基于UFLC-Triple-TOF-MS/MS技术的白虎加桂枝汤化学物质基础
|
摘要
目的:采用超快速高效液相色谱串联四级飞行时间质谱(UFLC-Triple-TOF-MS/MS)对白虎加桂枝汤的化学成分进行研究。方法:采用Phenomenex C_(18)色谱柱(3. 0 mm×100 mm,2. 6μm),以0. 1%甲酸乙腈溶液和0. 1%甲酸水溶液为流动相,梯度洗脱,柱温40℃,流速0. 3 m L·min~(-1),进样量5μL;采用高分辨三重四级杆飞行时间质谱,分别在正、负离子模式下进行检测;借助Peakview软件中Formula Finder功能计算化合物的元素组成,调用软件中IDA explore功能,获得该质量数的二级碎片离子,结合文献数据和对照品数据比对进行化合物结果鉴定。结果:通过精确相对分子质量的质谱裂解规律,定性白虎加桂枝汤的35个化合物,包括甾体皂苷类16个、三萜皂苷类6个、黄酮类7个、有机酸类4个和其他类2个,其中10个成分(知母皂苷AⅢ,知母皂苷Ⅰ,知母皂苷BⅢ,知母皂苷BⅡ,新芒果苷,芒果苷,芹糖甘草苷,甘草苷,甘草酸和肉桂酸)通过与对照品比对确认,并对化合物的药材来源进行归属。结论:UFLC-Triple-TOF-MS/MS可以简便、快速地对白虎加桂枝汤中化学成分进行定性。相关研究为该方的药效物质基础研究、质量控制以及作用原理阐释提供了参考。
Objective: To systematically identify the chemical constituents in extract of Baihu Jia Guizhi Tang( BHGZT) by ultra-fast liquid chromatography/triple time-of-flight tandem mass spectrometry( UFLC-TripleTOF-MS/MS). Method: Phenomenex C_(18)( 3. 0 mm × 100 mm,2. 6 μm) was eluted with acetonitrile( containing 0. 1% formic acid) and 0. 1% formic acid solution as the mobile phase in a gradient mode. The column temperature was set at 40 ℃,the flow rate was 0. 3 m L·min~(-1),and the injection volume was 5 μL. Highresolution triple quadrupole flight mass spectrometry was adopted to test in both positive and negative modes.Formula finder function in peakview software was used to calculate the element composition of the compounds,and IDA explore function in the software was invoked to obtain secondary fragmentation ions of this mass number. And the compounds were identified based on literature data and the reference substances. Result: On the basis of the mass fragmentation regularity of accurate molecular weight,35 compounds were identified in BHGZT,including 16 steroid saponins,6 triterpenoid saponins,7 flavonoids,and 4 organic acids and 2 others. Among them,10 components( timosaponin A Ⅲ, timosaponin I, timosaponin B Ⅲ, timosaponin B Ⅱ, new mangiferin,mangiferin,liquiritin apioside,liquiritin,glycyrrhizic acid and cinnamic acid) were confirmed by comparing with reference substances,and the sources of the compound were identified. Conclusion: UFLC-Triple-TOF-MS/MS can be used to characterize the chemical composition of BHGZT simply and quickly. Relevant researches provide references for the research of pharmacodynamic material basis, the quality control and the explanation of mechanism of action.
