Melaleuca alternifolia (Tea Tree) Oil: a Review of Antimicrobial and Other Medicinal Properties
ANTIMICROBIAL
ACTIVITY IN VITRO
Of all of the properties claimed for TTO, its antimicrobial
activity has received the most attention. The earliest reported use of
the M. alternifolia plant
that presumably exploited this property was the traditional use by the
Bundjalung Aborigines of northern New South Wales. Crushed leaves of “tea
trees” were inhaled to treat coughs and colds or were sprinkled on wounds,
after which a poultice was applied (135).
In addition, tea tree leaves were soaked to make an infusion to treat sore
throats or skin ailments (101, 135).
The oral history of Australian Aborigines also tells of healing lakes, which
were lagoons into which M.
alternifolia leaves had fallen and decayed over time (3).
Use of the oil itself, as opposed to the unextracted plant material, did not
become common practice until Penfold published the first reports of its
antimicrobial activity in a series of papers in the 1920s and 1930s. In
evaluating the antimicrobial activity of M. alternifolia oil and other oils, he
made comparisons with the disinfectant carbolic acid or phenol, the gold
standard of the day, in a test known as the Rideal-Walker (RW) coefficient. The
activity of TTO was compared directly with that of phenol and rated as 11 times
more active (121).
The RW coefficients of several TTO components were also reported, including 3.5
for cineole and 8 for cymene (122),
13 for linalool (123),
and 13.5 for terpinen-4-ol and 16 for terpineol (121).
As a result, TTO was promoted as a therapeutic agent (5-7). These publications, as well as several others (60, 70, 84, 102, 120, 124, 152),
describe a range of medicinal uses for TTO. However, in terms of the evidence
they provide for the medicinal properties of TTO, they are of limited value,
since by the standards of today the data they provide would be considered
mostly anecdotal.
In contrast, contemporary data clearly show that the
broad-spectrum activity of TTO includes antibacterial, antifungal, antiviral,
and antiprotozoal activities. Not all of the activity has been characterized
well in vitro, and in the few cases where clinical work has been done, data are
promising but thus far inadequate.
Evaluation
of the antimicrobial activity of TTO has been impeded by its physical
properties; TTO and its components are only sparingly soluble in water (Table (Table2),2), and this limits their miscibility in test media.
Different strategies have been used to counteract this problem, with the
addition of surfactants to broth and agar test media being used most widely (11, 13, 15, 31, 32, 61).
Dispersion of TTO in liquid media usually results in a turbid suspension that
makes determination of end points in susceptibility tests difficult.
Occasionally dyes have been used as visual indicators of the MIC, with mixed
success (31, 32, 40, 104).
Antibacterial Activity
The few reports of the antibacterial activity of TTO appearing in
the literature from the 1940s to the 1980s (11, 15, 100, 153)
have been reviewed elsewhere previously (35).
From the early 1990s onwards, many reports describing the antimicrobial
activity of TTO appeared in the scientific literature. Although there was still
a degree of discrepancy between the methods used in the different studies, the
MICs reported were often relatively similar. A broad range of bacteria have now
been tested for their susceptibilities to TTO, and some of the published
susceptibility data are summarized in Table Table3.3. While most bacteria are susceptible to TTO at
concentrations of 1.0% or less, MICs in excess of 2% have been reported for
organisms such as commensal skin staphylococci and micrococci, Enterococcus faecalis,
and Pseudomonas
aeruginosa (13, 79). TTO is for the most part bactericidal in nature,
although it may be bacteriostatic at lower concentrations.
The
activity of TTO against antibiotic-resistant bacteria has attracted
considerable interest, with methicillin-resistant Staphylococcus aureus (MRSA)
receiving the most attention thus far. Since the potential to use TTO against
MRSA was first hypothesized (153),
several groups have evaluated the activity of TTO against MRSA, beginning with
Carson et al. (31), who examined 64 MRSA isolates from Australia and
the United Kingdom, including 33 mupirocin-resistant isolates. The MICs and
minimal bactericidal concentrations (MBCs) for the Australian isolates were
0.25% and 0.5%, respectively, while those for the United Kingdom isolates were
0.312% and 0.625%, respectively. Subsequent reports on the susceptibility of
MRSA to TTO have similarly not shown great differences compared to
antibiotic-sensitive organisms (39, 58, 68, 106, 115).
For the
most part, antibacterial activity has been determined using agar or broth
dilution methods. However, activity has also been demonstrated using time-kill
assays (34, 48, 80, 106), suspension tests (107), and “ex vivo”-excised human skin (108). In addition, vaporized TTO can inhibit
bacteria, including Mycobacterium
avium ATCC 4676 (105), Escherichia coli, Haemophilus influenzae, Streptococcus pyogenes,
and Streptococcus
pneumoniae (85). There are anecdotal reports of aerosolized TTO
reducing hospital-acquired infections (L. Bowden, Abstr. Infect. Control Nurses
Assoc. Annu. Infect. Control Conf., p. 23, 2001) but no scientific data.
Mechanism of antibacterial action.
