FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2000; 15: 12±16

In vitro evaluation of antioxidant activity of essential oils

and their components
H. J. Damien Dorman,1 A. Christina Figueiredo,2 Jose G. Barroso2 and Stanley G. Deans1*
1
Aromatic and Medicinal Plant Group, Scottish Agricultural College, Auchincruive, South Ayrshire KA6 5HW, Scotland, UK
2Departamento de Biologia Vegetal, Faculdade de CieÃncias de Lisboa, Bloco C2, Campo Grande, 1749-016 Lisbon, Portugal

Received 2 December 1997

Revised 18 May 1999
Accepted 6 July 1999

ABSTRACT: Clove and nutmeg essential oils were analysed by GC and GC±MS. These oils, together with
16±18 components found to be present, were tested for antioxidant properties at ®nal concentrations of 0.05±
2.5  104 ppm in an egg yolk-based thiobarbituric acid reactive substances (TBARS) assay and also undiluted in
a b-carotene agar di€usion assay. Both the essential oils and the components tested in the TBARS assay
demonstrated some degree of antioxidant activity. Only the clove oil, the nutmeg oil, eugenol and terpinolene
demonstrated any ability to inhibit the oxidative bleaching of the b-carotene agar. The ability of the oil
components to inhibit malondialdehyde formation, and therefore lipid peroxidation, in the TBARS assay, yet
apparently to possess no activity in the b-carotene agar di€usion assay, demonstrates the importance in the
screening of plant material for bioactivity of using a bank of assays in vitro before assigning bioactivities. By
using a number of assays, not only should the number of false positives and negatives be greatly reduced, but
evidence pertaining to the mechanism of action may be obtained. Copyright # 2000 John Wiley & Sons, Ltd.

KEY WORDS: Myristica fragrans Houtt.; nutmeg; Syzygium aromaticum (L.) Merr. & Perry; clove; essential
oil; natural antioxidants; thiobarbituric acid reactive substances (TBARS) assay; b-carotene agar di€usion assay

Introduction chain, by irradiation, or generated as a product of

Lipid peroxidation may be described as the oxidative
deterioration of unsaturated fatty acids and the changes
resulting from this process. It is a paradox of aerobic
life that aerobic organisms require molecular oxygen
for their normal metabolic functions, yet oxygen can be
toxic to cellular material.1 Detrimental events which
may result from this process include membrane
fragmentation, disruption of membrane-bound enzyme
activity, disintegration and swelling of mitochondria,
and lysosomal lysis. The primary toxicological event of
lipid peroxidation, and the subsequent secondary and
tertiary events resulting from it, originate from the
interaction of free radicals with polyunsaturated fatty
acids. These reactive species may be endogenously
generated oxygen radicals, produced by the leakage of
electrons from the mitochondrial electron transport
* Correspondence to: S. G. Deans, Aromatic and Medicinal Plant Group,
Scottish Agricultural College, Auchincruive, South Ayrshire KA6 5HW,
Scotland, UK. Tel: ‡1292 52 5201; Fax: ‡1292 52 5071; e-mail: S.Deans
@au.sac.ac.uk
Contract grant sponsor: Ministry of Agriculture, Fisheries and Food, UK.
Contract grant sponsor: Agriculture, Environment and Fisheries Department
of the Scottish Oce, UK.
Contract grant sponsor: Centro de Biotecnologia Vegetal, Instituto de
Biotecnologia e Quimica Fina, Portugal.
cytochrome P450-mediated xenobiotic metabolism.
Plant volatile oils have been recognized since anti-
quity as possessing biological activities.2 Chief amongst
these are their antibacterial,3±6 antifungal5±11 and
antioxidant properties.3,5,12±21 Their ability to protect
highly unsaturated lipids in animal tissues has been the
subject of much interest.22,23 In addition to protecting
liver polyunsaturated fatty acids (PUFAs), these oils
have demonstrated a positive e€ect upon docosahex-
ñnoic acid (DHA, 22:6n-3) levels in ageing rodent
retinas.24 These studies and others25 have demonstrated
the bene®cial potential or dietary supplementation with
certain volatile oils upon the PUFAs, in particular the
C20 and C22 acids.
The thiobarbituric acid reactive substances (TBARS)
assay has been used as a technique for measuring the
auto-oxidation of foodstu€s and as an experimental
method for assessing lipid peroxidation. In order to
e€ect a rapid quanti®able screening process to identify
the extent of the antioxidative properties of essential
oils and their components in vitro prior to in vivo
evaluation, an avian-based modi®ed TBARS assay was
developed.15 The assay was used to measure the degree
of lipid peroxidation in a lipid-rich substrate, based

