Ihsan Ullah - prr.hec.gov.pk

EVALUATION OF SOME SELECTED MEDICINAL PLANTS AND

THEIR COMBINATIONS IN CISPLATIN INDUCED VOMITING IN

VOMIT MODEL(S); BEHAVIORAL NEUROCHEMICAL

CORRELATES

PhD Thesis

By

Ihsan Ullah

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR

(2013)

EVALUATION OF SOME SELECTED MEDICINAL PLANTS AND

THEIR COMBINATIONS IN CISPLATIN INDUCED VOMITING IN

VOMIT MODEL(S); BEHAVIORAL NEUROCHEMICAL

CORRELATES

Ihsan Ullah

This thesis is submitted to the University of Peshawar in partial fulfillment

of the requirements for the Degree of

Doctor of Philosophy (PhD) in Pharmaceutical Sciences



(2013)

DEDICATION

To my parents for their love, help, support, and prayers that have always

been a generous source of motivation for me

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CERTIFICATE OF APPROVAL:

This thesis, entitled, “Evaluation of some selected medicinal plants and their combinations

in cisplatin induced vomiting in vomit model(s); behavioral neurochemical correlates”

submitted by Mr. IhsanUllah is hereby approved for submission as partial fulfillment for the

award of Degree of “Doctor of Philosophy” in Pharmaceutical Sciences (Pharmacology).

Prof. Dr. Fazal Subhan __________________________

Research Supervisor,

Department of Pharmacy,

University of Peshawar.

Prof. Dr. Zafar Iqbal __________________________

Chairman,

Department of Pharmacy,

University of Peshawar.

External Examiner _________________________



(2013)

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AKNOWLEDGMENTS:

This research project would not have been possible without the blessings of Allah

(Subhanahu Wa Taala), Who sanctified me with the enthusiasm and understanding to

effectively accomplish my PhD studies, which is indeed a milestone in my life. This is

definitely a tough task to acknowledge the contribution of several individuals by name.

However, I am thankful to all my teachers since my school days.

I would like to express my greatest gratitude to my research supervisor Prof. Dr. Fazal

Subhan for his patience, guidance, persistent help, encouragement and excellent advice

throughout my study, who continually and realistically conveyed a spirit of adventure in

regard to research and provided me with very useful insights.

I am thankful to the members of the graduate studies committee (GSC) and Advance studies

and research board (ASRB) for their help and co-operation during my studies. I heartily

acknowledge the course tutors at Department of Pharmacy, University of Peshawar for their

support and care during my course work.

I am indebted to Prof. John A. Rudd for kindly providing me the opportunity to work in his

laboratory at School of BioMedical Sciences, Faculty of Medicine, the Chinese University

of Hong Kong (CUHK). Without his knowledge, facilitation and cooperation this study

would not have been completed. I would like to express my sincere appreciation to Mr. Jack

Lu for his guidance and advice on histological analysis. I am thankful to Mr. Man Keung

Wai, Mr. Man Pui Ngan, Ms. Corinna Au for their generous technical assistance during my

stay at School of BioMedical Sciences, CUHK.

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I am extremely obliged to Prof. Ikhlas Ahmad Khan, the national center for natural product

research, Mississippi, USA for the gift of bacosides HPLC standards. I am grateful to all of

my lab fellows including Dr. Khalid Rauf, Mr. Ikram ul Haq, Mr. Muhammad Ayaz, Mr.

Muzaffar Abbass Mr. Gowhar Ali, Mr. SamiUllah, Ms. Iffat Shaheen, Mr. Javaid Alam, Mr.

Rehmat Shah, Mr. Muhammad Shahid, Ms. Shehla Akbar, Ms. Urooj Amaan at Department

of Pharmacy, University of Peshawar and friends at the School of Biomedical Science

(SBS), the Chinese University of Hong Kong (CUHK) for their encouragement, support and

unforgettable moments of my life and making my stay comfortable in the laboratory and

campus during my study. I am really thankful to Dr. Ubaid Ullah, Ministry of health

Pakistan for his help in acquisition of cisplatin from Korea United Pharm. Inc Korea and

many thanks to Korea United Pharm. Inc Korea for the gift of cisplatin.

Last but not the least, I am forever indebted to my beloved parents and elder brother

NoorUllah Advocate for his understanding and endless love, throughout the duration of my

study. Many thanks to Higher education commission (HEC) of Pakistan for granting

indigenous PhD scholarship to complete my studies.

Ihsan Ullah

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ABSTRACT:

Cancer Chemotherapy Induced Vomiting (CIV) is one of the distressing untoward effects

and a cause of non compliance and even refusal of treatment by the patients undergoing

curative chemotherapy. Anti-emetics are therefore considered integral component of the

anti-cancer therapeutic regimen. Numerous anti-emetics and their combinations are in

clinical practice but none of them is capable of providing complete remission of CIV. The

mechanistically multifactorial CIV, a challenge in clinics especially considering the delayed

phase of vomiting necessitates the search for cost effective broad spectrum anti-emetic

regimen for the management of CIV for prolong time periods (upto many days).

In this study, extracts of some selected plants indigenous to Pakistan, were investigated for

anti-emetic activity employing well known vomit models of pigeon and Suncus murinus (S.

murinus). Vomiting was induced by highly emetogenic chemotherapeutic agent cisplatin in

both models. Anti-emetic studies in pigeons were conducted at the bioassay laboratory of

the Department of Pharmacy, University of Peshawar, Pakistan, while studies in S. murinus

were carried out at the School of Biomedical Sciences, The Chinese University of Hong

Kong, utilizing their state-of-the-art facilities.

First of all, extracts of Cannabis sativa (CS; Hexane, n-butanol & methanol), Bacopa

monniera (BM; methanol, & n-butanol) and Zingiber officinale (ZO; acetone) were prepared.

Cannabis sativa and Zingiber officinale extracts were prepared by simple maceration

method while Bacopa monniera was extracted by the method already developed by our

laboratory.

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The emetic, cisplatin was initially screened in a series of experiments to quantify its

vomiting inducing potential; as a result the dose of 7 mg/kg was selected for pigeons, while

30 mg/kg dose was used in S. murinus. The behavior of the animals was observed using

computer assisted video recording setup for each vomit model up to the desired period of

time. The Retching plus Vomiting (R + V) episodes were then quantified from the video

recordings for cisplatin control, standard & treatment groups. To find out the possible role of

gastrointestinal (GIT) pro-kinetic properties on CIV in pigeon, we examined the impact of

prokinetic/cholinergic agonist agents, utilizing charcoal propulsion method.

High Performance Liquid Chromatography (HPLC) with UV detection was used for the

quantification of bacosides whereas HPLC coupled with Electrochemical Detector (ECD)

was employed for the measurement of neurotransmitters and their metabolites in specific

brain areas and intestine involved in the act of vomiting of pigeons. Moreover, C-fos protein

expression, a marker of neuronal excitation was also analyzed in hind brain areas; area

postrema, nucleus tractus solitarius, dorsal motor nucleus of vagus nerve, and in the

forebrain areas including dorsomedial and ventromedial nucleus of hypothalamus in the S.

murinus model.

CS hexane fraction (CS-HexFr) at the dose of 10 mg/kg was found to be effective in

attenuating (P < 0.01) cisplatin induced R + V and has been proved to be superior to

standard metoclopramide (30 mg/kg), while CS n-butanol and methanol fractions failed (P >

0.05) to do so in pigeons. BM fractions; methanol (BM-MetFr; 10, 20 & 40 mg) and n-

butanol (BM-ButFr; 5, 10 & 20 mg), attenuated cisplatin induced R + V, dose dependently

in both pigeons and S. murinus. The BM-ButFr was however, found to be more potent as

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compared to BM-MetFr in the vomit models of pigeon and S. murinus, as it reduced the

number of R + V with high significance (P < 0.001) in pigeon (24 hr of observation period)

and provided significant remission (P < 0.05) in the S. murinus for prolong time period (48

hr of observation period). In the pigeon model the anti-emetic effect of BM-MetFr and BM-

ButFr was found to be pronounced as compared to standard metoclopramide (30 mg/kg),

while in S. murinus the results proved to be analogous with the standard palonosetron (0.5

mg/kg). The strong suppression of cisplatin induced R + V by BM-ButFr may be attributed

to the presence of high concentration of bacosides as HPLC - UV analysis revealed

BM-ButFr to be rich in bacosides as compared to BM-MetFr, where the quantities of

bacosides found were 115.74 µg/mg & 29.99 µg/mg of extract, respectively. The acetone

fraction of Zingiber officinale (ZO-ActFr) also showed R + V reduction in pigeon model

where, the dose of 50 mg/kg was found to be statistically significant (P < 0.05) while the

R + V suppression achieved was found to be equivalent to standard metoclopramide (30

mg/kg).

In combination studies; CS, BM & ZO showed variable protection against cisplatin induced

R + V in pigeons. The combined treatment of CS-HexFr (10 mg) and BM-ButFr (5 mg)

showed ~ 88.63 % protection (P < 0.001), where the protection provided by CS-HexFr (10

mg) and BM-ButFr (5 mg) alone were ~ 55.45 % (P < 0.01) & 68.08 % (P < 0.001),

respectively. In S. murinus, Δ9-THC synthetic analogue, WIN 55, 212-2 (10 mg) in

combination with BM-ButFr (5 mg) also enhanced protection against vomiting ~ 71.01 %

(P > 0.05), where when tested alone the protection was found to be ~ 55.71 % & 57.97 % (P

> 0.05) for WIN 55, 212-2 (10 mg) and BM-ButFr (5 mg), respectively.

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CS-HexFr at its effective anti-emetic dose (10 mg) suppressed GIT motility ~ 26.62 % as

compared to saline. The prokinetic agent metoclopramide (30 mg/kg) and cholinergic

agonist carbachol (0.1 mg/kg) antagonized the suppression (P < 0.001) caused by CS-HexFr.

