Study On Chromatographic Fingerprints And Anti-inflammatory Pharmacodynamics On Cells Of Tripterygium Wilfordii Preparations

Tripterygum wilfordii preparation, which has a significant effect, is widely used in the treatment of many autoimmune diseases such as rheumatoid arthritis, nephritic-syndrome, systemic lupus erythematosus, etc. But, it has a high incidence of adverse reactions applied to clinical. Adverse reactions and therapeutic effect of different preparations on the market vary considerably. Thus, how to make its clinical application safe and effective is the hot spot and the difficulty in research of Tripterygium wilfordii. As the complexity of the composition and structure of Tripterygium wilfordii, there is still a lack of quality standards which can effectively evaluate the quality of different Tripterygium wilfordii preparations. In order to resolve the problem, we established chromatographic fingerprints of the eight different manufacturers’Tripterygium wilfordii preparations. Furthermore, we investigated the anti-inflammatory effect of different Tripterygium wilfordii preparations on the lipopolysaccharide-stimulated RAW264.7cell model, which were applied to evaluate the relationship of anti-inflammatory effects and finger print profile.To get better characterization of Tripterygium wilfordii preparations information through the fingerprints, the conditions of extraction and purification were investigated and optimized. The effects of extraction solvents, extraction times, extraction methods, elution solvents and elution volumes on the total peak area of the common peaks in the HPLC analysis were evaluated. And the best optimal extraction proeess conditions were obtained: Tripterygium wilfordii preparations with ethyl acetate under ultrasonic for10min, neutral alumina column as purification column,10mL ethyl acetate extract.HPLC was used to analyze Tripteiygium wilfordii preparations from different manufactures. Mobile phase, chromatographic column and detected wavelength were optimized to complete the systemic methodology and establish the HPLC fingerprint of Tripterygium wilfordii preparation. What’s more, the similarity evaluation of chromatograms was processed by use of the software Similarity Evaluation System for Chromatographic Fingerprint of TCM (2004A), with11common peaks being confirmed. The results of fingerprints similarity analysis of8samples of Tripterygium wilfordii preparation indicated that there is significant differences from Tripterygium wilfordii preparation samples which are not from the same manufactures but under the same way of analysis.The inflammation model was constructed by treating RAW264.7cells with lipopolysaccharide (LPS).The levels of TNF-α,IL-6and NO were assayed to.evaluate the phannacodynamics. There were significant differences between anti-inflammatory activities of different Tripterygium wilfordii preparations, the anti-inflammatory effects of the extracts from Tripterygium wilfordii were dose dependent.Finally,on the basis of the peak areas of the HPLC fingerprint and its anti-inflammatory activities, the spectrum-effect relationships between HPLC fingerprint and anti-inflammatory activities were investigated using the partial least squares multiple linear regression analysis. The four multiple linear regression equations are:(1)Y=-0.052X1-0.078X2-0.060X3-0.046X4-0.027X5+0.002X6-0.055X7一0.050X8-0.106X9-0.048X10-0.164X11；(2)Y=0.071X1-0.028X2-0.033X3-0.004X4-0.012X5-0.018X6-0.039X7-0.058X8-0.173X9-0.148X10-0.230X11；(3)Y=-0.030X1-0.058X2-0.056X3-0.046X4-0.042X5-0.025X6-0.065X7-0.060X8-0.116X9-0.047X10-0.144X11；(4)Y=一0.012X10.024X2+0.050X3+0.052X4-0.162X5-0.220X6-0.120X7-0.045X8-0.138X9-0.093X10+0.004X11。.