PHYTOTHERAPY RESEARCH

Phytother. Res. 25: 12541256 (2011)

Published online 1 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3412

SHORT COMMUNICATION

Essential Oil of Clove (Eugenia caryophyllata)

augments the Humoral Immune Response but
Decreases Cell Mediated Immunity
Sumita Halder,1 Ashish K. Mehta,2 Pramod K. Mediratta1* and Krishna K. Sharma1
1

Department of Pharmacology, University College of Medical Sciences, Delhi 110095, India

Department of Physiology, University College of Medical Sciences, Delhi 110095, India

The present study was undertaken to explore the effect of the essential oil isolated from the buds of Eugenia
caryophyllata on some immunological parameters. Humoral immunity was assessed by measuring the
hemagglutination titre to sheep red blood cells and delayed type hypersensitivity was assessed by measuring
foot pad thickness. Clove oil administration produced a signicant increase in the primary as well as secondary
humoral immune response. In addition, it also produced a signicant decrease in foot pad thickness compared with
the control group. Thus, these results suggest that clove oil can modulate the immune response by augmenting
humoral immunity and decreasing cell mediated immunity. Copyright 2011 John Wiley & Sons, Ltd.
Keywords: Eugenia caryophyllata; humoral immunity; cell mediated immunity.

INTRODUCTION

MATERIALS AND METHODS

The essential oil isolated from the buds of Eugenia

caryophyllata L. Merr & Perry (Myrtaceae) is well
known for its medicinal properties. Traditional uses of
clove oil include use in dental care, as an antiseptic and
analgesic. It is also known to possess activity against
oral bacteria associated with dental caries and periodontal disease and is also effective against a large
number of other bacteria (Friedman et al., 2002;
Kalemba and Kunicka, 2003). Previous studies have
also reported antifungal, antiviral, antiallergic and
antioxidant properties of clove oil (Chami et al., 2005;
Ogata et al., 2000). Its action against mites in scabies
infection has also been reported (Pasay et al., 2010).
Clove oils antiseptic and aromatic properties make it a
vital ingredient in many skin care products. Clove oil
when applied with a cotton swab on eyelashes helps to
reduce sty inammation and helps to heal the infection.
People suffering from cough, colds, bronchitis, sinusitis
and asthma use clove oil infused in hot water as a
remedy (Lawless, 1995). It is not known whether its use
in these infections is due to its antimicrobial effect per se
or due to some immunomodulation induced in the body
by clove oil. The available reports strongly suggest that
clove oil could probably possess immunomodulatory
activity which could play a role in combating various
types of infections and allergies. The present study was
undertaken to evaluate the role of essential oil of
Eugenia caryophyllata in modulating the humoral
immune response and cell mediated immunity.

Animals. Male Wistar rats weighing 150200 g were used

in the study. The animals were procured from the Central
Animal House, University College of Medical Sciences,
Delhi. The animals were housed in groups of four
per cage, each cage having dimensions (28 22 14 cm)
with free access to a pellet diet and water in a
temperature controlled facility. All the experiments were
performed at daytime between 0930 and 1530 h. Care of
animals was according to the guidelines of Committee for
the Purpose of Control and Supervision of Experiments
on Animals (CPCSEA), Ministry of Environment and
Forest, Government of India, New Delhi. The study
was duly approved by the Institutional Animal Ethics
Committee, University College of Medical Sciences,
Delhi. The animals were divided into two groups. The
rst group was administered the vehicle (5 mL/kg/day,
i.p.). The second group was treated with clove oil
(0.1 mL/kg/day, i.p.).

* Correspondence to: Professor Pramod K. Mediratta, Department of

Pharmacology, University College of Medical Sciences (University of
Delhi), Delhi 110095, India.
E-mail: drpramod_k@yahoo.com

Copyright 2011 John Wiley & Sons, Ltd.

Plant material. Clove bud oil was obtained from M/s

Kancor Ingredients Limited, Kerala, India. As per the
literature provided by the manufacturer, the bud oil
was obtained by the steam distillation from the dried
ower buds of Eugenia caryophyllata to obtain a
colourless pale yellow liquid of specic gravity 1.0426
at 25 C and a refractive index of 1.533 at 20 C (sample
code no. 085407, Reference no. 1 of 730/0708). The
characterization of a sample of clove bud oil using GC
MS revealed a high concentration of eugenol (87.34%),
eugenyl acetate (5.18%) and betacaryophyllene
(2.01%).
For the purpose of study, the clove bud oil was
suspended in double distilled water along with 1%
Tween 80 to prepare suspensions of a required dose of
0.1 mL/kg.
Received 19 July 2010
Revised 28 December 2010
Accepted 28 December 2010

1255

IMMUNOMODULATORY ACTIVITY OF CLOVE OIL

Chemicals. Hemocyanin (Keyhole Limpets) was procured from Sigma, USA.

