基于糖谱法的半夏及其炮制品多糖结构特征分析及水/酶解前后抗氧化活性评价  

Structural Characteristics and Antioxidant Activity Analysis of Polysaccharides from Pinelliae Rhizoma and Its Processed Products Before and After Hydrolysis(Enzymolysis)by Sugar Spectrum

作　　者：胡美变 高奎旭 王瑶 彭溪 王静雅 孟祥龙 张朔生 黎江华 刘玉杰 HU Meibian;GAO Kuixu;WANG Yao;PENG Xi;WANG Jingya;MENG Xianglong;ZHANG Shuosheng;LI Jianghua;LIU Yujie(School of Traditional Chinese Medicine(TCM)and Food Engineering,Shanxi Provincial Key Laboratory of TCM Processing,Shanxi University of Chinese Medicine,Jinzhong 030619,China;School of Pharmacy,Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province,Chengdu University,Chengdu 610106,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 [2]成都大学药学院,抗生素研究与再评价四川省重点实验室,成都610106

出　　处：《中国实验方剂学杂志》2024年第11期192-201,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：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);山西省中医药管理局项目(2023ZYYC059);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);四川省自然科学基金项目(2022NSFSC0591)。

摘　　要：目的:采用糖谱法,通过部分酸水解和特异性糖苷酶酶解结合高效薄层色谱法(HPTLC)和荧光辅助凝胶电泳法(PACE),分析半夏及其炮制品多糖的糖苷键结构特征,以及其水/酶解前后的抗氧化活性变化。方法:半夏及其炮制品多糖加入淀粉酶除淀粉后,超滤法除去3000 Da以下的小分子成分;精制后的多糖以酸水解和5种特异性糖苷酶酶解制备样品,采用HPTLC和PACE进行分析;以2,2′-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)和2,2-联苯基-1-苦基肼基(DPPH)自由基清除实验评价多糖水/酶解前后抗氧化活性的变化。结果:通过HPTLC和PACE分析发现,半夏及其炮制品多糖能被β-半乳糖苷酶、β-甘露糖酶、纤维素酶和果胶酶水解,而几乎不能被葡聚糖酶水解,表明半夏及其炮制品的多糖含有β-吡喃半乳糖苷键、β-1,4-甘露糖苷键、β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键。通过体外抗氧化实验发现,半夏及其炮制品多糖经酸水解和果胶酶酶解后清除ABTS自由基能力减弱,而经纤维素酶,β-半乳糖苷酶和β-甘露糖酶酶解后,清除ABTS自由基能力增强;而半夏及其炮制品多糖经不同水/酶解后,清除DPPH自由基能力均明显增强。结论:该研究通过糖谱法分析了半夏及其炮制品多糖的糖苷键结构特征,并明确了其与抗氧化活性之间的关系,有利于进一步阐明半夏及其炮制品相关功效的物质基础,并为中药多糖结构特征分析提供新的思路与方法。Objective:The glycosidic linkage structural characteristics of polysaccharides from Pinelliae Rhizoma(PR)and its processed products were analyzed by sugar spectrum,high performance thin layer chromatography(HPTLC),fluorescence-assisted carbohydrate gel electrophoresis(PACE)based on partial acid hydrolysis and specific glycosidase hydrolysis,and the antioxidant activities of polysaccharides before and after hydrolysis(enzymolysis)were compared.Method:Polysaccharides from PR and its processed products were extracted by ultrasound extraction,starch was hydrolyzed byα-amylase,and small molecules below 3 kDa were removed by ultrafiltration.The purified polysaccharides were prepared by hydrolysis of acid and five different specific glycosidases,and the hydrolysates were analyzed by HPTLC and PACE.The antioxidant capacity of polysaccharides was analyzed by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS)and 2,2-diphenyl-1-picrylhydrazyl(DPPH)free radical scavenging experiment before and after different hydrolysis.Result:Through HPTLC and PACE analysis,it was found that polysaccharides from PR and its processed products could be hydrolyzed byβ-galactosidase,β-mannase,cellulase and pectinase,but hardly hydrolyzed by glucanase,indicating that the polysaccharides containedβ-galactopyranoside bond,β-1,4-mannoside bond,β-1,4-glucoside bond andα-1,4-galacturonic acid glycosidic bond.In vitro antioxidant experiments showed that the ABTS radical scavenging capacity of the polysaccharides from PR and its processed products was weakened after acid hydrolysis and pectinase enzymatic hydrolysis,while the ABTS radical scavenging capacity was enhanced after enzymatic hydrolysis with cellulase,β-galactosidase,andβ-mannase.And after different hydrolysis,the DPPH free radical scavenging capacity of polysaccharides from PR and its processed products was all significantly enhanced.Conclusion:The glycosidic linkage structural characteristics of polysaccharides from PR and its processed products was analyzed by sugar sp

关 键 词：半夏 炮制品 多糖 糖苷酶 糖谱法 抗氧化 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] R943.1[医药卫生—中医学] R28[理学—分析化学] O657[理学—化学]