基于阿尔茨海默病的茯苓中三萜类活性成分筛选、分离及鉴定研究

Screening,Isolation and Identification of Active Triterpenoids in Poria cocos based on Alzheimer’s Disease

建立了以活性为导向的快速筛选分离及鉴定茯苓中活性成分的分析方法。以茯苓总三萜得率为评价指标,应用星点设计-效应面法选取茯苓石油醚提取的最佳条件为:1-丁基-3-甲基咪唑四氟硼酸盐([Bmim][BF 4])浓度为0.4 mol/L,用量为10%,液料比为30∶1,提取时间为35 min,提取次数为3次;利用超滤-质谱技术(UF-LC-MS)共筛选得到5个潜在的乙酰胆碱酯酶(AChE)抑制剂和3个潜在的5-脂氧合酶(5-LOX)抑制剂。以活性为导向,经高速逆流色谱(HSCCC)结合半制备高效液相色谱(Semi-preparative HPLC)技术联合分离,从200 mg茯苓提取物中,分离制备得到2.36 mg 3-表去氢土莫酸、3.54 mg去氢茯苓酸,8.57 mg茯苓酸、5.34 mg松苓新酸、4.32 mg 3-氢化松苓酸,经高效液相色谱(HPLC)分析其纯度均达到90.0%以上;并采用超高效液相色谱-高分辨质谱仪及核磁共振波谱技术对单体活性成分进行鉴定。本研究所建立的方法简单、快速、高效,分离样品纯度高,适用于茯苓中三萜类活性化合物的分离纯化及鉴定研究,为天然药物的开发与应用提供了理论基础和技术支持。

An active-oriented method for rapid screening,isolation and identification of active components in Poria cocos(P.cocos)was established.Taking the total triterpene yield of P.cocos as the evaluation index,optimize with central composite design,the optimal extraction conditions of petroleum ether extract of P.cocos were as follows:concentration of 1-butyl-3-methylimidazole tetrafluoroborate([Bmim][BF 4])was 0.4 mol/L,dosage of[Bmim][BF 4]was 10%,liquid-solid ratio was 30:1,extraction time was 35 min,the extraction times was three and effect surface method of star point design and effect surface method.Five potential acetylcholinesterase(AChE)inhibitors and three potential 5-lipoxygenase(5-LOX)inhibitors were co-screened by ultrafiltration-liquid chromatography combined with mass spectrometry(UF-LC-MS).The active components were separated by high-speed counter current chromatography(HSCCC)in combination with Semi-preparative high performance liquid chromatography(Semi-preparative HPLC).From 200 mg extract of P.cocos,2.36 mg 3-epidehydrotumulosic acid,3.54 mg dehydropachymic acid,8.57 mg pachymic acid,5.34 mg dehydrotrimetrianolic acid,4.32 mg trametenolic acid were prepared.The purity of active compounds obtained by high performance liquid chromatography(HPLC)was over 90.0%.The purity of all the compounds was more than 90.0%by HPLC analysis.The active components of monomer were identified by UPLC-Q-Exactive and nuclear magnetic resonance(NMR)spectroscopy.The method established in this study was simple,rapid,efficient,and with high sample purity,which was suitable for the isolation,purification and identification of triterpenoid active compounds from P.cocos,and provided theoretical basis and technical support for the development and application of natural drugs.