Determination And Identification Of Cyclopeptide Toxin In Lepiota

In recent years,poisoning incidents caused by accidental eating of poisonous mushrooms occured frequently in China.The main cause of death is caused by Amanita species containing cyclopeptide toxins.In addition to the genus Amanita,genus Lepoita and Galerina also contain cyclopeptides.In recent years,the number of cases of poisoning caused by ingestion of Lepoita has been increasing,and there are few literatures on the peptide toxins of Lepoita.However,the studies on Lepoita in China mainly focused on the investigation and taxonomy of species resources,and the studies on the peptide toxins of Lepoita have never been reported.In the past two years,we have investigated three cases of acute liver damaging poisoning caused by Lepoita,and confirmed that two of three cases were caused by Lepiota brunneoincarnata,and one was caused by Lepiota venenata.The morphological and molecular identification of L.brunneoincarnata and L.venenata were studied in this paper.1.morphological identification of the Lepiota species: There are some differences in their morphological characteristics of L.brunneoincarnata and L.venenata.L.brunneoincarnata central disc with greyish-brown to dark brown scales,around center concentric crowded.light brown to pinkish brown in apical half,smooth in upper part,scaly in lower part;scales in bands forming incomplete girdles,non-persistent annulus.However,L.venenata center brown to dark brown,the scales is small,squamules on pileus cell elements lanceolate to subcylindrical,narrower in basal part,lower part covered with brown to reddish brown squamules,forming incomplete girdles.The basidiospores of L.brunneoincarnata are larger than those L.venenata.The shape of basidiospores of L.brunneoincarnata is oval to ovoid with almond shape.L.venenata spores are oval or cylindrical.2.Molecular identification based on ITS sequence: the phylogenetic analysis tree based on ITS sequence indicated that L.venenata was not on the same branch as L.brunneoincarnata,and L.venenata was a new species.3.Detection of cyclopeptide toxins in L.brunneoincarnata,and L.venenata: Determination of Chemical Components in two species of Lepoita by ultra high performance liquid chromatography-mass spectrometry.The results showed that L.brunneoincarnata contained?-amanitin(?-AMA),?-amanitin(?-AMA),amanin and amaninamide,L.venenata contained ?-amanitin,amanin II and an unknown compound,but ?-amanitin was absent in L.venenata.Phallotoxins and virotoxins were absent in L.brunneoincarnata and L.venenata.4.Quantitative analysis of two main peptides in L.brunneoincarnata and L.venenata: The linear regression of HPLC peak area and inject amount of two standard toxins(?-AMA,?-AMA)were established.The contents of toxins in samples of L.brunneoincarnata and L.venenata were calculated according to HPLC peak area.The results showed that the total amounts of toxins in L.brunneoincarnata were 1.3-2.661 mg/g dry weight,the ?-AMA concentration were 0.732-1.728 mg/g dry weight,?-AMA concentration were 0.568-0.933 mg/g dry weight.The ?-AMA concentration in L.venenata was 1.969 mg/g dry weight,and ?-AMA was absent in L.venenata.