Medicinal and Aromatic Plants—Industrial Profiles


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part of The Gordon and Breach Publishing Group.

IOLOGICAL ACTIVITY
173
between oils with similar MIC values could be demonstrated by studies of death kinetics
(Lattaoui and Tantaoui-Elaraki 1994).
Use of Solubilising and Emulsifying Agents
Broth and agar dilution methods have generally been developed for use with watersoluble
preparations and require modification for use with essential oils of low water solubility. To
ensure contact between the test organism and tea tree oil for the duration of the assay it is
necessary to use a solubilising or emulsifying agent. The agents most commonly used are
Tween 80 and Tween 20 at concentrations ranging from 0.001 to 20% (Beylier 1979; Walsh
and Longstaff 1987; Chand et al. 1994; Carson et al. 1995b; Griffin et al. 1998). DMSO
(Scortichini and Rossi 1991; Aboutabl et al. 1995), DMF (Kubo et al. 1991), ethanol (Morris
et al. 1979; Deans and Svoboda 1988; Biondi et al. 1993) and 0.15–0.2% agar have also
been used (Remmal et al. 1993; Mann and Markham 1998).
Use of emulsifying and solubilising agents may result in changes in the physicochemical
properties of the test system, even though they have no antimicrobial activity when tested
on their own. It has been reported that nonionic surfactants, such as the Tween compounds,
form micelles above a concentration known as the critical micelle concentration. Lipophilic
molecules, such as the components of tea tree oil, may become solubilised within the micelles
and thus partitioned out of the aqueous phase of the suspension (Schmolka 1973). Kazmi
and Mitchell (1978) have shown that antimicrobials solubilised within the surfactant do not
contribute to the activity as they do not come into direct contact with the microorganisms.
The amount of material solubilised increases at higher concentrations of surfactant (Van
Doorne 1990). Results from our laboratory support this view: the antimicrobial activity of
tea tree oil decreased as the concentration of Tween 20 in the test medium was increased
from 0.1% to 5% (Mann and Markham 1997). Premixing of the oil with Tween before
addition to the test medium would be expected to exacerbate this problem. The impact of
such effects must also be considered in the formulation of pharmaceutical and cosmetic
products, as the interaction between components may reduce the activity of the active
ingredients.
Other studies suggest that low concentrations of surface-active agents in the mixture
may actually enhance the activity of the antimicrobial through causing changes to the
permeability of the cell membrane of the microorganism (Denyer and Baird 1990). This
raises doubts as to the suitability of agents such as Tween 80 and 20 in the assay procedures.
Van Doorne (1990) reports that ethanol, in concentrations as low as 5%, can have a marked
potentiating effect on the activity of antimicrobial agents and these authors question its use
as a solubilising agent.
To obtain consistent, reproducible results it is important that contact between the oil and
the microorganism is maintained throughout the test period. Allegrini et al. (1973) reported
that emulsions of essential oils in water containing 1% Tween 80 or Tween 20 disintegrated
within one hour but that emulsions with 5% Tween were stable for 24 hours, the incubation
period of many assays. However, as stated above, Tweens at this concentration exert an
inhibitory effect. A more suitable dispersing agent for tests carried out in broth is
bacteriological agar at concentrations of 0.15–0.2% (w/v) (Mann and Markham 1998;
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

JULIE L.MARKHAM
174
Remmal et al. 1993). Emulsions containing 0.15% bacteriological agar were stable for 19
hours (Mann and Markham 1998). As well as the stability of the emulsion, the use of agar
as a stabiliser has the advantages of its lack of chemical reactivity and, as reported in the
study of Remmal et al. (1993), MIC values are lower than when Tween 20, Tween 80,
Triton X100 or ethanol are included as emulsifiers or solubilisers.
To conclude this section, several aspects of the methods which have been described
should be highlighted. Firstly, test conditions do not generally reflect the actual conditions
of use of the preparation. Thus, an agent may be active when in direct contact with an
organism in a liquid medium, but may be inactivated in the presence of blood or pus in a
wound, or be unable to penetrate unbroken skin and hence not reach the contaminated site
in sufficient concentration to be effective. Exposure time, particularly at concentrations
near the MIC, may not be sufficient, particularly in external use, unless the agent has residual
activity.
