| OBJECTIVESIn order to make full use of Radix Clematidis Henryi resource in China and exploit a medicine to cure RA, this research do some work in this area. First, we want to build a method to determine Total saponins of Radix Clematidis Henryi (TsCH). Second, we want to optimize the extraction, purification technique conditions of TsCH. Third, we investigate the effects of TsCH on collagen-induced arthritis Sprague-Dawley rats (CIAs), and preliminary study the mechanism.METHODS1. Content determination method of TsCHBy selecting Oleanolic acid as control index, colorating with 5% HClO4 and vanillin-CH3COOH, determine absorbability in the point of 550nm with ultraviolet-visible absorption spectroscopy and calculate content of TsCH.2,The optimization of extraction conditions for TsCHOn the basis of single factor experiment, designed L9(34) orthogonal test with the extraction rate of total saponins as index, to determine the rational microwave extraction process; then comparing it with heating reflux extraction and transonic extraction.3,The optimization of purification process for total saponins of TsCH5 kinds of macroporous resin were selected from different companys according to their different polarity. Static absorption indicated that D101 have a good absorptive character of TsCH.4,Anti-inflammatory effect of Clematis henryi Oliv. extract in collagen-induced arthritis rats The crude ethanolic extract of Radix Clematidis Henryi was subjected to macroporous resin. The extract was administrated to collagen-induced arthritis (CIA) Sprague-Dawley rats in the dose of 150 and 50mg·kg-1·d-1 by intragastrically administration, lasting 5 weeks, and the volume meter and thickness of paw were measured. The levels of IgG,IL1-β,TNF-αin their blood were detected and histopathology of arthritis were examined after execute rats at the end of the experiment.RESULTS1. Content determination method of TsCHThe results show that the method is linear in the range of 0.036～0.216 mg (R2=0.9994), absorbability stability within 120min, RSD 1.97%, moreover, the average recovery 97.9%, RSD 2.08%(n=9).2,The optimization of extraction conditions for TsCHThe optimum condition of microwave extraction was as follows: the microwave power was 300 W, the solvent was 70% ethanol, the proportion of raw material to solvent was 1:26, extracting 3 times and 6 min at each time. Under the optimal condition, the microwave extraction was up to about 95%.3,The optimization of purification process for total saponins of TsCHThe optimum technic showe as follows: the optimal concentration of the raw material was 0.2 g·mL-1, pH is 5, washed with 4 BV of 70% ethanol, and the absorption velocity of flow is 1 BV/h and elution velocity of flow is 2 BV/h. The experimental results showed that TsCH in the solid extract could reach 71.56%, well above unpurified of 29.95%, and their refine ratio and diversion ratio reach respectively 238.92% and 71.56%.4,Anti-inflammatory effect of Clematis henryi Oliv. extract in collagen-induced arthritis ratsThe results are as following: Intragastrically administration of Radix Clematidis Henryi extract exhibited anti-inflammatory activities (P<0.05) in a dose-dependent manner. It relieves symptoms and improves pathological changes to CIA rats. Compared with model group, Radix Clematidis Henryi extract reduce the volume meter and thickness of paw, lower the levels of IgG,IL1-β,TNF-α. Histopathological research shows that the extract of Radix Clematidis Henryi eases joint inflammation and reduces synovial hyperplasia. The anti-inflammatory effect is probably associated with regulating the levels of IgG,IL1-β,TNF-α.CONCLUSIONS1 This study provides a method to prepare TsCH.2 The animal experiment confirms the claimed utilization of Radix Clematidis Henryi against inflammatory complaints. |
PDF Full Text Request