| Traditional Chinese herbal medicine (TCHM) has been widely used for the treatment and prevention of various diseases for over thousands of years. In clinical practice, TCHM is prepared in decoctions with specific combination of different herbs as a formula to maximize synergistic effects and minimize adverse reactions.Cortex daphnes (CD, Zushima in Chinese) is the stem cortex and root bark of Daphne giraldii Nitsche, D. tangutica Maxim and D. retusa Hemsl, which is recorded in the first edition of China Pharmacopeia in1977. Clinically, CD has been used as a TCHM for treatment of ache, traumatic injury and rheumatoid arthritis (RA) in China for very long period, and also as a folk medicine for lumbago treatment and fever reduction in Turkey. Daphnetin, as a major component of CD, has many biological activities, such as anti-inflammatory, antioxidant, antimicrobial, and antimalarial properties as well as protein kinase inhibition.Glycyrrhizae uralenis (GU, Gancao in Chinese), derived from the root and rhizome of Glycyrrhizae uralenis Fisch., is prescribed in many Chinese traditional formulas for its medical potential in the treatment of anti-inflammation, immunoloregulatin, and anti-allergic effects. Phytochemical investigations revealed that revealed triterpenes and flavones are regarded as the principal components responsible for the main pharmacological activities of GU. Glycyrrhizin and liquiritin are the major active component of these triterpenes and flavones, respectively.The combined use of CD and GU in treating RA has been reported in various literatures, e.g. Handbook of Ningxia Chinese Herbal Medicine and Journal of Shaanxi Traditional Chinese Medicine, which are famous in the field of TCHM. CD is often clinically used in combination with GU to produce better efficacy in curing RA.To study the therapeutic effects and possible mechanism of different rate of Cortex Daphnes and Glycyrrhiza uralensis on rat adjuvant-induced arthritis (AA) model. The general state of health, paw edema, arthritis index (AI) and pathological changes of articular genu of the AA rats were evaluated. The contents of tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β) in AA rats serum were detected by ELISA. The expression of VEGF and MIF were detected with immunohistochemistry. The results indicated that the combined use of CD and GU can obviously relieve the arthritis syndrome in AA rats by reducing the expression of serum cytokines as well as inhibiting joint damage, and its therapeutic effect is better than used separately. Furthermore, the curative effect was the best when the compatibility of CD and GU reached3:2. The reducing TNF-a, IL-1β content in serum and VEGF, MIF in synovial membrane may be the drug treatment of rheumatoid arthritis mechanism.In order to clarify the rationality of herbaceous compatibility between CD and GU, the comparative evluations on pharmacokinetic behaviors of daphnetin (a predominantly active ingredient in CD) after intragastric administration of CD and CD-GU (combination of CD and GU) extract were studied, and the effects of glycyrrhizin and liquiritin, active ingredients of Glycyrrhiza triterpenes and Glycyrrhiza flavones respectively, on the pharmacokinetics of daphnetin were carried out below. Comparing with oral administration of CD extract, AUC and Tmax of daphnetin increased while CL decreased significantly after giving CD-GU. Besides, AUC, Cmax, Tmax increased as well as MRT and CL decreased significantly followed by co-administration of daphnetin and liquiritin compared to the daphnetin given alone. However, the pharmacokinetic parameters were not significantly changed as co-administered with glycyrrhizin.To study the influence of GU extract on the transport of daphnetin in CD using the well-characterized, human-based intestinal Caco-2cell model as a platform. Drug concentration was measured by HPLC and the apparent permeability coefficients (Papp) was calculated. Daphnetin was better absorbed in weak acid of pH6.8. The results showed that GU extract do not have significant influence on the absorption of daphnetin in CD. It indicated that GU extract can not enhance the intestinal absoption of daphnetin.The influence of GU extract on the metabolic process of daphnetin in CD extract in the rat liver microsomes was investigated. The results showed that the residual amount of daphnetin in CD-GU group increased significantly compared with CD group after the metabolism by the incubation system of liver microsomes. The metabolism of daphnetin was probable due to the metabolic enzymes inhibition by GU in the CD-GU formula, which increased the bioavailability of daphnetin in CD finally. The influences of different dosages of glycyrrhizin, glycyrrhetinic acid, liquiritin and liquiritigenin on the metabolic process of daphnetin in the rat liver microsomes were investigated. The results indicated that the residual amount of daphnetin decreased significantly in daphnetin with glycyrrhetinic acid group compared with the control group after the II phase metabolism by the incubation system of liver microsomes. While the residual amount of daphnetin increased significantly in daphnetin with glycyrrhizin, liquiritin and liquiritigenin group compared with the control group after the II phase metabolism. Therefore, we speculated that the glycyrrhetinic acid might promote the metabolism of daphnetin, while glycyrrhizin, liquiritin and liquiritigenin inhibit it. These results might explain the reason why glycyrrhizin did not affect the pharmacokinetics of daphnetin significantly while liquiritin did. And we hypothesized that the competition or inhibition between daphnetin and liquiritin may reduce the degradation and increase the concentration of daphnetin in plasma, which finally enhance its bioavailability.In this study, the rationale of the combined use of CD and GU was investigated from the perspective of pharmacology and biopharmaceutics by various models. We got the conclusion that the effect of CD in curing adjuvant-induced arthritis was increased when combining with GU in rat model. Meanwhile, the bioavailability of daphnetin in the CD-GU prescription was also significantly higher than that in CD alone, which could be due to liquiritin. Such findings clarified the rationale of the combined use of CD and GU and provided in vivo evidence for the clinical application of this combination formula. |
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