丹参和紫丹参脂溶性成分的UPLC及LC-MS/MS分析
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摘要
采用超高效液相色谱及液-质联用技术分析16批丹参和18批紫丹参脂溶性成分指纹图谱并进行比较。液相条件:Thermo Accucore C_(18)色谱柱(2.1 mm×100 mm,2.6μm);流动相为0.026%磷酸水(A)-乙腈(B),梯度洗脱;流速0.4 m L·min~(-1);检测波长270 nm;进样量2μL;柱温25℃。液质条件:Q Exactive Orbitrap LC-MS/MS,流动相为0.1%的甲酸水(A),0.1%甲酸和乙腈(B),梯度洗脱,采用电喷雾离子源,正离子模式下采集数据。丹参脂溶性成分有10个共有峰,16批不同产地丹参与丹参对照图谱的相似度大于0.942、平均相似度为0.973;紫丹参脂溶性成分有12个共有峰,18批紫丹参与紫丹参对照图谱的相似度大于0.937、平均相似度为0.976;丹参对照图谱和紫丹参对照图谱间的相似度为0.900。紫丹参中有3个丹参中没有的脂溶性成分,其中一个结构为α-拉帕醌;丹参中有1个紫丹参中没有的脂溶性成分丹参新酮;2种药材含有丹参酮Ⅱ_A、去甲丹参酮Ⅰ、隐丹参酮、丹参酮Ⅰ、去甲丹参酮在内的8个共同脂溶性成分,其中紫丹参的丹参酮Ⅱ_A(P<0.05)、丹参酮Ⅰ(P<0.05)、去甲丹参酮Ⅰ(P<0.05)、隐丹参酮(P<0.01)、二氢丹参酮Ⅰ(P<0.01)的含量显著低于丹参。丹参、紫丹参中脂溶性成分的种类及含量存在显著差异。
Fingerprints of lipophilic components in the roots of Salvia miltiorrhiza and S.yunnanensis were analyzed by UPLC-DADand UPLC coupled with mass spectroscopy to evaluate the differences and similarities of the lipophilic components in the two kinds of herbs.The UPLC analysis of 18 batches of S.miltiorrhiza and 16 batches of S.yunnanensis was performed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Shimadzu LC-20AD;mobile phase was 0.026%phosphoric acid(A)-acetonitrile(B)with gradient elution;flow rate was 0.4 m L·min~(-1);detection wavelength was set at 270 nm;injection volume was 2μL.The molecular structures of the lipophilic components were analyzed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Thermo U3000 UPLC Q Exactive Orbitrap LC-MS/MS with a mobile phaseconsisting of 0.1%formic acid water(A)and 0.1%formic acidacetonitrile(B).The mass spectrometry was acquired in positive modes using ESI.There are 10 common peaks in the lipophilic components of S.miltiorrhiza.The similarity between the 16 batches of S.miltiorrhiza and their own reference spectra was greater than 0.942,and the average similarity was 0.973.There are 12 common peaks in the lipophilic components of S.yunnanensis.The similarity between the 18 batches of S.yunnanensis and their own reference spectra was greater than 0.937,and the average similarity was 0.976.The similarity between the reference chromatograms of S.miltiorrhiza and S.yunnanensis was only 0.900.There are three lipophilic components in S.yunnanensis,which are not found in S.miltiorrhiza,and one of which isα-lapachone.There is a lipophilic component in S.miltiorrhiza not found in S.yunnanensis,which may be miltirone.The two herbs contain 8 common lipophilic components including dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠ,tanshinoneⅡ_A,nortanshinone in which the content of tanshinoneⅡ_A,dihydrotanshinoneⅠand cryptotanshinone of S.yunnanensisis significantly lower than that of S.miltiorrhiza(P<0.01),and the contents of tanshinoneⅠand nortanshinone are significantly lower than that of S.miltiorrhiza too(P<0.05).There are significant differences in the types and contents of lipophilic components between the roots of S.miltiorrhiza and S.yunnanensis,and the similarity between the fingerprints of interspecies is much lower than that between the same species.Therefore,the roots of S.miltiorrhiza and S.yunnanensis are two kinds of herbs which are quite different in chemical compounds and compositions.
