丹参酮Ⅰ和丹参酮Ⅱ_A在大鼠体内的代谢物研究
|
摘要
该文采用UHPLC-LTQ-Orbitrap高分辨质谱技术分析鉴定丹参酮Ⅰ和丹参酮ⅡA在大鼠体内的代谢产物。灌药后,收集大鼠血浆和尿液。经固相萃取法处理生物样品后,采用Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)色谱柱和0.1%甲酸水溶液(A)-乙腈(B)流动相系统进行梯度洗脱;在正离子模式下对含药血浆、尿液及空白组样品进行分析。最终通过分析精确质量数据、多级质谱信息与文献报道结果比对,共从大鼠生物样品中初步检测鉴定出26个代谢产物。其中7个为丹参酮Ⅰ的代谢产物,主要代谢途径为葡萄糖醛酸化、羟基化、还原反应、去甲基化反应、甲基化、硫酸酯化以及它们的复合反应等;19个为丹参酮ⅡA的代谢产物,其主要代谢途径为羟基化、还原反应、甲基化、硫酸酯化、葡萄糖结合、葡萄糖醛酸化以及它们的复合反应。结果表明,应用UHPLC-LTQ-Orbitrap高分辨液质联用技术可以较为全面地阐明丹参酮Ⅰ和丹参酮ⅡA在大鼠体内的代谢情况,可为进一步的药效学和毒理学再研究以及药物二次研发提供物质基础。
An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap(UHPLCLTQ-Orbitrap) mass spectrometer was established to elucidate the in vivo metabolites of tanshinone I and tanshinone ⅡAin rats.Urine and plasma samples were collected after oral gavage.After processing biological sample by solid phase extraction,Waters ACQUITY HPLC BEH C_(18) column(2.1 mm×100 mm,1.7 μm) was used with 0.1% formic acid(A)-acetonitrile(B) solution as the mobile phase for gradient elution.The plasma,urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode.On the basis of the accurate mass measurements,multiple mass spectra and comparison of data with published literature,a total of 26 metabolites were tentatively identified and characterized in the rat samples.Among them,7 metabolites were derived from tanshinone I through metabolic pathways of glucuronide conjugation,hydroxylation,reduction reaction,demethylation reaction,methylation,sulfate conjugation and their composite reactions.Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation,reduction reaction,methylation,sulfate conjugation,glucuronidation,glucosylation and their complicated reactions.The results showed that the metabolism of tanshinone I and tanshinone ⅡAin rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer,providing material basis for the further research in terms of pharmacodynamics,toxicology,and secondary development of Chinese medicine.
An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap(UHPLCLTQ-Orbitrap) mass spectrometer was established to elucidate the in vivo metabolites of tanshinone I and tanshinone ⅡAin rats.Urine and plasma samples were collected after oral gavage.After processing biological sample by solid phase extraction,Waters ACQUITY HPLC BEH C_(18) column(2.1 mm×100 mm,1.7 μm) was used with 0.1% formic acid(A)-acetonitrile(B) solution as the mobile phase for gradient elution.The plasma,urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode.On the basis of the accurate mass measurements,multiple mass spectra and comparison of data with published literature,a total of 26 metabolites were tentatively identified and characterized in the rat samples.Among them,7 metabolites were derived from tanshinone I through metabolic pathways of glucuronide conjugation,hydroxylation,reduction reaction,demethylation reaction,methylation,sulfate conjugation and their composite reactions.Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation,reduction reaction,methylation,sulfate conjugation,glucuronidation,glucosylation and their complicated reactions.The results showed that the metabolism of tanshinone I and tanshinone ⅡAin rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer,providing material basis for the further research in terms of pharmacodynamics,toxicology,and secondary development of Chinese medicine.
引文
[1]史顺敏,俞年军,于凡,等.丹参种质资源的研究进展[J].现代中医药,2016(6):113.
[2]马丙祥,董宠凯.丹参的药理作用研究新进展[J].中国药房,2014(7):663.
[3]张乙川.对丹参化学成分及临床药理的研究进展[J].中国医药指南,2015(11):214.
[4]张夏楠,关红雨,高伟,等.中药丹参资源开发现代研究进展[J].转化医学研究:电子版,2014,4(4):26.
[5]Jafari E,Andalib S,Abed A,et al.Neuroprotective,antimicrobial,antioxidant,chemotherapeutic,and antidiabetic properties of Salvia Reuterana:a mini review[J].Avicenna J Phytomed,2014,5(1):10.
