antimicrobic activity of ippu padang (ammannia octandra l.f.) leaves ethanol extract against skin pathogenic microbials muharni saputri*, vriezka mierza, nirwana putri department of pharmacy, faculty of pharmacy, universitas tjut nyak dhien, jl. gatot subroto, gg. rasmi, medan, north sumatra 20123, indonesia abstract ippu padang plant (ammannia octandra l.f.) belongs to the family of lythraceae, a hardy plant that can grow to a height of 50 cm. according to previous research, ippu padang leaves contain glycosides, alkaloids, flavonoids and tannins. the presence of alkaloid compounds, flavonoids and tannins is predicted to have potential as an antifungal agent against fungi and antibacterial agent. this research used an experimental method. the steps included collecting plant material, plant identification, processing plants into simplicia powder, phytochemical screening of simplicia powder, extracting simplicia by maceration method using ethanol solvent, antifungal and antibacterial activity test using agar diffusion method and determination of inhibition diameter of leaf ethanol extract. the results of phytochemical screening showed that ippu padang leaves contained secondary metabolites, namely alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. the results also revealed that the concentration with the largest inhibitory diameter was 400 mg/ml, namely 12.4 mm against the candida albicans, 17.46 mm against the dermacoccus nishinomiyaensis bacteria, 18.53 mm against the micrococcus luteus bacteria, 19.38 mm against the pseudomonas aeruginosa bacteria, and 17.71 mm against staphylococcus epidermidis bacteria. it was concluded that the ethanol extract of ippu padang leaves could inhibit the growth of the candida albicans, the bacteria dermacoccus nishinomiyaensis, micrococcus luteus, pseudomonas aeruginosa and staphylococcus epidermidis. keywords: ammannia octandra l.f.; candida albicans; dermacoccus nishinomiyaensis; micrococcus luteus; pseudomonas aeruginosa; staphylococcus epidermidis data of article received reviewed accepted : : : 28 dec 2021 17 feb 2022 21 feb 2022 doi 10.18196/jfaps.v2i1.13505 type of article: research introduction skin is the largest organ that covers the entire surface of the body. it is often in contact with safe and unsafe objects that can cause skin diseases. skin disease is a * corresponding author, e-mail: muharnisaputri@gmail.com skin disorder caused by interactions between fungi, bacteria, viruses, and others. this disease is common in society due to several factors such as climate, environment, unclean living habits, allergies and others.1 journal of fundamental and applied pharmaceutical science, 3(1), august 2022 2 the candida albicans fungus, as the cause of candidiasis, is commonly found in the oral cavity, digestive tract, reproductive tract and skin.2 the dermacoccus nishinomiyaensis bacteria, previously known as micrococcus nishinomiyaensis, is a gram-positive bacterium that exhibits the implication of peritonitis associated with peritoneal dialysis, catheter-use bacteremia, polymicrobial infections of the skin and urinary tract.3 micrococcus luteus is an opportunistic pathogenic bacterium or bacteria that will not cause disease if another disease does not precede it. this bacterium is one of the gram-positive bacteria that can cause complex dermatitis.4 the pseudomonas aeruginosa bacterium will become a pathogen if it is in a place with abnormal resistance, such as damaged skin due to tissue damage. these bacteria include gram-negative bacteria that can cause secondary infections in wounds, burns and soft tissue infections.5 staphylococcus epidermidis bacteria mostly live as normal flora on human skin. these bacteria include gram-positive bacteria that can cause acne when the amount is excessive on the skin6. diseases caused by fungi and bacteria are very common and can be treated with antifungals and antibacterials. however, excessive antifungal and antibacterial drugs and inappropriate antifungals and antibiotics can lead to resistance. resistance occurs when microorganisms change to turn infection drugs to become ineffective. resistance causes the microbe to fail to respond to the given drug, leading to the prolongation of the disease.7 ippu padang leaves have secondary metabolites of glycosides, alkaloids, flavonoids, and tannins secondary metabolites. secondary metabolites which are estimated to inhibit microbial growth are flavonoid alkaloids and tannins,8 but it is known that there has been no research on the activity of ethanol extract of ippu padang leaf extract against fungi and pathogenic skin bacteria; therefore, researchers are interested in conducting this research. method an antimicrobial activity test with the agar diffusion method was used to determine the activity of antimicrobial agents. a petri dish containing an antimicrobial agent was placed on an agar medium where microorganisms grew, which would diffuse into the agar medium. it was later incubated for 48 hours at a temperature of 25 ± 2ºc for fungi and incubated for 24 hours at a temperature of 35 ± 2ºc for bacteria. the clear zone indicated the growth inhibition of microorganisms on the surface of the agar medium.9 results and discussion determining the water content in the simplicia leaves of ippu padang showed 5.5%. fulfilling the requirements for simplicia content was carried out as the water content was not more than 10%. simplicia with excess water content stored for a long time will produce enzymes that can affect the content of chemical compounds and turn them into other products that may not have a pharmacological effect like the original compound.10 the results of phytochemical screening showed that the ethanol extract of ippu padang leaves contained secondary metabolites, which were positive for alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. table 1 shows the muharni saputri, vriezka mierza, & nirwana putri | antimicrobic activity of ippu padang (ammannia octandra l.f.) leaves ethanol extract against skin pathogenic microbials 3 results of phytochemical screening on ippu padang leaves. table 1. phytochemical screening no screening reagent observation results 1 alkaloids dragendorf chocolate precipitate + bouchardat chocolate precipitate mayer chocolate precipitate 2 flavonoids zn + hcl red + mg + hcl red 3 glycosides: + sugar molisch white precipitate fehling a+ b brick red precipitate non-sugar acetic acid anhydride + sulfuric acid purple 4 anthraquinone glycosides naoh red + 5 tannins fecl3 5% blackish green + 6 saponins hot water, shaken + hcl 1.5 cm high foam + 7 cyanogenic glycosides na. picrate yellow 8 steroidstriterpenoids acetic acid anhydride + sulfuric acid blackish green + description: (+) contains the test compound (-) does not contain the test compound an antimicrobial activity test was conducted to measure the response of microbial growth to antimicrobial materials. one of the uses of antimicrobial activity testing is to obtain an effective and efficient treatment system. the effectiveness of an antibacterial agent in inhibiting microbial growth depends on the nature of the test bacteria, concentration and length of contact time. measurement of antimicrobial activity was carried out using the agar well diffusion method, characterized by the formation of a clear part around the well (inhibitory diameter) if the tested extract could inhibit microbial growth. the area of the clear part was then measured in diameter.11 the diameter of the inhibition was divided into several categories based on the diameter of the clear part around the well, including weak (<5 mm), medium (5-10 mm), strong (10-20 mm) and very strong (>20 mm).12 journal of fundamental and applied pharmaceutical science, 3(1), august 2022 4 figure 1. antimicrobial activity test results based on figure 1, b(negative blank) does not have a diameter of inhibition for testing all microbes. the ethanol extract of ippu padang leaves tended to have an inhibitory diameter at a certain concentration even though it had no inhibitory diameter of 12.5 mg/ml. it indicated that the bused dmso: ethanol (3:4) could not inhibit microbial growth. the concentration of 400 mg/ml had the largest inhibitory diameter among all the tests carried out where the diameter of inhibition was almost the same as the diameter of the inhibitory b+ (positive blank) used, namely nystatin for fungi and chloramphenicol for bacteria. based on figure 1, it can be observed that the higher the concentration is, the larger the diameter of the formed inhibition will be. in line with it, according to the statement of surjowardojo et al. (2015), the greater the concentration is, the greater the interaction of the extract with the tested microbes will be. it causes a larger diameter of the inhibition as the extract with a large concentration contains a large number of chemical compounds that affect microbial growth.13 furthermore, alkaloids in ippu padang leaves can damage proteins that can harm enzyme activity and cause death in microbial cells. alkaloids can also arrange microbial cell walls so that they cannot be formed completely and cause death in these microbial cells, leading to the formation of an inhibitory diameter.14 flavonoids, as antifungals, work by damaging proteins, inhibiting the enzyme system, interfering with the formation of the end of hyphae, and constricting the cell wall so that the fungal cell wall died.15 flavonoids as antibacterial work by inhibiting the synthesis of nucleic acids, inhibiting cell membranes' function and inhibiting the metabolic energy of the bacteria.14 the mechanism of action of tannins as an antimicrobial is to inhibit the activation of the enzyme and disrupt transport protein in the cell's lining so that the antimicrobial cell cannot be formed. in addition, the antimicrobial effect of tannins also can be 0 5 10 15 20 25 30 12.5 25 50 100 200 300 400 b+ bin h ib it io n d ia m e te r consentration mg/ml antimicrobial activity test results candida albicans dermacoccus nishinomiyaensis micrococcus luteus pseudomonas aeruginosa staphylococcus epidermidis muharni saputri, vriezka mierza, & nirwana putri | antimicrobic activity of ippu padang (ammannia octandra l.f.) leaves ethanol extract against skin pathogenic microbials 5 through reaction with cell membranes and inactivation of the function of the genetic material of.14 conclusion based on the results of this research, it can be concluded that the results of phytochemical screening of ippu padang leaf simplicia powder showed the presence of alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins, saponins and steroids. ippu padang leaf ethanol extract had antimicrobial activity against the candida albicans, the dermacoccus nishinomiyaensis, micrococcus luteus, pseudomonas aeruginosa and staphylococcus epidermidis bacteria acknowledgment the authors would like to thank tjut nyak dhien university for their guidance and assistance in conducting this research. references 1. putri, d. d., furwan, m. t., perdana, r. s. klasifikasi penyakit kulit pada manusia menggunakan metode binary decision tree support vector machine (bdtsm). jurnal pengembangan teknologi informasi dan ilmu komputer. 2018; 2(5): 19121920. 2. alioes, y., kartika, a., zain, e. a., azzura, v. uji potensi antijamur candida albicans ekstrak daun gelinggang (cassia alata l.) dibandingkan dengan sediaan daun sirih yang beredar di pasaran secara in vitro. jurnal kimia riset. 2018; 3(2): 1-6. https://doi.org/10.20473/jkr.v3i2.12040 3. bolo, n. r., diamos, m. j. c., su, g. l. s., ocampo, m. a. b., suyom, l. m. isolation, identification, and evaluation of polyethylene glycol and low-density polyethylene-degrading bacteria from payatas dumpsite, quezon city, philippines. philippine journal of health research and development. 2015; 19(1): 50-59. 4. boro, s. e. e., suartha, i. n., sudimartini, l. m. efektivitas ekstrak daun mimba terhadap micrococcus luteus yang diisolasi dari anjing penderita dermatitis kompleks. indonesia medicus veterinus. 2018; 7(5):588-596. https://doi.org/10.24843/bulvet.2019. v11.i01.p05 5. aggraini, d., yulindra, u. g., savira, m., djojosugito, f. a., hidayat, n. prevalensi dan pola sensitivitas, antimikroba multidrug resistant pseudomonas aeruginosa di rsud arifin achmad. majalah kedokteran bandung. 2018; 50(1): 6-12. https://doi.org/10.15395/mkb.v50n1.1150 6. nugrahani, a. r., gunawan, f., khumaidi, a. aktivitas antibakteri ekstrak etanol daun kapas (gossypium barbadense l.) terhadap staphylococcus epidermidis dan propionibacterium acnes. jurnal farmasi udayana. 2020; 9(1): 52-61. https://doi.org/10.24843/jfu.2020.v0 9.i01.p08 7. humaida, r. strategy to handle resistance of antibiotics. j majority. 2014; 3(7): 113-119. 8. kavitha, g., sivakkumar, t., abraham, e. phytochemical screening anf anti-oxidant activity of https://doi.org/10.20473/jkr.v3i2.12040 https://doi.org/10.24843/bulvet.2019.v11.i01.p05 https://doi.org/10.24843/bulvet.2019.v11.i01.p05 https://doi.org/10.15395/mkb.v50n1.1150 https://doi.org/10.24843/jfu.2020.v09.i01.p08 https://doi.org/10.24843/jfu.2020.v09.i01.p08 journal of fundamental and applied pharmaceutical science, 3(1), august 2022 6 ammannia octandra l.f. jk welfare & pharmascope foundation. 2019; 10(3): 2470-2476. https://doi.org/10.26452/ijrps.v10i3.1496 9. mierza, v., rosidah., haro, g., suryanto, d. influence of variant extraction methods (clasical prosedure) for antibacterial activity of rarugadong (dioscorea pyrifolia kunth.) tuber. journal of inovation in applied pharmaceutical science (jiaps). 2019; 4.(1): 1-6. 10. siswati. analisa kadar air dan kadar abu pada simplisia temu giring (curcumae heyneana) dan simplisia kunyit (curcumae domestica) di balai riset standarisasi industri medan. tugas akhir. fakultas farmasi universitas sumatera utara. 2020. 11. trisia, a., philyris, r., toemon, a. n. uji aktivitas antibakteri ekstrak etanol daun kalanduyung (guazuma ulmifolia lam.) terhadap pertumbuhan staphylococcus aureus dengan metode difusi cakram (kirbybauer). anterior jurnal. 2018; 17(2): 136-143. https://doi.org/10.33084/anterior.v17i2.12 12. badaring, d. r., fiqriansyah, m., bahri, a. identifikasi morfologi mikroba pada ruangan water closet jurusan biologi universitas negeri makassar. prosiding seminar nasional biologi fmipa unm. makassar: program studi biologi, universitas negeri makassar. 2020:161-168. 13. surjowardoyo, p., susilorini, t. e., sirait, g. r. b. daya hambat dekok kulit apel manalagi (malus sylvestrs mill.) terhadap pertumbuhan staphylococcus aureus dan pseudomonas sp. penyebab mastitis pada sapi perah. j. ternak tropika. 2015; 16(2): 40-48. https://doi.org/10.21776/ub.jtapro.20 15.016.02.6 14. rijayanti, r. p. uji aktivitas antibakteri ekstrak etanol daun mangga bacang (mangifera feotida l.) terhadap staphylococcus aureus secara in vitro. naskah publikasi. fakultas kedokteran, universitas tanjung pura. 2014. 15. yanti, n., samingan., mudatsir. uji aktivitas antifungi ekstrak etanol gal manjakani (quercus infectoria) terhadap candida abicans. jurnal ilmiah mahasiswa pendidikan biologi. 2016; 1(1): 1-9 . https://doi.org/10.26452/ijrps.v10i3.1496 https://doi.org/10.33084/anterior.v17i2.12 https://doi.org/10.21776/ub.jtapro.2015.016.02.6 https://doi.org/10.21776/ub.jtapro.2015.016.02.6 determining caffeine in fat-burning supplements using thin layer chromatography-densitometry and uv-vis spectrophotometer muhammad luthfi maulana*, muhammad theza ghozali school of pharmacy, faculty of medicine and health science, universitas muhammadiyah yogyakarta, jl brawijaya, tamantirto, kasihan, bantul, yogyakarta 55183. abstract there are so many ways to lose weight, such as consuming fat burner supplements to burn fat faster. a fat burner supplement consists of various ingredients, such as caffeine. fat burner supplements are usually not registered in bpom ri. this study aims to evaluate caffeine in fat burner supplements qualitatively and quantitatively. these supplements were analyzed in the pharmaceutical laboratory in universitas muhammadiyah yogyakarta using thin layer chromatography (tlc)– densitometry and uv–vis spectrophotometer. sample preparation was conducted by separating caffeine from supplements by a separatory funnel with chloroform as an organic solvent. qualitative analysis was carried out by tlc and analyzing the standard spectrum, and the sampling technique was carried out with uv–vis spectrophotometer. the first quantitative analysis used densitometry by measuring the spot on the tlc plate. meanwhile, the second quantitative analysis used uv–vis spectrophotometer by observing absorbency value on samples with λ 273.5 nm. the result of the qualitative test using tlc was analyzed by comparing the rf value of the standard and the sample. the rf value of caffeine standard was 0.63, and every sample showed similar value with caffeine standard, indicating that all samples contain caffeine. the result of the quantitative test with tlc densitometry method revealed 1st sample 5.68 mg/ml, 2nd sample 5.74 mg/ml, 3rd sample 3.43 mg/ml, 4th sample 8.90 mg/ml, and 5th sample 1.88 mg/ml. the qualitative test result was analyzed using the uv–vis spectrophotometer method, and all of the caffeine standard spectra can be read at wavelength 273.5 nm, which means all samples contain caffeine. the second quantitative test result analyzed by using the uv–vis spectrophotometer method showed 1st sample 3.22 mg/g, 2nd sample 4.56 mg/g, 3rd sample 2.23 mg/g, 4th sample 11.22 mg/g, and 5th sample 0.26 mg/g. it can be concluded that all samples (fat burners) contained caffeine. keywords: caffeine; densitometry; fat burner; tlc; uv-vis spectrophotometer data of article received reviewed accepted : : : 6 mar 2020 12 may 2020 5 jun 2020 doi 10.18196/jfaps.010105 type of article: research * corresponding author, e-mail: luthfiiimaulanaa21@gmail.com muhammad luthfi maulana, muhammad theza ghozali | determining caffeine in fat-burning supplements using thin layer chromatography-densitometry and uv-vis spectrophotometer 38 introduction today's modern life currently focuses on healthy lifestyles, whether based on healthy food or exercise. lifestyle is a pattern of life expressed by someone through activity, interest, and opinion.1 it is carried out by paying attention to lifestyle related to fitness or individual bodyweight. a person will usually pay attention to his body weight in daily life. people do exercises to regulate their body weight, such as fitness or taking fatburned supplements. however, supplements are not necessary when someone wants a healthy fit body. a person can simply adopt a healthy lifestyle and adjust their diet to get their body fit. therefore, before deciding to take supplements, being informative about the products is a crucial thing to do. supplements are products intended to complete the nutritional needs of food, containing one or more ingredients in the form of vitamins, minerals, amino acids, and other elements having nutritional values and physiological effects in the concentrated amounts.2 an example of supplements used daily is a fat burner. fat-burner supplements are usually used to lose bodyweight in accelerating fat burning. in this supplement, there are a number of various kinds of ingredients, such as caffeine. this substance can mostly be identified in the content of coffee beans, tea leaves, chocolate.3 caffeine in fat-burners usually functions to accelerate fat burning. it is also used to improve performance to be active, suppress hunger, and increase the body's metabolism.4 it is one of the factors that someone uses fat-burning supplements to lose weight instantly.5 however, excessive consumption of caffeine has a number of adverse side effects for the body, such as nervousness, anxiety, insomnia, tremors, nausea, and vomiting. in indonesia, caffeine is one of the chemical substances categorized as psychoactive that has limitations on daily consumption, which is 150 milligrams a day.2 this study aims to identify the levels of caffeine in fatburning supplement products on indonesian markets by using thin layer chromatography – densitometry and uvvis spectrophotometry. the benefit of using this method is that the method is simple, fast, and inexpensive in its application. methods the supplements were analyzed in the pharmaceutical laboratory in the universitas muhammadiyah yogyakarta. the analytical methods used in this study were uv–vis spectrophotometer, and densitometry or tlc scanner (camag tlc scanner 4®), gf 254 silica gel (merck kgaa®), separatory funnel (pyrex®), analytical scales (mettler tolledo®), uv box, stirrer (well spencer®), glass beaker (pyrex®), measuring flask (pyrex®), glass funnel (pyrex®), volume pipette (pyrex®), erlenmeyer (pyrex®), cup, capillary tube (vitrex®), and a stove. meanwhile, materials used included caffeine anhydrous (pt brataco yogyakarta), distilled water (pt brataco yogyakarta), sodium carbonate/na2co3 (pt brataco yogyakarta), ethanol pa (pt brataco), pro-analysis chloroform / chcl3 (pt brataco yogyakarta), and commercial fat burners with brands (hhe, cshd, ur, hhng, fbl-c). they were then labeled 1 – 5. sample preparation a total of 2 grams of a fat burner sample was put into a glass beaker and then dissolved with 100 ml of boiling distilled journal of fundamental and applied pharmaceutical science, 1(1), august 2020 39 water and then filtered. the filtration was added and placed in a separatory funnel and extracted with 25 ml of chloroform in a row four times. the filtration obtained was then collected in a container. the chloroform sample was taken to be injected on a thin layer chromatography (tlc) plate. thin layer chromatography analysis a gf254 silica plate was cut to a size of 10 cm x 3 cm and then bordered 1 cm on the top edge and bottom, marked with a little pencil stroke. the mobile phase used in this study consisted of a mixture of proanalysis chloroform and pro-analysis ethanol by comparison 9: 1.6 preparation of a comparative solution to identify chemical substances using thin layer chromatography was conducted by dissolving 2 mg of caffeine powder in 10 ml of chloroform. the standard relative and sample solution were injected on each tlc plate as much as one using a capillary tube at 1 cm of the bottom. an analysis was then conducted by observing the elution leaking on the tlc plate. the stain on the tlc plate was identified with a 254-nm uv light, and then the stain was marked. the rf value was calculated and then compared to the rf value of the sample and standard for comparison. analysis with densitometry on the tlc plate that has been analyzed containing caffeine, the levels were calculated using a densitometer. the plate was then inserted into the densitometer to be detected with a-254 nm uv light. furthermore, an analysis using linear regression was carried out to obtain the value of the standard curve equation as shown in figure 1 and 2. analysis by uv -vis spectrophotometry in this study, a stock solution was prepared by weighing 100 mg of standardized caffeine and then put into a 1000 ml measuring flask and dissolved with distilled water to obtain a solution with a concentration of 100 ppm. the next step was to determine the maximum wavelength, conducted by taking 5 ml of the stock solution into a 100 ml measuring flask using a pipette, and then dissolved with distilled water to obtain a 5-ppm standard solution. the step was continued by measuring the absorption at wavelengths between 270-300 nm using a uv-vis spectrophotometer. in this study, a calibration curve was created using a series of concentration solutions, with the following concentrations 0; 2.5; 5; 7.5; 10; and 12.5 ppm. the next step was to measure the absorption using a maximum-absorbance uv-vis spectrophotometer, and the distilled water was used as a blank. in terms of samples analyzed by using uv-vis spectrophotometry, chloroform solvent in erlenmeyer was evaporated by utilizing a sublimation method in the cup; thus, the caffeine extract was obtained. the resulting caffeine extract was then put into a 100 ml volumetric flask and dissolved with distilled water to the mark, and then absorbance was measured. figure 1. densitometry value of standard, 1st sample, and 2nd sample muhammad luthfi maulana, muhammad theza ghozali | determining caffeine in fat-burning supplements using thin layer chromatography-densitometry and uv-vis spectrophotometer 40 figure 2. densitometry value of 3rd sample, 4th sample, and 5th sample results and discussion the results of this study showed that many commercial fat-burners contained caffeine. it was obtained from caffeine identification by utilizing thin layer chromatography. the mobile phase in this method was a mixture of chloroform and ethanol with a ratio of 9 : 1.6 this study used these solvents for the mobile phase as caffeine was very soluble in chloroform solvents.7 the identification of tlc was carried out by comparing the rf value of pure caffeine and the samples. furthermore, caffeine was analyzed by using densitometry; thus, the area under curve (auc) and chromatogram could be identified. table 1. rf value of sample no sample code rf value 1 a (pure caffeine) 0.63 2 hhe (1st sample) 0.58 3 cshd (2nd sample) 0.59 4 ur (3rd sample) 0.58 5 hhng (4th sample) 0.55 6 fbl-c (5th sample) 0.65 in determining the levels of each fatburner, a quantitative analysis was performed using densitometry. this study used a tlc densitometry as this analysis method was cheaper and easier to work on.7 table 2. the level of caffeine with tlcdesitometry method sample code auc* concentration (ppm) hhe 25921.4 1137.56 chsd 26158.6 1148.21 ur 15906.5 687.87 hhng 40258.3 1781.30 fbl-c 8961.2 376.02 *auc = area under curve the area obtained was put into the standard curve equation, y = 22.271x + 586.75, and then the concentration of each sample could be identified. each concentration that has been obtained was later converted, and the result of the 1st sample was 5.68 mg/ml; the 2nd was 5.74 mg/ml; the 3rd sample was 3.43 mg/ml; the 4th sample was 8, 90 mg/ml; and the 5th sample was 1.88 mg/ml.8 this study also used uv-vis spectrophotometry in determining caffeine in fat-burners that have been tested. in terms of calculating caffeine, samples were analyzed using uv-vis spectrophotometry, and then absorbance of each sample was obtained. this phase was replicated three times to reduce errors the moment the absorbance was read. the absorbance obtained was entered into the equation y = 0.0457x + 0.0034. furthermore, the concentration of each sample was found and then calculated. table 3. the level of caffeine with uv-vis spectrophotometry method no sample code average concentration 1 a (pure caffeine) 6.443 2 hhe (1st sample) 9.139 3 cshd (2nd sample) 4.472 4 ur (3rd sample) 4.491 5 hhng (4th sample) 5.386 each concentration was later converted, and the results showed as follows: the 1st sample was 3.22 mg/g; the 2nd sample was 4.56 mg/g; the 3rd sample was 2.23 mg/g; the 4th sample was 11, 22 mg/g; and the 5th sample was 0.26 mg/g. journal of fundamental and applied pharmaceutical science, 1(1), august 2020 41 conclusion based on the analysis, all samples (fat burners) contained caffeine with the result showed by tlc – densitometry as follows: the 1st sample was 5.68 mg/ml; the 2nd sample was 5.74 mg/ml; the 3rd sample was 3.43 mg/ml; the 4th sample was 8.90 mg/ml, and the 5th sample was 1.88 mg/ml. meanwhile, uv-vis spectrophotometry showed the results as follows: the 1st sample was 3.22 mg/g; the 2nd sample was 4.56 mg /g, the 3rd sample was 2.23 mg/g; the 4th sample was 11.22 mg/g, and the 5th sample was 0.26 mg/g. references 1. kotler, philip. (2002). manajemen pemasaran; edisi milenium, jilid 1. [versi elektronik]. jakarta: prenhallindo. 2. reyes, c. m., & cornelis, m. c. (2018). caffeine in the diet: country-level consumption and guidelines. nutrients, 10(11). p. 1772. 3. romandhoni, a. n., & arrosyid, m. (2019). penetapan kadar kafein pada teh oolong (camellia sinensis) menggunakan ekstraksi refluk dengan metode titrasi bebas air. cerata jurnal ilmu farmasi, 9(1). pp. 48-56. 4. weinberg, b. a., & bealer, b. (2002). the caffeine advantage: how to sharpen your mind, improve your physical performance, and achieve your goals--the healthy way. london: simon and schuster. 5. arwangga, a. f., asih, i. a. r. a., & sudiarta, i. w. (2016). analisis kandungan kafein pada kopi di desa sesaot narmada menggunakan spektrofotometri uv-vis. jurnal kimia, 10(1). pp. 110-114. 6. bean, anita. mei, (2009). fat burner, pentingkah?, (sport nutrition the complete guide 6th ed.). available at: https://www.apki.or.id/fat-burner perlu-kah/ [accessed: 20 may, 2018]. 7. kemenkes, r. i. (2014). farmakope indonesia edisi v. jakarta: direktorat jendral bina kefarmasian dan alat kesehatan republik indonesia. p. 195. 8. danasrayaningsih, v. s. (2007), penetapan kadar kafein dalam minuman berenergi merek “x” dengan metode spektrofotometri derivattif aplikasi peak-to-peak, skripsi, fakultas farmasi universitas sanata dharma, yogyakarta antioxidant activity evaluation from tomatoes’ nhexane, ethyl asetate, and water fraction with dpph ratna sari dewi*, desy ayu irma permatasari, tatiana siska wardani, muladi putra mahardika department of pharmacy, faculty of health science, universitas duta bangsa, jl. k.h samanhudi no.93, sondakan, laweyan, surakarta, central java 57147, indonesia abstract antioxidants are compounds that can stabilize free radicals in the body. free radicals are highly reactive molecules as they have unpaired electrons to interact with body cell molecules. tomatoes contain flavonoids, saponins, solanine tannins, folic acid, malic acid, citric acid, protein, fat, vitamins, minerals, and histamine, which can be used as antioxidants. this study aims to evaluate the antioxidant activity of n-hexane, ethyl acetate, water fraction, and ethanol extracts of tomatoes and to determine the greatest antioxidant activity between n-hexane, ethyl acetate, water and vitamin c. tomatoes (lycopersicum esculentum mill.) was extracted using the maceration method with ethanol followed by fractionation using nhexane and ethyl acetate solvents. the test of antioxidant activity to dpph radical was conducted on n-hexane, ethyl acetate, water, and vitamin c. the antioxidant activity results, expressed by ic50 value to the n-hexane, ethyl acetate, water fraction of tomatoes fruit, were 4.4603 ppm; 4.0868 ppm; and 4.0527 ppm, respectively. thus, the greatest antioxidant activity was the water fraction. keywords: tomatoes; antioxidant; fractionation; vitamin c; dpph data of article received reviewed accepted : : : 28 oct 2021 29 nov 2021 28 feb 2022 doi 10.18196/jfaps.v2i1.13023 type of article: research introduction tomato is one type of fruit with polyphenolic compounds, carotenoids, and vitamin c, which can act as antioxidants.1 tomatoes contain a carotenoid compound called lycopene.2 atherosclerosis is strongly related to lowdensity lipoprotein (ldl)-cholesterol. unsaturated lipid acid components in the ldl-cholesterol are easy to bind with free radicals and transform into oxidative ldl. it triggers sponge cells, releases reactive oxygen species (ros), and induces oxidative stress. ros can form * corresponding author, e-mail: sariratna339@gmail.com atherosclerosis, plague, lipid oxidation, and endothelial disorder. simultaneously oxidative stress will decrease the immune response to keep the body healthy. it can be avoided through high antioxidant food consumption. the antioxidant is easily obtained from various foods containing vitamin e, vitamin c, polyphenol, and carotenoid, especially lycopene and β-carotene. tomato is one of the abundant and affordable fruit or vegetables. tomato (lycopersicum esculentum mill.) is a species from the solanaceae family. it contains a ratna sari dewi, desy ayu irma permatasari, tatiana siska wardani, & muladi putra mahardika | antioxidant activity evaluation from tomatoes’ n-hexane, ethyl asetate, and water fraction with dpph 87 lot of natural antioxidants, such as vitamin e, vitamin c, and carotenoid. based on the chemical composition, carotenoid is divided into two chemical compound groups, namely, carotene and xanthophyll. the most abundant carotene in tomatoes is lycopene and β-carotene.3 carotene is an antioxidant that can reduce malignant cells of cancer. of all these carotenoid compounds, it turns out that lycopene is relatively more efficient as a singlet oxygen scavenger than other carotenoids (higher than beta-carotene and alphatocopherol).4 singlet oxygen is a nonradical electrophilic ros.5 lycopene is a special carotenoid that is potential to prevent prostate cancer and degenerative cardiovascular disease and anti-aging.6 lycopene is the main carotenoid in tomatoes which is a powerful antioxidant and has received much attention as it is associated with a lycopene-rich diet and reduces the risk of heart disease, cancer and disease in old age.7 aging is a biological process that occurs naturally and affects all living things, including all body organs such as the heart, lungs, brain, kidneys, and skin.8 the community prepares various techniques of tomato processing. some people eat fresh tomatoes directly without any treatment. meanwhile, most indonesian people always cook tomatoes in various processing, such as steaming, boiling and frying. the cooking process techniques are predicted to impact antioxidant amounts inside tomatoes. understanding the antioxidant capacity found in tomato fruits requires studying the antioxidant activity using the dpph method to identify the antioxidant compounds in the tomato sample. the research on samples of fruit tomato leaves (lycopersicon esculentum mill, var. pyriforme alef.) and vegetable tomato leaves (lycopersicon esculentum mill, var. commune bailey.) showed that it has antioxidant activity with ic50 values of 279.482 µg each. / ml and 280.190 µg / ml. it indicated the ethanol extract of fruit tomato leaves (lycopersicon esculentum mill, var. pyriforme alef.) and vegetable tomato leaves (lycopersicon esculentum mill, var. commune bailey.) have very weak activity as antioxidants.9 lycopene can hinder endometrial cancer growth, breast cancer, and lung cancer on cell culture with a higher activity than α and beta-carotene.10 generally, tomatoes have high antioxidants. however, high temperatures can reduce the antioxidant amount.11 the vitamin c was measured using ascorbic acid colorimetric assay kit, and the number of α-tocopherol analyses was performed using an elisa kit for αtocopherol. the antioxidant activities were measured by using 1-1-diphenyl-2pic-1-1-diphenyl-2-pic-rylhydrazyl (dpph) and stoichiometry method.12 dpph provided maximum absorption on 516 nm wavelength and resulted in purple color.13 this method was utilized due to its simple, easy, rapid, sensitive ability and only involved a minimum sample.14 method this research used 14 kg tomatoes (lycopersicum esculentum mill.) obtained from desa sepanjang, tawangmangu, karanganyar. centra java, indonesia and were collected through purposive random sampling. the tomatoes' criteria included ellipse form, red color, and approximately weighted 1,5-2 ons, fresh and peel. tomatoes processed in 6 different ways were then extracted by the same technique. this experimental study was conducted at the laboratory of biochemistry, department of biology, universitas ahmad dahlan. this research used a complete random design with a journal of fundamental and applied pharmaceutical science, 2(2), february 2022 88 post-test only randomized controlled group design. results and discussion the results of data analysis showed that all data were normally distributed and homogeneous. the data were then tested with ic50 (inhibition concentration 50). phytochemicals the below compounds were identified to determine the chemical compounds in tomatoes. phytochemical screening was carried out to determine the secondary metabolites in the extract and powder on tomatoes. secondary metabolites tested qualitatively included alkaloids, flavonoids, saponins, tannins, steroids/triterpenoids. table 1. identification of tomato extract chemical compounds no chemical content simplicia extract observation 1 alkaloids + + yellow-orange, no sediment with dragendrof dark orange with mayer reagent 2 flavonoids + + yellowish orange 3 tannins + + and brown orange with fecl3 reagent brownish orange, white sediment with gelatin 4 saponins + + the foam was formed with a foam test 5 steroids/ triterpenoids yellow with acetic acid anhydrous yellow-brown precipitated with acid fractionation the results of the extraction of tomato fruit samples (lycopersicum esculentum mill.) were then fractionated. fractionation was carried out to separate a class of compounds from other groups based on the difference in polarity of the solvent. n-hexane, ethyl acetate and water were the solvents used in fractionation in this study. table 2. tomato extract of fraction yield extract weight (gram) fraction fraction weight (gram) yield (%) b/b 1.6 n-hexane 0.48 30 1.6 ethyl acetate 0.65 40.625 1.6 water 1.53 95.625 antioxidants activity several methods can determine antioxidant activity. in this study, the method used was antioxidant testing with dpph free radical scavenger. antioxidant activity was characterized by trapping electrons by free radicals, which caused electrons in free radicals to become paired electrons, resulting in a color change from purple to yellow. ratna sari dewi, desy ayu irma permatasari, tatiana siska wardani, & muladi putra mahardika | antioxidant activity evaluation from tomatoes’ n-hexane, ethyl asetate, and water fraction with dpph 89 table 3. the absorbance average value of the test solution test solution concentration (ppm) average absorbance % inhibition fractio n-hexane 1.000 1.250 1.500 1.750 2.000 0.705 0.624 0.546 0.542 0.351 11.209 21.410 33.111 45.661 55.793 ethyl acetate fraction 250 500 750 1.000 1.250 0.676 0.597 0.504 0.415 0.300 14.861 24.811 36.523 47.695 62.216 water fraction 5.000 10.000 15.000 20.000 25.000 0.676 0.590 0.493 0.382 0.324 14.861 25.693 37.909 51.889 59.194 inhibition concentration 50 the results of the measuring absorbance were used to obtain the percentage of inhibition value. the antioxidant activity of the n-hexane fraction, ethyl acetate fraction, and the water fraction of the ethanol extract of tomato fruit can be expressed by ic50 (inhibition concentration 50). ic50 is the sample concentration needed to capture 50% of dpph free radicals during operating time. the data from n-hexane, ethyl acetate, and water fractions were then calculated for the ic50 value using a linear regression equation based on the formula y = a + bx. table 4. inhibition concentration 50 no test solution ic50 (ppm) value 1 n-hexane fraction 4.4603 2 ethyl acetate fraction 4.0868 3 water fraction 4.0527 the ic50 values were grouped into several groups, namely: ic50 values of less than 50 ppm were included in the very strong group, ic50 values of 50 ppm to 100 ppm were in the strong group, ic50 values of 101 ppm to 150 ppm were in the moderate class, ic50 values of more than 150 ppm were in the weak group while the ic50 values of more than 500 ppm were in the inactive group the n-hexane fraction had an ic50 value of 4.4603 ppm, indicating that the n-hexane fraction had the weakest antioxidant activity compared to the antioxidant activity of the ethyl acetate fraction, water fraction, and the vitamin c. meanwhile, hexane had strong antioxidant activity (ic value <50 ppm). ethyl acetate fraction had the greatest antioxidant activity compared to the antioxidant activity of the n-hexane fraction and water fraction with an ic50 value of 4.0868 ppm. it indicated that the ethyl acetate fraction had strong antioxidant activity as it contained flavonoids. it was stated that flavonoid compounds could inhibit oxidation reactions through a radical scavenger mechanism.15 furthermore, the water fraction had an ic50 value of 4.0527 ppm, indicating that the antioxidant activity of the water fraction was greater than the antioxidant activity of the n-hexane fraction due to its journal of fundamental and applied pharmaceutical science, 2(2), february 2022 90 triterpenoid compounds, tannins, saponins, and flavonoids that might capture radicals. conclusion based on the results of this research, it can be concluded that the n-hexane fraction had an antioxidant activity with an ic50 value of 4.4603 ppm; the ethyl acetate fraction had an antioxidant activity with an ic50 value of 4.0868, and the water fraction from the tomato extract had an antioxidant activity with an ic50 of 4.0527 ppm. therefore, the water fraction of the tomato fruit extract had the best antioxidant activity with an ic50 value of 4.0527 ppm. references 1. eveline, siregar, t. m., sanny. studi aktivitas antioksidan pada tomat (lycopersicon esculentum) konvensional dan organik selama penyimpanan. prosiding snst fakultas teknik. 2014;1(1):22-28. 2. mu’nisa, a. analisis kadar likopen dan uji aktivitas antioksidan pada tomat asal sulawesi selatan. jurnal bionatur. 2012 13(1):162-66. 3. sanhia, a. m., pangemanan, d. h. c., engka, j. n. a. gambaran kadar kolesterol low density lipoprotein (ldl) pada masyarakat perokok di pesisir pantai. j. e-biomedik. 2015;3(1):460-465. https://doi.org/10.35790/ebm.3.1.201 5.7425 4. damayanthi, e., kustiyah, l., khalid, m., farizal, h. aktivitas antioksidan bekatul lebih tinggi daripada jus tomat dan penurunan aktivitas antioksidan serum setelah intervensi minuman kaya antioksidan. journal of nutrition and food, 2010;5(3): 205–210. https://doi.org/10.25182/jgp.2010.5.3. 205-210 5. maong, r., rorong, j. a., fatimah, f. aktivitas ekstrak buah tomat (lycopersicum esculentum mill) sebagai penstabil oksigen singlet dalam reaksi fotooksidasi asam linoleat. jurnal mipa. 2016;5(1) 60-64. https://doi.org/10.35799/jm.5.1.2016. 12288 6. fiedor, j. & burda, k. potential role of carotenoids as antioxidants in human health and disease. nutrients. 2014;6(2):466-488. https://doi.org/10.3390/nu6020466 7. ma’sum, j. isnaeni., primaharinastiti, r., annuryanti, f. perbandingan aktivitas antioksidan ekstrak aseton tomat segar dan pasta tomat terhadap 1,1-diphenyl-2 picrylhidrazyl (dpph). jurnal farmasi dan ilmu kefarmasian indonesia. 2014;1(2):59-62. 8. surbakti, e. s. b., & berawi, k. n. tomat (lycopersicum esculentum mill.) sebagai anti penuaan kulit. majority. 2016;5(3):73-78. 9. pratama, m., baits, m. & yaqin, r. n. aktivitas antioksidan ekstrak etanol daun tomat buah (lycopersicon esculentum mill, var. pyriforme alef) dan daun tomat sayur (lycopersicon esculentum mill, var. commune bailey) dengan metode dpph (1,1diphenyl-2picryl hydrazil). 2015;2(1):76-82. https://doi.org/10.33096/jffi.v2i1.183 10. yuyun, y., seprililianti, yusriadi. pemanfaatan likopen tomat https://doi.org/10.35790/ebm.3.1.2015.7425 https://doi.org/10.35790/ebm.3.1.2015.7425 https://doi.org/10.25182/jgp.2010.5.3.205-210 https://doi.org/10.25182/jgp.2010.5.3.205-210 https://doi.org/10.35799/jm.5.1.2016.12288 https://doi.org/10.35799/jm.5.1.2016.12288 https://doi.org/10.3390/nu6020466 https://doi.org/10.33096/jffi.v2i1.183 ratna sari dewi, desy ayu irma permatasari, tatiana siska wardani, & muladi putra mahardika | antioxidant activity evaluation from tomatoes’ n-hexane, ethyl asetate, and water fraction with dpph 91 (lycopersicum esculentum mill) dalam sediaan soft candy sebagai suplemen antioksidan. jurnal pharmascience, 2016;3(2):95–106. http://dx.doi.org/10.20527/jps.v3i2.5744 11. suhartatik, n., & risnantoko, a. m. w., aktivitas antioksidan kurma tomat (solanum lycopersicum) dengan variasi jenis jahe dan lama pengeringan. jurnal teknologi dan industri pangan. 2019;4(1): 1–6. https://doi.org/10.33061/jitipari.v4i1.3012 12. iswari, r. s., susanti, r. antioxidant activity from various tomato processing. journal of biology & biology education. biosaintifika. 2016;8(1):129-134. https://doi.org/10.15294/biosaintifika. v8i1.4722 13. agustina, l., yulianti, m., shoviantari, f., sabban, i. f. formulation and evaluation of herbal liquid soap containing tomatoes (solanum lycopersicum l.) as antioxidants. jurnal wiyata. 2017;4(2);104-110. 14. setyawati, e., rahayu, c. k., haryanto, e. korelasi kadar likopen dengan aktivitas antioksidan pada buah semangka (citrullus lanatus) dan tomat (lycopersicum esculentum). jurnal analis kesehatan sains. 2019;8(2):717-724. 15. mariod, a. a., mustafa, m. m., nour, a., abdalla, m. a., salama, s. m., wajeeh, n. s. a. antioxidant activity and acute toxicity of two lagenaria siceraria (molina) standl. varieties from sudan. acta agric. slov. 2020;116(2), 261–271. https://doi.org/10.14720/aas.2020.116 .2.1031 http://dx.doi.org/10.20527/jps.v3i2.5744 https://doi.org/10.33061/jitipari.v4i1.3012 https://doi.org/10.15294/biosaintifika.v8i1.4722 https://doi.org/10.15294/biosaintifika.v8i1.4722 https://doi.org/10.14720/aas.2020.116.2.1031 https://doi.org/10.14720/aas.2020.116.2.1031 49 formula optimization of feminine liquid soap from garlic (allium sativum linn) extract ingenida hadning*, astri yunika school of pharmacy, faculty of medicine and health science, universitas muhammadiyah yogyakarta, jl brawijaya, tamantirto, kasihan, bantul, yogyakarta 55183. abstract leucorrhoea is a condition frequently experienced by women. if it is not appropriately treated, it can become a more severe problem. leucorrhoea is caused by “candida albicans” fungus infection. garlic (allium sativum linn) has antibacterial and antifungal activity, where garlic can inhibit the growth of the candida albicans. this study aims to determine the optimal formulation of feminine liquid soap from garlic extract (allium sativum linn) with good physical quality; thus, garlic in treating leucorrhoea can be practically easier. optimization of liquid soap formulation used carbopol and sodium lauryl sulfate with various concentration variations. the method included organoleptic observation, ph, and specific gravity for onemonth storage to obtain a good physical quality of liquid soap. the result showed that the formula with 10% sodium lauryl sulfate met the physical quality test requirements of feminine liquid soap; thus, the formulation of feminine liquid soap with 10% sodium lauryl sulfate was optimal. keywords: allium sativum linn; candida albicans; feminine liquid soap; garlic extract; leucorrhoea; physical evaluation data of article received reviewed accepted : : : 20 oct 2020 22 dec 2020 23 feb 2021 doi 10.18196/jfaps.v1i2.10295 type of article: research introduction leucorrhoea is a symptom of vaginal discharge other than menstrual blood, indicating a disease of the female reproductive organs.1 leucorrhoea can be caused by the infection of chlamydia trachomatis bacteria2, candida sp fungus3, trichomonas vaginalis parasite3, herpes4 virus, and hormonal balance disorders.5 candida albicans is part of the normal human flora that occupies the mucous membranes of the mouth, vagina, and intestinal tract.6 in pathogenic conditions, this fungus can proliferate and can cause fermentation. excessive consumption of antibiotics, steroids, and stamina * corresponding author, e-mail: ingenida.hadning@umy.ac.id enhancing drugs can increase the risk of vaginal discharge.7 there are many traditional medicines to treat vaginal discharge, and one of them is to use garlic (allium sativum linn). the part of garlic that is believed to have antifungal and antibacterial properties is the essential oil. the content of allyl sulfide or allicin (c3h5-s-sc3h5) in these essential oils can damage cell walls and inhibit bacterial protein synthesis.8 it has been proven in a study conducted by othman et al. (2008)9 regarding the effectiveness of garlic extract as an antibiotic in candida albicans species. the study stated that the garlic extract was effective ingenida hadning , astri yunika| formula optimization of feminine liquid soap from garlic (allium sativum linn) extract 50 as an anti-fungal for candida albicans species at a concentration of 20 mg/ml. along with the modernization of the medical world, treatments with the backto-nature method are still widely used. this method uses natural ingredients processed and developed into various kinds of traditional medicines with minimal side effects. this study aims to identify a feminine liquid soap from garlic extract (allium sativum linn) with good physical quality. method this study is experimental laboratory research in optimizing the formula for feminine liquid soap from garlic (allium sativum linn) extract. this study was conducted at the research laboratory of the faculty of medicine and health sciences, universitas muhammadiyah yogyakarta, for 1 month. research tools and materials the tools used in this study were 10-ml test tubes (pyrex), 4-ml test tubes (pyrex), tube ravls, erlenmeyer tubes (pyrex), volume pipettes (pyrex), analytical scales (casbee), blender (philips), filter papers, centrifuge, water bath, vortex, pycnometer (pyrex), and ph meter (mettler toledo). the materials used in this study were garlic (allium sativum linn) obtained from the traditional gamping market, tamantirto village, kasihan district, bantul regency, yogyakarta province, aqua dest, citric acid (brataco), rose oil perfume, sodium lauryl sulfate (brataco), methylparaben (brataco), propylparaben (brataco), propylene glycol (brataco) obtained from the pharmaceutical technology laboratory of universitas muhammadiyah yogyakarta, disodium hydrogen phosphate (brataco), and carbopol 980 (brataco) obtained from the technology laboratory ahmad dahlan university pharmacy. determination determination was carried out at the department of pharmaceutical biology, faculty of pharmacy, gadjah mada university by comparing the plants used in the study with a reference book, “flora of java (spermatophytes only)”.10 making garlic extract garlic extract was made with a solvent, aqua dest. one gram of garlic was mixed with 10 ml of aqua dest, then blended using a domestic blender for 1 minute so that the ingredients were smoother and easier to extract. the mixture was vortexed for 10 minutes and allowed to stand at room temperature for 30 minutes. the use of a vortex was to separate the less refined garlic extract. furthermore, the extract was centrifuged at 3500 rpm for 10 minutes to obtain a filtrate containing garlic extract, and the filtrate was filtered with filter paper to obtain a purer extract. the extract was placed in a water bath at 600ºc for 15 minutes so that the allicin compound was more soluble with water. the 600ºc temperature did not damage the allicin compound and was re-vortexed for 1 minute. the mixture was again centrifuged to separate the water produced from the garlic. the garlic extract obtained as a formulation was 10 ml. optimization of liquid soap formulations (vaginal douche) the formula development in this study used ingredients available and often used for liquid soap preparations in general. this study used carbopol 980 and sodium lauryl sulfate. the use of sodium lauryl journal of fundamental and applied pharmaceutical science, 1(2), february 2021 51 sulfate and carbopol 980 in this study was because the ingredients were easy to obtain and were more widely used in soap preparations than esaflor and viscolam. viscolam is a material that contains sodium polyacryladdimetyl taurate used as an emulgator and to increase the viscosity of the water phase.11 esaflor hm22 was used as a thickener in cosmetic preparations and as a stabilizer for oil-inwater emulsions.12 the pharmaceutical preparation of liquid soap with water as the primary solvent was a good medium to grow microbes quickly. the preservative used in this process was a combination of methylparaben and propylparaben. after obtaining the garlic extract, the soap’s pharmaceutical formulation would be made. first, disodium hydrogen phosphate was dissolved with water at ± 400ºc; thus, the sodium salt could dissolve completely. if the crystalline powder was not completely dissolved in cold water, disodium hydrogen phosphate was used as a buffering agent. after that, the sodium lauryl sulfate or carbopol 980 was put into a solution of disodium hydrogen phosphate, and was let to expand (± 15 minutes), then stirred homogeneously. carbopol 980 was dissolved with aqua dest, which had been heated to ± 600ºc. after the sodium lauryl sulfate and carbopol 980 expanded or were homogeneous in the disodium hydrogen phosphate solution, the garlic extract was mixed and homogenized. the citric acid solution was added until a suitable ph was obtained. the ph of the skin ranges from 5.0-6.5.13 then, propylparaben and methylparaben were added as preservatives. before being mixed into the formula, methylparaben and propylparaben were dissolved in 95% alcohol as they were insoluble in water. furthermore, propylene glycol was added as a cosolvent so that it can be thoroughly mixed in the preparation. the final stage was the addition of 100 ml aqua dest and the addition of oleum rose as a fragrance. table 1. formula optimization of garlic extract liquid soap formula f1 f2 f3 f4 f5 f6 garlic extract 1% 1% 1% 1% 1% 1% sodium hydrogen phosphate 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% na lauril sulfate 1% 5% 10% carbopol 980 0.1% 0.3% 0.5% propylene glycol 3% 3% 3% 3% 3% 3% methylparabens 0.18% 0.18% 0.18% 0.18% 0.18% 0.18% propylparabens 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% citric acid 5% 5% 5% 5% 5% 5% oleum rose 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% aquadest ad 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml explanation: f1: liquid soap formula with na lauryl sulfate 1% b/v f2: liquid soap formula with 5% na lauryl sulfate b/v f3: liquid soap formula with na lauryl sulfate 10% b/v f4: liquid soap formula with carbopol 980 0.1% b/v f5: liquid soap formula with carbopol 980 0.3% b/v f6: liquid soap formula with carbopol 980 0.5% b/v ingenida hadning , astri yunika| formula optimization of feminine liquid soap from garlic (allium sativum linn) extract 52 physical evaluation of liquid soap the physical evaluation was to test the stability and safety level of liquid soap preparations in a preclinical manner. the physical evaluation included organoleptic observations, ph measurements, and specific gravity measurements. organoleptic observations were carried out based on observations of color, odor, and liquid soap dosage form for 4 weeks. ph measurements were carried out using a ph meter; the ph value should be 5.06.5.13 ph measurements needed to be repeated to avoid mistakes. density measurements were carried out using a pycnometer and were carried out at a room temperature of 250c. density measurements also needed to be repeated to avoid mistakes. results and discussion organoleptic observation organoleptic observation showed that the formula with the sodium lauryl sulfate produced clear liquid soap, not too viscous, and foamy. meanwhile, the formula with the carbopol produced a slightly turbid, viscous liquid and not foamy. the results of organoleptic observations of liquid soap are shown in table 2. table 2. organoleptic observation result formula description organoleptic week 1 2 3 4 f1 shape lvl lvl lvl lvl color c c c c smell g g g g foam + + + + f2 shape lvl lvl lvl lvl color c c c c smell g g g g foam ++ ++ ++ ++ f3 shape lvl lvl lvl lvl color b b b b smell g g g g foam +++ +++ +++ +++ f4 shape vl vl vl vl color t t t t smell g g g g foam f5 shape vl vl vl vl color t t t t smell g g g g foam f6 shape vl vl vl vl color t t t t smell g g g g foam explanation: lvl : less viscous liquid vl : viscous liquid c : clear t : turbid g : garlic +/: foam intensity journal of fundamental and applied pharmaceutical science, 1(2), february 2021 53 the preparation of liquid soap formula containing sodium lauryl sulfate produced clear, not-too-thick, and foamy liquid soap. the higher the sodium lauryl sulfate concentration was, the foamier the soap would be. it was because sodium lauryl sulfate functioned as a detergent or foaming agent due to the saponification reaction. the saponification reaction occurred due to long hydrogen chain bonds (c12-c18) to form an alkaline carboxylate salt (rcoo-na). figure 1. chemical structure of sodium lauryl sulfate whereas in the liquid soap preparation formula containing carbopol, the soap was turbid, thick, and non-foaming. the higher the carbopol concentration was, the thicker the liquid soap preparation would be. carbopol did not have a hydrogen chain; thus, there was no saponification reaction. figure 2. chemical structure of carbopol ph observation the result of ph observation was that there was no significant change during the storage period, where the results were still in the desired ph range (5.0-6.5). the results of observing the ph of the preparations are shown in table 3. based on the results of the ph test, it shows that all formulas meet the requirements. table 3. the result of ph observation formula average ph day(during storage time) 1 3 6 9 12 15 18 21 24 27 f1 5.42 5.43 5.44 5.44 5.44 5.44 5.45 5.45 5.45 5.46 f2 5.43 5.52 5.52 5.53 5.54 5.54 5.54 5.54 5.55 5.55 f3 5.44 5.44 5.46 5.47 5.47 5.47 5.47 5.47 5.47 5.47 f4 5.53 5.53 5.53 5.54 5.54 5.55 5.55 5.55 5.57 5.57 f5 5.56 5.57 5.58 5.58 5.58 5.58 5.59 5.59 5.59 5.62 f6 5.53 5.53 5.54 5.54 5.55 5.56 5.56 5.57 5.57 5.58 comparison 4.5 density measurement density observation during storage time showed no significant changes. however, there were differences in density at each increase in the concentrations. according to the indonesian national standard test, the density value specified for liquid soap ranges from 1 1.10 gr/ml so that the soap can dissolve easily in water and does not leave residues on the skin surface to prevent environmental pollution.14 the result of density measurement of the liquid soap preparations is shown in table 4. the density measurement test results show that all formulas, both using sodium lauryl sulfate and carbopol, do not meet the requirements. ingenida hadning , astri yunika| formula optimization of feminine liquid soap from garlic (allium sativum linn) extract 54 table 4. density measurement results formula density (gram / ml) on day 1 3 6 9 12 15 18 21 24 27 f1 0.962 0.968 0.970 0.967 0.969 0.970 0.970 0.970 0.970 0.970 f2 0.968 0.969 0.968 0.970 0.970 0.971 0.970 0.970 0.971 0.971 f3 0.972 0.972 0.974 0.972 0.973 0.971 0.975 0.974 0.971 0.971 f4 0.967 0.969 0.970 0.970 0.970 0.970 0.973 0.972 0.971 0.971 f5 0.969 0.965 0.965 0.970 0.970 0.970 0.972 0.972 0.973 0.973 f6 0.976 0.974 0.974 0.974 0.973 0.975 0.976 0.974 0.973 0.970 comparis on 0.99 conclusion based on the physical quality test that has been carried out, it can be concluded that the formulation with a 10% concentration of sodium lauryl sulfate produced feminine liquid soap from garlic extract with good physical quality as it had met the requirements. acknowledgment this research was supported by universitas muhammadiyah yogyakarta. conflict of interest the author declares that there is no competing conflicts of interest. references 1. mansjoer, a. (2001). kapita selekta kedokteran. jakarta: media aesculapius. 2. sianturi, m. h. r. (2002). keputihan suatu kenyataan. jakarta: ui. 3. dalimartha, s. (2007). tumbuhan obat mengatasi keputihan. jakarta: puspaswara. 4. kesetyaningsih, t. w. (2003). prevalensi trikhomonasis pada leukhore dan faktor-faktor resiko yang berhubungan dengan kondisi keluarga. mutiara medika, 3(2). 5. havler, n. f., & moore, j. g. (2001). esensial obstetri dan ginekologi. jakarta: hipokrates. 6. haskel, r., & gayford, j. s. (1990). penyakit mulut (terj), ed. 2, ecg, jakarta, pp. 56-61. 7. sutarmi, r. (2006). taklukan penyakit dengan vco. depok: penebar swadaya. 8. rustama, m. m., rahayuningsih, s. r., kusmoro, j., & safitri, r. (2005). uji aktivitas antibakteri dari ekstrak air dan etanol bawang putih (allium sativum l.) terhadap bakteri gram negatif dan gram positif. biotika, 2, pp. 1-8. 9. fei, l. c., chen, p. y. v., ahmad, z., othman, f., & pei, c. p. (2008). antifungal properties of allium sativum extracts on candida species. journal of medicinal plants, 9(1). 10. backer, c. a & bakhuizen van den brink. jr, r. c. (1968). flora of java. vol. i, ii & iii. n.v.p. noordhoffgroningen-the netherlands. 11. bakhri, a. s. (2011). pengaruh emulgator november dan viscolam terhadap kestabilan fisik krim [skripsi]. makasar: universitas hassanudin makasar. 12. marsala, v. (2012). esaflor hm22. technical bulletin. https://asia.incosmetics.com/ 13. wasitaatmadja, s. (1997). penuntun ilmu kosmetik medik. jakarta: universitas indonesia press. https://asia.in-cosmetics.com/ https://asia.in-cosmetics.com/ journal of fundamental and applied pharmaceutical science, 1(2), february 2021 55 14. muhammad, a. (2012). studi pembuatan sabun cair mangrove pada komunitas wanita pesisir griya karya tiara kusuma rungkut surabaya [skripsi]. malang: universitas brawijaya malang. ethanol and methanol extract of analgesic activities of ganitri leaves (elaeocarpus ganitrus roxb) for in vivo naelaz zukhruf wakhidatul kiromah*; chondrosuro miyarso; yayu krisdiyanti departement of pharmacy, sekolah tinggi ilmu kesehatan muhammadiyah gombong, jl. yos sudarso no. 461, gombong, kebumen, jawa tengah 5441. abstract pain is a feeling of discomfort caused by intense or destructive stimuli, which can affect your daily routine if left untreated. pain can be treated with an analgesic. one of the plants that are considered to have analgesic effects is the leaves of the ganitri. this research aims to determine ethanol and methanol extracts' impact on decreasing analgesic activity and percent protection. the study began with collecting and processing the leaves of the ganitri into the ethanol and methanol extracts using the maceration method. the research was continued with in vivo analgesic activity testing of 24 mice induced by pain using 1% acetic acid. the induced mice were divided into eight treatment groups, where the mice in the first group served as a negative control group. in that group, they were given cmc at a dose of 0.5%. the second group was positive control, given mefenamic acid at a dose of 500 mg/kg bw. in contrast, the third until eighth groups were given ethanol and methanolic extracts of the ganitri leaves with consecutive doses of 100 mg/kg bw, 200 mg/kg bw dan 400 mg/kg bw. parameters measuring the effectiveness of the extracts used in this study included the amount of stretching, the percentage of analgesic power, and analgesic effectiveness. the results showed that the ethanolic and methanolic extract had the highest percentage of analgesic power at 400 mg/kg bw amounted to 91.3% and 88.3%. furthermore, based on the statistical analysis results using anova, it was found that the ethanol and methanolic extracts of the leaves of the ganitri dosage of 400 mg/kg bw had analgesic activity close to 500 mg/kg bw of mefenamic acid. keywords: acetic acid; analgetic; ganitri leaves; mice; mefenamic acid data of article received reviewed accepted : : : 29 june 2021 20 july 2021 28 aug 2021 doi 10.18196/jfaps.v2i1.12159 type of article: research introduction indonesia is rich in natural resources that can potentially become medicine precursors. more than 20.000 types of medicinal plants are scattered all around * corresponding author, e-mail: naela.zukhruf18@stikesmuhgombong.ac.id indonesia. about 1.000 plants have been identified, and only 300 have been used in traditional medicine.1 pain is one of the general problems that occur in society. it is a primary factor that naelaz zukhruf wakhidatul kiromah, chondrosuro miyarso, &yayu krisdiyanti | ethanol and methanol extract of analgesic activities of ganitri leaves (elaeocarpus ganitrus roxb) for in vivo 54 people come to see a doctor since pain disturbs sufferers' social function and life quality. pain is defined as sensory and emotional experience connected with tissue dysfunction (merskey and bogduk, 1994; salman, saputri and mustika, 2021). compounds in therapeutic dosage slow down or suppress pain without having general anesthesia, called analgesic. nonsteroidal anti-inflammatory drugs (nsaids) are commonly used to treat pain, analgesic, and inflammatory conditions.2 however, they have common side effects like ulcers, bleeding, and renal disorders.3 in recent years, study on herbal plants has been conducted concerning efficiency and side effects of chemical medicine.4 on the other hand, natural resources have advantages such as practical efficacy, good tolerance, fewer side effects, and allergy.5 one of the traditional plants that can be used as medicine is ganitri (elaeocarpus ganitrus roxb). ganitri contains alkaloids, flavonoids, polyphenol, tannins, saponins, and fatty acid 6. it is effectively used in the treatment of inflammation and analgesic.7 research by nain, garg, and dhahiya (2012) showed an analgesic of ganitri leaf aqueous extract at doses 100 mg/kg bw. based on the above findings, analgesic activities were evaluated in the present study. the purpose of this research is to see if there is an analgesic activity of ganitri leaf (elaeocarpus ganitrus roxb) ethanol and methanol extracts in mice (mus musculus). method preparation of extracts in this study, the plants utilized were the leaves. the leaves were collected from karanggayam, kebumen in central java. the sample was cleaned, cut into the stalk, and went through the air-dried at 250c using an oven. dried simplicial was then mashed with a blender to create powder simplicia and weight in the dried powder. simplicia powder was later covered in a closed container to avoid sunlight and moisture. this research used maceration as an extraction method using ethanol and methanol solvent. two hundred grams of dried powder simplicial were put into maceration container, and ethanol 70% and methanol 70% were added and left for 3 days. the mixture was clarified by filtration using whatman’s filter paper. maceration of ethanol 70% and methanol 70% were collected and concentrated using a rotary evaporator until achieving thick extract. after that, it was weightedin and placed in a closed glass container called ethanol extract of ganitri leaf (eeg) and methanol extract of ganitri leaf (meg). phytochemical screening various phytochemical tests were performed to determine alkaloids, flavonoids, tannins, and saponins in extracts. analgesic activity of ethanol and methanol extracts in mice experimental animals. male swiss mice with weights ranging from 20-30 g were used in this study. animal ethics committee approval was obtained for an animal experiment (registration number 022101002). the animals were maintained under environmental conditions and had free access to a standard diet and freshwater ad libitum. they were housed in animal cages in an air condition at 25 ± 2∘ c with 12 hours of light and dark conditions. prior to the commencement of the experiments, the animals were fasted journal of fundamental and applied pharmaceutical science, 2(1), august 2021 55 for twelve hours but still had access to water. making of na-cmc 0.5%. the researchers created na-cmc by using the following dosage, which was 0.5%. first, weight 0.5 g of na-cmc, then sprinkled at 100 ml of hot water, stirred vigorously inside mortar for homogeneous until the concentration of na-cmc was 0.5%. making of 1% of acetic acid. 1 ml of concentrated acetic acid was put into beaker glass. aquadest was added slowly until it reached volume 100 ml. it was then put into a vial then covered with aluminum foil dan sterilized in autoclave with 1210c for 15 minutes. 