INTRODUCTION:

The role of hair in humans is something very valuable. In addition to functioning as a protective exposure to sunlight, the hair also functions as thermoregulation and helps the transpiration process¹. In addition, hair (especially in the head) has an important role in social status, It is even believed that for the Romans, hair is a symbol of beauty and intellectual². Hair can cause and give a profound effect on confidence and quality of life³. The Food and Drug Administration (FDA) only allows two drugs, minoxidil, and finasteride for baldness handling. Minoxidil is the first FDA-approved topical product. It causes the stimulation and growth of follicles in the resting phase and also hair follicle enlargement.

Furthermore, it may also induce vascular endothelial growth factors resulting in sustained vascularization and increased dermal papilla size. Minoxidil also stimulates the production of prostaglandins in dermal papilla⁴. Finasteride is used orally with baldness due to androgen hormone (androgenic type). These two drugs have some side effects. Finasteride should not be for pregnant women as it will cause defects in the fetus while in men can cause prostate cancer⁵. The Other side effectsa include dermatitis, skin irritation or allergies, itching, and erythema^6,7. To overcome these side effects, herbal remedies are often an option to the hair loss and stimulate hair growth.

Some of the plants used empirically to overcome baldness have been proven to stimulate hair growth, including hibiscus flowers (Hibiscus rosa sinensis Linn.), Munding fern (Angiopteris evecta), pare (Momordica charantia), and dadap (Erythrina variegata)^8-11.

The Wera flower (Malvaviscus arboreus Cav) has usefulness and efficacy to treat various diseases such as to cure mucus and blood cough, cure mumps (parotitis)¹². As for the leaves of Wera, the community of Kampung Naga, Salawu District, Tasikmalaya, West Java as one of the efforts of herbal medicine to promote hair growth is done hereditary¹³. This article reports a scientific proof of the traditional use of the Wera leaf for alopecia treatment.

MATERIALS AND METHODS:

Material Plant:

The materials used in this study include Wera (Malvaviscus arboreus Cav.) leaves obtained from Manoko Experimental Garden, Bandung, West Java

Animal Test:

The test animal used was local rabbit males aged 3-5 months with a weight of 1.5-2 kg. The number of test animals used was determined using the formula of Federer: (n-1) (t-1) ≥ 15, where t was the number of treatments whereas n was the number of animals per treatment ¹⁴. According to the calculation of Federer's formula, the number of animals required was 7 male rabbits. It took three rabbits for testing ethanol extract and four rabbits for testing fractions (n-hexane, ethyl acetate, and water). Ethical approval for testing with animals was obtained from Research Ethics Committee, Universitas Padjadjaran No.03 / UN6.KEP / EC / 2018.

METHODS:

The research method included material collection, plant determination, simplicia phytochemical screening based on modifications of the Farnsworth method ¹⁵, making Wera leaf extract based on modifications of Mustarichie et.al method ¹⁶, phytochemical screening extract, extract parameter examination based on Indonesian Herbal Pharmacopeaia ¹⁷and BPOM RI ¹⁸, testing the stimulating activity of hair growth ethanol extract in rabbits based on modification of Tanaka et.al method ¹⁹, making n-hexane fraction; ethyl acetate fraction; and water fraction based on Mustarichie et.al method ¹⁶ testing fraction hair growth activity, fraction phytochemical screening, determination of thin layer extract and fraction chromatography patterns, and statistical data analysis ^{20, 21}.

RESULTS:

Leaf Collection and determination:

Simplicia of wera leaves was obtained from Manoko Experimental Garden, Lembang, West Java.

Maceration:

The Wera extract was made by maceration yielded of 11,6 %w/w viscous extract.

Phytochemical screening:

The results of phytochemical screening of simplicia and Wera leaf extract can be seen in Table 1.

Table 1 Phytochemical screening of Simplicia and Wera leaves extract

Secondary metabolites
Secondary metabolites	Simplicia	Ethanol Extract
Alkaloids	-	-
Flavonoids	+	+
Tannin	+	-
Saponins	+	+
Quinone	-	-
Polyphenols	+	+
Monoterpenoids	+	+
Sesquiterpenoids	+	+
Steroids	+	+
Triterpenoids	-	-

Notes: +: detected; -: not detected

Parameter Examination of Wera Leaf Extract:

Table 2. The result of observation of organoleptic of Wera Leaf Ethanol Extract criteria

Organoleptic criteria	Observation
Shape	Viscous
Color	Blackish green
Smell	Typical
Taste	No taste

Results of preparation of n-hexane fraction, Ethyl Acetate fraction, and water fraction of Wera leaf ethanol extract:

The yield of n-hexane, ethyl acetate, and water fractions from 100 g of viscous ethanol extract of M.arboreus were 30.68, 4.64, and 58.12 % w/w of n-hexane, ethyl acetate and water fractions, respectively.

