the effect of ꞵ -caryophyllene on isoproterenol- …
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United Arab Emirates University United Arab Emirates University
Scholarworks@UAEU Scholarworks@UAEU
Theses Electronic Theses and Dissertations
5-2020
THE EFFECT OF ꞵ -CARYOPHYLLENE ON ISOPROTERENOL-THE EFFECT OF ꞵ -CARYOPHYLLENE ON ISOPROTERENOL-
INDUCED MYOCARDIAL INFARCTION INDUCED MYOCARDIAL INFARCTION
Farah M. Jamal Laham
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Recommended Citation Recommended Citation Laham, Farah M. Jamal, "THE EFFECT OF ꞵ -CARYOPHYLLENE ON ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION" (2020). Theses. 828. https://scholarworks.uaeu.ac.ae/all_theses/828
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Declaration of Original Work
I, Farah M.Jamal Laham, the undersigned, a graduate student at the United Arab
Emirates University (UAEU), and the author of this thesis entitled “The Effect of β-
Caryophyllene on Isoproterenol-Induced Myocardial Infarction”, hereby, solemnly
declare that this thesis is my own original research work that has been done and
prepared by me under the supervision of Dr. Shreesh Kumar Ojha, in the College of
Medicine and Health Sciences at UAEU. This work has not previously been
presented or published, or formed the basis for the award of any academic degree,
diploma or a similar title at this or any other university. Any materials borrowed
from other sources (whether published or unpublished) and relied upon or included
in my thesis have been properly cited and acknowledged in accordance with
appropriate academic conventions. I further declare that there is no potential conflict
of interest with respect to the research, data collection, authorship, presentation
and/or publication of this thesis.
Student’s Signature: Date: ________________
30 - SEP - 2020
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Copyright
Copyright © 2020 Farah M.Jamal Laham
All Rights Reserved
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Advisory Committee
1) Advisor: Dr. Shreesh Kumar Ojha
Title: Associate Professor
Department of Pharmacology
College of Medicine and Health Sciences
2) Member: Prof. Ernest Adeghate
Title: Professor
Department of Anatomy
College of Medicine and Health Sciences
3) Member: Dr. Bassem Sadek
Title: Associate Professor
Department of Pharmacology
College of Medicine and Health Sciences
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This Master Thesis is accepted by:
Acting Dean of the College of Medicine and Health Sciences: Professor Juma Al Kaabi
Signature Date
Dean of the College of Graduate Studies: Professor Ali Al-Marzouqi
Signature Date
Copy ____ of ____
13 October 2020
13/10/2020
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Abstract
Cannabinoid type 2 receptors (CB2), a key member of the endocannabinoid
system has recently emerged as a crucial therapeutic target for cardiovascular diseases
(CVDs). Downregulation of CB2 receptors has been witnessed in various cancers,
neurological and cardiovascular disorders. Thus, the activation of CB2 receptors may
protect against ISO induced myocardial infarction (MI) in rats. The present study
investigates the cardioprotective effect of a selective cannabinoid type 2 receptor
agonist β-caryophyllene (BCP), a dietary phytocannabinoid and a natural bicyclic
sesquiterpene against isoproterenol (ISO)-induced MI in rats. Male albino Wistar rats
were pre- and co-treated with β-caryophyllene (50 mg/kg, orally) twice daily for 10
days along with the subcutaneous injection of ISO (85 mg/kg) at an interval of 24 h
for two days (9th and 10th day). AM630 (1 mg/kg), a CB2 receptor antagonist was
injected intraperitoneally as a pharmacological challenge prior to BCP treatment to
demonstrate CB2 receptor-mediated cardioprotective mechanisms of BCP. ISO
induced MI showed a significant decline in cardiac function, elevated levels of serum
cardiac marker enzymes and enhanced oxidative stress markers with increased lipid
peroxidation. Isoproterenol also induced pro-inflammatory cytokines release
following activation of the nuclear factor kappa-B. Furthermore, a significant rise was
also observed in the levels of inflammatory mediators, namely cyclooxygenase-2
(COX-2) and inducible nitric oxide synthase (iNOS) in ISO-challenged rats.
Additionally, Isoproterenol also increased expression of proapoptotic (Bax, and active
caspase-3) proteins along with the decreased expression of anti-apoptotic protein, Bcl2
and Bcl-xL in the myocardium. β-caryophyllene treatment resulted in significant
protective effects on all biochemical and molecular parameters analyzed.
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Histopathological and ultrastructural evidence was found in line with these findings.
Treatment with AM630, a potent CB2 receptor antagonist abrogates protective effects
of BCP on all the biochemical and molecular parameters analyzed in ISO induced MI
in rats. Thus, this study revealed that BCP protects the myocardium against ISO
induced MI by attenuating oxidative stress, apoptosis and inflammation and the
underlying mechanism of this protection is the activation of CB2 receptors. CB2
receptor selective compounds may provide a new potential class of cardioprotective
drugs. The pharmacophore of these compounds could be used for synthesizing leads
in drug discovery and development.
Keywords: Myocardial infarction, Phytochemical, Natural product, Cannabinoids, β-
Caryophyllene, Cannabinoid receptor type 2, Isoproterenol, Catecholamine.
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Title and Abstract (in Arabic)
إحتشاء عضلة القلب الناتج عن ىعل (-Caryophyllene)كاريوفايلين -تأثير بيتا
(Isoproterenol)الأيزوبرينالن
صالملخ
تعتبر مستقبلات الكانابينويد من النوع الثاني عضو أساسي في نظام الإندوكنابينويد. وقد
وقد شهد خفض تنظيم مستقبلات ظهر مؤخرا كهدف علاجي حاسم للأمراض القلبية الوعائية.
CB2 ات والاختلالات العصبية والقلبية. وبالتالي، قد يعمل تنشيط في مختلف أنواع السرطان
(Isoproterenol)على الحماية من إحتشاء عضلة القلب الناتج عن الأيزوبرينالن CB2مستقبلات
ناهض (-Caryophyllene)كاريوفايلين -في الجرذان. وتحقق هذه الدراسة في تأثير البيتا
، على وقاية عضلة القلب والأوعية الدموية من تأثير الأيزوبرينالن CB2لمستقبلات ال
(Isoproterenol) بب بإحتشاء عضلة القلب في الجرذان. وقد إستخدمنا جرذان ويستار الذكور المس
،ملغ/كج( 50بجرعة ) (-Caryophyllene)كاريوفايلين -وقد عولجت عن طريق الفم بالبيتا
(Isoproterenol)مرتين في اليوم لمدة عشرة أيام، بالإضافة الى الحقن تحت الجلد للأيزوبرينالن
ساعة لمدة يومين )اليوم 24قد أعطي جرعتين في فترة زمنية فاصلة تبلغ ملغ/كج( و 85بجرعة )
الذي يعتبر ضاد للمستقبل المذكور، وقد حقنت الجرذان (AM630) 630التاسع و العاشر(. اي.ام.
ملغ/كج( داخل الصفاق لتأكيد أليات الوقاية للقلب عبر نهض مستقبلات الكانابينويد من 1بجرعة )
نخفاض كبير بوظائف القلب، ارتفاع إب (Isoproterenol)سبب الأيزوبرينالن النوع الثاني.
مستويات الأنزيمات القلبية وارتفاع مؤشرات الإجهاد التأكسدي و بيروكسيدات الدهون. وقد سبب
nuclear factor)بي -بعد تفعيل العامل النووي كاباالسيتوكينات البادئة للالتهاب أيضا بحث إطلاق
kappa-B) ارتفاع كبير في مستويات الوسائط الالتهابية أي . وعلاوة على ذلك، لوحظ أيضا
بالإضافة الى ذلك، (.iNOSوحمض النتريك سينثاز المحرض ) (COX-2) 2-سيكلواكسجناز
بروتينات المؤيدة للإستماته من ارتفاع مستوى (Isoproterenol)سبب الأيزوبرينالن
(proapoptotic proteins)، باكس(Bax) النشط 3وكاسباز(active caspase-3) بالإضافة الى .
( Bcl-xLاكس ال )–وبي سي ال (Bcl2) 2نخفاض مقدار البروتين المضاد للإستماته بي سي ال ا
الى آثار وقائية كبيرة (-Caryophyllene)كاريوفايلين -في عضلة القلب. وقد أدى العلاج بالبيتا
الكيميائية الحيوية والجزيئية. وقد تماشت النتائج التي توصلنا لها مع الأدلة معاملاتعلى جميع ال
630والصور المأخوذة تحت المجهر. وعلاوة على ذلك، قد سبب حقن الجرذان ب اي.ام.
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(AM630)النوع الثاني، بإلغاء التأثيرات الوقائية ، وهو يعتبر ضاد لمستقبل الكانابينويد من
الكيميائية الحيوية و الجزيئية المدروسة في الجرذان المحقونة ب معاملاتعلى جميع ال BCPلل
يحمي عضلة القلب من الإحتشاء BCP. وهكذا كشفت دراستنا أن (Isoproterenol)الأيزوبرينالن
oxidative)لك من خلال تخفيف الإجهاد التأكسدي وذ (Isoproterenol)المصاحب للأيزوبرينالن
stress) والإستماته(apoptosis) والالتهاب(inflammation) والآلية الأساسية لهذه الحماية هي .
فئة جديدة من CB2المحفزة لمستقبلات ال مركبات. وقد توفر الCB2تنشيط مستقبلات ال
في اكتشاف وتطوير مركباتم حامل الخاصة الدوائية لهذه الالأدوية القلبية الوقائية ويمكن استخدا
العقاقير.
، المنتجات الطبيعية، كيميائيات نباتيةاحتشاء عضلة القلب، :مفاهيم البحث الرئيسية
.كاتيكولامينات، أيزوبرينالن، مستقبل كانابينويد من النوع الثاني، كاريوفايلين-بيتاات، كانابينويد
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Acknowledgements
In the name of Allah, the Most Gracious and the Most Merciful Alhamdulillah,
all praises to Allah for the strengths and His blessing in completing this thesis. First
off all, I would like to acknowledge everyone who played a role in my academic
accomplishments, my parents, my husband and my friends, who supported me with
love and understanding. Without you, I could never have reached this current level of
success.
Secondly, my supervisor, Dr. Shreesh Ojha for his continuous support,
motivation and untiring guidance have made this dream come true. His vast
knowledge, calm nature and positive criticism motivated me to starve for nice results.
It is my great opportunity to thank him for his kind co-operation, generous help and
constant encouragement for the successful completion of my thesis. I also would like
to thank the Chair of the Department Prof. Salim Bastaki and my thesis committee
members, each of whom has provided patient advice and guidance throughout the
research process. Thank you all for your unwavering support.
I am grateful to Dr. M.F. Nagoor Meeran. And I would like to thank him for
encouraging my research and for helping and mentoring me in the lab work and for
always being there when I required help.
Also, I would like to extend my thanks to Dr. Sheikh Azimullah, for his
constant advice and guidance in many issues that I faced during my research.
Special thanks also go to Dr. Rami Beiram, Assistant Dean for Scientific
Research and Graduate Studies, and all the other faculty members and non-teaching
staff members of the Department of Pharmacology and Therapeutics, UAEU for their
support and co-operation in all possible ways.
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Dedication
I dedicate this thesis to my beloved parents and family
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Table of Contents
Title ............................................................................................................................... i
Declaration of Original Work ...................................................................................... ii
Copyright .................................................................................................................... iii
Advisory Committee ................................................................................................... iv
Approval of the Master Thesis ..................................................................................... v
Abstract ...................................................................................................................... vii
Title and Abstract (in Arabic) ..................................................................................... ix
Acknowledgements ..................................................................................................... xi
Dedication .................................................................................................................. xii
Table of Contents ...................................................................................................... xiii
List of Tables.............................................................................................................. xv
List of Figures ........................................................................................................... xvi
List of Abbreviations................................................................................................ xvii
Chapter 1: Introduction ................................................................................................ 1
1.1 Cardiovascular diseases and myocardial infarction (MI)........................... 1
1.1.1 Myocardial infarction ......................................................................... 2
1.1.2 Free radicals ..................................................................................... 12
1.1.3 Antioxidants ..................................................................................... 15
1.1.4 Pro-inflammatory cytokines and myocardial infarction ................... 18
1.1.5 Apoptosis .......................................................................................... 20
1.1.6 Experimental myocardial infarction in animal models .................... 23
1.2 Cannabinoids as drug target for MI ......................................................... 25
1.2.1 Endocannabinoids ............................................................................ 26
1.2.2 Synthetic cannabinoid ...................................................................... 29
1.2.3 Phytocannabinoids ........................................................................... 31
1.3 Cannabinoid receptors .............................................................................. 34
1.4 CB2 receptors and heart diseases ............................................................. 36
1.5 Beta-Caryophyllene.................................................................................. 38
1.5.1 Pharmacological properties of BCP ................................................. 43
1.5.2 Chemical properties and SAR of BCP ............................................. 51
1.5.3 Safety and toxicity of BCP ............................................................... 52
1.5.4 AM630 ............................................................................................. 53
1.6 Aims and objectives of the study ............................................................. 54
Chapter 2: Materials and Methods ............................................................................. 56
2.1 Experimental animals ............................................................................... 56
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2.2 Induction of experimental myocardial infarction ..................................... 56
2.3 Dose dependent study .............................................................................. 56
2.4 Experimental design ................................................................................. 59
2.5 Experimental groups ................................................................................ 59
2.6 Hemodynamic measurements .................................................................. 60
2.7 Biochemical estimations .......................................................................... 61
2.7.1 Serological evaluation ...................................................................... 61
2.7.2 Assessment of oxidative stress and lipid peroxidation ..................... 61
2.7.3 Assessment of inflammatory markers in cardiac tissue ................... 66
2.7.4 Western Blot analysis ....................................................................... 66
2.8 Histopathological evaluation .................................................................... 67
2.9 Transmission electron microscopy (TEM)............................................... 68
2.10 Statistical analysis .................................................................................. 68
Chapter 3: Results ...................................................................................................... 69
3.1 Effect of BCP on serum CK (Dose dependent study) .............................. 69
3.2 Effect of BCP on cardiomyocyte injury marker enzymes in serum......... 70
3.3 Effect of BCP on hemodynamic measurements ...................................... 71
3.4 Effect of BCP on lipid peroxidation......................................................... 72
3.5 Effect of BCP on antioxidants ................................................................. 73
3.6 Effect of BCP on pro-inflammatory cytokines ........................................ 74
3.7 Effect of BCP on inflammatory mediators and NF-κB expressions ........ 75
3.9 Effect of BCP on CB2/PPARγ expressions and troponin-I ..................... 78
3.10 Effect of BCP on Histopathology of myocardium ................................. 79
3.11 Effect of BCP on cardiomyocyte ultrastructure (TEM) ......................... 81
Chapter 4: Discussion ................................................................................................ 83
4.1 Importance and relevance of BCP cardioprotictive effect findings ......... 83
Chapter 5: Conclusion ................................................................................................ 94
References .................................................................................................................. 95
List of Publications .................................................................................................. 116
xv
List of Tables
Table 1: Cardiovascular drugs .................................................................................. 11
Table 2: Selected radical and non-radical oxidants with significance in vivo ............... 13
Table 3: The pharmacological activity of endogenous cannabinoids ........................ 27
Table 4: Examples of synthetic cannabinoids ............................................................ 30
Table 5: Plant derived cannabinoids .......................................................................... 31
Table 6: Natural products that have been reported to interact with the ECS ............. 33
Table 7: Distribution of CB receptors ........................................................................ 35
Table 8: Plants that contain BCP (presented as % of BCP amongst constituent
phytochemicals) ........................................................................................... 39
Table 9: Physico-chemical properties and pharmacokinetics of BCP ....................... 43
Table 10: Molecular mechanisms that BCP has an effect on .................................... 45
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List of Figures
Figure 1: Structure of infarcted heart ........................................................................... 3
Figure 2: Risk factors of myocardial infarction ........................................................... 4
Figure 3: Electrocardiogram ........................................................................................ 7
Figure 4: The chemistry of oxygen radical induced membrane lipid peroxidation ... 15
Figure 5: Apoptosis .................................................................................................... 20
Figure 6: Intrinsic and extrinsic pathway of apoptosis .............................................. 22
Figure 7: Structure of isoproterenol ........................................................................... 23
Figure 8: Proposed mechanism of auto-oxidation of isoproterenol ........................... 25
Figure 9: Endocannabinoids synthesis and degradation ............................................ 29
Figure 10: Endocannabinoids structures .................................................................... 29
Figure 11: Phytocannabinoids possess at least some of these properties................... 32
Figure 12: Cannabinoid pathway ............................................................................... 34
Figure 13: Schematic showing the CB1 and CB2 receptors ...................................... 36
Figure 14: Some natural sources of BCP ................................................................... 38
Figure 15: Structure of β-Caryophyllene (BCP) ........................................................ 41
Figure 16: The multiple receptor targets of β-caryophyllene..................................... 44
Figure 17: Chemical structures of the bicyclic sesquiterpenes .................................. 51
Figure 18: In Silico Docking Analysis of the (E)-BCP CB2 Receptor Binding
Interaction .................................................................................................. 52
Figure 19: Structure of AM630 .................................................................................. 53
Figure 20: Activity of serum CK (dose dependent study). ........................................ 70
Figure 21: Effect of BCP on serum CK and LDH.. ................................................... 71
Figure 22: Effect of BCP on hemodynamics ............................................................. 72
Figure 23: Effect of BCP on lipid peroxidation status in the heart ............................ 73
Figure 24: Effect of BCP on endogenous antioxidant enzymes and non-enzymatic
antioxidant ................................................................................................. 74
Figure 25: Effect of BCP on pro-inflammatory cytokines activities ......................... 75
Figure 26: Effect of BCP on inflammatory mediators in the myocardium ................ 76
Figure 27: Effect of BCP on apoptosis ...................................................................... 77
Figure 28: Effect of BCP on the myocardial protein expressions .............................. 79
Figure 29: Effect of BCP on myocardial structure..................................................... 80
Figure 30: TEM study on heart mitochondria ............................................................ 82
xvii
List of Abbreviations
2-AG 2-Arachidonoylglycerol
ACEI Angiotensin-Converting Enzyme Inhibitors
ADP Adenosine Diphosphate
AEA Anandamide
AMP Adenosine Monophosphate
ATP Adenosine Triphosphate
BAX BCL2-Associated X Protein
BP Blood Pressure
BCP Beta Caryophyllene
Bcl-2 B-Cell Lymphoma 2
Bcl-xl B-Cell Lymphoma-Extra Large
cAMP Cyclic Adenosine Monophosphate
CAT Catalase
CB1, CB2 Cannabinoid Receptor 1, Cannabinoid Receptor 2
CBD Cannabidiol
CBR Cannabinoid Receptors
CCB Calcium Channel Blockers
CK Creatine Kinase
CNS Central Nervous System
COX-2 Cyclooxygenase-2
CVD Cardiovascular Disease
DP Diastolic Pressure
xviii
ECG Electrocardiogram
FAAH Fatty Acid Amide Hydrolase
GPx Glutathione Peroxidase
GSH Glutathione
GSTs Glutathione S-Transferases
HMG-COA β-Hydroxy β-Methylglutaryl-CoA
HR Heart Rate
IL-1β Interleukin 1 Beta
IL-6 Interleukin 6
iNOS Inducible Nitric Oxide Synthase
ISO Isoproterenol
LDH Lactate Dehydrogenase
LOOH Lipid Hydroperoxide
MAGL Monoacylglycerol Lipase
MAP Mean Arterial Pressure
MI Myocardial Infarction
NF-κB Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells
PPAR-γ Peroxisome Proliferator-Activated Receptor Gamma
PUFAs Polyunsaturated Fatty Acids
ROS Reactive Oxygen Species
SOD Superoxide Dismutase
SP Systolic Pressure
TBARS Thiobarbituric Acid Reactive Substances
xix
TLRs Toll-Like Receptors
TNF-α Tumor Necrosis Factor Alpha
TRBV1 Transient Receptor Potential Cation Channel Subfamily V
Member 1
VR1 Vanilloid Receptor 1
WBC White Blood Cells
Δ-9-THC Delta-9-Tetrahydrocannabinol
1
Chapter 1: Introduction
1.1 Cardiovascular diseases and myocardial infarction (MI)
Cardiovascular diseases (CVDs) are the leading causes of morbidity and
mortality worldwide and it accounts for 17.3 million deaths per year with expected
growth of more than 23.6 million by 2030 (Laslett et al., 2012). CVDs, which are
disorders of the heart and blood vessels, include:
• Coronary artery disease also known as coronary heart disease, or ischemic
heart disease, is the most common type of heart disease. The disease is caused
by the building up of plaques in the inner walls of the arteries of the heart,
which blocks blood flow and heightens the risk for heart attack and stroke.
• Peripheral vascular disease, also known as peripheral artery disease, refers to
the blockage of large arteries not within the coronary, aortic arch vasculature,
or brain. It can result from atherosclerosis, inflammatory processes leading to
the formation of thrombus. It causes either acute or chronic ischemia (lack of
blood supply).
• Rheumatic heart disease describes a group of short-term (acute) and long-term
(chronic) heart disorders that can occur because of rheumatic fever. One
common result of rheumatic fever is heart valve damage. This damage to the
heart valves may lead to valve disorders.
• Cerebrovascular disease refers to a group of conditions that affect the
circulation of blood to the brain, causing limited or no blood flow to the
affected areas of the brain.
2
• Congenital heart disease is a defect in the structure of the heart and vessels
which is present at birth (Wallace, 2011).
The deposition of fatty substances in the inner walls of the blood vessels causes
blockage and insufficient blood flow to the heart or brain results in heart attack and
stroke (Rauch et al., 2001). The coronary arteries supply nutrients and oxygen rich
blood to the heart muscle for the proper functioning of heart. A dearth of blood supply
to the heart due to the accumulation of fatty deposits such as cholesterol and fats called
plaques in the inner walls of the arteries resulting in pain or angina (Marcus et al.,
2007).
Myocardial infarction (MI) occurs when coronary blood flow is inadequate,
hence the oxygen/nutrients supply is insufficient for the demand of the myocardium
due to the formation of blood clot by the rupturing of lipid-laden plaques, resulting in
irreversible damage to the heart which in turn results in death of that portion of the
heart muscle (Rauch et al., 2001). The heart is a muscular organ about the size of a
closed fist that functions as the body’s circulatory pump which has four chambers that
pumps blood to all parts of the body (Iaizzo, 2009).
1.1.1 Myocardial infarction
MI is one of the major causes of mortality and morbidity in the world. It
commonly known as heart attack occurs when the blood supply to a part of the heart
is interrupted causing some heart cells to die. This is commonly due to the blockage
of a coronary artery following the rupture of a vulnerable atherosclerotic plaque, which
defined as an unstable collection of lipids (cholesterol) and WBC (especially
macrophage) in the wall of an artery. The resulting ischemia and oxygen shortage, if
left untreated for a sufficient period of time, can cause damage and/or death
3
(infarction) of the myocardium (Upaganlawar, Gandhi & Balaraman, 2009). The
structure of the infracted heart is shown in Figure 1.
1.1.1.1 Classification of myocardial infarction
There are two basic types of MI:
• Transmural: A transmural MI is characterized by ischemic necrosis of the full
thickness of the affected muscle segment(s), extending from the endocardium
through the myocardium to the epicardium. Transmural infarcts extend through
the whole thickness of the heart muscle and are usually a result of complete
occlusion of the area's blood supply (Fabricius et al., 1979).
• Subendocardial: A subendocardial MI involves a small area in the
subendocardial wall of the left ventricle, ventricular septum, or papillary
muscles. The subendocardial area is particularly susceptible to ischemia supply
(Fabricius et al., 1979).
Figure 1: Structure of infarcted heart
4
1.1.1.2 Signs and symptoms of myocardial infarction
Chest pain is the common symptom of MI and is often described as a sensation
of tightness, pressure, or squeezing. The pain may radiate to the left arm, neck,
shoulders, jaw and epigastrium where it may mimic heartburn (Arora & Bittner, 2015).
Other symptoms include diaphoresis (excessive sweating), weakness, light
headedness, nausea, vomiting and palpitations. Immediate administration of
thrombolytics (clot-dissolving drugs) after an acute MI reduces the risk of death. One-
fourth of all MI are silent which can be identified by electrocardiogram (ECG),
echocardiogram and enzyme tests (Valensi et al., 2011).
