Extraction, Separation And Screening Of Antioxidant Activity Of Polysaccharide TLH-3from Tricholoma Lobayense

Tricholoma Lobayensc Heim, is a layer of bacteria basidiomycotina genus Agaricus Mushroom Gang Division (white mushroom Branch). The nutritional and medicinal value of Tricholoma Lobayensc Heim originally revealed in a variety of compounds with pharmacological effects especially in polysaccharides is now being investigated on cell-wide level using recent technological advances, resulting in the discovery of a multiplicity of biochemical activities, including immune regulation, anti-oxidation, anti-tumor and so on. With the inexorable progress toward the pursuit of perfect health, Tricholoma Lobayensc Heim polysaccharides have become one of the most concerns because of its pharmacological effects. Tricholoma Lobayensc Heim contains protein27.56%,38.44%total sugar, fat9.58%, crude fiber8.20%according to the composition analysis per kg of dry product. Understanding how these compounds influence the spectrum of cell activities may lead to new dietary concept and insights into the pharmaceutical basis of food science and other areas. Hitherto, the present study about Tricholoma Lobayensc Heim has only focused on optimizing culture conditions and natural nutrition composition simply. Moreover, it is worth noting the research about Tricholoma Lobayensc Heim polysaccharides has not being carried out systematically. Although there is accumulating and compelling evidence that fungus polysaccharides have a great deal of biochemical activities, the relashionship between the spatial configuration and structure-activity of polysaccharides and biochemistry mechanism remain unclear. In this regard, we attempt to find potential biochemical pathways or processes of cell models in specific and relevant psychological tests that are coordinated with human dietary studies.In this regard, we attempt to find potential biochemical pathways or processes of cell models in specific and relevant psychological tests that are coordinated with human dietary studies. It will be essential to ask if specific components within the fungus polysaccharides have the above biochemical activities. Hence, three main parts of polysaccharides are got through sequential extraction, separation of polysaccharides and then select the most optimal composition, the polysaccharide TLH-3, based on the oxidation resistance. Next, the relashionship between the spatial configuration and structure-activity of TLH-3and biochemistry activies is the main aspect of our study, as follows.First, the traditional approach has relied on the solubility in different phase solvents that allow the isolation of Tricholoma Lobayensc Heim polysaccharide complex, including water extraction, alcohol precipitation and deproteinization by Sevag method sequentially. We first sought to get TLH-1、 TLH-2and TLH-3, three different parts of polysaccharide complex followed by the combination of gel-filtration chromatography and anion-exchange chromatography. We examined purification rate of the three based on determination of total polysaccharide and protein content by the ultraviolet absorption analysis and infrared spectrum analysis. The extraction yield of TLH-1, TLH-2and TLH-3is0.72±0.04%、0.41±0.10%and0.08±0.01%respectively. Based on the above results, we can see that the extraction and isolation of TLH-1with highest yield and that of TLH-3with lowest yield. Additionally, the protein content of TLH-1has the maximum value,1.31±0.05%and that of TLH-3is0.68±0.01%, that is to say, the three parts of polysaccharides have very low protein content. At last, sugar content of the three is greater than90%. In a word, the extraction method mentioned above is feasible and provides guarantee for the follow-up performance.Second, we assessed the free radical scavenging effects of TLH-1, TLH-2and TLH-3on DPPH and O2and anti-oxidant effects in human embryonic lung fibroblasts. Of these, TLH-1, TLH-2and TLH-3have the free radical scavenging capacity albeit with differing efficiencies. Notably, TLH-3has the best scavenging capacity especially on DPPH with roughly the same capacity as positive control of Vc, and that of TLH-2and TLH-3are very low. To further assesse the anti-oxidant effects in cell level, we surveyed TLH-1, TLH-2and TLH-3in human embryonic lung fibroblasts and found they have also the anti-oxidant capacity. Consistent with above results, TLH-3has the best anti-oxidant capacity and that of TLH-2and TLH-3are also very low. In addition, we tested polysaccharide administration in cell level, and found that TLH-1administration has hardly any ability of TLH-1to suppress Hela and HepG2growth, whereas the administration of TLH-2and TLH-3impaire cancer cell proliferation time-dependently and dose-dependently, especially TLH-3But they had no obvious effect on growth in the normal cells.In a word, TLH-2and TLH-3have the potential pharmacological action in the adjuvant treatment of cancer.Third, TLH-3shows a remarkable degree of anti-oxidant in vivo system as above mentioned. To validate the role of TLH-3in anti-oxidant, we confirm the spatial configuration and structure-activity of TLH-3after degradation and determination of molecular weight of TLH-3. We first confirm the molecular weight of TLH-3is5.134×103Da and consists of TLH-3A and TLH-3B by molecular sieve chromatography. The molecular weight of TLH-3A is3.193×103Da and that of TLH-3B is1.541×103Da, and the content of TLH-3A is also higer than TLH-3B. We further determine if TLH-3A is also functional in the free radical scavenging effects on DPPH and O2, we found that the free radical scavenging effects of TLH-3A is higer than that of TLH-3B, but lower than that of Vc. Above all, these results suggest active TLH-3structures are localized within TLH-3A.