Synergistic antifungal activity of tea tree (Melaleuca alternifolia) and lavender (Lavandula angustifolia) essential oils against dermatophyte infection

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Synergistic antifungal activity of tea tree (Melaleuca alternifolia) and lavender (Lavandula angustifolia) essential oils against dermatophyte infection

Dr John P. Cassella Reader in Biomedical Sciences, Division of Biological Sciences, School of Environmental and Applied Sciences, University of Derby, Kedleston Road, Derby, DE22 1GB, UK. E-mail: [email protected]

S . C a s s e l l a , J o h n P. C a s s e l l a and I. Smith

The antifungal potential of tea tree and lavender essential oils alone and in combination, against common causes of tinea infection in humans was investigated via in-vitro investigations, in order to determine a suitable dosage for use in clinical trials. The concept of synergy was considered, in the microbiological environment, and as a chemical phenomenon. Trichophyton rubrum and T. mentagrophytes var. interdigitale were studied, as the most prevalent causes of tinea and onychomycosis. Possible chemical interactions between essential oils were examined using gas chromatographymass spectrometry (GC-MS) infra-red (IR) spectroscopy and polarimetry. There was a clear antifungal action by both tea tree and lavender essential oils on these organisms grown in culture. In combination, appropriate blends demonstrated synergistic action. No changes in retention times or identified compounds were observed by GC-MS. Alterations were found using IR spectroscopy in some combinations of the essential oils. The inconsistency in findings between the two analytical techniques may in part be due to a difference in sensitivities of the techniques or the conditions used in the GC-MS equipment; different column parameters have yet to be trialled. These were time dependent and affected by changing temperature. The measurement of optical rotation was determined to be an inappropriate technique for the study of synergy in essential oil mixtures. The data from this study confirm that synergistic action does occur between these two commonly used essential oils in effecting antifungal activity. GC-MS analysis demonstrated that there was no chemical interaction resulting in a new compound that could be identified using the analytical equipment in this study. IR analysis supports the suggestion that synergistic action may be dependent upon reaction involving the numerous organic substances present in essential oils. These changes may be due to as yet unidentified transient chemical interactions between functional groups within the essential oil mixtures. © 2002 Elsevier Science Ltd. All rights reserved. 0962-4562/02/$ - see front matter © 2002 Elsevier Science Ltd.

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INTRODUCTION istorically, fungal infection

H

has taken low priority in the clinic, laboratory and medi-

cinal research. These infections are now occurring in ever-increasing numbers among neutropenic (low white blood cell count) cancer patients, transplant recipients and acquired immune deficiency syndrome (AIDS) patients, and are no longer uncommon disorders. Fungal infection of immunocompetent

subjects

is

considered

superficial, but can have a devastating psycho-social effect. In an arena where a holistic approach to health care is demanded, diseases such as onychomycosis and tinea capitis can no longer be viewed as simple ‘cosmetic problems’. Trichophyton rubrum and T. mentagrophytes var. interdigitale, both anthropophilic dermatophytes, are two of the most common causes of infection of the skin, nails, and sometimes hair, due to their propensity for keratin. Allopathic treatments for tinea infection and onychomycosis, including azoles and allylamines can have unpleasant side effects such as hepatoxicity and alopecia. There is a necessity for long periods of treatment (up to 18 months), exacerbating side effects, and they are considered by many GPs as prohibitively

expensive

(Kavalier,

1999; Rose & Wilson, 1999; Harris, 1999). The combination of these factors leads to a poor record for elimination of infection. These organisms may be viable in the environment for up to 15 months, presenting a significant public health issue. The UK, and indeed, world, population is showing an increasing desire to use natural alternatives to pharmaceutical products. With the known toxicity and side effects of antifungal drugs, the administration of these could be problematic in already compromized subjects. The development

of a ‘natural’ treatment/prophylaxis for

The word ‘synergy’ is derived

fungal infections would present an

from two Greek words meaning ‘work-

alternative where such drugs are con-

ing together’. It is claimed that whole

traindicated or refused. Fungal infec-

essential oils have been found in prac-

tions are also especially problematic in

tice to be more effective than their

warm, humid areas (equatorial/tropi-

isolated principal constituents, on

cal) and such communities in particu-

account of the synergistic effects (Hall,

lar would benefit from cheap natural

1904). Constituents present in very

products

small amounts are often found to be

rather

than

expensive

as active as or even more active than

imported drugs. The fungicidal effect of certain

the principal constituent. Apart from

essential oils was first described in

the synergy produced by the compo-

1927 by Myers. The antifungal effects

nents of a single oil, there is also an

of essential oils, particularly that of tea

enhancement of effect when two or

tree (Melaleuca alternifolia), have been

more whole oils are mixed together

widely studied in recent years (Tong

(Price & Price, 1999).

