新疆维药疏花蔷薇果调节脂质代谢的药效物质基础及作用机制研究

Rosa Laxa R. fruits is commonly used as a traditional Uygur medicine in xinjiang for treatment of diarrhea, spermatorrhea, waist and leg aches, cough and asthma, aphthae boil of the tongue. Previous research has shown that its ethyl acetate extraction and n-butanol extraction have obvious regulating blood lipids and antitumor activity in vitro. Meanwhile，the flavonoid content of the ethyl acetate extraction，including rutin, kaempferol，hyperin and so on，was 62%. At present，the effective constituents of regulating blood lipids of Rosa Laxa R. fruits 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 two hepatic steatosis cell models，will be applied to directed screening the active constituents of regulating lipids metabolism from Rosa Laxa R. fruits. The film absorption characteristics of active components were researched by Caco-2 cell model, which will provide the pharmacokinetic basis of effective constituents. To evaluate the safety and pharmacodynamics of effective parts in rats. Based on the methods of serum pharmacochemistry and serum pharmacology，the mechanism of regulating lipids metabolism will be systematically investigated based on the multi-site, multi-targe on the molecular level. To study the related gene expression influence of serum containing the active constituents by the main metabolism signal pathways of lipid synthesis, transformation，such as Adiponectin, AMPK, SREBP-1c, ACC, PPARα in hepatic steatosis cells. It will provide experimental basis for the research and development of preventing and treating for NAFLD oral drug from Rosa Laxa R. fruits in the future.

新疆维医临床常用疏花蔷薇果治疗腹泻遗精，腰腿酸痛，咳嗽气喘，口舌生疮等症，前期研究发现其乙酸乙酯和正丁醇提取部位具有明确的体外降脂、抗肿瘤活性，其中乙酸乙酯部位含芦丁、山奈酚、金丝桃苷等黄酮类成分含量达62%，但其调脂药效成分及作用机制未见报道。 .本研究以柱层析、制备液相、MS、NMR等分离鉴定技术结合体外肝脂肪变性模型筛选、追踪疏花蔷薇果调节NAFLD脂质代谢的活性成分；采用Caco-2细胞模型研究活性成分的膜吸收特性，为有效成分确定提供药动学依据；并在整体动物水平评价调脂有效部位的安全性和药效作用；结合“血清药物化学”和“血清药理学”方法，研究调脂有效部位含药血清对脂联素、AMPK、SREBP-1c、ACC、PPARα等与脂质合成、转化相关的各代谢信号通路基因表达的影响，从分子水平研究其多部位、多靶点的综合调节脂质代谢的机理，为开发防治NAFLD的口服制剂提供科学依据。

本课题系统研究了新疆疏花蔷薇果（FRL）的生药学、提取纯化工艺、成分鉴别、体外吸收特性、体内外的药效及其作用机制研究，为确定抗HLP的药效成分和剂型设计提供了研究基础。.对FRL进行性状鉴别、显微鉴别、理化特性和TLC鉴别等生药学研究，测定了总黄酮、总多酚、总三萜酸、总多糖、总氨基酸等含量，并完成微量元素的测定和评价。.优化疏花蔷薇果提取物（FRLE）的提取、纯化工艺，野鸦椿酸、椴树苷、熊果酸、委陵菜酸、儿茶素、没食子酸、金丝桃苷等主要成分含量约为33%。.在Caco-2细胞吸收模型中，FRLE中2种主要成分野鸦椿酸、椴树苷的摄取与时间、浓度、pH值、温度密切相关。FRLE及其2种主要成分双向转运的Qr值、Papp值与药物浓度成正比，Papp >1.0×10-5cm/s，属于吸收良好物质，以被动扩散方式为主，双向转运无明显方向性；野鸦椿酸不是P-gp底物，椴树苷可能为P-gp底物，且存在细胞旁路转运。弱酸性（pH 6.0）条件有利于2种成分的双向转运。.FRLE能减少L02、Bel-7402细胞体外脂肪堆积模型细胞内脂滴形成，降低TG、T-CHO含量（P＜0.05），增强LDH、SOD活性（P＜0.05），具有抑制脂质堆积的功效；FRLE可下调IL-6、TNF-α水平(P<0.01)，上调ADPN水平（P<0.01）；FRLE可上调P-AMPKα、P-ACC的蛋白表达（P<0.01），下调SREBP-lc 的蛋白表达（P<0.01），且具有剂量依赖性，FRLE可能通过SREBP-1c/AMPKα信号通路发挥调节脂质代谢作用。.FRLE无明显急性和亚慢性毒性，属无毒级药材资源。FRLE可增强实验性HLP大鼠模型肝组织SOD，GSH-PX酶活力（P<0.01），降低MDA含量（P<0.01），降低血清中TC、LDL-C、AIL、ALT和AST值（P<0.05），表明其具有抑制HLP大鼠脂质过氧化，调节血清生化指标、肝组织酶活力，可用于防治HLP。.分子对接预测RhoA/ROCK2信号通路中RhoA、ROCK2、AKT、PI3K为FRLE影响脂质代谢相关性较高的目标靶点，Western-blot体内验证结果与预测结果相符，表明FRLE通过下调RhoA/ROCK2通路中P-AKT、P-PI3K、RhoA、ROCK2等蛋白表达发挥体内调节脂质代谢作用，具有防治HLP作用。