【作者】 雷敏；

【导师】 薛长湖；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏工程， 2008， 博士

【摘要】 鱿鱼墨是鱿鱼加工过程中的废弃物,其主要化学成分是黑色素和蛋白多糖复合体。研究表明鱿鱼墨具有抗肿瘤、调节机体免疫功能、诱导多种细胞因子等生理活性,并一直认为其主要的活性成分为多肽片断、酸性多糖、酪氨酸酶以及微量元素。黑色素是以吲哚结构为主体的异聚物,能作为自由基的接受体而阻断自由基连锁反应,且能螯合多种金属离子。目前国内外对鱿鱼墨黑色素的研究多集中于其结构和理化性质,有关其生物活性的报道较少。为了系统评价鱿鱼墨黑色素的营养及药用功效,进一步合理开发利用鱿鱼墨提供理论依据,本文从鱿鱼墨中提取了高纯度黑色素,进一步降解制备了水溶性黑色素,并利用其螯合特性制备了黑色素螯合铁,并对其生物活性进行了研究。通过连续皮下注射氢化可的松建立小鼠免疫机能低下模型,以不同剂量的鱿鱼墨不溶性黑色素灌胃模型小鼠,探讨了鱿鱼墨不溶性黑色素对小鼠免疫机能的调节作用。结果表明,鱿鱼墨不溶性黑色素可显著提高免疫低下模型小鼠的脾及胸腺指数、血清溶血素含量(HC₅₀),小鼠迟发性变态反应及脾淋巴细胞转化能力,显著促进小鼠NK细胞杀伤活力及巨噬细胞吞噬能力。提示鱿鱼墨不溶性黑色素对机体特异性及非特异.性免疫机能具有显著调节作用。以颈背部皮下注射D-半乳糖建立小鼠衰老模型,通过灌胃不同剂量的鱿鱼墨不溶性黑色素,研究了鱿鱼墨不溶性黑色素的免疫调节作用。结果显示,鱿鱼墨不溶性黑色素能显著促进小鼠血清及肝脏中SOD和GSH-PX活力及皮肤中羟脯氨酸含量,降低MDA及脑脂褐质含量;提示鱿鱼墨不溶性黑色素具有显著的抗氧化、抗衰老功能。研究了鱿鱼墨不溶性黑色素对重金属的脱除作用。将小鼠随机分为正常组、黑色素组、重金属组、黑色素+重金属组。分别灌胃基础溶液、鱿鱼墨不溶性黑色素溶液、混合重金属溶液（Ba、Cd、Cr、Pb）鱿鱼墨不溶黑色素+混合重金属溶液。结果表明,与重金属组比较,黑色素+重金属组小鼠股骨、血样中重金属离子的含量显著降低,粪便中重金属排泄了显著升高,表明鱿鱼墨不溶性黑色素能有效阻碍食物中重金属离子在体内的吸收和累积,促进体内重金属离子排出体外,,减轻重金属对机体的危害。采用低铁饲料并辅以尾静脉定期放血法建立缺铁性贫血（Irondeficiency anemia,IDA）大鼠模型,灌胃不同剂量的鱿鱼墨黑色素铁。结果显示,鱿鱼墨黑色素铁能够显著提高IDA大鼠血红蛋白（Hb）含量、红细胞（RBC）数、红细胞压积（HCT）、红细胞平均体积（MCV）、血清铁（SI）含量,有效降低红细胞内游离原卟啉（FEP）浓度浓度,显著促进IDA大鼠贫血症状的恢复。鱿鱼墨黑色素铁对IDA大鼠红系造血具有促进作用。通过饲喂高脂饲料建立小鼠实验性高脂血症小鼠模型,研究了鱿鱼墨不溶性黑色素及水溶性黑色素对实验性高脂血症小鼠脂质代谢的调节作用及其机理。实验结果表明,鱿鱼墨不溶性黑色素及水溶性黑色素均能显著降低高胆固醇血症大鼠血清TC、TG、LDL-c含量及动脉粥样硬化指数（Atherosclerosis index,AI）（P＜0.05）;显著降低肝脏TC、TG含量;显著提高粪便中总脂质、胆固醇和胆汁酸的含量。并能显著提高肝脏中棕榈酰转移酶-1（Carnitine palmitoyltransterase-1,CPT-1）、β-过氧化物酶体（β-oxidationperoxisomal）和磷脂酸磷酸酶（Phosphatidic acidphophyhydrolase,PAP）的活力。提示鱿鱼墨黑色素及水溶性黑色素能有效改善添加胆固醇引起的脂质代谢紊乱,抑制脂质在消化道内的吸收,并促使其通过排泄途径排出体外。其主要作用机理是鱿鱼墨黑色素及水溶性黑色素能促进脂质在体内的β—氧化和异化作用。采用MTT法检测鱿鱼墨水溶性黑色素对H₂O₂损伤血管内皮细胞增殖活性的影响。结果表明鱿鱼墨水溶性黑色素能显著保护H₂O₂损伤血管内皮细胞的正常增殖,显著提高高脂血症小鼠血清中NO含量,提示其通过提高细胞的抗氧化能力,增强细胞膜的稳定性,对血管内皮细胞起到保护作用。选取4株具有代表性的肿瘤细胞,采用MTT法检测2种分子量的Me-W对不同肿瘤细胞株增殖活性的影响,结果表明,两种分子量的Me-W对悬浮生长的S180细胞均有显著抑制其增殖的作用,而对贴壁细胞Caco-2、Hela和B16增殖无显著性影响;分子量10～50Kda的Me-W对S180增殖的抑制作用显著优于分子量＞50Kda的Me-W。提示水溶性鱿鱼墨黑色素对肿瘤细胞的抑制作用有一定的选择性。其机理有待于进一步探讨。更多还原

