UFLC-MS/MS法同时测定5个不同发育时期牛蒡子胚薄壁细胞和内果皮石细胞中3种化学成分
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摘要
通过激光显微切割对5个不同发育时期牛蒡子胚薄壁细胞和内果皮石细胞进行精确收集。采用超快速液相色谱-质谱联用(UFLC-MS/MS)技术对样品中咖啡酸、牛蒡苷、牛蒡苷元进行定量分析。结果发现,胚薄壁细胞在花末期至成熟期产生和积累大量牛蒡苷,内果皮石细胞在成熟期也积累一定量的牛蒡苷,但其含量远小于胚薄壁细胞中的牛蒡苷含量,结果表明牛蒡子胚薄壁细胞和内果皮石细胞中的牛蒡苷的生物合成途径可能存在不同;而且发现牛蒡苷元在花末期胚薄壁细胞和内果皮石细胞中均有产生并且积累到成熟期,但是在内果皮石细胞中积累到花末期达到最高值,然后缓慢下降。实验建立了牛蒡子取材-冰冻制片-显微切割-液质联用分析的新方法,研究牛蒡子中主要化学成分牛蒡苷的动态积累规律。
In this study, we accurately collected the embryonic parenchyma cells and endocarp stone cells of Arctii Fructus at five different growth stages by laser microdissection. Quantitative analyse of caffeic acid, arctiin and arctigenin in these cells were performed using ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS). The results showed that a large amount of arctiin was produced and accumulated in embryonic parenchyma cells from the late flowering stage to mature stage, while much lower content of arctiin was produced and accumulated in endocarp stone cells at these stages. It suggested that the biosynthetic pathways of arctiin were different in embryonic parenchyma cells from endocarp stone cells of Arctii Fructus. Arctigenin was found to be produced and accumulated in both embryonic parenchyma cells and in endocarp stone cells from the late flowering stage to mature stage, but it reached a peak in endocarp stone cells at late flowering stage, then decreased slowly.The concentration of arctigenin was far less than that of arctiin regardless of embryonic parenchyma cells or endo‐carp stone cells. These results have validated the new method for analysis of dynamic accumulation of arctiin in Arctii Fructus by UFLC-MS/MS with frozen sections and microdissection.
In this study, we accurately collected the embryonic parenchyma cells and endocarp stone cells of Arctii Fructus at five different growth stages by laser microdissection. Quantitative analyse of caffeic acid, arctiin and arctigenin in these cells were performed using ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS). The results showed that a large amount of arctiin was produced and accumulated in embryonic parenchyma cells from the late flowering stage to mature stage, while much lower content of arctiin was produced and accumulated in endocarp stone cells at these stages. It suggested that the biosynthetic pathways of arctiin were different in embryonic parenchyma cells from endocarp stone cells of Arctii Fructus. Arctigenin was found to be produced and accumulated in both embryonic parenchyma cells and in endocarp stone cells from the late flowering stage to mature stage, but it reached a peak in endocarp stone cells at late flowering stage, then decreased slowly.The concentration of arctigenin was far less than that of arctiin regardless of embryonic parenchyma cells or endo‐carp stone cells. These results have validated the new method for analysis of dynamic accumulation of arctiin in Arctii Fructus by UFLC-MS/MS with frozen sections and microdissection.
引文
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[2]Ma TY,Chen YP,Cheng SP,et al.Research progress of Fructus Arctii[J].J Liaoning Univ Tradit Chin Med(辽宁中医药大学学报),2018,20:113-116.
[3]Fan XQ,Shen YF.Research progress on pharmacological activi‐ties of arctiin[J].Shanghai J Tradit Chin Med(上海中医药杂志),2017,51:113-116.
[4]Kang TG,Dou DQ,Hen GR,et al.Component comparison of the raw and processed Arctii Fructus and elucidation of its processing principle[J].Planta Med,2010,76:524-525.
[5]Xing YP,Chen SY,Xu L,et al.Study on high throughput sequencing identification of Fructus Arctii and five counterfeit species mix power[J].China J Chin Mater Med(中国中药杂志),2018,43:3862-3866.
[6]Kang TG,Dou DQ,Xu L.Establishment of a quality marker(Q-marker)system for Chinese herbal medicines using burdock as an example[J].Phytomedicine,2019,54:339-346.
