活细胞固相色谱法联合高分辨质谱快速筛选龙血竭中镇痛活性成分
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摘要
目的采用活细胞固相色谱法及高分辨液质联用技术快速筛选鉴定龙血竭中潜在的镇痛活性成分。方法首先采用HPLC-DAD-TOF-MS法对龙血竭药材中的主要化学成分信息进行表征,进一步选择以疼痛相关的离子通道受体表达丰富的小鼠背根神经元细胞为靶细胞,先使龙血竭提取液中的活性成分与靶细胞特异性结合,应用液质联用技术快速鉴定与细胞靶向亲和的化学成分。结果从龙血竭药材中鉴定出21个具有不同结构类型的主要成分,采用活细胞固相色谱法共筛选出10种能与背根神经元细胞亲和的潜在活性成分,包括二苯乙烯类2个、高异黄酮2个、高异黄烷1个、二氢查耳酮2个、黄酮寡聚体3个等。结论应用活细胞固相色谱法与高分辨质谱联用技术可快速筛选辨识龙血竭中活性成分,为龙血竭镇痛等活性成分的进一步研究奠定基础,也可为中药药效物质研究提供有益的方法学参考。
Objective To rapidly screen the potential analgesic ingredients from Draconis Resina by live cell immobilized chromatography coupled with HRMS. Methods An HPLC-DAD-ESI-TOF-MS technique was used to rapidly identify the main chemical constituents from Draconis Resina. Based on the bio-specific affinity adsorption of bioactive compounds with receptors or channels on cells, the potential bioactive components in Draconis Resina could be selectively bound to the target cells-mice dorsal root neurons cells, then the chemical constituents with cell target affinity were identified by LC-HRMS. Results A total of 21 compounds with various structures were tentatively identified and characterized by HPLC-DAD-ESI-TOF-MS, and among them, 10 potentially analgesic active ingredients in Draconis Resina extract combined with dorsal root neurons cells were successfully detected and identified, including two stilbene, two homoisoflavones, one homoisoflavone, two dihydrochalcone, and three flavonoid oligomers.Conclusion Live cell immobilized chromatography coupled with LC-DAD-HRMS analysis could provide a rapid and efficient tool for finding the potential bioactive components in Draconis Resina for the next pharmacology studies, which provide the reference for exploring of effective materials basis in Chinese medicines.
Objective To rapidly screen the potential analgesic ingredients from Draconis Resina by live cell immobilized chromatography coupled with HRMS. Methods An HPLC-DAD-ESI-TOF-MS technique was used to rapidly identify the main chemical constituents from Draconis Resina. Based on the bio-specific affinity adsorption of bioactive compounds with receptors or channels on cells, the potential bioactive components in Draconis Resina could be selectively bound to the target cells-mice dorsal root neurons cells, then the chemical constituents with cell target affinity were identified by LC-HRMS. Results A total of 21 compounds with various structures were tentatively identified and characterized by HPLC-DAD-ESI-TOF-MS, and among them, 10 potentially analgesic active ingredients in Draconis Resina extract combined with dorsal root neurons cells were successfully detected and identified, including two stilbene, two homoisoflavones, one homoisoflavone, two dihydrochalcone, and three flavonoid oligomers.Conclusion Live cell immobilized chromatography coupled with LC-DAD-HRMS analysis could provide a rapid and efficient tool for finding the potential bioactive components in Draconis Resina for the next pharmacology studies, which provide the reference for exploring of effective materials basis in Chinese medicines.
引文
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[23]Yu L,Wang S,Kogure Y,et al.Modulation of TRPchannels by resveratrol and other stilbenoids[J].Mol Pain,2013,9:3-13.
[24]Wei L S,Chen S,Huang X J,et al.Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1receptor currents in rat dorsal root ganglion neurons[J].Eur J Pharmacol,2013,702:275-284.
[25]刘向明,陈素,张玉霞,等.血竭对背根神经节细胞河豚毒素不敏感型钠电流的调制及其药效物质的确定[J].中国科学C辑:生命科学,2006,36(1):76-85.
[26]郭敏,陈素,刘向明.龙血竭抑制大鼠脊髓背角广动力范围神经元诱发放电的药效物质[J].中国科学C辑:生命科学,2008,38(12):1130-1142.
[2]Li Y S,Wang J X,Jia M M,et al.Dragon’s blood inhibits chronic inflammatory and neuropathic pain responses by blocking the synthesis and release of substance P in rats[J].J Pharmacol Sci,2012,118(1):43-54.