Objective: To systematically identify the chemical constituents in extract of Baihu Jia Guizhi Tang( BHGZT) by ultra-fast liquid chromatography/triple time-of-flight tandem mass spectrometry( UFLC-TripleTOF-MS/MS). Method: Phenomenex C_(18)( 3. 0 mm × 100 mm,2. 6 μm) was eluted with acetonitrile( containing 0. 1% formic acid) and 0. 1% formic acid solution as the mobile phase in a gradient mode. The column temperature was set at 40 ℃,the flow rate was 0. 3 m L·min~(-1),and the injection volume was 5 μL. Highresolution triple quadrupole flight mass spectrometry was adopted to test in both positive and negative modes.Formula finder function in peakview software was used to calculate the element composition of the compounds,and IDA explore function in the software was invoked to obtain secondary fragmentation ions of this mass number. And the compounds were identified based on literature data and the reference substances. Result: On the basis of the mass fragmentation regularity of accurate molecular weight,35 compounds were identified in BHGZT,including 16 steroid saponins,6 triterpenoid saponins,7 flavonoids,and 4 organic acids and 2 others. Among them,10 components( timosaponin A Ⅲ, timosaponin I, timosaponin B Ⅲ, timosaponin B Ⅱ, new mangiferin,mangiferin,liquiritin apioside,liquiritin,glycyrrhizic acid and cinnamic acid) were confirmed by comparing with reference substances,and the sources of the compound were identified. Conclusion: UFLC-Triple-TOF-MS/MS can be used to characterize the chemical composition of BHGZT simply and quickly. Relevant researches provide references for the research of pharmacodynamic material basis, the quality control and the explanation of mechanism of action.
引文
[1]马学玉.加味白虎加桂枝汤治疗类风湿关节炎湿热痹阻证临床研究[J].中医学报,2016,31(10):1573-1577.
[2]傅平,胡涛,杨明杰,等.白虎加桂枝汤配方颗粒炼膏外敷治疗手足痛风性关节炎30例[J].中医外治杂志,2016,25(1):11-12.
[3]胡爽杨.《金匮要略》白虎加桂枝汤治疗小儿咳嗽的临床应用心得[J].环球中医药,2013,6(S1):58.
[4]徐世军,李磊,张文生,等.基于方证相关理论的治“痹”经方药效学比较研究[J].中国中药杂志,2009,34(23):3131-3134.
[5]徐世军,代渊,李磊,等.基于“方证相关”理论的治“痹”经方调控T细胞亚群比较研究[J].中国中药杂志,2010,35(15):2030-2032.
[6]徐世军,李磊,张文生,等.基于“方证相关”理论的治“痹”经方调控TLR/TRAF信号通路的比较研究[J].中国中药杂志,2010,35(8):1025-1029.
[7]李玮婕,卢建秋,毛霞,等.基于靶标网络分析的治痹寒热经方抗类风湿关节炎作用机制对比研究[J].药学学报,2018,53(9):1387-1397.
[8]彭平,王梦婕,李晓盈,等.高效液相色谱法同时测定白虎加桂枝汤中5个有效指标性成分的含量[J].世界科学技术—中医药现代化,2017,19(8):1391-1395.
[9]李春来,李伟东,吴育,等. HPLC同时测定白虎汤中4种有效成分的含量[J].中国实验方剂学杂志,2012,18(12):78-80.
[10] ZHONG W F,TONG W S,ZHOU S S,et al. Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector[J]. J Food Drug Anal,2017,25(4):946-959.
[11] SHAN L,WU Y,YUAN L,et al. Rapid screening of chemical constituents in Rhizoma Anemarrhenaeby UPLC-Q-TOF/MS combined with data postprocessing techniques[J]. Evid Based Compl Alt,2017,2017:4032820.
[12] PENG Y,ZHAO L,LIN D,et al. Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by highperformance liquid chromatography quadrupole time-offlight mass spectrometry[J]. Biomed Chromatogra,2016,30(4):508-519.
[13]王秋萍,张承,李俐.两地桂枝挥发油化学成分分析[J].安徽农业科学,2015,43(11):113-115.
[14]赵艳敏,刘素香,张晨曦,等.基于HPLC-Q-TOF-MS技术的甘草化学成分分析[J].中草药,2016,47(12):2061-2068.
[15]马海娟,高简,张亚丽,等.基于HPLC-MSn的甘草成分快速鉴定及质谱裂解途径研究[J].中华中医药杂志,2018,33(3):1120-1123.