1.1.1.3 Risk factors of myocardial infarction
Diabetes (with or without insulin
resistance)
Tobacco smoking
Hypercholesterolemia (more accurately
Hyperlipoproteinemia, especially high (LDL-C),
(VLDL-C) and low (HDL-C)
Age: Men acquire an independent risk factor
at age 45; Women acquire an independent
risk factor at age 55
Obesity
Hypertension
Hyperhomocysteinemia
Stress (occupations with high stress index are
known to have susceptibility for MI)
Exposure to air pollution
Males are more at risk than females
Figure 2: Risk factors of myocardial infarction (Iaizzo, 2009)
5
Many of these risk factors that shown in Figure 2 are modifiable and so many
heart attacks can be prevented by maintaining a healthier lifestyle. Physical activity is
associated with a lower risk profile (Buttar et al., 2005).
1.1.1.4 Myocardial metabolism
Normal and Ischemic myocardium:
Carbohydrates and lipids are the major substrates of normal heart metabolism.
Normally, the increased levels of free fatty acids (FFAs) in the blood lead to the
inhibition of glucose oxidation by the heart. Fatty acids then become the major source
of energy and any glucose taken up is increasingly converted to glycogen by the
glucose-sparing effect of fatty acid oxidation. About 60-80% of the heart’s energy
supply is derived from fatty acid metabolism under normoxic conditions. About 20-
40% of the adenosine triphosphate (ATP) which is necessary for contractile function
is provided by glucose metabolism (Taegtmeyer, 1994).
The collapse of most of the cyclic processes is due to the sudden interruption
of oxygen. This collapse is apparent in the rapid cessation of the rhythmic contractions
of heart muscle in the area supplied by the affected coronary vessel. The collapse of
cycles which leads to the accumulation of the metabolic end-products is a salient
feature of MI. These include lactate, protons, succinate, alanine and fatty acid
metabolites fatty acyl carnitine, fatty acyl-CoA and 2-hydroxy fatty acids (Garfein,
1990).
The balance between ATP production and consumption is disturbed as in
myocardial ischemia when oxygen delivery is interrupted, leads to the ATP
concentration decline and a cascade phenomenon results (Zarco & Zarco, 1996).
6
• Adenosine monophosphate (AMP), adenosine diphosphate (ADP),
adenosine and Pi levels increase.
• Shift from aerobic respiration to anaerobic glycolysis
• Acidosis
• Osmolar load with increased levels of sodium (Na+) and calcium (Ca2+)
• Production of lactate
Intracellular acidosis:
A crucial role has been played by intracellular acidosis in contractile failure
and in glycolysis inhibition that leads to an irreversible ischemic damage. Cellular
acidosis reduces cardiac pressure and arouses cardiac arrhythmias. A major cause for
force reduction is an acidosis-induced decrease in the response of contractile proteins
to Ca2+. Protons compete with Ca2+ at Ca2+ binding sites resulting in decreased binding
of Ca2+ to troponin-C. Acidosis brings down the Ca2+ current which triggers the release
of Ca2+ from sarcoplasmic reticulum (Murphy et al., 1991).
1.1.1.5 Diagnosis of myocardial infarction
Diagnostic criteria:
The World Health Organization criteria have classically been used to diagnose
MI, a patient is diagnosed with MI, if two (probable) or three (definite) of the following
criteria are satisfied (Thygesen et al., 2012):
1. Clinical history of ischemic type chest pain lasting for more than 20 minutes.
2. Changes in serial ECG tracings.
3. Rise and fall of serum cardiac biomarkers such as creatine kinase-MB (CK-
MB) and troponins.
7
Physical examination:
The general appearance of patients may vary according to the experienced
symptoms; the patient may be uncomfortable, or restless and in severe distress with an
increased respiratory rate. A cool and pale skin is common and points to
vasoconstriction. Blood pressure may be elevated or decreased, and the pulse rate may
be irregular (Kasper, 2005).
Electrocardiography:
Electrocardiogram is a central tool used to establish the diagnosis of
myocardial ischemia or infarction. A simple, painless test records the heart's electrical
activity (Schweitzer, 1990). The increase in ST-segments could be due to ischaemic
or neurogenically mediated myocardial injury. Elevations of the ST-segment and
changes in the T wave are indication of MI. Q waves can be seen on the ECG
signifying damaged heart tissue after MI (Schweitzer, 1990). Electrocardiogram waves
are shown in Figure 3.
Figure 3: Electrocardiogram
8
Echocardiography:
Echocardiogram, often referred to as a cardiac echo is a sonogram of the heart.
Echocardiography uses standard two-dimensional, three-dimensional and doppler
ultrasound to create images of the heart. It has become routinely used in the diagnosis,
management and follow-up of patients with any suspected or known heart diseases.
Severe ischemia produces regional wall motion abnormalities that can be visualized
echocardiographically within seconds of coronary artery occlusion (Hauser et al.,
1985; Wohlgelernter et al., 1986). Immediately after coronary occlusion, myocardium
ceases to function in the area sub served by the involved vessel. The affected left
ventricular wall fails to thicken during systole and in fact may thin, producing systolic
bulging of the ischemic segment can be seen by using echocardiography. Such
abnormality of a regional wall motion can be detected in acute MI with approximately
90% sensitivity by echocardiography (Kloner & Parisi, 1987). So, it is one of the most
widely used diagnostic tests in cardiology.
Markers of myocardial infarction:
Myocardial infarction is accompanied by the release of cellular enzymes in the
blood stream. Thus, evaluation of these markers provides an excellent way to diagnose
MI.
• Total creatine kinase:
Creatine kinase (CK) is an intracellular enzyme present in large amounts in
skeletal muscle, brain and myocardium. Disruption of cell membranes due to hypoxia
or other injury releases CK from the cellular cytosol into the systemic circulation. On
this basis, elevated serum levels of CK have been used as a sensitive but nonspecific
test for MI. The poor specificity reflects the ubiquity of CK in many tissues other than
9
the myocardium (Cabaniss, 1990). A scientific study reports that the activity of total
CK is found to be increased in the serum of isoproterenol (ISO) induced myocardial
infarcted rats (Kannan & Quine, 2011a).
• Creatine kinase - MB:
Creatine kinase is subdivided into three isoenzymes: MM, MB, and BB. The
MM fraction is present in both cardiac and skeletal muscle, but the MB fraction is
much more specific for cardiac muscle. About 15 to 40% of CK in cardiac muscle is
MB, while less than 2% in skeletal muscle is MB. CK-MB is a very good marker for
acute myocardial injury, because of its excellent specificity, and it rises in serum
within 2 to 8 h of onset of acute MI. The CK-MB is also useful for diagnosis of
reinfarction or extensive of an MI, because it begins to fall after a day, dissipating in
1 to 3 days, so subsequent elevations are indicative of another event (Laboratory
Diagnosis of Myocardial Infarction, 1999). Serum CK-MB activity is found to be
raised in the ISO induced myocardial infarcted rats (Upaganlawar et al., 2009).
• Cardiac troponins:
Troponins-T and I are structural components of the muscle of the heart. They
are released into the blood stream in MI. They are specific for MI. Troponin levels
will begin to increase following MI within three to twelve h and remain elevated up to
five to nine days for troponin-I and up to two weeks for troponin-T. This makes troponins a
superior marker for diagnosing MI (Skeik & Patel, 2007). The levels of serum troponins (I
and T) are found to be elevated in ISO induced MI (Kumaran & Prince, 2010).
10
• Lactate dehydrogenase:
The enzyme lactate dehydrogenase (LDH) begins to rise in 12 to 24 h following
MI, and peaks in two to three days, gradually dissipating in 5 to 14 days. Measurement
of LDH is necessary for greater specificity for cardiac injury (Jaffe et al., 1996). The
LDH activity are found to be elevated in ISO induced myocardial infarcted rats
(Ouyang et al., 2019).
1.1.1.6 Prevention of MI
• Stop smoking.
• Regular exercise within tolerance level.
• Keep the body weight within recommended level.
• Sodium chloride intake should be less than 6 g per day.
• Control of diabetes mellitus.
• Intake of low levels of fat and cholesterol in diet (Doyle et al., 2012).
1.1.1.7 Treatment
Non-pharmacological therapy:
Dietary intake of fruits, vegetables (Law & Morris, 1998), fiber (Pietinen et al.,
1996) and fish (Albert et al., 1998) may able to protect the heart, while a high intake
of saturated fat (found in meat and dairy fat) and trans fatty acids (found in margarine
and processed foods containing hydrogenated vegetable oils) and its dietary intake
may contribute to heart disease (Hu et al., 1999).
Cardiovascular drugs:
The development of MI is a dynamic process, which reflects abnormal
hormonal, metabolic and hemodynamic effects, as well as increasing demand of
11
myocardial oxygen and decreasing coronary blood flow. Recent approaches include
the use of pharmacological agents to dissolve occluding thrombi and mechanical
procedures to recanalize occluded coronary arteries.
Most of the currently available cardiovascular drugs used in clinical practice
are divided into groups depending upon their action or what they treat, and it include
statins, diuretics, anticoagulants, beta blockers (BB), digitalis drugs, vasodilators,
calcium channel blockers (CCB) and angiotensin converting enzyme inhibitors
(ACEI). Examples of cardiovascular drugs and their pharmacological action are shown
in Table 1.
Table 1: Cardiovascular drugs
Category Drug Action Example
Statins Cholesterol lowering drugs, used to
control cholesterol levels. Act by
inhibiting HMG-CoA reductase
enzyme.
Atorvastatin
Diuretics Help to reduce fluid retention. These
may also reduce pressure. When the
body is retaining fluid, though, can
often make the heart harder, using
diuretics will reduce heart workload.
Furosemide
Anticoagulants
Lengthen the time it takes for blood to
clot, which can prevent the formation
of blood clots that might cause a
stroke. People who have artificial
valves, who have had a stroke or the
ones at risk for MI, may need an
anticoagulant to minimize further risk.
Warfarin
Anti-platelets Reduce the tendency of platelets in the
blood to clump and cause clotting.
Aspirin
12
Table 1: Cardiovascular drugs (continued)
Category Drug Action Example
Beta blockers Control pressure and reduce chest pain
associated with angina. BB slow
heartbeat that may control numerous
heart disease symptoms which may
reduce future risk of MI.
Metoprolol
Cardiac Glycosides Stimulates the heart to beat more
forcefully. Used for arrhythmia and in
congestive heart failure.
Digitalis
Vasodilators Are like beta-blockers may reduce the
work of heart and they are often
prescribed to treat chest pain resulting
from angina.
Nitroprusside,
nitroglycerin,
and hydralazine.
Calcium channel
blockers
Used in the treatment of some forms of
angina and to treat certain arrhythmias
or hypertension.
Nifedipine
ACEI Decrease some blood supply to the
heart, which reduces its work.
Cardiovascular drugs that fall into this
category might lower blood pressure
and increase heart function.
Captopril
1.1.2 Free radicals
Free radicals are defined as a group of atoms with an odd (unpaired) number of
electrons and it formed when oxygen interacts with certain molecules. Once formed they
will act as a highly reactive radicals and will start a chain or reactions. Their main danger
comes from the damage resulted from the reactions with important cellular components such
as DNA or cell membrane. Selected radical and non-radical oxidants in vivo are presented
in Table 2. The major contributors for oxidative stress in the human body (Huang et al.,
2005) are marked with an ash (#).
13
Table 2: Selected radical and non-radical oxidants with significance in vivo
Name Chemical formula
Superoxide radical# O2•-
Hydroxyl radical# OH•
Peroxynitrite# ONOO-
Nitric oxide radical NO•
Nitrogen dioxide radical NO2•
Alkoxyl radical RO•
Peroxyl radical# ROO•
Hydrogen peroxide* H2O2
Hypochlorous acid HOCl
Singlet oxygen# 1O2
1.1.2.1 Lipid peroxidation
Lipid peroxidation in vivo is a fundamentally deteriorative reaction occurs in
polyunsaturated fatty acids (PUFAs) and involves the direct reaction of oxygen and
lipids to form free radical intermediates and semi stable peroxides. The overall process
of lipid peroxidation consists of three stages: initiation, propagation and termination.
Which presented in Figure 4.
Initiation:
Lipid peroxidation is initiated by the attack on a fatty acid or fatty acyl side
chain of any chemical species that has sufficient reactivity to abstract a hydrogen atom
from a methylene carbon in the side chain. PUFAs are particularly susceptible to
peroxidation because of greater number of double bonds in a fatty acid side chain and
14
their hydrogen atoms are easily removable. The resulting carbon-centered lipid radical
can have several fates. But in aerobic cells, it undergoes molecular rearrangement and
form conjugated dienes, which are able to combine with O2 and produce peroxy
(ROO•) radical mutagens (Halliwell & Chirico, 1993).
Propagation:
Peroxyl radicals can combine with each other or they can attack the
membrane proteins. They are also capable of abstracting hydrogen from another
lipid molecule (adjacent fatty acid), especially in the presence of metals such as copper
or iron, thus causing an autocatalytic chain reaction and so propagating the chain
reaction of lipid peroxidation. The ROO• combines with free hydrogen molecule to
give a lipid hydroperoxide (LOOH). Probable alternative fates of ROO• are to be
transformed into cyclic peroxides or even cyclic endoperoxides (from PUFAs such as
arachidonic or eicosapentanoic acid). The length of the propagation chain depends on
many factors including the lipid-protein ratio in a membrane (the chance of a radical
reacting with a membrane protein will increase as the protein content of the membrane
rises), the fatty acid composition, oxygen concentration and the presence of chain
breaking antioxidant mutagens (Halliwell & Chirico, 1993).
Termination:
Termination (formation of a hydroperoxide) is most often achieved by the
reaction of a ROO• with α-tocopherol, which is the main lipophilic "chain-breaking
molecule" in the cell membranes. Furthermore, any kind of alkyl radical’s lipid free
radical (L•) can react with lipid peroxide radical (LOO•) to give non-initiating and non-
propagating species such as the relatively stable dimers or two peroxide molecules
combining to form hydroxylated derivatives. The resulting LOOH can easily
15
decompose into several reactive species including lipid alkoxyl radical (LO•),
aldehydes (e.g., malondialdehyde, HOC–CH2–CHO), alkanes, lipid epoxides and
alcohols in which most of them are toxic and active mutagens (Halliwell & Chirico,
1993).
1.1.3 Antioxidants
Compounds disposing the reactive oxygen species (ROS), scavenging them,
suppressing their formation or opposing their actions are antioxidants. There are two
types of antioxidants namely
(i) Enzymatic antioxidants, and
(ii) Non-enzymatic antioxidants
Figure 4: The chemistry of oxygen radical induced membrane lipid peroxidation
(Halliwell & Gutteridge, 1990)
16
1.1.3.1 Enzymatic antioxidants
Superoxide dismutase:
Superoxide dismutase is present in all aerobic organisms and in most
subcellular compartments that generates activated oxygen. It has a central role in the
defense against oxidative stress (Scandalios, 1993). It catalyzes the dismutation of
superoxide anion (O2•−) to hydrogen peroxide which will be subsequently removed by
the reaction of catalase or glutathione peroxidase (GPx).
The activity of superoxide dismutase is found to be decreased in ISO treated MI
(Saravanan et al., 2013).
Catalase:
Catalase is a tetrameric hemeprotein of molecular weight 240 kDa, consisting
of four identical subunits of 60 kDa. It is present in all mammalian cells and is
abundantly present in subcellular organelles such as the peroxisomes of the liver and
kidney. It catalyzes the reduction of hydrogen peroxide and thereby protects the
cellular constituents from oxidative damage by highly reactive hydroxyl radicals (OH•)
(Lalitha et al., 2013).
The activity of catalase is found to be decreased in ISO induced MI (Saravanan et al.,
2013)
17
Glutathione peroxidase:
Glutathione peroxidase, an important antioxidant enzyme whose foremost
biological function is to protect the organism from oxidative damage. It catalyses the
decomposition of both hydrogen peroxide and organic peroxides at the expense of
reduced glutathione (GSH) with the formation of glutathione disulfide, water and
organic alcohol (Vural et al., 2004).
A decrease in the heart GPx activity is reported in the ISO induced myocardial
infarcted rats (Saravanan et al., 2013).
Glutathione S-transferase:
The Glutathione S-transferases (GSTs) form a group of multi-gene isoenzymes
involved in the cellular detoxification of both xenobiotic and endobiotic compounds.
It catalyses the conjugation of reduced glutathione GSH via the sulfhydryl group to
electrophilic centers on a wide variety of substrates. This activity is involved in the
detoxification of endogenous compounds such as peroxidised lipids (Leaver &
George, 1998) as well as the metabolism of xenobiotics. The activity of GST is found
to be decreased in ISO treated MI (Saravanan et al., 2013).
1.1.3.2 Non-enzymatic antioxidants
Reduced Glutathione:
Glutathione is a tripeptide (Glu-Cys-Gly), whose antioxidant function is
facilitated by the sulphydryl groups of cysteine (Rennenberg, 1980). It can react
chemically with singlet oxygen, O2•−, and OH• and therefore function directly as a free
radical scavenger. It may stabilize membrane structure by removing acyl peroxides
18
formed by lipid peroxidation reactions. Decreased levels concentrations of GSH were
observed in ISO-induced myocardial infarcted rats (Saravanan et al., 2013).
Isoproterenol induces oxidative stress and results in alterations of cardiac
function and ultra-structure in experimental rats (Kumaran & Prince, 2010a). The
positive inotropic and positive chronotropic response of ISO augments myocardial
oxygen consumption. The increase in energy demand (Prabhu et al., 2000) and the
decrease in blood flow induce an energy imbalance by the overload of Ca2+ (Chagoya
de Sanchez et al., 1997). This is accompanied by the disruption of mitochondria (Xia
et al., 2002), with inactivation of tricarboxylic acid cycle enzymes and altered
mitochondrial respiration (Prabhu et al., 2006). In acute myocardial injury, ROS
toxicity shuts down mitochondrial oxidative phosphorylation and the tricarboxylic
acid cycle, blocking mitochondiral ATP production and cardiac contraction.
1.1.4 Pro-inflammatory cytokines and myocardial infarction
The release of proinflammatory cytokines such as TNF-α, interleukin-6 (IL-6)
and interleukin-1β (IL-1β) have been observed consistently in the experimental models
of MI. The functional pleiotropy and redundancy is a characteristic feature of
cytokines which indicates its wide range of biological effects on various cell types
(Kishimoto et al., 1995). The multifunctional, overlapping and often contradictory
effects of cytokines have been impeded because of its role in cardiac injury and repair.
Tumor necrosis factor-α:
Tumor necrosis factor-α is a potent proinflammatory cytokine has a crucial role
in cardiomyocytes injury and repair that acts on vascular endothelial cells to induce
expression of leukocyte adhesion molecules. Moreover, it decreases cardiac
19
contractility (Grandel et al., 2000) and enhances apoptosis in the cardiomyocytes
(Song et al., 2000). In cardiac fibroblasts, TNF-α regulates the extracellular matrix
metabolism by increasing the activity of matrix metalloproteinases (MMPs) and by
decreasing the synthesis of collagen (Siwik et al., 2000). Previous reports have shown
the effects of TNF-α signaling in cardiac dysfunction (Sugano et al., 2004).
Interleukin-1ß family:
Interleukin 1ß family is a group of cytokines, which plays a central role in the
regulation of immune and inflammatory responses to infections or sterile insults. It
consists of interleukin-lα, IL-1ß (agonists) and interleukin-1 receptor antagonist
(specific receptor antagonist) (Dinarello, 1996). The levels of IL-lß are increased in
the plasma of acute myocardial infarcted patients (Guillen et al., 1995). IL-1 shows an
injurious role in the ischaemic myocardium. This signaling activates inflammatory and
fibrogenic pathways in the healing infarct heart and may play a vital role in the
pathogenesis of postinfarction remodeling (Bujak et al., 2008).
Interleukin-6:
Interleukin-6 is a member of structurally related cytokines acts as both pro-
inflammatory and anti-inflammatory cytokine with overlapping physiological and
biological effects. It is secreted by T cells and macrophage to stimulate immune
response to trauma, especially burns or other tissue damage leading to inflammation.
Under hypoxic conditions, cardiomyocytes produce IL-6 which could contribute to the
myocardial dysfunction observed in ischemia reperfusion injury (Sharma & Das,
1997).
20
1.1.5 Apoptosis
Apoptosis, the programmed cell death is a process by which cells undergo
inducible non-necrotic cellular suicide that may occur in multicellular organisms
(Song et al., 2000). Biochemical events lead to characteristic cell changes
(morphology) and death, which shown in Figure 5. These changes include blebbing,
cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal
DNA fragmentation.
1.1.5.1 Apoptosis in myocardial infarction
Apoptosis, one of the major forms of cell death, has been implicated in
different CVDs. Several studies have reported cardiomyocyte apoptosis during human
MI (Baldi et al., 2002; Saraste et al., 1997). The discovery of apoptosis sheds a new
light on the role of cell death in MI and other CVDs. There is mounting evidence that
Figure 5: Apoptosis (Abou-Ghali & Stiban, 2015)
21
apoptosis plays an important role at multiple points in the evolution of MI, and
comprises not only cardiomyocytes but also inflammatory cells, as well as cells of
granulation tissue and fibrous tissue (Zidar et al., 2007). The two pathways that lead
to apoptosis are an extrinsic Pathway and an intrinsic Pathway, shown in Figure 1.6.
The extrinsic pathway begins outside the cell through activation of pro-apoptotic
receptors on the cell surface whereas the intrinsic or mitochondrial pathway is initiated
within the cell.
Involvement of reactive oxygen species in the pathogenesis of myocardial apoptosis:
Reactive oxygen species may play an important pathophysiological role in
cardiac diseases characterized by apoptotic cell death and suggest that different ROS-
induced activations of the apoptotic cell death program in cardiomyocytes involve
distinct signaling pathways (von Harsdorf et al., 1999). A scientific study has observed
significant linear relationship between apoptotic myocytes number, macrophages and
transmigrated neutrophils during early and prolonged reperfusion (Zhao et al., 2001).
Also, ischemia followed by reperfusion induces myocardial injury through apoptotic
pathway.
22
Figure 6: Intrinsic and extrinsic pathway of apoptosis (Kumar, 2015)
23
1.1.6 Experimental myocardial infarction in animal models
For the experimental MI studies, doxorubicin, cyclophosphamide and ISO are
used, but in this research, I will focus mainly on isoproterenol.
Isoproterenol:
Isoproterenol (1-[3, 4 dihydroxyphenyl]-2-isopropyl amino ethanol
hydrochloride) is a synthetic catecholamine and β-adrenergic agonist, which has been
found to cause severe stress in the myocardium resulting in infarct like necrosis of
heart muscles (Wexler & Greenberg, 1978). ISO-induced MI serves as a well-
standardized and common model to find out the beneficial effects of many drugs
(Ithayarasi & Devi, 1997). The supramaximal doses of catecholamines result in
necrotic lesions development in the myocardium of experimental animals (Knufman
et al., 1987). The pathophysiological changes that take place in rat's heart following
MI induced by ISO are comparable to those changes taking place in MI in humans
(Geng et al., 2004). Chemical structure of ISO is shown in Figure 7.
The hazardous effect of catecholamine is due to the oxidation of hydroxyl
groups leading to their conversion into quinones and the subsequent formation of
adrenochromes, as shown in Figure 8. ISO induced MI results in increased oxidative
stress, hypoxia, mitochondrial dysfunction, hyperlipidemia, intracellular depletion of
Figure 7: Structure of isoproterenol
24
ATP increased Ca2+ influx, and cyclic adenosine monophosphate levels. The free
radicals initiate the peroxidation of membrane bound PUFAs resulting in functional
and structural myocardial injury (Thompson & Hess, 1986). During ISO induced MI,
enhanced free radical formation and lipid peroxide accumulation have been proposed
as one of the possible biochemical mechanisms for myocardial damage
(Sushamakumari et al., 1989).
The enzymes, catechol-o-methyl transferase and monoamine oxidase are
involved in the degradation of catecholamines by methylation of the 3-hydroxyl group
of the catechol ring and removal of the amino group under physiological conditions
results in the formation of non-toxic metabolites such as metanephrine, dihydroxy
mandelic and vanillyl mandelic acids (Yates, Beamish & Dhalla, 1981). However,
high concentration of catecholamines saturates these enzyme systems. Free radicals
such as O2•- and hydrogen peroxide were formed by the metal catalyzed Fenton
reaction of OH• from the oxidation of catecholamines to quinones. Moreover,
adrenochrome, a toxic metabolite is formed by the intra cyclization of quinone (Yates
et al., 1981).
