Atividade in vitro e in vivo de compostos bioativos de plantas medicinais e nanopartículas de prata sobre Pythium insidiosum

Abstract

Pythium insidiosum is the etiologic agent of pythiosis. Despite research advancements, the treatment of this disease is not fully established. This study evaluated the in vitro antimicrobial activity of bioactive compounds from medicinal plants and biogenic silver nanoparticles (bioAgNP) against P. insidiosum isolates and verified the effect of Melaleuca alternifolia nanoemulsion and bioAgNP on the treatment of experimental pythiosis. In vitro susceptibility tests against bioAgNP, M. alternifolia, Origanum vulgare, Menthapiperita, Eugenia caryophyllata essential oils and á-terpineol, carvacrol, menthol and eugenol major compounds followed the CLSI M38-A2 protocol. BioAgNP cytotoxicity tests were performed on RK13 cells. Damage to the P. insidiosum hyphae ultrastructure treated with bioAgNP was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Dermal toxicity tests for M alternifolia essential oil were performed using 30 Wistar rats. For in vivo experiments, the disease was reproduced in fifteen female 90- day-old New Zealand rabbits, being divided into groups of three animals. Control group was daily and topically treated with anonionized gel-based formulation and 1 mL of sterile distilled water intralesion administered every 48 hours. In the bioAgNP experiment, three rabbits were daily and topically treated with gel-based formulation containing 0,001 mg/L bioAgNP and the other three animals received 1mL of bioAgNPin 0,001 mg/mL aqueous solution intralesion administered every 48 hours. In assay with M. alternifolia nanoemulsion, the same methodology was used, but animals were treated with 5g gel-based formulation containing 5mg/mL of M. alternifolia nanoemulsion and 1mL of M. alternifolia nanoemulsion at concentration of 5mg/mL in aqueous solution. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. In vitro P. insidiosum susceptibility (n=38) against bioAgNP showed MIC50 of 0.24mg/L and MIC90 of 0.47mg/L. BioAgNP toxicity threshold on RK13 cell was observed at concentrations of 0.6 to 5mg/L and 0.3 to 5mg/L in readings of 24 and 48 hours, respectively. In addition, it was observed through SEM that P. insidiosum hyphae treated with bioAgNP showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. TEM did not differentiate organelle structures, and the cell wall was rarefied, showing wrinkled and partly ruptured borders. P. insidiosum (n=34) susceptibility to essential oils and major compounds showed that α-terpineol was the most effective bioactive compound in inhibiting this oomycete. When MIC values of the major compound and essential oil were compared, it was observed that bioactive compounds α-terpineol and carvacrol had higher anti-Pythium insidiosum activity compared to M. alternifolia and O. vulgare essential oils, suggesting that these compounds may be responsible for the inhibitory activity. M. alternifolia essential oil did not show dermal toxicity. In the treatment of experimental pythiosis, with the exception of rabbits that received topical M. alternifolia nanoemulsion formulation, lesions observed in the other groups differed from the control group (P <0.05). It was observed that animal treated with topical bioAgNP formulation presented clinical cure at 35 days of treatment. The results of the present study expand prospects for the development of new drugs with effective activity in the control of pythiosis.