[16] WEI F,JIANG X,GAO H Y,et al. Liquiritin induces apoptosis and autophagy in cisplatin(DDP)-resistant gastric cancer cellsin vitroand xenograft nude micein vivo[J]. Int J Oncol,2017,51(5):1383-1394.
[17]陈丽敏,林辉,徐大量,等.桂枝甘草汤挥发性成分的GC-MS测定研究[J].中药新药与临床药理,2010,21(4):418-421.
[18] QIAO X,LI R,SONG W,et al. A targeted strategy to analyze untargeted mass spectral data:rapid chemical profiling of Scutellaria baicalensis using ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry and key ion filtering[J]. J Chromatogra A,2016,1441:83-95.
[19]许文,吴献,黄鸣清,等.栝楼桂枝汤的血清药物化学研究[J].中草药,2017,48(10):2033-2043.
[20]高厚明,胡兆流,郑平,等. HPLC-MS测定小儿安神补脑颗粒中8个成分的含量[J].中国实验方剂学杂志,2018,24(19):73-78.
[21]梁可,崔思娇,张琦,等. UPLC同时测定桂枝中5种活性成分的含量[J].中国中药杂志,2011,36(23):3298-3301.
[22]杨松,贾英,毕开顺. RP-HPLC法测定桂枝中4种活性成分的含量[J].药物分析杂志,2004,24(2):143-146.
[23] LIU X W,ZHANG F,GAO S H,et al. Metabolite profiling of Zi-Shen pill in rat biological specimens by UPLC-Q-TOF/MS[J]. Chin J Nat Med,2015,13(2):145-160.
[24]张宇伟,赵云芳,商婷婷,等.中药知母化学成分研究[J].亚太传统医药,2017,13(11):16-18.
[25] JI X,FENG Y F,RUI W. Rapid analysis of xanthones and steroidal saponins in extract of Rhizoma Anemarrhenae by ultra-performance liquid chromatography/Quadrupole-time-of-flightmass spectrometry[C]∥International Conference on Biomedical Engineering and Biotechnology. IEEE Computer Society,2012:477-485.
[26] ZHAO Y,KANG L,YU H,et al. Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry[J]. J Chin Pharm Sci,2013(3):226-233.
[27]邱昆成,孙振刚,何倩梅,等. LC-MS-MS测定配伍黄柏对知母中4种有效成分含量的影响[J].中国实验方剂学杂志,2016,22(14):84-88.
[28]季德,季德,苏晓楠,黄紫炎,等. HPLC-MS法测定知母盐炙前后8种成分量变化[J].中草药,2017,48(9):1784-1790.
[2]傅平,胡涛,杨明杰,等.白虎加桂枝汤配方颗粒炼膏外敷治疗手足痛风性关节炎30例[J].中医外治杂志,2016,25(1):11-12.
[3]胡爽杨.《金匮要略》白虎加桂枝汤治疗小儿咳嗽的临床应用心得[J].环球中医药,2013,6(S1):58.
[4]徐世军,李磊,张文生,等.基于方证相关理论的治“痹”经方药效学比较研究[J].中国中药杂志,2009,34(23):3131-3134.
[5]徐世军,代渊,李磊,等.基于“方证相关”理论的治“痹”经方调控T细胞亚群比较研究[J].中国中药杂志,2010,35(15):2030-2032.
[6]徐世军,李磊,张文生,等.基于“方证相关”理论的治“痹”经方调控TLR/TRAF信号通路的比较研究[J].中国中药杂志,2010,35(8):1025-1029.
[7]李玮婕,卢建秋,毛霞,等.基于靶标网络分析的治痹寒热经方抗类风湿关节炎作用机制对比研究[J].药学学报,2018,53(9):1387-1397.
[8]彭平,王梦婕,李晓盈,等.高效液相色谱法同时测定白虎加桂枝汤中5个有效指标性成分的含量[J].世界科学技术—中医药现代化,2017,19(8):1391-1395.
