除蛋白方式对湛江等鞭金藻多糖的组成、结构特征和体外抗肿瘤活性的影响
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摘要
为探索适宜的微藻多糖除蛋白方式,以湛江等鞭金藻Isochrysis zhanjianggensis为试验材料,通过水提醇沉法得到粗多糖(F),再分别采用Sevag法和三氯乙酸(TCA)法去除粗多糖蛋白得到样品F_S和F_T,然后对F、F_S和F_T进行总糖、硫酸根含量和单糖组成检测,并通过傅里叶变换红外光谱(FT-IR)和凝胶排阻层析(Sephacryl S-100)对其成分加以分析,另考察了3种组分体外对6株肿瘤细胞(MCF-7、HeLa、SW480、HT29、HepG2和A549)的抑制作用。结果表明:湛江等鞭金藻中主要的单糖组成包括甘露糖、葡萄糖、半乳糖、阿拉伯糖、木糖、葡萄糖醛酸、鼠李糖和岩藻糖;经不同除蛋白方式处理后所得样品F_S和F_T的单糖组成比例发生变化;粗提物F中高相对分子质量组分(80 000～90 000)在Sevag法处理后比例下降,但在TCA法处理后得以较好保留;而TCA法处理由于水解和透析的作用使得低相对分子质量组分比例下降;体外抗肿瘤活性试验中3种多糖样品同浓度下对肿瘤细胞的抑制作用相比较,TCA法所得样品F_T活性最强,粗提物F次之,Sevag法所得样品F_S最弱。研究表明,不同除蛋白方式所得的多糖在组成和活性上有所差异,在多糖商品化开发探索中应考虑以活性为导向进行工艺选择和优化。
The crude polysaccharide(F) was preparedfrom microalga Isochrysis zhanjianggensis through alcohol precipitation, and the crude fraction F was processed using Sevag reagent or trichloroacetic acid(TCA), with deproteinized preparations referred as F_S and F_T, respectively, in order to explore the suitable deproteinization method. The total sugar content, sulfate content and monosaccharide compositions were determined in F, F_S and F_T, in which the constituents were analyzed by Fourier transform infrared spectroscopy(FT-IR) and gel exclusion chromatography. In addition, inhibitory effects of the three fractions on six tumor cell lines including MCF-7, HeLa, SW480, HT29, HepG2 and A549 were assayed in vitro. It was found that the polysaccharides from the microalga were primarily consisted of mannose, glucose, galactose, arabinose, xylose, glucuronic acid, rhamnose and fucose. The ratios of monosaccharide composition were varied after the polysaccharides were processed using different deproteinizing methods. The high-molecular-weight component(80 000-90 000) was shown to be decreased in F administrated by Sevag, while it was better preserved through TCA treatment. In contrast, the proportion of low-molecular-weight components in F_T was reduced due to hydrolysis and dialysis. The antitumor experiment revealed that the maximal function of inhibiting tumor cells was observed in F_T, follows by F and the minimal in F_S. The findings indicated that polysaccharides treated with different deproteinizing methods showed different compositional characteristics and biological activities, and that the polysaccharide processing technique should be established and optimized based on activity preservation in future commercial exploration.
The crude polysaccharide(F) was preparedfrom microalga Isochrysis zhanjianggensis through alcohol precipitation, and the crude fraction F was processed using Sevag reagent or trichloroacetic acid(TCA), with deproteinized preparations referred as F_S and F_T, respectively, in order to explore the suitable deproteinization method. The total sugar content, sulfate content and monosaccharide compositions were determined in F, F_S and F_T, in which the constituents were analyzed by Fourier transform infrared spectroscopy(FT-IR) and gel exclusion chromatography. In addition, inhibitory effects of the three fractions on six tumor cell lines including MCF-7, HeLa, SW480, HT29, HepG2 and A549 were assayed in vitro. It was found that the polysaccharides from the microalga were primarily consisted of mannose, glucose, galactose, arabinose, xylose, glucuronic acid, rhamnose and fucose. The ratios of monosaccharide composition were varied after the polysaccharides were processed using different deproteinizing methods. The high-molecular-weight component(80 000-90 000) was shown to be decreased in F administrated by Sevag, while it was better preserved through TCA treatment. In contrast, the proportion of low-molecular-weight components in F_T was reduced due to hydrolysis and dialysis. The antitumor experiment revealed that the maximal function of inhibiting tumor cells was observed in F_T, follows by F and the minimal in F_S. The findings indicated that polysaccharides treated with different deproteinizing methods showed different compositional characteristics and biological activities, and that the polysaccharide processing technique should be established and optimized based on activity preservation in future commercial exploration.
引文
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[19] 杨海波,王茜,曹旭鹏,等.甘油对湛江等鞭金藻生长及细胞内多糖累积的影响[J].水产科学,2015,34(8):485-490.
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[22] 邢丽红,祝纯静,孙伟红,等.柱前衍生-高效液相色谱法测定岩藻多糖的单糖及糖醛酸含量[J].中国渔业质量与标准,2011,1(1):64-69.
[23] 陈清花,李文权,张赛金,等.海洋微藻多糖组成的气相色谱分析[J].厦门大学学报:自然科学版,2003,42(4):495-498.
