Effect of polycaprolactone nanocapsules loaded with essential oils on biofilm formation by Staphylococcus aureus strains isolated from bovine mastitis cases

Abstract Bovine infectious mastitis is largely resistant to antibacterial treatment, mainly due to mechanisms of bacterial resistance in the biofilms formed by Staphylococcus aureus. Melaleuca (MEO) and citronella essential oils (CEO) are promising agents for reducing or eliminating biofilms. Free melaleuca oil presented a medium Minimum Inhibitory Concentration (MIC) of 0.625% and a Minimum Bactericidal Concentration (MBC) of 1.250%, while free citronella oil showed medium MIC and MBC of 0.313%. Thus, free CEO and MEO demonstrate bacteriostatic and bactericidal potential. We generated polymeric nanocapsules containing MEO or CEO and evaluated their efficacy at reducing biofilms formed by S. aureus. Glass and polypropylene spheres were used as test surfaces. To compare the responses of free and encapsulated oils, strains were submitted to 10 different procedures, using free and nanoencapsulated essential oils (EOs) in vitro. We observed no biofilm reduction by MEO, free or nanoencapsulated. However, CEO nanocapsules reduced biofilm formation on glass (p=0.03) and showed a tendency to diminish biofilms on polypropylene (p=0.051). Despite nanoencapsulated CEO reducing biofilms in vitro, the formulation could be improved to modify the CEO component polarity and, including MEO, to obtain more interactions with surfaces and the biofilm matrix

In vitro anti-Candida activity of Melaleuca cajuputi extracts

Aims@#The rise of drug-resistant infectious diseases worldwide has spurred experts' interest in developing safe and effective alternative medicine. Melaleuca cajuputi extracts have been shown to exhibit antimicrobial activity in vitro against various bacterial species. This study evaluated the antimicrobial activity of local M. cajuputi leaf extracts (MCEs) against Candida albicans.@*Methodology and results@#Phytoconstituents of aqueous and ethanolic MCEs were screened conventionally using chemical tests. Broth microdilution assay and scanning electron microscope (SEM) were performed to study the anti-Candida activity of the extracts. Both MCEs contained terpenoids, phenols, flavonoids and tannins. Aqueous and ethanolic MCEs showed good fungicidal activity against the tested organism with minimum inhibitory concentration (MIC) values of 50 mg/mL and 25 mg/mL, respectively and a minimum fungicidal concentration (MFC) to MIC ratio of less or equal to 2. Scanning electron micrographs revealed yeast cell surface morphology alterations when treated with both MCEs at 1× MIC.@*Conclusion, significance and impact of study@#In conclusion, MCEs have anti-Candida properties and thus, M. cajuputi extract could be an excellent potential source of natural antimicrobial agents for disease remedies.

Melaleuca armillaris essential oil as an odor reducer in intestinal ostomy bags: a semi-experimental study / Aceite esencial de Melaleuca armillaris reductor de olores en bolsas de ostomía intestinal: estudio cuasi-experimental / Óleo essencial de Melaleuca armillaris redutor de odor em bolsas de estomia intestinal: estudo quase experimental

ABSTRACT Objective: Evaluate the effect of essential oil in odor reduction for intestinal ostomy bags. Method: Primary study, semi-experimental, prospective clinical trial with quantitative approach. A product prepared with Melaleuca armillaris leaves was tested in ostomy pouches, with and without effluents, for adhesion and odor control. Instrument: Labeled Magnitude Scale. Results: Colostomized participants, with a mean age of 73 ± 14.94, predominantly males; and informal caregivers, with a mean age of 44 ± 8.98, predominantly females. In the visual evaluation of the oil, 100% of the participants perceived its adherence. Regarding the evaluation of effluent odor by the colostomized, five reported "strong odor" before oil use and, six reported "weak" odor after use (p = 0.005). Five informal caregivers reported "very strong" effluent odor before oil use; and one reported "weak" and nine reported "moderate" after use (p = 0.0025). Conclusion: There was a reduction in the odor of effluents in the ostomy pouches with the essential oil of Melaleuca armillaris at 10%. Application for patent registration in the Brazilian National Institute of Industrial Property (INPI) under no. BR 10 2020 026987 9.

RESUMEN Objetivo: Evaluar el efecto del aceite esencial en la reducción del olor en bolsas de ostomía intestinal. Método: Estudio primario, ensayo clínico cuasi-experimental, prospectivo de tipo cuantitativo. Se evaluó un preparado con hojas de Melaleuca armillaris, probado en bolsas de recogida con y sin efluentes, en cuanto a adherencia y olor. Se aplicó como instrumento la Escala de Magnitud Etiquetada. Resultados: Los participantes colostomizados tenían edad media de 73 años ± 14,94, con predominio del sexo masculino, y los cuidadores informales, edad media de 44 años ± 8,98 y prevalencia mayor para el sexo femenino. En la evaluación visual del aceite, el 100% de los participantes notaron la adherencia. Respecto a la evaluación del olor del efluente por los pacientes colostomizados, cinco reportaron sentir un "olor fuerte" antes de usar el aceite y, después del uso, seis reportaron sentir un olor "débil" (p = 0,005). Cinco cuidadores informales detectaron un olor "muy fuerte" de los efluentes antes de usar el aceite y, después de usarlo, uno sintió un olor "débil" y nueve "moderado" (p = 0,0025). Conclusión: El olor de los efluentes en las bolsas de recolección con el aceite esencial de Melaleuca armillaris se redujo en un 10%. Solicitud de registro de patente ante el INPI bajo el n. BR 10 2020 026987 9.

