Extraction And Purification Of Polyphenols From Walnut Meal And Preliminary Study On Its Antioxidation And Lipid Lowering Activity

Objective: This paper studied walnut meal was as raw material to study on extraction and purification of polyphenols from walnut meal and its antioxidation and lipid lowering activity.Methods:1. Using single factor experiment and response surface analysis method studied the effects of the extraction temperature, extraction time, ethanol concentration and the liquid-solid ratio on polyphenols extracts and optimize the polyphenols process conditions from walnut meal. The purification process conditions of walnut meal polyphenols was optimized by using macroporous adsorption resin method.2. The antioxidant capacity of walnut meal polyphenols purification(WMP) by macroporous resin was studied through cleaning hydroxyl(·OH), hydrogen peroxide(H2O2), 2, 2-diphenyl-1-picrylhydrazyl(DPPH) and superoxide anion radical scavenging capacity(·O2-).3. Testing groups of mice serum TG and TC value was to study the WMP lipid-lowering effect on induceing by intraperitoneal injection of yolk emulsion of acute hyperlipemia mice.4. Effects of walnut on the obesity mice induced by sodium glutamate glucose and lipid metabolism research.The mice were tested blood glucose levels given by oral glucose tolerance test(OGTT) experiment. Serum levels of TC, TG, HDL-C, LDL-C, adiponectin, insulin, and glucose levels were analyzed. The TC, TG, HDL-C and LDL-C, aspartate aminotransferase(AST), alanine aminotransferase(ALT), glutathione peroxidase(GSH-PX), superoxide dismutase(SOD) activities and malondialdehyde(MDA) levels of sample liver were measured.5. MTT assay was used to detect the crude extract, the effect of different polar solvent extracts and WMP on the proliferation of 3T3-L1 cells. The 3T3-L1 preadipocyte cells were induced to build model into mature adipocytes and then were measured TG and TC contents to research crude extract, different polar solvent extraction and WMP from walnut meal on the influence of 3T3-L1 cells differentiation. The WMP was used to research the 3T3- L1 mature adipocyte with Annexin V-FITC/PI staining method.6. By ultra high performance liquid chromatography-electron spray ion source mass spectrometry UPLC-ESI-MS scanning, walnut meal polyphenols macroporous resin purification(WMP) relatively higher content, response better components were analyzed.Results:1. The results showed that the best extraction of walnut residue polyphenols was as follows: ethanol 50%, 60 min and 73℃. The verification experiment showed that the average yield of polyphenols was 6.95% in the best extraction conditions and polyphenol content of extract was 26.63%. Results also indicated that the HPD-100 resin was the best due to its highest adsorption and desorption properties. The optimum conditions were: the sample p H 4.0, concentration of the sample 4.0 mg/m L, current velocity 2 BV/h, ratio 1.8 BV of polyphenols volume to the bed volume, 75% alcoholas eluant for desorption, cu rrentvelocity 4 BV/h and eluant volume 3 BV. Under those conditions, the purity of walnut residue polyphenols increased from 26.63% to 81.10% and the recovery was 87.33%.2. The in vitro antioxidant assay results showed that the purified polyphenols dose-dependent scavenging activity against hydroxyl(?OH), hydrogen peroxide(H2O2), 2, 2-diphenyl-1-picrylhydrazyl(DPPH) and superoxide anion radical scavenging capacity(?O2-), with hemi-inhibiting concentrations(IC50) 481.18 μg/m L, 151.43 μg/m L, 8.19 μg/m L and 202.83 μg/m L, respectively, in which the ?DPPH and?OH scavenging stronger than Vc.3. Walnut meal polyphenols hypolipidemic action preliminary study3.1 The intraperitoneal injection of yolk emulsion of acute hyperlipemia mice results showed that the serum of triglyceride(TG) and total cholesterol(TC) levels were significantly higher than normal control group(p<0.01). Compared with model group, the fenofibrate group(p<0.01), WMP high dose group(p<0.05) and middle dose group(p<0.05) significantly decreased TG levels and Fenofibrate group(p<0.01), the WMP high-dose group(p<0.01) and middle dose group(p< 0.05) significantly decreased TC levels in serum.3.2 The efects of WMP on obesity mice induced by sodium glutamate results showed that the blood glucose values increased significantly glucose levels after oral glucose 30 or 60 min(p<0.01). Compared with MSG group, WMP 400 mg/kg/BW and WMP 800 mg/kg/BW administration reduced significantly glucose levels(p<0.01) after 120 min in the glucose tolerance of mice experiment, even closed to the normal group. Compared with normal control group, the MSG group of liver index, cardiac index, spleen index renal index, HDL-C and adiponectin significantly reduced(p<0.01); but body weight, Lee ’s index, fat index, TG, TC, LDL-C, glucose