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海带中岩藻黄素及其异构体的分离与生物活性研究
Alternative TitleIsolation, identification and biological functions of fucoxanthin stereoisomers from Saccharina japonica
吴素煌
Subtype硕士
Thesis Advisor张全斌
2019
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学硕士
Degree Discipline海洋生物学
Keyword岩藻黄素 顺反异构体 乳腺癌 抗肿瘤 抗氧化
Abstract近年来,我国对海洋资源的开发利用越来越重视,提高海洋资源加工水平迫在眉睫。传统的海带产业中,多以提取褐藻胶为主,海带色素等脂溶性物质多作为废料遗弃。岩藻黄素作为海带中类胡萝卜素的主要成分,具有良好的生物活性。目前已被食品、化妆品、药品等行业广泛关注,具有良好的商业前景与商业价值。 本实验以海带为原材料,采用乙醇提取海带中的色素,验证了使用大孔树脂富集岩藻黄素的可行性。结果表明大孔树脂X-5对岩藻黄素有较强的吸附作用,考察了使用乙醇和水作为洗脱剂对岩藻黄素的洗脱作用,结果表明60%以上的乙醇的水溶液能有效洗脱大孔树脂吸附的岩藻黄素。 建立了实验室通过减压柱层析快速富集富含岩藻黄素组分的方法,富集组分先通过硅胶柱层析纯化,最后用高效制备液相色谱法制备得到三种岩藻黄素的异构体。岩藻黄素及其异构体通过紫外可见光谱(UV–VIS)、核磁共振氢谱(1 H-NMR)和质谱(MS)进行结构表征和鉴定。采用乳腺癌细胞MDA-MB-231细胞系为模型,对岩藻黄素的体外抗肿瘤活性进行了评价。结果表明,岩藻黄素显著抑制乳腺癌细胞的增殖,13-顺和13’ -顺岩藻黄素组分对癌细胞具有最强的抑制活性,IC50值达到23.71 μg/mL。全反式岩藻黄素和9’ -顺岩藻黄素两种异构体IC50值分别为45.30 μg/mL和27.09 μg/mL。qPCR试验表明岩藻黄素各异构体可能通过抑制癌细胞NF-κB家族基因表达而抑制乳腺癌细胞增殖。考察了岩藻黄素异构体的自由基清除活力,其中全反式岩藻黄素清除DPPH自由基活性较高,13-顺和13’ -顺岩藻黄素组分对羟基自由基表现出较强清除能力,全反式岩藻黄素和13-顺和13’ -顺岩藻黄素组分对超氧自由基的清除能力要高于9’ -顺岩藻黄素。
Other AbstractIn recent years, our country attaches more and more attention to the exploitation and utilization of marine resources, and it is extremely urgent to improve the processing capacity of marine resources.In traditional kelp industry, kelp pigment and other fat-soluble substances are mostly abandoned as waste liquid. Fucoxanthin, as the main component of carotenoids in Saccharina japonica, has great biological activity. At present, it has been widely concerned by food, cosmetics, drugs and other industries, because it has good business prospects and commercial value. In this experiment, Saccharina japonica was used as raw material to extract pigments from it with ethanol, and the feasibility of enriching fucoxanthin with macroporous resin was verified. Results show that X-5 macroporous resin to fucoxanthin as strong adsorption, was investigated using ethanol and water as eluent to fucoxanthin elution effect, the results show that more than 60% ethanol aqueous solution can effectively elute fucoxanthin. The pigments from Saccharina japonica were extracted with ethyanol and the fucoxanthin stereisomers were purified by column chromatography. Finally, three fucoxanthin stereisomers were prepared by preparative high performance liquid chromatography (HPLC). The structures of the fucoxanthin stereisomers were characterized by ultraviolet-visible spectroscopy (UV-VIS), mass spectrometry (MS) and nuclear magnetic resonance (NMR). The in vitro antitumor activity of the fucoxanthin stereisomers was studied using MDA-MB-231 breast cancer cell line. The results showed that fucoxanthin isomers possessed great inhibitory effects against the growth of breast cancer cells. The 13-cis and 13’ -cis fucoxanthin mixtures showed the highest inhibitory activity, and the IC50 value was 23.71 μg/mL. In addition, the IC50 values of the all trans-fucoxanthin and the 9’ -cis-fucoxanthin were 45.30 μg/mL and 27.09 μg/mL, respectively. Further, the qPCR experiment suggested that the fucoxanthin stereisomers may inhibit the growth of breast cancer cells by down-regulating the expression of NF-κB related genes. The free radical scavenging activity of fucoxanthin isomers was investigated. All-trans fucoxanthin had higher DPPH radical scavenging activity, 13-cis and 13’ -cis fucoxanthin showed stronger scavenging ability to hydroxyl radicals, and all-trans fucoxanthin and 13’ -cis fucoxanthin had higher scavenging ability to superoxide radicals than 9’ -cis fucoxanthin.
