pharmacological investigations on some indigenous …

PHARMACOLOGICAL INVESTIGATIONS ON SOME

INDIGENOUS PLANTS OF CHOLISTAN DESERT

Thesis submitted in partial fulfillment of the requirement for the degree of

Ph.D. Pharmacy (Pharmacology)

By

NAVEED ASLAM Registration No. 2013-BZBP-10

Roll No. 02-PhDL-13, Session: 2013

Supervisor

PROF. DR. KHALID HUSSAIN JANBAZ Ph.D. (Pharmacology)

FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN,

PAKISTAN

CERTIFICATE OF DECLARATION

I hereby declare that this Ph.D. thesis entitled “Pharmacological investigations on some

indigenous plants of Cholistan desert” is written by me and no material included in this thesis

has been stolen or copied from anywhere. Proper references are cited wherever necessary.

Naveed Aslam Roll No. 02-PhDL-13 Session: 2013

V

DEDICATED

TO

MY PARENTS AND MY WIFE

VI

Acknowledgements

Thanks to Allah almighty for giving me power to concentrate and to complete my research.

In journey towards completing my research work, many people made important

contributions. I would like to extend my heartfelt gratitude to them.

Special thanks to my supervisor Prof. Dr. Khalid Hussain Janbaz for his continued guidance,

encouragement and having faith in my abilities. He extended his full support in planning and

carrying out my research work and ensured while observing that I don’t get detracted from

essential part of the study. Without his support, this research work would not have been

possible.

I felt lucky to be part of good support system provided to students by the Chairman,

Department of Pharmacy, Bahauddin Zakariya University, Multan for completion of research

work.

It gives me immense pleasure to share that Dr. Qaiser Jabeen (Associate Professor,

Department of Pharmacy, the Islamia University of Bahawalpur, Pakistan) remained a great

source of inspiration during experimentation and I thank her for her brainy ideas, for taking

out time from her busy schedule and showing me the way ahead.

I would also like to thanks to my class fellows Fatima Saquib, Ambreen Aleem, Irfan Hamid

and Laboratory staff, Mr. Tanveer Ahmed and Mr. Muhammad Ishfaq, for providing true

support during experiments. I could not have worked well without them. I knew that I could

always rely on them for assistance in Lab work.

Being an employee of Health Department, Government of the Punjab, I am highly grateful to

administration of Health Department for allowing me to enroll in and complete my Ph.D.

research work.

Last but not the least; I pay thanks to my family members for their unconditional support,

love, patience and encouragement. I am also thankful to everyone who help me directly or

indirectly during my whole work.

Naveed Aslam

VII

Contents

Acknowledgements .......................................................................................... VI

Contents ........................................................................................................... VII

Abstract .................................................................................................... IXXVII

List of abbreviations ..................................................................................... XIII

List of figures ................................................................................................... XV

List of tables............................................................................................... XXVII

1. General introduction ............................................................................ 1-12

1.1. Cholistan desert ...........................................................................................................2

1.2. Vegetation of Cholistan desert ....................................................................................2

1.3. Medicinal uses of plants ..............................................................................................4

1.4. Medicinal plant research .............................................................................................5

1.4.1. History and terminology...................................................................................5

1.4.2. Plants as source of new drug ............................................................................6

1.4.3. Positive interactions within phytomedicine .....................................................7

1.5. Gut, airway and cardiovascular disorders ...................................................................8

1.5.1. Gut motility disorders.......................................................................................8

1.5.2. Airway disease (Asthma) ...............................................................................10

1.5.3. Cardiovascular disorders ................................................................................11

1.6. Objectives of the present study .................................................................................12

2. Material and Methods ........................................................................ 13-23

2.1. Chemicals ..................................................................................................................14

2.2. Animals for experiments ...........................................................................................14

2.3. Test material ..............................................................................................................14

2.4. Phytochemical investigations on crude extracts.......................................................15

2.5. In vitro experimentation ...........................................................................................17

2.5.1. Isolated preparations of rabbit jejunum .........................................................17

2.5.2. Isolated preparations of rabbit trachea ..........................................................19

2.5.3. Isolated preparations of rabbit aorta ..............................................................20

VIII

2.5.4. Isolated preparations of rabbit paired atria ....................................................20

2.6. In vivo experimentation ............................................................................................21

2.6.1. Anti-diarrheal activity in mice ......................................................................21

2.6.2. Influence upon charcoal meal transit distance in mice .................................21

2.6.3. Laxative activity in mice ...............................................................................22

2.6.4. Influence on blood pressure in anesthetized rats ...........................................22

2.6.5. Diuretic activity in rats ..................................................................................23

2.7. Statistical analysis ....................................................................................................23

3. Asphodelus tenuifolius Cav. ............................................................... 24-53

3.1. Plant introduction ......................................................................................................25

3.2. Plant material and extraction .....................................................................................26

3.3. Results .......................................................................................................................27

3.3.1. Preliminary phytochemical analysis...............................................................27

3.3.2. In vitro experiments .......................................................................................27

3.3.3. In vivo experiments ........................................................................................41

3.4. Discussion .................................................................................................................49

3.5. Conclusion .................................................................................................................52

4. Corchorus depresuss Linn. ................................................................. 54-87



4.3. Results .......................................................................................................................57

4.3.1. Preliminary phytochemical analysis...............................................................57


4.3.3. In vivo experiments ........................................................................................75

4.4. Discussion .................................................................................................................84

4.5. Conclusion .................................................................................................................87

5. Gisekia pharnaceoides Linn. ............................................................ 88-131



5.3. Results .......................................................................................................................91

5.3.1. Preliminary phytochemicals analysis .............................................................91

IX


5.3.3. In vivo experiments ......................................................................................120

5.4. Discussion ...............................................................................................................127

5.5. Conclusion ...............................................................................................................131

6. Salsola imbricata Forssk. ................................................................ 132-168

6.1. Plant introduction ....................................................................................................133

6.2. Plant material and extraction ...................................................................................134

6.3. Results .....................................................................................................................135

6.3.1. Preliminary phytochemicals analysis ...........................................................135

6.3.2. In vitro experimentation ...............................................................................135

6.3.3. In vivo experimentation ................................................................................156

6.4. Discussion ...............................................................................................................164

6.5. Conclusion ...............................................................................................................167

7. General discussion and conclusion ............................................... 169-173

8. References ....................................................................................... 174-194

9. Publications ............................................................................................. 195

X

Abstract

Aims of study

The present study was undertaken to validate traditional medicinal claims and to further

explore pharmacological actions of four indigenous plants of Cholistan desert (Asphodelus

tenuifolius, Corchorus depressus, Gisekia pharnaceoides and Salsola imbricata) in relation to

gastrointestinal, respiratory and cardiovascular systems.

Material and methods

The dried and powdered plants materials were extracted by maceration using aqueous-ethanol

as solvent to obtain crude extracts. The crude extracts, their ethyl acetate and aqueous fractions

were tested for gastrointestinal, bronchodilator and cardiovascular activities; in vitro

experiments were performed upon isolated tissue preparations of rabbit using standard tissue

organ bath techniques and in vivo experiments were performed in mice and rats.

Results

The crude extract of Asphodelus tenuifolius (Cr.At) relaxed spontaneous, potassium (25 mM)

and potassium (80 mM) mediated contractions in isolated jejunum preparations, while being

equipotent in relaxing potassium (25 mM) and potassium (80 mM) mediated contractions, in

manner similar to verapamil. Pretreatment of jejunum preparations with Cr.At or verapamil

shifted calcium concentration response curves (CRCs) rightward with suppression of maximum

effect. Aqueous fraction of Cr.At exhibited contractile effect upon spontaneous contractions in

jejunum preparations. Oral administration of Cr.At to mice caused significant increase of

charcoal meal intestinal transit at dose of 100 mg per kg; enhanced wet and total feces counts at

doses of 50 and 100 mg per kg; but it significantly decreased charcoal meal intestinal transit at

500 mg per kg dose. Cr.At (300, 500 and 700 mg per kg; orally) reduced castor oil induced

diarrhea in mice. In rabbit isolated tracheal preparations, Cr.At was more potent against

potassium (80 mM) than carbachol (1 µM) mediated contractions and shifted carbachol CRCs

rightward in non-parallel fashion. In rabbit isolated aortic preparations (endothelium denuded),

Cr.At was more potent against potassium (80 mM) than phenylephrine (1 µM) mediated

contractions. In isolated rabbit paired atria, Cr.At caused overall negative inotopic and

chronotropic effects. Intravenous administration of Cr.At (3-30 mg per kg) to anesthetized

normotensive rats produced hypotensive effect, which remained unchanged on pre-treating the

XI

animals with atropine. Spasmolytic constituents were found partitioned in ethyl acetate fraction

of Cr.At, whereas aqueous fraction was found to contain spasmogenic activity.

The crude extract of Corchorus depressus (Cr.Cd) relaxed spontaneous, potassium (25 mM),

potassium (80 mM) and carbachol (1 µM) mediated contractions in rabbit isolated jejunum

preparations; being most potent in relaxing carbachol (1 µM) mediated contractions, and

equally potent in relaxing potassium (25 mM) and potassium (80 mM) mediated contractions,

similar to that of dicyclomine. It shifted calcium CRCs upon jejunum preparations rightward in

non-parallel manner, similar to that of dicyclomine. Aqueous fraction of Cr.Cd exhibited

atropine sensitive contractile effect on spontaneous contractions of rabbit jejunum preparations.

Oral administration of Cr.Cd to mice (50 and 100 mg per kg) caused significant increase in

charcoal meal intestinal travel, enhanced formation of wet and total stool; but at 300 and 500

mg per kg doses, it significantly decreased charcoal meal intestinal travel. It also reduced castor

oil induced diarrhea in mice at oral doses of 500 and 700 mg per kg. In isolated tracheal

preparations, Cr.Cd was more potent in relaxing carbachol (1 µM) than potassium (80 mM)

mediated contractions and shifted carbachol CRCs rightward, similar to that of dicyclomine. In

isolated rabbit aorta, Cr.Cd relaxed potassium (80 mM) mediated contractions. In isolated rabbit

paired atria, Cr.Cd was found to possess atropine sensitive cardio-depressant activity.

Intravenous administration of Cr.Cd (1-30 mg per kg) to anesthetized normotensive rats

produced hypotensive effect, which was reversed upon pre-treating animals with atropine.

Gisekia pharnaceoides extract (Cr.Gp) and its ethyl acetate fraction caused preferential

inhibition of potassium (25 mM) than potassium (80 mM) mediated contractions in rabbit

isolated jejunum, tracheal and aortic preparations; which was attenuated in presence of

glibenclamide (3 µM), in a manner similar to that of cromakalim. The aqueous fraction of

Cr.Gp produced contractions on baseline of rabbit jejunum, tracheal and aortic preparations;

which were attenuated subsequent to pre-incubation of tissues with caffeine (10 mM). Cr.Gp

was found to possess glibenclamide sensitive cardio-depressant effects upon spontaneously

beating rabbit paired atria. Oral administration of Cr.Gp to mice (50 and 100 mg per kg)

resulted significant inhibition of castor oil induced diarrhea as well as significant decrease in

charcoal meal intestinal travel; but at higher doses (300 and 500 mg per kg), it significantly

increased charcoal meal intestinal travel and increased formation of diarrheal feces in normal

mice. Intravenous administration of Cr.Gp (1-30 mg per kg) to anesthetized normotensive rats

produced hypotensive effect.

XII

The crude extract of Salsola imbricata (Cr.Si) relaxed spontaneous, potassium (25 mM),

potassium (80 mM) and carbachol (1 µM) mediated contractions in smooth muscle preparations

of rabbit isolated jejunum. Pretreatment of tissue preparations with Cr.Si shifted calcium CRCs

toward right with suppression of maximum effect, in a manner similar to verapamil.

Pretreatment of tissue with propranolol (1 µM) partially antagonized the relaxant activity of the

extract upon carbachol mediated contractions, similar to that of isoprenaline. Oral

administration of Cr.Si (100, 300 and 500 mg per kg) in mice caused inhibition of charcoal

meal intestinal travel and prevented castor oil induced diarrhea. In smooth muscle preparations

of rabbit isolated trachea, Cr.Si relaxed potassium (80 mM) and carbachol (1 µM) mediated

sustained contractions equipotently; shifted carbachol CRCs rightward in non-parallel fashion;

and propranolol (1 µM) decreased its relaxant effect upon carbachol (1 µM) mediated

contractions. In isolated aortic preparations, the Cr.Si caused doxazosin sensitive contractions

upon baseline tension and relaxed potassium (80 mM) mediated contractions. Cr.Si

demonstrated cardiotonic activity upon spontaneously beating paired atria, which was reversed

in presence of propranolol (1 µM). Intravenous administration of Cr.Si (1-30 mg per kg) to

normotensive anesthetized rats produced dose dependent hypertensive effect, which was

blocked in co-presence of propranolol and doxazosin. Calcium channel blocking constituents

were found to be concentrated in ethyl acetate fraction, whereas aqueous fraction of Cr.Si was

found responsible for aortic contractions.

The crude extracts, when administered orally to rats, increased urine and urinary electrolytes

(Na+, K+ and Cl-) excretion to different extent.

Conclusion

The study concludes that Cr.At, Cr.Cd and Cr.Gp possess gut modulatory, bronchorelaxant,

hypotensive and diuretic activities; whereas Cr.Si was found to exhibit gut relaxant,

bronchorelaxant, hypertensive and diuretic activities. Possible mechanisms responsible for the

observed pharmacological activities of the extracts include, but not limited to, calcium channel

blocking mechanism in Asphodelus tenuifolius; calcium channel blocking, muscarinic and anti-

muscarinic mechanisms in Corchorus depressus; ATP dependent potassium channel opening

and calcium channel blocking mechanisms in Gisekia pharnaceoides; and calcium channel

blocking and adrenergic receptors agonistic mechanisms in Salsola imbricata. The study

validated some of the ethnic medicinal claims of the plants along with mechanistic background.

XIII

List of abbreviations

Ach Acetylcholine

ANOVA Analysis of variance

Aq.At Aqueous fraction of Asphodelus tenuifolius extract

Aq.Cd Aqueous fraction of Corchorus depressus crude extract

Aq.Gp Aqueous fraction of Gisekia pharnaceoides crude extract

Aq.Si Aqueous fraction of Salsola imbricata crude extract

ATP Adenosine triphosphate

Ca+2 Calcium ion

CCh Carbachol

CI Confidence interval

Cl- Chloride ion

cm Centimeter

CO2 Carbon dioxide

Cr.At Crude extract of Asphodelus tenuifolius

Cr.Cd Crude extract of Corchorus depressus

Cr.Gp Crude extract of Gisekia pharnaceoides

Cr.Si Crude extract of Salsola imbricata

CRCs Concentration response curves / response versus concentration curves

DBP Diastolic blood pressure

DMSO Dimethyl sulfoxide

DPPH 2,2-diphenyl-1-picrylhydrazyl

Ea.At Ethyl acetate fraction of Asphodelus tenuifolius extract

Ea.Cd Ethyl acetate fraction of Corchorus depressus crude extract

Ea.Gp Ethyl acetate fraction of Gisekia pharnaceoides crude extract

Ea.Gp Ethyl acetate fraction of Salsola imbricata crude extract

EC50 Median effective concentration / median response concentration

EDTA Ethylene diamine tetra acetic acid

g Gram

H2O Water

HR Heart rate

hrs Hours

XIV

i.p. Intraperitoneal

K+ Potassium ion

KCl Potassium chloride

kg Kilogram

MBP Mean blood pressure

mg Milligram

min Minutes

ml Milliliter

mM Millimolar

n Number of observations

Na+ Sodium ion

NE Norepinephrine

O2 Oxygen

P p-value, significance level of the test

p.o. Per os / by oral route

PE Phenylephrine

Prop Propranolol

rpm Revolutions per minute

S.E.M. Standard error mean

SBP Systolic blood pressure

T.O.B.C. Tissue organ bath concentration

v/v Volume by volume

vs. Versus

w/v Wight by volume

WHO World Health Organization

α Alpha

β Beta

% Percent

& And

µg Microgram

µM Micro molar

µmol Micro mole

⁰C Centigrade

XV

List of figures

Fig. 1.1: Map of Cholistan desert ..............................................................................................3

Fig. 1.2: Flow chart for suggested scientific studies of medicinal plants used in traditional systems of medicine. ..................................................................................................................7

Fig. 3.1: Asphodeuls tenuifolius flowering plant. ....................................................................24

Fig. 3.2: Tracing showing spontaneous movements of smooth muscle preparations of rabbit isolated jejunum. ......................................................................................................................29

Fig. 3.3: Record of influence of cumulatively added Cr.At upon rhythmic periodic contractile movements in smooth muscle preparations of rabbit isolated jejunum. ..................................29

Fig. 3.4: Record of Cr.At influence upon cumulative addition to potassium (25 mM) mediated contractility of isolated preparation of rabbit jejunum. ............................................29

Fig. 3.5: Record of influence of cumulative addition of Cr.At upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum. ...................30

Fig. 3.6: Influence of Cr.At upon spontaneous, potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated jejunum. .................30

Fig. 3.7: Verapamil influence upon spontaneous contractions, potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated jejunum.. ..................................................................................................................................30

Fig. 3.8: Influence of Cr.At upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. ......................................................................................................................31

Fig. 3.9: Influence of verapamil upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum.. ...........................................................................................................31

Fig. 3.10: Record of influence of Ea.At upon rhythmic periodic contractility of isolated preparation of rabbit jejunum...................................................................................................32

Fig. 3.11: Record of influence of Ea.At upon potassium (80 mM) mediated contractility of isolated preparation of rabbit jejunum. ....................................................................................32

Fig. 3.12: Effects of Ea.At on spontaneous and potassium (80 mM) mediated contractions in isolated jejunum preparations. .................................................................................................32

Fig. 3.13: Effects of acetylcholine (Ach; 1 µM) as well as cumulative concentrations of Aq.At on rhythmic periodic contractions of isolated preparation of rabbit jejunum. ..............33

Fig. 3.14: Contractile activity of Aq.At upon rhythmic spontaneous contractions in smooth muscle preparations of rabbit isolated jejunum, alone and in presence of verapamil (1 µM)....................................................................................................................................................33

Fig. 3.15: Record demonstrating relaxant effect of Cr.At upon potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ..................................33

XVI

Fig. 3.16: The Cr.At demonstrated relaxation upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. .......................................................34

Fig. 3.17: Influence of Cr.At upon potassium (80 mM) and carbachol (1 µM) mediated contractions in rabbit isolated tracheal preparations. ...............................................................34

Fig. 3.18: Influence of verapamil upon potassium (80 mM) and carbachol (1 µM) mediated contractions of rabbit isolated tracheal preparations.. .............................................................34

Fig. 3.19: The Ea.At demonstrated relaxant effect upon potassium (80 mM) mediated contractility of isolated tracheal preparation of rabbit. ............................................................35

Fig. 3.20: The Ea.At demonstrated relaxant effect upon carbachol (1 μM) mediated contractility of isolated tracheal preparation of rabbit. ............................................................35

Fig. 3.21: Effects of Ea.At upon potassium (80 mM) and carbachol (1 μM) mediated contractions in rabbit isolated tracheal preparations. ...............................................................35

Fig. 3.22: Record showing inability of Aq.At to relax potassium (80 mM) mediated contractility of rabbit isolated tracheal preparation. ................................................................35

Fig. 3.23: Record showing influence of Aq.At upon carbachol (1 μM) mediated contractility of rabbit isolated tracheal preparation. .....................................................................................36

Fig. 3.24: Effects of Aq.At upon potassium (80 mM) and carbachol (1 μM) mediated contractile responses of smooth muscle preparations of rabbit isolated trachea.. ...................36

Fig. 3.25: Influence of Cr.At upon potassium (80 mM) mediated contractility of isolated denuded aortic preparation of rabbit. .......................................................................................37

Fig. 3.26: Influence of Cr.At upon phenylephrine (1 µM) mediated contractility of isolated denuded aortic preparation of rabbit. .......................................................................................37

Fig. 3.27: Influence of Cr.At upon phenylephrine (1 µM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated denuded aorta.. ........38

Fig. 3.28: Influence of verapamil upon phenylephrine (1 µM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated denuded aorta..........................................................................................................................................38

Fig. 3.29: Record demonstrating influence of Ea.At upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparation of rabbit. .....................................39

Fig. 3.30: Record showing effect of Ea.At on phenylephrine (1 μM) mediated contractile response in isolated denuded aortic preparation of rabbit. ......................................................39

Fig. 3.31: Effects of Ea.At on potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in smooth muscle preparations of rabbit isolated denuded aorta. .........39

Fig. 3.32: Tracing demonstrating Aq.At influence upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparation of rabbit. .....................................39

Fig. 3.33: Record showing effect of Aq.At on phenylephrine (1 μM) mediated contractile response in isolated denuded aortic preparation of rabbit. ......................................................40

XVII

Fig. 3.34: Effects of Aq.At upon potassium (80 mM) and phenylephrine (1 μM) mediated contractions in smooth muscle preparations of rabbit isolated denuded aorta. .......................40

Fig. 3.35: Record of Cr.At influence upon force of spontaneous contractions in isolated paired atrial preparation of rabbit. ...........................................................................................40

Fig. 3.36: Influence of Cr.At upon rhythmic periodic contractility of rabbit isolated preparations of paired atria. .....................................................................................................41

Fig. 3.37: Effect of intravenously administered acetylcholine (Ach, 1 µg per kg), solvent (5% DMSO in normal saline) and different doses of crude extract of Asphodelus tenuifolius (Cr.At) on blood pressure of normotensive anesthetized rat. ..................................................45

Fig. 3.38: Bar chart showing effects of acetylcholine (Ach), verapamil and different doses of crude extract of Asphodelus tenuifolius (Cr.At) on mean blood pressure (MBP) of normotensive anesthetized rats, with and without atropine (1 µM) pretraetment. ..................46

Fig. 4.1: Corchorus depressus plant ........................................................................................54

Fig. 4.2: Influence of cumulatively added Cr.Cd upon rhythmic periodic contractile movements in smooth muscle preparation of rabbit isolated jejunum. ...................................58

Fig. 4.3: The Cr.Cd influence upon cumulative addition to potassium (80 mM) mediated contractility in smooth muscle preparation of rabbit isolated jejunum. ...................................58

Fig. 4.4: The Cr.Cd influence upon cumulative addition to potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum. .................................................59

Fig. 4.5: The Cr.Cd influence upon cumulative addition to carbachol (1 µM) mediated contractile activity in isolated preparation of rabbit jejunum. .................................................59

Fig. 4.6: Influence of Cr.Cd and dicylomine upon rhythmic periodic contractions of rabbit isolated jejunum preparations. .................................................................................................59

Fig. 4.7: Influence of Cr.Cd upon carbachol (1 µM), potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. ...................................................................................................................................60

Fig. 4.8: Dicyclomine influence upon carbachol (1 µM), potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum.. ..................................................................................................................................60

Fig. 4.9: The Cr.Cd influence upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. ............................................................................................................61

Fig. 4.10: Dicyclomine influence upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum.. ...........................................................................................................61

Fig. 4.11: The Ea.Cd influence upon rhythmic periodic contractions of rabbit isolated jejunum preparation. ................................................................................................................62

Fig. 4.12: The Ea.Cd influence upon potassium (80 mM) mediated contractile response of rabbit isolated jejunum preparation. ........................................................................................62

XVIII

Fig. 4.13: The Ea.Cd influence upon carbachol (1 µM) mediated contractile response of smooth muscle preparation of rabbit isolated jejunum. ...........................................................62

Fig. 4.14: Influence of Ea.At upon spontaneous, carbachol (1 μM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. ...62

Fig. 4.15: Trace showing effects of acetylcholine (Ach, 1 µM) and Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum preparation.63

Fig. 4.16: Trace showing effects of acetylcholine (Ach, 1 µM) and Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum preparation in presence atropine (1 μM). ........................................................................................................63

Fig. 4.17: Effects Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum, without and with atropine (1 µM) pretreatment. ..........................63

Fig. 4.18: Record showing influence of Aq.Cd upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum. .......................................................64

Fig. 4.19: Record showing influence of Aq.Cd upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum. ..................................64

Fig. 4.20: Influence of Aq.Cd upon carbachol (1 μM) and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated jejunum. .................................64

Fig. 4.21: The Cr.Cd demonstrating relaxant effect upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated trachea. ....................................66

Fig. 4.22: The Cr.Cd demonstrating relaxant effect upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated trachea. ....................................66

Fig. 4.23: The Cr.Cd demonstrating relaxant effect carbachol (1 µM) and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ...................66

Fig. 4.24: Effects of dicyclomine on potassium (80 mM) and carbachol (1 µM) mediated contractile responses in smooth muscle preparations of rabbit isolated trachea. ....................67

Fig. 4.25: Effect of Cr.Cd upon CRCs for carbachol in smooth muscle preparations of rabbit isolated trachea.........................................................................................................................67

Fig. 4.26: Effect of dicyclomine upon CRCs for carbachol in smooth muscle preparations of rabbit isolated trachea.. ............................................................................................................68

Fig. 4.27: The Ea.Cd demonstrating relaxant effect upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated trachea. ....................................68

Fig. 4.28: The Ea.Cd demonstrating relaxant effect upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated trachea. ....................................68

Fig. 4.29: The Ea.Cd demonstrating relaxant effect upon potassium (80 mM) and carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ......69

Fig. 4.30: The influence of Aq.Cd upon potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ........................................................................69

XIX

Fig. 4.31: The influence of Aq.Cd upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ........................................................................69

Fig. 4.32: The influence of Aq.Cd upon potassium (80 mM) and carbachol (1 μM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ..................................70

Fig. 4.33: Effects of phenylephrine (PE) and Cr.Cd upon resting base line tension in rabbit isolated denuded aortic preparation. ........................................................................................71

Fig. 4.34: Influence of Cr.Cd upon baseline tension of rabbit isolated aorta preparations, without any pretreatment and with prazosin or verpamil pretreatments. .................................71

Fig. 4.35: Effects of Cr.Cd upon potassium (80 mM) mediated contractile response in rabbit isolated denuded aortic preparation. ........................................................................................72

Fig. 4.36: Effects of Cr.Cd upon phenylephrine (1 µM) mediated contractile response in rabbit isolated denuded aortic preparation. ..............................................................................72

Fig. 4.37: Effects of Cr.Cd upon potassium (80 mM) and phenylephrine (1 μM) mediated contractile responses in rabbit isolated denuded aortic preparations. ......................................72

Fig. 4.38: Influence of verapamil upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in tissue preparations of rabbit isolated denuded aorta. .........73

Fig. 4.39: Record of Cr.Cd influence upon force of spontaneous contractions of rabbit isolated paired atrial preparation. .............................................................................................73

Fig. 4.40: Influence of Cr.Cd upon force of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. ....................................74

Fig. 4.41: Influence of Cr.Cd upon rate of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. ....................................74

Fig. 4.42: Influence of acetylcholine upon force of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. .........................75

Fig. 4.43: Influence of acetylcholine upon rate of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment ..........................75

Fig. 4.44: Record showing effects of intravenously administrated solvent, acetylcholine (Ach), norepinephrine (NE) and different doses of Cr.Cd upon blood pressure of normotensive anesthetized rat. .................................................................................................80

Fig. 4.45: Record showing effects of intravenously administered solvent, acetylcholine (Ach), norepinephrine (NE) and Cr.Cd upon blood pressure of normotensive anesthetized rat subsequent to atropine (1 mg per kg) pretreatment. ................................................................80

Fig. 4.46: Effects of intravenous administration of solvent (5% DMSO in saline), acetylcholine and Cr.Cd upon MBP in anesthetized rats, with and without atropine (1 mg per kg) pretreatment. ......................................................................................................................81

Fig. 5.1: Gisekia pharnaceoides plant. ....................................................................................88

Fig. 5.2: Influence of cumulatively added Cr.Gp upon spontaneous rhythmic periodic contractile movements in smooth muscle preparations of rabbits isolated jejunum. ..............92

XX

Fig. 5.3: The influence of cumulatively added Cr.Gp upon potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum. .................................................93

Fig. 5.4: The influence of cumulatively added Cr.Gp upon potassium (80 mM) mediated contractile activity in isolated preparation of rabbit jejunum. .................................................93

Fig. 5.5: The influence of cumulatively added Cr.Gp upon potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum subsequent to pretreatment with glibenclamide (3 μM). .............................................................................................................93

Fig. 5.6: Influence of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated jejunum. ...................94

Fig. 5.7: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response of smooth muscle preparations of rabbit isolated jejunum, alone and after pre-incubation of tissue with glibenclamide (3 µM).. ..........................................................................................94

Fig. 5.8: Influence of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated jejunum. ...95

Fig. 5.9: Influence of cromakalim upon potassium (25 mM) mediated contractile response of smooth muscle preparations of rabbit isolated jejunum, alone and in presence of glibenclamide (3 µM).. ............................................................................................................95

Fig. 5.10: Influence of Cr.Gp upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum.. ...........................................................................................................96

Fig. 5.11: Influence of verapamil upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. ............................................................................................................96

Fig. 5.12: Influence of cromakalim upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum.). .........................................................................................................97

Fig. 5.13: Influence of Ea.Gp upon rhythmic periodic contractility of isolated preparations of rabbit jejunum. .........................................................................................................................97

Fig. 5.14: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparations of rabbit jejunum. ................................................................................................97

Fig. 5.15: Influence of Ea.Gp upon potassium (80 mM) mediated contractility of isolated preparations of rabbit jejunum. ................................................................................................98

Fig. 5.16: The influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparations of rabbit jejunum subsequent to pretreatment with glibenclamide (3 µM). ........98

Fig. 5.17: Influence of Ea.Gp upon spontaneous contractions, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum.. .....................................................................................................................98

Fig. 5.18: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response, alone and in presence of glibenclamide (3 µM), in smooth muscle preparations of rabbit isolated jejunum. ...................................................................................................................................99

XXI

Fig. 5.19: Effects of acetylcholine (Ach; 1 µM) as well as cumulative concentrations of Aq.Gp on rhythmic periodic contractions in isolated preparations of rabbit jejunum. ............99

Fig. 5.20: Influence of caffeine pretreatment upon Aq.Gp mediated enhancement in rhythmic periodic contractility in smooth muscle preparations of rabbit isolated jejunum. ...................99

Fig. 5.21: Contractile effects of Aq.Gp upon spontaneously contracting rabbit isolated jejunum preparations, alone and in presence of caffeine. ......................................................100

Fig. 5.22: Influence of Cr.Gp upon baseline tension of smooth muscle preparations of isolated rabbit trachea in comparison with carbachol (1 µM). ..............................................101

Fig. 5.23: Influence of Cr.Gp upon baseline tension of smooth muscle preparation of rabbit isolated trachea subsequent to pretreatment with caffeine (10 mM). ....................................101

Fig. 5.24: The influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea............................................................102

Fig. 5.25: The influence of Cr.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea............................................................102

Fig. 5.26: The influence of Cr.Gp upon carbachol (1 µM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea............................................................102

Fig. 5.27: The influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea subsequent to pretreatment with glibenclamide (3 µM). ...........................................................................................................102

Fig. 5.28: Influence of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated trachea. ................................103

Fig. 5.29: Influence of Cr.Gp upon potassium (25 mM) mediated contractility, alone and in presence of glibenclamide (3 µM), of smooth muscle preparations of rabbit isolated trachea. ................................................................................................................................................103

Fig. 5.30: Influence of Cr.Gp upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea.. .....................................................................104

Fig. 5.31: Influence of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractions of smooth muscle preparations of rabbit isolated trachea. ................104

Fig. 5.32: Influence of cromakalim upon potassium (25 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea, alone and in presence of glibenclamide (3 µM) . ..................................................................................................................................105

Fig. 5.33: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................105

Fig. 5.34: Influence of Ea.Gp upon potassium (80 mM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................105

Fig. 5.35: Influence of Ea.Gp upon carbachol (1 µM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................106

XXII

Fig. 5.36: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea subsequent to pretreatment with glibenclamide (3 µM). .........106

Fig. 5.37: Influence of Ea.Gp upon potassium (25 mM), potassium (80 mM) and carbachol (1 µM) mediated contractions of smooth muscle preparations of rabbit isolated trachea. ........106

Fig. 5.38: Influence of Ea.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. ...................................................................................................................................107

Fig. 5.39: Influence of Ea.Gp upon CRCs for carbachol in smooth muscle preparations of isolated preparations of rabbit trachea . .................................................................................107

Fig. 5.40: Influence of Aq.Gp upon baseline resting tension of isolated tracheal preparation of rabbit. .....................................................................................................................................108

Fig. 5.41: Influence of Aq.Gp upon baseline resting tension of isolated tracheal preparation of rabbit subsequent to pretreatment with caffeine (10 mM). ....................................................108

Fig. 5.42: Influence of Aq.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................108

Fig. 5.43: Influence of Aq.Gp upon potassium (80 mM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................108

Fig. 5.44: Influence of Aq.Gp upon carbachol (1 µM) mediated contractility of isolated preparation of rabbit trachea. .................................................................................................109

Fig. 5.45: Influence of Aq.Gp on potassium (25 mM), potassium (80 mM) and carbachol (1 μM) mediated contractions in rabbit isolated trachea preparations.. .....................................109

Fig. 5.46: Influence of Cr.Gp upon baseline tension in smooth muscle preparations of rabbit isolated denuded aorta............................................................................................................111

Fig. 5.47: Influence of Cr.Gp upon baseline tension in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with caffeine (10 mM). .........................111

Fig. 5.48: Influence of Cr.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................111

Fig. 5.49: Influence of Cr.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparations of rabbit isolated denuded aorta. ..............................................112

Fig. 5.50: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................112

Fig. 5.51: Influence of Cr.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................112

Fig. 5.52: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with glibenclamide (3 μM). ...........................................................................................................112

Fig. 5.53: Effects of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. ....................................113

XXIII

Fig. 5.54: Effects of Cr.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. .....113

Fig. 5.55: Effects of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. ....................................114

Fig. 5.56: Effects of cromakalim upon potassium (25 mM) mediated contractile response in rabbit isolated denuded aortic preparations, alone and in presence of glibenclamide (3 µM).................................................................................................................................................114

Fig. 5.57: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................115

Fig. 5.58: Influence of Ea.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................115

Fig. 5.59: Influence of Ea.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................115

Fig. 5.60: Effects of Ea.Gp upon potassium (25 mM), potassium (80 mM) and phenylephrine (1 μM) mediated contractile responses in rabbit isolated denuded aortic preparations. ........115

Fig. 5.61: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with glibenclamide (3 μM). ...........................................................................................................116

Fig. 5.62: Effects of Ea.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractile response in rabbit isolated denuded aortic preparations. ......116

Fig. 5.63: Influence of Aq.Gp upon baseline tension in smooth muscle preparations of rabbit isolated denuded aorta............................................................................................................116

Fig. 5.64: Influence of Aq.Gp upon baseline tension in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with caffeine (10 mM). .........................117

Fig. 5.65: Influence of Aq.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................117

Fig. 5.66: Influence of Aq.Gp upon potassium (25 μM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................117

Fig. 5.67: Influence of Aq.Gp upon phenylephrine (1 μM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta. ................................................118

Fig. 5.68: Influence of Aq.Gp upon potassium (25 mM), potassium (80 mM) and phenylephrine (1 μM) mediated contractile response in smooth muscle preparations of rabbit isolated denuded aorta............................................................................................................118

Fig. 5.69: Record of influence of Cr.Gp upon force of spontaneous contractions in rabbit isolated paired atrial preparation. ...........................................................................................118

Fig. 5.70: Influence of Cr.Gp, alone and in presence of glibenclamide, upon force of contractions of rhythmic periodic contractions in rabbit isolated preparation of paired atria.................................................................................................................................................119

XXIV

Fig. 5.71: Influence of Cr.Gp, alone and in presence of glibenclamide, upon rate of contractions of rhythmic periodic contractions in rabbit isolated preparation of paired atria.................................................................................................................................................119

Fig. 5.72: Record demonstrating effects of Cr.Gp upon blood pressure of an anesthetized normotensive rat.....................................................................................................................123

Fig. 6.1: Salsola imbricata plant ............................................................................................132

Fig. 6.2: Record of influence of cumulatively added Cr.Si upon rhythmic periodic contractile movements in smooth muscle preparation of isolated rabbit jejunum. .................................136

