| Poria cocos wolf, one of the traditional Chinese medicinal materials in china, is a kind of edible fungus attaching to the pine tree root which belongs to the polyporaceae plant. Polysaccharide from the poria cocos wolf is one of the most important biological active substance in the poria cocos wolf which has the activity of inducing diuresis and excreting dampness, strengthening spleen and being easy in mind, restraining and sterilizing bacteria, hypoglycemic effect, antivirus, modulateing immune function, inducing interferon and leukoregulin (LR) and so on. It is of high healthy function and medicinal value on human beings. At present, there have been papers about the effect of ultrasonication on extraction rate of polysaccharide from the poria cocos wolf, but there have been few papers about the systematic research of effect of ultrasonic enhanced extraction on extraction rate and structure of water-soluble polysaccharide and alkali-soluble polysaccharide from the sclerotium of poria cocos. Direct observation of microstructure of polysaccharides from the sclerotium of poria cocos has not been found by atomic force microscopy. Accordingly, water-soluble polysaccharide and alkali-soluble polysaccharide were extracted from the sclerotium of poria cocos by ultrasonic wave respectively in this study. Meanwhile, the extraction effect of ultrasonic wave was compare with that of traditional extraction in order to reveal the effect laws of ultrasonic extraction on extraction rate, monosaccharide composition and proportion among different components, structure of polysaccharide (water-soluble polysaccharide and alkali-soluble polysaccharide) from the sclerotium of poria cocos. Internal mechanism of extracting polysaccharide from the sclerotium of poria cocos by ultrasonic wave was preliminarily studied, which provided necessary basic data and experimental basis for the study of influence of ultrasonic extraction on structure and biological activity of polysaccharide from Chinese herbal medicine. The main research contents and corresponding results reported in the papers were summarized as follow: (1)The optimal extraction technology of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined. Taking polysaccharide extraction rate as the index, the optimal extraction conditions of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined with the orthogonal design on the basis of single factor experiment, namely ultrasonic function time: twenty five minutes, extraction temperature: 80℃, the ratio of liquid to solid: 60:1, which were compared with those of traditional hot water extracting method. At the same time, structural changes of poria cocos wolf cells were investigated before and after ultrasonic extraction by environmental scanning electron microscope. The results showed that poria cocos wolf cells without ultrasonic processing were intact and there were few cracks on their surfaces. Different cells were connected together by middle lamella. After twenty five minutes ultrasonic processing, there were craws in varying degrees on the surfaces of most of poria cocos wolf cells, some of which had been completely collapsed. Most of middle lamellas had also been destroyed.(2)Two kinds of water-soluble polysaccharides, which were named as PPTH and PPUH, were extracted from the sclerotium of poria cocos according to optimal extraction conditions by traditional hot water extracting method and ultrasound-assisted hot water extraction respectively. The polysaccharides functional groups were investigated by fourier transform infrared spectroscopy(FTIR). The monosaccharide compositions of polysaccharides were determined by gas chromatography. The structure of polysaccharides was analyzed by atomic force microscopy. Determination results of PPTH and PPUH were compared. The experimental results indicated that the components of PPUH were identified by GC as: ribose, xylose, mannose, fructose, galactose, glucose, which were the same as those of PPTH. Meanwhile, the infrared absorption spectras of PPUH and PPTH were basically the same. We found that PPTH on mica formed network-like structure by atomic force microscopy while PPUH existed in the form of different rod-like structure. It is presumed that ultrasonic radiation destructed network structure of water-soluble polysaccharide from the sclerotium of poria cocos, and made its macrochains degraded to small molecular fragments. Because of the aggregation of different small molecular fragments, they finally formed large cluster rod-like structure.(3)Two kinds of alkali-soluble polysaccharides, which were named as PPTA and PPUA, were extracted from the residue of sclerotium of poria cocos after ultrasound-assisted hot water extraction by traditional alkaline extracting method and ultrasound-assisted alkaline extraction respectively. The yields of PPTA and PPUA were 76.47% and 85.4%. Compared with using extraction methods Pan Qi, Huang Caihuan reported, using ultrasonic enhanced extraction shortened the extraction time and improved extraction efficiency. Meanwhile, it was found in the process of extraction that the extraction effects of extracting alkali-soluble polysaccharide by ultrasonic wave embodied in the first extraction, when most of polysaccharide transferred to alkaline solution. Functional groups and molecular morphologies of PPTA and PPUA were investigated by fourier transform infrared spectrometer and atomic force microscopy respectively. It was found by comparing with both of determination results that the infrared absorption spectras of PPTA and PPUA were basically the same, which basic structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos was not changed. The observation by atomic force microscopy showed that PPTA on mica formed network-like structure while PPUA existed in the form of different rod-like structure whose average height of chain was low. It was indicated that not only was network-like structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos destroyed, but also different intermolecular interactions of polysaccharide were influenced by ultrasonic enhanced extraction.(4)The structure of PPUA was modified by caboxymethyl group. The obtained polysaccharide, namely carboxymethylpachyman, was deproteinized by using Sevage method (repeated times 8). When there were no characteristic absorption peaks of pigments, protein and nucleic acid in its UV-Vis Absorption spectra, the modified PPUA was lyophilized and was reserved. The molecular weight of carboxymethylpachyman (CMP) was determined by high performance liquid chromatography. The structure of Carboxymethylpachymaran was characterized using fourier transform infrared spectroscopy. Morpological changes of carboxymethylpachymaran (CMP) were observed under different solution conditions by atomic force microscopy (AFM). After the carboxymethylation, the solubility of alkali-soluble polysaccharide from the sclerotium of poria cocos in the water was significantly increased, itsβ-D- glucan characteristic absorption peak at 890cm-1 became weak obviously, and its methylene vibration absorption peak and C=O antisymmetrical stretch vibration absorption peak appeared at 1333cm-1 and 1606cm-1 respectively, which indicated the carboxymethylation succeeded. The molecular weight of carboxymethylpachyman was 1.45×105 by high performance liquid chromatography. The analysis of AFM results showed that CMP molecules existed in different morphology under different solution conditions, and that the concentration, ionic strength and solvent physical chemistry characterisitics of polysaccharide solution had effects on the CMP chains conformation and the action mode between different molecular chains. The phenomena were considered to be related to hydrogen bond association and intramolecular and intermolecular electrostatic interactions of CMP. Meanwhile, the affinity and electrostatic interaction between CMP molecules and the mica substrate also had an influence on the CMP chains conformation and the image quality. |
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