Extracts of Hesperozygis ringens (Benth.) Epling: in vitro and in vivo antibacterial activity against fish pathogenic bacteria.

AIMS: This study investigated the in vitro antibacterial activity of Hesperozygis ringens (Benth.) Epling leaf extracts against fish pathogenic bacteria, as well as the in vivo activity of the most active extract in silver catfish (Rhamdia quelen) experimentally infected with Aeromonas hydrophila. Moreover, the chemical composition of the extract used in the survival assay was evaluated. METHODS AND RESULTS: Only hexane extract (HEHR) showed in vitro antibacterial activity (MIC and MBC ranging from 1600 to 3200 µg ml-1 ) against clinical isolates of A. hydrophila, Raoultella ornithinolytica and Citrobacter freundii, obtained from naturally infected silver catfish, and A. hydrophilaATCC 7966. The major compound of the volatile fraction of HEHR was determined as pulegone. HEHR promoted a 93·33% relative survival rate of silver catfish experimentally infected with A. hydrophila 7 days after a single therapeutic bath at 30 mg l-1 , while florfenicol at 4 mg l-1 , which promoted a 60% relative survival rate. CONCLUSIONS: The antibacterial activity of H. ringens (Benth.) Epling leaf extracts seems to be related to phytochemicals of apolar character, since HEHR promoted better survival rate of infected animals than florfenicol. SIGNIFICANCE AND IMPACT OF THE STUDY: The HEHR has potential to be used in the control and treatment of bacterial infections in organic aquaculture.

In vitro effects of plant essential oils on non-specific immune parameters of red drum, Sciaenops ocellatus L.

Phytochemicals such as plant essential oils (EOs) have been reported to favour various activities in the innate immune system of fish. Thus, the aim of this study was to verify the in vitro effect of three different plant EOs (Ocimum americanum, Cymbopogon flexuosus and Melaleuca alternifolia) on non-specific immune parameters and erythrocyte osmotic fragility of red drum, Sciaenops ocellatus. Concentrations of each plant EO evaluated in preparations of head-kidney macrophages, blood leucocytes and blood plasma were as follows: 0.0 (control), 1.0, 2.0, 4.0, 8.0, and 16.0 µg/ml. Red drum head-kidney macrophages significantly increased extracellular superoxide anion production when exposed (20 h) to O. americanum EO (1.0-8.0 µg/ml) and C. flexuosus EO (2.0 and 4.0 µg/ml). The respiratory burst of blood leucocytes (NBT test) significantly increased in all concentrations when compared to the respective control group, for all EOs. At the highest concentration (16.0 µg/ml), C. flexuosus EO significantly inhibited the haemolytic activity of complement system in red drum blood after 1 h exposure. None of the tested concentrations significantly altered plasma lysozyme activity or erythrocyte osmotic fragility after exposing (1 h) red drum whole blood to each EO. This study demonstrated that these plant EOs are capable of triggering superoxide anion production in red drum leucocytes (head-kidney macrophages and/or blood leucocytes). In vivo studies are warranted to address their potential as immunostimulants in the diet of red drum and other aquacultured species.

Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model.

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

Effects of anesthesia with the essential oil of Ocimum gratissimum L. in parameters of fish stress / Efeitos da anestesia com o óleo essencial de Ocimum gratissimum L. em parâmetros de estresse de peixes

