Kurdistan Regional Government-Iraq

Ministry of Higher Education and

Scientific Research
University of Zakho

A Thesis
Submitted to the Council of the Faculty of Science/University of Zakho,
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

in

MOLECULAR PHYSIOLOGY

by

B.Sc. (Biology), M.Sc. (Animal Physiology)

College of Science/University of Salahaddin-Erbil

Supervised by

2715 K. 1436 A.H. 2015 A.D.

(32)
 We certify that the thesis entitled “Phytochemical Screening and Anti-
contractile Effect of some Active Ingredients of Crataegus azarolus var. aronia on
Isolated Rat’s Aorta” was prepared under our supervision in the

Department of Biology, Faculty of Science, University of Zakho and

Department of Chemistry, University of Pavia as a partial requirement for
the degree of Doctor of Philosophy in Molecular Physiology.

Signature:
Name: Prof. Dr. Omar A. M. Al-Habib
Academic rank: Professor

Date: / /2015

Signature:
Name: Prof. Dr. Giovanni Vidari
Academic rank: Professor

Date: / /2015

Chairman's Certification

In view of recommendations, I forward this thesis for debate by the

Examining Committee.

Signature:

Name: Prof. Dr. Wijdan M. S. Mero

Academic rank: Professor

Date: / / 2015
 Examination Committee Certification

We are “the examination committee” certify that we have read this thesis
entitled “PHYTOCHEMICAL SCREENING AND ANTI-CONTRACTILE
EFFECT OF SOME ACTIVE INGRIEDIENTS OF Crataegus azarolus var.
aronia L. ON ISOLATED RAT’S AORTA” and we have examined the student
“Sarbast Ahmad Mahmud” in its content and in what is related to it, and in our
opinion, it meets the standards of a thesis for the degree of Doctor of Philosophy in
Molecular Physiology.

Prof. Dr. Ismail Salih Kakey Prof. Dr. Qasim Hasso Abdullah
University of Koya University of Duhok
(Chairman) (Member)
Date: 22 / 10 /2015 Date: 22 / 10 /2015

Prof. Dr. Faiq H.S. Hussain Dr. Baybeen Khorsheed AL-Selevany

University of Salahaddin University of Mosul
(Member)
(Member)
Date: 22 / 10 /2015
Date: 22 / 10 /2015

Dr. Mudhir S. Shekha

University of Salahaddin
(Member)
Date: 22 / 10 /2015

Prof. Dr. Omar A.M. Al-Habib Prof. Dr. Giovanni Vidari

University of Zakho University of Pavia/Italy
(Member and Supervisor) (Member and Supervisor)
Date: 22 / 10 /2015 Date: 22 / 10 /2015

Approved by the College Committee of Graduate Studies

Prof. Dr. Fadhil H. Easif
Dean of the Faculty of Science
Date: / /2015
 I would like to dedicate this study to my spouse and soulmate (Aveen), who
has been a constant source of support throughout the years of my postgraduate
study. In addition, I would like to thank my family, especially my mother and
father for constant support and encouragement.

Wonderful my lovely sons Muhammad, Mustafa and daughter Sara

for always bringing sunshine and smiles to my life.

To my Brothers and Sisters

To all whom I love

 Acknowledgement

Thanks God, the most merciful and compassionate, for giving me the strength and
guidance I needed to face today and not worry about tomorrow.
First and foremost, I would like to express the utmost gratitude to my supervisors Prof.
Dr. Omar A.M. Al-Habib (Vice president of Post-graduate and Scientific affairs, Zakho
University) and Prof. Dr. Vidari Giovani (in the Organic Chemistry Deparment, Pavia
University, Italy) for their time, support, wisdom, patience, understanding, generous and
valuable assistance in the preparation and completion of this research work. Through them,
I think I have learned a lot and accomplished more than ever possible.
I’m very thankful to President of Zakho University, Assist. Prof. Dr. Lazgin A. Jamil
for his considerable supports and facilitation which enabled me to finalize this study. I am
also deeply grateful to Prof. Dr. Fadhil H. Easif, Dean of the Faculty of Science and Prof.
Dr. Wijdan M. S. Mero, Head of Biology Dept. for their continuous encouragement and
support throughout my studies.
I extent my thanks to my dearest and great brother Dr. Mudhir S. Sheikha for his
promote, care with his inputs and many motivating discussions. My deep appreciation and
sincere thanks to Dr. Davide Gozzoni for his support and allowing me to use facilities for
performing the extraction part of the work in Dipartimento di Chimica e Centro CISTRE
dell'Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy. Furthermore, I want to
extend my thanks and appreciation to Dr. Zana R. Majid for an excellent guidance
alongside my research. I would also like to express my gratefulness to Mr. Sardar Saaid,
Dr. Fakhir Najim, Mr. Diler Shekhany and Mr. Bassim for having the pleasure to work
with them and for their invaluable supports, during all the phases of my work. In addition,
I would like to thank all the staff members in the Department of Biology and to Mr.
Thamer and Mr. Muhammad (Animal house staffs).
Finally, I have to say that the sincerity, the patience and the encouragement from my
spouse Aveen and our three children, Muhammad, Mustafa and Sara, have been my
inspiration as I hurdled all the obstacles in the completion of all the requirements needed
for my doctoral study.