0.3 ml acetic acid was inducted into mice with intraperitoneal.9 testing ganitri leaves’ analgesic activity. twenty fours male mice were divided into eight treatment groups. group i, as the negative control, was given cmc-na. as the positive control, group ii was given mefenamic acid with 500 mg/kg bw doses. group iii-v was given ganitri leaf ethanolic extract with doses of 100; 200, and 400 mg/kgbw, while group viviii was given ganitri leaf methanolic extract with three doses level respectively 100; 200, and 400 mg/kgbw. all of the treatments were given orally, and then after 30 minutes, they were given acetic acid of 1% doses of 50 mg/kg bw intraperitoneal (i.p). the data of the number of writhes obtained from the analgesic test result was analyzed by counting the protection percentage using the following equation: 10 . % protection = (100-(p/kx100))% note: p = cumulative number of tested animal writhes after given test compound k = median of the cumulative number of negative control tested animal writhes after given test compound statistical analysis the result was analyzed using the shapiro wilk test to obtain the number of data distribution. according to the test, it was seen that every group has a normal distribution (p>0.05). afterward, the variant test was conducted, and it resulted in a probability value as much as 0.405 (p>0.05), which showed that the tested data variant was the same. it was continued with a one-way anova test with a 95% trust level, which obtained a probability value as much as 0.000 (p.0.05), which showed that at least the total number of writhes was significant in the two groups. afterward, post hoc analysis was conducted using gameshowell or lsd test. results and discussion this research used ganitri leaves powder in the form of ethanolic and methanolic extract. plant determination was carried out in the plant systematics laboratory of gadjah mada university. based on the determination results, the tested plant was elaeocarpus ganitrus roxb. phytochemical screening of both ethanol and methanol extracts showed flavonoids, tannins, and alkaloids, as presented in table 1. table 1. phytochemical screening of ethanol and methanol extracts (+) and (-) signified presents and absent, respectively test ethanol methanol flavonoids + + tannins + + alkaloids + + saponin naelaz zukhruf wakhidatul kiromah, chondrosuro miyarso, &yayu krisdiyanti | ethanol and methanol extract of analgesic activities of ganitri leaves (elaeocarpus ganitrus roxb) for in vivo 56 analgesic activity test in this research used the chemical stimulation method using acetic acid 1% as the pain induction compound. acetic acid triggers the release of arachidonate from phospholipid tissue. cox enzyme will change arachidonate acid into prostaglandin, which will stimulate inflammation and pain. the mice showed pain response with writhes.11 figure 1. average graphic of mice writhes numbers based on figure one, the highest cumulative number of writhes is shown by the negative control. ganitri leaf extracts were more likely to decrease writhes compared to the negative control. it showed that at negative control of suspense given by na-cmc 0.5% did not have an inhibition rate towards writhes or analgesic rate. mefenamic acid suspense had cumulative of writhes number smaller than eeg at 100 and 200 mg/kg bw and all doses of meg; thus, the effectiveness of ganitri leaf extract in decreasing the number of writhes was eeg of 400 mg/kg bw dosage, indicating the effect was almost similar to mefenamic acid suspense of 500 mg. the number of writhes and protection percentage as well as eeg and meg are presented in table 2. the ministry of health of indonesia (1991) stated that there was an analgesic activity to the chemical stimulation method shown by the reduction of writhing ≥ 50% compared to the negative control groups. the mean of writhes of positive control mefenamic acid was significantly different compared to negative control cmc-na. it showed that mefenamic acid could give analgesic activity to mice induced with acetic acid with percentage inhibition of pain as 88.3%. 0 5 10 15 20 25 30 35 40 negative control positive control 100 mg/kg bw 200 mg/kg bw 400 mg/kg bw average of mice writhes numbers eeg meg journal of fundamental and applied pharmaceutical science, 2(1), august 2021 57 tabel 2. writhes and ganitri leaf protection percentage on mice test group writhes protection percentage (%) negative control (cmc-na) 34.15 0 positive control (mefenamic acid) 3.95 88.3 eeg 100 mg/kgbw 5.85 82.8 eeg 200 mg/kgbw 4.15 87.8 eeg 400 mg/kgbw 2.95 91.3 meg 100 mg/kgbw 9.85 71.2 meg 200 mg/kgbw 6.2 82.5 meg 400 mg/kgbw 4.4 87.2 in the treatment, the mean of writhes of ethanolic and methanolic extract of ganitri leaves with doses of 100; 200 and 400 mg/kgbw were significantly different (p<0.05) compared to the negative group of cmc-na as shown in table 2. it indicated that the three levels of extract doses could reduce writhing on mice induced with acetic acid. according to the ministry of health of indonesia (1991), all doses of eeg and meg showed analgesic activity since they showed percentage inhibition of pain to > 50% presented in table 2. this result is similar to the research reported by nain, garg, and dhahiya (2012), revealing that ganitri leaf aqueous extract had analgesic activity, indicating that organic extract both ethanol and methanol could attract compounds that gave analgesic activity. all in all, ganitri leaf extracts contain flavonoids that can act as an analgesic with cyclooxygenase enzyme by reducing the production of prostaglandins with arachidonic acid. furthermore, flavonoids also block out cytokines, free radicals, and enzymes in inflammation.13 conclusion the present study showed a significant analgesic effect of both ethanol and methanol extract at 100, 200, and 400 mg/kgbw of ganitri leaves; thus, it had analgesic activity towards acetic acidinduced mice. acknowledgments the authors would like to thank the gombong muhammadiyah university for guidance and financial assistance in conducting the research work. conflict of interest the authors have no conflict of interest references 1. mustika, i. (2021). ethanol extract analgesic activities of mahkota dewa (phaleria macrocarpa (scheff.) boerl) leaves for in vivo. j pharm sci. 4(1), pp.12-20. https://doi.org/10.36490/journaljps.com.v4i1.57 2. conforti, f., sosa, s., marrelli, m., menichini, f., statti, g. a., uzunov, d., tubaro, a., & menichini, f. (2009). the protective ability of mediterranean dietary plants against the oxidative damage: the role of radical oxygen species in inflammation and the polyphenol, flavonoid and sterol contents. food chem. 112(3), pp. 587-594. https://doi.org/10.36490/journal-jps.com.v4i1.57 https://doi.org/10.36490/journal-jps.com.v4i1.57 naelaz zukhruf wakhidatul kiromah, chondrosuro miyarso, &yayu krisdiyanti | ethanol and methanol extract of analgesic activities of ganitri leaves (elaeocarpus ganitrus roxb) for in vivo 58 3. saha, s., guria, t., singha, t., maity, t. k. (2013) evaluation of analgesic and anti-inflammatory activity of chloroform and methanol extracts of centella asiatica linn. isrn pharmacol. published online. 4. ripa, f. a., dash, p. r., faruk, m. o. (2015). preliminary report on antihyperglicemic and analgesic properties of methanol extract of brassica oleraceae l. var. italica sprouts. 3(5), pp. 121-125. 5. hasan, m. y., mahamud, r. a., rahman, s., ahmad, i., & rahmatullah, m. a. (2015). preliminary report on antihyperglicemic and analgesic properties of methanol extract of brassica oleraceae l. var. italica sprouts. world j pharm pharm sci. 4(9), pp. 225-234. 6. kumar, g., karthik, l., bhaskara, rao kv. (2014). a review on medicinal properties of elaeocarpus ganitrus roxb.ex g. don. (elaeocarpaceae). res j pharm technol. 7(10), pp. 11841186. 7. dubey, g. a. (2018). effect of extract of rudraksha (elaeocarpus ganitrus) on parkinson’s disease and depression. world j pharm res. 7(12), pp. 937-947. https://doi.org/10.20959/wjpr20181212697 8. nain, j., garg, k., dhahiya, s. (2012). analgesic and anti-inflammatory activity of elaeocarpus sphaericus leaf extract. int j pharm pharm sci. 4(1), pp. 379-381. 9. tatiya, a. u., saluja, a. k., kalaskar, m. g., surana, s. j., patil, p. h. (2017). evaluation of analgesic and anti_inflammatory activity of bridelia retusa (spreng) bark. j int complement med. 30, pp. 1-11. https://doi.org/10.1016/j.jtcme.2016.1 2.009 10. henny anggraeni f. (2010). uji analgetik ekstrak etanolik daun sambiloto (andrographis paniculata nees) padamencit betina swiss dengan metode rangsang kimia. published online, p. 210. 11. muhammad, n. (2014). in vivo models for management of pain. sci res. 5(1), pp. 92-96. https://doi.org/10.4236/pp.2014.5101 4 12. the ministry of health of indonesia. (1991). in: yayasan pengembangan obat bahan alam phyto medikapedoman pengujian dan pengembangan fitofarmaka, penapisan farmakologi, pengujian fitokimia dan pengujian klinik. departemen kesehatan republik indonesia. 13. sasongko, h.sugiyarto., yeni f, et al. (2016). analgesic activity of ethanolic extracts of kaarika leaves (carica pubescens) secara in vivo. j pharm sci clin res. 1, pp. 83-89. http://dx.doi.org/10.20959/wjpr201812-12697 http://dx.doi.org/10.20959/wjpr201812-12697 https://doi.org/10.1016/j.jtcme.2016.12.009 https://doi.org/10.1016/j.jtcme.2016.12.009 http://dx.doi.org/10.4236/pp.2014.51014 http://dx.doi.org/10.4236/pp.2014.51014 formulation and evaluation of body scrub using flour-based fruits of indramayu variety of cengkir mango (mangifera indica l) ismanurrahman hadi, dadan hidayattullah*, ade irawan departement of pharmacy, stikes muhammadiyah cirebon, jl. kalitanjung no.14 18 a, harjamukti, harjamukti, cirebon city, west java 45143, indonesia abstract the cengkir mango (mangifera indica l) is a variety of mango known in the northern part of west java and widely used as food, beverage, and natural-based pharmaceutical product. this fruit is potentially used as a body scrub ingredient due to its richness in polyphenols. this study aims to formulate natural-based body scrub using the cengkir mango as active ingredients. the formulation was divided into 3 groups with different concentrations of mango fruit flour, respectively 35, 46, and 50 grams (f1, f2, and f3). the product evaluation used were homogeneity, stability, ph, and hedonic test. the organoleptic of body scrub indicated mango scent and light brownish-darker colors, responding to an increase of concentration for each formulation. the homogeneity test showed that the body scrub had no granulation of mango flour. the evaluation of ph showed the product had ph respectively at 7.5, 7.5, and 7.0 (f1, f2, and f3). these results indicated that the body scrub had good physical properties. an investigation of body scrub stability was applied to evaluate the fragrance, color, and consistency of the formulation in storage for three weeks at room temperature. the results showed no change in color and consistency but a loss of mango fragrance after three weeks. the hedonic test indicated that most participants favored f1 (35 grams of mango flour). based on these results, the flour of cengkir mango had a good performance as an active ingredient in a natural-based body scrub. keywords: body scrub; cengkir mango; formulation data of article received reviewed accepted : : : 21 jul 2022 01 aug 2022 29 aug 2022 doi 10.18196/jfaps.v3i1.15569 type of article: research introduction the body scrub is one of the most popular skin cosmetics for men and women. this product is mainly used to exfoliate dead skin and open pores, resulting in clearer and more healthy skin.1 recently, natural compounds have been widely used as active ingredients due to better results on skin and safety. one of the species of * corresponding author, e-mail: hidayattullah1911@gmail.com mangoes in west java is the cengkir mango. the mango was commonly found in indramayu, the northern part of west java.2 it is often used for food, beverage, and active ingredients for natural productbased cosmetics.3 this fruit is rich in phytochemicals such as polyphenols.4 the compound can be used as a keratolytic agent to remove dead skin cells. it could weaken the bonds of dead skin tissue; journal of fundamental and applied pharmaceutical science, 3(1), august 2022 38 hence, the skin becomes easier to apart from the body skin.4,5 the natural-based body scrub used flour from various parts of plants. this study aims to formulate a body scrub with the active ingredient from the flour of cengkir mango material with good physical evaluation. method the tools included formulation set instruments (mortar, stamper, laboratory glassware, etc), digital scale instrument (ohaus), ph meter instrument, water bath, and oven (incucell). the used materials were cengkir mango obtained from the traditional markets in indramayu, methylparaben, stearic acid, triethanolamine, cetyl alcohol (brataco), propylene glycol (brataco), aquadest, vitamin e, fragrance, methanol (brataco), chloroform (brataco), ethyl acetate (brataco) and fecl3. the flour extraction of the cengkir mango the cengkir mango was obtained from the indramayu traditional market. the mango was sorted, peeled, and cleaned, followed by chopping the mango flesh. the chopped mangoes were cleaned with running water. after that, the sample was hydrated for 2x24 hours at a temperature of 50oc. the dried mango was powdered and sieved with a 40 mesh sieve.6,7 the percentage of mango fruit flour was calculated using the formula below: 𝑚𝑎𝑛𝑔𝑜 𝑐𝑒𝑛𝑔𝑘𝑖𝑟 𝐼𝑛𝑑𝑟𝑎𝑚𝑎𝑦𝑢 𝑓𝑙𝑜𝑢𝑟 𝑚𝑎𝑛𝑔𝑜 𝑐𝑒𝑛𝑔𝑘𝑖𝑟 𝐼𝑛𝑑𝑟𝑎𝑚𝑎𝑦𝑢 𝑓𝑟𝑒𝑠ℎ 𝑥 100 body scrub formulation of cengkir mango flour this study used a modified natural-based body scrub formulation from previous studies.8 the formulation used in this study is shown in table 1 below: table 1. formulation of the body scrub formulation reference formulation modification formulation 1 formulation 2 formulation 3 cengkir mango flour 35 gr 46 gr 50 gr cetyl alcohol 1 1 1 triethanolamine 2 2 2 propylene glycol 5 5 5 methylparaben 0.075 0.075 0.075 stearic acid 15 15 15 parfum 3 drops 3 drops 3 drops vitamin e 0.3 0.3 0.3 aquadest ad 50 ml 50 ml 50 ml the oil phase consisted of cetyl alcohol and stearic acid mixed in a water bath at a temperature of 70oc. the other phase comprised propylene glycol, triethanolamine, and methylparaben, mixed with hot aquadest. after that, the two phases were mixed until the soft cream mass was obtained, then mango fruit flour was added to the mixture until homogeneous. the vitamin e and the fragrance were added to the solution and stirred until homogeneous. ismanurrahman hadi, dadan hidayattullah, & ade irawan | formulation and evaluation of body scrub using flourbased fruits of indramayu variety of cengkir mango (mangifera indica l) 39 physical evaluation of cengkir mango body scrub organoleptic test of body scrub tests were carried out by observing changes in color, odor, and shape (consistency) of body scrub preparations. organoleptic tests such as color, aroma, and consistencies can use as qualitative indicators of the physical instability of arrangements which are directly related to the convenience of the preparation by consumers and the quality of the arrangements. 9 ph test of body scrub tests were carried out using a ph meter. a total of 1 gram of each preparation was put in a beaker and diluted in 100 ml of distilled water. the ph of preparation was measured using a ph meter. the ph that met the requirements of sni 16-43991996 was around 4.5 to 8.0.10 homogeneity test of body scrub a total of 0.5 grams of body scrub preparations were smeared on a slide, then observed for coarse particles by palpating and observing the preparation's texture. the homogeneity was indicated by the absence of coarse particles in the preparation and the uniform color of the preparation.8 furthermore, the phase dilution was carried out by diluting 0.5 grams of the preparation with 25 ml of water in a glass beaker. stability test of body scrub the stability of the body scrub formulation was used to investigate the product's storage stability. observations were conducted to evaluate body scrubs' scent, color, and consistency. this study was performed in a temperature room for three weeks.11 hedonic test valuation of preference for body scrub formulation (keratolytic effectiveness) of cengkir mango consisted of scent, color, and comfortability on the skin of 25 respondents. the hedonic scale test assessment applied the highest value of 5 (very liked) and the lowest value of 1 (very disliking).12 data analysis data analysis was performed using the chisquare method with a 95% confidence level. results and discussion the natural-based body scrub was one of the green cosmetics popular in public due to its safety and effectiveness on the skin. it uses many natural ingredients, such as phytochemicals, amylum, flour, and many more. this product can smooth the skin and lift damaged skin cells. 13 the naturalbased pumpkin (cucurbita moschata) in the form of a body scrub cream can improve the condition of rough skin for a better result. the body scrub did not change after stability testing with the cycling test method and had a ph value that met skin ph requirements. furthermore, it did not cause skin irritation.8 the previous study showed that mango cengkir is rich in phenol and flour. therefore, it was used as a flour-based ingredient on a pharmaceutical product. the active ingredient used in this formulation was the flour of cengkir mango. the fresh mango fruit was dehydrated at 50oc and then processed to make crude flour of cengkir mango. the flour obtained was 173 grams from 2 kg of fresh mango fruit (8.65% w/w). a previous study conducted by paramita (2012) obtained mango fruit flour weighing 54 grams from 500 grams of arumanis journal of fundamental and applied pharmaceutical science, 3(1), august 2022 40 mango with a percentage of 10.8% flour.14,15 it indicates that the cengkir mango had less flour than the other mango variety. a b c figure 1. the flour of cengkir mango process. the flour is made through a series of processes, including chopping, drying, and flouring. the drying temperature was adjusted to 50oc for two days. (a) the fresh cengkir mango; (b) the dry cengkir mango; (c) the flour of cengkir mango the body scrub formulations consisted of formulation 1 (35 grams of flour), formulation 2 (46 grams of flour), and formulation 3 (50 grams of flour). the components of the formulation were divided into two phases; active and non active excipients. in this formulation, several excipients were cetyl alcohol, methylparaben, triethanolamine, propylene glycol, stearic acid, and aquadest. meanwhile, the flour of cengkir mango was used as an active component in body scrub formulation. a b c figure 2. the formulation of body scrub. (a), formulation 1 (35 grams of mango flour) (b), formulation 2 (46 grams of mango flour) (c), formulation 3 (50 grams of mango flour) the evaluation of body scrub was used to determine the product's safety, quality, and efficacy. this study used organoleptic, ph, homogeneity, and stability tests to evaluate the physical and stability properties of the product. meanwhile, the hedonic test was used to measure the favorability level of the product. the organoleptic body scrub ismanurrahman hadi, dadan hidayattullah, & ade irawan | formulation and evaluation of body scrub using flourbased fruits of indramayu variety of cengkir mango (mangifera indica l) 41 showed that all formulations had a mango scent. color and consistency increased to darker and hard texture corresponded to increase active ingredients. furthermore, the stability test indicated that only fragrance decreased over time (3 weeks). the results are shown in table 2: table 2. the organoleptic and stability of body scrub after three weeks at room temperature formula parameter time (weekly) 0 1 2 f1 fragrance mango scent mango scent less mango scent color brownish-light brownish-light brownish-light consistency soft soft soft f2 fragrance mango scent mango scent less mango scent color brown brown brown consistency soft with a more solid texture soft with a more solid texture soft with a more solid texture f3 fragrance mango scent mango scent less mango scent color brownish-darker brownish-darker brownish-darker consistency slightly hard slightly hard slightly hard the acidity value of the product was used to ensure a non-irritant body scrub product. the evaluation showed a ph value of 7.5 (f1), 7.5 (f2), and 7.0 (f3). these results indicated that the ph value of the formulation was within the normal range for the product's acidity.11 test results showed the body scrub's ph according to the body scrub's normal ph criteria in table 3. the sampling in the homogeneity evaluation was carried out with random samples from the four sides of the body scrub; the surface, right side, left side, and bottom side of the product. the homogeneity was measured with the consistency of granules in the product. the evaluation showed no granules, indicating good product homogeneity (table 4). table 3. ph evaluation of the body scrub formula ph value normal value formula 1 7.5 ph between 4.5 to 8.0 formula 2 7.5 formula 3 7.0 table 4. homogeneity evaluation of the body scrub no formula homogeneity test surface right side left side bottom side 1 f1 no granule no granule no granule no granule 2 f2 no granule no granule no granule no granule 3 f3 no granule no granule no granule no granule this study also evaluates the product favorability in several people using the hedonic test. it determines the favorability of color, scent, and comfortability of body scrubs. the results of the hedonic test showed that the favorability of journal of fundamental and applied pharmaceutical science, 3(1), august 2022 36 0 20 40 60 a. favorability test of body scrub's scent *) p<0.05 * -5 15 35 55 75 b. favorability test of body scrub's color *)p<0.05 * respondents for color and comfortability in formula 1 was higher than in the others. moreover, the scent was less favorable to respondents. the results indicated that the f1 and f2 were more favorable than the f3 (figure 3). very unfavorable 0 unfavorable 0 rather favorable 50 favorable 25 very favorable 0 very unfavorable 0 unfavorable 0 rather favorable 34 favorable 41 very favorable 0 ismanurrahman hadi, dadan hidayattullah, & ade irawan | formulation and evaluation of body scrub using flourbased fruits of indramayu variety of cengkir mango (mangifera indica l) 43 -5 15 35 55 75 c. favorability test of body scrub's comfortability * * *)p<0.05 conclusion based on the result of this study, it can be concluded that formula 1 was the best formula for natural-based body scrub of cengkir mango flour with good physical properties and favorability. references 1. gitariastuti, n. k., mulyani, s. and wrasiati, l. p. the effect of moringa leaf powder addition and heating process temperature on the characteristics of body scrub, bali: journal of agroindustry engineering and management. 2020;8(1): 18-27. https://doi.org/10.24843/jrma.2020. v08.i01.p03 2. pertiwi, h. r. e. formulation and effectiveness test of body cream preparations scrub containing coffee grounds (coffea arabica l.). medan: program bachelor of pharmacy studies, faculty of pharmacy, university of north sumatra. 2018. 3. theeba p, c. g. and kumar r, sasi. phytochemical examination, antioxidant potential and in vitro antibacterial studies of crude extract of phartenium hysterophorus linn leaves. j. chem. pharm. res., 2015, 7(4):219-225 4. lestari, u., syamsurizal., trisna, y. formulation and physical properties test of activated charcoal body scrub from palm oil (elaeis guineensis jacq) as detoxification, journal of pharmaceutical science and technology 2017;19(1): 75-87. https://doi.org/10.24198/ijpst.v1i1.32664 5. apple a. bodemer md. integrative medicine (fourth edition). 2018. 6. happy, a., soetjipto, h., andini, s. optimization of oil extraction and antibacterial activity of mango seed chloroform extract (mangifera indica l. var arumanis). salatiga: faculty of science and mathematics, satya wacana christian university. 2015. 7. fitrah. study of mango flour (mangifera indica l) production with variations in temperature and drying very unfavorable 0 unfavorable 0 rather favorable 29 favorable 33 very favorable 13 https://doi.org/10.24843/jrma.2020.v08.i01.p03 https://doi.org/10.24843/jrma.2020.v08.i01.p03 https://doi.org/10.24198/ijpst.v1i1.32664 journal of fundamental and applied pharmaceutical science, 3(1), august 2022 44 time. pangkajene and islands: program agroindustry studies department of fishery products processing technology. 2018. 8. leny, ginting, i., tiary, s. n., hanum, s. f. hafiz, i. and iskandar, b. formulation and effectiveness test of pumpkin (cucurbita moschata) body scrub preparations. pharmacy magazine, 2021;6(4):375-385. https://doi.org/10.24198/mfarmasetik a.v6i4.35776 9. musdalipah., haisumanti., reymon. formulasi body scrub sari ubi jalar ungu (ipomoea batatas l.) varietas ayamurasaki. warta farmasi, 2016;5(1):88–98. https://doi.org/10.46356/wfarmasi.v5i2.16 10. andriyanti, p., indriati, d., wardatun, s. uji antioksidan sediaan sugar body scrud yang mengandung katekin gambir (uncaria gambir (hunter) roxb) dan essensial oil jeruk nipis (citrus aurantifolia l.) dengan metode dpph, bogor: pakuan university. 2018. 11. putri, n. r. a. formulasi sediaan hand and body lotion ekstrak etanol buah goji berry (lycium barbarum l.) medan: study program bachelor of pharmacy, faculty of pharmacy, university of north sumatra. 2020. 12. juliantoni, y., hajrin, w., subaidah, w. a., wirasya, d. g. formulation of ashitaba (angelica keiskei) herb ethanolic extract toothpaste, mataram. j pharm sci & practice, 2020, 7(2): 70 – 73. 13. fauzi, n. 2012, caring for the skin and face, jakarta: gramedia. 16-18. 14. octavianti, p. study of mango fruit flour production process (mangivera indica l) arumanis varieties with high soaking temperatures different. journal of renewable natural materials. 2012 15. rita, h., dorly, alex, h. 2020. diversity of cengkir mangoes indramayu regency. bogor: department of biology, fmipa ipb. 2020. https://doi.org/10.24198/mfarmasetika.v6i4.35776 https://doi.org/10.24198/mfarmasetika.v6i4.35776 https://doi.org/10.46356/wfarmasi.v5i2.16 total flavonoid content of lemongrass leaf (cymbogoncitratus (dc.) stapf) extract and antioxidant activity with frap ahda maulida ulufan nurinnafi’a*, kusumaningtyas siwi artini, desy ayu irma permatasari department of pharmacy, faculty of health science, universitas duta bangsa surakarta, jl. bhayangkara no.55, types, serengan, surakarta, central java 57154, indonesia abstract lemongrass (cymbogoncitratus (dc.) stapf) leaves contain alkaloids, saponins, tannins, anthraquinones, steroids, phenols and flavonoids. flavonoids act as antioxidants as they can reduce free radicals. this study aims to determine the total flavonoid content, antioxidant activity, and ic50 value of lemongrass leaf extract (cymbogoncitratus (dc.) stapf). extracts were made by maceration using 96% ethanol as solvent. testing of total flavonoid content with the alcl3 method using uv-vis spectrophotometry was carried out three times. the antioxidant activity test used the frap (ferric reducing antioxidant power) method on extracts containing 100, 200, 300, 400, and 500 ppm. the test results showed that the leaf extract of citronella (cymbogoncitratus (dc.) stapf) had a total flavonoid content of 22.60 mg qe/g extract. furthermore, there was antioxidant activity in the leaf extract of lemongrass (cymbogoncitratus (dc.) stapf indicated by the formation of a blue color purplish when reacted with frap solution, and ic50 extract value was 71.59 ppm and included in the category of strong antioxidants. keywords: antioxidant activity; lemongrass leaf extract (cymbogoncitratus (dc.) stapf); total flavonoid content data of article received reviewed accepted : : : 19 jul 2022 05 aug 2022 29 aug 2022 doi 10.18196/jfaps.v3i1.15556 type of article: research introduction indonesia is a country with a tropical climate where there are various kinds of plants that have therapeutic activity.1 the use of herbal medicines from plants increases disease prevention efforts as the side effects tend to be harmless and can be minimized.2 one of the wild plants widely used by the community is lemongrass (cymbogon citratus (dc.) stapf). the extract of lemongrass leaf (cymbopogon citratus (dc.) stapf) contains several * corresponding author, e-mail: ahdamaulida@gmail.com vegetable constituents, namely essential oils.3 lemongrass (cymbopogon citratus (dc.) stapf) can empirically be used as a medicine for headaches, coughs, stomach pains, diarrhea, body warmers, fever reducers, and mosquito repellents.4 it contains saponins, tannins, alkaloids, and flavonoids. flavonoids are secondary metabolites often found in various green plants so they can be found in every plant extract. flavonoids are found in many green plants, so they can be found in every plant extract. flavonoids contribute to ahda maulida ulufan nurinnafi’a, kusumaningtyas siwi artini, & desy ayu irma permatasari | total flavonoid content of lemongrass leaf (cymbogoncitratus (dc.) stapf) extract and antioxidant activity with frap 31 pigment production and belong to the water-soluble polyphenol family.5 flavonoids can counteract free radicals by liberating hydrogen atoms from their hydroxyl groups; thus, flavonoids are called antioxidants.6 antioxidants are compounds that can scavenge free radicals. free radicals are generated due to several factors, such as smoke, dust, pollution, and consuming fast food that is not balanced between carbohydrates, proteins, and fats.7 when free radical compounds meet other molecules, they can form new free radicals, resulting in chain reactions. this kind of reaction will continue and will stop if antioxidants reduce the reactivity.8 antioxidant compounds will donate one electron to unstable free radicals so that these free radicals can be neutralized and no longer interfere with body metabolism. total flavonoid content can be measured by identifying the absorbance value at a certain wavelength based on the lambertbeer principle of each herbal plant using a uv-vis spectrophotometer.9 the method used to determine total flavonoids is the alcl3 method, using uvvis spectrophotometry as flavonoids contain a conjugated aromatic system so that they show strong absorption bands in the ultraviolet and visible spectrum regions. the antioxidant activity test uses the frap (ferric reducing antioxidant power), which is used to determine the antioxidant activity of a material based on the ability of antioxidant compounds to reduce fe3+ ions (ferri-tripyridyl-trazine) to fe2+ (ferro-tripyridyl-trazine).3 in this study, the total flavonoid content of the lemongrass leaf extract (cymbogon citratus (dc.) stapf) will be tested using the uv-vis spectrophotometry method, and the antioxidant activity will be tested by using the frap method. method plastic bag, knife, scissors, label paper, oven (memmert), herb grinder, drop pipette, micro pipette, analytical balance (ohaus), test tube, test tube clamp, test tube rack, maceration jar, stirrer, spatula, 60 mesh sieve, filter paper, funnel, measuring cup, beaker, handscoon gloves, aluminum foil, rotary vacuum evaporator, tissue, cuvette, spectrophotometry uvvis (emclab), tlc chamber, silica gel 60 f tlc plate (merck), uv lamp 366 nm, citronella leaf (cymbopogon citratus), ethanol96%, potassium acetate 120mm (merck), aquadest, quercetin (sigma), alcl3(merck, 98%), concentrated h2so4(merck, 95-97%), sodium acetate trihydrate (merck 99-101%), acetic acid (merck, 99.8%), tptz powder (sigma), concentrated hcl (merck), fecl3.6h2o (merck), ascorbic acid (merck 99100.05%), oxalic acid1% (merck), chloroform, concentrated ammonia (merck), dragendorf reagent, mayer, wagner, lieberman burchard, fecl31%, ether, n-hexane, and ethyl acetate. lemongrass leaf samples were obtained from sribit village, delanggu, klaten, and then determined at the biology laboratory of universitas ahmad dahlan. lemongrass leaves were washed with running water, drained, dried in an oven, and ground by a herb grinder to become simplicia powder before being extracted. extraction was done by maceration method with 96% ethanol solvent for 3x24 hours. the filtrate was filtered and evaporated to obtain a thick extract. furthermore, phytochemical analysis was carried out on the lemongrass leaf extract, including the test for alkaloids, flavonoids, saponins, tannins, and steroids/ triterpenoids by tlc (thin layer chromatography). the total flavonoid content test in the extract was started by journal of fundamental and applied pharmaceutical science, 3(1), august 2022 32 determining the maximum wavelength of the quercetin standard solution, carried out by running the quercetin solution in the uv-vis wavelength range of 370 450 nm. the determined wavelength was 400 nm. furthermore, the standard curve of quercetin was made by making several concentrations, namely 10 ppm, 20 ppm, 25 ppm, 30 ppm, and 35 ppm. for each standard, 1 ml of quercetin was added with 1 ml of 2% alcl3 and 1 ml of 120 mm potassium acetate and incubated for 30 minutes. the absorbance was measured using uv-vis spectrophotometry with a determined wavelength (400 nm). the total flavonoid content of lemongrass leaf extract was determined using the alcl3 method by dissolving 10 mg of extract in 10 ml of 96% ethanol. 1 ml of extract was added with 1 ml of 2% alcl3 solution and 1 ml of 120mm potassium acetate and incubated for 30 minutes at room temperature. the absorbance was determined by uv-vis spectrophotometry. three replications were made for each analysis, and the average absorbance was then identified determination of the antioxidant activity of lemongrass leaf extract started with making a frap solution by mixing 25 ml of acetate buffer solution, 2.5 ml of tptz solution, and 2.5 ml of fecl3.6h2o solution, then added with aquadest to 100 ml in a volumetric flask. determination of the maximum wavelength of the frap solution was carried out by mixing 3 ml of frap reagent and 1 ml of distilled water and then measuring the absorption at a wavelength of 400-600 nm using uv-vis spectrophotometry. based on the result, the maximum wavelength was determined at 595 nm. ascorbic acid was used as a standard antioxidant solution also as a positive control. a standard curve solution of 100 ppm was prepared by dissolving 10 mg of ascorbic acid dissolved in 1% oxalic acid to the 100 ml volumetric flask limit. furthermore, the stock solution of 100 ppm was diluted into several concentrations, namely 10, 15, 20, 25, and 30 ppm. 1 ml of frap reagent was put and then added with 3 ml of ascorbic acid from each concentration, then homogenized and allowed to stand for 10 minutes at 37ºc. the absorbance was then observed at a determined wavelength of 595 nm. the ic50 value of lemongrass leaf extract was determined by taking 50 mg extract, dissolved in 50 ml of 96% ethanol, obtained a concentration of 1000 ppm, and then diluted into several concentrations, namely 500, 400, 300, 200, and 100 ppm. 1 ml of frap reagent was put in, and 3 ml of sample were added from each concentration. it was homogenized and allowed to stand for 10 minutes at 37ºc, then observed the absorbance at a determined wavelength of 595 nm. results and discussion the results of the extraction of lemongrass leaves (cymbopogon citratus (dc.) stapf) can be seen in table 1 below. table 1.the results of the extraction of lemongrass leaves (cymbopogon citratus (dc.) stapf) powder weight (g) extract weight (g) yield (%) (w/w) 426 57.37 13.46 ahda maulida ulufan nurinnafi’a, kusumaningtyas siwi artini, & desy ayu irma permatasari | total flavonoid content of lemongrass leaf (cymbogoncitratus (dc.) stapf) extract and antioxidant activity with frap 33 the yield of lemongrass leaf extract was 13.46% w/w. a good extract yield is indicated by a yield >10%.10 the higher the yield of the extract is, the higher the content of substances will be attracted to the raw material.11 it was also presumably due to the maceration time that has been done for three days. the remaceration was done two times so that the chemical compound extraction process could be maximized. phytochemicals based on the phytochemical identification, lemongrass leaf extract contained alkaloids, flavonoids, saponins, tannins, and steroids. the results of the flavonoid test using tlc showed the spots between the extract and quercetin were nearly by using eluents n-hexane: ethyl acetate: ethanol: water in a ratio of 2.5:1:0.5:6.5. the rf value of the extract was 0.51, and the rf value of quercetin was 0.56. the difference in the rf value was 0.05. the results would be declared positive containing quercetin if the rf value had a difference of 0.05.12 results of total flavonoid content test of lemongrass leaf extract (cymbopogon citratus (dc.) stapf quantitative tests were carried out to determine the total flavonoid content of the lemongrass leaf extract, using quercetin as a standard solution. the maximum wavelength of quercetin was 400 nm. furthermore, the standard curve of quercetin was made by making several concentrations, namely, 10 ppm, 20 ppm, 25 ppm, 30 ppm, and 35 ppm. each concentration was measured at a wavelength of 400 nm. based on these measurements, the linear regression equation for the standard solution of quercetin was y = 0.025x 0.072, which can be seen in figure 1 below. figure 1.the standard curve of quercetin y = 0.025x 0.072 r² = 0.969 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0 5 10 15 20 25 30 35 40 a b so rb a n ce quercetion concentration (ppm) the standard curve of quercetin y-values linear (y-values) ahda maulida ulufan nurinnafi’a, kusumaningtyas siwi artini, & desy ayu irma permatasari | total flavonoid content of lemongrass leaf (cymbogoncitratus (dc.) stapf) extract and antioxidant activity with frap 33 furthermore, the absorbance measurement of the lemongrass leaf extract was made for 3 replications. the sample solution was added with 1 ml of aluminum chloride (alcl3), which could form a complex, resulting in a shift in wavelength towards the visible indicated by the solution producing a more yellow color. 1 ml of potassium acetate was then added to maintain the wavelength in the visible region 13. it was measured by using uv-vis spectrophotometry to identify the extract absorbance (table 2). table 2. results of total flavonoid content of lemongrass leaf extract (cymbopogon citratus (dc.) stapf) replication sample weight (g) absorbance mg qe/g extract i 0.01 0.437 20.36 ii 0.01 0.427 19.96 iii 0.01 0.650 28.88 average 23.06 the results of this study showed that the total flavonoid content of lemongrass leaf extract (cymbopogon citratus (dc.) stapf was 23.06 mg qe/g extract, which can be seen in table 2. the higher the flavonoid content is, the higher the benefits of flavonoids as antioxidants will be.14 sample iii has a higher absorbance than the other two samples, probably due to the less precise determination of operating time. the three replications were analyzed in minutes of 10. in previous studies, it was shown that starting in the minutes of 30, the flavonoid compounds had completely reacted with the alcl3 reagent, and the absorbance reading was stable in the minutes of 30.15 results of antioxidant test of lemongrass leaf extract (cymbopogon citratus (dc.) stapf) an antioxidant activity test was carried out to determine the ic50 value of the lemongrass leaf extract. the maximum wavelength of the frap solution was 595 nm. qualitative analysis was carried out by taking a sample solution of lemongrass leaf extract. the frap reagent was added, and then color changes were observed. as a result, the sample turned purplish blue, indicating that the sample had antioxidant activity.3 furthermore, vitamin c was used to compare as it functions as a secondary antioxidant. it can reduce free radicals and prevent chain reactions.16 the ic50 value of vitamin c was 23.60 ppm. quantitative tests were conducted by making leaf extract of lemongrass (cymbopogon citratus (dc.) stapf) in five concentration series, namely, 100, 200, 300, 400, and 500 ppm. it was added with frap reagent at each concentration series and read the absorbance at the maximum wavelength of 595 nm. journal of fundamental and applied pharmaceutical science, 3(1), august 2022 34 table 3. results of antioxidant activity test of lemongrass leaf extract (cymbopogon citratus (dc.) stapf) extract concentration (ppm) absorbance average % inhibition 100 0.355 50.89 200 0.306 57.67 300 0.218 69.84 400 0.199 72.47 500 0.159 78.00 the results of the absorbance measurement were used to obtain the % inhibition value. the value of % inhibition was used to find the ic50 value to determine the strength of antioxidant activity in the sample. data in the form of concentration and % inhibition of lemongrass leaf extract showed a linear regression equation y = 0.069x + 45.06 (figure 2) to calculate the ic50 value. the calculation of the ic50 value of lemongrass leaf extract was as follows: y = 0.069 x +45.06 50 = 0.069 x +45.06 50 – 45.06 = 0.069x 𝑥 = 4.94 0.069 𝑥= 71.59 ppm the ic50 value of lemongrass leaf extract (cymbopogon citratus (dc.) stapf) was 71.59 ppm. specifically, phytochemicals are categorized as highly strong antioxidants when the ic50 value is <50 ppm, strong when the ic50 value is 50100ppm, moderate when the ic50 value is y = 0.069x + 45.06 r² = 0.956 0 10 20 30 40 50 60 70 80 90 0 100 200 300 400 500 600 % i n h ib it o n extract concentration the standard curve of 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universitas ngudi waluyo, ungaran, jl. diponegoro no.186, ngablak, gedanganak, ungaran timur, semarang, jawa tengah 50512. abstract hyperlipidemia is a risk factor for various diseases, which is still a big problem in indonesia. yellow pumpkin (cucurbita maxima d.) is a plant containing flavonoids and terpenoids, which can be used as an anti-hypercholesterolemic agent. this study aims to analyze the activity and dosage of yellow pumpkin extract, which can be used as an anti-hypercholesterolemic agent to reduce total cholesterol levels comparable to simvastatin by 1.8mg/kg bw/day. this research is pure experimental research with a pre and post-test group design approach. the number of samples was 25 male wistar rats induced by high-fat feed, quail egg yolk: pork oil (2:1) by 5 ml/200 grambw/day for 7 days. the extract dosage range was 200, 400, 600 mg/kg bw/ day for 7 days. data were analyzed using one way anova. the results showed that the administration of yellow pumpkin extract could reduce rats' total blood cholesterol levels with a dose of 600 mg/kg bw/day, comparable to simvastatin 1.8 mg/kg bw/day. the secondary metabolites of the extract were flavonoids and terpenoids. extract of yellow pumpkin (cucurbita maxima d.) could reduce total blood cholesterol levels of rats. the dose of 600 mg/kg bw/day could reduce blood cholesterol levels in rats comparable to simvastatin 1.8 mg/kg bw / day. keywords: antihypercholesterolemia; cucurbita maxima d; flavonoid; terpenoid data of article received reviewed accepted : : : 14 nov 2020 19 dec 2020 22 feb 2021 doi 10.18196/jfaps.v1i2.10819 type of article: research introduction hyperlipidemia is a condition of excess lipids or cholesterol metabolism disorders consisting of total cholesterol, triglycerides, hdl, and ldl caused by cholesterol levels in the blood exceeding normal limits1. one of the plants that can be used as medicine is yellow pumpkin (cucurbita maxima d.). yellow pumpkin flesh contains carbohydrates, protein, fiber, amino acids, tocopherols, and carotenoids2. in addition, yellow pumpkin * corresponding author, e-mail: istianahizna29@gmail.com flesh also contains flavonoids3. isoflavone, flavones, and flavanones are a group of flavonoids compounds that can reduce total cholesterol levels4. apart from flavonoids, terpenoids also have an activity to lower total serum cholesterol levels. method this research is purely a pre and post-test group design using the test animals model by taking blood samples before and after erna kustiyaningsih, istianatus sunnah, dian oktianti | yellow pumpkin (cucurbita maxima d.) extract as anti-hypercholesterolemia 43 treatment. the pre-test data were taken from the blood sample after induced with high-fat feed, while the post-test data were taken from the blood sample after induced with the extract. the animals were divided into 5 groups, showed in table (1). table 1. grouping of test animals model groups information normal group (kn) animals with standard feeding + cmc na positive control (k +) animals with standard feeding + simvastatin suspension 1.8mg/kgbw/day p 1 animals with standard feeding + extract suspension 200 mg / kgbw /day p 2 animals with standard feeding + extract suspension 400 mg / kgbw /day p3 animals with standard feeding +extract suspension 600 mg / kgbw /day making of yellow pumpkin extract yellow pumpkin (cucurbita maxima d.) was obtained from the kopeng area of semarang regency, made in simplicia and powdered. the extract was made from 500 grams of the pumpkin flesh, 500 grams of maceration with 96% ethanol solvent in a 1:10 ratio. maceration was carried out for 3 days, and re-maceration was done for 2 days. it was then evaporated using a rotary evaporator at a temperature of 60 ⁰c. phytochemical screening of the extract was carried out. the extract of pumpkin flesh detected flavonoids on tlc with silica gf254 stationary phase, which was then eluted using the mobile phase of nbutanol: acetic acid: water (3: 1: 1). as for terpenoids, the mobile phase of chloroform: methanol (9: 1) was selected. the selection of stationary and mobile phases was based on the polarity and properties of the flavonoids and terpenoids. induction of high cholesterol feed hypercholesterolemic feed was made from quail egg yolks and pork oil. the ratio of quail egg yolk: lard was 2:1. high cholesterol feed induction was given to rats for 7 days, and their cholesterol levels were monitored. pumpkin extract induction the experiment animals in this research were white male rats (rattus novergicus) aged 2-3 months, with an average weight of 200 grams. the number of samples was 25 male wistar rats induced with high-fat feed, quail egg yolk: pork oil (2: 1) 5 ml /200 grambw/day, for 7 days. the extract dosage ranges used were 200 mg/kg bw/day, 400 mg/kg bw/day, 600 mg/kg bw/day for 7 days. rat weighing weighing the rats was carried out to determine the effect of high-cholesterol feeding on their body weight. data analysis the data was in the form of the difference in total blood cholesterol levels in rats from pre and post-test. data were analyzed using one way anova. results and discussion table 1. identification ethanol-free test samples reagents result yellow pumpkin extract h2so4 pekat + ch3cooh was heated not smell ester yellow pumpkin extract k2cr2o7+h2so4 pekat no color change journal of fundamental and applied pharmaceutical science, 1(2), february 2021 44 a b c figure 1. the results of tlc flavonoids (a) observations on visible light, (b) observations at 254 nm uv after spraying ammonia vapor, (c) observations on uv light 366 nm after being sprayed with ammonia reagent a b c figure 2. tlc results for the terpenoid class compounds. (a) observing visible light, (b) observing uv light at 254 nm after being sprayed with libermanbuchard, (c) observing uv light at 366 nm after being sprayed with libermanbuchard table 2. identification of secondary metabolites secunder metabolities reagents color spot uv 254 nm uv 366 nm rf standart value rf informati on flavonoid ammonia yellow (254nm) blue (366nm) yellow blue 0.54-0.92 0.78 (+) terpenoid libermanbuchard yellow (254nm) green blue (366) yellow greenblue 0.08-0.96 0.95 (+) figure 3. graph of average weight gain of rats 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 w e ig h t ( g ra m s) average weight of rats ( day) normal groups positive control ( simvastatin 1,8 mg/kgbw) p1 200 mg/kgbw p2 400 mg/kgbw p3 600 mg/kgbw 1.8 erna kustiyaningsih, istianatus sunnah, dian oktianti | yellow pumpkin (cucurbita maxima d.) extract as anti-hypercholesterolemia 45 figure 4. diagram of difference in total cholesterol levels each group table 3. cholesterol level percentage groups cholesterol level at pretest cholesterol level at post-test cholesterol level differences % decrease level normal 113.800±3.493 112.000±2.345 1.800±1.924 1.58 positive 186.400±15.437 125.000±17.479 61.400±4.827 32.94 p1 190.000±15.572 176.200±18.606 13.800±3.421 7.26 p2 180.800±22.466 145.600±23.394 35.200±4.970 19.47 p3 182.000±22.249 125.400±23.734 56.600±3.286 31.90 yellow pumpkin has been widely known for its pharmacological benefits due to its metabolite content in the form of flavonoids and terpenoids. in this study, the pumpkin extraction was carried out to analyze the presence of antihypercholesterolemic activity. the extract was prepared by maceration of simplicia using ethanol 96% (1:10). maceration results obtained a thick reddish-brown extract of 90.149 grams, and the yield was 18.029%. this number has met the requirements of the indonesian herbal pharmacopeia (fhi), which is not less than 10.0%, as the higher the yield value, the greater the extract produced. phytochemicals screening the thick extract of pumpkin flesh was tested with the ethanol-free test to determine whether there was ethanol content in the extract not to affect the test. the test results showed that the thick extract of pumpkin flesh was free from ethanol as it did not smell ester, and there was no color change5 ( table 1). the result of phytochemical screening showed the presence of secondary metabolite compounds in the form of flavonoids and terpenoids in pumpkin extract (figure 1 and 2). in table (2), the result showed a presence of flavonoids with an rf value of 0.78, seen after sprayed 0 10 20 30 40 50 60 70 normal groups positive control p1 200 mg/kgbw p2 400 mg/kgbw p3 600 mg/kgbw cholesterol level differences ( mg/dl) 1.8 61.4 13.8 35.2 56.6 journal of fundamental and applied pharmaceutical science, 1(2), february 2021 46 with ammonia vapor reagent, and an rf value of 0.95 for terpenoids after sprayed with liberman buchard reagent. the mobile phase used to identify flavonoids was n-butanol: acetic acid: water (3: 1: 1), and the mobile phase for terpenoids used chloroform: methanol (9: 1). the solvent influence determined the metabolite compound extraction due to ‘like dissolve like’. rat weighing the rats fed high cholesterol in figure (3) showed a higher body weight gain compared to the normal control group. the increase in body weight in the normal group ( kn) was 37.43%, the positive group ( k+) was 48.91%, groups with 200 mg/ kg bw/ day dose ( p1) was 50.69%, groups with 400 mg/kg bw/ day dose (p2) was 56.20%, and groups with 600 mg/kg bw/ day dose (p3) was 54.59%. this result showed that high-fat feeding increased the weight of the test rats higher and faster. during the study, there were differences in the weight gain of the rats. the difference in weight gain occurred as these white rats had genetic differences, causing different treatment responses. increasing body weight was influenced by diet and lots of fat6. weight gain and increased cholesterol levels in rats were seen during fed fatty foods7. in the first stage of the study, the animal models, except the normal control group, were induced with a high cholesterol feed, raw quail egg yolk, and pork oil (2: 1) 5 ml/200 grambw/day for 7 days to be hypercholesterolemic. cholesterol levels could increase as quail egg yolks contained very high cholesterol compared to other eggs, which had 3,640 mg/100 grams of eggs, while pork oil contained 200 mg/100 grams of cholesterol. apart from containing very high cholesterol, quail eggs also had a composition of 31.85% saturated fatty acids8. in addition, pork oil also contained 21 % saturated fatty acids6. cholesterol level blood sampling for measuring total cholesterol levels was carried out through the tail, measured using the easy touch device. the advantage of using this tool is that it only requires a small amount of blood sample, is easy to use, and the results are obtained quickly (150 seconds). the results of measuring blood cholesterol levels after feeding high cholesterol were defined as pre-test total cholesterol levels, as showed in figure (4). the results showed a significant increase in the total blood cholesterol levels of rats. it means that the induction of high cholesterol feed using quail egg yolk and lard (2: 1) could increase the total cholesterol level of rats, indicated by total cholesterol level 98-148 mg/dl9. quail has 30.63% lipid content8. cholesterol levels of the tested animals showed a decrease after treatment with pumpkin extract and simvastatin. the difference between the highest and lowest decreases in a row was: the positive group was 61.40 mg/dl, the p3 group was 56.60 mg/dl, the p2 group was 35.20 mg/dl, the p1 group was 13.80 mg/dl, and the normal group was 1.80 mg/dl. the research data obtained in the pre and post test had a fairly large standard deviation, due to the limitations of researchers in controlling food intake in each tested animal. the results of lsd in the positive control group and the p3 group showed no significant difference (p = 0.062). in the positive group, the decrease in total blood cholesterol levels was 32.94%, which was the highest compared to other groups. whereas, in the p3 group, the decrease in total blood cholesterol levels was 31.09%, as showed in table (3). erna kustiyaningsih, istianatus sunnah, dian oktianti | yellow pumpkin (cucurbita maxima d.) extract as anti-hypercholesterolemia 47 based on the result, the ability of yellow pumpkin extract in reducing total blood cholesterol levels of rats was due to secondary metabolites, such as flavonoids and terpenoids. flavonoids had a mechanism of action to inhibit the enzyme hmg co-a reductase4. the mechanism of flavonoids has similarities with the mechanism of action of simvastatin in reducing cholesterol levels. flavonoids also have a role as antioxidants that act to reduce ldl in the body. yellow pumpkin contains a flavonoid of 4.433 mg/ml quercetin. it also contains flavon and flavonols as an antioxidant10 and antihyperlipidemic. flavonoid as antihyperlipidemic includes isoflavones, flavones and flavonols4. flavones groups in yellow pumpkin are viteksin, isoveteksin, krisoeriol and apigenin10 as antihyperlidemia4. quercetin and azaleatin are included in flavonols groups in yellow pumpkin extract10, having a role as antihyperlipidemic. apart from flavonoids, terpenoids also have activities to lower total blood cholesterol levels. it has antihypercholesterolemic activity by acting as ligands for ppar (peroxisome proliferator-activated receptor)7. conclusion extract of yellow pumpkin (cucurbita maxima d.) flesh affected the reduction of total cholesterol levels in hyperlipidemic male rats. a 600 mg/kg bw/day dose could reduce total cholesterol levels compared to a dose of simvastatin 1.8 mg/kg bw/day. this anti-hypercholesterolemic pharmacological activity was influenced by the content of secondary metabolite compounds in the form of flavonoids and terpenoids in yellow pumpkins. acknowledgment we would like to thank those who helped and were involved in this research. references 1. harikumar, k., althaf, s. a., kumar, b, k., ramunaik, m. & suvarna, c. h. (2013). a review on hyperlipidemic. international journal of novel trends in pharmaceutical sciences, 3(4), pp. 59– 70. 2. muchirah, p. n., waihenya, r., muya, s., abubakar, l., ozwara, h., & makokha, a. (2018). characterization and anti-oxidant activity of cucurbita maxima duchesne pulp and seed extracts. the journal of phytopharmacology, 7(2), pp. 134–140. 3. bhise, s. & kulkarni, s. (2015) determination of antioxidant activity and phytochemical screening of cucurbita maxima duch . fruit extracts in non polar to polar solvents. international journal of pharmtech research, 8(4), pp. 771–775. 4. zeka, k., ruparelia, k., arroo, r. r. j., budriesi, r., & micucci, m. (2017). flavonoids and their metabolites : prevention in cardiovascular diseases and diabetes. disease, 5(3), pp. 1–18. 5. tenda, p. e., lenggu, m. y., & ngale, m. s. (2017). antibacterial activity test of ethanol extract of faloak tree skin ( sterculia sp.) on staphylococcus aureus bacteria. jurnal info kesehatan, 15(1), pp. 227–239. 6. fitriarini, s. & rahayuningsih, h. m., (2014) perbedaan pengaruh antara ekstrak dan rebusan daun salam (eugenia polyantha) dalam journal of fundamental and applied pharmaceutical science, 1(2), february 2021 48 pencegahan penurunan kadar kolesterol hdl pada tikus sprague dawley. journal of nutrition college, 3(1), pp. 184–191. 7. azhari, b., luliana, s., & robiyanto (2017). uji aktivitas antihiperkolesterolemia ekstrak air buah belimbing wuluh (averrhoa bilimbi linn) pada pemodelan tikus jantan galur wistar hiperkolesterolemia. traditional medicine journal, 22(1), pp. 57–62. 8. polat, e. s., citil, o. b., & garip, m. (2013). fatty acid composition of yolk of nine poultry species kept in their natural environment. animal science papers and report, 31(4), pp. 363–368. 9. yin, w., carballo-jane, e., mclaren, d. g., mendoza, v. h., ..... (2012). plasma lipid profi ling across species for the identifi cation of optimal animal models of human dyslipidemia. journal of lipid researchsearch, 53(1), pp. 51–65. 10. sunnah, i., erwiyani, a. r., yunisa, k. o., & pratama, n. m. (2020). skreening fitokimia formula masker gel peel-off nano ekstrak daging labu kuning ( cucurbita maxima). indonesian journal of pharmacy and natural product, 3(1), pp. 19–24. antibacterial activity of fractions from extract ethanolic of hylocereus polyrhizus peel against e. coli and s. aureus tsania khusnul khotimah; annisa krisridwany*; salmah orbayinah ; sabtanti harimurti school of pharmacy, faculty of medicine and health science, universitas muhammadiyah yogyakarta, jl brawijaya, tamantirto, kasihan, bantul, yogyakarta 55183. abstract peel of red dragon fruit (hylocereus polyrhizus) is one of the plants used as an antibacterial agent as it contains saponin triterpenoid compounds, flavonoid compounds, and alkaloid compounds which can have antibacterial activity. this research aims to determine the antibacterial effect of n-hexane, ethyl acetate, and ethanol fraction of red dragon fruit’s peel against escherichia coli and staphylococcus aureus by the concentration of 10mg/ml, 20mg/ml, 40mg/ml, 80mg/ml dan 160mg/ml. this research was conducted by using laboratory experiments. the simplicia was macerated with 96% ethanol and fractionated by n-hexane and ethyl acetate. the phytochemical screening of the fraction was nhexane fraction containing saponin and alkaloid, while the ethyl acetate fraction contained saponin and flavonoid. kanamycin was used as a positive control, while dmso was used as a negative control. according to this research, the mic value of ethanol fraction, n-hexane fraction, and ethyl acetate fraction were 80mg/ml, 20mg/ml, and 80mg/ml, respectively, for e. coli and all fractions were 10mg/ml for s. aureus. based on the average diameter of the inhibition zone, the largest diameter zone in e. coli was ethyl acetate fraction with 160mg/ml concentration that was 10.33mm. meanwhile, in s. aureus n-hexane fraction, it was 160mg/ml, which was 11.20mm. this result showed that the nhexane fraction has good gram-positive activity while the ethyl acetate fraction has good activity on gram-negative. keywords: anti-bacterial; escherichia coli; fraction of hylocereus polyrhizus; staphylococcus aureus data of article received reviewed accepted : : : 30 dec 2020 27 jan 2021 11 feb 2021 doi 10.18196/jfaps.v1i2.11001 type of article: research introduction with the development of sciences and diseases, awareness of self-sanitation in indonesia increases; one of the causes of sanitation-related disease is bacteria. infection is an emergency problem around the world. the incidence of infection continues to increase from 1% to 40% in * corresponding author, e-mail: akrisridwany@umy.ac.id asia1. there is currently increasing interest in developing highly effective, not contain toxic agents and natural sources2. natural ingredients as medicine in indonesia are a part of cultural tradition and have been widely used by the community for centuries. one of the plants used as an antibacterial agent tsania khusnul khotimah, annisa krisridwany, salmah orbayinah, sabtanti harimurti | antibacterial activity of fractions from extract ethanolic of hylocereus polyrhizus peel against e. coli and s. aureus 66 derived from natural ingredients is dragon fruit. dragon fruit is a plant that has an excellent opportunity to be developed in indonesia. one of the dragon fruit variants is red dragon fruit, which has a delicious taste and many health benefits3. dragon fruit is a rich source of nutrients and minerals, such as vitamin b1, vitamin b2, vitamin b3 and vitamin c, protein, fat, carbohydrates, crude fiber, flavonoid, thiamine, niacin, pyridoxine, cobalamin, glucose, phenolic, betacyanin, polyphenols, carotene, phosphorus, iron, and phytoalbumin4. in addition, the red dragon fruit skin is no less beneficial than the fruit flesh. however, the benefits of dragon fruit peel are still not widely known. thus, currently, the fruit peels are only thrown away without being reprocessed. the previous research found that the peel of red dragon fruit contained flavonoid, alkaloid, terpenoids, thiamin, niacin, pyridoxine, cobalamin, phenolic, polyphenols, carotene, betalain4. based on the background above, this study aims to determine the antibacterial activity of red dragon fruit’s peel (hylocereus polyrhizus) in various fractions against e. coli and s. aureus. methods this research used an experimental laboratory method with several stages of research: sample preparation, fractionation of ethanolic extract of red dragon peel (hylocereus polyrhizus), phytochemical screening using thin-layer chromatography, and antibacterial activity test using the disc diffusion method (kirby-bauer test). this research was conducted in pharmaceutical technology laboratory and research laboratory in universitas muhammadiyah yogyakarta. the tools used in this research were oven (memmert®, germany), maceration vessel, rotary evaporation (heidolp®), waterbath (memmert®), tlc vessel (camag®), silica gel 60 f254 plate (merck®), analytical balance (ohaus®), separating funnel (iwaki®), measuring cup (iwaki®), erlenmeyer (iwaki®), test tube (iwaki®), test tube rack, micropipette (socorex®), dropper pipette, volume pipette (iwaki®), pipette measure (iwaki®), petri dishes (iwaki®), porcelain dishes (iwaki®), vortex mixer (gemmy®) calipers, handscoon (handseol®), masks (sensi®). the materials used were red dragon fruit (hylocereus polyrhizus) obtained from the kebun naga resto jogja, e. coli colony atcc 25922 (yogyakarta health laboratory center), colony s. aureus atcc 25923 (yogyakarta health laboratory center), 96% ethanol ( brataco®), 70% ethanol (brataco®), nhexane (brataco®), ethyl acetate (brataco®), methanol (brataco®), chlorophome pa (merck®), aquades (brataco®), cytoborate reagents, reagents nh3, liebermann-burchard reagent, 1% fecl3 reagent, dragendorff reagent, nutrient agar (na) media (merck®), nutrient broth (nb) media, kanamycin antibiotic (meiji®), dmso (merck®). procedure the red dragon fruit’s peel was separated from the flesh, cut into thin pieces, dried under the sun covered in a black cloth, and followed by oven at a temperature of 50oc for 7 days or more until dry. after that, it was blended to get dry powder of red dragon fruit’s peel. the dry powder of red dragon fruit’s peel was macerated with 96% ethanol with the journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 67 ingredient ratio: solvent (1:10) for 7 days with a 5-day maceration and 2 days of remaceration at room temperature and light-tight vessel5. the extract solution was filtered and concentrated using rotary evaporation at 50oc followed by a water bath at 50oc 70oc until the ethanolic extract of red dragon fruit’s peel was obtained. the ethanolic extract was later fractionated using the liquid-liquid method with ethanol, ethyl acetate, and nhexane as solvents. first, the ethanolic extract of red dragon fruit’s peel was dissolved in a mixture of aqua dest : ethanol (3:7) with the ratio of extract and solution (1:10)w/v6. furthermore, it was put into a separating funnel and added with n-hexane in a ratio of 1:1; then, it was shaken off and allowed to stand until separated, and the n-hexane fraction was taken. the ethanol extract resulted from the separation with n-hexane was then added ethyl acetate in a ratio of 1:1. it was later shaken off and allowed to stand until separated; thus, the ethyl acetate fraction and ethanol fraction were obtained. a qualitative analysis test of n-hexane fraction, ethyl acetate fraction, and ethanol fraction of red dragon fruit’s peel ethanolic extract in this study was carried out on saponins, tannins, flavonoids, and alkaloid compounds using thin layer chromatography (tlc) method and silica gel gf254. it was conducted with the mobile phases, namely chloroform: methanol (15:1) for saponin, tannins, and flavonoid, and chloroform: methanol (7:3) for alkaloid5. the result was obtained by observing the color of the spots appeared under 254nm of ultraviolet light and that the spraying reaction could clarify the spot. antibacterial activity test the antibacterial activity test was carried out using the disc diffusion method. first, the nutrient agar and nutrient broth were prepared, and then the tools and media were sterilized by autoclave at the temperature of 121oc for 15 minutes. media were poured into a petri dish and allowed until it hardened. after that, the bacterial suspension was made on the cold nutrient broth media and incubated at 37oc for 24 hours. after that, the bacterial suspension was added with nacl 0,9% until the turbidity level was the same as the mcfarland standard no 4. a series of concentration of each red dragon fruit’s peel fraction (160mg/ml, 80mg/ml, 40mg/ml, 20mg/ml, 10 mg/ml w/v) was made with a dilution system from the main concentration of 160mg/ml and obtained from 160mg viscous fraction dissolved in 15% dmso. after that, 0,5ml was taken and added 15% dmso until 1ml and homogenized to obtain the concentration of 80mg/ml and a concentration of 10mg/ml. the antibacterial test used the disc diffusion method by preparing a petri dish containing nutrient agar for e. coli and s. aureus bacteria. furthermore, using a sterile swab, each bacteria was spread on the surface of the nutrient medium until the bacteria covered the medium. the paper disc containing the test material was placed on the surface of the media that had been treated with bacteria and incubated for 24 hours at 37oc. the inhibition zone formed was calculated using a caliper. the test was replicated by 3 times. the minimum inhibitory concentration (mic) was determined based on the lowest sample concentration that could form an inhibition zone compared to negative controls. tsania khusnul khotimah, annisa krisridwany, salmah orbayinah, sabtanti harimurti | antibacterial activity of fractions from extract ethanolic of hylocereus polyrhizus peel against e. coli and s. aureus 68 results and discussion phytochemical screening the ethanol fraction, ethyl acetate fraction, and n-hexane fraction of the ethanolic extract of red dragon fruit’s peel were tested for phytochemistry using the thin layer chromatography (tlc) method to determine the compounds contained in each fraction with liebermann-burchard spots of saponin compounds identification. the fecl3 was for tannin compounds, dragendorff reagent for alkaloid compounds, and citroborate and nh3 for flavonoid compounds. phytochemical screening results are shown in table 1. table 1. result of phytochemical screening antibacterial activity test a red dragon fruit’s peel has antibacterial activity against e.coli and s. aureus bacteria. the results of the inhibition zone against e.coli are provided in table 2, and the inhibition zone against s. aureus can be seen in table 3 table 2. the measurement result of inhibition zone diameter against e. coli bacteria fraction series of concentration (mg/ml) replication (mm) average i ii n-hexane 10 8.50 8.05 8.28 ± 0.32 20 11.00 8.58 9.79 ± 1.71 40 10.50 9.40 9.95 ± 0.78 80 11.00 8.40 9.70 ± 1.84 160 7.03 7.03 7.03 ± 0.00 ethyl acetate 10 8.70 8.53 8.61 ± 0.12 20 8.50 8.60 8.55 ± 0.07 40 9.30 9.10 9.20 ± 0.14 80 8.70 10.15 9.43 ± 1.03 160 9.65 11.00 10.33 ± 0.95 ethanol 10 8.00 8.10 8.05 ± 0.07 20 7.50 8.83 8.16 ± 0.94 40 8.50 8.10 8.30 ± 0.28 80 9.60 10.13 9.86 ± 0.37 160 10.20 10.33 10.26 ± 0.09 positive control 27.70 25.70 26.70 ± 1.41 negative control 10.30 8.20 9.25 ± 1.48 fraction chemical content explanation n-hexane flavonoid (-) saponin (+) tannin (-) alkaloid (+) ethyl acetate flavonoid (+) saponin (+) tannin (-) alkaloid (-) ethanol flavonoid (-) saponin (-) tannin (-) alkaloid (-) journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 69 table 2. the measurement result of inhibition zone diameter against s. aureus bacteria fraction series of concentration (mg/ml) replication (mm) average i ii iii n-hexane 10 9.28 10.00 10.00 9.76 ± 0.42 20 9.20 9.15 10.95 9.77 ± 1.03 40 8.75 9.58 10.85 9.73 ± 1.06 80 11.73 10.10 10.10 10.64 ± 0.94 160 11.00 11.00 11.60 11.20 ± 0.35 ethyl acetate 10 8.70 7.55 9.25 8.50 ± 0.87 20 6.65 8.05 8.70 7.80 ± 1.05 40 7.20 9.40 9.40 8.67 ± 1.27 80 8.65 9.83 9.65 9.38 ± 0.63 160 6.75 10.70 9.55 9.00 ± 2.03 ethanol 10 7.50 7.10 8.50 7.70 ± 0.72 20 7.33 8.25 9.05 8.21 ± 0.86 40 8.83 9.10 8.10 8.68 ± 0.52 80 8.15 10.35 9.95 9.48 ± 1.17 160 8.40 10.40 8.83 9.21 ± 1.05 positive control 39.05 33.60 39.33 ± 3.85 negative control 6.25 7.00 6.62 ± 0.53 the test results showed differences in the inhibition zone's diameter produced against e. coli bacteria and s. aureus bacteria. the difference in the inhibition zone in gram-positive and gram-negative bacteria might be due to differences in the structure of the bacterial walls between gram-positive and gram-negative bacteria, which can affect fraction work because the fraction will affect if it enters the bacterial cell. gram-negative bacteria have a complex and three-layered cell wall structure; the outer layer is a lipoprotein, the middle layer is liposaccharide which blocks the entry of antibacterial bioactive materials, and the inner layer is peptidoglycan which has a high lipid content7. meanwhile, gram-positive bacteria have a simpler cell wall structure, which is single-layered with low lipid content, making it easier for bioactive compounds to enter the cell7. the results of phytochemical screening showed that the dragon fruit’s peel contained saponin triterpenoids, flavonoids, and alkaloids which could have bacterial activity. saponin triterpenoid has a bactericidal effect by causing protein and enzyme leakages in the cell. saponin can reduce bacterial cell surface tension, thereby increasing permeability and causing leakage in the bacterial cell membrane. due to the damage to the membrane cell, saponin will diffuse into the cytoplasm and cause the cytoplasm to leak and leave the cell, causing the death of bacteria8. meanwhile, alkaloids can interfere with the constituent components of peptidoglycan in bacterial cells so that the cell wall is not formed completely, which can lead to bacterial death9. in addition, alkaloids are also known as dna intercalators and can inhibit the bacterial topoisomerase enzyme10. flavonoid has an antibacterial effect. it forms complex compounds with proteins that damage the membrane and cause cell leakage11. flavonoids also interfere with tsania khusnul khotimah, annisa krisridwany, salmah orbayinah, sabtanti harimurti | antibacterial activity of fractions from extract ethanolic of hylocereus polyrhizus peel against e. coli and s. aureus 70 metabolism energy through inhibitory mechanisms of bacterial nucleic acid (dna and rna) synthesis and inhibition of cytoplasmic membrane function11. this mechanism is the same as the aminoglycoside class of antibiotics, which inhibit protein synthesis and cause damage to the cytoplasmic membrane. after that, a further test was carried out using mann-whitney to determine the difference between the control and test groups on s. aureus bacteria. the result was that each test concentration in the nhexane fraction had an inhibitory power against s. aureus bacteria. meanwhile, based on the test results on the ethyl acetate fraction, there was no significant difference between negative control at a concentration of 20 mg/ml. in contrast, there was no significant difference between negative control in the ethanol fraction with a concentration of 20 mg/ml. analysis of the result to see whether there was a significant difference between the test groups with the inhibitory power, the test was carried out using the non-parametric kruskal wallis test as the result of the data was not normal and homogenous. in terms of the e. coli bacteria, the value of p=0.065 (>0.05) indicated that there was no significant difference between the test group with inhibitory power. in contrast, for s. aureus bacteria, the value of p = 0.003 (<0.05) indicated a significant difference between the test groups with inhibition power. after that, a further test was carried out using mann-whitney to determine the difference between the control and test groups on s. aureus bacteria. conclusion 1. the ethanol fraction, n-hexane fraction, and ethyl acetate fraction of the red dragon fruit’s peel ethanolic extract (hylocereus polyrhizus) had antibacterial activity against e. coli and s. aureus bacteria. 