大黄炮制前后药性品质的差异表征
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摘要
炮制是中医药的一大特色,炮制不仅能改变药性,也能影响其品质。但是如何客观有效地评价炮制前后中药药性品质的变化,这不仅是长期制约中药炮制学科发展的瓶颈,也一直是困扰中药质量分析的难题。本文以大黄为模式药物,主要采用生物效价检测手段,结合化学成分分析,综合考察大黄炮制前后的药性品质的变化。在此基础上,探索建立主要基于生物效价检测的大黄及其炮制品药性品质评价方法。主要研究结果如下:
1.首次建立了基于复方地酚诺酯便秘小鼠动物模型的大黄生物效价检测方法。大黄道地药材在1200-5000U·kg~(-1)剂量范围内与小鼠排便质量呈良好的线性关系,R＞0.98,可采用量反应平行线2,2',2”法测定大黄不同炮制品的致泻效价。
2.首次建立基于生物热动力学表征的大黄生物效价检测方法。大黄道地药材在0.64U-3.84U剂量范围内与生物热动力学参数lg(t_2/p_2)呈良好的线性关系,R＞0.98。可采用一剂量法(标准曲线法)法测定大黄不同炮制品的生物效价。
3.采用生物效价检测方法和亚急性毒性试验、四膜虫生物热活性分析,较系统地考察了大黄炮制前后药性品质的生物效应差异。大黄炮制过后致泻效价明显降低,酒大黄降低19.98%,熟大黄降低62.90%,大黄炭降低91.49%。亚急性毒性试验表明生大黄和酒大黄的最大无毒剂量相当于人临床最大用量(30g·d~(-1))的76倍,经炮制后,可以明显提高最大无毒浓度,熟大黄和大黄炭最大无毒剂量为人临床最大用量的152倍。大黄不同炮制品对四膜虫均有不同程度的抑制生长作用,其抑制率分别为生大黄(50.59%)、酒大黄(46.20%)、熟大黄(32.31%)、大黄炭(21.28%)。表明大黄炮制具有一定的减毒作用。
4.通过化学成分定性定量分析,初步阐释了大黄炮制前后药性品质改变的物质基础。大黄炮制过后,总蒽醌和鞣质含量不同程度降低,游离蒽醌和结合蒽醌的比例也发生改变,生大黄中为1∶9.8,酒大黄为1∶6.7,熟大黄为1∶2.4,大黄炭为1∶0.8;随炮制程度游离蒽醌的比例加大。
5.采用判别分析,初步建立大黄及其不同炮制品的药性品质模式识别方法,并在此基础上提出了基于生物效价检测的大黄不同炮制品质量控制建议。以大黄不同炮制品的类别作为应变量,通过变量筛选得到由生物效价作为自变量建立的判别方程,正确率可以达到70.83%,提示生物效价可以作为评价大黄不同炮制品的药性品质差异的重要指标。以95%置信区间统计描述大黄不同炮制品的生物效价值波动范围,表明经过炮制后,大黄各炮制品的生物效价波动范围明显小于生大黄,品质趋于稳定和均一。但大黄不同炮制品在生物效价方面存在重叠区,亟待从生物效价角度建立大黄的炮制规范和标准。
综上,本文初步建立可用于表征大黄及其不同炮制品药性品质差异的生物效价检测方法和指标,在此基础上建立了大黄不同炮制品药性品质差异的模式识别方法,为进一步阐明大黄炮制机理、科学建立大黄炮制工艺规范和多元质量控制标准提供了实验支持。
Processing is an important component of TCM,not only changes the properties of Chinese meteria medica(CMM),but also affects its quality.But it has been a difficulty in the science of CMM processing development and quality analysis of Chinese medicine for a long time that how to effectively and objectively evaluate the property and quality differences of CMM before and after processing,Take rhubarb as the model drug,biological potency tests combined with chemical composition analysis, were used to explore the property and quality changes before and after processing rhubarb.On this basis,property and quality evaluation method of rhubarb processed product which mainly based on the biological potency was preliminarily established. The main results are as follows:
1.The purgative bioassay method of rhubarb was established for the first time, which based on constipation model caused by diphenoxylate complex.The concentration of genuine rhubarb ranged from 1200 U·kg~(-1)to 5000U·kg~(-1),the mass of feces in 10hours could show the significant dose-effect relationship(r＞0.98).The biopotency of rhubarb processed products could be detected according to the method of graded response parallel lines(2,2',2").
2.The bioassay method of rhubarb which based on biothermokinesics was established for the ftrst time.The concentration of genuine rhubarb ranged from 0.64 Uto 3.84U,the parameter lg(t_2/p_2)could show the significant dose-effect relationship (r＞0.98).The biopotency of rhubarb processed products could be determined according to the method of calibration curve.