I
 Abbreviations

LIST OF ABBREVIATIONS
Abbreviations Definitions
ACh Acetylcholine
ADP Adenosine diphosphate
App. Appendix
4-AP 4-Aminopyridine
ATP Adenosine triphosphate
α Alpha
BaCl2 Barium chloride
BKCa Large conductance calcium-activated potassium
β Beta
C. Crataegus
Ca++ Calcium
[Ca++]i Intracellular calcium concentration
CaCl2 Calcium chloride
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
CI 95% Confidence interval 95%
CO2 Carbon dioxide
CVD Cardiovascular disease
CVDs Cardiovascular diseases
DAG Diacylglycerol
DCM Dichloromethane
DNA Deoxyribonucleic acid
EA Euscaphic acid
EDHF Endothelial-derived hyperpolarizing factor
eNOS Endothelial nitric oxide synthase
ESI-MS Electrospray ionization-mass spectrometry
EtOAc Ethyl acetate
GLIB Glibenclamide
HPLC High performance liquid chromatography
IKCa Intermediate conductance calcium-activated potassium
IP3 Inositol 1,4,5-trisphosphate
K+ Potassium
KATP Adenosine triphosphate-sensitive potassium
KCa Calcium-activated potassium
Kir Inward-rectifier potassium

II
 Abbreviations

KV Voltage-dependent potassium
LC-MS Liquid Chromatography-Mass spectroscopy
L-NAME L-nitro arginine methyl ester
Log EC50 Logarithm half maximal effective concentration
MeOH Methanol
MLC Myosin light chain
MLC20 20-kDa myosin light chain
MLCK Myosin light chain kinase
MLCP Myosin light chain phosphatase
MPLC Middle performance liquid chromatography
MTBE Methyl tert-butyl ether
Na+ Sodium
Na+/K+-ATPase Sodium-potassium pump
NE Norepinephrine
NMR Nuclear magnetic resonance
NO Nitric oxide
NOS Nitric oxide synthases
O2 Dioxygen
PGI2 Prostaglandin I2
PKA Protein kinase A
PKC Protein kinase C
PKG Protein kinase G
ROCC Receptor operated Ca++ channel
ROCK Rho kinase
ROS Reactive oxygen species
RYR Ryanodine receptor
SERCA Sarcoplasmic reticulum Ca++-ATPase
SEM Standard error mean
sGC Soluble guanylyl cyclase
SKCa Small conductance calcium-activated potassium
SMCs Smooth muscle cells
SOCCs Store-operated Ca++ channels
SPE Solid phase extraction
SR Sarcoplasmic reticulum
SURs Sulphonylurea receptors
TEA Tetraethylammonium
TLC Thin layer chromatography
UV Ultraviolet
III
 Abbreviations

VDCCs Voltage-dependent calcium channels

Vm Membrane potential
VSMCs Vascular smooth muscle cells

IV
 ABSTRACT

The present study included the isolation, purification and identification of

bioactive compounds from the leaf of Crataegus (C.) azarolus var. aronia L.,
collected from Kurdistan Region- Iraq by using state-of-the-art organic and
analytical methods, including thin layer chromatography (TLC) (silica gel or

reverse phase C- 18), column chromatography, 1H and 13C nuclear magnetic

resonance (NMR) spectra, electrospray ionization mass spectrometry (ESI- MS)and
others.