Further, the combination of CS-HexFr (10 mg) with MCP (30 mg) or CS-HexFr (10 mg)

with carbachol (0.1 mg) resulted in the enhancement of anti-emetic profile of CS-HexFr at

delayed time point (12 hr +), however these combinations failed to show any

synergism/potentiating at the acute time point (01 hr +).

The neural data for acute vomiting response (03 hr) by cisplatin control group in this study

revealed a significant upsurge of 5-hydroxytryptamine (5HT, serotonin) in the brain stem

(BS; ~ 0.031 → 0.138 ng/mg tissue wet weight, P < 0.001) and intestine (~ 0.044 → 0.821

ng/mg tissue wet weight, P < 0.001) as compared to basal level, while for delayed response

(18 hr) the significant increase in the concentration of dopamine (~ 0.535 → 13.43 ng/mg

tissue wet weight, P < 0.001) and serotonin (~ 0.045 → 0.588 ng/mg tissue wet weight, P <

0.001) was observed in the area postrema (AP) and intestine, respectively in pigeon model.

CS-HexFr (10 mg), BM-MetFr (10, 20 & 40 mg), BM-ButFr (5, 10 & 20 mg), ZO-ActFr (50

mg) & combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) significantly decreased the

concentration of 5HT (~ 0.438 → 0.006 ng/mg tissue wet weight, P < 0.001) and its

metabolite; 5-hydroxy indole acetic acid (~ 5HIAA; 0.165 → 0.003 ng/mg tissue wet

weight, P < 0.001) in the brain area of BS and intestine at acute time point (03 hr) as

compared to cisplatin control. However, BM treatments failed to reduce 5HT concentration

in AP any significantly (P > 0.05), and 5HIAA concentration in all the brain areas (AP &

BS) and intestine. At the delayed time point (18 hr), BM-MetFr (10, 20 & 40 mg) and BM-

ButFr (5, 10 & 20 mg) significantly decreased the upsurge of dopamine caused by cisplatin

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(~ 13.43 → 0.007 ng/mg tissue wet weight, P < 0.001) in the brain area of AP & BS, while a

significant reduction in 5HT (~ 0.588 → 0.017 ng/mg tissue wet weight, P < 0.001) was

observed in the intestine. Furthermore, CS-HexFr (10 mg), ZO-ActFr (50 mg) and

combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) significantly decreased dopamine

(~ 7.36 → 0.098 ng/mg tissue wet weight, P < 0.001) in AP, while a significant decrease in

5HT (~ 0.292 → 0.002 ng/mg tissue wet weight, P < 0.001) was observed in the brain area

of BS and at the level of intestine. None of the treatment (CS, BM & ZO extract) and

combination (CS-HexFr 10 mg + BM-ButFr 5 mg) altered the basal neurotransmitter level

except the decrease in the concentration of 5HIAA in the brain area of BS, which was found

to be statistically significant.

In the S. murinus, cisplatin treatment induced C-fos protein expression in the hind brain

areas including area postrema, nucleus tractus solitarius, and dorsal motor nucleus of vagus

nerve and in the forebrain area of hypothalamus including dorsomedial and ventromedial

nucleus of hypothalamus. Treatment with BM-MetFr (10, 20 & 40 mg), BM-ButFr (5, 10 &

20 mg) & combination of WIN 55, 212-2 (10 mg) with BM-ButFr (5 mg) significantly

attenuated (P < 0.001) cisplatin induced C-fos activity in all the brain areas.

These findings highlight the intrinsic anti-emetic activity of CS hexane extract (rich in

cannabinoids), ZO acetone extract (rich in gingerols) and BM extract (rich in bacosides);

against cisplatin induced R + V in vomit models (pigeon & S. murinus). The significant

findings to suppress the behavioral signs of CIV, reduction in the upsurge of serotonin and

dopamine neurotransmitters caused by cisplatin and attenuation of C-fos immunoreactivity

in vomit model of S. murinus, especially the BM extracts that we found for the first time in

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this study, is an additional avenue to explore further for its anti-emetic potentials in other

animal models. Moreover, combination of BM extract with CS extract has shown promising

synergistic anti-emetic effect in vomit model of pigeon. Furthermore, no alteration observed

in the basal neurotransmitter level by these treatments is encouraging. The CS, BM & ZO

plant extracts and combination need to be further explored in gold standard vomit models of

ferret and dog and in clinics as well, keeping in view the safe and tolerable profile of these

extracts.

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LIST OF ABBREVIATIONS

Ach : Acetylcholine

AVP : Arginine vasopressin

AP : Area postrema

BM : Bacopa monniera

BM-MetFr : Bacopa monniera methanolic fraction

BM-ButFr : Bacopa monniera n-butanol fraction

BD : Bis in Die (Twice daily dosing)

BBB : Blood brain barrier

CIV : Chemotherapy induced vomiting

CINV : Chemotherapy induced nausea and vomiting

CS : Cannabis sativa

CS-HexFr : Cannabis sativa n-hexane fraction

CS-ButFr : Cannabis sativa n-butanol fraction

CS-MetFr : Cannabis sativa methanolic fraction

CNS : Central nervous system

CB1 : Cannabinoid receptor type 1

CB2 : Cannabinoid receptor type 2

CSF : Cerebrospinal fluid

CTZ : Chemoreceptor trigger zone

DA : Dopamine

D1- D5 : Dopamine receptor type 1-5

DMH : Dorsomedial nucleus of hypothalamus

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DOPAC : 3, 4-Dihydroxyphenylacetic acid

DMV/DMVN : Dorsal motor nucleus of vagus nerve

DVC : Dorsal vagal complex

EDTA : Ethylene diamine tetra acetic acid

EC cells : Enterochromaffin cells

GABA : Gamma-amino butyric acid

GIT : Gastrointestinal tract

GPCRs : G-protein coupled receptors

HVA : Homovanillic acid

H1 : Histamine receptor type 1

HPLC : High Performance Liquid Chromatography

HP : Hypothalamus

HEC : Highly emetogenic chemotherapy

i.m. : Intramuscular route of drug administration

i.v. : Intravenous route of drug administration

i.p. : Intraperitoneal route of drug administration

Kg : Kilogram

KH2PO4 : Potassium dihydrogen orthophosphate

KCl : Potassium chloride

M3 : Muscarinic receptor type 3

M4 : Muscarinic receptor type 4

MCP : Metoclopramide

MEC : Moderate emetogenic chemotherapy

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n : Number of animals in a group

NA : Noradrenaline

NaCl : Sodium chloride

NaH2PO4 : Sodium dihydrogen orthophosphate

NK1- NK3 : Neurokinin receptor type 1-3

NMDA : N-methyl-D-aspartate

NTS : Nucleus tractus solitarius

OD : Once daily dosing

OCT : Optimal temperature cutting compound

PalS : Palonosetron

PBS : Phosphate butter saline

PFA : Paraformaldehyde

S. murinus : Suncus murinus

s.c. : Subcutaneous route of drug administration

SEM : Standard error of mean

R + V : Retching plus Vomiting

VMH : Ventromedial nucleus of hypothalamus

ZO : Zingiber officinale

ZO-ActFr : Zingiber officinale acetone fraction

5HIAA : 5-Hydroxy indolacetic acid

5-HT : 5-Hydroxytryptamine (serotonin)

5HT1-5HT7 : 5-Hydroxytryptamine receptor type 1-7

Δ9-THC : Delta-9-tetrahydrocannabinol

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TABLE OF CONTENTS

CERTIFICATE OF APPROVAL .............................................................................................................................. I

AKNOWLEDGMENTS ............................................................................................................................................ II

ABSTRACT ............................................................................................................................................................... IV

LIST OF ABBREVIATIONS .................................................................................................................................... X

TABLE OF CONTENTS ....................................................................................................................................... XIII

LIST OF TABLES .............................................................................................................................................. XXIII

LIST OF FIGURES ............................................................................................................................................ XXVI

CHAPTER 1 ................................................................................................................................................................ 1

INTRODUCTION .......................................................................................................................................................... 1

1.1. General introduction:............................................................................................................................... 2

1.2. Physiology of nausea and vomiting: ....................................................................................................... 5

1.2.1. Nausea and vomiting: ................................................................................................................. 5

1.2.2. Emetic circuits: .......................................................................................................................... 6

1.2.2.1. Area postrema: ................................................................................................................ 7

1.2.2.2. Nucleus tractus solitarious: ............................................................................................. 7

1.2.2.3. Dorsal motor nucleus of vagus nerve: ............................................................................. 8

1.2.2.4. Hypothalamus: ................................................................................................................ 9

1.2.2.5. Abdominal vagal afferents: ............................................................................................. 9

1.3. Mechanisms of cisplatin induced nausea and vomiting: ....................................................................... 12

1.3.1. Neurotransmitters involved in cisplatin induced nausea and vomiting: ................................... 12

1.3.2. Receptors involved in cisplatin induced nausea and vomiting: ................................................ 14

1.4. Anti-emetics: ......................................................................................................................................... 16

1.4.1. Current anti-emetic drugs: ........................................................................................................ 16

1.5. Natural products in drug discovery: ...................................................................................................... 17

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1.5.1. Selected plants having anti-emetic potential: ....................................................................... 18

1.5.1.1. Cannabis sativa (cannabinoids): ................................................................................... 18

1.5.1.2. Zingiber officinale (gingerols): ..................................................................................... 20

1.5.1.3. Bacopa monniera (bacosides): ...................................................................................... 22

1.6. Vomit models: ....................................................................................................................................... 26

1.6.1. Pigeon: ......................................................................................................................... 27

1.6.2. Suncus murinus: ........................................................................................................... 28

1.7. Aims and objectives of the study: ......................................................................................................... 28

CHAPTER 2 .............................................................................................................................................................. 30

METHODS ................................................................................................................................................................ 30

2.1. Animal husbandry: .............................................................................................................................. 31

2.1.1. Pigeon: ............................................................................................................................................ 31

2.1.2. Suncus murinus: ............................................................................................................................. 33

2.2. Video recording setup: ........................................................................................................................ 33

2.2.1. Recording setup for pigeon experiments: .................................................................................. 33

2.2.2. Recording setup for Suncus murinus experiments: .................................................................... 35

