大蒜油的抗凝血作用

Anticoagulant Effect of Garlic Oil

为研究大蒜油的抗凝血作用,本实验从大蒜中分离提取大蒜油,经气相色谱-质谱联用仪鉴定该大蒜油中主要成分为二烯丙基硫醚(allyl sulfide,DAS)、二烯丙基二硫醚(diallyl disulfide,DADS)和二烯丙基三硫醚(diallyl trisulfide,DATS)。体外实验结果表明大蒜油、DAS、DADS、DATS均具有部分抑制凝血酶活性功能,且抑制作用由强到弱依次为DATS>大蒜油>DADS>DAS。利用Lineweaver-Burk双倒数作图法进行酶动力学分析,结果表明大蒜油、DAS、DADS、DATS对凝血酶的抑制作用属于混合性抑制(竞争性和非竞争性抑制)。分子荧光光谱分析结果表明大蒜油、DAS、DADS、DATS能与凝血酶蛋白中的发色基团相互作用导致荧光猝灭。三维分子对接分析结果显示出DATS与凝血酶结合性能良好;二维分子对接分析结果显示出DATS与凝血酶蛋白活性位点处Asp189、Gly219和Ala190形成氢键,与酶活性位点的Gly216、Ser195、Trp215形成疏水作用力,体现竞争性抑制作用,与非活性位点Gly226、Asp221A、Tyr225、Glu217、Cys191、Val213形成疏水作用,体现非竞争性抑制作用。体内实验结果表明,大蒜油、DAS、DADS、DATS分别通过正向调控大鼠凝血四项指标而直接展现出抗凝血功能,且抗凝血能力依次为DATS>DADS>DAS>大蒜油。大鼠体内抗氧化实验结果表明大蒜油、DAS、DADS、DATS均只有微弱抗氧化能力,大蒜油、DAS、DADS、DATS具有的抗凝血功能应与其抗氧化能力不相关。

To study the anticoagulant effect of garlic oil,garlic oil was extracted from garlic bulbs in this study and its major chemical components were allyl sulfide(DAS),diallyl disulfide(DADS)and diallyl trisulfide(DATS)as identified by gas chromatography-mass spectrometry(GC-MS).Experiments in vitro showed that garlic oil,DAS,DADS and DATS partially inhibited thrombin activity,and the inhibitory effect followed the decreasing order:DATS>garlic oil>DADS>DAS.Enzymatic kinetic analysis was carried out using the Lineweaver-Burk double reciprocal plotting method,which showed that garlic oil and its three components inhibited thrombin activity in both competitive and non-competitive mode.Molecular fluorescence analysis revealed that each inhibitor could interact with chromophores in thrombin,resulting in fluorescence quenching.Two-dimensional molecular docking results revealed that DATS could form hydrogen bonds with Asp189,Gly219 and Ala190 in the active site of thrombin and form hydrophobic interactions with Gly216,Ser195 and Trp215 for competitive inhibition;and also form hydrophobic interactions with Gly226,Asp221A,Tyr225,Glu217,Cys191 and Val213 in non-active sites for non-competitive inhibition.Garlic oil and its components exerted their anticoagulant effect by positively four blood coagulation parameters in rats,which decreased in the order of DATS>DADS>DAS>garlic oil.However,they could only weakly enhance antioxidant capacity in rats.Hence,their anticoagulant effect was not correlated with their antioxidant potential.