This paper reports the results of a screen for inhibitors of acetylcholinesterase and cAMP phosphodiesterase. Acetylcholinesterase inhibitors are clinically important in the treatment of myasthena gravisll], post-operative atony of smooth muscle [2] and potentially in the treatment of chronic pain [3]. In addition they also constitute two economically important groups of insecticides; organophosphorus and carbamate insecticides. The positive inotropic and relaxant effects of cAMP phosphodiesterase inhibitors have made these compounds potentially important in the treatment of congestive heart failure [4].As part of an on-going project to isolate compounds of potential clinical value from algae and cyanobacteria we have screened aqueous culture supernatants and methanolic cell extracts of 80 randomly selected freshwater and marine algae for these inhibitors. 100ml cultures of each of the algae tested were grown for 28 days in marine [5], blue-green [6] or M4[7] media as appropriate. During this time the cultures were shaken at 11 Orev./min and illuminated at 27001~. using flourescent strips for l6h/day. The algae were harvested by centrifugation (4000xg, 15min) and the culture supernatant retained. The algal pellet was extracted with methanol (6ml, HPLC grade) by shaking at 1lOrevJmin for 30 min at 25°C. Following centrifugation of the algal extract (4000xg, 15 min) the methanolic supernatant was retained and the algal pellet discarded. The methanolic cell extract and the aqueous culture supernatant were then screened for the presence of acetylcholinesterase inhibitors and cAMP phosphodiesterase inhibitors.A microtitre plate based method was employed for the detection of acetylcholinesterase inhibitors. 100~1 of aqueous test solution or 30y1 of methanolic cell extract with 70~~1 of water was added to each well and 25pl of 0.5M citrate buffer pH 7.7 and 100~1 of enzyme solution (Sigma, Electeric eel cholinesterase, 5 units/ml in 0.1 M citrate buffer) added. The plate was incubated at 37°C for 30 min and then 5Op1 of 2mg/ml 59dithiobis-(2-nitrobenzoic acid) and 25p1 of 1 mg/ml butrylthiocholine iodide both prepared in 0.1 M citrate buffer pH 7.7 added. The absorbance of the wells was read at 410 nm and the plate incubated at 37°C for a further hour after which time the absorbance was again determined. The presence of acetylcholinesterase inhibitors was indicated by i n h i b i t i o n o f c o l o u r d e v e l o p m e n t . Acetylcholinesterase inhibitors were detected in the culture supernatant of the cyanobacterium Audouinella parvula and the methanolic extract of the chlorophyte Cladophora globulina; and in both the culture supernatant and methanolic cell extract of the chrysophyte Ochromonas villosa. Cultures of these algae have been grown on at least 3 Occasions and been found positive on each occasion. Further purification of the active compounds from the rnethanolic cell extract of Ochromonas villosa is currently underway in our laboratory.A modified version of the method of Thompson and Appleman [8] has been ...
As part of an on-going project to detect extracellular algal compounds of potential medicinal value aqueous culture supernatants and methanolic cell extracts of randomly selected algae were screened for the presence of bioactive compounds using electrically stimulated guineapig ileum preparations[l] and a brine shrimp assay [2]. Bioactive compounds were detected in the methanolic extract of the cyanophyte Phormidium persicinurn in both screens, and in the aqueous culture supernatant using the brine shrimp assay. Correlation of growth and production of compounds toxic to brine shrimps showed two peaks of activity in the methanol cell extract; the first early in stationary phase and a second larger peak late in stationary phase. Two similar peaks of activity were also observed in the aqueous culture supernatant.The larger peak of activity from the methanolic extract has been fractionated into six constituent toxins using the following protocol. 2ml aliquots of stationary phase stock cultures were used to inoculate flasks containing 1 OOml aliquots of marine media[3] and the cultures placed on shaking incubators (1 1 Orev./min) illuminated at 27001~ for 16h/day at 25°C. On reaching a cell number of 4 . 7~1 0~ cells/ml the algae were harvested by centrifugation (4000xg), and the culture supernatant discarded. The algal pellet was extracted with methanol (6ml/l OOml algal culture) by shaking at 25°C for 30 rnin and the methanolic supernatant recovered by centrifugation for 15 rnin (4000xg). Following evaporation to dryness the algal extract was resuspended in an equivalent volume of Tris-HCI pH 7.5 and extracted by shaking with water saturated butan-1 -01 for 30 minutes. The aqueous and organic phases were separated and the organic solvent extract evaporated to dryness and resuspended in 1 ml methanol, prior to loading onto a Sephadex LH20 column ( 1 . 8~ 100cm) pre-equilibrated in methanol. The active fractions (5ml) were pooled and evaporated to dryness. Following resuspension in 1 ml methanol the active fractions were further purified by h.p.1.c. using a Spherisorb 5p amino column (25cm x 8mm) pre-equilibrated in methanol and run at 2mVmin. The active compounds passed through the column unretarded unlike much of the contaminating material. Following pooling of active fractions, evaporation to dryness and resuspension in 90% methanol the activity was applied to a C,, column (Spherisorb ODs2 25cmxl Omm) pre-equilibrated in the same solvent and run at 2mVmin. After loading the column was washed for 25 min with 90% methanol and then a linear gradient from 90-93% methanol applied over a 10 min period. The column was then washed using 93% methanol for a further 2 min prior to application of a second gradient from 93%-94% methanol over a 10 rnin period. The column was then washed again for 6 min using 94% methanol before a gradient from 94%-100% methanol was applied over a 7 min period. A final wash in 100% methanol was then carried out for 10 min. The activity was fractionated into 6 constituent toxins eluting at 19-20 m...
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