[[abstract]]In Taiwan, hepatocellular carcinoma (HCC) is the leading cause of cancer mortality. The development of potential liver protective agents and drugs from herbal medicines for the treatment of HCC deserves great attention. The purpose of this study is to elucidate the biological activities and antitumor pharmacological functions of Ganoderma australe (Fr.) Pat. (subgenus Elfvingia). For years, G. australe has been erroneously identified as G. applanatum in Taiwan. Until 1990 (Yeh, 1990), this species was identified as Ganoderma australe (Fr.) Pat. Comparing to G. lucidum (Fr.) Karst., a famous fungus in traditional Chinese medicine, studies on G. australe are very limited and the pharmacological potential of this fungus remains unknown. The fruiting bodies of G. australe were extracted by methanol (1:20, w/v) to obtain the triterpenoid-enriched crude extracts (designated as GA-M1-1206). Human hepatoma cell line (Hep 3B) was chosen as the in vitro model. Inhibition of hepatoma cell growth was used as a bioassay to guide the isolation of bioactive compounds from G. australe. Cell viability was determined by using the MTT assay. Separation of GA-M1-1206 by silica gel column chromatography gave 25 fractions (GA-M1-C1 to GA-M1-C25). The results of bioassay indicated that the fractions 4, 5, and 6 were the three most effective fractions to inhibit the growth of cultured Hep3B cells. These fractions were pooled and designated as GA-C46. Repeated bioassay was conducted to give the IC50 value (0.078 µg/mL). Male nude mice (BALB/c-nu/nu), inoculated subcutaneously with human hepatoma cells (Hep3B/T2), were used as the animal model to elucidate the antitumor pharmacological function of GA-C46. The results of in vitro assay were used to design the dose range in the animal model. Animals were randomly divided into two groups and treated for three cycles (7 days per cycle). Mice in the CT group (n=13) were fed with a normal diet (Purina 5010) and GA group (n=7) were treated with 20, 40, and 40 mg (pre kg body weight /day) of GA-C46 in cycles 1, 2, and 3, respectively. Tumor size (L×W2×0.52 cm3) was monitored every two days in the entire treatment period. The results showed that the tumor size of GA group (0.83±0.38 cm3), compared with the tumor size (1.28±0.48 cm3) of CT group, was reduced significantly (by 35.2%; p=0.0480) in the end of the first cycle. Reduction of tumor sizes in GA group was continuously observed in the three treatment cycles for 21 days (CT group 3.37±1.53 cm3; GA group 1.81±1.13 cm3) (46.3% reduction, p=0.0317 on day 21) until animals were sacrificed. Serum GOT, GPT, biochemical markers, and tumor weights were recorded and liver and lung metastasis was examined. Results showed that the tumor weight of GA group (0.67±0.37 g) was significantly reduced by 57.1%, compared with that of the CT group (1.56±0.70 g) (p=0.0064). The animal study established the antitumor pharmacological function of GA-C46. Further isolation of active components in GA-C46 was conducted by reversed-phase high performance liquid chromatography (RP-HPLC) (69 fractions were collected totally). By repeatedly using Hep 3B cells as the in vitro model, results showed that the biological activities appeared in seven low-polar fractions (tentatively designated as GA-C46-H43, 54, 57, 58, 60, 62, 63 fractions). These fractions will be further separated by semi-preparative RP-HPLC to obtain pure active compounds for structural elucidation. In conclusion, this study has incorporated in vitro and in vivo models to elucidate the potential of G. australe (GA-C46) to inhibit hepatoma cell growth and reduce implanted tumor. The antitumor potential of GA-C46 from G. australe was comparable or even better than those of G. lucidum and G. tsugae. This study provides valuable information for future evaluation of G. australe as an antitumor agent.
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