Chemical composition, antifungal activity, antibiofilm and citotoxicity of the essential oil of Citrus deliciosa tenore.

The leaves of Citrus deliciosa Tenore were collected in southern Brazil, dried, and subjected to the hydrodistillation process to obtain the essential oil. The extraction of essential oil yielded 0.97% ± 0.04. The chromatographic profile of this compound revealed 11 substances, being methyl N-methyl-anthranilate (75.1%), γ-terpinene (13.8%), and Limonene (7%) as major substances. The essential oil shows antifungal action against all tested yeasts, with promising action against Cryptococcus neoformans, Cryptococcus gattii, and Trichosporon asahii. This compound was also able to inhibit the biofilm production of Candida albicans, Candida glabrata, Candida parapsilosis, and T. asahii. The essential oil of tangerine showed weak antioxidant action. It did not show cytotoxicity in human mononuclear cells. It is hoped that these results may guide future studies for the production of formulations that can be used in the treatment of biofilms caused by yeasts, as well as in candidiasis, cryptococcosis, and/or trichosporonosis.

Antimycobacterial activity of Achyrocline flaccida (Asteraceae) aqueous extract from Southern Brazil.

Achyrocline flaccida aqueous extract was obtained by macerating wildflowers. The phytochemical profile present in the A. flaccida aqueous extract was elucidated by HPLC-ESI-MS/MS. Toxicity was evaluated in vitro by comet assay in peripheral blood mononuclear cells (PBMCs) and in vivo using Caenorhabditis elegans as a model. The antioxidant activity was also evaluated, and antimycobacterial activity was assessed by the broth microdilution method. The compounds present in the aqueous extract mainly belonged to the flavonoid class (89%). The concentrations that showed protective effects in C. elegans against oxidative stress and antimycobacterial activity had no toxic effects. The antimycobacterial activity test demonstrated that the concentration of 1,560 µg mL-1 inhibited the growth and eradication of the mycobacterial tested strains. Based on our findings, the A. flaccida aqueous extract presents a viable potential in developing new phytotherapeutic drugs against mycobacteria of clinical relevance.

Phytochemical characterization, genotoxicity, cytotoxicity, and antimicrobial activity of Gautheria procumbens essential oil.

This study investigated the chemical constituents of Gaultheria procumbens essential oil and is the first to relate cytogenotoxicity with oxidative metabolism and antimicrobial activity. Chromatographic analysis of the essential oil showed methyl salicylate (99.96%) and linalool (0.04%) as the major compounds. The essential oil showed no signs of cytogenotoxicity at different concentrations (1.82 to 58.34 mg mL-1). Furthermore, G. procumbens essential oil and methyl salicylate were used to evaluate the minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC). The results showed efficacy against several microorganisms, including Aeromonas caviae, Candida albicans, and Mycobacterium fortuitum with MIC values ranging from 1.82 to 3.64 mg mL-1 and MMC values ranging from 3.64 to 12.67 mg mL-1, which were confirmed by time-kill kinetics. Based on our results, the essential oil is a promising alternative to developing future formulations to treat infections caused by microorganisms.

Achyrocline flaccida essential oil from Brazil: phytochemical composition, genotoxicity, protective effects on Caenorhabditis elegans, and antimycobacterial activity.

Achyrocline flaccida (Weinm) DC. essential oil was obtained by hydrodistillation of wild flowers from southern Brazil. We explored, for the first time, the phytochemical composition, toxicity, resistance to oxidative stress in Caenorhabditis elegans, and antimycobacterial activities of A. flaccida essential oil. Twenty-four compounds were identified by GC-FID-MS and the major constituents identified were α-pinene (41.10%) and caryophyllene (30.52%). The essential oil showed no signs of genotoxicity in the comet assay and presented relevant antioxidant capacity since it prevented ROS production in the C. elegans model. Furthermore, the minimal inhibitory concentrations (MIC) results showed that M. abscessus, M. massiliense, and M. fortuitum had their growth inhibited by A. flaccida essential oil. Therefore, the essential oil of this plant is a promising alternative in the search for new compounds capable of decreasing oxidative stress and treating mycobacteriosis.