Test result activity stimulation hair growth ethanol extract Wera leaf on male rabbit by Tanaka method:

Table 3 Rabbit Hair Measurements in cm of the Test Activity of Wera Leaf Ethanol Extract

Test group		Days to-
Test group		3	6	9	12	15	18
Control Normal	Rabbit 1	0.5975	0.3033	0.4600	0.4633	0.6233	0.7833
	Rabbit 2	0.2750	0.2558	0.2275	0.2850	0.2950	0.3150
	Rabbit 3	0.2175	0.2367	0.3492	0.7508	0.8633	0.9758
	Means	0.3633	0.2653	0.3456	0.4997	0.5939	0.6914
	SD	0.2048	0.0343	0.1163	0.2350	0.2853	0.3399
Negative control	Rabbit 1	0.5583	0.4242	0.4600	0.4958	0.5316	0.5316
	Rabbit 2	0.3142	0.2792	0.2717	0.3342	0.4175	0.8542
	Rabbit 3	0.1775	0.2683	0.3467	0.7658	1.1858	1.6058
	Means	0.3500	0.3239	0.3594	0.5319	0.7116	0.9972
	SD	0.1929	0.0870	0.0948	0.2181	0.4146	0.5512
Extract 5%	Rabbit 1	0.6108	0.7692	0.4617	1.0008	1.8017	2.5508
	Rabbit 2	0.4575	0.3075	0.2717	0.3633	0.4508	1.2142
	Rabbit 3	0.2558	0.2133	0.5142	0.7842	1.7400	2.3850
	Means	0.4414	0.4300	0.4158	0.7161	1.3308	2.0500
	SD	0.1780	0.2975	0.1276	0.3242	0.7627	0.7286
Extract 10%	Rabbit 1	0.6075	0.9767	0.6892	0.9008	1.7758	2.5900
	Rabbit 2	0.2958	0.3317	0.2908	0.3308	0.4433	1.4167
	Rabbit 3	0.2258	0.2400	0.4333	0.9067	1.6550	2.8242
	Means	0.3764	0.5161	0.4711	0.7128	1.2914	2.2769
	SD	0.2032	0.4015	0.2018	0.3308	0.7369	0.7542
Extract 15%	Rabbit 1	0.4992	0.6258	0.5318	0.8708	1.8325	2.6983
	Rabbit 2	0.3558	0.2608	0.3550	0.4283	0.4092	1.7767
	Rabbit 3	0.2092	0.1375	0.4617	0.8975	1.6892	2.7317
	Means	0.3547	0.3414	0.4495	0.7322	1.3103	2.4022
	SD	0.1450	0.2539	0.0890	0.2635	0.7837	0.5420
Extract 20%	Rabbit 1	0.6775	0.4358	0.4075	0.9300	1.8367	2.7133
	Rabbit 2	0.3350	0.4075	0.4008	0.3325	0.5933	1.2667
	Rabbit 3	0.2033	0.3792	0.5158	0.9192	1.8667	2.7967
	Means	0.4053	0.4075	0.4414	0.7272	1.4322	2.2589
	SD	0.2448	0.0283	0.0646	0.3419	0.7267	0.8603

Fig. 1 Testing hair growth on the rabbit's back

Fig. 2 Hair Measurements in cm versus time of the Test Activity of Wera Leaf Ethanol Extract

Fig. 3 Hair Measurements in cm versus time of the Test Activity of Wera Leaf water, n-hexane, and ethyl acetate fractions

Table 4 ANOVA test result of Wera leaf ethanol extract

		Sum of Squares	df	Mean Square	F	Sig.
HariKe18	Between Groups	9.715	7	1.388	2.748	.045
	Within Groups	8.079	16	.505
	Total	17.794	23

Table 4.23. Test Continued Positive Control with Normal Control

	3^rd day	6^th day	9^th day	12^th day	15^th day	18^th day
Mann-Whitney U	6.000	2.000	.000	2.000	1.000	1.000
Wilcoxon W	16.000	12.000	10.000	12.000	11.000	11.000
Z	-.577	-1.732	-2.309	-1.732	-2.021	-2.021
Asymp. Sig. (2-tailed)	.564	.083	.021	.083	.043	.043
Exact Sig. [2*(1-tailed Sig.)]	.686^b	.114^b	.029^b	.114^b	.057^b	.057^b