【Abstract】 Squid ink is mainly composed of melanin and protein-polysaccharide compound. The physiological activities of squid ink,such as anti-tumor activity,immunity regulation or induction of many cytokines,have been widely researched in recent years.It has long been believed that the main active components are polypeptides fragments,acidic polysaccharide,tyrosinase and trae elements.Melanin is an irregular polymer consisting of indole structure,which can resist free radical chain reaction as an acceptor of free radicals,inhibit UVA/B and UVA-induced liposomal lipid peroxidation and chelate many kinds of metal ions.As to squid ink melanin,most researches focus on its structure and the physics and chemistry nature,there is few report concerns its biological activity,however.In this study,high-purity squid ink insoluble melanin（SM-I） was extracted from squid ink sac,then water-soluble melanin（SM-W） and squid ink melanin-Fe（SM-Fe） was prepared.The research on their bioaetivity were conducted in order to evaluate the nutritional and pharmaceutical virtue and encourage the reutilization of squid ink melanin.Hypoimmune model mice were induced by hydrocortisone subcutaneous injection and were given different dosages of SM-I,i.g.,respectively.The immunomodulatory effects of SM-I on hypoimmune mice were investigated.The results showed that Me-I significantly increased the spleen and thymus index,the amount of hemolytic antibody,enhanced the phagocytic activity of macrophages and response of Delayed-Type Hypersensitivity in model mice.It is suggested that insoluble squid ink melanin can stimulate both the non-specific and specific immuity in mice.Aged mice model were induced by injecting D-galactose(1000mg.kg^-1.d^-1).and were given Me-I of different dosages,respectively.The results showed that,ME-I significantly decreased the content of Malondialdehyde（MDA） and Lipofuscin （LIP）,enhenced the activity of SOD and GSH-PX both in serum and liver,increased thymus weight and the content of Hydroxyproline（HYP）,obviously（P＜0.05 or P＜0.01）.The results suggest that Me-I can enhance the anti-oxidation function and postpone the aging process in aged model mice.The heavy-metal expelling effect of Me-I were investigsted.Mice were divided randomly into 4 groups:control,Me-I,heavy metal（HM） and heavy metal+ melanin（HM+Me-I） group,and were given bisic solution,Me-I,mixed heavy metal, HM+Me-I solution,respectively.The results show that,in comparation with heavy metal group,the content of heavy-metal ion derease significantly in thighbone and blood while increased in fecal in HM+Me-I group.The results indicat that Me-I can effectively prevent the absorption and cumulation and reduce the poison of heavy-metal ion in mice.Iron deficiency anemia（IDA） model rats were established by feeding with iron-deprived food and bleeding via tail vein.Several dosages of squid ink melanin-Fe（Me-Fe） were given to the model rats via gastric administration.It was evident that Me-Fe significantly enhanced the contents of hemoglobin（Hb）,the number of red blood cell（RBC）,hematocrit（HCT）,mean corpuscular volume（MCV） and the content of serum iron（SI）,while reduced free erythrocyte proloporphyrin （FEP） content.It appeared to promote the restoration of iron deficiency anemia. Me-Fe also has significant effects on stimulating erythropoiesis in IDA rats.The experimental hyperlipidemia model mice were established by high-fat-diet feeding.The effects of Me-I and Me-W on the lipid metabolism in hyperlipidemic-hypercholesteremic mice and the mechanism underlying such effects were investigated.The results showed that both Me-I and Me-W significantly decreased the contents of TC,TG,LDL-c（P＜0.05） and the AI（P＜0.05） in serum,and reduced the accumulation of TC and TG in liver,increased the contents of total lipids, TC（P＜0.01） and bile acid（P＜0.01） in fecal.They also enhanced the activity of CPT-1（P＜0.01）,β-oxidation peroxisomal（P＜0.05） and PAP（P＜0.05） in liver markedly.It is suggested that both Me-I and Me-W can significantly improve lipid decompensation,inhibit the absorbtion of lipids in enteron and enhence the egestion of lipid in hyperlipidemia mice.The mechanisim is that Me-I and Me-W can enhance theβ-oxidation and dissimilation of lipids.MTT assay were used to test the influence of Me-W on proliferation activities of oxidative damaged endothelial cell.The results showed that Me-W could protect vascular endothellial cells from injury induced by H₂O₂.Cell proliferation activity and NO content were stimulated significantly（P＜0.05,P＜0.01）.MTT assay were used to test the influence of Me-W on proliferation activities of Hela,S180,Caco-2 and B16.The results showed that Me-W could inhibit Cell proliferation activity of S180 but has no significant effect on proliferation activity of Caco-2、Hela and B16 cells.更多还原