The Determination,Isolation,Identification And Function Of Main Peptide Toxins In Amanita Exitialis

Poisonous mushrooms was one of the most major sources of fatal food poisoning in China.Approximately 90%fatalities in mushroom poisoning were associated with the amanitins-containing species.Amanita exitialis was a lethal mushroom first described by Yang Zhuliang and Li Taihui in 2001,and which had led more than 20 fatalities since 2000.The aim of present thesis was to study systematically the toxins component,content,distribution,extraction and separation,purification and identification from A.exitialis,and to evaluate the hepatoprotectiveeffect of Ganoderma.lucidum aqueous extracts(GLE)on liver injuryinduced by a-amanitin(a-AMA)in mice and to analyse the possible hepatoprotective mechanisms related to radical scavenging activity,to assess the effect on viability of L02 cell treated with a-and ?-amanitin,and the effect of apoptosis and necrosis of L02 cell induced by a-and?-amanitin.The results were as follow:1.The a-and ?-amanitin contents and distribution in six differentmushroom tissues[gills,pileus(without gills),stipe,annulus,volva and Spores]of A.exitialis were analysed by reverse phase high-performance liquid chromatography.The highest concentrations of amatoxins were found in the gills and pileus(without gills)(5446.2 ±163.9 and 4907.0±155.9?g·g-1 dried tissues,respectively),followed by the stipe and annulus,with the lowest concentrations in the volva and spores(801.1±10.5 and 392.2±12.3?g·g-1 dried tissues,respectively).The a-amanitin/?-amanitin ratio varied significantly in different tissues.2.The analysis of a-and ?-amanitin contents in differentdevelopment stages[button(A),button with broken outer veil(B),pileus revealed from outer veil(C),stipe elongation(D),pileus open and flat with inner veil(E),matured fruit body(F)and wilted fruitbody with reflexed cap(G)]showed that the amatoxin content was relatively high and steady during A-D development stages,reached its peak(4204.0±137.3?g·g-1 dried fruit body)in the stage E,when the fruit body was in the vigorous growth stage and then decreased sharply when the mushroom entered its mature stage,only 1904.4±139.5 and 1152.8±48.8?g·g-1 dried fruit body in the stage F and stage G,respectively.3.The analysis of ?-and ?-amanitin contents in different mushrooms collected from different sites(South China Botaincal Garden,the forest of South China Agricultural University,near the south gate of Baiyun mountain park and near the south gate of Baiyun mountain park)in Guangzhou showed that amatoxin content was high in the fruit body from the region near the South China Botaincal Garden and the forest of South China Agricultural University(3550.6±88.6 and 3538.5±117.7?g·g-1 dried fruit body),while the lowest was found in A.exitialis collected in the region near the south gate of Baiyun mountain park(2054.6±57.9?g·g-1 dried fruit body).The variation of amatoxin level may relate to the geography and soil condition of collect sites.4.The capability of static state adsorption and adsorption stripping of four macroporous adsorptive resins(Amberlite XAD4,XAD16,XAD7HP and XAD1180)on the toxin components of A.exitialis were compared.The result showed that XAD16 possessed highest static state adsorbing and rather high adsorption stripping capability,while XAD7HP and XAD1180 possessed lower capability of static state adsorption and higher adsorption stripping capability.The adsorption and adsorption stripping velocity of the four resins for toxins components of A.exitialis were also analyzed,the result showed that XAD4 possessed the slowest adsorption velocity and XAD1180 possessed the fastest velocity;XAD4 and XAD16 possessed slower adsorption stripping velocity,while XAD1180 and XAD7HP possessed faster adsorption stripping velocity among the four resins.The results of comparison of the column chromatography effect of four resins showed that all four resins couldnot separate toxin components of A.exitialis,but could be used for richen toxins,and XAD16 was the favorite material for richen the toxin components of A.exitialis.5.A suit technique for separate and purify the peptide toxins from A.exitialis on laboratory scale were establised,which including macroporous adsorptive resins column chromatography,Sephadex LH20 column chromatography and semiprep HPLC.The yield ratio of ?-and?-AMA by Sephadex LH20 column chromatography were 89.55%and 94.07%respectively.Five high purified morphons of toxin components obtained from A.exitialis by the technique system.?-and ?-AMA were identified by mass spectrometry and nuclear magnetic resonance,and primary identified desoxoviroidin,phallisacin and phallacidin accord to molecular weight.6.Mice were treated with ?-AMA prepared from Amanita exitialis,then administrated with Ganoderma lucidum Aqueous Extract(GLE)after ?-AMA injection.The hepatoprotective activity of the GLE was compared with the reference drug silibinin(SIL).?-AMA induced a significant elevation in serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities and provoked a significant reduction of superoxide dismutase(SOD)and catalase(CAT)activities and a significant increment of malondialdehyde(MDA)content in liver homogenate.Treatment with GLE or SEL significantly decreased serum ALT and AST levels,significantly increased SOD and CAT activities and decreased MDA content in liver,as compared to the a-AMA control group.The histopathological examination of liver sections was consistent to that of biochemical parameters.The results demonstrated that GLE possesses hepatoprotective effects on acute liver injury induced by?-AMA and the protective effects may be in part related to its antioxidant properties.7.The influece on viability of L02 cell induce by different concentration of ?-and ?-AMA were evaluated.The result showed that the viability of L02 cell could be inhibit both by ?-and ?-AMA when the concentration of ?-and ?-AMA more than 1?M,and the the inhibition ratio increasing with ?-and ?-AMA concentration increased.8.The apoptosis of L02 cells induced by different concentration(1,2,5,10,20 and 40?M)?-and ?-AMA for different time(6,12 and 24 hours)were evaluated with flourchrome hoechst 33342 and propidium iodide stained and examinated by reversion fluorescence microscope.The results show that both ?-and P-amanitin can induce LO2 cell apoptosis,the effect of apoptosis was dose and time dependent.The LO2 cell co-cultured with more than 5?M ?-AMA presented necrosis apparently,and the higher concentration of ?-AMA,the more manifest of cell necrosis.The LO2 cell co-cultured with more than 10?M ?-AMA presented necrosis apparently,too.The nuclei chromatin of the cells which present apoptosis display condensaton and marginalization,the condensaton and marginalization become more and more potentiation,at last the nuclei display chip and disintegration.9.The DNA Laddering of LO2 cells induced by ?-and ?-AMA were analyzed by the means of electrophoresis on agarose gel.The results show that DNA fragmentation could be induced by more than 5?M ?-and ?-AMA.The amount of integrity DNA extracted from L02 cells decreasing with the increasing of ?-and ?-AMA concentration,it suggested that the DNA of LO2 cells decomposed accompanied L02 cells damaging when the concentration of ?-and ?-AMA increasing.10.The activity of caspase3,8 and 9,the key factor of cell apoptosis,in L02 cells treated with-and P-AMA were analyzed.The result show that the activity of three caspases significant elevation induced by more than 1?M a-and ?-amanitin.It suggested that ?-and P-amanitin priming L02 cell apoptosis which was caspase dependent.