Fig. 6.3: The Cr.Si influence upon cumulative addition to potassium (80 mM) mediated contractility in smooth muscle preparation of rabbit isolated jejunum. .................................137

Fig. 6.4: The Cr.Si influence upon cumulative addition to potassium (25 mM) mediated contractility of isolated preparation of rabbit jejunum. .........................................................137

Fig. 6.5: Influence of Cr.Si upon spontaneous contractility, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. ....................................................................................................................137

Fig. 6.6: Influence of verapamil upon spontaneous rhythmic periodic contractions, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum..................................................................................138

Fig. 6.7: Influence of Cr.Si upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. ....................................................................................................................138

Fig. 6.8: Verapamil influence upon CRCs for calcium in smooth muscle isolated preparations of rabbit jejunum. ...................................................................................................................139

Fig. 6.9: The influence of Cr.Si upon carbachol (1 µM) mediated contractility of isolated rabbit jejunum preparation. ....................................................................................................139

Fig. 6.10: Influence of Cr.Si upon carbachol (1 µM) mediated contractility of isolated preparations of rabbit jejunum, with and without propranolol (1 µM) pretreatment. ...........140

Fig. 6.11: Influence of verapamil upon carbachol (1 µM) mediated contractility of isolated preparations of rabbit jejunum, with and without propranolol (1 µM) pretreatment. ...........140

Fig. 6.12: The Ea.Si influence upon rhythmic periodic contractility of isolated preparation of rabbit jejunum. .......................................................................................................................141

Fig. 6.13: The Ea.Si influence upon potassium (80 mM) mediated contractility of isolated preparation of rabbit jejunum.................................................................................................141

Fig. 6.14: Influence of Ea.Si upon spontaneous rhythmic periodical contractility and potassium (80 mM) mediated contractile response in smooth muscle preparations of rabbit isolated jejunum. ....................................................................................................................141

Fig. 6.15: The Aq.Si influence upon rhythmic periodic contractility of isolated preparation of rabbit jejunum. .......................................................................................................................142

XXV

Fig. 6.16: The Aq.Si influence upon potassium (80 mM) mediated contractility of rabbit isolated jejunum preparation. .................................................................................................142

Fig. 6.17: Influence of Aq.Si upon spontaneous rhythmic periodical contractility and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated jejunum.). ...............................................................................................................................142

Fig. 6.18: Influence of Cr.Si upon potassium (80 mM) mediated contractile response in tissue preparation of rabbit isolated trachea. ....................................................................................144

Fig. 6.19: Influence of Cr.Si upon carbachol (1 µM) mediated contractile response in tissue preparation of rabbit isolated trachea. ....................................................................................144

Fig. 6.20: Influence of Cr.Si upon potassium (80 mM) and carbachol (1 µM) mediated contractility of tissue preparations of rabbit isolated trachea. Presence of propranolol (1 µM) attenuated relaxant effect of Cr.Si upon carbachol (1 µM) mediated contractions. ..............145

Fig. 6.21: Influence of verapamil upon potassium (80 mM) and carbachol (1 µM) mediated contractility of tissue preparations rabbit isolated trachea. Presence of propranolol (1 µM) did not alter relaxant effect of verapamil upon carbachol mediated contractility. ......................145

Fig. 6.22: Influence of isoprenaline upon carbachol (1 µM) mediated contractility of tissue preparations of rabbit isolated trachea, with and without propranolol (1 µM). .....................146

Fig. 6.23: Influence of Cr.Si upon CRCs for carbachol in tissue preparations of rabbit isolated trachea. ...................................................................................................................................146

Fig. 6.24: Influence of propranolol (Prop) upon Cr.Si mediated shift of CRCs for carbachol in rabbit isolated tracheal preparations.. ....................................................................................147

Fig. 6.25: The Ea.Si demonstrated relaxant effect upon potassium (80 mM) mediated contractility of isolated tracheal preparation of rabbit. ..........................................................147

Fig. 6.26: The Ea.Si demonstrated relaxant effect upon carbachol (1 µM) mediated contractility of isolated tracheal preparation of rabbit. ..........................................................147

Fig. 6.27: Effect of Ea.Si on potassium (80 mM) and carbachol (1 μM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Variable are mean ± S.E.M. (n=5).................................................................................................................................................148

Fig. 6.28: Influence of Aq.Si upon carbachol (1 µM) mediated contractile response in rabbit isolated tracheal preparation. .................................................................................................148

Fig. 6.29: Influence of Aq.Si upon potassium (80 mM) mediated contractility of rabbit isolated tracheal preparation. .................................................................................................148

Fig. 6.30: Influence of Aq.Si upon potassium (80 mM) and carbachol (1 μM) mediated contractions in smooth muscle preparations of rabbit isolated trachea. ................................149

Fig. 6.31: Tracing showing contractile responses of phenylephrine (PE) as well as cumulative addition of Cr.Si upon tissue preparation of rabbit isolated denuded aorta. ..........................150

Fig. 6.32: Influence of doxazosin upon CRCs for Cr.Si in tissue preparations of rabbit isolated denuded aorta............................................................................................................151

XXVI

Fig. 6.33: Influence of doxazosin upon CRCs for phenylephrine in tissue preparations of rabbit isolated denuded aorta.. ...............................................................................................151

Fig. 6.34: Influence of Cr.Si upon potassium (80 mM) mediated contractility in tissue preparation of rabbit isolated denuded aorta. .........................................................................152

Fig. 6.35: Influence of Cr.Si upon phenylephrine (PE) mediated contractility in tissue preparations of rabbit isolated denuded aorta. .......................................................................152

Fig. 6.36: Influence of Cr.Si upon phenylephrine (1 µM) and potassium (80 mM) mediated contractions in tissue preparations of rabbit isolated denuded aorta. ....................................152

Fig. 6.37: Influence of Ea.Si upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparations of rabbit. ......................................................................153

Fig. 6.38: Influence of Ea.Si upon phenylephrine (1 µM) mediated contractile response in isolated denuded aortic preparations of rabbit. ......................................................................153

Fig. 6.39: Influence of Ea.Si upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in tissue preparations of rabbit isolated denuded aorta. ......................153

Fig. 6.40: Influence of verapamil upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile response in tissue preparations of rabbit isolated endothelium denuded aorta........................................................................................................................................154

Fig. 6.41: Influence of Aq.Si upon baseline tension in tissue preparation of rabbit isolated denuded aorta. ........................................................................................................................154

Fig. 6.42: Tracings showing (A) effect of cumulatively added Cr.Si upon rhythmic contractility of rabbit isolated paired atria; (B) influence of pretreatment with propranolol (1 µM) on effect of cumulatively added Cr.Si upon rhythmic contractility of rabbit isolated paired atria. ............................................................................................................................155

Fig. 6.43: Influence of Cr.Si upon force of rhythmic periodic contractility of rabbit isolated paired atrial preparations, alone or with propranolol (1 µM) pretreatment. ..........................155

Fig. 6.44: Influence of Cr.Si upon rate of rhythmic periodic contractility of rabbit isolated paired atrial preparations, without and with propranolol (1 µM) pretreatment. ....................156

Fig. 6.45: Tracings showing effects of intravenous administration of norepinephrine (NE; 1 μg per kg) and Cr.Si (1.0, 3.0, 10.0 and 30.0 mg per kg) upon blood pressure of (A) normotensive anaesthetized rat without any pretreatment, (B) normotensive anaesthetized rat with doxazosin (1 mg per kg) pretreatment and (C) normotensive anaesthetized rat with doxazosin (1 mg per kg) and propranolol (1 mg per kg) pretreatments. ...............................160

Fig. 6.46: Presser responses of norepinephrine (NE) and Cr.Si in normotensive anesthetized rats, with and without doxazosin (1 mg per kg) as well as doxazosin (1 mg per kg) + propranolol (1 mg per kg) pretreatments. ..............................................................................161

XXVII

List of tables

Table 3.1: Effects of oral administrations of Cr.At, loperamide and carbachol upon charcoal meal intestinal transit in mice.. ................................................................................................42

Table 3.2: Effects of oral administration of Cr.At and loperamide upon castor oil caused diarrhea in mice........................................................................................................................43

Table 3.3: Laxative effects of Cr.At and carbachol in normal mice. ......................................44

Table 3.4: Effects of intravenous administration of solvent, crude extract of Asphodelus tenuifolius (Cr.At), acetylcholine and verapamil on mean blood pressure (MBP), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) of anesthetized rats. ...........................................................................................................................................45

Table 3.5: Diuretic activity of crude extract of Asphodelus tenuifolius (Cr.At) in rats. .........48

Table 4.1: Influence of Cr.Cd upon charcoal meal intestinal transit distance in mice............76

Table 4.2: Influences of Cr.Cd upon castor oil caused diarrhea in mice. ...............................78

Table 4.3: The Cr.Cd exerted laxative effect in normal mice as compared to carbachol. ......78

Table 4.4: Effects of intravenously administered solvent (5 % DMSO in saline), Cr.Cd, norepinephrine and acetylcholine upon SBP, DBP and MBP of normotensive anesthetized rats. ...........................................................................................................................................80

Table 4.5: Influence of saline, Cr.Cd and furosemide upon urinary volume and electrolytes contents in rats. ........................................................................................................................83

Table 5.1: Influence of Cr.Gp upon charcoal meal intestinal transit distance in mice. ........121

Table 5.2: Influence of Cr.Gp upon castor oil caused diarrhea in mice. ...............................122

Table 5.3: The Cr.Gp exerted laxative effect in mice as compared to carbachol. ................122

Table 5.4: Effects of intravenously administered vehicle (5% DMSO in saline), Cr.Gp and cromakalim upon SBP, DBP, MBP and HR of normotensive anesthetized rats. ..................124

Table 5.5: Effects of saline, Cr.Gp and furosemide upon urinary volume and urinary electrolytes contents in albino rats. ........................................................................................126

Table 6.1: Influence of Cr.Si upon charcoal meal intestinal transit distances in mice. ........157

Table 6.2: Effects of Cr.Si upon castor oil caused diarrhea in mice. ....................................158

Table 6.3: Effects of intravenously administered normal saline, Cr.Si (1, 3, 10 and 30 mg per kg), and norepinephrine (NE, 1 µg per kg) upon SBP, DBP, MBP and HR of normotensive anesthetized rats. ....................................................................................................................161

Table 6.4: Effects of crude extract of Salsola imbricata (Cr.Si) and furosemide on urinary volume and urinary electrolytes contents in albino rats. ........................................................163

1

1. General introduction

2

1.1. Cholistan desert

The word desert may be defined as an area with little rainfall and scanty greenery consisting

of plants adopted to harsh environment. Deserts occupies almost one quarter of earth surface,

which is about 75 million kilometers square (Abd El-Ghani et al., 2017).

Cholistan desert is situated in northern region of Punjab province of Pakistan, which extends

from latitudes 27°42' and 29°45' north and longitudes 69°52' and 75°24' east and covers

approximately 25,900 square kilometers (Baig et al., 1980). It is connected with Thar desert

spreading in Sindh province of Pakistan and with Rajasthan desert in India. The desert is

divided into lesser Cholistan (7,770 square kilometers) and greater Cholistan (18,130 square

kilometers). The lesser Cholistan (northern part) consists of alternating large areas of saline

compact plans and sandy ridges. Sand dunes may be enduring or shifting, but the plans are

mostly covered with yellow sand. A small part of lesser Cholsitan desert is irrigated by canal

system. The greater Cholistan (southern part) consist of large sand dunes with little plane

area. The sand dunes may reach up to height of 30-100 meters. Annual rainfall is erratic and

ranges about 1.0 cm to 2.5 cm, which chiefly occurs during moon soon season (July-August).

The underground water contains very high amount of dissolved salts and soil is poor in

organic matter containing high amount of salinity with alkaline pH (8.6-10.0). Temperature

may reach above 50°C with average temperature between 34-38°C during summer, whereas

in winter season mean temperature may ranges between 15-20°C (Akhter and Arshad, 2006;

Arshad et al., 2008; Wariss et al., 2013).

1.2. Vegetation of Cholistan desert

Vegetation of Cholistan consists of xeromorphic species, which accepted the harsh

environmental factors of high temperature, low water, extreme evaporation, salinity and low

nutrient soil. Several studies described distribution of plant species depending upon

variability of climate and soil within Cholistan (Arshad and Akbar, 2002; Dasti and Agnew,

1994; Hameed et al., 2011; Rafay et al., 2015).

Floristic composition of Cholistan desert is reported to comprises of 154 plant species

affiliated to 106 genera and 38 families, consisting of grasses, herbs, subshurbs, climbers and

trees (Wariss et al., 2013), many of which are used for medicinal purpose by local inhabitants

(Ahmed et al., 2014; Hameed et al., 2011).

3

Fig. 1.1: Map of Cholistan desert (Akhter and Arshad, 2006)

4

1.3. Medicinal uses of plants

Since ancient life, plants have extraordinary connection with human being as plants give

dress, houses and pharmaceuticals. Historical data reveals that human utilization of plants for

treatment or prevention of various ailments goes back to Middle Paleolithic age, around

60,000 years ago (Heinrich, 2015). Since then, societies built up method of using plants in

treatment of diseases along with progression of time. Almost all cultures have experts in

medicinal properties of locally available plants. Treatment of ailments with plant extracts has

been termed as “phytotherapy”, and it is well known that crude extracts of plants are of

medicinal importance; e.g., tincture Atropa belladonna as antispasmodic; Papaver somnifera

extract as analgesic (Houghton, 1995). Throughout World, many plants are used in traditional

medicine systems, as in Ayurvedic medicine, Chinese medicine and Unani medicine.

Traditional medicine systems encompass history of native beliefs and practices regarding use

of herbs and other indigenously available animal or mineral derived substances for medicinal

purpose in a particular culture. Whereas, complementary and alternative medicine includes

large number of non-indigenous non-conventional medical practices. According to an

estimate over 100 million people in Europe are using traditional, complementary and

alternative medicine including herbal medicine, and many more users are in Australia, Asia,

Africa and North America. In developing countries, about 80% of population relies on

traditional medicine systems, partly due to unavailability of modern medicine and partly due

to reasons of being acceptable culturally, availability close to home and affordability (WHO,

2013). In developed countries, medicinal use of botanicals as botanical drugs, dietary

supplements, functional foods and medicinal foods is increasing (Raskin et al., 2002),

particularly in chronic diseases (WHO, 2013). Estimated use of traditional, complimentary

and alternative medicine by multiple sclerosis patients was 41% in Spain to 70% in Canada

and 82% in Australia (Skovgaard et al., 2012). Worldwide botanical importation trade during

1990s was estimated to be 400,000 tons valued about US$ 1.22 billion with 80% imports

allotted to twelve countries, and Pakistan was ninth largest importer of the botanicals with

estimated value of US$ 9.81 million (Lange, 2004). Global business of botanicals is projected

to be greater than US$ 140 billion by year 2024 (Paine and Roe, 2018). Scientific

justifications regarding efficacious and safe use of herbal medicine are often demanded by

users and health care providers. In this regard, most of countries developed laws and started

implementing these laws to ensure proper and safe use of herbal medicine (Allkin, 2017;

Alostad et al., 2018).

5

1.4. Medicinal plant research

Out of about 250,000 known species of plants, only few have been assessed scientifically for

their medicinal activities (Fabricant and Farnsworth, 2001). Plants are utilized as natural

cures and some of these plants have toxic impact, however because of the absence of

scientific documentation, impacts of these plants on human wellbeing were not established.

Nonetheless, health impacts of many plants remained un-revealed.

1.4.1. History and terminology

Claude Bernard (1831-1878), French scientist’s work on medicinal value of plants gave birth

of a versatile multidisciplinary branch of science named "ethnopharmacology" aimed to

report customary uses of plants for medicinal purpose on logical and scientific grounds. He

worked on curare and found the reason of its being nontoxic when given by oral route,

justified its use in treatment of stomach problems by Indians instead of poisoning, and

reported that it is dangerous only when administered directly into the living tissue or

introduced into blood stream by some instrument, like in arrow poisoning. He described

muscle paralysis as main cause of death of animals poisoned with curare and observed that if

curare was applied to hind limb of a frog through injury while stopping blood circulation of

the limb via ligature, the animals survived. Consequent examinations permitted

comprehension of the pharmacological effects of curare in causing respiratory arrest.

Furthermore, secondary metabolite responsible for effect of curare was identified as d-

tubocurarine in 1947. This example portrays course of research actions, which

understandably combine ethnic knowledge and pharmacological research (Heinrich, 2015).

Various terms have been used to describe scientific research on medicinal value of plants.

Term ethnobotany was introduced in 1896 by William Harshberger (an American botanist) as

study of human’s medicinal uses of plants. Terms pharmakoëthnologie, pharmacoetnologia

and aboriginal botany were used at various times to describe study of medicinal uses of

plants; emphasis was on physical and chemical description of medicinal plants and their uses.

The term ethnopharmacology was first used in 1967 by Efron and colleagues in title of their

book, “Ethnopharmacological Search for Psychoactive Drug” (Heinrich, 2014). In modern

concept, the central approach of ethnopharmacology has been changed from simple curative

uses of medicinal plants towards better and safer use. Ethnopharmacology may be defined as

multidisciplinary approach toward scientific research on traditionally used agents in treatment

or prevention of diseases. Cultural beliefs on use of available agents for medical purpose are

6

not only documented, but also evaluated on scientific grounds. Ethnopharmacological

research usually encompass botany, chemistry, pharmacology and toxicology, however other

fields like anthropology, biotechnology, pharmaceutical technology may also make important

contribution. Integration of multiple fields of science is usually required and a specific filed

should not be considered as secondary to the other.

Particulars of cultural utilization of plants in treatment of diseases integrated with chemical

and pharmacological explorations may furnish a chance for tremendous community based

research on natural products. World Health Organization in 1985 highlighted the significance

of research and standardization of natural medicine being practiced in primary health care

system in developing countries and suggested a course for investigations on plants being

practiced in traditional medicine (Fig. 2) (Farnsworth et al., 1985).

1.4.2. Plants as source of new drug

Botanicals made a significant contribution in new drug invention and development. In

western medicines, 121 numbers of currently used molecules are derived from botanicals.

Basic and essential drug list of World Health Organization contains about 11% molecules

that come from plant origin (Veeresham, 2012). Some molecules, like salicylic acid being

one of the examples, were isolated from plants but subsequently synthesized in laboratory for

medical use. Some semi-synthetic molecules, like apomorphine, were derived from

phytochemicals. Still, chemical synthesis of many naturally occurring molecules, like

vincristine and vinblastine, is not economic and therefore obtained from plants (Newman et

al., 2003). An analysis of US FDA approved medicine during the period between 1981 to

2010 revealed that 34% of small molecules based medicine were either plant derived or direct

derivatives of plant derived molecules, including immunosuppressant drugs, anticancer drugs

and statins (Newman and Cragg, 2012). Enthusiasm of pharmaceutical firms for isolation of

new molecules from natural products decreased after modern techniques such as high through

put screening directed at molecular targets, problems in isolation of known molecules, ability

to synthesize molecules during pharmaceutical manufacture and expectations that chemical

techniques would synthesize all required molecules. The decline in natural product drug

discovery greatly reduced new leads in drug development and ultimately a downfall in new

drug registrations. Still, drug discoveries from natural products are making impact; 2015

Nobel Prize for physiology and medicine awarded to William C. Campbell and Satoschi

Omura for discovery of ivermectin (a derivative of avermactin) lowering incidents of

7

onchocerciasis and lymphatic filariasis, and to Youyou To for discovery of artemisinin

reducing mortality in malria patients. A large majority of natural sources are yet to be

explored for next drug discovery (Shen, 2015).

Fig. 1.2: Flow chart for suggested scientific studies of medicinal plants used in traditional systems of medicine (Fransworth et al., 1985).

1.4.3. Positive interactions within phytomedicine

Modern approach of using single purified molecule to cure a complicated ailment was

emphasized. The complicated diseases, like diabetes and cardiovascular diseases involve

various genetic, environmental and behavioral elements. Traditional medicine systems use

complex mixtures of chemicals in form of crude extracts of botanicals supposing that

complex chronic ailments can better be treated with composite mixture of chemicals

contained in crude natural origin medicine. Furthermore, interaction of multiple molecules in

botanical extract may contribute in attaining requisite outcome. This concept was

8

strengthened by some examples, for example, in Chinese medicine root extract of

Tripterygium wilfordii is used in treatment of rheumatoid arthritis. Its active constituent was

isolated and identified as triptolide, but the pure chemical was found to produce toxic effects,

when administered. It was proposed that the crude extract contain some unknown

phytochemicals that neutralize its toxic effects and perhaps enhance effectiveness of

treatment (Raskin et al., 2002). Similarly, crude extract of Berberis freemontii, containing

mixture of alkaloid berberine and two flavolignins, was found to possess greater antibacterial

activity against resistant strain of Staphylococcus aureus as compared to its pure antibacterial

alkaloid, berberine. It was found that the two flavolignins contained in the plant were devoid

of antibacterial activity but enhanced antibacterial activity of berberine (Stermitz et al.,

2000). Artemisia annua is well known for artemisinin contents and hence anti-malarial

activity. In a study, fresh juice of leaves of the plant was found to possess 6 to 18 fold less

minimum inhibitory concentration against Plasmodium falciparum than in terms of pure

artemisinin contents in the juice, meaning thereby that artemisinin could not be entirely

responsible for anti-malarial activity of the juice and some other compounds in the juice

enhanced anti-malarial activity of artemisinin through some interactions, which may be

pharmacodynamic or pharmacokinetic interaction (Rasoanaivo et al., 2011).

1.5. Gut, airway and cardiovascular disorders

1.5.1. Gut motility disorders

The gut perform several vital functions such as forward transfer of its contents, secretions,

absorption of nutrients, water and electrolytes, protection against pathogens, expulsion of un-

wanted and un-absorbed material from body. Motility of intestine is vital for its functions,

which is controlled by presence of food, enteric nervous system and hormones. Constipation,

abdominal distension, obstruction, spasmogenic pain, diarrhea and irritable bowel syndrome

are some of the important gut motility disorders, which involve loss of coordinated muscle

activity due to exogenous or endogenous causes. Constipation and diarrhea are opposite

motility disorders of gut (Barrett et al., 2016).

1.5.1.1. Constipation

All medical definitions of constipation unusually highlight few or problematic expulsion of

feces. However, normal range of bowel movements is difficult to define, and in a study 99%

of British population was having bowel movements in range of 3 per week to 3 per day. An

international expert committee defined constipation as 2 or less defecations per week and also

9

included straining, hard feces and incomplete defecation feeling (Longstreth et al., 2006).

Constipation is common throughout World but only few patients seek healthcare advice for

constipation, therefore, exact prevalence in not known, but estimated to be in range of 2 to

28% in general population (Locke et al., 2000). Healthy alterations in dietary and lifestyle

pattern are most important in treatment of chronic constipation. Different classes of drugs

used in management and treatment of constipation include bulk laxatives (psyllium,

polycarbophil, cellulose derivatives), lubricant laxatives (mineral oil), surfactant laxative

(docusate), stimulant laxative (bisacodyl, senna, aloe, cascara, castor oil), osmotic laxatives

(sodium phosphate, sodium sulphate, magnesium sulphate, magnesium citrate, lactulose,

sorbitol, glycerine, polyethylene glycol) and prokinetic agents (linaclotide, lubiprostone)

(Izzy et al., 2016; Tack et al., 2011).

1.5.1.2. Diarrhea

Diarrhea is described as incidence of increased loose stools without or with other

manifestations such as vomiting, abdominal cramps and fever. Worldwide diarrheal illness

resulted approximately 1.3 million deaths in year 2015, and 40% of diarrhea associated

deaths occurred in children under 5-year age. Diarrhea may be acute, sub-acute or chronic.

Causes of diarrhea may vary and may be primary related to specific gut ailment or secondary

to other disease not directly implicating gut. For example, infectious diarrhea directly affects

gut, but diabetes mellitus associated diarrhea is secondary to neurological disorder (Kotloff,

2017). There are numerous reasons of diarrhea as infectious agents (bacterial, viral,

protozoal), drugs, toxins, anxiety, un-absorbable food and inflammatory condition of gut

(Jankowiak and Ludwig, 2008). Various physiological mediators such as acetylcholine,

histamine, cholecystokinin, serotonin, prostaglandins and tachykinins are responsible for

enhanced gut motility (Olsson and Holmgren, 2001) via increase in cytosolic free calcium.

Drugs having potential to act on specific excitation contraction pathway (e.g., muscarinic

receptor antagonist, adrenergic and opioid receptor agonists, phosphodiesterase inhibitors) or

possessing non-specific inhibitory activity (e.g. calcium entry blocking drugs) may be

considered effective in hyper-motile gut state (Fedorak and Field, 1987).

Treatment of diarrhea and constipation with chemical drugs is associated with several types

of adverse effects, including that related to pharmacological actions of such drugs; use of

antidiarrheal drugs is associated with constipation, while treatment of constipation with

certain drugs (e.g., serotonergic drugs or bisacodyl) is associated with abdominal cramps and

diarrhea (Gattuso and Kamm, 1994). Many of herbal medicine are reported to contain both

10

spasmogenic and spasmolytic constituents, which intend to normalize gut function while

offsetting side effects associated with excessive stimulation or suppression of gut activity.

1.5.2. Airway disease (Asthma)

Airway apparatus consist of lungs, air passageway, muscles and bones of chest cavity. The

main tracheal tubes bifurcate into primary and then secondary branches which end in alveoli,

the functional unit of lung for exchange of gases with blood. During inspiration oxygen is

absorbed into blood, while expirations expel released carbon dioxide gas from blood into air

(Singh, 1997). Asthma, characterized by obstruction in airflow leading to difficulty breathing,

wheezing, cough and discomfort, is common and major disease of air passageway reported

for significant mortality and morbidity. Worldwide prevalence of clinical asthma is 4.5%,

lowest in Vietnam (1%) and highest is Australia (21%), increases with level of

industrialization. Asthma is associated with 250,000 annual deaths worldwide, and most of

deaths occur due to unavailability of medicine (Croisant, 2014). Several types of

neurotransmitters, hormones, mediators and drugs affect smooth muscle tone of airway;

interactions of inhibitory and excitatory receptors on smooth muscles determine

bronchoconstriction or bronchorelaxation. Adrenergic, vasoactive intestinal peptide (VIP)

and adenosine receptors activation cause bronchodilation via increase in cAMP. Activation of

muscarinic, histamine, tachykinins, calcitonin gene related peptides, prostanoids,

leukotrienes, bradykinin and platelet activating factor receptors have been implicated in

pathophysiology of asthma by causing bronchoconstriction (Barnes, 1989).

The drugs for management of asthma are classified into various groups including muscarinic

antagonists, antihistamines, β agonists, leukotrienes receptor inhibitors, mast cell

degranulation inhibitors, anti-inflammatory corticosteroids, phosphodiesterase inhibitors

(Barnes, 2006), potassium channel openers (Kocmalova et al., 2014) and calcium channel

blockers (Girodet et al., 2015).

The conventional allopathic medicine for asthma in form of inhalers are costly for patients of

developing World, whereas orally administered drugs are associated with undesirable effects;

hence patient satisfaction is low with available medicine. In a survey, 59% of patients used

complementary and alternative medicine to treat asthma and herbal medicine was third most

popular choice. In addition, β agonists, muscarinic antagonists and xanthine derivatives are

important classes of anti-asthmatic medicine originated from herbal origin (Ernst, 1998;

11

Huntley and Ernst, 2000). By continued research on herbal medicine, further effective

medicine for asthma may be found.

1.5.3. Cardiovascular disorders

Cardiovascular disorders result from malfunctioning of blood vessels and heart, and include

stroke, coronary artery disease, rheumatic heart disease, congestive cardiac failure,

hypertension and hypotension. Cardiovascular disorders are leading cause of morbidity and

mortality throughout the world (Luepker, 2011).

1.5.3.1. Hypertension

Hypertension in most common amongst cardiovascular disorders and is itself cause of other

cardiovascular diseases. Normal blood pressure is considered less than 120/80 mmHg.

Persistently elevated blood pressure is associated with organ damage due to increased risk of

damage of vascular beds of kidneys, brain, retina and heart. In most cases (95%), exact cause

of hypertension is not known and pathophysiology is complex involving several factors,

including genetic predisposition and lifestyle (Izzo et al., 2003). In 2000, 26.4% of adult

population of the world was having hypertension, which is predicted to be increased to 29.2%

by year 2025 (Labarthe, 2011).

Blood pressure is directly related to peripheral vascular resistance and cardiac output.

Diameter of arterioles, rather main arteries and capillaries, determine peripheral vascular

resistance; while cardiac output is derived from heart rate, myocardial force and end diastolic

pressure. Most pharmacological interventions in reducing blood pressure involve decreasing

total peripheral resistance or cardiac output or combination of both and may act by following

mechanisms (Izzo et al., 2003):

Reduction in sympathetic nervous system activity can be achieved by α- adrenoceptor

blocking drugs (e.g. prazosin), β-adrenoceptor blocking drugs (propranolol, atenolol),

neuron blocking drugs (reserpine), ganglion blocking drugs (hexamethonium) and

centrally acting drugs (clonidine, methyldopa).

Vasodilator drugs which may act through several mechanisms and include direct

vasodilators (e.g. diazoxide, hydralazine, nitrates), calcium entry blockers (e.g.

verapamil, diltiazem), angiotensin converting enzyme inhibitors (e.g. captopril,

enalapril, lisinopril), angiotensin receptor antagonists (e.g. losartan, valsartan),

potassium channel openers (e.g. nicorandil) and cholinomimetic drugs.

12

Diuretic drugs (e.g. hydrochlorothiazide, thiazide, furosemide, spironolactone)

decrease plasma volume and hence cardiac output by increasing water and

electrolytes excretion through kidneys. With continued use, diuretics decrease total

peripheral resistance due to unknown mechanism.

Studies documented that regular consumers of plant-based diet have lower blood pressure

than non-consumers. Vitamins, polyphenols, lignin, unsaturated fats present in plant food

have been reported to have cardiovascular protective effects by decreasing endothelium

dysfunction, oxidative damage, inflammation and thrombus formation (Appel et al., 2006;

Okoye, 2007). Reserpine is an example of well-known antihypertensive drug isolated from

plant Rauwolfia serpentina.

1.6. Objectives of the present study

Asphodelus tenuifolius, Corchorus depressus, Gisekia pharnaceoides and Salsola imbricata

are amongst medicinally important flora of the Cholistan desert (Ahmed et al., 2014; Hameed

et al., 2011). Although numerous reports have disclosed ethnomedical importance of these

plants in various disorders of gut, respiratory and cardiovascular systems, but, their

pharmacological activities have not been fully assessed on scientific basis. Efficacy of

traditionally used plants in treatment of various ailments must be evaluated and documented

so that folkloric uses of these plants may be justified. The present research was aimed:

To investigate actions of the selected plants extracts on gut, tracheal and

cardiovascular muscles in vitro.

To evaluate actions of the selected plants extracts on blood pressure, gut motility and

urinary excretion in vivo in laboratory animals.

To explore some possible modes of pharmacological actions of Asphodelus

tenuifolius, Corchorus depressus, Gisekia pharnaceoides and Salsola imbricata.

To provide pharmacological background for some of the traditional uses of the

selected plants or to identify new possible uses of the plants in gut, respiratory and

cardiovascular disorders.

13

2. Material and methods

14

2.1. Chemicals

The Sigma Aldrich USA supplied acetylcholine chloride, atropine sulphate, caffeine, castor

oil, carbachol chloride, carboxy methyl cellulose, cromakalim, dicylomine hydrochloride,

doxazosin mesylate, ethylene diamine tetra-acetic acid, furosemide hydrochloride, gallic acid,

isoprenaline hydrochloride, indomethacin, magnesium chloride, norepinephrine bitartrate,

phenylephrine hydrochloride, potassium chloride (KCl), propranolol hydrochloride,

pyrillamine maleate, quercetin, loperamide hydrochloride, and verapamil hydrochloride,

whereas, Merck, Darmstadt, Germany was provider of aluminum chloride (AlCl3), ethanol,

methanol, folin ciocalteu reagent, ferric chloride (FeCl3), calcium chloride (CaCl2), dimethyl

sulphoxide (DMSO), glucose, ethanol, potassium hydroxide (KOH), potassium-di-hydrogen

phosphate (KH2PO4), magnesium sulphate (MgSO4), sodium carbonate (Na2CO3), sodium

bicarbonate (NaHCO3), sodium nitrite (NaNO2) and sodium-di-hydrogen phosphate

(NaH2PO4). BDH, Laboratory Supplies, Poole, England was ordered to procure sodium

chloride (NaCl), sodium hydroxide (NaOH), ammonium hydroxide (NH4OH), charcoal,

hydrochloric acid (HCl) and sulphuric acid (H2SO4). VWR International Ltd. Poole, BH15,

1TD, England was supplier of ethyl acetate, chloroform, benzene and petroleum ether.

Glibenclamide was provided by Sanofi-Aventis Pakistan. Local pharmacy was visited to

purchase heparin, ketamine and diazepam injections. Solutions of caffeine, cromakalim,

glibenclamide, indomethacin and doxazosin mesylate were made in 10% DMSO; gallic acid

in methanol; quercetin in ethanol; other chemicals rendered soluble / suspended in either

distilled H2O or saline.

2.2. Animals for experiments

The experiments were conducted on (♂/♀) local albino breeds of rabbits (1.5-2.0 kg), rats

(200-250 g) and mice (20-25 g); caged individually and maintained at conducive environment

(i.e., 25 ± 2˚C; 12 hrs day-night cycles; free availability of drinking water and commercially

available feed) at the animal keeping facility of the institution concerned; in compliance with

directive of Institute of Laboratory Animal Resources, Commission on Life Sciences,

National Research Council, (1996). Permission to use animals was granted by Ethical

Committee of the institution concerned vide letter No. EC/02 PHDL/2013 dated 04.09.2013.

2.3. Test material

Crude extracts and fractions obtained from Asphodelus tenuifolius, Corchorus depressus,

Gisekia pharnaceoides and Salsola imbricata were used in experiments; details about plants

15

collection, extraction and fractionation are given in subsequent chapters. Storage of the

extract and fractions was achieved in amber colored wide mouth containers at -4°C for future

use.

2.4. Phytochemical investigations on crude extracts

Qualitative investigations regarding presence or absence of specific class of secondary

metabolites as well as quantitative determinations of phenolic as well as flavanoids amount

out of herbal extracts were performed according to standard methods (Harborne, 1998;

Hossain et al., 2013; Saeed et al., 2012) as described below:

2.4.1. Alkaloids

The alkaloids were detected as plant constituents on mixing of the concerned crude extract

(0.5 g) with 1% hydrochloric acid (8 ml); boiled for a few minutes, cooled, filtered and

divided into 3 glass tubes. The dragendorff’s reagent (0.5 ml) poured to the 1st tube, while,

Mayer’s reagent (0.5 ml) to the 2nd tube and color appeared in 1st and 2nd tube was matched

with filtrate in tube 3rd. The emergence of turbidity or precipitation in 1st and 2nd tube was

indication for existence of alkaloids among the constituents of concerned plant.

2.4.2. Saponins

The detection of saponins among the constituents of concerned plant was accomplished on

dissolving the crude extract (0.5 g) in 10 ml boiling H2O, cooled and was vigorously shaken.

Appearance of froth indicated presence of saponins.

2.4.3. Flavonoids

The crude extract was mixed and shaken with petroleum ether, which allows removal of fatty

material in petroleum ether layer. The residual de-fatted stuff subsequent to dissolution in

80% (30 ml) ethanol and filtration was treated as follows:

The dark yellow color developed subsequent to mixing of above-mentioned filtrate (3 ml)

with 1% potassium hydroxide (4 ml) indicated presence of flavonoids.

The yellow coloration observed subsequent to mixing of above-mentioned filtrate (3 ml) with

1% methanolic aluminum chloride (4 ml) indicated presence of flavonoids, flavones and/

chalcones.

2.4.4. Tannins and phenolic compounds

16

The addition of 1% aqueous or alcoholic solution of ferric chloride (2 ml) to solution of

extract (2 ml) of the concerned plant resulted in development of intense green, purple, blue or

black coloration as indication for existence of tannin or phenolics.

2.4.5. Anthraquinones

About 5 g extract added to 1% hydrochloric acid solution (10 ml), boiled, cooled and filtered;

extracted by benzene (5 ml). The benzene layer was removed and remained aqueous layer

was mixed with 10% ammonium hydroxide. Appearance of violet, pink or red coloration

indicated presence of anthraquinones.

