Black soybean promotes the formation of active components with antihepatoma activity in the fermentation product of Agaricus blazei

The antihepatoma activity and related active components in the fermentation products of Agaricus blazei (AB) cultured in the medium containing soybean (S) or black soybean (BS) were investigated. AB(BS)-pE and AB(S)-pE were the ethanolic extracts from the fermentation products of AB(BS) and AB(S), respectively. According to the IC₅₀ values, AB(BS)-pE (161.1 and 24.0 μg/mL for Hep 3B and Hep G2 cells, respectively) exhibited stronger cytotoxicities against hepatoma cells than AB(S)-pE (>200 and 99.9 μg/mL for Hep 3B and Hep G2 cells, respectively). AB(BS)-pE was separated by silica gel column chromatography and eluted with n-hexane/ethyl acetate/methanol gradient solvent system into 21 fractions. Fraction 3 [AB(BS)-pE-F3], eluted with n-hexane/ethyl acetate (97:3 and 19:1, v/v), was the most active fraction having inhibitory activity on the proliferation of Hep 3B and Hep G2 cells (IC₅₀ of 3.6 and 1.9 μg/mL, respectively). Three major compounds, compounds 1-3, were further isolated from the AB(BS)-pE-F3 fraction by reversed-phase semipreparative high-performance liquid chromatography. Compounds 2 and 3 gave better antihepatoma activity than that of compound 1. The IC ₅₀ values of compounds 2 and 3 were 2.8 and 4.5 μg/mL for Hep 3B cells and 1.4 and 2.0 μ/mL for Hep G2 cells, respectively. The structures of compounds 2 and 3 were identified by UV, IR, electron impact mass spectrometry, and ¹H and ¹³C NMR to be blazeispirols A and C, respectively. Blazeispirols A and C existed in the mycelia but not in the broth and were more in AB(BS)-pE (49.9 ± 8.9 and 14.2 ± 2.4 mg/g, respectively) than AB(S)-pE (15.9 ± 1.7 and 3.9 ± 0.6 mg/g, respectively). Additionally, the result shows that the production of blazeispirols A and C was increased after cultivation in the medium containing black soybean on day 6 and reached the maximum on day 12, and the contents of blazeispirols A and C were negatively correlated with Hep 3B and Hep G2 cell viabilities (r= -0.84 to -0.93, P < 0.01). It suggests that blazeispirols A and C could be used as biomarkers to produce the fermentation product of A. blazei with antihepatoma activity.