基于UPLC-Q-TOF-MS/MS分析粉葛芦头的化学成分及其对小鼠肝脏代谢物的影响

Analysis of Chemical Composition in Puerariae Thomsonii Stem Base and Investigation of Its Effect on Liver Metabolites in Mice Based on UPLC-Q-TOF-MS/MS

目的:系统对比粉葛芦头与粉葛根部化学成分差异,并运用肝脏代谢组学的分析方法进一步探讨粉葛芦头潜在的保肝作用。方法:使用超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS/MS)分析粉葛芦头与根部的化学成分,选取6~8周龄昆明小鼠20只,雌雄各半,随机分为空白组(无菌水)和粉葛芦头组(1.95 g·kg^(-1)),每组10只,灌胃给药14 d,每日称取体质量,于末次给药后麻醉小鼠,采集心、肝、脾、肺、肾等脏器,计算脏器指数;酶联免疫吸附测试法(ELISA)检测各组小鼠血清中天冬氨酸氨基转移酶(AST),丙氨酸氨基转移酶(ALT),总胆固醇(TC),甘油三酯(TG)含量;苏木素-伊红(HE)染色观察小鼠心、肝、脾、肺、肾各组织的形态变化;采用UPLC-Q-TOF-MS/MS分析粉葛芦头对小鼠肝脏代谢轮廓的调节作用,指认空白组与粉葛芦头组间的差异代谢物,并利用京都基因与基因组百科全书(KEGG)进行代谢通路富集。结果:从粉葛芦头、粉葛根部中鉴定出19个共有化学成分,均为粉葛主要药效物质。药效学结果表明,粉葛芦头可控制小鼠体质量的增长,降低小鼠血清中TC、TG、ALT、AST含量,HE染色观察及脏器指数表明,最高临床等效剂量下粉葛芦头对各主要脏器无明显影响。通过代谢组学分析共鉴定出38种差异代谢物,其中35个上调,3个下调。这些差异代谢物主要为氨基酸类、脂肪酸类、维生素类、甾体类、核苷类、嘧啶类及生物碱类等化合物。代谢通路分析共筛选得到3条关键代谢通路,包括酪氨酸代谢、维生素B_(6)代谢及色氨酸代谢。结论:粉葛芦头具有与粉葛根部相似的化学成分组成,其可能通过富含的黄酮类及异黄酮类成分调节氨基酸、维生素代谢,从而发挥保肝作用。该研究为粉葛芦头的临床应用与产品开发提供了数据基础。

Objective:To systematically compare the chemical compositional differences between Puerariae Thomsonii stem base(PTSB)and Puerariae Thomsonii Radix(PTR),and to explore the potential hepatoprotective effects of PTSB by liver metabolomics.Method:Ultra performance liquid chromatographyquadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS)was used to analyze the chemical compositions of PTSB and PTR.Twenty Kunming mice aged 6-8 weeks,half male and half female,were randomly divided into the blank group(sterile water)and PTSB group(1.95 g·kg^(-1)),with 10 mice in each group,and the drug was administered by gavage for 14 d,and the body mass was weighed once a day.After the last administration,mice were anesthetized,organs such as heart,liver,spleen,lungs and kidneys were collected,and the organ index was calculated.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC)and triglyceride(TG)in the serum of mice from each group,the morphological changes of heart,liver,spleen,lung and kidney tissues were observed by hematoxylin-eosin(HE)staining,and the regulation of PTSB for the hepatic metabolic profiles of mice was analyzed by UPLC-Q-TOF-MS/MS,then the differential metabolites between the blank group and PTSB group were designated,and the metabolic pathways was enriched by Kyoto Encyclopedia of Genes and Genomes(KEGG).Result:A total of 19 common chemical constituents were identified from PTSB and PTR,all of which were the main pharmacodynamic substances of PTR.The pharmacodynamic results showed that PTSB could control the growth of body mass of mice and reduce the contents of TC,TG,ALT and AST in serum of mice.HE staining observations and organ indexes showed that there was no significant effect of PTSB on all major organs at the highest clinically equivalent dose.A total of 38 differential metabolites were identified by metabolomics,of which 35 were up-regulated and 3 were down-regulated.These differential metabolites were mainly compounds such as amino acids,fatty acids,vitamins,steroids,nucleosides,pyrimidines and alkaloids.Three key metabolic pathways,including tyrosine metabolism,vitamin B_(6) metabolism and tryptophan metabolism,were screened by metabolic pathway analysis.Conclusion:PTSB has a similar chemical composition to that of PTR,and it may regulate the metabolism of amino acids and vitamins through the flavonoids and isoflavonoids,thus exerting a potential hepatoprotective effect.This study provides an experimental reference for the clinical application and product development of PTSB.