Anti-alopecia characteristic of Sauropus androgynus (L) Merr. ethanol extract and its fractions

Research Article Anti-alopecia characteristic of Sauropus androgynus (L) Merr. ethanol extract and its fractions Resmi Mustarichie 1 *, Rini Hendriani 2, Dila Triarini 1 ABSTRACT Objectives: This study aims to examine the anti-alopecia characteristics of ethanol extract and fraction of n-hexane, ethyl acetate, and water from katuk (Sauropus androgynus (L) Merr.) leaves. Materials and Methods: The katuk leaves were collected and macerated with ethanol 70% and fractionated with ethyl acetate and hexane solvents. The extract and its fractions tested its hair growth stimulator activity based on Tanaka modification method and using minoxidil 2% as a positive control. The analysis results were evaluated statistically. Results: It was found that katuk leaf extracts had provided significant hair growth activity at concentrations of 10%, 15%, 20%, and 25%. In testing the activity of katuk leaf fraction, water fraction showed positive hair growth activity compared to the fraction of n-hexane, ethyl acetate, and positive control. Statistically, it was found that the effect of hair growth was obtained from the concentration of 10% extract and found that the activity of katuk leaf water fraction significantly showed better hair growth activity compared to n-hexane, ethyl acetate, and positive control. Conclusions: It was concluded that katuk leaf scientifically had a potential in stimulating hair growth and could be used as anti-alopecia treatment. Further experiments are needed to find the chemical compound content that is responsible for the nature of hair growth. KEY WORDS: Anti-alopecia, Hair loss, Katuk, Maceration, Sauropus androgynus INTRODUCTION In humans, the role of hair cannot be underestimated. Hair has a function such as a protector of sun exposure, thermoregulation, and helps the process of transpiration. [1] In addition, hair (especially in the head) has an important role in the appearance and social interaction in individuals. In fact, for the Romans, hair is a symbol of beauty and intellectual. [2] Although hair loss is not life-threatening, it can have a profound effect on self-confidence and quality of life. [3] In the pharmacological handling of baldness, the Food and Drug Administration (FDA) only allows two drugs: Minoxidil and finasteride. Minoxidil is the first FDA-approved topical product. Minoxidil increases the duration of the anagen phase in the hair follicle. This causes the stimulation and growth of follicles in the resting phase and also hair follicle enlargement. In addition, the minoxidil used topically may also Access this article online Website: jprsolutions.info ISSN: 0975-7619 induce vascular endothelial growth factors resulting in sustained vascularization and increased dermal papilla size. Minoxidil also stimulates the production of prostaglandins in dermal papilla. [4] Finasteride is used orally with baldness due to androgen hormone (androgenic type) at a dose of 1 mg/day, for at least 3 months. Finasteride is a competitive inhibitor of 5-alfa hepatic and tissue reductase. These drugs can cause the conversion of testosterone to dihydrotestosterone (DHT) decrease and cause the serum DHT level to decrease significantly. Decreased serum DHT can improve symptoms caused by the presence of benign prostatic hyperplasia and improve hair growth and reduce hair loss rate. However, finasteride should not be for pregnant women as it will cause defects in the fetus while in men can cause prostate cancer. [5] Some other side effects that arise because of the use of these two synthetic drugs are dermatitis, skin irritation or allergies, itching, and erythema. [6,7] These side effects cause herbal remedies are often an option for overcoming hair loss and stimulate hair growth. Some of the plants used empirically to overcome baldness have been proven to stimulate hair growth, 1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia, 2 Department of Pharmacological and Clinic, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia *Corresponding author: Resmi Mustarichie, Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia. E-mail: resmi.mustarichie@unpad.ac.id Received on: 30-01-2017, Revised on: 26-02-2018, Accepted on: 22-04-2018 1302

