Bioautographic Method for the Detection of Xanthine Oxidase Inhibitors

RAMALLO, IA; ZACCHINO, SA; FURLAN, RLE

BIOCELL

IHEM-CONICET Fac. Cs. Médicas- UN Cuyo

Lugar: Mendoza; Año: 2004 vol. 28 p. 218 - 218

0327-9545

Natural products are an important source of new drugs (D.G. Newman J. Nat. Prod. 2003, 66,1022-1037). To screen plant extracts avoiding the time consuming isolation of known substances,assays able to detect active compounds present in complex plant matrices are needed (J.Chromatogr. A 2001, 915, 217). We describe here a bioauthographic assay suitable to localize Xanthine-Oxidase (XO) inhibitors absorbed on chromatography plates (TLC). This enzyme catalyzes the oxidation of hypoxanthine to xanthine and, in the presence of molecular oxygen, to uric acid and superoxide anions. Therefore, XO inhibition is a therapeutic approach for treating gout, kidney stones, and myocardial ischemia (H. Li J. Nat. Prod. 1999, 1053. For the development of the assay, enzyme and substrate were spread on the plates under different conditions, and several revealing agents for enzyme activity were tested. The best results were obtained when TLC plates were layered with agar solution containing XO and nitroblue tetrazolium chloride (NBT). After solidification the system was immersed in xanthine solution at 35°C for 20 minutes. Enzyme oxidation of xanthine produces superoxide which reduces soluble NBT to a purple precipitate that results in gel staining. Inhibitors of the enzyme are detected as white spots in a purple background. The detection limit for the commercial inhibitor allopurinol was 0.01 ıg. The bioassay is rapid and allows the screening of several samples at the same time. It is particularly suited for dereplication of plant extracts.