2. ethyl acetate fraction of red dragon fruit’s peel contained saponin triterpenoids and flavonoids, while the n-hexane fraction of red dragon fruit’s peel contained saponin triterpenoid and alkaloid compounds. 3. the ethanol fraction, n-hexane fraction, and ethyl acetate fraction of the red dragon fruit’s peel ethanolic extract showed minimal inhibitory concentration (mic) at the concentration of 80mg/ml, 20mg/ml, and 80mg/ml, respectively, against e. coli bacteria. whereas for s. aureus bacteria, each fraction showed mic at the concentration of 10mg/ml. 4. the ethanol fraction had the greatest inhibitory power against e. coli bacteria at a concentration of 160mg/ml and a concentration of 80mg/ml against s. aureus bacteria. ethyl acetate fraction had the greatest inhibitory power at a concentration of 160mg/ml against e. coli and at the concentration of 80mg/ml against s. aureus bacteria. furthermore, the n-hexane fraction had the greatest inhibition at a 40mg/ml concentration for each bacteria. acknowledgment we would like to thank the faculty of health, jenderal achmad yani university yogyakarta, for funding this research references 1. kemenkes ri. (2011). riset kesehatan dasar; riskesdas. jakarta: balitbang kemenkes ri. 2. shan, b., cai, y. z., brooks, j. d., & corke, h. (2007). the in vitro journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 71 antibacterial activity of dietary spice and medicinal herb extracts. international journal of food microbiology, 117(1), pp. 112-119. 3. zur, n. t., abbo, s., bar-zvi, d., & mizrahi, y. (2004). genetic relationships among hylocereus and selenicereus vine cacti (cactaceae): evidence from hybridization and cytological studies. annals of botany, 94(4), pp. 527-534. 4. bellec, f. l., vaillant, f., & imbert, e. (2006), pitahaya (hylocereus spp): a new fruit crop, a market with a future, fruits. the international journal of tropical & subtropical horticulture, 61(4), pp. 237-250. 5. hanani, e., (2015), analisis fitokimia. jakarta: penerbit buku kedokteran egc. 6. sari, n. s. (2016). uji aktivitas antioksidan dan fotoprotektif fraksi etilasetat ekstrak etanolik kulit buah naga merah (hylocereus polyrhizus) [skripsi]. yogyakarta: universitas muhammadiyah yogyakarta. 7. sani, r. n., nisa, f. c., andriani, r. d., & maligan, j. m. (2013). analisis rendmen dan skrining fitokimia ekstrak etanol mikroalga laut tetraselmis chuii. jurnal pangan dan agroindustri, 2(2), pp. 121-125. 8. cavalieri, s. j., rankin, i. d., harbeck, r. j., sautter, r. s., mccarter, y. s., sharp, s. e., ortez, j. h., & spiegel, c. a. u. (2005). manual of antimicrobial susceptibility testing. usa: american society for microbiology. 9. darsana, i. g. o., besung, i. n. k., mahatmi, h. (2012). potensi daun binahong (anredera cordifolia (tenore) steenis) dalam menghambat pertumbuhan bakteri escherichia coli secara in vitro indonesia medicus veterinus, 1(3), pp. 337-351. 10. karou, d., savadogo, a., canini, a., yameogo, s., montesano, c., simpore, j., colizzi, v., & traore, a. s. (2005). antibacterial activity of alkaloids from sida acuta. african journal of biotechnology, 4(12), pp. 1452-1457. 11. pelczar, m. j., & chan, e. c. s. (1988). dasar-dasar mikrobiologi. jakarta: universitas indonesia press. formulation and optimization of furosemide snedds with variation concentration of tween 80 and peg 400 sesilia putri nandita; ilham kuncahyo; reslely harjanti* departement of pharmacy, faculty of pharmacy, universitas setia budi, jl. letjen sutoyo, mojosongo, kec. jebres, kota surakarta, jawa tengah 57127. abstract furosemide is a potent diuretic drug that has low bioavailability. furosemide can be formulated into nanoemulsion preparations using the snedds method to increase its bioavailability as snedds can form stable nanoemulsions with droplet sizes < 200 nm. this study aims to identify the optimum formula for variations in the concentration of surfactant tween 80 and cosurfactant peg 400 based on the characterization tests of emulsification time, percent transmittance, and drug loading. the independent variables used in this study were tween 80 and peg 400. seven furosemide snedds formulas from the simplex lattice design (sld) method were tested for characterization in the form of emulsification time, percent transmittance, and drug loading. the characterization results were optimized using simplex lattice design. the optimum formula was re-characterized, including emulsification time, percent transmittance, drug loading, particle size, zeta potential, and in vitro dissolution. the results were then compared with theoretical values and analyzed using the one-sample t-test method. optimization results showed tween of 61.4922% and peg 400 of 18.5078% with the characterization of emulsification time 15.25 seconds, percentage transmittance 94.20%, drug loading 50 100.2 ppm, particle size 12.18 nm. furthermore, the zeta potential was -17.6 mv, and the in vitro dissolution rate reached 106.71% within 15 minutes. keywords: furosemide; snedds; tween 80; peg 400; sld data of article received reviewed accepted : : : 01 july 2021 20 july 2021 24 aug 2021 doi 10.18196/jfaps.v2i1.12180 type of article: research introduction furosemide is commonly used as a potent diuretic in treating edema associated with heart, renal, hepatic, and hypertension failure.1 furosemide belongs to the biopharmaceutical class system (bcs) of class iv with low permeability and solubility. low solubility and permeability are important factors responsible for the * corresponding author, e-mail: lely.harjanti@gmail.com low bioavailability of furosemide. the bioavailability of oral furosemide varies from 50-61%; thus, efforts are needed to overcome this problem.2 the low solubility of furosemide can be solved by using a lipid-based formulation with a nanosystem such as the self-nano emulsifying drug delivery system (snedds).3 snedds is an isotropic mixture system of the oil phase, abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 35 surfactant, and co-surfactant. when snedds is absorbed into the body, the preparation will quickly disperse to form droplets <200 nm in size and will form o/w (oil in water) nanoemulsions spontaneously in the gastrointestinal tract4. the components used in this study were oleic acid oil, surfactant tween 80, and cosurfactant peg 400. the oil component in the snedds formulation acts as a drug carrier or active substance. in contrast, the surfactant and cosurfactant components reduce droplet size, emulsion and retain the active substance at the absorption site without deposition in the gastrointestinal tract.5 a good snedds formula was determined from various parameters, including percent transmittance, particle size, drug loading, zeta potential, emulsification time, and in vitro dissolution. the steps before determining the optimum formula were carried out using the simplex lattice design (sld). the advantage of using this application is to increase the effectiveness in interpreting factors and interactions. the desired effect with no interaction can be predicted to make the study more efficient.6 based on the background above, this study aims to obtain the optimum formula using various concentrations of surfactant tween 80 and cosurfactant peg 400 according to the characterization test of emulsification time, percent transmittance, and drug loading. method this study is experimental laboratory research to identify the optimization of the formula of snedds furosemide with various concentrations of tween 80 and peg 400 using the simplex lattice design method. this study was conducted at the research laboratory of the faculty of pharmacy, setia budi university, for two months. research tools and materials this research utilized uv spectrophotometer (uv-1800 series), centrifuge (table top centrifuge plc-05 1601461), dissolution with basket stirrer type (erweka type dt 700), analytical balance (ohaus pa213 1 mg accuracy and ohaus av26422 0.1 mg accuracy), magnetic stirrer, and laboratory glassware. meanwhile, the materials used in this study were pure furosemide obtained from pt. graha farma, oleic acid, tween 80, and peg 400 obtained from pt. brataco chemika solo, aquadestilata, and methanol p.a obtained from the pharmaceutical technology laboratory of universitas setia budi. determination of snedds formulation preparation of the furosemide snedds formula used the simplex lattice design (sld), specifically design expert 10.0.3. the use of the sld method was based on two independent variables whose application was used to determine the optimal formula for the mixture of ingredients. the sld design in the study was made by choosing three combinations and observing the response obtained.7 preparation of furosemide snedds formulation of snedds furosemide of 3 ml was put into the vial by taking each ingredient according to run (7 runs). it was then stirred using a magnetic stirrer at a speed of 50 rpm. after forming an isotropic mixture, furosemide was added gradually until a cloudy solution was obtained. the stirring process was continued for 24 hours, and centrifugation journal of fundamental and applied pharmaceutical science, 2(1), august 2021 36 was carried out to separate the insoluble part of the furosemide at a speed of 5000 rpm. the clear supernatant was taken and then characterized, which included emulsification time, percent transmittance, drug loading, particle size, zeta potential, and in vitro dissolution test.8 furosemide snedds optimization the results of the snedds formula were entered in the simplex lattice design (sld) program so that one optimum formula was obtained for sneeds furosemide. the optimum formula was expected to achieve the set parameters consisting of emulsification time <3 minutes, particle size <200 nm, zeta potential ± 30mv, percent transmittance approaching 100%, high drug loading, and dissolution of less than 80%.8 characterization of the optimum formula of furosemide snedds emulsification time. 100.0 l of furosemide snedds was mixed into 10 ml of distilled water, then mixed using a magnetic stirrer at a speed of 50 rpm while observing the time for emulsification using a stopwatch.8 transmittance percent. the snedds formula resulting from the emulsification time was based on transmittance using uv-vis spectrophotometry at a 200-650 nm wavelength and aquadestilata blank.8 drug loading. the sample of furosemide snedds was pipetted 0.1 ml, and it was put into a 10 ml volumetric flask; then, the volume was added with methanol p.a to the limit mark and shaken. particle size and zeta potential. the snedds formulation has been taken 10 ml using a volume pipette then put into a 100.0 ml volumetric flask. the volume was adjusted to the mark with distilled water, homogenized using a magnetic stirrer, and then measured using a zetasizer.8 in vitro dissolution. the dissolution profile of snedds furosemide was compared with pure furosemide powder filled into soft shells. the dissolution test was carried out using a dissolution apparatus type 1 (basketball) with 900 ml of 0.1 n hcl media. the temperature was set at 37 ± 0.5°c, and the rotation speed was 50 rpm. at minute 0, the capsule was put into the medium. at minutes of 5, 10, 15, 20, 25, 30, 35, and 60, 5 ml of the solution was taken and re-replaced with an equivalent volume of the same type of dissolution medium at the same temperature.9 data analysis the optimum formulation of furosemide snedds obtained from the design expert was tested for characteristics including emulsification time, percent transmittance, and drug loading and then tested for normality.) with a 95% confidence level. the test was carried out by comparing the results obtained from the normality test with the theoretical requirements. it would meet the requirement if the sig value was > 0.05. results and discussion the furosemide snedds formula was obtained using the simplex lattice design (sld) by stating the upper and lower limits of the ingredients used in tween 80 with a range of 60% 65% and peg 400 with a range of 15% 20%. in comparison, it was set at 20% for oleic acid to obtain some suggested formulations. the furosemide drug delivery system from 7 formulas obtained using expert design was abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 37 characterized by the emulsification time, percent transmittance, and drug loading. the best emulsification time and percent transmittance were obtained by formula 7. the emulsification time was carried out to obtain an idea of the time required for furosemide snedds to form a precise proportion of nanoemulsion when interacting with the gastrointestinal tract in the body. the transmittance percentage was used to determine the furosemide snedds included in the nanoemulsion with clarity of consistency close to 100%. the highest drug loading produced by formula 3 was 58583.3 ppm since the most significant surfactant component was found in formula 3. surfactants were able to help the oil component dissolve the drug so that a high drug loading was obtained. table 1. furosemide snedds formulation and characterization tween 80 (%) peg 400 (%) emulsification time (seconds) percent transmittance (%) drug loading (ppm) 60 20 26±0.667 98.2±0.2 33670.6 63.75 16.25 43±0.12 82±11.99 49481.2 65 15 107±0.667 93±1.2 58583.3 62.5 17.5 23±0.333 81±11.86 44882.9 61.6667 18.3333 26±0.333 92.53±2,72 40001.2 63.3333 16.6667 29±0.12 79.2±6,17 43056.2 61.25 18.75 13±0.667 98.27±1.13 41575.9 contour plots of emulsification time and percent transmittance showed how the combination of surfactant and cosurfactant components influenced each other, as seen in the curved graph. meanwhile, the two did not affect each other for drug loading. the regression coefficient value obtained from the design expert showed that the interaction of the surfactant and cosurfactant components had an effect on decreasing the emulsification time; the surfactant component led to an increase in the transmittance percentage value while the cosurfactant component led to an increase in drug loading. figure1. contour plot of emulsification time journal of fundamental and applied pharmaceutical science, 2(1), august 2021 38 figure 2. contour plot of percent transmittance figure 3. contour plot of drug loading the optimum formula of furosemide snedds was determined based on the emulsification time, drug loading, and percent transmittance of characterization test results. based on the seven formulas, an optimization study was conducted using simplex lattice design in the design expert 10 program. the results obtained on the characterization of the emulsification time of the optimum formula of furosemide snedds did not significantly differ from the experiments carried out using the simplex lattice design. the emulsification time obtained from the optimum formula was included in the class 2 category, where the emulsification time formed was quite fast by producing a transparent emulsion system. it occurred since the more surfactant and cosurfactant compositions could accelerate the formation of emulsions abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 39 when in contact with the medium (aquadestylates). the transmittance percentage close to 100% indicated that the optimum formula produced a clear and transparent dispersion with a droplet size estimated to reach nanometers. when light passed through an emulsion system with tiny droplet sizes, the light beam would be transmitted so that the color of the solution looked transparent and the resulting transmittance was more excellent.10 meanwhile, drug loading was used to determine the ability of snedds in dissolving the drug until it was adequately saturated and determining the drug content in the snedds formula.11 the results of drug loading between predictions from simplex lattice design and the experiment were not highly different. the drug loading value obtained was relatively large. it was used to determine how much volume of snedds was needed for one use by comparing the usual dose of a one-time use of furosemide with the drug loading level. the results of the particle size characterization above showed that the optimum formula for snedds had entered the nano range. particle size analysis was an essential factor in evaluating a nanoemulsion formula. particle size was known to affect drug absorption, as studied by various researchers. the stability of nanoemulsions also depends on the size of the particles. the smaller the size of the particles is formed, the better the stability of the emulsion will be12 the most influential component in snedds in the formation of nanoparticles was the surfactant component (tween 80). high concentrations of surfactants would increase water penetration into the oil droplets, leading to its breakdown.13 furthermore, eta potential can be used to estimate the surface characteristics of nanoemulsions. the zeta potential value greater or farther from 0 will cause the preparation to be more stable due to the formation of an electric double layer, which prevents the incorporation of particles in the formula14. in the case of this study, the result obtained above showed that the potential zeta value of the optimum formula was adequate, indicating a stable system. the furosemide snedds dissolution test result was portrayed by a graph between the time and % dissolution of the drug dissolving in 0.1 n hcl medium, which described the in vitro drug release profile. this parameter was called the dissolution efficiency parameter15. the dissolution test results showed that the furosemide snedds formula already had a high concentration starting from the 5th minute of dissolution, 86.08%. moreover, the highest concentration was obtained at the 15th minute with a concentration of 106.71%. the high levels in the early minutes of dissolution were due to the snedds formulation, which could form spontaneous nano-emulsions when in direct contact with water or gastrointestinal fluids in the digestive tract. therefore, the body would quickly absorb the drug. verification of the optimum formula was carried out to determine whether there was a significant difference between the characterization of the experimental results and the characterization of the predictions of simplex lattice design. statistical analysis using a one-sample ttest showed that the emulsification time of characterization test, drug loading journal of fundamental and applied pharmaceutical science, 2(1), august 2021 40 determination, and percent transmittance in the experiment was compared with predictions using simplex lattice design (predicted results). the results were not significantly different, with a p-value > 0.05, indicating that the optimum formula was valid and verified. table 2. the characterization of furosemide snedds optimum formula parameters sld prediction result experiment result emulsification time (seconds) 13.9 15.25±0.99 percent transmittance (%) 95.32 94.20±1.51 drug loading (ppm) 52603.4 50100.2±5539.05 particle size (nm) 12.18 nm -17,6 106.71 (15 minutes) zeta potential (mv) in vitro dissolution (%) conclusion based on the optimization results, the optimum formula was at a concentration ratio of 61.4922% of tween 80 and 18.5078% of peg 400. it went with the characterization of emulsification time of 15.25 seconds, percent transmittance of 94.20%, drug loading of 50100.2 ppm, the particle size of 12.18 nm, the zeta potential of -17.6 mv, and the in vitro dissolution rate of 106.71% within 15 minutes. acknowledgment the author would like to thank those who helped and were involved in this research. conflict of interest the authors certify that they have no affiliations with or involvement in any 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(2011). study of cosurfactant effect on nanoemulsifying area and development of lercanidipine loaded (snedds) self nanoemulsifying drug delivery system. colloids and surface b: biointerfaces. elsevier. 86(2), pp. 327338. https://doi.org/10.1016/j.colsurfb.201 1.04.016 15. basalious, e. b., shawky, n., badreldin, s.m., (2010). snedds containing bioenhancers for improvement of dissolution and oral absorption of lacidipine. i: development and optimization. international journal of pharmaceutics. 391, pp. 203–211. https://doi.org/10.1016/j.ijpharm.201 0.03.008 https://doi.org/10.1016/j.colsurfb.2011.04.016 https://doi.org/10.1016/j.colsurfb.2011.04.016 https://doi.org/10.1016/j.ijpharm.2010.03.008 https://doi.org/10.1016/j.ijpharm.2010.03.008 screening of phytochemical secondary metabolites of muntingia calabura: a potential as hepatoprotector elasari dwi pratiwi1*, niluh puspita dewi2 1 school of pharmacy, faculty of health science, universitas muhammadiyah lamongan, jl raya plalangan plosohwahyu km 2 lamongan, surabaya 62211 2 school of pharmacy, sekolah tinggi ilmu farmasi pelita mas palu, jl wolter monginsidi no. 106a, palu 94236 abstract muntingia calabura is one of the plants employed to produce herbalbased treatments. muntingia calabura leaves are traditionally used as an alternative medicine due to their secondary metabolites. the maceration method extracted muntingia calabura leaves using 96% ethanol solvent for 3 x 24 hours. the fractionation process was carried out using a separating funnel method with different polarities, such as n-hexane, ethyl acetate, and ethanol-water. thinlayer chromatography (tlc) was used to confirm the phytochemical screening. tlc conditions under uv light 254 and 366 nm using solvents, such as chloroform: methanol (alkaloids), butanol: acetic acid: water (flavonoids), chloroform:methanol: water (saponins), and chloroform: methanol (phenolic). the phytochemical screening results of extracts and muntingia calabura fractions contained secondary metabolites, such as alkaloids, flavonoids, tannins, saponins, and phenolics. tlc results showed that n-hexane fraction contained flavonoid and saponin compounds; ethyl acetate fraction contained alkaloids, flavonoids, saponins and phenolic compounds; and ethanol-water fraction contained alkaloids, flavonoids, saponins, and phenolics. muntingia calabura leaves indicated the potential as herbal medicine by containing secondary metabolites. keywords: fractionation; muntingia calabura; phytochemical screening; tlc data of article received reviewed accepted : : : 21 july 2021 26 aug 2021 13 jan 2022 doi 10.18196/jfaps.v2i1.12364 type of article: research introduction natural resources have benefits for various purposes, such as developing herbalbased medicines1. the utilization of natural ingredients for traditional medicine has been consumed by a large number of people in the world for many years2. in addition, traditional medicine is also used as a health supplement by 80% of the population in developing countries around the world, and about 85% of ingredients in traditional medicine involve * corresponding author, e-mail: edpratiwi8@gmail.com plant extracts3. one of the plants used as traditional medicine is muntingia calabura. muntingia calabura belongs to the elaeocarpaceae family, widely used in traditional medicine. people in indonesia usually eat the fruit directly due to its sweet taste4. meanwhile, the peruvian people have used the flowers and stem as an antiseptic to reduce swelling of the prostate gland and reduce headaches and fever5. several studies have shown that extracts of muntingia calabura had a elasari dwi pratiwi & niluh puspita dewi | screening of phytochemical secondary metabolites of muntingia calabura: a potential as hepatoprotector 60 variety of activities that had been tested, such as antioxidant6, anticancer7, antidiabetic8, antibacterial9, and antiinflammatory10. based on this information, this study aims to determine the secondary metabolites contained in the muntingia calabura leaf fraction so that further tests can be carried out both in vivo and in vitro as hepatoprotection. method this study is a laboratory experiment aiming to discover the secondary metabolites found in muntingia calabura plants. this study was conducted at the pelita mas palu school of pharmacy's phytochemical laboratory. research tools and materials the tools used in this study were an oven (memmert universal), rotary evaporator (ryela n-100), water bath (eyela sb-1000), tlc plate, photometer 5010 (rielegermany), uv lamp, and glassware (pyrex). the materials used in this study were muntingia calabura leave, ammonia (merck), aquadest, anhydrous acetic acid (merck), hcl (merck), concentrated hcl (merck), h2so4 (merck), butanol (merck), ethanol 96% (merck), ethyl acetate (bratachem), fecl3 (merck), chloroform (merck), methanol (merck), nacl (merck), n-hexane (merck), ragendroff merck), magnesium powder p (merck). determination the identification of muntingia calabura was carried out at upt. biological resources tadulako university, central sulawesi. preparation of muntingia calabura extracts and fractions the maceration method extracted muntingia calabura simplicia using 96 percent ethanol solvent for 3 x 24 hours. to obtain a thick muntingia calabura extract, the maceration results were filtered and concentrated using a rotary evaporator at a temperature of 40-50°c11. the thick ethanol extract of muuntingia calabura leaves was fractionated using non-polar, semi-polar and polar solvents (n-hexane, ethyl acetate and water). first, the thick muntingia calabura extract was fractionated in a separating funnel with nhexane and water (1:3) and thoroughly shaken. after that, it was left until two layers, the n-hexane layer and the water layer had formed. to get the n-hexane fraction, the process was repeated three times. the water layer was fractionated three times with ethyl acetate (3:1), yielding the water and ethyl acetate fractions. to obtain a thick fraction of muntingia calabura leaves, the results of the n-hexane and ethyl acetate fractions were condensed with a rotary evaporator at a temperature of 40-50°c11. phytochemical screening of muntingia calabura extract and fraction alkaloids test 0.5 grams of the extract was weighed, and the thick fraction of muntingia calabura leaves were weighed and placed in an erlenmeyer with 5 ml chloroform and 5 ml ammonia. it was then heated on a water bath, shaken and filtered. 5 drops of 2n h2so4 were added to the filtrate in a test tube. dragendorff's reagent was added to each tube containing the filtrate, along with several ml of hcl, and the tubes were filtered. dragendorff's reagent produced red-orange precipitation, which indicated a positive reaction12. journal of fundamental and applied pharmaceutical science, 2(2), february 2022 61 flavonoids test 0.5 grams of the extract was weighed, and the thick fraction of muntingia calabura leaves was added with 10 ml of distilled water and heated for 1 minute on a water bath. it was then filtered and dissolved in 1 ml of ethanol (95%) with magnesium p powder. if a crimson hue appeared after being dissolved in 10 ml of strong hydrochloric acid, it would indicate the presence of flavonoids12. saponins test 0.5 grams of the extract was weighed, and a fraction of muntingia calabura leaves were put into a test tube. after that, the 10 ml of hot water was added, let cool, and was shaken vigorously for 10 seconds. saponins would be present if a foam formed and lasted for at least 1 minute at the height of 10 cm or if 1 drop of 2n hydrochloric acid did not evaporate after being added12. tannins test 0.5 grams of extract and a thick fraction of muntingia calabura leaves were weighed and placed in a test tube with 20 ml hot water and 3 drops of a 10% nacl solution. if a blue-black color appeared after adding a fecl3 solution, it would mean the presence of tannin12. phenolic test 0.5 grams of extract and a thick fraction of muntingia calabura leaves were weighed and dissolved in 5 ml of water. if a dark green color shift occurred after adding a few drops of 5% fecl3 solution, it would mean the existence of phenolic12. thin-layer chromatography of qualitative test alkaloids test in the development of chloroform: methanol (85:15), the sample was detected on a silica gel plate. detection of the presence of alkaloids was conducted under uv light 254-366 nm. ensure that the plate was sprayed with dragendorff's reagent, and spots would appear if it were positive for alkaloids13. flavonoids test in the development of butanol: acetic acid: water (4:1:5), the sample was detected on a silica gel plate. detection of the presence of flavonoids was conducted under uv light 254-366 nm. it was then sprayed with alcl3 staining solution in chloroform; yellow color indicated the presence of flavonoids13. saponins test the sample was detected on a silica gel plate in developing chloroform: methanol: water (40:50:10). it was then sprayed with a mixture of anisaldehyde and sulfuric acid reagent, which produced a purplish-blue tint with a hint of yellow13. phenolic test in the development of chloroform: methanol (9:1), the sample was detected on a silica gel plate. it was then sprayed with fecl3 reagent and formed a blackish blue, green or turquoise color13. results and discussion the test material in this study was muntingia calabura leaves obtained from the city of palu and was started from june to august 2020. the determination aimed to determine the accuracy of the test material employed. determination of muntingia calabura leaves was carried out at upt. biological resources tadulako university, central sulawesi. the analysis revealed that the muntingia calabura leaves utilized in the study belonged to the genus elaeocarpaceae. muntingia calabura leaves were made by maceration using ethanol as a solvent14. elasari dwi pratiwi & niluh puspita dewi | screening of phytochemical secondary metabolites of muntingia calabura: a potential as hepatoprotector 62 the thick extract of muntingia calabura leaves obtained from the simplicia maceration was 45 grams with a yield value of 6.4%. furthermore, the thick extract of muntingia calabura leaves was fractionated using 3 solvents, namely nhexane, ethyl acetate and ethanol-water. the weight of each fraction obtained was the n-hexane fraction of muntingia calabura leaves of 20.29 grams with a yield value of 4.05%, the ethyl acetate fraction of muntingia calabura leaves of 13.91 grams with a yield value of 2.7% and the ethanol-water fraction of muntingia calabura leaves of 29.17 grams with a yield value of 5.83%. the class of secondary metabolites found in muntingia calabura leaves extract as bioactive chemicals expected to have a role in delivering hepatoprotective effects was determined through phytochemical testing. the ethanol extract of muntingia calabura leaves contained positive alkaloids, flavonoids, saponins, tannins, and phenolics, according to the results of phytochemical screening (table i). table 1. the results of phytochemical screening of muntingia calabura extract chemical compounds reagent result exp. alkaloids dragendorf red (+) flavonoids mg powder + hcl red yellow (+) saponins water + hcl foam ± 1 cm (+) tannins fecl3 + nacl 10% blackish green (+) phenolic fecl31% blackish green (+) note: (+) : detected (-) : not detected as muntingia calabura leaves extract contained a wide range of polar, semipolar, and non-polar secondary metabolite chemicals, the fractionation method was used to separate some of these compounds based on their polarity. fractionation was carried out in stages, starting with separating non-polar compounds using n-hexane as a non-polar solvent, then semi-polar compounds using ethyl acetate as a semi-polar solvent, and finally using water as a polar solvent to attract polar compounds15. the phytochemical screening of the obtained fractions revealed that alkaloids, flavonoids, saponins, tannins, and polyphenols were present in the muntingia calabura fractions (table 2). table 2. the results of phytochemical screening of muntingia calabura fractionation chemical compound reagent result n-hexane fraction ethyl acetate fraction ethanol-water fraction alkaloids dragendorf + + flavonoids mg and hcl + + + saponins water and hcl + + + tannins fecl3 + nacl + + phenolic fecl3 + + note: (+) : detected (-) : not detected thin-layer chromatography (tlc) was performed to observe the chromatogram pattern of the muntingia calabura fraction. tlc can be identified by separating the journal of fundamental and applied pharmaceutical science, 2(2), february 2022 63 muntingia calabura fraction and the eluent. the eluents used in determining the chromatogram of the muntingia calabura fraction were alkaloid compounds using a mixture of chloroform: methanol (85:15), flavonoid compounds using a mixture of butanol: acetic acid: water (4: 1: 5), saponin compounds using a mixture of chloroform: methanol: water (40:50:10) and phenolic compounds using a mixture of chloroform: ethanol (9:1). the observations were conducted under uv light 254 and 366 nm. at uv 254 nm, the plate fluoresced while the sample appeared dark in color. meanwhile, the stain fluoresced at uv 366 nm, and the plate appeared dark in color. it was then followed by spraying using 10% h2so4 in methanol. the results of tlc showed that the muntingia calabura fraction contained alkaloids, flavonoids, saponins and phenols, which were indicated by purple spots (table 3 and figure 1). table 3. the results of thin-layer chromatography chemical compound eluent n-hexane fraction ethyl acetate fraction ethanol-water fraction alkaloids chloroform: methanol (85: 15) + flavonoids butanol:acetic acid:water (4 :1 : 5) + + + saponins chloroform:methanol:water (40: 50: 10) + + + phenolic chloroform: methanol (9: 1) + + note: (+) : detected (-) : not detected (a) (b) (c) (d) figure 1. thin-layer chromatography test using uv light with a wavelength of 254 nm and 366 nm (a) alkaloids; (b) flavonoids; (c) saponins; (d) phenolic elasari dwi pratiwi & niluh puspita dewi | screening of phytochemical secondary metabolites of muntingia calabura: a potential as hepatoprotector 64 the result of tlc profiling is summarized in figure 1. n-hexane fraction muntingia calabura showed the presence of flavonoid (rf 0.8) and saponin (rf 0.8). ethyl acetate fraction muntingia calabura showed the presence of alkaloid (rf 0.7), flavonoid (rf 0.6), saponin (rf 0.8) and phenolic (rf 0.8). ethanol-water fraction muntingia calabura showed the presence of flavonoid (rf 0.5), saponin (rf 0.7), and phenolic (rf 0.3). tlc is usually done for better identification of the bioactive compounds. different rf values of the compounds provide an idea about their polartity that may also help select a particular solvent system to isolate any compound from the plant fraction further using chromatographic and spectroscopic. compounds with a high rf value in a less polar solvent system have low polarity while those with a low rf value have high. these findings suggested that muntingia calabura fractions could be evaluated in vivo and in vitro for hepatoprotective properties. research conducted by (haki, 2019) revealed that the muntingia calabura extract could reduce the alt enzyme of mice although it had not reached the normal value16. furthermore, (hakim, 2012) research stated that the muntingia calabura extract dose of 420 mg/200 gram and 84 mg/200 gram could prevent increased levels of the enzyme alt in acetaminophen-induced rats17. this information is critical for a simple standardization in evaluating activity as herbal-based medicines. conclusion based on the research results, it is confirmed that muntingia calabura contained alkaloids, tannins, saponins, and phenolics, as evidenced by tlc results. as a result, muntingia calabura played hepatoprotective herbal 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sgpt pada mencit yang diinduksi karbon tetraklorida. skripsi. fakultas kedokteran universitas sebelas maret. surakarta. 17. hakim, w. r. 2012. uji efek daun kersen (muntingia calabura l.) terhadap kadar alanin aminotransferase (alt) pada tikus yang diinduksi asetaminofen. fakultas kedokteran. universitas muhammadiyah surakarta. p. 12. https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 https://doi.org/10.20961/alchemy.15.1.23362.57-78 72 antimicrobial test of 1-(2.5-dihydroxi phenyl)-(3pyridine-2-il) -propanone compound in enterococcus faecalis and escherichia coli bacteria using a well diffusion method andy eko wibowo*; rivaldy rifai hatala; dan awaluddin m edang school of pharmacy, faculty of medicine and health science, universitas muhammadiyah yogyakarta, jl brawijaya, tamantirto, kasihan, bantul, yogyakarta 55183. abstract 1-(2.5-dihydroxy phenyl)-(3-pyridine-2-il)-propenone compound is a compound synthesized by reacting the pyridine-2-carbaldehyde and 2.5-dihydroxyacetophenone compound without solvent with k2co3 (potassium carbonate) catalyst in the microwave. the 1(2.5-dihydroxy phenyl)-(3-pyridine-2-il)-propenone compound is a chalcone derivative compound substituted by two hydroxy groups on ring a and has 2-pyridyl groups on ring b. chalcone is a secondary metabolite compound from the flavonoid group, which has several activities as anti-platelet, anti-bacterial, immunomodulator, anti-hyperglycemic, and anti-inflammatory. this study aims to determine the antibacterial effect of 1-(2.5dihydroxifenil)-(3-pyridine-2-il)-propenone compound against enterococcus faecalis and escherichia coli bacteria. this study used tlc (thin layer chromatography) and melting point test to analyze the purity of 1-(2.5-dihydroxy phenyl)-(3-pyridine-2-il)propenone compound. meanwhile, the test for antibacterial activity used a well diffusion method. concentration variation for 1-(2.5-dihydroxifenyl)-(3-pyridine-2-il)-propenone compound as antibacterial in escherichia coli were 0.25 mg/100 μl, 0.5 mg/200 μl, and 0.75 mg/300 μl. meanwhile, the concentration variation for enterococcus faecalis bacteria was 5%, 2.5%, 1.25% and was replicated three times. the results of the compound purity test using the melting point test and thin layer chromatography (tcl) showed that the 1-(2.5-dihydroxy phenyl)-(3-pyridine-2-il)propenone compound was pure. the results of the antibacterial activity test for 1-(2.5-dihydroxiphenyl)-(3-pyridine-2-il)propenone compound showed no zone of inhibition at each test concentration. in conclusion, the 1-(2.5-dihydroxifenyl)-(3pyridine-2-il)-propenone compound did not have an antibacterial effect on enterococcus faecalis and escherichia coli bacteria. keywords: 1-(2.5-dihydroxiphenyl)-(3-pyridine-2-yl) -propenon; antibacterial; chalcone compounds; enterococcus faecalis; escherichia coli data of article received reviewed accepted : : : 28 dec 2020 20 jan 2021 22 feb 2021 doi 10.18196/jfaps.v1i2.10983 type of article: research * corresponding author, e-mail: andyew@umy.ac.id andy eko wibowo, rivaldy rifai hatala, awaluddin m edang | antimicrobial test of 1-(2.5-dihydroxi phenyl)-(3pyridine-2-il) -propanone compound in enterococcus faecalis and escherichia coli bacteria using a well diffusion method 73 introduction there have been many studies on new compounds whose efficacy is still not significantly known all this time. one of them is 1-(2.5-hydroxyphenyl)-(3pyridine-2-il)-propenone compound, a synthesis of chalcone derivative compounds that have several benefits in it3. chalcone is one of the flavonoids which has a c6-c3-c6 framework. derivatives of this compound have an essential role in nature and the world of health. chalcone and its derivatives have several activities that can be utilized in the pharmaceutical field, such as anti-platelet, anti-bacterial, immunomodulatory, anti-hyperglycemic, and anti-inflammatory1. therefore, these compounds' biological activity and potential are significant and beneficial for drug development, and some efforts are needed to develop the synthesis of chalcone and its derivatives2. one example of a compound that contains chalcone is the ashitaba plant, which also contains alkaloids. the 1-(2.5-dihydroxy phenyl)-(3-pyridine2-il)-propenone compound is a chalcone derivative compound substituted by two hydroxy groups in ring a and has 2-pyridyl groups on ring b. the compound is obtained from synthesis using the microwave method. the synthesis is from 2.5-dihydroxyacetophenone and pyridine2-carbaldehyde compound with k2co3 catalyst without solvent, taking 4 minutes using a 140-watt microwave power3. based on this background, this study aims to determine the antimicrobial activity of 1-(2.5-dihydroxyphenyl)-(3-piridin-2-il)propenone compound, which was tested on enterococcus faecalis and escherichia coli by the well diffusion method. method tools and materials the tools used in this study were aluminum foil (klin pak®), label (brand®), blue tip (pipette tip®), yellow tip (pipette tip®), glassware (prex®), analytical scales (casbee®), ose, tweezers, micropipette (gilson®), incubator (memmert®), laminar airflow (laf), hot plate (thermo scienific®), autoclave (all american®), spatel, capillary tube, chamber, mortir stamper, melting point apparatus, uv viewing cabinet, and vortex mixer. meanwhile, the materials used in the study were dry simplicia of 1-(2.5-dihydroxy phenyl)-(3-pyridine-2-il)-propenone compound, dmso, distilled water (brataco®), chloroform (chcl3), sodium chloride (nacl), n-hexane, ethyl acetate, ethanol 70%, and silica gel 60 gf 254. the test microbes used in this study were escherichia coli atcc 25922 and enterococcus faecalis atcc 29212. the comparative antibiotic was amoxicillin, and the media was nutrient agar (na). this study was conducted in the pharmaceutical technology laboratory and microbiology laboratory at university muhammadiyah yogyakarta. method of compound purity test 1. purity test by thin layer chromatography (tlc) the compound purity test was to determine the purity of the compound, 1(2.5-dihydroxy phenyl) -3-pyridine-2il-propenone, which was obtained from the synthesis of pyridine-2carabaldehyde and 2.5 dihydroxyacetophenone3. the mobile phases used were chloroform, nhexane: ethanol (10:1), and n-hexane: journal of fundamental and applied pharmaceutical science, 1(2), february 2021 74 ethanol (1:2), and the stationary phase used was silica gf254. the comparison solutions used in this study were 2.5 dihydroxyacetophenone and pyridine2-carbaldehyde compounds. three tlc plates were prepared with each measuring 10 cm long and 4 cm wide, and on each tlc plate, the test solution and comparison solution were placed separately using a micropipette. the tlc plates were inserted into the tlc chambers filled with the mobile phase. after the mobile phase rose to the upper edge of tlc plates, the tlc plates were dried to be read under uv light at a wavelength of 254 nm. 2. purity test with melting point test test for purity with a melting point used the melting point apparatus. this tool is commonly used to measure the value of the melting point or melting point of a compound. this tool was easy to use. the first thing to do was determining the compound's melting point to be tested; compound 1-(2.5-dihydroxy phenyl)-(3-pyridine-2-yl)-propenone has a melting point of 190.1o c. the 1 (2.5-dihydroxiphenyl) (3-pyridine-2-il) -propenone compound was crushed using a mortar and a stamper until the test sample became smooth, and the 3mm test sample was inserted into the capillary tube used in measuring the melting point test. furthermore, the melting point apparatus was set at 180.1o c; thus, when the temperature reached 180.1o c, the capillary tube that had been filled with the test sample was inserted into the hole to identify at what temperature the 1-(2.5dihydroxiphenyl)-(3-pyridine-2-il)propenone compound would melt. antimicrobial activity test method with well diffusion method 1. tool sterilization tool sterilization was necessary to ensure there was no contamination during the test process. the tools were first wrapped in aluminum foil and then sterilized in an autoclave at 121oc for 15 minutes. ose and tweezers were burned with bunsen before being used. 2. the making of growth media the media used in this study was nutrient agar (na), which was put into 10 petri dishes, totaling 15 ml. the method used was by weighing 4.2 grams of na, then dissolving the na with 150 ml of distilled water. once it was mixed, it was heated until it dissolved completely. after that, the media was sterilized using autoclave at 121oc for 15 minutes, and the media was poured into 10 sterilized petri dishes. 3. inoculum preparation the bacteria used in the study were escherichia coli atcc 25922 and enterococcus faecalis atcc 29212. a. rejuvenation of tested bacteria the bacteria were rejuvenated on nutrient agar (na) media. the test microbes were inoculated for one ose into na and incubated at 37oc for 24 hours. the rejuvenation of bacteria was carried out sterilely in laminar air flow (laf). andy eko wibowo, rivaldy rifai hatala, awaluddin m edang | antimicrobial test of 1-(2.5-dihydroxi phenyl)-(3pyridine-2-il) -propanone compound in enterococcus faecalis and escherichia coli bacteria using a well diffusion method 75 b. the making of bacterial suspensions the rejuvenated bacteria were taken for one ose, suspended into 15 ml of sterile 0.9% nacl solution, and then homogenized. 4. the making of test solution for escherichia coli atcc 25922 bacteria the preparation of the test solution was carried out by making several variations in volume levels, by weighing 25 mg of 1-(2.5-dihydroxy phenyl)-(3-pyridine-2yl)-propenone compound and adding 10 ml of dmso 100%. it was later vortexed until it became homogeneous. it was divided into 3 parts: a (100 μl), b (200 μl), and c (300 μl). each part received dmso 100% up to 1000 μl (1 ml), and it was re-vortexed until it became homogeneous. 5. the making of test solution for enterococcus faecalis atcc 29212 bacteria the test solution was made into three levels: 5%, 2.5%, and 1.25%. firstly, the base solution was prepared by weighing 10 mg of the test compound, dissolved in 10ml of dmso 100%, and vortexed until it was homogeneous. a total of 5%, 2.5%, and 1.25% of the base solution were taken, and each solution was later added dmso 100% up to 1 ml and was vortexed to make it homogeneous. 6. the making of positive control solutions the preparation of positive control solutions used amoxicillin trihydrate antibiotics as much as 1mg/1ml. in producing the test solution, 1 mg of amoxicillin was dissolved in 1 ml of sterile aqua dest and vortexed until it became homogeneous. 7. the making of negative control solutions the preparation of negative control solutions used dmso 100%. the negative control solution's volume was 1 ml, and 1ml of dmso 100% was taken. 8. antibacterial activity determination by using a well method the method used to determine the antimicrobial activity of 1-(2.5dihydroxiphenyl)-(3-pyridine-2-il)propenone compound was the well method. what needs to be prepared first was a sterile petri dish containing na media, the test solution, the positive control solution (the antibiotic), and the negative control solution. the test was carried out after all tools had been sterilized in laminar air flow (laf). three petri dishes containing na media were evenly rubbed with the bacterial suspension solution using cotton buds (sterilized using autoclaving). the petri cups containing na media were perforated; each cup had 5 holes. the holes were aimed for the test solution, positive control solution, and negative control solution. journal of fundamental and applied pharmaceutical science, 1(2), february 2021 76 each hole was inserted with a test solution of 20 μl using a micropipette. furthermore, the petri dishes were put into the incubator for 24 hours at 37oc. after 24 hours, the inhibition zone diameter was observed and measured by three measurement methods. results and discussion purity test using thin-layer chromatography tlc test used a stationary phase (silica gel 60 f254) and a mobile phase, adjusted to research by wibowo (2013), namely, hexane: ethanol (10: 1); hexane: ethanol (1: 2); chloroform. a 0.50 μl of 2.5 dihydroxyacetophenone and pyridine-2carbaldehyde compound were spotted on tlc plates with a size of 3 x 10 cm. the plate that has been spotted with the sample was developed in a tlc chamber filled with hexane: ethanol (10: 1); hexane: ethanol (1: 2); chloroform. the results of tlc that have been developed in the chamber were then observed under 254nm uv light (figure i) figure i. purity test with tlc. hexane mobile phase: ethanol (10: 1) (a); hexane: ethanol (1: 2) (b); chloroform (c). the purity of the compounds from the analysis using tlc showed that the test compound had a difference in rf with the material compound, and the test compound spots had only 1 spot. thus, it can be concluded that the compound was pure by tlc. purity test using the melting point the results of the melting point test were 190.50c, 190.40c, and 190.50c. these results were similar to the research by wibowo (2013), which was 190.1. a compound is considered pure if it has a sharp melting point and the melting distance does not exceed 0.5-10c. therefore, the 1-(2.5-dihydroxiphenyl)-(3pyridine-2-il)-propenone compound used in this study was pure, based on the melting point test. table i. results of the purity test of 1-(2.5dihydroxifenyl)-(3-pyridine-2-il)propenone by melting point test replication melting point (oc) 1 190.5 2 190.4 3 190.5 in addition, the 1-(2.5-dihydroxifenyl)-(3pyridine-2-il)-propenone compound used andy eko wibowo, rivaldy rifai hatala, awaluddin m edang | antimicrobial test of 1-(2.5-dihydroxi phenyl)-(3pyridine-2-il) -propanone compound in enterococcus faecalis and escherichia coli bacteria using a well diffusion method 77 in this study had the same characteristics as those used by wibowo (2013), for example, in the form of a dark orange solid, sticky, easily soluble in dmso, insoluble in water and slightly soluble in ethanol. antibacterial activity test for escherichia coli atcc 25922 the 1-(2.5-dihydroxiphenyl) antibacterial activity test-(3-pyridine-2-il)-propenone compound was carried out by dividing it into 3 volumes: 100 μl, 200 μl, and 300 μl. the solvent used to carry out the antibacterial activity test was dmso 100%. dmso 100% was also used as a negative control, while amoxicillin was used as a positive control. each volume variant of the 1-(2.5-dihydroxiphenyl)-(3pyridine-2-il)-propenone compound was replicated 3 times. the method was well diffusion, followed by basting the bacteria that have been made into a suspension. furthermore, holes were made in the media as a place for the test compound, positive control, and negative control. the inhibition zone diameter obtained from the antibacterial activity test can be seen in the following table. table ii. the result of the antibacterial activity test of the 1-(2.5-dihydroxifenil)-(3-pyridine2-il)-propenone compound against the growth of the escherichia coli atcc 25922 bacteria volume replication (mm) average (mm) ±sd i ii iii 300 μl 0 0 0 0 0 200 μl 0 0 0 0 0 100 μl 0 0 0 0 0 k – (dmso) 0 0 0 0 0 k+ (antibiotic) 12.3 12.3 10.7 11.7 ±0.930 *each replication hole had 3-time measurements. figure ii. the test results for the antibacterial activity of 1(2.5-dihydroxiphenyl) (3pyridine-2-il) -propenone compound on the growth of escherichia coli in experiment i, ii, and iii. i ii iii journal of fundamental and applied pharmaceutical science, 1(2), february 2021 78 escherichia coli is a gram-negative bacteria, a cumulative anaerobic which can grow in the presence or absence of oxygen. escherichia coli is also a gramnegative bacterium with multi-layered and complex cell walls, and its outer membrane can work as a variety of compounds, including the antibacterial compound. antibacterial activity test of enterococcus faecalis atcc 29212 enterococcus faecalis is a gram-positive bacteria belonging to the facultative anaerobic group. amoxicillin was used as a positive control as it is a broad-spectrum antibiotic that can be used as an antibacterial for gram-negative and grampositive bacteria. amoxicillin is a semisynthetic penicillin antibiotic that has a β-lactam ring. the zone of inhibition formed after incubation for 24 hours on media smeared with bacteria was measured using a caliper. the result of the antibacterial activity test can be seen in figure iii. after obtaining the results from the incubation with three repetitions, the inhibition zone was measured, and the results are shown in table iii. figure 3. results of antibacterial activity test of 1-(2.5-dihydroxiphenyl)-3-pyridine-2-ilpropenone the average inhibition rate of amoxicillin positive control with a concentration of 0.1% was 26 mm. the criteria for the antibacterial power are as follows: the inhibition zone diameter of 5 mm or less is categorized as weak, the inhibition zone 5 10 mm is categorized as moderate, the inhibition zone 10-20 mm is categorized as strong, and the inhibition zone of 20 mm or more is categorized as very strong4. based on the results, amoxicillin antibacterial inhibition against enterococcus faecalis bacteria was in the category “strong” (≥20mm). control + 1.25% 2.5% 5% control control control 1.25% 2.5% 2.5% 1.25% 5% 5% andy eko wibowo, rivaldy rifai hatala, awaluddin m edang | antimicrobial test of 1-(2.5-dihydroxi phenyl)-(3pyridine-2-il) -propanone compound in enterococcus faecalis and escherichia coli bacteria using a well diffusion method 79 table iii. results of antibacterial activity test of 1-(2.5-dihydroxiphenyl)-3-pyridine-2-ilpropenone the inhibition of the 1-(2.5dihydroxyphenyl)-3-pyridine-2-ilpropenone from three concentrations made in three replications was 0 mm. it indicated none of the three kinds of concentrations of 1-(2.5-dihydroxifenyl)3-pyridine-2-il-propenone (5%, 2.5%, 1.25%) caused the effect of inhibiting bacterial growth enterococcus faecalis. this result was different from the initial hypothesis of this study, where the 1-(2.5dihydroxiphenyl)-3-pyridine-2-ilpropenone compound was considered a chalcone derivative that had the effect of inhibiting the growth of enterococcus faecalis bacteria. it was probably due to the functional group in the compound that was unable to inhibit bacterial growth5. the lack of sensitivity of chalcone-derived compounds in inhibiting the growth of gram-positive bacteria was due to the absence of specific receptors (protein molecules that receive chemical signals) for the entry of test compounds into bacterial cells6. the antibacterial properties of chalcone depend on the group attached to the two aromatic rings, such as the cl, br, and oh groups7. chalcone is one of the secondary metabolic compounds from the flavonoid group. flavonoids can inhibit bacterial growth by damaging bacterial cell walls, deactivating enzymes, binding to adhesins, and damaging cell membranes8. conclusion the 1-(2.5-dihydroxy phenyl)-(3-pyridine2-il) -propenone compounds used in the study were proven to be pure. there was one spot on tlc, and the solid was dark orange, sticky, easily dissolved in dmso, insoluble in water, and slightly soluble in ethanol. compound 1-(2.5-dihydroxifenil)(3-pyridine-2-il)-propenone, in the antimicrobial test using the well diffusion method, could not inhibit the growth of escherichia coli and enterococcus faecalis bacteria. conflict of interest there are no conflicts of interest in the research. references 1. gaikwad, k. v., gaikwad, s., jadhav, s. b. (2010). synthesis of some novel chalcones of phthalimidoester concentration replication (mm) average sd 1 2 3 5% 0 0 0 0 0 2,5% 0 0 0 0 0 25% 0 0 0 0 0 control 0 0 0 0 0 control + 26 26 26 26 0 journal of fundamental and applied pharmaceutical science, 1(2), february 2021 80 possessing good anti-inflammatory and antimicrobial activity. indian journal of chemistry-b, 49, pp. 131-136. 2. diedrich, d. f. (1962). some new synthetic flavonoid glycosides related in structure to phlorizin. journal of medicinal and pharmaceutical chemistry, 5, pp. 1054-1062. 3. wibowo, a. e. (2013). sintesis dan uji aktifitas antiinflamasi senyawa 1(2.5dihidroksifenil)-(3-piridin-2-il) propenon. tesis. program studi ilmu farmasi, fakultas farmasi, universitas gajah mada. 4. davis, w. w., & stout, t. r. (1971). disc plate method of microbiological antibiotic assay. applied microbiology , 22(4), pp. 659–665. 5. eryanti, y., zamri, a., jasril & rahmita. 2010. sintesis turunan 2’-hidroksi kalkon melalui kondensasi claisenschmidt dan uji aktivitasnya sebagai antimikroba. jurnal natur indonesia. 12(2), pp. 223-227. 6. nilawati, a., & ansory, h. m. (2017). aktivitas antibakteri pada senyawa turunan kalkon hasil sintesis dari miristisin buah pala. jurnal farmasi indonesia. 14 (2), pp. 154-159. 7. prasad, y. r., kumar, p. r., deepti, c. a., & ramana, m. v. (2006). synthesis and antimicrobial activity of some novel chalcones of 2-hydroxy-1acetonapthone and 3-acetyl coumarin. journal of chemistry, 3(4), pp. 236-241. 8. cowan, m. m. (1999). plant products as antimicrobial agents. clinical microbiology reviews, 12(4), pp. 564– 582. validation of uv-vis spectrophotometric method of quercetin in ethanol extract of tamarind leaf erma yunita*, deni yulianto, siti fatimah, tirsa firanita akademi farmasi indonesia, jl. veteran gg. jambu, pandeyan, umbulharjo, kota yogyakarta, daerah istimewa yogyakarta 55161. abstract tamarindus indica l is a medicinal plant that has many benefits. one of the chemical compounds contained therein is the flavonoid quercetin type. the number of herbal products on the market makes the quality assurance of herbal products need to be done by performing the assay of the active compounds using validated methods. this study aims to validate the assay method quercetin in the extract of tamarind leaves. tamarind leaf extract was macerated with hexane; then, it was re-macerated with 70% ethanol. the extract was concentrated using a rotary evaporator. the assay was performed using the uv-vis spectrophotometry method, and parameter validation specified in this study, including linearity, lod, loq, precision, and accuracy. quercetin level obtained in extracts of tamarind leaves was at 21.52 mg/g. based on the test method validation, the correlation coefficient (r) was 0.9999, the regression function coefficients (vx0) was 0.59545%, lod 0.1515 ppm, loq 0.4592 ppm, coefficient of variation precision was < 2%, and recoveries range was in 97-103%. keywords: tamarind; tamarindus indica l.; uv-vis spectrophotometric; validation data of article received reviewed accepted : : : 24 feb 2020 12 may 2020 4 jul 2020 doi 10.18196/jfaps.010102 type of article: research * corresponding author, e-mail: ermayunita@afi.ac.id erma yunita , deni yulianto, siti fatimah, tirsa firanita | validation of uv-vis spectrophotometric method of quercetin in ethanol extract of tamarind leaf 12 introduction tamarindus indica l. can be used for many purposes, such as herbs, medicinal materials, and cosmetics. furthermore, tamarind fruit pulp can be used as a mixture of traditional medicine and herbs.1,2 flavonoid compounds contained in tamarind leaves are a kind of flavonoid quercetin. quercetin has potential as antiviral, antibacterial, and antiinflammatory.1 quercetin has a good antibacterial activity for the phenol group with protein coagulation mechanism by deactivating enzymes and disrupting the cell wall; thus, it has good bactericidal properties.3 quercetin in plants is in various forms of glycosides.4 there are a considerable amount of benefits of tamarind leaves, which can increase the development of product innovation.5 quality assurance of herbal products can be done by performing the assay of the active compounds using methods that have been validated.6 the current widely used analytical method is the uv-vis spectrophotometry method. the method provides a simple way to determine the quantity of a little substance. the method used in the assay should be validated. validation of the methods of analysis is an assessment of the actions of certain parameters based on laboratory experiments. this activity is carried out to prove the parameters used to meet the requirements for its use. validation can also be used to identify the source of the unwanted variability.7 methods the material used in this study was tamarind leaves (tamarindus indica l.) from mantrijeron, yogyakarta. other materials used included ethanol pro analysis (merck), 70% ethanol (cv. general labora), filter paper, quercetin (sigma aldrich), aquadest (cv. general labora), and aluminum foil. the analysis instrument used in this study was the genesys 10s uv-vis spectrophotometer. preparation of tamarind leaves extract tamarind leaf was macerated with nhexane then stirred using an electric stirrer for 1 hour. it was then soaked for 24 hours. the results of the maceration were filtered. the residue obtained was then dried and macerated by 70% ethanol and then stirred using an electric stirrer for 1 hour. the soaking process was conducted for 24 hours to protect it from light, while it was occasionally stirred. the results of maceration were filtered to separate the pulp and filtrate. the macerating process was concentrated by a rotary evaporator and thickened above the water bath until a thick extract was formed. the ethanol extract could be calculated for its yield. rendemen calculation = g extract g simplicia x 100% preparation of stock solutions the stock solution of quercetin and tamarind leaf extract was made at a concentration of 10,000 ppm. stock solutions were created using ethanol pa. determination of maximum wavelength the stock concentration of 100 ppm as much as ± 3 ml was measured with a spectrophotometer at a wavelength of 200-800 nm. preparation of quercetin standard curve the standard curve was created by connecting the standard solution concentration in the series concentration of 4 ppm, 6 ppm, 8 ppm, 10 ppm, and 12 ppm with the absorption results obtained from the measurement using uv-vis spectrophotometry at maximum wavelength.8 journal of fundamental and applied pharmaceutical science, 1(1), august 2020 13 determination of my quercetin levels in the tamarind leaf extract tamarind leaf extract of a concentration of 500 ppm measured its absorbance using uv-vis spectrophotometry at a maximum wavelength. sample solutions were made in 3 trials. validation of analysis methods linearity was conducted by measuring the absorbance of the comparison solution. furthermore, the curve of the relationship between concentration vs. absorption and the linear regression equation and the correlation coefficient (x) and the correlation coefficient of the function (vxo) were determined. the regression function coefficient (vxo) can be calculated using the formula below.6 sy/x = √ σ(yr−y)2 n−2 sxo = syfx b vxo = sy/x x̅ x 100% information: yr : absorbance sy/x : residual standard deviation the correlation coefficient is considered to be good if r has a value of ≥ 0.998. the regression function coefficient (vxo) is deemed to be good if it has a value of ≤ 5%. limit of detection and limit of quantitation can be calculated statistically through linear regression lines from the calibration curve. the measurement value will be the same as the value of b in the linear line equation y = a + bx, while the standard deviation of the blank is the same as the residual standard deviation (sy/x) or by the formula below.6 lod = 3,3 x sy/x b loq = 10 x sy/x b information: sy/x : residual standard deviation b : response from slope (slope in line equation y = bx + a) precision is carried out by measuring the absorbance of the test solution, and then the percentage of correlation coefficients was calculated to identify the reproducibility of the system used cv requirements <2%6. testing was conducted 7 times. accuracy in this study was carried out by the standard addition method. the comparison between the extract and the standard is 70:30 with a range of 80%, 100%, and 120%. the absorbance of the solution was measured using a uv spectrophotometer at a maximum wavelength, and the experiment was carried out 3 times. accuracy testing was calculated as percent recovery using the formula: percent recovery = 𝐶𝐹−𝐶𝐴 𝐶 𝐴 ∗ x 100% information: cf : total sample concentration obtained from measurements ca : the actual sample concentration c*a : the concentration of the analyte added results and discussion tamarind leaf extract was carried out using the maceration method. maceration is the most widely used simple method. this method is carried out by inserting the appropriate plant powder and solvent into an inert container, which is tightly closed at room temperature.9 maceration was conducted using n-hexane solvent and then macerated again using 70% ethanol. erma yunita , deni yulianto, siti fatimah, tirsa firanita | validation of uv-vis spectrophotometric method of quercetin in ethanol extract of tamarind leaf 14 extraction with n-hexane solvent aimed to dissolve chlorofil contained in tamarind leaves as chlorophyll would interfere at absorbance readings. n-hexane solvents were selected as chlorophyll is insoluble in water. however, it can dissolve in various types of organic solvents10; thus, it can be seen that chlorophyll is non-polar, while solvents are chosen to have the same properties as chlorophyll. the residue from maceration was then added with 70% ethanol until the entire powder was submerged. the filtrate was then evaporated using a rotary evaporator at 60˚c with a speed of 80 rpm to separate the extract from the solvent. the extract obtained was then evaporated again using a water bath at a temperature of 60˚c until a thick extract was obtained. the thick extract obtained was 11.98 grams. the extract yield was calculated by comparing the weight of the thick extract with the weight of dry simplicia in percent.11 the calculation of extract yield aimed to estimate the amount of material to be used to obtain the desired amount of extract. the yield value of tamarind leaf extract obtained was 9.74%. determination of the maximum wavelength is needed to increase sensitivity and minimize errors when it repeated measurements.12 in this study, the determination of maximum wavelength was carried out using a standard solution of a quercetin concentration of 100 ppm, which was measured in the wavelength range of 200-800 nm using uv-vis spectrophotometry. the measurements were carried out at that range as sibling uv, and visible rays had wavelengths ranging from 200-800 nm. based on optimization screening results, the maximum wavelength found two-peak spectra at 210 nm and 361.8 nm (figure 1). ethanol, as a solvent, is known to have maximum absorption at a wavelength of 210 nm.13 therefore, it can be concluded that the maximum wavelength of quercetin was 361.8 nm. meanwhile, other studies showed that quercetin has a maximum wavelength of 380 nm.14 a standard curve was carried out by using a standard solution of quercetin with series concentrations of 4 ppm, 6 ppm, 8 ppm, 10 ppm, and 12 ppm. the absorbance results obtained from each series of concentrations were then made linear regression line equations, which can be used to calculate the quaternary levels of quercetin in the tamarind leaf extract. the results of the quercetin standard curve are shown in figure 2. figure 1. determination of quercetin maximum wavelength λmax of quersetin 361,8 nm λmax of ethanol 210nm journal of fundamental and applied pharmaceutical science, 1(1), august 2020 15 figure 2. quercetin standard curve the results of the quercetin standard curve obtained the intercept axis value 0.0616 and the slop value of -0.018; thus, the linear regression line equation derived was y = 0.0616 x –0.018. determination of quercetin levels was carried out using a solution of tamarind leaves extract with a concentration of 500 ppm. the test was carried out three times, and it then calculated the average level of quercetin in the tamarind leaf extract. the results of level measurements are shown in table 1. quercetin levels can be calculated by processing data from the results of absorption measurements with the linear regression line equation y = 0.0616x – 0.018. the measurement results showed that quercetin levels were 10.76 ppm in 500 ppm tamarind leaf extract; thus, it can be seen that in 1 gram of tamarind leaf extract, there was 21.52 mg quercetin. measurement of this level can be useful to determine the amount of tamarind leaf extract needed in producing drugs with natural ingredients so that the desired pharmacological effect can be achieved. validation of the assay method is conducted to ensure that the analytical methods are accurate, specific, table 1. quercetin levels in tamarind extracts replication absorbance (a) quercetin levels (ppm) 1 0.645 10,763 2 0.644 19,747 3 0.645 10,763 𝒙 10.758 reproducible, and resistant to the analyte range to be analyzed15. the validation parameters of the method observed in this study are as follows: linearity is conveyed in terms of variance around the direction of the regression line, which is calculated based on mathematical equations of data obtained from the test results of analytes in samples with various series of concentration analytes.6 the parameter used in linearity testing is the correlation coefficient (r) in linear regression analysis y = a + bx. the linear test results are shown in table 2. the linear regression line equation obtained is y = 0.06160.018x with a correlation coefficient (r) 0.9999. linearity can also be determined by calculating the value of vxo, which is 0.59545%. based on the results of the correlation coefficient and the calculation of vxo, it can be seen that the spectrophotometric method has good linearity for vxo value is ≤ 5% with a correlation coefficient of 0.9999.16 table 2. linearity of quercetin concentration (ppm) absorbance (a) 4 0.231 6 0.349 8 0.474 10 0,600 12 0.722 slop (b) -0,018 intercept axis (a) 0.0616 correlation coefficient (r) 0.9999 y = 0.0616x 0.018 r = 0.9999 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0 5 10 15 a b so rb a n ce ( a ) concentration (ppm) erma yunita , deni yulianto, siti fatimah, tirsa firanita | validation of uv-vis spectrophotometric method of quercetin in ethanol extract of tamarind leaf 16 limit of detection is the smallest number of analytes in the sample, which still gives a significant response compared to others. limit of detection is a limit test parameter. meanwhile, the limit of quantification can be interpreted as the smallest quantity of analytes in a sample that had fulfilled the criteria of precision and accuracy6. the lod and loq can be calculated statistically based on the standard deviation response and the standard slope (s) curve. lod results obtained in this test are 0.1515 ppm. the loq results obtained were 0.4592 ppm. the result showed that the standard curve and the determination of quercetin content in tamarind leaf extract had fulfilled the lod and loq parameters as the standard curve used the lowest concentration of 4 ppm and quercetin levels above the lod and loq values of 9.34 ppm in 500 ppm tamarind leaf extract. a precision method was conducted repeatedly by the same analyst in a shorter time interval; thus, the precision can also be described as repeatability or reproducibility. precision was measured as a standard deviation or coefficient of variation6. precision parameters were carried out using various series of concentrations in making quercetin raw curves, namely 4 ppm, 6 ppm, 8 ppm, 10 ppm, and 12 ppm. the precision results in this study are shown in table 3. it shows that the variation of coefficient values of each concentration has a value of less than 2%. accuracy is a measure that shows the degree of closeness of the analyst's results to the actual levels of analyte. accuracy is described as a percentage of the recovery. accuracy testing performed in this study is a standard addition method for the tested compounds suk terms of secondary metabolite classes. tests were carried out in the range of 80%, 100%, and 120%, with differences in extract concentrations and standard used. the range of % recovery received for analyte levels of more than 1% is 97-103%6. the accuracy results in table 4 show the value of percent recovery that has met the requirements. it indicates that the determination of levels using the spectrophotometric method fulfills the parameters of accuracy so that it can provide accurate results. table 3. precision of quercetin replication absorbance (a) 4 ppm 6 ppm 8 ppm 10 ppm 12 ppm 1 0.232 0.343 0.470 0.601 0.731 2 0.226 0.343 0.476 0.594 0.716 3 0.226 0.348 0.476 0.598 0.725 4 0.229 0.349 0.474 0.607 0.716 5 0.231 0.356 0.472 0.596 0.721 6 0.235 0.353 0.471 0.604 0.724 7 0.235 0.353 0.480 0.597 0.722 𝒙 0.231 0.349 0.474 0,600 0.722 cv (%) 1.629 1.452 0.736 0.779 0.731 journal of fundamental and applied pharmaceutical science, 1(1), august 2020 17 table 4. percent recovery of quercetin range (%) absorbance (a) ca c*a cf % recovery 80 0.474 5.6 2.4 7.987 99.45 0.479 5.6 2.4 8.068 102.83 0.472 5.6 2.4 7.954 98.08 𝒙 100.12 100 0.601 7.0 3.0 10.048 101.60 0.595 7.0 3.0 9.951 98.36 0.597 7.0 3.0 9.983 99.43 𝒙 99.80 120 0.720 8.4 3.6 11.980 99.44 0.719 8.4 3.6 11.964 99.00 0.725 8.4 3.6 12.061 101.69 𝒙 100.04 ca : the actual sample concentration (ppm) c*a : the concentration of the analyte added (ppm) cf : total sample concentration obtained from measurements (ppm) based on the results of the study, it can be identified that the uv-vis spectrophotometry method has fulfilled several parameters for the determination of the content of quercetin from tamarind leaf extract. these parameters are linearity, lod, loq, precision, and accuracy. conclusion based on the results of the study, it can be concluded that the uv-vis spectrophotometry method has fulfilled several parameters for the determination of the concentration of quercetin from tamarind leaf extract. the quercetin level in the extract in tamarind was 21.52 mg in 1 g extract. the correlation coefficient is 0.9999, and the regression function coefficient (vxo) ≤ 5% is 0.59545%. the lod is 0.1515 ppm, and loq is 0.4592 ppm. the variation coefficient values for precision parameters have been less than 2%. meawnhile, the accuracy values have a range of 97-103%. acknowledgment the authors would like to thank the ministry of research, technology and higher education republic of indonesia (ristekdikti) for funding this research through the beginner lecturer research grant in 2019. conflict of interest there is no potential for conflict of interest. references 1. fakhrurrazi, f., hakim, r. f., & keumala, c. n. (2016). pengaruh daun asam jawa (tamarindus indica linn) terhadap pertumbuhan candida albicans. journal of syiah kuala dentistry society, 1(1), pp. 29-34. 