柴胡及相關成分與製劑抑制病毒複製及誘導免疫機轉探討之體外研究

The mechanism of the inhibitive replication and inducible immunization of Bupleuri Radix and its related ingredients in virus in vitro

鄭珮妏(Pei-Wen Cheng)

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/博士(2008年)

摘要

柴胡(Bupleuri Radix)為治療肝炎、瘧疾、間歇性發燒等重要的傳統生藥之一。而高氏柴胡(Bupleurum kaoi)為台灣特有種之柴胡。本實驗分別利用高氏柴胡及北柴胡粗抽物(crude extract)、粗多醣體(crude polysaccharide)、粗皂素(crude saponin)、相關成分(saikosaponin A、B2、C、D)及分別含高氏柴胡與北柴胡之小柴胡湯比較對於疱疹病毒(HSV)、冠狀病毒(coronavirus 229E)與B群克沙奇病毒(coxsackie B virus type 1)的毒殺作用之初步篩選。
由柴胡及其相關成分與製劑抗疱疹病毒(HSV-1)實驗結果顯示，高氏柴胡多醣抑制疱疹病毒的活性最好，EC50、CC50和SI値分別為63.93、1591.72 μg/ml 與24.23。又由藥物抑制病毒的時間效應發現，高氏柴胡多醣抑制病毒感染前與同時感染的活性比病毒感染後好。除此之外，從實驗的結果發現高氏柴胡多醣不具有將病毒去活化的能力也不影響病毒與細胞結合。卻發現高氏柴胡多醣可誘導干擾素-β(IFN-β)的增加而達到抑制病毒複製蛋白 (ICP4、ICP8、UL42和gD)的表現，且加入干擾素-β抑制劑之後又使結果更加明確。進一步發現高氏柴胡多醣也可活化宿主細胞病毒感染後核內干擾素調控因子(IRF-3)的表現。
另由抗人類冠狀病毒(HcoV-229E)實驗結果顯示，柴胡類生藥所含saikosaponin B2抑制HcoV-229E的活性最好，其EC50、CC50和SI値分別為1.7、 383.3 μM與221.9。由藥物抑制病毒的時間效應發現，saikosaponin A、B2、C、D對病毒感染前與後皆有良好的抑制活性。除此之外，從實驗的結果也發現以saikosaponin B2可干擾病毒吸附及穿透進入細胞感染宿主細胞的能力最佳。
又由柴胡及其相關成分與製劑抗B型克沙奇病毒(CVB1)實驗結果顯示，高氏柴胡與含北柴胡之小柴胡湯抑制B型克沙奇病毒的活性最好，IC50値分別為12.38和 12.39 μg/ml、CC50値分別為883.56和 945.7 μg/ml與SI 値分別為17.34和 18.92。除此之外，實驗結果也顯示高氏柴胡和含北柴胡之小柴胡湯在濃度250 μg/ml時對標的細胞(CCFS-1)的存活率與生長並不具有影響。由藥物抑制病毒的時間效應發現，高氏柴胡和含北柴胡之小柴胡湯對病毒感染前與後皆有良好抑制活性。除此之外，從實驗的結果也發現高氏柴胡多醣和含北柴胡之小柴胡湯可中和(Neutralization)病毒感染細胞後產生的病理現象(CPE)。進一步也發現高氏柴胡和含北柴胡之小柴胡湯可誘導干擾素-α/β(IFN-α/β)的表現而達到抑制病毒複製的作用，且加入干擾素-α/β抑制劑之後又更加確証此結果。由此可得知高氏柴胡和含北柴胡之小柴胡湯可經由誘導第一型干擾素(type I interferon)表現而達到抑制病毒複製的作用。
綜合以上的初步研究成果發現，高氏柴胡多醣、高氏柴胡、含北柴胡之小柴胡湯、含高氏柴胡的小柴胡湯及saikosaponin B2對疱疹病毒 (HSV-1)、人類冠狀病毒(HcoV-229E)和B型克沙奇病毒(CVB1)具有雙重抑制活性，除了可以直接抑制病毒感染之外還可以誘導免疫反應而減少病毒對細胞的傷害，同時具有預防及治療功效。除此之外，對標的細胞不具有明顯的毒殺及敏感性。由這些種種優勢，建議高氏柴胡多醣、高氏柴胡、含北柴胡之小柴胡湯、含高氏柴胡的小柴胡湯及saikosaponin B2可深入研究以期發展成抗疱疹病(HSV-1)、人類冠狀病毒(HcoV-229E)和B型克沙奇病毒(CVB1)之臨床用藥。

