Carbohydrate analysis of functional ingredients of plant materials from roots, leaves, stem bark, and seed

다국어 초록 (Multilingual Abstract)

The carbohydrates in natural plant materials obtained from roots, leaves, stem barks, and seed were investigated. All of the plant materials were dried, and their constituents were isolated by room temperature-water or hot-water extraction. Ethanol precipitation was also used to produce solutions containing the constituents for analysis. Mono- and disaccharides were separated using a high-pH anion-exchange chromatography system equipped with a CarboPac PA10 column and an AminoTrap column working at an isocratic NaOH concentration of 20 mM and at a flow rate of 0.5 ml/min at room temperature for 40 min. Carbohydrates were detected by a pulsed amperometric detector. The concentration of each carbohydrate was quantified by calculation from a calibration curve constructed using a standard mixture containing twofold dilutions from 1,600 pmol of each carbohydrate. Enzymatic deglycosylation of the plant extracts was also performed. Namely, each sample was incubated in the presence of 40 μM trypsin and 40 μM chymotrypsin in 10 mM Tris-HCl buffer (pH 8.0) at 37°C for 18 h. N-glycans were then released from glycopeptides after reaction with 0.1 mU of glycoamidaseA in citrate-phosphate buffer (pH 5.0) at 37°C for 18h. Peptides and salts were removed using graphitized carbon cartridges. The released glycans were lyophilized and stored at –20°C. The N-glycan structures were analyzed by normal-phase high-performance liquid chromatography of 2-aminobenzamide-labeled glycans in combination with exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The functions of the identified and isolated chemical constituents were also investigated.