2.3. Quantification of vomiting: ................................................................................................................. 37

2.3.1. Quantification of vomiting in Pigeon:................................................................................... 37

2.3.2. Quantification of vomiting in Suncus murinus: .................................................................... 38

2.4. Measurement of locomotor activity in Suncus murinus: ..................................................................... 39

2.5. Plant collection and extraction: ........................................................................................................... 40

2.5.1. Cannabis sativa: ................................................................................................................ 40

2.5.2. Bacopa monniera: .............................................................................................................. 41

2.5.3. Zingiber officinale:............................................................................................................. 44

2.6. Chemicals and drugs: .......................................................................................................................... 45

2.7. Instruments and apparatus: .................................................................................................................. 47

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2.8. Drug formulation: ................................................................................................................................ 49

2.9. Drugs administration: .......................................................................................................................... 49

2.9.1. Intravenous administration: ........................................................................................... 49

2.9.2. Intramuscular administration: ....................................................................................... 50

2.9.3. Intraperitoneal administration: ...................................................................................... 50

2.9.4. Subcutaneous administration: ....................................................................................... 51

2.10. Measurement of gastrointestinal motility: ........................................................................................... 51

2.11. Standardization of Bacopa monniera extracts for bacoside “A” major components: ......................... 52

2.11.1. High Performance Liquid Chromatography (HPLC) system for bacoside

quantification: .......................................................................................................................... 52

2.11.2. Preparation of standards: ........................................................................................ 52

2.11.3. Preparation of samples:........................................................................................... 53

2.11.4. Chromatographic conditions: .................................................................................. 53

2.12. High Performance Liquid Chromatography (HPLC) method for neurotransmitter analysis:.............. 54

2.12.1. Sample handling: ............................................................................................................... 54

2.12.2. Preparation of stock solutions: ........................................................................................... 54

2.12.3. Sample preparation: ........................................................................................................... 55

2.12.4. Chromatography: ............................................................................................................... 55

2.13. C-fos immunohistochemistry: ............................................................................................................. 56

2.13.1. Immunohistochemical procedure: ...................................................................................... 56

2.13.2. Quantification of C-fos immunoreactivity: ........................................................................ 57

2.13.3. Image acquisition and processing: ..................................................................................... 57

2.14. Ethical approval: ................................................................................................................................. 58

CHAPTER 3 .............................................................................................................................................................. 59

STUDIES ON THE EMETIC POTENTIAL OF CISPLATIN IN PIGEON AND SUNCUS MURINUS ............................................. 59

3.1 Introduction: ............................................................................................................................................. 60

3.1.1. Cisplatin induced vomiting in the pigeon: ...................................................................................... 61

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3.1.2. Cisplatin induced vomiting in the Suncus murinus: ....................................................................... 62

3.2 Aims and objectives of the study: ............................................................................................................ 62

3.3 Materials and methods: ............................................................................................................................ 63

3.3.1. Animals: .................................................................................................................................... 63

3.3.1.1. Pigeon: ................................................................................................................................. 63

3.3.1.2. Suncus murinus (House musk shrew): ................................................................................. 63

3.3.2. Video recording setup: ............................................................................................................... 63

3.3.3. Drug formulation and administration: ....................................................................................... 63

3.3.4. Quantification of vomiting: ....................................................................................................... 64

3.4 Results:..................................................................................................................................................... 64

3.4.1. Induction of vomiting by i.v. administration of cisplatin in pigeon: .................................. 64

3.4.2. Induction of vomiting by i.p. administration of cisplatin in Suncus murinus: ................... 66

3.5 Discussion: ............................................................................................................................................... 67

CHAPTER 4 .............................................................................................................................................................. 69

STANDARDIZATION OF BACOPA MONNIERA FRACTIONS FOR BACOSIDE “A” MAJOR COMPONENTS ........................... 69

4.1. Introduction: ........................................................................................................................................ 70

4.2. Aims and objectives of the study: ....................................................................................................... 71

4.3. Materials and methods: ....................................................................................................................... 71

4.3.1. Chemicals and reagents: ................................................................................................................. 71

4.3.2. High Performance Liquid Chromatography (HPLC) system: ........................................................ 71

4.3.3. Sample handling: ............................................................................................................................ 72

4.3.4. Preparation of stock solutions: ....................................................................................................... 72

4.3.5. Chromatography: ............................................................................................................................ 72

4.4. Results: ................................................................................................................................................ 72

4.4.1. Standardization of Bacopa monniera methanolic fraction (BM-MetFr): .................................. 72

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4.4.2. Standardization of Bacopa monniera n-butanol fraction (BM-ButFr):...................................... 73

4.5. Discussion: .......................................................................................................................................... 74

CHAPTER 5 ............................................................................................................................................................. 76

EFFECT OF CANNABIS SATIVA, BACOPA MONNIERA OR ZINGIBER OFFICINALE (GINGER)

EXTRACTS AND THEIR COMBINATIONS ON CISPLATIN INDUCED RETCHING PLUS VOMITING IN

PIGEON…..……………………….………………………………………………………………………..……..76

5.1. Introduction: ........................................................................................................................................ 77

5.2. Aims and objectives: ........................................................................................................................... 79

5.3. Materials and methods: ....................................................................................................................... 79

5.3.1. Animals: ......................................................................................................................................... 79

5.3.2. Plants extraction: ............................................................................................................................ 80

5.3.3. Drugs and chemicals: ..................................................................................................................... 80

5.3.4. Drug formulation: ........................................................................................................................... 80

5.3.5. Drug administration: ....................................................................................................................... 80

5.3.6. Video recording setup & quantification of vomiting and retching: ................................................ 80

5.3.7. Data analysis: ................................................................................................................................. 81

5.4. Results: ................................................................................................................................................ 81

5.4.1. Anti-emetic effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction

(CS-ButFr) & methanol fraction (CS-MetFr): ........................................................................................... 81

5.4.2. Effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction (CS-ButFr) &

methanol fraction (CS-MetFr) on cisplatin induced jerking and weight loss: ........................................... 85

5.4.3. Anti-emetic effect of Bacopa monniera methanol fraction (BM-MetFr) & n-butanol

fraction (BM-ButFr): ................................................................................................................................. 86

xviii

5.4.4. Effect of standard metoclopramide (MCP), anti-oxidant N-(2- mercaptoprpionyl) glycine

(MCP), Bacopa monniera methanol fraction (BM-MetFr) & n-butanol fraction (BM-ButFr) on

cisplatin induced jerking and weight loss: ................................................................................................. 91

5.4.5. Anti-emetic effect of Zingiber officinale acetone fraction (ZO-ActFr): .................................... 92

5.4.6. Effect of Zingiber officinale acetone fraction (ZO-ActFr) & metoclopramide (MCP) on

cisplatin-induced jerks and weight loss: .................................................................................................... 94

5.4.7. Anti-emetic effect of CS-HexFr 10 mg + BM-MetFr 10 mg (combination 1), BM-ButFr 5

mg + ZO-ActFr 25 mg (combination 2), ZO-ActFr 25 mg + CS-HexFr 10 mg (combination 3) or

CS-HexFr 10 mg + BM-ButFr 5 mg (combination 4): .............................................................................. 95

5.4.8. Effect of CS-HexFr 10 mg + BM-MetFr 10 mg (combination 1), BM-ButFr 5 mg + ZO-

ActFr 25 mg (combination 2), ZO-ActFr 25 mg + CS-HexFr 10 mg (combination 3) or CS-HexFr

10 mg + BM-ButFr 5 mg (combination 4) on cisplatin-induced jerks and weight loss: ......................... 103

5.5. Discussion: ........................................................................................................................................ 104

CHAPTER 6 ............................................................................................................................................................ 109

EFFECT OF CANNABIS SATIVA ON GASTROINTESTINAL MOTILITY AND CONSEQUENT INFLUENCE ON CISPLATIN

INDUCED RETCHING PLUS VOMITING (R + V) IN PIGOEN ....................................................................................... 109

6.1. Introduction: ...................................................................................................................................... 110

6.2. Aims and Objectives: ........................................................................................................................ 111

6.3. Materials and methods: ..................................................................................................................... 111

6.3.1. Animals: ....................................................................................................................................... 111

6.3.2. Materials and drugs: ..................................................................................................................... 111

6.3.3. Extraction of Cannabis sativa: ..................................................................................................... 112

6.3.4. Drug administration: ..................................................................................................................... 112

6.3.5. Video recording setup & Quantification of vomiting: .................................................................. 112

6.3.6. Measurement of Gastrointestinal motility: ................................................................................... 112

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6.4. Results: .............................................................................................................................................. 113

6.4.1. Gastrointestinal suppression caused by Cannabis sativa hexane fraction (CS-HexFr) and

its antagonism by metoclopramide and carbachol: .................................................................................. 113

6.4.2. Impact of Cannabis sativa hexane fraction (CS-HexFr) in combination with

metoclopramide (MCP) and carbachol on cisplatin induced R + V: ....................................................... 114

6.5. Discussion: ........................................................................................................................................ 118

CHAPTER 7 ........................................................................................................................................................... 121

EFFECT OF CANNABIS SATIVA, BACOPA MONNIERA OR ZINGIBER OFFICINALE EXTRACTS ON

NEUROTRANSMITTERS IMPLICATED IN VOMITING CIRCUITS IN PIGEON .................................................................. 121

7.1. Introduction: ...................................................................................................................................... 122



7.3.1. Chemicals and reagents: ............................................................................................................... 125

7.3.2. High performance liquid chromatography system: ...................................................................... 125

7.3.3. Sample preparation, handling and Preparation of stock solutions: ............................................... 125

7.3.4. Chromatography: .......................................................................................................................... 126

7.4. Results: .............................................................................................................................................. 126

7.4.1. High Performance Liquid Chromatography (HPLC), method reproducibility: ....................... 126

7.4.2. Effect of MCP, BM-MetFr & BM-ButFr, CS-HexFr, ZO-ActFr or combination of CS-HexFr

(10 mg) with BM-ButFr (5 mg) on Basal level of neurotransmitters and their metabolites at specific brain

areas (AP + BS) and intestine of pigeon: ................................................................................................. 129

