while bad seeds would float when soaked in salt solution. furthermore, good quality rice seeds were dried under sunlight for about 3 days until dry grains were obtained. it aimed to avoid possible contamination of moist rice seeds. furthermore, rice seeds were spread in a nursery area of 2x2 m2 with inundated soil. in this condition, seeds would experience imbibition, or the process of water seeping into the seeds. water would activate enzymes that play roles in the germination process. the seeds were grown until 20 days. at this age, the rooting system of the rice seedlings were strong enough and the number of leaves were more than 4, so it could be stated that the rice seedlings were ready to be moved to the rice field. prior to the process of moving rice seedlings to the rice field, the land were processed with biogas sludge. biogas sludge used were 0; 1; 1.5; 2; and 2.5 liters per 100 m2 of the rice field and 0 ml; 4 ml; 8 ml; 12 ml and 24 ml per 5 kg of soil on polybags. data were taken on three repetitions. the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, root, filled grains, empty siswanti et al. – physiological response of 'segreng' rice plant (oryza sativa l.) … 23 grains, and total weight and number of filled grains, empty rains, and the number of panicles. the treatment of rice crops included the opening of irrigation flows, weeding and eradicating pests. measurements of growth parameters were done weekly. the measurement of plant height was done by medline from the base of stem to the highest leaf tip. the number of seedlings is the number of new clumps that appears next to the parent plant. the total number of leaves is the number of leaves of the parent plant and the number of leaves of the seedlings or clumps. the measurement of chlorophyll content was done on the 4th week, the leaf samples used were parent plant leaves, taken ± 5 cm in the middle of the leaf. this was because the section was assumed to be mesophyll tissue where the process of photosynthesis happens, so the chlorophyll content obtained were expected to be more optimum than at the edges. the measurement of chlorophyll content was done by spectrophotometric method. the principle of the spectrophotometric method is measuring the absorbance value of the solution to a particular wavelength. in this study, the wavelength used were 642,5 nm and 660 nm, because chlorophyll a is able to absorb wavelength of 673 nm, while chlorophyll b is able to absorb wavelength at 455-640 nm. meanwhile, nra test was done when the rice entered the generative stage, marked by busted panicles. the nra test was performed because the activity of enzyme nitrate reductase could show how much the rate of metabolism in the plant body is increased due to the preparation of building molecules (proteins) to be transported to the grain of rice. nitrate reductase is the key enzyme that first converts nitrate ions which are absorbed by the roots into nitrites, which are then converted to ammonia. ammonia will react with glutamine to form amino acid glutamate and form other amino acids. these amino acids are used as cell constituents and transported to the grain. therefore, according to alnopri (2004), nra levels usually show predictions of the productivity of panicle-busted rice. in the nra test, the rice leaves used were ‘flag leaves’ or the leaves located just above the grains. the flag leaves are the topmost leaves and the youngest leaves among the other leaves. young leaves have a higher activity than the old leaves. according to hartiko (1983), the leaf that has a higher position has a higher nitrate reductase activity than the leaves below it. results and discussion based on the research, the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, root, filled grains, empty grains, and total weight and number of filled grains, empty rains, and the number of panicles, was present below: note for figure 1 – 8: treatment 1= control treatment 2= 1 liter sludge/100 m2 or 4 ml sludge/polybag treatment 3= 1,5 liters sludge/100 m2 or 8 ml sludge/polybag treatment 4= 2 liters sludge/100 m2 or 12 ml sludge/polybag treatment 5= 2,5 liters sludge/100 m2 or 24 ml sludge/polybag figure 1. the average of plant height on 9th weeks in, (a) rice field; (b) polybags. figure 2. the average of number of leaves on 9th weeks in, (a) rice field; (b) polybags. a, b b a, b a, b a 65 70 75 80 85 90 95 1 2 3 4 5 p la n t h e ig h t (c m ) treatments a, b b a, b b a, b 50 55 60 65 70 1 2 3 4 5 p la n t h e ig h t (c m ) treatments a, b b a, b a, b a 0 2 4 6 1 2 3 4 5 n u m b e r o f le a v e s treatments b a a, b a, b a, b 4 4.5 5 5.5 6 1 2 3 4 5 n u m b e r o f le a v e s treatments 24 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 figure 3. the average of number of seedling on 9th in, (a) rice field; (b) polybags. figure 4. the chlorophyl content of each treatment in (a) rice field and (b) polybags. figure 5. the average of dried mass in (a) rice field and (b) polybags. figure 6. the average number of grains in (a) rice field (b) polybag. a b a, b b a, b 0 0.5 1 1.5 2 2.5 1 2 3 4 5 n u m b e r o f se e d li n g s treatments a b a, b a, b a, b 0 0.5 1 1.5 2 2.5 1 2 3 4 5 n u m b e r o f s e e d li n g s treatments 0 2 4 6 8 10 1 2 3 4 5 c h lo ro p h y l c o n te n t (m g /g le a f) treatments chlorophyl a chlorophyl b 0 5 10 15 20 1 2 3 4 5 c h lo ro p h y l c o n te n t (m g /g le a f) treatments chlorophyl a chlorophyl b a,b a, b b a a, ba a a a a a, b a,b b a a,b 0 10 20 30 40 50 1 2 3 4 5 d ri e d b io m a ss ( g ) treatments total biomass stem biomass root biomass 0 5 10 15 20 25 1 2 3 4 5 d ri e d b io m a ss ( g ) treatments total biomass stem biomass root biomass 461 266 483 326 407 209 278 143 126 138 0 100 200 300 400 500 600 1 2 3 4 5 n u m b e r o f g ra in s treatments filled grains empty grains a b b b b a a a a a 0 50 100 150 200 1 2 3 4 5 n u m b e r o f g ra in s treatments filled grains empty grains siswanti et al. – physiological response of 'segreng' rice plant (oryza sativa l.) … 25 figure 7. the average of grains mass in (a) rice field and (b) polybags. figure 8. nitrate reductase analysis levels of sample in (a) rice field and (b) polybags. discussion the rice cultivation system conducted in this research is the legowo jajar planting system or commonly known as tajarwo. tajarwo system is a planting system with spaces between rows : 25 cm, in rows : 12.5 cm, and between legowo/groups of rows : 50 cm. the spacing is arranged by narrowing the distance of the rice inside the row and widening the distance between the rows. tajarwo's unique principle is the arrangement of rice plants that affects those at the outer sides. generally, the plants grown at the outer sides provide better growth than plants in the middle because of the reduced interrow plant competition (pahruddin et al., 2004). tajarwo was chosen because it has several advantages, i.e. sunlight can be absorbed by the stem better so that the rate of photosynthesis is higher, fertilization and crop pest control are easier to be done because there is a wide distance (legowo) between rows. in addition, the tajarwo system can also increase the population of plants per hectare (pahruddin et al., 2004). a previous research by anggraini et al. (2013) also shows that the productivity of rice grown by tajarwo planting system is better than by conventional/tile system. the tajarwo system has the advantage of having wide open space between two groups of crop rows, increasing the intensity of sunlight entering the clumps, increasing rice yield to 10-15%, facilitating plant maintenance, and reducing the risk of pests and diseases (abdulrachman et al. 2013). the height of segreng rice crops tend to increase every week. the height drop of rice plants with treatment of 24 ml / 5kg at week 4 was possibly due to the pest attack or the torn out leaves at several centimeters from the top. from table 2 on environmental parameters data, it can be seen that there was a considerable decrease in air humidity, it can also indicate a decrease in the height of rice plants. figure 2 is given to clarify the observations for plant height. based on the observations, the highest plant height was found in rice plants grown in 4 ml sludge / 5kg of rice field soil. based on dmrt test (α = 0,05), the effect of biogas sludge dosage on plant height was not significantly different. this can happen if the rice plants have reach the vegetative phase at the 9th week, because according to vergara (1976), the rice plant at the 4th week until before the 7th week is actively propagating to produce seedlings and after the 7th week, the plant is ready to enter the reproductive phase by initiating the formation of panicles. after going through the planting period, the rice plants at week 7 have entered the reproductive phase. the rice plant has passed through a phase where the cells of active plants divide and enlarge. of the five treatments, the best biogas sludge concentration for the vegetative parameters of segreng rice was p2 (1 l/100 m2). based on anova test result, the result of treatment 2 (p2) was significantly different to treatment 5 (p5). 9 4 9 6 7 1 2 1 0 1 -2 0 2 4 6 8 10 12 1 2 3 4 5 g ra in s m a ss ( g ) treatments filled grains empty grains 0.5 2.27 1.98 2.28 2.34 0.15 0.31 0.2 0.24 0.3 0 0.5 1 1.5 2 2.5 3 1 2 3 4 5 g ra in s m a ss ( g ) treatments filled grains empty grains a a a a a 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 2 3 4 5 n r a l e v e ls treatments 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 a b c d e n r a l e v e ls treatments 26 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 meanwhile, the number of leaves were obtained on the 9th week. the treatment that gave the highest total number of leaves was treatment 2 (p2) that was 1 l/100 m2 or 100 l/ha. based on anova test result, it was found that treatment 2 (p2) was significantly different to treatment 5 (p5). this is because at that concentration, the n and mg content is optimally absorbed by the rice plant for chlorophyll synthesis, to obtain more leaf number than other treatments. plant growth is affected by various factors, both internal factors and external factors. internal factors derived from the seeds of rice itself (e.g. genetic and seed conditions), while external factors derived from the surrounding environment around the rice which certainly has an effect on rice growth. each place has a different environment, be it air temperature, water temperature, soil moisture, soil ph, other soil conditions, pests and diseases, and rainfall. the existence of these variations of environmental conditions certainly result in the different growth and development of rice among regions, although given the same treatment. conclusion conclusion the biogas sludge could increase the vegetative and generative growth of ‘segreng’ rice plant on rice field or polybags in wukirsari village, cangkringan, sleman. based on the dmrt analysis with one way anova, the treatment 2 viz 1 liter/100 m2 or 4 ml/polybag gave the best result on vegetative growth and chlorophyll content of the ‘segreng’ rice plant. meanwhile, treatment 3 viz 1,5 liters/100 m2 or 8 ml/polybag gave the best result for generative growth and nra levels of the ‘segreng’ rice plant. suggestion based on the research that has been done, there are some suggestions to support the next research of organic farming, such as we need to increase the biogas sludge concentration and need to add the treatment range to get significantly differences by one way anova analyze. references abdulrachman, s., m. j. mejaya, n. agustiani, i. gunawan, p. sasmita, a. guswara. 2013. legowo cultivation system. badan penelitian dan pengembangan pertanian kementerian pertanian. subang. p: 8 alavan, ade, rita hayat, dan erita hayati. 2015. pengarhun pemupukan terhadap pertumbuhan beberapa varietas padi gogo (oryza sativa l.). jurnal floratek 10: 61-68. badan pusat statistik indonesia. 2016. rata-rata konsumsi per kapita seminggu beberapa macam makanan penting. https://www.bps.go.id/linktabelstatis/view/id/950. accessed on 1st april 2016. ginting. 2007. teknologi pengolahan limbah peternakan. fakultas pertanian universitas sumatra utara. medan. hartiko, h. 1983. leaf and root in vivo nitrate reductase activities of coconut (cocos nucifera l.) cultivars and hybrids. ph.d dissertation of university of the phillipines. los banos, p: 132 pahruddin, a., maripul dan p. rido. 2004. cara tanam padi sistem legowo mendukung usaha tani di desa bojong, cikembar sukabumi. buletin teknik pertanian, 9(1): 10-12 redono, cucuk. 2016. respon petani terhadap penggunaan pupuk organik pada tanaman padi sawah di kelurahan bokoharjo kecamatan prambanan kabupaten sleman. jurnal agrica ekstensia. vol. 10 no.1st, june 2016. p. 29 rpjm pemerintah desa wukirsari. 2014. rencana pembangunan jangka menengah desa (rpjmdes) wukirsari, cangkringan, sleman. unpublished. siswanti, d.u. 2015. pertanian organik terpadu di desa wukirsari, sleman, yogyakarta sebagai usaha pemulihan kesuburan lahan terimbas erupsi merapi 2010 dan pencapaian desa mandiri sejahtera. indonesian journal of community engagement. vol. 01 no. 01, september 2015. steenis, dr. c.g.g.j van. 1988. flora: untuk sekolah indonesia. pt. pradnya paramita. jakarta. p. 127. sutejo, m.m. 1995. pupuk dan cara pemupukan. jakarta: rineka cipta. suzuki, k, takesi w and volum.2001. concentration and critalization of phosphate, ammonium and mineral in the effluent of biogas digester in the mekong delta. jerrrean and contho university vietnam. vietnam. p.3 physiological response of 'segreng' rice plant (oryza sativa l.) to biogas sludge at wukirsari village, cangkringan, sleman biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 5-13 | doi: 10.14421/biomedich.2018.71.5-13 issn 2540-9328 (online) stability of t-dna integration in phalaenopsis “sogo vivien” transgenic orchid carrying 35s::gal4::atrkd4::gr endang semiarti1,*, exsyupransia mursyanti2, ahmad suyoko1, faiza senja widya perdana1, catharina tri widyastuti1, aditya nur subchan1 1faculty of biology, universitas gadjah mada, jl. teknika selatan, sekip utara, yogyakarta 55281, indonesia. 2faculty of technobiology, universitas atma jaya, jl. babarsari, yogyakarta, indonesia author correspondency*: endsemi@ugm.ac.id abstract orchid is an elegant ornamental plant and favoured by the society. phalaenopsis "sogo vivien" is a mini-sized orchid with an interesting white-striped purple petals. this study was aimed to analyze the stability of the integration of embryonic gene carrier t-dna from arabidobsis atrkd4 into the p. "sogo vivien" genome produced in 2016. the study was conducted in 3 stages: 1) transgenic plant phenotype analysis (1 year old); 2) examination of t-dna integration in orchid genotypes using pcr. 3) analysis of transgenic plant leaf explants’ ability to produce somatic embryo in vitro. in vitro cultures were performed on the base medium of new phalaenopsis (np), plus various concentrations of tdz (0, 1, 2 mg.l-1) and iba (0, 1, 2 mg.l-1) or without tdz and iba as controls. the transgenic phalaenopsis "sogo vivien" were transferred to pot mediums via ex vitro with two treatments: the first leaves were cut as explants for in vitro culture, and the plants were transferred to the mixture of fern medium with bark shavings. the integration of t-dna in the genome was detected by dna genome amplification from the second leaves using the atrkd4 gene primers and the poh1 gene. the results showed that the highest number of somatic embryo (se) propagules or protocorm like bodies (plbs) amounted to 27 were derived from transgenic plant # 2 cultured on np + 2 mg.l-1 tdz +1 mg.l-1 iba medium. the presence of atrkd4 transgenes were detected with the amplification of 380 bp of the rkd4 gene from the genome of transgenic plant # 2 by using pcr. there were 2 out of 15 plants that positively carry the atrkd4 gene and produce se. thus, the stability of the atrkd4 carrier t-dna integration in the genomes of transgenic plants was 13.3%. keywords: phalaenopsis “sogo vivien”; atrkd4; somatic embryo; stable transgenics introduction indonesia is home of tropical orchids, more than 5000 of 30,000 species of orchids in the world exist in indonesia. orchids are much favored by the society because of their beauty and attractive appearances. one of the popular ornamental orchids is phalaenospsis "sogo vivien" that has a beautiful, unique, and long lasting blooming period of about 3 months (mckinley, 2005). p. "sogo vivien" has minito medium-sized flowers with purple as the main colour of its petals. one uniqueness of this orchid is the genetic mutation that causes the colour of its leaves’ borders white, called as variegata. however, the percentage of mutation frequencies for the formation of this phenotype is only about 0.007% (fig. 1, mursyanti et al., 2016). orchid propagation by seed is naturally difficult because most of orchid seeds do not have endosperm, thus it requires symbiosis and have a low seed survival percentage. in vitro culture is the best solution for the cultivation of orchid plants. in vitro propagation can be performed using seed parts or other parts of plants such as leaves, roots and internodes of flower stems (vendrame et al., 2007). in a previous study, 17 t-1 plantlets from 2648 protocorms were produced through the formation of somatic embryos, which later developed into 413 t-2 transgenic plantlets (mursyanti et al., 2015). however, the phenotypes resulting from the genetic transformation are still variable and unstable because of the possibility of genetic segregation from crossover results. to see the stability of transgenes integration in p. "sogo vivien", a molecular analysis is required to see the stability of the gene. in this study, the object of our research is confirming the stability of 35s :: atrkd4 integrationin the transgenic plant genome originated from t-2 generation and its ability to induce somatic embryogenesis in vitro through the overexpression of atrkd4 gene in the transformant plants. figure 1. the morphology of phalaenopsis ‘sogo vivien’. (a) normal plant with green leaves; (b) mutated plant with white borders on the leaves. bar: 1 cm (mursyanti et al., 2016). https://doi.org/10.14421/biomedich.2018.71.5-13 6 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 mursyanti et al. (2015) has successfully produced transgenic plantlets that has been transfered with 35s:: atrkd4 gene. through the insertion of atrkd4 gene, it was expected to increase germination rate due to the rwp-rk motif causes the atrkd4 gene to work in the very early stage of embryogenesis (waki et al., 2011). regarding to the potential of the transformed plants, it is necessary to do propagation efforts to maintain the plant’s phenotypes. this research was conducted to acquire p. "sogo vivien" transgenic plants which carry 35s :: atrkd4 containingt-dna; to understand the growth of the transgenic p. "sogo vivien" and the nontransgenic plants, to obtain stable transgenic plants of phalaenopsis "sogo vivien" that carry 35s :: atrkd4 containingt-dna for induction of somatic embryogenesis; and to determine the most effective medium for induction of somatic embryogenesis using the leaf explants from phalaenopsis “sogo vivien” that carry the atrkd4 gene. the transgenic orchid plant p. "sogo vivien" can be used for the development of somatic embryos using the whole plant to meet the market demand. materials and methods plant materials and culture conditions the one year old transgenic plants p. "sogo vivien" carrying 35s :: gal4 :: atrkd4 :: gr that obtained from the previous research at the laboratory of biotechnology, faculty of biology ugm were used as a source of explants for in vitro induction of ses (plbs). plantlets were overplanted into a hormone-free np medium for refreshment. cultures were maintained under continuous white light, at 25-28° celcius. isolation of genomic dna genes and detection of tdna integration into the genomes of transgenic plants plant genomic dna was isolated using a method as described by semiarti et al. (2007), and the isolated dna genome of transgenic plants were purified using the dna kit dna mytaqtm hs following procedure in the instruction manual described by bioline (uk). based on the structure oft-dna (figure 2), detection of t-dna integration was performed by amplifying atrkd4 gene fragments with specific primers for atrkd4: atrkd4 f1 and atrkd4 r1, that a mplified 380 bp dna fragments. the primer sequences would be served upon request. in vitro induction of somatic embryos from leaf explants of transgenic plants the first leaves of the transgenic plants p. "sogo vivien" were cut into two parts in the middle, the distal and proximal parts of leaf were used as explants. the explants were introduced into np medium with addition of various concentration of thidiazuron (tdz) growth regulator (0, 1, 2 mg.l-1) and indole butyric acid (iba) (0, 1, 2 mg.l-1). cultures were maintained with continuous white light at 25-28ºc. the growth of explants were observed until the propagules were formed, which then developed into se or called as protocorm-like body (plb), the term of ‘protocorm’ is a special term for the development of spherical orchid embryos. the calculated se and the stability of t-dna integration in the orchid genome were indicated by the detection/amplification of the atrkd4 gene from the genomic dna of the transgenic plants, indicating stable plant genomes that carry 35s :: gal4 :: atrkd4 :: gr containing t-dna (figure 2). figure 2. the structure of t-dna carrying 35s :: gal4 :: atrkd4 :: gr (mursyanti et al, 2015). results and discussion results phenotypes of transgenic plants carrying 35s :: gal4 :: atrkd4 :: gr in this study, 15 transgenic plants lines of p. "sogo vivien" which carries 35s :: gal4 :: atrkd4 :: gr were obtained (figure 4 and table1). the phenotypes of the transgenic plants showed smaller size compared to the control of non-transgenic plants with normal plant size (figure 3), especially in leaf length, leaf width, and plant height. semiarti et al. – stable orchid transgenic carrying atrkd4 7 nt#1 nt#2 nt#3 nt#4 nt#5 nt#6 nt#7 nt#8 nt#9 nt#10 nt#11 figure 3. phenotypes of non-transgenic p.”sogo vivien” plants, 8 weeks after transplanted into the pots. bars: 1 cm. t#1 t#2 t#3 t#4 t#5 t#6 t#7 t#8 t#9 t#10 t#11 t#12 t#13 t#14 t#15 figure 4. phenotypes of transgenic p. “sogo vivien” plants that carry 35s::gal4::atrkd4::gr. t = transgenic plant lines # 1# 15; bars: 1cm. 8 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 table 1. the vegetative growth of transgenic p. “sogo vivien” carrying 35s::gal4::atrkd4::gr on 8 weeks after transplanted into pots. scale (cm) plant height first leaf length first leaf width the last leaf length the last leaf width non transformant 2.0±0.5 4.7±0.6 2.3±0.6 4.7±0.8 2.8±0.4 transformant 1.6±0.59 2.4±0.72 1.32±0.42 2.2±0.32 1.2±0.22 as shown in table 1, transgenic plants produced higher number of leaves and roots compare to that of non-transgenic/normal plants. the detection of t-dna integration into the orchid genome using pcr analysis showed that 380 bp of atrkd4 dna fragments were detected from the genomic dna of transgenic plant line # 1 and line # 2, thus it proved that the t-dna is stably integrated in the genomes of orchid transgenic (figure 6). table 2. detection of atrkd4 transgene in the genome of transgenic orchid. no. pcr detection somatic embryos of atrkd4 poh1 tdz:iba (2:1) nt#1 0 t#1 + 0 t#2 ++ 27 t#3 t#4 ++ notes: iba= indol butyric acid (ppm), tdz= thiadizuron (ppm), nt: non transgenic, t#1-t#4: transgenic plant lines#1line #4 table 3. percentage of living explants, somatic embryos and the colour change of in vitro transgenic t#2 orchid p. “sogo vivien” carrying atrkd4 gene in various media with the combination of tdz and iba, 4 weeks after plantation. treatment media ∑ explants observed ∑ and the percentage of living explants (%) ∑ and the percentage of explants forming somatic embryo candidates (%) ∑ explants changing colours green yellow brown np +t0i0 9 5 (55.6 %) 4 (44.4 %) 6 1 2 np+t0i1 9 5 (55.6 %) 1 (11.1 %) 5 1 3 np+t0i2 9 8 (88.9 %) 0 (0 %) 8 1 0 np+t1i0 9 0 (0 %) 0 (0 %) 0 9 0 np+t1i1 9 1 (11.1 %) 0 (0 %) 1 3 5 np+t1i2 9 1 (11.1 %) 0 (0 %) 1 2 6 np+t2i0 9 4 (44.4 %) 0 (0 %) 4 1 4 np+t2i1 9 8 (88.9 %) 6 (66.7 %) 8 0 1 np+t2i2 9 5 (55.6 %) 0 (0 %) 5 0 4 table 4. effect of growth regulator on the speed of somatic embryogenesis induction 4 weeks after explant plantation. plants time of somatic embryogenesis induction (day) media np+t0i0 np+t0i1 np+t0i2 np+t1i0 np+t1i1 np+t1i2 np+t2i0 np+t2i1 np+t2i1 t#1 7 21 0 0 0 0 0 14 0 t#2 7 0 0 0 0 0 0 3.5 0 nt1 0 0 0 0 0 0 0 0 0 notes: np= new phalaenopsis medium, t= thidiazuron, i = indol butyric acid nt: non transfromant, t#1,t#2: transfromants semiarti et al. – stable orchid transgenic carrying atrkd4 9 table 5. propagule formation on the leaf explants from non transgenic and transgenic p. “sogo vivien”, 4 weeks after explant plantation on media containing tdz and iba. average number of propagule formation on explants iba tdz 0 ppm 1 ppm 2 ppm numbers morphology numbers morphology numbers morphology 0 ppm nt1 0 0 0 t1 20.5 0 0 t2 2 0 0 1 ppm nt1 0 0 3.4 t1 3 0 11 t2 0 0 27 2 ppm nt1 0 0 0 t1 0 0 0 t2 0 0 0 notes: iba= indole butyric acid (ppm), tdz= thidiazuron (ppm), nt: non transfromant, t1, t2: transformant, bars= 5 mm. 10 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 figure 5. the process of somatic embryogenesis in the surface of leaf explants from transgenic orchid p. "sogo vivien" carrying t-dna 35s :: gal4 :: atrkd4 :: gr. (a) the formation of somatic pre-embryo/ plb (arrow) on the part of the cuts 1 week after culturing on np 0. (b) formation of somatic pre-embryo (arrow) 4 weeks after culturing on np 0. (c, d) formation of somatic pre-embryo (arrow) 1 week after culturing on tdz 2 ppm and iba 1 ppm. (e) formation of somatic pre-embryo (arrow) 4 weeks after culturing on tdz medium 2 ppm and iba 1 ppm, the colour turned into yellowish green. (bars: 1 mm). m nt#1 nt#2 nt#3 t#1 t#2 t#3 t#4 figure 6. electrophoregraph of genomic dna (whole genome dna) from the leaf of phalaenopsis “sogo vivien”. non transformants(nt#1, nt#2, nt#3) and transformant with t-dna containing 35s::atrkd4 (t#1, t#2, t#3, t#4) and λ dna/hindiii as dna markers (m). . figure 7. electrophoregraph of amplified dna by using pcr of genome dna of p. “sogo vivien”, both non-transformantand transformant carrying 35s::gal4::atrkd4::gr. the amplified dna from non transformant plants (nt#1, nt#2, nt#3) and trasnformant plants (t#1, t#2, t#3, t#4); marker (m): 100 bp dna ladder; amplification of genome dna using poh1 primer produced 300 bp sized band; degrkd4 primer produced 380 bp sized band of dna fragments. 23130 bp 9416 bp 4361 bp 6557 bp 2322 bp 2027 bp semiarti et al. – stable orchid transgenic carrying atrkd4 11 discussion the characteristics of vegetative growth in plant that are inserted by foreign genes (transgenic) and normal/nontransgenic plants may be the same or different depending on the expression level of the inserted gene. in most transgenic plants, the vegetative growth character is similar to that of normal plants. more than 100 apple cultivars of the royal gala carrying the uida, als, and nptiii genes showed no difference at the morphological level, growth type, or response to environmental factors compared to non-transgenic plants (bajaj, 2012: 242). in transgenic soybean plants carrying the atpg7 gene, plant height is the same or more than non-transgenic plants (kim et al., 2017: 237-242). however, this depends on the character of the genes inserted. for example, the broccoli plant (brassica oleracea subsp. italica) that carries the heat-tolerant gene, athsp101 is slightly shorter than nontransgenic plants as the effect of position (ravanfar et al., 2013: 14). the gene insertion site has an effect on gene expression when its location on the chromosome changes, and can cause silencing genes as well as dna regulation. the atrkd4 gene is a gene that regulates gamet cell differentiation processes (koi et al., 2016: 1775-1781) and can induce somatic embryogenesis in p. "sogo vivien" (mursyanti et al., 2015: 26-37). this study was aimed to study the vegetative growth character of p. "sogo vivien" that carries atrkd4 gene compared to normal orchid plants. in general, there was no difference in the number of roots. plant height differed from first week to seventh week but itswere no different at the eight week. this indicated that the growth of transgenic plant stems and normal plants become stable from the eighth week. the length of the basal leaf did not differ in the first week and the second week but were different until the eighth week. the same phenomenon happened on the basal leaf width. it suggests that there is a slightly difference of growth in basal leaves between transgenic and normal plants. the length of terminal leaf did not differ from the first week to the third week, but it began to be differed after the eighth week. the width of the terminal leaf did not differ in the first and second weeks and began to be differed from the third week to the eighth week. the growth difference between the terminal leaf of transgenic and normal plantsstarted from the third week. vegetative growth of transformantplants was slower than that of non-transformant plants that proved by anova test for some growth parameters including plant height, basal leaf length and width, and terminal leaf length and width. based on the anova test, differences in the length and width of the first leaf and the length and width of the last leaf were detected. the observation results on the number and percentage of live explants (table 2) showed that, from the whole treatments, only the tdz:iba (1: 0) media was not fit for survival, whereby all of the leaves had chlorosis. the different life-force responses of the explants may be due to the adaptability of the nonuniform explants and the physiological conditions of the explant source. during four weeks of planting, the explants changed colour to yellowish brown. table 2 showed yellowish discoloration in all treatment mediums except tdz: iba (2: 1) and tdz: iba (2: 2), whereas for brown colour change occured in all treatment mediums except tdz: iba 0: 2) and tdz: iba (1: 0). change of explants colour into yellow was caused by the occurrence of chlorosis. the phenomenon of chlorosis is resulted from the decreasing chlorophyll content in the explant. the decrease of chlorophyll content on media may be caused by the relativily low explants’ ability to utilize iron micronutients. in a previous study, during in vitro culture of leaf explants, the chlorophyll content value was linear with iron concentration in culture medium (sivanesan et al., 2008: 4482-4490). some explants in some media changed its colour into brown. this indicated the occurence of browning symptoms of leaf explants. the process of browning might due to the oxidation of polyphenol compounds and the formation of quinon compounds. both of these compounds are inhibited the growth of leaf tissue explants (sukumar et al., 2008: 361). the emergence of the oxidation process is activated by oxidase enzyme after injury, the enzyme may leave the cell and possibly create bonds between hydrogen and proteins, increasing the activity of phenylalanine ammonia lyase, an enzyme that promotes the production of phenylpropanoid, resulting in the brownish color of the leaves (hutami, 2008: 8388). of the total media with variation of hormones/growth regulators, there were 3 media treatments that successfully performed somatic embryogenesis induction (table 2.). in induction media, 4 explants (np 0), 1 explant (tdz: iba; 0: 1) and 6 explants (tdz: iba; 2: 1) were able to form candidates of somatic embryo. compared with hormon-free media (44.4%), media with a combination of tdz: iba (2:1) resulted in a larger percentage (66.7%). these results support the results of previous studies, which suggested that the combination of exogenous auxin and cytokinin hormones can effectively induce direct somatic embryogenesis on phalaenopsis orchid leaf explants (feng and chen, 2014: 4). the velocity induction of somatic embryogenesis processes varies (tables 3 and 4.). transgenic explants were successfully induced whereas non-transgenic explants did not undergo somatic embryogenesis formation. transgenic plant t#2 took 7 days in np 0medium, and 3.5 days in tdz: iba (2:1) containing medium to form somatic embryos. meanwhile, t#1 plant took 7 days in np 0medium, 21 days in tdz: iba (0: 1) containing medium and 14 days in tdz; iba (2: 1) containing medium. the shortest time for se induction occured on the transgenic planlet leaf explant in medium with tdz: iba (2: 1), which required a mean time of 3.5 days, twice faster than the 12 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 se induction in the control medium. the induction velocity was relatively faster compared to previous studies (kasi and semiarti, 2016: 31-40) using a wildtype p. "sogo vivien" which took 8 weeks after inoculation to form somatic embryos. table 5 showed that the results of somatic embryogenesis induction can be known directly through the observation of average propagule appearance on leaf explants, at 4 weeks after planting. the propagules produced from this induction process will develop into protocorm-like bodies (plb), an orchid embryo derived from somatic cells, forming a swollen tuber, which then potentially form in vitro shoots (kumar et al., 2008: 718). the propagules appeared in transgenic leaf explants t#1 and t#2, but not observed in nontransformantleaf explants. t#1 transgenic leaf explants produced a mean of 20.5 propagules in np 0 medium, 3 pieces in tdz : iba (0: 1) medium, whereas the explants of t#2 transgenic leaves produced 2 propagules in np 0 medium and 27 propagules in tdz: iba (2: 1) medium. the results of this study showed that the transgenic leaf that carries atrkd4 with a 35s promoter was able to undergo somatic embryogenesis process directly on the medium without hormon and produce more embryos on tdz: iba (2:1) medium. the tdz growth regulator requirement is relatively lower, which is ≤2 ppm, compared to a previous study (kasi and semiarti, 2016: 31-40) where wildtype p. "sogo vivien" required tdz concentration of 10 ppm for induction of somatic embryogenesis from leaves in vitro. the results of in vitro inoculation of leaf explants of transgenic plants showed faster in induction speed of somatic embryogenesis (table 4), with lower tdz concentration requirement (table 3, table 4) and higher capability to form propagules in np hormon-free medium table 3, figure 5). this is in line with our previous research which showed that high activity of atrkd gene can induce somatic embryogenesis process on leaf explants (mursyanti et al., 2016: 45-53). based on all the results obtained, it was shown that the media with a combination of tdz: iba produced the highest number of propagules. these results suggest that the presence of exogenous hormones may support somatic embryogenesis induction in orchid. molecular analysis showed that the isolated dna fragments from both non-transformant and transformant plants (figure 6) indicated good quality of genomic dna with no smear appearance in the electrophoresis gel. the genomic dna amplification results with atrkd4 specific primers and degrkd4 degenerate primers indicated that the atrkd4 transgene has been integrated into genomes of p. "sogo vivien" plant and the stably integrated in the orchid plant genome. figure 7 showed that a 300 bp dna fragment was amplified from t#1 genome using specific primers of poh1 gene and 380 bp dna fragment were amplified from t#2 and t#4 genomes using primers of atrkd4 gene. its indicates that the atrkd4 transgene has only stably integrated in transgenic plant t#2 and t#4, finally, we got the two stable transgenic lines t#2 and t#4, which are being acclimatize in the greenhouse and maintain for the next generation. conclusion integration of the t-dna carrying 35s::gal4::atrkd4::gr in the genome of transgenic plant phalaenopsis "sogo vivien" can be detected in 2 of 15 strains, thus it means the stability of t-dna integration is 13.3%. the morphology of transgenic plants did not change significantly in root quantities compared with non-transformant plants, but experienced significant differences in plant height, length, basal and terminal leaf width and length compared with nontransformant plants. the development of somatic preembryogenesis occurred after 4 weeks of culture characterized by the appearance of propagules on the surface of plant leaf explants. acknowledgements this research was funded by a research grant hibah penelitian biodiversitas tropika dosen untuk pengembangan materi pembelajaran bpptnbh from the faculty of biology, universitas gadjah mada on 2017 with contract number: ugm/bi/1734/um/05/01. references bajaj, y. p. s. 2012. biotechnology in agriculture and forestry 47: transgenic crops ii. springer. new delhi. p. 242. feng j and cheng j.2014. ‘a novel in vitro protocol for inducing direct somatic embryogenesis in phalaenopsis aphrodite without taking explants’. the scientific world journal. vol. 2014, article id 263642, 7 pages, 2014. doi:10.1155/2014/263642. hutami, s.2008. ‘masalah pencoklatan pada kultur jaringan.jurnal agrobiogen. 4(2).83-88 kasi, p. d. dan semiarti e.2008.’pengaruh thiadizuron dan naphtalene acetic acid untuk induksi embriogenesis somatik dari daun anggrek phalaenopsis “sogo vivien”. jurnal dinamika. 7(1):31-40. kim, h. j. cho, h. s. pak, j. h. kim, k. j. lee, d. h. and chung, y. s. 2017. overexpression of a chromatin architecturecontrolling atpg7 has positive effect on yield components in transgenic soybean. plant breed. biotech. 5(3):237-242. kumar a, neumann k and sopory s k. 2008. recent advances in plant biotechnology and its applications.i.k. international. new delhi. p. 718. koi, s. hisanaga, t. sato, k. shimamura, m. yamato, k. t. ishizaki, k. kohchi, y. and nakajima, k. 2016. an evolutionary conserved plant rkd factor controls germ cell differentiation. curr. biol. 26(13): 1775-1781. semiarti et al. – stable orchid transgenic carrying atrkd4 13 mckinley, m. 2005. complete guide to orchids: ortho books. american orchid society. usa. p.137. mursyanti, e., aziz-purwantoro, s. moeljopawiro and e. semiarti. 2015. induction of somatic embryogenesis through overexpression of atrkd4 genes in phalaenopsis ‘sogo vivien’. indonesian journal of biotechnology. 20(1): 26-37. mursyanti, e., aziz-purwantoro, s. moeljopawiro and e. semiarti. 2016. micropropagation of mini orchid hybrid phalaenopsis ‘sogo vivien’, journal of tropical biodiversity and biotechnology. 1(1): 45-53. ravanfar, s. a. aziz, m. a. shabanimofrad, m. and samarfard, s. 2013. greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (issr) markers. electronic journal of biotechnology. 16(5): 1-4. semiarti e., indrianto a., purwantoro a., isminingsih s., suseno, n., ishikawa t., yoshioka y., machida y.,and machida c. 2007. agrobacterium-mediated transformation of the wild orchid species phlaenopsis amabilis. plant biotechnology 24: 265-272. sivanesan i, hwang sj and jeong b r.2008. ‘influence of plant growth regulators on axillary shoot multiplication and iron source on growth of scrophularia takesimensis nakaia rare enemic medicinal plant’.african journal of biotechnology. 7(24):4484-4490. sukumar e, essa m m and manickavasagan a. 2012. dates: production, processing, food and medicinal values. crc press. new york. p. 361. vendrame, w. a. maguire, i. and carvalho, v. s. 2007. in vitro propagation and plantlet regeneration from doritaenopsis purple gem ‘ching hua’ flower explants. hort. science. 42:1256-1258. waki, t., t. hiki, r. watanabe, t. hashimoto & k. nakajima. 2011. the arabidopsis rwp-rk protein rkd4 triggers gene expression and pattern formation in early embryogenesis. current biology. 21(15): 1277-1281. this page intentionally left blank stability of t-dna integration in phalaenopsis “sogo vivien” transgenic orchid carrying 35s::gal4::atrkd4::gr biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 9-12 | doi: 10.14421/biomedich.2017.61.9-12 issn 2540-9328 (online) comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani1, muhammad jafar luthfi2 1center for integrative zoology; 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: nurulsafitri89@gmail.com1 abstract black pomfret and nile tilapia are belonging to order perciformes. both fish are live in different habitat. black pomfret is marine water whereas nile tilapia is in freshwater. the purpose of the study was to determine anatomy and histology of the kidneys structures in both fishes. histological preparations were done using paraffin method, with hematoxylin-eosin (he) staining. the results, showed that black pomfret and nile tilapia have y-shape kidney. nile tilapia has darker red colour and softer texture than black pomfret kidney. histologically, black pomfret and nile tilapia kidneys have a distal tubule, proximal tubule, glomerulus and lymphoid tissue. glomerular diameter of nile tilapia (69,22 µm) was larger than pomfret (61,25 µm). it can be concluded that differences between anatomical and histological structure of kidney are affected by habitat differences. keywords: anatomy; black pomfret (formio niger); histology; kidney; nile tilapia (oreochromis niloticus). introduction fishes (pisces) are members of subphylum vertebrates that have species number more than 27,000 species in the world. fish has the most diversity in terms of morphology (bond, 1979). there are also anatomical and histological variations which is manifest in its form and function. this could be caused by different habitats, even in fish in the same order, such as black pomfret (formio niger) and nile tilapia (oreochromis niloticus). black pomfret and nile tilapia are belonging to the order perciformes. these fish live in different habitats. black pomfret (formio niger) is one of many fishes species found in marine areas in indonesia, especially in the southern sea. nile tilapia is a freshwater fish. it can be found in lakes, river, and water reservoirs (suyanto, 1994; djarijah, 1995; taufik et al., 2002). osmoregulation is the capacity of aquatic animals to control the water and ion balance between the body and its environment, or a process of osmoregulation (nicol, 1967; fujaya, 2004). the organ that has an important role in osmoregulation process in the body is kidney (karl, 1962). kidney is an excretory organ that has an important role in the filtration and excretion process. the fish of kidney absorb some salt and release the salt into the bloodstream. the fish kidney also has a pump that will take salt from water and release ammonia and other waste products (karl, 1962). the function of each parts of the kidneys that involved in the osmoregulation process will show some differences between the renal fish structure that living in marine or freshwater. comparative anatomical study was conducted to determine the differences anatomical and histological renal organ of black pomfret and tilapia in the same order level. the comparisons including the different in histological and anatomical structures of renal fish organ that may affected by the physiological factors of the fishes due to habitat difference. materials and methods in this study, we used three adults black pomfret (formio niger), obtained from pangandaran beach, west java and three adults nile tilapia (oreochromis niloticus) from nila cultivation center, cangkringan yogyakarta. anatomical observation were done by observing the topography, colour, texture, shape and length of the kidney. histological observation were done on histological slides stained with using hematoxylin-eosin (he). histological slides were made using paraffin method. results and discussion black pomfret (formio niger) and nile tilapia (oreochromis niloticus) (figure 1 & figure 2) have similarities as common perciformes fish, which have dorsal, caudal, anal, pelvic and pectoral fins. dorsal fin of nile tilapia has a spiny fin or a hard fin called spinal fin. pomfret and tilapia have difference in its caudal fin shape. pomfret has a fork-shaped, whereas tilapia fish http://dx.doi.org/10.14421/biomedich.2017.61.9-12 10 biology, medicine, & natural product chemistry 6 (1), 2017: 9-12 caudal fin is rounded shaped. mouth of pomfret has a terminal type, whereas in tilapia fish is sub-terminal and tapered shaped. pomfret body is slim, with the dorsal and ventral body is strong and equally convex, whereas tilapia body is flat-shaped toward the vertical (compressed). scales on both fish also have differences. black pomfret has small size, tight scales covering the dorsal and ventral parts of the body, whereas tilapia has large, rough and well-scales (kottelat et al., 1993). figure 1. morphology of black pomfret (formio niger). (a). dorsal; (b). caudal; (c). anal; (d). pelvic; (e). pectoral. figure 2. morphology of nile tilapia (oreochromis niloticus). (a). dorsal; (b). caudal; (c). anal; (d). pelvic; (e). pectoral. kidney anatomy of adult black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney of the pomfret has a longitudinal shape along the abdominal cavity (figure 3). similar to lamprey, marine kidney generally elongated along the underside of vertebrae (spine) and slightly lobuled (karl, 1962; ferguson, 1989 in damayanti, 2010). in pomfret and tilapia fish the kidney and reproductive organ are located adjacent to the top of the celoem, and are divided into two specific system aisles (kent, 1987). figure 3. topography of black pomfret (formio niger) kidney. (x). kidney, (a). original topography of pomfret kidney, (b). sketch topography of pomfret kidney. figure 4. topography of tilapia (oreochromis niloticus) kidney. (x). kidney, (a). original topography tilapia kidney, (b). sketch topography of tilapia kidney. apriliani & luthfi – comparative anatomy and histology of black pomfret (formio niger) … 11 the result of the research showed that kidney black pomfret and nile tilapia have a similarity to kidney of perciformes fish in general, which is y-shaped form. fish kidneys generally have different shapes to each class. type of kidney and pomfret's configuration kidney is included in the same type of yellow-tail fish, namely y-shape. figure 5. anatomy of adult black pomfret (formio niger) kidney, elongated shape and dark red colour. (a). head kidney (anterior); (b). body kidney (transition); (c). tail kidney (posterior). figure 6. anatomy of adult nile tilapia (oreochromis niloticus) kidney, elongated and bright red colour. (a). head kidney (anterior); (b). body kidney (transition); (c). tail kidney (posterior). black pomfret kidney has a deep red color and has a soft kidney texture (karl, 1962). meanwhile, the tilapia kidney has a slightly softer texture than the pomfret's kidney, and has bright red-colored, compared to darker pomfret kidney. the differences in color and texture of the kidney are affected by pigment, number of capillary and connective tissue. histology of adult black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney fish kidney has histological structure like the other marine vertebrate kidney, which have glomerulus and tubules (fujaya, 2004). glomerulus plays an important role in the process of excretion. the magnitude of the diameter of the glomerulus and the density of the tubules is related to filtration and filter blood. based on the results of the study, there is a difference between the glomerular diameter of pomfret and tilapia fish. the histology of pomfret and tilapia kidney is presented in figure 7 and figure 8. figure 7. histology of kidney of black pomfret (formio niger). (a). distal tubule; (b). proximal tubule; (c). lymphoid tissue; (d). glomerulus. he staining. (40x10 magnification). figure 8. histology of kidney of nile tilapia (oreochromis niloticus). (a). distal tubule; (b). glomerulus; (c). proximal tubule. he staining. (40x10 magnification). based on histological observations on serial preparations, adult black pomfret (formio niger) kidney has smaller glomerular diameter (61.25 μm) compared to adult tilapia (69.22 μm). this finding is consistent with marshall & smith (1930), which stated that fish living in water with low salinity content having a larger glomerular size than fish living with water with high salinity. karl (1962), also stated that freshwater fish kidney have more glomeruli than fish. based on the result it can be concluded that there is similar kidney anatomy yet different size of glomerular diameter in pomfret and tilapia. this is an adaptive character due to different environmental factors. regarding to the way of osmoregulation process, different habitats greatly affect the structure and function of the kidney (margaret, 1957; karl, 1962). 12 biology, medicine, & natural product chemistry 6 (1), 2017: 9-12 conclusion based on the result of research, we drew conclusion as follows: 1. pomfret and tilapia have the same anatomical kidney structure, consisting of head, body and tail of the kidney. but the tail/posterior kidney pomfret is longer than the body part (transition). the location of the same kidney that is attached to ventral part of vertebrae, but different in the direction of the posterior part of the kidney. the color and texture of the kidneys are different. relatively softer on tilapia than pomfret. the kidneys of tilapia fish have a bright red-red color, while the pomfret's kidney has dark red color. in terms of morphometry, the weight and length of the kidney are relatively larger in tilapia. 2. pomfret and tilapia have the same histological structure of the kidney, which consists of distal tubules, proximal tubules, and glomeruli and lymphoid tissue, but different in glomerular diameter. references bond, c.e. 1979. biology of fishes. philadelphia: w. b. saunders company. damayanti, f. n. 2010. pengaruh pencemaran logam berat terhadap kondisi histologi ikan nila (oreocromis niloticus linn) dalam karamba jaring apung di blok jangari waduk cirata. thesis. universitas padjajaran, jatinangor. djarijah, a. s. 1995. nila merah: pembenihan dan pembesaran secara intensif. kanisius, yogyakarta. ferguson, hugh. 1989. systematic pathology of fish, a test and atlas of comparative tissue responses in diseases of teleosts. iowa state univ. press. ames, iowa. fujaya, yushita. 2004. fisiologi ikan dasar pengembangan teknik perikanan. jakarta: pt rineka cipta. karl l. 1962. ichtyology. john wiley & sons. inc: new york. kent g. c. 1987. comparative anatomy of the vertebrates. 6th ed. times mirror/mosby toronto: college publishing. kottelat m, whitten aj, kartikasari sn, wirjoatmojo s. 1993. freshwater fishes of western indonesia and sulawesi. hong kong: periplus editions. hlm: 344 margaret e. b. 1957. the physiology of fishes: behavior. new york: academic press inc, publisher. nicol, j.a.c, 1967. the biology of marine animals. 2nd ed. new york: wiley-interscience. suyanto. 1994. nila. jakarta: penebar swadaya. taufik, i., s. koesoemadinata, sutrisno, & nugroho. 2002. potensi akumulasi insektisida klorpiricosetil dalam jaringan tubuh ikan nila (oreochromis niloticus). jurnal penelitian perikanan indonesia, 8 (3): 37–44. comparative anatomy and histology of black pomfret (formio niger) 1and nile tilapia (oreochromis niloticus) kidney volume 5 number 1 2016 i s s n 2 0 8 9 6 5 1 4 biology, medicine, & natural product chemistry volume 5 – number 1 – 2016 issn: 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university 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jafar luthfi local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini determination of levels leisure village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural village green and environmentally friendly supriatna, thaqibul fikri niyartama, iwan kuswidi modified alizarin red s-alcian blue staining for reptilian skeleton muhammad jafar luthfi checklist of macroalgae in waisai coast, raja ampat retno suryandari, widodo 1-8 9-14 15-18 19-22 23-32 volume 5 number 1 2016 published twice a year printed in indonesia front cover: tacca palmata (family taccaceae) (photo: widodo) issn 2089-6514 cover jurnal biomenaprochy vol 5 num 1 2016.pdf (p.1) editorial j.biomedich v5 n1.pdf (p.2) guidance for authors j.biomedich.pdf (p.3) cover jurnal biomenaprochy vol 5 num 1 2016 2.pdf (p.4) biology, medicine, & natural product chemistry volume 3 – number 2 – 2014 issn 2089-6514 contents a discovery and characteristics description of telosma puberula (asclepiadoideae) in mount gedang atas and mount ijo, baturagung mountain yogyakarta widodo 47 52 larvicidal activity of a mixtureof cashew nut shell liquid and water-soluble extract of soapnut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati, sudarsono, budi mulyaningsih 53 57 substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cytbofindonesian swamp buffalo (bubalus bubalis) akhmad sukri, mohamad.amin, aris winaya, abdul gofur 59 63 the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a., arief r.h., sudarsono 65 67 biology, medicine, & natural product chemistry volume 4 – number 2 – 2015 issn 2089-6514 contents krokot (portulaca oleracea. l) as a natural sensitizer for tio2 dye-sensitized solar cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi, didik krisdiyanto 25 29 effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja'far luthfi 31 33 antioxidant potential of black, green and oolong tea methanol extracts wahyu widowati, tati herlina, hana ratnawati, gabriella constantia, i dewa gde sathya deva, maesaroh maesaroh 35 39 medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja'far luthfi, mahanem mat noor, jalifah latip 41 47 tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik 49 51 biology, medicine, & natural product chemistry volume 7 – number 2 – 2018 issn 2089-6514 (paper) | issn 2540-9328 (online) contents degradation study of biodegradable plastic using nata de coco as a filler tiara nur elfiana, anisa nur izza fitria, endaruji sedyadi, susy yunita prabawati, irwan nugraha 33 38 comparative anatomy of labyrinth and gill of catfish (clarias gariepinus) (burchell, 1822) and snakehead fish (channa striata) (bloch, 1793) ina karlina, muhammad ja’far luthfi 39 43 diversity of angiospermae plant class liliopsida in mount nglanggeran bayu setya aji nugraha, widodo, rendi yuntara, normalita 45 49 link of nasopharyngeal carcinoma and epstein-barr virus sugiyanto, lina aryati, fajar adi kusumo, mardiah suci hardianti 51 55 alizarin red s-alcian blue staining for regenerated tail of common house gecko (hemidactylus frenatus) rakhmiyati, muhammad ja’far luthfi 57 59 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 41-47 | doi: 10.14421/biomedich.2016.52.41-47 issn 2540-9328 (online) optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono biology laboratory, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: sutriyono@uin-suka.ac.id abstract binocular microscope optimization using micro digital camera to measure seminiferous tubules epithelium of mouse testis (mus muculus) was completed. this research was conducted at biology laboratory of faculty of science and technology uin sunan kalijaga. the purpose of this study was to determine the height of the seminiferous tubules epithelium of mouse testis using micro digital camera. a computer was connected with binocular microscope and optilab advance, then calibration of optilab advance were done on objective lens magnification at 4x, 10x and 40x with 10x ocular lens. the analysis used was descriptive analysis. the mean of the seminiferous tubules epithelium height is 105.6 μm. optimization on computer and binocular microscope with micro digital camera can be used to measure seminiferous tubules epithelium. keywords: seminiferous tubules epithelium; binocular microscope; micro digital camera; mouse testis. introduction binocular microscopes are commonly used to view microscopic objects at magnification of 4x, 10x, 40x, 100x objective lenses and of the 10x ocular lens, resulting in a maximum magnification of 1000x. the microscope can be used to measure the size of organ or cell. measurement of length or height of cells or organs can be done using micrometer slide. after the organ or cell image are taken, the measurement were done using micrometer photomicrograph at the same magnification. we can also measure the size of the cell or organ directly by using a camera in which there is a ruler menu that works to scale or measure cells or organs. there are two shortcuts in optilab, optilab viewer and raster image. inside the optilab viewer there are tools (buttons) for image snippets (photos), video recording (for recording in video form) and successive snippets (for sequential photos). for raster image shortcuts there are facilities for enumerators (counting by marking), scale (which can be dropped freely), there is also a marking facility that can be either arrow, circle, plus sign or box, there is also a ruler button that can be used to measure height, length or diameter of the cell or organ. knowing the height of epithelial tissue in the seminiferous tubules is important as seminiferous tubules can be used as indicators in the process of spermatogenesis. one of the important parameters in histology is the size of organ and cells, for example measuring the diameter of the seminiferous tubule and the height of mouse epithelium tubulus seminiferous. for microscope to be able to measure, it is required additional tools namely micro digital camera that have facilities to measure on a micron scale. therefore, a combination of binocular microscopy with a micro digital camera can be used to measure the height of the seminiferous tubule of epithelial cells. this paper will described the optimization of binocular microscope with micro digital camera for measuring seminiferous tubule epithelium height. materials and methods the equipment and materials used in this study are a set of pc or laptop, binocular microscope, miconos optical advanced digital micro camera, histological slide of testis. the research was conducted in biological laboratory, faculty of science & technology uin sunan kalijaga. this research began with assembling a computer or laptop with a binocular microscope and connecting a digital micro optilab advace camera. before taking measurements, optilab advande needs to be calibrated first, ie at the magnification of the objective lens 4x, 10x and 40x, so that the maximal magnification used is 400x. objective lens calibration process begins by calibrating the smallest magnification of 4x, by clicking the raster image then select the open button then select the micrometer image with 4x magnification, then click the calibration button, the objective lens is worth 4x then select the micrometer image again with 4x magnification then calibration mode 4x flashing, click and drag the micron and input http://dx.doi.org/10.14421/biomedich.2016.52.41-47 42 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 the measuring value then saved. take the same steps for 10x and 40x objective lens magnification. then measurements of epithelial tubular epithelial cells testis and recording could be done. of the many slide preparations, we selected three slide, in each slide selected three slices of testicular organ, in each slice of the testicular organ selected three seminiferous tubules, in each seminiferous tubule an epithelium cell measurement was measured three times. figure 1. a set of microscope and optilab advance. results and discussion appearance of testis before measurement: figure 2. mus musculus testis.10x10 magnification. this study measured the length or height of epithelium cells in the seminiferous tubules. prior to measurement the height of the tubular epithelium seminiferus testis, we select three intact seminiferous tubules. the corresponding cells are marked (figure 3). figure 3. marked tubulus seminiferous to be measured. selected tubulus seminiferuos were measured (figure 4-figure 9). figure 4. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 123,9 µm; 137,7 µm; 88,6 µm, (b). 81,6 µm; 84,8 µm; 89,2 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 43 figure 5. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 91,9 µm; 134,1 µm; 127,3 µm, (b). 114,7 µm; 102,9 µm; 90,9 µm; (c). 117,7 µm; 106,4 µm; 137,2 µm, (d). 142,3 µm; 115,1 µm; 132,4 µm, (e). 97,6 µm; 86,1 µm; 98,3 µm, (f). 132,7 µm; 104,6 µm; 95,3 µm. 44 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 figure 6. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 119,6 µm; 144,8 µm; 101,1 µm, (b). 91,7 µm; 96,2 µm; 107,6 µm, (c). 87,0 µm; 111,9 µm; 150,8 µm, (d). 79,4 µm; 125,1 µm; 122,1 µm; (e). 78,5 µm; 106,2 µm; 93,3 µm, (f). 93,4 µm; 82,5 µm; 124,3 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 45 figure 7. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 94,8 µm; 100,9 µm; 109,7 µm, (b). 132,0 µm; 106,3 µm; 75,6 µm, (c). 134,8 µm; 92,9 µm; 140,5 µm, (d). 78,4 µm; 104,2 µm; 93,2 µm, (e). 103,1 µm; 115,7 µm; 75,8 µm, (f). 159,1 µm; 71,6 µm; 106,3 µm. 46 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 figure 8. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 117,1 µm; 92,2 µm; 89,6 µm, (b). 97,3 µm; 99,4 µm; 113,4 µm, (c). 82,7 µm; 123,0 µm; 91,3 µm, (d). 78,1 µm; 132,5 µm; 81,8 µm, (e). 99,2 µm; 113,0 µm; 49,8 µm, (f). 