麦冬多糖修复荷瘤大鼠肠道菌群结构谱促进甾体皂苷体内吸收的机制研究

Most of the previous studies on the metabolic transformation of TCMs via gut microbiota are performed under normal conditions without considering the influence of diseases. Previous study revealed that steroidal saponins from Ophiopogon japonicas show strong antitumor activities in vitro with poor absorption in vivo in tumor-bearing rats. To study the metabolic transformation of steroidal saponins on the basis of intestinal dysbacteriosis of tumor-bearing rats, a hypothesis was proposed as follows: the intestinal dysbacteriosis of tumor-bearing rats affects the absorption of the steroidal saponins in vivo to some extent, whereas polysaccrides from Ophiopogon japonicus, which are a kind of natural prebiotics, can restore the intestinal dysbacteriosis and enhance the absorption of the steroidal saponins in tumor-bearing rats. The differences between metabolic transformations in vivo and in vitro of the saponins via gut microbiota of normal and tumor-bearing rats, as well as the influences from compatibilities with the polysaccrides are to be studied by using LC-HR-MS/MS and metabonomics methods. Meanwhile, the structural spectra of different gut microbiota determined by PCR-DGGE and the glycosidase activity will be correlated to reveal the mechanism in the polysaccrides enhancing in vivo absorption of the saponins. The present study will also provide a new idea for the metabolic transformation of TCMs via gut microbiota, and a new method for clarifying the material basis of TCMs acting through multi-component, multi-target and multi-channel pattern, as well as the mechanism of action and compatible regularity of TCMs.

中药化学成分肠道菌群代谢研究均在机体正常状态下开展，未考虑疾病状态对代谢的影响。前期研究发现，麦冬皂苷具有很好的体外抗肿瘤活性，但在荷瘤大鼠体内代谢吸收效果较差。以荷瘤大鼠失调肠道菌群作为研究麦冬皂苷代谢吸收的切入点，提出假说：荷瘤大鼠失调肠道菌群影响机体对甾体皂苷的吸收过程，而多糖作为天然益生元可以通过修复失调菌群结构谱促进皂苷的体内吸收，从而更好地发挥抗肿瘤作用。课题应用LC-HR-MS/MS以及代谢组学方法比较正常大鼠与荷瘤大鼠肠道菌群对甾体皂苷的体内外代谢谱差异，并考察麦冬多糖对代谢处置过程的影响；同时，采用PCR-DGGE技术表征不同状态下的菌群结构谱，并与糖苷酶的活性相关联，探讨麦冬多糖促进皂苷肠道代谢吸收的机制。课题的完成，将为中药化学成分的肠道菌群代谢转化研究提供新的思路，也为中药"多成分、多靶点、多途径"作用的物质基础、作用机理、配伍规律的阐明提供新的方法。

中药化学成分肠道菌群代谢研究均在机体正常状态下开展，未考虑疾病状态对代谢的影响。前期研究发现，麦冬皂苷具有很好的体外抗肿瘤活性，但在荷瘤大鼠体内代谢吸收效果较差。以荷瘤大鼠失调肠道菌群作为研究麦冬皂苷代谢吸收的切入点，提出假说：荷瘤大鼠失调肠道菌群影响机体对甾体皂苷的吸收过程，而多糖作为天然益生元可以通过修复失调菌群结构谱促进皂苷的体内吸收，从而更好地发挥抗肿瘤作用。课题组首先对麦冬类药材的本草信息、化学成分以及药理作用进行了详细的文献调研；应用多种柱色谱技术，制备了麦冬甾体皂苷和多糖药效部位；建立了麦冬甾体皂苷专属的指纹图谱控制方法；构建了一系列基于LC-MS、NIR等技术的分析方法，用于阐明麦冬干燥、硫熏等过程的化学物质基础变化；靶向富集分离了8个主要的甾体皂苷类成分；建立了荷Walker256肿瘤的大鼠模型；比较了正常大鼠与荷瘤大鼠肠道菌群结构谱的差异，同时研究了麦冬多糖对于荷瘤大鼠肠道菌群的作用；比较了麦冬甾体皂苷在正常大鼠、荷瘤大鼠以及连续给予麦冬多糖的大鼠体内代谢转化状况。通过研究发现，荷瘤大鼠肠道菌群结构谱异于正常大鼠；麦冬多糖对于该失调肠道菌群具有修复调整作用，主要表现在有益菌数量升高，有害菌数量降低；麦冬总甾体皂苷经正常大鼠和荷瘤大鼠体内代谢转化后，主要以极其微量的原形成分入血，且在两种大鼠的体内并无显著性差异；麦冬皂苷D的代谢过程也与此类似。上述结果表明，尽管麦冬多糖对于荷瘤大鼠失调肠道菌群具有修复调整作用，但其对于麦冬甾体皂苷的体内代谢转化影响很小，究其主要原因可能在于麦冬甾体皂苷自身结构特殊，难以被吸收入血。上述结果还提示，麦冬甾体皂苷可能是通过诸如调整“失调肠道菌群-内毒素-炎症因子”等致病途径来发挥抗肿瘤药效的。本课题的完成，不仅为中药化学成分的肠道菌群代谢转化研究提供新的思路，也为中药“多成分、多靶点、多途径”作用的物质基础、作用机理、配伍规律的阐明提供新的研究方向。