基于整合AUC与代谢组学的栀子-连翘药对配伍协同增效机理研究

Herb couples are the unique combinations of two relatively fixed herbs, which have been frequently used for achieving mutual enhancement and assistance in clinical practice of traditional Chinese medicine (TCM). Previously, we found that oral administration of Fructus Gardeniae (FG)-Fructus Forsythiae (FF) herb couple markedly attenuated acute lung injury (ALI), and the efficacy was significantly better than that of single herb. However, the synergistic mechanism is unclear. In order to elucidate the mechanism underlying the synergistic effects, we will innovatively develop a two-step screening UPLC-Q-Tof-MS method with mass defect filtering (MDF) for rapid capture, identification and verification of components in vivo from known consistuents of different chemical structure types, which will be used to establish active components database in FG-FF, FG and FF groups. The integrated AUC of the components in FG-FF, FG or FF group will be evaluated by integrated pharmacokinetics (PK)-pharmacodynamics (PD) model, where the integrated weighting factors will be set up by the normalized 1/IC50 values. Besides the underlying mechanism of anti-ALI after oral administration of FG-FF herb couple group, FG group and FF group will be investigated using plasma and lung-specific metabolomics by LC-MS combined with GC-MS. The comparison of relative distance between treated groups (FG-FF herb couple, FG, FF) and control group in OPLS-DA score plots will be performed to evaluate their correction differences of metabolic disorders. These differences will be illustrated by the changes in metabolic pathways corresponding to metabolic markers, where the metabolic markers will be obtained from comparison between treated groups and non-treated group using OPLS-DA. The outcome of the study would further provide information on synergistic mechanistic studies of TCM herb couples as well as serve as a template for further studies.

“药对”是两味中药相对固定的配对，相须、相使是药对在中医临床中常用的配伍形式。本项目以栀子-连翘药对为例，发现该药对配伍可显著抑制小鼠急性肺损伤，且效果显著优于单味药，但其协同增效机理不明。本课题为阐明其机理，拟开展以下工作：一方面创新性地提出UPLC-Q-TOF/MS两步筛查法结合线性梯度质量亏损过滤技术，从已知不同母核类型中快速捕获、鉴定、验证并建立体内成分群，以1/IC50归一化法设置权重系数的整合PK-PD评价模式，构建体内成分综合量化指标“整合AUC”，通过比较整合AUC来评判栀子、连翘配伍前后物质基础的差异；另一方面采用LC-MS与GC-MS相结合的代谢组学手段比较栀子、连翘配伍前后纠偏代谢紊乱的差异，并通过研究其对机体代谢调节的影响，分析代谢标志物变化规律所对应的代谢通路变化来解释纠偏差异，从而揭示药对配伍前后作用机制。本课题为进一步阐明药对配伍协同增效机理提供了思路与方法。

“药对”是两味中药相对固定的配对，相须、相使是药对在中医临床中常用的配伍形式。本项目以栀子-连翘药对为例，发现该药对配伍可显著抑制小鼠急性肺损伤，且效果显著优于单味药，但其协同增效机理不明。我们通过两步筛查UPLC-Q-Tof/MS结合线性梯度MDF法联合二元logistic回归建立的皂苷质谱数据库，快速从不同母核类型中捕获到栀子-连翘药对提取液共64个成分，包括了环烯醚萜类16个，西红花苷类10个，单环单萜类2个，有机酸类8个，苯乙醇苷类7个，黄酮类11个，木脂素类7个和皂苷类3个；血中捕获到31个成分，其中包括环烯醚萜类6个，西红花苷类3个，单环单萜类2个，有机酸类7个，苯乙醇苷类4个，黄酮类4个，木脂素类4个和皂苷1个；肺组织中发现4个成分，包括栀子苷、京尼平、连翘酯苷A与连翘酯苷E。以1/IC50归一化法设置权重系数，得栀子-连翘药对整合AUC(blood)约为59427，栀子组整合AUC(blood)约为43656，连翘组整合AUC(blood)约为5300；栀子-连翘药对整合AUC(lung)约为5807，栀子组整合AUC(lung)约为4642，连翘组整合AUC(lung)约为1705，结果表明栀子-连翘药对配伍组整合AUC优于栀子组、更优于连翘组。再者，我们通过LC-MS的脂质组学与GC-MS的代谢组学，均发现栀子-连翘药对配伍可显著回调紊乱的差异脂质（TG18:2/20:4/22:6，TG16:0/22:5/22:6，LPC18:3，TG16:0/14:1/16:1，TG16:0/16:1/16:1，LPC18:0与LPC16:0）和代谢物（behenic acid与palmitic acid），且效果优于栀子组、连翘组。研究成果以通讯作者发表在Journal of Chromatography A、Journal of Pharmaceutical and Biomedical Analysis、Evidence-Based Complementary and Alternative Medicine；以第一发明人授权中国发明专利2 项。该研究工作将为揭示中药药对配伍协同增效机理提供思路与方法。