Fingerprints of lipophilic components in the roots of Salvia miltiorrhiza and S.yunnanensis were analyzed by UPLC-DADand UPLC coupled with mass spectroscopy to evaluate the differences and similarities of the lipophilic components in the two kinds of herbs.The UPLC analysis of 18 batches of S.miltiorrhiza and 16 batches of S.yunnanensis was performed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Shimadzu LC-20AD;mobile phase was 0.026%phosphoric acid(A)-acetonitrile(B)with gradient elution;flow rate was 0.4 m L·min~(-1);detection wavelength was set at 270 nm;injection volume was 2μL.The molecular structures of the lipophilic components were analyzed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Thermo U3000 UPLC Q Exactive Orbitrap LC-MS/MS with a mobile phaseconsisting of 0.1%formic acid water(A)and 0.1%formic acidacetonitrile(B).The mass spectrometry was acquired in positive modes using ESI.There are 10 common peaks in the lipophilic components of S.miltiorrhiza.The similarity between the 16 batches of S.miltiorrhiza and their own reference spectra was greater than 0.942,and the average similarity was 0.973.There are 12 common peaks in the lipophilic components of S.yunnanensis.The similarity between the 18 batches of S.yunnanensis and their own reference spectra was greater than 0.937,and the average similarity was 0.976.The similarity between the reference chromatograms of S.miltiorrhiza and S.yunnanensis was only 0.900.There are three lipophilic components in S.yunnanensis,which are not found in S.miltiorrhiza,and one of which isα-lapachone.There is a lipophilic component in S.miltiorrhiza not found in S.yunnanensis,which may be miltirone.The two herbs contain 8 common lipophilic components including dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠ,tanshinoneⅡ_A,nortanshinone in which the content of tanshinoneⅡ_A,dihydrotanshinoneⅠand cryptotanshinone of S.yunnanensisis significantly lower than that of S.miltiorrhiza(P<0.01),and the contents of tanshinoneⅠand nortanshinone are significantly lower than that of S.miltiorrhiza too(P<0.05).There are significant differences in the types and contents of lipophilic components between the roots of S.miltiorrhiza and S.yunnanensis,and the similarity between the fingerprints of interspecies is much lower than that between the same species.Therefore,the roots of S.miltiorrhiza and S.yunnanensis are two kinds of herbs which are quite different in chemical compounds and compositions.
引文
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[29]黄超,陈科力.滇丹参与丹参脂溶性成分指纹图谱的比较研究[J].中国药师,2015,18(8):1256.
[30]韩凤梅,张玲,陈怀侠,等.丹参脂溶性成分的ESI-MS行为及其特征图谱研究[J].中草药,2006,37(1):122.
[31]郭增军,龙丽辉,徐颖,等.UPLC-ESI-MS分析丹参药材、脂溶性及水溶性提取物的色谱、质谱行为及相关性[J].中药材,2010,33(6):925.
[32]Zhou Y,Xu G,Choi F F,et al.Qualitative and quantitative analysis of diterpenoids in Salvia species by liquidchromatography coupled with electrospray ionization quadrupole time-of-flighttandem mass spectrometry[J].J Chromatogr A,2009,1216(24):4847.
[33]倪力军,张立国,基础化学计量学及其应用[M].上海:华东理工大学出版社,2011.
[2]Wu Y B,Ni Z Y,Shi Q W,et al.Constituents from Salvia species and their biological activities[J].Chem Rev,2012,112(11):5967.
[3]Lu L L,Hou S,Zheng T T,et al.Simultaneous determination of five hydrophilic and lipophilic components from roots of Salvia miltiorrhiza by HPLC[J].Chin Herb Med,2015,7(1):75.
[4]刘艾林,李铭源,王一涛,等.丹参药理学活性物质基础研究现[J].中国药学杂志,2007,42(9):641.
[5]何治,潘志红,鲁文红,等.丹参酮ⅡA对血管性痴呆大鼠的神经保护作用机制[J].中国中药杂志,2010,35(14):1883.
[6]曾金,张志荣,缪萍,等.隐丹参酮的药理作用研究进展[J].中成药,2015,37(6):1309.
[7]秦飞,王娴,李晓,等.丹参酮ⅡA对术后肠黏连大鼠模型防治作用研究[J].今日药学,2014,24(5):316.
[8]Qin J H,Shi H B,Xu Y J,et al.TanshinoneⅡAinhibits cervix carcinoma stem cells migration and invasion via inhibiting YAPtranscriptional activity[J].Biomed Pharmacother,2018,105:758.
[9]Lee W Y,Liu K W,Yeung J H.Reactive oxygen speciesmediated kinase activation by dihydrotanshinone in tanshinonesinduced apoptosis in HepG2 cells[J].Cancer Lett,2009,285(1):46.