[6]闫俊,冯娟,杨雪,等.丹参酮ⅡA的药理作用及疾病治疗的最新进展[J].实用药物与临床,2015(8):972.
[7]Mahendran R,Lim H A,Tan J Y,et al.Salvia divinorum:an overview of the usage,misuse,and addiction processes[J].Asia-Pac Psychiat,2015,8(1):23.
[8]Delamare A P L,Moschen-Pistorello I T,Artico L,et al.Antibacterial activity of the essential oils of Salvia officinalis L.and Salvia triloba L.cultivated in South Brazil[J].Food Chem,2007,100(2):603.
[9]Wang C,Ma F J,Tian J,et al.Protective effect of salvianic acid a on acute liver injury induced by carbon tetrachloride in rats[J].Biol Pharm Bull,2007,30(1):44.
[10]Li P,Wang G J,Li J,et al.Identification of tanshinoneⅡAmetabolites in rat liver microsomes by liquid chromatography-tandem mass spectrometry[J].J Chromatogr A,2006,1104(1):366.
[11]Zhang J Y,Wang Z J,Zhang Q,et al.Rapid screening and identification of target constituents using full scan-parent ions listdynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer[J].Talanta,2014,124:111.
[12]Zhang J Y,Wang Z J,Li Y,et al.A strategy for comprehensive identification of sequential constituents using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer,application study on chlorogenic acids in Flos Lonicerae Japonicae[J].Talanta,2016,147:16.
[13]Zhang J Y,Cai W,Zhou Y,et al.Profiling and identification of the metabolites of baicalin and study on their tissue distribution in rats by ultra-high-performance liquid chromatography with linear ion trap-orbitrap mass spectrometer[J].J Chromatogr B,2015,985:91.
[14]Xie T,Liang Y,Hao H P,et al.Rapid identification of ophiopogonins and ophiopogonones in ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry[J].J Chromatogr A,2012,1227:234.
[15]Cai W,Zhang J Y,Dong L Y,et al.Identification of the metabolites of Ixerin Z from Ixeris sonchifolia Hance in rats by HPLC-LTQ-Orbitrap mass spectrometry[J].J Pharmaceut Biomed,2015,107:290.
[16]Shang Z P,Wang F,Dai S Y,et al.Profiling and identification of(-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-orbitrap mass spectrometer[J].Drug Test Anal,2017,doi:10.1002/dta.2155.
[17]刘思燚,张秀平,尚展鹏,等.UHPLC-LTQ-Orbitrap鉴定苦碟子注射液化学成分及其在大鼠血浆中的移行成分[J].中国中药杂志,2016,41(12):2235.
[18]戴海学,李晓蓉,李宇航,等.丹参酮ⅡA和丹参酮Ⅰ的电子轰击与电喷雾电离质谱分析[J].分析试验室,2008,27(5):24.
[19]刘劼,杨黄浩,黎先春,等.高效液相色谱-电喷雾飞行时间质谱分析丹参中的丹参酮类化合物[J].质谱学报,2008,29(5):261.
[20]Wang M,Dai H,Li X,et al.Structural elucidation of metabolites of tanshinone I and its analogue dihydrotanshinone I in rats by HPLC-ESI-MSn[J].J Chromatogr B,2010,878(13/14):915.
[21]Zhao X,Yang D,Xu F,et al.The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLCwith electrospray ionization tandem ion trap and time-of-flight mass spectrometry[J].Biomed Chromatogr,2015,29(2):285.
[22]Wei Y,Li P,Wang C,et al.Metabolism of tanshinoneⅡA,cryptotanshinone and tanshinone I from Radix Salvia miltiorrhiza in zebrafish[J].Molecules,2012,17(7):8617.
[23]Sun J,Yang M,Han J,et al.Profiling the metabolic difference of seven tanshinones using high-performance liquid chromatography/multi-stage mass spectrometry with data-dependent acquisition[J].Rapid Commun Mass Sp,2007,21(14):2211.
[24]Chan W,Cui L,Xu G,et al.Study of the phase I and phaseⅡmetabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry[J].Rapid Commun Mass Sp,2006,20(11):1755.
[2]马丙祥,董宠凯.丹参的药理作用研究新进展[J].中国药房,2014(7):663.
[3]张乙川.对丹参化学成分及临床药理的研究进展[J].中国医药指南,2015(11):214.
[4]张夏楠,关红雨,高伟,等.中药丹参资源开发现代研究进展[J].转化医学研究:电子版,2014,4(4):26.
