基于谱-效结合、药动学--药效学结合比较研究菟丝子酒炙、盐炙增强补肝肾作用的异同及增效机制

Semen Cuscutae (Tu-Si-Zi), is a well-known traditional Chinese medicine used to nourish liver and kidney. Its processing method ‘soaking with wine ’ was first recorded in the Jin dynasty, and Tu-Si-Zi processed with wine was mostly used in the history, however YanZhiPin (YZP) has been recorded since the 1990 edition pharmacopeia. To compare the efficacy of tonifying liver and kidney and study the processing mechanism between JiuZhiPin (JZP) and YZP, a research proposal has be made. First, The pharmacodynamical study on the kidney-yang deficiency rats induced by hydrocortisone and discussion the mechanism on reproductive system in rats will be compared with the crude, JZP and YZP, while the study on liver injury in the rat induced by CCl4 and ethanol will be compared with them, too. Second, Comparative study on the similarities and differences between the main chemical composition of JZP and YZP, the contents and constituents of flavonoids and polysaccharides will be compared. Many morden techniques for analysis, isolation and identification, such as HPLC-MSn, high performance gel permeation chromatography (HPGPC) coupled with chemometrics, etc. will be used. HPLC fingerprints of the JZP and YZP will be established and chromatogram - effect analysis will be made respectively and each component's contribution to the effectiveness will be analyzed coupled with chemometrics methods. Third, the simultaneous determination of main efficacy components in plasma will be respectively established by HPLC-MS–MS. Dose - effect - time relationship will be explained and compared by PK- PD combined model on liver injury rats induced by CCl4. Fourth, methods with specificity constituents, such as total polysaccharides, total flavonoids, hyperoside, quercetin, kaempferol, astragalus as the markers, will be established, and the contents of above components will be determined in the crude and processed products. The study will have the importance on explanation on the processing theory of tonifying the kidney to arrest spontaneous emission and protect liver between JZP and YAP, reasonable usage of the processed products, quality control on the processing products and deeply developing on Semen Cuscutae.

菟丝子为补肝肾之要药，其炮制始载于晋，为“酒浸”，且历史上多为酒制，但自1990版药典收载的炮制品却为盐炙品。为比较其酒炙和盐炙品补肝肾作用的异同及增效机制，我们拟对二者在肝损伤大鼠保肝作用和肾阳虚大鼠补肾作用进行药效对比研究及机制探讨。比较研究二者主要化学成分的异同，如采用HPLC-MSn比较黄酮组分，HPLC、凝胶色谱等比较二者多糖组分组成、分子量等的异同；并分别建立各炮制品的特征图谱，结合化学计量学进行谱—效分析，分析各成分对药效的贡献度，明确主要药效成分。采用HPLC-MS-MS建立主要药效成分在血浆中的分析方法，PK-PD结合模型比较二者主要药效成分在肝损伤大鼠中的剂量—效应—时间关系。采用主要药效成分完善菟丝子及炮制品的质量评价体系。上述研究对于揭示菟丝子炮制理论，为临床合理选用炮制品规格，有效控制炮制品质量具有重要意义，对于深入开发菟丝子也具有重要价值。

菟丝子具有滋补肝肾、固精缩尿等作用，其炮制方法首载于晋代葛洪著的《肘后备急方》：“酒浸服”，菟丝子盐炙始见于宋代《圣济总录》：“入盐少许炒，乘热捣末”，《中国药典》（2015版）收载的菟丝子炮制品规格为生菟丝子和盐菟丝子。前期研究了菟丝子盐炙增强“补肾固精”的传统炮制理论，阐明了药效物质基础，在前期研究的基础上，进一步深入了研究，1. 采用 HPLC-MSn、凝胶色谱等多种分析方法对菟丝子生品、酒炙品、盐炙品的化学成分进行比较研究，并分别建立特征图谱；结果表明菟丝子炮制后黄酮类及有机酸类成分种类没有发生变化但含量发生了变化，除槲皮素、阿魏酸外盐炙品中各成分含量均高于酒炙品中各成分的含量。2. 比较研究三种炮制品对肝损伤大鼠的保肝作用和肾阳虚大鼠保护作用，并探讨其作用机制，进行谱——效分析，阐明各炮制品补肝肾的特点及作用机制的异同，找出主要药效成分；结果表明三种炮制品均能改善肾阳虚大鼠的生殖机能，改善大鼠肝损伤，盐炙品的效果优于酒炙品。3. 分别建立三种炮制品中主要药效成分在血浆中的分析方法，PK-PD结合模型比较研究主要黄酮成分在CCl4肝损伤大鼠体内的浓度-效应-时间的关系。菟丝子保肝药效的发挥与绿原酸、金丝桃苷和紫云英苷三种成分相关，三种成分的血药浓度较高时，AST和ALT下降也较为明显，在1小时左右药效和血药浓度有较为明显的相关性。4. 对三种炮制品建立系统的质量评价体系。5.采用均匀设计法建立了酒炙菟丝子最佳炮制工艺为20%浓度的黄酒闷润6小时，以100℃炒制18分钟。6. 进行了菟丝子不同炮制品对化学性肝损伤大鼠的代谢组学研究；三种炮制品都可以改善CCl4导致的代谢异常，但生物标记物类型基本相同，主要为脂质类化合物。三种炮制品对CCl4化学性肝损伤大鼠的保肝作用可能主要影响了脂质代谢及能量代谢。以上对于阐明菟丝子不同炮制品补肝肾异同、控制饮片质量，充分发挥其临床疗效具有重要意义。