α-Glucosidase inhibitory activities from freshwater green algae

Abstract

The screening of microbial culture filtrates for the presence of secondary metabolites of use to the pharmaceutical industry is well established. Such studies, especially on Actinomycetes. have resulted in the identification of a wide spectrum of compounds with biological activity, including antibiotics. antifungal agents and a range of enzyme inhibitors with pharmacological activities (Schindler, 1980; Demain. 1983). However, such large-scale screening programmes for pharmacological activity have not yet been applied to algae. Reports of antibacterial and antifungal activities from algae exist (Reichelt & Borowitzka, 1984; Accorinti, 1983). but reports of enzyme inhibitors from algae are rare. We have, therefore, developed a range of rapid throughput assays for screening for the presence of enzyme inhibitors in culture filtrates and organic solvent extracts of algae. The majority of these assays are simple colorimetric microtitre plate assays and allow for the screening of hundreds of samples in a normal working day. The efficacy of such assays has been demonstrated in an initial screen of 300 marine and freshwater algal cultures, where a total of 76 enzyme inhibitors have been detected either in the aqueous growth medium or in organic solvent extracts of biomass. Included within these results, methanol extracts of four freshwater members of the order Conjugales were found to possess a-glucosidase inhibitory properties. a-Glucosidase activity was monitored by measuring the hydrolysis of p-nitrophenyl-a-I>-glucopyranoside by the change in absorbance at 410nm. These four algae were Spirogyru variuns, Zr<yticwiu c~~~lindricunr, M sotaenium caldariorum and Mou<gcwtici s p . The algal cultures were grown in Medium M 4 (Asher I % Spalding. 1982). an inorganic minimal medium, for 4-6 weeks. harvested by centrifugation, and the biomass extracted with methanol (5 ml/g wet weight). The cr-glucosidase inhibitor has been purified from Spirogyru extract by the following procedure. The methanol extract was dried under vacuum. redissolved in 0.05 M-citrate buffer, pH 6.8, and the solution extracted sequentially three times with diethyl ether. The aqueous solution was then extracted three times with butanol and the butanol extract containing the inhibitor evaporated to dryness and redissolved in methanol. This was then run in methanol on a Sephadex LH20 column (IOcm x 0.75cm, 0.5ml/min), from which the inhibitor was eluted between 5 and 8 column volumes. This sample was further purified by h.p.1.c. gel filtration (Zorbax PSM 6OS, 95% methanol solvent, 0.5 mlimin). The elution volume indicates a molecular mass in the range 2 O W O O daltons, which is in the range of nearly all the low molecular mass enzyme inhibitors identified from microorganisms, (Umezawa, 1982). Interestingly, the fractionation by Sephadex LH20 also resulted in the identification of a fraction possessing r-glucosidase activation properties, which is being further investigated. The purified inhibitor has absorbance maxima at 280 nm and 220nm and shows a single spot ( R , = 0.8) on 1.l.c. when run on silica in ethyl acetatelbutanonefformic acid/ water ( 5 : 3 : 1 : I , by vol.) and identified by 50% sulphuric acid staining with heating to 140' C. The inhibitor is alkali labile (24h, pH lo), thermostable (5mIn. 100 C) and ninhydrin negative. The purified compound also possessed antibacterial activity against the following Gram-positive and Gramnegative bacteria: Stuphylococcus uurws, Eschcvichiu c d i . Aerobacter aerogmes, Mycohucterium phlri, Micwcocws ,flavus, Proteus vulguris and Pseudomonus ueruginosu. Collaborative work with Astra Alab AB, Sweden, has also shown the Spirogyra extract to possess cytotoxic activity, (1.5 p1 of extract in 2.7 cm' of confluent monolayer Vero cells). Kinetic studies have shown the inhibition of cr-glucosidase to be of a competitive nature. In addition to inhibiting cr-glucosidase, the active fraction also showed some inhibitory activity against r-amylase, albeit to a considerably lesser degree, but n o activity against /I-galactosidase. The inhibitors from the other three algal extracts are presently being studied to see if they are of a similar nature. In conclusion, this work confirms that algae have potential as a source of novel and possibly useful compounds, and are worthy of considerable further study.