2. doughari, j. h. (2006). antimicrobial activity of tamarindus indica linn. tropical journal of pharmaceutical research, 5(2), pp. 597-603. 3. nwodo, u. u., obiiyeke, g. e., chigor, v. n., & okoh, a. i. (2011). erma yunita , deni yulianto, siti fatimah, tirsa firanita | validation of uv-vis spectrophotometric method of quercetin in ethanol extract of tamarind leaf 18 assessment of tamarindus indica extracts for antibacterial activity. international journal of molecular sciences, 12(10), pp. 63856396. 4. kelly, g. s. (2011). quercetin. alternative medicine review, 16(2), pp. 172-195. 5. hendra, a. r. (2010). isolasi dan identifikasi golongan flavonoid daun dandang gendis (clinacanthus nutans) berpotensi sebagai antioksidan. [skripsi]. bandung: ipb. 6. harmita, h. (2012). petunjuk pelaksanaan validasi metode dan cara perhitungannya. pharmaceutical sciences and research (psr), 1(3), pp. 117-135. 7. torbeck, l. (2010). pharmaceutical and medical device validation by experimental design. journal of validation technology, 16(3), pp. 9-11. 8. alwi, h. (2017). validasi metode analisis flavonoid dari ekstrak etanol kasumba turate (carthamus tinctorius l.) secara spektrofotometri. [doctoral dissertation]. makasar: universitas islam negeri alauddin makasar. 9. tetti, m. (2014). ekstraksi, pemisahan senyawa, dan identifikasi senyawa aktif. jurnal kesehatan, 7(2), pp. 361367. 10. riyono, s. h. (2007). beberapa sifat umum dari klorofil fitoplankton. oseana, 32(1), pp. 2331. 11. pratomo, n. a., yunita, e., widyarini, s., & anshory, h. (2014). efek anti angiogenesis temu kunci (boesenbergia pandurata,(roxb.) schlecht) pada membran korio alantois embrio ayam yang diinduksi basic fibroblast growth factor (bfgf). khazanah: jurnal mahasiswa, 6(2), pp. 35-45. 12. yunita, e., arifah, e. n., & tamara, v. f. (2019). validasi metode penetapan kadar vitamin c kulit jeruk keprok (citrus reticulata) secara spekteofotometri uvvis. pharmacy: jurnal farmasi indonesia (pharmaceutical journal of indonesia), 16(1), pp. 118-131. 13. moffat, a. c., osselton, m. d., widdop, b., & watts, j. (2011). clarke's analysis of drugs and poisons (vol. 3). london: pharmaceutical press. 14. bancirova, m. (2015). changes of the quercetin absorption spectra in dependence on solvent. chemistry journal, 1(2), pp. 31-34. 15. pharmacopeia, u. s. (2007). national formulary 25. in rockville, md: us pharmacopeial convention (p. 1654). 16. prabowo, m. h., wibowo, a., & fauziyah, l. (2012). pengembangan dan validasi metode analisis rifampicin isoniazid-pirazinamid dalam fixed dose combination dengan metode kromatografi lapis tipis-densitometri. jurnal ilmiah farmasi, 9(2). antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis eva kholifah*, dewi nurazizah, fajrin noviyanto departement of pharmacy, sekolah tinggi ilmu kesehatan salsabila serang, banten, indonesia abstract antioxidants are chemical compounds that donate one or more electrons to free radicals to inhibit free radical reactions. one of the potential antioxidant sources is gandaria fruit (bouea macrophylla griff). this research aims to determine the antioxidant activity and vitamin c content of the gandaria fruit extract. gandaria fruit was macerated with ethanol as a solvent. furthermore, the rotated extract was carried out for the phytochemical screening. the results of the phytochemical screening of the gandaria fruit extract showed a positive of flavonoids, terpenoids, tannins, saponins, and phenolics. the gandaria fruit extract was tested for antioxidants and quantitative analysis of vitamin c levels to obtain ic50 values and vitamin c levels using spectrophotometry uv-vis at a wavelength of 517nm with vitamin c as a positive control. the results of spectrophotometric measurements revealed that the gandaria fruit extract had an ic50 value of 5.72 g/ml, and vitamin c had an ic50 value of 2.260 g/ml, indicating that the gandaria fruit extract and vitamin c had very strong antioxidants. the value of vitamin c levels was 0.526 mg. keywords: antioxidants; gandaria fruit; spectrophotometry uv-vis; vitamin c data of article received reviewed accepted : : : 25 aug 2022 09 dec 2022 30 jan 2023 doi 10.18196/jfaps.v2i1.15992 type of article: research introduction oxidative stress is a state with a high level the territory of indonesia has various natural potentials/assets that benefit indonesian people's lives. however, many natural assets in indonesia have not been fully explored and extracted optimally. indonesia is the third largest wealth of biological resources after brazil and zaire. according to who records, only about 20,000 plant species have been used as medicinal ingredients.1 * corresponding author, e-mail: evakholifah.apt@gmai.com antioxidants are compounds that can neutralize the bad effects of free radicals in the body. based on the source, there are two kinds of antioxidants: natural and synthetic (artificial). natural antioxidants are more desirable than synthetic antioxidants as they are considered safer or harmless. synthetic antioxidants such as bht (butylated hydroxytoluene) and bha (butylated hydroxy anisole) have been questioned as they have big side effects and can cause liver damage. it makes natural antioxidants an alternative needed by today's society.2 eva kholifah, dewi nurazizah, fajrin noviyanto | antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis 55 since ancient times, indonesian people have used natural resources to survive, one of them is as medicine. indonesian people have used plants as medicines to solve health problems. the knowledge and skills of the indonesian people to use plants as medicine has become an ancestral heritage from generation to generation that remains useful today.3 one kind of traditional medicinal plant that the medical industry has begun to look at is gandaria (bouea macrophylla griff). gandaria is the most abundant plant in malaysia, sumatra, west java, ambon and banten. this plant, known as exotic fruit, is generally spread on the island of ambon. in indonesia, this plant is called gandaria, jatake (sunda). some research showed gandaria (bouea macrophylla griff) has antibacterial, anticancer and antiaging activity. gandaria fruit (bouea macrophylla griff) contains high vitamin c.1 vitamin c is a water-soluble vitamin. insufficient consumption of vitamin c will cause some effects such as weakness, shortness of breath, muscle spasms, sore bones and joints and reduced appetite, dry, rough and itchy skin, and dry lips. some research revealed that gandaria has several pharmacological activity such as anticancer, antiphotoaging, antimicrobial, anti-hyperglycemic, and improved vegetable intake and blood beta carotene concentration. this research focuses on analyzing antioxidant activity and vitamin c concentration in gandaria fruit. method extraction gandaria fruit was obtained from pandeglang, banten, indonesia. fresh fruit was washed and dried. 400 g of powder was extracted in ethanol (1:7) for 3 days and macerated 3 times. the ethanol extracts were filtered and evaporated with a rotary evaporator. phytochemical screening alkaloid test 2 ml of extract was added to mayer and dragendorf reagent through the wall of the test tube. the presence of a white precipitate indicated the presence of alkaloids. phenolic test the extract was dissolved in water, then added to a lead acetate solution (10%). a white precipitate indicated the presence of phenolic compounds. steroid test acetic anhydride was added to 0.5 ml crude extract of plant sample with h2so4. the color change from violet to blue or green in the sample indicated the presence of steroids. flavonoid test the extract was dissolved in ethanol 96%, then 0.1 g magnesium and hcl were added. the yellow or violet coloration disappeared on standing. saponin test approximately 3 ml of gandaria extract was added to 3 ml of aqua dest and shaken vigorously. stable, persistent froth formation was taken as a positive test for saponin. tannin test the gandaria extract was added to 1% pb(ch3cooh) solution. tannins are present a yellow precipitate formed during the reaction. steroid test approximately 2 ml of chloroform and h2so4 were added to 5 ml of gandaria journal of fundamental and applied pharmaceutical science, 3(2), february 2023 56 extract. a red layer indicated the presence of steroids in the low chloroform. antioxidant activity analysis the method used to determine antioxidant activity by 1,1-di[phenyl-2 picrylhydrazyl (dpph) and spectroscopy uv-vis. a volume of 2 ml dpph solution, freshly prepared in 96% ethanol, was mixed with 2 ml gandaria fruit extract, and the maximal absorbance was measured at 517 nm. the antioxidant activity (ic50) was analyzed using a calibration curve using y=ax+b. the value of y has been replaced with 50. the ic50 value was classified by blois in table 1 determination of vitamin c concentration a standard 100 ppm ascorbic acid solution was prepared by dissolving 0.5 mg of ascorbic acid and diluted to 25 ml with aqua dest. next, the solution was determined to analyze maximal absorbance at 200 – 400 nm. the next step was calibration curve analysis. 5 different concentration (1 ppm, 2 ppm, 4 ppm, 8 ppm and 16 ppm) was prepared and measured at 216 nm. table 1. parameter of antioxidant activity based on the value of ic50 no value of ic50 antioxidant properties 1 <50 ppm very strong 2 50-100 ppm strong 3 101-150 ppm moderate 4 151-200 ppm weak 5 >200 ppm very weak results and discussion gandaria fruit was observed to determine its plant identity. this plant was determined at the biology learning laboratory of the faculty of applied science and technology ahmad dahlan university (uad). the determination result showed that the sample used was bouea macrophylla grif. extraction is the process of separating the active compound using a certain solvent. the extraction process aims to obtain the active compounds of a sample.4 the extraction results can be seen in table 2. table 2. extract yield gandaria fruit simplicia extract weight yield extract characteristics appearance color scent 3 kg 74.5gram 2.483% thick brown unique table 2 shows that the simplicia weight of gandaria fruit (bouea macrophylla griff) is 3 kg with a total solvent is 3.5 liters. after being concentrated with a rotary evaporator, the gandaria extract is thick and brown in color, weighing 74.5 grams with a yield of 2.483%. the results of the phytochemical screening test aim to identify the content of secondary metabolites that can potentially have antioxidant activity. the results of the phytochemical screening carried out can be seen in table 3. eva kholifah, dewi nurazizah, fajrin noviyanto | antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis 57 table 3. gandaria fruit phytochemical screening results (bouea macrophylla griff) examination reactant observation results alkaloid sample+ammonia+chloroform+mayer no white sediment flavonoid sample + alcohol + hydrochloric acid the color changes to orange + tanin sample + fecl3 color change + steroid& terpenoid sample + h2so4 + acetic anhydrous the color changes to dark red + saponin sample + aqua dest foam doesn't disappear + description: (+) contains the test compound (-) does not contain the test compound based on table 3, the results of the phytochemical screening conducted, it is known that the extract of gandaria fruit (bouea macrophylla griff) contained secondary metabolites, including alkaloids, flavonoids, tannins, triterpenoids, and saponins,5 which states that gandaria fruit contained secondary metabolites including flavonoids, triterpenoids, and saponins. we used the metal reagent mg and concentrated hcl for the flavonoid test, reducing the benzopyran core in the flavonoid structure and forming an orange flavilium salt. the results of the flavonoid sample test and the bouea macrophylla griff extract did not contain flavonoids (negative) by showing a red-orange color change in the extracted sample. the reaction that occurs is between flavonoid compounds with hcl and mg metal. tannins are chemical compounds of the phenol group soluble in water and polar solvents. testing for the existence of tannins in the ethanol extract was carried out with a fecl3 reagent. after the addition of fecl3, the color of the solution became blackish-green. the formation of a green-black color was caused by tannins forming a complex with fe3+. catechol tannins were green-black after adding fecl3.5 the results of the tannin test on the bouea macrophylla griff extract did not contain tannins (negative) by showing a blackishgreen color change so that the type of tannin contained in the bouea macrophylla griff extract was catechol tannin. for the saponin glycosides test, it can form foam in water when stirred. the saponin test was carried out based on the forth test by adding distilled water to the ethanol extract and then shaking it with vortex. if the bouea macrophylla griff extract contains saponins, then after shaking, it will form a foam.5 the bouea extract macrophylla griff test results revealed that it did not contain saponins, showing the presence of foam after shaking. in this study, the antioxidant activity of the extract of gandaria fruit bouea macrophilia griff was tested using the dpph method quantitatively using a uvvis spectrophotometer at a wavelength of 517 nm. journal of fundamental and applied pharmaceutical science, 3(2), february 2023 58 picture 3. absorbance curve wavelength dpph the purpose of determining the maximum wavelength is to determine the absorption area that can be produced in the form of the absorbance value of the gandaria fruit extract dissolved in ethanol and then measure the wavelength of 200-600 nm. testing of antioxidant activity was carried out using the dpph method. the dpph method is a method that can be used to determine antioxidant activity in the sample to be tested by looking at its ability to ward off dpph free radicals. this method was chosen as it was a simple, easy, fast, and sensitive method and only required a small sample. it is easy to apply as the dpph radical compound is relatively stable compared to other methods.6 the absorbance measurement in this study was carried out at a maximum wavelength of 517 nm dpph solution with an absorbance of (2,000). the wavelength is in accordance with the maximum wavelength range that can be used for measurements with the dpph method stated by londo,5 namely, from 517 nm to 522 nm, which can be seen in table 4. table 4. gandaria fruit bouea macrophilia griff antioxidant test results concentration (ppm) average of absorbance (a) = 517 nm inhibitory activity (%) linear linear regression equation ic50 value (μg/ml) 1 0.012 18.627 y = 40.384x + 19.412 5.72 2 0.016 32.353 4 0.020 44.118 8 0.024 55.882 16 0.073 67.647 blanko 0.035 eva kholifah, dewi nurazizah, fajrin noviyanto | antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis 59 the antioxidant activity of the fruit of gandaria bouea macophylla griff was measured at 5 dilution concentrations. analysis of antioxidant activity was carried out three times. it can be seen from the data in table 4 that all sample concentrations showed a reduced absorbance value, and % dpph radical inhibition (% inhibition) increased with increasing concentration. it indicated a reduction in the concentration of dpph after being reacted with the sample, which can be seen in figure 4. figure 4. linear regression curve of gandaria fruit extract (bouea macrophylla griff) the regression equation between the concentration of the extract of gandaria fruit (bouea macrophylla griff) on the xaxis and the percent (%) of antioxidant activity on the y-axis is y = 40,384x+19,412 with a correlation coefficient value of 0.999. the positive correlation coefficient value illustrated that increasing the concentration of the extract of gandaria fruit (bouea macrophylla griff) also raises the antioxidant activity. the parameter used to show antioxidant activity is inhibition concentration (ic50) which is the concentration of an antioxidant substance that can cause 50% of dpph to lose its radical character or the concentration of an antioxidant substance that gives 50% inhibition. the smaller the ic50 value is, the higher the antioxidant activity will be. the ic50 value of the sample extract was obtained based on the calculation of the linear regression equation from the absorbance curve of the sample. in this research, the antioxidant test utilized a comparison of vitamin c since the compounds contained in vitamin c could reduce or ward off free radicals, which were very good and are widely used by research as a comparison to other compounds, especially in antioxidants of gandaria fruit. the measurement of the antioxidant activity of vitamin c is shown in table 5. y = 40.384x + 19.412 r² = 0.999 0,000 10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 0,000 0,200 0,400 0,600 0,800 1,000 1,200 1,400 % in h ib it io n concentration µg/ml journal of fundamental and applied pharmaceutical science, 3(2), february 2023 60 table 5. vitamin c antioxidant test results concentration (ppm) average of absorbance (a) = 50nm inhibitory activity (%) linear regression equation ic50 value (μg/ml) 1 0.329 48.424 y = 4.4511x + 48.424 2.260 2 0.338 49.754 4 0.347 51.133 8 0.356 52.414 16 0.365 53.793 blanko 0.678 in measuring the antioxidant activity of vitamin c, the largest percentage at a concentration of 1 ppm revealed a free radical inhibition value of 48.424%. in comparison, the largest percentage at a concentration of 16 ppm showed a free radical inhibition value of 53.793%, so it can be seen that vitamin c had a strong activity from the large value—free radical inhibition from the smallest concentration to the highest concentration. analysis of antioxidant activity was carried out three times. the data from the antioxidant activity test results were then associated with the concentration of vitamin c on the absorbance curve so that a linear equation was obtained to achieve the ic50 value. the absorbance curve of vitamin c can be seen in figure 5. figure 5. linear regression curve of vitamin c y = 4.4511x + 48.424 r² = 0.9999 48,000 49,000 50,000 51,000 52,000 53,000 54,000 55,000 0,000 0,200 0,400 0,600 0,800 1,000 1,200 1,400 % in h ib it io n antioxidant activity eva kholifah, dewi nurazizah, fajrin noviyanto | antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis 61 gandaria fruit bouea macrophylla griff contains secondary metabolites, namely phenol. antioxidant activity in gandaria fruit is due to the presence of phenol components. in the human body, phenolic compounds can bind to active oxygen. in this case, these phenolic compounds act as antioxidants and prevent the effects of active oxygen, which can damage biological components such as proteins, lipids, vitamins and dna. a compound is classified as a very strong antioxidant if its ic50 value is less than 50 g/ml, strong if its ic50 value is between 50100 g/ml, moderate if its ic50 value is between 100-150 g/ml, and weak if the ic50 value is between 150-200 g/ml. the ic50 value of gandaria fruit bouea macrophylla griff is 5.72 µg/ml, which is classified as a strong antioxidant agent. however, vitamin c's antioxidant activity (ic50 value 2.260 µg/ml) has more potency than gandaria fruit bouea macrophylla griff. gandaria fruit bouea macrophylla griff is an extract with several components in small concentrations. at the same time, vitamin c is an isolated compound and single component with high concentration. determination of vitamin c concentration in the juice of gandaria fruit flesh was carried out using the uv vis spectrophotometry method. the linearity of vitamin c compounds was determined by making a standard series of 5 concentrations in the range of 0.25 ppm to 4 ppm at a wavelength of 265 nm. the following is a table and graph of the linear equation of standard vitamin c with distilled water at a wavelength of 265 nm for low concentrations (1 ppm to 16 ppm), as shown in table 6. table 6. absorbance analysis of vitamin c standard curve concentration (ppm) average of absorbance linear regression equation 1 0.042 y= 0.0122+ 0.0577x r² = 0.9326 2 0.085 4 0.128 8 0.171 16 0.242 based on the data above, the absorbance value was in the allowable linear range, namely y = 0.0122 + 0.0577x, with a correlation coefficient of r2 = 0.9326, indicating the linearity of the equation. in determining vitamin c levels, the results obtained were 0.526 mg/g. the value of vitamin c content of gandaria fruit indicated the measurement's accuracy level. accuracy is a measure that shows the degree of correspondence between individual test results and the average; if the procedure is repeated, the value used is 2%.7 conclusion this study revealed phytochemical compounds of gandaria (bouea macrophylla griff) were flavonoid, tannin, steroid, terpenoid, and saponin. the ic50 value of the extract of gandaria (bouea macrophylla griff) was 5.720 µg/ml, classified as a strong antioxidant agent. the vitamin c concentration of gandaria (bouea macrophylla griff) was 0.526 mg/ml. journal of fundamental and applied pharmaceutical science, 3(2), february 2023 62 references 1. hanifa d, susilawati y. potensi tanaman gandaria (bouea macrophylla griff) sebagai obat herbal yang beraktivitas antioksidan. farmaka. 2017; 15(3): 134-142. 2. kurniawan a. uji aktivitas antioksidan menggunakan radikal 1,1-difenil-2-pikrilhidrazil (dpph) dan penetapan kandungan fenolik total fraksi etil asetat ekstrak etanolik herba seledri. skripsi thesis, sanata dharma university. 2011. 3. harsono, t. tinjauan ekologi dan etnobotani gandaria (bouea macrophylla griffith). jurnal biosains, 2017; 3(2), 119. https://doi.org/10.24114/jbio.v3i2.7584 4. cahyani ai. uji aktivitas antioksidan dari ekstrak kulit batang kayu jawa ( lannea coromandelica ) dengan metode dpph (2,2-difenil-1pikrilhidrazil). skripsi. uin syarif hidayatullah jakarta. 2017. 5. 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https://doi.org/10.26418/jurkeswa.v1i2.42997 https://doi.org/10.36341/jops.v2i1.1015 eva kholifah, dewi nurazizah, fajrin noviyanto | antioxidant activity and vitamin c concentration analysis of gandaria (bouae macrophylla griff) ethanol extract using spectrophotometry uv vis 63 antioksidan kandungan fitokimia jus buah gandaria ( bouea macrophylla griffith). pharmacon. 2013; 2(2): 1–8. 15. murwanti r, rahmadani a, kholifah e, hermawan a. effect of curcumin on mmp-9 activity of 4t1 murine triplenegative breast cancer cells effect of curcumin on mmp-9 activity of 4t1 murine triple-negative breast cancer cells. in aip conference proceedings 2020; 2260(1): 060017). https://doi.org/10.1063/5.0016424 16. rusmiati. pengaruh metode ekstrak terhadap aktivitas antimikroba ekstrak metanol daun mimba. skripsi. universitas islam negeri alauddin makassar. 2010. https://doi.org/10.1063/5.0016424 hair tonic formulation of black tea extract (camellia sinensis) as hair growth nurul arfiyanti yusuf*; besse hardianti; rahma departement of pharmacy, sekolah tinggi ilmu farmasi makassar, jl. perintis kemerdekaan, pai, kec. biringkanaya, kota makassar, sulawesi selatan 90242. abstract black tea contains flavonoid and its derivate which is a mechanism of actions to stimulate and increase cell proliferation of hair dermal papilla cell to suppress the production of tumor necrosis factor alpha (tnf alpha) that trigger baldness. this study aimed to determine the stable hair tonic formula and to investigate the effectiveness of black tea extract hair tonic in rabbits (oryctolagus cuniculus) as animal testing. there were 3 different concentrations used in this study namely formula 1 (1.25 % of black tea extract), formula 2 (2.5 % of black tea extract) and formula 3 (5 % of black tea extract). the physical stability evaluation was conducted using some tests such as organoleptic, ph, density determination and viscosity test. the result of this evaluation showed that all the three formulas were stable, its effectiveness as hair restorer then was tested and the result of this test indicated that black tea extract has activity as a hair fertilizer and the optimum extract was shown by the formula 3 with black tea extract concentration of 5% with the hair growth in the 18th day i.e., 9.62 ± 0.365 mm. keywords: black tea; hair tonic; hair growth data of article received reviewed accepted : : : 31 july 2021 09 aug 2021 25 aug 2021 doi 10.18196/jfaps.v2i1.12451 type of article: research introduction hair is a horn that is obliquely embedded in the hair follicle. hair is known as a crown for women. having beautiful and healthy hair will make a woman look beautiful. hair beauty and health can be obtained from hair cleanliness; therefore, the scalp and hair need care to remain clean and healthy. problems that arise due to not taking care of the scalp and hair include hair loss.1 hair loss is a disorder in which the hair is shed less or more than normal, with or without visible thinning. the normal number of hair follicles on the head is around 100.000, and it is called abnormal if * corresponding author, e-mail: ikhlasiyahyusufnurul@gmail.com the number only reaches 50%, which means 50.000 strands. normally, 80-120 strands of hair are shed per day2. hair loss occurs in many people to reduce its function and protection against the body and head environment. it is not lifethreatening but affects self-confidence and can even be a psychological stressor.3 haircare is not enough to use shampoo and conditioner because hair is a living cell, so it needs to be cared for and given fertilizer to grow healthy and beautiful. one way to do this is to use a hair grower, commonly known as a hair tonic.4 hair tonic is a preparation of hair cosmetics used to thicken or stimulate hair growth in baldness or hair loss. hair tonic nurul arfiyanti yusuf, besse hardianti, & rahma | hair tonic formulation of black tea extract (camellia sinensis) as hair growth 44 contains solvents, active substances, and vasodilators that enlarge blood vessels to trigger hair growth, including pilocarpine and minoxidil, sebum gland stimulants, hair conditioner substances, and perfumes. the benefits of cosmetic hair tonic include increasing hair growth, preventing hair loss, eliminating dandruff, and being a source of hair nutrition.5,6 in this case, black tea (camellia sinensis) is one of the plants that are useful for fertilizing hair. the active compounds in black tea are polyphenols, theophylline, flavonoids, tannins, caffeine, vitamins c and e, and several minerals, such as zn, se, mo, ge, mg, which are beneficial for hair care7. black tea leaf catechin has major components: epicatechin (ec), epicatechin gallate (ecg), epigallocatechin gallate (egcg), gallocatechin gallate (gcg), gallocatechin (gc), catechin gallate (cg), catechin (c), theaflavins, and thearubigins. epigallocatechin gallate influences hair follicles and dermal papilla cells, indicating that hair follicle cultures have increased hair elongation and growth; dermal papilla cells also have more substantial proliferation than controls.8 for this reason, this study aims to obtain data on the hair tonic formula of black tea extract, which is effective for hair growth. method the materials used in this study included distilled water, 70% ethanol, propylene glycol, menthol, sodium metabisulfite, sodium benzoate, minoxidil, and black tea (camellia sinensis). samples of black tea leaves processed in the form of black tea products were obtained from a tea processing factory located in malino, gowa regency (pt. malino highlands). preparation of experimental animals before testing the effectiveness of hair growth in rabbits, rabbits had to be adapted for seven days. the rabbits consisted of four rabbits; one experimental animal on the back was divided into six boxes, three left and three right sections with an area of 2x2 cm2 each with a considerable distance of 2 cm2, while three experimental animals were divided into two boxes, one left and one right with 2x2 each. the hair in each box was shaved until bald and then smeared with ethanol to prevent irritation.9 figure 1. the back of the treated rabbit making black tea extract the extracts were made by maceration with a ratio of 1:7.5. simplicia powder of 300 grams was put in a container containing 2.250 ml of 70% ethanol. the container was tightly closed and protected from sunlight for three days while stirring regularly every day. after three days, the maceration was filtered through filter paper. remaceration was carried out twice. the filtrate was then accommodated in a beaker glass and then concentrated with a rotary evaporator until the solvent was wholly lost and a thick extract was obtained.10 making formula of black tea extract hair tonic the black tea extract was weighed, then the extract was dissolved in distilled water until completely dissolved. then, propylene glycol was added and stirred journal of fundamental and applied pharmaceutical science, 2(1), august 2021 45 until homogeneous. sodium metabisulfite and sodium benzoate were dissolved in distilled water respectively until dissolved; then, the black tea extract solution, which had been dissolved in propylene glycol, was added and stirred until homogeneous. after that, the menthol was dissolved in ethanol until dissolved, and the black tea extract solution, sodium metabisulfite solution, and sodium benzoate solution were added little by little and stirred until homogeneous. table 1. formula design for hair tonic of black tea extract information: f1= hair tonic preparation with 1.25% black tea extract f2= hair tonic preparation with 2.5% black tea extract f3= hair tonic preparation with 5% black tea extract evaluation of black tea extract hair tonic formula accelerated storage test one way to speed up stability evaluation is to conduct tests before and after storage for several periods at higher and normal temperatures. this test can be carried out using a climatic chamber, consisting of one cycle with a temperature of 5ºc for 12 hours and 35ºc for 12 hours. in this study, the test was carried out for ten cycles.11 organoleptic test the organoleptic test on the preparation was observed visually, including the preparation's color, smell, and shape before and after the accelerated storage test. ph test the ph test was carried out using a ph meter; the electrode on the ph meter was calibrated with standard buffers of ph 4 and ph 7. then, the electrode was dipped into the preparation being examined and then waited for a stable value to appear and recorded the ph value that appeared on the screen. the ph of the hair tonic preparation was adjusted to the head ph, which is in the ph range of 4.5-6.5.4,5 density test density was measured using a clean and dry pycnometer. at room temperature, ingredients function concentration (%) negative control positive control f1 f2 f3 black tea ethanol extract active ingredient 1.25 2.5 5 minoxidil vasodilator 2 ethanol 70 % solvent 60 60 60 60 60 propylene glycol enhancer 10 1 10 10 10 sodium benzoate preservative 0.25 0.25 0.25 0.25 0.25 menthol cooling sensation 0.1 0.1 0.1 0.1 0.1 sodium metabisulfite antioxidant 0.2 0.2 0.2 0.2 0.2 distilled water solvent ad 100 ad 100 ad 100 ad 100 ad 100 nurul arfiyanti yusuf, besse hardianti, & rahma | hair tonic formulation of black tea extract (camellia sinensis) as hair growth 46 the pycnometer (w1) was weighed and then filled with distilled water; the outside of the pycnometer was wiped dry and weighed (w2). the distilled water was discarded. the pycnometer was dried and then filled with hair tonic preparations, whose density was measured and then weighed (w3).12 viscosity test viscometer measurements of hair tonic preparations were carried out using an ostwald viscometer. the hair tonic preparation was pipetted into the ostwald viscometer utilizing a pipette, and then the liquid was sucked in using a bull pump until it crossed two limits. the time for the liquid for the first limit was measured using a stopwatch, and the results were recorded. the samples measured before and after the accelerated storage test was carried out and then calculated using the following formula.12 effectiveness test for black tea extract hair tonic giving hair tonic was done twice a day, as much as 1 ml. the first day of giving was considered day zero. observations were made for 18 days, and the effectiveness tests were carried out on the 6th, 12th, and 18th days. group 1 was not smeared with preparations as normal control. group 2 was smeared with a base as a negative control. group 3 was smeared with formula 1, with an extract concentration of 1.25%. group 4 was smeared with formula 2, namely preparations with an extract concentration of 2.5%. group 5 was smeared with formula 3, namely preparations with an extract concentration of 5%. group 6 was smeared with minoxidil as a positive control.13 in addition, observations were made by taking six strands of rabbit hair by pulling them out using straightened tweezers. they were placed on a dark base, pasted with tape, and then the longest rabbit hair was measured utilizing a caliper. the average hair length data obtained were statistically processed to see the difference between the test and control areas. data analysis the effectiveness test of hair fertilizer included the rate of hair growth. the rate of hair growth was obtained from the length of the hair. the hair length data obtained were then statistically processed using one way anova. results and discussion the plant used in this research was camellia sinensis or better known as black tea. the black tea used came from tea plantations in malino, gowa regency. black tea is the result of plants processed that are empirically widely used to nourish hair. in this study, black tea was made in hair tonic preparations to test its effectiveness in hair growth. in this research, propylene glycol was used as a penetration enhancer in the skin. menthol was meant to provide a cooling sensation to the skin. sodium metabisulfite was used as an antioxidant to prevent oxidation in black tea extracts. meanwhile, sodium benzoate was used as a preservative because its water content could be used as a medium for microbial growth. then, in this study, formulations with different extract concentrations but the same carrier were made to find the best formulation to increase optimal hair growth and hair density. journal of fundamental and applied pharmaceutical science, 2(1), august 2021 47 physical stability test organoleptic test results table 2. organoleptic test results of black tea extract hair tonic formulation before accelerated storage conditions after accelerated storage conditions color smell shape color smell shape f1 brown typical tea liquid brown typical tea liquid f2 brown typical tea liquid brown typical tea liquid f3 almost black, brown typical tea liquid almost black, brown typical tea liquid the organoleptic test of hair tonic preparations was carried out to determine each formula's color, smell, and shape. organoleptic observations of the three hair tonic formulas showed that the greater the concentration of black tea extract used in the preparation, the preparation became almost blackish browner. preparations subjected to accelerated storage conditions did not change in color, smell, and shape. if there is a change in the color of the hair tonic after testing, it is possible that a chemical reaction will occur, which will determine the hair tonic. ph test results table 3. ph test results of hair tonic black tea extract hair tonic formulation before accelerated storage conditions after accelerated storage conditions ph ph f1 6.5 6.0 f2 6.1 5.9 f3 5.9 5.7 in the observation data, the addition of black tea extract influenced the ph value of the preparation, where the higher the concentration of the added extract, the lower the ph value tended to be. the ph measurement results before and after accelerated storage conditions revealed that the three formulas experienced a decrease in ph values but still had ph values that met the ph range for the scalp (4.5-6.5) so that the hair tonic preparations could be said to be stable.4,5 nurul arfiyanti yusuf, besse hardianti, & rahma | hair tonic formulation of black tea extract (camellia sinensis) as hair growth 48 density test results table 4. density results of hair tonic black tea extract hair tonic formulation before accelerated storage conditions after accelerated storage conditions density (g/ml) density (g/ml) f1 0.9565 0.9188 f2 0.9474 0.9230 f3 0.9366 0.9330 in this study, the density measurement of hair tonic preparations used a pycnometer. measurements were carried out before and after being given accelerated storage conditions. the test results showed that the density of each formula was less than 1. the three formulas showed different results, where the higher the extract concentration, the lower the density of the hair tonic preparation. related to this, the density of a substance should remain stable in storage until used to be stable. viscosity test results table 5. viscosity results of hair tonic black tea extract hair tonic formulation before accelerated storage conditions after accelerated storage conditions f1 0.0057 poise 0.0054 poise f2 0.0054 poise 0.0052 poise f3 0.0053 poise 0.0054 poise viscosity testing was performed utilizing an ostwald viscometer on preparations before and after being given accelerated storage conditions. viscosity affects the density value—the greater the value of viscosity, the greater the density of the preparation. viscosity is a measure of physical properties that are usually measured to estimate the effect of pressure conditions on the preparation and can be used as a parameter to indicate the stability of cosmetic products during storage. viscosity is a statement of the resistance of a liquid to flow; the higher the viscosity, the higher the resistance4,5. from the test data, the differences in the viscosity values of each formula were obtained; the higher the amount of extract added, the lower the viscosity value of the preparation. based on the statistical value for the viscosity test, it was found that the viscosity values before and after the accelerated storage condition were not significant (p < 0.05). it signifies that the hair tonic formula was stable under conditions before and after the accelerated storage conditions. effectiveness test of hair tonic preparations the hair growth effectiveness test of the formula of black tea extract hair tonic was on male rabbit hair. the rabbits used consisted of four male rabbits. observations were made for 18 days, and the effectiveness test was carried out on the 6th, 12th, and 18th days. from the journal of fundamental and applied pharmaceutical science, 2(1), august 2021 49 data obtained from hair length measurement, the average rabbit hair length was calculated for each treatment. the calculation results of the average hair extension per treatment per six days can be seen in table 6. table 6. the effectiveness test results of preparations of black tea extract hair tonic no. group hair length (mm) 6th day 12th day 18th day 1 normal control 1.64 ± 0.131 3.81 ± 0.275 6.60 ± 0.355 2 negative control 1.66 ± 0.134 3.82 ± 0.307 6.43 ± 0.305 3 f1 1.65 ± 0.088 5.11 ± 0.375 7.87 ± 0.386 4 f2 1.75 ± 0.126 5.80 ± 0.305 8.94± 0.417 5 f3 1.69 ± 0.126 6.13 ± 0.234 9.62 ± 0.365 6 positive control 1.78 ± 0.13 6.62 ± 0.221 10.54 ± 0.363 based on the results of the average hair length measurement, it is known that the lowest rabbit hair growth was in the negative control group and then the untreated group, followed sequentially by formula 1 (1.25%), formula 2 (2.5%), and formula 3 (5%). meanwhile, the highest hair growth was in the positive control group (minoxidil). group 6th day 12th day 18th day untreated negative control f1 f2 f3 positive control figure 2. test area of black tea extract hair tonic preparations on rabbit’s back nurul arfiyanti yusuf, besse hardianti, & rahma | hair tonic formulation of black tea extract (camellia sinensis) as hair growth 50 figure 3. diagram of preparation effectiveness of black tea extract hair tonic based on the measurement data of the rabbit hair length, it is known that the six treatments given between treatments had very significantly different activities in accelerating hair length growth. for the hair growth effect of each treatment, the addition of black tea extract could increase hair growth activity. based on the hair growth diagram, each treatment group was negative control, which was applied a base without an active substance, formula 1 (1.25%), formula 2 (2.5%), formula 3 (5%), and positive control group (minoxidil), which was compared to the untreated group. increased hair growth occurred every day; the increase in hair growth was significantly different on the 18th day, and the highest increase was in formula 3, with a 5% black tea extract concentration. based on the follow-up test with the f-test on anova, each treatment showed different results. formula 1, formula 2, formula 3, and positive control gave significantly different results. however, only negative and normal controls gave results that were not significantly different. moreover, derivatives of flavonoid compounds consist of epigallatocatechin gallate (egcg), epigallatocatechin (egc), epicatechin gallate (ecg), epicatechin (ec) and quercetin. in black tea, the polyphenols have a mechanism of action by stimulating and increasing cell proliferation in hair dermal papilla cells, suppressing the production of tumor necrosis factor-alpha (tnf alpha), which triggers baldness14,15. meanwhile, saponins are compounds that can stimulate hair growth, seen from their properties as counterirritants. saponins can also increase peripheral blood circulation to increase hair growth. in this study, flavonoid and saponin compounds in black tea extract were suspected of playing a role in accelerating hair growth in rabbits, where the more extracts added to hair tonic preparations, the higher the effect in accelerating hair growth. negative control 18th day 12th day 6th day initial length untreate d group positive control f1 f2 f3 journal of fundamental and applied pharmaceutical science, 2(1), august 2021 51 conclusion black tea extract formulated as a hair tonic preparation gave physically stable results for all three formulas. the formula of black tea extract hair tonic, which is effective for hair growth, is formula 3, with a 5% black tea extract concentration. references 1. bariqina, e., & ideawati, z., (2011). perawatan & penataan rambut. yogyakarta: adicita karya nusa. 2. horev l. (2007). environmental and cosmetic factors in hair loss and destruction. curr probl dermatol. 35, 103-17. https://doi.org/10.1159/0000106418 3. rassman, w. r., & bernstein, r. m., szaniawski, w., & halperin, a. (2009). hair loss & replacement for dummies. indianapolis: wiley publishing inc. 4. tranggono, r. (2007). buku panduan ilmu pengetahuan kosmetik. jakarta: pt.gramedia pustaka utama 5. riska, n. h., dolih, g., rini, h., resmi, m. (2020). formulasi dan evaluasi sediaan herbal hair tonic sebagai perangsang pertumbuhan rambut. majalah farmasetika, 5 (5), 218-232. https://doi.org/10.24198/mfarmasetik a.v5i5.27555 6. rejeki, e. s. (2010). analisis etanol dalam hair tonic dan hair spray secara kromatografi gas. surakarta: universitas setia budi 7. kartodimedjo, s. (2013). cantik dengan herbal rahasia puteri keraton. yogyakarta: citra media pustaka. 8. longo, g., karan, m., sharma, p. d rakesh d.d, vyas s, vasist. k. (2008). quantitative analysis of green tea polyphenosin indian cultivars, in economic crisis in tea industry. studium press llc: houston usa. 9. putra, h. t. p. (2013). formulasi dan uji efektivitas sediaan emulsi perangsang pertumbuhan rambut ekstrak seledri (apium graveolens linn). skripsi. universitas pakuan. bogor 10. ditjen pom ri. (1986). sediaan galenik. departemen kesehatan republik indonesia: jakarta 11. rahman, l., marzuki, a., sukamto & musdalifah azis. (2021). antibacterial cream activity test of banyuru extract combination (pterospermum celebicum miq) dan bee pollen. eco. env. & cons. 27 (2), pp. 709-715. 12. martin, alfred. (2008). farmasi fisika dasar-dasar farmasi fisika dalam ilmu farmasetik ed. ketiga. jakarta: uipress. 13. mu’ani, h., & purwati. (2019). uji stabilitas fisik dan uji aktivitas sediaan hair tonic dari ekstrak etanol 96% daun kangkung (ipomoea aquatica forsk.) pada rambut kelinci jantan (new zealand white). іndоnеѕіа nаturаl rеѕеаrсh рhаrmасеutісаl jоurnаl, 4(2), pp. 23-31 14. purwantini, i., munawaroh, r., darwati n., b.s. (2008). combination of the and mangkokan leaves extract to promote hair growth, traditional medicine journal 13, p. 43. 15. amin, j. simamora, e. l. p., effionora, a. & djajadisastra, j. (2014). green tea (camellia sinensis l) ethanolic extract as hair tonic in nutraceutical; physical stability, hair growth activity on rats, and safety test. international journal of pharmacy and pharmaceutical sciences. 6 (5), pp. 9499. https://doi.org/10.1159/0000106418 https://doi.org/10.24198/mfarmasetika.v5i5.27555 https://doi.org/10.24198/mfarmasetika.v5i5.27555 36 acute toxicity study of the combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. leave extracts nofran putra pratama*, kurnia rahayu purnomo sari; ririn irma marliana faculty of health, universitas jenderal achmad yani yogyakarta, jl. ringroad barat, gamping kidul, ambarketawang, gamping, sleman, yogyakarta 55294. abstract azadirachta indica a.juss. and gynura procumbens (merr.) are medicinal herbs widely used in traditional medicine. recent research on the combination of these two plants showed aggressive antioxidant activity. the combination results can improve insulin and beta-cell morphology and can increase insulin expression. the variety of activities performed is the basis for conducting acute toxicity tests on the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. to increase public confidence in its efficacy and ensure the safety of its use. the acute toxicity test on the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. was carried out on female wistar rats by following the 423 procedures of oecd (organization for economic cooperation and development) guideline with five groups of experimental animals, namely normal treatment, aquadest solvent treatment and 0.5 nacmc 0.5%, a separate treatment of the ethanol extract of azadirachta indica a. juss., a separate treatment of the ethanol extract of gynura procumbens (lour.) merr., and combination treatment of the ethanol extract of azadirachta indica a. juss. and leaves of gynura procumbens (lour.) merr. furthermore, it was proceeded by observing the liver of the experimental animals histopathologically. the acute toxicity test results utilizing the 423 procedures of the oecd did not confirm any death or signs of toxicity in the experimental animals, and histopathological observations did not show any major histopathological damage. based on these results, according to globally harmonized classification system (ghs), the combination of the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. is included in the unclassified category (> 2,000 mg/kg bw. keywords: acute toxicity; azadirachta indica a. juss. and gynura procumbens (lour.) merr; histopathology; oecd data of article received reviewed accepted : : : 21 nov 2020 12 dec 2020 22 feb 2021 doi 10.18196/jfaps.v1i2.9868 type of article: research * corresponding author, e-mail: nofranputrapratama@gmail.com nofran putra pratama, kurnia rahayu purnomo sari, ririn irma marliana | acute toxicity study of the combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. leave extracts 57 introduction the use of natural materials, especially those derived from plant materials, has been carried out by the indonesian people from generation to generation to prevent and treat diseases. this natural ingredient is known as traditional medicine/herbs as its manufacture, and use principles are still traditional, and most of its properties to date have merely been based on empirical experience. the use of traditional medicine is considered an alternative treatment with few side effects. therefore, further research is required on the safety testing of these traditional medicines/herbs. research related to traditional medicines/herbs derived from plants is still mainly in the form of separated extracts, while the combined extracts are still rarely conducted. the combined extracts can be an alternative in the development of medicinal plants to obtain better results. one example of a combination of extracts that has been widely researched is the combination of the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. having been known to have many properties, including antioxidant activity1, decreased blood glucose levels2, decreased glycemia and improving insulin morphology and beta cells and increasing insulin expression3, significantly decreased glycemia in streptozotocin-induced rats4, and hypoglycemic effect in mice5. an acute toxicity test is needed to determine the safety of the combination of the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. this test is intended to obtain information regarding the symptoms of poisoning, the cause of death, the sequence of the death process, and the lethal dose range for the experimental animals (ld50)6. based on the data above, an acute toxicity test was carried out utilizing a method adopted from the organization for economic co-operation and development (oecd) and histopathological observation on the lab rats' liver. methods materials the materials for the extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. were obtained from moyudan sleman yogyakarta, such as 70% ethanol (onemed). cmc na 0.5%, buffer formalin 10%, nacl physiology (otsuka), hematoxillin eosin (merck)1, female wistar rats. research process 1. animal grouping an acute toxicity test of a combination of the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. utilized the 423 procedures of oecd guidelines7. the experimental animals handled were 15 female wistar rats aged 8-12 weeks, not pregnant, with a bodyweight of 150-250 grams. female experimental animals were more sensitive than males, allowing toxic effects to appear in female animals. the experimental animals were divided into five groups, consisting of 3 animals in each group. the control groups (without treatment) were the solvent control group (aquades and 0.5% nacmc), the group of the ethanol extract of azadirachta indica a. juss., the group journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 58 of the ethanol extract of gynura procumbens (lour.) merr, and the group of the combined extract of both. 2. provision of test preparation in the first stage of the test, the experimental animals were fasted overnight before administrating the test preparation, and its weighed. after that, the pressure was carried out on three experimental animals with an initial 300 mg/kg bw dose. furthermore, the toxic symptoms were observed, especially during the first 4 hours, and were continued during the first 24 hours after the treatment. observation of toxic symptoms was continued every day until the 14th day. when 2-3 experimental animals died, the dose was reduced to 50 mg/kg bb. meanwhile, when 0-1 experimental animals died, administration of test preparations of 300 mg/kg bw was continued. when 0-1 experimental animals died in the second round, the test was continued by administering a test dosage of 2000 mg/kg bb to 3 test animals, as shown in figure 1. the weight of the experimental animals was weighed on day 1, 2, 5, 8, 11, 14. on the 14th day, the tested animals were dynecropsied, and a histopathological examination was performed. 3. observation of toxic symptoms the physical observation was carried out on toxic symptoms in all rats from the five test groups. this examination was carried out continuously during the first four hours after giving the test preparation. observation of toxic symptoms qualitatively followed the criteria in the balazs table8. however, not all criteria in the balazs table were observed in this study. the criteria observed were those that could be seen with the naked eye, such as changes in behavior, body weight, seizures, tremors, paralysis, sedation, coma, and salivation. 4. histopathological observations histopathological observations were carried out in several stages, namely organ harvesting, making histopathological preparations carried out at balai besar veteriner yogyakarta, and histopathological examinations by comparing them with the organ's histological preparations and histology of the treated groups of experimental animals qualitatively. results and discussion data on experimental animals' body weight were used to determine whether the extract treatment affected the experimental animals' weight. in addition, one of the symptoms of toxicity can be weight loss8. based on the results of body weight observations, a graph of the experimental animal weight's average was created to illustrate the experimental animals' changes in body weight for 14 days (figures 1 and 2). nofran putra pratama, kurnia rahayu purnomo sari, ririn irma marliana | acute toxicity study of the combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. leave extracts 59 figure 1. a graph of the average weight of the experimental animal after the administration of test preparation of 300 mg/kg bw for 14 days of observation. figure 2. a graph of the average weight of the experimental animal after the administration of test preparation of 2,000 mg/kg bw for 14 days of observation based on the experiment's weight average figure after administrating the test preparation, there was no decrease in the experimental animals' weight. it indicates one of the toxic symptoms, namely weight loss, was not found. the difference in weight between each treatment might be due to each experimental animal's amount of food consumed. the amount of food consumed was different as the amount of feed given was not weighted. dosage for the azadirachta indica a. juss test was 50 mg/kg bw and 112.5 mg/kg bw was gynura procumbens (lour.) merr3. for the essential dosage of acute toxicity, the selection of the dosage was carried out based on the 423 procedures of oecd guideline consisting of dosages of 5, 50, 300, and 2,000 mg/kg bw, as well as additional tests with a dose of 5.000mg/kg bw when it was needed and possible7. the initial dose for the test preparation used in 0 50 100 150 200 250 1 2 5 8 11 14 w e ig h t (g ra m ) control solvent control gynura procumbens extract azadirachta indica extract combination extract of both journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 60 this study was 300 mg/kg bw. according to the 423 procedures of the oecd guideline, the initial dose for the test preparation was unknown, or there was no information related to its toxicity that was 300 mg/kg bw. the test preparation was administrated at an initial dose of 300 mg/kg bw to 3 tested animals per group. upon the treatment with this dosage, no toxic symptoms and death signs were found in the experimental animals. the experiment was then continued at a dose of 2.000 mg/kg bw, and the condition of the experimental animals was reobserved. up to 14 days after the treatment, there were no toxic symptoms and death signs in the experimental animals. table 1. the number of dead animals in the observation for 14 days after the administration of the test preparations at a dose of 300 mg/kg bw and 2,000 mg/kg bw treatment group number of early rats number of final rats, the dosage of 300 mg/kg bw number of final rats, the dosage of 2,000 mg/kg bw ld50 cut off (mg/kg bw) dead live dead live control 3 0 3 0 3 solvent control 3 0 3 0 3 gynura procumbens extract 3 0 3 0 3 >2,000 azadirachta indica extract 3 0 3 0 3 combination extract of both 3 0 3 0 3 total 15 0 3 0 3 table 1 shows that administering the test preparation at a 300 mg/kg bw dose did not cause death in the experimental animals until the 14th day. based on these results, the potential for acute toxicity or ld50 cut-off of the test preparation administered to female wistar rats falls into category 5 or unclassified category7. the 423 procedures of the oecd guideline were used to calculate the ld50 cut-off because this method was relatively simple; the number of experimental animals used was small, and the test results' validity was not inferior compared to other methods. observation of toxic symptoms is one of the qualitative measures of toxicity. based on the observed toxic symptoms, it did not display any toxic symptoms appearing after treatment. hence, it can be perceived that the test preparations administered either single or in combination between the extracts of azadirachta indica a. juss. and gynura procumbens (lour.) merr. were declared safe in terms of acute toxicity. in the final stage, the experimental animals were sacrificed by means of neck nofran putra pratama, kurnia rahayu purnomo sari, ririn irma marliana | acute toxicity study of the combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. leave extracts 61 dislocation. prior to the surgery, rats' heart rate and breath were re-observed to ensure the experimental animal had actually died. furthermore, the liver organ was taken. the reason for examining the liver is because the liver has various vital functions in the body, like neutralizing toxic substances, synthesizing serum protein, regulating nutrition and secreting bile salts9. the taken liver organs were then weighed and preserved in a 10% formalin solution. observation of the experimental animal organs was carried out macroscopically and microscopically. macroscopic observations were employed to determine any visible damage to the organs due to the test's provision that did not appear visually when the experimental animal was still alive. based on the results of macroscopic observations, no damage and abnormalities were found after treatment. based on the vision, there was no difference between the organs of each group. furthermore, organ weighing was carried out to determine the effect of treatment on the experimental animal organs seen from their weight. the average results of each treatment showed no significant difference from the experimental animals' organ weights. table 2. the weight of the experimental animal's liver no treatment rat weight (gram) average weight 1 control 1 5.6277 2 7.5499 7.1289 3 8.2091 2 solvent control 1 7.6987 2 6.2049 7.3045 3 8.0099 3 gynura procumbens extract 1 10.3372 2 7.6726 8.8553 3 8.5562 4 azadirachta indica extract 1 7.2171 2 7.2091 7.4488 3 7.9202 5 combination extract of both 1 5.6412 2 7.1454 7.2778 3 9.0470 the formalin solution's liver organs were then transferred into histopathological preparations at the pathology laboratory of balai besar veteriner, wates, yogyakarta. the dye used was hematoxylin-eosin (he). he is a common coloring compound used for cells and tissues. hematoxylin would color the cell nucleus blue, while eosin would color the cytoplasm and extracellular matrix red. the results of observations on the liver are as follows: journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 62 figure 3. histopathological microscopic profile of the rats’ liver of the dosage of 2.000 mg/kg bw the histological profile of normal treated rats, namely the group of rats without administering the test preparation, includes a) the cell nucleus structure is located in the middle and shows a radial arrangement pattern. (40 x 10) (figure 3.1). the histological profile of the solventtreated rats was a group of rats that were given distilled water and 0.5% na-cmc. a) the structure of the cell nucleus is located in the middle, b) sinusoid (40 x 10) (figure 3.2). the rats' histological profile was treated with the ethanol extract of leaves of longevity spinach (gynura procumbens (lour.) merr.). a) the nucleus is located in the center, b) dilation of the central vein, c) sinusoid. (40 x 10) (figure 3.3). histological profile of rats treated with ethanol extract of neem leaves (azadirachta indica a. juss.). a) the nucleus is located in the middle, b) dilation of the central vein, c) fat hepatocytes, d) sinusoids (40 x 10) (figure 3.4). histological profile of rats treated with a combination of ethanol extracts of neem leaves (azadirachta indica a. juss.) and leaves of longevity spinach (gynura procumbens (lour.) merr.), a) dilation of the central vein, b) sinusoids (40 x 10) (figure 3.5). table 2. the score of hepatocyte damage test hepatocyte damage (%) total normal inflammation degradation necrosis control 60 0 0 0 60 solvent control 52 0 0 0 52 gynura procumbens extract 58 22 0 4 84 azadirachta indica extract 58 20 0 4 82 combination extract of both 58 14 0 8 80 determination of liver damage score is a modification of manja roenigk’s scoring histopathology. the interpretation of the histological preparations on the liver organs of the experimental animals did not show any damage to the cells in the nofran putra pratama, kurnia rahayu purnomo sari, ririn irma marliana | acute toxicity study of the combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. leave extracts 63 normal and solvent group. the control group showed the highest percentage of the normal cell than the other groups. in the group treated with the ethanol extract of gynura procumbens (lour.) merr., there was no liver cell damage, indicating that there were only a few widenings of the central veins. it was probably due to the high content of flavonoids in the extract. in the group treated with the ethanol extract azadirachta indica a. juss., it did not show any significant damage and only a few widenings of the central veins leading to inflammation, and a little fatty hepatocyte was shown as empty vacuoles in the cytoplasm of the liver cells. this fatty process was reversible and caused by an inhibited release of lipid transfer from inside cells and an imbalance in synthesis and the release of triglycerides carried by parenchyma cells into the circulation10. in the combination treatment group, azadirachta indica a. juss's extract and leaves of gynura procumbens (lour.) merr. was seen to widen the central vein. in all treatments except the normal treatment group, several sinusoids were indicated by the presence of a flat and dark cell nucleus with little cytoplasm. in the experimental animals' liver cells, the possibility of necrosis was indicated by the destruction or loss of the nucleus (cell nucleus). however, the amount of necrosis found was not too much, and no tested animals had died during the treatment. moreover, in general, there was no significant damage to the liver of the experimental animal. it was likely due to the presence of flavonoid and phenolic compounds in both extracts. hence, these compounds can act as antioxidants that can minimize toxin compounds' harmful effects1. conclusion the test compound of a combination of the ethanol extract of azadirachta indica a. juss. and gynura procumbens (lour.) merr. after acute toxicity test with the 423 procedures of oecd up to a dose of 2,000 mg/kg bw did not show any death or signs of toxicity in the experimental animals; hence, the combined extract was categorized as unclassified category according to ghs. acknowledgment we would like to thank the faculty of health, jenderal achmad yani university yogyakarta, for funding this research references 1. pratama, n. p., sari, k. r. p., mutya, e. (2019). effect of azadirachta indica a.juss. and gynura procumbens (merr.) leaf extract combination towards free-radical scavenging activity. international journal of advanced research, 7(9), pp. 737-741. 