INTRODUCTION:

Herbal medicine is still considered the primary form of health care, primarily in developing countries, which account for approximately 75-80% of the global population. There is a widespread belief that herbal drugs have no harmful side effects, aside from being inexpensive and easily accessible¹.

Hair loss is one of the signs of unhealthy hair. Hair loss can be caused by heredity and hormonal influences, lack of nutrition in the hair, free radicals, drug side effects, stress, and genetics^2,3.

Alopecia can be caused by abnormal hair loss (baldness). Hair loss is influenced by a lack of hair nutrients such as water, protein, vitamins, B vitamins, vitamin E, and iron^4,5.

Saponins, alkaloids, terpenoids, quinones, and flavonoids are found in Capsicum frutescens L. leaves⁶. Saponins and flavonoids play a role in hair growth stimulation⁷. Saponins have the ability to increase blood flow to hair follicles; when blood flow to hair follicles is reduced, the hair follicles suffer and hair loss occurs⁸. While flavonoid compounds' antioxidant effect can promote hair growth and inhibit free radicals, which are one of the causes of hair loss⁹. According to Neelam Gurnani et al., the antioxidant activity of crude extracts of red chilli seeds (Capsicum frutescens L.) is modest when compared to normal ascorbic acid 10. The levels of total phenolic content and total flavonoid content in the same extracts are 7.95-26.15 gallic acid equivalents (GAEmg/g) and 4.64-12.84 rutin equivalents (RU mg/g), respectively, of dry weight of extract¹⁰. Capsicum frutescens is widely recognized for its pungency and nutritional value, as well as being an excellent source of phenolic compounds. Gilvanda et al found that chilli pepper contained a wide range of phenolics, ranging from phenolic acids (benzoic and cinnamic acids) to flavonoids (flavonols and flavan-3-ols)¹¹.

Extract delipidation aims to remove interfering compounds including fat, resin, sugar, fibre, and starch that can cause losses during the formulation process. Crude extracts are needed to be delipidated to minimize interference with pharmacological tests and are aimed at obtaining biopharmaceutical products with high content of active compounds¹². Hair tonic solution product, which is already widely available on the market, is one of the cosmetic products that have been developed to solve the problem of hair loss and baldness¹³. The advantages of hair tonic cosmetics include keeping hair colour from fading, providing hair nutrition, and helping make dry and dull hair shinier¹⁴. Because hair tonic cosmetics are not rinsed, they take longer to soak the scalp and thus reduce the growth of microorganisms that cause hair loss. The hair tonic formula is made up of both basic and active ingredients¹⁵. Hair tonic designed to make scalp application easier. Furthermore, as compared to other semisolid dose hair tonics, it is not oily and leaves no residue on the scalp¹⁶. Based on this description, the researchers intend to test the efficacy of a delipidated ethanol extract of cayenne pepper (Capsicum frutescens L.) formulated in the form of a hair tonic for rabbit hair growth (Oryctolagus cuniculus) using concentration variations of 5%, 10%, and 20%.

METHODS:

Preparation of Capsicum frutescens L. leaves Ethanol Extract:

Leaves of Capsicum frutescens L. were obtained in Bandar Lampung.Leaf samples were washed and washed under running water to remove any remaining materials¹⁷, dried in oven at 60^oC, made into powderand sievedthrough meshwith size of 80¹⁸.The powder was gathered in an airtight jar and stored away from sunlight in a cold, dry spot¹⁹. A total of 500 grams of simplicia was macerated with 96% ethanol for 3 days²⁰, every 24 hours stirring and replacing 96% ethanol with the same amount²¹. The use of ethanol as a solvent may inhibit the growth of fungus and bacteria in the extract, reducing the likelihood of contamination. Furthermore, the secondary metabolites were insoluble, non-toxic, and inert, ethanol was utilized as a solvent in the extraction procedure²². The macerate was filtered using a clean white handkerchief, followed by No. 1 Whatman filter paper²³. The macerate was evaporated using a rotary evaporator at a temperature of 60°C to obtain a thick extract^24,25.

Delipidation of Ethanol Extract of Capsicum frutescens L. Leaves.

A liquid-liquid extraction method with a separating funnel was used in the extract delipidation process. The viscous extract was delipidated by adding n-hexane solvent, which formed two layers, the top layer being n-hexane and the bottom layer being acetone because acetone has a higher density than n-hexane. The purification steps were repeated until the green colour in the n-hexane solvent was removed or the top layer was clear²⁶.

