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Masthead Logo Pakistan Journal of Neurological Sciences (PJNS)

Volume 14 | Issue 1 Article 9

3-2019

Antioxidative activity of clove (syzygium

aromaticum) oil administration in Middle cerebral
artery occlusion (mcao) Models of acute focal
cerebral ischemia
Hira Jawed
University of Karachi

Mufzala Shamim
University of Karachi

Sumera Sohail
University of Karachi.

Uzma Firdous
University of Karachi.

Nazish Iqbal Khan

University of Karachi.

Follow this and additional works at: https://ecommons.aku.edu/pjns

Part of the Neurology Commons

Recommended Citation
Jawed, Hira; Shamim, Mufzala; Sohail, Sumera; Firdous, Uzma; and Iqbal Khan, Nazish (2019) "Antioxidative activity of clove
(syzygium aromaticum) oil administration in Middle cerebral artery occlusion (mcao) Models of acute focal cerebral ischemia,"
Pakistan Journal of Neurological Sciences (PJNS): Vol. 14 : Iss. 1 , Article 9.
Available at: https://ecommons.aku.edu/pjns/vol14/iss1/9
 O R I G I N A L A R T I C L E

ANTIOXIDATIVE ACTIVITY OF CLOVE

(SYZYGIUM AROMATICUM) OIL ADMINISTRATION IN
MIDDLE CEREBRAL ARTERY OCCLUSION (MCAO)
MODELS OF ACUTE FOCAL CEREBRAL ISCHEMIA
Hira Jawed1, Mufzala Shamim1, Sumera Sohail1, Uzma Firdous1, Nazish Iqbal Khan1
1
Pathophysiology Research Unit, Department of Physiology, University of Karachi.

Correspondence to: Mufzala Shamim Email: mufzalashamim@yahoo.com

Date of submission: November 12, 2018 Date of revision: December 28, 2018 Date of acceptance: February 20, 2019

ABSTRACT

Objective: Stroke is a multifactorial neurological deficit syndrome. Oxidative stress is the principal underlying
pathophysiological mechanism of ischemic stroke associated with neuronal damage and neuroinflammation.

Methodology: The purpose of present study is to investigate the preventive effects of clove (Syzygium aromaticum) oil
pre-stroke and post-stroke administration against cerebral ischemia/reperfusion injury. Total of forty Wistar rats were
divided into five groups as control, sham, stroke, pre-stroke treated (receive clove oil (33mg/kg body weight) for 15
days once daily, stroke given after completion of 15 days pretreatment regime) and post-stroke treated group (receive
two doses of clove oil (33mg/kg body weight) one at 0 h (immediately after stroke induction) and second dose after 6
h of stroke). Stroke was induced via middle cerebral artery occlusion method (MCAO) (15 minutes occlusion followed
by 24 h reperfusion). At end of experimentation, animals were tested for sensorimotor functioning via neurological
deficit score and brain antioxidants including superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were
estimated in all groups

Results: Results showed significant betterment in neurological deficit score in treated groups (P<0.05) compared to
stroke group. MCAO induction significantly (P<0.01) increase oxidative stress in brain tissue of stroke group.
Pre-stroke and post-stroke treatment regime significantly (P<0.05) enhance brain endogenous antioxidants (SOD,
CAT, and GSH).

Conclusion: Suggesting the possible preventive role of clove oil against oxidative neuronal damage in acute focal
cerebral ischemia.

Keywords: Oxidative stress, eugenol, antioxidants, clove oil, neurological deficit, stroke.

