Abstract: Emblica (Phyllanthus emblica Linn.) has been used to promote the growth of hair in traditional medicine, however less is known regarding its pharmacological activities in hair follicles. To investigate the effects of emblica fruit extracts in terms of hair growth stimulation, the proliferative effects of extract in HaCaT keratinocytes and Dermal Papilla (DP) cells of human hair follicles were determined by MTT assay and cell counts. The results show that emblica extract stimulated proliferation of DP cells in a concentration-dependent manner, whereas it showed minimal effect on keratinocytes, suggesting that the extract might promote hair growth by prolonging the anagen phase through the proliferative effect on DP cells.
INTRODUCTION
Dermal Papilla (DP) cells, specialized mesenchymal cells, play an important part in hair cycle mediating hair growth and loss (Rogers and Hynd, 2001; Botchkarev and Kishimoto, 2003; Alonso and Fuchs, 2006). The DP cells not only provide nutrient supply but also produce a number of growth factors and cytokines which influence proliferations and functions of several follicular cells namely keratinocytes (Matsuzaki and Yoshizato, 1998; Stenn and Paus, 2001; Fujie et al., 2001). Anagen is an active growth phase of hair follicle, thereby the length and strength of hair is depended on the duration of this phase. In addition, prolongation of anagen is clinically shown to maintain the hair follicle growth (Paus and Cotsarelis, 1999). Many studies have reported the positive correlation between DP cell number and follicle size during anagen (Ishino et al., 1997; Elliott et al., 1999). In particular, cultured DP cells are currently used for evaluation of potential hair growth stimulators in several studies (Rho et al., 2005; Sung et al., 2006).
Phyllanthus emblica Linn., commonly known as emblica or amla, is distributed over tropical and subtropical areas of China, India, Indonesia and Thailand. The emblica fruit extract represents several pharmacological activities, such as anti-oxidant, anti-tumor and anti-inflammation, attributable to its high vitamin C and polyphenolic content (Scartezzini et al., 2006; Liu et al., 2008). Importantly, emblica fruits have been long used as a hair growth nourishment in traditional Tibetan and Ayurvedic medicine (Dweck and Mitchell, 2005). Recent in vivo studies suggested emblica as one of the herbs that acclaimed with hair growth promoting activity as it is composed in the herbal formulations that effectively enlarge size and prolong the anagen phase of hair follicles (Purwal et al., 2008; Jadhav et al., 2009). However, its biological effects on follicular cells are still largely unknown.
In the present study, we performed in vitro experiments using cultured human DP cells of healthy hair follicles and HaCaT keratinocytes to investigate the effect of emblica fruit extract.
MATERIALS AND METHODS
Plant materials: Fresh emblica fruits were collected from Srakaew Province of Thailand, where the climate is tropical. The samples were picked at commercial harvest time (May 2007) and selected for the uniformity of shape and maturity.
Extraction and sample preparation: Fresh emblica fruits were cut into small pieces. The materials (1 kg) were macerated and agitated in distilled water (5 L) and then filtered. The filtrate was converted to powder form by spray drying. Yield of the extract was approximately 35%. The extract contained 308-320 mg g-1 of total phenolics calculated as Gallic Acid Equivalent (GAE) using Folin-Ciocalteu’s reaction. Various samples of emblica extract were prepared by dissolving the dry emblica extract powder in deionized water to the indicated concentrations (10-500 μg mL-1).
Chemical reagents: Vitamin C (L-ascorbic acid) was obtained from Ajax Finechem (Auckland, New Zealand). Ketoconazole was obtained from Crosschem International Company, Derb and Co. (Lugano, Switzerland). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Chemical, Inc. (St. Louis, MO, USA).
Cells and cell culture: Human follicle dermal papilla cells (DP cells) were obtained from PromoCell (Heidelberg, Germany). Cells were cultured in DP cell growth medium (PromoCell) containing 100 units mL-1 of penicillin and 100 μg mL-1 of streptomycin (Gibco, Gaithersburg, MA, USA) in a 5% CO2 environment at 37°C.
Human immortalized keratinocyte HaCaT cells were obtained from Cell Lines Service (Heidelberg, Germany) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MA, USA) supplemented with 2 mmol L-1 L-glutamine, 10% fetal bovine serum, 100 units mL-1 of penicillin and 100 μg mL-1 of streptomycin in a 5% CO2 at 37°C.
Cell proliferation: Cell proliferation was determined by MTT colorimetric assay (Mosmann, 1983) and cell counts. For MTT assay, cells at a density of 5x103 cells per well (3 replicates per treatment) were seeded in a 96-well plate and incubated with emblica fruit extracts (0-500 μg mL-1) for 24 h. After that cells were incubated with 500 μg mL-1 of MTT for 4 h at 37°C. The supernatant was then removed and dimethylsulfoxide (DMSO) was added to dissolve the formazan product. The intensity was spectrophotometrically measured at 550 nm using an ELISA reader (Anthros, Durham, NC, USA). All analyses were performed in three independent replicate cultures. The intensity readings were normalized to a non-treated control and represented in the terms of relative cell proliferation.
For cell counts, cells at a density of 2x104 cells well-1 were plated in 24-well plates. Cells were cultured in a control medium or emblica extract-containing medium. At each time point (0, 24 and 48 h), the number of DP cells was counted using hemocytometer under light microscope.
Statistical analysis: Statistical analysis is performed using the Student’s t-test. A p-value of less than 0.05 would be considered as statistically significant.