Humoral immune response. The humoral immune
response was estimated by the method described by
Halder et al. (2009). The rats were immunized with
sheep red blood cells (SRBC; 0.5 109cells/mL/100 g,
i.p.) on day 0. The animals were divided into two
groups. One group received vehicle only, while the
other group received clove oil (0.1 mL/kg/day, i.p.) from
day 1 to day 6. On day 7, animals were lightly
anesthetized with ether and blood was collected from
retroorbital plexus using a microcapillary technique.
The serum was separated and the hemagglutination
titre was estimated as follows: Twofold dilutions
(0.025 mL) of sera were made in the microtitre plates
with normal saline. To each well, 0.025 mL of 1% (v/v)
SRBC was added. The plates were incubated at 37 C
for 1 h, and then observed for hemagglutination. The
highest dilution giving hemagglutination was taken as
the primary antibody titre and expressed in a graded
manner, the minimum dilution (1/2) being ranked as 1,
and mean ranks of different groups were compared for
statistical analysis.
To estimate the secondary immune response, on day 7,
the animals received the same dose of antigen again
(booster dose). The animals received treatment with
clove (0.1 mL/kg, i.p.) from day 1 to day 8. The control
group received vehicle only. On day 9, blood was
collected from the retroorbital plexus and the secondary
antiSRBC titre was estimated by the same method as
described above.
Cell mediated immune response. The delayed type
hypersensitivity response was estimated by the method
described by Institoris et al. (2001). Animals were
sensitized by keyhole limpet hemocyanin (KLH). Each
rat was immunized subcutaneously at the base of the tail
by 0.3 mL of KLH (containing 0.5 mg of KLH in
0.15 mL 0.9% saline and mixed with 0.15 mL Freunds
complete adjuvant) on day 0 of treatment. The animals
were administered vehicle (control group) and clove oil
(0.1 mL/kg/day, i.p.) on day 1 to day 14. On day 14, the
reaction was challenged by injecting 20 g KLH in
50 L normal saline in the left hind foot pad. The foot
pad thickness was measured before and 24 h after
challenge by antigen using dial calipers. The results
were expressed as the percentage change in foot pad
thickness.
Statistical analysis. The results were expressed as mean
SEM. The data were analysed statistically using the
MannWhitney Utest (twotailed) and Students ttest
(unpaired).

The secondary immune response was also increased

as shown by a signicant increase in the secondary anti
SRBC titre measured on day 9 following a booster dose
of antigen on day 7 (p < 0.05) (Table 1).
Cell mediated immune response
In the delayed type hypersensitivity test, clove oil
(0.1 mL/kg/day, i.p.) when administered for 14 days,
produced a signicant decrease in foot pad thickness
when compared with the control group (p < 0.01)
(Table 2).

DISCUSSION
The essential oil extracted from clove is topically used
to relieve pain and promote healing in dental medicine
(Prashar et al., 2006). The major component of clove oil
is considered to be eugenol, and lesser amounts of beta
caryophyllene and eugenol acetate (Prashar et al., 2006;
Chaieb et al., 2007).
In the present study, it was demonstrated that clove
oil (0.1 mL/kg) increases the primary and secondary
immune response as shown by an increase in the anti
SRBC titre. This indicates that clove oil increases the
humoral immunity and produces a positive effect on the
lymphocyte cell function as an immunostimulant.
However, treatment with clove oil was observed to
suppress delayed type hypersensitivity as shown by a
decrease in foot pad thickness. It is known that during
the cell mediated immune response, the sensitized
Tlymphocytes, when challenged with the antigen,
secrete a number of lymphokines, such as macrophage
migration inhibition factor, leukocyte migration inhibition factor, etc. (Agrawal and Reynolds, 1991). The
factors, in turn, attract more scavenger cells to the site
of reaction. The inltrating cells are then immobilized
to promote a defensive (inammatory) reaction. The
present ndings indicate that there was probably an
inhibition of the release of lymphokines, on clove oil
administration allowing the inammatory cells to move
Table 1. Effect of clove oil on hemagglutination titre to sheep red
blood cells (SRBC) in rats
Treatment
(mL/kg)

Vehicle
Clove oil (0.1)

Primary response
(antiSRBC titre
on day 7)

Secondary response
(antiSRBC titre
on day 9)

6 1.02
8.85 0.5a

6.8 1.06
9.4 0.04b

MannWhitney test. ap < 0.05 vs vehicle (primary response);

b
p < 0.05 vs vehicle (secondary response).