The mechanism of action of TTO against bacteria has now been
partly elucidated. Prior to the availability of data, assumptions about its
mechanism of action were made on the basis of its hydrocarbon structure and
attendant lipophilicity. Since hydrocarbons partition preferentially into
biological membranes and disrupt their vital functions (138), TTO and its components were also presumed to
behave in this manner. This premise is further supported by data showing that
TTO permeabilizes model liposomal systems (49). In previous work with hydrocarbons not found in
TTO (90, 146a) and with terpenes found at low concentrations
in TTO (4, 146), lysis and the loss of membrane integrity and
function manifested by the leakage of ions and the inhibition of respiration
were demonstrated. Treatment of S.
aureus with TTO resulted in the leakage of potassium ions (49, 69) and 260-nm-light-absorbing materials (34) and inhibited respiration (49). Treatment with TTO also sensitized S. aureus cells to
sodium chloride (34) and produced morphological changes apparent under
electron microscopy (127).
However, no significant lysis of whole cells was observed
spectrophotometrically (34) or by electron microscopy (127).
Furthermore, no cytoplasmic membrane damage could be detected using the lactate
dehydrogenase release assay (127),
and only modest uptake of propidium iodide was observed (50)
after treatment with TTO.
In E. coli, detrimental
effects on potassium homeostasis (47), glucose-dependent respiration (47), morphology (67), and ability to exclude propidium iodide (50)
have been observed. A modest loss of 280-nm-light-absorbing material has also
been reported (50).
In contrast to the absence of whole-cell lysis seen in S. aureus treated
with TTO, lysis occurs in E.
coli treated with TTO (67), and this effect is exacerbated by cotreatment
with EDTA (C. Carson, unpublished data). All of these effects confirm that TTO
compromises the structural and functional integrity of bacterial membranes.
The loss
of viability, inhibition of glucose-dependent respiration, and induction of
lysis seen after TTO treatment all occur to a greater degree with organisms in
the exponential rather than the stationary phase of growth (67; S. D. Cox, J. L. Markham, C. M. Mann, S. G.
Wyllie, J. E. Gustafson, and J. R. Warmington, Abstr. 28th Int. Symp. Essential
Oils, p. 201-213, 1997). The increased vulnerability of actively growing cells
was also apparent in the greater degree of morphological changes seen in these
cells by electron microscopy (S. D. Cox et al. Abstr. 28th Int. Symp. Essential
Oils, p. 201-213). The differences in susceptibility of bacteria in different
phases of growth suggest that targets other than the cell membrane may be
involved.
When the
effects of terpinen-4-ol, α-terpineol, and 1,8-cineole on S. aureus were
examined, none was found to induce autolysis but all were found to cause the
leakage of 260-nm-light-absorbing material and to render cells susceptible to
sodium chloride (34). Interestingly, the greatest effects were seen
with 1,8-cineole, a component often considered to have marginal antimicrobial
activity. This raises the possibility that while cineole may not be one of the
primary antimicrobial components, it may permeabilize bacterial membranes and
facilitate the entry of other, more active components. Little work on the
effects of TTO components on cell morphology has been reported. Electron
microscopy of terpinen-4-ol-treated S.
aureus cells (34) revealed lesions similar to those seen after TTO
treatment (127), including mesosome-like structures.
Mechanism
of action studies analogous to those described above have not been conducted
with P. aeruginosa. Instead,
research has concentrated on how this organism is able to tolerate higher
concentrations of TTO and/or components. These studies have indicated that
tolerance is associated with the outer membrane by showing that when P. aeruginosa cells
are pretreated with the outer membrane permeabilizer polymyxin B nonapeptide or
EDTA, cells become more susceptible to the bactericidal effects of TTO,
terpinen-4-ol, or γ-terpinene (99, 103).
In summary, the loss of intracellular material, inability to
maintain homeostasis, and inhibition of respiration after treatment with TTO
and/or components are consistent with a mechanism of action involving the loss
of membrane integrity and function.
Antifungal
Activity
Comprehensive
investigations of the susceptibility of fungi to TTO have only recently been
completed. Prior to this, data were somewhat piecemeal and fragmentary. Early
data were also largely limited to Candida albicans, which
was a commonly chosen model test organism. Data now show that a range of
yeasts, dermatophytes, and other filamentous fungi are susceptible to TTO (14, 42, 52, 61, 116, 128, 140)
(Table (Table4).4). Although test methods differ, MICs
generally range between 0.03 and 0.5%, and fungicidal concentrations generally
range from 0.12 to 2%. The notable exception is Aspergillus
niger, with minimal fungicidal concentrations (MFCs) of as high as 8%
reported for this organism (74). However, these assays were performed with
fungal conidia, which are known to be relatively impervious to chemical agents.
Subsequent assays have shown that germinated conidia are significantly more
susceptible to TTO than nongerminated conidia (74), suggesting that the intact conidial wall
confers considerable protection. TTO vapors have also been demonstrated to
inhibit fungal growth (86, 87) and affect sporulation (88).
Mechanism of antifungal
action.
Studies investigating the mechanism(s) of antifungal action have
focused almost exclusively on C. albicans. Similar
to results found for bacteria, TTO also alters the permeability of C. albicans cells.