7.4.2.1. Effect of standard metoclopramide (MCP) on basal neurotransmitters and their metabolites

in the brain areas (AP & BS) and intestine: …………….…………………………………………….129

7.4.2.2. Effect of Bacopa monniera methanolic (BM-MetFr) and butanolic fraction (BM-

ButFr) on basal neurotransmitters and their metabolites in the brain areas (AP & BS) and

intestine:… .......................................................................................................................................... 129

xx

7.4.2.3. Effect of Cannabis Sativa Hexane fraction (CS-HexFr) on basal neurotransmitters and

their metabolites in the brain areas (AP & BS) and intestine: ..... …………………………………….130

7.4.2.4. Effect of Zingiber officinale acetone fraction (ZO-ActFr) on basal neurotransmitters

and their metabolites in the brain areas (AP & BS) and intestine: ...................................................... 130

7.4.2.5. Effect of combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) on basal

neurotransmitters and their metabolites in the brain areas (AP & BS) and intestine: ......................... 130

7.4.3. Effect of MCP, CS-HexFr, BM-MetFr & BM-ButFr, ZO-ActFr or combination of CS-

HexFr (10 mg) with BM-ButFr (5 mg) on level of neurotransmitters and their metabolites at specific

brain areas (AP + BS) and intestine of pigeon at acute time point (3rd

hour): …………………………..134

7.4.3.1. Effect of standard metoclopramide (MCP) on neurotransmitters and their

metabolites in the brain areas (AP & BS) and intestine at 3rd

hour of cisplatin treatment: ............. 134

7.4.3.2. Effect of Cannabis Sativa Hexane fraction (CS-HexFr) on neurotransmitters and

their metabolites in the brain areas (AP & BS) and intestine at 3rd

hour of cisplatin treatment:..... 134

7.4.3.3. Effect of Bacopa monniera methanolic (BM-MetFr) or butanolic fraction (BM-

ButFr) on neurotransmitters and their metabolites in the brain areas (AP & BS) and intestine at

3rd

hour of cisplatin treatment: ........................................................................................................ 135

7.4.3.4. Effect of Zingiber officinale acetone fraction (ZO-ActFr) on neurotransmitters and

their metabolites in the brain areas (AP & BS) and intestine at 3rd

hour of cisplatin treatment:..... 139

7.4.3.5. Effect of CS-HexFr (10 mg) in combination with BM-ButFr (5 mg) on

neurotransmitters and their metabolites in the brain areas (AP & BS) and intestine at 3rd

hour

of cisplatin treatment: ..................................................................................................................... 139

7.4.4. Effect of MCP, CS-HexFr, BM-MetFr & BM-ButFr, ZO-ActFr or combination of CS-

HexFr (10 mg) with BM-ButFr (5 mg) on level of neurotransmitters and their metabolites at specific

brain areas (AP + BS) and intestine of pigeon at delayed time point (18th

hour): ................................... 143

7.4.4.1. Effect of standard metoclopramide (MCP) on neurotransmitters and their

metabolites in the brain areas (AP & BS) and intestine at 18th

hour of cisplatin treatment: ....... 143

xxi

7.4.4.2. Effect of Cannabis Sativa Hexane fraction (CS-HexFr) on neurotransmitters and

their metabolites in the brain areas (AP & BS) and intestine at 18th

hour of cisplatin

treatment:.. .................................................................................................................................. 144

7.4.4.3. Effect of Bacopa monniera methanolic (BM-MetFr) or butanolic fraction (BM-

ButFr) on neurotransmitters and their metabolites in the brain areas (AP & BS) and intestine

at 18th

hour of cisplatin treatment: .............................................................................................. 144

7.4.4.4. Effect of Zingiber officinale acetone fraction (ZO-ActFr) on neurotransmitters

and their metabolites in the brain areas (AP & BS) and intestine at 18th

hour of cisplatin

treatment:.. .................................................................................................................................. 148


neurotransmitters and their metabolites in the brain areas (AP & BS) and intestine at 18th

hour of cisplatin treatment: ......................................................................................................... 148

7.5. Discussion: ........................................................................................................................................ 152

CHAPTER 8 ............................................................................................................................................................ 157

ATTENUATION OF CISPLATIN INDUCED RETCHING PLUS VOMITING (R + V) AND C-FOS IMMUNOREACTIVITY

(C-FOS-IR)BY BACOSIDES CONTAINING BACOPA MONNIERA FRACTIONS IN SUNCUS MURINUS .............................. 157

8.1. Introduction: ...................................................................................................................................... 158



8.3.1. Animals: ....................................................................................................................................... 159

8.3.2. Materials and drugs: ..................................................................................................................... 159

8.3.3. Extraction of Bacopa monniera: ................................................................................................... 160

8.3.4. Drug formulation: ......................................................................................................................... 160

8.3.5. Drug administration: ..................................................................................................................... 160

8.3.6. Experimental setup for behavioral studies: ................................................................................... 160

8.3.7. Measurement of Retching plus Vomiting (R + V) and locomotor activity: ................................. 160

xxii

8.3.8. C-fos immunohistochemistry:....................................................................................................... 161

8.3.9. Data analysis: ............................................................................................................................... 161

8.4. Results: .............................................................................................................................................. 162

8.4.1. Effect of palonosetron, Bacopa monniera methanol fraction (BM-MetFr) and n-butanol

fraction (BMButFr) on cisplatin induced Retching plus Vomiting (R + V): ........................................... 162

8.4.2. Induction of C-fos by cisplatin: ............................................................................................... 167

8.4.3. Effect of palonosetron, Bacopa monniera methanol fraction (BM-MetFr) & n-butanol

fraction (BMButFr) on cisplatin induced C-fos expression: .................................................................... 170

8.5. Discussion: ........................................................................................................................................ 173

CHAPTER 9 ............................................................................................................................................................ 178

GENERAL DISCUSSION ........................................................................................................................................... 178

9.1. General discussion: ........................................................................................................................... 179

9.2. Future work: ...................................................................................................................................... 189

REFERENCES ........................................................................................................................................................ 193

APPENDICES ......................................................................................................................................................... 216

xxiii

LIST OF TABLES

2.1 Chemicals and drugs ..................................................................................................................................... 45

2.2 Instruments and apparatus ............................................................................................................................. 47

5.1 Effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction (CS-ButFr), and methanol fraction

(CS-MetFr) on cisplatin-induced R + V in pigeons ............................................................................................ 83

5.2 Effect of Bacopa monniera methanol fraction & n-butanol fraction on cisplatin-induced R + V in pigeons

............................................................................................................................................................................ 88

5.3 Effect of Zingiber officinale acetone fraction (ZO-ActFr) on cisplatin-induced R + V in pigeons .............. 93

5.4 Effect of various combinations of CS Hexane fraction, BM methanolic and bacoside rich n-butanol fraction

and ZO acetone fraction on cisplatin induced R + V in pigeons ........................................................................ .99

6.1 Effect of CS hexane fraction (CS-HexFr) and its combinations on cisplatin induced R + V in pigeons .... 115

7.1A Effect of MCP, BM-MetFr & BM-ButFr, CS-HexFr, ZO-ActFr and combination (CS-HexFr 10 mg +

BM-ButFr 5 mg) on basal level of neurotransmitters and their metabolites at the brain level of AP in pigeons

.......................................................................................................................................................................... 131

7.1B Effect of MCP, BM-MetFr & BM-ButFr, CS-HexFr, ZO-ActFr and combination (CS-HexFr 10 mg +

BM-ButFr 5 mg) on basal level of neurotransmitters and their metabolites at the brain level of BS in pigeons

.......................................................................................................................................................................... 132

7.1C Effect of MCP, BM-MetFr & BM-ButFr, CS-HexFr, ZO-ActFr and combination (CS-HexFr 10 mg +

BM-ButFr 5 mg) on basal level of neurotransmitters and their metabolites at the level of intestine in pigeons

.......................................................................................................................................................................... 133

7.2A Effect of standard MCP, BM-MetFr & BM-ButFr on neurotransmitters and their metabolites at the brain

level of AP at 3rd hour of cisplatin treatment .................................................................................................. 136

xxiv

7.2B Effect of standard MCP, BM-MetFr & BM-ButFr on neurotransmitters and their metabolites at the brain

level of BS at 3rd hour of cisplatin treatment .................................................................................................. 137

7.2C Effect of standard MCP, BM-MetFr & BM-ButFr on neurotransmitters and their metabolites at the level

of intestine at 3rd hour of cisplatin treatment ................................................................................................... 138

7.3A Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites at the brain area of AP at 3rd hour of cisplatin treatment ..... 140

7.3B Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites at the brain area of BS at 3rd hour of cisplatin treatment ..... 141

7.3C Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites in the intestine at 3rd hour of cisplatin treatment .................. 142

7.4A Effect of standard MCP, BM-MetFr & BM-ButFr on neurotransmitters and their metabolites in the brain

area of AP at 18th hour of cisplatin treatment: ................................................................................................ 145

7.4B Effect of standard MCP, BM-MetFr & BM-ButFr on neurotransmitters and their metabolites in the brain

area of BS at 18th hour of cisplatin treatment: ................................................................................................. 146

7.4C Effect of standard MCP, BM-MetFr & BM-ButFr) on neurotransmitters and their metabolites in the

intestine at 18th hour of cisplatin treatment: .................................................................................................... 147

7.5A Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites in the brain area of AP at 18th hour of cisplatin treatment: .. 149

7.5B Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites in the brain area of BS at 18th hour of cisplatin treatment: .. 150

7.5C Effect of standard MCP, CS-HexFr, ZO-ActFr and combination of CS-HexFr (10 mg) with BM-ButFr (5

mg) on neurotransmitters and their metabolites in the intestine at 18th hour of cisplatin treatment: ............... 151

xxv

8.1A Effect of Bacopa monniera methanol fraction (BM-MetFr) on cisplatin induced R + V in Suncus

murinus. ........................................................................................................................................................... 165

