糖谱法结合多元色谱分析比较铁皮石斛功能性多糖结构特征

目的：比较不同产地铁皮石斛功能性多糖结构特征，为其质量控制奠定基础。方法：高效凝胶色谱联用多角度激光散射和示差折光仪法（HPSEC-MALLS-RID法）用于测定多糖的重均相对分子质量、回旋半径及含量，色谱柱为TSK-GEL G5000 PW_XL串联TSK Gel G3000PW_XL，柱温35℃，流动相为9 mg·mL^-1氯化钠水溶液，流速0.5 mL·min^-1。气相色谱联用质谱法用于分析单糖组成及糖苷键，毛细管色谱柱为Agilent HP-5MS（30 m×0.25 mm×0.25 μm），进样量2 μL，用于单糖组成分析的程序升温条件为起始温度165℃，保持7 min，以5℃·min^-1升温至185℃，保持5 min，以4℃·min^-1升温至200℃，以20℃·min^-1升至280℃；用于糖苷键分析的程序升温条件为起始温度120℃，以5℃·min^-1升温至200℃，以8℃·min^-1升温至250℃，以20℃·min^-1升至280℃。基于荧光辅助凝胶电泳、高效薄层色谱-糖谱法分析功能性多糖部分酸水解和酶解产物特征，荧光辅助凝胶电泳采用200 V分离10 min，再调整电压至700 V分离40 min，凝胶在UV 365 nm下成像；TLC展开剂为正丁醇-异丙醇-水-醋酸（7：5：2：1），显色剂为苯胺-二苯胺。结果：不同产地铁皮石斛多糖重均相对分子质量、回旋半径和含量分别为0.533×10⁵~4.575×10⁵、19.8~71.7 nm和6.28%~28.72%；铁皮石斛功能性多糖的单糖组成主要为（乙酰）甘露糖和葡萄糖，摩尔比（3.86~6.99）：1.00，糖苷键类型主要为1，4-Manp和1，4-Glcp，摩尔比（4.51~7.89）：1.00；不同铁皮石斛多糖的部分酸水解和酶解产物糖谱特征相似。结论：不同产地铁皮石斛功能性多糖结构具有较高的一致性，功能性多糖是铁皮石斛较好的质量控制指标。

Objective:To characterize and compare specific polysaccharides in Dendrobium officinale from different habitats for its quality control.Methods:HPSEC-MALLS-RID was adopted for the analysis of molecular weights,radius of gyrations and contents of specific polysaccharide.Columns of TSK-GEL G5000 PW_XL and TSK Gel G3000PW_XL in series were employed with the temperature of 35.The mobile phase was 9 mg·mL^-1 NaCl aqueous solution at a flow rate of 0.5 mL·min^-1.GC-MS was performed on an Agilent HP-5MS capillary column (30 m×0.25 mm×0.25 μm) to analyze compositional monosaccharides and glycosidic linkages.Injection volume was 2 μL.For compositional monosaccharides analysis,the column temperature was set at 165℃ and held for 7 min then programmed at 5℃·min^-1 to 185℃ and held for 5 min,then at 4℃·min^-1 to 200,and finally,at 20·min^-1 to 280.For glycosidic linkages analysis,the column temperature was set at 120℃ and then programmed at 5℃·min^-1 to 200℃,then at 8℃·min^-1 to 250℃,and finally,at 20℃·min^-1 to 280℃.Saccharide mapping based on PACE and TLC was employed for fingerprinting of partial acid and enzymatic hydrolysates of specific polysaccharides.For PACE analysis,samples were electrophoresed first at 200 V for 10 min,then at 700 V for 40 min and gels were imaged under UV 365 nm.The TLC plate was developed with 1-butanol-isopropanol-acedicacid-water(7:5:2:1) and the colorized with aniline-diphenylamine.Results:The weight-average relative molecular mass,radius of gyration,and the content of specific polysaccharide in D. officinale from different locations were 0.533×10⁵-4.575×10⁵,19.8-71.7 nm,and 6.28%-28.72%,respectively.The main compositional monosaccharides were (acetylated) mannose and glucose (3.86-6.99):1.00 with glycosidic linkages of 1,4-Manp and 1,4-Glcp (4.51-7.89):1.00.Saccharide mappings of partial acid and enzymatic hydrolysates from specific polysaccharides of different samples were similar.Conclusion:Similarity of specific polysaccharides in D. officinale from different habitats was high,which could be a good marker for the quality control of D. officinale.