基于热力学表达的黄连炮制与配伍变化的生物活性差异及化学表征研究
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摘要
中药药性理论是中药研究与应用的基础,也是当今中医药研究的热点和难点之一。作为可表征中药生物活性之一的药性理论是中药的基础与核心,传统炮制和复方配伍可不同程度地改变中药药性,从而改变中药的生物活性,这也是中医用药的两大特色。为了客观地评价中药炮制和配伍对中药活性/药性的改变,探明其作用机理及其可能的物质基础,本论文以临床常用、在药性表达上有代表性意义的中药黄连炮制品及其配伍类方为研究对象,应用生物热力学方法,并结合常规药理和化学分析法,对黄连炮制与配伍变化的生物活性差异及化学表征进行了较系统的研究。主要研究结果如下:
1.采用生物热力学方法,定性定量地刻画了黄连、黄连不同炮制品(醋制黄连、胆汁制黄连、盐制黄连、吴萸黄连、姜制黄连、酒制黄连)及其与吴茱萸配伍系列经典方(左金丸、甘露散、茱萸丸、反左金丸)对大肠杆菌、金黄色葡萄球菌及痢疾杆菌等微生物生长代谢的作用。研究结果表明:黄连不同炮制品和不同配伍复方,寒热药性不同,其作用于细菌生长代谢的热谱及热动力学参数有不同程度的改变,具体表现为细菌在寒凉药作用下的生长速率常数k减小,且代谢产热量Q也趋于下降,这与传统中医对方药的赋性基本吻合。此外,首次用生物热力学方法研究了黄连的主要活性部位——生物碱成分的的抑菌活性,结果显示,小檗碱的抑制活性最强。
2.采用常规药理实验方法,分别考察了黄连不同炮制品和不同配伍类方的抑菌和解热作用及其差异。常规药理试验也支持了生物热力学法试验所得结果,提示生物热力学法可作为抗菌活性筛选方法之一。
3.采用高效液相色谱法,同时测定了黄连不同炮制品中小檗碱、巴马汀、黄连碱、表小檗碱、药根碱五种生物碱成分的含量;还同时测定了黄连与吴茱萸配伍系列经典类方中小檗碱、巴马汀、黄连碱、表小檗碱、药根碱及吴茱萸次碱的含量;最后,分别建立了黄连不同炮制品和不同配伍类方的HPLC指纹图谱。
4.采用多元统计分析方法,综合分析了黄连不同炮制品和不同配伍经典方的生物热谱图与常规药理活性、化学组分之间的相关性,结果表明:黄连不同样品作用于细菌的热力学结果与常规药理实验得到的最小抑菌浓度(MIC)相关性非常显著,相关系数所对应的P值小于0.0001;黄连不同炮制品作用于细菌的热动力学参数与其所含的的小檗碱、巴马汀及黄连碱的含量典型相关;而黄连与吴茱萸配伍系列经典类方作用于细菌的热动力学参数与其所含的小檗碱和药根碱的含量典型相关,这可能是黄连经炮制与配伍后,体现其生物活性差异的主要物质基础之所在。提示生物热力学方法可作为评价中药活性/药性的新的有效手段,且该方法具有普适性好、实时、在线、微量、高效等特点。
本文初步阐明了中药黄连炮制与配伍变化的生物活性差异及其可能的物质基础,为探讨炮制机理和配伍规律提供了理论依据。本文所建立的方法可望在中医药现代化研究特别是中药四性、中医治则、复方配伍、品质评价、药效物质筛选和新药开发等方面发挥独到的作用。
Theory of property of Traditional Chinese Medicine(TCM) is the foundation of research and application of TCM,and be also considered as focus and bottlenecks in TCM modernization.Property that is the core of TCM can explain bioactivity of TCD clearly.The traditional preparation and compatibility can change the property of TCD in different degree,while it change the bioactivity of TCD.In order to evaluate the effect of preparation and compatibility on bioactivity/property of TCD and discover its mechanism and possible material basis,based on thermodynamic theory, associated with pharmacological and chemical method,the bioactive difference and active essences in different processed products of Rhizoma Coptidis and complex prescription combined Rhizoma Coptidis with Fructus Evodiae were studied in this dissertation.The main results of research were as follows:
1.The action of different processed products of Rhizoma Coptidis (Cuzhihuanglian,Danzhihuanglian,Yanzhihuanglian,Wuyuhuanglian, Jiangzhihuanglian and Jiuzhihuanglian) and the complex prescription combined Rhizoma Coptidis with Fructus Evodiae(Zuojinwan,Ganlusan,Zhuyuwan, Fanzuojinwan) on three bacteria(Escherichia coli,Staphylococcus aureus,Shigella dysenteriae) was investigated with biothermodynamics in this research.All the samples could restrain the bacteria growth and metabolism in different levels. According to thermokinetic equation,the rate constant k and the heat output were showed quantitatively.The results showed that all the samples could restrain the growth of bacteria and the biothermokinetic parameters were changed in different levels.Drags with cool property could make the organisms decreasing the rate constant k and decreasing heat output(Q) remarkably,while drugs with warm property could make the organisms increasing the rate constant k and increasing heat output(Q) a little.There was a stable difference between them.Additionally,the inhibitory effect of alkaloids of Rhizoma Coptidis on Shigella dysenteriae was quantitatively investigated by microcalorimetry.The experimental results indicated that the sequence of anti-microbial activities of the three alkaloids was:berberine＞coptisine＞palmatine.All the results above-mentioned were matched with the conclusions reported in literatures.