et al., 1992; Buck, 1994; Nenoff et al.,

Whilst the evidence for the benefi-

1996; Hammer et al., 1998, 2000;

cial therapeutic use of synergistic blends

Lis-Balchin et al., 2000; Inouye et al.,

is scarce, the documented evidence of

2001; D’Auria et al., 2001). These

potential dangers is virtually non-

could provide a viable treatment for

existent. Numerous uses of essential oils

tinea infection. However, a rigorous

and their blends is based largely on

study of the safety and efficacy of

anecdotal evidence as to their effective-

essential oils for such a clinical use has

ness and have not been published in

not yet occurred (Ernst & Huntley,

scientifically accepted or peer-reviewed

2000). In a small published trial

literature (Lis-Balchin et al., 1997).

(Goodwin & Hardiman, 2000), tea tree

There is also a misconception that

oil was reported as an efficacious treat-

any ‘blend’ of essential oils will exhibit

ment for tinea infection. However, this

a synergistic effect. Lis-Balchin et al.

trial used undiluted essential oil, with

(1997) noted that the synergy/antago-

serious consequences (lower limb

nism question is a complex one, and

dermatitis) for some participants.

that a ‘blend’ demonstrating one type

The main aim of the current study

of synergy, e.g. antimicrobial, may not

was to determine the optimum dosage

give synergistic action in other ways,

range of essential oils for potential use

e.g. smooth muscle activity.

in a clinical trial. This preliminary

As well as being described as an

in-vitro study will provide baseline data

antifungal

for lavender and tea tree oils upon

1997), lavender (Lavandula angustifolia)

which to build ex-vivo and in-vivo stud-

essential oil is often considered as the

ies of tinea infection of human tissue.

ideal essential oil to use in synergistic

(Inouye,

2001;

Caddy,

In using essential oils, it is a

blends, due to its range of properties

widely established practice of aroma-

and effects. Therefore, the current

therapists to mix (or blend) these for

study investigated the individual, and

administration by various techniques.

combined antifungal effects of laven-

It is an accepted premise by practicing

der and tea tree essential oils. In order

aromatherapists (Price & Price, 1999)

to elucidate the possibility of synergistic

that essential oils act in a more benefi-

activity, the in-vitro antifungal effects of

cial manner when ‘mixed’ or ‘blended’

these two essential oils, both singly, and

in this way; this effect is known as

as various blends, was studied in paral-

‘synergy’.

lel with chemical analysis of the blends.

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The comparison of the efficacy of

encountered with other techniques,

detailed analysis of essential oil com-

antifungal agents is routinely assessed

and ensures greater exposure of the

ponents can be made, by reference to a

by means of their minimum inhibitory

full surface area of the fungal culture to

database of organic chemicals. If any

concentration, or minimum fungicidal

the essential oils, this method utilized

synergistic, or indeed antagonistic,

concentration. The determination of

a sealed system. Whilst the report

effect resulting from the blending of

these values is notoriously problematic

demonstrated that the growth of the

essential oils has a chemical basis, it

for filamentous fungi (Guarro et al.,

organisms was unaffected, there was no

would be expected that a ‘new’ chemi-

1997; Manavathu et al., 1999; Pujol

clear demonstration that the action of

cal moiety be detectable if the GC-MS

et al., 1996, 1997). Variations in a

essential oils, or the interaction of

detector, column and other analytical

number of technical factors, such as

essential oils and fungi may be altered

conditions were appropriate.

inoculum size, type of medium, incu-

in the absence of adequate gaseous

The use of IR analysis alone in the

bation temperature and time of read-

exchange. Also, with a view to clinical

analysis of essential oils may be consid-

ing, can have a major effect on results

use, the necessity of enclosing the

ered of little use, due to the complex

obtained. The National Committee for

affected area in a sealed chamber

mixture of organic chemicals present

Clinical Laboratory Standards (USA)

might prove to be unattractive to

in a single oil. Fourier transform IR is

has established a standard method for

clients, resulting in problems with

very useful nevertheless; the finger-

antifungal susceptibility testing of

compliance.

print region of the IR absorption spec-

yeasts (NCCLS M27-A, 1997). Whilst

With all of these factors in mind,

trum will be unique for each essential

this has been amended for use with

the microbiological aspect of the cur-

oil (Williams, 1997). Therefore, IR

filamentous fungi (NCCLS M38-P),

rent study was designed to examine the

analysis of blends of essential oils

this method is to date a proposed stan-

effect of single, timed exposures to

might elucidate the possibility of inter-

dard only, and a reference technique

essential oils, singly or as mixtures.

actions between oils in a blend.