INTRODUCTION: ischemic tissue results in free radicals generation which

augments neuronal injury followed by initiation of
Cerebrovascåular accidents are highly prevalent global inflammatory mechanism (3). Uncontrolled calcium
problem. Stroke is the fourth most common cause of influx, glutamate excitotoxicity, reactive oxygen species
death and second most leading cause of disability (ROS) over production are the underlying
around the globe (1). Among other forms of stroke, pathophysiological mechanisms of ischemic stroke (4).
ischemic stroke accounts for the majority (approx. Among all pathophysiological mechanisms of ischemic
85%) of all stroke cases. Stroke is the focal stroke, ROS over-production crucially contributes to
neurological deficit syndrome results from interrupted neuronal oxidative burden, disruption of blood brain
cerebral blood flow (CBF) with subsequent oxygen and barrier, neuroinflammation and activation & recruitment
glucose deprivation in nervous tissue (2). Reperfusion in of immune mediators in ischemic tissue (5,6). The only

PAKISTAN JOURNAL OF NEUROLOGICAL SCIENCES 10 VOL. 14 (1) J A N U A R Y- M A R C H 2 0 1 9

approved stroke therapy is recombinant tissue CLOVE OIL:
plasminogen activator (rtPA) but it has very narrow
Commercially available analytical grade Clove Oil
therapeutic window with high risk of thrombolysis limits
(catalog no. C8392) was purchased from
its clinical use (7).
Sigma-Aldrich (St Louis, MO, USA).
Dietary antioxidants and antioxidant therapies has been
Experimental Protocol:
long investigated for their promising neuroprotective,
neuronal survival and restorative potentials(8). A total of forty animals were randomly divided into
following groups:
Clove (Syzygium aromaticum), an aromatic flower buds
harvest from tree belongs of Myrtaceae family. Cloves’ Control Group: (n=8) Normal, untreated animals.
essential oil (aromatic, pale yellowish fluid) extracted
Stroke Group (n=8): Stroke inducted group
via distillation from air-dried, un-opened flower buds(9).
For centuries, clove flowers, buds and essential oil of Stroke Induction: Focal acute cerebral ischemia was
clove have been used in traditional medicinal systems established via MCAO technique as mentioned earlier
to treat variety of health problems. (12). Briefly, following anesthesia (via ketamine
(50mg/kg body weight) + xylazine (10mg/kg
A single clove bud contains 14-20% of essential oil. bodyweight) intraperitoneal injection) neck incision was
Eugenol is a principal bioactive phytochemical richly made to expose common carotid artery (CCA), external
found in clove oil(10). Therapeutic potentials of clove are carotid artery (ECA) and internal carotid artery (ICA). A
credited to eugenol including analgesic, silicon coated 4/0 nylon filament was introduce in left
anti-spasmodic, antioxidant, anti-inflammatory, ECA to the lumen of ICA and advanced until it occluded
anti-thrombotic, neuroprotective, anti-convulsant, the origin of middle cerebral artery (MCA). Filament was
anti-carcinogenic, anti-diabetic, anti-bacterial and removed after 15 minutes to allow reperfusion (24 h).
antifungal properties(9,10). To maintain body temperature animal was placed on
heating pad and carefully monitored throughout the
Therefore, the present study aimed to investigate the procedure.
antioxidative and possible neuroprotective effects of
Sham Group (n=8): Sham operated group.
clove (Syzygium aromaticum) oil administration against
ischemia-reperfusion (IR) injury in middle cerebral Sham surgery: Sham group animals also underwent
artery occlusion (MCAO) rat models of acute focal identical MCAO procedure but without the occlusion of
cerebral ischemia. MCA
Pre-stroke Treated Group (n=8): Animals of this
MATERIALS & METHODS:
group were intraperitoneally administered with clove oil
Ethics Statement: Animal handling, care and all (33mg/kg body weight per day) for 15 days. After the
experimental procedures were performed according to pre-treatment period, animals were inducted with
the guidelines of animal care and use (11). stroke.

