Journal of Ethnopharmacology 88 (2003) 235239

In vivo and in vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn.
N. Adhirajan, T. Ravi Kumar, N. Shanmugasundaram, Mary Babu
Biomaterials Division, Central Leather Research Institute, Adyar, Chennai 600 020, India Received 8 July 2002; received in revised form 18 June 2003; accepted 4 July 2003

Abstract Petroleum ether extract of leaves and owers of Hibiscus rosa-sinensis was evaluated for its potential on hair growth by in vivo and in vitro methods. In vivo, 1% extract of leaves and owers in liquid parafn was applied topically over the shaved skin of albino rats and monitored and assessed for 30 days. The length of hair and the different cyclic phases of hair follicles, like anagen and telogen phases, were determined at different time periods. In vitro, the hair follicles from albino rat neonates were isolated and cultured in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves and owers. From the study it is concluded that the leaf extract, when compared to ower extract, exhibits more potency on hair growth. 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hibiscus rosa-sinensis; Hair growth; Anagen; Telogen; Hair follicle

1. Introduction The herb Hibiscus rosa-sinensis Linn. (Malvaceae) is a glabrous shrub widely cultivated in the tropics as an ornamental plant and has several forms with varying colours of owers. In medicine, however, the red owered variety is preferred. The leaves and owers are observed to be promoters of hair growth and aid in healing of ulcers (Nadkarni, 1954; Ali and Ansari, 1997; Kurup et al., 1979). Flowers have been found to be effective in the treatment of arterial hypertension (Dwivedi et al., 1977) and to have signicant antifertility effect (Singh et al., 1982; Sethi et al., 1986). According to traditional texts (Nadkarni, 1954; Kumar et al., 1994), it is well accepted that the leaves and owers of Hibiscus rosa-sinensis have hair growth promoting and anti-greying properties. Moreover, in India the herbal products in the market intended for hair growth include the extract of various parts of Hibiscus rosa-sinensis. Hence, the present study is focused on the scientic investigation of the hair growth potential of the herb Hibiscus rosasinensis.

2. Materials and methods 2.1. Plant material Fresh leaves and owers of Hibiscus rosa-sinensis Linn. were collected in the month of October and authenticated by Anna Sidha Research Institute, Chennai, India. The collected leaves and owers were dried in the shade, powdered to a coarse consistency and stored in an airtight container at room temperature (35 C). 2.2. Preparation of extract The dried, powdered leaves and owers were Soxhlet extracted with petroleum ether (6080 C). The extract was weighed after solvent elimination under reduced pressure. The percent yield of the extract was found to be 5% (w/w) and 3% (w/w) of the leaves and owers, respectively. One gram of each extract was dissolved in 100 ml of liquid parafn to produce the 1% active compound and it was further used for the evaluation of potential hair growth effects in vivo. 2.3. Animals

author. Tel.: +91-44-2442-0709; fax: +91-44-2491-1589. E-mail address: marybabu@hotmail.com (M. Babu).

Corresponding

Female Wistar albino rats, weighing 120150 g, from Vetenary University, Chennai, were used for hair growth