Copyright # 2000 John Wiley & Sons, Ltd. Flavour Fragr. J. 2000; 15: 12±16
 ANTIOXIDANT ACTIVITY OF ESSENTIAL OILS 13

upon the spectrophotometric measurement of malon- Table 1. Percentage composition of the essential oils
dialdehyde-thiobarbituric acid (MDA)2-TBA coloured isolated from nutmeg (Myristica fragrans) and clove
complex. MDA is a secondary product of lipid (Syzygium aromaticum)
peroxidation. Compound RIa Nutmeg Clove
Tricyclene 921 t ±
a-Thujene 924 1.5 ±
Experimental a-Pinene 930 27.9 ±
Camphene 938 0.4 ±
Sabinene 958 16.0 ±
Plant Material b-Pinene 963 27.1 ±
Myrcene 975 2.2 ±
The essential oils used in this study were isolated by a-Phellandrene 995 0.8 ±
D3-Carene 1000 0.9 ±
hydrodistillation using the British Pharmacopoeia a-Terpinene 1002 1.8 ±
essential oil distillation apparatus [`Quick Fit'].26 p-Cymene 1003 1.1 ±
Commercial supplies of the plant material and essential b-Phellandrene 1005 2.2 ±
Limonene 1009 3.8 ±
oil components were used throughout. The individual g-Terpinene 1035 2.6 ±
oil constituents were purchased either from Sigma or trans-Sabinene hydrate 1037 0.2 ±
Fluka Chemicals. Serial dilutions of the oils and their Terpinolene 1064 1.0 ±
cis-Sabinene hydrate 1066 0.1 ±
components were made with either methanol or Linalol 1074 0.4 ±
absolute alcohol, depending upon solubility. trans-p-Menthen-1-ol 1095 0.1 ±
Borneol 1134 0.2 ±
Terpinene-4-ol 1148 3.3 ±
a-Terpineol 1159 0.4 ±
Gas Chromatography (GC) Eugenol 1327 0.3 91.2
a-Terpenyl acetate 1334 0.1 ±
Geranyl acetate 1370 0.1 ±
GC analyses were performed using a Perkin-Elmer a-Copaene 1375 0.1 ±
8700 gas chromatograph equipped with two FIDs, a Methyleugenol 1337 0.3 ±
data-handling system and a vaporizing injector port b-Caryophyllene 1414 0.1 4.1
trans-Isoeugenol 1422 0.2 ±
into which two columns of di€erent polarities were a-Humulene 1447 ± 0.6
installed: a DB-1 fused-silica column (30 m  0.25 mm Eugenyl acetate 1493 ± 2.9
i.d., ®lm thickness 0.25 mm; J & W Scienti®c Inc., Myristicin 1493 3.1 ±
Elemicin 1525 0.1 ±
Rancho Cordova, CA, USA) and a DB-Wax fused- b-Caryophyllene epoxide 1561 ± 0.5
silica column (30 m  0.25 mm i.d., ®lm thickness
% Identi®cation 98.4 99.3
0.25 mm; J & W Scienti®c Inc.). Oven temperature was Monoterpene hydrocarbons 89.4 ±
programmed to 45±1758C at 38C/min, subsequently at Oxygen-containing monoterpenes 4.9 ±
158C/min up to 2408C, and then held isothermal for Sesquiterpene hydrocarbons 0.2 4.7
Oxygen-containing sesquiterpenes ± 0.5
10 min. Injector and detector temperatures were 2208C Phenlypropanoids 4.0 94.1
and 2408C, respectively. Hydrogen was used as the a Retention index, relative to C9 ±C16 n-alkanes on the DB-1 column.
carrier gas at a linear velocity of 30 cm/s. The samples
were injected using a split sampling technique, ratio
1:50. The percentage composition of the oils was
computed by the normalization method from the GC energy, 70 eV; ionization current, 60 mA; scan range,
peak areas, calculated as mean values of two injections, 40±300 u; scan time, 1 s. The identity of the com-
without using correction factors. ponents was assigned by comparison of their retention
indices relative to C9 ±C16 n-alkanes, and mass spectra
with corresponding data of components of reference
Gas Chromatography±Mass Spectrometry oils.
(GC±MS)

