| There are many ways to exact polysaccharide, and each has its own advantages and disadvantages. In addition to the polysaccharide extraction rate and the polysaccharide content of such indicators, such as monosaccharide composition of polysaccharides obtained by different extraction methods, whether there are significant differences in structure and biological activity and how about structure-activity relationship all determine the conditions of the extraction method and basis. Therefore, research the different extraction methods on the structure and activity of the polysaccharide to provide a theoretical basis for the choice of the polysaccharide extraction methods.In this paper, polysaccharides were extracted from Agrocybe aegerita by hot water and alkali. Using gas chromatography (GC), high performance liquid chromatography (HPLC), fourier transform Infrared spectra (FT-IR), nuclear magnetic resonance (NMR) determined the primary structure and advanced structure, and particle size analyzer, thermal analyzer, environmental scanning electron microscope (ESEM) and atomic force microscopy (AFM) were used to study the solution conformation and the sugar chain structure. What's more, the differences between4kinds of polysaccharides biological activity, and alkali extraction of polysaccharides were analyzed. The results showed that:1. Polysaccharides were extracted from Agrocybe aegerita by hot water and alkali, the obtained polysaccharides were designated as S-ACP and J-ACP, respectively. The content of the protein and the total sugars were determined by Coomassic brilliant blue method and phenol-sulfuric acid method; The monosaccharide compositions of polysaccharides were analyzed by gas chromatography (GC); The structure of polysaccharides were investigated by atomic force microscopy (AFM); Meanwhile,the antioxidant activity in vitro of polysaccharides also were tested. The crude polysaccharides were purified by CTAB and Sephadex G-150in sequence, and the obtained ones were designated as S-ACP2-1, S-ACP2-2and J-ACP2-1, J-ACP2-2, respectively. Comparative analysis:structure, solution conformation and biological activity of these four components.2. Comparison of water extraction and alkaline extraction on crude polysaccharides of the Agrocybe aegerita:J-ACP extraction rate of4.86%is far greater than S-ACP extraction rate of1.83%. Compared the monosaccharide composition and molar ratio shows that:rhamnose can be extracted by the lye water, which can't be extracted by water extraction, and increase the amount of glucose. Therefore, the extraction rate of the alkaline extraction is greatly increased. The antioxidant activity results showed that S-ACP activity is higher than J-ACP, and the results may be related to the content of the crude polysaccharide containing protein.3. Influences of Comparison of water extraction and alkaline extraction on the purified Agrocybe aegerita polysaccharide in structure:The4kinds of polysaccharides were all composed of xylose, mannose, glucose and galactose. Among them, S-ACP2-1, J-ACP2-1, molecular weight larger, which are8.72×105Dal and6.56×105Dal; S-ACP2-2and J-ACP2-2molecular weight smaller, which are2.09x104Dal, and2.05×104Dal.4kinds of sugar residue are mainly β-pyran configuration. The polysaccharide didn't have many changes in structure of monosaccharide composition, the sugar chain and so on, which were extracted by alkali extraction and water extraction. It showed that the purified polysaccharide, which was purified by water extraction and alkaline extraction, had similarity in the structure.4. Comparison of water extraction and alkaline extraction on the purified Agrocybe aegerita polysaccharide in solution behavior and chain structure:the granularity determination of results shows that H+and OH" have different influences on solution conformation of the4kinds of polysaccharides. The polysaccharide granularity extracted by water mention in acidic condition was minimum, and the polysaccharide granularity extracted by alkaline extraction in the neutral environment was the least prone to aggregation. S-ACP2-2and J-ACP2-2was mainly disordered structure in water solution, and a small amount of S-ACP2-2was ordered conformation. S-ACP2-2conformation changed less in the acidic environment, but will be a amount in alkaline environment; J-ACP2-2changed a lot both in acidic and alkaline environment, especially in alkaline environment. Congo red test results showed that S-ACP2-2and J-ACP2-2in aqueous solution were free to curl conformation exists.Thermal analysis showed similar thermal properties of4kinds of polysaccharides. J-ACP2-1and J-ACP2-2's thermal properties, which was extracted by alkaline, was slightly higher than S-ACP2-1and S-ACP2-2's, which was extracted by water. Alkali extracted polysaccharide's enthalpy change values in the process of thermal degradation is more than2times than water extracted polysaccharide.The polysaccharide appearance morphology of the ESEM observations showed that alkali extraction had certain damaging effects, making larger molecules to produce more small fragments and debris. Compared with the AFM observations, alkali extracted polysaccharide molecular chain connection was shorter, the distribution of dispersed in a neutral environment, but water extracted polysaccharide was woven into a flexible long-chain by the intermolecular interaction.So alkali extraction had certain impact on solution properties and chain structure of the polysaccharide.5. Comparison of water extraction and alkaline extraction on the purified Agrocybe aegerita polysaccharide in activity:The test of antioxidant activity in vitro show that, all of S-ACP2-1, S-ACP2-2, J-ACP2-1and J-ACP2-2exhibited obvious scavenging effects against DPPH·, and their antioxidant activity increased gradually with increasing concentrations. In addition, compared with S-ACPP2-2, the clearance rates of J-ACP2-2reached5%significant level when the polysaccharide concentration exceeded0.4mg mL"1, and at the concentration of0.8,1.0mg mL-1, they reached1%significant level. The test of anti-tumor activity in vitro show that,4kinds of polysaccharides had potent inhibitory action on K562cell, the inhibition rate of J-ACP2-2was significantly higher than S-ACP2-2, in addition, at the concentration of100,200,400ug-mL-1, the inhibition rate reached1%significant level. Results of these studies demonstrated that J-ACP2-2which was been treated by alkaline had stronger activity than S-ACP2-2. Compared to the structure and solution properties of the4kinds of polysaccharides, shows that the alkali-extracted polysaccharides had basically similar primary structure with water extraction, but their solution properties are quite different, thus it created the significant difference of biological activity of polysaccharide. 6. Non-isothermal kinetics of thermal decomposition of S-ACP2-2was studie d by the multiple heating rate method:the activation energy Eα and frequency factor lnA of the second thermal degradation of S-ACP2-2was respectively115.947kJ·moL-1and27.461s-1, the most likely kinetic equation coul-d be expressed as:... |
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