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

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 spectrum in this paper,and the relationship between glycosidic bond types and their antioxidant activity was clarified through in vitro antioxidant experiments,which is beneficial for further elucidating the material basis of the related efficacy of PR and its processed products,and providing new ideas and methods for analyzing the structural characteristics of polysaccharides in Chinese medicines.