建立UPLC-MS联合HPLC方法探究外源物质对三七叶皂苷含量和生长的影响
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摘要
【目的】为更加精确探究外源物质赤霉素(GA3)、6-苄基腺嘌呤(6-BA)、脱落酸(ABA)、茉莉酸甲酯(MeJA)、水杨酸(SA)、萘乙酸(NAA)、乙烯(ETH)、褪黑素(MT)、亚精胺(Spd)、一氧化氮供体硝普钠(SNP)和过氧化氢(H2O2)对三七[Panax notoginseng (Burk.) F.H.Chen]叶皂苷含量的影响。【方法】开发超高效液相色谱—四极杆串联飞行时间质谱(UPLC-MS)结合高效液相色谱(HPLC)的新方法,测定三七叶中7种皂苷单体含量和生长指标。【结果】(1)建立的HPLC法可在52 min内准确测出三七叶中人参皂苷Rb1、Rc、Rb2和Rb3、三七皂苷Fa、Fe及七叶胆苷XVII等7种皂苷单体含量;(2) MeJA显著提高三七叶人参皂苷Rb1、Rc、Rb2、Rb3、三七皂苷Fa、Fe及七叶胆苷XVII的含量和总含量,且对三七生长无显著影响;而NAA、6-BA、H2O2、SNP、Spd和ABA显著提高三七皂苷Fe含量,ABA显著提高七叶胆苷XVII含量,但总含量均显著减少,且对三七生长有显著影响。【结论】建立了三七叶皂苷单体快速简便、稳定可靠的新检测方法,基于该方法明确了外源物质MeJA能大幅诱导三七叶皂苷单体的积累。
[Purpose]We aimed to explore the effect of exogenous substances, including gibberellin(GA3), 6-benzyladenine(6-BA), abscisic acid(ABA), methyl jasmonate(MeJA), salicylic acid(SA), naphthylacetate(NAA), ethylene(ETH), melatonin(MT), spermidine(Spd), sodium nitroprusside(SNP) and hydrogen peroxide(H2 O2) on saponin contents of Panax notoginseng leaves.[Method]We developed a new method of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-MS) together with high performance liquid chromatography(HPLC), and determined 7 saponin monomer contents and growth indexes.[Results](1) The established HPLC method could accurately measure 7 saponin monomer contents, including ginsenoside Rb1, Rc, Rb2, Rb3, notoginsenoside Fa, Fe and gypenoside XVII in P. notoginsen leaves within 52 minutes within a single run;(2) MeJA treatment significantly increased the contents of 7 monomer saponins, including ginsenoside Rb1, Rc, Rb2, Rb3, notoginsenoside Fa, Fe and gypenoside XVII in leaves, and had no effect on P. notoginseng growth. Other exogenous substances, including NAA, 6-BA, H2 O2, SNP and Spd significantly increased the content of notoginsenoside Fa, while ABA significantly improved the content of gypenoside XVII, but all of them significantly decreased the total saponin contents, and had effect on P. notoginseng growth.[ Conclusion] A rapid and simple, stable and reliable detection method for saponin monomer of P. notoginseng leaves was established. Based on this method, it is clear that exogenous substance MeJA could significantly induce saponin monomer contents of P. notoginseng leaves.
[Purpose]We aimed to explore the effect of exogenous substances, including gibberellin(GA3), 6-benzyladenine(6-BA), abscisic acid(ABA), methyl jasmonate(MeJA), salicylic acid(SA), naphthylacetate(NAA), ethylene(ETH), melatonin(MT), spermidine(Spd), sodium nitroprusside(SNP) and hydrogen peroxide(H2 O2) on saponin contents of Panax notoginseng leaves.[Method]We developed a new method of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-MS) together with high performance liquid chromatography(HPLC), and determined 7 saponin monomer contents and growth indexes.[Results](1) The established HPLC method could accurately measure 7 saponin monomer contents, including ginsenoside Rb1, Rc, Rb2, Rb3, notoginsenoside Fa, Fe and gypenoside XVII in P. notoginsen leaves within 52 minutes within a single run;(2) MeJA treatment significantly increased the contents of 7 monomer saponins, including ginsenoside Rb1, Rc, Rb2, Rb3, notoginsenoside Fa, Fe and gypenoside XVII in leaves, and had no effect on P. notoginseng growth. Other exogenous substances, including NAA, 6-BA, H2 O2, SNP and Spd significantly increased the content of notoginsenoside Fa, while ABA significantly improved the content of gypenoside XVII, but all of them significantly decreased the total saponin contents, and had effect on P. notoginseng growth.[ Conclusion] A rapid and simple, stable and reliable detection method for saponin monomer of P. notoginseng leaves was established. Based on this method, it is clear that exogenous substance MeJA could significantly induce saponin monomer contents of P. notoginseng leaves.