and insulin significantly increased(p<0.05). The MSG group mice appeared the typical centrality obesity dwarfism signs through sodium glutamate induced. Compared with MSG group mice were administrated by gastric gavage WMP for 10 weeks, body weight, Lee ’s index, fat index, glucose, TG, TC, LDL-C and insulin levels were significantly decreased(p<0.05), but the marked increased in HDL-C and adiponectin(p<0.05). WMP group and fenofibrate group significantly decreased insulin levels(p<0.05). The effects of all groups on liver were shown that compared with normal control group, MSG group of ALT, AST and MDA, TG, TC levels significantly increased(p<0.05), GSH-PX and SOD activities significantly decreased(p<0.05) and simvastatin group of GSH-PX and SOD activities were significantly increased(p<0.05). Compared with MSG group, WMP high dose group was significantly decreased ALT, AST and MDA(p<0.05). The WMP high dose group, fenofibrate group and simvastatin group significantly increased GSH-PX(p<0.05), simvastatin group significantly increased SOD(p<0.01). The WMP high dose group, fenofibrate group and simvastatin group significantly decreased TG(p<0.05). WMP high dose group and medium dose group significantly decreased TC(p< 0.01) levels. Fenofibrate treatment and simvastatin treatment reduced LDL-C(p<0.01) levels, but only fenofibrate treatment increase HDL-C level(p<0.01).3.3 Effects on the proliferation, differentiation and apoptosis of 3T3-L1 cells by the crude extract, different polar solvents and WMP from walnut meal results showed that crude extract, different polar solvent extraction and WMP all inhibited proliferation of 3T3-L1 preadipocyte. The IC50 was crude extract 964.2 μg/m L, WMP 439110 μg/m L, water extract 559.2 μg/m L, n-butyl alcohol extract 1116 μg/m L, ethyl acetate extract 4675 μg/m L, chloroform extracts 564.5 μg/m L, respectively, but petroleum extract promoted proliferation of 3T3-L1 preadipocyte. The crude extract, WMP and different polar solvent extraction(except petroleum extract) significantly reduced both TG and TC levels in 3T3- L1 mature adipocytes and inhibited 3T3-L1 preadipocytes to differentiate into mature adipocytes with a dose-dependent manner. Compared with normal group, the TG and TC levels were significantly increased(p<0.01) in model group. Compared with model group, crude extract, WMP, water extract, n-butyl alcohol extract, the ethyl acetate extract, chloroform extract and fenofibrate were significantly decreased TG and TC levels(p<0.05), but petroleum extract significantly increased TG levels(p<0.01). Under the same concentration, compared with crude extract, water extract, n-butyl alcohol extract, the ethyl acetate extract and chloroform extract, the effection of WMP was the best decreased TG and TC levels. Compared with control group, WMP significantly induced 3T3-L1 mature adipocytes apoptosis(p<0.01).4. UPLC-ESI-MS analysis of WMP result showed in WMP liquid chromatography, the relative content the 12 peaks, through the analysis of the structure of the sample spectrum information and literature were as follows: ellagic glucose, single gallic acid acyl acyl glucose, gallic acid, ellagic glucose, two gallic acid acyl acyl combined with glucose compounds(1, 6- two gallic acid acyl glucose or 2, 6- two gallic acid acyl glucose), gallic acid, ellagic acyl acyl-- glucose(small wood ephedrine or different small wood ephedrine), two gallic acid acyl- ellagic acyl- glucose(terry Ma SuⅠor terry Ma Su Ⅱ), gallic acid ethyl ester, quercetin, Glansreginin A pentose derivatives, ellagic acid.Conclusion:This paper studies have shown that the polyphenols of walnut meal have stronger antioxidant activity and lipid-lowering effect. The WMP scavenging activity against hydroxyl(?OH), hydrogen peroxide(H2O2), 2, 2-diphenyl-1-picrylhydrazyl(DPPH) and superoxide anion radical scavenging capacity(?O2-). WMP could effectively prevent the egg yolk emulsion induced acute hyperglycemia mice serum TG and TC levels. WMP significantly reduced the weight, Lee ’s index, fat index, serum of TG, TC, LDL- C level; but increased HDL-C and adiponectin levels, which improved the obese mice induced by sodium glutamate lipid disorders.WMP reduced the TG, TC, ALT, AST and MDA levels and significantly improved the activity of GSH-PX and SOD enzymein liver,which suggested it improved liver antioxidant capacity and the function of liver injury.WMP significantly reduced the blood sugar level and effectively improved the obese mice induced by sodium glutamate sugar disorders. The polyphenols of crude extracts, different polar solvent extraction(except petroleum ether extraction store) and WMP from walnut meal were significantly inhibited the proliferation and differentiation of 3T3-L1 cells and also significantly.induced 3T3-L1 mature adipocytes apoptosis.