Eastern connecticut state university

The effect of the essential oil and its components

Senior Honors Thesis

by

Rachel E. Schmid

of the Requirements of the

University Honors Program

May 2010

Ross E. Koning

Honors Council Member Date

Phillip F. Elliott

Director, University Honors Program Date

Phillip F. Elliott

Abstract

The oil of Melaleuca alternifolia, tea tree oil, has been shown to exhibit broad spectrum antimicrobial activity. By using Bacillus cereus as a model organism for B. anthracis, tea tree oil’s ability to inhibit germination of endospores along with the component(s) of the oil responsible for this action was tested. The activity of eight of the main components of the oil including terpinen-4-ol, γ-terpinene, α-terpinene, 1,8-cineole, α-pinene, p-cymene, α-terpineol, and limonene was tested using a disc diffusion method. Synergisms between the active and active and active and inactive components were also evaluated. Terpinen-4-ol, α-terpinene, and α-terpineol were active against endospore germination. Significant synergisms were observed between terpinen-4-ol and α-terpineol, α-terpinene and 1,8-cineole, α-terpinene and p-cymene, α-terpineol and 1,8-cineole, and α-terpineol and γ-terpinene. Parallel work with B. anthracis endospores is suggested as well as altered concentrations for some of the required standards for commercial production of the oil.

Introduction

History

As researchers examine naturally occurring antimicrobial agents, the essential oil of Melaleuca alternifolia, commonly known as tea tree oil (TTO), has exhibited promising results. Native to Australia, M. alternifolia, is a small, summer flowering tree with tapered leaves that grow up to a length of 20 mm (Carson and Riley 1993). It was originally found in a small region on the northeast coast of Australia called New South Wales, which is characterized by a low elevation and swampy terrain (Carson and Riley 1993). Since Australia’s colonization in 1788, the medicinal use by Bundjalong aborigines in this region has been documented; they historically used the leaves and small branches of the Melaleuca plant to treat headaches and minor sickness, as an insect repellant, and to produce vapors to treat respiration aliments (Carson and Riley 1993). Additionally, damaged leaves were soaked in water and either ingested orally or poured on the body (Carson and Riley 1993). It was not until the early 1900s that the antimicrobial action of the distilled oil was discovered (Penfold and Grant 1925). Since that discovery, commercial production of the oil and various testing has been ongoing.

Numerous studies have shown that TTO not only exhibits antibacterial properties, but also antifungal, antiviral, antiprotozoan, and anti-inflammatory properties (Table 1). Its bactericidal abilities encompass Gram positive and negative bacteria of various genera. In addition to TTO’s broad-spectrum antibacterial capabilities, work with antibiotic resistant Staphylococcus aureus (MRSA) has shown sensitivity to topical applications of TTO in concentrations of 1-2% (Carson et al. 1995).

Tea tree oil is produced by steam distillation or hydrodistillation of the subepidermal oil glands in the leaves of the Melaleuca tree and is light yellow in color with a distinct odor (Baker et al. 2000; Carson and Riley 1993). Brophy et al. (1989) tested over 800 samples of tea tree oil and identified about 100 components in M. alternifolia oil. They identified terpinen-4-ol to be the most abundant and also identified the other major components in the oil (Table 2). Although the original standard was set specifically for the ‘Oil of Melaleuca alternifolia,’ the current industry standard, which can encompass other species of Melaleuca, is called ‘Oil of Melaleuca' (Carson and Riley 1993; Carson et al. 2006). The 6 varieties of Melaleuca alternifolia oil include a terpinen-4-ol chemotype, a terpinolene chemotype, and four 1,8-cineole chemotypes, each exhibiting similar effects on the pathogenesis of organisms (Homer et al. 2000). The name ‘Oil of Melaleuca (terpinen-4-ol)’ is used in commercial TTO production today, and the International Organization for Standardization (ISO) has regulations on the minimum and maximum percentages for 14 components of the oil, most notably ≥30% terpinen-4-ol and ≤15% 1,8-cineole (ISO 2004) (Table 2). Originally, these regulations were promulgated because terpinen-4-ol was believed to be the active ingredient, and 1,8-cineole was falsely reported to be a skin irritant (Carson et al. 2006). Currently, the standard remains in place because 1,8-cineole and terpinen-4-ol are generally found in inverse proportions in the oil, and the oil with higher concentrations of terpinen-4-ol are found to exhibit more antimicrobial activity (Brophy et al. 1989; Carson et al. 2006).

