蒲黄"生行熟止"的物质基础及质量评价

The opinion of "Sheng Xing Shu Zhi" for Typhae Pollen was recorded in books on Chinese materia medica and has been validated by clinical. Generally，flavonoids in Typhae Pollen are considered to have hemostasis effect. But contents of flavonoids in Typhae Pollen carbonized are significantly lower, while its hemostasis effect was significantly enhanced. The unclear effective substances lead to quality control has not been fundamentally resolved yet. Our research shows that phenolic acids did not change significantly and flavonoids changes significantly in Typhae Pollen carbonized. Phenolic acids prolong hemostaisis time, and flavonoids shorten the hemostasis time. Hemostasis effect was enhanced with reducing the ratios of flavonoids/phenolic acids. Therefore, this project is first proposed research ideas on substances foundation based on the theory of "drug-drug interaction". We will explore mechanism of the different proportions of phenolic acids and flavonoids in Caco2 cells absorption, plasma protein binding, and CYP450 enzyme inhibition, investigate correlation between pharmacokinetic and pharmacodynamic, find out substances foundation and build quality evaluation strategy about "Sheng Xing Shu Zhi" of Typhae Pollen by molecular biology, enzymology and separation and analysis techniques. And the research will find new constituents in treatment of blood stasis syndrome and hemorrhagic syndrome, and provide strong scientific evidence for guiding using of drugs accurately in clinical, and also provide a new model and reference for the research of similar traditional Chinese medicine.

本草记载蒲黄"生行熟止"，现已被临床公认，即蒲黄行血，炒炭止血。一般认为蒲黄黄酮类有止血作用，但蒲黄炭黄酮类明显下降，而止血作用增强。这种药效物质基础不明确，导致质量控制没有得到根本解决。我们研究表明，蒲黄炭中酚酸类变化不明显，黄酮类变化显著，酚酸类延长凝血时间，黄酮类缩短凝血时间。当黄酮/酚酸比例减低，则凝血作用增强。因此，本课题首次提出了基于"药物-药物相互作用"理论研究中药药效物质基础的科学假说，探讨黄酮与酚酸不同配比在Caco2细胞吸收、血浆蛋白结合率、CYP450酶抑制作用的机制，考察酚酸类与黄酮类成分配比的药动学-药效学相关性，构建适合"生行熟止"的蒲黄及蒲黄炭质量评价策略。通过应用药物代谢学、酶学和分离分析等技术，系统地探讨蒲黄"生行熟止"的物质基础。本课题可能发现治疗血瘀证及出血证新的组分配比，为指导临床准确用药提供有力的科学证据，并为同类中药的研究提供新的模式和借鉴。

中药蒲黄“生行熟止”已被中医临床验证，但其药效物质基础、作用机理、质量评价还不清楚。本研究开展以下内容：（1）建立了蒲黄和蒲黄炭的体内外分析方法，对其在蒲黄和蒲黄炭及其在大鼠血浆、人工胃液、人工肠液、肠道菌群及肝微粒体中的成分进行分析和鉴定，形成了本团队的分析技术平台。（2）对蒲黄及活血部位进行急性血瘀状态下药效学研究，阐明了蒲黄的活血作用，进一步通过PK-PD相关性研究，阐明了蒲黄的活血成分为酚酸和黄酮苷类，并用一测多评法进行测定，为新版药典蒲黄的质量控制提供依据。（3）对蒲黄炭新生成的主要成分进行结构确证，通过药效实验证明蒲黄炭及其新生成的部位对凝血障碍大鼠具有显著的止血作用，进一步对蒲黄炭进行正常及模型大鼠中药动学研究及药动学参数模式识别分析，阐明了蒲黄炭的止血成分为香蒲胺类。通过建立色度差-成分-药效相关性的方法，用于蒲黄炭的质量评价。（4）通过体外肠吸收、血浆蛋白结合率及代谢三个环节分析，表明蒲黄中黄酮苷在肠菌中代谢成苷元，其与血浆蛋白结合率低并降低酚酸的血浆蛋白结合率，推测蒲黄中黄酮和酚酸类在体内更好地发挥活血作用，炒炭后黄酮大量降低又生成新的止血部位，导致蒲黄炭具有止血作用，从而阐明了蒲黄“生行熟止”的物质基础及作用机制。发表论文2篇，其中SCI 论文1篇，中文核心期刊1篇，会议论文1篇，申请专利1项。本项目的骨干教师潘英妮副教授获得“辽宁省高水平创新团队国（境）外项目资助（负责人），赴日本进行访问和交流，展现了本研究团队影响力；本研究共培养硕士研究生3名， 2名已毕业并取得学位，1名在读；参与该项目的博士研究生和硕士研究生获得国家奖学金、企业奖学金及多次获得学业奖学金。本项研究为同类中药炮制的研究提供借鉴，进一步深化“生熟异治”理论的科学内涵，推动学科发展与创新。同时，提高了团队对中药药效物质、作用机理、质量评价研究的整体技术水平和研究实力，为进一步开展相关研究奠定基础。