198 M. MIYAZAWA

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. 2006; 21: 198–201

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2006; 21: 198–201Published online 31 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1580

Inhibition of acetylcholinesterase activity by tea tree oiland constituent terpenoids

Mitsuo Miyazawa* and Chikako Yamafuji

Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosakashi, Osaka577-8502, Japan

Received 10 January 2004; Revised 20 May 2004; Accepted 8 June 2004

ABSTRACT: In vitro inhibition of bovine erythrocyte acetylcholinesterase (AChE) activity by tea tree oil was investi-

gated. The main constituents in the tea tree oil batch used for the analysis of AChE inhibition were terpinen-4-ol (35.6%),

γγγγγ -terpinene (19.5%), ααααα-terpinene (8.3%), p-cymene (7.2%) and 1,8-cineole (4.4%). AChE was measured by a colorimetric

method. IC50 values were obtained for tea tree oil and ααααα-pinene and were 51.2 µµµµµg/ml and 57.1 µµµµµg/ml, respectively. Tea tree

oil was found to contain mixed-type inhibitors; a mixture of main constituents and main constituents showed competi-

tive inhibition. Copyright © 2005 John Wiley & Sons, Ltd.

KEY WORDS: acetylcholinesterase; tea tree oil; Melaleuca alternifolia; inhibitory activity; synergistic effect

* Correspondence to: M. Miyazawa, Department of Applied Chemistry,

Faculty of Science and Engineering, Kinki University, Kowakae,

Higashiosaka-shi, Osaka 577-8502, Japan.

E-mail: miyazawa apch.kindai.ac.jp

Introduction

Australian tea tree oil, usually obtained from Melaleuca

alternifolia Cheel, is becoming an increasingly important

commercial oil due to its biological activity. Tea tree

oil has powerful antiseptic properties1 and bactericidal

properties against a wide range of Gram-negative and

Gram-positive bacteria, yeast and fungi.2 Tea tree oil has

therefore been used in perfumes and cosmetics. The com-

position of tea tree oil has been reported to be a mixture

of terpenoids.1,3,4 The biological activity has been found

to be related to terpinene-4-ol, the major component of

tea tree oil.5–7 It is of interest to search for bioactive com-

pounds from tea tree oil.

Reversible inhibitors of cholinesterase are currently

used in clinical trials examining the treatment of

Alzheimer’s disease. The treatment of Alzheimer’s dis-

ease was based on inhibition of the acetylcholinesterase

(AChE) which hydrolyses acetylcholine, increasing

the acetylcholine available for transmission at the

cholinergistic synapse. Some AChE inhibitors have been

found to occur naturally in plants. Recently, galantamine

and amaryllidaceae alkaloid have shown effective results

for Alzheimer’s disease and safety of treatment.8–11 The

effects of Salvia lavandulaefolia Vhal (Spanish sage)

essential oil and some of its constituent terpenoids on

human erythrocyte acetylcholinesterase have also been

reported.12 In our previous paper, the inhibition of AChE

by monoterpenoids having a p-menthane skeleton,13

essential oils of Mentha species,14 volatile α,β-

unsaturated ketones15 and the essential oil from Citrus

paradisi16 were reported.

As a part of our continuing programme to investigate

AChE inhibitory activity by fragrance components, we

report here the inhibition of AChE from bovine erythro-

cytes by tea tree oil and a comparison between essential

oil and terpenoids as main components in the oils.

Materials and Methods

General Procedure

Electron impact mass spectra (EI–MS) were obtained

by gas chromatography–mass spectrometry (GC–MS),

which was performed on a Hewlett-Packard 5972A

mass selective detector (70 eV ion source; 180 °C) inter-

faced with a Hewlett-Packard 5890E Series II Plus gas

chromatograph fitted with a capillary column (TC-WAX,

60 m × 0.25 mm i.d.). Chromatographic conditions were

as follows: column temperature, raised from 60 °C to

240 °C at 2 °C/min; injector temperature, 240 °C; detector

temperature, 270 °C; carrier gas, He at 1.85 ml/min.

