| 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. L16(45) 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 C18 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 C18 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 Tmax of glycyrrhetic acid were 10.4±0.9 h and 11.2±1.1 h respectively. The Cmax 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. AUC0～t of glycyrrhetic acid were 43.8±7.8μg·h·L-1 and 24.0±11.6μg·h·L<sup>-1 respectively, AUC0～∞ 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.
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