Analgesic effect of clove essential oil in mice

Abstract

Objective: Results obtained from literature reviews and human studies have shown the analgesic effects of clove plant in toothache. The present work was undertaken in order to investigate the possible analgesic effect of clove oil in mice.
Materials and Methods: Fifty mice were divided into 5 groups: 1) Saline; 2) Essential oil (Ess) 2%, 3) Ess 5%, 4) Ess10% and 5) Ess 20%. The hot plate test (55±0.2 °C; Cut-off 60 sec) was performed as a base record 15 min before injection of drugs (Saline or 2, 5, 10 and 20% concentrations of Essential oil) and consequently repeated every 15 minutes after injection.
Results: Repeated measures ANOVA test showed that maximal percent effect (MPE) in animal groups treated by 5, 10 and 20% essential oil was significantly higher than saline group. Comparison between 4 treated groups showed that MPE in 10% essential group was higher than 2 and 5% groups however; there was no significant difference between 10% and 20% groups.
Conclusion: The result of present study showed that clove essential oil has analgesic effect inmice using hot plate test. More investigations are needed to elucidate the exact mechanism (s).

Full text

Avicenna Journal of Phytomedicine Received: Feb 20, 2011; Accepted: Apr 18, 2011 Vol. 1, No. 1, Summer 2011, 1-6
1AJP, Vol. 1, No. 1, Summer 2011
Analgesic effect of clove essential oil in mice
Mahmoud Hosseini
1,*
,
Mina Kamkar Asl
1
, Hassan Rakhshandeh
2
Abstract
Objective: Results obtained from literature reviews and human studies have shown the analgesic
effects of
clove
plant in toothache. The present work was undertaken in order to investigate the
possible analgesic effect of clove oil in mice.
Materials and Methods: Fifty mice were divided into 5 groups: 1) Saline; 2) Essential oil (Ess)
2%, 3) Ess 5%, 4) Ess10% and 5) Ess 20%. The hot plate test (55±0.2 °C; Cut-off 60 sec) was
performed as a base record 15 min before injection of drugs (Saline or 2, 5, 10 and 20%
concentrations of Essential oil) and consequently repeated every 15 minutes after injection.
Results: Repeated measures ANOVA test showed that maximal percent effect (MPE) in animal
groups treated by 5, 10 and 20% essential oil was significantly higher than saline group.
Comparison between 4 treated groups showed that MPE in 10% essential group was higher than 2
and 5% groups however; there was no significant difference between 10% and 20% groups.
Conclusion: The result of present study showed that clove essential oil has analgesic effect inmice
using hot plate test. More investigations are needed to elucidate the exact mechanism (s).
Keywords:
Clove, Essential oil, Analgesia, Mice, Hot plate
1- Neuroscience Research Center and Department of Physiology, School of medicine, Mashhad
University of Medical Sciences, Mashhad, I. R. Iran
2- Pharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad
University of Medical Sciences, Mashhad, I. R. Iran
*Corresponding Author:
Tel: +985118002222; Fax: +985118828564
E-mail: Hosseinim@mums.ac.ir