3.The biopotency method was used to indicate the property and quality diffences before and after rhubarb processing.Purgative bioassay degraded to varying degrees, Jiudahuang rhubarb(stir-fried with wine) lowered down by 19.98%,Shudahuang rhubarb(Steamed with wine)rhubarb lowered down by 62.90%,rhubarb charcoal lowered down by 91.49%.The experimental research of subacute toxicity indicated the largest safe dose of extract of shengdahuang and jiudahuang was 38g·kg~(-1) in 14 days which was equal to 76 times of 30g·d~(-1)(max dose in clinic),and the largest safe dose of extract of shudahuang and dahuangtan was 76g·kg~(-1) which was equal to 152 times of 30g(max dose in clinic).Rhubarb processed products have different levels of inhibition effects to tetrahymena,the inhibition rate were respectively 50.59% (rhubarb),46.20%(Jiudahuang rhubarb),32.31%(Shudahuang rhubarb),21.28% (rhubarb charcoal).Processing of rhubarb has attenuation effect.
4.Material basis of Rhubarb quality and properties changes before and after processing was studied by qualitative and quantitative analysis of chemical composition.After processing the anthraquinone and tannin content lowered down, the proportion of anthraquinone and conjugated anthraquinone changed too,which ranged from 1:9.8 to 1:0.8(rhubarb1:9.8,Jiudahuang rhubarb 1:6.7,Shudahuang rhubarb1:2.4,rhubarb charcoal 1:0.8);and the ratio of anthraquinone increased with the extent of processing.
5.Discriminant analysis was used in pattern recognition research on rhubarb different processed products' properties and quality differences and based on this proposed rhubarb processed products quality control recommendations based on the biological potency.The correct rate of the discriminant equation established by setting the categories of rhubarb different processed products as dependent variable,the biological potency as independent variable through variables screened was 70.83%, suggesting that biological potency of rhubarb can be used as key indicators to evaluate the properties and quality differences of rhubarb processed products.95% confidence interval was used to descript biological effect fluctuations of rhubarb different processed products,the volatility of processed products biological potency was significantly less than rhubarb,quality has been stabilized and homogenization. But rhubarb different processed products exist overlap in the biological potency,It is necessary to form biopotency standard for rhubarb different processed products
To sum up,in this article biological potency method and index were established to indicate rhubarb different processed products' properties and quality differences,and based on which pattern recognition mathematical model was studied.All the research provided experimental support for further clarify the mechanism of rhubarb processing,scientific establishment multiple quality control standards of rhubarb different processed products.
1.首次建立了基于复方地酚诺酯便秘小鼠动物模型的大黄生物效价检测方法。大黄道地药材在1200-5000U·kg~(-1)剂量范围内与小鼠排便质量呈良好的线性关系,R＞0.98,可采用量反应平行线2,2',2”法测定大黄不同炮制品的致泻效价。
2.首次建立基于生物热动力学表征的大黄生物效价检测方法。大黄道地药材在0.64U-3.84U剂量范围内与生物热动力学参数lg(t_2/p_2)呈良好的线性关系,R＞0.98。可采用一剂量法(标准曲线法)法测定大黄不同炮制品的生物效价。
3.采用生物效价检测方法和亚急性毒性试验、四膜虫生物热活性分析,较系统地考察了大黄炮制前后药性品质的生物效应差异。大黄炮制过后致泻效价明显降低,酒大黄降低19.98%,熟大黄降低62.90%,大黄炭降低91.49%。亚急性毒性试验表明生大黄和酒大黄的最大无毒剂量相当于人临床最大用量(30g·d~(-1))的76倍,经炮制后,可以明显提高最大无毒浓度,熟大黄和大黄炭最大无毒剂量为人临床最大用量的152倍。大黄不同炮制品对四膜虫均有不同程度的抑制生长作用,其抑制率分别为生大黄(50.59%)、酒大黄(46.20%)、熟大黄(32.31%)、大黄炭(21.28%)。表明大黄炮制具有一定的减毒作用。
4.通过化学成分定性定量分析,初步阐释了大黄炮制前后药性品质改变的物质基础。大黄炮制过后,总蒽醌和鞣质含量不同程度降低,游离蒽醌和结合蒽醌的比例也发生改变,生大黄中为1∶9.8,酒大黄为1∶6.7,熟大黄为1∶2.4,大黄炭为1∶0.8;随炮制程度游离蒽醌的比例加大。
5.采用判别分析,初步建立大黄及其不同炮制品的药性品质模式识别方法,并在此基础上提出了基于生物效价检测的大黄不同炮制品质量控制建议。以大黄不同炮制品的类别作为应变量,通过变量筛选得到由生物效价作为自变量建立的判别方程,正确率可以达到70.83%,提示生物效价可以作为评价大黄不同炮制品的药性品质差异的重要指标。以95%置信区间统计描述大黄不同炮制品的生物效价值波动范围,表明经过炮制后,大黄各炮制品的生物效价波动范围明显小于生大黄,品质趋于稳定和均一。但大黄不同炮制品在生物效价方面存在重叠区,亟待从生物效价角度建立大黄的炮制规范和标准。
综上,本文初步建立可用于表征大黄及其不同炮制品药性品质差异的生物效价检测方法和指标,在此基础上建立了大黄不同炮制品药性品质差异的模式识别方法,为进一步阐明大黄炮制机理、科学建立大黄炮制工艺规范和多元质量控制标准提供了实验支持。
Processing is an important component of TCM,not only changes the properties of Chinese meteria medica(CMM),but also affects its quality.But it has been a difficulty in the science of CMM processing development and quality analysis of Chinese medicine for a long time that how to effectively and objectively evaluate the property and quality differences of CMM before and after processing,Take rhubarb as the model drug,biological potency tests combined with chemical composition analysis, were used to explore the property and quality changes before and after processing rhubarb.On this basis,property and quality evaluation method of rhubarb processed product which mainly based on the biological potency was preliminarily established. The main results are as follows:
1.The purgative bioassay method of rhubarb was established for the first time, which based on constipation model caused by diphenoxylate complex.The concentration of genuine rhubarb ranged from 1200 U·kg~(-1)to 5000U·kg~(-1),the mass of feces in 10hours could show the significant dose-effect relationship(r＞0.98).The biopotency of rhubarb processed products could be detected according to the method of graded response parallel lines(2,2',2").