including hibiscus flowers (Hibiscus rosa-sinensis Linn.), Munding fern (Angiopteris evecta), pare (Momordica charantia), and dadap (Erythrina variegata). [8-11] Katuk leaf is one of the plants used empirically by Kampung Mak Kemas community, Malaysia, to overcome the problem of baldness and nourish hair. The leaves of katuk are processed by pounding with milk and used topically. [12] This article reports scientific proof of the traditional use of the katuk leaf. MATERIALS AND METHODS Material Plant The material used in the form of leaf katuk (Sauropus androgynus [L.] Merr.) simplicia obtained from Manoko Experimental Garden, Lembang, West Java. Animal Test The test animal used was a local rabbit (local) strain (Oryctolagus cuniculus) weighing 1.5 2 kg. Rabbits obtained from Ranch Rabbit Rajawali Farm, Bandung. Before being used for testing, rabbits were adapted and acclimatized first for 1 week to observe their health. Rabbits were fed a special feed with adequate nutrition. Ethical approval for testing with animals was obtained from Research Ethics Committee, Universitas Padjadjaran, No.04/UN6. KEP/EC/2018. Methods The research method that was used in this research was as follows: 1. Collection of materials and determination of katuk plants 2. Processing of katuk plants 3. Phytochemical screening of leaf and viscous katuk leaf extract, based on modifications of the Farnsworth method [13] 4. The extraction of katuk leaf (S. androgynous (L.) Merr.) used 96% ethanol solvent by maceration method. The maceration process was based on modifications of Mustarichie et al. method [11] 5. Examinations of ethanol extract parameters of katuk leaf (S. androgynus (L.) Merr.) Based on Indonesia Pharmacopoeia IV [14,15] 6. Fractionation of ethanol extract of katuk leaf was done by liquid-liquid extraction method (ECC). Modifications of the Mustarichie et al. method [16] were applied 7. Examination of thin layer chromatography (TLC) profile and the fraction 8. Testing of hair growth stimulation activity of ethanol extract and its fractions on male rabbit test animal using Tanaka et al. [17] 9. Statistical analysis of data with analysis of variance (ANOVA) method. [18,19] RESULTS AND DISCUSSION The Result of Material Collection and Determination of Katuk Plant Simplicia katuk leaf collected from Manoko Experimental Garden, Lembang, West Java. The katuk plants were detected in the Laboratory of Plant Taxonomy, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. The results of determination indicated that the plants used for this study came from the Plantae kingdom, the Magnoliophyta division, the Magnoliopsida class, the Malphigiales order, the Phyllanthaceae family, the genus Sauropus, and the species S. androgynus (L.) Processing of Katuk Plants Dry sorting was carried out on the simplicia to separate simplicia from foreign matter and other impurities left in dry simplicia. After dry sorting, the leaves leaf simplicia was reduced to smaller size. This was done to expand the surface of the simplicia, thereby increasing the contact of the simplicia with the solvent and facilitating the solvent penetrating into the cell. [20] Phytochemical Screening The results of phytochemical screening of simplicia and katuk leaf extract can be seen in Table 1. Susanti et al. [21] claimed that they found ethanol extract 90% of katuk (S. androgynus (L.) Merr.) leaf positively contains alkaloid group compounds, triterpenoids, saponins, tannins, polyphenols, glycosides, and flavonoids. Putranto et al. [22] reported that in their S. androgynus sample contained steroids. [22] These researchers did not explain the origin of the katuk plant they used. Djamil and Zaidan [23] claimed to have found flavonoids in extract methanol from katuk leaves. Bunawan et al. [24] mentioned that different paper reported difference phytochemical screening results. Table 1: Phytochemical screening of simplicia and katuk leaf extract Secondary metabolites Simplicia Ethanol extract Alkaloids Flavonoids + + Tannin Saponins + + Quinone + + Polyphenols + + Monoterpenoids + + Sesquiterpenoids + + Steroids Triterpenoids +: Detected; : Not detected 1303

We could say, the differences in secondary metabolite content could also be affected by differences in plant origin and extraction methods used. Parameter Examination of Katuk Leaf Extract Examination of leaf katuk extract parameters included moisture content test, total ash content, and acid soluble ash content. [14,15] Moisture Content Measurement of moisture content was performed to determine the residual water after the extracting process of extract, so as to avoid the possibility of microbial contamination and mold growth. The water content of leaf extract katuk was done by toluene distillation method. Percentage of the water content of katuk leaf extract obtained was 7.929%. According to BPOM, [15] water content for katuk leaves was not more than 6.3%. Voight [25] stated that a viscous extract was an extract with a moisture content of not more than 30% and in