[9]李春来,李伟东,吴育,等. HPLC同时测定白虎汤中4种有效成分的含量[J].中国实验方剂学杂志,2012,18(12):78-80.
[10] ZHONG W F,TONG W S,ZHOU S S,et al. Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector[J]. J Food Drug Anal,2017,25(4):946-959.
[11] SHAN L,WU Y,YUAN L,et al. Rapid screening of chemical constituents in Rhizoma Anemarrhenaeby UPLC-Q-TOF/MS combined with data postprocessing techniques[J]. Evid Based Compl Alt,2017,2017:4032820.
[12] PENG Y,ZHAO L,LIN D,et al. Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by highperformance liquid chromatography quadrupole time-offlight mass spectrometry[J]. Biomed Chromatogra,2016,30(4):508-519.
[13]王秋萍,张承,李俐.两地桂枝挥发油化学成分分析[J].安徽农业科学,2015,43(11):113-115.
[14]赵艳敏,刘素香,张晨曦,等.基于HPLC-Q-TOF-MS技术的甘草化学成分分析[J].中草药,2016,47(12):2061-2068.
[15]马海娟,高简,张亚丽,等.基于HPLC-MSn的甘草成分快速鉴定及质谱裂解途径研究[J].中华中医药杂志,2018,33(3):1120-1123.
[16] WEI F,JIANG X,GAO H Y,et al. Liquiritin induces apoptosis and autophagy in cisplatin(DDP)-resistant gastric cancer cellsin vitroand xenograft nude micein vivo[J]. Int J Oncol,2017,51(5):1383-1394.
[17]陈丽敏,林辉,徐大量,等.桂枝甘草汤挥发性成分的GC-MS测定研究[J].中药新药与临床药理,2010,21(4):418-421.
[18] QIAO X,LI R,SONG W,et al. A targeted strategy to analyze untargeted mass spectral data:rapid chemical profiling of Scutellaria baicalensis using ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry and key ion filtering[J]. J Chromatogra A,2016,1441:83-95.
[19]许文,吴献,黄鸣清,等.栝楼桂枝汤的血清药物化学研究[J].中草药,2017,48(10):2033-2043.
[20]高厚明,胡兆流,郑平,等. HPLC-MS测定小儿安神补脑颗粒中8个成分的含量[J].中国实验方剂学杂志,2018,24(19):73-78.
[21]梁可,崔思娇,张琦,等. UPLC同时测定桂枝中5种活性成分的含量[J].中国中药杂志,2011,36(23):3298-3301.
[22]杨松,贾英,毕开顺. RP-HPLC法测定桂枝中4种活性成分的含量[J].药物分析杂志,2004,24(2):143-146.
[23] LIU X W,ZHANG F,GAO S H,et al. Metabolite profiling of Zi-Shen pill in rat biological specimens by UPLC-Q-TOF/MS[J]. Chin J Nat Med,2015,13(2):145-160.
[24]张宇伟,赵云芳,商婷婷,等.中药知母化学成分研究[J].亚太传统医药,2017,13(11):16-18.
[25] JI X,FENG Y F,RUI W. Rapid analysis of xanthones and steroidal saponins in extract of Rhizoma Anemarrhenae by ultra-performance liquid chromatography/Quadrupole-time-of-flightmass spectrometry[C]∥International Conference on Biomedical Engineering and Biotechnology. IEEE Computer Society,2012:477-485.
[26] ZHAO Y,KANG L,YU H,et al. Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry[J]. J Chin Pharm Sci,2013(3):226-233.
[27]邱昆成,孙振刚,何倩梅,等. LC-MS-MS测定配伍黄柏对知母中4种有效成分含量的影响[J].中国实验方剂学杂志,2016,22(14):84-88.
[28]季德,季德,苏晓楠,黄紫炎,等. HPLC-MS法测定知母盐炙前后8种成分量变化[J].中草药,2017,48(9):1784-1790.