Original Contribution

O R I G I NA Blackwell Publishing Inc L C O N T R I BU T I O N S

Development and evaluation of polyherbal formulation for hair growthpromoting activity

Ram Kumar Roy, PhD, Mayank Thakur, MPharm & V K Dixit, PhD, LLB
Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar, Madhya Pradesh, India

Summary

Background Cuscuta reexa (Roxb.), Citrullus colocynthis (Schrad.), and Eclipta alba (Hassk.) are traditionally acclaimed herbs for their hair growthpromoting potential. Aim In the present study, it was envisaged to prepare herbal formulations containing petroleum ether extracts of the three herbs in varying ratio and evaluating the formulations for the hair growthpromoting activity. Methods The formulations as well as minoxidil (2%) solution (positive control) were applied topically on shaved skin of rats, and the time required for initiation and completion of hair growth cycle was recorded. Results and conclusions Hair growth initiation time was markedly reduced to one third on treatment with the prepared formulation compared with control animals. The time required for complete hair growth was also reduced by 32%. Quantitative analysis of hair growth cycle after treatment with formulations and minoxidil (2%) exhibited greater number of hair follicles in anagenic phase compared with control. The results thus corroborate with the traditionally acclaimed hair growthpromoting capabilities of the plants. The prepared formulation also holds potential for treatment of alopecia. Keywords: alopecia, Citrullus colocynthis, Cuscuta reexa, Eclipta alba, hair growth

Introduction
Hair loss is a dermatologic disorder, and the surge for discovering natural products with hair growth promoting potential is continuous.1,2 Hair loss, or alopecia, is a common patient complaint and a source of signicant psychologic and physical distress.3 Androgens are considered to be one of the most important causes for alopecia apart from a variety of other factors.4 Natural products in the form of herbal formulations are available on the market and are used as hair tonic,

Correspondence: V. K. Dixit, Department of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.), India 470003. E-mail: vkdixit2011@rediffmail.com Accepted for publication November 6, 2006

hair growth promoter, hair conditioner, hair-cleansing agent, antidandruff agents, as well as for the treatment of alopecia and lice infection.5 A number of herbal products have been acclaimed with hair growthpromoting activity.6 The traditional system of medicine in India acclaims a number of herbal drugs for hair growth promotion. In our previous studies, we have found that the petroleum ether extracts of Cuscuta reexa Roxb. (Cuscutaceae), Citrullis colocynthis Schrad (Cucurbitaceae), and Eclipta alba Hassk. (Asteraceae) are useful in treating Indralupta (i.e., loss of hair).7,8 The present study was, therefore, undertaken to develop a formulation containing petroleum ether extracts of these drugs in the form of herbal hair creams in varying ratios and evaluating the creams for their hair growthinitiating and hair growth promoting activity.

108

2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6, 108112

Herbal formulation for hair growth R K Roy et al.

Materials and methods

Plant material

Aerial parts of E. alba were collected in the months of January and February in and around Sagar, Madhya Pradesh, India. The stems of C. reexa were collected from Modinagar, Uttar Pradesh, and C. colocynthis fruits freed from rind were purchased from the local market. The plants were authenticated at the Department of Pharmaceutical Sciences, Dr. H.S. Gour University Sagar. A voucher specimen for the herbarium has been submitted.
Preparation of extracts

animals were housed at room temperature (24 2 C) on a normal daynight cycle (06:00 h to 18:00 h). Permission from the institutional ethical committee of Dr. H.S. Gour Vishwavidyalaya, Sagar, India was taken before animal experimentation. Hair growth studies were carried out on rats that were divided into ve groups of six animals each. Rats of control group (group I) were given topical application of the cream base and served as control. Group II was treated with 2% minoxidil and served as positive control. Daily, animals were given topical application of CET1 (group III), CET2 (group IV), and CET3 (group V).
Preliminary skin irritation test