RESUMO Objetivo: Avaliar o efeito do óleo essencial na redução de odor em bolsas de estomia intestinal. Método: Estudo primário, ensaio clínico quase experimental, prospectivo, com abordagem quantitativa. Preparado com folhas de Melaleuca armillaris, foi testado em bolsas coletoras, sem e com efluentes, quanto à aderência e odor. Instrumento: escala de magnitude rotulada. Resultados: Participantes colostomizados, com média de 73 anos ± 14,94, predomínio do sexo masculino; e cuidadores informais, com média de 44 anos ± 8,98, predomínio do sexo feminino. Na avaliação visual do óleo, 100% dos participantes perceberam a aderência. Em relação à avaliação do odor dos efluentes pelos colostomizados, cinco relataram "odor forte" antes do uso do óleo e, após o uso, seis relataram odor "fraco" (p = 0,005). Cinco cuidadores informais detectaram odor "muito forte" dos efluentes antes do uso do óleo e, após o uso, um "fraco" e nove "moderado" (p = 0,0025). Conclusão: Houve redução do odor dos efluentes nas bolsas coletoras com o óleo essencial de Melaleuca armillaris a 10%. Pedido registro de patente no INPI sob nº. BR 10 2020 026987 9.

Essential oils in pathogen resistance induction of Eucalyptus benthamii Maiden et Cambage / Óleos essenciais na indução de resistência a patógenos em Eucalyptus benthamii Maiden et Cambage

ABSTRACT: This study evaluated the essential oils of Melaleuca alternifolia, Casearia sylvestris and Eugenia uniflora as inducers of defense mechanisms in Eucalyptus benthamii seedlings. Four mL of each oil, with a concentration of 0.75% were sprayed in E. benthamii seedlings and two bioassays were performed, in the first, the essential oils were sprayed and after 30 days, sugars, proteins, peroxidases, phenylalanine ammonia (PAL), and phenols were evaluated and; in the second, seven days after the first analysis, the essential oils were again sprayed and after three days, the same variables were evaluated. The essential oils of M. alternifolia, C. sylvestris, and E. uniflora sprayed had no significant effects on E. benthamii seedlings after 30 days in terms of total sugars, proteins, peroxidase, PAL activity, and phenols. However, when M. alternifolia and E. uniflora essential oils were sprayed seven days after the first analysis with evaluation after 3 days, an increase in total sugars was observed. After these days, all essential oils promoted an increase in protein levels. The oils of E. uniflora and C. sylvestris also increased peroxidase levels. The PAL defense enzyme not showed increased when essential oils were used. The essential oils of M. alternifolia and C. sylvestris had potential as inducers of defense mechanisms on E. benthamii seedlings after 3 days of their application, what it demonstrated not be permanent.

RESUMO: Este trabalho teve como objetivo avaliar os óleos essenciais de Melaleuca alternifolia, Casearia sylvestris e Eugenia uniflora como indutores de mecanismos de defesa em mudas de Eucalyptus benthamii. Quatro mL de cada óleo, com concentração de 0,75%, foram pulverizados em mudas de E. benthamii e foram realizados dois bioensaios. No primeiro foram pulverizados os óleos essenciais e após 30 dias, açúcares, proteínas, peroxidases, fenilalanina amônia (PAL) , e fenóis foram avaliados e; na segunda, sete dias após a primeira análise, os óleos essenciais foram novamente pulverizados e após três dias, as mesmas variáveis foram avaliadas. Os óleos essenciais de M. alternifolia, C. sylvestris e E. uniflora pulverizados não tiveram efeito significativo sobre as mudas de E. benthamii após 30 dias em termos de açúcares totais, proteínas, peroxidase, atividade PAL e fenóis. Entretanto, quando os óleos essenciais de M. alternifolia e E. uniflora foram pulverizados sete dias após a primeira análise com avaliação após três dias, foi observado um aumento nos açúcares totais. Depois desses dias, todos os óleos essenciais promoveram aumento nos níveis de proteínas. Os óleos de E. uniflora e C. sylvestris também aumentaram os níveis de peroxidase. A enzima de defesa PAL não se mostrou aumentada quando óleos essenciais foram usados. Os óleos essenciais de M. alternifolia e C. sylvestris apresentaram potencial como indutores de mecanismos de defesa em mudas de E. benthamii após três dias de sua aplicação, o que demonstrou não ser permanente.

Methyl Eugenol, 1,8-Cineole and Nerolidol Rich Essential Oils with their Biological Activities from three Melaleuca Species Growing in Tarai Region of North India

Abstract The essential oils from the fresh leaves of three Melalecua spp. viz; Melaleuca leucadendron (L.) Melaleuca linariifolia Sm. and Melaleuca bracteata F. Muell. growing in Tarai region of North India were analyzed by a combination of gas chromatography/mass spectrometry. The analysis revealed the presence of several constituents of industrial and pharmacological importance. M. leucadendron essential oil was found to be dominated by E-nerolidol (85.7%) rich chemotype. 1,8-cineole (61.1%) along with significant presence of α-terpineol (12.3%), α-pinene (4.0%), β-myrcene (3.8%), and E-caryophyllene (1.7%) were identified in the essential oil from M. linariifolia Similarly M. bracteata was dominated by the presence of phenylpropanoids viz; methyl eugenol (74.8%) and methyl cinnamate (8.0%). The essential oils were studied for their in-vitro antioxidant, anti-inflammatory and antimicrobial potential. All the oils revealed potential antioxidant activity with maximum in M. bracteata essential oil. All the oils exhibited significant antibacterial activity against Bacillus megaterium, Staphylococcus aureus, Escherichia coli,Salmonella typhimurium and anti-fungal activity against phytopathogenic fungi Fusarium oxysporum, Sclerotinia sclerotiorum, Exserohilum turcicum and Curvularia lunata. The observations from present study suggest further cultivation of Melaleucas and its commercialization as industrial crops.

