甘草附子汤药效物质基础研究
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摘要
本研究以《伤寒论》中祛风除湿的经典名方甘草附子汤为研究对象，对其药效物质基础进行了一系列研究。
对甘草附子汤的提取溶剂进行了优化，选择巴豆油致小鼠耳肿胀反应和醋酸致小鼠扭体反应两个模型分别作为抗炎和镇痛药理指标，考察了水、30％、50％、70％和95％乙醇五种提取溶剂，结果表明以50％乙醇提取效果较好，抗炎和镇痛抑制率分别达到27.5％和69.6％。以甘草附子汤50％乙醇提取液在2.5、5、10g／kg三个剂量灌胃给药，抗炎抑制率达到15.6％-46.2％，镇痛抑制率达到52.8％-79.8％，且表现出了良好的量效关系，说明甘草附子汤具有良好的抗炎和镇痛作用。
从甘草附子汤君、臣、佐、使配伍关系出发，利用正交实验设计，对甘草附子汤进行拆方研究，探讨甘草附子汤配伍机制。选择L_(16)(4~5)正交表，以甘草附子汤中4味药为因素，分别选取原方0、0.5、1、2倍量构成4个水平。对所得16个处方分别进行药理实验；采用直观分析、方差分析和逐步回归分析，将所得药理数据和处方组成相关联，探讨甘草附子汤的配伍机制。结果确证了方中以甘草为君，以附子为臣，白术为佐，桂枝为使的配伍关系，与甘草附子汤的传统方解相一致。
利用RP-HPLC梯度洗脱技术，对16个处方进行了分析，共得到26个色谱峰。将药理数据和各指纹峰峰面积相关联，通过逐步回归分析方法处理，共有14个色谱峰被引入到回归方程。表明这14个色谱峰所代表的化学成分对其药效有显著作用，确定为甘草附子汤的药效物质基础。
对甘草附子汤的提取工艺进行了优化研究。选取浸膏得率、甘草酸含量和桂皮酸含量三个指标，对溶剂量、提取时间、提取次数三个因素进行考察，确定甘草附子汤最佳提取工艺为以10倍量50％乙醇提取2次，每次1h。
采用反相高效液相色谱法对甘草附子汤中甘草酸、桂皮酸、甘草苷和香豆素等4种成分进行了定量分析。采用Hypersil C_(18)柱，在254nm检测，其中甘草酸以甲醇：乙腈：水：冰醋酸(19：30：51：1)为流动相，在0.055～1.10mg·mL~(-1)范围内成良好线性关系，r=0.9999，平均回收率为95.9％。桂皮酸以甲醇：乙腈：水：冰醋酸(37：1：63：0.3)为流动相，在2.0～30.1μg·mL~(-1)范围内成良好线性关系，r=0.9998，平均回收率为94.8％。甘草苷和香豆素以甲醇：乙腈：水：冰醋酸(5∶15∶80∶1)为流动相，分别在20～300μg·mL~(-1)和1.7～16.7μg·mL~(-1)范围内成良好线性关系，相关系数均为0.9999，回收率分别为95.9％和95.9％。方法简便、准确，为甘草附子汤质量控制方法提供了定量依据。
建立了大鼠血浆中甘草酸及其代谢产物甘草次酸的RP-HPLC测定方法，以联苯和苯丙酸诺龙为内标，血浆样品经异丙醇-乙醚(1∶1)萃取后取上清液，氮气吹干，甲醇定容后分析。使用Hypersil C_(18)色谱柱，测定甘草酸时采用流动相：甲醇-乙腈-水-冰醋酸=24∶29∶47∶1；测定甘草次酸时采用甲醇-乙腈-水-冰醋酸=58∶18∶24∶1；流速0.8 mL·min~(-1)；检测波长254 nm。结果表明，甘草酸在1.25～200μg·mL~(-1)范围内线性关系良好(r=0.9942)；甘草次酸在0.506～81μg·mL~(-1)范围内线性关系良好(r=0.9936)。低、中、高浓度的甘草酸和甘草次酸日内、日间精密度均小于10.7％。甘草酸和甘草次酸的回收率分别高于94.7％和88.9％。
研究了大鼠口服甘草附子汤和甘草酸后甘草酸的药物动力学行为，结果表明甘草酸在体内以代谢物甘草次酸的形式存在。给药甘草附子汤和甘草酸后甘草次酸的T_(max)分别为10.4±0.9 h和11.2±1.1 h，C_(max)分别为1.40±0.40μg·mL~(-1)和1.64±0.77μg·mL~(-1)。二者没有显著差异。AUC_(0～t)分别为43.8±7.8μg·h·L~(-1)和24.0±11.6μg·h·L~(-1)，AUC_(0～∞)分别为122.8±46.7μg·h·L~(-1)和40.9±9.6μg·h·L~(-1)，MRT分别为27.6±1.6 h和15.0±2.0 h。说明甘草在配伍附子、白术、桂枝后，有利于甘草次酸在体内的吸收，使其生物利用度增加，作用时间延长。
以甘草附子汤中君药甘草为研究对象，对其进行了血清药理学研究。选择一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)两个指标，考察大鼠口服甘草后不同时间点(1 h、2 h、4 h、8 h、10 h、12 h和24 h)，不同浓度(0.1％、1％、5％)的血清对LPS诱导的大鼠腹腔巨噬细胞NO和TNF-α释放的影响。结果表明，低浓度(0.1％)和中浓度(1％)的血清可以显著降低NO和TNF-α的释放。随着给药剂量的增加，其作用发生翻转，高浓度(5％)的血清可以增加NO和TNF-α的释放。口服甘草后对NO和TNF-α表现出了双向调节的作用，且对二者的作用趋势相似。揭示甘草对炎症的调节作用机理可能是通过调节TNF-α的产生，进而影响NO合酶(iNOS)的活性、调节NO的释放而达到的。
