AromaChemistry and Applications of Tea Tree Oil – Part 2 of 4
Potential Modes of Action
Even after substantial research, the mode of action of Tea Tree Oil is still not properly understood. Numerous studies have been conducted involving a variety of assays to hypothesize the mechanism behind Tea Tree Oil’s efficacy. Potential modes of action, as have been identified in bacteria, include inhibition of respiration, alterations to the cellular membrane, cellular leakage, and salt intolerance. The bacteria and fungi utilized in research are frequently human pathogenic agents; consequently, their efficacy may provide a pivotal role in medicine and medicinal uses (1, 5, 6, 7, 8, 9).
One study looked at the potential mechanism of Tea Tree Oil on the bacteria, Staphylococcus aureus (ATCC 9144). This study not only looked at the effects of Tea Tree Oil itself, but also included three of its components: terpinen-4-ol, α-terpineol, and 1,8-cineole. Several assays were implemented, including those observing bacteriolysis, loss of 260-nm absorbing material, bacterial killing, salt tolerance, and electron microscopy differentiations (6).
Before the study was carried out, the Minimum Inhibitory Concentration values (MICs) for these four treatment groups were determined via a broth microdilution method. This enabled the attainment of appropriate concentration levels, which could then be used throughout the entirety of the experiment. The MIC values for Tea Tree Oil, terpinen-4-ol, and α-terpineol were determined to be 0.25%, whereas 1,8-cineole was 0.50% (6).
Nucleic acids within the cell absorb wavelengths of 260-nm; thus, with their loss, there is likely leakage from the cell. Differences in 260-nm absorbing material were analyzed via spectrophotometry between experimental and control groups. Samples were taken and tested at 0, 30, and 60 minutes at the MIC levels for each treatment group. After 30 minutes, only 1,8-cineole exhibited significant differences in wavelength readings. All treatment groups displayed significant difference after 60 minutes. Mean ratios were determined for each experimental group and compared to the mean ratios for the untreated sample using the two-tailed Student t test (Figure 1). Thus, as can be seen in Figure 1, 1,8-cineole provided the greatest leakage of 260-nm absorbing material, likely corresponding to leakage of nucleic acids (6).
Another assay observed salt tolerance changes in S. aureus. Three plates were used for both control and experimental groups: 0, 50, and 75 g/L NaCl. The 50 and 75 g/L concentrations were chosen as they only moderately compromised the colony-forming abilities of untreated organisms in a preliminary study. The introduction of the treatment groups at varying levels based on the MICs, .5x, and 1x for terpinen-4-ol and α-terpineol, and .5x, 1x, and 2x for TTO and 1,8-cineole, enabled the determination of Colony-Forming Units (CFU). Exposure to TTO and its components produced extreme decreases in CFU (Figure 2). Surprisingly, 1,8-cineole produced drastic reductions in CFU, especially at 1x and 2x its MIC value (6).
A Time-Kill Assay was incorporated to evaluate the changes in CFU counts before and after treatment, thereby enabling the efficacy to be determined. The assay observed the effects of the treatment groups over 2-hours at varying amounts based on the respective MIC values: .5x, 1x, and 2x the MIC. Samples were taken at 0, 30, 60, 90, and 120 minutes, with additional samples to be taken after witnessing rapid killing. The CFU values were recorded, and reduction of CFU counts was observed after an overnight incubation. All treatments were relatively ineffective at half of their MIC values, and were comparable to the control group. Treatment with α-terpineol was inconsistent with other treatments in that 1x the MIC did not reduce viability in a constant manner, where the other treatment groups were moreso consistent. 1,8-cineole also portrayed strange results in that 1x and 2x the MIC elicited almost the same reductions in CFU. The other three components demonstrated significant differences between these changes in concentrations. Terpinen-4-ol provided the most rapid reduction at 2x its MIC within approximately 10 minutes. Terpinen-4-ol achieved in ~10 minutes, what others still had not achieved within 120 minutes (See Figure 3) (6).