The results of the ethyl acetate (EtOAc) extract of C. azarolus leaves revealed
the presence of nine different triterpenoids and flavonoids, one o f them was
novel triterpenoid and discovered for the first time in the field of chemistry and
isolated from a plant source, namely azarolic acid (5). Furthermore, additional four
triterpenoids, namely euscaphic acid (EA) (jacaranoic acid) (2), 2α,
19α dihydroxy 3 oxo urs 12 en 28 oic acid (3), 2- oxopomolic acid (4) and arjunic
acid (7) were isolated for the first time from C. azarolus. In addition, the current
study also re- confirmed the presence of (- )- epicatechin(1),
4”’ acetylvitexin 2” O rhamnoside (8), vitexin 2” O- rhamnoside (9) and vitexin
(10) in C. azarolus which were already previously detected in Crataegus spp.

Furthermore, the physiological part of the current work included the

anti-vasoconstriction effects of EtOAc, methanol (MeOH) extracts, and EA,
prepared from C. azarolus on isolated aortic rings. PowerLab Data Acquisition
Organ- Bath System(ADI) was used to measure the isometric tension resulted from

the aortic contraction. Prior to the experiment, the organ bath was set at 37 oC for
at least one hour, followed by the addition 10 ml of Kreb’s or calcium (Ca++)
free solutions to the glass tissue chamber. The preparation was aerated
V
continuously with 95 % dioxygen (O2) and 5 % carbon dioxide (CO2). Aortic rings
were connected to the base of the chamber from one end and to the force
transducer from the other end. The initial tension was set at 2 g weight and left for
60- 90minutes with changing the solution every 15 minutes until a stable resting
tone was recorded, and then experiments were conducted.

The first part of the work aimed to study the role of endothelium derived
relaxing factors in anti-vasoconstriction effects induced by EtOAc, MeOH extracts
and EA in aortic rings using different blockers and drugs of different molecular
targets, such as L- nitro arginine methyl ester (L- NAME), indomethacin and
methylene blue. Furthermore, the study also included the role of potassium (K+)
channel subtypes and L- type Ca++ channels in mediating anti-vasoconstriction
effects of EtOAc, MeOH extracts and EA on rat aorta to obtain insights into
underlying the mechanisms of anti-vasoconstriction action of the above extracts and
compound.

Ethyl acetate extract produced a significant anti-vasoconstriction effect on

endothelium intact and denuded aortic rings and significantly reduced the
contraction induced by norepinephrine (NE) as compared to control rings.
Furthermore, EtOAc extract at a low concentration (1X10- 2 mg/ml) produced a
weak anti-vasoconstriction effect on aortic rings pre- incubatedwith indomethacin
(prostaglandin I2 (PGI2) inhibitor), whereas, at a higher concentration (3X10- 2
mg/ml) had a more potent anti- vasoconstrictioneffect as compared with control
group. On the other hand, EtOAc extract showed a higher anti- vasoconstriction
effect on aortic rings pre- incubated with methylene blue (cyclic guanosine
monophosphate (cGMP) inhibitor) and significantly reduced the contraction in both
cases, pre- incubated with indomethacin and methylene blue. Whereas,

VI
endothelial- derived nitric oxide (NO) did not play a noticeable role in
anti- vasoconstriction action of EtOAc extract since L- NAME application, the
endothelial nitric oxide synthase (eNOS) inhibitor, did not change the dose-
response curves of NE in absence and presence of extract.

In aortic rings pre- incubated with barium chloride (BaCl2) (inwardly

rectifying potassium (KIR) channel blocker) a low concentration of EtOAc extract
produced weak anti- vasoconstrictioneffects; whereas, at a higher concentration, it
produced a more potent effect, as compared to control rings, and reduced the
contraction. In contrast, the EtOAc extract failed to change the contraction
responses of aortic rings that pre- incubated with tetraethylammonium (TEA)
(calcium activate potassium (KCa) channel blocker), glibenclamide (GLIB)
(adenosine triphosphate sensitive potassium (KATP) channel blocker) and
4- aminopyridine (4- AP)(voltage- gatedpotassium (KV) channel blocker).