2.4.6. Coumarins

The plant extract was heated on placing test tube in boiling water bath. The vapors of boiling

exact were trapped on covering mouth of the vessel with a piece of filter paper soaked in

dilute sodium hydroxide solution. After a few minutes, the filter paper was viewed on

exposure to ultraviolet light and emission of yellow florescence out of filter paper was

indication for presence of coumarins.

2.4.7. Steroides

The chloroform solution of herbal extract (10%; w/v) from concerned plant was taken in a

test tube to which concentrated sulphuric acid was added gently along wall of test tube. The

emergence of red colour at the top layer and appearance of greenish fluorescence in bottom

layer indicated presence of steroids among constituents.

2.4.8. Determination of phenolic contents

The extract was got dissolved in 50% (v/v) aqueous methanol at the rate of 1 mg per ml.

About 0.5 ml of solution was mixed to equal volume of 50% Folin-Ciocalteu reagent and

allowed to stand for 2-5 minutes, added with 20% sodium carbonate solution (1 ml), mixed

and placed in dark location for 10 minutes. Subjected to centrifugation at 4000 rpm for 8 min;

supernatant solution was separated and subjected to absorbance measurement at 730 nm by

means of a spectrophotometer (Model U2020, IRMECO, Germany). Concentration versus

absorbance curve for known concentrations of gallic acid was prepared using same method.

The phenolic contents of the extract were calculated as gallic acid equivalent per gram.

2.4.9. Determination of flavonoids contents

The solution of extract in 50% (v/v) aqueous ethanol was made (1 mg per ml). The extract

solution (3.7 ml) was mixed with 0.5 molar sodium nitrite (0.15 ml) and 0.3 molar aluminum

17

chloride (0.15 ml). After 5 min, 1 molar sodium hydroxide solution (1 ml) was added, mixed

and subjected to measurement of absorbance at 506 nm by means of a spectrophotometer

(Model U2020, IRMECO, Germany). The standard concentration versus absorbance for

quercetin under the same procedure was constructed. The flavonoids contents as quercetin

equivalent upon gram basis of the crude extract were subsequently determined from the

standard curve.

2.5. In vitro experimentation

In vitro experimentation was based on already reported methods (Gilani et al., 2006; Janbaz

et al., 2011; Janbaz et al., 2013a, b).

2.5.1. Isolated preparations of rabbit jejunum

The crude extracts as well as fractions of the concerned plants were tested on smooth muscle

preparations of rabbit isolated jejunum for possible spasmolytic or spasmogenic activity. The

jejunum was dissected out from rabbit, transferred into oxygenated Tyrode’s solution in a

Petri plate, cut into 2 cm length portions and cleaned from attached tissues. Each smooth

muscle preparation was tied with help of cotton thread at both ends and hanged in tissue

organ bath (l5 ml) filled by Tyrode’s solution at 37°C and blown by mixture of gases O2 and

CO2 (95:05). Composition of Tyrode’s solution was (mM): magnesium chloride (1.05),

sodium chloride (136.90), sodium bi carbonate (11.90), sodium dihydrogen phosphate (0.42),

potassium chloride (2.68), calcium chloride (1.80) and glucose (5.55). About 1 g preload was

applied on the rabbit preparation of isolated jejunum and contractility was assessed via

isotonic transducer (MLT0015) connected with Power Lab® data Acquisition System (AD

Instruments, Sydney, Australia) on Lab Chart® Pro (Version 7) software.

The suspended tissue preparations were incubated for 30 min for achievement of

equilibration; however, the bath fluid was replaced by fresh fluid after every 10 min.

Subsequently, the tissue was subjected repeatedly to acetylcholine (0.3 µM) exposure for

restoration of rhythmic periodic contractility. Possible influence upon contractility of

spontaneous rhythmic contractions was recorded on addition of test agents to tissue organ

bath in cumulative fashion. Additionally, the treatment of the smooth muscle preparations of

rabbit isolated jejunum with potassium (80 mM) and potassium (25 mM) exerted sustained

contractile effects, upon which test agents were applied on adding to tissue organ bath in

cumulative manner. The relaxation demonstrated by test agents was calculated on basis of

percent changes noted in rhythmic periodic contractility of isolated preparations of rabbit

18

jejunum; taking the contractile response recorded immediately prior to addition of first dose

of test material as 100% (Janbaz et al., 2013b).

The test agents capable to demonstrate potassium (80 mM) and potassium (25 mM) mediated

contractile responses to equal extent were considered to be blockers of channels for calcium.

The blocking activity for calcium channels of any test agent was confirmed further on

stabilizing the isolated preparation of rabbit jejunum in Tyrode; followed by incubation up to

30 min in Tyrode without calcium and added EDTA (0.1 mM) for removal of calcium out of

the tissue preparation. The above-mentioned solution was further substituted by Tyrode’s

solution of high potassium and devoid of calcium with contents (mM): glucose (5.55),

potassium chloride (50), sodium chloride (91.04), sodium bi carbonate (11.90), sodium

dihydrogen phosphate (0.42), magnesium chloride (1.05) and EDTA (0.1). After

equilibration, concentration response curves (CRCs) for calcium were constructed. On

achieving the reproducibility of CRCs; the isolated preparations of rabbit jejunum were

treated with different concentrations of test agent, incubated for 60 min and CRCs of calcium

were again constructed in presence of test agent.

On the other hand, test agent demonstrated relaxation of potassium (25 mM) mediated

contractile response in preference over potassium (80 mM) mediated contractile response in

smooth muscle preparations of rabbit isolated jejunum was taken as opener of potassium

channels. The confirmation of the opening of potassium channel activity was achieved as pre-

treatment with specific blocker of potassium channel resulted in blunting of test agent

demonstrated relaxant effect upon potassium (25 mM) mediated contractile response in

isolated preparations of rabbit jejunum (Gilani et al., 2006).

The antispasmodic test agent, which either failed to relax potassium mediated contractile

responses or failed to demonstrate potent relaxant activity against potassium mediated

contractile responses in comparison with spontaneous contractions of smooth muscle

preparations of rabbit isolated jejunum, were tested for possible involvement of muscarinic

receptor antagonism on application to carbachol mediated contractile response (Janbaz et al.,

2011; Khan and Gilani, 2009). Carbachol (1 µM) added to tissue organ bath demonstrated a

sustained contractile response in rabbit preparations of isolated jejunum through activation of

muscarinic receptors upon which test agent was added in cumulative tissue bath

concentration for possible relaxant response. The possible involvement of β adrenoceptor

agonistic activity was assessed upon carbachol (1 μM) mediated contractions following pre-

treatment of isolated jejunum preparations by propranolol (1 μM), which may result in

19

mitigation of the relaxant effect of the test material upon carbachol (1 μM) mediated

contractile response (Chaudhary et al., 2012).

2.5.2. Isolated preparations of rabbit trachea

The thoracic cavity of sacrificed rabbits was incised and trachea was excised, transformed

into rings comprising at least two cartilages. An incision made to cartilaginous portion on

ventral side of ring just opposite to muscle portion straightened out the tissue appearing to be

a layer of muscle embedded between two cartilaginous ends. It was loaded to tissue organ

bath (10 ml) filled with Krebs Henseleit solution of composition (mM); glucose (11.7),

sodium chloride (118.2), potassium chloride (4.7), sodium bicarbonate (25.0), calcium

chloride (2.5), potassium dihydrogen phosphate (1.3) and magnesium sulphate (1.2); steadily

blown by a gas mixture of O2 and CO2 (95:05) and kept at 37°C with help of thermo

circulator. 1.0 g preload was attached to isolated preparations of rabbit trachea and was

incubated (45 min) for achievement of equilibration; during this period bathing fluid was

replaced by fresh Krebs Henseleit solution after every 10 min intervals. The isometric

contractility of the tissue preparation was assessed on attachment with transducer linked to

Power Lab Data Acquisition System (AD Instruments, Australia). Subsequent to

equilibration, tracheal strip was exposed to carbachol (0.3 µM) for induction of sustained

contractile response and preparation was considered stable when two consecutive responses

to carbachol (0.3 µM) were found to be identical. The possible effect of test agent on baseline

tension was assessed following cumulative addition to tissue organ bath (0.01-10.0 mg per

ml); while, smooth muscle relaxant potential of test agents were screened upon carbachol (1

µM) or potassium (80 mM) mediated sustained contractile response on cumulative addition

to tissue organ bath of isolated preparations of rabbit trachea. The relaxation demonstrated by

test agent was taken as percent of control response induced by added carbachol (1 µM) or

potassium (80 mM). The CRCs for carbachol were prepared in the absence as well as

presence of test agent for assessment of plausible mode of activity.

The possible involvement of β adrenoceptor agonistic activity was assessed upon carbachol

(1 μM) mediated contractions following pre-treatment of the rabbit isolated preparations by

propranolol (1 μM), which may result in mitigation of the relaxant effect of the test material

upon carbachol (1 μM) mediated contractile response (Janbaz et al., 2011; Chaudhary et al.,

2012).

20

2.5.3. Isolated preparations of rabbit aorta

Incision to thoracic cavity of scarified rabbit was made to excise the descending thoracic

aorta, retained in a petri dish filled by Krebs; cleaned from extraneous fatty material in

careful manner. Thoracic aorta was cut into rings of 2-3 mm width, which was loaded to

tissue organ bath (10 ml) filled by Krebs solution, blown by mixture of O2 + CO2 (95: 05);

while maintaining at 37°C by thermo circulator pump. About 2 g preload was applied and

incubated for 60 min to achieve equilibration, while replacing the tissue bath fluid after every

20 min intervals and quantification of the extent of contraction in rabbit preparations of

isolated aorta was accomplished in isometric manner by means of a transducer (Model

FORT100, WPI, USA) linked with Power Lab® Data Acquisition Systems (AD Instruments,

Sydney, Australia) attached to computer running Lab Chart® Pro (Version 7).

Following incubation, rabbit preparations of isolated aorta were several times challenged by

phenylephrine (0.3 μM) to achieve stabilization as demonstrated by reproducible contractile

responses. The status of endothelial integrity of aortic preparations was assessed following

exposure to acetylcholine (1 µM) as most of the rabbit isolated aortic preparations were

without relaxant effect on imposed tone, hence considered to be having denuded

endothelium. The isolated preparations of rabbit denuded aorta have been used for study of

test agents capable to cause contractions in vascular smooth muscles on adding at cumulative

tissue organ bath concentration at the baseline tension. Whereas, relaxant potential of the test

agent was explored upon application to potassium (80 mM) or phenylephrine (1 μM)

mediated contractile responses in rabbit preparations of isolated denuded aorta on cumulative

addition to tissue organ bath; relaxation caused by each concentration of test material was

expressed as parentage of initial control contraction mediated by respective contractile agent.

2.5.4. Isolated preparations of rabbit paired atria

The thoracic cavity of sacrificed rabbit was incised and spontaneously beating heart was

excised. The paired atria were isolated on carefully removal of ventricles by means of sharp

scissors. The paired atria were loaded to tissue organ bath (15 ml) filled by Krebs maintained

at 34°C, blown persistently by gas mixture of O2 + CO2 (95 + 05). One gram pre-load was

attached to spontaneously contracting isolated rabbit paired atria preparation and contractile

response in terms of force and rate of contraction was measured via isometric transducer

linked to PowerLab data acquisition system (AD Instruments, Australia). The isolated rabbit

paired atria was incubated for 30 min for equilibration during which nothing was added to

preparation except bathing fluid was replace after every 10 min. The possible influence of

21

test agent upon contractile behavior of isolated rabbit paired atria was observed on adding to

tissue organ bath in cumulative manner and recorded in terms of percent change in force and

rate of base line control contractions.

2.6. In vivo experimentation

2.6.1. Anti-diarrheal activity in mice

The anti-diarrheal activities on the part of herbal extracts were demonstrated upon castor oil

caused diarrheal model (Mehmood et al., 2011). Albino mice (♂/♀; 20-25 g) were

accommodated individually in cages lined by filter papers and randomly allocated to six

groups (n=5). Mice were subjected to overnight fasting but availability of drinking water was

ensured. The 1st group of mice was served orally by saline (10 ml per kg) as well as 1 hr after

by orally administered olive oil (10 ml per kg) and was designated as control group. The 2nd

group of mice received oral treatment with saline (10 ml per kg) as well as 1 hr after by

castor oil (10 ml per ml) and was designated diarrheal control group. The 3rd, 4th and 5th

groups received oral treatment of herbal extract at various doses as well as 1 hr after by castor

oil (10 ml per ml) and were designated as Treated 1, Treated 2 and Treated 3 groups,

respectively. The 6th group of mice received oral treatment with loperamide (10 mg per kg) as

well as 1 hr after by castor oil (10 ml per ml) and was designated as standard group. After 4

hrs of the last treatment, diarrheal feces were counted from cages and absence or reduced

number of diarrheal drooping was considered to be indication for protection from castor oil

mediated diarrheal effect. Percent protection in wet feces counts for each treatment group was

calculated, taking fecal mass count in treated control group as 100%.

2.6.2. Influence upon charcoal meal transit distance in mice

Influence of herbal extracts upon charcoal meal travel in small intestine of mice was assessed

by slight modification of already reported procedure (Gilani and Ghayur, 2004; Akindele et

al., 2014). The albino mice (♂/♀; 20-25 g) were randomly allocated to groups, each

consisting five animals. Mice were subjected to overnight fasting but availability of drinking

water was ensured. The control group mice were given oral treatment with saline (10 ml per

kg) and designated as control group. The three or four groups of mice were given oral

treatment with herbal extracts at various doses and designated as Treated groups. Standard

groups of mice were given oral treatments with loperamide (10 mg per kg) or carbachol (1

mg per kg). Afterward, 15 min after the above treatments, all the animals were orally

administered with charcoal meal (5% charcoal suspended in 2% methylcellulose) at a rate of

22

0.2 ml per mouse. After 30 min of last treatment, the animals were killed, abdominal cavity

was incised, distance travelled by charcoal in small intestine was measured in cm and %

change in charcoal movement in comparison with control group was calculated.

2.6.3. Laxative activity in mice

The possible laxative effect of herbal extracts was assessed by the procedure reported by

Chaudhary et al., (2012). The albino mice (♂/♀; 20-25 g) were accommodated individually

in filter paper lined cages and randomly divided to four groups (n=6). Mice were subjected to

overnight fasting but availability of drinking water was ensured. The 1st group of mice

designated as saline control was treated orally by saline (10 ml per kg). The 2nd and 3rd group

mice, designated as Treated 1 and Treated 2, were treated orally by different doses of herbal

extract dissolved in same volume of saline. The 4th groups of mice, designated as standard

control was treated orally by carbachol (1 mg per kg). All the groups were monitored for 6

hrs; mean number of wet and total feces were calculated for all the 4 groups and compared

with control.

2.6.4. Influence on blood pressure in anesthetized rats

The possible influence of herbal extracts upon blood pressure was assessed in normotensive

anaesthetized rats on adoption of earlier reported methods (Jabeen et al., 2009; Jabeen and

Aslam, 2013). The study was conducted in rats (♂/♀; 200-250 g); subjected to overnight

fasting but availability of drinking water was ensured. Anesthesia was induced by means of

ketamine (i.p.; 50-80 mg per kg) and diazepam (i.p.; 10 mg per kg) and fixed on dissecting

board in supine posture while exposed to tungsten lamp to avoid hypothermia. The trachea

was exposed following 2 cm long incision and cannulated by polyethylene tube (PE 90) to

avoid possible interference in spontaneous respiratory activity. The cannulation of right

jugular vein was achieved by saline filled polyethylene tube (PE 50) to facilitate systemic

injection of test agents or saline. Blood pressure was measured following cannulation of left

carotid artery by polyethylene tubing (PE 50) filled by heparinized saline (60 i.u. per ml)

connected to pressure transducer attached with Power Lab® data Acquisition System (AD

Instruments, Australia) and computer running Lab Chart® Pro (Version 7).

The heparized saline (60 i.u. per ml) was injected (i.v.; 0.1 ml) to animal for prevention of

intravascular blood clot formation. The animal was allowed to be stabilized prior to

commencement of experiment to obtain constant levels of blood pressure and heart rate. The

test agents were injected intravenously dissolved in 0.1 ml volume and after that 0.1 ml saline

23

was injected intravenously. The responses were measured at the lowest or highest point after

intravenous administration of test agent and expressed as per cent changes in blood pressure

and heart rate from steady state values taken immediately prior to injection. The blood

pressure and heart rate of animal were allowed to be returned to basal values prior to injection

of test agents. The control responses to saline, acetylcholine and norepinephrine injections

were recorded prior to injections of test agent.

2.6.5. Diuretic activity in rats

The possible diuretic effect of herbal extract was assessed by slight modification of

previously reported methods (Jabeen and Aslam, 2013; Chen et al., 2014). The albino rats

(♂/♀; 200-220 g) were used in experiments. Rats were subjected to overnight fasting but

availability of drinking water was ensured and randomly allocated into five groups. The

animals were orally treated by saline (20 ml per kg) for proper uniform hydration, 30 min

before any treatment. The 1st group of rats were designated as control group and treated orally

by saline (10 ml per kg). The 2nd, 3rd and 4th groups of animals designated as Treated 1,

Treated 2 and Treated 3 groups, respectively, were treated by herbal extracts at various oral

doses. The 5th group of rats designated as standard group was treated orally by furosemide at

a dose of 20 mg per kg. Subsequent to dosing, animals were accommodated in diuretic cages

(Techniplast, Italy) individually. Voided urine by rats was collected up to 6 hrs and total

voided urinary volume per 100 g body weight of animals was estimated. The Na+ and K+

contents of urinary samples were determined by means of flame photometery (Model 410C,

Sherwood, UK), while Cl- contents were estimated by mean of commercially available kit

(Chloride FS, DiaSys Diagnostic Systems GmbH, Germany).

2.7. Statistical analysis

The variables were enumerated as arithmetic mean ± standard error of mean and median

effective concentrations (EC50) with 95% confidence interval (CI). The data analysis as well

as graphic presentation was accomplished by means of GraphPad Prism Software (GraphPad,

San Diego, CA, USA). Analysis of variance was the statistical technique employed (one way

or two way) along with Dunnett’s test for comparison among empirical findings and p < 0.05

was taken as statistically significant.

24

3. Asphodelus tenuifolius Cav.

Fig. 3.1: Asphodelus tenuifolius flowering plant.

25

3.1. Plant introduction

Asphodelus tenuifolius Cav. (family Asphodelaceae); known by several vernacular names,

i.e., Piazi in Urdu; Asphodel and Onion weed in English; Tazia and Barwag in Arabic;

bokhat, dangro, jangli piaz and piazai in Indian. Asphodelaceae is family of flowering plants,

comprising of approximately 39 genera and 900 species, having wide scattered distribution

throughout tropics and temperate zones of world (Christenhusz and Byng, 2016). It is one of

indigenous plants of Mediterranean region, Asia and Mascarene Islands and found throughout

Punjab, Pakistan.

It is an annual herb, seen as rosette of radical, erect, hollow, cylindrical and ribbed leaves

having reduced or underground stem and fibrous roots. Scapes are longer than leaves, several

in number, branched and hollow. Flowers racemose and bractiate appears in spring season.

Bracts, 4-6 mm, boat shaped, scarious with brownish keel. Sepals, 3-6 mm long, have white

color with pinkish central stripe and six in number. Stamens six, 3-3.5 mm long, arranged in

whorls of three each. Stigma subcapitate and three lobbed. Capsules globose and 3-4 mm in

diameter. Seed about 3 mm in circumference, rugose, trigonous and black (Verma, 2011).

Asphodelus tenuifolius has been used for culinary purpose; i.e., shoots are used as taste

enhancer, leaves are consumed boiled or fried in oil, seeds are added to flour for bread

making, seeds are also sprinkled to dates as delicacy (IUCN, 2005). The whole plant and its

parts are reported to have medicinal properties, i.e., liver protective (Yaseen et al., 2015),

antihyptensive (Mahmood et al., 2011; Ouhaddou et al., 2015), antidiabetic, antidiarrheal,

antiepileptic, antiparalytic, antipyretic, antitussive, narcotic, sedative and aphrodisiac,

(Ahmed et al., 2014), and used to treat inflammation, constipation, cold, insomnia, oedema,

piles and skin problems (Qureshi and Bhatti, 2008; Quattrocchi, 2012)

The alkaloids, anthraquinones, flavonoids, phenols, tannins and steroids are among the

reported plant constituents (Eddine et al., 2015). The isolation and characterization of β-

sitosterol, β-sitosterol 3-O-β-D-glucosides, hexadecanoic and 3-hydroxybenzoic acids, 1-

octacosanol, 1-triacontanol, tetracosanoic acid and triacontanoic acid from chloroform

fraction, and asphorin A, asphorin B, asphorodin and 2-hentriacontyl-8-methyl-5,7-

dihydroxy-4H-1-benzopyran-4-one from ethyl acetate fraction were reported (Safder et al.,

2009b; Safder et al., 2012). A number of polyphenols with variable structure, e.g., apigenin,

caffeic acid, chrysoeriol, luteolin, luteolin-7-O-β-D-glucoside, rutin, trans-N-

feruloyltyramine and vanillin are reported to be plant constituents (Khaled et al., 2014;

26

Eddine et al., 2015). The β-sitosterol, sigmasterol, 1,8-dimethoxynaphthalene, 2-acetyl-3-

methyl -8-methoxy- 1-naphthol, 2-acetyl-3-methyl-1,8-dimethoxynephthalene and

anthraquinones (chrysophanol and fallacinal) were isolated from rhizomes (Basaif and Abdel-

Mogib, 2002); while, triglycerides and fucosterol were reported from seeds (Khaqn et al.,

1961).

The plant extract demonstrated antioxidant potential (i.e., DPPH·, ABTS·+, nitric oxide,

hydroxyl, superoxide and peroxynitrite radicals scavenging activities), which was reported to

based upon high phenolics and flavonoids contents (Kalim et al., 2010). Moreover, triterpene

diglycoside (asphorodin) exhibited lipoxygenase inhibitory activity (Safder et al., 2009a).

The plant constituents were reported to boost up immune response in immune-compromised

human individuals (Papadakis and Papadakis, 1992).

The antibacterial and antifungal activities on the part of plant extracts were demonstrated

against pathogenic and non-pathogenic strains (Panghal et al., 2011; Eddine et al., 2015;

Snoussi et al., 2016).

Based upon the prevailing folkloric repute as effective remedy for management of multiple

ailments; the plant extracts were subjected to extensive scientific evaluation to validate its

existing folkloric status.

3.2. Plant material and extraction

Collection of Asphodelus tenuifolius (whole plants) was accomplished in April 2015 from

territorial jurisdiction of Bahawalpur district, Pakistan; followed by authenticated from Dr.

Zafar Ullah Zafar (plant taxonomist); Institute of Pure and Applied Biology, Bahauddin

Zakariya University, Multan, on submission of a representative specimen (Fl-PK-50-3) in

herbarium. Removal of possible adulterants was done manually and placed under shade to be

dried.

Specially designed electric grinder was used for grinding of dried herbal stuff into coarse

powder and 1 kg pulverized material was soaked at ambient room temperature (25-30°C) for

3 days in aqueous-ethanol (30:70 v/v) by using wide mouthed dull glass container with casual

stirring; followed after by straining and filtration through respective muslin cloth and filter

paper (Whatman grade 1). The above-mentioned process was reproduced twice on using new

menstruum; three aliquots of filtrates were mixed and evaporated at 37°C in a rotary

evaporator (BUCHI, Switzerland) to get a brownish green semisolid mass; i.e. crude extract

of Asphodelus tenuifolius (Cr.At) and estimated yield was 9.20 %. About 40 g of Cr.At was

27

added to 80 ml of H2O taken in a separating funnel; shaken vigorously and extracted thrice in

succession by 80 ml aliquots of petroleum ether for solvent-solvent fractionation. All the

collected aliquots of petroleum ether were mixed and solvent was removed by means of

rotary evaporator. The aqueous layer left in separating funnel was extracted thrice by 80 ml

aliquots of ethyl acetate by using above-mentioned procedure and the separated fractions

were added up and was dried in rotary evaporator. The aqueous layer remained in separating

funnel was dried through freeze drying. The yield obtained for respective petroleum ether

(Pe.At), ethyl acetate (Ea.At) and aqueous (Aq.At.) fractions was 3.52, 5.22 and 85.70 % of

the crude extract. Pe.At was not studied due to solubility issues and minute quantity. The

Cr.At was administered orally to animals on suspending in normal saline and administered

intravenously to animals while soluble in 5% DMSO-saline; Cr.At and Ea.At were used for in

vitro experiments on dissolving in 10% DMSO (Jabeen et al., 2009) to make stock solution

of 300 mg per ml, from which further dilutions were made in distilled water. Aq.At was

soluble in distilled water. Previously, time and volume matched solvent control were run for

in vitro experiments; the solvents used in dissolving the extract and fractions were without

any notable effect on baseline or induced contractions of isolated tissue preparations.

3.3. Results

3.3.1. Preliminary phytochemical analysis

The alkaloids, steroids, tannin, phenols, anthraquinones, saponins and flavonoids were

detected to be among the plant constituents. The estimation of total phenolic and flavonoids

contents demonstrated it to be 57.67 ± 4.48 and 35.00 ± 1.73 mg per g extract, respectively,

based upon respective gallic acid and quercetin equivalents.

3.3.2. In vitro experiments

3.3.2.1. Influence upon isolated preparations of rabbit jejunum

The rhythmic periodic contractions, potassium (80 mM) and potassium (25 mM) mediated

consistent contractile responses in smooth muscle preparations of rabbits isolated jejunum

were relaxed by crude extract of Asphodelus tenuifolius (Cr.At) at respective EC50 (median

response concentration) of 1.34 mg per ml [CI(95%) of 1.11-1.61 mg per ml; n=5], 0.61 mg

per ml [CI (95%) of 0.48-0.76 mg per ml; n=5] and 0.67 mg per ml [CI(95%) of 0.57-078 mg

per ml; n=5], respectively (Fig. 3.2 to 3.6). The perusal of median response concentrations

indicated potent activity of Cr.At upon potassium (80 mM) and potassium (25 mM) mediated

contractions in smooth muscle preparations of rabbit isolated jejunum compared to rhythmic

28

periodic contractile movements of smooth muscle preparations of rabbit isolated jejunum

(Fig. 3.6). Standard calcium channel blocking agent (verapamil) exhibited similar pattern of

relaxation upon potassium (80 mM), potassium (25 mM) mediated contractions and rhythmic

periodic contractions in smooth muscle preparations of rabbit isolated jejunum at median

response concentrations 0.06 µM [CI (95%) of 0.05-0.07 µM; n=5], 0.06 µM [CI (95%) of

0.04-0.07 µM; n=5] and 0.17 µM [CI (95%) of 0.15-0.19 µM; n=5], respectively (Fig. 3.7).

Furthermore, in smooth muscle preparations of rabbit isolated jejunum subsequent to

pretreatment by Cr.At at tissue organ bath concentrations (T.O.B.C.) of 0.3 and 1.0 mg per ml

resulted in shifting of response versus concentration curves (CRCs) for calcium rightward in

non-parallel manner and decreasing maximally achieved responses in significant (p < 0.05)

manner (Fig. 3.8). Similarly, verapamil at T.O.B.C. of 0.1 and 0.3 μM caused shifting of

CRCs for calcium in non-parallel fashion with mitigation of maximally achievable response

in significant (p < 0.05) manner in smooth muscle preparations of rabbit isolated jejunum

(Fig. 3.9).

Moreover, ethyl acetate fraction of Cr.At (Ea.At) caused relaxation of rhythmic periodic

contractions and potassium (80 mM) mediated contractile response in smooth muscle

preparations of rabbit isolated jejunum (Fig. 3.10 & 3.11) at median response concentrations

of 0.26 mg per ml [CI(95%) of 0.20-0.33 mg per ml; n=5] and 0.05 mg per ml [CI(95%) of

0.04-0.06 mg per ml; n=5], respectively (Fig. 3.12). The Aq.At (aqueous fraction of Cr.At)

augmented the pattern of rhythmic periodic contractility of smooth muscle preparations of

rabbit isolated jejunum (Fig. 3.13); which remained unaffected in presence of atropine (1

µM), pyrilamine (1 µM) or indomethacin (1 µM) (data not shown) but were abolished

completely following pretreatment by 1 µM verapamil (Fig. 3.14).

3.3.2.2. Influence upon isolated preparations of rabbit trachea

Application of Cr.At and verapamil to isolated tracheal preparations of rabbit did not

demonstrate any contractile response on resting baseline (data not shown). However, Cr.At

demonstrated inhibition of carbachol (1 µM) and potassium (80 mM) mediated contractile

responses in isolated tracheal preparations of rabbit at median response concentrations of

3.68 mg per ml [CI(95%) of 3.39-3.98 mg per ml; n=5] and 0.79 mg per ml [CI(95%) of

0.75-0.84 mg per ml; n=5], respectively (Fig. 3.15, 3.16 & 3.17). On comparing the median

response concentrations, Cr.At was found to be more potent in demonstration of relaxant

effect on potassium (80 mM) than carbachol (1 µM) mediated contractile response. In a

similar manner, verapamil was found to be more potent in relaxing potassium (80 mM)

29

mediated contractile response in comparison with carbachol (1 µM) mediated contractile

response in isolated tracheal preparations of rabbit at respective median response

concentrations of 0.02 µM [CI(95%) of 0.01-0.03 µM; n=5] and 0.40 µM [CI(95%) of 0.33-

0.49 µM; n=5] (Fig. 3.18). Moreover, Ea.At also exerted more potent relaxant effect on

potassium (80 mM) mediated contractile response of isolated preparations of rabbit trachea

with median response concentration of 0.04 mg per ml [CI(95%) of 0.03-0.05 mg per ml;

n=5] in comparison with carbachol (1 µM) mediated contractile response with median

response concentration of 0.58 mg per ml [CI(95%) of 0.47-0.71 mg per ml; n=5] (Fig. 3.19,

3.20 & 3.21). Whereas, Aq.At was found to demonstrate incomplete relaxant response upon

potassium (80 mM) and carbachol (1 µM) mediated contractile activities (Fig. 3.22, 3.23 &

3.24).

Fig. 3.2: Tracing showing spontaneous movements of smooth muscle preparations of rabbit isolated jejunum.

Fig. 3.3: Record of influence of cumulatively added Cr.At upon rhythmic periodic contractile movements in smooth muscle preparations of rabbit isolated jejunum.

Fig. 3.4: Record of Cr.At influence upon cumulative addition to potassium (25 mM) mediated contractility of isolated preparation of rabbit jejunum.

30

Fig. 3.5: Record of influence of cumulative addition of Cr.At upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum.

Fig. 3.6: Influence of Cr.At upon spontaneous, potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5).

Fig. 3.7: Verapamil influence upon spontaneous contractions, potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated jejunum. Variable are mean ± S.E.M. (n=5).

31

Fig. 3.8: Influence of Cr.At upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5). * indicates statistically significant (p < 0.05) difference from maximum response of control.

Fig. 3.9: Influence of verapamil upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5). * indicates statistically significant (p < 0.05) difference from maximum response of control.

32

Fig. 3.10: Record of influence of Ea.At upon rhythmic periodic contractility of isolated preparation of rabbit jejunum.

Fig. 3.11: Record of influence of Ea.At upon potassium (80 mM) mediated contractility of isolated preparation of rabbit jejunum.

Fig. 3.12: Effects of Ea.At on spontaneous and potassium (80 mM) mediated contractions in isolated jejunum preparations. Values are mean ± S.E.M. (n=5).

33

Fig. 3.13: Effects of acetylcholine (Ach; 1 µM) as well as cumulative concentrations of Aq.At on rhythmic periodic contractions of isolated preparation of rabbit jejunum.

Fig. 3.14: Contractile activity of Aq.At upon rhythmic spontaneous contractions in smooth muscle preparations of rabbit isolated jejunum, alone and in presence of verapamil (1 µM). Variables are mean ± S.E.M. (n=5).

Fig. 3.15: Record demonstrating relaxant effect of Cr.At upon potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea.

34

Fig. 3.16: The Cr.At demonstrated relaxation upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea.

Fig. 3.17: Influence of Cr.At upon potassium (80 mM) and carbachol (1 µM) mediated contractions in rabbit isolated tracheal preparations. Variable are mean ± S.E.M. (n=5).

Fig. 3.18: Influence of verapamil upon potassium (80 mM) and carbachol (1 µM) mediated contractions of rabbit isolated tracheal preparations. Variable are mean ± S.E.M. (n=5).

35

Fig. 3.19: The Ea.At demonstrated relaxant effect upon potassium (80 mM) mediated contractility of isolated tracheal preparation of rabbit.

Fig. 3.20: The Ea.At demonstrated relaxant effect upon carbachol (1 μM) mediated contractility of isolated tracheal preparation of rabbit.

Fig. 3.21: Effects of Ea.At upon potassium (80 mM) and carbachol (1 μM) mediated contractions in rabbit isolated tracheal preparations. Variable are mean ± S.E.M. (n=5).

Fig. 3.22: Record showing inability of Aq.At to relax potassium (80 mM) mediated contractility of rabbit isolated tracheal preparation.

36

Fig. 3.23: Record showing influence of Aq.At upon carbachol (1 μM) mediated contractility of rabbit isolated tracheal preparation.

Fig. 3.24: Effects of Aq.At upon potassium (80 mM) and carbachol (1 μM) mediated contractile responses of smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5).

3.3.2.3. Influence upon isolated denuded aortic preparations of rabbit

As acetylcholine (1 µM), on application to isolated aortic preparations of rabbit, was unable

to exert any relaxant effect on the imposed tone; hence, preparations were found to be

denuded because endothelium was proved nonfunctional. The Cr.At demonstrated relaxant

effect upon phenylephrine (1 µM) as well as potassium (80 mM) mediated contractile

responses of isolated denuded aortic preparations of rabbit with respective median response

concentrations of 5.55 mg per ml [CI(95%) of 2.59-11.85 mg per ml; n=5] and 0.77 mg per

ml [CI(95%) of 0.73-0.82 mg per ml; n=5) (Fig. 3.25, 3.26 & 3.27). In a similar manner,

verapamil exerted relaxation of phenylephrine (1 µM) as well as potassium (80 mM)

mediated contractile response of isolated denuded aortic preparations of rabbit at median

response concentrations of 1.0 µM [CI(95%) of 0.88-1.14 µM; n=5] and 0.28 µM [CI(95%)

of 0.24-0.34 µM; n=5], respectively (Fig. 3.28). Moreover, Ea.At exerted relaxant effect upon

37

phenylephrine (1 µM) as well as potassium (80 mM) mediated contractile responses in

denuded aortic preparations of rabbit at respective median response concentrations of 1.82

mg per ml [CI(95%) of 01.62-2.06 mg per ml; n=5] and 0.38 mg per ml [CI(95%) of 0.29-

0.49 mg per ml; n=5] (Fig. 3.29, 3.30 & 3.31). Furthermore, Aq.At also demonstrated

relaxation of potassium (80 mM) mediated contractile response in isolated denuded aortic

preparations of rabbit at median response concentration of 1.01 mg per ml [CI(95%) of 0.82-

1.22 mg per ml; n=5], but only partially relaxed (about 20%) the phenylephrine (1 µM)

mediated contractions when tested at T.O.B.C. of 0.01-10 mg per ml (Fig. 3.32, 3.33 & 3.34).

Moreover, Cr.At, Ea.At and verapamil were found to be more effective in relaxing potassium

(80 mM) mediated contractile response compared to phenylephrine (1 µM) mediated

contractile response in isolated denuded aortic preparations of rabbit.

3.3.2.4. Influence upon isolated paired atrial preparations of rabbit

The isolated paired atrial preparations of rabbit demonstrated positive inotropic activity when

exposed to Cr.At at T.O.B.C. range of 0.01-1.0 mg per ml; however, it exhibited negative

inotropic activity at higher T.O.B.C. of 3.0-10.0 mg per ml. The Cr.At was without any

change to the heart rate at T.O.B.C. range of 0.01-1.0 mg per ml but demonstrated negative

chronotropic activity at T.O.B.C. range of 3.0-10.0 mg per ml in isolated paired atrial

preparations of rabbit (Fig. 3.35 & 3.36).

Fig. 3.25: Influence of Cr.At upon potassium (80 mM) mediated contractility of isolated denuded aortic preparation of rabbit.

