DETERMINATION OF OPTIMIZED PROTOCOLS FOR THE EXTRACTION OF ANTICHOLINESTERASIC COMPOUNDS IN ENVIRONMENTAL SAMPLES CONTAINING CYANOBACTERIA SPECIES

摘要

The effects of temperature and pH during extraction of toxin(s) from the cyanobacterial genus Anabaena were studied. Different extracts were compared for their anticholinesterase potency against purified eel acetylcholinesterase. A lyophilized powder from Anabaeba spiroides cells was dissolved in ethanol, sonicated, and filtered. This solution was then evaporated at three different temperatures (40, 60, or 80℃), resuspended in chloroform, and washed in deionized water at three different pH values (3.3, 5.0, or 8.5). At 40℃, the estimated 50% inhibition concentration (IC50) was 2.06, 2.19, and 8.64 mg lyophilized A. spiroides cells ml~(-1) for the extraction pH of 3.3, 5.0, and 8.5, respectively. At 60℃, the values were 4.82, 3.06, and 12.0 mg/ml at increasing pH values. Finally, at 80℃, the IC50 values were 3.52, 4.35, and 30.1 mg/ml at the same pH values. These results indicate that pH 3.3 and a temperature of 40℃ should be employed to preserve toxin content. The optimized protocol was employed to evaluate the anticholinesterase potency of environmental samples of water containing different numbers of filaments of cyanobacterial species. Registered inhibition ranged from 5.11 to 11.1%, supporting the feasibility of the method.