引文
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[9]魏莉,杜奕,周浩.不同产地和生长年限三七花蕾中总皂苷及皂苷单体的含量测定[J].上海中医药杂志,2008,42(4):76.DOI:10.3969/j.issn.1007-1334.2008.04.031.
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[11]朱洁,杨蓉,张洪彬.HPLC-ELSD法测定三七叶中人参皂苷Rb3、Rc、Rb1的含量[J].中草药,2004,35(12):1366.DOI:10.3321/j.issn:0253-2670.2004.12.019.
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[13]郝岗平,杜希华,史仁玖.NO对银杏悬浮细胞生长及黄酮类物质合成的影响[J].西北植物学报,2007,27(2):272.DOI:10.3321/j.issn:1000-4025.2007.02.011.
[14]徐仰仓,徐金,李飞飞,等.H2O2对药用植物活性成分含量的影响[J].四川中医,2011(9):43.
[15]田娇,刘园,房敏峰.外源茉莉酸类激素对药用植物次生代谢的影响研究[J].天然产物研究与开发,2015,27(1):185.DOI:10.16333/j.1001-6880.2015.01.037.
[16]赵寿经,侯艳,贾冬梅,等.西洋参发根的诱导及不同外源物质对发根生长和皂苷含量的影响[J].天然产物研究与开发,2010,22(1):98.DOI:10.3969/j.issn.1001-6880.2010.01.024.
[17]徐立新.植物激素和诱导子对人参发根皂苷含量的影响研究[D].长春:吉林大学,2007.
[18]PAEK K Y,MURTHY H N,HAHN E J,et al.Large scale culture of ginseng adventitious roots for production of ginsenosides[M]//ZHONG J J,BAI F,ZHANGW.Biotechnology in China I.Advances in biochemical engineering/biotechnology,Vol.113.Berlin,Heidelberg:Springer,2009.
[19]MURTHY H N,GEORGIEV M I,KIM Y S,et al.Ginsenosides:prospective for sustainable biotechnological production[J].Applied Microbiology&Biotechnology,2014,98(14):6243.DOI:10.1007/s00253-014-5801-9.
[20]朱宏涛,李江,李元,等.茉莉酸甲酯对三七组培苗中总皂苷含量的影响[J].西部林业科学,2014(2):72.DOI:10.3969/j.issn.1672-8246.2014.02.012.
[21]滕红梅,谢旭蓉,王玲丽,等.外源物质对远志根中皂苷含量的影响[J].南方农业学报,2014,45(12):2143.DOI:10.3969/j:issn.2095-1191.2014.12.2143.
[22]崔秀明,王朝梁.三七生长及干物质积累动态的研究[J].中药材,1991(9):9.DOI:10.3321/j.issn:1001-4454.1991.09.017.
[23]国家药典委员会.中华人民共和国药典:2010年版:一部[M].北京:中国医药科技出版社,2010.
[24]LIANG Z T,CHEN Y J,XU L,et al.Localization of ginsenosides in the rhizome and root of Panax ginseng,by laser microdissection and liquid chromatographyquadrupole/time of flight-mass spectrometry[J].Journal of Pharmaceutical&Biomedical Analysis,2015,105:121.DOI:10.1016/j.jpba.2014.12.005.
[25]CHEN Y J,XU L,ZHAO Y C,et al.Tissue-specific metabolite profiling and quantitative analysis of ginsenosides in Panax quinquefolium using laser microdissection and liquid chromatography-quadrupole/time offlight-mass spectrometry[J].Chemistry Central Journal,2015,9(1):66.DOI:10.1186/s13065-015-0141-0.
[26]匡双便,张广辉,陈中坚,等.不同光照条件下三七幼苗形态及生长指标的变化[J].植物资源与环境学报,2014(2):54.DOI:10.3969/j.issn.1674-7895.2014.02.08.
[27]宾金华,潘瑞炽.茉莉酸甲酯的生理生化及在植物抗病中的作用[J].植物学报,1995,12(4):17.
