the effect of ꞵ -caryophyllene on isoproterenol- …

United Arab Emirates University United Arab Emirates University

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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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ii

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from other sources (whether published or unpublished) and relied upon or included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

iii

Copyright

Copyright © 2020 Farah M.Jamal Laham

All Rights Reserved

iv

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


3) Member: Dr. Bassem Sadek

Title: Associate Professor

Department of Pharmacology


vi

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

vii

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.

viii

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.

ix

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.

x

(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

xi

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.

xii

Dedication

I dedicate this thesis to my beloved parents and family

xiii

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

xiv

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

xvi

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

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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.


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


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)

https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=2449371_zpq9990838760003.jpg

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)



9th and 10th day).

Group VI: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)



9th and 10th day).

Group VI: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)



9th and 10th day).

Group VII: Rats were orally pre and co-treated with BCP (25 mg/kg body weight)



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)






Group IX: Rats were orally pre and co-treated with BCP (100 mg/kg body weight)






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)



9th and 10th day).

Group V: Rats were orally pre and co-treated with BCP (50 mg/kg body weight, BID)






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


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


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.

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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

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List of Publications

Meeran, M., Laham, F., Azimullah, S., Tariq, S., & Ojha, S. (2019). α-Bisabolol

abrogates isoproterenol-induced myocardial infarction by inhibiting mitochondrial

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evidences. Journal of biochemical and molecular toxicology, 32(10), e22200.

https://doi.org/10.1002/jbt.22200.

https://doi.org/10.1007/s11010-018-3434-5

https://doi.org/10.1002/jbt.22200