The GC±MS unit consisted of a Carlo Erba 6000 Vega
gas chromatograph equipped with a DB-1 fused-silica
column (30 m  0.25 mm i.d., ®lm thickness 0.25 mm;
J & W Scienti®c Inc.) and interfaced with a Finnigan
MAT 800 Ion Trap Detector (ITD; software version
4.1). Oven temperature was as above; transfer line
temperature was 2808C; ion trap temperature, 2208C.
Helium was used as the carrier gas and was adjusted to
a linear velocity of 30 cm/s; split ratio, 1:40; ionization
Medium for the Antioxidant Assay
Egg yolk was separated from the albumin and the yolk
membrane prior to its storage at 48C until use. The
average lipid composition in the egg yolk was 63%
triacyglycerides, 28% phospholipids, 5% free choles-
terol and 1% cholesterol ester. For the assay, an aliquot
of yolk material was made up to a concentration of
10% (w/v) in KCl (1.15% w/v). The yolk was then

Copyright # 2000 John Wiley & Sons, Ltd. Flavour Fragr. J. 2000; 15: 12±16
14 H. J. DAMIEN DORMAN ET AL.
Figure 1. A dose±response curve of the antioxidant capacity of clove ( ± h ± ) and nutmeg ( ± r ± ) essential oils, as
measured by their ability to inhibit malondialdehyde formation. Each data point is the mean+SEM of three
independent experiments
homogenized for 30 s followed by ultrasonication for a
period of 3 min.
TBARS Assay
The TBARS assay was carried out as described pre-
viously.15 The antioxidant index percentage (AI%)+
SEM was plotted as a dose±response curve against the
concentration (in ppm) of the ®nal reaction volume.
This was used to demonstrate the comparative anti-
oxidative capacities of the essential oils and their
components. The oils and the oil components were
used at concentrations of 0.05±25 000 ppm.
b -Carotene Agar Diffusion Assay
The screening for antioxidant capacities of the essential
oils using the b-carotene agar di€usion technique was
adapted from that of Araujo and Pratt.27 A sample of
2.0 g of agar (Bacto-agar) was dissolved in 100 ml of
boiling water. The agar solution was allowed to cool to
ca. 508C, then 2.0 ml of linoleic acid in ethanol (2.0 mg/
ml) and 10 ml of b-carotene in acetone (2.0 mg/ml)
were ¯ushed into the agar. The agar was poured into
Petri dishes and allowed to set for 30 min. As acetone is
used to solubilize the b-carotene, the agar does not
completely set hard but remains jelly-like. Two holes or
wells (4 mm diameter) were punched into the agar of
each Petri dish, and into each well 15 ml of test sample
Copyright # 2000 John Wiley & Sons, Ltd.
was added. Plates with test samples were incubated at
458C overnight until the background colour had
bleached. The zones of yellow colour surrounding the
test sample wells were marked by pen, and the diameter
of the circle measured using Vernier callipers. Yellow
colour/intensity and/or persistence of colour, on a scale
of * to *** (see Table 2), around the wells containing
the test agent were indicative of the antioxidant
capacity of the test substances.
Results and Discussion
The percentage composition of the essential oils from
nutmeg and clove are shown in Table 1. The mono-
terpene hydrocarbons (89%) constituted the main
fraction of nutmeg oil, a-pinene (28%), b-pinene
(27%) and sabinene (16%) being its main components,
whereas phenylpropanoids were the major fraction
(94%) of clove oil, eugenol being its main constituent
(91%).
The antioxidant capacities of the nutmeg and clove
oils and their main components, as determined by the
modi®ed TBARS assay, are shown in Figures 1 and 2.
Their abilities to prevent the oxidative bleaching of b-
carotene is shown in Table 2.
All the essential oils and components demonstrated
some degree of antioxidant capacity over the concen-
tration range tested in the TBARS assay. Both total
essential oils tested (Figure 1) demonstrated clear
sigmoidal dose±response curves over the concentration
Flavour Fragr. J. 2000; 15: 12±16
 ANTIOXIDANT ACTIVITY OF ESSENTIAL OILS 15