154,1 µm; 68,2 µm; 120,5 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 47 figure 9. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: 86,3 µm; 139,3 µm; 77,3 µm. the results of this study were data of the height of tubular epithelium seminiferous testis of mice (mus muculus). from the measurement of eighty-one of measurement of the tubulus seminiferous epithelium, the average of the height of the tubulus seminiferous tubule epithelium was 105.6 μm. conclusion the optimization of binoculer microscopy with optilab advance can be done to measure diameter of the tubulus seminiferous. the height of epithelium of tubulus seminiferous in this study was 105.6 μm references alatlabmiconos.co.id retrieved at 05 march 2017 luiz r. franca and christiane l. godinho. 2003. testis morphometry, seminiferous epithelium cycle length, and daily sprem production in domestic cats (felis catus). biology or reproduction. federal university of minas gerais, brazil rizka arifianti. 2013. berat testis dan struktur histologi testis mencit (mus musculus l.) akibat paparan kebisingan. skipsi. universitas lampung. lampung. reza anindita dkk. 2009. diameter dan tebal lapisan tubulus seminiferus serta bobot testis mencit (mus muculus) setelah pemberian tauge kacang hijau (vigna radiata). laboratorium biologi. universitas diponegoro semarang. sutriyono. 2015. optimasi mikroskop binokuler dengan menggunakan optilab advance untuk mengukur diameter organ testis mencit (mus muculus). integrated lab journal. universitas islam negeri sunan kalijaga yogyakarta. tri suciati dkk. 2012. pengaruh likopen terhadap gambaran tubulus seminiferus dan kualitas sperma mencit (mus muculus) yang terpapar asap rokok. fakultas kedokteran universitas sriwijaya palembang. tri widjatmaka dan sonki prasetya, “perancangan dan pembuatan peralatan laboratorium pengkonversi gambar struktur mikro dari mikroskop ke komputer sebagai sarana praktikum metalografi”. jurnal politeknologi. 10 (3). tung yang wing and kent christensen. 1982. morphometric studies on rat seminferous tubules. the american journal of anatomy. the university of michigan. this page intentionally left blank optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height untitled-1 volume 6 number 1 2017 i s s n 2 0 8 9 6 5 1 4 ( p a p e r ) i s s n 2 5 4 0 9 3 2 8 ( o n l i n e ) biology, medicine, & natural product chemistry volume 6 – number 1 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original full-length research papers, reviews, and short communication. manuscript of original research should be written in no more than 8,000 words (including tables and picture), or proportional with articles in this publication number. review articles will be accommodated, while, short 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate dyah ayu widyastuti, praptining rahayu analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani, muhammad jafar luthfi anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro, muhammad jafar luthfi checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo, muhammad jafar luthfi 1-4 5-8 9-12 13-17 19-36 volume 6 number 1 2017 published twice a year printed in indonesia front cover: melastoma malabathricum (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back untitled-1 volume 6 number 2 2017 i s s n 2 0 8 9 6 5 1 4 ( p a p e r ) i s s n 2 5 4 0 9 3 2 8 ( o n l i n e ) biology, medicine, & natural product chemistry volume 6 – number 2 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original full-length research papers, reviews, and short communication. manuscript of original research should be written in no more than 8,000 words (including tables and picture), or proportional with articles in this publication number. review articles will be accommodated, while, short communication should be written at least 2,000 words, except for pre-study. submission the journal accepts online submission, through ojs website system and/ or email to the editors at jafarluthfi@yahoo.com. submitted manuscripts should be the original works of the author(s). the manuscript must be accompanied by a cover letter containing the article title, the first name and last name of all the authors, a paragraph describing the claimed novelty of the findings versus current knowledge. submission of a manuscript implies that the submitted work has not been published before (except as part of a thesis or report, or abstract); and is not being considered for publication elsewhere. when a manuscript written by a group, all authors should read and approve the final version of the submitted manuscript and its revision; and agree the submission of manuscripts for this journal. all authors should have made substantial contributions to the concept and design of the research, acquisition of the data and its analysis; drafting of the manuscript and correcting of the revision. all authors must be responsible for the quality, accuracy, and ethics of the work. ethics author(s) must obedient to the law and/or ethics in treating the object of research and pay attention to the legality of material sources and intellectual property rights. copyright if and when the manuscript is accepted for publication, the author(s) still hold the copyright and retain publishing rights without restrictions. authors or others are allowed to multiply article as long as not for commercial purposes. for the new invention, authors are suggested to manage its patent before published. open access the journal is committed to free-open access that does not charge readers or their institutions for access. readers are entitled to read, download, copy, distribute, print, search, or link to the full texts of articles, as long as not for commercial purposes. the license type is cc-by-nc. acceptance the only articles written in english (u.s. english) are accepted for publication. manuscripts will be reviewed by editors and invited reviewers 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini clustering of 18 local black rice base on total anthocyanin kristamtini, endang wisnu wiranti vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari, kristamtini, endang wisnu wiranti alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati, ahya zhilalikbar amin, mohamad amin on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi, riyanto 37-45 47-51 53-57 59-62 63-68 volume 6 number 2 2017 published twice a year printed in indonesia front cover: commelina benghalensis (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry volume 4 – number 1 – 2015 issn 2089-6514 contents a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja'far luthfi 1 3 the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani 5 9 the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi, maratun janah 11 15 krokot extract (portulaca oleracea. l) as natural light-harvesting pigments for dye-sensitized solar cells (dsscs): influence of dye acidity cici nurfaizah, didik krisdiyanto, khamidinal, sudarlin 17 24 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 457-461 | doi: 10.14421/biomedich.2023.122.457-461 issn 2540-9328 (online) proximate and mineral composition of atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus) joseph adaviruku sanni1,*, grace omayoza sanni2, rufus ranmilowo awoniyi2, remi osanyinlusi2, yvonne ego richards3 1department of biochemistry, faculty of science, adekunle ajasin university akungba-akoko, ondo state nigeria. 2science laboratory technology, faculty of applied science, rufus giwa polytechnic, owo, ondo state nigeria. 3department of crop science, faculty of agriculture, ladoke akintola university of technology, ogbomoso oyo state, nigeria. corresponding author* josephsanni59@gmail.com abstract atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus), locally known as kote, are fishery species consumed in nigeria due to their high nutritional values. this research determined the nutritional composition of the local dried fish, scomber scombrus and trachurus trachurus. results for scomber scombrus shows the mean value of the moisture, ash, crude fat, crude fiber, and crude protein contents in percent (%) as: 5.26±0.00, 5.20±0.10, 35.60±0.00, 2.90±0.10 and 46.30±0.01 respectively. for trachurus trachurus, the moisture, ash, crude fat, crude fiber, and crude protein contents in percent (%) were 9.52±0.00, 32.26±0.01, 24.14±0.00, 11.91±0.85 and 40.95±0.00 respectively. the mineral composition of scomber scombrus was as follows: sodium (na) had the highest mineral composition with 78.90 mg/100g, followed by magnesium (mg) with 15.90 mg/100g, manganese (mn) with 0.86 mg/100g, zinc (zn) with 0.282 mg/100g, and iron (fe) with 0.10 mg/100g. trachurus trachurus has magnesium (mg) at 27.00 mg/100g as its highest mineral composition, followed by sodium (na) at 22.50 mg/100g, zinc (zn), iron (fe) at 0.17 mg/100g, and manganese (mn) at 0.09 mg/100g. it is concluded from the study, that scomber scombrus and trachurus trachurus are good sources of essential nitrates, fat, proteins containing essential amino acids, and other micronutrients that are beneficial to human health. keywords: proximate composition; mineral content; scomber scombrus; trachurus trachurus. introduction fish is an essential food consumed in developing countries like nigeria because of its abundance and nutritional values, like its high protein, unsaturated fatty acids, carbohydrate, and mineral contents. it is cheap and available in urban and rural areas (bene and heck, 2005). it is acceptable worldwide because of its high nutritional value and low cholesterol content when compared with meat and is thus often recommended for consumption, especially among the adult population (eyo, 2001). several studies have recorded the importance of including fish in diets. fish contains essential nutrients, especially protein, vitamins, and minerals, which enhance the management of cardiovascular and other related diseases (damsgaard et al., 2006). according to (damsgaard, 2006), fish contains eicosapentaenoic and docosahexaenoic acids, long-chain n-3 polyunsaturated fatty acids that supply essential nutrients and promote good health. atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus), locally known as kote are the most predominant fishery species in various markets and are well consumed among the nigerian populace. trachurus trachurus originates from portugal and is consumed widely because of its availability and nutritional value (nadeisa et al., 2001). scomber scombrus is known for its high protein and fat content (ackman, 1990). proximate analysis or nutritional evaluation of fish indicates the percentage composition of essential nutrients or constituents such as proteins, crude fats, and other minerals in fish products. the chemical constituents are naturally used as a pointer to the nutritional value found in fish (moghaddam et al., 2007; aberoumand, 2011). fish generally differ from species to species. the differences can result from seasonal variation, feeding habits, or sex (islam et al., 2005). this study evaluates two samples of commercially available fish species obtained from a market in southwestern nigeria. therefore, this work determines the proximate and mineral compositions of scomber scombrus and trachurus trachurus to identify similarities and differences in their nutritional content. manuscript received: 14 may, 2023. revision accepted: 10 august, 2023. published: 15 august, 2023. https://doi.org/10.14421/biomedich.2023.122.457-461 458 biology, medicine, & natural product chemistry 12 (2), 2023: 457-461 materials and method materials powdered scomber scombrus and trachurus trachurus, hcl, h2so4, naoh, weighing balance, filter paper, heating mantle, crucible, thread, beaker, conical flask, distilled water, reagent bottle, chloroform, water bath, acetic acid, and pipette. figure 1. pictorial representation of scomber scombrus. figure 2. pictorial representation of trachurus trachurus. method sample preparation the materials were obtained from oja oba, owo local government area of ondo state. the fish was cut into small pieces, sundried for a month, and placed in an oven for further drying. the dried sample was crushed into powder using an el-850w blender and packed into a container. proximate analysis fat content determination a clean fat-free filter paper was weighed (w1). 5g of the sample was added to the filter and weighed (w2). the weighed sample was tied with a piece of thread and dropped into the thimble of the soxhlet apparatus. 250 ml of petroleum ether was poured into the round bottom flask of the apparatus. soxhlet was set up on the heating mantle, and the extraction process was done for four hours to extract the fat with the help of the solvent. petroleum ether was siphoned over the barrel, the condenser was detached, and the thimble was removed. the solvent extract (lipid) mixture was carefully poured into a clean, dried petri dish and transferred into a fume cupboard for two hours. the solvent evaporated, leaving behind the extracted fat. the filter paper containing the residue was dropped into a beaker and transferred into an oven at 50°c. it was then dried to a constant weight and cooled in a desiccator and reweighed (w3). the percentage of fat was calculated. % 𝐹𝑎𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 moisture content determination the moisture content was determined using a drying method based on weight loss. a clean and dry crucible was weighed using a weighing balance, and its weight was recorded (w1). samples were added to the empty crucible, and their weight was recorded (w2). the crucible containing the sample was transferred into the oven, maintained at 105°c, and dried for four hours. the dish was placed in a desiccator, cooled for one hour, and reweighed (w3). the percentage moisture content was calculated. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 ash content an ash-free crucible was weighed, and the weight was recorded (w1). 2g of sample was weighed into the crucible (w2) and transferred into the muffle furnace. the muffle furnace was then ignited at 600 °c for about four hours until a grayish-white substance was obtained. the crucible was transferred into a desiccator, cooled, and reweighed (w3). the percentage ash content was calculated. % 𝑎𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊3 − 𝑊1 × 100 crude-fiber content determination 5g of the defatted sample was weighed (w1) into a 2500 ml conical flask. 200 ml of 1.25% h2so4 was added. the mixture was heated for 30 minutes, then cooled and filtered through poplin cloth by suction using a bunchier funnel. the residue was rinsed in hot, distilled water and scraped back into a flask. 200 ml of 1.25% naoh was added, and the mixture was heated for 30 minutes. it was cooled, filtered, and washed once with hot, distilled water, once with 10% hcl, four times with hot water, and twice with methylated spirit. the residue was drained, placed in a crucible, and dried in an oven at 105ºc. after drying in an oven, it was then cooled in a desiccator and weighed (w2). the crucible containing the residue was placed in a muffle furnace at 300°c for 30 minutes, placed in a desiccator to cool to room temperature, and weighed (w3). % 𝑐𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑒𝑟 = 𝑊2 − 𝑊3 𝑊1 × 100 determination of protein content 2g of sample was weighed into a 50-ml kjeldahl flask, and 12.5 ml of concentrated h2so4 was added with one kjeldahl catalyst tablet. the flask was heated on low heat sanni et al. – proximate and mineral composition of atlantic mackerel … 459 for about 15 minutes, on medium heat for 30 minutes, and then on high heat until digested. the flask was rotated at intervals until the digest was clear, and the heating continued for a few minutes to ensure complete digestion. the flask was allowed to cool, and the sample residue was washed and filtered to make the digest up to 50 ml (v1). after the digestion was completed, 5 ml of 2% boric acid (h3bo3) was placed into a 100-ml conical flask, and 3 drops of the mixed indicator were added. the receiving flask was placed so that the tip of the condenser tube was below the surface of the boric acid. 5 ml of the digest (v2) was pipetted into the distillation tube, and 10 ml of 40% naoh was added. the heater was turned on, and the distillation continued until approximately 50 ml of distillate was collected into the receiving flask. the distillate was titrated with 0.01m hcl, and the blank was titrated with the acid. %𝑁 = 𝑀 × 𝑇 × 0.014 𝑊 × 𝑉1 𝑉2 × 100 % 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = % 𝑁 𝑥 6.25 results and discussion the result of proximate analysis of the nutritional composition of scomber scombrus and trachurus trachurus showed the presence of moisture, ash, crude fat, crude fiber, and crude protein in the following proportions, respectively: the result of the mineral analysis of the nutritional composition of scomber scombrus and trachurus trachurus showed the presence of sodium (na), zinc (zn), magnesium (mg), iron (fe), and manganese (mn), respectively: proximate composition table 1. shows the type and quantity of the nutritional composition of scomber scombrus and trachurus trachurus. s/n parameters measured scomber scombrus trachurus trachurus 1 moisture content (%) 5.26±0.00 9.52±0.00 2 ash content (%) 5.20±0.10 32.26±0.01 3 crude fat (%) 35.60±0.00 24.14±0.00 4 crude fibre (%) 2.90±0.10 11.91±0.85 5 crude protein (%) 46.30±0.01 40.95±0.00 table 2. showing the result of metal (mg/100g) analysis of scomber scombrus and trachurus trachurus. s/n parameters measured scomber scombrus trachurus trachurus 1 sodium(mg/100g) 78.90±0.02 22.50±0.01 2 zinc(mg/100g) 0.28±0.00 0.17±0.00 3 magnesium(mg/100g) 15.90±0.01 27.00±0.02 4 iron(mg/100g) 0.10±0.00 0.17±0.00 5 manganese(mg/100g) 0.09±0.00 0.09±0.00 discussion the proximate composition of both scomber scombrus and trachurus trachurus are in table 1. proteins and fats are known to be the major nutrients found in fish. considering the various species, there are variations depending on age, sex, and environment. the variations help to determine their nutritional status (aberoumad and pourshafi, 2010). the mean value of crude protein obtained for scomber scombrus was 46.29%. this was slightly higher than the value obtained from trachurus trachurus, which was 40.95%. this value was found to be lower when compared to 57.80% obtained from scomber scombrus reported when the sample was smoked with an electric oven (aremu et al., 2014); lower compared with the 70.24% for melon husk heat treatment reported (aremu et al., 2014); 62.14% for clarias gariepinus as observed by kumolu and ndimele, 2010; and higher than 26.25% reported for scomber scombrus (agu and bhandary, 2004). the value of the crude fat of scomber scombrus was observed to be 35.60%, while that of trachurus trachurus was recorded to be 24.14%. the mean value of crude fat for both scomber scombrus and trachurus trachurus was higher than the range of crude fat for scomber scombrus, ranging between 7.41 and 17.51% using different heat sources, as reported by (aremu et al., 2014). a moisture content of 5.26% was observed in scomber scombrus, while 9.52% was recorded for trachurus trachurus. the result shows that the moisture content found in trachurus trachurus was higher than that obtained in scomber scombrus. (olusola et al., 2011) reported the moisture content of scomber scombrus at 56.5% and roughear scad at 66.7%, which was seen to have a higher mean value. this can be a result of the type of the environment in which the 460 biology, medicine, & natural product chemistry 12 (2), 2023: 457-461 samples were collected or the season in which the fish was collected. the 5.26% of scomber scombrus is almost the same as the 5.28% reported by aremu et al. (2014) in scomber scombrus dried using an electric oven. another study reported by adeyi et al. (2010) gives a moisture content of 5.43% when coconut husk was used as a heat treatment. the result is almost similar, though it has about a 0.17% difference, which may imply that the heat generated from an electric oven dries the fish better and is stronger than the heat generated from the husk of a coconut. the ash content of scomber scombrus is 5.20%, and that of trachurus trachurus is 32.26%; this shows that trachurus trachurus has a higher ash content compared to scomber scombrus. the 5.20% of scomber scombrus obtained using an electric oven was compared to the ash content of 5.70% obtained for heat treatment using sawdust by (aremu et al., 2014) and 6.01% reported for mackerel fish (agu and bhandary, 2004). the mineral composition indicates certain organic matter that is not denatured by high heat intensity and is known for its low volatility compared to other food classes (remi, 2023). the mineral composition in mg/100g of scomber scombrus and trachurus trachurus is shown in table 2. from the result for scomber scombrus, sodium (na) was shown to be more abundant in the dried fish with 78.90 mg/100g, followed by magnesium (mg) with 15.90 mg/100g, manganese (mn) with 0.86 mg/100g, zinc (zn) with 0.282 mg/100 g, and iron (fe) with 0.10 mg/100g. for trachurus trachurus, magnesium is more abundant at 27.00 mg/100g, followed by sodium at 15.90 mg/100g. this value is higher than the 7.43 mg/100g reported by (aremu, 2014). magnesium is an important element in connection with the treatment of diseases of the circulatory system, such as ischaemic heart disease, and calcium metabolism in bones (ishida et al., 2000). iron (fe), zinc (zn), and manganese (mn) were low, albeit available for biological functions. conclusion it is concluded from this study that scomber scombrus and trachurus trachurus are high in fat, proteins, and some essential micronutrients, which are good sources of nutrients for the growth of the body. scomber scombrus was reported to have high protein and fat content, which implies that it would be a good source of protein for growing children and adults. authors’ contributions: rra, gos, and jas conceptualized the study, carried out the research, and wrote the manuscript draft. ro, gos, jas, and yer contributed to the study design and manuscript editing. all authors read and approved the final manuscript. competing interests: the authors declared that they have no competing interests. funding: not applicable. availability of data and materials: not applicable. ethics approval and consent to participate: not applicable. consent for publication: not applicable. references aberoumand, a. 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(2010). the effects of smoking on the nutritional qualities and shelf-life of clarias gariepinus (burchell 1822). african journal of biotechnology, 9(1). moghaddam, h. n., mesgaran, m. d., najafabadi, h. j., & najafabadi, r. j. (2007). determination of chemical composition, mineral contents, and protein quality of iranian kilka fish meal. international journal of poultry science, 6(5), 354-361. odiko, a. e., & obirenfoju, j. (2017). proximate composition and mineral contents of different brands of canned fishes marketed in edo state nigeria. int. j. fisher. aquacult. res, 3(2), 30-38. olaofe, o., & sanni, c. o. (1988). mineral contents of agricultural products. food chemistry, 30(1), 73-77. https://doi.org/10.1016/0308-8146(88)90026-x olusola, o. a. (2011). technological properties and proximate composition of two imported fish species in nigeria. electronic journal of environmental, agricultural and food chemistry (ejeafche), 10(6), 2398-2403. remi, o. (2023). nutritional composition of processed and unprocessed samples of unripe plantain (musa paradisiaca). journal of advanced education and sciences, 3(1), 75-81. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 437-440 | doi: 10.14421/biomedich.2023.122.437-440 issn 2540-9328 (online) chemical properties of liquid broth extracted from freshwater and marine shrimp shells waste esa ghanim fadhallah*, dyah koesoemawardani, lathifa indraningtyas department of agricultural product technology, faculty of agriculture, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* esa.ghanim@fp.unila.ac.id manuscript received: 30 mei, 2023. revision accepted: 27 july, 2023. published: 12 august, 2023. abstract indonesia's shrimp industry is growing rapidly, but a surge in shrimp waste such as shells and heads’ shrimps are increasing as well. these waste products contain important components such as protein, minerals, and amino acids. this study aims to determine the chemical properties of liquid broth extracted from freshwater and marine shrimp shells, including ash, protein, fat, monosodium glutamate (msg), and antioxidant. the liquid broth was extracted by boiling shrimp shells and heads in water with a ratio of 1:2 for 1 hour at 80oc. results indicate that the type of shrimp used did not affect the broth's ash, fat, protein, msg, or antioxidant content. marine and freshwater shrimp liquid broths contain 0.56% and 0.28% ash, 0.10% and 0.50% fat, 2.19% and 1.97% protein, 1.5291% and 1.6274 % msg, and 2263.73 ppm and 2786.2 ppm antioxidant. keywords: chemical; liquid broth; protein; shrimp shell; waste. introduction shrimp is one of the leading commodities of indonesia's fisheries industry. according to the data, shrimp ranks first with the largest export value of 239.28 million kg and us$ 2.04 billion. shrimp export volume increased by 28.96% compared to 2019, reaching 207.70 million kg. shrimp also contributed 18.95% of the total export volume of fishery products last year (ministry of marine affairs and fisheries republic of indonesia, 2021). however, as shrimp production increases, the waste generated from shrimp shells and heads also rises since these byproducts in the industry are approximately 60% of total production (abdollahi & undeland, 2020). the shrimp shells and heads have been used as additives in aquaculture feeds but are commonly discarded and polluting the environment (saini et al., 2020). shrimp shells and heads still contain important components such as protein, astaxanthin pigment, minerals, and amino acids (liu et al., 2021), making utilizing these contents as products possible. the broth is a result of an extracting certain food ingredients such as chicken meat, beef, seafood, and vegetables which can be used to enhance the umami taste of dishes (mosby, 2009; rostini & pratama, 2018). the extract can be obtained by boiling to dissolve the watersoluble components inside the ingredients into the water. marine and freshwater shrimp differ in taste and texture, whereas marine shrimp has a richer and sweeter taste than freshwater shrimp (liang et al., 2008). moreover, the shrimp pigment called astaxanthin is believed to have an important antioxidant activity that is beneficial to human health (fakhri et al., 2018). the previous study (saleh et al., 1996) applied the liquid broth extracted from shrimp heads into crackers, but there is no further investigation on the various chemical properties. the study on characteristics differences of liquid broth obtained from marine and freshwater shrimp shells has yet to be reported. therefore, this study aims to determine the chemical properties of liquid broth obtained from the shells and heads of marine and freshwater shrimp. materials and methods materials and equipment the raw materials used in this study were the shells and heads of marine and freshwater shrimp obtained from the gudang lelang market in bandar lampung, indonesia. other materials used included chemicals for chemical analysis and titration. the equipment included a digital scale, beaker, hot magnetic stirrer, stirring rod, and stainless-steel strainer. the chemical analysis of the broth was carried out using a set of titration tools, chemical glassware, a furnace oven, a soxhlet apparatus, https://doi.org/10.14421/biomedich.2023.122.437-440 438 biology, medicine, & natural product chemistry 12 (2), 2023: 437-440 a kjeldahl apparatus, and a uv-vis spectrophotometer (hitachi u-2010). liquid broth preparation this study was carried out in several steps, including preparing shrimp shells and heads, the boiling process, and the characterization of the boiled shrimp shell and head broth. all stages of the research were carried out on samples of marine and freshwater shrimp. the shrimp shells and heads were washed with running water until clean and drained, then placed in a beaker glass. water was added at a ratio of shrimp to the water 1:2 and then boiled using a hot magnetic stirrer at 80°c for 60 minutes (meiyani et al., 2014). the broth was then filtered to separate the shrimp shells and heads from the broth, and then cooled before being characterized. the characteristics of the broth analyzed included the proximate analysis of protein, fat, ash (aoac, 2005), monosodium glutamate (sulastri, 2017), and antioxidant activity (rahmawati et al., 2016). data analysis the results of several analyses were processed, and the significance of the differences was analyzed using a ttest with the ibm spss version 23 application. results and discussion chemical properties of liquid broth the parameters analyzed to obtain the characteristics of the broth from freshwater and marine shrimp shells included ash, fat, protein, msg, and antioxidants. the results of these parameter tests are presented in table 1. based on the results presented in table 1, it is known that the ash, fat, protein, msg, and antioxidant content of the broth from freshwater shrimp shells were 0.28%, 0.50%, 1.97%, 1.6274%, and 2786.2 ppm, respectively. meanwhile, the values for the broth from marine shrimp shells were 0.56%, 0.10%, 2.19%, 1.5291%, and 2263.73 ppm, respectively. the t-test statistical analysis showed no significant difference in chemical properties between the broth from freshwater and marine shrimp shells. table 1. chemical properties of liquid broth from freshwater and marine shrimp shells analysis freshwater shrimp marine shrimp ash (%) 0.28±0.01 a 0.56±0.03 a fat (%) 0.50±0.04 a 0.10±0.01 a protein (%) 1.97±0.31 a 2.19±0.02 a msg (%) 1.6274±0,02 a 1.5291±0.04 a antioxidant (ppm) 2786.2±125.27 a 2263.73±201.67 a note: the numbers followed by different letters on the same row indicate significant differences based on the t-test. ash content the ash content of the liquid broth obtained from the shells and heads of freshwater shrimp is lower than marine shrimp. specifically, the ash content of the liquid broth from freshwater shrimp is 0.28%, while from marine shrimp is 0.56%. however, a t-test analysis shows no significant difference in the ash content of the broth between marine and freshwater shrimp. this indicates that the type of shrimp does not significantly impact the ash content in the shrimp broth, and it is inferred that the ash content was more likely affected by the boiling period. another study (silab et al., 2022) reported that the longer boiling period of pig bones resulted in higher ash content. ash content in broth is related to the presence of minerals in a substance. the boiling process using water can only dissolve watersoluble minerals. the longer boiling period makes more water-soluble minerals come out of the tissue and dissolve in the water (liu et al., 2021). fat content the fat content of the liquid broth obtained from the shells and heads of freshwater shrimp is higher than marine shrimp, specifically from freshwater shrimp and marine shrimp, which are 0.50% and 0.10%, respectively. since the t-test shows no significant difference in the fat content, it indicated that the type of shrimp does not affect the fat content in the shrimp broth produced. the fat content in the broth can be more influenced by the length of boiling time. another finding (silab et al., 2022) showed a significant increase in the fat content of broth produced from boiling pig bones with rising boiling time. the fat will leach outside the tissue and move into the water media. the longer boiling process will break down more fat components and dissolve them into the boiling water, increasing the fat content in the broth. the boiling process with water causes hydrolysis to break down fat into glycerol, soluble in water, and fatty acids, and also significantly changes the saturated and unsaturated fatty acids profile (saborowski et al., 2022). protein content the protein content of the liquid broth obtained from the shells and heads of freshwater shrimp is lower than marine shrimp. specifically, the protein content of the broth from freshwater shrimp is 1.97%, while from marine shrimp is 2.19%. the t-test results show no significant difference in the protein and indicate that the type of shrimp does not affect the protein content in the broth. the protein content in this study is lower than in another study. the protein content of liquid broth from marine shrimp heads, black tiger shrimp (penaeus monodon), ranges from 2.24% to 3.73% and varies depending on the boiling time and temperature (saleh et al., 1996). the protein content of the liquid broth is affected by the length of the boiling time. this statement is supported by other findings (silab et al., 2022) that showed the protein content of broth produced from boiling pig bones showed a significant increase with fadhallah et al. – chemical properties of liquid broth 439 increasing boiling time. the longer the boiling time, it is suspected to soften the tissue on the shrimp shell so that the protein components in the shrimp connective tissue will be extracted into the broth liquid. processing temperature could affect the protein content of boiled water at the same boiling time. another study (saleh et al., 1996) reported that boiled water from the heads of tiger prawns at 100°c had a protein content of 2.68% and significantly increased to 3.73% at a boiling temperature of 115°c. this is suspected by the increase in protein solubility at the higher processing temperature. monosodium glutamate (msg) the monosodium glutamate (msg) content in broth from marine shrimp is slightly lower than freshwater shrimp, which are 1.5291% and 1.6274%, respectively. however, a t-test analysis shows no significant difference in the ash content of the broth between marine and freshwater shrimp, which indicates that the type of shrimp does not affect the msg content in the shrimp broth produced. the msg concentration in the liquid broth correlates with the glutamic acid content of shrimp shells and heads. according to (liu et al., 2021), the combined total glutamic acid concentration of shrimp heads (1.56% 1.76%) and shells (1.69% 1.83%) is more than that of the flesh (3.00% 3.10%). the msg content in the broth can be more influenced by the length of boiling time. the longer boiling time is suspected of causing the protein to be denatured and break down into amino acids, including glutamic acid, which has a high solubility in water (kingwascharapong & benjakul, 2016). boiling process will cause protein breakdown, producing amino acids that are easier to absorb by the body (anwa et al., 2007). antioxidant antioxidants in shrimp play a role in delaying, slowing down, and preventing free radical oxidation in lipid oxidation (nikoo et al., 2021). astaxanthin, a xanthophilic carotenoid compound with two hydroxyl groups, is one of the antioxidants found in shrimp skin. this compound is more soluble in methanol and ethanol and can be extracted by bleaching method against βcarotene. astaxanthin extracts from shrimp shells have an antioxidant activity of 88% against β-carotene (saleha & murniana, 2009). in this study, the antioxidant content of the freshwater shrimp broth extracted from shrimp shells and heads was 2786.2 ppm and 2263.73 ppm. according to the t-test analysis, it did not show a significant difference. during boiling, the antioxidant content correlated with the astaxanthin pigment extracted from shrimp shells and head tissue into the water. this result is higher than another study (liu et al., 2021), which reported the astaxanthin content of shrimp heads, shells, and tails that ranges from 2.91 ppm to 19.20 ppm. during soaking and boiling, when shrimp parts come into contact with water, some proteins, including carotenoproteins (such as astaxanthin), were partially leached out. as a result, fewer pigments were retained in the meat, making the boiling water reddish (kingwascharapong & benjakul, 2016). the amount of astaxanthin in shrimp is impacted by the composition of their feed, as proven by research (ruangdej & laohavisuti, 2014) that showed shrimp fed with astaxanthin-enriched diets had higher pigmentation and total carotenoid levels than those given without it. conclusions the chemical properties of liquid broth produced from freshwater and marine shrimp shells, including ash, fat, protein, msg, and antioxidants, have been studied. the liquid broth obtained from marine shrimp shells has higher protein content, msg content, and antioxidants than the broth obtained from freshwater shrimp shells. however, the ash and fat content are lower in marine shrimp shell broth than in freshwater shrimp shell broth. these chemical properties are not affected by the type of shrimp used. further study is needed to vary the time and temperature boiling process with another analysis, such as amino acids and fatty acid profiles. acknowledgements: the authors would like to thank the faculty of agriculture, universitas lampung, for financial support of this work. authors’ contributions: dyah koesoemawardani designed the study. esa ghanim fadhallah and lathifa indraningtyas carried out the laboratory work. esa ghanim fadhallah analyzed the data. esa ghanim fadhallah and lathifa indraningtyas wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the funding was granted by the faculty of agriculture, universitas lampung. references abdollahi, m., & undeland, i. 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(2017). analisis kadar monosodium glutamat (msg) pada bumbu mie instan yang diperjualbelikan di koperasi wisata universitas indonesia timur. jurnal media laboran, 7(1), 5–9. https://uit.e-journal.id/medlab/article/view/347 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 191-196 | doi: 10.14421/biomedich.2023.121.191-196 issn 2540-9328 (online) antihypertensive drugs therapy in hypertension and covid-19 comorbidity emuesiri goodies moke1,*, endurance efe ahama2, pere-ebi yabrade toloyai3, mamerhi taniyohwo enaohwo2, ekuerhare basil4, emuesiri kohworho umukoro5, anthony taghogho eduviere1, ikuesirioghene udumebraye1, choice udufowe1 1department of pharmacology, faculty of basic medical sciences; 2department of anatomy and cell biology, faculty of basic medical sciences; 3department of medical biochemistry, faculty of basic medical sciences; 4university health services; 5department of pharmacology and therapeutics, faculty of basic clinical sciences, delta state university, abraka, nigeria. corresponding author* hiligoodies@gmail.com manuscript received: 06 january, 2023. revision accepted: 07 february, 2023. published: 11 february, 2023. abstract the novel coronavirus (cov) severe acute respiratory syndrome (sars)-cov-2 outbreak began at the end of 2019 in wuhan, china, and has spread to over 200 countries. many comorbidities have shown to be associated with the severity of the viral infection wit h hypertension being one of the highest rated comorbidities since loss of the ace2 receptor due to sars-cov-2 infection can lead to increased blood pressure. the effects and clinical characteristics associated with the use of beta-blockers, angiotensin receptor blockers (arb), and calcium-channel blockers (ccb) shows not to affect the outcome of covid-19, except in angiotensin converting enzyme inhibitor (acei) which may have negative outcomes on covid-19 infected patients. many comorbidities have shown to be associated with the severity of the viral infection. keywords: blood pressure; comorbidity; covid-19; renin-angiotensin system. introduction hypertension (htn) is a dangerous medical condition that raises the risk of disorders of the heart, brain, kidneys, and other organs (who, 2021). hypertension is characterized as persistently increased blood pressure with a systolic blood pressure of 140 mmhg and/or a diastolic blood pressure of 90 mmhg (mills et al., 2020). globally, an estimated 1.13 billion people do have hypertension, with over 700 million people reported to with untreated hypertension (who, 2021). worldwide, hypertension is the biggest avoidable risk factor for cvd and all-cause death (stanaway et al., 2018). globally, the incidence of hypertension is increasing due to population aging and increased exposure to lifestyle risk factors such as bad diets (i.e. excessive salt and low potassium consumption, as well as a lack of physical exercise) (mills, 2016). another reason may be as a result of suboptimal outcome even with wellconstructed antihypertensive treatment regimens (resistant hypertension) (acelajado et al., 2019; carey 2020; moke et al., 2022). however, variations in hypertension prevalence levels are not consistent over the world. high-income countries (hics) had a minor decline in hypertension prevalence during the last two decades, whereas poor and middle-income countries (lmics) saw large rises (mills, 2016). antihypertensive medications are chemical substances used to prevent and treat excessive blood pressure (jackson and bellamy, 2015; alawode et al., 2021). angiotensin-converting enzyme (ace) inhibitors, angiotensin ii receptor blockers (arbs), diuretics, calcium channel blockers (ccbs), and beta-blockers are the most widely prescribed antihypertensive medication groups (khalil and zeitser, 2020; moke et al., 2022). the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory viral disease that first appeared in december 2019 in wuhan, the capital of the chinese province hubei (ruscitti et al., 2020). it is a highly contagious illness caused by the severe acute respiratory syndrome coronavirus 2 (sarscov-2) that causes respiratory infections and discomfort. covid-19 possesses a wide range of clinical symptoms, from asymptomatic disease to severe pulmonary infections (carfora et al., 2020; omo-aghoja et al., 2021). various statistics on covid-19 patients surfaced, revealing that some populations may be at higher risk than others (booth et al., 2021; enaohwo et al., 2021). with covid-19 infections, patients with pre-existing https://doi.org/10.14421/biomedich.2023.121.191-196 mailto:hiligoodies@gmail.com 192 biology, medicine, & natural product chemistry 12 (1), 2023: 191-196 cardiac disease are among the three greatest risk categories (chan et al., 2020). different comorbidities with hypertension exists, including covid-19, and these have been reported (schmieder and ruilope, 2008; noh et al., 2016, unger et al., 2020; batiha et al., 2021; okonofua et al., 2021). according to preliminary findings, hypertension (htn) is more common in critically sick, hospitalized covid19 patients, with total htn rates ranging from 50 to 56 percent (richardson et al., 2020). it was unclear if this link was causative or masked by age and other htnrelated comorbidities such as obesity, diabetes, and chronic kidney disease. furthermore, covid-19affected hypertensive individuals exhibited a greater mortality risk than non-hypertensive patients (barrera et al., 2020). concerns about the use of angiotensin-converting enzyme inhibitors (aceis) in these patients arose as a result of the identification of angiotensin-converting enzyme 2 (ace2), the monocarboxypeptidase that inactivates angiotensin ii, thus, counteracts the activation of the classic renin–angiotensin–aldosterone system (raas), as the functional receptor for the severe acute respiratory syndrome coronavirus 2 (vadughanathan et al., 2020). because hypertension is a major risk factor for developing covid-19, selecting the right medication for successful blood pressure management is critical. this review briefly discusses the drug therapy with antihypertensive drugs in hypertensive patients with covid-19 comorbidity. articles used for this review ranged from 2008 to 2022, and over 100 articles were obtained following literature search, amongst which 64 were adapted for this article. other articles whose scope were not applicable to the review were excluded. the articles were retrieved following searches using search engines and databases including medline, elsevier, medscape, emedicine, google, pubmed, and others. corona virus disease 2019 (covid-19) the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory viral disease that first appeared in december 2019 in wuhan, the capital of the chinese province hubei (ruscitti et al., 2020). it is a highly contagious illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that causes respiratory infections and discomfort. corona virus disease 2019 (covid-19) is an rna virus with a crown-like appearance under an electron microscope due to glycoprotein spikes on its envelope (malik, 2020). covid-19 possesses a wide range of clinical symptoms, from asymptomatic disease to severe pulmonary infections (carfora et al., 2020; omo-aghoja et al., 2021). it is not the first time that a coronavirus has caused an epidemic, as an outbreak of coronaviruses (covs) with severe acute respiratory syndrome (sars)cov began in the chinese province of guangdong in november 2019, while on september 2012, the middle east respiratory syndrome (mers)-cov appeared (lu et al., 2020). the coronaviruses (covs) are classified into four genera: (a) α-coronavirus (alphacov), (b) β-coronavirus (betacov), (c) δ-coronavirus (deltacov), and (d) γcoronavirus (gammacov), which are most likely found in birds (pal et al., 2020). the origin of the virus is natural and zoonotic in nature. the clinical severity of the disease is varies from displaying no symptom to being very fatal, as its medical features and risk factors are so inconstant (phan, 2020). molecular structure of corona virus coronaviruses are enclosed positive strand rna viruses with the biggest known rna genomes (30–32 kb), a 5'cap structure, and a 3'-poly-a tail. the production of polyprotein 1a/1ab (pp1a/pp1ab) in the host begins with viral rna (lei et al., 2018). transcription is carried out by the replication-transcription complex (rct), which is structured in double-membrane vesicles, and by the synthesis of subgenomic rnas (sgrnas). it is worth noting that transcription termination occurs at transcription regulatory sequences, which are positioned between the so-called open reading frames (orfs), which serve as templates for the creation of subgenomic mrnas (letko et al., 2020). at least six orfs can be found in the atypical cov genome. among these, a frameshift between orf1a and orf1b directs the synthesis of both pp1a and pp1ab polypeptides, which are then processed by a virally encoded chymotrypsin-like protease (3clpro) or main protease (mpro), as well as one or two papain-like proteases for the synthesis of 16 non-structural proteins (nsps) (letko et al., 2020). other orfs, in addition to orf1a and orf1b, encode structural proteins such as spike, membrane, envelope, and nucleocapsid proteins, as well as auxiliary proteic chains (lei et al., 2020). different covs have unique structural and auxiliary proteins that are translated by specialized sgrnas. the role of the nsps and structural proteins is linked to the pathophysiology and virulence processes of covs, and hence to sars-cov-2. among the structural proteins' activities, the envelope plays a critical role in virus pathogenicity by promoting viral assembly and release (yadav et al., 2021). prevalence of hypertension in people with covid-19  prevalence of hypertension in people with covid-19 was 25.4% in africa, 31% in china, 49% in italy, 21% in india, 32% in oman, and 10% in iran (cook, 2020).  the prevalence of hypertension in patients with covid-19 was 4.7%, and 24.37% of covid-19 moke et al. – antihypertensive drugs therapy in hypertension and … 193 related deaths occurred in individuals in iran (moftakhar et al., 2021).  hypertension was the most prevalent reported comorbidity in covid-19 patients in wuhan; the reported prevalence rates ranged from 15.0% to 36.5% (haung et al., 2020).  the prevalence of hypertension was also higher in covid-19 deceased patients; 34.0% vs. those who were discharged alive; 28.0% (leiva et al., 2020).  along with the increased risk of infection and worsened outcomes among hypertensives, there is a growing concern that some medications used in the treatment may influence mortality in patients with covid-19 (patel and verma, 2020).  studies have reported that covid-19 deaths were mostly among people with comorbidities (99%), the majority of these were hypertensive (guan et al, 2020; osibogun et al., 2021).  in nigeria, the prevalence of hypertension, the most common comorbidity of covid-19, was 17.8% followed by diabetes (7.2%) and asthma (2.0%) (abayomi et al., 2021).  in nigeria, overall mortality was 4.2% while mortality among the hypertensives was 13.7%. severe symptoms and mortality were significantly higher among the hypertensives and survival rates were significantly lowered by the presence of additional comorbidity to 50% (abayomi et al., 2021).  studies have shown that hypertension imposes on those who suffer from it an increased risk of getting infected with covid-19, experiencing worse symptomatology and complications and a 2-fold risk of dying from the infection (cancarevic and malik, 2020; abayomi et al., 2021). some anti-hypertensive drugs in the treatment of hypertensive patients with covid-19 comorbidity anti-hypertensive medications are a type of medication used to treat hypertension (high blood pressure) (ettehad et al., 2016). antihypertensive treatment aims to avoid high blood pressure problems such as stroke and myocardial infarction. evidence shows that lowering blood pressure by 5 mmhg can reduce the risk of stroke by 34%, ischemic heart disease by 21%, and the chance of dementia, heart failure, and cardiovascular disease death by 34% (nelson, 2010). there are several kinds of anti-hypertensives, each of which works in a different way to reduce blood pressure. thiazide diuretics, calcium channel blockers, ace inhibitors, angiotensin ii receptor antagonists (arbs), and beta blockers are among the most significant and extensively used drugs (armstrong, 2014; ettehad et al., 2016). beta-adrenoreceptor blockers beta-blockers lower blood pressure by preventing catecholamines from binding to beta-adrenergic receptors, resulting in coronary and peripheral artery vasodilation (farzam and jan, 2020). betaadrenoreceptor blockers reduce blood pressure via decreasing cardiac output, heart rate, renin release, and adrenergic nerve system effects (farzam and jan, 2020). they improve outcomes after an acute myocardial infarction and in patients with heart failure who have a low left ventricular ejection fraction, but in the absence of these comorbidities, beta-adrenoreceptor blockers are inferior to other first-line antihypertensives in terms of reducing cvd morbidity and mortality (wiysonse et al., 2017). this impact has been related to lower aortic blood pressure and negative effects on body weight and glucose metabolism with beta-adrenoreceptor blockage (frishman et al., 2011). vasanthakumar's study suggested that using betablockers to treat covid-19 could provide numerous benefits, including reducing sars-cov-2 cell entry via downregulation of the angiotensin-converting enzyme 2 (ace-2), reducing the expression of proinflammatory cytokines, and reducing complications such as pulmonary embolism, ards, and septic shock (vasanthakumar, 2020). a research found that betablockers had no effect on the severity of covid-19 (bauer et al., 2021). another study found that using betablockers greatly lowered the chance of severe consequences (kjeldsen et al., 2021). patients on betablockers had a decreased risk of testing positive for covid-19 (reynold et al., 2020). calcium channel blockers (ccbs). calcium channel blockers (ccbs) inhibit calcium entry into cells by binding to l-type voltage-gated calcium channels found in organs such as the heart and vascular smooth muscle (mckeever and hamilton, 2020). a drop in the intracellular concentration of calcium causes smooth muscle cell relaxation and, as a result, a fall in blood pressure (kuo and ehrlich, 2022). headaches, flushing, palpitations, peripheral edema, hypotension, atrioventricular block, constipation, and nausea are the most common side effects associated with this category (solanki et al., 2021). calcium channel blockers, particularly verapamil, inhibit cardiac calcium channels, which can lower heart rate and cardiac contractility (elliott and ram, 2011). according to reports, ccbs can reduce sarscov-2 multiplication (zhang et al., 2020; loas et al., 2022). amlodipine besylate was observed to decrease the risk of death in hypertension individuals (zhang et al., 2020). another research found that nifedipine and amlodipine significantly lowered the mortality rate as well as the risk of intubation and mechanical ventilation in elderly covid-19 patients (solaimanzadeh, 2020). one research observed no significant changes in the administration of amlodipine in covid-19 patients with 194 biology, medicine, & natural product chemistry 12 (1), 2023: 191-196 primary hypertension in terms of mortality and length of hospital and intensive care unit (icu) stay (nourivaskeh et al., 2021). angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) the renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors and arbs, are among the most often prescribed blood pressure medications (wang et al., 2020). ace medications reduce blood pressure by inhibiting the angiotensin-converting enzyme, resulting in less angiotensin ii production and vasodilation. the mechanism of action of arbs is to prevent angiotensin ii from attaching to angiotensin-1 (at1) receptors (khalil and zeitser, 2020). the most serious side effects of this class include hyperkalemia, renal failure, coughing, and first-dose hypotension. there is growing concern about the use of antihypertensives in covid-19 patients, owing to the fact that angiotensin-converting enzyme 2 (ace2), a negative regulator of the renin–angiotensin–aldosterone system (raas), is a co-receptor for viral entry into human cells by sars-cov-2 (hui, 2020). ace cleaves angiotensin i to produce angiotensin ii, whereas ace2 converts angiotensin ii to angiotensin iii (zou, 2014). ace2 plays an important role in maintaining blood pressure homeostasis, fluid and salt balance by counteracting the impact of ace (forrester, 2018). ace inhibitors and arbs may increase the expression of ace-2, a cellular receptor for sars-cov2. these medications, on the other hand, have been found to protect against acute lung damage (guo et al., 2020). concerns have been raised about the use of angiotensin-converting enzyme inhibitors (aceis) in patients following the identification of angiotensinconverting enzyme 2 (ace2), a monocarboxypeptidase that inactivates angiotensin ii and thus counteracts the activation of the classic renin–angiotensin–aldosterone system (raas), as the functional receptor for the severe acute respiratory syndrome coronavirus 2 (sars-cov2) (vadughanathan et al., 2020). as a result, discontinuing aceis or arbs may result in poorer results than continuing to use them in individuals with covid-19. most of the world’s professional societies either recommend or strongly encourage continuing aceis/arbs in covid-19 infected patients (hoffman et al., 2020). the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory illness that causes respiratory distress among other symptoms. one of the major comorbidities of covid-19 is hypertension, which is defined by persistently high blood pressure. the use of several anti-hypertensive classes, including as beta-blockers, calcium channel blockers, diuretics, and angiotensin converting enzyme inhibitors (acei), to treat hypertension in patients with covid-19 may have side effects on the patients. while other antihypertensive classes have no to beneficial effects, acei have been demonstrated in multiple studies to be a source of worry since they may potentially enhance covid-19 symptoms and promote viral entry into the cell. conclusion most anti-hypertensive drugs have no detrimental impact on the outcome of covid-19 therapy, and a few classes have favorable benefits in alleviating symptoms in patients infected with covid-19. aceis are the only class of anti-hypertensive that may have an influence on the result of covid-19 among the anti-hypertensive classes evaluated. more studies and clinical trial are recommended to be carried out with more common antihypertensive to evaluate the potential effects on covid19. competing interests: the authors declare that there are no competing interests. references abayomi a, osibogun a, kanma-okafor o, idris j, bowale a, wright o, adebayo b, balogun m, ogboye s, adeseun r, abdus-salam i, mutiu b, saka b, lajide d, yenyi s, agbolagorite r, onasanya o, erinosho e, obasanya j, adejumo o, adesola s, oshodi y, akase ie, ogunbiyi s, omosun a, erinoso f, abdur-razzaq h, osa n, akinroye k (2021). morbidity and mortality outcomes of covid-19 patients with and without hypertension in lagos, nigeria: a retrospective cohort study. glob 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| pages: 55-59 | doi: 10.14421/biomedich.2016.52.55-59 issn 2540-9328 (online) effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti1, endaruji sedyadi2, susy yunita prabawati3 1,2,3chemistry department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: endaruji@yahoo.com2 abstract this study aimed to determine the effect of addition of soursop leaf extract to edible film and its effect on the storage time of red grapes. this research had three main steps, soursop leaf extraction, edible film making, and application on red grapes storage time. soursop leaves extract variation used in this research are 0.5; 1; and 1.5% (w/w total) and the best result was used to coat the red grapes. the results showed that the optimum composition was obtained on the edible film with the addition of 0.5% (w/w total) soursop leaf extract. the composition increased film thickness from 0.03 to 0.08 mm, decreased film’s tensile strength from 11.89 to 8.42 mpa, decreased elongation from 12.71 to 11.03%, decreased the young modulus from 0.935 to 0.764 mpa, and decreased the vapor transmission rate from 7.45 to 6.55 g/m2.hour. the 50% shrinkage of weight and 50% texture damage are used as parameters to measure the red grapes storage time. based on weight shrinkage, red grapes storage time change from 24 days to 29 days by using an edible film coating without addition of extract, an extend to 32 days while using an edible film coating with addition of soursop leaf extract. based on texture damage, red grapes storage time increased from 13 to 41 days if using an edible film coating without addition of extract, and increased to 40 days using an edible film coating with the addition of soursop leaf extract. keywords: antioxidant; edible film; soursop leaf extract; aloe vera l.; ganyong starch. introduction indonesia is a tropical country where many tropical plants such as pineapple, grapes, mango, and citrus are cultivated. grape is a fruit that very common in many occasion. the fruit has round shape with a sweet taste and thin-skinned. grape is known to have many benefits as anti-cancer and contain a, b, c, and e vitamins (suwarto, 2010). grape plant is a seasonal crop with harvest 2-3 times per year. the plant in the harvest season yielding 10 tons fruit per hectare. the abundant supply of grape during this harvest season caused problems for the farmer, i.e., rotten grapes. it is caused by the storage time of grape only reaches 14 days at room temperature. it takes a given preservation method for the grape to last longer. some ways that can be done to extend the storage time of grape are by cooling, coating with wax, and coating with active packaging. the use of wax as a coating are not suggested due to negative effect on human health. the preservation technique using cooling process requires a greater cost. therefore, in this research we will carried out an active packaging coating to extend the storage time of grape. one of the active packaging is edible film. edible film is a thin and continuous layer made of edible materials. layers are formed to coat the food components (coating) or placed between food components (film). a layer acts as a barrier to mass transfer (e.g. moisture, oxygen, lipids, light and solutes). the coating may also serve as a carrier of groceries and additives, as well as to improve the handling of a food (krochta & johnston, 1997). the main components of edible film can be grouped into three categories, namely hydrocolloids, lipids, and composites (donhowe, 1994). some types of hydrocolloids that can be used as edible films are proteins (gelatin, casein, soy protein, corn protein, and wheat gluten) and carbohydrates (starch, alginate, pectin, arabian gum, and other carbohydrate modifications), whereas the lipid used is wax, glycerol and fatty acids (irianto et al., 2006). according to suryadi (2011), application of ganyong starch and glycerol as edible film on strawberries (fragaria ananasa) can extend the term life of strawberry fruit at room temperature that has a storage time of 2 days longer. kismaryanti (2007) explained that the application of aloe vera l. gel as an edible film can inhibit the deterioration of the quality of tomato fruit that is prolong the storage time up to 3 days at room temperature. this research conducted an application of edible film of ganyong starch and aloe vera l. which is expected to inhibit damage and prolong the storage time of grape at room temperature. http://dx.doi.org/10.14421/biomedich.2016.52.55-59 56 biology, medicine, & natural product chemistry 5 (2), 2016: 55-59 edible film as an active packaging can be a carrier of flavors, dyes, antimicrobial agents and antioxidants (murdianto, 2005). soursop leaves have chemical content such as alkaloids, flavonoids, carbohydrates, saponins, tannins, phytosterols, terpenoids, and proteins. flavonoids in soursop leaves can serve as antioxidants, antimicrobials, anti virals, photosynthetic regulators, and growth regulators (edeoga et al., 2005). antioxidants are one of the additives that can protect the food from damage due to the reaction of fat or oil oxidation. antioxidants can also extend storage time by protecting foods against oxidation-induced deterioration such as rancidity, discoloration and loss of nutritional value (harikedua, 2012). materials and methods materials the materials used in this study were ganyong starch obtained from ugm agro plaza, aloe vera l. and soursop leaf from ngampilan’s market of yogyakarta, glycerol p.a., red grape obtained from ugm mirota campus, 96% ethanol, nacl, silica gel. the tools used for analysis were screw micrometers, universal testing machine, ftir, uv-vis spectrophotometer, and konica minolta cr-400 chromameter. soursop leaf extraction soursop leaf was washed thoroughly and dried. dried soursop leaves then blend until yielding a fine powder. the sample extraction was done by maceration method (immersion). soursop leaf powder weighed as much as 100 g then put into the macerator and added 10 parts solvent. the solvent used is 96% ethanol. the samples were soaked for 5 days and once a day the samples were stirred for homogenization. the macerated extract is separated by precipitation and filtration. it was repeated at least twice with the same type and amount of solvent. the macerated extract was collected, then evaporated with a vacuum evaporator at 55 ºc to obtain a viscous extract (rivai et al., 2013). soursop leaf extract obtained then tested for its antioxidant activity using a dpph method and its functional group using a ftir instrument. edible film preparation edible film preparation was made by 3% of aloe vera gel, 6% of ganyong starch, and 15% glycerol (w/w total) of aloe vera gel. starch weight was suspensioned by addition of 100 ml distilled water. further stirred and heated for 30 minutes at 70 °c ± 5 ° c (afriyah et al., 2015). the suspension was cooled while stirring and then added soursop leaf extract as much as 0.5%, 1% and 1.5% at 50 oc then cooled to 37 ºc. 25 ml of suspension then cast onto a 13x18 cm mica plastic. the suspension of the film was dried at room temperature for 48 hours to allow the edible film to dry and easily removed. edible films produced were tested for chemical properties (ftir) and their physical properties include: thickness, tensile strength, elongation, young modulus, and water vapor transmission rate (wvtr). application to red grape the grape was cleaned with running water and then dried. furthermore, red grape was dipped into 2 edible film solutions. first, its dipped in a solution of ganyong starch and aloe vera l. without addition. other grape was dipped in a solution of ganyong starch and aloe vera l. with addition of soursop leaves extract with best characteristic (0.5%). red grape was dipped in solution at 40 ºc and aerated to dry. the grape that has been coated with edible film was stored at room temperature (25-27 ºc) (lathifa, 2013). data collection was done on days 1, 4, 7, 10 and 13 respectively. red grape samples were tested for weight loss, texture, and color. red grape not coated with edible film used as a control. results and discussion soursop leaf extraction the extraction done in this research yielded the thick extract as much as 10.23 g and the yield of 9.49%. the extract of soursop leaf was then analyzed using ftir spectrophotometer to know the functional groups contained in the extract and tested the antioxidant activity using dpph method. ftir analysis results (figure 1) showed soursop leaf extract gave spectrum on wave number 3387 cm-1 (oh), 2924,09 cm-1 (ch aliphatic), 1735,93 (c = o), 1612,49 cm1 (c = c), 1072.42cm-1 (co). these functional groups are identical with flavonoid compounds such as oh, c-h aliphatic, c = o, c = c, and c-o (suteja et al., 2016) groups. the results of testing of antioxidant activity obtained ic50 value 86.11 ppm and classified as having active antioxidant activity. figure 1. spectrum of ftir test result of soursop leaf extract. widyastuti et al. – effect of addition of soursop leaf extract to … 57 edible film making ganyong and aloe vera l. edible film without addition of soursop leaf extract have a thickness of 0.0325 mm, tensile strength 11,89 mpa, elongation 12,71%, young modulus 0,935 mpa, and water vapor transmission rate 7,45 g/m2hour. the thickness value of ganyong starch and aloe vera l. edible films with the addition of soursop leaf extract ranged from 0,071-0,091 mm as shown in table 1. it showed that the addition of soursop leaf extract effect the resulting film thickness. the greater concentration of the material causes the total amount of solids contained in the edible film to become larger, so that after the edible film suspension is drying the edible film obtained is thicker (sulistiana and widya, 2015). table 1 also shows that the tensile strength of ganyong and aloe vera l. edible films with the addition of soursop leaf extract ranged from 7,578-10,558 mpa. the increased concentration of soursop leaf extract added will increase the tensile strength value of the edible film. it showed that the resulting edible film has a non-fragile nature. the results are similar to murdianto's (2005) who studied the addition of janggelan leaf extract to the tapioca edible film. addition the extract of janggelan leaves causing the increase of tensile strength of the resulting edible film. the addition of janggelan leaf extract to edible film causes more components available to bind to starch molecules, so the resulting edible film becomes more robust and requires greater tensile force to break edible film. table 1. physical properties test result of edible film bulbs ganyong and aloe vera l. with the addition of soursop leaf extract. properties concentrations of soursoup leaf extract 0,5 % 1 % 1,5 % thickness (mm) 0,088 0,091 0,071 tensile strength (mpa) 8,424 7,578 10,558 elongation (%) 11,033 8,719 6,867 modulus young 0,764 0,869 1,538 water vapor transmission rate (g/m2.hour) 6,55 7,88 8,02 elongation of edible film decreased as the concentration of soursop leaf extract added. the same result are the study of edible corn starch film which was added black temu as antioxidant by kusumawati & putri (2013) i.e. where the higher temu hitam added will reduce elongation. the presence of starch still bound to the temu hitam can increase the total solids that can strengthen the film matrix and reduce the elongation of the edible film. young modulus of ganyong starch and aloe vera l. edible film with the addition of soursop leaf extract ranged from 0.764-1.538 mpa. the increase in young's modulus shows the resulting edible film's declining flexibility (nahwi, 2016). based on table 1, the highest young modulus score of 1,538 mpa showed edible film with addition of soursop leaf extract 1.5% (b/b total) more rigid compared with edible film with addition of soursop leaf extract 0,5% and 1%. the rate of water vapor transmission (wvtr) is the amount of moisture lost per unit time divided by the area of the plastic. factors influencing the rate of water vapor transmission are thickness, plasticizer concentration, and type of plastic base material (ekawati, 2015). the increased concentration of soursop leaf extract added will increase the value of water vapor transmission rate from edible film. addition of soursop leaf extract may reduce the hydrogen bonds in the intermolecular bond matrix and allow for the breaking of bonds between the amylose chains (sari et al., 2013). reduced amylose chains that make up the edible film will have an impact on the matrix of the film that has become less dense. the dense film matrix will be easily penetrated by water vapor (santoso et al., 2011). according to afriyah et al. (2015) the value of the vapor transmission rate of the edible film is influenced by aw, rh, temperature, type, edible film-forming properties and the arrangement of starch granules. edible film with flour base tends to have a high rate of water vapor transmission rate. figure 2. ftir spectra of (a) ganyong starch and aloe vera l. edible film without the addition of soursop leaf extract and (b) with the addition of soursop leaf extract. ftir spectra of ganyong and aloe vera l. edible film without soursop leaf extract and with addition of soursop leaf extract are showed in figure 2. its yielded a wide absorption at 3425,58 indicating the vibration of oh group and some shift of wave number that is at 2931,80 cm-1 to 2924,09 cm-1 (ch stretching), 1658,78 cm-1 to 1635,64 cm-1 (c = o), and 1026,13 cm-1 become 1033,85 cm-1 (co). application to red grape the percentage of red grape weight loss (figure 3.) increases with the length of red grape storage. during 58 biology, medicine, & natural product chemistry 5 (2), 2016: 55-59 storage, the shrinkage of fruit weight tends to increase and cannot be prevented. this is due to the physiological processes of respiration and transpiration during storage (lathifa, 2013). figure 3. graph of measurement results of red grape wrap during storage. red grape storage time is determined based on red grape damage against losing weight of 50% using the equation of the line. red grape loses 50% weight loss on 24th day on red grape control, 29th day on red grape coated with edible film without addition of extract, and day 32 on red grape coated edible film with addition of soursop leaf extract. figure 4. graph of red grape grape texture test result during storage. the value of red grape texture test results showed in figure 4 shows that edible film coating on red grape can reduce red grape texture decrease. this is supported by research of sari et al. (2015), which informed that the texture of strawberries decreased during storage and hardness value of strawberries without coating more quickly decreased compared with the coated strawberries. the reduction of red grape texture during storage is caused by the loss of water content in the cells resulting from the respiration and transpiration of red grapes so that the water-deficient cells become softer and weaker. fruit coatings using edible film can suppress high transpiration and respiration in order to inhibit the loss of water content in the fruit tissue (sari et al., 2015). figure 5. graph of test result of l values of red grape during storage. red grape storage period is determined based on texture softening by 50% using the equation of the line. red grape controls were subjected to 50% texture softening on day 13, red grape coated with edible films without extract additions were able to retain 50% of the texture for up to 41 days, and in red grape coated with edible film with the addition of soursop leaf extract subjected to as much texture softening 50% on day 40. figure 6. graph of test result of a red grape value during storage. observations of discoloration on red grape samples were performed using a chromameter tool. interpretation of data to color is translated through the scale l * a * b. the value of l denotes the brightness of the red grape, a scale represents the color red-yellow, and scale b denotes a blue yellow color (kismaryanti, 2007). 0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 w e ig h t lo ss ( % ) strorage time (days) kontrol edible tanpa ekstrak edible +ekstrak 0,5% 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 m a x im u m f o r c e ( n ) storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% 22 24 26 28 30 32 0 1 2 3 4 5 6 7 8 9 10 11 12 13 l v a lu e s storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 10 11 12 13 a r e d g r a p e v a lu e storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% widyastuti et al. – effect of addition of soursop leaf extract to … 59 figure 7. graph of test result of b value of red grape during storage. based on these data, the longer time red grape storage, the value of l, a and b red grape decreased. the observations of the color test showed that the treatment of red grape coating with edible film without the addition of extract and with the addition of extract had not been able to inhibit the decrease of l, a, and b values during storage. conclusion based on the research that has been done, it can be concluded that the best edible film are from the addition of soursop leaves extract 0.5% (w/w total). the addition of soursop leaf extract of 0.5% (w/w total) to edible film of ganyong starch and aloe vera l. can affect the physical properties of edible film, i.e. increasing film thickness from 0,03 to 0,08 mm, decreasing tensile strength of 11,89 to 8,42 mpa, decreased elongation value from 12,71 to 11,03%, young modulus decreased from 0,935 to 0,764 mpa, and decreased vapor transmission rate from 7,45 to 6,55 g/m2.hour. red grape storage time is elongated in terms of 50% shrinkage and 50% texture damage. references afriyah, y., putri, widya dwi r. & wijayanti, sudarma d.. 2015. penambahan aloe vera l. dengan tepung sukun (artocarpus communis) dan ganyong (canna edulis ker.) terhadap karakteristik edible film. jurnal pangan dan agroindustri, 3(4): 1313-1324. donhowe, g. & fennema, o.. 1994. edible film and coating: characteristic, formation, definitions and testing methods. in krochta, j.m., baldwin, e.a. and nisperos-carriedo, m.o. (eds.). edible coating and film to improve food quality. technomic publ. co. inc. lancaster, pennsylvania. p. 378. edeoga, h.o & a. gomina. 2000. nutritional values of some nonconventional leafy vegetables of nigeria, j.econ, taxon, bot, 24 dalam febriani, diana., et al. 2015. karakterisasi simplisia dan ekstrak etanol daun sirsak (annona muricata linn). prosiding penelitian spesia unisba, p. 457480. harikedua, s.d.. 2012. penhambatan oksidasi lipida ikan tuna oleh air jahe selama penyimpanan dingin. jurnal perikanan dan kelautan tropis, viii-1. irianto, h.e., darmawan, m. & mindarwati, e., 2006. pembuatan edible film dari komposit karaginan, tepung tapioka dan lilin lebah (beeswax). j. penel. perik. indonesia, 1 (2): 93– 101. kismaryanti, andiny. 2007. aplikasi gel lidah buaya (aloe vera l.) sebagai edible coating pada pengawetan tomat (lycopersicon esculentum mill.). thesis: fakultas teknologi pertanian, institut pertanian bogor. krochta, j.m & johnston, c.m.. 1997. edible and biodegradable polymer films. j. food technology, 51 (2): 61– 74. kusumawati, d., h., & w. d. r. putri. 2013. karakteristik fisik dan kimia edible film pati jagung yang diinkorporasi dengan perasan temu hitam. jurnal pangan dan agroindustri, 1 (1): 90-100. lathifa, hafidzatul. 2013. pengeruh jenis pati sebagai bahan dasar edible coating dan suhu penyimpanan terhadap kualitas buah tomat (lycopersicon esculentum mill.). thesis: fakultas sains dan teknologi, universitas islam negeri maulana malik ibrahim. murdianto, w.. 2005. sifat fisik dan mekanik edible film ekstrak daun janggelan. universitas gajah mada yogyakarta. nahwi, naufal fadli. 2016. analisis pengaruh penambahan plasticizer gliserol pada karakteristik edible film dari pari kulit pisang raja, tongkol jagung, dan bonggol enceng gondok. thesis: fakultas sains dan teknologi, uin maulana malik ibrahim malang. rivai, h., ernita widiya s, & rusdi. 2013. pengaruh perbandingan pelarut etanol-air terhadap kadar senyawa fenolat total dan daya antioksidan dari ekstrak daun sirsak (annona muricata l.). jurnal sains dan teknologi farmasi, 18 (1): 35-42. santoso, budi, filli pratama, basumi hamzah & rindit pambayun, 2011. pengembangan edible film dengan menggunakan pati ganyong termodifikasi ikatan silang. jurnal teknol dan industri pangan, xxii (2): 105-109. sari, r.p., wulandari, septia t., wardhani & dyah h.. 2013. pengaruh penambahan ekstrak bawang putih (allium sativum) terhadap karakteristik edible film pati ganyong (canna edulis kerr.). jurnal teknologi kimia dan industri, 2 (3): 82-87. sari, rita n., dwi dian novita & cicih sugianti. 2015. pengaruh konsentrasi tepung karagenan dan gliserol sebagai edible coating terhadap perubahan mutu buah stroberi (fragaria x ananassa) selama penyimpanan. jurnal teknik pertanian lampung, 4 (4): 305-314. sulistiana, evi erizha & widya dwi rukmi putri. 2015. komparasi penggunaan tepung ganyong dan tepung sukun terhadap karakteristik edible film kulit jeruk bali. jurnal pangan dan agroindustri, 3 (4): 1325-1336. suryadi, meliani octavia. 2011. aplikasi pati ganyong dan gliserol sebagai edible coating pada stroberi (fragaria ananasa). thesis: fakultas teknologi industri, universitas pelita harapan. suteja, kadek p., rita, wiwik s. & gunawan, i.w.g.. 2016. identifikasi dan uji aktivitas senyawa flavonoid dari ekstrak daun trembesi (albizia saman (jacq.) merr) sebagai antibakteri escherichia coli. jurnal kimia, 10: 141-148. suwarto, agus. 2010. 9 buah dan sayur sakti tangkal penyakit. yogyakarta: liberplus. -0.5 0 0.5 1 1.5 2 2.5 3 3.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 b v a lu e s storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% this page intentionally left blank effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 13-17 | doi: 10.14421/biomedich.2017.61.13-17 issn 2540-9328 (online) anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro1, muhammad jafar luthfi2 1sdi ma’arif tawangsari garum, kompleks perguruan ma'arif garum, tawangsari, garum, tawangsari, garum, blitar, east java 66182, indonesia 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: anisatuzz@gmail.com1 abstract bats are one of the mammals of chiroptera order. chiroptera order has two sub-orders, megachiroptera and microchiroptera. one of the species of megachiroptera is cynopterus titthaecheilus (c. titthaecheilus). local name for c. titthaecheilus is the indonesian short-nosed fruit bat. characteristics of c. titthaecheilus are dark brown body hair and no tragus in the ear. population of c. titthaecheilus in indonesia are relatively abundant, but research about anatomy of male reproduction organs of this species is still rare. the purpose of this study was to determine the anatomical features of male c. tittahecheilus reproductive organs. observations of macroscopic anatomy include observation of morphology, size, weight and volume of penis, testiscle, epididymis, vas defferens, prostate and seminal vesicles. observation on microscopical anatomy of male reproduction organ is using histological slide preparations stained by hematoxylin-eosin. keywords: anatomy; histology; cynopterus titthaecheilus; male reproductive organ. introduction the indonesian short-nosed fruit bat is the local name of the fruit-eating bat of cynopterus titthaecheilus species. bat is living in many areas or islands in sumatra, java, and nusa tenggara. the cynopterus titthaecheilus species are belonging to sub-order megachiroptera of chiroptera order (maharadatunkamsi, 2011). suyanto (2003) stated that bats consist of several species and occupy a second position in number of species related to the population of the mammalian class after rodentia. the majority of these bats live in tropical and semitropical areas. there are 4000 species of mammals, 963 of them are bat species. this species has an important role in the ecosystem as controlling insect pest agent, pollinators of flowers and grain spreaders and guano producers that can be used as fertilizer. its ability to fly and cruising are as far as about 20 km. bats that live in asia and africa have small body size (anonim, 1997). bats live and actively searching for food only at night because bats are very sensitive to dehydration. in the daylight, bat hanging upside down. these animals are nocturnal and therefore require a roosting area during the day. there were also crepuscular bats, they go out from cave during afternoon till dark (prasetyo et al., 2011). there are two factors that can affect the survival of bats, namely external and internal factors. outside factors consist of environment, food and photoperiodicity, whereas inner factor consists of nerves and hormones. photoperiodicity has a serious impact on the size of reproductive organs, the more exposed to the sun exposure the better effect to reproductive organs (isnaeni, 2006). morphology of male reproduction organs of bats differs during hibernation and active period. the testis volume is smaller when it comes to hibernation. at the time of hibernation no spermatogenesis occurs because the body temperature and environment are unstable and incompatible (entwistle et al., 1998). materials and methods this research was carry on for 3 months at zoological laboratory in integrated laboratory of state islamic university sunan kalijaga yogyakarta, and pathology laboratory of veterinary center, wates, yogyakarta. the equipments used in this study were a set of surgical instruments (consisting surgical scissors, tweezers, razors and scalpels), flacon bottles, surgical or paraffin box, needles, digital cameras, ruler, embedding proccessor, tissue proccessor, embedding cassette, base mold, staining jar, incubator, microtome, slide glass, cover glass, toothpick, mask, gloves and optilab® microscope. materials used in this study were hvs paper, ether or chloroform solution, 0.9% nacl physiological solution, fixative bouin, alcohol, hematoxcylin-eosin http://dx.doi.org/10.14421/biomedich.2017.61.13-17 14 biology, medicine, & natural product chemistry 6 (1), 2017: 13-17 dye, albumin, xylol, distilled water, canada balsam, paraffin. in this study, the test animals were ten fruit-eating bat (cynopterus titthaecheilus) of with a weight size ranging from 78.88 ± 3.88 grams. objects observed include testes, epididymis, penis, prostate, vas deferens, seminal vesicles and bulbourethral gland. reproductive organs of test animals were dissect and then put in flacon bottles containing bouin for overnight. the next step is dehydration using stratified alcohol, clearing using xylol and infiltration using paraffin. the next step were embedding and cutting block of paraffined organs. observations were done using optilab® microscope with magnification of 40, 100 and 400 times, and taking picture for each sample. results and discussion description of macroscopic anatomy of male c. titthaecheilus reproductive organs a common characteristic of c. titthaecheilus is dark brown hair color on its entire body and light brown in the neck area. determination of adolescent and adult bats is by looking at the color of the hairs that found on the neck. the darker the brown color of hairs, the more mature the bat. bat tail is not long. the ears are small and slightly elongated. the shape of the face is rather tapered forward (figure 1). figure 1. morphology of bat c. titthaecheilus. (a). ears are slightly tapered; (b). hair on the neck; (c). modification of thumb; (d). clawed toes; (e) short tail. testes of c. tithaecheilus species have a yellowish white color and there are irregular streaks of blood vessels (figure 2). the position of testes of c. titthaecheilus species remains in the abdomen. this is similar to that of the rhinipoma kinneari wroughton species (singwi & lall, 1983). in other mammals such as mice and squirrels, the testes are located in the outer body in scrotum. visible ducts are attached to the testes (figure 3). this duct is called epididymal duct. figure 2. reproductive organs seen from the outside after the integument is removed. (a). testes; (b). epididymis; (c). penis. figure 3. accessory glands of c. titthaecheilus. (a). testis; (b). epididymis; (c). vas deferent vas; (d). seminal vesicles; (e). prostate gland. penis is located in the outside of body cavity, like mammals in general. penis is an external reproduction organ because its location is outside the body. description of microscopic anatomy of male c. titthaecheilus reproductive organs histological or microscopic anatomical characters of c. titthaecheilus reproductive organs are not much different from other bats. in general, testes have functions as a reproductive organ and regulator of the hormonal anisatuzzahro & luthfi – anatomical study of male reproductive organs of the indonesian short-nosed … 15 system. testes of c. titthaecheilus are protected by 2 layers, including tunica vaginalis which is the outermost layer and tunica albuginea which lies beneath tunica vaginalis (figure 4). the tunica albuginea is a layer formed from connective tissue. in tunica albuginea there are septa that form some lobules. the lobule is called the testicular lobule, where in the lobule there is a long that forms the sperm called the seminiferous tubule (figure 5). figure 4. cross-section of testes c. titthaecheilus with he. 100x magnification. (ts). seminiferous tubule; (l). leydig cells; (ta). tunica albuginea; (tv) tunica vaginalis. figure 5. cross-sectional of seminiferous tubule of c. titthaecheilus with he. 400x magnification. (a). sperm; (b). secondary spermatocytes; (c). primary spermatocytes; (d). spermatogonia; (e). thin myoid cells; (f). leydig cells; (g). connective tissue; (h). fibroblast layer; (i). sertoli cells; (j). spermatids; (k). lumen. seminferuos tubule is a canal that has an arch-shaped and forming a coil in a lobule. intersitial space between the seminiferous tubule fills with connective tissue and leydig cells that play a role in producing testosterone hormone. in seminiferous tubule occurs a process of sperm production called spermatogenesis. spermatogenesis involves a large number of epithelial germ cells or epithelial cells of the germ. germinal epithelial cells consist of spermatogonia, spematocytes and spermatids. the position of the spermatogonia are in the basal layer of the seminiferous tubule (figure 5). sometimes these secondary spermatocyte cells are rarely found because the transition of development from primary spermatocytes to secondary spermatocytes is so rapid that it is rather difficult to observe (dreef et al., 2007). process of sperm maturation will occur in the duct of epididymis. epididymis is a duct that delivers spermatozoa to the next duct called vas defferens or commonly called vas deferens. epididymal duct consists of several layers. the outermost layer is composed of smooth, circular muscles, beneath it there is a network of semi-shaped columnar epithelium with stereocillia. the tissue of tunica vaginalis (figure 6) acts as a protective layer. figure 6. cross sectional of epididymis of c. titthaecheilus with he. 100x magnification. (ed). epididymal duct; (e). semi-shaped columnar epithelium with long stereosilia; (tv). tunica vaginalis. figure 7. cross-sectional of vas deferens of c. titthaecheilus stain with he. 100x magnification. (c-sm). circular muscle; (e). epithelial layer; (l-sm). longitudinal smooth muscle; (s). lumen containing sperm. 16 biology, medicine, & natural product chemistry 6 (1), 2017: 13-17 sperm movement is assist by stereocillia that move unidirectional. sperm will move through the epididymis to the vas deferens by peristaltic contraction of smooth muscle tissue connective wall (sinaga, 2011). vas deferens is also known as ductus deferens. vas deferens is a duct that bring sperm to the urethra. this duct is surrounded by a thick layer of muscle. there is a longitudinal smooth muscle and smooth circular muscle (figure 7). there is a layer of epithelium that surrounds the lumen containing sperm. lumen in the deferens duct is narrower than lumen in the duct of the epididymis. there are accessory glands in this species such as seminal vesicles, prostate, and bulbouretralis. this seminal vesicle is one of the accessory glands of the male mammal. between species threre may have different morphological or anatomical forms of this seminal vesicle gland (hafez, 2000). the seminal vesicle gland consists of a mucous that characterized the duct. the mucous occupies almost all of the lumen. this gland is surrounded by a layer of smooth muscle in its outermost. the lumen is in the central part surrounded by the ducts. figure 8. cross-sectional of seminal vesicle gland of c. titthaecheilus stained with he. 400x magnification. (a). lumen; (b). mucous; (c). smooth muscle. figure 9. cross-sectional of prostate gland of c. titthaecheilus stained with he. 100x magnification (a) and 400 times magnification (b). (a). plain muscle; (b). columnar epithelium; (c). lamina propia; (g). tubuloalveolar gland; (s). solid fibromuscular stroma. in addition to seminal vesicle glands, the second gland is prostate gland. in general, the results of previous studies in the prostate gland have solid fibromuscular stroma. most of the prostate gland is occupied by the tubulo-alveolar glands. tubulo-alveolar gland is a form of secretion that is a combination of tubular forms and alveolar (like grape). in this tubuloalveolar gland there is a stratified columnar epithelial cell. in addition there is also a lamina propia that surrounds the epithelial cells. as well as the smooth muscle tissue surrounding the lamina propia. the third accessory gland after seminal vesicle and prostate gland is the bulbourethral gland or commonly known as cowper’s gland. in general, this gland has a function as a secrete producer therefore called exocrine gland (wahyuni et al., 2013). observation of microscopic anatomy from the bulbourethral gland of c. titthaecheilus shows that in this gland there were tubuloalveolar glands that occupy most of the bulbourethral gland space. in tubulo-alveolar gland, a columnar epithelial cell lies at the edge of the tubulo-alveolar lobe (figure 10). the smooth muscle tissue seen around the tubulo-alveolar gland and the secretions inside the tubulo-alveolar lumen appear on the bulbourethral gland preparations. figure 10. cross-sectional of bulbourethral gland of c. titthaecheilus stained with he. 100x magnification. (a). tubulo-alveolar glands; (b). columnar cells of the lining; (c). smooth muscle; (d). secretions in the lumen of the gland. penis is a copulation organ for mammals that has the function of transferring sperm from the male reproductive tract to the female reproductive tract. in this microscopic anatomy there is a major component found in the penis namely two corpus cavernosun and one corpus spongiosum and penis urethra. as a major component, corpus cavernosum and corpus spongiosum is an erectile network. anisatuzzahro & luthfi – anatomical study of male reproductive organs of the indonesian short-nosed … 17 figure 11. cross-sectional of penis of c. titthaecheilus stained with he. 40x magnification. (cc). corpora cavernosa, (cs). corpus spongiosum; (p). veins; (ta). tunica albuginea; (u). urethra. cynopterus titthaecheilus penis do not have fibrocartilago and os penis or baculum tissue (figure 11) which are usually present in chiropteran mammals. baculum or os penis has a function as a supporting assistance to penetrate the vaginal canal of female mammal animals as well as improve transport and deposition during mating (vamburkar, 1957 in danmaigoro, 2014). conclusion species of fruit-eating bats (cynopterus titthaecheilus) has no baculum (os penis and fibrocartilago). the mature animal of this species do not descent its testes to scrotum, instead of retain in body celoem. the testes and its accessories gland have similar histological structure to other mammal in general. references anonim.1997. bat facts and amazing trivia. beautifortia, 40: 111177. dreef, h.c., van esch e., de rijk e.p.c.t. 2007.spermatogenesis in cynomolgus monkey (macaca fascicularis): a practical guide forroutine morphological staging. toxicolpathol, 35: 395-404. entwistle, a.c., p.a. racey & j.r. speakman. 1998. the reproductive and determination of sexual matury in male brown long-eared bats, plecotus auritus (chiroptera: vespertilionidae). (abstract) journal of zoology, 244:63-70. hafez, e.s.e. 2000. reproduction in farm animals. hafez (7 th ed.). lippincott william & wilkins. a wolter kluwer company. isnaini, wiwi. 2006. fisiologi hewan. yogyakarta: penerbit kanisius. maharadatunkamsi. 2011. profil fauna mamalia kecil gunung. slamet. jurnal biologi indonesia, 7 (1): 171-185. prasetyo, pandam nugroho., s. noerfahmy & h.l. tata. 2011. jenis-jenis kelelawar khas sumatera. world agroforestry centre icraf, sea regional office: 75. sinaga,h.2011.http://repository.usu.ac.id/bitstream/123456789/25 485/4/chapter%20ii.pdf/ retrieved at 07/08/2015 time 04.29 wib. singwi m.s. & lall s.b. 1983. spermatogenesis in the nonscrotal bat-rhinopoma kinneari wroughton (microchiroptera: mammalia). (abstract). 116 (3): 136-145. suyanto, a. 2003. kelelawar pemakan buah dan taman nasional gunung halimun. zoo indonesia, 5 (2): 31-40. vamburkar, s. 1957. the male genital tract of the indian megachiropteran bat cynopterus sphinx gangeticus anderson. (summary) proceeding of the zoological society of london, 130: (57-77). wahyuni, s., l.e.m manik., srihadi a., m. agil., t.l yusuf., hamny., i ketut mudite a. 2013. morfologi kelenjar aksesori kelamin muncak (muntiatus muntjak muntjak) jantan. acta veterinaria indonesia, 1 (2): 84-93. this page intentionally left blank anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 1-4 | doi: 10.14421/biomedich.2018.71.1-4 issn 2540-9328 (online) leukocytes description of mudskipper (periophthalmodon schlosseri) of barito river estuary, desa tanipah, kalimantan selatan heri budi santoso*, hidayaturrahmah, muhamat biology department, mathematics and science faculty, lambung mangkurat university, south borneo jl. brigjen h. hasan basri, banjarmasin utara, banjarmasin, kalimantan selatan, indonesia author correspondency*: heri_budisantoso@yahoo.com abstract leukocytes have an important role in driving away infections from pathogen microorganism by phagocytosis together with macrophages. the aim of this research was to analyze the leukocytes’ differentiation (eosinophil, basophil, neutrophil, lymphocytes, and monocytes) of mudskipper‘s blood (periophthalmodon schlosseri). samples were taken from desa tanipah kalimantan selatan. the sampling locations were decided by purposive or taking the data intentionally according to the consideration of mudskipper‘s location which is not homogenous. the data collection were conducted using line transect which means the sample was taken according to how many encounters can be taken. there were 15 mudskippers taken from desa tanipah used in this research. the parameter observed was the differentiation of leukocytes which consists of the percentages of monocytes, lymphocytes, eosinophil, basophil, and heterophil. the result showed that the percentage of lymphocytes in mudskippers is 62+4,1% and monocytes 24,7+0,8%, and the neutrophil is 0,5+0,1% and eosinophil 0,6+0,1% and no basophils were found. according to the result, it can be concluded that lymphocytes is the most dominant one amongst others. keywords: leukocytes’ differentiation; desa tanipah; periathalmodon schlossery; mudskipper introduction the description of differentiation of leukocytes is one of several parameters to complete the blood’s profile, because the leukocytes’ differentiation is component which has important function as first self-defense when diseases or patogen struck dellman & brown, 1989). the leukocytes’ differentiation consists of lymphocytes, monocytes, neutrophil, basophil, and eosinophil. leukocytes have a role in driving away the infections from pathogen microorganism by phagocytosis together with macrophage. the number of leukocytes in fish is more than the number of leukocytes in human’s blood (fujaya, 2004). the number of leukocytes of human’s blood is about 5.000-9.000 cells/µl, whereas the number of leukocytes in fish’s blood is 20.000-150.000 sel/µl. if the infective materials come in, the number of leukocytes in blood will increase (moyle & cech, 1988). mudskipper/ikan timpakul/ikan gelodok (periophthalmodon schlosseri) is found along the coastal beaches and the habitat is spread in indonesia, thailand, peninsular of malaysia, singapore and some coastal beaches of indonesia (baker, 2011). this animal is included as threatened species because the condition of coastal mangroves increasingly damaged everyday (bay science foundation, 2009; fadli, 2010). for some provinces in indonesia, the flesh of mudskipper is used as the medicine for asthma (fadli, 2010). moreover, p. schlosseri is known has potency as bio-indicator in coastal ecosystem (shirani et al., 2010). the estuary of barito river is the mouth’s river with mangrove forest which is more than 30 km along the west coastal and around 20 km along the river to the inland. the swamps of mangroves forest which are rich of fauna who can adapt to the mud which is changing from the inundated to opened (flux and reflux). one of the faunas that can be found in the coastal beaches at the barito river estuary kalimantan selatan is mudskipper (periopthalmodon schlosseri) (mackinnon et al., 2000; shirani et al., 2014). according to lavabetha et al. (2015) mudskipper’s (p. schlosseri) blood profile from muara sungai barito kalimantan is the number of erythrocytes is 3,36±0,1x10 6sel/µl; the he level is 12,38±0,56 gr%; the hematocrit is 41,53±0,60 %; the number of mcv is 123,78±3,94 μm3 ; the number of mch is 36,88±1,82 pg/sel; the number of mchc is 29,80±1,18 g/dl. the average of leukocytes’ total of mudskipper (p. schlosseri) is 22,62±5,09 x103 cells/µl, whereas the number of leukocytes in common fish is between 30.000 until 150.000 cells/µl. it can be said that the amount of leukocytes of p. schlosseri is higher than the leukocytes’ number in other animals (hrubec & smith, 2000). the research was done to describe differentiation of leukocytes of mudskipper at barito river estuary, https://doi.org/10.14421/biomedich.2018.71.1-4 2 biology, medicine, & natural product chemistry 7 (1), 2018: 1-4 especially at desa tanipah, has never been reported before. based on that consideration, this research was focused on the differentiation of leukocytes (eosinophil, basophil, neutrophil, lymphocytes and monocytes) of p. schlosseri from barito river estuary, kalimantan selatan. the objective of this research was to analyze the differentiation of leukocytes (eosinophil, basophil, neutrophil, lymphocytes and monocytes) of mudskipper’s blood (periophthalmodon schlosseri) at barito river estuary, desa tanipah, kalimantan selatan. materials and methods tools and materials the tools used in this study were a set of fishing equipment, bucket, drain, analytic balance, tray, tissue paper, object’s glass, syringe, haemacytometer, camera, and microscope. the materials were blood (periophthalmodon schlosseri), edta solution (ethylene diamine tetra acid), giemsa solution 10%, alcohol 70%, methanol and clean water. the procedure of research the decision of location in taking samples the determination of sampling location in taking samples was conducted in purposive or taking the samples incidentally according to the consideration of heterogeny of the sample location. the samples collected in a region flux and reflux of bahagia river, desa tanipah kecamatan tabunganen kabupaten barito kuala, south borneo. the samples was taken by line transect method namely the samples were taken according to its encounter. preparation of blood samples the blood’s samples of mudskipper were taken from caudal vein between the squama near the tail using syringe which was dampened with anticoagulant edta (ethylene diamine tetra acid). syringe’s needle was inserted from the anal into vertebra until the needle touched the bone. the blood was suctioned for 1 ml then the needle will be put off, and the blood’s samples was moved to the tube (erika, 2008). preparation of blood cell slides blood cell preparation was conducted by placing a drop of blood in object glass. the second object’s glass was put with angle 45° above the first one, then moved it to the back and touched the blood so the blood was spread. the second object glass was moved to the opposite direction so it made a thin layer of blood. the blood cell slide preparation was then air dried. after that, fixation would be conducted by saturated the preparation in methanol for 5 minutes, then it would be dried the slide was then immersed to giemsa solution for 30 minutes. after that, it was washed with clean water and let it dry. then, the preparation was observed under light microscope with strong magnification, and it was counted for each type of leukocytes (roberts, 1992). the observation of leukocytes’ differentation the observation of leukocytes’ differentiation was conducted to decide the percentage of each leukocyte’s type in blood, by observed the blood cell slides under microscope. the micro setting in microscope was used, then it was counted from the side to the underside, then it moved to the right and to the top and so on. the calculation of leukocytes was conducted with strong magnification. the method used was shilling method which the calculation was conducted with different viewing field as 10 viewing fields or until the leukocytes’ number was reached 100 (mitruka & rawnsley, 1977). the technique of data collection the data collection was quantitative. quantitative data consisted of the number of leukocytes’ blood of mudskipper (eosinophil, basophil, neutrophil, lymphocytes and monocytes). the quantitative data taken in this research was processed statistically. the data was served in mean and standard deviation. results and discussion results the result of laboratory’s analysis of the measurement of leukocytes’ differentiation of mudskipper in barito river estuary was served in table 1 and figure 1 as follows: table 1. description of percentages (%) leukocytes’ differentiation of mudskipper. no fish (n) length (cm) weight (g) lymphocytes monocytes neutrophils basophil eosinophil p1 5 23,8 157,4 58,6 25,4 0,6 0 0 p2 5 24,12 162,6 60,8 25 0,2 0 0,8 p3 5 23.32 153,64 66,6 23,8 0,8 0 1,0 average 5 16,30 + 13,27 157,88+ 4.5 62+4,13 24,73+0,83 0,53+0,1 0 0,6+0,1 santoso et al. – leukocytes description of mudskipper … 3 figure 1. description of differentiation of mudskipper leukocytes. magnification 40 x. discussion lymphocytes percentage from the observation of lymphocytes percentage, it was found around 62+4,13%. according to the observation’s result, the taken average percentage was under normal range. the normal percentage of lymphocytes in fish was between 71,12 82,88% (robert, 1978). this condition showed the reduction of lymphocytes’ number which called as lymphocytosis. lymphocytes found in mudskipper were more than human lymphocytes around 20% 40%. the more lymphocytes in body the more body can attack the disease. lymphocytes had an important role in immune’s respond and it produced the antibody. according to jain (1986) & jain (1993), lymphocytes had important roles in producing the hormonal and cellular immunity to attack and destroy the disease agent. the increase of leukocytes number in blood circulation was called lymphocytosis; however, the decrease was called leucopenia. the decrease of lymphocytes’ number in peripheral blood occured because most lymphocytes were pulled from the circulation and concentrated into the tissue where there was inflammation (jain, 1993). moyle & cech (1988) reported that lymphocytes functioned as antibody’s production to face foreign materials from outside if there were a decreasing percentage of lymphocytes in circulation when an infection occured, it predicted as lymphocytes activity in antibody production was disturbed. the response of leukocytes’ number and leukocytes’ differentiation will be iaffected by several stressor, such as, temperature, season, activities, hunger’s condition, self-defense, maintenance, and density, diseases, infections, parasites, poisoned and metals. the leukocytes’ number and leukocytes’ differentiation will be increased or decreased (schalms, 2000). mudskipper had higher activities if we compared to other fish because it is air-breathing and it can adapt in land and water. the difference of haematological parameter of fish reflected the ecological condition in their habitat and it was their physiological adaptation in their life’s way. monocytes’ percentages from observation, the monocytes found was 24,73% which the monocytes percentage in fish commonly was 0,1% (robert, 1978). according to moyle & cech (1988), there would be less monocyt in leukocyt unless there were infection in blood circulation. roberts (1978) reported that the monocytes percentage in fish was 0,1% from the total population of circulated leukocytes. monocytes had a role as macrophage and it was found in an infection’s area (dellman & brown, 1989). monocytes and macrophage of the tissues will do the phagocytosis to the tissues’ cells and the disease agent (nabib & pasaribu, 1989). neutrophil’s percentage from observation, the neutrophil found was 0,53% which the monocytes percentage was below the normal average. the percentage of normal average of neutrophil of fish was around 6-8% (roberts, 1978). the neutrophil’s cell functioned in the blood’s vein in phagocytosis the bacteria faster because p. schlosseri was spending more time outside the water. in this case, it can be said that neutrophil cell functioned in blood’s veins in phagocytosis the bacteria faster because p. schlosseri was spending more time outside the water. 4 biology, medicine, & natural product chemistry 7 (1), 2018: 1-4 eosinophil and basophil percentage the observation result from mudskipper blood samples were there was no basophil cell (0) and the eosinophil was 0,6%. the average percentage of basophil cell of mudskipper was under the normal average, which the normal average was between 0.5–1%. nabib & pasaribu (1989) reported that eosinophil and basophil was rarely participated in fish blood circulation. the eosinophil and basophil play a role in parasitic infections and allergic response which connected to the critical disease. conclusion according to the result of this research, the lymphocytes percentage was found as the most dominant amongst other leukocyt types, which was 62%. the monocyt percentage was 24.73% and the eosinophil percentage was 0.6%. the neutrophil percentage was found as the lowest one amongst all the population of leukocyt type which was 0.5% whereas basophil was not found. references bay science foundation. 2009. periophthalmodon schloseryi (pug-headed mud skipper). zip code zoo index to als. http://zipcodezoo.com/animals/p/periophthalmodon%5fschl osseri/default.asp dellman, h.d & e.m. brown. 1989. textbook of veterinary histology. hartono's translation. ui press, jakarta. erika, y. 2008. description of leukocyte differentiation in mujair fish (oreochromis mossambicus) in ciampea region bogor. essay. faculty of veterinary medicine. ipb, bogor. fadli, a. 2010. timpakul, from aspiration. e-paper compass. january 8, 2008. http://m.kompas.com/iphone/read/data/2008.01.08.18214974 fujaya, y. 2004. fish physiology, basic development of fishery technique. pt rineka cipta. jakarta hrubec, t.c. & s.a. smith. 2000. hematology of fish. in feldman, b.f., j.g. zinkl, & n.c. jain (editor). schalm's veterinary hematology fifth jain, n.c. 1993. essentials of veterinary hematology. lea & febiger. philadelphia. 417 pp. lavabetha, a.r.r.r. hidayaturrahmah, muhamat, heri budi santoso. 2014, timpakul fish blood profile (periophthalmodon schlosseri) from barito river estuary south kalimantan thesis, bioscientiae volume 12, number 1, january 2015, page 78 89 mulyanti. w. 2013, differentiation differentiation of tilapia leucocytes (oreochromis niloticus l.) in river waters of riam kanan district of banjar thesis, faculty of mathematics and natural sciences. university of lambung mangkurat. banjarbaru mitruka, b.m & h.m. rawnsley. 1977. clinical biochemical and hematological refferent value in normal experimental animal. mason publishing, usa.165-181 pp. moyle, p.b. & j.j. cech. 1988. fish an introduction to ichthyology second edition. prentice hall, new jersey. nabib, r & f.h. pasaribu. 1989. pathology and fish disease. department of education and culture. directorate general of higher education. inter-university center of biotechnology. bogor agricultural university, bogor. roberts, r.j. 1978. fish pathology. ballier tindall london philadelphia sydney shirani, m., a. mirvaghefi, h. farahmand, & m. abdollahi. 2010. biomarker responses in mudskipper (periophthalmus waltoni) from the coastal areas of the persian gulf with oil pollution, environmental toxicology and pharmacology 10: 4-29 schalm, o.w. 1965. veterinary hematology, 2th ed. lea & febiger, philadelphia. leukocytes description of mudskipper(periophthalmodon schlosseri) of barito river estuary, desa tanipah, kalimantan selatan biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 45-49 | doi: 10.14421/biomedich.2018.72.45-49 issn 2540-9328 (online) diversity of angiospermae plant class liliopsida in mount nglanggeran bayu setya aji nugraha*, widodo, rendi yuntara, normalita department of biology education, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: bayu.08114@gmail.com abstract nglanggeran is a place that has a high plant diversity and there are many unique and unidentified wild plants. this study aims to list liliopside class plants which found around the climbing route. the angiosperm plants in the liliopside class found around the climbing route were successfully identified and consisted of 40 species belong to 17 families. keywords: diversity plants; liliopsida; mount nglanggeran introduction indonesia is a country with a high level of plant diversity of approximately 30 thousand species of 40 thousand species of plants that exist in the world (fahrurozi, 2014). the level endemicity of indonesian flora is recorded between 40-50% of the total flora species on each island except sumatra island which is estimated at only 23% (lipi, 2014). according to bappenas (2016), this data is not accurate and data collection and name validation are still needed. the species that have been identified and recorded only reach 50% of the total number of flora recorded, that is 19,112 species. but the rate of extinction and reduced diversity of plant species has accelerated. this is a problem and a challenge that must be solved. another problem where the majority of research focuses only on horticulture plants and ignores wild plants that have not been identified. the data about wild plants can be applied in other field as health, food, environmental and etc. (widodo, 2015). mount nglanggeran is a place that has high plant diversity and there are many wild plants that have not been identified. widodo (2015) in his exploration on mount nglanggeran found various types of unique plants and are rarely found in residential areas. some of these plants are also found around the region of baturagung. the plant is not recognized by the people and its local name is unknown. probably, the local name of the plant had known in the past but the knowledge does not pass to the next generation. this shows why taxonomic and systematic studies are needed so that knowledge of plants can be passed onto the next generation. this paper aimed to list the diversity of liliopsida plants wich found around the climbing route of ngelanggeran volcano. materials and methods this research is a field research that uses survey and exploration methods. equipments for observation and collection consist of: nikon d320 digital camera and fuji mirrorless, smartphones, stationery, observation sheets, maps applications, and complete plant books. this research is divided into three stages, namely exploration of climbing routes, collection of photo specimens, and discovery of plants. plant were indentify using various literature and matching with herbariums and illustrations/images in the literature for the discovery of specimens found. result and discussion angiosperms of the liliopsida class found around the nglanggeran volcano were 39 species grouped in 16 families (table 1). the species are grouped into family araceae, two species from commelinaceae, one species from costaceae, one species from hypoxidaceae, four species from zingiberaceae, six species from orchidaceae, five species from dioscoreaceae, one species from pandanaceae, three species from smiles, one species of agavaceae, one species of tacaceae, one species of liliaceae, and one species of colchiaceae (figure 1). https://doi.org/10.14421/biomedich.2018.72.45-49 46 biology, medicine, & natural product chemistry 7 (2), 2018: 45-49 table 1. liliopsida class plants found around the nglanggeran volcano climbing trail. family species name family species name araceae alocasia crassifolia flagellariaceae flagellaria indica amorphophallus variabilis hypoxidaceae curculigo latifolia anadendrum latifolium liliaceae zephyranthes rosea epripemnum aureum orchidaceae dendrobium crumenatum caladium sp liparis sp pothos scandens nervilia plicata typonium trilobatum pholidota imbricata syngonium podophyllum pecteilis susannae arecaceae arenga pinnata coelogyne trinervis colchicaceae gloriosa superba pandanaceae pandanus bouetii commelinaceae cyanotis cristata smilacaceae smilax spinosa commelina difusa smilax anceps costaceae costus speciosus smilax celebica cyperaceae kyllinga nemoralis tacaceae taca palmata scleria laevis zingiberaceae curcuma longa cyperus imbricatus globba racemosa cyperus rotundus zingiber zerumbet dioscoreaceae dioscore bulbifera dioscorea alata disocorea hispida dioscorea oppositifolia dioscorea pentaphylla figure 1. diagram of plant family class of liliopsida araceae is the most abundant family of species, among the 8 species found, alocasia crassifolia and amorphophallus variabilis are abundant species and are often found around climbing routes. alocasia crassifolia has large leafy features, up to 1.2 x 2 m round shape, the leaf veins are very clear. the leaves are dark green, while the bottom is dull green, has a long leaf stalk reaching 0.4 1 m. male and female flowering is located on a long-stemmed cob which exits at the end of the stem. egg-shaped fruit with orange color when ripe. the fruit contains only one seed. this plant prefers in wet environment and can be found on the banks of rivers, lakes and mountain slopes which are rather humid (suhono et al., 2010). whereas amorphophallus variabilis has the characteristics of the leaf stalk morphology varies widely, the surface of the leaf stalk is flat or rough, the color of the tuber skin is white, green, gray or purple. (afifah et al, 2014). figure 2. alocasia crassifolia. figure 3. amorphophallus variabilis. some unique species that are rarely found include pothos scandens, curculigo latifolia, nervilia plicata, pholidota imbricata, and smilax anceps. nugraha et al. – diversity of angiospermae plant class liliopsida in mount nglanggeran 47 figure 4. pholidota imbricate. figure 5. smilax anceps. conclusion the diversity of plants in the liliopasida class at the ancient volcano nglanggeran consists of 39 species belong to 16 families. eight species are grouped into family araceae, two species from commelinaceae, one species from costaceae, one species from hypoxidaceae, four species from zingiberaceae, six species from orchidaceae, five species from dioscoreaceae, one species from pandanaceae, three species from smiles, one species of agavaceae, one species of tacaceae, one species of liliaceae, and one species of colchiaceae. araceae is the most abundant family of species, among the 8 species found, alocasia crassifolia and amorphophallus variabilis are abundant species and are often found around climbing routes. some unique species that are rarely found include pothos scandens, curculigo latifolia, nervilia plicata, pholidota imbricata, dan smilax anceps. acknowledgements the authors wish to thank to m. ja'far luthfi for the discussion, didik zulfahmi akbar and all student who accompanied researcher during the research. references afifah, e., nugrahani, m. o. & setiono, 2014. peluang budidaya iles-iles (amorphophallus spp.) sebagai tanaman sela. warta perkaretan 2014, pp. 35-46. bappenas. 2016. indonesian biodiversity strategy and action plan (ibsap) 2015-2020. kementerian perencanaan pembangunan nasional/bappenas. indonesia fahrurozi, irpan. 2014. keanekaragaman tumbuhan obat di taman nasional gunung gede pangrango dan di hutan terfragmentasi kebun raya cibodas serta pemanfaatannya oleh masyarakat lokal. (skripsi). universitas islam negeri syarif hidayatullah jakarta lipi [lembaga ilmu pengetahuan indonesia]. 2014. kekinian keanekaragaman hayati indonesia 2014. kerjasama kementrian ppn/bapennas, klh dan lipi. bogor: lipi press. suhono, budi dan tim lipi. 2010. ensiklopedia flora jilid 1. bogor: pt kharisma ilmu. widodo. 