Ethyl acetate extract significantly suppressed the contraction induced by

calcium chloride (CaCl2) in aortic rings pre- incubatedwith verapamil (L- typeCa++
channel blocker) and significantly reduced the contraction as compared to control
group.

Methanol extract at concentrations (1X10- 2 and 3X10- 2 mg/ml) produced

anti- vasoconstrictioneffect on NE induced contraction in both endothelium intact
and denuded aortic rings in which the maximum contraction was significantly
reduced as compared with the control group. In aortic rings pre- incubated with
indomethacin and methylene blue, MeOH extract shifted the NE dose-response
curves to the right and the contraction was significantly decreased as compared to
the control groups. Similarly to EtOAc extract, endothelial- derivedNO did not play

VII
a noticeable role in anti- vasoconstriction action of MeOH extract, and did not
change the anti- vasoconstrictionaction of the extract.

Furthermore, in aortic rings pre- incubated with GLIB and 4- AP, MeOH
extract showed a potent inhibitory effect on NE induced contraction, in which
the contraction was significantly decreased as compared to the control group. In
aortic rings pre- incubated with TEA, MeOH extract at a low concentration
produced a moderate anti- contractioneffect on NE induced contractions, whereas
at a higher concentration, it produced a more potent anti-vasoconstriction effect.
Also, in aortic rings pre- incubated with BaCl2, MeOH extract at a concentration
(1X10- 2 mg/ml) produced a weak anti-vasoconstriction effect, whereas at a higher
concentration produced a more potent anti-vasoconstriction effect, as compared
with the results of the control experiments. In addition, MeOH extract also reduced
the contraction in aortic rings pre- incubatedwith TEA and BaCl2.

Both doses of MeOH extract (1X10-2 and 3X10-2 mg/ml) produced a potent
inhibitory effect on CaCl2 contracted aortic rings pre- incubated with verapamil,
and significantly reduced the maximum contraction as compared to control rings.

Euscaphic acid also shifted the NE dose-response curves to the right in

endothelium- intact and denuded aortic rings, and significantly reduced the
contractions in both cases, as compared to control groups.

In aortic rings pre- incubatedwith indomethacin, EA significantly suppressed

contraction of aortic rings induced by NE and reduced the contraction than did in
control group. On the other hand, a low concentration of EA produced a weak
anti- vasoconstriction effect on aortic rings pre- incubated with methylene blue,
whereas, a higher concentration, produced a more potent anti- vasoconstriction
effect, as compared with the control rings, and also reduced the contraction. On
VIII
the other hand, NO did not play any role in anti- vasoconstrictioneffect induced by
EA.

The current results showed that in aortic rings pre- incubatedwith TEA, EA at a
low concentration produced a weak anti- vasoconstriction effect, whereas, at a
higher concentration, a more potent anti- vasoconstrictioneffect was observed, as
compared with control. Thus, amplitude of contraction was reduced from (0.875 ±
0.038) to (0.776 ± 0.044) and (0.705 ± 0.034), respectively. While, in aortic
rings pre- incubatedwith other K+ channel subtypes blockers such as GLIB, BaCl2
and 4- AP,the EA did not affect the contraction induced by NE.

In aortic rings pre- incubated with verapamil, both doses of EA produced

potent inhibitory effects on CaCl2 induced contraction as compared with the control
group, and also reduced the maximum contraction from (0.257 ± 0.012) to
(0.239 ± 0.021) and (0.164 ± 0.015), respectively.

From the results of the current study, it can be concluded that EtOAc, MeOH
extracts and EA have potent anti- vasoconstriction effects on aortic rings.
Endothelium- derived relaxant factors, K+ and Ca++ channels subtypes play
indispensable roles in anti- vasoconstrictionaction of EtOAc, MeOH extracts and
EA, which are mediated, partly, by the enhancement of PGI2 and cGMP
biosynthesis, modulating different K+ channels and L- types Ca++ channels
activities. Furthermore, the results of current study provides the mechanism of
action of some constituents of medicinal plant C. azarolus var. aronia L. which
can be exploited to develop more specific drugs to be used for the treatment of
various vascular diseases.