The plants were dried under sunlight, crushed to moderately coarse power, and stored in airtight container. The dried powdered drugs were extracted with petroleum ether (6080 C) using Soxhlet extractor until completely exhausted. The solvent from the extract was eliminated under reduced pressure, and dried extracts were collected. The percentage yield for the extracts was 2.9% (w/w), 4.6% (w/w), and 4.7% (w/w) for E. alba, C. colocynthis, and C. reexa, respectively. The extracts were used as such based on our previous study, whereby the petroleum ether extract of the individual extracts were found to be most effective when compared with the ethanolic extract.6,7 The extracts were characterized by thin layer chromatography on silica gel Gprecoated plates (E Merck, Germany) (20 20), using hexane/ethyl acetate (8.4:1.6, v/v) solvent system that showed best resolution. The spots were visualized after derivatization using 50% ethanolic H2SO4.
Preparation of test sample

The method reported in ATSM10 was used for carrying out preliminary skin irritation test on Himalayan albino rabbits weighing between 2 and 2.5 kg. In brief, the skin from the back of six rabbits was shaved on both sides of the back using hair clipper and electric shaver to expose test areas. This was done with a view to provide three test sites on the back of each rabbit. On cleaned test sites, the prepared cream formulations were applied, and visual observation were made for the appearance of any irritation or erythema for a total period of 72 h after the application of test preparations.
Evaluation of stability of extracts

The petroleum ether extracts were incorporated into hair cream base prepared by fusion method using o/w base (Adhirajan et al.).7 The cream base consisted of glyceryl monostearate (9%, w/w), light liquid parafn (20%, w/ w), cetyl alcohol (15%, w/w), bees wax (15%), propyl and methyl paraben (0.15%, w/w), glycerol (4.5%, w/w), and water (59%, w/w). A mixture of 5% (w/w) of all the three proportionate extracts ET1, ET2, and ET3 were incorporated in the base to obtain 5% herbal creams coded as CET1, CET2, and CET3, respectively. The creams were prepared were using general method reported for preparation of the cream.9
Animals

The prepared formulations were stored at a room temperature of 25 2 C and 40 2 C. One gram of stored formulation was mixed with petroleum ether and mixture was centrifuged at 1750 g for 5 min. The supernatant was separated, collected, and applied on precoated silica gel-G TLC plates (20 20). The plates were developed in a twin trough chamber (Camag, Swizerland) using hexane/ethylacetate (8.4:1.6, v/v) as mobile phase. Along with the formulations, individual combinations of extracts ET1, ET2, and ET3 were also run on the plates. Identical spots at the same Rf values were obtained. This conrms the stability of extracts in the prepared formulation and also afrms that preparation of cream by fusion method does not result in any degradation of the extracts or their combination.
Application of test formulation

Wistar strain albino rats of either sex weighing 120 to 150 g were fed on standard diet and water ad libitum. The

Hair clippers and electric shavers were used to remove the hair from dorsal portion of all test animals. Hair remover (Anne French) was also used to ensure complete removal of hairs from denuded area, which was nearly 6 cm2. Finally, denuded skin was wiped off with surgical spirit. Equal quantities of prepared creams and standard

2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6, 108 112

109

Herbal formulation for hair growth R K Roy et al.

Figure 1 Percentage of anagenic and telogenic follicles after 30 days of treatment.

solution were applied to the denuded area of albino rats once a day up to 30 days.
Qualitative hair growth study

early anagen, mid anagen, and late anagen) were recorded. Percentage frequency distribution of hair follicles of different length has been graphically presented (Fig. 1).

Qualitative hair growth analysis was undertaken by visual observation of two parameters: hair growth initiation time (i.e., minimum time to initiate hair growth on denuded skin region) and hair growth completion time (i.e., minimum time taken to complete cover the denuded skin region with new hair).7,8
Quantitative hair growth study

Results
The stability of the formulations was studied using TLC. When the extracts ET1, ET2, and ET3 were run along with the formulations CET1, CET2, and CET3, which were stored at 25 2 C and 40 2 C in vessels, identical spots and Rf values were obtained, indicating good chemical stability of the formulations. The preliminary skin irritation test conducted on Himalayan albino rabbits provided validation for the safety of the prepared formulations. No erythema and/or edema was observed in Himalayan albino rabbits after application of CET1, CET2, and CET3 in skin irritation test. Thus, it can be concluded that the prepared formulations are free from observable irritant effect.
Qualitative studies on hair growth

