UHPLC-LTQ-Orbitrap质谱法鉴定染料木素在大鼠体内的代谢产物
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摘要
为深入研究染料木素在大鼠体内的效应物质基础,采用超高效液相色谱-离子阱-静电轨道场质谱(UHPLC-LTQ-Orbitrap MS)技术分析染料木素在大鼠体内可能的代谢产物。大鼠单次灌胃染料木素的CMC-Na混悬液后,收集不同时间的血浆及24 h内的尿液,样品经固相萃取法处理后,采用Waters Acquity UPLC BEH C18色谱柱(1.7μm×2.1 mm×100 mm)分离,0.1%甲酸水溶液(A)-乙腈(B)流动相系统进行梯度洗脱;在负离子扫描模式下对含药血浆、尿液及空白组样品进行质谱分析。通过分析大鼠生物样品的质谱信息,并结合相关文献,共筛选鉴定出31种染料木素代谢产物。结果表明,染料木素在大鼠体内的主要代谢途径为葡萄糖醛酸化、葡萄糖结合、羟基化、还原反应、甲基化、硫酸酯化及其复合反应。本研究初步阐明了染料木素在大鼠体内的代谢情况,推测了体内发挥药效的物质基础,可为进一步药理作用机制研究提供依据。
For further study of bioactive components of Genistein in vivo, a method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was established to comprehensively profile its metabolism in rats. Genistein was suspended in 0.5% carboxymethylcellulose sodium(CMC-Na) solution. Rats in drug group were given a dose of 200 mg/kg body weight orally. 0.5% CMC-Na aqueous solution(2 mL) was administrated to rats in control group. The obtained biological samples were pretreated using solid phase extraction method to exclude protein and solid residue precipitation. Samples were separated on a Waters Acquity BEH C18 column(2.1 mm×100 mm×1.7 μm) with 0.1% formic acid aqueous solution-acetonitrile solution as the mobile phases in gradient elution, and then analyzed by LTQ-Orbitrap mass spectrometry equipped with an ESI ion source in negative mode. According to the obtained accurate mass measurements and mass fragmentation patterns, a total of 31 metabolites(including Genistein) were tentatively or unambiguously identified. The results demonstrated that Genistein underwent multiple in vivo metabolic reactions, including dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. The results not only provided novel and useful data to comprehensively understand the metabolic mechanism and material basis of Genistein for further researches of safety, toxicity and efficacy, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.
For further study of bioactive components of Genistein in vivo, a method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was established to comprehensively profile its metabolism in rats. Genistein was suspended in 0.5% carboxymethylcellulose sodium(CMC-Na) solution. Rats in drug group were given a dose of 200 mg/kg body weight orally. 0.5% CMC-Na aqueous solution(2 mL) was administrated to rats in control group. The obtained biological samples were pretreated using solid phase extraction method to exclude protein and solid residue precipitation. Samples were separated on a Waters Acquity BEH C18 column(2.1 mm×100 mm×1.7 μm) with 0.1% formic acid aqueous solution-acetonitrile solution as the mobile phases in gradient elution, and then analyzed by LTQ-Orbitrap mass spectrometry equipped with an ESI ion source in negative mode. According to the obtained accurate mass measurements and mass fragmentation patterns, a total of 31 metabolites(including Genistein) were tentatively or unambiguously identified. The results demonstrated that Genistein underwent multiple in vivo metabolic reactions, including dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. The results not only provided novel and useful data to comprehensively understand the metabolic mechanism and material basis of Genistein for further researches of safety, toxicity and efficacy, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.
引文
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[6] 杨维霞,梁宗锁,王四旺,张磊,谢艳华,贺中民. 染料木素对Ⅱ型糖尿病大鼠模型血糖和血脂的影响[J]. 西北农林科技大学学报:自然科学版, 2009, 37(9): 1-6. YANG Weixia, LIANG Zongsuo, WANG Siwang, ZHANG Lei, XIE Yanhua, HE Zhongmin. Effect of genistein to FPG and blood lipids on type II diabetes rats[J]. Journal of Northwest A&F University, 2009, 37(9): 1-6 (in Chinese).
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[8] KALUDJEROVIC J, WARD W E. Neonatal exposure to daidzein, genistein, or the combination modulates bone development in female CD-1 mice[J]. Journal of Nutrition, 2009, 139(3): 467-473.