在对大鼠血清的LC-MS分析中共检测出9个色谱峰。逐步回归分析结果表明，化合物3、6、7、9和化合物1、2、5、7、8、9分别对NO和TNF-α的产生有显著影响，综合作用表现出对二者的双向调节作用。确定为甘草在体内发挥药效的物质基础。
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、药物动力学、化学计量学和计算机技术相结合，对传统复方甘草附子汤进行了现代化研究，为复方中药的药效物质基础研究做了有意义的探索。
Gancaofuzi Decoction was first recorded in Treatise on Cold-induced Febrile Diseases written by Zhang Zhongjing, which has been used for the treatment of wind dampness over a long period of time and produced quite a favorable effect. The therapeutic material basis of Gancaofuzi Decoction was studied in this paper.The extraction solvent of Gancaofuzi Decoction was optimized. Two models of croton oil induced mouse ear edema and acetic acid induced writhing were selected as the models to evaluate the anti-inflammatory and analgesic effects of the decoction respectively. Five solvents of water, 30%, 50%, 70% and 95% ethanol were investigated. Extracted with 50% ethanol yielded best anti-inflammatory and analgesic results, the anti-inflammatory inhibition rate was 27.5% and analgesic inhibition rate was 69.6%. After mice were orally treated with Gancaofuzi Decoction at the dose of 2.5, 5 and 10 g/kg, the anti-inflammatory inhibition rate and analgesic inhibition rate were 15.6-46.2% and 52.8-79.8% respectively in good dose-dependent manner. The results showed that Gancaofuzi Decoction exhibited good anti-inflammatory and analgesic effects.The compatibility of Gancaofuzi Decoction was investigated using orthogonal design. The four herbs of Gancao, Fuzi, Baizhu and Guizhi were selected as 4 factors and 0, 0.5, 1 and 2 folds of the original prescription amounts were selected as 4 levels. L_(16)(4~5) orthogonal array was utilized. Anti-inflammatory and analgesic effects were selected as pharmacological indices to evaluate the 16 combinations of Gancaofuzi Decoction. The results were processed with intuitionistic analysis, analysis of variance and stepwise regression analysis. The compatibility of Gancaofuzi Decoction was found to be precise and reasonable. It was suggested Gancao as imperial, Fuzi as ministerial, Baizhu as assistant and Guizhi as servant drug respectively, which was consistent with the traditional interpretation.
