大承气汤方药物质基础及其配伍规律研究
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摘要
大承气汤出自张仲景《伤寒论》,为泻下的代表方剂,本研究对其提取工艺、配伍前后化学成分和药效的变化规律、物质基础以及剂量配伍进行研究。
(1)本实验首先建立了大黄蒽醌类成分(芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚)HPLC含量测定方法,枳实黄酮类成分(柚皮苷、橙皮苷和新橙皮苷)HPLC含量测定方法以及厚朴类成分(和厚朴酚、厚朴酚)HPLC含量测定方法。所建立的方法简便、快速、数据真实、可靠、准确。在单因素考察试验的基础上,采用星点设计-响应面法,分别以大黄蒽醌类、枳实黄酮类、厚朴类成分为考察指标,对大承气汤的提取工艺进行优化。由实验数据所绘制的曲面图可以看出,乙醇体积分数、提取时间对考察指标的影响较大,而溶媒比对考察指标的影响相对较小。最终确定最佳工艺为60%乙醇、提取时间为90min,溶媒比为20倍量。
(2)考察了大承气汤中单味药以及复方配伍后的化学成分变化,对大承气汤复方配伍的化学物质基础进行研究。针对中药化学成分的复杂性,对色谱流动相、比例,检测波长,色谱柱温度,进样量等色谱条件进行优化,建立了全方HPLC统一分析方法,并对分析方法做了仪器精密度、样品稳定性和方法重复性等考察。结果表明所建立的方法稳定、可靠、重复性好。统一的HPLC分析方法的建立是进行复方配伍前后成分变化研究的基础。利用在该统一HPLC分析方法下得到的色谱图的保留时间对大承气汤中49个峰的来源进行归属,并对配伍前后化学成分含量的变化进行了研究。结果说明:大承气汤配伍中有12个峰来自枳实,12个峰来自厚朴,17个峰来自大黄,1个峰是大黄、厚朴和枳实的共流出峰,5个峰是大黄、枳实的共流出峰,2个峰是大黄、厚朴的共流出峰,在目前的研究条件下考察,没有新峰产生。对全方配伍前后化学成分含量的变化,有24个峰的峰面积增加,8个峰的峰面积降低,这些含量发生变化的峰所对应的成分,可能就是大承气汤配伍作用的化学物质基础。全方配伍后柚皮苷、新橙皮苷、和厚朴酚、厚朴酚的峰面积没有变化,大黄素甲醚的峰面积降低,而橙皮苷、芦荟大黄素、大黄酸、大黄素、大黄酚的峰面积都是增加的,说明了大承气汤四味药材合煎后,能显著增加有效成分的溶出率,达到增强药效的结果。
(3)将大承气汤中的四味药,以君药大黄为核心,依次配伍其它两味中药,进行拆方药对设计,以及不同煎煮方法的实验,通过测定不同药对配伍,不同煎煮方法后芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚、柚皮苷、橙皮苷、新橙皮苷、厚朴酚、和厚朴酚的含量变化,对药对配伍方法有效成分变化规律以及全方配伍中不同煎煮方法有效成分变化规律进行研究。结果表明,不同药对配伍,以及不同煎煮方法对厚朴类成分和枳实黄酮类成分溶出的影响不大。但芒硝、大黄后下,在煎煮过程中可以提高蒽醌类成分的溶出率;厚朴、芒硝、枳实与大黄进行配伍,也能显著增加大黄中蒽醌类成分的溶出率。这说明了大承气汤传统煎法要求“先煎枳、朴,后下大黄,纳芒硝,溶化服”,其科学性就在于有效地提高主要有效成分的溶存量而为其药效作用提供物质基础,从而可以达到增强药效的目的。
(4)实验利用黑色墨汁作为指示剂,观察墨汁在肠道的推进距离,对大承气汤泻下作用的配伍机制研究,同时,选用家兔离体肠,考察各给药组对肠平滑肌收缩的影响情况。本实验中,相对空白组,大承气汤各不同配伍组对小鼠小肠的推进率均较高,对家兔肠管张力、兔肠管收缩频率均有显著性差异或非常显著性差异(P＜0.01,P＜0.05)。大黄+芒硝组的推进率、对家兔肠管张力、兔肠管收缩频率较高,说明了芒硝的芒硝与大黄配伍,有较强的泻下作用。经典煎煮方法的大承气汤,泻下作用最强。
(5)研究了复方大承气汤的化学成分相应色谱峰与药效物质基础,对49个色谱峰与药效进行相关性分析,其中11个色谱峰与药理效应Y_1(小鼠小肠碳末推进率)有显著性(P＜0.05)或非常显著性(P＜0.01);3个色谱峰与药理效应Y_3显著性差异(P＜0.05),2号峰为负相关。从实验的相关分析结果来看,可以说明厚朴酚、芦荟大黄素、大黄酸、大黄素、大黄酚等化学成分为大承气汤发挥药效作用的物质基础,也说明了这些化学成分作为质量控制和工艺优化指标的合理性和科学性。
(6)应用Doehlert设计-渴求函数.响应曲面优化法,研究枳实、厚朴和芒硝的剂量配伍变化对大黄蒽醌类成分在整方中溶出的影响。通过对3个自变量(枳实,厚朴,芒硝)进行配伍试验及其结果预测,优化二项式方程的变量值,得到具有良好拟合性的模型,自变量的变化与指标性成分之间的相关分析说明3个自变量(枳实,厚朴,芒硝)对大黄蒽醌成分的提取百分含量有较高的显著性影响,特别是厚朴。以得到的方程为分析模型,可以得出当剂量配比为大黄:枳实:厚朴:芒硝(1∶4∶2.31∶2)时,D有预测的最大值0.85。
本研究在中医药学理论和实践的指导下将中药学、分析化学、药理学、统计学和计算机技术相结合,优化了大承气汤的提取工艺,探讨了其复方的配伍机制、药效物质基础,为大承气汤方药配伍及中药复方配伍的方法学研究提供了理论依据。
Dachangqi decoction,which derived from Treatise on FebrHe Disease,is a famous prescription that has been used to treat constipation over a very long period of time and proved quite a good effect.The extracted technology, chemical components and pharmacological activities and the compatibility of dosage change of Dachengqi decoction was study on this research
