花椒多糖抗阿尔茨海默病作用及其糖谱法结构特征分析  

Anti-Alzheimer's Disease Effect of Polysaccharides from Zanthoxyli Pericarpium and Analysis of Its Structural Characteristics by Sugar Spectrum

作　　者：胡美变 孟祥龙 王璞 张朔生 刘玉杰 HU Meibian;MENG Xianglong;WANG Pu;ZAHNG Shuosheng;LIU Yujie(College of Traditional Chinese Medicine(TCM)and Food Engineering,Shanxi Provincial Key Laboratory of TCM Processing,Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学中药与食品工程学院,中药炮制山西省重点实验室,山西晋中030619

出　　处：《中国实验方剂学杂志》2023年第10期174-182,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：山西中医药大学科技创新能力培育计划基础研究专项(2021PY-JC-12);山西中医药大学博士科研启动基金项目(2022BK09);山西中医药大学优秀博士毕业生来晋工作奖励经费科研启动基金项目(2022BKS06);国家自然科学基金青年科学基金项目(82104547);山西省科技厅基础研究计划项目(20210302124278);山西省高等学校科技创新项目(2021L358)。

摘　　要：目的:建立阿尔茨海默病(AD)小鼠模型,评价花椒多糖对AD的治疗作用并采用糖谱法分析其结构特征。方法:采用腹腔注射D-半乳糖联合灌胃三氯化铝建立快速衰老的AD小鼠模型,通过Morris水迷宫实验评价小鼠的学习记忆能力,苏木素-伊红(HE)染色和尼氏染色观察小鼠脑组织病理学状况和神经元损伤情况。对花椒多糖进行酸水解和不同糖苷酶酶解后,采用高效薄层色谱法(HPTLC)和荧光辅助凝胶电泳法(PACE)分析水解产物的结构特征。HPTLC色谱条件为采用硅胶60高效薄层色谱板,点样量5μL,展开剂乙酸乙酯-冰乙酸-水(2∶2∶1),展开2次,苯胺-二苯胺-磷酸溶液显色,在105℃加热10 min后日光下检视。PACE检测条件为34%分离胶和8%浓缩胶,电泳缓冲液为0.1 mol·L^(-1)三羟甲基氨基甲烷(Tris)-硼酸缓冲液(pH 8.2),电泳在0℃环境下进行,上样量3~6μL,用15 mA恒流跑至样品达凝胶前端,紫外灯365 nm下检视。结果:Morris水迷宫实验结果表明,花椒多糖明显改善了AD模型小鼠的学习和记忆能力,逃避潜伏期缩短,穿梭平台次数和目标象限停留时间明显增加。组织病理学实验结果表明,花椒多糖能改善AD小鼠海马组织CA1、CA3区的病理情况和神经元的损伤,且尼氏小体数量明显增加。糖谱法分析结果表明,HPTLC和PACE分析结果基本一致,花椒多糖主要能被纤维素酶和果胶酶催化酶解成小分子糖类,表明其主要含有β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键,能被葡聚糖酶、β-半乳糖苷酶和β-甘露糖酶少量酶解,表明其含有少量α-1,6-葡萄糖苷键、β-吡喃半乳糖苷键和β-1,4-甘露糖苷键。结论:花椒多糖对AD小鼠有明显治疗作用,其结构中主要含有β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键,可为中药多糖的结构解析提供有益借鉴。Objective:The therapeutic effect of polysaccharides from Zanthoxyli Pericarpium on Alzheimer's disease(AD) was evaluated through establishing a mouse model of AD,and the structural characteristics of the polysaccharides was analyzed by sugar spectrum.Method:The AD model of mice with rapid aging was established by intraperitoneal injection of D-galactose combined with gavage of aluminum trichloride,and the learning and memory ability of mice was evaluated by Morris water maze test,the histopathological status of brain and neuronal damage were observed by hematoxylin-eosin(HE) staining and Nissl staining.After hydrolysis of polysaccharides from Zanthoxyli Pericarpium with acid and different glycosidases,the characteristics of hydrolysates were analyzed by high performance thin layer chromatography(HPTLC) and fluorescence assisted carbohydrate gel electrophoresis(PACE).HPTLC chromatography was performed on a silica gel 60 plate with sampling volume of 5 μL,developing solvent of ethyl acetate-glacial acetic acid-water(2∶2∶1),developing twice,aniline-diphenylamine-phosphoric acid solution as chromogenic agent,and heating at 105 ℃ for 10 min,and then observed under sunlight.PACE experimental conditions were 34% separation gel and 8% concentration gel,electrophoresis buffer was 0.1 mol·L~(-1) tris(hydroxymethyl) aminomethane(Tris)-boric acid buffer(pH 8.2).Electrophoresis was carried out at 0 ℃ and the loading amount was 3-6 μL.The sample ran to the front of the gel with a constant current of 15 mA,and imaged under ultraviolet 365 nm.Result:The results of Morris water maze test showed that polysaccharides from Zanthoxyli Pericarpium significantly improved the learning and memory ability of AD model mice,shortened the escape latency,and significantly increased the number of crossing and the residence time in the target quadrant.The results of histopathological experiments showed that polysaccharides from Zanthoxyli Pericarpium could improve the pathological conditions and neuronal damage in the CA1 and CA3 reg

关 键 词：花椒多糖 阿尔茨海默病(AD) 糖谱法 糖苷酶 高效薄层色谱法(HPTLC) 荧光辅助凝胶电泳法(PACE) 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R96[理学—分析化学] O657[理学—化学]