RESUMO: The effects of anesthesia with the essential oil of Ocimum gratissimum (EOO) in parameters of stress after handling were investigated in silver catfish (Rhamdia quelen). EOO was obtained from the aerial parts by hydrodistillation. Juveniles were anesthetized with 70 or 300 mg L-1 EOO and submitted to air exposure for 1 minute. The fishes were sampled immediately or transferred to anesthetic-free aquaria until sampling. In the first experiment, juveniles had their blood collected at 0, 1, 4, and 8 h after handling to assay plasma cortisol and blood glucose levels. The unanesthetized animals were restrained manually for blood collection. In the second experiment, water samples of the recovery aquaria were collected to evaluate net ion fluxes at 0 - 4 h and 4 - 8 h. Water and ethanol controls were also performed under the same conditions. The results showed that the cortisol levels did not differ among the treatments. Hyperglycemia was verified in fish exposed to 70 and 300 mg L-1 EOO at 1 h and 4 h after handling. After 8 h, cortisol and glucose concentrations were lower or similar than those from immediately after handling for all treatments. EOO anesthesia prevented Na+ efflux observed in the control groups in both flux periods. There were net Cl- and K+ effluxes at 0 - 4 h and influxes at 4 - 8 h after handling in most treatments, and these fluxes did not differ among the treatments. The results suggest that EOO did not impair stress recovery and did not act as an additional handling stressor in silver catfish.

RESUMO: Os efeitos da anestesia com o óleo essencial de Ocimum gratissimum (EOO) em parâmetros de estresse após manuseio foram investigados em jundiás (Rhamdia quelen). EOO foi obtido a partir das partes aéreas por hidrodestilação. Os juvenis foram anestesiados com 70 ou 300 mg L-1 de EOO e expostos ao ar por 1 minuto. Os peixes foram amostrados imediatamente ou transferidos para aquários sem anestésico até amostragem. No primeiro experimento, os juvenis tiveram seu sangue coletado em 0, 1, 4, e 8 h após manuseio para avaliar os níveis de cortisol e glicemia. Os animais não anestesiados foram contidos manualmente para coleta sanguínea. No segundo experimento, amostras de água foram coletadas do aquário de recuperação dos animais para avaliação do fluxo iônico entre 0 - 4 h e 4 - 8 h. Grupos controles em água e etanol também foram realizados sobre as mesmas condições. Os resultados demonstraram que os níveis de cortisol não diferiram entre os tratamentos. Hiperglicemia foi detectada em peixes expostos a 70 e 300 mg L-1 de EOO em 1 h e 4 h após o manuseio. Após 8 h, os teores de cortisol e glicose foram menores ou similares aqueles imediatamente após o manuseio para todos os tratamentos. A anestesia com EOO preveniu o efluxo de Na+ observado para os grupos controle em ambos os períodos avaliados. Ocorreram efluxos de Cl- and K+ entre 0 - 4 h e influxos entre 4 - 8 h após o manuseio para a maioria dos tratamentos, e estes eventos não diferiram entre os tratamentos. Os resultados sugerem que o EOO não prejudica a recuperação do animal frente ao evento estressor ou atua como estressor adicional ao manuseio em jundiás.

Plant essential oils against Aeromonas hydrophila: in vitro activity and their use in experimentally infected fish.

AIMS: The aims of this study were to investigate the in vitro antibacterial activity of the essential oils (EOs) of Hesperozygis ringens (HREO), popularly known as 'espanta-pulga' and two different species of basil, Ocimum gratissimum (OGEO) and Ocimum americanum (OAEO), as well as, the potential of these products to be used in silver catfish (Rhamdia quelen) infected with Aeromonas hydrophila. METHODS AND RESULTS: OGEO and HREO showed better antibacterial activity in vitro. Subinhibitory concentrations of all EOs inhibited haemolysis caused by Aer. hydrophila in fish erythrocytes (100% reduction for OAEO at 100 µg ml(-1) and more than 90% for HREO and OGEO at 150 µg ml(-1) ). However, OAEO and HREO showed the best survival results (75 and 70% respectively) after their use as treatment (therapeutic baths-1 h daily/5 days) in silver catfish experimentally infected with Aer. hydrophila. A second in vivo assay using healthy fish was conducted to verify the potential of the EOs (preventive baths-1 h daily/5 days) to promote fish survival. Fish exposed to HREO and OAEO and their diluent (ethanol) showed significant lower haematocrit values and higher complement system activity compared to control. Plasma cortisol level was significantly higher in the groups exposed to both EOs. There was no significant difference in survival of silver catfish challenged with Aer. hydrophila after preventive baths with HREO, OAEO and control group. CONCLUSIONS: All tested EOs showed in vitro antibacterial properties against Aer. hydrophila and HREO and OAEO showed potential to be used in the treatment of infected fish. SIGNIFICANCE AND IMPACT OF THE STUDY: These products can be used in aquaculture as therapeutic and prophylactic agents against fish pathogens, with antimicrobial and/or immunostimulant properties.

