基于广泛靶向代谢组学研究甘草不同年限差异代谢物  

Study on differential metabolites of Glycyrrhiza uralensis Fisch. in different years based on extensive targeted metabonomics

作　　者：周德来 王苗 冯金梁 赵鲲鹏 李运 程显隆[5] 杨扶德 ZHOU De-lai;WANG Miao;FENG Jin-liang;ZHAO Kun-peng;LI Yun;CHENG Xian-long;YANG Fu-de(College of Pharmacy,Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Kangle Pharmaceutical Co.,Ltd.,Lanzhou 730300,China;Clinical College of Traditional Chinese Medicine,Cansu University of Chinese Medicine,Lanzhou 730000,China;Lanzhou Institute of Food and Drug Control,Lanzhou 730050,China;National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]甘肃中医药大学药学院,兰州730000 [2]甘肃康乐药业有限责任公司,兰州730300 [3]甘肃中医药大学中医临床学院,兰州730000 [4]兰州食品药品检验检测研究院,兰州730050 [5]中国食品药品检定研究院,北京102629

出　　处：《药物分析杂志》2024年第1期144-157,共14页Chinese Journal of Pharmaceutical Analysis

基　　金：2021年度甘肃高等学校产业支撑计划项目(2021CYZC-40);甘肃省科技小巨人企业培育计划项目(17CXIJA084);甘肃省委组织部人才发展专项资金项目(2018年);2018年兰州市人才创新创业科技计划项目(2017-RC-112)。

摘　　要：目的:通过对不同栽培年限甘草中的代谢成分进行定性定量分析,寻找其差异代谢物,探究甘草体内代谢物的累积规律。方法:采用Agilent SB-C_(18)(100 mm×2.1 mm, 1.8μm)色谱柱,以0.1%甲酸水溶液为流动相A,0.1%甲酸乙腈溶液为流动相B,梯度洗脱,流速0.35 mL·min^(-1),柱温40℃,进样量4μL;质谱采用正负离子扫描,多反应监测模式,进行样品质谱信号采集,基于自建二级质谱数据库对不同年限甘草体内的代谢物进行定性与定量分析,结合主成分分析、正交偏最小二乘法判别分析、聚类热图分析等手段对不同年限甘草的代谢物进行多元统计分析。结果:(1)从不同年限甘草样品共检测到1 038个代谢物,其中一年生与二年生甘草间存在201个差异代谢物,125个上调,76个下调;二年生与三年生之间存在223个差异代谢物,64个上调,159个下调;一年生与三年生之间存在185个差异代谢物,59个上调,126个下调;发现一年生甘草特有代谢物4个,二年生6个,三年生1个。(2)对差异代谢物进行K-均值聚类分析,按照积累趋势不同将差异代谢物进行分类,发现大多数黄酮、酚酸、萜、木脂素及香豆素等类代谢物在二年生甘草中达到峰值,大多数生物碱、氨基酸及其衍生物等类代谢物在一年生甘草中达到峰值,一部分黄酮、酚酸等类代谢物在三年生甘草中达到峰值,提示甘草体内代谢物含量变化存在一定的规律。(3)在京都基因与基因组百科全书(KEGG)数据库注释得到160个差异代谢物,黄酮类、氨基酸及其衍生物、有机酸是其中占比较多的差异代谢物。不同对比组间共富集到79条差异代谢通路,其中极显著富集的差异代谢通路(P<0.01)6条,显著富集的通路(P<0.05)23条,参与上述通路的化合物在不同年限对比中的分布与富集前基本一致。结论:本研究阐明了不同生长年限甘草代谢组分间的差异,并通过差异代谢物进一步分析了�Objective:To investigate the accumulation pattern of metabolites in Glycyrrhiza uralensis by qualitative and quantitative analyses of metabolic constituents in Glycyrrhiza uralensis with different cultivation years, and to search for its differential metabolites. Methods: The separation was performed on an Agilent SB-C_(18)(100 mm×2.1 mm, 1.8 μm) column with 0.1% formic acid aqueous solution as the mobile phase A and 0.1% formic acid acetonitrile solution as the mobile phase B. The gradient elution was carried out at a flow rate of 0.35 mL·min^(-1), and the column temperature was 40 ℃ with an injection volume of 4 μL. The mass spectrometry was performed with positive and negative ions scanning in multiple reaction monitoring mode. The mass spectrometry was performed in multi-response monitoring mode with positive and negative ion scanning. The qualitative and quantitative analyses of the metabolites in Glycyrrhiza uralensis were carried out on the basis of the self-constructed secondary mass spectrometry database, and the multivariate statistical analyses of the metabolites of Glycyrrhiza uralensis with different cultivation were combined with principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and cluster heat map analyses. Results:(1) A total of 1 038 metabolites were detected from the samples of Glycyrrhiza uralensis with different cultivation years, among which 201 differential metabolites existed between annual and biennial Glycyrrhiza uralensis, 125 up-regulated and 76 down-regulated;223 differential metabolites existed between biennial and three years old Glycyrrhiza uralensis, 64 up-regulated and 159 down-regulated;185 differential metabolites existed between annual and three years old Glycyrrhiza uralensis, 59 up-regulated and 126 down-regulated. Four metabolites specific to annual Glycyrrhiza uralensis, six to biennial and one to three-year old were found.(2) K-mean cluster analysis was performed on the differential metabolites, and the differential

关 键 词：甘草 栽培年限 广泛靶向代谢组学 超高效液相色谱-串联四极杆/线性离子阱质谱法 差异代谢物 KEGG通路 正交偏最小二乘法判别分析 

分 类 号：R917[医药卫生—药物分析学]