Hair Growth Stimulants activity from Sterculia urceolata JE Smith Ethanol Extract
Resmi Mustarichie1*, Imam Adi Wicaksono2
1Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty Pharmacy, Universitas Padjadjaran, Indonesia
2Department of Pharmacology, Faculty Pharmacy, Universitas Padjadjaran, Indonesia
*Corresponding Author E-mail: resmi.mustarichie@unpad.ac.id
ABSTRACT:
The Kampung Naga community, Salawu District, Tasikmalaya, West Java uses plants to clean and strengthen hair for a long time. Until now, they have thick and strong hair. This is considered as the effect of the plants they use when shampooing, namely Sterculia urceolata. The objective of this work was to investigate the hair growth stimulants activity from Sterculia urceolata JE Smith’s ethanol extract and its fractions. Leaves of S. urceolata were macerated thrice a week in petroleum ether, then in ethanol. Powdered leaves and ethanolic extract were subjected to phytochemical investigation. The ethanol extract was fractionated by ethyl acetate and n-hexane. Ethanol extract and fractions obtained were tested on male rabbits for 18 days a day twice every morning and evening using a modified Tanaka method. Minoxidil 2% was used as control positive. It was found that the extract concentration of 15% was the best concentration compared to the positive control. All fractions had hair growth stimulant activity, but the fraction that had a statistically significant difference was the water fraction with a concentration of 15%. From the results of this study, it concluded that the ethanol extract of S. urceolata and its fractions showed hair growth stimulant activity in male rabbits and statistically the fraction of water with 15% extract concentration was the best fraction.. It suggest that research on volunteers needs to be done before it can be used in humans.
KEYWORDS: Sterculia urceolata, hair growth, minoxidil, maceration, fractionation.
INTRODUCTION:
In addition to androgenetic alopecia, there are two common hair disorders, namely telogen effluvium, and alopecia areata (hair loss that commonly occurs in childhood and adulthood)3. One of the factors triggering telogen effluvium is stress2.
Alopecia can be experienced by all people, both men, women, and children. The risk of alopecia abnormalities increases with age. The scale of the classifications in clinical studies and the general population that applies in general are Hamilton, Norwood, and Ludwig. In a population study conducted on Caucasian men, the prevalence of alopecia occurred 46-92% of those aged around 20-70 years. The prevalence of alopecia in the Hamilton-Norwood classification is around 40%, while the Ludwig classification that occurs in women is lower but increases after menopause1. The prevalence of androgenetic alopecia is lower in black and Asian men than Caucasian men4.
Some common treatments for hair loss include hair care using a hair mask, hair tonic, shampoo anti-hair fall, cream bath, hair spa, and hair vitamins5. In addition to using various hair cosmetics, it can be handled using pharmaceutical preparations. Only two pharmaceutical preparations have been allowed by the FDA to treat hair loss and baldness, namely Minoxidil and Finasteride. Minoxidil is a pharmaceutical preparation that can stop hair loss and increase hair growth by widening blood vessels so that a lot of blood flows to the scalp6.
Today the society is more inclined to return to the era of herbal medicine, where people use plants that are effective as hair growers, hair fertilizers, and hair enhancers to deal with hair loss. These plants include aloe vera7, Peria leaf8, hibiscus9, candlenut10, celery11, green tea leaves12, and mangkokan leaves (Polyscias scutellaria)11, fern roots munding (Angiopteris evecta) 13.
The Kampung Naga community, Salawu District, Tasikmalaya, West Java also uses plants to clean and strengthen hair long ago. Until now, they seemed to have thick and strong hair. This is thought to be the effect of the plants they use when shampooing. One of the plants that they usually use when shampooing is the hantap leaf (Sterculia urceolata JE Smith)14. However, until now there has been no scientific testing of the activity of stimulating hair growth from leaves. This study reports the testing of the activity of S. urceolata as a stimulator of hair growth.
MATERIALS AND METHODS:
Collection of plant materials:
Leaves of Sterculia urceolata were collected from Salawu Village, Tasikmalaya, West Java in December 2017, the plant was determined at the Taxonomy Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran.
