Effect of Citrullus coclocynthis Schrad. on hair growth activity of albino rats.
Subject: Alopecia (Control)
Alopecia (Research)
Baldness (Control)
Baldness (Research)
Materia medica, Vegetable (Health aspects)
Materia medica, Vegetable (Usage)
Plant extracts (Health aspects)
Plant extracts (Usage)
Watermelons (Usage)
Watermelons (Physiological aspects)
Watermelons (Research)
Authors: Roy, R.K.
Thakur, Mayank
Dixit, V.K.
Pub Date: 06/22/2007
Publication: Name: Australian Journal of Medical Herbalism Publisher: National Herbalists Association of Australia Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2007 National Herbalists Association of Australia ISSN: 1033-8330
Issue: Date: Summer, 2007 Source Volume: 19 Source Issue: 2
Topic: Event Code: 310 Science & research
Product: Product Code: 0160231 Watermelons NAICS Code: 111219 Other Vegetable (except Potato) and Melon Farming SIC Code: 0161 Vegetables and melons
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 174818478
Full Text: Citrullus colocynthis Schrad is a traditionally acclaimed hair tonic in Indian system of medicine. Studies were therefore undertaken in order to evaluate petroleum ether and ethanolic extract 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 positive control. Hair growth initiation time was significantly reduced to half on treatment with the extracts compared to 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%) in anagenic phase than standard minoxidil (67%). The results of treatment with 2% and 5% petroleum ether extracts were comparable to the positive control minoxidil.

Key words: alopecia, hair growth, Citrullus colocynthis, bitter apple

Introduction

Hair loss is a dermatological disorder that has been recognised for more than 2000 years. It is common throughout the world and has been estimated to effect nearly 2% of the world's population (Bertolino 2000, Olsen 1993). Apart from metabolic and hereditary causes alopecia has been observed as a major side effect of anticancer, immunosuppressant and many others drug treatments. Presently minoxidil (useful in both male and female pattern baldness) (Goodman 1996) and finasteride (useful in male pattern baldness) (Libecco 2004) are two USFDA approved synthetic drugs finding concomitant use for treatment of androgenic alopecia although their side effects have abbreviated their usage.

Natural products are unequivocally advocated in the cosmetic and hair care industry and about 1000 different plant extracts have been examined with respect to hair growth activity; proanthocynidine from grape seeds (Vitis Vinifera) and [beta]-sitosterol in saw palmetto (Serenoa serrulata) have shown remarkable effect (Takahashi 1998). There are many products available prepared by combination of one or more herbal drug that find acceptability as hair tonics, hair growth promoters, hair conditioners, hair cleansing agents, antidandruff agents and for the treatment of alopecia and lice infection (Saraf 1991).

The traditional system of medicine in India acclaims a number of herbal drugs for hair growth promotion however lack of sound scientific evidence limits their use. Citrullus colocynthis (Cucurbitaceae) is a herb which has been recommended as a hair growth promoter in traditional literature. The dried pulp of the unripe but full grown fruit freed from the rind, constitutes the drug colocynth and was included in the Pharmacopoeia of India for treating loss of hair (The Wealth of India 1992). The oil from the seeds of the plant has reportedly been used by ethnic tribes for putting check to premature falling and greying of hairs (Kirtikar 1975, Hamdard Pharmacopoeia 1998).

Bhavpraksh, an Ayurvedic treatise, mentions the use of a drug for the treatment of indralupta i.e. a drug used in the treatment of hair loss, probably the Sanskrit name Indrayan signifying the hair growth rejuvenating property of the drug (Chunekar 2002). Besides its use as a hair growth promoter C. colocynthis is used for variety of purposes including a hypoglycaemic agent (Al-Ghaithi 2004), an antifertility agent (Chaturvedi 2003) and an anti-inflammatory agent (Memon 2003).

In the present study the effect of ethanolic and petroleum ether extract of C. colocynthis on hair growth initiation and promotion was investigated.

Material and methods

Plant material

C. colocynthis fruits were procured from the local market and authenticated by comparing the characteristics in the Indian Pharmacopoeia. Moderately coarse powder of the drug was used for the purpose of extraction.

