Susceptibility of Propionibacterium acnes to the essential oil of Melaleuca alternifolia

The susceptibility of 32 strains of Propionibacterium acnes to the essential oil of Melaleuca alternifolia , tea tree oil, was examined using a broth dilution method. The minimum bactericidal concentration of tea tree oil for five strains was 0.25% or less while, for the remainder, it was 0.50%.     

Interactions between components of the essential oil of Melaleuca alternifolia

AIMS: This study compared the antimicrobial activity of Melaleuca alternifolia (tea tree) oil with that of some of its components, both individually and in two-component combinations. METHODS AND RESULTS: Minimum inhibitory concentration and time-kill assays revealed that terpinen-4-ol, the principal active component of tea tree oil, was more active on its own than when present in tea tree oil. Combinations of terpinen-4-ol and either gamma-terpinene or p-cymene produced similar activities to tea tree oil. Concentration-dependent reductions in terpinen-4-ol activity and solubility also occurred in the presence of gamma-terpinene. CONCLUSION: Non-oxygenated terpenes in tea tree oil appear to reduce terpinen-4-ol efficacy by lowering its aqueous solubility. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings explain why tea tree oil can be less active in vitro than terpinen-4-ol alone and further suggest that the presence of a non-aqueous phase in tea tree oil formulations may limit the microbial availability of its active components.     

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.     

Role of the MexAB-OprM efflux pump of Pseudomonas aeruginosa in tolerance to tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol

Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organism's tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.     

Isolation and partial characterisation of a putative monoterpene synthase from Melaleuca alternifolia.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.     

Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae)

The composition and yield of oil in 615 trees representing the natural populations of Melaleuca alternifolia, or tea tree, was investigated. A sixth distinct oil chemotype was identified. Of the six chemotypes, one chemotype is dominated by terpinen-4-ol, one by 1,8-cineole, one by terpinolene and the remaining three chemotypes are all dominated by 1,8-cineole and differ in either terpinen-4-ol or terpinolene content. Whilst most chemotypes are present throughout the distribution range, a definite correspondence of oil types with geographic location was found. Terpinen-4-ol types predominate in and around the Bungawalbin basin in the Casino area of northern New South Wales (NSW), high 1,8-cineole types predominate toward the southern end of the distribution around Grafton and terpinolene types predominate in southern Queensland. Preliminary formulae have been developed to allow comparisons of oil data obtained by steam distillation with a static headspace gas chromatography method.     

Influence of organic matter, cations and surfactants on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil in vitro

The effect of some potentially interfering substances and conditions on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil was investigated. Agar and broth dilution methods were used to determine minimum inhibitory and cidal concentrations of tea tree oil in the presence and absence of each potentially interfering substance. Activity was determined against Gram-positive and -negative bacteria, and Candida albicans. Minimum inhibitory or cidal concentrations differed from controls by two or more dilutions, for one or more organisms, where Tween-20, Tween-80, skim-milk powder and bovine serum albumin were assessed. These differences were not seen when assays were performed in anaerobic conditions, or in the presence of calcium and magnesium ions. The effect of organic matter on the antimicrobial activity of tea tree oil was also investigated by an organic soil neutralization test. Organisms were exposed to lethal concentrations of tea tree oil ranging from 1-10% (v/v), in the presence of 1-30% (w/v) dry bakers' yeast. After 10 min contact time, viability was determined. At > or = 1%, organic matter compromised the activity of each concentration of tea tree oil against Staphylococcus aureus and C. albicans. At 10% or more, organic matter compromised the activity of each tea tree oil concentration against Pseudomonas aeruginosa. Organic matter affected 1 and 2% tea tree oil, but not 4 and 8%, against Escherichia coli. In conclusion, organic matter and surfactants compromise the antimicrobial activity of tea tree oil, although these effects vary between organisms.     

Antimycotic activity of Melaleuca alternifolia essential oil and its major components

AIMS: The aim of this study was to analyse the antimycotic properties of Melaleuca alternifolia essential oil (tea tree oil, TTO) and its principal components and to compare them with the activity of 5-fluorocytosine and amphotericin B. METHODS AND RESULTS: The screening for the antimycotic activity was performed by serial twofold dilutions in Roswell Park Memorial Institute medium with the inclusion of Tween-80 (0.5%). TTO and terpinen-4-olo were the most active compounds. CONCLUSIONS: The majority of the organisms were sensitive to the essential oil, with TTO and terpinen-4-olo being the most active oils showing antifungal activity at minimum inhibitory concentration values lower than other drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a sample large enough to determine the antifungal properties of TTO and terpinen-4-olo and suggests further studies for a possible therapeutic use.     

Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil

AIMS: To investigate the in vitro antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. METHODS AND RESULTS: Activity was investigated by broth microdilution and macrodilution, and time kill methods. Components showing the most activity, with minimum inhibitory concentrations and minimum fungicidal concentrations of < or =0.25%, were terpinen-4-ol, alpha-terpineol, linalool, alpha-pinene and beta-pinene, followed by 1,8-cineole. The remaining components showed slightly less activity and had values ranging from 0.5 to 2%, with the exception of beta-myrcene which showed no detectable activity. Susceptibility data generated for several of the least water-soluble components were two or more dilutions lower by macrodilution, compared with microdilution. CONCLUSIONS: All tea tree oil components, except beta-myrcene, had antifungal activity. The lack of activity reported for some components by microdilution may be due to these components becoming absorbed into the polystyrene of the microtitre tray. This indicates that plastics are unsuitable as assay vessels for tests with these or similar components. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified that most components of tea tree oil have activity against a range of fungi. However, the measurement of antifungal activity may be significantly influenced by the test method.     

Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia

Tea tree oil, or the essential oil of Melaleuca alternifolia , is becoming increasingly popular as a naturally occurring antimicrobial agent. The antimicrobial activity of eight components of tea tree oil was evaluated using disc diffusion and broth microdilution methods. Attempts were also made to overcome methodological problems encountered with testing compounds which have limited solubility in aqueous media. After assessing media with and without solubilizing agents, the disc diffusion method was used to determine the susceptibility of a range of micro-organisms to 1,8-cineole, 1-terpinen-4-ol, ρ-cymene, linalool, α-terpinene, γ-terpinene, α-terpineol and terpinolene. While the disc diffusion method lacked reproducibility, it was considered useful as a procedure for screening for antimicrobial activity. Terpinen-4-ol was active against all the test organisms while ρ-cymene demonstrated no antimicrobial activity. Linalool and α-terpineol were active against all organisms with the exception of Pseudomonas aeruginosa. Minimum inhibitory and minimum cidal concentrations of each component against Candida albicans, Escherichia coli and Staphylococcus aureus were determined using a broth microdilution method. Modifications to this method overcame solubility and turbidity problems associated with the oil components and allowed the antimicrobial activity of each of the components to be quantified reproducibly. There was reasonable agreement between minimum inhibitory concentrations and zones of inhibition. These results may have significant implications for the future development of tea tree oil as an antimicrobial agent.     

A new method for determining the minimum inhibitory concentration of essential oils

A new microdilution method has been developed for determining the minimum inhibitory concentration (MIC) of oil-based compounds. The redox dye resazurin was used to determine the MIC of a sample of the essential oil of Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of 0·15% (w/v) agar as a stabilizer overcame the problem of adequate contact between the oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid version of the assay was also developed for use as a screening method. A comparison of visual and photometric reading of the microtitre plates showed that results could be assessed without instrumentation; moreover, if the rapid assay format was used, rigorous asepsis was not necessary. Accuracy of the resazurin method was confirmed by plate counting from microwells and MIC values were compared with results obtained using an agar dilution assay. The MIC results obtained by the resazurin method were slightly lower than those obtained by agar dilution.     

Effect of Australian tea tree oil on Gyrodactylus spp. infection of the three-spined stickleback Gasterosteus aculeatus

Gyrodactylus spp. infections of commercially farmed fishes are responsible for significant economic losses. Existing treatments have proved uneconomic, stressful to the fishes, and ecologically damaging. Essential oils are naturally occurring compounds that exhibit a wide range of anti-microbial and anti-fungal activities. This study explored the possibility of using Australian tea tree (Melaleuca alternifolia) oil (TTO) to treat Gyrodactylus spp. infection on the three-spined stickleback Gasterosteus aculeatus. In the presence of 0.01 % Tween 80 as an emulsifier, TTO treatments at concentrations between 3 and 30 ppmv (parts per million by volume) lowered the prevalence and significantly reduced the parasite burden of sticklebacks naturally infected with Gyrodactylus spp. In addition, Tween 80 alone exhibited parasiticidal activity against Gyrodactylus spp. These findings show the potential of TTO in combination with Tween 80 as an effective treatment of Gyrodactylus spp. infection of fishes.     

Monoterpenoid accumulation in Melaleuca alternifolia seedlings

Individual leaves of the commercial terpinen-4-ol type of Melaleuca alternifolia were examined both quantitatively and qualitatively for volatile constituents from the emergence of the first true leaves, through to 6-week-old tenth leaf set material. A GC internal standard addition method was used to measure changes in oil composition and the accumulation of volatile constituents expressed on a dry weight, unit leaf area and whole leaf basis. In the early stages of seedling growth, leaves contained higher concentrations of terpinolene, alpha-pinene and beta-pinene and lower concentrations of terpinen-4-ol, sabinene and cis-sabinene hydrate than mature leaf. Concentrations of the former constituents fell and the latter rose by the time leaf set 10 was 6 weeks old. Key constituent, 1,8-cineole remained in similar concentration throughout ontogeny. The variation in concentration of other key constituents during early stages of seedling development suggests that caution is required in extrapolating seedling leaf data to mature tree oil quality.     