DISCUSSIONS:

Leaf Collection and determination:

To ensure the identity of the plants carried out, the determination of Wera plants at the Herbarium Laboratory, the School of Biological Technology, Bandung Institute of Technology, Bandung. The result of the determination showed that the plants used to belong to the division of Magnoliophyta, class Magnoliopsida (Dicots), Malvales, and family Malvaceae with the name Malvaviscus arboreus Cav.

Wera Leaf Processing Results:

Simplified Wera leaves that had been collected and dried, sorted to separate the other undesirable parts of the plant and other impurities left behind in dried simplicia²². Simplicia was further reduced in size by using scissors or blenders to increase the surface area of the simplicia so as to facilitate the extraction process because the more surface area allows the solvent to reach the cell^23,24. The small particle size of simplicia expands the contact of the simplicia with the solvent, resulting in more secondary metabolites being attracted ²⁵.

Maceration:

The process of Wera leaf extraction was done by maceration method using 96% ethanol. In the maceration process, the solvent flowing into the cell could cause the protoplasm to swell and the cell content would dissolve in accordance with its solubility. Immersion in ethanol was carried out 3 x 24 hours where the solvent was replaced every 24 hours. Immersion of simplicia results in differences in pressure outside and inside the cell that caused the breaking of walls and cell membranes. Secondary metabolites in the cytoplasm would be attracted to the organic solvent used Ethanol was selected as a solvent in the extraction process because it had high solubility, most of the secondary metabolites were insoluble, not toxic, and were inert so they do not interfere with other components. The use of ethanol as a solvent could prevent the growth of molds and bacteria in the extract, so as to minimize the occurrence of contamination in the extract. The low ethanol boiling point facilitated the evaporation process in the process of extracting extracts and extract contact with less heat of^27,28.

Phytochemical screening:

Seen in Table 1, that alkaloids, quinone, and triterpenoids were not detected either in simplicia or in the ethanol extract of Wera leaf. The results of the inspection were consistent with the compounds reported by Chooi ²⁹ that the Wera leaf contains the tannin compound. Kaisoon et.al³⁰ and Cahiuch et.al³¹ mentioned some phenolic acid compounds and eugenol compounds belonging to the class of polyphenols contained in Wera leaves, and Rakhmani et.al³² reported saponins were also present in this plant. These differences might be due to the origin of the plant being examined. Negative results of tannins in Table 1 could be caused by the number of tannin compounds that were not much and the nature of tannin was more soluble in water solvent than ethanol so that the tannin extract could not be detected.

Parameter Examination of Wera Leaf Extract: Examination of Wera leaf extract parameters performed included organoleptic examination, the yield of extract, ash content, moisture content, and thin layer chromatography extract pattern. The examination was based on Government Standard Parameter³³. Regarding ash content, it aimed to give a good mineral description derived from the initial process until the formation of extract from both inside and outside contained in the extract. There were two kinds of mineral salts in a material that is organic salts, such as salts of oxalic acid, acetate, malate, etc. and inorganic salts, such as phosphates, chlorides, phosphates, alkali metals, and nitrate sulfates. The result of the total ash content of Wera leaf extracts 4,32% while the ash content of Wera leaf acid 0,4983%. The low acid soluble ash content indicates a low impurity content such as sand and silicate.

Determination of moisture content was done by distillation method by using a solvent type which could not mix with water (immiscible). The solvent used was toluene, this solvent had a boiling point higher than that of 100.6 °C and a lower specific gravity of 0.866. The smaller the water content in the extract would be less likely to be contaminated by bacteria and fungi. The results of calculation of Wera leaf content showed the result of 8.33%. These levels meet the literature requirements for viscous extracts of 5-30%³⁴.

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 fractionation³⁵. The principle of this fractionation method was the difference in the partition coefficient of the two phases of the compound, ie the phases of organic compounds and the non-intermingling water phase³⁶. When the compound was added to the non-mixed mixture, the compound would be distributed between the solvent mixture 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 non-polar solvent, and ethyl acetate with a solubility value of 8.7% in water so it was a semipolar solvent³⁷.