2. akowuah, g. a., sadikun, a. & mariam, a. (2002). flavonoid identification and hypoglycemic studies of butanol fraction from gynura procumbens. pharmaceutical biology, 40(6), pp. 405410. 3. sunarwidhi, a. l., sudarsono, s., & nugroho, a. e. (2014). hypoglicemic effect of combination of azadirachta indica a. juss. and gynura procumbens (lour.) merr. ethanolic extracts standardized by rutin and quersetin in alloxan-induced hyperglycemic rats. advanced journal of fundamental and applied pharmaceutical science, 1(2), febuary 2021 64 pharmaceutical bulletin, 4(2), pp. 613618. 4. zhang, x. f., & tan, b. k. (2000). effects of an ethanolic extract of gynura procumbens on serum glucose, cholesterol and triglyceride levels in normal and streptozotocininduced diabetic rats. singapore medical journal, 41(1), pp. 9-13. 5. pillai, n. r., & santhakumari, g. (1981). hypoglycemic activity of melia azadirachta linn (neem), indian journal of medical research, 74, pp. 931-933. 6. ngatidjan. (1997). metode laboratorium dalam toksikologi. yogyakarta: pusat antar universitas bioteknologi universitas gadjah mada. 7. oecd. (2001). oecd guideline 423 for testing of chemicals, acute oral toxicity-acutetoxic class method. no. 423, oecd, paris. 8. balasz, t. (1970). measurement of acute toxicity. oxford and edinburgh: blackwell scientific publications. 9. longo, d. l., kasper, d. l., jameson, j. l., fauci, a. s., loscalzo, j., & hauser, s. (2011). harrison's principles of internal medicine, 18th ed. new york: mcgrawhill. 10. francisco, j. s., waldo, l., garcia, j., isadora, l., & karin, s. (2018). histopathological and immunohistochemical characterisation of hepatic granulomas in leishmania donovaniinfected balb/c mice: a time-course study. parasites vector journal, 11(73), pp. 1–9. phytochemical analysis and antioxidant potential of ethylacetate extract of tamarindus indica (tamarind) leaves by frap assay mubarak muhammad dahiru*, hadiza ahmadi, maimuna umar faruk, huzaifa aminu hamman, abreme gahana charles department of science laboratory technology, adamawa state polytechnic yola, nigeria abstract oxidative stress is characterized by an imbalance in the generation of free radicals and their subsequent elimination by endogenous antioxidants. it is a characteristic of several diseases, especially during the progression stage, which can lead to fatal effects. this study aims to investigate the phytochemical components and antioxidant capability of tamarindus indica and assess its capability as a candidate for managing diseases associated with oxidative stress. the gravimetric method detected and quantified phytochemicals, while the reducing power assay determined the antioxidant potential. saponins, steroids, and flavonoids were detected in 6.83 ±0.44, 4.30 ±0.60, and 10.17% ±0.60, respectively, without alkaloids, glycosides, and terpenoids. the antioxidant test showed a concentration-dependent increase in absorbance of both the extract and standard (ascorbic acid). however, ascorbic acid had higher absorbance. at 100% concentration, the sample had an absorbance of 0.388 ±0.022, which was lower than the absorbance of ascorbic acid (0.411 ±0.009) at 40% concentration. it can be concluded that tamarind leaves could be utilized to manage diseases associated with oxidative stress, evidenced by their antioxidant potential credited to the phytochemical content of the leaves. however, there is a need for further studies to ascertain the exact compounds and their modes of action. keywords: antioxidant; ethylacetate; oxidative stress; phytochemical; tamarindus indica data of article received reviewed accepted : : : 01 nov 2022 22 dec 2022 30 jan 2023 doi 10.18196/jfaps.v2i1.16708 type of article: research introduction oxidative stress is a state with a high level of reactive oxygen species (ros) accompanied by low levels of endogenous antioxidants due to disturbance in the balance for production and neutralization of the ros by the antioxidants.1 several diseases, including diabetes, neurodegenerative, and cancer, are * corresponding author, e-mail: mubaraq93@gmail.com associated with oxidative stress, notably during the progression of the diseases where there is continuous damage to dna, lipids, and proteins.2-4 in proteins, oxidative damage goes through carbonylation, modification of the side chain, and integrity of the protein molecule. in contrast, in dna damage, 8hydroxydeoxyguanosine is formed, which binds to thymidine instead of cytosine, mailto:mubaraq93@gmail.com journal of fundamental and applied pharmaceutical science, 3(2), february 2023 46 causing mutagenesis.5,6 some antioxidants in the form of enzymes found within cells are crucial in maintaining cellular homeostasis, including catalase, superoxide dismutase, and glutathione peroxidases. however, non-enzymatic forms of antioxidants also exist, which include ascorbic acid, glutathione, and vitamin e.7 in type 2 diabetes, a generation of ros causes the development and progression of diabetes by disrupting the β-cell signaling and regulation pathway, subsequently causing β-cell dysfunction and insulin resistance.8 generation of ros is a characteristic of cancer cells due to the rapid cellular division. however, strategies are adopted by these cells to stay beneath a threshold for activation of apoptosis, leading to their proliferation. during cancer progression, there is an overexpression of antioxidant enzyme genes and the production of nadph, thus emphasizing the effects of oxidative stress on cancer progression.2 in cardiovascular diseases, ros is an important part of signaling in the heart, acting as a second messenger. however, oxidative stress sets in when they are in excess, leading to cardiac dysfunction, hypertrophy, apoptosis, and heart failure.9 in neurodegenerative disease, oxidative stress causes target macromolecules and the oxidation of these molecules; proteins, lipids, and nucleic acids, disturbance in proteasome and mitochondrial function, production of cytokines and inflammatory responses, formation of amyloid β deposition, plaque, and advanced glycation end products, and cell death.10 plant and their products are applied in managing diseases through several mechanisms, sometimes attributed to their antioxidant potential.11-13 phytochemicals from plants are implicated with therapeutic roles of ailments associated with oxidative stress, which is credited to their several pharmacological effects acting individually or synergistically.14 for example, phytochemicals were previously implicated in managing cancer progression by suppressing dna damage caused by oxidative stress and modulating signaling pathways leading to carcinogenesis due to their antioxidant effects.15 considering the background mentioned above, this study aims to investigate the phytochemical components and antioxidant potential of tamarind (tamarindus indica) leaves to assess their capability for managing diseases linked to oxidative stress. it considers the effects of oxidative stress in the progression of several diseases, including diabetes, cardiovascular and neurodegenerative diseases, and cancer. method reagent all the reagents utilized in this study were of anarlar. plant material tamarind leaves were collected from the yolde pate area of yola south local government, adamawa state, nigeria. it was identified by a forest technologist from the forestry technology department of adamawa state polytechnic, yola. a voucher specimen was maintained in the departmental herbarium with voucher number asp/ft/118. the leaves were air-dried and ground to powder using a blender. extraction the plant sample was extracted by maceration of 300 g of the leave powder in 1l of 70% ethyl acetate for 48 h, followed by filtration and concentration to dryness mubarak muhammad dahiru, hadiza ahmadi, maimuna umar faruk, huzaifa huzaifa aminu hamman,abreme gahana charles | phytochemical analysis and antioxidant potential of ethylacetate extract of tamarindus indica (tamarind) leaves by frap assay 47 under reduced pressure yielding 9.2 g extract.16 qualitative phytochemical analysis phytochemical tests and identification in ethyl acetate extract of tamarind leaves (eetl) were carried out to detect alkaloids, saponins, steroids, glycosides, terpenoids, and flavonoids, as previously reported.16 quantitative phytochemical analysis saponins content saponins were quantified by the gravimetric method.17 steroids content steroids were quantified by the gravimetric method.18 flavonoids content flavonoids were quantified according to a method described previously.18 reducing power assay the reducing power of eetl was carried out by a previously reported method.19 the plant extract varying concentrations of 20, 40, 60, 80, and 100% were prepared in distilled water. 2.5 ml of 0.2 m phosphate buffer (ph 6.6) and 2.5 ml of 1% potassium ferricyanide were added. it was then incubated at 50 °c for 20 min. after incubation, 2.5 ml of 10% trichloroacetic acid was added, then centrifugated for 10 min at 3000 rpm, and the upper layer was collected. lastly, 2.5 ml of distilled water was added to 2.5 ml of the upper layer solution, followed by adding 0.5 ml of 0.1% fecl3 solution. the absorbance was measured at 700 nm against a blank using a uv-vis spectrophotometer (752 uv-vis spectrometer, shanghai yoke instruments co., ltd, china). ascorbic acid was used as standard. statistical analysis data obtained in the study were expressed as mean ± standard error of triplicate determinations' mean (± sem) evaluated with statistical package for the social sciences (spss) version 22 software. results and discussion phytochemical analysis of eetl the results of the quantitative determination of the phytochemical composition of the ethylacetate extract of tamarind leaves are presented in table 1. saponins, steroids, and flavonoids were detected, while alkaloids, glycosides, and terpenoids were absent. table 1: phytochemicals detected in eetl phytochemical inference alkaloids absent saponins present steroids present glycosides absent terpenoids absent flavonoids present the phytochemical reported in this study correlates with a previous report on an ethyl acetate extract of tamarind leaves. however, in their study, alkaloids were detected as absent.20-22 a similar study on the ethanol extract of tamarind reported the absence of saponins, although alkaloids were detected.23 besides, in the other study, saponins, steroids, and flavonoids were detected in the ethanol extract of tamarind leaves at the moment alkaloids were also found.24 in the same study, vitamin e was detected as a good antioxidant. the present study partially agreed with this study for detecting saponins, steroids, and flavonoids. the polarity of ethyl acetate was lower than that of ethanol; thus, the difference in journal of fundamental and applied pharmaceutical science, 3(2), february 2023 48 solvent might be the reason for not being able to detect alkaloids.25 the phytochemicals quantified in the ethylacetate extract of tamarind leaves are shown in table 2. flavonoids were quantified in the highest (10.17% ±0.60) concentration, followed by saponins which were present in a concentration of 6.83% ±0.44. meanwhile, steroids were quantified in the least concentration (4.30% ±0.60). table 2: quantitation of phytochemical analysis of eetl phytochemical concentration (%) saponins 6.83 ±0.44 steroids 4.30 ±0.60 flavonoids 10.17 ±0.60 values are in triplicate determinations ± sem. phytochemicals exert different pharmacological effects, including antioxidant activities through several mechanisms of action. saponins exert their antioxidant effect by raising the concentration of antioxidant enzymes, decreasing the release of malondialdehyde or lactate dehydrogenase, and restoring glutathione homeostasis. thus, they were implicated in repairing oxidative damage in cells and inhibiting cell death.26 saponins were previously reported to exert an antioxidant effect on alzheimer’s disease (ad) by preventing dna damage due to the formation of 8hydroxydeoxyguanosine and increasing the expression of endogenous antioxidant enzymes in the brain of mice, making it a potential therapeutic in the therapy of ad.27 saponins were also reported to possess hepatoprotective effects on alcohol induced-liver injury by reducing the level of ethanol-induced oxidative stress.28 polymyxin e-induced nephrotoxicity was previously reported to be decreased by saponins, and a possible mechanism of action was postulated to be inhibiting oxidative stress and cell death through the mitochondrial pathway.29 furthermore, flavonoids were previously postulated as a crucial metabolite for managing central nervous system disorders by acting as gammaaminobutyric acid (gaba) receptors.30 the flavonoid naringenin exerts an antioxidant effect by neutralizing ros and promoting the activities of endogenous antioxidant enzymes in diabetes, cardiovascular and neurodegenerative diseases.31 in a similar study, flavonoids were reported to exert antioxidant and antiradical effects against ros and were postulated for application in managing oxidative stress.32 flavonoids were previously reported to exert an antioxidant effect on streptozotocininduced diabetic rats by direct antiradical effects.33 hepatoprotective effects of flavonoids against oxidative stress induced by high glucose were previously reported to regulate antioxidant enzymes.34 reducing the power of eetl the reducing power of the eetl is presented in table 3. a concentrationdependent increase in absorbance by the ethyl acetate extract of tamarind leaves was observed, with the lowest absorbance at 20% concentration (0.235 ±0.008), while the highest was observed at 100% concentration (0.388 ±0.022). however, the absorption of ascorbic acid was higher than the extract, even at 40% concentration (0.411 ±0.009). mubarak muhammad dahiru, hadiza ahmadi, maimuna umar faruk, huzaifa huzaifa aminu hamman,abreme gahana charles | phytochemical analysis and antioxidant potential of ethylacetate extract of tamarindus indica (tamarind) leaves by frap assay 49 table 3: reducing power of eetl concentration (%) absorbance tamarind leaves ascorbic acid 20 0.235 ±0.008 0.243 ±0.012 40 0.245 ±0.014 0.411 ±0.009 60 0.259 ±0.008 0.644 ±0.016 80 0.298 ±0.017 0.815 ±0.026 100 0.388 ±0.022 1.131 ±0.018 values are in triplicate determinations ± sem. several studies reported different pharmacological effects of tamarind, which were attributed to the antioxidant potential of the plant. tamarind leaves revealed a high radical scavenging effect in vitro by dpph and abts. they were suggested to be a natural antioxidant with anti-diabetic properties.35 the antioxidant activity of tamarind was reported in a similar study and was attributed to the phenolic compounds detected due to an observed correlation between the total phenolic compounds and dpph radical scavenging inhibition.36 a previous study on the antioxidant potential of tamarind leaves using frap reported a concentration-dependent increase in absorbance, although it was lower compared to ascorbic acid, which aligns with the result of our study.37 in this study, the absorbance of eetl increased along with concentration. however, the absorbance of the extract was lower than that of ascorbic acid. besides, several studies reported similar results for frap of tamarind leaves.38-42 conclusion this research investigated the phytochemical components and antioxidant potential of ethylacetate extract of tamarind leaves 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narasimhacharya avrl. ameliorative effect of tamarind leaf on fluoride-induced metabolic alterations. environ health prevent med. 2012;17(6):484-93. https://doi.org/10.1007/s12199-0120277-7 https://doi.org/10.5897/ajbr2016.0896 https://doi.org/10.4103/0974-8490.129043 https://doi.org/10.4103/0974-8490.129043 https://doi.org/10.5455/javar.2015.b103 https://doi.org/10.1371/journal.pone.0070058 https://doi.org/10.1371/journal.pone.0070058 https://doi.org/10.1038/s41598-022-13716-x https://doi.org/10.1038/s41598-022-13716-x https://doi.org/10.1007/s12199-012-0277-7 https://doi.org/10.1007/s12199-012-0277-7 qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) – densitometry desy ayu irma permatasari1*; novita kurniasri2; muladi putra mahardika3 1,2departement of pharmacy, faculty of health science, universitas duta bangsa surakarta, jl. pinang, cemani, grogol, sukoharjo, jawa tengah 57552. 3 departement of pharmacy, politeknik harapan bersama, jl. mataram, no 9, tegal, jawa tengah 52147. abstract chemical medicine is chemical substances that is used as the main ingredient of chemical drugs. this compound is usually added to herbal medicine preparation to strengthen the indication of the traditional medicine.chemical medicine was prohibited from being added to traditional medicinal preparations. but in reality, a lot of medicinal herbs could have contained medicinal chemicals on the market sale. this purpose of this research was to know the chemical contamination of the dexamethasone also the concentration contained in the rheumatic pain herbal medicine. this research was used three kind of rheumatic pain herbal medicine sample to identify dexamethasone, the sample code is s1, s2, and s3. the analysis of qualitative method are organoleptic test, ftir characteristic test, tlc evaluation. the analysis of quantitative were purposed to know the dexamethasone concentration contained on the rheumatic pain herbal medicine using tlc-densitometric method. the result show that the three sample of rheumathic pain herbal medicine were contaminated by dexamethasone chemical medicine. based on organoleptic test, the results show color, smell, and taste. characterization of the dexamethasone using ftir were to know functional groups of dexamethasone contained in the rheumatic pain herbal medicine sample, the functional groups of the sample s1, s2, and s3 were compared to dexamethasone standard. to identify of tlc method, obtained rf value of dexamethasone standard and the sample, visualizing a stain color purplefluorescence on the uv 254 nm. the analysis of quantitative dexamethasone concentration using tlc-densitometric showed the presence of dexamethasone in the sample for s1, s2 and s3. the concetration of dexamethasone obtained of sample s1, s2, s3 were 1014.64 µg/g ; 131.15 µg/g ; 135.54 µg/g respectively. keywords: dexamethasone; rheumatic pain herbal medicine; tlcdensitometric; quantitave analysis, qualitative analysis data of article received reviewed accepted : : : 31 july 2021 05 aug 2021 24 aug 2021 doi 10.18196/jfaps.v2i1.12450 type of article: research * corresponding author, e-mail: desyayu_permatasari@udb.ac.id desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 11 introduction the lower middle class widely uses traditional medicines and medicinal plants due to their affordable price. another reason people use traditional medicine is that medicinal plants or traditional medicine are relatively safer than synthetic medicine. the natural efficacy and purity of traditional medicines are frequently tainted by irresponsible parties, especially traditional medicine manufacturers who are only looking for financial gain without considering the purity and risks of the content of traditional medicines. mixing herbs with medicinal chemicals is also done to make these herbs more efficacious instantly1. the regulations in indonesia require that natural medicines and herbs are not allowed to contain chemical medicine. it is hazardous as natural medicines in herbal medicine are often used for a long time and at doses that cannot be ascertained, although the healing effect is immediately felt. however, the uncontrolled use of bko with doses that cannot be ascertained can cause serious side effects, ranging from nausea, diarrhea, dizziness, headache, visual disturbances, chest pain to severe organ damage such as liver damage, kidney failure, heart failure, and even death2. furthermore, thin-layer chromatography is the separation of the components of a compound from another compound where the components will be distributed between two phases, namely the stationary phase and the mobile phase. the compound's polarity will last longer; otherwise, the component interacts less with the faster-moving mobile phase. the stages in tlc, the analysis stages, start from spotting samples and standards on the tlc plate until direct visual observation under 254 nm uv light for spotting separation components3. based on this background, this research aims to analyze dexamethasone's qualitative and quantitative contamination in rheumatic pain herbal medicine sold in mungkid district, magelang. dexamethasone was chosen as it is often added to herbal pain relievers such as rheumatic herbs and herbs for aches and pains. this research provided new information about the contamination of chemical medicine in traditional medicine. method tools and materials the tools used in this study included a weight analytical (ohaus), 10 ml volumetric flask (pyrex), 20 ml measuring cup (pyrex), beaker glass (pyrex), capillary tube, boiling stone, micropipette, chamber, tlc plate gf254 (merck), uv light 254nm, vortex mixer (b-one), centrifuge, ftir (shimadzu), and tlcdensitometry scanner (camag). materials used in this research included rheumatic pain herbal medicine, standard dexamethasone pro analysis (bpom), 96 % ethanol pro analysis (merck), chloroform pro analysis (merck), toluene pro analysis (merck), ethyl acetate pro analysis (merck), methanol pro analysis (merck), and n-hexane pro analysis (merck). in the mobile phase, the material used was a mixture of ethyl acetate, toluene, and methanol ratio of 45:55:1. meanwhile, in the stationary phase, it used a silica gel tlc plate gf254. sample the sample in this study were three kinds of rheumatic herbal medicine obtained from different manufacturers purchased from several market stalls for traditional journal of fundamental and applied pharmaceutical science, 2(1), august 2021 12 herbal aches and pains. this research is experimental research conducted in the pharmacy laboratory at universitas duta bangsa surakarta, pharmacy laboratory at politeknik indonusa surakarta, and ugm integrated research and testing laboratory4. organoleptic test each herbal medicine product included ingredients and organoleptically tested shape, color, and taste5. sample preparation the sample in the herbal medicine weighed approximately 100 mg. it was put into a beaker, and then a solution of chloroform: methanol (9:1) approximately 10 ml was added. it was sonicated for 20 minutes and filtered. furthermore, the liquid extract from the herbal medicine sample was collected. the extract was evaporated on a water bath to dry. the remaining evaporation was dissolved with 5 ml of methanol6. preparation of dexamethasone comparative standards the standard for dexamethasone weighed 10 mg and was put in a volumetric flask. it was dissolved with 96% ethanol to 10 ml and then homogenized6. qualitative test with thin layer chromatography method sample solution and dexamethasone standard were spotted on the gf254 silica gel tlc plate. the eluent was a mixed solvent of ethyl acetate: toluene: methanol (45:55:1) that was put into the chamber. after the eluent reached the mark, it was removed and dried. the resulting chromatogram was observed for stains under ultraviolet (uv) light with a wavelength of 254 nm. furthermore, the stains were found in the sample and the standard. the rf value of the sample and standard was later calculated2. quantitative analysis of dexamethasone by tlc-densitometry a. preparation of standard solutions the standard solution was prepared carefully by weighing approximately 10 mg of dexamethasone and dissolving it with ethanol up to 10 ml to obtain a dexamethasone stock solution with a concentration of 1000 ppm. furthermore, a 2.5 ml pipette was put into a 5 ml measuring flask filled with 96% of ethanol 2.5 ml so that a standard solution with a concentration of 500 ppm was obtained7. b. preparation of sample solution the sample of macerated herbal medicine was weighed 100 mg and then dissolved in 1 ml of 96% ethanol. it was mixed with vortex for 2 minutes and was sonicated for 60 minutes. samples were macerated overnight. it was put into vortex and centrifuge for 5 minutes to take the supernatant4. c. determination of dexamethasone levels in samples the standard solutions and samples included concentrations of 100 µg, 200 µg, 400 µg, 600 µg, 800 µg, 1000 µg. the sample spot was then done by micropipette with a concentration of 5 µl on the tlc gf254 plate. the plates were eluted in a chamber containing eluent ethyl acetate: toluene: ethanol: n-hexane (51:12:3:15)4. separated spots were observed with a uv lamp of 254 desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 13 nm and measured by tlcdensitometry at a maximum wavelength of 240 nm, and the scan results were analyzed8. characterization with ftir the characterization of the ftir spectrum aimed to determine the functional groups of the identified organic compounds, namely dexamethasone in rheumatic pain herbal medicine. the sample s1, s2, and s3 powder containing dexamethasone were grounded using kbr into a solid pallet. spectra were recorded at a wavelength of 4000-650 cm-1 to obtain functional group bands from the sample. the spectra were recorded first to read the background (air background). furthermore, the sample was inserted into the sample holder and left for a few moments for the sample scanning to finish and get detailed spectra data. results and discussion sample description the description below is the difference based on the brand, composition, efficacy or use, dosage, and the registration status with bpom. table 1. description of the samples of jamu aches and pains sample and picture ingredients (s1) royal jelly foeniculi fructus liqustici radic conidii radic (s2) kaempferiae rhizome curcumae rhizome retrofracti fructus zingiberis purpurae rhizome parkiae semen (s3) kaempferiae rhizome curcumae rhizome retrofracti fructus zingiberis purpurae rhizome zingiberis aromaticae rhizome parkiae semen dexamethasone qualitative analysis organoleptic test the first test carried out in the qualitative analysis was the organoleptic test. in the organoleptic test, the aim is to determine the tested sample's color, taste, dosage form, and odor5. journal of fundamental and applied pharmaceutical science, 2(1), august 2021 14 table 2. organoleptic test results of rheumatic pain herbal medicine samples herbal samples form color scent flavor picture sample s1 powder pale-yellow herb plain sample s2 powder pale-yellow herb sweet sample s3 powder lightbrown herb bittersweet characterization with ftir the characterization of the ftir spectrum aims to determine the functional groups of the identified organic compounds, namely dexamethasone in rheumatic pain herbal medicine. basically, the infrared spectrum is used to determine the type of functional group of a compound. in the standard compound of dexamethasone, several groups can show peaks in the ir spectrum. figure 1. ftir spectrum on dexamethasone standard the rheumatic pain herbal medicine sample carried out using ftir spectrophotometer analysis should not contain dexamethasone as it will affect the predicted concentration value to be greater than the concentration intentionally added by the researcher when reading ftir and that the oh c-h c=o c=c c-x c-h desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 15 concentration value is not valid. thus, it is necessary to conduct qualitative analysis to prove that the sample of herbal pain relief does not contain chemicals drugs, considering that many herbal products sold in the market have been mixed with chemicals drugs such as dexamethasone9. based on the results of standard characterization of dexamethasone and samples of rheumatic pain herbal medicine using ftir, it was shown that there was a possibility that all samples of rheumatic pain herbal medicine contained dexamethasone, as evidenced by the functional group comparisons to dexamethasone standard obtaining the same functional groups, namely ch, oh, c= o, c=c and cx8. the results of the ftir spectra and the description of the identification of the functional groups from the standard dexamethasone and samples of rheumatic pain herbal medicine s1, s2, and s3 are shown in picture 1, 2, 3, and 4, respectively. table 3. interpretation of ftir spectra on s1, s2, s3, and dexamethasone standard wavelength (cm-1) [9] sample wavelength (cm-1) intencity functional group s1 s2 s3 standard 3570 – 3200 3282.74 3318.07 3283.76 3393.67 strong o-h (hydroxyl) 3000 – 2850 2921.12 2924.37 2925.05 2932.76 strong c-h (alkene) 1820 – 1600 1635.09 1636.43 1633.45 1705.82 strong c=o (ketone) 1400 – 1600 1418.12 1431.55 1418.33 1436.56 medium – weak c=c (aromatic) 1400 – 1000 1076.02 1075.57 1075.27 1091.93 strong c-x (flouride) 1400 – 1000 1012.54 1011.22 1011.43 1033.84 strong c-x (flouride) 1000 – 650 666.14 666.90 666.30 661.98 strong c-h (alkene) figure 2. ftir spectrum on sample s1 o-h c-h c= o c= c c-x c-h journal of fundamental and applied pharmaceutical science, 2(1), august 2021 16 figure 3. ftir spectrum on sample s2 figure 4. ftir spectrum on sample s3 figure 5. overlay spectra of ftir on sample s1, s2, and s3 o-h c-h c=o c=c c-x c-h o-h c-h c=o c=c c-x c-h desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 17 the overlayed spectra of ftir on samples s1, s2, and s3 showed that all the samples had the same peak. it is expected that all the samples were contaminated with chemical medicine, dexamethasone. thin layer chromatography test the third test using the tlc method was carried out to separate the substances contained in the sample and the standard. it has the advantage of better identification accuracy and determines the rf value between the sample and the dexamethasone standard10. the test was carried out by spotting the dexamethasone standard solution and samples s1, s2, s3 on one plate with six replications of the standard dexamethasone solution and each sample s1, s2, s3 with two replications. the following are the results of detecting the tlc plate generated by the spot on uv 254nm observation. description : , = spot standard dexamethason and sample = spot standard dexamethasone with six replications = spot sample with two replications figure 6. tlc evaluation on rheumatic pain herbal medicine samples chromatograms resulting from qualitative analysis by tlc-densitometry consisting of std 1-6 is standard dexamethasone with the concentration of 100 µg, 200 µg, 400 µg, 600 µg, 800 µg, 1000 µg, respectively. the s1-s1a is a sample of herbal medicine s1 with two replications. meanwhile, s2s2a is herbal medicine sample s2 with two replications, and s3-s3a is herbal medicine sample s3 with two replications. journal of fundamental and applied pharmaceutical science, 2(1), august 2021 18 quantitative analysis table 4. thin layer chromatography (tlc) test results of rheumatic pain herbal medicine samples 0. sample and standard visual color uv 254 nm solute (cm) eluent (cm) rf value result dexamethason standard dexamethason pale-purple flouresence 4.80 8.50 0.56 + sampel s1 spot 1 pale-purple flouresence 3.90 8.50 0.45 spot 2 pale-purple flouresence 4.90 8.50 0.57 + spot 3 pale-purple flouresence 6.40 8.50 0.75 spot 4 pale-purple flouresence 7.00 8.50 0.82 spot 5 pale-purple flouresence 8.30 8.50 0.97 sample 2 spot 1 pale-purple flouresence 2.10 8.50 0.24 spot 2 pale-purple flouresence 4.00 8.50 0.47 spot 3 pale-purple flouresence 4.90 8.50 0.57 + spot 4 pale-purple flouresence 7.00 8.50 0.82 spot 5 pale-purple flouresence 8.30 8.50 0.97 sample 3 spot 1 pale-purple flouresence 2.10 8.50 0.24 spot 2 pale-purple flouresence 4.00 8.50 0.47 spot 3 pale-purple flouresence 4.90 8.50 0.57 + spot 4 pale-purple flouresence 7.00 8.50 0.82 spot 5 pale-purple flouresence 8.30 8.50 0.97 desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 19 tlc-densitometry evaluation the determination of the standard curve aims to obtain a line equation which can later be used for quantitative analysis of a compound. the standard curve of dexamethasone can be seen in picture 7. the results of making the standard curve of dexamethasone showed r2 = 0.9937 with a linear equation y = 13.845x + 942.46. the best r-value is close to 0.99 [11]. meanwhile, the b-value obtained on the standard curve of dexamethasone was 13.845. the value of b is the slope indicating the sensitivity. it means that the greater value of b indicates the sensitive result of the method. moreover, the value of a, on the standard curve of dexamethasone, was 942.4. the value of a means selectivity, indicating that the smaller the value of a is, the more selective the measurement will be. the tlcdensitometry method is selective for determining the concentration of dexamethasone. figure 7. dexamethasone standard curve the alignment of the regression model can be explained using the value of r2. if the r2 value is close to 1, it means the regression model is getting better. r2 value has the characteristic where the value is always positive. the maximum r2 value is 1. the result obtained in this study showed an r2 value of 0.9937, indicating a good meaning of conformity. after obtaining the line equation to identify the dexamethasone content in the sample, the testing was carried out to determine the area of the sample so that the “y” value can be obtained. the result of the analysis of the samples is then interpreted in the form of chromatograms. based on table 5, in finding out the dexamethasone content in the sample, the area value entered as the “y” value in the line equation obtained previously. the dexamethasone level in the sample can be calculated using the line equation based on the previous line equation. in the s1 herbal medicine sample, the dexamethasone level was 1014.64 µg/g, and in the s2 herbal medicine, the concentration was 131.15 µg/g. in the s3 herbal medicine, the levels obtained were 135.54 µg/g. y = 13,845x + 942,46 r² = 0,9937 0 2000 4000 6000 8000 10000 12000 14000 16000 0 200 400 600 800 1000 1200 a re a ( a u ) concentration (µg) dexamethasone standard curve journal of fundamental and applied pharmaceutical science, 2(1), august 2021 20 table 5. result of the qualitative analysis using tlc-densitometry sample replication concentration sample (µg) auc dexamethason in sample (µg) concentration of dexamethason (µg/g) mean (µg/g) s1 1 505 7905.33 0.503 995.87 1014.64 2 504.5 8160.74 0.521 1033.42 s2 1 501 2188.62 0.090 179.65 131.15 2 504 1519.28 0.042 82.66 s3 1 514 1694.84 0.054 105.72 135.54 2 513.5 2118.13 0.085 165.36 validation of the analytical method is used to show that the analytical method is feasible and is expected to obtain reliable results. furthermore, the precision parameters are used to identify the closeness of the measurement results under the same conditions. in this study, standard solutions used a concentration of 100 µg, 200 µg, 400 µg, 600 µg, 800 µg, 1000 µg. this research was conducted with two replications. based on the table above, the three rheumatic pain herbal medicine samples contained the chemical medicine of dexamethasone, whereas there should be no medicinal chemicals in herbal medicine. table 6. the results of the organoleptic test, ftir test, and tlc-densitometry test sample organoleptic test ftir tlc tlcdensitometry conclusion s1 + + + (positive) + s2 + + + (positive) + s3 + + + (positive) + based on the result in table 6 above, the observation of organoleptic tests, tlc tests, ftir tests, and tlc-densitometry test can conclude that the three samples had positive results containing the chemical contamination of the dexamethasone. in terms of the safety aspect, it is an absolute requirement that must be met by herbal medicine, according to the minister of health regulation no. 007 of 2012 concerning the registration of traditional medicines, including using materials that meet safety and quality requirements. it explains that the efficacy has to be empirically and scientifically proven. furthermore, the production process must meet the requirements for a suitable traditional medicine manufacturing method. it must not contain any medicinal chemicals, narcotics or psychotropic substances, and other materials that, based on health considerations or based on research, can endanger health [16]. in relation to it, the factor of herbal medicine manufacturers adding medicinal chemicals is to increase the efficacy of herbal medicine and provide a more instant herbal effect than those that do not contain medicinal chemicals. conclusion traditional herbal medicine basically should not be mixed with chemical desy ayu irma permatasari, novita kurniasri, & muladi putra mahardika | qualitative and quantitative analysis of dexamethasone in rheumatic pain herbal medicine using thin layer chromatography (tlc) –densitometry 21 medicine, such as dexamethasone. based on the result of this study, there was contamination of the chemical medicine, dexamethasone, in the samples of herbal aches and pains s1, s2, and s3 after the evaluation with qualitative and quantitative analysis such as organoleptic tests, characterization with ftir, thin layer chromatography, and tlcdensitometry. the concentration of dexamethasone in the s1, s2, and s3 herbal pain relief were 1014.64 µg/g, 131.15 µg/g, and 135.54 µg/g. furthermore, the highest concentration of the chemical medicine of dexamethasone was found in 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(2011). keputusan kepala badan obat dan makanan no. 00.05.41.1384 tentang kriteria dan tata laksana pendaftaran obat tradisional, obat herbal terstandar, dan fitofarmaka. jakarta https://doi.org/10.33096/jffi.v3i1.172 hematological parameters in subchronic toxicity test of black garlic ethanol extract in rats ellen stephanie rumaseuw1*, yoppi iskandar2, eli halimah3 1department of phytochemical, saint borromeus college of health sciences, jl. parahyangan lot 8 block b no.1, kota baru parahyangan, cipeundeuy, padalarang, cipeundeuy, padalarang, west bandung regency, west java 40553, indonesia 2department of pharmacy biology, faculty of pharmacy, universitas padjadjaran, jl. raya bandung sumedang km 21, jatinangor, sumedang, west java 45363, indonesia 3department of pharmacology and clinical pharmacy, faculty of pharmacy, universitas padjadjaran, jl. raya bandung sumedang km 21, jatinangor, sumedang, west java 45363, indonesia abstract the community has used black garlic since ancient times for hypercholesterolemic. until now, people still consume both raw and cooked black garlic. black garlic is included in processed garlic products. people on the asian continent have used black garlic for the past 10 years. this study aims to determine the sub-chronic toxic effect of black garlic ethanol extract on male and female white rats using hematological parameters consisting of hematocrit, hemoglobin, erythrocytes, leukocytes, platelets, mcv, mch, and mchc. this research method was a completely randomized design with the administration of ethanolic extract of black garlic to rats in 5 treatment groups, including a negative control group, a dose group of 1000 mg/kg bw, a dose group of 2000 mg/kg bw, a negative control satellite group and a satellite group with a dose of 2000 mg/kg bw. data were analyzed statistically using one-way anova with a 95% confidence level and spss version 20. the study results of black garlic ethanol extract showed no significant effect or were at normal levels on hematological parameters. therefore, it can be concluded that the ethanolic extract of black garlic is safe for human use as a treatment for hypercholesterolemia. keywords: black garlic; hematology; subchronic toxicity test data of article received reviewed accepted : : : 01 nov 2021 24 dec 2021 17 feb 2022 doi 10.18196/jfaps.v2i1.13755 type of article: research introduction the community has used black garlic for hypercholesterolemia. until now, people still consume both raw and cooked black garlic. black garlic is one of the processed garlic products. people on the asian it has a sweet, chewy taste and a distinctive aroma.1 black garlic contains carbohydrates, amino acids, total polyphenols, and flavonoids.2 in addition, there are changes in several bioactive compounds such as s-allyl cysteine, * corresponding author, e-mail: rumaseuwellen@gmail.com vitamins, phenolic acids and flavonoids in black garlic during the heating process. sallyl cysteine, one of the main components of sulfur-containing amino acid compounds, is five to six times higher than fresh garlic.3 the decrease in alliin content in black garlic is that alliin is converted to s-allyl cysteine. s-allylmercapto-cysteine, arginine and other compounds are not defined when the heating process.4 according to the 2016 minister of health regulation, garlic is included as a native plant of indonesia that can be used as ellen stephanie rumaseuw, yoppi iskandar, & eli halimah | hematological parameters in subchronic toxicity test of black garlic ethanol extract in rats 77 herbal medicine. it has conditions proven to be safe, efficacious and of good quality.5,6 the use of black garlic as a processed garlic product is a native indonesian herbal medicine included in the herbal medicine category called jamu. several studies of black garlic in vivo have also been carried out over the last 20 years, including prof. dr. jin ichi sasaki in japan, revealing that black garlic has antitumor activity.7 black garlic initially resulted in many japanese people producing black garlic using simple tools such as rice cookers and other heating devices so that black onions can be consumed.7 in addition to black garlic as an anti-tumor, many other activities were investigated by several researchers, such as research by wang et al. in 2012 related to the content of black garlic, namely sallyl-cysteine , which was found to be able to reduce 50% of the size of fibrosarcoma in mice.8 other research also investigated that black garlic has other biological activities such as antioxidant activity,9 anticancer on human leukemic cells,10 antiobesity where black garlic is given to obese mice,11 anti-inflammatories,12 hypoallergenic,13. research conducted by nuristika in 2018 concluded that black garlic did not cause death in mice, so the ld50 was unknown. another study revealed that the administration of dayak onion ethanol extract did not affect the hematological profile in white rats.14 the development of an herbal medicinal product that has been tested in vivo, apart from toxicity testing, parameters can also be studied to obtain a toxic effect. a natural ingredient can also be investigated for its toxicity, including changes in body weight, clinical symptoms, hematological parameters, clinical biochemistry, macro pathology, histopathology, target organs, mortality, and other general or specific effects.15 testing of a natural ingredient was carried out on a biological system such as oral subchronic toxicity testing on rats for 28 days or 90 days with five dose groups, namely the negative control group, a dose of 1000 and 2000 mg/kg bw, as well as the negative control satellite group and satellite group of 2000 mg/kg bw. at the end of the period of administering the test preparations, all living experimental animals were autopsied and subjected to mactopathological, hematological, clinical biochemical and histopathological observations. the test was carried out to determine the cumulative effect and reversibility effect after repeated exposure to the test preparation for a certain period.15 this study aims to identify if people with a history of hypercholesterolemia consuming black garlic continuously or in the long term causes toxic effects, as seen from the description of the blood hematology parameters. method the research design for the oral subchronic toxicity test was a completely randomized design (crd), in which there were treatment and control groups with homogeneous environmental factors. the research was conducted at the phytochemical laboratory of santo borromeus college of health and the pharmacology and toxicology laboratory of padjadjaran university. this research was started from february to may 2019. the sample used was garlic (allium sativum, l) determined in advance at the taxonomy laboratory of the department of biology, faculty of mathematics and natural sciences, padjadjaran university. 1 kg of garlic was processed into black garlic by the maceration method for 5x24 hours with 70% ethanol solvent. the garlic was processed with a rice cooker at 70oc heating for 21 days. the preparation of tested animals used were 25 male rats and journal of fundamental and applied pharmaceutical science, 2(2), february 2022 78 25 female wistar rats with a bodyweight of approximately 100-200 grams according to the cage space guidelines for animals used in biomedical research (2008), adapted for 1 week. before carrying out subchronic toxicity testing, it is necessary to have a research code of ethics (ethical clearance) from the research ethics commission of the faculty of medicine, the university of padjadjaran, with number 528/un6.kep/ec/2019. the black garlic ethanol extract was administered for 28 days (measurement of rats’ body weight) and 29 days for the negative control group, and 43 days for the satellite test examination. hematological examination on mice took 1 ml of blood into a k2edta vacutainer tube containing 0.5 ml of ethylene diamine tetraacetate (edta). the blood was then examined using a hematological analyzer (xp-100). the data obtained included hemoglobin, erythrocytes, leukocytes, platelets, mcv, mch, and mchc. the graph was created to determine the increase in the body weight of rats. statistical analysis of blood hematological parameter data used one way anova and spss version 20. the research materials included garlic obtained from lembang-west java. all other chemicals and reagents were sourced commercially, such as ethanol from merck (germany) and pulvis gummi arabicum from j. trading co. ltd. (thailand). research tools included thermostatic water bath (china), rotary evaporator re 100-s dlab (china) and hematological analyzer xp-100 (china). results and discussion ethanol extract of black garlic the maceration results were revealed after being conducted for 5 days. this method was used to extract the active substance that has pharmacological activity. hypercholesterolemia obtained a yield of 49.584%, as shown in table 1. table 1. yield value of black garlic ethanol extract black garlic extract of black garlic yield value of black garlic 1.000 gram 495.84 gram 49.584% oral subchronic toxicity test the graph of the weight development of male rats is shown below. in administering test preparations at a dose of 1000 mg/kg bw and 2000 mg/kg bw, the bodyweight development of rats fluctuated every day, as shown in figure 1. figure 1. bodyweight development of male rats ellen stephanie rumaseuw, yoppi iskandar, & eli halimah | hematological parameters in subchronic toxicity test of black garlic ethanol extract in rats 79 there were fluctuations in the body weight of rats in the negative control group and doses of 1000 and 2000 mg/kg bw until the 28th day. an increase in body weight of rats occurred in the negative control satellite group, and the satellite dose of 2000 mg/kg bw significantly until the 42nd day. the graph of the bodyweight development of female rats is shown below. in administering test preparations at a dose of 1000 mg/kg bw and 2000 mg/kg bw, the bodyweight development of rats fluctuated every day, shown in figure 2. figure 2. bodyweight development of female rats there were fluctuations in the body weight of female rats in the negative control group and doses of 1000 and 2000 mg/kg bw until the 28th day. an increase in body weight of rats occurred in the negative control satellite group, and the satellite dose of 2000 mg/kg bw significantly until the 42nd day. in addition to weighing the body weight of rats, the average increase in body weight was also observed to determine the magnitude of administering black garlic ethanol extract with certain doses, as shown in table 2. table 2. average body weight of male and female rats groups n male rats n female rats average average negative control 5 191.6±14.18 5 175.6±12.29 1000 mg/kg bw 5 187.6±11.09 5 166.7±12.97 2000 mg/kg bw 5 184.7±12.95 5 151.8±13.15 satellite 2000 mg/kg bw 5 194.3±17.34 5 174.42±9.09 satellite negative control 5 191.07±16.87 5 169.21±10.45 note: n = number of rats in each treatment group journal of fundamental and applied pharmaceutical science, 2(2), february 2022 80 the statistical data analysis was carried out regardless of the difference in the increase in body weight of rats among groups, as shown in table 3. table 3. result of statistical analysis of body weight of male and female rats groups statistical analysis results (p>0.05) male rats female rats day 1-28 negative control 1000 mg/kg bb 2000 mg/kg bb anova 0.034 0.144 0.312 anova 0.345 0.434 0.146 day 29-42 satellite negative control satellite 2000 mg/kg bb kruskal wallis 0.140 kruskal wallis 0.337 the research data were tested for normality and homogeneity using anova and non-parametric analysis methods for k independent samples (kruskal wallis). if the significance is more than 0.05 (p>0.05), there is no significant difference, and no further test is needed. if the significance is less than 0.05 (p<0.05), a significant difference can be further identified by conducting the tukey test. the administration of black garlic ethanol extract in the negative control group, a dose of 1000 and a dose of 2000 mg/kg bw of male and female rats for 28 days, did not significantly increase the bodyweight of the tested rats (28 days) and the satellite group (42 days). hematological parameter test the hematological examination was carried out to determine abnormalities in the quantity and quality of blood cells. it also examined changes in plasma that played a role in the blood clotting process and identified if there was inflammation or infection, as indicated in table 4 and table 5 below. ellen stephanie rumaseuw, yoppi iskandar, & eli halimah | hematological parameters in subchronic toxicity test of black garlic ethanol extract in rats 81 table 4. value of hematological parameters analysis of male rat parameter groups normal level male grade value anova (p>0.05) hemoglobin (g/dl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 13.7-17.6 12.5 13.3 14.9 14.1 16.8 0.259 hematocrit (%) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 39.6-52.5 33.46 40.01 41.10 40.81 49.87 0.239 leukocytes (103/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 1.96-8.25 10.31 1.96 9.98 10.04 9.29 0.565 erythrocytes (106/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 7.27-9.65 5.86 7.83 7.96 7.71 8.51 0.280 platelets (103/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 638-1.177 784 638 487 645 676 0.487 mcv (fl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 48.9-57.9 57 53 52 53 59 0.550 mch (pg) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 17.1-20.4 21,4 17,8 18,7 18,3 19,7 0.679 mchc (g/dl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 32.9-37.5 37.5 32.9 36.2 34.6 33.7 0.755 journal of fundamental and applied pharmaceutical science, 2(2), february 2022 82 table 5. value of hematological parameters analysis of female rat parameter groups normal level female grade value anova (p>0.05) hemoglobin (g/dl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 13.7-16.8 12.4 13.2 14.3 13.0 14.4 0.520 hematocrit (%) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 37.9-49.9 34.42 38.74 38.57 38.86 44.11 0.122 leukocytes (103/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 1.13-7.49 7.24 5.82 6.17 6.07 5.74 0.437 erythrocytes (106/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 7.07-9.03 5,87 6,02 7,23 6,35 7,27 0.552 platelets (103/μl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 680-1.200 442 743 394 688 537 0.077 mcv (fl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 49.9-58.3 59 56 53 61 61 0.111 mch (pg) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 17.8-20.9 21.1 20.2 19.8 20.4 19.8 0.658 mchc (g/dl) negative control 1000 mg/kg bw 2000 mg/kg bw satellite2000 mg/kg bw satellite negative control 33.2-37.9 35.9 36.0 37.1 33.4 32.7 0.010 ellen stephanie rumaseuw, yoppi iskandar, & eli halimah | hematological parameters in subchronic toxicity test of black garlic ethanol extract in rats 83 male rats in the 2000 mg/kg bw and satellite 2000 mg/kg bw groups experienced an increase in the production of leukocytes to fight infection or inflammation caused by immune system disorders16,17 . in female rats in the 2000 mg/kg bw satellite dose group, the mcv value increased due to a lack of folate/vitamin b12 nutrition and the possibility of liver infection, which could be examined through further histopathological examination.18,19 the suspension of ethanolic extract of black garlic in the negative control group, doses of 1000 mg/kg bw, 2000 mg/kg bw, the negative control satellite group and the satellite group of 2000 mg/kg bw in male and female white rats for 28 days to 42 days did not significantly affect blood levels, such as hemoglobin, hematocrit, leukocytes, erythrocytes, platelets, mcv, mch, and mchc. a study on black 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(bactericidal and fungicidal). betel leaf (piper betle) can be used in the treatment of tinea versicolor or known in the community as panu. however, its use in the form of leaves is considered impractical by the public and also difficult to obtain. this research formulated betel leaf (piper betle) into lotion preparations, so its use in the community can be more practical. this study aims to determine the type and concentration of the suspending agent used to produce lotion preparations from betel leaf extract (piper betle), which has optimal physical quality. this research was an experimental study. the distillation of betel leaf (piper betle) was carried out to get essential oils. the essential oils of betel leaf (piper betle), which was used as an active substance in formulations, was 5%. suspending agents that were used in this study were arabic gum with concentrations of 10%, 15%, 20%, and cmc na, with concentrations of 0.25%, 0.5%, and 1%. the formulation of lotion preparations included organoleptic observation, ph test, homogeneity test, and adhesion test. based on the physical quality test of the lotion preparations that had been conducted, it can be concluded that the lotion preparations of betel leaf extract with 20% concentration of arabic gum suspending agent had physical qualities in accordance with lotion dosage criteria. keywords: betel leaf extract; formulation; lotion; piper betle data of article received reviewed accepted : : : 28 feb 2020 9 may 2020 23 jul 2020 doi 10.18196/jfaps.010104 type of article: research * corresponding author, e-mail: ingenida.hadning@umy.ac.id journal of fundamental and applied pharmaceutical science, 1(1), august 2020 29 introduction tinea versicolor is a non-inflammatory chronic disorder in which the type depends on the specific features, etiology, or place. tinea versicolor is characterized by the presence of multiple macular spots that usually seen in the tropics area and caused by malasseziafurfur.1 tinea versicolor is a disease caused by a fungus that lodged in the skin, because the body neither maintained nor cleaned regularly.2 the community has known tinea versicolor as panu. panu is a skin disease that often occurs, both in women and men, especially because it is associated with problems of hygiene and poor sanitation. the prevalence of tinea versicolor 50% occurs in tropical communities, 5% in subtropical regions and <1% in female populations 20.8% in the cold areas.3 the public has long known the plant-based treatment. the effort to develop plants for treatment is required considering that in indonesia, plants are easily obtained, and also at low prices. however, the use of plants for treatment needs to be supported by research data from these plants; thus, the efficacy is not scientifically doubtful, and it could be justified. it certainly can encourage the widespread use of the plants as medicine by the community.4 piper betle is one of the medicinal plants that grows a lot in indonesia and is known as daun sirih. betel leaf is traditionally used as a mouth ulcer medicine, sore throat, cough medicine, eyewash medication, leucorrhoea, bleeding in the nose/nosebleeds, accelerate wound healing, eliminate bad breath, and treat toothache. betel leaf has a typical aroma as it contains essential oils of 1-4.2%, water protein, fat, carbohydrates, calcium, phosphorus, vitamins a, b, c iodine, sugar, and starch. based on these various contents, in essential oils, there are natural phenols which have extreme antiseptic power (bactericidal and fungicidal) but are not sporosid.5,6 however, the use of medicinal plants in their original form is considered impractical and also challenging to obtain. thus, in this study, betel leaf (piper betle) has been formulated into a lotion preparation, so its use in the community can be more practical. this research aims to determine the type and concentration of suspending agent used to produce lotion preparations from betel leaf extract (piper betle) which has optimal physical quality. methods this study is an experimental study. the research was conducted for one month at the laboratory of pharmacy technology, faculty of medicine and health sciences, muhammadiyah university of yogyakarta. materials the tools needed in this research were homogenizer (t 25 digital ultra-turrax®), ph meter (mettler toledo®), and analytical balance (mettler toledo®). meanwhile, the materials used in this study were betel leaf (piper betle) obtained from nasaran village, cangkreplor, purworejo district, purworejo regency, propylene glycol / peg 400 (brataco), cmc na (brataco), arabic gum (brataco), methylparaben/nipagin (brataco), oleumrosae (brataco), 70% ethanol (brataco) and aqua dest. collection of test materials the betel leaves used in this study were obtained from the village of narget, cangkrep lor, purworejo district, purworejo regency. the betel leaves were ingenida hadning, putri kurnyaningtyas, muhammad thesa ghozali | the formulation of lotion preparations of betel leaf extract (piper betle) 30 fresh and free from pesticides and plant pests. they were separated from the stem and were washed with running water and cleaned from the dirt. plant determination the purpose of determination is to identify the correctness of the sample used in the study. determination of betel leaf plants was carried out in the division of pharmaceutical biology, faculty of pharmacy of gadjah mada university by matching the morphological characteristics that exist in the betel leaf plants against the literature of flora of java volume i.7 making betel leaf extract (piper betle) six kilograms of fresh betel leaves were cut into pieces and put into cormorant filled with water. the distillation apparatus was then assembled with a cooler condenser. sodium chloride was added to the distillate so that the emulsified oil was separated. the water phase was accommodated with erlenmeyer. in the water phase, sodium chloride was added and separated by a separating funnel. this step was carried out repeatedly until all the oil was separated. in the oil phase obtained, anhydrous calcium chloride was added, decanted and weighed.8 optimization of lotion base formulation the process of producing lotion preparations in this study used six treatment formulas, such as arabic gum 10%, 15%, 20%, and cmc na 0.25%, 0.5%, 1%, as shown in table 1. the producing process was that the betel leaves were separated from other ingredients. suspending agent ingredients, such as arabic gum and cmc na, were developed first. the suspension material used was developed by producing the hydrocolloid dispersion stock and by sprinkling the cmc na / arabic gum powder slowly and little by little into a mortar that had been filled with hot water. after that, the cmc na powder / arabic gum was soaked, then stirred quickly. furthermore, the peg 400 was poured, and then betel leaf extract was added. methylparaben ingredient was dissolved using 70% ethanol. the 60 ml of distilled water was then added into the components that had been slowly mixed while stirring. it was later put in a beaker glass. the stirring process was continued by using ultraturrax for 2 minutes. table 1. the formula development name of substance (%) function of substance g1 g2 g3 c1 c2 c3 betel leaf extract active substance 5 5 5 5 5 5 nipagin preervative 1 1 1 1 1 1 peg 400 binder 3 3 3 3 3 3 cmc na suspending agent 0,25 0,5 1 arabic gum suspending agent 10 15 20 oleum rose deodorizer 1 1 1 1 1 1 aquadest solvent ad 100 ad 100 ad 100 ad 100 ad 100 ad 100 note : g1: lotion formulation with arabic gum 10% g2: lotion formulation with arabic gum 15% g3: lotion formulation with arabic gum 20% c1: lotion formulation with cmc na 0.25% c2: lotion formulation with cmc na 0.5% c3: lotion formulation with cmc na 1% journal of fundamental and applied pharmaceutical science, 1(1), august 2020 31 selection of lotion formulations in this part, ph concentration measurements were taken for each lotion formula with a different betel leaf extract composition. the formula with the ph of the preparation must be consistent with the ph requirements of the antifungal lotion, which is 6.6-7.5. formulations were made on a large scale, and evaluation was then carried out. physical quality test of losio preparations evaluation for lotion preparations was carried out to determine the stability of the preparation and the level of safety in pre-clinical use. the evaluation of lotion preparations included organoleptic observation, ph changes, homogeneity test, and adherence test. organoleptic observation included observing changes in shape, color, and an odor that occurred at specific timescale for 28 days. the organoleptic observation was carried out on days 1, 7, 14, and day 28.9 ph measurements of the lotion formula that had been produced were carried out using a ph meter, and it was then dipped into lotion preparations. upon the proper immersion process, the color change in the ph meter was observed and adjusted to the standard color on the device. measurements were taken on days 1, 7, 14, 21, and 28.10. the lotion homogeneity test was conducted by applying each formula sufficiently on a glass plate, then touched and rubbed. the mass of the lotion must show its homogeneous arrangement proved by the absence of solid material on the glass. the replication was carried out three times. furthermore, the adhesive strength test was conducted using a glass object which was bound using two statives. one of the ends of the glass bonding object was given a 60-gram ballast. then, the resulting lotion was smeared on one of the glass objects and covered with another glass object and then suppressed with a weight of 1 kg for 5 minutes. the glass object was well positioned so that the two strings which bound the two glass objects stiffened up to release the ballast. calculating the time was needed to take the two glass objects to release their attachment.7 results and discussion extraction betel leaf extract was produced by taking fresh betel leaf essential oil and by referring to a study entitled determination of eugenol levels in essential oils from red betel leaves (piper cf fragile benth.) and green betel (piper betle) by gas chromatography.8 steam distillation method was chosen to take the betel leaf essential oil to be used as an active substance on the betel leaf lotion preparations. betel leaf extract was made with 70% ethanol solvent. a total of 6 kg was used for the extraction process. before the extraction process, betel leaves were chopped into ±5 cm, so that the essential oil contained in the betel leaf was easily produced. then, the distillation process was carried out using water vapor for 6 hours with seven replications. it was intended that the betel leaf essential oil contained in the betel leaf could be extracted as a whole. after the distillation process was complete, the result of essential oils was produced. at first, the oil produced was murky yellow and still contained water. after adding anhydrous sodium sulfate to remove water in essential oils, clear yellow essential oils were obtained. then essential oils that have been produced were used in this study. the formulation of lotion preparations of betel leaf extract the process of producing lotion preparations from betel leaf extract used ingenida hadning, putri kurnyaningtyas, muhammad thesa ghozali | the formulation of lotion preparations of betel leaf extract (piper betle) 32 three concentration ratios of a suspending agent of arabic gum, which was 10%, 15%, 20%. meanwhile, cmc na was 0.25%, 0.5%, 1%, and the concentration of betel leaf essential oil was 5%.12. in addition, producing lotion preparations used peg 400 as a binder. it has the advantages of being non-irritating, having good adhesion, and distribution to the skin and does not inhibit gas exchange and sweat production. thus, it was easy to wash with water and can be used on hairy body parts.13 moreover, in this study, nipagin was used as a preservative as an antibacterial.14 before being used in formulas, arabic gum or cmc na was developed. it aimed to thicken the viscosity of the liquid when the material was mixed.15 after the development of cmc na was added, the betel leaf extract, which was an essential oil role was as the oil phase. after all the ingredients had been put into the mortar, nipagin that had been dissolved with 70% ethanol was added. then, the aquadest was added up to 60 ml. after that, it was proceeded with the homogenization process using ultraturrax to dissolve all the ingredients in the container evenly. after all the procedures were finished, the container should tightly be closed and stored at room temperature. in the formulation of a lotion preparation, it is necessary to have certain ingredients to support the formation of the desired lotion.16in this research, as a formula development, suspending agents were used, such as arabic gum and cmc na, with different concentration variations. in terms of arabic gum, the concentration was 10%, 15%, 20%, while cmc na was 0.25%, 0.5%, 1%. the suspending agents function to slow down deposition, prevent clumping of resin and fatty material. the process was by increasing the viscosity of the liquid. physical evaluation results organoleptic observations showed that the concentration of arabic gum resulted in the form of betel leaf lotion. the concentration of arabic gum on the formulation process of betel leaf lotion conducted for one month can be seen in table 2. table 2. the organoleptic observation results formula description organoleptic week 1 2 3 4 g1 shape c c c c color p p p p odor ads ads ads ads g2 shape c+ c+ c+ c+ color p p p p odor ads ads ads ads g3 shape k k k k color p p p p odor ads ads ads ads c1 shape c, ks c, ks c, ks c, ks color pk pk pk pk odor ads ads ads ads c2 shape c+, ks c+, ks c+, ks c+, ks color pk pk pk pk odor ads ads ads ads c3 shape c++, ks c++, ks c++, ks c++, ks color pk pk pk pk odor ads ads ads ads note : c : liquid; + and ++ show the intensity of increased viscosity ks : rough texture k : thick p : white pk : yellowish-white ads : aromatic betel leaf the higher the concentration of arabic gum was, the thicker the form of the lotion would be. the highest form of viscosity level was obtained at 20% arabic gum concentration, while the lowest one was obtained at 10% arabic gum journal of fundamental and applied pharmaceutical science, 1(1), august 2020 33 concentration. arabic gum has a function as a suspending agent; thus, the more significant the use is, the thicker the liquid will be. the thickness produced from arabic gum was caused by high molecular weight which was around 240,000 580,000.14 the arabic gum structure (figure 1) consists of complex polysaccharides and branched molecules with different mass variations. it contains amino acids and some sugar monomers, such as galactose and arabinose.14 the product had a white color, which had an aromatic odor of betel leaf as, in this formulation, essential oil of 2 ml was added. this essential oil contained phenols, which caused lotion preparations to have an odor. meanwhile, formulas with cmc na suspending agents (figure 2) also produced different forms at each concentration. the highest viscosity was obtained at a concentration of 1%, while the lowest one was obtained at a concentration of 0.25%. compared to arabic gum, the lotion form with cmc na suspending agent with the highest concentration produced a very coarse lotion form. it was due to the smaller molecular weight of arabic gum, which was 90,000 700,000, and the viscosity of liquid cmc na. at higher concentrations, it can be used to prevent evaporation. figure1.the chemical structure of arabic gum figure 2. the chemical structure of cmc na based on the observations of ph obtained during the storage period, there was a change that the results obtained were close to the desired ph range. the ph requirement of betel leaf lotion, based on literature, was in the range of 5.5.17. the ph values are shown in table 3 displayed variations in the betel leaf lotion concentration, which affected the length of time storage. it was because there was no addition of a buffer solution to the betel leaf lotion formulation. a buffer solution was a mixture of weak acids or bases with their conjugate acids or bases, which could maintain the ph around the buffer capacity area. the function of this solution as a buffer solution was to maintain the ph. the preparations on the market (neutral body lotion) that had been measured had a ph of 5.5. it must be stable in the storage period as the preparations were not directly used. similarly, betel leaf lotion preparations in this study were to be stable in mass storage. the homogeneity test was conducted by applying 2x2 cm of the lotion on a glass plate based on the concentration of each lotion. it was then touched and rubbed. the mass of the lotion showed how much the homogeneous distribution was on the glass plate. the six formulas can be seen in table 4, with the average smoothest ingenida hadning, putri kurnyaningtyas, muhammad thesa ghozali | the formulation of lotion preparations of betel leaf extract (piper betle) 34 table 3. ph observation results formula the average ph during storage time day 1 3 6 9 12 15 18 21 24 27 g1 4,75 4,72 5,36 4,76 4,61 4,63 4,65 4,63 4,61 4,61 g2 4,74 4,75 4,65 4,68 4,63 4,66 4,69 4,66 4,68 4,62 g3 4,73 4,61 4,59 4,56 4,56 4,71 4,63 4,55 4,58 4,55 c1 6,72 6,44 6,51 6,05 5,84 5,82 5,76 5,77 5,67 5,65 c2 6,63 6,45 6,43 6,08 5,93 5,94 5,87 5,86 5,77 5,75 c3 6,90 6,78 6,82 6,55 6,35 6,38 6,31 6,30 6,17 6,16 comparison 5,5 table 4. the average of lotion homogeneity formula the average of lotion homogeneity day 1 3 6 9 12 15 18 21 24 27 g1 h h h h h h h h h h g2 h h h h h h h h h h g3 sh sh sh sh sh sh sh sh sh sh c1 h h h h h h h h h h c2 h h h h h h h h h h c3 k k k k k k k k k k note: k : rough h : soft sh : very soft -homogeneity in formula g3. g3 was lotion using 20% arabic gum concentration. whereas, the roughest homogeneity in the c3 formula was lotion using 1% cmc na concentration. the results of the study can be seen in table 4. the lotion homogeneity test was carried out to find out whether the lotion was truly mixed after the mixing process.11 it was to ensure that the active substances contained therein have been distributed equally. homogeneity was one of the factors that influenced the quality of the preparation of the lotion. homogeneity affects the distribution of betel leaf extract active ingredients in the lotion. the active ingredients of betel leaf extract must be dispersed to provide maximum effectiveness as an antifungal. an adhesion test is a test used to determine the maximum ability of the adhesive power of the lotion on the skin when it is used. the aim was to find out how strong the lotion preparations can be attached to the application area, such as the skin and to coat the surface of the skin in an impervious way, and not to clog pores and physiological functions of the skin.13 the adhesion test result was obtained by calculating the length of glass plates sticking or shifting that had been smeared with lotion. it was later overwritten with a load of 1 kg for 5 minutes and was released. the shift showed that the lotion was less attached journal of fundamental and applied pharmaceutical science, 1(1), august 2020 35 table 5.the results of the adhesion test formula the average of the adhesion test in a minute day 1 3 6 9 12 15 18 21 24 27 g1 ‘33 ‘38 ‘27 ‘46 ‘34 ‘29 ‘28 ‘27 ‘29 ‘28 g2 ‘35 ‘37 ‘26 ‘24 ‘38 ‘28 ‘38 ‘35 ‘28 ‘25 g3 ‘52 ‘38 ‘27 ‘24 ‘50 ‘47 ‘37 ‘34 ‘30 ‘31 c1 ‘32 ‘34 ‘45 ‘33 ‘32 ‘29 ‘37 ‘33 ‘24 ‘29 c2 1.63 7.34 ‘40 1.88 ‘49 ‘42 ‘36 ‘31 ‘33 ‘34 c3 1.32 2.27 ‘25 ‘39 ‘36 ‘38 ‘26 ‘24 ‘38 ‘39 -to the applied skin area, and it was then smeared off. the results of the adhesion observation test can be seen in table 5. the stickiness profile of each lotion was stored for four weeks after finding out that all lotion formulations had the same tendency. the longer the storage duration was, the more decreasing the time of the adhesion would be. the most significant decrease in the duration of the adhesive power of lotion was in formulas with 0.5% cmc-na suspending agent and 1% cmcna. formula with arabic gum suspending agent decreased the duration of adhesion compared to a formula with cmc-na suspending agent. conclusion based on the physical quality test of lotion preparations that have been conducted, it can be concluded that the lotion preparations from betel leaf extracts with 20% arabic gum suspending agent had a physical quality that matched the required lotion dosage criteria. acknowledgment the authors would like to thank the universitas muhammadiyah yogyakarta for supporting this research. conflict of interest the author declares that there is no conflict of interest. references 1. dorland, w. n. (2012). kamus saku kedokteran dorland edisi 28. jakarta: egc. 2. sahoo, a. k., & mahajan, r. (2016). management of tinea corporis, tinea cruris, and tinea pedis: a comprehensive review. indian dermatology online journal, 7(2), p. 77. 