25
1.2 Cannabinoids as drug target for MI
Cannabinoids are mainly defined as the active compounds in marijuana, which
extracted from different part of the hemp plant Cannabis sativa, including flowers,
leaves and resin. It has been used in China for medical purposes throughout the history
from around five-thousand years ago (Mendizabal & Adler-Graschinsky, 2007). The
medical marijuana has been documented to be used as analgesic, anti-inflammation,
muscle relaxant, anticonvulsant, antiemetic, appetite stimulants and as recreational use
due to its euphoric effect, which has limited its medical application (Iversen, 2001;
Mechoulam, 1986).
Cannabinoids could also be defined as a class of diverse chemical compounds
that act on cannabinoid receptors (endocannabinoid system), which alter the release of
Figure 8: Proposed mechanism of auto-oxidation of isoproterenol (Singal et
al., 1983)
26
neurotransmitters in the brain. The ligands for these receptors are often grouped into
three types.
1.2.1 Endocannabinoids
Endocannabinoids, are the endogenous lipids that bind cannabinoid receptors
and lead to affecting the behavior in a way partially mimicked by the effects produced
by the psychoactive components of cannabis, mainly Delta 9-THC ((-) trans-delta-9-
tetrahydrocannabinol) (Lu & Mackie, 2016). The first discovered endocanabinoid is
anandamide (N-arachidonoylethanolamine; AEA), it has been discovered by (Devane
et al., 1992). According to this report, anandamide is an arachidonic acid derivative
that function as a natural ligand for the cannabinoid receptor. Three years later another
endocanabinoid was discovered by (Mechoulam et al., 1995), 2-arachidonoylglycerol
(2-AG). Since then, a number of other endocanabinoid-like-activity lipids was
discovered and reported in Table 3, but the most well studied and best characterized
are AEA and 2-AG.
27
Table 3: The pharmacological activity of endogenous cannabinoids
Type Activity References
Anandamide (AEA) CB1>>CB2 agonist
TRBV1 agonist
(Mechoulam et al.,
1995)
(Khanolkar et al.,
1996)
2-Arachidonoylglycerol (2-
AG)
CB1≈CB2 agonist (Mechoulam et al.,
1995)
2-Arachidonyl glyceryl ether CB1>>CB2 agonist
TRPV1 partial agonist
(Hanus et al., 2001)
(Duncan et al.,
2004)
O-Arachidonoyl ethanolamine
(Virodhamine)
CB1 antagonist, CB2
agonist
(Porter et al., 2002)
N-Arachidonoyl dopamine CB1>>CB2 agonist
TRBV1 agonist
(Bisogno et al.,
2000)
(Huang et al., 2002)
Anandamide is an N- (polyunsaturated fatty acyl) ethanolamine that formed
from the formal condensation of carboxy group of arachidonic acid with amino group
of ethanolamine. It play a role as a neurotransmitter, vasodilator and as a blood serum
metabolite (PubChem, 2019b).
Anandamide is a highly potent endogenous agonist of both cannabinoid
receptors (CB1 and CB2 receptors) (PubChem, 2019b). Previous studies show that
AEA might inhibit tumor cell proliferation or induce apoptosis independently of CB1
and CB2 receptors, this action mediated by interaction with the type 1 vanilloid
receptor (VR1). VR1 is an ion channel expressed almost exclusively by sensory
neurons, it’s activated by pH, noxious heat (>48 degree °C) and plant toxins and it
28
plays an important role in nociception. Cervical cancer cells are sensitive to AEA-
induced apoptosis via VR1 that is aberrantly expressed in vitro and in vivo while CB1
and CB2 receptors shows to play a protective role (Contassot et al., 2004).
2-Arachidonoylglycerol (2-AG), is an endocannabinoid and an endogenous
agonist of both cb1 and cb2 cannabinoid receptors. It is isolated in 1995 from rat brain
and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is an ester
formed from omega-6-arachidonic acid and glycerol. It play a role as a human
metabolite and it’s derived from arachidonic acid-containing phospholipids.
(PubChem, 2019a).
A vital feature of these endocannabinoids is that their precursors are present in
lipid membranes. Upon demand (typically by activation of certain G protein-coupled
receptors or by depolarization), endocannabinoids are synthesized in one or two rapid
enzymatic steps and then released into the extracellular space. Unlike the classical
neurotransmitters that are synthesized ahead of time and stored in vesicles. Intrinsic
efficacy of the endogenous cannabinoids varies, THE 2-AG show a high efficacy
agonist for both CB1 and CB2 receptors, while AEA show a low efficacy agonist at
CB1 receptors and a very low efficacy at CB2 receptors (Gonsiorek et al., 2000). The
endocannabinoids structures are represented in Figure 9.
The activity of both AEA and 2-AG at their receptors is limited by cellular
uptake. This action mediated by anandamide membrane transporter (AMT), followed
by intracellular degradation. Fatty acid amide hydrolase (FAAH) is the main enzyme
that degrade AEA, whereas a monoacylglycerol lipase (MAGL) is critical in degrading
2-AG (Sugiura et al., 2006). The Endocannabinoids synthesis and degradation are
represented in Figure 10.
29
1.2.2 Synthetic cannabinoid
Synthetic CBR agonists are a large group of legal and illegal compounds that
bind to one or both known CBR. Many of these substances were initially researched in
the 1950s and 1960s for their potential therapeutic action in a variety of diseases.
Adverse effects, such as depression and increased potential of suicide stopped these
substances from reaching therapeutic use (Tai & Fantegrossi, 2017). Examples of
synthetic cannabinoids are shown in Table 4.
Figure 10: Endocannabinoids structures
Figure 9: Endocannabinoids synthesis and degradation
30
Synthetic cannabinoids have been developed which can act as selective
agonists or antagonists to CB receptors. Synthetic analogues are produced to either
stimulate or block the CB receptors specifically. Specific CB2 agonists are useful in
avoiding the undesirable effects caused by CB1 stimulation (Xavier, 2013).
The endocannabinoid levels can be increased by:
• Inhibiting the degrading enzymes (FAAH inhibitors).
• Inhibiting the reuptake of endocannabinoids (Reuptake inhibitors- Inhibiting
reuptake transporters).
• Specific agonists and antagonists are useful in the research in finding out the
receptors involved in specific function.
Table 4: Examples of synthetic cannabinoids (Xavier, 2013)
Group Synthetic analogues
Cannabinoid receptor agonists Classical cannabinoids:
Delta-8-THC, HU-210, AM411
Non-classical cannabinoids: CP-55940
Specific CB2 agonists: AM-1241, HU-308
Cannabinoid receptor antagonists SR141716A (Rimonabant): CB1
antagonist.
AM251: CB1 antagonist.
SR147778: CB1 antagonist.
Uptake blockers AM404, UCM707, AM1172, VDM 11
Inhibitors of fatty acid amide
hydrolase (FAAH)
OL-135, URB 597
31
1.2.3 Phytocannabinoids
More than 60 organic compounds are derived from the cannabis plant and
called cannabinoids. Among the plant cannabinoids extracted from cannabis, THC is
the most active ingredient and considered the main psychotropic constituent of
cannabis. Δ-THC and cannabidiol (CBD) are the mostly studied. Other cannabinoids
are cannabinol, Δ9-tetrahydrocannabivarin, cannabigerol. Amount of the cannabinoids
present in each plant strain are different. For example, THC levels of the plant strains
bred for recreational purpose is much more than that of the plants bred for the hemp.
The affinity of the cannabinoids to their receptors is different since their effect (Xavier,
2013). Examples of plant derived cannabinoids are summarized in Table 5.
• THC act as CB1 and CB2 partial agonist and show equal affinity to both CB1
and CB2 receptors.
• CDB act as CB2 receptor antagonist (inverse agonist).
Table 5: Plant derived cannabinoids (Xavier, 2013)
Phytocannabinoids
Δ 9-tetrahyrocannabinol type Cannabicyclol type
Δ 8- tetrahyrocannabinol type Cannabielsoin type
Cannabidiol type Cannabitriol type
Cannabigerol type Miscellaneous type
Cannabichromene type Cannabinol and cannabinodiol type
32
Phytocannabinoids found in plants other than cannabis:
In the last few years, several non-cannabinoid plant products have been
discovered and reported to act as cannabinoid receptor ligands, this allow us to define
the “phytocannabinoids” as any plant constituents not specifically from cannabis sativa
capable to interact directly with CB receptors or share chemical similarity of
cannabinoids or both (Gertsch, Pertwee & Marzo, 2010).
In Table 6, some examples of natural products are summarized that have been
reported to interact with the ECS:
Direct CB receptor ligands
Compounds that show high binding affinities for cannabinoid
receptors
Exert discrete functional effects (i.e. agonism, neutral antagonism
or inverse agonism)
Indirect CB receptor ligands
Target key proteins within the ECS that regulate tissue levels of
endocannabinoids.
OR target allosteric sites on the CB receptor.
Figure 11: Phytocannabinoids possess at least some of these properties (Gertsch et
al., 2010).
33
Table 6: Natural products that have been reported to interact with the ECS
Name Structure Description and Function
β-caryophyllene • Selectively target the CB2
receptor at nM
concentrations (Ki = 155
nM) and to act as a full
agonist (Gertsch, 2008a).
• Orally administration (<5
mg·kg-1), strong anti-
inflammatory and
analgesic effects in
wildtype mice but not in
CB2 receptor knockout
mice (Gertsch et al.,
2010).
Pristimerin
• Naturally occurring
quinonoid triterpenoids
• Potent reversible
monoacylglycerol lipase
inhibitor (IC50 < 100 nM)
(King et al., 2009)
Rutamarin • Coumarin derivative from
the medicinal plant Ruta
graveolens L (Rollinger et
al., 2009).
• Selective CB2 affinity (Ki
value <10 mM) in human
(Rollinger et al., 2009).
DIM
3,3-diindolylmethane
metabolite from
indole-3-carbinol
• Anti-carcinogenic
metabolite generated by
ingestion of indole-3-
carbinol (Yin et al., 2009).
• It shown to act as a weak
CB2 receptor partial
agonist (Yin et al., 2009).
34
1.3 Cannabinoid receptors
Initially it was thought that the psychotropic effects mediated by cannabinoids
are due to their high lipophilicity. At the middle of 1980’s, they found that the
cannabinoids activity is highly stereospecific and this led them to discover the both
types of CB receptors, cannabinoid type 1 (CB1) and type 2 (CB2) receptors (Pertwee,
2006). Among many new therapeutic targets, the endogenous cannabinoid system
represents one of the newest and most promising drug targets (Dias Dde et al., 2012).
Both receptors are members of the GPCR family, and its activation leads to adenylate
cyclase inhibition (Gi/o) or MAPK activation, as shown in Figure 12. CB1 receptors
are mainly distributed in the brain and CB2 in the periphery, as shown in Table 7.
(Pertwee, 2006).
Figure 12: Cannabinoid pathway (Xavier, 2013)
35
Table 7: Distribution of CB receptors
CB2 receptors are encoded by the gene CNR2, which consists of 360 amino
acid in humans. At the protein level, it shares 44% sequence homology with CB1
receptors. The CB2 receptors show a greater species differences among humans and
rodents in comparison to CB1 receptors, as the amino acid sequence homology is
slightly above 80% between humans and rodents (Liu et al., 2009; Zhang et al., 2015).
Structurally both CB1 and CB2 receptors composed of seven transmembrane
protein domains (Shao et al., 2016), shown in Figure 13, which predominantly couple
to the inhibitory G proteins Gi and Go (Mackie, 2008). Activation of G protein causes
intracellular consequences include the inhibition of adenylate cyclase and certain
voltage gated calcium channels, the activation of mitogen activated protein kinase
(MAP kinase) and of inwardly rectifying potassium channels (Howlett et al., 2002). In
the central and peripheral nervous system, the net effect of these processes is to lessen
neuronal excitability and to negatively modulate neurotransmission. In immune cells,
activation of CB2 receptor results in a suppressive effects which including impaired
Receptor Distribution
Cannabinoid 1 In CNS: Hippocampus, basal ganglia, cerebellum,
cerebral cortex, periaqueductal gray, certain parts of
spinal cord.
In periphery: Heart, GIT, urinary tract, leukocytes,
spleen, endocrine glands, salivary glands,
reproductive tract.
Cannabinoid 2 Immune cells (leukocytes), spleen, tonsils, thymus,
and bone.
36
antigen presentation, reduced cytokine release and disruption of immunocyte
migration (Eisenstein & Meissler, 2015).
1.4 CB2 receptors and heart diseases
Cardioprotective effects of endocannabinoid-mediated CB2 receptor activation
were first reported in LPS-induced preconditioning (Sultana & Saify, 2013).
Thereafter, several recent reports using synthetic CB2 receptor agonists in vitro, ex
vivo or in vivo showed protective effects on remote preconditioning, infarct size,
arrhythmias while counterbalancing chronic heart failure-induced structural changes,
inhibiting atherogenesis and preventing myocyte enlargement (Liu et al., 2013; Oda et
al., 2011; Wang et al., 2010). The anti-inflammatory effects of CB2 receptor activation
in the endothelium and its inhibitory effect on monocytes/macrophages and/or
Figure 13: Schematic showing the CB1 and CB2 receptors
37
leukocyte migration were reversed by pharmacological antagonism of CB2 receptors
with AM630 (Liu et al., 2015). Pretreatment with the CB2 receptor antagonist AM630
or SR144528 abolished the cardioprotective effects and reversed cardioprotection.
CB2 receptors have also been implicated in the modulation of endoplasmic reticulum
stress and immune cell migration involving the MAPK, PI3K/Akt and ERK½
pathways (Liu et al., 2015; Liu et al., 2013; Oda et al., 2011; Wang et al., 2010). Many
studies with synthetic cannabinoid ligands of CB2 receptors paved the way for CB2
receptor activation as a strategy in cardioprotection.
The adverse effects, abuse liability and addictive properties of synthetic CB2
receptor ligands in humans generated interest in targeting CB2 receptors by
phytocannabinoids, which are ligands of natural source. To date, several
phytocannabinoids have been pharmaceutically developed and this is demonstrated by
the availability of Sativex, nabilone and dronabinol. Phytocannabinoids have been
widely studied as potential protective agents in chronic diseases because of their
multiple targets, time tested efficacy and low cytotoxicity (Bento et al., 2011).
However, in general, the adverse psychoactive effects of phytocannabinoids derived
from cannabis also limit their therapeutic use and bring legal concerns. Recently,
several of the phytocannabinoids derived from plants other than cannabis have
received interest for their favorable physicochemical, pharmacokinetic and
pharmacological properties and their anti-inflammatory and antioxidant effects which
make them promising for pharmaceutical development (Wu et al., 2014).
38
1.5 Beta-Caryophyllene
Beta-caryophyllene (BCP) is a volatile bicyclic sesquiterpene which found
abundantly in the essential oils of many edible plants and spices such as cloves
(Syzygium aromaticum), cinnamon (Cinnamomum spp.), oregano (Origanum vulgare),
rosemary (Rosmarinus officinalis), hemp (Cannabis sativa), germander (Teucrium
spp.), Copaiba oil (Copaiba spp.), hops (Humulus lupulus) and black pepper (Piper
nigrum) (Sharma et al., 2016). Sources of BCP are shown in Figure 14.
BCP has been approved by USFDA and European Food Safety Authority
(EFSA) for use as food additive and flavoring agent (Sharma et al., 2016), and this
attracted the scientific community to explore it for its additional possible therapeutic
benefits (Gertsch, 2008a; Gertsch et al., 2008b). It exhibited significant therapeutic
potential in numerous diseases due to multiple pharmacological properties to name a
few like antioxidant, anti-inflammatory (Gertsch et al., 2008b), antimicrobial (Donati
et al., 2015), anticancer and chemopreventive properties (Sain et al., 2014).
Figure 14: Some natural sources of BCP
39
BCP is mainly found in the essential oils obtained from different parts or whole
plant mainly aromatic plants. It has been reported to exist in more than one thousand
plants which provide an extensive natural availability and accessibility. It is also one
of the significant constituents (about 35%) in the essential oil of Cannabis sativa L,
which considered a major source of plant derived cannabinoids or phytocannabinoids
(Calleja et al., 2013; Hendriks et al., 1975). Table 8 shows the medicinal plants that
are recognized to contain the amount of BCP equal to or more than 30% of total
phytoconstituents.
Table 8: Plants that contain BCP (presented as % of BCP amongst constituent
phytochemicals) (Sharma et al., 2016)
Plant name Parts % BCP
Acanthospermum hispidum Oil 35.2
Callicarpa integerrima Leaves 33.7
Canarium parvum Leen Stem 30.4
Copaifera multijuga Trunks 36.0
Copaifera multijuga Hayne Trunks 57.5
Copaifera reticulata Ducke Trunks 40.9
Croton heliotropiifolius Oil 35.8
Euphorbia cotinifolia Leaves 39.3
Glechon marifolia Oil 32.2
Guarea humaitensis Leaves 33.3
Hyptis pectinata Oil 54.1
Lantana montevidensis Leaves 31.5
Leucas indica Aerial parts 51.1
Leucas aspera Aerial parts 34.2
40
Table 8: Plants that contain BCP (presented as % of BCP amongst constituent
phytochemicals) (Sharma et al., 2016) (continued)
Plant name Parts % BCP
Melampodium divaricatum Aerial parts 56.0
Mydocarpus fraxinifolius Leaves 63.0
Murraya paniculata Leaves 30.0
Myrica rubra Leaves 89.9
Newbouldia laevis Leaves 36.0
Peristrophe bicalyculata Oil 33.9
Phoenix dactylifera Buds 44.2
Pinus halepensis Oils 30.0
Piper guineense, white Fruits 51.7
Piper guineense, black Fruits 57.6
Piper duckei Leaves 30.0
Plectranthus rugosus Aerial parts 36.0
Plectranthus neochilus Leaves 30. 0
Stachys cretica Aerial parts 51.0
Tabernaemontana
catharinensis
Leaves 56.9
Teucrium chamaedrys Aerial parts 47.6
Teucrium polium Aerial parts 52.0
Teucrium flavum Aerial parts 32.5
Teucrium divaricatum Aerial parts 30.1
Valeriana alliariifolia Aerial parts 38.9
41
BCP is usually found to be concentrated mostly in the aerial parts, leaves,
flowers and florescence and in traces in roots, rhizomes, stems and barks of different
plants (Dias Dde et al., 2012; Lucca et al., 2015).
Chemically, BCP is a bicyclic sesquiterpene which is named (trans-(1R,9S)-8-
Methylene-4,11,11-trimethylbicyclo (7.2.0) undecene), it is also recognized by several
other synonyms such as caryophyllene, trans caryophyllene, (-)-trans-caryophyllene,
β-(E)-caryophyllene and L-caryophyllene (Budavari et al., 1996). BCP present in
plants as a mixture of two pharmacologically active isomers E-BCP and Z-BCP,
together with substantially inactive sesquiterpenes such as alpha-humulene and
derivatives such as BCP oxide. Natural sources include a greater proportion of E-BCP
than Z-BCP (Gertsch et al., 2008c). Optically, β-Caryophyllene and iso-caryophyllene
are trans- and cis- double isomers respectively, while α-humulene is a ring-opened
isomer. In plants, the BCP is generally found in their essential oils as a mixture with
iso-caryophyllene or α-humulene (Budavari et al., 1996). Structure of β-Caryophyllene
is shown in Figure 15.
BCP is being used since 1930 in different forms in foods and cosmetics,
however the first total synthesis was in 1964 and to date several approaches including
semi-synthetic and microbial biotransformation have been adopted in order to meet
the industrious demand for the production and supply of BCP (Oda et al., 2011; Sultana
& Saify, 2013).
Figure 15: Structure of β-Caryophyllene (BCP) (Javed et al., 2016)
42
BCP, which is a sesquiterpene, is effective on the endocannabinoid system in
terms of binding to the CB2 receptor. Due to its lipophilicity, BCP can cross through
the plasma membranes (Gertsch et al., 2008b; Sharma et al., 2016). CB2 receptor
agonists have been reported to repress inflammation (Gertsch et al., 2008b). BCP and
-Caryophyllene oxide (BCPO), which is an oxide of BCP, show both anticancer and
analgesic features (Fidyt et al., 2016). Moreover, BCP has anti-inflammatory
(Medeiros et al., 2007; Sain et al., 2014), antimicrobial (Sabulal et al., 2006),
antioxidative (Kubo et al., 1996; Singh et al., 2006), antispasmodic (Leonhardt et al.,
2010), and antiviral (Astani et al., 2011) effects. A research which was carried out in
2012 revealed that BCP was effective in alleviating cisplatin-induced nephrotoxicity
(Horvath et al., 2012b). The researchers found CB2 receptors in the rat heart using the
Western Blot method (Bouchard, Lépicier & Lamontagne, 2003). The research also
revealed that endocannabinoids (endogenous cannabinoids) exhibited a strong
cardioprotective effect mainly by the activation of a CB2 receptor in a rat model of
cardiac ischemia-reperfusion (I/R) injury (Bouchard et al., 2003). Besides this, CB2
expression was upregulated in cardiomyocytes of the ischemic mouse hearts (Duerr et
al., 2014). Due to all these promising features of BCP, it has been chosen as a perfect
candidate to be used in this study.
BCP is not only a volatile substance but also its solubility in water is quite low.
It is also quite sensitive to ambient factors such as temperature, humidity, light,
oxygen, and moisture (Sköld et al., 2006; Wang et al., 2010). The animal studies show
that BCP, which is orally bioavailable, reaches its peak concentration in serum/plasma
within an hour after administration (Liu et al., 2013). BCP is not metabolized instantly
like the popularly used polyphenolic compounds which have been recently researched.
However, its volatility and poor solubility in the water cause a decrease in the
43
bioavailability of BCP (Liu et al., 2015). The pharmacokinetic characteristics of BCP,
including its bioavailability, have been enhanced by delivering BCP in a β-
cyclodextrin inclusion complex. Thus it renders a hydrophilic surface to BCP
improving its dissolution and solubility in the water (Liu et al., 2015). The Physico-
chemical properties and pharmacokinetics of BCP are summarized in Table 9.
Table 9: Physico-chemical properties and pharmacokinetics of BCP (Sharma et al.,
2016)
1.5.1 Pharmacological properties of BCP
BCP is considered as a multifunctional and poly pharmacological agent for
therapeutic development in complex diseases (Chicca et al., 2014). The
pharmacological targets of BCP are exemplified in Figure 16.
Chemical name -Caryophyllene
Synonym: Caryophyllene
Physical state: Colorless to yellow oily liquid
Solubility: Insoluble in water
Odor: Woody/terpene and cloves odor
Density: 0.90 g/mL at 20oC
Boiling point: 256-259oC at 760 mmHg
Specific gravity: 0.899 to 0.908 at 25oC
Molecular weight: 204.35 g/mol
Molecular formula: C15H25
CAS number: 87-44-5
XLogP3-AA: 4.4
44
It is seen that BCP regulate the expression and release of a variety of pro-
inflammatory cytokines, chemokine’s, growth factors, transcription factors, genes,
enzymes, adhesion molecules, receptors and heat shock proteins and apoptosis and
also proteins associated with the cell cycle (Bento et al., 2011; Gertsch et al., 2008c;
Horvath, et al., 2012b; Jung et al., 2015; Sain et al., 2014). It has been stated that BCP
regulated multiple molecular targets in various diseases and disorders and this is
performed by modifying gene expression, molecular and cellular signaling pathways
or by direct interaction with the targets to thwart the progression and development of
various disease processes (Sharma et al., 2016).
β-Caryophyllene
PPAR α/γ
CB2
receptor
Toll-like receptors
(TLRs)
μ opioid receptors
Figure 16: The multiple receptor targets of β-caryophyllene
45
It has been manifested that BCP has various pharmacological targets. BCP
functions by adjusting cellular and molecular signaling tracts, modifying gene
expression and also interacting with biochemical and/or molecular targets (Sharma et
al., 2016). Table 10, display some of the molecular mechanisms, which BCP is
involved in.
