白及有效部位中主要成分在Caco-2细胞中的吸收特性研究
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摘要
为研究白及有效部位中主要成分在Caco-2细胞中的吸收特性,初步阐明白及有效部位的口服吸收机制。该实验采用Caco-2细胞单层细胞吸收模型,通过UPLC-Q-TOF分析白及有效部位在Caco-2细胞中的摄取成分,并建立UPLC-TQD分析方法测定白及有效部位主要成分在Caco-2细胞中的含量,分别考察时间、浓度、pH和P-gp抑制剂对各成分的吸收影响。经过UPLC-Q-TOF指认白及有效部位中的主要成分B6,B12,B14,B17,B19,B23,研究发现该6个成分能够摄取进入Caco-2细胞中,并且在Caco-2细胞中的吸收呈现浓度依赖性。其中B12,B14的吸收在0～180 min为上升趋势,未呈现饱和,B6,B17,B19,B23在60 min后吸收趋于饱和;各成分在偏酸性或酸性条件下吸收较好,其中B6在pH 6.0环境中吸收较好,其余成分B12,B14,B17,B19,B23在pH 4.0环境中吸收较好;且加入维拉帕米P-gp抑制剂后和空白相比,B6,B12的吸收无差异,而B14,B17,B19,B23的Caco-2细胞摄取量增加,但无显著性差异,说明B6等6个成分的摄取过程中没有P-gp的参与,即B6等6个成分不是P-gp底物。由此结果可知白及有效部位主要成分进入Caco-2细胞以被动扩散为主,且吸收过程不受P-糖蛋白的外排作用影响。
To investigate the active fraction from Bletilla striata in Caco-2 cell monolayer,so as to explore its absorption mechanism of oral administration preliminarily.Active fraction from B.striata in Caco-2 cell monolayer was analyzed by UPLC-Q-TOF and detected by UPLC-MS/MS,and the effects of different concentrations,pH and P-glycoprotein inhibitors on Caco-2 cells Monolayer were investigated.Six compounds were isolated from the active fraction of B.striata in Caco-2 cell monolayer by UPLC-Q-TOF,and identified as B6,B12,B14,B17,B19 and B23,with concentration dependence.Within the 0-180 min,the uptake of B12 and B14 had a time dependence,while B6,B17,B19 and B23 tended to saturate after 60 min.All of the components had a good absorption in an acidic environment.B6 had a good absorption at pH 6.0,while the other components B12,B14,B17,B19 and B23 had a good absorption at pH4.0.The absorption of the 6 main components of B.striata were not be affected by P-glycoprotein inhibitors(verapamil/cyclosporin A).Compared with the control group,there was no difference in the absorption of B6 and B12,and the absorption of B14,B17,B19 and B23 increased,but with no significant difference.The absorption characteristic of B.striata extract across the Caco-2 cell monolayer is probably passive diffusion,and the absorption process was not affected by P-glycoprotein.
To investigate the active fraction from Bletilla striata in Caco-2 cell monolayer,so as to explore its absorption mechanism of oral administration preliminarily.Active fraction from B.striata in Caco-2 cell monolayer was analyzed by UPLC-Q-TOF and detected by UPLC-MS/MS,and the effects of different concentrations,pH and P-glycoprotein inhibitors on Caco-2 cells Monolayer were investigated.Six compounds were isolated from the active fraction of B.striata in Caco-2 cell monolayer by UPLC-Q-TOF,and identified as B6,B12,B14,B17,B19 and B23,with concentration dependence.Within the 0-180 min,the uptake of B12 and B14 had a time dependence,while B6,B17,B19 and B23 tended to saturate after 60 min.All of the components had a good absorption in an acidic environment.B6 had a good absorption at pH 6.0,while the other components B12,B14,B17,B19 and B23 had a good absorption at pH4.0.The absorption of the 6 main components of B.striata were not be affected by P-glycoprotein inhibitors(verapamil/cyclosporin A).Compared with the control group,there was no difference in the absorption of B6 and B12,and the absorption of B14,B17,B19 and B23 increased,but with no significant difference.The absorption characteristic of B.striata extract across the Caco-2 cell monolayer is probably passive diffusion,and the absorption process was not affected by P-glycoprotein.
