| Fungi have influenced human life for thousands of years, either as a direct food source or as a medicine. The nutritive and medicinal values of mushrooms have long been recognized all over the world. Many researches showed that fungi have several biological activities such as anticancer, boost in the immune system, hypoglycemic action, lipid-lowering, anti-hypertension and reduce in coronary heart disease.Tricholoma matsutake is a kind of rare, edible and medicinal fungus in the world. It can be found in many regions of Africa, America, Europe and Asia. It is a valuable species throughout the world, exhibiting a characteristic delicate flavour as well as several biological activities, such as lower cholesterol, anti-oxidant, immuno-modulating and antitumor effects in humans.In this paper, the chemical composition of T. matsutake was analyzed. Besides, the separation and purification, physicochemical properties, structure characterization and anti-tumor activity of the polysaccharides from T. matsutake were investigatedIn the first part, the chemical compositions of T. matsutake were determined by routine laboratory assays, including crude protein, crude fibre, crude fat, carbohydrate, ash and mineral elements. Moreover, the amino acid and fatty acid profiles of proteins and lipids were examined to establish a preliminary guide for assessing the relative nutritive qualities and provide a basis for taping the nutritive potential of the natural resource. The analysis results indicated that the wild edible mushroom contained rich sources of crude protein (20.3%), crude fibre (29.10%) and carbohydrate (36.67%) but little fat (5.04%) in which oleic and linoleic acids accounted for more than 75%. In general, the analysis revealed that T. matsutake had rich sources of protein and fibre and had low amounts of fat. The essential amino acid accounted for 34.65% of the total amino acid which was similar to the ideal protein requirements suggested by the FAO/WHO. The first limited amino acid was identified as methionine and the score of ratio coefficient of amino acid of its protein was 80.16. Additionally, the mushroom had been found to contain good amounts of minerals. The results showed that T. matsutake was a good nutritional plant for their unique contributions to health.The second part was the preparation technology of the polysaccharides. In this section, the contents of polysaccharides and protein were determined by the reaction with phenol in the presence of sulfuric acid and the coomasssie brilliant blue method, respectively. The polysaccharides were extracted with water extraction and alcohol precipitate (WEA). The effects of several parameters such as temperature, time, liquid-solid ratio and frequency on the extraction of the polysaccharides were observed by the yield determination of the polysaccharides with odd factor experiment and orthogonal experiment. The optimal technology for precipitation with ethanol was confirmed by the yield determination of the polysaccharides. The optimal technology for deproteinizing was obtained by comparing the effects of different methods on the loss of polysaccharides and protein. The parameter of decolorizing technology was optimized by the determination of the polysaccharides and the color comparison. Moreover, D101 macroporous resin column chromatography and sephadex G-100 gel filtration chromatography were employed to obtain the reasonable preparation technology of the polysaccharides. Finally three kinds of polysaccharides MTS-1, MTS-2 and MTS-3 were obtained from T. matsutake.In the third part, the physicochemical properties and structure characterization of polysaccharides were investigated. The glucuronic acid content and SO42" content were determined by means of m-hydroxydiphenyl colorimetry and turbidimetry method, respectively. The specific rotation and C, H compositions were determined by automatic polarimeter and elementary analyzer. The molecular weight and purity of polysaccharides were analyzed by high performance gel permeation chromatography (HPGPC). After completely acid-hydro lyzed and trimethylsilyl derivatized, component analysis of monosaccharides in polysaccharides were analyzed by gas chromatography (GC). Besides, the structural investigation of polysaccharides were synthesized and characterized by ultraviolet-visible spectrophotometry (UV-VIS), Fourier transform infrared spectrophotometry (FTIR), one and two-dimensional nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS).The results showed that the homogeneity of these three polysaccharides was confirmed, their molecular weights were 5939,180357,232372, respectively. Polysaccharide MTS-1 from T. matsutake contain glucose, xylose and galactose in a molar ratio:Glu:Xyl:Gal=12.89:1.20:1. Polysaccharide MTS-2 was confirmed to contain the same monosaccharides as MTS-1, but the molar ratio of monosaccharides was 16.77:1.63:1. The main backbone of the MTS-2 was found to be composed of a-D-glucose linked by 1→6 glucoside bond, along with small amounts of a-D-xylose and P-D-galactose could be in the side chain. MTS-3 was identified to be composed of glucose units. According to the comprehensive analysis, the backbone of MTS-3 was consisted of 36-37 units (3-D-glucoses combined by 1→6 glycosidic linkage and the structure of MTS-3 was as follows.In the forth part the anti-tumor activity was assayed by MTT method together with morphological observation under inversed light microscope. The cytotoxic activities of different concentrations and different time of MTS in vitro were investigate against human tongue carcinoma cells Tca8113, human liver cancer cells HepG-2, human pancreatic cancer cells HS766T, human cervical cancer cells Hela and human breast cancer cells MCF-7. The results showed that the low concentration of polysaccharides from T. matsutake weakly inhibited the growth of HepG-2 and HS766T but obviously affected the growth of Tca8113, Hela and MCF-7. It showed potential antiproliferative activity with dose-dependent and time-dependent relation in vitro. Maximum inhibition ratios of MTS under the 100μg·mL-1 and 72h to Tca8113, HepG-2, HS766T, Hela and MCF-7 were 67.71%,74.89%,83.83%,75.57%,76.15%, repectively. From the cell morphology observation, polysaccharides of T. matsutake can give rise to different degrees of apoptosis on the five tumor cells mentioned above, the mechanism needs further study.In summary, this study investigated not only the basic chemical composition of T. matsutake, but the extraction and purification, physicochemical properties, structural characterization and the anti-tumor activity of mushroom polysaccharides. These systematic studies of the rare and valuable of edible-medicinal fungi provide a theoretical basis for the further development of T. matsutake. |
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