Avaliação do potencial antimicrobiano dos extratos proteicos das sementes de Inga laurina (Fabaceae) e sua aplicação em uma preparação farmacêutica

Abstract

The investigation of molecules from plants with potential antimicrobial activity has gained increasing relevance in the prevention and treatment of different diseases. Inga laurina trypsin inhibitor (ILTI), present in the seeds of this specie, is stable under a wide range of pH and temperature. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of the extract from the seeds of I. laurina (EBS), to investigate the potential mechanisms of action associated as well as incorporate, for the first time, EBS in mesoporous silica nanoparticles (SiO2NPs) to compare the antimicrobial activity in the absence and presence of the nanocarrier and develop and evaluate the antimicrobial activity of a semi-solid topical preparation containing the extract. We collected ripe fruits of I. laurina and we separated the seeds from the pulp. After dehydrated and macerated, we extract the proteins from seeds flour with 0.1 M potassium phosphate buffer 0.1 M + NaCl 0.15 M, pH 7.6, at 25°C, for 2 h, under magnetic stirring. After centrifugation, the supernatant was named EBS. The antimicrobial activity was determined in vitro by broth microdilution method, by determining the minimum inhibitory concentration (MIC) values. To evaluate the acute toxicity of EBS, we used the invertebrate model of Galleria mellonella. Different semi-solid preparations containing EBS (1%, w/w) were produced and antimicrobial activity was evaluated in vitro by well plate diffusion method against Candida albicans. Commercially available Nystatin cream was used as reference compound. Different concentrations of EBS in preparation (1, 5, 10, 15 and 20%) were tested. After selecting the best concentration, an accelerated stability study was carried out at a controlled temperature of 40°C ± 2°C during selected intervals up to 30 days, in which the characteristics of homogeneity, odor, color, pH and microbiological activity were analyzed. EBS was active at a concentration of 4 mg/mL, showing specificity against fungi and not inhibiting bacterial growth. The lowest MIC was reported to C. gattii, (31.25 µg/mL) and against different Candida species, the MIC ranged between 250 and 1000 µg/mL. After the synthesis of SiO2NPs, about 50.34% of the EBS was incorporated into the nanoparticles, through thermogravimetric analysis. However, the nanoparticles incorporated into EBS did not show antimicrobial activity. In the assay with G. mellonella, EBS did not demonstrate toxicity at the doses tested. Among the preparations, the one with the best activity was the carbopol-based gel containing the extract. There was a significant increase (p<0.05) in the antifungal activity of the gel at the concentration of 5% (w/w) of EBS. Above this concentration, there was no significant increase in antifungal activity. In the accelerated stability study, the gel preparation (5%) showed an inhibition zone of 14.66 ± 1.22 mm at day zero, which was higher than the zone of Nystatin cream. However, throughout the study, there was a loss of antifungal activity after 21 days. The pH, odor and homogeneity characteristics did not change significantly after 30 days. This is the first study that proposes the development of a semi-solid preparation containing the extract of I. laurina seeds and besides being non-toxic, the extract has potential technological application in the treatment of topical infections associated with Candida, encouraging future studies that evaluate the reformulation of semi-solid base to increase its activity in the preparation.