ANIMALS: Post-stroke Treated Group (n=8): Animals of this group

received two intraperitoneal doses of clove oil
Adult female Wistar rats (200-250g) were purchased
(33mg/kg body weight i.p). First dose was administered
from animal care facility of ICCBS, university of Karachi,
immediately after stroke induction and second dose
Karachi Pakistan.
was administered 6 hours of stroke induction.
Wistar Adult Female Rats (200-250gms) were used in
At the end of experimentation period (after 24 h of
all the experimental groups, purchased from the animal
reperfusion), neurological deficit score and brain tissue
house of ICCBS Karachi, Pakistan.
antioxidants (superoxide dismutase, catalase and
glutathione) were investigated in all groups
Animals were housed as one animal per cage in a
well-aired room (12h light/dark cycles) with food
NEUROLOGICAL ASSESSMENT
(standard laboratory diet) and water ad libitum. Animals
were allowed to acclimatize for a week prior to any Neurological deficits were evaluated 24 h after
experimentation. reperfusion according the methods as described earlier

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(13,14). The neurological deficit (ND) score comprised RESULTS
of three tests: body twisting, forelimb flexion and body
Pre and Post Treatments with Clove Oil Reduces
balance as described earlier (15).
Neurological Functional Deficit in MCAO Rats In the
present study, we investigate whether clove oil
Final ND score was calculated as follow: ND score=
administration is neuroprotective against acute IR injury
score of Test 1 + Test 2 + Test 3
by using MCAO rat model and pre and post-treatment
ND score rates neurological deficit on a scale of 0-9
regimen. As shown in Figure 1, no neurological deficits
where 0, no deficit and 9 maximum neurological deficit.
were observed in control and sham operated animals.
Whereas MCAO mediated IR injury significantly affect
Biochemical Assessment and Analytical
sensorimotor function as indicated by the highest
Procedures
neurological scores stroke group animals (ND
Tissue Sample score=7.83≈8, P<0.0001), after MCAO. Compared
to stroke group, animals pretreated with clove oil for 15
Under deep anesthesia brain was removed immediately
days showed a significant decrease in neurological
rinsed with ice-cold saline (0.9% NaCl) weighed and
deficit score (P<0.01) after MCAO. While ND score of
stored at -80ºC till further investigation.
animals post-treated with clove oil with two dose after
Preparation of Brain Tissue Homogenate: stroke also exhibit significant (P<0.05) decrease in ND
score when compared with stroke group (Table 1).
Brain tissue homogenate was prepared according to
Comparing with stroke ND score, pre-treated group
the method as described in previous study (5).
showed more improvements then post-treated group.
Assessment of Brain Antioxidants: However, no statistically significant change was
observed in pre and post-treated groups (P>0.05)
Brain tissue antioxidants including superoxide
(Figure1, Table 1).
dismutase (SOD), catalase (CAT) and glutathione
(GSH) was estimated according to the recommended
procedures. Pre and Post Treatments with Clove Oil
Ameliorates Oxidative stress in MCAO rats’ Brain
Superoxide Dismutase Assay: Tissue SOD Tissue Brain weights of all experimental groups remain
concentration was estimated spectrophotometrically by close to baseline vale except a nonsignificant increase
the method of Kono (16) based on nitroblue was observed in brain weights of MCAO stroke group
tetrazolium (NBT) reduction rate recorded at 560nm compared to control (P>0.05). As shown in Table 2,
using Schimadzu UV spectrophotometer. SOD activity MCAO mediated IR injury significantly decrease brain
was expressed as U/g of tissue. Catalase Assay: antioxidants including superoxide dismutase, catalase
Concentration of catalase in brain tissue was and glutathione activities when compared with animals
determined by the spectrophotometric procedure as of control (SOD and CAT P<0.05; GSH: P<0.01) and
mentioned by Sinha, 1972. The reaction mixture sham (SOD and CAT P<0.05; GSH: P<0.01) groups.
comprised of hydrogen peroxide and dichromate acetic Effect of Pretreatment Regimen: Per day i.p. clove oil
acid solution and read at 570nm wavelength. Tissue administration at dose of 33mg/kg body weight for 15
CAT activity was recorded as µmol/g of tissue. days found to significantly increase brain SOD
(P<0.01) CAT (P<0.01) and GSH (P<0.05)
Glutathione Assay: GSH activity in brain tissue was
concentrations when compared with MCAO untreated
estimated according to previously described method
rats (Table 2).
(18). GSH concentration was recorded on kinetic
spectrophotometer at 25ºC and results were Effect of Post-treatment Regimen: Intraperitoneal
represented as U/g of tissue. administration of clove oil doses one at 0 h and second
at 6 h post-stroke induction improve brain antioxidant
Statistical Analysis:
(CAT, SOD, GSH) status in animals of post-treated
Data were presented as mean ± SEM (standard error
group (SOD: P<0.05) however these increase were not
of mean) Statistical analysis was performed by one-way
statistically significant for CAT and GSH (P>0.05) as
analysis of variance (ANOVA) with Tukey’s post hoc test
compared to stroke group (Table 2). Moreover,
using the Statistical Package for the Social Sciences
pretreatment found to improve tissue antioxidants more
(SPSS) 16.0 software (SPSS Inc., Chicago, IL, USA).
effectively then post-stroke clove oil doses (Table 2).
Difference with P<0.05 was considered statistically
significant.

