【作者】 王鹏；

【导师】 李秋红；

【作者基本信息】 黑龙江中医药大学 ， 药剂学， 2016， 硕士

【摘要】 研究目的:以CYP450酶介导的代谢性相互作用作为理论依据,研究大黄附子汤中附子配伍减毒规律,进而阐明中药复方大黄附子汤的配伍规律。研究方法:将大黄附子汤拆方分为8组:大黄组、附子组、细辛组、大黄+附子组、大黄+细辛组、附子+细辛组、大黄附子汤全方组以及空白对照组。采用高效液相色谱法建立同时测定肝微粒体中特异性探针药物咖啡因(CYP1A2酶底物)和咪达唑仑(CYP3A4酶底物)的检测方法。借鉴氯化钙沉淀法用于制备肝微粒体,并应用BCA法测定肝微粒体中蛋白含量。实验采用Cocktail探针药物体外孵育法测定CYP1A2和CYP3A4酶活性。利用一氧化碳示差法测定肝微粒体中CYP450酶总含量。采用RT-PCR检测CYP1A2和CYP3A4酶mRNA表达调控。研究结果:①建立同时测定肝微粒体中探针底物咖啡因和咪达唑仑的高效液相方法色谱柱:Topsil.TM.C1.分析柱(250mm×4.6mm,5μm),C18.预.柱(10mm×4.6mm);流动相:甲醇和磷酸氢二胺缓冲溶液以51:49(V/V)混合;流速:0.8ml/min;检测波长:254nm;柱温:35℃;进样量:20μL。该方法的专一性、灵敏度、精确度、稳定性等均符合方法学检测要求。②大黄附子汤全方及拆方对CYP1A2和CYP3A4酶活性的影响全方或拆方对CYP1A2活性的影响:实验结果表明大黄、附子+大黄、大黄+细辛组对CYP1A2酶活性具有显著的诱导作用(p<0.05,p<0.05,p<0.05)。附子+细辛组对CYP1A2酶活性具有显著的抑制作用(p<0.05),大黄附子汤全方组对CYP1A2酶活性具有轻微的诱导作用,但无显著性差异。附子组、细辛组对CYP1A2酶活性无影响。全方或拆方对CYP3A4酶活性的影响:实验结果表明细辛组,附子+细辛组对CYP3A4酶活性具有显著的抑制作用(p<0.05,p<0.05)。大黄组、大黄+细辛组以及大黄附子全方对CYP3A4酶活性具有显著的诱导作用(p<0.01,p<0.05,p<0.05),附子组、附子+大黄组对CYP3A4酶活性无影响。③大黄附子汤全方及拆方对CYP450酶总含量以及mRNA基因表达的影响全方或拆方对CYP450酶总含量的影响:结果表明大黄组显著诱导P450酶总含量(p<0.05),细辛组和附子+细辛组抑制CYP450酶总含量(p<0.05,p<0.05,P<0.01),附子+大黄组,大黄+细辛组,大黄附子汤对CYP450总含量有轻微的诱导作用,但无显著性差异。全方或拆方对CYP1A2和CYP3A4酶mRNA基因表达调控的影响:结果显示大黄组、大黄+细辛组、大黄附子汤组可以上调CYP1A2酶mRNA表达(P<0.05;P<0.05;P<0.05)。其余各组对CYP1A2酶mRNA表达无影响。另外,大黄附子汤全方、大黄组可以上调CYP3A4酶的mRNA表达(P<0.05,P<0.05)。细辛组和附子+细辛组对CYP3A4酶的mRNA表达具有下调作用(P<0.05,P<0.05)。大黄+细辛组、附子组和附子+大黄组对CYP3A4酶mRNA基因表达无影响。结论:由于CYP1A2和CYP3A4酶是附子中毒性成分乌头碱的主要代谢酶,大黄对于全方诱导CYP1A2和CYP3A4酶具有突出的贡献。而此贡献对于降低全方中附子毒性成分乌头碱具有至关重要的作用。这体现了大黄在全方中君药的主导地位。另外,中药复方组合弱化了大黄单独用药对CYP1A2和CYP3A4酶的强诱导作用,体现了中药复方的整体观念。大黄附子汤对于CYP3A4酶活性的影响很可能通过CYP3A4酶mRNA水平来调控。基于药性理论的寒热配伍作用与CYP450酶的诱导或抑制作用之间是否具有相关性还需进一步研究。更多还原