[9] 邱奇琦,胡晓鹃,许春莲,申明月,舒翔,聂少平,谢明勇. 小鼠血浆中染料木素水溶性代谢产物和染料木素脂肪酸酯的分离及测定[J]. 分析科学学报,2012,28(1):155-159. QIU Qiqi, HU Xiaojuan, XU Chunlian, SHEN Mingyue, SHU Xiang, NIE Shaoping, XIE Mingyong. Separation and determination of genistein and its fatty acid ester in mice's plasma[J]. Journal of Analytical Science, 2012, 28(1): 155-159(in Chinese).
[10] 邢萌萌,聂少平,杨希,杜国,谢敏,李文娟,谢明勇. UPLC/Q-TOF 鉴定染料木素在小鼠血浆中的代谢产物[J]. 南昌大学学报:理科版,2014,(1):79-85. XING Mengmeng, NIE Shaoping, YANG Xi, DU Guo, XIE Min, LI Wenjuan, XIE Ming-yong. Determination of genistein metabolites in plasma of mice by UPLC/Q-TOF[J]. Journal of Nanchang University: Natural Science, 2014, (1): 79-85(in Chinese).
[11] ZHANG J Y, CAI W, ZHOU Y, LIU Y, WU X D, LI Y, LU J Q, QIAO Y J. 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]. Journal of Chromatography B, 2015, (985): 91-102.
[12] ZHANG J Y, WANG Z J, ZHANG Q, WANG F, MA Q, LIN Z Z, LU J Q, QIAO Y J . Rapid screening and identification of target constituents using full scan-parent ions list-dynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer[J]. Talanta, 2014, (124): 111-122.
[13] LIANG J, XU F, ZHANG Y Z, HUANG S, ZANG X Y, ZHAO X, ZHANG L, SHANG M Y, YANG D H, WANG X, CAI S Q. The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC-DAD-ESI-IT-TOF-MSn technique: a novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines[J]. Journal of Pharmaceutical and Biomedical Analysis, 2013, 83(5): 108-121.
[14] LIU M Y, ZHAO S H, WANG Z Q, WANG Y F, LIU T, LI S, WANG C C, WANG H T, TU P F. Identification of metabolites of deoxyschizandrin in rats by UPLC-Q-TOF-MS/MS based on multiple mass defect filter data acquisition and multiple data processing techniques[J]. Journal of Chromatography B, 2014, 949-950(4): 115-126.
[15] RUAN Q, PETERMAN S, SZEWC M A, MA L, CUI D, HUMPHREYS W G, ZHU M S. An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection[J]. Journal of Mass Spectrometry, 2010, 43(2): 251-261.
[16] XIE T, LIANG Y, HAO H P, A J Y, XIE L, GONG P, DAI C, LIU L S, KANG A, ZHWNG X, WANG G J. 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]. Journal of Chromatography A, 2012, 1 227(5): 234-244.
[17] SHANG Z P, CAI W, CAO Y F, WANG F, WANG Z B, LU J Q, ZHANG J Y. An integrated strategy for rapid discovery and identification of the sequential piperine metabolites in rats using ultra high-performance liquid chromatography/high resolution mass spectrometery[J]. Journal of Pharmaceutical and Biomedical Analysis, 2017, (146): 387.
[18] LEE D H, MIN J K, SONG E J, JIN K H, JIYUN A, NAM Y D, JANG Y J, HA T Y, CHANG H J. Nutrikinetic study of genistein metabolites in ovariectomized mice[J]. Plos One, 2017, 161(10): 83-93.
[19] TIAN Z M, WAN M X, WANG Z L, WANG B. The preparation of genistein and LC-MS/MS on-line analysis[J]. Drug Development Research, 2010, 61(1): 6-12.
[20] SANG G S, HEE Y, SEUNG H S, SOYUN M, KIM Y A, YU J G, LEE D E, CHUNG M Y, HEO Y S, KWON J Y, YUE S H, KIM K H, CHENG J X, LEE K W, LEE H J. A metabolite of daidzein, 6,7,4′-trihydroxyisoflavone, suppresses adipogenesis in 3T3-L1 preadipocytes via ATP-competitive inhibition of PI3K[J]. Molecular Nutrition and Food Research, 2013, 57(8): 1 446-1 455.
[21] CHEN Y C, SUGIYAMA Y, HIROTA A. Isolation of a new metabolite from biotransformation of daidzein by Aspergillus oryzae[J]. Bioscience Biotechnology and Biochemistry, 2009, 73(8): 1 877-1 879.
[22] QIU F, CHEN X Y, SONG B, ZHONG D F, LIU C X. Influence of dosage forms on pharmacokinetics of daidzein and its main metabolite daidzein-7-O-glucuronide in rats[J]. Acta Pharmacologica Sinica, 2005, 26(9): 1 145-1 152.