Chemical constituents of Melaleuca alternifolia and its antitumor activities / 中草药

Objective: To investigate the chemical constituents of the barks and stems of Melaleuca alternifolia and its antitumor activities. Methods: The chemical constituents were separated and purified consecutively by silica gel, Sephadex LH-20 column chromatography, recrystallization as well as preparative HPLC. Their structures were determined by physicochemical properties and spectral analyses. Furthermore, the cytotoxicity of some compounds against PC-9 (human non-small cell lung cancer cell line), HT29 (human colon cancer cell line) and MCF-7 (human breast cancer cell line) were tested by CCK8 method. Results: Eleven compounds were isolated and identified as ursolic acid 3-O-β-cis-caffeate (1), ursolic acid 3-O-β-trans-caffeate (2), tricontyl ferulates (3), 3-O-acetyl-11(12)-en-urs-28,13β-olide (4), 3-O-acetyl-ursolic acid (5), tricontyl caffeate (6), ursolic acid (7), n-nonacosanol (8), urs-12(13)-en-3-one-28-oic acid (9), 3β-O-acetyl-11α,12α-epoxy-oleanane-28,13β-olide (10), and betulin (11). Conclusion: Compound 1 and 2 are cis-trans isomers. Compound 1 is isolated from natural product for the first time, all the compounds are isolated from this plant for the first time except for compound 11. Compound 1 exhibited moderate inhibited effect on the proliferation of three human cancer cell lines.

The sensitivity of Demodex canis (Acari: Demodicidae) to the essential oil of Melaleuca alternifolia - an in vitro study / A sensibilidade do Demodex canis (Acari: Demodicidae) ao óleo essencial de Melaleuca alternifolia - um estudo in vitro

Abstract The essential oil of the Melaleuca alternifolia (Maiden & Betche) (tea tree oil) has been effective in previous studies, in the treatment of infestation by Demodex mites in humans. The present study aimed at evaluating the in vitro acaricidal effect of this herbal medicine on Demodex canis. For the parasitological examination, samples were collected from a dog's skin and examined using optical microscopy. Only samples with intact mites and with evident movement of chelicerae and tarsi were selected. Twenty-one samples were tested with the oil, in seven different concentrations: 100%; 50%; 25%; 12.5%; 6.25%; 5.0% and 3.13%. Three samples were tested with the positive control amitraz, and three with the negative control neutral shampoo. The interval between the time the solution was added and the moment the movement of the last mite ceased defined the survival time in the sample. By comparing the times at different concentrations and controls, the results showed that the higher the concentration of the oil, the more quickly its lethal effect occurred, and that the survival times in the controls were longer than in the different concentrations of melaleuca oil.

Resumo O óleo essencial de Melaleuca alternifolia (Maiden & Betche) (óleo da árvore do chá) foi eficaz em estudos anteriores, no tratamento da infestação por ácaros Demodex em humanos. O presente estudo teve por objetivo avaliar o tempo do efeito acaricida, in vitro, desse fitoterápico sobre Demodex canis. Para o exame parasitológico, amostras foram coletadas da pele de um cão e examinadas por microscopia óptica. Apenas amostras com ácaros íntegros e com evidente movimentação de quelíceras e tarsos foram selecionadas. Vinte e uma amostras foram direcionadas para teste com o óleo, em sete concentrações diferentes: 100%; 50%; 25%; 12,5%; 6,25%; 5,0% e 3,13%. Três amostras foram testadas com o controle positivo, amitraz, e três com o controle negativo, xampu neutro. O intervalo entre o momento em que a solução foi adicionada e o instante em que cessou o movimento do último ácaro definiu o tempo de sobrevivência na amostra. Os tempos nas diferentes concentrações e nos controles foram comparados. Quanto mais alta a concentração do óleo, mais rapidamente ocorreu seu efeito letal. Os tempos de sobrevivência nos controles foram maiores que nas diferentes concentrações do óleo de melaleuca.

Efeito antibiofilme e citotoxicidade do hidróxido de cálcio associado ao óleo essencial de Melaleuca alternifolia / Antibiofilm effect and cytotoxicity of calcium hydroxide associated with the Melaleuca alternifolia essential oil