A gradient elution RP-HPLC method was developed for the analysis of 16 combinations of Gancaofuzi Decoction. 26 chromatographic peaks were detected. By the stepwise regression analysis between the biological information and chemical information, 14 peaks were introduced in the regression equation, which were suggested to be the main constituents that could impact the pharmacological results remarkably and were considered as the therapeutic material basis of Gancaofuzi Decoction.
The extraction process of Gancaofuzi Decoction was optimized by orthogonal design. The three factors of amount of solvent, extraction time and times of extraction were investigated at three levels by the three indices of extraction yield, content of glycyrrhizic acid and content of glycyrrhetic acid. The optimized extraction process was 2 times of extraction for 1h each time and the extraction solvent was 10 folds of the amounts of material.
RP-HPLC methods were developed for the determination of glycyrrhizic acid, cinnamic acid, liquiritin and coumarin in Gancaofuzi Decoction. Hypersil C_(18) column was used and the detected wavelength was 254nm. Glycyrrhizic acid was analyzed with methanol-acetonitrile-water-HAC(19: 30: 51: 1) as mobile phase. The calibration curve was linear in the range of 0.055～1.10 mg·mL~(-1) and the correlation coefficient was 0.9999 with recovery 95.9%. Cinnamic acid was analyzed with methanol-acetonitrile-water-HAC (37: 1: 63: 0.3) as mobile phase. The calibration curve was linear in the range of 2.0～30.1μg·mL~(-1) and the correlation coefficient was 0.9998 with recovery 94.8%. Liquiritin and coumarin was analyzed with methanol-acetonitrile-water-HAC(5: 15: 80: 1) as mobile phase. The calibration curve was linear in the range of 20～300μg·mL~(-1) and 1.7～16.7μg·mL~(-1) respectively and the correlation coefficient was both 0.9999. The recovery of liquiritin and coumarin was 95.9% and 95.9% respectively. The methods were simple and accurate and could be used as the quality control methods of Gancaofuzi Decoction.
RP-HPLC methods were developed to determine the concentration of glycyrrhizic acid and glycyrrhetic acid in rat plasma. Diphenyl and nandrolone phenylpropionate were used as internal standards. The plasma sample was extracted with isopropanol-ethyl ether (1: 1). The supernatant was dried under the stream of nitrogen and the residue was reconstituted with methanol for analysis. Seperation was achieved on a Hypersil C_(18) column (4.6mm×200mm, 5μm) with themobile phase of methanol-acetonitrile-water-acetic acid (24: 29: 47: 1) for the detection of glycyrrhizic acid and methanol-acetonitrile-water-acetic acid (58: 18: 24: 1) for the detection of glycyrrhetic acid and the detection wavelength was set at 254 nm. The linear calibration curve of glycyrrhizic acid was obtained in the concentration range of 1.25～200μg·mL~(-1) (r=0.9942) and glycyrrhetic acid in the concentration range of 0.506～81μg·mL~(-1) (r=0.9936). The within day precision and between day precision of GL and GA at low, medium and high concentrations were found to be less than 10.7%. The recoveries of GL and GA were above 94.7% and 88.9% respectively.
The pharmacokinetic behavior of glycyrrhizic acid was investigated after rats were orally treated with Gancaofuzi Decoction and glycyrrhizic acid respectively. It was found that glycyrrhizic acid was metabolized to glycyrrhetic acid in vivo. After Gancaofuzi Decoction and glycyrrhizic acid were orally treated, the T_(max) of glycyrrhetic acid were 10.4±0.9 h and 11.2±1.1 h respectively. The C_(max) were 1.40±0.40μg·mL~(-1) and 1.64±0.77μg·mL~(-1) respectively. There was no significant difference between the above two parameters. AUC_(0～t) of glycyrrhetic acid were 43.8±7.8μg·h·L~(-1) and 24.0±11.6μg·h·L~~(-1) respectively, AUC_(0～∞) were 122.8±46.7μg·h·L~(-1) and 40.9±9.6μg·h·L~(-1) respectively and MRT were 27.6±1.6 h and 15.0±2.0 h respectively, which suggested a prolonged action time and increased bioavailability after Gancaofuzi Decoction was orally administered.