Preparation of extracts

The dried coarse powder of C. colocynthis was fed in a Soxhlet extractor and extracted initially with petroleum ether (60-80oC) till completely exhausted. The solvent from the extract was eliminated under reduced pressure (yield 4.7% w/w). The marc was further extracted in Soxhlet extractor with ethanol (95%) till completely exhausted and was freed of solvent under reduced pressure (yield 4.1% w/w).

Characterisation of extracts

Thin Layer Chromatography was performed on precoated silica gel-G plates (10X10) (Emerck, Germany) for characterisation of the extracts. Chlorofrom : methanol : water (7:2.1:0.9 v/v) gave best resolution for ethanolic extract. Petroleum ether extract was best resolved in toluene : ethylacetate (84:16 v/v) as mobile phase (Wagner 2002). The spots were visualised using 50% ethanolic H2SO4 as derivatising agent.

Animals

Wistar strain albino rats of either sex weighing between 120-150 g were fed on standard diet and water ad libitum. The animals were housed at room temperature (24 [+ or -] 2 0 C) on a normal day-night cycle (06:00 hrs to 18:00 hrs).

Preparation of samples

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

Treatment

Animals were divided in groups of 6 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% ethanolic extract in ointment base; Group V topical application of 5% ethanolic extract in ointment base; Group VI topical application 2% alcoholic solution of minoxidil (Mintop(r)).

Hairs on the dorsal side of the rats were removed using hair clippers and electrical shavers. A marketed hair remover (Anne French) was also used to assure complete denual of 6 sq cm area (Roy 2006).

Toxicity studies

Toxicity studies were carried out and the ethanolic and petroleum extracts when applied in a concentration of up to 10% did not show any toxic side effects or erythema on skin surface. Thus the prepared extracts were considered safe for topical administration.

Permission from the institutional ethical committee was taken before animal experimentation.

Statistical treatment

Data is reported as mean [+ or -] SEM. Statistical analysis of data was carried out by one way ANOVA comparing all test groups vs control followed by Dunnet's test using Instat v 2.1 software run on Windows Xp residing in Pentium IV processor.

Qualitative hair growth study

Qualitative hair growth was evaluated by visual observation of two parameters a. hair growth initiation time i.e. minimum time to initiate perceptible hair growth and b.) hair growth completion time i.e. 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 euthanicated after 10, 20 and 30 days of treatment. Skin biopsies were taken from the shaved area and specimens were preserved in 10% formalin. The specimens were fixed on paraffin wax and blocks prepared for microtomy. After fixation vertical sections of the skin were cut with the help of semiautomatic rotary microtome (Remi-1871, India).

The sections were stained with heamatoxylin and eosin. The number of hair follicles per mm area of skin and ratio of hair follicles in different cyclic phases i.e. anagen (active growth phase) and telogen (resting phase) were determined using the microscope. Hair folliculogram was prepared by observing growth cycle of 100 hairs and length of hair follicle.

Global dispensary

Results

Qualititative 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 denuded area in the second week, whereas it was noted in the first week in the petroleum ether extract treated groups and minoxidil treated groups. Hair growth was initiated on the 4th day with 5% ointment of petroleum ether extract of C. colocynthis. Hair growth initiation was recorded on the 5th day with 2% ointment of C. colocynthis whereas it was the 6th day in the minoxidil treated control group. Although reduction in hair growth initiation time was recorded in the 2% and 5% ethanolic extract treated groups it was not as pronounced as with the petroleum ether extract treated groups (Table 1).

The time taken for complete hair growth on the shaved area was also affected with 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; on the 19th day with minoxidil treatment. In ethanolic extract and vehicle control group animals complete hair growth was noted after 24 days. It was further observed in the petroleum ether extract treated group that the texture of hair was coarse, rough and hard compared to the hairs of the minoxidil 2% treated group which were short and silky (Fig I).

The results clearly show that the 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 to 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 ethanolic extract treated group a similar scenario is visible with most of the follicles in telogenic phase and only a few in anagenic phase with no anagenic hair follicles (PM II).There is a complete reversal of the picture in petroleum ether extract and minoxidil treated group 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 the percentage anagenic population (Table 2). Anagenic population of 47 [+ or -] 0.3% was found in the vehicle treated control group, whereas with 2% petroleum ether extract it was 72 [+ or -] 0.2% and with 5% petroleum ether extract treatment 75 [+ or -] 0.2% hair follicles were found in anagenic phase. With minoxidil treatment, 67 [+ or -] 0.5% hair follicle were observed in anagenic phase whereas. Treatment with ethanolic extract also improved population of anagenic hair follicles but it was not as appreciable as the petroleum ether extract treatment (Table 2).