[3]刘向明,陈素.龙血竭外周镇痛效应的药理研究和反向药理学[J].中南民族大学学报:自然科学版,2008,27(3):40-46.
[4]陈素,吴水才,曾毅,等.龙血竭总黄酮抗炎镇痛作用及其镇痛机制探讨[J].时珍国医国药,2013,24(5):1030-1032.
[5]陈福锋,郑亚男,杜文杰,等.龙血竭胶囊抗慢性血瘀证作用的研究[J].药物评价研究,2015,38(3):274-278.
[6]万青,王文清,方建国,等.一测多评法测定龙血竭中5种有效成分[J].中草药,2017,48(21):4541-4545.
[7]苏小琴,李曼曼,顾宇凡,等.龙血竭酚类成分研究[J].中草药,2014,45(11):1511-1514.
[8]刘芳,戴荣继,邓玉林,等.龙血竭化学成分研究进展[J].中国药房,2010,21(15):1437-1439.
[9]秦建平,林夏,潘有智,等.基于指纹图谱分析和多成分同时定量的龙血通络胶囊质量评价研究[J].中草药,2015,46(20):3028-3033.
[10]朱荃.细胞膜固相色谱及其在中药效应-物质基础研究中的应用[J].南京中医药大学学报,2006,22(1):8-13.
[11]毕肖林,马世堂,狄留庆.中药药效物质筛选与辨识的研究思路及进展[J].中草药,2018,49(22):5229-5234.
[12]罗涛,李力燕.外周神经系统中背根神经节的研究进展[J].解剖科学进展,2015,21(5):543-546.
[13]Hara T,Chiba T,Abe K,et al.Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion[J].Pain,2013,154(6):882-889.
[14]Kobayashi K,Yamanaka H,Noguchi K.Expression of ATP receptors in the rat dorsal root ganglion and spinal cord[J].Anat Sci Int,2013,88(1):10-16.
[15]Sun J,Song Y,Sun H,et al.Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by HPLC-DAD-IT-TOF-MS[J].J Pharm Biomed Anal,2017,145:462-472.
[16]Xu H,Zhang Y,Lei Y,et al.A systems biology-based approach to uncovering the molecular mechanisms underlying the effects of dragon′s blood tablet in colitis,involving the integration of chemical analysis,ADMEprediction,and network pharmacology[J].PLoS One,2014,9(7):e101432.
[17]秦建平,潘有智,郎悦,等.龙血通络胶囊中化学成分的RRLC-Q-TOF MS分析[J].药物分析杂志,2017,37(10):1782-1790.
[18]贺浪冲,杨广德,耿信笃.固定在硅胶表面细胞膜的酶活性及其色谱特性[J].科学通报,1999,44(6):632-637.
[19]焦姣姣,杨园珍,袁恩,等.UPLC-Q-TOF-MS筛选穿心莲中亲和Caco-2细胞的活性成分[J].中草药,2018,49(12):2946-2952.
[20]刘思燚,张红柱,范菁,等.内皮细胞提取和UHPLC-LTQ-Orbitrap法分析筛选苦碟子注射液中的活性成分[J].中国中药杂志,2017,42(17):3362-3367.
[21]樊宏伟,朱荃,洪敏,等.血小板细胞膜固相色谱法在脉络宁注射液效应物质分析中的应用[J].中国药学杂志,2006,41(1):63-66.
[22]樊宏伟,余黎,洪敏,等.血小板细胞膜固相色谱法的建立及其对丹参效应物质的初步分析[J].中国药学杂志,2004,39(5):62-65.
[23]Yu L,Wang S,Kogure Y,et al.Modulation of TRPchannels by resveratrol and other stilbenoids[J].Mol Pain,2013,9:3-13.
[24]Wei L S,Chen S,Huang X J,et al.Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1receptor currents in rat dorsal root ganglion neurons[J].Eur J Pharmacol,2013,702:275-284.
[25]刘向明,陈素,张玉霞,等.血竭对背根神经节细胞河豚毒素不敏感型钠电流的调制及其药效物质的确定[J].中国科学C辑:生命科学,2006,36(1):76-85.
[26]郭敏,陈素,刘向明.龙血竭抑制大鼠脊髓背角广动力范围神经元诱发放电的药效物质[J].中国科学C辑:生命科学,2008,38(12):1130-1142.