Serum pharmacology studies were performed on the licorice. After rats were orally treated with licorice, the effects of sera collected at different time (1h, 2h, 4h, 8h, 10h, 12h and 24h) with different concentration (0.1%, 1% and 5%) were investigated on the production of nitric oxide (NO) and tumor necrosis factor-α(TNF-α) induced by lipopolysaccharide (LPS) in rat peritoneal macrophages. The production of NO and TNF-αwere significantly reduced by lower concentration of the sera (0.1%, 1%), while both levels of NO and TNF-αwere markedly increased by higher concentration of the sera (5%). Similar dual regulatory effects were exhibited on NO and TNF-αproduction after licorice was orally administered, suggested that the regulatory effects of licorice on the inflammatory process might be mediated through the activation of TNF-αproduction and the followed activation of NO.
By the LC-MS analysis, 9 chromatographic peaks were detected in the sera colleted after licorice was orally treated. Compared with the chromatograms of licorice extract at the same chromatographic condition, both the 9 compounds were found to be the metabolites of licorice. The results of the stepwise regression analysis revealed that compound 3, 6, 7, 9 and compound 1, 2, 5, 7, 8, 9 acted synergistically on the NO and TNF-αproduction respectively, causing an overall dual effect. These 8 compounds were considered as the therapeutic material basis of Gancaofuzi Decoction in vivo.
Under the direction of the theory and clinical practice of Traditional Chinese Medicine, utilizing the technique of Chinese material medica science, analytical chemistry, pharmacology, pharmacokinetics and chemometrics, the therapeutic material basis of Gancaofuzi Decoction was studied in the present paper. This work provided an exploration for the therapeutic material basis research of Traditional Chinese Medicine.
对甘草附子汤的提取溶剂进行了优化，选择巴豆油致小鼠耳肿胀反应和醋酸致小鼠扭体反应两个模型分别作为抗炎和镇痛药理指标，考察了水、30％、50％、70％和95％乙醇五种提取溶剂，结果表明以50％乙醇提取效果较好，抗炎和镇痛抑制率分别达到27.5％和69.6％。以甘草附子汤50％乙醇提取液在2.5、5、10g／kg三个剂量灌胃给药，抗炎抑制率达到15.6％-46.2％，镇痛抑制率达到52.8％-79.8％，且表现出了良好的量效关系，说明甘草附子汤具有良好的抗炎和镇痛作用。
从甘草附子汤君、臣、佐、使配伍关系出发，利用正交实验设计，对甘草附子汤进行拆方研究，探讨甘草附子汤配伍机制。选择L_(16)(4~5)正交表，以甘草附子汤中4味药为因素，分别选取原方0、0.5、1、2倍量构成4个水平。对所得16个处方分别进行药理实验；采用直观分析、方差分析和逐步回归分析，将所得药理数据和处方组成相关联，探讨甘草附子汤的配伍机制。结果确证了方中以甘草为君，以附子为臣，白术为佐，桂枝为使的配伍关系，与甘草附子汤的传统方解相一致。
利用RP-HPLC梯度洗脱技术，对16个处方进行了分析，共得到26个色谱峰。将药理数据和各指纹峰峰面积相关联，通过逐步回归分析方法处理，共有14个色谱峰被引入到回归方程。表明这14个色谱峰所代表的化学成分对其药效有显著作用，确定为甘草附子汤的药效物质基础。
对甘草附子汤的提取工艺进行了优化研究。选取浸膏得率、甘草酸含量和桂皮酸含量三个指标，对溶剂量、提取时间、提取次数三个因素进行考察，确定甘草附子汤最佳提取工艺为以10倍量50％乙醇提取2次，每次1h。
