基于受体配体结合技术研究酸枣仁镇静催眠活性成分

Effective components of Semen Ziziphi Spinosae for sedative-hypnotic based on receptor ligand binding assay

目的利用受体配体结合法探索酸枣仁中镇静催眠的活性成分,以及该成分的镇静催眠作用机制与苯二氮卓受体的关系。方法首先,对酸枣仁中的化学成分进行乙醇粗提;其次,制备Wistar大鼠脑组织匀浆液;根据受体-配体结合原理,采用体外饱和分子竞争的方法,利用高浓度的地西泮作为阳性竞争药,竞争低浓度的酸枣仁中可与脑组织中苯二氮卓受体结合的配体化合物;收集这些镇静催眠有效化合物,利用HPLC和LC-MS方法对酸枣仁与脑组织孵育提取化合物、地西泮与脑组织孵育提取化合物、地西泮+酸枣仁与脑组织孵育提取化合物进行谱图表征差异性分析鉴定,以确认被地西泮竞争置换出的酸枣仁中的有效镇静催眠化合物。结果 HPLC检测结果显示,与地西泮组、酸枣仁+地西泮组对比,酸枣仁作用组在出峰时间2.71min和46.87min处出现特殊峰。LC-MS检测结果表明,酸枣仁中与苯二氮卓受体结合的配体化合物分别为274.28 m/z、453.34 m/z、496.34 m/z和608.38 m/z,根据酸枣仁指纹图谱的比对这些化合物可能是脂肪酸类物质和三萜类化合物。结论利用受体配体结合技术的原理,结合HPLC和HPLC-MS分析技术研究酸枣仁中镇静催眠活性成分,实验结果发现,酸枣仁中镇静催眠的活性成分可能是脂肪酸类物质棕榈酸(palmitic acid,C_(16)H_(32)O_2)、三萜类化合物麦珠子酸(alphitolic acid,C_(30)H_(48)O_4)和斯皮诺素(spinosin,C_(28)H_(32)O_(15)),这3种化合物由于与地西泮竞争结合苯二氮卓受体,可能具有类似的镇静催眠作用,这为进一步开发新型镇静催眠药奠定了理论基础。

Aim To determine the effective components of Semen Ziziphi Spinosae for sedative-hypnotic and its mechanism. Methods The extraction of Semen Ziziphi Spinosae and the rat brain homogenates were prepared. High concentrations of Diazepam competitively replaced the ligand compounds of Semen Ziziphi Spinosae combining BDZ receptor in brain tissue,and all the compounds with sedative and hypnotic effects were collected and identified by HPLC and LCMS technique,as the compounds extracted from the brain tissue were administered with Semen Ziziphi Spinosae. The brain tissue was administered with Diazepam,and with Semen Ziziphi Spinosae and Diazepam.Results The HPLC chromatograms show that the peak time of BDZ receptor ligand compounds was 2. 71 min and 46. 87 min,when compared with Diazepam. And the LC-MS chromatograms display the relative molecular weight of the ligand compounds was 274. 28 m / z,453. 34 m / z,496. 34 m / z and 608. 38 m / z respectively. According to the fingerprint of Semen Ziziphi Spinosae,these compounds may be fatty acid substances and lupine pill triterpene compounds. Conclusions On the basis of the principle of receptor ligand binding,we established a way to quickly analyze and identify the role of natural products in the same drug target compounds. The method not only can clearly define the effective components of natural products,but also clarify the mechanism of action of the compounds. The active ingredient of calm hypnosis in Semen Ziziphi Spinosae may be fatty acid substances Palmitic acid( C_(16)H_(32)O_2) and lupine pill triterpene compounds Alphitolic acid( C_(30)H_(48)O_4) and Spinosin( C_(28)H_(32)O_(15)). They exert their sedative and hypnotic effects by combining with BDZ receptor,and the research has laid a theoretical foundation for the further study about mechanism of Semen Ziziphi Spinosae.