Effect of Citrullus colocynthis. on Hair Growth in Albino Rats

Abstract

Citrullus colocynthis. Schrad (Cucurbitaceae) is a traditionally acclaimed hair tonic in Ayurveda (the traditional Indian system of medicine). Studies were therefore undertaken to evaluate petroleum ether and ethanol extracts of C. colocynthis. for their effect on hair growth in albino rats. The extracts incorporated into oleaginous ointment base were applied topically on shaved denuded skin of albino rats. The time required for initiation of hair growth as well as completion of hair growth cycle was recorded. Minoxidil 2% solution was applied topically and served as the standard. Hair growth initiation time was significantly reduced to half on treatment with the petroleum ether extracts compared with untreated control animals. The time required for complete hair growth was also considerably reduced. The treatment was successful in bringing a greater number of hair follicles (>70%) to anagenic phase than standard minoxidil (67%). The result of treatment with 2 and 5% petroleum ether extracts were comparable with the standard minoxidil.

Keywords:

• Alopecia
• bitter apple
• Citrullus colocynthis
• hair growth

Introduction

Hair loss is a dermatologic disorder that has been recognized for more than 2000 years. It is common throughout the world and has been estimated to affect nearly 2% of the world population (Olsen, 1993; Bertolino, 2000). Apart from metabolic and hereditary causes, alopecia has also been observed as a major side effect of anticancer, immunosuppressant, and many others drug treatments. Currently, minoxidil (Goodman & Gilman, 1996) (useful in both male and female pattern baldness) and finasteride (useful in male pattern baldness) (Libecco & Bergfeld, 2004) are two U.S. FDA approved synthetic drugs finding concomitant use for treatment of androgenic alopecia, but their side effects have reduced their usage.

Natural products are unequivocally advocated in the cosmetic and hair care industries and about 1000 plant extracts have been examined for hair care usage. Proanthocynidine from grape seeds and β-sitosterol in saw palmetto have shown remarkable hair growth–promoting activity (Takahashi et al., 1998). There are many products available in the market, which are prepared by combination of one or more herbal drugs and find acceptability as hair tonic, hair growth promoter, hair conditioner, hair cleansing agent, antidandruff agents, and for the treatment of alopecia and lice infection (Saraf et al., 1991; Roy et al., 2007).

The traditional system of medicine in India (Ayurveda and Unani) recommends numerous herbal drugs for hair growth promotion. However, lack of sound scientific evidence limits their usage. Citrullus colocynthis. Schrad (Cucurbitaceae) is a herb that has been recommended in traditional literature as a hair growth promoter (Mukerji, 1953). The dried pulp of the unripe but full-grown fruit freed from the rind constitutes the drug, colocynth of commerce, and is used for treating loss of hair (Anon, 1992). The oil from the seeds of the plant has been reported and is used by ethnic tribes for controlling premature falling and graying of hairs (Kirtikar & Basu, 2003; Anon, 1998). Bhavprakash, an Ayurvedic treatise, mentions the use of the drug for the treatment of “Indralupta”, that is, the drug used in the treatment of hair loss. The Sanskrit name “Indrayan” probably signifies the attributed hair growth rejuvenating property of the drug (Chunekar & Hota, 2002). The drug was included in the Indian Pharmaceutical Codex. and Martindales Extra Pharmacopoeia..

Besides its use as a hair growth promoter, C. colocynthis. is used for variety of purposes, viz, as a hypoglycemic (Al-Ghaithi et al., 2004), antifertility agent (Chaturvedi et al., 2003), and anti-inflammatory agent (Memon et al., 2003).

In the current study, we examined the effect of ethanol and petroleum ether extracts of C. colocynthis. on hair growth initiation and promotion.

Materials and Methods

Plant material and preparation of extracts

C. colocynthis. fruits were procured in the month of November from the local market at Sagar (M.P., India) and authenticated by comparing the characteristics in the Indian Pharmaceutical Codex. (Mukerji, 1953). A voucher specimen for the plant has been deposited in the herbarium at the Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar. The plant material was dried under the sun, and moderately coarse powder (all the particles passed through a sieve of 710 µm and not more than 40% through a sieve of 250 µm) of the drug was used for the purpose of extraction. All the chemicals and reagents used were of analytical grade.

