Separation And Purification Of The Antioxidant Compounds From Mushrooms By Molecularly Imprinting Technique And The Protective Mechanism Of The Antioxidant Compounds To Oxidative Stress

Natural products with antioxidant activity have been paid much attention by many researchers because of its efficiency and low toxicity. Mushroom, as an important natural resource, is one of the main sources of antioxidant components.In this study, a method to separate and enrich the active compounds from mushrooms using molecularly imprinted technology (MIT) is reported. The molecularly imprinted polymers (MIPs) were prepared by bulk polymerization and the antioxidants were the template. The effects of porogenic solvents, functional monomers and cross linkers on the MIPs were investigated. MIPs coupled with solid phase extraction (SPE) provided an approach for separation and enrichment active compounds from mushrooms. The synthesis and application of the natural active compound-MIPs consist of three segments:(1) The MIPs were synthesized using caffeic acid phenethyl ester (CAPE) as the template,4-vinyl pyridine (4-vp) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross linker and a mixture of tetrahydrofuran (THF) and isooctane as porogenic solvent. The selectivty, the sorption kinetic and the adsorption isotherm were investigated. MIP as the adsorbent phase for SPE (MISPE) was used to separate and enrich CAPE and its analog caffeic acid (CA) from25species of mushrooms. CA was detected in7out of the25mushroom species and the concentration was range from0.004to0.340μg g-1(dw). This is the first report describing the isolation of CA from Schizophyllum commune, Agaricus blazei, Pleurotus eryngii, P. adiposa, and P. cystidiosus. In comparison with C18-SPE, MISPE displayed high selectivity and good affinity for the target compounds.(2) The MIPs was synthesized by gallic acid (GA) as the template,4-vp as functional monomer, EDMA as cross-linker and a mixture of THF and isooctane as porogenic solvent. The selectivity of the MIPs was evaluated by GA and its structure analogs (pyrogallic acid,1,3-dihydroxybenzene, protocatechuic acid,3,5-Dihydroxytoluene) and its adsorption behavior was investigated in detail. The results showed that the MIPs exhibited good selective ability, offered a faster kinetics for the adsorption and desorption of GA. The adsorption equilibrium was almost reached within120min at GA concentration of25mg L-1in50mg MIPs. GA was detected in9out of the25mushroom species and the concentration was in range of0.044-0.528μg g-1(dw). The structure analog protocatechuic acid (PA) was also detected in Ramaria formosa. Comparison with C18-SPE, the separation of GA from the extract of mushrooms with MISPE demonstrated high affinity and good recognition. The C18-SPE exhibited much lower extraction yields to the extract of R. formosa.(3) The MIPs were prepared by hispidin as the template, acrylamide (AA) as functional monomer, EDMA as cross-linker and a mixture of THF and isooctane as porogenic solvent. Comparing with the two analogs of hispidin, hispolon and inotilone, the template hispidin exhibited good affinity to the MIPs. The adsorption behaviors were evaluated by the sorption kinetic and the adsorption isotherm. The prepared MIPs were employed as an adsorbent phase for convenient direct separation and enrichment hispidin from8species mushrooms. Hispidin was detected in3out of the8mushroom species and the highest concentration of hispidin was detected in Phellinus igniarius,8.44μg g-1(dw). Comparison with commercial C18-SPE, MISPE displayed high selectivity and good affinity for hispidin for extract of P. igniarius and most of interfering components present in the extract of the mushroom were removed using MISPE column.The antioxidant activity of the extracts after using MIPs was evaluated by inhibitions of erythrocyte hemolysis and lipid peroxidation, the scavenging activity of free radical. The extracts contained target compounds after perconcentration using the MIPs exhibited higher inhibition activity than those before using MIPs. It indicated that the extracts of the mushrooms after extracted by MIPs have been clean up and the active ingredients have been enriched.More and more researchers pay attention to the occurrence and development of the disease caused by oxidative stress-induced cell oxidant damage. Therefore, the mechanism of the oxidative damage caused by reactive oxygen species (ROS) and the antioxidant pathway of the active compounds are particularly important. The antioxidants Be, Rf and Pi, which was extracted from Boletus edulis, Ramaria formosa and Phellinus igniarius by MIPs, used to assess the efficacy against hydrogen peroxide (H2O2)-induced mouse splenocytes oxidative stress. The H2O2-induced cell death in dose-and time-dependent was more pronounced. The extracts of the mushrooms showed moderate protective effects against H2O2induced cell death. The cell viability increased from63.9to73.7-77.7%and the protective effects of the active compounds were dose dependent. Intracellular ROS levels in the cells was enhanced after H2O2treatment but effectively suppressed in the cells pretreated with the extracts. In order to study the antioxidant mechanism of the active compounds, the mitochondria-dependent signal transduction pathway and the death receptor-mediated signal transduction pathway were studied. The oxidant damage induced by H2O2seems to be through the mitochondrial dependent signaling pathway, did not activate Fas/FasL pathway in the death receptor-mediated apoptosis pathway. The protective effect of the active compounds to H2O2-induced oxidative damage of mouse splenocytes is closely related its protective effect on mitochondria. Be, Rf and Pi could significantly increased the disruption of mitochondrial membrane potential caused by H2O2, restored the mitochondrial physiological function and inhibited the release of cytochrome c from the mitochondria into the cytosol. The pretreatment with the extracts significantly increased the mRNA and protein level of Bcl-2and inhibited caspase-3and caspase-9cleavage and block H2O2induced cell apoptosis.In the present researches, MIT was applied to the separation and purification of the antioxidant compounds form mushrooms. In order to study the antioxidant mechanism, the antioxidant activity in vitro and the protection effect of the antioxidant compounds H2O2against induced oxidate damage were discussed. MIT provided a rapid and effective approach for separation and enrichment active compounds from mushrooms, which is the basis to the development of mushroom resources.