Ultrasound Polysaccharides Structure, Behavior And Biological Activity Of The Solution Right

In this paper, polysaccharides were extracted from ophiopogon japonicus by hot water and ultrasound. The polysaccharides extracted by hot water were subjected to ultrasonic treatment. The effect on primary structure of the polysaccharides extracted by hot water and subjected to ultrasonic treatment was studied. Morphology, conformation and biological activity of the polysaccharides extracted by hot water and ultrasound were studied to reveal the effect of ultrasound on the structure and activity of ophiopogon japonicus polysaccharides. The main research contents and results are as follows:(1) The effect of ultrasonic power on the structure and activity of ophiopogon japonicus polysaccharides was studied. We selected five kinds of ultrasonic power to extract polysaccharides from ophiopogon japonicus root. The polysaccharides of API and AP3were obtained applying quaternary ammonium salt to isolate the five kinds of polysaccharides, respectively. Then molecular weight distribution and monosaccharide composition of API and AP3were detected by high performance liquid chromatography and gas chromatography, respectively. The results showed that larger molecular weight polysaccharides were obtained with the increasing of ultrasonic power, but degradation phenomenon that large molecular weight cut in half and degraded to small molecular weight emerged with the further increasing of ultrasonic power. Gas chromatography studies showed that, monosaccharide components, such as rhamnose, arabinose and xylose, which were steadily embedded in ophiopogon japonicus raw material and difficult to extract using small power obtained with the increase of the ultrasonic power. Study on antioxidant activity showed that antioxidant activity of the polysaccharides increased to the maximum and then decreased with the increasing ultrasonic power. The antioxidant activity was strongest when the ultrasonic power was400W. Therefore, the ultrasonic power of400W was selected in follow-up experiments.(2) Structure and biological activity of polysaccharides WPOJ extracted by hot water and UPOJ extracted by ultrasound were studied. The results showed that the extraction rate of UPOJ was significantly higher than that of WPOJ. The functional groups of WPOJ and UPOJ were detected with IR spectrum. The result showed (3-glucopyranose was detected in WPO but not in UPOJ, indicating that the structure of ophiopogon japonicus polysaccharides was affected with ultrasound. The result of bioactivity showed the antioxidant activity and antidiabetic activity in vitro of UPOJ were higher than that of WPOJ. But the antitumor activity on K562cells of UPOJ was lower than that of WPOJ.(3) The homogeneity of WPOJ-DS and UPOJ-DS were purified by a combination of DEAE-52anion-exchange column chromatography and repeated Sephacryl S-300HR gel chromatography from WPOJ and UPOJ. The primary structure of WPOJ-DS and UPOJ-DS were studied by chemical analysis (including periodate oxidation, Smith degradation, hydrolysis with acid and methylation analysis) and instrumental analysis (including GC, HPLC, UV, IR, MS and NMR). The results showed that the backbone of WPOJ-DS was different with UPOJ-DS. The backbone of WPOJ-DS was1,6-α-D-glucopyranose,1,6-β-D-glucopyranose,1,3,6-α-D-glucofuranose and1,3-α-D-arabinofuranose in the molar ratio of5:1:3:2, while the backbone of UPOJ-DS was1,6-α-D-glucopyranose,1,3,6-α-D-glucofuranose and1,3-a-D-arabinofuranose in the molar ratio of5:4:2, indicating1,6-β-D-glucopyranose in WPOJ-DS was instead of1,3,6-α-D-glucofuranose in UPOJ-DS. The branched chains and the ends of the backbone of WPOJ-DS were mainly composed of1,6-α-D-glucopyranose,1,2-α-D-fructofuranose and1-α-D-arabinofuranose, in the molar ratio of2:3:5, while the branched chains and the ends of the backbone were mainly composed of1,6-α-D-glucopyranose,1,2-α-D-fructofuranose and1-α-D-arabinofuranose, in the molar ratio of3:4:6, demonstrating the branched chains of UPOJ-DS were richer than that of WPOJ-DS. Therefore, the polysaccharides including richer branched chains, embedded in ophiopogon japonicus could be extracted by ultrasound but not by hot water.(4) WPOJ-DS was treated by ultrasound and named as WPOJ-DS-U. The primary structures of WPOJ-DS-U and WPOJ-DS were studied comparatively. Analysis showed that the molecular weight, monosaccharide composition, molar ratio, and chain connections of WPOJ-DS-U were not in the least different with WPOJ-DS. β-configuration in WPOJ-DS was affected with ultrasound. β-configuration in WPOJ-DS had changed to α-configuration in WPOJ-DS-U. The main effect was→6)-β-D-Glcp(1→in WPOJ-DS had changed to→6)-a-D-Glcp(1→in WPOJ-DS-U. Generally, β-D-Glc is more stable than α-D-Glc. Therefore, the tremendous energy of ultrasound causes the structure of ophiopogon japonicus polysaccharide in a metastable state and the increasing of the biological activity.(5) The solution behavior and chain conformation of WPOJ-DS and UPOJ-DS were studied comparatively. The analysis result of particle size showed the effective diameter of UPOJ-DS was smaller than that of WPOJ-DS. Both of them had smaller effective diameter under acidic conditions and aggregation under alkaline conditions. The result of Congo red experiment showed UPOJ-DS had single spiral structure, while the WPOJ-DS had not. The result of circular dichroism showed it had larger effects on the conformation of UPOJ-DS but smaller effects on the conformation of WPOJ-DS when Ca2+and Congo red were joined in. It had larger effects on the conformation and increasing the asymmetry of both WPOJ-DS and UPOJ-DS when DMSO was joined in. The conformation of WPOJ-DS and UPOJ-DS changeed in acid and alkaline solution and the change was more obvious in alkaline solution. The result of X ray diffraction showed WPOJ-DS and UPOJ-DS had crystal structure, and the crystal lattice constant of UPOJ-DS was slightly larger than that of WPOJ-DS. TG-DTG and DSC curve and the thermal stability of the two kinds of polysaccharides were determined. Results indicated that thermal properties of the two kinds of polysaccharides were basically similar, but the UPOJ-DS relative to WPOJ-DS had a good thermal stability.The results of atomic force microscopy and scanning electron microscopy showed the molecules aggregation of UPOJ-DS significantly reduced relative to WPOJ-DS. The spiral structure was observed in UPOJ-DS by atomic force microscopy, which was consistent with the result of Congo red experiment. The results of SEM (includes mica samples and powder sample s) showed molecular aggregation behavior of UPOJ-DS significantly weakened relative to WPOJ-DS, indicating the tremendous energy of ultrasound had changed the structure of ophiopogon japonicus polysaccharide. The intermolecular force and molecular cross-linking degree in UPOJ-DS reduced, causing the change on the aggregates of the polysaccharide.(6) Antioxidant activity results of WPOJ-DS and UPOJ-DS showed that both of them had scavenging effects on·OH, O2-· and DPPH·. The scavenging effect of UPOJ-DS was better than that of UPOJ-DS. Combine the results of primary structure, morphology and solution behavior of the two kinds of polysaccharides that the branched chains of UPOJ-DS were richer than that of WPOJ-DS, UPOJ-DS had the spiral structure observed by atomic force microscopy, and the molecular distribution was more dispersed. Therefore, it was speculated that the higher antioxidant activity is closely related to the structure, morphology and solution behavior.