Subject Area生物学
MOST Discipline Catalogue理学
Pages74
Language中文
Table of Contents第1章 引言 1 1.1 研究背景 1 1.2 类胡萝卜素的顺反异构 1 1.2.1 类胡萝卜素顺反异构的基本原理和结果 2 1.2.2 类胡萝卜素顺反异构体分析 3 1.2.3 功能食品和营养补充剂中的类胡萝卜素顺式异构体 4 1.3 岩藻黄素分离纯化研究进展 5 1.3.1 岩藻黄素的分离纯化与结构鉴定 5 1.3.2 褐藻中岩藻黄素含量 6 1.3.3 岩藻黄素的提取方法 7 1.4 岩藻黄素生物活性研究进展 9 1.4.1 抗氧化活性 9 1.4.2 减肥活性 11 1.4.3 抗糖尿病活性 12 1.4.4 抗血管生成作用 13 1.4.5 抗肿瘤活性 13 1.4.6 抗炎活性 13 1.4.7 岩藻黄素的潜在健康保护作用 14 1.4.8 岩藻黄素的生物安全性与生物利用度 15 1.5岩藻黄素抗肿瘤机制研究现状 17 1.5.1 岩藻黄素诱导肿瘤细胞细胞周期阻滞 18 1.5.2 岩藻黄素诱导肿瘤细胞凋亡 19 1.5.3 岩藻黄素在小鼠模型中的抗肿瘤作用 19 1.6 抗乳腺癌天然产物研究现状 20 1.6.1 传统的治疗方法及其缺点 21 1.6.2 植物源化合物对癌症治疗的作用 21 1.7 研究目的与意义 23 第2章 材料与方法 25 2.1实验仪器与材料 25 2.1.1 实验材料 25 2.1.2 实验仪器 25 2.2 实验方法 25 2.2.1 海带色素提取 25 2.2.2 岩藻黄素树脂吸附 26 2.2.3 岩藻黄素及其异构体分离纯化 27 2.2.4 岩藻黄素异构体结构表征及鉴定 28 2.2.5 岩藻黄素异构体抗肿瘤活性研究 28 2.2.6 抗氧化活性研究 29 2.2.7 显著性分析 30 第3章 结果 31 3.1 岩藻黄素树脂吸附实验 31 3.1.1 大孔树脂的选择 31 3.1.2 岩藻黄素静态吸附曲线与乙醇解吸附能力考察 32 3.1.3 大孔树脂对岩藻黄素的动态吸附与解吸附 33 3.2 岩藻黄素及其异构体分离纯化 34 3.3 岩藻黄素异构体结构表征及鉴定 37 3.4 岩藻黄素异构体抗肿瘤活性 39 3.5 抗氧化活性 41 第4章讨论 45 第5章 结论与展望 47 参考文献 51 致谢 71 作者简历及攻读学位期间发表的学术论文与研究成果 73
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156878
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
吴素煌. 海带中岩藻黄素及其异构体的分离与生物活性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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