Hair Growth Promoting Effect of Radish Crude Saponin Extract on Athymic Nude Mice

Abstract

This study investigates the hair restoration efficacy of selected radish saponin extracts on nude mice. Nude mice genetically predisposed to pattern balding were used in this study. Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of radish saponin may represent a novel strategy for the management and therapy of certain forms of alopecia. The term of hair density of PEE treated nude mice were significantly increase as compared with of control nude mice. Histological observation of skin sample showed no hair follicle or only distorted hair follicles were observed in the control samples, in contrast, by the PEE treatment groups showed a fully formed and increased the number of hair follicles up to three times higher than that of control group in terms of the number of hair follicles in nude mouse skin.PEE treated mice the number of BrdU-labeled keratinocytes per anagen follicle increased significantly, especially in the follicular bulbs and outer root sheath compared with the control mice. Moreover, PEE-treated nude mice also exhibited a significant increase in the number of BrdU-labeled epidermal keratinocyte proliferation.

Figure 1. Stimulation of hair growth by PEE of radish crude saponin in nude mice wasa dministered for 16 consecutive days. A) Vehicle (control) treated nude mice skin on Days 0, 08, 10, 16. Control mouse exhibiting nearly complete alopecia on Day 16. B) PEE treated nude mice skin on Days 0, 08, 10, 16 respectively, while PEE of radish crude saponin induced Profound effect is evident and having the dense hair coat with most hair coverage on Day 16. C) Skin surface reveals that the only a few hair shaft emerging from skin surface and leads to bending in control nude mice on Day 16 (arrow head). D) PEE fraction has a normal dense hair coat on Day 16. Four mice /group were evaluated for each treatment group. Digital image were taken from skin surface of vehicle (C) and PEE (D) treated nude mice on Day 8 and 16 by KONG, Bom-Viewer Plus, 80X magnification lens.

Figure 2. Monitoring of hair coverage area in athymic nude mice. Mice were topically treated with PEE, HeF, BuF, WaF versus vehicle control and or 5% Minoxidil for 20 consecutive days. Hair coverage area of different treatment groups on nude mice were evaluated by hair monitoring scale as illustrated in Table 1. All experimental groups were statistically analyzed for differences of hair coverage by the analysis of variance at 1% levelof significance (p < 0.01).

Figure 3. Hair density in athymic nude mice on experimental day 8 and day 16, treated for 20 consecutive days with PEE, HeF, BuF, WaF versus vehicle control and or 5% Minoxidil. All experimental groups were statistically analyzed for differences in hair density by theanalysis of variance at 1% level of significance; PEE induces a statistically significant increase in hair density. Digital image were taken on day 8 and 16 by KONG, Bom-Viewer Plus (Seoul, Korea) with a 80X and 200X magnification lens.

Figure 4. H&E- stained section of the skin from control and PEE treated athymic nude mice. A) Cystically dilated hair follicles filled with fragmented hair shafts and keratinized debris (arrows) that are normally present in nude mouse skin. E) PEE treated skin exhibits prominent follicular hypertrophy (arrows) as well as moderate sebaceous gland hypertrophy (arrowheads) with a normalization of follicular morphology and the presence of hair shafts above the skin's surface (arrows and inset). Note that PEE treated HFs were uniformly in late anagen phase of the hair cycle. D) Vehicle treated nude follicle, cortex formation is severely injured: IRS: inner root sheath, ORS: outer root sheath. F) Skin from PEE treated hair shaftregularly formed, intact and coated by a clearly discernible hair cuticle. (B, C) Skin of control nude mice shows hair shaft twist and coil at the level of sebaceous gland (B, C). (G)PEE treated mouse shows well differentiated straight hair shaft penetrating the skin surfaceshows no substantial difference with that of wild type. For further explanation, see text; RS: inner root sheath, ORS: outer root sheath, M: medulla, C: cortex, SG: sebaceous gland, HF: hair follicle, OST: Osteum. Scale bars: A, B = 100μm; C, D, E, F = 50μm.

Figure 5. Number of hair follicle varies remarkably between a skin section from a control nude nouse with that of PE treated mice on day 16 (A and B). A) Control mouse with abortive and less number of hair follicle. B) Test nude mouse reveal normal and increase in number of hair follicles in the dorsal skin of PEE treated mice. The sections were stained with hematoxylin and eosin (original magnification, X100).

Figure 6. BrdU Immunohistochemistry for keratinocytes proliferation in control (Figure A, B, C) and PEE treated (Figure D, E, F) athymic nude mice skin. BrdU positive cells were detected in both the hair matrix and the outer root sheath (arrows). Arrow indicates follicular keratinocytes of the outer root sheath of hair bulb D: A moderate increase in BrdU-labeled follicular keratinocytes, particularly within follicular bulbs and outer root sheats (filled arrowheads), as well asa slight increase in BrdU-labeled peripheral epithelial cells in sebaceous glands (open arrowhead) in PEE treated nude mouse skin versus control nude mouse skin (C). Scale bar in A, 100 μm.

Table 1. Effect of PE extraction of radish crude saponin on follicle count in nude mouse skin.

Table 2. Proliferation of BrdU positive keratinosites in PEE of radish saponin treated Nude Mice.