贻贝蒸煮液水溶性多糖的分离纯化、结构分析和活性研究
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摘要
贻贝蒸煮液由新鲜贻贝的煮汁经24小时加热浓缩而成，其中含有大量的可溶性蛋白质、糖类等营养物质和功能性成份。将贻贝蒸煮液中的水溶性多糖进行分离纯化，进而研究它们的理化性质、结构及活性，以期达到资源的有效利用、经济效益的提高和新功能性食品的开发等目的。
首先，通过化学分析法对贻贝蒸煮液原料中基本成分进行分析。接着，采用乙醇沉淀、Sevage+木瓜蛋白酶法除蛋白、活性炭脱色从贻贝蒸煮液中分离出水溶性粗多糖MJPs，再经琼脂糖凝胶Sepharose4B柱层析和透析，纯化得到两种贻贝蒸煮液水溶性多糖MJPsⅠ和MJPsⅡ。经紫外扫描、醋酸纤维薄膜电泳、高效凝胶渗透色谱等方法检验它们的均一性。将得到的均一多糖MJPsⅠ和MJPsⅡ用苯酚-硫酸法测定总糖含量，高效凝胶渗透色谱法测定分子量，并利用气相色谱、红外光谱和核磁共振光谱，确定它们的初级结构。通过抑菌杯法、标准曲线法和DPPH·抑制率法分别测定贻贝蒸煮液和水溶性粗多糖MJPs的体外抑菌、体外NaNO_2清除及NDMA合成阻断以及体外抗氧化活性，并对MJPsⅠ和MJPsⅡ的免疫学活性、抗肿瘤活性和抑制黑色素生成活性进行测定。
实验结果表明，贻贝蒸煮液原料中糖类主要以非还原糖的形式存在，纯化得到的水溶性多糖MJPsⅠ和MJPsⅡ经检验为均一多糖。通过结构分析得到，MJPsⅠ主要由葡萄糖组成，平均分子量大于2．0×10~6Da，推测其结构为：MJPsⅡ主要由葡萄糖组成，平均分子量亦大于2．0×10~6Da，推测其结构为：{→2)-α-D-Glcp-(1→}n。
在抑菌活性实验中，贻贝蒸煮液及MJPs对4种供试菌金黄色葡萄球菌、酵母菌、大肠杆菌和副溶血性弧菌均无明显抑制作用；在体外NaNO_2清除及NDMA合成阻断实验中，贻贝蒸煮液对NaNO_2的清除作用和NDMA合成阻断作用极为显著，但是MJPs对NaNO_2的清除和NDMA合成阻断活性不显著；在体外抗氧化实验中，贻贝蒸煮液(以可溶性固形物计)的IC_(50)为3．80μg／mL，MJPs的IC_(50)为1．12μg/mL，TBHQ的IC_(50)为7．55μg/mL，MJPs的最大抑制率59．9％不如TBHQ的最大抑制率89．9％高，但是其IC_(50)明显优于TBHQ。MJPsⅠ对小鼠T、B淋巴细胞有显著的正向调节作用，MJPsⅡ在低剂量下对小鼠B淋巴细胞有显著的负向调节作用，在高剂量下对小鼠T淋巴细胞表现出正向调节作用。此外，MJPsⅠ和MJPsⅡ均能显著抑制Raji细胞的增长和抑制黑色素的生成且对细胞毒性较小。
贻贝蒸煮液粗多糖MJPs在低浓度下具有较强的抑制DPPH·的活性。分离纯化得到的贻贝蒸煮液均一多糖MJPsⅠ和MJPsⅡ具有免疫调节作用，抑制Raji细胞增殖和抑制黑色素生成的活性，并且它们的低毒性使其在保健品和美白化妆品的开发以及结合其他强抗癌制剂以降低药物的细胞毒性方面具有极大的应用价值。
Mussel boiling Juice (MJ) was concentrated from decoction of fresh mussel which was boiled for 24 hours, and contained many nutritional and functional components like proteins, saccharide and so on. In this study, the water-soluble polysaccharides (MJPs) from MJ were isolated, and then their physical and chemical properties, structures and activities were investigated to help to make full use of the resources, to develop new functional foods and to improve the economic benefits.
