口服独参汤中人参多糖影响人参皂苷代谢和吸收及肠道菌介导机制研究

Decoction is the major prescription of traditional Chinese medicines. Decoction is generally rich in polysaccharides. Unravelling the synergistic interactions between polysaccharides and coexisting small molecules in decoction and the mechanisms involved is key for a better understanding and modern applications of this tradition.Our previous studies (supported by NSFC small grant No.81274068) found that the main components of Du-Shen-Tang, a commonly-used decoction derived from the water extract of ginseng,are polysaccharides and ginsenosides, and ginseng polysacchrides could affect the metabolism of ginsenosides by gut microbiota in the same pattern with fructo-oligosaccharide, a well-known prebiotics (stimulating growth of benificial gut microbiota),indicating that ginseng polysaccharides are potential prebiotics, and the biotransformation of ginsenosides by gut microbiota and the consequent systemic exposure may subject to changes due to an alteration of gut microbiota. There might be potential synergistic interactions between polysaccharides and ginsenosides in Du-Shen-Tang decoction and the mechanisms involved need to be further investigated. In this proposal, the associations between gut absorption/systemic exposure of ginsenosides and coexisting ginseng polysaccharides and the involvement of gut microbiota will be investigated using GC-MS based metabolomic study to characterize overall alterations of endogenous metabolic network, 16S rRNA-based RT-PCR metagenomic analysis in combination with in vitro biotransformation and enzymatic activity determination to identify compositional and functional changes of gut microbiota in response to ginseng polysaccharides intervention, and UPLC-QTOF-MS/MS and LC-TQ-MS/MS based chemical profiling to monitor ginsenosides profiles in gut cotents, blood and urine. And the following hypothesis will be verified: When Du-Shen-Tang decoction is taken orally, ginseng polysaccharides can act as prebiotics to stimulate the growth and reproduction of beneficial bacteria in the gut and reinstate the balance of gut microbiota. As a consequence, the ginsenosides biotransformation by gut microbiota will be facilitated to form the more absorbable secondary ginsenosides or aglycons. From this study, the gut microbiota-mediated synergistic interactions between ginseng polysaccharides and ginsenosides in Du-Shen-Tang decoction will be demonstrated, and the role of gut microbiota in the holistic actions of Du-Shen-Tang decoction will be revealed. The present proposal also offers a novel strategy for modernization study of other decoctions of traditional Chinese medicines.

汤剂中多糖与其它成分的协同作用和机制研究对汤剂的继承创新具有重要意义。我们前期及小额资助项目（81274068）研究发现，独参汤主要含人参多糖和人参皂苷，人参多糖能明显影响人参皂苷的肠道菌代谢，提示独参汤中人参多糖与人参皂苷存在肠道菌活性关联，但关联作用和机制有待深入研究。本项目拟应用基于GC-MS的代谢组学方法、16S rRNA RT-PCR的肠道菌群组成表征方法、体外生物转化和代谢酶活性测定的肠道菌功能评价方法，以及LC-MS的化学轮廓表征方法，研究人参多糖影响人参皂苷肠内代谢和吸收与其维持肠道菌群平衡的关联性，验证如下假设：口服独参汤后，其中的多糖起益生元作用，维持肠道菌群平衡，进而影响人参皂苷的肠道菌代谢，转化成更易吸收的次生苷或苷元发挥药效。通过此假设的验证，阐明独参汤中多糖与皂苷协同作用的肠道菌介导机制，揭示独参汤多成分整体作用的科学内涵，为中药汤剂的科学化研究提供新思路。

本项目通过研究独参汤中人参多糖（GP）对人参皂苷吸收代谢的影响，验证GP通过调节肠道菌群影响人参皂苷吸收、代谢的科学假说，揭示独参汤中GP与人参皂苷的协同作用。研究分三个部分进行：(一)人参药材、多糖和皂苷质量控制。建立并应用UPLC-Q-TOF-MS/MS快速筛查的方法，筛选非硫熏人参以进行后续研究；建立并应用HPGPC方法发现GP分子量范围分布在为1.00-1308.98 kDa之间，建立并应用HPLC-DAD方法测定GP单糖组成，发现其主要由Man, Rha, GlaA, Glc, Gal, Ara和Fuc按摩尔比1.11: 1.00: 7.11: 15.98: 4.40: 6.12: 2.42组成；建立并应用UPLC-Q-TOF-MS/MS方法对30个人参皂苷进行定性定量控制。(二) 建立动物模型用于GP糖益生元作用研究。应用强迫游泳加冷刺激法创建非药物诱导的脾气虚伴随肠道菌群紊乱大鼠模型，利用基于UPLC-Q-TOF-MS和16S rRNA RT-PCR的技术研究肠道菌群代谢及多样性，发现肠道菌群紊乱状态下有益共生菌Bacteroides spp. 和 Lactobacillus spp.等丰度有降低趋势，而GP可以促进Bacteroides spp. 和 Lactobacillus spp.等增殖，调节肠道菌群整体结构，揭示GP具有益生元作用。(三) GP对人参皂苷吸收和代谢的影响研究。采用HPLC-TQ-MS技术研究血浆和粪便中11种主要人参皂苷及其次生苷的药代动力学特征；应用UPLC-Q-TOF-MS/MS技术表征其粪便化学轮廓。结果显示，模型组大鼠血浆中次生苷20(S)-Rg3的Cmax和20(S)-Rg2, Rd, 20(S)-Rg3的AUC显著低于正常组，粪便中原生苷Re, Rc和次生苷20(S)-Rg2的含量显著高于正常组，次生苷Rg1和Rd显著低于正常组；GP可以选择性地回调这些人参皂苷的相应参数，即显著提高血浆中Rg1, Rd, 20(S)-Rg3的Cmax和20(S)-Rg2, Rd, 20(S)-Rg3的AUC，并显著降低粪便中Re, 20(S)-Rg2, Rc, Rd, 20(S)-Rg3, CK的含量；揭示GP可以调节肠道菌群结构及相关代谢通路影响人参皂苷的肠内吸收和代谢，在体内发挥协同作用。