2015. apocynoideae dan asclepiadoideae dari pegunungan baturagung (gunung nglanggeran, gunung mintorogo, gunung parangan, gunung gedang, gunung ijo): inisiasi pencirian dan konservasi. seminar nasional konservasi dan pemanfaatan sumber daya alam. fkip uns. 48 biology, medicine, & natural product chemistry 7 (2), 2018: 45-49 alocasia crassifolia amorphophallus variabilis anadendrum latifolium caladium sp commelina difusa costus speciosus curculigo latifolia curcuma longa cyanotis cristata cyperus imbricatus cyrtococum sp. dendrobium crumenatum dioscorea sp. dioscorea bulbifera dioscorea alata dioscorea hispida dioscorea oppositifolia dioscprea pentaphylla epripemnum aureum globba racemose gloriosa superba kyllinga nemoralis liparis sp. nervilia plicata nugraha et al. – diversity of angiospermae plant class liliopsida in mount nglanggeran 49 pandanus bouetii pholidota imbricate photos scandens scleria laevis smilax smilax spinose syngonium podophyllum taca palmata typhonium trilobatum zephyranthes rosea zingiber zerumbet biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 47-52 | doi: 10.14421/biomedich.2019.82.47-52 issn 2540-9328 (online) liver profile of atazanavir/ritonavir in pregnant albino rats elias adikwu1,*, james kemelayefa1, winifred ocheiga2 1department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, rivers state, nigeria corresponding author* adikwuelias@gmail.com abstract medication use during pregnancy is challenging due to the occurrence of maternal or fetal toxicities. atazanavir/ritonavir (atv/r) has hepatotoxic potential hence; use in pregnant patients living with human immunodeficiency virus may cause maternal hepatotoxicity. this study assessed the liver profile of atv/r in pregnant albino rats. thirty pregnant albino rats randomized into groups were orally treated daily with atv/r (4.28/1.43 mg/kg-34.3/11.4 mg/kg) for 16 days. after treatment, the rats were weighed and sacrificed. blood samples were collected and examined for serum biochemical parameters. liver samples were weighed and assessed for biochemical and histological changes. body and liver weights were normal (p>0.05) in atv/r-treated pregnant rats when compared to control. serum total cholesterol, triglyceride, low density lipoprotein cholesterol and blood glucose levels were significantly (p<0.01) elevated whereas high density lipoprotein cholesterol level was significantly (p<0.01) decreased in rats treated with atv/r (34.3/11.4 mg/kg) when compared to control. liver and serum aminotransferases, alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase, total bilirubin, and conjugated bilirubin levels were significantly increased in a dose-dependent fashion in rats treated with atv/r; 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control. liver superoxide dismutase, catalase, glutathione and glutathione peroxidase levels were significantly decreased whereas malondialdehyde levels were significantly increased in a dosedependent fashion in rats treated with atv/r; 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control. necrotic hepatocytes were observed at higher doses of atv/r. atv/r may not be hepatotoxic in pregnant women living with hiv at the clinical dose. keywords: atazanavir/ritonavir; pregnancy; liver; toxicity; rat introduction human immunodeficiency (hiv) infection is a leading cause of maternal and neonatal morbidity and mortality (abdol-karim et al., 2010). pregnancy, whether actual or anticipated, has been a critical driver for the diagnosis, treatment and care of women living with hiv to prevent transmission from mother to child. this necessitates the use of highly active antiretroviral therapy (haart) in pregnant women living with hiv and neonates exposed to hiv. this strategy has drastically reduced mother to child transmission of hiv (coovadia et al., 2007). most haart have demonstrated favorable safety profiles for mothers and infants during trial follow-up (coovadia et al., 2007). however, toxicities have been reported in some quarters with growing concern on hepatotoxicity in pregnant women (bera et al., 2012). the use of medication during pregnancy has been a serious clinical challenge in terms of maternal and fetal safety due to physiological changes and complications that do arise during pregnancy. hepatitis, intrahepatic cholestasis, and acute fatty liver, are some complications that may occur during pregnancy (hammoud et al., 2014). also, pregnancy could increase the incidence of treatment related adverse effect including hepatotoxicity (joy et al., 2015; clark, 2015) and has a significant impact on the efficacy and safety of a drug (abduljalil et al., 2012). atazanavir/ritonavir (atv/r) is use as a component of haart in the management of hiv (mandelbrot et al., 2011). its use in hiv management has decreased mortality associated with hiv. it has a high genetic barrier to resistance, favorable adherence profile and low effects on lipids and glucose metabolism (achenbach et al., 2011). however, it has been associated with hyperglycemia, alterations in cardiac conduction and renal toxicity (clayden, 2009; conradie et al., 2011; hamada et al., 2012). furthermore, elevations in serum liver enzymes and indirect hyperbilirubinemia have been reported hence, use in pregnant women living with hiv raises toxicological concern such as hepatotoxicity (eholié et al., 2013). this study assessed the liver profile of atv/r in a pregnant albino rat model. material and methods animal and drugs pregnant albino rats (200g-250g) used for this study were obtained from the animal house of the department of pharmacology and toxicology, madonna university, nigeria. the rats were housed under room temperature https://doi.org/10.14421/biomedich.2019.82.47-52 48 biology, medicine, & natural product chemistry 8 (2), 2019: 47-52 with a 12:12 light: dark cycle. the rats were randomized into 6 rats per cage according to their weights and had free access to diet and water. atv/r used for this study was manufactured by mylan laboratories limited india. the doses of atv/r (4.28/1.43, 8.57/2.86, 17.1/5.72, and 34.3/11.4 mg/kg) used for this study represent clinical dose, 2, 4, and 8, times the clinical dose respectively (von hentig et al., 2007). experimental protocol the reproductive status and estrous period of the rats were determined by obtaining their virginal smears. after two complete regular cycles, timed mating of female rats was done on the night of the pro-estrous (n) phase of the cycle. in the morning following mating, vaginal smears were taken again. the presence of spermatozoa and squamus cells in the smear confirmed mating and fertilization of ovule. the sperm – positive morning was thus designated day 0 of pregnancy. thirty pregnant albino rats used were weighed and divided into five groups labeled a–e of six rats each. group a (control) orally received normal saline (0.2ml) whereas groups b-e orally received atv/r (4.28/1.43 mg/kg, 8.57/2.86 mg/kg, 17.1/5.72 mg/kg and 34.3/11.4 mg/kg) daily for 16 days respectively. collection of samples and biochemical analyses the rats were sacrificed on day 17 after exposure to inhalational diethyl ether. blood samples were collected and centrifuged at 1200g for 15 minutes and sera extracted. sera were analyzed for biochemical parameters. liver samples were collected, weighed and washed in a cold 1.15 % kcl solution and homogenized in 0.1 m tris-hcl buffer, ph 7.4. the homogenates were centrifuged at 1200 g speed for 15 minutes and the supernatants were collected and evaluated for biochemical parameters. alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), gamma-glutamyl transferase (ggt), lactate dehydrogenase (ldh), total bilirubin (tb), conjugated bilirubin (cb), triglyceride (tg), total cholesterol (tc), and high density lipoprotein cholesterol (hdl-c) were evaluated using commercial test kits (randox diagnostics, crumlin, uk). blood glucose (g) was estimated with the aid of a glucometer whereas low density lipoprotein cholesterol (ldl-c) was estimated as reported by friedewald et al. (1972). liver superoxide dismutase (sod) was determined as described by sun and zigma, (1978). catalase (cat) was assayed according to aebi, (1984). glutathione (gsh) was estimated according to sedlak and lindsay, (1968). malondialdehyde (mda) was determined as reported by buege and aust, (1978). the method of rotruck et al. (1973) was used for the evaluation of glutathione peroxidase (gpx) whereas total protein was evaluated according to gonall et al. (1949). histological examination of the liver liver samples were collected, cleaned and weighed. liver samples were fixed in10% buffered neutral formalin for 24h. liver samples were dehydrated in increasing ethanol concentrations and mounted in paraffin block. paraffin sections (3-5 μm) were prepared, deperaffinized and stained with hematoxylin and eosin dye (h&e). stained sections were examined using a light microscope for histological changes. statistical analysis results are expressed as mean ± sem and were evaluated using one way analysis of variance (anova) followed by dunnett’s post hoc tests. results were considered to be significant at p<0.05; 0.01; 0.001. results the body and liver weights of pregnant rats treated with atv/r were not significantly (p>0.05) different when compared to control (table 1). significant increases in a dose-dependent fashion occurred in the liver and serum ast, alt, alp, ggt, ldh, tb and cb levels in rats treated with atv/r (tables 2 and 3). the increases in the levels of the aforementioned parameters were not significant (p>0.05) at the clinical dose (4.28/1.43 mg/kg), but were significant at 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4mg/kg (p<0.001) when compared to control (tables 2 and 3). serum tg, tc, ldl-c and blood g levels were significantly (p<0.01) increased whereas serum hdl-c level was significantly (p<0.01) decreased in pregnant rats treated with atv/r (34.3/11.4 mg/kg) when compared to control (table 4). treatment with atv/r produced decreases in liver sod, cat, gsh and gpx levels with increases in mda levels in a dose-dependent fashion (table 5). the effects on liver sod, cat, gsh, gpx and mda levels were not significant (p>0.05) at the clinical dose (4.28/1.43 mg/kg), but were significant at 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control (table 5). the liver of control rat showed normal hepatocytes (figure a). the liver of rats treated with atv/r (4.28/1.43 mg/kg) showed inflammatory cell infiltration (figure b). in contrast, the liver of rats treated with atv/r; 8.57/2.86 mg/kg, 17.1/5.74 mg/kg and 34.3/11.4 mg/kg showed hepatocyte necroses respectively (figures c, d and e). adikwu et al. – liver profile of atazanavir/ritonavir in pregnant albino rats 49 table 1. effects of atazanavir/ritonavir on body and liver weights of pregnant albino rats. group body weight (g) absolute liver weight (g) relative liver weight (%) a b c d e 250.7±12.4 257.6 ±13.2 255.1±10.9 260.6±11.2 250.4± 10.7 7.88±1.14 7.58±1.65 7.46±0.57 7.91±0.32 7.80 ±1.42 3.15±0.27 2.95±0.14 2.93±0.09 3.04±0.43 3.12±0.83 tv/r =atazanavir/ritonavir, data are expressed as mean± sem. n=6 table 2. effect of atazanavir/ritonavir on serum liver function parameters of pregnant albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) cb(g/dl) tb(g/dl) a 45.5±3.99 34.2±2.15 47.3±3.56 48.5±3.12 0.73±0.04 4.50±0.18 8.54±0.71 b 50.3±3.46 38.8±2.25 51.9±4.06 52.1±4.00 0.80±0.02 4.87±0.25 9.01±0.36 c 66.7±5.00a 45.8±4.70a 70.4±5.36a 78.2±5.31a 1.31±0.19a 6.44±1.22a 12.7±1.11a d 96.3±7.87b 63.3±2.62b 99.4±8.66b 121.1±10.6b 1.90±0.12b 8.95±1.06b 15.5±1.40b e 140.6±10.3c 128.8±7.00c 155.3±10.3c 180.4±12.5c 2.60±0.52c 13.3±1.01c 23.5±2.22c atv/r: atazanavir/ritonavir, alt: alanine aminotransferase, alp: alkaline phosphatase, ast: aspartate aminotransferase, ggt: gamma-glutamyl transferase, ldh: lactate dehydrogenase, cb: conjugated bilirubin, tb: total bilirubin. data are expressed as mean± sem. n=6 . a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control. table 3. effect of atazanavir/ritonavir on some biochemical parameters in the liver tissue of pregnant albino rats. group alt(u/l) ast(u/l) alp(u/l) ggt(u/l) ldh(u/l) a 185.0±15.5 173.8±14.1 187.2±15.34 192.5±12.2 3.25±0.18 b 200.5±19.1 211.2±16.1 201.4±11.4 224.7±16.2 3.76±0.12 c 321.3±20.2a 338.2±22.2a 351.0±18.4a 330.1±20.6a 4.84±0.85a d 463.8±22.6b 548.1±18.7b 474.2±19.0b 580.1±17.1b 6.95±0.32b e 695.2±27.4c 789.2±20.6c 690.0±21.9c 768.8±22.8c 9.97±0.71c atv/r: atazanavir/ritonavir, alt: alanine aminotransferase, alp: alkaline phosphatase, ast: aspartate aminotransferase, ggt: gamma-glutamyl transferase, ldh: lactate dehydrogenase. data are expressed as mean± sem. n=6. a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control. table 4. effect of atazanavir/ritonavir on lipid profile of pregnant albino rats. group g (mg/dl) tg(mg/dl) tc(mg/dl) hdl-c (mg/dl) ldl-c (mg/dl) a 95.0±6.41 70.5±6.78 110.1±12.8 30.7±3.44 65.7±6.00 b 92.0±9.00 74.9±6.70 112.0±13.6 32.6±2.61 64.5±6.42 c 89.3±6.42 73.2±7.53 109.8±12.0 32.5±3.72 63.8±5.11 d 100.9±9.19 81.1±7.56 110.3±11.1 29.4±2.63 65.7±7.62 e 152.0±8.00a 132.7±9.21a 161.0±14.1a 21.3±2.10a 113.4±8.32a data are express as mean± sem. n=6 g: glucose, tg: triglyceride tc: total cholesterol hdl-c: high density lipoprotein cholesterol, ldl-c: low density lipoprotein cholesterol. a significant (p<0.01) when compared to control table 5. effect of atazanavir/ritonavir on liver oxidative stress indices of pregnant albino rats. group mda (nmol/mg protein) sod (u/mg protein) cat (u/mg rotein) gsh (µmol/mg protein) gpx (u/mg protein) a 0.32±0.05 16.2±2.09 26.4±3.77 8.97±0.15 9.44±0.12 b 0.36±0.01 14.6±1.04 23.6±2.87 8.25±0.70 9.10±0.12 c 0.50±0.08a 10.4±1.06a 15.5±3.05a 5.88±0.54a 7.42±0.34a d 0.68±0.03b 7.06±1.15b 10 .6±1.37b 3.10±0.79b 5.02±0.44b e 0.98±0.01c 3.16±0.27c 20.5±3.07c 1.06±0.58c 2.11±0.53c atv/r: atazanavir/ritonavir, mda: malondialdehyde, cat: catalase, gsh: glutathione, sod: superoxide dismutase, gpx: glutathione peroxidase. data are expressed as mean± sem. n=6. a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control 50 biology, medicine, & natural product chemistry 8 (2), 2019: 47-52 figure 1. fig a: liver of control pregnant albino rat showing normal hepatocytes. fig b: liver of pregnant albino rat treated with atv/r (4.28/1.43 mg/kg) showing inflammatory cell infiltration. fig c: liver of pregnant albino rat treated with atv/r (8.57/2.86 mg/kg) showing hepatocyte necrosis. fig d: liver of pregnant albino rat treated with atv/r (17.1/5.74 mg/kg) showing hepatocyte necrosis. fig e: liver of pregnant albino rat treated with atv/r (34.2/11.4 mg/kg) showing hepatocyte necrosis. (h and e x100). fig c fig e fig d fig a fig b adikwu et al. – liver profile of atazanavir/ritonavir in pregnant albino rats 51 discussion drug-induced liver injury is a potential complication of nearly every prescribed drug and many fatal and nearfatal drug reactions occurred each year (lee, 1995). pregnancy-related liver disease is the most frequent cause of liver dysfunction in pregnancy and provides a real threat to fetal and maternal survival (joshi, 2010) and could be aggravated by some medications (eyal et al., 2010). this makes the provision of antiretroviral therapy during pregnancy a serious clinical challenge due to the possible occurrence of hepatotoxicity. the present study evaluated the liver profile of atv/r in pregnant albino rats. the evaluation of organ weight in toxicology studies is an integral component in the assessments of pharmaceuticals, chemicals, and medical devices (wooley, 2003). this study did not observe significant changes in the body and liver weights of atv/r-treated pregnant rats. serum tg, tc, ldl-c, hdl-c and blood g levels are yardsticks for hyperlipidemia and hyperglycemia caused by drugs (ehigiator and adikwu, 2019). this study, observed elevated levels of serum tg, tc, ldlc and blood g with decreased hdlc level at the highest atv/r dose. these observations are possible signs of hyperlidemia and hyperglycemia (ehigiator and adikwu, 2019). the serum levels of alt, ast, alp, ldh and ggt are effective clinical tools for the assessment of liver status (navarro and senior, 2006). ast is found in mitochondria and cytosol of hepatocytes, alt is localized to the cytosol whereas alp is found on the sinusoidal surface of hepatocytes (rosen and keefe, 2000). ldh is a cytoplasmic marker enzyme whereas ggt is a membrane bound enzyme. higher serum activities of the aforementioned parameters are often associated with hepatic damage (kim et al., 2001). this study observed elevations in the serum and liver levels of alt, ast, alp, ldh and ggt in a dose-dependent fashion in atv/r-treated pregnant rats. this observation may be due to the damage of liver hepatocyte membrane leading to the release of the aforementioned parameters into the blood. the measurement of bilirubin is a very sensitive test to substantiate the functional integrity of the liver and severity of necrosis (singh et al., 1989). this study observed elevations in serum cb and tb levels in a dose-dependent fashion in atv/r-treated pregnant rats. this may be due to decreased disposal of bilirubin by the liver or the blockade of the excretory duct of the liver. reactive oxygen species (ros) could be toxic or beneficial for cellular functions. the toxic effects of ros can occur as a result of excess activity due to overload or accumulation leading to oxidative stress (os). os plays a major part in the development of chronic and degenerative diseases. antioxidants such as sod, cat, gsh and gpx are inhibitors of os even at relatively low concentrations, but their functions can be incapacitated by insurmountable activities of ros (sultan, 2014). in this study, hepatic sod, cat, gsh and gpx levels were depleted in a dose-dependent fashion in atv/rtreated pregnant rats. malondialdehyde (mda) is a highly reactive three carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid oxidation and during arachidonic acid metabolism for the synthesis of prostaglandins. the monitoring of mda level in biological systems has been an important indicator of lipid peroxidation (lpo) in-vitro and in-vivo (janero, 1990). this study observed increases in hepatic mda levels in a dose-dependent fashion in atv/r-treated pregnant rats which indicate hepatic lpo. lpo is the oxidative destruction of lipids containing carbon-carbon double bond resulting in lipid peroxyl radicals and hydro peroxides production. lipid peroxyl radicals and hydroperoxides can stimulate alterations in cell signal transduction and functions leading to cell necrosis and apoptosis, which may facilitate the development of various pathological states (yin et al., 2011). liver histological examination contributes to diagnostic accuracy in drug-induced liver damage and remains a valuable tool for the evaluation of druginduced liver damage. as a clinical test, it provides more information about the state of the liver than any other single assay (kleiner, 2014). histopathological examination of the liver of atv/r-treated pregnant rats showed necrotic hepatocytes at higher doses. the observed necrotic hepatocytes correlate with changes in evaluated biochemical parameters. hepatic necroses observed in atv/r-treated rats could be attributed to os, because os as a consequence of ros production can cause progressive modification or degradation of biomolecules such as dna, proteins, lipids and carbohydrates. this can lead to loss of cell function, cell necrosis and cell death (halliwell and gutteridge, 1999). conclusion the use of atv/r in pregnant women living with hiv may 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(2011). free radical lipid peroxidation: mechanisms and analysis chem rev, 111; 10; 5944–5972 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 1-6 | doi: 10.14421/biomedich.2020.91.1-6 issn 2540-9328 (online) antioxidant activities of the leaf extract and fractions of dryopteris filix-mas (l.) schott could be attributed to the abundance of polyphenol compounds earnest oghenesuvwe erhirhie1,2,*, emmanuel emeka ilodigwe1, daniel lotanna ajaghaku3, blessing ogechukwu umeokoli4, peter maduabuchi eze5, festus basden chiedu okoye4 1department of pharmacology and toxicology, faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 2department of pharmacology and toxicology, faculty of pharmaceutical sciences, chukwuemeka odumegwu ojukwu university, igbariam, nigeria. 3department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu state, nigeria. 4department of pharmaceutical and medicinal chemistry faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 5department of environmental health science, faculty of health sciences and technology, nnamdi azikiwe university, nnewi campus, anambra state, nigeria. corresponding author* erhirhieochuko@yahoo.com, tel: +234-7060434974 manuscript received: 17 august, 2019. revision accepted: 06 february, 2020. published: 10 april, 2020. abstract dryopteris filix mas (d filix-mas) is wildly used in ethnomedicine for the management of rheumatoid arthritis, wounds and other diseases. we investigated the anti-oxidant activities of its leaf extract, and chromatographic fractions. the ethanol leaf extract was partitioned into four fractions; n-hexane, ethyl acetate, n-butanol and water. ferric reducing anti-oxidant power (frap), 1, 1-diphenyl-2-picrylhydrazil (dpph) and nitric oxide (no) scavenging in vitro assays were carried out on the extract and fractions at 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml. the most active fraction (ethyl acetate fraction) was further purified using chromatographic techniques to isolate its major compound whose structure was elucidated using id nuclear magnetic resonance (nmr) and mass spectrometry. the ethyl acetate fraction produced the highest free radical scavenging activity among the other fractions. the fraction (vlc-e7) from which the bioactive compound, quercetin-3-o-αl-rhamnopyranoside, was isolated had the best frap and dpph scavenging activities with ec50 and ic50 values of 88.81 ± 3.41 and 26.87 ± 0.24 respectively more than the ethyl acetate fraction. this study revealed that the polyphenol flavonoid, quercetin-3-o-αl-rhamnopyranoside could be responsible for antioxidant activity of ethno-medicinal property of d filix-mas leaf. keywords: dryopteris filix-mas; wounds; rheumatoid arthritis; quercetin-3-o-αl-rhamnopyranoside; anti-oxidant properties introduction free radicals are constantly produced in all normal living cells (kumar et al., 2014). imbalance between the production of these radicals and endogenous antioxidants creates a state of oxidative stress with its associated damaging effects on endogenous molecules such as lipids, proteins and nucleic acids (yan et al., 2015). unbridled oxidative stress has been reported to be implicated in several chronic and degenerative diseases such as diabetes, rheumatoid arthritis, cancer, neurodegenerative diseases, atherosclerosis, ischemic heart disease, ageing among others (khatoon et al., 2013). in attempt to ameliorating oxidative damages caused by free radicals, synthetic antioxidants such as butylated hydroxytoluene (bht), butylated hydroxyanisole (bha), and tert-butylhydroquinone (tbhq) had been developed. however, these synthetic antioxidants left much to be desired due to their side effects, such as liver toxicity, carcinogenicity as well as their high cost and inaccessibility (deepa et al., 2014). these limitations have inspired the search for more effective antioxidant from natural source, especially medicinal plants, which are biodegradable, less toxic, affordable and accessible (ezeja et al., 2015; ahlem et al., 2015). d filix-mas is an evergreen fern belonging to the family of dryopteridaceae. male fern, worm fern, aspidium and shield fern are common names assigned to it. it grows between 60-150 cm high and it is habitat to stream and moist environments (uwumarongi et al., 2016). it is native to europe, asia, and north america. its leaves are bipinnated and consist of 20-35 pinnae on each side of the rachis (fig 1). its stalks are covered with orange-brown scales (bafor et al., 2017). the leaf decoction is ethno medicinally used in rheumatoid arthritis, wounds, bleeding disorders, ulcers, worm infestation and malaria (tagarelli et al., 2010; erhirhie et al., 2019). it is also reported as one of the ferns with https://doi.org/10.14421/biomedich.2020.91.1-6 2 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 useful secondary metabolites against chronic diseases and aging (valentyna et al., 2017). previous studies have found that it possesses antihelmintic activity (urban et al., 2014), antimicrobial activity (mandal and mondal, 2014), antidiarrheal (uwumarongi et al., 2016), uterine relaxant (bafor et al., 2017) and anti-inflammatory (erhirhie et al., 2019) activities. a preliminary study by sekender et al, (2012) revealed that it possesses anti-oxidant activity. in this present study, we evaluated the antioxidant activity of the leaf extract and fractions. the bioactive compound responsible for its antioxidant activity was isolated and characterized. figure 1. d. filix-mas photograph. methodology materials visible spectrophotometer (721g, zhejiang, china), thermostatic water bath (equitron mumbai india), analytical weighing balance (ohaus corp. nj usa), quercetin (institute of pharmaceutical biology and biotechnology, heinerich-heine university, dusselforf, germany), 2,2-diphennyl-1-picrylhydrazyl, dpph (sigma aldrich), ascorbic acid (sigmaaldrich). procedure plant collection, extraction, fractionation and purification of active compound plant collection, authentication, extraction, fractionation, chromatographic separation, and structural elucidation techniques were carried out using similar method as described in our earlier study (erhirhie et al., 2019). the purification and structural elucidation was based on bioactivity guided antioxidant screening of liquid-liquid fractions (n-hexane, ethyl acetate, butanol, and water fractions), vacuum liquid chromatographic fractions and sephadex fractions. dpph scavenging assay the test was carried out based on the method described by ajaghaku et al (2017). the reaction mixture contained 0.1 ml of various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml) of sample, 0.1 ml of 0.6 mmol of dpph and 0.8 ml of methanol. the mixture was incubated in the dark for 30 minutes at room temperature. the absorbance of the sample was measured at 517 nm against blank (methanol) using a spectrophotometer. ascorbic acid and quercetin were used as standards. a tube containing 0.1 ml of dpph solution and 0.9 ml of methanol served as control. experiments were carried out in duplicate. free radical scavenging activities of each sample were determined as follows: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (𝐴𝐶– 𝐴𝑆)/𝐴𝐶) × 100 ac : average absorbance of control as : average absorbance of sample a graph of percentage inhibition against concentration was plotted and the concentration that produced 50% inhibition (ic50) was extrapolated using a regression analyses equation. ferric reducing antioxidant power (frap) assay frap assay was carried out following the method described by habibur et al, (2013). two hundred and fifty microliter (0.25 ml) of various concentrations, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml of samples were mixed with 0.625 ml of phosphate buffer and 0.625 ml of 1% potassium ferricyanide [k3fecn6]. the mixtures were heated at 50oc for 20 minutes. then, 0.625 ml of 10% trichloroacetic acid (tca) was added and the mixtures were centrifuged at 3000 rpm for 10 minutes. from the upper layer, 0.625 ml was pipetted and mixed with 0.625 ml of distilled water and 0.125 ml of 0.1% (w/v) ferric chloride (fecl3) solution. absorbances of the mixtures were measured at 700 nm against air using a spectrophotometer. ascorbic acid and quercetin were used as standards. tests were performed in duplicate and percentage inhibition was calculated using the formula below. % inhibition = average absorbance of sample−average absorbance of blank 1 x 100 1 erhirhie et al. – antioxidant activities of the leaf extract and fractions … 3 a graph of percentage inhibition against concentration was plotted and the effective concentration (ec50) was extrapolated using a regression analyses equation. nitric oxide scavenging activity this assay was carried out following the method described by ezeja et al., (2015). two milliliter of 10 mm sodium nitroprusside prepared with phosphate buffer saline (ph 7.4) was mixed with 0.5 ml of samples at various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml). the mixture was incubated at room temperature for 150 minutes. thereafter, 0.5 ml of the reaction mixture was withdrawn and mixed with 0.5 ml of griess reagent (1% sulphanilamide + 0.1% naphthylethylenediamine dichloride + 3% phosphoric acid) was added to each test tube. the absorbance of the pink chromophore formed was measured at 540 nm. ascorbic acid and quercetin were used as standards. assays were carried out in duplicates. percentage inhibition was calculated using the formula below abs control−abs test abs control × 100 1 inhibitory concentration, ic50 was estimated from graph of % inhibition against concentration using regression analyses equation statistical analysis results were presented as mean ± standard error of mean (sem) using statistical package for social science (spss, version 20). calculation of fifty percent inhibitory concentration (ic50) of the extracts and fractions was carried out using regression equation in microsoft excel, 2010. results dpph, scavenging activity of extract and fractions in dpph scavenging assay, 50% inhibitory concentration (ic50) of 134.12 ± 2.88, 94.97 ±0.14, 63.91 ± 1.29, 62.09 ± 0.21and 45.72 ± 0.16 µg/ml were recorded in n-hexane, water fractions, extract, butanol and ethyl acetate fractions respectively. the ethyl acetate fraction produced the lowest ic50 value among other samples, except ascorbic acid and quercertin which showed ic50 values of 12.26 ± 0.20 µg/ml and 10.46 ± 0.15 µg/ml respectively (table 1). inhibition against dpph at 100 µg/ml by vlc fractions ethyl acetate vlc fractions, vlc-e1 (78.68%), vlce3 (52.33%), vlc-e7 (72.38%), vlc-e6 (73.84%), vlc-e14 (65.60%), vlc-e8 (73.08%), vlc-e10 (74.81%), vlc-e12 (72.09%), vlc-e16 (69.67%) produced more than 50 % inhibition against dpph (table 2). table 1. free radical (dpph) scavenging activities of extract and fractions of d filix-mas. dpph, scavenging activity, ic50(ug/ml) extract 63.91 ± 1.29 n-hexane fraction 134.12 ± 2.88 ethyl-acetate fraction 45.72 ± 0.16 butanol fraction 62.09 ± 0.21 water fraction 94.97 ±0.14 ascorbic acid 12.26 ± 0.20 quertcetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. table 2. dpph scavenging activities of ethyl acetate vacuum liquid chromatographic fractions of d filix-mas. solvent ratio sample code inhibition against dpph (%) ethyl acetate fraction 73.26 n(500): e (0) vlc-e1 78.68 n(450): e (50) vlc-e2 19.38 n(400): e(100) vlc-e9 18.12 n(350): e(150) vlc-e13 23.06 n(300):e(200) vlc-e4 28.00 n(250): e(250) vlc-e11 36.34 n(200):e(300) vlc-e3 52.33 n(150):e(350) vlc-e15 16.86 n(100):e(400) vlc-e5 12.79 n(50): e(450) vlc-e17 37.79 n(0):e(500) vlc-e7 72.38 d(500):m(0) vlc-e6 73.84 d(450):m(50) vlc-e14 65.60 d(350):m(150) vlc-e8 73.06 d(250):m(250) vlc-e10 74.81 d(100):m(400) vlc-e12 72.09 d(0):m(500) vlc-e16 69.67 n = n-hexane, e = ethyl acetate, m = methanol. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract selected fractions. frap activity in frap assay, quercetin and ascorbic acid produced the lowest ec50 values followed by vlc-e7, ethyl acetate fraction, extract and butanol fraction. in nitric oxide scavenging assay, ascorbic acid exhibited the lowest ic50 value (14.74 ± 0.11 µg/ml) followed by ethyl acetate fractions (15.38 ± 3.65 µg/ml) and butanol fractions (15.45 ± 2.72 µg/ml), followed by vlc-e7 fraction (16.66 ± 0.48 µg/ml), quercetin (18.14 ± 2.57 µg/ml) and extract (1854.60 ± 200.25 µg/ml). in dpph assay, quercetin and ascorbic acid produced the lowest ic50 values (11.50 ± 0.08 and 14.19 ± 0.43µg/ml) followed by vlc-e7 (26.87 ± 0.24 µg/ml), ethyl acetate fraction (50.25 ± 0.40 µg/ml), butanol fraction (62.09 ± 0.21 µg/ml) and crude extract (63.91 ± 1.29 µg/ml) (table 3). 4 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 dpph, scavenging activity of sephadex fractions dpph scavenging activities of samples tested decreased in the following order; quercetin (10.46 ± 0.15 µg/ml) > ascorbic acid (12.26 ± 0.20 µg/ml) > sph-e3 (26.35 ± 1.38 µg/ml) > vlc-e7 (26.87 ± 0.24 µg/ml) > sphe6 (33.44 ± 0.38 µg/ml) > sph-e5 (37.30 ± 0.30 µg/ml) > sph-e7 (46.95 ± 0.61 µg/ml) > sph-e4 (86.10 ± 7.69 µg/ml) (table 4). table 3. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract and fractions of d filix-mas. sample frap, ec50 (µg/ml) nitric oxide, ic50 (µg/ml) dpph, ic50 (ug/ml) extract 1854.60 ± 200.25 38.51 ± 2.23 63.91 ± 1.29 ethyl acetate fraction 461.75 ± 22.91 15.38 ± 3.65 50.25 ± 0.40 butanol fraction 1911.03 ± 137.44 15.45 ± 2.72 62.09 ± 0.21 vlc-e7 88.81 ± 3.41 16.66 ± 0.48 26.87 ± 0.24 ascorbic acid 69.34 ± 2.41 14.74 ± 0.11 14.19 ± 0.43 quercetin 30.81 ± 0.86 18.14 ± 2.57 11.50 ± 0.08 frap: ferric reducing antioxidant power. values are expressed as mean ± standard error of mean. table 4. dpph scavenging activity, of sephadex (sph) fractions of d filix-mas. dpph, scavenging activity, ic50( µg/ml) vlc-e7 26.87 ± 0.24 sph-e1 sph-e2 22.10 ± 0.62 sph-e3 26.35 ± 1.38 sph-e4 86.10 ± 7.69 sph-e5 37.30 ± 0.30 sph-e6 33.44 ± 0.38 sph-e7 46.95 ± 0.61 ascorbic acid 12.26 ± 0.20 quercetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. discussion it is well established that by-products of oxygen metabolism produce free radicals such as reactive oxygen and nitrogen species which results to cellular damage and pathogenesis of several diseases such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases among others (li et al., 2016). there is a revitalization of interest in the search for natural anti-oxidant that could ameliorate reactive oxygen species which are implicated in numerous disease conditions (ahlem et al., 2015; deepa et al. 2014). in this study, we investigated the antioxidant properties of the extract and fractions of d filix-mas and also elucidated the major compound responsible for its antioxidant activity using in vitro bioassay guided isolation approach. dpph test is a widely acceptable and reproducible assay for screening potential antioxidants (patel et al., 2010). this method involves the ability of anti-oxidants to donate electrons to dpph thereby causing discoloration of the reaction mixture from deep violet color to yellow color (patel et al., 2010). the yellow color change observed in this study substantiates the ability of the extract and fractions of d filix-mas to scavenge free radicals generation by dpph. this further suggests that the constituents present in the extract and fractions could ameliorate oxidative damages by free radicals. reduction of ferric (fe3+) to ferrous (fe2+) ions is a documented method of determining antioxidants with reducing power, capable of breaking free radical chain progression in lipid peroxidation (habibur et al., 2013). from this study, reduction of iron (iii) complex to the ferrous form, iron (ii) could be a possible antioxidant mechanism as a result of the presence of reducing agents in the extract and fractions of d filix-mas. phenolic compounds such as quercetin has been reported to prevent free radicals by forming complex and chelating metal ions such as iron and cupper, thereby preventing auto-oxidative damage to the living system (nimse and pal, 2015). nitric oxide generated following incubation of sodium nitroprusside with phosphate buffer in the presence of oxygen is known to cause toxicity to biomolecules (deepa et al., 2013). the scavenging capacities of the extract and fractions against nitric oxide generation suggest that d filix-mas could prevent nitric oxide generation in various disease conditions. polyphenols such as quercetin has been reported to interact with nitric oxide synthase resulting in modulation of nitric oxide production (hussain et al., 2016). the roles of phenolic compounds from medicinal plants as supplement in the treatment of oxidative stress and free radicals mediated tissue damage cannot the overemphasized (li et al., 2016). flavonoids and phenolic acids from polyphenols have been reported to play a significant role in scavenging free radicals and prevention of oxidative damage to cells. phenolic compounds have capacity to neutralize several forms of oxidizing free radicals due to their electron donating abilities (habibur et al., 2013; ajaghaku et al., 2017). ojo et al., (2013) reported that flavonoids inhibit lipid peroxidation in various biological systems. presence of hydroxyl groups in flavonoids allows them to disconnect free radical chain reactions via the formation of intramolecular hydrogen bonds (monika et al., 2011). erhirhie et al. – antioxidant activities of the leaf extract and fractions … 5 from the forgoing, antioxidant properties elicited by the extract and fractions of d filix-mas could be attributed to presence of flavonoids. this is substantiated by presence of the flavonoid, quercitrin as the major compound in the hplc chromatogram of the extract and fractions of d filix-mas as documented in our earlier study (erhirhie et al., 2019). the ethyl acetate vlc fraction (vlc-e7), where the compound, quercetin-3-o-αl-rhamnopyranoside was isolated exhibited the highest anti-oxidant activity compared to the extract and other fractions. in line with this finding, quercetin-3-o-αl-rhamnopyranoside isolated from other medicinal plants was found to elicit significant antioxidant activity (zhang et al., 2014). it is possible that possible that quercetin-3-o-αlrhamnopyranoside may be the key antioxidant compound of d filix-mas. quercetin, the most widely distributed flavonoid in nature, existing mostly in its glycoside form quercitrin has been reported to possess various properties such as antioxidant, anti-inflammatory, neuroprotective, antiviral, anticancer, hepatoprotective, cardioprotective, antimicrobial and anti-obesity properties (maalik et al., 2014). mir, et al., (2017) reported that flavonoids from tridax procumbens possess antioxidant properties. phenolic compounds, catechin, epicathechin, dihydroquercetin isolated from alchornea floribunda leaf were also found to elicit antioxidant properties (ajaghaku et al., 2017). in this study, the ability of extract and fractions of d filixmas to scavenge generation of free radicals from dpph, nitric oxide as well as its chelating capacity could be attributed to the phenolic compound, quercetin3-o-αl-rhamnopyranoside, which was postulated to be responsible for its anti-inflammatory activity as earlier reported (erhirhie et al., 2019). conclusion the isolated flavonoid, quercetin-3-o-αlrhamnopyranoside from ethyl acetate chromatographed fraction of d filix-mas could be responsible for its antioxidant activity, which validates its folkloric use in the management of rheumatoid arthritis and other free radical mediated diseases. acknowledgments the authors are thankful to prof. dr. peter proksch of the institute of pharmaceutical biology and biotechnology, heinrich-heine university, düsseldolf, germany for making their facilities available for the high performance liquid chromatography (hplc) and nmr analyses that were carried out. we are also thankful to dr. h.a. akinnibosun for assisting in the authentication of the plant specimen used for this study. references ahlem, r., souad, i.b., béatrice, b., valérie, m.l., fathi, m., evelyne, o., jamila, k.c and malika, t.a., 2015, total phenolic, total flavonoid, tannin content, and antioxidant capacity of halimium halimifolium (cistaceae), japs, 5 (01): 052-057. ajaghaku, d.l., obasi, o., umeokoli, b.o., ogbuatu, p., nworu, c.s., ilodigwe, e.e., and okoye, f.b.c., 2017, in vitro and in vivo antioxidant potentials of alchornea floribunda leaf extract, fractions and isolated bioactive compounds, avicenna j phytomed, 7 (1): 80-92. bafor, e.e., 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activity of dryopteris filix-mas (l.) schott (dryopteridaceae), ew j herbal chempharmacol res, 2(1): 19-25. valentyna, m., iryna, t., t et yan a, d . an d la r ys a, m . , 2 0 1 7 , a review of the medicinal ferns of ukraine, scr. sci. pharm, 4 (1): 46-62. yan, h., yuan, y., xi, z., kun, z., shaohua, c. and zhiyun, d., 2015. curcumin, inflammation, and chronic diseases: how are they linked? molecules, 20, 9183-9213; doi:10.3390/molecules20059183. zhang, y., wang, d., yang, l., zhou, d., zhang, j. 2014, purification and characterization of flavonoids from the leaves of zanthoxylum bungeanum and correlation between their structure and antioxidant activity, plos one, 9(8): e105725. doi: 10.1371/journal.pone.010572. biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 23-32 | doi: 10.14421/biomedich.2016.51.23-32 checklist of macroalgae in waisai coast, raja ampat retno suryandari1, widodo departement of biological education, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: suryandari44@gmail.com abstract macroalgae are very abundant organisms in indonesian coastal zone. they comprise 8.6% of the total marine organisms.the aim of the research was to identify macroalgae in waisai coast raja ampat. the results showed that 38 macroalgae were found in waisai coast raja ampat but only 29 species of macroalgae can be identified. macroalgae found in waisai coast raja ampat are green algae, red algae and brown algae. green algae found and identified are caulerpa macra (weber-van bosse) draisma & prud’homme, caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), caulerpa cupressoides (vahl) c. agardh, halimeda discoidea decaisne, halimeda opuntia (linnaeus) j.v. lamoroux, halimeda tuna (j. ellis & solander) j.v. lamoroux, halimeda cylindraceae decaisne, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c.schmidt, boergesenia forbesii (hardvey) feldmann, valonia ventricosa j. agardh, dictyosphaeria cavernosa (forsskål) børgesen, chaetomorpha spiralisokamura, anadyomene wrightii harvey ex. j. e. gray, neomeris annulata dickie. red algae species found and successfully identified areacanthophora spicifera (m. vahl) børgesen, laurencia papilosa (c. agardh) greville, gracilaria salicornia (c. agardh) e.y. dawson, amphiora fragilissima (linnaeus) j.v. lamoroux, hypnea pannosa j. agardh. brown algae species found and identified are hormophysa cuneiformis (j.f. gmelin) p.c. silva, sargassum aquifolium (turner) c. agardh, sargassum polycystum c. agardh, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & de clerck hydroclathrus clatratus (c. agardh ) m. howe. the only species found in indonesia is sargassum aquifolium. keywords: macroalgae, waisai coast, raja ampat, green algae, red algae, brown algae introduction indonesia have more than 17,504 islands. approximately three-quarters of indonesian territory are ocean (5.8 million km2) with 95.161 km coastline. indonesia have the second longest coastline in the world (lasabuda, 2013). indonesia's marine wealth runs between the indian ocean to the pacific ocean with an area of coral reefs achieve 50.875 km2 or 18% of the world's coral reefs. most of these reefs are located in the eastern part of indonesia on the coral triangle region. one of the center of coral reef biodiversity are in raja ampat (solihin, batungbacal, & nasution, 2013). raja ampat islands is an area located in the western part of papua. the islands are known as one of the marine world heritage site because of its marine biodiversity. in 2002, the nature conservation (tnc) and the research center for oceanography lipi reported data that the marine ecosystem consist of 537 species of corals (75% of coral species in the world), and 828 species of fish (setiawan, 2011). in raja ampat, coral reef ecosystems exposed in the shallow areas in most of islands.coral reefs are usually covered with abundance of seaweed that is not only diverse if compared with other living beings but also more complex in morphology and ecological role in coral reef ecosystems (mambrisaw, wurlianty, liuw, & hamel, 2006). waisai coast is located around the port of raja ampat, south waigeo district, raja ampat, west papua.this area can be reached by sea from sorong city and overland from waisai city.waisai coastzonation started from the mainland that made up of mangrove forest dominated by rhizophora genus, then towards the shallow water, seagrass species dominate byenhalus sp. with various sizes up to 40 cm. among the seagrasses are found living macroalgae associated with seagrass and coral. this coast has a mixed substrate consist of dead corals and mud sand. according to dahuri (1998) in suparmi & sahri (2009) seaweed is a biological resource that is very abundant in indonesian ocean which is comprised of 8.6% of the total marine organisms. the seaweed habitat in indonesia reached 1.2 hectares, one of the largest in the world (wawa, 2005) in suparmi & sahri (2009). based on the report by vamn bosse, indonesia has 555 of 8642 world seaweed species (suparmi & sahri, 2009). mambrisaw et al (2006) stated that in the area of raja ampat misool particularly in the south east, south and ayau waigeo, seaweed has been cultivated and are the main commodity in the region. however, checklist of macroalgae in raja ampat have not been done. seaweeds (macroalgae) are organisms like plants that live in marine ecosystems which is a primary producer because it has the ability to photosynthesize (autotrof). macroalgae are classified into three main groups, namely green algae (chlorophyta), brown algae (phaeophyta) and red algae (rhodophyta) (dhalgarkar & kavlekar, 2004). in addition to a taxonomic grouping, macroalgae can be distinguished by the character of the http://dx.doi.org/10.14421/biomedich.2016.51.23-32 24 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 organism and its ecological characteristics. based on this category of macroalgae are grouped into three main categories: 1.turf algae, 2. fleshy algae (fleshy and calcified) 3. crustose corraline algae (mambrisaw, wurlianty, liuw, & hamel, 2006). this research aims to determine the types of macroalgae found in waisai coast of raja ampat. materials and methods the research was conducted on 4-5 april 2016 in waisai coast, south waigeo district, raja ampat. figure 1. map of waigeo island of raja ampat. the equipment used in this research include waterproof paper, clipboard, pencils 2b, mask snorkel, fins, frame squares size 1x1 meter, gps (global positioning system), a digital camera, underwater camera, identification book, refractometer, ph meter and thermometer, materials used in this research are specimens and 10% formalin. sampling vegetation was done using transect quadrat method. transect line was divided into three stations in one location with the distance of each station is 25 meters. the steps of this research are: 1) determining the location for line transect stations 1, 2, and 3 with a length of each transect is 50 meters. the distance of each transect lines are installed parallellyand its lengthis 25 meters. each transect mounted perpendicular to the shoreline, 2) in each transect line is placed a quadrat frame with size 1 m x 1 m of 10 quadrat frame with a distance of each quadrat frame is 5 meters.first squares are laid in the area closest to the mainland where macroalgae was found, 3) calculate all macroalgae in each plot, 4) the amounts found are recorded on paper underwater based on the location of the squares, 5) sampling macroalgae and placed in plastic sample was labeled , 6) samples were found to note the type of the substrates, 7) took data of environmental parameters such as: the coordinates of global positioning system (gps), the measurement of temperature using a thermometer, measuring salinity of seawater using a refractometer and ph measurements of water using a ph-meter. 8) samples were taken washed clean of the substrate and then preserved with formalin 10% for identification purposes. result and discussion based on the research that has been done, macroalgae were found in waisai coast at coordinates s 00˚26'12,3 ", e 130˚48'38,7" are as many as 38 species. macroalgae that can be identified to species level are 29 species consisting of 17 species of green algae (chlorophyta), 5 species of red algae (rhodophyta), 7 species of brown algae (phaeophyceae), and 9 species unidentified. green macroalgae (clorophyta) class, order and family of green macroalgae that found at waisai coast shown in the table 1 below. retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 25 table 1. checklist of green macroalgae that found at waisai coast. class order family species ulvophyceae bryopsidales caulerpaceae caulerpa macra (weber-van bosse) draisma & prud’homme caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus) caulerpa cupressoides (vahl) c. agardh halimedaceae halimeda discoidea decaisne halimeda opuntia (linnaeus) j.v. lamoroux halimeda tuna (j. ellis & solander) j.v. lamoroux halimeda cylindraceae decaisne halimeda macroloba decaisne udoteaceae avrainvillea erecta (berkeley) a. gepp & e.s. gepp codiaceae codium geppiorum o.c.schmidt cladophorales valoniaceae boergesenia forbesii (hardvey) feldmann valonia ventricosa j. agardh dictyosphaeria cavernosa (forsskål) børgesen cladophoraceae chaetomorpha spiralis okamura anadyomenaceae anadyomene wrightii harvey ex. j. e. gray dasycladales dasycladaceae neomeris annulata dickie caulerpa macra (weber-van bosse) draisma & prud’homme (fig. 2a) description: large caulerpa with long thick stolons, these several decimeters long, 3– 5 mm in width but often over 5 mm. rhizoidal pillars well developed, arising from stolons at irregular distances, up to 10 cm long and several mm in diameter, often with thick terminal clumps of branched rhizoids. erect assimilators up to 10 cm in height, with irregularly to regularly placed ramuli arranged distichously or radially and opposite or alternate. ramuli oviform, pyriform, more rarely claviform, usually with some having a form like the head of a golf club, to 12 mm long and 6 mm in width (belton et al, 2014) distribution in southeast asia: indonesia, java, philippines (guiry & guiry, 2017). caulerpa racemosa var. macrophysa (kiitzing) taylor (fig. 2b) description: plants large, to 100 mm tall, erect branches simple or occasionally branched, few and distantly spaced on stout, creeping stolon, branches bearing ramelli expanded into a hemispherical end and arranged irregularly on terete rachis (menez & calumpong, 1982). distribution in southeast asia: indonesia, singapore, thailand, vietnam (guiry & guiry, 2017) philippines (menez & calumpong, 1982). caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus) (fig.2c) description: bright to dark green in colour, tubular with delicate, branched stolon and erect assimilators, plumose or feathery resembling palm leaves, up to 13 cm tall, 812 mm broad, rarely branched, branching generally at the base of assimilators, branchlets opposite, incurve above, needle shaped with pointed apices (jha et al, 2009). distribution in southeast asia: indonesia, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). caulerpa cupressoides (vahl) c. agardh (fig. 2d) description: dark green, the stolon on the macroalga forms a branching with an elongated shape of ramuli and jagged edges (atmadja et al, 1996). distribution in southeast asia: indonesia, philippines, singapore, vietnam (guiry & guiry, 2017). halimeda discoidea decaisne (fig. 2e) description: compact tallus growth, dichotomous or trichotomous branching, rough surface segments, squiggly edges, base segments slightly elongated and thicker (atmadja et al, 1996). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). halimeda opuntia (linnaeus) j.v. lamoroux (fig. 2f) description: compact tallus growth, bifurcation of crumpled segments spreading and forming new growth. the relatively small segments are round-shaped, rounded-oval, kidney, and wavy. high carbonate contents. basal segment is not visible. the non-bulbous holdfast consists of filamentous mass (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). 26 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 halimeda tuna (j. ellis & solander) j.v. lamoroux (fig. 2g) description: dark green in colour, generally tufted, moderately calcified and attached by compressed conical holdfast, branching in one plane, dichotomous or trichotomous upper segments, cuneate remiform discoid, 6-13 mm high, 5-23 mm broad,medullary filaments entangled, fusing 2-3 together at the nodes, dichotomously branched above the fused filaments (jhaet al, 1996). distribution in southeast asia: indonesia, malaysia, philippines, singapore, vietnam (guiry & guiry, 2017). a b c d e f g h i j k l m n o p figure 2. macroalgae of chlorophyta at waisai coast (a. caulerpa macra (weber-van bosse) draisma & prud’homme, b. caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, c. caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), d. caulerpa cupressoides (vahl) c. agardh, e. halimeda discoidea decaisne, f. halimeda opuntia (linnaeus) j.v. lamoroux, g. halimeda tuna (j. ellis & solander) j.v. lamoroux, h. halimeda cylindraceae decaisne, i. halimeda macroloba decaisne, j. avrainvillea erecta (berkeley) a. gepp & e.s. gepp, k. codium geppiorum o.c.schmidt, l. boergesenia forbesii (hardvey) feldmann, m. valonia ventricosa j. agardh, n. dictyosphaeria cavernosa (forsskål) børgesen, o. chaetomorpha spiralis okamura, p. anadyomene wrightii harvey ex. j. e. gray, q. neomeris annulata dickie). q halimeda cylindraceae decaisne (fig. 2h) description: perpendicular tallus growth consists of cylindrical segments or prisms. main dichotomous branching, tallus growth toward small bud, width segment size 18 mm, length 10 mm. between the basal segment and the segment sometimes there is a segment bearing as a place for the formation of new segment retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 27 growth or segment basal branching. holdfast tuber or tuber (atmadja et al, 1996). distribution in southeast asia: indonesia, philippines, vietnam (guiry & guiry, 2017). halimeda macroloba decaisne (fig. 2i) description: green in colour when not calcified, solitary, erect, 7-12 cm long, branched in one plane giving it a flat appearance, attached by a cylindrical holdfast, upper segments discoid, reniform, rounded, 0.5-1.5 cm high, 1.5-2.0 cm broad, margins thick entire, branching dito tri-chotomous above and polychotomous at the basal part, cortex commonly composed of 3-4 layers of utricles, outermost utricles slightly attached when young and separating on decalcification when fertile. it is abundant on dead corals (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). avrainvillea erecta (berkeley) a. gepp & e.s. gepp (fig. 2j) description: dark to dull green in colour, erect, up to 8 cm tall, attached by elongated, densely interwoven basal mass of rhizoids with apical fan-shaped sessile and filamentous foliar position, foliar portions composed of branched filaments about 4cm long and 6 cm broad, spongy to somewhat hairy. it commonly found in muddy substratum with still waters (jha et al, 2009). distribution southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). codium geppiorum o.c.schmidt (fig. 2k) description: dark green in colour, procumbent, attached by holdfast, 3-5 cm tall, thallus sub-dichotomously divided and creeping type, branches cylindrical, divergent and compressed, utricles abovate to pyriform with rounded tips, 330 – 550 μm long and 100 – 300 μm broad with slight flattened apices, gametangia fusiform, pedicellate, 1 – 2 born on each fertile utricle. common in tide pools on sheltered rocks (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). boergesenia forbesii (hardvey) feldmann (fig. 2l) description: yellowish green in colour, 3-5 cm tall forming patches on the substratum, vesicles slightly curved, clavate, filled with fluid, club-shaped in younger stage enlarged in he upper part giving grape-like appearance in older stage. basal branched filamentous parts of the thallus septate. common in intertidal rock pools (jha et al, 2009). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). valonia ventricosa j. agardh (fig. 2m) description: dark green colour, single round and unbranched thallus, fluid-filled thallus(olsen & j.a., 1988). distribution in southeast asia: philippines, singapore, vietnam (guiry & guiry, 2017). indonesia (sukiman et al, 2014). dictyosphaeria cavernosa (forsskål) børgesen (fig. 2n) description: dark green, round hollow rounded with harsh spheres, stiff and slightly thick (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017) chaetomorpha spiralis okamura (fig. 2o) description: plant bluish green in colour, up to 5cm long, rigid, spirally coiled, attached by rhizoidal basal cells with simple branched short and blunt rhizoids, cells 400-600 μm in diameter and 2-3 times longer than broad, moniliform or cylindrical in shape (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). anadyomene wrightii harvey ex. j. e. gray (fig. 2p) description: light green coloured, no stipe, blade shaped like a corrugated fan with serrated blade edge, on a blade that looks like a leaf-like loop (anonymous, 2004). distribution in southeast asia: indonesia, philippines, singapore, vietnam (guiry & guiry, 2017). neomeris annulata dickie (fig. 2q) description: light green colour, thallus cylindrical and unbranched, calcified basal part, the apical portion consists of green fibers (jha et al, 2009). distribution southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). red macroalgae (rodhophyta) class, order and family of redmacroalgae that found at waisai coast shown in the table 2 below. 28 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 table 2. checklist of red macroalgae that found at waisai coast. class order family species florideophyceae ceramiales rhodomelaceae acanthophora spicifera (m. vahl) børgesen laurencia papilosa (c. agardh) greville gracilaria salicornia (c. agardh) e.y. dawson corallinales corallinaceae amphiora fragilissima (linnaeus) j.v. lamoroux gigartinales cyatocloniaceae hypnea pannosa j. agardh acanthophora spicifera (m. vahl) børgesen (fig. 3a) description: yellowish brown colour, erect plants, to 40 cm tall, with solid cylindrical branches, 2 3 mm wide, branched either sparingly to repeatedly. main branches have short, determinate branches, irregularly shaped and spinose, with spines numerous and radially arranged. there are no spines on main axes. the plant grows from a large, irregularly shaped holdfast. (anonymous, 2001). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). laurencia papilosa (c. agardh) greville (fig. 3b) description: plants about 5-16 tall, growing in dense clusters. the lower parts of the plants are smooth, but toward the ends, the branches are sparsely crowded with short, truncate to tuberculate branchlets. plants olive green to greenish purple in colour, consistency somewhat cartilaginous (kluijvver, gijswijt, leon, and cunda, 2012) distribution in southeast asia: indonesia, philippines, vietnam (guiry & guiry, 2017). gracilaria salicornia (c. agardh) e.y. dawson (fig. 3c) description: brownish to yellowish red in colour, up to 16 cm in height, attached by small discs, thallus bushy with irregularly branched, cylindrical axes, lower branches cylindrical, not attenuated at the base, upper branches attenuated below, elongate clavate, swollen at the apex and showing apical depressions, one or two branchlets arising from the depressions, cortex 1-2 layered, medulla with large central cells, cystocarps scattered all over the thallus, rostrate (jha, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). amphiora fragilissima (linnaeus) j.v. lamoroux (fig. 3d) description: purple red in colour, up to 3 cm tall, calcified, erect, fragile, regularly dichotomously or trichotomously branched, sometimes with adventitious branches, apices obtuse, segments or intergenicula cylindrical or slightly compressed, several times longer than broad, sometimes with pad-like swellings at the tip, conceptacles lateral, hemispherical, prominent (jhaet al, 1996). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). hypnea pannosa j. agardh (fig. 3e) description: h. pannosa has densely entangled plants and tetrasporangial sori usually growing on one side of branchlets, than gradually completely surrounding the branchlets. this macroalgae are most commonly found growing abundantly on stable rocks of middle to lower intertidal zones, but sometimes they can be found growing in tide pools, branchlets are thick almost all the way to the apex, and the branch tips are often divaricate (chiang, 1990). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). a b c d figure 3. macroalgae of rhodophyta at waisai coast (a. acanthophora spicifera (m. vahl) børgesen, b. caulerpa laurencia papilosa (c. agardh) greville, c. gracilaria salicornia (c. agardh) e.y. dawson, d. gracilaria salicornia (c. agardh) e.y. dawson, d. amphiora fragilissima (linnaeus) j.v. lamoroux, e. hypnea pannosa j. agardh.). e retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 29 brown macroalgae (phaeophyceae) class, order and family of brown macroalgae that found at waisai coast shown in the table 3 below. table 3. checklist of brown macroalgae that found at waisai coast. class order family species phaeophyceae fucales sargassaceae hormophysa cuneiformis (j.f. gmelin) p.c. silva sargassum aquifolium (turner) c. agardh sargassum polycystum c. agardh turbinaria ornata (turner) j. agardh dictyotales dictyotaceae padina australis hauck canistrocarpus cervicornis (kutzing) de paula & de clerck ectocarpales scytosiphonaceae hydroclathrus clatratus (c. agardh) m. howe hormophysa cuneiformis (j.f. gmelin) p.c. silva (fig. 4a) description: dark brown in colour, 30 cm in height or more, sparsely branched, pseudo-dichotomously branched, branches articulated, triangular articulation narrow at the base, 3-5 mm broad, margins dentate, vesicles ellipsoid or oblong, embedded in the middle of swollen wings, solitary (jha et al, 2009). irregularly branched thallus, blade-shaped blade-like wings with three sides and a serrated edge, holdfast filament-shaped filaments (tsuda, 2004). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). sargassum aquifolium (turner) c. agardh (fig. 4b) description: thallus medium brown, 10-45 cm tall, with a discoid-crustose holdfast 7-11 mm wide. primary axes terete, smooth, 3-4 mm long, 1.25-2.00 mm diameter. primary branches smooth to wrinkled, compressed, with sharp edges, 2-4 mm wide. primary laterals alternate, linear lanceolate to ovate-elongate, 13-30 mm long, 5-11 mm wide, margins undulate to deeply dentate, apices obtuse, base subsymmetrical, cuneate, arising directly from the stem and lacking a distinct stalk,margins vanishing 80% of the way to the apex (huisman & parker, 2016). distribution in southeast asia: indonesia (guiry & guiry, 2017). sargassum polycystum c. agardh (fig. 4c) description: thallus yellowish brown in colour, attached with discoid holdfast, main axis cylindrical and rough due to the presence of numerous outgrowth, supporting alternately arranged branches bearing blade and vesicles. in young thalli, blades are longer and broader measuring 13-42 mm long including the stipe and 2.5 – 11.5 mm wide, blades are generally oblong slightly tapered, retuse (slightly rounded) or emarginated at the tip finally serrated throughout the margin, mature thalli fewer leaves smaller. cryptostomates are scattered on the surface of the blade (mattio & payri, 2009). this macroalgae has secondary holdfast that is transformed from the stolon and heavily muricate on main branch (noiraksa, ajisaka, & kaewsuralikhil, 2006). distribution in southeast asia: indonesia, philippines, singapore (guiry & guiry, 2017). turbinaria ornata (turner) j. agardh (fig. 4d) description: dark brown in colour, up to 50 cm tall, bushy, axes arising from dichotomously branched holdfast, main axes erect and cylindrical and irregularly branched, leaves closely arranged, turbinate to obconical, coarse, 0.5-1.5 cm long 10-15 mm broad at the distal ends, distal ends of the leaves triangular, subconcave with double row of spines on the surface with terete stalks, vesicles immerged in the leaves, receptacles racemose, arising on the stalks of the upper leaves(jha et al, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). padina australis hauck (fig. 4e) description: brownish yellow, fan-shaped thallus, texture like a membrane, there is a concentric spike that follows the shape of the thallus, calcareous surface part, rhizoid holdfast (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). canistrocarpus cervicornis (kutzing) de paula & de clerck (fig. 4f) description: growth from ascending, resupinate, attached to tha substrate by broad, erect part more slender and supple, epiphytic specimens generally lack a conspicuous basal part, color yellow to dark brown, dry specimens apical part pale brown, basal part turn a bit darker. branching isotomous dichotomous up to the middle parts typically cervicorn, recurved branches often with cervicorn, branching angle (30) 60-90 (120). margins and surface margins smooth and sinoidally curved (darakrai, 2012). 