基于特征图谱和一测多评法的蟾酥药材质量控制研究
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摘要
蟾酥为蟾蜍科动物中华大蟾蜍Bufo bufo gargarizans或黑眶蟾蜍B.melanostictus耳后腺及皮肤腺的干燥分泌物。目前商品蟾酥质量问题突出,混伪、掺假现象普遍存在。亟需构建科学完善的质量控制方法,以提升蟾酥药材、饮片及其中成药制剂的质量控制水平,保障临床用药安全有效。该研究采用高效液相色谱法建立蟾酥药材的特征图谱,采用一测多评法(QAMS)测定5种主要蟾毒配基的含量。通过不同批次市售蟾酥样品的分析,建立了以五羟色胺、日蟾毒它灵、沙蟾毒精、嚏根草配基、远华蟾毒精、蟾毒它灵、华蟾毒它灵、蟾毒灵、华蟾酥毒基和脂蟾毒配基为特征峰的对照特征图谱;建立了以华蟾酥毒基为内参物,同步测定蟾酥药材中5种蟾毒配基含量的一测多评法,并对相对校正因子(RCF)的耐用性和系统适用性进行了系统研究。华蟾酥毒基对日蟾毒它灵、蟾毒它灵、蟾毒灵和脂蟾毒配基的RCF分别为1.05,0.895,1.09,0.913。建立的特征图谱和QAMS法,可以有效控制蟾酥药材的质量,为2020年版《中国药典》蟾酥药材质量标准的完善与提高提供参考。
Toad venom(Chansu) is prepared from the dried secretion of parotid gland and skin gland from Bufo bufo gargarizans or B. melanostictus. Up to now,much attention shall be paid to the poor quality of commercial toad venom because of the adulteration.So,it is urgent to establish a scientific and perfect quality control method to improve the quality of toad venom and guarantee its safety and effectiveness in clinical application. The different batches of toad venom samples were assayed by high performance liquid chromatography(HPLC) and the quantitative analysis of multi-components by single marker(QAMS) was used to detect the contents of five bufagenins. As a result,the reference characteristic chromatogram was established,displaying serotonin,gamabufotalin,arenobufagin,hellebrigenin,telocinobufagin,bufotalin,cinobufotalin,bufalin,cinobufagin and resibufogenin as characteristic peaks. Taking cinobufagin as an internal reference substance,QAMS was verified for the determination of five bufagenins(gamabufotalin,bufotalin,bufalin,cinobufagin,resibufogenin) in toad venom samples. The durability and applicability of the relative correction factor(RCF) were also studied systematically. RCFs of cinobufagin to gamabufotalin,bufotalin,bufalin and resibufogenin were determined as 1. 05,0.895,1. 09 and 0. 913,respectively. The characteristic chromatogram and QAMS established in this study could effectively control the quality of toad venom and provide scientific evidence for the improvement of the quality standard of the toad venom to be described in Chinese Pharmacopoeia(2020 edition).
Toad venom(Chansu) is prepared from the dried secretion of parotid gland and skin gland from Bufo bufo gargarizans or B. melanostictus. Up to now,much attention shall be paid to the poor quality of commercial toad venom because of the adulteration.So,it is urgent to establish a scientific and perfect quality control method to improve the quality of toad venom and guarantee its safety and effectiveness in clinical application. The different batches of toad venom samples were assayed by high performance liquid chromatography(HPLC) and the quantitative analysis of multi-components by single marker(QAMS) was used to detect the contents of five bufagenins. As a result,the reference characteristic chromatogram was established,displaying serotonin,gamabufotalin,arenobufagin,hellebrigenin,telocinobufagin,bufotalin,cinobufotalin,bufalin,cinobufagin and resibufogenin as characteristic peaks. Taking cinobufagin as an internal reference substance,QAMS was verified for the determination of five bufagenins(gamabufotalin,bufotalin,bufalin,cinobufagin,resibufogenin) in toad venom samples. The durability and applicability of the relative correction factor(RCF) were also studied systematically. RCFs of cinobufagin to gamabufotalin,bufotalin,bufalin and resibufogenin were determined as 1. 05,0.895,1. 09 and 0. 913,respectively. The characteristic chromatogram and QAMS established in this study could effectively control the quality of toad venom and provide scientific evidence for the improvement of the quality standard of the toad venom to be described in Chinese Pharmacopoeia(2020 edition).
引文
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[2]李宗云,曲婷,王鹏飞,等.毒性中药蟾酥质量研究现状及关键影响因素分析[J].中国中药杂志,2017,42(5):863.
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[7]Zhang D M,Liu J S,Deng L J,et al.Arenobufagin,a natural bufadienolide from toad venom,induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/m TOR pathway[J].Carcinogenesis,2013,34(6):1331.
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[11]Zhu Y R,Xu Y,Fang J F,et al.Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation[J].Biochem Biophys Res Commun,2014,451(1):112.
[12]Chen Y Y,Lu H F,Hsu S C,et al.Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-κB and matrix metalloproteinase-2/-9-signaling pathways[J].Environ Toxicol,2015,30(1):74.