The dried coarse powder (500 g) of C. colocynthis. was fed in to Soxhlet extractor and extracted initially with petroleum ether (60–80°C) until completely exhausted. The marc was further extracted in a Soxhlet extractor with ethanol (95%) until completely exhausted. Removal of solvent under reduced pressure gave yields of 4.7% w/w and 4.1% w/w for petroleum ether and ethanol extracts, respectively.

Characterization of extracts

Thin-layer chromatography was performed on precoated silica gel-G plates (10 × 10) (E. Merck, Darmstadt, Germany) for characterization of the extracts. Chloroform:methanol:water (7:2.1:0.9 v/v) gave best resolution for ethanol extract. Petroleum ether extract was best resolved in toluene:ethyl acetate (84:16 v/v) as mobile phase (Wagner & Bladt, 1992). The spots were visualized using 50% ethanol H₂SO₄ as derivatizing agent.

Animals

Wistar strain albino rats of either sex weighing between 120 and 150 g were fed standard diet and water ad libitum.. The animals were housed at room temperature (24 ± 2°C) on a normal day-night cycle (0600 h to 1800 h). All animal experimentation was carried out after approval of the protocol by the Institutional Ethical Committee of Dr. H.S. Gour University. The guidelines of CPCSEA, India, were strictly followed.

Preparation of samples

The petroleum ether and ethanol extracts were incorporated into ointment base in concentrations of 2% and 5% (w/w), respectively. Ointments were made following the procedure given in the Pharmacopoeia of India. (Anonymous, 1996). The extracts were then incorporated in the prepared oleaginous base.

Treatment

Animals were divided into groups of six rats each. The following treatment was given to animals of different groups:

• Group I: Vehicle only and served as control.

• Group II: Topical application of 2% petroleum ether extract in ointment base.

• Group III: Topical application of 5% petroleum ether extract in ointment base.

• Group IV: Topical application of 2% ethanol extract in ointment base.

• Group V: Topical application of 5% ethanol extract in ointment base.

• Group VI (standard): Topical application 2% alcohol solution of minoxidil (Mintop, Dr. Reddys Laboratory, Hyderabad, India).

Hair on the dorsal sides of the rats was removed using hair clippers and electric shavers. A marketed hair remover (Anne French) was also used to ensure complete denual of a 6 cm² area (Roy et al., 2006).

Toxicity studies

The ethanol and petroleum ether extracts were applied on the surface of denuded skin for 7 days at 5% concentration and observation made for skin rashes, inflammation, or allergic reactions (Adhirajan et al., 2003). Thus, the prepared extracts were safe for topical administration. Permission from the institutional ethical committee was obtained for animal experimentation.

Statistical analysis

Data are reported as mean ± SEM. Statistical analysis of data was carried out by one-way ANOVA comparing all test groups versus control followed by Dunnett's test using Instat v 2.1 software.

Qualitative hair growth study

Qualitative hair growth was evaluated by visual observation of two parameters: (a) hair growth initiation time, that is minimum time to initiate perceptible hair growth, and (b) hair growth completion time, that is, minimum time taken to cover the denuded skin region with new hair completely (Adhirajan et al., 2001). Hair growth initiation and completion time was recorded for each group of animals.

Quantitative hair growth study

The method reported by Uno (1991) was followed for the quantitative evaluation of C. colocynthis. extract. One rat from each group was euthanized after 10, 20, and 30 days of treatment, skin biopsies were taken from the shaved area, and specimens preserved in 10% formalin. The specimens were fixed on paraffin wax, and blocks were prepared for microtomy. After fixation, vertical sections of the skin were cut with the help of a semiautomatic rotary microtome (Remi-1871, Mumbai, India). The sections were stained with hematoxylin and eosin. The number of hair follicles per mm area of skin and ratio of hair follicles in different cyclic phases, that is, anagen (active growth phase) and telogen (resting phase), were determined using a microscope. Hair folliculogram was prepared by observing growth cycle of 100 hairs and length of hair follicle (Uno & Kurata, 1993).

