Hair is one of the vital parts of body derived from ectoderm of the skin and it is one of the protective appendages on the body [1]. It is an important of the overall appeal of the human body [2,3]. Many people suffer from hair loss or hair thinning, despite the development of several medical treatments. Therefore, it is important to develop new therapies that prevent hair loss and increase hair growth. In this respect, alternative medicine has attracted interest, although it has not yet been included into mainstream of medical care, due to the limited scientific proofs and incomplete knowledge of the mechanisms involved [4].

Many plant extracts have been traditionally used for treating hair loss in oriental medicine. However, there is no report available on hair growth promoting activity of N. crenulata leaves. Therefore, the present study was focused on the scientific investigation of the hair growth potential of the leaf extracts of N. crenulata.

Leaf collection and identification

The leaf specimens were collected in the month of August from kumbakonam, Tamil Nadu, India. The leaf specimens were authenticated by Professor N. Raaman, Herbal Science Laboratory and centre for Advanced Studies in Botany, University of Madras, Chennai. After a thorough investigation leaves were checked for any pathological disorders and contamination of other plants and were washed with distilled water.

Preparation of extracts

The fresh leaves (300 grams) were grounded into paste and were extracted with water for 12 h at room temperature. This process was repeated successively with chloroform and acetone for 72 h at room temperature until the color of the extract becomes pale. The extracts obtained were filtered separately using Whatmann No. 1 filter paper. This was repeated for 2 to 3 times and similar extracts were pooled together and dried on water bath until the constant weight with dry mass was obtained for solvent extracts. The residual extracts were stored in refrigerator at 4°C in small and sterile glass bottles.

Estimation of total phenolic content

The amount of total phenolics in leaf extracts was determined with the Folin-Ciocalteau reagent using the method of Spanos and Wrolstad (1990) [5], as modified by [6]. To 0.50 ml of each sample (three replicates), 2.5 ml of 1/10 dilution of Folin-Ciocalteau’s reagent and 2 ml of Na₂CO₃ (7.5%, w/v) were added and incubated at 45°C for 15 min. The absorbance of all samples was measured at 765 nm using a Spectronic 20D+ spectrophotometer. Results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw). A standard curve was prepared from 0 to 100 mg/l gallic acid (Figure 1). Total phenolic content was expressed in mg gallic acid equivalents/g dry matter using the following equation based on the calibration curve: y=0.001x+0.002, r²=0.996, where x was the gallic acid equivalent (mg GAE/g dw) and y was the absorbance.

Figure 1: Calibration curve of gallic acid used to estimate the total phenolic content in solvent extracts, plotted with a 95% confidence interval.

Primary skin irritation test and treatment for hair growth activity in vivo

Wistar albino rats of either sex, weighing 180-200 g, were obtained from National Institute of Nutrition (NIN), Hyderabad and reared in Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University. All the animals were housed in cages at a room temperature of 25 ± 2°C with 12 h dark/12 h light cycles. The rats were fed with rat pellets and water ad libitum. The experiments were approved by the Institutional Animal Ethics Committee, Annamalai University (Proposal Number: 841). Hairs from the dorsal side of the rats were removed using a commercially available hair remover (Veet) for complete removal of hair from 4 sq cm area [7]. The shaved area was cleaned with surgical spirit and the animals did not show any toxic effects when acetone, water and chloroform extracts were applied for 48 hours post application. Hence the prepared extracts were considered safe for topical application [8]. Twenty albino rats were divided into five groups of four animals in each group. Starting the following day (day 1), 0.2 ml of leaf extracts dissolved in 3.5% DMSO was applied topically, on a daily basis, for 30 days.

Hair length determination

Hairs were plucked randomly from the shaved area of all rats on 10, 20, and 30 day after beginning the treatment. The length of 10 hairs was measured and the average length was determined. The results are expressed as the mean length ± S.D. of 10 hairs.

Hair weight determination

After 30 days, the rats were sacrificed by cervical dislocation. 1 cm² area of dorsal skin with hair and without hair was cut from all the rats of each group and weighed by using analytical balance. After measuring, hair weight was calculated by subtracting skin weight from skin with hair weight.

Three rats from each group were authenticated after 30 days of treatment. Skin biopsies were obtained from the shaved portion and preserved in 10% formalin. Pieces of skin tissues were implanted in paraffin wax and sectioned into a thickness of 7 μm. The sectioned tissues were stained with haematoxylin and eosin and the follicular phases of hairs were examined under microscope with an ocular micrometer [9].

All experimental measurements were carried out in triplicate and are expressed as average of three analyses ± standard deviation. Correlation between the antioxidant activity and total phenolic contents was carried out using the correlation and regression in the Excel program (Microsoft Excel v. 2007). The data were analyzed using one way analysis of variance (ANOVA) and DMRT on SPSS/PC (statistical package for social sciences, personal computer) Ver. 15 and the results were considered statistically significant if the p value is less than or equal to 0.05.

