The Anti-Inflammatory Activity of Eugenia Caryophllata Essential Oil: An animal model of anti-inflammatory activity

Abstract

Aim: The aim of this study is gas chromatographic analysis of Eugenia caryaphyllata (clove) essential oil and investigation of its anti-inflammatory effects. Methods: The study involved eight groups; Serum physiologic, ethyl alcohol, indomethacin (3 mg/kg), etodolac (50 mg/kg), cardamom (0.05 mL/kg), EC-I (0.025 mL/kg), EC-II (0.050 mL/kg), EC-III (0.100 mL/kg) and EC-IV (0.200mL/kg). After measuring the volumes of right hind-paws of rats using a plethysmometer, drugs were injected intraperitoneally and lambda-carrageenan were injected subcutaneously into the plantar region. Three hours after the injections the volume measurements of the right hind-paws were repeated and the difference between the volumes were compared. Results: The composition of the essential oil was as follows: β-caryophyllen % 44.7, eugenol % 44.2, α-humulen % 3.5, eugenyl acetate % 1.3 and α-copaen % 1.0. It was found that indomethazin reduced the inflammation by 95.70%, etodolac by 43.42 %, EC-I by 46.55 %, EC-II by 90.15 %, EC-III by 66.94 % and EC-IV by 82.78 %. The essential oil of Eugenia caryophyllata had an anti-inflammatory effect matching to that of etodolac at 0.025 and 0.1 mL/kg and to that of indomethacin at 0.05 and 0.2 mL/kg doses. Conclusion: As a result Eugenia caryophyllata essential oil extract was shown to have an anti-inflammatory effect.

Full text

Correspondence: Hanefi Özbek, MD, PhD.
Yüzüncü Yıl Üniversitesi Tıp Fakültesi Farmakoloji AD
65300 VAN / TURKEY
Fax: 904322168352
E-mail: hanefiozbek@hotmail.com
Aim: The aim of this study is gas chromatographic analysis of Eugenia caryaphyllata (clove)
essential oil and investigation of its anti-inflammatory effects.
Methods: The study involved eight groups; Serum physiologic, ethyl alcohol, indomethacin (3
mg/kg), etodolac (50 mg/kg), cardamom (0.05 mL/kg), EC-I (0.025 mL/kg), EC-II (0.050 mL/kg),
EC-III (0.100 mL/kg) and EC-IV (0.200mL/kg). After measuring the volumes of right hind-paws
of rats using a plethysmometer, drugs were injected intraperitoneally and lambda-carrageenan
were injected subcutaneously into the plantar region. Three hours after the injections the volume
measurements of the right hind-paws were repeated and the difference between the volumes were
compared.
Results: The composition of the essential oil was as follows: ß-caryophyllen % 44.7,
eugenol % 44.2, α-humulen % 3.5, eugenyl acetate % 1.3 and α-copaen % 1.0. It was found that
indomethazin reduced the inflammation by 95.70%, etodolac by 43.42 %, EC-I by 46.55 %, EC-II
by 90.15 %, EC-III by 66.94 % and EC-IV by 82.78 %. The essential oil of Eugenia caryophyllata
had an anti-inflammatory effect matching to that of etodolac at 0.025 and 0.1 mL/kg and to that
of indomethacin at 0.05 and 0.2 mL/kg doses.
Conclusion: As a result Eugenia caryophyllata essential oil extract was shown to have an anti-
inflammatory effect.
Key words: Eugenia caryophyllata, essential oil, anti-inflammatory activity, rats.
Eur J Gen Med 2005; 2(4):159-163
THE ANTI-INFLAMMATORY ACTIVITY OF EUGENIA
CARYOPHYLLATA ESSENTIAL OIL: AN ANIMAL MODEL OF ANTI-
INFLAMMATORY ACTIVITY
Abdurrahman Öztürk, Hanefi Özbek
Yüzüncü Yıl University, Faculty of Medicine, Department of Pharmacology, Van, Turkey
INTRODUCTION
Eugenia caryophyllata (clove) plant
belongs to Myrtaceae families (1). Clove
spice is a nail-shaped dried flower bud of
Eugenia caryophyllata Thunb. species. It is an
ever-green plant of ten-to-twenty centimeters
in height with spear-shaped leaves and
racemiferous yellowish flowers. It’s grown
naturally in Moluku Islands of Indonesia
and cultivated in Tanzania, Madagascar, Sri
Lanka, India, Indonesia, Malaysia, Brazil,
Jamaica and Guinea. The plant has a strong
phenolic smell and sharp acrid taste. It
contains essential oil at 15-20%, tanene 13%
and fixed oil 10%. Non-essential ether extract
constitutes 6-12%. Essential oil of clove is a
colorless or light yellowish fluid extracted
from dried flower buds by steam distillation.
The main constituents are eugenol (80-90%),
β-caryophyllene (9%), eugenyl acetate (7%),
α-humulen, ylangen, metoxy benzaldehyde,
benzyl alchohol, benzaldehyde and cavychole.
Clove is used in cooking, food processing,
pharmacy, parfumery and cosmetics (2).
Eugenia caryophyllata (EC) was found
to be effective against egg and adult of
Pediculus capitis (3). It has antiseptic
as well as bacteriostatic and bactericidal
activity against several bacteria including
Escherichia coli and Staphylococcus aureus
(4;5). Growth of Helicobacter pylori being
one of the major causes of peptic ulcer
disease has been shown to be inhibited by
EC (6). Clove oil also showed the acaricidal
activity against Dermatophagoides farinae
and Dermatophagoides pteronyssinus (7). Its
anesthetic and spazmolytic properties have
also been reported (5). In mouse macrophage
cultures, methanol extract of EC inhibits
cyclooxigenase by 80% (8) without affecting