3. wantini, s., violita, y., & sulistianingsih, e. (2017). perbandingan uji efektivitas air perasan lengkuas merah (alpinia purpurata k. schum) dengan air perasan lengkuas putih (alpinia galnga l. wild) terhadap pertumbuhan jamur malassezia furfur penyebab panu. jurnal analis kesehatan, 2(2), pp. 282-289. 4. ardelia, p. i., andrini, f., & hamidy, m. y. (2017). aktivitas antijamur air perasan daun seledri (apium graveolens l.) terhadap candida albicans secara in vitro. jurnal ilmu kedokteran, 4(2), pp. 102-107. 5. hariyanto, s., 2012. ensiklopedia tanaman obat indonesia. jakarta: mitra setia. ingenida hadning, putri kurnyaningtyas, muhammad thesa ghozali | the formulation of lotion preparations of betel leaf extract (piper betle) 36 6. yuniarti, t, 2008. ensiklopedia tanaman obat tradisional. yogyakarta: media presindo. 7. erwiyani, a. r., luhurningtyas, f. p., & sunnah, i. (2017). optimasi formula sediaan krim ekstrak etanol daun alpukat (persea americana mill) dan daun sirih hijau (piper betle linn). cendekia journal of pharmacy, 1(1), pp. 77-86. 8. nurhidayati, l. y. d., & mariani, s. (2012). penetapan kadar eugenol dalam minyak atsiri dari daun sirih merah ( piper cf fragile benth.) dan sirih hijau ( piper betle l.) secara kromatografi gas. in seminar nasional pokjanas toi xlii. 9. octavia, m. d., halim, a., & afrinda, m. (2017). karakterisasi kompleks inklusi simvastatin–β-siklodekstrin metoda co-grinding dengan variasi waktu penggilingan. jurnal farmasi higea, 7(1), pp. 30-43. 10. anggraini, d., & kasmawati, l. y. (2017). formulasi gel sarang burung walet putih (aerodramus fushipagus) dan uji penyembuhan luka bakar derajat ii pada mencit. jurnal sains farmasi & klinis, 4(1), pp. 55-60. 11. fajriyah, u., astuti, i. y., & hartanti, d. (2010). formulasi losion ekstrak herba tali putri (cuscuta australis r. br.) dan aktivitas antioksidan secara in vitro. pharmacy: jurnal farmasi indonesia (pharmaceutical journal of indonesia), 7(01), pp. 24-34. 12. fitriana, a. y., wahyuningrum, r., & sudarso, s. (2012). daya repelan dan uji iritasi formula lotion ekstrak etanol daun sirih (piper betle linn) dengan variasi basis lanolin terhadap nyamuk aedes aegypti. pharmacy: jurnal farmasi indonesia (pharmaceutical journal of indonesia), 9(02), pp. 39-57. 13. siregar, c. j., & wikarsa, s. (2010). teknologi farmasi sediaan tablet dasar-dasar praktis. jakarta: egc. 14. rowe, r. c., sheskey, p., & quinn, m. (2009). handbook of pharmaceutical excipients. london: libros digitalespharmaceutical press. 15. hadning, i., (2016). formulasi dan uji stabilita fisik sediaan oral emulsi virgin coconut oil. mutiara medika: jurnal kedokteran dan kesehatan, 11(2), pp. 88-100. 16. nurlina, n., tomagola, m. i., hasyim, n., & rahman, f. (2014). formulasi suspensi kering kombinasi ekstrak etanol kunyit (curcuma longa l.) dan serbuk daging buah pisang kepok (musa balbisiana colla.) dengan variasi bahan pensuspensi. as-syifaa jurnal farmasi, 6(2), pp. 166-177. 17. yuliana, a., nurdianti, l., fitriani, f., & amin, s. (2020). formulasi dan evaluasi kosmetik dekoratif perona pipi dari ekstrak angkak (monascus purpureus) sebagai pewarna dengan menggunakan lesitin sebagai pelembab kulit. fitofarmaka: jurnal ilmiah farmasi, 10(1), pp. 1-11. the chemopreventive effects of the combination between tea leaf and mandarine peel extract on breast cancer cell ega hida prabowo, rifki febriansah* school of pharmacy, faculty of medicine and health science, universitas muhammadiyah yogyakarta, jl brawijaya, tamantirto, kasihan, bantul, yogyakarta 55183. abstract the number of breast cancer patients is increasing high in the world. this study aims to determine the chemopreventive effect of a combination of ethanolic extracts of tea leaves and mandarin peel in silico and in vitro on t47d breast cancer cells. extraction by the maceration method used ethanol solvent 70%. the research is an in silico molecular docking utilized software of autodock vina to determine the binding affinity of tangeretin compounds and epigallocatechin gallate (egcg) on her-2 protein. the antioxidant test used the 2,2-diphenyl-1-picrylhydrazyl (dpph) method to determine the antioxidant activity of the combination of tea leaf and mandarine peel (ctm) extract. the in vitro test used the method of 3-(4,5-dimethyltiazol-2-il) -2,5-diphenyltetrazolium bromide (mtt) to determine the value of ic50 from ctm on t47d breast cancer cells. the result of this study showed that ctm had a vigorous antioxidant activity with an ic50 value of 83 μg/ml. ctm had a weak cytotoxic activity with an ic50 value of 1889 μg/ml. molecular tethering results of tangeretin and egcg compounds produced a docking score of -6.6 and -5.0 kcal/mol with docking score proportion consisting of -4.9 kcal/mol of original ligand, -6.1 kcal/mol of doxorubicin and -4.5 kcal/mol of 5-fluorouracil. ctm had potential as a chemopreventive agent based on the robust antioxidant activity data on t47d breast cancer cells and molecular docking on the her-2 protein. keywords: antioxidant test; camellia sinensis; citrus reticulate; mtt assay; molecular docking; t47d breast cancer cells data of article received reviewed accepted : : : 18 feb 2020 12 may 2020 23 jul 2020 doi 10.18196/jfaps.010101 type of article: research * corresponding author, e-mail: rifki.febriansah@umy.ac.id ega hida prabowo, rifki febriansah | the chemopreventive effects of the combination between tea leaf and mandarine peel extract on breast cancer cell 2 introduction breast cancer is a deadly cancer attacking women with a high prevalence rate. in 2012, there were 26 cases per 100,000 women.1 one of the proteins that mainly causes breast cancer cell growth is her-2. in the majority of breast cancer patients, her-2 protein experiences uncontrolled expression through gene amplification that increases her-2 protein expression up to 40-100 times.2-5 breast cancer can grow and spread into its surrounding tissue, such as lymph nodes, and then enter the blood vessels and even to other organs (lungs, bones, liver, and brain).6 common treatments for breast cancer patients are such as chemotherapy, surgery, and radiation.7 however, these treatments are expensive and also often cause adverse side effects, which can kill the healthy surrounding tissue and even the effects of immunosuppression. it is necessary to innovate in defeating cancer, for example, using safer alternative treatments, such as plant extracts.8 natural ingredients that have the potential as a chemopreventive agent are tea leaves and mandarin (mandarin orange) peels. tea leaves contain a flavonoid-polyphenol compound, epigallocatechin gallate (egcg), which is proven to have a substantial antioxidant property. mandarin (citrus reticulata) peel contains a compound called ‘tangeretin’ that can potentially inhibit tumor cell growth, which is proven to be able to induce cell-cycle g1 arrest processes.9 considering all those benefits, this study aims to determine the chemopreventive effect of a combination of ethanolic extracts of tea leaves and mandarine peels in silico and in vitro. the combination of both extracts is expected to have a good synergy effect that can inhibit the development of cancer cells from being more significant. methods plant determination tea leaves (camellia sinensis) were obtained from the kaliurang area, yogyakarta, and mandarin peel (citrus reticulata) were obtained from the bantul region, yogyakarta. the determination of both plants was carried out in the laboratory of pharmacy biology, faculty of pharmacy, ugm yogyakarta. macerating extraction the selection of the extraction method was based on the natures and compositions to be isolated.10 extraction was conducted using ethanol solvent 70% and stirred routinely every day for five days. after that, the pulp was remacerated for two days. after two days, the maceration was filtered again and was then evaporated using a rotary evaporator until a thick extract was obtained.11 antioxidant test of dpph the dpph method was based on the freeradical reduction of dpph with maximum absorption at a wavelength of 517 nm, which produced a purple color in ethanol.12 after that, 2 ml of vitamin c solution was taken, which was the sample solution at various concentration series made into a 10-ml volumetric flask. the solution was added with 2 ml of dpph and a methanol solution into the limit mark. the solution was vortexed for 30 seconds and left alone within the operating time. uptake was read at  maximum and replicated three times. cytotoxic test of mtt assay this method was based on the breakdown of tetrazolium salt or yellow journal of fundamental and applied pharmaceutical science, 1(1), august 2020 3 mtt (3(4,5-dimethylthyazol-2-yl) -2,5diphenyl tetrazolium bromide) by dehydrogenase succinate enzyme contained in the cell mitochondria to turn into purplish blue formazan crystals.13 cells with a density of 70-80% (confluent) were distributed into 96 well plates, observed under an inverted microscope to identify the cells’ distribution, and then incubated for 48 hours so that the cells could adapt and attach to the bottom of the wells. on the following day, the media in the wells were dumped by turning the well plates over the dump, then drained with a tissue. the wells were washed using 100 µl pbs which was added to all wells filled with cells, and then they were discarded. furthermore, a series of concentrations of the test sample and culture media containing 0.2% dmso (control) was added and then re-incubated for 24 hours. at the end of the incubation, culture media containing the sample were removed and washed with 100 µl pbs, then added with the mtt reagent. in this case, living cells would react with mtt to form purple formazan crystals. after 4 hours of incubation, the condition of the cells was examined under an inverted microscope. when formazan was created, a 200 μl sds stopper solution was added into 0.1% hcl to dissolve the formazan crystals. the well plates were then covered with aluminum foil and incubated in a dark place at room temperature for 3 hours. upon that process, the aluminum foil was opened and shaken for 10 minutes. the absorption was read with an elisa reader at a wavelength of 595 nm. based on the absorbance data, the percentage of living cells and ic50 values could be calculated. molecular docking test molecular docking is a computational method used to predict the interaction between ligands and receptors or proteins to obtain the value which describes their total bond energy. the protein structure selected as the target of molecular docking in this study was her-2 (pdb id: 3pp0). the structure was downloaded from the protein data bank (pdb) through www.rcsb.org; thus, the protein’s pdb id could be identified. the application used for protein and ligand preparation in this study was ds visualizer. rmsd value was determined by filling in the windows command prompt according to the code; therefore, some conformations would appear. each conformation showed its rmsd affinity value, then the conformation, which had an rmsd value of less than 2 å, was selected. the visualization process of docking results used the ds visualizer app. the visualization was to determine the position and the image of the bond between proteins and ligands in 3d. results and discussion plant determination the results of the determination showed that the simplisia used in this study was tea leaf (camellia sinensis) and mandarin orange peel (citrus reticulata). macerating extraction the maceration method in this study used ethanol solvent 70%, 10 l for 1 kg of dried tea leaf powder, and 1 kg of dried mandarin peel powder. based on the filtering result, 2,450 ml of liquid extract was obtained. it was then dried using a rotary evaporator and produced 27.3 grams of thick tea leaf extract (tle) with a dark green color and 26.4 grams of a concentrated mandarin peel extract (mpe) with a dark yellowish-green color. both tea leaf extract and mandarin peel extract were mixed to get a combined extract. ega hida prabowo, rifki febriansah | the chemopreventive effects of the combination between tea leaf and mandarine peel extract on breast cancer cell 4 antioxidant test of dpph absorbance data of dpph antioxidant test results can be seen in tables 1 and 2, and the graphic is shown in figures 1 and 2. also, the ic50 value of the antioxidant test is shown in table 3. table 1. data of comparative inhibition percentage for vitamin c concentration (μg/ml) average absorbance blank absorbance % inhibition 0.5 0.641 0.702 8.73 1.0 0.638 0.702 9.11 2.0 0.625 0.702 11.01 5.0 0.557 0.702 20.76 10.0 0.402 0.702 42.76 20.0 0.189 0.702 73.04 30.0 0.055 0.702 92.22 table 2. data of inhibition percentage for ctm extract concentration (μg/ml) average absorbance blank absorbance % inhibition 5.0 0.721 0.702 -2.61 7.5 0.703 0.702 -0.09 10.0 0.664 0.702 5.46 15.0 0.633 0.702 9.87 20.0 0.472 0.702 32.87 30.0 0.381 0.702 45.80 40.0 0.346 0.702 50.78 table 3. ic50 value of antioxidant test for vitamin c and ctm extract compounds linear regression equation ic50 value (μg/ml) description vitamin c y=2.9913x+7.5319 14.19 very strong r2= 0.9859 ctm y=0.6427x-3.3458 83.00 strong r2=0.9698 table 4. the result of the molecular docking between ligands and her-2 receptors compounds rmsd value docking score conformation native ligand 1.957 -4.9 7 egcg 1.343 -6.6 2 tangeretin 1.197 -5.0 4 doxorubicin 1.542 -6.1 4 5-fluorouracil 1.576 -4.5 3 molecular docking with autodock vina molecular docking was conducted using the autodock vina application. open babel, and the shape of the structure were then visualized in 2d and 3d with the ds visualizer. the data on the molecular docking results can be seen in table 4. cytotoxic test of mtt assay the cytotoxic test was performed to determine mtt assay activity towards t47d breast cancer cells by giving ctm extract treatment shown in table 5. table 5. data of living cell after ctm extract treatment (%) content (μg/ml) average sample absorbance standard deviation % living cells 100 0.130 0.014 4.308 400 0.128 0.003 3.798 800 0.124 0.002 3.122 1000 0.118 0.001 2.009 average absorbance cell control 0.663 media 0.103 equation y = -0.0024x + 4.6713 r2 = 0.9233 ic50=1889 μg/ml cytotoxic test of mtt assay the cytotoxic test was performed to determine mtt assay activity towards t47d breast cancer cells by giving ctm extract treatment, as shown in table 6. table 6. data of living cell after ctm extract treatment (%) content (μg/ml) average sample absorbance standard deviation % living cells 100 0.130 0.014 4.308 400 0.128 0.003 3.798 800 0.124 0.002 3.122 1000 0.118 0.001 2.009 average absorbance cell control 0.663 media 0.103 equation y = -0.0024x + 4.6713 r2 = 0.9233 ic50=1889 μg/ml journal of fundamental and applied pharmaceutical science, 1(1), august 2020 5 figure 1. chart of vitamin c inhibition figure 2. chart of ctm extract inhibition figure 3. 3d structure of geometry optimization results: (a) native ligand (b) egcg (c)tangeretin (d) doxorubicin (e) 5-fluorouracil y = 2,9913x + 7,5319 r² = 0,9859 0,00 20,00 40,00 60,00 80,00 100,00 120,00 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 % i n h ib it io n concentration (μg/ml) y = 0,6427x 3,3458 r² = 0,9698 (10,00) 0,00 10,00 20,00 30,00 40,00 50,00 60,00 0,0 20,0 40,0 60,0 80,0 100,0 % i n h ib it io n concentration (μg/ml) (a) (b) (c) (d) (e) ega hida prabowo, rifki febriansah | the chemopreventive effects of the combination between tea leaf and mandarine peel extract on breast cancer cell 6 figure 4. 2d structure of interaction of test compounds on her-2 in atomic pockets: (a) native ligand (b) egcg (c) tangeretin (d) doxorubicin (e) 5-fluorouracil figure 5. graphic of living cells t47d with ctm extract treatment (%) figure 6. changes in t47d cell morphology (a) before the extract treatment (b) shortly after the extract treatment (c) after the extract treatment (sign ‘’ indicates damaged or dead cells) y = -0,0024x + 4,6713 r² = 0,9233 0,000 0,500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 0 200 400 600 800 1000 1200 % l iv in g c e ll s concentration (μg/ml) (d) (e) (b) (c) (a) (c) (a) (b) journal of fundamental and applied pharmaceutical science, 1(1), august 2020 7 antioxidant test results showed an ic50 value of vitamin c compounds was 14.87 μg/ml. whereas, the ic50 value of tle and mpe combination was 83.00 μg/ml, which was weaker in capturing radicals compared to vitamin c. it occurred as the combination sample (ctm) was still in the form of crude extracts; thus, the number of active compounds that could inhibit free radical activity was likely to be smaller. however, ic50 < 100 μg/ml was obtained and classified as antioxidants with vigorous activity. therefore, the combination of ctm extract had strong potential in the activity of capturing free radical compounds. referring to a similar study, an antioxidant test of kemuning extract (murraya paniculata (l) jack) produced an ic50 value of 126.17 μg/ml, and antioxidant analysis of mangosteen peel extract (garcinia mangostana l.) resulted in an ic50 value of 44.49 μg/ml.15 compared to this, ctm extract had more substantial antioxidant potential than kemuning extract. however, it was weaker compared to mangosteen peel extract.16 the results of molecular docking showed that the egcg compound had the most vigorous inhibitory activity on the her-2 protein with a docking score of -6.6 kcal/mol, compared to other comparative compounds. visualization of the docking results showed that egcg compounds were comparable to doxorubicin which had 7 bounds interacting with amino acid glutamine at position 407 (gln 407), glutamic acid at position 299 (glu 299), glutamic acid at position 383 (glu 383), threonine at position 301 (thr 301), glutamic acid at position 382 (glu 382), lysine at position 347 (lys 347), and phenylalanine at position 349 (phe 349). it was higher than the original ligand, which only had five bonds. the tangeretin compound interacted by binding to 6 bonds interacting with the amino acid threonine at position 301 (thr 301), lysine at position 346 (lys 346), lysine at position 347 (lys 347), glutamine at position 383 (glu 383), and valine at position 300 (val 300).17-20 the 5-fu ligand and tangeretin interacted with five bonds of amino acids, as many as the original ligand. the more the bonds are formed between the ligand and the target protein, the stronger the potential activity of a compound will be. it indicates that the egcg compound in silico has a strong potential to inhibit her-2 protein.21 based on the cytotoxic test results, ctm extract had a weak potential in inhibiting the viability of t47d breast cancer cells with ic50 values of 1889 μg/ml. it was probably due to the small amount of tangeretin compound content in ctm extracts, so the cytotoxic effect was not reliable. the tangeretin flavonoid compound, which was suspected of having an effect as a chemopreventive agent, had a mechanism to induce cell cycle g1 arrest when the cell was in the restriction point phase.22-24 a restriction point is a point where hyperphosphorylation occurs that causes the p27 inhibitor, a p53 expression gene (an apoptosis gene), to be degraded. at this point, the reparation of damaged dna occurs. if dna damage is severe and cannot be repaired, it will be immediately eliminated to the g0 phase. the mechanism of the cell cycle starts with the entry of it from the g0 phase to the g1 phase due to the growth factor stimuli. it then causes retinoblastoma protein (prb) hyperphosphorylation and dna to become loose. furthermore, it later enters the restriction point and the s ega hida prabowo, rifki febriansah | the chemopreventive effects of the combination between tea leaf and mandarine peel extract on breast cancer cell 8 phase to replicate dna. if there is damage during the synthesis, it can be repaired before it finally enters the m phase. in cancer cells, the p53 gene is mutated. conclusion it can be concluded that the combination of ethanolic extract of tea leaves and mandarin orange peel (ctm) had strong potential as an antioxidant agent in chemopreventive activity based on dpph and molecular docking tests. however, the results of the cytotoxic test showed a weak potential to inhibit the development of t47d breast cancer cells. some compounds that were considered to play an essential role in the chemopreventive mechanism were tangeretin and egcg compounds. acknowledgment the authors would like to thank the ministry of research, technology, and higher education republic of indonesia (ristekdikti) for funding this research through the beginner lecturer research grant in 2019. conflict of interest there is no potential for conflict of interest. references 1. waks, a. g., & winer, e. p. 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(2019). polymethoxyflavones: chemistry and molecular mechanisms for cancer prevention and treatment. current pharmacology reports, 5(2), pp. 98113. stability testing of compounding capsule combination between paracetamol and tramadol in a private hospital semarang shirley candra kurniawan, michael raharja gani*, sri hartati yuliani department of pharmacy, sanata dharma university, paingan, maguwoharjo, depok, yogyakarta-55284. abstract a combination of paracetamol and tramadol is used in mild to severe pain management. in a private hospital semarang, this combination is included in fast-moving drugs that it is frequently compounded and prepared in advance. the study aims to determine beyond use date (bud) in compounding capsules combination between paracetamol and tramadol samples. beyond use date is a time limit indicating that a medicine beyond the expiration date must not be used and determined based on the results of stability testing. samples were stored for 14 days in a tightly closed container far from direct sunlight in room temperature without ac (280c) and without silica gel, room temperature with ac (250c) without silica gel, and room temperature with ac (250c) with silica gel. furthermore, samples underwent physical and chemical stability testing. physical stability testing was conducted using organoleptic testing by observing direct changes from the powder color in capsules, capsules form, and scent on the samples. meanwhile, chemical stability testing was conducted by determining the content of active ingredient from the samples using reversed-phase hplc method and c18 as the stationary phase, methanol and aquabidest (40:60) as the mobile phase, wavelength 271 nm, flow rate 0.6 ml/min and injection volume 10 μl. the result shows that samples were physically stable for being able to retain the original physical properties showed by consistent powder color and capsules form, and there was no available scent. however, the chemical stability testing method was unable to separate and quantify the content of paracetamol, tramadol, and formed degradation products. it can be concluded that beyond use date based on organoleptic and chemical stability testing for 14 days of compounding capsules combination between paracetamol and tramadol could not be determined. nevertheless, this study showed favorable storage conditions in a tightly closed container far from direct sunlight in room temperature with ac (250c) with silica gel. keywords: beyond use date; paracetamol; reversed-phase hplc; tramadol data of article received reviewed accepted : : : 25 feb 2020 7 apr 2020 4 jul 2020 doi 10.18196/jfaps.010103 type of article: research * corresponding author, e-mail: mr_gani@usd.ac.id shirley candra kurniawan, michael raharja gani, sri hartati yuliani | stability testing of compounding capsule combination between paracetamol and tramadol in a private hospital semarang 20 introduction compounding is the process of combining, admixing, diluting, pooling, reconstituting other than as provided in the manufacturer’s labeling, or otherwise altering a drug or bulk drug substance to create a non-sterile medication.1 limitations of extemporaneous compounding practice in drug formulas are not available on the market, limitations of the patient’s condition in consuming the dosage forms, limited supply of drug preparations, patient compliance, and medical costs in the market.2 stability is the extent to which a product or preparation retains physical and chemical properties and characteristics within specified limits throughout its expiration or beyond use date.1 the stability of a pharmaceutical product may be defined as the capability of a particular formulation in a specific container/closure system to remain within its physical, chemical, microbiological, toxicological, protective, and informational specifications.3 stability studies are carried out to establish a beyond use date for the compounded preparation. the beyond use date indicates the days after the compounded formula is prepared in which the product is no longer safe to be used.1 high-performance liquid chromatography (hplc) is one of the separation methods in a column containing a stationary phase that will be flowed by the mobile step.4 reversedphase will elute samples with higher polarity earlier as the characteristic of the mobile phase is more polar than the stationary phase.5 hplc method is used since the chromatography technique can separate a mixture of compounds and is used to identify, quantify and purify the individual components of the mixture with better, faster, and more efficient compound separation results.4,6 reversed-phase of hplc with a uv detector provides the best available reliability, analysis time, repeatability, sensitivity, and capable of monitoring several wavelengths concurrently.7 combination of paracetamol and tramadol works synergistically with different mechanisms of action and used as a second-line medication for the treatment of moderate to severe pain management.8 in a private hospital, semarang, this combination is included in fast-moving drugs. yet, the prices are quite high on the market, that it is frequently compounded to lower the cost of the medication. real-time stability testing was carried out to assign beyond use date; thus, it can be used as a reference in storing and maintaining compounding capsules combination between paracetamol and tramadol. methods the materials were working standards of the pharmaceutical-grade of paracetamol (batch no. ws-qc-400395378) and tramadol (batch no. 100209203). it was used without further purification and certified to contain 99.3 % of paracetamol and 99.2 % of tramadol on a dry weight basis. the materials were such as paracetamol tablet 500 mg (promedrahardjo pharmaceutical industries, batch no. 00818l0110) and tramadol capsules 50 mg (otto pharmaceutical industries, batch no. 8j 0935). methanol gradient grade for liquid chromatography e.merck®, aquabidest was obtained from water purification system easy pure ii rf, hard capsules shell, and silica gel. journal of fundamental and applied pharmaceutical science, 1(1), april 2020 21 instrumentation and chromatographic conditions used was the hplc system (shimadzu®, lc-2010ht), consisting of a pump with a dual serial plunger, microvolume (10 μl on the primary side, 5 μl on secondary along with autosampler injector programmed at 10 µl capacity per injection, was used. the detector consisting of uv. lc separations was performed on a phenomenex c18 column (250×4.6 mm i.d., 5 μm particle size). the mobile phase consisted of 40: 60 (v/v); methanol: aquabidest. the flow rate was set to 0.6 ml/min, and uv detection was carried out at 271 nm. other materials and tools were such as ultrasonication retsch® t460, analytical scales ohaus® paj1003 (max 120g, min 0.001g), micropipette socorex® (0.1-2μl; 10100μl; and 100-1000μl), millipore filter (0.45μm), solvent membrane filter whatman® (0.45μm), microtube 1ml, injection spuit 5ml, pyrex glassware, tightly closed container, mortar, and pestle. there were two kinds of samples made, which were compounding capsules combination between paracetamol and tramadol and compounding capsules of tramadol. the first samples were prepared by finely powdered a tablet of paracetamol and a pill of tramadol separately. the accurate weight of the powder, which was 25 mg of paracetamol and 10 mg of tramadol, was weighed and mixed before being inserted into an empty capsule shell. the second samples were made by 10 mg of a finely powdered capsule of tramadol and were inserted into an empty capsule shell. furthermore, samples were stored for 14 days in a tightly closed container far from direct sunlight with three different storage conditions such as room temperature without ac (280c) and without silica gel, room temperature with ac (250c) without silica gel, and room temperature with ac (250c) and with silica gel. the third condition conformed to private hospital semarang conditions. physical and chemical stability testing was conducted to the samples on day 0, 1, 3, 7, 10,14. organoleptic testing the characteristic of the physical stability of the samples was determined by observing direct changes from the powder color in capsules, capsules form, and scent on the samples. the parameter used was the result on day 0 as the sample was freshly made and expected to have higher stability than the sample with a longer storage period. chemical stability testing the chemical analysis performed in this study was conducted by determining the content of active ingredient from the samples using reversed-phase hplc method and using phenomenex c18 (5 µm) as the stationary phase, methanol and aquabidest (40:60) as the mobile phase, wavelength 271 nm, flow rate 0.6 ml/min and injection volume 10 μl [9]. standard solutions and calibration a stock standard solution containing paracetamol (2500 µg/ml) and tramadol (1000 µg/ml) was prepared by dissolving 25 mg of paracetamol and 10 mg of tramadol in 10 ml volumetric flask with methanol. it was further diluted to obtain working standard solutions in a concentration range of 0.05 – 2.45 µg/ml (i.e. 0.05; 0.45 ; 0.85 ; 1.25 ; 1.65 ; 2.05 ; and 2.45 μg/ml) for paracetamol and 1 μg/ml for tramadol. this solution was filtered through a 0.45 μm millipore then transferred to hplc vials and sonicated for 10 min. a constant volume of 10 µl injections was made for each concentration of three replications and chromatography under the aboveshirley candra kurniawan, michael raharja gani, sri hartati yuliani | stability testing of compounding capsule combination between paracetamol and tramadol in a private hospital semarang 22 mentioned conditions. the peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. linear calibration curves were generated using least-squares linear regression analysis. sample preparation samples that had been stored were taken and transferred into 10 ml volumetric flask and diluted to 10 ml with mobile phase methanol and aquabidest (40:60). 0.286 μl of compounding capsules combination between paracetamol and tramadol was transferred to 1 ml microtube, and 999.714 μl of the mobile phase was added. 1 μl of compounding capsules of tramadol was then transferred to 1 ml microtube, and 999 μl of the mobile phase was added. the concentration achieved after the abovementioned dilution was 1 μg/ml. this solution was filtered through a 0.45 μm millipore, then transferred to hplc vials and sonicated for 10 min. a constant 10 µl volume of sample solution was injected under the above-mentioned conditions. the peak areas were measured at 271 nm, and their contents in the samples were determined using a multilevel calibration curve developed on the same hplc system under the same conditions using the linear regression equation. results and discussion organoleptic testing this test is carried out to identify the physical stability of the compounded preparation and to establish a beyond use date. table 1 and figure 1 show that within 14 days of storage, compounding capsules combination between paracetamol and tramadol, compounding tablet of paracetamol, and compounding capsules of tramadol remained consistent in powder color and capsules form, and there is no available scent on the samples. it indicates that the compounded preparation is physically stable for being able to retain the original physical properties.10 table 1. organoleptic studies sample storage time room temp. without ac (280c) and silica gel room temp. with ac (250c) and without silica gel room temp. with ac (250c) and silica gel day 0 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : day 1 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : day 3 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : day 7 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : day 10 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : day 14 tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : tb : white pc : white cf : rigid scent : information: powder color in tablets (tb), capsules (pc), capsules form (cf) journal of fundamental and applied pharmaceutical science, 1(1), april 2020 23 prior to storage after storage figure 1. the physical appearance of the sample figure 2. chromatogram of paracetamol standard 0,45 μg/ml figure 3. chromatogram of tramadol standard 1 μg/ml figure 4. chromatogram of compounding capsules combination between paracetamol and tramadol 1 μg/ml figure 5. chromatogram of compounding capsules of tramadol 1 μg/ml however, this testing has a high subjectivity; thus, chemical stability testing is required to determine the stability of compounded preparation. chemical stability testing chemical stability is the ability of each active ingredient to retain its chemical integrity and labeled potency within the shirley candra kurniawan, michael raharja gani, sri hartati yuliani | stability testing of compounding capsule combination between paracetamol and tramadol in a private hospital semarang 24 specified limits.10 this chemical stability testing is carried out to establish a beyond use date. linearity linear relationships were observed by plotting paracetamol concentration against obtained peak areas. paracetamol showed linear response in the concentration of 0.05; 0.45; 0.85; 1.25; 1.65; 2.05; and 2.45 μg/ml. the corresponding linear regression equation was y = 22746x + 9210.3 with correlation coefficient (r) of 0.9927 indicating the linear relationship between increased concentration and instrument response. the retention time for paracetamol standard 0.45 μg/ml, tramadol standard 1 μg/ml, compounding capsules combination between paracetamol and tramadol 1 μg/ml, and compounding capsules of tramadol 1 μg/ml was found to be 6.504; 6.502; 6.541; and 0 minutes respectively. retention time is a measure of the time taken by solute to pass through a column to the detector. in this study, reversed-phase chromatography was used with the mobile phase, which was more polar than the stationary phase; thus, the more polar compounds would have earlier retention time. the retention time variation requirement was ≤0.05 minutes.11 the time difference obtained from the time retention of paracetamol standard and compounding capsules combination between paracetamol and tramadol was 0.037 minutes; therefore, paracetamol was found in the compounded preparation samples. in figure 5, the auc and the time retention cannot be determined as, at 1 μg/ml, the tramadol samples also contain excipients, which make the concentration measured lower than 1 μg/ml. based on figure 6, it can be concluded that paracetamol is more stable at room temperature with ac (250c) and silica gel as, in the other two conditions, there is an increased paracetamol content in day 7, 10, 14. figure 6. paracetamol content in three different storage conditions journal of fundamental and applied pharmaceutical science, 1(1), april 2020 25 decreased paracetamol content there is a decreased paracetamol content since the physical or chemical characteristics of the compounded preparation can change over time.1 humidity will be more controlled at room temperature with ac (250c) because ac will absorb humid air in the room. humidity in room temperature without ac (280c) will vary based on the environmental humidity. moreover, silica gel can help to slow down decreased paracetamol content as the microporous structure in silica gel has the capacity to absorb the water vapor into those cavities. there is no chemical reaction taken place in the way silica gel adsorbs moisture. it happens due to the difference in water vapor gradient between the surrounding environment and the microcavities in silica gel. the moisture absorption is taken place until the cavities are saturated or until the water vapor pressure of the surrounding environment, and microcavities have gained an equilibrium, which is mostly around 12 weeks.12 increased paracetamol content there is increased paracetamol content because, during storage, gelatin capsules can absorb or release moisture. when stored in a high relative humidity environment, hard gelatin capsules can absorb moisture and lose their rigid shape and become distorted. whereas, in a different environment of extreme dryness, capsules may become too brittle.13 paracetamol contains the amide group, which may be sensitive to hydrolysis degradation.14 hydrolysis takes place in labile carbonyl function groups, which include amides, esters, arylamines, imides, imines alcohols, and carbamates.15 during the storage process, hydrolysis degradation of paracetamol may occur and produce paminophenol, which is stated to be a very toxic substance and has the ability to cause nephrotoxicity, teratogenicity, and methemoglobinemia.16 paracetamol degradation product can be measured in the hplc system as it has a similar structure to paracetamol and because the hplc system for degradation product is not optimized. mechanism of the paracetamol oxidation pathway is favored by the hydroxylation of the benzene ring, leading to the formation of 3-hydroxyacetaminophen and releasing acetamide as a reaction of byproduct. the breakdown of the bond between the amino group and the substituted methanol in the paracetamol molecule can lead to degradation of byproducts with substituted nitrogens, such as 4aminophenol and nitrophenol. the mineralization of paracetamol appears through ortho, meta, and parasubstituted oxidation pathways, leading to the formation of dihydroxy aromatic rings such as catechol, resorcinol, hydroquinone as well as trihydroxylated rings such as pyrogallol, phloroglucinol, and hydroxyhydroquinone.17 moreover, the time difference obtained from the time retention of paracetamol standard (6.504) and tramadol standard (6.502) is 0.002 minutes. therefore, there is a possibility that tramadol is also measured in the area of paracetamol due to the slight difference in time retention, which is ≤0.05 minutes.12 beyond use date in the absence of a usp-nf compounded preparation of monograph or specific stability information for solid dosage forms (capsules, tablets, granules, powders), the maximum bud packaged in tight, light-resistant containers in controlled room temperature (200-250 c) shirley candra kurniawan, michael raharja gani, sri hartati yuliani | stability testing of compounding capsule combination between paracetamol and tramadol in a private hospital semarang 26 is 180 days.1 in this study, beyond use date based on organoleptic and chemical stability testing for 14 days of compounding capsules combination between paracetamol and tramadol is unable to be determined as the chemical stability testing method cannot separate and quantify the content of paracetamol, tramadol, and formed degradation product. nevertheless, this study showed favorable storage conditions in a tightly closed container far from direct sunlight at room temperature with ac (250c) and silica gel. conclusion beyond use date based on organoleptic and chemical stability testing for 14 days of compounding capsules combination between paracetamol and tramadol is unable to be determined. nevertheless, this study showed favorable storage conditions in a tightly closed container far from direct sunlight in room temperature with ac (250c) and silica gel. acknowledgment the authors would like to thank the faculty of pharmacy, sanata dharma university, yogyakarta, indonesia, for providing facilities. this research was financially funded by the young lecturer research grant of 2018, institute for research and community services, sanata dharma university, with the contract number of 032/penel./lppmusd/vii/2018 awarded to michael raharja gani. conflict of interest the authors declare there is no potential of conflict of interest with the research, authorship, and article publication. references 1. usp., (2019). united states pharmacopeial convention 795 pharmaceutical compounding nonsterile preparations. united states pharmacopeia 42. national formulary 37. 2. widyaswari, r., & wiedyaningsih, c. (2012). evaluasi profil peresepan obat racikan dan ketersediaan formula obat untuk anak di puskesmas propinsi diy. majalah farmaseutik, 8(3), pp. 227-234. 3. bajaj, s., singla, d., & sakhuja, n. (2012). stability testing of pharmaceutical products. j app pharm sci, 2(3), pp. 129-138. 4. raeni, s. f., haresmawati, u., mulyasuryani, a., & sabarudin, a. (2018). evaluasi pemisahan alkilbenzena menggunakan kolom monolith berbasis polimer organik secara kromatografi cair kinerja tinggi. alchemy jurnal penelitian kimia, 14(1), pp. 37-50. 5. aulia, s. s., & muchtaridi, m. (2016). penetapan kadar simvastatin menggunakan kromatorafi cair kinerja tinggi (kckt). farmaka, 14(4), pp. 70-78. 6. boligon, a. a., & athayde, m. l. (2014). importance of hplc in analysis of plants extracts. austin chromatogr, 1(3), pp. 1-2. 7. siddiqui, m. r., alothman, z. a., & rahman, n. (2017). analytical techniques in pharmaceutical analysis: a review. arabian journal of chemistry, 10, pp. s1409-s1421. 8. dewi, g. p., & nugroho, t. e. (2016). pengaruh pemberian analgesik kombinasi parasetamol dan tramadol terhadap kadar kreatinin serum tikus wistar. jurnal kedokteran diponegoro, 5(4), pp. 917925. journal of fundamental and applied pharmaceutical science, 1(1), april 2020 27 9. chandra, p., rathore, a. s., lohidasan, s., & mahadik, k. r. (2012). application of hplc for the simultaneous determination of aceclofenac, paracetamol and tramadol hydrochloride in pharmaceutical dosage form. scientia pharmaceutica, 80(2), pp. 337-352. 10. bokser ad, & o’donnell pb., (2013). stability of pharmaceutical products. in: remington : essentials of pharmaceutics. london, uk: pharmaceutical press. pp. 37 – 50. 11. snyder rl, kirkland jj, & dolan jw. (2010). introducing to modern liquid chromatography. 3th ed. new jersey : john wiley & sons inc 12. amarakoon, a. s. h., & navaratne, s. (2017). evaluation of the effectiveness of silica gel desiccant in improving the keeping quality of rice crackers. international journal of science and research, 6(1), pp. 2163 – 2168. 13. srividya, b., & reddy, c. s. (2014). capsules and it’s technology: an overview. international journal of pharmaceutical drug analysis, 2(9), pp. 727-33. 14. golonka, i., kawacki, a., & musial, w. (2015). stability studies of a mixture of paracetamol and ascorbic acid, prepared extempore, at elevated temperature and humidity conditions. tropical journal of pharmaceutical research, 14(8), pp. 1315-1321. 15. naveed, s., ishaq, h., & urooj, s. (2016). degradation study of different brands of paracetamol by uv spectroscopy. journal of coastal life medicine, 4(5), pp. 377-379. 16. abdelwahab, n. s., abdelrahman, m. m., boshra, j. m., & taha, a. a. (2019). different stability‐indicating chromatographic methods for specific determination of paracetamol, dantrolene sodium, their toxic impurities and degradation products. biomedical chromatography, 33(9), p. e4598. 17. villota, n., lomas, j. m., & camarero, l. m. (2016). study of the paracetamol degradation pathway that generates color and turbidity in oxidized wastewaters by photofenton technology. journal of photochemistry and photobiology a: chemistry, 329, pp. 113-119. clove oil (syzygium aromaticum) edible film formulation and antibacterial activity test against streptococcus mutans wida ningsih1*, afdhil arel2 1universitas baiturrahmah, jalan raya by pass, aie pacah, koto tangah, aie pacah, kec. koto tangah, kota padang, sumatera barat 25586 2universitas muhammadiyah sumatera barat, jl. pasir jambak no.4, pasie nan tigo, kec. koto tangah, kota padang, sumatera barat 25172 abstract clove oil contains eugenol as an antibacterial. meanwhile, products containing clove oil have been widely used as toothpaste and mouthwash. in this study, clove oil was formulated in the form of edible film because it is practical, easy to use, and could be used without water like other oral hygiene preparations. the edible film is a thin layer film made of consumable materials used as a carrier of antibacterial compounds. clove oil edible film was then formulated with clove oil concentrations of 1%, 1.5%, and 2% and determined for its antimicrobial activity against streptococcus mutans. clove oil edible film preparations were evaluated under their physical properties, including friability, drying shrinkage, ph, thickness, and swelling ability. antibacterial activity testing of clove oil edible film was conducted, employing the blood agar diffusion method against streptococcus mutans. the physical evaluation of the clove oil edible film showed almost the same physical properties as the comparison (gf). clove oil edible film test results revealed the greatest inhibition at f1 of 18.6 mm ± 0.577, f2 of 22.3 mm ± 2.081, and f3 of 25.3 mm ± 1.527. according to david and stout, the inhibition activity of bacteria on f3 was categorized as a very strong group inhibition response. in addition, anova test analysis results uncovered that the concentration of clove oil affected the inhibition of the streptococcus mutans bacteria with a significance value of 0.000 (p <0.05). also, duncan's test exhibited that each concentration of clove oil had a significant difference in the inhibition of streptococcus mutans bacteria. keywords: antibacterial, edible film, eugenol, agar diffusion, clove oil data of article received reviewed accepted : : : 28 apr 2021 23 july 2021 18 aug 2021 doi 10.18196/jfaps.v2i1.11640 type of article: research introduction clove oil has secondary metabolites in the form of essential oil with the highest eugenol content so that it has biological activity as an antiseptic and analgesia in the treatment of teeth and mouth, * corresponding author, e-mail: nwida777@gmail.com antifungal, antibacterial, antioxidant, and anticarcinogenic1. clove oil can be isolated from the leaves (1-4%), stems (5-10%), and clove flowers (10-20%). previous research was about the bactericidal effect of clove oil on streptococcus mutans and streptococcus pyogenes bacteria, which wida ningsih, afdhil arel | clove oil (syzygium aromaticum) edible film formulation and antibacterial activity test against streptococcus mutans 2 were tested on mouthwash preparations at 0.5%, 0.75%, and 1% clove oil concentrations. it was shown that there was no minimum bactericidal concentration (mbc) at a concentration of 0.5%, so it was concluded that it could inhibit bacterial growth (bacteriostatic). meanwhile, at concentrations of 0.75% and 1%, clove oil had an mbc value, so that it was denoted to have bacteria-killing activity (bactericidal)2. moreover, oral hygiene preparations that have been widely circulated are in the form of toothpaste, mouthwash, and edible film as mouth fresheners containing menthol. the edible film is a thin layer made of safefor-consumption materials and used as a wrapper to prevent the process of fat oxidation, organoleptic changes, microbial growth, or absorption of moisture. in addition, edible films function as a barrier against the mass transfer of solutes, as a carrier for additives, and improving the handling of food3. the formation of edible films can also prevent the evaporation of eugenol in clove oil since one of its functions is as a barrier to mass transfer. many studies on edible films have been conducted, including the use of betel leaf extract as an anti-halitosis formulated in the form of edible films4 and areca seed extract edible films as antibacterial5. on the other hand, streptococcus mutans is a bacterium capable of attaching to the tooth surface that produces glucosyltransferase enzyme. these enzymes produce insoluble glucans in water and play a role in causing plaque and colonies on the tooth surface6. the growth of streptococcus mutans must be inhibited so that it does not become pathogenic and cause caries by giving antibacterial agents. one way to prevent caries is to limit the plaque formation on the tooth surface, either by preventing its formation or regularly cleaning plaque. plaque control can be done by mechanical and chemical cleaning of plaque containing antibacterial ingredients, suppressing streptococcus mutans growth. based on the description above, the formulation of clove oil in the form of edible film was carried out, which could prevent the evaporation of clove oil compared to the dosage form of toothpaste and mouthwash and tested its antibacterial activity against streptococcus mutans. method materials and tools 1. materials clove oil (pt. lansida), corn starch (maizenaku), hpmc (brataco), sorbitol (brataco), na-saccharin, mint oil, menthol (brataco), sodium metabisulfite (brataco), nipagin (brataco), nipasol (brataco), and aqua dest culture of streptococcus mutans atcc 31987, 70% ethanol (brataco), crystal violet solution, lugol's solution, safarin solution, physiological nacl solution (widatra), blood agar media, comparative (gf®), and dimethylsulfoxide (merck). 2. tools the tools used were digital scale (shimadzu), refrigerator (panasonic), hot plate and magnetic stirrer (boeco), oven (memmert), desiccator, modified edible film printing equipment, caliper (kenmaster), ph meter (ohaus), roche friabilator (amtast cs-2), incubator (memmert), laf (laminar air flow) (robust), autoclave (memmert), and micropipette (soccorex). journal of fundamental and applied pharmaceutical science, 2(1), august 2021 3 clove oil (syzygium aromaticum) edible film formula the edible film formula used referred to the formula made by harmely et al. (2014) because this formula gave good results in previous studies. table 1. formula edible film composition (%) formula f0 f1 f2 f3 clove oil 0 1 1.5 2 corn starch 6 6 6 6 hpmc 4 4 4 4 sorbitol 70% 4 4 4 4 na saccharin 0.25 0.25 0.25 0.25 menthol 0.1 0.1 0.1 0.1 candy oil 1 1 1 1 nipagin 0.18 0.18 0.18 0.18 nipasol 0.02 0.02 0.02 0.02 na metabisulfite 0.02 0.02 0.02 0.02 distilled water up to 100 100 100 100 description: f1: clove oil concentration of 0% f2: clove oil concentration of 1% f3: clove oil concentration of 1.5% f4: clove oil concentration of 2 % corn starch was dispersed into 20 parts of aqua dest, heated at ± 60ºc, and stirred until a clear gel was formed. hpmc was developed in distilled water, which had added sorbitol and sodium saccharin, and then was stirred at a temperature maintained at ± 60ºc. the two gels were mixed at a temperature of ± 60ºc by adding other additives (nipagin, nipasol, menthol, mint oil, na metabisulfite, and clove oil) at room temperature; the mixture was stirred homogeneously, then was poured and leveled on the mold (26 x 20 cm). the preparation was dried in the oven at a temperature of 40-50 ºc for 24 hours. then, the edible film formed was released from the mold and cut into pieces with 2.2 x 3.2 cm. edible film evaluation 1. organoleptic examination the organoleptic examination included visual observations using the five senses on the edible film's shape, smell, taste, and color. this examination was carried out at room temperature (15-30ºc)7. 2. edible film friability examination the friability of the edible film was carried out according to the friability test of the tablet, 8,9 utilizing a roche friabilator. first, 20 sheets of edible film free of dust (w1) were weighed, then put into the roche friabilator. the tool was run for four minutes with a rotation speed of 25 rpm. the 20 edible films were cleaned of dust and weighed again (w2). edible film friability can be calculated by the formula: friability = x 100% 3. drying shrinkage examination the porcelain dish was dried in an oven at 105oc until a constant weight (a) was obtained. edible film weighed 2g in a porcelain dish (b), then dried in an oven for 2-5 hours until a constant weight was obtained (c). the drying shrinkage was then determined in percent by weight of the sample used10. % drying shrinkage = x 100 % 4. ph examination this examination was performed using the inolab ph meter. first, this instrument was calibrated employing a buffer of ph 4 and ph 7. the electrodes were rinsed with distilled water and dried. the ph measurement of the clove oil edible film was carried out by dissolving 1g of the edible film in distilled water up to 10 ml. the electrode was immersed in the wida ningsih, afdhil arel | clove oil (syzygium aromaticum) edible film formulation and antibacterial activity test against streptococcus mutans 4 container, and the number on the ph meter represented the ph value of the clove oil edible film.7 5. edible film thickness examination11 the thickness of the edible film produced was measured utilizing a micrometer with an instrument accuracy of 1 m. measurements were made at five different places. 6. swelling test the swelling test was carried out by inserting one sheet of edible film into a glass beaker, then expanded with 10 ml of distilled water. then, it was determined how long it took the preparation to expand.12 antimicrobial activity test by diffusion method (kirby bauer method) this method was used to determine the activity of antimicrobial agents. a plate containing an antimicrobial agent was placed on an agar medium on which microorganisms had been grown, which would diffuse into the agar medium. then, it was incubated at 37ºc for 18-24 hours. the clear zone indicated the presence of inhibition of the growth of microorganisms on the surface of the agar medium13. clove oil antibacterial activity test 1. sterilization of tools and materials the tools utilized were washed and dried before being sterilized. some tools, such as petri dishes, were wrapped in newspaper, while the mouthpieces, test tubes, and droppers were covered with cotton and then wrapped one by one with newspaper. all tools were sterilized in an autoclave at 121ºc for 15 minutes. laminar air flow was cleaned and then sprayed with 70% ethanol. after that, it was sterilized by turning on a uv lamp for five minutes. 2. making blood agar media the media powder of blood agar base was weighed as much as 4 grams, then dissolved with 100 ml of distilled water and heated to boiling while stirring. then, it was sterilized by autoclave at 121oc for 15 minutes. the sterile media was cooled to a temperature of 45-50oc. then, 10 ml of fresh sheep blood was added and stirred until smooth. then, it was poured into a petri dish and waited for it to solidify. once frozen, the media was ready to use. 3. preparation of test microbial suspension bacterial colonies were suspended in sterile physiological nacl solution in sterile test tubes and homogenized by the vortex. then, the turbidity of the equivalent suspension was measured by standard turbidity uv-vis spectrophotometry. thus, a suspension with a transmittance of 25% at a wavelength of 580nm was obtained13. 