在台灣，‘肝癌’連續多年名列癌症死因之首，肝癌對國人的威脅不可忽視。在慢性病毒肝炎、肝硬化，肝癌等疾病治療上，西方醫學尚缺乏有效之藥物，因此，利用中草藥發展出具有保肝、抑制肝癌等藥物，是極具開發潛力之領域。本研究以台灣產南方靈芝Ganoderma australe (Fr.) Pat.為對象，研究其三萜類天然物之抗腫瘤生物活性與藥理功效。靈芝三萜類與固醇類之薄層色層分析(TLC)分布模式在物種間具有獨特性(specificity)，相較於研究資料豐富的赤芝而言，南方靈芝內應還有許多未被開發的活性三萜類與固醇類，值得進一步地研究與開發。
南方靈芝子實體經甲醇萃取 (1:20, w/v)，得到富含三萜類之甲醇抽出物 (編號為GA-M1-1206)。GA-M1-1206經由矽膠管柱層析得到分離產物共25個劃分物，依序編號為GA-M1-C1~GA-M1-C25。在離體模式(in vitro)中，利用人類肝癌細胞株(human hepatoma cell line，Hep 3B)之生長受抑制程度作為篩選指標，經由MTT呈色分析法推算細胞存活率，追蹤出南方靈芝抑制Hep 3B細胞生長之生物活性主要分布於GA-M1-C4至GA-M1-C6三個管柱層析劃分物中，因此，將三者合併後編號為GA-C46，其IC50=0.078 μg/ mL。利用離體模式之結果延伸進入活體試驗 (in vivo)，並藉以推算動物之合理使用劑量。
在活體試驗(in vivo)中，利用先天免疫缺陷之裸鼠(nude mice BALB/c-nu/nu)，採用皮下注射的方式接種人類肝癌細胞(Hep 3B/ T2)形成腫瘤以作為動物模式，探討南方靈芝生物活性成分GA-C46是否具有抗腫瘤之藥理功效。對照組(CT group) (n=13)與南方靈芝活性成分GA-C46處理組(GA group)(n=7 )均分別處理三個週期，每個週期7天(共21天)。CT組以口服餵食正常飼料，GA組則於三個週期中分別以口服餵食GA-C46以5倍、10倍、10倍之劑量(一倍GA-C46劑量為3.97 mg/ kg b.w./ day)，即分別飼以20、40、40 mg/ kg b.w./ day。試驗期間，每兩天測量一次腫瘤大小 (tumor size= L×W2×0.52 cm3)，並全程追蹤腫瘤之生長趨勢。結果顯示：於第一個週期結束時(第7天)，GA組之腫瘤體積(0.83±0.38 cm3)相對於CT組之腫瘤者(1.28±0.48 cm3)為小，且已達到統計差異(p=0.0480)，腫瘤抑制率為35.2%，並且腫瘤生長被持續抑制至第21天(GA組腫瘤體積為1.81±1.13 cm3，CT組3.37±1.53 cm3，p=0.0317，腫瘤抑制率為46.3%)。動物犧牲當日，採取血液樣本測定肝功能指數(GOT與GPT值)與三項血液生化值、摘取腫瘤紀錄重量、並詳細觀察肝、肺臟之轉移情形。結果顯示：GA組之腫瘤重量(0.67±0.37 g)相對於CT組之腫瘤重量(1.56±0.70 g)為輕，腫瘤重量減少57.1%，達到明顯之統計差異(p=0.0064)。
由於南方靈芝抗腫瘤之藥理功效於動物模式中得到驗證，故進而將生物活性成分GA-C46進行逆向式高效液相層析(RP-HPLC)分離，共收集得到69個分離產物，再重複以Hep 3B細胞存活率進行離體模式之篩選。結果顯示生物活性集中在較低極性區之7個訊號峰，編號為GA-C46-H43, 54, 57, 58, 60, 62, 63。此活性成分進一步地以製備型高效液相層析進行純化分離工作，獲致南方靈芝之活性化合物 (pure bioactive compounds)以進行結構鑑定工作。
本研究綜合了離體與活體模式之結果，藉以評估南方靈芝活性成分(GA-C46)對抑制肝癌細胞生長與抑制肝腫瘤之潛力。南方靈芝GA-C46抗腫瘤之功效可比擬或甚至優於赤芝與松杉靈芝，本研究將有助於評估南方靈芝能否進而開發以用於癌症治療藥物。

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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