Eugenia caryophyllata 160
Table 1. Analysis of Eugenia caryophyllata
essential oil
Component %
ß-caryophyllen 44.7
Eugenol 44.2
α-humulen 3.5
Eugenyl acetate 1.3
α-copaen 1.0
COX-1 enzyme (7). Some components of
virus-cell fusion inhibitors were isolated from
its extracts (9). Pourgholami and colleagues
(10) found the essential oil of EC had
anticolvulsive effect in tonic seizures but not
in clonic seizures in mice. The terpens, beta-
caryophyllene, beta-caryophyllene oxide,
alpha-humulene, alpha-humulene epoxide I
and eugenol from EC induces glutathione S-
transferase enzyme which plays an important
role in detoxification in liver and intestines.
Induction of glutathione S-transferase
has been suggested to inhibit chemical
carcinogens, hence these terpens are promising
anticarcinogens (11). Özbek et al reported
that median lethal dose of EC and Eugenia
caryophyllata etheric oil were 0.613 mL/kg
and 0.863 mL/kg in mice respectively (12).
In traditional public medicine, EC has been
used as antipyretic, aphrodisiac, appetizer
(13,14), expectorant, antiemetic, anksiolytic,
myorelaxant, analgesic, decongestant, anti-
inflammatory and hypnotic (15,16).
In this study, we investigated whether EC
had an anti-inflammatory effect on the anti-
inflammatory animal model as supposed by
traditional medicine.
MATERIALS AND METHODS
Animals
Male, outbred, Sprague-Dawley rats were
maintained in the Animal House of Yüzüncü
Yıl University, Faculty of Medicine. The
rats were bred in our institutional animal
house but the lineage originally obtained
from Ankara Health Protection Institute (a
governmental organisation). The animals were
reared in standard cages with food and water
ad libitum, at room temperature (22±2 0C)
with artificial light from 7.00 am to 7.00 pm,
and provided with pelleted food (Van Animal
Feed Factory, Van-TURKEY). Ambient temp
was 22±2 0C and RH was % 55-60. The rats
housed in groups. The approval of Animal
Ethics Committee was obtained.
Chemicals
Lambda-carrageenan Type IV and
indomethacin were obtained from Sigma
(Steinheim, Germany), and Etodolac from
FAKO Pharmacy (İstanbul, Turkey). EC
used was purchased at local market from
Van in Turkey. It was identified by Dr. Fevzi
Özgökçe, a plant taxonomist from Department
of Biology in Faculty of Science and Art,
Yüzüncü Yıl University. A voucher specimen
(B-12) has been kept in our laboratory for
future reference.
Isolation of tested material
EC was powdered in a mixer, placed in
a distillation flask (50 g) and the oil was
collected by steam distillation (100 0C, 2
hours). The yield of essential oil was 1.06%
(v/w). Lambda-carrageenan was prepared in
isotonic saline solution (0.9 % NaCl) and
indomethacine was prepared in ethyl alcohol
just before use.
Analysis of essential oil
Analysis of essential oil was carried out
by Anadolu University Research Center for
Medical and Aromatic Plants and Drugs,
Eskisehir, Turkey. Gas chromatography
analysis was carried out on a Shimadzu GC-
9A gas chromatograph with FID detector and
a Thermon-600 T capillary column (50 mL,
0.25 mm I.D.). The operating condition was as
follows. Carrier gas was nitrogen with a split
rate of 60:1, the oven temperature for first 10
min was kept at 70°C-10’ and then increased
at a rate of 2°C:min until 180°C-30’. Injector
and detector temperature was set at 250°C.
Anti-inflammatory activity
The method of Winter et al (17) with
slight modification was used. Eighty
rats of either sex were divided into eight
groups of ten animals each. The rats were
fasted for 12 h and deprived of water only
during the experiment. Deprivation of
water was to ensure uniform hydration and
minimize variability in edematous response.
Inflammation of the hind paw was induced by
injecting 0.05 mL fresh lambda carrageenan
(phlogistic agent) into the subplantar surface
of the right hind paw. The control group
I was given normal saline and the control
group II was given ethyl alcohol. The third
(reference group-I) and fourth (reference
group-II) group received indomethacin (3
mg/kg, i.p.) (18) and etodolac (50 mg/kg, i.p.)
(19), respectively. The remaining four groups
received the extract at doses of 0.025 mL/kg,

