鞣質抗單純皰疹病毒第二型活性及其作用機轉的研究

The Study of Anti-Herpes Simplex Virus Type-2 Activity of Several Tannins

曾華友(Hua-Yew Cheng)

指導教授 ： 林俊清

高雄醫學大學/藥學院/藥學研究所/博士(2004年)

摘要

皰疹病毒所致疾患，雖然迄今已有數種相當有效的治療藥物，但由於抗藥性病毒的出現以及病毒潛伏的種種特性，導致有研究及發掘新抗皰疹病毒藥物的需求。本次研究的目的是為了開發單純皰疹病毒治療生藥的資源。在經過初步篩選了二十幾種天然物之純化合物的抗單純皰疹病毒活性後，發現其中casuarinin、ent-epiafzelechin- (4β→8)-epiafzelechin（EEE）、epiafzelechin-(4β→8, 2β→O→7)-epiafzelechin-(4α→8)-epiafzelechin（EOEE）、proanthocyanidins A-1、3’-O-galloyl prodelphinidin B-2、3, 3’-di-O-galloyl prodelphinidin B-2、pterocarnin A和putranjivain A這八種鞣質具抗單純皰疹病毒第二型的活性。因此，本研究主要探討上述八種鞣質其抗單純皰疹病毒第二型的活性及作用機轉，以期開發具抗皰疹病毒活性的生藥資源。
在XTT分析法及溶斑減少分析法這兩種方法的檢測下，所探討的八種鞣質皆顯示了不同程度的抗單純皰疹病毒第二型之活性；其在XTT分析法及溶斑減少分析法所得的IC50值（抑制50%病毒生長的藥物濃度）分別介於3.6~73.5 ?M及0.4~182.8 ?M之間。當MOI（Multiplicity of Infection）增加時，其IC50值也跟隨上升。另外，溶斑減少分析法的IC90值（抑制90%病毒生長的藥物濃度）介於2.7~62.8 ?M之間。所測試的鞣質對細胞毒性的CC50值（毒殺死50%細胞的藥物濃度），皆大於其IC50值；其CC50值介於31.7 ?M至大於1000.0 ?M之間。依據CC50值和IC50值，可計算出其選擇指數（Selectivity Index；SI = CC50值/IC50值）；SI值介於3.8~59.3。
作用機轉的研究結果顯示了所測試的鞣質會抑制單純皰疹病毒第二型吸附至Vero細胞膜、阻止單純皰疹病毒第二型穿透過Vero細胞和干擾單純皰疹病毒第二型的晚期感染。另外，在高濃度時，除了EEE和proanthocyanidins A-1外，大部份的鞣質皆能將單純皰疹病毒第二型去活化，。在與ACV（acyclovir）併用時，casuarinin、3’-O-galloyl prodelphinidin B-2、3, 3’-di-O-galloyl prodelphinidin B-2、pterocarnin A和putranjivain A皆能增強ACV的抗單純皰疹病毒第二型活性，其併用效果主要是呈現副協同作用。
由上述的研究結果可結論出，所測試的八種鞣質各具不同程度的抗單純皰疹病毒第二型之活性，其抗病毒的作用機轉為多樣性的。對於與ACV併用後呈現副協同作用之抗病毒效果的鞣質，其是值得日後更深入的研究及探討。

關鍵字

天然物 ； 鞣質 ； 抗單純皰疹病毒第二型活性 ； 作用機轉 ； 副協同作用

並列摘要

In this study, eight tannins, namely casuarinin, ent-epiafzelechin- (4β→8)-epiafzelechin (EEE), epiafzelechin-(4β→8, 2β→O→7)-epiafzelechin-(4α→8)-epiafzelechin (EOEE), proanthocyanidins A-1、3’-O-galloyl prodelphinidin B-2、3, 3’-di-O-galloyl prodelphinidin B-2、pterocarnin A and putranjivain A, were investigated for their in vitro anti-HSV-2 activity. Results demonstrated that tested tannins inhibited HSV-2 replication at different magnitudes of activity. The 50% inhibitory concentrations (IC50) against HSV-2 infected on Vero cells were in the range of 3.6~73.5 and 0.4~182.8 ?M for XTT and plaque reduction assays (PRA), respectively. The IC50 values shifted high as MOI (Multiplicity of Infection) increased. IC90 values for PRA were between 2.7~62.8 ?M. All tannins exhibited less cell cytotoxic effect at antiviral concentrations. The 50% cell cytotoxic concentrations (CC50) against Vero cells were in the range of 31.7~> 1000.0 ?M. As a result, the selectivity indexes (SI, calculated as the ratio of CC50 value to IC50 value) were between 3.8~59.3.
Mechanism studies showed that tested tannins inhibited HSV-2 from attaching to cell membrane, prevented HSV-2 from penetrating into cell, and suppressed late stage of HSV-2 infection. The tannins also inactivated the HSV-2 infectivity at high concentrations. EEE and proanthocyanidins A-1 were the two compounds that exhibited no virucidal activity. When combined together with ACV (acyclovir), casuarinin, 3’-O-galloyl prodelphinidin B-2, 3, 3’-di-O-galloyl prodelphinidin B-2, pterocarnin A and putranjivain A acted subsynergistically with ACV in suppressing HSV-2 multiplication.
In summary, the tested tannins were concluded to suppress HSV-2 infection at many modes of action. Those that acted with ACV at subsynergistic manner were, therefore, merit further investigation.

並列關鍵字

natural products ； tannins ； anti-HSV-2 activity ； mode of action ； subsynergistic manner

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