Length of hair follicles

The treatment with 2% and 5% petroleum ether extract of C. colocynthis had a remarkable effect on the length of hair follicles. In the control group only 34 [+ or -] 0.4% had an average length of 0.5mm, whereas in the extract treated groups 46 [+ or -] 0.3% and 48 [+ or -] 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 [+ or -] 0.1% hair population had a length of 0.5 mm and above (Fig. 3).

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 resulted in soft and silky hair.

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

Petroleum ether extract was the best in inducing hair growth initiation, with the minoxidil treatment next. The study confirms that the petroleum ether extract (5%) treatment is at par with the 2% minoxidil treatment in revitalising the growth of hair in male 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, characterised by continuous miniaturisation of androgen reactive hair follicles and accompanied by perifollicular fibrosis of follicular units in histological examination (Yoo 2006). Retention of late anagenic follicles as well as increase in follicular length and prevention of their miniaturisation may therefore be attributed to 5[alpha]-reductase inhibitory activity. Although 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 the 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 was augmented and their continuous growth and differentiation may have resulted in construction of new anagenic follicles (Tanaka 1980).

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

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

References

Adhirajan N, Dixit VK, Gowri C. 2001. Development and evaluation of herbal formulations for hair growth. Indian Drugs 38:11;559-63.

Adhirajan N, Ravi Kumar T, Shanmugasundaram N, Mary B. 2003. In vivo and in vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn. J. Ethnopharmacol 88;235-9.

Al-Ghaithi F, El-Ridid MR, Adeghate E, Amid MH. 2004. Biochemical effects of Citrullus colocynthis in normal and diabetic rats. Mol Cell Biochem 261:1-2;143-9.

Chaturvedi M, Mali PC, Ansari AS. 2003. Induction of reversible antifertility with a crude ethanol extract of Citrullus colocynthis Schrad. fruit in male rats. Pharmacology 68:1;38-48.

Chunekar KC, Hota NP. 2003. Plants of Bhavprakash. National Academy of Ayurveda New Delhi 1; 116.

Goodman LS, Gilman A. 1996. The Pharmacological Basis of Therapeutics. New York: McGraw Hills Inc.

Hamdard Pharamceopeia of Eastern Medicine 1990. Ed. Hakim MD Said 2nd reprint. New Delhi: Sri Satguru Publications.

Kirtikar KR, Basu BD. 2003. Indian Medicinal Plants. 3rd ed. Vol 8. Delhi: Sri Satguru Publication.

Libecco J F, Bergfeld WF. 2004. Finasteride in the treatment of alopecia. Expert Opin Pharmacother 5:4;933-40.

Memon U, Brohi AH, Ahmed SW, Azhar I, Bano H. 2003. Antibacterial screening of Citrullus colocynthis. Pak J Pharm Sci 16:1;1-6.

Olsen EA. 1993. Androgenetic alopecia. In: E.A. Olsen (Ed.), Disorders of hair growth: Diagnosis and treatment. New York: MacGraw Hill Inc.

Pharmacopoeia of India. 1996. Vol II. New Delhi: Ministry of Health.

Saraf S, Pathak AK, Dixit VK. 1991. Hair growth promoting activity of Tridax procumbens. Fitoterapia 62:495-8.

Takahashi T, Kamiya T, Yokoo Y. 1998. Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle in vivo. Acta Derm Venereol 78:428-32.

Tanaka S, Saito M, Tabata M. 1980. Bioassay of crude drugs for hair growth promoting activity in mice by a new simple method. Planta Med S1:84-90.

The Wealth of India. 1992. Vol C. New Delhi: National Institute of Scientific Communication and Research New Delhi.

Uno H, Kurata S. 1993. Chemical agents and peptides affect hair growth. J Invest Dermatol 101;143-7S.

