栽培菊苣籽总黄酮的细胞毒性及清除羟自由基活性成分的识别
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摘要
采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT)法评价栽培菊苣籽总黄酮对小鼠巨噬细胞RAW264.7的毒性。从总抗氧化能力、清除2,2’-联氮-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐自由基(2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt cation radical,ABTS+·)能力、清除O_2~-·能力以及抑制Fe~(2+)诱发的卵黄脂蛋白脂质过氧化能力4个方面评价栽培菊苣籽总黄酮的抗氧化活性,并与常见的抗氧化剂VC和叔丁基对苯二酚(tertbutylhydroquinone,TBHQ)作比较。之后采用高效液相色谱(high-performance liquid chromatography,HPLC)法对栽培菊苣籽总黄酮中清除·OH的活性成分进行了识别。MTT实验结果显示,菊苣籽总黄酮质量浓度为1 mg/mL时,对RAW264.7细胞具有一定的毒性,而当其质量浓度适当减小后,则基本无毒性。抗氧化实验结果表明:栽培菊苣籽总黄酮的总抗氧化活性和清除ABTS~+·的能力弱于VC和TBHQ,但在低质量浓度时,其总抗氧化活性与TBHQ非常接近;在清除O_2~-·方面,活性由强到弱为VC>菊苣籽总黄酮>TBHQ;在抑制卵黄脂蛋白脂质过氧化实验中,栽培菊苣籽总黄酮强于VC,但弱于TBHQ。HPLC法结果显示,绿原酸比洋蓟素具有更高的清除·OH的活性,且二者对·OH的清除率分别为66.42%和46.00%。
The cytotoxicity of total flavonoids from Cichorium endivia L. seeds towards RAW264.7 cells was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay and the antioxidant activities were investigated based on total antioxidant activity, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt cation radical(ABTS~+·) scavenging capacity, superoxide anion radical scavenging capacity and inhibition on Fe~(2+)-induced lipid peroxidation in egg yolk. These activities were compared with those of the common antioxidants vitamin C(VC) and tert-butylhydroquinone(TBHQ). Furthermore, the total flavonoids extracted from C. endivia seeds were screened for those scavenging hydroxyl radical by a high-performance liquid chromatography(HPLC) method. MTT assay showed that the total flavonoids were toxic towards RAW264.7 cells at a concentration of 1 mg/mL, but they were hardly toxic at a certain lower temperature. Antioxidant assays revealed that the total antioxidant activity and ABTS~+· scavenging activity of these compounds were lower than those of VC and TBHQ. However, at low concentration, the total antioxidant activity was similar to that of TBHQ. The superoxide anion scavenging capacity of these samples was in the decreasing order of VC > total flavonoids from C. endivia seeds > TBHQ. The lipid peroxidation inhibitory activity of the total flavonoids was higher than that of VC, but lower than that of TBHQ. The HPLC analysis indicated that hydroxyl radical scavenging capacity of chlorogenic acid was higher than that of cynarin, which showed a scavenging percentage of 66.42% and 46.00%, respectively.
The cytotoxicity of total flavonoids from Cichorium endivia L. seeds towards RAW264.7 cells was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay and the antioxidant activities were investigated based on total antioxidant activity, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt cation radical(ABTS~+·) scavenging capacity, superoxide anion radical scavenging capacity and inhibition on Fe~(2+)-induced lipid peroxidation in egg yolk. These activities were compared with those of the common antioxidants vitamin C(VC) and tert-butylhydroquinone(TBHQ). Furthermore, the total flavonoids extracted from C. endivia seeds were screened for those scavenging hydroxyl radical by a high-performance liquid chromatography(HPLC) method. MTT assay showed that the total flavonoids were toxic towards RAW264.7 cells at a concentration of 1 mg/mL, but they were hardly toxic at a certain lower temperature. Antioxidant assays revealed that the total antioxidant activity and ABTS~+· scavenging activity of these compounds were lower than those of VC and TBHQ. However, at low concentration, the total antioxidant activity was similar to that of TBHQ. The superoxide anion scavenging capacity of these samples was in the decreasing order of VC > total flavonoids from C. endivia seeds > TBHQ. The lipid peroxidation inhibitory activity of the total flavonoids was higher than that of VC, but lower than that of TBHQ. The HPLC analysis indicated that hydroxyl radical scavenging capacity of chlorogenic acid was higher than that of cynarin, which showed a scavenging percentage of 66.42% and 46.00%, respectively.