采用反相高效液相色谱法对甘草附子汤中甘草酸、桂皮酸、甘草苷和香豆素等4种成分进行了定量分析。采用Hypersil C_(18)柱，在254nm检测，其中甘草酸以甲醇：乙腈：水：冰醋酸(19：30：51：1)为流动相，在0.055～1.10mg·mL~(-1)范围内成良好线性关系，r=0.9999，平均回收率为95.9％。桂皮酸以甲醇：乙腈：水：冰醋酸(37：1：63：0.3)为流动相，在2.0～30.1μg·mL~(-1)范围内成良好线性关系，r=0.9998，平均回收率为94.8％。甘草苷和香豆素以甲醇：乙腈：水：冰醋酸(5∶15∶80∶1)为流动相，分别在20～300μg·mL~(-1)和1.7～16.7μg·mL~(-1)范围内成良好线性关系，相关系数均为0.9999，回收率分别为95.9％和95.9％。方法简便、准确，为甘草附子汤质量控制方法提供了定量依据。
建立了大鼠血浆中甘草酸及其代谢产物甘草次酸的RP-HPLC测定方法，以联苯和苯丙酸诺龙为内标，血浆样品经异丙醇-乙醚(1∶1)萃取后取上清液，氮气吹干，甲醇定容后分析。使用Hypersil C_(18)色谱柱，测定甘草酸时采用流动相：甲醇-乙腈-水-冰醋酸=24∶29∶47∶1；测定甘草次酸时采用甲醇-乙腈-水-冰醋酸=58∶18∶24∶1；流速0.8 mL·min~(-1)；检测波长254 nm。结果表明，甘草酸在1.25～200μg·mL~(-1)范围内线性关系良好(r=0.9942)；甘草次酸在0.506～81μg·mL~(-1)范围内线性关系良好(r=0.9936)。低、中、高浓度的甘草酸和甘草次酸日内、日间精密度均小于10.7％。甘草酸和甘草次酸的回收率分别高于94.7％和88.9％。
研究了大鼠口服甘草附子汤和甘草酸后甘草酸的药物动力学行为，结果表明甘草酸在体内以代谢物甘草次酸的形式存在。给药甘草附子汤和甘草酸后甘草次酸的T_(max)分别为10.4±0.9 h和11.2±1.1 h，C_(max)分别为1.40±0.40μg·mL~(-1)和1.64±0.77μg·mL~(-1)。二者没有显著差异。AUC_(0～t)分别为43.8±7.8μg·h·L~(-1)和24.0±11.6μg·h·L~(-1)，AUC_(0～∞)分别为122.8±46.7μg·h·L~(-1)和40.9±9.6μg·h·L~(-1)，MRT分别为27.6±1.6 h和15.0±2.0 h。说明甘草在配伍附子、白术、桂枝后，有利于甘草次酸在体内的吸收，使其生物利用度增加，作用时间延长。
以甘草附子汤中君药甘草为研究对象，对其进行了血清药理学研究。选择一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)两个指标，考察大鼠口服甘草后不同时间点(1 h、2 h、4 h、8 h、10 h、12 h和24 h)，不同浓度(0.1％、1％、5％)的血清对LPS诱导的大鼠腹腔巨噬细胞NO和TNF-α释放的影响。结果表明，低浓度(0.1％)和中浓度(1％)的血清可以显著降低NO和TNF-α的释放。随着给药剂量的增加，其作用发生翻转，高浓度(5％)的血清可以增加NO和TNF-α的释放。口服甘草后对NO和TNF-α表现出了双向调节的作用，且对二者的作用趋势相似。揭示甘草对炎症的调节作用机理可能是通过调节TNF-α的产生，进而影响NO合酶(iNOS)的活性、调节NO的释放而达到的。
在对大鼠血清的LC-MS分析中共检测出9个色谱峰。逐步回归分析结果表明，化合物3、6、7、9和化合物1、2、5、7、8、9分别对NO和TNF-α的产生有显著影响，综合作用表现出对二者的双向调节作用。确定为甘草在体内发挥药效的物质基础。
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、药物动力学、化学计量学和计算机技术相结合，对传统复方甘草附子汤进行了现代化研究，为复方中药的药效物质基础研究做了有意义的探索。
Gancaofuzi Decoction was first recorded in Treatise on Cold-induced Febrile Diseases written by Zhang Zhongjing, which has been used for the treatment of wind dampness over a long period of time and produced quite a favorable effect. The therapeutic material basis of Gancaofuzi Decoction was studied in this paper.The extraction solvent of Gancaofuzi Decoction was optimized. Two models of croton oil induced mouse ear edema and acetic acid induced writhing were selected as the models to evaluate the anti-inflammatory and analgesic effects of the decoction respectively. Five solvents of water, 30%, 50%, 70% and 95% ethanol were investigated. Extracted with 50% ethanol yielded best anti-inflammatory and analgesic results, the anti-inflammatory inhibition rate was 27.5% and analgesic inhibition rate was 69.6%. After mice were orally treated with Gancaofuzi Decoction at the dose of 2.5, 5 and 10 g/kg, the anti-inflammatory inhibition rate and analgesic inhibition rate were 15.6-46.2% and 52.8-79.8% respectively in good dose-dependent manner. The results showed that Gancaofuzi Decoction exhibited good anti-inflammatory and analgesic effects.The compatibility of Gancaofuzi Decoction was investigated using orthogonal design. The four