Uno H. 1991. Quantitative models for the study of hair growth in vivo. In: Stenn KS, Messenger AY, Baden H P. Molecular and Structural Biology of hairs. New York: New York Academy of Science.

Yoo HG, Kim JS, Lee SR, Pyo HK, Moon HI, Lee JH et al. 2006. Perifollicular fibrosis: pathogenetic role in androgenetic alopecia. Biol Pharm Bull 29:6;1246-50.

RK Roy, Mayank Thakur and VK Dixit *

Department of Pharmaceutical Sciences

Dr HS Gour University

Sagar (MP) 470 003 India

* Corresponding Author

Prof VK Dixit, Department of Pharmaceutical Sciences Dr HS Gour University, Sagar (MP) 470 003 India Telephone: +91 7582 264 582 +91 9826 497 030 Email: vkdixitl 11 @rediffmail.com
Table 1
Effect of Citrullus colocynthis Schrad extracts on qualitative
hair growth

S.                                        Hair Growth (Days)

No.   Treatment (topical)      Initiation Time       Completion Time

1.    Control (vehicle       12 [+ or -] 0.82      24 [+ or -] 1.02
      only)
2.    Standard (% minoxi-    6 [+ or -] 0.41 ***   20 [+ or -] 1.08 ***
      dil solution)
3.    Petroleum ether        5 [+ or -] 0.28 ***   18 [+ or -] 0.56 ***
      extract (2%
      ointment)
4.    Petroleum ether        4 [+ or -] 0.12 ***   18 [+ or -] 0.42 ***
      extract (5%
      ointment)
5.    Ethanolic extract      11 [+ or -] 0.46      24 [+ or -] 1.34
      (2% ointment)
6.    Ethanolic extract      10 [+ or -] 0.68 *    24 [+ or -] 1.21
      (5% ointment)

Value are mean [+ or -] SEM., n=6,

* p <0.05, ** p <0.01 and *** p <0.001, significance vs control

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

                            Percentage of hair follicles

                                   After 10 Days

Treatment *                   Telogen           Anagen

Control (Vehicle)         58 [+ or -] 0.4   42 [+ or -] 0.4
Standard (2% minoxidil)   54 [+ or -] 0.3   46 [+ or -] 0.3
2% pet. ether extract     52 [+ or -] 0.3   48 [+ or -] 0.3
5% pet. ether extract     46 [+ or -] 0.4   54 [+ or -] 0.4
2% ethanolic extract      55 [+ or -] 0.5   45 [+ or -] 0.5
5% ethanolic extract      54 [+ or -] 0.4   46 [+ or -] 0.5

                            Percentage of hair follicles

                                   After 20 Days

Treatment *                  Telogen               Anagen

Control (Vehicle)         55 [+ or -] 0.4      45 [+ or -] 0.4
Standard (2% minoxidil)   37 [+ or -] 0.9      62 [+ or -] 0.9
2% pet. ether extract     37 [+ or -] 0.1      63 [+ or -] 0.1
5% pet. ether extract     34 [+ or -] 0.2      66 [+ or -] 0.2
2% ethanolic extract      50 [+ or -] 0.6      50 [+ or -] 0.6
5% ethanolic extract      46 [+ or -] 0.2      54 [+ or -] 0.2

                            Percentage of hair follicles

                                   After 30 Days
                                                                  T/A
Treatment *                  Telogen            Anagen           ratio

Control (Vehicle)         53 [+ or -] 0.3   47 [+ or -] 0.3      1.21
Standard (2% minoxidil)   33 [+ or -] 0.5   67 [+ or -] 0.5 **   0.49
2% pet. ether extract     28 [+ or -] 0.2   72 [+ or -] 0.2 **   0.38
5% pet. ether extract     25 [+ or -] 0.2   75 [+ or -] 0.2 **   0.33
2% ethanolic extract      46 [+ or -] 0.8   54 [+ or -] 0.8      0.85
5% ethanolic extract      43 [+ or -] 0.6   57 [+ or -] 0.6      0.75

T/A--Telogenic/Anagenic ratio after 30 days of treatment

* All extracts were applied topically
Value are% mean [+ or -] SEM.

** P< 0.05 (considered significant)
Gale Copyright: Copyright 2007 Gale, Cengage Learning. All rights reserved.