[13]Ye M,Guo H,Guo H Z,et al.Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine Chan Su by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections[J].J Chromatogr B,2006,838:86.
[14]Zhang P,Cui Z,Liu Y S,et al.Quality evaluation of traditional Chinese drug Toad Venom from different origins through a simultaneous determination of Bufogenins and indole alkaloids by HPLC[J].Chem Pharm Bull,2005,53(12):1582.
[15]王智民,高慧敏,付雪涛,等.“一测多评”法中药质量评价模式方法学研究[J].中国中药杂志,2006,31(23):1925.
[16]高慧敏,宋宗华,王智民,等.适合中药特点的质量评价模式QAMS研究概述[J].中国中药杂志,2012,37(4):405.
[17]朱晶晶,王智民,高慧敏,等.一测多评法在中药质量评价中的应用研究进展[J].中国实验方剂学杂志,2016,22(16):220.
[18]Gao H M,Zehl M,Leitner A,et al.Comparison of toad venoms from different Bufo species by HPLC and LC-DAD-MS/MS[J].J Ethnopharmacol,2010,131:368.
[19]朱玲英,钱大玮,段金傲,等.蟾酥药材HPLC指纹图谱研究[J].中成药,2008,30(5):625.
[20]姬诚,郭万周.蟾酥的高效液相指纹图谱研究[J].光明中医,2016,31(12):1727.
[2]李宗云,曲婷,王鹏飞,等.毒性中药蟾酥质量研究现状及关键影响因素分析[J].中国中药杂志,2017,42(5):863.
[3]曲婷,高慧敏,陈两绵,等.蟾蜍类药材中吲哚生物碱和蟾毒配基类成分的含量[J].中国中药杂志,2012,37(20):3086.
[4]Yu Z L,Guo W,Ma X C,et al.Gamabufotalin,a bufadienolide compound from toad venom,suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in lung cancer cells[J].Mol Cancer,2014,13(1):203.
[5]Tang N,Shi L,Yu Z L,et al.Gamabufotalin,a major derivative of bufadienolide,inhibits VEGF-induced angiogenesis by suppressing VEGFR-2 signaling pathway[J].Oncotarget,2016,7(3):3533.
[6]Zhang L Y,Yu Z L,Wang Y,et al.Quantitative proteomics reveals molecular mechanism of gamabufotalin and its potential inhibition on Hsp90 in lung cancer[J].Oncotarget,2016,7(47):76551.
[7]Zhang D M,Liu J S,Deng L J,et al.Arenobufagin,a natural bufadienolide from toad venom,induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/m TOR pathway[J].Carcinogenesis,2013,34(6):1331.
[8]Li M M,Wu S,Liu Z,et al.Arenobufagin,a bufadienolide compound from toad venom,inhibits VEGF mediated angiogenesis through suppression of VEGFR-2 signaling pathway[J].Biochem Pharmacol,2012,83:1251.
[9]Waiwut P,Inujima A,Inoue H,et al.Bufotalin sensitizes death receptor-induced apoptosis via Bid-and STAT1-dependent pathways[J].Int J Oncol,2012,40(1):203.
[10]Zhang D M,Liu J S,Tang M K,et al.Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant Hep G2 cells through G2/M cell cycle arrest and apoptosis[J].Eur J Pharmacol,2012,692(1-3):19.
[11]Zhu Y R,Xu Y,Fang J F,et al.Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation[J].Biochem Biophys Res Commun,2014,451(1):112.
[12]Chen Y Y,Lu H F,Hsu S C,et al.Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-κB and matrix metalloproteinase-2/-9-signaling pathways[J].Environ Toxicol,2015,30(1):74.
[13]Ye M,Guo H,Guo H Z,et al.Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine Chan Su by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections[J].J Chromatogr B,2006,838:86.
[14]Zhang P,Cui Z,Liu Y S,et al.Quality evaluation of traditional Chinese drug Toad Venom from different origins through a simultaneous determination of Bufogenins and indole alkaloids by HPLC[J].Chem Pharm Bull,2005,53(12):1582.
[15]王智民,高慧敏,付雪涛,等.“一测多评”法中药质量评价模式方法学研究[J].中国中药杂志,2006,31(23):1925.
[16]高慧敏,宋宗华,王智民,等.适合中药特点的质量评价模式QAMS研究概述[J].中国中药杂志,2012,37(4):405.
[17]朱晶晶,王智民,高慧敏,等.一测多评法在中药质量评价中的应用研究进展[J].中国实验方剂学杂志,2016,22(16):220.
[18]Gao H M,Zehl M,Leitner A,et al.Comparison of toad venoms from different Bufo species by HPLC and LC-DAD-MS/MS[J].J Ethnopharmacol,2010,131:368.
[19]朱玲英,钱大玮,段金傲,等.蟾酥药材HPLC指纹图谱研究[J].中成药,2008,30(5):625.
[20]姬诚,郭万周.蟾酥的高效液相指纹图谱研究[J].光明中医,2016,31(12):1727.
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