Figure 2. A dose±response curve of the antioxidant capacity of individual components found in clove and nutmeg
essential oils, as measured by their ability to inhibit malondialdehyde formation. Each data point is the mean+SEM of
three independent experiments

range tested. The IC50-values were 20 ppm and
100 ppm for clove and nutmeg oils, respectively. The
plateau for nutmeg oil was slightly higher than for clove
oil. Both, however, gradually declined in measured
antioxidant capacity from the same concentration
point. The slope of this decline was greatest for clove
oil. All oil components tested demonstrated some
degree of antioxidant capacity, Figure 2. Two types of
response may explain the observed components pro®le:
it can be seen from Figure 2 that some components
(e.g. eugenol and terpinolene) exhibit a marked
sigmoidal e€ect across the concentration range, while
others were more linear in their response (e.g. D3-carene
and a-pinene).
Clove and nutmeg oils were also able to inhibit the
oxidative bleaching of b-carotene, Table 2. The relative
antioxidant capacity of these two oils compared to the
oils isolated from monarda (Monarda citriodora var.
citriodora Cerv. ex Lag.), geranium (Pelargonium
graveolens L'HeÂrit.), oregano (Origanum vulgare spp.
hirtum (Link) (Ietsw.) and black pepper (Piper nigrum
L.) was: black pepper 4 monarda 4 oregano 4
geranium 4 clove 4 nutmeg 4 , as tested in the
b-carotene assay.25 In the case of the oil components,

Copyright # 2000 John Wiley & Sons, Ltd. Flavour Fragr. J. 2000; 15: 12±16
16 H. J. DAMIEN DORMAN ET AL.