Hossieni et al.
2
AJP, Vol. 1, No. 1, Summer 2011
Introduction
The clove (Eugenia caryophyllata) is
a tree from Myrtaceae family with a
height ranging from 10 to 20 meters
which is growing in islands of
Indonesia, Tanzania, Sri Lanka,
Madagascar, India and Malaysia (Arung
et al., 2011) (Tyler et al., 1988).
Traditionally, several parts of the plant
such as leaves and buds are used in
cooking, food processing, pharmacy,
perfumery and cosmetics (Daniel et al.,
2009). It has also been used to treat
many diseases such as disorder of
digestive systems (Baytop, 1984). It has
been shown that some components of
clove are useful in bacterial and fungal
infections (Zheng et al., 1992; Zhang
and Chen, 1997). The cytotoxic and anti
cancerogenic effects of the plant and its
components have also been reported
(Zheng et al., 1992; Zhang and Chen,
1997; Kouidhi et al., 2010). Several
antimicrobial agents are present in clove
and therefore, the extracts of this plant
have been frequently used to treat the
oral bacteria which are commonly
associated with dental caries and
periodontal disease (Cai and Wu, 1996).
The antimicrobial and anti-fungal
properties of clove oil allow its use for
acne, warts, scars and parasites (Saeed
and Tariq, 2008). Vaso-relaxant as well
as smooth muscle relaxant effects of the
essential oil has also been demonstrated
(Nishijima et al., 1999; Damiani et al.,
2003). Beneficial effects of the plant in
asthma as well as various allergic
disorders has also been reported (Kim et
al., 1998).
Results obtained from human studies
also confirmed analgesic effects of the
plant in toothache and in patients
suffering from anal fissure (Tyler et al.,
1988; Elwakeel et al., 2007). In
experimental researches it has been
reported that essential oil from several
parts of this plant has anesthetic effects
in fish (Park et al., 2011).
Phytochemical analysis of clove
essential oil has shown the presence of
eugenol as a main component (Yu and
Hungju, 1981; Daniel et al., 2009). The
anesthetic effects of eugenol in dental
pain as well as the analgesic and anti-
inflammatory effects of this component
in animal models has been well
documented (Diaz and Sembrano, 1985;
oztürk and ozbek, 2005; Kurian et al.,
2006; Daniel et al., 2009).
Pharmacological studies have also
demonstrated the anticonvulsant and
anti-stress properties of eugenol
(Dallmeier and Carlini, 1981) (Sen et
al., 1992). In Iranian folk medicine, the
buds of this plant have been used as an
antiepileptic remedy (Avicenna, 1988).
Therefore, the present work was
undertaken in order to investigate the
possible analgesic effect of clove oil in
mice.
Material and Methods
Animals and drugs
50 male mice (27-32 g) were used.
All mice were housed in 4–6 per
standard cages, at room temperature 2±1
°C) on a 12 h light/dark cycle. Food and
water were available ad libitum. Animal
handling and all related procedures were
carried out in accordance with Mashhad
University of Medical Sciences, Ethical
Committee Acts. Essential oil was
kindly provided by Eksir Gol Sorkh
Company, Mashhad, Iran. Different
concentrations of Essential oil were
prepared in 10 ml saline.
Nociceptive test
To assess nociceptive responses, hot
plate method was used. In hot plate
method, animals were placed on the hot
plate with temperature setting controlled
at 55±0.2 °C. Cut-off time was 60 seconds
(Hosseini et al., 2011). Nociceptive
response was defined as licking forepaws
or moving hindpaws. Time duration
between placing the animals on hot plate
and licking forepaws or moving hind paws

Analgesic effect of clove essential oil
3
AJP, Vol. 1, No. 1, Summer 2011
was considered as reaction time. The hot
plate test was performed as a 3 base
records (10 min interval) 15 min before
injection of essential oil or saline (10
ml/kg; i.p.) and consequently was
repeated 5 times , every 10 minutes after
injection.
Experimental design
Fifty mice were divided into 5 groups:
1) Saline; 2) Essential oil (Ess) 2%, 3) Ess
5%, 4)Ess 10% and 5) Ess 20%. The
animals were treated with saline or
different concentrations of essential ( 2, 5,
10 and 20 %) oil 30 min before testing in
hot plate. The volume of injection was 10
ml/ kg;(i.p.).
Statistical analysis
Analgesic effect of essential oil or
saline was calculated as maximal possible
effect (MPE) [MPE (%) = [(test response
time-basal response time)/(cut-off time-
basal response time) × 100%] (Sepehri
and Shafeiee, 2006). All data were
presented as mean ± S.E.M of %MPE.
Statistical comparison of base reaction
latency time between groups was done
with one-way analysis of variance
(ANOVA) and post hoc Tukey’s HSD
test. Repeated measures ANOVA
followed by post hoc Tukey’s HSD test
was used for comparison of %MPE after
injection of drugs. Differences were
considered statistically significant when
p<0.05.
Results
The basal reaction time in saline group
was 4.29 ± 0.8 sec. As the Figure 1 shows,
basal reaction latency times in Ess 2%,
Ess 5%, Ess 10% and Ess 20 % groups
were 3.26 ± 0.7, 3.53 ± 0.8, 4.93 ± 0.9 and
3.52 ± 0.8 sec respectively. There was no
significant difference between 5 groups
(Figure 1). Repeated measures ANOVA
test showed that MPE in animal groups
treated by 5, 10 and 20% essential oil was
significantly higher than saline group
(p<0.05 and p<0.001, Figure 2).
Comparison between 4 treated groups
showed that MPE in 10% essential oil
group was higher than 2 and 5% groups
(p<0.001, Figure 2). There was no
significant difference between 10 and 20
groups
.
Figure 1. Comparison of basal reaction time
between the animals of different groups. Data
were shown as mean± SEM (n=10 in each
group). There was no significant difference
between groups.
Figure 2. Comparison of MPE between the
groups which received different
concentrations of essential oil or Saline. Data
were shown as mean± SEM (n=10 in each
group). Repeated measures ANOVA test
showed that MPE in animal groups treated by
5, 10 and 20% essential oil was significantly
higher than saline group (p<0.05 and
p<0.001). Comparison between 4 treated
groups showed that MPE in 10% essential oil
group was higher than 2 and 5% groups
(p<0.001).There was no significant difference
between 10 and 20 groups.
0
2
4
6
8
saline Ess 2% Ess 5% Ess 10% Ess 20%
latency time(sec)
groups