The HPLC contents determination method of Anthraquinone Components (Aloe-emodin,Rhein,Emodin,Chrysophanol,Physcion),Flavonoids Components (Naringin,Hesperidin,Neohesperidin),Phenols Components(Honokiol, Magnolol) were establish,these method are simple,reliable,accurate.The traditional 'one - factor at a time' technique was used to identify the level and extent of all 3 factors of ethanol concentration,reflux time,and solvent ratio,then a central composite design-response surface methodology was used to optimize the extracting process for Dachengqi decoction,a two-order polynomial equation was fitted to the data and resultant model was used to predict the response in the optimal region.Analytical results indicated the optimum conditions were 60%ethanol,90 min for once reflux,20-fold solvent for extraction.It shows that the optimum process is simple and more convenient for extracting Anthraquinone,Flavonoids and Phenols components with an higher yields.
A stable,reliable,uniform HPLC analytical method with good reproducibility was developed to study the changes of chemical ingredients between single herbs of Dahuang,Houpo,Zhishi,Mangxiao and the compound recipe of Dachengqi decoction.The results indicated that in the 49 peaks of Dachengqi decoction,there are 12 peaks contributed from Zhishi,12 peaks contributed from Houpo and 17 peaks contributed from Dahuang,but no new peaks produced.Comparing Dachengqi decoction with single herbs of Dahuang,Houpo and Zhishi,the areas of 24 peaks are increased while the areas of 8 peaks are decreased.The peaks areas of Naringin,Hesperidin,Neohesperidin, Honokiol and Magnolol didn't increase,but yields of Aloe-emodin,Rhein, Emodin,Chrysophanol were increased when four herbs decoct together,which also indicated that traditional decoction method of Dachengqi decoction was increase the Anthraquinone components of the medicine so as to enhance the effect to treat constipation.
The quantitative contents of Aloe-emodin,Rhein,Emodin,Chrysophanol, Physcion,Naringin,Hesperidin,Neohesperidin,Honokiol and Magnolol were detested by HPLC in different compatibility of Dachengqi decoction as to study the active ingredient changed regularity of Chinese material medicine.The results show that Flavonoids and Phenols Components were hardly influence by different compatibility of Dachangqi decoction,but the contents of Anthraquinone components could become higher when Mangxiao and Dahuang were decocted later,and Houpo,Mangxiao,Zhisbi were compatible with Dahuang together can also improved the yields of Anthraquinone components,which proved that traditional decoction method of Dachengqi decoction was reasonable for increase the Anthraquinone components of the medicine as to enhance the effect to treat constipation.