The MJPs was extracted by alcohol fractional precipitation, free proteins and colors were removed by Sevage with papain method and charcoal, respectively. The homogeneity of MJPsⅠand MJPsⅡwhich were isolated and purified by Sepharose 4B column and dialysis membrane was determined by UV absorption spectrum, aceto-cellulose-thin layer-electrophoresis and HPGPC. And then their primary structures were analyzed by GC, IR and NMR. Furthermore, the anti-bacteria activity, blocking synthesis of NDMA and anti-oxidation activity of MJ and MJPs were measured by anti-bacteria cup method, standard curve method and DPPH·method. Lastly, the immunological activity, anti-tumor activity and inhibitory effect on melanin of MJPsⅠand MJPsⅡwere tested.
The results showed that saccharide in MJ were nonreducing sugar mostly, and purified MJPsⅠand MJPsⅡwere tested to be homogenous. The structural analysis indicated that MJPsⅠ, with a molecular weight of over 2.0×10~6Da, was composed of glucose as its major components. Its structure was speculated as follows.MJPsⅡ, with a molecular weight of over 2.0×10 Da, was also composed of glucose as its major components. Its structure was speculated as follows.{→2)-α-D-Glcp-(1→}n.
The MJ and MJPs did not display significant inhibitory effects on Staphylococcus aureus, Saccharomyces carlsbergengensis Hansen, Escherichia coli Castellani et Chalmers and Vibrio parahaemolyticus. In the experiment of scavenging NaNO_2 and blocking synthesis of NDMA, MJ showed strong activities, while MJPs only showed low activities; In the experiment of scavenging of DPPH·, MJ's IC_(50)=3.80μg/mL measured by soluble solid, MJPs'IC_(50)=1.12μg/mL, while the TBHQ's IC_(50)=7.55μg/mL. For MJPs, the strongest Inhibitory Rate was 59.9%, which was lower than TBHQ, of which the strongest Inhibitory Rate was 89.9%. However, the IC_(50) of MJPs was much lower than TBHQ. MJPsⅠshowed significant positive adjusting effect on T cells and B cells. MJPsⅡexhibited significant negative adjusting effect on B cells in low concentration and adjusting effect on T cells in high concentration. Both of them showed the inhibitory effects on EBV activation and inhibitory effects on melanin, with low toxicity on Raji and melanoma cells.
MJPs displayed strong inhibition of DPPH·in low concentration. MJPsⅠand MJPsⅡdisplayed immunological activities, anti-tumor activities and inhibitory effects on melanin with low toxicity on Raji and melanoma cells, which indicated that the Mussel Juice Polysaccharides may be valuable on developing the white-makeup ingredients and functional foods and inducing the cytotoxicity when added in other strong anti-cancer agents with high cytotoxicity.
首先，通过化学分析法对贻贝蒸煮液原料中基本成分进行分析。接着，采用乙醇沉淀、Sevage+木瓜蛋白酶法除蛋白、活性炭脱色从贻贝蒸煮液中分离出水溶性粗多糖MJPs，再经琼脂糖凝胶Sepharose4B柱层析和透析，纯化得到两种贻贝蒸煮液水溶性多糖MJPsⅠ和MJPsⅡ。经紫外扫描、醋酸纤维薄膜电泳、高效凝胶渗透色谱等方法检验它们的均一性。将得到的均一多糖MJPsⅠ和MJPsⅡ用苯酚-硫酸法测定总糖含量，高效凝胶渗透色谱法测定分子量，并利用气相色谱、红外光谱和核磁共振光谱，确定它们的初级结构。通过抑菌杯法、标准曲线法和DPPH·抑制率法分别测定贻贝蒸煮液和水溶性粗多糖MJPs的体外抑菌、体外NaNO_2清除及NDMA合成阻断以及体外抗氧化活性，并对MJPsⅠ和MJPsⅡ的免疫学活性、抗肿瘤活性和抑制黑色素生成活性进行测定。
实验结果表明，贻贝蒸煮液原料中糖类主要以非还原糖的形式存在，纯化得到的水溶性多糖MJPsⅠ和MJPsⅡ经检验为均一多糖。通过结构分析得到，MJPsⅠ主要由葡萄糖组成，平均分子量大于2．0×10~6Da，推测其结构为：MJPsⅡ主要由葡萄糖组成，平均分子量亦大于2．0×10~6Da，推测其结构为：{→2)-α-D-Glcp-(1→}n。
在抑菌活性实验中，贻贝蒸煮液及MJPs对4种供试菌金黄色葡萄球菌、酵母菌、大肠杆菌和副溶血性弧菌均无明显抑制作用；在体外NaNO_2清除及NDMA合成阻断实验中，贻贝蒸煮液对NaNO_2的清除作用和NDMA合成阻断作用极为显著，但是MJPs对NaNO_2的清除和NDMA合成阻断活性不显著；在体外抗氧化实验中，贻贝蒸煮液(以可溶性固形物计)的IC_(50)为3．80μg／mL，MJPs的IC_(50)为1．12μg/mL，TBHQ的IC_(50)为7．55μg/mL，MJPs的最大抑制率59．9％不如TBHQ的最大抑制率89．9％高，但是其IC_(50)明显优于TBHQ。MJPsⅠ对小鼠T、B淋巴细胞有显著的正向调节作用，MJPsⅡ在低剂量下对小鼠B淋巴细胞有显著的负向调节作用，在高剂量下对小鼠T淋巴细胞表现出正向调节作用。此外，MJPsⅠ和MJPsⅡ均能显著抑制Raji细胞的增长和抑制黑色素的生成且对细胞毒性较小。