8.1B Effect of Bacopa monniera n-butanol fraction (BM-ButFr) and combination of WIN 55, 212-2 (10 mg)

with BM-ButFr (5 mg) on cisplatin induced R + V in Suncus murinus. .......................................................... 166

xxvi

LIST OF FIGURES

1.1 Emetic circutiry for chemotherapy induced vomiting ................................................................................... 11

1.2 Delta-9-tetrahydrocannabinol........................................................................................................................ 19

1.3 A photograph of Cannabis sativa.................................................................................................................. 20

1.4 Gingerol ........................................................................................................................................................ 21

1.5 A photograph of Zingiber officinale.............................................................................................................. 22

1.6 A photograph of Bacopa monniera ............................................................................................................... 23

1.7 Bacoside II .................................................................................................................................................... 24

1.8 Bacoside A3 .................................................................................................................................................. 25

1.9 Bacosaponin C .............................................................................................................................................. 25

2.1 Pigeon breeding facility at Department of Pharmacy, University of Peshawar ............................................ 32

2.2 Specially designed confining cages for video recording of pigeon experiments: ........................................ 34

2.3 Video recording setup for pigeon experiments ............................................................................................. 35

2.4 Video recording setup for Suncus murinus experiments ............................................................................... 36

2.5 Pigeon showing the act of vomiting .............................................................................................................. 38

2.6 Suncus murinus showing the act of vomiting ................................................................................................ 39

2.7 Extraction scheme for Cannabis sativa ......................................................................................................... 41

2.8 Extraction scheme for Bacopa monniera ...................................................................................................... 43

2.9 Extraction scheme for Zingiber officinale ..................................................................................................... 44

xxvii

2.10 Vectastain Elite ABC kit ............................................................................................................................. 57

3.1A Dose response relationship of cisplatin ..................................................................................................... 65

3.1B 24 hr sketch of cisplatin induced R+ V in pigeon ...................................................................................... 65

3.2 48 hr sketch of cisplatin induced R + V in Suncus murinus .......................................................................... 66

4.1A HPLC chromatogram showing peaks of standard Bacosides ..................................................................... 73

4.1B HPLC chromatogram showing peaks of bacosides in sample .................................................................... 74

5.1 Percent protection provided by Cannabis sativa hexane fraction ................................................................. 82

5.2 Effect of CS hexane fraction, n-butanol fraction and methanol fraction on cisplatin-induced R + V in

pigeons ................................................................................................................................................................ 85

5.3 Dose response relationship of Bacopa monniera .......................................................................................... 87

5.4 Effect of standard metoclopramide, antioxidant N-(2- mercaptoprpionyl) glycine, Bacopa monniera

methanolic fraction and n-butanol fraction on cisplatin-induced R + V in pigeons ............................................ 91

5.5 Effect of Zingiber officinale acetone fraction on cisplatin induced R + V in pigeons .................................. 94

5.6 Effect of various combinations of CS Hexane fraction, BM methanolic and bacoside rich n-butanol fraction

and ZO acetone fraction on cisplatin-induced V + R in pigeons ....................................................................... 100

5.7 Emesis suppression sketch of various combinations of CS Hexane fraction, BM methanolic and bacoside

rich n-butanol fraction and ZO acetone fraction on cisplatin induced R + V in pigeons .................................. 103

6.1 Gastrointestinal suppression caused by Cannabis sativa hexane fraction ................................................... 113

6.2 Effect of Cannabis sativa hexane fraction (CS-HexFr) 10mg and its combination with MCP and carbachol

on cisplatin-induced R + V ............................................................................................................................... 117

6.3 Effect of Cannabis sativa hexane fraction and its combinations, on cisplatin-induced R + V in pigeon ... 118

xxviii

7.1A HPLC chromatogram showing peaks of standard neurotransmitters and their metabolites ..................... 127

7.1B HPLC chromatogram showing peaks of neurotransmitters and their metabolites in sample ................... 128

8.1 Dose response relationship of Bacopa monniera extracts ........................................................................... 164

8.2 Representative photomicrographs showing C-fos immunoreactivity .......................................................... 170

8.3 Effect of palonosetron, Bacopa monniera methanolic fraction (BM-MetFr) or n-butanolic fraction (BM-

ButFr) on cisplatin induced C-fos count ........................................................................................................... 173

Chapter 1 Introduction

1

Chapter 1

Introduction


2

1.1. General introduction:

Nausea and vomiting are considered the most distressing side effects of cancer

chemotherapeutic agents. Despite of recent progress in understanding the mechanisms of

adverse effects of drugs, the side effects of anticancer agents are not well understood yet.

The severity of Chemotherapy Induced Nausea and Vomiting (CINV) depends upon various

factors including intrinsic emetogenecity of the chemotherapeutic agent, the administered

dosage and patient characteristics such as previous exposure, sex and alcohol intake history

etc (Hesketh, 2008; Markman, 2002). These stressful side effects often lead to poor

compliance and even refusal of curative treatment (Hesketh, 2008; Tanihata et al., 2000).

One of the bizarre aspects of CINV is the presence of two phases; an acute phase and a

delayed phase. The acute vomiting phase lasts for 24 hours in humans and ferrets (Kris and

Tonato, 2011; Sam et al., 2001), 8 hours in pigeons (Tanihata et al., 2000) and 16-18 hours

in piglets (Grelot and Esteve, 2009), while delayed phase extends up to 72 hours in humans,

ferrets and dogs (Fabi and Barduagni, 2003; Sam et al., 2001; Yamakuni et al., 2002b), 48

hours in pigeons (Tanihata et al., 2000) and 58 hours in piglets (Milano et al., 1995).

The traditional anti-emetics such as dopamine receptor antagonists, antihistaminics and

anticholinergics have modest efficacy against Chemotherapy Induced Vomiting (CIV) when

used alone or in combination (Sharma et al., 1997). The introduction of 5HT3 receptor

antagonists (ondansetron, granisetron & palonosetron) was a milestone in the clinical

management of CIV, however they are also found to be ineffective in achieving complete

remission particularly against delayed phase of vomiting (Rossel et al., 1992), signifying the

involvement of other mediators/mechanisms such as neuropeptide “Substance P” (Saito et

al., 2003), 5-hydroxytryptamine-4 (5HT4) receptors (Nakayama et al., 2005), D2 and D3


3

receptors (Darmani et al., 1999) in the etiology of nausea and vomiting. Substantial progress

resulted in the development of neurokinin 1 (NK1) receptor antagonists like aprepitant,

natupitant and vofopitant, which appeared to be no more effective than 5HT3 receptor

antagonists during acute phase though effective during delayed phase when used in

combination with other anti-emetics (Hesketh, 2001).

The identification of cannabinoid receptors resulted in the discovery of endocannabinoids

(Pacher et al., 2006). Delta-9-Tetrahydrocannabinol (Δ9-THC) and synthetic cannabinoids

exert their cannabimimetic effects via CB1 and CB2 receptors (Mackie and Stella, 2006).

CB1 receptors are primarily located centrally and peripherally while CB2 receptors occur

mainly on immune cells (Pertwee, 2006). Cannabinoids have been shown to affect neuronal

circuits that modulate nausea, vomiting and other gastrointestinal functions. Evidences are

emerging regarding the interaction of cannabinoid (CB1), serotonin (5HT3), neurokinin-1

(NK1) and dopamine receptors (D2 & D3) implicating an important role for cannabinoids in

vomiting circuits.

Cancer chemotherapeutic agents bump up the release of free radicals including superoxide

anions, hydroxyl radicals and hydrogen peroxide (Sangeetha et al., 1990) responsible at least

partly for vomiting induction. Accordingly, N-(2-mercaptopropionyl)glycine, an antioxidant,

proved effective against cisplatin induced vomiting in Suncus murinus (Torii et al., 1993)

thus connecting the role of antioxidants in the management of CIV.

The expression of C-fos protein is considered a marker for neuronal excitation and can be

labelled by immunohistochemical procedures. The basal level of C-fos is low but can be

rapidly induced by different stimuli. Cisplatin induces acute C-fos in vomiting species in the


4

hind brain areas including area postrema (AP), nucleus tractus solitarius (NTS) and dorsal

motor nucleus of vagus nerve (DMV) and in the hypothalamus of forebrain area (Ariumi et

al., 2000; De Jonghe and Horn, 2009; Miller and Ruggiero, 1994). Moreover, the studies in

rodents are also providing evidences for the expression of C-fos by cisplatin in hind brain

areas (Endo and Minami, 2004).

Synthesis of new compounds is laborious, expensive and up-till now no satisfactory

synthetic anti-emetic remedy is available that completely ameliorates both the acute and

delayed phases of vomiting. Plants are proving themselves as important therapeutic entities

that are economical, safe and readily available particularly in rural communities.

Cannabis sativa, Bacopa monniera and Zingiber officinale are unique sources of compounds

known as cannabinoids (Pertwee, 2006), bacosides (Gohil and Patel, 2010; Roodenrys and

Booth D. Bulzomi, 2002) and gingerols (Qiu-hai et al., 2010) respectively. Cannabis sativa

the natural source of cannabinoids has been used as medicine, for religious and recreational

purposes since long. The therapeutic indications of cannabinoids include as anti-emetic,

anti-spasmodic, analgesic and appetite stimulant (Carlini, 2004). Bacopa monniera the

natural source of bacosides have a long history of neuropharmocological profile and usage

in both ayurvedic and local folk therapies (Russo and Borrelli, 2005). The plant has been

shown to be anti-oxidant (Bhattacharya et al., 2000), neuroprotective (Limpeanchob et al.,

2008) and memory enhancer (Roodenrys and Booth D. Bulzomi, 2002).

Zingiber officinale (Ginger) has also been used as a natural herb in the treatment of vomiting

for more than 2000 years in China and as common spice for cooking in Asian countries

(Qiu-hai et al., 2010). One of its therapeutic indications has always been in the treatment of


5

nausea and vomiting, as its carminative, spasmolytic and aromatic properties suggest its

direct effects on the gastrointestinal system. Gingerols, in particular 6-gingerol has been

identified as the major active constituent responsible for its characteristic taste and is

reported to enhance gastrointestinal motility and have capability to antagonize 5HT3

receptors in the gastrointestinal tract (GIT) (Ernst and Pittler, 2000), which supports its anti-

emetic property.