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Table 1: Effect of Clove Oil administration on from natural products for stroke or any other disease
Neurological Deficit Score among different management is the current horizon of research.
experimental groups Bioactive components from herbal origin and plant
polyphenols are multipotent therapeutic agent to treat
Neurological Deficit Score
several health ailments (20).
Control Sham Stroke Pre-Treated Post-Treated
0 0 7.83±1.06 4.83±1.07** 5.83±1.2*/NS
The current study also designed to investigate the
Values are presented as mean±SEM. *P<0.05, therapeutic potentials of clove oil administration
**P<0.01, NS: non-significant (compared with against focal cerebral ischemia induced neuronal
stroke/compared with Pre-treated) damage. To study the protective effects of clove oil
against cerebral IR injury, we used MCAO intraluminal
Figure 1: ND score comaprision among technique to reproduce acute focal cerebral ischemia in
experimental groups rats (15 min occlusion and 24 h reperfusion). During
reperfusion phase of ischemic stroke, ROS
Effect of Clove Oil on Neurological Deficit Score overproduction plays crucial role in destructing brain’s
+ anatomical regions (neuronal damage) and its
*
) associated sensorimotor dysfunction(15).
ND Score

(
'
& In our study we evaluate the post stroke neurological
% function and effects of treatment regimens via
$
#
neurological deficit score. ND score is a composite
" score to assess sensorimotor functioning in rodent
,-./0-1 2345 2/0-67 8079:074/7; 8-</9:074/7;
models of ischemic stroke or other neurological
Experimental Groups
damage. Results of present study showed that MCAO
untreated rats score maximum ND score (Table 1)
Table 2: Effect of Clove Oil Pre and Post Treatments on while neurological functioning get better with pre and
Brain organ weight, total protein and Antioxidants post treatments with clove oil possibly attributable to
(SOD, CAT, and GSH) levels in MCAO Rats antioxidative and anti-spasmodic potentials of eugenol
(bioactive component of clove oil). A study reported
Control (n=8) Sham (n=8) Stroke (n=8) Pre-Treated (n=8) Post-Treated (n=8)
Organ Weight (g) 2.015±0.21 2.04±0.23NS 2.29±0.15NS/NS 2.07±0.24NS/NS/NS 2.14±0.24NS/NS/NS/NS that eugenol effectively protect cortical cells from
oxidative damage, excitotoxicity and ischemic injury via
NS NS/NS NS/NS/NS
Total Protein (g/dL) 5.19±0.28 5.16±0.23 5.27±0.27 5.07±0.25 5.15±0.27NS/NS/NS/NS
SOD (U/g tissue) 73.99±6.64 71.94±5.99NS 50.842±2.89*/* 63.65±1.69NS/NS/** 59.89±2.01NS/NS/*/NS
CAT ( mol/g tissue) 20.36±1.81 20.53±1.66NS 13.79±1.05*/* 18.807±0.86NS/NS/** 15.38±1.78NS/NS/NS/NS modulation of N-methyl-D-aspartate (NMDA) receptors
GSH (U/g tissue) 0.084±0.006 0.083±0.005NS 0.056±0.004**/** 0.077±0.006NS/NS/* 0.067±0.006NS/NS/NS/NS
& superoxide radicals (21).