Fig. 3.26: Influence of Cr.At upon phenylephrine (1 µM) mediated contractility of isolated denuded aortic preparation of rabbit.

38

0.01 0.1 1 10

0

25

50

75

100

PE (1 µM)

K+ (80 mM)

[Cr.At] mg/ml

Co

ntr

ol

(%)

Fig. 3.27: Influence of Cr.At upon phenylephrine (1 µM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated denuded aorta. Variables are mean ± S.E.M. (n=5).

Fig. 3.28: Influence of verapamil upon phenylephrine (1 µM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated denuded aorta. Variables are mean ± S.E.M. (n=5).

39

Fig. 3.29: Record demonstrating influence of Ea.At upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparation of rabbit.

Fig. 3.30: Record showing effect of Ea.At on phenylephrine (1 μM) mediated contractile response in isolated denuded aortic preparation of rabbit.

Fig. 3.31: Effects of Ea.At on potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in smooth muscle preparations of rabbit isolated denuded aorta. Variables are mean ± S.E.M. (n=5).

Fig. 3.32: Tracing demonstrating Aq.At influence upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparation of rabbit.

40

Fig. 3.33: Record showing effect of Aq.At on phenylephrine (1 μM) mediated contractile response in isolated denuded aortic preparation of rabbit.

Fig. 3.34: Effects of Aq.At upon potassium (80 mM) and phenylephrine (1 μM) mediated contractions in smooth muscle preparations of rabbit isolated denuded aorta. Variables are mean ± S.E.M. (n=5).

Fig. 3.35: Record of Cr.At influence upon force of spontaneous contractions in isolated paired atrial preparation of rabbit.

41

Fig. 3.36: Influence of Cr.At upon rhythmic periodic contractility of rabbit isolated preparations of paired atria. Variables are mean ± S.E.M. (n=5).

3.3.3. In vivo experiments

3.3.3.1. Influence upon charcoal meal transit distance in mice

The charcoal meal transit distance in control group of animals (n=5) was assessed to be 31.20

± 0.85 cm. The administration of Cr.At to Treated 1 group of animals (n=5) at an oral dose of

100 mg per kg resulted significant (p < 0.05) increase in intestinal transit distance of charcoal

as compared to control group and was assessed to be 35.84 ± 0.75 cm. However, Cr.At

administration (300 mg per kg; orally) to Treated 2 group of animals (n=5) resulted

insignificant decrease (p > 0.05) in intestinal charcoal transit distance as compared to control

and was assessed to be 29.88 ± 1.80 cm. Moreover, the Cr.At administration (500 mg per kg;

orally) to Treated 3 group of mice (n=5) resulted significant decrease (p < 0.05) in intestinal

transit distance of charcoal meal in comparison with control group and was measured to be

23.46 ± 0.75 cm. Furthermore, intestinal transit distance of charcoal meal in loperamide

treatment (10 mg per kg; orally) standard group of mice (n=5) was significantly (p < 0.05)

decreased as compared to control group and was measured to be 7.40 ± 0.29 cm.

Furthermore, carbachol administration (1 mg per kg, orally) to another standard group of

mice (n=5) caused significantly increased (p < 0.05) charcoal intestinal transit distance in

comparison with control and was measured to be 44.82 ± 1.05 cm (Table 3.1).

42

Group Treatment

Charcoal meal transit distance

Mean ± S.E.M. (cm)

% Change

Control Saline + Charcoal meal

(10 ml per kg + 0.2 ml) 31.20 ± 0.85 --

Treated 1 Cr.At + Charcoal meal

(100 mg per kg + 0.2 ml) 35.84 ± 0.75* +11.20


(300 mg per kg + 0.2 ml) 29.88 ± 1.80ns -7.29


(500 mg per kg + 0.2 ml) 23.46 ± 0.75* -27.21

Standard 1 Loperamide + Charcoal meal

(10 mg per kg + 0.2 ml) 7.40 ± 0.29* -77.04

Standard 2 Carbachol + Charcoal meal

(1 mg per kg + 0.2 ml) 44.82 ± 1.05* +39.06

Table 3.1: Effects of oral administrations of Cr.At, loperamide and carbachol upon charcoal meal intestinal transit in mice. Values are mean ± S.E.M. (n=5); (+) indicates increase and (–) indicate decrease in intestinal travel distance relative to the control group. Statistic: one way ANOVA followed by Dunnett test (nsp ≥ 0.05, *p < 0.05 vs. control group).

3.3.3.2. Anti-diarrheal effect in mice

The mean number of wet feces in saline control group of mice (n=6), administered orally

with saline (10 ml per kg) and olive oil (10 ml per kg), was found to be 0.33 ± 0.21; whereas,

wet feces count in treated control group of animal (n=6), administered orally with saline (10

ml per kg) and castor oil (10 ml per kg), was 8.20 ± 0.31. The Treatment 1 group of mice

(n=6), administered orally with Cr.At and castor oil at respective doses of 300 mg per kg and

10 ml per kg, resulted insignificantly decreased wet feces count to 7.40 ± 0.24 (p > 0.05 vs.

treated control group). However, Treatment 2 group (n=6) of mice, administered orally with

Cr.At and castor oil at respective doses of 500 mg per kg and 10 ml per kg, resulted

significantly decreased (p < 0.05) wet feces count to 5.00 ± 0.32. Similarly, oral

administration of Cr.At (700 mg per kg) and castor oil (10 ml per kg) to Treated 3 group of

mice (n=6) resulted significant decrease (p < 0.05) in mean wet feces count to 3.80 ± 0.37,

43

when compared to treated control group. Mean diarrheal feces count in the standard treatment

group of mice (n=6), administered orally with loperamide (10 mg per kg) and castor oil (10

ml per kg), was significantly decreased to 0.80 ± 0.37 (p < 0.05 vs. treated control) (Table

3.2).

Group Treatment

(ml per kg)

Wet feces count

Mean ± S.E.M.

Protection (%age)

Saline Control

Saline + Olive oil

(10 ml per kg + 10 ml per kg) 0.33 ± 0.21 -

Treated Control Saline + Castor oil

(10 ml per kg + 10 ml per kg) 8.20 ± 0.31 0

Treatment 1 Cr.At + Castor oil

(300 mg per kg + 10 ml per kg) 7.40 ± 0.24ns 9.76


(500 mg per kg + 10 ml per kg) 5.00 ± 0.32* 39.02


(700 mg per kg + 10 ml per kg) 3.80 ± 0.37* 53.65

Standard Treatment

Loperamide + Castor oil

(10 mg per kg + 10 ml per kg) 0.50 ± 0.22* 93.90

Table 3.2: Effects of oral administration of Cr.At and loperamide upon castor oil caused diarrhea in mice. Statistics: one way ANOVA and Dunnett’s test (nsp ≥ 0.05 and *p < 0.05 vs. treated control group).

3.3.3.3. Laxative activity in mice

A group of mice (n=6) designated as saline control was administered with saline (10 ml per

kg; orally); and wet feces as well as total feces counts after 6 hrs were found to be 0.50 ± 0.22

and 4.83 ± 0.31, respectively. The Treated 1 group of mice (n=6) was orally administered

with Cr.At (50 mg per kg); the wet and total feces counts after 6 hrs were significantly (p <

0.05) increased to 2.00 ± 0.26 and 6.50 ± 0.22, respectively, as compared to saline control

group. Similarly, Treated 2 group of mice were treated orally by Cr.At (100 mg per kg); wet

and total feces counts after 6 hrs were significantly (p < 0.05) increased to 2.50 ± 0.22 and

7.50 ± 0.22, respectively, as compared to saline control group. The wet and total fecal mass

44

counts in standard control group of mice, to whom carbachol (1 mg per kg) was administered,

were significantly increased (p < 0.05) to 7.67 ± 0.33 and 11.17 ± 0.40, respectively, as

compared to saline control group (Table 3.3).

Groups Treatment Wet feces count

Mean ± S.E.M.

Total feces count

Mean ± S.E.M.

Saline control Normal saline

10 ml per kg 0.50 ± 0.22 4.83 ± 0.31

Treated 1 Cr.At

50 mg per kg 2.00 ± 0.26* 6.50 ± 0.22*

Treated 2 Cr.At

100 mg per kg 2.50 ± 0.22* 7.50 ± 0.22*

Standard control Carbachol

1 mg per kg 7.67 ± 0.33* 11.17 ± 0.40*

Table 3.3: Laxative effects of Cr.At and carbachol in normal mice. Values are mean ± S.E.M. (n=6). Statistics: one way ANOVA followed by Dunnett test (*p < 0.05 vs. saline control group).

3.3.3.4. Hypotensive effect in normotensive anaesthetized rats

Vehicle (5% DMSO in saline) administered intravenously to normotensive anesthetized rats

caused 1.06 ± 0.35, 1.46 ± 0.43, 1.58 ± 0.55 and 0.35 ± 0.16 % decrease in systolic blood

pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate

(HR) from baseline values, respectively. The Cr.At administered intravenously to

normotensive anesthetized rats at a dose of 3 mg per kg resulted in insignificant (p ˃ 0.05)

decrease in SBP (9.23 ± 0.50 %) and HR (0.90 ± 0.27 %) but demonstrated significant (p <

0.05) decrease in DBP (22.32 ± 0.52 %) and MBP (14.16 ± 0.53 %) as compared with vehicle

treated group. Moreover, Cr.At administration to normotensive anesthetized rats at

intravenous dose of 10 mg per kg resulted significantly (p < 0.05) decreased SBP, DBP, MBP

and HR; and % decrease in values was recorded to be 16.52 ± 0.51, 34.15 ± 1.01, 25.57 ±

0.56 and 4.80 ± 0.46, respectively. Furthermore, intravenous administration of Cr.At (30 mg

per kg) also demonstrated significant decrease (p < 0.05 vs. vehicle treated values) in SBP,

DBP, MBP and HR, which were recorded to be decreased by 29.42 ± 0.80, 52.93 ± 1.89,

34.67 ± 1.05 and 6.48 ± 0.47 %, respectively. The hypotensive and bradycardiac effects of

45

Cr.At were transitory, which returned to normal baseline values within 2-3 minutes.

Similarly, intravenous administration of acetylcholine (1 µg per kg) to normotensive

anaesthetized rats demonstrated significant fall (p < 0.05 vs. vehicle) in SBP, DBP, MBP and

HR, values were recorded to be decreased by 41.25 ± 1.32, 54.57 ± 0.97, 46.55 ± 2.04 and

21.00 ± 1.81 %, respectively. Intravenous administration of verapamil (1 mg per kg) to

normotensive anaesthetized rats also demonstrated significant (p < 0.05) decrease in SBP,

DBP, MBP and HR in comparison with solvent treatment values; decrease in values was

recorded 32.87 ± 1.21, 37.63 ± 1.76, 34.78 ± 1.36 and 12.85 ± 1.26 %, respectively (Fig.

3.37; Table 3.4). The pretreatment of animals with atropine (1 mg per kg) abolished the

acetylcholine (1 µg per kg) induced hypotensive effect, but did not affect verapamil (1 mg

per kg) and Cr.At induced hypotensive effects (Fig. 3.38).

Fig. 3.37: Effect of intravenously administered acetylcholine (Ach, 1 µg per kg), solvent (5% DMSO in normal saline) and different doses of crude extract of Asphodelus tenuifolius (Cr.At) on blood pressure of normotensive anesthetized rat.

Treatment % decrease from basal values

SBP DBP MBP HR

Solvent (5% DMSO in saline) 1.13 ± 0.44 1.08 ± 0.52 1.15 ± 0.49 0.35 ± 0.16

Cr.At (3 mg per kg) 9.23 ± 0.56 22.32 ± 0.52* 14.16 ± 0.53* 0.90 ± 0.27

Cr.At (10 mg per kg) 16.52 ± 0.51* 34.15 ± 1.01* 25.57 ± 0.56* 4.80 ± 0.46*

Cr.At (30 mg per kg) 29.42 ± 0.80* 52.93 ± 1.89* 34.67 ± 1.05* 6.48 ± 0.47*

Acetylcholine (1 µg per kg) 41.25 ± 1.32* 54.57 ± 0.97* 46.55 ± 2.04* 21.00 ± 1.81*

Verapamil (1 mg per kg) 32.87 ± 1.21* 37.63 ± 1.76* 34.78 ± 1.36* 12.85 ± 1.26*

Table 3.4: Effects of intravenous administration of solvent, crude extract of Asphodelus tenuifolius (Cr.At), acetylcholine and verapamil on mean blood pressure (MBP), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) of anesthetized rats. Variables are mean ± SEM (n=6), *p < 0.05 vs. respective vehicle treatment, one way ANOVA followed by Dunnett test.

46

Fig. 3.38: Bar chart showing effects of acetylcholine (Ach), verapamil and different doses of crude extract of Asphodelus tenuifolius (Cr.At) on mean blood pressure (MBP) of normotensive anesthetized rats, with and without atropine (1 µM) pretreatment. Values are mean ± S.E.M. (n=5).

3.3.3.5. Diuretic activity in rats

The rats in control group (n=8) were treated by normal saline (10 ml per kg; orally); the

volume of voided urine was measured to be 1.01 ± 0.05 ml per 100 g body weight in 6 hrs;

the excretion of urinary electrolytes estimated, i.e., Na+, K+ and Cl- were 122.00 ± 3.79, 7.70

± 0.20 and 128.10 ± 2.00 µmol per 100 g in 6 hrs, respectively. The Cr.At (100 mg per kg)

treatment to a group of rats (n=8) caused increase in volume of the voided urine (1.16 ± 0.05

ml per 100 g in 6 hrs) and Cl- contents (139.10 ± 2.24 µmol per 100 g in 6 hrs) in non-

significant (p > 0.05) manner, but it increased significantly (p < 0.05) the respective Na+ and

K+ excretion to 155.40 ± 5.22 and 10.09 ± 0.04 µmol per 100 g in 6 hrs, as compared to

saline control group of rats. The Cr.At (300 mg per kg) treatment to a group of rats (n=8)

caused significant (p < 0.05) increase in volume of the voided urine (1.55 ± 0.06 ml per 100 g

in 6 hrs) as well as respective Na+, K+ and Cl- urinary excretion to 190.00 ± 4.75, 13.96 ±

0.22 and 220.60 ± 5.55 µmol per 100 g in 6 hrs in comparison with saline control group of

47

rats. The Cr.At (500 mg per kg) treatment to a group of rats caused significant increase (p <

0.05) in volume of the voided urine (2.32 ± 0.05 ml per 100 g in 6 hrs) as well as respective

Na+, K+ and Cl- excretion to 313.50 ± 6.02, 23.98 ± 0.60 and 364.10 ± 7.82 µmol per 100 g in

6 hrs in comparison with saline control group of rats. The furosemide (20 mg per kg)

treatment to a group of rats (n=8) caused significant increase (p < 0.05) in volume of the

voided urine (4.09 ± 0.09 ml per 100 g in 6 hrs) as well as respective Na+, K+ and Cl- urinary

excretion to 375.40 ± 4.88, 31.50 ± 0.45 and 512.80 ± 7.33 µmol per 100 g in 6 hrs, in

comparison with saline control group of rats. Derived parameters including diuretic index,

Na+/K+ ratio and ion quotient were also calculated (Table 3.5).

48

Treatment group (dose) Volume of urine

ml/100 g/6 hrs

Diuretic index

Na+ K+ Cl-

Na+/K+ Ion quotient (µmol/100 g/6 hrs)

Saline (10 ml per kg) 1.01 ± 0.05 - 122.00 ± 3.79 7.70 ± 0.20 128.10 ± 2.00 15.95 ± 0.77 0.99 ± 0.02

Cr.At (100 mg per kg) 1.16 ± 0.05ns 1.15 155.40 ± 5.22* 10.09 ± 0.04* 155.50 ± 4.67* 15.40 ± 0.49 ns 0.93 ± 0.04

Cr.At (300 mg per kg) 1.57 ± 0.07* 1.55 190.00 ± 4.75* 13.96 ± 0.22* 220.60 ± 5.55* 13.68 ± 0.38 ns 1.01 ± 0.05

Cr.At (500 mg per kg) 2.34 ± 0.07* 2.31 313.50 ± 6.02* 23.98 ± 0.60* 364.10 ± 7.82* 13.12 ± 0.35* 1.08 ± 0.03

Furosemide (20 mg per kg) 4.09 ± 0.11* 4.05 375.40 ± 4.88* 31.50 ± 0.45* 512.80 ± 7.33* 11.94 ± 0.29* 1.27 ± 0.02

Table 3.5: Diuretic activity of crude extract of Asphodelus tenuifolius (Cr.At) in rats. Diuretic index was calculated by dividing urinary excretion of treatment group by urinary execration of saline group, Ion quotient = Cl-/ (Na+ +K+). Data shown as mean ± S.E.M. (n=8). p values compared to control saline group as nsp ≥ 0.05, *p < 0.05.

49

3.4. Discussion

The Asphodelus tenuifolius is one of the indigenous plants possesses folkloric reputes to be

used by traditional healers in the management of common ailments, i.e., asthma, constipation,

diarrhea and hypertension (Qureshi and Bhatti, 2008; Mahmood et al., 2011; Quattrocchi,

2012; Ouhaddou et al., 2015). In present study, the plant was subjected to extensive

pharmacological investigations for scientific authentication of its folkloric utility to manage

gut, respiration and cardiovascular disorders.

Chemical investigations upon crude extract of Asphodelus tenuifolius (Cr.At) detected

presence of alkaloids, steroids, tannin, phenols, anthraquinones, saponins and flavonoids

among the plant constituents.

The Cr.At as well as its Ea.At (ethyl acetate) and Aq.At (aqueous) fractions were studied on

rhythmic periodical contractile activity in smooth muscle preparations of isolated rabbit

jejunum. The Cr.At and Ea.At were found to exert relaxant effect upon rhythmic periodic

contractions in smooth muscle preparations of isolated rabbit jejunum. It has been reported

earlier that isolated tissue preparations subsequent to potassium ion exposure exhibited

contractile response, mediated primarily through opening of voltage dependent calcium

channels (Karaki et al. 1984, Grasa et al., 2004; Ratz et. al., 2005). Agents capable to relax

potassium (80 mM) and potassium (25 mM) mediated contractions in smooth muscle

preparations of isolated rabbit jejunum at comparable fashion are known to do so on blocking

of calcium channels; whereas, substances capable to relax selectively potassium (25 mM)

mediated contractions in smooth muscle preparations of isolated rabbit jejunum are known to

be potassium channel openers (Gilani et al., 2006; Jabeen et al., 2009). Therefore, the

possible mechanisms of the observed relaxant activity on the part of Cr.At was explored on

testing upon potassium (80 mM) and potassium (25 mM) mediated contractions in smooth

muscle preparations of isolated rabbit jejunum. The Cr.At relaxed potassium (80 mM) and

potassium (25 mM) mediated contractility of smooth muscle preparations of isolated rabbit

jejunum at comparable T.O.B.C. in similar fashion to verapamil, a standard voltage

dependent calcium channel blocking drug (Scholz, 1997); hence, presence of voltage

dependent calcium channel blocking activity was indicated. The shifting of CRCs for calcium

rightward with mitigation of the optimal response following exposure of smooth muscle

preparations of isolated rabbit jejunum to Cr.At confirmed presence of noncompetitive

calcium antagonist activity in the extract (Daniel et al., 2001; Jabeen et al., 2009).Verapamil

also caused rightward displacement of CRCs for calcium in manner similar to Cr.At. The

50

calcium channel blockers, which exert smooth muscle relaxant effect by inhibition of calcium

influx via voltage dependent calcium channels, are valuable therapeutic modality for

treatment of multiple ailments (Godfraind, et al., 1986; Katzung and Chatterjee, 2009).

In contrast to the crude extract (Cr.At), its aqueous fraction (Aq.At) exerted contractile

activity upon smooth muscle preparations of isolated rabbit jejunum, indicating separation of

some contractile components of the plant in Aq.At. The contractile effect was apparent at

T.O.B.C. range of 0.3-10.0 mg per ml. The water based preparations of Asphodelus

tenuifolius (infusion or decoction) possess medicinal properties and have been effective in the

management of constipation (Quattrocchi, 2012). The Aq.At induced contractile response in

smooth muscle preparations of isolated rabbit jejunum was explored for underlying cause of

the demonstrated contractile response through pre-incubation of the tissue with antagonistic

drugs like atropine (muscarinic receptor antagonist), pyrilamine (histamine receptor

antagonist), indomethacin (cycloxygenase inhibitor) and verapamil (calcium channel blocker)

(Mard et al., 2011; Najeeb-ur-Rehman et al., 2011). The observed contractile activity of

Aq.At was suppressed subsequent to exposure of the smooth muscle preparations of isolated

rabbit jejunum to verapamil reflecting that contractile activity may possibly be mediated

through opening of voltage dependent calcium channels (Mard et al., 2011). The presence of

both contractile and relaxant activities in a same plant extract have already been reported

(Khan and Ahmad, 2007; Mehmood et al., 2011; Qin et al., 2011; Ahmad et al., 2012) and

may be regarded as a beneficial mechanism to keep excessive contractile or relaxant bodily

activities within limits. The effect of Cr.At on gastrointestinal motility was assessed in

charcoal meal transit distance study in mice by the measurement of charcoal propulsion

through small intestine. The Cr.At exerted dual effect on charcoal propulsion, i.e., stimulatory

at low dose and inhibitory at high doses. The possible anti-diarrheal activity of Cr.At was

assessed on diarrhea caused by castor oil in mice. As castor oil is known to produce diarrhea

due to hydrolysis by pancreatic enzymes into ricinoleic acid in gut, which produces diarrhea

following alteration in water as well as electrolytes transportation leading to enhancement in

contractile activities in colon (Stewart et al., 1975). The oral administration of Cr.At (500 and

700 mg per kg) resulted significant (p < 0.05) inhibition of castor oil exerted diarrhea in mice

in comparison with saline treated control group. The calcium channel blockers may have

beneficial effect in diarrhea because of holding luminal fluid due to relaxant activity in gut

smooth muscles (Lee et al., 1997). The anti-diarrheal potential of Cr.At may possibly be

assigned to calcium channel blocking activity.

51

The Asphodelus tenuifolius possesses ethnopharmacological reputation for effectiveness in

the management of diseases pertaining to respiratory system (Ahmed et al., 2014); hence,

Cr.At, Ea.At and Aq.At were screened upon carbachol (1 μM) and potassium (80 mM)

mediated sustained contractions in smooth muscle preparations of rabbit isolated trachea. The

Cr.At and Ea.At caused relaxation of potassium (80 mM) and carbachol (1 μM) mediated

contractile responses. The relaxant effects of Cr.At as well as Ea.At were noted to be

achieved potently on potassium (80 mM) mediated contractile effect as compared to

carbachol (1 μM) exerted contractile response in a fashion comparable with verapamil

(standard calcium channel blocker); hence blockade of calcium channels may be the plausible

explanation. Whereas, Aq.At partially relaxed the potassium (80 mM) as well as carbachol (1

μM) mediated contractile responses in smooth muscle preparation of isolated rabbit trachea.

The hypertension is commonly managed on employing vasodilator and cardio-depressant

drugs (Benowitz, 2009). The possible vasodilator potential of Cr.At was screened upon

potassium (80 mM) as well as phenylephrine (1 µM) exerted contractile responses in smooth

muscle preparations of rabbit isolated endothelium denuded aorta. The smooth muscle

preparations of rabbit isolated denuded aorta following treatment with potassium (80 mM)

exposure undergoes depolarization of cell membrane; causing calcium channels opening;

resulting in extracellular calcium influx; consequently increased free calcium in cytoplasm

produced sustained contractions (Karaki et al., 1997). The Cr.At caused relaxation of

potassium (80 mM) mediated contractions, reflecting possible blockade of voltage dependent

calcium channels in observed relaxant effect. Whereas, phenylephrine activates α1-adrenergic

receptors; causing activation of membrane associated phospholipase, release of inositol-1,4,5-

triphosphate and diacylglycerol (DAG) from phosphatidylinositol 2,3-bisphosphate; hence,

increased free calcium in cytoplasm either due to release of calcium from sarcoplasmic

reticulum, or calcium influxed from calcium channels; leading ultimately to contraction of

aorta (Karaki et al., 1997). So, observed relaxant effect on the phenylephrine mediated

contractile response by Cr.At was speculated to be mediated either via blockade of calcium

channels or blockade of calcium released from sarcoplasmic reticulum. Since, Cr.At caused

relaxation of potassium (80 mM) exerted contractile response in isolated denuded aortic

preparations of rabbit in potent manner in comparison with phenylephrine (1 µM) exerted

contractile response in a comparable fashion to verapamil (standard calcium channel blocker)

suggesting that Cr.At exerted relaxant effect may be convened via calcium channels

blockade. Calcium channel blocking agents, possessing vasodilator and cardio-depressant

52

activities, as therapeutic modalities serve to manage hypertension and angina (Eisenberg et

al., 2004).

On application to the spontaneously contracting muscular preparations of isolated rabbit

paired atria, the Cr.At at T.O.B.C. range of 0.01-1.0 mg per ml, exhibited positive inotropic

activity without affecting atrial rate of contractions. However, it exhibited negative inotropic

and chronotropic activities at T.O.B.C. range of 3-10 mg per ml. The demonstrated decrease

in rate and force of contraction of spontaneously contracting muscular preparations of

isolated rabbit paired atria on the part of Cr.At may be assigned to proposed calcium channel

blocking potential. The combined presence of mild cardio-tonic along with calcium channel

blocking activities in the extract of Asphodelus tenuifolius may be envisioned as a measure to

minimize the side effects on heart due to cardio-depressive activity of calcium channel

blockers (Benowitz, 2009).

The Cr.At caused fall in systolic, diastolic and mean blood pressure, and heart rate on

intravenous administration to normotensive anesthetized rats. The observed response is in

good agreement with its folkloric uses in the management hypertension (Mahmood et al.,

2011). As cardiac output and peripheral vascular resistance are main determinants of blood

pressure (Benowitz, 2009); hence, observed hypotensive response of Cr.At may be assigned

to both of these components. The observed hypotensive activity can be attributed to observed

presence of calcium channel blocking activity on the part of Cr.At.

The Asphodelus tenuifolius possesses folkloric repute as a diuretic; hence, subjected to

diuretic assay. The Cr.At administered orally to rats resulted in increased urinary excretion of

electrolytes (Na+, K+, Cl-) and water. Hence, possible mechanism of the observed diuretic

activity on the part of Cr.At was investigated in terms of urinary electrolytes and their ratios.

A large Na+/K+ ratio illustrates potassium conserving ability, and ion quotient less than 0.8

indicates carbonic anhydrase inhibitory potential of diuretics (Vogel, 2008). The results

suggested that constituents available in Cr.At exhibited pattern of electrolyte and water

excretion in a manner similar to furosemide. The diuretics as a therapeutic modality are

effective in management of cardiovascular diseases, i.e., hypertension and congestive heart

failure (Ives, 2009).

3.5. Conclusion

In conclusion, Asphodelus tenuifolius extract possesses relaxant effects on smooth muscles

possibly medicated through calcium entry blocking activity, which through scientific

53

authentication validated the folkloric uses of the plant in treating diarrhea, asthma and

hypertension. The aqueous fraction was also found to exert contractile activity in smooth

muscle preparations of isolated rabbit jejunum. Moreover, in vivo studies on plant extract

revealed dual activity, i.e., laxative and antidiarrheal activities. The laxative activity was

observed in small doses, whereas, antidiarrheal activity at higher doses in mice. The Cr.At

also exhibited hypotensive and diuretic activities in rats. Study on fractions revealed that

relaxant and calcium channel blocking activities were concentrated in ethyl acetate fraction.

54

4. Corchorus depressus Linn.

Fig. 4.1: Corchorus depressus plant

55


Corchorus depressus Linn. (Synonyms; Antichorus depressus Linn, Corchorus antichorus

Raeusch and Corchorus prostratus Royle) belongs to family Tiliaceae. The plant is

commonly found in sandy clay and saline areas of arid and semi-arid parts of the World and

grows luxuriously in Afghanistan, Arabia, India, Pakistan, and Tropical Africa (Mathur and

Sundaramoorthy, 2008).

The plant is a perennial, prostate mat forming, densely branched woody herb. Leaves are

about 1 cm in width and 2 cm in length, elliptical in shape, ribbed and glabrous, having

rounded or wedge shaped bases and irregular serrate margins connected with filiform

petioles. The plant has cymose inflorescence with up to 1 cm across yellow flowers. Fruits

are cylindrical capsules, straight or curved, containing blackish triangular seeds (Mathur and

Sundaramoorthy, 2008; Sinha et al., 2011).

In subcontinent, the plant is sold by herbalists under the name of “Bhauphali” and “Munderi”

(Ahmad et al., 2000). Traditionally, the plant is used in urinary problems (Goodman and

Ghafoor, 1992; Kakrani and Saluja, 2001), swellings, diarrhea, dysentery, gonorrhea (Tareen

et al., 2010) and as aphrodisiac (Ahmad et al., 2014). Infusion of leaves is demulcent,

carminative, laxative, stimulant and appetizer (Singh and Panday, 1983). The plant is crushed

in water and used as emollient, tonic and cooling agent in summer season (Goodman and

Ghafoor, 1992). Stone triturated roots are applied on forehead for the treatment of migraine.

The pulverized mixtures of plant parts of Corchorus depressus and Prosopis cineraria

subsequent to sugar addition are consumed as a remedy for bodily pains, uterine protrusion,

urinary inflammations and protection of pregnancy; while, fruits taken along milk is used to

treat leucorrhea (Upadhyay et al., 2010).

The plant contains alkaloids, steroids, flavonoids, tannins, glycosides, saponins and fixed oils

(Kataria et al., 2013b). Detailed phytochemical instigations revealed presence of

depressosides, cordepressin, cordeprssic acid, cordepressinic acid, quercetin, kaemferol,

apigenin, luteolin, depressonol A, depressonol B and sitosterol glucoside in the plant (Ahmad

al et., 2000; Khan et al., 2006).

Ethanol extract of Corchorus depressus exhibited antioxidant potential vide DPPH, ABTS

radicals and hydrogen peroxide tests. The plant extract orally administered to rats resulted in

provision of protection against carbon tetra chloride exerted liver toxicity (Pareek et al.,

2013). Methanol, ethanol, acetone, ether and n-hexane extracts obtained from leaves and root

56

of the plant were studied for mineral profile, antioxidant and antimicrobial activities (Bokhari

et al., 2014; Jabeen et al., 2014). Plant extracts were reported to exhibit antioxidant, anti-

malarial, antibacterial, antifungal (Simonsen et al., 2001); diuretic (Akhtar, 1985); analgesic,

antipyretic (Vohora et al., 1981; Ikram et al., 1987) and aphrodisiac activities (Kataria et al.,

2013a).

Literature review on Corchorus depressus reflected that plant material has not been

investigated to reveal its possible cardiovascular, respiratory and gastrointestinal activities.

Keeping in mind the folkloric uses of plant in various ailments in traditional systems of

medicine, the crude extract of the plant was screened on in vitro and in vivo models to assess

possible pharmacological along with exploration of plausible mechanism(s) of the exhibited

activities.


Collection of Corchorus depressus (whole plants) was accomplished in October 2015 from



Zakariya University, Multan, on submission of a representative specimen (Fl.P.472-4) in


dried.


powder and 1.2 kg pulverized material was soaked at ambient room temperature (25-30°C)

for 3 days in aqueous-ethanol (30:70 v/v) in a wide mouthed dull glass container with casual

stirring; followed after by straining and filtration through respective muslin cloth and filter

paper (Whatman grade 1). The above-mentioned process was reproduced twice on using new

menstruum; three aliquots of filtrates were mixed and evaporated at 37°C in a rotary

evaporator (BUCHI, Switzerland) to get a brownish green semisolid mass; i.e. crude extract

of Corchorus depressus (Cr.Cd) and estimated yield was 7.60%. About 40 g of Cr.Cd was

added to 80 ml of H2O taken in a separating funnel; shaken vigorously and extracted thrice in

succession by 80 ml aliquots of ethyl acetate for solvent-solvent fractionation. All the

collected aliquots of ethyl acetate were mixed and solvent was removed by means of rotary

evaporator. The aqueous layer remained in separating funnel was dried through freeze drying.

The respective yield for ethyl acetate (Ea.Cd) and aqueous (Aq.Cd) fractions was 12.20 and

86.57 % of the crude extract. The Cr.Cd was administered orally to animals on dissolving in

57

normal saline and intravenously to animals after solubilizing in 5% DMSO-saline; Cr.Cd and

Aq.Cd were dissolved in distilled water for in vitro experiments; while Ea.Cd was used for in

vitro experiments on dissolving in 10% DMSO-saline to make stock solution (300 mg per

ml), from which further dilutions were made in distilled water. The solvents as used to

administer test material during in vitro experimentation were without any notable effect on

isolated tissue preparations.

4.3. Results

4.3.1. Preliminary phytochemical analysis

The alkaloids, flavonoids, saponins, tannins, steroids and cardiac glycosides were detected to

be among the plant constituents. The estimation of total phenolic and flavonoids contents per

gram of Cr.Cd were demonstrated to be 113.76 ± 6.05 and 47.02 ± 4.02 mg, respectively,

based upon respective gallic acid and quercetin equivalents.


4.3.2.1. Influence upon smooth muscle preparations of rabbit isolated jejunum

The rhythmic periodic contractions, potassium (80 mM), potassium (25 mM) and carbachol

(1 μM) mediated consistent contractile responses in smooth muscle preparations of rabbit

isolated jejunum were blocked by crude extract of Corchorus depressus (Cr.Cd) at respective

tissue organ bath concentrations (T.O.B.C.) of 5.0, 10.0, 10.0 and 1.0 mg per ml (Fig. 4.2-

4.5), at EC50 (median response concentration) values of 0.42 mg per ml [CI(95%) of 0.37-

0.48 mg per ml; n=5], 3.24 mg per ml [CI (95%) of 2.01-5.22 mg per ml; n=5], 3.32 mg per

ml [CI(95%) of 2.23-4.95 mg per ml; n=5] and 0.34 mg per ml [CI(95%) of 0.29-0.40 mg per

ml; n=5], respectively (Fig. 4.6 & 4.7). The perusal of median response concentrations

indicated potent activity of Cr.Cd upon carbachol (1 μM) mediated contractions as compared

to rhythmic periodic contractile movements, potassium (80 mM) and potassium (25 mM)

mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum (Fig.

4.7). Moreover, standard drug dicyclomine, having dual calcium channel and muscarinic

receptor blocking activities, exhibited similar pattern of relaxation upon rhythmic periodic

contractions, potassium (80 mM), potassium (25 mM) and carbachol (1 μM) mediated

contractions in smooth muscle preparations of rabbit isolated jejunum at median response

concentrations 1.46 µM [CI (95%) of 1.22-1.73 µM; n=5], 2.15 µM [CI (95%) of 1.80-2.56

µM; n=5], 2.21 µM [CI (95%) of 1.86-2.62 µM; n=5] and 0.21 µM [CI (95%) of 0.17-0.26

µM; n=5], respectively (Fig. 4.6 & 4.8).

58

Furthermore, smooth muscle preparations of rabbit isolated jejunum subsequent to

pretreatment by Cr.Cd resulted in shifting of response versus concentration curves (CRCs)

for calcium rightward in non-parallel manner and decreasing maximally achieved responses

in significant (p < 0.05) manner (Fig. 4.9). Similarly, dicyclomine caused shifting of CRCs

for calcium in non-parallel fashion with mitigation of maximally achievable response in

significant (p < 0.05) manner in smooth muscle preparations of rabbits isolated jejunum (Fig.

4.10).

Similarly, ethyl acetate fraction of Cr.Cd (Ea.Cd) caused relaxation of rhythmic periodic

contractions, potassium (80 mM) and carbachol (1 μM) mediated contractile responses in

smooth muscle preparations of rabbits isolated jejunum (Fig. 4.11, 4.12 & 4.13) at median

response concentrations of 0.14 mg per ml [CI(95%) of 0.12-0.17 mg per ml; n=5], 0.05 mg

per ml [CI(95%) of 0.04-0.06 mg per ml; n=5] and 0.33 mg per ml [CI(95%) of 0.29-0.38 mg

per ml; n=5], respectively (Fig. 4.14). The Aq.Cd (aqueous fraction of Cr.Cd) augmented the

pattern of rhythmic periodic contractility of in smooth muscle preparations of rabbit isolated

jejunum at T.O.B.C. of 0.01-3.0 mg per ml, which was abolished completely following

pretreatment of tissues by atropine (1 µM) (Fig. 4.15, 4.16 & 4.17). The Aq.Cd was unable to

relax potassium (80 mM) mediated contractile response but demonstrated relaxation of

carbachol (1 µM) mediated contractile response at median response concentration of 1.14 mg

per ml [CI(95%) of 0.48-2.68 mg per ml; n=5] (Fig.4.18, 4.19 & 4.20).

