Renata P. Raffin

Trypanocidal action of tea tree oil (Melaleuca alternifolia) against Trypanosoma evansi in vitro and in vivo used mice as experimental model

This study aimed to evaluate the Trypanosoma evansi susceptibility to tea tree oil (TTO - Melaleuca alternifolia) and tea tree oil nanocapsules (TTO nanocapsules) in vitro and in vivo tests. In vitro, we observed a mortality curve of trypomastigotes proportional to dose, i.e., the TTO and TTO nanocapsules have trypanocidal effect. Treatment with TTO in vivo was assessed in experiments (I and II). For Experiment I, T. evansi infected mice were treated with TTO and/or combinations of essential oil with chemotherapy (diminazene aceturate - D.A.). Treatment with TTO at a dose of 1mLkg(-1) was able to extend animal longevity, but had no curative efficacy. However, when TTO was combined with D.A. a disease curative efficacy of 100% for disease was observed, a much better result than the D.A. treatment (33.3%). In Experiment II, T. evansi infected mice were treated with TTO nanocapsules with doses of 0.3, 0.6 and 0.9mLkg(-1). Animals treated with 0.9mLkg(-1) showed higher longevity however without curative effect. Active compounds present in natural products, such as M. alternifolia, may potentiate the treatment of trypanosomosis when associated with other trypanocidal drugs.

Influence of tea tree oil (Melaleuca alternifolia) on the cattle tick Rhipicephalus microplus

The aim of this study was to verify the influence of tea tree oil (TTO) (Melaleuca alternifolia) tested in its pure and nanostructured (TTO nanoparticles) forms on the reproduction of female Rhipicephalusmicroplus. For our purpose, female ticks were collected from naturally infected animals and treated in vitro with TTO (1, 5, and 10xa0%) and TTO nanoparticles (0.075, 0.375, and 0.75xa0%). In order to validate the tests, they were performed in triplicate using positive (amitraz) and negative (untreated) controls. It was possible to observe that pure TTO (5 and 10xa0%) and TTO nanoparticles (0.375 and 0.75xa0%) showed 100xa0% reproductive inhibition on female ticks. Additionally, pure TTO (1xa0%) also showed an acaricide effect (70xa0%), similarly to the positive control (78.3xa0%). This is the first study demonstrating the activity of pure TTO and TTO nanoparticles on female ticks. Therefore, based on these results, we were able to show that both forms and all concentrations of M. alternifolia affected tick reproduction by inhibiting egg laying and hatching. We were also able to show that TTO nanoparticles potentiated the inhibitor effect of pure TTO on the reproduction of R. microplus.

Influence of Melaleuca alternifolia oil nanoparticles on aspects of Pseudomonas aeruginosa biofilm

The Pseudomonas aeruginosa is a gram-negative bacillus and frequent cause of infection. This microorganism is resistant intrinsically to various drugs. The P. aeruginosa is associated with the biofilm formation, which causes worsen the prognosis and difficulty the treatment. The influence of Melaleuca alternifolia oil or tree of tee oil (TTO) and TTO nanoparticles on adhesion of P. aeruginosa in buccal epithelial cells was investigated. Also was determined the antimicrobial and antibiofilm activity against this microorganism. The TTO nanoparticles were produced by deposition of preformed polymer and the physic-chemical properties of nanoparticles were measured by electrophoresis and dynamic light scattering. The characterization of nanoparticle showed acceptable values for diameter and zeta potential. The evaluation of antimicrobial and antibiofilm activity against P. aeruginosa PAO1 was performed by microdilution indicating the minimal inhibitory concentration, and the potential antibiofilm. It was verified the action on virulence factors such the motility, besides the influence on adhesion in buccal epithelial cells. Both oil and nanoparticles showed a decrease in adhesion of microorganisms to buccal cells, decrease of biofilm and interfering on P. aeruginosa PAO1 motility. The nanostructuration of TTO, shows be a viable alternative against formed biofilm microorganisms.