Branchial bioenergetics dysfunction as a relevant pathophysiological mechanism in freshwater silver catfish (Rhamdia quelen) experimentally infected with Flavobacterium columnare.

Flavobacterium columnare, the causative agent of columnaris disease, is a serious bacterial disease responsible for causing devastating mortality rates in several species of freshwater fish, leading to severe economic losses in the aquaculture industry. Notwithstanding the enormous impacts this disease can have, very little is known regarding the interaction between the host and bacterium in terms of the mortality rate of silver catfish (Rhamdia quelen), as well its linkage to gill energetic homeostasis. Therefore, we conducted independent experiments to evaluate the mortality rates caused by F. columnare in silver catfish, as well as whether columnaris disease impairs the enzymes of the phosphoryl transfer network in gills of silver catfish and the pathways involved in this inhibition. Experiment I revealed that clinical signs started to appear 72 h post-infection (hpi), manifesting as lethargy, skin necrosis, fin erosion and gill discoloration. Silver catfish began to die at 96 hpi, and 100% mortality was observed at 120 hpi. Experiment II revealed that creatine kinase (CK, cytosolic and mitochondrial) and pyruvate kinase (PK) activities were inhibited in silver catfish experimentally infected with F. columnare, while no significant difference was observed between experimental and control groups with respect to adenylate kinase activity. Activity of the branchial sodium-potassium pump (Na+, K+-ATPase) was inhibited while reactive oxygen species (ROS) and lipid peroxidation levels were higher in silver catfish experimentally infected with F. columnare than in the control group at 72 hpi. Based on these data, the impairment of CK activity elicited by F. columnare caused a disruption in branchial energetic balance, possibly reducing ATP availability in the gills and provoking impairment of Na+, K +ATPase activity. The inhibition of CK and PK activities appears to be mediated by ROS overproduction and lipid peroxidation, both of which contribute to disease pathogenesis associated with branchial tissue.

Diphenyl Diselenide and Clotrimazole Co-loaded into Eudragit® RS 100 Nanocapsules Formulation Has Superior Antioxidant Potential and Promising Anti-candida Activity

Abstract In the current study, nanocapsules (NC) formulations containing a co-load of clotrimazole (C), a highly prescribed antifungal drug, and diphenyl diselenide [(PhSe)2], an organoselenium compound with a promising scope of pharmacological actions, were prepared. Formulations were characterized as well as the potential toxicity, antioxidant action, and antifungal effect were assessed using in vitro techniques. The NCs were prepared employing Eudragit® RS 100 as polymeric wall and medium chain triglycerides or virgin coconut oil (CO) as core. All NC suspensions had pH around acid range, compound content close to theoretical value (1 mg/mL/drug), average diameter in nanometric range, positive values of zeta potential as well as high encapsulation efficacy and mucoadhesive property. Physicochemical stability was performed over a 30-day period and showed no modification in the aforementioned parameters to all samples. Preliminary screening of toxicological potential performed by the hen's egg test chorioallantoic membrane technique classified the formulations as non-irritant. The DPPH radical assay revealed that nanoencapsulated compounds had superior antioxidant action in comparison to their free forms (concentration range tested 1.0-25.0 µg/mL). Importantly, the formulation composed of CO and containing C and (PhSe)2 showed the highest antioxidant potential and was selected for further investigation regarding antifungal effect against some Candida spp strains. Results of in vitro antifungal assay demonstrated that the C and (PhSe)2 co-encapsulation had a minimum inhibitory concentration (MIC) values around 60. Thus, our study supplies additional data about advantages achieved by encapsulating active compounds.

Oxidative stress and antioxidant responses in Nile tilapia Oreochromis niloticus experimentally infected by Providencia rettgeri.