Stimulate hair growth ethanol extract and its fractions:

The test of hair growth stimulator of Tanaka et.al¹⁹ and Mustarichie et.al methods^10,11. The test was carried out with the following steps: Preparation of testing of Wera 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 rabbits used are ensured in a healthy state and enter in an age sufficient for testing so as not to interfere during the process of research takes place. The use of rabbits as test animals in this study should take into account the rights and welfare of test animals and animals treated animal ally so that this research should be accompanied by the objectives of the Ethics Committee.

On the back of the shaved rabbit was divided into 8 areas for different treatments: normal control, negative control (1% Na-CMC), positive control (Minoxidil 2%), 5%, 10%, 15%, 20%, and 25% extracts (Fig.1). A normal control area was included to see the difference in hair growth between the treated area and the untreated area and to see the effect of the preparation used in the study. Na-CMC was used as a dosage base for extracts. Na CMC which has a higher viscosity level than water, so the extract would be able to stick and contact longer after applying. The negative control area treated only with NaCM solution aimed to find the base used to have an effect when compared to the normal control of hair growth. Minoxidil used as a positive dose preparation in the study was a topical preparation that has been FDA approved as a remedy for hair loss. Minoxidil increases the amount of intracellular Ca²⁺, which had been shown to regulate enzyme adenosine triphosphate (ATP) enzymes that played a role in differentiation processes that facilitate hair growth³⁸. The test lasts for 18 days and once every three days a hair length was measured as six strands from each treatment area of each test rabbit. Hair length measurements can be seen in Table 3.

On the 12^th-day hair growth in the extracting area of 25% extract showed a difference compared to other treatment areas. In the growth of hair on the 15^th day already seen growth experienced extracts had differences with normal control and negative control. On the 18th day showed that the highest growth was seen in the 15% extract which had hair growth almost equal to the positive control. Because the best growth was shown in the extract of 15%, so this concentration was used for testing the fraction of the leaves of Wera to find out which fraction had the most optimal activity as a stimulator of hair growth. The test results of hair growth of ethanol extract can be seen in Figure 2 whereas the results of the effects of fractions can be seen in Fig. 3. Based on the data in Fig. 3, it could be seen the difference in hair growth on day 9 with growth was best aimed at fraction of water. In the 18th day, growth was best demonstrated by the n-hexane fraction and had better growth activity compared with positive control hair growth (Minoxidil). To verify all the data statistical analysis was then carried out.

Statistical analysis:

The statistical method used to see the difference in hair growth data between groups was ANOVA (Analysis of Variance). Statistical analysis was performed with the help of IBM SPSS Statistic 24.0 software^20,21,39. To use this method, the data used must be normally distributed and the data was homogeneous. Data could be said to be normally distributed and homogeneous if the value of significance> 0.05. Hair growth data gave a significance value <0.05 which indicated there was a significant difference in the test group (see Table 4). The LSD (Least Significant Difference) method showed the smallest significance was the 15% extract which had the same significance as the positive control (Minoxidil).

The statistical results of the normality and homogeneity test of hair growth data on fractions test showed not normally distributed and homogeneous because of significance value <0,05, so could not use the ANOVA method. Kruskall Wallis non-parametric method is used to see differences between groups with significance values <0.05. On the 9th day and the 18th day, there is a noticeable difference in the fractional test data, but data on day 9 can not be used to identify the fraction activity of Wera leaves as a hair growth stimulator. In the advanced test the Mann Withney method showed that the water fraction and n-hexane fraction showed significant differences on day 15, but on the 18th day only the n-hexane fraction had a significance value <0.05 and showed similar growth with the positive control (see Table 5).

CONCLUSION:

It was found ethanol extract, n-hexane fraction, ethyl acetate fraction and water fraction of Wera leaf (Malvaviscus arboreus Cav.) had hair growth stimulant activity. The best extract of ethanol was seen in extracts with 15% concentration and the Wera n-hexane fraction was the most optimum hair growth fraction. Overall results suggest that the M.arboreus has an anti-alopecia characteristic. It is recommended that there is further research in the form of structural elucidation to know the chemical content responsible for this hair growth activity.

ACKNOWLEDGMENT:

We thank ALG (Academic Leadership Grant) Universitas Padjadjaran to support and finance this project.

CONFLICT OF INTEREST:

There is no conflict of interest among authors

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Received on 30.07.2018 Modified on 10.09.2018

Research J. Pharm. and Tech 2018; 11(11): 5066-5072.

DOI: 10.5958/0974-360X.2018.00924.1