Insecticidal and repellent effects of tea tree and andiroba oils on flies associated with livestock

This study aimed to evaluate the insecticidal and repellent effects of tea tree, Melaleuca alternifolia (Myrtales: Myrtaceae), and andiroba, Carapa guianensis (Sapindales: Meliaceae), essential oils on two species of fly. For in vitro studies, free‐living adult flies were captured and reared in the laboratory. To evaluate the insecticidal effects of the oils, adult flies of Haematobia irritans (L.) and Musca domestica L. (both: Diptera: Muscidae) were separated by species in test cages (nu2009=u200910 per group), and subsequently tested with oils at concentrations of 1.0% and 5.0% using a negative control to validate the test. Both oils showed insecticidal activity. Tea tree oil at a concentration of 5.0% was able to kill M.u2009domestica with 100.0% efficacy after 12u2009h of exposure. However, the effectiveness of andiroba oil at a concentration of 5.0% was only 67.0%. The insecticidal efficacy (100.0%) of both oils against H.u2009irritans was observed at both concentrations for up to 4u2009h. The repellency effects of the oils at concentrations of 5.0% were tested in vivo on Holstein cows naturally infested by H.u2009irritans. Both oils demonstrated repellency at 24u2009h, when the numbers of flies on cows treated with tea tree and andiroba oil were 61.6% and 57.7%, respectively, lower than the number of flies on control animals. It is possible to conclude that these essential oils have insecticidal and repellent effects against the species of fly used in this study.

In vivo bactericidal effect of Melaleuca alternifolia essential oil against Aeromonas hydrophila: Silver catfish (Rhamdia quelen) as an experimental model

Aeromonas hydrophila is one of the main causative agent of high mortality and significative economic losses in aquaculture and has become increasingly resistant to conventional antibiotics. One feasible alternative to control and treat it is the use of essential oils. This study aimed to evaluate A.xa0hydrophila susceptibility to tea tree oil (TTO-Melaleuca alternifolia) inxa0vivo, and the effect of this treatment. Inxa0vivo tests were performed using silver catfish (Rhamdia quelen) as the experimental model. Silver catfish were treated with TTO at 25 and 50xa0μL/L for seven days before infection. After seven days, the fish were inoculated with A.xa0hydrophila via intramuscularly. Treatment with TTO at 50xa0μL/L was able to extend longevity of infected fish, and showed 88% of therapeutic success, even though it did not show curative efficacy. TTO treatment was not toxic under these tested concentrations, since biomarkers of hepatic and renal functions were not affected, and the concentration of 50xa0μL/L was able to prevent increased levels of aspartate aminotransferase. There was no significative differences regarding hematological parameters (pxa0<xa00.05). Treatment with TTO 50xa0μL/L was able to reduce histopathological alterations usually caused by this type of bacteria in the gills, but it was unable to reduce hepatic histopathological alterations. Our results showed, for the first time, that TTO has high activity against A.xa0hydrophila and proved to be a natural alternative to prevent and control this pathogen.

Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi

This study aimed to evaluate the effect of tea tree oil (TTO - Melaleuca alternifolia) on hepatic and renal functions, and the immune response of rats infected by Trypanosoma evansi. A pilot study has shown that rats treated with TTO orally (1u2009mlu2009kg(-1)) had increased survival rate without curative effect. In order to verify if increased longevity was related to a better immune response against T. evansi when using tea tree oil, a second experiment was conducted. Thus, twenty-four rats were divided into four groups. The groups A and B were composed of uninfected animals, and the groups C and D had rats experimentally infected by T. evansi. Animals from the groups B and D were treated orally with TTO (1u2009mlu2009kg(-1)) for three days. Blood samples were collected to verify humoral response analysis for immunoglobulins (IgA, IgM, IgE, and IgG) and cytokines (TNF-α, INF-γ, IL-1, IL-6, IL-4, and IL-10) at days 0, 3, 5 and 15 post-infection (PI). TTO treatment caused changes in the immunoglobulins and cytokines profile, as well as the course of T. evansi infection in rats. It was found that the TTO was not toxic, i.e., hepatic and renal functions were not affected. Therefore, it is possible to conclude that TTO influences the levels of inflammatory mediators and has trypanocidal effect, increasing life expectancy of rats infected by T. evansi.

In vitro activity of essential oils of free and nanostructured Melaleuca alternifolia and of terpinen-4-ol on eggs and larvae of Haemonchus contortus.

Haemonchus contortus is one of the major gastrointestinal nematodes responsible for significant economic and production losses of sheep. Diseases caused by this species lack effective anthelmintic products, and the search for new compounds to replace synthetic anthelmintics has been extensive. The present investigation assesses the in vitro activity of the essential oil of melaleuca (Melaleuca alternifolia), both free (TTO) and nanostructured (nanoTTO), and terpinen-4-ol (terp-4-ol) on eggs and larvae of H. contortus. Tests of egg hatching (EHT) and inhibition of larval migration (LMIT) were used to assess the in vitro efficacy of TTO, nanoTTO and terp-4-ol. Using EHT, at a concentration of 3.5 mg/ml, 100% inhibition occurred using TTO and terp-4-ol, with LC50 values of 0.43 and 0.63 mg/ml, and LC90 values of 1.75 mg/ml and 3.12 mg/ml, respectively. NanoTTO had lower activity, with 82.6% inhibition at the same concentration. Using LMIT, TTO and nanoTTO had a similar activity with 88.0% and 84.8% inhibition, respectively, at a concentration of 56 mg/ml. Terp-4-ol had a greater effect on larvae, with 85.7% inhibition at a concentration of 56 mg/ml and 82.4% at 3.5 mg/ml, demonstrating high activity at the lowest concentration tested. Therefore, the results indicate that all substances tested showed ovicidal and larvicidal activity against H. contortus. TTO, terp-4-ol and, mainly, nanoTTO may be targeted in in vivo studies, besides being a promising line of research into the control and treatment of veterinary important helminths.