cold conditions could not be poured. The smaller the water content of an extract, the better because it was associated with microbial growth. Determination of Total Ash Content Determination of total ash content of katuk leaf extract was done to give an overview of internal and external mineral content of katuk leaf extract. [26] Based on the requirements for the total ash content limit of katuk leaf extract according to BPOM [15] was not more than 0.37%. The result of the total ash content of katuk leaves was 7.8987% and has exceeded the requirement limit which might be caused by the less optimum factor of dry sorting process and high contamination factor. Determination of Absorbable Ash Content Determination of acid soluble ash content was carried out by permitting the washed ash product dissolved in dilute hydrochloric acid. BPOM [15] stated that the limit of acid soluble ash content for katuk leaf extract was not more than 0.07%. Determination of acid soluble ash content katuk leaf extract that has been done gives a result of 0.4418% and possibly due to less optimum factor dry sorting process and high contamination factor. Fractionation Result The method of liquid-liquid extraction (LLC) used was a modification of the method of Mustarichie et al. [16] The LLC method was a simple, fast, economical, and commonly used as a method of fractionation. [27] The principle of this fractionation method was the difference in the partition coefficient of the two phases of the compound, i.e., the phases of organic compounds and the nonintermingling water phases. [28] When the compound was added to the nonmixed mixture, the compound would be distributed between the solvent mixtures according to its polarity. The solvent used in the LLC was water which was a polar solvent, n-hexane with a 0.014% solubility value in water which makes it a nonpolar solvent, and ethyl acetate with a solubility value of 8.7% in water, so it was a semipolar solvent. [29] The result of fractionation obtained by n-hexane fraction 44.9%, ethyl acetate fraction 7.4%, and water fraction 52.1%. The results of extraction with methanol solvent, which Suprayogi et al. [30] reported that they re found a value of 18.06%, 10.04%, and 71.90%, respectively. This can be caused by the use of different solvents during the extraction process. TLC Result TLC examination on ethanol extraction, n-hexane fraction, ethyl acetate, and water was conducted to know the separation pattern of compound and component of the compound contained in extract and katuk leaf fractions. The TLC pattern examination was performed using silica gel GF254 as a stationary phase and two kinds of the solvent mixture as the mobile phase. Spots on the development of TLC were observed in visible light, ultraviolet light 254 nm and 366 nm, and 10% sulfuric acid removal in methanol. The TLC profile of ethanol extract and katuk leaf fractions with chloroform phase of methanol (9.5: 0.5) can be seen in Table 2. Based on Table 2, the TLC profile results using chloroform phase methanol (9.5: 0.5) showed the presence of seven patches on ethanol extract and ethyl acetate fraction, six spots on the n-hexane fraction, and four spots on the water fraction, which meant that the phase of motion could already attract compounds contained in the water fraction. The extract and fraction TLC results showed the presence of several spots of compounds with the same Rf, which meant the compounds had been separated according to polarity in each fraction. Stimulate Hair Growth Ethanol Extract The test of hair growth stimulator of katuk leaf extract was performed on male rabbit test animals based on a modification of Mustarichie et al. and Tanaka et al. methods. [11,17] The test was carried out with the following steps: Preparation of testing of katuk leaf extract, preparation of test animal. In this study, male rabbits were used as test animals because male rabbits were hormonally more stable than female rabbits. The selected male rabbit was a healthy, unregulated, healthy rabbit aged 4 5 months because it had excellent and perfect physiological function. The study has obtained approval from the Research Ethics Committee to protect the rights and welfare of test animals. Before the test, rabbits acclimatized 1304