30 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, vietnam (guiry & guiry, 2017), thailand (darakrai, 2012). hydroclathrus clatratus (c. agardh) m. howe (fig. 4g) description: yellowish to dark brown in colour, up to 12 cm long, 4-8 cm broad, vesicular or irregularly ovate, un-perforated when young, hollow and perforated as net like structures when mature, perforations variable in size, rounded, margins around perforations involute, thallus composed of an epidermal layer and a zone of colourless cells bellow the epidermis, plurolocular sori scattered all over the surface of the thallus, hairs grouped in shallow depression. (jha et al, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). a b c d e f figure 4. macroalgae of phaeophyceae at waisai coast (a. hormophysa cuneiformis (j.f. gmelin) p.c. silva,b. sargassum aquifolium (turner) c. agardh, c. sargassum polycystum c. agardh, d. turbinaria ornata (turner) j. agardh, e. padina australis hauck, f. canistrocarpus cervicornis (kutzing) de paula & de clerck , g. hydroclathrus clatratus (c. agardh) m. howe. g there are nine specimens of macroalgae that can’t be identified to species level, namely: sp. 3. brown colour, brown colour, flat stipe with irregular blade branches, thin blade and branches, penumatocyst found on the base of the blade (figure 5a). eucheuma sp. dark green in colour, irregular branching, cylindrical blade tip of each branch and the main branch off two or three (figure 5b). gracilaria sp. 1.colour pale brown, small thallus with bumps smooth surface (figure 5c). gracilaria sp. 2. the green colour, thin cylinder-shaped thallus with sparse branching, the tip of the needleshaped thallus (figure 5d). gracilaria sp. 3. colour brown, thallus like a needle with a larger size, thick and rigid, irregular branching (figure 5e). sp. 1. the colour brown, thallus branching from the base about 5 branches with each branch are spina-bifida short (figure 5f). sp. 2. dark green colour, thick thallus and looked porous, spherical and elongated like a tool at the thallus filled with fluid (figure 5g). sp. 3. the colour is yellowbrown, primary branches cylindical, opposite branches arrangement, the blade is lanceolate and the margin is jagged (figure 5h). sp. 4. the colour is dark brown, primary branches cylindical, opposite branches arrangement, the blade is linear like spinose with the apex rounded (figure 5. g, i). retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 31 a b c d e f g h i figure 5. macroalgae that cannot be identified to species level at waisai coast (a.sp. 3, b. eucheuma sp., c.gracilaria sp. 1, d.gracilaria sp. 2, e.gracilaria sp. 3, f.sp. 1, g.sp. 2, h. sp. 3, i. sp. 4. table 4. environmental parameters of waisai coast. environmental parameters site 1 site 2 site 3 average salinity 32 ‰ 30 ‰ 31 ‰ 31 ‰ temperature 29,4 ˚c 29,7 ˚c 30,7 ˚c 29.9 ˚c ph 8,05 8,22 8,23 8.17 twenty nine identified macroalgae species have been found in previous studies in southeast asia. southeast asia region where the species are found, among others indonesia, malaysia, myanmar, philippines, singapore, thailand, and vietnam. species that can be found in the seven countries of southeast asia region are halimeda opuntia (linnaeus) j.v. lamoroux, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c. schmidt, dictyosphaeria cavernosa (forsskål) børgesen, acanthophora spicifera (m. vahl) børgesen, gracilaria salicornia (c. agardh) e.y. dawson, hormophysa cuneiformis (j.f. gmelin) p.c. silva, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & de clerck hydroclathrus clatratus (c. agardh) m. howe. halimeda cylindraceae decaisne, laurencia papilosa (c. agardh) greville found only in indonesia, philippines, and vietnam. as for species found only in indonesia, philippines, and singapore is sargassum polycystum c. agardh. the only species found in indonesia is sargassum aquifolium (turner) c. agardh (guiry & guiry, 2017). halimeda cylindraceae is a species of the genus halimeda. halimeda is a genus that has a characteristic thallus form of segments that contain calcium caco3. the content of the lime produced from the process of calcification is an important role for the environment. when individuals macroalgae of the genus halimeda is 32 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 dead, then the content of calcium will be major contributors to the formation of coral reefs (wizemann & meyer, 2014). genus halimeda addition, some other species also contribute to the environment. laurencia papilosa which are still found in three countries of asia also have a role for the environment. macroalgae have been tested have the ability to eliminate waste water colour stain on the textile industry (el maghraby, 2013). the methanol extract of s. aquifolium has the greatest free radical inhibiting activity and the antioxidant activity index is 0.54. based on the rules of the antioxidant activity index, the methanol extract of s. aquifolium is classified as moderate antioxidant activity (muhamad, 2013) this research shows the water temperature at the location of the study had an average of 29.9 ° c, salinity 31 ‰ and an average ph of 8.23. basically every type of macroalgae have standardized optimum temperature for growth vary. according to amalia (2013), the optimum temperature for growth of particular species of macroalgae gracillaria verrucosa ranged 22-27˚c but at a temperature of 31c can still grow although not with good quality. according raiker (2001) in the chung (2007) states that the optimal salinity for the growth of macroalgae that are at normal salinity of seawater is 35 psu. as for ph, according marianingsih et al (2013) seaweed can grow continuously at ph 7-8. based on environmental factors shows that the temperature and salinity of the water obtained in the studies are not optimal location to support the growth of algae. while the ph found in the study site can be classified include the appropriate ph for growth of macroalgae. conclusion based on the research, macroalgae found in waisai coast raja ampat are green algae, red algae and brown algae. green algae found and identified are caulerpa macra (weber-van bosse) draisma & prud’homme, caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), caulerpa cupressoides (vahl) c. agardh, halimeda discoidea decaisne, halimeda opuntia (linnaeus) j.v. lamoroux, halimeda tuna (j. ellis & solander) j.v. lamoroux, halimeda cylindraceae decaisne, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c.schmidt, boergesenia forbesii (hardvey) feldmann, valonia ventricosa j. agardh, dictyosphaeria cavernosa (forsskål) børgesen, chaetomorpha spiralisokamura, anadyomene wrightii harvey ex. j. e. gray, neomeris annulata dickie. red algae species found and successfully identified areacanthophora spicifera (m. vahl) børgesen, laurencia papilosa (c. agardh) greville, gracilaria salicornia (c. agardh) e.y. dawson, amphiora fragilissima (linnaeus) j.v. lamoroux, hypnea pannosa j. agardh. brown algae species found and identified are hormophysa cuneiformis (j.f. gmelin) p.c. silva, sargassum aquifolium (turner) c. agardh, sargassum polycystum c. agardh, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & de clerck hydroclathrus clatratus (c. agardh ) m. howe. the only species found in indonesia is sargassum aquifolium. references anonim. 2001. algae: invasive alien. www.hawaii.edu. retrieved at 22 february 2017 anonim. 2004. seaweed of africa. www.seaweedafrica.org. retrieved at 20 february 2017 atmadja, w., kadi, a., sulistijo, & r, s. (1996). pengenalan jenis-jenis rumput laut indonesia. jakarta: lipi. castro, p., & huber, m. e. (2010). marine biology. new york: mcgrawhill. chiang, y. (1990). species of hypnea lamoroux (gigartinales, rhodophyta) from taiwan. oxford: national sea grant library. darakrai, a. (2012). diversity, species composition and habitat of the genus dictyota j.v. lamour. and canistrocarpus de paula & de clerck in the peninsular thailand. thesis. prince of songkla university el maghraby, d. m. (2013). evaluation of non-viable biomass of laurencia papilosa for decolorization of dye waste water. african journal of biotechnology, 12, 2215-2223. huisman, j., & parker. (2016). florabase. www.florabase.dpaw.wa.gov.au. accessed march 14th, 2017 jha, b., reddy c.r., t. m., & m.u, r. (2009). seaweeds of india. new york: springer. jha, b., reddy, c., thakur, m. c., & rao, m. u. (2009). seaweeds of india. london: springer. kluijvver, m., gijswijt, g., leon, r. d., & cunda, i. (2012). interactive guide to carribean diving. www.speciesidentification.org. lasabuda, r. (2013). pembangunan wilayah pesisir dan lautan dalam perspektif negara kepulauan republik indonesia. jurnal ilmiah platax , 92-101. mambrisaw, a., wurlianty, b., liuw, f., & hamel, s. (2006). atlas sumber daya pesisir kabupaten raja ampat. raja ampat: conservation international indonesia. mattio, & payri. (2009). macoi : university of coimbra. retrieved april 3, 2017, from portuguese seaweeds website: http://macoi.ci.uc.ptf noiraksa, t., ajisaka, t., & kaewsuralikhil, c. (2006). species sargassum in the east coast of the gulf of thailand. science asia, 32, 99-106. olsen, j., & j.a., w. (1988). ventricara (siphonocladalescladophorales complex, chlorophyta). a new genus for valonia ventricosa. department of botany, 56, 103-108. solihin, a., batungbacal, e., & nasution, a. m. (2013). laut indonesia dalam krisis. jakarta: greenpeace. sukiman, muspiah, a., astuti, s. p., ahyadi, h., & aryanti, e. (2014). keanekaragaman dan distribusi spesies makroalga di wilayah sekotong lombok barat. jurnal penelitian unram, 18, 71-81. tsuda, r. (2004). hormophysa cuneiformis (phaeophyta: fucales) in micronesia. pasific sciennce, 58, 23-26. wizemann, a., & meyer, f. w. (2014). a new model for the calcification of the calcification of the green macro-alga halimeda opuntia (lamoroux) . berlin: springer. yagci, m., & turna, i. (2002). a new record for the algal floral of turkey: chaetomorpha crassa (c. ag.) kutz. (cladophoraceae, chlorophyceae). turk j bot, 26, 171-174. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 37-40 | doi: 10.14421/biomedich.2019.82.37-40 issn 2540-9328 (online) anatomical and histological study of shark (carcharhinus sorrah) kidney hikmah supriyati1,*, rakhmiyati2, muhammad ja’far luthfi3 1postgraduate biology education program, yogyakarta state university, indonesia. 2postgraduate biosciences program, sebelas maret university, indonesia. 3biology education department, faculty of science and technology, uin sunan kalijaga, indonesia. author correspondency*: hikmahsupriyati@gmail.com abstract sharks are sea water fishes belong to the class chondrichthyes, subclass elasmobranchii. sharks are cartilaginous fish that have a different osmoregulation process than any other sea water fish. cartilaginous fish is the only vertebrate that can maintain urea. this study aims to determine the anatomical and histological structure of the kidney in the anterior, medial and posterior parts of kidney. the study was conducted by observing anatomy of the kidney. histological preparations were made using the paraffin method. qualitative descriptive data analysis was done. research results show that shark kidneys consist of three parts, namely the head kidney, the body kidney, and the tail kidney. kidney sharks are brownish red with a size of 18 cm long. histological observations of shark kidney in the head kidney reveals many glomerulus, body kidney reveals many distal and tubule proximal contractile tubules whereas tail kidney reveals stroma that is rarely found in vertebrate kidney. keywords: sharks; kidney; anatomy; histology introduction pisces are belonging to poikilothermic vertebrate (cold blooded) that live in water and breathe with gills. every aquatic animal has osmotic pressure that is different from its environment, therefore aquatic animals must prevent excess water or lack of water so that the physiological processes in the body take place normally. pisces is one of the taxa that has the largest member. they inhabit sea water, fresh water, mud and semi aquatic habitats. therefore, the fish's kidney has a different structure and its histology is reflected by its habitat. the osmoregulation mechanism in pisces also varies depending on the environment or habitat (fujaya, 2004). osmoregulation is a process of homeostasis to keep body fluids in a stable state. excretion plays an important role in the removal of metabolic waste substances, especially those containing nitrogen so that these substances do not accumulate in the body. these metabolic waste substances may be able to disrupt homeostasis of the internal environment, especially those related to osmotic pressure and the stability ratio of ions in body fluids (suripto, 1998). the main excretory organ in vertebrates is kidney. kidneys in vertebrates generally a pair. embryologically, kidney originate from mesoderm. kidneys in pisces are different of those in frogs, lizards, birds, especially when compared to mammalian kidneys. there are three types of kidneys namely pronephros, mesonephros and metanephros. fish kidney is the most primitive kidney among other vertebrate kidneys (soesilo et al, 1994). each type of environment provides a variety of supporting factors typical for animals that live in it. the abilities and types of body organs possessed by each animal are different. therefore, the osmoregulation mechanism by animals is also different and shows a very wide variation depending on the ability, osmoregulation organs that are owned, and the environmental conditions of each (isnaeni, 2006). like aquatic animals, freshwater and sea water fishes have differences in osmoregulation and excretion. freshwater fish have osmotic levels of body fluids that are hyperosmotic compared to their environment. thus the fish will excrete a lot of water and hold ions (lagler et al., 1997), whereas sea water fish have osmotic levels of body fluids that are hypo-osmotic in their environment. therefore, fish must drink a lot of water (1/5 to 1/2 of their body weight) and remove a lot of salt from their bodies (suripto, 1998). sharks are water fishes and are included in the class chondrichthyes (cartilaginous fish), subclass elasmobranchii. seawater fish in general will drink a lot of water because it is to maintain bodyi fluids that are hypoosmatic on the environment. in contrast to elasmobranchii which has a problem that is too little water intake. to overcome this animal produces a little urine. even if only a little excreted, the urine can also be used to remove excess nacl (ville, 1988). https://doi.org/10.14421/biomedich.2019.82.37-40 38 biology, medicine, & natural product chemistry 8 (2), 2019: 37-40 these differences allow the organs involved in the osmoregulation process in sharks to have differences with kidney organs in other marine fish. besides this research is also to find out the type of shark kidney. references about the anatomy and histology of sharks are still very rare, so researchers are interested in doing the research. previous research has been carried out under the title anatomical and histological comparisons of the catfish uropoetic organs (pangasius hypophtalmus) and sharks (carcharhinus sorrah). this study discusses one field of view on shark kidney organs and catfish. the histological structure of the shark and catfish consists of glomerulus, distal tubules, proximal tubules and hematopoietic tissue. this research emphasizes the difference in shark glomerular diameter length which is smaller than that of catfish. this researchs focus on the anatomy and histological structure of the kidney in the anterior, transition and posterior parts. this research discussed each histology structure of shark kidney, starting from many glomerulus and tubules in each part of kidney. in addition, this study discussed the histological structure that is likely to be rarely found in other vertebrates. material and methods material test animals used in this study were five sharks obtained from depok beach, yogyakarta. the materials used in this study are hvs paper, bouin solution, graded alcohol (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and absolute), hematoxylin-eosin dyes, albumin, xylol, distilled water, entelan, toluene, absolute alcohol, paraffin, glassware, mask, gloves, needles and tweezers. tool the tools used in this study were a set of surgical instruments, flakon bottles, paraffin tanks, paraffin ovens, beaker glasses, slide warmers, microtomes, staining jars, microscopes and computers. procedure 1. shark dissection this research was conducted in april 2019 in the zoology laboratory of the faculty of science and technology of uin sunan kalijaga yogyakarta. the sharks that has been sacrificed were then observed morphologically. morphological observations included the gills, pectoral fins, dorsal fins, caudal fins, abdominal fins. then the standard length, total length and fish height were measured. 2. anatomical observation anatomical observation started with dissection performed from the posterior ventral to the anterior ventral. after the internal organs were observed, kidneys were taken and cut on three different parts, namely anterior (head), medial (body), and posterior (tail). 3. histological preparations this research was conducted from april to june 2019 in the zoology laboratory of the faculty of science and technology of uin sunan kalijaga yogyakarta. the preparation of histology started with process of fixation which is the process of immersion of organs using a bouin fixative solution. then the dehydration process was carried out with multilevel alcohol, clearing, infiltration, embedding, sectioning, staining with hematoxylin-eosin, and mounting process. then observations using a binocular microscope (suntoro, 1983). results and discussion shark morphology figure 1. general shape and external parts of the shark's body: (1) nostrils, (2) eyes, (3) first dorsal fin, (4) second dorsal fin, (5) tail, (6) fins rectum, (7) pectoral fins, (8) gills, (9) body height, (10) standard length, (11) total length (manik, 2004). based on observations that had been done, sharks have an elongated body shape. total length 74 cm, standard length 53 cm and body height 21 cm. the head is symmetrical and slightly small, has a long, rounded snout. sharks have double dorsal fins. the anterior dorsal fin is larger than the posterior dorsal fin. the caudal fin is pointed, the dorsal caudal fin is longer than the ventral (phillips, 1991). the type of shark's tail is heterocercal (kardong, 2009). there are cloaca holes between the pelvis fins. in male sharks, caudal fins will turn into claspers (organs to hug female sharks when mating) (brotowijoyo, 1994). the dominant body color is black-brown, especially the dorsal, abdomen and caudal fins are rather brightly colored. rows of teeth in the upper jaw are more triangular shaped. the gills open out with 5 gill slits located on the side of the head (jones & larson; 1974). anatomy of shark kidney figure 2. anatomy of a shark kidney: (a) the anterior part (head kidney), (b) the transition part (body kidney), (c) the posterior part (tail kidney). supriyati et al. – anatomical and histological study of shark … 39 anatomy observations show that shark kidney consists of a pair of kidneys that are located along body cavities, ventral to vertebrae. shark's kidney has an elongated shape and is brown in color. the shark's length is 18 cm. shark's kidney consists of three parts, namely the anterior part (head kidney), the medial part (body kidney), the posterior part (tail kidney). adult fish kidney is mesonephros kidney which consists of 70-80 tubules that form the mesonephric duct (vize, 2012). sharks are cartilaginous fish which differ in the osmoregulation process compared to other sea water fish. cartilaginous fish is the only vertebrate that can maintain urea. excess urea will be excreted by the kidneys and gills. cartilage is able to restore blood urea from the filtrate. as a result, blood contains urea that is sufficient to be hyperosmotic to seawater (hildebrand, 1995). the existence of this process makes blood concentration closer to seawater concentration. so that sharks do not need to drink excess sea water. whereas to overcome dehydration sharks also absorb water through the gills and can produce runny urine (castro & huber, 2005). in most animals, urea is poisonous and excreted, but sharks excrete only small amounts. urea and related compounds are not so toxic to cartilaginous fish. the concentration of urea in shark's body fluids can reach 100 times the level of mammalian urea (villee, 1999). excess salt is excreted by the kidneys, intestines, and special glands near the anus called the rectal glands. this gland can help regulate how to deal with excess ions, especially urea. excess urea in the shark's body will be removed through the gills and rectal glands (castro & huber, 2005). histology of shark kidney figure 3. histology of anterior shark kidney: (1) medulla, (2) bowman capsules, (3) glomerulus, (4) cortex, (5) radius medullaris, (6) lymphoid tissue, (7) distal tubule, (8) proximal tubule based on histological observations that have been made that there is a cortex in the kidneys of sharks. the cortex is in a capsule and is dark color (yatim, 1996). kidney capsules in the form of irregular solid connective tissue. the cortex contains the proximal tubule, distal tubule, glomeruli and medullary radius. the medullaris radius is formed by straight nephrons, blood vessels, and collagen tubules that fuse in the medulla (eroschenko, 2010). medulla is the innermost part of the kidney, under the cortex, light colored. medulla contains bundles of vessels and urinary collection vessels, which number in millions according to many malphigian bodies. a malphigian body along with its veins is an autonomous unit of disposal equipment, called a nephron. it is estimated that each hemisphere of the left-right kidney contains two million nephrons. the direction to the medulla renal sinus forms many bulges, called the malphigi’s pyramid. the top of each pyramid is called a papilla. apart from the medulla and cortex on the anterior part of the shark kidney there are also many glomerulus. glomerulus is an anastomotic clot of capillaries. the part that covers the glomerulus is the bowman capsule. the bowman capsule is a ball shape basin that is not full (yatim, 1996). in addition to the anterior part of the kidney there is also a lymphoid tissue (hematopoietic). this network is composed of reticular fibers (subowo, 1992). figure 4. histology of the transitional shark kidney (body kidney: (1) distal contour tubules, (2) proximal tubule tubules. based on observations that have been made that in the transition (body) histology of shark kidney there are many proximal tubule and distal contour tubules. the proximal tubule is longer than the distal tubule. the proximal tubule has one layer of cuboid cells with a granular and eosinophilic cytoplasm. whereas the distal tubule is shorter and the number is less in the cortex. the distal tubule has a larger lumen within cells (eroschenko, 2010). the number of proximal tubules and distal tubules in the transitional part of the kidney (body kidney) allows the process of reabsorption to occur. the absorbed substances are glucose, protein, 40 biology, medicine, & natural product chemistry 8 (2), 2019: 37-40 amino acids and carbohydrates. related to its function the presence of distal tubules also results in ion transport. this is related to the maintenance of acid-base in the blood (ramaley & bevelender, 1979). figure 5. histology of the posterior shark kidney: (1) stroma, (2) blood vessels. based on histological observation at the posterior kidney there is a stroma. renal stroma is rarely found in vertebrate, stroma consists of loose connective tissue that accompanies blood vessels, lymph vessels and nerves. in the stroma there are cells that occupy the gaps between the 3 dimensional webbing which are composed of reticular fibers, reticular cells and macrophage cells (subowo, 1992). basically, the vertebrate kidney has the same structure, namely the glomerulus and tubules. but in each class there are different arrangements. the anterior part of shark kidneys has a lot of glomerulus which in this part allows the filtration process to occur. while in the transition (body kidney) there are many proximal tubules and distal tubules. the results of this research are consistent with the theory by villee (1999) that in the kidney of the fish the anterior tubules have disappeared, several tubules in the middle region are related to the testes, and there is a concentration and multiplying of the posterior tube. in addition there are also archinephric ducts that function as excretion and sperm ducts in males. while the tail of the kidney (posterior kidney) there is a stroma. stroma is usually found in mammals and is rarely found in vertebrate kidneys (subowo, 1992). conclusion based on research that has been done, it can be concluded that, shark kidneys consist of three parts, namely the head kidney (anterior), body kidney (transition), and tail kidney (posterior). kidney shark brownish red with a size of 18 cm. while histological observations of the anterior kidney there are many glomerulus, transition kidney there are many distal tubule, proximal tubule and in the tail kidney there is a stroma, where the stroma is very rarely found in vertebrate kidney. references brotowidjoyo, mukayat djarubito. 1994. zoologi dasar. jakarta: erlangga castro peter dan huber michael. 2005. marine biology fifth edition. new york: mc graw hill companies inc dellmann, h dan brown esther.1989. textbook of veterinary histology. amerika: lea & febiger eroschenko, victor. 2010. difiore’s atlas of histology with functional correlation, 11 th ed. usa: egc medical publisher fujaya, y. 2004. fisiologi ikan dasar pengembangan teknik perikanan. jakarta: pt rineka cipta hildebrsnd, milton.1995. analysis of vertebrate structure fourth edition. united states of america: r.r donnelleycrawfordsville isnaeni, wiwi. 2006. fisiologi hewan. yogyakarta: pt kanisius jones, r, t and h.k, larson 1974. a key to the families of fishes as recorded from guam. univ. of guam, the marine laboratory, tech. rep. (10): 1-4. kisia, seh m. 2009. vertebrates: structures and functions. usa: science publishers lagler, k. f., j.e. bardach, r. r. miller dan d. r. m. passino. 1997. ichtyologi 2nd ed. john wiley & sons. new york. manik, nurdin. 2004. mengenal beberapa jenis hiu. jurnal oseana, volume xxix no (1): 9-17 mescher, anthony. 2010. junquera’s basic histology: text and atlas, 12th ed. new york: mc graw hill companies inc and egc medical publisher ramaley dan bevelender. 1979. dasar-dasar histologi: edisi kedelapan. (alih bahasa oleh ir wisnu gunarso). jakarta: erlangga soesilo, nyoman dkk. 1994. anatomi hewan. jakarta: universitas terbuka soewolo. 2000. pengantar fisiologi hewan. jakarta: proyek pengembangan guru sekolah menengah ibrd loan no 3979, direktorat jenderal pendidikan tinggi departemen pendidikan nasional subowo. 1992. histologi umum. jakarta: bumi aksara suntoro, s. h, 1983. metode pewarnaan histologi dan histokimia. jakarta: bhratara karya aksara. suripto. 1998. fisiologi hewan. bandung: itb press villee, c. a; w. f walker dan r. d burner. 1988. zoologi umum. jakarta: erlangga. villee, claude a et.all. 1999. general zoology: sixth edition (alih bahasa prof. dr. nawangsari sugiri). jakarta: erlangga yatim, wildan. 1996. biologi modern: histologi.bandung: pt tarsito. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 27-31 | doi: 10.14421/biomedich.2018.71.27-31 issn 2540-9328 (online) quercetin: the bioactive compound from allium cepa l. as anti-inflammation based on in silico screening mohamad amin1,*, kurniawan setia putra2, ihya fakhrurizal amin3, nanda earlia4, dina maulina5, betty lukiati1, umie lestari1 1department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia 2graduate school for biology education study program, universitas negeri malang, indonesia 3faculty of medicine, university of indonesia, indonesia 4graduate school of mathematics and applied sciences, universitas syiah kuala, banda aceh, indonesia 5biology education, faculty of teacher training and education, university of lampung, lampung, indonesia author correspondency*: mohamad.amin.fmipa@um.ac.id abstract inflammation is a tissue injury that occurs due to physical trauma or microbiological substances that involve the activities of many cell types. inflammation can be prevented using the natural medicines from allium cepa l. quercetin is one of the bioactive compounds found in allium cepa l and has been reported to have anti-inflammatory activity. the natural medicines have been used to minimize nonsteroidal anti-inflammatory drugs. this study aims to investigate the modeling structures and the protein receptor from quecertin in inflammation mechanism and their optimization of the effectiveness in the human body. the bioinformatics tools used in this study are the database of quercetin compounds, pubchem and swis target prediction protein prediction databases, pyrx 0.8 molecular docking software, ligand docking, and binding site analysis with pymol and ligplus software. the results from in silico show that quercetin compounds can interact with muscleblind-like protein 1 target protein with a binding affinity minus value which is not much different from the dexamethasone compound. dexamethason is a standart because it is a corticosteroid drug that can be used as an antiinflammatory to reduce inflammation, allergic reactions, arthritis and other inflammatory diseases. keywords: allium cepa l.; anti inflamation; in silico, quercetin introduction quercetin compounds are found in the allium cepa l. that contain compounds including carbohydrates (11.0 g), protein (1.2 g), fiber (0.6 g), fat (0.30%) and several vitamins such as vitamin a (0.012 mg), vitamin c (11 mg), thiamin (0.08 mg), riboflavin (0.01 mg), and niacin (0.2 mg), and some minerals such as phosphorus, calcium, sodium, iron and potassium (rodrigues et al., 2003). quercetin has a molar mass of 302,236 g/mol, in the form of yellow crystalline powder, a density of 1,799 g/cm3 and a melting point of 316 ° c (smith et al., 2003). inflammation is a local reaction to tissue infection or injury and involves more mediators. inflammation has a fairly high incidence, where inflammation can be caused by physical trauma, infection or antigenic reactions from an illness. the natural secondary metabolite compound that has the potential for inflammatory treatment is quercetin, which is abundant in the allium cepa. chronic inflammation means long-term inflammation, which can last for several months and even years. this is due to the failure to remove anything that causes acute inflammation, an autoimmune response to self-antigens (the immune system attacks healthy tissue), an existing low-intensity chronic irritation. the research on allium cepa has been done before, however the mechanism of quercetin compounds can prevent the occurrence of inflammatory processes is unknown. therefore, this study aims to identify natural bioactive compounds from onion allium cepa for antiinflammatory based on in silico. we investigate the modeling structures and the protein receptor from quercetin in inflammation mechanism and their optimization of the effectiveness in the human body. materials and methods in silico screening in this research was conducted following the previous methods (maulina, et. al., 2018; pangastuti et.al, 2016; 2018 and ilmawati, 2017). retrieval of sample this research used the spesific compound from allium cepa l. the quecertin is the most abundant compound in allium cepa l (fredotović, z. et al., 2017) and it has been reported to have anti-inflammatory activity. therefore, quecertin was selected as a specific sample for analysis. in addition, the compositions of quercetin were obtained from the compounds database of pubchem. https://doi.org/10.14421/biomedich.2018.71.27-31 28 biology, medicine, & natural product chemistry 7 (1), 2018: 27-31 preparation of ligand the structure of quercetin 3d chemical compounds and smiles ligands is taken from the database on the pubcem server online (https://pubchem.ncbi.nlm.nih.gov/) with id number: cid: 5280804. target selection target protein selection is predicted by using the help of a number of servers to check and ensure the target protein is selected correctly. first, enter the ligand quercetin smiles value on the pharmmapper server online (http://59.78.96.61/pharmmapper/) to identify potential target proteins. second, observing potential target proteins on the swiss target prediction server online (http://www.swisstargetprediction.ch/) to look for similar target proteins which found on the first server. third, observing potential target proteins on the superpret server online (http://prediction.charite.de/) to adjust the similarity of target proteins which found on the first and second servers. the process of observing the target protein is carried out repeatedly to ensure the similarity of the target protein is correct, so could be not to affect the failure or inaccuracy during the docking process. docking molecules docking of quercetin molecules, target proteins, and control compounds (dexamethasone) which are chemical drugs in the treatment of inflammation using pyrx 0.8 software. visualization and molecular interaction the interaction between quercetin, target proteins, and control compounds known based on protein visualization was then analyzed using pymol and ligplus software and discovery studio 2016 client. results and discussion target selection results using pharmmapper (job id: cid 5280804), swiss target prediction and superpret found that quercetin interacts with the muscleblind-like protein 1 receptor. quercetin is a group of flavonoids produced by plants and can be used to treat inflammatory diseases and these compounds can be isolated from bulb onions (allium cepa). based on the results of the study it was found that quercetin compounds were able to bind to muscleblind-like protein 1 receptor protein (fig. 1) by forming bonds at one end of the side. visualization results using pymol and ligplus software show the following images (figure 1). the bonding between muscleblind-like protein 1 receptor as a target protein and quercetin compound has the same position as chemical compounds which have generally been used as inflammatory drugs, namely dexamethasone (figure. 2). figure 1. binding visualization between protein target (muscleblind-like protein 1 receptor) and natural bioactive quercetin (brown is protein target; green is quercetin). figure 2. visualization of binding between protein target, quercetin as bioactive compound and dexamethasone as control compound. note: brown color for protein target; green for quercetin and purple for dexamethason). dexamethasone is a corticosteroid drug that can be used as an anti-inflammatory to reduce inflammation, allergic reactions, arthritis and other rheumatic diseases, skin diseases, intestinal diseases such as ulcerative colitis and multiple sclerosis or myasthenia gravis. in general, dexamethasone is widely used in the therapy of chronic inflammatory diseases for its pain-modulating effects (laste et al, 2013). moreover, dexamethasone significantly inhibited the levels of tnf-α and il-6, suggesting a key role for these cytokines in sickness behavior (plessers et al. 2015). the results showed in figure 2 indicate that the natural compound of quercetin has the same molecular interaction as dexamethasone, a chemical compound that is often used as an anti-inflammatory drug. in addition, the results of the interaction analysis of the natural compound quercetin and the muscleblind like protein 1 protein were compared with the dexamethasone amin et al. – quercetin: the bioactive compound from allium cepa l.… 29 chemical compound with the muscleblind like protein 1 protein, which showed no significant difference. this shows that the natural compound quercetin is very potential to be used as an anti-inflammatory drug. the results of the calculation analysis of the target protein ligand bond interaction is described in table 1 and 2 as following. table 1. the analysis of the interaction of quercetin compounds with muscleblind-like protein 1 target protein. ligand binding affinity rmsd/ub rmsd/lb quercetinmuscleblind-like protein 1 -6.5 0 0 quercetinmuscleblind-like protein 1 -6.3 21.774 17.9 quercetinmuscleblind-like protein 1 -6.1 4.053 2.596 quercetinmuscleblind-like protein 1 -6 8.163 4.193 quercetinmuscleblind-like protein 1 -5.8 22.599 18.725 quercetinmuscleblind-like protein 1 -5.8 22.149 18.221 quercetinmuscleblind-like protein 1 -5.8 22.272 19.67 quercetinmuscleblind-like protein 1 -5.7 6.343 1.724 quercetinmuscleblind-like protein 1 -5.7 4.238 2.877 table 2. the analysis of the interaction of dexamethasone compounds with muscleblind-like protein 1 target protein. ligand binding affinity rmsd/ub rmsd/lb dexamethasone muscleblind like protein 1 -6.5 0 0 dexamethasone muscleblind like protein 1 -6.2 3.953 2.41 dexamethasone muscleblind like protein 1 -6 7.669 2.149 dexamethasone muscleblind like protein 1 -5.8 21.751 19.441 dexamethasone muscleblind like protein 1 -5.8 7.481 1.846 dexamethasone muscleblind like protein 1 -5.7 25.176 23.523 dexamethasone muscleblind like protein 1 -5.7 21.778 19.947 dexamethasone muscleblind like protein 1 -5.7 16.298 13.067 dexamethasone muscleblind like protein 1 -5.7 19.714 16.467 analysis based on data in tables 1 and 2 shows that it is not significantly different from the chemical bonds between quercetin and dexamethasone, with the binding affinity value of the two compounds in the similar ranges. the binding affinity value shows the best bond between the ligand and the target protein. the minus value shows that the ligand is easier to bind to the target protein because it requires less energy to bind. based on the range of binding affinity values showed that many ligand residues that bind to the target protein, especially in dexamethasone compounds as control compounds have better bond range than natural compounds, quercetin compounds. but from the range of values these two compounds show not much different. visualization of the results of inter-molecular interactions using the discovery studio 2016 client software shows that the bond distance between molecules and the type of bond that occurs on each residues of the ligand molecule and target protein more details show in figure 3 and 4. cyclooxygenase (cox) or prostate glandinendo peroxide synthase (pghs) is a bifunctional enzyme that initially converts arachidonic acid to prostaglandin g2 (pgg2) through oxygenated, then catalyzes pgg2 peroxidase to pgh2. pgh2 is a precursor to the formation of several important mediators for pain, fever and inflammation. two forms of cyclooxygenase enzyme isoforms are known, namely cox-1 and cox2. cox-1 is a constitutive enzyme that have a role for play in the regulation of several cellular processes, including homeostasis vascular, protection of the gastrointestinal tract and kidney function. cox-2 is induced and is in inflamed tissue. inhibition of this cox enzyme is the main working mechanism of non-steroidal anti-inflammatory drugs that are widely used (kartasasmita et al, 2009). 30 biology, medicine, & natural product chemistry 7 (1), 2018: 27-31 figure 3. distance of intra interaction between quercetin (bioactive compound) and protein target (muscleblind-like protein 1) and the type of binding. figure 4. distance of intra interaction between dexamethasone (control compound) and protein target (muscleblind-like protein 1) and the type of binding. cyclooxygenase (cox) or prostate glandinendo peroxide synthase (pghs) is a bifunctional enzyme that initially converts arachidonic acid to prostaglandin g2 (pgg2) through oxygenated, then catalyzes pgg2 peroxidase to pgh2. pgh2 is a precursor to the formation of several important mediators for pain, fever and inflammation. two forms of cyclooxygenase enzyme isoforms are known, namely cox-1 and cox2. cox-1 is a constitutive enzyme that have a role for play in the regulation of several cellular processes, including homeostasis vascular, protection of the gastrointestinal tract and kidney function. cox-2 is induced and is in inflamed tissue. inhibition of this cox enzyme is the main working mechanism of non-steroidal anti-inflammatory drugs that are widely used (kartasasmita et al, 2009). conclusion this study proves that the natural compound quercetin found in onion plants (allium cepa l) has the potential as an anti-inflammatory drug. the type of chemical bounding between protein ligand and control compound (dexamethasone) is similiar. there is no to significant the bond distance and type of chemical bond formed. references fredotovic, ž., šprung, m., soldo, b., ljubenkov, i., budi´c-leto, i., bilušic´, t., cˇ ikeš-ˇulic´, v., and puizina, j. 2017. chemical composition and biological activity of allium cepa l. and allium _ cornutum(clementi ex visiani 1842) methanolic extracts. molecules, 22, 448; doi:10.3390/molecules22030448. amin et al. – quercetin: the bioactive compound from allium cepa l.… 31 kartasasmita, r.e., herowati, r., harmastuti, n., & gusdinar, t. (2009). quercetin derivatives docking based on study of flavonoids interaction to cyclooxygenase-2. indo. j. chem. 9 (2):297-302. maulina, d., amin, m., lestari, s.r., & aziz, m. 2018. alanine as natural biopesticide from mirabilis jalapa and its interaction with glutamate as an inhibitor in insects immune system. journal of biological researches, vol. 23: 2. doi: 10.23869/bphjbr.23.2.20185. pangastuti, a., amin, i.f., zhilalikbar, a., amin, m. 2016. natural bioactive compound from moringa oleifera against cancer based on in silico screening. jurnal teknologi (sciences & engineering). 78(5): 315-318 pharmmapper database. http://59.78.96.61/pharmmapper/ (accessed on march 2018) plessers, e.; watteyn. a.; wyns, h.; pardon, b.; de backer, p. and croubels, s. 2015. study of the immunomodulatory properties of gamithromycin and dexamethasone in a lipopolysaccharide inflammation model in calves. res vet sci. 2015 dec; 103:218-23. doi: 10.1016/j.rvsc.2015.10.014. epub 2015 oct 28 pubchem database. https://pubchem.ncbi.nlm.nih.gov/ (accessed on march 2018) puspita sari, 2015.studi in silico daun salam (syzygium polyanthum) terhadap angio tensin converting enzyme. jurnal kedokteranvol 3, no 1 ringo maeda, 2013. isolasi senyawa flavonoid dari kulit bawang merah (allium cepa l).skripsi fakultas matematika dan ilmu pengetahuan alam, universitas sumatra utara medan. rodrigues a., fogliano v., graziani g., mendes, s., vale, a. and goncalves, c., 2003. nutrition value of onion regional varieties in northwest portugal, ejeafche 2(4): 519-524. ryfai, ea 2012, ‘penapisan in silico anti malaria dari basis data tanaman obat indonesia terhadap target plasmepsin’, (skripsi) fakultas matematika dan ilmu pengetahuan alam, universitas indonesia. smith, c., lombard, k.a., peffley, e.b.,liu, w. 2003. genetic analysis of quercetin in onion (allium cepa l.) ‘lady raider’. the texas journal of agriculture and natural resource 16:24-28. soemari yulistia, 2016. uji aktivitas anti inflamasi quercetin bawang merah (allium cepa l).pada mencit putih jantan (musmusculus) superpret server. http://prediction.charite.de/ (accessed on march 2018) swiss target prediction. http://www.swisstargetprediction.ch/ (accessed on march 2018) teresita, g., alejandra, e.r., americo, o. j., &lilian, e.p. 2001. anti-inflammatory properties of plant flavonoids. effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. elsevier ilfarmaco 56:683-687. https://www.ncbi.nlm.nih.gov/pubmed/?term=plessers%20e%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=watteyn%20a%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=wyns%20h%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=pardon%20b%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20backer%20p%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=croubels%20s%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/26679821 https://www.ncbi.nlm.nih.gov/pubmed/26679821 this page intentionally left blank quercetin: the bioactive compound from allium cepa l. as anti-inflammation based on in silico screening biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 47-51 | doi: 10.14421/biomedich.2017.62.47-51 issn 2540-9328 (online) clustering of 18 local black rice base on total anthocyanin kristamtini1, endang wisnu wiranti2 1,2assessment institute for agricultural technology (aiat) yogyakarta jln. stadion maguwoharjo no. 22, wedomartani, ngemplak, sleman, yogyakarta, indonesia author correspondency: krisniur@yahoo.co.id1 abstract black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color. anthocyanin serve as an antioxidant that control cholesterol level in the blood, prevent anemia, potentially improve the body's resistance to disease, improve damage to liver cells (hepatitis and chirrosis), prevent impaired kidney function, prevent cancer/tumors, slows down antiaging, and prevent atherosclerosis and cardiovascular disease. exploration results at aiat yogyakarta, indonesia from 2011 to 2014 obtained 18 cultivar of local black rice indonesia. the names of the rice are related to the color (black, red or purple) formed by anthocyanin deposits in the pericarp layer, seed coat or aleuron. the objective of the study was to classify several types of local black rice from explorations based on the total anthocyanin content. the study was conducted by clustering analyzing the total anthocyanin content of 18 local black rice cultivars in indonesia. cluster analysis of total anthocyanin content were done using sas ver. 9.2. clustering dendogram shows that there were 4 groups of black rice cultivars based on the total anthocyanin content. group i consists of melik black rice, patalan black rice, yunianto black rice, muharjo black rice, ngatijo black rice, short life of tugiyo black rice, andel hitam 1, jlitheng, and sragen black rice. group ii consists of pari ireng, magelang black hairy rice, banjarnegara-wonosobo black rice, and banjarnegara black rice. group iii consists of ntt black rice, magelang non hairy black rice, sembada hitam, and longevity tugiyo black rice. group iv consist only one type of black rice namely cempo ireng. the grouping result indicate the existence of duplicate names among the black rice namely patalan with yunianto black rice, and short life tugiyo with andel hitam 1 black rice. keywords: clustering; black rice; exploration; total anthocyanin. introduction black rice is a type of rice, along with white rice and red rice. black rice is not consumed as a staple food, but consumed as functional food by some communities. functional food is a food which naturally or through a certain process contains one or more compounds considered to have physiological functions that are beneficial to health (wijayanti, 2004). black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color (ryu et al., 1998; takashi et al., 2001). black rice is one of the functional foods because of its anthocyanin content. anthocyanin pigments effectively reduce cholesterol levels in the human body (lee et al., 2008). anthocyanin has been recognized as functional health foodstuff due to its antioxidant activity (satue-gracia et al., 1997; nam et al., 2006; philpot et al., 2006). anthocyanin serve as an antioxidant that cleans blood cholesterol, prevent anemia, potentially increase body resistance to disease, improve liver cell damage (hepatitis and chirrosis), prevent impaired kidney function, prevents cancer/tumors, slows down antiaging (harmanto, 2008), as well as to prevent narrowing of the arteries (atherosclerosis) and cardiovascular disease (ling et al., 2001 and ling et al., 2002). indonesia is a megabiodiversity country. black rice is one of the wealth of indonesia genetic resources that need to be conserved. therefore, the explorations of black rice in the region of yogyakarta and other regions in indonesia are important. eighteen black rice cultivars were collected by aiat yogyakarta, indonesia. the name of the black rice was different, some even have no name, just given the name according to the farmers who cultivate it. the difference in the name of black rice is allegedly caused by the diversity of the color of the rice, from bright black to black. the color difference of rice occurs as a result of differences in anthocyanin content. characterization of morphological properties have been done, while the study of the diversity of anthocyanin content in some black rice has not been done. solouki et al., (2008) suggests that the study of genetic diversity can be done with morphological, biochemical, and molecular markers. biochemical studies of anthocyanin content in some black rice complement the morphological characterization, and was expected to help plant breeders to choose cultivars as parent in plant breeding programs. based on these considerations, this study aimed to classify the 18 black rice cultivars from indonesia based on the total anthocyanin content. http://dx.doi.org/10.14421/biomedich.2017.62.47-51 mailto:krisniur@yahoo.co.id 48 biology, medicine, & natural product chemistry 6 (2), 2017: 47-51 materials and methods the research began by planting 18 black rice cultivars from exploration (table 1), and maintained until harvest. the black rice from the harvest were analyzed for the total anthocyanin content in the agricultural production technology laboratory of gadjah mada university yogyakarta according to lees & francis method (1972) (appendix 1). clustering analysis of total anthocyanin were done using sas ver software. 9.2. cluster method was analyzed using hierarchical cluster analysis to know the kinship relationship. hierarchical cluster analysis was a common way of grouping objects in groups that share resemblance to each other. table 1. eighteen black rice cultivars were collected by aiat yogyakarta, indonesia. black rice cultivar code origin 1. melik a bantul, yogyakarta, indonesia 2. jlitheng b sleman, yogyakarta, indonesia 3. cempo ireng c sleman, yogyakarta, indonesia 4. pari ireng d sleman, yogyakarta, indonesia 5. beras hitam ntt e ntt, indonesia 6. beras hitam bantul o bantul, yogyakarta, indonesia 7. beras hitam magelang berbulu r magelang, jawa tengah, indonesia 8. beras hitam magelang tak berbulu s magelang, jawa tengah, indonesia 9. beras hitam sragen t sragen, jawa tengah, indonesia 10. beras hitam banjarnegara berbatasan wonosobo w banjarnegara, jawa tengah, indonesia 11. beras hitam banjarnegara y banjarnegara, jawa tengah, indonesia 12. beras hitam tugiyo umur panjang aa bantul, yogyakarta, indonesia 13. sembada hitam ab sleman, yogyakarta, indonesia 14. beras hitam muharjo ac bantul, yogyakarta, indonesia 15. beras hitam patalan ad bantul, yogyakarta, indonesia 16. beras hitam tugiyo umur pendek ae bantul, yogyakarta, indonesia 17. andel hitam 1 af kulon progo, yogyakarta, indonesia 18. beras hitam yunianto ag bantul, yogyakarta, indonesia results and discussion black rice was one of functional foods due to anthocyanin content that expressed in rice color. black rice is one of anthocyanin sources. other anthocyanin source are fruits, vegetables, and grains that have given color pigments. healthy foods usually have a black or purple color, but in terms of appearance, this color less appealing. most plants have the largest anthocyanin content in fruit (houghton & hendry 1995). some plants, such as tea, cocoa, cereals, beans, red cabbage, and petunias have anthocyanins in plant par ts other than fruits (lila 2004; sterling 2011). anthocyanins also found in foods that have red, purple to black pigments such as black soybeans, black sesame, blackberry, and grapes. foods consumed daily are often source s of free radicals that harmful to the body. free radicals can accelerate the aging process and increase the risk of diseases such as heart disease, stroke, diabetes, arthritis, and cancer. body damage due to free radical attack can be prevented by antioxidants. free radicals are atoms or molecules that are very active and unstable because the structure of an atom or its molecule has an unpaired electron. in order to be stable, these atoms or molecules need to get another electron by taking an electron from another molecule. anthocyanin can serve as an antioxidant, a compound capable of donating its electrons to neutralize the damaging properties of free radicals (bustanul 2007). based on these matters then conducted analysis of total anthocyanin content on 18 local black rice collection of aiat of yogyakarta. the total anthocyanin content of 18 black rice cultivars is presented in table 2. table 2 shows that each black rice cultivar contains anthocyanin content varying from 53.22 to 428.38 mg/ 100 g, according to its genetic potential. the total anthocyanin content is expressed as the color of rice, and the color of rice from each rice cultivar is presented in figure 1. kristamtini & wiranti – clustering of 18 local black rice base on total anthocyanin 49 table 2. total anthocyanin content in 18 black rice cultivars. black rice cultivar code total anthocyanin (mg/100 g) 1. melik a 100.06 2. jlitheng b 53.22 3. cempo ireng c 428.38 4. pari ireng d 230.48 5. beras hitam ntt e 264.43 6. beras hitam bantul o 90.22 7. beras hitam magelang berbulu r 196.34 8. beras hitam magelang tak berbulu s 288.53 9. beras hitam sragen t 65.04 10. beras hitam banjarnegara berbatasan wonosobo w 179.09 11. beras hitam banjarnegara y 165.78 12. beras hitam tugiyo umur panjang aa 298.93 13. sembada hitam ab 284.05 14. beras hitam muharjo ac 122.45 15. beras hitam patalan ad 107.22 16. beras hitam tugiyo umur pendek ae 81.81 17. andel hitam 1 af 82.55 18. beras hitam yunianto ag 107.39 figure 1. appearance of rice color from 18 black rice cultivars. (a). melik; (b). jlitheng; (c). cempo ireng; (d). pari ireng; (e). beras hitam ntt; (f). beras hitam bantul; (g). beras hitam magelang berbulu; (h). beras hitam magelang tak berbulu; (i). beras hitam sragen; (j). beras hitam banjarnegara berbatasan wonosobo; (k). beras hitam banjarnegara; (l). beras hitam tugiyo umur panjang; (m). sembada hitam; (n). beras hitam muharjo; (o). beras hitam patalan; (p). beras hitam tugiyo umur pendek; (q). andel hitam 1; (r). beras hitam yunianto. black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color. the color difference of rice is regulated genetically. according to chang & bardenas (1965), the color of rice is determined by the pigments in the pericarp and the tegmen, namely white, bright red, red, reddish brown, brown, grayish, gold, purple, and purple (almost black), while endosperm from all rice varieties was white. ayoola & faluyi (2007), said that pericarp rice seeds are usually white but may be red, brown or almost purple, pigmentations/colorations are no deeper than pericarp and endosperm was always white. clustering of black rice based on total anthocyanin content in 18 black rice cultivars in the form of dendogram in figure 2. 50 biology, medicine, & natural product chemistry 6 (2), 2017: 47-51 information: melik : a beras hitam banjarnegara berbatasan wonosobo : w jlitheng : b beras hitam banjarnegara : y cempo ireng : c beras hitam tugiyo umur panjang : aa pari ireng : d sembada hitam : ab beras hitam ntt : e beras hitam muharjo : ac beras hitam bantul : o beras hitam patalan : ad beras hitam magelang berbulu : r beras hitam tugiyo umur pendek : ae beras hitam magelang tak berbulu : s andel hitam 1 : af beras hitam sragen : t beras hitam yunianto : ag figure 2. dendogram of clustering of 18 black rice cultivars based on the total anthocyanin content. dendogram in figure 2 shows that 18 black rice cultivars based on their total anthocyanin content were divided into 3 groups. group i consists of a (melik), ad (beras hitam patalan), ag (beras hitam yunianto), ac (beras hitam muharjo), o (beras hitam bantul), ae (beras hitam tugiyo umur pendek), af (andel hitam 1), b (jlitheng), and t (beras hitam sragen). group ii consists of d (pari ireng), r (beras hitam magelang berbulu), w (beras hitam banjarnegara berbatasan wonosobo), y (beras hitam banjarnegara), e (beras hitam ntt), s (beras hitam magelang tak berbulu ab (sembada hitam) and aa (beras hitam tugiyo umur panjang). group iii consists of only cultivar c = cempo ireng, because cempo ireng has the highest anthocyanin content of 428.38 mg / 100 g. it appears in figure 2 that, in group 1, there are 2 cultivars that have the same genetic distance as the total antioxidant content, that was ad (beras hitam patalan) and ag (beras hitam yunianto). two other cultivars in group i also have the same genetic distance, thas was ae (beras hitam tugiyo umur pendek) and af (andel hitam 1). the cultivars are allegedly the same or there was duplication of names based on their total anthocyanin content. the result of clustering based on the total anthocyanin content still needs to be supported by clustering based on plant morphology, such as plant height, plant age, stem color, leaf color, leaf tongue color, leaf ear color and so forth. conclusion the total anthocyanin content in 18 black rice cultivars of aiat of yogyakarta collection varies according to its genetic potential. there are 3 groups of black rice based on the total anthocyanin content, some cultivars have the same genetic distance (close kinship relationship) and there were suspected duplication of the name based on the total anthocyanin content. name of observation or cluster c aa ab s e y w r d t b af ae o ac ag ad a average distance between clusters 0 25 50 75 100 125 150 175 200 225 250 275 300 kristamtini & wiranti – clustering of 18 local black rice base on total anthocyanin 51 suggestion required clustering of black rice cultivars based on morphological properties to support of clustering based on the total anthocyanin content. references ayoola, a.o. and j.o. faluyi. 2007. inheritance of caryopsis/ ripened-hull colour in a selection from land races of rice oryza sativa (linn.). agric. j. 2 (3): 412-414. bustanul. 2007. manfaat beras hitam. http://bumiganesa.com. harian metro online friday, 08 june 2007. retrieved at 28 april 2011. chang, te-tzu and e.a. bardenas. 1965. the morphology and varietas characteristics of the rice plant. technical bulletin 4 december, 1965. irri.los baños, laguna, the philippines. 27 31. harmanto, a. 2008. varietas beras organik berdasarkan warna. http://aghribisnis-ganesha.com. p. 146. retrieved at 26 september 2008. houghton, j.d. and g.a.f. hendry. 1995. natural food colorants. springer. pp. 53-59. lee, j.c., j.d. kim, f.h. hsieh, and j.b. eun. 2008. production of black rice cake using ground black rice and medium-grain brown rice. int'l. j. food sci. technol. 43 (6): 1078-1082 lila, m.a. 2004. anthocyanins and human health: an in vitro investigative approach. j. biomed. biotechnol. 5: 306–313. doi: 10.1155/s111072430440401x. http://www.ncbi.nlm. nih.gov/pmc/articles/pmc1082894/. ling, w. h., cheng, q. x., ma, j. and wang, t. 2001. red and black rice decrease artherosclerotic plaque formation and increase antioxidant status in rabbits. j. nutr. 131: 1421 1426. ling, w.h., l.l. wang and j. ma. 2002. supplementation of black rice outer layer fraction to rabbits decreases the atherosclerotic plaque formation and increases antioxidant status. j. nutr. 132: 20 26. nam, s.h., s.p. choi, m.y. kang, h.j. koh, n. kozukue, and m. friedman. 2006. antioxidative activities of bran from twenty one pigmented rice cultivars. food chem. 94: 613-620. philpot, m., k.s. gould, c. lim, and l.r. ferguson. 2006. in situ and in vitro antioxidant activity of sweet potato anthocyanins. j. agric. food chem. 54: 1710-1715. ryu, s.n., s.z. park, and c.t. ho. 1998. high performances liquid chromatographic determination of anthocyanin pigments in some varieties of black rice. j. food drug analysis 6: 1710-1715. satue-gracia, m., i.m. heinonen, and e.n. frankel. 1997. anthocyanins as antioxidants on human low-density lipoprotein and lechthin-liposome system. j. agric. food chem. 45: 3362–3367. solouki, m., h., mehdikhani, h. zeinali & a.a. emamjomeh, 2008. study of genetik diversity in chamomile (matricaria chamomilla) based on morphological traits and molecular markers. sci. hortic. sterling, r.d.m. 2011. anthocyanins. the chiropractic resource organization, 19 march 2011. takashi, i., x. bing, y. yoichi, n. masaharu and k. tetsuya. 