超滤亲和质谱结合分子对接探讨丹参活血化瘀功效成分群的显效形式

Ultra-filtration affinity mass spectrometry coupled with molecular docking to explore effective form of blood-activating and stasis-resolving active ingredient group in Salvia miltiorrhiza

目的 采用亲和超滤-液质联用技术(ultra-filtration affinity-liquid chromatography-mass spectrometry,UF-LC-MS)结合体外酶活性实验和分子对接虚拟筛选,发现不同丹参提取物中凝血酶抑制活性成分,并靶向凝血酶探讨丹参活血化瘀功效成分群的显效形式。方法 UF-LC-MS分析结合体外凝血酶活性测定,筛选和鉴定丹参总酚酸提取物和丹参酮提取物的凝血酶抑制活性成分群;结合谱效关系探讨不同活性成分对丹参提取物抗凝活性的贡献;进一步通过分子对接虚拟筛选阐明主要活性成分作用于凝血酶的分子机制。结果 UF-LC-MS分析结合体外凝血酶活性测定,发现丹参中丹酚酸C、紫草酸、丹酚酸A、15,16-二氢丹参酮I、隐丹参酮、丹参酮IIA和丹参酮I 7种具有直接凝血酶抑制活性的成分;灰色关联分析结果显示,在丹参总酚酸提取物中,紫草酸和丹酚酸A对抗凝活性具有正向促进作用,而在丹参酚酸和丹参酮同时存在的丹参酮提取物中,丹参酮类成分是主要抗凝活性成分,紫草酸和丹酚酸A对抗凝活性具有反向贡献;通过改变不同化合物配比考察多组分同时作用于凝血酶的共同效应,当高浓度(均大于IC_(50))的丹参酚酸类与丹参酮类成分同时存在时,两组成分会竞争凝血酶活性位点,使得整体抗凝活性降低;当丹参酚酸类与丹参酮类成分组的浓度均低于IC_(50)时,凝血酶活性位点充足,两组成分间存在协同增效作用。分子对接虚拟筛选研究发现这种竞争性抑制作用可能与不同活性成分竞争性结合凝血酶同一氨基酸残基有关。结论 采用超滤亲和质谱与分子对接虚拟筛选相结合,表征丹参活血化瘀功效成分群作用于凝血酶共同效应,发现活性成分的种类、含量及不同成分的含量比例等均是影响其显效的关键因素,为中药功效机制的探索和质量控制提供了有价值的参考。

Objective Ultra-filtration affinity-liquid chromatography-mass spectrometry(UF-LC-MS)was utilized in conjunction with enzyme activity assays in vitro and molecular docking virtual screening methodologies.These approaches were applied for the purpose of pinpointing active ingredient for thrombin inhibition in different Salvia miltiorrhiza extracts and for the specific investigation of the“effective form”for these bioactive component group responsible for the blood-activating and stasis-resolving properties within S.miltiorrhiza.Methods UF-LC-MS analysis and in vitro thrombin activity assay were employed for the screening and identification of thrombin inhibitors in total phenolic extracts and tanshinone extracts.Spectrum-effect relationship was utilized to assess the contribution of different active ingredients to the anticoagulant activity of S.miltiorrhiza extracts.Additionally,molecular docking virtual screening was conducted to elucidate the underlying molecular mechanisms of the principal active components targeting thrombin.Results The combination of UF-LC-MS analysis and thrombin activity assay conducted in vitro led to the identification of seven components in S.miltiorrhiza with thrombin inhibitory activity.These components consist of salvianolic acid C,lithospermic acid,salvianolic acid A,15,16-dihydrotanshinone I,cryptotanshinone,tanshinone IIA,and tanshinone I.Gray correlation analysis results indicated that lithospermic acid and salvianolic acid A positively enhanced the anticoagulant effect within the total phenolic acid extract of S.miltiorrhiza.Conversely,tanshinone was identified as the primary anticoagulant element in the tanshinone extracts,which containing salvianolic acid and tanshinone concurrently,while lithospermic acid and salvianolic acid A exhibited an opposing impact on the anticoagulant effect.Subsequently,an exploration of the collective impact of multi-components targeting thrombin was conducted by altering the compound ratios.At high concentrations(all exceeding IC50)of salvianolic acid and tanshinones simultaneously present,these components vied for the thrombin’s active site,resulting in an overall reduction in anticoagulant activity.Conversely,when the concentrations of salvianolic acid and tanshinone components were below IC50,an obvious synergistic effect was observed due to sufficient thrombin active sites.Molecular docking virtual screening technology was utilized to confirm that the competitive inhibition may be related to the different active ingredients competitively bind to the same amino acid residue of thrombin.Conclusion Ultra-filtration affinity mass spectrometry,in conjunction with molecular docking virtual screening,was employed to delineate the collective impact of the blood-activating and stasis-resolving functional component group in S.miltiorrhiza on thrombin.The investigation revealed that the type and content of active ingredients and the content proportion of different ingredients played pivotal roles in determining their efficacy.These findings provide significant contributions to elucidating the pharmacological mechanisms and ensuring the quality assurance of traditional Chinese medicine.