基于“针捕集-肠道微生态学-代谢标志物”探讨白术芍药散治疗溃疡性结肠炎炮制增效机理

The processing of taditional chinese medicine (TCM) has an important function in the clinical applications of Chinese medicine. However, studies on the action mechanism of the processing in the compounds are rare. In the early study stage of the National Natural Science Foundation Youth Fund, the research team carried out the process of stir-frying the bran of monarch drug Atractylodis Macrocephalae Rhizoma and adjuvant drug Paeoniae Radix Alba in Baizhu Shaoyao San. The differences in the chemical composition and efficacy before and after processing and combination, as well as the metabolism of the effective components, were studied on a rat model established with TCM symptoms of ulcerative colitis with liver stagnation and spleen deficiency. Atractylodis Macrocephalae Rhizoma and Paeoniae Radix Alba were found to function better in strengthening the liver and spleen after the combination. Based on previous studies, the present research probes into the effect of Baizhu Shaoyao San before and after processing on the efficacy of ulcerative colitis, metabolic markers, and intestinal flora. The molecular biological and overall in vivo evaluations were performed via needle trap-high performance liquid chromatography /mass spectrometry NT–HPLC/MS, 16S rDNA microbial ecology, high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) metanomics, and other techniques. The idea of biological effects–feature components–metabolic markers–gut microflora was considered, with Baizhu Shaoyao San as representative, to explain clearly its physical basis and synergistic mechanism. The study introduces the scientific connotation of processing efficiency with respect to the compound to establish a new model and idea for the processing of Chinese medicines and to propose a compound mechanism based on the overall concept.

炮制在中药方剂临床用药中起着关键作用，但炮制在复方中的作用规律研究甚少。课题组前期在国家自然基金青年基金项目研究中，已完成“白术芍药散”中君药白术和臣药白芍的麸炒工艺、炮制配伍前后化学成分和药效学差异及有效组分体内代谢，建立了肝郁脾虚型溃疡性结肠炎中医症候大鼠模型等工作，发现白术和白芍炮制配伍后柔肝健脾作用增强。本课题在前期工作基础上，以"白术芍药散"为代表方，采用炮制前后的饮片组方，遵循“生物效应—特征成分—代谢标志物—肠道微生态”的思路，从分子生物学水平、体内整体评价入手，运用NT-HPLC/MS、16S rDNA的微生物分子生态学及LC-QTOF/MS代谢组学等技术手段，探索白术芍药散中药物炮制前后对溃疡性结肠炎的药效、代谢标志物及肠道菌群的影响，阐明其作用的物质基础及增效机理，从复方角度阐释药物炮制增效的科学内涵，并最终建立基于整体观的中药炮制和复方作用机理研究的新模式和思路。

本课题系统考察了组方药材的炮制对白术芍药散所含化学成分、药效及血中移行成分的影响，并充分利用谱-效相关的思路探究了血中移行成分与药效之间的相关性，旨为全面研究炮制对白术芍药散全方的药效变化及其相应的科学内涵奠定坚实的基础。采用UHPLC-Q-TOF/MS结合主成分分析统计方法探究了白术芍药散炮制前后化学成分的变化。在炮制前后白术芍药散全方的70%乙醇提取液中共鉴定出186种化学成分，筛选获得白术芍药散生品和炮制品中共有62种具有显著性差异的化合物，其中23种成分在炮制后显著降低，39种成分在炮制后显著升高。从整体上看，炮制使得白术芍药散中主要药效成分的含量明显增加，推测这些成分是全方炮制后增效的物质基础。. 通过Needle Trap Device/GC-MS快速测定了白术芍药散生品与炮制品中的挥发性化学组分，并采用多元数据分析方法鉴别了具有显著性差异的化学成分。通过与NIST 数据库的匹配，在白术芍药散生品中共鉴别出121种挥发性化学成分，在白术芍药散制品中共鉴别出123种挥发性化学成分，PCA和OPLS-DA可将白术芍药散生品与制品明显区分为两类。进一步的S-PLOT和VIP分析结果确认了21种具有显著性差异的化合物，其中具有燥性作用的苍术酮含量显著降低，具有焦香健脾作用的糠醛和5-甲基糠醛含量显著升高。通过研究白术芍药散炮制前后对UC大鼠的药效作用，发现了生品与炮制品均具有一定的抗炎的作用，均能降低血清中IL-6和TNF-α的水平，升高IL-10和TGF-β1的水平，其中炮制品的作用明显优于生品，差异具有统计学意义。采用Western Blot技术检测结肠组织的蛋白表达，结果发现白术芍药散治疗溃疡性结肠炎的作用可能与抑制TLR4/MyD88/NF-κBp65通路的激活有关。通过谱效相关的研究方法，共鉴定出134种成分，其中24种为原型成分，110种为代谢产物，采用灰色关联度的统计方法对白术芍药散生品与炮制品不同时间点的血中移行成分图谱和药效指标进行相关性分析，筛选出关联度较高的成分。结果显示，对于选定的不同药效学指标，各成分与之相关联的程度不尽相同，关联度较高的血中移行成分来源于白术芍药散中各味组方饮片的原型成分及其代谢产物，且部分为炮制后复方中含量显著增加的化学成分。实验结果充分体现了传统中医药复方配伍的君臣佐使理论和炮制理论。