The method of charcoal powder propelling ratio was used to investigate the effects of Dachengqi decoction on the intestinal propulsive movements in mice,and observe the influence of Dachengqi decoction on the intestinal smooth muscle of the experimental rabbits.The results show that,compare the blank group,the compatibility of different groups all show the Correlation were significant at the 0.05 level or 0.01 for charcoal powder propelling ratio, tonicity and contraction of the rabbits' s isolated intestinal muscle.The groups of Dahuang combine with Mangxiao hava a good pharmacological activities indicate that Dahuang and Mangxiao have a sronger effect to treat constipation, as well as the group of tradictional method.
The correlation between chemical components and pharmacological activities was carries on in order to study the therapeutic basis of Dachengqi decoction.49 peaks correspond to pharmacological activities were analysis by bivariate with the software of SPSS 16.0 for windows,there 11 peaks correlated to the pharmacological activity of Y_1(charcoal powder propelling ratio) with Correlation is significant at the 0.05 level or 1.01 level;3 peaks correlated to the pharmacological activity of Y_3(contracted frequency of isolated intestinal muscle) with Correlation is significant at the 0.05 level. The results indicated that Aloe-emodin,Rhein,Emodin,Chrysophanol are the therapeutic basis of Dachengqi decoction,and also explain the rationality and mechanism that these chemical components are used as to control the quality of herbs and recipe.
Response surface methodology(RSM) involving Doehlert design was adopted to evaluate the yields of Anthraquinone components in Dachengqi decoction by change of dosage of Zhishi,Houpo and Mangxiao and shorten analysis time through a desirability function.The results of this research show that Anthraquinone components were got a high yields of predicable value of 0.85 when the dosage ratio of Dachengqi decoction were compatible as follows: Dahuang:Zhishi:Houpo:Mangxiao(1:4:2.31:2).The Doehlert design and desirability function,which allow a sequential response methodology,are a good methods,and,of cause,the mathematical model can be further popular and apply for the compatibility research of Chinese material medicine.
This research was conduct under the theory of traditional Chinese medicine incorporated with science of Chinese materia medica,analytical chemistry, physics,statistics and technology of computer.In this research,the extracted technology were optimize by CCD,probe into the mechanism of compatibility,therapeutic basis of Dachengqi decoction,which provided a significative exploration for the modernization of compatibility of traditional Chinese medicine.
(1)本实验首先建立了大黄蒽醌类成分(芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚)HPLC含量测定方法,枳实黄酮类成分(柚皮苷、橙皮苷和新橙皮苷)HPLC含量测定方法以及厚朴类成分(和厚朴酚、厚朴酚)HPLC含量测定方法。所建立的方法简便、快速、数据真实、可靠、准确。在单因素考察试验的基础上,采用星点设计-响应面法,分别以大黄蒽醌类、枳实黄酮类、厚朴类成分为考察指标,对大承气汤的提取工艺进行优化。由实验数据所绘制的曲面图可以看出,乙醇体积分数、提取时间对考察指标的影响较大,而溶媒比对考察指标的影响相对较小。最终确定最佳工艺为60%乙醇、提取时间为90min,溶媒比为20倍量。
