Study On The Therapeutic Basis Of Traditional Mongolian Medicinal Preparation Sendeng-4 Decoction

Sendeng-4 decoction, was first recorded in guan-zhe-zhi-xi which was written by Jigemude-danjinzhamusu, a well-known traditional Mongolian medicinal (TMM) preparation, has been included in the Drug Standard of Ministry of Public Health of the People's Republic of China (Mongolian Drugs Fascicule, 1998 edition). The decoction is prepared by boiling Wenguanmu, Chuanlianzi, Zhizi and Hezi. In the clinical practice of TMM science, the decoction has been used to tread dampness over a very long period of time and produced quite a favorable effects. The therapeutic material basis of Sendeng-4 Decoction was studied in this paper.The extraction solvent of Sendeng-4 Decoction was optimized. Two models of Xylene 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, 35%, 55%, 75% and 95% ethanol were investigated. The extracting yielded with 95% ethanol had the best anti-inflammatory and analgesic results, the anti-inflammatory inhibition rate was 60% and analgesic inhibition rate was 44.1%.The compatibility of Sendeng-4 Decoction was investigated using orthogonal design. The four herbs of Wenguanmu, Chuanlianzi, Zhizi and Hezi 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 Sendeng-4 Decoction. The results were processed with intuitionistic analysis, analysis of variance and stepwise regression analysis. The compatibility of Sendeng-4 Decoction was found to be precise and reasonable. It was suggested Wenguanmu as monarchic, Chuanlianzi as ministerial, Zhizi as assistant and Hezi as guide drug respectively, which was consistent with the traditional interpretation.A gradient elution RP-HPLC method was developed for the analysis of 16 combinations of Sendeng-4 Decoction. 32 chromatographic peaks were detected. By the stepwise regression analysis between the biological information and chemical information, 15 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 Sendeng-4 Decoction, and 3 compounds among them were selected as the quality control indices of Sendeng-4 decoction.The extraction process of Sendeng-4 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 four indices of extraction yield, content of geniposide content of quercetin, and content of gallic acid . The optimized extraction process was 3 times of extraction for 1 h each time and the extraction solvent was 10 folds of the amounts of material.RP-HPLC methods were developed for the determination of geniposide, quercetin, and gallic acid in Sendeng-4 Decoction. Hypersil C₁₈ ODS column was used and the detection wavelength was 238nm. Geniposide was analyzed with acetonitrile-water(l3.5:86.5, v/v) as mobile phase. The calibration curve was linear in the range of 13.25～66.25μg·mL^-1 and the correlation coefficient was 0.9996 with recovery 97.1% (RSD=1.7%) . Quercetin was analyzed on Century SIL C₁₈ BDS column with methanol-water-H₃PO₄ (60:40 :0.02, v/v) as mobile phase. The calibration curve was linear in the range of 27～137μg·mL^-1 and the correlation coefficient was 0.9993 with recovery 96.9%, (RSD=1.8%) . Gallic acid was analyzed with acetonitrile-water- H₃PO₄ (5:95:0.02, v/v) as mobile phase. The calibration curve was linear in the range of 22～110μg·mL^-1 and the correlation coefficient was 0.9991 with recovery 98.7% (RSD =3.0%) . The methods were simple and accurate and could be used as the quality control methods of Sendeng-4 Decoction.RP-HPLC methods were developed to determine the concentration of geniposide,rutin and quercetin in rat plasma. Separation of geniposide was accomplished on a reversed-phase ODS C₁₈ column (250×4.6mm-i.d. 5μm particles) and mobile phase of acetronitrile-water (12:88, v/v) with UV detection at 238 nm. Paeoniflorin was used as the internal standard (IS). The calibration plot was linear over the range 0.0848-7.42μg·mL^-1. The lower limit of quantification was 0.0848μg·mL^-1. The intra-day precision better than 11.4% and inter-day precision better than 9.3%. Mean extraction recovery was determined to be 87.1%. Plasma samples of rutin and quercetin taken from rats were pretreated by protein precipitation with methyl- acetic acid (9:1,v/v). HPLC analysis of the extracts is performed on a hypersil BDS C₁₈ analytical column using methyl-water-phosphoric acid (47:53 :0.02,v/v) as mobile phase with UV detector at 360nm. Vanallic aldehyde was used as the internal standard (IS).The linear range of the calibration curves were 0.068-8.60μg·mL^-1 (r²=0.997) for rutin and 0.052-13.0μg·mL^-1 (r²=0.996) for quercetin respectively. The intra- and inter-day precisions better than 13.2% for rutin and better than 14.4% for quercetin , respectively.The lower limit of quantification was 0.068μg·mL^-1 for rutin and 0.052μg·mL^-1 for quercetin. Mean extraction recovery was determined to be 84.8% for Rutin and 85.1% for Quercetin.The pharmacokinetic behavior of geniposide ,rutin and quercetin was investigated after rats were orally treated with Sendeng-4 Decoction. After Sendeng-4 Decoction was orally treated, the T_max of geniposide ,rutin and quercetin were 1.013±0.184 h,7.0±0.75 h, and 7.0±0.63 h respectively. The C_max were 3.342±0.1689μg·mL^-1,15.49±0.861μg·mL^-1 ,and 10.65±0.658μg·mL^-1 respectively.AUC_0～t of geniposide ,rutin and quercetin were 4.65±1.51μg·mL^-1,128.6±10.99μg·mL^-1, and 96.67±0.658μg·mL^-1 respectively, AUC_{0～∞} were 4.753±0.1505μg·mL^-1, 121.6±12.53μg·mL^-1, and 83.6±5.32μg·mL^-1 respectively, and MRT were 1.389±0.195h, 8.819±0.3722h, and 9.702±0.8778h respectively. Under the direction of the theory and clinical practice of Traditional Mongolian Medicine, utilizing the technique of Mongolian material medica science, analytical chemistry, pharmacology, pharmacokinetics and chemometrics, the therapeutic material basis of Sendeng-4 Decoction was studied in the present paper. This work provided an exploration for the therapeutic material basis research of Traditional Mongolian Medicine.