Gynostemma Polysaccharide Purification, Structural Analysis, And Its Antioxidant Activity

Gynostemma Gynostemma pentaphyllum (Thunb.) Makino, one of most critical wild Chinese medicinal materials is one of kind of a perennial herbaceous vine which belongs to Cucurbitaceae: As the one of higher active components, Gynostemma pentaphyllum polysaccharides is attracting of many scholars at home and abroad. Studies have shown that Gynostemma pentaphyllum polysaccharides have significant anti-tumor, enhancement immunity, lower blood pressure, lower blood lipids, antioxidation and so on.This experiment mainly researches extraction, purification, detection and antioxidant activity and other aspects of wild Gynostemma pentaphyllum. Two polysaccharides was refined by water extraction and alcohol precipitation to optimize extraction process and through a series of separation and purification methods.They were also analysed preliminary structure and activity. The main results are as followed:1. Polysaccharides from Gynostemma pentaphyllum were obtained through hot water extraction, microwave and ultrasonic extraction and optimized parameters of solid-liquid ratio, extraction temperature (microwave power, ultrasonic power), extraction time and number by a single factor and orthogonal test method to determine the effect of the extraction rate of rude polysaccharides. The results showed that the optimal conditions of traditional hot water extraction was the temperature of 100℃, extraction 2 times, solid-liquid ratio of 1:20 and extraction time of 2h, the optimal conditions of microwave extraction process was extraction power of 800W, extraction 2 times, solid-liquid ratio of 1:35 and extraction time of 15min, the optimal conditions of ultrasonic extraction process was extraction power of 900W, extraction 2 times, solid-liquid ratio of 1:25 and extraction time of 40min. Three different methods can effectively extract the polysaccharide and the most efficient method was microwave extraction.The optimum extraction rate reached 8.61% and taked the shortest time of 15 min to get themost obvious effect. The least efficient method was traditional hot water extraction and extraction rate was only 6.35% under optimum extraction condition spending 2h.2. The technology for removing protein was researched by using Sevage method,TCA-Sevag method and papain-Sevag method from polysaccharides of Gynostemma pentaphyllum. The technology for decolorization was researched using activated carbon adsorption, H2O2 bleaching method, DA201-C resin decolorization from polysaccharides of Gynostemma pentaphyllum. Polysaccharide from removing proteins and pigments through DEAE-52 cellulose column chromatography obtained further purification to get four elution peaks. The more elution components of 0.05M and 0.3M NaCl were pured by G-200 dextran gel column chromatography and washed for homogeneous elution peak. The results showed that the best removing protein was protein-Sevag method, the removal rate of 63.08% and polysaccharide retention rate of 77.34% by total 2 times. DA201-C resin decolorization was best to remove pigment at the maximum extent and minimize the loss of polysaccharide. Amount of resin was 1:20, temperature of 50℃, the adsorption time of 4h, decolorization rate of 71.13% and the loss rate of polysaccharide of 19.90%. Column chromatography got two homogeneous elution peaks, dialysing by freeze-dried.There were two refined polysaccharides, named GPM-1, and GPM-2.3. Physical and chemical properties and relative molecular weight of physical properties of the two refined polysaccharide was determined respectively. Sugar key configuration of two polysaccharides was scaned by infrared spectra. Monosaccharide composition and molar ratio were determined and calculated using gas chromatography. Both sugar surface and morphology were observed by environmental scanning electron microscope. The results showed that the two refined polysaccharides didn't contain starch, uronic acid, protein, amino acids, peptides and nucleic acids and other impurities. The relative molecular weight of GPM-1 and GPM-2 was 200,576 and 166,861, respectively, using High performance liquid gel chromatography. Two polysaccharides had the characteristic absorption peak of polysaccharides material by infrared spectroscopy. Monosaccharide composition of GPM-1 with Gas chromatography analysis of the the molar ratio of rhamnose was rhamnose:arabinose:xylose:mannose:glucose:galactose=1.78:1.99:1.00:1.11:6.00:6.89, which the highest concentrations was galactose. The molar ratio of monosaccharide composition of GPM-2 was rhamnose:arabinose:xylose:mannose:glucose:galactose=3.23:7.70:1.00:2.29:2.88:14.82, which galactose content was significantly higher than other monosaccharide.Two kinds of polysaccharide distributed debris shape and lamellar through environmental scanning electron microscopy.4. Free radical-scavenging activities of Gynostemma pentaphyllum polysaccharide (GPM) were separately analyzed for DPPH radical, superoxide anion radical and hydroxyl radical. The results showed that the GPM-1 and GPM-2 could scavenge free radical and the scavenging capacities increased with the increasing of its concentrations, but the capacities are less than the capacities of Vc. Free radical-scavenging effects was very obvious for·OH radical. When the concentration was 3.0mg/mL, scavenging rate of GPM-1 and GPM-2 for·OH radical reached 72.9% and 77.3%, respectively. Experiments also showed that the polysaccharides of Gynostemma pentaphyllum had good potential in the removal of·OH radicals and illustrated the antioxidant effect of Gynostemma pentaphyllum polysaccharides.