新疆维药芹菜根抗高脂血症的药效物质基础及其作用机制研究

Apium graveolens L. belongs to umbelliferous plant. The dry root of Apium graveolens L. is commonly used as a traditional Uygur medicine in xinjiang for treatment of headache，dizziness，numbness of limbs，high blood pressure. Previous research has shown that the ethyl acetate extraction and n-butanol extraction of Apium graveolens L. root have obvious regulating blood lipids，protect liver and antihypertensive activity. Meanwhile，ten chemical constituents，including flavonoids，sterol and glycosides，were isolated. At present，the effective constituents of anti-hyperlipidmia of Apium graveolens L. root and its action mechanism are still unclear. Based on previous research results，some separation and detection technology，including column chromatography，preparative HPLC，MS、NMR and so on，combined yeast two-hybrid system，which carry apolipoprotein AI (apo-AI) and scavenger receptors class B type I (SR-BI) genes of rat，will be applied to directed screening the active constituents of anti-hyperlipidemia from anti-hyperlipidmia effective part of Apium graveolens L. root. The film absorption characteristics of active components were researched by Caco-2 cell model, which will provide the pharmacokinetic basis of effective constituents. The mechanism of regulating blood lipids will be systematically investigated based on the multi-site, multi-targe on the molecular level. RT-PCR technology was adopted to study the influence of serum containing the active constituents on the main parts of the cholesterol metabolism such as the absorption, synthesis and transofrmation in liver cells by means of observing the expressions of LDL-R, HMG-CoA reductase and CYP7A1 mRNAs at different time points. It will provide experimental basis for the research and development of anti-hyperlipidmia oral drug from Apium graveolens L. root in the future.

新疆维医临床习用伞形科植物旱芹(Apium graveolens L.)的干燥根治疗头痛头晕，四肢麻木，高血压等症。前期研究发现维药芹菜根的乙酸乙酯和正丁醇萃取部位具有明显的调脂护肝、降压作用，并分离获得了黄酮、甾醇、苷类等10个成分，但其抗高脂血症的有效成分和作用机理尚不清楚。 . 本研究以柱层析、制备液相、MS、NMR等分离鉴定技术结合含大鼠载脂蛋白apo-AⅠ及其受体SR-BⅠ基因的酵母双杂交系统，定向筛选、追踪芹菜根抗高脂血症有效部位中的活性成分；采用Caco-2细胞模型研究活性成分的膜吸收特性，为有效成分的确定提供药动学依据；采用RT-PCR技术研究有效成分含药血清对肝细胞胆固醇代谢的吸收、合成、转化各环节相应靶点LDL-R、HMG-CoA 还原酶和CYP7A1 mRNA表达的影响，从分子水平研究其多部位、多靶点的综合调脂机理，为从维药芹菜根中开发防治高脂血症的口服药物提供研究基础。

本课题系统研究了新疆芹菜根抗HLP精制物及其主要成分体内外的吸收特性、调脂作用和机理研究，为药效成分确定和剂型设计提供了研究基础。. 优化新疆芹菜根抗HLP症精制物的提取、分离、纯化工艺，确定主要成分为芹菜素、芹菜苷、3′-甲氧基芹菜苷、大波斯菊苷，4种成分总含量51.13%。.芹菜根精制物中4种成分在Caco-2细胞单层吸收模型的摄取和累计转运量与时间、浓度、pH值、温度、P-gp抑制剂、EGTA螯合剂密切相关，并与时间、浓度呈正相关，吸收明显强于外排，且均有RB-A/A-B＜2，主动转运与被动扩散共同参与转运过程，双向转运无明显方向性；芹菜苷可能是P-gp的底物；在设定给药浓度范围内各成分吸收较好，无跨细胞通道转运。.大鼠灌服芹菜根精制物后在体内迅速分布，达峰时间较短，双峰现象明显，呈二室模型分布，表明可能存在肝肠循环，未检测到相关代谢产物。.构建酵母双杂交系统，芹菜根提取物、芹菜素、芹菜苷、3΄-甲氧基芹菜苷可增强报告基因ɑ，β-半乳糖苷酶活力，表明能增强两种蛋白在apo-AⅠ与SR-BⅠ之间互相作用力，具有一定的调脂活性。.芹菜根提取物、芹菜素、芹菜苷、3΄-甲氧基芹菜苷均能减少L02、HepG2和Bel-7402细胞体外脂肪堆积模型胞内脂滴的形成，降低TG、TC含量(P<0.05)，增强LDH、SOD活性 (P<0.05)，具有体外调脂及抑制脂质堆积的作用；可显著降低TNF-α and IL-6水平(P<0.05)；在脂质代谢AMPK/SREBP-1c信号通路中，剂量依赖性的升高P-AMPKα蛋白的表达（P<0.05），降低SREBP-lc的表达（P<0.05）。.芹菜根提取物可降低实验性HLP症大鼠模型的TC、TG含量，升高HDL-C含量，并增强肝组织SOD活力，MDA含量，GSH-PX酶活力，对HLP具有防治作用。.分子对接虚拟筛选了芹菜根精制物主要成分的调脂作用靶点，采用RT-PCR和Western-blot技术发现其体内调脂机理是抑制了HLP大鼠肝脏组织脂质代谢途径的HMGCR、SREBP蛋白表达。.以上体内外的吸收、药效及机制研究结果相互之间得到了印证与支持，新疆芹菜根精制物具有确定的调脂护肝的作用，可用于防治HLP症。