Phytochemical Screeningof Delipidated Ethanol Extractof Capsicum frutescens L. Leaves

a) Flavonoid Test:

A total of 2mL of Delipidated Ethanol Extract was placed in a test tube and heated for 5 minutes. Mg metal of 0.1g and 5 drops of concentrated HCl were added. If a red, yellow, or orange solution formed, the extract contained flavonoids²⁷.

b) Alkaloid Test:

In a test tube, 2mL of delipidated Ethanol Extract was added, followed by 5 drops of Dragendorff's reagent. The solutioncontained alkaloids if it produced an orange or brick-red precipitate^28,29.

c) Tannin Test:

A total of 2mL of delipidated Ethanol Extract was put into a test tube and then 1mL of 1% FeCl3 was added. Positive extract contains tannins if a greenish-brown, blackish green or blue-black colour is formed^30,31.

d) Saponin Test:

A total of 1mL of the extract was placed in a test tube, followed by 1mL of warm aquadest, shaken vigorously vertically for 1 minute, and then a few drops of 2 N HCl were added. If a stable foam is formed, it indicates that the extract contains saponins³².

Hair Tonic formulation from delipidated ethanol extract of Capsicum frutescens L. leaves:

Delipidated ethanol extract of Capsicum frutescens L. leaves was formulated into hair tonic with various concentrations of 5%, 10% and 20% which can be seen in Table 1.

Table 1: Hair Tonic Formula Design of delipidated ethanol extract of Capsicum frutescens L. leaves

Ingredients	Purposes	Concentration (%) (b/b)
Ingredients	Purposes	F I	F II	F III
Extract	Active Ingredient	5	10	20
Glycerol	Cosolvent	2.5	2.5	2.5
Propylene glycol	Humectants	7.5	7.5	7.5
Methyl-paraben	Preservative	0.009	0.009	0.009
Ethanol 96%	Solvent	20	20	20
Menthol	Cooling sensation	0.05	0.05	0.05
BHA	Antioxidant	0.005	0.005	0.005
Aquadest	Solvent	Add 100	Add 100	Add 100

The formulation of Hair Tonic preparations used the principle of dissolving and stirring without involving heating. Delipidated ethanol extract of Capsicum frutescens L. leaveswas dissolved in propylene glycol, and then stirred until homogeneous (Solution I). Methylparaben was dissolved in glycerol plus BHA (Solution II), and Menthol was dissolved in ethanol (Solution III). Solutions I, II, and III are mixed and added with distilled water up to 50 ml.

Hair Tonic Physical Characterization Test:

Organoleptic test:

The organoleptic test was the initial test. The purpose of this test was to observe the physical qualities of the hair tonic by visual identification using the senses and covering the scent and colour of the hair tonic.

Acidity Test:

The pH measurement was carried out using Jenway pH meter which was calibrated with buffer solutions at pH 4 and 7. The hair tonic was taken as much as 10 ml then the electrode was immersed in the hair tonic for a few minutes to allow the pH to stabilize. The number shown on the pHmeter is the level of acidity of the hair tonic³³.

Homogeneity Test:

Visual inspection was used to assess the homogeneity of all hair tonic compositions³⁴. The hair tonic was dripped onto a cleaned and dried objectglass, which was then covered with another object glass. The presence of any coarse particle was used to assess homogeneity conducted under light.

Hair growth activity test:

This tests were performed on three areas of the rabbits' shaved back, one on each side, right and left, each measuring 2.5 x 2.5cm and separated by 1cm. The depilatory cream (Veet® cream) was then applied for 3-5minutes before the area was rinsed with water until it was free of hair. As an antiseptic, the rabbit's back extract was smeared with 70% ethanol before application. Before any activity tests were performed on the rabbit, it was left for 24 hours. Area 1 was the standard control, as no intervention was performed.Area 2 acted as the negative control, where hair tonic containing no test substance was applied and Delipidated ethanol extract of Capsicum frutescens L. 5%, 10%, and 20% was applied to areas 3, 4, and 5, respectively. Last, hair tonic containing minoxidil 2% as the positive control was applied on area 6. The rabbit was then given 0.1ml of each therapy twice a day for three weeks. The first day of using the hair tonic was labelled day 0³³.