161 Öztürk and Özbek
Table 2. Symptoms and findings
Groups Dose Paw edema (% mL) In h i bi t io n
(%)
Control-I (ISS) 0.1 mL 1.043±0.084 -
Control-II (ethyl alcohol) 0.1 mL 0.988±0.075 -
Indomethacin 3mg/kg 0.042±0.015b 95.70
Etodolac 50mg/kg 0.572±0.033bd 43.42
E. caryophyllata-I 0.025mL/kg 0.555±0.056ad 46.5 5
E. caryophyllata-II 0.050mL/kg 0.102±0.037afh 90.15
E. caryophyllata-III 0.100 mL/kg 0.476±0.090ac 66 . 94
E. caryophyllata-IV 0.200 mL/kg 0.179±0.047aeg 82 .78
F value 50.151
p value 0.000
0.050 mL/kg, 0.100 mL/kg, and 0.200 mL/kg,
i.p. These doses of the extract utilized in the
current study has been chosen accordingly
LD 1 value (LD1: 0.20014 mL/kg) (12).
The measurement of foot volume was
accomplished immediately by displacement
technique using the plethysmometer (Ugo
Basile 7140 plethysmometer, Italy) before
the drug injection and three hours after the
carrageenan injection.
The inhibition percentage of the
inflammatory reaction was determined for
each animal by comparison with controls and
calculated by the formula (20): I%: [(1-(dt/
dc)]x100dt is the difference in paw volume in
the drug-treated group and dc the difference
in paw volume in the control group.
Statistical analysis
Results of the paw edema of the rats were
reported as mean±standard error of mean
(SEM). The total variation was analyzed by
performing one-way analysis of variance
(ANOVA). Tukey’s HSD test (Tukey’s
honestly significant difference test) was used
for determining significance. Probability
levels of less than 0.05 were considered
significant.
RESULTS
Analysis of essential oil
The results of gas chromatographic
analysis was shown in Table 1.
Anti-inflammatory activity
Table 2 shows antiedema effect of
intraperitoneally administered EC on
carrageenan paw oedema in rats. Essential
oil of EC extract showed significant anti-
inflammatory effect at doses of 0.05 mL/
kg (90.15% inhibition) and 0.200 mL/kg
(82.78% inhibition). However, the activity
at doses of 0.05, 0.100 and 0.200 mL/kg
were similar (p>0.05). Therefore the order
of anti-inflammatory activity was not
0.05>0.200>0.100 mL/kg. It can be say the
order of anti-inflammatory activity were
0.05=0.100=0.200 mL/kg. Compared to the
controls, strong anti-inflammatory activity
was observed in indomethacin group with a
95.7 % regression of the inflammation.
Etodolac, the second reference agent
showed significant but weaker anti-
inflammatory activity with 43.42 % regression
of edema. Essential oil extract of EC has
significantly lower anti-inflammatory effect
compared to indomethacin at 0.025 mL/kg
and 0.100 mL/kg doses and comparable effect
at 0.050 mL/kg and 0.200 mL/kg doses.
When compared to etodolac the extract had
statistically similar effect at 0.025 mL/kg and
0.100 mL/kg and higher activity at 0.050 mL/
kg and 0.200 mL/kg.
Essential oil extract of EC had significantly
lower anti-inflammatory activity at 0.025 mL/
kg compared to the other doses of EC.
DISCUSSION
EC has been used in traditional public
medicine to relieve nasal obstruction and
musculoskeletal pain which imply anti-
inflammatory activity for the plant (16).
Analgesic, anesthetic, spasmolytic and
antibacterial effects of EC were demonstrated
by several scientific studies (3-6). Molecular
evidence for its anti-inflammatory activity
comes from the studies indicating COX-2
inhibition without affecting COX-1 in mice
macrophage cell cultures (8,21). On the
other hand in vitro studies are validated
Data presented as mean ± standard error mean (n=10).
a: p<0.001 compared to control-I (SF) group, b: p<0.001 compared to control-II (ethyl alchohol) group,
c: p<0.05 compared to indomethacin group, d: p<0.001 compared to indomethacin group,
e: p<0.01 compared to etodolac group, f: p<0.001 compared to etodolac group,
g: p<0.01 compared to E. Caryophyllata-I group, h: p<0.001 compared to E. caryophyllata-I group.