The treatment of C. albicans with 0.25% TTO resulted in
the uptake of propidium iodide after 30 min (50),
and after 6 h significant staining with methylene blue and loss of
260-nm-light-absorbing materials had occurred (72). TTO also alters the permeability of Candida
glabrata (72). Further research demonstrating that the
membrane fluidity of C. albicans cells treated with 0.25%
TTO is significantly increased confirms that the oil substantially alters the
membrane properties of C. albicans (72).
TTO also
inhibits respiration in C. albicans in
a dose-dependent manner (49). Respiration was inhibited by
approximately 95% after treatment with 1.0% TTO and by approximately 40% after
treatment with 0.25% TTO. The respiration rate of Fusarium
solani is inhibited by 50% at a concentration of 0.023% TTO (88). TTO also inhibits glucose-induced medium
acidification by C. albicans, C. glabrata,
and Saccharomyces cerevisiae (72). Medium acidification occurs largely by
the expulsion of protons by the plasma membrane ATPase, which is fuelled by ATP
derived from the mitochondria. The inhibition of this function suggests that
the plasma and/or mitochondrial membranes have been adversely affected. These
results are consistent with a proposed mechanism of antifungal action whereby
TTO causes changes or damage to the functioning of fungal membranes. This
proposed mechanism is further supported by work showing that the terpene
eugenol inhibits mitochondrial respiration and energy production (46).
Additional
studies have shown that when cells of C. albicans are
treated with diethylstilbestrol to inhibit the plasma membrane ATPase, they
then have a much greater susceptibility to TTO than do control cells (72), suggesting that the plasma membrane
ATPase has a role in protecting cells against the destabilizing or lethal
effects of TTO.
TTO inhibits the formation of germ tubes, or mycelial conversion,
in C. albicans (52, 78). Two studies have shown that germ tube
formation was completely inhibited in the presence of 0.25 and 0.125% TTO, and
it was further observed that treatment with 0.125% TTO resulted in a trend of
blastospores changing from single or singly budding morphologies to multiply
budding morphologies over the 4-h test period (78). These cells were actively growing but
were not forming germ tubes, implying that morphogenesis is specifically
inhibited, rather than all growth being inhibited. Interestingly, the
inhibition of germ tube formation was shown to be reversible, since cells were
able to form germ tubes after the removal of the TTO (78). However, there was a delay in germ tube
formation, indicating that TTO causes a postantifungal effect.
Antiviral Activity
The antiviral activity of TTO was first shown using tobacco mosaic
virus and tobacco plants (18).
In field trials with Nicotiniana glutinosa, plants
were sprayed with 100, 250, or 500 ppm TTO or control solutions and were then
experimentally infected with tobacco mosaic virus. After 10 days, there were
significantly fewer lesions per square centimeter of leaf in plants treated
with TTO than in controls (18).
Next, Schnitzler et al. (132) examined the activity of TTO and eucalyptus
oil against herpes simplex virus (HSV). The effects of TTO were investigated by
incubating viruses with various concentrations of TTO and then using these
treated viruses to infect cell monolayers. After 4 days, the numbers of plaques
formed by TTO-treated virus and untreated control virus were determined and
compared. The concentration of TTO inhibiting 50% of plaque formation was
0.0009% for HSV type 1 (HSV-1) and 0.0008% for HSV-2, relative to controls.
These studies also showed that at the higher concentration of 0.003%, TTO
reduced HSV-1 titers by 98.2% and HSV-2 titers by 93.0%. In addition, by
applying TTO at different stages in the virus replicative cycle, TTO was shown
to have the greatest effect on free virus (prior to infection of cells), although
when TTO was applied during the adsorption period, a slight reduction in plaque
formation was also seen (132). Another study evaluated the activities
of 12 essential oils, including TTO, for activity against HSV-1 in Vero cells (110). Again, TTO was found to exert most of
its antiviral activity on free virus, with 1% oil inhibiting plaque formation
completely and 0.1% TTO reducing plaque formation by approximately 10%.
Pretreatment of the Vero cells prior to virus addition or posttreatment with
0.1% TTO after viral absorption did not significantly alter plaque formation.
Some
activity against bacteriophages has also been reported, with exposure to 50%
TTO at 4°C for 24 h reducing the number of SA and T7 plaques formed on lawns
of S. aureus and E. coli,
respectively (41).
The results of these studies indicate that TTO may act against
enveloped and nonenveloped viruses, although the range of viruses tested to
date is very limited.
Antiprotozoal Activity
Two publications show that TTO has antiprotozoal activity. TTO
caused a 50% reduction in growth (compared to controls) of the protozoa Leishmania
major and Trypanosoma brucei at
concentrations of 403 mg/ml and 0.5 mg/ml, respectively (109). Further investigation showed that
terpinen-4-ol contributed significantly to this activity. In another study, TTO
at 300 mg/ml killed all cells of Trichomonas vaginalis (151).
There is also anecdotal in vivo evidence that TTO may be effective in
treating Trichomonas vaginalis infections (120).
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