[28]LIU H C,WU W,HOU K,et al.Deep sequencing reveals transcriptome reprogramming of Polygonum multiflorumb thunb.roots to the elicitation with methyl jasmonate[J].Molecular Genetics&Genomics,2016,291(1):337.DOI:10.1007/s00438-015-1112-9.
[29]CHEN R B,LIU J H,YING X,et al.Deep sequencing reveals the effect of MeJA on scutellarin biosynthesis in Erigeron breviscapus[J].PLoS One,2015,10(12):e0143881.DOI:10.1371/journal.pone.0143881.
[30]HU F X,ZHONG J J.Jasmonic acid mediates gene transcription of ginsenoside biosynthesis in cell cultures of Panax notoginseng treated with chemically synthesized2-hydroxyethyl jasmonate[J].Process Biochemistry,2008,43(1):113.DOI:10.1016/j.procbio.2007.10.010.
[31]HAN J Y,IN J G,KWON Y S,et al.Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J].Phytochemistry,2010,71(1):36.DOI:10.1016/j.phytochem.2009.09.031.
[32]潘瑞炽.茉莉酸甲酯对水分胁迫下花生幼苗SOD活性和膜脂过氧化作用的影响[J].花生科技,1996(4):221.
[33]桂连友,刘树生,陈宗懋.外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理[J].昆虫学报,2004,47(4):507.DOI:10.3321/j.issn:0454-6296.2004.04.016.
[2]侯茂奇.三七、人参和西洋参的化学成分比较研究[D].上海:上海中医药大学,2010.
[3]王超群,贾秀虹,王璇,等.三七中11种皂苷类活性成分外标法和一测多评法含量测定及其与药材质量的关联性研究[C]//2014年中国药学大会暨第十四届中国药师周论文集.中国药学会,2014.
[4]李海舟,张颖君,杨崇仁.三七叶化学成分的进一步研究[J].天然产物研究与开发,2006,18(4):552.DOI:10.3969/j.issn.1001-6880.2006.04.005.
[5]韩凌.三七叶皂苷活性成分的化学研究[D].沈阳:辽宁中医药大学,2007.
[6]KIM Y S,YEUNG E C,HAHN E J,et al.Combined effects of phytohormone,indole-3-butyric acid,and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A.Meyer[J].Biotechnology Letters,2007,29(11):1789.DOI:10.1007/s10529-007-9442-2.
[7]黄凤,向飞军,伍杰雄.三七叶皂苷成分及其单体提取分离研究进展[J].中药材,2009,32(6):1002.DOI:10.3321/j.issn:1001-4454.2009.06.057.
[8]刘英,崔秀明,杨野,等.不同产地三七茎叶的质量评价[J].中国医药工业杂志,2015,46(7):701.DOI:10.16522/j.cnki.cjph.2015.07.009.
[9]魏莉,杜奕,周浩.不同产地和生长年限三七花蕾中总皂苷及皂苷单体的含量测定[J].上海中医药杂志,2008,42(4):76.DOI:10.3969/j.issn.1007-1334.2008.04.031.
[10]王隶书,程东岩,程东红.HPLC法测定不同产地的三七叶中人参皂苷Rb3的含量[J].中国药师,2003,6(5):285.DOI:10.3969/j.issn.1008-049X.2003.05.013.
[11]朱洁,杨蓉,张洪彬.HPLC-ELSD法测定三七叶中人参皂苷Rb3、Rc、Rb1的含量[J].中草药,2004,35(12):1366.DOI:10.3321/j.issn:0253-2670.2004.12.019.
[12]刘佳,曹瑞霞,吴能表.外源物质对半夏愈伤氮代谢关键酶及有效成分积累的影响[J].西南大学学报(自然科学版),2017,39(5):15.DOI:10.13718/j.cnki.xdzk.2017.05.003.
[13]郝岗平,杜希华,史仁玖.NO对银杏悬浮细胞生长及黄酮类物质合成的影响[J].西北植物学报,2007,27(2):272.DOI:10.3321/j.issn:1000-4025.2007.02.011.
[14]徐仰仓,徐金,李飞飞,等.H2O2对药用植物活性成分含量的影响[J].四川中医,2011(9):43.
[15]田娇,刘园,房敏峰.外源茉莉酸类激素对药用植物次生代谢的影响研究[J].天然产物研究与开发,2015,27(1):185.DOI:10.16333/j.1001-6880.2015.01.037.