Equipment and chemicals:
The instruments used were macerator, Analytical balance (Mettler Toledo), Water bath (Memmert), Rotary evaporator (IKA® RV 10 Basic), Spektrofotometri UV-Betrachte (Lamag). Chemicals used were ammonia (Merck), amyl alcohol (Merck), alcohol, aluminum chloride, hydrochloric acid (PT Brataco), concentrated sulfuric acid (CV Agung Menara Abdi), iron (III) chloride (FeCl3), ether (Global Pratama Science CV), ethyl acetate (CV Dutaraya), gelatin, potassium hydroxide (PT Brataco), carboxyl methyl cellulose (PT Brataco), chloroform (Merck), minoxidil 2% (Regrou®), n-hexane (CV Dutaraya), Dragendroff reagent (mixture of solution Bi (NO3) 3.H2O in HNO3), Lieberman-Burchard reagent (mixture of anhydrous acetic acid and concentrated sulfuric acid), Mayer reagent (mixture of HgCl2 solution in water and KI in water), magnesium (Mg) powder (CV Agung Menara Abdi), vanillin sulfate, and toluene (PT Brataco). Unless stated otherwise, all chemicals were analytical grades.
Animals:
The experimental animals used were white male rabbits, 3-5 months old, healthy, normal activities, having a body weight of 1.5-2 kg. All rabbits were adapted first before being treated for about two weeks by regulating their environmental conditions so as not to be stressed, still being fed, and given drinks. For two weeks his health was monitored. The number of white male rabbits used in this study was determined using the Federer formula12. Ethical approval no. 06/UN6.KEP/EC/2018 issued for this study by Research Ethic Committee, Universitas Padjadjaran.
Methods:
Extraction:
The extraction method used in this study was maceration. The selection of this method was done to prevent the occurrence of damage to the thermolabile chemical compounds contained in the S. urceolata leaf. This maceration was done by soaking the sample in the macerator then leaving it for 24 hours at room temperature with stirring occasionally. The timing was effective for the process of withdrawal of secondary metabolites in plant cells by solvents. The solvent replacement was carried out during 3x24 hours13,15,16.
Phytochemical investigation:
Phytochemical screening was applied to the powder leaf and crude extract of S. urceolata based on Farnsworth's method17 for secondary metabolites.
General Standard Parameters of Extracts:
Examination of standard extract parameters based on the general standard of extract parameters18. The general standard parameters of extracts carried out included organoleptic extract criteria, extract yield, ash content, and moisture content.
Fractionation of crude ethanolic extract:
The fractionation method of leaves ethanol extract used was liquid-liquid extraction (LLE) using two or more non-mixed solvents. This fraction used ethyl acetate and n-hexane solvents with reference to the Mustarichie et.al 19 method.
Test the activity of stimulating hair growth from the ethanol extract:
This test was based on a modification of the Tanaka et.al 20. method. The test aimed to find effective concentrations that were efficacious as hair growth.
Test of Hair Growth Stimulating Activity from the Ethanol Extract fraction:
This test was similar to a test of ethanol extract. It was based on a modified method of Tanaka et.al20. This test aimed to find the most effective fraction of hair growth.
Data analysis:
Rabbit hair length data obtained from the results of the study per three up to eighteen days were averaged, then processed statistically using the One-way Variant Analysis (ANOVA) and Kruskal-Wallis methods21.
RESULTS:
Table 1. Phytochemical screening results of S. urceolata
S. No |
Chemical constituents |
Folium |
Ethanol extract |
1 |
Alkaloids |
- |
- |
2 |
Polyphenols |
+ |
+ |
3 |
Tannins |
- |
- |
4 |
Flavonoids |
+ |
+ |
5 |
Quinone |
- |
- |
6 |
Saponins |
+ |
- |
7 |
Monoterpenoid and sesquiterpenoid |
+ |
+ |
8 |
Steroids |
- |
- |
9 |
Triterpenoid |
- |
- |
Notes: (+): detected, (-): not detected
Table 2 Characteristics of S. urceolata leaves ethanol extract
Criteria |
