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To determine proper range of each extracts concentration for in vitro efficacy evaluation, neutral red assay was performed. Within the test range of 0.1~10 μg/mL, all extracts did not show any significant effect on cell proliferation or cytotoxicity. However, theses extracts showed cytotoxicity at 100 μg/mL.
Testosterone has been considered as a factor which induces apoptosis of dermal papilla cell. It was evaluated through neutral red assay and microscopic observation. However, apparent evidences how testosterone induces apoptosis were not found. RT-PCR was performed to determine the effects of each herbal extracts on growth factor expression. When dermal papilla cells were cultured with predetermined concentration of extracts for 48 h, the expression of VEGF
was up-regulated by A. radix and S. flavescens, KGF was up-regulated by S. flavescens and T. occidentalis, respectively. As type Ⅱ 5α-Reductase is crucial factor which induces androgenic alopecia, the effects of each herbal extracts on type Ⅱ 5α-Reductase expression was evaluated through western blot analysis.
All herbal extracts significantly down regulated the expression of type Ⅱ 5α-Reductase. Espite reactive oxygen species (ROS) is not the direct reason of androgenic alopecia, ROS deteriorates it. DPPH assay was performed to evaluate free radical scavenging effect of each extracts. All herbal extracts showed significant antioxidative capacity and among this A. radix and S. flavescens showed almost the same level of antioxidative capacity of positive control, α-Tochopherol. The efficacy of herbal extracts was reconfirmed through in vivo experiment using B6CBAF1/j mice. Liposomal formulation was selected as a drug delivery system to maximize the medicinal treatment and the stability of the formulation was evaluated before the in vivo experiment was performed.
Liposomal formulation was prepared by thin-film hydration method. Phosphatidylcholine, cholesterol, and other constituents were dissolved in 100% ethanol, then thin-film was obtained by dehydrating in a round flask. The film was rehydrated in distilled water and sonicated or/and homogenized by high pressure homogenizer. The diameter of liposome prepared by this protocol was in range of 160~200 nm. The stability evaluated by time and temperature was
not much changed. Mice were subcutaneously injected with testosterone, which was suspended in an aqueous solution. Injections were given daily for 14 weeks, then hair loss was induced. Each formulations were applied on the upper dorsum once a day for 12 weeks. At the end of the study, the mice were sacrificed by ether inhalation. Upper and lower dorsum samples of mouse were taken and stained for microscopic analysis. The herbal extracts showed similar
degree of hair loss inhibition effects compared with positive control, cyproterone acetate, in the aspects of number and depth of hair follicles. Consequently, the herbal extracts and the liposomal formulation used in this study could be useful for treating hair loss.