Bacterial diseases are one of the major problems in freshwater fish culture and have been linked to significant losses and high mortality rate. In this study, Nile tilapia Oreochromis niloticus was infected by Providencia rettgeri to evaluate the oxidative stress and antioxidant responses in the fish tissues. Juvenile Nile tilapia was divided into two groups, as follow: control (uninfected) and experimentally infected with 100 µL of P. rettgeri suspension containing 2.4 × 107 viable cells/fish, and the liver and kidney tissues were collected on days 7 and 14 post-infection (PI). Liver and kidney ROS and lipid peroxidation levels were high in infected fish on day 14 PI compared to control group, while superoxide dismutase activity was lower in liver (days 7 and 14 PI) and kidney (day 14 PI) compared to their respective control groups. Liver and kidney antioxidant capacity against peroxyl radicals, non-proteic, and proteic thiols levels was lower in infected tilapia on day 14 PI compared to control group. Based on these results, P. rettgeri infection may elicit oxidative damage via increased ROS production, decreased ROS elimination and inhibits enzymatic and non-enzymatic antioxidant defense systems; which may contribute directly to disease pathophysiology of infected animals.

The disturbance of antioxidant/oxidant balance in fish experimentally infected by Aeromonas caviae: Relationship with disease pathophysiology.

Aeromonas caviae is a Gram-negative bacterium rarely found in fish but it can be associated to high mortality of infected animals. The disease pathogenesis in fish associated to liver and kidney lesions directly linked to the initiation and progression of the disease remains poorly understood. Thus, the aim of this study was to evaluate whether A. caviae infection causes oxidative stress in liver and kidney of silver catfish Rhamdia quelen, and its involvement in disease pathogenesis. Reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) levels increased in liver and kidney of fish experimentally infected by A. caviae compared to the control uninfected group. On the other hand, non-protein sulfhydryl (NPSH) levels decreased in both tissues of infected animals, while the glutathione S-transferase (GST) activity decreased only in the hepatic tissue. No difference was observed between groups in both tissues regarding superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) activities and glutathione (GSH) levels. In summary, the disturbance of hepatic and renal antioxidant/oxidant equilibrium contributes to the pathophysiology of the disease in fish experimentally infected by A. caviae.

Antimicrobial activity of Melaleuca alternifolia nanoparticles in polymicrobial biofilm in situ.

Microbial biofilms represent a challenge in the treatment of infections, due to the low efficacy of the antimicrobials. This study evaluated the antimicrobial effect of nanoparticles of Melaleuca alternifolia (TTO) in dental biofilm. Thirty-eight volunteers used an oral device in situ in situ including four bovine enamel specimens for 07 days. From the fifth day four solutions were applied randomly for each specimen: Physiological Saline Solution (0.85% NaCl) (C+), Chlorhexidine 0.12% (CHX), M. alternifolia oil 0.3% (TTO), and a nanoparticle solution of 0.3% M. alternifolia oil (NPTTO). The nanoparticles of TTO were characterized for pH, IPD, medium size, zeta potential and Transmission Electron Microscopy. Antimicrobial activity was evaluated by viable microorganisms count and the structure of the biofilm by atomic force microscopy. The NPTTO presented pH 6.4, particle diameter of 197.9 ± 1 nm, polydispersion index of 0.242 ± 0.005, zeta potential of -7.12 mV and ±0:27 spherical shape. The C+ resulted in 100% of bacterial vitality, while CHX, TTO and NPTTO showed 34.2%, 51.4% and 25.8%, respectively. The AFM images showed biofilms with an average roughness of 350 nm for C+, 275 nm for CHX, 500 nm for TTO and 100 nm for NPTTO. The NPTTO demonstrated excellent antimicrobial activity in the biofilm formed in situ and will possibly be used in future for the treatment/prevention of oral biofilms.

Xanthine oxidase activity affects pro-oxidative and pro-inflammatory profiles in spleen of silver catfish experimentally infected with Aeromonas caviae.