herbs of Gancao, Fuzi, Baizhu and Guizhi were selected as 4 factors and 0, 0.5, 1 and 2 folds of the original prescription amounts were selected as 4 levels. L_(16)(4~5) orthogonal array was utilized. Anti-inflammatory and analgesic effects were selected as pharmacological indices to evaluate the 16 combinations of Gancaofuzi Decoction. The results were processed with intuitionistic analysis, analysis of variance and stepwise regression analysis. The compatibility of Gancaofuzi Decoction was found to be precise and reasonable. It was suggested Gancao as imperial, Fuzi as ministerial, Baizhu as assistant and Guizhi as servant drug respectively, which was consistent with the traditional interpretation.
A gradient elution RP-HPLC method was developed for the analysis of 16 combinations of Gancaofuzi Decoction. 26 chromatographic peaks were detected. By the stepwise regression analysis between the biological information and chemical information, 14 peaks were introduced in the regression equation, which were suggested to be the main constituents that could impact the pharmacological results remarkably and were considered as the therapeutic material basis of Gancaofuzi Decoction.
The extraction process of Gancaofuzi Decoction was optimized by orthogonal design. The three factors of amount of solvent, extraction time and times of extraction were investigated at three levels by the three indices of extraction yield, content of glycyrrhizic acid and content of glycyrrhetic acid. The optimized extraction process was 2 times of extraction for 1h each time and the extraction solvent was 10 folds of the amounts of material.
RP-HPLC methods were developed for the determination of glycyrrhizic acid, cinnamic acid, liquiritin and coumarin in Gancaofuzi Decoction. Hypersil C_(18) column was used and the detected wavelength was 254nm. Glycyrrhizic acid was analyzed with methanol-acetonitrile-water-HAC(19: 30: 51: 1) as mobile phase. The calibration curve was linear in the range of 0.055～1.10 mg·mL~(-1) and the correlation coefficient was 0.9999 with recovery 95.9%. Cinnamic acid was analyzed with methanol-acetonitrile-water-HAC (37: 1: 63: 0.3) as mobile phase. The calibration curve was linear in the range of 2.0～30.1μg·mL~(-1) and the correlation coefficient was 0.9998 with recovery 94.8%. Liquiritin and coumarin was analyzed with methanol-acetonitrile-water-HAC(5: 15: 80: 1) as mobile phase. The calibration curve was linear in the range of 20～300μg·mL~(-1) and 1.7～16.7μg·mL~(-1) respectively and the correlation coefficient was both 0.9999. The recovery of liquiritin and coumarin was 95.9% and 95.9% respectively. The methods were simple and accurate and could be used as the quality control methods of Gancaofuzi Decoction.