[7]Xu L,Dou DQ,Wang B,et al.Identification of traditional medicine"Fructus Arctii"by nuclear ribosomal DNA ITSsequences[J].China J Chin Mater Med(中国中药杂志),2011,36:338-341.
[8]Zhang F,Cai YF.Laser microdissection technology and its application in botany[J].Bull Biol(生物学通报),2010,45:11-12.
[9]Jiang YB,Shan JH,Wang Y,et al.Application progress of LC-MS/MS technology in drug metabolism research[J].Chin JPharm Anal(药物分析杂志),2014,34:385-391.
[10]Hu J,Qin KM,Zhu TT,et al.Qualitative and quantitative analysis of major constituents of raw and processed Arctii Fructus by UHPLC-UV-Q-TOF-MS/MS[J].Acta Pharm Sin(药学学报),2017,52:603-608.
[11]Zhou A,Li QL,Peng DY,et al.Analysis of xanthones in gamboge by HPLC-PDA-ESI/MS[J].Acta Pharm Sin(药学学报),2008,43:838-842.
[12]Xue Y,Li XW,Li ZY,et al.UPLC/Q-TOF MS and NMR plant metabolomics approach in studying the effect of growth year on the quality of Polygala tenuifolia[J].Acta Pharm Sin(药学学报),2015,50:340-347.
[13]Liang Z,Chen Y,Xu L,et al.Localization of ginsenosides in the rhizome and root of Panax ginseng by laser microdissection and liquid chromatography-quadrupole/time of flight-mass spec‐trometry[J].J Pharm Biomed Anal,2015,105:121-133.
[14]Zhou W,Liang Z,Li P,et al.Tissue-specific chemical profiling and quantitative analysis of bioactive components of Cinnamomum cassia by combining laser-microdissection with UPLC-Q/TOF-MS[J].Chem Central J,2018,12:71.
[15]Qiao Q,Xiao YP,Wang ZZ.Anatomical structure and histo‐chemical localization of the drupe of Macrocarpium officinacle[J].Acta Bot Yunnan(云南植物研究),2004,26:651-655.
[16]Liang Z,Oh K,Wang Y,et al.Cell type-specific qualitative and quantitative analysis of saikosaponins in three Bupleurum species using laser microdissection and liquid chromatography-quadru‐pole/time of flight-mass spectrometry[J].J Pharm Biomed Anal,2014,97:157-165.
[17]Yi L,Liang ZT,Peng Y,et al.Tissue-specific metabolite profiling of alkaloids in Sinomenii Caulis using laser microdissection and liquid chromatography-quadrupole/time of flight-mass spectrom‐etry[J].J Chromatogr A,2012,1248:93-103.
[18]Wang Q,Liang Z,Peng Y,et al.Whole transverse section and specific-tissue analysis of secondary metabolites in seven different grades of root of Paeonia lactiflora using laser microdissection and liquid chromatography-quadrupole/time of flight-mass spec‐trometry[J].J Pharm Biomed Anal,2015,103:7-16.
[19]Liang Z,Sham T,Yang G,et al.Profiling of secondary metabo‐lites in tissues from Rheum palmatum L.using laser microdissec‐tion and liquid chromatography mass spectrometry[J].Anal Bioanal Chem,2013,405:4199-4212.
[20]Chen YJ,Liang ZT,Zhu Y,et al.Tissue-specific metabolites profiling and quantitative analyses of flavonoids in the rhizome of Belamcanda chinensis by combining laser-microdissection with UHPLC-Q/TOF-MS and UHPLC-QqQ-MS[J].Talanta,2014,130:585-597.
[21]Zhai MZ.Disease Resistance Activity and Structure-Activity Analysis of Plant Secondary Metabolites(植物次生产物的抗病活性及构效分析)[D].Fuzhou:Fujian Agriculture and Forestry University,2003.
[22]Liu DL,Xu SX,Liu FS.Advances in biosynthesis of lignans from Forsythia[J].Chin J Med Chem(中国药物化学杂志),1996,6:68-73.
[23]Kim HJ,Ono E,Morimoto K,et al.Metabolic engineering of lignan biosynthesis in forsythia cell culture[J].Plant Cell Physiol,2009,50:2200-2209.
[24]Boerjan W,Ralph J,Baucher M.Lignin biosynthesis[J].Ann Rev Plant Biol,2003,54:519-546.
[25]Umezawa T.Diversity in lignan biosynthesis[J].Phytochem Rev,2003,2:371-390.
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