【Abstract】 Purpose:The study was based on the theory of metabolic drug interactions base on cytochrome P450,was to research the mechanism that Fuzi Toxicity reducing and then to clarify law of compatibility of medicines in Dahuang-fuzi-tang prescriptions.Methods:Rats were randomly divided into eight groups:da-hang group(DH);fu-zi group(FZ);xi-xin group(XX);da-hang+fu-zi group(DHFZ);da-hang+xi-xin group(DHXX);fu-zi+xi-xin group(FZXX);Dahuang-fuzi-tang group(DHFZT)and blank control group.development and utilization the HPLC method for simultaneous determination the concentrations of caffeine(CYP1A2 enzyme substrates)and midazolam(CYP3A4 enzyme substrates)in liver microsomes.Using calcium chloride precipitation method to obtain liver microsomes.The concentration of protein in liver microsomes was determined with a commercially available kit.using BSA as a standard.The CYP1A2和CYP3A4 enzyme activity was quantified by Cocktail method in vitro,The CYP450 enzyme total content was measured by carbon monoxide-binding difference spectra analysis.Meanwhile,the mRNA expression of CYP1A2 and CYP3A4 in the liver was detected by reverse transcription PCR(RT-PCR).Results:①Simultaneous determination of caffeine and midazolam by HPLC.All samples(20μL)were separated on a Diamonsil C18 reversed-phase column(150mm×4.6mm,5 mm)by HPLC system.The mobile phase consisted of methyl alcohol and Diammonium phosphate buffer solution(51:49 V/V)at a flow rate was 0.8 mL/min.The separation was carried out at 35℃.UV detection wavelength was 254nm.Specificity,sensitivity,accuracy and stability of the method met the requirements of biological sample measurement.②Effect of different groups on CYP1A2 and CYP3A4 enzyme activity DH、DHFZ、DHXX could induce the enzyme activity of CYP1A2 significantly(p<0.05,p<0.05,p<0.05).FZXX could inhibit the enzyme activity significantly(p<0.05).DHFZT could induce enzyme activity of CYP1A2 slightly,but the effects was no statistically significant.FZ and XX showed no effects on enzyme activity of CYP1A2.XX and FZXX could inhibit the enzyme activity of CYP3A4(p<0.05,p<0.05).DH、DHXX and DHFZT could induce the enzyme activity of CYP3A4 significantly(p<0.01,p<0.05,p<0.05).FZ and DHFZ showed no effects on the enzyme activity of CYP3A4.③Effect of different groups on CYP1A2 and CYP3A4 mRNA Expression in the LiverDH could induce the total CYP450 enzyme contents significantly(p<0.05),while XX and FZXX could inhibit the total CYP450 enzyme contents(p<0.05,P<0.01).DHFZ、DHXX and DHFZT could induce the total CYP450 enzyme contents slightly,but the effects was no statistically significant.Compared to the control,DH、DHXX and DHFZT could raise the hepatic mRNA levels of CYP1A2 expression(P<0.05;P<0.05;P<0.05),when DHFI and FZXX showed no effects on it.In addition,DH and DHFZT increased the hepatic mRNA levels of CYP3A4 expression(P<0.05,P<0.05).However,XX and FZXX decreased the hepatic mRNA levels of CYP3A4 expression(P<0.05,P<0.05).The others showed no effects on the hepatic mRNA levels of CYP3A4 expression.Conclusions:CYP1A2 and CYP3A4 were mainly involved in the metabolic of aconitine which is the main toxic ingredients in FZ.The results from the present study suggested that DH make a contribution to induction of CYP1A2 and CYP3A4 in DHFZT.This could reduce toxicity of DHFZT.it shows that DH as monarch drug in a prescription was importance.in addition,dahaungfuzitang prescription could weaken the strong induction of CYP1A2 and CYP3A4 caused by DH.It suggested that comprehensive therapeutic effect of traditional Chinese medicine.However,further research is needed to understand the relationship between properties theory of Chinese medicinal Herbs and CYP450 enzyme.更多还原