2001. antioxidant activity of anthocyanin extract from purple black rice. j. med. food. 4: 211218. wijayanti, e. 2004. potensi dan prospek pangan fungsional indigenous indonesia. presented at seminar nasional pangan fungsional indigenous indonesia: potensi, regulasi, keamanan, efikasi dan peluang pasar. bandung, 6-7 october 2004. http://bumiganesa.com/ http://www.ncbi.nlm/ this page intentionally left blank clustering of 18 local black rice base on total anthocyanin biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 17-21 | doi: 10.14421/biomedich.2019.81.17-21 issn 2540-9328 (online) a simple method for clearing and staining specimens for the demonstration of animal skeleton muhammad ja’far luthfi1,*, nyoman puniawati soesilo2 1department of biological education, faculty of science & technology uin sunan kalijaga yogyakarta, indonesia 2faculty of biology, universitas gadjah mada yogyakarta, indonesia. author correspondency*: jafarluthfi@yahoo.com abstract teaching skeletal system would be more attractive using real/preserved specimen compared to those using only book. the aim of the research was to develop clearing and staining method of animal skeleton specimen using alizarin red s-alcian blue as teaching aid tool. the specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. the spesimen will increase effectiveness in elementary school science teaching and learning. keywords: skeleton; alizarin red s; alcian blue; elementary school; teaching aid introduction skeleton is a supporting system for vertebrate bodies. skeleton serves to give shape and support the body, as a means for movement, as a protector of internal organs and delicate organs (blood vessels and nerve tissue), and as a place for muscle attachment. skeleton is divided into axial skeleton and appendicular skeleton. the axial skeleton consists of skull and vertebrae (spine), while the appendicular skeleton consists of bones in the fore limbs and hind limbs (kardong, 2002). vertebrates are animals that have an endoskeleton and from one of its components the name is taken (vertebra/spine). the skeletal system is one of the subject taught in high school. this material is usually taught by using books or pictures. however, according to prokop et al (2007), teaching skeletal systems will be more attractive if using original specimens or preserved material compared to books. direct observation plays an important role in students' understanding of biological phenomena. students' mental models of a biological phenomenon are strongly influenced by the learning process (prokop et al, 2007). the national science education standards in the usa emphasize that scientific inquiry is the basis of science education, where students at all levels should have the opportunity to conduct scientific inquiry. experiments are not only provide information about an organism's system, but can also develop the ability to observe, think critically and experimentally. students who follow the science process will better understand science concepts, appreciate how science concepts are discovered, and enhance the skills needed to investigate nature (greene & greene, 2001; newman et al., 2004). clearing and staining of the skeleton aims to demonstrate bone structure and cartilage in animals (erdogan et al., 1995; inouye, 1976). this method is commonly used in the field of teratology to study fetal defects. in teratological studies, experimental animals used are rat and mouse embryos. since bone tissue and cartilage in embryos and in adult animals are similar histologically, and so do the small vertebrates and the large ones, theoretically the method for mouse embryos can be applied to demonstrate skeletons in adult animals or in larger vertebrates (junquiera & carniero, 1988; leeson & leeson, 1981). from its ability to demonstrate bone and cartilage structure, this method has the potential to be applied for developing skeletal learning media. the method described in this article is a procedure for clearing and staining animal skeleton specimens using alizarin red s-alcian blue as a tool in teaching the skeletal system. the procedure can be followed by students themselves. materials and methods the animals used in this study were goldfish (cyprinus carpio), oriental whip-snake (ahaetulla prasina), skink (mabouya multifasciata), turtle (trachemys scripta elegans), and flying dragon (draco volans). the equipments used in this study were surgical scissors, scalpels, tweezers, and glass jars. the chemical used in this study were alizarin red s-alcian blue, ethanol, acetone, aquades, glacial acetic acid, glycerin, and koh. the method is a modification of the inouye (1976) originally used for small vertebrates/embryo. inouye is https://doi.org/10.14421/biomedich.2019.81.17-21 mailto:jafarluthfi@yahoo.com 18 biology, medicine, & natural product chemistry 8 (1), 2019: 17-21 one of the first researchers that successfully use the method for bones and cartilage staining. this is a double staining method for bone and cartilage of mice for teratological studies. using this method, cartilage stained blue, bone stained red, whereas muscles and other tissues become transparent. preparations for animal larger than mice and not embryos will need modification of a more massive skin and muscle removal, additional fixation time, fat removal, and extensive clearing and staining due to the thickness and robustness of tissue of mature and larger vertebrates. clearing and staining specimens (carp, snake, gecko tail, turtle tail, flying dragon) include several stages. the first step is exfoliation. after the scales were removed, the specimens were fixed in a tube containing 95% alcohol for 3 days. then the skin and muscles in the specimen are removed using scissors, tweezers and scalpel. then fixed again in 95% new ethanol for 3 days. to remove fat, the fixed specimen was immersed in a tube containing acetone for 4 days with the replacement of new acetone on the second day. after that the specimens were immersed for 5 days in a dye solution at 37 ° c (1 volume 0.3% alcian blue in ethanol 70% + 1 volume 0.1% alizarin red s in 95% ethanol + 1 volume glacial acetic acid + 17 volume ethanol 70%). the specimens were washed in water and then immersed in a solution of 1% koh for 5 days (to make it transparent) and then soaked in succession of a mixture of glycerin and 1% koh at a ratio of 20%: 80%; 50%: 50%; 80%: 20%. finally, the specimen were stored in pure glycerin. results and discussion this research has successfully develop method for clearing and staining animal skeleton specimens with alizarin red s-alcian blue (figure 1, figure 2, figure 3, figure 4 and figure 5). figure 1 shows a carp skeleton. the preparation shows the bones of the skull, vertebrae (backbone), and the ray fin of tail of fish that are suitable for live in water. figure 2 is skeleton specimen of oriental whipsnake. the snake specimens show a special adaptation of vertebrate skeletons, that is, very large numbers of vertebrae (+ 300 vertebrae) suitable for terrestrial life without limbs. figure 3 shows a skink's tail skeleton. the preparation shows the structure of the tail’s vertebrae that can autotomized. figure 4 shows a turtle's tail skeleton. figure 5 shows the skeleton of flying dragon. the turtle and flying dragon specimens show the structure of the tail vertebra in animals that cannot be autotomized. using these specimens, students can directly observe the structure of the real specimen, can figure out its morphology, know the difference in structure related to its function, and gain direct experience about the skeleton. students can more easily understand the way of life and movement of snakes and the appearance of their elongated morphology by looking at the structure of the snake's vertebrae and its enormous number of segments compared to fish. likewise, students will be able to understand the relationship between structure and function, where an autotomized tail has a fracture plane in its vertebrae while animals that cannot autotomy do not have a fracture plane in their tail vertebrae. according to eshach & fried (2005), in science learning real specimens have advantages compared to sketches, drawings, or books. real specimens will attract more students. in addition, original specimens have the advantage of 3-dimensional detail that is not possessed by sketches or photographs. while skeleton props often do not have the details according to the original preparation. figure 1. structure of a carp skeleton. visible skull bones, spine, and rays of fish tails. (a) tail rays; (b) vertebra. figure 2. (a) complete view of snake skeleton. (b) higher magnification. skeletal structure of an oriental whip-snake. there are skull bones, vertebrae with very large amounts of ribs. note the spine with ribs. (a. ribs; b. vertebra). b a b a luthfi & soesilo – a simple method for clearing and staining specimens for … 19 figure 3. (a) skink's tail skeleton structure. visible vertebrae/tail bones with their processes. note that there are fracture plane in the bone where the tail breaks off during an autotomy. (b) skeleton of regenerated skink tail. after autotomy, skink will develep regenerated tail which the cartilaginous tube (stained blue) supporting tail instead of bony vertebrae. (a. neural spine; b. centrum; c. fracture plane; d. intervertebral pad; e. chevron bone; f. vertebra; g. cartilaginous tube). figure 4. high magnification of the turtle tail skeleton structure. the tail consists of a series of vertebrae with their processes. the turtle's tail cannot autotomy and therefore the turtle's tail vertebrae do not have a fracture plane. (a. centrum; b. chevron bone). figure 5. high magnification of the flying dragon tail skeleton structure. the tail consists of a series of vertebrae with their processes. the flying dragon tail cannot autotomy and therefore do not have a fracture plane. (a). dorsal view. (b). ventral view. (c). lateral view. (a. neural spin; b. tranverse process; c. centrum; d. chevron bone). the results show clearly visible bone structure in goldfish, oriental whip-snake, gecko tails, flying dragon tail and turtle tail. the bones stained red while the cartilage stained blue. muscle and connective tissue become transparent/clear by the clearing process using koh. in these preparations the details of the skeleton can also be observed such as the processus of the vertebrae, intervertebral discs, ribs, fracture plane, and other details. figure 1 shows a fish's skeleton. students can observe the presence of skull bones in fish, vertebrae, and fin rays of fish. from these specimens, it is known that the fin rays of the fish are composed of bone material. from the snake preparation (figure 2) we can observe the structure of the snake vertebrae that has ribs on its body vertebrae. only in the tail, the snake vertebrae do not have ribs. the ribs serve as muscles attachment and help the movement of snakes. the skink tail (figure 3), turtle tail (figure 4) and flying lizard tail (figure 5) provide structures that are ideal for studying skeleton anatomy, and to some extend student can learn the relationship between form and function. skink's tail can autotomized its tail, whereas the tail of a turtle and flying lizard cannot autotomy. this functional difference is also reflected by differences in structure, namely the existence of fracture plane in the skink tail vertebrae. it is on these fracture planes that the tail can break. this autotomy ability is a form of gecko's self-defense to avoid predator. turtles and flying lizard cannot autotomized its tail so that it can be understood if there are no fracture plain in the vertebrae. adapting its way of life, the tail of the flying lizard is needed to help the movement on the tree and when it gliding. therefore, breaking the tail would certainly be less profitable. the same is true for turtles. turtles have very hard carapace and plastrons. if there is a threat or an enemy, the turtle g f e b a d c b a d cba b 20 biology, medicine, & natural product chemistry 8 (1), 2019: 17-21 will insert its head and motion apparatus into the carapace and plastron. learning in this way will be more interesting for students. this bone specimen makes it easier for students to understand skeletal structure. understanding the anatomical structure will facilitate the understanding of its function. students will be able to observe the components of the skeleton, the skeleton differences in its way of life, the skeleton differences between animal classes, and the relationship between structure and function (a given structure reflects certain function). in addition, using real specimens will be more interesting for students related to their 3-dimensional shapes, attractive colors, and direct experience of seeing original specimens from a visual, tactile and motoric perspective. appleton (2008) stated that often school teachers are hesitant in teaching science. the main reason is the limited knowledge of teachers about science content. this paper provides a teaching of animal skeleton by directly involving teachers and students in the science process so that they can see directly the science process. thus the confidence of teachers and students will arise to learn science. matthews et al (1997) stated that students have a high curiosity about the world around them, and nature offers a variety of materials in which the teacher becomes a mentor to help students to satisfy their curiosity by studying the natural surroundings. this study used local animals that are widely found around us, namely, goldfish/carp (cyprinus carpio), oriental whip-snake (ahaetulla prasina), gecko (gecko gecko), turtle (trachemys scripta elegans), and flying dragon. this certainly makes it easier for teachers to develop science learning media in accordance with school availability and abilities. this is in accordance with the opinion of matthews et al (1997) which stated that nature provides the appropriate material to satisfy students' curiosity that is so high towards the natural surroundings. according to eshach & fried (2005), the superiority of the real specimens for learning as mentioned above is very suitable for children's learning. children (including elementary school students) have a high curiosity. this character is very suitable for science considering that one of the factors driving science is curiosity. children really like to observe and think about the environment. exposing scientific processes to young persons will develop positive attitudes towards science (eshach & fried, 2005; appleton, 2008). greene et al (1993) stated that real specimens are very important in many aspects of cognitive learning in the classroom. in this study the most important aspect in the success of staining is skin and muscle removal, and the stages of fixation. the skin and muscles must be removed as much as possible so that only a little muscle remains for the attachment of each bones. but it should be noted that skin and muscle removal should not cut the bone/skeleton itself. the vertebrae have many processes. often during the removal of skin and muscles, the process of the vertebrae is unintentionally cut off. this will certainly change the vertebral morphology caused by the artifact, as a result of error in cutting. fixation and fat removal were done longer than the inouye method, because the structure of the skin and muscles of the mature animals are thicker and stronger than the embryo so that the fixative solution takes longer to penetrate the tissue. different specimens require different time periods for fixation, fat removal, clearing and staining. this depends on the species, maturity (embryo or adult), body size, and organs/body parts made for preparations. in general, larger and more mature animals require a longer period of fixation, fat removal, clearing and staining. the length of time for fixation, fat removal, clearing and staining in this study can be used as a guide in staining for other animals. this research has succeeded in developing a skeleton (bone and cartilage) staining method for learning skeleton systems for high schools. learning in this way will involve the teacher and students in the science process. in addition, the use of real specimens will make it easy to understand the skeleton stucture and more attractive to students. the process and materials needed in this study are very simple, and can be applied to other local animals that are available according to the availability in the environment around the school. conclusions the study of organisms including the skeletal structure is an important part of the basic education science curriculum. the use of pictures, sketches, visual aids, and books to explain the skeletal structure is a common method used in the teaching of skeletal systems. however, this method often causes less student interest. the skeleton system is also more difficult to understand with the use of instructional media. the author provides alternative for high school teachers in teaching the skeletal system. making skeletal preparations by involving students is not commonly carry out in the learning of primary school framework systems, but teachers can combine them with the existing curriculum to expand the scope of basic education. acknowledments the authors would like to thank the assistants of animal anatomy lab work at the faculty of science and technology of uin sunan kalijaga, members of the apprenticeship program at the faculty of science and technology of uin sunan kalijaga, and members of the center for integrative zoology of uin sunan kalijaga for their role to make this research possible. the authors also would like to thank to mr. riyanto (staff of the faculty of science of technology) for his assistance in pictures processing. luthfi & soesilo – a simple method for clearing and staining specimens for … 21 references appleton, k. 2008. developing science pedagogical content knowledge through mentoring elementary teachers. j sci teacher educ 19: 523–545. erdogan, d., kadioglu, d., peker, t. 1995. visualization of the fetal skeletal system by double staining with alizarin red and alcian blue. gazi medical journal 6: 55-58. eshach, h., fried, m. n. (2005). should science betaught in early childhood? journal of science education and technology, 14: 315–336. greene, j.s., b.d. greene. 2001. using amphibians and reptiles to learn the process of science. science activities 37 (4): 2932. greene, e. a., k. r. smith, j. s. pendergraft, r. h. raub, and, m. j. arns. 1993. technical note: equine skeletal preservation techniques to enhance teaching effectiveness’. journal of animal science. 71:2270-2274. inouye, m. 1976. differential staining of cartilage and bone in mouse skeleton by alcian blue and alizarin red s. congenital anomalies 16: 171-173. junquiera, l.c. & carneiro, j. 1988. histologi dasar. terjemahan. edisi ke-3. egc penerbit buku kedokteran. jakarta. kardong, k. v. 2002. vertebrates comparative anatomy, function, and evolution. international edition. third edition. mcgraw-hill higher education. new york. usa. hal: 233236. leeson, t. s., & c.r. leeson. 1981. histology. fourth edition. w.b. saunders company. philadelphia. 514-534. matthews, r.w., l. r. flage, and j. r. matthews. 1997. insects as teaching tools in primary and secondary education. annual reviews of entomology 42:269–89. newman, w.j., s. k. abell, p.d. hubbard, j. mcdonald, j. otaala, m. martini. 2004. dilemmas of teaching inquiry in elementary science methods. journal of science teacher education 15(4): 257–279. prokop, p., m. prokop, s.d. tunnicliffe, c. diran. 2007. children’s ideas of animals’internal structures. jbe 41 (2): 62-67 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 11-15 | doi: 10.14421/biomedich.2019.81.11-15 issn 2540-9328 (online) mathematical model of cervical cancer treatment using chemotherapy drug murtono1,*, meksianis zadrak ndii2, sugiyanto3 1department of physics education; 3department of mathematics, universitas islam negeri sunan kalijaga, 55281, yogyakarta, indonesia. 2department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia. author correspondency*: hasnamur@yahoo.co.id abstract cervical cancer is a malignant disease that causes problems in women's health, espe cially in developing countries such as indonesia. cervical cancer cells will develop quickly, uncontrollably, and will continue to divide and then infiltrate the surrounding t issue and continue to spread to connective tissue, blood, and attack important organs and spinal nerves. the aim of the research is to study the mathematical model of cervical cancer by chemotherapy treatment. the results of this study are that cervical cancer treatment using chemotherapy is effective enough to kill abnormal cells such as infected cells, pre-cancerous cells and cancer cells, although there are side effects, namely the killing of normal cells due to chemotherapy drugs. keywords: cervical cancer; infected cells; infected cells; cancer cells; chemotherapy introduction cervical cancer is an excessive and uncontrolled cell growth around the cervix (walboomers et. al., 1999). cervical cancer originated from cells in the cervix (naganawa et al., 2005). most cervical cancers begin in the transformation zone which is a shift from squamous cell type to cylindrical cell type. these cells do not directly turn into cervical cancer. normal cervical cells due to the influence of carcinogenic substances can develop gradually into pre-cancerous cells and then become cancer cells (sari et al., 2016). the main trigger for the emergence of cervical cancer is infection of several types of high-risk human papilloma virus (hpv) which causes proliferation of the epidermal surface and cervical mucosa (bosch, 1995). the types of hpv that are very common in cases of cervical cancer are types 16 and 18, which is more than 70% of all cervical cancers reported. the results of a study of 1,000 samples from 22 countries proved the presence of hpv infection in 99.7% of cervical cancer cases (wuryanti et. al., 2015). cervical cancer is the second most common type of cancer in women worldwide to breast cancer (boice et. al., 2002). chemotherapy is a kind of cancer treatment that uses drugs to destroy cancer cells. chemotherapy works by stopping or slowing the growth of cancer cells, which grow and divide rapidly. chemotherapy can also harm healthy cells that divide rapidly, such as the lines of the mouth and intestines or cells that affect growth. damage to healthy cells can cause side effects. often, side effects will be disappear after chemotherapy is complete (rose et. al., 1999). reduction of the mass of cervical cancer can be used as used to measure the effectiveness of treatment because chemotherapy can cause shrinkage of the mass of cervical cancer. cancer mass has an important role to detect the prognosis of a cervical cancer. formulation of model this model developed frpm the past research (asih, et. al., 2015) about the development of cervical cancer and pillis et al. (2007) about the model of treating cancer in general with chemotherapy. figure 1. cervical cancer treatment diagram by chemotherapy. https://doi.org/10.14421/biomedich.2019.81.11-15 12 biology, medicine, & natural product chemistry 8 (1), 2019: 11-15 table 1. subpopulations, parameters and units. symbol symbol explanation unit unit s(t) normal cell density cell/mm2 i(t) infected cell density cell/mm2 p(t) pre-cancerous cell density cell/mm2 c(t) cancer cell density cell/mm2 v(t) virus density virus/mm2 m(t) concentration of chemotherapy drugs mg/m2 r growth rate of normal cell 1/day n homeostatic carrying capacity cell/mm3  the rate of infection 1/(day.virus) 1a the rate of proliferation of infected cells 1/day 1d the rate of infected cell apoptosis 1/day  the rate of progression, from infection to pre-cancer 1/day 2a the rate of proliferation of precancerous cell 1/day 2d the rate of pre-cancerous cell apoptosis 1/day  the maximum invasion rate, from precancerous to cancer 1/day k half-saturation consentration cell/mm3 3a cancer cell proliferation rate 1/day 3d summing the rate of apoptosis and the rate of cancer cell metastasis 1/day n the average number of viruses produced by an infected cell constant 4d the rate of virus death 1/day sk fractional susceptible cells kill by chemotherapy 1/day ik fractional infected cells kill by chemotherapy 1/day pk fractional pre-cancerous cells kill by chemotherapy 1/day ck fractional cancer cells kill by chemotherapy 1/day  the rate of chemotherapy drug decay 1/day mv the rate of chemotherapy drug intake mg/m2.day the dynamics of changes in cervical cells from normal cells to cancer cells are given in the following system of differential equations: 1 s ds s i rs sv k ms dt n           (1a) 1 1 i di sv a i d i i k mi dt       (1b) 1 1 4 dv n d i d v dt   (1c) 2 2 2 2 2 p dp p i a p d p k mp dt k p        (1d) 2 3 32 2 c dc p a c d c k mc dt k p       (1e) m dm m v dt    (1f) let 1 1 2 2 3 3 1 1 1 1 1 1 4 , , , , , , , , , , s i a d a b a d k d a s i n n p c n p c n n d n c d p k k k k                 by non-conventionalizing the system (1a) (1f) to be   1 1 1 1 1 11 s dsds rs s i s v k ms dt dt       (2a) 1 i didi sv ai i k mi dt dt       (2b) dv ni cv dt   (2c) 2 1 12 1 p dp p pi bp k mp dt p       (2d) 2 2 1 c dc p kc k mc dt p     (2e) m dm m v dt    (2f) by eliminating the variable index, system (2a) (2f) which is non-professional becomes   1 s ds rs s i sv k ms dt      (3a) i di sv ai i k mi dt      (3b) dv ni cv dt   (3c) 2 2 1 p dp p pi bp k mp dt p       (3d) 2 2 1 c dc p kc k mc dt p      (3e) m dm m v dt    (3f) murtono et al. – mathematical model of cervical cancer treatment … 13 equilibrium point theorem 1. let 3 22 9 27 , 3u x xy z v x y     . the equilibrium point with chemotherapy in cervical cancer from system (3a) (3f) is     * * * * * * 3 4 5 5 2 5 1 , , , , , , , , , ,i i ep s i p c v m p c          1 2 3i , , , where 1 0 mv    , 2 0 n c    , 1 3 1 sk r     , 2 4 1 0 r            , 1 2 3 5 2 4 ia k          , 5 1 , p p x k b        51 1 1 , .p p p pk b y z k b k b           proof. from equation (3f) is obtained 1 0 mvm      . (4) from equation (3c) is obtained 2 ni v i c   , (5) where 2 0 n c    . if equation (4) and equation (5) are substituted into equation (3a), then we obtain 3 4s i   , (6) where 13 1 sk r     and 24 1 r           . if equations (4), (5) and (6) are substituted in equation (3b), they are obtained 0i  or 1 2 3 5 2 4 ia ki             , (7) where 1 2 35 2 4 ia k          . for 𝐼 = 0 impossible, because the virus is the cause of cervical cancer, so it was chosen 𝐼 = 𝜉5. if equation (4) and equation (7) are substituted into equation (1d), then they are obtained 𝑃3 + 𝑥𝑃2 + 𝑦𝑃 + 𝑧 = 0, where 5 51 1 1 1 , , .p p p p p pk b x y z k b k b k b                  the roots are 2 33 1 2 33 1 1 4 3 3 2 1 1 4 , 3 2 x p u u v u u v                 , 2 33 2 2 33 1 3 1 4 3 6 2 1 3 1 4 6 2 x i p u u v i u u v                   2 3 3 3 2 3 3 1 3 1 4 3 6 2 1 3 1 4 . 6 2 x i p u u v i u u v                 (8) if equation (4) and equation (8) are substituted into equation (3e), then they are obtained 𝐶 = 𝜃𝑝𝑖 2 (𝑘+𝑘𝐶𝜉1)(1+𝑝𝑖 2) = 𝑐𝑖, where 𝑖 = 1,2,3.■ theorem 2. if 1 2 3 0ia k       , 3 4 5 0    and if one 𝑝𝑖 > 0 and real, then the equilibrium point with chemotherapy or value 𝐸𝑃 exist. proof. from theorem 1 is obtained 𝑀 = 𝑣𝑀 𝛾 = 𝜉1 > 0. because 𝑎 + 𝛿 + 𝑘𝐼𝜉1 − 𝑎𝜉2𝜉3 > 0, then 𝐼 = 𝑎+𝛿+𝑘𝐼𝜉1−𝑎𝜉2𝜉3 𝑎𝜉2𝜉4 = 𝜉5 > 0. because 𝐼 = 𝜉5 > 0, then 𝑉 = 𝑛𝐼 𝑐 = 𝜉2𝐼 > 0. because 𝜉3 + 𝜉4𝜉5 > 0, then 𝑆 = 𝜉3 + 𝜉4𝜉5 > 0. one is 𝑝𝑖 > 0, where 𝑖 = 1,2,3 and real. value is 𝜃𝑝𝑖 2 (𝑘+𝑘𝐶𝜉1)(1+𝑝𝑖 2) = 𝑐𝑖 > 0. so, the equilibrium point with chemotherapy or value 𝐸𝑃 exist.■ stability theorem 3. let 𝑢 = 2𝑥3 − 9𝑥𝑦 + 27𝑧, 𝑣 = 𝑥2 − 3𝑦. if −𝑘 − 𝑘𝐶𝜉1 < 0, 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2) − 𝑘𝑃𝜉1 < 0, where 𝑖 = 1,2,3, 𝑥 > 0, 𝑢 > 0, 𝑢2 = 4𝑣3, and 𝑢 < 2𝑥3, then equilibrium point 𝐸𝑃 = (𝜉3 + 𝜉4𝜉5,𝜉5,𝜉2𝜉5,𝑝𝑖, 𝑐𝑖,𝜉1) asymptotic stable. proof. from system (3a) (3f) the jacobian matrix with eigenvalue is obtained 1   , 2 1ck k    ,   3 12 2 2 1 i p i p b k p       , 14 biology, medicine, & natural product chemistry 8 (1), 2019: 11-15 2 33 4 2 33 1 1 4 3 3 2 1 1 4 , 3 2 x u u v u u v                  2 33 5 2 33 1 3 1 4 3 6 2 1 3 1 4 6 2 x i u u v i u u v                    2 33 6 2 33 1 3 1 4 3 6 2 1 3 1 4 . 6 2 x i u u v i u u v                    value is 𝜆1 = −𝛾 < 0. because −𝑘 − 𝑘𝐶𝜉1 < 0, then 𝜆2 = −𝑘 − 𝑘𝐶𝜉1 < 0. because 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2)2 − 𝑘𝑃𝜉1 < 0, then 𝜆2 = 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2)2 − 𝑘𝑃𝜉1 < 0. because 𝑣 = 0, then 𝜆4 = − 𝑥 3 − 1 3 √𝑢 3 , 𝜆5 = − 𝑥 3 − 1 3 √ 1 2 𝑢 3 , 𝜆6 = − 𝑥 3 + 1 3 √ 1 2 𝑢 3 . because 𝑢 > 0, then 𝜆4 < 0. because 𝑢 < 𝑥 3, then 𝜆5 = 𝜆6 < 0. because 𝜆1,𝜆2,𝜆3,𝜆4,𝜆5 and 𝜆6, then the equilibrium point 𝐸𝑃 asymptotic stable.■ simulation in this section we will discuss numerical simulations and medical interpretations of the mathematical model of cervical cancer affected by chemotherapy. first, the parameter values used and the initial values for each variable are given first. taking parameter values for numerical simulations is still in the form of assumptions based on the rate of growth of cancer cells in general. sources and interpretations of parameter values can be seen in the reference. the parameter values for this case are given in table 2. table 2. case parameter value of the effects of chemotherapy. symbol value reference 𝑟 0.02 asih et al. (2015) 𝛼 0.0001 asih et al. (2015) 𝑎 0.01 asih et al. (2015) 𝛿 0.0082 asih et al. (2015) 𝑛 10000 asih et al. (2015) 𝑐 50 asih et al. (2015) 𝑝 13.44 asih et al. (2015) 𝑏 1 asih et al. (2015) 𝜃 2.03 asih et al. (2015) 𝑘 1.01 asih et al. (2015) 𝑘𝑆 0.0006 estimation 𝑘𝐼 0.6 pillis et al. (2007) 𝑘𝑃 0.6 pillis et al. (2007) 𝑘𝐶 0.8 pillis et al. (2007) 𝛾 0.9 pillis et al. (2007) 𝑣𝑀 1 pillis et al. (2007) figure 2. system simulation (3a) (3f) with parameter values 𝑟 = 0, 𝛼 = 0.0001, 𝑎 = 0.01, 𝛿 = 0.0082, 𝑛 = 10000, 𝑐 = 50, 𝑝 = 13.44, 𝑏 = 1, 𝜃 = 2.03, 𝑘 = 1.01, 𝑘𝑆 = 0.0006, 𝑘𝐼 = 0.6, 𝑘𝑃 = 0.6, 𝑘𝐶 = 0.8, 𝛾 = 0.9, 𝛾 = 1, and initial conditions (13, 13, 13, 13, 13, 13). the dynamics due to normal cell chemotherapy drugs, infected cells, precancerous cells, cancer cells, cancer cells, and viruses declined in three days. (a) normal cells. (b) infected cells, precancerous cells, and cancer cells. (c) viruses. (d) chemotherapy drug concentration. murtono et al. – mathematical model of cervical cancer treatment … 15 figure 2 shows a trajectory with the influence of chemotherapy drugs, on cervical cancer patients. normal cells in the first 3 days showed a decrease, from 13 cells/mm2 to 7,254 cells/mm2. this makes a severe effect for cervical cancer patients with this chemotherapy drug. infected cells, precancerous cells and cancer cells drop very quickly, in the first 3 days of chemotherapy, from 13 cells/mm2 to 0.01308 cells/mm2. the initial virus rose would derease after chemotherapy. the virus in 3 days is from 13 viruses/mm2 to 0.3771 viruses/mm2. this is because, many infected cells die because of the effects of chemotherapy drugs. the chemotherapy drug on the third day still contained 1.91mg/m2. it means that chemotherapy drugs still have an effect on normal cells and other abnormal cells, including cancer cells. conclusion cervical cancer is one type of malignant cancer and is most prevalent in women compared to other cancers. treatment of cervical cancer with chemotherapy is quite effective. abnormal cancer cells such as infected cells, precancerous cells, and many cancer cells die from this treatment. because normal cells are dead, this is the side effect of the of this treatment. this research needs to be continued with other treatments that are more effective, especially to reduce the adverse effects to the normal cell. references asih, t. s. n., lenhart, s., wise, s., aryati, l., adi-kusumo, f., hardianti, m. s., & forde, j. (2016). the dynamics of hpv infection and cervical cancer cells. bulletin of mathematical biology, 78(1), 4-20. boice jr, j. d., day, n. e., andersen, a., brinton, l. a., brown, r., choi, n. w., ... & hakama, m. (1985). second cancers following radiation treatment for cervical cancer. an international collaboration among cancer registries. journal of the national cancer institute, 74(5), 955-975. bosch, f. x., manos, m. m., muñoz, n., sherman, m., jansen, a. m., peto, j., ... & shan, k. v. (1995). prevalence of human papillomavirus in cervical cancer: a worldwide perspective. jnci: journal of the national cancer institute, 87(11), 796-802. de pillis, l. g., gu, w., fister, k. r., head, t. a., maples, k., murugan, a., ... & yoshida, k. (2007). chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls. mathematical biosciences, 209(1), 292-315. naganawa, s., sato, c., kumada, h., ishigaki, t., miura, s., & takizawa, o. (2005). apparent diffusion coefficient in cervical cancer of the uterus: comparison with the normal uterine cervix. european radiology, 15(1), 71-78. rose, p. g., bundy, b. n., watkins, e. b., thigpen, j. t., deppe, g., maiman, m. a., ... & insalaco, s. (1999). concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. new england journal of medicine, 340(15), 1144-1153. sari, h. e., mudigdo, a., & demartoto, a. (2016). multilevel analysis on the social determinants of cervical cancer in yogyakarta. journal of epidemiology and public health, 1(2), 100-107. walboomers, j. m., jacobs, m. v., manos, m. m., bosch, f. x., kummer, j. a., shah, k. v., ... & muñoz, n. (1999). human papillomavirus is a necessary cause of invasive cervical cancer worldwide. the journal of pathology, 189(1), 12-19. wuryanti, s., andrijono, a., susworo, s., & witjaksono, f. (2015). the effect of high poly unsaturated fatty acid (pufa) dietary supplementation on inflammatory status of patients with advanced cervical cancer on radiation treatment. acta medica indonesiana, 47(1). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 33-36 | doi: 10.14421/biomedich.2019.82.33-36 issn 2540-9328 (online) the effects of wet cupping therapy in blood pressure, glucose, uric acid and total cholesterol levels sutriyono1,*, muhammad rodham robbina2, meksianis zadrak ndii3 1department of biology; 2department of mathematics, faculty of science and technology, uin sunan kalijaga, yogyakarta, indonesia 3department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia author correspondency*: sutriyono@uin-suka.ac.id abstract wet cupping therapy is a simple, cheap, and effective alternative medical treatment. in china wet cupping therapy become a formal treatment in the hospital. 14 centuries ago prophet muhammad implemented wet cupping therapy or hejamah as a curing treatment and become sunnah for muslim. today in a modern world, many studies proved the advantages of wet cupping therapy, which is a sunnah since 14 centuries ago. the aims of this study were to measure the effects of wet cupping therapy treatment on blood pressure, blood glucose, uric acid, and total cholesterol level. 21 participants were treated with hejamah/wet cupping, blood pressure and blood samples were collected from all the participants one week before and one week after hejamah. blood pressure, either systolic or diastolic was significantly decreased following therapy (p|<|0.05), blood glucose had a slight decreased but not significant (p|>|0.05). uric acid and total cholesterol level was also significantly decreased following therapy (p|<|0.05). with only just one time treatment, hejamah had a significant effects on blood pressure, uric acid and total cholesterol level, that is proved the advantages of hejamah. keywords: hejamah; wet cupping therapy; cardiovascular diseases; hematology; blood pressure; blood glucose; uric acid; cholesterol introduction cardiovascular diseases are major health problems and they are associated with mortality and morbidity (refaat et al, 2014). the world health organization (who) has reported that cardiovascular diseases are responsible for 30% of all deaths, which is equivalent to the combined death rates associated with several other medical conditions (rehm et al, 2009). the estimated number of death due to cardiovascular diseases by the who is 17.3 million people per year and the number is expected to increase to 23.3 million/year in 2030 (ng et al, 2012). several benefits of complementary medicine/alternative medicine, also known as traditional medicine, have been shown and it is effective in the prevention of disease, treatment of noncommunicable diseases, and improvement of the quality of life for persons living with chronic diseases (burge and albright, 2002). the who has been encouraging the implementation of traditional medicine since many patients are not satisfied by the outcomes of modern medicine particularly those related to chronic diseases (who, 2002). one of the alternative medicines is islamic hejamah/wet cupping. in the middle east, arabic writers report that cupping therapy dates back to 3500 b.c. (5500 years ago), where assyrians were the first arab population to use primitive tools as animal horns and bamboo wood for cupping therapy then the chinese physician, jee hong (381281 b.c.) was among the leaders in that art. arabic civilization termed cupping therapy, al-hijamah therapy (which means in arabic to restore to the original size), where it was used in treating hypertension, polycythemia, headache, migraine and drug intoxication. they diagnosed polycythemia whenever there was an exaggeration of the pink color of the skin (omar, 2009). interestingly, venesection (phlebotomy) is still being used currently in hospitals for treating polycythemia, where blood is drawn out and is replaced by saline infusion (mcmullin et al, 2005). cupping therapy is being practiced nowadays in many countries all over the world including germany, norway, denmark, saudi arabia, egypt, india, china and other countries. german people are familiar with cupping therapy (michalsen et al, 2009) and so are danish and norwegian peoples where those european societies already have a shift in attitude to include complementary medicine within the conventional health care system (salomonsen et al, 2011). the exact origin of cupping therapy is a matter of controversy. chinese scientists report in their literature that cupping therapy is a part of the traditional chinese medicine dating back to at least 2,000 years (chirali, 2014). materials and methods this study was a true experimental research. 21 adult men were recruited into the study, wet cupping was performed on the study subjects one time according to https://doi.org/10.14421/biomedich.2019.82.33-36 34 biology, medicine, & natural product chemistry 8 (2), 2019: 33-36 islamic protocol, the subject were asked to be fasting at least 3 hours before the initiation of the procedure. blood pressure and blood sample were obtained from the subject before the procedure. we run a test to the blood sample to measure blood glucose, uric acid, and total cholesterol level. a second blood pressure and blood sample were obtained from the subjects 7 days after wet cupping therapy. figure 1. wet cupping therapy. wet cupping therapy were performed using seven points on the back of each subjects, the cup was sterilized using alcohol. the cups were placed on the chosen sites and a negative pressure was created by manual suction using the provided pump with the kit. the cups were left for a period of 7-10 minutes after ensuring their firm attachment to the skin. the cups were removed and 12-15 superficial incisions were made on each chosen area of the skin using sterile surgical blades. after soaking the cups, they were replaced back on the chosen areas of the skin and negative pressure was created as previously described. the cups were left on the skin till they were filled with blood from the capillary vessels. the cups were removed approximately after 3 minutes and new cups were placed on the same areas as previously mentioned. the used cups were socked in betadine solution for sterilization. the process of blood-letting was repeated for two times in total. result and discussion statistical analysis was performed using spss version 15. normality and homogeneity of data were assessed with the shapiro-wilk test. table 1. the effect of wet cupping therapy to blood pressure. mean (mmhg) p systolic pretest 132.33 0.001 posttest 124.81 diastolic pretest 86.29 0.001 posttest 81.19 according table 1 that using t test showing that there is a different on blood pressure (systolic and diastolic) between before the wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of systolic pressure in amount of 7.5 mmhg from 132.33 mmhg becomes 124.81 mmhg, while for the diastolic pressure there is some reduction in amount of 5.1 mmhg from 86.29 mmhg becomes 81.19 mmhg. the analysis result using t test to systolic pressure and diastolic pressure showing the p value is 0.001 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects blood pressure after wet cupping therapy is significant. the study of refaat et al (2014) showing the same result that wet cupping therapy can reduce blood pressure significantly. the positive effect that given by wet cupping therapy into the reduction of blood pressure is because wet cupping therapy has benefit to remove all waste and sediment in blood artery that connected to the circulation of blood (ica, 2011). the blood pressure reduction also happen because wet cupping therapy in a skin (cutaneous), under skin tissue (sub-cutaneous), fascia, and the muscle will cause the damage of mast cell, the result of this damage will release a few substance like serotonin, histamine, bradikin, slowreacting substance (srs), and few other substance, this substance will cause capillar and arteriole dilatation also flare reaction in the place of wet cupping therapy point. capillar dilatation also happen in a place who far from wet cupping therapy point, this is cause the blood artery microcirculation repair, then will show relaxation effect in clumsy muscle also because of vasodilatation will reduce blood pressure (refaat et al, 2014) table 2. the effect of wet cupping therapy to blood glucose. blood glucose mean (mgdl-1) p pre-test 98.48 0.311 post-test 93.33 according to table 2 that using t test, it is showing that there is a different on blood glucose level between sutriyono et al. – the effects of wet cupping therapy in blood pressure … 35 before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of blood glucose level between before wet cupping therapy and after the wet cupping therapy in amount of 5.15 mgdl-1 from 98.48 mgdl-1 becomes 93.33 mgdl-1. the analysis result using t test to blood glucose level showing the p value is 0.311 (p|>|0.05), then the effect of wet cupping therapy to the reduction of subjects blood glucose level after wet cupping therapy is not significant, even though the effect of wet cupping therapy still make some reduction on blood glucose level. wet cupping therapy enhances insulin sensitivity in healthy subjects with normal glucose levels and normoferritinemia (vakilinia et al, 2016). bleeding due to hijamah was found to decrease glucose serum and triglycerides in patient with diabetes (lowe, 2017). some study has proved the efficacy of wet cupping therapy, on of it is the study by farahmand et al (2012), this study prove that wet cupping therapy an diet have significant effect to lipid profile improvement. another study about efficacy of wet cupping therapy is the study by refaat et al (2014) who mention that wet cupping therapy has positive effect to blood glucose level on subject who has diabetes mellitus disease. table 3. the effect of wet cupping therapy to uric acid. uric acid mean (mgdl-1) p pretest 7.15 0.001 posttest 5.91 according to table 3 that using t test showing that there is a different on uric acid level between before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of uric acid level between before wet cupping therapy and after the wet cupping therapy in amount of 1.24 mgdl-1 from 7.15 mgdl-1 becomes 5.91 mgdl-1. the analysis result using t test to uric acid level showing the p value is 0.001 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects urid acid level after wet cupping therapy is significant. this reduction is positive enough to prevent cardiovascular disease because high level of uric acid or commonly called hyperuricemia according to euser et al (2008) can rise the risk of cardiovascular disease. table 4. the effect of wet cupping therapy to total cholesterol. cholesterol mean (mgdl-1) p pretest 177.05 0.022 posttest 160.43 according to table 4 that using t test showing that there is a different on total cholesterol level between before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of total cholesterol level between before wet cupping therapy and after the wet cupping therapy in amount of 16.62 mgdl-1 from 177.05 mgdl-1 becomes 160.43 mgdl-1. the analysis result using t test to total cholesterol level showing the p value is 0.022 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects total cholesterol level after wet cupping therapy is significant. the reduction of total cholesterol level has shown the effect of wet cupping therapy to total cholesterol level, which mean that if wet cupping therapy is doing regularly, wet cupping therapy can be alternative treatment beside conventional treatment for stroke patients to reduce total cholesterol level. the reduction of total cholesterol level after wet cupping therapy is because there is some effect from hematology system mechanism who give main effect through the regulation system pathways of coagulantanticoagulation with the improvement of blood flow and organ oxygenation (ahmadi et al, 2008). considering hepar is a place that blood filtration from various toxic substances who entering the body happen, through ti=his hematology system mechanism and immune system mechanism the total cholesterol level can be reduced (ahmadi et al, 2008). however, there are some consideration that need to be consider. first the sample we were taken is small, and we didn’t consider the health factor of subjects of this study. second, in the process of our study we didn’t consider factors who maybe have a big impact to this study, like subjects diet, sleep patterns, etc. despite the shortcomings of this study, hejamah has been proven have some benefits in the prevention of cardiovascular diseases. study of bassem refaat (2014) shown that although hejamah have no significant impact on decreasing total cholesterol level, but significantly raised hdl (high density lippoprotein) level and decreased ldl (low density lippoprotein) level. with lower ldl level can lower the risk of stroke attack. conclusion overall, wet cupping therapy is a traditional treatment that is a sunnah from prophet muhammad. first the sample we are taken is small, and we didn’t consider the health factor of subjects of this study. second in the procces of our study we didn’t consider factors who maybe have a big impact to this study, like subjects diet, sleep patterns, etc. despite the shortcomings of this study, hejamah has been proven have some benefits in the prevention of cardiovascular diseases. study of bassem refaat (2014) shown that although hejamah have no significant impact on decreasing total cholesterol level, but significantly raised hdl (high density lippoprotein) level and decreased ldl (low density lippoprotein) level. with lower ldl level can lower the risk of stroke attack. the conclusion is hejamah or wet cupping theraphy have a great chance to become a medical treatment who 36 biology, medicine, & natural product chemistry 8 (2), 2019: 33-36 can reduce blood pressure, blood glucose level, urid acid level, and total cholesterol level. hejamah also can prevent cardiovascular diseases because it can raise the level of hdl and reduce the level of ldl. references ahmadi, a., schwebelb, d. c., & rezaei, m. 2008. the efficacy of wet-cupping in the treatment of tension and migraine headache. the american journal of chinese medicine, 36(01), 37-44. burge s. k., albright t. l., & residency research network of south texas (rrnest) investigators. 2002. use of complementary and alternative medicine among family practice patients in south texas. american journal of public health 92.10: 16141616. chirali, i. z. 2014. traditional chinese medicine cupping therapy. elsevier health sciences. euser, s. m., hofman, a., westendorp, r. g. j., & breteler, m. m. (2008). serum uric acid and cognitive function and dementia. brain 132(2), 377-382. farahmand, s. k., gang, l. z., saghebi, s. a., mohammadi, m., mohammadi, s., mohammadi, g., & ghayour-mobarhan, m. 2012. the effects of wet cupping on coronary risk factors in patients with metabolic syndrome: a randomized controlled trial. the american journal of chinese medicine, 40(02), 269-277. indonesian cupping association. 2011. standard operating procedure hejamah. bogor: research and development division ica. ng, n., johnson, o., lindahl, b., & norberg, m. 2012. a reversal of decreasing trends in population cholesterol levels in västerbotten county, sweden. global health action, 5(1), 10367. lee, m. s., choi, t. y., shin, b. c., kim, j. i., & nam, s. s. 2010. cupping for hypertension: a systematic review. clinical and experimental hypertension 32(7), 423-425. mcmullin, m. f., bareford, d., campbell, p., green, a. r., harrison, c., hunt b., & ryan, k. 2005. guidelines for the diagnosis, investigation and management of polycythaemia/ erythrocytosis. british journal of haematology 130(2), 174195. michalsen, a., bock, s., lüdtke, r., rampp, t., baecker, m., bachman, j., & dobos, g. j. 2009. effects of traditional cupping therapy in patients with carpal tunnel syndrome: a randomized controlled trial. the journal of pain 10(6), 601608. omar, s. a. h. 2009. al-hijamah (cupping therapy): sunnah and therapy. dar ommah for publication, jeddah. refaat, b., el-shemi, a. g., ebid, a. a., ashshi, a., & basalamah, m. a. 2014. islamic wet cupping and risk factors of cardiovascular diseases: effects on blood pressure, metabolic profile and serum electrolytes in healthy young adult men. alternative and integrative medicine, 3(1): 151. rehm, j., mathers, c., popova, s., thavorncharoensap, m., taerawattananon, y., & patra, j. 2009. global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. the lancet 373(9682), 22232233. salomonsen, l. j., skovgaard, l., lacour, s., nyborg, l., launsø, l., & fonnebo, v. 2011. use of complementary and alternative medicine at norwegian and danish hospitals. bmc complementary and alternative medicine, 11(1), 4. vakilinia, s. r., bayat d., & asghari, m. 2016. hijama (wet cupping or dry cupping) for diabetes treatment. iranian journal of medical sciences 4(3 suppl), s37. lowe, d. t. 2017. cupping therapy: an analysis of the effects of suction on skin and the possible influence on human health. complementary therapies in clinical practice, 29, 162-8. who. 2002. who traditional medicine strategy 2002-2005. world health organization, geneva, switzerland. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 39-43 | doi: 10.14421/biomedich.2018.72.39-43 issn 2540-9328 (online) comparative anatomy of labyrinth and gill of catfish (clarias gariepinus) (burchell, 1822) and snakehead fish (channa striata) (bloch, 1793) ina karlina1,*, muhammad ja’far luthfi2 1center for integrative zoology, 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: ikarlina017@gmail.com abstract dumbo catfish (clarias gariepinus) and gabus (channa striata) have additional organ respiratory system structures in the gills, called labyrinth. this organ is a tool for fish to take oxygen directly from the air so that it can live in low oxygen habitats. both fish have differences at the order level. catfish is an order of siluriformes as gabus is an order of perciformes. the purpose of this study was to distinguish the anatomy and histology of the gill structure and labyrinth in both fish. the macroanatomy observation was conveyed by dissection to determine the gill topography in the two fish species. histology preparations were carried out using the paraffin method and using hematoxylin-eosin (he) staining that showed in the labyrinth organ there were many blood vessels to bind oxygen then distributed to all parts of the fish's body. keywords: anatomy; catfish (clarias gariepinus); snakehead fish (channa striata); hematoxylin-eosin (he); histology; labyrinth introduction catfish (clarias gariepinus) and snakehead fish (channa striata) have an additional structure in their gill organs but both were grouped into different orders. catfish belonging to order siluriformes while snakehead fish belonging to order perciformes. labyrinth is a multiplied membrane that filled with blood capillaries located at the top of second and thirdgill arches, and organ shaped like a sponge. fujaya (2008) stated that some species of fish have a labyrinth which is an upward expansion of gills forming folds and so become irregular cavities. explanation other sources that gills of catfish have a small size that located on the back head (najiyati, 2010 in utomo 2006). according to jayaram, 1981 (in utomo, 2006) this size causes catfish had trouble to filter water in, therefore catfish need a labyrinth as an additional respiration tool. the labyrinth allowed catfish and snakehead fish to extract oxygen directly from the air so they can live in low content oxygen waters (susanto, 1989; angka et al., 1990; suyanto, 1992). insight into the development of organs in pisces is still lack and limited (rather limited), so it required in-depth study to find out more extensively (fumio & takashi, university of tokyo, 2010). based on the facts it is necessary to have a more in-depth study about labyrinth structure in gills of catfish and snakehead fish. this confirmed that groupings of pisces members are generally not only based on morphological features and characteristics but also anatomy and histology aspects that interesting to be studied further. not only to refer to those additional structures in helping and sustain its life-related adaptation aspects to the environment, but also the study of anatomy and histology. method this study used three adults catfish (clarias gariepinus) and snakehead fish (channa striata). the anatomical observation was done by observing the color, shape, and length of the gill and labyrinth-gill. histological slides used paraffin method and stained with using hematoxylin-eosin (he). results and discussion gill and labyrinth-gill anatomy of adult catfish (clarias gariepinus) and snakehead fish (channa striata) based on the results obtained from the study gill color on catfish and bright red bright snakehead fish, this indicates the number of blood vessels capillaries and blood cells contained inside. gills of catfish and snakehead fish consist four pairs of sheets, while the shape is like a raker, besides it has three main structures namely gill arch, gill racker and gill filament. gills catfish and snakehead fish are located on the side-head https://doi.org/10.14421/biomedich.2018.72.39-43 40 biology, medicine, & natural product chemistry 7 (2), 2018: 39-43 precisely on right and left of the pharynx and outside covered by an operculum. gill arch or branchialis archus structure in catfish and snakehead fish are colored white is a place of attachment gill filament and filament. the differences between catfish are the gills surface which smaller and narrower than snakehead fish which wider.  gill figure 1. catfish gill and snakehead fish. gill filament or hemibranchia in catfish and snakehead fish resembles a raker and attaches on gill arch. each gill sheet consists of pair filaments, and on each filament contains many thin layers called lamela. lagler et al (1997) stated that gills consist of pair gill filaments, each filament consists of thin transverse fiber covered by a thin epithelium called lamella. the lamella is a filament composer. another structure is the gill or gill rakers, which is a pair of rows cartilage rods are short and serrated small, attached to the front of gill arch and has a function to filter the water respiration. according to achmad (2005), gills on the anterior middle edge are equipped with a structure (gill rakers) that function filtering particles. figure 2. portions of catfish and snakehead fish gill.  labyrinth-gill the results from the research, the labyrinth on the catfish has bright-pink color, while snakehead fish has reddish cream color. the pink color is caused by capillary blood vessels, which are not much different from gills. catfish labyrinth has sponge-like shaped (arborescent) with irregular structures. this signifies many structures resembling vesicle gas as air reserves that support respiratory process, especially when lacking oxygen. while snakehead fish labyrinth has solid-shape with a serrated edge. labyrinth is located on the first-gill sheets catfish and the longest sheet attached to the edges. according to kisia (2010), the labyrinth in clarias cluster gills is covered by respiratory epithelial folds and derived from the first-gills of the dorsolateral head bone of gill cover. while labyrinth of snakehead fish is located lies in the pharyngeal. according to sukiya (2010), the labyrinth of snakehead fish is a development in dorsal part a pair of the pharynx and an extension known as suprapharyngeal space, this room is supported when in lack oxygen and the process breathes. figure 3. catfish and snakehead fish labyrinth-gill. macroanatomically, the gills structure of catfish and snakehead fish are generally same, they have three main parts i.e gill raker, gill arch, and gill filament. these three structures have the same functions. a form of gill raker dumbo catfish serrated small dense and tightly neat, while the form of gill raker snakehead fish is larger, uneven and irregular. the striking difference is shown in terms of color, structure, and location or topography of the labyrinth. all anabantoid/anabyroid species have specialized labyrinth organs to support gas exchange and involved in the surface water respiratory behavior (graham, 1997). reviewing in terms of complexity, resilience and the surface area catfish has more survive than snakehead fish. the more complex and larger surface area of the respiratory tool structure then accommodated oxygen reserve will more plenty. so that fish can survive longer when lack oxygen in the water. according to kisia (2008), catfish can survive in last minutes or even several hours because it has an additional respiratory tool called arboresence, this tool is a folded membrane which is full of blood capillaries located in the upper cavity above the gills. histology of gill and labyrinth-gill  gill results showed that catfish and snakehead fish gills have a thin epithelial layer for the efficiency of oxygen and gas exchange (absorption and release). in addition, gills have a function to regulate salt and water exchange and function in excretion of nitrogenous waste products, gill gill labyrinth gill raker gill raker gill arch gill arch gill filament karlina & luthfi – comparative anatomy of labyrinth and gill of catfish … 41 especially ammonia. gill is composed by gill raker, gill arch and gill filament. this gill arch composed by primary lamella that has the parallel lamella secondary sheets along primary lamella. this secondary lamella functions to take oxygen from water. histologically, primary lamella is composed of pillar cells lined up and cells are covered in a thin epidermal membrane semipermeable. spaces between pillar cells are called lacuna as the blood pass-over place. figure 4. gill of catfish and snakehead fish (40x10 magnification). hematoxylin-eosin (he). according to lagler et al (1997), lamella is composed of thin epidermal cells and stem-shaped support cells (pillar cells) that helps blood-flow to the gills. the thickness of lamella are varied depends on species and its activity. gas exchange occurs in secondary lamella which is the fold of epithelial cells with a single supports cell layer and separated by pillar cells. a thin layer of blood vessels lies between the pillar cells and epidermis which is become gas exchange site, nitrogenous wastes of metabolites remains and some electrolyte exchanges. pseudobrankhia is located at the bottom of the upper operculum. this organ is a gill arch with a series of filaments. the function of pseudobranchial is not yet known, but it is thought the structure that supplies oxygen-rich blood to chloroid cells optical and retinal, it may play a role in baroreceptor and thermoregulation functions. in addition, gills are equipped with a number of glands known as the branchial gland i.e specialized gill epithelial cells. these glands are a mucous gland and the acidophilic glands (chloride cells). marrison (2007 in prasetyo, 2011), stated that the gill arch consists of the primary lamella. each primary lamella has a secondary lamella located in a perpendicular to the primary lamella. gill arch is covered by epidermal tissue and contained many mucosal cells. in the primary lamella, contained chloride cells that commonly found in basal (proximal) of lamellae. these cells function in ion transport and detoxification. gas exchange occurs throughout the surface of secondary lamella primarily through the exchange of blood and water coming from the environment. the secondary lamellar surface consists by squamous cell epithelial overlap, usually, one layer supported and separated by pillar cells.  labyrinth figure 6. histology of labyrinth-gill of catfish (clarias gariepinus). he staining. (40x10 magnification). 1. microvili, 2. goblet cell, 3. epithelium cell, 4. granula, 5. lamina basal, 6. loose connective tissue, 7. capillary blood vessels. the outer surface covered by epithelial cells and restrict two internal parts as almost all layers of the body or the organs are covered epithelial cells. epithelial cells construct the labyrinth of dumbo catfish that very dense and almost no intercellular space. flat-shaped and belonging to a simple squamous cell type epithelium, mostly in the respiratory organs that have primary functions to exchange substances. according to orphans (1996), in some types of epithelium form a bulge or crease to expand its surface area. figure 7. histology of labyrinth-gill of dumbo catfish (clarias gariepinus). he staining. (40x10 magnification). 