Results

Qualitative studies on hair growth

Hair growth initiation and completion time was considerably reduced upon treatment with petroleum ether extract of C. colocynthis.. In the control group animals, hair growth was initiated in the denuded area in the second week, whereas it was noted in the first week in petroleum ether extract–treated groups and minoxidil-treated groups. Hair growth was initiated on the fourth day with 5% ointment of petroleum ether extract of C. colocynthis.. Hair growth initiation was recorded on the fifth day with 2% ointment of C. colocynthis., whereas it was on the sixth day in minoxidil-treated standard group. Although hair growth initiation time was reduced in 2% and 5% ethanol extract–treated groups, it was only marginal and not as pronounced as in the petroleum ether extract–treated group (Table 1).

Table 1. Effect of Citrullus colocynthis. Schrad extracts on qualitative hair growth.

		Hair growth (days)
Sample no.	Treatment (topical)	Initiation time	Completion time
1.	Control (vehicle only)	12 ± 0.82	24 ± 1.02
2.	Standard (2% minoxidil solution)	6 ± 0.41***	20 ± 1.08***
3.	Petroleum ether extract (2% ointment)	5 ± 0.28***	18 ± 0.56***
4.	Petroleum ether extract (5% ointment)	4 ± 0.12***	18 ± 0.42***
5.	Ethanol extract (2% ointment)	11 ± 0.46	24 ± 1.34
6.	Ethanol extract (5% ointment)	10 ± 0.68*	24 ± 1.21

Values are mean ± SEM, n = 6.

*p < 0.05.

**p < 0.01.

***p < 0.001, significance versus control.

The time taken for complete hair growth on shaved area was also significantly affected with the petroleum ether extract of C. colocynthis.. Complete hair growth was observed on the 18th day with 2% and the 16th day with 5% petroleum ether extract treatment. With standard minoxidil, complete hair growth was observed on the 19th day. In ethanol extract and vehicle control group animals, complete hair growth was noted after 24 days. It was further observed that in the petroleum ether extract–treated group, the texture of hair was coarse, rough, and hard, compared with the hairs of the minoxidil (2%)-treated group, which were short and silky, and the hairs of the ethanol extract treated–animals were coarse and rough as well (Fig. 1).

Figure 1 Qualitative effect of extracts of Citrullus colocynthis. Schrad on hair growth.

The results clearly show that the petroleum ether extract was successful in reducing the time taken for hair growth initiation and completion. The results further suggest that petroleum ether extract of C. colocynthis. has better activity compared with minoxidil as far as hair growth initiation and completion is concerned.

Quantitative studies on hair growth

A considerable difference in cyclic phase of hair growth was observed in groups treated with minoxidil and petroleum ether extract of C. colocynthis.. In vehicle-treated control group animals, most of the hair follicles are in telogenic phase, only one or two are in catagenic phase (PM I). In the ethanol extract–treated group, a similar scenario is visible with most of the follicles in telogenic phase and only few in anagenic phase with no anagenic hair follicles (PM II). There is a complete reversal of the picture in the petroleum ether extract–treated and minoxidil-treated groups where the majority of hair follicles are in anagenic phase with only a few catagenic hair follicles. Telogenic follicles are almost negligible (PM III–IV).

An appreciable increase was noted in percent anagenic population (Table 2). An anagenic population of 47 ± 0.3% was found in vehicle-treated control group, whereas, with 2% petroleum ether extract, it was 72 ± 0.2%, and with 5% petroleum ether extract treatment, 75 ± 0.2% hair follicles were found in anagenic phase. With minoxidil treatment, 67 ± 0.5% hair follicles were observed in anagenic phase, whereas treatment with ethanol extract also improved population of anagenic hair follicles, but it was not as appreciable as petroleum ether extract treatment (Table 2).