Observation result |
Organoleptic |
Crystal-shaped, greenish-brown in color, distinctive smell, and bitter taste |
Extraction yield |
9.3 % w/w |
Ash content |
Total ash content: 5.548 ± 0,260433 %, Acid-insoluble ash level: 0,5913 ± 0,196054 % |
Water content |
10.5 ± 3.05505 % |
Fig. 1 Rabbit hair measurement in S. urceolata ethanol extract
Table 3 Results of Measurement of Rabbit Hair Length on Activity Test of n-Hexane Fraction, Ethyl Acetate Fraction, Water Fraction
Groups |
Animal |
|
|
Days to- |
|
|
|
|
|
3 |
6 |
9 |
12 |
15 |
18 |
Normal control |
Rabbit 1 |
0.0883 |
0.1283 |
0.1617 |
0.2167 |
0.2583 |
0.2717 |
|
Rabbit 2 |
0.2467 |
0.3083 |
0.4100 |
0.5800 |
0.7233 |
0.7380 |
|
Rabbit 3 |
0.0850 |
0.1150 |
0.1383 |
0.1767 |
0.2350 |
0.2650 |
|
Rabbit 4 |
0.0608 |
0.0808 |
0.1133 |
0.1483 |
0.2350 |
0.2800 |
|
Mean |
0.1400 |
0.1838 |
0,2366 |
0.3244 |
0.4055 |
0.4249 |
|
SD |
0.0924 |
0.1079 |
0,1505 |
0.2222 |
0.2754 |
0.2712 |
Negative control |
Rabbit 1 |
0.1250 |
0.1250 |
0.1467 |
0.2350 |
0.2867 |
0.3120 |
|
Rabbit 2 |
0.2433 |
0.2950 |
0.4400 |
0.6133 |
0.7267 |
0.7617 |
|
Rabbit 3 |
0.0933 |
0.1300 |
0.1408 |
0.1717 |
0.2317 |
0.3660 |
|
Rabbit 4 |
0.0700 |
0.0975 |
0.1633 |
0.2067 |
0.2733 |
0.3075 |
|
Mean |
0.1538 |
0.1833 |
0.2425 |
0.3400 |
0.4150 |
0.3120 |
|
SD |
0.0790 |
0.0967 |
0.1710 |
0.2387 |
0.2713 |
0.7617 |
Positive |
Rabbit 1 |
0.1333 |
0.2050 |
0.2475 |
0.3550 |
0.533 |
0.8267 |
control |
Rabbit 2 |
0.2850 |
0.4483 |
0.6017 |
0.7917 |
0.9500 |
1.4550 |
|
Rabbit 3 |
0,1217 |
0.2300 |
0.2942 |
0.4200 |
0.6067 |
0.8733 |
|
Rabbit 4 |
0.0800 |
0.1117 |
0.1633 |
0.2733 |
0.4250 |
0.6350 |
|
Mean |
0.1550 |
0.2487 |
0.3266 |
0.4600 |
0.6286 |
0.9475 |
|
SD |
0.0896 |
0.1424 |
0.1911 |
0.2291 |
0.2268 |
0.3537 |
n-hexane fraction 15% |
Rabbit 1 |
0.1758 |
0.1883 |
0.1933 |
0.2383 |
0.4000 |
0.7700 |
|
Rabbit 2 |
0.3167 |
0.4517 |
0.7617 |
0.8200 |
1.0467 |
1.5500 |
|
Rabbit 3 |
0.0883 |
0.1017 |
0.1450 |
0.2000 |
0.5283 |
0.7533 |
|
Rabbit 4 |
0.1000 |
0.1225 |
0.1617 |
0.2350 |
0.4050 |
0.7180 |
|
Mean |
0.1936 |
0.2472 |
0.3667 |
0.4194 |
0.6583 |
1.0244 |
|
SD |
0.1152 |
0.1822 |
0.3429 |
0.3474 |
0.3423 |
0.4552 |
Ethyl acetate fraction 15% |
Rabbit 1 |
0.1083 |
0.1133 |
0.1483 |
0.2617 |
0.4267 |
0.7433 |
|
Rabbit 2 |
0.2450 |
0.3100 |
0.4283 |
0.7417 |
1.0817 |
1.4050 |
|
Rabbit 3 |
0.0750 |
0.1192 |
0.1392 |
0.4917 |
0.4917 |
0.6683 |
|
Rabbit 4 |
0.0667 |
0.1367 |
0.1750 |
0.4017 |
0.4017 |
0.7040 |
|
Mean |
0.1427 |
0.1808 |
0.2386 |
0.4100 |
0.6667 |
0.9389 |
|
SD |
0.0900 |
0.1119 |
0.1643 |
0.2877 |
0.3608 |
0.4054 |
Water fraction 15% |
Rabbit 1 |
0.1983 |
0.2100 |
0,2317 |
0.2667 |
0.4933 |
0.8500 |
|
Rabbit 2 |
0.3017 |
0.3983 |
0.7367 |
0.8600 |
12.417 |
1.617 |
|
Rabbit 3 |
0.1383 |
0.1450 |
0.2117 |
0.3133 |
0,5817 |
0,900 |
|
Rabbit 4 |
0.0933 |
0.1283 |
0.1783 |
0.2733 |
0,7420 |
0,7423 |
|
Mean |
0.2127 |
0.2511 |
0.3933 |
0.4800 |
0.7722 |
1.1206 |
|
SD |
0.0826 |
0.1315 |
0.2975 |
0.3213 |
0,4089 |
0.4261 |
Fig. 2 Results of measurement of rabbit hair on testing of n-hexane, ethyl acetate, and water fractions
DISCUSSION:
Plant Leaves Process:
The obtained leaves were chopped and washed with running water to clean, this process was called wet sorting which aimed to be free of dirt such as dust and soil. Sorting was also done to separate leaves that were moldy, rotten, or which were not suitable (eg too young or too old). Then the leaves were dried without sunlight for about one week. Drying of sample materials aimed to reduce water content and stop enzymatic reactions so that the sample was not easily damaged and prolongs storage time. Dried leaves that had been dried were sorted for preventing contamination, separating unused parts in examiners, as well as to get the best quality sample chopped up. This was done to increase the surface area of the extraction process so that the penetration of the solvent was spread evenly to all parts of the sample so that the withdrawal of secondary metabolites was more optimal16.