Materials

Acetylcholinesterase (AChE) from bovine erythrocytes

was purchased from and 5,5′-dithiobis (2-nitrobenzoic

acid) (DTNB) and acetylthiocholine iodide (ATC) from

Tokyo Chemical Industry Co. Ltd (TCI). Tea tree oil

was gifted from Yamamoto Perfumery Co. Ltd (Osaka,

Japan). Terpenoids were purchased from Fluka Co.

(Tokyo, Japan) and Shiono Koryo Kaisha Ltd (Osaka).

INHIBITION OF AChE ACTIVITY BY TEA TREE OIL 199

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. 2006; 21: 198–201

Preparatory Solutions

AChE (0.04 units/ml) and ATC (75 mM) were each

dissolved in 0.1 M phosphate buffer (pH 8.0). DTNB

(0.01 M) was made up in 10 ml 0.1 M phosphate buffer

(pH 7.0) containing 15 mg NaHCO3. Tea tree oil was

dissloved in ethanol. The final ethanol concentrations

in all assays were maintained at 5% (v/v), including

controls.

Inhibition of AChE Activity

Inhibition of AChE was assessed by the colorimetric

method of Ellman.17 Inhibitor solution (50 µl) and AChE

(0.5 ml) were mixed in a test tube and buffer (2.4 ml)

was added to the tube. The tube was pre-incubated

at 25 °C for 5 min. The reaction was started by adding

ATC (40 µl) and mixture was incubated at 25 °C for

20 min. The absorbance at 412 nm was measured spectro-

photometrically (Spectronic 20D, Milton Roy Co., NY)

and all test and control (without essential oil) assays

were corrected by blanks for non-enzymic hydrolysis.

Each assay was run in triplicate, at a minimum.

Results and Discussion

As shown in Table 1, the main consituents of tea tree

oil were terpinene-4-ol (35.6%), γ-terpinene (19.5%),

α-terpinene (8.3%), p-cymene (7.2%) and 1,8-cineole

(4.4%). Tea tree oil efficiently inhibited AChE activity at

an IC50 value of 51.2 µg/ml. This oil was fractionated by

SiO2 column chromatography with pentane:diethyl ether

and a hydrocarbon fraction and an oxygen-containing

fraction were obtained. However, these fractions were

shown to be less potent than tea tree oil (Table 2). To

clarify the cause of the inhibition effect of the tea tree oil,

the main terpenoids mentioned above were investigated.

As shown in Table 2, the most potent monoterpenoid

inhibitor tested was 1,8-cineole, with IC50 = 49.0 µg/ml.

The other main terpenoids showed identical inhibition

of AChE at 50 µg/ml, but these terpenoids were not

as potent as tea tree oil (Figure 1). The concentrations

of the main components in tea tree oil (100 µg/ml)

Table 1. Main components of tea tree oil

Compounds Peak areaa (%)

Terpinen-4-ol 35.6

γ-Terpinene 19.5

α-Terpinene 8.3

p-Cymene 7.2

1,8-Cineole 4.4

a Peak areas were quantified using a HP 3396 Series II integrator.

Table 2. Inhibition of AChE activity by tea tree oil,main terpenoids and mixture

Compounds IC50 (µg/mL)a or% inhibitory activity (50 µg/ml)b

Tea tree oil 51.2

Hydrocarbon fr. (33.3%)

Oxygen-containing fr. (25.5%)

Terpinen-4-ol (32.0%)

γ-Terpinene (31.7%)

α-Terpinene (34.0%)

p-Cymene (38.3%)

1,8-Cineole 49.0

Mixturec 65.5

a Concentration of compound (treatment) required for 50% enzyme inhibi-

tion as calculated from the dose–response curve.b Percentage AChE inhibition values (50 µg/mL) were calculated as com-

pared to control (without terpenoids) enzyme activity (assumed to be 0%

inhibition).c Terpinen-4-ol:γ-terpinene:α-terpinene:p-cymene:1,8-cineole = 36:20:8:7:4.

were: terpinene-4-ol, 35.6 µg/ml; γ-terpinene, 19.5 µg/ml;