RESULTS
Table 2. Effect of clove oil on delayed type hypersensitivity in rats

Humoral immune response

Clove oil (0.1 mL/kg, i.p.) when given on day 1 to day 6
after sensitization with SRBC on day 0, caused a
signicant increase in the primary antiSRBC titre
measured on day 7 compared with the control (p < 0.05)
(Table 1).
Copyright 2011 John Wiley & Sons, Ltd.

Treatment (mL/kg/day 14 days, i.p.)

Vehicle
Clove oil (0.1)

Increase in paw
thickness (%)
21.6 1.48
11.81 1.10a

Unpaired ttest. ap < 0.01 vs vehicle.

Phytother. Res. 25: 12541256 (2011)

1256

S. HALDER ET AL.

away from the site of reaction. These results predict that

there will be probably less inammatory response after
clove oil administration. It has been reported earlier
that eugenol, the major constituent of clove oil,
possesses antiinammatory activity (Markowitz et al.,
1992). The present observation demonstrates that clove
oil, besides having a direct effect as an antiinammatory
substance, may also reduce inammation indirectly
through the inhibition of cell mediated immune
response.
It is known that clove oil causes damage to the
bacterial and fungal cell wall, reduces the fungal
inoculum size and also inhibits viral DNA polymerase
activity, thus resulting in its antimicrobial action
(Chaieb et al., 2007). The present study shows that

clove oil decreases the cellmediated immune response

probably by reducing secretion of lymphokines and
augments humoral immunity. Thus, it can be concluded
that the essential oil of buds of Eugenia caryophyllata
inuences some immunological parameters in animals
and these effects along with its antimicrobial action per
se could be responsible for its role as an agent to combat
various bacterial, viral and fungal infections in the body
and also as an antiinammatory agent.

Conflict of Interest
The authors have declared that there is no conict of interest.

REFERENCES
Agrawal PK, Reynolds DL. 1991. Reynolds evaluation of the cell
mediated immune response of chickens vaccinated with
Newcastle disease virus as determined by the under
agarose leukocytemigrationinhibition technique. Avian Dis
35: 360364.
Chaieb K, Hajlaoui H, Zmantar T et al. 2007. The chemical
composition and biological activity of clove essential oil,
Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a
short review. Phytother Res 21: 501506.
Chami F, Chami N, Bennis S, Bouchikhi T, Remmal A. 2005.
Oregano and clove essential oils induce surface alteration of
Saccharomyces cerevisiae. Phytother Res 19: 405408.
Friedman M, Henika PR, Mandrell RE. 2002. Bactericidal activities
of plant essential oils and some of their isolated constituents
against Campylobacter jejuni, Escherichia coli, Listeria
monocytogenes, and Salmonella enterica. J Food Prot 65:
15451560.
Halder S, Bharal N, Mediratta PK, Kaur I, Sharma KK. 2009. Anti
inflammatory, immunomodulatory and antinociceptive activity
of Terminalia arjuna Roxb bark powder in mice and rats. Indian
J Exp Biol 47: 577583.

Copyright 2011 John Wiley & Sons, Ltd.

Institoris L, Siroki O, Undeger U, Basaran N, Desi I. 2001.

Immunotoxicological investigations on rats treated subacutely
with dimethoate, As3+ and Mg2+ in combination. Hum Exp
Toxicol 20: 329336.
Kalemba D, Kunicka A. 2003. Antibacterial and antifungal
properties of essential oils. Curr Med Chem 10: 813829.
Lawless J. 1995. The Illustrated Encyclopaedia of Essential Oils.
Element Books: England.
Markowitz K, Moynihan M, Liu M, Kim S. 1992. Biologic
properties of eugenol and zinc oxideeugenol. A clinically
oriented review. Oral Surg Oral Med Oral Pathol 73:
729737.
Ogata M, Hoshi M, Urano S, Endo T. 2000. Antioxidant activity of
eugenol and related monomeric and dimeric compounds.
Chem Pharm Bull 48: 14671469.
Pasay C, Mounsey K, Stevenson G et al. 2010. Acaricidal activity
of eugenol based compounds against scabies mites. PLoS One
5: e12079.
Prashar A, Locke IC, Evans CS. 2006. Cytotoxicity of clove
(Syzygium aromaticum) oil and its major components to
human skin cells. Cell Prolif 39: 241248.

Phytother. Res. 25: 12541256 (2011)