基于UPLC-LTQ-Orbitrap高分辨质谱的中药瓜蒌化学成分分析
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摘要
目的:基于超高效液相色谱-线性离子阱-静电场轨道阱高分辨质谱系统(UPLC-LTQ-Orbitrap-MS)分析鉴定中药瓜蒌的化学成分。方法:瓜蒌样品粉碎过筛后采用70%甲醇超声提取,Waters HSS T3-C18色谱柱(2. 1 mm×100 mm,1. 8μm),流动相0. 1%甲酸水溶液-甲醇梯度洗脱,流速0. 3 mL·min-1,采用电喷雾离子源(ESI),正、负离子分别进行一级、二级质谱数据采集。为了获得更多的化合物信息,根据色谱峰分离情况、质谱响应强度以及一级质谱解卷积后提取得到的准分子离子数量,优化样品提取条件、色谱及质谱参数。根据精确相对分子质量、离子碎片信息、相关对照品比对,并结合瓜蒌化学成分文献信息,对其中的化学成分进行分析鉴定。结果:从瓜蒌中共分析鉴定了91个化学成分,包括14个氨基酸类化合物,5个单萜类化合物,5个四环三萜类化合物,1个五环三萜类化合物,14个黄酮类化合物,17个有机酸类化合物,3个多糖类化合物,7个核苷类化合物,7个生物碱及含氮化合物,12个挥发性成分,1个植物甾醇,及其他5种化合物。结论:该方法具有高效分离、高灵敏检测优势,可较为全面地对瓜蒌中不同类型化合物进行分析鉴定,为瓜蒌药效物质基础及质量标准研究提供参考。
Objective: To analysis and identify the chemical components in Trichosanthis Fructus by UPLC-LTQ-Orbitrap-MS. Method: Samples of Trichosanthis Fructus were extracted by ultrasonic with 70%methanol after smashing and sifting by 40 mesh sieve. Thermo ScientificTMDionexTMUlti MateTM3000 Rapid Separation LC system performed UPLC separations with Waters HSS T3-C18(2. 1 mm × 100 mm,1. 8 μm)column. The mobile phase was 0. 1% formic acid water(A)-methanol(B) with a gradient elution. The volume flow was 0. 3 mL·min-1. A Thermo ScientificTMLTQ-Orbitrap mass spectrometer equipped with a ESI probe was employed. The samples were respectively scanned in MS1 and MS2 mode of positive and negative ions. According to the chromatographic peak separation,mass signal intensity,and the number of molecular ions in MS1 model,the extraction condition,chromatogram and mass spectrum parameters were optimized. The chemical compounds were identified by the accurate mass measurement of molecular ions and fragment ion and comparation with reference substance. Result: 91 chemical compositions in Trichosanthis Fructus were totally identified,including 14 amino acids,5 monoterpenoids,5 tetracyclic triterpenoids,1 pentacyclic triterpene,14 flavonoids,17 organic acids,3 polysaccharides,7 nucleotides,7 alkaloids and nitrogen compounds,2 volatile components,1 phytosterol,5 other compositions. Conclusion: The established UPLC-LTQ-Orbitrap-MS method can be used to quickly analyze and identify the main chemical constituents of Trichosanthis Fructus. The chemical information concerning the constituents in Trichosanthis Fructus could be helpful to the quality control and further studies of Trichosanthis Fructus.
Objective: To analysis and identify the chemical components in Trichosanthis Fructus by UPLC-LTQ-Orbitrap-MS. Method: Samples of Trichosanthis Fructus were extracted by ultrasonic with 70%methanol after smashing and sifting by 40 mesh sieve. Thermo ScientificTMDionexTMUlti MateTM3000 Rapid Separation LC system performed UPLC separations with Waters HSS T3-C18(2. 1 mm × 100 mm,1. 8 μm)column. The mobile phase was 0. 1% formic acid water(A)-methanol(B) with a gradient elution. The volume flow was 0. 3 mL·min-1. A Thermo ScientificTMLTQ-Orbitrap mass spectrometer equipped with a ESI probe was employed. The samples were respectively scanned in MS1 and MS2 mode of positive and negative ions. According to the chromatographic peak separation,mass signal intensity,and the number of molecular ions in MS1 model,the extraction condition,chromatogram and mass spectrum parameters were optimized. The chemical compounds were identified by the accurate mass measurement of molecular ions and fragment ion and comparation with reference substance. Result: 91 chemical compositions in Trichosanthis Fructus were totally identified,including 14 amino acids,5 monoterpenoids,5 tetracyclic triterpenoids,1 pentacyclic triterpene,14 flavonoids,17 organic acids,3 polysaccharides,7 nucleotides,7 alkaloids and nitrogen compounds,2 volatile components,1 phytosterol,5 other compositions. Conclusion: The established UPLC-LTQ-Orbitrap-MS method can be used to quickly analyze and identify the main chemical constituents of Trichosanthis Fructus. The chemical information concerning the constituents in Trichosanthis Fructus could be helpful to the quality control and further studies of Trichosanthis Fructus.