Fig. 4.2: Influence of cumulatively added Cr.Cd upon rhythmic periodic contractile movements in smooth muscle preparation of rabbit isolated jejunum.

Fig. 4.3: The Cr.Cd influence upon cumulative addition to potassium (80 mM) mediated contractility in smooth muscle preparation of rabbit isolated jejunum.

59

Fig. 4.4: The Cr.Cd influence upon cumulative addition to potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum.

Fig. 4.5: The Cr.Cd influence upon cumulative addition to carbachol (1 µM) mediated contractile activity in isolated preparation of rabbit jejunum.

Fig. 4.6: Influence of Cr.Cd and dicylomine upon rhythmic periodic contractions of rabbit isolated jejunum preparations. Variables are mean ± S.E.M., n=5.

60

Fig. 4.7: Influence of Cr.Cd upon carbachol (1 µM), potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M., n=5.

Fig. 4.8: Dicyclomine influence upon carbachol (1 µM), potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variable are mean ± S.E.M. (n=5).

61

Fig. 4.9: The Cr.Cd influence upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables were mean ± S.E.M. (n=5). * indicates significant (p < 0.05) difference as compared to control maximum.

Fig. 4.10: Dicyclomine influence upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5). * indicates significant (p < 0.05) difference as compared to control maximum.

62

Fig. 4.11: The Ea.Cd influence upon rhythmic periodic contractions of rabbit isolated jejunum preparation.

Fig. 4.12: The Ea.Cd influence upon potassium (80 mM) mediated contractile response of rabbit isolated jejunum preparation.

Fig. 4.13: The Ea.Cd influence upon carbachol (1 µM) mediated contractile response of smooth muscle preparation of rabbit isolated jejunum.

Fig. 4.14: Influence of Ea.At upon spontaneous, carbachol (1 μM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variables are S.E.M. (n=5).

63

Fig. 4.15: Trace showing effects of acetylcholine (Ach, 1 µM) and Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum preparation.

Fig. 4.16: Trace showing effects of acetylcholine (Ach, 1 µM) and Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum preparation in presence atropine (1 μM).

Fig. 4.17: Effects Aq.Cd upon rhythmic periodic contractions in smooth muscle preparations of rabbit isolated jejunum, without and with atropine (1 µM) pretreatment. Variable were mean ± S.E.M., n=5.

64

Fig. 4.18: Record showing influence of Aq.Cd upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum.

Fig. 4.19: Record showing influence of Aq.Cd upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated jejunum.

Fig. 4.20: Influence of Aq.Cd upon carbachol (1 μM) and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated jejunum. Variables were mean ± S.E.M., n=5.


Application of Cr.Cd and dicyclomine to smooth muscles preparations of rabbit trachea did

not demonstrate any contractile response on resting baseline. However, Cr.Cd demonstrated

inhibition of carbachol (1 µM) and potassium (80 mM) mediated contractile responses in

isolated tracheal preparations of rabbit at median response concentrations of 0.23 mg per ml

[CI(95%) of 0.19-0.27 mg per ml; n=5] and 1.98 mg per ml [CI(95%) of 1.62-2.41 mg per

65

ml; n=5], respectively (Fig. 4.21, 4.22 & 4.23). On comparing the median response

concentrations; Cr.Cd was found to be more potent in demonstration of relaxation upon

carbachol (1 µM) than potassium (80 mM) mediated contractile response (Fig. 4.23). In a

similar manner, dicyclomine was found to be more potent in relaxing carbachol (1 µM)

mediated contractile response in comparison with potassium (80 mM) mediated contractile

response in isolated tracheal preparations of rabbit at respective median response

concentrations of 0.27 µM [CI(95%) of 0.21-0.34 µM; n=5] and 1.68 µM [CI(95%) of 1.51-

1.87 µM; n=5] (Fig. 4.24).

Furthermore, smooth muscle preparations of rabbit isolated trachea subsequent to

pretreatment by Cr.Cd at T.O.B.C. of 0.03 mg per ml resulted in shifting of CRCs for

carbachol rightward in parallel manner without decreasing maximally achieved response but

further increase in T.O.B.C. to 0.1 mg per ml caused shifting of CRCs for carbachol

rightward in non-parallel fashion with significant (p < 0.05) decrease in maximally achieved

response (Fig. 4.25). Similarly, dicyclomine at T.O.B.C. of 0.3 μM caused shifting of CRCs

for carbachol in parallel fashion without mitigation of maximally achievable response,

however, non-parallel shifting toward right of CRCs for carbachol was attained with

significant (p < 0.05) decrease in maximally achieved response on increasing T.O.B.C. to 1.0

µM in smooth muscle preparations of rabbit isolated trachea (Fig. 4.26).

Moreover, Ea.Cd exerted more potent relaxant effect on potassium (80 mM) mediated

contractile response of isolated preparations of rabbit trachea with median response

concentration of 0.09 mg per ml [CI(95%) of 0.08-0.10 mg per ml; n=5] in comparison with

carbachol (1 µM) mediated contractile response with median response concentration of 0.36

mg per ml [CI(95%) of 0.29-0.45 mg per ml; n=5] (Fig. 4.27, 4.28 & 4.29). Whereas, Aq.Cd

was found to demonstrate incomplete relaxant response upon potassium (80 mM) mediated

contractions but relaxed carbachol (1 µM) exerted contractile response at median response

concentration of 0.80 mg per ml [CI(95%) of 0.66-0.97 mg per ml; n=5] (Fig. 4.30, 4.31 &

4.32).

66

Fig. 4.21: The Cr.Cd demonstrating relaxant effect upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated trachea.

Fig. 4.22: The Cr.Cd demonstrating relaxant effect upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated trachea.

Fig. 4.23: The Cr.Cd demonstrating relaxant effect carbachol (1 µM) and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Variable are mean ± S.E.M. (n=5).

67

Fig. 4.24: Effects of dicyclomine on potassium (80 mM) and carbachol (1 µM) mediated contractile responses in smooth muscle preparations of rabbit isolated trachea. Variables were mean ± S.E.M., n=5.

Fig. 4.25: Effect of Cr.Cd upon CRCs for carbachol in smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5), *indicates significant (p < 0.05) difference as compared to control maximum.

68

Fig. 4.26: Effect of dicyclomine upon CRCs for carbachol in smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5), *indicates significant (p < 0.05) difference as compared to control maximum.

Fig. 4.27: The Ea.Cd demonstrating relaxant effect upon potassium (80 mM) mediated contractility of smooth muscle preparation of rabbit isolated trachea.

Fig. 4.28: The Ea.Cd demonstrating relaxant effect upon carbachol (1 µM) mediated contractility of smooth muscle preparation of rabbit isolated trachea.

69

Fig. 4.29: The Ea.Cd demonstrating relaxant effect upon potassium (80 mM) and carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5).

Fig. 4.30: The influence of Aq.Cd upon potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea.

Fig. 4.31: The influence of Aq.Cd upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea.

70

Fig. 4.32: The influence of Aq.Cd upon potassium (80 mM) and carbachol (1 μM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5).

4.3.2.3. Influence on rabbit preparation of isolated denuded aorta

Cumulative application of Cr.Cd to rabbit isolated denuded aorta preparations at T.O.B.C.

range of 0.01-10.0 mg per ml demonstrated contractile effect at T.O.B.C. range of 0.01-5.0

mg per ml, followed by partial relaxation at 10 mg per ml (Fig. 4.33). The above-mentioned

Cr.Cd exerted contractile response of rabbit isolated denuded aorta was found reversible

because repeated washing with bathing fluid restored the base line tension. Moreover,

pretreatment of rabbit isolated denuded aorta with doxazosin (1 µM) resulted in partial

suppression of the Cr.Cd exerted contractile activity; whereas, pretreatment with verapamil (1

µM) caused complete blockade of Cr.Cd mediated contractile effect (Fig. 4.34).

Moreover, Cr.Cd demonstrated relaxation of potassium (80 mM) mediated contractile

response upon rabbit isolated denuded aortic preparations at T.O.B.C. of 10 mg per ml with

median response concentration of 4.28 mg per ml [CI(95%) of 4.18-4.37 mg per ml; n=5];

whereas, phenylephrine (1 μM) mediated contractions were relaxed partially (Fig. 4.35, 4.36

& 4.37). The standard calcium channel blocker (verapamil) failed to exhibit contractile

response upon resting baseline tension (data not shown); exerted relaxant effect upon

potassium (80 mM) and phenylephrine (1 μM) mediated contractile responses at median

71

response concentrations of 0.28 µM [CI(95%) of 0.24–0.33 µM; n=5] and 1.01 µM [CI(95%)

of 0.88–1.14 µM; n=5], respectively. The comparison of median response concentrations

reflected that verapamil exerted relaxant effect was more potent upon potassium (80 mM)

than phenylephrine (1 μM) mediated contractile response upon rabbit isolated denuded aortic

preparations (Fig. 4.38).

Fig. 4.33: Effects of phenylephrine (PE) and Cr.Cd upon resting base line tension in rabbit isolated denuded aortic preparation.

Fig. 4.34: Influence of Cr.Cd upon baseline tension of rabbit isolated aorta preparations, without any pretreatment and with prazosin or verapamil pretreatments. Variables were mean ± S.E.M., n=5.

72

Fig. 4.35: Effects of Cr.Cd upon potassium (80 mM) mediated contractile response in rabbit isolated denuded aortic preparation.

Fig. 4.36: Effects of Cr.Cd upon phenylephrine (1 µM) mediated contractile response in rabbit isolated denuded aortic preparation.

Fig. 4.37: Effects of Cr.Cd upon potassium (80 mM) and phenylephrine (1 μM) mediated contractile responses in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

73

Co

ntr

ol

(%)

Fig. 4.38: Influence of verapamil upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in tissue preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).


The isolated paired atrial preparations of rabbit demonstrated negative inotropic and negative

chronotropic activities when exposed to Cr.Cd at T.O.B.C. ranging 0.01-3.0 mg per ml;

however, pre-treatment with atropine (1 μM) suppressed the Cr.Cd mediated negative

inotropic and chronotropic activities (Fig. 4.39, 4.40 & 4.41). Similarly, cumulative addition

of acetylcholine upon isolated paired atrial preparations of rabbit demonstrated negative

inotropic and negative chronotropic activities; pre-treatment with atropine (1 μM) suppressed

the acetylcholine mediated negative inotropic and chronotropic activities (Fig. 4.42 & 4.43).

Fig. 4.39: Record of Cr.Cd influence upon force of spontaneous contractions of rabbit isolated paired atrial preparation.

74

Fig. 4.40: Influence of Cr.Cd upon force of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. Variables were mean ± S.E.M., n=5.

Fig. 4.41: Influence of Cr.Cd upon rate of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. Variables were mean ± S.E.M., n=5.

75

Fig. 4.42: Influence of acetylcholine upon force of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. Variables were mean ± S.E.M., n=5.

Fig. 4.43: Influence of acetylcholine upon rate of rhythmic periodic contractions in isolated paired atrial preparations of rabbit, with and without atropine pretreatment. Variables were mean ± S.E.M. (n=5).


4.3.3.1. Influence of Cr.Cd upon charcoal meal transit distance in mice


± 0.85 cm. The Cr.Cd administration to Treated 1 and 2 groups of animals (n=5) at respective

oral doses of 50 and 100 mg per kg caused significant (p < 0.05) enhancement of intestinal

transit distances of charcoal as compared to control and were assessed to be 36.66 ± 1.21 and

38.78 ± 1.04 cm, respectively; however, Cr.Cd administered orally at doses of 500 and 700

76

mg per kg to Treated 3 and 4 groups of animals (n=5) caused significantly decreased (p <

0.05) intestinal charcoal transit distances as compared to control and were assessed to be

24.96 ± 0.74 and 24.36 ± 0.91 cm, respectively. Moreover, loperamide treatment (10 mg per

kg; orally) to standard group of mice (n=5), the intestinal transit distance of charcoal was

found to be 7.40 ± 0.29 cm; which was found to be decreased significantly (p < 0.05) in

comparison with control. Furthermore, oral carbachol administration (1 mg per kg) to another

standard group of mice (n=5) caused significantly increased (p < 0.05) charcoal intestinal

transit distance in comparison with control and was measured to be 44.82 ± 1.05 cm (Table

4.1).

Groups

Treatment

(given orally)

Charcoal movement

Mean ± S.E.M. (cm)


10 ml per kg + 0.2 ml 31.20 ± 0.85

Treated 1 Cr.Cd + Charcoal meal

50 mg per kg + 0.2 ml 36.66 ± 1.21*


100 mg per kg + 0.2 ml 38.78 ± 1.04*


500 mg per kg + 0.2 ml 24.96 ± 0.74*


700 mg per kg + 0.2 ml 24.36 ± 0.91*


10 mg per kg + 0.2 ml 7.40 ± 0.29*


1 mg per kg + 0.2 ml 44.82 ± 1.05*

Table 4.1: Influence of Cr.Cd upon charcoal meal intestinal transit distance in mice. Values are mean ± S.E.M. (n=5); * denotes statistically significant difference (p < 0.05 vs. control group) as analyzed by ANOVA (one way) along with Dunnett test.

77


The mean number of wet feces in saline control group (n=6) of mice (administered saline and

olive oil orally at rate of 10 ml per kg) were found to be 0.33 ± 0.21; whereas, wet feces in

treated control group (n=6) of animal (administered saline and castor oil orally at rate of 10

ml per kg) were counted to be 8.20 ± 0.31. The Treatment 1 group (n=6) of mice

(administered Cr.Cd and castor oil orally at respective doses of 300 mg per kg and 10 ml per

kg) resulted mean wet feces count to be 7.67 ± 0.33, which was not significantly different

from the saline control group. However, Treatment 2 group (n=6) of mice (administered

Cr.Cd and castor oil orally at respective rate of 500 mg per kg and 10 ml per kg) significantly

(p < 0.05) decreased mean wet feces count to 6.83 ± 0.31. Similarly, Treatment 3 group (n=6)

of mice (administered Cr.Cd and castor oil orally at respective rate of 700 mg per kg and 10

ml per kg) significantly (p < 0.05) decreased mean wet feces count (6.00 ± 0.37). The

standard control group (n = 6) of mice (administered loperamide and castor oil orally at

respective rate of 10 mg per kg and 10 ml per kg) significantly (p < 0.05) decreased mean wet

feces count was evident to be 0.50 ± 0.22 (Table 4.2).

4.3.3.3. Laxative activity among mice

The total feces and wet feces produced by mice as result of various treatments were counted.

The saline control group of mice to whom normal saline was administered (10 ml per kg); the

total and wet feces counts for 6 hrs were found to be 4.83 ± 0.31 and 0.5 ± 0.22, respectively.

The treated 1 group of mice (n=6) were orally treated with Cr.Cd (50 mg per kg); the total

and wet feces count for 6 hrs was significantly (p < 0.05) increased to 6.83 ± 0.31 and 2.00 ±

0.25, respectively, as compared to that of saline control group. Similarly, Treated 2 group of

mice received orally Cr.Cd (100 mg per kg); the total and wet feces count was significantly

(p < 0.05) increased to 8.17 ± 0.40 and 3.33 ± 0.21, respectively, as compared to that of

saline control group. The standard control group of mice to whom carbachol was

administered orally (1 mg per kg); the total and wet feces count was significantly (p < 0.05)

increased to 11.17 ± 0.40 and 7.67 ± 0.33, respectively, as compared to that of saline control

group (Table 4.3).

78

Groups Treatment

(given orally)

Wet feces count

Mean ± S.E.M.

Protection (%age)

Saline Control

Saline + Olive oil

(10 ml per kg + 10 ml per kg) 0.33 ± 0.21 -


(10 ml per kg + 10 ml per kg) 8.20 ± 0.31 -

Treatment 1 Cr.Cd + Castor oil



(500 mg per kg + 10 ml per kg) 6.83 ± 0.31* 16.71


(700 mg per kg + 10 ml per kg) 6.00 ± 0.37* 26.83

Standard Treatment


(10 mg per kg + 10 ml per kg) 0.50 ± 0.22* 93.90

Table 4.2: Influences of Cr.Cd upon castor oil caused diarrhea in mice. Values are mean ± S.E.M. (n=6); ns denotes insignificant difference (p ≥ 0.05) and * denotes statistically significant difference (p < 0.05) vs. control group as analyzed by ANOVA (one way) along with Dunnett test.

Groups Treatment Wet feces count

Mean ± S.E.M.

Total feces count

Mean ± S.E.M.

Saline Control Normal saline (10 ml per kg) 0.50 ± 0.22 4.83 ± 0.31

Treated 1 Cr.Cd (50 mg per kg) 2.00 ± 0.25* 6.83 ± 0.31*

Treated 2 Cr.Cd (100 mg per kg) 3.33 ± 0.21* 8.17 ± 0.40*

Standard Control Carbachol (1 mg per kg) 7.67 ± 0.33* 11.17 ± 0.40*

Table 4.3: The Cr.Cd exerted laxative effect in normal mice as compared to carbachol. Values are mean ± S.E.M. (n=6). * denotes statistically significant difference (p < 0.05) vs. control group as analyzed by ANOVA (one way) along with Dunnett test.

79

4.3.3.4. Influence upon blood pressure of rats

Vehicle (5% DMSO in normal saline) administered intravenously to normotensive

anesthetized rats caused 1.06 ± 0.35, 1.46 ± 0.43, 1.58 ± 0.55 % decrease in systolic blood

pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP),

respectively. The decrease in SBP, DBP and MBP by vehicle was not significant from

respective basal values. The Cr.Cd administered intravenously to normotensive anesthetized

rats at a dose of 3 mg per kg resulted in insignificant (p > 0.05) decrease in SBP (3.72 ± 0.72

%) as well as MBP (7.70 ± 1.74 %) but demonstrated significant (p < 0.05) decrease in DBP

(11.20 ± 2.24 %) in comparison with respective vehicle treated values. Moreover, Cr.Cd

administration to normotensive anesthetized rats at respective intravenous dose of 10 mg per

kg resulted in 12.16 ± 1.29, 17.22 ± 2.25 and 15.20 ± 1.85 % decrease of SBP, DBP and

MBP, respectively; decrease was significant (p < 0.05) in comparison with respective vehicle

treated values. Furthermore, intravenous administration of Cr.Cd (30 mg per kg) also

demonstrated significantly (p < 0.05) decreased SBP, DBP and MBP; and decrease in values

was recorded 23.20 ± 1.39, 25.22 ± 1.61 and 25.80 ± 1.68 %, respectively. Similarly,

intravenous administration of acetylcholine (1 µg per kg) to normotensive anaesthetized rats

(n=5) demonstrated 31.76 ± 2.49, 48.10 ± 2.33 and 40.10 ± 1.17 % decrease in SBP, DBP

and MBP, respectively; and decrease was significant (p < 0.05) in comparison with respective

values achieved following vehicle treatment. Whereas, intravenous administration of

norepinephrine (1 µg per kg) to normotensive anaesthetized rats (n=5) demonstrated

increased SBP, DBP and MBP by 40.92 ± 1.65, 50.26 ± 3.98 and 46.16 ± 4.89 %,

respectively; and percent increase was significant (p < 0.05) in comparison with respective

values achieved following vehicle treatment (Fig. 4.44; Table 4.4). However, pretreatment of

animals with atropine (1 mg per kg, i.v.) resulted in blockade of acetylcholine as well as

Cr.Cd exerted hypotensive effects in normotensive anesthetized rats (Fig. 4.44, 4.45 & 4.46).

The vehicle and Cr.Cd did not alter heart rate of anesthetized rats in significant manner (data

not shown).

80

Fig. 4.44: Record showing effects of intravenously administrated solvent, acetylcholine (Ach), norepinephrine (NE) and different doses of Cr.Cd upon blood pressure of normotensive anesthetized rat.

Fig. 4.45: Record showing effects of intravenously administered solvent, acetylcholine (Ach), norepinephrine (NE) and Cr.Cd upon blood pressure of normotensive anesthetized rat subsequent to atropine (1 mg per kg) pretreatment.

Treatments % Change

SBP DBP MBP

Solvent (5% DMSO) -1.06 ± 0.35 -1.46 ± 0.43 -1.58 ± 0.55

Cr.Cd (3 mg per kg) -3.72 ± 0.72 -11.20 ± 2.24* -7.70 ± 1.74

Cr.Cd (10 mg per kg) -12.16 ± 1.29* -17.22 ± 2.25* -15.20 ± 1.85*

Cr.Cd (30 mg per kg) -23.20 ± 1.39* -25.22 ± 1.61* -25.80 ± 1.68*

Acetylcholine (1µg per kg) -31.76± 2.49* -48.10 ± 2.33* -40.10 ± 1.17*

Norepinephrine (1µg per kg) +40.92 ± 1.65* +50.26 ± 3.98* +46.16 ± 4.89*

Table 4.4: Effects of intravenously administered solvent (5 % DMSO in saline), Cr.Cd, norepinephrine and acetylcholine upon SBP, DBP and MBP of normotensive anesthetized rats. Variables were mean ± S.E.M., n=5. (-) indicates decrease and (+) indicates increase from baseline values.* denotes statistically significant difference (p < 0.05) vs. vehicle treatment values as analyzed by ANOVA (two way) along with Dunnett test.

81

Fig. 4.46: Effects of intravenous administration of solvent (5% DMSO in saline), acetylcholine and Cr.Cd upon MBP in anesthetized rats, with and without atropine (1 mg per kg) pretreatment. Variables were mean ± S.E.M. (n=5).


The comparison among different groups of rats on different treatments was made in terms of

voided urinary volume per 100 g body weight in 6 hrs and amount of excreted electrolytes

(Na+, K+ & Cl-) in urine on µmol per 100 g body weight in 6 hrs. The urine volume in group

of rats treated by saline (control) was measured to be 1.02 ± 0.06 ml per 100 g in 6 hrs; Na+,

K+ and Cl- excreted were 121.00 ± 4.05, 7.78 ± 0.23 and 127.50 ± 2.20 µmol per 100 g in 6

hrs, respectively. The group of rats treated by Cr.Cd (100 mg per kg) demonstrated

significant (p < 0.05) increase in urinary volume to 1.46 ± 0.62 ml per 100 g in 6 hrs as well

as significant (p < 0.05) increase in respective Na+, K+ and Cl- contents to 153.62 ± 2.98, 9.57

± 0.39 and 158.98 ± 4.59 µmol per 100 g in 6 hrs in comparison with saline control group.

The group of rats treated by Cr.Cd (300 mg per kg) demonstrated significant (p < 0.05)

increase in urinary volume to 1.96 ± 0.87 ml per 100 g in 6 hrs as well as significant increase

in respective Na+, K+ and Cl- contents to 184.83 ± 3.85, 11.35 ± 0.32 and 197.73 ± 2.42 µmol

per 100 g in 6 hrs, respectively, in comparison with saline control group of rats. The group of

82

rats treated by Cr.Cd (500 mg per kg) demonstrated significant (p < 0.05) increase in voided

urinary volume to 2.33 ± 0.10 ml per 100 g in 6 hrs and significant (p < 0.05) increase in

respective Na+, K+ and Cl- excretion to 216.45 ± 4.92, 12.50 ± 0.77 and 236.53 ± 5.11 µmol

per 100 g in 6 hrs in comparison with saline control group of rats. The group of rats treated

by furosemide (20 mg per kg) demonstrated significant (p < 0.05) increase in urinary volume

to 4.10 ± 0.11 ml per 100 g in 6 hrs and significant (p < 0.05) increase in respective Na+, K+

and Cl- to 397.17 ± 5.64, 31.42 ± 0.58 and 516.50 ± 9.39 µmol per 100 g in 6 hrs in


Na+/K+ ratio and ion quotient were also calculated (Table 4.5). The diuretic indices following

Cr.Cd administration at doses of 100, 300 and 500 mg per kg were calculated to be 1.43, 1.92

and 2.28, respectively, whereas diuretic index of the standard drug furosemide was found

4.02. Oral administration of furosemide resulted in significantly (p < 0.05) decreased urinary

Na+/K+ ratio to 12.10 ± 0.36 in comparison with saline treated group (control). However,

Cr.Cd administration at doses of 100, 300 and 500 mg per kg demonstrated insignificant (p >

0.05) increased Na+/K+ to 16.22 ± 0.90, 16.38 ± 0.71 and 17.63 ± 1.09, respectively (Table

4.5).

83

Treatment group (dose)

Volume of urine (ml/100

g/6 hrs)

Diuretic index

Na+ K+ Cl-

Na+/K+ Ion quotient

(µmol/100 g/6 hrs)

Saline

(10 ml per kg) 1.02 ± 0.06 - 121.00 ± 4.05 7.78 ± 0.23 127.50 ± 2.20 15.63 ± 0.80 0.99 ± 0.03

Cr.Cd

(100 mg per kg) 1.46 ± 0.62* 1.43 153.62 ± 2.98* 9.57 ± 0.39ns 158.98 ± 4.59* 16.22 ± 0.90 ns 0.97 ± 0.04

Cr.Cd

(300 mg per kg) 1.96 ± 0.87* 1.92 184.83 ±3.85* 11.35 ± 0.32* 197.73 ± 2.42* 16.38 ± 0.71 ns 1.01 ± 0.02

Cr.Cd

(500 mg per kg) 2.33 ± 0.10* 2.28 216.45 ± 4.92* 12.50 ± 0.77* 236.53 ± 5.11* 17.63 ± 1.09 ns 1.04 ± 0.04

Furosemide

(20 mg per kg) 4.10 ± 0.11* 4.02 379.17 ± 5.64* 31.42 ± 0.58 * 516.50 ± 9.39* 12.10 ± 0.36* 1.25 ± 0.03

Table 4.5: Influence of saline, Cr.Cd and furosemide upon urinary volume and electrolytes contents in rats. Diuretic index was calculated as volume of urine in treatment group/volume of urine in control (saline treated group); whereas ion quotient was calculated as Cl-/ (Na+ + K+). The values were mean ± S.E.M. (n=6). nsp ≥ 0.05, * p < 0.05 in comparison with saline treated group.

84

4.4. Discussion

Corchorus depressus has been used to manage constipation, diarrhea and dysentery by native

natural healers throughout Indian subcontinent (Singh and Panday, 1983; Tareen et al., 2010),

therefore, Cr.Cd and its fractions were subjected to pharmacological screening on multiple in

vitro and in vivo models to authenticate scientifically its ethno-pharmacological repute in

indigenous herbal literature. The isolated tissue preparations of rabbit jejunum have been

regarded convenient to explore spasmolytic and spasmogenic activities of herbal extracts

(Akhlaq et al., 2016). The crude extract of Corchorus depressus (Cr.Cd) and its fractions, on

application to the rhythmic periodic contractility of rabbit isolated jejunum preparations,

exhibited suppression of the amplitude of rhythmic periodic contractions. The rhythmic

periodic contractions in rabbit isolated jejunum preparations are originated on repeated

depolarization and repolarization (Huizinga et al., 2000); leading to increased cytoplasmic

calcium concentration on opening of voltage dependent calcium channels or calcium released

from intracellular storage sites. The rhythmic periodic contractions in smooth muscle

preparations of rabbit isolated jejunum are reported to be suppressed on application of

calcium channel blockers, potassium channel openers and anti-muscarinic agents. Hence, the

status of Cr.Cd in this regard was decided upon testing on potassium (80 mM), potassium (25

mM) and carbachol (1 μM) exerted contractile responses in isolated tissue preparations of

rabbit jejunum (Gilani et al., 2006; Gilani et al., 2008; Jabeen et al., 2009). The smooth

muscle preparations exhibit contractile response on exposure to potassium (20-80 mM);

chiefly because of opening of voltage dependent calcium channels (Ratz et al., 2005). The

agents capable to relax preferentially potassium (25 mM) mediated contractile response

without any influence upon potassium (80mM) mediated contractile response are known to be

viewed as potassium channel opener (Gilani et al., 2006). Calcium channels blockers are

equally competent to exert relaxant effect upon potassium (80 mM) and potassium (25 mM)

mediated contractile responses (Okumura, et al., 1997). The Cr.Cd caused complete

relaxation of potassium (80 mM) and potassium (25 mM) mediated contractions at

comparable efficacy in isolated tissue preparations of rabbit jejunum; hence, observed

relaxing potential of Cr.Cd may be outcome of calcium channel blocking activity.

Furthermore, typical calcium channel blockers (diltiazem and verapamil) are reported to be

more potent against potassium (80 mM) mediated contractions than spontaneous contractions

in rabbit jejunum preparations (Chaudhary et al., 2012; Janbaz et al., 2013b). But, contrary to

typical calcium channel blockers, the Cr.Cd was found to be more potent against spontaneous

85

contractions than potassium mediated contractions; hence involvement of some additional

mechanism was also postulated. When tested against carbachol (1 µM) demonstrated

contractile response in isolated jejunum preparations of rabbit, the Cr.Cd relaxed carbachol (1

µM) mediated contractions most potently reflecting presence of some muscarinic antagonist

constituents among on the part of Cr.Cd. Therefore, Cr.Cd exhibited anti-muscarinic as well

as calcium channel blocking activities in a comparable manner to dicyclomine, a dual blocker

of muscarinic cholinergic receptors and calcium channels (Janbaz et al., 2015). Muscarinic

(M3) receptors are involved in regulation of gastrointestinal movement; stimulation of which

is linked to phospholipase C activation, hydrolysis of phosphatidylinositol to diacylglycerol

and inositol triphosphate within cytoplasm, in turn releasing calcium from stores of

sarcoplasmic reticulum to cause contraction of smooth muscles (Gerthoffer, 2005).

The confirmation of calcium channel blocking potential of Cr.Cd was attempted as tissue

preparation from rabbit isolated jejunum following pretreatment with Cr.Cd resulted in

rightward shifting of CRCs for calcium in non-parallel manner with suppression of

maximally achieved responses in a comparable fashion to dicylomine and verapamil. The

ethyl acetate fraction of Cr.Cd (Ea.Cd) exhibited more potent action on potassium (80 mM)

exerted contractile response as compared to carbachol (1 μM) exerted contractions in isolated

preparations of rabbit jejunum; indicating that calcium channel blocking activity was

partitioned into organic solvent. On the other hand aqueous fraction of Cr.Cd (Aq.Cd) was

found to exhibit anti-muscarinic activity as it relaxed carbachol mediated contractions

without affecting potassium mediated contractions in isolated jejunum preparations (Gilani et

al., 2008). Moreover, Aq.Cd was also observed to exhibit atropine sensitive mild contractile

response on periodic rhythmic contractions in isolated preparations of rabbit jejunum;

indication for simultaneous presence of muscarinic agonistic and muscarinic antagonistic

activities in the same plant extract. The presence of dual muscarinic agonistic and muscarinic

antagonistic activities have already been reported on the part of some crude extracts (Akhlaq

et al., 2016) as well as in pure agents (Hamilton and Rubinstein, 1968; Strycker and Long,

1969; Dretchen et al., 1971).

The Cr.Cd was subjected to screening of laxative and anti-diarrheal activities on in vivo

models of mice. The Cr.Cd following oral administration (50 and 100 mg per kg) to mice

demonstrated significant increase in charcoal meal transit distance through small intestine as

well as increased wet feces count and total feces count; thus, indicating presence of laxative

activity of the plant. Whereas, on increasing the oral dose to 500 and 700 mg per kg, the

86

Cr.Cd caused decrease in charcoal meal transit distance through small intestine of mice and

also demonstrated protection against castor oil caused diarrhea. The castor oil is known to

produce diarrhea due to its hydrolysis into ricinoleic acid by means of pancreatic lipases; and

it is the ricinoleic acid which produce diarrhea through modification of electrolytes and water

transport (Stewart et al., 1975). The Cr.Cd demonstrated anti-diarrheal activity can be

attributed to the observed anti-muscarinic and calcium channel blocking activities (Lee et al.,

1997).

The Cr.Cd was tested on carbachol (1 µM) and potassium (80 mM) exerted contractile

responses in tissue preparations of rabbit isolated trachea to explore possible presence of

bronchodilator activity. The Cr.Cd exhibited relaxing activities upon carbachol (1 µM) and

potassium (80 mM) mediated contractions; however, relaxant effect was observed to be more

potent upon carbachol (1 µM) than potassium (80 mM) mediated contractions reflecting

dominant presence of anti-muscarinic constituents in Cr.Cd. The muscarinic antagonistic

potential of Cr.Cd was confirmed further as it caused parallel rightward shifting of CRCs for

carbachol at lower concentrations of tissue organ baths (Arunlaksana and Schild, 1959).

However, further increase in tissue bath concentration of Cr.Cd demonstrated rightward

shifting of CRCs for carbachol in un-parallel pattern with mitigation of maximally achievable

response in tissue preparations of rabbit isolated trachea in comparable fashion with

dicyclomine possessing dual activities, i.e., muscarinic receptor antagonist and calcium

channel blocker (Janbaz et al., 2015).

The Cr.Cd applied to tissue preparations of rabbit isolated aortic rings in cumulative manner

induced contractility at concentrations of tissue organ bath ranging from 0.01-5.0 mg per ml;

found to be partially antagonized following pre-treatment with doxazosin; reflecting that

observed vaso-contractile effect was likely to be mediated through α1 agonistic activity. The

Cr.Cd was also tested on potassium (80 mM) and phenylephrine (1 µM) mediated contractile

responses in tissue preparations of rabbit isolated aorta. The Cr.Cd demonstrated complete

relaxation of potassium (80 mM) mediated contractions; whereas phenylephrine (1 μM)

demonstrated contractions were only partially relaxed. Thus, presence of calcium channel

blocking potential of Cr.Cd was also confirmed in case of isolated preparations of rabbit

aorta. The Cr.Cd, when applied upon periodic rhythmic contractile pattern of tissue

preparations of rabbit isolated paired atria, exhibited negative inotropic and negative

chronotropic effects at tissue organ bath concentration range of 0.01-3.0 mg per ml. The

negative inotropic and negative chronotropic effects of Cr.Cd were found to be blocked

87

partially following pretreatment of the rabbit isolated paired atria preparations with atropine;

indication for presence of muscarinic agonistic activity to be held, at least partially,

responsible for observed negative inotropic and negative chronotropic activities. The Cr.Cd

administered intravenously at 3, 10 and 30 mg per kg to normotensive anesthetized rats

resulted in decreased mean arterial blood pressure; however, pretreatment of animals with

atropine blocked the hypotensive effect; hence, indication for involvement of muscarinic

agonistic activity for the hypotensive effect on the part of Cr.Cd.

The Cr.Cd was tested for possible diuretic effect in rats and found to possess significant

diuretic activity in terms of enhancement of voided urinary volume and electrolyte contents at

oral doses of 100, 300 and 500 mg per kg. These results are in confirmations of previously

reported diuretic activity on the part of plant extract (Akhtar, 1985). However, diuretic

activity of Cr.Cd was found less potent as compared to furosemide (standard).

4.5. Conclusion

It can be concluded that crude extract of Corchorus depressus demonstrated spasmolytic,

anti-diarrheal and bronchodilator activities possibly mediated through calcium channel

blocking and muscarinic receptor antagonistic activities. The extract was also found to

possess laxative activity possibly medicated through muscarinic agonistic activity. The

hypotensive effect may be mediated through calcium channel blocking and muscarinic

receptor agonistic activities. The study provided scientific authentications for folkloric repute

for utilization of Corchorus depressus in constipation, diarrhea, respiratory problems and

edema.

88

5. Gisekia pharnaceoides Linn.

Fig. 5.1: Gisekia pharnaceoides plant.

89


Gisekia pharnaceoides Linn. (synonym: Gisekia molluginoides Wight; family: Gisekiaceae)

has been known by multiple local names including Baluka and Baluka sag in India and

Pakistan; Ji su cao in China (Quattrocchi, 2012); Gant ka lar in Mayanmar (Myatt et al.,

2005); Manalikkirai in Sri Lanka; Melanguar, Takka cijla, Kona basi, Stroke’s henna and

Herdsmann’s henna in West African countries (Burkill, 1985). The plant grows in dry sandy

places and is distributed in Afghanistan, Africa, Arabia, India, Sri Lanka and Pakistan

(Kritikar and Basu, 1987; Jain et al., 2010).

