基于LC-MS/MS分析板芪清肺组方发酵代谢差异

doi:10.16431/j.cnki.1671-7236.2023.10.038

摘要/Abstract

摘要： 【目的】试验从代谢组学水平探讨贝莱斯芽孢杆菌发酵板芪清肺组方中药材的代谢差异。【方法】采用液相色谱质谱/质谱联用(LC-MS/MS)技术,通过主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)等多元统计和代谢途径分析探讨板芪清肺组方中药材粉末和经贝莱斯芽孢杆菌固态发酵的板芪清肺组方药材差异代谢产物和代谢通路。【结果】发酵板芪清肺颗粒组方组(Enzyme组)和未发酵板芪清肺颗粒组方对照组(Water组)在正离子模式下共筛选出34 624个差异代谢产物,在负离子模式下共筛选出15 762个差异代谢产物。将差异代谢物进行聚类分析,鉴定出的差异代谢物可分为203类,正、负离子模式组间关系PCA图的区分效果良好;发酵后主要上调差异代谢物包括雌马酚、二乙酸乙二醇酯、香草乙二醇、D-鸟氨酸、喹雌酚、胆固醇和乙酰胆碱等,下调差异代谢产物包括黄曲霉素、芥酸、肉豆蔻酸和黄嘌呤等;KEGG代谢通路富集分析筛选到27条显著差异代谢通路,包括近端小管碳酸氢盐回收、精氨酸生物合成、组氨酸和嘌呤衍生生物碱的生物合成、植物次生代谢物的生物合成、苯丙烷的生物合成、蛋白质的消化和吸收等。【结论】板芪清肺组方发酵后差异代谢产物脂类、酮类等氨基酸显著增加,酸类氨基酸显著降低,涉及到多种氨基酸生物合成和嘌呤代谢等途径,为板芪清肺组方发酵产品提供了理论依据。

关键词: 板芪清肺组方; 发酵; LC-MS/MS; 代谢组学

Abstract: 【Objective】 The experiment aimed to explore the metabolic differences of traditional Chinese medicine in the fermentation of Banqi Qingfei prescription by Bacillus subtilis at the metabolomics level.【Method】 Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technology,multiple statistical analysis and metabolic pathway analysis such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA),the differential metabolites and metabolic pathways between the traditional Chinese medicine powder of Banqi Qingfei prescription and the traditional Chinese medicine powder of Banqi Qingfei prescription fermented by Bacillus subtilis were explored.【Result】 A total of 34 624 differential metabolites were screened out in the fermentation group of Banqi Qingfei granules (Enzyme group) and the control group of unfermented Banqi Qingfei granules (Water group) in the positive ion mode,and 15 762 differential metabolites were screened out in the negative ion mode.Cluster analysis was conducted on the differential metabolites,and the identified differential metabolites could be divided into 203 categories.The PCA diagram of the relationship between the positive and negative ion mode groups showed good differentiation effect.After fermentation,the main up-regulated differential metabolites include equol,ethynodiol diacetate,vanylglycol,D-ornithine,quinestrol,cholesterol and acetylcholine,and the down-regulated differential metabolites include Aspergillus flavus,erucic acid,myristic acid and xanthine.After enrichment analysis of KEGG metabolic pathways,27 significantly different metabolic pathways were screened,including proximal tubule bicarbonate recovery,arginine biosynthesis,biosynthesis of histidine and purine derived alkaloids,biosynthesis of plant secondary metabolites,phenylpropane biosynthesis,protein digestion and absorption,etc.【Conclusion】 After the fermentation of Banqi Qingfei prescription,amino acids such as lipids and ketones of differential metabolites increased significantly,and acid amino acids decreased significantly,involving multiple amino acid biosynthesis and purine metabolism,providing a theoretical basis for the fermentation products of Banqi Qingfei prescription.

Key words: Banqi Qingfei prescription; fermentation; LC-MS/MS; metabonomics

中图分类号:

S853.7

张艳, 刘雪松, 薛沾枚, 杨昊天, 张国华, 张备, 李莉, 冯万宇, 王志强, 钟鹏, 史同瑞. 基于LC-MS/MS分析板芪清肺组方发酵代谢差异[J]. 中国畜牧兽医, 2023, 50(10): 4243-4251.