Values are presented as mean±SEM. *P<0.05, Endogenous antioxidants like glutathione, SOD and CAT
**P<0.01, NS: non-significant (compared with are necessary to maintain tissue oxidant/antioxidant
control/compared with sham/compared with balance and to protect tissues from ROS damage (5,8).
stroke/compared with Pre-treated). Results of present study showed marked decrease in
brain SOD, CAT and GSH concentrations in MCAO
DISCUSSION animals while 15 days clove oil pretreatment and two
doses of clove oil post-stroke both treatments found to
The global stroke burden has been raised alarmingly in
significantly improve the antioxidant status following
past few years equally affecting young and geriatric
stroke induction. Consistent to our results, several
population and both genders. Moreover ischemic stroke
experimental studies also reported the potent
is the second most leading cause of permanent
antioxidative activity of eugenol against oxidative injury
disabilities in adult (19). Alteplase (rtPA) is the only
due to eugenol free radical scavenging capabilities(9).
available pharmacological therapy for ischemic stroke
but it has its own therapeutic limitations. Post-stroke
Study of Farias and colleagues reported that that
physical disabilities are matter of serious concern as
eugenol and eugenol derivatives effectively inhibit
most of the stroke victims suffer some degree of
protein and lipid peroxidation in liver and brain tissues
physical disability after stroke incident. Due to side (22)
. According to another study, eugenol effectively
effects and other pharmacological therapeutic
inhibiting oxygen glucose deprivation (OGD) and NMDA
tribulations, identification of effective and safe therapy
induced neurotoxicity by rapidly scavenging superoxide

PAKISTAN JOURNAL OF NEUROLOGICAL SCIENCES 13 VOL. 14 (1) J A N U A R Y- M A R C H 2 0 1 9

radicals and controlling calcium influx and neuronal treatments against IR injury in MCAO rat model of acute
apoptosis (23). focal cerebral ischemia as exhibited by better
neurological function and antioxidants activities. Clove
CONCLUSION: oil or eugenol could be a preventive treatment of stroke
however further detailed experimental studies at
In summary, results of the present study demonstrate
different doses are recommended.
the antioxidant mediated protective effects of clove oil

REFERENCES

1. WHO | Global Health Observatory (GHO) data Review Review Article. Int J Pharm Pharm Sci.
[Internet]. WHO. World Health Organization; 2018 2014;6(8):67–72.
[cited 2018 Apr 1]. Available from:
http://www.who.int/gho/en/ 10. Bhowmik D, Sampath Kumar KP, Yadav A,
Srivastava S, Paswan S, Dutta AS. Recent Trends
2. Hankey GJ. The global and regional burden of in Indian Traditional Herbs Syzygium aromaticum
stroke. Vol. 1, The Lancet Global Health. 2013. p. and its Health Benefits. J Pharmacogn Phytochem.
e239–40. 2012;1(1):13–23.

3. Gan Y, Liu Q, Wu W, Yin J-X, Bai X-F, Shen R, et al. 11. National Research Council (U.S.) C for the U of the
Ischemic neurons recruit natural killer cells that G for the C and U of LAI for LAR (U. S. Guide for the
accelerate brain infarction. Proc Natl Acad Sci. care and use of laboratory animals. National
2014 Feb 18;111(7):2704–9. Academies Press; 2011. 220 p.

