去氢骆驼蓬碱在秀丽隐杆线虫体内代谢研究
|
摘要
去氢骆驼蓬碱是一种β-咔啉类生物碱,能够显著地抑制秀丽隐杆线虫let-60(n1046gf)的多产卵器表型,具有治疗胰腺癌的潜在能力。本研究采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-QTOF MS)法分析去氢骆驼蓬碱,确定其可能的裂解途径及特征碎片离子,同时研究其单体在线虫体内的代谢产物,并对给药后不同时间点的线虫成虫体内去氢骆驼蓬碱的含量进行半定量分析。结果显示,去氢骆驼蓬碱的裂解途径主要为RDA裂解,包括甲基、羟基等基团的丢失以及吲哚环的开裂。在电喷雾正离子模式下,检测到去氢骆驼蓬碱(m/z213)的去甲基化(m/z199)、羟基化(m/z229)的代谢产物。半定量分析结果表明:0~5h,线虫代谢旺盛,线虫体内去氢骆驼蓬碱的含量下降约80%;5h后,代谢速率减慢。该方法适用于去氢骆驼蓬碱在线虫体内代谢产物的研究,能够为去氢骆驼蓬碱治疗胰腺癌的新药研发提供数据支持。
Harmine is one ofβ-carboline alkaloid that significantly inhibits the multivulva phenotype of let-60(n1046gf),a worm called Caenorhabditiselegans.Therefore,harmine has an important potential application in pancreatic cancer.Metabolic research in vivois one of the important ways of pharmacodynamic substance research,which can illuminate the existent form medicine in vivo and confirm the real active constituent of medicine.The fragmentation pathway of harmine was analyzed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF MS),and the possible fragmentation pathways and characteristic fragment ions were deter-mined.The metabolites of harmane in Caenorhabditis elegans were studied by UPLC-QTOF MS.The MRM data acquisition model was used to study the content of harmine in the nematode worms at different time after administration by the semi-quantitative analysis.The results show that the fragment pathways of harmine are mainly RDA cleavage,including the loss of methyl groups and hydroxyl groups,and the cracking of indole ring.The metabolites of demethylated(m/z 199)and hydroxylated(m/z 229)of harmine(m/z213)were detected in positive ion mode.The results of semi-quantitative study show that the metabolism of nematodes is strong in 0-5h,and the content of harmine is decreased by about 80%,the metabolism rate slows down after 5h.The study can lay the foundation for the development of new drugs for the treatment of pancreatic cancer.
Harmine is one ofβ-carboline alkaloid that significantly inhibits the multivulva phenotype of let-60(n1046gf),a worm called Caenorhabditiselegans.Therefore,harmine has an important potential application in pancreatic cancer.Metabolic research in vivois one of the important ways of pharmacodynamic substance research,which can illuminate the existent form medicine in vivo and confirm the real active constituent of medicine.The fragmentation pathway of harmine was analyzed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF MS),and the possible fragmentation pathways and characteristic fragment ions were deter-mined.The metabolites of harmane in Caenorhabditis elegans were studied by UPLC-QTOF MS.The MRM data acquisition model was used to study the content of harmine in the nematode worms at different time after administration by the semi-quantitative analysis.The results show that the fragment pathways of harmine are mainly RDA cleavage,including the loss of methyl groups and hydroxyl groups,and the cracking of indole ring.The metabolites of demethylated(m/z 199)and hydroxylated(m/z 229)of harmine(m/z213)were detected in positive ion mode.The results of semi-quantitative study show that the metabolism of nematodes is strong in 0-5h,and the content of harmine is decreased by about 80%,the metabolism rate slows down after 5h.The study can lay the foundation for the development of new drugs for the treatment of pancreatic cancer.
引文
[1]付瑾,赵爽,王贝贝,等.骆驼蓬种子及其炮制品中去氢骆驼蓬碱的含量测定[J].现代中药研究与实践,2015,(4):35-36.FU Jin,ZHAO Shuang,WANG Beibei,et al.Determination of harmine in seeds of ethnic medicine and its processed products of Peganum harmala[J].Research and Practice of Chinese Medicines,2015,(4):35-36(in Chinese).