ZHANG Yan, LIU Xuesong, XUE Zhanmei, YANG Haotian, ZHANG Guohua, ZHANG Bei, LI Li, FENG Wanyu, WANG Zhiqiang, ZHONG Peng, SHI Tongrui. Analysis of Difference in Fermentation Metabolism of Banqi Qingfei Prescription Based on LC-MS/MS[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4243-4251.

参考文献

[1] 黄敏繁,于滢,李文凯,等.中兽药在兽医临床中的应用及创新发展研究[J].饲料博览,2022,3:77-80. HUANG M F,YU Y,LI W K,et al.Study on application and innovative development of Chinese veterinary medicine in veterinary clinical[J].Feed Review,2022,3:77-80.(in Chinese)
[2] 张馨予,苏建青,褚秀玲.发酵中药在畜禽高效养殖中的应用研究进展[J].中国兽医杂志,2022,58(5):121-124. ZHANG X Y,SU J Q,CHU X L.Research progress in the application of fermented traditional Chinese medicine in efficient breeding of livestock and poultry[J].Chinese Journal of Veterinary Medicine,2022,58(5):121-124.(in Chinese)
[3] 刘秋瑾,尹珺伊,张军,等.黄芪、板蓝根固态发酵中有效成分变化[J].现代畜牧科技,2022,4:9-11. LIU Q J,YIN J Y,ZHANG J,et al.Changes of active components in solid-state fermentation of Radix astragali and Radix isatidis[J].Modern Animal Husbandry Science & Technology,2022,4:9-11.(in Chinese)
[4] 陈银翠,杜静,王琪琪,等.冠突散囊菌固态发酵对黄芪化学成分和生物活性的影响[J].中成药,2022,44(7):2202-2207. CHEN Y C,DU J,WANG Q Q,et al.Effects of solid-state fermentation of Saccharomyces coronatus on chemical components and biological activities of Astragalus[J].Chinese Traditional Patent Medicine,2022,44(7):2202-2207.(in Chinese)
[5] 乔宏兴,张立恒,张晓静,等.基于LC-MS代谢组学的植物乳杆菌发酵黄芪的代谢产物分析[J].中国畜牧兽医,2021,48(9):3283-3292. QIAO H X,ZHANG L H,ZHANG X J,et al.Metabolites analysis of Astragalus membranaceus fermented by Lactobacillus plantarum based on LC-MS metabolomics[J].China Animal Husbandry & Veterinary Medicine,2021,48(9):3283-3292.(in Chinese)
[6] 谢静,李云鹃,缪兴龙,等.基于代谢组学技术的中药复方药效物质基础与作用机制研究[J].药物评价研究,2020,43(7):1439-1445. XIE J,LI Y J,MIU X L,et al.Study on the material basis and mechanism of efficacy of traditional Chinese medicine based on metabonomics technology[J].Drug Evaluation Research,2020,43(7):1439-1445.(in Chinese)
[7] VASILEV N,BOCCARD J,LANG G,et al.Structured plant metabolomics for the simultaneous exploration of multiple factors[J].Scientific Reports,2016,6:37390.
[8] ZELENA E,DUNN W B,BROADHURST D,et al.Development of a robust and repeatable UPLC-MS method for the long-term metabolomic study of human serum[J].Analytical Chemistry,2009,81(4):1357-1364.
[9] WANT E J,MASSON P,MICHOPOULOS F,et al.Global metabolic profiling of animal and human tissues via UPLC-MS[J].Nature Protocols,2013,8(1):17-32.
[10] THÉVENOT E A,ROUX A,XU Y,et al.Analysis of the human adult urinary metabolome variations with age,body mass index,and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses[J].Journal of Proteome Research,2015,14(8):3322-3335.
[11] XIA J,WISHART D S.Web-based inference of biological patterns,functions and pathways from metabolomic data using MetaboAnalyst[J].Nature Protocols,2011,6(6):743-60.
[12] GAO S W,WEI D Z,HOUKAI L,et al.Application of metabolomics for unveiling the therapeutic role of traditional Chinese medicine in metabolic diseases[J]. Journal of Ethnopharmacology,2019,10(5):112057-112106.