引文
[1]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:112-113.
[2] Jang K C,Lee J H,Kim S C,et al. Antibacterial and radical scavenging activities of 1-C-(p-hydroxyphenyl)-glycerol from Trichosanthes kirilowii[J]. J Appl Bio Chem,2007,50(1):17-21.
[3]苏雪芬,李先涛,谢蓉,等.冠心病痰瘀互结证临床用药规律的文献研究[J].中国实验方剂学杂志,2015,21(14):191-198.
[4]吴波,曹红,陈思维,等.瓜蒌提取物对缺血缺氧及缺血后再灌注损伤心肌的保护作用[J].沈阳药科大学学报,2000,17(6):450-451,465.
[5]刘金娜,温春秀,刘铭,等.瓜蒌的化学成分和药理活性研究进展[J].中药材,2013,36(5):843-848.
[6]巢志茂,何波,敖平.瓜蒌的化学成分研究进展[J].国外医学:中医中药分册,1998,20(2):7-10.
[7]滕勇荣,张永清.瓜蒌化学成分研究进展[J].山东中医药大学学报,2011,35(1):85-86.
[8]刘荣霞,果德安,叶敏,等.液质联用技术(LC/MS)在中药现代研究中的应用[J].世界科学技术—中医药现代化,2005,7(5):33-40,84.
[9]周立分,赵栋云,刘霞,等. LTQ-Orbitrap MS在中药研发中的应用[J].江西中医药,2017,48(12):75-78.
[10]王勇为. LTQ-Orbitrap Velos双分压线性阱和静电场轨道阱组合式高分辨质谱性能及应用[J].现代仪器与医疗,2010,16(5):15-19.
[11]贡瑞生,张黎明,郑建科.山东栝楼氨基酸及微量元素的分析[J].中国中药杂志,1989,14(6):43-44.
[12]巢志茂,何波.栝楼果实的化学成分研究[J].中国中药杂志,1999,24(10):612.
[13]李爱峰,孙爱玲,柳仁民,等.栝楼果皮水溶性部位化学成分研究(英文)[J].中华中医药杂志,2014,29(11):3420-3423.
[14] CHAI X,LI S,ZHU L,et al. Chemical constituents of the roots of Trichosanthes kirilowii[J]. Chem Nat Compd,2014,50(5):965-967.
[15]李爱峰.栝楼(Trichosanthes kirilowii Maxim.)果皮化学成分研究[D].济南:山东中医药大学,2014.
[16]刘岱琳,曲戈霞,王乃利,等.瓜蒌的抗血小板聚集活性成分研究[J].中草药,2004,35(12):1334-1336.
[17]范雪梅,陈刚,郭丽娜,等.瓜蒌化学成分的分离与鉴定[J].沈阳药科大学学报,2011,28(11):871-874.
[18]时岩鹏,姚庆强,刘拥军,等.栝楼化学成分的研究及其α-菠菜甾醇的含量测定(Ⅰ)[J].中草药,2002,33(1):16-18.
[19]陈亮.瓜蒌化学成分与质量标准研究[D].上海:上海中医药大学,2011.
[20]徐美霞.瓜蒌皮化学成分分离与鉴定[D].泰安:山东农业大学,2013.
[21] FAN X,CHEN G,SHA Y,et al. Chemical constituents from the fruits of Trichosanthes kirilowii[J]. J Asian Nat Prod Res,2012,14(6):528-532.
