The Determination,Purification And Identification Of Peptide Toxins In Amanita Fuliginea

Amanita fuliginea is a lethal amanita species,which is mainly distributed in Japan and south China.Since the 1990 s,the majority of all fatal cases of human mushroom poisoning was caused by eating Amanita species in China,particularly by A.fuliginea.The cycle peptide toxin is main factor of death in A.fuliginea,at the same time,these toxin s has important potential value in biomedical research and tumor treatment.In this paper,we compared the contents and distribution of the main toxin in A.fuliginea were compared and the main toxins were isolated and purifed from A.fuliginea.A method was established for the detetion and measurement of cyclic peptide toxin in A.fuliginea using the UPLC-Q-TOF-MS technique.The results are as follows:1.The content and distribution of the main peptide toxin in A.fuliginea: The contents of amatoxins and phallotoxins in A.fuliginea from different tissues,development stages and collection sites were evaluated by high-performance liquid chromatography(HPLC).High amounts of total toxins were found in the gills,pileus and stipe,whereas relatively small amounts of total toxins were found in the annulus and volva and minimal amounts of toxins were detected in the spores.In all tissues,there was a higher amount of amatoxins than the phallotoxins in all tissues and the ratios of phallotoxins/amatoxins(P/A)were higher in the pileus,gills and stipe than those in the annulus,volva and spores.Further analysis of the toxins at different development stages showed that the amatoxin and phallotoxin contents were relatively high and constant during early development,reached maximum when the fruit body was in the vigorous growth stage and then decreased during the maturity stage.Collection sites had a significant effect on the amount of total toxins but minor effect on the P/A ratios.2.The separation and purification of the main peptide toxins in A.fuliginea: A suit method for separation and purification the peptide toxins from A.fuliginea in laboratory were established,which including the macroporous adsorptive resins column chromatography(XAD16),Sephadex LH20 column chromatography and semipreparative HPLC.The most of toxin component can be separated by twice column chromatography,and the purity of ?-amanitin can reach more than 85%,in the end,six kinds of toxins were obtained,there were ?-amanitin(?-AMA),?-amanitin(?-AMA),phallacidin(PCD),phalloidin(PHD)and Amaninamide(after appraisal)respectively.3.The identification of the main peptide toxin in A.fuliginea: Using UPLC-Q-TOF-MS technology,combined with secondary mass spectrometry to analysis peptide toxin of A.fuliginea,eleven kinds of components were identified,of which,eight kinds were known components,there were ?-AMA,?-AMA,PHS,Amaninamide,PCD,PHD,PHDII and PHN respectively.two components had the samemolecular weight,according to the PHD retention time in HPLC,we extrapolate that the two components were isomers.On this basis,we also analyzed MS/MS,the results showed that 9 components similar to the PHD in multiple fragments,so we speculated that it may be to a PHD derivatives.The other two unknown components did not find the similar characteristics to amanita peptide toxin.