基于UPLC-Q TOF-MS和HPLC-QQQ-MS技术的赤豆酚类化合物定性、定量分析及其抗氧化能力分析
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摘要
为深入了解赤豆的营养价值,用80%甲醇溶液提取酚类物质,对提取物中不同存在形态的酚类化合物含量及其抗氧化活性进行测定,并对其80%甲醇提取物中酚类物质进行定性、定量分析。结果表明,赤豆80%甲醇提取物中酚酸含量为3.44 mg/g,黄酮含量为2.50 mg/g,其中3种存在形式酚类萃取物含量及抗氧化能力不同,糖苷键合态酚酸含量最高,可达1.44 mg/g,游离态黄酮含量最高,可达0.85 mg/g;糖苷键合态亚铁离子还原能力最强,可达15.76 mmol/g,酯键合态1,1-二苯基-2-三硝基苯肼自由基、2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基清除能力最强;80%甲醇提取物通过超高效液相色谱串联四极杆飞行时间质谱仪定性分析,初步鉴定出25种化合物,包括3种有机酸、6种酚酸、16种黄酮;高效液相色谱串联三重四极杆质谱联用仪定量分析可得,芦丁(327.40?μg/g)、儿茶素类(210.70?μg/g)含量丰富。
Adzuki bean(the seed of Vigna angularis) is a medicinal and edible legume. In order to better understand its nutritional value, phenolics from adzuki bean were extracted with 80% methanol and separated into free, glycoside and esterified forms. The phenolics were qualitatively and quantitatively analyzed. The results showed that the 80%methanol extract contained 3.44 mg/g phenolic acid and 2.50 mg/g flavonoid. The contents of the three forms of phenolics and their antioxidant activities varied significantly, among which the glycosidically bound fraction was found to be the richest in phenolic acid, up to 1.44 mg/g, whereas the free fraction was had the highest flavonoid content of 0.85 mg/g.The glycosidically bound fraction showed the strongest ferric reducing antioxidant power(FRAP)(15.76 mmol/g),while the ester bound fraction presented the strongest 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonate sulfonic acid)(ABTS) radical cation scavenging abilities. The 80% methanol extract was analyzed by high performance liquid chromatography coupled with triple quadruple mass spectrometry(UPLC-Q TOF-MS),and a total of 25 compounds including 3 organic acids, 6 phenolic acids and 16 flavonoids were identified. Quantitative analysis by HPLC-QQQ-MS showed that adzuki bean was rich in both rutin(327.40 μg/g) and catechin(210.70 μg/g).
Adzuki bean(the seed of Vigna angularis) is a medicinal and edible legume. In order to better understand its nutritional value, phenolics from adzuki bean were extracted with 80% methanol and separated into free, glycoside and esterified forms. The phenolics were qualitatively and quantitatively analyzed. The results showed that the 80%methanol extract contained 3.44 mg/g phenolic acid and 2.50 mg/g flavonoid. The contents of the three forms of phenolics and their antioxidant activities varied significantly, among which the glycosidically bound fraction was found to be the richest in phenolic acid, up to 1.44 mg/g, whereas the free fraction was had the highest flavonoid content of 0.85 mg/g.The glycosidically bound fraction showed the strongest ferric reducing antioxidant power(FRAP)(15.76 mmol/g),while the ester bound fraction presented the strongest 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonate sulfonic acid)(ABTS) radical cation scavenging abilities. The 80% methanol extract was analyzed by high performance liquid chromatography coupled with triple quadruple mass spectrometry(UPLC-Q TOF-MS),and a total of 25 compounds including 3 organic acids, 6 phenolic acids and 16 flavonoids were identified. Quantitative analysis by HPLC-QQQ-MS showed that adzuki bean was rich in both rutin(327.40 μg/g) and catechin(210.70 μg/g).
引文
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[21]ZHANG B,DENG Z,RAMDATH D D,et al.Phenolic profiles of20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects onα-glucosidase and pancreatic lipase[J].Food Chemistry,2015,172:862-872.DOI:10.1016/j.foodchem.2014.09.144.
[22]SUN Y,QIN Y,LI H,et al.Rapid characterization of chemical constituents in Radix Tetrastigma,a functional herbal mixture,before and after metabolism and their antioxidant/antiproliferative activities[J].Journal of Functional Foods,2015,18:300-318.DOI:10.1016/j.jff.2015.07.009.