Extraction:
The initial step to get secondary metabolites from a part of the plant was extraction. The extraction method used in this study was maceration. The selection of this method was done to prevent the occurrence of damage to the thermolabile chemical compounds contained in the leaf This maceration was done by soaking the sample in the macerator then leaving it for 24hours at room temperature with occasional stirring. The timing was effective for the process of withdrawal of secondary metabolites in plant cells by solvents16,22. 96% ethanol replacement is carried out for 3x24 hours. The choice of this solvent was because ethanol was a safe and non-toxic universal solvent, which could dissolve and attract almost all secondary metabolites in leaves with broad polarity ranging from nonpolar to polar. According to Harborne23, ethanol could also prevent the occurrence of hydrolysis and oxidation in the extraction process by depositing proteins and inhibiting the work of enzymes contained in leaf leaves. Macerates obtained were concentrated using a rotary evaporator at a speed of 75rpm and a temperature of 60°C to minimize the risk of compound damage (secondary metabolites) by heating. This concentration was done by evaporating the solvent based on the boiling point of the solvent. This Ethanol 96% has a boiling point of 78.29°C. Evaporation of the solvent produces a thick extract which was then concentrated again on a water bath at 60°C to get a thicker extract.
Phytochemical screening:
Phytochemical screening consisting of seven test groups of compounds, namely testing of compounds of alkaloid groups, tannins and polyphenols, flavonoids, monoterpenoids and sesquiter penoids, steroids and terpenoids, quinones, and saponins. The method used was based on the Farnsworth method17. Phytochemical screening was carried out on the S. urceolata folium and its ethanol extract to find out the qualitative content of the chemical compounds. The results can be seen in Table 1.
From other species of Sterculiaceae, Mari et.al reported that they found tannins, alkaloids, and steroids from powder bark and ethanol extract of S.foetida24. Phytochemical screening of the powdered material of S.tragacantha showed that alkaloid, flavonoid and reducing sugar were present while tannin, cardiac glycosides, saponins, and anthraquinones were absent25. Polyphenols and Flavonoids were detected in the leaves of S.setigera26. In both ethanolic extract of S. urens and S. villosa was the absence of saponins27. The bark of S.urceolata contained flavonoid, anthraquinone, saponin, cardenolide, and tannin while the presence of saponin and triterpenoid in the acetone extract of S. comosa bark were mentioned by Triani et.al28.
General Standard Parameters of Extracts:
Examination of general standard parameters carried out on thick leaves of hantap extract aims to ensure extract quality as well as a guarantee of quality stability so that it can be developed as pharmaceutical products in general18. The results of the examination show that the ethanol extract of the leaves of S. urceolata has a characteristic as shown in Table 2.
According to WHO29, the required limit for ash content was not more than 1% while the total ash content obtained exceeds the requirements limit and acid insoluble ash content met the requirements. The water content in the thick extract obtained met the requirements of no more than 30%30. The water content in the extract was related to the purity of the extract, the less water content in the extract, the smaller the possibility of extract contaminated with microbes or fungi31.
Test Results for Stimulating Hair Growth in Ethanol Extract of S. urceolata leaves:
Testing of hair growth stimulant activity on ethanol extract of S. urceolata leaves was carried out on male rabbits using modified Tanaka method by shaving off the back hair of the rabbit. The ethanol extract used was extract with a concentration of 5%, 10%, 15%, 20%, 25%. The reason male rabbits were chosen as test animals because male rabbits had a more stable hormonal system compared to female rabbits that were easily affected by psychological factors from the menstrual cycle, pregnancy, and breastfeeding. In addition, the selected rabbit ought to be an adult rabbit with an age of 3-5 months, healthy without a disability, and weighing around 1.5-2 kg. Adult rabbits had good and perfect physiological functions that were expected to not interfere with the research process. Rabbits used should be healthy without defects because it was feared that there were physiological defects affecting the results of this hair growth study. The use of test animals in research must be accompanied by ethical agreements[32] to protect the welfare and human rights of test animals so that researchers do not treat test animals arbitrarily.
