红毛藻多糖的化学组成及其体外免疫诱导活性研究

目的:对红毛藻中多糖组分进行提取、分离纯化,分析其化学组成和体外免疫诱导活性,阐明红毛藻具有提高免疫力食药用功效机制,为红毛藻的高值化利用提供科学依据。方法:通过水提醇沉法从红毛藻中提取红毛藻粗多糖,经阳离子交换柱层析和Sephadex G75柱层析纯化得到红毛藻多糖(BFP),再用DEAE-cellulose52柱层析对BFP进行分级纯化得到3个多糖组分F1、F2和F3;采用PMP柱前衍生、HPLC及化学方法分析其单糖组成和化学成分;通过RAW264.7细胞模型分析BFP、F1、F2和F3的体外免疫诱导活性和其相关细胞信号传导通路。结果:组成分析结果表明BFP、F1、F2和F3均为杂多糖且单糖组成存在较大差异。细胞模型结果表明,BFP、F1、F2和F3在所测定质量浓度范围(0~100μg/mL)均显著诱导RAW264.7细胞活化,释放NO和分泌TNF-α,而不诱导ROS的产生。细胞信号传导通路抑制试验结果表明BFP具有体外免疫诱导活性与细胞的NF-κB、JNK MAPK、ERK MAPK和p38 MAPK信号通路的激活相关;F1与细胞的NF-κB、JNK MAPK、ERK MAPK和p38 MAPK信号通路的激活有关;而F2和F3作用机制相似,分别与细胞的NF-κB、JNK MAPK和ERK MAPK信号通路的激活相关。结论:红毛藻多糖具有显著的体外免疫诱导活性,其中分级纯化多糖组分F1和F2是BFP中主要的免疫诱导活性组分;BFP、F1、F2和F3诱导RAW264.7细胞活化释放NO的共同的信号传导通路主要有两条:即NF-κB和JNK MAPK信号途径;而诱导RAW264.7细胞分泌TNF-α的共同信号通路主要是JNK MAPK和ERK MAPK信号途径。

Studies on Composition,in Vitro Immune-induced Activities of Polysaccharides Isolated from Bangia fusco-purpure

Objective:In this study,the polysaccharide derived from Bangia fusco-purpurea was separated and purified,and its chemical composition and in vitro immune-induced activity were analyzed to illuminate food and medicine mechanism of Bangia fusco-purpurea,which provided scientific basis for the high-efficient utilization of Bangia fusco-purpurea.Methods:Crude polysaccharide was extracted from Bangia fusco-purpurea by hot water extraction and ethanol precipitation.The polysaccharide fractions(F1,F2 and F3)were obtained with isolating and purifying polysaccharide BFP by cation exchange column chromatography,Sephadex G75 chromatography and DEAE-cellulose 52 exchange chromatography.The monosaccharide composition and chemical component of BFP,F1,F2 and F3 were analyzed by PMP pre-column derivatization high performance liquid chromatography(HPLC)and the chemical methods.Further,the in vitro immune-induced activity and signal transduction pathway of BFP,F1,F2 and F3 were analyzed by mouse macrophage RAW264.7 cells model.Results:The results of chemical component and monosaccharide composition showed that BFP,F1,F2 and F3 were heteropolysaccharides with significant differences in monosaccharide compositions.The in vitro immune-induced activity cells model revealed that BFP,F1,F2 and F3 significantly induced RAW264.7 cells activation to release NO and TNF-αin the measured concentration range(0~100μg/mL),but did not induce the generation of ROS.Cellular signal pathway inhibition experimental results show that the immune-induced activity of BFP was related to the cellular signal transduction pathways activation of NF-κB,JNK MAPK,ERK MAPK,and p38 MAPK;F1 was associated with the cellular signal transduction pathways activation of NF-κB,JNK MAPK,ERK MAPK and p38 MAPK;however,the mechanisms of action of F2 and F3 were similar,which were related to the activation of NF-κB,JNK MAPK and ERK MAPK signal transduction.Conclusion:The BFP possessed strong in vitro immune-induced activity,of which fractional purified polysaccharide fractions F1 and F2 were the major immune-induced components.The common signal transduction pathways of BFP,F1,F2 and F3 induced RAW264.7 cells release NO were NF-κB and JNK MAPK.However,the common signal pathways inducing TNF-αsecretion in RAW264.7 cells were mainly JNK and ERK MAPK signaling pathways.