鱿鱼墨多糖和黑色素的研究
摘要
本文选用的北太平洋鱿鱼是最重要的鱿鱼加工品种之一,年加工量在15-30万吨之间,在中国和日本,北太平洋鱿鱼的加工量尤其的多,在加工过程中,其废弃物往往被丢弃,而很少有报道鱿鱼墨中生物活性的利用和研究。因此,本文以北太平洋鱿鱼墨为原料,探讨鱿鱼墨中多糖和黑色素的结构和功能,并展开了以下几个方面的研究:
1)鱿鱼墨粗多糖的分离纯化和化学组成分析。鱿鱼墨经过酶法提取、乙醇沉淀和三氯乙酸除杂蛋白的方法提取得到了一种类似于糖胺聚糖的多糖粗品,得率为1.5%,粗品通过sephacryl凝胶渗透色谱和Q-Sepharose FF阴离子交换色谱进行分级纯化,得到两个糖组成相对单一的组分,采用液相色谱和气相色谱的方法鉴定了其糖组成,发现他们分别含有相同摩尔比例的葡萄糖醛酸,氨基半乳糖(或乙酰氨基半乳糖)。创新性的发现该种多糖不含硫酸根,和文献报道不一致。采用醋酸纤维膜电泳和凝胶渗透色谱鉴定两个组分的纯度和分子量,SIP I和SIP II的分子量分别为52000和61000,纯度为96.1%和94.5%,且为同一类多糖,我们以SIP I进一步研究对象。
2)鱿鱼墨黑色素的提取和活性研究。分析比较了不同提取方法对鱿鱼墨黑色素结构和化学性质的影响,结果发现碱法和酸法能够导致鱿鱼墨黑色素的脱羧和脱氨基现象,而且能导致鱿鱼墨结构的破坏。最终我们以高速离心法制备黑色素。考察了鱿鱼墨黑色素的体外自由基清除活性发现,鱿鱼墨黑色素清除超氧阴离子自由基和羟基自由基的IC50值分别为0.2 mg·mL-1和0.015 mg·mL-1,远低于肌肽的0.53mg·mL-1和0.1mg·mL-1.小鼠体内免疫活性实验的各项指标均表明,鱿鱼墨黑色素体内给药能增强小鼠的细胞免疫和体液免疫功能以及巨噬细胞的吞噬能力,且不影响小鼠的正常体重,可开发为一种新的人体免疫增强保健食品。
3)鱿鱼墨黑色素与金属离子的螯合作用。黑色素对Fe(III)、Pb(II)、Cd(II)最大吸附量分别为1.43mM/g, 0.93mM/g,以及0.65mM/g。温度对于黑色素螯合金属离子没有太大的影响。pH为4吸附率较高,用EDTA和HCl对于洗脱已与黑色素螯合的金属离子有很好的效果,研究了不同浓度的盐对黑色素螯合金属离子的影响,发现盐对Fe(III)及Pb(II)的影响较小,而Cd在低浓度条件下,吸附率也能达到85%以上。进一步采用紫外光谱红外光谱的结果表明,鱿鱼墨黑色素在与Cd(II) ,Pb(II), Fe(III)三种金属离子的螯合过程中,我们都发现了羧基,羟基和亚胺基这三个鱿鱼墨黑色素与金属离子结合位点的特征峰的减弱,这三个官能团都参与了黑色素与金属离子的螯合。采用扫描电镜技术(SEM)表明,鱿鱼墨黑色素黑色素与金属离子Cd(II) ,Pb(II)螯合后,对其结构形态影响不大;而Fe(III)可能由于鱿鱼墨黑色素发生了氧化还原反应,鱿鱼墨黑色素表明结构遭到一定的破坏。黑色素的DSC曲线则表明,黑色素与金属离子螯合后,能够增强其结构稳定性。
1. Purification of polysaccharide from squid ink. Crude melanin-free polysaccharide of Ommastrephes bartrami was obtained with a yield of 1.5%, followed centrifugation, papain digestion and ethanol precipitation. Crude polysaccharide was then fractionated by Sephacryl S-300 gel chromatography and DEAE anion-exchange chromatography, two major fractions SIP I and SIP II were collected. Chemical compositions analysis indicated that both of them had the same proportion of Fuc, GlcA, and GalNAc, and no sulfate was detected. The purity of the SIP I and SIP II were determined by cellulose-acetate membrane electrophoresis and either of the electrophoretogram showed a single band at the same position, which migrated more slowly than chondroitin sulfate A and B. The molecular mass of SIP I and SIP II were determined to be 52 KD and 61KD by gel filtration chromatography on a TSK G3000PWXL column.
