基于高效液相色谱-串联质谱和线粒体呼吸功能对开心散药效物质基础研究

Research on the Pharmacodynamic Material Basis of Kaixinsan Based on High Performance Liquid Chromatography-Tandem Mass Spectrometry and Mitochondrial Respiratory Function

目的基于高效液相色谱-串联质谱(HPLC-MS/MS)技术和“化学成分谱-血浆暴露成分谱-线粒体功能”联合分析,揭示开心散药效物质基础。方法通过研究文献、数据库及前期研究成果,系统梳理人参、远志、茯苓和石菖蒲的化学成分,建立开心散水提物的化学成分定性分析方法,并对开心散水提物化学成分进行鉴定。建立基于HPLC-MS/MS大鼠血浆定性分析方法,并利用该法对大鼠灌胃开心散水提取物后的血浆暴露成分谱进行分析。使用Seahorse细胞能量代谢分析仪进行有氧呼吸检测,评估开心散关键成分对线粒体有氧呼吸的影响。结果对开心散水提取物中4类主要成分(皂苷、寡糖酯、[口山]酮和三萜)进行鉴定,共鉴定出化合物231个。对大鼠灌胃给予开心散30 min时的血浆进行分析,共鉴定开心散相关成分55个。利用体外细胞实验对入血的4类成分进行有氧呼吸检测,发现人参皂苷Rg1、3,6'-二芥子酰基蔗糖、远志[口山]酮Ⅲ、茯苓新酸B均可显著提高线粒体呼吸能力。结论皂苷、寡糖酯、[口山]酮和三萜类成分可能是开心散通过改善线粒体功能发挥药效的物质基础。“化学成分谱-血浆暴露成分谱-线粒体功能”联合分析为深入研究中药药效物质基础提供了新思路。

Objective To reveal the pharmacodynamic material basis of Kaixinsan by using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)and the integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function".Methods Through a review of literature,databases,and previous studies,the chemical components of ginseng,polygala,poria,and acorus were systematically cataloged.A qualitative analysis method for the chemical constituents in the aqueous extract of Kaixinsan was developed,allowing for the identification of its chemical components.A qualitative analysis for rat plasma based on HPLC-MS/MS was established,which was applied to analyze the plasma exposure component spectrum following oral administration of Kaixinsan aqueous extract in rats.Aerobic respiration was evaluated using a seahorse cell energy metabolism analyzer,and the effect of key components of Kaixinsan on mitochondrial aerobic respiration was assessed.Results Four main types of components were identified in the Kaixinsan aqueous extract,including saponins,oligosaccharide esters,xanthones,and triterpenes,comprising a total of 231 identified compounds.Analysis of rat plasma 30 minutes after gavage with Kaixinsan identified 55 compounds.The analysis revealed that ginsenoside Rg1,3,6'-disinapoylsucrose,polygalaxanthoneⅢand poricoic acid B could significantly enhance mitochondrial respiratory capacity using in vitro cellular assays to detect aerobic respiration of four main components entered blood.Conclusions Saponins,oligosaccharide esters,xanthones,and triterpenes may be the material basis for the pharmacological effect of Kaixinsan by improving mitochondrial function.The integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function"provides a new approach for in-depth exploration of the material basis underlying the efficacy of traditional Chinese medicine.