Studies on a toxin produced by the cyanobacterium Phormidium persicinum.

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 min (90% methanol), 21-22 rnin (90% methanol), 23-24 min (90-90.1% methanol), 26-29 min. (90.3-91.2% methanol), 30-33 min (91 592 .4% methanol) and 35-37 min (93% methanol) respectively. The toxin eluted from the C,, column at 23-24 min was finally purified following a further gel filtration purification step using a (Zorbax PSM 60S 25cmx6.2mm) column pre-equilibrated in 100% methanol and run at 1 ml/min. Electron impact spectroscopy, fast atom bombardment mass spectroscopy and H’ n.m.r. have failed to yield any structural information or estimate of molecular weight since the active compound appears to be present in residual quantities only. This does however highlight the potency of the toxin isolated. The toxins produced by this alga have been screened for insecticidal activity by topical application and injection of Musca domestica and Blattella germanica. No insecticidal activity has been observed, although this may be attributed to the limited quantity of material available for testing. This is the first report of toxin production by P.persicinum. Antitumour[4], cytotoxic[4], healing[5] and stimulatory[5] activities have previously been attributed to crude extracts or culture supernatants of Phormidium sp. but the active compounds have never been identified. The production of toxins by this alga is not however totally unexpected since P.persicinum belongs to the order Nostocales which includes other acknowledged toxin producers such as Lyngbya sp.[6], Anabaena sp.[7,8] and Aphanizomenon sp.191.