Electron Microscopy was incorporated to determine if cellular lysis took place. Electron Microscopy compared S. aureus cells treated with terpinen-4-ol, as well as those that were untreated. This part of the experiment used heart infusion broth as a medium, and was supplemented with Tween 80 (a detergent). The experimental group was treated with 0.3% terpinen-4-ol, and was allowed to sit for 10 minutes, whereas the control group sat for 10 minutes without treatment. After centrifugation, pellets were treated and fixed overnight. Ultrathin sections were taken from the groups and were stained with uranyl acetate and lead citrate. As can be seen in the pictures from electron microscopy (Figure 4), there were various alterations once treated, including an amorphous and depleted appearance and the presence of mesosome-like structures. Electron microscopy displayed that cellular lysis did not take place. Therefore, it is suggested that TTO and its constituents follow a different mode of action than outright lysis (6).
This study suggests that the mode of action of Tea Tree Oil on S. aureus lies in its ability to alter the cellular membrane in some manner. This modification has to enable leakage from the cytoplasm, as seen in the loss of 260-nm absorbing material. Additionally, it seems the cellular alteration lead to salt intolerance of relatively harmless concentrations. Furthermore, electron microscopy indicated that terpinen-4-ol did not lyse cells, but did alter their shape and appearance. It would thereby appear that the primary mechanism of Tea Tree Oil is to compromise the integrity of the cellular membrane compared to lysis of the cells, which is the mode of action of other antimicrobial agents (6). The research also confutes previous statements that 1,8-cineole may have little to no antimicrobial activity (1).
Another study, conducted by S.D. Cox, et al, looked at the mode of action of Tea Tree Oil on Escherichia coli strain AG100, by performing respiration and K+ leakage assays. K+ leakage assays were performed to determine if TTO acts on alterations of the cellular membrane and, most specifically, on the associated K+ ion channels. A previous study determined the minimum bactericidal concentration (MBC) of TTO on this strain to be 0.25%. A standardized bacteria culture of E. coli was prepared on Iso-sensitest media at 37ºC. Cultures were harvested after either 4 hours of growth (indicating exponential phase cells) or 48 hours (indicating stationary phase cells), to enable comparison between the differing growth rates for the assays. The cells were washed in sodium phosphate buffer to maintain pH and were resuspended with 0.5% Tween-20, a detergent (7).
The respiration assay utilized a Clark-type oxygen and oxygen meter to ultimately measure respiration rates. E. coli undergoes aerobic respiration and, as a result, will utilize available oxygen. With decreases in oxygen levels for a closed system, it would be noted that these decreases are a direct result of the bacteria undergoing aerobic respiration, for which E. coli uses glucose. Various Tea Tree Oil concentrations were added to cell suspensions and were pre-incubated for 5 minutes with stirring at 25°C. D-glucose was added to initiate glucose-dependent aerobic respiration, and after 5 minutes, oxygen concentration was measured in each of the treatment groups. Accordingly, a lack of consumed oxygen correlated with an inhibition of this glucose-dependent respiration (7).
TTO was not effective at concentrations less than 0.125%. However, at double the MBC, or 0.5%, Tea Tree Oil completely inhibited cellular respiration in exponential phase cell suspensions. Additionally, suspensions for both stationary and exponential phases at 0.25 and 0.5% were associated with rapid cell death. Thereby, the results from the respiration assay indicate that Tea Tree Oil inhibits the glucose-dependent respiration at its MBC level in both the exponential phase cell and stationary phase cell suspensions (7).
The K+ leakage assay was executed by determining the initial extracellular K+ concentrations and comparing those values to post-treatment concentrations. Readings were performed by using a combination potassium ion selective/reference electrode that was connected to a pH/mV meter. The net difference was expressed as a percentage of total free K+ concentrations in 109 CFU cells. The total free K+ was determined by sonication-induced lysis, as with cell death, cells are no longer able to retain K+ and skew equilibrium concentrations. There were four experimental groups, including the combinations of stationary vs. exponential, as well as 0.25% vs. 0.5% (7).