α-terpinene, 8.3 µg/ml; p-cymene, 7.2 µg/ml; and 1,8-

cineole, 4.4 µg/ml. At these concentrations, these com-

ponents showed slight inhibitory activity. The inhibitory

activity of tea tree oil can be considered to be due to not

a single strong inhibitor but a synergistic activity caused

by a combination of the components. To clarify the cause

of the inhibitory effect of tea tree oil, the synergistic

effect of a mixture of the main terpenoids, a mixture

of terpinen-4-ol:γ-terpinene:α-terpinene:p-cymene:1,8-

cineole (36:20:8:7:4) was tested. This mixture showed a

Figure 1. Effect of tea tree oil main terpenoidson AChE activity. The percentage enzyme activityvalues for the inhibitors were calculated as comparedwith control activity (assumed to be 100%). �, tea treeoil; �, terpinen-4-ol; �, α-terpinene; �, γ -terpinene;�, p-cymene; �, 1,8-cineole

200 M. MIYAZAWA

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. 2006; 21: 198–201

synergistic effect and IC50 value was 65.5 µg/mL. The

inhibitory activity of the mixture was weaker than that

of tea tree oil. However, the inhibitory activity of the

mixture reflected that of the main terpenoids.

The inhibition of AChE by tea tree oil may be more

potent than that of the primary monoterpenoid con-

stituents (a mixture of the major constituents gave an

IC50 of 65.5 µg/ml, whereas the IC50 of the whole oil

was 51.2 µg/ml). Although it can be proposed that the

monoterpenoids act synergistically to inhibit AChE, it

cannot be excluded that a minor, as yet unidentified

constituent of the tea tree oil is more potent.

In conclusion, the inhibition of AChE by tea tree

oil is likely to be due to the presence of more than one

terpenoid present in tea tree oil, the main compounds

responsible being terpinen-4-ol, γ-terpinene, α-terpinene,

p-cymene and 1,8-cineole.

AChE Inhibition Kinetics

Tea tree oil showed mixed type inhibition of AChE

(by intersection in the Lineweaver–Burke plot). The

inhibition constant of tea tree oil was 54.7 µg/ml (by

intersection in the Dixon plot; Figure 2). On the other

hand, the main terpenoids showed competitive inhibition

of AChE (Figure 3). As shown in Figure 4, 1,8-cineole,

the most potent of the main terpenoids tested, was a com-

petitive inhibitor, as indicated by the increasing inhibition

associated with decreasing substrate concentration and by

the intersections in the Dixon plots. The plots of the other

main monoterpenoids tested were similar to that of 1,8-

cineole. These terpenoids and the mixture compete with

Figure 2. Lineweaver–Burke plots derived from theinhibition of AChE by tea tree oil. The concentra-tions of inhibitor were: �, no inhibitor; �, 12.5 µg/ml;�, 50.00 µg/ml

Figure 3. Lineweaver–Burke plots derived from theinhibition of AChE by the mixture. The concentra-tions of inhibitor were: �, no inhibitor; �, 12.5 µg/ml;�, 50.00 µg/ml

Figure 4. Dixon plots derived from the inhibition ofAChE by 1,8-cineole. The concentrations of ATC were:�, 0.163 mM; �, 0.325 mM; �, 0.650 mM

the substrate for its active centre on the enzyme. The Ki

values determined by the intersections in the Dixon plots

are shown in Table 3.

From this study, it is expected that tea tree oil can

be applied as an AChE antagonist, although it remains

to be determined whether tea tree oil inhibits brain AChE

in vivo with relevant potency.

Acknowledgement—The authors thank Yamamoto Perfumery Co. Ltd(Osaka, Japan) for providing the tea tree oil.

INHIBITION OF AChE ACTIVITY BY TEA TREE OIL 201

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. 2006; 21: 198–201

Table 3. Inhibition constant (Ki) for tea tree oil, mainterpemnoids and mixture Inhibition of AChE

Compounds Ki

Tea tree oil 54.7 (µg/ml)

Terpiene-4-ol 2.0 (mM)

γ-Terpinene 1.6 (mM)

α-Terpinene 0.8 (mM)

p-Cymene 1.5 (mM)

1,8-Cineole 0.1 (mM)

Mixturea 62.3 (µg/ml)

a Terpinen-4-ol:γ-terpinene:α-terpinene:p-cymene:1,8-cineole = 36:20:8:7:4.

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