引文
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[18]李雪莲,王嘉馨,王静,等.不同浓度葛根芩连水煎液对Caco-2细胞P-糖蛋白及多药耐药蛋白转运体表达的影响[J].中国药学杂志,2018,43(15):3135.
[19]袁紫朔,张婷婷,金波,等.异丹叶大黄素在Caco-2细胞模型上转运机制的研究[J].中国中药杂志,2017,42(3):587.
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[21]何丹,颜苗,李焕德,等.甘草提取物及其主要成分对Caco-2细胞膜上P-gp功能和表达的影响[J].中国药学杂志,2010,54(10):751.
[2]中国药典.一部[S].2010.
[3]庞素秋,王国权,吴双,等.白及多糖对大鼠深Ⅱ度烫伤创面的保护作用[J].中药材,2013,36(11):1819.
[4]吴久健,孟岳良,邹俪华,等.白及不同提取部位对小鼠止血活性实验[J].药学实践杂志,2011,29(3):206.
[5]Wu X,Xin M,Chen H,et al.Novel mucoadhesive polysaccharide isolated from Bletilla striata improves the intraocular penetration and efficacy of levofloxacin in the topical treatment of experimental bacterial keratitis[J].J Pharm Pharmacol,2010,62(9):1152.
[6]刘明志,唐建洲,张建社,等.白芨萜类化合物对人脐静脉内皮细胞凋亡和细胞骨架的作用[J].生命科学研究,2009,13(6):482.
[7]Dong L,Xia S,Luo Y,et al.Targeting deliveryoligonucleotide into macrophages by cationic polysaccharide from Bletilla striata successfully inhibitedthe expression of TNF-α[J].J Controlled Release,2009,134(3):214.
[8]王蒨,李东华,李继坤,等.白及胶对体外培养兔胆管成纤维细胞形态及活性的影响[J].河北中医,2007,29(8):752.
[9]国家中医药管理局中华本草编委会.中华本草·苗药卷[M].贵阳:贵州科技出版社,2005.
[10]赵菲菲,杨馨,徐丹,等.白及非多糖组分的止血作用及其机制的初步研究[J].中国药理学通报,2016,32(8):1121.
[11]王爱民,鄢艳,兰波,等.UPLC同时测定白及药材中9种指标成分的含量[J].中国中药杂志,2014,39(11):2051.
[12]戴鸥,仰莲,周勤梅,等.白及化学成分分离鉴定[J].中国实验方剂学杂志,2018,24(14):43.
[13]马先杰,崔保松,韩少伟,等.中药白及的化学成分研究[J].中国中药杂志,2017,42(8):1578.
[14]Hubatsch I,Ragnarsson E G,Artursson P.Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers[J].Nat Protoc,2007,2(9):2111.
[15]杨延平,杨勇.Caco-2细胞模型的应用进展[J].今日药学,2012,22(3):189.
[16]廖沙,谢剑炜.Caco-2细胞模型在药物体外研究中的应用[J].中国药学杂志,2005,14(4):416.
[17]Diao H,Li X,Chen J,et al.Bletilla striata polysaccharide stimulates inducible nitric oxide synthase and proinflammatory cytokine expression in macrophages[J].J Biosci Bioeng,2008,105(2):85.
[18]李雪莲,王嘉馨,王静,等.不同浓度葛根芩连水煎液对Caco-2细胞P-糖蛋白及多药耐药蛋白转运体表达的影响[J].中国药学杂志,2018,43(15):3135.
[19]袁紫朔,张婷婷,金波,等.异丹叶大黄素在Caco-2细胞模型上转运机制的研究[J].中国中药杂志,2017,42(3):587.
[20]曾宝,吴安国,陈建南,等.HPLC考察和厚朴酚在Caco-2细胞模型的转运特征[J].中国中药杂志,2011,36(10):1286.
[21]何丹,颜苗,李焕德,等.甘草提取物及其主要成分对Caco-2细胞膜上P-gp功能和表达的影响[J].中国药学杂志,2010,54(10):751.