Table 2. Ability of essential oils isolated from nutmeg Acknowledgements Ð HJDD gratefully acknowledges the award of a
MAFF Studentship. SAC received ®nancial support from the
(Myristica fragrans) and clove (Syzygium aromaticum)
Scottish Oce Agriculture, Environment and Fisheries Department.
and their components to inhibit the oxidative bleaching The Portuguese team acknowledges the Centro de Biotecnologia
of b-carotene, as measured by the b-carotene agar Vegetal, Instituto de Biotecnologia e Quimica Fina (IBQF), for
diffusion assay. Each value is the mean of two measur- ®nancial support.
ements+SEM
Oil/oil component Diameter (mm)+SEM/colour intensity
Clove 15.55+0.55*** References
Nutmeg 12.64+1.64***
a-Pinene 0.00 1. K. J. A. Davies, in Oxidative Stress: The Paradox of Aerobic Life,
Camphene 0.00 Vol. 61, p. 1, ed. C. Rice-Evans, B. Halliwell and G. G. Lunt,
Sabinene 0.00 Portland Press, London (1995).
b-Pinene 0.00 2. S. G. Deans and P. G. Waterman, in Volatile Oil Crops: Their
Myrcene 0.00 Biology, Biochemistry and Production, p. 113, ed. R. K. M. Hay
a-Phellandrene 0.00 and P. G. Waterman, Longman, London (1993).
D3-Carene 0.00 3. R. Piccaglia, M. Marotti, E. Giovanelli, S. G. Deans and E.
a-Terpinene 0.00 Eaglesham, Industrial Crops Products, 2, 47 (1993).
p-Cymene 0.00 4. B. J. Juven, J. Kanner, F. Schved and H. Weisslowicz, J. Appl.
Limonene 0.00 Bact., 76, 626 (1994).
g-Terpinene 0.00 5. R. Tisserand and T. Balacs, Int. J. Aromatherapy, 6, 28 (1994).
Terpinolene 8.10+0.10** 6. J. M. Kim, M. R. Marshall, J. A. Cornell, J. F. Preston and C. I.
Linalol 0.00 Wei, J. Food Sci., 60, 1364 (1995).
Borneol 0.00 7. R. R. Carlton, A. I. Gray, P. G. Waterman and S. G. Deans,
Terpinen-4-ol 0.00 Chemoecology, 3, 55 (1992).
Eugenol 16.20+1.10*** 8. A. Tantouielaraki and A. Erri®, Grasas Aceites, 45, 363 (1994).
b-Caryophyllene 0.00 9. N. Paster, M. Menasherov, U. Ravid and B. Juven, J. Food
a-Humulene 0.00 Protect., 58, 81 (1995).
10. H. Martini, W. Weidenborner, S. Adams and B. Kunz, Ital. J.
** and ***: see text. Food Sci., 8, 63 (1996).
11. D. P. Thompson, J. Food Protect., 59, 412 (1996).
12. K. Miura and N. Nakatani, Agric. Biol. Chem., 53, 3043 (1989).
only terpinolene and eugenol demonstrated any anti- 13. N. Deighton, S. M. Glidewell, S. G. Deans and B. A. Goodman,
oxidant capacity; their order of e€ectiveness being J. Sci. Food Agric., 63, 221 (1993).
eugenol 4 terpinolene. Their e€ectiveness related to 14. A. M. Campos and E. A. Lissi, Boletin Soc. Chilena Quimica, 40,
375 (1995).
carvacrol, an essential oil component with antioxidant 15. H. J. D. Dorman, S. G. Deans, R. C. Noble and P. Surai, J.
properties was: eugenol 4 carvacrol 4 terpinolene.25 Essent. Oil Res., 7, 645 (1995).
When the composition of the oils is considered and 16. M. A. Ozcan, Acta Aliment., 24, 81 (1995).
17. M. Takacsova, A. Pribela and M. Faktorova, Nahrung-Food, 39,
the activity of these two components, the di€erence in 241 (1995).
the antioxidant capacity of the oils may be due to the 18. N. V. Yanishlieva and E. M. Marinova, Nahrung-Food, 39, 458
di€ering amounts of these two components. Eugenol (1995).
19. S. K. Chung, T. Osawa and S. Kawakishi, Biosci. Biotechnol.
comprises ca. 90% the total clove oil, while only 1.0% Biochem., 61, 118 (1997).
of the total nutmeg oil consists of terpinolene. Perhaps 20. E. M. Marinova and N. V. Yanishlieva, Food Chem., 58, 245
more interesting, however, is the lack of activity (1997).
21. D. A. Pearson, E. N. Frankel, R. Aeschbach and J. B. German, J.
demonstrated by the other components. Based upon Agric. Food Chem., 45, 578 (1997).
the data generated from this assay alone, we would 22. S. G. Deans, R. C. Noble, L. G. PeÂnzes and S. G. Imre, Age, 16,
conclude that these components, under these experi- 71 (1993).
23. S. G. Deans, R. C. Noble, A. MacPherson, L. G. PeÂnzes and S. G.
mental conditions, do not appear to possess anti- Imre, in Aspects of Ageing and Disease, Vol. 4, p. 173, ed. G.
oxidant properties. Yet, when one looks at the data of Hofecker and M. Skalicky, Vienna Ageing Series, Facultas Press,
the TBARS assay, one would conclude that all the Vienna (1994).
24. Z. Recsan, G. Pagliuca, M. V. Piretti, L. G. PeÂnzes, K. A.
components have antioxidant activity, under the Youdim, R. C. Noble and S. G. Deans, J. Essent. Oil Res., 9, 53
experimental conditions applied. This demonstrates (1997).
the importance in a screening of plant material for 25. H. J. D. Dorman, Phytochemistry and Bioactive Properties of
Plant Volatile Oils: Antibacterial, Antifungal and Antioxidant
bioactivity to use a bank of assays in vitro before Activities, PhD Dissertation, University of Strathclyde (1999).
assigning bioactivities. By the use of a number of assays 26. S. G. Deans, in Modern Methods of Plant Analysis, Vol. 12:
not only should the number of false positives and Essential Oils and Waxes, p. 309, ed. H. F. Linskens and J. F.
Jackson, Springer-Verlag, Heidelberg (1991).
negatives be greatly reduced, but evidence pertaining to 27. J. M. A. Araujo and D. E. Pratt, CieÃncia Tecnollogia Alimentos, 5,
the mechanism of action may be obtained. 57 (1985).

Copyright # 2000 John Wiley & Sons, Ltd. Flavour Fragr. J. 2000; 15: 12±16