Secondly, some tests measure only inhibition of growth of the test organisms, not a
lethal effect. Whilst the former are the tests most commonly reported in the literature, the
link between these measurements, which generally involve prolonged contact (18 hours or
more) between the organism and the agent, and in vivo use, are questionable. Even where
such methods are adapted to also measure MBC no indication is given of the death kinetics
(rate of kill).
Thirdly, the test methods which have been published in the literature have been
developed for water-soluble compounds, and they do not always give reliable results with
non-water-soluble oils and may underestimate the true antimicrobial activity. Adaptations
are needed to ensure adequate and consistent contact between the oil and the test organism
throughout the period of the test. The importance of standardisation of methods must be
stressed.
Lastly, the method of formulation of the product can profoundly affect the physical and
biological properties of the active agent. Thus, it cannot be assumed that a formulation will
be effective simply because a specific amount of active ingredient has been included. This
also highlights the need for microbiological testing of products to ensure that they are effective
at inhibiting or eliminating microorganisms: chemical tests to measure concentrations of
active ingredients are more precise, but do not, on their own, provide evidence of the efficacy
of the product. Laboratory tests are important, but results of these tests can still only be
regarded as a useful preliminary to clinical trials.
Antimicrobial Activity of Tea Tree Oil
Penfold and Grant (1925) first demonstrated the activity of tea tree oil in the RidealWalker
test, a standard test of the period, which employed Bacillus typhosus (now known as
Salmonella typhi) as the test organism. The Rideal-Walker coefficient was reported to be
11, indicating that tea tree oil is 11 times more effective than phenol. The results in 
Table 1
show that tea tree oil also compares very favourably with a number of other essential oils
when tested by this method.
Very little further work was done until the 1970s when Low et al. (1974) reported MIC
values of 1:16 against S. aureus and 1:32 against Salmonella typhi, and Beylier (1979)
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IOLOGICAL ACTIVITY 175
included tea tree oil in a study of the antimicrobial activity of ten essential oils derived from
Australian native plants. Tea tree oil was effective against the five test organisms, which
included both Gram-positive and Gram-negative bacteria, a yeast and a mould. The MIC
value against S. aureus (0.25–0.2%) was considerably lower than that reported by Low et
al. (1974).
Both tea tree oil and formulations containing tea tree oil at concentrations of 5% have
been reported to pass the Therapeutic Goods Act (TGA) test for antiseptics and disinfectants
(Graham 1978), which includes type strains of the Gram-positive bacterium Staphylococcus
aureus and the Gram-negative Escherichia coli, Proteus vulgaris and Pseudomonas
aeruginosa as test organisms (Altman 1989). The oil has also been reported to pass both
USP and BP preservative efficiency tests in semi-solid formulations at concentrations of
approximately 0.5–1% (Altman 1991), although higher concentrations may be needed to
satisfy the requirements of the test in relation to Aspergillus niger (this volume, 
Chapter
12
).
The very broad spectrum of activity of tea tree oil, a highly desirable characteristic in an
antiseptic or disinfectant, has been confirmed by a number of studies published during the
last decade. 
Table 2
 identifies the organisms which have been tested by various authors and
presents MIC values for a large number of organisms from a single study (Griffin et al.
1998). These results suggest the enormous potential of tea tree oil in a variety of applications,
including the treatment of external conditions such as acne, tinea, thrush and staphylococcal
and streptococcal infections, in oral hygiene products, in the disinfection of cooling towers
contaminated with Legionella, and in agricultural uses. Not only has the sensitivity of many
species of bacteria and fungi to tea tree oil been demonstrated, but some studies have
examined the susceptibility of large numbers of recent clinical isolates, as well as type
strains, of particular species (see Table 2 for references). Such data provides valuable
information about the variability in sensitivity of organisms likely to be encountered in
therapeutic use of the oil.