[22] DUAN W,PAN S,YU Z,et al. Studies on chemical constituents of twigs of Trichosanthes kirilowii Maxim[J]. Asian J Chem,2015,27(8):2756-2758.
[23] Rahman M A A,Moon S. Isoetin 5'-methyl ether,A cytotoxic flavone from Trichosanthes kirilowii[J]. Bull Korean Chem Soc,2007,28(8):1261-1264.
[24]安亮,何侃,张沿军,等.双边栝楼果实化学成分研究[J].中南药学,2015,13(1):37-39.
[25]韦兴光,杨春芳,时岩鹏,等.栝楼化学成分的研究(Ⅱ)[J].中草药,2005,36(增刊):67-68.
[26] Minh C V,Nhiem N X,Yen H T,et al. Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities[J]. Arch Pharm Res,2015,38(8):1443-1448.
[27]李爱峰,孙爱玲,柳仁民,等.栝楼果皮化学成分研究[J].中药材,2014,37(3):428-431.
[28]巢志茂,刘静明.双边栝楼皮挥发油的化学成分研究[J].中国中药杂志,1996,21(6):37-39.
[29]孙晓业,吴红华,付爱珍,等.瓜蒌的化学成分研究[J].药学学报,2012,47(7):922-925.
[30]马跃平,高健,傅克玲,等.瓜蒌霜化学成分的分离与鉴定[J].沈阳药科大学学报,2010,27(11):876-879.
[31] XU Y,CHEN G,LU X,et al. Chemical constituents from Trichosanthes kirilowii Maxim.[J]. Biochem Syst Ecol,2012,43:114-116.
[32] Oh H,Mun Y J,Im S J,et al. Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10melanoma cells[J]. Planta Med,2002,68(9):832-833.
[33]黄庆勇.天花粉中总皂苷提取工艺及乙酸乙酯部位活性筛选研究[D].福州:福建中医药大学,2015.
[34] Ryu S,Lee S,Choi S,et al. Antitumor activity of Trichosanthes kirilowii[J]. Arch Pharm Res,1994,17(5):348-353.
[35] Dat N T,JIN X,HONG Y,et al. An isoaurone and other constituents from Trichosanthes kirilowii seeds inhibit hypoxia-inducible factor-1 and nuclear factor-KB[J]. J Nat Prod,2010,73(6):1167-1169.
[36]安亮.双边栝楼果实化学成分及其对3T3-L1前脂肪细胞增殖和分化影响的研究[D].长沙:湖南中医药大学,2015.
[37] WU T,CHENG X,S. W. Annie B,et al. Multiflorane triterpene esters from the seeds of Trichosanthes kirilowii[J]. Helv Chim Acta,2005,88(10):2617-2623.
[38]范雪梅,陈刚,苏姗姗,等.瓜蒌化学成分的分离与鉴定[J].沈阳药科大学学报,2011,28(12):947-948,954.
[39] XU Y,WU J,XIANG Z,et al. Three flavonoids from Trichosanthes kirilowii Maxim[J]. J Inst Ind Appl Eng,2014,2(3):127-130.
[40]曾益坤,黄秀娟,王兴国.栝楼籽油理化性质及其脂肪酸组成分析[J].中国油脂,2007,32(10):80-82.
[41]巢志茂,刘静明.双边栝楼化学成分研究[J].中国中药杂志,1991,16(2):97-99,127.
[42]霍启录,陈振德,许重远,等.双边栝蒌种子油含量及其脂肪酸的测定[J].华西药学杂志,2002,17(5):351-352.
[43] JIANG X,WU S,ZHOU Z,et al. Physicochemical properties and volatile profiles of cold-pressed Trichosanthes kirilowii Maxim seed oils[J]. Int J Food Properties,2016,19(8):1765-1775.
[44]王玲娜,于京平,张永清.栝楼化学成分研究概述[J].环球中医药,2014,7(1):72-76.
[45]王艳芳,王新华,朱宇同.槲皮素药理作用研究进展[J].天然产物研究与开发,2003,15(2):171-173.
[46]王祝枝,王晓华,朱玉,等.不同产地瓜蒌皮药材中槲皮素及总黄酮含量测定[J].中国实验方剂学杂志,2014,20(19):86-89.
[47]蓝天,刘卫国,王晓稼.葫芦素B抗肿瘤作用的研究进展[J].中华中医药学刊,2013,31(12):2801-2803.
