虎杖萃取物抗B型肝炎病毒及木犀草素抗肝癌活性之研究

The Biological Activity of Polygonum cuspidatum Sieb. et Zucc. against Hepatitis B Virus and Luteolin against Hepatocellular Carcinoma

張榮叁(Jung-San Chang)

指導教授 ： 劉宏文 ； 林俊清

高雄醫學大學/醫學院/醫學研究所/博士(2005年)

摘要

慢性B型肝炎盛行於台灣，而且又是肝癌之主要致病因子。慢性B型肝炎及肝癌目前皆為傷害國人健康的頭號殺手之一，但是這二種嚴重疾病都缺乏有效且令人滿意的治療方法。為了發展有效的抗B型肝炎病毒藥物，在努力的篩選數十種醫學文獻及傳統醫學認為可能有效的藥物後，使用體外培養的Hep G2 2.2.15細胞上，經同步定量聚合酶鏈反應(real time polymerase chain reaction; PCR)方法，反覆證實虎杖(Polygonum cuspidatum Sieb. et Zucc.)萃取物具有顯著抑制體外培養的Hep G2 2.2.15細胞上清液內B型肝炎病毒的病毒量(p<0.0001)，此抑制反應具有時間依賴性(time-dependent)及濃度依賴性(concentration-dependent)效果。同時使用酵素免疫分析法(enzyme linked immunosorbent assay; ELISA)檢定虎杖萃取物對B型肝炎病毒抗原(HBeAg及HBsAg)的影響時發現，虎杖的乙醇萃取物在最低有效劑量10 µg/ml以上，具有濃度依賴性的抑制B型肝炎病毒的複製(p<0.0001)。虎杖的水萃取物也在30 µg/ml以上的劑量，具有抑制B型肝炎病毒複製的能力(p<0.05)。虎杖的乙醇萃取物與水萃取物則具有時間依賴性地(p<0.0001)大量增加B型肝炎病毒表面抗原(HBsAg)的表現。虎杖的乙醇萃取物在30 µg/ml以上，則具有抑制B型肝炎病毒e抗原(HBeAg)的表現(p<0.05)。因此推論虎杖內可能含有數種成分，將來可被分離後對控制慢性B型肝炎作出一定的貢獻。但同時必須注意其是否具有細胞毒性及是否會增加B型肝炎病毒表面抗原的表現。 此外對發展有效的抗肝癌藥物治療方面，使用XTT分析法及flow cytometry證明luteolin可有效的抑制體外肝癌細胞株(HepG2、SK-Hep-1、PLC/PRF/5、Hep3B、HA22T/VGH)的成長。Luteolin 在1 µg/ml時即有效抑制PLC/PRF/5、Hep3B及HA22T/VGH肝癌細胞株(P<0.05)；在5 µg/ml時有效抑制HepG2肝癌細胞株(P<0.05)；但需10 µg/ml 才能抑制SK-Hep1肝癌細胞株(P<0.05)。Luteolin的肝癌細胞50％抑制濃度(IC50)介於7.29 µg/ml 和32.59 µg/ml之間，和5-FU(15.35 µg/ml至 32.84 µg/ml)相當。有趣的是對luteolin最沒效應的肝癌細胞株(SK-Hep1)是對5-FU最有效應的，而對5-FU最沒效的肝癌細胞株(HA22T/VGH)卻可被luteolin有效的抑制，luteolin和5-FU對肝癌細胞似乎有互補作用。Luteolin抑制PLC/PRF/5肝癌細胞是經由增加Bax蛋白質同時抑制Bcl-XL蛋白質，使Bax/Bcl-XL比值增加、及活化casepase-3而造成細胞凋亡(apoptosis)而來。此外luteolin也可誘導細胞週期停止於G0/G1期，因此luteolin經由細胞凋亡與細胞週期的停止而能有效抑制肝癌細胞的生長，luteolin和5-FU對肝癌細胞的互補作用在將來可能具有臨床價值。

關鍵字

慢性B型肝炎 ； 虎杖 ； 表面抗原 ； e抗原 ； 抗病毒 ； 治療 ； 木犀草素 ； 細胞週期 ； 肝癌 ； 細胞凋亡 ； 同步定量聚合?鏈反應 ； 西方點墨法

並列摘要

Endemic chronic viral hepatitis B and one of its sequelae, hepatocellular carcinoma (HCC), are among the major health problems in Taiwan. No therapeutic modality is satisfactory to manage them. To explore effective therapies to deal with HBV infection and HCC, we screened pure compounds and crude extracts that had been suggested to have possible anti-HBV and/or anti-HCC effect in medical literatures and tranditional medicine. For control of HBV infection, by using HepG2 2.2.15 cell culture model and quantitative real time polymerase chain reaction, we found that the ethanol extract of Polygonum cuspidatum Sieb. et Zucc. (P. cuspidatum) could dose-dependently inhibit the production of HBV (p<0.0001) with an effective minimal dosage of 10 µg/ml. Hot water extract of P. cuspidatum at 30 µg/ml also might inhibit the replication of HBV. The expression of HBsAg, determined by enzyme linked immunosorbent assay (ELISA), was significantly increased by both ethanol extract and water extract of P. cuspidatum dose-dependently (p<0.0001) and time-dependently (p<0.0001). Hot water extract of P. cuspidatum at 30 µg/ml could inhibit the expression of HBeAg (p<0.05). Further purification of the active compounds, identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are warranted. For the control of HCC, we used several HCC cell lines for flow cytometry and XTT assay. We found that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 µg/ml, but it needed 5 µg/ml to inhibit HepG2 and 10 µg/ml for SK-Hep1 (P<0.05). The inhibitory concentrations of 50％ (IC50) of luteolin were between 7.29 µg/ml and 32.59 µg/ml, which were comparable with those of 5-FU (15.35 µg/ml to 32.84 µg/ml). The most resistant cell line to luteolin (SK-Hep1) was the most susceptible one to 5-FU. The least affected cell line of 5-FU (HA22T/VGH) was effectively affected by luteolin. Luteolin seemed complementary to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/Bcl-XL ratio and activation of caspase-3 were consistent with the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of both cell cycle arrest and apoptosis.

並列關鍵字

P. cuspidatum ； surface antigen ； chronic hepatitis B ； therapy ； anti-viral ； e antigen ； treatment ； apoptosis ； western blotting ； PCR ； cell cycle ； hepatocellular carcinoma ； luteolin ； polymerase chain reaction