Hossieni et al.
4
AJP, Vol. 1, No. 1, Summer 2011
Discussion
The result of present study showed that
essential oil from clove had analgesic
effects tested in hot plate. The results also
showed that 10% concentration of
essential oil was the most effective in
comparison with other concentrations. A
maximum effect of 10% concentration
was seen 70 min after injection. There is
evidence that different parts of clove are
beneficial in toothache (Yu and Hungju,
1981). The extracts of this plant has been
frequently used to anesthetize the fish
which was comparable to lidocaine
(Anderson et al., 1997; Waterstrat, 1999;
Oryzias dancena ; Park et al., 2011). It
was also demonstrated that topical
application of clove oil cream had a
significant beneficial effect in patients
suffering from chronic anal fissure
(Elwakeel et al., 2007). Essential oil of
clove is a colorless or light yellowish fluid
extract from dried flower buds by steam
distillation. The results obtained by
GC/MS analysis showed that clove
essential oil contains 36 components. The
highest concentration was of eugenol
(88.58%), eugenyl acetate (5.62%) and β-
cariophyllene (1.38%). (oztürk and ozbek,
2005; Chaieb et al., 2007).
The analgesic
effects of the essential oil which was seen
in the present study may be due to this
component. Daniel et al (2009) and kurain
et al (2006) also confirmed the
antinociceptive activity of eugenol against
chemical (acetic acid tests), as well
thermal stimuli. They suggested that
eugenol predominantly inhibits the
peripheral pain mechanism (Kurian et al.,
2006; Daniel et al., 2009). In several
studies the analgesic effects of eugenol
has been attributed to its capability to
suppress prostaglandins and other
inflammatory mediators such as
leukotrienes (Raghavenra et al., 2006).
Anti inflammatory, antipyretic and anti
allergic effects of this compound may
confirm this hypothesis (Feng and Lipton,
1987; Murakami et al., 2003). It has been
reported that eugenol reduces paw edema
in carrageenan induced inflammation test
and pleural exudates in carrageenan-
induced pleurisy in rats .(Daniel et al.,
2009) Eugenol is also believed to depress
the sensory receptors involved in pain
perception (Robbers and Tyler, 1999).
The results of present study also showed
that the essential olive of clove has
analgesic effects with mechanism(s)
which are different from previous studies.
Eugenol also inhibits the conduction of
action potential in sciatic nerves (Kozam,
1977). Eugenol produces anesthesia in
rodents similar to propofol (Sell and
Carlini, 1976; Guénette et al., 2007), and
alleviates thermal hypersensitivity in an
experimental model of neuropathic pain in
rats (Guénette et al., 2007). Eugenol
inhibits N-methyl-
D
-aspartate (NMDA)
receptors but potentiates ionotropic γ-
aminobutyric acid (GABA
A
) receptors,
which are both involved in pain sensitivity
(Aoshima and Hamamoto, 1999). Eugenol
depresses compound action potentials in
both A and C fibers which may explain its
analgesic effects (Brodin, 1985). Eugenol is
similar in chemical structure to capsaicin
and therefore its effect on a vanilloid
receptor should not be ignored (Yang et al.,
2003). It was also shown that eugenol
inhibits Na
+
currents in rat dorsal root
ganglion neurons (Cho et al., 2008). β-
Caryophyllene, the other main component
of clove oil, showed anti-inflammatory
activity in several animal models, including
carrageenan- and PGE-induced hind paw
edema (Ghelardini et al., 2001). The role of
this component in analgesic effects of clove
essential oil which was shown in the present
study should not be ignored.
It is concluded that different
concentrations of the essential oil from
clove have analgesic effects however the
exact mechanism (s) need to be
investigated.
Acknowledgments
Authors would like to thank the Vice
Chancellery of Research of Mashhad
University of Medical Sciences for financial
supports
.