Tea tree oil is composed mostly of cyclic monoterpenes, with about half being oxygenated and the other half being non-oxygenated terpenoid hydrocarbons (Cox et al. 2000). By using liposomes, terpenes have been shown to work by gathering in the membrane bilayer to cause a loss of membrane integrity and disruption of the proton-motive force (Sikkema et al. 1995; Cox et al. 1998). Other studies indicate that terpenes are able to disturb or pass through lipid layers (Cox et al. 1998). Using several test bacteria, many studies indicate that the mechanism by which TTO works in bacteria is similar to that by which terpenes work, to disrupt the structure and normal function of membranes (Cox et al. 1998; Cox et al. 2000; Carson et al. 2002; Carson et al. 2006).

The complexity of the ratio of components found in the oil indicates that a combination of some of the components is likely to contribute to the broad-spectrum antimicrobial activity. Terpinen-4-ol and α-terpineol constitute the majority of the antibacterial and antifungal action (Carson et al. 2006). Along with terpine-4-ol and α-terpineol, α-pinene has been shown to be active against bacteria (Raman et al. 1995; Sikkema et al. 1995). Additionally, the components linalool and limonene have shown antimicrobial activity (Janssen 1989 from Sikkema et al. 1995; Cox et al. 1998). The varying concentrations of the components in the oil along with the incompletely elucidated synergistic and antagonist relationships among these components make identifying all of the active ingredients of the oil difficult.

Terpinen-4-ol, the major component of TTO, has been acknowledged as the prime antimicrobial component of the oil because it is generally present in the highest concentration (Carson and Riley 1995). It has a Rideal-Walker (RW) coefficient (a measure of the disinfecting power of a substance) of 13, which is higher than that of TTO alone having a RW coefficient of 11 (Penfold and Grant 1925). α-terpineol has an RW coefficient of 16 (Penfold and Grant 1925), but is only found in relatively small percentages in the oil (Carson and Riley 1993) (Table 2). Additionally, TTO that contains higher levels of this component have shown reduced antimicrobial activity, because those samples also generally contain higher 1,8-cineole and lower terpinen-4-ol levels (Carson and Riley 1993). Terpinen-4-ol has shown more antimicrobial activity than TTO itself, because the non-oxygenated components found in the oil (for example p-cymene and γ-terpinene) reduce its solubility in water (Cox et al. 2001). To complicate matters more, cineole does not have an accepted role in antimicrobial action, but it is thought to play a role in permeating the cell membrane allowing other components of the oil to enter (Carson et al. 2002, Carson et al. 2006).

The broad antimicrobial capabilities of M. alternifolia oil have been explored using many organisms (Table 1). One piece missing from the literature is the potential ability to inhibit endospore germination. Specific genera of bacteria, including the aerobic heterotrophs of genus Bacillus, are able to form endospores when they come under environmental stress (Nicholson et al. 2000). Endospores are resistant to many agents including: heat, radiation, and various cleaning components (Nicholson et al. 2000). Although dormant in their spore form, they are able to germinate when conditions improve (Nicholson et al. 2000). This mechanism allows these organisms an effective strategy to survive unharmed through hostile conditions (Nicholson et al. 2000). Endospores are important in biological studies because they are thought to be some of the most durable propagules of living organisms (Nicholson et al. 2000). Because TTO is able to safely and effectively eliminate many species of live bacteria, fungi, and other pathogens, the present study explores its ability to inhibit germination of dormant, resistant endospores using Bacillus cereus.

This study uses B. cereus as a model organism for B. anthracis due to the lack of a proper containment laboratory for work with the latter. Although presenting vastly different phenotypes, the genomes of both bacteria are highly similar, with their functional differences present on plasmids (Helgason 2000). B. cereus and B. anthracis are argued to belong to a single species (Helgason 2000). The pathogenicity of B. cereus is much milder than that of B. anthracis. B. cereus is a ubiquitous soil bacterium and opportunistic human pathogen, causing food poisoning primarily in the dairy industry (Helgason 2000). B. anthracis is an acute infection that can be initiated via three avenues: cutaneous, gastrointestinal, and by inhalation (Brook 2002). Although generally a zoonotic disease, it has received recent media attention due to the use of anthrax endospores in biological warfare (Brook 2002).

In 2001, twenty-two mail workers were infected and five were killed after coming in contact with B. anthracis endospores on the envelopes of mailed letters (Shane 2010). Although some survived, the fatal infections were due to inhalation of the endospores. However many victims exhibited cutaneous infections as well (Brook 2002). The panic caused over that fine white powder resulted in a swift change in the bioterrorism budget, with the Postal Service spending hundreds of millions of dollars in the clean-up and the federal government increasing its biodefense budget drastically (Shane 2010). The suspect was a microbiologist working for a biodefense laboratory of the U.S. Army who produced the weapons-grade endospores (Shane 2010). Naturally produced anthrax endospores can be lethal, too. Anthrax is a common barnyard inhabitant, and in 2006 and 2008 U.K drum-makers died of exposure to inhaled endospores found on imported animal skins used to make drum heads (BBC 2008). Less deadly anthrax infections are those caused by the cutaneous mode, resulting in about 20% mortality without antibiotics and 1% with them (Brook 2002). These types of infections are the most common and are generally seen in agricultural regions where the presence of the bacterium is not adequately controlled in livestock (Brook 2002). One of the potential outcomes of this study is to promote further research into the effectiveness of TTO in bacterial spore germination, more specifically with the cutaneous infections of B. anthracis.