引文
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[26]MAZAREI F,JOOYANDEH H,NOSHAD M,et al.Polysaccharide of caper(Capparis spinosa L.)leaf:extraction optimization,antioxidant potential and antimicrobial activity[J].International Journal of Biological Macromolecules,2017,95:224-231.DOI:10.1016/j.ijbiomac.2016.11.049.
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[30]ZHANG J J,MENG G Y,ZHAI G Y,et al.Extraction,characteristisation and antioxidant activity of polysaccharides of spent mushroom compost of Ganoderma lucidum[J].International Journal of Biological Macromolecules,2016,82:432-439.DOI:10.1016/j.ijbiomac.2015.10.016.
[2]王福鑫,林铌,邓安珺,等.苦菊提取物的肝保护作用研究[J].食品与药品,2013,15(3):153-155.DOI:10.3969/j.issn.1672-979x.2013.03.001.
[3]陈超杰,秦海林,邓安珺,等.苦菊提取物的抗氧化活性研究[J].食品与药品,2011,13(3):93-96.DOI:10.3969/j.issn.1672-979X.2011.02.005.
[4]KANG H W.Antioxidative activity of extracts from Cichorium endivia L.[J].Journal of the Korean Society of Food Science and Nutrition,2012,41(11):1487-1492.DOI:10.3746/jkfn.2012.41.11.1487.
[5]MASCHERPA D,CARAZZONE C,MARRUBINI G,et al.Identification of phenolic constituents in Cichorium endivia var.crispum and var.latifolium salads by high-performance liquid chromatography with diode array detection and electrospray ioniziation tandem mass spectrometry[J].Journal of Agricultural and Food Chemistry,2012,60(49):12142-12150.DOI:10.1021/jf3034754.
[6]DUPONT M S,MONDIN Z,WILLAMSON G,et al.Effect of variety,processing,and storage on the flavonoid glycoside content and composition of lettuce and endive[J].Journal of Agricultural and Food Chemistry,2000,48(9):3957-3964.DOI:10.1021/jf0002387.
[7]El-SHAFEY N M,ABDELGAWAD H.Luteolin,a bioactive flavone compound extracted from Cichorium endivia L.subsp.divaricatum alleviates the harmful effect of salinity on maize[J].Acta Physiologiae Plantarum,2012,34(6):2165-2177.DOI:10.1007/s11738-012-1017-8.
[8]陈超杰,李万芳,秦海林,等.苦菊乙酸乙酯提取物的体外抗氧化活性研究[J].中国药房,2011,22(15):1347-1350.
[9]陈超杰,展丽娟,魏金锋,等.苦菊提取物对Hep G2细胞的抗氧化作用及其机制研究[J].中国中药杂志,2014,39(14):2716-2720.DOI:10.4268/cjcmm20141424.
[10]?赵月,李荣,姜子涛.栽培菊苣籽总黄酮的提取、成分鉴定及抗氧化活性成分的识别[J].食品科学,2016,37(16):36-42.DOI:10.7506/spkx1002-6630-201616006.
[11]ZHAO M H,JIANG Z T,LIU T,et al.Flavonoids in Juglans regia L.leaves and evaluation of in vitro antioxidant activity via intracellular and chemical methods[J].Scientific World Journal,2014,2014:1-6.DOI:10.1155/2014/303878.
[12]ZHANG T T,WANG M,YANG L,et al.Flavonoid glycosides from Rubus chingii Hu fruits display anti-inflammatory activity through suppressing MAPKs activation in macrophages[J].Journal of Functional Foods,2015,18:235-243.DOI:10.1016/j.jff.2015.07.006.
[13]LI T T,ZHANG H,WU C E.Screening of antioxidant and antitumor activities of major ingredients from defatted Camellia oleifera seeds[J].Food Science and Biotechnology,2014,23(3):873-880.DOI:10.1007/s10068-014-0117-1.
[14]DUAN X,LI M,MA H J,et al.Physicochemical properties and antioxidant potential of phosvitin-resveratrol complexes in emulsion system[J].Food Chemistry,2016,206:102-109.DOI:10.1016/j.foodchem.2016.03.055.