Objetivo: Avaliar e comparar os efeitos antimicrobiano e antibiofilme, e a citotoxicidade promovida pela associação do hidróxido de cálcio ao óleo essencial de Melaleuca alternifolia (MA), em diferentes concentrações, e ao propilenoglicol (PG). Métodos: As seguintes medicações compuseram os grupos experimentais: G1) HC/MA 1%; G2) HC/MA 5%; G3) HC/MA 10%; G4) HC/MA 20%; e G5) HC/PG. Solução salina 0,85% e meio DMEM serviram como controle nos testes antimicrobianos e de citotoxidade em fibroblastos do ligamento periodontal humano (FbLP), respectivamente. A atividade antimicrobiana (n = 12) foi avaliada por meio do teste de difusão em ágar. O efeito antibiofilme (n = 12) imediato das medicações foi avaliado por meio do teste de viabilidade bacteriana em biofilmes de 72 horas de E. faecalis, formados sobre discos de dentina e tratados por sete dias com as medicações. Após a coleta microbiológica do biofilme remanescente, os discos de dentina foram imersos em meio estéril e armazenados por mais sete dias, para a análise do efeito antibiofilme residual das medicações, quando nova coleta microbiológica foi realizada. A atividade metabólica de FbLP foi avaliada por meio do ensaio colorimétrico MTS (n = 9). Os valores médios dos halos de inibição, em mm, das unidades formadoras de colônia, e o percentual de atividade metabólica celular foram analisados pelos testes Kruskal-Wallis e post hoc Dunn (α = 5%). Resultados:Todas as medicações experimentais apresentaram superior ação antimicrobiana e antibiofilme comparadas ao controle, solução salina (p < 0,05), e mantiveram viáveis os FbLP, semelhante ao controle DMEM (p > 0,05). Conclusão: A associação do óleo essencial de Melaleuca alternifolia, nas concentrações de 1%, 5%, 10% e 20%, ao hidróxido de cálcio promoveu excelente ação antimicrobiana, antibiofilme e biocompatibilidade com fibroblastos, de forma semelhante à associação com propilenoglicol.

Aim:To evaluate and compare the antimicrobial and antibiofilm effect, as well as the cytotoxicity of calcium hydroxide (CH) associated with the Melaleuca alternifolia (MA)essential oil, in different concentrations, and with propylene glycol. Methods: The following medications composed the experimental groups: G1) CH/MA 1%; G2) CH/MA 5%; G3) CH/MA 10%; G4) CH/MA 20%; and G5) CH/PG. Saline solution and culture medium DMEM were used as a control in antimicrobial and cytotoxicity tests in human periodontal ligament fibroblasts (PDLF), respectively. The antimicrobial activity (n = 12) was evaluated by the disk-diffusion agar method. The immediate antibiofilm effect (n = 12) of the medications was evaluated for bacterial viability in 72 hours-biofilms of E. faecalis, formed on the dentin disc surface and treated for seven days with medications. After microbiological sampling of the remaining biofilm, the dentin discs were immersed in sterile culture medium and stored for another seven days, for analysis of the residual antibiofilm effect of the medications, when a new microbiological sampling was performed. PDLF viability was evaluated by MTS colorimetric assay (n = 9). The mean values of the inhibition halos, in mm, the colony forming units, and the metabolic cell activity percentage were analyzed by means of Kruskal-Wallis and post hoc Dunn (α = 5%) tests. Results:All of the experimental medications presented higher antimicrobial and antibiofilm effects, when compared to the saline solution control (p < 0.05), and maintained the PDLF feasible, similar to the DMEM control (p > 0.05). Conclusions:The association of the Melaleuca alternifolia essential oil, at concentrations of 1%, 5%, 10%, and 20%, with calcium hydroxide promoted an excellent antimicrobial and antibiofilm activity, and biocompatibility with fibroblasts, similarly to the association with propylene glycol.

Basil, tea tree and clove essential oils as analgesics and anaesthetics in Amphiprion clarkii (Bennett, 1830) / Óleos essenciais de manjericão, melaleuca e cravo como anestésicos e analgésicos em Amphiprion clarkii (Bennett, 1830)

Abstract In this study were evaluated the anaesthesia and analgesic effects of clove Eugenia caryophyllata, tea tree Melaleuca alternifolia and basil Ocimum basilicum essential oils (EO) during handling of yellowtail clownfish Amphiprion clarkii. Juveniles (3.70 ± 0.75 cm and 1.03 ± 0.50 g; mean ± standard deviation) were submitted to concentrations of 40, 50, 60, 70 and 80 µl L-1 of clove, 150, 200, 250, 300 and 350 µl L-1 of basil and 200, 300, 400, 500 and 600 µl L-1 of tea tree oils (n=10/concentration), previously defined in pilot tests. Individually and only once, fish from each treatment were placed in a glass recipient containing 1 L of seawater at a temperature of 25 °C, salinity of 35 g L-1 and the specific concentration of diluted EO (stock solution). Control (only seawater) and blank (seawater and ethanol at the highest concentration used to dilute the oils) treatments were also conducted. After reaching the stage of surgical anaesthesia, fish were submitted to biometry and a sensibility test. After that, they were transferred to clean seawater for anaesthesia recovery. The times of induction needed to reach each anaesthesia stage and anaesthesia recovery were recorded. Animals were observed for 72 hours after the procedures. All the EO provoked anaesthesia and analgesic effects in A. clarkii, but basil oil is not recommended because it caused involuntary muscle contractions and mortality in 100% and 12% of fish, respectively. The lower concentrations that promote suitable induction and recovery times are 50 µl L-1 of clove oil and 500 µl L-1 of tea tree oil. However, due to its complementary high analgesic efficiency, clove oil is recommended as the ideal anaesthetic for A. clarkii.

