The anti-hepatoma activity and its mechanism of extract and its active components from the fermentation mycelia of Ganoderma lucidum cultivated in the medium containing Glycine max (L) Merr. and Astragalus membranaceus

Ganoderma lucidum is a famous medicinal fungus that has been used as an herbal medicine in Asian regions for thousands of years. Several lines of evidence indicate that G. lucidum possess anti-allergic, anti-mutagenic, anti-viral, anti-inflammatory, antitumour and hypoglycaemic properties. On the other hand, Glycine max (L.) Merr. and Astragalus membranaceus have been reported that contain several important components with various physiological and biological activities. However, the liver protective function of fermentation mycelia of G. lucidum cultivated in the medium containing leguminous plants (G. max (L.) Merr. and A. membranaceus) has not been studied yet. The objective of our study was to investigate the anti-cancer effect of ethanolic extract of mycelia of G. lucidum (EMG) cultivated in the medium containing leguminous plants on human hepatocellular carcinoma cells (Hep3B) in vitro and in vivo assays. Furthermore, the active components of the fraction of EMG and their anti-hepatoma activity were determined.he anti-proliferation effect was observed in vitro growth of Hep3B cell lines after incubation with ethanolic extract of mycelia of G. lucidum (EMG) cultivated in the medium containing G. max (L.) Merr. and A. membranaceus using 200 L fermentor for 12 days. The results indicated that EMG inhibited cell viability in a dose- and time-dependent manner, and the IC50 treated with EMG for 24, 48 and 72 hr were 156.8, 89.9 and 70.1 μg/mL, respectively. EMG inhibited the growth of Hep3B cells via the sub-G1 peak rise in cell cycle analysis, DNA ladder, and increasing of caspase-3, caspase-8 and caspase-9 activities. These results suggested that EMG might suppress Hep3B cells growth through apoptosis in vitro.n vivo, we studied the anti-tumor activity of EMG in Hep3B tumor-bearing nude mice model. EMG was administered for 2 wk before inoculation of Hep3B cells and was continued for 9 wk. The results indicated that oral administration of 40 and 200 mg/kg bw/day of EMG significantly reduced the tumor volume and weight in xenografted nude mice; tumor volume was inhibited by 48 and 50% (p<0.05); tumor weight was inhibited by 60 and 61% (p<0.05), respectively. Immunohistochemistry showed that apoptosis is visualized by detecting cleaved caspase-3. Furthermore, there was no significant difference in body weights among the various groups at the end of the study. The AST and ALT values showed no significant difference between treatment groups and control group. The results implied that EMG feeding to mice did not appear to induce any adverse effects.n the pre-experiment of separation and identification of active components from EMG, the EMG was separated further by gel filtration chromatography to obtain 29 fractions. The effects of various fractions on cell viability of Hep 3B cells were analyzed by MTT assay. The results showed that fraction 8 (PF8) and 13 (PF13) had higher growth inhibition on Hep3B cells. The IC50 values of PF8 and PF13 for Hep3B cells were 15.7 and 21.8 μg/mL, respectively. Cell cycle analysis showed that the growth inhibition effect was associated with sub-G1 peak rising. Moreover, DNA ladder was observed from Hep3B cells treated by PF8 and PF13. Our results indicate that the fractions (PF8 and PF13) of EMG could inhibit growth of Hep3B cells through apoptosis. But the amounts of PF8 and PF13 were too few to identify.urthermore, EMG was fractionated into seven fractions (F1-F7). We found that F5 and F6 had higher growth inhibitory effects on Hep 3B cells than the other fractions, and F6 possessed enough amounts (about 2.1 g) to carry out a more detailed study. The F6 was separated further by HPLC to obtain two compounds, and was identified as 5α,8α-epidioxy-22E-ergosta-6,9(11),22-trien-3β-ol (9,11-dehydro- ergosterol peroxide, 9(11)-DHEP) and 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (ergosterol peroxide, EP) by NMR. 9(11)-DHEP and EP showed growth inhibition effect on Hep3B cells and DNA ladder was observed with 20 μg/mL of treatment. Thus, it was suggested that 9(11)-DHEP and EP should be the major active compounds in EMG to induce apoptosis of hepatoma cells.verall, these finding demonstrated that EMG could suppress Hep3B cells growth through apoptosis in vitro and exhibit anti-tumor effects in vivo model, and suggested that it has a useful potency in cancer prevention. The anti-cancer activity of EMG might attribute to 9(11)-DHEP and EP.