海洋硅藻中金藻昆布多糖提取纯化工艺和定量检测方法优化

Optimization of Extraction-purification Process and Quantitative Detection for Chrysolaminarin from Marine Diatoms

摘要:

金藻昆布多糖（Chrysolaminarin）是一种具有抗氧化、抗肿瘤、降血糖、降血脂和免疫调节等多种功效的β-1,3-葡聚糖。海洋硅藻中富含金藻昆布多糖，但提取纯化工艺和准确定量分析方法是目前研究和开发的瓶颈。该研究对海洋硅藻三角褐指藻胞内金藻昆布多糖的提取纯化工艺进行了优化，建立的最佳条件为：以Tris-EDTA缓冲液提取冻干藻粉4次后，采用终浓度为10%的三氯乙酸（Trichloroacetic Acid，TCA）除蛋白后再进行醇沉回收多糖。随后系统比较了苯胺蓝荧光检测法、苯酚-硫酸法、凝胶液相色谱法检测其含量的方法学。结果表明，苯胺蓝荧光检测法的最佳检测范围为1~40 μg/mL，具有最低检测下限（4.12 μg/mL）、最高精密度（相对标准偏差1.27%）和最好重现性（相对标准偏差2.49%），加样平均回收率高达100.85%，显著优于其他两种方法。该研究建立的优化工艺和检测新方法为金藻昆布多糖的开发利用提供了技术支持。

Abstract:

Chrysolaminarin is a kind of β-1,3-glucan possessing many functions such as anti-oxidative, anti-tumor, hypoglycemic, hypolipidemic and immunomodulatory activities. Marine diatoms are rich in chrysolaminarin, but extraction-purification process and accurate quantitative determination method are the bottlenecks of current research and development. In this study, the extraction-purification process for the intracellular chrysolaminarin from marine diatom Phaeodactylum tricornutum was optimized, and the optimal conditions were established as follows: The freeze-dried algal powder was extracted four times with the Tris-EDTA buffer, and the protein was removed using trichloroacetic acid (TCA; final concentration 10%) before the polysaccharide was recovered via alcohol precipitation. The aniline blue fluorescence method, phenol-sulfuric acid method and gel chromatography method were used to compare systematically the determined contents. The results showed that the aniline blue fluorescence method had the optimal detection range of 1~40 μg/mL and the lowest detection limit (4.12 μg/mL), the highest precision (relative standard deviation (RSD) 1.27%), the best reproducibility (RSD 2.49%) and high average recovery rate in spiked samples (100.85%), thereby being superior to other two methods. The optimized process and new detection method established in this study provide technical support for the development and utilization of chrysolaminarin.