| ObjectiveTCM decoction Wu Tou Tang (WTT for short) is from Jin Kui Yao Lue which was written by Zhang Zhongjing the famous physician of Traditional Chinese Medicine. This prescription is composed of radix aconiti preparata, ephedra, peony, astragalus membranaceus, and honey-fried licorice root. The main treatment of Wu Tou Tang is to reduce joint pain that now been called rheumatoid arthritis or arthritis and other rheumatic diseases. TCM decoction Wu Tou Tang has been changed and got some innovation after several generation of famous doctors, while some people add other drugs in, while spawned many other new prescriptions, for example, Lee’s improved TCM decoction Wu Tou Tang, Chen’s TCM decoction Wu Tou Tang with asarum, and Mao’s TCM decoction Wu Tou Tang with Caulis spatholobi and honeysuckle stem. But WTT is still the root and main composition of these prescriptions. Visiblly, TCM decoction Wu Tou Tang has good curative effect which has complitely proved in clinical treament.Before this research we had carried out the experiment of compatibility for attenuation synergia and spectral studies. On the basis of exploration, we do further study of anti-inflammatory effect that total alkaloids of radix aconiti preparata and ephedra have. Further more, we also compared anti-inflammatory effect that other components with total alkaloids in WTT. The result shows that total alkaloids have better effect of anti-inflammatory than other components in WTT. So we continute the fingerprint and refined study of total alkaloids in WTT, which is to provide the reference for follow-up research.Methods This study is operated by culturing rheumatoid arthritis synovial fibroblasts to screen the effect of anti-inflammatory with ingredients including aconitine, ephedrine hydrochloride, paeoniflorin, licorice glycosides, monoammonium glycyrrhizinate, astragaloside and total alkaloid of WTT. In the part of anti-inflammatory effects in vitro screening, it was conducted by thiazole blue colorimetry cytotoxicity experiment (MTT method), that analyzing proliferation inhibitory rate of human rheumatoid arthritis synovial fibroblasts. Afer it was proved that total alkaloids have better effect of anti-inflammatory, we do further study in the extraction and purification process of WTT, in order to improve the purity of the total alkaloid, and ensure its drug effect. On the extraction and purification research part, it was used content of total alkaloid, ephedrine hydrochloride, single ester type aconitine as examining index, and Acid-base Titration and HPLC analysis. At the same time, it also had investigated the extraction solvent concentration, solvent quantity, time which have effect on the index component content to optimize extraction process. Through the selection of macroporous resin type and dosage in eluent concentration, conducting the orthogonal experiment, it was to optimize the purification process. To ensure the consistency of total alkaloid area, this study also used HPLC method to analyze the characteristic fingerprint, in order to achieve better quality control.ResultsThrough initial drug screening in vitro cell research, results showed that ephedrine hydrochloride (202.00-808.00μg/ml), paeoniflorin (1.2-24.0mg/ml), liquorice glycosides (26.00-400.00μg/ml) and total alkaloid (3.0-12.0mg/ml) have of obvious inhibiting effect on proliferation of human rheumatoid arthritis synovial fibroblasts, and presented dose-dependent. It is important that the drug concentration of ephedrine hydrochloride is low, but shown the obivious proliferation inhibition, and the effective drug concentration change range is narrow. Total alkaloid parts mainly contain ephedrine hydrochloride with single ester type of aconitine, and its inhibitory effect is also obviously. Aconitine, ammonium glycyrrhetate, astragalus glycosides have inhibitory effect in the experimental is not obvious that may be related to its poor water solubility. In total alkaloid extraction purification section of the study, it was established the acid-base titration for determination of the content of total alkaloid, and HPLC to simultaneous determination of ephedrine hydrochloride and single ester type of aconitine. After all, we successfully mapped out the area of total alkaloid of HPLC fingerprint and analyzed the chromatographic peak of main component.ConclusionEphedrine hydrochloride, paeoniflorin, liquorice glycosides and total alkaloid in WTT have capacity for proliferation inhibition of human rheumatoid arthritis synovial fibroblasts. Inhibitory effect of aconitine, ammonium glycyrrhetate, astragalus glycosides is not obvious and their anti-inflammatory mechanism may different from the composition such as ephedrine hydrochloride. |
PDF Full Text Request