2.Using routine pharmacological method,the effect of inhibiting bacteria and eliminating the heat syndrome of different processed products of Rhizoma Coptidis and the complex prescription combined Rhizoma Coptidis with Fructus Evodiae was studied.The results were consistent with the above results of biothermodynamics.So, the thermodynamic method could be used as an antibiotic active screening method in TCD study.
3.By HPLC,five kinds of berberine type alkaloids:berberine,palmatine, coptisine,epiberberine and jatrorrhizine in different processed products of Coptidis Rhizoma can be separated completely,and the contents of alkaloids were determined in this paper.In the meantime,the contents of six main alkaloids including berberine, palmatine,coptisine,epiberberine,jatrorrhizine and rutaecarpine of different complex prescription were determined.What's more,HPLC fingerprints of different samples of Coptidis Rhizoma were established with different HPLC method.
4.Using statistical method,the relation among threee experimental methods (Biothermodynamics,chemical analysis and routine pharmacology) was analyzed in this study.The correlation analysis of P-t curve of different processed products of Coptidis Rhizoma and HPLC plots of five alkaloids indicated the growth rate constant k of bacteria inhibited by preparation was interrelated with contents of berberine, palmatine and coptisine.While the correlation analysis of P-t curve of different complex prescription of Coptidis Rhizoma and HPLC plots of the main alkaloids indicated the growth rate constant k of bacteria inhibited was interrelated with the contents of berberine and jatrorrhizine.Both of which could be regarded as the main active essences.The results showed that biothermodynamics,whose characteristics are micromount,high effective,real-time and so on,could be an effective method to evaluate the bioactivity of TCD.
In this dissertation,the differences of bioactivity of preparation and compatibility of Coptidis Rhizoma were explained.The method combined with TCM theory and biothermodynamic theory could provide new method for Four Property evaluation, treatment principle,quality evaluation,effective substances screening and research and development of new drug.
1.采用生物热力学方法,定性定量地刻画了黄连、黄连不同炮制品(醋制黄连、胆汁制黄连、盐制黄连、吴萸黄连、姜制黄连、酒制黄连)及其与吴茱萸配伍系列经典方(左金丸、甘露散、茱萸丸、反左金丸)对大肠杆菌、金黄色葡萄球菌及痢疾杆菌等微生物生长代谢的作用。研究结果表明:黄连不同炮制品和不同配伍复方,寒热药性不同,其作用于细菌生长代谢的热谱及热动力学参数有不同程度的改变,具体表现为细菌在寒凉药作用下的生长速率常数k减小,且代谢产热量Q也趋于下降,这与传统中医对方药的赋性基本吻合。此外,首次用生物热力学方法研究了黄连的主要活性部位——生物碱成分的的抑菌活性,结果显示,小檗碱的抑制活性最强。
2.采用常规药理实验方法,分别考察了黄连不同炮制品和不同配伍类方的抑菌和解热作用及其差异。常规药理试验也支持了生物热力学法试验所得结果,提示生物热力学法可作为抗菌活性筛选方法之一。
3.采用高效液相色谱法,同时测定了黄连不同炮制品中小檗碱、巴马汀、黄连碱、表小檗碱、药根碱五种生物碱成分的含量;还同时测定了黄连与吴茱萸配伍系列经典类方中小檗碱、巴马汀、黄连碱、表小檗碱、药根碱及吴茱萸次碱的含量;最后,分别建立了黄连不同炮制品和不同配伍类方的HPLC指纹图谱。
4.采用多元统计分析方法,综合分析了黄连不同炮制品和不同配伍经典方的生物热谱图与常规药理活性、化学组分之间的相关性,结果表明:黄连不同样品作用于细菌的热力学结果与常规药理实验得到的最小抑菌浓度(MIC)相关性非常显著,相关系数所对应的P值小于0.0001;黄连不同炮制品作用于细菌的热动力学参数与其所含的的小檗碱、巴马汀及黄连碱的含量典型相关;而黄连与吴茱萸配伍系列经典类方作用于细菌的热动力学参数与其所含的小檗碱和药根碱的含量典型相关,这可能是黄连经炮制与配伍后,体现其生物活性差异的主要物质基础之所在。提示生物热力学方法可作为评价中药活性/药性的新的有效手段,且该方法具有普适性好、实时、在线、微量、高效等特点。
本文初步阐明了中药黄连炮制与配伍变化的生物活性差异及其可能的物质基础,为探讨炮制机理和配伍规律提供了理论依据。本文所建立的方法可望在中医药现代化研究特别是中药四性、中医治则、复方配伍、品质评价、药效物质筛选和新药开发等方面发挥独到的作用。
Theory of property of Traditional Chinese Medicine(TCM) is the foundation of research and application of TCM,and be also considered as focus and bottlenecks in TCM modernization.Property that is the core of TCM can explain bioactivity of TCD clearly.The traditional preparation and compatibility can change the property of TCD in different degree,while it change the bioactivity of TCD.In order to evaluate the effect of preparation and compatibility on bioactivity/property of TCD and discover its mechanism and possible material basis,based on thermodynamic theory, associated with pharmacological and chemical method,the bioactive difference and active essences in different processed products of Rhizoma Coptidis and complex prescription combined Rhizoma Coptidis with Fructus Evodiae were studied in this dissertation.The main results of research were as follows:
