超滤离心质谱法筛选和鉴定当归四逆汤中的直接凝血酶抑制剂

Screening and identification of sirectthrombin inhibitors in DangguiSini decoction by ultrafiltration centrifugation mass spectrometry

目的:采用超滤离心质谱法筛选和鉴定当归四逆汤(DSD)中直接凝血酶(THR)抑制剂。方法:采用体外酶活性实验结合超滤离心质谱技术(UF-UPLC/Q-TOF/MS)筛选和鉴定当归四逆汤中THR的特异性结合配体。结果:当归四逆汤水提物表现出了较强的THR抑制活性,其IC50为0.119 41 mg/mL。使用UF-UPLC-DAD/LC-MS筛选并鉴定了17种潜在直接THR抑制剂,分别为藁本内脂、丁基苯酞、香芹酮、辛醇葡萄糖苷、Valenciaxanthin、当归内酯、甘草宁、Apo-10’-violaxanthal、香豆基棕榈酸、亚麻酸、环四(异)亮氨酸、环六(异)亮氨酸、环七(异)亮氨酸、氧化芍药苷、Kanzonol P、苯甲酰芍药苷、白芍苷R3。结论:超滤离心质谱法可以有效地筛选当归四逆汤中的潜在THR抑制剂,并为进一步确定天然产物中直接THR抑制剂提供实验依据。

Objective: Direct thrombin(THR) inhibitors inDangguiSini decoction(DSD) were screened and identified by ultrafiltration centrifugation mass spectrometry.Methods: The specific binding ligands of THR in DangguiSini decoction were screened and identified by in vitro enzyme activity test combined with ultrafiltration centrifugation mass spectrometry(UF-UPLC/Q-TOF/MS).Results: The aqueous extract of DangguiSini decoction showed strong THR inhibitory activity, and its IC50 was 0.11941 mg/mL.Seventeen potential direct THR inhibitors were screened and identified by UF-UPLC-DAD/LC-MS. They were ligustilide, butylphthalide, carvone, octanolglucoside, valenciaxanthin, angelica lactone, gancaonin, apo-10’-violaxanthal, coumaricpalmitic acid, linolenic acid, cyclotetra(isoleucine), cyclohexane(isoleucine), cycloheptane(isoleucine), oxypaeoniflorin, Kanzonol P, benzoylpaeoniflorin and paeoniflorin R3.Conclusion: Ultrafiltration centrifugation mass spectrometry can effectively screen potential THR inhibitors in DangguiSini decoction, and provide study evidence for the further determination of direct THR inhibitors in natural products.