關鍵字

柴胡

並列摘要

Bupleuri Radix (Chai-hu in Chinese and Saiko in Japanese) is one of the most important traditional Chinese crude drugs for treating hepatitis, malaria and intermittent fever. Bupleurum kaoi is one of the Bupleurum spp. families endemic in Taiwan. The aim of this study was to explore the antiviral activities of Bupleurum Radiex and its related ingredients against herpes simplex virus type 1 (HSV-1) , human coronavirus 229E (HcoV-229E) and Coxsackie B virus type 1 (CVB1) infection and to elucidate the mechanism of drug action.
Our study showed the antiviral activities of Bupleurum kaoi polysaccharide (BKP) against HSV-1 infection and to elucidate the mechanism of drug action. At a 50% effective concentration (EC50) of 63.93 μg/ml, BKP did not significantly affect cell viability and growth, and had a CC50 and SI values of 1591.72 μg/ml and 24.23, respectively. Interestingly, our initial results from the time-of-addition studies indicated that BKP could inhibit HSV-1 infection whether added at times of pre-infection and infection more batter than post-infection. We showed that BKP could not effect of HSV-1 inactivation and binding to target cells. However, subsequent studies revealed that BKP could impede HSV-1 post-infection stages by inhibiting viral proteins HSV ICP4, ICP8, UL42, and gD expression, and by inducing interferon-beta (IFN-β) expression. The addition of IFN-β inhibitor could abrogate these effects. Furthermore, the effect of BKP in inducing nuclear IRF-3 is strongest in the presence of HSV-1, implying that BKP appears to potentiate host antiviral response during viral infection.
The antiviral activities of saikosaponinB2 against HcoV-229E infection and to elucidate the mechanism of drug action. At a 50% effective concentration (EC50) of 1.7 μM, saikosaponin B2 did not significantly affect cell viability and growth, and had a CC50 and SI values of 383.3 μM and 221.9, respectively. Interestingly, our initial results from the time-of-addition studies indicated that saikosaponin b2 could inhibit HcoV-229E infection whether added at times of pre-infection, infection and post-infection. We showed that saikosaponin B2 could inhibit the HcoV-229E attachment and penetration to target cells.
The antiviral activities of Bupleurum kaoi (BK) and Xiao Chai Hu Tang (XCHT) against CVB1 infection and to elucidate the mechanism of drug action. Our data showed that BK and XCHT neutralized the CVB1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC50 and EC50 values around 12.38, 12.39 and 50.93 μg/ml, respectively. Its CC50 and SI values were 883.56, 945.7 μg/ml and 17.34, 18.92 respectively. These results suggest that BK and XCHT possessed anti-CVB1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 μg/ml. The time-of-addition studies showed that BK and XCHT added at various time of preinfection, coinfection and postinfection could inhibit CVB1 infection. Interestingly, BK and XCHT also showed an inhibition on viral replication through the induction of IFN-α/β expression. In conclusion, BK and XCHT possessed antiviral activity against CVB1 infection. It interfered the early stage of viral replication and viral replication after infection through the induction of type I interferon expression.
Together, these results suggested that BKP, BK, XCHT (BK and BC) and saikosaponin b2 possesses antiviral activities that can interfere with HSV-1, HcoV-229E and CVB1 infection at multiple levels, including directly averting viral attachment and penetration (preventive effect), and inhibiting viral protein expression as well as viral replication through induction IFN-β expression (therapeutic effect). The antiviral mechanisms observed along with its low cytotoxicity level and high selectivity at effective concentrations, suggest that the use of BKP, BK, XCHT (BK and BC) and saikosaponin B2 as direct or supportive treatment for HSV-1, HcoV-229E and CVB1 infection merits to be developed.

並列關鍵字

Bupleuri Radix

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