Protective Effects Of Vitexin Of Trollions On Oxidative Damaged Erythrocyte

The Trollius Chinensis Bunge belonged to Trollius chinensis of the ranunculaceae. The total flavonoids of trollius chinensis Bunge has antibacterial, anti-viral, anti-tumor, anti-oxidation effects, and so on. Vitexin is Trollius flavonoids monomer, belonging to the flavonoids carbon glycosides. Vitexin has antiviral, antioxidant and anticancer effects in vivo and in vitro, which has a protective effect on ischemic myocardial injury by inhibiting the formation of thrombosis and lowering blood viscosity.In recent years, the study of free radicals and their scavenger has become a hot spot, searching, screening the antioxidant drugs such as flavonoids that have blocked the formation of free radicals or inhibited cell membrane peroxidation activity, which has been paid more and more attention to. At present, there is more study of polyphenols, quercetin, soyisoflavone, that belong to flavonoids antioxidant activity and mechanism. However, currently polyphenols compounds such as apigenin, quercetin, tea polyphenols research on erythrocytes protection has been wideiy studied, and yet the vitexin role of oxidative damage to human erythrocytes research has not been reported.In this paper, human erythrocytes was uesed as experimental material, the model of hydrogen peroxide on erythrocyte oxidative damage was established, taking vitamin C(VC) as a positive control, researching the impact of vitexin and apigenin of the protection and restoration of the role of oxidative damage on human erythrocytes, observing the protective structure of vitexin and apigenin on erythrocytes morphology by scanning electron microscopy(SEM) and transmission electron microscopy(TEM), and comparing the antioxidant capacity and structure-activity relationship of vitexin glycosides and apigenin.Establish the oxidative damage model:Taking erythrocytes hemolysis rate as the index, prepare2%erythrocytes suspension, with the addition of a series of different concentrations of H2O2saline solution to incubate at37℃, sampling to determine hemolysis ratio every30min. Ultimately, the damage concentration of H2O2being400mmol·L-1and incubation time1.5h were determined.The experiment was divided into normal control group, model group, the vitexin of different dosage groups(15,30,60μg·mL-1), apigenin of different dosage groups(15,30,60μg·mL-1) and VC(30μg·mL-1) positive control group. Use ultraviolet spectrophotometer to detect the hemolysis rate; through the kits to detect the activity of antioxidant enzymes (including GSH-Px, SOD and CAT) and ATPs enzyme, the content of lipid peroxidation product, MetHb, hydrosulphonyl(-SH), and ROS; using SDS-PAGE to analyze erythrocytes membrane protein and observe the erythrocytes surface morphology and superfine structure by SEM and TEM.The results showed as follows:Compared with normal group:the erythrocyte hemolysis rate, Malondialdehyde(MDA) content, MetHb content, ROS content of model group were increased with significant difference (P<0.01); Antioxidant enzymes activity and mercapto group content and ATPs enzyme activity was significantly decreased, with a significant difference(P<0.01); SDS-PAGE analysis showed that model group of membrane protein has aggregated, some protein bands has reduced or even disappeared; SEM observations showed that the cell surface shape deformation, shrinkage, perforation, aggregation of pathological has changed; TEM observations showed that the membrane skeleton in the junctional complex (J) has disappeared, contractile proteins four polymers (SP4) has fractured, and network structure has lost.Compared with model group:each drug group can effectively reduce hemolysis rate and the content of MDA, ROS and MetHb, with a significant difference(P<0.01); except for vitexin15μg·mL-1dosage group, it has no significant effection to ATPs activity, each drug group could improve the antioxidant enzymes, ATPs enzyme activity and content of mercapto group, with a significant difference(P<0.01, P<0.05); the results of SDS-PAGE showed that each drug group can inhibit the membrane protein aggregation in a certain extent, prevent the decrease of spectrin and band3protein, of which only apigenin and VC can inhibit actin bands disappearing; the results of SEM showed that each drug group could significantly improve the erythrocyte shrinkage, spinous process, perforation, aggregation phenomenon; the results of TEM showed that each dosage drug group can be recovered to J point at different extents, gradually make SP4become complete and orderly, and reshape the network structure of the membrane skeleton, and this improvement capacity was positively correlated to the concentration of the drug.Compared with Apigenin group:the capability of reduce hemolysis rate and content of MDA, ROS and MetHb, increased antioxidant enzymes, the ATPs enzyme activity and mercapto group content, vitexin15μg·mL-1and30μg·mL-1dosage group activity was lower than the apigenin equivalent dosage group with a significant difference (P<0.01, P<0.05); the activity of vitexin60μg·mL-1dosage group was higher than the apigenin equivalent dosage group, with a significant difference (P<0.01). The results of SDS-PAGE revealed that vitexin activity was weaker than the apigenin group to recovery of spectrin, ankyrin and actin proteins. The results of SEM showed that, the ability of15μg·mL-1and30μg·mL-1concentration vitexin on erythrocytes morphology resilience was weaker than the same dosage of apigenin group, the vitexin60μg·mL-1dosage group compared at the same dosage of apigenin group can better restore the surface morphology of the erythrocytes. The results of TEM showed that, the vitexin60μg·mL-1dosage group can significantly restore the unique network structure of the erythrocytes membrane skeleton that has the similar effect as Apigenin30μg·mL-1dosage group.Compared with VC group:there is no significant difference of CAT enzyme,60μg·mL-1dosage group of vitexin,15μg·mL-1and30μg·mL dosage group of Apigenin and the VC group on the protective effect; there is no significant difference of60μg·mL-1dosage group of vitexin and30μg·mL-11dosage group of Apigenin and the VC group on reducing hemolysis rate, the content of MDA, ROS and MetHb, increasing the GSH-Px, SOD, ATPs enzyme activity and protect of sulfhydryl, but the activity of15μg·mL-1and30μg·mL-1dosage group of vitexin and15μg·mL-1and60μg·mL-1dosage group of Apigenin were weaker than the VC group, with significant difference(P<0.01, P<0.05). SDS-PAGE analysis revealed that the capability of inhibiting membrane proteins aggregation and disappearance of vitexin and apigenin were weaker than the VC group. SEM showed that, the30μg·mL-1dosage group of VC is better than same dosage of vitexin and apigenin group in the capability of repairing the surface morphology of erythrocyte. TEM showed that the60μg·mL-1dosage group of vitexin, the30μg·mL-1dosage group of apigenin and the30μg·mL-1dosage group of VC can effectively restore erythrocyte membrane skeleton structure.In summary, vitexin and apigenin have protective effects on erythrocyte which kept it from oxidative damages. The protective effect on erythrocyte was achieved through inhibiting erythrocyte hemolysis, enhancing the activity of antioxidant enzymes in the cell, protecting ATPase and sulfhydryl, reducing the lipid peroxidation product and the formation of MetHb, quenching excess ROS in the cell, inhibting membrane protein aggregation, keeping the normal surface morphology of erythrocyte and membrane skeleton structure.Compared with Vitexin, the apigenin has stronger antioxidant activity at low concentrations. With the increase of concentration, apigenin’s antioxidant activity was reduced, and it may be associated with pro-oxidation effect of flavonoid compounds, when the concentraton reaches a certain threshold, apigenin itself occurs oxidization or some metal ions or peroxide enzyme in the experiment system inducing oxidation, producing free radicals, and then appears prooxidane effect.The antioxidant ability of vitexin’s low, middle concentration group and apigenin’s low, high concentration group were weaker than the group of VC, the antioxidant ability of vitexin’s high concentration group and apigenin’s middle concentration group were similar to that of VC.