A calibration curve used to estimate the total phenolic content is shown in Figure 1. The total phenolic contents (TPC) of leaf extracts of N. crenulata are shown in Figure 2. The total phenolic content of aqueous, acetone and chloroform solvent extracts were 161 ± 1.62, 34 ± 1.81 and 1 ± 2.06 mg/g of GAE. Among all the extracts analyzed, a significant phenolic content was found for aqueous extract.

Figure 2: Comparison between the total phenolic contents of the solvent extracts of N. crenulata leaves.

Hair growth initiation from the shaved area was observed in all the groups. Topical use of leaf extracts of N. crenulata onto the shaved area on the animals, produced a significant increase in the rate of hair growth, compared with the vehicle (DMSO) treated and control animals. Rats treated with 0.2 ml of acetone extract produced a significant effect on the hair growth when compared to other groups (Figure 3).

Values are expressed as mean + SD, [n=3]. Values not sharing a common superscript significantly differ at P < 0.05. (Duncan’s multiple range tests)

Figure 3: Hair growth promoting effect of Naringi crenulata leaf extract in wistar rats. The back skins of rats were shaved, and then different extracts of Naringi crenulata were topically applied for 30 days. (A) Control (B) DMSO treated (C) chloroform extract treated (D) acetone extract treated and (E) water extract treated.

The length of hair for acetone, chloroform and water treated groups were 8.2, 7.5 and 5.99 mm, respectively, compared to the vehicle treated (5.75 mm) and control (5.7 mm) at the end of the experiment (Figure 4).

Figure 4: Effect of Naringi crenulata leaf extracts on the hair length of wistar rats.

The weight of newly grown hairs in all the treated groups were measured and compared with that of the control group. It was found that weight of hair was highest for chloroform treated group and was measured to be 0.040, 0.039, 0.038, 0.037 and 0.035 mg/cm² area of dorsal skin for chloroform, acetone, water, DMSO treated and control rats (Figure 5). Statistical analysis (ANOVA) has shown that this increase in weight of hair in treated groups is not significantly different when compared to control and DMSO treated groups (P >0.05).

Values are expressed as mean + SD. Values not sharing a common superscript significantly differ at P < 0.05. [DMRT]

Figure 5: Effect of Naringi crenulata leaf extracts on the weight of hair of wistar rats.

The reverse side of the dorsal skin of treated rats showed more vascularisation and colour changes as compared to control groups (Figure 6). Normally, the colour of the reverse side of telogen-phase skin is white, because hair follicles at this phase are very small. This denotes that normal hair growth cycle was stimulated. Moreover, the histological appearance of skin observed was similar in all the treated groups. There was no much difference in the number of hair follicles per millimetre of skin of the chloroform, water, and control rats. The number of hair follicles was found to be 4-7 per mm of skin of chloroform, water, and control rats and was 3-5 in acetone treated group (Figure 7).

Values are expressed as mean + SD. Values not sharing a common superscript significantly differ at P < 0.05. [DMRT]

Figure 6: Effect of Naringi crenulata leaf extracts on the telogen-phase and vascularisation of back skin of wistar rats (A) Control (B) DMSO treated (C) water extract (D) acetone extract treated and (E) chloroform extract treated.

Figure 7: Histology section of albino rat skin after 10 days of treatment with leaf extract of Naringi crenulata, (A) control (B) acetone extract treated (C) water extract treated (D) DSMO treated (magnification x 400).

Hair loss is a distressing condition for an increasing number of men and women; therefore, to develop new therapies for the treatment of hair loss is of great importance. In the present study, hair growth promoting potential of the crude chloroform and acetone extracts of N. crenulata was demonstrated in rodent models. The exact mechanism of action or the component(s) responsible for the hair growth promoting activity in the plant extract could not be established in this study. However, a number of investigators have shown that flavonoids and terpenoids possess hair growth promoting activity by strengthening the capillary wall of the smaller blood vessels supplying hair follicles, improve blood circulation to nourish the hair follicles and thereby promoting hair growth [10-12]. In the present study increased blood circulation and skin colouration was observed hence the present study is in agreement with the study of the above authors. Other workers also implicate flavonoids in stimulating telogen to anagen phase, a process involved in hair growth [13]. Sampath kumar and Ramakrishnan isolated flavonoids and terpenoids compounds from N. crenulata plant extracts which were found to be responsible for the various biological activities [14]. It may therefore, be possible that these flavonoids compounds and other chemical components such as triterpenes contained in these extracts may be responsible for the observed hair growth promoting effect and that, these compounds may also have stimulatory actions on these hair growth factors.

Finally, this study concluded that the crude chloroform and acetone leaf extracts of N. crenulata possesses hair growth promoting activity and further work needs to be carried out in order to establish the mechanism of action and the exact chemical component responsible for the hair growth potential of N. crenulata.