Julie L. Markham

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

S.D. COX, C.M. MANN, J.L. MARKHAM, H.C. BELL, J.E. GUSTAFSON, J.R. WARMINGTON and S.G. WYLLIE.2000.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.

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.

The role of structure and molecular properties of terpenoids in determining their antimicrobial activity

The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into five groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to confirm these groupings and to show the differences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans. Copyright

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.

The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil)

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism’s susceptibility to tea tree oil and to its normally inert hydrocarbons, p‐cymene and γ‐terpinene.

Determining the Antimicrobial Actions of Tea Tree Oil

Research into the mode of action of the essential oil of Melaleuca alternifolia (Tea tree oil) is briefly reviewed. Its mode of action is interpreted in terms of the membrane-toxicity of its monoterpenoid components and different approaches for determining cell membrane damage are discussed.

Determination of octanol-water partition coefficient for terpenoids using reversed-phase high-performance liquid chromatography.

Octanol-water partition coefficients (Kow) for 57 terpenoids were measured using a RP-HPLC method. Sample detection was achieved with standard UV and refractive index detectors and required no special column treatment. Measured log Kow values for the terpenoids ranged from 1.81 to 4.48 with a standard error of between 0.03 and 0.08 over the entire range. Partition coefficients determined by the RP-HPLC method were compared against shake flask, atom/fragment contribution, fragment and atomistic methods. The HPLC values were found to give the best correlation with shake flask results. Log Kow values calculated by the atom/fragment contribution method gave the best correlation with the HPLC values when compared to fragment and atomistic methods.

An agar dilution method for the determination of the minimum inhibitory concentration of essential oils

Abstract Methods for the measurement of the minimum inhibitory concentration (MIC) of antibacterial agents have been developed for water-soluble substances and require adaptation for use with water-insoluble essential oils. This paper reports a standardized agar dilution MIC method, using 0.5% v/v Tween 20 as a dispersant, which provides a reliable and reproducible technique. The method was tested using two Melaleuca alternifolia oil samples with two different levels of terpinen-4-ol (37% and 45% v/v). The MIC values of die tea tree oil samples against a wide selection of bacteria, moulds and yeast are reported.

Xanthine oxidase inhibitory activity of selected Australian native plants

Twenty-eight extracts from 17 species of Australian native plants traditionally used as general anti-inflammatory medicines by Australian Aboriginal people were examined for inhibition of the enzyme xanthine oxidase (XO). The extracts from nine species were found to have more than 25% inhibition at a concentration of 100 microg/ml in the assay mixture. Extracts from three species Clerodendrum floribundum R. Br. (Verbenaceae), Eremophila maculata (Ker Gawler) (Myoporaceae) and Stemodia grossa Benth. (Scrophulariaceae) showed the greatest activity with inhibitions of 84, 61 and 57%, respectively, at 50 microg/ml, with four other species having more than 40% inhibitory activity at this concentration.

In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines

Abstract Cytotoxicity of Australian tea tree oil (oil of Melaleuca alternifolia) and its major oxygenated monoterpenes: terpinen-4-ol, 1,8-cineole and α-terpineol were investigated using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was α-terpineol>tea tree oil>terpinen-4-ol> 1,8-cineole and with comparison with the controls used mercuric chloride>tea tree oil>aspirin. Antimicrobial activity (MICs) displayed a similar pattern where α-terpineol>terpinen-4-ol>tea tree oil> 1,8-cin...