| Walnuts, the seeds of Juglans regia L.(Juglandaceae), are a highly nutritious food. It contains rich nutrients, good anti-oxidation, anti-aging, enhance immunity health care efficacy, etc. Walnut cultivation in our country is widespread, resources are very rich. In this article, the different parts of the walnut kernel were prepared and evaluated for their in vitro and in vivo antioxidant, anti-hypertensive activity. And the antioxidant, anti-hypertensive extracts of extraction technology was studied. The specific research works were as follows:The different parts of the walnut kernel were evaluated for their in vitro antioxidant by using ferric ion reducing capacity(FRAP), DPPH radical scavenging ability and ABTS radical scavenging capacity of three kinds of different methods of in vitro antioxidant. Three different antioxidant evaluation method showed a trend of the antioxidant capacity of the consistent, walnut oil, walnut kernel and nonfat walnut powder parts have antioxidant effect in vitro. To evaluate the antioxidant effect in vivo, aging mice model was established by D-galactose induction. Four groups of mice were treated with vitamin C positive control group, walnut kernel extract group, nonfat walnut powder extract group and walnut oil group at doses of 0.20, 0.50, 0.20, and 2.00 g kg-1 D respectively. The antioxidant status in the aging mice was measured by determining the activities of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), catalase(CAT) and total anti-oxidant capability(T-AOC) in the serum and liver and malondialdehyde(MDA) content in the serum, kidney, brain, liver and heart. Compared with model group, three extracts from different parts of walnut group and vitamin C positive control group significantly enhanced activities of SOD, GSH-Px, CAT and decreased MDA content in the serum, kidney, brain, liver and heart at the tested doses. In vivo experiment results show that walnuts, walnut oil group and nonfat walnut powder way between antioxidant activity was no significant difference(P > 0.05). So, in vivo and in vitro experiment results show that three extracts from different parts of walnut demonstrated the potent antioxidant activity.Response surface methodology(RSM) was used to estimate the optimum extraction parameters, in which the antioxidant activity of the extract from the defatted walnut powder, it was investigated by the radical scavenging activity assays DPPH, ABTS, FRAP ferric ion reducing power trolox equivalent antioxidant capacity(TEAC, Trolox equivalent antioxidant capacity, μM) and extract the polyphenols content(Total Polyphenols, mg g-1). The independent processing variables were ethanol concentration, temperature and time. The optimal extraction parameters of defatted walnut powder extracts for the highest antioxidant activity and for the total polyphenols: ethanol concentration was 57.67%, temperature was 47.81 ℃, time was 76.91 minutes.The effects of three independents variables in terms of extraction time, temperature, and liquid- solid ratio on the walnut oil yield was determined and the optimal conditions for walnut oli was evaluated by means of response surface methodology. The optimal extraction parameters of walnut oil for the highest oil yield: time was 69.98 min, extraction temperature was 45℃, and liquid- solid ratio was 11.25 m L g-1.Established a simultaneous determination of three kinds of unsaturated fatty acid in the walnut oil content measurement method, this method was simple, highly selective. Chromatographic analysis using ZORBAX Eclipse XDB- C8 column(4.6 × 150 mm, 5 microns), composition of mobile phase of acetonitrile: water(90:10), such as degree of elution, the flow rate of 1 m L/min. The method of linear range(γ > 0.9992), good intra- and inter-day of precision are all less than 3.0%, show that method for quantitative determination of unsaturated fatty acids in walnut oil with a good reproducibility.The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine(HHL), as model peptide, and HPLC-UV, as analytical method. The experimental results show that the defatted walnut powder ethanol extract has good anti-hypertensive effect in vitro. To evaluate the anti-hypertensive effect in vivo, hypertension rat model was established by drinking water 10% fructose induction. Four groups of rats were treated with captopril positive control group, walnut kernel extract group, nonfat walnut powder extract group and walnut oil group at doses of 0.10, 0.25, 0.10, and 1.00 g kg-1 D respectively. The anti-hypertensive status in the hypertension rat was measured by determining the rat tail artery blood pressure once a week. After four weeks, compared with blank group, model group rats blood pressure was significantly increased(P < 0.01). Compared with model group, only defatted walnut powder group can significantly reduce the blood pressure of hypertensive rats(P < 0.01). So, in vivo and in in vitro experiment results show that defatted walnut powder demonstrated the potent anti-hypertensive activity.Response surface methodology(RSM) was used to estimate the optimum extraction parameters, in which the anti-hypertensive activity of the extract from the defatted walnut powder, it was investigated by ACE inhibition. The effects of three independent variables of ultrasound time(min), ethanol concentration(v/v, %), and liquid- solid ratio(m L g-1) by central composite rotatable design were investigated in vitro. According to response surface analysis, the optimum conditions for the highest ACE inhibition were ultrasound time, ethanol concentration and liquid- solid ratio of 39.19 min, 40.95% and 7.1m L g-1, respectively. Predict the ACE inhibition rate was 76.00%, the measured values was 75.96%. |
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