Resumo Neste estudo foram avaliados os efeitos anestésicos e analgésicos dos óleos essenciais (OE) de cravo Eugenia caryophyllata, melaleuca Melaleuca alternifolia e manjericão Ocimum basilicum durante manejo de peixes-palhaços Amphiprion clarkii. Juvenis (3.70 ± 0.75 cm e 1.03 ± 0.50 g; média ± desvio padrão) foram submetidos às concentrações de 40, 50, 60, 70 e 80 µl L-1 de cravo, 150, 200, 250, 300 e 350 µl L-1 de manjericão e 200, 300, 400, 500 e 600 µl L-1 de melaleuca (n=10/concentração), previamente definidas em testes pilotos. Individualmente e somente uma vez, os peixes de cada tratamento foram colocados em recipiente de vidro contendo 1 L de água salgada, em temperatura de 25 °C, salinidade de 35 g L-1 e a concentração específica de OE diluída (solução estoque). Tratamentos controle (apenas água marinha) e branco (água marinha e a maior concentração de etanol utilizada para diluição dos óleos) também foram conduzidos. Após atingirem o estágio de anestesia cirúrgica, os peixes foram submetidos à biometria e teste de sensibilidade. Em seguida, foram transferidos para água marinha limpa. Os tempos necessários para atingir cada estágio anestésico e recuperação foram registrados. Os animais foram observados por 72 horas após os procedimentos. Todos os OE provocaram anestesia e analgesia em A. clarkii, porém o óleo de manjericão não é recomendado, pois causou contrações musculares involuntárias e mortalidade em 100% e 12% dos animais, respectivamente. As menores concentrações que promovem indução anestésica e recuperação em tempos adequados são 50 µl L-1 de óleo de cravo e 500 µl L-1 de óleo de melaleuca. Entretanto, devido à sua alta eficiência analgésica complementar, o óleo de cravo é recomendado como o anestésico ideal para A. clarkii.

In vitro and in vivo efficacy of tea tree essential oil for bacterial and yeast ear infections in dogs / Uso in vitro e in vivo do óleo essencial de Melaleuca (Melaleuca alternifolia) em otites bacterianas e por leveduras

Otitis externa is a common complaint in dogs. Bacteria and yeasts are commonly involved and may perpetuate inflammatory reactions inside the ear canal. Otoscopy, cytological examination of secretion and microbiological culture embody forms of diagnosis. Cytology also has great use in accessing treatment evolution. Therapy usually consists of cleaning ear canals and subsequent use of antibiotics or antifungal products. As some of them may cause hypersensitivity and even ototoxicity, searching for new pharmacological bases is currently necessary and justifies this study, which aimed to evaluate in vitro and in vivo efficacy of tea tree essential oil for bacterial and yeast ear infections in dogs. Twenty-eight dogs from a particular shelter in Cuiabá (Mato Grosso, Brazil), presenting clinical signs of otitis externa, were enrolled in this clinical trial. In all of them, clinical and cytological evaluations, as well as culture and susceptibility testing of the affected ears were carried out. From each dog, one ear was treated with 5% tea tree essential oil lotion and the other with standard otic formulation, according to the type of infection (bacterial, yeast or both). In vitro susceptibility testings of all ear cultures, to the same drugs used in treatment, were also carried out. Culture results showed 62.5% bacterial and fungal infection, 33.9% bacterial infection and 3.6% fungal infection, from the 56 ear samples collected. The most common microorganisms isolated were Staphylococcus intermedius, Staphylococcus aureus, Proteus mirabilis and Malassezia pachydermatis. Gram-positive bacteria were susceptible to gentamycin in 60.5% and resistant in 16.3% of the samples. Five percent tea tree essential oil formulation produced a 5mm clear zone of inhibition around the disks in one of the 63 samples evaluated. Pure (100%) tea tree essential oil formulation produced a 10mm clear zone of inhibition around the disks in four of the 63 samples evaluated, a 9mm zone in three samples, an 8mm zone in 16 samples, a 7mm zone in seven samples, a 6mm zone in two samples and there was no clear zone in 31 samples. Inhibition zones were produced by strains of Staphylococcus intermedius, Staphylococcus hyicus, Corynebacterium sp., Proteus mirabilis and Enterobacter sp. tea tree essential oil ear solution significantly induced remission of clinical signs both in bacterial and yeast ear infections. It also reduced as much Malassezia pachydermatis ear infection as the nystatin solution used in this study, while gentamycin solution showed better antibacterial effect. More studies should be conducted to evaluate in vitro diffusion properties of tea tree essential oil. Good antimicrobial spectrum and the absence of adverse reactions confirm the importance of developing a tea tree formulation as an alternative therapy for ear infections in dogs.(AU)

