Resumo

The bullfrog (Rana catesbeiana Shaw) oil (BO), which is known due to its therapeutic applications, including its antimicrobial and anti-inflammatory activities, may be an alternative for future treatments of infectious diseases caused by many microorganisms. The aim of this study was to identify the antimicrobial compounds of BO by Gas Chromatography-Mass Spectrometry (CG-MS) using the bioautography assay. A Thin Layer Chromatography (TLC) was developed with BO/methanol (2:8) and the mixture of hexane/ethyl acetate (9:1) was used as the eluent system. Staphylococcus aureus ATCC 29213, S. epidermides ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 90027, C. parapsilosis ATCC 22019, C. glabrata ATCC 2001, C. krusei ATCC 6258 and C. tropicalis ATCC 13803 were used. A bioautography assay was performed loading the TLC into culture plates containing microorganisms in suspension. After an incubation period of 24 and 48 hours for bacteria and fungi, respectively, at 35°C the inhibition halos using tetrazolium chloride (TTC) were observed. The TLC plates were reproduced and the silica corresponding to inhibition halo was removed and chemical compounds were extracted by ethyl acetate in ultrasound bath for 20 min. The sample was also methylated to investigate the components major improving the separation and volatilization of the carboxylic acid compounds. The analyses were performed in a GC-MS Hewlett-Packard 5975, injector split/splitless, with capilar column HP-5. Temperatures: injector=250°C, detector= 300°C, column= 110°C, 5°C.min^-1, 280°C (26min). Super dry Helium flow = 1.0 mL.min^-1. The bioautography showed that BO has an anti- P. aeruginosa activity. The CG-MS analyses were performed by comparison to NIST MS library. Fatty acids and esters were found and 2-mono-oleoyl glycerol was the major compound (46.55%), besides others less representatives such as 1,2-dipalmitin and oleic acid. The methylated sample revealed the presence of stearic, phtalic and palmitic acid methyl esters, alcohols and hydrocarbons. This study predicts the presence of isolable antimicrobial compounds in BO, fatty acids, which are the major compounds of the oil. Also, the presence of antimicrobial fatty acids suggests that these compounds can be used to develop drug delivery systems with antimicrobial activity in addition to the current therapeutic system.