4. Deb P, Sharma S, Hassan KM. Pathophysiologic 12. Fluri F, Schuhmann M, Kleinschnitz C. Animal
mechanisms of acute ischemic stroke : An overview models of ischemic stroke and their application in
with emphasis on therapeutic significance beyond clinical research. Drug Des Devel Ther. 2015
thrombolysis. Pathophysiology. Jul;9:3445.
2010;17(3):197–218.
13. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano
5. Shamim M, Khan NI. Neuroprotective effect of K. Behavioral changes after focal cerebral ischemia
Panax ginseng extract against cerebral by left middle cerebral artery occlusion in rats.
ischemia–reperfusion-injury-induced oxidative Brain Res. 1988 Jun 14;452(1–2):323–8.
stress in middle cerebral artery occlusion models.
Facets. 2019;4(1):52–68. 14. Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G.
Behavioral tests after intracerebral hemorrhage in
6. Bang OY. Considerations When Subtyping Ischemic the rat. Stroke. 2002;33(10):2478–84.
Stroke in Asian Patients. J Clin Neurol. 2016
Apr;12(2):129–36. 15. Shamim M, Khan NI. Preemptive Effect of Panax
Ginseng Extract on Sensorimotor Dysfunction in
7. Bansal S, Sangha KS, Khatri P. Drug treatment of Experimentally Induced Middle Cerebral Artery
acute ischemic stroke. Am J Cardiovasc Drugs. Occlusion-Reperfusion Injury Model of Ischemic
2013 Feb;13(1):57–69. Stroke. FUUAST J Biol. 2018;8(2):271–8.

8. Lee J-C, Won M-H. Neuroprotection of antioxidant 16. Kono Y. Generation of superoxide radical during
enzymes against transient global cerebral ischemia autoxidation of hydroxylamine and an assay for
in gerbils. Anat Cell Biol. 2014 superoxide dismutase. Arch Biochem Biophys.
Sep;47(3):149–56. 1978 Feb;186(1):189–95.

9. Mittal M, Gupta N, Parashar P, Mehra V, Khatri M. 17. Sinha AK. Colorimetric assay of catalase. Anal
Phytochemical Evaluation And Pharmacological Biochem. 1972 Jun;47(2):389–94.
Activity Of Syzygium Aromaticum: A Comprehensive

PAKISTAN JOURNAL OF NEUROLOGICAL SCIENCES 14 VOL. 14 (1) J A N U A R Y- M A R C H 2 0 1 9

18. Carlberg I, Mannervik B. Glutathione reductase. 22. d’Avila Farias M, Oliveira PS, Dutra FSP, Fernandes
Methods Enzymol. 1985;113:484–90. TJ, de Pereira CMP, de Oliveira SQ, et al. Eugenol
derivatives as potential anti-oxidants: is phenolic
19. Suwanwela NC, Poungvarin N. Stroke burden and hydroxyl necessary to obtain an effect? J Pharm
stroke care system in Asia. Neurol India. 2016;64 Pharmacol. 2014 May;66(5):733–46.
Suppl(7):S46-51.
23. Wie M-B, Cheon B-H, Lee S-Y, Son K-H, Song D-K,
20. Feigin VL. Herbal medicine in stroke: does it have a Shin T-K, et al. Eugenol inhibits
future? Stroke. 2007 Jun 1;38(6):1734–6. excitotoxins-induced delayed neurotoxicity,
oxidative injury and convulsion. Toxicol Res.
21. Wie MB, Won MH, Lee KH, Shin JH, Lee JC, Suh 2006;22(3):275–82.
HW, et al. Eugenol protects neuronal cells from
excitotoxic and oxidative injury in primary cortical
cultures. Neurosci Lett. 1997 Apr 4;225(2):93–6.

Conflict of interest: Author declares no conflict of interest.

Funding disclosure: Nil
Author’s contribution:
Hira Jawed; data collection, data analysis, manuscript writing, manuscript review
Mufzala shamim; concept, data collection, data analysis, manuscript writing, manuscript review
Sumera Sohail; data analysis, manuscript writing, manuscript review
Uzma Firdous; contributed to data management and literature review
Nazish Iqbal Khan; data analysis, manuscript writing, manuscript review

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