specifically for use with dermatophytes

The essential oils were diluted in a

Polarimetry, or analysis of optical

has yet to be established (Fernandez-

standard carrier oil, as might be used

rotational properties of essential oils, is

Torres et al., 2000; Jessup et al., 2000).

in aromatherapy practise. Short-term

also considered to be a useful indicator

Susceptibility testing with essential

exposure eliminated problems due to

of essential oil quality and it is one of a

oils is further complicated by inherent

volatility of the essential oils and the

number of physio-chemical techniques

difficulties due to their volatility, com-

necessity for the homogenous disper-

used routinely to help establish the

plexity and water insolubility. Various

sion of essential oils in nutrient media

authenticity of an essential oil. Hence

techniques have been utilized, includ-

was obviated. As with exposure to

this technique was also included for

ing agar overlay techniques, broth

vapour, the surface area of the fungus

the analysis of essential oil blends, to

macro- and microdilution techniques

exposed to essential oil was maximized

investigate the possibility of synergistic

and agar incorporation methods.

by this approach. In a clinical situation,

activity.

Owing to the volatility of essential

a product applied to an affected area

oils, coupled with the long incubation

might remain in-situ for a similarly

period necessary for the study of

short exposure time, before being

dermatophytes, the first of these meth-

removed by contact with clothing.

Essential oil source

ods was considered unsuitable for the

Likewise, a short exposure time,

Tea tree and lavender essential oils

current study. Many studies have used

followed by a long incubation period

were obtained from New Horizon

emulsifiers, surfactants or solvents to

better mimics the clinical situation

(Southampton, UK), Purple Flame

achieve dispersion of essential oils in

encountered due to client compliance.

(Kenilworth, UK) and Fragrant Earth

the culture medium. However, it has

The (physico-chemical) volatile

(Glastonbury, UK). Oils of each type

been demonstrated that the use of

nature of essential oils makes them well

were blended in equal volumes imme-

such agents may significantly decrease

suited for chemical analysis. Gas chro-

diately before use.

the antimicrobial action of some essen-

matographic analysis of essential oils in

tial oils (Remmal et al., 1993; Hill

comparison with reference compounds

Microbiology

et al., 1997). Inouye et al. (2001) uti-

allows identification of the components

Clinical (wild type) isolates of T. rubrum

lized a novel technique using exposure

within a blend or isolated essential oil

and T. mentagrophytes var. interdigitale

to vaporized essential oils. Whilst this

by comparison of retention times.

(referred to as T. mentagrophytes in

overcomes many of the problems

Coupled with mass spectrometry, a

this report) were obtained from the

MATERIALS AND METHODS

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The results were compared with

diagnostic microbiology service at the Derbyshire

Royal

Infirmary,

UK.

Equivalent 7 mm diameter plugs of each species were excised from the

Table 1

margin of a 14-day-old culture grown

Percentage mixtures of tea tree and lavender essential oils for IR spectroscopy and GC-MS

the individual chromatograms for each oil. The internal library database (Wiley database) was used to identify the peaks. Quinoline (0.05% v/v) was included in each of the analyses as an

on Sabouraud dextrose agar (SDA). These were immersed in dilutions of essential oils in sweet almond oil (New Horizon, Southampton, UK). Excess oil was blotted from the plugs using sterile filter paper; they were then transferred to SDA in 9 cm Petri dishes, sealed with gas-permeable tape, and incubated at 28 ⬚C for 4 weeks. For the preliminary experiments, fungi were exposed to dilutions of essential oils from 0–30% v/v at 5% intervals, for exposure times of 30 sec-

Tea tree oil (%)

Lavender oil (%)

0 10 20 30 40 50 60 70 80 90 100

100 90 80 70 60 50 40 30 20 10 0

A half-shadow polarimeter (Lippich type) was used to determine if there was any change in the specific rotation of the blended oils with reference to the values of the pure essential tea tree and lavender essential oils. Freshly prepared blends (as in Table 1) were placed in the polarimeter and the

study, a typical ‘checkerboard’ layout

range of dilutions (v/v, Table 1) were

was used to give dilutions of each essen-

prepared, stored in dark bottles and

tial oil from 0–100% at 10% intervals,

analyzed within 1 hour of preparation.

with an exposure time of 30 minutes.