[23] ZHAO W J, SHANG Z P, LI Q Q, HUANG M R, HE W B, WANG Z B, ZHANG J Y. Rapid screening and identification of daidzein metabolites in rats based on UHPLC-LTQ-Orbitrap mass spectrometry coupled with data-mining technologies[J]. Molecules, 2018, 23(1): 151.
[24] TOTTA P, ACCONCIA F F, VIRGILI F, CASSIDY A, WEINBERG P D, RIMBACH G, MARINO M. Daidzein-sulfate metabolites affect transcriptional and antiproliferative activities of estrogen receptor-beta in cultured human cancer cells[J]. Journal of Nutrition, 2005, 135(11): 2 687-2 693.
[25] MUTHYALA R S, JU Y H, SHENG S, WILLIAMS L D, DOERGE D R, KATZENELLENBOGEN B S, HELFERICH W G, KATZENELLENBOGEN J A. Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta[J]. Bioorganic and Medicinal Chemistry, 2004, 12(6): 1 559-1 567.
[2] SZKUDELSKA K, NOGOWSKI L, SZKUDELSKI T. Genistein affects lipogenesis and lipolysis in isolated rat adipocytes[J]. Journal of Steroid Biochemistry and Molecular Biology, 2000, 75(4): 265-271.
[3] ZHENG X, LEE S K, CHUN O K. Soy isoflavones and osteoporotic bone loss: a review with an emphasis on modulation of bone remodeling[J]. Journal of Medicinal Food, 2015, 19(1): 1-14.
[4] ZHENG Q L, NIE S P, LI W J, HU X J, XIE M Y. Effect of genistein on proliferation of human cervical cancer cell (HeLa)[J]. Chinese Pharmacological Bulletin, 2013, 29(11): 1 577-1 581.
[5] 李蔚辉. 染料木素对小鼠抗氧化作用的研究[J]. 抗感染药学,2014,11(3):200-202. LI Weihui. Study on antioxidation of genistein in mice[J]. Anti-infection Pharmacy, 2014, 11(3): 200-202(in Chinese).
[6] 杨维霞,梁宗锁,王四旺,张磊,谢艳华,贺中民. 染料木素对Ⅱ型糖尿病大鼠模型血糖和血脂的影响[J]. 西北农林科技大学学报:自然科学版, 2009, 37(9): 1-6. YANG Weixia, LIANG Zongsuo, WANG Siwang, ZHANG Lei, XIE Yanhua, HE Zhongmin. Effect of genistein to FPG and blood lipids on type II diabetes rats[J]. Journal of Northwest A&F University, 2009, 37(9): 1-6 (in Chinese).
[7] PAVESE J M, FARMER R L, BERGAN R C. Inhibition of cancer cell invasion and metastasis by genistein[J]. Cancer Metastasis Rev, 2010, 29(3): 465-482.
[8] KALUDJEROVIC J, WARD W E. Neonatal exposure to daidzein, genistein, or the combination modulates bone development in female CD-1 mice[J]. Journal of Nutrition, 2009, 139(3): 467-473.
[9] 邱奇琦,胡晓鹃,许春莲,申明月,舒翔,聂少平,谢明勇. 小鼠血浆中染料木素水溶性代谢产物和染料木素脂肪酸酯的分离及测定[J]. 分析科学学报,2012,28(1):155-159. QIU Qiqi, HU Xiaojuan, XU Chunlian, SHEN Mingyue, SHU Xiang, NIE Shaoping, XIE Mingyong. Separation and determination of genistein and its fatty acid ester in mice's plasma[J]. Journal of Analytical Science, 2012, 28(1): 155-159(in Chinese).
[10] 邢萌萌,聂少平,杨希,杜国,谢敏,李文娟,谢明勇. UPLC/Q-TOF 鉴定染料木素在小鼠血浆中的代谢产物[J]. 南昌大学学报:理科版,2014,(1):79-85. XING Mengmeng, NIE Shaoping, YANG Xi, DU Guo, XIE Min, LI Wenjuan, XIE Ming-yong. Determination of genistein metabolites in plasma of mice by UPLC/Q-TOF[J]. Journal of Nanchang University: Natural Science, 2014, (1): 79-85(in Chinese).
[11] ZHANG J Y, CAI W, ZHOU Y, LIU Y, WU X D, LI Y, LU J Q, QIAO Y J. 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]. Journal of Chromatography B, 2015, (985): 91-102.