Eugenia caryophyllata 162
only with consistent in vivo studies as many
compensatory mechanisms may interfere
with the drug effects resulting in weaker
physiological response.
The current study clearly demonstrated
anti-inflammatory effect of EC essential oil in
vivo, which equals to that of etodolac at 0.025
mL/kg and 0.100 mL/kg doses and to that of
indomethacin at 0.050 mL/kg and 0.200 mL/
kg doses.
Anti-inflammatory effect of EC was thought
to be due to COX-2 inhibition (7,8). However,
the effective molecule of the extract has to be
identified by testing each of the constituents
after being purified. Eugenol which makes up
44.2 % of the extract is sold in pharmacies as
an analgesic. Analgesic effect of the eugenol
may imply anti-inflammatory effect as well.
In an experimental model of arthritis in
rats induced by deactivated Mycobacterium
tuberculosis bacilli, eugenol suppressed the
inflammation significantly when administered
at a dose of 33 mg/kg, orally for 26 days (22).
It was also shown to inhibit COX-2 enzyme
in mouse macrophage RAW264.7 cell lines
induced by lipopolisaccaride (21). These
studies favor eugenol as the effective molecule
of EC extract providing an anti-inflammatory
activity comparable to indomethacin.
However, all other constituents listed in Table
1 may contribute to that activity. These are
mixed together in the essential oil extract so
they may have some enhancing or interfering
effects on one another. Therefore, in the next
step of investigation of therapeutic potential
of EC, each constituent molecule should be
tested for its anti-inflammatory activity and
undergone all necessary stages of modern
drug development process.
The current study proves the anti-
inflammatory activity of EC in vivo besides
its antibacterial, analgesic, spasmolytic
and anesthetic actions. EC appears to be a
promising agent for drug research.
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