Study On The Effect Of Oil Bases On Cellular Antioxidant Activities Of Lipid Concomitants

Three active ingredients,?-tocopherol,?-oryzanol and sterols,as minor constituents in vegetable oils,have good antioxidant function.The highest proportion of glycerides in vegetable oils has large differences in properties due to carbon chains with different lengths and saturations.The effect and mechanism of this difference on the lipid concomitant exerting antioxidant function in cells are not yet clear.In view of this,two typical vegetable oil bases,rice bran oil?RBO?rich in long-chain unsaturated fatty acid glycerides and coconut oil?CNO?rich in medium-chain saturated fatty acid glycerides were selected in this study.The emulsions containing lipid concomitant with different concentrations were prepared by the high-pressure homogenization method and were delivered to HepG2 cells.The effect and mechanism of different types of oil-based emulsions on the cellular antioxidant activity?CAA?of three lipid concomitants of?-tocopherol,?-oryzanol and sterol in cell models were studied.The main research contents were as follows:Firstly,the high-pressure homogenization process conditions were optimized,two typical oils of rice bran oil and coconut oil were selected,and homogeneous and stable emulsions were prepared.The effects of oil compositions on zeta potential,particle size distribution,and physical storage stability of the emulsions were studied.The results showed that the mean particle size of the emulsions were small,and the minimum polydispersity coefficient?PDI?value was less than 0.2 when the oil-water ratio was 1:9?w/w?;As the homogenization pressure and the cycle times increased,the mean particle size of the emulsion decreased in the range of250 nm-300 nm;When the homogeneous pressure were 60 MPa and the cycle times were 3,the mean particle size were about 250 nm,the PDI values were less than 0.2,the zeta potentials were about-30 mV,and the emulsion systems were uniform and stable.The zeta potential values of RBO emulsions with long-chain unsaturated glyceride was significantly lower than that of CNO emulsions with medium-chain saturated glyceride?p<0.05?;In the particle size distribution diagram,the droplet size distributions of the three RBO?RBO-D,RBO-P,and RBO-S?emulsions were unimodal and the peak width gradually increased with the increase of lipid concomitant content,while that of the CNO?CNO-C,and CNO-J?emulsions were unimodal and the peak widths were narrow,which meant RBO was more likely to form larger droplets;When stored at 4?for 25 d,the mean particle sizes of RBO emulsions did not change significantly but that of CNO emulsions increased by about 8%,which meant the physical storage stability of RBO emulsions were better than that of CNO emulsions.Secondly,a HepG2 cell model was constructed to evaluate the antioxidant function of the emulsions.Cellular uptake and fluorescence signal labeling of dichlorofluorescein?DCF?confirmed that HepG2 cells could effectively evaluate the antioxidant function of the lipid concomitants in the emulsion system.Cellular uptake experimental data showed that compared with the blank cell group?C16:0/C18:2=2.92?,RBO?C16:0/C18:2=0.46?emulsion-treated cells?C16:0/C18:2=1.08?significantly decreased?p<0.05?,the intracellular C12:0/C16:1 of CNO emulsion treatment increased significantly from 0.67 to 23.13?p<0.05?;After treatment for 1 h,1.6%of oryzanol entered the cells;After oil red O staining,the shape of HepG2 cells after emulsions treatment was relatively smooth,and bright red lipid droplets appeared;Fatty acid/concomitant data and morphological characterization results proved that the lipid had entered the cells in the form of emulsions.In HepG2 cells,RBO emulsions and CNO emulsions inhibited the increase in fluorescence produced by DCF oxidation in a dose-dependent manner,and R²>0.9,showing a good dose-response relationship.The half effective concentration(EC₅₀)of RBO emulsions and CNO emulsions were 5.87 mg/mL?RBO-D?,4.63 mg/mL?RBO-P?,0.58 mg/mL?RBO-S?,4.58 mg/mL?CNO-C?and 6.40 mg/mL?CNO-J?.The CAA value of CNO-C emulsion was not significantly different from that of RBO-P emulsion?p>0.05?,but was significantly higher than that of RBO-D emulsion?p<0.05?.In the oxidative stress model induced by 400?M concentration of H₂O₂,the intracellular antioxidant enzyme activities showed that the glutathione peroxidase?GSH-Px?enzyme activities of the cells treated with CNO-C emulsion were significantly larger than those treated with RBO-P emulsion?p<0.05?.Finally,the lipid concomitants were removed by the preparative chromatography column to obtain oil-based.The lipid concomitant with different concentrations was added back to the oil bases,due to investigate the effect of oil base on the cellular antioxidant activities of lipid concomitants.Subsequently,two kind of idealized oil bases of glyceryl trioleate?GLT?and medium chain glyceride?MCT?were selected to clarify the cellular antioxidant mechanism of lipid concomitant in different types of oil bases.The results showed that the CAA unit values of?-tocopherol and?-oryzanol in CNO oil-based emulsions were significantly higher than those in RBO oil-based emulsions?p<0.05?,and the CAA unit values of sterols gradually increased with increasing content in RBO oil-based emulsions,while there were no significant changes in CNO oil-based emulsions?p>0.05?.However,unlike cell systems,the oxygen radical absorption capacity?ORAC?values of?-tocopherol in RBO oil-based emulsions were significantly higher than that in CNO oil-based emulsions?p<0.05?,and there were no significant differences in the ORAC values of?-oryzanol and sterols in the two oil-based emulsions?p>0.05?.It was further verified that the cellular uptakes of?-tocopherol and?-oryzanol were significantly greater in GLT oil-based emulsions than in MCT oil-based emulsions?p<0.05?,but the CAA unit values of them were significantly higher in MCT oil-based emulsions than in GLT oil-based emulsions?p<0.05?.It was speculated that long-chain unsaturated glycerides?RBO and GLT oil bases?were more conducive to cell delivery for?-tocopherol and?-oryzanol than medium-chain saturated glycerides?CNO and MCT oil bases?;But the glycerides with unsaturated bonds were easily attacked by free radicals,and antioxidants were needed.Therefore,?-tocopherol and?-oryzanol reflected lower cellular antioxidant activities in long-chain unsaturated glycerides than that in medium-chain saturated glycerides.In summary,the cellular antioxidant capacity of the lipid concomitant in the medium-chain saturated glyceride was higher than that in the long-chain unsaturated glyceride.