IX
 Title Pages
ACKNOWLEDGEMENT I
LIST OF ABBREVIATIONS II
ABSTRACT V
CONTENTS X
LIST OF FIGURES XVII
LIST OF TABLES XXIV

1. INTRODUCTION 1-6

2. LITERATURE REVIEW 7-44

2.1 Herbal Medicine 7
2.2 The Genus Crataegus (Hawthorn) 8
2.3 Phytochemical Screening of Crataegus 10
2.4 Crataegus and Cardiovascular Disorders 11
2.5 Other Physiological Activities of Crataegus 13
2.6 Phenolic compounds 15
2.7 Euscaphic Acid (Jacaranoic Acid) 17
2.8 Vascular System 18
2.8.1 Vascular Structure 19
2.8.1.1 Vascular Endothelium 19
2.8.1.1.1 Endothelial-Derived Hyperpolarizing Factors 22
2.8.1.1.1.1 Nitric Oxide/ Cyclic-Guanosin Monophosphate Pathway 22
2.8.1.1.1.2 Prostaglandin I2/ cAMP Pathway 24
2.8.1.2 Smooth Muscle 25
2.8.1.2.1 Types of Smooth Muscle 27
2.8.1.2.1.1 Visceral (Unitary or Single-Unit) Smooth Muscle 27
2.8.1.2.1.2 Multi-Unit of Smooth Muscle 28
2.8.1.2.2 Physiology of Smooth Muscle 29
2.8.1.2.2.1 Smooth Muscle Contraction and Relaxation 29
2.8.1.2.2.2 Molecular Basis of Smooth Muscle Contraction 29
2.8.1.2.2.3 Molecular Basis of Smooth Muscle Relaxation 32
2.8.1.3 Ion Channels 33
2.8.1.3.1 Potassium Channels 33
2.8.1.3.1.1 Voltage-Dependent Potassium Channels 34

X
2.8.1.3.1.2 Calcium-Activated Potassium Channels 36
2.8.1.3.1.3 Adenosine Triphosphate-Sensitive Potassium Channels 38
2.8.1.3.1.4 Inward Rectifier Potassium channels 40
2.8.1.3.2 Voltage-Dependent Calcium Channels 41
2.8.1.4 Vascular Contractile Responses 43
2.8.1.4.1 Norepinephrine 43
2.8.1.4.2 Calcium chloride 43
2.8.1.4.3 Vascular Relaxation Responses to Acetylcholine 44

3. MATERIALS AND METHODS 45-65

3.1 Phytochemical Studies 45
3.1.1 Plant Materials 45
3.1.2 Classification 45
3.1.3 Plant Extraction 46
3.1.3.1 Solvent Extraction 46
3.1.3.1.1 Hexane Extraction 46
3.1.3.1.2 Ethyl Acetate Extraction 46
3.1.3.1.3 Absolute Methanol Extraction 47
3.1.3.1.4 Methanol 60% Extraction 47
3.1.4 Chromatography 49
3.1.4.1 Thin Layer Chromatography 49
3.1.4.2 Reagent Mechanism of Coloration 49
3.1.4.2.1 Vanillin/H2SO4 Mechanism 49
3.1.4.3 Solid Phase Extraction 49
3.1.4.4 Middle Performance Liquid Chromatography 50
3.1.4.4.1 Instrumentation 50
3.1.4.4.2 Procedure 50
3.1.4.4.3 Assay conditions 50
3.1.4.4.4 Gradient Elution 51
3.1.4.5 Column Chromatography 51
3.1.4.5.1 Introduction 51
3.1.4.5.2 Stationary Phase Selection 52
3.1.4.5.3 Mobile Phase Selection 53
3.1.4.5.4 Packing the Column 53
3.1.4.5.5 Sample Loading 54
3.1.4.5.6 Monitoring the Column 55
3.1.4.5.7 Isolation of the Separated Compounds 55
3.1.4.5.8 Flash Chromatography 55