2. Extraction and bioactivities of squid melanin. By comparing different isolation protocols on the structure and activities of squid melanin, it was found that repeated treatments with concentrated acid and base could cause lose of carboxyl and amido. Finally, the squid melanin was isolated by high-speed centrifugation method. The activities of melanin to scavenge superoxide anion radical (O2.-) and hydroxyl radical (·OH) were determined by Luminol photochemical reaction system, and the IC50 was 0.015mg·mL-1and 0.2mg·mL-1, respectively, lower than carnosine’s 0.1 mg·mL-1and 0.53mg·mL-1. Further research in vivo carried out indicated that squid melanin have eminent effect on improving the phagocytic capability of Mφ, humoral immune and cell-mediated immunity function, but have no obvious effect on the body weight of the rat, indicating that it could be further developed to be a new health food to enhance the immunity function.
3. Characterization of metal ions binding with squid melanin. The results revealed that the binding capability of squid melanin at pH 4.0 for Fe(III), Pb(II), Cd(II) were 1.43mM/g, 0.93mM/g, and 0.65mM/g, respectively. Temperature has no significant effect on the absorption capability. And at the pH value around 4, melanin had a great binding content of all the three metals. EDTA and HCl have a excellent effect on removing the bonded heavy metals in melanin. Even by exposing squid eumelanin to aqueous solutions of salts, it can still hold its binding capacity to Fe(III), Pb(II); And to low concentration Cd(II), the binding rate could also be kept above 85%. Further examining the effect of metal concentration on transition frequencies associated with the COOH, NH and OH moieties of the pigment in IR and UV spectra, the functional groups of the melanin granules interacting with the bound metal ions could be determined to be all the three kinds of groups. Scanning electron microscopy (SEM) images of ion-binded squid melanin showed that Pb(II) and Cd(II) had no obvious effect on the surface structure of melanin; however, Fe(III) was supported to be reduced by the squid melanin ,which induced little breakage of the granules structure. Differential scanning calorimetry curve of ion-riched melanin shown that the structure of melanin was stabilized after binding with metals.
1)鱿鱼墨粗多糖的分离纯化和化学组成分析。鱿鱼墨经过酶法提取、乙醇沉淀和三氯乙酸除杂蛋白的方法提取得到了一种类似于糖胺聚糖的多糖粗品,得率为1.5%,粗品通过sephacryl凝胶渗透色谱和Q-Sepharose FF阴离子交换色谱进行分级纯化,得到两个糖组成相对单一的组分,采用液相色谱和气相色谱的方法鉴定了其糖组成,发现他们分别含有相同摩尔比例的葡萄糖醛酸,氨基半乳糖(或乙酰氨基半乳糖)。创新性的发现该种多糖不含硫酸根,和文献报道不一致。采用醋酸纤维膜电泳和凝胶渗透色谱鉴定两个组分的纯度和分子量,SIP I和SIP II的分子量分别为52000和61000,纯度为96.1%和94.5%,且为同一类多糖,我们以SIP I进一步研究对象。
2)鱿鱼墨黑色素的提取和活性研究。分析比较了不同提取方法对鱿鱼墨黑色素结构和化学性质的影响,结果发现碱法和酸法能够导致鱿鱼墨黑色素的脱羧和脱氨基现象,而且能导致鱿鱼墨结构的破坏。最终我们以高速离心法制备黑色素。考察了鱿鱼墨黑色素的体外自由基清除活性发现,鱿鱼墨黑色素清除超氧阴离子自由基和羟基自由基的IC50值分别为0.2 mg·mL-1和0.015 mg·mL-1,远低于肌肽的0.53mg·mL-1和0.1mg·mL-1.小鼠体内免疫活性实验的各项指标均表明,鱿鱼墨黑色素体内给药能增强小鼠的细胞免疫和体液免疫功能以及巨噬细胞的吞噬能力,且不影响小鼠的正常体重,可开发为一种新的人体免疫增强保健食品。
3)鱿鱼墨黑色素与金属离子的螯合作用。黑色素对Fe(III)、Pb(II)、Cd(II)最大吸附量分别为1.43mM/g, 0.93mM/g,以及0.65mM/g。温度对于黑色素螯合金属离子没有太大的影响。pH为4吸附率较高,用EDTA和HCl对于洗脱已与黑色素螯合的金属离子有很好的效果,研究了不同浓度的盐对黑色素螯合金属离子的影响,发现盐对Fe(III)及Pb(II)的影响较小,而Cd在低浓度条件下,吸附率也能达到85%以上。进一步采用紫外光谱红外光谱的结果表明,鱿鱼墨黑色素在与Cd(II) ,Pb(II), Fe(III)三种金属离子的螯合过程中,我们都发现了羧基,羟基和亚胺基这三个鱿鱼墨黑色素与金属离子结合位点的特征峰的减弱,这三个官能团都参与了黑色素与金属离子的螯合。采用扫描电镜技术(SEM)表明,鱿鱼墨黑色素黑色素与金属离子Cd(II) ,Pb(II)螯合后,对其结构形态影响不大;而Fe(III)可能由于鱿鱼墨黑色素发生了氧化还原反应,鱿鱼墨黑色素表明结构遭到一定的破坏。黑色素的DSC曲线则表明,黑色素与金属离子螯合后,能够增强其结构稳定性。
