雨生红球藻多糖的分离纯化和免疫活性组分鉴定
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摘要
以虾青素提取后的雨生红球藻渣为原料,在体外免疫细胞模型活性跟踪下,对超声辅助提取的雨生红球藻粗多糖(Haematococcus pluvialis polysaccharide,HPP)采用DEAE-52纤维素阴离子交换柱层析和Sephacryl S400葡聚糖凝胶柱层析进行分离纯化,使用高效凝胶渗透色谱法测定各组分多糖分子质量。结果表明:HPP经DEAE-52柱层析分离得到HPP-c1、HPP-c2、HPP-c3、HPP-c4和HPP-c5五个多糖组分,其中HPP-c3免疫活性较好且含量最高;HPP-c3经Sephacryl S400柱层析分离得到HPP-c3-s1、HPP-c3-s2和HPP-c3-s3,其中HPP-c3-s1在0～250μg/mL质量浓度范围内极显著刺激小鼠脾细胞的增殖(P<0.01),并呈现剂量效应,最高刺激指数达1.34,高于HPP-c3-s2和HPP-c3-s3。经纯度鉴定,HPP-c3-s1为均一多糖,分子质量为23 413 kDa。研究结果可为雨生红球藻的高值化综合利用提供有利指导。
An in vitro immune cell model was used for bioactivity-guided fractionation of the crude polysaccharides ultrasonically extracted from Haematococcus pluvialis residue left after the extraction of astaxanthin by sequential DEAE-52 anion exchange and Sephacryl S400 column chromatographies. The molecular masses of the isolated polysaccharides were determined by high performance gel permeation chromatography (HPGPC). The results showed that five polysaccharides, named HPP-c1, HPP-c2, HPP-c3, HPP-c4 and HPP-c5, were obtained by DEAE-52 column chromatography. Of these, HPP-c3 had the highest immunological activity. Further, HPP-c3-s1, HPP-c3-s2, and HPP-c3-s3 were isolated from HPP-c3 by Sephacryl S400 column chromatography. HPP-c3-s1 could significantly stimulate the proliferation of spleen cells (P < 0.01) in the concentration range of 0–250 μg/mL in a dose-dependent fashion. HPP-c3-s1 had the highest stimulation index (SI) of 1.34 among the HPP-c3 fractions. The purity of HPP-c3-s1 was found to be homologous with a molecular mass of 23 413 kDa. These results provide a valuable guidance for the high-value comprehensive utilization of H. pluvialis.
An in vitro immune cell model was used for bioactivity-guided fractionation of the crude polysaccharides ultrasonically extracted from Haematococcus pluvialis residue left after the extraction of astaxanthin by sequential DEAE-52 anion exchange and Sephacryl S400 column chromatographies. The molecular masses of the isolated polysaccharides were determined by high performance gel permeation chromatography (HPGPC). The results showed that five polysaccharides, named HPP-c1, HPP-c2, HPP-c3, HPP-c4 and HPP-c5, were obtained by DEAE-52 column chromatography. Of these, HPP-c3 had the highest immunological activity. Further, HPP-c3-s1, HPP-c3-s2, and HPP-c3-s3 were isolated from HPP-c3 by Sephacryl S400 column chromatography. HPP-c3-s1 could significantly stimulate the proliferation of spleen cells (P < 0.01) in the concentration range of 0–250 μg/mL in a dose-dependent fashion. HPP-c3-s1 had the highest stimulation index (SI) of 1.34 among the HPP-c3 fractions. The purity of HPP-c3-s1 was found to be homologous with a molecular mass of 23 413 kDa. These results provide a valuable guidance for the high-value comprehensive utilization of H. pluvialis.
引文
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[13]马超.微藻中功能性物质的放大提取制备与生物活性评价[D].广州:华南理工大学,2016.
[14]吕小华,陈文青,罗世英,等.螺旋藻多糖对C H B患者PBMC免疫功能的影响[J].中国药理学通报,2015,31(8):1121-1125.DOI:10.3969/j.issn.1001-1978.2015.08.019.
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[28]席波.海洋微藻的活性筛选及活性成分的功能研究[D].天津:天津科技大学,2015.
[29]李志平.巢湖蓝藻多糖的分离、纯化及理化性质研究[D].合肥:安徽大学,2014.
[30]蔡冰娜,陈纯馨,陈忻,等.抗氧化钝顶螺旋藻多糖的分离纯化[J].食品科学,2013,34(23):83-87.DOI:10.7506/spkx1002-6630-201323018.
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[32]孙颖颖,周豹,徐深圳,等.球等鞭金藻胞内和胞外多糖的分离纯化及其抑菌活性[J].食品科学,2012,33(11):137-141.
[33]熊双丽,李安林.酸性多糖的最新研究进展[J].食品科技,2010,35(5):80-83.DOI:10.13684/j.cnki.spkj.2010.05.073.
[34]张岩,马丽娜,陶遵威.中药多糖的分离纯化方法研究进展[J].天津药学,2011,23(3):72-74.DOI:10.3969/j.issn.1006-5687.2011.03.030.
[35]宫春宇,唐庆九,张劲松,等.龙须菜免疫活性多糖的分离纯化及结构鉴定[J].天然产物研究与开发,2010,22(4):603-606;624.DOI:10.3969/j.issn.1001-6880.2010.04.016.
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