inhibition of acetylcholinesterase activity by tea tree oil and constituent terpenoids

INHIBITION OF ACETYLCHOLINESTERASE ACTIVITY 617

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. 2005; 20: 617–620

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2005; 20: 617–620Published online 26 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1505

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 activity by tea tree oil was investigated. The

main constituents in the tea tree oil batch used for analysis of acetylcholinesterase 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 and 57.1 µµµµµg/ml, respectively. Tea tree

oil was found to comprise of mixed type inhibitors as the main constituents. The main constituents were competitive

inhibitors. 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: [email protected]

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

positive bacteria, yeast and fungi.2 It has also been used

in perfumes and cosmetics. The composition of tea tree

oil has been reported to be a mixture of terpenoids.1,3,4

The biological ativity has been found to be related to

terpinene-4-ol, the major component.5–7 It is of interest to

search for bioactive compounds 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 ace-

tylcholine available for transmission at the cholinergistic

synapse. Some acetylcholinesterase inhibitors have been

found to occur naturally in plants. Recently, galantamine,

an amaryllidaceae alkaloid, has shown effective results

for Alzheimer’s disease with good safety.8–11 The effects

of Salvia lavandulaefolia Vhal (Spanish sage) essential

oil and some of its constituent terpenoids on human

erythrocyte acetylcholinesterase have been reported

as well.12 In our previous paper, the inhibition of AChE

by monoterpenoids with a p-menthane skeleton,13 essen-

tial oils of Mentha species,14 volatile α,β-unsaturated

ketones15 and essential oil from Citrus paradisi16 were

reported.

As part of our continuing program to investigate AChE

inhibitory activity by fragrance components, we report

here the inhibition of AChE from bovine erythrocytes

by tea tree oil and a comparison of 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).

GC-MS was performed on a Hewlett-Packard 5972A

massselevtive 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 increased from 60 to

240 °C at 2 °C/min; injector temperature, 240 °C; detec-

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

Materials

AChE from bovine erythrocytes was purchased from

Sigma Co., Ltd (Tokyo). 5, 5′-Dithiobis (2-nitrobenzoic

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

perchased from Tokyo Chemical Industry Co. Ltd (TCI).

618 M. MIYAZAWA AND C. YAMAFUJI


Tea tree oil

Tea tree oil was gifted from Yamamoto Perfumery

Co. Ltd (Osaka, Japan).

Terpenoids

Terpenoids were purchased from Fluka Co. (Tokyo,

Japan) and Shiono Koryo Kaisha Ltd (Osaka).

Preparatory solutions

AChE (0.04 units/mL) and ATC (75 mM) were dissolved

in 0.1 M phosphate buffer (pH 8.0), respectively. DTNB

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

(pH 7.0) containing 15 mg of 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 et al.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

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

by adding ATC (40 µL) and the mixture was incubated

at 25 ° C for 20 min. The absorbance at 412 nm was

measured spectrophotometrically (Spectronic 20D, Milton

Roy Co., NY, USA), and all test and control (without

essential oil) assays were corrected by blanks for non-

enzymic hydrolysis. Each assay was run in triplicate, at

least.

Results and Discussion

As shown 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 act-

ivity, with an IC50 value of 51.2 µg/mL. This oil

was fractionated by SiO2 column chromatography with

pentane–diethylether and a hydrocarbon fraction and

an oxygen-containing fraction were obtained. However,

these fractions were less potent than tea tree oil (Table 2).

To clarity the cause of the inhibition effect of the tea tree

oil, the main terpenoids mentioned above were inves-

tigated. As shown in Table 2, the most potent of the

monoterpenoid inhibitors tested was 1,8-cineole, with an

IC50 value of 49.0 µg/mL. The other main terpenoids

showed identical inhibition of AChE at 50 µg/mL. How-

ever, these terpenoids were not as potent as tea tree oil

(Fig. 1). The concentration of each main component in

tea tree oil (100 µg/mL) was as follows; 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 this concentration, these components

showed slight inhbitory activity. Inhibitory activity of tea

tree oil can be considered to be not one strong inhibitor,

but synergistic activity in combination with each com-

ponent. To clarify the cause of the inhibitory effect of tea

tree oil, the synergistic effect of a mixture of the main

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 percentageinhibitory 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 The percentage AChE inhibition values (50 µg/mL) were calculated as

compared with 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.

Figure 1. Effect of tea tree oil, main terpenoids onAChE activity. The percentage enzyme activity valuesfor the inhibitors were calculated compared with thecontrol activity, assumed to be 100%: (�) tea treeoil; (�) terpinen-4-ol; (�) γ -terpinene; (�) α-terpinene;(�) p-cymene; (�) 1,8-cineole.

INHIBITION OF ACETYLCHOLINESTERASE ACTIVITY 619


terpenoids, a 36:20:8:7:4 mixture of terpinen-4-ol, γ -

terpinene, α-terpinene, p-cymene and 1,8-cineole, was

tested. This mixture showed a synergistic effect and the

IC50 value was 65.5 µg/mL. The inhibitory activity of

mixture was weaker than that of tea tree oil. However,

the inhibitory activity of the mixture was affected by the

main terpenoids.

The inhibition of AChE by tea tree oil may be more

potent than that of the primary monoterpenoid constitu-

ents (the 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 is proposed that the

monoterpenoids act synergistically to inhibit AChE, the

possibility cannot be excluded that a minor, as yet uni-

dentified, 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

terpenoids 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 inhibition of AChE (by inter-

section on the Lineweaver–Burk plot). The inhibition

constant of tea tree oil was 54.7 µg/mL (by intersection

on the Dixon plot; Fig. 2). On the other hand, the

main terpenoids showed competitive inhibition of AChE

(Fig. 3). As shown in Fig. 4, 1,8-cineole, the most potent

main terpenoid tesed, was a competitive inhibitor, as

indicated by increasing inhibition associated with decreas-

ing substrate concentration and by the intersections on

the Dixon plots. The plots of the other main mono-

terpenoids tested were similar to that of 1,8-cineole.

These terpenoids and mixture compete with the substrate

for its active centre on the enzyme. The Ki values deter-

mined by the intersections in the Dixon plots are shown

in Table 3.

Figure 2. Lineweaver–Burk plots derived from theinhibition of AChE by tea tree oil. The concentrationsof inhibitor are: (�) no inhibitor; (�) 12.5 µg/mL; and(�) 50.0 µg/mL.

Figure 3. Lineweaver–Burk plots derived from theinhibition of AChE by mixture. The concentrationsof inhibitor are: (�) no inhibitor; (�) 12.5 µg/mL; and(�) 50.0 µg/mL.

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

used as an AChE antagonist, although it remains to be

determined whether the tea tree oil inhibited brain AChE

in vivo with relevant potency.

Acknowledgements—The authors thank Yamamoto Perfumery Co. Ltd(Osaka, Japan) for providing the tea tree oil. This work was supportedby ‘High-Tech Research Centre’ project for Private Universities: match-ing fund subsidy from MEXT (Ministry of Education, Culture, Sports,Science and Technology), 2004–2008.

Figure 4. Dixon plots derived from the inhibitionof AChE by 1,8-cineole. The concentrations of ATC are:(�) 0.163 mM; (�) 0.325 mM; and (�) 0.650 mM.

Table 3. Inhibition constants (Ki) for tea tree oil, mainterpenoids 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.

620 M. MIYAZAWA AND C. YAMAFUJI


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