The majority of studies report MIC values and a comparison of results for commonly
tested organisms from different studies is presented in 
Table 3
. Although there is variation
in MIC values against different strains within a species and between studies, the majority of
values are less than 1%. Lack of consistency of results may be accounted for by differences
in oil composition, in test organisms and methods of determining MIC values. For example,
Table 1 Antimicrobial acitivity of some essential
oils (Rideal-Walker coefficient data adapted from
Penfold and Grant (1925) and Schilcher (1985))
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

JULIE L.MARKHAM
176
the relatively high MIC values reported by Raman et al. (1995) may result from leaving oil/
agar combinations to stand overnight at 55°C to allow mixing before preparing dilutions
and inoculating, a practice likely to result in the loss of volatile active ingredients.
Overall, there are no apparent differences in sensitivity between Gram-positive and Gram-
negative bacteria in most studies. In studies where MBC values are also reported (for example,
see Hammer et al. 1996) they are similar to the MIC value for some organisms, but several
dilutions higher against other organisms. Generally, MIC and MBC values tend to be closer
Table 2 Spectrum of antimicrobial activity of tea tree oil
1
The numbers in brackets refer to the number of isolates tested.
2
a Beylier (1979); b Bassett et al. (1990); c Williams et al. (1993); d Southwell
et al. (1993); e Raman et al. (1995); f Carson and Riley (1994); g Carson et al.
(1995a); h Carson et al. (1995b); i Hammer (1996); j Hammer et al. (1996); k
Griffin et al. (1998); l Carson et al. (1996); m Nenoff et al. (1996).
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IOLOGICAL ACTIVITY
177
for Gram-negative than for Gram-positive bacteria, suggesting that there may be differences
in the mechanism of action between the two groups of organisms.
Pseudomonas aeruginosa has been consistently shown to be far more resistant to tea tree
oil, as well as to a number of other disinfectants or antiseptics, than other organisms tested
(Table 3; Janssen et al. 1986; Williams et al. 1993). Some variation does occur between
strains, but MIC values are generally above 2%. It has been reported that oils high in sabinene-
hydrate, the precursor of terpinen-4-ol in the plant, are more active against Pseudomonas
aeruginosa than standard tea tree oil (Markham et al. 1995), as are some aged oils (Markham
et al. 1996).
The study of Hammer et al. (1996) reports an interesting difference between the sensitivity
of bacteria which are part of the normal skin flora, such as Micrococcus spp. and coagulase-
negative staphylococci, and some pathogenic organisms, termed transients, which may be
transmitted via the hands to people and surfaces. When a large number of strains were
examined, MBC
90
 values for the first group were higher than for some of the transients
organisms, suggesting that handwashes containing tea-tree oil may reduce transmission of
pathogens via the hands, while causing minimal disturbance to the normal flora.
In order to assess the potential of tea tree oil in oral hygiene products, two studies of the
sensitivity of anaerobic and facultatively anaerobic oral bacteria, including species of
Actinomyces, Bacteroides, Fusobacterium, Peptostreptococcus and Streptococcus to tea tree
oil have been reported. Walsh and Longstaff (1987) used both broth and agar dilution methods
Table 3 Comparison of tea tree oil minimum inhibitory concentration (MIC) data determined by an
Agar
a
 or Broth
b
 dilution method and expressed as %, v/v
nd not determined. *n refers to the number of isolates tested.
1
Griffin et al. (1998); 
2.1
Carson et al. (1995b); 
2
.
2
Hammer et al. (1996); 
2.3
Carson and Riley (1994);
2.4
Hammer (1996); 
3
Beylier (1979); 
4
Walsh and Longstaff (1987); 
5
Raman et al. (1995).
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

JULIE L.MARKHAM
178
and recorded MIC values in the range 0.02 to 0.08%, indicating a greater sensitivity of
these organisms than the aerobic organisms included in the study. Shapiro et al. (1994)
report higher MIC values (0.1 to >0.6%) for a similar group of organisms. There are a
number of possible explanations of these differences. Firstly, different isolates of the same
species and different species in the same genus do not always have the same sensitivity to
an antimicrobial agent; secondly, as reviewed earlier in this chapter, results are affected by
variations in test methodology and thirdly, the composition of the oils were not clearly
defined.
