摘要
目的阐明桑叶中具有降血糖活性的多糖结构基础,为桑叶的开发和利用提供理论基础。方法采用水提醇沉法获得桑叶粗多糖,经离子交换色谱和凝胶渗透色谱分离纯化获得SY02、SY02-3、SY02-3A及SY02-3B等多糖组分,并测定均一多糖的理化性质;联合单糖组成分析、糖残基连接方式分析和核磁分析对桑叶多糖进行结构鉴定;采用棕榈酸(Palimitate,PA)诱导INS-1细胞凋亡模型、Caspase3/7活性试剂盒测定桑叶多糖对INS-1E细胞凋亡的保护作用,并运用Western blot法检测桑叶多糖对INS-1E细胞凋亡关键蛋白Cleaved Caspase 3表达的影响。结果采用水提醇沉法获得桑叶粗多糖SY,经离子交换树脂柱分离再由凝胶柱纯化得到SY02-3,再次纯化后得到均一酸性多糖SY02-3A与蛋白多糖SY02-3B,两者分子量分别为3.7×10^(4)Da和1.03×10^(4)Da。单糖组成分析结果表明SY02-3A含有鼠李糖、半乳糖醛酸、半乳糖、木糖、阿拉伯糖,摩尔比分别为23.97:33.85:11.73:5.04:25.41,得率为0.32%;SY02-3B由鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖与阿拉伯糖组成,其摩尔比19.83:10.34:45.42:9.01:2.23:13.17,得率为2.00%;SY02-3B含有14种氨基酸,其中天冬氨酸、谷氨酸、丙氨酸和甘氨酸含量相对较高。生物活性研究表明SY02-3可以保护PA诱导的INS-1E细胞凋亡,这种作用可能与降低Caspase 3活性和下调cleaved Caspase3蛋白表达有关。结论桑叶中含有酸性肽聚糖(SY02-3A)和蛋白聚糖(SY02-3B)的桑叶多糖SY02-3具有抑制PA诱导的INS-1E细胞凋亡的作用。
Objective To elucidate the structural basis of polysaccharides with hypoglycemic activity in mulberry leaves,and provide a theoretical basis for the development and utilization of mulberry leaves.Methods The crude polysaccharides from mulberry leaves were extracted by hot water.The components of SY02,SY02-3,SY02-3A and SY02-3B were obtained by ion-exchange chromatography and gel permeation chromatography.We determined the physicochemical properties of homogeneous polysaccharide.The structure of mulberry leaf polysaccharide was identified by monosaccharide composition analysis,sugar residue linkage analysis and nuclear magnetic analysis.Palimitate(PA)induced INS-1 cell apoptosis model was used to determine the protective effect of mulberry leaf polysaccharide on INS-1E cell apoptosis,and Caspase3/7 activity kit was used to detect mulberry leaf polysaccharide on the key protein of INS-1E cell apoptosis Cleaved by Western blot method effect of Caspase 3 expression.Results The crude polysaccharide SY was extracted from mulberry leaves by hot water,which was separated by ion exchange resin column and purified by gel column to obtain SY02-3.After purification,we obtained homogeneous polysaccharide SY02-3A and proteoglycan SY02-3B,with molecular weights of 3.7×10^(4)Da and 1.03×10^(4)Da.The results of monosaccharide composition analysis showed that SY02-3A contained rhamnose,galacturonic acid,galactose,xylose and arabinose.The molar ratio of SY02-3A was 23.97:33.85:11.73:5.04:25.41,and the yield was 0.32%.SY02-3B is composed of rhamnose,galacturonic acid,glucose,galactose and arabinose.The molar ratio of SY02-3B is 19.83:10.34:45.42:9.01:2.23:13.17 with a yield of 2.00%.SY02-3B contains 14 amino acids,among which the content of aspartate,glutamic acid,alanine and glycine is relatively high.We also found that SY02-3 protects palmitic acid-induced INS-1E cell apoptosis,which may be associated with reduced Caspase 3 activity and down-regulated cleaved Caspase 3 protein expression.Conclusion Mulberry leaf polysaccharide SY02-3,which contains acid peptidoglycan(SY02-3A)and proteoglycan(SY02-3B)in mulberry leaves,has the effect of inhibiting PA-induced apoptosis of INS-1E cells.
作者
邵赛聪
李茂婷
靳灿
王海颖
丁侃
SHAO Saicong;LI Maoting;JIN Can;WANG Haiying;DING Kan(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Chinese Academy of Sciences Shanghai Institute of Materia Medica,Shanghai 201203,China;Zhongshan Institute for Drug Discovery/Shanghai Institute of Materia Medica,Zhongshan 528400,China;Guizhou Medical University,Guiyang 550025,China;Jiangsu Lianyungang College of Higher Traditional Chinese Medicine,Lianyungang 222007,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2024年第5期1192-1201,共10页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会面上项目(31870801):蛋白聚糖Glypican-6在胰腺癌及其肿瘤组织纤维化发生中的功能机制研究,负责人:丁侃
上海市“超级博士后”(2022686),负责人:靳灿
广东省高水平创新研究院项目(2021B0909050003)
作者简介
通讯作者:靳灿,博士后,主要研究方向:多糖和寡糖的结构解析,构效关系和作用机制研究,基于糖的药物新靶标和新药发现研究;王海颖,副教授,硕士研究生导师,主要研究方向:糖尿病及肾病相关的科研课题研究。
