| The black rice of China is widely cultivated,and its varieties account for 90.8%of the world’s black rice resources.The main producing areas are distributed in Shaanxi,Yunnan,Hunan and other places.Black rice is rich in phenolic active ingredients and has preventive and health effects on chronic diseases such as cancer,cardiovascular disease and diabetes caused by oxidative stress in cells.At present,studies on black rice phenolics mainly focus on total polyphenol extracts,and few studies on specific free and bound phenolic compounds.In this study,black rice was used as raw material to extract total,free and bound phenolic compounds.The differences in the content of active components in extracts and their relationship with antioxidant activity were compared,and the specific phenolic compounds were qualified and quantified.Meanwhile,the protective effect of polyphenol extracts in black rice on Caco-2 cells oxidative stress were studied by cell model.The main findings are as follows:(1)The total,free and bound phenolic compounds in black rice were extracted by acidified methanol ultrasonic extraction,solvent extraction(70%acetone)and ethyl acetate extraction,and purified by AB-8 macroporous resin.The extracts before and after purification were further freeze-dried,and the highest yield of lyophilized powder was unpurified free phenol extract(FP),which reached 5.84%.Except for bound phenol extract before and after purification(BP,PBP),the content of antioxidants in the purified lyophilized powder per unit of purified extract was significantly higher than that before purification(P<0.05).The purified total phenolic extract(PTP)had the highest total polyphenols and total flavonoids,which were 45.86±0.06 mg GAE/g P,107.50±1.23 mg CE/g P,the purified free phenolic extract(PFP)had the highest anthocyanin content,reaching 22.03±0.31 mg Cy-3-G/g P.The polyphenol and anthocyanin content in the bound phenol extract were the least.(2)Three in vitro antioxidant methods were used to detect the activity of black rice polyphenol extract.The results showed that total polyphenols,total flavonoids and anthocyanin content were significantly correlated with antioxidant capacity.The results of each test were expressed in terms of Trolox equivalent per unit mass of lyophilized powder(μmol TE/g P),and the antioxidant capacity of each extract after purification was significantly increased(P<0.05).PTP had the best antioxidant capacity,and its DPPH,ABTS radical scavenging ability and ferric ion reducing antioxidant power were 240.98±6.31 μmol TE/g P,413.35±4.08 μmol TE/g P and 439.80±6.35 μmol TE/g P,respectively+The antioxidant activity of the purified black rice polyphenol extract showed a trend of PTP>PFP>PBP.(3)The results of UPLC-ESI-MS/MS showed that the black rice polyphenol extract contained protocatechuic acid,chlorogenic acid,p-hydroxybenzoic acid,caffeic acid,p-coumaric acid and vanillic acid,and there were two kinds of anthocyanins in the compound(cyanidin-3-glucoside,peonidin-3-glucoside).Among the eight phenolic compounds,protocatechuic acid and cyanidin-3-glucoside were the main components.The content of anthocyanin detected in the purified free phenol extract was the highest,reaching 31069.51±305.37μg/g P(1812.91±17.82 μg/g black rice),and the content of phenolic compounds in each lyophilized powder showed a trend of PFP>PTP>unpurified total phenol extract(TP)>FP>PBP>BP.(4)Purified total and free phenolic extracts with better antioxidation effect in vitro were used for cell experiments.The results showed that PTP and PFP treatments showed protective effects against oxidative stress damage in Caco-2 cells at selected concentrations(62.5 μg/mL,125 μg/mL,250 μg/mL),which significantly increased the survival ofinjured cells,reduced MDA levels,improved SOD enzyme activity,and work best at 250μg/mL.PTP and PFP could reduce ROS levels to varying degrees and significantly increase Glutathione reductase(GR)enzyme activity at 250 μg/mL. |
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