Analgesic effect of clove essential oil
5
AJP, Vol. 1, No. 1, Summer 2011
References
Anderson WG, McKinley RS, Colavecchia M.
1997. The use of clove oil as an
anestheticfor rainbow trout and its effects
on swimming performance. North
American Journal of Fisheries
Management, 17:301-307.
Aoshima H, Hamamoto K. 1999. Potentiation
of GABAA receptors expressed in
Xenopus oocytes by perfume and
phytoncid. Biosci Biotechnol Biochem,
63:743-748.
Arung ET, Matsubara E, Kusuma IW,
Sukaton E, Shimizu K, Kondo R. 2011.
Inhibitory components from the buds of
clove (Syzygium aromaticum) on melanin
formation in B16 melanoma cells.
Fitoterapia, 82:198-202.
Avicenna A. 1988. In: Ghanoon dar Teb.
Soroosh, Tehran:244–251. Baytop T.
1984. Therapy with medicinal plants in
Turkey (Past and Present). Nobel T p Bas
mevi. Brodin P. 1985. Differential
inhibition of A, B and C fibres in the rat
vagus nerve by lidocaine, eugenol and
formaldehyde. Arch Oral Biol, 30:477-
480.
Cai L, Wu CD. 1996. Compounds from
Syzygium aromaticum possessing growth
inhibitory activity against oral pathogens.
J. Nat. Prod, 59:987- 990.
Chaieb K, Hajlaoui H, Zmantar T, Kahla
Nakbi AB, Rouabhia M, Mahdouani K,
Bakhrouf A. 2007. The chemical
composition and biological activity of
clove essential oil, Eugenia caryophyllata
(Syzigium aromaticum L. Myrtaceae): a
short review. Phytotherapy research,
21:501-506.
Cho JS, Kim TH, Lim JM, Song JH. 2008.
Effects of eugenol on Na+ currents in rat
dorsal root ganglion neurons. Brain Res,
1243:53-62.
Dallmeier K, Carlini EA. 1981. Anesthetic,
hypothermic, myorelaxant and
anticonvulsant effects of synthetic eugenol
derivatives and natural analogues.
Pharmacology, 22:113.
Damiani CEN, Rossoni LV, Vassallo DV.
2003. Vasorelaxant effects of eugenol on
rat thoracic aorta. Vascular
pharmacology, 40:59-66.
Daniel AN, Sartoretto SM, Schmidt G,
Caparroz-Assef SM, Bersani-Amado CA,
Cuman RKN. 2009. Anti- inflammatory
and antinociceptive activities A of eugenol
essential oil in experimental animal
models. Revista Brasileira de
Farmacognosia, 19:212- 217.
Diaz MR, Sembrano JM. 1985. A
comparative study of the efficacy of garlic
and eugenol as palliative agents against
dental pain of pulpal origin. The Journal of
the Philippine Dental Association, 35:3-
10.
Elwakeel HA, Moneim HA, Farid M, Gohar
AA. 2007. Clove oil cream: a new
effective treatment for chronic anal fissure.
Colorectal Dis 9:549-552.
Feng J, Lipton JM. 1987. Eugenol:Antipyretic
activity in rabbits. Neuropharmacology,
26:1775-1778.
Ghelardini C, Galeotti N, Di Cesare Mannelli
L, Mazzanti G, Bartolini A. 2001. Local
anaesthetic activity of [beta]-
caryophyllene 1. Il Farmaco, 56:387-389.
Guénette SA, Ross A, Marier JF, Beaudry F,
Vachon P. 2007. Pharmacokinetics of
eugenol and its effects on thermal
hypersensitivity in rats. European journal
of pharmacology, 562:60-67.
Hosseini M, Taiarani Z, Hadjzadeh MA,
Salehabadi S, Tehranipour M, Alaei HA.
2011. Different responses of nitric oxide
synthase inhibition on morphine- induced
antinociception in male and female rats.
Pathophysiology, 18:143- 149.
Kim HM, Lee EH, Hong SH, Song HJ, Shin
MK, Kim SH, Shin TY. 1998. Effect of
Syzygium aromaticum extract on
immediate hypersensitivity in rats. Journal
of ethnopharmacology, 60:125- 131.
Kouidhi B, Zmantar T, Bakhrouf A. 2010.
Anticariogenic and cytotoxic activity of
clove essential oil (Eugenia caryophyllata)
against a large number of oral pathogens.
Annals of Microbiology, 60:1-6.
Kozam G. 1977. The effect of eugenol on
nerve transmission. Oral Surgery, Oral
Medicine, Oral Pathology, 44:799-805.
Kurian R, Arulmozhi DK, Veeranjaneyulu A,
Bodhankar SL. 2006. Effect of eugenol on
animal models of nociception. Indian
Journal of Pharmacology, 38:341.
Murakami Y, Shoji M, Hanazawa S, Tanaka
S, Fujisawa S. 2003. Preventive effect of
bis- eugenol, a eugenol ortho dimer, on
lipopolysaccharide-stimulated nuclear
factor kappa B activation and