Evaluation of antimicrobial activity of glycerol monolaurate nanocapsules against American foulbrood disease agent and toxicity on bees

The American Foulbrood Disease (AFB) is a fatal larval bee infection. The etiologic agent is the bacterium Paenibacillus larvae. The treatment involves incineration of all contaminated materials, leading to high losses. The Glycerol Monolaurate (GML) is a known antimicrobial potential compound, however its use is reduced due to its low solubility in water and high melting point. The nanoencapsulation of some drugs offers several advantages like improved stability and solubility in water. The present study aimed to evaluate the antimicrobial activity against P. larvae and the toxicity in bees of GML nanoparticles. The nanocapsules were produced and presented mean diameter of 210xa0nm, polydispersity index of 0.044, and zeta potential of -23.4xa0mV demonstrating the acceptable values to predict a stable system. The microdilution assay showed that it is necessary 142 and 285xa0μg/mL of GML nanocapsules to obtain a bacteriostatic and bactericidal effect respectively. The time-kill curve showed the controlled release of compound, exterminating the microorganism after 24xa0h. The GML nanocapsules were able to kill the spore form of Paenibacillus larvae while the GML do not cause any effect. The assay in bees showed that the GML has a high toxicity while the GML nanoparticles showed a decrease on toxic effects. Concluding, the formulation shows positive results in the action to combat AFB besides not causing damage to bees.

Melaleuca alternifolia nanoparticles against Candida species biofilms

Candida infection is an important cause of morbidity and mortality on immunosuppressed patients. This growing trend has been associated with resistance to the antimicrobial therapy and the ability of microorganism to form biofilms. TTO oil is used as antimicrobial which shows antibiofilm activity against Candida species. However, it presents problems due to its poor solubility and high volatility. The present study aimed to evaluate inxa0vitro antibiofilm activity of TTO nanoparticles against many Candida species. It was performed the characterization of the oil and nanoparticles. The levels of exopolysaccharides, proteins, and the biomass of biofilms were measured. The chromatographic profile demonstrated that the TTO oil is in accordance with ISO 4730 with major constituents of 41.9% Terpinen-4-ol, 20.1% of γ-Terpinene, 9,8% of α-Terpinene, and 6,0% of 1,8-Cineole. The TTO nanoparticles showed pH of 6.3, mean diameter of 158.2xa0±xa02xa0nm, polydispersion index of 0.213xa0±xa00.017, and zeta potential ofxa0-8.69xa0±xa00.80xa0mV. The addition of TTO and its nanoparticles represented a significant reduction of biofilm formed by all Candida species, as well as a reduction of proteins and exopolysaccharides levels. It was possible to visualize the reduction of biofilm in presence of TTO nanoparticles by Calcofluor White method.

Simultaneous separation and sensitive detection of naringin and naringenin in nanoparticles by chromatographic method indicating stability and photodegradation kinetics

A simple, sensitive, precise and linear method by liquid chromatography was established for simultaneous determination and quantification of naringin and naringenin in polymeric nanoparticles. The method results in excellent separation in <11 min and with a peak purity of both flavonoids. The analyses were performed using a C18 column (4.6 × 150 mm, 5 µm), at a 1 mL/min flow rate. The mobile phase consisted of a gradient of acetonitrile-water (pH 4.0; v/v) at a temperature of 25°C. The nanoparticles were prepared according to the method of interfacial deposition of a pre-formed polymer. The method were validated in compliance with guidelines, and was found to be linear in the 1-40 µg/mL concentration range for both naringin and naringenin (r > 0.99). Repeatability was determined at three concentration levels, obtaining an RSD (%) <0.9%, and the accuracy of the method was >98%. The photodegradation kinetics was determined for naringin; the coefficient that best represents degradation was of first order and naringenin presented a zero-order kinetics. To our knowledge, a rapid and sensitive method for naringin and naringenin in polymeric nanoparticles has not been published elsewhere and this method is applicable to simultaneous evaluation of flavonoids.