XI
3.1.5 Spectroscopic Methods 56
3.1.5.1 Nuclear Magnetic Resonance Spectroscopy 56
3.1.5.1.1 Theory 56
3.1.5.1.2 Sample Preparation for 1H, 13C NMR Measurement 57
3.1.5.2 Liquid Chromatography-Mass spectroscopy (LC-MS) 57
3.2. Physiological Studies 57
3.2.1 Animal Housing and Breeding 57
3.2.2 Chemicals and Preparation of Solutions 58
3.2.2.1 Chemicals 58
3.2.2.2 Instruments 59
3.2.2.3 Preparation of Solutions 60
3.2.2.3.1 Kreb’s and Kreb’s Calcium Free Solutions 60
3.2.2.3.2 Stock Solutions 61
3.2.3 Animal Experiments 61
3.2.3.1 Preparation of Aorta 61
3.2.3.2 Measurement of Isometric Contraction in Isolated Rat Aorta 61
3.2.3.3 Experimental Protocols 63
3.2.3.3.1 Group I 63
3.2.3.3.2 Group II 63
3.2.3.3.3 Group III 64
3.2.3.3.4 Group IV 64
3.2.3.3.5 Group V 65
3.2.4 Statistical Analysis 65

4. RESULTS AND DISCUSSIONS 66-146

4.1 Phytochemical Results and Discussions 66
4.1.1 Preliminary Phytochemical Analysis 66
4.1.2 Chromatographic Separation of the Ethyl Acetate Fraction by Middle
67
Performance Liquid Chromatography
4.1.2.1 Purification and Identification of Epicatechin 71
4.1.2.2 Purification and Identification of Euscaphic acid 73
4.1.2.3 Purification and Identification of 2α, 19α-dihydroxy-3-oxo-urs-12-
78
en-28-oic Acid
4.1.2.4 Isolation and Identification of 2-Oxopomolic Acid 80
4.1.2.5 Isolation and Structure Determination of Azarolic Acid 81
4.1.2.6 Isolation and Identification of Arjunic Acid 86
4.1.2.7 Flavonoid Compounds 89
4.2 Physiological Results 97

XII
4.2.1 Anti-vasoconstriction Effects of Ethyl Acetate Extract on Aortic
97
Rings contracted with Norepinephrine or Calcium Chloride
4.2.1.1 Anti-vasoconstriction Effect of Ethyl Acetate Extract on
Endothelium-intact Aortic Rings 97

4.2.1.2 Anti-vasoconstriction Effect of Ethyl Acetate Extract on

Endothelium-denuded Aortic Rings 100

4.2.1.3 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Rat’s

Aortic Rings Pre-incubated with each of L-NAME, Indomethacin and 102
Methylene blue

4.2.1.3.1 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

102
Rings Pre-incubated with L-NAME

4.2.1.3.2 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Rat

103
Aortic Rings Pre-incubated with Indomethacin

4.2.1.3.3 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

105
Rings Pre-incubated with Methylene blue

4.2.1.4 Anti-vasoconstriction Effect of Ethyl Acetate Extract on

Endothelium-intact Rat Aortic Rings Pre-incubated with each of Potassium 107
Channels Sub-type Blockers

4.2.1.4.1 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

107
Rings Pre-incubated with Tetraethylammonium

4.2.1.4.2 Anti-vasoconstriction Effect of Ethyl Acetate Extract on

108
Endothelium-intact Rat Aortic Rings Pre-incubated with Glibenclamide

4.2.1.4.3 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

109
Rings Pre-incubated with Barium Chloride

4.2.1.4.4 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

110
Rings Pre-incubated with 4-Aminopyridine

4.2.1.5 Anti-vasoconstriction Effect of Ethyl Acetate Extract on Aortic

111
Rings Pre-incubated with Verapamil (L-type Ca++ Channel Blocker)
4.2.2 Anti-vasoconstriction Effect of Crataegus azarolus Methanol Extract
113
on Rat’s Aortic Rings Contracted with Norepinephrine or Calcium

XIII
Chloride
4.2.2.1 Anti-vasoconstriction Effect of Methanol Extract on Endothelium-
113
intact Rat Aortic Rings
4.2.2.2 Anti-vasoconstriction Effect of Methanol Extract on Endothelium-
115
denuded Rat Aortic Rings
4.2.2.3 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings
Pre-incubated Separately with each of L-NAME, Indomethacin and 117
Methylene blue