Since numerous mediators acting on different sites/receptors are implicated in vomiting

induced by cancer chemotherapy, combination treatment is proven to be more efficacious

than a single drug though at the expense of increased cost. Therefore, there is a need of

further research in this area to get economically useful remedy such as herbal combinations

for the management of both acute and delayed phases of CIV.

1.2. Physiology of nausea and vomiting:

1.2.1. Nausea and vomiting:

Nausea and vomiting are the natural defense mechanisms/reflexes mediated by central

nervous system (CNS) of the body through which the body gets rid of toxic substances from

the gastrointestinal tract (GIT) and to prevent further ingestion of such substances (Hesketh

and Van Belle, 2003). Nausea and vomiting occur in many diseased conditions due to

different causes including medical treatments like cancer chemotherapy, radiotherapy,

anesthesia, post surgery and in some other conditions like motion sickness and pregnancy.

Nausea and vomiting occur separately, as nausea refers to the less intense stimulation of the

vomiting system, if stimulated more intensely leads to vomiting response. Counter

intuitively, nausea is more difficult to control than vomiting even the current


6

pharmacological therapies fail to control nausea. These evidences indicate that the

neurobiological systems responsible for these two acts are partially separate. The neural

circuit responsible for nausea is largely unknown but evidences indicate the involvement of

forebrain areas like hypothalamus, amygdala etc in the mediation of nausea. Anticipatory

nausea and vomiting is another type of condition which is not adequately controlled by

using anti-emetics like 5HT3 receptor antagonists. Interestingly, cancer chemotherapeutic

agents cause a biphasic vomiting response; the initial phase is known as acute phase which

lasts for 24 h followed by delayed phase which continues for several days, both are different

mechanistically as the ligands effective in the acute phase fail at the delayed phase of

vomiting (Markman, 2002).

1.2.2. Emetic circuits:

In some cases the principal input pathways responsible for the vomiting act have been

identified, but the neuronal substrate underlying the coordination of the vomiting response

remains incompletely understood. The previously proposed organization schemes have

included a vomiting center, a pattern generator and some more distributed control systems. It

has been proposed that the vomiting reflex is initiated by a sequential activation of effector

nuclei, and it is suggested, that the paraventricular system of nuclei defined by their

connections with area postrema and each other, can collectively account for most of the

phenomenon associated with the act of nausea and vomiting. Some of the areas identified

are confirmed to be the parts of vomiting circuit are described in the following sections.


7

1.2.2.1. Area postrema:

In classic studies, the Area Postrema (AP) is also known as the Chemoreceptor Trigger Zone

(CTZ) (Borison et al., 1984; Carpenter, 1989) for vomiting since 40 years, which is one of

the circumventricular organs that serves as a boundary between the brain parenchyma and

the Cerebrospinal Fluid (CSF) containing ventricles. Anatomically, AP is located on the

dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. The AP is

lacking Blood Brain Barrier (BBB) and is anatomically positioned in such a way to detect

toxins in blood and CSF (Duvernoy and Risold, 2007). There are evidences for the presence

of dopaminergic (Yoshikawa et al., 1996), serotonergic (Higgins et al., 2012) and

neurokininergic (Ariumi et al., 2000) receptors in the AP whose stimulations lead to the

initiation of vomiting reflex. The excitation originating in the AP is conveyed to NTS via

glutaminergic neurons which elaborate the significance of neuronal connections among AP

and NTS in the emetic reflex (Migita and Hori, 1998). The C-fos protein expression in the

area postrema by cancer chemotherapeutic agents and X-irradiation is authenticating the

involvement of area postrema in vomiting circuits (De Jonghe and Horn, 2009; Ito et al.,

2003).

1.2.2.2. Nucleus tractus solitarius:

The recognition of the significance of Nucleus Tractus Solitarius (NTS) in vomiting circuit

has grown in prominence, particularly the realization that the neuronal dendrites from NTS

projects to AP. NTS is situated adjacent to the AP and is implicated in the coordination of

the emetic response. Furthermore, NTS with the neighboring nucleus of DMN forms the

dorsomedial medulla or vagal complex (Duvernoy and Risold, 2007). The AP and NTS are

connected so adjacently that the surgical ablation of the AP leads to damage of the neuronal


8

dendrites from NTS, therefore lesioning which is directed towards area postrema may lead

to an erroneous conclusion that the AP is the primary site for the detection of toxins.

Moreover, the AP and NTS are interlinked to each other and the excitation produced in the

AP is conveyed to NTS via a non-NMDA receptors and is modified by NMDA receptors

activation secondly (Migita and Hori, 1998). The neurons in the NTS were found to be

activated by X-irradiation and anti-cancer agents in rats (Yamada et al., 2000) and Suncus

murinus (De Jonghe and Horn, 2009; Ito et al., 2003) evidenced by C-fos

immunohistochemistry (De Jonghe and Horn, 2009; Endo and Minami, 2004) certifying the

role of NTS in the mediation of vomiting response. As NTS receives inputs form the AP,

vestibular system and vagus nerve (Yates et al., 1994), it may serve as the start of the final

common pathway by which different emetogenic substances trigger vomiting (Miller and

Leslie, 1994).

1.2.2.3. Dorsal motor nucleus of vagus nerve:

Dorsal Motor nucleus of Vagus nerve (DMV) in combination with NTS is known as Dorsal

Vagal Complex (DVC). The DMV is located dorsolateral to the hypoglossal nucleus and

lateral to the fourth ventricle rostrally. The DMV contains pre-ganglionic neurons which

targets the stomach and in this way control gastric motility and secretion (Rogers et al.,

1999; Rogers et al., 1996). The anti-emetic site of NK1 receptor antagonist has been

proposed to be in the DMV, where NK1 receptors located on pre-ganglionic cholinergic

vagal neurons in the DMV mediate the inhibition of gastric relaxation, which is an important

prodromal component of vomiting (Krowicki and Hornby, 2000). Moreover, Substance P is

highly expressed in the DMV of all species including humans (Navari and Reinhardt, 1999).


9

1.2.2.4. Hypothalamus:

The involvement of hindbrain areas is well understood in the mediation of vomiting. In this

regard, hypothalamus (HP) of the forebrain area is also known to be involved partly in the

alteration of homeostatic processes of the body during nausea and vomiting. The act of

nausea and vomiting have also got outputs from the autonomic nervous system which leads

to tachycardia, increased salivation, hypotension, anorexia and increased level of

vasopressin (Billig et al., 2001; Morrow et al., 1992). Moreover, increased level of

vasopressin is a biomarker of nausea and vomiting and in brain the receptors for vasopressin

are widely distributed in the medulla oblongata, thalamus, hypothalamus and amygdala

(Ikegaya and Matsuki, 2002). The areas of thalamus and hypothalamus are the potential

targets for the emetogenic Arginine Vasopressin (AVP) particularly, in motion sickness and

chemically induced vomiting (Ikegaya and Matsuki, 2002; Migita and Hori, 1998).

1.2.2.5. Abdominal vagal afferents:

The abdominal vagal afferents provide the sensory pathway from the Gastrointestinal Tract

(GIT) to the Central Nervous System (CNS). The vagus nerve mainly receives input from

the stomach and intestine while splanchnic nerve receives input from the small intestine

(Andrews, 1986; Andrews and Sanger, 2002). A number of stimuli which may lead to the

activation of vagal afferents include over-distention of the stomach (due to food), presence

of gastric irritants (e.g. hypertonic solution, copper sulphate, ipecac), and toxic substances in

food (e.g. bacterial/viral toxins). Many chemotherapeutic agents such as cisplatin also

causes the release of serotonin (5HT) from Enterochromaffin (EC) cells of the intestine

which causes the activation of 5HT3 receptors ultimately leading to the activation of vagus

nerve and vomiting is initiated (Hillsley and Grundy, 1999). Abdominal vagal afferents have


10

proved to be having supreme relevance for chemotherapy induced nausea and vomiting

because of the presence of variety of receptors including 5-hydroxytryptamine type 3 (5HT3)

and neurokinin 1 (NK1) receptors (Krowicki and Hornby, 2000; Minami and Endo, 2003)

which are important in the mediation of vomiting response (Figure 1.1).


11

Emetic circuitry for chemotherapy induced vomiting:

Circulatory system

Figure 1.1 The emetic circuitry for chemotherapy induced vomiting. Vomiting is a complex

process mediated in the vomiting center nuclei. All the inputs from the gastrointestinal tract

by the vagus nerve, in the circulation via area postrema and from the higher brain regions

(involved in nausea and anticipatory vomiting) are integrated in the Nucleus Tractus

Area postrema

Nucleus tractus solitarous

Dorsal motor nucleus of vagus nerve

1. Gastric relaxation

2. Giant retrograde contractions

GIT

Vagal efferents

Vagal afferents

Enterochromaffin cells containing

5HT & substance P

Chemotherapy

5HT SP

Higher brain centers

Hind brain D

5HT

SP

Vasopressin

Nausea

3. Abdominal contraction

&diaphragm contraction

4. Crural fiber relaxation

5. Vomit

BBB

Phrenic nerve


12

Solitarius (NTS) in the brain stem. The subsequent autonomic and somatic motor outputs

from the Dorsal Motor nucleus of Vagus (DMV) and ventral regions of brain stem trigger

the act of vomiting in sequential order. The cholinergic and histaminergic systems are not

shown because of any relevance with chemotherapy induced vomiting. Abbreviations are

5HT; 5-hydroxytryptamine, SP; substance P, D; dopamine, GIT; gastrointestinal tract, BBB;

blood brain barrier.