贻贝蒸煮液粗多糖MJPs在低浓度下具有较强的抑制DPPH·的活性。分离纯化得到的贻贝蒸煮液均一多糖MJPsⅠ和MJPsⅡ具有免疫调节作用，抑制Raji细胞增殖和抑制黑色素生成的活性，并且它们的低毒性使其在保健品和美白化妆品的开发以及结合其他强抗癌制剂以降低药物的细胞毒性方面具有极大的应用价值。
Mussel boiling Juice (MJ) was concentrated from decoction of fresh mussel which was boiled for 24 hours, and contained many nutritional and functional components like proteins, saccharide and so on. In this study, the water-soluble polysaccharides (MJPs) from MJ were isolated, and then their physical and chemical properties, structures and activities were investigated to help to make full use of the resources, to develop new functional foods and to improve the economic benefits.
The MJPs was extracted by alcohol fractional precipitation, free proteins and colors were removed by Sevage with papain method and charcoal, respectively. The homogeneity of MJPsⅠand MJPsⅡwhich were isolated and purified by Sepharose 4B column and dialysis membrane was determined by UV absorption spectrum, aceto-cellulose-thin layer-electrophoresis and HPGPC. And then their primary structures were analyzed by GC, IR and NMR. Furthermore, the anti-bacteria activity, blocking synthesis of NDMA and anti-oxidation activity of MJ and MJPs were measured by anti-bacteria cup method, standard curve method and DPPH·method. Lastly, the immunological activity, anti-tumor activity and inhibitory effect on melanin of MJPsⅠand MJPsⅡwere tested.
The results showed that saccharide in MJ were nonreducing sugar mostly, and purified MJPsⅠand MJPsⅡwere tested to be homogenous. The structural analysis indicated that MJPsⅠ, with a molecular weight of over 2.0×10~6Da, was composed of glucose as its major components. Its structure was speculated as follows.MJPsⅡ, with a molecular weight of over 2.0×10 Da, was also composed of glucose as its major components. Its structure was speculated as follows.{→2)-α-D-Glcp-(1→}n.
The MJ and MJPs did not display significant inhibitory effects on Staphylococcus aureus, Saccharomyces carlsbergengensis Hansen, Escherichia coli Castellani et Chalmers and Vibrio parahaemolyticus. In the experiment of scavenging NaNO_2 and blocking synthesis of NDMA, MJ showed strong activities, while MJPs only showed low activities; In the experiment of scavenging of DPPH·, MJ's IC_(50)=3.80μg/mL measured by soluble solid, MJPs'IC_(50)=1.12μg/mL, while the TBHQ's IC_(50)=7.55μg/mL. For MJPs, the strongest Inhibitory Rate was 59.9%, which was lower than TBHQ, of which the strongest Inhibitory Rate was 89.9%. However, the IC_(50) of MJPs was much lower than TBHQ. MJPsⅠshowed significant positive adjusting effect on T cells and B cells. MJPsⅡexhibited significant negative adjusting effect on B cells in low concentration and adjusting effect on T cells in high concentration. Both of them showed the inhibitory effects on EBV activation and inhibitory effects on melanin, with low toxicity on Raji and melanoma cells.
MJPs displayed strong inhibition of DPPH·in low concentration. MJPsⅠand MJPsⅡdisplayed immunological activities, anti-tumor activities and inhibitory effects on melanin with low toxicity on Raji and melanoma cells, which indicated that the Mussel Juice Polysaccharides may be valuable on developing the white-makeup ingredients and functional foods and inducing the cytotoxicity when added in other strong anti-cancer agents with high cytotoxicity.
引文
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