[13] XIE B G,ZHANG Z R,GONG T,et al.Application of metabonomic strategy to discover an unreported active ingredient in Liuweidihuang pills suppressing beta-glucuronidase[J].Analytical and Bioanalytical Chemistry,2015,407(2):609-614.
[14] 李雅倩,李克鑫,李道明,等.黄芪的活性成分、生物学功能及其在猪养殖中的应用[J].猪业科学,2022,39(7):102-106. LI Y Q,LI K X,LI D M,et al.Active components,biological functions and application in pig breeding of Astragalus membranaceus[J].Swine Industry Science,2022,39(7):102-106.(in Chinese)
[15] 黄远,董福越,李楚源.板蓝根中主要化学成分含量测定方法研究进展[J].中国药业,2020,29(7):150-156. HUANG Y,DONG F Y,LI C Y,et al.Research progress in determination methods of main chemical components in Isatis indigotica[J].China Pharmaceuticals,2020,29(7):150-156.(in Chinese)
[16] 孔祥琳,李运伦,阚东方,等.黄芪甲苷调控动脉粥样硬化小鼠肝脏代谢组学研究[J].中华中医药杂志,2022,37(7):3779-3784. KONG X L,LI Y L,KAN D F,et al.Metabolomic study of Astragaloside Ⅳ regulating liver metabolism in atherosclerotic mice[J].China Journal of Traditional Chinese Medicine and Pharmacy,2022,37(7):3779-3784.(in Chinese)
[17] 曾涵芳,林红,裴明财,等.基于非靶向代谢组学分析黄芪多糖对热应激奶牛血清代谢的影响[J].中国兽医学报,2022,42(2):319-325. ZENG H F,LIN H,PEI M C,et al.Effect of Astragalus polysaccharide on serum metabolism of heat stress cows based on non-targeted metabolomics[J].Chinese Journal of Veterinary Science,2022,42(2):319-325.(in Chinese)
[18] 赵美雪,马丽,盛钰,等.脂质体仿生膜联合LC-MS筛选板蓝根有效成分[J].中南药学,2015,13(11):1168-1171. ZHAO M X,MA L,SHENG Y,et al.Screening of effective components of Isatis indigotica by liposome biomimetic membrane combined with LC-MS[J].Central South Pharmacy,2015,13(11):1168-1171.(in Chinese)
[19] 侯宪邦.板蓝根潜在药效成分的发现及其作用机制的研究[D].南京:南京中医药大学,2017. HOU X B.Discovery of potential pharmacological components of Isatis indigotica and study on their mechanism of action[D].Nanjing:Nanjing University of Traditional Chinese Medicine,2017.(in Chinese)
[20] WU G,BAZER F W,DAVIS T A,et al.Arginine metabolism and nutrition in growth,health and disease[J].Amino Acids,2009,37(1):153-168.
[21] 胡孟,武书庚,王晶,等.组氨酸的生理功能及在动物生产中的应用[J].中国饲料,2018,7:38-45. HU M,WU S G,WANG J,et al.Physiological function and application of histidine in animal production[J].China Feed,2018,7:38-45.(in Chinese)
[22] KRIENGSINYOS W,RAFII M,WYKES L J,et al.Long-term effects of histidine depletion on whole-body protein metabolism in healthy adults[J].The Journal of Nutrition,2002,132(11):3340-3348.
[23] KOPEC W,WILICZKIEWICZ A,JAMROZ D,et al.Antioxidant status of turkey breast meat and blood after feeding a diet enriched with histidine[J].Polutry Science,2016,95(1):53-61.
[24] 陈梦涵,于立辉,李丽爽,等.生物碱类化合物抗菌及逆转细菌耐药性研究进展[J].动物医学进展,2023,44(4):109-113. CHEN M H,YU L H,LI L S,et al.Progress on antibacterial and drug resistance reversal effects of alkaloids[J].Progress in Veterinary Medicine,2023,44(4):109-113.(in Chinese)
[25] MITRA S,PROVA S R,SULTANA S A,et al.Therapeutic potential of indole alkaloids in respiratory diseases:A comprehensive review[J].Phytomedicine,2021,90:153649-153672.
[26] 周雨亭.贝莱斯芽孢杆菌纤溶酶的纯化、性质研究及体内外溶栓效果的评价[D].南宁:广西大学,2022. ZHOU Y T.Biological characteristics of Bacillus velezehsis and its effects on growth performance and mycotoxin metabolism of broilers[D].Nanning:Guangxi University,2022.(in Chinese)