[48]张延亭,欧阳东云,何贤辉.葫芦素B抗肿瘤作用及其机制研究进展[J].中国药理学与毒理学杂志,2012,26(1):112-115.
[49]孙文,巢志茂,王淳,吴等. HPLC-UV对栝楼各部位中葫芦素B的含量测定[J].中国实验方剂学杂志,2014,20(23):86-88.
[2] Jang K C,Lee J H,Kim S C,et al. Antibacterial and radical scavenging activities of 1-C-(p-hydroxyphenyl)-glycerol from Trichosanthes kirilowii[J]. J Appl Bio Chem,2007,50(1):17-21.
[3]苏雪芬,李先涛,谢蓉,等.冠心病痰瘀互结证临床用药规律的文献研究[J].中国实验方剂学杂志,2015,21(14):191-198.
[4]吴波,曹红,陈思维,等.瓜蒌提取物对缺血缺氧及缺血后再灌注损伤心肌的保护作用[J].沈阳药科大学学报,2000,17(6):450-451,465.
[5]刘金娜,温春秀,刘铭,等.瓜蒌的化学成分和药理活性研究进展[J].中药材,2013,36(5):843-848.
[6]巢志茂,何波,敖平.瓜蒌的化学成分研究进展[J].国外医学:中医中药分册,1998,20(2):7-10.
[7]滕勇荣,张永清.瓜蒌化学成分研究进展[J].山东中医药大学学报,2011,35(1):85-86.
[8]刘荣霞,果德安,叶敏,等.液质联用技术(LC/MS)在中药现代研究中的应用[J].世界科学技术—中医药现代化,2005,7(5):33-40,84.
[9]周立分,赵栋云,刘霞,等. LTQ-Orbitrap MS在中药研发中的应用[J].江西中医药,2017,48(12):75-78.
[10]王勇为. LTQ-Orbitrap Velos双分压线性阱和静电场轨道阱组合式高分辨质谱性能及应用[J].现代仪器与医疗,2010,16(5):15-19.
[11]贡瑞生,张黎明,郑建科.山东栝楼氨基酸及微量元素的分析[J].中国中药杂志,1989,14(6):43-44.
[12]巢志茂,何波.栝楼果实的化学成分研究[J].中国中药杂志,1999,24(10):612.
[13]李爱峰,孙爱玲,柳仁民,等.栝楼果皮水溶性部位化学成分研究(英文)[J].中华中医药杂志,2014,29(11):3420-3423.
[14] CHAI X,LI S,ZHU L,et al. Chemical constituents of the roots of Trichosanthes kirilowii[J]. Chem Nat Compd,2014,50(5):965-967.
[15]李爱峰.栝楼(Trichosanthes kirilowii Maxim.)果皮化学成分研究[D].济南:山东中医药大学,2014.
[16]刘岱琳,曲戈霞,王乃利,等.瓜蒌的抗血小板聚集活性成分研究[J].中草药,2004,35(12):1334-1336.
[17]范雪梅,陈刚,郭丽娜,等.瓜蒌化学成分的分离与鉴定[J].沈阳药科大学学报,2011,28(11):871-874.
[18]时岩鹏,姚庆强,刘拥军,等.栝楼化学成分的研究及其α-菠菜甾醇的含量测定(Ⅰ)[J].中草药,2002,33(1):16-18.
[19]陈亮.瓜蒌化学成分与质量标准研究[D].上海:上海中医药大学,2011.
[20]徐美霞.瓜蒌皮化学成分分离与鉴定[D].泰安:山东农业大学,2013.
[21] FAN X,CHEN G,SHA Y,et al. Chemical constituents from the fruits of Trichosanthes kirilowii[J]. J Asian Nat Prod Res,2012,14(6):528-532.
[22] DUAN W,PAN S,YU Z,et al. Studies on chemical constituents of twigs of Trichosanthes kirilowii Maxim[J]. Asian J Chem,2015,27(8):2756-2758.
[23] Rahman M A A,Moon S. Isoetin 5'-methyl ether,A cytotoxic flavone from Trichosanthes kirilowii[J]. Bull Korean Chem Soc,2007,28(8):1261-1264.
[24]安亮,何侃,张沿军,等.双边栝楼果实化学成分研究[J].中南药学,2015,13(1):37-39.