Table 10: Molecular mechanisms that BCP has an effect on (Sharma et al., 2016)
In several experimental studies (in vitro, in vivo and in silico), it has been
manifested that BCP interacts with and binds to CB2 receptors and serves as a full
selective functional agonist (Gertsch, 2008a). It was revealed that BCP specifically
binds with Δ9-tetrahydrocannabinol binding site in the CB2 receptor. It is also
exceptional in terms of having an atypical cyclobutane ring, a unique scaffold that
enables an interacting geometry for its binding to CB2 receptors (Gertsch et al.,
Inhibition of intracellular Ca²⁺
Voltage-dependent Ca²⁺ channel blockade
Inhibition of proinflammatory cytokines
Inhibition of activation of the toll-like receptor complex CD14/TLR4/MD2
Activates the mitogen-activated kinases Erk1/2 and p38
Blocker of STAT3 signaling cascade
Enhanced phosphorylation of AMP-activated protein kinase (AMPK)
Enhanced phosphorylation of cAMP responsive element-binding protein (CREB)
Stimulates SIRT1/PGC-1α-dependent mechanism
Down-regulation of anti-apoptotic genes (bcl-2, mdm2, cox2 and cmyb)
Up-regulation of pro-apoptotic genes (bax, bak1, caspase-8, caspase-9 and ATM)
46
2008c). The CB2 receptors are G-protein coupled receptors that generally bind with
G-proteins in the Gi/o family. Triggering of the CB2 receptors leads to multiple
intracellular effects that may be of cell type and ligand specific and that involve the
suppression of diverse voltage gated Ca2+ channels and adenylate cyclase activity and
the triggering of K+ channels, leading to lower levels of cAMP along with the
triggering of MAPK pathways (Felder et al., 1995). The CB2 receptor combined with
Gi intercedes their cellular effects through the suppression of adenylyl cyclase and
regulation of transcription factors (Felder et al., 1995). Activation of CB2 receptors
symbolize an important therapeutic target in numerous diseases (Calleja et al., 2013;
Gertsch et al., 2008c; Horvath et al., 2012b). The cyclobutane pharmacophore of BCP
was further utilized as a template for the discovery of new drugs and exposed to
structural modifications. The modifications brought forth a series of new monocyclic
amides which preserved CB2 receptor agonism property in addition to its quality in
order to suppress fatty acid amide hydrolase (FAAH), an endocannabinoid degrading
enzyme of the endocannabinoid system that improves the tone of endocannabinoid
signaling. The generated molecules also evoke the suppressive properties on
endocannabinoid substrate-specific metabolizing enzyme, cyclooxygenase-2 (COX-
2), which is an important interceder of inflammatory pathways in the metabolism of
arachidonic acid for the generation of prostanglandins (Chicca et al., 2014). This
shows that the pharmacophore of BCP is disposed to preserve the polypharmacological
nature.
It has also been determined that BCP regulate the nuclear receptors and
transcription factor, peroxisome proliferator-activated receptors (PPARs) subtype,
PPAR-γ and PPAR-α. The triggering of PPARs by cannabinoid affiliated molecules
evokes multiple beneficial physiological effects and therapeutic advantages (Bento et
47
al., 2011; Guo et al., 2014; Wu et al., 2014). The PPARs are among the significant
members of nuclear receptor superfamily, which function as ligand triggered
transcription factors and play a vital role in the differentiation and proliferation of cells,
organogenesis as well as inflammation. They also found to regulate the expression of
hepatic enzymes and participate in glucose homeostasis, insulin sensitivity as well as
lipid metabolism (Kota et al., 2005). There are three separate isoforms of PPARs
namely PPAR-α, PPAR-γ and PPAR-δ which show different ligand selectivity and
specific distribution in the tissues. Among these different isoforms, PPAR-α is
primarily expressed in heart, liver, intestine and macrophages and is triggered by
polyunsaturated fatty acids and leukotrienes. While PPAR-γ is essentially expressed
in adipocytes, but its transcript has also been specified in various other tissues, though
in low abundance. PPAR-γ plays a critical role in adipocyte differentiation and lipid
accumulation as it is triggered by polyunsaturated fatty acids and 15d-prostaglandin
J2 (Inagaki et al., 2007). The PPAR isoforms are like those of the steroid receptors and
are associated with several functions started by nutrients, nutraceuticals and
phytochemicals.
The ligand-binding to cannabinoid receptors activate mitogen-activated protein
kinase (MAPK), which adjusts the activation of PPAR even more through direct
phosphorylation. Although the accurate mechanisms about cannabinoid and PPARs
interactions are not known, the PPAR stimulation by cannabinoid ligands materialize
as partners that act together to obtain the therapeutic benefits (O'Sullivan & Kendall,
2010). This presents a basis through which cannabinoids can regulate gene
transcription triggered by the dietary interferences. Since BCP is highly lipophilic, it
can go across the membranes and access the nuclear receptors; that is, it has the
capacity to regulate both the surface and nuclear receptors.
48
BCP was also observed to suppress the pathways activated by the initiation of
the toll like receptor complex (CD14/TLR4/MD2) and curtailed the immune-
inflammatory processes in multiple autoimmune diseases (Gertsch, 2008a). The extra
μ-opioid receptor activity (Katsuyama et al., 2013; Paula-Freire et al., 2014) and
effective antagonist activity on homomeric nicotinic acetylcholine receptors (α7-
nAChRs) strengthen its potent anti-inflammatory activity. Multiple studies have
shown the in vitro (Donati et al., 2015; Lee et al., 2013; Vinholes et al., 2014) and in
vivo antioxidant mediated protection of different organs (Calleja et al., 2013; Guo et
al., 2014; Horvath et al., 2012b; Molina-Jasso et al., 2009). BCP displayed an effective
chain breaking antioxidant and free radical scavenger activity against the highly
reactive free radicals such as hydroxyl and superoxide anions (Molina-Jasso et al.,
2009; Vinholes et al., 2014). BCP has also been observed to regenerate the glutathione
redox cycle and efficiently impede lipid peroxidation, a significant pathogenic event
and a critical culprit in most of the chronic degenerative and acute organ injuries. BCP
has also been observed to be distinguished compared to various standard antioxidants
such vitamin C and E, available for clinical use (Vinholes et al., 2014). BCP (32.5%)
extracted from essential oil of Teucrium flavum L. subsp. flavum displayed free radical
scavenging activity and antioxidant potential in DPPH assay (Hammami et al., 2015).
It has also been observed to impede 5-lipoxygenase, an important enzyme of
arachidonic acid pathways that is efficiently involved in the production of
inflammatory mediators and institution of inflammation in multiple inflammatory
diseases (Sain et al., 2014). The GST inhibitory and lipoxygenase inhibitory activity
of BCP was further supported by in silico data (Babu et al., 2012). The antioxidant
activities interceded by the CB2 receptors were also displayed and they implied the
49
additional antioxidant properties of BCP in therapeutic benefits other than anti-
inflammatory activity (Assis et al., 2014; Vinholes et al., 2014).
Regarding BCP, it has been stated that it exercises strong anti-inflammatory
effects in various in vitro and in vivo studies (Bento et al., 2011; Gertsch et al., 2008c;
Ma et al., 2015; Paula-Freire et al., 2014; Rufino et al., 2015). Moreover, these effects
positively alter many of its biological and therapeutic activities in diseases where low
grade and long term inflammation play a significant role in etiopathogenesis (Fine &
Rosenfeld, 2013; Guo et al., 2014). The mostly attested favorable effects of BCP are
its capacity to regulate pro-inflammatory cytokines and chemokines and avoid the
occurrence and progression of immune-inflammatory disorders interceded by CB2
receptors (Gertsch et al., 2008a).
Several of the studies demonstrate that the anti-inflammatory effects of BCP
are mediated by the activation of CB2 receptors. BCP upon binding to CB2 receptors
inhibit the enzyme, adenylate cyclase which leads to intracellular Ca2+ transients and
further activates the signaling pathways mediated by Erk1/2 and p38 (Gertsch, 2008a).
As is the case with various known CB2 receptor specific ligands, BCP also impedes
the pathways triggered by the activation of the toll-like receptor (TLR) complex;
CD14/TLR4/MD2, which results in the expression of pro-inflammatory cytokines (IL-
1β, IL-6, IL-8 and TNF-α) and incites TH1 interceded immune response interceded by
CB2 receptor mechanism (Gertsch, 2008b; Gertsch et al., 2008c). Moreover, BCP
exhibited significant anti-inflammatory effects in multiple in vivo models such as
xylene-induced mice ear edema (Okoye et al., 2011), lipopolysaccharides (LPS)-
induced inflammation (Medeiros et al., 2007) and carrageenan-induced rat paw
inflammation (Gertsch et al., 2008c). The anti-inflammatory activity of various plants
including Copaifera multijuga has been associated with the presence of a high amount
50
of BCP (Dias Dde et al., 2012). The essential oil of Hyptis pectinate, which involves
54.07% of BCP was developed in an inclusion complex with β-cyclodextrin and
exhibited intensified analgesic and anti-inflammatory pharmacological effects in
formalin-incited pain protocol in mice (Menezes Pdos et al., 2015). BCP is observed
to be an important natural agent that treats diseases where immune-inflammatory
alterations are the common accompaniment of the pathogenesis of diseases because of
its multimodal anti-inflammatory mechanisms (Gertsch et al., 2008c). Besides, in
addition to the antioxidant and anti-inflammatory activities, BCP exhibited effective
immunomodulatory potential through the inhibition of T-cell immune responses in
mouse primary splenocytes (Ku & Lin, 2013). The repressive effects of BCP on both,
TH1/TH2 cytokines point to the potential possible benefits of BCP in various
autoimmune diseases interceded by CB2 receptors activation and subsequent
inhibition of toll like receptors (Ku & Lin, 2013). The CB2 receptors are mainly found
on the cells of immune origin involving spleen and thymus and also circulating
inflammatory cells including T and B-lymphocytes, natural killer cells, monocytes and
neutrophils, which indicates a significant possible role of BCP in multiple diseases
associated with the modulation of the immune system (Galiègue et al., 1995). As a
result, the specific activation of CB2 receptors by BCP could offer encouraging
therapeutic applications in multiple autoimmune and immune-inflammatory diseases.
51
1.5.2 Chemical properties and SAR of BCP
(E)-BCP is generally found together with small amounts of its isomers (Z)-β
caryophyllene ((Z)-BCP) and α-humulene or in a mixture with its oxidation product,
BCP oxide (Gertsch et al., 2008c). Chemical structures of the bicyclic sesquiterpenes
is manifested in Figure 17.
Quantitative radioligand binding experiments performed by Gertsch et al.
(2008c) revealed that (E)-BCP and its isomer, (Z)-BCP, dose-dependently displaced
[3H]CP55,940 from hCB2 receptors expressed in HEK293 cells with apparent Ki
values in the nM range. (E)-BCP exhibited a slightly higher CB2 receptor binding
affinity (Ki=155 ± 4 nM) than (Z)-BCP (Ki= 485 ± 36 nM). Nevertheless, BCP oxide
and the ring-opened isomer α-humulene did not remove [3H]CP55,940 from the hCB2
receptor (Ki > 20 µM). In line with the data obtained with Cannabis essential oils, BCP
isomers did not exhibit significant binding affinity to the hCB1 receptor.
In silico docking Analysis of the (E)-BCP CB2, Receptor Binding Interaction
was performed by Gertsch et al. (2008c). It indicates that (E)-BCP binds into the
hydrophobic region of the water-accessible cavity. The apparent binding site of CB2
Figure 17: Chemical structures of the bicyclic sesquiterpenes
(Gertsch et al., 2008c)
52
receptor ligands is situated next to helices III, V, VI, and VII at the near extracellular
site of the 7TM bundles, as shown in Figure 18. In this model, the lipophilic (E)-BCP
docks into the hydrophobic cavity of the amphiphatic binding pocket and the binding
mode of (E)-BCP seems to be alleviated by π–π stacking interactions with residues
F117 and W258. Moreover, (E)-BCP closely interacts with the hydrophobic residues
I198, V113, and M265, as shown in Figure 18.
1.5.3 Safety and toxicity of BCP
The FDA has classified BCP as a flavoring substances and it is designated
“generally recognized as safe” by the USFDA and several other regulatory agencies
worldwide for human consumption (Gertsch et al., 2008c). Recently, BCP has been
approved by European Union (EU) for food preservation and adopted in EU for food
additive legislation. The safety profile of BCP has been shown by several in vitro and
in vivo studies revealing low toxicity for humans (Di Sotto et al., 2008; Di Sotto et al.,
Figure 18: In Silico Docking Analysis of the (E)-BCP CB2 Receptor
Binding Interaction (Gertsch et al., 2008c)
53
2013; Di Sotto et al., 2010; LaVoie et al., 1986; Molina-Jasso et al., 2009; Seifried et
al., 2006). Its reported as a safe functional non-psychoactive and a macrocyclic anti-
inflammatory CB2 receptor ligand in foodstuffs (Gertsch, 2008a).
The LD50 of acute oral doses of BCP in rats and the LD50 of acute dermal
doses in rabbits found more than 5000 mg/kg (Opdyke, 1973). BCP in intratracheal
doses (12-48 mg/kg) was found non-toxic to the respiratory system including lungs in
rats. Also, BCP in ointments upon dermal exposure did not found to cause skin
irritation or sensitization in humans at concentrations up to 4% (LaVoie et al., 1986).
Most of findings in literature have reported absence of genotoxic or mutagenic effects
with BCP in preclinical studies (Di Sotto et al., 2008; Di Sotto et al., 2010; Molina-
Jasso et al., 2009).
1.5.4 AM630
AM630 (iodopravadoline) is an aminoalkylindole which act as a CB2 receptor
antagonist. It is commonly used in research to demonstrate the effect, or lack thereof,
of CB2 receptor inhibition (Javed et al., 2016). In this study AM630 was chosen as a
CB2 receptor antagonist to demonstrate the protective effects of BCP mediated by
CB2 receptors. Structure of AM630 is shown in Figure 19.
Figure 19: Structure of AM630 (Javed et al., 2016)
54
1.6 Aims and objectives of the study
A search for novel pharmacotherapy for MI is in progress for last decade. This
is reflected by a large number of studies on natural as well as synthetic compounds to
find out a suitable substitute for those drugs that have been used nowadays to treat
myocardial infarction.
In recent years, the endocannabinoid system consisting of cannabinoid type 1
and 2 receptors (CB1 & CB2) has emerged as an important therapeutic target for
cardioprotection (Pacher et al., 2006). Among the cannabinoid receptors, the CB2
receptor subtype has gained enormous attention as an important therapeutic target due
to its multipharmacological properties and lack of psychoactivity, a common feature
which manifest with cannabinoids (Mukhopadhyay et al., 2010). Among the ligands
which target CB2 receptors, β caryophyllene (BCP), a dietary sesquiterpene garnered
attention being widely found in various essential oils including cinnamon, oregano,
black pepper, basil and cloves (Gertsch et al., 2008c) and pharmacologically showing
a fully selective CB2 receptor agonist activity with a Ki value of 155nmol/L for human
CB2 receptors, with no affinity for CB1 receptors, which causes activation of Gi/Go
subtype of G-proteins (Gertsch et al., 2008c). The wide availability, dietary
accessibility and favourable physicochemical and potent pharmacological properties
makes it an outstanding candidate for therapeutic interventions. The therapeutic
benefits of BCP through the activation of CB2 receptors have been revealed in various
diseases affecting numerous organs such as liver (Calleja et al., 2013), kidney (Horvath
et al., 2012b; Javed et al., 2016) and brain (Javed et al., 2016).
55
Aim 1: To determine the optimal dose of BCP from different doses (25, 50 and 100
mg/kg) in ISO-induced MI model in rats.
• To study the effect of BCP at different doses on cardiomyocyte injury marker
(CK) in the serum, and to determine the optimum effective dose for further
studies.
Aim 2: To investigate the therapeutic potential of natural CB2 receptor agonist, BCP
in ISO induced MI in rats.
• To study the effect of BCP on cardiomyocyte injury markers in serum.
• To study the effect of BCP on hemodynamic measurements.
• To study the effect of BCP on oxidative stress and lipid peroxidation in the
heart.
• To study the effect of BCP on myocardial pro-inflammatory cytokines and
inflammatory mediators.
• To study the effect of BCP on apoptosis in the heart.
• To study the effect of BCP on NF-κB expressions in the myocardium.
• To study the effect of BCP on CB2/PPARγ expressions.
• To study the effect of BCP on structural changes in heart tissue.
• To elucidate the CB2 receptor mediated mechanism of BCP.
• Pretreatment with CB2 receptor antagonist AM630 should abolish/reverse the
cardioprotective effects of BCP.
56
Chapter 2: Materials and Methods
2.1 Experimental animals
Male albino Wistar rats weighing (200-250 g) acquired from the experimental
animal research facility in the College of Medicine and Health Sciences (CMHS),
United Arab Emirates University. Maximum of four rats were housed per cage in
polypropylene cages (47 × 34 × 20 cm3) lined with husk (renewed every 24 h) in a 12‐
hour light/dark cycle at pathogen free environment and controlled temperature around
22°C. Rats were fed a standard rodent chow diet (National Feed and Flour Production
and Marketing Company LLC., Abu Dhabi, UAE) and water ad libitum. The
experimental protocol and procedures for animal experimentation was approved by the
Animal Ethics Committee of United Arab Emirates University.
2.2 Induction of experimental myocardial infarction
Isoproterenol (85 mg/kg body weight) dissolved in saline and injected
subcutaneously to rats at an interval of 24 h for two days (Malik et al., 2011; Ojha et
al., 2011; Ojha et al., 2012; Ojha et al., 2013). ISO-induced MI was confirmed by
increased activities of serum creatine kinase (CK) and lactate dehydrogenase (LDH)
in rats.
2.3 Dose dependent study
A total of 60 rats divided into 10 groups of 6 rats in each group were used in
the dose dependent study. Blood was collected after cervical decapitation; serum was
separated and used for the assay CK to reveal the dose dependent effect of BCP.
57
Group I: Rats were orally given light olive oil in a similar quantity to BCP treatment
and used as a normal control.
Groups II: Rats were orally treated with BCP (100 mg/kg body weight,) dissolved in
light olive oil daily for a period of 10 days.
Group III: Rats were subcutaneously injected with ISO alone (85 mg/kg body weight)
at an interval of 24 h for 2 days (on 9th and 10th day).
Group IV: Rats were orally pre and co-treated with BCP (25 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day).
Group V: Rats were orally pre and co-treated with BCP (50 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day).
Group VI: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day).
Group VI: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day).
Group VII: Rats were orally pre and co-treated with BCP (25 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
58
9th and 10th day). Rats were treated with AM630 (1 mg/kg), a selective
CB2 receptor antagonist prior to the administration of BCP to reveal
the functional CB2 receptor dependent mechanisms of BCP.
Group VIII: Rats were orally pre and co-treated with BCP (50 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day). Rats were treated with AM630 (1 mg/kg), a selective
CB2 receptor antagonist prior to the administration of BCP to reveal
the functional CB2 receptor dependent mechanisms of BCP.
Group IX: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day). Rats were treated with AM630 (1 mg/kg), a selective
CB2 receptor antagonist prior to the administration of BCP to reveal
the functional CB2 receptor dependent mechanisms of BCP.
Group X: Rats were intraperitoneally injected with AM630 (1 mg/kg body weight) for
a period of 10 days.
Assay of creatine kinase:
Serum CK activity was assayed by using VetTest® 8008 Chemistry Analyzer
(IDEXX Laboratories, Hoofddrop, Netherland).
Statistical analysis:
Statistical analysis was performed by one-way analysis of variance (ANOVA)
followed by Duncan's multiple range test (DMRT) using Statistical Package for the
Social Science (SPSS) software package version 12.0. Results were expressed as mean
59
± standard deviation (S.D) for 6 rats in each group. P values <0.05 were considered
significant.
2.4 Experimental design
The animals were randomly divided into six experimental groups each
containing 15 rats. BCP (Sigma-Aldrich, St. LOUIS, MO, USA) was diluted in
scientific grade light olive oil which has been used as a vehicle, based upon previous
research studies. AM630 (Sigma-Aldrich, St. LOUIS, MO, USA) a selective CB2
receptor antagonist, was prepared by using Dimethyl sulfoxide (DMSO) as solvent and
saline as vehicle. ISO (Sigma-Aldrich, St. LOUIS, MO, USA) was used to induce
myocardial infraction. BCP, AM630 and ISO were administered orally,
intraperitoneally and subcutaneously, respectively. All treatments were prepared fresh
just before dosing.
2.5 Experimental groups
The dose of BCP was given as (50 mg/kg body weight) and the dose of ISO
was (85 mg/kg body weight). The dose selection in this experimental protocol was
based in dose dependent study.
Group I: Rats were orally given light olive oil in a similar quantity to BCP treatment
and used as a normal control.
Groups II: Rats were orally treated with BCP (50 mg/kg body weight) dissolved in
light olive oil daily for a period of 10 days.
Group III: Rats were subcutaneously injected with ISO alone (85 mg/kg body weight)
at an interval of 24 h for 2 days (on 9th and 10th day).
60
Group IV: Rats were orally pre and co-treated with BCP (50 mg/kg body weight, BID)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day).
Group V: Rats were orally pre and co-treated with BCP (50 mg/kg body weight, BID)
dissolved in light olive oil daily for a period of 10 days and were
subcutaneously injected with ISO at an interval of 24 h for 2 days (on
9th and 10th day). Rats were treated with AM630 (1 mg/kg), a selective
CB2 receptor antagonist prior to the administration of BCP to reveal
the functional CB2 receptor dependent mechanisms of BCP.
Group VI: Rats were intraperitoneally injected with AM630 (1 mg/kg body weight,
OD) for a period of 10 days.
12 hours after the second dose of ISO injection (i.e. on 11th day), the rats were
anesthetized by pentobarbital sodium (60 mg/kg body weight) and then sacrificed by
cervical decapitation. For serum, blood was collected in dry tubes without
anticoagulant and serum was separated by centrifugation at room temperature for 15-
20 minutes at 3000 rpm. The heart was excised immediately, rinsed in ice chilled saline
then snap frozen in liquid nitrogen and stored at -80°C, for biochemical analysis. Some
hearts were collected and fixed in both 10% neutral buffered formalin and karnovsky
fixative and stored at 4°C for histological studies such as light microscopy and
transition electron microscopy respectively.
2.6 Hemodynamic measurements
The measurement of systolic blood pressure (SP), diastolic blood pressure (DP)
and mean arterial pressure (MAP) were measured by tail cuff method using CODA
61
Non-invasive Blood Pressure System (Kent Scientific Corporation., U.S.A). Heart rate
(bpm) was also measured using the CODATM monitor equipped with a volume
pressure recording sensor. The conscious rats were well placed in a restrainer that was
positioned on a heating pad to maintain the body temperature and establish ample
blood flow to the tails. Each rat was allowed to adjust to the restrainer for 5-7 minutes
and thereafter blood pressure was measured. The volume-pressure recording
determines the tail blood volume using a volume pressure recording sensor and an
occlusion tail-cuff. Twenty continuous cycles were performed and the average values
were used for data analysis as mentioned in the Statistical Analysis section. 15 seconds
between each cycle was programmed. The CODATM monitor system includes a
controller, laptop computer, software, cuffs, animal holders, infrared warming pads
and an infrared thermometer.
2.7 Biochemical estimations
2.7.1 Serological evaluation
Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activity was
assayed by using VetTest® 8008 Chemistry Analyzer (IDEXX Laboratories,
Hoofddrop, Netherland).
2.7.2 Assessment of oxidative stress and lipid peroxidation
The concentrations of heart thiobarbituric acid reactive substances (TBARS),
lipid hydroperoxides (LOOH), superoxide dismutase (SOD), catalase (CAT) and
reduced glutathione (GSH) were estimated.
62
Estimation of heart thiobarbituric acid reactive substances:
The concentration of TBARS in the heart was estimated by the method of
(Fraga et al., 1988).
Reagents:
1. Thiobarbituric acid - 0.38% in hot distilled water.
2. Trichloro acetic acid - 15%.
3. Hydrochloric acid - 0.25 N.
4. Thiobarbituric acid-trichloro acetic acid-hydrochloric acid reagent: Solutions
(1), (2) and (3) were mixed freshly in the ratio of 1:1:1.
5. Stock standard: 4.8 mM: 0.079 ml of 1, 1", 3, 3' tetra methoxy propane was
diluted to 100 ml.
6. Working standard: Stock solution was diluted to get a concentration of 4.8
nM/ml.
