海带多糖的分离纯化及化学结构的初步研究
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摘要
以海带为原料,研究了海带多糖的提取方法、纯化工艺和粗多糖的分级,确定了两种海带多糖的分子量并对其结构进行了初步研究。
确定了硫酸-苯酚法测定多糖的工艺条件:吸收波长为487nm,多糖溶液为1ml,苯酚为1.2ml,硫酸为6ml,用蒸馏水定容至10ml,不需要水浴加热。根据单因素实验和L_9(3~3)正交实验,确定了提取工艺条件:pH值为2、温度为65℃、液固比为40:1、提取时间为3h、提取次数2次,该条件下粗多糖的提取率为8.094%,并用浸提理论解释了海带多糖浸出速率的变化趋势。
比较了Sevag法、三氯乙酸法、木瓜蛋白酶法对蛋白的脱色效果,结果表明木瓜蛋白酶法可有效脱除蛋白,该方法的工艺条件为:酶底比1.2%、pH值5.0,体系在70℃水浴中水解1.5h,蛋白的去除率为66.67%。实验发现木瓜蛋白酶脱蛋白的条件与多糖的提取条件相似,按照以上确定的方法在热水提取海带多糖的工艺条件下提取1.5h,再加木瓜蛋白酶提取1.5h,粗多糖的提取率达到13.17%,蛋白去除率为65.5%。
活性炭、DA201树脂、DA202树脂和DA203树脂脱色结果表明: DA202树脂用量最小,色素去除率最高,多糖的损失率最低。
DEAEC-52纤维素离子交换柱分级纯化粗多糖得到三种多糖L1、L2和L3,其中L2和L3含量高,为海带多糖的主要成分。将此两种多糖进行DEAE-SephadexA-75凝胶柱层析、紫外检测和冻融分析,证明了L2和L3为单一多糖。DEAE-SephadexA-200凝胶柱层析得到L2和L3的分子量分别为131.8kD和93.3kD。红外光谱分析表明:L2含有α、β两种糖苷键,是带有硫酸根的酸性多糖,L3是一种黏度很大的酸性多糖。
The extraction, purification technology, refined polysaccharide classi-fication of Laminaria Japonica (LJ) have been studied. Two of Laminaria Japonica polysaccharide (LJP) molecular weight was identified and structure of LJ was preliminary studied.
The technics condition of the sulfate-phenol method to mensurate polysaccharide was determined. The result of experiment indicated that there were polysaccharide solution of 1 ml, phenol solution of 1.2 ml, concentrated sulfuric acid of 6mL, 10 ml of distilled water to supplement, natural cooling, scanning in the 487 nm. The optimal conditions were obtained on the basis of studying orthogonal experiment. The result of experiment indicated that pH at 2 and extracting temperature at 65℃, leaching rate of LJP was up to the maximum by extracting 3h at 40:1.Leaching rate of LJP was fixed after extracting 2 times and leaching rate of LJP was 8.094%. Meanwhile the leaching trend of polysaccharide was explained with extraction theory.
The sevag method, TCA method and papain method were studied and the optimal conditions were obtained in deproteinization from LJP. The result of experiment indicated that: ratio of enzyme to base material 1.2%, pH 5.0, the eliminating rate of protein was up to 66.67 % by reacting 1.5h at 70℃. The leaching rate of polysaccharide increased to 13.17 % by adding papain to extraction process, and the eliminating rate of protein was up to 65.5 %.
Activated carbon, DA201 resin, DA202 resin, DA203 resin four different columniation was researched in the process of decolorization. The results showed that DA202 resin is one of resin with the smallest amount, the highest pigment removal and the lowest rate of losing polysaccharide,
Three components L1, L2 and L3 were obtained through DEAEC-52 celluloseion exchange column. The contents of L2 and L3 were higher, and were the major components of LJP. L2 and L3 were analysized by DEAE-SephadexA-75 gel column chromatography, UV detection, freeze-thaw analysis. L2 and L3 components were proved to be homogeneous components. By DEAE-SephadexA-200 gel column chromatography, the molecular weight of L2 and L3 were 131.8 kD and 93.3kD respectively. By Infrared spectrometry, L2 containedα、βglycosidic bond, and was a kind of acidic sulfate polysaccharide. L3 was a high viscosity acidic polysaccharides.