0378-8741/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00231-9

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studies. Based on the guidelines of the ethical committee of CLRI, the rats were placed in cages and kept in standard environmental conditions, fed with standard diet ad libitum and allowed free access to drinking water. 2.4. Hair growth activity in vivo The rats were divided into four groups of six rats each. A 4-cm2 area of the hair from dorsal portion of all the rats was shaved off and wiped with surgical spirit. One millilitre of the prepared oil and the placebo (liquid parafn) were applied to the denuded area of the respective groups once a day and a control group received no treatment. This treatment was continued for 30 days during which time, hair growth pattern was observed visually and recorded. Skin biopsies were taken on the 10th, 20th and 30th day of sample application for follicular analysis. 2.4.1. Hair length determination Hair was plucked randomly from the shaved area of selected rats, from each group on 15th, 20th, 25th and 30th day of the treatment. The length of 25 hairs was measured and the average length was determined. The results are expressed as the mean length S.D. of 25 hairs. 2.4.2. Histological studies One rat from each group was euthanicated on the 10th, 20th and 30th day of drug treatment. Skin biopsies were taken from the shaved area and xed in 10% formalin buffer. Tissues were embedded in parafn wax and sectioned into uniform thickness of 10 m and were stained with haematoxylin and eosin. From the sections, the number of hair follicles per millimetre of the skin (Sawada et al., 1987), and the percentage ratio of different cyclic phases, like anagen and telogen, of hair follicles were determined using the microscope tted with an ocular micrometer facility. 2.5. Hair growth in vitro Wistar albino rat hair follicles, isolated from the neonates, were used for the present study. The neonates were killed by cervical dislocation and the dorsal portion of the skin was dissected out and washed thoroughly in phosphate buffered saline (PBS). The skin was cut into small pieces, approximately 0.5 cm2 in size, and individual pieces were placed in a petri dish containing PBS. The skin pieces were chopped thoroughly until the intact follicles came out from the skin. The separated intact hair follicles were isolated using a ne Pasteur pipette under binocular dissecting microscope. Individual, freshly isolated hair follicles were placed in separate wells of 96-well plates containing 150 l of Dulbeccos Modied Eagles Medium (Himedia) supplemented with 10% FCS (Sigma), 4 mM l-Glutamine, 100 g/ml Streptomycin, Penicillin 10 units/ml and Gentamycin 30 g/ml (all antibiotics were purchased from Himedia). Finally 1.5 l of 1% (0.01 mg/ml) solution of

extracts in dimethyl sulfoxide (DMSO) was added to the corresponding wells and the plates were maintained at 37 C. After 24, 48 and 72 h the increase in hair follicle length was measured using a binocular inverted microscope equipped with an eyepiece measuring graticule.

3. Results Hair growth was observed from the denuded area at the end of the 2nd week and the length of the hair began to increase until the end of the treatment course (Table 1). In comparison to the control, for all the other groups the whole denuded area was covered with hair during the 4th week (Plate 1eg). Moreover, the hair growth was sparse in all the groups except the leaf extract-treated group and there was no considerable change in hair texture. It was found that the leaf and ower extract-treated groups produced a signicant effect with respect to the placebo and control. However, in the leaf extract-treated group the length of hair was signicantly higher than that of the ower extract-treated group. 3.1. Histological studies The histological appearance observed was similar in the skin and hair of both the control and treated rats. There was no difference in the number of hair follicles per millimetre of skin of the treated and control rats. The number of hair follicles was found to be 47 hair follicles per mm of skin. However, a marked difference in the different cyclic phases (anagen and telogen) of hair follicles in treated and control rats was observed (Plate 1a and b). On 10th day, nearly 40% of the follicles were in anagen phase in all the groups and at the end of the course the leaf extract-treated groups showed the maximum number of anagen follicles (67%) when compared to ower, placebo and control. The ower extract also produced a higher value than the placebo and control, but to a lesser extent than the leaf extract (Table 2). 3.2. In vitro hair follicle culture It was observed that while chopping the skin pieces thoroughly, a greater number of intact hair follicles could
Table 1 Effect of petroleum ether extract of Hibiscus rosa-sinensis on hair length of female albino rats Compound name Leaves Flowers Placebo Control Length of hair (mm) Day 15 6.0 4.5 4.2 3.5 1.0 0.8 0.5 1.08 Day 20 9.8 8.2 7.7 6.6 1.0 0.9 1.0 1.0 Day 25 14.6 12.4 11.0 10.4 1.2 1.2 1.2 1.2 Day 30 17.0 15.8 14.5 13.6 1.2 1.2 1.5 1.5

P < 0.05, when compared to respective placebo and control values by Students t-test (n = 25 hairs).

