Optimal Extraction Of Flavonoids From Sea Buckthorn Fruits And Their Protective Mechanism Against Oxidative Stress RPE Cells

Sea buckthorn is widely distributed in China,but its medicinal and edible value still needs to be explored.The flavonoids in sea buckthorn have good antioxidant activity and can be used as a natural source of antioxidants.The discovery of its active ingredients and the further development of high-value-added products will also help to improve the application value of sea buckthorn.In this study,the total flavonoids of sea buckthorn were extracted from sea buckthorn fruits using cellulase-microwave synergistic extraction method,and the extraction conditions were optimized.Subsequently,an ultraviolet B(UVB)-induced oxidative damage model of human retinal pigment epithelium(ARPE-19)cells was established,and the effective polar fraction of sea buckthorn fruit total flavonoids were screened by assessing the protective effect of the samples on damaged ARPE-19 cells.Then the main components of the effective fraction were identified by high performance liquid chromatography.Finally,the effects and mechanisms of action of the effective fraction and its main components on oxidative stress in ARPE-19 cells were investigated.The extraction method of flavonoid components from sea buckthorn fruits was optimized,and the active fractions and components were screened.Through the study of cellular oxidation products,antioxidant enzymes and signaling pathways,the protective mechanism of flavonoids of sea buckthorn on ARPE-19 cells was preliminarily revealed.The main findings were as follows:Synergistic extraction of flavonoids from sea buckthorn fruits using cellulase-microwave.Based on the single-factor test,the influencing factors were enzyme addition,liquid-to-material ratio,and ethanol volume fraction,and the extraction process was optimized using response surface methodology,yielding the optimal extraction conditions for total flavonoids as follows:enzyme addition of 15 mg/g,microwave power of 280 W,liquid-to-material ratio of 50:1,ethanol volume fraction of 50%,microwave extraction time of 6 min,and the extraction rate of total flavonoids was 31.25 mg/g,which was similar to the predicted value.The crude extract obtained under the optimized conditions was extracted in successive steps with petroleum ether,dichloromethane,ethyl acetate and n-butanol,and the extract obtained from the different fractions showed some antioxidant activity with good ferric ion reduction and radical scavenging.Dichloromethane,ethyl acetate and n-butanol were found to have better antioxidant activity in vitro.A UVB-induced oxidative damage model of ARPE-19 cells was successfully established,and the effective fraction of sea buckthorn fruit flavonoids was screened as the n-butanol fraction by assessing the protective effect of the samples on oxidatively damaged ARPE-19 cells.The composition of this site was identified by ultra high performance liquid phase tandem mass spectrometry(UPLC-MS/MS)and high performance liquid chromatography(HPLC),and its main components were identified as isorhamnetin-3-O-glucoside-7-Orhamnoside,rutin and isorhamnetin-3-O-glucoside.The total flavonoid n-butanol component of sea buckthorn fruit and its principal component have been shown to be effective in attenuating UVB-induced oxidative damage in ARPE-19 cells,as indicated by increased cell survival,enhanced antioxidant enzyme activity,reduced lipid peroxidation levels,scavenged intracellular reactive oxygen species,and reduced apoptosis.Protein immunoblotting(Western blot)results indicated that this protective effect might be accomplished by inhibiting Caspase-3 activation,inducing the phosphorylation process of Akt,and activating the Keap1-Nrf2 signaling pathway.