确定了硫酸-苯酚法测定多糖的工艺条件:吸收波长为487nm,多糖溶液为1ml,苯酚为1.2ml,硫酸为6ml,用蒸馏水定容至10ml,不需要水浴加热。根据单因素实验和L_9(3~3)正交实验,确定了提取工艺条件:pH值为2、温度为65℃、液固比为40:1、提取时间为3h、提取次数2次,该条件下粗多糖的提取率为8.094%,并用浸提理论解释了海带多糖浸出速率的变化趋势。
比较了Sevag法、三氯乙酸法、木瓜蛋白酶法对蛋白的脱色效果,结果表明木瓜蛋白酶法可有效脱除蛋白,该方法的工艺条件为:酶底比1.2%、pH值5.0,体系在70℃水浴中水解1.5h,蛋白的去除率为66.67%。实验发现木瓜蛋白酶脱蛋白的条件与多糖的提取条件相似,按照以上确定的方法在热水提取海带多糖的工艺条件下提取1.5h,再加木瓜蛋白酶提取1.5h,粗多糖的提取率达到13.17%,蛋白去除率为65.5%。
活性炭、DA201树脂、DA202树脂和DA203树脂脱色结果表明: DA202树脂用量最小,色素去除率最高,多糖的损失率最低。
DEAEC-52纤维素离子交换柱分级纯化粗多糖得到三种多糖L1、L2和L3,其中L2和L3含量高,为海带多糖的主要成分。将此两种多糖进行DEAE-SephadexA-75凝胶柱层析、紫外检测和冻融分析,证明了L2和L3为单一多糖。DEAE-SephadexA-200凝胶柱层析得到L2和L3的分子量分别为131.8kD和93.3kD。红外光谱分析表明:L2含有α、β两种糖苷键,是带有硫酸根的酸性多糖,L3是一种黏度很大的酸性多糖。
The extraction, purification technology, refined polysaccharide classi-fication of Laminaria Japonica (LJ) have been studied. Two of Laminaria Japonica polysaccharide (LJP) molecular weight was identified and structure of LJ was preliminary studied.
The technics condition of the sulfate-phenol method to mensurate polysaccharide was determined. The result of experiment indicated that there were polysaccharide solution of 1 ml, phenol solution of 1.2 ml, concentrated sulfuric acid of 6mL, 10 ml of distilled water to supplement, natural cooling, scanning in the 487 nm. The optimal conditions were obtained on the basis of studying orthogonal experiment. The result of experiment indicated that pH at 2 and extracting temperature at 65℃, leaching rate of LJP was up to the maximum by extracting 3h at 40:1.Leaching rate of LJP was fixed after extracting 2 times and leaching rate of LJP was 8.094%. Meanwhile the leaching trend of polysaccharide was explained with extraction theory.
The sevag method, TCA method and papain method were studied and the optimal conditions were obtained in deproteinization from LJP. The result of experiment indicated that: ratio of enzyme to base material 1.2%, pH 5.0, the eliminating rate of protein was up to 66.67 % by reacting 1.5h at 70℃. The leaching rate of polysaccharide increased to 13.17 % by adding papain to extraction process, and the eliminating rate of protein was up to 65.5 %.
Activated carbon, DA201 resin, DA202 resin, DA203 resin four different columniation was researched in the process of decolorization. The results showed that DA202 resin is one of resin with the smallest amount, the highest pigment removal and the lowest rate of losing polysaccharide,
Three components L1, L2 and L3 were obtained through DEAEC-52 celluloseion exchange column. The contents of L2 and L3 were higher, and were the major components of LJP. L2 and L3 were analysized by DEAE-SephadexA-75 gel column chromatography, UV detection, freeze-thaw analysis. L2 and L3 components were proved to be homogeneous components. By DEAE-SephadexA-200 gel column chromatography, the molecular weight of L2 and L3 were 131.8 kD and 93.3kD respectively. By Infrared spectrometry, L2 containedα、βglycosidic bond, and was a kind of acidic sulfate polysaccharide. L3 was a high viscosity acidic polysaccharides.
