Exploring the Fluconazole-Resistance Modifying Activity and Potential Mechanism of Action of Fixed Oil from Caryocar coriaceum Wittm. (Caryocaraceae) against Candida Species.

The fixed oil from the inner mesocarp of Caryocar coriaceum Wittm. is used in the Chapada do Araripe region of Brazil for the treatment of genitourinary candidiasis. This study aimed to evaluate the chemical composition, antifungal activity, reduction of fungal virulence, and the preliminary toxicity of the fixed oil from the inner mesocarp of C. coriaceum tested against three Candida yeasts. The oil was characterized by gas chromatography (GC-MS and GC-FID). Antifungal activity was assessed using the serial microdilution method. Additionally, the potential of the oil as an enhancer of fluconazole action was tested at sub-inhibitory concentrations (MIC/8). The mechanism of action of C. coriaceum fixed oil was determined by evaluating the inhibition of morphological transition in Candida spp. The chemical composition of the fixed oil of C. coriaceum comprised both unsaturated and saturated fatty acids. Oleic (61 %) and palmitic (33 %) acids were the major constituents. Regarding its anti-Candida activity, the oil inhibited the growth of C. albicans (IC50 : 371 µg/mL) and C. tropicalis (IC50 : 830 µg/mL). Furthermore, the oil reversed the antifungal resistance of C. albicans and C. tropicalis, restoring the susceptibility to fluconazole and reducing their IC50 from 12.33 µg/mL and 362 µg/mL to 0.22 µg/mL and 13.93 µg/mL, respectively. The fixed oil of C. coriaceum completely inhibited the morphological transition of C. albicans and C. tropicalis at a concentration of 512 µg/mL, but exhibited limited low antifungal potential against C. krusei. The observed antifungal activity may be attributed to the overproduction of reactive oxygen species. Additionally, the oil showed no toxic effect on the Drosophila melanogaster in vivo model. The fixed oil from the inner mesocarp of C. coriaceum emerge as a strong candidate for the development of new pharmaceutical formulations to treat infections caused by Candida spp.

Composition, Antibiofilm, and Antibacterial Potential of Volatile Oils from Geopropolis of Different Stingless Bees' Species.

We aimed to characterize and investigate the antibacterial potential of the native stingless bees geopropolis volatile oils (VO) for the search of potentially new bioactive compounds. Geopropolis samples from Melipona bicolor schencki, M. compressipes manaosensis, M. fasciculata, M. quadrifasciata, M. marginata and M. seminigra merrillae were collected from hives in South Brazil. VO were obtained by hydrodistillation and characterised by gas chromatography coupled to mass spectrometry (GC/MS). Antimicrobial activity was assessed by microplate dilution method. The lowest MIC against cell walled bacteria was 219±0 µg mL-1 from M. quadrifasciata geopropolis VO with Staphylococcus aureus. The M. b. schencki geopropolis VO minimal inhibition concentration (MIC) was 424±0 µg mL-1 against all the mycoplasma strains evaluated. Fractionation resulted in the reduction of 50 % of the MIC value from the original oil. However, its compounds' synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25 % eradication activity and 13.20 % inhibition of biofilm formation after 24 h for one subfraction at 2× its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity.

Mollicute Anti-Adhesive and Growth Inhibition Properties of the Methanolic Extract of Propolis from the Brazilian Native Bee Melipona quadrifasciata.

Hydroalcoholic propolis extracts from the bee species Melipona quadrifasciata have been shown to possess antimicrobial activity against different mollicute strains, but a methanolic extract (ME) could contain an increased diversity of nonpolar bioactive components with a potentially higher antimicrobial activity. The ME obtained by maceration of the propolis sample was fractionated with solvents of different polarities and then, purified by silica gel column chromatography through biomonitoring of its antimicrobial activity against mollicute strains. Analysis by gas chromatography-mass spectrometry (GC/MS) enabled the identification of compounds using the NIST library. Minimum inhibitory concentrations (MICs) of the samples were determined by broth microdilution. Anti-adhesive assays were performed with Mycoplasma pneumoniae cells. The hexane (MIC=62.5 mg/L) and dichloromethane (MIC=125 mg/L) fractions presented the most promising results against M. pneumoniae. They were fractionated into 74 subfractions, and even the best ones did not show better results (MIC>250 mg/L) than their original fractions, likely due to the loss of terpene compounds that seem to act in synergy. The dichloromethane subfraction FD4 was highlighted in the anti-adhesive assay with an inhibitory activity of 21.6 %. A synergistic effect of the nonpolar compounds in M. quadrifasciata propolis may be responsible for its antibacterial activity, but several purified components can improve its anti-adhesive properties.

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper.

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30 min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20 min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.