1.The action of different processed products of Rhizoma Coptidis (Cuzhihuanglian,Danzhihuanglian,Yanzhihuanglian,Wuyuhuanglian, Jiangzhihuanglian and Jiuzhihuanglian) and the complex prescription combined Rhizoma Coptidis with Fructus Evodiae(Zuojinwan,Ganlusan,Zhuyuwan, Fanzuojinwan) on three bacteria(Escherichia coli,Staphylococcus aureus,Shigella dysenteriae) was investigated with biothermodynamics in this research.All the samples could restrain the bacteria growth and metabolism in different levels. According to thermokinetic equation,the rate constant k and the heat output were showed quantitatively.The results showed that all the samples could restrain the growth of bacteria and the biothermokinetic parameters were changed in different levels.Drags with cool property could make the organisms decreasing the rate constant k and decreasing heat output(Q) remarkably,while drugs with warm property could make the organisms increasing the rate constant k and increasing heat output(Q) a little.There was a stable difference between them.Additionally,the inhibitory effect of alkaloids of Rhizoma Coptidis on Shigella dysenteriae was quantitatively investigated by microcalorimetry.The experimental results indicated that the sequence of anti-microbial activities of the three alkaloids was:berberine＞coptisine＞palmatine.All the results above-mentioned were matched with the conclusions reported in literatures.
2.Using routine pharmacological method,the effect of inhibiting bacteria and eliminating the heat syndrome of different processed products of Rhizoma Coptidis and the complex prescription combined Rhizoma Coptidis with Fructus Evodiae was studied.The results were consistent with the above results of biothermodynamics.So, the thermodynamic method could be used as an antibiotic active screening method in TCD study.
3.By HPLC,five kinds of berberine type alkaloids:berberine,palmatine, coptisine,epiberberine and jatrorrhizine in different processed products of Coptidis Rhizoma can be separated completely,and the contents of alkaloids were determined in this paper.In the meantime,the contents of six main alkaloids including berberine, palmatine,coptisine,epiberberine,jatrorrhizine and rutaecarpine of different complex prescription were determined.What's more,HPLC fingerprints of different samples of Coptidis Rhizoma were established with different HPLC method.
4.Using statistical method,the relation among threee experimental methods (Biothermodynamics,chemical analysis and routine pharmacology) was analyzed in this study.The correlation analysis of P-t curve of different processed products of Coptidis Rhizoma and HPLC plots of five alkaloids indicated the growth rate constant k of bacteria inhibited by preparation was interrelated with contents of berberine, palmatine and coptisine.While the correlation analysis of P-t curve of different complex prescription of Coptidis Rhizoma and HPLC plots of the main alkaloids indicated the growth rate constant k of bacteria inhibited was interrelated with the contents of berberine and jatrorrhizine.Both of which could be regarded as the main active essences.The results showed that biothermodynamics,whose characteristics are micromount,high effective,real-time and so on,could be an effective method to evaluate the bioactivity of TCD.
In this dissertation,the differences of bioactivity of preparation and compatibility of Coptidis Rhizoma were explained.The method combined with TCM theory and biothermodynamic theory could provide new method for Four Property evaluation, treatment principle,quality evaluation,effective substances screening and research and development of new drug.
引文
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