软枣猕猴桃黄酮对过氧化氢诱导HaCaT细胞损伤的保护作用
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摘要
研究软枣猕猴桃中黄酮类化合物对过氧化氢(H_2O_2)损伤人永生化表皮细胞(HaCaT细胞)的保护作用。以湖南省浏阳市大围山软枣猕猴桃为实验材料,以体外培养的HaCaT细胞为实验对象。实验分为空白对照组、H_2O_2模型组、阳性对照组(VC组)和黄酮处理组(软枣猕猴桃黄酮0.1~1.0 mg/mL预处理),以细胞内超氧化物歧化酶(superoxide dismutase,SOD)活力、活性氧簇(reactive oxygen species,ROS)水平为综合评价指标,并以VC作为阳性对照评价其抗氧化活性。实验结果表明:当H_2O_2浓度为30μmol/L时,细胞相对存活率为40%左右,较好地诱导了HaCaT细胞氧化损伤模型。当加入黄酮类化合物对细胞进行预保护后,质量浓度0.3、0.6 mg/mL的黄酮处理组细胞相对存活率为60%左右,较模型组均提高了20%左右。0.3 mg/mL黄酮处理组细胞内SOD活力(6.33 U/mg)较模型组升高了约1倍,且存在明显差异(P<0.05);ROS水平显著下降,与模型组(677.80)相比下降了13%左右。说明软枣猕猴桃中黄酮类化合物对H_2O_2造成的氧化损伤具有一定的预保护作用,可进一步将其开发成相应的天然抗氧化剂产品投入市场。
The protective effect of Actinidia arguta flavonoids(AAF) on H_2O_2-induced oxidative injury in human immortalized epidermal cells(HaCaT) was investigated in this study. The flavonoids were extracted from A. arguta grown in Da Wei Mountain, Liuyang, Hunan provice. Ha Ca T cells were cultured in vitro. The experiment was divided into control, hydrogen peroxide damage, positive control(VC), and AAF treatment groups(at concentrations of 0.1–1.0 mg/m L). Superoxide dismutase(SOD) activity and reactive oxygen species(ROS) levels in cells were measured to evaluate the cytoprotective effect of AAF. VC was used as a positive control to evaluate its antioxidant activity. The results showed that at a H_2O_2 concentration of 30 μmol/L, the survival rate of the cells was approximately 40% and the oxidative damage model was established. When AAF at concentrations of 0.3 and 0.6 mg/mL was used to protect the cells, the survival rate was about 60%, which increased by about 20% compared with the model group. SOD activity(6.33 U/mg) in the treatment group(0.3 mg/m L) was about 2 times higher than that in the model group, with a significant difference(P < 0.05). ROS level decreased significantly by 13% when compared with the model group(677.80). These data suggested a protective effect of AAF on H_2O_2-indued oxidative damage. Therefore, it can be further developed into a potent natural antioxidant product.
The protective effect of Actinidia arguta flavonoids(AAF) on H_2O_2-induced oxidative injury in human immortalized epidermal cells(HaCaT) was investigated in this study. The flavonoids were extracted from A. arguta grown in Da Wei Mountain, Liuyang, Hunan provice. Ha Ca T cells were cultured in vitro. The experiment was divided into control, hydrogen peroxide damage, positive control(VC), and AAF treatment groups(at concentrations of 0.1–1.0 mg/m L). Superoxide dismutase(SOD) activity and reactive oxygen species(ROS) levels in cells were measured to evaluate the cytoprotective effect of AAF. VC was used as a positive control to evaluate its antioxidant activity. The results showed that at a H_2O_2 concentration of 30 μmol/L, the survival rate of the cells was approximately 40% and the oxidative damage model was established. When AAF at concentrations of 0.3 and 0.6 mg/mL was used to protect the cells, the survival rate was about 60%, which increased by about 20% compared with the model group. SOD activity(6.33 U/mg) in the treatment group(0.3 mg/m L) was about 2 times higher than that in the model group, with a significant difference(P < 0.05). ROS level decreased significantly by 13% when compared with the model group(677.80). These data suggested a protective effect of AAF on H_2O_2-indued oxidative damage. Therefore, it can be further developed into a potent natural antioxidant product.