It is a diffuse, succulent, fleshy smooth, annual herb. Branches of plant ( up to 80 cm long)

spreads along the ground or moving up in air; leaves are 2.0-3.8 cm long, 0.3-0.6 cm wide,

shape elliptic-lanceolate, subacute or obtuse, tapering base, underside showing raphides;

flowering occurs from August to September, in umbel cymes; seeds are shiny black minutely

punctuated 0.1 cm long (Gilbert, 1993; Jain et al., 2010).

The plant has been used as animal fodder, leaves are eaten raw as emergency food, cooked

with meat; used as condiment and generally considered strength restorer (Burkill, 1985; Raza

et al., 2014). In native systems of medicine, it has been considered as analgesic, anthelmintic,

antipyretic, antiseptic, bitter, digestible, diuretic and used for asthma, bronchitis, constipation,

diarrhea, edema, heart troubles, jaundice, leprosy, miscarriage, pain, rhinitis, urinary

problems, vitiligo and wound healing (Watt and Breyer-Barndwijk, 1962; Kritikar and Basu,

1987; Malik et al., 2015).

Phytochemical studies on the plant revealed alkaloids, glycosides, steroids, flavonoids,

saponins and tannins among the constituents (Stella et al., 2004; Myatt et al., 2005; Jain et

al., 2010). The triacontane, myristone, tetracosanol and dotriacontane were isolated from the

plant (Iyer et al., 1972); whereas, proanthocyanidins were detected in unripe seeds of the

plant (Bittrich and Amaral, 1991).

The herbal extract demonstrated anti-inflammatory (Gandhimathi et al., 2011), analgesic

(Gandhimathi, 2007), hypotensive (Myatt et al., 2005) and antibacterial as well as antifungal

activities (Robertson et al., 2013).

Literature review on Gisekia pharnaceoides reflected that plant material has not been

investigated to reveal its possible respiratory and gastrointestinal activities, whereas only one

study related to hypotensive activity of the plant is reported (Myatt et al., 2005). However,

90

mode of action of antihypertensive activity of the plant is yet to be explored. Based upon the

folkloric repute as remedy for management of multiple ailments pertaining to cardiovascular,

gut and respiratory diseases; the plant extracts were subjected to extensive scientific

evaluation to validate its existing folkloric status.


Collection of Gisekia pharnaceoides (young plants) was accomplished in August 2015 from



Zakariya University, Multan, on submission of a representative specimen (Fl-PK-42-13) in


dried.


powder and 1.5 kg pulverized material was soaked at room temperature (25-30°C) for 3 days

in hydro-alcoholic mixture (30:70 v/v) in a wide mouthed brown colored glass container with

daily periodic stirring; followed after by straining via fine cotton cloth and subsequently

filtration via Whatman grade 1 filter paper. The above-mentioned process was repeated on

marc twice on using new menstruum; the resulting three aliquots of filtrates were mixed and

evaporated at 37°C in a rotary evaporator (BUCHI, Switzerland) to get a brownish semisolid

mass; i.e. crude extract of Gisekia pharnaceoides (Cr.Gp) and estimated yield was 11.94 %.

About 75 g of Cr.Gp was mixed to 150 ml of distilled water taken in a separating funnel;

shaken vigorously and extracted thrice in succession by 150 ml aliquots of ethyl acetate for

solvent-solvent fractionation. All the collected aliquots of ethyl acetate were mixed and

solvent was removed by means of rotary evaporator. The aqueous layer left in separating

funnel was dried through freeze drying. The yield obtained was 4.60 and 94.95 % for

respective ethyl acetate (Ea.Gp) and aqueous (Aq.Gp) fractions, calculated on basis of crude

extract. The Cr.Gp was suspended in normal saline for oral administration to animals;

administered intravenously to animals while soluble in 5% DMSO-saline. Cr.Gp and Ea.Gp

were used for in vitro experiments on dissolving in 5% DMSO-distilled water to make stock

solutions of 300 mg per ml, from which further dilutions were made in distilled water. Aq.Gp

was soluble in distilled water. Previously, time and volume matched solvent control were run

for in vitro experiments; the solvents used in dissolving the extract and fractions were without

any notable effect on baseline or induced contractions of isolated tissue preparations.

91

5.3. Results

5.3.1. Preliminary phytochemicals analysis

The alkaloids, flavonoids, tannin, phenols, steroids, coumarins and saponins were detected to

be among the plant constituents, while test for anthraquinones was negative. The estimation

of total phenolic and flavonoids contents demonstrated it to be 71.04 ± 3.31 and 62.03 ± 2.74

mg, respectively, based upon respective gallic acid and quercetin equivalents per gram of the

crude extract.


5.3.2.1. Influence upon isolated preparations of rabbit jejunum

Application of crude extract of Gisekia pharnaceoides (Cr.Gp) on rhythmic periodic

contractions in smooth muscle preparations of rabbits isolated jejunum did not cause

significant change in rhythmic periodic contractions at tissue organ bath concentration

(T.O.B.C.) range of 0.01-0.1 mg per ml, but demonstrated gradual inhibition in amplitude of

rhythmic periodic contractions at T.O.B.C. of 0.3 mg per ml (Fig. 5.2). The Cr.Gp

demonstrated relaxant activity upon potassium (80 mM) and potassium (25 mM) mediated

persistent contractile responses in smooth muscle preparations of rabbits isolated jejunum at

EC50 values of 3.05 mg per ml [CI(95%) of 2.28-4.06 mg per ml; n=5] and 0.91 mg per ml

[CI(95%) of 0.65-1.29 mg per ml; n=5], respectively (Fig. 5.3, 5.4 & 5.6). The comparison of

the EC50 values reflected that Cr.Gp demonstrated potent relaxant activity upon potassium

(25 mM) mediated contractions than potassium (80 mM) mediated contractions in smooth

muscle preparations of rabbits isolated jejunum. However, pretreatment of smooth muscle

preparations of rabbit isolated jejunum with glibenclamide (standard ATP dependent

potassium channel blocker; 3 µM), resulted in attenuation of the Cr.Gp exerted relaxant effect

upon potassium (25 mM) mediated contractile response with median response concentration

of 3.01 mg per ml [CI(95%) of 2.08-4.36 mg per ml; n=5] (Fig. 5.5 & 5.7).

Moreover, cromakalim (a standard ATP dependent potassium channel opener) relaxed

rhythmic periodic contractions as well as potassium (25 mM) mediated contractile response

in smooth muscle preparations of rabbits isolated jejunum at median response concentrations

of 0.81 µM [CI (95%) of 0.67-0.98 µM; n=5] and 0.53 µM [CI (95%) of 0.45-0.63 µM; n=5],

respectively; but failed to relax potassium (80 mM) mediated contractile response (Fig. 5.8).

Whereas, pretreatment with glibenclamide (3 µM) of smooth muscle preparations of rabbit

92

isolated jejunum resulted in suppression of cromakalim exerted relaxant effect upon

potassium (25 mM) mediated contractile response (Fig. 5.9).

Furthermore, pretreatment by Cr.Gp at T.O.B.C. of 0.3-1.0 mg per ml of smooth muscle

preparations of rabbits isolated jejunum resulted in shifting of response versus concentration

curves (CRCs) for calcium rightward in non-parallel manner and decreasing maximally

achieved responses in significant (p < 0.05) manner (Fig. 5.10). Similarly, pretreatment of

smooth muscle preparations of rabbits isolated jejunum with verapamil at T.O.B.C. range of

0.1-0.3 μM also caused shifting of CRCs for calcium in non-parallel fashion with mitigation

of maximally achievable response in significant (p < 0.05) manner (Fig. 5.11). However,

pretreatment by cromakalim at T.O.B.C. of 0.1-0.3 μM did not modify CRCs for calcium in

smooth muscle preparations of rabbit isolated jejunum (Fig. 5.12).

Moreover, ethyl acetate fraction of Cr.Gp (Ea.Gp) caused relaxation of rhythmic periodic

contractions and potassium (25 mM) as well as potassium (80 mM) mediated contractile

responses in smooth muscle preparations of rabbit isolated jejunum (Fig. 5.13, 5.14 & 5.15)

at median response concentrations of 0.15 mg per ml [CI(95%) of 0.14-0.16 mg per ml; n=5],

0.11 mg per ml [CI(95%) of 0.08-0.13 mg per ml; n=5] and 0.54 mg per ml [CI(95%) of

0.42-0.69 mg per ml; n=5], respectively (Fig. 5.17). However, pretreatment of tissues

preparations with glibenclamide (3 µM) decreased Ea.Gp mediated relaxant effect upon

potassium (25 mM) mediated contractions (Fig. 5.16 & 5.18).

The aqueous fraction of Cr.Gp (Aq.Gp) demonstrated contractile response upon spontaneous

rhythmic contractions in smooth muscle preparations of rabbit isolated jejunum, which was

about 110% of acetylcholine (1 µM) mediated response (Fig. 5.19). The contractile effect of

Aq.Gp remained unchanged in presence of atropine (1 µM), pyrilamine (1 µM),

indomethacin (1 µM), verapamil (1 µM) or in calcium free Tyrode solution (data not shown),

but it was found to be inhibited following pre-treating the tissue with caffeine (10 mM) (Fig.

5.20 & 5.21).

Fig. 5.2: Influence of cumulatively added Cr.Gp upon spontaneous rhythmic periodic contractile movements in smooth muscle preparations of rabbits isolated jejunum.

93

Fig. 5.3: The influence of cumulatively added Cr.Gp upon potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum.

Fig. 5.4: The influence of cumulatively added Cr.Gp upon potassium (80 mM) mediated contractile activity in isolated preparation of rabbit jejunum.

Fig. 5.5: The influence of cumulatively added Cr.Gp upon potassium (25 mM) mediated contractile activity in isolated preparation of rabbit jejunum subsequent to pretreatment with glibenclamide (3 μM).

94

Fig. 5.6: Influence of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5).

Fig. 5.7: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response of smooth muscle preparations of rabbit isolated jejunum, alone and after pre-incubation of tissue with glibenclamide (3 µM). Variables are mean ± S.E.M. (n=5).

95

Fig. 5.8: Influence of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractile responses of smooth muscle preparations of rabbit isolated jejunum. Variable are mean ± S.E.M. (n=5).

Fig. 5.9: Influence of cromakalim upon potassium (25 mM) mediated contractile response of smooth muscle preparations of rabbit isolated jejunum, alone and in presence of glibenclamide (3 µM). Variable are mean ± S.E.M. (n=5).

96

-4.5 -3.5 -2.5 -1.5

0

25

50

75

100

Cr.Gp (0.3 mg/ml)

Cr.Gp (1.0 mg/ml)

Control

Log [Ca+2] M

*

*

Fig. 5.10: Influence of Cr.Gp upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. * indicates significant difference (p < 0.05) from control maximum. Variables are mean ± S.E.M. (n=5).

Fig. 5.11: Influence of verapamil upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. * indicates significant difference (p < 0.05) from control maximum. Variables are mean ± S.E.M. (n=5).

97

% o

f co

ntr

ol

ma

xim

um

Fig. 5.12: Influence of cromakalim upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=5).

Fig. 5.13: Influence of Ea.Gp upon rhythmic periodic contractility of isolated preparations of rabbit jejunum.

Fig. 5.14: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparations of rabbit jejunum.

98

Fig. 5.15: Influence of Ea.Gp upon potassium (80 mM) mediated contractility of isolated preparations of rabbit jejunum.

Fig. 5.16: The influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparations of rabbit jejunum subsequent to pretreatment with glibenclamide (3 µM).

Fig. 5.17: Influence of Ea.Gp upon spontaneous contractions, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variables are S.E.M. (n=5).

99

Fig. 5.18: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response, alone and in presence of glibenclamide (3 µM), in smooth muscle preparations of rabbit isolated jejunum. Variables are S.E.M. (n=5).

Fig. 5.19: Record showing influence of Ach and cumulative concentrations of Aq.Gp upon spontaneous contractions in isolated preparations of rabbit jejunum.

Fig. 5.20: Record showing influence of caffeine (10 mM) upon spontaneous and Aq.Gp mediated contractions in rabbit isolated jejunum preparation. Caffeine caused transient contraction followed by relaxation of rabbit isolated jejunum preparation and Aq.Gp failed to elicit contraction in caffeine treated tissue.

100

Fig. 5.21: Contractile effects of Aq.Gp upon spontaneously contracting rabbit isolated jejunum preparations, without and with caffeine (20 mM) pretreatment. Variables are mean ± S.E.M. (n=5).


Application of Cr.Gp at 0.01-5.0 mg per ml to isolated preparation of rabbit trachea did not

demonstrate any contractile response on resting baseline; but produced contractile response at

T.O.B.C. of 10 mg per ml (Fig. 5.22); which was found to be mitigated upon pretreatment of

the isolated preparations with caffeine (10 mM) (Fig. 5.23). However, Cr.Gp demonstrated

relaxant response upon carbachol (1 µM), potassium (25 mM), and potassium (80 mM)

mediated contractile responses in isolated preparations of rabbit trachea (Fig. 5.24, 5.25 &

5.26) at median response concentrations of 0.85 mg per ml [CI(95%) of 0.68-1.05 mg per ml;

n=5], 0.21 mg per ml [CI(95%) of 0.17-0.25 mg per ml; n=5] and 1.23 mg per ml [CI(95%)

of 1.03-1.47 mg per ml; n=5], respectively (Fig. 5.28 & 5.30). Hence, Cr.Gp demonstrated

more potent relaxant effect upon potassium (25 mM) mediated contractile response as

compared to potassium (80 mM) and carbachol (1 µM) mediated contractile responses.

Whereas, the Cr.Gp demonstrated relaxant effect upon potassium (25 mM) mediated

contractile response was found to be mitigated subsequent to pretreatment with glibenclamide

(3 µM) (Fig. 5.27 & 5.29). Likewise, cromakalim, (ATP dependent potassium channel

opener) demonstrated relaxant effect upon potassium (25 mM) mediated contractile response

with median response concentration of 0.87 µM [CI(95%) of 0.65-1.18 µM; n=5]; but failed

to relax potassium (80 mM) mediated contractile response in isolated preparations of rabbit

trachea (Fig. 5.31). Moreover, cromakalim exerted relaxant activity upon potassium (25 mM)

101

induced contractions was mitigated following pretreatment with glibenclamide (3 µM) (Fig.

5.32). Furthermore, ethyl acetate fraction of the Cr.Gp (Ea.Gp) exerted relaxant effect upon

potassium (25 mM), potassium (80 mM) and carbachol (1 µM) mediated contractile

responses in isolated preparations of rabbit trachea with median response concentrations of

0.04 mg per ml [CI(95%) of 0.03-0.05 mg per ml; n=5], 7.22 mg per ml [CI(95%) of 6.09-

8.55 mg per ml; n=5] and 0.37 mg per ml [CI(95%) of 0.29-0.47 mg per ml; n=5],

respectively (Fig. 5.33, 5.34, 5.35 & 5.37). Additionally, Ea.Gp exerted potent relaxant effect

upon potassium (25 mM) mediated contractile response was blunted subsequent to

pretreatment with glibenclamide (3 μM) (Fig. 5.36 & 5.38). The Ea.Gp also shifted CRCs of

carbachol rightward with suppression of maximal response (Fig. 5.39). On the other hand,

aqueous fraction of the Cr.Gp (Aq.Gp) enhanced baseline tension of isolated preparations of

rabbit trachea (Fig. 5.40), which was attenuated following pretreatment with caffeine (10

mM) (Fig. 5.41). Aq.Gp was found to demonstrate partial relaxation of potassium (25 mM),

potassium (80 mM) and carbachol (1 µM) mediated contractile activities (Fig. 5.42 to 5.45).

Fig. 5.22: Influence of Cr.Gp upon baseline tension of smooth muscle preparation of isolated rabbit trachea without any pretreatment.

Fig. 5.23: Influence of Cr.Gp upon baseline tension of smooth muscle preparations of rabbit isolated trachea, without and with caffeine (10 mM) pretreatment. Variables are mean ± S.E.M. (n=5).

102

Fig. 5.24: The influence of Cr.Gp upon potassium (25 mM) mediated contractile response in

smooth muscle preparation of isolated rabbit trachea.

Fig. 5.25: The influence of Cr.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea.

Fig. 5.26: The influence of Cr.Gp upon carbachol (1 µM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea.

Fig. 5.27: The influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of isolated rabbit trachea subsequent to pretreatment with glibenclamide (3 µM).

103

Fig. 5.28: Influence of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractions in smooth muscle preparations of rabbit isolated trachea. Values are mean ± S.E.M.; n=5.

Fig. 5.29: Influence of Cr.Gp upon potassium (25 mM) mediated contractility, alone and in presence of glibenclamide (3 µM), of smooth muscle preparations of rabbit isolated trachea. Values are mean ± S.E.M.; n=5.

104

Co

ntr

ol

(%)

Fig. 5.30: Influence of Cr.Gp upon carbachol (1 µM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Values are mean ± S.E.M.; n=5.

Fig. 5.31: Influence of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractions of smooth muscle preparations of rabbit isolated trachea. Values are mean ± S.E.M.; n=5.

105

Fig. 5.32: Influence of cromakalim upon potassium (25 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea, alone and in presence of glibenclamide (3 µM) (mean ± S.E.M.; n=5).

Fig. 5.33: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea.

Fig. 5.34: Influence of Ea.Gp upon potassium (80 mM) mediated contractility of isolated preparation of rabbit trachea.

106

Fig. 5.35: Influence of Ea.Gp upon carbachol (1 µM) mediated contractility of isolated preparation of rabbit trachea.

Fig. 5.36: Influence of Ea.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea subsequent to pretreatment with glibenclamide (3 µM).

Fig. 5.37: Influence of Ea.Gp upon potassium (25 mM), potassium (80 mM) and carbachol (1 µM) mediated contractions of smooth muscle preparations of rabbit isolated trachea. (Mean ± S.E.M.; n=5).

107

Fig. 5.38: Influence of Ea.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractility of smooth muscle preparations of rabbit isolated trachea (mean ± S.E.M.; n=5).

Fig. 5.39: Influence of Ea.Gp upon CRCs for carbachol in smooth muscle preparations of isolated preparations of rabbit trachea (mean ± S.E.M.; n=5).

108

Fig. 5.40: Record of influence of Aq.Gp upon baseline resting tension of isolated tracheal preparation of rabbit without any pretreatment.

Fig. 5.41: Influence of Aq.Gp upon baseline resting tension of isolated tracheal preparations of rabbit, without and with caffeine (10 mM) pretreatment (mean ± S.E.M.; n=5).

Fig. 5.42: Influence of Aq.Gp upon potassium (25 mM) mediated contractility of isolated preparation of rabbit trachea.

Fig. 5.43: Influence of Aq.Gp upon potassium (80 mM) mediated contractility of isolated preparation of rabbit trachea.

109

Fig. 5.44: Influence of Aq.Gp upon carbachol (1 µM) mediated contractility of isolated preparation of rabbit trachea.

Fig. 5.45: Influence of Aq.Gp on potassium (25 mM), potassium (80 mM) and carbachol (1 μM) mediated contractions in rabbit isolated trachea preparations. Variable are mean ± S.E.M. (n=5).

5.3.2.3. Influence upon isolated denuded aortic preparations of rabbit

The Cr.Gp was without any effect upon tissue preparations of rabbit isolated endothelium

denuded aorta at T.O.B.C. range of 0.01-0.3 mg per ml, but exerted contractile response at

T.O.B.C. range of 1.0-5.0, followed by relaxation at T.O.B.C. of 10.0 mg per ml (Fig. 5.46 &

5.47). However, Cr.Gp mediated contractile response in smooth muscle preparations of

isolated rabbit denuded aorta was blunted subsequent to pretreatment with caffeine (10 mM)

(Fig. 5.47).

Moreover, Cr.Gp demonstrated relaxant effect upon application to phenylephrine (1 μM),

potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle

110

preparations of isolated rabbit denuded aorta at median response concentrations of 3.80 mg

per ml [CI(95%) of 2.82-5.14 mg per ml; n=5], 0.30 mg per ml [CI(95%) of 0.21-0.44 mg per

ml; n=5] and 5.75 mg per ml [CI(95%) of 3.10-8.67 mg per ml; n=5], respectively (Fig. 5.48

to 5.51 & 5.53). On the other hand, pretreatment of tissue with glibenclamide (3 µM)

attenuated Cr.Gp exerted relaxant effect upon potassium (25 mM) mediated contractile

response with resulting median response concentration of 4.87 mg per ml [CI(95%) of 3.45-

6.87 mg per ml; n=5] (Fig. 5.52 & 5.54). Whereas, cromakalim relaxed potassium (25 mM)

mediated contractile response in potent fashion at median response concentration of 0.28 µM

[CI(95%) of 0.21–0.37 µM; n=5]; however, it failed to relax potassium (80 mM) exerted

contractile response; whereas, presence of glibenclamide (3 µM) abolished the cromakalim

exerted relaxant effect upon potassium (25 mM) mediated contractile response in smooth

muscle preparations of rabbit isolated denuded aorta (Fig. 5.55 & 5.56).

The Ea.Gp failed to demonstrate contractile response upon application to the smooth

preparations of rabbit isolated denuded aorta but demonstrated relaxant response upon

potassium (25 mM), potassium (80 mM) and phenylephrine (1 µM) mediated contractile

responses at median response concentrations of 0.31 mg per ml [CI(95%) of 0.20-0.48 mg

per ml; n=5], 1.07 mg per ml [CI(95%) of 0.58-1.96 mg per ml; n=5] and 8.68 mg per ml

[CI(95%) of 3.59-13.76 mg per ml; n=5], respectively (Fig. 5.57 to 5.60). Furthermore,

pretreatment with glibenclamide (3 µM) attenuated Ea.Gp induced relaxant effect upon

potassium (25 mM) mediated contractile response with resulting median response

concentration value of 1.12 mg per ml [CI(95%) of 0.63-1.91 mg per ml; n=5] (Fig. 5.61 &

5.62). Furthermore, Aq.Gp demonstrated contractile response on smooth muscle preparations

of rabbit isolated denuded aorta, which was abolished subsequent to pretreatment with

caffeine (10 mM) (Fig. 5.63 & 5.64). Additionally, Aq.Gp (0.01-10.0 mg per ml)

demonstrated partial relaxation of potassium (25 mM), potassium (80 mM) and

phenylephrine (1 µM) mediated contractile responses in smooth muscle preparations of rabbit

isolated denuded aorta to variable extent (Fig. 5.65 to 5.68).


The Cr.Gp at T.O.B.C. range of 0.01-3.0 mg per ml demonstrated decrease in force as well as

rate of contractions in spontaneously contracting isolated preparations of rabbit paired atria;

which was found to be mitigated subsequent to pretreatment of the tissue with glibenclamide

(3 µM) (Fig. 5.69. 5.70 & 5.71).

111

Fig. 5.46: Influence of Cr.Gp upon baseline tension in smooth muscle preparation of rabbit isolated denuded aorta without any pretreatment.

% o

f p

hen

yle

ph

rin

e (1

M)

resp

on

se

Fig. 5.47: Influence of Cr.Gp upon baseline tension in smooth muscle preparations of rabbit isolated denuded aorta, without and with caffeine (10 mM) pretreatment. Values were mean ± S.E.M. (n=5).

Fig. 5.48: Influence of Cr.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

112

Fig. 5.49: Influence of Cr.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).

Fig. 5.50: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.51: Influence of Cr.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.52: Influence of Cr.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with glibenclamide (3 μM).

113

Fig. 5.53: Effects of Cr.Gp upon potassium (25 mM) and potassium (80 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

% o

f co

ntr

ol m

ax

imu

m

Fig. 5.54: Effects of Cr.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

114

Fig. 5.55: Effects of cromakalim upon potassium (25 mM) and potassium (80 mM) mediated contractile responses in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

% o

f co

ntr

ol

ma

xim

um

Fig. 5.56: Effects of cromakalim upon potassium (25 mM) mediated contractile response in rabbit isolated denuded aortic preparations, alone and in presence of glibenclamide (3 µM). Values were mean ± S.E.M. (n=5).

115

Fig. 5.57: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.58: Influence of Ea.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.59: Influence of Ea.Gp upon phenylephrine (1 µM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.60: Effects of Ea.Gp upon potassium (25 mM), potassium (80 mM) and phenylephrine (1 μM) mediated contractile responses in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

116

Fig. 5.61: Influence of Ea.Gp upon potassium (25 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta subsequent to pretreatment with glibenclamide (3 μM).

Fig. 5.62: Effects of Ea.Gp, alone and in presence of glibenclamide (3 µM), upon potassium (25 mM) mediated contractile response in rabbit isolated denuded aortic preparations. Values were mean ± S.E.M. (n=5).

Fig. 5.63: Influence of Aq.Gp upon baseline tension in smooth muscle preparation of rabbit isolated denuded aorta.

117

Fig. 5.64: Influence of Aq.Gp upon baseline tension in smooth muscle preparations of rabbit isolated denuded aorta, without and with caffeine (10 mM) pretreatment. Values were mean ± S.E.M. (n=5).

Fig. 5.65: Influence of Aq.Gp upon potassium (80 mM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.66: Influence of Aq.Gp upon potassium (25 μM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

118

Fig. 5.67: Influence of Aq.Gp upon phenylephrine (1 μM) mediated contractile response in smooth muscle preparation of rabbit isolated denuded aorta.

Fig. 5.68: Influence of Aq.Gp upon potassium (25 mM), potassium (80 mM) and phenylephrine (1 μM) mediated contractile response in smooth muscle preparations of rabbit isolated denuded aorta. Values were mean ± S.E.M. (n=5).

Fig. 5.69: Record of influence of Cr.Gp upon force of spontaneous contractions in rabbit isolated paired atrial preparation.

119

Fig. 5.70: Influence of Cr.Gp, alone and in presence of glibenclamide, upon force of contractions of rhythmic periodic contractions in rabbit isolated preparation of paired atria. Variables were mean ± S.E.M., n=5.

Fig. 5.71: Influence of Cr.Gp, alone and in presence of glibenclamide, upon rate of contractions of rhythmic periodic contractions in rabbit isolated preparation of paired atria. Variables were mean ± S.E.M., n=5.

120


5.3.3.1. Influence upon charcoal meal transit distance in mice


± 0.80 cm. The Cr.Gp administration to Treated 1 and 2 groups of animals (n=5) at respective

oral doses of 50 and 100 mg per kg caused significant (p < 0.05) decrease in intestinal transit

distances of charcoal meal as compared to control and were assessed to be 25.64 ± 1.44 and

21.76 ± 1.31 cm, respectively. However, Cr.Gp administered orally at doses of 300 and 500

mg per kg to Treated 3 and 4 groups of animals (n=5), respectively, caused significantly

increased (p < 0.05) intestinal charcoal meal transit distances as compared to control and

were assessed to be 34.48 ± 0.60 and 37.62 ± 0.41cm. Moreover, loperamide treatment (10

mg per kg; orally) to standard group of mice (n=5), the intestinal transit distance of charcoal

meal was found to be 7.40 ± 0.29 cm; which was found to decrease in significant (p < 0.05)

fashion in comparison with control. Furthermore, carbachol administration (1 mg per kg) to

another standard group of mice (n=5) caused significantly increased (p < 0.05) charcoal meal

intestinal transit distance in comparison with control and was measured to be 44.82 ± 1.05 cm

(Table 5.1).


The mean wet fecal masses count in saline control group (n=6) of mice (administered saline

and olive oil orally at rate of 10 ml per kg) was found to be 0.33 ± 0.21; whereas, mean wet

fecal masses count in treated control group (n=6) of animals (received saline and castor oil

orally at rate of 10 ml per kg) was found to be 8.20 ± 0.31. The Treatment 1 group (n=6) of

mice (administered Cr.Gp and castor oil orally at respective rate of 50 mg per kg and 10 ml

per kg) resulted significantly (p < 0.05) decreased mean wet fecal masses count, which was

found to be 6.83 ± 0.31. However, mean wet fecal masses count in Treatment 2 group (n=6)

of mice (administered Cr.Gp and castor oil orally at respective rate of 100 mg per kg and 10

ml per kg) was significantly (p < 0.05) decreased to 6.00 ± 0.26. Similarly, mean wet fecal

masses count in Treatment 3 group (n=6) of mice (administered Cr.Gp and castor oil orally at

respective rate of 300 mg per kg and 10 ml per kg) was insignificantly (p > 0.05) decreased to

7.17 ± 0.31. Mean wet fecal masses count in the standard control group (n=6) of mice

(administered loperamide and castor oil orally at respective rate of 10 mg per kg and 10 ml

per kg) was significantly (p < 0.05) decreased to 0.50 ± 0.22. (Table 5.2).

121

Groups Treatment

(given orally)

Charcoal movement

Mean ± S.E.M. (cm)


10 ml per kg + 0.2 ml per mouse 31.23 ± 0.80

Treated 1 Cr.Gp + Charcoal meal

50 mg per kg + 0.2 ml per mouse 25.64 ± 1.44*











Table 5.1: Influence of Cr.Gp upon charcoal meal intestinal transit distance in mice. Values are mean ± S.E.M. (n=5); Statistics: ANOVA (one way) along with Dunnett’s test; *p < 0.05 vs. control group.

5.3.3.3. Laxative activity among mice

The saline control group of mice to whom normal saline was administered (10 ml per kg); the

total and wet feces counts for 6 hrs were found to be 4.83 ± 0.31 and 0.50 ± 0.22,

respectively. The Treated 1 group of mice (n=6) was orally treated with Cr.Gp (300 mg per

kg); the total and wet feces counts for 6 hrs were significantly (p < 0.05) increased to 7.17 ±

0.31 and 2.67 ± 0.33, respectively, as compared to that of saline control group. Similarly,

Treated 2 group of mice was treated orally by Cr.Gp (500 mg per kg); the total and wet feces

counts were significantly (p < 0.05) increased to 8.50 ± 0.22 and 4.50 ± 0.42, respectively, as

compared to that of saline control group. The standard control group of mice to whom

carbachol was administered orally (1 mg per kg); the total and wet feces counts were

significantly (p < 0.05) increased to 11.17 ± 0.40 and 7.67 ± 0.33, respectively, as compared

to that of saline control group (Table 5.3).

122

Group Treatment

(given orally)

Wet feces count

Mean ± S.E.M.

Protection (%age)

Saline Control Saline + Olive oil

(10 ml per kg + 10 ml per kg) 0.33 ± 0.21 -


(10 ml per kg + 10 ml per kg) 8.20 ± 0.31 -

Treatment 1 Cr.GP + Castor oil

(50 mg per kg + 10 ml per kg) 6.83 ± 0.31* 16.29

Treatment 2 Cr.GP + Castor oil

(100 mg per kg + 10 ml per kg) 6.00 ± 0.26* 26.47

Treatment 3 Cr.Gp + Castor oil


Standard Treatment


(10 mg per kg + 10 ml per kg) 0.50 ± 0.22* 93.90

Table 5.2: Influence of Cr.Gp upon castor oil caused diarrhea in mice. Values are mean ± S.E.M. (n=6). Statistics: ANOVA (one way) along with Dunnett’s test; nsp ˃ 0.05 and *p < 0.05 compared to treated control group.

Groups Treatment

(given orally)

Wet feces count

Mean ± S.E.M.

Total feces count

Mean ± S.E.M.

Saline Control Normal saline (10 ml per kg) 0.50 ± 0.22 4.83 ± 0.31

Treated 1 Cr.Gp (300 mg per kg) 2.67 ± 0.33* 7.17 ± 0.31*

Treated 2 Cr.Gp (500 mg per kg) 4.50 ± 0.42* 8.5 ± 0.22*

Standard Control Carbachol (1 mg per kg) 7.67 ± 0.33* 11.17 ± 0.40*

Table 5.3: The Cr.Gp exerted laxative effect in mice as compared to carbachol. Values are mean ± S.E.M. (n=6). Statistics: ANOVA (one way) along with Dunnett’s test; *p < 0.05 as compared to saline control group.

123

5.3.3.4. Influence upon blood pressure of rats

Vehicle (5% DMSO in saline) administered intravenously to normotensive anesthetized rats

did not cause significant changes in blood pressure and heart rate from basal values as

percent change in systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood

pressure (MBP) and heart rate (HR) were recorded to be 1.06 ± 0.35, 1.46 ± 0.43, 1.58 ± 0.55

and 0.30 ± 0.10 %, respectively. The Cr.Gp administered intravenously to normotensive

anesthetized rats at a dose of 1 mg per kg resulted in insignificant (p > 0.05) decrease in SBP

(9.50 ± 1.67 %) and MBP (14.00 ± 2.51%), but demonstrated significant (p < 0.05) decrease

in DBP (18.64 ± 2.58 %). Moreover, Cr.Gp administration at intravenous dose of 3 mg per kg

resulted in insignificant decrease (p > 0.05 ) in SBP (14.72 ± 1.34 %), but caused significant

decrease (p < 0.05) in DBP and MBP, which were recorded to be 25.10 ± 1.23 and 20.78 ±

0.74 %, respectively. Intravenous administration of Cr.Gp (10 mg per kg) demonstrated

significantly decreased (p < 0.05) SBP, DBP and MBP, and decrease in respective values

were recorded to be 28.16 ± 2.04, 39.56 ± 2.42 and 39.92 ± 2.37 %. Furthermore, Cr.Gp at 30

mg per kg dose also demonstrated significantly (p < 0.05) decreased SBP, DBP and MBP,

and percent decrease from baseline values recorded were 38.5 ± 2.36, 47.70 ± 2.19 and 45.96

± 2.05, respectively. Similarly, intravenous administration of cromakalim (3 µg per kg) to

normotensive anaesthetized rats (n=5) demonstrated significantly (p < 0.05) decreased SBP,

DBP and MBP, and percent decrease from baseline values recorded were 17.36 ± 1.51, 22.10

± 0.78 and 18.16 ± 0.47, respectively. Intravenous administration of cromakalim (3 µg per

kg) caused insignificant increase (p > 0.05) in HR of anesthetized rats. Intravenous

administration of Cr.Gp (1, 3, 10 and 30 mg per kg) decreased HR of anesthetized rats, but

decrease was not significant (p > 0.05) in comparison to vehicle treated values (Fig. 5.62;

Table 5.3).

Fig. 5.72: Record demonstrating effects of Cr.Gp upon blood pressure of an anesthetized normotensive rat.

124

Treatments

(intravenous)

% change

SBP DBP MBP HR

Vehicle (0.1 ml) -1.06 ± 0.35 -1.46 ± 0.43 -1.58 ± 0.55 -0.30 ± 0.10

Cr.Gp (1 mg per kg) -9.50 ± 1.67 -18.64 ± 2.58* -14.00 ± 2.51 -2.52 ± 0.29

Cr.Gp (3 mg per kg) -14.72 ± 1.34 -25.10 ± 1.23* -20.78 ± 0.74* -6.23 ± 0.55

Cr.Gp (10 mg per kg) -28.16 ± 2.04* -39.56 ± 2.42* -39.92 ± 2.37* -7.08 ± 0.83

Cr.Gp (30 mg per kg) -38.5 ± 2.36* -47.70 ± 2.19* -45.96 ± 2.05* -7.36 ± 1.33

Cromakalim (3 µg per kg)

-17.36 ± 1.51* -22.10 ± 0.78* -18.16 ± 0.47* +1.86 ± 1.14

Table 5.4: Effects of intravenously administered vehicle (5% DMSO in saline), Cr.Gp and cromakalim upon SBP, DBP, MBP and HR of normotensive anesthetized rats. (-) indicates decrease and (+) indicates increase in values. Values are mean ± S.E.M. (n=5). Statistics: ANOVA (two way) along with Dunnett test; *p < 0.05 as compared to values of vehicle treatment values.