[25]韦兴光,杨春芳,时岩鹏,等.栝楼化学成分的研究(Ⅱ)[J].中草药,2005,36(增刊):67-68.
[26] Minh C V,Nhiem N X,Yen H T,et al. Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities[J]. Arch Pharm Res,2015,38(8):1443-1448.
[27]李爱峰,孙爱玲,柳仁民,等.栝楼果皮化学成分研究[J].中药材,2014,37(3):428-431.
[28]巢志茂,刘静明.双边栝楼皮挥发油的化学成分研究[J].中国中药杂志,1996,21(6):37-39.
[29]孙晓业,吴红华,付爱珍,等.瓜蒌的化学成分研究[J].药学学报,2012,47(7):922-925.
[30]马跃平,高健,傅克玲,等.瓜蒌霜化学成分的分离与鉴定[J].沈阳药科大学学报,2010,27(11):876-879.
[31] XU Y,CHEN G,LU X,et al. Chemical constituents from Trichosanthes kirilowii Maxim.[J]. Biochem Syst Ecol,2012,43:114-116.
[32] Oh H,Mun Y J,Im S J,et al. Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10melanoma cells[J]. Planta Med,2002,68(9):832-833.
[33]黄庆勇.天花粉中总皂苷提取工艺及乙酸乙酯部位活性筛选研究[D].福州:福建中医药大学,2015.
[34] Ryu S,Lee S,Choi S,et al. Antitumor activity of Trichosanthes kirilowii[J]. Arch Pharm Res,1994,17(5):348-353.
[35] Dat N T,JIN X,HONG Y,et al. An isoaurone and other constituents from Trichosanthes kirilowii seeds inhibit hypoxia-inducible factor-1 and nuclear factor-KB[J]. J Nat Prod,2010,73(6):1167-1169.
[36]安亮.双边栝楼果实化学成分及其对3T3-L1前脂肪细胞增殖和分化影响的研究[D].长沙:湖南中医药大学,2015.
[37] WU T,CHENG X,S. W. Annie B,et al. Multiflorane triterpene esters from the seeds of Trichosanthes kirilowii[J]. Helv Chim Acta,2005,88(10):2617-2623.
[38]范雪梅,陈刚,苏姗姗,等.瓜蒌化学成分的分离与鉴定[J].沈阳药科大学学报,2011,28(12):947-948,954.
[39] XU Y,WU J,XIANG Z,et al. Three flavonoids from Trichosanthes kirilowii Maxim[J]. J Inst Ind Appl Eng,2014,2(3):127-130.
[40]曾益坤,黄秀娟,王兴国.栝楼籽油理化性质及其脂肪酸组成分析[J].中国油脂,2007,32(10):80-82.
[41]巢志茂,刘静明.双边栝楼化学成分研究[J].中国中药杂志,1991,16(2):97-99,127.
[42]霍启录,陈振德,许重远,等.双边栝蒌种子油含量及其脂肪酸的测定[J].华西药学杂志,2002,17(5):351-352.
[43] JIANG X,WU S,ZHOU Z,et al. Physicochemical properties and volatile profiles of cold-pressed Trichosanthes kirilowii Maxim seed oils[J]. Int J Food Properties,2016,19(8):1765-1775.
[44]王玲娜,于京平,张永清.栝楼化学成分研究概述[J].环球中医药,2014,7(1):72-76.
[45]王艳芳,王新华,朱宇同.槲皮素药理作用研究进展[J].天然产物研究与开发,2003,15(2):171-173.
[46]王祝枝,王晓华,朱玉,等.不同产地瓜蒌皮药材中槲皮素及总黄酮含量测定[J].中国实验方剂学杂志,2014,20(19):86-89.
[47]蓝天,刘卫国,王晓稼.葫芦素B抗肿瘤作用的研究进展[J].中华中医药学刊,2013,31(12):2801-2803.
[48]张延亭,欧阳东云,何贤辉.葫芦素B抗肿瘤作用及其机制研究进展[J].中国药理学与毒理学杂志,2012,26(1):112-115.
[49]孙文,巢志茂,王淳,吴等. HPLC-UV对栝楼各部位中葫芦素B的含量测定[J].中国实验方剂学杂志,2014,20(23):86-88.