Figure 1: Areas of Hair growth activity test of Hair Tonic of Capsicum frutescens L. Leaves Extracton Male Rabbit (Oryctolagus cuniculus)

RESULT AND DISCUSSION:

Phytochemicalsof Delipidated Ethanol Extractof Capsicum frutescens L. leaves

The bioactive compounds contained in the extract can be identified through phytochemical screening. One of the methods used for the phytochemical screening stage is the tube method which is based on the colour testing reaction using a certain reagent³⁵. Delipidated Ethanol Extract of Capsicum frutescens L. leaves contained flavonoids, alkaloids, tannins, and saponins (Table 3). The content of phytochemical compounds contained in plant extracts can be influenced by the level of polarity of the solvent which will affect the bioactive activity of the plant extract³⁶.

Table 2. Results of Phytochemical Screening of Delipidated Ethanol Extract of Capsicum frutescens L. Leaves

Phytochemicals Identification	Reaction	Presence
Flavonoid	Yellowish Orange	+
Alkaloid	Brick red precipitate	+
Tanin	Blackish green	+
Saponin	Foam	+

Hair Tonic Physical Characterization:

Physical Characterization for organoleptic evaluation properties of the hair tonic by the addition of 5%, 10%, and 20% Delipidated Ethanol Extract of Capsicum frutescens L. leaves produced homogeneous results. However, there was a colour difference between each hair tonic. Formula I (Extract 5%) is light brown, Formula II (Extract 10%) is brown, and Formula III (Extract 20%) is dark brown. The browner it was, the higher the concentration of extract used. This was influenced by the colour of the ethanol extract (Figure. 1).

Figure 2: Hair Tonic of Capsicum frutescens L. Leaves Extract

The homogeneity test revealed that all hair tonics were homogeneous and could be easily dropped and spread on the skin. The pH values of the formulas FI 5%, FII 10%, and F III 20% were 6, 5, 6, 2, and 5, 9, respectively. This acidity is within the normal pH range for human skin, which is 4.5 to 6.5/pH balance³⁷. A topical product with a pH value less than 4.5 will cause skin irritation, whereas a pH value greater than 6.5 or too alkaline will cause dry skin³⁸.

Table 3: Physical Characterization of Hair Tonic

Hair Tonic Formula	Colour	Smell	Homogeneity	pH
F I	Light Brown	Distinctive	Homogenous	4.5
F II	Brown	Distinctive	Homogenous	5.2
F III	Dark brown	Distinctive	Homogenous	5.9

The pH of the preparation increased after the addition of Delipidated Ethanol Extract from Capsicum frutescens L. leaves. The concentration of the added extract determines the pH of the Hair Tonic preparation. As a result of the alkaloid content extracted in the solvent, the extract has an alkaline tendency. Because of the presence of the -NH group, alkaloid compounds are alkaline³⁹.

Hair growth activity:

Table 4 showed that the normal control performed very similarly to the negative control in the first week, while F I was 5%, F II 10%, F III 20%, and minoxidil did not. There was, however, a significant difference in hair length between the normal control and the negative control, as well as hairs in areas treated with F I 5%, F II 10%, F III 20%, and minoxidil. When the normal control and negative control, as well as the F I 5%, F II 10%, and F III 20%, were compared in the second week, no significant difference was found. When hair growth in areas treated with K10% and minoxidil was compared, there was no significant difference.

Table 4: Average Hair Growth of Male Rabbits (Oryctolagus cuniculus) for 3 weeks

Treatment	Average Lenght (mm)
Treatment	7^th day	14^th day	21^stday
Normal control	2.23 ± 0.02	5.21 ± 0.17	6.88 ± 0.50
Negative control	2.70 ± 0.58	6.08 ± 0.55	7.59 ± 0.94
Positive control	12.60 ± 0.89	27.53 ± 0.49	34.54 ± 0.93
F I	5.71 ± 0.63	12.35 ± 0.78	18.28 ± 0.74
F II	7.51 ± 0.57	17.04 ± 0.53	24.53 ± 0.88
F III	11.84 ± 0.54	25.83 ± 0.55	33.33 ± 0.86

Normal control is area I that does not receive treatment.

