## 4 Discussion  
Hair growth was rapidly increased from day 9 after hair removal and waxing, similar with other studies [28, 29, 30]. The kinetic hair growth on each day was not significantly different among the groups, probably due to small sample sizes or limitation of the measurements to detect the subtle changes on each day. In addition, the individual differences shown in the same group might be involved in the incomplete anagen synchronization, which may result in a failure to approach the significant differences. However, the higher doses of fRGM and PH increased the final grown hair regions on day 0 to 15 post-treatment. Hair length and thickness increased for all doses of fRGM and PH, and hair weight increased for the treatment groups except the low-dose groups. The combination treatments also showed beneficial effects on hair growth and hair quality, even though the combination dose was half the high doses of fRGM and PH. Hair weighing and microscopic measurements of hair length and width are generally used in clinical studies to evaluate the effects of drugs or cosmetic molecules as a hair tonic [33]. Further, clinical diagnosis for hair loss needs scalp biopsy to observe the follicular quantity and diameter and other morphometric data for abnormal hair cell growth. The current histopathological analyses revealed that components of fRGM and PH contribute to increasing follicular numbers, diameters and proliferation, in particular in the hair follicles located at the deep subcutis. These suggest that the fRGM and PH may promote hair-growth cycle, probably by enhancing anagen-phased cell division, as well as functions as a hair tonic. The length and thickness of a hair shaft depend on the duration of the hair follicular cycle, in particular duration of anagen phase [34]. Indeed, androgenetic alopecia produces thinning of scalp hair, and the miniaturization of hair follicles involves shortened anagen phase and reduced sizes of the hair matrix [3]. In this context, enhancing the induction of anagen or prolonging the duration can be a therapeutic strategy for hair loss. The fRGM, PH and the combination increased the hair follicles closed to the deep layer of subcutis, suggesting promoting effects on development of the follicles in anagen III to VI or catagen stages [34]. It might result in the increased total hair follicles in the treatments of the higher doses of fRGM, PH and the combination. The increases in anagen-phased follicles might be due to induction of an earlier anagen phase or transition of hair growth from telogen to anagen [35,36]. The transformation from telogen- to anagen-phased hair follicles undergoes through rapid proliferation of follicular keratinocytes and elongation and thickening of the hair shaft [37]. Here, considering that the fRGM and PH and the combination increased Ki-67 positive cells in the bulb of the subcutis, the anagen-like hair follicles could be regenerated by rapid proliferation of the matrix keratinocytes, producing increased follicular sizes for a new hair fiber. It suggests that the components contained in the fRGM and PH may contribute to induction of anagen-phased follicles and improve the altered hair-growth cycles or destroyed hair follicles in hair loss [2,38]. Hair growth is regulated by various hormones, cytokines and growth factors. For example, endorsogens reduce the anagen phase, increasing the other phased follicles and delaying the telogen to anagen transitions [39]. Basic FGF promotes proliferation of the papilla cells, contributing to elongate the hair shaft [40], and hepatocyte growth factor and vascular endothelial growth factor (VEGF) stimulate growth of the hair follicle and hair shaft [41,42]. The growth factors and others including epidermal growth factor, keratinocyte growth factor (KGF), insulin-like growth factor-I and transforming growth factor have shown mitogenic and motogenic effects on keratinocyte [43]. The traditional polyherb formula used here is composed of seven herbs, and the components may contribute to enhancing the hair-growth cycle.