Otite externa é queixa frequente em cães. Bactérias e leveduras estão comumente envolvidas e podem perpetuar as reações inflamatórias dentro do canal auditivo. Dentre as formas de diagnóstico, encontram-se a otoscopia, o exame citológico da secreção e a cultura microbiológica. Citologia também tem grande utilidade no acesso à evolução do tratamento. A terapia consiste de limpeza dos canais auditivos e posterior utilização de antibióticos ou produtos antifúngicos. Como alguns antimicrobianos utilizados no tratamento podem causar hipersensibilidade e até mesmo ototoxicidade, a busca por novas bases farmacológicas justifica a existência deste estudo, que teve como objetivo avaliar in vitro e in vivo a eficiência do óleo de Melaleuca alternifolia em otites bacterianas e fúngicas de cães. Vinte e oito cães, de um abrigo particular, apresentando sinais clínicos de otite externa, foram incluídos neste estudo clínico. Todos passaram por avaliação clínica, citologia e cultura de material das orelhas afetadas. De cada animal, uma orelha foi tratada com óleo de Melaleuca 5% e a outra com formulação ótica padrão, de acordo com a afecção (bacteriana, fúngica ou mista). As culturas também foram submetidas a testes de susceptibilidade in vitro aos mesmos agentes utilizados no tratamento in vivo. Os resultados da cultura mostraram 62,5% de infecção mista (bacteriana e fúngica), 33,9% de infecção bacteriana e 3,6%, de infecção fúngica a partir das 56 orelhas. Os micro-organismos mais isolados foram Staphylococcus intermedius, Staphylococcus aureus, Proteus mirabilis e Malassezia pachydermatis. As bactérias GRAM-positivas foram sensíveis à gentamicina em 60,5% e resistentes em 16,3% das amostras. A formulação com 5% de óleo essencial de Melaleuca produziu uma zona de inibição de 5mm em torno dos discos em uma das 63 amostras avaliadas. A formulação pura (100%) do mesmo produto produziu uma zona de 10mm de inibição em quatro das 63 amostras analisadas, uma zona de 9 mm em três amostras, uma zona de 8mm em 16 amostras, uma zona de 7mm em sete amostras, uma zona de 6mm em duas amostras e não havia nenhuma zona clara em 31 amostras. Zonas de inibição foram produzidas por estirpes de Staphylococcus intermedius, Staphylococcus hyicus, Corynebacterium sp., Proteus mirabilis e Enterobacter sp. Clinicamente, a formulação com o óleo essencial de melaleuca induziu significativamente uma melhora nas manifestações, tanto nas otites bacterianas quanto nas causadas por leveduras, sendo estatisticamente semelhante ao tratamento com nistatina (nas otites por levedura), mas menos eficaz que a solução de gentamicina nas otites bacterianas. Mais estudos devem ser realizados para avaliar as propriedades de difusão in vitro do óleo essencial de melaleuca. O bom espectro antimicrobiano, a boa resposta clínica e a ausência de reações adversas confirmam a possibilidade de desenvolvimento de formulação ótica com o óleo essencial de melaleuca, como uma alternativa para a terapia de infecções do ouvido em cães.(AU)

Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

Abstract The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

Research progress on chemical constituents in plants of Melaleuca L. and their pharmacological activities / 中草药

Plants in Melaleuca L. contain volatile oils, triterpenoids, tannins, flavonoids, glycosides, and other ingredients, which show a variety of pharmacological activities, such as antimicrobial, antifungal, anti-inflammatory, antitumor, antiviral, and anti-oxidant. This review summarized the research advances on the chemical constituents and pharmacological activities of plants from Melaleuca L. systematically. It may provide the reference and scientific basis for the further study and development of the plants in this genus.

Diversity and functional characterization of antifungal-producing Streptomyces-like microbes isolated from the rhizosphere of cajuput plants (Melaleuca leucodendron L.)

Aims@#The study was undertaken to evaluate the diversity of actinomycetes from the rhizosphere of the cajuput plant (Melaleuca leucodendron L.) using ARDRA, and to examine their in vitro antifungal potency against selected fungi. @*Methodology and results@#A total of 78 Streptomyces-like microbes were isolated from the limestone rhizosphere of cajuput plants and cultured in SN agar medium. The ability to inhibit fungal growth was observed using a dual culture assay. The diversity of the isolates was examined by morphological and genotype profiling using ARDRA (Amplified 16S ribosomal DNA restriction analysis), following which they were assigned to eight groups. Seventeen (21.8%) strains showed the ability to produce the antifungal compound as evidenced by their antagonism in vitro against the tested fungi, namely Saccharomyces cerevisiae BY 47420, Candida albicans CGMCC 2538, Aspergillus flavus NRLL 3357, and Fusarium oxysporum KFCC 11363P in the dual culture assay. One isolate, GMR22, which showed potent antagonism against unicellular and filamentous fungi, displayed 97% 16S rRNA sequence similarity to Streptomyces vayuensis. @*Conclusion, significance and impact of study@#This study demonstrated that a diversity of Streptomyces-like antifungal producing microbes were present in the rhizosphere of cajuput plants in the Wanagama Forests of Indonesia. This rhizosphere represents a potential new source of actinomycetes that produce biologically active compounds. One selected strain (GMR22) has the potential to be developed as a commercial biofungicide.

Efeitos do extrato de própolis e do óleo de melaleuca na formação do biofilme e na desmineralização dental: estudo in situ

Avaliar os efeitos de soluções naturais na formação do biofilme e na prevenção da desmineraliza- ção dos dentes. Sujeitos e método: realizou-se um estudo piloto, in situ, com 12 voluntários, que utilizaram dispositivos de acrílico removível, contendo 4 blocos de dente cada. Os blocos de dente foram submetidos ao desafio cariogênico, quatro vezes ao dia, e testados com uma solução experimental, duas vezes ao dia, durante 14 dias. A amostra foi dividida em quatro grupos, com três indivíduos em cada grupo, os quais testaram as seguintes soluções: soro fisiológico (Grupo 1), clorexidina 0,12% (Grupo 2), óleo de melaleuca 0,2% (Grupo 3) e extrato de própolis 30% (Grupo 4). Ao final de 14 dias o biofilme formado foi coletado e semeado em três meios de cultura diferentes, os blocos de dente foram pesados em balança de precisão e testados quanto à microdureza superficial. Resultados: os resultados mostraram que houve um grande crescimento microbiano em todos os meios de cultura; houve uma tendência de perda de peso mineral nos blocos dos Grupos 1, 2 e 3, enquanto o grupo 4 mostrou uma tendência de ganho de peso. Na análise da microdureza superficial, observou-se uma tendência de menor resistência da dentina após o desafio cariogênico, em todos os grupos, comparados ao grupo controle que não foi submetido ao desafio cariogênico. Conclusão: o óleo de Melaleuca 0,2% não influenciou na formação de placa, na dureza superficial e no peso dos blocos, enquanto que o extrato de própolis mostrou uma tendência de interferir na formação de placa e no peso mineral dos dentes.

