| In recent years,plant polysaccharides have gradually attracted more and more attention.Pueraria is the dried root of the legume Pueraria Lobate（Willd.）Ohwi.It is rich in isoflavones,polysaccharides,amino acids and trace elements,and has outstanding medicinal value.Pueraria polysaccharide has been proven to have biological activities such as bacteriostasis,scavenging free radicals,anti-oxidation,anti-tumor,and improving immunity.The different structure,composition and content of monosaccharides in polysaccharides may lead to different spatial structures of polysaccharides and different biological activities.Therefore,extraction and purification of Pueraria polysaccharides and determination of the monosaccharide composition and structure of Pueraria polysaccharides are of great significance for its activity analysis and quality control.Saccharides are not directly detectable because they do not have UV absorption and have a strong polarity.At present,the commonly used methods are mainly derivatized liquid chromatography-ultraviolet detector method,gas chromatography-ultraviolet detector method,or by mass spectrometry.ELSD is a"universal"and"mass"detector.The detection method does not rely on the optical characteristics of the substance itself and can be used to detect most non-volatile or semi-volatile compounds.Pressurized capillary electrochromatography is a high-efficiency micro-separation technology that combines the advantages of capillary electrophoresis and capillary liquid chromatography to achieve rapid and efficient separation of substances.This study will establish new methods for the analysis of saccharides and identify and analyze actual Pueraria polysaccharide samples:The first chapter mainly introduces the current research progress of plant polysaccharides,including the extraction,purification,structure identification and biological activity of plant polysaccharides at home and abroad.At the same time,pressurized capillary electrochromatography and evaporative light scattering detector are introduced.In the second chapter,a new Halo-Glycan column was used to establish a HPLC-ELSD method.The separation of monosaccharides was quickly explored without derivation.Finally,the separation conditions are determined:mobile phase:acetonitrile-water 86:14,flow:1.2 m L/min,drift tube temperature:90°C,carrier gas flow rate 1.8 L/min,under these conditions,6 kinds of monosaccharides containing two sets of isomers can be separated in 8 minutes.Chapter 3 establishes a new method of PMP derivatization combined with pressurized capillary electrochromatography-ultraviolet detection.The buffer concentration,buffer p H,applied voltage,column,and flow rate were optimized to achieve fast and efficient separation of 8 neutral monosaccharides in 24 minutes,which was more than half the time compared with traditional LC methods.The pre-column-derived p CEC-UV method was used to identify the monosaccharide composition of the two Pueraria polysaccharides and provide a new method and method for the separation of monosaccharides.The fourth chapter uses single-factor experiments combined with response surface analysis to explore the ultrasonic-assisted extraction process of two kinds of Pueraria polysaccharides,and analyzes the effect of ultrasonic time,temperature,liquid-material ratio and power to the extraction rate of polysaccharide,finally,predict the optimal process conditions.Chapter 5 is concerned with purification of crude polysaccharides of Pueraria.Four kinds of pure polysaccharides were obtained by anion exchange chromatography and high performance gel permeation chromatography.The molecular weights of the four kinds of polysaccharides were determined and analyzed.Ultraviolet full-wavelength scanning characterization showed that the four purified Pueraria polysaccharides did not contain protein and nucleic acid components,and the purification effect was good;the infrared spectral characterization showed that they all had characteristic peaks of polysaccharides;and the antioxidant tests of two pure polysaccharides were carried out.The test results showed that the Pueraria polysaccharide has the antioxidant effect of scavenging free radicals and certain antioxidant properties,the crude polysaccharide has the strongest antioxidant effect,and the PS-2 effect is stronger than PS-1.In Chapter 6,the structure of the main active ingredient PS-2 was identified.The pre-column derivatized GC-MS method was used to determine the monosaccharide composition of the hydrolyzed purified polysaccharide,and the results showed that the PS-2 monosaccharide composition was only glucose.It contains mainly glucose and a small amount of rhamnose.PS-2 was subjected to nuclear magnetic resonance（NMR）.The structure of the purified polysaccharide was identified by one-dimensional 1H-NMR and 13C-NMR,and it was judged to be an α-configuration pyranose-based hybrid,and made a preliminary judgment on the connection of glycosidic bonds. |