The method reported by Uno11 was followed for the quantitative evaluation of treatment. One rat from each group was euthanized after 30 days of treatment; skin biopsies were taken from the shaved area; and specimen was preserved in 10% formalin. Tissues were embedded in parafn wax and sectioned into uniform thickness of 10 m and stained with hematoxylin and eosin. Sections from all the groups were evaluated for the number of hair follicles per mm area of skin, and percentage ratio of hair follicles in different cyclic phases, like anagen (growth phase) and telogen (resting phase), was determined microscopically.
Length and percentage population of hair growth cyclic phases (folliculogram)

After staining of prepared slides as mentioned above, the growth cycle of 100 hair follicles was observed, and hair follicles present in different phases (catagen, telogen,

Initiation of hair growth time in denuded area was considerably reduced by all the cream formulations. In control group animals, data recording was done in the second week, whereas data were recorded in rst week in groups treated with developed formulations and minoxidil. As against 12 days in control animals, hair growth initiation time was 4 days in CET3, 5 days in CET1, and 7 days in CET2 group. In minoxidil-treated control group, hair growth initiated after 6 days. Data clearly exhibit better performance of CET3 and CET1 over minoxidil in this parameter.

110

2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6, 108112

Herbal formulation for hair growth R K Roy et al.

Figure 2 Percentage population of hair follicles of different length (mm) after 30 days.

The developed formulation and minoxidil also inuenced time taken for complete growth and reduced it considerably. Completion of hair growth was recorded on the 17th day in CET3, the 18th day in CET1, and the 20th day in CET2. Date were recorded on the 20th day in minoxidiltreated group. The observations show that CET3 was best followed by CET1 and CET2. CET2 was comparable with minoxidil-treated group, whereas, CET1 and CET3 exhibited better performance for this parameter. In the control group, where only base was applied, 24 days were taken for complete hair growth to occur.
Quantitative studies on hair growth

Length and percentage population of hair growth cyclic phases

A substantial increase in hair follicles with greater length (>0.5 mm) was observed after 30 days of treatment with the developed formulations (Fig. 3). In the control group, the percentage population of hair follicles above 0.5 mm length was 34 1.2%. In the CET2 group, 36 2.1% hair follicles are above the length of 0.5 mm; CET1 group is the next to follow, with 46 0.9%. In the CET3 group, the percentage is increased to 52 2.1%, whereas in minoxidil, only 36 1.8% hair follicles are above 0.5 mm length. The observations clearly reect the growth of follicle by treatment over control.

A signicant difference in cyclic phase of hair growth was observed after the treatment with developed formulations for 30 days. In the control group (cream base only), treated animals most of the hair follicles are in the telogenic phase; only one or two follicles are in the catagenic phase. In the treated group, the picture is reversed where the majority of follicles are in the anagenic phase, with few of them in the catagenic phase. Telogenic-phase follicles are negligible. A prominent and signicant increase in percentage population of anagenic hair follicles was also observed after 30 days of treatment with the developed formulations. It was only 47 1.6% in the control group, whereas in CET1 group, the percentage anagenic population was 75 1.2%. Percentage was 66 1.5% in CET2 group. Maximum anagenic follicles were observed in the CET3treated group where their percentage population was 83 2.1%. With minoxidil treatment, only 67 1.1% hair follicles were in the anagenic phase of hair growth (Fig. 2).

Discussion and conclusions

Treatment given to the denuded skin of albino rats with herbal formulation had pronounced effect on hair growth initiation and completion time. The treatment not only reduced the time of hair growth initiation over control but was even better than the standard minoxidil treatment. The quality of hairs was soft, silky, and shiny. This is remarkable considering the fact that individually the quality of hairs reported by treatment of petroleum ether extracts C. reexa and C. colocynthis are rough and coarse,7,12 whereas E. alba has been traditionally acclaimed for improving the shine and texture of the hairs.1315 Thus, the study provides an interesting observation for the developed formulations. Among the prepared formulation, CET3 was the best as evidenced by least hair growth initiation and completion time; maximum number of hair follicles in anagenic phase were present in CET3 group, and it was superior to

2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6, 108 112

111

Herbal formulation for hair growth R K Roy et al.