Volatile oil comparison of cotyledon leaves of chemotypes of Melaleuca alternifolia

Oil quantity and quality were measured for the cotyledon leaves of the commercial terpinen-4-ol chemical variety of Melaleuca alternifolia. Oil yield obtained by ethanolic extraction was 3.8 micrograms per leaf or 2.6% (dry weight basis). The major components of the oil were alpha-pinene (7.4%), beta-pinene (12.0%) and terpinolene (27.3%). The non-commercial terpinolene chemical variety was found to be rich in 1,8-cineole (12.5%) and terpinolene (25.4%). The non-commercial 1,8-cineole chemical variety was rich in 1,8-cineole (37%) with significant quantities of alpha-pinene (15.5%), beta-pinene (23.3%) and terpinolene (10.9%). The cotyledon leaf composition, when compared with that of mature leaf from the same chemical variety, was found to be biased toward pinene and terpinolene biogenetic pathway constituents and hence not a good indicator of mature tree quality especially for the commercial terpinen-4-ol chemical variety. The implications of these analyses for the determination of M. alternifolia plantation quality and the understanding of oil formation, are discussed.     

互叶白千层油化学成分的研究

采用水蒸汽蒸馏收集互叶白千层芳香油,对该油进行ＧＣ／ＭＳ／ＤＳ定性分析及其总离于流图的面积归一化定量分析,鉴定出２５种化合物,其中含氧化合物６种,碳氢化合物１９种。直接影响该油商品价值的化学成分４－萜品醇含量约为１７％,桧樟脑含量约为２．４％。     

广东地区互叶白千层栽培技术规程

互叶白千层Melaleuca alternifolia是一种生产周期短、收益快的药用和香料树种。文中总结广东地区互叶白千层的栽培技术规程,包括种苗繁育、种植方法、田间管理、采收等,为提高互叶白千层品质、保障精油质量提供标准化技术指导。     

淹水深度对互叶白千层幼苗气体交换、叶绿素荧光和生长的影响

实验设置对照、浅淹(水位高出土壤表面5cm)和深淹(水位高出土壤表面20cm)3种处理,研究了淹水深度对互叶白千层幼苗的气体交换、叶绿素荧光和生长状况的影响。研究结果表明,随着淹水深度的增加,互叶白千层受到的胁迫程度有所增强。经过270d的淹水处理,浅淹组和深淹组的株高和生物量有所下降,分别为对照的90.86%、64.58%和74.52%、36.46%。浅淹组植株叶绿素含量、净光合速率、气孔传导率和蒸腾速率略有下降,分别为对照的95.39%、94.26%、90.02%和88.94%。深淹组植株在淹水后180d内上述参数显著下降,分别为对照的79.44%、73.54%、61.79%和71.46%,随后逐渐接近对照组。浅淹组PSⅡ的最大光化学量子效率(Fv/Fm)比较稳定,与对照组基本相同。深淹组在淹水后150d内Fv/Fm稍有下降,随后恢复到对照水平。浅淹组植株光化学淬灭系数(qP)稍有下降,非光化学淬灭(NPQ)略有上升,分别为对照的96.63%和105.66%。深淹组植株在淹水后120d内qP显著下降,NPQ明显上升,分别为对照的94.51%和126.66%,随后逐渐接近对照组。另外,淹水过程中,互叶白千层形成不定根和产生发达的通气组织,淹水植株的根孔隙度显著高于对照。     

The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials

Aims:  Due to the emergence of multi-drug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to investigate the in vitro pharmacological interactions between essential oils (considered valuable as natural therapeutic treatments) and conventional antimicrobials (ciprofloxacin/amphotericin B) when used in combination.
Methods and Results:  Interactions of the essential oils ( Melaleuca alternifolia , Thymus vulgaris , Mentha piperita and Rosmarinus officinalis ) when combined with ciprofloxacin against Staphylococcus aureus indicate mainly antagonistic profiles. When tested against Klebsiella pneumoniae the isobolograms show antagonistic, synergistic and additive interactions depending on the combined ratio. The R. officinalis/ ciprofloxacin combination against K. pneumoniae displayed the most favourable synergistic pattern. The interactions of M. alternifolia (tea tree), T.   vulgaris (thyme), M. piperita (peppermint) and R. officinalis (rosemary) essential oils with amphotericin B indicate mainly antagonistic profiles when tested against Candida albicans.
Conclusion:  While a number of interactions show complete antagonism, others show varied (synergistic, additive and/or antagonistic) interactions, thus the efficacy is dependent on the ratio in which the two components co-exist.
Significance and Impact of the Study:  The predominant antagonistic interactions noted here, suggests that some natural therapies containing essential oils should be used with caution when combined with antibiotics.     

Antifungal Activity of Nanocapsule Suspensions Containing Tea Tree Oil on the Growth of Trichophyton rubrum

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL^?1 (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm², 14.59 ± 2.01 mm², 40.98 ± 2.76 mm² and 38.72 ± 1.22 mm² for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.