Optimization Of The Extraction Process Of Polysaccharide From Tibetan Medicine Cremanthodium Discoideum And Its Effect On Macrophage Function

Cremanthodium discoideum Maxim is a compositae perennial alpine meadow vegetation,growing in the Himalayas,endemic to 3600～5300 m above sea level(concentrated in the Tibetan Plateau and southwestern mountains).At present,research on Cremanthodium discoideum is mainly focused on the antibacterial,antiinflammatory,antitumor and antiviral effects of sesquiterpenoids and flavonoids as active ingredients,while the research on other active ingredients is less reported.In this study,the main factors affecting the extraction of polysaccharides were extraction time,extraction temperature and material-to-liquid ratio as the independent variables,and the polysaccharide yield of the aqueous extract as the response value,using the Tibetan medicine Cremanthodium discoideum as the object of study.Optimization of the aqueous extraction process of Cremanthodium discoideum polysaccharides based on single-factor tests using a three-factor,three-level response surface analysis(RSM)with a central combination design.Cremanthodium discoideum polysaccharide was used as the study material,and mouse peritoneal macrophages RAW264.7 were used as the study object.Detection of the effect of Cremanthodium discoideum polysaccharide on macrophage toxicity or proliferative function by CCK-8.EA/YC wreaths elaborate the effect of Cremanthodium discoideum polysaccharide on macrophage Fc/C3 b receptor function.Neutral red test to detect the effect of Cremanthodium discoideum polysaccharide on macrophage phagocytosis.The effects of Cremanthodium discoideum polysaccharides on the secretory function of macrophages(NO,IL-6,IL-1β,TNF-α,IFN-γ,LZ)were determined by ELISA Cremanthodium discoideum polysaccharide was used as the study material and mouse peritoneal macrophage J774 A.1 was used as the study object,Hoechst/PI staining combined with ELISA were used to study the effect of Cremanthodium discoideum polysaccharide on LPS+ATP-induced system macrophage pyroptosis(Caspase-1,Caspase-3,IL-18,IL-1β,LDH).The results are as follows:(1)In the single-factor test combined with response surface analysis,the best aqueous extraction process for the Tibetan drug Cremanthodium discoideum polysaccharide was 2.5h extraction time,1:20 material-to-liquid ratio in 75℃,and the actual average extraction rate is 4.99%,similar to the theoretical value of 5.17%.(2)CCK-8 cytotoxicity and proliferation assay showed that the maximal non-toxic concentration of Cremanthodium discoideum polysaccharide in macrophages RAW264.7 was 1000 μg/m L in the range of cellular non-toxic concentration,where31.25～1000 μg/m L all promoted macrophage RAW264.7 proliferation(500μg/m L,the highest value-added rate).Cremanthodium discoideum polysaccharide at 31.25～500μg/m L mass concentration had a promotive effect on macrophage Fc/C3 b receptor activity(500μg/m L,EA and YC wreath rate were the highest,63.38% and 74.25%,respectively);31.25～500 μg/m L mass concentration Cremanthodium discoideum polysaccharide enhances macrophage phagocytosis and exhibits dose-dependent characteristics.(3)Griess and ELISA assays showed that Cremanthodium discoideum polysaccharides at mass concentrations of 31.25～1000 μg/m L promoted the secretion of NO,IL-6,TNF-α,IL-1β,IFN-γ and LZ by macrophages and exhibited dosedependent characteristics.(4)Hoechst/PI and ELISA assays showed that Cremanthodium discoideum polysaccharides at 62.50～500 μg/m L mass concentrations had varying degrees of inhibition of macrophage pyroptosis induced by LPS+ATP(inhibition of macrophage secretion of Caspase-1,IL-18,IL-1β and LDH),Cremanthodium discoideum polysaccharide at 125～500 μg/m L mass concentration inhibits the activation of inflammatory vesicles NLRP3 and thus macrophage scorching by enhancing the enzymatic activity of AC and PKA in the c AMP-PKA pathway.In conclusion,the Tibetan medicine Cremanthodium discoideum polysaccharide enhances the immune adhesion ability and phagocytosis of macrophages by enhancing the activity of macrophage Fc/C3 b receptors,promotes the secretion of macrophages to regulate the immune function of the body,and inhibits the occurrence of macrophage pyroptosis to enhance the anti-inflammatory ability of the body.The results of the study provide data to support the clinical application and in-depth development of the Tibetan medicine Cremanthodium discoideum.