Optimization Of Extraction Process Of Isoflavones From Trifolium Pratense And Study On Their Anti-inflammatory Activit

Red clover(Trifolium pretense L.)is rich in isoflavones and polysaccharides,which have anti-inflammatory,antioxidant,cardiovascular disease prevention,and estrogen-like effects.Therefore,it is important to improve the extraction method and explore the pharmacological mechanism of isoflavones of Red clover.to promote the medicinal development of isoflavones of Red clover.1.The method was optimized for the quantification and extraction of the isoflavones of red clover by High-performance liquid chromatography-diode array detection(HPLC-DAD)combined with response surface methodology.The results showed that the HPLC-DAD method was able to accurately quantify the daidzein,genistein,and Biochanin A,which are the main components of isoflavones in red clover.A single-factor test screening followed by a three-factor,three-level response surface test was conducted to optimize the extraction method of isoflavonoids from red clover.The results showed that the relevant factors affecting the extraction amount of isoflavones from red clover were: material-to-liquid ratio(29)herb particle size(29)ethanol volume fraction,the best extraction process conditions were: ethanol volume fraction86%,material-to-liquid ratio 1:29(g/m L)and herb particle size 40 mesh,and the relative error between the extraction amount of isoflavones and the theoretical value was The relative error between the extraction amount of isoflavones and the theoretical value was 0.403%,and the process model was well fitted.2.An in vitro inflammation model of 3D4/2 cells induced by Lipopolysaccharide(LPS)and Porcine circovirus type 2(PCV2)was established,and the mechanism of in vitro anti-inflammatory activity of red clover isoflavones was investigated by RT-q PCR and ELISA to detect the changes of cellular inflammatory factors.The results showed that in the macrophage inflammation induced by LPS and PCV2,the isoflavones of red clover could dose-dependently inhibit the m RNA expression of three inflammatory factors,IL-1β,IL-6,and TNF-α,and promote the m RNA expression of antiinflammatory cytokine IL-10,and the secretion of related inflammatory factors showed the same trend.3.Western Blot technique was used to detect the changes in the expression of pp65,p65,IκB,and p38 MAPK proteins,which are key proteins in immune regulation,to explore the mechanism of the effect of red plantain isoflavones on inflammatory pathways.The results showed that red clover isoflavones have good anti-inflammatory activity,and their effects were related to their down-regulation of the NF-κB signaling pathway and p38 MAPK signaling pathway.