黑芝麻黑色素的分离纯化、结构表征及体外抗氧化活性

doi:10.3864/j.issn.0578-1752.2020.12.014

中国农业科学 ›› 2020, Vol. 53 ›› Issue (12): 2477-2492.doi: 10.3864/j.issn.0578-1752.2020.12.014

黑芝麻黑色素的分离纯化、结构表征及体外抗氧化活性

李杰^1,²,贾栩超²,张瑞芬²,刘磊²,池建伟²,黄菲²,董丽红²,张名位^1,²()

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

收稿日期:2019-10-27 接受日期:2020-02-25 出版日期:2020-06-16 发布日期:2020-06-25
通讯作者: 张名位
作者简介:李杰,E-mail：771906135@qq.com。
基金资助:
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);科技创新战略专项资金-高水平农科院建设(R2018QD-081)

Isolation, Structural Characterization and Antioxidant Activity of Black Sesame Melanin

LI Jie^1,²,JIA XuChao²,ZHANG RuiFen²,LIU Lei²,CHI JianWei²,HUANG Fei²,DONG LiHong²,ZHANG MingWei^1,²()

¹ College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350000;
² 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

Received:2019-10-27 Accepted:2020-02-25 Online:2020-06-16 Published:2020-06-25
Contact: MingWei ZHANG

摘要/Abstract

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38 800 Da和6 000 Da,Fr1的黑色素含量为782.16 mg SME?g^-1DW,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。EPR结果显示两个级分均具有较强的顺磁共振特性,Fr1和Fr2的g值分别为2.0078和2.0085,ΔHpp分别为0.7430和0.6950 mT;X-射线衍射表明黑芝麻黑色素的两个级分均为非晶态化合物,Fr1存在平面堆叠结构。Fr1和Fr2的DPPH自由基清除的IC₅₀值分别为83.00和54.00 μg·mL^-1,ABTS自由基清除的IC₅₀值分别为53.00和30.00 μg·mL^-1。Fr1和Fr2的FRAP值分别为1.05和1.62 mmol FeE·g^-1 DW,ORAC值分别为3 141.80和4 143.76 μmol TE·g ^-1 DW。【结论】Fr1是黑芝麻黑色素的主要级分;Fr1主要是真黑色素,而Fr2可能是异黑色素。Fr1和Fr2结构中均含有羰基、羟基、氨基、芳环和氮杂环等官能团,Fr2结构芳香性较Fr1高;Fr2的DDPH和ABTS自由基清除能力、FRAP和ORAC抗氧化能力均高于Fr1。

关键词: 黑芝麻, 黑色素, 分离纯化, 结构表征, 体外抗氧化活性

Abstract:

【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 38 800 Da and 6 000 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 of¹H-NMR indicated that Fr1 possessed more aliphatic hydrogen and substituted aromatic ring compared with Fr2. ¹³C-NMR spectrum indicated that there were more aliphatic carbon and carbonyl and less aromatic carbon in Fr1 compared with Fr2. The results of XPS showed that the content of functional groups of two fractions was different, C1s spectra indicated that the ratio of C-C(H) and C=O group of Fr1 was higher than that of Fr2, but the ratio of C-OH/C-N and O-C=O was lower than that of Fr2. N1s spectra showed that the ratio of C-NH of Fr1 was higher than that of Fr2, the ratio of aromatic N was lower than that of Fr2, and there was no C-NH₃⁺ in Fr1. O1s spectra indicated that the ratio of C-OH of Fr1 was higher than that of Fr2, but the ratio of C=O of Fr1 was slightly lower than that of Fr2, and there was no absorbed H₂O in Fr1. The results of EPR showed that these two fractions demonstrated strong paramagnetic resonance properties, the g values of Fr1 and Fr2 were 2.0078 and 2.0085, respectively, and the ΔHpp of Fr1 and Fr2 were 0.7430 and 0.6950 mT, respectively. The results of X-ray diffraction showed that both fractions of black sesame melanin were amorphous compounds, and there was a planar stack structure in Fr1. The IC₅₀ values of DPPH free radical scavenging of Fr1 and Fr2 were 83.00 and 54.00 μg·mL^-1, respectively. The IC₅₀values of ABTS free radical scavenging of Fr1 and Fr2 were 53.00 and 30.00 μg·mL^-1, respectively. The FRAP values of Fr1 and Fr2 were 1.05 and 1.62 mmol FeE·g^-1 DW, respectively. The ORAC values of Fr1 and Fr2 were 3 141.80 and 4 143.76 μmol TE·g ^-1DW, respectively. 【Conclusion】 Above results indicated that Fr1 was the main fraction of black sesame melanin, and spectroscopic analysis indicated that Fr1 was eumelanin and Fr2 might be allomelanin. Functional groups, such as carbonyl, hydroxyl, amino, aromatic ring and nitrogen heterocycle, existed in both fractions, and the aromaticity of Fr2 was higher than that of Fr1. The DDPH and ABTS free radical scavenging ability, and FRAP and ORAC antioxidant capacity of Fr2 were higher than that of Fr1.

Key words: black sesame, melanin, purification, structural characterization, antioxidant activity

李杰,贾栩超,张瑞芬,刘磊,池建伟,黄菲,董丽红,张名位. 黑芝麻黑色素的分离纯化、结构表征及体外抗氧化活性[J]. 中国农业科学, 2020, 53(12): 2477-2492.

LI Jie,JIA XuChao,ZHANG RuiFen,LIU Lei,CHI JianWei,HUANG Fei,DONG LiHong,ZHANG MingWei. Isolation, Structural Characterization and Antioxidant Activity of Black Sesame Melanin[J]. Scientia Agricultura Sinica, 2020, 53(12): 2477-2492.

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样品 Sample	得率 Yield (%)	色价 Color value (U·g^-1)
粗黑色素 Crude melanin	--	180.54±8.85b
Fr1	60.97±4.79b	155.55±6.22a
Fr2	24.26±2.64a	183.65±4.98b

样品 Sample	乌贼墨黑色素当量 Sepia melanin equivalent (mg SME·g^-1 DW)
粗黑色素 Crude melanin	578.18±1.15a
Fr1	782.16±3.33b
Fr2	884.66±2.93c

样品 Sample	数均分子量 (Mn) Number average molecular weight (Da)	重均分子量(Mw) Weight average molecular weight (Da)	分布宽度(D) Wide distribution
Fr1	1.82×10⁴	3.88×10⁴	2.13
Fr2	3.60×10³	6.00×10³	1.67

样品 Sample	元素组成 Elemental composition				元素比例 Element ratio
样品 Sample	C%	N%	H%	S%	N/C	H/C
粗黑色素 Crude melanin	47.6525	3.6625	5.0225	0.2365	0.0769	0.1054
Fr1	46.1900	7.7750	6.0300	0.2080	0.1683	0.1305
Fr2	30.4900	1.1600	3.3400	0	0.0380	0.1095

样品 Sample	DPPH (IC₅₀ μg·mL^-1)	ABTS (IC₅₀ μg·mL^-1)	FRAP (mmol FeE·g^-1 DW)	ORAC (μmol TE·g^-1 DW)
Vc	13.00±1.04a	12.00±1.12a	13.27±0.25c	9 091.75±347.74d
粗黑色素 Crude melanin	45.00±1.40b	23.00±2.60b	1.22±0.09a	3 730.19±191.38b
Fr1	83.00±2.14d	53.00±1.41d	1.05±0.15a	3 141.80±195.74a
Fr2	54.00±2.13c	30.00±1.04c	1.62±0.05b	4 143.76±99.96c

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

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