Study On Mao Inhibitor From The Fermention Broth Of Beauveria Amorpha

A screening of monoamine oxidase (MAO) inhibitive activity of metabolite of entomogenous fungi revealed that the ethylacetate extract from fermentation broth of a strain of beauveria amorpha Ba02 exhibited strong inhibitory activity on MAO. The extract was tested for its IC50, kinetics trait, and some related factors including optimal fermentation time, separation, extraction and purification of bioactive components, in vivo activity of methanol extract and structure identification of purified compound.The inhibition on MAO-A and B of the ethylacetate extract from Ba02 showed a dose-dependent manner with IC50 at 18.3 μg.ml-1 and 28.2 μg.ml-1, respectively. The inhibitory manner also showed that the ethylacetate extract from Ba02 on MAO-A was competitive and that on MAO-B was both competitive and noncompetitive with Km at 0.47×10-5mol.L-1 and 0.11×10-5mol.L-1, respectively.Based on active component yield from the ethylacetate phase of the fermentation broth by means of TLC analysis, the fermentation time of the strain Ba02 was determined at 10.5 days. Before extracted with organic solvent, the fermentation broth was condensed at a proper temperature, then type of organic reagent, the ratio of reagent and the broth and extraction times were optimized. Based on the results a protocol was determined as the followings: the broth is condensed at 50℃ to 1/5 volume of the broth, 3 times of 95% ethanol is used to remove big molecule impurity, the condensed broth is extracted with ethylacetate twice with a exact ratio ethylacetate to condensed broth at 2:1, dissolve the extract and freezing-dry the extract. The raw extract was separated through a silica gel column chromatogram (LCC) with the mobile phase which was optimized with TLC experiment of ethylacetate : methanol : water = 85 : 10 : 5. The purified components from the LCC were submitted to high performance liquid chromatogram (HPLC) for further purification. Four pure components JL-1, JL-2, JL-3 and JL-4 were derived obtained finally. The purity of the 4 components were determined by TLC and HPLC. Among the quantity of all the 4 purified compounds, only JL-1 with the content of 86% was enough for structure identification.JL-1 was submitted for structure analysis with MS, NMR, IR and UV, respectively. The results revealed its molecular weight at 306, its molecule formula at C14H10O8. The structure proved to be a known compound Oosporein, by structure analysis and Champman & Hall Natural Productions Data Bank search. At the same time, its described characters in database were identified with those of JL-1. Though Oosporein is a known compound, its inhibitory activities on MAO-A and MAO-B were unreported till now. In vivo experiments showed that the methanol extract of Ba02 exerted a substantial inhibitory activity on MAO-A. In addition, the result of the experiment on chronic unpredictable stress mouse models revealed that it had distinct inhibition on MAO-A and MAO-B of brain mitochondria at a dose of 25, 50 and 100mg.kg-1, respectively. These results were consistent well with those in the in vitro tests at the same time.