基于多成分序贯代谢的石榴皮中枢神经系统保护作用物质基础研究

将序贯代谢法与超高效液相色谱-串联质谱（UPLC-MS/MS）相结合，研究石榴皮发挥中枢神经系统保护作用的物质基础。首先制备石榴皮提取液，通过肠灌流和封闭肠环法收集肠壁代谢、肠道菌群代谢的血浆样品，经液相色谱-串联质谱分析以原型吸收入血成分和代谢产物；综合代谢组大鼠灌胃给药后，鉴定其血中移行成分；再结合以上结果对脑脊液样品进行解析，鉴定出入脑成分。结果表明，从提取液中共检测到鞣质类、黄酮类、酚酸类和异香豆素类等38个化学成分。肠壁代谢、肠道菌群代谢情况基本一致，检测到30个原型入血成分和12个代谢产物；综合代谢组血浆中检测到7个原型入血成分和16个代谢产物；在脑脊液中发现2个原型成分和4个代谢产物，分别是柠檬酸、鞣花酸和2个甲基鞣花酸同分异构体、二甲基鞣花酸、二甲基鞣花酸葡萄糖醛酸化物。本研究基于多成分序贯代谢靶向识别出石榴皮及其代谢产物中入脑成分，可为阐明石榴皮中枢神经系统保护作用提供证据。

Material Basis Study on the Protective Effect of Punica Granatum L. on Central Nervous System Based on Multicomponent Sequential Metabolism

The blood-brain barrier (BBB) is a critical biological structure which maintains the bathing microenvironment of brain but prevents the therapeutic drugs for central nervous system (CNS) work at the same time. Traditional Chinese medicine (TCM) shows advantages of fewer adverse reactions, mild and long-lasting effects in treating CNS diseases, and screening brain-penetrable constituents from TCM is a promising way for neuroprotective drug discovery. The purpose of the research is to study the material basis on the protective effect of Punica Granatum L. on CNS by combining sequential metabolism with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Many in situ models have been developed and widely used to study drug intestinal absorption, such as in situ closed-loop method, intestinal single-pass perfusion, intestinal recirculating perfusion, and intestinal perfusion with venous sampling (IPVS) method. In this study, the plasma samples of intestinal wall metabolism and intestinal flora metabolism were collected by intestinal perfusion and in situ closed-loop method, respectively, and the protoplasmic components and metabolites in the plasma sample were identified by UPLC-MS/MS after the extract of Punica granatum L. was prepared. Then, components in plasma were identified after the rats of comprehensive metabolism group were given the extract by intragastric administration. Finally, the components into the brain were found in the cerebrospinal fluid. A total of 38 chemical components were detected in the extract, including tannins, flavonoids, phenolic acids, and isocoumarins. The situations for intestinal wall metabolism and intestinal flora metabolism groups were similar, as the same 30 protoplasmic components and 12 metabolites were found. For the comprehensive metabolism group, 7 protoplasmic components and 18 metabolites were detected. 2 protoplasmic components and 4 metabolites were found in cerebrospinal fluid samples, including citric acid, ellagic acid, and two methyl ellagic acid isomers, dimethyl ellagic acid, and dimethyl ellagic acid glucuronide. The components into the brain of Punica Granatum L. and its metabolites were identified using multicomponent sequential metabolism, which provided evidence to clarify the protective effect of Punica Granatum L. on central nervous system. This study provides evidence that the intestinal perfusion and in situ closed-loop method can help filter out part of the unabsorbed components and reduce the workload of comprehensive metabolic component identification, and it is also a supplement to the missing components in the plasma of gavage group. In a word, compared with detecting the components in cerebrospinal fluid directly, the addition of multicomponent sequential metabolism can improve identification speed and accuracy greatly.