Using a Perkin-Elmer 881IR spectrophotometer,

equal

volumes

of

sured at several different points for

essential oils singly, or as percent vol-

each culture and percentage inhibition

ume per volume mixtures (% v/v), were

of growth calculated using an adapta-

placed between sodium chloride plates

tion of the method published by Nidiry

and analyzed across an IR spectrum of

(1998). At day 28, the original ‘plugs’

4000–600 cm91. Ten subsequent scans

were taken from the plates where ‘no

were performed, in order to determine

growth’ was observed, washed in nor-

whether any transient changes in peak

mal saline solution, macerated, and

expression occurred.

subcultured onto fresh SDA plates.

temperature of the essential oils or

gistic antifungal activity was occurring,

mixtures thereof was raised to 37⬚C,

two

and the series of scans repeated.

of

analysis

were

specific optical rotation determined using the formula:

RESULTS Microbiology In the preliminary experiment, with exposure to single essential oils, 100% inhibition of T. rubrum was achieved with 25% v/v dilutions of lavender or tea tree oil respectively, with both 30-second and 30-minute exposure times. For T. mentagrophytes, none of the dilutions tested gave 100% inhibition

Using a heated air current, the

In order to assess whether synermethods

analysis of results.

Polarimetry

onds and 30 minutes. For the synergy

Diameters of growth were mea-

internal standard to allow quantitative

with either essential oil, the maximum inhibition being 80% (Fig. 1). By day 14, there was growth on all plates. By day 21 all plates had growth equivalent

employed, as described by Warnock

to control cultures.

ting isobolagrams for each of the

Gas chromatography mass spectrometry (GC-MS)

exposure to each essential oil individu-

fungal species. Secondly, fractional

Dilutions prepared for IR spectroscopy

ally confirmed the preliminary findings.

inhibitory concentrations (FICs) were

(Table 1) were also analyzed for GC-MS.

For T. rubrum at day 7, 30% v/v lavender

calculated for the components of those

A 5% solution of each mixture was

or 20% v/v tea tree oil gave 100% inhi-

blends of essential oils that appeared

prepared by dilution with dichlo-

bition. By comparison, the equivalent

visually to have synergistic action.

romethane and a 2 ␮l aliquot analyzed

effect upon T. mentagrophytes required

using GC-MS. The GC used was a

50% v/v lavender or 60% v/v tea tree oil.

Hewlett Packard 5890 series II with a

By day 14, the minimum dilution main-

Infra-red (IR) spectroscopy

Zebron ZB-1 30 m
0.25 mmID liquid

taining 100% inhibition for T. rubrum

To look at the potential chemical syn-

phase 100% methylpolysiloxane col-

was 40% v/v lavender or 30% v/v tea tree

ergy between different blends of tea

umn. A Hewlett Packard 5971 series

oil. For T. mentagrophytes, 100% inhibi-

tree and lavender essential oils, a

mass selective detector was used.

tion was not maintained by lavender

(1989). The first of these involved plot-

In the synergy experiment (Fig. 2),

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(B)

100 90

80

80

70

70 % Inhibition of growth

90

60 50 40

60

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% Inhibition of growth

100

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(A)

50 40

30

30

20

20

10

10

6 0

0 0.05

0.1

1

10

15

20

25

30

0.05

0.1

Dilution (%v/v) of essential oils in carrier oil Lavender

10

Tea tree (D)

80

15

20

25

30

70

60

60

50

40

30

25

30

Lavender

80

70

% Inhibition of growth

% Inhibition of growth

T h e I n t e r n a t i o n a l J o u r n a l o f A r o m a t h e r a p y 2002

Tea tree (C)

1

Dilution (%v/v) of essential oils in carrier oil

50

40

30

20

20

10

10

0

0 0.05

0.1

1

10

15

20

Dilution (%v/v) of essential oils in carrier oil

vol

Tea tree

Lavender

25

30

0.05

0.1

1

10

15

20

Dilution (%v/v) of essential oils in carrier oil Tea tree

Lavender

12

no 1

Fig. 1 Graphs demonstrating the effect of single essential oils upon the two species studied, with single, 30-second and 30-minute exposures. (A) Trichophyton rubrum 30-second exposure. (B) Trichophyton rubrum 30-minute exposure. (C) Trichophyton mentagrophytes var. interdigitale 30-second exposure. (D) Trichophyton mentagrophytes var. interdigitale 30-minute exposure.

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A)

(B)

Fig. 3 Isobolagrams for: (A) Trichophyton rubrum. (B) Trichophyton mentagrophytes var. interdigitale.

14 for both blends with T. rubrum. For T. mentagrophytes, the sum of fractional Fig. 2 Fungal cultures demonstrating the antifungal effect of essential oil blends at day 14 postexposure. 1. Trichophyton mentagrophytes var. interdigitale control. 2. Trichophyton mentagrophytes var. interdigitale. (20% lavender/30% tea tree). 3. Trichophyton mentagrophytes var. interdigitale. (20% lavender/40% tea tree). 4. Trichophyton rubrum control. 5. Trichophyton rubrum (10% lavender/10% tea tree). 6. Trichophyton rubrum (20% lavender/10% tea tree).

inhibitory concentrations for potential synergistic blends both at days 7 and 14 was 1 in all cases.