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Plate 1. (a) Histology section of albino rat skin treated with leaf extract of Hibiscus rosa-sinensis for 30 days showing more number of anagen hair follicles (10) than control (b) (10). (c) Light microscopic picture of hair follicles treated with petroleum ether extract of leaves of Hibiscus rosa-sinensis showing intact morphology at 72 h (10) and control (d) (20) showing the shrinkage of the bulb region at 72 h. (e) Initially shaved albino rat and control group (f) after 30 days. (g) Albino rat treated with leaf extract for 30 days showing complete hair growth.

Table 2 Effect of petroleum ether extract of Hibiscus rosa-sinensis on anagen/telogen ratio Compound name Percentage of hair follicles Day 10 Anagen Leaves Flowers DMSO Control 44 40 40 38 Telogen 56 60 60 62 Day 20 Anagen 59 52 48 45 Telogen 41 48 52 55 Day 30 Anagen 67 60 54 50 Telogen 33 40 46 50

be removed from the skin and easily isolated using a ne Pasteur pipette. When observed under an inverted binocular microscope at higher magnication, hair follicles showed an apparently intact morphology. When maintained free oating in individual wells of 96-well plates, the leaf extract-treated groups showed a signicant increase in length over 72 h in culture (0.11 mm/day) (P < 0.05) and the rate of growth was 0.34 0.04 mm per 72 h (mean S.D.). This value was signicantly higher than that for follicles maintained in the ower extract, placebo or control. However, after 72 h the rate of increase in length was reduced and subsequently the growth ceased.

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Table 3 Effect of petroleum ether extract of Hibiscus rosa-sinensis on in vitro hair follicle culture Compound name Leaves Flowers DMSO Control

Increase in hair follicle length (mm) S.D. 24 h 0.15 0.08 0.04 0.08 0.03 0.01 0.03 0.03 48 h 0.28 0.11 0.07 0.11 0.03 0.02 0.03 0.04 72 h 0.34 0.12 0.07 0.11 0.04 0.03 0.02 0.03

P < 0.05, when compared to respective placebo and control values by Students t-test (n = 10 hair follicles).

For hair follicles maintained in ower extract, the rate of growth was 0.12 0.03 mm per 72 h. This value was significantly less (P < 0.05) than that of hair follicles maintained with the leaf extract and there was no signicant difference with the placebo or control (Table 3). 4. Discussions and conclusion The shaved skin of albino rats when treated with the topical application of leaf and ower extract and placebo for 30 days, it was observed that the hair growth initiated from the shaved area at the end of the 2nd week in all the groups, including control, and the whole denuded area has been covered at the end of the course, including placebo, in comparison to the control. This may be due to the gentle rubbing of the shaved skin while application of extracts and placebo (liquid parafn) and this enhances the blood circulation in the local area and thus may exert some effect on hair growth. It was also evident that the rats when treated similarly with water, the whole denuded area has been covered at the end of the course (data not shown). The leaf extract-treated groups produced a greater effect on the length of hair when compared to other groups being 17 mm at the end of the course, compared to 13.6 mm in the control and 14.5 mm in the placebo. This may be due to the premature switching of follicles from the telogen to anagen phase of hair growth cycle as observed by Philpot et al. (1992). In another study, Uno and Kurata (1993) reported that the topical application of fuzzy rat with minoxidil, diazoxide and copper peptide produced a conversion of short vellus hairs to long terminal hairs and an enlargement of the follicular size with prolongation of anagen phase by enhancing the rate of cell proliferation. Moreover, in all the groups except the leaf extract-treated group, the hair was looking sparse. This explains the presence of greater number of hair follicles in the anagen phase of the hair growth cycle in leaf extract-treated groups. The histological study proved that there was no difference in the number of hair follicles in the treated and control groups and it was found to be 47 hair follicles per mm of the skin. This clearly indicates that no new hair follicle has been formed in the treated area. Uno (1991) studied the quantitative evaluation of hair growth potential of minoxidil on macaque monkey and fuzzy