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[26]SREERAMA Y N,TAKAHASHI Y,YAMAKI K.Phenolic Antioxidants in some Vigna species of legumes and their distinct inhibitory effects onα-glucosidase and pancreatic lipase activities[J].Journal of Food Science,2012,77(9):927-933.DOI:10.1111/j.1750-3841.2012.02848.x.
[27]YAHIA E M,GUTIéRREZ-OROZCO F,MORENO-PéREZ M A.Identification of phenolic compounds by liquid chromatographymass spectrometry in seventeen species of wild mushrooms in central mexico and determination of their antioxidant activity and bioactive compounds[J].Food Chemistry,2017,226:14-22.DOI:10.1016/j.foodchem.2017.01.044.
[28]HU X,CHEN L,SHI S,et al.Antioxidant capacity and phenolic compounds of Lonicerae macranthoides by HPLC-DAD-Q TOF-MS/MS[J].Journal of Pharmaceutical and Biomedical Analysis,2016,124:254-260.DOI:10.1016/j.jpba.2016.03.008.
[29]BASKARAN R,PULLENCHERI D,SOMASUNDARAM R.Characterization of free,esterified and bound phenolics in custard apple(Annona squamosa L.)fruit pulp by UPLC-ESI-MS/MS[J].Food Research International,2016,82:121-127.DOI:10.1016/j.foodres.2016.02.001.
[30]LI H,ZHANG D,TAN L H,et al.Comparison of the antioxidant properties of various solvent extracts from Dipsacus asperoides and identification of phenolic compounds by LC-ESI-Q TOF-MS-MS[J].South African Journal of Botany,2017,109:1-8.DOI:10.1016/j.sajb.2016.12.018.
[31]KELEBEK H.LC-DAD-ESI-MS/MS characterization of phenolic constituents in Turkish black tea:effect of infusion time and temperature[J].Food Chemistry,2016,204:227-238.DOI:10.1016/j.foodchem.2016.02.132.
[32]WANG L,CHEN C,SU A,et al.Structural characterization of phenolic compounds and antioxidant activity of the phenolicrich fraction from defatted adlay(Coix lachryma-jobi L.var.mayuen Stapf)seed meal[J].Food Chemistry,2016,196:509-517.DOI:10.1016/j.foodchem.2015.09.083.
[33]BELGUITH-HADRICHE O,AMMAR S,CONTRERAS M D M,et al.HPLC-DAD-QTOF-MS profiling of phenolics from leaf extracts of two tunisian fig cultivars:potential as a functional food[J].Biomedicine&Pharmacotherapy,2017,89:185-193.DOI:10.1016/j.biopha.2017.02.004.
[34]INBARAJ B S,LU H,KAO T H,et al.Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC-DAD-ESI-MS[J].Journal of Pharmaceutical and Biomedical Analysis,2010,51(3):549-556.DOI:10.1016/j.jpba.2009.09.006.
[35]AMAROWICZ R,ESTRELLA I,HERNANDEZ T,et al.Antioxidant activity of extract of adzuki bean and its fractions[J].Journal of Food Lipids,2008,15(1):119-136.DOI:10.1111/j.1745-4522.2007.00106.x.
[36]TAKAHAMA U,YAMAUCHI R,HIROTA S.Antioxidative flavonoids in adzuki-meshi(rice boiled with adzuki bean)react with nitrite under simulated stomach conditions[J].Journal of Functional Foods,2016,26:657-666.DOI:10.1016/j.jff.2016.08.032.
[37]BAI Y,XU Y,WANG B,et al.Comparison of phenolic compounds,antioxidant and antidiabetic activities between selected edible beans and their different growth periods leaves[J].Journal of Functional Foods,2017,35:694-702.DOI:10.1016/j.jff.2017.06.036.
[2]孙丽丽,董银卯,李丽,等.红豆生物活性成分及其制备工艺研究进展[J].食品工业科技,2013,34(4):390-392.DOI:10.13386/j.issn1002-0306.2013.04.034.
[3]卫莹芳,闫婕,王化东,等.赤小豆总黄酮分光光度分析方法建立及全国不同产地药材含量测定[J].时珍国医国药,2010,21(11):2729-2731.DOI:10.3969/j.issn.1008-0805.2010.11.003.
[4]MARUYAMA C,ARAKI R,KAWAMURA M,et al.Azuki bean juice lowers serum triglyceride concentrations in healthy young women[J].Journal of Clinical Biochemistry and Nutrition,2008,43(1):19-25.DOI:10.3164/jcbn.2008039.