引文
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[24]SMITS E,BURVEENICH C,HEYNEMAN R.Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood[J].Veterinary Immunology&Immunopathology,1997,56(3/4):259-269.DOI:10.1016/S0165-2427(96)05739-X.
[25]步犁.利用多种细胞筛选抗氧化物质方法的研究[D].广州:广东药学院,2013:16-21.
[26]张泽生,卢亚莉,高云峰,等.常见生物模型抗氧化活性评价方法的研究进展[J].中国食品添加剂,2015(3):178-183.DOI:10.3969/j.issn.1006-2513.2015.03.021.
[27]WOLFE K L,LIU R H.Structure-activity relationships of flavonoids in the cellular antioxidant activity assay[J].Journal of Agricultural and Food Chemistry,2008,56(18):8404-8411.DOI:10.1021/jf8013074.
[28]WOLFE K L,KANG X,HE X,et al.Cellular antioxidant activity of common fruits[J].Journal of Agricultural and Food Chemistry,2008,56(18):8418-8426.DOI:10.1021/jf801381y.
[29]付维维,刘娟,王业秋,等.甘草黄酮对UVB损伤HDF细胞保护作用研究[J].安徽农业科学,2016,44(4):170-171.DOI:10.13989/j.cnki.0517-6611.2016.04.056.
[30]薛丽,王常利,陈慧,等.大豆异黄酮对骨肉瘤MG-63细胞的代谢和抗氧化应激研究[J].西北国防医学杂志,2016,37(5):284-287.DOI:10.16021/j.cnki.1007-8622.2016.05.002.
[2]李坤明,胡忠荣,陈伟.昭通地区野生猕猴桃资源及其利用评价[J].中国野生植物资源,2006,25(2):39-41.DOI:10.3969/j.issn.1006-9690.2006.02.012.
[3]张兰杰,辛广,谷昊,等.双波长分光光度法测定软枣猕猴中总黄酮的含量[J].食品科学,2005,26(9):434-436.DOI:10.3321/j.issn:1002-6630.2005.09.114.
[4]LIU Y Y,LIU C J.Determination of total alkaloids in different parts of Actinidia arguta by spectro-photography[C]//2015 International Conference on Materials,Environmental and Biological Engineering,Paris:Atlantis Press,2015:127-130.DOI:10.2991/mebe-15.2015.31.
[5]穆晶晶,张博,李书倩,等.失水处理对软枣猕猴桃贮藏期间褐变相关因子的影响[J].食品科学,2013,34(20):307-311.DOI:10.7506/spkx 1002-6630-201320064.
[6]YU X,LIU C,LIU Y,et al.Actinidia arguta polysaccharide induces apoptosis in Hep G2 cells[J].Advance Journal of Food Science&Technology,2015,7:857-863.DOI:10.19026/ajfst.7.2522.
[7]辛广,张博,冯帆,等.软枣猕猴桃果实香气成分分析[J].食品科学,2009,30(4):230-232.DOI:10.3321/j.issn:1002-6630.2009.04.050.
[8]MATICH A J,YOUNG H,ALLEN J M,et al.Actinidia arguta:volatile compounds in fruit and flowers[J].Phytochemistry,2003,63(3):285-301.DOI:10.1016/S0031-9422(03)00142-0.
[9]姜爱丽,申新,胡文忠,等.软枣猕猴桃籽油的超临界萃取及成分分析[J].中国油脂,2008,33(9):77-79.DOI:10.3321/j.issn:1003-7969.2008.09.025.
[10]栾云峰.软枣猕猴桃黄酮的提取分离纯化及结构鉴定[D].沈阳:沈阳农业大学,2011:61-67.
[11]ZUO L L,WANG Z Y,FAN Z L,et al.Evaluation of antioxidant and antiproliferative properties of three Actinidia(Actinidia kolomikta,Actinidia arguta,Actinidia chinensis)extracts in vitro[J].International Journal of Molecular Sciences,2012,13(5):5506-5518.DOI:10.3390/ijms13055506.
[12]高扬,易若琨,宋家乐.竹叶总黄酮对UVB诱导HaCaT细胞氧化损伤的保护作用[J].南京中医药大学学报,2015,31(2):165-169.DOI:10.14148/j.issn.1672-0482.2015.0165.
[13]曾泰,马磊,王伟,等.枸杞黄酮对H_2O_2致血管内皮细胞损伤的保护作用[J].食品研究与开发,2015,36(15):13-15.DOI:10.3969/j.issn.1005-6521.2015.15.004.
