肠道菌介导的口服独参汤中人参多糖促进人参皂苷体内吸收及机制研究

Decoction is the major prescription of traditional Chinese medicines. Decoction is generally rich in polysaccharides. Unravelling the synergistic interactions between polysaccharides and co-existing small molecules in decoction and the mechanisms involved is key for a better understanding and modern applications of this tradition.Previous studies found that the main components of Du-Shen-Tang, a commonly-used decoction derived from the water extract of ginseng, are polysaccharides and ginsenosides. Polysaccharides are potential prebiotics (stimulating growth of beneficial bacteria), and the biotransformation of ginsenosides by gut microbiota and the consequent systemic exposure may subject to changes due to an alteration of gut microbiota. Thus, there might be potential synergistic interactions between polysaccharides and ginsenosides in Du-Shen-Tang and the mechanisms involved need to be investigated. In this proposal, the associations between gut absorption/systemic exposure of ginsenosides and co-existing 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 and blood. 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。前期研究发现，独参汤主含多糖和人参皂苷；多糖是肠道菌潜在的益生元；苷类成分肠内代谢存在肠道菌群平衡依赖性；独参汤中两类成分可能存在活性关联，但协同作用及机制有待深入研究。本项目拟应用基于GC-MS的代谢组学方法、基于16S rRNA RT-PCR的肠道菌群组成表征方法、基于体外生物转化和代谢酶活性测定的肠道菌功能评价方法，以及基于LC-MS的化学轮廓表征方法，研究人参多糖促进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，扶植肠道益生菌生长繁殖，维持肠道菌群平衡，进而促进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，阐明独参汤中多糖促进人参皂苷吸收的肠道菌介导机制，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。

本课题主要通过研究独参汤中人参多糖对人参皂苷Rb1吸收和代谢的影响，以肠道菌为介导，阐明多糖对汤剂中小分子皂苷类化合物吸收代谢影响的可能机制。首先对独参汤中人参多糖和人参总皂苷制备工艺进行标准化研究，分析多糖理化性质并建立人参总皂苷的定性定量方法，确保研究结果的可靠性和可重复性。通过对正常组、溃疡性结肠炎（UC）组及人参多糖给药组大鼠Rb1口服药代动学研究，发现多糖给药组大鼠血浆中Rb1的Cmax和AUC0-t是正常组和UC组的2倍，其肠道菌代谢产物Rd，F2，CK及原人参二醇PPD药动学参数组间无差异，但较未治疗组也有略微上升。为阐明相关机制，首先采用Caco-2单层细胞模型研究Rb1生物转运与多糖的关系。结果表明，人参多糖可使Rb1表观渗透系数 Papp 从1.1~2.6 ×10-7 cm/s 提高至10-6 cm/s，可有效改善其生物利用度，与体内药动学研究结果即体内血药浓度水平提高相吻合，并且发现人参多糖具有促进上皮细胞生长的作用。Rb1肠道菌体外温孵代谢研究结果表明，肠道菌失衡状态下人参多糖主要通过糖苷键水解提供碳源扶植益生菌的繁殖，与Rb1水解构成竞争，使其代谢停留在CK阶段，直接影响其肠道菌代谢。同时应用16S-rRNA RT-PCR方法对不同组别大鼠粪便中总菌、双歧杆菌、乳酸菌、肠杆菌、脆弱拟杆菌、柔嫩梭菌和球状梭菌进行定量，发现人参多糖在UC大鼠肠道中显示出益生元作用，可促进益生菌生长，并能降低有害菌的增殖，维持优势菌的数量，具有调节肠道菌群平衡的作用。而在正常动物中无益生元作用。同时进一步检测β-葡萄糖苷酶和β-葡萄糖醛酸苷酶活性，发现人参多糖可降低β-葡萄糖醛酸苷酶活性，改善炎症；增强β-葡萄糖苷酶活性，影响人参皂苷肠道菌代谢。综合以上结果说明人参多糖具有益生元作用，通过调节UC大鼠肠道菌群影响Rb1的肠道菌代谢，同时因肠道菌的改变影响肠道黏膜层结构及上皮细胞生长，促进Rb1吸收。由于人参皂苷类化合物结构相似，推测人参多糖极可能以肠道菌为介导影响Rb1或其他小分子皂苷类化合物的体内吸收代谢，因此在传统口服汤剂中不可或缺。本课题的完成为深入探索中药汤剂中大分子成分的作用机理奠定基础，为开拓新的中药有效物质研究方法和思路提供理论基础。.本课题发表SCI论文3篇（IF总计大于10分）。培养研究生2名。