2.The bioassay method of rhubarb which based on biothermokinesics was established for the ftrst time.The concentration of genuine rhubarb ranged from 0.64 Uto 3.84U,the parameter lg(t_2/p_2)could show the significant dose-effect relationship (r＞0.98).The biopotency of rhubarb processed products could be determined according to the method of calibration curve.
3.The biopotency method was used to indicate the property and quality diffences before and after rhubarb processing.Purgative bioassay degraded to varying degrees, Jiudahuang rhubarb(stir-fried with wine) lowered down by 19.98%,Shudahuang rhubarb(Steamed with wine)rhubarb lowered down by 62.90%,rhubarb charcoal lowered down by 91.49%.The experimental research of subacute toxicity indicated the largest safe dose of extract of shengdahuang and jiudahuang was 38g·kg~(-1) in 14 days which was equal to 76 times of 30g·d~(-1)(max dose in clinic),and the largest safe dose of extract of shudahuang and dahuangtan was 76g·kg~(-1) which was equal to 152 times of 30g(max dose in clinic).Rhubarb processed products have different levels of inhibition effects to tetrahymena,the inhibition rate were respectively 50.59% (rhubarb),46.20%(Jiudahuang rhubarb),32.31%(Shudahuang rhubarb),21.28% (rhubarb charcoal).Processing of rhubarb has attenuation effect.
4.Material basis of Rhubarb quality and properties changes before and after processing was studied by qualitative and quantitative analysis of chemical composition.After processing the anthraquinone and tannin content lowered down, the proportion of anthraquinone and conjugated anthraquinone changed too,which ranged from 1:9.8 to 1:0.8(rhubarb1:9.8,Jiudahuang rhubarb 1:6.7,Shudahuang rhubarb1:2.4,rhubarb charcoal 1:0.8);and the ratio of anthraquinone increased with the extent of processing.
5.Discriminant analysis was used in pattern recognition research on rhubarb different processed products' properties and quality differences and based on this proposed rhubarb processed products quality control recommendations based on the biological potency.The correct rate of the discriminant equation established by setting the categories of rhubarb different processed products as dependent variable,the biological potency as independent variable through variables screened was 70.83%, suggesting that biological potency of rhubarb can be used as key indicators to evaluate the properties and quality differences of rhubarb processed products.95% confidence interval was used to descript biological effect fluctuations of rhubarb different processed products,the volatility of processed products biological potency was significantly less than rhubarb,quality has been stabilized and homogenization. But rhubarb different processed products exist overlap in the biological potency,It is necessary to form biopotency standard for rhubarb different processed products
To sum up,in this article biological potency method and index were established to indicate rhubarb different processed products' properties and quality differences,and based on which pattern recognition mathematical model was studied.All the research provided experimental support for further clarify the mechanism of rhubarb processing,scientific establishment multiple quality control standards of rhubarb different processed products.
引文
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2.高同银,崔同淑,崔淑婷.论中药炮制对药性药效的影响.河北中医,1993,15(5):30-31
3.肖小河,金城,赵中振,等.论中药质量控制与评价模式的创新与发展.中国中药杂志,2007,38(7):1-5.
4.鄢丹,肖小河,金城,等.论中药质量管理模式的挑战与发展.中草药,2006,37(6):806
5.吴燕芳.药性刚柔辨.上海中医药杂志,1993,(6):29-30
6.穆京生浅谈中药药性应有缓急之分上海中医药杂志1996(3):35
7.邓家刚,秦华珍,秦海洗中药药性效应及物质基础的文献研究河南中医2007,27(5)82-84
8.王普霞,周春祥.基于“证-药效-药性观念及药性本质多元化”假说探讨中药药性本质南京中医药大学学报,2006,22(6):345-347
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