真空提取核桃粕多酚的抗氧化活性研究

Study on the antioxidant activity of polyphenols from walnut meal extracted by vacuum

目的 探究真空提取的核桃粕(walnut meal)多酚的抗氧化活性。方法 以非真空条件下提取的多酚为对照,采用超声波辅助真空法提取核桃粕多酚,用HPD-100型大孔树脂对多酚进行纯化,福林酚(Folin-Ciocalte)法测定多酚含量,通过试剂盒检测多酚对1,1-二苯基-2-苦基肼自由基(1,1-diphenyl-2-picrylhydrazyl,DPPH)、超氧阴离子自由基的清除率及Fe3+还原能力(ferric reducing antioxidant power,FRAP)来评价其体外抗氧化活性,用cck8(cell countingkit-8)法测定不同浓度的核桃粕多酚对HepG2细胞的毒性作用,确定3个无毒性作用浓度,以770μmol/L的H_(2)O_(2)诱导HepG2细胞建立氧化应激损伤模型,通过测定HepG2细胞中丙二醛(malonicdialdehyde, MDA)含量、超氧化物歧化酶(superoxide dismutase, SOD)活力、谷胱甘肽(glutathione, GSH)含量探究12.5、25.0、50.0μg/mL核桃粕多酚对HepG2细胞氧化损伤的保护作用。结果 真空条件下提取的多酚含量为26.96mg/g,比非真空条件下提取的多酚含量高出14.50%;经HPD-100型大孔树脂纯化后,对照组和真空组核桃粕多酚纯度分别由22.69%、26.60%提高至73.87%、77.53%;纯化后,对照组与真空组多酚质量浓度为0.5 mg/mL时对DPPH自由基的清除率高达93.83%、93.67%,高于维生素C的清除率91.03%;同一浓度范围内,真空条件下提取的核桃粕多酚对超氧阴离子自由基的清除率和FRAP均显著高于对照组多酚(P<0.05),其中多酚质量浓度为2.0 mg/mL时,对照组与真空组多酚的FRAP分别为8.91μmol/L、9.92μmol/L,均高于维生素C的FRAP(7.91μmol/L);核桃粕多酚可以使受氧化损伤的HepG2细胞内MDA含量明显减少,并能提高受氧化损伤细胞内SOD活力及GSH含量。结论 真空条件有利于核桃粕多酚的提取,提取纯度较高,纯化后核桃粕多酚具有较好的体外氧化活性,对HepG2细胞氧化损伤有一定的保护作用。

Objective To investigate the antioxidant activity of polyphenols extracted from walnut meal under vacuum conditions.Methods Polyphenols extracted under non-vacuum conditions were used as control,and the polyphenols were extracted by ultrasonic-assisted method under vacuum conditions,and the polyphenols were purified by HPD-100 type macroporous resin.The content of polyphenols was determined by Folin-Ciocaltea method.The antioxidant activity in vitro of the polyphenols was evaluated by measuring the scavenging rates of 1,1-diphenyl-2-picrylhydrazyl(DPPH)radicals,superoxide anion radicals and ferric reducing antioxidant power(FRAP)by kit,the toxic effects of different concentrations of walnut meal polyphenols on HepG2 cells were determinated by the cell counting kit-8(cck8)method,the 3 non-toxic effect concentrations were determined,oxidative stress injury model was established by inducing HepG2 cells with 770µmol/L H_(2)O_(2).The content malondialdehyde(MDA)content,superoxide dismutase(SOD)activity and glutathione(GSH)content in HepG2 cells were measured to explore the protective effect of walnut meal polyphenols on oxidative damage of HepG2 cells at 12.5,25.0 and 50.0μg/mL.Results The content of polyphenols extracted under vacuum conditions was 26.96 mg/g,which was 14.50%higher than that of polyphenols extracted under non-vacuum conditions.After purification by HPD-100 macroporous resin,the purity of walnut meal polyphenols in the control and vacuum groups increased from 22.69%and 26.60%to 73.87%and 77.53%,respectively.The scavenging rates of DPPH radicals were 93.83%and 93.67%in the control and vacuum groups after purification at a mass concentration of 0.5 mg/mL,which was higher than that of vitamin C at 91.03%.In the same concentration range,the scavenging rate of superoxide anion radicals and Fe3+reducing antioxidant power of walnut meal polyphenols extracted under vacuum conditions were significantly higher than that of control polyphenols(P<0.05),when the mass concentration of polyphenols was 2.0 mg/mL,the Fe3+reducing antioxidant power of polyphenols in the control and vacuum groups were 8.91 and 9.92μmol/L,respectively,which were higher than that of vitamin C(7.91μmol/L).Walnut meal polyphenols significantly reduced the MDA content of oxidatively damaged HepG2 cells,and increased the SOD activity and GSH content in oxidatively damaged cells.Conclusion The vacuum condition is conducive to the extraction of polyphenols from walnut meal with high extraction purity.Purified walnut meal polyphenols have good oxidative activity in vitro,and have certain protective effect on oxidative damage in HepG2 cells.