基于谱效相关及代谢组学技术解析甘草蜜炙增效炮制原理

Licorice is a commonly used traditional Chinese medicine in clinical, which has a long history as a drug. In clinical,there are two specification most commonly used, including crude and product processed with honey.Traditional theory holds that,after being processed with honey, efficacy of licorice changes from Qing to Bu,tonic effect enhances, which was confirmed using pharmacological experiment research by the project team.In addition,we compared the composition of raw and processed product by TLC and HPLC method. The study found that the proportion of the main chemical components of licorice changed in certain after being processed. 5-Hydroxymethylfurfural emerged and the content of flavonoids and triterpene acids have decreased. In addition, there were some changes in content of some unknown ingredients. But the correlation of component changing and enhanced tonic efficancy is not clear. Accordingly, we present the following hypothesis: After being processed,chemical composition changed in qualitative as well as in quantitative, causing the change of endogenous metabolites and enable its tonic effect enhanced. The project team intent to study the changing composition of licorice after being processed by establishing fingerprint by UPLC-DAD-ELSD-MS(N) method.By spectrum-effect correlation analysis, material base of pharmacal efficacy will be clarified. Using metabonomics technology,we will further study the influence of licorice and active components on animal model of spleen deficiency, to study the metabolic trend and reveal the enhanced tonic effect mechanism, and finally clarify the processing principle of licorice being fried with honey .

甘草为临床最常用的传统中药之一，多以生品和蜜炙品入药。传统炮制理论认为，蜜炙后甘草功效由清转补，补益作用增强。前期通过对非特异性免疫功能影响、抗疲劳试验等研究证实甘草蜜炙后确能增强补脾益气功能,通过开展化学研究发现，蜜炙后黄酮和三萜等主要成分含量降低，成分比例关系发生变化，增加蜂蜜成分和5-HMF，另有其它未知成分也有含量增减变化，但目前这些变化与药效增强相关性尚不明确。基于此，我们提出假说：甘草蜜炙后功效物质发生量变与质变，调整机体代谢引起内源性代谢物的变化，从而使补益作用增强。项目拟以甘草蜜炙后量/质变成分为目标化合物，运用UPLC-DAD-ELSD-MS(N)技术研究生、炙品成分谱差异，结合药效试验，通过谱效相关筛选甘草炙后补益作用增强的活性物质。运用代谢组学技术，进一步开展生、炙饮片及活性成分干预脾虚动物模型标志物代谢趋势研究，从整体角度为解析甘草蜜炙增效炮制原理提供实验依据。

项目以传统中医药理论为指导，融合多学科理论和技术，拟通过开展谱效相关和代谢组学研究，为解析甘草蜜炙原理提供数据支持。通过对生、炙甘草化学成分开展系统的比较研究，对生炙甘草成分差异有了较为系统的了解，并筛选出对甘草生品和蜜炙品分组有重要贡献的 27个化合物。结合药效学实验结果，进一步通过谱效相关性分析筛选出其中8个成分对蜜炙后补益增强具有较大的影响（VIP＞1.0），研究较明确的解析了甘草蜜炙后成分的总体变化情况以及成分变化与补益增效之间的相关性。蜜炙后其他成分变化还包括具有免疫活性的甘草多糖含量增加，具有免疫抑制作用甘草酸含量降低，推测也与甘草蜜炙后免疫活性增强有关。同时甘草次酸还是导致甘草长期服用皮质激素样副作用的主要成分，因此甘草酸含量的降低，也与蜜炙减轻甘草不良反应有关。釆用RRLC-Q-TOF/MS代谢组学技术，筛选出38种潜在的脾虚潜在生物标志物，其中炙甘草水提物干预25种，甘草水提物干预13种。分析和筛选出5种甘草和炙甘草水提物补脾作用的生物标志物的代谢途径，分别为鞘脂代谢，甘油磷脂代谢，花生四烯酸代谢，α-亚麻酸代谢，不饱和脂肪酸的生物合成，其中甘草水提物对不饱和脂肪酸的生物合成代谢途径干预明显，炙甘草水提物则对另外4个代谢通路的干预效果更为显著。. 项目首次将植物代谢组学手段用于分析生炙甘草成分差异，筛选出的差异化合物，可作为区分生甘草和蜜炙甘草的炮制标记物，并分别用于甘草和蜜炙甘草饮片专属性质量评价，可为完善饮片质量标准提供数据支持。谱效相关分析较为准确的解释了甘草多个化学成分与药效指标的相关性大小和方向，为进一步开展甘草蜜炙增强补益功效的物质基础研究明确了方向。代谢组学研究结果筛选出的脾虚大鼠的潜在生物标志物，可用于评价甘草蜜炙前后干预脾虚大鼠模型作用，同时揭示蜜炙甘草生熟异治补脾的作用机制。