Negative control is area II that applied hair tonic does not contain extract

Positive control served as areaIII received a hair tonic containing 2% of minoxidil

F I, II, and III are areas IV, V, dan VI that apply hair tonic containing 5%; 10%; and 20% of Extract

All hair Growth promotion values of all treatments from weeks 1 to 3 were analyzed for their statistical normality. The Shapiro-Wilk normality test was used to assess the data's normality; data were considered normally distributed if the p-value was greater than 0.05. The normality test results for all treatments revealed that all data were normally distributed because of p> 0.05 (Table 5). If the data were normally distributed, the process was carried on by comparing the differences between groups.

Table 5: Tests of Normality

Treatment	Statistic	df	Sig.
Normal control	0.63	4	0.051
Negative control	0.877	4	0.328
Positive control	0.906	4	0.462
F I	0.966	4	0.815
F II	0.890	4	0.381
F III	0.979	4	0.895

The homogeneity test is used to determine whether or not several population variants are identical. This test was performed as a prerequisite for the independent sample t-test and ANOVA analysis. If the data group has a normal distribution, a homogeneity test can be performed⁴⁰. The homogeneity test was used to determine whether or not the hair growth data from the different treatment groups had the same variance. In this study, the value used in the homogeneity test was the value of hair growth at week III (day 21).Based on the homogeneity test calculation (Table 6), it is known that the significance value (p-value) for the homogeneity test is > 0.05, indicating that there was no difference in variance between the experimental treatment groups and thus the homogeneity assumption can be met⁴¹.

Table 6. Test of Homogeneity of Hair Growth Promotion

Homogeneity	Levene Statistic	df1	df2	Sig.
Based on Mean	1.438	5	18	0.258
Based on Median	1.251	5	18	0.327
Based on trimmed mean	1.251	5	14.894	0.335

The statistical analysis was continued with the Tukey-HSD test to see if there was a significant difference between each test solution, and the results were shown in Table 7 below.

Table 7: Differences in Treatment Solutions for Hair Growth Promotion

Between Treatment		Sig. Value	Conclusion
Normal control	Negative control	0.309	not significant
	Positive control	0.000	significant
	F I	0.000	significant
	F II	0.000	significant
	F III	0.000	significant
Negative control	Positive control	0.000	significant
	F I	0.000	significant
	F II	0.000	significant
	F III	0.000	significant
Positive control	F I	0.000	significant
	F II	0.000	significant
	F III	0.309	not significant
F I	F II	0.000	significant
F I	F III	0.000	significant

Because the sig value in the Tukey HSD test table was 0.05 (Table 7), the hair tonic containing 20% extract (F III) was not significantly different from the positive control, namely Minoxidil 2%. Similarly, the negative control did not differ significantly from the normal control; the sig value was greater than 0.05. All formulations of hair tonic preparations differed significantly (p 0,05) from the negative and normal controls. This implied that hair tonic preparations containing Delipidated Ethanol Extract of Capsicum frutescens L. Leaves had significantly higher hair growth potential than negative and normal controls, and hair tonic with extract content of 20% (FIII) had hair growth promoting activity roughly equivalent to hair tonic containing 2% Minoxidil.

CONCLUSION:

Flavonoids, tannins, alkaloids, and saponins were among the bioactive compounds found in Delipidated Ethanol Extract of Capsicum frutescens L. Leaves. This extract-based hair tonic had appropriate physical properties in terms of acidity (pH), homogeneity, and organoleptic. Hair Tonic with delipidated ethanol extract of Capsicum frutescens L. at 5%, 10% and 20% had potential for hair-promoting growth when compared to placebo (Negative control) and Normal control. With concentration of 20%, the formulation of Hair Tonic Delipidated Ethanol Extract of Capsicum frutescens L. Leaves hadhair growth-promoting activity roughly equivalent to hair tonic containing 2% Minoxidil (Positive control).

ACKNOWLEDGEMENT:

Thanks to AVICENNA Institute of Technology and Health, UniversitasHalu Oleo, and Universitas Lampung that had provided support in the form of funding and laboratory facilities so that this research could run and produced data that were appropriate and worthy of publication so that it could become a reference source in the field of Pharmaceutical and Health Technology.

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Received on 05.10.2022 Modified on 13.11.2022

Research J. Pharm. and Tech 2023; 16(7):3305-3310.

DOI: 10.52711/0974-360X.2023.00545