厚叶海带岩藻聚糖硫酸酯分离纯化及其体外肿瘤抑制活性研究

Study on separation and purification of fucoidan from Kjellmaniella crassifolia and antitumor activity in vitro

本研究以日本厚叶海带(Kjellmaniella crassifolia)中提取的岩藻聚糖硫酸酯(Fuciodan)为原料,采用DEAESepharose Fast Flow弱阴离子交换柱层析分离纯化,通过苯酚-硫酸法和氯化钡-明胶法检测各组分中多糖及硫酸根(SO_4~2)含量,MTT法分析50、200、800μg/m L的日本厚叶海带岩藻聚糖硫酸酯粗品(F)及分离组分对肝癌细胞Hep G2、肺癌细胞A549和结肠癌细胞HT-29细胞存活率影响。研究结果表明,DEAE-Sepharose Fast Flow弱阴离子交换柱对粗品F的分离纯化效果良好,可以得到5个组分(F-0、F-1、F-2、F-3和F-4);组分F-4多糖与SO_4^(2-)含量最高,分别为64.25%和26.32%。MTT实验结果显示,岩藻聚糖硫酸酯粗品及各分离组分对三种肿瘤细胞均具有肿瘤抑制活性,不同组分对HepG2、A549、HT-29的最低细胞存活率分别为67.69%、56.03%和27.56%。基于以上研究结果,表明日本厚叶海带岩藻聚糖硫酸酯具有很大的发展潜力,开发成为一种有效的天然抗肿瘤药物。

In this study, the crude fucoidan extracted from Kjellmaniella crassifolia was taken as raw material, and then purified by DEAE- Sepharose Fast Flow weak anion exchange column. The contents of total polysaccharide and sulfate （ SOl- ） of fucoidan in each fraction were determined by phenol-sulfuric acid colorimetry method and barium chloride-gelatin method, respectively.The antitumor activities for all in the crude and separated fractions with 50,200,800 μg/mL concentrations against HepG2 cells, A549 cells and HT- 29 ceils were determined by MTT assay. The results showed that, crude fucoidan from Kjellmaniella crassifolia could be well separated and purified by DEAE-Sepharose Fast Flow weak anion exchange column, and five different fractions（ F-0, F-1, F-2, F-3, F-4）were obtained.Total polysaecharide and SOl- content in F-4 were the highest, which were 64.25% and 26.32%, respectively.The results of MTT assay indicated that the crude fucoidan and the fractions had significant inhibitory activities on HepG2, A549 and HT-29cells.The lowest cell viability of 67.69% ,56.03% and 27.56% were obtained for HepG2, A549 and HT-29cells respectively.Based on those findings,it suggested that fucoidan from Kjellmaniella crassifolia had great potential to be a kind of effective natural antineoplastic drugs.