[12] ZHANG J Y, WANG Z J, ZHANG Q, WANG F, MA Q, LIN Z Z, LU J Q, QIAO Y J . Rapid screening and identification of target constituents using full scan-parent ions list-dynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer[J]. Talanta, 2014, (124): 111-122.
[13] LIANG J, XU F, ZHANG Y Z, HUANG S, ZANG X Y, ZHAO X, ZHANG L, SHANG M Y, YANG D H, WANG X, CAI S Q. The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC-DAD-ESI-IT-TOF-MSn technique: a novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines[J]. Journal of Pharmaceutical and Biomedical Analysis, 2013, 83(5): 108-121.
[14] LIU M Y, ZHAO S H, WANG Z Q, WANG Y F, LIU T, LI S, WANG C C, WANG H T, TU P F. Identification of metabolites of deoxyschizandrin in rats by UPLC-Q-TOF-MS/MS based on multiple mass defect filter data acquisition and multiple data processing techniques[J]. Journal of Chromatography B, 2014, 949-950(4): 115-126.
[15] RUAN Q, PETERMAN S, SZEWC M A, MA L, CUI D, HUMPHREYS W G, ZHU M S. An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection[J]. Journal of Mass Spectrometry, 2010, 43(2): 251-261.
[16] XIE T, LIANG Y, HAO H P, A J Y, XIE L, GONG P, DAI C, LIU L S, KANG A, ZHWNG X, WANG G J. 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]. Journal of Chromatography A, 2012, 1 227(5): 234-244.
[17] SHANG Z P, CAI W, CAO Y F, WANG F, WANG Z B, LU J Q, ZHANG J Y. An integrated strategy for rapid discovery and identification of the sequential piperine metabolites in rats using ultra high-performance liquid chromatography/high resolution mass spectrometery[J]. Journal of Pharmaceutical and Biomedical Analysis, 2017, (146): 387.
[18] LEE D H, MIN J K, SONG E J, JIN K H, JIYUN A, NAM Y D, JANG Y J, HA T Y, CHANG H J. Nutrikinetic study of genistein metabolites in ovariectomized mice[J]. Plos One, 2017, 161(10): 83-93.
[19] TIAN Z M, WAN M X, WANG Z L, WANG B. The preparation of genistein and LC-MS/MS on-line analysis[J]. Drug Development Research, 2010, 61(1): 6-12.
[20] SANG G S, HEE Y, SEUNG H S, SOYUN M, KIM Y A, YU J G, LEE D E, CHUNG M Y, HEO Y S, KWON J Y, YUE S H, KIM K H, CHENG J X, LEE K W, LEE H J. A metabolite of daidzein, 6,7,4′-trihydroxyisoflavone, suppresses adipogenesis in 3T3-L1 preadipocytes via ATP-competitive inhibition of PI3K[J]. Molecular Nutrition and Food Research, 2013, 57(8): 1 446-1 455.
[21] CHEN Y C, SUGIYAMA Y, HIROTA A. Isolation of a new metabolite from biotransformation of daidzein by Aspergillus oryzae[J]. Bioscience Biotechnology and Biochemistry, 2009, 73(8): 1 877-1 879.
[22] QIU F, CHEN X Y, SONG B, ZHONG D F, LIU C X. Influence of dosage forms on pharmacokinetics of daidzein and its main metabolite daidzein-7-O-glucuronide in rats[J]. Acta Pharmacologica Sinica, 2005, 26(9): 1 145-1 152.
[23] ZHAO W J, SHANG Z P, LI Q Q, HUANG M R, HE W B, WANG Z B, ZHANG J Y. Rapid screening and identification of daidzein metabolites in rats based on UHPLC-LTQ-Orbitrap mass spectrometry coupled with data-mining technologies[J]. Molecules, 2018, 23(1): 151.
[24] TOTTA P, ACCONCIA F F, VIRGILI F, CASSIDY A, WEINBERG P D, RIMBACH G, MARINO M. Daidzein-sulfate metabolites affect transcriptional and antiproliferative activities of estrogen receptor-beta in cultured human cancer cells[J]. Journal of Nutrition, 2005, 135(11): 2 687-2 693.
[25] MUTHYALA R S, JU Y H, SHENG S, WILLIAMS L D, DOERGE D R, KATZENELLENBOGEN B S, HELFERICH W G, KATZENELLENBOGEN J A. Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta[J]. Bioorganic and Medicinal Chemistry, 2004, 12(6): 1 559-1 567.
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