| The accumulation of reactive oxygen species(ROS)in cells will lead to oxidative stress,result in oxidative damage of cells or tissues,and cause a variety of diseases.α-Tocopherol andγ-oryzanol are natural minor constituents in vegetable oils,which exert great antioxidant activity.Previous research found that,α-tocopherol and γ-oryzanol have certain antioxidant interaction in vitro,and the effects in different media are different.However,whether there is antioxidant interaction in cells after digestion and absorption,and the mechanism is not clear.Based on this,in vitro digestion and cellular antioxidant evaluation model in Hep G2 cell were used to study the antioxidant interaction of α-tocopherol and γ-oryzanol and interaction mechanism between the two compounds were also illustrated.The results are as follows:First of all,the in vitro digestion model was used to investigate the changes of physical stability and antioxidant activity of α-tocopherol and γ-oryzanol during digestion.The results showed that in the process of digestion,the concentrations of α-tocopherol and γ-oryzanol were decreasing and the contents were lost,indicating that digestion will affect the stability of minor constituents.On one hand,it is due to the influence of digestive fluid and p H value at different stages.On the other hand,the addition of minor constituents increased the particle size(184.5～274.6 nm)and zeta-potential(-48.9～-33.5 m V)of emulsion,indicating the aggregation of emulsion droplets,which reduced the binding site between lipase and emulsion droplets,reducing the degree of lipolysis(92%～62%),and finally reduced the content minor constituents in the process of digestion.However,α-tocopherol and γ-oryzanol showed good antioxidant activity during in vitro digestion,up to 477.94 ± 45.3 μmol TE/m L digests.Besides,the antioxidant activity of the mixed group(341.77 ± 66.5～418.14 ± 14.3 μmol TE/m L digests)was always higher than that of single addition group(213.77 ± 19.2～371.85 ± 52.0 μmol TE/m L digests).Secondly,cellular antioxidant assay model and combined index method were used to explore the antioxidant activity and interaction between α-tocopherol and γ-oryzanol in Hep G2 cells.The results showed that the cellular antioxidant activity(CAA)of α-tocopherol or γ-oryzanol showed an increasing trend with the increase addition of minor constituents(α-tocopherol of 0.2～4 μg/m L and γ-oryzanol of 2～40 μg/m L),the addition concentration was positively correlated with CAA value.However,when α-tocopherol and γ-oryzanol were mixed with the same ratio of 1:10 at different concentrations,the CAA value showed an increased trend first and then decreased,and in TO11(1 μg/m L α-tocopherol and 10 μg/m L γ-oryzanol),the maximum peak value reached up to 94.84 ± 4.2.In addition,after calculation,the combined index(CI)value of the mixed group showed a trend of first decreasing and then increasing,indicating that the synergistic effect between the two increased first and then decreased.In the combination of TO11,the CI value is 0.32(CI > 1 indicates antagonism,CI < 1 indicates synergy),showing the strongest synergistic effect.However,in the combination of high concentration TO44,the CI value was 3.3,showing a strong antagonistic effect.Finally,the mechanism of synergistic antioxidant effect of α-tocopherol and γ-oryzanol was explained from three aspects including cellular uptake,intracellular ROS level and endogenous enzyme activity.The results showed that the cellular uptake increased with the single addition of α-tocopherol and γ-oryzanol increasing.But when mixed,the cellular uptake of α-tocopherol and γ-oryzanol increased first and then decreased,and reached the maximum in the combination of TO11,up to 0.65 μg/mg pro and 11.87 μg/mg pro respectively,which was consistent with the results of CAA,indicating that the cellular antioxidant activity is related to the cellular uptake of minor constituents,and the cellular uptake of γ-oryzanol in the mixed group was significantly higher than that in the single addition group(p < 0.05),indicating thatα-tocopherol could promote the cellular uptake of γ-oryzanol.The results of ROS level and endogenous enzyme activity including superoxide dismutase(SOD),catalase(CAT)and Glutathione peroxidase(GSH-Px)showed that after entering cell,in the combination of TO11,α-tocopherol and γ-oryzanol played a synergistic antioxidant effect by scavenging ROS and up regulating GSH-Px enzyme activity,resulting in the strongest cellular antioxidant activity.In the combination of TO44,cellular uptakes of mixed group were lower than that of the single addition group(p < 0.05).Although the combination of α-tocopherol and γ-oryzanol showed a synergistic effect on the activities of the three endogenous enzymes,the ROS scavenging ability showed strong antagonistic effect,which made the combination show antagonism.In all,the antioxidant interaction of α-tocopherol and γ-oryzanol in cells is related to cellular uptake of minor constituents,and finally affect the antioxidant activity of minor constituents in cells.In conclusion,the digestion process had a certain effect on the stability and antioxidant activity of α-tocopherol and γ-oryzanol.After digestion and enter into cells,The combination of 1 μg/m L α-tocopherol and 10 μg/m L γ-oryzanol exert greatest antioxidant activity and synergistic effect,which was positively correlated with the cellular uptake of minor constituents.It played a synergistic effect in scavenging ROS and up regulating GSH-Px activity in cells.This paper explained the synergistic effect of minor constituents in cells,which can provide a theoretical basis for the development of related functional foods and rational dietary collocation. |
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