[2]顾帆,顾月清.去氢骆驼蓬碱通过诱导凋亡发挥抗肿瘤药理作用研究进展[J].中成药,2014,36(6):1 277-1 280.GU Fan,GU Yueqing.Research progress on antitumor pharmacological effects of harmine through inducing apoptosis[J].Chinese Traditional Patent Medicine,2014,36(6):1 277-1 280(in Chinese).
[3]王锦军,张秀梅,罗磊.去氢骆驼蓬碱体外抗消化道肿瘤活性和对小鼠脾细胞增殖反应的抑制作用[J].中国现代应用药学,2008,25(s1):607-608.WANG Jinjun,ZHANG Xiumei,LUO Lei.In vitro anti tumor activity and inhibition of proliferation of splenocytes in mice induced by hydrogen peroxide[J].Chinese Journal of Modern Applied Pharmacy,2008,25(s1):607-608(in Chinese).
[4]PATEL K,GADEWAR M,TRIPATHI R,et al.A review on medicinal importance,pharmacological activity and bioanalytical aspects of beta-carboline alkaloid‘Harmine’[J].Asian Pacific Journal of Tropical Biomedicine,2012,2(8):660-664.
[5]宋红健.天然产物骆驼蓬碱和去氢骆驼蓬碱及其衍生物的合成、生物活性和构效关系研究[D].天津:南开大学,2014.
[6]郭亮,孙洁,范文玺,等.去氢骆驼蓬碱衍生物的合成和抗肿瘤活性研究[J].中国现代应用药学,2012,(5):385-388.GUO Liang,SUN Jie,FAN Wenxi,et al.Synthesis and antitumor activities of harmine derivatives[J].Chinese Journal of Modern Applied Pharmacy,2012,(5):385-388(in Chinese).
[7]周雨朦,陈代杰.秀丽隐杆线虫在药物筛选中的应用[J].上海医药,2011,32(11):566-571.ZHOU Yumeng,CHEN Daijie.Applications of Caenorhabditis elegans in drug screening[J].Shanghai Medical&Pharmaceutical Journal,2011,32(11):566-571(in Chinese).
[8]杨再昌,杨小生.秀丽隐杆线虫(Caenorhabditis elegans)在药物筛选中的应用[J].生命科学,2009,21(4):593-598.YANG Zaichang,YANG Xiaosheng.An application of Caenorhabditis elegans for drug screening[J].Chinese Bulletin of Life Sciences,2009,21(4):593-598(in Chinese).
[9]KOBET R A,PAN X,ZHANG B,et al.Caenorhabditis elegans:a model system for anti-cancer drug discovery and therapeutic target identification[J].Biomolecules&Therapeutics,2014,22(5):371-383.
[10]PEROZICH J,HEMPEL J,Jr M S.Roles of conserved residues in the arginase family[J].Biochimica et Biophysica Acta,1998,1 382(1):23-37.
[11]FUNKHOUSER J D,Jr A N.Chitinase family GH18:evolutionary insights from the genomic history of a diverse protein family[J].BMC Evolutionary Biology,2007,7(1):96.
[12]SINGH S,BROCKER C,KOPPAKA V,et al.Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress[J].Free Radical Biology&Medicine,2013,56(3):89-101.
[13]AARNIO V,LEHTONEN M,STORVIK M,et al.Caenorhabditis elegans mutants predict regulation of fatty acids and endocannabinoids by the CYP-35Agene family[J].Frontiers in Pharmacology,2011,2:12.
[14]MDI A,MIKKAT S,KOY C,et al.Mass spectrometric proteome analysis suggests anaerobic shift in metabolism of Dauer larvae of Caenorhabditis elegans[J].Biochimica et Biophysica Acta,2008,1 784(11):1 763-1 770.
[15]STIERNAGLE T.Maintenance of C.elegans[J].Wormbook the Online Review of C Elegans Biology,2006:1-11.
[16]李娜,李圣坤,张前亮,等.骆驼蓬中主要生物碱的电喷雾多级质谱裂解行为研究[J].西北大学学报:自然科学版,2013,43(6):909-911.LI Na,LI Shengkun,ZHANG Qianliang,et al.Fragmentation pathway of main alkaloids from Peganum harmala L.by electrospray ionization tandem mass spectrometry[J].Journal of Northwest University,2013,43(6):909-911(in Chinese).
