紫贻贝多糖提取分离、结构鉴定及其生物活性的初步研究
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摘要
贻贝是重要的世界性养殖品种,以紫贻贝产量占有最大,并以新鲜贻贝为主要销售形式,经济效益低。贻贝不但作为传统的滋补食品,且具有较高的营养价值和多种药理功效。文献报道,贻贝及其提取物具有抗氧化、免疫调节、抗肿瘤、降血脂等功能活性。其中,多糖是紫贻贝中的主要活性成分之一,目前对其研究主要集中在紫贻贝粗多糖上,而对紫贻贝多糖组分的提取、分离纯化及结构功能研究报道较少。因此,开发利用紫贻贝多糖具有理论价值和实际意义。本课题以嵊泗枸杞所产紫贻贝为试验材料,研究了紫贻贝多糖的提取、脱蛋白、分离纯化、结构鉴定及其免疫抗肿瘤活性。主要研究结果如下：
采用响应面方法优化紫贻贝多糖的热水提取工艺。实验以冷冻干燥后的紫贻贝粉末为原料,研究了浸取温度、浸取时间及液固比对紫贻贝多糖得率的影响,确定了提取的最佳工艺参数为：浸提温度93℃,浸提时间4.2h,液固比44.3：1,在此条件下多糖得率可达19.73%。
对热水提取醇沉后所得紫贻贝粗多糖MES进行脱蛋白工艺的研究。本实验采用三种不同方法—Sevag法、酶法和酶法与Sevag联用法并比较了对MES的脱蛋白的效果。结果表明酶法与Sevag联用法为最合适的方法,其工艺条件为：酶法：酶种为碱性蛋白酶,最优酶解参数：酶量[E]/[S]1.79%,pH9.19,酶解温度61.79℃；Sevag法：氯仿：正丁醇=4：1(V/V),料液比=3：1(V/V),通过三次脱蛋白处理。
对经酶法与Seveg联用法脱蛋白后的紫贻贝多糖MES-I进行分离纯化研究。本实验先将MES-I溶液过0.45μm微孔滤膜抽滤,其后采用10000的超滤膜进行超滤处理。再通过Marco-Prep DEAE离子交换层析和Sephrose 4B凝胶柱层析进一步分离纯化得到三个组分(MES-I-I1、MES-I-12和MES-I-2)。采用Sephrose 6B凝胶过滤层析法、高效液相色谱(HPLC)和醋酸薄膜电泳三种方法对三个组分进行纯度分析,确定MES-I-11、MES-I-12和MES-I-2为均一多糖组分。
研究了MES-I-I1、MES-I-12和MES-I-2三个均一组分的理化性质和初级结构。通过苯酚-硫酸法测定,MES-I-I1、MES-I-12和MES-I-2的总糖含量分别为95.2%、91.2%和94.8%。颜色反应和紫外光谱(UV)法检测推断三个均一组分都不含有蛋白质和核酸。HPLC法测定了MES-I-I1、MES-I-12和MES-I-2的分子量分别为572.8KD、18.20KD和69.50KD。采用傅里叶红外光谱(FT-IR)法初步判断三个组分为糖类化合物,且其多糖结构均含有a-D-葡萄吡喃糖苷键。气质联用(GC-MS)法分析表明MES-I-11、MES-I-12和MES-I-2分别由不同摩尔比的3种单糖—甘露糖、半乳糖和葡萄糖组成。其中,MES-I-11的组成成分甘露糖、半乳糖和葡萄糖的摩尔比为2.74：1：18.06;MES-I-12的摩尔比为2.2：1：19.44；MES-I-2的摩尔比为2.87：1：3.63。核磁共振法(NMR)分析结果是MES-I-I1、MES-I-12和MES-I-2均有α-D-葡萄糖吡喃糖苷,且在C6上有发生羟基被取代,主要以α-D-Glc(1→4)-为主要的连接方式。
采用噻唑盐(MTT)比色法研究MES-I-1、MES-I-2、MES-I-11和MES-I-12的免疫活性。结果表明,在ConA诱导小鼠T淋巴细胞的体外增殖中,各组分均不同程度抑制细胞的增殖反应。在高浓度时有明显抑制作用,显著性分别为P<0.01、P<0.001、P<0.05、P<0.01。在LPS诱导小鼠B淋巴细胞的体外增殖反应中,MES-I-1和MES-I-12没有显著影响,MES-I-2在高浓度下呈促进作用(P<0.05),MES-I-11在中浓度下也呈促进作用,在低浓度条件下则呈增殖抑制作用(P<0.05)。对K562肿瘤细胞的抗肿瘤活性研究实验表明,MES-I-1、MES-I-2、MES-I-11和MES-I-12在各浓度下均表现出不同程度的抗肿瘤活性,并呈一定的量效关系,其中MES-I-2在低浓度和高浓度时的抑瘤率分别为24.48±1.72%和62.82±8.57%,表现出较强的抗肿瘤作用。
Mussel is an important aquaculture species worldwide, the ratio of Mytilus edulis (ME) is the largest. But people could not make more profits if only mussel was sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects. According to related materials, both mussel and its extracts have variety of functional effects, such as anti-oxidant, immune regulation, anti-tumor, antihypertensive effect and so on. Polysaccharides are one of the major chemical ingredients of ME. And now more researches were focused on the crude polysaccharides from ME, however, the studies about the extractions, purifications and structures and functional researched systemly have not yet reported. Therefore, ME were obtained from an island named wolfberry in Zhoushan and then employed to extract polysaccharides. Extraction, isolation, structure identification and bioactivity of polysaccharides from ME were further studied. The major results obtained in this study were as follows:
Response surface method (RSM) was applied to optimize extractions of polysaccharides from ME by hot-water extraction. The effects of extraction temperature, extraction time and ratio of liquid to solid on the yields of polysaccharides were investigated. The optimum extraction conditions were obtained as follows:extraction temperature 93℃, extraction time 4.2h, ratio of liquid to solid 44.3:1. The yield of polysaccharides can be up to 19.73% under the optimal extraction conditions.
Three different deprotein-methods (seveg method, enzymatic method and enzymatic-seveg method) were used to deproteinize crude polysaccharides from MES. The results showed that enzymatic-seveg method was the best method to remove protein. The optimum conditons were as follow:Alkaline protease was as enzyme:the amount of [E]/[S]1.79%, pH9.19, temperature of 61.79℃; Sevag method: chloroform:butanol= 4:1 (V/V), liquid-Solvent ratio= 3:1 (V/V), the times of removing-protein processing was three.
In this study, MES-I were isolated and purified by several approaches. After using 0.45μm microporous membrane and ultra-filtration, the obtained polysaccharides were further isolated with Marco-Prep DEAE ion-exchange chromatography and Sephrose 4B gel column chromatography. In the end, three components (MES-I-11, MES-I-12 and MES-I-2) were obtained. They were pure determined by Sephrose 6B gel column chromatography, HPLC and cellulose acetate membrane electrophoresis (CAME).
The Physical, chemical properties and the structures of MES-I-11, MES-I-12 and MES-I-2 was analyzed with chemical methods and instrumental analysis methods. According to Phenol-sulfuric acid method, the total sugar contents of MES-I-11, MES-I-12 and MES-I-2 were 95.2%, 91.2% and 94.8% respectively. Color reactions and UV detection inferred that the three homogeneous samples contained no protein and nucleic acids. From HPLC analysis, molecular weight of MES-I-11, MES-I-12 and MES-I-2 were 572.8KD,18.20KD and 69.50KD respectively. From FT-IR, MES-I-11, MES-I-12 and MES-I-2 were polysaccharides, and all their polysaccharides structures contained a-D-glucopyranoside bond. From GC-MS analysis, MES-I-11, MES-I-12 and MES-I-2 were composition of mannose, galactose and glucose with different molar ratios. MES-I-11 was composition of Man, Gal and Glc with the molar ratio of 2.74:1:18.06; MES-I-12 with molar ratio of 2.2:1:19.44; MES-I-2 with molar ratio of 2.87:1:3.63. Nuclear magnetic resonance analysis results was that the structures of MES-I-11, MES-I-12 and MES-I-2 were mainly composed with the presence of a-D-Glc(1→4)-linkage glycoside, and in the C6 where had been replaced.