4. clove oil antibacterial activity testing the sterilized blood agar medium was poured into a ±20ml petri dish. after the media solidified, a sterile cotton swab was dipped into the bacterial suspension and then smeared evenly over the media. sterile disc paper that had been previously dripped with clove oil with concentrations of 1%, 1.5%, 2%, and as a negative control dimethyl sulfoxide (dmso) was taken using a 10µl micropipette. it was incubated for 24 hours at 37oc. then, it was observed for bacterial growth. the inhibition area was also measured, indicated by the appearance of a clear area around the disc utilizing a caliper. journal of fundamental and applied pharmaceutical science, 2(1), august 2021 5 5. edible film antibacterial activity testing blood agar media sterilized was poured into a petri dish as much as ± 20 ml. after the media solidified, a 5 mm diameter film sheet was placed on the agar medium and then incubated at 37oc for ±24 hours. the growth of bacteria was observed, and the inhibition area diameter was measured, indicated by the presence of an area not overgrown by bacteria. tests were carried out on f1, f2, f3 preparations. f0 was used as a negative control based on the edible film, while (gf®) was employed as a comparison14. data analysis the data on the antibacterial activity of clove oil in edible film preparations were processed by one-way analysis of variation (anova) utilizing the spss program. the results will be meaningful if the comparison of inhibition in each formula gives a real and meaningful difference. results and discussion this study aimed to formulate a preparation of clove oil (syzygium aromaticum) in the form of an edible film as a deodorizer and determine its antibacterial activity against streptococcus mutans. clove oil contains eugenol as much as 78-98%. the substance is produced from the oil glands on the surface of the clove flower body. in addition, clove oil also contains eugenol acetate, caryophyllene, and other minor compounds, in small amounts15. edible clove oil film was made by casting method on a mold (26 x 20 cm). the edible film formed was removed from the mold and cut into pieces with 2.2 x 3.2 cm (figure 1). figure 1. preparation of clove oil edible film description: p = comparative preparation formula “gf®” f0 = edible film base formula with 0% clove oil concentration f1 = edible film base formula with 1% clove oil concentration f2 = edible film base formula with 1.5% clove oil concentration f3 = edible film base formula with 2% clove oil concentration the edible clove oil film formed was evaluated for physical properties and activities against streptococcus mutans. evaluation of the clove oil edible film physical properties was then compared to the circulating physical properties (gf). p f 0 f 1 f 2 f 3 wida ningsih, afdhil arel | clove oil (syzygium aromaticum) edible film formulation and antibacterial activity test against streptococcus mutans 6 table 2. evaluation results of clove oil edible film evaluation observation f0 f1 f2 f3 p description -shape -smell -taste -color plt km mm pt plt kmmc mapm pt plt kmmc mapm pt plt kmmc mapm pt plt km mm u friability (%) 0.98 ± 0.004 0.94 ±0.004 1.02 ±0.003 1.17 ±0.007 1.06 ±0.006 drying shrinkage (%) 13.91 ±0.016 13.23 ±0.013 12.98 ±0.014 12.69 ±0.013 14.32 ±0.017 ph 5.98 ±0.232 6.33 ±0.156 5.96 ±0.167 5.81 ±0.143 6.23 ±0.186 thickness (mm) 0.035 ±0.0037 0.0392 ±0.0037 0.044 ±0.0038 0.050 ±0.0060 0.010 ±0 swelling test (second) 08.48 10.31 12.22 14.15 05.93 antibacterial activity (mm) 10 ± 0.5 18.6 ± 0.577 22.3 ± 2.081 25.3 ± 1.527 10.5 ± 0.5 description plt : thin layer solid km : typical mint kmmc : typical mint clove oil mm : refreshing sweet mapm : sweet but a bit refreshing bitter pt : transparent white u : purple the organoleptic examination of the clove oil edible film was carried out for six weeks. every week, the organoleptic examination results of edible films showed no change in shape, smell, taste, and color. it indicates that the edible film preparation was stable during storage. in addition, the friability examination of edible films utilizing the "roche friabilator" friability test equipment did not appear to have changed shape or broken the edible film during the test. however, there was a reduction in the dosage weight caused by the edible film experiencing friction during testing. the % friability test results of the edible film also revealed that the % friability of edible film f3 (1.17%) was higher than the comparison (1.06%). this friability test describes the resistance of the preparation surface to the friction experienced during packaging, shipping, and storage.16 the results of the edible film drying shrinkage examination uncovered that the highest drying shrinkage was shown in the formula f0, which was 13.91%, whereas the lowest was displayed in the comparison preparation, which was 12.69%. meanwhile, according to previous research, the percentage of good drying shrinkage for edible films was less than 9.29%.17 moreover, the ph evaluation of the edible film was performed for six weeks. the test results exposed that the ph changed every week but still met the physiological ph range of the mouth. weekly changes in ph values might be caused by environmental factors, such as temperature, storage, and the sensitivity of the ph meter. the ph value of the resulting edible film must be in the ph range of the oral cavity, which ranges from 5.5 to 7.91818, so as not to journal of fundamental and applied pharmaceutical science, 2(1), august 2021 7 irritate the oral mucosa when the preparation is consumed. next, the thickness of the edible film was examined employing a screw micrometer with an accuracy of 0.01 mm at five different places, and the results were then averaged. the thickness difference occurred due to clove oil concentration differences in each preparation, where f3 was the thickest compared to f1, f2, and f0. the measuring results of the edible film thickness in all formulas met the requirements of a good thickness of the edible film, which was <0.25 mm.19this thickness test was carried out to see that the edible film produced is thin but not easily torn, which is an indicator of uniformity and quality control of the edible film. further, in the swelling test, the mean swelling time results of the edible film were obtained. the measurement results disclosed that f1, f2, and f3 required a longer time for the edible film to swell because it contained clove oil, which is difficult to dissolve in water with different concentrations. it is suspected that the mechanism of film destruction is through swelling and wicking mechanisms. the swelling process is that after the film is in contact with water, water penetration occurs due to capillarization (wicking) so that the film swells. meanwhile, the wicking mechanism is the presence of water drawn by the disintegrant, where water will penetrate the film pores. as a result, the bonds between the particles become weak, and the film swells.11 furthermore, the antibacterial activity testing of clove oil edible film against streptococcus mutans bacteria was conducted using the agar diffusion method. this method is most commonly used to determine the activity of the test material against bacteria. the 24-hour incubation was carried out to give time for bacteria to multiply rapidly and carry out metabolic activities. the results showed that after 24 hours of incubation, all clove oil groups’ concentrations could inhibit the bacteria streptococcus mutans growth. the higher the concentration, the higher the zone of inhibition. it aligns with pelczer and chan’s opinion that the higher the concentration of an antibacterial agent, the stronger the antibacterial activity.20 in this study, the test bacteria were suspended in a 0.9% physiological nacl solution because the physiological nacl solution was an isotonic environment for the test bacteria. the suspension was homogenized by vortex, and the turbidity was measured utilizing a uv-vis spectrophotometer with a wavelength of 580 nm and transmitter 25%, describing the optimal cell density for antibacterial activity testing.13 from the antibacterial activity test analysis, homogeneous and normal data were obtained, which showed that the data obtained were parametric, followed by one-way anova statistics utilizing spss 16 and duncan's test. the results of the one-way anova statistical test revealed the p-value (<0.005). it signifies a significant difference between all formulas. the difference in the concentration of clove oil at a 2% concentration did not show a significant difference with a 1.5% concentration but showed greater inhibition at a 2% concentration. in the edible film preparation, between the comparisons of the gf® edible film, a significant difference was shown with the edible film preparation in varying concentrations. however, on 1.5% clove oil edible film, it did not show a significant difference with 1% and 2% edible film, but 2% clove oil wida ningsih, afdhil arel | clove oil (syzygium aromaticum) edible film formulation and antibacterial activity test against streptococcus mutans 8 edible film had greater antibacterial inhibition. based on the response table of bacterial growth inhibition according to davis and stout, the inhibition classification was divided into four categories: very strong = 20 mm, strong = 10-20 mm, moderate = 510 mm, and weak = 5 mm. from the results obtained for each clove oil edible film formula, f1 = 18.6 mm, f2 = 22.3 mm, and f3 = 25.3 mm. it was categorized into a very strong bacterial growth inhibition response. according to a study,21 factors that affect the size of the inhibition zone include the organism sensitivity, the culture medium, the incubation conditions, and the rate of agar diffusion. meanwhile, the factors that influence the rate of agar diffusion consist of the microorganism concentration, the medium composition, the incubation temperature, the incubation time, and the ph value of the medium. in this regard, some antibacterials work well under acidic conditions and others under alkaline conditions. conclusion clove oil with a concentration of 1% (f1), 1.5% (f2), and 2% (f3) can be formulated in the form of edible film. from the physical evaluation results, almost the same results as the comparison of gf® in the form of edible film containing menthol were obtained. in addition, the antibacterial activity test results showed significant differences between f1, f2, and f3. of the three formulas containing clove oil, f3 gave the greatest inhibitory power of 25.3mm (p<0.05) and was included in the very strong bacterial growth inhibition response. references 1. prianto, h., retnowati, r., juswono, u.p. isolasi dan karakterisasi dari minyak bungan cengkeh (syzigium aromaticum) kering hasil distilasi uap. j ilmu kimia univ. brawijaya. 2013;1(2):269–75. 2. riyadi, e. efek bakterisid dari berbagai konsentrasi minyak cengkeh dalam sediaan obat kumur dengan tween 80 sebagai surfaktan terhadap streptococcus mutans dan streptococcus pyogenes. universitas katalik widya mandala surabaya; 2010. 3. krochta, j. control of mass transfer in foods with edible coating and film. new york: elsevier sci, publ; 1992. 4. arifin, m.f., hidayati, ln., syarmalina, s., rensy, r. formulasi edible film ekstrak daun sirih (piper betle l.) sebagai antihalitosis. j ilmu kefarmasian indones. 2010;8(1): 6168. 5. ningsih, w. formulasi dan uji efektivitas antibakteri edible film ekstrak biji pinang (areca catechu linn). jiffk j ilmu farm dan farm klin. 2018; 15(2): 71. http://dx.doi.org/10.31942/jiffk.v15i2. 2569 6. pratiwi, r. perbedaan daya hambat terhadap streptococcus mutans dari beberapa pasta gigi yang mengandung herbal (the difference of inhibition zones toward streptococcus mutans among several herbal toothpaste). dent j (majalah kedokt gigi). 2005; 38(2): 64. http://dx.doi.org/10.31942/jiffk.v15i2.2569 http://dx.doi.org/10.31942/jiffk.v15i2.2569 journal of fundamental and applied pharmaceutical science, 2(1), august 2021 9 7. depkes. farmakope indonesia. edisi iii. jakarta; 1979. 8. voigt, r., noerono, s. buku pelajaran teknologi farmasi. ed.3, peny. yogyakarta: gadjah mada university press; 1994. 9. harmely, f., deviarny, c., yenni, w.s. formulasi dan evaluasi sediaan edible film dari ekstrak daun kemangi (ocimum americanum l.) sebagai penyegar mulut. j sains farm klin. 2015;1(1):38. 10. depkes. farmakope indonesia. edisi iv. jakarta; 1995. 11. krochta, j. m., baldwin, e. a. nisperos-carriedo, m. edible coatings and films to improve food quality. new york: technomic publishing company; 1994. 12. djaya, a. halitosis: nafas tak sedap. jakarta: dental lintas mediatama; 2000. 13. jasril, j., teruna, h.y., zamri, a., alfatos, d., yuslinda, e., nurulita, y. sintesis dan uji antibakteri senyawa bromo kalkon piridin. j natur indones. 2013;14(3):172. 14. amaliya, r. r., dwi, w., putri, r. karakterisasi edible film daripati jagung dengan penambahan filtrat kunyit putih sebagai antibakteri characterization edible film of corn starch with the addition of white saffron filtrateas antibacterial. j pangan dan agroindustri. 2014;2(3):43–53. 15. towaha, j. manfaat eugenol cengkeh dalam berbagai industri di indonesia. perspektif. 2012;11(2):323–77. 16. leon, l., lieberman, a. h., kanig, j. l. teori dan praktek farmasi industri. edisi iii. jakarta: universitas indonesia; 1994. 17. kavitha k, rajendra mr. design and evaluation of transdermal films of lornoxicam k . 2011;2(2):54–62. 18. barman i, g ucp. effects of habitual arecanut and tobacco chewing on resting salivary flow rate and ph. int j oral heal med reserach. 2015;2(1):13–8. 19. susanto t, budi s. teknologi pengolahan hasil pertanian. surabaya; 1994. 20. jamaluddin n, pulungan mh, warsito. uji aktivitas antibakteri minyak atsiri jeruk purut ( citrus hystrix dc ) terhadap klebsiella pneumoniae atcc antibacterial activity test of kaffir lime ( citrus hystrix dc ) essential oil against klebsiella pneumoniae atcc. j teknol dan manaj agroindustri. 2017;6(2):61–66. https://doi.org/10.21776/ub.industria. 2017.006.02.1 21. schlegel hg. mikrobiologi umum. yogyakarta: gadjah mada univ. press; 1994. https://doi.org/10.21776/ub.industria.2017.006.02.1 https://doi.org/10.21776/ub.industria.2017.006.02.1 comparison between the suspension and capsule preparation from waste of avocado seeds as antidiarrhea in induced mouse muharni saputri*, nilsya febrika zebua, fivi nur asih, ghera fakhira putri department of pharmacy, faculty of pharmacy, universitas tjut nyak dhien, gg. rasmi no.28, sei sikambing c. ii, medan helvetia, medan, north sumatra 20123, indonesia abstract diarrhea is one type of disease with the most sufferers every year. thus, it is considered an endemic disease in indonesia and the potential disease of extraordinary events accompanied by mortality. one of the plants that can be used as a traditional medicine for diarrhea is the avocado seed, as it contains tannins, alkaloids, flavonoids, steroids and glycosides, which act as antidiarrheals. this study aims to determine the antidiarrheal effect of avocado seed extract suspension on mice induced by oleum ricini and the optimum concentration of avocado seed extract suspension and capsules with an antidiarrheal effect in white male mice induced by oleum ricini. the dosage forms chosen were suspension and capsules. this study used an experimental method with a test sample of the avocado seed. avocado seed simplicia was extracted by the percolation method, then an oral suspension and capsule formulation were made from the avocado seed methanol extract. it was evaluated and tested for its effectiveness with mice to cure diarrhea. normal data were analyzed by one way anova and post hoc tuckey method. the results of this study showed that the administration of a suspension of avocado seed methanol extract at a dose of 800 mg/kgbb had the most optimum effect as an antidiarrheal against white male mice with a stool weight of 0.39 grams and a duration of diarrhea for 74 minutes. furthermore, the administration of avocado seed extract capsules at a dose of 75 mg/kgbb had the most optimum effect as antidiarrheal against white male mice with loperamide as a positive control. therefore, it can be concluded that all suspension formulations and capsules of avocado seed methanol extract met the requirements for preparation evaluation. suspension and avocado seed methanol extract capsules can cure diarrhea in white male mice. keywords: antidiarrheal; avocado seeds; oleum ricini; suspension; capsule data of article received reviewed accepted : : : 28 dec 2021 28 jan 2022 14 feb 2022 doi 10.18196/jfaps.v2i1.13504 type of article: research introduction diarrhea in indonesia has become an endemic disease and is a potential * corresponding author, e-mail: muharnisaputri@gmail.com extraordinary event (klb) accompanied by death.1 there are various kinds of treatment to treat this diarrheal disease, ranging from traditional medicine to muharni saputri , nilsya febrika zebua, fivi nur asih, & ghera fakhira putri | comparison between the suspension and capsule preparation from waste of avocado seeds as antidiarrhea in induced mouse 67 modern medicine. people prefer traditional medicine that uses plants as this type of treatment has fewer side effects than conventional preparations. one of the traditional treatments is using avocado (persea americana mill.) avocado seeds contain alkaloids, tannins, triterpenes, and quinones.2 avocado seed waste can be used for traditional medicine by drying and then mashing. empirically, avocado seeds are used as medicine to treat diarrhea, diabetes medicine, cholesterol medicine and treat toothache. there are various pharmaceutical preparations, such as capsules and suspensions. researchers chose capsules and suspension preparations to investigate the antidiarrheal effect of methanol extract of avocado seeds (persea americana mill.). capsules that mask unpleasant tastes and odors are easy to consume and prepare. the medicinal ingredients are protected from external influences (light, humidity). powder formulations often require the addition of fillers, lubricants, and glides to the active ingredients to facilitate the capsule filling process. capsules are solid preparations consisting of a soluble hard or soft shell drug; the shell is generally made of gelatin, starch, or other suitable materials. the extracts were made using the percolation method.3 meanwhile, the suspension is widely used as it is easy to use for children, infants, and adults who have difficulty swallowing tablets or capsules. suspensions can also be added with additives to mask the unpleasant taste of the active substance. in general, the liquid form is preferred over the tablet or capsule form as it is easy to swallow and adjust the dose for children.4 the advantages of these two preparations are that they are practical to provide comfort for drug consumers and can cover unpleasant tastes and odors in drugs.5 based on the description of the background, this study aims to identify whether there is a difference between the suspension and capsules of avocado seed methanol extract that meets the physical quality requirements of the preparation and to determine whether the suspension and capsules of avocado seed methanol extract (persea americana mill.) had an antidiarrheal effect. furthermore, this study also aims to identify the optimum concentration of avocado seed methanol extract that can have an antidiarrheal effect on mice. method this research is an experimental study with a sample of avocado seeds (persea americana mill.). the chemicals used in this study were avocado seed, na cmc, simple syrup, loperamide hcl, oleum ricini, distilled water, hcl, bouchardat reagent, dragendroff reagent, fehling's reagent a, fehling's reagent b, hno3, mayer's reagent, molish's reagent, naoh, pb(c2h3o2), lieberman-bouchardat reagent and hno3.𝐹𝑒𝐶𝑙3 making simplicia and extract simplicia was made by cleaning avocado seeds from dirt, then dried in a drying cabinet at a temperature of ± 400c. it was then mashed and weighed of the dry powder obtained. the next step was to make methanol extract of avocado seeds by the percolation method. the procedure for making extracts in the percolation process was that the dry powder of avocado seeds was weighed as much as 500 grams and first soaked for 24 hours with 1200 ml of methanol in a closed glass vessel and dark in color. the extraction process was continued in a percolator, and the solvent used was 2.305 l to the liquid that dripped from the clear percolator. the percolate liquid was allowed to drip while the filtered journal of fundamental and applied pharmaceutical science, 2(2), february 2022 68 fluid was added repeatedly; thus, the filtered fluid limit remained 10 cm above the simplicia powder. the percolate yield obtained was 2.100 l. the percolation process was carried out until the percolate liquid no longer gave a cloudy color. the percolation results were evaporated in a vaporizer cup covered with aluminum foil and given a small hole. it was left in an open room for a week until the solvent evaporated and obtained a thick extract. the extract was later weighed. phytochemical screening phytochemical screening was carried out qualitatively on avocado seed simplicia powder (persea americana mill.), including examination of alkaloids, flavonoids, tannins, saponins, glycosides, cyanogenic glycosides, anthraquinone glycosides, steroids/triterpenoids. preparation evaluation organoleptic examination organoleptic examination of the suspension and capsules of avocado seed methanol extract included color, odor, taste and texture. an avocado seed methanol extract suspension was an organoleptic examination that included a light brown color, characteristic aromatic odor, sweet taste, and sandy texture. organoleptic examination of avocado seed methanol extract capsules included dark brown color, aromatic odor, bitter taste and gritty texture. stability test the stability evaluation for the suspension was carried out by the cycling test method. it was accelerated storage under forced conditions, carried out by storing at 40c. furthermore, it was put in the refrigerator and stored at 400c in the oven alternately for 24 hours for 6 cycles with organoleptic test parameters (color), odor, dosage form, and the ph of the preparation. viscosity test the suspension viscosity measurement was carried out after reconstituted using a brookfield viscometer spindle number 1 at a speed of 30 rpm. before and after accelerated storage conditions, the shear stress was calculated. sedimentation test the measurement of the sedimentation volume of the suspension has been made before and after accelerated storage conditions. it was carried out by comparing the sediment's final volume (vu) with the original (v0) before deposition. the redispersion ability would be good if the suspension was completely dispersed when shaken by hand for a maximum of 30 seconds. specific weight test specific gravity is the ratio of the weight of the substance to water in the same volume, which is weighed at room temperature before and after being given accelerated storage conditions at 50c and 350c for 12 hours each for 5 cycles. a. use a clean and empty pycnometer, then fill it with distilled water; the outside of the pycnometer is dried and weighed b. discard the distilled water, dry the pycnometer and then fill it with liquid syrup at the same temperature and at the time of measurement of distilled water and weigh it. formula:  = 𝑚 v information :  = density (g/cm3 ) or (g/ml) m = mass of object (g) v = volume of object (cm3 ) or (ml) muharni saputri , nilsya febrika zebua, fivi nur asih, & ghera fakhira putri | comparison between the suspension and capsule preparation from waste of avocado seeds as antidiarrhea in induced mouse 69 ph test preparation evaluation of the preparation's ph was carried out using a calibrated ph meter. the suspension was put in a beaker glass, and then the ph meter was dipped into the suspension. the ph value of the suspension was identified by looking at the numbers listed on the ph meter. weight uniformity test thoroughly, 10 capsules were weighed one at a time and identified. the contents of each capsule in an appropriate manner were then removed. destroyed time test 6 capsules were inserted in each tube in the basket under the 10 mesh steel plates. the medium water temperature of 370c was then used. in the observation of the capsules, all capsules must be crushed, except for part of the capsule shell. hygroscopicity test a total of 3 capsules was placed in a brown bottle and stored in a desiccator. each treatment was observed every day for seven days and every week for a month. observations were made on changes in capsule weight, capsule shape, and capsule contents. experimental animal preparation the test animals used in this study were adult mice (mus musculus) weighing 20-30 grams. the total sample was 25 mice divided into 5 groups. each treatment group consisted of 5 mice. group 1 was a negative control, group 2 was a positive control, and groups 3, 4, and 5 were treatment groups. two weeks before the experiment, the mice were adapted to the experimental environment. antidiarrheal effectiveness test the dose of avocado seed methanol extract given to experimental animals used a dose ratio with loperamide. in a positive control group, loperamide hcl was used. oleum ricini was used as induction, and four test groups were given suspension and capsules of avocado seed methanol extract with three dose ratios. it was given to 25 mice adapted to the research environment for 1 week. before the study was conducted, the mice fasted for 18 hours while still being given water before testing. they were grouped into 5 groups; each group consisted of 5 mice. all mice were given oleum ricini as a diarrhea induction of 0.75 ml per mouse orally except for the normal group. grafic 1. grafic of duration of diarrhea in suspensions preparations 0,00 50,00 100,00 150,00 200,00 250,00 na-cmc loperamid dose 200 mg/kg bb dose 400 mg/kg bb dose 800 mg/kg bb t re a tm e n t t im e ( m in u te s) the duration of diarrhea with ​​suspension preparations journal of fundamental and applied pharmaceutical science, 2(2), february 2022 70 grafic 2. grafic of duration of diarrhea in capsule preparations after being given oleum ricini as much as 0.75 ml per head as an inducer, the capsule and suspension were left for 30 minutes. it was then given the test material orally according to each group of animals. the antidiarrheal effect was determined by observing the onset of diarrhea, the frequency of feces, the weight of the feces in grams, and the duration of diarrhea. in this experiment, the antidiarrheal effect was observed for 5 hours. results and discussion phytochemical screening results table 1. phytochemical screening results compound group content of dried avocado seed simplicia phytochemical screening results alkaloids + mayer: dark chocolate draendroff: muddy chocolate bouchardat: cloudy yellow saponins + white foam flavonoids + red orange tannins + blackish green glycoside + glycon: purplish brown/purple in the middle aglycone: reddish purple anthraquinone glycosides pink/red cyanogenic glycoside blood red on filter paper steroids + green 0 100 200 300 400 500 600 700 na-cmc loperamid dose 25 mg dose 50 mg dosis 75 mg t r e a tm e n t t im e ( m in u te s) the duration of diarrhea with capsul preparations na-cmc loperamid dose 25 mg dose 50 mg dosis 75 mg muharni saputri , nilsya febrika zebua, fivi nur asih, & ghera fakhira putri | comparison between the suspension and capsule preparation from waste of avocado seeds as antidiarrhea in induced mouse 71 test results of avocado seed methanol extract suspension on mice with post tukey table 2. test results of fecal weight when given avocado seed methanol extract sustenance treatment fecal weight (gram) amount average statistical result ral repetition (gram) 1 2 3 na-cmc 0.62 0.6 0.41 1.63 0.543333333 0.526 loperamid hcl 0.31 0.31 0.24 0.86 0.286666667 dosage of 200 mg/kgbb 0.29 0.4 0.61 1.3 0.433333333 dosage of 400 mg/kg bb 0.27 0.38 0.73 1.38 0.46 dosage of 800 mg/kg bb 0.25 0.26 0.67 1.18 0.393333333 table 3. results of the post tukey fecal frequency test when suspended with avocado seed methanol extract treatment n subset for alpha=0.05 1 dosage of 400 mg 3 9.6667 dosage of 800 mg 3 10.0000 loperamide hcl 3 10.3333 dosage of 200mg 3 10.3333 na_cmc 3 12.3333 sig. 0.299 table 4. post tukey test results in fecal weight when given avocado seed methanol extract sustenance treatment n subset for alpha=0.05 1 loperamide hcl 3 0.2867 dosage of 800 mg 3 0.3933 dosage of 200mg 3 0.4333 dosage of 400 mg 3 0.4600 na_cmc 3 0.5433 sig. 0.436 test results of avocado seed methanol extract suspension on mice with post tukey table 5. diarrhea results when given avocado seed methanol extract capsules treatment fecal weight (gram) amount average repetitation (gram) 1 2 3 na-cmc 0.75 0.72 0.42 1.89 0.63 loperamid 0.23 0.23 0.17 0.63 0.21 dosage of 25 mg 0.22 0.25 0.61 1.08 0.36 dosage of 50 mg 0.27 0.32 0.61 1.2 0.4 dosage of 75 mg 0.27 0.39 0.36 1.02 0.34 journal of fundamental and applied pharmaceutical science, 2(2), february 2022 72 table 6. results of the post tukey test of feces frequency when given avocado seed methanol extract capsules treatment n subset for alpha=0.05 1 loperamide hcl 3 8.0000 dosage of 800 mg 3 8.0000 dosage of 200mg 3 9.0000 dosage of 400 mg 3 10.0000 na_cmc 3 12.0000 sig. 0.211 table 7. post tukey test results in fecal weight when given avocado seed methanol extract capsules treatment n subset for alpha=0.05 1 2 loperamide hcl 3 0.2100 dosage of 800 mg 3 0.3400 dosage of 200mg 3 0.3600 dosage of 400 mg 3 0.4000 na_cmc 3 0.6300 sig. 0.581 0.221 based on the results of phytochemical screening of avocado seed methanol extract, it is known that avocado seeds contain a group of compounds such as alkaloids, saponins, flavonoids and tannins. alkaloid compounds work by inhibiting the growth of salmonella typhimurium bacteria which have been known to have the potential to cause diarrhea.6 meanwhile, tannin compounds are astringent, which can help stop diarrhea.7 flavonoids are antidiarrhea with the mechanism of inhibiting intestinal motility by reducing fluid and electrolytes. another flavonoid activity (quercetin) is by inhibiting the release of acetylcholine in channel.8 furthermore, the results of organoleptic observations included odor, color, and taste which were observed every 14 days. in each suspension formula of avocado seed methanol extract, organoleptic changes occurred in the suspension. there is a change in taste in the table due to a fermentation reaction. fermentation is the main energy-producing process of various microorganisms. the following table measuring ph meter. table 8. table measuring ph mater replication ph meter before the suspension was made after the suspension was made (ph) (ph) f1 4.8 4 f2 4.7 3.7 f3 4.5 3.8 average 4.75 3.83 muharni saputri , nilsya febrika zebua, fivi nur asih, & ghera fakhira putri | comparison between the suspension and capsule preparation from waste of avocado seeds as antidiarrhea in induced mouse 73 the ph test results were tested by shelflife. it revealed the suspension formulation with the suspending agent cmc na. the ph decreased from 4.75 to 3.83 during the storage cycle, and the optimum suspension ph was 5-6.9 the ph of the preparation was acidic due to the presence of additives (preservatives). it was used in the form of benzoic acid with a ph <4.5 to affect the ph of the preparation. meanwhile, the specific gravity measurement results showed a change in the specific gravity of the suspension before and after the accelerated storage conditions from 1.03 to 1.02, which met the suspension-specific gravity requirements, namely >1.00 g/ml.10 table 9. table measuring ph meter replication ph meter before the suspension was made after accelerated storage conditions (ph) (ph) f1 4.8 4 f2 4.7 3.7 f3 4.5 3.8 average 4.75 3.83 based on the results of statistical tests on observing the initial diarrhea time with anova testing, a significance value was 0.366 (p> 0.05).11 furthermore, ho was accepted, indicating that the five treatments had unequal variances (not homogeneous), which could be concluded to accept ho. it meant that the average value of diarrhea in each of these treatments was not significantly different.12 the decision-making of the tukey method can be carried out by looking at the placement of the statistical test results in the column. the test results for the duration of diarrhea at a dose of 800 mg were in the same column as loperamide which concluded that the diarrhea value in each treatment was not significantly different. meanwhile, a dose of 200 mg, 400 mg and na-cmc are in different columns, indicating that the diarrhea value in each treatment was significantly different from the positive control group.13 furthermore, in the statistical test results in the column, the results of the frequency of diarrhea at a dose of 400 mg, a dose of 800 mg, loperamide, a dose of 200 mg and na-cmc were in the same column, concluding that the value of diarrhea in each of these treatments was not significantly different. the results of the loperamide stool weight at a dose of 800 mg, 200 mg, 400 mg and na-cmc were in the same column, concluding that the value of diarrhea in each of these treatments was not significantly different. meanwhile, the results of the frequency of diarrhea at a dose of 50 mg, loperamide, a dose of 75 mg, 25 mg and na-cmc were in the same column, concluding that the diarrhea value in each treatment was not significantly different.14 the results of the 75 mg dose of feces and loperamide were in the same column, indicating that, in each treatment, it was not significantly different (significant). however, it was significantly different at 25 mg, 50 mg and na-cmc. meanwhile, the 50 mg dose, 25 mg dose and na-cmc were in the same column, indicating that the diarrhea value at 50 mg dose, 25 mg dose and na-cmc was not significantly journal of fundamental and applied pharmaceutical science, 2(2), february 2022 74 different. the results of the frequency of diarrhea at a dose of 50 mg, loperamide, a dose of 75 mg, a dose of 25 mg and nacmc were in the same column, indicating that the diarrhea value in each treatment was not significantly different.15 conclusion based on the result of this study, it can be concluded that the results of the study indicated that avocado seeds contained alkaloids, flavonoids, glycosides, and saponins compounds. the extract yield obtained was 17.23%, and the water content was 3.7. the results of the preparation evaluation tests, such as ph testing on formulas i, ii, and iii, respectively, were 4.8; 4.7; 4.5. the average formula-specific gravity measurement was 1.03 before accelerated storage. after accelerated storage, it was 1.02. furthermore, the result of the viscosity measurement was 199.8, while the result of the sedimentation volume measurement was 0.76; 0.8; 0.8. %. the administration of avocado seed methanol extract suspension at a dose of 800 mg/kg bw had the most optimum effect as an antidiarrheal against white male mice with a stool weight of 0.39 grams and a duration of diarrhea of 74 minutes. the test results were slightly different between the dose of 800 mg/kg bb and loperamide as a positive 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https://doi.org/10.35799/jm.1.1.2012.423 https://doi.org/10.35799/jm.1.1.2012.423 https://doi.org/10.22487/j24428744.2020.v6.i1.13929 https://doi.org/10.22487/j24428744.2020.v6.i1.13929 https://doi.org/10.30595/pharmacy.v14i2.1948 https://doi.org/10.30595/pharmacy.v14i2.1948 https://doi.org/10.33096/ja.v12i2.638 https://doi.org/10.31596/cjp.v4i2.107 identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis abdul aziz; veggy nadya yuliawan; paula mariana kustiawan* departement of pharmacy, faculty of pharmacy, universitas muhammadiyah kalimantan timur, jl. ir. h. juanda no.15, samarinda, kalimantan timur 75124. abstract propolis is one of the natural products produced by kelulut bees and is still not widely used. the type of stingless bee that is the prima donna in the community is heterotrigona itama. this study aims to determine the phytochemical content of the n-hexane fraction of heterotrigona itama bee propolis collected from kutai kartanegara, east kalimantan. the n-hexane fraction was obtained from the methanol extract of h. itama propolis by the liquid-liquid partition method. after obtaining the n-hexane fraction, the research continued with a qualitative phytochemical test to identify the compound and total phenolic content. antibacterial activity was determined by the agar well diffusion method with a serial concentration in escherichia coli bacteria. qualitative phytochemical analysis in the form of color changes showed that the n-hexane fraction of h. itama propolis contained flavonoids, alkaloids, saponins, and tannins. based on the results, the total phenolic content of the n-hexane fraction sample was 490 mggae/100 g. it caused the n-hexane fraction to have lower phenolic content than the methanol extract (792 mg gae100 g). furthermore, this result indicated that the non-polar fraction was not substantial enough to extracted phenolic compounds. it correlated to the antibacterial activity of the n-hexane fraction, which was very weak (2 mm ± 1.5) at 200µg/ml concentration. keywords: heterotrigona itama; n-hexane; phenolic content; propolis data of article received reviewed accepted : : : 30 july 2021 04 aug 2021 24 aug 2021 doi 10.18196/jfaps.v2i1.12406 type of article: research introduction indonesia has incredible biodiversity, but non-timber forest products' potential is still limited and not managed properly. bee product is one of the non-timber forest products that have a potential effect on health. nowadays, in the pandemic era, stingless bee (kelulut bee) products have more attention for maintaining our * corresponding author, e-mail: pmk195@umkt.ac.id health than utilized as a biodiversity-based economic potential1. this honey is even more expensive than honey from apis spp bees. however, the potential use of another product, such as propolis, is still limited. if this potential is appropriately managed, it can contribute to the community's economy, especially the villagers. according to fao (food and agriculture organization), beekeeping is abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 24 one of the best economic opportunities for people living in forest areas2. these benefits are directly obtained in the form of beekeeping products. generally, propolis was generated by different resinous substances with antimicrobial activities from plants near the hive and collected to protect the hive. bee colonies make a barrier immunity in the hive against pathogens and parasites3, while propolis has prospective as a natural antibacterial. escherichia coli, one of the anaerobic facultative gram-negative bacteria, was abnormal flora in the large intestine of humans. most of the strains of this bacterium are harmless, but some strains have acquired the dna encoding for plasmid bacteriophage or enterotoxin or invasion factor and become pathogenic. strains that contain this virulence factor are responsible for the incidence of global diarrhea, neonatal meningitis, sepsis, and urinary tract infection4. if the bacteria are located outside the intestine or in their abnormal habitat, e. coli can become pathogenic bacteria. furthermore, e. coli with certain strains usually cause acute diarrhea. this bacteria can be transmitted through water, food, or people who have bad sanitation5,6. to overcome this, natural ingredients are used, such as propolis which is usually naturally used by bees to protect their hives from bacteria and fungi. heterotrigona itama is the most cultivated species of kelulut bee in indonesia. in east kalimantan, people are starting to be interested in cultivating kelulut bees. the community around pltgu (pembangkit listrik tenaga gas dan uap) in kutai kartanegara region try to develop kelulut bee farms as additional income in this pandemic era. previous research showed that the ethyl acetate fraction of propolis from that location contains an alkaloid, saponin, and terpenoid7. this study was conducted to identify the secondary metabolite of n-hexane fraction and antibacterial activity against e. coli. the nhexane fraction from methanolic extract of plants source indicated has inhibition to multidrug resistance human pathogenic strains8,9,10. propolis is collected by a bee from plant resin to prevent bacteria and fungi from their hive. based on the background above, this study aims to evaluate the antibacterial activity of the nhexane fraction of stingless bee propolis from that area. method this study used heterotrigona itama propolis obtained from bukit biru, kutai kartanegara, east kalimantan. furthermore, the used materials also included methanol, folin ciocalteu reagent (merck), sodium carbonate (na2co3) (merck), n-hexane (ch3(ch2)4ch3), distilled water, gallic acid (sigma-aldrich), ethanol pro analysis (merck), 70% ethanol (cv. mulawarman medika), filter paper, quercetin (sigma aldrich), aqua dest (cv. mulawarman medika), and aluminum foil. the genesys 10s uv-vis spectrophotometer was used as instrument analysis. sample preparation fresh propolis was collected from kelulut bee apiary, then kept at -4oc until used. the dirt, bees, and larva were removed out from propolis, then cut into pieces. the 100 grams of propolis were kept in containers, and methanol solvent was added and mixed. the propolis mixture that has been left for 24 hours was filtered, and the solvent was changed 3 times. the filtrate was evaporated at low pressure at a temperature of 60-70oc using an evaporator to obtain a thick extract11. journal of fundamental and applied pharmaceutical science, 2(1), august 2021 25 specimen of stingless bee was determined at laboratory of faculty of forestry, mulawarman university and identify as h. itama. fractionation the 20 g of methanol extract was put into a container then added 400 ml of hot water. the extract was mixed, added in a separating funnel, and 400 ml of n-hexane solvent was added in a ratio (1:1). it was mixed by shaking for one minute then allowed to stand until the solution separated and formed two layers. furthermore, n-hexane fraction filtrate was obtained and evaporated until a thick extract was obtained, and the yield was calculated. phytochemical assay flavonoid test identification the extract was pipetted as much as three drops into the drip plate. the extract was then added with 1 drop of h2so4 p.a. positive samples contain flavonoids if the solution undergoes a very striking color change to yellow, red, or brown.12 tannin test identification the 1 ml methanol extract of propolis was added into a test tube and added 2-3 drops of 1% fecl3. samples were contained tannins when there was a color change from light green to blackish green.13 alkaloid test identification a sample of 1 ml was put in a test tube, then 1 drop of hcl was added and homogenized. next, 3 drops of meyer's reagent were added. the test results were considered positive for containing alkaloids if the precipitate was yellowishwhite.14 terpenoid test identification the 1 ml of ethyl acetate fraction was taken and put into a test tube, and 0.5 ml of chloroform was added. a few drops of h2so4 concentrated were later added to the side of the tube. if it showed reddishbrown color between the surfaces, it indicated the presence of triterpenoid compounds.14 saponins test identification a sample of 1 ml was put in a test tube. the 1 ml of distilled water was added, then shake, and let stand for 30 seconds. furthermore, the 5 drops of 1% hcl were mixed into the solution. the presence of saponin compounded with the formation of foam.14 total phenolic content assay first, the 10 % na2 co3 solution was prepared 24 hours before use. on the next day, the n-hexane fraction of propolis was dissolved with 10 % methanol and distilled water to make a 10 mg/ml concentration. the gallic acid was used as a standard curve with a 3-50 µg/ml concentration. the phenolic content of the standard curve was determined with a 10 ml solution of 500 µl gallic acid, 500 µl of folin-ciocalteureagent, 3 ml of 10% na2co3 solution distilled water. it was then incubated for 2 hours in a dark condition and measured on a uv-vis spectrophotometer at a wavelength of 765 nm. the total phenolic content was calculated as natural compound (gallic acid) equivalent (gae) by the following equation: t= c xv/m. t is the total phenolic content in mg/g of the extracts as gae, while c is the concentration of gallic acid established from the calibration curve in mg/ml. v is the volume of the extract solution in ml, and m is the weight of the extract in g. the total phenolic content analysis results were expressed in units of g gallic acid equivalent (gae)/ abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 26 100g samples. the n-hexane fraction was carried out in the same step of total phenolic determination.15 antibacterial assay the antibacterial test used the agar well disk of diffusion method with some modification16. first, the media agar was prepared to culture bacteria and sterilized, then 20ml nutrient agar solution was poured into petri dishes and wait until solid. after that, the 20 µl e.coli bacteria solution was added and spread on the surface of the media on the plates. media agar plates were divided into five holes with a size four-mm diameter. the 20 µl of 25-200 µg extracts in acetone solution was added into the hole. a concentration of 30 µg/20 µl of positive control (chloramphenicol) was added to each petri disk. acetone was used as a negative control and then incubated in the dark at 32oc for 24 h. inhibition of bacteria was measured (mm) by calipers. antibacterial activity was calculated as the mean inhibition zone for the test sample divided by the mean inhibition zone for the standard drug. results and discussion phytochemical screening the phytochemical screening process is one of the preliminary stages in providing an overview of the class of compounds contained in h. itama propolis to overview the sample. the results of phytochemical screening of stingless bee propolis extract contained secondary metabolite compounds, which can be seen in table 1. table 1. phytochemical screening of n-hexane fraction from heterotrigona itama propolis compounds result indicator flavonoid + color change to yellow, red to brown alkaloid terpenoid + yellowish-white precipitate no color change between the surfaces tanin + color change from light green to blackish green saponin + changes in the initial solution and showed foam based on the data in table 1, stingless bee propolis extract contains flavonoids, alkaloids, tannins, and saponins. these compounds are secondary metabolites produced from plants. this condition is related to the food source of the bees17,18. stingless bees visit the several flowers of plants for nectar and pollen19. in addition, stingless bees take plant sap to build their nests20,21,22. the n-hexane fraction was obtained for the phenolic determination. compounds of phenol are described by the presence of a light green or slightly blue color, and the sample shows a blackish green stain so that it is suspected to be positive for phenolic23. phenolic responds with 1% fecl3 to frame intense red, blue, purple, or dark colors as fecl3 will respond with –oh. aromatics identification of tannins can also be carried out with ferric chloride expansion. one of them is because tannins are polyphenolic compounds, which means they have a place with a group of phenolic compounds. both examples show positive tannins by showing a blackish green stain24. furthermore, a few drops of concentrated hcl and magnesium were added to the sample during the flavonoid test. flavonoids were indicated by the appearance of maroon or pink color within 3 minutes. both samples showed a red journal of fundamental and applied pharmaceutical science, 2(1), august 2021 27 color suspected to be positive for flavonoids. besides, in the saponin test, distilled water is added to the sample, then bubbled using a water shower and shaken to form a stable foam. a fixed foam formation indicates the presence of saponins. two examples showed positive results. saponin compounds have a glycosyl group as a polar group and a steroid or triterpenoid group as a nonpolar group. thus, they surfaced dynamically and formed micelles when it is shaken with water. in micellar structure, the polar groups face outward while the nonpolar groups face inwards, which looks like foam25. total phenolic content the total phenolic content results showed phenolic compounds from the n-hexane fraction of h. itama propolis with absorbance values (table 2). the data generated from the total phenolic test content was processed in absorbance tables and standard curves (figure 1). figure1. standard curve of gallic acid to determine phenolic content furthermore, the researchers calculated the total phenolic content of the n-hexane fraction of propolis with gallic acid equivalent in table 2. table 2. total phenolic content of nhexane fraction from h. itama propolis. the results of the measurement of the phenolic content of propolis extract depend on the solvent as the solvent can affect the total phenolic content. when mixed in food, the phenolic compounds contained in propolis extract will be absorbed. however, it does not change in structure to increase the function of the food as an antioxidant. the phenol content of the kelulut bee h.itama propolis was determined by uv-vis spectrophotometry, with the standard used gallic acid as gallic acid is a derivative of hydroxyl benzoic acid indicating a simple phenolic acid. the researchers used gallic acid as a standard based on the 0,101 0,178 0,314 0,58 1,08 1,977 y = 0,3529x 0,5302 r² = 0,8482 -0,5 0 0,5 1 1,5 2 2,5 0,5 μg/ml 1 μg/ml 2 μg/ml 4 μg/ml 8 μg/ml 16 μg/ml a b so rb a n ce concentration absorba nce mean of absorba nce gallic acid equivale nt (μggae/ ml) total phenolic content (mggae /100 g) 0.896 0.3375 2.45 490 0.889 abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 28 availability of a stable and pure substance. another factor is that gallic acid is cheaper than other standard compounds. the total phenol content was calculated by putting the sample absorption value at a wavelength of 767 nm into the linear equation y= ax+b obtained from the gallic acid calibration curve. the yield is expressed in mg gallic acid equivalent per 100 grams(mg gae/100 grams). standard curves were made with a concentration of 0.5; 1; 2; 4; 8; 16 g/ml. the calibration curve was made from the level of gallic acid done in duplicate, and the r2 value was searched. the r2 or coefficient of determination value is a number whose value ranges from 0 to 1, indicating how close the estimated value for regression analysis represents the actual data. regression analysis will be the closest and most reliable one if the r2 value is equal to or close to 1. based on the calibration curve measurement results, the r2 value was 0.8482. the tested phenolic content had the highest concentration in the nhexane fraction, which was 490 mggae/g, while the phenolic content in the methanol extract was 792 mggae/g. the total phenolic content test results were influenced by the high methanol content and could also affect the withdrawal process of phenolic compounds. it means the higher the methanol content is, the more the absorbed phenolic compounds will be. it occurs because h.itama propolis has a short hydrocarbon chain so that the non-polar solvent ability of n-hexane in absorbing extracts from propolis is still lacking. based on the total phenolic test results, the total phenolic content in the nhexane fraction sample was lower than the methanol extract (table 3.) table 3. comparison of total phenolic content this result aligns with the research of26,27, stating that the total phenolic content of methanol extract was more significant than the n-hexane fraction. it could be due to the polar nature of methanol, which could attract phenolic compounds well in the extraction process. moreover, it also aligns with the results of previous studies revealing that methanol was a polar solvent that did not have a high boiling point so that compounds susceptible to high temperatures would not be damaged.28 furthermore, the chemical composition of propolis varies qualitatively and quantitatively due to the diversity of plant resins, in addition to the various geographic and climatic characteristics of the environment around the beehive.29,30 antibacterial activity antibacterial activity of non-polar fraction from h. itama propolis was determined with agar well diffusion. acetone was used as a negative control, and broad-spectrum antibacterial drug (chloramphenicol) was used as a positive control. e. coli is a gramnegative bacterium and is well known for its resistance to many antibiotics due to the permeability barrier provided by its outer membrane31. the result of antibacterial activity of n-hexane fraction of h. itama propolis against e. coli showed a weak level of inhibition (table 4). sample total phenolic content n-hexane fraction 490 mggae/100 g methanol extract 792 mggae/100 g journal of fundamental and applied pharmaceutical science, 2(1), august 2021 29 table 4. antibacterial activity of n-hexane fraction of h. itama propolis against e. coli concentration (µg/ml) inhibition zone (mm) antibacterial category rep. 1 rep. 2 rep. 3 mean ± sd 25 1 0 1 1 ± 0.5 weak 50 3 1 1 1 ± 1.1 weak 100 3 2 1 2 ± 1 weak 200 3 4 1 2 ± 1.5 weak chloramphenicol 21 20 17 19 ± 2 very strong control (-) the antibacterial activity was categorized by diameter inhibition. if the inhibition diameter regions on 5 mm or less, antibacterial activities are categorized as weak. 5-10 mm is categorized as medium; 10-19 mm is categorized as strong, while 20 mm or more is categorized as very strong32. the maximum concentration of 200 µg/ml only showed inhibition of 2 mm width. table 4 shows the inhibition in the weak category as it is around 1-2 mm. the non-polar fraction of propolis was had weak activity against e.coli. this gramnegative bacteria activity was caused by differences in the structure of the outer membrane of bacteria and the production of hydrolytic enzymes that caused degradation of the active components of propolis33. most of the polyphenol compound was detected in polar solvent rather than non-polar solvent34. propolis was collected by a bee from plant resin and correlated to a specific compound from that plant. bioactivities of plants were related to total phenolic compounds, such as flavonoids and lignin as an antioxidant, alkaloid as antibacteria35. the low content of phenolic components in the n-hexane fraction of h.itama propolis was correlated with antibacterial activity against e.coli. another research about the antibacterial activity of the nhexane fraction of plant extract showed relatively low phenolic content.36 the nhexane fraction also indicates it had good gram-positive activity rather than gramnegative bacteria37. that present of secondary metabolite in n-hexane fraction and phenolic content also had a contribution to antibacterial activity. it is was influenced by the time of sample collected, climate, and plants near the sample collected38. the chemical composition of propolis was determined by the composition of the plant source39. most of the activity was not directly affected by the presence of pltgu conclusion the n-hexane fraction of h. itama propolis contained alkaloid, flavonoid, saponin, and tannin, indicating potential compounds. this non-polar fraction was insufficient to extract phenolic compounds (490 mg gae/100 g). the total phenolic content of the non-polar fraction, which was less than the crude extract, indicated that some phenolic compounds were present in polar fractions. it correlated to the weak antibacterial activity of n-hexane fraction (2 mm ± 1.5) at 200µg/ml concentration. furthermore, the results of this study are preliminary studies. further research is suggested to carry out the bioactivity of the kelulut bee propolis h.itama from kutai kartanegara to benefit the community for the use of kelulut bee propolis. acknowledgment the authors would like to thank the research institute and community service abdul aziz, veggy nadya yuliawan, & paula mariana kustiawan | identification of secondary metabolites and antibacterial activity of non polar fraction from heterotrigona itama propolis 30 universitas muhammadiyah kalimantan timur (kdm grant) for their support in this study. conflict of interest all authors 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(2008). composition and in vitro antimicrobial activity of populus buds and poplar-type propolis. world j microbiol biotechnol, 24 (27), pp. 1011– 1017. https://doi.org/10.1007/s11274007-9566-5 http://dx.doi.org/10.18196/jfaps.v1i2.11001 http://dx.doi.org/10.18196/jfaps.v1i2.11001 https://dx.doi.org/10.3390%2fplants10010022 https://dx.doi.org/10.3390%2fplants10010022 http://dx.doi.org/10.1007/s11274-007-9566-5 http://dx.doi.org/10.1007/s11274-007-9566-5 antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria nadia aisy andika*, kusumaningtyas siwi artini, tatiana siska wardani faculty oh healthy sciences, universitas duta bangsa surakarta, jl. pinang no. 47, jati, cemani, kec. grogol, kabupaten sukoharjo, jawa tengah abstract one of the herbal plants that have medicinal properties is abrus precatorius l or commonly known as saga in indonesia. empirically, the boiled water of saga leaves is widely used as an ingredient in cough medicine, cancer sores and swollen tonsils. the chemical constituents of antibacterial activity in the saga leaves are glycosides (abrusoside ad and abrusgenin), flavonoids and saponins (glycerin). this study aims to determine the antibacterial activity of extracts and fractions from saga leaves and the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of the most active fraction of saga leaves on the growth of streptococcus mutans atcc 25175. saga leaf powder was macerated using 96% ethanol, then fractionated using n-hexane, ethyl acetate, water as solvent, 96% ethanol extract, and n-hexane fraction. the antibacterial activity test using the diffusion method showed that the extract, n-hexane fraction, ethyl acetate fraction, and water fraction of saga leaves had antibacterial activity against streptococcus mutans. the most active fraction was the ethyl acetate fraction, with a concentration of 50% with an average inhibition zone diameter of 12.2 mm. the ethyl acetate fraction from saga leaves had the most active antibacterial activity compared to ethanol extract, n-hexane fraction, ethyl acetate fraction, and water fraction, as seen from the average diameter of the inhibition zone obtained. the test results of the dilution method of the ethyl acetate fraction of saga leaves showed a minimum inhibitory concentration of 12.5% and a minimum killing concentration of 25%. keywords: abrus precatorius. l; antibacterial; extract and fraction; streptococcus mutansatcc25175 data of article received reviewed accepted : : : 17 sept 2022 22 dec 2022 27 mar 2023 doi 10.18196/jfaps.v3i2.16206 type of article: research introduction one of the herbal plants that have medicinal properties is abrus precatorius l. the saga plant belongs to the leguminosae plant family and is a type of herbaceous plant with small stems that propagates to the host in a twisted * corresponding author, e-mail: nadiayaya216@gmail.com manner. this plant grows wild in forests and fields or is deliberately kept in the yard.1 saga plants contain flavonoid and steroid compounds in the leaves. the chemical constituents of antibacterial activity in saga leaves are glycosides (abrusoside ad journal of fundamental and applied pharmaceutical science, 3(2), february 2023 100 and abrusgenin), flavonoids and saponins (glycerin). empirically, saga leaves boiled in hot are widely used as a cough medicine for cancer sores and swollen tonsils.2 among the 300 species of bacteria in the oral cavity, streptococcus mutants are the bacteria that mostly cause dental caries.3,4 streptococcus mutant is a gram-positive bacterium that forms dental plaque in the form of a sticky substance containing bacteria and products that form on the tooth surface.5 in previous studies, it was known that saga leaf extract ((abrus precatorius l.) was able to inhibit the growth of gram-positive bacteria, namely staphylococcus aureus, streptococcus beta-hemolytic, streptococcus pneumonia, so that it could be seen that saga leaves had the potential as antibacterial.6,7 based on the description above, a study was conducted on the antibacterial extract and the active fraction of abrus precatorius l. leaf against the growth of streptococcus mutans atcc 25175 to determine which of the solvents had the greatest inhibitory power and to determine the value of the minimum inhibitory concentration (mic) and its minimum bactericidal concentration (mbc). method materials this research utilized aluminum foil, bacterial culture of streptococcus mutans atcc 25175, abrus precatorius l leaf powder, antibiotic disc ciprofloxacin 5µg/ml, tlc plate, cotton, sterile gauze, sterile disc paper, filter paper, mueller hinton agar (mha) media, nutrien broth media (nb), 0.9% nacl, 10% dmso, 96% ethanol solvent, ethyl acetate solvent, nhexane solvent, aqua dest, tissue, and cotton. plant determination the abrus precatorius l. leaves were taken from the tawangmangu area, karanganyar regency, central java. the abrus precatorius l. was determined at the biology laboratory of setia budi university, surakarta. simplicia preparation the abrus precatorius l. leaves were washed using running water and separated from the attached dirt. drying was done directly under the hot sun covered with black cloth. pollination was done using a blender. they were sifted using mesh number 40.6 extraction the simplicial powder was weighed as much as 550 grams, then put into a maceration bottle and added with 96% ethanol as a solvent in a ratio (1:10). the bottle was stored in a place protected from sunlight for 5 days. the macerate was concentrated using a rotary evaporator at a temperature of approximately 40oc to obtain a thick ethanol extract.6 the concentrated extract from the maceration was then fractionated using a solvent of different polarity. fractionation was carried out using the llf (liquidliquid fractionation) method with nhexane (non-polar solvent), ethyl acetate (semi-polar solvent), and water (polar solvent) using a separating funnel. the thick extract of saga leaves was weighed as much as 10 g, then dissolved with 75 ml of water solvent (replicated 3 times), and fractionated with 75 ml of n-hexane solvent (replicated 3 times), the residue obtained from the n-hexane fraction followed by fractionation with 75 ml of ethyl acetate solvent each (replicated 3 nadia aisy andika, kusumaningtyas siwi artini, tatiana siska wardani | antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria 101 times). the result was the ethyl acetate fraction, and the residue obtained from the ethyl acetate fraction is the water fraction. the results of each fraction were evaporated with a water bath.8 phytochemical screening test phytochemical tests of the ethanolic extract of saga leaves were carried out, including tests for alkaloids, flavonoids, saponins, tannins, steroids, and triterpenoids. antibacterial activity test bacterial rejuvenation culture streptococcus mutans atcc 25175 was rejuvenated by using the method to tilt it by scratching it on the surface of the media mueller hinton so that it tilted in a zig-zag manner and then incubated at 37°c for 24 hours using an incubator. preparation of streptococcus mutans suspension atcc 25175 culture streptococcus mutans atcc 25175 was suspended in a tube containing sterile 0.9% nacl solution and incubated at 37oc for 15 minutes, then compared the turbidity of the suspension with standard mc. farland.9 making variable concentration of saga leaf extract a 50% concentration of mother liquor was made from the thick extract of saga leaves, and each fraction with 10% dmso as solvent. the dilution was continued until the test solution concentration series was 25% w/v, 12.5% w/v, and 6.25% w/v. furthermore, for the mic and mbc testing, the sample with the greatest antibacterial activity was selected, then the concentration was made at 25% w/v, 12.5% w/v, 6.25% w/v, 3.12% w/v, 1, 56% w/v. determination of bacterial inhibitory zones disc diffusion method mueller hinton agar (mha) media was poured into 10 ml petri dishes each and allowed to solidify as a base layer. furthermore, streptococcus mutans bacteria was suspended in a petri dish with a micropipette under aseptic conditions. furthermore, the disc paper dipped in the test material was placed on the plate, positive control disk ciprofloxacin and negative control dmso 10%. the petri dish was incubated for 24 hours at 30– 37oc. the inhibition zone formed around the paper disc was measured with a caliper. determination of minimum inhibitory level (mic) and minimum bactericidal concentration (mbc) in the most active fraction by dilution method prepared 8 test tubes. the stock solution concentration was 50%, then diluted with 10% dmso solvent. aseptically from the stock solution, a series of concentrations were made below that of 25% (tube 1); 12.5% (tube 2); 6.25% (tube 3); 3.12% (tube 4); 1.56% (tube 5); positive control (tube 6) and negative control (tube 7), the nb media used was put in 2 ml in each tube. furthermore, aseptically, 4 ml of the stock solution to be tested was put into the stock tube, then 2 ml of the tube was pipetted and put into tube 1. 