Composition and evaluation of the antimicrobial activity of the essential oil of Senecio selloi Spreng DC / Composição e avaliação da atividade antimicrobiana do óleo essencial de Senecio selloi Spreng DC

The essential oil of the aerial parts of Senecio selloi Spreng. DC. was extracted by hydrodistillation and analyzed by GC/MS. Nineteen compounds were identified, representing 99.9% of the total. The main compounds were found to be sesquiterpene hydrocarbons (71.3%), most of them with a bisabolane skeleton (59.4%). The major constituent was α-zingiberene (54%), followed by monoterpene α-isolimonene (16%). The essential oil was also tested against two Gram-positive and two Gram-negative bacterial species, three yeasts, and an algae. From the strains assayed, only Bacillus subtilis ATCC 6633 showed susceptibility (MIC and MBC = 4400 µg/mL) to the essential oil.

O óleo essencial das partes aéreas de Senecio selloi Spreng DC. foi extraído por hidrodestilação e analisado por CG/EM. Dezenove constituintes foram identificados, representando 99,9% do total. Os principais compostos fornecidos foram sesquiterpenos hidrocarbonetos (71,3%), a maioria destes com esqueleto bisabolano (59,4%). O constituinte majoritário foi a-zingibereno (54%), seguido do monoterpeno a-isolimoneno (16%). O óleo essencial foi testado contra duas cepas Gram-positivas e duas Gram-negativas, três fungos e uma alga. De todas as linhagens testadas somente Bacillus subtilis ATCC 6633 mostrou suscetibilidade (CIM e CBM = 4400 µg/mL) para o óleo essencial.

Biological studies on Brazilian plants used in wound healing.

AIM OF THE STUDY: n-Hexanic and ethanolic extracts from twelve plants (Brugmansia suaveolens Brecht. et Presl., Eupatorium laevigatum Lam., Galinsoga parviflora Cav., Iresine herbstii Hook., Kalanchöe tubiflora Hamet-Ahti, Petiveria alliacea L., Pluchea sagittalis (Lam.) Cabrera, Piper regnellii DC., Schinus molle L., Sedum dendroideum Moç et Sessé ex DC., Waltheria douradinha St. Hill., Xanthium cavanillesii Schouw.) used in traditional South Brazilian medicine as wound healing agents were investigated in various biological assays, targeting different aspects in this complex process. MATERIALS AND METHODS: The extracts were investigated on NF-kappaB DNA binding, p38alpha MAPK, TNF-alpha release, direct elastase inhibition and its release as well as on caspase-3. Fibroblasts migration to and proliferation into the wounded monolayers were evaluated in the scratch assay, the agar diffusion test for antibacterial and the MTT assay for cytotoxic effects. RESULTS: The hydrophilic extracts from Galinsoga parviflora, Petiveria alliacea, Schinus molle, Waltheria douradinha and Xanthium cavanillesii as well as the lipophilic extract of Waltheria douradinha turned out to be the most active ones. CONCLUSIONS: These results increase our knowledge on the wound healing effects of the investigated medicinal plants. Further studies are necessary to find out the effective secondary metabolites responsible for the observed effects.

Sesquiterpene peroxides from Senecio selloi and Eupatorium rufescens.

Two bisabolane-type sesquiterpenes, zingiberene-3,6-beta-endoperoxide (1) and zingiberene-3,6-alpha-endoperoxide (2) have been isolated from the aerial parts of Senecio selloi and Eupatorium rufescens. Their structures were determined by spectroscopic methods. Both compounds show schizonticidal activity against Plasmodium falciparum (EC50 = 10 microg/ml).