rats by determining the percentage transformation of hair follicles from telogen to anagen. The study revealed that the topical application of 5% minoxidil produced approximately 10% conversion of telogen follicles into the anagen follicles. Though it is an antihypertensive drug, it was postulated that they readily stimulate the telogen buds and transform them to larger anagen follicles than those in previous cycles. Savin and Atton (1993) reported that the minoxidil induces proliferation of epithelial cells near the base of the hair follicle and may induce the vasodilation of scalp blood vessels. However, the exact mechanism of stimulation of hair growth was not known. Similarly, in our study, we have observed that the hair follicles periodically transformed from telogen to anagen phase in all the groups. Shortly after treatment, the secondary germs associated with aggregated dermal papilla cells in telogen follicles began to proliferate and their continuous growth and differentiation may result in construction of new anagen follicles. At the end of the course, the leaf extract-treated group showed 67% of the follicles in anagen phase whereas in ower extract-treated groups it was 60% and in placebo and control it was 54 and 50% of the anagen follicles, respectively. From this study, it is apparent that the leaf extract exhibited a more substantial effect compared to ower extract and placebo groups. Philpot et al. (1990) reported for the rst time the successful maintenance and growth of human hair follicles in vitro and the number of authors (Philpot et al., 1992; Bhul et al., 1989; Waldon et al., 1993; Jindo et al., 1994) have reported the organ culture technique to culture hair follicles from rodents with mixed success. It has been shown that the human hair follicles can be maintained for at least 9 days (Westgate et al., 1991) whereas the rat hair follicles do not continue to grow for more than 48 h in culture. It is also known that when hair follicles are stressed, they respond by premature entry into catagen and moreover it is also possible that as a result of the trauma of isolation they are more likely to enter a catagen-like state prematurely (Philpot et al., 1992). The hair follicles isolated from the skin of the rat neonates were found to possess intact morphology under an inverted binocular microscope. The isolated rat hair follicles could be maintained in vitro up to 72 h in the leaf extract-treated group and up to 48 h in the ower extract-treated group, placebo and control (Plate 1c and d). During this period, the hair follicle length increased signicantly and after 72 h there was no further growth. The increase in hair follicle length with time in culture may be attributed to the production of a keratinized hair shaft. After 48 h the follicles may enter into pseudocatagen phase. Despite the length of hair follicle increased in culture, the rate of growth is markedly less than in vivo. Since the culture conditions do not entirely mimic the complex in vivo environment, the in vitro growth rate may not correlate with the in vivo growth rate. The in vitro study revealed that the leaf extract has direct impact on hair follicles and thus may improve the hair growth. Inaoka et al. (1994) studied the effects of methanolic extracts of 80 herbs on hair growth and they reported that

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18 of the extracts apparently promoted hair regrowth on the mice. It was shown that the petroleum fraction of ethanolic extract of Tridax procumbens Linn. when administered orally as well as topically to the albino rats produced an effective hair growth (Saraf et al., 1991). In another study, effective hair growth potential of 70% ethanolic extract of leaves of Ginkgo biloba was reported and suggested to be used as a hair tonic (Kabyashi et al., 1993). However, the exact mechanism of such extracts on hair growth has not been discussed. Finally, it is concluded that the leaf extract of Hibiscus rosa-sinensis has a potential effect on maintaining the hair growth in in vivo and in vitro methods and it is suggested that the leaf extract of Hibiscus rosa-sinensis could be included as a constituent in the hair growth formulations. Acknowledgements We express our sincere thanks to CSIR, New Delhi, and Dr. T. Ramasami, Director, CLRI, for giving us the opportunity and encouragement to carry out the work. We are also thankful to Anna Sidha Research Institute for plant identication. References
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