1. Purification of polysaccharide from squid ink. Crude melanin-free polysaccharide of Ommastrephes bartrami was obtained with a yield of 1.5%, followed centrifugation, papain digestion and ethanol precipitation. Crude polysaccharide was then fractionated by Sephacryl S-300 gel chromatography and DEAE anion-exchange chromatography, two major fractions SIP I and SIP II were collected. Chemical compositions analysis indicated that both of them had the same proportion of Fuc, GlcA, and GalNAc, and no sulfate was detected. The purity of the SIP I and SIP II were determined by cellulose-acetate membrane electrophoresis and either of the electrophoretogram showed a single band at the same position, which migrated more slowly than chondroitin sulfate A and B. The molecular mass of SIP I and SIP II were determined to be 52 KD and 61KD by gel filtration chromatography on a TSK G3000PWXL column.
2. Extraction and bioactivities of squid melanin. By comparing different isolation protocols on the structure and activities of squid melanin, it was found that repeated treatments with concentrated acid and base could cause lose of carboxyl and amido. Finally, the squid melanin was isolated by high-speed centrifugation method. The activities of melanin to scavenge superoxide anion radical (O2.-) and hydroxyl radical (·OH) were determined by Luminol photochemical reaction system, and the IC50 was 0.015mg·mL-1and 0.2mg·mL-1, respectively, lower than carnosine’s 0.1 mg·mL-1and 0.53mg·mL-1. Further research in vivo carried out indicated that squid melanin have eminent effect on improving the phagocytic capability of Mφ, humoral immune and cell-mediated immunity function, but have no obvious effect on the body weight of the rat, indicating that it could be further developed to be a new health food to enhance the immunity function.
3. Characterization of metal ions binding with squid melanin. The results revealed that the binding capability of squid melanin at pH 4.0 for Fe(III), Pb(II), Cd(II) were 1.43mM/g, 0.93mM/g, and 0.65mM/g, respectively. Temperature has no significant effect on the absorption capability. And at the pH value around 4, melanin had a great binding content of all the three metals. EDTA and HCl have a excellent effect on removing the bonded heavy metals in melanin. Even by exposing squid eumelanin to aqueous solutions of salts, it can still hold its binding capacity to Fe(III), Pb(II); And to low concentration Cd(II), the binding rate could also be kept above 85%. Further examining the effect of metal concentration on transition frequencies associated with the COOH, NH and OH moieties of the pigment in IR and UV spectra, the functional groups of the melanin granules interacting with the bound metal ions could be determined to be all the three kinds of groups. Scanning electron microscopy (SEM) images of ion-binded squid melanin showed that Pb(II) and Cd(II) had no obvious effect on the surface structure of melanin; however, Fe(III) was supported to be reduced by the squid melanin ,which induced little breakage of the granules structure. Differential scanning calorimetry curve of ion-riched melanin shown that the structure of melanin was stabilized after binding with metals.
引文
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