[16]赵寿经,侯艳,贾冬梅,等.西洋参发根的诱导及不同外源物质对发根生长和皂苷含量的影响[J].天然产物研究与开发,2010,22(1):98.DOI:10.3969/j.issn.1001-6880.2010.01.024.
[17]徐立新.植物激素和诱导子对人参发根皂苷含量的影响研究[D].长春:吉林大学,2007.
[18]PAEK K Y,MURTHY H N,HAHN E J,et al.Large scale culture of ginseng adventitious roots for production of ginsenosides[M]//ZHONG J J,BAI F,ZHANGW.Biotechnology in China I.Advances in biochemical engineering/biotechnology,Vol.113.Berlin,Heidelberg:Springer,2009.
[19]MURTHY H N,GEORGIEV M I,KIM Y S,et al.Ginsenosides:prospective for sustainable biotechnological production[J].Applied Microbiology&Biotechnology,2014,98(14):6243.DOI:10.1007/s00253-014-5801-9.
[20]朱宏涛,李江,李元,等.茉莉酸甲酯对三七组培苗中总皂苷含量的影响[J].西部林业科学,2014(2):72.DOI:10.3969/j.issn.1672-8246.2014.02.012.
[21]滕红梅,谢旭蓉,王玲丽,等.外源物质对远志根中皂苷含量的影响[J].南方农业学报,2014,45(12):2143.DOI:10.3969/j:issn.2095-1191.2014.12.2143.
[22]崔秀明,王朝梁.三七生长及干物质积累动态的研究[J].中药材,1991(9):9.DOI:10.3321/j.issn:1001-4454.1991.09.017.
[23]国家药典委员会.中华人民共和国药典:2010年版:一部[M].北京:中国医药科技出版社,2010.
[24]LIANG Z T,CHEN Y J,XU L,et al.Localization of ginsenosides in the rhizome and root of Panax ginseng,by laser microdissection and liquid chromatographyquadrupole/time of flight-mass spectrometry[J].Journal of Pharmaceutical&Biomedical Analysis,2015,105:121.DOI:10.1016/j.jpba.2014.12.005.
[25]CHEN Y J,XU L,ZHAO Y C,et al.Tissue-specific metabolite profiling and quantitative analysis of ginsenosides in Panax quinquefolium using laser microdissection and liquid chromatography-quadrupole/time offlight-mass spectrometry[J].Chemistry Central Journal,2015,9(1):66.DOI:10.1186/s13065-015-0141-0.
[26]匡双便,张广辉,陈中坚,等.不同光照条件下三七幼苗形态及生长指标的变化[J].植物资源与环境学报,2014(2):54.DOI:10.3969/j.issn.1674-7895.2014.02.08.
[27]宾金华,潘瑞炽.茉莉酸甲酯的生理生化及在植物抗病中的作用[J].植物学报,1995,12(4):17.
[28]LIU H C,WU W,HOU K,et al.Deep sequencing reveals transcriptome reprogramming of Polygonum multiflorumb thunb.roots to the elicitation with methyl jasmonate[J].Molecular Genetics&Genomics,2016,291(1):337.DOI:10.1007/s00438-015-1112-9.
[29]CHEN R B,LIU J H,YING X,et al.Deep sequencing reveals the effect of MeJA on scutellarin biosynthesis in Erigeron breviscapus[J].PLoS One,2015,10(12):e0143881.DOI:10.1371/journal.pone.0143881.
[30]HU F X,ZHONG J J.Jasmonic acid mediates gene transcription of ginsenoside biosynthesis in cell cultures of Panax notoginseng treated with chemically synthesized2-hydroxyethyl jasmonate[J].Process Biochemistry,2008,43(1):113.DOI:10.1016/j.procbio.2007.10.010.
[31]HAN J Y,IN J G,KWON Y S,et al.Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J].Phytochemistry,2010,71(1):36.DOI:10.1016/j.phytochem.2009.09.031.
[32]潘瑞炽.茉莉酸甲酯对水分胁迫下花生幼苗SOD活性和膜脂过氧化作用的影响[J].花生科技,1996(4):221.
[33]桂连友,刘树生,陈宗懋.外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理[J].昆虫学报,2004,47(4):507.DOI:10.3321/j.issn:0454-6296.2004.04.016.