The prepared preparations were immediately tested on three male rabbits whose backs were shaved and divided into 8 plots consisting of normal controls, negative controls (NaCMC 0.5%), positive controls (Minoxidil 2%), extract, extract of 5, 10, 15, 20, and 25%. Normal control functions as a comparison to determine differences in hair growth in plots that were given normal control and treatment (not treated) and to know the effect (activity) of preparations on hair growth. In the negative control plot, 0.5% NaCMC was applied as the basis of the extract preparation. This negative control was used as a comparison to prove that NaCMC had no activity to stimulate hair growth during testing. Positive controls used were synthetic drugs that had been circulating in the market, namely 2% Minoxidil. This drug was chosen because it was an FDA-approved hair growth drug and has been shown to had hair growth activities and its use topically.
Test parameters measured in testing the activity of hair growth stimulant of ethanol extract of leaf leaves, namely rabbit hair length which grows every three days for 18 days, measured using a caliper with an accuracy of 0.05 mm. The measurement of rabbit hair length was carried out on six strands of hair per plot, the more hair has taken the more representative results of hair growth measurements per plot in three rabbits7. The measurement results can be seen in Fig. 1.
Analysis with statistical methods for ethanol extract with the following stages:
1 In the Normality test, conclusions were obtained: Ho was accepted, so the data on the average rabbit hair length of each group was normally distributed.
2 In the Homogeneity test, the results of statistical analysis of normality and homogeneity of the data obtained showed a significant value> 0.05 so that the samples were declared normal and homogeneous so AVONA tests could be carried out.
3 In the ANOVA test, statistically, significantly different results were obtained starting with 15% extract.
Test Results for Hair Growth Activity of, n-Hexane, Ethyl Acetate, and Water Fractions in Male Rabbits with Tanaka Method:
Testing the activity of hair growth stimulator on n-hexane, ethyl acetate fraction, and water fractions of S. urceolata leaf carried out on male rabbits using the modification of Tanaka method by shaving off the back hair of the rabbit. Each fraction with a concentration of 15% was tested on four male rabbits that had been shaved back and divided into six parts. Test parameters measured in the testing of hair growth stimulant activity of n-hexane, ethyl acetate, and water fractions, which was rabbit hair length which grew every three days for 18 days, measured using a caliper with the accuracy of 0.05 mm. Hair length measurements carried out every three days for 18 days13. This was considered an adequate time for observation and was expected to represent the cycle of hair growth in rabbits. The measurement of rabbit hair length was carried out on six strands of hair per plot, the more hair has taken the more measured results of measurement of hair growth per plot in four rabbits7.
Table 3 and Fig. 2 shows the measurement results of rabbit hair length on the activity test of the n-hexane, ethyl acetate, and water fractions of S. urceolata leaves.
In Table 3 on the third day, it had been seen that there was a difference in hair growth per plot but not yet significant. After 18 days of testing, it was found that the activity of hair growth stimulator in each fraction and hair growth was more effective than the positive control which was shown in rabbit hair length which was smeared leaf fraction was longer than those given positive control. The results of testing the activity of hair growth stimulator n-hexane, ethyl acetate, and water fractions can be seen in Fig. 2
Rabbit hair growth data as a result of testing the activity of n-hexane, ethyl acetate, and water fractions as a stimulator of hair growth were analyzed using the Kruskal-Wallis method (One-way Nonparametric ANOVA) because fraction testing data were not normally distributed with a significance value <0, 05 so that it includes nonparametric data. Then the data were statistically tested using IBM SPSS Statistic Version 22.0 Software with the Kruskal-Wallis method, obtained significant results on the 18th day. Because the test results were significant so it was necessary to do further testing with the Mann-Whitney method, obtained significant results on the n-hexane fraction and water fraction, but from these results, the best fraction for hair growth was the water fraction.
CONCLUSION:
The present study provides enough information regarding Sterculia urceolata ethanol extract. Characteristics of its ethanol extract fulfill requirements of the general standard of extract parameters. The results of phytochemical screening can complement existing scientific data from family Sterculiaceae. 15% Water fraction of its ethanol extract was the best fraction for hair growth. The study suggest that research on volunteers needs to be done before it can be used in humans
ACKNOWLEDGMENT:
We are deeply grateful to the ALG (Academic Leadership Grant) – Universitas Padjadjaran for giving us the opportunity and funding this work. We thanks to Reni Hernawati for the technical support.
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Received on 17.04.2019 Modified on 21.05.2019
Accepted on 29.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2019; 12(9):4111-4116.
DOI: 10.5958/0974-360X.2019.00709.1