Cellular immunity was tested by MTT colormetry. The results showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could affect the proliferation of Lymphocyte induced by ConA differently. And in high concentrations, all samples could inhibit the proliferation, with the respective significances were P<0.01, P<0.001, P<0.05,P<0.01. In Lymphocyte proliferation induced by LPS, MES-I-1 and MES-I-12 did not show significant proliferation effects. And MES-I-2, at high concentrations, showed promotive effects, also did MES-I-11 at middle concentrations. But it showed depressant effects in low concentrations(P <0.05). The results of antitumor test of K562 tumor cells showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could inhibit proliferation of cancer cell differently. And the effects were enhanced with the increasing of sample doses. MES-I-2, at low concentrations and high concentrations, displayed growth inhibitory effects with 24.48±1.72% and 62.82±8.57% respectively, the results showed that the inhibition effects of MES-I-2 on K562 tumor cells were better than the effects of other samples.
采用响应面方法优化紫贻贝多糖的热水提取工艺。实验以冷冻干燥后的紫贻贝粉末为原料,研究了浸取温度、浸取时间及液固比对紫贻贝多糖得率的影响,确定了提取的最佳工艺参数为：浸提温度93℃,浸提时间4.2h,液固比44.3：1,在此条件下多糖得率可达19.73%。
对热水提取醇沉后所得紫贻贝粗多糖MES进行脱蛋白工艺的研究。本实验采用三种不同方法—Sevag法、酶法和酶法与Sevag联用法并比较了对MES的脱蛋白的效果。结果表明酶法与Sevag联用法为最合适的方法,其工艺条件为：酶法：酶种为碱性蛋白酶,最优酶解参数：酶量[E]/[S]1.79%,pH9.19,酶解温度61.79℃；Sevag法：氯仿：正丁醇=4：1(V/V),料液比=3：1(V/V),通过三次脱蛋白处理。
对经酶法与Seveg联用法脱蛋白后的紫贻贝多糖MES-I进行分离纯化研究。本实验先将MES-I溶液过0.45μm微孔滤膜抽滤,其后采用10000的超滤膜进行超滤处理。再通过Marco-Prep DEAE离子交换层析和Sephrose 4B凝胶柱层析进一步分离纯化得到三个组分(MES-I-I1、MES-I-12和MES-I-2)。采用Sephrose 6B凝胶过滤层析法、高效液相色谱(HPLC)和醋酸薄膜电泳三种方法对三个组分进行纯度分析,确定MES-I-11、MES-I-12和MES-I-2为均一多糖组分。
研究了MES-I-I1、MES-I-12和MES-I-2三个均一组分的理化性质和初级结构。通过苯酚-硫酸法测定,MES-I-I1、MES-I-12和MES-I-2的总糖含量分别为95.2%、91.2%和94.8%。颜色反应和紫外光谱(UV)法检测推断三个均一组分都不含有蛋白质和核酸。HPLC法测定了MES-I-I1、MES-I-12和MES-I-2的分子量分别为572.8KD、18.20KD和69.50KD。采用傅里叶红外光谱(FT-IR)法初步判断三个组分为糖类化合物,且其多糖结构均含有a-D-葡萄吡喃糖苷键。气质联用(GC-MS)法分析表明MES-I-11、MES-I-12和MES-I-2分别由不同摩尔比的3种单糖—甘露糖、半乳糖和葡萄糖组成。其中,MES-I-11的组成成分甘露糖、半乳糖和葡萄糖的摩尔比为2.74：1：18.06;MES-I-12的摩尔比为2.2：1：19.44；MES-I-2的摩尔比为2.87：1：3.63。核磁共振法(NMR)分析结果是MES-I-I1、MES-I-12和MES-I-2均有α-D-葡萄糖吡喃糖苷,且在C6上有发生羟基被取代,主要以α-D-Glc(1→4)-为主要的连接方式。
采用噻唑盐(MTT)比色法研究MES-I-1、MES-I-2、MES-I-11和MES-I-12的免疫活性。结果表明,在ConA诱导小鼠T淋巴细胞的体外增殖中,各组分均不同程度抑制细胞的增殖反应。在高浓度时有明显抑制作用,显著性分别为P<0.01、P<0.001、P<0.05、P<0.01。在LPS诱导小鼠B淋巴细胞的体外增殖反应中,MES-I-1和MES-I-12没有显著影响,MES-I-2在高浓度下呈促进作用(P<0.05),MES-I-11在中浓度下也呈促进作用,在低浓度条件下则呈增殖抑制作用(P<0.05)。对K562肿瘤细胞的抗肿瘤活性研究实验表明,MES-I-1、MES-I-2、MES-I-11和MES-I-12在各浓度下均表现出不同程度的抗肿瘤活性,并呈一定的量效关系,其中MES-I-2在低浓度和高浓度时的抑瘤率分别为24.48±1.72%和62.82±8.57%,表现出较强的抗肿瘤作用。
Mussel is an important aquaculture species worldwide, the ratio of Mytilus edulis (ME) is the largest. But people could not make more profits if only mussel was sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects. According to related materials, both mussel and its extracts have variety of functional effects, such as anti-oxidant, immune regulation, anti-tumor, antihypertensive effect and so on. Polysaccharides are one of the major chemical ingredients of ME. And now more researches were focused on the crude polysaccharides from ME, however, the studies about the extractions, purifications and structures and functional researched systemly have not yet reported. Therefore, ME were obtained from an island named wolfberry in Zhoushan and then employed to extract polysaccharides. Extraction, isolation, structure identification and bioactivity of polysaccharides from ME were further studied. The major results obtained in this study were as follows:
Response surface method (RSM) was applied to optimize extractions of polysaccharides from ME by hot-water extraction. The effects of extraction temperature, extraction time and ratio of liquid to solid on the yields of polysaccharides were investigated. The optimum extraction conditions were obtained as follows:extraction temperature 93℃, extraction time 4.2h, ratio of liquid to solid 44.3:1. The yield of polysaccharides can be up to 19.73% under the optimal extraction conditions.
