酸枣仁皂苷的人体肠道菌群体外代谢转化及其转化产物改善睡眠活性研究

Semen Ziziphi Spinosae, the seed of Ziziphus jujuba Mill var. spinosa (Bunge) Hu ex. H.F. Chou, is the first choice to tranquilize mind and nourish the heart in traditional Chinese medicine(TCM). Jujubosides the maior effective components of Semen Ziziphi Spinosae have obvious hypnotic effects. Nevertheless, very few side-effects (impairment on learning and memory) are found. Jujubosides may be considered to be a novel lead compound. The low oral bioavailability does not match the high bioactivity, which is the critical scientific question not yet solved for Semen Ziziphi Spinosae. The “Microbiota-Gut-Brain axis” interactions and implication for sphysiological-function had been gradually illuminated. The human intestinal microflora might be the one of the main ways to improve the quality of sleep forJujubosides. Firstly, this research will integrate “Microbiota-Gut-Brain axis” with “Translational research”, and use the intestinal microflora of insomniac that was caused by the deficiency of Yin and blood as research tools. A stabilized metabolism and biotransformation system of human intestinal microflora should be established in vitro. Secondly, liquid chromatography-tandem mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) are adopted as the effective technique developed to understand the chemical nature of metabolites and biotransformation products. Finally, the pharmacological study of biotransformation products is investigated in mice, cells and moleculars in order to identify the active components, as well as determine the action of metabolites from jujubosides.. It is significant and valuable for the new drug research and development of jujuboside based on “Microbiota-Gut-Brain axis”. This project will provide the ideas and methods of effective material basic study of TCM based on the human intestinal microflora.

酸枣仁是中医药养心安神的首选药物之一。酸枣仁皂苷是其发挥改善睡眠作用的主要有效成分，具有疗效确切副作用低等特点，有望成为新颖的先导化合物。然而较低的生物利用度与较高的药理活性相矛盾的现象，是酸枣仁皂苷研究中尚待解决的科学问题。随着“微生物-肠-脑轴”对人体生理功能调控作用的日益明确，提示肠道菌群可能是酸枣仁皂苷改善睡眠的作用途径。本项目整合“微生物-肠-脑轴”与“转化医学”的研究思路，以血亏阴虚型失眠病人肠道菌群为研究工具，构建稳定的体外代谢转化体系；整合液质联用技术和天然产物结构鉴定技术，确定酸枣仁皂苷的肠道菌群代谢转化产物结构；整合分子、细胞、整体动物等药效评价方法与代谢组学技术，评价转化产物改善睡眠活性。以期明确酸枣仁皂苷改善睡眠的药效基础。本研究对基于“微生物-脑-肠轴”的酸枣仁皂苷新药研究具有重要意义和潜在价值，同时为基于肠道菌群代谢转化环节的中药药效物质研究做出有益的方法探索。

酸枣仁是中医药养心安神的首选药物之一。酸枣仁皂苷（Ziziphi Spinosae Semen saponin，ZSSS）是酸枣仁发挥改善睡眠作用的主要有效成分，然而现有研究表明ZSSS存在较低的生物利用度与其较高药理活性相矛盾的现象。针对上述问题，项目在中医药理论的指导下，基于“微生物-肠-脑轴”研究策略，开展以下研究：以肠道菌群结构和ZSSS转化率为评价指标，对六种常用培养基进行筛选，研究发现GAM培养基可有效保持菌种活力、稳定菌种结构和保持ZSSS转化率。利用UPLC-Q-Orbitrap-MS技术对ZSSS成分进行全面表征；结果表明自制ZSSS纯度为45.75%，共鉴定酸枣仁皂苷20个，其中四环三萜类17个，五环三萜类3个。收集并培养正常和失眠病人肠道菌群开展代谢转化研究。利用UPLC-Q-Orbitrap-MS技术比较分析ZSSS的肠道菌群转化产物，共鉴定ZSSS代谢产物38个。其中正常人菌群代谢产物34个，主要发生糖基水解反应，其次为生糖基化、去氧和脱羧反应；病人菌群代谢产物36个，主要发生糖基水解反应，其次为去氧反应和氧化反应。利用Illumina MiSeq测序平台对肠道菌群16S rRNA高通量测序，表明ZSSS可显著回调肠道菌群alpha多样性，门水平ZSSS可显著回调占主要丰度的厚壁菌门、变形菌门、放线菌门和拟杆菌门，属水平ZSSS可显著上调15个属，下调7个属，ZSSS可能通过影响短链脂肪酸代谢进一步调控中枢神经系统。利用戊巴比妥钠小鼠模型研究表明酸枣仁总皂苷及转化产物均有镇静催眠作用，ZSSS代谢转化产物整体好于ZSSS。采用PC12细胞模型研究表明ZSSS代谢转化产物对神经细胞具有保护作用。利用分子对接技术研究表明ZSSS代谢转化产物与GABAA受体结合能力强于ZSSS。本研究建立失眠病人肠道菌群培养体系，构建基于UPLC-Q-Orbitrap-MS技术的ZSSS分析方法，阐明正常和失眠人肠道菌群对ZSSS的代谢转化规律；明确了ZSSS通过调控肠道菌群改善失眠的作用机制。从分子、细胞和动物水平，确证了ZSSS代谢产物是改善睡眠的潜在效应成分。本研究初步阐明了酸枣仁皂苷的效应成分，为基于肠道菌群代谢转化的中药药效物质基础研究做出有益的方法探索。