RESULTS
Effects of emblica fruit extract on cell proliferation: The cell growth promoting effect of emblica fruit extract was investigated in immortalized keratinocytes (HaCaT) and primary human hair DP cells using MTT assay. Cells were incubated with various concentrations (10-500 μg mL-1) of emblica extract and cell proliferation was determined after 24 h. The results indicated that emblica extract caused a dose-dependent increase in DP cell proliferation (1.11±0.18-fold, 1.19±0.19-fold, 1.88±0.05-fold, 2.31±0.11-fold and 2.67±0.39-fold at 10, 50, 100, 250 and 500 μg mL-1 of extract, respectively), whereas, it had minimal proliferative effect on HaCaT keratinocytes (Fig. 1a). At various concentrations ranging from 100-500 μg mL-1, the extract significantly increased DP cell growth with the maximal effect by approximately 3-fold compared to the non-treated control. Notably, at the treatment dose of 500 μg mL-1, the proliferative effect of emblica extract was only slightly increased compared to that of 250 μg mL-1.
To confirm the proliferative effect of emblica extract on DP cells, DP cell numbers were counted after cultivation in emblica extract (100 μg mL-1)-containing medium at various times. Consistent with the MTT assay, the number of DP cells markedly increased in the emblica-treated group over a non-treated control (Fig. 1b).
| Fig. 1: | Emblica fruit extract increases DP cell proliferation. (a) HaCaT and DP cells were treated with various concentrations of emblica extract (10-500 μg mL-1) for 24 h and cell proliferation was determined by MTT assay and (b) DP cells were cultured in emblica extract (100 μg mL-1)-containing medium. Cell numbers were counted using hemocytometer at 24 and 48 h. Values are mean of triplicate samples±SD. *p<0.05 versus non-treated control |
| Fig. 2: | Proliferative effects of emblica extract, vitamin C and ketoconazole on DP cells. Cells were treated with emblica extract (100 μg mL-1), vitamin C (50 μg mL-1) or ketoconazole (50 μg mL-1) for 24 h and cell proliferation was determined by MTT assay. Values are mean of triplicate samples±SD. *p<0.05 versus non-treated control |
Proliferative effects of emblica fruit extract and hair growth stimulators on DP cells: Previous study has shown that vitamin C derivative can induce hair growth in vivo and in vitro. This action of vitamin C derivative was shown to be caused by its ability to induce DP cell growth, while having a minimal effect on other follicular cells (Sung et al., 2006). Moreover, clinical evidence indicated that anti-fungal ketoconazole possesses an ability to promote hair growth (Pierard-Franchimont et at., 1998; Ellis and Sinclair, 2008). Therefore, we compared the proliferative effect of emblica extract with vitamin C and ketoconazole.
Figure 2 shows that emblica extract, vitamin C (L-ascorbic acid) and ketoconazole exhibited significant proliferative effects on DP cells. The proliferative effect of emblica extract at its minimal effective concentration (100 μg mL-1) was stronger than those of vitamin C and ketoconazole at the concentration of 50 μg mL-1 (2.03±0.21-fold vs. 1.25±0.10-fold and 1.15±0.09-fold, respectively) (Fig. 2). Notably, higher concentrations (>50 μg mL-1) of vitamin C and ketoconazole could not be used in the present study due to their cytotoxic effect.
DISCUSSION
Emblica extract has been used for centuries in traditional medicines to stimulate hair growth (Dweck and Mitchell, 2005). However, the mechanisms underlying its activity are largely unknown. We document for the first time that emblica fruit extract stimulated the growth of cultured DP cells. The increase in DP cell growth could be extrapolated to the hair growth stimulating potential since: (1) DP cells secrete several cytokines which are required for follicular cell growth and functions; (2) an increase of DP cell number or activity is tightly related to enlarged hair follicle and prolonged anagen phase and (3) a reduction of DP cell number caused by several stimuli contributed to hair loss (Ishino et al., 1997; Elliott et al., 1999; Hoffmann, 2002; Rho et al., 2005; Krause and Foitzik, 2006). Thus, the results of this study provide evidence supporting emblica fruit extract as a good candidate to be developed for the treatment of hair loss. Long-term use of available synthetic drugs, minoxidil and finesteride, is limited by its side effects. In this regard, biologically active herbal extracts such as emblica fruit extract along with the suitable hair follicle-targeted delivery system are currently in demand in the industry. Since, it is widely known that emblica extract contains high content of vitamin C and polyphenolic compounds (Scartezzini et al., 2006; Liu et al., 2008) and vitamin C derivative has been reported to stimulate DP cell growth in vitro (Sung et al., 2006), vitamin C may at least in part contribute to an enhancing effect of emblica extract on DP cell proliferation. However, our comparative study on DP cell proliferation revealed that emblica extract, at the minimal effective dose, had a higher proliferative effect compared to vitamin C, suggesting that the effect of emblica extract might not be attributed to vitamin C in the form of ascorbic acid but to its derivatives contained in emblica extract and/or other components such as polyphenols. In addition, we first reported the direct enhancing effect of ketoconazole on DP cell proliferation; however, this effect of ketoconazole seemed weak when compared to the effect of emblica extract.
In summary, the present study shows the strong proliferative effect of emblica extract on DP cells, which may prolong the anagen phase of hair cycle. The pharmacologic activities particularly its effects on DP cell-derived growth factors may be further investigated.
ACKNOWLEDGMENTS
This research was supported by Thailand Research Fund RGJ Grant 5.Q.CU./49/A1 (U. Nimmannit) and grant for development of a new staff, Chulalongkorn University (P. Chanvorachote).