4.2.2.3.1 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

117
Pre-incubated with L-NAME

4.2.2.3.2 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

118
Pre-incubated with Indomethacin

4.2.2.3.3 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

120
Pre-incubated with Methylene blue

4.2.2.4 Anti-vasoconstriction Effect of Methanol Extract on Rat’s Aortic

122
Rings Pre-incubated with each of Potassium Channels Sub-type Blockers

4.2.2.4.1 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

122
Pre-incubated with Tetraethylammonium

4.2.2.4.2 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

124
Pre-incubated with Glibenclamide

4.2.2.4.3 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

126
Pre-incubated with Barium Chloride

4.2.2.4.4 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

128
Pre-incubated with 4-Aminopyridine

4.2.2.5 Anti-vasoconstriction Effect of Methanol Extract on Aortic Rings

130
Pre-incubated with Verapamil (L-type Ca++ Channel Blocker)
4.2.3 Anti-vasoconstriction Effects of Euscaphic Acid on Endothelium-
intact Rat Aortic Rings contracted with Norepinephrine or Calcium 132
Chloride
4.2.3.1 Anti-vasoconstriction Effect of Euscaphic Acid on Endothelium- 132

XIV
intact Aortic Rings
4.2.3.2 Anti-vasoconstriction Effect of Euscaphic Acid on Endothelium-
denuded Rat Aortic Rings 133

4.2.3.3 Anti-vasoconstriction Effect of Euscaphic Acid on Endothelium-

intact Rat Aortic Rings Pre-incubated with each of L-NAME, 135
Indomethacin and Methylene blue

4.2.3.3.1 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

135
Pre-incubated with L-NAME

4.2.3.3.2 Anti-vasoconstriction Effect of Euscaphic Acid on Rat Aortic

136
Rings Pre-incubated with Indomethacin

4.2.3.3.3 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

138
Pre-incubated with Methylene blue

4.2.3.4 Anti-vasoconstriction Effect of Euscaphic Acid on Endothelium-

intact Rat Aortic Rings Pre-incubated with some of Potassium Channels 140
Sub-type Blockers

4.2.3.4.1 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

140
Pre-incubated with Tetraethylammonium

4.2.3.4.2 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

141
Pre-incubated with Glibenclamide

4.2.3.4.3 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

142
Pre-incubated with Barium Chloride

4.2.3.4.4 Anti-vasoconstriction Effect of Euscaphic Acid on Aortic Rings

143
Pre-incubated with 4-Aminopyridine

4.2.3.5 Anti-vasoconstriction Effect of Euscaphic Acid on Endothelium-

intact Rat Aortic Rings Pre-incubated with Verapamil (L-type Ca++ 144
Channel Blocker)

5. PHYSIOLOGICAL DISCUSSIONS 147-158

5.1 Anti-vasoconstriction Effects of Ethyl Acetate Extract on Rat Aortic 147

XV
Rings

5.1.1 Role of Endothelium and Endothelium Derived Relaxing Factors in

147
Ethyl Acetate Extract Anti-vasoconstriction Effects

5.1.2 Role of Potassium Channel Subtypes in Anti-vasoconstriction

148
Effects of Ethyl Acetate Extract

5.1.3 Role of Calcium Channels (L-type VDCCs) in Anti-vasoconstriction

149
Effects of Ethyl Acetate Extract

5.2 Anti-vasoconstriction Effects of Methanol Extract on Rat Aortic Rings 150

5.2.1 Role of Endothelium and Endothelium Derived Relaxing Factors in

150
Anti-vasoconstriction Effects of Methanol Extract
5.2.2 Role of Potassium Channel Subtypes in Anti-vasoconstriction
Effects of Methanol Extract 153

5.2.3 Role of Calcium Channels (L-type VDCCs) in Anti-vasoconstriction

154
Effects of Methanol Extract

5.3 Anti-vasoconstriction Effects of Euscaphic Acid on Norepinephrine or

155
Calcium Chloride-induced Contraction in Rat Aortic Rings

5.3.1 Role of Endothelium and Endothelium Derived Relaxing Factors in

155
Euscaphic Acid Anti-vasoconstriction Effects

5.3.2 Effect of Euscaphic Acid on the Activity of Potassium Channel

156
Subtypes

5.3.3 Effect of Euscaphic Acid on the Activity of Calcium Channels (L-

157
type Ca++ Channels)
CONCLUSIONS AND RECOMMENDATIONS 159-161
CONCLUSIONS 159
RECOMMENDATIONS 161
REFERENCES 162-198
APPENDIX I 199
APPENDIX II 209
ABSTRACT IN ARABIC -
ABSTRACT IN KURDISH A-F

XVI