Isolation And Identification Of Swainsonine In The Mycelia Of Fungal Endophytes From Locoweeds

Locoweeds are the poisonous species of Astragalus and Oxytropis （leguminosae）, andhas been worldwidely recognized as one of the most significant toxic weeds damaging tolivestock on grasslands and resulting in valuable economic losses to animal husbandry atpresent. The primary reason for animal poisoning caused by locoweeds is the bioactivesubstance （Swainsonine, SW） within locoweeds. However, SW possess an potentialpharmacological effects including anti-tumor and immune regulation in addition to its toxicity.Low SW concentrations in locoweeds and incomplete extraction methods lead to expensivecost but low inefficient in yield and artificial synthesis of SW also requires complexprocedures resulting from difficulties in extracting, isolating and purifying. Currently, SW isfound to produced by fungi including Metarhizium anisopliae, Rhizoctonia leguminicola andUndifilum endophytes frequently isolated from locoweeds by the researchers at home andabroad and is detected through a wealthy of different methods. However, there is no studyfocusing on extracting SW with high purity from fungal endophytes exsiting in locoweeds. Toprovide a new source of SW, meet the demand of scientific research and medicine, and lay thefoundation for batch production of SW on industrial level, the cryopreserved FEL4-F5isolated from O. ochrocepala were selected as research object recovered, transferred andharvested pure fungal cultures in this experiment. Organic crude extractings were obtainedafter extracting mycelia in the methanol using a Soxhlet apparatus, and then removingimpurities through adsorption and exchange of cationic resin. SW in the crude extract wasqualitatively detected by TLC, and subsequently was quantificationally determinated byHPLC-ELSD. The crude extract were performed liquid-liquid extraction and purification, theextracted crystals were identified and verified. The achieved results were as follow:1.0.1g of total alkaloid extractum were abtained from lyophilized FEL4-F5mycelia （30g of sampling0.5g） growing on PDA for45d. The results of TLC indicated that SW werepresent in the secondary metabolites of FEL4-F5based upon the consistent purple spots andmigration distances appeared on the lamina, which confirmed the ability of the chronicallypreserved locoweed’s endophytic fungi synthesizing SW and provided a guarantee for thesubsequent research on determinating SW concentrations.2. The crude extracts were determined by HPLC-ELSD after treated by a BioRad AG50W ion-exchange resin. The consistent peaks at much the same rentention time （3.083min） with SW standard were appeared on the liquid chromatographic of the sample, and SWcontent was4338.42μg/g calculated in compliance with the relationship betweenconcentrations and their peak area values.3.10mg of white needle crystals were isolated from30g of dry FEL4-F5mycelia andthen subjected to structural identification. The measured results indicated that the MP ofthe crystal was144～145℃; It was concluded that functional groups including-OH (3431.37cm^-1), C-H (2947.39cm^-1,2891.21cm^-1) and C-O (1072.85cm^-1) were present in the crystalfrom IRmax (KBr, cm^-1) spectrogram. Especially, the trans CH-N absorption (2828.51cm^-1～2727.86cm^-1) suggested that indole ring was also contained in the extracted crystal; The dataof ESI-MS indicated that the molecular weight of the crystal was173.1132; GC-MS spectrumshowed that the GC retention time of crystal was7.93min and the characteristic molecularion peak of its trimethyl silane derivates was374.40after the MS fracture;¹H-NMR,¹³-NMR and DEPT135spectrum demonstrated that there were four CH2and CH groups inthe molecular structure of the crystal while¹H-¹H COSY and¹H-¹³ HMQC spectrum furtherrevealed the structural information including connection mode and order among elements;Consequently, the isolated crystal was proved to be SW.4. Trial for verifing biological toxicity function stated that the crystal were capable ofinhibiting α-mannosidase activity. Despite of the appearance, inherent structure or function ofextracted crystal, We did not discover that there was any difference between pure toxinisolated from fungi and pure toxin SW derived from locoweeds by comparison. They were thesame compound.