基于微透析及多光谱成像技术的白芍和白术饮片配伍治疗肝郁脾虚证机理研究

The syndrome of liver qi stagnation and spleen deficiency is defined as liver dysfunction and dys-splenism characterized by painful distension of the hypochondrium, abdominal distension, and passing of loose stool. In clinics, compatibility of Radix Paeoniae Alba and Atractylodis Macrocephalae by frying is often prescribed to treat this condition. Practitioners of traditional medicine believe that frying enhances the efficacy of the medicine. Radix Paeoniae Alba nourishes the blood and liver, and Atractylodis Macrocephalae helps invigorate the spleen and eliminate dampness. Thus, the compatibility of these 2 medicines could help achieve the goal of purging wood from the earth, regulating functions of the liver and spleen, nourishing the blood, and benefiting qi. Although the compositions of these 2 medicines have been extensively studied, the appropriate method for processing these medicines and the mechanism underlying their compatibility remain unclear. The objective of this study is to investigate the quantitative, qualitative, pre-processing, and post-processing changes in the composition and compatibility of Radix Paeoniae Alba and Atractylodis Macrocephalae and to determine the effects of these 2 medicines by using an animal model of liver qi stagnation and spleen deficiency. Further, this study attempts to confirm the therapeutic material basis of these 2 medicines for the treatment of syndrome of liver qi stagnation and spleen deficiency and to determine the appropriate processing method and mechanism underlying the compatibility of these medicines. The pharmaceutical ingredients in these 2 medicines were analyzed by microdialysis technique coupled with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and multi-spectral imaging microscopy. This work could serve as a theoretical basis for the development of medicines from white peony root and white atractylodes rhizome, reasonable pharmacy, and clinical medication. Furthermore, it provides new insights into the investigation of "softening of the liver and nourishing of the spleen" in traditional Chinese medicine and for the study of the appropriate processing method for these medicines and mechanism underlying their compatibility in a humor drugged tissue biological effects pattern.

肝郁脾虚证是指肝失疏泄，脾失健运而表现以胸胁胀痛、腹胀、便溏等为主症的证候。又称肝脾不和证。临床上多以炒白芍和炒白术配伍治疗此证。传统理论认为炒制可引药入脾，且白芍长于养血柔肝，白术善于健脾化湿，二药配对，则相使为用，达到土中泻木，共奏调肝和脾、补气养血的目的。虽然二药的化学成分研究较为深入，但其炮制、配伍的机制则远未明晰。本项目以治疗肝郁脾虚证的典型药对白芍-白术为研究对象，采用体液微透析-UPLC /MS联用及多光谱成像分析技术，研究白芍、白术炒制前后、配伍前后物质成分的量变与质变规律以及作用于肝郁脾虚模型动物的生物学特性规律，确定与白芍-白术药对治疗肝郁脾虚证密切相关的药效物质基础，阐明药物炮制、配伍以及作用机理。本项目的开展，为白芍、白术的开发、合理制药和临床用药提供理论依据，并探讨中药"柔肝健脾"作用机理及"体液药化-组织生物效应模式"研究中药炮制与配伍机理的新思路。

肝郁脾虚证是指肝失疏泄，脾失健运而表现以胸胁胀痛、腹胀、便溏等为主症 的证候。临床上多以炒白芍和炒白术配伍治疗此证。传统理论认为炒制可引药入脾，且白芍长于养血柔肝，白术善于健脾化湿，二药配对，则相使为用，达到土中泻木，共奏调肝和脾、补气养血的目的。虽然二药的化学成分研究较为深入，但其炮制配伍的机制则远未明晰。本课题系统考察了白芍、白术最佳炮制工艺，药对炮制和配伍对整体化学成分，体内化学成分及药效等的影响。本项目以8个指标性成分为考察指标，运用正交试验和多指标综合加权评分法，考察了白术-白芍药对提取工艺，即加入10倍量的70%甲醇溶液，回流提取2次，每次1h。通过对白芍-白术药对炮制和配伍前后体外化学成分的HLPC、LC-TOF/MS和IMS定性、定量分析，结果发现研究发现，白术和白芍炮制配伍前后，主要成分含量发生了不同程度的改变。采用MD-Q Exactive LC-MS/MS联用技术对白芍和白术饮片炮制配伍前后血液和肾脏中的原型成分及代谢产物进行了鉴定和分析。结果发现血透析液和肾脏透析液共鉴定出27 种原形药物，结合常见的I 相和II 相代谢类型，共鉴定出55 种Paeoniflorin 的代谢产物，从中共鉴定到60 种atractylenolide II 的主要代谢产物。采用光谱成像法建立了白芍和白术饮片炮制配伍前后成分变化的快速检测技术，GC-IMS可以通过检测传统中药的挥发性组分的变化来监控中药饮片质量。通过药效和分子生物学方法揭示白术-白芍炮制配伍前后治疗肝郁脾虚型UC的药效作用和机制。研究结果显示，药物治疗组（SASP、制品配伍和生品配伍）大鼠结肠组织TNF-α, IL-1, MPO和5-HT水平明显下降（P＜0.01，P＜0.05）；IL-10水平显著升高（P＜0.01，P＜0.05），且制品配伍组疗效优于生品配伍组。Wb研究结果显示，模型组大鼠结肠组织TLR4、MyD88和NF-κB p65蛋白水平表达显著增高，给药后，蛋白表达水平明显降低（P＜0.01，P＜0.05）。而关于Tollip蛋白表达水平，UC大鼠模型组显著下降（P＜0.01），给药后，可以明显升高Tollip蛋白表达水平（P＜0.05），但生品配伍组升高优于制品配伍组。给药后TLR4、MyD88和NF-κB p65mRNA水平明显降低、可以明显升高Tollip mRNA表达水平。