Studies on the liver protective function and its mechanism of the fermentation products of Ganoderma lucidum and Agaricus blazei cultivated in the medium containing leguminous plants

Liver is the major organ in human being for metabolism, detoxification and antioxidation. However, the annual report of the Department of Health, Executive Yuan, Taiwan, indicated chronic liver disease and liver cancer are the leading causes of death in 2007. Recently, many researches have reported that fungi, such as Ganoderma lucidum (GL) and Agaricus blazei (AB), and leguminous plants, such as black soybean and Astragalus membranaceus, contained many active compounds which can manipulate biological activities, including antitumor activity, induction of apoptosis, and hepato-protection etc. However, there is almost no research on the fermentation products of GL and AB cultivated in the medium containing leguminous plants. The objectives of this project were to investigate the anti-hepatoma activity and liver protective function of the fermentation products of GL and AB cultivated in the medium containing black soybean and/or A. membranaceus; to evaluate the potential active compunds and their anti-hepatoma activities; and to study their anti-hepatoma mechanisms. The study may provide the important information for the development of health food in the near future.irstly, the anti-hepatoma activity and liver protective function of the fermentation products (5 L fermenter) of GL and AB cultivated in the medium containing black soybean and/or A. membranaceus were investigated. Human hepatoma Hep 3B cells and primary rat hepatocytes were used as experimental models. The results indicated that the ethanolic extracts of mycelia from the fermentation products with best anti-hepatoma activity were GL-3-mE (medium containing 50 g/L black soybean and 20 g/L A. membranaceus, 24℃, 0.75 vvm, 11 days) (IC50 26.6 μg/mL) and AB-9-m-E (medium without leguminous plants, 28℃, 0.05 vvm, 45 days) (IC50 14.3 μg/mL). GL-3-mE or AB-9-mE at 10 and 100 μg/mL reduced the CCl4-induced damage in primary rat hepatocytes, while neither extracts at a dose as high as 200 μg/mL caused any effect on the growth of the primary rat hepatocytes. Furthermore, the anti-hepatoma activity of the fermentation products (500 L fermenter) of AB with different fermentation conditions was investigated. The ethanolic extract of AB(GF3)-pE inhibited the growth of Hep 3B and G2 cells (IC50 161.1 and 86.9 μg/mL, respectively) most efficiently. Its fermentation condition was two stages: the initial medium contained 20 g/L soybean, and the inoculum was transferred to new medium containing 10 g/L black soybean on day 6. The total fermentation period was 17 days. AB(BS)-pE was further 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 to inhibit the proliferation of Hep 3B and G2 cells (IC50 3.6 and 1.9 μg/mL, respectively). Two active compounds from AB(BS)-pE-F3 were seperated by RP-HPLC and identified by UV, IR, EIMS, and 1H and 13C NMR to be blazeispirols A and C, and they were increased after feeding the medium containing black soybean at day 6 and reached the maximum at day 12. The contents of blazeispirols A and C were highly negatively correlated with Hep 3B and G2 cell viabilities. It showed that blazeispirol A inhibited the growth of Hep 3B cells by induction of apoptois. Decrease in phosphor-Bcl-2 and Bcl-xL proteins, increase in Bax protein, disruption of mitochondrial membrane potential, activation of caspases-3 and -9 and poly(ADP-ribose)polymerase (PARP), and DNA fragmentation were involved with blazeispirol A treatment. Additionally, HtrA2/Omi and AIF were also released from mitochondria to cytosol. The results indicated that blazeispirol A can induce caspase-dependent and -independent apoptosis in Hep 3B cells. It also suggested that blazeispirol A is the major contributor in AB(GF3)-pE and AB(GF3)-pE-F3 to induce apoptosis in human hepatoma Hep 3B cells. The effects of AB(GF3)-pE on normal primary rat hepatocytes ex vivo, primary rat hepatocytes damaged by carbon tetrachloride ex vivo, and carbon tetrachloride-induced liver damage in rats in vivo were investigated. The results showed that 10 and 100 μg/mL of AB(GF3)-pE reduced the CCl4-induced damage in primary rat hepatocytes, while 200 μg/mL didn’t cause any effect on the growth of normal primary rat hepatocytes. In addition, AB(GF3)-pE reduced the inflammation and necrosis of hepatocytes, and up-regulated glutathione peroxidase (GPx)、glutathione reductase (GRd), and glutathione S-transferase (GST) to preserve the level of reduced glutathione in the liver.