Study On Antioxidant Activity Of Polyphenol Compounds In Wheat Bran

Polyphenol compounds, which are found in nature, are very important natural antioxidants. They achieve antioxidant effects through direct scavenging of free radicals, forming complex metal ions, producing antioxidant enzymes in the body, regenerating and improving immunity. These ways included directly scavenging free radicals, complexing metal ions, acting on antioxidant enzymes in the body, regenerating and synergies of antioxidants and improve immunity in the body. Recent studies showed that wheat bran, a byproduct of wheat flour processing, contains wheat cortex, which includes aleurone and endosperm part, both of which are rich in nutrients, antioxidants, and polyphenols. However, wheat bran has not been used extensively in the antioxidative applications. In this study, wheat bran was chosen as the raw material for its polyphenol compounds. Experimental conditions were optimized for the isolation and purification of polyphenol compounds from wheat bran. The antioxidant activity of extracted polyphenol compounds were studied in vivo to determine its suitability for commercial applications.In current study, different solvent extraction methods were used during the preparation of wheat bran polyphenol. The methods of total phenolic content and DPPH? radical scavenging activity as comparing parameters were used to determine the optimal ratio of solvent and degrees. Through our study, 80% aqueous ethanol was determined as the best extraction method for the polyphenol compounds. The resins, which are suitable for enriching wheat bran polyphenol compounds, were screened through static adsorption experiments and dynamic adsorption experiments from four macroporous resin. The results of static adsorption experiment showed that the adsorption capacity of four resins listed in decreasing order as H1020> DA201-C> NKA-II> NKA-9; whereas the desorption rate in descending order of NKA-9> DA201-C> H1020> NKA-II. The optimum adsorption conditions of dynamic adsorption experiments were as follow: column bed volume(BV) was 350 m L, the concentration of sample was 3 mg/m L, the rate of the column was 2 m L/min, the injection volume was 400 m L. Ethanol was used in gradient for elution after loading with samples. The concentrations(V / V) of ethanol were 20%, 40%, 60%, 80%. UV detector was selected at 310 nm for detection and collection of the sample solution and eluent samples. Sixteen kinds of enrichments, which were defined as wheat bran polyphenol compounds(WBPE) were obtained. All the samples were carried out in accordance to the following experiments to determine the best method of enrichment.Many reports showed that wheat bran polyphenol compounds in wheat bran was mainly phenol acid, dominated by ferulic acid. In this paper, Acquity UPLC-TQD technology was used for detection and analysis of polyphenols known in 16 kinds of WBPE. The starting condition for each experiment was 5:95 mobile phase A(acetonitrile):mobile phase B(water+0.1%(v/v) formic acid) held for 0.8 min, with a ramp-up to 10:90(A:B) by 1.2 min, then to 15:85 by 2.4 min, and held for 1.3 min. Further gradient increases were carried out to 21:79 by 4.0 min and to 50:50 by 7.8 min, with a change to 100:0(A:B) by 8.8 min and held for 0.5 min, and finally, reconditioned to initial starting conditions. The mass acquisition parameters of standard samples as follows: m/z 163.08> 119.64 3-coumaric acid; m/z 163.08> 119.15 4-coumaric acid; m/z 193.00> 134.10 ferulic acid; m/z 169.05> 125.00 gallic acid; m/z 181.08 > 43.03 olivtol. ES+ was used for olivetol, others used ES-. The analysis and comparison of the contents of several polyphenolic compounds in samples were showed that ferulic acid had the highest concentration, 4-coumaric acid as follow, the contents of other three polyphenol compounds were few. WBPE-N9-4 had showed the highest value of ferulic acid, 4-coumaric acid and the amount of five kinds of polyphenol compounds 16 kinds of WBPE.Subsequent study focused on the levels of cell on the antioxidant activity of WBPE. The study was used to construct H2O2-induced HEK293 cells oxidative injury model, which ultimately resulted in the injury model that was based on the concentration of H2O2 at 1 mmol/L, and the cells seeding per well at 3 × 104, and the reaction time was 2 h. The antioxidant activities of 16 kinds of WBPE were analyzed in this cell model through comparing the results of the cell viability and ROS. The results showed that the antioxidant capacity of WBPE-N9-4 was the strongest for experiments. The morphological changes of injured cells were examined using inverted phase contrast microscopy. The results showed that after treatment with WBPE-N9-4 the number of damaged cells was remarkable reduction, maintaining the morphological of the cells, reducing the cells shrink and DNA fragmentation. The antioxidant acitvity of intracellular antioxidant enzymatic system and the content of lipid peroxidation products were measured using assay kits. The results showed that WBPE-N9-4 significantly attenuated the increase the activities of SOD, CAT, GSH-Px in HEK293 cells and decreased MDA levels. H2O2 could cause damage on HEK293 cells, resulting in a significant increase in the rate of LDH release(13.09%). However, pretreatment with WBPE-N9-4(0.10 mg/m L-1.00 mg/m L), reduced the rate of LDH release in HEK293 cells from 13.09 ± 1.14%(model group) to 11.86 ± 1.05% to 7.64 ± 0.40%, LDH release was significantly lower(p <0.05). The cell cycle, cell apoptosis and mitochondrial membrane potential were measured using flow cytometry. The results showed that WBPE-N9-4 could regulate the proportions of cell cycle, reducing the proportion of Sub-G1 peak; while reducing cell apoptosis and inhibiting the decrease of mitochondrial membrane potential.The experiments for in vivo feeding of WBPE-N9-4 in high-fat diet mice, showed that the mice feeding WBPE-N9-4 can improve the antioxidant capacity. As compared to the Control group, high fat diet group(A group) had significantly improve the body weight of mice(p <0.05), the visceral weight changes of mouse(p <0.05), TC, TG and LDL-C in A group were higher than that of the Control group, HDL-C was lower than that of the Control group(p <0.05). The results of tissue sections showed that liver and kidney tissue sections in A group had clearly steatosis vacuoles, cell swelling, although deformation of the nucleus is not clear. However, the polyphenol compounds can effectively control the weight of high-fat diet mice(p >0.05), as compared to A group, the body weight of WBPE-N9-4 group(B, C and D group) were significantly lower, the results were significant(p <0.05). As compared to A group, the mice visceral in group B has no significant change(p >0.05), C, D group has significant change(p <0.05), as compared to the Control group, C group have no statistically significant(p >0.05). The results showed that wheat bran polyphenol compounds can effectively control the visceral changes of high-fat diet mice. Meanwhile, the results of TC, TG, LDL-C in group C and group D were lower than that of group A, and the data were significantly different(p <0.05). The results showed that wheat bran polyphenol compounds have a good effect on lipid regulator of their significant regulatory role on TG and HDL-C. The results of tissue sections showed that B, C and D groups were gradually returning to normal, reducing vacuoles, suggesting that polyphenol compounds have an inhibition effects on liver and kidney tissue fatty disease in high-fat diet mouse.