黑芝麻黑色素的分离纯化、结构表征及体外抗氧化活性  

Isolation,Structural Characterization and Antioxidant Activity of Black Sesame Melanin

作　　者：李杰 贾栩超[2] 张瑞芬 刘磊[2] 池建伟[2] 黄菲[2] 董丽红[2] 张名位[1,2] LI Jie;JIA XuChao;ZHANG RuiFen;LIU Lei;CHI JianWei;HUANG Fei;DONG LiHong;ZHANG MingWei(College of Food Science,Fujian Agriculture and Forestry University,Fuzhou 350000;Sericultural and Agri-Food Research Institute,Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods,Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Agricultural Products Processing,Guangzhou 510610)

机构地区：[1]福建农林大学食品科学学院,福州350000 [2]广东省农业科学院蚕业与农产品加工研究所/农业农村部功能食品重点实验室/广东省农产品加工重点实验室,广州510610

出　　处：《中国农业科学》2020年第12期2477-2492,共16页Scientia Agricultura Sinica

基　　金：广州市科技计划(201909020001);广东省现代农业产业技术体系创新团队建设专项资金(2019KJ117);科技创新战略专项资金-高水平农科院建设(R2017YJ-YB2006,R2018QD-081)。

摘　　要：【目的】对黑芝麻黑色素进行分离纯化,并对分离得到的不同级分进行结构表征,评价各级分的体外抗氧化活性,为黑芝麻黑色素的精细结构解析及功能活性研究提供理论依据。【方法】以黑芝麻皮为原料,通过碱提酸沉法得到黑芝麻黑色素。黑芝麻黑色素经HW-40C尺寸排阻色谱柱分离纯化,测定所得不同级分的得率、色价和黑色素含量,并采用紫外可见光谱(UV-Vis)、元素分析(EA)、傅里叶红外光谱(FT-IR)、核磁共振氢谱(1H-NMR)和碳谱(13C-NMR)、X-射线光电子能谱(XPS)、电子顺磁共振(EPR)、X-射线衍射(XRD)等手段表征黑色素不同级分的结构,通过DPPH(2,2-diphenyl-1-picrylhydrazyl)、ABTS(2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate))自由基清除率、铁离子还原能力(ferric reducing antioxidant power,FRAP)和氧自由基吸收能力(oxygen radical absorption capacity,ORAC)评价不同级分的体外抗氧化活性。【结果】黑芝麻黑色素经过分离纯化得到黑色的Fr1和棕褐色的Fr2两个级分,得率分别为60%和24%,两个级分的分子量分别为38800 Da和6000 Da,Fr1的黑色素含量为782.16 mg SME·g^-1 DW,Fr2的黑色素含量为884.66 mg SME·g^-1 DW。元素分析结果表明Fr1为真黑色素而Fr2可能是异黑色素;Fr1和Fr2的紫外可见吸收光谱及红外光谱显示,两个级分均含有苯环、-OH、-NH2及-COOH等官能团结构;1H-NMR结果表明Fr1中含有更多的脂肪氢且芳香环上的氢大多数被取代,Fr2的芳香氢含量较Fr1高;固体核磁13C-NMR结果表明Fr1结构中含有更多的脂肪碳和羰基,Fr2含有更多的芳香碳;XPS结果显示两个级分黑色素的官能团含量不同,C1s结果表明Fr1的C-C(H)和C=O官能团的比例高于Fr2,但C-OH/C-N和O-C=O的比例低于Fr2;N1s结果表明Fr1的C-NH官能团比例高于Fr2,但芳香N的比例低于Fr2,且Fr1不含C-NH3+;O1s结果表明Fr1的C-OH官能团比例高于Fr2,但C=O官能团的比例略低于Fr2,且Fr1中不含吸收的H2O【Objective】In order to provide more knowledge about the chemical structure and biological activity of black sesame melanin,black sesame melanin extract was isolated,and the structure of obtained fractions was elucidated by multiple spectroscopic methods.The in vitro antioxidant activity of obtained fractions was also evaluated.【Method】Melanin was extracted from black sesame hull by the methods of alkali extraction and acid precipitation.Crude black sesame melanin extract was then isolated by HW-40C size exclusion column,and the yields,color value and melanin content of obtained fractions were determined.The structure of each fraction was studied by extensive spectroscopic methods,including UV-Vis,elemental analysis(EA),fourier transform infrared spectroscopy(FT-TR),nuclear magnetic resonance spectroscopy(1H and 13C),X-ray photoelectron spectroscopy(XPS),electron paramagnetic resonance(EPR),and X-ray diffraction(XRD).Four methods,including 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging assay,2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS)radical scavenging assay,ferric reducing antioxidant power(FRAP)and oxygen radical absorbance capacity(ORAC)assay,were used to evaluate the in vitro antioxidant activity of each fraction of black sesame melanin.【Result】Black Fr1 and brown Fr2 were isolated from black sesame melanin extract,with the yields of 60%and 24%,respectively;the molecular weights of these two fractions were 38800 Da and 6000 Da,respectively,and the melanin content of two fractions was 782.16 mg SME·g^-1 DW and 884.66 mg SME·g^-1 DW,respectively.Elemental analysis showed that Fr1 was eumelanin and Fr2 might be allomelanin.The UV-visible and infrared spectrum of Fr1 and Fr2 demonstrated that there were functional groups such as benzene ring,-OH,-NH2,-COOH and nitrogen heterocycle in the structure of both fractions.The results of 1H-NMR indicated that Fr1 possessed more aliphatic hydrogen and substituted aromatic ring compared with Fr2.13C-NMR spectrum indicated that there wer

关 键 词：黑芝麻 黑色素 分离纯化 结构表征 体外抗氧化活性 

分 类 号：TS264.4[轻工技术与工程—发酵工程]