Evaluation of antifungal activity of extracts of Punica granatum L. on Malassezia pachydermatis

Fungal external otitis is a major diseases in small animals and the infection by Malassezia pachydermatis is the most frequent. The growing demand for medicinal plants is due to the indiscriminate use of antibiotics, and high recurrence rates. This work aimed to evaluate the aqueous and alcoholic extracts of Punica granatum L., at different concentrations, on M. pachydermatis. The fungus was isolated from a dog holder of chronic otitis and the diagnostic was confirming from imprint on glass slide and Gram stain. The suspension of M. pachydermatis was adjusted to concentration of 1 × 10⁶ CFU/mL. The sensitivity of the microbial isolates was determined in quintuplicate by the technique of disc diffusion. The minimum inhibitory concentration was determined by inhibition zone greater than 15 mm. The aqueous extract was made by the cold extraction (24 hours), in water bath (60 minutes), boiling (10 minutes) and by infusion (60 minutes). The alcoholic extract was made only by the cold extraction at different extraction times: 15, 30, 60 and 90 days. After obtaining the extracts at 50% (w/v), additional concentrations were prepared at 40, 30, 20 and 10%. The aqueous extract obtained by boiling was more efficient in inhibiting the growth of M. pachydermatis, proceeded by the extracts obtained by water bath, cold extraction and infusion. The extracts in concentrations of 50% regardless of the method used, showed greater zones of inhibition, however, all of aqueous extracts showed zone greater than 15 mm. Regarding the alcoholic extracts all of them presented inhibition of M. pachydermatis, except extracts at 10% and processed by 15 of extraction days. Thus, the results obtained by in vitro experiments, allows us to conclude that the extracts of Punica granatum L. presents potential clinical use for the control of otitis by M. pachydermatis in dogs.