RP-HPLC methods were developed to determine the concentration of glycyrrhizic acid and glycyrrhetic acid in rat plasma. Diphenyl and nandrolone phenylpropionate were used as internal standards. The plasma sample was extracted with isopropanol-ethyl ether (1: 1). The supernatant was dried under the stream of nitrogen and the residue was reconstituted with methanol for analysis. Seperation was achieved on a Hypersil C_(18) column (4.6mm×200mm, 5μm) with themobile phase of methanol-acetonitrile-water-acetic acid (24: 29: 47: 1) for the detection of glycyrrhizic acid and methanol-acetonitrile-water-acetic acid (58: 18: 24: 1) for the detection of glycyrrhetic acid and the detection wavelength was set at 254 nm. The linear calibration curve of glycyrrhizic acid was obtained in the concentration range of 1.25～200μg·mL~(-1) (r=0.9942) and glycyrrhetic acid in the concentration range of 0.506～81μg·mL~(-1) (r=0.9936). The within day precision and between day precision of GL and GA at low, medium and high concentrations were found to be less than 10.7%. The recoveries of GL and GA were above 94.7% and 88.9% respectively.
The pharmacokinetic behavior of glycyrrhizic acid was investigated after rats were orally treated with Gancaofuzi Decoction and glycyrrhizic acid respectively. It was found that glycyrrhizic acid was metabolized to glycyrrhetic acid in vivo. After Gancaofuzi Decoction and glycyrrhizic acid were orally treated, the T_(max) of glycyrrhetic acid were 10.4±0.9 h and 11.2±1.1 h respectively. The C_(max) were 1.40±0.40μg·mL~(-1) and 1.64±0.77μg·mL~(-1) respectively. There was no significant difference between the above two parameters. AUC_(0～t) of glycyrrhetic acid were 43.8±7.8μg·h·L~(-1) and 24.0±11.6μg·h·L~~(-1) respectively, AUC_(0～∞) were 122.8±46.7μg·h·L~(-1) and 40.9±9.6μg·h·L~(-1) respectively and MRT were 27.6±1.6 h and 15.0±2.0 h respectively, which suggested a prolonged action time and increased bioavailability after Gancaofuzi Decoction was orally administered.
Serum pharmacology studies were performed on the licorice. After rats were orally treated with licorice, the effects of sera collected at different time (1h, 2h, 4h, 8h, 10h, 12h and 24h) with different concentration (0.1%, 1% and 5%) were investigated on the production of nitric oxide (NO) and tumor necrosis factor-α(TNF-α) induced by lipopolysaccharide (LPS) in rat peritoneal macrophages. The production of NO and TNF-αwere significantly reduced by lower concentration of the sera (0.1%, 1%), while both levels of NO and TNF-αwere markedly increased by higher concentration of the sera (5%). Similar dual regulatory effects were exhibited on NO and TNF-αproduction after licorice was orally administered, suggested that the regulatory effects of licorice on the inflammatory process might be mediated through the activation of TNF-αproduction and the followed activation of NO.
By the LC-MS analysis, 9 chromatographic peaks were detected in the sera colleted after licorice was orally treated. Compared with the chromatograms of licorice extract at the same chromatographic condition, both the 9 compounds were found to be the metabolites of licorice. The results of the stepwise regression analysis revealed that compound 3, 6, 7, 9 and compound 1, 2, 5, 7, 8, 9 acted synergistically on the NO and TNF-αproduction respectively, causing an overall dual effect. These 8 compounds were considered as the therapeutic material basis of Gancaofuzi Decoction in vivo.
Under the direction of the theory and clinical practice of Traditional Chinese Medicine, utilizing the technique of Chinese material medica science, analytical chemistry, pharmacology, pharmacokinetics and chemometrics, the therapeutic material basis of Gancaofuzi Decoction was studied in the present paper. This work provided an exploration for the therapeutic material basis research of Traditional Chinese Medicine.
引文
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