[10]Liu X X,Meng J.TanshinoneⅡAameliorates lipopolysaccharideinduced inflammatory response in bronchial epithelium cell line BEAS-2B by down-regulating miR-27a[J].Biomed Pharm,2018,104:158.
[11]中国科学院昆明植物研究所.云南植物志.第1卷[M].北京:科学出版社,1977:656.
[12]Lin F W,Amooru G D,Wu T S.Abietane diterpene alkaloids from Salvia yunnanensis[J].J Nat Prod,2006,69(1):93.
[13]Wu C Y,Liao Y,Yang Z G.et al.Cytotoxic diterpenoids from Salvia yunnanensis[J].Phytochemistry,2014,6:171.
[14]Fraga B M,Díaz C E,Guadano A,et al.Diterpenes from Salvia broussonetii transformed roots and their insecticidal activity[J].Food Chem,2005,53(13):5200.
[15]罗庆祎,李建美.滇丹参对冠心病的防治作用[J].临床误诊误治,2007,20(5):23.
[16]张小方,云鹰.丹参注射液对抗Thy-1系膜增生性肾炎模型大鼠TNF-α、IV-C水平的影响[J].中成药,2009,31(10):1512.
[17]史海龙,崔亚亚,李军,等.基于分子对接技术探讨复方丹参方多靶点治疗冠心病的作用机制[J].中国实验方剂学杂志,2016,22(18):176.
[18]Si Y Y,Li N,Tong L,et al.Bioactive minor components of the total salvianolic acids injection prepared from Salvia miltiorrhiza Bge[J].Bioorg Med Chem Lett,2016,26(1):82.
[19]刘春娟,石俊英.复方丹参片药理与临床研究的近年进展[J].食品与药品,2011,13(11):434.
[20]宋敏,杭太俊,张正行,等.丹参脂溶性成分指纹图谱标准的对照品对照法研究[J].药物分析杂志,2007,27(1):8.
[21]王立萍,周凤琴,郭庆梅,等.山东丹参脂溶性组分HPLC指纹图谱及有效成分含量测定[J].中华中医药学刊,2009,27(1):133.
[22]丁晓彦,刘青,李岩,等.丹参脂溶性成分的HPLC指纹图谱及模式识别研究[J].中华中医药杂志,2016,31(6):2254.
[23]金樟照,祝明,张文婷,等.不同产地丹参水溶性成分和脂溶性成分指纹图谱测定及相关性研究[J].中草药,2004,35(10):1174.
[24]Zhang J L,Cui M,He Y,et al.Chemical fingerprint and metabolic fingerprint analysis of Danshen injection by HPLC-UV and HPLC-MS methods[J].J Pharm Biomed Anal,2005,36(5):1029.
[25]Jin X F,Y H,Wei D Z,et al.Chemical fingerprint and quantitative analysis of Salvia plebeia R.Br.by high-performance liquid chromatography[J].J Pharm Biomed Anal,2008,48(1):100.
[26]Liu A H,Lin Y H,Yang M,et al.Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC-MS[J].J Chromatogr B,2007,846(1/2):32.
[27]Ma L J,Zhang X Z,Zhang H P,et al.Development of a fingerprint of Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometricelectrode array system[J].J Chromatogr B,2007,846:139.
[28]张晓灿,罗丹丹,陶爱恩,等.基于指纹图谱和化学计量学的丹参及紫丹参质量评价研究[J].中药材,2017,40(5):1061.
[29]黄超,陈科力.滇丹参与丹参脂溶性成分指纹图谱的比较研究[J].中国药师,2015,18(8):1256.
[30]韩凤梅,张玲,陈怀侠,等.丹参脂溶性成分的ESI-MS行为及其特征图谱研究[J].中草药,2006,37(1):122.
[31]郭增军,龙丽辉,徐颖,等.UPLC-ESI-MS分析丹参药材、脂溶性及水溶性提取物的色谱、质谱行为及相关性[J].中药材,2010,33(6):925.
[32]Zhou Y,Xu G,Choi F F,et al.Qualitative and quantitative analysis of diterpenoids in Salvia species by liquidchromatography coupled with electrospray ionization quadrupole time-of-flighttandem mass spectrometry[J].J Chromatogr A,2009,1216(24):4847.
[33]倪力军,张立国,基础化学计量学及其应用[M].上海:华东理工大学出版社,2011.
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