Extraction,Purification And Identification Of Polyphenols From Chaenomeles Speciosa(Sweet) Nakai And Its Antioxidant And Anti-inflammatory Activities

Chaenomeles speciosa(Sweet)Nakai(C.speciosa)belongs to the genus Rosaceae,and has a unique medicinal and health value with abundant polyphenols.Further study on the extraction,purification and biological activities of polyphenols from C.speciosa is of great significance for promoting the development of the C.speciosa industry.This study used C.speciosa as raw material,the surfactant-assisted ultrasound extraction was used to extract the C.speciosa polyphenols,which was then purified with LSA-900C macroporous resin.The chemical composition of the phenolic extract from C.speciosa was analyzed by UPLC-Q-TOF-MS/MS and HPLC.In addition,the total reducing power,DPPH,ABTS,·OH methods,and HepG2 cell oxidative damage model were used to study the antioxidant capacity of phenolic extract,the RAW 264.7 cell inflammation model was used to study the anti-inflammatory effect and mechanism of phenolic extract from C.speciosa.The research results were as follows:(1)Single-factor and response surface experiments were used to optimize the extraction process of phenolic extract from C.speciosa with surfactant-assisted ultrasound.The optimal extraction process was as follows:SDS concentration 0.40 g/L,ethanol concentration 58%,material-to-liquid ratio 1:33 g/m L,ultrasonic temperature 51?,time 30 min,power 200 W.Under these conditions,the yield of C.speciosa polyphenols was 32.42 mg/g.(2)The crude phenolic extract of C.speciosa was purified by the macroporous resins.Among the 6 selected macroporous resins,LSA-900C macroporous resin had the strongest adsorption and desorption capacity.The best adsorption and desorption conditions were as follows:sample solution pH 3,sample concentration of 2.0 mg/m L,the loading flow rate of1.0 m L/min,the loading volume of 100 m L,ethanol concentration of 50%,the elution flow rate of 1.5 m L/min,and the elution volume of 120 m L.After purification,the content of phenolic extract increased from 6.49%to 57.00%,which was about 8.78 times than the crude phenolic extract.(3)The purified phenolic extract of C.speciosa was identified by UPLC-Q-TOF-MS/MS,and then three main polyphenol compounds were preliminarily identified as procyanidin B1,catechins and chlorogenic acid,and their concents were detected by HPLC as 43.73,25.96,and 71.96 mg/g,respectively.(4)The antioxidant ability of phenolic extract of C.speciosa was tested using the methods of total reducing power,DPPH,ABTS and·OH.The results indicated that the total reducing power of phenolic extract of C.speciosa showed a significant dose-dependent effect.The EC₅₀values of Vc,purified phenolic extract and unpurified phenolic extract on DPPH,ABTS,and·OH were 8.10,8.67,55.78 and 9.51,9.97,24.75 and 48.52,272.81,and 1358.77?g/m L,respectively.The results showed that the antioxidant capacity of the purified phenolic extract of C.speciosa was significantly higher than that of the unpurified phenolic extract.(5)HepG2 cells were stimulated with 500?mol/L TBHP to establish an oxidative damage model,and then the antioxidant mechanism of phenolic extract from C.speciosa was studied.The results showed that the treatment with phenolic extract from C.speciosa significantly suppressed the production of ROS and MDA,while the activities of antioxidant enzymes such as SOD and CAT were significantly increased.The mechanism underlying such antioxidant actions might increase the expression of Nrf2 and HO-1 protein and reduce the expression of Keap1 protein,thereby showing an antioxidant effect.(6)A cellular inflammation model was established by RAW 264.7 cells stimulated with 1?g/m L LPS and the underlying anti-inflammatory mechanism of phenolic extract from C.speciosa was studied.The results showed that the phenolic extract exhibited dose-dependent anti-inflammatory effects on LPS-treated RAW 264.7 cells.The extract at 30?g/m L was most potent and enabled most cells in normal morphology under LPS stimulation without causing cytotoxicity.The extract suppressed the levels of NO,TNF-?,IL-6,and IL-1?,as well as the m RNA and protein expressions of iNOS and COX-2.The potential mechanism of this anti-inflammatory effect could inhibit the phosphorylation of I?B?and p65 proteins,thus preventing NF-?B dimer nuclear transport.Besides,it could also inhibit the phosphorylation of p38 and JNK proteins,thereby inhibiting the inflammatory signal transduction in the MAPK signaling pathway.