and allowed to adapt to the condition of the cage so that the rabbit was not stressed. On the 7 th day of acclimatization, the rabbit s back was shaved clean. The tested solution on the rabbit s back divided into eight parts consisted of Na CMC (negative control), minoxidil 2% (positive control), 5% katuk leaf ethanol extract (test solution 1), ethanol extract of 10% katuk leaf (test solution 2), 15% katuk ethanol leaf extract (test solution 3), 20% katuk leaf ethanol extract (test solution 4), 25% katuk leaf ethanol extract (test solution 5), and untested portion given test solution (normal control). The Na CMC solution as a negative control functioned to know that the base used (Na CMC) in the manufacture of test solution did not give effect to the animal test and did not have the effect of stimulating hair growth. The parameter used in this test was the length of the rabbit s hair. The test solution was given to the rabbit s back at a different location on each animal to reduce the measurement bias. The hair length of each rabbit of each part was averaged. The results of measurement of rabbit hair length on ethanol test of katuk leaf extract can be seen in Table 3. From Table 3, after 18 days of testing, among the test group, katuk leaf extract at 15% concentration showed the highest hair growth result that was 2474 cm, while the ethanol extract test group of Table 2: TLC profile of ethanol extract, n hexane, ethyl acetate, and water fractions katuk leaf with mobile phase chloroform: methanol (9.5: 0.5) Sample Spot Rf Observed with Visible light UV 254 UV 366 Sulfuric acid 10% Ethanol extract 1 0.21 Green Light green Red orange Green 2 0.34 Green Light green Red orange Green 3 0.49 Red orange 4 0.52 Green Light green Red orange Green 5 0.64 Red orange 6 0.76 Green Light green Red orange Green 7 0.87 Purple Red orange n hexane fraction 1 0.21 Green Light green Red orange Green 2 0.34 Green Light green Red orange Green 3 0.51 Green Light green Red orange Dark green 4 0.61 Red orange 5 0.73 Green Light green Red orange Green 6 0.87 Red orange Ethyl acetate fraction 1 0.21 Green Light green Red orange Dark green 2 0.34 Dark green Light green Red orange Dark green 3 0.49 Green Light green Red orange 4 0.56 Dark green Light green Red orange Dark green 5 0.64 Dark green Light green Red orange Dark green 6 0.76 Dark green Light green Red orange Green 7 0.89 Red orange Water fraction 1 0.23 Red orange 2 0.34 Red orange 3 0.51 Green Light green Red orange Green 4 0.61 Red orange UV: Ultraviolet, TLC: Thin layer chromatography Figure 1: Graph of rabbit hair growth on the test of katuk ethanol extract 1305

Table 3: Rabbit hair length on katuk leaf ethanol extract test Days to Rabbit no Rabbit hair length per group (cm) Normal Control ( ) Control (+) 5% extract 10% extract 15% extract 20% extract 25% extract 3 1 0.070 0.208 0.143 0.284 0.378 0.305 0.256 0.219 2 0.300 0.137 0.302 0.348 0.487 0.604 0.486 0.533 3 0.275 0.386 0.381 0.395 0.439 0.494 0.422 0.486 Mean±SD 0.215±0.126 0.243±0.128 0.275±0.121 0.342±0.056 0.434±0.055 0.468±0.151 0.388±0.119 0.413±0.169 6 1 0.108 0.199 0.248 0.275 0.343 0.289 0.276 0.236 2 0.242 0.143 0.358 0.425 0.448 0.666 0.457 0.544 3 0.405 0.411 0.630 0.660 0.574 0.927 0.728 1.005 Mean±SD 0.251±0.149 0.251±0.141 0.412±0.196 0.453±0.194 0.455±0.116 0.627±0.320 0.487±0.228 0.595±0.387 9 1 0.126 0.228 0.293 0.333 0.483 0.258 0.577 0.362 2 0.304 0.333 0.397 0.413 0.684 0.889 0.791 0.888 3 0.555 0.507 0.779 0.929 0.860 1.213 1.032 1.136 Mean±SD 0.328±0.216 0.356±0.141 0.489±0.256 0.559±0.323 0.676±0.188 0.787±0.485 0.800±0.228 0.795±0.395 12 1 0.203 0.140 0.263 0.292 0.548 0.412 0.319 0.474 2 0.315 0.358 0.394 0.571 0.773 1.663 1.355 1.090 3 0.533 0.588 1.393 1.100 1.616 1.852 1.773 1.749 Mean±SD 0.350±0.168 0.362±0.224 0.683±0.618 0.654±0.411 0.979±0.563 1.309±0.783 1.149±0.748 1.104±0.638 15 1 0.244 0.318 0.524 0.451 0.741 0.995 0.849 0.628 2 0.582 0.422 0.945 0.774 1.108 1.828 1.545 1.296 3 0.618 0.786 1.415 1.148 1.739 2.107 1.894 1.573 Mean±SD 0.481±0.206 0.509±0.246 0.961±0.446 0.791±0.349 1.196±0.505 1.643±0.578 1.429±0.532 1.165±0.486 18 1 0.951 0.832 1.159 1.048 1.544 1.698 1.301 1.438 2 0.970 1.194 1.726 1.446 2.089 3.023 2.073 2.693 3 0.962 1.085 2.183 1.672 1.982 2.703 2.374 2.536 Mean±SD 0.961±0.010 1.037±0.186 1.689±0.513 1.388±0.316 1.872±0.289 2.474±0.692 1.916±0.554 2.222±0.684 SD: Standard deviation 1306