[5]Jafari E,Andalib S,Abed A,et al.Neuroprotective,antimicrobial,antioxidant,chemotherapeutic,and antidiabetic properties of Salvia Reuterana:a mini review[J].Avicenna J Phytomed,2014,5(1):10.
[6]闫俊,冯娟,杨雪,等.丹参酮ⅡA的药理作用及疾病治疗的最新进展[J].实用药物与临床,2015(8):972.
[7]Mahendran R,Lim H A,Tan J Y,et al.Salvia divinorum:an overview of the usage,misuse,and addiction processes[J].Asia-Pac Psychiat,2015,8(1):23.
[8]Delamare A P L,Moschen-Pistorello I T,Artico L,et al.Antibacterial activity of the essential oils of Salvia officinalis L.and Salvia triloba L.cultivated in South Brazil[J].Food Chem,2007,100(2):603.
[9]Wang C,Ma F J,Tian J,et al.Protective effect of salvianic acid a on acute liver injury induced by carbon tetrachloride in rats[J].Biol Pharm Bull,2007,30(1):44.
[10]Li P,Wang G J,Li J,et al.Identification of tanshinoneⅡAmetabolites in rat liver microsomes by liquid chromatography-tandem mass spectrometry[J].J Chromatogr A,2006,1104(1):366.
[11]Zhang J Y,Wang Z J,Zhang Q,et al.Rapid screening and identification of target constituents using full scan-parent ions listdynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer[J].Talanta,2014,124:111.
[12]Zhang J Y,Wang Z J,Li Y,et al.A strategy for comprehensive identification of sequential constituents using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer,application study on chlorogenic acids in Flos Lonicerae Japonicae[J].Talanta,2016,147:16.
[13]Zhang J Y,Cai W,Zhou Y,et al.Profiling and identification of the metabolites of baicalin and study on their tissue distribution in rats by ultra-high-performance liquid chromatography with linear ion trap-orbitrap mass spectrometer[J].J Chromatogr B,2015,985:91.
[14]Xie T,Liang Y,Hao H P,et al.Rapid identification of ophiopogonins and ophiopogonones in ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry[J].J Chromatogr A,2012,1227:234.
[15]Cai W,Zhang J Y,Dong L Y,et al.Identification of the metabolites of Ixerin Z from Ixeris sonchifolia Hance in rats by HPLC-LTQ-Orbitrap mass spectrometry[J].J Pharmaceut Biomed,2015,107:290.
[16]Shang Z P,Wang F,Dai S Y,et al.Profiling and identification of(-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-orbitrap mass spectrometer[J].Drug Test Anal,2017,doi:10.1002/dta.2155.
[17]刘思燚,张秀平,尚展鹏,等.UHPLC-LTQ-Orbitrap鉴定苦碟子注射液化学成分及其在大鼠血浆中的移行成分[J].中国中药杂志,2016,41(12):2235.
[18]戴海学,李晓蓉,李宇航,等.丹参酮ⅡA和丹参酮Ⅰ的电子轰击与电喷雾电离质谱分析[J].分析试验室,2008,27(5):24.
[19]刘劼,杨黄浩,黎先春,等.高效液相色谱-电喷雾飞行时间质谱分析丹参中的丹参酮类化合物[J].质谱学报,2008,29(5):261.
[20]Wang M,Dai H,Li X,et al.Structural elucidation of metabolites of tanshinone I and its analogue dihydrotanshinone I in rats by HPLC-ESI-MSn[J].J Chromatogr B,2010,878(13/14):915.
[21]Zhao X,Yang D,Xu F,et al.The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLCwith electrospray ionization tandem ion trap and time-of-flight mass spectrometry[J].Biomed Chromatogr,2015,29(2):285.
[22]Wei Y,Li P,Wang C,et al.Metabolism of tanshinoneⅡA,cryptotanshinone and tanshinone I from Radix Salvia miltiorrhiza in zebrafish[J].Molecules,2012,17(7):8617.
[23]Sun J,Yang M,Han J,et al.Profiling the metabolic difference of seven tanshinones using high-performance liquid chromatography/multi-stage mass spectrometry with data-dependent acquisition[J].Rapid Commun Mass Sp,2007,21(14):2211.
[24]Chan W,Cui L,Xu G,et al.Study of the phase I and phaseⅡmetabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry[J].Rapid Commun Mass Sp,2006,20(11):1755.