Hossieni et al.
6
AJP, Vol. 1, No. 1, Summer 2011
inflammatory cytokine expression in
macrophages. Biochemical pharmacology,
66:1061-1066.
Nishijima H, Uchida R, Kameyama K,
Kawakami N, Ohkubo T, Kitamura K.
1999. Mechanisms mediating the
vasorelaxing action of eugenol, a pungent
oil, on rabbit arterial tissue. The Japanese
Journal of Pharmacology, 79:327-334.
oztürk A, ozbek H. 2005. The anti-
inflammatory activity of Eugenia
caryophyllata essential oil: an
animalmodel of anti-inflammatory
activity. European Journal of General
Medicine, 2:159-163.
Park IS, Park SJ, Gil HW, Nam YK, Kim
DS.2011. Anesthetic effects of clove oil
and lidocaine-HCl on marine medaka
(Oryzias dancena). Lab animal, 40:45-51.
Raghavenra H, Diwakr BT, Lokesh BR,
Naidu KA. 2006. Eugenol--The active
principle from cloves inhibits 5-
lipoxygenase activity and leukotriene- C4
in human PMNL cells. Prostaglandins,
Leukotrienes and Essential Fatty Acids,
74:23-27.
Robbers JE, Tyler VE. 1999. Tyler's Herbs of
choice: the therapeutic use of
phytomedicinals: CRC. Saeed S,
Tariq P. 2008. In vitro antibacterial activity of
clove against Gram negative bacteria.
Pakistan J. Botany, 40:2157- 2160.
Sell AB, Carlini EA. 1976. Anesthetic action
of methyleugenol and other eugenol
derivatives. Pharmacology, 14:367-377.
Sen P, Maiti PC, Puri S, Ray A, Audulov
NA,Valdman AV. 1992. Mechanism of
anti- stress activity of Ocimum sanctum
Linn, eugenol and Tinospora malabarica
inexperimental animals. Indian journal of
experimental biology, 30:592.
Sepehri G, Shafeiee MN. 2006. Effect of
Cuneiformis Nucleus Inactivation by
Lidocaine Microinjection on the Analgesic
Response of Morphine in Rats. Iranian
Biomedical Journal, 10:21-26.
Tyler VE, Brady LR, Robbers JE. 1988.
Pharmacognosy. Lea & Febriger,
Philadelphia, PA. Links.
Waterstrat PR. 1999. Induction and recovery
from anesthesia in channel catfish
Ictalurus punctatus fingerlings exposed to
clove oil.Journal of the World Aquaculture
Society, 30:250- 255.
Yang BH, Piao ZG, Kim YB, Lee CH, LeeJK,
Park K, Kim JS, Oh SB. 2003. Activation
of vanilloid receptor 1 (VR1) by eugenol. J
Dent Res, 82:781- 785.
Yu J, Hungju F. 1981. Studies on the essential
oils of clove buds and clove leaves. Zhong
Caoyao, 12:339–342.
Zhang Y, Chen Y. 1997. Isobiflorin,
achromone C-glucoside from cloves
(Eugenia caryophyllata). Phytochemistry,
45:401-403.
Zheng GQ, Kenney PM, Lam LKT. 1992.
Sesquiterpenes from clove (Eugenia
caryophyllata) as potential
anticarcinogenic agents. Journal of natural
products, 55:999-1003.