Procedure:
The heart tissue homogenate was prepared in Tris-HCL buffer (pH 7.5). 100
l of the tissue homogenate was treated with 200 l of thiobarbituric acid-trichloro
acetic acid-hydrochloric acid reagent and mixed thoroughly. The mixture was kept in
a boiling water bath for 15 minutes. After cooling, the tubes were centrifuged for 10
minutes and the supernatant was taken for measurement. The absorbance was read at
535 nm against the reagent blank. The TBARS values were expressed as mmoles/100g
wet tissue.
63
Estimation of lipid hydroperoxides:
The levels of LOOH in the heart was estimated by the method of (Jiang et al.,
1992).
Reagents:
1. Fox reagent: 88 mg of butylated hydroxy toluene, 7.6 mg of xylenol orange
and 9.8 mg of ammonium iron (II) sulphate were added to 90 ml of methanol
and 10 ml of concentrated sulphuric acid.
2. Standard - 0.2 M, hydrogen peroxide.
Procedure:
180 l of the Fox reagent was mixed with 20 l of the heart
homogenate/plasma and incubated for 30 minutes at room temperature. The colour
developed was read at 560 nm.
The LOOH values were expressed as mmoles/100 g wet tissue for heart.
Assay of superoxide dismutase:
The activity of superoxide dismutase in the heart was assayed according to the
procedure of (Kakkar et al., 1984).
Reagents:
1. Sodium pyrophosphate buffer - 0.025 M, pH 8.3
2. Phenazine methosulphate - 186 M
3. Nitroblue tetrazolium - 300 M
4. Nicotinamide adenine dinucleotide (reduced) - 780 M
5. Glacial acetic acid
6. n-Butanol
64
7. Chloroform
8. Ethanol
Procedure:
20 l of heart tissue homogenate was diluted to 100 l with distilled water.
Then, 250 l of ethanol and 150 l of chloroform (all reagents chilled) were added.
This mixture was shaken for one minute at 4°C and then centrifuged. The enzyme
activity in the supernatant was assayed. The assay mixture contained 120 l of sodium
pyrophosphate buffer (0.025 M, pH 8.3), 10 l of 186 M phenazine methosulphate,
30 l of 30 M nitroblue tetrazolium, 20 l of 780 M nicotinamide adenine
dinucleotide (reduced), appropriately diluted enzyme preparation and distilled water
in a total volume of 300 l. The reaction was started by the addition of nicotinamide
adenine dinucleotide (reduced). After incubation at 30°C for 90 seconds, the reaction
was arrested by the addition of 100 l of glacial acetic acid. The reaction mixture was
stirred vigorously and shaken with 400 l of n-butanol. The intensity of the chromogen
in the butanol layer was measured at 560 nm against a blank containing n-butanol. The
activity of superoxide dismutase was expressed as U/mg protein. One unit of the
enzyme activity is defined as the enzyme concentration required inhibiting the optical
density at 560 nm of chromogen production by 50% in one minute.
Assay of catalase:
The activity of catalase in the heart was assayed by the method of (Sinha,
1972).
Reagents:
1. Phosphate buffer - 0.01 M, pH 7.0.
2. Hydrogen peroxide - 0.2 M.
65
3. Potassium dichromate - 5%.
4. Dichromate-acetic acid reagent: Potassium dichromate and glacial acetic acid
were mixed in the ratio of 1:3. From this, 1.0 ml was diluted again with 4.0 ml
of glacial acetic acid.
5. Standard hydrogen peroxide: 0.1 ml of 0.2 M hydrogen peroxide was diluted
to 100 ml with distilled water.
Procedure:
To 50 l of phosphate buffer, 50 l of heart tissue homogenate and 50 l of
hydrogen peroxide were added. After 60 seconds, 200 l of dichromate-acetic acid
mixture was added. The tubes were kept in a boiling water bath for 10 minutes and the
color developed was read at 620 nm. The activity of catalase was expressed as moles
of hydrogen peroxide consumed/minute/mg protein.
Estimation of reduced glutathione:
The levels of GSH in the heart was estimated by the method of (Ellman, 1959).
Reagents:
1. Phosphate buffer - 0.2 M, pH 8.0.
2. Trichloro acetic acid - 5%.
3. Ellman's reagent: 19.8 mg of dithio nitro bisbenzoic acid in 100 ml of 1%
sodium citrate solution.
4. Disodium hydrogen phosphate - 0.3 M.
5. Standard glutathione solution: 10 mg of reduced glutathione was dissolved in
100 ml of distilled water.
66
Procedure:
A known weight of heart tissue was homogenized in phosphate buffer. From
this, 50 l of tissue homogenate was pipetted out and precipitated with 200 l of 5%
trichloro acetic acid. After centrifugation, 100 l of the supernatant was taken. To this,
50 l of Ellman’s reagent and 300 l of phosphate buffer were added. The yellow color
developed was read at 412 nm. The concentrations of GSH was expressed as mmoles/g
wet tissue for heart.
2.7.3 Assessment of inflammatory markers in cardiac tissue
Heart tissue was homogenized in lysis buffer using Tris–HCl buffer (0.1 M;
pH 7.4) and 1X HaltTM phosphatase inhibitor cocktail (Thermo Fisher Scientific). A
handheld homogenizer (TH tissue homogenizer, OMNI, USA) was used and the
homogenate was centrifuged at 4C for 30 minutes at 12,000 rpm. Supernatant was
collected and used to quantify inflammatory markers via ELISA assay kits for TNF-
, IL-6 and IL1 (R&D Systems, Minneapolis, MN, USA). The assays were carried
out as instructed by the manuals provided in the respective kits.
2.7.4 Western Blot analysis
Protein extractions have been obtained by homogenizing heart samples in an
ice-cold RIPA buffer (Sigma Aldrich, St. Louis, MO, USA). The homogenate was
centrifuged at 4C for 30 minutes at 12,000 rpm. The clear supernatant was mixed with
laemmlli sample buffer (Bio-Rad, USA) along with 2-mercaptoethanol (Sigma
Aldrich, St. Louis, MO, USA). The protein contents in the sample were estimated
using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL,
USA). Samples containing equal amount of proteins has been loaded and separated by
67
gel electrophoresis using SDS polyacrylamide (12%). Then it transferred onto PVDF
membranes (Amersham Hybond P 0.45, PVDF, GE Healthcare Life Sciences,
Germany) and incubated overnight at 4C with primary antibodies against COX-2
(1:500), iNOS (1:2000), (Sigma Aldrich, St. Louis, MO, USA), B-cell lymphoma-2
associated-x (Bax) (1:1000), anti-apoptotic B-cell lymphoma-2 (Bcl-2) (1:1000), B-
cell lymphoma-extra-large (bcl-xL) (1:1000), active caspase-3 (1:500), (Abcam,
Cambridge, MA, USA), CB2 receptor ((1:500) dilution; Abcam, Cambridge, UK)
PPARγ (1:500) (Cell signaling technology, USA), NF-κB-p65 (1:1000), p-NF-κB-p65
(1:1000), cTnI (1:1000) and -actin ((1:2000) dilution; MERCK Millipore, USA) was
used as a loading control. The samples were then incubated with their corresponding
secondary antibodies (anti-rabbit or anti-mouse IgG conjugated to horseradish
peroxidase) for one hour at room temperature and the proteins were visualized by using
an enhanced chemiluminescence pico kit (Thermo Fisher Scientific, Rockford, IL,
USA). The intensity of bands was quantified using Image J software public domain
Java image processing software.
2.8 Histopathological evaluation
After fixation of heart tissue in 10% neutral buffered formalin for a week, the
tissue was gradually dehydrated in increasing concentrations of ethanol, cleared in xylene
and finally embedded in paraffin wax. Sections of 10 µm thickness were cut using a
microtome (RM2125 RTS, Leica Biosystems, Nussloch, Germany) and stained with
hematoxylin and eosin (H&E). The H&E sections were mounted on glass slides and
evaluated under a light microscope (BX41, Olympus).
68
2.9 Transmission electron microscopy (TEM)
Tissue samples from the apex of the heart were fixed in Karnovsky’s fixative for
TEM imaging. After fixation and subsequent washing in buffer solution, samples were
dehydrated in increasing concentration of ethanol. This was followed by treatment with
propylene oxide and staining with osmium tetroxide. Samples were treated with propylene
oxide and resin mixtures before finally being embedded in resin for ultrathin slicing and
examination.
2.10 Statistical analysis
All results are expressed as mean ± standard error of the mean (SEM). Statistical
analysis was done using one-way analysis of variance (ANOVA), followed by the
Duncan's Multiple Range Test (DMRT) using IBM SPSS statistics v24.0. A value of P <
0.05 was considered statistically significant.
69
Chapter 3: Results
3.1 Effect of BCP on serum CK (Dose dependent study)
A pilot study was conducted with three different doses of BCP (25, 50, and 100
mg/kg body weight) to determine its dose dependent effect in ISO induced myocardial
infarcted rats, as shown in Figure 20. ISO injections showed a significant rise in the
serum levels of CK. However, treatment with BCP at all the doses prevented the
leakage of CK into the serum as evidenced by the reduced serum CK levels in the
treatment groups as compared to the ISO group. BCP (50 mg/kg body weight)
treatment showed more promising effect compared to the other doses of BCP on the
activity of serum CK in ISO induced myocardial infarcted rats compared to ISO alone
induced myocardial infarcted rats. BCP (100 mg/kg body weight) alone treated rats
showed no significant effects compared to normal control rats. Since, 50 mg/kg body
weight of BCP showed the highest effect, this dose has been chosen for the further
studies.
70
3.2 Effect of BCP on cardiomyocyte injury marker enzymes in serum
The effects of BCP on the activities of CK and LDH in the serum of normal
and ISO induced rats is presented in Figure 21. ISO injections increased the serum
levels of CK and LDH as compared to the normal control rats. However, treatment
with BCP prevented the injury induced release of CK, and LDH into the serum as
evidenced by the significant (P<0.05) decrease in the levels of these cardiac marker
enzymes in the serum compared to ISO control rats. Interestingly, this protective effect
of BCP was significantly (P<0.05) reversed by prior administration of CB2 receptor
antagonist AM630 to the rats treated with ISO and BCP. Rats treated with AM630
alone did not show any alterations in the levels of CK and LDH as compared to normal
control rats.
Figure 20: Activity of serum CK (dose dependent study). Each column is mean ± SEM
for six rats in each group; *P<0.05 compared to normal control, $P<0.05 compared to
ISO control, #P<0.05 compared to BCP+ISO (DMRT).
71
3.3 Effect of BCP on hemodynamic measurements
The effect of BCP on hemodynamic parameters was presented in Figure 22.
ISO injections to the rats caused a significant (P<0.05) decrease in the heart rate,
systolic, diastolic, and mean arterial pressure compared to the normal control rats. BCP
treatment significantly (P<0.05) restored the heart rate, systolic, diastolic, and mean
arterial pressure compared to ISO control rats. However, treatment with AM630 prior
to the administration of BCP considerably (P<0.05) alters the hemodynamics of rats
administered ISO when compared to ISO control rats. Rats treated with AM630 alone
did not show significant alterations in the hemodynamics of rats.
Figure 21: Effect of BCP on serum (A) CK and (B) LDH. Each column is mean ±
SEM for eight rats in each group; *P<0.05 compared to normal control, $P<0.05
compared to ISO control, #P<0.05 compared to BCP+ISO (DMRT).
72
3.4 Effect of BCP on lipid peroxidation
The levels of lipid peroxidation in the heart of normal and experimental rats is
presented in Figure 23. Rats induced with ISO showed significant (P<0.05) increase
in the levels of lipid peroxidation products in the heart compared to normal control
rats. Treatment with BCP to ISO induced rats near normalized the levels of heart lipid
peroxidation products such as TBARS and LOOH compared to ISO alone induced
rats. The administration of AM630 before BCP treatment significantly abrogated the
effect of BCP on heart TBARS and LOOH. Furthermore, rats treated with AM630
alone did not showed any alterations in the lipid peroxidation parameters compared to
normal control rats.
Figure 22: Effect of BCP on hemodynamics. (A) HR, (B) SP, (C) DP and (D) MAP.
Each column is mean ± SEM for eight rats in each group; *P<0.05 compared to normal
control, $P<0.05 compared to ISO control, #P<0.05 compared to BCP+ISO (DMRT).
73
3.5 Effect of BCP on antioxidants
The activities of the antioxidant enzymes (catalase and SOD) and non-
enzymatic antioxidant (GSH) are presented in Figure 24. Rats challenged with ISO
showed a significant (P<0.05) decrease in the activities/concentrations of SOD,
catalase and GSH compared to normal control rats. However, rats treated with BCP
showed significant (P<0.05) increase in the activities/concentration of SOD, catalase
and GSH compared to ISO control rats. Administration of AM630 before BCP
treatment significantly reduced the impact of BCP in ISO challenged rats.
Furthermore, rats treated with AM630 alone did not showed any alterations in the
above-mentioned parameters compared to normal control rats.
(A) (B)
Figure 23: Effect of BCP on lipid peroxidation status in the heart. (A) TBARS and (B)
LOOH. Each column is mean ± SEM for eight rats in each group; *P<0.05 compared
to normal control, $P<0.05 compared to ISO control, #P<0.05 compared to BCP+ISO
(DMRT).
74
3.6 Effect of BCP on pro-inflammatory cytokines
The effect of BCP on pro-inflammatory cytokine levels is presented in Figure
25. The levels of TNF-α, IL-6, IL-1β were significantly (P<0.05) increased in ISO
induced myocardial infarcted rats compared to normal control rats. BCP treatment
showed significant (P<0.05) decrease in the levels of TNF-α, IL-1β, IL-6 in ISO
induced myocardial infarcted rats compared to ISO alone induced rats. Administration
of AM630 before BCP treatment significantly (P<0.05) decreased the effect of BCP
in ISO-induced MI in rats. Rats treated with AM630 alone did not show any alterations
in the levels/expressions of cytokines and inflammatory mediators compared to normal
control rats.
(A) (B)
(C)
Figure 24: Effect of BCP on endogenous antioxidant enzymes and non-enzymatic
antioxidant. (A) catalase, (B) SOD, (C) GSH in heart tissue. Each column is mean ±
SEM for eight rats in each group; *P<0.05 compared to normal control, $P<0.05
compared to ISO control, #P<0.05 compared to BCP+ISO (DMRT).
75
3.7 Effect of BCP on inflammatory mediators and NF-κB expressions
The expression of iNOS and COX-2 is presented in Figure 26. The expression
of COX-2 and iNOS increased in ISO-challenged rats as compared to control rats.
However, BCP treatment significantly decreased the elevated expression of COX-2
and iNOS as compared to ISO-injected animals. AM630 pretreatment abrogated the
protective effect of BCP with respect to COX-2 expression. The expression of NF-κB
is presented in Figure 26, (A, D). Administration of ISO caused a remarkable increase
in the expression of NF-κB while treatment with BCP significantly reduced NF-κB
expression.
(A) (B)
(C)
Figure 25: Effect of BCP on pro-inflammatory cytokines activities. (A) TNF-α, (B)
IL-6 and (C) IL-1β in the myocardium. Each column is mean ± SEM for eight rats in
each group; *P<0.05 compared to normal control, $P<0.05 compared to ISO control,
#P<0.05 compared to BCP+ISO (DMRT).
76
3.8 Effect of BCP on apoptosis
The effect of BCP on apoptosis is presented in Figure 27. ISO-induced MI in
rats showed a significant (P<0.05) increase in the myocardial protein expressions of
Bax and active caspase-3 along with significant (P<0.05) decrease in the expressions
of Bcl-2 and bcl-xL compared to normal control rats. Treatment with BCP to ISO-
induced MI in rats showed significant (P<0.05) decrease in the expressions of Bax and
active caspase-3 with significant (P<0.05) increase in the expressions of Bcl-2 and bcl-
Figure 26: Effect of BCP on inflammatory mediators in the myocardium. (A)
represents the images of Western immunoblot analysis for iNOS, COX-2, NF-κB-P65
and p-NF-κBP65. (B-D), represents the densitometric analysis of myocardial protein
expressions of (B) iNOS, (C) COX-2, (D) NF-κB-P65 and p-NF-κBP65 assessed by
Western blot analysis. *P<0.05 compared to normal control, $P<0.05 compared to ISO
control, #P<0.05 compared to BCP+ISO (Group-III) (DMRT).
77
xL which in turn was significantly (P<0.05) abrogated by the CB2 receptor antagonist,
AM630. AM630 alone treated rats did not show any alterations in the apoptotic
markers compared to normal control rats.
(D)
Figure 27: Effect of BCP on apoptosis. (A), represents the images of Western
immunoblot analysis for Bax, Bcl2, Bcl-xL and active caspase-3 in the myocardium.
(B-E), represents the densitometric analysis of myocardial protein expressions of (B)
Bax, (C) Bcl2, (D) Bcl-xL and (E) active caspase-3 assessed by Western blot analysis.
*P<0.05 compared to normal control, $P<0.05 compared to ISO control, #P<0.05
compared to BCP+ISO (DMRT).
78
3.9 Effect of BCP on CB2/PPARγ expressions and troponin-I
The effect of BCP on CB2/PPARγ expressions and troponin- I is presented in
Figure 28, (A-D). ISO-induced MI in rats exhibited a significant (P<0.05) decrease in
the myocardial expression of CB2 receptors, PPARγ and troponin-I compared to
normal control rats, whereas BCP treatment showed significant (P<0.05) increase in
the expression of CB2 receptors, PPARγ and troponin-I compared to ISO control rats.
However, treatment with the selective CB2 receptor antagonist, AM630, prior to the
administration of BCP caused a considerable decline in the expression of CB2
receptors, PPARγ and troponin-I in ISO-induced MI in rats. Additionally, rats treated
with AM630 alone showed a subtle insignificant decrease in the expression of CB2
and PPARγ in the myocardium.
79
Figure 28: Effect of BCP on the myocardial protein expressions. (A), represents the
images of Western immunoblot analysis for CB2, PPARγ and troponin-I. (B-D),
represents the densitometric analysis of myocardial protein expressions of (B) CB2,
(C) PPARγ and (D) troponin-I. *P<0.05 compared to normal control, $P<0.05
compared to ISO control, #P<0.05 compared to BCP+ISO (DMRT).
3.10 Effect of BCP on Histopathology of myocardium
Histopathological changes in the heart tissues of all groups were shown in
Figure 29. The heart of normal control rats (i) showed a normal intact muscle with no
edema, and inflammation. BCP alone treated rat's heart (ii) also showing normal intact
muscle fibers without degradation and inflammatory cells. However, rats treated with
ISO (iii) showed extensive muscle fiber degradation with inflammatory cells.
Treatment with BCP to ISO-induced MI in rats (iv) protects the myocardial
(B)
(C)
80
architecture as evidenced by less muscle fiber degradation without inflammatory cells.
Furthermore, treatment with CB2 receptor antagonist AM630 (v) abrogated the
protective effects of BCP against ISO-induced pathological changes in the
myocardium. Treatment with AM630 (vi) did not show any pathological changes
compared to normal control rats.
Figure 29: Effect of BCP on myocardial structure. (i)NOR, (ii) BCP, (iii) ISO,
(iv) ISO+BCP, (v) ISO+BCP+AM630 and (vi) AM630. (X10 magnification)
81
3.11 Effect of BCP on cardiomyocyte ultrastructure (TEM)
The ultrastructure of heart tissue examined by electron microscopy is shown in
Figure 30. The heart of normal and BCP alone treated rats, (i) and (ii), showed intact
myofibrils without mitochondrial damage. However, rats treated with ISO (iii) showed
extensive myofibrillar loss and mitochondrial degeneration. ISO-induced MI in rats
treated with BCP (iv) showed near normal mitochondrial architecture without any
degeneration. Treatment with CB2 receptor antagonist AM630 (v) abrogated the
protective effects of BCP against ISO induced mitochondrial damage as evidenced by
mitochondrial degradation with myofibrillar loss in the myocardium clearly revealed
the functional CB2 receptor dependent mechanism behind the protective effects of
BCP on heart mitochondria. Treatment with CB2 receptor agonist AM630 alone (vi)
did not show any changes in the mitochondrial architecture compared to normal
control rat's heart mitochondria.
82
Figure 30: TEM study on heart mitochondria. (i) NOR, (ii) BCP, (iii) ISO, (iv)
ISO+BCP, (v) ISO+BCP+AM630 and (vi) AM630. (X16,500 Magnification), (↑
Myofibril, ↑ Mitochondria)
83
Chapter 4: Discussion
4.1 Importance and relevance of BCP cardioprotictive effect findings
Myocardial infarction is the irreversible necrosis of heart muscle occurs as a
result of imbalance between coronary blood supply and myocardial demand.
Production of toxic ROS such as O2•− and OH• causes myocardial cell damage (Vaage
& Valen, 1993). Catecholamines undergo auto-oxidation which results in the
generation of highly cytotoxic free radicals (Cohen & Heikkila, 1974), which could
initiate peroxidation of membrane bound PUFAs, leading to both functional and
structural myocardial injury (Thompson & Hess, 1986). In this study, ISO was used to
induce MI in male Wistar rats. The effects of ISO on heart are mediated through B1
and B2 adrenoceptors and they mediate the positive inotropic and chronotropic effects
of b-adrenoceptor agonists (Brodde, 1991). Thus, ISO produces relative ischemia or
hypoxia due to coronary hypotension, myocardial hyperactivity and induce myocardial
ischemia due to overload of cytosolic Ca2+ (Senthil et al., 2007). The toxic dosage of
ISO causes characteristic myocardial damage that subsequently leads to heart failure
(Grimm et al., 1998).
Among the many new therapeutic targets, the endogenous cannabinoid system,
which comprises cannabinoid ligands and cannabinoid type 1 (CB1) and type 2 (CB2)
receptors represents one of the newest and most promising drug targets. Among
cannabinoid receptors, CB2 receptors are G-protein coupled receptors that, upon
activation, induce members of the MAPK family, which trigger expression of genes
including those involved in stress response, inflammation, cell survival or proliferation
(Howlett, 2005). CB2 receptor activation is also associated with nuclear translocation
of the transcription factor NF-κB (Derocq et al., 2000). In contrast to CB1 receptors,
84
CB2 receptor activation does not modulate ion channel function (Bosier et al., 2010).
As a result, CB2 receptor mediated Ca2+ responses are less pronounced (Schuehly et
al., 2011) than the potent CB1 receptor-mediated effects on Ca2+ fluxes (Bosier et al.,
2010). Cardioprotective effects of endocannabinoid-mediated CB2 receptor activation
were first reported in LPS-induced preconditioning (Lagneux & Lamontagne, 2001).
Thereafter, several recent reports using synthetic CB2 receptor agonists in vitro, ex
vivo or in vivo showed protective effects on remote preconditioning, infarct size,
arrhythmias and counterbalanced chronic heart failure-induced structural changes,
inhibited atherogenesis and prevented myocyte enlargement (Horvath et al., 2012b;
Montecucco et al., 2009; Ramirez et al., 2012; Steffens et al., 2005; Weis et al., 2010;
Zarruk et al., 2012). The anti-inflammatory effects of CB2 receptor activation in the
endothelium, and its inhibitory effect on monocytes/macrophages and/or leukocyte
migration were diminished by pharmacological antagonism of CB2 receptors with
AM630 (Zhao et al., 2010). Pretreatment with the CB2 receptor antagonist AM630 or
SR144528 abolished the cardioprotective effects and reversed cardioprotection. CB2
receptors have also been implicated in the modulation of endoplasmic reticulum stress
and immune cell migration (Miller & Stella, 2008) involving the PI3K/Akt and
ERK1/2 pathways. Many studies with synthetic cannabinoid ligands of CB2 receptor
paved the way for CB2 receptor activation as a strategy in cardioprotection.
In the present study, the cardioprotective effect of BCP has been studied, a
dietary phytocannabinoid that has attracted attention after its approval by European
regulatory agencies and the USFDA for use as food additive and flavoring agent.
Chemically, BCP is a bicyclic sesquiterpene and pharmacologically, it is a selective
CB2 receptor agonist. It is found abundantly in various flowering plants and spices
which makes it one of the more widely available and accessible agents devoid of
85
psychoactive properties (Gertsch et al., 2008c). It has been recognized as a key
ingredient in several traditional Chinese, European, Eastern and Indian medicines and
is reported to possess a wide range of potent biological activities, including potent anti-
inflammatory, antioxidant and analgesic effects. BCP’s structure contains a
cyclobutane ring which provides a potent and rigid geometry to this molecule for
binding to cannabinoid receptors and largely contributes to BCP’s anti-inflammatory
properties. The therapeutic benefits of BCP in inflammation has been shown to be
mediated by potent CB2 receptor activation (Gertsch, 2008a). Additionally, the
neuroprotective effects of BCP in Parkinson’s disease, cerebral ischemia, epilepsy,
Alzheimer’s disease, depression, anxiety and addiction have been reported recently
(Donati et al., 2015; Sain et al., 2014).
