Isolation And Characterization Of Aconitine Producing Endophytic Fungus And Its Medium Optimization

Endophytic fungi are known as potential resources for producing active compounds.Sometimes, endophytic fungi have been found to produce same or similar chemicalcompounds to those produced by its host, and also have been recognized as a repository ofnovel compounds of immense value in agriculture, industry, and medicine. Aconitine, is amain content in aconitum plant and has been shown to have analgesic, anti-inflammatory andanti-tumor activity. However, it has apparently high cardio-and neuro-toxicity, and showsreliable and durable arrhythmogenic effects, it has also commonly been used as a classic toolfor inducing arrhythmias in experimental animals. The main natural source of aconitine isfound in the aconitum plants that limited distribution and exists in extremely lowconcentrations. Therefore, the isolation of aconitine from aconitum plants is an inefficientmeans of production. Our study was prompted by isolating and identificating of aconitineproducing endophytic fungi, this has led us to discover novel aconitine sources for its moreavailable and some basic researches. We carry out further work to increase aconitine yield byoptimizing media for large scale aconitine production. The results are as follows:1.22strains of endophytes were obtained from the roots of Aconitum leucostomumgrown of Xinjiang Uygur Autonomous Region in China, one fungus isolates was found toproduce aconitine. The presence of aconitine was confirmed by the chromatographic andspectroscopic analyses. The yield of aconitine was recorded as236.4μg/g （aconitine per drywt of mycelium） by high performance liquid chromatography. Strain XJ-AC03was chosenfor further characterisation, and its structure was also confirmed with the MS and NMRspectroscopic analysis. The1H NMR and13C NMR spectrum of the fungal aconitine wasfound to be identical in comparison with authentic aconitine.2. Basing on our observation of the morphological characteristics of XJ-AC03, it waspreliminarily identified as Cladosporium cladosporioides. The552bp DNA were obtained byamplifying ITS fragments. The result shows that ITS sequence homology of XJ-AC03withNCBI Cladosporium sp.LH-CAF5（HQ717404） can reach99%. Strain XJ-AC03wasidentified as Cladosporium cladosporioides by its characteristic culture morphology and ITSrDNA sequence analysis. 3. Based on the single factor test, Plackett-Burman design was undertaken to evaluate theeffects of the six factors. By the statistical regression analysis, the significant factors affectingthe aconitine by the strain XJ-AC03were determined as follows: sucrose, NaNO3, KCl. Themedium consist with sucrose34.44g/L, NaNO35.12g/L, KCl0.97g/L, MgSO₄·7H₂O0.5g/L, FeSO40.01g/L, K₂HPO₄1g/L, were defined as the medium for strain XJ-AC03producing aconitine, through the center combination of design and response surface analysis.Under these conditions, the aconitine can be enhanced to377.48μg/g, increasing59.68%thanbefore optimization.