The susceptibility of fungi and viruses to tea tree oil has been less extensively studied.
With the fungi the focus has been on species included in standard tests such as Aspergillus
niger and Candida albicans, on post-harvest fungal pathogens (Bishop and Thornton 1997)
and on several dermatophytes implicated as causative agents of skin conditions such as
tinea (
Table 2
). Published MIC data indicates that tea tree oil is inhibitory to the dermatophytes
at concentrations up to 1%. Data of minimal fungicidal concentrations is lacking, but the
results of preservative efficiency tests suggest that fungicidal concentrations will be at least
several times higher than the MIC values. This is also suggested by the clinical study of
Tong et al. (1992) in which a formulation containing 10% tea tree oil was evaluated in the
treatment of tinea of the foot. Although subjects reported an improvement in symptoms,
there was no mycological cure.
Inactivation of viruses has not been reported, however, a recent study has reported that
plants treated with tea tree oil were more resistant to infection with tobacco mosaic virus
than untreated controls (Bishop 1995). Testing of viruses, such as herpes simplex virus and
human papilloma viruses, which cause infection of the skin and mucous membranes in
humans, would be useful in determining whether there is a potential for products aimed at
the treatment of cold sores, genital herpes or warts.
Relationship Between Chemical Composition and Antimicrobial Activity
Gas chromatography studies have shown that tea tree oil is a complex mixture of
approximately 100 terpenes, the concentrations of which can vary widely between oils
(Brophy et al. 1989). Although the composition which optimises antimicrobial activity has
not been completely defined, there is strong evidence that terpinen-4-ol, a monoterpene
alcohol, is the most significant antimicrobial component and the ISO Standard 4730 “Oil of
Melaleuca Terpinen-4-ol Type” (International Standards Organisation 1996) stipulates a
minimum concentration of 30% of this component. Antimicrobial activity increases rapidly
as the concentration of terpinen-4-ol increases up to approximately 40%, with only a slight
further increase at concentrations above this (Williams et al. 1993; Southwell et al. 1993;
Griffin et al. 1998). MIC values begin to rise when the concentration of terpinen-4-ol drops
below 30%. Terpinen-4-ol has a Rideal-Walker coefficient of 12–13.5 (Penfold and Grant
1925), and MIC values of 0.2–0.6% against S. aureus, 0.06–0.2% against E. coli and 0.2–
0.25% against C albicans have been reported (Carson and Riley 1995; Griffin 1995; Raman
et al. 1995).
The significance of the other components is less clear. The antimicrobial activity of a-
terpineol, which is also a monoterpene alcohol, and hence more water soluble than the
hydrocarbon components of the oil, has been reported in a number of studies, with MIC
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IOLOGICAL ACTIVITY
179
values in a similar range to those for terpinen-4-ol (Deans and Svoboda 1989; Nguyen et al.
1994; Carson and Riley 1995; Griffin 1995; Raman et al. 1995). This compound is only a
minor component in tea tree oil (typically approximately 3%) and, as a result, its contribution
to the oil’s antimicrobial activity is probably limited.
Knobloch et al. (1989) reports a correlation between water solubility and antimicrobial
activity of terpenoids, however, the diffusion method used in the study to determine
antimicrobial activity does not adequately assess the activity of non-water-soluble
components (Southwell et al. 1993). Other studies report that a number of the hydrocarbon
components present in tea tree oil, including 
α- and γ-terpinene, terpinolene and p-cymene
do possess varying degrees of antimicrobial activity, but the results are conflicting and
difficult to compare because of differences in the purity of compounds, test methods and
test organisms (Deans and Svoboda 1989; Nguyen et al. 1994; Williams and Lusunzi 1994;
Carson and Riley 1995).
The contribution of the minor components to the properties of the oil is not well
understood, but given the complexity of the composition of the oil, there is considerable
potential for interactions between various components. There are reports in the literature
that certain monoterpenes affect the functioning of cell membranes (Brown et al. 1987), but
whether this is the primary effect of tea tree oil on microbial cells is yet to be determined. It
is important to determine the mechanism of action before an understanding of the significance
of the minor components can be achieved. In determining the optimal antimicrobial chemistry
of tea tree oil, it will also be important to maintain other valuable properties of the oil,
especially the reported antiinflammatory and mild anaesthetic effects, in the treatment of
conditions of the skin and mucous membranes.
