Study On The Purification, Physicochemical Properties And Monosaccharide Composition Of The Polysaccharide From Momordica Charantia L. Vines

In our country, Momordica charantia L.vines is rich in source and have high medicinal value. Momordica charantia L.vines polysaccharide (MVP) is one of the main ingredient in Momordica charantia L.vines, the studies on the Purification, physicochemical properties and monosaccharide Composition of the Polysaccharide from Momordica charantia L.vines is of great significance to exploit and utilize Momordica charantia L.vines.In this paper, the extraction and purification technics, physicochemical properties and monosaccharide Composition of MVP were studied systematically. The main process parameters and conclusions were as follows:After the dry grinding, Momordica charantia L.vines was extracted by ultrasonic extraction process or ordinary hot water extraction process, phenol-sulfuric acid method was used to determine the crude polysaccharide content, and further the coarse polysaccharide extraction yield was calculated. Used single factor and orthogonal experiment to optimize the two parameters of extraction process, then compared the polysaccharide yield of the two optimum extraction processes. The results showed that, in the ultrasonic extraction process, the significance of factors affecting the yield of polysaccharide followed the sequence:temperature> ultrasonic time>pH>solid-liquid ratio. The optimal MVP extraction conditions optimized by L9(34) orthogonal experiment were:extraction temperature65±2℃, solid-liquid ratio1:25(g/mL), ultrasound time35min, and pH7, with polysaccharides yield of3.08%after twice extractions. In the ordinary hot water extraction process, the significance of factors affecting the yield of polysaccharide followed the sequence:solid-liquid ratio>temperature>ultrasonic time. The optimal MVP extraction conditions optimized by L9(33) orthogonal experiment were:extraction temperature75±2℃, solid-liquid ratio1:55(g/mL), ultrasound time90min, twice extractions. Compared to hot water extraction method, ultrasonic extraction had about2.5times higher efficiency.In the study of deproteinization of MVP, Sevag method and LSA-10method were used to remove the protein. The Phenol-sulfuric acid method was used to determined the damage rate of the polysaccharide, and the coomassie brilliant blue method was used to determined the damage rate of the protein. Finally, the two methods to remove protein were compared through UV scanning after deproteinization process. It was found that the reaction was modest by using Sevag method to remove protein, the protein was not removed entirely. After five times of deproteinization, the total removal rate of protein was55.99%, while the MVP loss reached48.81%. LSA-10resin showed a good effect on removing protein with the deproteinization rate83%, and the MVP loss rate was3.02%. Comparing with Sevag method, it had a higher deproteinization rate and lower polysaccharide los(?)ate. Since LSA-10macroporous resin had a good selection on the adsorption of free protein and glycoprotein conjugates, it was suitable for the deproteinization of MVP.In the research of decolorization, the static adsorption ability to pigment and polysaccharides of five resins were studied, including DA-201, LSA-10, D-101, AB-8and XDA-6. The adsorption rate of pigment and polysaccharides were determined by Ultraviolet spectrophotometry method and phenol-sulfuric acid method. And used the comprehensive evaluation mechanism to find the optimal resin depigmentation, which was applicated to remove polysaccharides liquid pigment. The results showed that, LSA-10resin was chosen as the optimal resin of decolorization. Through dynamic decolorization study, it was found that the decolorization ability of LSA-10resin was77.85%.In the separation and purification of MVP, DEAE-52Cellulose and Sephadex G-200column chromatography were used to separate and purify MVP. The elution was conducted in turn with distilled water,0.15mol/L and0.3mol/L sodium chloride solution of isocratic elution, in which MVP was separated, and tracked by phenol-sulfuric acid method. Then, Sephadex G-200column chromatography purification was used to separate the components to ensure their refinement. The results showed that MVP was eluted completely from column in0.15mol/L sodium chloride solution where two main components, neutral polysaccharide MVP-1and acidic polysaccharide MVP-2, were obtained. Sephadex G-200column chromatography was used in the further purification of the two polysaccharides, and it was considered preliminarily that MVP-1and MVP-2both were single polysaccharides, which confirmed that DEAE-52cellulose had a good separation and purification effect.In the analysis of physical and chemical properties of MVP, anthrone-sulfuric acid method, sulfuric acid-carbazole, coomassie brilliant blue method, and iodine-potassium iodide were used, then the monosaccharide composition, molecular weight and structure were identified by GC, IR, BI-MwA/BI-DNDC absolute molecular weight characterization system. Color reactions showed that MVP-1and MVP-2were non-starch polysaccharides without binding protein. The monosaccharide composition of polysaccharides MVP-1and MVP-2were analyzed by. GC, they were, mainly composed of Rha, Ara, Man, Glu and Gal. The result showed that MVP-1was composed of Gal, Man, Glu, Rha and Ara in a molar ratio of1,0.92,0.54,0.43,0.21; MVP-2was composed of Gal, Man, Glu, Ara and Rha in a molar ratio of1,0.71,0.63,0.32,0.27. Mw of MVP-1was42230daltons(Da), and the Mw of MVP-2was24460daltons(Da). Infrared spectrum of polysaccharides MVP-1and MVP-2indicates that they had typical characteristic absorption peaks.