2 ml of tube 1 was pipetted and put into tube 2, and so on until the test tube 5. on test tube 5, 2 ml was discarded. test tube 6 was added with 1 ml of ciprofloxacin as a positive control, and test tube 7 was filled with 1 ml of 10% dmso as a negative control. the entire test tube was then added to the bacterial suspension streptococcus mutans atcc 25175 and incubated for 24 hours at 37°c in an incubator. the smallest concentration of test material in the tube that showed the absence of turbidity in the tube was journal of fundamental and applied pharmaceutical science, 3(2), february 2023 102 called the minimum inhibitory concentration (mic). the minimum bactericidal concentration (mbc) was calculated by inoculating the sample in the tube on mueller hinton agar (mha) media in a petri dish, then incubated at 37°c for 24 hours. data processing and analysis method the data obtained from the test results are presented in tabular form and compared with the table classification of bacterial growth inhibition response.10 table 1. classification of bacterial growth inhibitory responses bright zone diameter growth inhibition response > 17 mm strong 12–16 mm medium 7–11 mm weak 0 mm no inhibition the data was then processed using spss (statistical product for service solutions) version 23 with the one-way anova (analysis of variance) test. results and discussion determination results plant determination aims to determine the correctness of the plant and avoid errors in the material collection. the determination was carried out at the biology laboratory, setia budi university, surakarata. based on the determination results, the sample was abrus precatorius l or commonly known as saga in indonesia drying loss the test used an oven at a temperature of 105oc for 30 minutes with a drying shrinkage of 3.33%. the test results met the drying shrinkage parameter of not more than 10%.11 extraction results the results of the extraction of saga leaf powder were 550 grams, which was found to weigh 69.43 grams with a yield of 12%. the yield obtained met the requirements of the indonesian herbal pharmacopeia, which was not less than 7.2%.11 the results of the calculation of the average percentage yield of saga leaf extract, namely the n-hexane fraction of saga leaves of 22.121%, ethyl acetate fraction of saga leaves of 24.401% and water fraction of saga leaves of 25.861%. water content test results the moisture content was tested with a moisture balance at a temperature of 105oc for 5 minutes. the result showed the water content of the saga leaf extract was 9.02%. these results met the requirements of the indonesian ministry of health (2000), which stated that the percentage of water content should be not more than 10%. ethanol free test results ethanol-free testing on saga leaf extract has been carried out utilizing the esterification test. the results were positive for ethanol-free saga leaf extract as it did not smell the distinctive odor of ethanol esters when tested.12 phytochemical screening test results based on the phytochemical screening test results, the ethanolic extract of saga leaves was positive for flavonoid compounds, saponins, steroids, phenols and tannins. the results of the phytochemical screening test can be seen in table 2. nadia aisy andika, kusumaningtyas siwi artini, tatiana siska wardani | antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria 103 table 2. phytochemical screening test result, saga result, saga leaf extract ((abrus precatorius linn.) determination results determination results determination results plant determination aims to determine the correctness of the plant and avoid errors in the material collection. the determination was carried out at the biology laboratory, setia budi university, surakarata. based on the determination results, the sample was abrus precatorius l or commonly known as saga in indonesia plant determination aims to determine the correctness of the plant and avoid errors in the material collection. the determination was carried out at the biology laboratory, setia budi university, surakarata. based on the determination results, the sample was abrus precatorius l or commonly known as saga in indonesia plant determination aims to determine the correctness of the plant and avoid errors in the material collection. the determination was carried out at the biology laboratory, setia budi university, surakarata. based on the determination results, the sample was abrus precatorius l or commonly known as saga in indonesia drying loss drying loss drying loss the test used an oven at a temperature of 105oc for 30 minutes with a drying shrinkage of 3.33%. the test results met the drying shrinkage parameter of not more than 10%.11 the test used an oven at a temperature of 105oc for 30 minutes with a drying shrinkage of 3.33%. the test results met the drying shrinkage parameter of not more than 10%.11 the test used an oven at a temperature of 105oc for 30 minutes with a drying shrinkage of 3.33%. the test results met the drying shrinkage parameter of not more than 10%.11 extraction results extraction results extraction results the results of the extraction of saga leaf powder were 550 grams, which was found to weigh 69.43 grams with a yield of 12%. the yield obtained met the requirements of the indonesian herbal pharmacopeia, which was not less than 7.2%.11 the results of the calculation of the average percentage yield of saga leaf extract, namely the n-hexane fraction of saga leaves of 22.121%, ethyl acetate fraction of saga leaves of 24.401% and water the results of the extraction of saga leaf powder were 550 grams, which was found to weigh 69.43 grams with a yield of 12%. the yield obtained met the requirements of the indonesian herbal pharmacopeia, which was not less than 7.2%.11 the results of the calculation of the average percentage yield of saga leaf extract, namely the n-hexane fraction of saga leaves of 22.121%, ethyl acetate fraction of saga leaves of 24.401% and water the results of the extraction of saga leaf powder were 550 grams, which was found to weigh 69.43 grams with a yield of 12%. the yield obtained met the requirements of the indonesian herbal pharmacopeia, which was not less than 7.2%.11 the results of the calculation of the average percentage yield of saga leaf extract, namely the n-hexane fraction of saga leaves of 22.121%, ethyl acetate fraction of saga leaves of 24.401% and water journal of fundamental and applied pharmaceutical science, 3(2), february 2023 104 chromatography (tlc) test results thin layer chromatography (tlc) test was carried out to confirm the presence of secondary metabolites in the extract and the fraction of saga leaves. table 3. thin layer chromatography test result of saga leaf extract and fraction ((abrus precatorius linn.) chemical content test material mobile phase stain color uv 366 rf reference alkaloids (dragondorf stain viewer) extract toluene: ethyl acetate: diethylamine (7:2:1) brownishyellow brownishyellow 0.45 13 f. water toluene: ethyl acetate: diethylamine (7:2:1) brownishyellow brownishyellow 0.59 13 f. ethyl acetate toluene: ethyl acetate: diethylamine (7:2:1) brownishyellow brownishyellow 0.61 13 f. nhexane toluene: ethyl acetate: diethylamine (7:2:1) 13 flavonoids (ammoniac vapor stain viewer) extract butanol : acetic acid : water (4:5:1) yellowish green greenish blue 0.80 0.61 14 f. water butanol : acetic acid : water (4:5:1) yellowish green greenish blue 0.76 14 f. ethyl acetate butanol : acetic acid : water (4:5:1) yellowish green greenish blue 0.78 0.58 14 f. nhexane butanol : acetic acid : water (4:5:1) yellowish green greenish blue 0.62 14 tannins (fecl3 stain viewer) extract toluene: ethyl acetate (3:1) black dark blue 0.61 14 f. water toluene: ethyl acetate (3:1) black dark blue 0.64 14 determination results determination results determination results fraction of saga leaves of 25.861%. fraction of saga leaves of 25.861%. fraction of saga leaves of 25.861%. nadia aisy andika, kusumaningtyas siwi artini, tatiana siska wardani | antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria 105 chemical content test material mobile phase stain color uv 366 rf reference f. ethyl acetate toluene: ethyl acetate (3:1) black dark blue 0.72 14 f. nhexane toluene: ethyl acetate (3:1) black dark blue 0.60 14 antibacterial activity test of saga leaf extract and fraction ((abrus precatorius l.) disc diffusion method antibacterial activity tests were carried out on each extract and fraction of saga leaves from concentrations of 50%, 25%, 12.5%, and 6.25%. the measurement results are listed in table 4. table 4. activity test result of saga leaves ((abrus precatorius l.) against streptococcus mutans bacteria using the diffusion method test material concentration (%) barrier zone diameter (mm) average category p1 p2 p3 extract 50% 10.0 10.5 10.5 10.3 weak 25% 9.5 9.5 10 9.6 weak 12.5% 8.0 8.5 8.5 8.3 weak 6.25% 8.0 8.0 8.5 8.2 weak water faction 50% 10.0 10.0 10.5 10.2 weak 25% 9.0 9.5 9.5 9.3 weak 12.5% 8.0 8.0 8.5 8.2 weak 6.25% 7.0 7.0 7.5 7.2 weak ethyl acetate fraction 50% 12.0 12.0 12.5 12.2 medium 25% 9.5 10.0 10.0 9.8 weak 12.5% 8.0 8.0 8.5 8.2 weak 6.25% 7.0 7.0 7.5 7.2 weak nhexane fraction 50% 8.0 8.5 8.5 8.3 weak 25% 7.5 8.0 8.0 7.8 weak 12.5% 7.0 7.0 7.5 7.1 weak 6.25% 5.0 5.0 6.5 5.5 weak + control (ciprofloxacin) 0.0005% 21 21 22 21.3 strong control – 10% 0 0 0 0 no inhibited information: p1: repetition 1 p2: repetition 2 p3: repetition 3 positive control: ciprofloxacin disk 5 g/ml (0.0005%) negative control: dmso 10% journal of fundamental and applied pharmaceutical science, 3(2), february 2023 106 according to the response classification table of bacterial growth inhibition zones, the inhibitory power of positive control disk ciprofloxacin of 21.3 mm belongs to the strong category.10 the ethyl acetate fraction of saga leaves at a concentration of 50% of 12.2 mm belonged to the medium category. in comparison, the ethanol extract of saga leaves at a concentration of 50% of 10.3 was in the weak category, the water fraction of saga leaves at a concentration of 50% of 10.2 mm was in the weak category, and the nhexane fraction of saga leaves at a concentration of 50% of 8.3 mm was categorized as weak. the negative control dmso 10% of 0 mm had no inhibition. these results revealed that the most active fraction was the ethyl acetate fraction. ethyl acetate solvent attracted antibacterial compounds in saga leaves, namely flavonoids, phenols, and glycosides. the higher the concentration series of saga leaf extract and fraction is, the stronger the inhibitory response to the growth of streptococcus mutants bacteria will be. it aligns with the statement of frazier and westhof,15 denoting that concentration can affect the effectiveness of an antimicrobial substance. the increase in the concentration of the extract causes a greater number of antimicrobial compounds to diffuse into the agar media, so an increase in the inhibition zone is expected chemical compounds in the extract and fraction of saga leaves that are considered to have antibacterial activity against streptococcus mutants bacteria are alkaloids, flavonoids, and saponins. the ability of flavonoid compounds as antibacterial is influenced by the difference in polarity between the lipids that make up bacterial dna and the alcohol groups on the flavonoid compounds, which cause damage to the bacterial dna structure so that bacterial cells undergo lysis and die.16 in alkaloid compounds, there are nitrogen groups that, when in contact with bacteria, will change the genetic balance in bacterial dna. thus, the bacterial cell nucleus will be damaged and lysed, leading them to die.17 the data obtained were tested for data analysis. data analysis used the one-way anova (analysis of variances) test and continued with the post hoc test using the tukey method. the kolmogorov-smirnov one sample test results obtained a significant result of 0.201 > 0.05, indicating the hypothesis is accepted. it was concluded that the data were normally distributed, so the anova analysis of variance could continue. the homogeneity of variances test results were 0.056 > 0.05, indicating that ho is accepted. it means that the four samples had the same variance or were homogeneous. the results of the significance of the anova test data were 0.000 <0.05, indicating that the four samples had differences in the diameter of the inhibition zone, followed by the post hoc test. the post hoc tukey test revealed no significant difference in comparing 50% saga leaf extract with 50% water fraction (p>0.05). in comparison, the ethyl acetate fraction and 50% n-hexane fraction had a significant difference (p<0.05). post hoc tukey test of extract, n-hexane fraction, ethyl acetate fraction and water fraction of saga leaf with positive ciprofloxacin control showed significant differences (p<0.05). the factor that influenced the formation of a greater inhibitory power of ciprofloxacin was because ciprofloxacin had an antibacterial effect (broad spectrum). it was categorized in the bactericidal quinolone group (kills nadia aisy andika, kusumaningtyas siwi artini, tatiana siska wardani | antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria 107 bacteria) and was quite effective for grampositive bacteria.18 results of mic and mbc test of the most active fraction of saga leaves ((abrus precatorius l.) dilution method the mic and mbc tests used the ethyl acetate fraction from saga leaves. the mic test was carried out to identify the smallest amount of active antibacterial substances that could inhibit the growth of the tested organisms. the results of the mic observations can be seen in table table 5. results of observation of mic ethyl acetate fraction of saga leaves ((abrus precatorius linn.) against streptococcus mutans by liquid dilution method no. tube information 1 25% clear 2 12.5% clear️-mic 3 6.25% turbid 4 3.125% turbid 5 1.5625% turbid 6 c+ (ciprofloxacin 0.0005%) clear 7 c(dmso 10%) turbid these results concluded that the minimum inhibitory concentration (mic) of the ethyl acetate fraction of saga leaves was 12.5%, as it showed clear tube results. a clear tube was obtained in the positive control of ciprofloxacin, indicating that the antibiotic ciprofloxacin could inhibit the growth of streptococcus mutans atcc 25175. meanwhile, the negative control of dmso 10% produced a tube that looked cloudy, proving that the solvent could not inhibit the growth of streptococcus mutans atcc 25175 bacteria. after obtaining the mic value, the minimum bactericidal concentration (mbc) test continued. the inoculation results to determine the mbc are summarized in table 6 and figure 1. table 6. inoculation results of antibacterial activity of ethyl acetate fraction by dilution against bacteria streptococcus mutans atcc 25175 the concentration of ethyl acetate fraction replication i ii iii 25% 12.5% + + + 6.25% + + + 3.12% + + + 1.56% + + + control (-) + + + control (+) description (+) : there is bacterial growth (-) : no bacterial growth positive control : ciprofloxacin negative control : dmso 10% journal of fundamental and applied pharmaceutical science, 3(2), february 2023 108 figure 1. results streaking on media mueller hinton agar to determine mbc (personal documentation, 2022) based on the table and figure above, it is known that the three replications revealed the same results, namely, the ethyl acetate fraction with a concentration of 25% and the positive control of ciprofloxacin produced a clear area. therefore, it can be concluded that the minimum kill concentration (kbm) of the ethyl acetate fraction of saga leaves against streptococcus mutans bacteria was 25%. conclusion based on the research results, it can be concluded that the ethanol extract, nhexane fraction, ethyl acetate fraction and water fraction of saga leaf (abrus precatorius l.) had antibacterial power against the growth of streptococcus mutans atcc 25175. the ethyl acetate fraction of saga leaf was the most active fraction in inhibiting the growth of streptococcus mutans bacteria atcc 25175 with an inhibitory value of 11.87 mm, categorized in the strong category at a concentration of 50%. the minimal inhibitory concentration (mic) of the ethyl acetate fraction of saga leaves was 12.5% , while the minimum kill concentration (mbc) of the ethyl acetate fraction of saga leaves was 25% against streptococcus mutans atcc 25175. acknowledgment with sincerity and humility, the author would like to thank mrs. tatiana siska wardani, s.farm., m.farm, as head of the undergraduate study program pharmacy at universitas duta bangsa surakarta as well as a mentor who has given lots of constructive advice and mrs. apt. kusumaningtyas siwi artini, s.farm., m.sc as the supervisor who has provided much direction and motivation for the author. references 1. pramiastuti o, sri rejeki d. uji antibakteri kombinasi ekstrak daun belimbing wuluh (averrhoa bilimbi l) dan daun kersen (muntingia calabura l) terhadap staphylococcus aureus. j ilm farm. 2020;9(2):2020–53. https://doi.org/10.30591/pjif.v9i2.2026 2. osmeli d, yuhernita y. kandungan senyawa kimia, uji toksisitas (brine shrimp lethality test) dan antioksidan (1,1-diphenyl-2pikrilhydrazyl) dari ekstrak daun saga (abrus precatorius l.). makara j sci [internet]. 2009;13(1). https://doi.org/10.7454/mss.v13i1.378 3. alhassan a, ahmed q. averrhoa bilimbi linn.: a review of its ethnomedicinal uses, phytochemistry, and pharmacology. j pharm bioallied 25% k 6.25 % 12.5 % 3.12 % 1.56 % k+ 25% k 6.25 % 12.5 % 1.56 % 3.12 % k+ k 6.25 % 25% 12.5 % 1.56 % 3.12 % k+ https://doi.org/10.30591/pjif.v9i2.2026 https://doi.org/10.7454/mss.v13i1.378 nadia aisy andika, kusumaningtyas siwi artini, tatiana siska wardani | antibacterial activity of abrus precatorius l. leaves against streptococcus mutans atcc 25175 bacteria 109 sci [internet]. 2016;8(4):265. https://doi.org/10.4103/09757406.199342 4. silva br da, freitas vaa de, nascimento-neto lg, carneiro va, arruda fvs, aguiar asw de, et al. antimicrobial peptide control of pathogenic microorganisms of the oral cavity: a review of the literature. peptides. 2012 aug 1;36(2):315–21. https://doi.org/10.1016/j.peptides.201 2.05.015 5. lemos ja, quivey rg, koo h, abranches j. streptococcus mutans: a new gram-positive paradigm? microbiology [internet]. 2013;159(pt 3):436. https://doi.org/10.1099/mic.0.066134-0 6. nisak sk, pambudi db, waznah u, slamet s. uji antibakteri ekstrak etanol daun saga (abrus precatorius l.) terhadap bakteri streptococcus mutans atcc 31987 dan staphylococcus aureus atcc 25923pk/5. pros semin nas kesehat [internet]. 2021;1:2031–7. 7. pertiwi rd, kristanto j, praptiwi ga. uji aktivitas antibakteri formulasi gel untuk sariawan dari ekstrak daun saga ( abrus precatorius linn. ) terhadap bakteri staphylococcus aureus. j ilm manuntung [internet]. 2016;2(2):239–47. https://doi.org/10.51352/jim.v2i2.72 8. sulistyarini ii, sari da, rahardian mrr. anti-bacterial activity test of ethanol extract, n-hexane fraction, ethyl acetate fraction and water fraction from dragon fruit stem (hylocereus polyrhizus) against methicillin-resistant staphylococcus aureus (mrsa). j ilmu kesehat [internet]. 2021;9(2):162–71. https://doi.org/10.30650/jik.v9i2.2284 9. wardani ts, nisa tc, artini ks. antibacterial activity test of n-hexan, ethyl acetate and water from ethanol extract of kitolod leaf (isotoma longiflora (l.) c. presl.) against staphylococcus aureus atcc 25923. proc int conf nurs heal sci [internet]. 2022;3(1):9–16. 10. lesmana h, saleh m, thioritz e, miko h, sopianah y. the resistance of bandotan (ageratum conyzoides) leaf extract and siwak stem extract on the growth of butterial streptococcus mutans. j phys conf ser [internet]. 2020;1477(6):062027. https://doi.org/10.1088/17426596/1477/6/062027 11. depkes ri. parameter standar umum ekstrak tumbuhan obat (edisi 1). jakarta: direktorat jenderal pengawasan obat dan makanan. direktorat pengawasan obat tradisional,; 2000. 12. raymon m, taebe b, ali a, khairuddin k. antibacterial activity test of manila sawo fruit extract (achras zapota l.) with various liquid filters against salmonella typhimurium. j pharm med sci. 2016;1(1):6–11. 13. harborne j. metode fitokimia: penentuan cara modern menganalisa tumbuhan (edisi ii). bandung: institut teknologi bandung; 2003. 14. zaini m, shofia v, studi p, farmasi d-i, kalimantan pu, analis d-i, et al. skrining fitokimia ekstrak carica papaya radix, piper ornatum folium dan nephelium lappaceum semen asal kalimantan selatan. j kaji ilm kesehat dan teknol [internet]. 2020;2(1):15–27. https://doi.org/10.52674/jkikt.v2i1.30 15. frazier w, westhoff d. food microbiology. hill m, editor. new york; 1998. 16. dzoyem jp, hamamoto h, ngameni b, ngadjui bt, sekimizu k. antimicrobial action mechanism of flavonoids from https://doi.org/10.4103/0975-7406.199342 https://doi.org/10.4103/0975-7406.199342 https://doi.org/10.1016/j.peptides.2012.05.015 https://doi.org/10.1016/j.peptides.2012.05.015 https://doi.org/10.1099/mic.0.066134-0 https://doi.org/10.51352/jim.v2i2.72 https://doi.org/10.30650/jik.v9i2.2284 https://doi.org/10.1088/1742-6596/1477/6/062027 https://doi.org/10.1088/1742-6596/1477/6/062027 https://doi.org/10.52674/jkikt.v2i1.30 journal of fundamental and applied pharmaceutical science, 3(2), february 2023 110 dorstenia species. drug discov ther. 2013 apr 30;7(2):66–72. 17. fauzia la. testing the effect of water from avocado leaves (persea gratissima) against streptococcus mutans from saliva by thin layer chromatography (tlc) and minimum inhibitory concentration (mic). nusant med mag. 2008;41(3):173–8. 18. andersson mi, macgowan ap. development of the quinolones. j antimicrob chemother [internet]. 2003 may 1;51(suppl_1):1–11. https://doi.org/10.1093/jac/dkg212 https://doi.org/10.1093/jac/dkg212 aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats bridget osamuyimen ikponmwosa & usunobun usunomena* department of biochemistry, faculty of basic medical sciences, edo state university uzairue, edo state, nigeria abstract methotrexate (mtx) usage, despite its toxicity in body organs, has increased steadily over the years due to its broad applicability for treating different ailments, including various forms of cancer. certain plant species have been shown to possess therapeutic properties by offering a protective effect against drug side effects. thus, the current study was carried out to evaluate the potential of aqueous chromolaena odorata leaf extract (aeoc) to attenuate the effect of mtx-induced hepatotoxicity in wistar rats. the study divided thirty (30) male wistar rats into five groups consisting of six each: group i (control), group ii (aeoc at 250 mg/kg bw), group iii (mtx at 7 mg/kg bw), group iv (aeoc at 250 mg/kg bw + mtx at 7 mg/kg bw), and group v (vitamin c (100 mg/kg bw) + mtx at 7 mg/kg bw). chromolaena odorata and vitamin c was administered for ten consecutive days, while mtx was administered on day 8 for three consecutive days. rats were sacrificed 24hrs after the last administration. serum collected was used for the determination of aspartate aminotransferase (ast), alanine transaminase (alt), albumin (alb), total bilirubin (tb), and total protein (tp), while liver tissue was used for assessment of superoxide dismutase (sod), malondialdehyde (mda) and catalase (cat) as well as histopathological analysis. the result showed a significant increase in the level of sod, cat and a significant reduction in mda in chromolaena odorata or vitamin c treated groups compared with mtx. furthermore, chromolaena odorata or vitamin c significantly reduced liver function enzymes and total bilirubin levels while increasing synthetic molecules compared to the mtx group. chromolaena odorata attenuated the toxic effect of mtx, which was corroborated by histopathological analysis. in conclusion, chromolaena odorata attenuated mtx-induced hepatotoxicity by enhancing antioxidant status; thus, scavenging free radicals and reducing oxidative stress. keywords: chromolaena odorata; hepatotoxicity; methotrexate; oxidative stress; wistar rats data of article received reviewed accepted : : : 29 jul 2022 10 aug 2022 29 aug 2022 doi 10.18196/jfaps.v3i1.15652 type of article: research * corresponding author, e-mail: usunobun.usunomena@edouniversity.edu.ng bridget osamuyimen ikponmwosa & usunobun usunomena | aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats 17 introduction methotrexate (mtx) is an anti-metabolic agent that affects the metabolism of folic acid.1. in medical practice, methotrexate is indicated for a plethora of clinical conditions, including autoimmune rheumatic conditions; rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, juvenile idiopathic arthritis, inflammatory myopathies, sarcoidosis, rheumatic polymyalgia, arthritis related to secondary amyloidosis and others. it is also indicated by other autoimmune conditions, such as sjögren syndrome, inflammatory bowel disease, vasculitis, and some neoplasms.2 the importance of mtx as an effective chemotherapy agent for cancer treatment cannot be overemphasized as it has successfully been used in treating breast cancer, leukemia, lung cancer, lymphoma, gestational trophoblastic disease, and osteosarcoma.3 however, side effects abound, affecting various body systems, including the gastrointestinal system,4 hematopoietic system,2 central nervous system, respiratory system5, and cardiovascular system.2 mtx also presents renal toxicity and decreases creatinine clearance and glomerular filtration rate.2 furthermore, there is evidence that mtx can be oncogenic, notably in lymphomas and leukemias.5 the use of herbal medicines has continued to gain momentum, particularly in africa, where 70-80 % of its people depend either totally or partially on it. certain herbs have been reported to have the potential to alleviate the side effects of most synthetic drugs.6 chromolaena odorata, a pantropic herb, possess phytochemicals and antioxidant enzymes that activate defense mechanisms and stress-sensing transcription factors to prevent oxidative damage.7 the dried leaf of ch. odorata contains active phytochemical substances, such as flavonoid aglycones (flavanones, flavonols, flavones). it includes acacetin, chalcones, eupatilin, luteolin, naringenin, kaempferol, quercetin, quercetagetin, sinensetin, terpenes and terpenoids, essential oils, alkaloids (pyrrolizidine, saponins, and tannins), phenolic acids (ferulic acid, protocatechuic acid), and phytoprostane compound including chromomeric acid.8,9,10 ch. odorata has been reported to be used to treat wounds, burns, and skin infections and possess anticancer, antidiabetic, anti-inflammatory, antimicrobial, anti-diarrheal, astringent, antispasmodic, antihypertensive, antiinflammatory, diuretic, tonic, antipyretic and heart tonic and also cough remedy agent.11,12 therefore, the present study aims to evaluate the potential of ch. odorata to reduce the effect of mtxinduced toxicity in rats. method chemical and reagent methotrexate (liquid) of 50 mg (zuvius life sciences, india) was purchased from medvalik pharmaceuticals limited, lagos, nigeria. all reagents used were of analytical grade and had the highest purity. collection and identification of plant material fresh leaves of ch. odorata were collected from within the locality of iyamho community, uzairue, in etsako local government area of edo state, nigeria, and taxonomically authenticated at the department of plant biology and biotechnology herbarium, edo state university, uzairue, edo state nigeria with voucher number euh/00066. journal of fundamental and applied pharmaceutical science, 3(1), august 2022 18 preparation and extraction of plant material the fresh leaves of ch. odorata were thoroughly rinsed and air-dried at room temperature for one month, then pulverized, crushed into a fine powder using an electric blender, and weighed with an electric weighing balance. an aqueous extract of the plant was prepared by soaking 1000g of the dried powdered plant materials in 5 liters of doubledistilled water and then kept at room temperature for 48 hours to ensure a thorough extraction process. at the end of the 48 hours, the extracts were filtered first through a whatman filter paper no. 42 (125mm) and then cotton wool. the resultant filtrate was concentrated using a rotary evaporator set at 40oc to one-tenth of its original volume and then reduced to solid form using a water bath. the solid residue (crude extract) was stored at 4oc. aliquot portions of the crude plant extract residue were weighed and dissolved in normal saline on each experiment day. experimental animal and design thirty (30) male wistar rats (180-200g) of the speciesrattus norvegicus were purchased from the animal house, department of zoology, ambrose alli university, nigeria. the animals were housed in a well-lit, adequately ventilated room using a wood-gauze cage in the animal house of the department of biochemistry, edo state university uzairue, edo state. standard environmental conditions were used (12 hours light and 12 hours dark) in acclimatizing the animals to the new environment. animals were fed with standard laboratory pellets and given free access to water. this study was approved by the ethics committee of the faculty of basic medical sciences, edo state university uzairue, and followed the guidelines for ethical conduct in the care and use of non-human animals in research.13 after acclimatization for seven days, the rats were randomly distributed into the following groups: group i: served as a control and only received normal saline orally once daily. group ii: rats were given aqueous extract of ch. odorata at a dose of 250 mg/kg bw orally once daily for ten days. group iii: rats were given mtx intraperitoneally at a dose of 7 mg/kg bw on day 8 of the experiment for three consecutive days. group iv: rats were given an aqueous extract of ch. odorata (250 mg/kg bw) orally once daily for ten days and then mtx intraperitoneally (7 mg/kg bw) on day 8 of the experiment for three consecutive days. group v: rats were given vitamin c (100 mg/kg bw) orally once daily for ten days and mtx intraperitoneally (7 mg/kg bw) on day 8 for three consecutive days. mtx was dissolved in saline and injected intraperitoneally (i.p.) at 7 mg/kg bw dose.14 ch. odorata at a 250 mg/kg bw dose was based on another study15. vitamin c, an antioxidant, was chosen as a hepatoprotection; thus, it was a positive control. at the end of the experiment, after 24hrs of the last administration, the rats were sacrificed, and blood was collected in plain tubes. it was allowed to stand for 45 minutes before being centrifuged at 4000 rpm for 25 min to obtain serum for analysis. the serum was used for the determination of aspartate aminotransferase (ast), alanine transaminase (alt), albumin (alb), total bilirubin (tb), and total protein (tp). the liver was immediately excised, washed in ice-cold saline, and weighted. a portion was fixed in 10% phosphatebuffered formalin for histopathological bridget osamuyimen ikponmwosa & usunobun usunomena | aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats 19 examination, while the remaining portion was stored at -20oc to determine oxidative stress and endogenous enzymes. 10 % tissue homogenate of the stored liver tissues was prepared using phosphate buffer solution at ph 7.34. the homogenate was centrifuged at 5000 rpm for 15 minutes, and a clear supernatant obtained used to determine superoxide dismutase (sod), malondialdehyde (mda), and catalase (cat). biochemical parameter alanine aminotransferase (alt) and aspartate aminotransferase (ast) activity were determined using the randox kit according to the manufacturer’s instructions.16 total bilirubin was determined using the randox kit according to the manufacturer’s instructions.17 total protein was determined by using the randox kit according to the manufacturer’s instructions as described.18 according to the manufacturer's instructions, albumin was determined using the randox kit based on the bromocresol green (bcg) method as described.19 the reaction of thiobarbituric acid determined malondialdehyde (mda) as an indicator of lipid peroxidation according to the method of.20 the level of superoxide dismutase (sod) activity was according to the method of.21 while the method of22 was used to determine catalase (cat). histopathological studies rats were sacrificed after, and liver samples were excised and washed with normal saline (0.9% nacl). the isolated livers were fixed in 10% buffered formalin and were further processed for histopathological investigations. histopathologically the liver tissues were stained with hematoxylin and eosin (h&e), then sections were examined under a light microscope, leitz (biomed), and histopathological changes were captured by a nikon camera, eos700d, 18–55 lens. statistical analysis all the data in the treatment groups were presented as mean ± standard error of the mean (sem), and statistical analysis was carried out using statistical package (spss) version 20, windows 10. mean values of the different treatment groups were compared using one-way analysis of variance (anova), followed by duncan multiple range post hoc tests. the p<0.05 was considered statistically significant. results and discussion the biomarkers for liver damage in wistar rats treated with aqueous extract of ch. odorata and administered with mtx are presented in table 1. administration of mtx significantly increased (p > 0.05) ast (113.67 u/l) and alt (127.08 u/l) enzymes as well as total bilirubin (17.31 mg/dl), while total protein (3.54 g/dl) and albumin (1.56 g/dl) were reduced significantly when compared with a control group and other groups in this study. however, treatment with ch. odorata or vitamin c to rats administered mtx significantly (p > 0.05) restored the level of ast, alt, total bilirubin, total protein, and albumin towards normalcy. journal of fundamental and applied pharmaceutical science, 3(1), august 2022 20 table 1. effects of aqueous leaf extract of chromolaena odorata on liver function and synthetic molecules in methotrexate-induced wistar rats treatment group ast (u/l) alt (u/l) alb (g/dl) tbil (mg/dl) tp (g/dl) control 41.33a ± 3.33 36.67a ±3.17 7.47c ± 0.99 2.39a ± 0.45 9.65d ± 0.91 ch. odorata (250 mg/kg bw) 43.67a ± 3.03 40.70a±3.31 6.04c ± 0.70 1.25d ± 0.10 7.79d ± 0.93 mtx (7mg /kg bw) 113.67c ±6.67 127.08c±7.55 1.56a ± 0.32 17.31c ±1.71 3.54a ± 0.11 mtx (7 mg/kg bw) + ch. odorata (250 mg/kg bw) 61.33b ± 4.07 62.08b±5.04 3.66b ± 0.72 7.23b ± 0.96 5.39b ± 0.14 mtx (7 mg/kg bw) + vit. c (100 mg/kg bw) 56.33b ±4.88 73. 01b±5.57 3.82b ± 0.60 8.87b ± 0.87 6.07c ± 0.61 values are expressed as mean ± standard error of the mean, n=6. values with different superscripts down the column differ significantly at (p<0.05). ast-aspartate aminotransferase; alt-alanine aminotransferase; albalbumin; tbiltotal bilirubin; tp total protein; vit. cvitamin c; mtxmethotrexate. the present study indicated a significant effect of aqueous leaf extract of ch. odorata on liver function enzymes, ast, and alt in methotrexate-induced wistar rats. the administration of mtx caused significant liver toxicity marked by elevated serum levels of ast and alt, similar to previous studies.23,24,25 uraz et al.26 reported that an increased level of ast indicated damage caused by methotrexate toxicity to the visceral organs. in this present study, mtx at a dose of 7mg/kg bw was recorded to have a higher concentration of ast and alt in the serum, which vividly indicated hepatotoxicity against other treatment groups with values towards normalcy. however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx was found to improve liver functions, evidenced by the reduction in the ast and alt values similar to previous findings of patel et al.27 there was no significant difference in the improvement of liver function between vitamin c and ch. odorata against mtx-induced toxicity. the ability of ch. odorata or vitamin c treatment groups to reduce the ast and alt levels may result from the antioxidant activity of vitamin c or bioactive constituents of ch. odorata leaves such as flavonoid.10 furthermore, xu et al.28 reported that alt is a more specific indicator of liver damage, particularly liver inflammation, than ast. in the present study, mtx elevated alt more than ast, as observed by ozogula et al.29 the present study also showed a significant effect of aqueous leaf extract of ch. odorata on synthetic liver molecules, albumin (alb), total bilirubin (tbil), and total protein (tp) in methotrexate-induced rats. although vitamin c improved in terms of tp level, there was no significant difference in tbil and alb levels between vitamin c and ch. odorata against mtx-induced toxicity. these parameters are used as reliable bridget osamuyimen ikponmwosa & usunobun usunomena | aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats 21 checks to indicate liver damage. based on the results, the low concentration of alb in blood serum for mtx-administered rats is a clear sign of hepatic impairment compared to the control group. however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx recorded much higher values which could be attributed to the protective effect of ch. odorata and vitamin c due to their antioxidant capacity. the findings of this study on the albumin level align with swayeh et al.30 according to swayeh et al.,30 these consistent observations are probably due to an indirect effect of methotrexate on protein synthesis by declining the amount of tetrahydrofolate. this study also recorded a reduction in total protein (tp) due to mtx toxicity. generally, it is expected that the body must constantly produce proteins to fight infections to aid the health and growth of the body's cells and tissues, among others. the level of total proteins shows how well the liver works appropriately to produce these proteins. conversely, significantly lower values obtained in mtx (7mg/kg bw) group further indicated the extent to which the liver was damaged and could not properly function. the reduction in total protein and albumin could be due to damage to the liver by mtx, increased intestinal protein loss, and protein-losing nephropathy.31 similar findings of a decrease in total protein have been previously reported by.23,32,33,34 however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx gradually increased total protein concentration; thus, it demonstrated the protective and antioxidant potential of ch. odorata and vitamin c. furthermore, bilirubin analysis is carried out to ascertain the liver's health or monitor the progression of an affected liver. the elevated levels of total bilirubin recorded for the mtx (7mg/kg bw) administered group indicated liver damage or disease. it signified that the ability of the liver to clear bilirubin had been impacted; hence, it observed toxicity. this finding aligns with previous studies of.35,36,37,38 however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx reduced total bilirubin concentration to the value recorded by mtx untreated group. this decrease could be attributed to the protection of the liver against oxidative damage caused by mtx. mda, an end product of lipid peroxidation, is often used as a marker of lipid peroxidation. in this study, mtx injection for three consecutive days at a dose of 7mg/kg bw significantly increased mda in the intoxicated group. in contrast, treatment with ch. odorata or vitamin c significantly reduced mda; thus, hepatic damage indicated hepatoprotection (table 2). antioxidant enzymes, such as sod and cat, were estimated in the present study. there was a significant decrease in cat content in the liver homogenates of mtxtreated groups compared to the control at 𝑃 < 0.05. the supplementation of ch. odorata with mtx caused a significant increase in cat compared to the mtx group (table 2). mtx at a dose of 7mg/kg for three consecutive days also decreased the activity of sod compared to the control group (figure 2). however, rats that received ch. odorata or vitamin c together with methotrexate experienced a significant increase in sod activity compared to the mtx-treated group. journal of fundamental and applied pharmaceutical science, 3(1), august 2022 22 table 2. effect of aqueous leaf extract of chromolaena odorata on hepatic oxidative stress parameters in methotrexate-induced wistar rats treatment group sod (u/mg protein) mda (µmol/mg protein) cat (u/mg protein) control 89.47a ± 3.13 3.29a ± 1.54 2.87a ± 0.21 ch. odorata (250 mg/kg bw) 93.59a ± 5.71 6.92b ± 0.85 3.50a ± 0.58 mtx (7 mg/kg bw) 29.94b ± 4.79 13.99c ± 1.23 0.93b ± 0.12 mtx (7 mg/kg bw) + ch. odorata (250 mg/kg bw) 73.22c ± 3.72 8.63d ± 1.37 1.17c ± 0.06 mtx (7 mg/kg bw) + vit. c (100 mg/kg bw) 56.10d ± 3.87 9.22d ± 0.95 1.42c ± 0.19 values are expressed as mean ± standard error of the mean, n=3. values with different superscripts down the column differ significantly at (p<0.05); sodsuperoxide dismutase mdamalondialdehyde, catcatalase, mtxmethotrexate, vit cvitamin c the body's antioxidant defense system is the primary line of defense that counteracts the deleterious effects of free radicals, oxidative damage, and oxidative stress. data from this study indicated a remarkable decrease in the activity of cat and sod in the group administered with mtx (7 mg/kg bw) compared to the control group (table 2). according to sener et al.,39 oxidative stress leads to pathological and cellular damage to liver tissues. however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx showed a significant increase in the activity of cat and sod in the liver. this result conforms to a previously reported study of.40-41 unlike sod and cat, an increase in mda values was observed for the group administered with mtx. it occurred because lipid peroxidation decreased membrane fluidity42 and could compromise the integrity and function of the plasma membrane, thereby leading to leakages of materials from hepatocytes into the blood. however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx had recorded a much-reduced mda compared to the group administered with mtx (7 mg/kg bw). it is similar to previous studies of.43,44,45,40 although ch. odorata had better improvement in increased sod levels, there was no significant difference in mda and cat levels between vitamin c and ch. odorata against mtx-induced toxicity. furthermore, the histopathological analysis showed that mtx (7 mg/kg bw) caused distortion necrosis, congestion, cell infiltration, irregular loss of hepatocytes architecture with dilated central veins and hepatic sinusoid vacuolar degeneration (figure 2). however, administration of aqueous leaf extract of ch. odorata or vitamin c for ten days with an intraperitoneal injection of mtx showed a reduction in hepatic lesions with moderately spaced central veins surrounded by uniform hepatocytes distribution (figures 4 and 5). bridget osamuyimen ikponmwosa & usunobun usunomena | aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats 23 figure 1. photomicrograph of liver of control rats that received normal saline showing normal liver architecture figure 2. photomicrograph of liver given mtx at a dose of 7 mg/kg bw for three consecutive days showing necrosis, cell infiltration dilation, and congestion figure 3. photomicrograph of liver of rats that received 250 mg/kg bw ch. odorata for ten consecutive days showing normal liver architecture journal of fundamental and applied pharmaceutical science, 3(1), august 2022 24 figure 4. photomicrograph of liver of rats given 100 mg/kg bw vitamin c for ten consecutive days and 7 mg/kg bw mtx for three consecutive days showing a reduction in hepatic lesions figure 5. photomicrograph of liver of rats given 250 mg/kg bw ch. odorata for ten consecutive days and 7 mg/kg bw mtx for three consecutive days showing a reduction in the histopathological lesions histopathological analysis showed that mtx (7 mg/kg bw) had significant distortion on the hepatocytes as seen with observed necrosis, congestion, cell infiltration, irregular loss of hepatocytes architecture with dilated central veins, dilated hepatic sinusoid as well as vacuolar degeneration similar to findings of.46 however, administration of aqueous leaf extract of ch. or vitamin c for ten days with an intraperitoneal injection of mtx showed a reduction in hepatic lesions with moderately spaced central veins surrounded by uniform hepatocytes distribution. conclusion based on the result of this study, it can be concluded that ch. odorata attenuated mtx-induced hepatotoxicity by bridget osamuyimen ikponmwosa & usunobun usunomena | aqueous leaf extract of chromolaena odorata attenuates methotrexate-induced hepatotoxicity in wistar rats 25 scavenging free radicals, reducing oxidative damage and oxidative stress, thus enhancing antioxidant status. acknowledgment the authors would like to thank the animal house staff, department of biochemistry, edo state university uzairue, for all their support in this research. references 1. benedek, t. g. methotrexate: from its introduction to non-oncologic therapeutics to anti tnf-α. clin exp rheumatol 2010;5:3-8. 2. kozuch, p., & hanauer, s. treatment of inflammatory bowel disease: a review of medical therapy. world j gastroenterol 2008;14:354-377. https://doi.org/10.3748/wjg.14.354 3. matera, c., gomila, a. m., camarero, n., libergoli, m., soler, c., & gorostiza, p. photoswitchable antimetabolite for targeted photoactivated chemotherapy. journal of the american chemical society. 2018;140:15764–15773. https://doi.org/10.1021/jacs.8b08249 4. canhão, h., santos, m., 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vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease benjamin z. gbolo1,2*, amandine nachtergael2, damien s. t. tshibangu3, nicole m. misengabu4, nsabatien victoire1, patrick b. memvanga5, dorothée d. tshilanda2, k. n. ngbolua1, pius t. mpiana3, pierre duez2 1department of biology, faculty of sciences, university of kinshasa, kinshasa, democratic republic of the congo 2unit of therapeutic chemistry and pharmacognosy, faculty of medicine and pharmacy, university of mons (umons), 7000 mons, belgium 3department of chemistry, faculty of sciences, university of kinshasa, kinshasa, democratic republic of the congo 4department of biology, institut de recherche en sciences de la santé (irss), kinshasa, democratic republic of the congo 5faculty of pharmaceutical sciences, university of kinshasa, democratic republic of the congo abstract sickle cell disease (scd) is an autosomal recessive blood disorder characterized by red blood cells that assume an abnormal, rigid sickle shape under low-oxygen conditions. these sickle-shaped erythrocytes tend to lyse, aggregate, and obstruct small blood vessels, leading to major complications. the present study aims to investigate properties that may underlie the activity of drepanoalphaâ, an antisickling herbal formulation developed in the democratic republic of congo (drc) for the prevention and symptomatic treatment of sickle cell disease crises. the drepanoalpha® ethanolic extract (dee) is a dry extract (drug-extract ratio, der, 100/11) prepared from ethanol (96 %, v/v) percolation of a 1:1 mixture of 2 food plants, justicia secunda vahl and moringa oleifera lam. sickling was classically measured by light microscopy on diluted washed erythrocytes obtained from homozygote patients; erythrocytes were treated with 2 % na2s2o5 in the presence of dee (suspension in 9 ‰ nacl), 9 ‰ nacl (negative control) or disodium cromoglycate (dscg, positive control). for all tested conditions, the sickle hemoglobin polymerization, the fe2+/fe3+ ratio, and the median corpuscular fragility were measured by spectrophotometry. the dee reversed sickling by 89.1 %, comparable to dscg (87.7 %; 60.3 µg/ml), inhibiting sickle cell hemoglobin polymerization of 77.8 % and 74.4 %, respectively. the fe2+/fe3+ ratio was improved by 18.0 % for dee and 15.9 % for dscg. the median corpuscular fragility values were 0.602, 0.714, and 0.732 for nacl 9 ‰, dscg, and dee, respectively. the measured in vitro parameters validate an effective antisickling effect of dee and confirm the value of this improved traditional herbal formulation for the management of scd. keywords: antisickling activity; drepanoalpha®; erythrocytes; fe2+/fe3+ ratio; hemoglobin polymerization; sickle cell disease data of article received reviewed accepted : : : 27 aug 2022 30 dec 2022 23 feb 2023 doi 10.18196/jfaps.v2i1.16000 type of article: research * corresponding author, e-mail: benjamin.gbolo@unikin.ac.cd benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 65 introduction sickle cell disease (scd), or sickle cell anemia or drepanocytosis, is a genetic disease that affects hemoglobin and leads to the synthesis of hemoglobin s (hbs) instead of normal hemoglobin (hba). in their oxygenated forms, the solubility of hbs decreases 50 times, resulting in its precipitation and intracellular polymerization, which modifies the structure of red blood cells that take a "sickle shape," which tend to lyse but also aggregate and obstruct small blood vessels, leading to major complications.1,2 the disease is not only most prevalent in black people from africa but is also prevalent around the mediterranean and in india.3 it is estimated that more than 300,000 babies are born worldwide each year with severe forms of this hemoglobinopathy.1,4,5 management of the disease is difficult in developing countries, particularly in the democratic republic of congo (drc), where, with some 25 % as genotypes, nearly 2 % of the population is affected with ss genotypes.6 indeed, poverty conjugates to the absence of a welfare system, with huge difficulties in meeting medical costs.5,7 as for many diseases, the relative costs of treatments and their associated adverse effects make the recourse to herbal medicines attractive or even essential, especially for rural populations.4,8 effectively, an estimated 80 % of the sub-saharan population uses traditional medicine for health care; some plants have already proven their effectiveness, and bioactive molecules have been identified.1,4,9,10 the growing interest and use of phytomedicines to treat sickle cell disease are also probably linked to the assumption that medicinal plants are "natural" and "safe". 4,11–13 in drc, a vast bio-prospecting program has identified a hundred antisickling plants; based on an in vitro antisickling assay, the most active plants were developed in drepanoalpha®, an improved traditional herbal formulation.1,5,14,15 the drepanoalpha® powder is a mixture 1:1 (w/w) of 2 edible plant leaves powder, justicia secunda vahl (acanthaceae) and moringa oleifera lam. justicia secunda, a native tropical herbaceous plant originating from south america, is nowadays grown in tropical or subtropical african countries. in the past, this plant was considered ornamental until locals discovered the medicinal properties of its leaves, notably for the treatment of anemia, hypertension16,17 and sickle cell disease.14,18 the phytochemical study of leaves from various j. secunda cultivars revealed the presence of polyphenols, including tannins, leucoanthocyanins, anthocyanins and, mainly, flavonoids.16 the moringa genus comprises 14 species, among which m. oleifera is sometimes designated as the "tree of life" or "miracle vegetable". this tropical tree, native to the sub-himalayan mountains, is widely distributed in tropical and sub-tropical areas, both dry and humid.19–21 the leaves used for animal and human feeding, given their alleged richness in proteins, vitamins, b-carotene, and amino acids, are now considered a functional food.14,19,21–25 a wide variety of medicinal uses have been attributed to m. oleifera's various organs for anti-inflammatory, anti-infectious, cardiovascular, gastrointestinal and hematological properties, including the management of sickle cell disease.14,19,21,24 phytochemical studies reported the highest level of phenolic compounds, mainly flavonoids, in m. oleifera leaves.26 the originality of this study resides in the extraction that allowed to prepare of an journal of fundamental and applied pharmaceutical science, 3(2), february 2023 66 improved phytomedicine, easier to manage compared to traditional decoctions, and the combination of a series of anti-sickling tests (on cells and hemoglobin) to evaluate, in vitro, the effectiveness of this extract. the present study was carried out on a polar extract of the 2 plants mixture to investigate properties that may underlie the use of drepanoalpha in the prevention and symptomatic treatment of sickle cell disease crises. method chemicals and reagents all reagents were of analytical grade. 2aminoethyl diphenylborinate (97 %) and quercetin hydrate (≥ 95 %) were purchased from sigma-aldrich (merck); polyethyleneglycol 400 (peg 400) (for laboratory use), methanol (99 %), absolute ethanol (≥ 99.8 %), methyl ethyl ketone (gpr reactapur), formic acid (98 %) and ethyl acetate (acs reagent) were obtained from vwr chemicals. a 10 g/l solution of diphenyl boric acid 2-amino ethyl ester (np reagent) and a 50 g/l solution of peg 400 were prepared in methanol to detect polyphenols. herbal material the drepanoalpha® powder, a 1:1 (w/w) mixture of the leaves from 2 food plants, justicia secunda vahl (herbarium mnhnp-p00719831) and moringa oleifera lam. (herbarium mnhn-p-p05401821) has been provided by research for sustainable development (resud, kinshasa, drc), approved by producer and distributor of the phytomedicine. extraction the dry extract (drug-extract ratio, der, 100/11) was obtained by percolating 100 g of the herbal material mixture with 1000 ml ethanol 96 % for 48 h and drying under vacuum at 40°c. the resulting drepanoalpha® ethanolic extract (dee) was stored at  4°c for a maximum of 3 months. the leaves of j. secunda and m. oleifera were individually extracted similarly. high-performance thin-layer chromatography (hptlc) hptlc was performed according to the procedure of the european pharmacopeia 10,27 using automatic tlc sampler (ats 4), automatic developing chamber 2 (adc 2), derivatizer and tlc visualizer 2 (camag, muttenz, switzerland). the systems were driven by the software visioncats version 2.5. the hptlc was performed on silica gel 60 f254 hptlc plates (merck, germany); 2 µl of quercetin (1 mg/ml metoh), 10 µl of dee (30 mg/ml metoh) and 5 µl of j. secunda and m. oleifera (30 mg/ml metoh) extracts were applied in 6-mm wide bands, the plates were activated on mgcl2 (~ 33 % rh) and the tank saturated for 20 min; the solvent system was ethyl acetate methylethylketone formic acid -water (60:30:5:5, v/v/v/v) and the plate was developed over a path of 60 mm (fig.1). the plate was heated at 100 °c for 3 min, sprayed with 2 ml of the np reagent (derivatizer with green nozzle, level 3) and peg (derivatizer with blue nozzle, level 2), and photographed immediately after derivatization under uv365nm, using the visualizer system. benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 67 figure 1. hptlc profile of the ethanolic extracts of drepanoalpha® and its constituent plants. stationary phase: hptlc plate (silica gel 60 f254) mobile phase: ethyl acetate methylethylketone formic acid water (60:30:5:5, v/v/v/v); detection: np-peg-400; 1% solution of 2-aminoethyl diphenylborinate in methanol, followed by 5% polyethylene glycol in ethanol; examination under uv365nm. legend: tracks: 1-quercetin, 2-justicia secunda, 3-moringa oleifera, 4drepanoalpha®. blood samples blood samples were left-overs of specimens sampled for the regular monitoring of known sickle cell disease homozygote patients attending the “centre de médecine mixte et d’anémie ss” (kalamu district, kinshasa, drc) and the “hôpital civil marie curie” (charleroi, belgium). none of these patients had experienced a recent transfusion with hb aa blood. all antisickling experiments were carried out with blood freshly collected on citrate and stored at ± 4 °c for a maximum of 72 h. red cell pellets, obtained by centrifugation (1500 g, 10 min) of 0.5 ml of ss blood, were washed thrice with nacl 9 ‰, in a 1:10 (v/v) ratio, centrifuged (1500g, 10 min) and resuspended in 4 ml nacl 9 ‰. the study was conducted after receiving the approval of the ethical and scientific committee of the school of public health, faculty of medicine, university of kinshasa, kinshasa-drc (approval no.: esp/ce/237/2019) and of the i.s.p.p.c. om008 ethical committee of “c.h.u. charleroi”, charleroi, belgium (approval no p19/55-23/10 chrau: umons ccb: b325201941714). in vitro antisickling activities induction of sickling and inhibition of falciformation samples of 950 µl of blood, obtained from homozygote patients, diluted 1:10 in 9 ‰ nacl, were added with 10 µl of drepanoalpha ethanolic extract (dee) (suspension in 9 ‰ nacl), 9 ‰ nacl 1 2 3 4 journal of fundamental and applied pharmaceutical science, 3(2), february 2023 68 (negative control) or disodium cromoglycate (dscg, positive control) and homogenized. upon adding 50 µl of 2 % na2s2o5 and homogenizing, 5 µl of samples were placed on a microscope slide, covered and smeared with clear varnish to isolate from oxygen and induce hypoxia and sickling. the sickled/normal erythrocyte ratios were measured in light microscopy at times 0 and 60 min on images of 5 different microscopic fields acquired with a digital camera (olympus u-cmad3): f (%) = srb trb 𝑥100 where f: sickling cell rate; srb: sickled red blood cell count and trb: total red blood cell count. the proportion of sickle cell inhibition si was calculated as follows (28): 𝑆𝐼 = 𝐹0 − 𝐹𝑛 𝐹0 𝑥 100 where si is the percentage of sickling inhibition, 𝐹0 is the % of sickling of the mixture [ss blood + na2s2o5] (negative control) and 𝐹𝑛 is the % of sickling of the mixture [ss blood + tested extract or compound + na2s2o5]. for a test to be considered valid, the ratio trb60 min/trb0 min should be over 80  5 % to control that only limited hemolysis has been induced by the tested extract or compound. the capacity of extracts to prevent, reverse or protect against falciformation to understand whether the tested extract or compound prevents or reverses falciformation, 3 procedures were assessed: a) a mixture of 950 µl diluted blood and 50 µl na2s2o5 was incubated for 60 min in a closed vial, then added with 10 µl of dee, nacl 9 ‰ or dscg and homogenized; b) a mixture of 950 µl diluted blood and 10 µl dee, nacl 9 ‰ or dscg was incubated for 60 min in a closed vial, then added with 50 µl na2s2o5 and homogenized; c) a mixture of 950 µl diluted blood, 10 µl of dee, nacl 9 ‰ or dscg and 50 µl na2s2o5 was incubated for 60 min in a closed vial. for the different procedures, the proportions of sickle cell inhibition were assessed as previously described. determination of the minimum reversibility concentrations (mrc) for the mrc determination, the proportions of sickle cell inhibition (= reversibility rate) were measured as described here above by varying the concentrations of dee and dscg (50 to 250 g/ml). for each concentration, the proportion of sickle cell inhibition (si) was calculated as above to determine the rate of reversibility r as: 𝑅 = 𝑆𝐼0 − 𝑆𝐼𝑛 𝑆𝐼0 𝑥 100 where r is the reversibility rate (%) and 𝑆𝐼0 and 𝑆𝐼𝑛 are the proportions of sickling inhibition for the control (nacl 9 ‰) and the tested concentration, respectively. dose-effect curves were obtained by fitting data to the equation y = 𝐴1−𝐴2 1+(𝑥 𝑥0⁄ ) 𝑝 + 𝐴2 using the origin 8.5 software (originlab, northampton, ma, united states). inhibition of sickle hemoglobin (hbs) polymerization according to the original method of (29), the hbss polymerization was assessed at 700 nm from the turbidity of a polymerizing mixture. 200 µl of a red cell pellet were hemolyzed by adding 400 µl of distilled water, incubated for 30 min in the presence or absence of the drug (400 µl) and primed for polymerization by deoxygenating with 3000 µl of a 2 % benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 69 sodium metabisulphite solution. the optical densities were measured after centrifuging at 3500 rpm for 5 min. their difference yields the measure of turbidity. the rate of polymerization inhibition was estimated by the tangent of the graph "absorbance versus time". the relative polymerization and relative inhibition were determined concerning the control (24) as 𝑅𝑝 = 𝑂𝐷𝑡 − 𝑂𝐷𝑖 𝑡 where 𝑅𝑝 = rate of polymerization, 𝑂𝐷𝑡 = optical density at time t, 𝑂𝐷𝑖 = initial optical density, t=time determination of erythrocyte membrane stability the osmotic fragility of erythrocytes allows the measurement of eventual membrane-stabilizing effects by a 60 min incubation in osmotic stress conditions. 10 µl of a red cell pellet were diluted in 1990 µl of a series of buffered hypotonic saline solutions at different concentrations (0.2 0.8 % nacl), added with 10 µl of dee, dscg, or nacl 9 ‰ and homogenized. the effect of the different extracts on hemolysis was observed in light microscopy with a digital camera (olympus u-cmad3). total cells were counted from 5 different fields across each slide at 0 and 60 min. for each nacl concentration and extract, the percentage of hemolysis was calculated as follows: % 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝑁0 − 𝑁60 𝑁0 𝑥 100 where 𝑁0 and 𝑁60 are the numbers of red blood cells at 0 and 60 min, respectively. the median corpuscular fragility (mcf), the nacl concentration that causes 50 % erythrocyte hemolysis, was estimated from the linear regression "% hemolysis versus nacl concentration" using the origin 8.5 software. determination of methemoglobin concentration the red cell pellet was hemolysed with distilled water in a 1: 2 ratio (v/v) and centrifuged (1500 g, 10 min). the hemolysate was incubated at room temperature in the presence or absence of the dee/dscg. the evolution of absorbances was measured at 540 and 630 nm for hemoglobin (fe2+) and methemoglobin (fe3+), respectively. the proportion of methemoglobin was calculated at each time as follows: 𝐹𝑒3+ = (𝐴630) 2 (𝐴540) 2 + (𝐴630) 2 𝑥100 𝐹𝑒3+, 𝐴540 and 𝐴630 are the proportion of methemoglobin and the absorbances at 540 and 630 nm, respectively. to appreciate the kinetics of the reaction in the presence or absence of extract, 𝐹𝑒3+-time curves were obtained by fitting data to the equation y = 𝐴1−𝐴2 1+(𝑥 𝑥0⁄ ) 𝑝 + 𝐴2 using the origin 8.5 software. statistical analysis all the experiments were conducted in triplicate; the data were expressed as mean ± standard deviation (s.d) and analyzed using origin 8.5 software with a chi-square test. the level of significance was classically set at 0.05. journal of fundamental and applied pharmaceutical science, 3(2), february 2023 70 results and discussion sickling induction table 1: sickling induction assay (60 min contact with sodium metabisulphite in airtight conditions; measurement over 5 microscopic fields) patient date of assay sample code total red blood cells sickled red blood cells % sickling test considered as 1 31/10/19 ss_70 168 139 80.4 positive 2 31/10/19 hetero_40 136 4 4.4 negative 3 31/10/19 hetero_58 185 6 3.2 negative 4 05/11/19 ss_91 189 21 11.1 negative 5 05/11/19 ss_54 240 240 100.0 positive 6 08/11/19 ss_40 152 14 9.2 negative 7 12/11/19 ss_59 144 6 4.2 negative 8 12/11/19 ss_759 138 14 10.1 negative 9 12/11/19 ss_36 93 78 83.9 positive 10 12/11/19 ss_68 127 15 11.8 negative 11 14/11/19 ss_07 100 13 13.0 negative 12 18/11/19 ss_396 126 117 92.9 positive 13 18/11/19 ss_597 195 162 83.1 positive 14 12/12/19 ss_237 234 207 88.4 positive fourteen samples were received from hôpital civil marie curie and tested for their ability to falciform in deoxygenation conditions (table 1). figure 2 shows phenotypic micrographs of representative samples. as expected, the samples from heterozygote patients yielded a very low proportion of sickled cells in our experimental conditions. however, 6 samples from homozygote patients were also weakly falciform, indicating a possible treatment by hydroxyurea (induction of non-sickling fetal hemoglobin) and antioxidants (scavenging na2s2o5). these heterozygotes and non-falciform samples (<50 % sickling) were not considered for the following experiments. figure 2. sickling induction assay. phenotypic micrographs of representative samples (60 min contact with sodium metabisulphite in air-tight conditions; 500 x) legends: hetero: heterozygous blood; ss: ss homozygous blood; 40, 58, 396 are the identification numbers of the samples attributed by the laboratory of the supplying hospitals. normal rbcs sickle cells benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 71 reversibility assay table 2 details the sickling reversal of ss patient erythrocytes untreated (control) and treated with dscg and j. secunda, m. oleifera, and dee under hypoxic conditions. figure 3 shows representative phenotypic micrographs of untreated and treated erythrocytes. figure 3. morphology of drepanocytes of ss blood (sample ss_54), a: untreated (0.9% nacl), and upon treatment with sodium cromoglycate (b: 250 µg/ml); ethanolic extracts of j. secunda (c), m. oleifera (d) and drepanolpha® (e) (125 µg/ml); 60 min contact with sodium metabisulphite in air-tight conditions; 500 x) table 2. anti-sickling effects on ss erythrocytes (60 min contact with sodium metabisulphite in air-tight conditions; measurement over 5 microscopic fields) blood sample negative control cromoglycate ethanolic extracts justicia secunda moringa oleifera drepanoalpha® trb(a) srb(b) si(c) trb srb si trb srb si trb srb si trb srb si ss_70 168 139 82.7 159 31 19.5 168 10 6.0 162 11 6.8 156 30 19.2 ss_54 240 240 100.0 186 28 15.1 204 18 8.8 225 20 8.9 219 24 11.0 ss_36 93 78 83.9 93 9 9.7 95 7 7.4 95 7 7.4 93 9 9.7 ss_396 126 117 92.9 177 11 6.2 177 8 4.5 120 8 6.7 102 8 7.8 ss_597 195 162 83.1 174 18 10.3 189 11 5.8 189 18 9.5 183 15 8.2 ss_237 234 207 88.5 183 15 8.2 231 15 6.5 234 20 8.6 210 18 8.6 mean 88.5 11.5 6.5 8.0 10.8 (sd) (6.9) (4.9) (1.5) (1.2) (4.3) p vs. negative control(e) -- <0.001 <0.001 <0.001 <0.001 description (a) trb: total red blood cell count (b) srd: sickled red blood cells count (c) si: percentage of sickling induction (e) anova one-way with posthoc t-tests (tukey); there were no statistical differences between the treatments cromoglycate justicia secunda moringa oleifera drepanoalpha® a b c d e journal of fundamental and applied pharmaceutical science, 3(2), february 2023 72 figure 4. reversibility rate of sickled red blood cells according to the concentration of dee and dscg. (60 min contact with sodium metabisulphite in air-tight conditions). (data from 3 biological tests in triplicate, bars represent the mean ± sd) description dee: drepanoalpha® ethanolic extract dscg: disodium cromoglycate mrc: minimum reversibility concentration mrr: maximum reversibility rate figure 4 presents the reversibility rate of sickled red blood cells according to dee and cromoglycate concentration. the rate of reversibility of sickle red blood cells in hypoxic conditions increased with the concentration of dee or dscg until reaching a maximum threshold (mmr, maximum reversibility rate), above which the reversibility remained constant, regardless of the increase in concentration. the minimum reversibility concentration (mrc) was defined as the extract concentration for which 50 % of the sickled cell population was normalized. mmr and mrc were evaluated by non-linear regression using origin 8.5 software the capacity of extracts to prevent, reverse, and protect against falciformation to verify whether dee prevents or reverses falciformation, the ss_54 sample was treated in 3 different protocols (dee treatment followed by deoxygenation, i.e., prevention; deoxygenation followed by dee treatment, i.e., reversal; concomitant dee treatment and deoxygenation, i.e., protection) compared with cromoglycate. figure 5 shows the phenotypic micrographs of treated samples. benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 73 figure 5. morphology of drepanocytes of ss blood (sample ss_54) upon different schemes of treatment with drepanolpha® ethanolic extract (dee) or cromoglycate (dscg) (125 g/ml; 60 min contact with sodium metabisulphite in air-tight conditions; 500 x) these experiments indicated that the dee had the ability to prevent, reverse and protect against erythrocyte sickling. although dscg also reversed and protected against sickle cell formation, the preventive dscg treatment appeared inefficient in averting sickling. inhibition of sickle hemoglobin (hbs) polymerization the polymerization rates of hbs and its inhibition are presented in table 3 journal of fundamental and applied pharmaceutical science, 3(2), february 2023 74 table 3. polymerization rates of hbs and its inhibition by the drepanoalpha® extract (dee) and cromoglycate (dscg) (n = 3 technical replicates) sample rate of polymerization relative % inhibition vs. negative control negative control 0.65  0.01 --- dscg 0.17  0.0 74.4  0.0 dee 0.14  0.02 77.8  0.0 table 3 indicates that the polymerization of sickle hemoglobin (hbs) is partly inhibited in the presence of either dscg or dee in a similar proportion. this property is well known to contribute to antisickling activities. stabilization of erythrocyte membranes figure 6 indicates an effective hypoosmolarity-induced lysis of sickle erythrocytes when decreasing the nacl concentration. although this effect was less marked in the presence of drepanoalpha® extract or cromoglycate, the protection afforded was not statistically significant. figure 6. lysis susceptibility of sickle erythrocytes, according to osmolarity, in the presence of drepanoalpha® ethanolic extract or cromoglycate (125 g/ml; 60 min incubation at room temperature) description mcf: median corpuscular fragility 0,0 0,2 0,4 0,6 0,8 0 25 50 75 100 h e m o ly s is r a te ( % ) nacl concentration(%) negative control : mcf= 0.669 disodium cromoglycate : mcf= 0.714 drepanoalpha ® ethanolic extract : mcf= 0.732 0.0 0.2 0.4 0.6 0.8 benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 75 modulation of methemoglobin formation figure 7 and table 4 show the evolution of methemoglobin as a function of time; dee and dscg significantly reduced the formation of methemoglobin in hbss blood, preventing the oxidation of fe2+ into fe3+. figure 7. evolution of methemoglobin proportion versus time. bars represent the mean ± sd for n=3 technical replicates. table 4. modulation of methemoglobin formation in the presence of dee and dscg (mean ± sd; n = 3 technical replicates) sample %hemoglobin (fe2+) %methemoglobin (fe3+) fe2+/fe3+ % increase negative control 84.90.3 15.1  0.3 5.6  1.0 ---- dscg 94.10.4 5.9  0.4 15.9  1.0 64.641.3 dee 94.70.6 5.3  0.6 18.0  1.0 68.851.0 results and discussion the bioactivity was assessed based on the phenotypic sickling of ss red blood cells (rbcs) in the presence of an oxygenscavenging agent. the individual plant extracts and dee displayed remarkable sickling inhibitory effects, reverting sickle erythrocytes to a typically normal morphology in the same proportions as cromoglycate, a well-known positive control (18,24,30). it confirms that the dee extract conserves the previously shown antisickling effects of moringa oleifera extracts24 and drepanoalpha® herbal powder.1 the sickling of red blood cells is a process that results from the polymerization of hb s under conditions of hypoxia and cellular dehydration by loss of ions (k+, cl-, mg++) and water (31,32). the antisickling effect indicated that dee could rehydrate deoxygenated sicked red cells, thus preventing the increase of intracellular hb s concentration. anthocyanins, part of dee secondary metabolites1, have been shown to be 0 60 120 180 240 4 6 8 10 12 14 16 p ro p o rt io n o f m e th e m o g o lb in ( f e 3 + ) time (min) negative controle disodium cromoglycate drepanoalpha ethanolic extract journal of fundamental and applied pharmaceutical science, 3(2), february 2023 76 responsible for most plants' biological activities against sickle cell disease in traditional congolese medicine by weakening hydrophobic interactions at the intermolecular contact sites of different deoxyhemoglobin s molecules.1,15,33–37 the antisickling effect of dee is higher compared to some antioxidants and micronutrients often combined in the management of sickle cell disease, i.e., magnesium (0.1 mm;48.4 % reversal of in vitro falciformation), zinc (0.1 mm; 89.7 %), vitamins a (100 iu; 30.9 %), c (1 mg/ml; 38.1%) and e (1 mg/ml; 30.9 %) (38). the action of dee on the inhibition of polymerization could be a synergistic effect of its (phyto)chemical constituents such as anthocyanins, flavonoids and micronutrients, including the mg++, zn++, and vit a .1 however, the copper identified among the micronutrients of this phytomedicine (1) is a negative agent for sickle cell disease whose level should be controlled in the final product. it is well established that dense and dehydrated red blood cells can contain hbs polymers under conditions of moderate hypoxia, and even in arterial blood, due to the particularly high intracellular concentration of hbs. these dense and dehydrated red blood cells thus play a central role in sickle cell disease's acute and chronic manifestations based on reduced blood flow and vasoocclusions in small vessels (31). the erythrocyte membrane stability test performed on sickled red cells in hypotonic nacl condition indicates a slight but insignificant increase of sickle cell resistance as measured by the mcf. it would be interesting to repeat the test with the preincubation of erythrocytes before hypotonic stressing to evaluate an eventual membrane protective effect. given the high red cell oxidative status (39,40), hemoglobin can oxidize to methemoglobin and thus lose the property of combining with oxygen (41– 44). in normal blood, only a very small amount of methemoglobin exists. an effective system based on nicotinamide adenine dinucleotide phosphate (nadph), methemoglobin reductase and cytochrome b5 to reduce the heme fe3+ to fe2+, the metabolic shunt pathway of pentose phosphates in erythrocyte is necessary for the synthesis of nadph that protects hemoglobin and membrane lipids from oxidation.45 however, this reduction system is less efficient in cases of glucose6-phosphate dehydrogenase (g-6-pd) deficiency, an erythrocytic enzymopathy often associated with sickle cell disease. a treatment inducing a decrease in methemoglobin level would indicate an effective antioxidant effect on sickle red blood cells,46 likely to protect from sickling and senescence. as shown here, both dscg and dee effectively improve the fe2+/fe3+ ratio, a mechanism likely to increase the oxygen affinity of drepanocytes and so to reverse sickle improving the fe2+/fe3+ ratio erythrocytes to their original biconcave structure. our results indicated that, in vitro, dee effectively prevented both erythrocyte sickling and hemoglobin oxidation. here again, this activity could be the result of the dee content in polyphenols, including flavonoids, and trace elements such as mg++, zn++ and vit a (1,38,47). this study will likely impact transfusion treatments as our previous clinical studies on the phytomedicine decoction have shown an increase in hb level and protection against early hemolysis, which would prevent anemia and avoid transfusion (48,49). benjamin z. gbolo, amandine nachtergael, damien s. t. tshibangu,nicole m. misengabu,nsabatien victoire, patrick b. memvanga,et al | in vitro biological activities of drepanoalpha® ethanolic extract, a justicia secunda and moringa oleifera-based phytomedicine proposed for the symptomatic treatment of sickle cell disease 77 conclusion this research depicted in vitro anti-sickling activity of drepanoalpha® ethanolic extract. the results revealed that dee preventing sickling and reversing sickled hbss red blood cells had a membrane stabilizing effect on sickled red blood cells, possessed abilities to inhibit sickle cell hemoglobin polymerization, and improved the oxidant status of erythrocytes by increasing the fe2+/fe3+ ratio in a sickled red blood cell. it highlighted that this traditional improved herbal formulation had medicinal benefits, confirming its use in managing sickle cell disease (scd). future studies are suggested to identify and isolate the active principle of this phytomedicine by bio-guided fractionation, which could enhance the standardization of this antisickling recipe. acknowledgments this research received funding from the academy of research and higher education (ares) in a program entitled "institutional support (ia) 2018-2020". grant number: coop-conv-18-004, ai ares unikin. the authors would like to thank resud for providing the herbal material and dr. nicole tasiaux, mr. charles chevalier of “c.h.u. charleroi” and mr. didier bosolo efunda «of “centre de médecine mixte et d’anémie ss” for their collaboration. references 1. gbolo bz, asamboa ls, bongo gn, tshibangu dst, kasali f, memvanga p, et al. 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