katuk leaf at concentration 5% showed the most hair growth results in low, i.e. 1.388 cm. Figure 1 showed that the growth of rabbit hair length at normal control and the negative control was not too fast compared with positive control and test group. The ethanol extract test group leaves 15% katuk concentration has the highest average hair length at 2.474 cm. The ethanol extract of katuk leaves at concentrations of 10%, 15%, 20%, and 25% had shown better hair growth results than negative controls and normal controls. The following test for hair growth stimulant activity at katuk leaf fraction was done at 15% concentration because, at this concentration, ethanol extract concentration of katuk leaf was not too small and had shown good hair growth activity. Figure 2 showing the graph of rabbit hair growth on the test of katuk leaf fractions. Seen on the 18 th day, the length of the rabbit hairs in the three test groups was longer than the positive controls. The water fraction test group showed the best hair growth effect that could be seen from the highest hair length when compared to other test groups that were equal to 1.659 cm. The ethyl acetate fraction test group showed the lowest hair growth compared to the other test group, which was 1.361 cm. Statistical Analysis From the average length of rabbit hair on ethanol extract testing, it was seen an increase in the length of rabbit hair. The average data of rabbit hair length on katuk leaf extract test was then processed statistically. [18] The statistical analysis showed that the average data of rabbit hair lengths were normal and homogeneous (P > 0.05) so that the statistical analysis test was continued with the ANOVA test and further test in the least significant difference (LSD) test. [19] The results of statistical analysis showed no significant difference (P > 0.05) between normal control and negative control, which meant that negative control and normal control had equal hair growth and showed that Na CMC as the test solution carrier had no activity on hair growth. Based on statistical analysis with ANOVA test and LSD advanced test, ethanol extract at 10%, 15%, 20%, and 25% concentrations showed significant difference (P < 0.05) to normal control and negative control, meaning that at those concentrations the ethanol extract katuk leaf has a stimulant activity of hair growth and give effect of hair growth faster. Leaf ethanol extract of katuk leaves at a concentration of 15% had greater concentration and also statistically showed good hair growth stimulating activity when compared with negative control and normal control, so 15% ethanol extract was chosen as the dose for subsequent katuk leaf fraction testing. Similarly, in the statistic test of extract ethanol effect, in testing the activity of katuk leaf fraction in stimulating hair growth, normality test and homogeneity test of the average length of rabbit hair each group showed that average data of rabbit hair lengths were normal and varied homogeneous (P > 0.05). Statistical analysis of the results of katuk leaf fraction testing was done by ANOVA test to see whether there was any significant difference between each test group and continued with LSD advanced test. From the results of this test, it was found that the fractions of n-hexane and ethyl acetate fractions, statistically, did not show any significant difference to positive control (P > 0.05), which meant that the activity of n-hexane and ethyl acetate fractions in stimulating hair growth was equivalent to minoxidil as a positive control. Statistically, however, the water Figure 2: Graph of rabbit hair growth on the test of katuk ethanol extract fractions 1307

fraction showed significant differences (P < 0.05) when compared with positive controls, which meant that the water fraction had better and faster hair growth than positive controls. From the data collected, it was seen that the water fraction shows the longest rabbit hair growth, which was 1.659 cm, compared to positive control and other test groups. Except for hair growth, the study of S. androgynus for health has been widely studied. This includes Khoo et al. [31] stated that S. androgynus contained a fairly high amount of bioactive antioxidants. These antioxidants could prevent inflammation, antimicrobial, and anticancer effects. Hayati et al. [32] studied the use of S. androgynus (L.) Merr in some traditional societies in East Java. By using in-depth interviews with purposive sampling technique, they found that katuk in traditional societies was used as vegetables that enhancing breast milk, relieve fever, cough, food coloring, goats feeding, and for the salvation ritual traditions. Roosdiana et al. [33] studied the potency of ethanolic extract of S. androgynus (L.) Merr leaves as therapeutic herbs of rats (Rattus norvegicus) peptic ulcer model induced by Aspirin. CONCLUSION It was found that ethanol extract katuk (S. androgynus (L.) Merr.) Leaves had activity in stimulating hair growth from 10% w/v ethanol extract concentration and best at 15% w/v concentration. The fraction of n-hexane, ethyl acetate, and water had hair growth stimulant activity. It was found that the leaves fraction of katuk which gave the best activity in stimulating hair growth was water fraction. 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