聚合草多肽的酶法制备及其免疫活性研究

为了提高聚合草利用价值,开发聚合草免疫活性肽,利用Box-Behnken设计原理,以多肽含量为响应值,用响应面法构建数学模型,确定制备聚合草多肽的最优工艺条件,并建立Con A诱导的小鼠脾淋巴细胞增殖的体系(MTT法)和LPS促进小鼠腹腔巨噬细胞吞噬功能的体系(中性红法),探究所制备的聚合草多肽的免疫活性。结果表明,将聚合草以8%的料水比打浆,添加3%纤维素酶,调节pH值至6.0,45℃酶解2 h后,再添加4.57%的碱性蛋白酶,调节pH值至8.91,43.4℃酶解56 min,酶解液中的多肽含量达0.664 mg·m L~(-1)。用乙醇沉淀法收集酶解液中的多肽,制成多肽液,结果发现,66、0.66和0.066μg·m L~(-1)的聚合草多肽对小鼠脾淋巴细胞的增殖均具有显著促进作用;66、6.6和0.66μg·m L~(-1)的聚合草多肽对小鼠腹腔巨噬细胞的吞噬功能均具有显著增强作用。由此可见,本研究所制备的聚合草多肽具有免疫增强活性,这为聚合草的高值利用提供了理论依据。

Enzymatic Preparation and Immuno-enhancing Activities of Symphytum officinale L.Polypeptides

The present study aimed to prepare immunomodulatory peptides of Symphytum officinale L.to improve its utilization values.Based on the basic principles of Box-Behnken design,a mathematical model was established by response surface method and the optimum conditions for preparing Symphytum officinale L.polypeptides were determined.The ConA-induced spleen lymphocyte proliferation system (MTT method) and the LPS-promoted celiac macrophages phagocytosis system (neutral red method) in mice were established to explore the immuno-enhancing activity of the resulted Symphytum officinale L.polypeptides.The results showed that the polypeptide content in the hydrolysate reached to 0.664mg· mL-1 and the optimized condition for Symphytum officinale L.polypeptides preparation was as following:smashing the leaves at a ratio of 8％ (material to water),adding 3％ of cellulose,adjusting initial pH to 6.0,water-bathing at 45℃ for 2h,then adding 4.57％ of alcalase to the system and adjusting initial pH to 8.91,maintaining at 43.4℃ for 56min,then the ethonal precipitated polypeptides were used for later study.Results suggested that 66,0.66 and 0.066μg· mL-1 Symphytum officinale L.polypeptides significantly enhanced the proliferation of mice spleen lymphocytes.66,0.66 and 0.066μg·mL-1 polypeptides dramatically increased the phagocytic capacity of mice celiac macrophages.A conclusion can be drew that the Symphytum officinale L.polypeptides were immunocompetent and the present study provided a theoretical basis for future high-value utilization of Symphytum officinale L..