Analysis of pH and release of calcium of association between melaleuca alternifolia oil and calcium hydroxide / Análise de pH e liberação de cálcio da associação entre óleo de melaleuca alternifólia e hidróxido de cálcio

Introduction: The use of intracanal medications with antimicrobial properties is essential for decontaminating root canals during endodontic treatment. Calcium hydroxide is used for this because of its excellent properties. Melaleuca alternifolia oil has shown medicinal importance by demonstrating antifungal and bactericidal action against proven human pathogens. Objective: To evaluate the physical and chemical aspects such as pH and calcium release, of Melaleuca alternifolia oil associated with calcium hydroxide, during different time intervals. Material and method: Calcium hydroxide powder was added to vehicles to reach a concentration of 72mg / 0.1mL. Three groups were formed: Group I: Calcium Hydroxide + Distilled Water; Group II: Calcium hydroxide + Propylene Glycol; Group III: Calcium hydroxide + Melaleuca oil. The pH of each group was measured after time intervals of 10 minutes; 24 and 48 hours; 7, 15 and 30 days after tooling by a pH meter. Calcium release was analyzed by atomic absorption spectrometry equipped with a calcium hollow cathode lamp. Data were statistically analyzed by using the Kruskall-Wallis and Dunn test. Result: Group II showed high pH, similar to group III that remained uniform at 15 and 30 days. Calcium release that began after 24 hours, was similar in Groups II and III, and showed a peak release in 48 hours. Conclusion: The association of Melaleuca oil with calcium hydroxide showed good results in the pH and calcium release analyses, and showed action similar to that of propylene glycol + calcium hydroxide.

Introdução: O uso de medicações intracanais com propriedades antimicrobianas é fundamental para descontaminação dos canais radiculares durante o tratamento endodôntico. O hidróxido de cálcio é utilizado como medicação intracanal por apresentar excelentes propriedades. O óleo de Melaleuca alternifólia apresenta importância medicinal demonstrando ação antifúngica e bactericida comprovada contra patógenos humanos. Objetivo: Avaliar aspectos físico-químicos da associação do óleo de Melaleuca Alternifólia com hidróxido de cálcio, como: pH e liberação de cálcio durante diferentes períodos. Material e método: O pó do hidróxido de cálcio foi adicionado aos veículos até a concentração de 72mg/0,1mL. Foram divididos três grupos: Grupo I: Hidróxido de Cálcio + Água Destilada; Grupo II: Hidróxido de Cálcio + Propilenoglicol; Grupo III: Hidróxido de Cálcio + Óleo de Melaleuca. O pH de cada grupo foi medido após 10 minutos, 24, 48 horas, 7, 15 e 30 dias após a espatulação por um pHmetro. A liberação de cálcio foi analisada através da espectrometria de absorção atômica equipada com uma lâmpada cátodo para cálcio. Os dados foram analisados estatisticamente pelos testes de Kruskall-Wallis e Dunn. Resultado: O grupo II apresentou pH elevado, semelhante ao grupo III, permanecendo uniforme aos 15 e 30 dias. A liberação de cálcio iniciou em 24 horas, de forma semelhante nos grupos II e III e seu pico de liberação deu-se em 48 horas. Conclusão: A associação do Óleo de Melaleuca com hidróxido de cálcio apresentou bons resultados quanto à análise de pH e liberação de cálcio, demonstrando ação semelhante ao propilenoglicol + hidróxido de cálcio.

Basil, tea tree and clove essential oils as analgesics and anaesthetics in Amphiprion clarkii (Bennett, 1830)

Abstract In this study were evaluated the anaesthesia and analgesic effects of clove Eugenia caryophyllata, tea tree Melaleuca alternifolia and basil Ocimum basilicum essential oils (EO) during handling of yellowtail clownfish Amphiprion clarkii. Juveniles (3.70 ± 0.75 cm and 1.03 ± 0.50 g; mean ± standard deviation) were submitted to concentrations of 40, 50, 60, 70 and 80 µl L-1 of clove, 150, 200, 250, 300 and 350 µl L-1 of basil and 200, 300, 400, 500 and 600 µl L-1 of tea tree oils (n=10/concentration), previously defined in pilot tests. Individually and only once, fish from each treatment were placed in a glass recipient containing 1 L of seawater at a temperature of 25 °C, salinity of 35 g L-1 and the specific concentration of diluted EO (stock solution). Control (only seawater) and blank (seawater and ethanol at the highest concentration used to dilute the oils) treatments were also conducted. After reaching the stage of surgical anaesthesia, fish were submitted to biometry and a sensibility test. After that, they were transferred to clean seawater for anaesthesia recovery. The times of induction needed to reach each anaesthesia stage and anaesthesia recovery were recorded. Animals were observed for 72 hours after the procedures. All the EO provoked anaesthesia and analgesic effects in A. clarkii, but basil oil is not recommended because it caused involuntary muscle contractions and mortality in 100% and 12% of fish, respectively. The lower concentrations that promote suitable induction and recovery times are 50 µl L-1 of clove oil and 500 µl L-1 of tea tree oil. However, due to its complementary high analgesic efficiency, clove oil is recommended as the ideal anaesthetic for A. clarkii.

