基于受体配体-HPLC-MS联用技术筛选五味子镇静催眠成分方法学的研究

A novel method for screening effective constituents of Schisandra chinensis( Turcz.)Baill. by receptor ligand-HPLC-MS techniques

目的建立基于受体配体结合竞争试验和HPLC-MS分析技术联用初步确认五味子镇静催眠成分的方法学,为快速高效地分析五味子镇静催眠活性有效成分并确认作用靶标奠定方法学基础,也为简便迅速地研究中药中有效物质基础和作用机理提供新的研究思路和方法。方法运用受体配体结合原理,先制备大鼠脑组织中包含GABA(γ-氨基丁酸)受体的蛋白质提取物,构建供药物与GABA受体结合的体外模型,采用已知具有通过结合GABA受体发挥镇静催眠作用的地西泮,与五味子竞争性结合同一受体成分GABA的方法,用地西泮竞争置换出结合GABA受体的五味子中单体成分,通过HPLC-MS分析表征,鉴定结合GABA受体的五味子成分,再通过检索文献确认该成分是否具有镇静催眠活性的作用。结果利用受体配体结合技术和HPLC-MS联用技术成功建立了快速筛选单味中药中结合已知药靶成分的方法。利用该方法研究五味子中镇静催眠活性有效成分,通过模型药地西泮与五味子提取液竞争结合脑组织中的GABA受体,结合HPLC-MS检测分析结果,初步筛选出五味子中结合GABA受体的成分可能为五味子甲素(分子式:C24H32O6,相对分子质量:416.51),对比文献结果提示五味子甲素为五味子产生镇静催眠作用的有效成分。结论初步建立了基于受体配体结合-HPLC-MS联用技术筛选单味/复方中药中有效成分的方法学,同时利用该方法初步分析出了五味子中结合GABA受体的成分为五味子甲素。

Objective To establish a new method for screening effective components of Schisandra chinensis（ Turcz.） Baill. by receptor ligand binding assays and HPLC-MS analysis techniques,which could lay the foundation for the rapid analysis and confirm method of the effective components in the single or compound traditional Chinese Medicine prescription. This method even provided a new research idea and method for the study of the effective material foundation and the mechanism or drug target of the traditional Chinese Medicine. Methods The protein extracts contained GABA receptors of brain tissue of rats were the prepared under the receptor ligand binding principle,which was used to establish the in vitro model of the combination of the GABA receptor and the drug. Diazepam was used to replace the monomer composition of S. chinensis combined with the GABA receptor competitively. The experiment was based on the method that diazepam played sedative and hypnotic effects by combining with GABA receptor and was competitively combined the same receptor as S. chinensis. The effective monomer of the S. chinensis which had combined with the GABA receptor was identified by means of HPLC-MS analysis techniques. The effect of the monomer on the activity of sedative and hypnotic was confirmed by literature retrieval. Results A rapid screening method for the effective components of the single traditional Chinese herb was established by receptor ligand binding assays and HPLC-MS analysis techniques. The application of this method to study the effective components with sedative and hypnotic activity of S. chinensis, showed that diazepam competitively replaced the aqueous extract of S. chinensis combined GABA receptor,and the HPLC-MS analysis result revealed that deoxyschizandrin（ Molecular formula： C24H32O6,molecular weight： 416.51）maybe the monomer of the schisandra chinensis which combined with the GABA receptor. The literature retrieval result showed that deoxyschizandrin was the effective component with sedative and hypnotic effects of S. chinensis. Conclusion A novel method for screening effective components in the single or compound prescription of traditional Chinese Medicine by receptor ligand binding techniques has been initially established. Meanwhile,the method has preliminary confirmed that deoxyschizandrin maybe the monomer of the S.chinensis that combined with the GABA receptor.