(2)考察了大承气汤中单味药以及复方配伍后的化学成分变化,对大承气汤复方配伍的化学物质基础进行研究。针对中药化学成分的复杂性,对色谱流动相、比例,检测波长,色谱柱温度,进样量等色谱条件进行优化,建立了全方HPLC统一分析方法,并对分析方法做了仪器精密度、样品稳定性和方法重复性等考察。结果表明所建立的方法稳定、可靠、重复性好。统一的HPLC分析方法的建立是进行复方配伍前后成分变化研究的基础。利用在该统一HPLC分析方法下得到的色谱图的保留时间对大承气汤中49个峰的来源进行归属,并对配伍前后化学成分含量的变化进行了研究。结果说明:大承气汤配伍中有12个峰来自枳实,12个峰来自厚朴,17个峰来自大黄,1个峰是大黄、厚朴和枳实的共流出峰,5个峰是大黄、枳实的共流出峰,2个峰是大黄、厚朴的共流出峰,在目前的研究条件下考察,没有新峰产生。对全方配伍前后化学成分含量的变化,有24个峰的峰面积增加,8个峰的峰面积降低,这些含量发生变化的峰所对应的成分,可能就是大承气汤配伍作用的化学物质基础。全方配伍后柚皮苷、新橙皮苷、和厚朴酚、厚朴酚的峰面积没有变化,大黄素甲醚的峰面积降低,而橙皮苷、芦荟大黄素、大黄酸、大黄素、大黄酚的峰面积都是增加的,说明了大承气汤四味药材合煎后,能显著增加有效成分的溶出率,达到增强药效的结果。
(3)将大承气汤中的四味药,以君药大黄为核心,依次配伍其它两味中药,进行拆方药对设计,以及不同煎煮方法的实验,通过测定不同药对配伍,不同煎煮方法后芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚、柚皮苷、橙皮苷、新橙皮苷、厚朴酚、和厚朴酚的含量变化,对药对配伍方法有效成分变化规律以及全方配伍中不同煎煮方法有效成分变化规律进行研究。结果表明,不同药对配伍,以及不同煎煮方法对厚朴类成分和枳实黄酮类成分溶出的影响不大。但芒硝、大黄后下,在煎煮过程中可以提高蒽醌类成分的溶出率;厚朴、芒硝、枳实与大黄进行配伍,也能显著增加大黄中蒽醌类成分的溶出率。这说明了大承气汤传统煎法要求“先煎枳、朴,后下大黄,纳芒硝,溶化服”,其科学性就在于有效地提高主要有效成分的溶存量而为其药效作用提供物质基础,从而可以达到增强药效的目的。
(4)实验利用黑色墨汁作为指示剂,观察墨汁在肠道的推进距离,对大承气汤泻下作用的配伍机制研究,同时,选用家兔离体肠,考察各给药组对肠平滑肌收缩的影响情况。本实验中,相对空白组,大承气汤各不同配伍组对小鼠小肠的推进率均较高,对家兔肠管张力、兔肠管收缩频率均有显著性差异或非常显著性差异(P＜0.01,P＜0.05)。大黄+芒硝组的推进率、对家兔肠管张力、兔肠管收缩频率较高,说明了芒硝的芒硝与大黄配伍,有较强的泻下作用。经典煎煮方法的大承气汤,泻下作用最强。
(5)研究了复方大承气汤的化学成分相应色谱峰与药效物质基础,对49个色谱峰与药效进行相关性分析,其中11个色谱峰与药理效应Y_1(小鼠小肠碳末推进率)有显著性(P＜0.05)或非常显著性(P＜0.01);3个色谱峰与药理效应Y_3显著性差异(P＜0.05),2号峰为负相关。从实验的相关分析结果来看,可以说明厚朴酚、芦荟大黄素、大黄酸、大黄素、大黄酚等化学成分为大承气汤发挥药效作用的物质基础,也说明了这些化学成分作为质量控制和工艺优化指标的合理性和科学性。
(6)应用Doehlert设计-渴求函数.响应曲面优化法,研究枳实、厚朴和芒硝的剂量配伍变化对大黄蒽醌类成分在整方中溶出的影响。通过对3个自变量(枳实,厚朴,芒硝)进行配伍试验及其结果预测,优化二项式方程的变量值,得到具有良好拟合性的模型,自变量的变化与指标性成分之间的相关分析说明3个自变量(枳实,厚朴,芒硝)对大黄蒽醌成分的提取百分含量有较高的显著性影响,特别是厚朴。以得到的方程为分析模型,可以得出当剂量配比为大黄:枳实:厚朴:芒硝(1∶4∶2.31∶2)时,D有预测的最大值0.85。
本研究在中医药学理论和实践的指导下将中药学、分析化学、药理学、统计学和计算机技术相结合,优化了大承气汤的提取工艺,探讨了其复方的配伍机制、药效物质基础,为大承气汤方药配伍及中药复方配伍的方法学研究提供了理论依据。
Dachangqi decoction,which derived from Treatise on FebrHe Disease,is a famous prescription that has been used to treat constipation over a very long period of time and proved quite a good effect.The extracted technology, chemical components and pharmacological activities and the compatibility of dosage change of Dachengqi decoction was study on this research
The HPLC contents determination method of Anthraquinone Components (Aloe-emodin,Rhein,Emodin,Chrysophanol,Physcion),Flavonoids Components (Naringin,Hesperidin,Neohesperidin),Phenols Components(Honokiol, Magnolol) were establish,these method are simple,reliable,accurate.The traditional 'one - factor at a time' technique was used to identify the level and extent of all 3 factors of ethanol concentration,reflux time,and solvent ratio,then a central composite design-response surface methodology was used to optimize the extracting process for Dachengqi decoction,a two-order polynomial equation was fitted to the data and resultant model was used to predict the response in the optimal region.Analytical results indicated the optimum conditions were 60%ethanol,90 min for once reflux,20-fold solvent for extraction.It shows that the optimum process is simple and more convenient for extracting Anthraquinone,Flavonoids and Phenols components with an higher yields.