IR spectroscopy Figure 4 shows the spectra produced

essential oil, even at 100% concentra-

70% v/v lavender with 10% v/v tea tree

for the neat essential oils and some of

tion, whilst only a 90% v/v dilution of

oil, and 20% v/v lavender with 40% v/v

the blends examined. On first exami-

tea tree oil gave 100% inhibition.

tea tree oil.

nation, all spectra appeared to be quite similar. However, on further analysis it

For T. rubrum, the minimum dilu-

At days 21 and 28, those cultures

tion of essential oil blends giving 100%

that had shown no growth at day 14,

was

inhibition was 10% v/v lavender with

for both species, still maintained 100%

appeared/disappeared in subsequent

20% v/v tea tree oil, or 20% v/v lavender

inhibition, macroscopically. Washing

scans in some blends of lavender and

with 10% v/v tea tree oil. At day 14, the

and maceration of the initial plug at

tea tree essential oils. This did not

cultures that had been exposed to these

day 28 followed by subculture also gave

occur for subsequent scans of indivi-

blends maintained 100% inhibition.

no growth in all cases.

dual oils. Changes were also observed in

found

that

certain

peaks

some blends when the temperature was

For T. mentagrophytes at day 7,

Figure 3 shows the isobolagrams

essential oil blends giving 100% inhibi-

for each species. (Note: for T. mentagro-

raised to 37⬚C. These are too extensive

tion were 30% v/v lavender with 10% v/v

phytes, the minimum inhibitory dilu-

to be fully described here. However, as

tea tree oil and 10% lavender with 30%

tion of lavender essential oil at day 14

an example, results for blends of 40%

tea tree oil. Unlike the situation with T.

has been plotted as 100% v/v, to allow

tea tree and 60% lavender oil and 60%

rubrum, these dilutions did not main-

for visualization of potential synergistic

tea tree and 40% lavender oil follow.

tain efficacy by day 14. At day 14, the

effect). The sum of fractional inhibitory

A peak was present at 1560 cm91

minimum

blended

concentrations for potential synergistic

in all consecutive scans of 40% tea

oils giving 100% inhibition were

blends was 1 at day 7 and 1 at day

tree/60% lavender oil at ambient room

dilutions

of

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(B) 95.140

95.140

85.061

85.061

85.061

85.061

74.982

74.982

74.982

74.982

64.903

64.903

64.903

64.903

54.824

54.824

54.824

54.824

44.745

44.745

44.745

44.745

34.666

34.666

34.666

34.666

24.587

24.587

24.587

24.587

14.508

14.508

14.508

14.508

4.429

4.429

4.429

4.429

T h e I n t e r n a t i o n a l J o u r n a l o f A r o m a t h e r a p y 2002

–5.650 4000 3600 3200 2800 2400 2000

1700

1400

1100

800

–5.650 500

–5.650 4000 3600 3200 2800 2400 2000

1700

1400

1100

–5.650 500

800

(C) 100

100

(D) 100

100

90

90

90

90

80

80

80

80

70

70

70

70

60

60

60

60

50

50

50

50

40

40

40

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30

30

20

20

20

20

10

10

10

10

0 4000 3600 3200 2800 2400 2000

1700

1400

1100

800

0 500

0 4000 3600 3200 2800 2400 2000

1700

1400

1100

800

0 500

vol 12

Fig. 4 IR spectra for: (A) Tea tree essential oil. (B) Lavender essential oil. (C) 40% tea tree/60% lavender essential oil blend. (D) 60% tea tree/40% lavender essential oil blend.

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temperature (18⬚C). However, in the

tree/40% lavender oil blend at room

giving 100% inhibition confirm that

60% tea tree/40% lavender oil mixture

temperature. This peak was absent

these two essential oils have a synergistic

at room temperature, the presence of

in the first, third, fourth fifth, sixth,

antimycotic effect, particularly against

this peak was intermittent, i.e. it was

seventh, ninth and tenth scans, and

the more resistant T. mentagrophytes.

missing in the first, fifth, sixth and

present in the second and eighth scans.