To the best of my knowledge, this is the first study to report the
cardioprotective effects of BCP against ISO-induced MI via activation of CB2
receptors in rats. It has been found that ISO administration downregulates the
activation of myocardial CB2 receptors which proves that CB2 receptors activation is
an important strategy to safeguard the myocardium against MI. BCP, natural CB2
receptor agonist upregulates the CB2 receptors activation and protects the myocardium
against ISO-induced MI in rats. The crosstalk between CB2 and PPARγ pathways has
been demonstrated recently in which the binding of natural and synthetic cannabinoids
to PPARγ has been demonstrated (Burstein, 2005; Meeran et al., 2019). This study has
revealed that ISO-challenge downregulates the expression of PPARγ whereas BCP
treatment unregulates its expression in ISO-induced MI in rats. Upregulation of
PPARγ along with the expression of CB2 receptors clearly revealed the CB2/PPARγ
receptor mediated cardioprotection offered by BCP against ISO-induced MI in rats.
86
The myocardium contains plentiful concentrations of diagnostic markers of MI
and once metabolically damaged, it releases its contents into the extra cellular fluid
(Upaganlawar et al., 2009). Serum CK-MB, a golden standard diagnostic marker of
MI along with CK, LDH and troponins are the other diagnostic markers of myocyte
injury or death are found to be increased in ISO induced rats. Due to poor oxygen or
glucose supply the myocardial cells are damaged making the cardiac membrane
permeable resulting in the leakage of these enzymes. These enzymes enter into the
blood stream thus increasing their concentration in the serum (Mathew, Menon &
Kurup, 1985). The activities of CK, LDH and troponin- I are declined in the heart
tissue of ISO induced rats. This might be due to the damage caused to the sarcolemma
by the β-agonist, which has rendered it leaky (Mathew et al., 1985). BCP defends the
myocardium by inhibiting the leakage of cardiac diagnostic marker enzymes into the
circulation in ISO induced myocardial infarcted rats by virtue of its potent membrane
stabilizing property. The protective effect of BCP was abrogated by the administration
of CB2 antagonist AM630 has clearly revealed the CB2 receptor dependent
mechanism behind the protective effects of BCP against MI in rats.
Administration of ISO induces impaired hemodynamics (decreased SP, DP,
MAP and heart rate) followed by the left ventricular dysfunction (Ojha et al., 2008).
Alterations in hemodynamics after ISO injections reflects systolic and diastolic
dysfunction in rats (Malik et al., 2011). BCP treatment reinstates normal heart rate and
hemodynamics in ISO-induced MI in rats whereas the protective effects of BCP were
attenuated by the prior administration of CB2 antagonist AM630. Thus, BCP protects
the myocardium in a CB2 dependent mechanism.
Lipid peroxidation is an important pathogenic event that has been linked to
altered membrane structure and enzyme inactivation in MI. Lipid peroxidation of
87
membranes is regulated by the availability of inducers such as free radicals, and the
increased levels of lipid peroxides in ISO induced damage might be due to free radical-
mediated chain reactions that could damage the myocardium. It is an indication of the
severity of ISO induced necrotic damage of the heart (Albayrak et al., 2009). The
degree of lipid peroxidation was reflected by increased levels of TBARS, LOOH and
conjugated dienes in the plasma and heart of ISO induced myocardial infarcted rats.
An increase in the lipid peroxidation products level injures blood vessels, thereby
increasing adherence and platelets collection in the injured sites. Pre and co-treatment
with BCP near normalized excess lipid peroxidation products level in ISO induced
rats. Hence, BCP scavenges the excess lipid peroxidation products generated by ISO
and safeguards the heart by its anti-lipid peroxidation effect. The protective effect of
BCP was abrogated by the administration of CB2 antagonist AM630 which has clearly
revealed the CB2 receptor dependent mechanism behind the protective effects of BCP
against MI in rats.
Pharmacological augmentation of endogenous myocardial antioxidants has
been identified as a promising therapeutic approach in diseases associated with
increased oxidative stress. Endogenous antioxidant enzymatic defense is a very
important source to neutralize the oxygen free radical-mediated tissue injury
(Saravanan et al., 2013). Superoxide dismutase and catalase, the primary free radical
scavenging enzymes, are the first line of cellular defense against oxidative injury,
decomposing oxygen and hydrogen peroxide before their interaction to form the more
reactive OH•. In this study, significantly lowered activities of superoxide dismutase
and catalase were observed in heart of ISO induced rats. The observed decrease in the
activities of these enzymes might be due to their increased utilization for scavenging
ROS and their inactivation by excessive ISO oxidants. Treatment with BCP improved
88
the activities of superoxide dismutase and catalase by scavenging O2•− and OH•
produced by ISO. The protective effect of BCP was abrogated by the administration
of CB2 antagonist AM630 which has clearly revealed the CB2 receptor dependent
mechanism behind the protective effects of BCP against MI in rats.
GSH level is commonly used marker for oxidative stress. The decreased GSH
levels during ISO induction inactivate GSH related enzymes (Sathish et al., 2002)
which are responsible for degrading hydrogen peroxide and O2•− during ischemia or
injury and significantly reduce the activities of GPx and GST (Kumaran & Prince,
2010b). GSH is important in protecting the myocardium against oxygen free radical
injury and thus a reduction in cellular glutathione content could impair recovery after
short period of ischemia (Saravanan & Prakash, 2004). The observed decrease in GSH
levels might be due to increased utilization in protecting thiol ROS oxygen species.
Treatment with BCP near normalized the activities/concentrations of GSH in ISO
induced rats. This shows the antioxidant property of BCP. The protective effect of
BCP was abrogated by the administration of CB2 antagonist AM630 has clearly
revealed the CB2 receptor dependent mechanism behind the protective effects of BCP
against MI in rats.
Mediators of myocardial inflammation predominantly cytokines in ISO
induced MI were evaluated, because cytokines have been implicated in the healing
process after infarction. Pro-inflammatory cytokines, namely TNF-α, IL-6 and IL-1β
are elevated in acute myocardial injury and infarction. (Cusack et al., 2002) reported
that the cytokines have been implicated in the initiation and maintenance of vascular
and systemic inflammation associated with the coronary artery disease. Furthermore,
pro-inflammatory cytokines alter proliferation of cells, collagen extra-cellular matrix
production, and generation of mediator substances by the cardiac fibroblast (Koudssi
89
et al., 1998). Pro-inflammatory cytokines have been implicated as pathological cardiac
remodeling mediators with evidence linking these molecules to heart failure
progression in chronic heart failure (Ono et al., 1998). In coronary heart disease, TNF-
α is a relevant cytokine in the course of the inflammation process and affects a number
of mediators of the pathological process involved in MI. Recently, cardio myocytes
have been shown to be a main source of TNF-α production in conditions such as MI
and heart failure (Kannan & Quine, 2011a).
TNF-α inhibits contractility of myocardium, induces cardio myocyte apoptosis
and acts directly on cardio myocytes as well as the vascular endothelium to increase
the adhesion of leukocytes during inflammation (Sharma & Das, 1997). Moreover, it
is thought to contribute to the progression of the atheroma by augmenting the local
inflammatory response (Olivieri et al., 2006).
IL-6 is another proinflammatory cytokine involved in cardiodepression. There
is evidence that in patients suffering from acute MI, IL-6 may affect the progression
and the healing process of this CVDs, because serum IL-6 levels to be elevated in these
myocardial infarcted patients (Ikeda et al., 1992). IL-6 is predominantly produced by
myocardial fibroblasts, although it has also been reported to be produced by
cardiomyocytes as a result of myocardial ischemia or cardiotoxicity by beta-adrenergic
agonists. Also, IL-6 up-regulation is an early and important phenomenon in ISO-
induced MI (Mikaelian et al., 2008).
Interleukin-1 is a multifunctional cytokine, primarily involved in the regulation
of inflammatory process. Two genes are expressed for IL-1: IL-1α and IL-1β. IL-1β
gene is one of the key pro-inflammatory cytokines because its elevated levels are
correlated with disease severity. An enhanced level of IL-1β mRNAs in ischemic
90
myocardium indicates the role of cytokine in myocardial inflammation (Sharma &
Zimmerman, 1993).
Higher expressions of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β)
has been observed in the myocardium of ISO-induced myocardial infarcted rats. The
observed increased expression of these proinflammatory cytokines mediates
inflammation in the myocardium thereby reducing cardiac muscle function and
extensive damage to the myocardium (Kannan & Quine, 2011b). The present study
confirmed that treatment with BCP downregulated the expression of pro-inflammatory
cytokines in the myocardium of ISO-induced myocardial infarcted rats, thereby
inhibiting inflammation by its anti-inflammatory effect. The protective effect of BCP
was abrogated by the administration of CB2 antagonist AM630 has clearly revealed
the CB2 receptor dependent mechanism behind the protective effects of BCP against
MI in rats.
Although the inflammatory response considered as a defense mechanism
against infection or injury, sustained inflammation is a pathological condition. Over
expression of several pro-inflammatory enzymes and reactive species like superoxide
(O2•−) and nitric oxide (•NO) radicals, are produced during inflammation (Okoli &
Akah, 2004). Nitric oxide is formed from an oxygen and L-arginine by inducible nitric
oxide synthase (iNOS), this enzyme become up-regulated during the inflammatory
process (Tayeh & Marletta, 1989).
Prostaglandins (PGs) is a group of physiologically active mediators which also
over-expressed during inflammation. Prostaglandins are bioactive signaling molecules
derived from cyclooxygenase (COX) and subsequent PG synthase activity on
arachidonic acid. Like iNOS, COX-2 is highly up-regulated in response to infection,
atherosclerosis and a number of cancers (Simmons et al., 2004). It plays a critical role
91
in the mediation of inflammation, and catalyzes the rate limiting step in prostaglandin
biosynthesis. PG synthases will form important signaling molecules, including PGI2,
thromboxane A2, PGE2, PGD2 and PGF2α (Buczynski, Dumlao & Dennis, 2009). The
up-regulation of iNOS and COX-2 during inflammation is controlled by NF-κB which
is a pro-inflammatory transcription factor (Kim et al., 2008; Raso et al., 2001).
BCP treatment reduced COX-2, iNOS and proinflammatory cytokine levels.
This reduction in proinflammatory cytokines seems to be in line with the results of
other studies conducted with BCP (Cho et al., 2015; Horvath et al., 2012a). The
protective effect of BCP was abrogated by the administration of CB2 antagonist
AM630 which has clearly revealed the CB2 receptor dependent mechanism behind the
protective effects of BCP against MI in rats. CB2 receptors are expressed in immune
cells and many studies have shown that the CB2 receptor plays a role in
immunomodulation (via both endocannabinoids and cannabinoids) (Turcotte et al.,
2016). However the exact mechanisms and pathways are still not fully understood or
known (Pacher & Mechoulam, 2011).
Cardiomyocyte apoptosis produces hypoxia and ischemia after increased
myocardial contractions during β1-adrenergic receptor activation (Nagoor Meeran et
al., 2019). Bax activation due to the overproduction of ROS plays a major role in
promoting apoptosis by creating pore in the mitochondria which leads to the release of
mitochondrial cytochrome-C into the cytosol (Sahu et al., 2014). CHOP is known to
down regulate the expression of Bcl2 and triggers apoptosis through cytochrome-C
release by inhibiting mitochondrial Bcl2 family proteins and by creating mitochondrial
permeability transition pore (MPTP) opening (Roy et al., 2009). Mst1 triggers
apoptosis by inhibiting YAP by promoting the phosphorylation of LATS1/2. During
ischemic conditions under severe oxidative stress, activation of MST1 activates
92
intrinsic pathway of apoptosis by dissociating Bcl-xL from Bax through Bcl-xL
phosphorylation (Nakamura et al., 2016). Released cytochrome-C activates
executioner caspases by the formation of apoptosomes (Radhiga et al., 2012).
Isoproterenol activates intrinsic pathway of apoptosis by releasing cytochrome-C
followed by the activation of caspases whereas BCP treatment protects the
myocardium against ISO induced apoptotic cell death of the myocardium. The
antagonistic effects of AM630 against the anti-apoptotic effects of BCP clearly
revealed the functional CB2 receptor dependent mechanisms behind the anti-apoptotic
properties of BCP against ISO induced myocardial apoptosis in rats.
ISO causes myocardial injury which is characterized by various morphological
features which include necrosis, myocardial hypertrophy, blood capillary distortion,
and interstitial edema (Al-Rasheed et al., 2015). The histopathological findings show
extensive muscle degradation in ISO induced myocardial infarcted heart, which shows
necrosis, edema, and inflammatory cells. The histopathological findings of BCP
treated ISO induced myocardial infarcted heart displayed a near normal morphology
of cardiac muscle without necrosis, edema and inflammatory cells as compared to ISO
induced myocardial infarcted heart. The histopathology of BCP treated myocardium
established the protective nature of BCP in ISO induced rats.
BCP could be used for nutritional supplementation over other
phytocannabinoids due to its unique properties as being natural, dietary, readily
bioavailable, non-psychoactive and safe with a wide presence in numerous plants.
Translating the outcomes in humans would be promising as these agents have not only
shown the ability to activate CB2 receptors only, but they also activate PPAR-γ, which
are the target of thiazolidinedione class of drugs that used clinically to treat diabetes.
BCP is of more therapeutic value than the other phytocannabinoids due to it being
93
found in plants other than cannabis which may help in the legal logistics of its use.
CB2 receptor selective compounds may provide a new potential class of
cardioprotective drugs. The pharmacophore of these compounds could be used for
synthesizing leads in drug discovery and development.
94
Chapter 5: Conclusion
The present study revealed that BCP, a plant-derived sesquiterpene have
pharmacotherapeutic significance due to its multi pharmacological properties.
Activation of CB2 receptors is the major underlying mechanism behind the
cardioprotective property of BCP which has shown to attenuate oxidative stress,
inflammation and apoptosis in ISO induced MI.
This study is the first study so far that report the cardioprotective effects of
BCP against ISO-induced MI via activation of CB2 receptors in rats. I observed that
ISO administration down-regulates the activation of myocardial CB2 receptors which
proves that CB2 receptors activation is an important strategy to safeguard the
myocardium against MI. BCP also demonstrated a positive effect by reducing
oxidative stress, inflammation and inflammatory mediators, apoptosis along with
reducing the serum levels of the cardiomyocyte injury marker enzymes CK and LDH.
The histological and ultra-structural evidences are found in line with the biochemical
and molecular findings. Treatment with AM630, a potent CB2 receptor antagonist
abrogates protective effects of BCP on all the biochemical and molecular parameters
analyzed in ISO-induced MI in rats. The present study findings clearly demonstrate
that BCP may be an attractive natural compound which can bestow protective effects
to the myocardium via CB2 receptor-dependent manner against ISO induced MI.
Furthermore, BCP could be the first plant-derived CB2 receptor agonist in the quest
for development of novel class of drugs that might improve conventional therapies as
well as provide novel disease modifying agents.
95
References
Abou-Ghali, M., & Stiban, J. (2015). Regulation of Ceramide Channel Formation and
Disassembly: Insights on the Initiation of Apoptosis. Saudi Journal of
Biological Sciences, 274, 760-772.
Al-Rasheed, N. M., Al-Oteibi, M. M., Al-Manee, R. Z., Al-Shareef, S. A., Hasan, I.
H., Mohamad, R. A., & Mahmoud, A. M. (2015). Simvastatin prevents
isoproterenol-induced cardiac hypertrophy through modulation of the
JAK/STAT pathway. Drug Des. Devel. Ther., 2015:9, 3217-3229.
Albayrak, F., Bayir, Y., Halici, Z., Kabalar, E., Bayram, E., Ozturk, C., Bakan, E.
(2009). Preventive effect of amiodarone during acute period in isoproterenol-
induced myocardial injury in Wistar rats. Cardiovasc. Toxicol., 9(4), 161-168.
Albert, C. M., Hennekens, C. H., O'Donnell, C. J., Ajani, U. A., Carey, V. J., Willett,
W. C., Manson, J. E. (1998). Fish consumption and risk of sudden cardiac
death. Jama, 279(1), 23-28.
Arora, G., & Bittner, V. (2015). Chest pain characteristics and gender in the
early diagnosis of acute myocardial infarction. Curr. Cardiol. Rep.,
17(2), 5. doi: 10.1007/s11886-014-0557-5.
Assis, L. C., Straliotto, M. R., Engel, D., Hort, M. A., Dutra, R. C., & de Bem, A. F.
(2014). β-Caryophyllene protects the C6 glioma cells against glutamate-
induced excitotoxicity through the Nrf2 pathway. Neuroscience, 279, 220-231.
Astani, A., Reichling, J., & Schnitzler, P. (2011). Screening for antiviral activities of
isolated compounds from essential oils. Evid. Based Complement. Alternat.
Med., Vol 2011, 253643. doi:10.1093/ecam/nep187.
Babu, R. O., Moorkoth, D., Azeez, S., & Eapen, S. J. (2012). Virtual screening and in
vitro assay of potential drug like inhibitors from spices against Glutathione-S-
Transferase of Meloidogyne incognita. Bioinformation, 8(7), 319-325.
Baldi, A., Abbate, A., Bussani, R., Patti, G., Melfi, R., Angelini, A., Di Sciascio, G.
(2002). Apoptosis and post-infarction left ventricular remodeling. J Mol. Cell.
Cardiol., 34(2), 165-174.
Ben-Shabat, S., Fride, E., Sheskin, T., Tamiri, T., Rhee, M. H., Vogel, Z., Mechoulam,
R. (1998). An entourage effect: inactive endogenous fatty acid glycerol esters
enhance 2-arachidonoyl-glycerol cannabinoid activity. Eur. J Pharmacol.,
353(1), 23-31.
96
Bento, A. F., Marcon, R., Dutra, R. C., Claudino, R. F., Cola, M., Leite, D. F., &
Calixto, J. B. (2011). beta-Caryophyllene inhibits dextran sulfate sodium-
induced colitis in mice through CB2 receptor activation and PPARgamma
pathway. Am. J Pathol., 178(3), 1153-1166.
Bisogno, T., Melck, D., Bobrov, M., Gretskaya, N. M., Bezuglov, V. V., De
Petrocellis, L., & Di Marzo, V. (2000). N-acyl-dopamines: novel synthetic
CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation
with cannabimimetic activity in vitro and in vivo. Biochem. J, 351, 817-824.
Bosier, B., Muccioli, G. G., Hermans, E., & Lambert, D. M. (2010). Functionally
selective cannabinoid receptor signalling: therapeutic implications and
opportunities. Biochem. Pharmacol., 80(1), 1-12.
Bouchard, J. F., Lépicier, P., & Lamontagne, D. (2003). Contribution of
endocannabinoids in the endothelial protection afforded by ischemic
preconditioning in the isolated rat heart. Life Sci., 72(16), 1859-1870.
Brodde, O. E. (1991). Beta 1- and beta 2-adrenoceptors in the human heart: properties,
function, and alterations in chronic heart failure. Pharmacol. Rev., 43(2), 203-
242.
Buczynski, M. W., Dumlao, D. S., & Dennis, E. A. (2009). Thematic Review Series:
Proteomics. An integrated omics analysis of eicosanoid biology. J Lipid Res.,
50(6), 1015-1038.
Budavari S, O. N. M., Smith A, Heckelman P, Obenchain J. (1996). The Merck Index,
Print Version. (12th Ed.). USA: CRC Press.
Bujak, M., Dobaczewski, M., Chatila, K., Mendoza, L. H., Li, N., Reddy, A., &
Frangogiannis, N. G. (2008). Interleukin-1 receptor type I signaling critically
regulates infarct healing and cardiac remodeling. Am J Pathol, 173(1), 57-67.
Burstein, S. (2005). PPAR-gamma: a nuclear receptor with affinity for cannabinoids.
Life Sci, 77(14), 1674-1684.
Buttar, H. S., Li, T., & Ravi, N. (2005). Prevention of cardiovascular diseases: Role of
exercise, dietary interventions, obesity and smoking cessation. In Exp. Clin.
Cardiol., 10(4), 229-249.
Cabaniss, C. D. (1990). Clinical Methods: The History, Physical, and Laboratory
Examinations (3rd Ed.), Boston: Butterworths, 876-877.
Calleja, M. A., Vieites, J. M., Montero-Melendez, T., Torres, M. I., Faus, M. J., Gil,
A., & Suarez, A. (2013). The antioxidant effect of beta-caryophyllene protects
rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic
stellate cell activation. Br. J. Nutr., 109(3), 394-401.
97
Capasso, R., Borrelli, F., Cascio, M. G., Aviello, G., Huben, K., Zjawiony, J. K., Izzo,
A. A. (2008). Inhibitory effect of salvinorin A, from Salvia divinorum, on
ileitis-induced hypermotility: cross-talk between kappa-opioid and
cannabinoid CB(1) receptors. Br. J. Pharmacol., 155(5), 681-689.
Chagoya de Sanchez, V., Hernandez-Munoz, R., Lopez-Barrera, F., Yanez, L., Vidrio,
S., Suarez, J., Cruz, D. (1997). Sequential changes of energy metabolism and
mitochondrial function in myocardial infarction induced by isoproterenol in
rats: a long-term and integrative study. Can. J. Physiol. Pharmacol., 75(12),
1300-1311.
Chicca, A., Caprioglio, D., Minassi, A., Petrucci, V., Appendino, G., Taglialatela-
Scafati, O., & Gertsch, J. (2014). Functionalization of β-caryophyllene
generates novel polypharmacology in the endocannabinoid system. ACS
Chem. Biol., 9(7), 1499-1507.
Cho, J. Y., Kim, H. Y., Kim, S. K., Park, J. H. Y., Lee, H. J., & Chun, H. S. (2015).
beta-Caryophyllene attenuates dextran sulfate sodium-induced colitis in mice
via modulation of gene expression associated mainly with colon inflammation.
Toxicol. Rep., 2, 1039-1045.
Cohen, G., & Heikkila, R. E. (1974). The generation of hydrogen peroxide, superoxide
radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related
cytotoxic agents. J. Biol. Chem., 249(8), 2447-2452.
Contassot, E., Tenan, M., Schnuriger, V., Pelte, M. F., & Dietrich, P. Y. (2004).
Arachidonyl ethanolamide induces apoptosis of uterine cervix cancer cells via
aberrantly expressed vanilloid receptor-1. Gynecol. Oncol., 93(1), 182-188.
Cusack, M. R., Marber, M. S., Lambiase, P. D., Bucknall, C. A., & Redwood, S. R.
(2002). Systemic inflammation in unstable angina is the result of myocardial
necrosis. J. Am. Coll. Cardiol., 39(12), 1917-1923.
Derocq, J. M., Jbilo, O., Bouaboula, M., Segui, M., Clere, C., & Casellas, P. (2000).
Genomic and functional changes induced by the activation of the peripheral
cannabinoid receptor CB2 in the promyelocytic cells HL-60. Possible
involvement of the CB2 receptor in cell differentiation. J. Biol. Chem.,
275(21), 15621-15628.
Devane, W. A., Hanus, L., Breuer, A., Pertwee, R. G., Stevenson, L. A., Griffin, G.,
Mechoulam, R. (1992). Isolation and structure of a brain constituent that binds
to the cannabinoid receptor. Science, 258(5090), 1946-1949.
Di Sotto, A., Evandri, M. G., & Mazzanti, G. (2008). Antimutagenic and mutagenic
activities of some terpenes in the bacterial reverse mutation assay. Mutat. Res.,
653(1-2), 130-133.
98
Di Sotto, A., Maffei, F., Hrelia, P., Castelli, F., Sarpietro, M. G., & Mazzanti, G.
(2013). Genotoxicity assessment of beta-caryophyllene oxide. Regul. Toxicol.
Pharmacol., 66(3), 264-268.
Di Sotto, A., Mazzanti, G., Carbone, F., Hrelia, P., & Maffei, F. (2010). Inhibition by
beta-caryophyllene of ethyl methanesulfonate-induced clastogenicity in
cultured human lymphocytes. Mutat. Res., 699(1-2), 23-28.