Three different deprotein-methods (seveg method, enzymatic method and enzymatic-seveg method) were used to deproteinize crude polysaccharides from MES. The results showed that enzymatic-seveg method was the best method to remove protein. The optimum conditons were as follow:Alkaline protease was as enzyme:the amount of [E]/[S]1.79%, pH9.19, temperature of 61.79℃; Sevag method: chloroform:butanol= 4:1 (V/V), liquid-Solvent ratio= 3:1 (V/V), the times of removing-protein processing was three.
In this study, MES-I were isolated and purified by several approaches. After using 0.45μm microporous membrane and ultra-filtration, the obtained polysaccharides were further isolated with Marco-Prep DEAE ion-exchange chromatography and Sephrose 4B gel column chromatography. In the end, three components (MES-I-11, MES-I-12 and MES-I-2) were obtained. They were pure determined by Sephrose 6B gel column chromatography, HPLC and cellulose acetate membrane electrophoresis (CAME).
The Physical, chemical properties and the structures of MES-I-11, MES-I-12 and MES-I-2 was analyzed with chemical methods and instrumental analysis methods. According to Phenol-sulfuric acid method, the total sugar contents of MES-I-11, MES-I-12 and MES-I-2 were 95.2%, 91.2% and 94.8% respectively. Color reactions and UV detection inferred that the three homogeneous samples contained no protein and nucleic acids. From HPLC analysis, molecular weight of MES-I-11, MES-I-12 and MES-I-2 were 572.8KD,18.20KD and 69.50KD respectively. From FT-IR, MES-I-11, MES-I-12 and MES-I-2 were polysaccharides, and all their polysaccharides structures contained a-D-glucopyranoside bond. From GC-MS analysis, MES-I-11, MES-I-12 and MES-I-2 were composition of mannose, galactose and glucose with different molar ratios. MES-I-11 was composition of Man, Gal and Glc with the molar ratio of 2.74:1:18.06; MES-I-12 with molar ratio of 2.2:1:19.44; MES-I-2 with molar ratio of 2.87:1:3.63. Nuclear magnetic resonance analysis results was that the structures of MES-I-11, MES-I-12 and MES-I-2 were mainly composed with the presence of a-D-Glc(1→4)-linkage glycoside, and in the C6 where had been replaced.
Cellular immunity was tested by MTT colormetry. The results showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could affect the proliferation of Lymphocyte induced by ConA differently. And in high concentrations, all samples could inhibit the proliferation, with the respective significances were P<0.01, P<0.001, P<0.05,P<0.01. In Lymphocyte proliferation induced by LPS, MES-I-1 and MES-I-12 did not show significant proliferation effects. And MES-I-2, at high concentrations, showed promotive effects, also did MES-I-11 at middle concentrations. But it showed depressant effects in low concentrations(P <0.05). The results of antitumor test of K562 tumor cells showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could inhibit proliferation of cancer cell differently. And the effects were enhanced with the increasing of sample doses. MES-I-2, at low concentrations and high concentrations, displayed growth inhibitory effects with 24.48±1.72% and 62.82±8.57% respectively, the results showed that the inhibition effects of MES-I-2 on K562 tumor cells were better than the effects of other samples.
引文
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