1.3. Mechanisms of cisplatin induced nausea and vomiting:

1.3.1. Neurotransmitters involved in cisplatin induced nausea and vomiting:

Many neurotransmitters have been implicated in the pathogenesis of vomiting act, which

includes dopamine (Darmani and Crim, 2005), acetylcholine (Wood et al., 1993), histamine

(Lucot, 1989), serotonin (Javid and Naylor, 2002; Minami and Endo, 2003), opioids

(Hesketh and Van Belle, 2003) and substance P (Saito et al., 2003). A thorough

understanding of these neurotransmitters and their inter-relationship in the act of vomiting is

necessary to develop more effective approaches for the prevention and management of

chemotherapy induced vomiting.

The neurotransmitter serotonin (5HT) has been proved clinically and in animal assays as

well, to be the primary mediator involved in acute phase of CIV. Pre-clinical studies are

indicative for calcium dependant exocytic release of serotonin by cisplatin from the EC cells

of the GIT (Andrews et al., 1998; Minami, 1995). The released serotonin activates 5HT3

receptors on vagal afferents that stimulates vomiting center and induction of vomiting

(Andrews et al., 1998; Matsuki et al., 1993).


13

Substance P, a member of the tachykinin family of neuropeptides, was first elucidated by

Amin and colleagues in the area postrema of dogs (Amin et al., 1954). Subsequently, the

studies in ferrets showed anti-emetic activity of capsaicin analogue resinferotoxin against

the centrally and peripherally acting emetogens (Yamakuni et al., 2002a). It was suggested

that this anti-emetic activity was mediated by resinferotoxin-induced depletion of substance

P in the brain area of NTS. Substance P is found in parallel with serotonin in the EC cells of

the GIT and the elevated levels of substance P has been reported in patients after cisplatin

administration (Hesketh, 2008).

Dopamine is the predominant catecholamine neurotransmitter in the brain, where it plays a

variety of roles including cognition, positive reinforcement, emotion, locomotor activity and

behavior (Missale et al., 1998). Endogenously increased release of dopamine acts as

dopamine receptor agonist and causes vomiting. The emetic action of dopamine is assumed

to be mediated by its action on dopamine D2 receptors located in the Chemoreceptor Trigger

Zone (CTZ). These emetic actions are well inhibited by dopamine receptor antagonists like

metoclopramide and domperidone (Ferrari and Donlon, 1992; Minami and Endo, 2003).

Other neurotransmitters that are involved in the vomiting reflex include histamine,

acetylcholine (Ach), endorphins, Gamma-Amino Butyric Acid (GABA) and

endocannabinoids. Histamine and acetylcholine are important in the induction of motion

sickness. Consequently, studies investigating anti-histaminics and anti-cholinergics showed

little or no effect

against chemotherapy-induced nausea and vomiting (Herrstedt, 1997).


14

1.3.2. Receptors involved in cisplatin induced nausea and vomiting:

As discussed above, several neurotransmitters (histamine. acetylcholine, dopamine,

serotonin) and neuropeptides (substance P) are involved in the vomiting reflex, these

neurotransmitters act via specific receptors to induce vomiting (Grelot and Esteve, 2009;

Grunberg and Koeller, 2003; Ray et al., 2009).

Indeed selective activation of dopamine D2 receptors induces vomiting. Upto 1990s the

dopamine receptors were considered to be of two types i.e. D1 and D2 (Clark and White,

1987). Apomorphine, a non-selective dopaminergic agonist is reported to cause emesis in

animals and man (Andrews et al., 1990). The D2 receptors located in the CTZ/AP of the

vomiting center are thought to be involved in apomorphine induced vomiting (King, 1990).

The advent of more advance molecular techniques has led to the discovery of additional

subtypes of D1 and D2 receptors where the D1 receptor family consists of D1 and D5 sites

while D2 receptor consists of D2, D3 and D4 sites (Missale et al., 1998).

There are now much evidences about the role of 5HT receptors and, in particular, 5HT3

receptors in the mediation of vomiting induced by cytotoxic agents (Higgins et al., 2012).

Many serotonin receptor subtypes ranging from 5HT1 through 5HT7 have been identified

(Humphrey et al., 1993). 5HT3 receptors are located both centrally and peripherally with

particularly high concentrations being found in GIT. Emesis induced by cisplatin is

associated with the increase in concentration of 5HT in intestinal lumen; the released 5HT

activates the 5HT3 receptors present on vagal afferent terminals which ultimately, leads to

the genesis of vomiting (Hesketh, 2008).


15

Tachykinin receptor antagonists have been proved to possess broad spectrum anti-emetic

activity than the 5HT3 receptor antagonists - the therapy of choice to control vomiting

associated with cancer chemotherapy, that acts centrally (Grelot and Esteve, 2009). The term

tachykinin was invented in the early 1970s (Maggi, 1995). These peptides exert a plethora of

biological effects in the body mediated through three types of neurokinin receptors

identified as NK1, NK2 and NK3 receptors. Compounds that are antagonists at NK1 receptors

minimize vomiting induced by ipecac, cisplatin, apomorphine and radiation (Ariumi et al.,

2000; Diemunsch and Grelot, 2000; Hesketh, 2001; Saito et al., 2003).

Cholinergic and histaminergic (H1) receptors have been reported to be present in the

brainstem (Pollard et al., 1993; Wamsley et al., 1981), which are involved in the mediation

of motion sickness (Lucot, 1989). Histamine receptor antagonists like diphenhydramine,

promethazine, dimenhydrinate etc, the selective muscarinic receptor antagonist hyoscine

(scopolamine) and the non-selective M3/M5 receptor antagonist zemifenacin are in practice

for the treatment of motion induced vomiting.

The anti-emetic activity of the Phyto and synthetic cannabinoids has been investigated for

several decades and are useful anti-emetics for the treatment of chemotherapy induced

vomiting in clinical setups. The anti-emetic efficacy of cannabinoids has been proved to be

superior or equivalent to dopamine D2 receptor antagonists however, the efficacy of tested

compounds is not as good as that of 5HT3 receptor antagonists (Gralla et al., 1999). Several

cannabinoids can block cisplatin and apomorphine induced vomiting in different animal

models including pigeon, cat and least schrew (Cryptotis parva) (Darmani and Pandya,

2000; Ferrari et al., 1999; McCarthy and Borison, 1981; Stark, 1982). Upto date two types


16

of cannabinoid receptors named CB1 and CB2 have been identified and investigated.

Cannabinoid CB1 receptors have been found on central and peripheral neurons, while CB2

receptors are mainly found peripherally. Recent studies have shown that diverse range of

cannabinoids exerts their anti-emetic activity against cisplatin induced vomiting via agonism

of CB1 receptors, as their anti-emetic activity is reversed by CB1 receptor antagonist, SR

141716A (Darmani N.A and Sim-Selley L.J, 2003; Darmani, 2001b).

1.4. Anti-emetics:

The purpose of anti-emetic therapy is to abolish nausea and vomiting. Over the past twenty

years nausea and vomiting were the common side effects of cancer chemotherapy that

forced ~ 20% of patients to refuse the curative chemotherapy (Herrstedt, 2002). Basic and

clinical research over the last 20 - 25 years has led to the development of remedies in control

of CIV.

1.4.1. Current anti-emetic drugs:

The anti-emetic treatment has been improved, with the increased number of agents

available. Selection of the proper anti-emetic depends on patient’s emetic risk. Newer agents

are used not only to control CIV but also are helping to reduce hospitalization and time

required in ambulatory setting. Considerations of enhancing quality of life and to reduce

costs of treatment while achieving maximal efficacy are appropriate. The identification of

different neurotransmitter systems and related receptors that play key roles in the mediation

of drug induced vomiting has contributed progress in the development of anti-emetics. In

early clinical practice agents that bind to dopamine receptors such as Phenothiazines were

the drugs of choice, as dopamine receptors were found in high concentration in CTZ/AP


17

(Darmani and Crim, 2005). However, the introduction of newer chemotherapeutic agents

with their prominent adverse effects of nausea and vomiting resulted in failure of dopamine

receptor antagonists.

During the past 15 years, there has been much advancement in the control and management

of CIV. The introduction of 5HT3 receptor antagonists in 1990s dramatically revolutionized

the treatment of chemotherapy induced vomiting in clinical setups. 5HT3 receptors are

present both centrally and peripherally with particularly high concentration being found in

GIT. Subsequently, dexamethasone in combination with 5HT3 receptor antagonists

improved the results for patients receiving high to moderate emetogenic chemotherapy

(Einhorn and Rapoport, 2005; Wang et al., 2009).

Recently, special attention has been paid on the role of neuropeptides such as tachykinins,

since they have been identified immunohistologically in the DVC of the ferret. These

peptides participate in many body functions through G-protein coupled receptors (GPCRs)

known as NK1, NK2 and NK3 receptors (Maggi, 1995). Substance P has been proved to be

involved in the induction of vomiting through activation of NK1 receptors (Hesketh and

Grunberg, 2003). Aprepitant, vofopitant, ezlopitant and natupitant are the NK1 receptor

antagonists currently in use for the management of CIV especially in combination with other

anti-emetics (Diemunsch and Grelot, 2000).

1.5. Natural products in drug discovery:

Nature is an in-exhaustible source of novel chemical entities and has been a source of

medicinal agents for thousands of years, as a remarkable number of modern drugs find their

derivation from natural products. The nature’s terrestrial flora and fauna constitute a


18

sophisticated system of traditional medicine that has been in existence for thousands of years

and the intrinsic dependence of human beings on nature has invoked tremendous interest in

the scientific world, which ultimately led to the isolation of large number of chemical

entities with significant biological activities (Balunas and Kinghorn, 2005; Kinghorn, 2001;

Newman et al., 2000). In the more recent history, the use of plants as medicine has involved

the extraction and isolation of active constituents, beginning with the isolation of morphine

from Papaver somniferum in the 19th

century (Kinghorn, 2001).

1.5.1. Selected plants having anti-emetic potential:

1.5.1.1. Cannabis sativa (cannabinoids):

Uptill now no plant has been extensively studied as Cannabis sativa (CS, family;

Cannabinaceae) and it may be stated that this plant is the most controversial in the history

of mankind. Cannabis is the most psychoactive drug and is the most single popular illegal

drug. Worldwide more than 160 million people are abusing this drug and their number is

increasing day by day (Mechoulam, 1986).