Dias Dde, O., Colombo, M., Kelmann, R. G., De Souza, T. P., Bassani, V. L., Teixeira,
H. F., Koester, L. S. (2012). Optimization of headspace solid-phase
microextraction for analysis of beta-caryophyllene in a nanoemulsion dosage
form prepared with copaiba (Copaifera multijuga Hayne) oil. Anal. Chim.
Acta., 721, 79-84.
Dinarello, C. A. (1996). Biologic basis for interleukin-1 in disease. Blood, 87(6), 2095-
2147.
Donati, M., Mondin, A., Chen, Z., Miranda, F. M., do Nascimento, B. B., Jr., Schirato,
G., Froldi, G. (2015). Radical scavenging and antimicrobial activities of Croton
zehntneri, Pterodon emarginatus and Schinopsis brasiliensis essential oils and
their major constituents: estragole, trans-anethole, beta-caryophyllene and
myrcene. Nat. Prod. Res., 29(10), 939-946.
Doyle, B., Fitzsimons, D., McKeown, P., & McAloon, T. (2012). Understanding
dietary decision-making in patients attending a secondary prevention clinic
following myocardial infarction. J. Clin. Nurs., 21(1-2), 32-41.
Duerr, G. D., Heinemann, J. C., Suchan, G., Kolobara, E., Wenzel, D., Geisen, C.,
Dewald, O. (2014). The endocannabinoid-CB2 receptor axis protects the
ischemic heart at the early stage of cardiomyopathy. Basic Res. Cardiol.,
109(4), 425. doi: 10.1007/s00395-014-0425-x.
Duncan, M., Millns, P., Smart, D., Wright, J. E., Kendall, D. A., & Ralevic, V. (2004).
Noladin ether, a putative endocannabinoid, attenuates sensory
neurotransmission in the rat isolated mesenteric arterial bed via a non-
CB1/CB2 G(i/o) linked receptor. Br. J. Pharmacol., 142(3), 509-518.
Eisenstein, T. K., & Meissler, J. J. (2015). Effects of Cannabinoids on T-cell Function
and Resistance to Infection. J. Neuroimmune Pharmacol., 10(2), 204-216.
Ellman, G. L. (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82(1), 70-
77.
Fabricius-Bjerre, N., Munkvad, M., & Knudsen, J. B. (1979). Subendocardial and
transmural myocardial infarction: a five-year survival study. Am. J. Med.,
66(6), 986-990.
99
Felder, C. C., Joyce, K. E., Briley, E. M., Mansouri, J., Mackie, K., Blond, O.,
Mitchell, R. L. (1995). Comparison of the pharmacology and signal
transduction of the human cannabinoid CB1 and CB2 receptors. Mol.
Pharmacol., 48(3), 443-450.
Fichna, J., Schicho, R., Andrews, C. N., Bashashati, M., Klompus, M., McKay, D. M.,
Storr, M. A. (2009). Salvinorin A inhibits colonic transit and neurogenic ion
transport in mice by activating kappa-opioid and cannabinoid receptors.
Neurogastroenterol. Motil., 21(12), 1326-e1128.
Fidyt, K., Fiedorowicz, A., Strzadala, L., & Szumny, A. (2016). beta-caryophyllene
and beta-caryophyllene oxide-natural compounds of anticancer and analgesic
properties. Cancer Med., 5(10), 3007-3017.
Fine, P. G., & Rosenfeld, M. J. (2013). The endocannabinoid system,
cannabinoids, and pain. Rambam Maimonides Med. J., 4(4), e0022.
doi: 10.5041/RMMJ.10129.
Fraga, C. G., Leibovitz, B. E., & Tappel, A. L. (1988). Lipid peroxidation measured
as thiobarbituric acid-reactive substances in tissue slices: characterization and
comparison with homogenates and microsomes. Free Radic. Biol. Med., 4(3),
155-161.
Galiègue, S., Mary, S., Marchand, J., Dussossoy, D., Carrière, D., Carayon, P.,
Casellas, P. (1995). Expression of central and peripheral cannabinoid receptors
in human immune tissues and leukocyte subpopulations. Eur. J. Biochem.,
232(1), 54-61.
Garfein, O. B. (1990). Current Concepts in Cardiovascular Physiology (1st Ed.),
Academic Press.
Geng, B., Chang, L., Pan, C., Qi, Y., Zhao, J., Pang, Y., Tang, C. (2004). Endogenous
hydrogen sulfide regulation of myocardial injury induced by isoproterenol.
Biochem. Biophys. Res. Commun., 318(3), 756-763.
Gertsch, J. (2008a). Anti-inflammatory cannabinoids in diet: Towards a better
understanding of CB(2) receptor action. Commun. Integr. Biol., 1(1), 26-28.
Gertsch, J. (2008b). Immunomodulatory lipids in plants: plant fatty acid amides and
the human endocannabinoid system. Planta. Med., 74(6), 638-650.
Gertsch, J., Leonti, M., Raduner, S., Racz, I., Chen, J. Z., Xie, X. Q., Zimmer, A.
(2008c). Beta-caryophyllene is a dietary cannabinoid. Proc. Natl. Acad. Sci.
USA, 105(26), 9099-9104.
Gertsch, J., Pertwee, R. G., & Di Marzo, V. (2010). Phytocannabinoids beyond the
Cannabis plant - do they exist? Br. J. Pharmacol., 160(3), 523-529.
100
Gonsiorek, W., Lunn, C., Fan, X., Narula, S., Lundell, D., & Hipkin, R. W. (2000).
Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type
2 cannabinoid receptor: antagonism by anandamide. Mol. Pharmacol., 57(5),
1045-1050.
Grandel, U., Fink, L., Blum, A., Heep, M., Buerke, M., Kraemer, H. J., Sibelius, U.
(2000). Endotoxin-induced myocardial tumor necrosis factor-alpha synthesis
depresses contractility of isolated rat hearts: evidence for a role of sphingosine
and cyclooxygenase-2-derived thromboxane production. Circulation, 102(22),
2758-2764.
Grimm, D., Elsner, D., Schunkert, H., Pfeifer, M., Griese, D., Bruckschlegel, G.,
Kromer, E. P. (1998). Development of heart failure following isoproterenol
administration in the rat: role of the renin-angiotensin system. Cardiovasc.
Res., 37(1), 91-100.
Guillen, I., Blanes, M., Gomez-Lechon, M. J., & Castell, J. V. (1995). Cytokine
signaling during myocardial infarction: sequential appearance of IL-1 beta and
IL-6. Am. J. Physiol., 269(2 Pt 2), R229-235.
Guo, K., Mou, X., Huang, J., Xiong, N., & Li, H. (2014). Trans-caryophyllene
suppresses hypoxia-induced neuroinflammatory responses by inhibiting NF-
κB activation in microglia. J. Mol. Neurosci., 54(1), 41-48.
Halliwell, B., & Chirico, S. (1993). Lipid peroxidation: its mechanism, measurement,
and significance. Am. J. Clin. Nutr., 57(5 Suppl), 715S-724S; discussion 724S-
725S.
Halliwell, B., & Gutteridge, J. M. (1990). Role of free radicals and catalytic metal ions
in human disease: an overview. Methods Enzymol., 186, 1-85.
Hammami, S., El Mokni, R., Faidi, K., Falconieri, D., Piras, A., Procedda, S., El
Aouni, M. H. (2015). Chemical composition and antioxidant activity of
essential oil from aerial parts of Teucrium flavum L. subsp. flavum growing
spontaneously in Tunisia. Nat. Prod. Res., 29(24), 2336-2340.
Hanus, L., Abu-Lafi, S., Fride, E., Breuer, A., Vogel, Z., Shalev, D. E., Mechoulam,
R. (2001). 2-arachidonyl glyceryl ether, an endogenous agonist of the
cannabinoid CB1 receptor. Proc. Natl. Acad. Sci. U S A, 98(7), 3662-3665.
Hauser, A. M., Gangadharan, V., Ramos, R. G., Gordon, S., & Timmis, G. C. (1985).
Sequence of mechanical, electrocardiographic and clinical effects of repeated
coronary artery occlusion in human beings: echocardiographic observations
during coronary angioplasty. J. Am. Coll. Cardiol., 5(2 Pt 1), 193-197.
101
Hendriks, H., Malingré, T. M., Batterman, S., & Bos, R. (1975). Mono-and sesqui-
terpene hydrocarbons of the essential oil of Cannabis sativa. Phytochemistry,
14(3), 814-815.
Horvath, B., Magid, L., Mukhopadhyay, P., Batkai, S., Rajesh, M., Park, O., Pacher,
P. (2012a). A new cannabinoid CB2 receptor agonist HU-910 attenuates
oxidative stress, inflammation and cell death associated with hepatic
ischaemia/reperfusion injury. Br. J. Pharmacol., 165(8), 2462-2478.
Horvath, B., Mukhopadhyay, P., Kechrid, M., Patel, V., Tanchian, G., Wink, D. A.,
Pacher, P. (2012b). beta-Caryophyllene ameliorates cisplatin-induced
nephrotoxicity in a cannabinoid 2 receptor-dependent manner. Free Radic.
Biol. Med., 52(8), 1325-1333.
Howlett, A. C. (2005). Cannabinoid receptor signaling. Handb. Exp. Pharmacol.,
(168), 53-79.
Howlett, A. C., Barth, F., Bonner, T. I., Cabral, G., Casellas, P., Devane, W. A.,
Pertwee, R. G. (2002). International Union of Pharmacology. XXVII.
Classification of cannabinoid receptors. Pharmacol. Rev., 54(2), 161-202.
Hu, F. B., Stampfer, M. J., Rimm, E., Ascherio, A., Rosner, B. A., Spiegelman, D., &
Willett, W. C. (1999). Dietary fat and coronary heart disease: a comparison of
approaches for adjusting for total energy intake and modeling repeated dietary
measurements. Am. J. Epidemiol., 149(6), 531-540.
Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity
assays. J. Agric. Food Chem., 53(6), 1841-1856.
Huang, S. M., Bisogno, T., Trevisani, M., Al-Hayani, A., De Petrocellis, L., Fezza, F.,
Di Marzo, V. (2002). An endogenous capsaicin-like substance with high
potency at recombinant and native vanilloid VR1 receptors. Proc. Natl. Acad.
Sci. U S A, 99(12), 8400-8405.
Iaizzo, P. (2009). Handbook of Cardiac Anatomy, Physiology, and Devices (3rd Ed.),
Springer International.
Ikeda, U., Ohkawa, F., Seino, Y., Yamamoto, K., Hidaka, Y., Kasahara, T., Shimada,
K. (1992). Serum interleukin 6 levels become elevated in acute myocardial
infarction. J. Mol. Cell. Cardiol., 24(6), 579-584.
Inagaki, T., Dutchak, P., Zhao, G., Ding, X., Gautron, L., Parameswara, V., Kliewer,
S. A. (2007). Endocrine regulation of the fasting response by PPARalpha-
mediated induction of fibroblast growth factor 21. Cell Metab., 5(6), 415-425.
102
Ithayarasi, A. P., & Devi, C. S. (1997). Effect of alpha-tocopherol on lipid peroxidation
in isoproterenol induced myocardial infarction in rats. Indian J. Physiol.
Pharmacol., 41(4), 369-376.
Iversen, L. L. (2001). The science of marijuana (2nd Ed.), Oxford University Press.
Jaffe, A. S., Landt, Y., Parvin, C. A., Abendschein, D. R., Geltman, E. M., &
Ladenson, J. H. (1996). Comparative sensitivity of cardiac troponin I and
lactate dehydrogenase isoenzymes for diagnosing acute myocardial infarction.
Clin. Chem., 42(11), 1770-1776.
Javed, H., Azimullah, S., Haque, M. E., & Ojha, S. K. (2016). Cannabinoid
Type 2 (CB2) Receptors Activation Protects against Oxidative Stress
and Neuroinflammation Associated Dopaminergic Neurodegeneration
in Rotenone Model of Parkinson's Disease. Front. Neurosci., 2(10),
321. doi: 10.3389/fnins.2016.00321.
Jiang, Z. Y., Hunt, J. V., & Wolff, S. P. (1992). Ferrous ion oxidation in the presence
of xylenol orange for detection of lipid hydroperoxide in low density
lipoprotein. Anal. Biochem., 202(2), 384-389.
Jung, J. I., Kim, E. J., Kwon, G. T., Jung, Y. J., Park, T., Kim, Y., Park, J. H. (2015).
β-Caryophyllene potently inhibits solid tumor growth and lymph node
metastasis of B16F10 melanoma cells in high-fat diet-induced obese
C57BL/6N mice. Carcinogenesis, 36(9), 1028-1039.
Kakkar, P., Das, B., & Viswanathan, P. N. (1984). A modified spectrophotometric
assay of superoxide dismutase. Indian J. Biochem. Biophys., 21(2), 130-132.
Kannan, M. M., & Quine, S. D. (2011a). Ellagic acid ameliorates isoproterenol
induced oxidative stress: Evidence from electrocardiological, biochemical and
histological study. Eur. J. Pharmacol., 659(1), 45-52.
Kannan, M. M., & Darlin Quine, S. (2011b). Pharmacodynamics of ellagic acid on
cardiac troponin-T, lyosomal enzymes and membrane bound ATPases:
mechanistic clues from biochemical, cytokine and in vitro studies. Chem. Biol.
Interact., 193(2), 154-161.
Kasper, D. L. (2005). Harrison's Principles of Internal Medicine (16th Ed.), New York
: McGraw-Hill, Medical Pub.
Katsuyama, S., Mizoguchi, H., Kuwahata, H., Komatsu, T., Nagaoka, K., Nakamura,
H., Sakurada, S. (2013). Involvement of peripheral cannabinoid and opioid
receptors in β-caryophyllene-induced antinociception. Eur. J. Pain, 17(5), 664-
675.
103
Khanolkar, A. D., Abadji, V., Lin, S., Hill, W. A., Taha, G., Abouzid, K., Makriyannis,
A. (1996). Head group analogs of arachidonylethanolamide, the endogenous
cannabinoid ligand. J. Med. Chem., 39(22), 4515-4519.
Kim, J. Y., Park, S. J., Yun, K. J., Cho, Y. W., Park, H. J., & Lee, K. T. (2008).
Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-
induced iNOS and COX-2 expression via the attenuation of NF-kappaB in
RAW 264.7 macrophages. Eur. J. Pharmacol., 584(1), 175-184.
King, A. R., Dotsey, E. Y., Lodola, A., Jung, K. M., Ghomian, A., Qiu, Y., Piomelli,
D. (2009). Discovery of Potent and Reversible Monoacylglycerol Lipase
Inhibitors. Chem. Biol., 16(10), 1045-1052.
Kishimoto, T., Akira, S., Narazaki, M., & Taga, T. (1995). Interleukin-6 family of
cytokines and gp130. Blood, 86(4), 1243-1254.
Kloner, R. A., & Parisi, A. F. (1987). Acute myocardial infarction: diagnostic and
prognostic applications of two-dimensional echocardiography. Circulation,
75(3), 521-524.
Knufman, N. M., van der Laarse, A., Vliegen, H. W., & Brinkman, C. J. (1987).
Quantification of myocardial necrosis and cardiac hypertrophy in
isoproterenol-treated rats. Res. Commun. Chem. Pathol. Pharmacol., 57(1), 15-
32.
Kota, B. P., Huang, T. H., & Roufogalis, B. D. (2005). An overview on biological
mechanisms of PPARs. Pharmacol. Res., 51(2), 85-94.
Koudssi, F., Lopez, J. E., Villegas, S., & Long, C. S. (1998). Cardiac fibroblasts arrest
at the G1/S restriction point in response to interleukin (IL)-1beta. Evidence for
IL-1beta-induced hypophosphorylation of the retinoblastoma protein. J. Biol.
Chem., 273(40), 25796-25803.
Ku, C. M., & Lin, J. Y. (2013). Anti-inflammatory effects of 27 selected terpenoid
compounds tested through modulating Th1/Th2 cytokine secretion profiles
using murine primary splenocytes. Food Chem., 141(2), 1104-1113.
Kubo, I., Chaudhuri, S. K., Kubo, Y., Sanchez, Y., Ogura, T., Saito, T., Haraguchi, H.
(1996). Cytotoxic and antioxidative sesquiterpenoids from Heterotheca
inuloides. Planta. Med., 62(5), 427-430.
Kumar, V. (2015). Robbins and Cotran pathologic basis of disease (9th Ed.), Elsevier/
Saunders.
Kumaran, K. S., & Prince, P. S. (2010a). Caffeic acid protects rat heart mitochondria
against isoproterenol-induced oxidative damage. Cell Stress Chaperones,
15(6), 791-806.
104
Kumaran, K. S., & Prince, P. S. (2010b). Protective effect of caffeic acid on cardiac
markers and lipid peroxide metabolism in cardiotoxic rats: an in vivo and in
vitro study. Metabolism, 59(8), 1172-1180.
Kumaran, K. S., & Prince, P. S. M. (2010). Caffeic acid protects rat heart mitochondria
against isoproterenol-induced oxidative damage. Cell Stress Chaperones.
2010;15(6):791-806.
Laboratory Diagnosis of Myocardial Infarction. (1999). Retrieved from
https://labmed.hallym.ac.kr/chemistry/MI-Diagnisis.htm. Accessed 13 Nov.
2019.
Lagneux, C., & Lamontagne, D. (2001). Involvement of cannabinoids in the
cardioprotection induced by lipopolysaccharide. Br. J. Pharmacol., 132(4),
793-796.
Lalitha, G., Poornima, P., Archanah, A., & Padma, V. V. (2013). Protective effect of
neferine against isoproterenol-induced cardiac toxicity. Cardiovasc. Toxicol.,
13(2), 168-179.
Laslett, L. J., Alagona, P., Jr., Clark, B. A., 3rd, Drozda, J. P., Jr., Saldivar, F., Wilson,
S. R., Hart, M. (2012). The worldwide environment of cardiovascular disease:
prevalence, diagnosis, therapy, and policy issues: a report from the American
College of Cardiology. J. Am. Coll. Cardiol., 60(25 Suppl), S1-49.
LaVoie, E. J., Adams, J. D., Reinhardt, J., Rivenson, A., & Hoffmann, D. (1986).
Toxicity studies on clove cigarette smoke and constituents of clove:
determination of the LD50 of eugenol by intratracheal instillation in rats and
hamsters. Arch. Toxicol., 59(2), 78-81.
Law, M. R., & Morris, J. K. (1998). By how much does fruit and vegetable
consumption reduce the risk of ischaemic heart disease?. Eur. J. Clin. Nutr.,
52(8), 549-556.
Leaver, M. J., & George, S. G. (1998). A piscine glutathione S-transferase which
efficiently conjugates the end-products of lipid peroxidation. Marine
Environmental Research, 46(1), 71-74.
Lee, W., Ham, J., Kwon, H. C., Kim, Y. K., & Kim, S. N. (2013). Anti-diabetic effect
of amorphastilbol through PPARα/γ dual activation in db/db mice. Biochem.
Biophys. Res. Commun., 432(1), 73-79.
Leonhardt, V., Leal-Cardoso, J. H., Lahlou, S., Albuquerque, A. A., Porto, R. S.,
Celedônio, N. R., Coelho-de-Souza, A. N. (2010). Antispasmodic effects of
essential oil of Pterodon polygalaeflorus and its main constituent β-
caryophyllene on rat isolated ileum. Fundam. Clin. Pharmacol., 24(6), 749-
758.
105
Liu, H., Song, Z., Liao, D., Zhang, T., Liu, F., Zhuang, K.,Yang, L. (2015).
Neuroprotective effects of trans-caryophyllene against kainic acid induced
seizure activity and oxidative stress in mice. Neurochem. Res., 40(1), 118-123.
Liu, H., Yang, G., Tang, Y., Cao, D., Qi, T., Qi, Y., & Fan, G. (2013). Physicochemical
characterization and pharmacokinetics evaluation of beta-caryophyllene/beta-
cyclodextrin inclusion complex. Int. J. Pharm., 450(1-2), 304-310.
Liu, Q. R., Pan, C. H., Hishimoto, A., Li, C. Y., Xi, Z. X., Llorente-Berzal, A., Uhl,
G. R. (2009). Species differences in cannabinoid receptor 2 (CNR2 gene):
identification of novel human and rodent CB2 isoforms, differential tissue
expression and regulation by cannabinoid receptor ligands. Genes Brain
Behav., 8(5), 519-530.
Lu, H. C., & Mackie, K. (2016). An Introduction to the Endogenous Cannabinoid
System. Biol. Psychiatry, 79(7), 516-525.
Lucca, L. G., de Matos, S. P., Borille, B. T., de, O. D. D., Teixeira, H. F., Veiga, V.
F., Jr., Koester, L. S. (2015). Determination of beta-caryophyllene skin
permeation/retention from crude copaiba oil (Copaifera multijuga Hayne) and
respective oil-based nanoemulsion using a novel HS-GC/MS method. J.
Pharm. Biomed. Anal., 104, 144-148.
Ma, L., Jia, J., Liu, X., Bai, F., Wang, Q., & Xiong, L. (2015). Activation of murine
microglial N9 cells is attenuated through cannabinoid receptor CB2 signaling.
Biochem. Biophys. Res. Commun., 458(1), 92-97.
Mackie, K. (2008). Cannabinoid receptors: where they are and what they do. J.
Neuroendocrinol., 20 Suppl 1, 10-14.
Malik, S., Goyal, S., Ojha, S. K., Bharti, S., Nepali, S., Kumari, S., Arya, D. S. (2011).
Seabuckthorn attenuates cardiac dysfunction and oxidative stress in
isoproterenol-induced cardiotoxicity in rats. Int. J. Toxicol., 30(6), 671-680.
Marcus, G. M., Cohen, J., Varosy, P. D., Vessey, J., Rose, E., Massie, B. M.,Waters,
D. (2007). The utility of gestures in patients with chest discomfort. Am. J.
Med., 120(1), 83-89.
Mathew, S., Menon, P. V., & Kurup, P. A. (1985). Effect of administration of vitamin
A, ascorbic acid and nicotinamide adenine dinucleotide + flavin adenine
dinucleotide on severity of myocardial infarction induced by isoproterenol in
rats. Indian. J. Exp. Biol., 23(9), 500-504.
Mechoulam, R. (1986). The pharmacohistory of Cannabis sativa. Cannabinoids as
therapeutic agents (1st Ed.), CRC-Press.
106
Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N. E., Schatz,
A. R., et al. (1995). Identification of an endogenous 2-monoglyceride, present
in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol., 50(1),
83-90.
Medeiros, R., Passos, G. F., Vitor, C. E., Koepp, J., Mazzuco, T. L., Pianowski, L. F.,
Calixto, J. B. (2007). Effect of two active compounds obtained from the
essential oil of Cordia verbenacea on the acute inflammatory responses elicited
by LPS in the rat paw. Br. J. Pharmacol., 151(5), 618-627.
Meeran, M. F. N., Al Taee, H., Azimullah, S., Tariq, S., Adeghate, E., & Ojha, S.
(2019). β-Caryophyllene, a natural bicyclic sesquiterpene attenuates
doxorubicin-induced chronic cardiotoxicity via activation of myocardial
cannabinoid type-2 (CB2) receptors in rats. Chemico-Biological Interactions,
304, 158-167.
Mendizabal, V. E., & Adler-Graschinsky, E. (2007). Cannabinoids as therapeutic
agents in cardiovascular disease: a tale of passions and illusions. Br. J.
Pharmacol., 151(4), 427-440.
Menezes Pdos, P., Araujo, A. A., Doria, G. A., Quintans-Junior, L. J., de Oliveira, M.
G., dos Santos, M. R., Serafini, M. R. (2015). Physicochemical
characterization and analgesic effect of inclusion complexes of essential oil
from Hyptis pectinata L. Poit leaves with β-cyclodextrin. Curr. Pharm.
Biotechnol., 16(5), 440-450.
Mikaelian, I., Coluccio, D., Morgan, K. T., Johnson, T., Ryan, A. L., Rasmussen, E.,
Wheeldon, E. B. (2008). Temporal gene expression profiling indicates early
up-regulation of interleukin-6 in isoproterenol-induced myocardial necrosis in
rat. Toxicol. Pathol., 36(2), 256-264.
Miller, A. M., & Stella, N. (2008). CB2 receptor-mediated migration of immune cells:
it can go either way. Br. J. Pharmacol., 153(2), 299-308.
Molina-Jasso, D., Alvarez-Gonzalez, I., & Madrigal-Bujaidar, E. (2009).