There is a clear difference in the

tenth scans, but present in the second,

This peak was absent in the same blend

susceptibility of the two Trichophyton

third, fourth, seventh, eighth and

heated to 37 ⬚C. It was also absent in

species to the two essential oils used in

ninth scans. When this mixture was

the 40% tea tree/60% lavender oil

this study. This concurs with the obser-

heated to 37⬚C, and the series of scans

blend at room temperature, but was

vation that T. mentagrophytes is less

was repeated, this peak was present in

present in this blend at 37 ⬚C. It was

susceptible to antifungal drugs than is

all 10 consecutive scans. This peak was

absent from neat tea tree oil, but pre-

T. rubrum (Macura, 1993). This sug-

absent in all scans of neat lavender and

sent in lavender oil.

gests that clinically, it is desirable to confirm the causative agent of infec-

tea tree oils at room temperature and at 37⬚C when analyzed singly. Peaks at 948 cm91, 1092 cm91, 91

91

tion prior to treatment. It is also

GC-MS Figure

5

shows

chromatograms

apparent from this study that the levels

were pre-

obtained for neat tea tree and neat

of dilution of essential oils generally

sent in the 60% tea tree/40% lavender

lavender oils and for some of the

used in routine aromatherapy practise

blend in all consecutive scans, but

blends analyzed. Comparison of these

(which are particularly variable), are

absent in all consecutive scans of 40%

gave no indication of additional peaks

probably insufficient to have fungicidal

tea tree/60% lavender oil. With the

in blends of essential oils compared to

activity.

1070 cm

and 1050 cm

exception of the peak at 1092 cm91,

Clinically, susceptibility testing is

neat oils.

rarely employed for dermatophyte

the other peaks were all present in all scans of 60% tea tree/40% lavender oil at 37⬚C. In the 40% tea tree/60% lavender oil blend, the peak at 948 cm91 was present, whilst the rest were absent at 37 ⬚C. A peak at 834 cm91 showed inter-

infection (COLLINS, personal communi-

Polarimetry Specific rotation of the blends of the essential oils fell within the normal range of variation anticipated for the essential oils used.

cation). Nevertheless, for novel antifungal agents for such infections to be considered medically, their efficacy must be measured in this standard manner. The methodology used for the

mittent appearance in the 60% tea tree/40% lavender blend, being pre-

DISCUSSION

current study may be considered to

sent in the first, fourth, fifth, sixth, and

Tea tree and lavender essential oils

give only semiquantitative results. For

tenth scans, but absent in the second,

clearly

demonstrated

antimycotic

absolute quantitative determination, it

third, seventh, eighth and ninth scans.

activity

against

Trichophyton

is necessary to utilize a standardized

This peak was present in all scans of

species in-vitro. A previous study

number of ‘colony-forming units’ for

lavender essential oil, but not in tea

suggested that for T. rubrum this activ-

each species under consideration.

tree oil. At 37 ⬚C, this peak was present

ity is fungistatic rather than fungicidal

Also, the current study has utilized

in the 40% tea tree/60% lavender oil

(Cassella et al., 2001). However, further

only a single clinical isolate of each

blend and absent in the 60% tea

evaluation reported here has demon-

species.

tree/40% lavender oil blend.

strated that at lower dilutions, the

Further investigation is necessary

both

In the 40% tea tree/60% lavender

action is simply fungistatic. At higher

to determine minimum inhibitory and

blend at 37 ⬚C, peaks were consistently

concentrations, both oils seem to

minimum fungicidal concentrations

present at 738 cm91 and 782 cm91,

demonstrate fungicidal effects against

(MICs and MFCs), for essential oils, in

that were absent in the same blend at

T. rubrum. Against T. mentagrophytes,

parallel with pharmaceutical antifungal

room temperature. The peak at

lavender essential oil is fungistatic

agents for comparison. Ex-vivo and

738 cm91 was present in neat lavender

only, whilst a high concentration of tea

in-vivo clinical trials of essential oil

oil, but not tea tree oil, and that at

tree essential oil (90% v/v) is required

blends, for the treatment dermatophyte

782 cm91 was present in neat tea tree

for fungicidal activity.

infection, will indicate how well in-vitro

The isobolagrams and the sum of

MIC and MFC determination correlate

Finally, at 684 cm91 an intermit-

fractional inhibitory concentrations for

with clinically effective dosage. The

tent peak was observed in the 60% tea

the minimum blends of essential oils

possibility of the emergence of resistant

oil, but not lavender oil.

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(A)

(B)

(C)

(D)

Fig. 5 GC-MS chromatographs for: (A) Tea tree essential oil. (B) Lavender essential oil. (C) 40% tea tree/60% lavender essential oil blend. (D) 60% tea tree/40% lavender essential oil blend.

strains must also be considered. Care

Dermatophyte infection is partic-

in practise, as many sufferers may not

must be taken that the use of subopti-

ularly common, with some 15% of the

have identified (or revealed) that they

mal concentrations of essential oil

population being affected by athlete’s

have such conditions. The importance

blends does not contribute to such a

foot (Gentles & Evans, 1973). Massage

of the ability of practitioners to recog-

phenomenon. Further studies will uti-

and other manipulative techniques

nize potential symptoms of fungal

lize quantitative analysis, with large

have obvious potential for the spread

infection should be stressed. The

numbers of clinical isolates from differ-

of infection.

observed antimycotic synergy may

Aromatherapists and other practi-

have a chemical basis. Using a number

tioners should be particularly observant

of analytical methods, this study has

ent geographical locations, in order to address these issues.