[14]温钢,刘海燕,杨梅.软枣猕猴桃黄酮的提取及体外抗氧化研究[J].北方园艺,2015(4):140-143.DOI:10.11937/bfyy.201504033.
[15]CHEN J J,PENG Z X,SHI M Y,et al.Advances in on flavonoid composition and metabolism in citrus[J].Acta Horticulturae Sinica,2016,43(2):384-400.DOI:10.16420/j.issn.0513-353x.2015-0689.
[16]VALKO M,LEIBFRITZ D,MONCOL J,et al.Free radicals and antioxidants in normal physiological functions and human disease[J].International Journal of Biochemistry&Cell Biology,2007,39(1):44-84.DOI:10.1016/j.biocel.2006.07.001.
[17]POMPELLA A,COMPORTI M.Xenobiotics,free radicals and cell membranes[J].Pathophysiology,1998,5:93.DOI:10.1016/S0928-4680(98)80644-5.
[18]ASEERVATHAM S B G,SIVASUDHA T,JEYADEVI R,et al.Environmental factors and unhealthy lifestyle influence oxidative stress in humans:an overview[J].Environmental Science and Pollution Research,2013,20(7):4356-4369.DOI:10.1007/s11356-013-1748-0.
[19]GETOFF N.Anti-aging and aging factors in life.the role of free radicals[J].Radiation Physics and Chemistry,2007,76(10):1577-1586.DOI:10.1016/j.radphyschem.2007.01.002.
[20]AFAQ F,SYED D N,MALIK A,et al.Delphinidin,an anthocyanidin in pigmented fruits and vegetables,protects human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis[J].Journal of Investigative Dermatology,2007,127(1):222-232.DOI:10.1038/sj.jid.5700510.
[21]ZAID M A,AFAQ F,SYED D N,et al.Inhibition of UVB-mediated oxidative stress and markers of photoaging in immortalized HaCaT keratinocytes by pomegranate polyphenol extract POMx[J].Hotochemistry&Photobiology,2007,83(4):882-888.DOI:10.1111/j.1751-1097.2007.00157.x.
[22]LEE E R,KANG Y J,KIM J H,et al.Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids[J].Journal of Biological Chemistry,2005,280(36):31498-31507.DOI:10.1074/jbc.m505537200.
[23]NGUYEN C N,KIM H E,LEE S G.Caffeoylserotonin protects human keratinocyte HaCaT cells against H_2O22-induced oxidative stress and apoptosis through upregulation of HO-1 expression via activation of the PI3K/Akt/Nrf2 pathway[J].Phytotherapy Research,2013,27(12):1810-1818.DOI:10.1002/ptr.4931.
[24]SMITS E,BURVEENICH C,HEYNEMAN R.Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood[J].Veterinary Immunology&Immunopathology,1997,56(3/4):259-269.DOI:10.1016/S0165-2427(96)05739-X.
[25]步犁.利用多种细胞筛选抗氧化物质方法的研究[D].广州:广东药学院,2013:16-21.
[26]张泽生,卢亚莉,高云峰,等.常见生物模型抗氧化活性评价方法的研究进展[J].中国食品添加剂,2015(3):178-183.DOI:10.3969/j.issn.1006-2513.2015.03.021.
[27]WOLFE K L,LIU R H.Structure-activity relationships of flavonoids in the cellular antioxidant activity assay[J].Journal of Agricultural and Food Chemistry,2008,56(18):8404-8411.DOI:10.1021/jf8013074.
[28]WOLFE K L,KANG X,HE X,et al.Cellular antioxidant activity of common fruits[J].Journal of Agricultural and Food Chemistry,2008,56(18):8418-8426.DOI:10.1021/jf801381y.
[29]付维维,刘娟,王业秋,等.甘草黄酮对UVB损伤HDF细胞保护作用研究[J].安徽农业科学,2016,44(4):170-171.DOI:10.13989/j.cnki.0517-6611.2016.04.056.
[30]薛丽,王常利,陈慧,等.大豆异黄酮对骨肉瘤MG-63细胞的代谢和抗氧化应激研究[J].西北国防医学杂志,2016,37(5):284-287.DOI:10.16021/j.cnki.1007-8622.2016.05.002.