Two aspects of the chemistry of tea tree oil which deserve further consideration are the
significance of 1,8-cineole and changes which occur in oils with aging. These are discussed
below.
Significance of 1,8-cineole
In recent years there has been a great deal of controversy over the significance of 1,8cineole
in tea tree oil. The Standard stipulates a maximum concentration of 15% for 1,8-cineole.
The purpose of this maximum limit for cineole is to exclude oils from the high cineole
chemotypes of Melaleuca alternifolia, which have low levels of terpinen-4-ol, as such oils
are less effective antimicrobials. In recent years, there has been a push towards minimising
the level of cineole and as a result low cineole oils (<5%) have become popular in the
market place.
The most commonly stated reason for minimising cineole concentrations in tea tree oil is
that it is claimed to be a skin irritant (Lassak and McCarthy 1983; Barnes 1990; Carson et
al. 1995b; Raman et al. 1995). However, these claims are unsubstantiated. A recent study
has reported no irritancy or allergenicity due to 1,8-cineole in any of the 25 participants,
and, although there were three cases of allergy, none were due to cineole (Southwell et al.
1997). In another study, in which twenty-eight women were treated daily with vaginal
pessaries impregnated with tea tree oil containing 9.1% 1,8-cineole, no irritation of the
mucous membranes was reported (Bélaiche 1985a). The long-term use of eucalyptus oil,
which contains approximately 75% 1,8-cineole, as a chest rub also supports this view. There
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

JULIE L.MARKHAM
180
is evidence that a small number of individuals do have an adverse skin reaction to tea tree
oil, but the component responsible varies from person to person and there is only one
documented case of a skin reaction to cineole (de Groot and Weyland 1992). There is also
a report of irritancy due to terpinen-4-ol (Knight and Hausen 1994). The toxicity of tea tree
oil is discussed in 
Chapter 10
 of this monograph and readers are referred to it for further
information.
Another possible reason for maintaining levels of cineole below 5% would be that such
oils were more effective as antimicrobials. When tested as a pure compound, conflicting
results for MIC values of 1,8-cineole are reported. A number of workers report low activity
(Low et al. 1974; Cruz et al. 1989; Williams et al. 1993; Raman et al. 1995; Griffin 1995),
while other studies report zones of inhibition comparable to those obtained with terpinen-4-
ol and a-terpineol and MIC values between 0.16 and 0.5% against various test organisms
(Deans and Svoboda 1989; Nguyen et al. 1994; Carson and Riley 1995). Williams et al.
(1988, 1993) used the disc diffusion assay to compare several oils with different levels of
1,8-cineole and terpinen-4-ol and report that oils become less active against C. albicans as
cineole concentrations increase. The difference was less marked when the same oils were
tested against E. coli and S. aureus. However, the reported effects may be attributable to
changes in concentration of terpinen-4-ol rather than 1,8-cineole. The lack of reproducibility
of the disc diffusion method used also limits the significance of these results.
In a more comprehensive study of eight oils blended to contain various concentrations of
1,8-cineole between 1.5 and 28%, there were no significant differences found in MIC values
(Table 4, Southwell et al. 1996). By comparing the results for Oil 6 with Oils 7 and 8, it can
be seen that it is important that the level of terpinen-4-ol remains above 30%. Similar results
were obtained with a further 12 Gram-positive and Gram-negative bacteria (Mann and
Markham 1997), providing convincing evidence for the view that oils with ultra low levels
of cineole are not superior in terms of antimicrobial activity. Thus there is no scientific basis
for the market push for ultra low cineole oils. In fact, it has been reported that 1,8-cineole is
Table 4 Effect of 1,8-cineole on MIC values determined by an agar
dilution method using Isosensitest agar with 0.25% Tween 20
*Tea tree oil samples were mixed with 1,8-cineole to give final concentrations of
1,8-cineole ranging from 1.5–28.5%.
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