含高氏柴胡之小柴胡湯與柴胡的指標成分Saikosaponins抑制B型肝炎病毒之活性研究

Evaluation of antiviral effects from Xiao-Chai-Hu-Tang contining Bupleurum kaoi and saikosaponins against hepatitis B virus infections in vitro

蕭維中(Wei-Chung Hsiao)

指導教授 ： 林俊清

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2009年)

摘要

B型肝炎是全世界主要的公共衛生問題之一，全球大約有3億多的人口感染慢性B型肝炎；在台灣大約有80%肝癌的患者與B型肝炎有關。目前用於治療B型肝炎的藥物以核苷酸衍生物如lamivudine (3TC)、adefovir和entecavir等及干擾素(Interferon)為主要用藥。但因成本高，有副作用大等問題，且會使病毒產生抗藥性突變，使得這些藥物治療B型肝炎的效果降低。本研究係利用含高氏柴胡之小柴胡湯與含北柴胡之小柴胡湯的水萃取物和乙醇萃取物及柴胡的主要成份saikosaponins a、b2、c、d於HepG2 2.2.15細胞株評估其抗B型肝炎病毒的效果。首先利用XTT法偵測小柴胡湯與saikosaponins對HepG2 2.2.15細胞的細胞毒性，再以ELISA方式偵測病毒的表面抗原(HBsAg)和e抗原(HBeAg)的表現，並進一步使用Quantitative real-time-PCR(q PCR)測定細胞內的B型肝炎病毒DNA(HBV DNA)表現，再以polymerase chain reaction(PCR)偵測INF-β的表現。由研究結果顯示，在低於細胞毒性的藥物濃度下，含高氏柴胡之小柴胡湯、含北柴胡之小柴胡湯的水萃取物與含高氏柴胡之小柴胡湯的乙醇萃取物不僅能抑制e抗原的表現，也能抑制表面抗原的表現；且含高氏柴胡之小柴胡湯效果比含北柴胡之小柴胡湯顯著。在抑制HBV DNA表現方面，含高氏柴胡之小柴胡湯的水萃取物、含北柴胡之小柴胡湯的水萃取物與saikosaponin b2皆能有效抑制HBV DNA，且saikosaponin b2抑制HBV DNA效果與Lamivudine (3TC)相近。小柴胡湯與saikosaponin b2也能促進INF-β的表現。因此推論小柴胡湯與saikosaponins能直接抑制B型肝炎表面抗原、e抗原、HBV DNA外，尚經由促進INF-β的表現而有增強免疫的現象，其藥物作用機轉值得進一步去探討。

關鍵字

小柴胡湯 ； 高氏柴胡 ； 北柴胡 ； saikosaponin b2 ； B型肝炎病毒 ； 表面抗原 ； e抗原 ； B型肝炎病毒DNA ； INF-β

並列摘要

Hepatitis B virus (HBV) infection is one of the major public health problems in the world. In Taiwan, 80 % of patients who develop hepatocellular carcinoma have etiology related to chronic hepatitis B. Interferon-α (IFN-α) and nucleotide analogs such as lamivudine (3TC), adefovir, and entecavir are currently used for the treatment of HBV infection. However, the high cost, side effects, and drug resistance have limited their application. In this study, the efficacy of water and ethanol extracts from two Xiao-Chai-Hu-Tang formulae, one including Bupleurum kaoi and the other consisting of B. chinense, and saikosaponins a, b2, c, d were examined for their ability to inhibit HBV in HepG2 2.2.15 cell lines. The cytotoxicity of Xiao-Chai-Hu-Tang extracts and saikosaponins on HepG2 2.2.15 cells were assessed using the XTT method. Expression of HBV viral antigens, HBsAg and HBeAg, was examined using Enzyme-linked immuno-absorbent assay (ELISA). Expression of HBV DNA was examined using Quantitative real-time-PCR (q PCR). Expression of INF-β was examined using Polymerase Chain Reaction (PCR). Our results indicated that Xiao-Chai-Hu-Tang water and ethanol extracts consisting of not only B. kaoi but also using B. chinense can inhibit the expression of HBsAg and HBeAg at concentration that are non-toxic to cells. The water extracts of these two Xiao-Chai-Hu-Tang formulae and saikosaponin b2 can inhibit the HBV DNA expression. The water extracts of these two Xiao-Chai-Hu-Tang formulae and saikosaponin b2 also can induce the expression of INF-β. The low cytotoxicity level and selectivity at effective concentration suggested that Xiao-Chai-Hu-Tang extracts consisting of either B. kaoi or B. chinense merit to be further investigated for their antiviral mechanism.

並列關鍵字

Xiao-Chai-Hu-Tang ； Bupleurum kaoi ； B. chinense ； saikosaponin b2 ； Hepatitis B virus ； HBsAg ； HBeAg ； HBV DNA ； INF-β

參考文獻

1.Allen, M. I., M. Deslauriers, C. W. Andrews, G. A. Tipples, K. A. Walters, D. L. Tyrrell, N. Brown, and L. D. Condreay. 1998. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 27:1670-7.

2.Alter, H. J., R. H. Purcell, J. L. Gerin, W. T. London, P. M. Kaplan, V. J. McAuliffe, J. Wagner, and P. V. Holland. 1977. Transmission of hepatitis B to chimpanzees by hepatitis B surface antigen-positive saliva and semen. Infect Immun 16:928-33.

3.Bermejo Benito, P., M. J. Abad Martinez, A. M. Silvan Sen, A. Sanz Gomez, L. Fernandez Matellano, S. Sanchez Contreras, and A. M. Diaz Lanza. 1998. In vivo and in vitro antiinflammatory activity of saikosaponins. Life Sci 63:1147-56.

4.Bond, W. W., M. S. Favero, N. J. Petersen, C. R. Gravelle, J. W. Ebert, and J. E. Maynard. 1981. Survival of hepatitis B virus after drying and storage for one week. Lancet 1:550-1.

5.Chiang, L. C., L. T. Ng, L. T. Liu, D. E. Shieh, and C. C. Lin. 2003. Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species. Planta Med 69:705-9.