A stable,reliable,uniform HPLC analytical method with good reproducibility was developed to study the changes of chemical ingredients between single herbs of Dahuang,Houpo,Zhishi,Mangxiao and the compound recipe of Dachengqi decoction.The results indicated that in the 49 peaks of Dachengqi decoction,there are 12 peaks contributed from Zhishi,12 peaks contributed from Houpo and 17 peaks contributed from Dahuang,but no new peaks produced.Comparing Dachengqi decoction with single herbs of Dahuang,Houpo and Zhishi,the areas of 24 peaks are increased while the areas of 8 peaks are decreased.The peaks areas of Naringin,Hesperidin,Neohesperidin, Honokiol and Magnolol didn't increase,but yields of Aloe-emodin,Rhein, Emodin,Chrysophanol were increased when four herbs decoct together,which also indicated that traditional decoction method of Dachengqi decoction was increase the Anthraquinone components of the medicine so as to enhance the effect to treat constipation.
The quantitative contents of Aloe-emodin,Rhein,Emodin,Chrysophanol, Physcion,Naringin,Hesperidin,Neohesperidin,Honokiol and Magnolol were detested by HPLC in different compatibility of Dachengqi decoction as to study the active ingredient changed regularity of Chinese material medicine.The results show that Flavonoids and Phenols Components were hardly influence by different compatibility of Dachangqi decoction,but the contents of Anthraquinone components could become higher when Mangxiao and Dahuang were decocted later,and Houpo,Mangxiao,Zhisbi were compatible with Dahuang together can also improved the yields of Anthraquinone components,which proved that traditional decoction method of Dachengqi decoction was reasonable for increase the Anthraquinone components of the medicine as to enhance the effect to treat constipation.
The method of charcoal powder propelling ratio was used to investigate the effects of Dachengqi decoction on the intestinal propulsive movements in mice,and observe the influence of Dachengqi decoction on the intestinal smooth muscle of the experimental rabbits.The results show that,compare the blank group,the compatibility of different groups all show the Correlation were significant at the 0.05 level or 0.01 for charcoal powder propelling ratio, tonicity and contraction of the rabbits' s isolated intestinal muscle.The groups of Dahuang combine with Mangxiao hava a good pharmacological activities indicate that Dahuang and Mangxiao have a sronger effect to treat constipation, as well as the group of tradictional method.
The correlation between chemical components and pharmacological activities was carries on in order to study the therapeutic basis of Dachengqi decoction.49 peaks correspond to pharmacological activities were analysis by bivariate with the software of SPSS 16.0 for windows,there 11 peaks correlated to the pharmacological activity of Y_1(charcoal powder propelling ratio) with Correlation is significant at the 0.05 level or 1.01 level;3 peaks correlated to the pharmacological activity of Y_3(contracted frequency of isolated intestinal muscle) with Correlation is significant at the 0.05 level. The results indicated that Aloe-emodin,Rhein,Emodin,Chrysophanol are the therapeutic basis of Dachengqi decoction,and also explain the rationality and mechanism that these chemical components are used as to control the quality of herbs and recipe.
Response surface methodology(RSM) involving Doehlert design was adopted to evaluate the yields of Anthraquinone components in Dachengqi decoction by change of dosage of Zhishi,Houpo and Mangxiao and shorten analysis time through a desirability function.The results of this research show that Anthraquinone components were got a high yields of predicable value of 0.85 when the dosage ratio of Dachengqi decoction were compatible as follows: Dahuang:Zhishi:Houpo:Mangxiao(1:4:2.31:2).The Doehlert design and desirability function,which allow a sequential response methodology,are a good methods,and,of cause,the mathematical model can be further popular and apply for the compatibility research of Chinese material medicine.
This research was conduct under the theory of traditional Chinese medicine incorporated with science of Chinese materia medica,analytical chemistry, physics,statistics and technology of computer.In this research,the extracted technology were optimize by CCD,probe into the mechanism of compatibility,therapeutic basis of Dachengqi decoction,which provided a significative exploration for the modernization of compatibility of traditional Chinese medicine.
引文
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