引文
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2张哲,张筠.海带中的生理活性多糖.食品科技. 1999,3:31~34
3 C.K. Veena, Anthony Josephine and S.P. Preetha, et al. Beneficial Role of Sulfated Polysaccharides from Edible Seaweed Fucus Vesiculosus in Experimental Hyperoxaluria. Food Chemistry. 2007,100(4):1552~1559
4李福川,唐志红,崔博文.三种海带多糖的降糖作用.中国海洋药物. 2000,19(5):12~16
5胡颖红,李向荣.海带对高血压的降压作用观察.浙江中西医结合杂志. 1997,7(5):266~267
6侯毓礼.海带根药理实验研究.中草药. 1984,12(12):19~23
7廖建民,沈子龙,张瑾.海带多糖中不同组分降血脂及抗肿瘤作用的研究.中国药科大学学报. 2002,(01):22~25
8李兆杰,薛长湖,陈磊等.低分子量海带岩藻聚糖硫酸酯的清除活性氧自由基和体内抗氧化作用.水产学报. 2001(01):11~15
9 Yasantha Athukorala, K.W. Lee and S.K. Kim, et al. Anticoagulant Activity of Marine Green and Brown Algae Collected from Jeju Island in Korea. Bioresource Technology. 2007,98(9):1711~1716
10 M.G.. Pereira, M.B. Norma and Benevides, et al. Structure and Anticoagulant Activity of a Sulfated Galactan from the Alga, Gelidium Crinale. Is There a Specific Structural Requirement for the Anticoagulant Action? Carbohydrate Research. 2005,340(12):2015~2023
11 M.S. Pereira, E.S. Cristina and A.P. Valente, et al. A 2-sulfated,3-linkedα- -galactan is an Anticoagulant Polysaccharide. Carbohydrate Research. 2002,337(21-23):2231~2238
12 Sabine Matou, Dominique Helley and Delphine Chabut, et al. Effect of Fucoidan on Fibroblast Growth Factor-2-induced Angiogenesis in Vitro. Thrombosis Research. 2002,106(4-5):213~221
13潘家祜,陈阳,常建杰.褐藻多糖硫酸酯抗PAF作用研究.中国海洋药物杂志. 2003,93(3):7~11
14彭波.褐藻多糖硫酸酯的抗凝和纤溶活性.中草药. 2001,32(11):1015~1018
15 T. Nishino and T. Nagumo. Structural Characterization of a New Anticoagulant Fucan Sulfate from the Brown Seaweed Eclonia kurome. Carbohydr Res. 1991,211:77~86
16 M.J. Kwona, T.J. Nam. A Polysaccharide of the Marine Alga Capso-siphon Fulvescens Induces Apoptosis in AGS Gastric Cancer Cells Via an IGF-IR-mediated PI3K/Akt Pathway. Cell Biology International. 2007,31(8): 768~775
17薛静波,刘希英,张鸿芬.海带多糖对小鼠腹腔巨噬细胞的激活作用.中国海洋药物. 1999,18(3):23~27
18宋剑秋,徐誉泰,张华坤.海带硫酸多糖对小鼠腹腔巨噬细胞的免疫调节作用.中国免疫学杂志. 2000,16(2):70~75
19张英慧,曲爱琴,宋剑秋等.海带多糖FGS对小鼠巨噬细胞细胞毒活性的影响.免疫学杂志. 2002,(05):22~26
20范曼芳,陈琼华.褐藻淀粉和褐藻淀粉硫酸酯的制取、分析及生物活性比较.中国药科大学学报. 1988,(01):12~16
21滕霞,丛建波,田晓华等.海藻硫酸多糖抗氧化与抗肿瘤作用的实验研究.营养学报. 1998,(01):21~26
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