Materials and Methods

Preparation of Bacteria

Stock cultures of Bacillus megaterium and Bacillus cereus were obtained from Carolina Biological Company. The bacteria were cultured on Lysogeny Broth phytagel (Sigma Chemical Co.) plates for 24 hours at 32 °C. Single colony isolates of these bacteria were subcultured under the same conditions. From these isolates, broth cultures were routinely prepared in 2 ml of Lysogeny Broth in sterile transformation tubes. These were placed on an Eppendorf Thermomixer R shaker at 32°C at 1000 rpm for 8 days. This long-term culture is needed to increase the number of cells, to deplete the LB medium, and to initiate endospore production (Piggot and Coote 1976).

Single colony isolates of B. cereus were placed in 2 mL LB broth in an Eppendorf shaker at 32°C at 1000 rpm for 10 hours. 3 µL of this was placed in 2 mL of fresh LB broth and allowed multiply for 6 hours. This ensured that the bacteria were rapidly growing and the innoculum was devoid of ungerminated endospores. 100 µL of this young broth culture was spread on an LB plate and 250 µL was heat treated at 85°C for 15 minutes. 100 µL of the heat treated broth culture was spread on LB plates. This process was repeated 6 hours later. Both plates were incubated at 32°C for 16 hours. Vegetative cells were effectively eliminated during the heat treatment. A Schaeffer-Fulton stain was used on each treatment.

Mature LB broth cultures of bacteria were heat-treated at 85°C for 15 minutes to kill the viable vegetative cells and leave only viable endospores. 100 µL of the mature both cultures were spread on LB phytagel plates. After 10 minutes of drying in the laminar flow hood, autoclaved Whatman 3M chromatography paper discs were placed in contact with the phytagel surface. A small volume (1-2.5 µL) of tea tree oil or its components was placed on each disc. Because the oils are volatile, the cultures were placed in Ziploc plastic bags, separated by the treatment to prevent cross-treatments, and incubated at 32°C for at least18 hours. The width of any clear halo or static growth around each disc in the lawn of germinated bacterial spores was measured in mm with a metric ruler. It was measured from the edge of the paper disc to the edge of the halo. This value was considered a measure of the effectiveness of the treatment in inhibiting spore germination. This process was repeated for all subsequent procedures.

The general endospore selection and assay procedure was followed for each tube containing B. cereus and B. megaterium. The tea tree oil, made by NOW® Personal Care in Bloomingdale, IL, was obtained from a retail store just prior to beginning the experiment. Each 3M disc was dosed with 3 µl of either tea tree oil, terpinen-4-ol, γ-terpinene, α-terpinene, 1,8-cineole, α-pinene, p -cymene, α-terpineol, or limonene, all obtained from Sigma Chemical Co. Control discs did not receive an oil or component treatment. All discs on each plate were dosed with the same component.

After this step, B. megaterium was no longer used due to insufficient lawn growth.

Determination of dose effects for each active component

Each 3M disc received 0.5 µl, 1.0 µl, 1.5 µl, or 2.0 µl of terpinen-4-ol, α-terpinene, or α-terpineol in one LB phytagel plate. Four replicate plates were prepared. This test was repeated two more times on six plates for each component using 1.0 µl, 1.5 µl, 2.0 µl, or 2.5 µl of each active component.

Four 3M discs per plate were treated with 1.0 µl of terpinen-4-ol, 1.0 µl of α-terpinene, 1.0 µl of α-terpineol, a combination of two of these, or nothing (a dry control disc). Six replicate plates were prepared for each combination test.

A similar procedure was followed to test for interaction between the three active components: terpinen-4-ol, α-terpinene, and α-terpineol with the major inactive components of the oil: limonene, γ-terpinene, 1,8-cineole, α-pinene, p-cymene. Four 3M discs per plate were treated with 1.0 µl of an active component, 1.0 µl of an inactive component, a combination of the two, or was left as an untreated control disc, respectively. Six replicate plates were prepared for each combination test.