Resumo Neste estudo foram avaliados os efeitos anestésicos e analgésicos dos óleos essenciais (OE) de cravo Eugenia caryophyllata, melaleuca Melaleuca alternifolia e manjericão Ocimum basilicum durante manejo de peixes-palhaços Amphiprion clarkii. Juvenis (3.70 ± 0.75 cm e 1.03 ± 0.50 g; média ± desvio padrão) foram submetidos às concentrações de 40, 50, 60, 70 e 80 µl L-1 de cravo, 150, 200, 250, 300 e 350 µl L-1 de manjericão e 200, 300, 400, 500 e 600 µl L-1 de melaleuca (n=10/concentração), previamente definidas em testes pilotos. Individualmente e somente uma vez, os peixes de cada tratamento foram colocados em recipiente de vidro contendo 1 L de água salgada, em temperatura de 25 °C, salinidade de 35 g L-1 e a concentração específica de OE diluída (solução estoque). Tratamentos controle (apenas água marinha) e branco (água marinha e a maior concentração de etanol utilizada para diluição dos óleos) também foram conduzidos. Após atingirem o estágio de anestesia cirúrgica, os peixes foram submetidos à biometria e teste de sensibilidade. Em seguida, foram transferidos para água marinha limpa. Os tempos necessários para atingir cada estágio anestésico e recuperação foram registrados. Os animais foram observados por 72 horas após os procedimentos. Todos os OE provocaram anestesia e analgesia em A. clarkii, porém o óleo de manjericão não é recomendado, pois causou contrações musculares involuntárias e mortalidade em 100% e 12% dos animais, respectivamente. As menores concentrações que promovem indução anestésica e recuperação em tempos adequados são 50 µl L-1 de óleo de cravo e 500 µl L-1 de óleo de melaleuca. Entretanto, devido à sua alta eficiência analgésica complementar, o óleo de cravo é recomendado como o anestésico ideal para A. clarkii.

Chemical characterization of essential oil from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.)

Objective:To isolate and identify the compounds in the essential oils from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.) collected in Dakar,Senegal.Methods:The essential oils from the leaves of these two myrtaceaes were extracted by steam distillation and analyzed by gas chromatograph and gas chromatography-mass spectrometer.Results:A total of 34 constituents were identified in the oil of Callistemon viminalis and the major compounds were 1.8-cineole (58.12％),limonene (9.72％),α-terpineol (9.56％),geranial (6.02％),δ-elemene (3.53％),myrcene (2.96％) and α-pinene (2.49％).For the essential oil of Melaleuca leucadendron,43 constituents were identified,and 1.8-cineole (28.87％),epiglobulol (23.06％),α-pinene (12.22％),limonene (11.65％) and α-terpineol (7.06％) were major compounds.Conclusions:Considering properties of the identified major compounds,essential oils of both studied myrtaceae could be used in the medicine field including the food,pharmaceutical and cosmetic industry.

Chemical characterization of essential oil from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.)

Objective To isolate and identify the compounds in the essential oils from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.) collected in Dakar, Senegal. Methods The essential oils from the leaves of these two myrtaceaes were extracted by steam distillation and analyzed by gas chromatograph and gas chromatography–mass spectrometer. Results A total of 34 constituents were identified in the oil of Callistemon viminalis and the major compounds were 1.8-cineole (58.12%), limonene (9.72%), α-terpineol (9.56%), geranial (6.02%), δ-elemene (3.53%), myrcene (2.96%) and α-pinene (2.49%). For the essential oil of Melaleuca leucadendron, 43 constituents were identified, and 1.8-cineole (28.87%), epiglobulol (23.06%), α-pinene (12.22%), limonene (11.65%) and α-terpineol (7.06%) were major compounds. Conclusions Considering properties of the identified major compounds, essential oils of both studied myrtaceae could be used in the medicine field including the food, pharmaceutical and cosmetic industry.

Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae): Natural alternative for mosquito control

OBJECTIVE@#To evaluate an essential oil with larvicide, adulticide and growth inhibitory activity against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, of medical importance.@*METHODS@#Standardized methodology by WHO was used to determine the levels of susceptibility of mosquito larvae exposed to the essential oil. To evaluate the adulticide activity with the essential oil at different doses, bottles were impregnated according to the methodology CDC. To determine the development inhibitory activity of Melaleuca quinquenervia (M. quinquenervia) oil in three mosquito species, third instar larvae were exposed to the LC and LC dose (calculated for each population) of M. quinquenervia oil in glass containers with a capacity of 500 mL. After 24 h exposure, the dead larvae were discarded. The mortality of larvae and pupae were recorded on a daily basis.@*RESULTS@#The calculated LC indicates an order of effectiveness of preferential oil for Culex quinquefasciatus (LC = 0.0021%), Aedes aegypti (LC = 0.0047%) and Aedes albopictus (LC = 0.0049%).@*CONCLUSIONS@#The adulticide activity was achieved with impregnated bottles at 40 and 50 mg/mL with the three mosquitoes species. In larvae, a growth inhibition was detected when exposed to sublethal doses. The results indicate that M. quinquenervia is a plant with promising environmentally sustainable source for vector control.

Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae): Natural alternative for mosquito control

Objective To evaluate an essential oil with larvicide, adulticide and growth inhibitory activity against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, of medical importance. Methods Standardized methodology by WHO was used to determine the levels of susceptibility of mosquito larvae exposed to the essential oil. To evaluate the adulticide activity with the essential oil at different doses, bottles were impregnated according to the methodology CDC. To determine the development inhibitory activity of Melaleuca quinquenervia (M. quinquenervia) oil in three mosquito species, third instar larvae were exposed to the LC