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examined the effect of mixing tea tree

various groups of organic chemicals

For lavender essential oil alone,

and lavender essential oils.

within the oils (Franchomme and

the levels of alcohols and sequiter-

The absence of any obvious

Pénoël, 1990). It has been suggested

penes in the dilutions giving 100%

changes in retention times or identified

that effects attributable to specific

inhibition of T. rubrum at days 7 and 14

components by GC-MS, for any of the

chemical groups could be due to the

are comparable to those of the syner-

blends examined, as compared with

interaction of these groups with partic-

gistic blends.

neat essential oils would suggest that

ular cell surface receptors (Balacs,

This is also the case for lavender

no chemical reaction occurs between

1991). As a note of caution, it has been

vs T. mentagrophytes at day 7. However,

the two essential oils. However, clear

stressed that essential oils are very

at day 14, lavender essential oil failed

changes in vibrational energy levels

complex mixtures of organic chemicals

to give 100% inhibition of T. mentagro-

have been demonstrated using IR spec-

and such grouping may be an oversim-

phytes, even when undiluted. The levels

troscopy. Some of these changes were

plification. ‘Not enough is yet under-

of alcohols and sesquiterpenes in undi-

intermittent with time, and others were

stood about the pharmacological

luted lavender essential oil are 36%

temperature dependent. The pattern

effects of essential oils to know how

and 5%, respectively. In the synergistic

of changes observed is a complex one

each type may differ in effect’ (Price &

blends effective against T. mentagro-

and requires further analysis. However,

Price 1999).

phytes at day 14, these are 25.2%/29.9%

these findings would suggest that there

The antifungal properties of

(alcohols) and 3.4%/4.1% (sesquiter-

is a degree of transient chemical inter-

essential oils have been attributed to

penes), respectively. The case is similar

action between certain components of

aldehyde and ester content (Price &

with tea tree essential oil, with the

essential oil mixtures.

Price, 1999), and to alcohols, ketones

levels of alcohols and sesquiterpenes in

These transient changes in the

and sesquiterpenes (Caddy, 1997;

that dilution giving 100% inhibition of

chemical make up of the composition

Larrondo et al., 1995; Kurita et al.,

T. mentagrophytes at day 14 being 40.5%

of blends of essential oils give some

1981; Knobloch et al., 1989; Carson &

and 5.4%, respectively.

credence to the hypothesis that inter-

Riley, 1995). The antifungal effect of

The higher level of these two

actions between discrete chemical moi-

tea tree essential oil, in particular is

groups of chemicals in the concentra-

eties may contribute to the beneficial

attributed largely to its high alcohol

tion of single essential oils giving com-

therapeutic effects of essential oils

content (Caddy, 1997). It is often

plete inhibition of growth supports the

upon the client. Many of the tech-

claimed that the observed ‘synergistic

hypothesis that there is a factor other

niques used in the application of

action’ is due simply to the additive

than a direct additive effect occurring

essential oils (e.g. massage, vaporiza-

effect of combining groups of chemi-

within the synergistic blends.

tion, hot and cold compresses and

cals with similar actions.

Just as synergy can occur in single

addition to baths) exploit the use of

Analysis of the percentages of

essential oils or in blends, it is possible

temperatures other than ambient

each chemical group for lavender and

for certain components of a single oil,

room temperature.

tea tree oils individually (Caddy, 1997)

or blend to ‘quench’ undesirable

The changes in IR spectra as a

and for those blends resulting in syner-

effects of other components (Price &

result of changing ambient tempera-

gistic antifungal effect in the current

Price, 1999). It may also be possible

ture deserve further investigation as a

study suggests that this is not the case.

therefore that certain chemical groups

contributory factor to the phenome-

Tables 2–4 indicate the percentages of

in a single essential oil or blend might

non of synergistic action.

the most common constituents of

reduce the positive effects of some

There are a number of major

lavender and tea tree essential oils, for

other components. It has been demon-

components to lavender and tea tree

neat oils, for dilutions of the individual

strated that the antimicrobial efficacy

essential oils. These comprise a com-

oils, and for those blends showing syn-

of terpinen-4-ol in tea tree oil is

plex series of organic molecules with

ergistic antifungal activity. It is notable

lowered by the presence of non-

the potential for either internal syner-

that the blends showing synergistic

oxygenated terpenes, due to their low-

gistic reactions within a single oil

activity against a particular species at a

ering of the aqueous solubility of the

and/or between mixes or blends of the

particular timepoint have similar levels

essential oil as compared to the iso-

two different oils.

of alcohols and sesquiterpenes. This

lated chemical (Cox et al., 2001). This

might suggest that these two chemical

might partially explain the greater effi-

effects of essential oils may be catego-

groups

cacy of the essential oil blends in this

rized according to the presence of

observed antifungal activity.