Results from the K+ leakage assay indicated that both levels, 0.25% and 0.5%, were efficacious when acting on both the exponential and stationary phases. Leakage of K+ ions began 1 minute after addition of TTO. Furthermore, the results indicated that the K+ ion leakage was much slower in the stationary phase cells, compared to the exponential phase; this was true for both percentage levels (7).
This study suggests that an important mode of action of Tea Tree Oil includes its ability to inhibit glucose-dependent respiration and K+ retention within E. coli. The efficacy of these effects is strongly correlated to the stability of the cellular membrane structures. As previously mentioned, TTO effect of K+ leakage was considerably slower in the stationary phase of cell suspensions. This is believed to be due to alterations in the cell membrane that occur in transitioning from the exponential to the stationary phase. This shift is associated with alterations in the composition of fatty acids in the inner membrane, as well as an increase on the charge of the membrane due to an increase in the production of lipopolysaccharide. Therefore, it is believed that these changes increase the resistance to the effects of TTO (7).
In a similar experiment performed on E. coli (AG 100), whole cell autolysis was taken into account to determine if TTO’s mode of action on E. coli is outright lysis. After the addition of 1x and 2x the MBC in samples, it was observed that the E. coli cells died much more rapidly than they autolysed. Therefore, it was suggested that autolysis is a secondary occurrence, following TTO-induced death. Loss of electron-dense material and observation of coagulated material (potentially representing denatured membranes or proteins) were observed via electron microscopy (8). Similar findings were also found in the aforementioned study on S. aureus, conducted by C.F. Carson et al. In this study, TTO was not believed to perform outright lysis, but was believed to work via alteration in the cellular membrane and induce loss of cytoplasmic material (6).
Pseudomonas aeruginosa (NCTC 6749) has proven to be an interesting bacterium in that it demonstrates strong resistance to a wide range of biocides (9). This resistance is attributed to the unique lipopolysaccharide composition of its outer membrane. Lipopolysaccharide is typically associated with a negative charge, and it is hypothesized that this charge affects how TTO acts on the cell. Mann, et al (2000) conducted a study to observe the effects of TTO, alone, and with polymyxin B nonapeptide (PMBN). PBMN has a permeabilizing action and interacts with the lipopolysaccharide layer without releasing the lipopolysaccharide or the outer membrane protein. This interaction should enable TTO, or a similar agent, to display an increased ease of penetration, and thereby effect on P. aeruginosa (9).
The MICs and MBCs were determined with and without the addition of PMBN for TTO, and three of its components: y-terpinene, p-cymene, and 1,8-cineole. The results showed that the addition of PMBN significantly reduced the MIC and MBC values for all of the test components (Table 3). It is also notable that the initial MIC and MBC for P. aeruginosa was considerably high, compared to that of other bacteria. TTO has an MIC for S. aureus of 0.25%, and an MBC for E. coli of 0.25%, compared to P. aeruginosa where TTO has an MIC of 4.0% and an MBC of 8.0%. Both of these bacteria lack an outer membrane similar to P. aeruginosa, suggesting that the outer membrane composition is important to its resistance, and how important it is that TTO diffuses across the cellular membrane for its action to take effect. This can be noted as TTO exhibits efficacy on S. aureus and E. coli, both of which lack an outer membrane of lipopolysaccharide. TTO can be efficacious on P. aeruginosa, but only in the presence of PMBN, which permeabilizes the outer membrane. However, P. aeruginosa with an intact outer membrane, is resistant to TTO (9).
Effect of 10 μg/mL PMBN on the MIC/MBC of TTO and
Some of its Monoterpene Components Against P. aeruginosa NCTC 6749 (9)
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