牛黄上清丸治疗实热证的药效物质基础研究

As a representative classic preparation treating “internal fire” clinically, the complex preparation Niuhuang Shangqing pill has a history of usage of more than 400 years; however, with the big prescription, multiple composing medicines and massive constitutions, its effective material basis remains unknown. Our previous work demonstrated that the “divide and conquer” strategy is a perfect solution to this problem. Employing this strategy, the current project plans to divide the complex preparation into three segments according to polarity; and then, we plan to develop multi-dimensional fingerprints for these segments by the advanced technique of comprehensive two-dimension chromatography; afterwards, we are to conduct effect-spectrum analysis on the antiviral activity against respiratory syncytial virus and antibacterial activity against diplococcus pneumonia by chemometrics methods, principal component analysis and orthogonal partial least squares. On the above basis, the bioactive components peaks of curing the internal fire could be located by a comprehensive analysis with the result of anti-internal-fire activity. Finally, with the MS qualitative identification method developed by previous work, these peaks could be identified, which will reveal its effective material basis for curing the internal fire. The current research will build up good basis for the upcoming research such as modem composing of effective components, serum pharmaceutical chemistry and offer approaches and examples to the study of other complex preparations.

大复方牛黄上清丸是中医临床治疗“上焦”之火的代表性中药,作为经典方剂，已有400多年的历史；但因其组方大、药味多、成分复杂，因而药效物质基础尚不明确。我们的前期工作表明，采用“分而治之”策略可以有效解决这一难题。本项目拟采用该策略，按极性划将大复方分成三段，其后通过先进的全二维色谱技术，建立三个极性部分的多维指纹图谱；接着，借助化学计量学方法——主成分分析和正交投影偏最小二乘分析法，对各极性段的抗肺炎双球菌的活性进行多维谱效关系分析；在此基础之上，结合去火活性结果进行综合比较分析，确定治疗上焦火的活性峰群；最后，利用前期研究已经建立的MS定性鉴别方法，对活性峰群进行鉴别，从而揭示其治疗“上焦”之火的药效物质基础。本研究可为接下来的现代化有效组分配伍、血清药物化学等研究奠定良好基础；同时，可为其他大复方的药效物质基础研究提供方法和借鉴。

牛黄上清丸由19味药材组成，为中医临床治疗实热证“上焦”之火的常用代表性方剂。但因其组方大、药味多、成分复杂，因而药效物质基础尚不明确。本研究首先将牛黄上清丸按极性分离成5%、30%、60%、95%乙醇部位，之后采用以中医舌诊为观察指标的实热证小鼠模型对各部进行筛选，发现30%乙醇部位为牛黄上清丸治疗实热证的药效部位。其次，采用基于微量热法的抗肺炎链球菌的试验对各部位进行筛选，结果表明，牛黄上清丸不同部位均有不同程度的抑菌作用，以60%乙醇部位相对更强。经过综合分析，确定60%乙醇部位为有效部位。再次，通过建立市售10批次牛黄上清丸60%乙醇部位的三维指纹图谱，确定了17个共有峰（X1-X17）。最后，采用化学计量学方法主成分分析（PCA）、正交偏最小二乘法（OPLS）和正交偏最小二乘判别分析（OPLS-DA）对得到的三维指纹图谱与抗菌活性数据进行谱-效关系分析。结果显示，化合物X17、X14、X15和X10为治疗实热证的主要活性峰群。对活性峰群采用LC×LC-Triple-Quadrupole-MS与UPLC-Q-TOF-MS分析，并通过对照品验证，鉴定了其中的3个化学成分：黄芩素（X10）、汉黄芩素（X14）、甘草酸（X15），从而阐明了其治疗实热证的药效物质基础。本研究所建立的思路和方法，为接下来的现代化有效组分配伍、血清药物化学等研究奠定良好基础；同时可为其他大复方的药效物质基础研究提供方法和借鉴。