It has been claimed that the

are

responsible

for

the

study as compared with oils used singly.

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Table 2

Major components of lavender (L. angustifolia) essential oil

Dilution (%)

Esters (%)

Aliphatic aldehydes (%)

Aromatic aldehydes (%)

Ketones (%)

Sesquiterpenes Alcohols (%) (%)

Oxides (%)

Monoterpenes (%)

Neat 10 20 30 40 50 60 70 80 90

45.0 4.5 9.0 13.5 18.0 22.5 27.0 31.5 36.0 40.5

1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

4.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6

5.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

2.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

4.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6

Table 3

36.0 3.6 7.2 10.8 14.4 18.0 21.6 25.4 29.0 32.6

Major components of tea tree (M. alternifolia) essential oil

Dilution (%)

Esters (%)

Aliphatic aldehydes (%)

Aromatic aldehydes (%)

Ketones (%)

Sesquiterpenes (%)

Alcohols (%)

Oxides (%)

Monoterpenes (%)

Neat 10 20 30 40 50 60 70 80 90

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

6.0 0.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4

45.0 4.5 9.0 13.5 18.0 22.5 27.0 31.5 36.0 40.5

7.0 0.7 1.4 2.1 2.8 3.5 4.2 4.9 5.6 6.3

41.0 4.1 8.2 12.3 16.4 20.5 24.6 28.7 32.8 36.9

Table 4

Chemical composition of synergistic antifungal blends of lavender and tea tree essential oils (assuming no chemical reaction)

Vs T. rubrum Day 14 Lavender (%v/v) Tea tree (%v/v) Esters (%) Aliphatic aldehydes (%) Aromatic aldehydes (%) Ketones (%) Sesquiterpenes (%) Alcohols (%) Oxides (%) Monoterpenes (%)

Vs T. mentagrophytes Day 7

Day 14

10 20 4.5 0.1

20 10 9.0 0.2

10 30 4.5 0.1

30 10 13.5 0.3

20 40 9.0 0.2

70 10 31.5 0.7

0.1

0.2

0.1

0.3

0.2

0.7

0.4 1.7 12.6 1.6 8.6

0.8 1.6 11.7 1.1 4.9

0.4 2.3 17.1 2.3 12.7

1.2 2.1 15.3 1.3 5.3

0.8 3.4 25.2 3.2 17.2

2.8 4.1 29.9 2.1 6.9

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For example, the 90% dilution of tea

Further conditions that should be

might be longer lived in-vivo due to an as

tree oil giving 100% inhibition of T.

considered involve catalysis using

yet undetermined catalytic requirement.

mentagrophytes at day 14 contains 36.9%

metals, usually in the form of salts,

Clearly further investigation at the mol-

monoterpenes. However, the 20%

which may be present in the media or

ecular–chemical level is also warranted.

lavender, 40% tea tree blend contains

cell membranes of organisms tested

only 17.2% monoterpenes. A more

in-vitro or on the skin surface of

likely explanation is Clinically, this

patients. Sodium, potassium, copper

might also be advantageous, since the

salts etc. are abundant on the surface

The authors wish to acknowledge

degradation products of monoterpenes

of the skin and in the body. Potential

Professor Alan Dronsfield (Professor of

may be the cause of sensitization to tea

contamination from the metal vessels

Organic Chemistry) for invaluable

tree essential oil (Hausen et al., 1999).

used in the distillation process may

comments and advice with respect to

The observed transient changes in

result in the presence of metal ions

the chemistry aspect of this work.

IR spectra in some blends of essential

such as copper, iron and aluminium.

oils raises a possibility for the contribu-

Equally there is the possibility of enzy-

tion of intermediate chemical moieties

matically or biochemically oriented

to synergistic effects in essential oil

catalysis from skin commensal bacte-

blends. It is well known that the esterifi-

ria, or even from the fungi themselves.

cation reaction between carboxylic acids

In conclusion, this study confirms

Buck, D.S. et al. (1994) Comparison of

and alcohols is reversible within single

that synergy does occur in the antifun-

two topical preparations for the treat-

essential oils (Price & Price, 1999).

gal activity of blends of tea tree and

ment of onychomycosis Melaleuca

There is also a clear potential for

lavender essential oils against the

alternifolia (tea tree) oil and clotrima-

transesterification reactions, where one

species used in this study. This demon-

alcohol group replaces another in an

strates the clear potential of appropri-

Caddy, R. (1997) Essential Oils in

ester in the general reaction.

ate essential oil blends as antimycotic

Colour. Amberwood Publishing Ltd,

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