板蓝根多糖酶法脱蛋白工艺、组成分析与免疫调节活性研究  

Study on the Enzymatic Deproteinization Technology,Composition Analysis and Immunomodulatory Activity of Isatidis Radix Polysaccharides

作　　者：赖梦亭 麦麦提敏·麦提萨伍尔 李彤[1] 肖平[1] 宿树兰[1] 段金廒[1] LAI Mengting;Memitimin Metsawur;LI Tong;XIAO Ping;SU Shulan;DUAN Jinao(Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine,Key Laboratory of Chinese Medicinal Resources Recycling Utilization,National Administration of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学江苏省中药资源产业化过程协同创新中心/中药资源产业化与方剂创新药物国家地方联合工程研究中心/国家中医药管理局中药资源循环利用重点研究室,江苏南京210023

出　　处：《南京中医药大学学报》2024年第4期379-390,共12页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：江苏省中医药科技发展计划项目(MS2021004);中央本级重大增减支项目(2060302);国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005);国家中医药管理局高水平中医药重点学科建设项目-中药资源化学(ZYYZDXK-2023083);南京中医药大学自然科学基金青年项目(NZY81703642);康缘中药学院创新创业重点项目(kyxysc08);宁夏回族自治区重点研发计划重点项目(2020BFH02013)。

摘　　要：目的优化板蓝根多糖脱蛋白工艺,并进一步探讨其免疫调节活性,为板蓝根多糖的开发利用提供科学依据。方法通过单因素结合Box-Behnken响应面法优化酶法脱蛋白的最佳工艺条件;利用紫外可见光谱、傅里叶变换红外光谱、高效凝胶渗透色谱、高效液相色谱和扫描电镜等方法对板蓝根多糖化学组成与结构特征进行分析;采用斑马鱼免疫低下模型探讨脱蛋白板蓝根多糖对斑马鱼体内中性粒细胞、巨噬细胞、IL-1β和IL-6含量的影响。结果酶法脱蛋白最佳工艺为:胰蛋白酶500 U·mL^(-1)、pH 8.0、酶解时间5 h、酶解温度37℃,脱蛋白率为(86.39±0.07)%,综合评分(91.15±0.37)%。紫外、红外光谱扫描和电镜扫描显示酶法可以除去粗多糖中含有的蛋白质,脱蛋白后相对分子量在5.82~60.26 kDa之间,单糖摩尔组成为甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=2.17∶0.96∶2.90∶83.25∶4.88∶5.84。免疫活性评价结果表明,脱蛋白后的板蓝根多糖浓度在50~300μg·mL^(-1)时,能显著增加斑马鱼免疫细胞密度,增加巨噬细胞增殖,降低免疫低下斑马鱼体内IL-1β和IL-6含量,从而发挥免疫调节作用。结论酶法可以有效去除板蓝根粗多糖中的蛋白质,脱蛋白后的板蓝根多糖具有一定的免疫调节作用。OBJECTIVE To optimize the deproteinization process of Isatidis Radix polysaccharides and further explore its immunomodulatory activity,and to provide a scientific basis for the development and utilization of it.METHODS The optimum conditions of enzymatic deproteinization were optimized by a single factor combined with the Box-Behnken response surface method.The chemical composition and structural characteristics of deproteinized Isatidis Radix polysaccharides were analyzed by UV-visible spectrum,Fourier transform-infrared spectroscopy,high-performance gel permeation chromatography,high-performance liquid chromatography and scanning electron microscopy.The effects of deproteinized Isatidis Radix Polysaccharide on neutrophils,macrophages,IL-1βand IL-6 in zebrafish were investigated by using a zebrafish immunocompromised model.RESULTS The optimal enzymatic deproteinization process was as follows:trypsin 500 U·mL^(-1),pH 8.0,enzymatic hydrolysis time 5 h,enzymatic hydrolysis temperature 37℃.The deproteinization rate was(86.39±0.07)%,and the comprehensive score was(91.15±0.37)%.Ultraviolet,infrared spectroscopy scanning and scanning electron microscopy showed that the protein contained in the crude polysaccharide could be removed by enzymatic method.The relative molecular weight of the polysaccharides were between 5.82 and 60.26 kDa.The monosaccharide mole composition was mannose∶rhamnose∶galacturonic acid∶glucose∶galactose∶arabinose=2.17∶0.96∶2.90∶83.25∶4.88∶5.84.The results of immune activity evaluation showed that when the concentration of deproteinized Radix Isatidis polysaccharides was 50~300μg·mL^(-1),it could significantly increase the density of zebrafish immune cells,increase the number of macrophages,and reduce the content of IL-1βand IL-6 in immunocompromised zebrafish,thus exerting immunomodulatory effects.CONCLUAION The enzymatic method can effectively remove the proteins contained in the crude polysaccharides of Isatidis Radix,and the deproteinized Isatidis Radix polysacchari

关 键 词：板蓝根多糖 酶法除蛋白 多糖结构 免疫调节 斑马鱼 

分 类 号：R283[医药卫生—中药学]