| This study was supported by the National Natural Science Foundation of China (NO.81273655).Zushima, a kind of folk medicines in the western China, is the dry bark and root bark of Daphne giraldii Nitsche., Daphne tangutica Maxim., Daphne retusa Hemsl..This herb has a great effect of activating blood circulation, analgesia and eliminating wind and dampness, which is used to treat rheumatic, arthritic diseases and rheumatoid arthritis. According to the clinical research, combined with Gancao can decrease toxicity and increase efficacy of Zushima. Our previous study has shown that the optimal ratio of compatibility is3:2in the zushima and gancao prescription. Based on these researches, we carried out a study on pharmacokinetic and metabonomics of the prescription on treatment of rheumatoid arthritis:Firstly, we compared the pharmacokinetics behavior of multi-components from the prescription between normal and adjuvant-induced arthritis rats. Secondly, we found the biomarkers of AA by qualitative and quantitative analysis of endogenous metabolites in rat plasma. Lastly, we combined with the pharmacological activity evaluation to explore the mechanisms of disease and drug effects.Coumarins and diterpenoids are the main ingredients of Zushima, but the later have some side effect of mucosa and skin irritation. The pharmacological activity of daphnetin is more clear for the widely studies. This paper chose three coumarins as the indicators of Zushima for their high contents and strong activities. Gancao is extremely complex chemical composition, flavonoids and triterpenoids as its main active ingredient. Three pairs of glycosides and aglycones were choosed in our study, which were flavonoids (liquiritin and liquiritigenin), isoflavonoids (isoliquiritin and isoliquiritigenin) and triterpenes (glycyrrhizin and glycyrrhetinic acid) components.A LC-MS/MS method was established to simultaneously analyse main active components (daphnetin, daphnoretin,7-hydroxycoumarin, liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, glycyrrhizin and glycyrrhetinic acid). The method was also applied to study the comparative pharmacokinetics of ZG in rat at different physiological states. Compared with normal group, the bioavailabilities of daphnetin, liquiritin, isoliquiritin, and glycyrrhizin were decreased significantly in AA group, but the change of glycyrrhetinic acid was the opposite. AUC(o-t), AUC(o-∞) and Cmax of liquiritin and isoliquiritin in normal group were45.45±3.89ng·h·mL-1,6.72±0.60ng·h·mL-1,45.55±3.90ng·h·mL-1,6.74±0.60ng·h·mL-158.75±12.54ng-mL-1and8.92±1.68ng-mL-1, which were significantly decreased by27.13%(P<0.01),39.88%(P<0.05),24.57%(P<0.05),39.02%(P<0.01),48.75%(P<0.05) and48.09% (P<0.05) in AA group, respectively. AUC(o-∞) of daphnetin (255.68±54.77ng-h·mL-1) and glycyrrhizin (266.68±49.71ng-h·mL-1) were decreased by34.56%and29.89%(P<0.05), and Cmax of daphnoretin (14.40±3.31ng-mL-1) was significantly decreased by50.97%(P<0.05). However, AUC(o-t) and AUC(o-∞) of glycyrrhetinic acid increased by1.2times in AA group (P<0.05), and Tmax of isoliquiritin, isoliquiritigenin and glycyrrhetinic acid were increased as well. While there were no significant change in7-hydroxycoumarin and isoliquiritin.These results proved that the process of drug in vivo was affected by many factors, so we should apply some appropriate disease model to study drug pharmacokinetics, which can provide the basis for the clinical medicine.This study also used the metabolomics method combined with a series of physiological indicators (weight, paw edema, arthritis index and pathological section) to describe the dynamic process of AA and be applied to study the treatment of ZG on AA rats. A robust PC A model was established to explain the dynamic progress of AA. An supervised multivariate analysis model (OPLS-DA) were adopted to show the metabolites changes of different stage between normal group and AA model group and87metabolites were found related to the diease. Meanwhile,30abnormal metabolites were identified as the potential biomarkers, and the related pathways of AA mainly involved amino acids, lipids, nucleic acids and vitamins metabolism. ZG had great effect on AA by improving12biomarkers. Multiple pathological indicators and small molecule metabolites in AA rats were deviated from the normal ones, which indicated that AA model was successfully established in this experiment. The effects of positive medicine (tripterygium glycoside) and ZG on AA were similar, but the effect mechanisms of them were different. |
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