1. intercapillary space, 2. perisite (pervascular cells), 3. capillary blood, 4. endotel. blood vessels blood vessels secondary lamella secondary lamella primary lamella spaces between lamella primary lamella 42 biology, medicine, & natural product chemistry 7 (2), 2018: 39-43 the surface epithelial type of labyrinth catfish is simple squamous epithelial cells. these cells consist of a single layer of epithelial cells as the point of contact for the epithelial tissue with the basement membrane. types can be in face rapid diffusions areas such as lung alveoli lining (respiratory unit called airbags), endothelium (blood vessel lining), capillaries, kidneys, the main body cavity (mesothelium), and other major areas where the little activity occurred. the respiratory mucosa is composed of epithelial cells and there is a lamina propria. the epithelium is a tall columnar with cilia and goblet cells. supporters of lamina propria under the epithelium contain elastin. the function of simple squamous epithelial cells is responsible for allowing the exchange of oxygen. they are also responsible for blood filtering, and diffusion that allows passing oxygen in the blood. in addition, there is also a connective tissue that connects with the network under it, the type of loose connective tissue which is the most widely spread connective tissue in the body of vertebrate animals. histological description showed that labyrinth of dumbo catfish has a loose connective tissue which is a network of rare cells. part of tissue composed of a matrix which located beneath the matrix epithelial layer of mucous fluid, the deepest part which is capillary blood vessels. this capillary vessel is limited by endothelial cells that are flattened, located long in accordance with the blood flow, are blue and prominent into the lumen. figure 8. histology of labyrinth-gill of gabus (channa striata). he staining. (10x10 magnification). 1. microvilli, 2. epithelium layer, 3. goblet cell, 4. lamina basal, 5. connective tissue, 6. capillary blood vessel. labyrinth in snakehead fish is known diverticula although it functions equally with the labyrinth in catfish as an additional tool when fish is in lack oxygen. diverticula is in the oral cavity and pharynx. diverticula is in a group of snakehead fish (channa). according to john, karl and robert (1997), diverticula is coated by respiratory epithelium with a numerous blood supply, usually from afferent gill circulation. this showed that the coating and composition of the constituent are same as the labyrinth in dumbo catfish. figure 8, showed the tissue and constituent cells of the snakehead fish labyrinth, the outermost part of respiratory epithelial cells and blood vessels. there is a type of respiratory epithelium and also equipped with microvilli, which functions from microvilli to exchange and absorb substances. there is a goblet cell that serves to convert mucus into latex/gum and equipped by basal lamina at the base-part. figure 9. histology of labyrinth-gill of snakehead fish (channa striata). he staining. (10x10 magnification). 1. loose connective tissue, 2. tight connective tissue and, 3. blood vessel. lamina basal as a barrier between epithelial tissue with underlying tissue, the deepest part of connective tissue and capillary blood vessels. seen on the histology of labyrinth snakehead fish there is connective tissue that connects with other parts. although catfish and snakehead fish have additional breathing apparatus, it can be seen from the development of aspect and different surface area, the endurance if compared between them is different. seen on the histologic picture both in dumbo catfish is more complex than snakehead fish. labyrinth of catfish has a mucous-stratified epithelium, whereas snakehead fish has a pseudostratified epithelium. the difference is striking shown at 40 times magnification, the tissue quite clears constituent cells and their shape. in the labyrinth of snakehead fish, the surface area is not as wide as in catfish. reviewing from the aspects, the surface area and structure cell and its constituent tissue on catfish are more complex than snakehead fish. measurement morphometric of gill and labyrinth table 1. calculation standard deviation of gill and labyrinth. no measured aspects standard deviation value catfish snakehead fish 1 mass left gill (gram) 5,01 ± 0,004 17,83 ± 0,55 2 mass right gill (gram) 5,34 ± 0,045 17,74 ± 0,55 3 filament length 1 (cm) 4,07 ± 0,002 4,83 ± 0 4 filament length 2 (cm) 3,84 ± 0 4,68 ± 0,004 5 filament length 3 (cm) 2,90 ± 0 2,89 ± 0 6 filament length 4 (cm) 1,97 ± 0 2,43 ± 0 7 gill arch length 1 (cm) 15,56 ± 0,08 32,24 ± 0 8 gill arch length 2 (cm) 13,67 ± 0,01 26,18 ± 0,004 9 gill arch length 3 (cm) 11,80 ± 0,01 12,75 ± 0 10 gill arch length 4 (cm) 10,08 ± 0,002 11,58 ± 0 11 mass labyrinth (gram) 3,81 ± 0 2,44 ± 0 karlina & luthfi – comparative anatomy of labyrinth and gill of catfish … 43 measured aspects include mass and length of fish, right and left labyrinth mass, the overall of labyrinth and gills mass, the right and left labyrinth mass, the right and left gills of each gill sheet, and length of filament gill and gill arch. this study focused on gill and labyrinth comparisons. so the standard deviation calculation is more focused on calculation aspects of the left and right gill morphology, right and left labyrinth volume, long morphometry of filament and gill arch. value of standard deviation that was measured on calculation gill mass in catfish was 0.004 and 0,045 on right gill. while on the left and right gills of snakehead fish were 0,55. measuring the mass of labyrinth catfish and snakehead fish is 0. morphometry length of four filament gill in catfish is 0, whereas in snakehead fish is 0 and measurement on the four-gill arch length of catfish is 0.08, 0.01, 0.01 and 0.002 while in the snakehead fish is 0. data was indicated that the diversity of samples under study is small, the smaller standard deviation values obtained indicate that the sample is same. mass between left and right gill is same, it is seen from the mass of the two is similar to maintain balance. based on the morphometric data showed that the smaller standard deviation calculation value, the sample diversity is small, meaning sample used in this study is the same. length of each gill sheet is also different. it is seen from the development of labyrinth in catfish is more developed than snakehead fish. conclusion gills of catfish and snakehead fish are bright-red color, four-gill sheets with three main structures: gill arch, raker, and filament, located on side of the head and protected by an operculum. catfish labyrinth has a pink color, shaped like a sponge, attached to the first gill, solid in shape with jagged edges, located near the pharynx. catfish and snakehead fish gills consist of primary lamella, secondary lamella, inter-lamella, blood vessels, epithelial cells, and pole cells. catfish labyrinth has microvilli, goblet cells, epithelium lining, granules, basal lamina, loose connective tissue, capillaries, perivascular and endothelium, whereas in snakeheadfish there are microvilli, epithelium lining, goblet cells, basal laminae, connective tissue, and capillary blood vessels. references achmad dkk, 2005. struktur hewan. universitas terbuka. jakarta. fujaya, 2008. fisiologi ikan. pt rineka cipta, jakarta. fumio & takashi, 2010, histology of fish. university of tokyo. tokyo press. john e. bardach, lagler, dan miller, 1997. ichtyology. university of michigan. wiley internasional publisher. usa. kisia, seth m, 2010. structures and funtions. markono print media pte ltd, usa an imprint of edenbrige ltd., british channel island. prasetyo, 2011. proses pernafasan pada ikan. jurnal sistem regulasi pada hewan perairan. diunduh dari web http//www.sistem.regulasi.hewan.perairan. accessed on a date march 20th 2015, at 05.30 wib sukiya, 2003. biologi vertebrata. universitas negeri yogyakarta: jica susanto, 1989; murhananto, 2002. jenis perairan sesuai ikan lele. diunduh dari web http///www.perairan.ikan.lele.com. accessed on a date april 11th 2015, at 13:45 wib. suyanto, 1986. suyanto, s.r., 2003. ikan. penebar swadaya. jakarta utomo 2006. letak dan morfometri insang pada ikan lele. jurnal penelitian insang ikan. diunduh dari web http//www.insang.ikan.lele.com. accessed on a date march 12th 2015, at 10:00 wib. wildan yatim, 1996. biologi modern histologi. tarsito bandung, anggota ikapi: bandung. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 15-20 | doi: 10.14421/biomedich.2018.71.15-20 issn 2540-9328 (online) comparison of detergent and ctab method for isolation of dna from salak (salacca zalacca (gaert.) voss. ‘pondoh’) namira nur arfa1,*, budi setiadi daryono2, reflinur3 1,2faculty of biology, universitas gadjah mada, jl. bulaksumur, yogyakarta, 55281, indonesia tel. +62-274-6492599, fax. +62-274-565223 3laboratory of molecular biology, balai besar bioteknologi dan sumber daya genetik pertanian (bb-biogen), bogor, indonesia author correspondency*: namiranurarfa@rocketmail.com abstract this study conducted in laboratory of molecular biology, balai besar bioteknologi dan sumberdaya genetik pertanian (bbbiogen) bogor. the aims of this study were to determine and comparing the quantity, quality and the efficiency of dna isolation result using detergent method and ctab method. the parameters observed in this study were the value of dna concentration, purity, and visualization result using gel electrophoresis. the samples were the leaves of salak ‘pondoh’ (salacca zalacca (gaert.) voss.). detergent method is a method which was developed by faculty of biology ugm, it has simple procedure and relatively affordable cost. meanwhile, ctab method is one of the commonly used methods of dna isolation protocol with relatively expensive cost. detergent method used detergent in the cell wall separation and protein removal in the sample. the ctab method used cetyltrimethyl ammonium bromide (ctab) for cell membrane separation in the sample. the research methods included dna isolation with detergent and ctab methods, pcr analysis and electrophoresis. data analysis was done quantitatively using spectrophotometric method and qualitative used electrophoresis method. the result of the study showed that dna isolation using ctab method showed higher purity compared with detergent method with the purity values ranging from 1,31,4 . meanwhile, the concentration of dna in the detergent method was higher than that of ctab with the highest concentration of 1730 µg/ml. there is no difference between the quality of genomic dna isolated by ctab and detergent methods. keywords: ctab; detergent; dna isolation; salak (salacca zalacca (gaert.) voss.) introduction indonesia is a megabiodiversity country which has enormous exotic tropical fruits, such as pinneapple, mangosteen and salak (suhandy et al., 2010). salak or known as snake fruit, salak palm and snake palm (lim 2012) belong to genus salacca reinwardt. so far 21 species of salacca have been identified (supapvanich et al., 2011) and dispersed naturally in malesia area, such as birma, thailand, malaysia, philliphines, cambodia, sumatera, java and borneo (mogea 1980). salacca growth on primary tropical rain forest with optimal temperatures 220c320c and high humidity (ni made 2005). salak has sweet taste, crunchy texture and high nutritional and economical value. salak plant classified into palmae family and can be planted in many regions in indonesia, either by cultivation or growing as a wild plants (haryanto 2010). salak fruit can be made as cuisine, such as salad, pickles, jam or crackers (lim 2012). three kinds of salak which already cultivated are salacca sumaterana becc. in sidempuan, (salacca zalacca (gaert.) voss.) in bali, ambon and also salacca wallichiana in thailand. salacca zalacca var zalacca divided into some local cultivar, such as salak bongkok in sumedang, salak petruk in ambarawa, salak pondoh, salak gading and salak kembang arum in sleman, salak condet in condet and salak nglumut in magelang (harsono & hartana 2003). salak ‘pondoh’ is a new cultivar originated from yogyakarta (supriyadi et al., 2002). the varieties of salak pondoh that had been cultivated in indonesia are salak pondoh hitam, salak pondoh merah, salak pondoh kuning and salak pondoh super (purnomo, 2001). indonesia exported high quality salak fruit to some countries such as singapore, middle east country, netherlands, hongkong and china (lim, 2012: 433). salak plants need rain fall about 200-400mm/month and usually growth undershades with sun intensity range from 50%-70%. salak growth optimal in ph 6,5 and temperature between 200c300c (suskendriyati et al., 2000). salak ‘pondoh’ (salacca zalacca (gaert.) voss.) is a dioecious plants which are spiny, clustered and grows as a dense clump. the fruit is ovoid shaped, has brown skin which is look like a snake skin (supriyadi et al., 2002). the leaves are pinnate or palmate with range from 4-7m long (ni made, 2012:8) the stem are partly erect and short. biodiversity of salak plants are high in each cultivar. variation in skin type, fruit color, taste and size showed that the plants has high genetic variability. genetic condition is an important factor in plant breeding, such as crossing over and to extend genetic variability (annisaurrohmah et al., 2014). genetic condition of a https://doi.org/10.14421/biomedich.2018.71.15-20 16 biology, medicine, & natural product chemistry 7 (1), 2018: 15-20 plant is inherited by dna. in salak plant, dna isolation is needed to obtain the pure dna for genetic analysis in salak plant breeding. dna isolation technique is needed to obtain dna from organism with high purity and concentration. dna isolation can conduct using sds method (sambrook et al. 1982:29), ctab (mulyani et al., 2011:2), phenol chloroform method (tenriulo et al. 2001:2) and detergent method (nasiri et al., 2005). dna isolation technique consists of three main procedures, they are cell lysis, removing the contaminant and purification of dna (milligan, 1992: 66). dna isolation method was chose based on some consideration, such as amount and the molecular weight, purity, time, species and the cost. general method of dna isolation is ctab methods, a dna isolation method which can produce high concentration and purity of dna, but the cost is expensive. faculty of biology universitas gadjah mada develop a dna isolation technique using detergent method, which is relative easy and cheap. the aim of this study was to compare the result of dna isolation of salak ‘pondoh’ using detergent method and ctab method. the method can be used to get dna isolation which is effective, easy, cheap and simple. materials and methods procedures materials the material used in this research were the leaves of salak ‘pondoh’ plant from turi, sleman, yogyakarta, aquades, pvp (polyvinylpyrrolidone), mercaptoethanol, ctab (cetyl trimethyl ammonium bromide), taq dna polymerase, taq dna buffer, dntps mix, nuclease free water, detergent, ddh2o, chloroform: isoamyl alcohol (chisam), agarose, ethidium bromide, tbe (tris boric acid edta) buffer 0,5 x, primer opa 13, loading dye, dna ladder, dna marker, mineral oil, naoac, te + rnase tools that used in this study were scissor, mortar, glassware, volumetric pipette, pipette tips, pcr thermocycler, vortex, waterbath, sentrifuge, freezer, refrigerator, autoclave, magnetic stirrer, electrophoresis machine, microwave, microtube, microplate, chemidoc software and spectrophotometer. methods the source of dna were leaves of salak ‘pondoh’ from sleman, yogyakarta. the result of dna isolation between ctab methods and detergent method were compared by seeing the optical density and the visualization of dna band after electrophoresis. 1. detergent method 0,5 gram samples of salak ‘pondoh’ leaves were extracted using mortar and stored in microtubes. after that, 1ml detergent buffer (1,4m nacl, detergent, 100 µltris hcl ph 8, 20mm edta) are added to the leaves extract. after that, the sample were separated by sentrifuge about 10 minutes at 12.000 rpm. after that the supernatant were taken from the juice and filtered using filter paper and added with isopropanol from the wall of the tubes. then the mixture are shook slowly then the dna will appear as a white fiber which is dna band. the isopropanol removed from the bottle, then the dna were added by ddh2o and stored in refrigerator. 2. ctab method preheat ctab isolation buffer, after that 0.5 grams of salak ‘pondoh’ leaves were extracted using mortar and added by 500µl buffer ctab (tris hcl ph 8,0; 1,4 m nacl; 20mm edta; 0,2% ctab; 0,2% mercaptoethanol). after that, the sample are homogenized using vortex and incubated in waterbath for 30 minutes at 650 celcius. every 10 minutes, the tube are turned upside down. after 30 minutes, the mixture are taken from waterbath and added by 500µl chisam (chloroform: isoamyl alcohol) with ratio 24:1, mixed gently and the samples are turned upside and down for 200 times until the samples are homogeneous. then, the samples are centrifuged for 10 minutes in 12.000 rpm. supernatant that formed, moved into the another microtubes. supernatant added by naoac 50µl and added cold isopropanol 500µl to precipitate the dna. then the samples were centrifuged in 12000 rpm for 10 minutes to precipitate the dna. remove the liquid and wash the dna using 70% ethanol and centrifuges by 5 minutes. remove the liquid and dry the dna for a night. add the dna with 100µl te rnase and incubated in 370c about 30-60 minutes. 3. pcr analysis amplification of dna was performed by pcr type my cycler machine from biorad, using primer opa 13 and total pcr reaction of 20 μl. rapd condition setting was initial denaturation at 94ºc for 4 min followed by 44 cycles from 94ºc for 1 minute, 37ºc for 1 minutes, and 72ºc for 2 minutes. after that the temperature was maintained at 72° c for 10 minutes, then the temperature was lowered to 4° c. table 1. pcr composition table. 1 ddh2o 13,55 µl 2 10x buffer 2 µl 3 10 mm dntps 0,2 µl 4 taq polymerase 0,25 µl 5 dna 2 µl 6 5 um primer 2 µl total volume 20 µl arfa et al. – comparison of detergent and ctab method for isolation of dna … 17 figure 1. the settings of pcr program. 4. electrophoresis  1 making buffer solution 10xtris borat edta (tbe) 108 grams tris, 55,2 grams boric acid were added into a 1000 ml beaker glass and added by aquades up to 700 ml. the solution was stirred with a magnetic stirrer until homogen. next, 9,2 grams of edta was added and stirred back with a magnetic stirrer and fed into a 500 ml measuring flask and added to a volume of 500 ml. the solution is shaken out to homogeneous. the available buffer solution was 10x concentrated buffer solution, to obtain 0,5x buffer solution, 10x buffer solution is taken 50ml and fed into 1000 ml of measuring flask. then plus aquades up to 1000 ml and the solution shaken until homogen.  preparation of agarose gel 2% 2 grams of agarose was weighed in 100 ml of 0,5x tbe solution and allowed to stand at room temperature for 1 minute. the solution was heated by microwave about 2 minutes to a perfect solute solution. the solution is allowed to stand at room temperature to keep it from overheating. the agarose solution is poured onto a mold with a combed comb to make a well on the gel. 0,5 x tbe solution was added to the surface of the solid gel and then the comb was lifted slowly. place agarose then placed on electrophoresis tank in submerged condition of 0,5x tbe solution.  electrophoresis dna the dna samples and dna markers were prepared. a 5 μl dna marker was inserted into the left most edge well. the diluted dna in te buffer 5 μl then mixed with 3 μl of loading dye, then inserted into the 2nd well to the next. after all samples were put into a well, the electrophoresis apparatus was closed and connected to a power source. the power supply was set to 100 v for a 60 minutes and turned on by pressing the on button. once it was done, the tool was turned off. gel was removed along with its mold from electrophoresis fittings. the gel was stained with ethidium bromide for 25 minutes then rinsed with aquades. gel was observed with uv transilluminator. the dna bands will glow and observed with chemidoc software. data analysis data analysis conducted by quantitative dan descriptive qualitative of dna profile using detergent and ctab method. the factor compared are as follows: 1. quantitative factor quantitative factor which measured were optical density from both dna profile, including dna total, rna total, protein, purity and concentration. 2. qualitative factor qualitative factor that compared were the result of electrophoresis of dna profile using detergent and ctab method after pcr. results and discussion table 1. optical density of dna isolation product from salak (salacca zalacca (gaert.) voss.) was done using detergent method. no concentration (µg/ ml) purity λ260 λ280 1. 725,0433 1,1798 0,073 0,061 2. 767,4141 1,1666 0,077 0,0 3. 486, 2714 1,1989 0,049 0,041 4. 1025,8585 1,1842 0,103 0,087 5. 1076,1797 1,1406 0,108 0,094 6. 1102,3815 1,1891 0,110 0,093 7. 1730,4355 1,1255 0,173 0,154 8. 854,9668 1,0139 0,085 0.080 table 1 showed optical density of dna isolation product from salak leaves using detergent method, the purity of dna isolation product range from 1,01391,1989 with dna concentration has value 486,2713 1730,4355(µg/ ml). 18 biology, medicine, & natural product chemistry 7 (1), 2018: 15-20 table 2. optical density of dna isolation product from salak (salacca zalacca (gaert.) voss.) by ctab method. no concentration (µg/ ml) purity λ260 λ280 1. 850,3145 1,333 0,085 0,064 2. 937,99 1,4926 0,094 0,063 3. 1112,24 1,4388 0,111 0,077 4. 909,6155 1,441 0,091 0,063 5. 858,0715 1,5533 0,086 0,055 6. 690,5455 1,4703 0,069 0,047 7. 687,5588 1,4232 0,069 0,048 8. 491,976 1,4700 0,049 0,033 table 2 showed optical density of dna isolation product from salak leaves using ctab method, the purity of dna isolation product range from 1,333-1,553 with dna concentration value 491,9761112,24 (µg/ ml). detergent ctab figure 1. result of dna isolation product after pcr using primer opa 13 (5’-cagcacccac-3’). discussion dna isolation is the first stage in genetic engineering (faatih, 2009). dna isolation defines as a procces to extract dna from cell organism, and get a pure dna from another organelles and materials in cells, such as polysaccharides, lipid, rna and protein. the process of dna isolation consists of several stages: cell separation, separation of dna from other components and purification of dna (yusdiana, 2008). the process of dna separation is done by mechanical or physical means using mortar, this is done to extract dna, cell wall, cell membrane and nuclear membrane so that expected dna can come out of cell. the next process is the separation of dna from various organelles and other cell components such as proteins, cellulose, lipids, rna. the last process is a dna purification process that aims to purify the dna so that the results of dna isolation easily observable and can be judged both in quantity and quality. optimization of dna isolation method can be conducted by arranging the lysis buffer composition and method in extract dna from another compounds in cells (milligan, 1992). dna isolation methods are of several types, such as simple methods using detergents, nucleon ™ phytopure ™ genomic dna extraction kit method and ctab (cetyl trimethyl ammonium bromide) method. ctab/nacl dan sds method are conventional and popular dna isolation method (fitriya et al., 2011) in extraction of plant which contains high polyphenol and polysaccharides (ardiana, 2009). in this study we used two methods of dna isolation and compared the results. detergent method is using detergent in the process of cell separation, while the ctab method used ctab buffer for cell separation process. the buffer solution in the detergent method consists of edta, detergent and nacl. salt solution is involved in regulating osmotic pressure in cells. the salt content of the buffer will cause hypertonic conditions outside the cell so that the cells will experience plasmolysis. detergent will damage the cell wall of the organism. after that the sample was centrifuged by using a centrifuge with a speed of 12000 rpm. this centrifugation serves to separate the cell debris or the larger part of the pellet and the smaller, dna-containing supernatant (langga et al., 2012). the difference methods of dna isolation can produce different dna isolation product. furthermore, the supernatant was taken using a micropipette and inserted into a new microtube. after that, the supernatant were filtered using filter paper and moved into new microtubes. then isopropanol were added from the microtubes wall, this addition was done to prevent isopropanol from evaporating and to avoid spontaneous reactions between the dna with isopropanol. after that the tubes were turned back slowly to form dna threads. after the dna threads were visible, the tube was centrifuged at a rate of 12000 rpm for 5 minutes. the isopropanol were removed then the dna pellet was dried and dissolved in a tebuffer solution that acts as a dna solvent and stored in the freezer. in the use of isolation method with ctab buffer, the process of separation was done by ctab (cetyl trimethyl ammonium bromide). first, ctab buffers were made with a composition of 2% ctab, 0.5 m edta of 20 μm, 1m tris hcl 1 μl, 2%pvp, 0,2 % mercaptoethanol and 5 m nacl of 1.4 μl. first ctab buffer was preheated into waterbath at 65ºc. the sample was extracted using mortar until soft. the samples were moved into a 1.5 ml micro-tube and added 500μl of the heated extraction buffer and then mixed well. incubate the at 65ºc for 30 minutes and every 10 minutes the tube was reversed for the process. after 30 min, the mixture was taken from the waterbath and allowed to stand at room temperature for 2 minutes and added with 500 μl of chloroform and isoamic alcohol mixture at a ratio of 24: 1. after that the material spinned down for 5 minutes, it aims to mixed the samples with chloroform. after that, centrifuged for 15 minutes at a speed of 12000 rpm. the supernatant formed was then removed and transferred into a new micro tube. the function of incubation in 650c and presipitation using chloroform: isoamyl alcohol was to remove proteins in the cells (semagn et al., 2006). the functions of adding polyvinylpyrrolidone (pvp) in the arfa et al. – comparison of detergent and ctab method for isolation of dna … 19 early stage of dna isolation in the ctab method was to bind the phenolic compounds and to minimize the oxzidation of phenolic compounds (utami et al., 2012). pvp also can produce thick and clear dna bands. the supernatant was then added 1/10 ch3coona and then added with 2 / 3x isopropanol volume for dna precipitation. the sample was then centrifuged at a rate of 12000 rpm for 10 minutes to precipitate dna. the liquid was then removed and the precipitate washed with 70% ethanol for 10 minutes to precipitate the dna and centrifuged again for 5 minutes. the liquid was removed and dried. after that the dna was dissolved with 50 μl of te buffer. after the isolation process was complete, a total quantitative dna analysis was performed using spectrophotometry and qualitative analysis using electrophoresis. table 1 showed that the purity of dna isolated results with ctab method is better than detergent method. the detergent method has purity value 1,3-1,5 meanwhile detergent method has value 1,0-1,19. a pure dna has ratio odλ 260/λ280 between 1,8-2,0 (sambrook et al., 1989). optical density with value <1 showed that the dna isolation product was contaminated by compounds such as polysaccharides, protein or phenolic compounds. meanwhile, optical density with value >2 showed high rna accumulation in dna isolation product. the result showed that the samples are still contaminated with compounds such as proteins or polysaccharides. dna concentration in the detergent method has a higher concentration than the dna concentration by the ctab method. in the detergent method, the highest concentration value is 1730,4355 (µg/ml) while the ctab method has highest value 1112, 24 (µg/ml). based on the isolated dna concentration, it is known that dna isolation using detergent method is better than using ctab method. the total dna was qualitatively done using electrophoresis technique. stages of electrophoresis technique is done by gel preparation of 2% agarose. in the running stage electrophoresis was done by using 2 μl samples, loading dye 2 μl and h2o as much as 1 μl. loading dye plays a weight and as a marker of dna to enter into the wells. running electrophoresis was performed with a 100 volt voltage for 30 minutes. after running finished, electrophoresis results are inserted into etbr (ethidium bromide) to color the dna. ethidium bromide is capable of inserting between the nitrogen bases in the dna helix. once stained, electrophoresis results are seen under ultraviolet light by using a uv transluminator. pcr rapd are conducted to make sure the quality of dna. pcr is a process of multiplying nucleotide sequences by enzymatic methods at a certain temperature. there are several major components in pcr, including dna fragments, primers, deoxyribonucleotides triphosphate (dntp) and dna polymerase enzymes. the pcr stage consists of several stages: predenaturation, denaturation, elongating and post elongation. the time required is about 4 hours 31 minutes and there are 44 cycles. pcr was performed using pct-100 programmable thermal controller with rapd program. thermocycler is a device capable of rapidly changing the different temperatures required in the pcr cycle (louie et al., 2000: 475). in addition, another primer is used for pcr mj research machine with pcr time 04:31:36. in pcr used primer opa 13 (5'-cagcacccac-3') .in addition to the opa primer 13, there were several primers used for the analysis of rapd of salak plants, such as opa 20 (ediwirman, 2015). the results show that electrophoresis results after in pcr using detergent method and ctab method was not much different. this was characterized by the number of bands and the appearance of dna bands. the results showed the presence of dna at 200bp to 1100 bp. conclusion from the result of the research, it can be concluded that the result of dna isolation (salacca zalacca (gaert.) voss.) using ctab method has better purity but using detergent method has higher dna concentration. there is no difference in the quality of the detergent method and ctab method. further research about the modification of the detergent method is needed to obtain higher purity. acknowledgements the authors gratefully acknowledge balai besar bioteknologi dan sumber daya genetik pertanian (bb biogen), bogor, indonesia for supported this study. 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[indonesian]. comparison of detergent and ctab method for isolation of dna from salak (salacca zalacca (gaert.) voss. ‘pondoh’) biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 35-46 | doi: 10.14421/biomedich.2014.31.35-46 morpho-anatomical analysis of cosmostigma racemosum (asclepiadoideae) flowers widodo1,2*, mohamad amin1, mimien henie irawati al-muhdar1 and muhammad ja’far luthfi2 1biology education-graduate program, state university of malang. jalan semarang 5 malang 65145, east java, indonesia 2faculty of science and technology, uin sunan kalijaga. jl. marsda adisucipto no.1 yogyakarta 55281, indonesia author correspondency*: wwidodo594@gmail.com, phone: +62 815 4856 6820 abstract cosmostigma racemosum is a plant species belonging to the family apocynaceae, the subfamily asclepiadoideae. cosmostigma racemosum is found in nglanggeran mountain gunungkidul, yogyakarta. the local name and its original distribution are not known. information or study of cosmostigma racemosum in indonesia is not available. comprehensive characterization of this species is important for authentication and addition of data base. characterization was conducted by analyzing the morphology and anatomy of flower. the objectives of this study were to describe and analyze the morphology and anatomy of c. racemosum flowers. the method of research was based on observation method and exploration of plant systematics evidence or taxonomy evidence, including analyses and description of morphology and anatomy of flower structure and its development. the results showed that the characteristics of flower morphology are in accordance with the existing description in literatures. characteristics of pollinia are specific characters of morphological aspect of flower. data of anatomy of flower and its parts development are the new ones which confirm the position of c.racemosum as a member of the tribe marsdenieae. the data of anatomy also show new information of the ontogeny of the important parts of flower: pollinia formation, pollinia corpusculum, anther wall, anther sac, stigma, stamen, staminal tube, stigmatic chamber, and structure of ovary in asclepiadoideae. keywords: morpho-anatomy, cosmostigma racemosum, asclepiadoideae introduction cosmostigma racemosum is a species belonging to the family apocynaceae, subfamily asclepiadoideae (takhtajan, 2009: 522; apg iii, 2009: 105-121). backer and bakhuizen (1963: 274) state that this species is native to sri lanka and india. according to trimen (1895: 177), this species is found in india, burma and java. hooker (1885: 46) states that c. racemosum is common in sri lanka but its range of distribution includes java. this species was listed in the appendix 1 of threatened species in sri lanka (ganashan et al., 1995: 61) and in 2009 it became the protected plant because it was critically endangered (the ministry of forestry and environment, republic of sri lanka, 1999:105; parlement of the democratic socialist republic of sri lanka, 2009: 29). cosmostigma racemosum is also found in nglanggeran mountain in pathuk, gunung kidul, yogyakarta as a component of unique vegetation dominated by shrubs and lianas (widodo, 2012: 1-15). until now, there has been no information of the origin and the cause of its presence in nglanggeran mountain. this mountain is the remnant of tertiary volcano (oligomiosen) or approximately 0.6-70 million years ago (adhie, 2009: 1-3). only few sources of information, such as writings, books, atlas of flora, journal as well as website regarding c. racemosum are available. because there is no species data base and information of the presence of c. racemosum in indonesia, it is necessary to compile data of structure characteristics of this species for authentication of its identity and systematics. based on the results of research by widodo (2012:1-15), it is necessary for the characterization of morphology and anatomy structure of c. racemosum. in the subfamily asclepiadoideae, cosmostigma belongs to the tribe marsdeniaeae. subfamili asclepiadoideae consists of three tribes: marsdeniae, ceropegieae and asclepiadeae (takhtajan, 2009: 529). in the classic classification, the division of tribes is based on the position of pollinia and flower. pollinia are erect in the tribe marsdeniae, horizontal in the tribe gonolobinae (ceropegieae) and pendulous in the tribe asclepiadeae (civeyrel 1998: 523). the aspect of flower structure of asclepiadoideae can be used to confirm the position of specimen in genus, subtribe, tribe and subfamily. the characteristics of flower parts, its ontogeny and development are important for tracing and confirmation of evolutionary phase and its relative position in phylogeny. kunze (1995: 8) has made the schematic diagram of evolutionary phase of asclepiadoideae flower based on the structure of corona, kunze (1995: 1-24) has analyzed the structure and function of flower in asclepiadoideae, especially the corona and gynostegium. his study revealed the morphology and evolution of corona and morphology of pollinia translator in apocynoideae, periplocoideae, and asclepiadoideae. the morphological complexity of flowers in asclepiadoideae has confused yet, at the same time, instilled curiosity among botanists in the last two centuries (ollerton and liede, 2003: 107). morphology is the most important basis for taxonomic description (simpson, 2006: 348). morphological characters are the outer appearance of plants. these characters provide 36 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 characteristics which can easily be used for identification or supposition of the phylogenic relationship. morphological characters have long been used more often than anatomical and molecular characters because the morphological characters are easier to observe and more practical to use. flower morphology is important marking characters in identification. morphology is considered to be old fashioned but it is still the basis for the solution of taxonomical problems (stuessy, 2009: 232). figure 1. flower of cosmostigma racemosum. a, b. inflorescence. c. calyx and ovary. d individual flower. e. gynostegium. f. position of pollinia at gynestegium when the coronaria and anther wall removed. g. pollinia unit and their parts. the characters of plant inner structure have made significant contribution to plant systematics for more than 150 years (judd, 2002: 55-100). anatomical characters and inner structure of plants have been used for identification and determination of phylogenic relationship. anatomical characters are observed using widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 37 light microscope with simple techniques as well as electron microscope. some important aspects of anatomical characters which have taxonomical values are flower anatomy and its development, anther development and pollen structure. anatomical characteristics play a role in explaining the phylogenic relationship (singh, 1999: 165). cervantes and diego (2010: 1) state that description of morphological characteristics in plants is the fundamental part of botanical study and as a key to taxonomy or systematics. currently, the studies to describe development are mostly those of gene interaction and protein control. these indicate that biology as a discipline has reached its ultimate maturity with experimental procedures and biochemistry point of view. however, all procedures based on experiments have caused the decline in descriptive aspects of morphology. molecular data do provide information related to morphology, but the role of genes and protein in controlling morphology can only be understood if the description of morphology of plant and its parts have been understood first. de craenne and wanntorp (2011: 1) state that the decline of knowledge of morphology and systematics of plansts has ocured in the last decades. the studies of organisms continue to increase, but with limited knowledge of structure. this results in inaccuracy of interpretation and limitedness of research horizon. flower morphology shows the evolutionary processes which have been occurring since 130 million years ago. flower morphology is the best explaining component in plant systematics researches and the key for creating phylogeny which broadens the understanding of evolution and other related organs. data of morphology are useful in clarifying molecular characters in phylogeny studies and as sources of data that help to clarify flower evolution and the underlying mechanism of flower development (de craenne dan wanntorp, 2011: 3). the objective of this study was to reveal morphoanatomical structure of cosmostigma racemosum flower. the morpho-anatomical characteristics of c. racemosum flower can be used for confirmation and checking at the levels of species, genus, and tribe in the existing classification hierarchy and for clarification of its phylogenic relationship in each level of taxon. methodology flowers of c. racemosum were collected from the nglanggeran mountain which lies at a latitude of s.07o5”25.5 and a longitude of e.110o32”20.’ the samples were subsequently kept in biology laboratory of sunan kalijaga state islamic university, yogyakarta. samples were kept in collection bags, and some of which were put in fixative faa (formalin acetic-alcohol). samples were collected as dry specimens, wet specimens and fresh material. the tools for observation and collection were digital cameras sony nex f3, sony cyber-shot dsc-w180, canon dslr, rulers, micrometers, calipers, small measuring tapes, collection bags, scissors, cutters, labeling paper, a gps (global positioning system), tools for dried herbarium collection, flacon bottles, stereo microscopes nikon smz 1500 equipped with a camera, light microscope nikon eclipse 50 equipped with a camera nikon dsf1. materials for observation and collection were distillated water, alcohol 70 %, faa (formalin acetic alcohol) solution. tools for plant tissue dissection method consisted of: microtome leica, oven, slide hot plate, becker glasses, measuring glasses, flacon bottles, object glasses, deck glasses, labeling paper, cutters, tweezers, spatula, and slide boxes. materials for plant tissue dissection were: distillled water, alcohol (30 %, 50%, 70%, 80%, 96%), xylol, parafin, glycerin-albumin solution, safranin, hematoxylin, fast green, canadian balm. the techniques and procedures for plant tissue section (slice) preparation followed cutler et al. (2007: 170-193) and schweingruber et al. (2011: 7). preparation and observation of flowers were done by observation, photographing, measurement and description of characteristics of parts of flowers, fruits and seeds. identification of morphological characteristics was based on cullen (2006: 1-357) and haris and haris (2006: 1-206). photographing was conducted with digital cameras sony nex f3, canon dslr, and sony cybershot dsc-w180. ruler or other tools were put beside the photographed materials in order to show the size of the materials. photographing was replicated 15 times. measurements were done using caliper, tape meter and graph paper. observation and photographing of detailed morphology of flower’s parts was done using stereo microscope nikon smz 1500 equipped with a camera nikon. preparation and observation of pollinia were conducted by picking gynostegium, then opening carefully the corona covering pollinia using preparation needles under stereoscopic microscope. a unit of complete pollinia was picked and isolated to get the detailed parts of pollinia and their orientation. observation of pollinia morphology was done under a stereoscopic microscope nikon smz 1500 equipped with a camera. pollinia were photographed in their natural condition, which were attached to one side of gynostegium and their orientation was determined. measurements taken for each unit of pollinia were the length and width of pollinia lobes, the length and width of corpusculum, the length of caudicula or translator arm.the measurement of pollinia’s parts was done using objective micrometer in the stereoscopic microscope nikon smz 1500 equipped with a camera and the results were then calibrated using paint program, windows picture manager and corel photo paint 12. slides of flower anatomy were made using parafin method with double coloring of fast green safranin. observation of section (slice) preparation was done by observation and photographing using light microscope nikon eclipse 50 equipped with a camera nikon dsf1. observation and photographing with weak magnification 38 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 were done for identification of shape, configuration of tissues in calyx, corolla, gynostegium, pollinia and other organs, and the configuration of an organ in the whole inflorescence. observation and strong magnification were done for identification of structure and configuration and other details of cells which compose epidermal tissues, mesophyll, vascular tissues in calyx, corolla gynostegium, style and stamen. the measurement of cells which compose tissues was done using objective micrometer scale calibration method and the results were then analyzed using program paint, windows picture manager and corel photo paint 12. data analyses refered to the description and semiquantive method according to albrechtova (2004: 13). data in the forms of macroscopic and microscopic pictures were analyzed descriptively and comparatively. the pictures were visually presented and described or accompanied with explanation. the presentation of data in the forms of pictures, numbers, description and explanation were compared with literaures and related studies. this research was conducted from march to july 2013 in biology laboratory, a part of integrated laboratory of islamic state university of sunan kalijaga, yogyakarta. results and discussions flower morphology the flowers of cosmostigma racemosum is inflorescence of racemose to corymbose type; peduncle is between the petioles of leaf pair (interpetiolaris); peduncle is brightly green; the transversal section is round, 6-8 cm in lenght. pedicel is also brightly green; transversal section is round, the tip wider than the base, 1 cm long. the width of inflorescence is 3-4 cm (figure 1a, b). the individual flower of cosmostigma racemosum is bisexual, 3-4 mm in height without pedicel; 9 mm in width when blossoming. the individual flower is shown in figure 1c. the sepal consists of 5 parts, united at the base, 1 mm in length, tip obtuse; margin pubescent, brighlt green. corrolla has 5 petals, forming into a bellshape short tube, 3-4 mm in length, 2-3 m in width, tip acuminate, surface glaborous, greenish yellow, reddishdotted. flower has 5 corona-scales, 1 1,5 mm in height, tip bilobed, whitish grees. stamen and pistil form a gynostegium (figure 1e). 5 anthers are attached to the lateral side of stigma. anther is protected by corollines corona. the tips of corollines corona wrap the stigma. diameter of stigma is 2 mm. the ovary has 2 carpels, 1 mm in length. the morphological characteristics of c. racemosum flower in this study match the decription in sivalaya farm (2011: 1), page (2008: 1), backer & bakhuizen (1963: 274), brandis (1906:468), trimen, (1895:177), hooker (1885: 46), drury (1866: 228). the structure of corona is important for distinguishing cosmostigma from non cosmostigma. according to ping-tao et al., (1995:4) the characteristics of cosmostigma corona is that it is relatively small and thin like a membrane. the visual presentations of c. racemosum flowers as sketches have been available in old literatures or monographs, namely de lessert and de candole (1846: 84), wight (1846: 591), hendrik (1686: 32). the visual presentations in the forms of ancient herbarium photographs were uploaded in the web site of herbarium musei parisiensis, in 2012. description and visual presentation in this study are important and needed because the characteristics of flower have high identity value and the information of the presence of c. racemosum in indonesia currently is not known except in the ancient literature of trimen (1895: 177) in flora of ceylon, hooker (1885: 46) in flora of british india, and drury (1866: 228) flora of india. the additional description data of c. racemosum flower morphology contributed by this study are the position of petals (aestivatio) toward each other and the structure of corollines corona in anther. the aestivation type in c. racemosum is dextrostum contortus or rightward contortion. the tip and middle of the corolla lobe reduplicate or folded with margin folded outward. de craene and wanntorp (2011: 2) state that data of flower morphology are very good clarifying components in systematic research and the key or the main tool for developing phylogeny concept, expanding and enriching the understanding of evolution and significance of organs in evolution. morphology of pollinia pollinia are produced by an anther. an anther consists of two theca or pollen sacs, each forming one pollinium, so there are 2 pollinia in each anther. corona corollines form at the dorsal side of anther. an anther is fused to the lateral side of stigma forming a structural unit called gynostegium (figure 1e). corpusculum is shiny brown located between anthers. corpusculum is connected by caudicule to two pollinia lobes from two different anthers, namely one on its right and the other on its left side (figure 1f). pollinia lobe is erect, obovate, golden yellow (figures 1f, g), 550-600 mµ in length, 200-250 mµ in width. caudicule or translator leg is clyndrical, 500-600 mµ in length, without membrane. corspusculum is ovate, cleft in ventral side, 250 mµ in length, 150-200 mµ in width, shiny blackish brown. the color and structure of corpusculum of c. racemosum ensure its position as a member of the subfamily asclepiadoideae. the characteristics of pollinia lobe of c. racemosum, namely erect, ovate, relatively long caudicule without membrane, ensure its position as a member of the tribe marsdenieae. shape, size, location, orientation, and location of line, are important criteria of pollinia morphology (sreenath et al., 2012:45-53; sinha dan mondal, 2011:7981-7986; rahman, 1990: 83), which play an important role in identification of tribe and genus. the results of this study confirm the classification takhtajan (2009: 522) which put cosmostigma racemosum in the tribe marsdenieae. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 39 anatomy of floral development longitunal section of inflorescence in figure 2 shows that c. racemosum inflorescence is of racemose type (inflorescentia racemosa). the development of inflorescence unit starts from the edge or bottom of the inflorescence. there is a small bractea on each base of inflorescence unit. trichomate are on the bractea and leaf surface. figure 2 b shows the early development of inflorescence unit. the characterics of early inflorescence development indicate that sepals occupy the main proportion of flower; colleter gland in calyx base is clearly visible. stigma also occupies a large proportion of flower. stamens are cleary visible, consisting of filaments and anther. the bases of filaments are attached to the base of corolla. the wall of pollen sac (theca) begins to differentiate simultaneously with the formation of pollen. the next step is the enlargement of flower (figure 2d), indicating the following characteristics: elongation of corolla base, the expansion of stigma to the periphery so that it press and lift the anther. the anther’s position is precisely on the lateral side of stigma. the development of ovary is clear; the wall of anther and theca (pollen sac) which contain pollinia are clearly visible. the maturation of pollinia is happening.the wall of anther clearly shows the layers of tapetum, middle layers and endotesium. the stage of blossoming is shown in figures 2d, e with the following characteristics: the corolla dominates the proportion of the flower; the stigma is lfted by the uniting tissues of corolla base, calyx and stamen. pollinia and their components (tapetum, middle layer, endotesium) adhere to upper lateral side of stigma. the remaining structure of anther sticks to stigma and becomes corollines corona which protects pollinia; the stigma is lifted away from ovary. the presence of bractea at the primodial stage of infloresence development confirms the position of c. racemosum as a member of the subfamily asclepiadoideae which ontogenically has bractea like inflorescence in apocynoideae. the data of the presence of braktea during primordial phase of inflorescence development also rectify the statement of hooker (1885: 42) that the bractea in cosmostigma racemosum is zero (0). but the same data may also confirm the statement of hooker if what he means by (0) is that ontogenically c.racemosum has bractea. figure 2. longitunal section of cosmostigma racemosum flower. a. section of young inflorescence insert: bractea and trichoma. b, c, d, e. flower development. data of flower development anatomy clarify the proccess of gynostegium structure formation (the fusion of stigma and anther) in c. racemosum. the early development shows that the structure of stamen is not united with stigma, so the fusion of stigma and anther is difficult to explain. data of flower development can complement, clarify or support the concept of flower phylogeny of asclepiadoideae presented by kunze (1995:1-24). the early stage of flower structure of asclepiadoideae is similar to that of apocynoideae so that phylogenically apocynoideae precedes asclepiadoideae. data of floral development also show that gynostegium is formed and then lifted due to the elongation of staminal tube and ovary. data of flower development show the development of the wall of anther and theca (pollen sac) and the ontogeny of pollinia. 40 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 anatomy of longitudinal section of receptacle, calyx, corolla, stamen and pistil the longitudinal section of blossoming inflorescence of c. racemosum shows the flower parts, namely receptacle, calyx, corolla and gynostegium (the fusion of stamen and pistil). the parts and the tissue structures are shown in figure 3. figures 3b-c show tissues of receptacle and calyx. receptacle consists of large cells and its outer side is covered by flat epidermal cells with stomata among which. the tissues of calyx consist of upper (adaxial) epidermis, mesophyll, and lower (abaxial) epidermis. the upper epidermis in calyx consists of a layer of flat elongated cells (15-25 μm). the mesophyll of calyx is composed of rounded cells and it has small vascular tissues. the abaxial epidermis consists of cuboid cells (20x20 μm) and stomata among which. colleter gland is found at the abaxial surface of base of calyx (figure 3d). the border between calyx and receptacle tissues is not clear. the tissues in calyx are more differentiated than those in receptacle. the mesophyll cells in tissues of receptacle are larger than those in calyx tissues. a. longitudinal section of flower b. receptacle c. sepal f. basal of petald. coleter glands e. sepal g. anther wall h. pollinium i. stigma head j. ovary figure 3. tissues in longitudinal section of cosmortigma racemosum flower. characteristics of calyx of c. racemosum are: the presence of colleter gland at the base of the connection of two callyces, so there are five colleter glands. the colleter gland produces sticky slime or resin, insoluble in water. the function of this gland is to protect dormant buds and meristem development, leaf buds, and young stipules (evert, 2006: 459). the colleter gland in the callyx of c. racemosum flower is of the general standard type, namely long head and short stalk. the head is covered with epidermal cells that look like palisade. the colleter type of c. racemosum belongs to the type of the family apocynaceae. the presence of colleter in the callyx of apocynoideae was reported by martins et al. (2013: 21) in secondatia densiflora, whereas the presence of colleter in the calyx of asclepiadoideae was reported by silva et al. (2008: 924) in oxypetalum. the presence of colleter gland or glandula emergentia in the base of calyx of the tribe marsdenieae of asclepiadoideae was receptacle sepal petal antherstigma head ovary gynostegium adaksial epiderm abaxial epiderm epidermis mesophyllpollinium mesophyll adaxial mesophyll abaxial mesophyll adaxial epiderm abaxial epiderm epidermal glands carpel ovule epidermis mesophyl widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 41 found by valente dan costa (2005: 53) in marsdenia loniceroides. the presence of colleter gland in the calyx of cosmostigma racemosum shows that the petals and sexual apparatus of flower have been and prevented from drought since they are still buds. figure 3e shows the arrangement of corolla tissues. the corolla is composed of several tissues: adxial (upper) epidermis, mesophyll (the densely-packed and spongy mesophylls) and abaxial (lower) epidermis. adaxial epidermal tissues at the tip of corolla consist of short papilla cells while abaxial epidermis consists of a layer of flat cells with stomata. the papilla cells of adaxial epidermis get more elongated in the middle and base of corolla. the structure of papilla cells at the adaxial epidermis at the base of corolla is of glandular type (figure 3f). the structure of papilarry epidermis like palisade at the adaxial surface of corolla base of c. racemosum indicate is function as nectariferous tissue. in this tissue, epidermal cells function as secretory gland. the characteristic of nectar epidermis is the presence of trichomate cells or the elongated cells like palisade (evert, 2006: 453). figure 4. a-d the development of stamen and other structures. d. parts of microsporangium wall during the development of pollinia. e. parts of pollinia. 42 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 the mesophyll tissues of c. racemosum flower are differentiated based on cell size and density. the difference between the the dense and spongy tissues can be observed by safranin-fast green double coloration. the dense mesophyll tissues consist of densely-packed round cells with limited intercellular spaces, and the cells contain chloroplast. the presence of cholorplast in densely packed mesophyll indicates that the petals of c. racemosum still have photosynthetic function. the spongy mesophyll tissues consist of long-rounded cells larger than the cells in densely packed mesophyll tissues. there are more intercellular spaces and vascular tissues at the border with densely packed mesophyll tissues. figure 3g shows the arrangement of tissues of anther wall. figure 3h shows the mature pollinia in the broken anther sac. the base of anther sticks to the lateral side of stigma. anther and pollinia are at the depression of lateral side of stigma. the upper tip of anther wall is additional corona or corollines corona. the additional corona is composed of thin layer of outer and inner epidermis, the reduced mesophyll containing druse crystal. parts of the whole stamen can be seen in figure 3a. the lower part of an anther is a filament which is the extension of stamina tube, which in turn is the extension of corolla base which wrap the ovary. the term staminal tube is adopted from tubo estaminal (silva et al., 2008: 929; valente dan costa, 2005: 53). the cells at the periphery of staminal tube look larger than those in the middle. the staminal tube wall facing the base of filament shows epidermal gland. the longitudinal section crossing the middle of corona shows that the tissues of stamen base and staminal tube are united (indistinguisble), while the tissues of corona base tend to separate from staminal tube. these data show the ontogenical difference between the two tissues. the tissues of anther consist of upper epidermis, mesophyll, and inner epidermis. the upper epidermis is composed of densely-packed longitudinally elongated flat cells. the mesophyll consists of densely packed round cells. the inner epidermis consists of papilla cells larger than those in mesophyll. the tissues of filament are composed of outer epidermis, mesophyll and inner epidermis. the outer epidermis consists of densely packed small flat cells. the mesophyll is composed of densely-packed, small rounded cells. the inner epidermis is composed of glandular cells which can be sheded. the inner side of filament seems to be asscociated with the base of stigma and the tip of stamina tube forming a secretion-laden sac. the arrangement of stigma is shown in figure 3i while the arrangement of ovary is shown in figure 4j. the stigma consists of epidermal tissues and mesophyll tissues. the outer epidermis of stigma is composed of short papilla-shaped cells like those in adaxial epidermis of corolla. the shape of mesophyll cells look like spongy tissues but with limited intercellular spaces. many druse crystalls are found in the mesophyll tissues of stigma. in the lower middle part of mesophyll of stigma there are xylem vessels with thickened ring. the part of stigma which sticks to anther is composed of glandular cells producing secretion. ovary consists of wall and sacs containing many ovules. anatomy of stamen and pollinia development figures 4a-d show the development of stamen of cosmostigma racemosum. transversal section of young flower (figure 4a) shows that the base of stamen is at the junction between ovary and corolla. anther and filament are separate parts from stigma so that gynostegium has not been formed. theca or pollen sacs have been formed but the wall differentiation has not been complete. corollines corona at the tip of anther is still at the early stage of development. orderly layers of pollen as the precursors of pollinia are seen. staminal tube has not been formed in this immature flower. the maturation process of stamen is shown in figures 4c-d. anther is lifted at the same time with the elongation of filament. the base of filament is also lifted and forms staminal tube.the base of anther sticks to the lower lateral side of stigma, so the gynostegium has been formed. the wall of theca (pollen sacs) shows the layers of tissues of tapetum, middle layer and endotesium. pollinia have been formed and there is space between pollinia and the anther wall. corollines corona has been elongated. the mature stamen and pollinia are shown in figure 4d. the tissues of theca wall have been broken but the tissues of anther and corolines corona are still present. the staminal tube has been maximally elongated. the stamina tube function as the supporting structure for stigma and its tip function as the base of filament. gynostegium structure has been completely formed at the blossoming flower. pollen in mature pollinia emits shiny blue fluorenscent light while the wall of sporopollenin emits orange fluorescent light. data of stamen development clarify the origin of staminal tube and the formation of gynostegium in c. racemosum flower wich can be traced ontogenically. filament structure in gynostegium is maintained until mature stage. this indicates that c. racemosum belongs to a group closely related to apocynoideae or the lower group of the sub-family asclepiadoideae. the lower groups of the sub-famili asclepiadoideae are the tribes fockeeae and marsdenieae. polinia of c. racemosum are the aggregation of pollen grains. polinia of asclepiadaceae (asclepiadoideae) are wrapped by sporopollenin. the sporopollenin wall extends entering each group of pollen grains so that all pollen grains are not united. the outer wall of sporopollenin is called pollinial wall, while the extension of wall into the units of pollen grain group is called extension of pollinian wall (srideevi et al., 1990: 324). in general sporopollenin wall is soluble in hot 2aminoethanol solution. srideevi et al. (1990: 324) states that young pollinial wall in tylophora (asclepiadaceae) is soluble in hot 2-aminoethanol solution but the wall of mature pollinia is not. pollen grains in intact pollen can be observed using light without coloring because they emit fluorescent light or autofluorescence. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 43 (pacini dan franchi, 1999: 303). the emission of fluorescence indicates the maturation stage of pollnia. anatomy of transversal section of flower transversal sections of cosmostigma racemosum flower from the top to the base of stigma were observed at 8 points (figure 5). the first point of section crossing the the upper stigma in figure 5a shows the tissues of corolla, anther and stigma. the tissues of corolla consist of abaxial epidermis, mesophyll cells at the abaxial side, mesophyll cells at adaxial side and adaxial epidermis (figure 5e). epidermal cells and mesophyll cells at abaxial side are larger than those in mesophyll cells at adaxial side. vascular bunddels are located between abaxial and adaxial mesophylls. each petal shows three vascular bundles. stomata are located in outer abaxial epidermis. the tissues of stigma are composed of ground tissues with one vascular bundle and two pollen sacs (figures 5g, h). pollen sac is round and and each sac has one pollinium. the tissues of stigma consist of extensive mesophyll tissues, in the middle of which are circles of vascular bundles (figure i). the epidermis or the outer layer is composed of densely packed tube-shaped epidermal cells, some of which are glandular (figure 5j). figure 5. the transversal sections of tissues of cosmostigma racemosum flower. a. sketches and point of transversal section, b-e. transversal section, f. corolla tissues, g. vacular bundle in anther, h. anther sac containing pollinia, j. tissues of stigma, k. section of corpuskullum of pollinia, l. stigmatic sac, m. ovule. the second point of section crossed the middle of pollinia lobe. in this section, the pollen sacs look flat, oval to round-shaped. the tissues of vascular bundles at the pistil show two sliced circles. at the periphery of the stigma there is an attachment of transversal section of pollinia corpusculum as shown in figure 5k. there are two shapes of epidermal cells at the lateral side of stigma, namely: a) dense smaller columnar shape and b) secretory 44 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 large, columnar shape. the smaller epidermal cells are at the lateral side of stigma facing anther, while the large, secretory cells seem to be the components of corpusculum and caudicule. the third point of section crossing the lower part of stigma base shows the characteristics of the edge of anther which are extending and folded outward. the junction of two edges of anther base forms a stigmatic chamber as shown in figure 5l. the stigmatic chamber is located under corpusculum. the transversal section of style look rounded with two circles of vascular bundles tightly close to each other. the tissues of mesophyll are rounded-ovate and densely packed. epidermis is composed of densely packed tube-shaped cells. in this section style is surrounded by five stigmatic chambers. the transversal section of corolla has 8-9 vascular bundles. the fourth point of section crossed the ovary. this section shows that the transversal section of corolla tissues has 3 (x5) vascular bundles. the transversal section of corolla is attached to the base of staminal tube. the staminal tube has 1 (x5) kidney-shaped vascular bundle. this section shows two ovaries, each having ovules. each carpel wall has 8-12 vascular bundles, one of which is larger than the others. each ovary has 8-14 rows of ovules. the transversal section of the base of staminal tube circles the ovules. what remains of the stigmatic chamber is a narrow curve as the border between the remains of anther base. five tissues of transversal section of calyx are outside of corolla tube circle. at the junction among calyx there is a colleter gland. data of structure of flower organ and tissues of cosmostigma racemosum in transversal section strengthen and clarify the data obtained from longitudinal section. the transversal section through stigma shows: lay out and orientation of petals from one another, arrangement of corolla tissues at the tip or distal, number of layers of anther structure, location of anther, arrangement of anther tissues, number of theca or anther sacs, structure of theca, structure of anther wall, location and position of stigma, structure of stigma, arrangement of stigma tissues. the structure of stigmatic chamber of cosmostigma racemosum as a member of the tribe marsdenieae is relatively similar to that of marsdenia loniceroides presented in the result of research by valente et al. (2005: 55). the stigmatic structure and its extension vertically downward under staminal tube widen the pollination reach. staminal tube is a set of tissues, forming rings made into 5 main lobes. each has three sub-lobes (a large one in the middle accompanied by two small ones). the junction of main lobes is a downward longitudinal vessel and an extension of stigmatic chamber. epidermal cells at the vessel between the main lobes or small lobes are secretory or glandular. the arrangement of staminal tube is essentially the same as that of vascular bundle which extends laterally. stigamaric chamber is directly connected to the tip of ovary with no staminal tube in between. the transversal section crossing ovary shows two seperate ovaries or apocarps, each having half-rounded shape transversally. the two ovaries are not the same in sizes; one is smaller than the other. each ovary has many ovules. analyses of observation and confirmation of carpel types according to esau in suradinata (1998: 235) show that each visible ovary consists of one carpel arranged in conduplicate involution with placenta of parietal ovules. each ovary has one ovary chamber. tissues of carpel is composed of three (1+2) large vascular bundles and 12 (6+6) larger ones. colleter gland is found in the edge of intersection among sepals, the structure of pistil with the ring of vascular bundle inside shows that stigma has two vascular bundles which have been fused. toward the base of stigma, the vascular bundle get separated from each other and become two sliced rings. the fact that pistil has two apocarps seems to be contradicted with the evolutional trend of style and ovary. apocarp is supposed to have its own style. morphology of female sexual organ and its terminology are still disputable (hidayat, 1995: 226). the presence of two apocarps but only one stigma in c. racemosum and asclepiadoieae in general confirm that statement. a phenomenon that a flower has apocarps but the stigmas are united is explained by kunze (1995: 5) as post genital fusion. study of kunze (2005: 347) also found this post genital fusion in the formation of stamina corona and corollines corona. conclusion and suggestion morphological characteristics of cosmostigma racemosum flower in this study are in accordance with the existing description in literatures. new data are found regarding specific structure of anatomy or flower parts and tissues, developments of flower, stamen, anther wall, anther chamber and pollinia, formation of pollinia, ontogeny of pollinia corpusculum, position of stigma, and structures of staminal tube, stigmatic chamber and ovary. characteristics of pollinia are specific morphological characters of anther for species identification. new data of flower structure anatomy confirm the position of c. racemosum in the tribe marsdenieae. data of flower development anatomy especially stamen development of c. racemosum shows its ontogeny and clarify in the tribe marsdenieae, subfamily asclepiadoideae as well as filogeny of the tribe mardenieae in the subfamili asclepiadaceae. species authentification of c. racemosum and other members of asclepiadoideae can be done by matching data of pollinia, structure of morpho-anatomy of flower. all data of shape and qualitative characters of structure are primary characteristics for cross checking and authentication. data of morpho-anatomy of c. racemosum flower can be used to complement the evolutionary analyses of asclepiadoideae, tribe marsdenieaae currently in trend. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 45 acknowledgment the author is grateful to all parties who have helped him to conduct this study, especially the directorate general of higher education, the ministry of religion of the republic of indonesia which funded this study. references angiosperm phylogeny group iii. 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february 7, 2013 – february 8, 2013. widodo, amin, m., al-muhdar, m. h. i., luthfi, m. j. 2013. profil fitokimia metabolit sekunder cosmostigma racemosum (asclepiadoideae). makalah disajikan dalam seminar nasional biodiversitas. studi, pemanfaatan dan konservasi keanekaragaman hayati nusantara dalam bidang kesehatan. uns, 9 november 2013. wight. 1846. icones plantarum orientalis. 1.2 (1). madras: j. b. pharoah. zhengyi, w., raven, p. h. 1996. flora of china. chicago: science press. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 23-26 | doi: 10.14421/biomedich.2019.81.23-26 issn 2540-9328 (online) echinoderms diversity and abundance in gunung kidul beach yogyakarta hikmah supriyati1,*, setyawati dwi k2, ahzami2, farhani sodiq3, septiana khoiriyah4 1postgraduate biology education program, yogyakarta state university, indonesia. 2biology education department, faculty of science and technology, uin sunan kalijaga, yogyakarta, indonesia. 3smait ihsanul fikri, pabelan, mungkid, magelang, central java 56551, indonesia. 4mts nu miftahul huda, gembong colo street, glagah kulon village, kudus, indonesia. author correspondency*: hikmahsupriyati@gmail.com abstract echinoderms are one of marine invertebrate animals that have an important role, as a recycle of nutrients in the functioning of ecosystems. echinoderms usually appear in the intertidal zone of the coast. the existence of echinoderms in the intertidal zone is affected by external and internal factors. this study aims to determine the level of abundance and diversity of echinoderms in the intertidal zone in the krakal and drini coastal areas, gunung kidul, yogyakarta. data were collected using transect method. the main transect along 50 meters was divided into 5 sampling points, and the distance between sampling points was 10 meters. after that, each point was taken for 3 observation plots with an area of 1x1 meters and the distance between plots is 5 meters. the results showed that, the diversity index on the krakal coast had a higher value (0.139) when compared to the drini coast (0.129). calculation of echinonderm abundance index shows that the drini beach location has a high abundance value when compared to the abundance value at the krakal beach loc ation. the parameter measurement results can be seen that both the ph and temperature parameters on both the krakal and drini coast have values that correspond to the optimum parameter in the waters. keywords: echinoderms; diversity; abundance; krakal coast; drini coast introduction echinoderms are one of marine invertebrate animals that have an important role in the functioning of ecosystems (supono et.al., 2014). echinoderms have a role as nutrient recycling (triana et al., 2015). it can be used as a parameter (bioindicator) quality in marine waters (marine ecosystems) (jalaluddin, 2017). echinoderm habitat is beach and sea to a depth of 366 m. in addition, echinoderm can live in a variety of habitats such as reef flat zones, algal growth areas, seagrass beds, live coral colonies and dead coral colonies (yusron, 2009). echinoderms are very common in sandy areas, especially those that are overgrown with seaweed (aziz, 1996). one area that has an environment like this is the area of krakal and drini beach gunung kidul, yogyakarta. the intertidal zone of krakal and drini beach has different types of substrates, both whether it is sandy, muddy, or rocky. the intertidal zone in the area occupies a very wide area, and when sea water reaches the lowest ebb, the length of the dried water base can reach hundreds of meters. this condition is utilized by the local coastal community in search of marine animals to meet their daily needs. the growth of marine biota in the coastal intertidal zone is very high, due to this area being a place of life, shelter, and a place to look for food. in addition, environmental conditions in this area are very favorable for the growth of marine biota because of the support of marine physical, chemical and biological factors. soemodhiharjo (1990) revealed that marine physical-chemical factors including salinity, ph, currents, temperature, and ever-changing brightness greatly affect the life of organisms in coastal intertidal zones. furthermore, rowe & doty (in hasan, 2004) said that another important factor influencing the distribution of echinoderms is the average topography of an island. density of marine animals depends on temperature, salinity, and pressure. in addition, aziz (1996) revealed that the condition of the substrate and habitat largely determines the distribution of echinoderms. the existence of echinoderms in the gunung kidul area, especially in the krakal and drini coast areas, has not yet been identified and is well known for its habitat trends, so research is needed to determine the diversity and abundance in the intertidal zone of the krakal and drini beach of gunung kidul, yogyakarta. materials and methods materials and tools the equipment used in this study were ph meter to measure the ph of seawater, a thermometer to measure water temperature, a flakon bottle to place echinoderms that have not been identified, 70% alcohol as a https://doi.org/10.14421/biomedich.2019.81.23-26 mailto:hikmahsupriyati@gmail.com 24 biology, medicine, & natural product chemistry 8 (1), 2019: 23-26 preservative for echinoderms, a rope for making transects, stakes as a barrier, gauge for measuring, scissors, tweezers and stationery. determination of transect and sampling the transect used in this study is the comb transect. the use of comb transects is due to the highest echinoderms in the intertidal zone. each area of the research site is made of a main transect with a length of 50 meters. then the main transect is divided into 5 sampling points, the distance between the sampling points is 10 meters. after that, each point was taken 3 observation plots with an area of 1x1 meters and the distance between plots 5 meters. figure 1. transect and sampling design. calculation of echinoderms diversity and abundance calculation of the diversity of echinoderms on the krakal and drini coast used a diversity index. the diversity index formula is (shannon and wienner, 1963): h’ : shannon wiener diversity index pi : ni/n = relative abundance of species h’ < 1 : low diversity and low community stability 1 < h’< 3 : medium diversity and stable community h’ > 3 : high diversity and high community stability meanwhile, to determine the abundance of types of echinoderms performed calculations with abundance index. the formula is as follows (rahma and fitriana, 2006): d = 𝑛𝑖 𝐴 d : the abundance of individual species i ni : number of individuals of the i-th species a : sampling plot area result and discussions table 1. number of species of echinoderms phylum. no species krakal beach drini beach 1 ophiotrix sp 594 876 2 echinometra sp 58 85 3 holothuria sp 1 0 4 crinoidea 1 0 based on the results of research conducted there were 4 types of species found on krakal beach. whereas 2 types of species found on the drini coast. the species most commonly found in the krakal coast and the drini coast are the ophiotrix sp. this is because the ophioridea class can be found starting from the intertidal area to a depth of more than 6500 meters (stohr, et al., 2012). the abundance index of phylum echinoderms on krakal and drini beaches can be seen in figure 2. figure 2. graph of the abundance of echinoderms on the krakal and drini beach. the results of the calculation of the abundance index of echinonderms on both beaches show that ophiuroidea and echinoidea are the most abundant class of echindermata found. when viewed from the substrate, krakal and drini beaches are beaches composed of coral. the abundance of corals on both coasts causes the abundance of echinoderms. according to yusron (2016) states that the ophiuroidea class and echinodea class are generally found in flat reef areas. both of these classes are commonly found on flats as a place to hide and find food in the rocks. likewise, research conducted by sugiarto and supardi (1995) which states that ophiuroidea and echinoidea are a class of phylum echinoderms have a tendency to attach to corals. in addition, ophiuroidea and echinodea classes have the ability to grip rocks and be able to refrain from waves so that they are able to adapt in coral areas (aziz, 1996). 5.11 0.88 0 0 3.96 0.3867 0.0067 0.0067 0 1 2 3 4 5 6 ophiotrix sp echinometra sp holothuria sp crinoidea abundance of echinoderms on krakal and drini beach drini krakal h’ = ∑ pi log pi supriyati et al. – echinoderms diversity and abundance in gunung kidul beach … 25 it is contrast to the holothuridea class which is very rarely found on krakal and drini beaches. this is because species in this class prefer habitats that are always inundated. meanwhile, based on observations on both beaches at low tide, the water content in the intertidal area is very small. these conditions make the cause of at least holothuridea species found. according to aziz (1996) holothuridea occupy habitats that are always flooded even at low tide, and for their life, these animals prefer clear water habitats and are relatively calm. crinoidea species are also very few found, this is because crinoidea usually live in coastal areas (yusron, 2016). while the research location does not pass through the edge. figure 3. graph the diversity of echinoderms on the krakal and drini beach. from this diagram it can be seen that waters on the krakal coast have a higher diversity index (0.139) when compared to waters at the drini coast (0.129). the difference in diversity obtained on the two beaches can be influenced by several internal and external factors. according to (shannon and wienner, (1963), the diversity of echinoderms on the drini and krakal beaches can be categorized as low because the results of the calculation of h 'is less than 1. the low diversity of echinoderms on the krakal and drini beaches indicates that the productivity level at both beaches is very low, so it can be said that the ecosystems on both coasts are unstable. the imbalance of ecosystems on the two beaches can be seen from the presence of a very dominant individual, the ophiothrichidae family. table 2. results of measurement of environmental parameters on krakal and drini beach. parameter krakal beach drini beach parameter stability ph 8 8 7,5-8,6 temperature 30oc 27oc 20oc-30oc temperature measurements on the drini and krakal beaches are 27º c and 30º c. the results of temperature measurements on both beaches are still classified as the optimum temperature that allows echinoderms to live and develop properly. thus echinoderms can be found abundantly on the coast of krakal and drini. the good temperature for the life of echinoderms is 20º c 30º c (aziz, 1998). the ph value describes the acidity and alkalinity intensity of a waters which is indicated by the presence of hydrogen ions. the value of the ph range in the study is still relatively good for seawater parameters. the results of ph measurements at the drini and krakal beaches are 8. the measurement results are in accordance with research conducted by romimohtarto and juwana (2007) where the ph of sea water in indonesia varies between 6.0-8.5. as according to aziz (1996), a good ph value for the life of echinoderms is 7.5-8.6. from the parameter measurements it can be seen that both the ph and temperature parameters at both the krakal and drini coast have values that correspond to the optimum parameter theory in the waters. this allows the existence of other factors that influence the differences in the results of the diversity of echinoderms on the krakal and drini beaches. based on observations, there are more seaweed on the krakal beach compared to the drini beach. the amount of seaweed in an ecosystem will affect the number of echinoderms. krakal beach with a large amount of seaweed has few echinometra species compared to the drini beach. the results of this study are in accordance with research conducted by maila (2017) which states that the amount of seaweed in an ecosystem is negatively correlated with the amount of echinometra sp. conclusions the diversity index on the krakal coast has a higher value (0.139) when compared to the waters on the drini coast (0.129). while the calculation of echinondermal abundance index shows that the drini beach location has a high abundance value when compared to the abundance value at the krakal beach location. from the results of parameter measurements, it can be seen that both the ph and temperature parameters at both the krakal and drini coast have values that correspond to the optimum parameter theory in the waters. interaction with biotic factors in the form of seaweed found in both locations still shows a range of tolerance that can support the life of echinoderms. references aziz, a. 1996. habitat dan zonasi fauna echinodermata di ekosistem terumbukarang. oseana 1. 24(2): 33-43. 0.124 0.126 0.128 0.13 0.132 0.134 0.136 0.138 0.14 krakal drini diversity index of echinoderms on krakal and drini beach diversity index 26 biology, medicine, & natural product chemistry 8 (1), 2019: 23-26 aziz, a. 1998. pengaruh tekanan panas terhadap fauna echinodermata. jurnaloseana, 13(3): 125-132 brower je, & zar jh. 1977. field and laboratory methods for general ecology. wm. j. brown company publ, iowa. p.288. hasan, s. 2004. kepadatan dan pola distribusi echinodermata di zona intertidal pantai pulau ternate. media ilmiah mipa. jalaluddin & ardeslan, 2017. identifikasi dan klasifikasi phylum echinodermata di perairan laut desa sembilan kecamatan simeulue barat kabupaten simeulue. jurnal biology education, vol 6 no, page 82-97 rahma, yulia & fitriana. 2006. keanekaragaman dan kemelimpahan makrozoo-benthos di hutan mangrove hasil reha-bilitasi taman hutan raya ngurah rai bali. jurnal biodiversitas vol. 7, no. 1. hal: 67-72. romimohtarto, k. & juwana, s. 2007. biologi laut: ilmu pengetahuan tentang biota laut. jakarta: djambatan: 540 prowe, f. e. w. and j. e. doty. 1977. the shallow-water holothuirans of guam. micronesia. shannon c. e., & wiener. 1963. the mathematical theory of communications. univ. illinois. urbana. p.117. soemodhiharjo. 1990. teluk ambon. ambon: balai penelitiandan pengembangan sumberdaya laut (lipi) ambon. sugiarto, h. & supardi 1995. beberapa catatan tentang bulu babi marga diadema. oseana 20(4): 35 triana r, dkk, 2015. identifikasi echinodermata di selatan pulau tikus, gugusan pulau pari, kepulauan seribu, jakarta. (jurnal pros sem nas masy biodis indon). vol 01 no. 03. program studi biologi, universitas al azhar indonesia. jakarta yusron, e. 2009. keanekaragaman jenis ekhinodermata di perairan teluk kuta, nusa tenggara barat. makara, sains, vol. 13: 45-49. yusron, e. 2010. keanekaragaman species ekhinodermata di perairan likupang, minahasa utara, sulawesi utara. ilmu kelautan (indonesian journal of marine sciences) juni 2010. vol. 15 (2) 85-90 yusron. 2016. struktur komunitas ekhinodermata (asteroidea, ophiuroidea, echinoidea dan holothuroidea) di perairan taman nasional wakatobi sulawesi tenggara. jurnal ilmu dan teknologi kelautan tropis, vol. 8, no. 1, hlm. 357-366 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 41-45 | doi: 10.14421/biomedich.2019.82.41-45 issn 2540-9328 (online) hepatotoxic assessment of tramadol-diclofenac use: a study in a rat model elias adikwu*, ebinyo clemente nelson department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* adikwuelias@gmail.com abstract the concurrent use of tramadol and diclofenac may increase hepatotoxic risk due to their individual hepatotoxic effects. this study assessed the hepatotoxic effect of tramadol-diclofenac administration in albino rats. twenty-four adult male albino rats (200-220g) randomized into four groups were orally administered with tramadol (12mg/kg/day), diclofenac (6mg/kg/day) and tramadol-diclofenac for 14 days respectively. the rats were anesthetized, blood samples were collected and evaluated for serum liver function and lipid parameters. liver samples were weighed and evaluated for biochemical parameters and histology. the effects of tramadol-diclofenac on the body and liver weights did not differ significantly (p>0.05) when compared to control. also, effects were not significant (p>0.05) on blood glucose, and serum cholesterol, triglyceride, low and high density lipoprotein cholesterol levels when compared to control. liver and serum levels of aminotransferases, alkaline phosphatase, lactate dehydrogenase, gamma–glutamyl transferase, conjugated bilirubin and total bilirubin increased significantly in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control. furthermore, significant decreases in liver catalase, glutathione, superoxide dismutase, glutathione peroxidase levels with significant increases in malondialdehyde levels occurred in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control. hepatocyte necrosis was observed in rats treated with tramadol-diclofenac. tramadol-diclofenac may increase hepatotoxic risk at doses used for this study. keywords: tramadol; diclofenac; co-treatment; toxicity; liver; rat introduction most medical conditions are associated with pain. pain remains the most inadequately treated symptom due to different cultural, attitudinal, educational, legal, and system-related reasons (connors et al., 1995). pain has a multifactorial origin; hence it may be difficult to achieve effective pain control with a single drug. combination therapy with analgesics from different groups is advantageous in targeting both peripheral and central pain pathways (rawal et al., 2011). the world health organization (who) analgesic ladder recommends the combination of acetaminophen or nsaids with opioids as the second step in the treatment of pain, based on increasing pain severity (blondel and azadfard, 2013). tramadol-diclofenac can be used for the treatment or management of chronic pain (mitra et al., 2012). diclofenac is a phenylacetic acid derivative which belongs to the acetic acid class of nsaids. it acts by inhibiting cellular cyclooxygenases (cox-1 and cox-2), which results to decreases in the production of proinflammatory prostaglandin, prostacyclin and thromboxane products which are important mediators of inflammation and pain (zimmerman, 1999; lewis and stine, 2013). tramadol is a synthetic codeine analog that acts as a weak opioid agonist in addition to mildly inhibiting serotonin and norepinephrine reuptake. tramadol is effective against mild-to-moderate pain. clinically, tramadol-diclofenac use could advantageously target both peripheral and central pain pathways and leverage on the ability of individual drug to reduce pain and fasten recovery (raffa, 2001). however, tramadol-diclofenac use may increase hepatotoxic risk since both drugs have hepatotoxic potential. clinically, apparent liver injury due to diclofenac has been reported and it ranks in the top 10 causes of drug-induced liver injury (dunk et al., 1982). tramadol can cause respiratory arrest as well as acute liver failure, which several fatal instances reported (loughrey et al., 2003). the use of tramadol-diclofenac calls for hepatotoxic assessment which this study evaluated in a rat model. material and methods animals and drugs adult male albino rats of weight 200-220g were used. the rats were supplied by the animal house of the department of pharmacology and toxicology niger, delta university, nigeria. the rats were housed in four cages (6 per cage) and allowed to acclimatize for 2 weeks in a well-ventilated room, maintained at a room temperature of 28 ± 2°c, under natural lighting condition. the rats were fed with standard rodents chow https://doi.org/10.14421/biomedich.2019.82.41-45 42 biology, medicine, & natural product chemistry 8 (2), 2019: 41-45 and given water ad libitum. tramadol hydrochloride (zim laboratories ltd india) and diclofenac potassium (adpharm nigeria ltd) were used for this study. all other chemical substances used for this study are of analytical grade. higher doses of tramadol (12mg/kg/day) and diclofenac (6mg/kg/day) dissolved in normal saline were used for this study. grouping of animals and drug treatment twenty-four adult male albino rats were divided into four (4) groups a-d of 6 rats each.  group a (control) was orally administered with normal saline (0.2ml) for 14 days.  group b was orally administered with tramadol (12 mg/kg/day) for 14 days.  group c was orally administered with diclofenac (6mg/kg/day) for 14 days.  group d was orally administered with tramadol (12mg/kg/day) and diclofenac (6mg/kg/day) for 14 days. collection of sample the rats were sacrificed with inhalational diethyl ether after drug administration and blood samples were collected from the heart. at 1500g for 15 minutes, the blood samples were centrifuged, serum samples extracted and evaluated for liver function parameters. liver samples were harvested and washed in a cold 1.15% kcl solution and homogenized in 0.1 m trishcl buffer, ph 7.4. the homogenates were centrifuged at 1500g for 15 minutes and the supernatants were decanted and evaluated for biochemical parameters. evaluation of biochemical and oxidative stress indices aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), total bilirubin (tb), conjugated bilirubin (cb), gammaglutamyl transferase (ggt), lactate dehydrogenase (ldh), total cholesterol (tc), triglyceride (tg) and high density lipoprotein cholesterol (hdl-c) were evaluated using standard laboratory test kits. blood glucose (g) was evaluated using glucometer while low density lipoprotein cholesterol (ldl-c) was determined using friedewald equation. liver protein was evaluated according to gornall et al. (1949). the method described by sun and zigma, (1978) was used for the evaluation of superoxide dismutase (sod) whereas catalase (cat) was determined according to aebi, (1984). glutathione (gsh) was estimated as described by sedlak and lindsay, (1968) whereas glutathione peroxidase (gpx) was evaluated according to the method of rotruck, et al, (1973). malondialdehyde (mda) was determined as reported by buege and aust, (1978). histological examination of the liver liver samples were fixed in 10% neutral buffered formalin, processed and embedded in paraffin wax. sections of 5 μm thickness were cut, stained with haematoxylin and eosin and examined under a light microscope and relevant sections photographed. statistical analysis data are expressed as mean ± sem. data was subjected to student t test. results were considered to be significant at p<0.05; 0.01; 0.001. table 1. effects of tramadol-diclofenac on body and liver weights of albino rats. groups body weight (g) absolute liver weight(g) relative liver weight (%) a b c d 290±11.3 295±14.2 275±10.6 295±11.5 6.03±0.13 6.22±0.25 5.99±0.31 6.21±0.19 2.07±0.01 2.10±0.73 2.17±0.59 2.10±0.77 n=6. data are expressed as mean ± sem. table 2. effects of tramadol-diclofenac on blood glucose and serum lipids of albino rats. group g(mg/dl) tg(mg/dl) tc(mg/dl) hdlc(mg/dl) ldlc(mg/dl) a 95.0±6.41 70.5±6.78 110.1±10.8 30.7±3.44 65.7±6.00 b 90.7± 7.85 72.1±7.59 106.8±10.3 31.8±3.35 61.6±5.32 c 92.0±6.42 74.9±6.70 112.0±10.6 32.6±2.61 64.5±6.42 d 100.9±9.19 81.1±7.56 110.3±11.1 32.4±2.63 64.7±7.62 n=6, data are expressed as mean ± sem. table 3. effects of tramadol-diclofenac on serum liver function indices of albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) cb(g/dl) tb(g/dl) a 41.5±5.57 34.2±3.15 42.3±3.56 45.5±3.11 0.75±1.53 2.66±0.18 4.21±0.05 b 60.5±6.96a 55.1±4.06a 69.5±6.11a 66.0±6.01a 1.60±0.71a 3.95±0.06a 6.6±0.02a c 89.3±6.33b 79.2±5.25b 87.7±7.71b 88.1±9.43b 2.67±0.52b 5.73±0.65b 9.8±0.06b d 220.6±10.7c 181.3±9.70c 211.3±9.37c 220.2±14.2c 6.21±0.37c 15.4±1.22c 22.8±0.76c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control. adikwu & nelson – hepatotoxic effect of tramadol-diclofenac 43 table 4. effects of tramadol-diclofenac on liver tissue biochemical parameters of albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) a 244.8±12.0 246.9±12.1 247.2±12.3 250.5±14.2 22.9±4.03 b 373.8± 17.6a 374.6±20.4a 390.0±20.0a 399.0±17.4a 45.2±7.09a c 499.6±25.0b 470.3±26.0b 488.4±23.4b 475.1±21.2b 68.1±8.16b d 960.0±23.7c 983.3±41.8c 901.0±43.7c 888.2±30.6c 190.5±12.8c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control table 5. effect of tramadol-diclofenac on liver oxidative stress indices of albino rats. group mda (nmol/mg protein) sod (u/mg protein) cat (u/mg protein) gsh (µmol/mg protein) gpx (u/mg protein) a 0.17±0.06 16.2±1.89 26.4±2.77 8.96±0.15 11.7±0.16 b 0.39± 0.04a 11.6±0.15a 17.0±0.79a 5.02±0.32a 5.25±0.01a c 0.52±0.04b 9.98±0.04b 13.6±1.16b 3.91±0.73b 4.00±0.18b d 1.37±0.32c 3.63±0.06c 5.61±0.83c 1.30±0.22c 2.57±0.82c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control figure 1. (a-e) showed the liver of control rat, rats treated with diclofenac, tramadol and diclofenac-tramadol (h &e stain 400x). (a): the liver of control rat showing normal hepatocytes (b): liver of rat treated with tramadol (12 mg/kg/day) showing hepatocyte necrosis (c): liver of rat treated with diclofenac (6 mg/kg/day) showing hepatocyte necrosis. (d): liver of rat treated with diclofenac-tramadol showing hepatocyte necrosis results the effects of tramadol-diclofenac were not significant (p>0.05) on the body and liver weights of treated rats when compared to control (table 1). also, serum tc, tg, hdl cholesterol, ldl cholesterol and blood g levels were normal (p>0.05) in rats treated with tramadoldiclofenac when compared to control (table 2). serum ast, alp, alt, ggt, ldh, ct and tb levels were significantly increased in tramadol (p<0.05) and diclofenac (p<0.01) treated rats when compared to control. however, elevations in the serum levels of the aforementioned parameters were most significant (p<0.001) in rats treated with tramadol-diclofenac when compared to control (table 3). the liver levels of ast, alp, alt, ggt and ldh were significantly increased in rats treated with tramadol (p<0.05) and diclofenac (p<0.01), but significant increases occurred at p<0.001 in rats treated with tramadol-diclofenac when compared to control (table 4). furthermore, liver sod, cat, gsh and gpx levels were significantly decreased whereas mda levels were significantly increased in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control (table 5). the liver of control rat showed normal hepatocytes (fig a) whereas the liver of rat treated with tramadol and diclofenac showed hepatocyte necroses respectively (fig b and c). the liver of rats treated with tramadol-diclofenac also showed hepatocyte necrosis (fig d). discussion the present study evaluated the hepatotoxic effect of tramadol-diclofenac in a rat model. the indices evaluated in this study are useful parameters to indicate impairment in the functional capacity of the liver. analysis of organ weight in toxicology studies is an important endpoint for the identification of potentially harmful effects of chemicals. it is one of the most sensitive drug toxicity indicators, and its changes often precede morphological changes (bailey et al., 2004). in 44 biology, medicine, & natural product chemistry 8 (2), 2019: 41-45 the present study, tramadol-diclofenac had no effects on the body and liver weights of treated rats. serum alp, ast, alt, ggt, and ldh are excellent biomarkers of hepatocellular injury. ast and alt participate in gluconeogenesis by catalyzing the transfer of amino groups from aspartic acid or alanine to ketoglutaric acid to produce oxaloacetic acid and pyruvic acid respectively. ast is present in cytosolic and mitochondrial isoenzymes and is found in the liver while alt, a cytosolic enzyme is found in its highest concentration in the liver and is more specific to the liver. alp is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes (rosalki and mcintyre, 1999). ggt is a microsomal enzyme that is abundant in hepatocytes and biliary epithelial cells. it is involved in the transfer of γglutamyl groups from peptides to amino acids and the metabolism of glutathione conjugates (friedman et al., 1996). these liver biomarkers are usually released into circulation causing elevated serum levels with the advent of hepatocellular injury (yousef et al., 2010). this study observed elevated serum and liver levels of alp, ast, alt, ggt, and ldh in rats treated with tramadol-diclofenac which is a sign of hepatotoxicity. bilirubin is an endogenous anion derived from the regular degradation of haemoglobin from the red blood cells and excreted from the liver in the bile (saukkonen et al., 2006). toxic insult to the liver can impair its excretory capacity to dispose bilirubin stimulating serum accumulation (gaw et al., 1999; jain et al., 2008). this study observed elevated serum cb and tb levels in rats treated with tramadol-diclofenac. the anti-oxidative defense system which includes sod, cat, gsh and gpx is necessary for the maintenance of redox homeostasis in organisms. it terminates or prevents free radicals such as reactive oxygen species (ros) from incapacitating the functions of biomolecules through oxidative stress (os) (borković et al., 2005). however, the functions of anti-oxidative system can be surmounted or incapacitated via depletion by over whelming actions of free radicals. this study observed hepatic depletions of sod, cat, gsh and gpx in rats treated with tramadol-diclofenac. this is an evidence which shows that os is a factor in hepatotoxicity induced by tramadol-diclofenac. lipid peroxidation (lpo) is an oxidative degradation of polyunsaturated fatty acids which can cause impairment in membrane structure and function. mda level which is an important indicator of lpo can indirectly reflect the extent of hepatic lpo in-vivo and in-vitro (jafari et al., 2012). in this study, hepatic mda levels were increased in rats treated with tramadol-diclofenac. this observation attests to the involvement of lpo in hepatotoxicity induced by tramadol-diclofenac. furthermore, the current study observed hepatocyte necrosis in rats treated with tramadol-diclofenac which correlates with changes observed in evaluated biochemical parameters. this study was able to show that tramadol-diclofenac use may increase the risk of hepatotoxicity. the use of diclofenac has been associated with hepatotoxicity (hussein et al., 2016) and hepatic os (das and roy, 2012) which is consistent with findings in this study. the exact mechanism by which diclofenac causes hepatotoxicity is not well understood, but diclofenac is metabolized in the liver (castel et al., 1997) and its hepatotoxic effect has been related to its metabolites (4hydroxy 3 diclofenac, 5 hydroxy 4 diclofenac and 5 hydroxy 6 diclofenac) (tang et al., 1999). also, studies have associated tramadol with hepatotoxicity (el-wessemy, 2008) characterized by os (rukhshanda et al., 2014) which is in agreement with observation in the present study. the mechanism by which tramadol causes hepatotoxicity is not fully known, but studies have speculated that tramadol and/or its active metabolite can stimulate hepatic ros production leading to os and hepatic biomolecular damage (singal et al., 1998). conclusion tramadol-diclofenac use may be associated with hepatotoxicity at the doses use for this study. conflict of interest: none references aebi h. catalase in vitro, in method in enzymology, s. p. colowick and n. o. kaplane, eds., 1984; 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(1999). drugs used to treat rheumatic and musculospastic disease. the nsaids.in, zimmerman hj. hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. 2nd ed. philadelphia: lippincott, pp. 1999; 517-41. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 27-32 | doi: 10.14421/biomedich.2019.82.27-32 issn 2540-9328 (online) digital anthropometer development for improving the measurement quality of human body dimensions trio yonathan teja kusuma*, arya wirabhuana**, faurosi syafa’atul yusuf department of industrial engineering, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency: trio.yonathan@gmail.com*, arya.wirabhuana@uin-suka.ac.id** abstract digital information technology has become the nerve of information for industry-based companies. however, there are still data or information that retrieved manually, one of them is measurement of the human body dimension. the problem is seemed solved by invention of digital anthropometer application. yet, the application requires a further improvement especially in its accuracy and user interface. the improvement of accuracy quality of its measurement uses the capability process analysis method. as a result, the current cp and cpk is 0.26 and -0.209 while in the developed application is 2.56 and 1.218. thus, the developed application is stated that quality of measurement increases to meet the criteria of good process capability. the user interface is improved based on the user voices. data storage databases and percentile calculation in the application was added as one on the significant user-interface improvement. keywords: application, digital anthropometer, quality, process capability introduction technology is an important factor for industrial development in world today. the development of industrial sector requires technology to improve its productivity. one of widely used technology in industry today is information and communication technology. it has been developing rapidly so that it can help people solve many problems. digital information technology has also become nerve information for companies today. in the current digital age, there are still fields for manual data or information retrieval, one of them is measurement of human dimension. its measuring device is called anthropometric chairs. yet, these measuring instrument is still far from user-friendly. however, this deficiency has been limited by existing research. previous research makes digital and automatic measuring devices. however, there are still deficiencies in this measurement tool. therefore, it is necessary to develop the current digital measuring instrument. the development plan for current application is to improve quality of measurement by testing ability of current application process and developing application to see the improvement in the application process capability. and will be added a place to store human data in application development and added percentiles of human dimension data that has been stored. apart from that it also enhances user interface also needed. literature review anthropometry according to wignjosoebroto (2000), anthropometry comes from "anthro" which means human and "metri" which means size. anthropometry can be definitively expressed as a study relating to the measurement of the human body dimensions including areas of size, strength, and other aspects of body movement. anthropometry is a part of ergonomics that specifically studies body size which includes linear dimensions, weight, contents, and also includes areas of size, strength, speed, and other aspects of body movement. eko nurmianto (nurminato, 2004) uses the term “the fallacy of the average man or average woman”. the term states that it is a mistake to design a workplace or product if it is only based on a hypothetical dimension, which is to assume that all dimensions are average values. the use of one dimension, e.g forward reach, using an average (50% percentile) in adjusting the installation of a control device will cause 50% of the population will not be able to reach it. in addition, if a certain dimension of a person (for example: height) is in the population average, then not necessarily other body dimensions of that person (for example: shoulder width) are in the population average as well. common percentile distribution values are applied in anthropometric data calculations. following is the percentile distribution table. https://doi.org/10.14421/biomedich.2019.82.27-32 mailto:trio.yonathan@gmail.com mailto:arya.wirabhuana@uin-suka.ac.id 28 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 table 1. percentile. percentil calculation 1 �̅� − 2,325𝜎𝑥 2,5 �̅� − 1,96𝜎𝑥 5 �̅� − 1,645𝜎𝑥 10 �̅� − 1,28𝜎𝑥 50 �̅� 90 �̅� + 1,28𝜎𝑥 95 �̅� + 1,645𝜎𝑥 97,5 �̅� + 1,96𝜎𝑥 99 �̅� + 2,325𝜎𝑥 (source: wignjosoebroto, 2008) image processing image processing according to munir (2004) is any form of signal processing where the input is an image, such as a photo or video, while the output of image processing can be in the form of images or a number of characteristics or parameters related to images. most image processing techniques involve or treat photos as two-dimensional signals and apply standard signal processing techniques to them, usually referring to digital image processing, but can also be used for optical and analog image processing. image acquisition or that produces input image in the first place is referred to as imaging. generally digital images are rectangular or square (in some imaging systems some are hexagonal) which have a certain width and height. this size is usually expressed in terms of points or pixels so that the size of the image is always round. each point has coordinates according to their position in the image. these coordinates are usually expressed in positive integers, which can start from 0 or 1 depending on the system used. each point also has a value in form of a digital number that represents the information represented by that point. the format of digital image data is closely related to color. in most cases, especially for the purpose of visual appearance, digital data values represent colors of processed image. the most widely used digital image formats are binary image (monochrome), gray scale image, true color image, and indexed color image. vitruvian man theory anthropometric variables have a lot of influence, so the selection of anthropometric variables is adjusted to purpose of using anthropometric data. in the study of stancic, et al (2011) in rachmatullah (2016) using 8 body dimensions to perform human anthropometric measurements using video. research by karuppiah et al (2011) in rachmatullah (2016) used 11 dimensions of the human body for anthropometric measurements in motorists. the research shows that the use of anthropometric variables does not have to be entirely, more efficient on the variables needed only. figure 1. vitruvian man, leonardo da vinci, 1490. (source: leonardo da vinci in kelly, 2005) based on the vitruvian man concept that was coined by leonardo da vinci, stating that human body size is a proportion of body size in other parts, the proportion of vitruvian man's body is described below (kelly, 2005): 1. the width of the palm is 4 fingers wide 2. foot length is the width of 4 palms 3. one cubit is the width of six palms 4. a person's height is four cubits (and thus 24 palms) 5. the step length is four cubits 6. the length of a man's arms is the same as his height 7. the distance from the hairline to the bottom of the chin is one tenth of the height of a human 8. the distance from the crown of the head to the bottom of the chin is one-eighth of the height of a human 9. the distance from the hairline to the top of the breast is one seventh of human height 10. the distance from the crown of the head to the nipple is a quarter of the height of a human 11. maximum shoulder width is a quarter of human height 12. the distance from the elbow to the tip of the hand is a quarter of the height of a human 13. the distance from the elbow to the armpit is 1/8 of the height of a human 14. the length of the hand is one tenth of the height of a human 15. the distance from the bottom of the chin to the nose is one third of the face length 16. the distance from the hairline to the eyebrows is one third of the face length 17. ear length is one third of the face length kusuma et al. – digital anthropometer development for improving the … 29 there are three additional dimensions for this study that originated from maccurdy (1955), namely: 1. the distance from the footwear to the pubic root is half the height of a human 2. the distance from the footwear to the bottom of the knee is a quarter of human height 3. the distance from below the knee to the pubic root is a quarter of human height process capability analysis according to ariani (2004) an analysis of process capability is carried out to meet various reasons, for example responding to customer requests regarding the company's process capability index or conducting an evaluation of the process for carrying out quality improvement. process capability analysis is also a procedure used to predict long-term performance that is within the limits of a statistical process controller. typical process capabilities are formulated in ± 3σ or 6σ (six sigma). where σ expresses the standard deviation of controlled process. this means assuming that normal distribution is 99.73% of products within the ± 3σ limit. to be able to stick the product specifications process capability is calculated by quantifying process capability. for under controlled conditions, the ppa can be done by: (rasyidin et. al., 2012) 1. capability process (cp) 𝐶𝑝 = 𝐵𝑆𝐴 − 𝐵𝑆𝐵 6𝜎 cp : capability process bsa : upper specification limit bsb : lower specification limits assessment criteria:  if cp> 1.33, the capability process is very good  if 1,00 ≤ cp ≤ 1,33 then the capability process is good, but it needs tight repairs of cp 1,00.  if cp <1.00 then the process capability is low, the performance needs to be improved through improving the process. 2. top process capability index/bottom process capability index. 𝐾𝑃𝐴 = 𝐵𝑆𝐴 − 𝜇 3𝜎 𝐾𝑃𝐵 = 𝜇 − 𝐵𝑆𝐵 3𝜎 kpa/kpb : top/bottom process capability index bsa : upper specification limit bsb : lower specification limit 𝜇 : average 𝜎 : standard deviation 3. process capability index (cpk) 𝐶𝑝𝑘 = 𝑚𝑖𝑛 { 𝐵𝑆𝐴 − 𝜇 3𝜎 , 𝜇 − 𝐵𝑆𝐵 3𝜎 }  if 𝐶𝑝𝑘 >= 1  capable  if 𝐶𝑝𝑘 <= 1  not capable  if 𝐶𝑝𝑘 >> fewer products are outside the specification limits research methodology object of research the object of this study is implementing the application development concept research that has been conducted before by rachmatullah (2016) into a ready to use digital anthropometer device. data type the types of data used are: 1. data obtained from direct observation and field study are called primary data. primary data of this study are height, body width, palm length, palm width, length of one cubit, distance from elbow to armpit, distance from elbow to tip of hand, length of arm extended, distance from top of head to chin, distance from line hair to the chin, distance from footwear to pubic roots, distance from footwear to below the knee, distance from below the knee to pubic roots and the length of the feet. 2. secondary data is data that is not obtained through direct observation or measurement of the object under study. the secondary data of this study is size of vitruvian man. method of collecting data data collection methods used in this study are as follows: 1. observation observation is a method of collecting data by observing directly on research object. 2. literature study the method used to find resources related to development needed in the current application. 3. interview interviews were conducted on user to improve user interface to support and facilitate measurement with the application made by user. 30 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 flowchart start initial observation and problem identification literature review development of digital anthropometer with image processing method 1. is data in control limit ? 2.is good capability index value end data collection data processing literature review literature review no yes figure 2. flowchart. results and discussion anticipating causes of inaccurate measurement in current application with fishbone diagram fishbone diagram is used to identify the potential causes of inaccurate measurements in current application. therefore, to reduce the inaccurate measurements potentials, an application development is carried out to close the gaps from potential causes of accurate measurements. the cause of human measurement in using current application is that point measurement is difficult to be exact and precise because it only uses points only for measurement, there is no clarity to straighten the height measurement point. so in the application that has been developed, drawing a line after getting the object click on the top, after that it can straighten the measurement of body height by drawing a line straight to the bottom point of object. so the tendency of causes of accurate measurements has been minimized by drawing a straight line. the method in measuring is still unclear, therefore potential causes for inaccurate measurements also occur. because the measurement point of current application is still just a point from the object to the bottom of object. then the development of a new application is done by line drawing after clicking on the top object. with the help line, measurement of the point is more accurate on the object. the environment is also one of the factors causing inaccurate measurements. the environment will not have a big impact because it is still light even though it is too bright or dark but because the measurement is straight from top to bottom the object is not affected. however, light or dark environment only affects the click point of top most object point or click point of bottom object point. there is no guidebook also included one of the factors that influence the measurement of acute curating, therefore the development application is given a help menu to facilitate and understand the application. latest application digital measurement methods in current application done by clicking on the top and bottom of an object only. after that the calculation is done to get the other dimensions. there are 11 dimensions of the human body that are used, namely: 1. height (tb) 2. palm length (ptt) 3. talraw width (ltt) 4. length of one cubit (psh) 5. stretched arm length (plt) 6. maximum shoulder width (lmb) 7. distance from the peak of the head to the chin (jpkd) 8. distance from hairline to chin (jgrd) 9. distance from elbows to armpits (jsk) 10. distance from elbows to the end of the hand (jsku) 11. foot length (ptk) application that exist today are still many gaps to be developed. one of them is improving the quality of measurement. previous studies had not yet been measured using a quality testing tool that uses process capacity index measurements. therefore, to see the potential of the application a sample search is performed to see the measurement quality of the current application. the results obtained with 30 samples that have a value of process capability (cp) is 0.26. judging from the value is still below 1, the process capability is still low and needs to be improved performance. and the value of kane process capability (cpk) is -0.209, so because the value of kane process capability is less than equal to 1 (cpk <= 1), the results are not capable. judging from the results of the measurement of the kusuma et al. – digital anthropometer development for improving the … 31 process capability index, it is necessary to develop and improve the quality of the accuracy of this application. application development this development application has been a bit of a method of taking measurements of body dimensions that are different from previous application. this developed application minimizes the causes of inaccurate measurements. namely with points and straight lines aiding to straighten points or automatically scan and get straight lines also from automatic scans performed. measurable dimensions are: 1. height (tb) 2. body width (lb) 3. palm length (ptt) 4. handprint width (ltt) 5. length of one cubit (psh) 6. distance from elbows to armpits (jsk) 7. distance from elbows to the end of the hand (jsku) 8. outstretched arm length (plt) 9. distance from the peak of the head to the chin (jpkd) 10. distance from hairline to chin (jgrd) 11. distance from footwear to pubic root (jakak) 12. distance from footwear to the knee (jakbl) 13. distance from below knee to pubic root (jblak) 14. foot length (ptk) before calculating the process capability in sample, data that has been obtained from measurement application that has been developed is carried out by controlling data using one of the quality tools, namely the x-bar s. control map. upper and lower control limits of x-bar s. map control chart these following are the calculation of x-bar chart (ucl & lcl) and s chart (ucl&lcl):  x-bar chart upper control limit x-bar chart 𝑈𝐶𝐿�̅� = 0.702 + 1.427 × 0.446 𝑈𝐶𝐿�̅� = 0.702 + 0.636 𝑈𝐶𝐿�̅� = 1.339 cm lower control limit x-bar chart 𝐿𝐶𝐿�̅� = 0.702 − 1.427 × 0.446 𝐿𝐶𝐿�̅� = 0.702 − 0.636 𝐿𝐶𝐿�̅� = 0.065 cm  s chart upper control limit s chart 𝑈𝐶𝐿𝑆 = 2.089 × 0.446 𝑈𝐶𝐿𝑆 = 0.932 cm lower control limit s chart 𝐿𝐶𝐿𝑆 = 0 × 0.446 𝐿𝐶𝐿𝑆 = 0 cm based on data processing using x-bar chart and s chart, the results of measurement with digital antthropometer is still within the control limits. it shows that the measurement is stable and normal. process capability  process capability (cp) the following is a calculation of process capability: 𝐶𝑝 = 1.339 − (−1.339) 6(0.174) 𝐶𝑝 = 2.678 1.044 𝐶𝑝 = 2.561 because the value of process capability is more than 1.33 (cp> 1.33), the process capability is very good.  kane process capability (cpk) the following is a calculation of kane process capability (cpk): 𝑐𝑝𝑢 = 1.336 − 0.702 3(0.174) 𝑐𝑝𝑢 = 1.218 𝑐𝑝𝑢 = 0.702 − (−1.339) 3(0.174) 𝑐𝑝𝑙 = 3.904 𝐶𝑝𝑘 = 𝑚𝑖𝑛 {𝑐𝑝𝑢; 𝑐𝑝𝑙} 𝐶𝑝𝑘 = 𝑚𝑖𝑛 {1.218; 3.904} 𝐶𝑝𝑘 = 1.218 because the value of kane process capability is more than equal to 1 (𝐶𝑝𝑘 > = 1), the results are capable. judging from value of the ability process, this application is good and the quality of measurement accuracy is also good and growing. additional menus in this application are the database for storing data after taking measurements, percentile menu, this menu makes it easy to perform percentile calculations from data in the database and help menu which functions to provide assistance and explanations for all buttons and functions of everything on user interface. 32 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 user interface comparison between current application and application development comparison of user interfaces before and after development: 1. latest application figure 3. user interface latest application (source: rachmatullah, 2016) 2. development application figure 4. user interface of development application. conclusion the following is the conclusion of the study: 1. calculation of process capability from the current application has cp and cpk values respectively 0.26 and -0.209. while the process capability of the development application has cp and cpk values respectively 2.56 and 1,218. thus, the developed application is stated that the quality of measurement increases to meet the criteria of good process capability. 2. application made from previous studies can already measure quickly and can reduce activity of manual measurement. however, development application can measure quickly, accurately and also reduce measurement activities manually. 3. development application have additional specifications namely databases and percentile calculations. database of application were performed using microsoft access. percentile calculations in the application use data from the database. references ariani, dorothea wahyu. 2004. pengendalian kualitas 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