[5]KITANO-OKADA T,ITO A,KOIDE A,et al.Anti-obesity role of adzuki bean extract containing polyphenols:in vivo and in vitro effects[J].Journal of the Science of Food and Agriculture,2012,92(13):2644-2651.DOI:10.1002/jsfa.5680.
[6]MORINA F,TAKAHAMA U,YAMAUCHI R,et al.Quercetin7-O-glucoside suppresses nitrite-induced formation of dinitrosocatechins and their quinones in catechin/nitrite systems under stomach simulating conditions[J].Food&Function,2015,6(1):218-228.DOI:10.1039/C4FO00695J.
[7]NISHI S,SATIO Y,SOUMA C,et al.Suppression of serum cholesterol levels in mice by adzuki bean polyphenols[J].Food Science and Technology Research,2008,14(2):217-220.DOI:10.3136/fstr.14.217.
[8]ITOH T,KITA N,KUROKAWA Y,et al.Suppressive effect of a hot water extract of adzuki beans(Vigna angularis)on hyperglycemia after sucrose loading in mice and diabetic rats[J].Bioscience,Biotechnology,and Biochemistry,2004,68(12):2421-2426.DOI:10.1271/bbb.68.2421.
[9]李波,赵青威,楼宜嘉.赤豆荚果总黄酮提取物对原代培养大鼠肝细胞氧化损伤的保护作用[J].营养学报,2005,27(5):397-400.DOI:10.3321/j.issn:0512-7955.2005.05.012.
[10]聂芊,廖顺雯,刘涛.四种粮豆作物的花色苷抗氧化性能比较[J].食品科学,2007,28(9):46-48.DOI:10.3321/j.issn:1002-6630.2007.09.004.
[11]徐金瑞,张名位,刘兴华.黑大豆种皮花色苷体外抗氧化活性研究[J].营养学报,2007,29(1):54-57.DOI:10.3321/j.issn:0512-7955.2007.01.015.
[12]周威,王璐,范志红.小粒黑大豆和红小豆提取物的体外抗氧化活性研究[J].食品科技,2008,33(9):145-148.DOI:10.3969/j.issn.1005-9989.2008.09.041.
[13]张兵.小扁豆植物化学物组成及其抗氧化,抗炎活性研究[D].南昌:南昌大学,2014.
[14]FRATIANNI A,GIUZIO L,DI CRISCIO T,et al.Response of carotenoids and tocols of durum wheat in relation to water stress and sulfur fertilization[J].Journal of Agricultural and Food Chemistry,2013,61(11):2583-2590.DOI:10.1021/jf304168r.
[15]BOUDJOU S,OOMAH B D,ZAIDI F,et al.Phenolics content and antioxidant and anti-inflammatory activities of legume fractions[J].Food Chemistry,2013,138(2/3):1543-1550.DOI:10.1016/j.foodchem.2012.11.108.
[16]王毕妮.红枣多酚的种类及抗氧化活性研究[D].杨凌:西北农林科技大学,2011.
[17]ROBBINS R J.Phenolic acids in foods:an overview of analytical methodology[J].Journal of Agricultural and Food Chemistry,2003,51(10):2866-2887.DOI:10.1021/jf026182t.
[18]WANG B N,LIU H F,ZHENG J B,et al.Distribution of phenolic acids in different tissues of jujube and their antioxidant activity[J].Journal of Agricultural and Food Chemistry,2011,59(4):1288-1292.DOI:10.1021/jf103982q.
[19]WANG B,HUANG Q,VENKITASAMY C,et al.Changes in phenolic compounds and their antioxidant capacities in jujube(Ziziphus jujuba Miller)during three edible maturity stages[J].LWT-Food Science and Technology,2016,66:56-62.DOI:10.1016/j.lwt.2015.10.005.
[20]祝爱艳,杨延峰,周凯鹏,等.赣南脐橙幼果中的多酚与抗氧化活性[J].食品科技,2017,42(4):207-211.
[21]ZHANG B,DENG Z,RAMDATH D D,et al.Phenolic profiles of20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects onα-glucosidase and pancreatic lipase[J].Food Chemistry,2015,172:862-872.DOI:10.1016/j.foodchem.2014.09.144.
[22]SUN Y,QIN Y,LI H,et al.Rapid characterization of chemical constituents in Radix Tetrastigma,a functional herbal mixture,before and after metabolism and their antioxidant/antiproliferative activities[J].Journal of Functional Foods,2015,18:300-318.DOI:10.1016/j.jff.2015.07.009.