The Melaleuca alternifolia oil sample and each individual component was checked for purity using GC/MS. 50 µl of each oil or component was dissolved in 1.5 ml of optima grade dichloromethane (CH₂Cl₂) and placed in a PTFE- capped auto sampler vial. The analysis of the contents of each vial was carried out using a Shimadzu GCMS-QP2010 Plus quadrupole mass spectrometer. Separation was achieved using a capillary RTX-5MS 30 m x 0.25 mm i.d., 0.25 µm column. Helium was used as the mobile phase at 0.99 ml/min column velocity. The injector was held at 250°C with a split ratio of 100:1. The temperature was increased linearly at 7.5°C/min from 50°C to 250°C with a ten minute hold. Post column ions were generated by electron impact (70kEv) and data were collected from 3.5 min to 30 min in scanning mode (TIC) at 384 data scans/sec.

An ANOVA was used to determine the statistical significance (p≤0.05) observed among the dose effects of the active components. An ANOVA was also used to evaluate the differences observed among the components used in the synergisms between active and between the active and the inactive components. A Tukey-Kramer Post Hoc test was performed as a follow-up in order to reveal which results were significantly different from others.

Results

Temperature Sensitivity
The vegetative Bacillus cereus control cell suspension placed in 85°C for 15 minutes resulted in no colony growth. The suspension of bacterial cells that was depleted of nutrients had endospores emerge that were resistant to the heat treatment. By using the heat treatment, any bacterial cells that remained were killed so the study could concentrate on endospore germination. The spores were able to germinate after they were exposed to fresh LB media in plates, and placed in a 32°C environment for 24 hours.


Figure 1. An example of the zones of inhibition created in the lawn of germinated endospores around Whatman 3M discs dosed with 3 µL of TTO.

Dose Effects
The oil of Melaleuca alternifolia was active against endospore germination in B. cereus. It was determined that three of the eight most-abundant components of the oil were active against endospore germination: terpinen-4-ol, α-terpinene, and α-terpineol (Fig. 2). The other five components gave no significant response. None of the active components were significantly more active than another or than the TTO sample at each given dose.
Synergistic Effects of Active Components

A significant synergism between terpinen-4-ol and α-terpineol was observed (Fig. 3).

Further analysis with a Tukey-Kramer Post Hoc test revealed that the treatment with both components showed significantly more inhibition than either one of the individual components on its own.
Synergistic Effects between Inactive and Active Components

A significant synergism between 1,8-cineole and α-terpinene was observed (Fig. 4). Further analysis with a Tukey-Kramer Post Hoc test revealed that all three were significantly different from each other. The combination of the two components was more effective than either of the single components. Another significant interaction between α-terpinene and p-cymene was observed (Fig. 5). A Tukey-Kramer Post Hoc test revealed the combination treatment was significantly different from both single treatments. A significant synergism between 1,8-cineole and α-terpineol was observed (Fig. 6). A Tukey-Kramer Post Hoc test revealed that all three components tested for this interaction were significantly different from each other. 1,8-cineole showed significant interactions with all active ingredients but terpinen-4-ol. -Terpineol and γ-terpinene also showed significant synergistic effects (Fig. 7). A Tukey-Kramer Post Hoc test shows that all three of these components gave significantly different results from each other.

Most of the commercially available individual components of tea tree oil tested had minor components that could yield significant effects on inhibition of endospore germination. We also found that the individual-component α-terpinene commercial sample was only 76.46% pure with o-cymene and 1,8-cineole as major contaminants (Table 4). The only pure component found to lack contaminants was 1,8-cineole (eucalyptol) (Table 4).

Discussion

terpinen-4-ol	α-terpineol	α-terpinene

1,8-cineole and γ-terpinene were not shown to be active on their own against endospore germination, but they significantly enhanced the performance of α-terpineol and α-terpinene (Fig. 4 and Fig. 6-7). This suggests they have important roles in the overall sporicidal action. 1,8-cineole has more impact because of its synergistic effect with two of the active components (Fig.3 and Fig. 6). In bacteria, it has been shown that cineole disrupts membranes to allow entry of the active components (Carson et al. 2002; Carson et al. 2006). α-terpinene showed significant interactions with two of the major components found in the sample, possibly contributing to its overall action. It was shown that almost 20% of the α-terpinene oil sample was composed of 1,8-cineole and o-cymene, and both of these exhibit significant synergisms with α-terpinene (Table 4).

γ-terpinene	1,8-cineole	p-cymene

As finding alternatives in the treatment of infectious diseases becomes increasingly vital, TTO may have an important role to play. This study suggests promise in the oil’s role in preventing germination of endospores. Additionally, the lipophilic property of the TTO allows it to penetrate the skin, suggesting it may be particularly suitable in the treatment of cutaneous infections (Calcabrini et al. 2004). Further research in a containment laboratory with endospores of B. anthracis is suggested with hopes of finding promise in preventing or healing endospore-based cutaneous infections, particularly in countries where refrigeration of heat-labile antibiotics is impractical or impossible.