Table 2. Hair growth population (anagen/telogen ratio) after treatment with extracts of Citrullus colocynthis. Schrad.

	Percentage of hair follicles (%)
	After 10 days		After 20 days		After 30 days
Treatment^a.	Telogen	Anagen	Telogen	Anagen	Telogen	Anagen	T/A ratio
Control (vehicle)	58 ± 0.4	42 ± 0.4	55 ± 0.4	45 ± 0.4	53 ± 0.3	47 ± 0.3	1.21
Standard (2% minoxidil)	54 ± 0.3	46 ± 0.3	37 ± 0.9	62 ± 0.9	33 ± 0.5	67 ± 0.5*	0.49
2% pet. ether extract	52 ± 0.3	48 ± 0.3	37 ± 0.1	63 ± 0.1	28 ± 0.2	72 ± 0.2*	0.38
5% pet. ether extract	46 ± 0.4	54 ± 0.4	34 ± 0.2	66 ± 0.2	25 ± 0.2	75 ± 0.2*	0.33
2% ethanol extract	55 ± 0.5	45 ± 0.5	50 ± 0.6	50 ± 0.6	46 ± 0.8	54 ± 0.8	0.85
5% ethanol extract	54 ± 0.4	46 ± 0.5	46 ± 0.2	54 ± 0.2	43 ± 0.6	57 ± 0.6	0.75

Values are % mean ± SEM.

T/A, telogenic/anagenic ratio after 30 days of treatment.

^a.All extracts were applied topically.

*p < 0.05 (considered significant).

Length of hair follicles

Treatment with 2% and 5% petroleum ether extract of C. colocynthis. had a remarkable effect on the length of hair follicle. In the control group, only 34 ± 0.4% had average length of 0.5 mm, whereas in extract-treated groups, 46 ± 0.3% and 48 ± 0.1% hair population with more than 0.5 mm was observed with 2% and 5% extract treatment, respectively. The results of treatment were comparable with the minoxidil group, where 49 ± 0.1% hair population had length of 0.5 mm and above (Fig. 2).

Figure 2 Percent frequency distribution of hair follicle population after 30 days of treatment with C. colocynthis..

Discussion and Conclusions

Topical application with the petroleum ether extract of C. colocynthis. fruit reduced the time required for hair growth initiation and was superior to standard (minoxidil 2%) solution. The quality of hair in the petroleum ether extract–treated group was coarse, rough, and hard, whereas the minoxidil-treated group.

Petroleum ether extract treatment caused premature switching of hair follicle from resting telogenic phase to active anagenic phase. It also helped the treated animals to retain anagenic hair follicles. Thus, percent hair follicle population in anagenic phase exhibited marked improvement over the control group.

Petroleum ether extract was the best in inducing hair growth initiation as well as completion. Minoxidil treatment was the next to follow. The study thus confirms that the petroleum ether extract (5%) treatment is on par with 2% minoxidil treatment in hair growth in rats. The remarkable improvement in length of hair follicles also supports the hair growth–promoting effects of the herb.

Androgenetic alopecia (AGA) is a dihydrotestosterone (DHT)-mediated process, characterized by continuous miniaturization of androgen reactive hair follicles and accompanied by perifollicular fibrosis of follicular units in histologic examination (Yoo et al., 2006). Retention of late anagenic follicles as well as an increase in follicular length and prevention of their miniaturization may therefore be attributed due to 5α-reductase inhibitory activity. However, detailed investigations in this regard are necessary to explore the possibility of this mechanism.

The periodic transformation of hair follicles from telogen to anagen phase in petroleum ether extract–treated group is a healthy validation of hair growth potential of C. colocynthis.. During the treatment, the secondary germ associated with aggregated dermal papilla cells in telogen follicles augmented, and their continuous growth and differentiation may have resulted in construction of new anagenic follicles (Tanaka et al., 1980).

The current study validates the ethnomedical use of this plant for hair loss treatment. Further utilization of the petroleum ether extract and its incorporation in a formulation are warranted for commercial utilization of C. colocynthis..