Figure 3 Qualitative effect of herbal formulation on hair growth.

minoxidil treatment. The activity shown by CET1 and CET2 was comparable with minoxidil. The promising activity is due to the switching of hair follicle from telogen phase to anagen phase and retention of the late anagenic hair follicles as evidence by increased percentage population of hair follicles above 0.5 mm in the treated groups. The two formulations (i.e., CET1 and CET3) containing higher amounts of E. alba extract resulted in hairs of better quality, indicating its possible role in producing soft and silky hairs. 5-Reductase has been implicated as one of the major causes of hair loss.16 It may be rewarding if studies to unfold the mechanism of action of herbal extracts are undertaken using this bed.

References
1 Arakawa T, Emoto K, Utsnomiya S, Hagiwara Y, Shimizu T. Effect of Swertinogen in hair growth with special reference to its activities on skin function. Tokushima J Exp Med 1962; 9: 3759. 2 Adhirajan N, Ravi Kumar T, Shanmugasundaram N, Babu M. In vivo and in vitro evaluation of hair growth potential of Hibiscus rosasinensis Linn. J Ethnopharmacol 2003; 88: 2359. 3 Han A, Mirmirani P. Clinical approach to the patient with alopecia. Semin Cutan Med Surg, 2006; 25: 1123. 4 Bagatell C, Bremner WJ. Androgens in men uses and abuses. New Engl J Med 1996; 334: 70715. 5 Olsen EA. Androgenetic alopecia. In: EA Olsen, ed. Disorders of Hair Growth: Diagnosis and Treatment. New York: McGraw Hill, Inc; 1993: 257 87.

6 Takahashi T, Kamiya T, Yokoo Y. Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle in vivo. Acta Derm Venereol 1998; 78: 42832. 7 Adhirajan N, Dixit VK, Gowri C. Development and evaluation of herbal formulations for hair growth. Indian Drugs 1999; 38: 55963. 8 Roy RK, Thakur M, Dixit VK. Effect of Cuscuta reexa Roxb. on hair growth in albino rats. Indian Drugs 2006; 43 (12): 951 6. 9 Lehne RK. Hair grooming preparation in balsams. In: MS Sagarin, E Gershon, SD Rieger, JS Strianse, eds. Cosmetic Science and Technology, 2nd edn. New York: Wiley Interscience; 1972; 2: 148. 10 A.S.T.M. Standard Practice for testing biomaterials in rabbits for primary skin irritation, A.S.T.M. designation F 719-81; Philadelphia: American Society for Testing of Materials, 1998: 1789. 11 Uno H. Quantitative models for the study of hair growth in vivo. In: KS Stenn, AG Messenger, HP Baden, eds. Molecular and Structural Biology of Hairs, Vol. 642. New York: New York Academy of Science; 1991: 10724. 12 Adam SE, Al Yhya MA, AlFarhan AH. Combined toxicity of Cassia senna and Citrullus colocynthis in rats. Vet Hum Toxicol 2001; 43: 702. 13 Chopra RN, Nayer SL, Chopra IC. Glossary of Indian Medicinal Plants. New Delhi: Publication and Information Directorate, CSIR; 1992: 85. 14 Kirtikar KR, Basu BD. Indian Medicinal Plants, 3rd edn, Vol. 8. Delhi: Sri Satguru Publication; 2003: 24013. 15 Sharma SK, Chunekar KC, Pondel K. Plants of Sharangdhar Samhita, National Academy of Ayurveda, 1999; Vol. 1: 289. 16 Park WS, Lee CH, Lee B, Chang I. The extract of Thujae occidentalis semen inhibited 5alpha-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse. J Dermatol Sci 2003; 31 (2): 91 8.

112

2007 Blackwell Publishing Journal of Cosmetic Dermatology, 6, 108112