5.3.3.5. Diuretic activities in rats



(Na+, K+ & Cl-) in urine as µmol per 100 g body weight in 6 hrs. The urine volume in group



hrs, respectively. The group of rats treated by Cr.Gp (50 mg per kg) demonstrated significant

(p < 0.05) increase in urinary volume to 1.71 ± 0.08 ml per 100 g in 6 hrs as well as

significant increase in respective Na+, K+ and Cl- urinary contents to 163.05 ± 6.39, 10.60 ±

0.74 and 200.63 ± 7.07 µmol per 100 g in 6 hrs in comparison with saline control group. The

group of rats treated by Cr.Gp (100 mg per kg) demonstrated significant (p < 0.05) increase

in urinary volume to 1.99 ± 0.80 ml per 100 g in 6 hrs as well as significant increase in

respective Na+, K+ and Cl- excretion to 188.18 ± 5.17, 12.43 ± 0.39 and 222.83 ± 2.52 µmol

125

per 100 g in 6 hrs in comparison with saline control group of rats. The group of rats treated

by Cr.Gp (300 mg per kg) demonstrated significant (p < 0.05) increase in voided urinary

volume to 2.27 ± 0.13 ml per 100 g in 6 hrs and significant (p < 0.05) increase in respective

Na+, K+ and Cl- excretion to 226.53 ± 6.10, 15.57 ± 0.71 and 253.10 ± 2.57 µmol per 100 g in

6 hrs in comparison with saline control group of rats. The group of rats treated by furosemide

(20 mg per kg) demonstrated significant (p < 0.05) increase in urinary volume to 4.10 ± 0.11

ml per 100 g in 6 hrs and significant (p < 0.05) increase in respective Na+, K+ and Cl- urinary

contents to 397.17 ± 5.64, 31.42± 0.58 and 516.50 ± 9.34 µmol per 100 g in 6 hrs in


Na+/K+ ratio and ion quotient were also calculated (Table 5.5). The diuretic indices following

Cr.Gp administration at doses of 50, 100 and 300 mg per kg were calculated to be 1.68, 1.95

and 2.23 respectively, whereas, diuretic index of the standard drug furosemide was found

4.10. Oral administration of furosemide resulted significantly (p < 0.05) decreased urinary

Na+/K+ ratio to 12.10 ± 0.36 in comparison with saline treated group (control). However,

Cr.Gp administration at doses of 50, 100 and 300 mg per kg demonstrated insignificant (p >

0.05) change of Na+/K+ to 15.85 ± 1.49, 15.16 ± 0.36 and 14.70 ± 0.74 respectively, as

compared to the saline group (Table 5.5).

126

Treatment groups (dose)

Volume of

urine (ml/100

g/6 hrs)

Diuretic

index

Na+ K+ Cl-

Na+/K+ Ion

quotient (µmol/100 g/6 hrs)

Normal saline (10 ml per kg) 1.02 ± 0.06 - 121.00 ± 4.05 7.78 ± 0.23 127.50 ± 2.20 15.63 ± 0.80 0.99 ± 0.03

Cr.Gp (50 mg per kg) 1.71 ± 0.08* 1.68 163.05 ± 6.39* 10.60 ± 0.74* 200.63 ± 7.07* 15.85 ± 1.49 1.16 ± 0.06

Cr.Gp (100 mg per kg) 1.99 ± 0.80* 1.95 188.18 ± 5.17* 12.43 ± 0.39* 222.83 ± 2.52* 15.16 ± 0.36 1.11 ± 0.03

Cr.Gp (300 mg per kg) 2.27 ± 0.13* 2.23 226.53 ± 6.10* 15.57 ± 0.71* 253.10 ± 2.57* 14.70 ± 0.74 1.05 ± 0.03

Furosemide (20 mg per kg) 4.10 ± 0.11* 4.02 379.17 ± 5.64* 31.42 ± 0.58 * 516.50 ± 9.34* 12.10 ± 0.36* 1.25 ± 0.03

Table 5.5: Effects of saline, Cr.Gp and furosemide upon urinary volume and urinary electrolytes contents in albino rats. Diuretic index was calculated as volume of urine in treatment group/volume of urine in control (saline treated group); whereas ion quotient was calculated as Cl-/ (Na+ + K+). The values were mean ± S.E.M. (n=6). * p < 0.05 on comparison with control (saline treated) group.

127

5.4. Discussion

Based on folkloric reports regarding uses of Gisekia pharnaceoides in management of diarrhea,

constipation, respiratory and heart troubles and as diuretic (Kritikar and Basu, 1987; Raza et al.,

2014; Malik et al., 2015), the plant was selected for this study. The aqueous-ethanol (30:70)

crude extract (Cr.Gp), its aqueous fraction (Aq.Gp) and ethyl acetate fraction (Ea.Gp) were

applied on spontaneous rhythmic contractions of rabbit jejunum preparations. Cr.Gp and Ea.Gp

relaxed rhythmic contractions of smooth muscle preparations of rabbit jejunum, thus confirming

antispasmodic activity. To study mechanism of spasmolytic activity of the extract, smooth

muscle preparations of rabbit isolated jejunum were contracted by potassium (80 mM) or

potassium (25 mM), and after attaining sustained contractions, the extract was applied in

cumulative doses. Cr.Gp was found to possess weak inhibitory effect upon potassium (80 mM)

than potassium (25 mM) mediated contractions of smooth muscle preparations of rabbit isolated

jejunum. Previous studies reported that substance relaxing potassium (25 mM) mediated

contractions in preference over potassium (80 mM) mediated contractions of smooth muscle

preparations is considered potassium channel opener (Mehmood et al., 2015). Potassium channel

openers constitute and important class of therapeutic agents capable of being used in verity of

diseased conditions involving smooth muscle hyperactivity, like diarrhea, hypertension, asthma,

heart and urinary troubles (Andersson, 1992; Fozard and Manley, 2001; dela Pena et al., 2009).

Opening of potassium channels causes hyperpolarization of membranes via efflux of potassium

ions, creating greater voltage barrier to achieve action potential and consequently inhibiting

opening of voltage dependent calcium channels, thus decreasing intracellular free calcium

leading to smooth muscle relaxation (Andersson, 1992). ATP dependent potassium channel

activity of the extract was confirmed when pretreatment of tissue with standard ATP dependent

potassium channel blocker, glibenclamide (Franck et al., 1994) attenuated the relaxing effect of

Cr.Gp upon potassium (25 mM) mediated contractions of smooth muscle preparations of rabbit

isolated jejunum. Cromakalim, a standard ATP dependent potassium channel opener (Hamilton

and Weston, 1989) was also found to exhibit potent relaxant activity against potassium (25 mM)

mediated contractions of smooth muscle preparations of rabbit isolated jejunum, which was

blocked in presence of glibenclamide. In contrast, verapamil, a calcium channel blocker, was

found equally effective in relaxing potassium (25 mM) and potassium (80 mM) mediated

128

contractions of smooth muscle preparations; and relaxant activity of verapamil against potassium

(25 mM) mediated contractions remained unaffected in presence of glibenclamide (3 µM).

Potassium ions at high concentrations are familiar for causing contractions in smooth muscle via

opening of voltage operated calcium channels (Ratz et al., 2005). Thus, a substance capable of

relaxing potassium (80 mM) mediated contractions of smooth muscle preparations is regarded as

voltage operated calcium entry blocker (Jabeen et al., 2009). The extract, at tissue organ bath

concentrations of 0.3 and 1.0 mg per ml, displaced the CRCs of calcium toward right with

suppression of maximum contractions in a manner similar to verapamil, a standard calcium entry

blocker. In contrast, cromakalim was found devoid of any effect upon CRCs of calcium.

Therefore, in addition to potassium channel opening effect, calcium channel blocking activity is

also confirmed in Cr.Gp.

The ethyl acetate fraction of Cr.Gp (Ea.Gp) also exhibited potent relaxant action upon potassium

(25 mM) than potassium (80 mM) mediated contractile responses in isolated preparations of

rabbit jejunum, indicating that spasmolytic and channel blocking activities were partitioned into

organic solvent. On the other hand, aqueous fraction of Cr.Gp (Aq.Gp) was found to devoid of

relaxant effect upon potassium (25 mM) or potassium (80 mM) induced contractions and

exhibited contractile response on periodic rhythmic contractions in isolated preparations of rabbit

jejunum; thus indicating simultaneous presence of spasmolytic and spasmogenic activities in the

same plant extract. The spasmogenic effect of Aq.Gp remained unaffected in presence of either

atropine, a muscarinic receptor antagonist (Akhlaq et al., 2016); indomethacin, a cyclooxygenase

inhibitor (Ferreira et al., 1976); verapamil, a calcium entry blocker; pyrilamine, histamine

receptor antagonist (Gilani et al., 2010) or calcium free tyrode solution (Mard et al., 2011); thus

excluding contribution of muscarinic receptors, histamine receptors, cyclooxygenase products

and calcium ion influx in contractile activity of the Aq.Gp. Treatment of tissues with caffeine (10

mM) caused transient spike of contraction followed by relaxation. Contractile effect of Aq.Gp

was blocked in presence of millimolar concentration of caffeine. In addition to other possible

modes of action, caffeine (at millimolar concentrations) is reported to act on intracellular sources

of calcium; where it causes to deplete ryanodine sensitive calcium stores, blockade of IP3

receptor mediated calcium release and intracellular calcium release channels in smooth muscle

cells (Ahn et al., 1988; Oh et al., 1997). Therefore, the contractile activity of Aq.Gp possibly

involves intracellular stores of calcium (Grasa et al., 2004).

129

The Cr.Gp was subjected to screening of laxative and anti-diarrheal activities on in vivo models

of mice. The Cr.Gp following oral administration (300 and 500 mg per kg) to mice demonstrated

significant increase in charcoal meal transit distance through small intestine as well as increased

wet feces count and total feces count; thus, indicating presence of laxative activity of the plant.

Whereas, at lower doses of 50 and 100 mg per kg, the Cr.Gp caused decrease in charcoal meal

transit distance through small intestine and also demonstrated protection against castor oil caused

diarrhea in mice. The castor oil is known to produce diarrhea due to its hydrolysis into ricinoleic

acid by means of pancreatic lipases; and it is the ricinoleic acid which produce diarrhea through

modification of electrolytes and water transport (Stewart et al., 1975). Potassium channel

openers are reported to possess antidiarrheal activity due to smooth muscle relaxation and

retention of luminal fluid in bowl (Poggioli et al., 1995). The anti-diarrheal activity in mice

against castor oil induced diarrhea of Cr.Gp can be assigned to its spasmolytic, potassium

channel opening and calcium channel blocking activities.

The possible bronchodilator potential of the Cr.Gp was investigated upon potassium (25 mM),

potassium (80 mM) and carbachol (1 µM) elicited sustained contractions in isolated preparations

of rabbit trachea. The Cr.Gp exhibited relaxant activities upon all contractions; however, relaxant

effect was observed to be most potent upon potassium (25 mM) elicited contractions in

comparison with potassium (80 mM) or carbachol (1 µM) mediated contractions, reflecting

dominant presence of potassium channel opening constituents in Cr.Gp. The potassium channel

opening potential of Cr.Gp was confirmed further as pretreatment of tissue with glibenclamide (3

µM), a known ATP dependent potassium channel blocker (Franck et al., 1994), caused decrease

in relaxant activity of the extract against potassium (25 mM) mediated contractions in isolated

preparations of rabbit trachea. Whereas, cromakalim, a standard ATP dependent potassium

channel opening drug (Hamilton and Weston, 1989) was also found to relax potassium (25 mM)

elicited contractions potently, which was antagonized in presence of glibenclamide. Cr.Gp and

Aq.Gp, when tested upon baseline tension of isolated rabbit tracheal preparations at tissue organ

bath concentration range of 0.01-10 mg per ml, resulted contractions at high T.O.B.C. of 5 and

10 mg per ml. The contractile effects of Cr.Gp and Aq.Gp were blocked in presence of caffeine

(10 mM), indicating possible mobilizing effect of the extract on intracellular stores of calcium.

The Ea.Gp fraction did not alter baseline tension of isolated rabbit tracheal preparations (data not

shown) and relaxed potassium (25 mM) mediated contractions most potently in comparison with

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potassium (80 mM) and carbachol (1 µM) elicited sustained contractions in isolated preparations

of rabbit trachea; indicating that relaxant components are concentrated in organic fraction. The

relaxant activity of extract was found concentrated in organic fraction (Ea.Gp), and to test

possibility of muscarinic receptor antagonistic potential, CRCs for carbachol in absence or

presence of the fraction was constructed; upon which the fraction caused rightward shift with

suppression of the maximally achieved response of CRCs for carbachol in isolated preparations

of rabbit trachea, indicating noncompetitive or nonspecific anti-muscarinic activity in rabbit

trachea (Kenakin, 2009). The tracheal constrictor effect of the extract is portioned in aqueous

fraction indicating polar nature of constrictor constituents, which might possibly not be get

absorbed into systemic circulation during ordinary use of the plant and further that constrictor

activity was observed at very high concentration.

The Cr.Gp, on application to rabbit isolated aortic rings preparations in cumulative manner, did

not alter baseline tension at T.O.B.C. range of 0.01-0.3 mg per ml, but it exhibited contractile

response at T.O.B.C. range of 1.0-5.0 mg per ml. The contractile effect of the extract was found

to be antagonized following pretreatment the tissue with caffeine (10 mM); reflecting that

observed vaso-contractile effect was likely to be mediated through modulation of intracellular

calcium stores by the plant constituents (Ahn et al., 1988). The Cr.Gp was also tested upon

potassium (25 mM), potassium (80 mM) and phenylephrine (1 µM) mediated contractile

responses in isolated rabbit aorta preparations. The Cr.Gp was found to exhibit complete

relaxation of potassium (25 mM), potassium (80 mM) and phenylephrine (1 µM) mediated

contractions to various potencies; while being most potent against potassium (25 mM) mediated

sustained contractions and pretreatment of tissue with glibenclamide (ATP dependent potassium

channel blocker) attenuated relaxant activity of the extract against potassium (25 mM) mediated

contractions. Thus, presence of ATP dependent potassium channel opening potential of Cr.Gp

was also confirmed in case of isolated preparations of rabbit aorta. The Ea.Cd also exhibited

more potent relaxant activity upon potassium (25 mM) exerted contractions than upon potassium

(80 mM) and phenylephrine (1 µM) mediated contractions. The Cr.Gp on application to periodic

rhythmic contractions of isolated rabbit paired atria preparations exhibited negative inotropic and

negative chronotropic effects at tissue bath concentration range of 0.01-3.0 mg per ml. The

negative inotropic and negative chronotropic effects of Cr.Gp were found to be decreased

following pretreatment of the paired atrial preparations with glibenclamide (3 µM); which also

131

indicates presence of ATP dependent potassium channel agonistic activity. The Cr.Gp, on

intravenous administration at the doses of 1, 3, 10 and 30 mg per kg to normotensive

anesthetized rats, resulted in decreased blood pressure; thus supporting traditional use of the

plant in cardiovascular protection. The typical ATP dependent potassium channel openers are

known to be associated with reflux tachycardia (Andersson, 1992), but Cr.Gp was not found to

increase heart rate of anesthetized rats, which may be attributed to presence of dual potassium

channel opening and calcium channel blocking activities in the same extract.

The Cr.Gp was tested for possible diuretic effect in rats and it was found to possess significant

diuretic activity in terms of enhanced urinary volume and electrolyte excretion at oral doses of

50, 100 and 300 mg per kg. These results are in confirmations of previously reported diuretic

activity on the part of plant traditional uses in urinary problems, in swellings and as diuretic

(Kritikar and Basu, 1987; Murthy and Madhav, 2015). However, diuretic activity of Cr.Gp was

found less potent as compared to furosemide (standard).

5.5. Conclusion

It can be concluded that crude extract of Gisekia pharnaceoides demonstrated spasmolytic, anti-

diarrheal, bronchodilator and hypotensive activities possibly mediated through potassium

channel opening and calcium channel blocking activities. The extract was also found to possess

laxative activity possibly medicated through action on mobilization of intracellular calcium

stores. The study provided scientific authentications and mechanistic grounds for folkloric repute

for utilization of Gisekia pharnaceoides in constipation, diarrhea, bronchoconstriction,

cardiovascular and urinary problems.

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6. Salsola imbricata Forssk.

Fig. 6.1: Salsola imbricata plant

133


Salsola imbricata Forssk. (family: Chenopodiaceae; synonyms; Chenopodium baryosmum

Schult., Caroxylon foetidum Moquin., Salsola foetida Del., and Salsola baryosma Schult.); is

known by multiple vernacular names, i.e., Khareet and Haram (Arabic), Lani and Lana

(Punjabi). It abundantly grows in hot and dried desert areas of Africa and Asia (Afghanistan,

India, Iran and Pakistan) (Boulos, 1991).

The plant is a shrub up to 1 m high and 3 m across, stems and lower leaves covered with

spreading or ascending, curved warty hairs up to 1.5 mm long. Stems selendr, filiform,

irregularly densely branched, up to 2 cm in diameter, nodulated, gray or reddish in color.

Leaves minute, fleshy and subglobose shape. Flowers are in short cylindrical spikes, made of

long and imbricate floral leaves. Bracts are unequal in shape and size, up to 1.5 × 1.5 mm

size, usually glabrous shorter than parienth. Fruits perianth portion is glabrous 3-5 mm in

diameter with dorsal membranous silvery white wings up to 2-3.5 mm, margins often

overlapping to form round or obtuse apex. Seeds semi-globular, flattened at top, 1-1.2 mm

diameter. The plant smells unpleasant and fish like when crushed (Boulos, 1991).

The plant is used as camel fodder, laundering cloths and burnt to obtain crude sodium

carbonate. Flowers have been employed as diuretic and remedy for inflammatory conditions

by Arabian population (Phondani et al., 2016). In United Arab Emirates, the powdered plant

is sniffed to relieve sinus congestion (Handa et al., 2006). In Central Sahara countries,

infusion of whole plant is taken internally for treatment of hypertension (Hammiche and

Maiza, 2006). In Pakistan the plant is used as anthelmintic and blood purifier; in treatment of

skin diseases, vertigo, migraine, headache, dyspepsia, abdominal distension, constipation,

dysentery, cold and asthma (Ahmad et al., 2014; Ahmed et al., 2014b, c; Wariss et al., 2014).

In veterinary medicine, plant shoots are mixed with common salt and drenched for treatment

of helminthiasis (Farooq et al., 2008).

Chemical investigations on S. imbricata reported alkaloids, anthraquinones, carbohydrates,

tannins, terpenoids, saponins, flavonoids and cardiac glycosides among the constituents

(Hamed et al., 2011; Munir et al., 2014).

Pharmacological investigations on plant extracts and constituents reported antioxidant,

spasmolytic, antibacterial, antifungal (Ahmad et al., 2006; Ahmed et al., 2006; Kaur and

Bains, 2012; Ahmed et al., 2014a), tyrosinase inhibitory (Khan et al., 2003), α- amylase and

α- glucosidase inhibitory (Khacheba et al., 2014), inhibitory to inflammations (Osman et al.,

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2016), hepatoprotective (Shehab et al., 2015) and male contraceptive (Shehab and Abu-

Gharbieh, 2014) activities.

The Salsola imbricata was subjected to thorough scientific investigations to validate its

folkloric use for treatment of gastrointestinal, respiratory and cardiovascular malfunctioning.


Collection of Salsola imbricata (aerial plants) was accomplished in May 2014 from territorial

jurisdiction of Bahawalpur district (Cholistan), Pakistan; followed by authenticated from

Prof. Dr. Altaf Ahmed Dasti (plant taxonomist); Institute of Pure and Applied Biology.,

Bahauddin Zakariya University, Multan, on submission of a representative specimen

(Fl.P.225-9) in herbarium. Removal of possible adulterants was done manually and placed

under shade to be dried for period of two weeks.

Purpose build electric grinder was used for grinding of dried herbal stuff into coarse powder

and 1 kg pulverized plant material was soaked at ambient room temperature (25-30°C) for 3

days in 70% aqueous-ethanol by using wide mouthed colored glass container with casual

stirring; followed after by straining through a piece of fine cloth and filtration (Whatman

grade 1; filter paper). Above-described process was reproduced twice using new solvent;

three aliquots of filtrates were added up and dried at 37°C using rotary evaporator (BUCHI,

Switzerland) to get a brownish green semisolid mass designated as crude extract of Salsola

imbricata (Cr.Si); calculated yield was 8.90 % of pulverized herbal stuff. About 60 g of Cr.Si

was added to 120 ml of H2O taken in a separating funnel; shaken vigorously and extracted

thrice in succession by 120 ml aliquots of petroleum ether for solvent-solvent fractionation.

All the collected aliquots of petroleum ether were mixed and solvent was removed by means

of rotary evaporator. The aqueous layer left in separating funnel was extracted thrice by 120

ml aliquots of ethyl acetate by using above-mentioned procedure and the separated fractions

were added up and was dried in rotary evaporator. The aqueous layer remained in separating

funnel was dried through freeze drying. The yield obtained for respective petroleum ether

(Pe.Si), ethyl acetate (Ea.Si) and aqueous (Aq.Si) fractions was 2.16, 3.34 and 93.30 % of the

crude extract. The Cr.Si was administered orally or intravenously to animals while soluble in

saline. Cr.Si and Aq.Si were used for in vitro experiments on dissolving in normal saline.

Pe.Si was not studied due to poor solubility and limited availability. Ea.Si was used for in

vitro experiments on dissolving in DMSO-saline (10%) for making of stock solution (300 mg

per ml); of which further dilutions were made by adding normal saline. The solvents as used

135

to dissolve test material were without any pharmacodynamic effects upon isolated tissue

preparations.

6.3. Results

6.3.1. Preliminary phytochemicals analysis

The alkaloids, steroids, tannin, phenols, flavonoids, coumarins and saponins were detected to

be among the plant constituents, while test for the presence of anthraquinones was negative.

The estimation of total phenolic and flavonoids contents per gram of Cr.Si were demonstrated

to be 136.56 ± 7.52 and 110.0 ± 4.36 mg respectively based upon respective equivalent of

gallic acid and quercetin.

6.3.2. In vitro experimentation

6.3.2.1. Influence upon tissue preparations of rabbit isolated jejunum

The rhythmic periodic contractions, potassium (80 mM) and potassium (25 mM) mediated

consistent contractile responses in isolated smooth muscle preparations of rabbit jejunum

were attenuated by crude extract of Salsola imbricata (Cr.Si) with respective tissue organ

bath median response concentrations (EC50) of 0.39 mg per ml [CI(95%) of 0.35–0.44 mg per

ml; n=6], 0.69 mg per ml [CI(95%) of 0.61–0.78 mg per ml; n=6] and 0.67 mg per ml

[CI(95%) of 0.61–0.74 mg per ml; n=6], respectively (Fig. 6.2, 6.3, 6.4 & 6.5). The perusal of

EC50 values indicated equal activity of Cr.Si in relaxing potassium (80 mM) and potassium

(25 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated

jejunum (Fig. 6.5). Calcium channel blocking agent, verapamil exhibited similar pattern of

relaxation to the extent of being equally potent on potassium (80 mM) and potassium (25

mM) mediated contractions in smooth muscle preparations of rabbit isolated jejunum at

median response concentrations 0.06 µM [CI(95%) of 0.05-0.07 µM; n=5] and 0.06 µM

[CI(95%) of 0.05-0.07 µM; n=5], respectively, however, it was less potent in relaxing

rhythmic periodic contractions in smooth muscle preparations of tissue preparations of rabbit

isolated jejunum with median response concentration 0.17 µM [CI(95%) of 0.15-0.19 µM;

n=5] (Fig. 6.6). Furthermore, smooth muscle preparations of isolated rabbit jejunum

subsequent to pretreatment by Cr.Si resulted in shifting of response versus concentration

curves (CRCs) for calcium rightward in non-parallel manner and decreasing maximally

achieved responses in significant (p < 0.05) manner (Fig. 6.7). Similarly, verapamil caused

shifting of CRCs for calcium in non-parallel fashion with mitigation of maximally achievable

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response in significant (p < 0.05) manner in smooth muscle preparations of rabbits isolated

jejunum (Fig. 6.8).

The Cr.Si demonstrated relaxation of carbachol (1 µM) mediated contractile response in

isolated preparations of rabbits jejunum (Fig. 6.9) with median response concentration of

0.67 mg per ml [CI(95%) of 0.57-0,75 mg per ml; n=5], which was attenuated on

pretreatment with propranolol (1 µM) resulting in enhancement in median response

concentration to 1.61 mg per ml [CI (95%) of 1.38-1.87 mg per ml; n=5] (Fig. 6.10).

Verapamil also demonstrated relaxation of carbachol (1 µM) mediated contractile response in

smooth muscle preparations of rabbit isolated jejunum at median response concentration to

0.42 µM [CI (95%) of 0.33-0.54 µM; n=5], which remained un-affected subsequent to

pretreatment with propranolol (Fig. 6.11)

Ethyl acetate fraction of Cr.Si (Ea.Si), of fashion similar with verapamil, demonstrated

relaxing of potassium (80 mM) mediated contractile response more potently than rhythmic

periodic contractions in smooth muscle preparations of rabbits isolated jejunum (Fig. 6.12 &

6.13) at median response concentrations of 0.04 mg per ml [CI(95%) of 0.03–0.05 mg per ml;

n=5] and 0.30 mg per ml [CI(95%) of 0.27–0.35 mg per ml; n=5], respectively (Fig. 6.14).

The Aq.Si (aqueous fraction of Cr.Si) exerted relaxant effect on rhythmic periodic

contractility more potently than on potassium (80 mM) mediated contraction in smooth

muscle preparations of rabbit isolated jejunum (Fig. 6.15 & 6.16) at respective median

response concentrations of 1.14 mg per ml [CI(95%) of 0.87–01.49 mg per ml; n=5] and 6.24

mg per ml [CI(95%) of 4.41-8.82 mg per ml; n=5] (Fig. 6.17).

Fig. 6.2: Record of influence of cumulatively added Cr.Si upon rhythmic periodic contractile movements in smooth muscle preparation of isolated rabbit jejunum.

137

Fig. 6.3: The Cr.Si influence upon cumulative addition to potassium (80 mM) mediated contractility in smooth muscle preparation of rabbit isolated jejunum.

Fig. 6.4: The Cr.Si influence upon cumulative addition to potassium (25 mM) mediated contractility of isolated preparation of rabbit jejunum.

Fig. 6.5: Influence of Cr.Si upon spontaneous contractility, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variables are mean ± S.E.M. (n=6).

138

Fig. 6.6: Influence of verapamil upon spontaneous rhythmic periodic contractions, potassium (25 mM) and potassium (80 mM) mediated contractile responses in smooth muscle preparations of rabbit isolated jejunum. Variable were mean ± S.E.M. (n=5).

Fig. 6.7: Influence of Cr.Si upon CRCs for calcium in smooth muscle preparations of rabbit isolated jejunum. Variables were mean ± S.E.M. (n=5). *p < 0.05 compared to control maximum.

139

Fig. 6.8: Verapamil influence upon CRCs for calcium in smooth muscle isolated preparations of rabbit jejunum. Variables were mean ± S.E.M. (n=5). *p < 0.05 compared to control maximum.

Fig. 6.9: The influence of Cr.Si upon carbachol (1 µM) mediated contractility of isolated rabbit jejunum preparation.

140

Fig. 6.10: Influence of Cr.Si upon carbachol (1 µM) mediated contractility of isolated preparations of rabbit jejunum, with and without propranolol (1 µM) pretreatment. Variables were mean ± S.E.M. (n=5).

Fig. 6.11: Influence of verapamil upon carbachol (1 µM) mediated contractility of isolated preparations of rabbit jejunum, with and without propranolol (1 µM) pretreatment. Variables were mean ± S.E.M. (n=5).

141

Fig. 6.12: The Ea.Si influence upon rhythmic periodic contractility of isolated preparation of rabbit jejunum.

Fig. 6.13: The Ea.Si influence upon potassium (80 mM) mediated contractility of isolated preparation of rabbit jejunum.

Fig. 6.14: Influence of Ea.Si upon spontaneous rhythmic periodical contractility and potassium (80 mM) mediated contractile response in smooth muscle preparations of rabbit isolated jejunum. Variables were mean ± S.E.M. (n=5).

142

Fig. 6.15: The Aq.Si influence upon rhythmic periodic contractility of isolated preparation of rabbit jejunum.

Fig. 6.16: The Aq.Si influence upon potassium (80 mM) mediated contractility of rabbit isolated jejunum preparation.

Fig. 6.17: Influence of Aq.Si upon spontaneous rhythmic periodical contractility and potassium (80 mM) mediated contractility of smooth muscle preparations of rabbit isolated jejunum. Variables were mean ± S.E.M. (n=5).

143

6.3.2.2. Influence upon tissue preparations of rabbit isolated trachea

The Cr.Si was without any relaxant or constrictor effect on the baseline tension of tissue

preparations of rabbit isolated trachea at T.O.B.C. of 0.01-10.0 mg per ml (data not shown).

Whereas, it exerted relaxant effect upon potassium (80 mM) as well as carbachol (1 μM)

mediated contractile responses at T.O.B.C. of 0.01-5.0 mg per ml at median response

concentrations of 0.86 mg per ml [CI(95%) of 0.75-0.98 mg per ml; n=6] and 0.82 mg per ml

[CI(95%) of 0.74-0.91 mg per ml; n=6], respectively (Fig. 6.18, 6.19 & 6.20). In presence of

propranolol (1 μM), the relaxing effect of Cr.Si upon carbachol (1 μM) mediated contractile

response was decreased at median response concentration of 4.47 mg per ml [CI(95%) of

4.14-4.83 mg per ml; n=5] (Fig. 6.20).

Verapamil was found more potent in relaxing potassium (80 mM) mediated contractile

response in comparison with carbachol (1 µM) mediated contractile response in tissue

preparation of rabbit isolated trachea at median response concentrations of 0.02 µM [CI(95%)

of 0.01-0.02 µM; n=5] and 0.40 µM [CI(95%) of 0.33-0.49 µM; n=5], respectively. However,

pretreatment with propranolol (1 μM) did not affect verapamil exerted relaxant activity upon

carbachol (1 µM) mediated contractile response in rabbit isolated tracheal preparations (Fig.

6.21).

Isoprenaline, a β adrenergic agonist drug, was found to relax carbachol (1 μM) mediated

contractile response in tissue preparations of rabbit isolated trachea at median response

concentration of 1.17 µM [CI(95%) of 0.99-1.39 µM; n=5], however, propranolol (1 μM)

pretreatment of tissue preparation of rabbit isolated trachea mitigated the isoprenaline exerted

relaxing response with median response concentration of 51.24 µM [CI(95%) of 41.57-

63.17µM; n=5] (Fig. 6.22).

The possibility of competitive muscarinic antagonist activity was explored, while Cr.Si was

applied at T.O.B.C. of 1 and 3 mg per ml to CRCs for carbachol on tissue preparations of

rabbit isolated trachea and shifted it rightward with suppression of the maximally achievable

response; hence, indicative involvement of non-competitive mechanism in relaxation of

carbachol (1 µM) mediated contractile response in tissue preparations of rabbit isolated

trachea (Fig. 6.23). Whereas, pretreatment with propranolol (1 μM) resulted in blunting of the

Cr.Si mediated rightward shifting in CRCs for carbachol in isolated preparations of rabbit

trachea (Fig. 6.24).

144

The Ea.Si relaxed carbachol (1 μM) and potassium (80 mM) mediated contractile responses

in isolated preparations of rabbit trachea at median response concentrations of 2.01 mg per ml

[CI(95%) of 1.51-2.66 mg per ml; n=5] and 0.3 mg per ml [CI(95%) of 0.28-0.33 mg per ml;

n=5], respectively (Fig. 6.25, 6.26 & 6.27). The potassium (80 mM) mediated contractions in

smooth muscle preparations of rabbit isolated trachea were relaxed at much lower value of

median response concentration in comparison with carbachol (1 µM) mediated contractile

response in a pattern comparable with verapamil; hence, partitioning of calcium channel

blocking activity to non-polar organic solvent was confirmed.

The Aq.Si exerted relaxant effect upon carbachol (1 μM) mediated contractile response upon

tissue preparations of rabbit isolated trachea at median response concentration of 2.76 mg per

ml [CI(95%) of 2.45-3.11; n=5]; however, demonstrated only partial relaxing of potassium

(80 mM) mediated contractile response in tissue preparations of rabbit isolated trachea at

median response concentration of 8.87 mg per ml [CI(95%) of 8.29-9.49; n=5] (Fig. 6.28,

6.29 & 6.30). As Aq.Si relaxed carbachol (1 µM) induced contractions at much lower values

of median response concentrations in comparison with potassium (80 mM) mediated

contractile response; hence, muscarinic antagonist or β-adrenoceptor agonistic activity may

be speculated.

Fig. 6.18: Influence of Cr.Si upon potassium (80 mM) mediated contractile response in tissue preparation of rabbit isolated trachea.

Fig. 6.19: Influence of Cr.Si upon carbachol (1 µM) mediated contractile response in tissue preparation of rabbit isolated trachea.

145

Fig. 6.20: Influence of Cr.Si upon potassium (80 mM) and carbachol (1 µM) mediated contractility of tissue preparations of rabbit isolated trachea. Presence of propranolol (1 µM) attenuated relaxant effect of Cr.Si upon carbachol (1 µM) mediated contractions. Variable were mean ± S.E.M. (n=6).

Fig. 6.21: Influence of verapamil upon potassium (80 mM) and carbachol (1 µM) mediated contractility of tissue preparations rabbit isolated trachea. Presence of propranolol (1 µM) did not alter relaxant effect of verapamil upon carbachol mediated contractility. Variable were mean ± S.E.M. (n=5).

146

Fig. 6.22: Influence of isoprenaline upon carbachol (1 µM) mediated contractility of tissue preparations of rabbit isolated trachea, with and without propranolol (1 µM). Variable were mean ± S.E.M. (n=5).

Fig. 6.23: Influence of Cr.Si upon CRCs for carbachol in tissue preparations of rabbit isolated trachea. Values were mean ± S.E.M. (n=5).

147

Fig. 6.24: Influence of propranolol (Prop) upon Cr.Si mediated shift of CRCs for carbachol in rabbit isolated tracheal preparations. Values are mean ± S.E.M. (n=5).

Fig. 6.25: The Ea.Si demonstrated relaxant effect upon potassium (80 mM) mediated contractility of isolated tracheal preparation of rabbit.

Fig. 6.26: The Ea.Si demonstrated relaxant effect upon carbachol (1 µM) mediated contractility of isolated tracheal preparation of rabbit.

148

Fig. 6.27: Effect of Ea.Si on potassium (80 mM) and carbachol (1 μM) mediated contractility of smooth muscle preparations of rabbit isolated trachea. Variable are mean ± S.E.M. (n=5).

Fig. 6.28: Influence of Aq.Si upon carbachol (1 µM) mediated contractile response in rabbit isolated tracheal preparation.

Fig. 6.29: Influence of Aq.Si upon potassium (80 mM) mediated contractility of rabbit isolated tracheal preparation.

149

Fig. 6.30: Influence of Aq.Si upon potassium (80 mM) and carbachol (1 μM) mediated contractions in smooth muscle preparations of rabbit isolated trachea. Variables are mean ± S.E.M. (n=5).

6.3.2.3. Influence upon tissue preparations of rabbit isolated aorta

The Cr.Si exerted contractile response upon tissue preparations of rabbit isolated denuded

aorta at T.O.B.C. range of 0.03-0.3 mg per ml (Fig. 6.31) at median response concentration of

0.08 mg per ml [CI(95%) of 0.06-0.11; n=5] at a comparable efficacy of phenylephrine (1

µM). The observed contractile response on the part of Cr.Si was of reversible nature as tissue

regained baseline tension on repeated washings. Pretreatment of tissue preparations of rabbit

isolate denuded aorta with doxazosin resulted in rightward parallel shifting of CRCs for Cr.Si

(Fig. 6.32). Phenylephrine is one of α-agonistic drugs, demonstrating contractile response on

tissue preparations of rabbit isolated denuded aorta; and pretreatment of tissue preparation

with doxazosin (α1-antagonistic drugs) caused parallel rightward shifting of CRCs for

phenylephrine (Fig. 6.33).

Moreover, Cr.Si on application to potassium (80 mM) mediated contractile response in tissue

preparations of rabbit isolated denuded aorta demonstrated relaxant effect at median response

concentration of 0.61 mg per ml [CI(95%) of 0.52-0.70 mg per ml ; n=5]. Whereas it caused

partial relaxation of phenylephrine (1 μM) mediated contractile response at higher T.O.B.C.

3.0-10.0 mg per ml (Fig. 6.34, 6.35 & 6.36).

150

The Ea.Si was unable to exhibit contractile response on application to the tissue preparations

of rabbit isolated denuded aorta but was found competent to demonstrate relaxant response

upon potassium (80 mM) and phenylephrine (1 µM) mediated contractility at median

response concentrations of 0.18 mg per ml [CI(95%) of 0.16-0.19 mg per ml; n=5] and 1.63

mg per ml [CI(95%) of 1.53-1.74 mg per ml; n=5], respectively (Fig. 6.37, 6.38 & 6.39).