[2]顾帆,顾月清.去氢骆驼蓬碱通过诱导凋亡发挥抗肿瘤药理作用研究进展[J].中成药,2014,36(6):1 277-1 280.GU Fan,GU Yueqing.Research progress on antitumor pharmacological effects of harmine through inducing apoptosis[J].Chinese Traditional Patent Medicine,2014,36(6):1 277-1 280(in Chinese).
[3]王锦军,张秀梅,罗磊.去氢骆驼蓬碱体外抗消化道肿瘤活性和对小鼠脾细胞增殖反应的抑制作用[J].中国现代应用药学,2008,25(s1):607-608.WANG Jinjun,ZHANG Xiumei,LUO Lei.In vitro anti tumor activity and inhibition of proliferation of splenocytes in mice induced by hydrogen peroxide[J].Chinese Journal of Modern Applied Pharmacy,2008,25(s1):607-608(in Chinese).
[4]PATEL K,GADEWAR M,TRIPATHI R,et al.A review on medicinal importance,pharmacological activity and bioanalytical aspects of beta-carboline alkaloid‘Harmine’[J].Asian Pacific Journal of Tropical Biomedicine,2012,2(8):660-664.
[5]宋红健.天然产物骆驼蓬碱和去氢骆驼蓬碱及其衍生物的合成、生物活性和构效关系研究[D].天津:南开大学,2014.
[6]郭亮,孙洁,范文玺,等.去氢骆驼蓬碱衍生物的合成和抗肿瘤活性研究[J].中国现代应用药学,2012,(5):385-388.GUO Liang,SUN Jie,FAN Wenxi,et al.Synthesis and antitumor activities of harmine derivatives[J].Chinese Journal of Modern Applied Pharmacy,2012,(5):385-388(in Chinese).
[7]周雨朦,陈代杰.秀丽隐杆线虫在药物筛选中的应用[J].上海医药,2011,32(11):566-571.ZHOU Yumeng,CHEN Daijie.Applications of Caenorhabditis elegans in drug screening[J].Shanghai Medical&Pharmaceutical Journal,2011,32(11):566-571(in Chinese).
[8]杨再昌,杨小生.秀丽隐杆线虫(Caenorhabditis elegans)在药物筛选中的应用[J].生命科学,2009,21(4):593-598.YANG Zaichang,YANG Xiaosheng.An application of Caenorhabditis elegans for drug screening[J].Chinese Bulletin of Life Sciences,2009,21(4):593-598(in Chinese).
[9]KOBET R A,PAN X,ZHANG B,et al.Caenorhabditis elegans:a model system for anti-cancer drug discovery and therapeutic target identification[J].Biomolecules&Therapeutics,2014,22(5):371-383.
[10]PEROZICH J,HEMPEL J,Jr M S.Roles of conserved residues in the arginase family[J].Biochimica et Biophysica Acta,1998,1 382(1):23-37.
[11]FUNKHOUSER J D,Jr A N.Chitinase family GH18:evolutionary insights from the genomic history of a diverse protein family[J].BMC Evolutionary Biology,2007,7(1):96.
[12]SINGH S,BROCKER C,KOPPAKA V,et al.Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress[J].Free Radical Biology&Medicine,2013,56(3):89-101.
[13]AARNIO V,LEHTONEN M,STORVIK M,et al.Caenorhabditis elegans mutants predict regulation of fatty acids and endocannabinoids by the CYP-35Agene family[J].Frontiers in Pharmacology,2011,2:12.
[14]MDI A,MIKKAT S,KOY C,et al.Mass spectrometric proteome analysis suggests anaerobic shift in metabolism of Dauer larvae of Caenorhabditis elegans[J].Biochimica et Biophysica Acta,2008,1 784(11):1 763-1 770.
[15]STIERNAGLE T.Maintenance of C.elegans[J].Wormbook the Online Review of C Elegans Biology,2006:1-11.
[16]李娜,李圣坤,张前亮,等.骆驼蓬中主要生物碱的电喷雾多级质谱裂解行为研究[J].西北大学学报:自然科学版,2013,43(6):909-911.LI Na,LI Shengkun,ZHANG Qianliang,et al.Fragmentation pathway of main alkaloids from Peganum harmala L.by electrospray ionization tandem mass spectrometry[J].Journal of Northwest University,2013,43(6):909-911(in Chinese).