[23]PENG H,LI W,LI H,et al.Extractable and non-extractable bound phenolic compositions and their antioxidant properties in seed coat and cotyledon of black soybean(Glycine max(L.)Merr.)[J].Journal of Functional Foods,2017,32:296-312.DOI:10.1016/j.jff.2017.03.003.
[24]李思荻,赵永焕.东北几种豆类提取物的体外抗氧化活性研究[J].粮油加工,2010(4):54-57.
[25]GAN R Y,DENG Z Q,YAN A X,et al.Pigmented edible bean coats as natural sources of polyphenols with antioxidant and antibacterial effects[J].LWT-Food Science and Technology,2016,73:168-177.DOI:10.1016/j.lwt.2016.06.012.
[26]SREERAMA Y N,TAKAHASHI Y,YAMAKI K.Phenolic Antioxidants in some Vigna species of legumes and their distinct inhibitory effects onα-glucosidase and pancreatic lipase activities[J].Journal of Food Science,2012,77(9):927-933.DOI:10.1111/j.1750-3841.2012.02848.x.
[27]YAHIA E M,GUTIéRREZ-OROZCO F,MORENO-PéREZ M A.Identification of phenolic compounds by liquid chromatographymass spectrometry in seventeen species of wild mushrooms in central mexico and determination of their antioxidant activity and bioactive compounds[J].Food Chemistry,2017,226:14-22.DOI:10.1016/j.foodchem.2017.01.044.
[28]HU X,CHEN L,SHI S,et al.Antioxidant capacity and phenolic compounds of Lonicerae macranthoides by HPLC-DAD-Q TOF-MS/MS[J].Journal of Pharmaceutical and Biomedical Analysis,2016,124:254-260.DOI:10.1016/j.jpba.2016.03.008.
[29]BASKARAN R,PULLENCHERI D,SOMASUNDARAM R.Characterization of free,esterified and bound phenolics in custard apple(Annona squamosa L.)fruit pulp by UPLC-ESI-MS/MS[J].Food Research International,2016,82:121-127.DOI:10.1016/j.foodres.2016.02.001.
[30]LI H,ZHANG D,TAN L H,et al.Comparison of the antioxidant properties of various solvent extracts from Dipsacus asperoides and identification of phenolic compounds by LC-ESI-Q TOF-MS-MS[J].South African Journal of Botany,2017,109:1-8.DOI:10.1016/j.sajb.2016.12.018.
[31]KELEBEK H.LC-DAD-ESI-MS/MS characterization of phenolic constituents in Turkish black tea:effect of infusion time and temperature[J].Food Chemistry,2016,204:227-238.DOI:10.1016/j.foodchem.2016.02.132.
[32]WANG L,CHEN C,SU A,et al.Structural characterization of phenolic compounds and antioxidant activity of the phenolicrich fraction from defatted adlay(Coix lachryma-jobi L.var.mayuen Stapf)seed meal[J].Food Chemistry,2016,196:509-517.DOI:10.1016/j.foodchem.2015.09.083.
[33]BELGUITH-HADRICHE O,AMMAR S,CONTRERAS M D M,et al.HPLC-DAD-QTOF-MS profiling of phenolics from leaf extracts of two tunisian fig cultivars:potential as a functional food[J].Biomedicine&Pharmacotherapy,2017,89:185-193.DOI:10.1016/j.biopha.2017.02.004.
[34]INBARAJ B S,LU H,KAO T H,et al.Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC-DAD-ESI-MS[J].Journal of Pharmaceutical and Biomedical Analysis,2010,51(3):549-556.DOI:10.1016/j.jpba.2009.09.006.
[35]AMAROWICZ R,ESTRELLA I,HERNANDEZ T,et al.Antioxidant activity of extract of adzuki bean and its fractions[J].Journal of Food Lipids,2008,15(1):119-136.DOI:10.1111/j.1745-4522.2007.00106.x.
[36]TAKAHAMA U,YAMAUCHI R,HIROTA S.Antioxidative flavonoids in adzuki-meshi(rice boiled with adzuki bean)react with nitrite under simulated stomach conditions[J].Journal of Functional Foods,2016,26:657-666.DOI:10.1016/j.jff.2016.08.032.
[37]BAI Y,XU Y,WANG B,et al.Comparison of phenolic compounds,antioxidant and antidiabetic activities between selected edible beans and their different growth periods leaves[J].Journal of Functional Foods,2017,35:694-702.DOI:10.1016/j.jff.2017.06.036.