Verapamil, relaxed potassium (80 mM) mediated contractile response in potent fashion as

comparison with phenylephrine (1 µM) exerted contractile response at median response

concentrations of 0.28 µM [CI(95%) of 0.24–0.34 µM; n=5] and 1.01 µM [CI(95%) of 0.88-

1.14 µM; n=5], respectively (Fig. 6.40).

Furthermore, Aq.Si exhibited contractile response on tissue preparations of rabbit isolated

denuded aorta (Fig. 6.41) and also augmented potassium (80 mM) and phenylephrine (1 µM)

mediated contractile responses in tissue preparations of rabbit isolated denuded aorta

preparations to variable extent (data not shown).

6.3.2.4. Influence upon tissue preparations of rabbit isolated paired atria

The Cr.Si exerted positive inotropic and positive chronotropic effects upon cumulative

addition at T.O.B.C. range of 0.01-1.0 mg per ml to tissue preparation of rabbit isolated

paired atria. The pretreatment with propranolol (1 µM) of tissue preparation of rabbit isolated

paired atria demonstrated blunting of the positive inotropic and positive chronotropic effects

on the part of Cr.Si (Fig. 6.42, 6.43 & 6.44). However, at elevated T.O.B.C. the Cr.Si

demonstrated variable and inconsistent results having low reproducibility.

Fig. 6.31: Tracing showing contractile responses of phenylephrine (PE) as well as cumulative addition of Cr.Si upon tissue preparation of rabbit isolated denuded aorta.

151

Fig. 6.32: Influence of doxazosin upon CRCs for Cr.Si in tissue preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).

% o

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ol m

axim

um

Fig. 6.33: Influence of doxazosin upon CRCs for phenylephrine in tissue preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).

152

Fig. 6.34: Influence of Cr.Si upon potassium (80 mM) mediated contractility in tissue preparation of rabbit isolated denuded aorta.

Fig. 6.35: Influence of Cr.Si upon phenylephrine (PE) mediated contractility in tissue preparations of rabbit isolated denuded aorta.

Fig. 6.36: Influence of Cr.Si upon phenylephrine (1 µM) and potassium (80 mM) mediated contractions in tissue preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).

153

Fig. 6.37: Influence of Ea.Si upon potassium (80 mM) mediated contractile response in isolated denuded aortic preparations of rabbit.

Fig. 6.38: Influence of Ea.Si upon phenylephrine (1 µM) mediated contractile response in isolated denuded aortic preparations of rabbit.

Fig. 6.39: Influence of Ea.Si upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile responses in tissue preparations of rabbit isolated denuded aorta. Variables were mean ± S.E.M. (n=5).

154

0.01 0.1 1 10

0

20

40

60

80

100

PE (1 µM)

K+ (80 mM)

[Verapamil] µM

Fig. 6.40: Influence of verapamil upon potassium (80 mM) and phenylephrine (1 µM) mediated contractile response in tissue preparations of rabbit isolated endothelium denuded aorta. Variables were mean ± S.E.M. (n=5).

Fig. 6.41: Influence of Aq.Si upon baseline tension in tissue preparation of rabbit isolated denuded aorta. Variables are mean ± S.E.M. (n=5).

155

Fig. 6.42: Tracings showing (A) effect of cumulatively added Cr.Si upon rhythmic contractility of rabbit isolated paired atria; (B) influence of pretreatment with propranolol (1 µM) on effect of cumulatively added Cr.Si upon rhythmic contractility of rabbit isolated paired atria.

Fig. 6.43: Influence of Cr.Si upon force of rhythmic periodic contractility of rabbit isolated paired atrial preparations, alone or with propranolol pretreatment. Variables were mean ± S.E.M. (n=5).

(A)

(B)

156

Fig. 6.44: Influence of Cr.Si upon rate of rhythmic periodic contractility of rabbit isolated paired atrial preparations, without and with propranolol pretreatment. Variables were mean ± S.E.M. (n=5).

6.3.3. In vivo experimentation

6.3.3.1. Influence upon charcoal meal intestinal transit distance in mice


± 0.85 cm. The Cr.Si administration to Treated 1 and 2 groups of animals (n=5) at respective

oral doses of 300 and 500 mg per kg resulted significantly (p < 0.05) decreased intestinal

transit distances of charcoal as compared to control and were assessed to be 26.80 ± 0.66 and

20.72 ± 0.62 cm, respectively. Moreover, loperamide treatment (10 mg per kg; orally) to

standard group of mice (n=5), the intestinal transit distance of charcoal was found to be 7.40

± 0.29 cm; which was significantly decreased (p < 0.05) as compared to control (Table 6.1).


The mean number of wet fecal masses in saline control group (n=6) of mice (administered

saline and olive oil orally at rate of 10 ml per kg) were found to be 0.33 ± 0.21; whereas, wet

fecal mass count in treated control group (n=6) of animal (administered saline and castor oil

orally at rate of 10 ml per kg) was counted to be 8.20 ± 0.31. The Treatment 1, 2 and 3

groups of mice (n=6 in each group), administered Cr.Si orally at respective doses of 300, 500

and 700 mg per kg followed by castor oil (10 ml per kg) produced significantly (p < 0.05 vs.

157

treated control group) decreased number of diarrheal wet feces, which were counted to be

3.83 ± 0.31, 1.50 ± 0.62 and 0.83 ± 0.31, respectively. The standard control group (n=6) of

mice (administered loperamide and castor oil orally at respective rate of 10 mg per kg and 10

ml per kg) significantly (p < 0.05) decreased mean wet fecal mass count was evident to be

0.50 ± 0.22. (Table 6.2).

Groups

Treatments

(given orally)

Charcoal movement

Mean ± S.E.M. (cm)


10 ml per kg + 0.2 ml 31.20 ± 0.85

Treated 1 Cr.Si + Charcoal meal

300 mg per kg + 0.2 ml 26.80 ± 0.66*

Treated 2 Cr.Si + Charcoal meal

500 mg per kg + 0.2 ml 20.72 ± 0.62*

Standard Loperamide + Charcoal meal

10 mg per kg + 0.2 ml

7.40 ± 0.29*

Table 6.1: Influence of crude extract of Salsola imbricata (Cr.Si) upon charcoal meal intestinal transit distances in mice. Values are mean ± S.E.M. (n=5); * indicates statistically significant difference (p < 0.05 vs. control group) as analyzed by ANOVA (one way) along with Dunnett test.

158

Group Treatments

Wet fecal mass count

Mean ± SEM

Protection (%age)

Saline Control

Saline + Olive oil

(10 ml per kg +10 ml per kg) 0.33 ± 0.21 -


(10 ml per kg + 10 ml per kg) 8.20 ± 0.31 -

Treatment 1 Cr.Si + Castor oil

(100 mg per kg + 10 ml per kg) 3.83 ± 0.31* 53.29


(300 mg per kg + 10 ml per kg) 1.50 ± 0.62* 81.70


(500 mg per kg + 10 ml per kg) 0.83 ± 0.31* 89.87

Standard Treatment Loperamide + Castor oil

(10 mg per kg + 10 ml per kg) 0.50 ± 0.22* 93.90

Table 6.2: Effects of crude extract of Salsola imbricata (Cr.Si) upon castor oil caused diarrhea in mice. Values are mean ± S.E.M. (n=6); * indicates statistically significant difference (p < 0.05) vs. treated control group, ANOVA (one way) along with Dunnett’s test.

159

6.3.3.3. Influence upon blood pressure of anesthetized rats

Saline (vehicle) administered intravenously to normotensive anesthetized rats did not cause

significant changes in SBP (systolic blood pressure), DBP (diastolic blood pressure), MBP

(mean blood pressure) and HR (heart rate) from respective baseline values; percent changes

in respective values were recorded to be 0.91 ± 0.47, 1.57 ± 0.98, 0.51 ± 0.31 and 0.43 ±

0.05. The Cr.Si administered intravenously to normotensive anesthetized rats at a dose of 1.0

mg per kg demonstrated statistically significant (p < 0.05) increased SBP, DBP, MBP and

HR as compared to saline treatment; percent increase in respective values were recorded to be

35.00 ± 2.48 , 42.80 ± 1.86, 27.50 ± 4.37 and 13.85 ± 0.13. Moreover, Cr.Si administered

intravenously to normotensive anesthetized rats at the dose of 3.0 mg per kg resulted in

significantly (p < 0.05) increased SBP, DBP, MBP and HR as compared to respective values

in vehicle treated group and values (% increase) were recorded to be 61.00 ± 1.35, 55.63 ±

1.03, 48.93 ± 3.97 and 15.18 ± 0.23, respectively. Furthermore, intravenous administration of

Cr.Si (10 mg per kg) also demonstrated significantly (p < 0.05) increased SBP, DBP, MBP

and HR as compared to respective values achieved following saline treatment, and percent

increase in values recorded were 82.20 ± 2.28, 65.43 ± 2.02, 69.23 ± 3.62 and 18.83 ± 0.48,

respectively. Similarly intravenous administration of Cr.Si (30 mg per kg) to normotensive

anaesthetized rats (n=5) demonstrated significantly (p < 0.05) increased SBP, DBP, MBP and

HR, and percent increase in values recorded were 97.75 ± 1.25, 82.64 ± 2.06, 85.25 ± 1.32

and 20.83 ± 0.37, respectively. The intravenous administration of norepinephrine (1 μg per

kg) to normotensive anaesthetized rats (n=5) demonstrated significantly (p < 0.05) increased

SBP, DBP, MBP and HR as compared to respective values achieved following saline

treatment, and percent increase in the values were 48.65 ± 3.14, 46.33 ± 3.70, 50.30 ± 3.09

and 13.20 ± 0.27, respectively (Fig. 6.45(A); Table 6.3).

However, intravenous pretreatment with doxazosin (1 mg per kg) as well as doxazosin (1 mg

per kg) plus propranolol (1 mg per kg) resulted in mitigation of norepinephrine and Cr.Si

exerted hypertensive effects in normotensive anesthetized rats (Fig. 6.45 & 6.46).

160

(A)

(B)

(C)

Fig. 6.45: Tracings showing effects of intravenous administration of norepinephrine (NE) and crude extract of Salsola imbricata (Cr.Si) upon blood pressure of (A) normotensive anaesthetized rat without any pretreatment, (B) normotensive anaesthetized rat with doxazosin (1 mg per kg) pretreatment and (C) normotensive anaesthetized rat with doxazosin (1 mg per kg) and propranolol (1 mg per kg) pretreatments.

161

Treatments % Change

SBP DBP MBP HR

Normal Saline 0.91 ± 0.47 1.57 ± 0.98 0.51 ± 0.31 0.43 ± 0.05

Cr.Si (1 mg per kg) 35.00 ± 2.48* 42.80 ± 1.86* 27.50 ± 4.37* 13.85 ± 0.13*

Cr.Si (3 mg per kg) 61.00 ± 1.35* 55.63 ± 1.03* 48.93 ± 3.97* 15.18 ± 0.23*

Cr.Si (10 mg per kg) 82.20 ± 2.28* 65.43 ± 2.02* 69.23 ± 3.62* 18.83 ± 0.48*

Cr.Si (30 mg per kg) 97.75 ± 1.25* 82.64 ± 2.06* 85.25 ± 1.32* 20.83 ± 0.37*

NE (1 µg per kg) 48.65 ± 3.14* 46.33 ± 3.70* 50.30 ± 3.09* 13.20 ± 0.27*

Table 6.3: Effects of intravenously administered normal saline, crude extract of Salsola imbricata (Cr.Si) and norepinephrine (NE) upon systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR) of normotensive anesthetized rats. Variables were mean ± S.E.M. (n=5). Statistics: ANOVA (two way) along with Dunnett test; * indicates statistically significant difference (p < 0.05) from values after saline injections.

NE (1

g/

kg)

Cr.S

i (1

mg/

kg)

Cr.S

i (3

mg/

kg)

Cr.S

i (10

mg/

kg)

Cr.S

i (30

mg/

kg)

% i

ncr

ease

in

MB

P

Fig. 6.46: Presser responses of norepinephrine (NE) and crude extract of Salsola imbricata (Cr.Si) in normotensive anesthetized rats, with and without doxazosin (1 mg per kg) as well as doxazosin (1 mg per kg) + propranolol (1 mg per kg) pretreatments. Variables were mean ± S.E.M. (n=5). * indicates significant difference (p < 0.05) in comparison with group not pre-treated with any antagonist. # indicates significant difference (p < 0.05) in comparison with group pre-treated with doxazosin (1 mg per kg).

162




(Na+, K+ & Cl-) in urine on µmol per 100 g body weight in 6 hrs. The urine volume in group



hrs, respectively. The rats of group treated by Cr.Si (100 mg per kg) demonstrated

insignificantly (p > 0.05) enhanced voided urinary volume to 1.13 ± 0.06 ml per 100 g in 6

hrs as well as insignificant (p > 0.05) increase in respective Na+, K+ and Cl- to 144.70 ± 7.54,

9.08 ± 0.26 and 142.50 ± 3.40 µmol per 100 g in 6 hrs versus saline control. The rats of group

treated by Cr.Si (300 mg per kg) demonstrated significantly (p < 0.05) enhance voided

urinary volume to 1.39 ± 0.04 ml per 100 g in 6 hrs as well as significantly (p < 0.05)

enhanced respective Na+, K+ and Cl- contents to 146.00 ± 5.65, 9.27 ± 0.28 and 157.00 ± 4.24

µmol per 100 g in 6 hrs versus control. The rats of group treated by Cr.Si (500 mg per kg)

demonstrated significantly (p < 0.05) enhanced voided urinary volume, i.e., 1.89 ± 0.12 ml

per 100 g in 6 hrs as well as significantly (p < 0.05) enhanced respective Na+, K+ and Cl-

contents to 207.70 ± 8.32, 14.30 ± 0.36 and 182.70 ± 5.40 µmol per 100 g in 6 hrs versus

control. The rats of group treated by furosemide (20 mg per kg) demonstrated significantly (p

< 0.05) enhanced voided urinary volume to 4.10 ± 0.11 ml per 100 g in 6 hrs and

significantly (p < 0.05) enhanced respective Na+, K+ and Cl- contents to 379.17 ± 5.64,

31.42± 0.58 and 516.50 ± 9.39 µmol per 100 g in 6 hrs versus control. Derived parameters

including diuretic index, Na+/K+ ratio and ion quotient were also calculated. The diuretic

indices following administration of Cr.Si (100, 300 and 500 mg per kg) were calculated as

1.11, 1.36 and 1.85 respectively, whereas, diuretic index of the standard drug furosemide was

found to be 4.02. The Na+/K+ ratio in saline treated group was 15.63 ± 0.80; oral

administration of Cr.Si at 100, 300 and 500 mg per kg demonstrated insignificant (p > 0.05)

change in Na+/K+ ratios to 15.98 ± 0.89, 15.89 ± 1.02 and 14.64 ± 0.96, respectively.

Whereas, oral administration of furosemide resulted significantly (p < 0.05) decreased

urinary Na+/K+ ratio to 12.10 ± 0.36 versus control (Table 6.4).

163

Treatment group

(dose)

Urinary volume

(ml/100 g/6 hrs)

Diuretic index

Na+ K+ Cl- Na+/K+ Ion quotient

(µmol/ 100 g/ 6 hrs)

Saline

(10 ml per kg) 1.02 ± 0.06 - 121.00 ± 4.05 7.78 ± 0.20 127.50 ± 2.20 15.63 ± 0.80 0.99 ± 0.03

Cr.Si

(100 mg per kg) 1.13 ± 0.06ns 1.11 144.70 ± 7.54ns 9.08 ± 0.26ns 142.50 ± 3.40ns 15.98 ± 0.89ns 0.94 ± 0.06

Cr.Si

(300 mg per kg) 1.39 ± 0.04* 1.36 167.70 ± 9.99* 10.77 ± 0.62* 163.00 ± 6.84* 15.93 ± 1.46ns 1.00 ± 0.08

Cr.Si

(500 mg per kg) 1.89 ± 0.12* 1.85 220.00 ± 12.67* 15.17 ± 0.97* 198.70 ± 9.37* 14.89 ± 1.47ns 0.91 ± 0.07

Furosemide

(20 mg per kg) 4.10 ± 0.11* 4.02 379.17 ± 5.64* 31.42 ± 0.58 * 516.50 ± 9.39* 12.10 ± 0.36* 1.25 ± 0.05

Table 6.4: Effects of crude extract of Salsola imbricata (Cr.Si) and furosemide on urinary volume and urinary electrolytes contents in albino rats. Diuretic index = Urinary volume in treated groups/ Urinary volume in control group; Ion quotient = Cl-/ (Na+ + K+). The variables were mean ± S.E.M. (n=6); nsp ≥ 0.05, *p < 0.05 in comparison with values of control group.

164

6.4. Discussion

The Salsola imbricata has been known to possess folkloric repute to be effective to treat for

ailments involving digestive tract, respiratory tract, heart and circulatory systems (Hammiche

and Maiza, 2006; Handa et al., 2006; Ahmed et al., 2014c); the current scientific

investigations on the plant were aimed for scientific authentication of some traditional uses.

The alkaloids, flavonoids, phenols, tannins, saponins and steroids are among the detected

constituents out of crude extract of Salsola imbricata (Cr.Si). The Cr.Si and its factions (i.e.,

Ea.Si and Aq.Si) demonstrated relaxation of rhythmic periodic contractility in rabbit isolated

jejunum preparations, thus demonstrated antispasmodic activity. Since, rhythmic periodic

contractility of jejunum is based upon sequential depolarization and repolarization among

smooth muscle cells (Huizinga et al., 2000); which mediates contractile phenomenon vide

opened channels for calcium during the depolarization phase resulting in increased cytosolic

free calcium (Bolton, 1979). The free calcium subsequent to binding with calmodulin resulted

in activation of kinase for phosphorylation of myosin light chain, resulting finally to

contractions of contractile proteins. Increased intracellular free calcium may be outcome of

either influx of calcium through ligand /voltage gated channels for calcium or calcium

released out of sarcoplasmic reticulum (Karaki et al., 1997). The antispasmodic activities out

of plant sources have been known to be convened either following blocking of channels for

calcium or opening channels for potassium (Gilani et al., 2006; Janbaz et al., 2013). The

possible involvement of calcium channel blocking activity in observed antispasmodic activity

was assessed on application of Cr.Si to potassium (80 mM) mediated contractile activity in

rabbit isolated jejunum preparations. Whereas, potassium channels opening potential was

assessed upon testing upon potassium (25 mM) mediated contraction in rabbit isolated

jejunum preparations (Gilani et al., 2006). The Cr.Si exhibited calcium channel blocking

potential through demonstration of relaxant activity upon potassium (80 mM) and potassium

(25 mM) mediated contractile responses in equipotent fashion comparable to verapamil, a

standard calcium channel blocking drug (Katzung and Chatterjee, 2009). The confirmation of

the proposed mechanism, i.e., blockade of channels for calcium by Cr.Si was achieved as

pretreatment with Cr.Si demonstrated shifting of CRCs for calcium toward right as non-

parallel fashion, causing mitigation of maximally achievable response in isolated preparation

of rabbit jejunum (Daniel et al., 2001).

165

The relaxant potential of Cr.Si was also assessed on carbachol exerted contractile response in

rabbit isolated jejunum preparations (Chaudhary et al., 2012). Since, carbachol is an agonist

for muscarinic receptor, capable to generate persistent contractile response in preparations of

rabbit isolated jejunum subsequent to increased intracellular calcium concentration either

influxed via voltage dependent calcium channels as well as released out of cellular stores for

calcium (Grasa et al., 2004). The Cr.Si relaxed carbachol (1 μM) and potassium (80 mM)

exerted contractile responses in rabbit isolated jejunum preparations at comparable potencies.

The blockers for calcium channel are known to be competent for demonstration of relaxant

response upon potassium (80 mM) as well as carbachol (1 μM) mediated contractions in

rabbit isolated jejunum preparations (Gilani et al., 2008; Mushtaq et al., 2015); but pure anti-

muscarinic agents are capable only to relax carbachol (1 μM) mediated contractile response

and not effective in exerting relaxant response upon potassium (80 mM) mediated

contractility of rabbit isolated jejunum preparations (Gilani et al., 2008; Akhlaq et al., 2016).

It is also known that adrenergic receptors stimulation can cause inhibition of rabbit jejunum

(Bowman and Hall, 1970). The likely involvement of β-adrenergic activity in jejunum

relaxant activity on the part of Cr.Si was assessed on carbachol (1 μM) mediated contractility

in rabbit jejunum preparations, alone and in presence of propranolol (Chaudhary et al., 2012).

The pretreatment with propranolol (1 μM) of rabbit isolated jejunum preparations resulted in

mitigation of Cr.Si exhibited relaxant activity upon carbachol (1 μM) mediated contractile

response, indicating possible participation of β-agonistic activity in relaxant activity of the

extract.

The gut relaxant activity of Cr.Si was confirmed further through in vivo studies; as oral

administration of Cr.Si (300 and 500 mg per kg) caused decrease in charcoal transit distance

in small intestine of mice. The Cr.Si also caused inhibition of castor oil caused diarrhea in

mice; hence, likely to be attributed to the suppressant effect on gut contractility due to its

antispasmodic, β-agonistic and calcium channel blocking activities. Thus, decrease in

charcoal meal transit distance and protection from castor oil exerted diarrhea by Cr.Si

authenticates scientifically the folkloric use of Salsola imbricata as anti-diarrheal remedy.

Since, S. imbricata has been known to be used to alleviate asthma, cough and chest

congestion (Ahmad et al., 2014; Handa et al., 2006); hence, bronchodilator potential of Cr.Si,

Ea.Si and Aq.Si was assessed upon carbachol (1 μM) and potassium (80 mM) mediated

contractility of isolated preparations of rabbit trachea (Chaudhary et al., 2012). The Cr.Si

relaxed potassium (80 mM) and carbachol (1 μM) mediated contractile responses at

166

comparable median response concentrations, whereas, verapamil as a pure calcium channel

blockers was more potent against potassium (80 mM) exerted contractile response (Gilani

et al. 2008). Anti-muscarinic mechanism was speculated be involved in manifestation of

bronchodilator activity on the part of Cr.Si in addition to calcium channel blocking activity.

The proposed anti-muscarinic potential was tried to be confirmed further on application of

Cr.Si to CRCs for carbachol; which caused shifting toward right in non-parallel way with

mitigation of maximally achievable response, suggestive of involvement of some non-

muscarinic activity (Kenakin, 2009). It is also known that bronchial smooth muscle contains

β2-receptors, whose stimulation cause bronchodilation (Bristow et al., 1970; Gan et al.,

2003). The possible involvement of β2-agonistic activity in relaxation of carbachol (1 μM)

mediated contractile activity on the part of Cr.Si was assessed on rabbit isolated tracheal

preparation. The pretreatment with propranolol (1 μM) of carbachol (1 μM) mediated

contractile response in rabbit isolated tracheal preparations resulted in mitigation of Cr.Si

exhibited relaxant activity and confirmation of which was achieved as pretreatment with

propranolol (1 μM) of isolated preparations of rabbit trachea demonstrated blunting of the

Cr.Si mediated right ward shifting in CRCs for carbachol (Kenakin, 2009; Janbaz et al.,

2011). The β-adrenoceptors coupled with G-proteins being linked to adenylyl cyclase; the

activation of which resulted in increased intracellular level of cAMP, leading to inhibition of

kinases for myosin light chain as well as blockade of channels for calcium leading ultimately

to smooth muscle relaxation (Wikberg, 1977). The β2 adrenoceptor agonistic drugs as

therapeutic modality are useful in the management of asthma and COPD (Nelson, 1995).

The Ea.Si exhibited potent relaxant effect upon potassium (80 mM) and carbachol (1 μM)

mediated contractile responses in rabbit isolated jejunum as well as rabbit isolated tracheal

preparations, reflecting partitioning of calcium channel blocking constituents in non-polar

organic fractions, as reported previously (Gilani et al., 2008). The existence of blockers for

calcium channel along with β2-adrenoceptor agonistic activities would have synergistic

impact upon relaxation of spasmodic ailments pertaining to respiratory system (Gilani, 2005).

The Cr.Si was also subjected to evaluations regarding management of cardiovascular

ailments by using in vitro as well as in vivo animal models. The Cr.Si demonstrated

contractility upon the base line of rabbit isolated denuded aorta, and exhibited relaxant effect

upon potassium (80 mM) mediated contractile response. The Cr.Si mediated contractile

response upon rabbit isolated denuded aorta was attenuated following pretreatment of the

tissue preparation with doxazosin, α1-adrenoceptor antagonistic drug (Hamilton et al., 1985).

167

The α1-adrenoceptor agonistic activity of Cr.Si was confirmed further as doxazosin

demonstrated parallel rightward shifting of CRCs for Cr.Si and phenylephrine in aortic

preparations. Moreover, Cr.Si demonstrated positive inotropic and positive chronotropic

effects; which were found to be blocked subsequent to pretreatment with propranolol, a non-

selective β-adrenoceptor antagonist (Nies and Shand, 1975). Hence, it was speculated that

positive inotropic and positive chronotropic effects of Cr.Si upon heart would be mediated

through β1-adrenoceptors activation; as β1 adrenoceptors are known to be located in rabbit

atria (Costin et al., 1983; Wilson and Lincoln, 1984). The Cr.Si administered intravenously to

normotensive anesthetized rats exerted presser response in terms of enhancements in SBP,

DBP, MBP and HR. The Cr.Si mediated presser response in normotensive anaesthetized rats

was mitigated upon pretreatment of rats with doxazosin (1 mg per kg) plus propranolol (1 mg

per kg). Thus, confirming presence of constituents possessing α1 and β-adrenoceptors

agonistic activities in the extract. The α-agonistic drugs are known to be effective in cough,

cold and congestion (Fitzpatrick and Rasheed, 2017) through activation of α1-adrenoceptors

located on pre-capillary as well as post-capillary blood vessels of the nasal mucosa, resulting

in vasoconstriction, local tissue shrinkage and air passage opening (Johnson and Hricik,

1993). Presence of α1-agonist activity in Cr.Si provides scientific justification for reported

usefulness of the plant to relive sinus congestion (Handa et al., 2006). The Salsola imbricata

has been used internally for treatment of hypertension in some countries, but, contrary to

previous considerations, it has been noted to possess vasoconstrictor, cardiotonic and

hypertensive effects in animal models. The controversy may partially be resolved on the fact

that for the management of hypertension, the plant extract has been administered orally;

while in present study, it was administered intravenously. There may be variation among

plant secondary metabolites depending upon, altitude, nature of soil, availability of water,

climatic conditions, season of collections and variation among different varieties of same

species (Steinberg, 1989; Metlen et al., 2009; Moore et al., 2014). The hypertensive and

vaso-constrictive activities were found to be portioned into aqueous fraction; indicating its

polar nature and due to polar nature, hypertensive ingredient may not be able to get access to

systemic circulation or likely to be destroyed by gastrointestinal enzymes in a manner similar

to tyramine (Rice et al., 1976).

The Cr.Si was tested for possible diuretic activity in rats and caused increase in voided

urinary volume but observed diuretic activity was found less than that of furosemide.

168

6.5. Conclusion

It is concluded that Cr.Si possesses anti-spasmodic, anti-diarrheal, bronchodilator and

diuretic activities, possibly mediated through combination of calcium channel blocking and

adrenergic agonistic actions, thus authenticate scientifically some of the folkloric claims of

Salsola imbricata in traditional systems of medicine. The crude extract was found to possess

vasoconstrictor, cardio-stimulant and hypertensive activities mediated through adrenergic

agonistic mechanisms.

169

7. General discussion and conclusion

170

Four indigenous plants of Cholistan desert, namely Asphodelus tenuifolius, Corchorus

depressus, Gisekia pharnaceoides and Salsola imbricata, were chosen for pharmacological

investigations. The selection was based upon medicinal usage reputability of the plants in one

or several ailments pertaining to gastrointestinal, cardiovascular and respiratory systems.

Thorough internet literature search was undertaken to ensure that these plants have not been

reported for their mechanisms of purported ethnobotanical activities.

Extraction of plant material was carried out with aqueous-ethanol (Gilani et al., 2005).

Previous studies documented that combination of aqueous-ethanol as extracting solvent,

while being environmentally friendly and nontoxic, gives better yields resulting extraction of

large variety of bioactive phytochemicals (Bergeron et al., 2005; Cowan, 1999; Sultana et al.,

2009). Medicinal properties of plants depend upon unique secondary metabolite referred as

phytochemicals. More than 100,000 phytochemicals have been identified in plants.

Phytochemicals are hypothesized to be result of plant evolution as defense against herbivores,

against microorganisms and against competing plants. Some phytochemicals play role in

growth regulation, gene expression and signal transduction or protect plant against stresses

related with water derivation, starvation, extreme temperature changes and minerals excess

(Kaufman et al., 1999). Toxic effects of phytochemicals against animals depend upon

characteristics of individual compound, mode of action, dosage and exposure time; and may

be harmful or desirable depending upon ecological or pharmacological context (Matsuura and

Fett-Neto, 2015; Wink, 1988).

Calcium channel blocking and potassium channel opening activities of all the four plants

extracts were assessed in animal models. Voltage dependent calcium channel blocking agents

are useful in managing several ailments pertaining to cardiovascular, respiratory and gut

disorders (Eisenberg et al., 2004; Girodet et al., 2015; Lee et al., 1997). Like in animal cells,

free calcium is important single transducer that regulates vital cellular events in plants. In

plant cells, calcium is low in cytosol and sequestered in specific subcellular compartments

(mitochondria, endoplasmic reticulum and vacuoles), where several types of calcium specific

channels and pumps regulate calcium hemostasis in plants. Plant derived secondary

metabolites have regulatory role upon calcium channels and hence intracellular calcium

hemostasis (Maffei et al., 2007). Medicinally used calcium channel blockers, verapamil and

nifedipine, have been reported to act on plant calcium channels in many plant cells (White,

2000). Research on animal smooth muscle preparations revealed widespread distribution of

171

voltage dependent calcium channel blockers in plants extracts. Potassium channels are

present in all cells of animals and plants (Hedrich and Schroeder, 1989) and research revealed

some similarities between animals and plants potassium ion channels (Cherel, 2004).

Potassium channel opening agents are useful in management of cardiovascular diseases and

have potential to be used in several other diseases involving smooth muscle hyperactivity

(Andersson, 1992).

In vitro studies were performed on rabbit isolated jejunum, trachea, aorta and paired atria

preparations. Smoot muscle cells are basic components of gut, airway and blood vessels,

which own various ion channels and receptors responsible for regulation of normal tone.

Crude extracts of all the four selected plants exhibited spasmolytic activity upon spontaneous

rhythmic contractions of smooth muscle preparations of rabbit jejunum. To study

involvement of calcium channel blocking or potassium channel opening activities in

spasmolytic activity, the extracts were applied to rabbit jejunum preparations pre-contracted

with either potassium (25 mM) or potassium (80 mM) (Gilani et al., 2006). Depending upon

potencies (median response concentration values) of the extracts in inhibition of spontaneous

rhythmic contractions, potassium (25 mM) and potassium (80 mM) mediated contractions,

inferences were made for possible mechanisms of actions. Typical calcium channel blockers

(like verapamil) are reported to be equally effective in inhibition of potassium (25 mM) and

potassium (80 mM) mediated contractions in rabbit isolated jejunum preparations (Gilani et

al., 2006), but their potency to relax spontaneous contractions was found less than that for

potassium mediated contractions (Gilani et al., 2008; Jabeen et al., 2009). In present study,

Asphodelus tenuifolius extract was found equally potent in relaxing potassium (25 mM) and

potassium (80 mM) mediated contractions, and was relatively less potent in relaxing

spontaneous contractions of rabbit jejunum preparations, in a manner similar to verapamil.

Corchorus depressus and Salsola imbricata extracts were found equally potent in relaxing

potassium (25 mM) and potassium (80 mM) mediated contractions suggesting calcium

channel blocking activity in the extracts. However, unlike pure calcium channel blockers,

these extracts were found relatively more potent in inhibition of spontaneous contractions of

rabbit jejunum preparations. Therefore, possibility of involvement of additional

mechanism(s) was also explored in these extracts; anti-muscarinic activity was found in

Corchorus depressus extract as it relaxed carbachol (1 µM) mediated contractions of rabbit

isolated jejunum and tracheal preparations potently (Gilani et al., 2008); β-adrenergic

agonistic activity of the Salsola imbricata extract was indicated by ability of propranolol to

172

mitigate effects of the extract upon induced contractions in jejunum and tracheal tissues

preparations (Chaudhary et al., 2012) as well as upon spontaneous contractions of paired

atrial preparations. Concentration response curves for calcium were constructed upon

jejunum isolated preparations and confirmation of calcium antagonistic activity in extracts

was achieved following rightward displacement of concentration response curves for calcium

constructed in presence of the extracts (Gilani et al., 2008; Jabeen et al., 2009).

Potassium channel openers (like cromakalim) are known to relax potassium (25 mM)

mediated but not potassium (80 mM) mediated contractions (Gilani et al., 2006). Gisekia

pharnaceoides extract was found to exhibit potent activity in relaxing potassium (25 mM)

mediated contractions in compression to potassium (80 mM) mediated contractions of

smooth muscle preparations. Pretreatment of tissues with ATP dependent potassium channel

blocker (glibenclamide) partially antagonized relaxant activity of the extract against

potassium (25 mM) mediated contractions, thus postulating presence of ATP dependent

potassium channel opening activity in the extract. At higher tissue organ bath concentrations,

Gisekia pharnaceoides extract displaced concentration versus response curves for calcium,

constructed upon isolated jejunum preparations, rightward with suppression of maximal

response, which indicates co-existence of calcium channel blocking and potassium channel

opening activities in the same extract (Mehmood et al., 2015). Bronchorelaxant activates of

extracts were explored upon baseline, potassium and carbachol mediated sustained

contractions of rabbit tracheal preparations. Vascular activities were studied upon baseline

tension, potassium and phenylephrine mediated sustained contractions of rabbit isolated

endothelium denuded aorta preparations. Effects of crude extracts upon spontaneous

contractions of paired atria of rabbit were also investigated (Janbaz et al., 2011).

Studies on fractions revealed separation of spasmogenic constituents in aqueous fractions of

Asphodelus tenuifolius, Corchorus depressus and Gisekia pharnaceoides. Presence of smooth

muscle relaxant and contractile components in the same plant may be beneficial in offsetting

side effects associated with excessive stimulation or depression of bodily functions.

Keeping in view of the actions of extracts and fractions upon isolated smooth muscle

preparations and ethnobotanical reports, the crude extracts were studied in vivo for

confirmation of anti-diarrheal, laxative, cardiovascular and diuretic activities. In vivo studies

confirmed anti-diarrheal, hypotensive and diuretic activities of Asphodelus tenuifolius,

Corchorus depressus and Gisekia pharnaceoides extracts. Salsola imbricata was found to

173

possess hypertensive, anti-diarrheal and diuretic activities. In addition to anti-diarrheal

activity, the crude extracts of Asphodelus tenuifolius, Corchorus depressus and Gisekia

pharnaceoides also showed laxative activity in mice at doses different from those producing

anti-diarrheal effects.

In conclusion, in vitro and in vivo studies revealed that Asphodelus tenuifolius, Corchorus

depressus and Gisekia pharnaceoides possess gut modulatory, bronchorelaxant, hypotensive

and diuretic activities. While Salsola imbricata was found to possess gut relaxant,

bronchorelaxant, hypertensive and diuretic activities. The data mentioned in this dissertation

stipulates that spasmolytic, bronchorelaxant and cardiovascular effects of selected plants are

mediated through one or more mechanisms, such as calcium channel blocking mechanism in

Asphodelus tenuifolius; calcium channel blocking, muscarinic and anti-muscarinic

mechanisms in Corchorus depressus; ATP dependent potassium channel opener and weak

calcium entry blocking mechanisms in Gisekia pharnaceoides; and calcium channel blocking

and adrenergic receptors agonistic mechanisms in Salsola imbricata. The present research

furnishes authentication of some of the ethnic medicinal claims of the selected plants along

with mechanistic background. However, further research for recognition and separation of

pure phytochemicals responsible for each activity along with standardization of crude

extracts using phytochemicals techniques is also required.

174

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pharmacological investigations on some indigenous …

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