[24] 李洁琼,刘红全,袁莎.微藻多糖的研究进展[J].现代化工,2016,36(6):60-62.
[25] 汤梦瑶,王海波,祁艳霞,等.蛤类多糖的研究进展[J].大连海洋大学学报,2018,33(1):130-136.
[26] Pugh N,Ross S A,ElSohly H N,et al.Isolation of three high molecular weight polysaccharide preparations with potent immunostimulatory activity from Spirulina platensis,Aphanizomenon flos-aquae and Chlorella pyrenoidosa[J].Planta Medica,2001,67(8):737-742.
[2] 杨福利,李秀辰,白晓磊,等.小球藻脱氮除磷及其生物量增殖潜力的研究[J].大连海洋大学学报,2014,29(2):193-197.
[3] Raja R,Hemaiswarya S,Kumar N A,et al.A perspective on the biotechnological potential of microalgae[J].Critical Reviews in Microbiology,2008,34(2):77-88.
[4] 夏嵩,万凌琳,李爱芬,等.微藻生物质产品和生物活性物质的研究与开发[J].天然产物研究与开发,2014,26(3):463-469,453.
[5] 陈玮,刘启顺,李曙光,等.微藻多糖生物活性研究进展[J].中国海洋药物杂志,2012,31(3):55-60.
[6] Sun Liqin,Wang Ling,Zhou Yan.Immunomodulation and antitumor activities of different-molecular-weight polysaccharides from Porphyridium cruentum[J].Carbohydrate Polymers,2012,87(2):1206-1210.
[7] Sun Liqin,Chu Jinling,Sun Zhongliang,et al.Physicochemical properties,immunomodulation and antitumor activities of polysaccharide from Pavlova viridis[J].Life Sciences,2016,144:156-161.
[8] Kim M,Yim J H,Kim S Y,et al.In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03[J].Antiviral Research,2012,93(2):253-259.
[9] Mustopa A Z,Lages A C,Ridwan M,et al.Purification and characterization of polysaccharide from microalgae btm 11 as inhibitor of hepatitis c virus rna helicase[J].Indonesian Journal of Pharmacy,2015,26(3):134-140.
[10] 周妍,王凌,孙利芹,等.5种海洋微藻多糖体外免疫调节活性的筛选[J].海洋通报,2010,29(2):194-198.
[11] Park J K,Kim Z H,Lee C G,et al.Characterization and immunostimulating activity of a water-soluble polysaccharide isolated from Haematococcus lacustris[J].Biotechnology and Bioprocess Engineering,2011,16(6):1090-1098.
[12] 孙颖颖,王辉.球等鞭金藻胞外多糖的体外抗氧化活性和理化性质的初步分析[J].海洋科学,2013,37(5):45-49.
[13] 王凌,孙利芹,赵小惠.一种微藻多糖的理化性质及抗氧化和保湿活性[J].精细化工,2012,29(1):20-24,29.
[14] 陈晓清,郑怡,林雄平.二种微藻多糖与蛋白质提取物的抗菌活性[J].福建师范大学学报:自然科学版,2005,21(2):76-79.
[15] De Jesus Raposo M F,De Morais A M M B,De Morais R M S C.Influence of sulphate on the composition and antibacterial and antiviral properties of the exopolysaccharide from Porphyridium cruentum[J].Life Sciences,2014,101(1-2):56-63.
[16] Wu Qinghua,Liu Lian,Miron A,et al.The antioxidant,immunomodulatory,and anti-inflammatory activities of Spirulina:an overview[J].Archives of Toxicology,2016,90(8):1817-1840.
[17] 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.
[18] 周妍,王凌,孙利芹,等.叉鞭金藻多糖的分离纯化及体外抗氧化作用研究[J].烟台大学学报:自然科学与工程版,2010,23(4):289-293.
[19] 杨海波,王茜,曹旭鹏,等.甘油对湛江等鞭金藻生长及细胞内多糖累积的影响[J].水产科学,2015,34(8):485-490.
[20] DuBois M,Gilles K A,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances[J].Analytical Chemistry,1956,28(3):350-356.
[21] 张惟杰.糖复合物生化研究技术[M].杭州:浙江大学出版社,1999.
[22] 邢丽红,祝纯静,孙伟红,等.柱前衍生-高效液相色谱法测定岩藻多糖的单糖及糖醛酸含量[J].中国渔业质量与标准,2011,1(1):64-69.
[23] 陈清花,李文权,张赛金,等.海洋微藻多糖组成的气相色谱分析[J].厦门大学学报:自然科学版,2003,42(4):495-498.
[24] 李洁琼,刘红全,袁莎.微藻多糖的研究进展[J].现代化工,2016,36(6):60-62.
[25] 汤梦瑶,王海波,祁艳霞,等.蛤类多糖的研究进展[J].大连海洋大学学报,2018,33(1):130-136.
[26] Pugh N,Ross S A,ElSohly H N,et al.Isolation of three high molecular weight polysaccharide preparations with potent immunostimulatory activity from Spirulina platensis,Aphanizomenon flos-aquae and Chlorella pyrenoidosa[J].Planta Medica,2001,67(8):737-742.