Inhibition of Rat 5α-Reductase Activity and Testosterone-Induced Sebum Synthesis in Hamster Sebocytes by an Extract of Quercus acutissima Cortex

Abstract

Objective. Bokusoku (BK) is an extract from the Quercus cortex used in folk medicine for treatment of skin disorders and convergence, and is present in jumihaidokuto, a traditional Japanese medicine that is prescribed for purulent skin diseases like acne vulgaris. The excess of sebum production induced by androgen is involved in the development of acne. Our aim is to examine whether BK and its constituents inhibit testosterone metabolism and testosterone-induced sebum synthesis. Methods. Measurements of 5α-reductase activity and lipogenesis were performed using rat liver microsomes and hamster sebocytes, respectively. Results. BK dose-dependently reduced the conversion of testosterone to a more active androgen, dihydrotestosterone in a 5α-reductase enzymatic reaction. Twenty polyphenols in BK categorized as gallotannin, ellagitannin, and flavonoid were identified by LC-MS/MS. Nine polyphenols with gallate group, tetragalloyl glucose, pentagalloyl glucose, eugeniin, 1-desgalloyl eugeniin, casuarinin, castalagin, stenophyllanin C, (-)-epicatechin gallate, and (-)-epigallocatechin gallate, inhibited testosterone metabolism. In particular, pentagalloyl glucose showed the strongest activity. BK and pentagalloyl glucose suppressed testosterone-induced lipogenesis, whereas they weakly inhibited the lipogenic action of insulin. Conclusions. BK inhibited androgen-related pathogenesis of acne, testosterone conversion, and sebum synthesis, partially through 5α-reductase inhibition, and has potential to be a useful agent in the therapeutic strategy of acne.

Figure 1

Representative TLC-image showing inhibition of rat liver microsomal 5α-reductase in testosterone metabolism by BK treatment. Testosterone (T.) was incubated at a final concentration of 3.5 μmol/L for 30 min in the presence or absence of rat liver microsomes (40 μg/mL) and the cofactors. Bokusoku (BK) was added at 30 μg/mL before starting the reaction. The androgens were extracted with ethyl acetate after the incubation and analyzed by TLC. All androgen standards were applied at 0.1 μg/μL/spot. DHT: dihydrotestosterone, A. diol: 5α-androstane-3α,17β-diol.

Figure 2

Quantification of inhibitory effect of BK on rat liver microsomal 5α-reductase activity. Testosterone was incubated at a final concentration of 3.5 μmol/L for 30 min in the presence or absence of rat liver microsomes (40 μg/mL) and the cofactors. Bokusoku (BK) was added to the reactions at 3, 10, 30, or 100 μg/mL before starting the reaction. Testosterone remaining was extracted with ethyl acetate after the incubation and quantified by HPLC technique. Data are shown as relative amounts of testosterone, which are a percentage of testosterone alone control reaction. N = 3.

Figure 3

Suppression of testosterone-induced sebum synthesis of hamster primary sebocytes by BK treatment. Hamster-derived sebaceous gland cells (Ha-SE) were precultured, until confluent in the presence of epidermal growth factor, and further treated with the indicated concentrations of bokusoku (BK) and a differentiation factor, 10 μmol/L testosterone or 10 μg/mL insulin. Eight days later, the sebum synthesis was determined using a lipogenesis detecting assay kit. N = 3.

Figure 4

Representative images of hamster primary sebocytes treated with or without BK in the presence of a differentiation factor. Hamster-derived sebaceous gland cells (Ha-SE) were precultured until confluent in the presence of epidermal growth factor, and further treated with 10 μg/mL bokusoku (BK) or vehicle in the presence of a differentiation factor, 10 μmol/L testosterone or 10 μg/mL insulin. Eight days later, cells were stained with Oil Red O. The representative images of sebum droplets were shown for ((A), (a)) none, ((B), (b)) testosterone + vehicle, ((C), (c)) testosterone + BK, ((D), (d)) insulin + vehicle, and ((E), (e)) insulin + BK. Scale bars: 20 μm. The images shown in (A), (B), (C), (D), and (E) were enlargements of those shown in (a), (b), (c), (d), and (e), respectively.

Figure 5

Suppression of testosterone-induced sebum synthesis of hamster sebocytes by pentagalloyl glucose, a main active constituent of BK. Hamster-derived sebaceous gland cells (Ha-SE) were precultured until confluent in the presence of epidermal growth factor and further treated with the indicated concentrations of pentagalloyl glucose or bokusoku (BK) (30 μg/mL) and a differentiation factor, 10 μmol/L testosterone or 10 μg/mL insulin. Eight days later, the sebum synthesis was determined using a lipogenesis detecting assay kit. N = 3.