Study On Extraction Of Active Ingredients From Rosa Davurica Pall.and Their Anti-Inflammation And Immune-Enhancing Activities

Rosa davurica Pall.,a subgenus of genus Rosa genus with high nutritional and medical values.Its mature frute was regarded as,,special medicine for the treatment of scurvy''in many European countries.In Chinese ethnomedicine,Rosa davurica Pall.was used to make medicinal wine,decoction and tea to treat conditions such as indigestion,heartburn,diarrhea and irregular menstruation.It has been reported that flavonoids,saponins and polysaccharides are the main bioactive components of Rosa davurica Pall.Besides,it has been proved by morden researches that Rosa davurica Pall.is of great pharmacological activities such as anti-oxidation,anti-tumor,anti-aging,protection of cardiovascular system,hypoglycemic and immunity system enhancement.In this study,natural product separation and extraction technology were introduced to obtain the major substances of Rosa davurica Pall.,modern molecular biology methods were carried out to evaluate the bioactivities of Rosa davurica Pall.and to explore the mechanisms.The main results of this work are as below:Major substances were obtained from Rosa davurica Pall.,including water extract,70%alcohol extract,chloroform extracts(CHL),ethyl acetate extracts(EA),n-butyl alcohol extracts(n-Bu),water phase residues,flavonoids(30%Fla,50%Fla,70%Fla,90%Fla),saponins(30%Sap,50%Sap,70%Sap,95%Sap)and polysaccharides.It was showed that30%Fla,50%Fla,30%Sap and 50%Sap,which were of higher contents of flavonoids and saponins,were capable of exerting more significant antioxidant activities.The flavonoids and saponins of Rosa davurica Pall.exhibited favorable activities in alleviating CCL₄-induced and H₂O₂-induced hepatocyte injury;HepG2,A549,MDA-MB-231and MCF-7 cancer cell lines were introduced to evaluate the anti-tumor capicities.It was suggested that the active substances with significant antitumor abilities were mainly concentrated in EA and n-Bu fraction;3T3-L1 preadipocytes was induced by cocktail method and it was showed that flavonoids and saponins could extert certain inhibitory effects on lipid accumulation in 3T3-L1 cells,among which the effect of 30%Sap was of the best;Polysaccharide showed more significant immune-enhancing potency in cytophagocytic neutral red test;LPS-induced RAW264.7 cell inflammation model was introduced to test the anti-inflammation effect,flavonoids and saponins were determined as the main ati-inflammatory components.The results of ELISA,RT-qPCR and Western Blot showed that the flavonoids and saponins of Rosa davurica Pall.could significantly inhibit the secretion of IL-6 and TNF-?,down-regulate the mRNA expression of iNOS,IL-6,TNF-?and IL-1?,regulate the protein expression of MAPKs/NF-?B signaling pathway,thus inhibiting the inflammatory response and exerting anti-inflammatory activity.LC-MS/MS results indicated that the 50%Fla of Rosa davurica Pall.contained kaempferol-3-O-rutin and ursolic acid.Meanwhile,hyperoside,luteolin,ursolic acid and euscaphic acid were found in 30%Sap of Rosa davurica Pall.Polysaccharides were separted and purified by DEAE ion chromatography column,and three homogeneous polysaccharides(P-?,P-?,P-?)and three heterogeneous polysaccharides were obtained.Physical and chemical properties of polysaccharide samples were further analyzed by spectrophotometry and infrared detection.It was proven by NO releasing test of macrophages that P-?,P-?,P-?,P-?,P-?were of satisfying immune-enhancing activities.RT-q PCR results suggested that purified polysaccharides could up-regulate the m RNA expression of cytokins including IL-1?,IL-6,iNOS and TNF-?.Finally,the protein expressions of IL-1?,IL-6 and TNF-?and the phosphorylation levels of ERK,JNK and P38 proteins(which played key roles in immune regulation)were evaluated by Western Blot analysis.It was shown that purified polysaccharides from Rosa davurica Pall.played an immunomodulatory role by regulating the expression of inflammatory cytokine proteins and regulating the phosphorylation levels of MAPKs pathway proteins.