Several pieces of evidence have linked the involvement of xanthine oxidase (XO), a source of uric acid and reactive oxygen species (ROS), to pro-oxidative and pro-inflammatory effects observed during bacterial fish diseases. Thus, the aim of this study was to evaluate whether upregulation of splenic XO activity contributes to disease pathogenesis of Aeromonas caviae infection, as well as whether it may be considered a pathway involved in ROS and nitric oxide (NO) production. XO activity increased in the spleen of infected animals, as did the splenic levels of uric acid, ROS and metabolites of nitric oxide (NOx), compared to the uninfected control group. Based on this evidence, upregulation of splenic XO activity induces pro-oxidant and pro-inflammatory profiles in the spleen of fish infected by A. caviae due to excessive formation of uric acid. Moreover, excessive uric acid induces the release of pro-inflammatory mediators, such as ROS and NOx, which contribute to disease pathophysiology. In summary, upregulation of XO activity may be considered a pathway involved in ROS and NOx production.

Antimicrobial and antibiofilm activities of nanoemulsions containing Eucalyptus globulus oil against Pseudomonas aeruginosa and Candida spp.

Candida species are the main responsible microorganisms for causing fungal infections worldwide, and Candida albicans is most frequently associated with infectious processes. Pseudomonas aeruginosa is a gram-negative bacterium commonly found in immunocompromised patients. The infection persistence caused by these microorganisms is often related to antimicrobial resistance and biofilm formation. In this context, the objective of the present study was to prepare and characterize nanoemulsions containing Eucalyptus globulus oil and to verify its antimicrobial and antibiofilm activities against P. aeruginosa and Candida spp. The nanoemulsions had a size of approximately 76 nm, a polydispersity index of 0.22, a zeta potential of - 9,42 mV and a pH of approximately 5.0. The E. globulus oil was characterized by gas chromatography, being possible to observe its main components, such as 1-8-Cineol (75.8%), p- Cymene (7.5%), α-Pinene (7.4%) and Limonene (6.4%). The antimicrobial activity of the nanoemulsion was determined from the macrodilution tests and the cell viability curve, where the minimum fungicidal concentration of 0.7 mg/mL for C. albicans and 1.4 mg/mL for C. tropicalis and C. glabrata were obtained. However, the nanoemulsions did not present antimicrobial activity against P. aeruginosa, since it contains only 5% of the oil, being ineffective for this microorganism. The nanoencapsulated oil action against the formed biofilm was evaluated by atomic force microscopy and calcofluor staining, and the nanoemulsion was more efficient for two of the three Candida species when compared to free oil.

Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na+, K+-ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na+, K+-ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis.

In vitro susceptibility of the oomycete Pythium insidiosum to metallic compounds containing cadmium, lead, copper, manganese or zinc.

Pythium insidiosum is an aquatic oomycete that causes pythiosis, an important and severe disease of difficult treatment that affects humans, domestic and wild animals. This infection is often described in horses in Brazil and humans in Thailand. In clinical practice, we have observed many cases that do not respond to available therapies, indicating the need to explore alternative therapeutic approaches. In this sense, studies using metal compounds in conjunction with available antimicrobial agents have been demonstrated greater antimicrobial activity. Thus, in this research, we tested in vitro activities of metallic compounds containing cadmium, lead, copper, manganese, or zinc against 23 isolates of P. insidiosum. The assays were performed by broth microdilution based on CLSI M38-A2 document. The minimum inhibitory and fungicidal concentrations were established for all isolates. Copper acetate and cadmium acetate showed the highest inhibitory effects, with minimal inhibitory concentration ranging from 4-64 µg/ml and 16-256 µg/ml, respectively. The mean geometric for minimal fungicidal concentrations were, respectively, 26 µg/ml and 111.43 µg/ml for copper acetate and cadmium acetate. These results suggest that copper and cadmium can inhibit P. insidiosum growth, highlighting the greater inhibitory activity of copper acetate. In addition, our results propose that copper and/or cadmium compounds can be used in upcoming researches to formulate effective new complexed drugs against P. insidiosum in in vitro and in vivo experimental models.