[15]杨云舒,姜子涛,李荣.广枣黄酮清除自由基能力及抗氧化性能的细胞模型法评价[J].食品科学,2016,37(9):92-97.DOI:10.7506/spkx1002-6630-201609018.
[16]党娅,刘水英.紫甘蓝花青苷提取工艺及抗氧化性研究[J].北方园艺,2015(1):128-136.DOI:10.11937/bfyy.201501035.
[17]SAEED N,KHAN M R,SHABBIR M.Antioxidant activity,total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L.[J].BMC Complementary and Alternative Medicine,2012,12(2):221.DOI:10.1186/1472-6882-12-221.
[18]XIN X Y,FAN R,GONG Y,et al.On-line HPLC-ABTS+·evaluation and HPLC-MSn identification of bioactive compounds in hot pepper peel residues[J].European Food Research and Technology,2014,238:837-844.DOI:10.1007/s00217-014-2153-8.
[19]HONG S,LIU S B.Targeted acylation for all the hydroxyls of(+)-catechin and evaluation of their individual contribution to radical scavenging activity[J].Food Chemistry,2016,197:415-421.DOI:10.1016/j.foodchem.2015.10.134.
[20]LOCATELLI D A,NAZARENO M A,FUSARI C M,et al.Cooked garlic and antioxidant activity:correlation with organosulfur compound composition[J].Food Chemistry,2017,220:219-224.DOI:10.1016/j.foodchem.2016.10.001.
[21]WANG L Y,DING L,WANG Y,et al.Isolation and characterisation of in vitro and cellular free radical scavenging peptides from corn peptide fractions[J].Molecules,2015,20(2):3221-3237.DOI:10.3390/molecules20023221.
[22]周媛,李荣,姜子涛.食用委陵菜黄酮的抗氧化性及清除自由基能力研究[J].食品工业科技,2012,33(17):102-105.DOI:10.13386/j.issn1002-0306.2012.17.070.
[23]张禄捷,李荣,姜子涛.茼蒿叶中总黄酮的提取纯化及抗氧化活性分析[J].食品科学,2015,36(24):40-45.DOI:10.7506/spkx1002-6630-201524007.
[24]WANG F,YANG J X.A comparative study of caffeic acid and a novel caffeic acid conjugate SMND-309 on antioxidant properties in vitro[J].LWT-Food Science and Technology,2012,46(1):239-244.DOI:10.1016/j.lwt.2011.09.025.
[25]MARKOVI?J M D,PEJIN B,MILENKOVI?D,et al.Antiradical activity of delphinidin,pelargonidin and malvin towards hydroxyl and nitric oxide radicals:the energy requirements calculations as a prediction of the possible antiradical mechanisms[J].Food Chemistry,2017,218:440-446.DOI:10.1016/j.foodchem.2016.09.106.
[26]MAZAREI F,JOOYANDEH H,NOSHAD M,et al.Polysaccharide of caper(Capparis spinosa L.)leaf:extraction optimization,antioxidant potential and antimicrobial activity[J].International Journal of Biological Macromolecules,2017,95:224-231.DOI:10.1016/j.ijbiomac.2016.11.049.
[27]TLILI N,MEJRI H,ANOUER F,et al.Phenolic profile and antioxidant activity of Capparis spinosa seeds harvested from different wild habitats[J].Industrial Crops and Products,2015,76:930-935.DOI:10.1016/j.indcrop.2015.07.040.
[28]XU J K,LI M F,SUN R C.Identifying the impact of ultrasoundassisted extraction on polysaccharides and natural antioxidants from Eucommia ulmoides Oliver[J].Process Biochemistry,2014,50(3):473-481.DOI:10.1016/j.procbio.2014.12.021.
[29]YANG J X,GUO J,YUAN J F.In vitro antioxidant properties of rutin[J].LWT-Food Science and Technology,2008,41(6):1060-1066.DOI:10.1016/j.lwt.2007.06.010.
[30]ZHANG J J,MENG G Y,ZHAI G Y,et al.Extraction,characteristisation and antioxidant activity of polysaccharides of spent mushroom compost of Ganoderma lucidum[J].International Journal of Biological Macromolecules,2016,82:432-439.DOI:10.1016/j.ijbiomac.2015.10.016.