Clastogenicity of beta-caryophyllene in mouse. Biol. Pharm. Bull., 32(3), 520-
522.
Montecucco, F., Lenglet, S., Braunersreuther, V., Burger, F., Pelli, G., Bertolotto, M.,
Steffens, S. (2009). CB(2) cannabinoid receptor activation is cardioprotective
in a mouse model of ischemia/reperfusion. J. Mol. Cell. Cardiol., 46(5), 612-
620.
107
Mukhopadhyay, P., Rajesh, M., Pan, H., Patel, V., Mukhopadhyay, B., Batkai, S.,
Pacher, P. (2010). Cannabinoid-2 receptor limits inflammation,
oxidative/nitrosative stress, and cell death in nephropathy. Free Radic. Biol.
Med., 48(3), 457-467.
Murphy, E., Perlman, M., London, R. E., & Steenbergen, C. (1991). Amiloride delays
the ischemia-induced rise in cytosolic free calcium. Circ. Res., 68(5), 1250-
1258.
Nagoor Meeran, M. F., Laham, F., Azimullah, S., Tariq, S., & Ojha, S. (2019). alpha-
Bisabolol abrogates isoproterenol-induced myocardial infarction by inhibiting
mitochondrial dysfunction and intrinsic pathway of apoptosis in rats. Mol. Cell.
Biochem., 453(1-2), 89-102.
Nakamura, M., Zhai, P., Del Re, D. P., Maejima, Y., & Sadoshima, J. (2016). Mst1-
mediated phosphorylation of Bcl-xL is required for myocardial reperfusion
injury. JCI Insight, 1(5), e86217. doi:10.1172/jci.insight.e86217.
O'Sullivan, S. E., & Kendall, D. A. (2010). Cannabinoid activation of peroxisome
proliferator-activated receptors: potential for modulation of inflammatory
disease. Immunobiology, 215(8), 611-616.
Oda, S., Fujinuma, K., Inoue, A., & Ohashi, S. (2011). Synthesis of (-)-beta-
caryophyllene oxide via regio- and stereoselective endocyclic epoxidation of
beta-caryophyllene with Nemania aenea SF 10099-1 in a liquid-liquid interface
bioreactor (L-L IBR). J. Biosci. Bioeng., 112(6), 561-565.
Ojha, S., Bhatia, J., Arora, S., Golechha, M., Kumari, S., & Arya, D. S. (2011).
Cardioprotective effects of Commiphora mukul against isoprenaline-induced
cardiotoxicity: a biochemical and histopathological evaluation. J. Environ.
Biol., 32(6), 731-738.
Ojha, S., Goyal, S., Kumari, S., & Arya, D. S. (2012). Pyruvate attenuates cardiac
dysfunction and oxidative stress in isoproterenol-induced cardiotoxicity. Exp.
Toxicol. Pathol., 64(4), 393-399.
Ojha, S., Goyal, S., Sharma, C., Arora, S., Kumari, S., & Arya, D. S. (2013).
Cardioprotective effect of lycopene against isoproterenol-induced myocardial
infarction in rats. Hum. Exp. Toxicol., 32(5), 492-503.
Ojha, S. K., Nandave, M., Arora, S., Narang, R., Dinda, A. K., & Arya, D. S. (2008).
Chronic administration of Tribulus terrestris Linn extract improves cardiac
function and attenuates myocardial infarction in rats. Intern. Journal. of
Pharmacol., 4(1), 1-10.
108
Okoli, C. O., & Akah, P. A. (2004). Mechanisms of the anti-inflammatory activity of
the leaf extracts of Culcasia scandens P. Beauv (Araceae). Pharmacol.
Biochem. Behav., 79(3), 473-481.
Okoye, F. B., Osadebe, P. O., Nworu, C. S., Okoye, N. N., Omeje, E. O., & Esimone,
C. O. (2011). Topical anti-inflammatory constituents of lipophilic leaf
fractions of Alchornea floribunda and Alchornea cordifolia. Nat. Prod. Res.,
25(20), 1941-1949.
Olivieri, F., Antonicelli, R., Cardelli, M., Marchegiani, F., Cavallone, L.,
Mocchegiani, E., & Franceschi, C. (2006). Genetic polymorphisms of
inflammatory cytokines and myocardial infarction in the elderly. Mech.
Ageing Dev., 127(6), 552-559.
Ono, K., Matsumori, A., Shioi, T., Furukawa, Y., & Sasayama, S. (1998). Cytokine
gene expression after myocardial infarction in rat hearts: possible implication
in left ventricular remodeling. Circulation, 98(2), 149-156.
Opdyke, D. L. (1973). Monographs on fragrance raw materials. Food Cosmet.
Toxicol., 11(6), 1011-1081.
Ouyang, B., Li, Z., Ji, X., Huang, J., Zhang, H., & Jiang, C. (2019). The protective
role of lutein on isoproterenol-induced cardiac failure rat model through
improving cardiac morphology, antioxidant status via positively regulating
Nrf2/HO-1 signalling pathway. Pharm. Biol., 57(1), 529-535.
Pacher, P., Batkai, S., & Kunos, G. (2006). The endocannabinoid system as an
emerging target of pharmacotherapy. Pharmacol. Rev., 58(3), 389-462.
Pacher, P., & Mechoulam, R. (2011). Is lipid signaling through cannabinoid 2
receptors part of a protective system?. Prog. Lipid. Res., 50(2), 193-211.
Paula-Freire, L. I., Andersen, M. L., Gama, V. S., Molska, G. R., & Carlini, E. L.
(2014). The oral administration of trans-caryophyllene attenuates acute and
chronic pain in mice. Phytomedicine, 21(3), 356-362.
Pertwee, R. G. (2006). Cannabinoid pharmacology: the first 66 years. Br. J.
Pharmacol., 147 Suppl 1, S163-171.
Pietinen, P., Rimm, E. B., Korhonen, P., Hartman, A. M., Willett, W. C., Albanes, D.,
& Virtamo, J. (1996). Intake of dietary fiber and risk of coronary heart disease
in a cohort of Finnish men. The Alpha-Tocopherol, Beta-Carotene Cancer
Prevention Study. Circulation, 94(11), 2720-2727.
109
Porter, A. C., Sauer, J. M., Knierman, M. D., Becker, G. W., Berna, M. J., Bao, J.,
Felder, C. C. (2002). Characterization of a novel endocannabinoid,
virodhamine, with antagonist activity at the CB1 receptor. J. Pharmacol. Exp.
Ther., 301(3), 1020-1024.
Prabhu, S., Jainu, M., Sabitha, K. E., & Shyamala Devi, C. S. (2006). Effect of
mangiferin on mitochondrial energy production in experimentally induced
myocardial infarcted rats. Vascul. Pharmacol., 44(6), 519-525.
Prabhu, S. D., Chandrasekar, B., Murray, D. R., & Freeman, G. L. (2000). beta-
adrenergic blockade in developing heart failure: effects on myocardial
inflammatory cytokines, nitric oxide, and remodeling. Circulation, 101(17),
2103-2109.
PubChem. (2019a). 2-AG. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/.
Accessed 3 Dec. 2019.
PubChem. (2019b). Anandamide. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/. Accessed 3 Dec. 2019.
Radhiga, T., Rajamanickam, C., Sundaresan, A., Ezhumalai, M., & Pugalendi, K. V.
(2012). Effect of ursolic acid treatment on apoptosis and DNA damage in
isoproterenol-induced myocardial infarction. Biochimie., 94(5), 1135-1142.
Ramirez, S. H., Hasko, J., Skuba, A., Fan, S., Dykstra, H., McCormick, R., Persidsky,
Y. (2012). Activation of cannabinoid receptor 2 attenuates leukocyte-
endothelial cell interactions and blood-brain barrier dysfunction under
inflammatory conditions. J. Neurosci., 32(12), 4004-4016.
Raso, G. M., Meli, R., Di Carlo, G., Pacilio, M., & Di Carlo, R. (2001). Inhibition of
inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids
in macrophage J774A.1. Life Sci., 68(8), 921-931.
Rauch, U., Osende, J. I., Fuster, V., Badimon, J. J., Fayad, Z., & Chesebro, J. H.
(2001). Thrombus formation on atherosclerotic plaques: pathogenesis and
clinical consequences. Ann. Intern. Med., 134(3), 224-238.
Rennenberg, H. (1980). Glutathione metabolism and possible biological roles in higher
plants. Phytochemistry, 21(12), 2771-2781.
Rollinger, J. M., Schuster, D., Danzl, B., Schwaiger, S., Markt, P., Schmidtke, M.,
Stuppner, H. (2009). In silico target fishing for rationalized ligand discovery
exemplified on constituents of Ruta graveolens. Planta Medeca, 75(3), 195-
204.
110
Roy, S. S., Madesh, M., Davies, E., Antonsson, B., Danial, N., & Hajnoczky, G.
(2009). Bad targets the permeability transition pore independent of Bax or Bak
to switch between Ca2+-dependent cell survival and death. Mol. Cell, 33(3),
377-388.
Rufino, A. T., Ribeiro, M., Sousa, C., Judas, F., Salgueiro, L., Cavaleiro, C., &
Mendes, A. F. (2015). Evaluation of the anti-inflammatory, anti-catabolic and
pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model
of osteoarthritis. Eur. J. Pharmacol., 750, 141-150.
Sabulal, B., Dan, M., J, A. J., Kurup, R., Pradeep, N. S., Valsamma, R. K., & George,
V. (2006). Caryophyllene-rich rhizome oil of Zingiber nimmonii from South
India: Chemical characterization and antimicrobial activity. Phytochemistry,
67(22), 2469-2473.
Sahu, B. D., Anubolu, H., Koneru, M., Kumar, J. M., Kuncha, M., Rachamalla, S. S.,
& Sistla, R. (2014). Cardioprotective effect of embelin on isoproterenol-
induced myocardial injury in rats: possible involvement of mitochondrial
dysfunction and apoptosis. Life. Sci., 107(1-2), 59-67.
Sain, S., Naoghare, P. K., Devi, S. S., Daiwile, A., Krishnamurthi, K., Arrigo, P., &
Chakrabarti, T. (2014). Beta caryophyllene and caryophyllene oxide, isolated
from Aegle marmelos, as the potent anti-inflammatory agents against
lymphoma and neuroblastoma cells. Antiinflamm. Antiallergy Agents Med.
Chem., 13(1), 45-55.
Saraste, A., Pulkki, K., Kallajoki, M., Henriksen, K., Parvinen, M., & Voipio-Pulkki,
L. M. (1997). Apoptosis in human acute myocardial infarction. Circulation,
95(2), 320-323.
Saravanan, G., Ponmurugan, P., Sathiyavathi, M., Vadivukkarasi, S., & Sengottuvelu,
S. (2013). Cardioprotective activity of Amaranthus viridis Linn: effect on
serum marker enzymes, cardiac troponin and antioxidant system in
experimental myocardial infarcted rats. Int. J. Cardiol., 165(3), 494-498.
Saravanan, G., & Prakash, J. (2004). Effect of garlic (Allium sativum) on lipid
peroxidation in experimental myocardial infarction in rats. J. Ethnopharmacol.,
94(1), 155-158.
Sathish, V., Vimal, V., Ebenezar, K. K., & Devaki, T. (2002). Synergistic effect of
nicorandil and amlodipine on mitochondrial function during isoproterenol-
induced myocardial infarction in rats. J. Pharm. Pharmacol., 54(1), 133-137.
Scandalios, J. G. (1993). Oxygen Stress and Superoxide Dismutases. Plant Physiol.,
101(1), 7-12.
111
Schuehly, W., Paredes, J. M. V., Kleyer, J., Huefner, A., Anavi-Goffer, S., Raduner,
S., Gertsch, J. (2011). Mechanisms of osteoclastogenesis inhibition by a novel
class of biphenyl-type cannabinoid CB(2) receptor inverse agonists. Chemistry
& biology, 18(8), 1053-1064.
Schweitzer, P. (1990). The electrocardiographic diagnosis of acute myocardial
infarction in the thrombolytic era. Am. Heart J., 119(3 Pt 1), 642-654.
Seifried, H. E., Seifried, R. M., Clarke, J. J., Junghans, T. B., & San, R. H. (2006). A
compilation of two decades of mutagenicity test results with the Ames
Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays.
Chem. Res. Toxicol., 19(5), 627-644.
Senthil, S., Sridevi, M., & Pugalendi, K. V. (2007). Cardioprotective effect of
oleanolic acid on isoproterenol-induced myocardial ischemia in rats. Toxicol.
Pathol., 35(3), 418-423.
Shao, Z., Yin, J., Chapman, K., Grzemska, M., Clark, L., Wang, J., & Rosenbaum, D.
M. (2016). High-resolution crystal structure of the human CB1 cannabinoid
receptor. Nature, 540(7634), 602-606.
Sharma, C., Al Kaabi, J. M., Nurulain, S. M., Goyal, S. N., Kamal, M. A., & Ojha, S.
(2016). Polypharmacological Properties and Therapeutic Potential of beta-
Caryophyllene: A Dietary Phytocannabinoid of Pharmaceutical Promise. Curr.
Pharm. Des., 22(21), 3237-3264.
Sharma, H. S., & Das, D. K. (1997). Role of cytokines in myocardial ischemia and
reperfusion. Mediators Inflamm., 6(3), 175-183.
Sharma, H. S., & Zimmerman, R. (1993). Growth factors and development of coronary
collaterals. In P. Cummins (Ed.). Boston, MA: Springer US. 119-148.
Simmons, D. L., Botting, R. M., & Hla, T. (2004). Cyclooxygenase isozymes: the
biology of prostaglandin synthesis and inhibition. Pharmacol. Rev., 56(3), 387-
437.
Singal, P. K., Beamish, R. E., & Dhalla, N. S. (1983). Potential oxidative pathways of
catecholamines in the formation of lipid peroxides and genesis of heart disease.
Adv. Exp. Med. Biol., 161, 391-401.
Singh, G., Marimuthu, P., de Heluani, C. S., & Catalan, C. A. (2006). Antioxidant and
biocidal activities of Carum nigrum (seed) essential oil, oleoresin, and their
selected components. J. Agric. Food Chem., 54(1), 174-181.
112
Sinha, A. K. (1972). Colorimetric assay of catalase. Anal. Biochem., 47(2), 389-394.
Siwik, D. A., Chang, D. L., & Colucci, W. S. (2000). Interleukin-1beta and tumor
necrosis factor-alpha decrease collagen synthesis and increase matrix
metalloproteinase activity in cardiac fibroblasts in vitro. Circ. Res., 86(12),
1259-1265.
Skeik, N., & Patel, D. C. (2007). A review of troponins in ischemic heart disease and
other conditions. Int. J. Angiol., 2007;16(2):53-58.
Sköld, M., Karlberg, A. T., Matura, M., & Börje, A. (2006). The fragrance chemical
beta-caryophyllene-air oxidation and skin sensitization. Food Chem. Toxicol.,
44(4), 538-545.
Song, W., Lu, X., & Feng, Q. (2000). Tumor necrosis factor-alpha induces apoptosis
via inducible nitric oxide synthase in neonatal mouse cardiomyocytes.
Cardiovasc. Res., 45(3), 595-602.
Steffens, S., Veillard, N. R., Arnaud, C., Pelli, G., Burger, F., Staub, C., Mach, F.
(2005). Low dose oral cannabinoid therapy reduces progression of
atherosclerosis in mice. Nature, 434(7034), 782-786.
Sugano, M., Tsuchida, K., Hata, T., & Makino, N. (2004). In vivo transfer of soluble
TNF-alpha receptor 1 gene improves cardiac function and reduces infarct size
after myocardial infarction in rats. Faseb j., 18(7), 911-913.
Sugiura, T., Kishimoto, S., Oka, S., & Gokoh, M. (2006). Biochemistry, pharmacology
and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid
receptor ligand. Prog. Lipid Res., 45(5), 405-446.
Sultana, N., & Saify, Z. S. (2013). Enzymatic biotransformation of terpenes as
bioactive agents. J. Enzyme Inhib. Med. Chem., 28(6), 1113-1128.
Sushamakumari, S., Jayadeep, A., Kumar, J. S., & Menon, V. P. (1989). Effect of
carnitine on malondialdehyde, taurine and glutathione levels in heart of rats
subjected to myocardial stress by isoproterenol. Indian J. Exp. Biol., 27(2),
134-137.
Taegtmeyer, H. (1994). Energy metabolism of the heart: from basic concepts to
clinical applications. Curr. Probl. Cardiol., 19(2), 59-113.
Tai, S., & Fantegrossi, W. E. (2017). Pharmacological and Toxicological Effects of
Synthetic Cannabinoids and Their Metabolites. Curr. Top. Behav. Neurosci.,
32, 249-262.
113
Tayeh, M. A., & Marletta, M. A. (1989). Macrophage oxidation of L-arginine to nitric
oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J. Biol.
Chem., 264(33), 19654-19658.
Thompson, J. A., & Hess, M. L. (1986). The oxygen free radical system: a fundamental
mechanism in the production of myocardial necrosis. Prog. Cardiovasc. Dis.,
28(6), 449-462.
Thygesen, K., Alpert, J. S., Jaffe, A. S., Simoons, M. L., Chaitman, B. R., White, H.
D., Mendis, S. (2012). Third universal definition of myocardial infarction.
Circulation, 126(16), 2020-2035.
Turcotte, C., Blanchet, M. R., Laviolette, M., & Flamand, N. (2016). The CB2 receptor
and its role as a regulator of inflammation. Cell. Mol. Life Sci., 73(23), 4449-
4470.
Upaganlawar, A., Gandhi, C., & Balaraman, R. (2009). Effect of green tea and vitamin
E combination in isoproterenol induced myocardial infarction in rats. Plant
Foods Hum. Nutr., 64(1), 75-80.
Vaage, J., & Valen, G. (1993). Pathophysiology and mediators of ischemia-
reperfusion injury with special reference to cardiac surgery. A review. Scand
J. Thorac. Cardiovasc. Surg. Suppl., 41, 1-18.
Valensi, P., Lorgis, L., & Cottin, Y. (2011). Prevalence, incidence, predictive factors
and prognosis of silent myocardial infarction: a review of the literature. Arch.
Cardiovasc. Dis., 104(3), 178-188.
Vinholes, J., Gonçalves, P., Martel, F., Coimbra, M. A., & Rocha, S. M. (2014).
Assessment of the antioxidant and antiproliferative effects of sesquiterpenic
compounds in in vitro Caco-2 cell models. Food Chem., 156, 204-211.
von Harsdorf, R., Li, P. F., & Dietz, R. (1999). Signaling pathways in reactive oxygen
species-induced cardiomyocyte apoptosis. Circulation, 99(22), 2934-2941.
Vural, H., Aksoy, N., & Ozbilge, H. (2004). Alterations of oxidative-antioxidative
status in human cutaneous leishmaniasis. Cell Biochem. Funct., 22(3), 153-
156.
Wallace, T. C. (2011). Anthocyanins in cardiovascular disease. Adv. Nutr., 2(1), 1-7.
Wang, R. L., Staehelin, C., Peng, S. L., Wang, W. T., Xie, X. M., & Lu, H. N. (2010).
Responses of Mikania micrantha, an invasive weed to elevated CO(2):
induction of beta-caryophyllene synthase, changes in emission capability and
allelopathic potential of beta-caryophyllene. J. Chem. Ecol., 36(10), 1076-
1082.
114
Weis, F., Beiras-Fernandez, A., Sodian, R., Kaczmarek, I., Reichart, B., Beiras, A.,
Kreth, S. (2010). Substantially altered expression pattern of cannabinoid
receptor 2 and activated endocannabinoid system in patients with severe heart
failure. J. Mol. Cell. Cardiol., 48(6), 1187-1193.
Wexler, B. C., & Greenberg, B. P. (1978). Protective effects of clofibrate on
isoproterenol-induced myocardial infarction in arteriosclerotic and non-
arteriosclerotic rats. Atherosclerosis, 29(3), 373-395.
Wohlgelernter, D., Cleman, M., Highman, H. A., Fetterman, R. C., Duncan, J. S.,
Zaret, B. L., & Jaffe, C. C. (1986). Regional myocardial dysfunction during
coronary angioplasty: evaluation by two-dimensional echocardiography and 12
lead electrocardiography. J. Am. Coll. Cardiol., 7(6), 1245-1254.
Wu, C., Jia, Y., Lee, J. H., Jun, H. J., Lee, H. S., Hwang, K. Y., & Lee, S. J. (2014).
trans-Caryophyllene is a natural agonistic ligand for peroxisome proliferator-
activated receptor-alpha. Bioorg. Med. Chem. Lett., 24(14), 3168-3174.
Xavier, A. (2013). Cannabinoids- Pharmacology and their potential therapeutic
applications. ResearchGate. Retrieved from
https://www.researchgate.net/publication/324943141_Cannabinoids-
_Pharmacology_and_their_potential_therapeutic_applications. Accessed 12
Dec 2019
Xia, T., Jiang, C., Li, L., Wu, C., Chen, Q., & Liu, S.-s. (2002). A study on
permeability transition pore opening and cytochrome c release from
mitochondria, induced by caspase-3 in vitro. FEBS Letters, 510(1), 62-66.
Yates, J. C., Beamish, R. E., & Dhalla, N. S. (1981). Ventricular dysfunction and
necrosis produced by adrenochrome metabolite of epinephrine: Relation to
pathogenesis of catecholamine cardiomyopathy. American Heart Journal,
102(2), 210-221.
Yin, H., Chu, A., Li, W., Wang, B., Shelton, F., Otero, F., Chen, Y. A. (2009). Lipid
G protein-coupled receptor ligand identification using beta-arrestin PathHunter
assay. J Biol Chem, 284(18), 12328-12338.
Zarco, P., & Zarco, M. N. (1996). Biochemical aspects of cardioprotection.
Medicographia, 18(2), 18-21.
Zarruk, J. G., Fernandez-Lopez, D., Garcia-Yebenes, I., Garcia-Gutierrez, M. S.,
Vivancos, J., Nombela, F., Moro, M. A. (2012). Cannabinoid type 2 receptor
activation downregulates stroke-induced classic and alternative brain
macrophage/microglial activation concomitant to neuroprotection. Stroke,
43(1), 211-219.
115
Zhang, H. Y., Bi, G. H., Li, X., Li, J., Qu, H., Zhang, S. J., Liu, Q. R. (2015). Species
differences in cannabinoid receptor 2 and receptor responses to cocaine self-
administration in mice and rats. Neuropsychopharmacology, 40(4), 1037-1051.
Zhao, Y., Liu, Y., Zhang, W., Xue, J., Wu, Y. Z., Xu, W., Yuan, Z. (2010). WIN55212-
2 ameliorates atherosclerosis associated with suppression of pro-inflammatory
responses in ApoE-knockout mice. Eur. J. Pharmacol., 649(1-3), 285-292.
Zhao, Z. Q., Budde, J. M., Morris, C., Wang, N. P., Velez, D. A., Muraki, S.,Vinten-
Johansen, J. (2001). Adenosine attenuates reperfusion-induced apoptotic cell
death by modulating expression of Bcl-2 and Bax proteins. J. Mol. Cell.
Cardiol., 33(1), 57-68.
Zidar, N., Jera, J., Maja, J., & Dusan, S. (2007). Caspases in myocardial infarction.
Adv. Clin. Chem., 44, 1-33.
Zygmunt, P. M., Petersson, J., Andersson, D. A., Chuang, H., Sorgard, M., Di Marzo,
V., Hogestatt, E. D. (1999). Vanilloid receptors on sensory nerves mediate the
vasodilator action of anandamide. Nature, 400(6743), 452-457.
116
List of Publications
Meeran, M., Laham, F., Azimullah, S., Tariq, S., & Ojha, S. (2019). α-Bisabolol
abrogates isoproterenol-induced myocardial infarction by inhibiting mitochondrial
dysfunction and intrinsic pathway of apoptosis in rats. Molecular and cellular
biochemistry, 453(1-2), 89-102. https://doi.org/10.1007/s11010-018-3434-5.
Meeran, M., Laham, F., Al-Taee, H., Azimullah, S., & Ojha, S. (2018). Protective
effects of α-bisabolol on altered hemodynamics, lipid peroxidation, and nonenzymatic
antioxidants in isoproterenol-induced myocardial infarction: In vivo and in vitro
evidences. Journal of biochemical and molecular toxicology, 32(10), e22200.
https://doi.org/10.1002/jbt.22200.