The plant CS contains a lot of organic chemicals as true with other botanical species; CS

contains mono and sesquiterpenes, aromatics, carbohydrates and a variety of nitrogenous

compounds and can be successfully extracted with hexane, petroleum ether and benzene

(Doorenbos et al., 1971). Interest has been focused on the resinous material found on the

flowering tops of female plant and as microscopic exudates on the surface of ariel parts of

either sex (Shulgin, 1968). The group of compounds isolated from this plant is known as

cannabinoids uptill now, already 70 different types of cannabinoids have been identified,

several of which are found active biologically in the one way or other (Mechoulam, 1986).


19

During the past 25 years, a lot of literature has been reported on the therapeutic potential of

CS; even the potential of cannabis was largely ignored until the discovery of

endocannabinoid system. Now-a-days it is believed that many of our body functions are

controlled by endocannabinoid system. The anti-emetic action of CS was not anticipated for

years despite of anecdotal evidences, although the anti-emetic action of Delta-9-

tetrahydrocannabinol (Δ9-THC, Figure 1.2) had been suggested in 1972. Comparison with

other anti-emetics like prochlorperazine and metoclopramide has been done and was found

to be superior (Russo, 2001). It has been known that cannabinoid receptors are present in the

brain, in the immune system and other organs of the body. Uptill now two types of

cannabinoid receptors, CB1 and CB2 have been cloned and characterized for their biological

functions (Howlett and Barth, 2002). Cannabinoid CB1 receptors are primarily present on

central nervous system and peripheral neurons, while CB2 receptors are mainly present on

immune cells. There are evidences for the involvement of CB1 receptors in the mediation of

its anti-emetic effect whose stimulation leads to the inhibition of transmitter release

(Darmani, 2001a).

Figure 1.2 Delta-9-Tetrahydrocannabinal


20

Figure 1.3 A photograph of Cannabis sativa:

1.5.1.2. Zingiber officinale (gingerols):

Zingiber officinale (ZO) which is commonly known as “ginger” belongs to family

Zingiberaceae. This plant is widely distributed in India, Pakistan and Malaysia and is

cultivated in Bangladesh, Taiwan, China, India and Nigeria (Shadmani et al., 2004). In

Chinese and Unani’s Tibb system ginger has been used for the treatment of nervous

diseases, rheumatism, constipation and gingivitis while in Asian medicine it is used as

carminative, diuretic, appetite stimulant and anti-emetic (Tyler, 1993). Throughout the

world ginger rhizome is used as a spice and flavoring agent (Tyler, 1988). The characteristic

aroma of ginger rhizome is due to volatile oils present in concentration of 1-3 % and the

pungent smell is attributed to oleoresin (Tyler, 1993).


21

Figure 1.4 Gingerol

The major components present in ginger extract which is a mixture of homologues having

10, 20 and 14 carbon atoms in side chain that are designated as gingerols (Tyler, 1988)

(Figure 1.4). Acetone fraction of ginger (rich in gingerols) have been proved to be effective

against cisplatin induced vomiting in dogs (Sharma et al., 1997) advocating the medicinal

usefulness of the acetone fraction. Ginger have been screened for a lot of pharmacological

activities including molluscicidal (Adewunmi et al., 1990), antitussive, antipyretic,

analgesic, anti-emetic (Frisch et al., 1995) and cardiotonic activities. The notion that ginger

may be effective for the control of nausea and vomiting is supported by several lines of

evidences. Animal studies are indicative for its anti-emetic activity (Frisch et al., 1995)

when induced by cisplatin (Qiu-hai et al., 2010; Sharma et al., 1997) or cyclophosphamide

(Yamahara et al., 1989)


22

Figure 1.5 A photograph of Zingiber officinale (ginger):

1.5.1.3. Bacopa monniera (bacosides):

Bacopa monniera (BM), locally known as Jal Neem bootee, water hyssop, Herpestis

monniera and Brahmi in India (Sanskrit word), is a small creeping succulent herb, having

oblong small leaves, numerous branches, and whitish flowers, found in marshy places in

Europe, Asia, including Pakistan (Qureshi and Raza Bhatti, 2008; Subhan et al., 2010b).

This plant belongs to family “Scrophulariaceae”, has been reported to have 220 genera and

3000 species. BM has a century’s old clinical utility in Ayurvedic system of medicine for

various pathological conditions, like anxiety (Bhattacharya and Ghosal, 1998), epilepsy

(Mathew et al., 2010), memory deficits (Roodenrys and Booth D. Bulzomi, 2002), as cardiac

and nervous tonic, anti-inflammatory (Channa et al., 2006) and anti-nociceptive (Rauf et al.,

2011; Subhan; et al., 2010). BM is also reported to be having antidepressant activity

comparable to selective serotonin reuptake inhibitor-imipramine (Sairam et al., 2002). BM


23

major ayurvedic indication is for the management of poor cognition, and lack of

concentration, as nootropic and gastric aid. Furthermore, it has been confirmed that BM

reduces the effects of morphine withdrawal in guinea pig ileum (Sumathi et al., 2002) that is

suggestive for its usefulness in reducing withdrawal symptoms induced by morphine.

Additionally, BM also suppresses acquisition and expression of morphine tolerance (Rauf et

al., 2011). BM has been proved to be having antioxidant activity and protects kidney, liver

and brain from morphine toxicity (Ghosh et al., 2007). In addition BM has been reported to

be having anti-dopaminergic effect reported by others (Sumathi et al., 2007) and our

laboratory as well (Rauf et al., 2012).

Figure 1.6 A photograph of Bacopa monniera:

BM phytochemical study reveals the presence of many active moieties including tannins,

flavonoids, triterpenoids and saponins (Subhan et al., 2010a). The major bioactive

component of BM, is Bacoside "A" & "B", the "B" component is in fact an optical artifact of

"A" produced during isolation. Bacoside “A” is actually a mixture of four major


24

components, i.e. bacoside II (Figure 1.7), bacoside A3 (Figure 1.8), bacosaponin C (Figure

1.9) and an isomer of bacopasaponin C (Deepak et al., 2005). The pharmacological profile

of BM is mainly attributed to bacosides which are present in high concentration in the n-

butanol fraction (Kahol et al., 2004).

Figure 1.7 Bacoside II


25

Figure 1.8 Bacoside A3

Figure 1.9 Bacosaponin C


26

1.6. Vomit models:

Ethically humans cannot be used as first line experimental model for studying various

diseases, and their treatment probabilities to screen new legends, synthetic or herbal

molecules. Animals are therefore used as models to study prognosis, pathology, physiology

and responses of newer molecules as treatment options (Mitruka et al., 1982).

Since 18th

century, animal models were inducted to assess newer agents/molecules for

prevention and treatment of various diseases/complications. Furthermore, attempts were

made to induce pathologies with similarity to human diseases to better understand molecular

level prognosis and treatment options (Nomura, 1997). In 19th

century, animal usage

underwent a real boom and newer models were screened and approved to study various

pathological disorders (Mitruka et al., 1982).

Animal models are selected on the basis of their capability to induce behavior, normative

biology, or pathology and have both behavioral, neuro histochemical resemblance of the

pathology, signs and progression of the disease which are comparable to humans. The

induced pathology or behavior in animals must have resemblance in common with human

diseases. The model is considered suitable when it is affordable, easy to breed, and have

similarity with human subjects (Mitruka et al., 1982; Nomura, 1997).

Now-a-days all potential legend molecules as future candidates need preclinical screening in

various approved preclinical animal models with approved standards both qualitatively and

quantitatively (McBride and Li, 1998).

The criterion to be accepted as animal model of disease is that it should be isomorphic or

homologous. An animal model is said to be Isomorphic when it exhibits holistically or in

part the clinical picture (behavioral, neurochemical, etc) observed in human disease, even


27

though the cause of disease may be different in animals than humans (McBride and Li,

1998; Medina and Reiner, 1995). While in homologous system both cause and induced

changes are same in both humans and in the animal; the homologous models are more

preferred and authentic for preclinical assessment of novel molecules/therapies (Emmett-

Oglesby et al., 1990).

In research on vomiting, there are important species differences, In general, vomiting

phenomenon is not so important for survival but is highly advantageous for the body to get

rid of toxic substances which have been ingested. The animals which are commonly used in

laboratory research including rats, mice, rabbit, guinea pig and hamster are lacking the

vomiting reflex therefore, the alternative options are cats (Lucot, 1989), dogs (Topal et al.,

2005), pigeon (Preziosi et al., 1992), Suncus murinus (Matsuki et al., 1988), least schrew

(Darmani, 2001b) and some other species capable of vomiting response.

Now-a-days many national/international laws ensure the ethical use of animals to avoid

unwarranted distress during experimental procedures and regulate bodies and structures that

ensure Helsinki declaration. Additionally international research communities ensure abiding

by such ethical laws and procedure, by not accepting the experimental data for publication,

in which Helsinki declaration is either violated or procedures not validated and verified by

ethical committees of concerned University or research institution (Mitruka et al., 1976).

1.6.1. Pigeon:

The pigeon is a specie that has been used in emesis research for many years and responds to

a number of different emetic stimuli including cardiac glycosides (Hanzlik and Wood, 1929;

Hildebrandt and Paas, 1953), reserpine (Gupta and Dhawan, 1960), sigma ligands (Hudzik,

1992), 5-HT3 receptor agonists (Wolff and Leander, 1995) and the chemotherapeutic drugs


28

cyclophosphamide (Wolff and Leander, 1997) and cisplatin (Navarra et al., 1992). In terms

of its translational value, it can be used to assay the anti-emetic activity of several classes of

drugs like NK1 receptor blockers (Tanihata et al., 2003; Wolff and Leander, 1995) and

glucocorticoids (Tanihata et al., 2004). A few studies have looked at lower doses of cisplatin

(4 mg/kg), where the response can continue for several days and is mediated by vagal and

reserpine-sensitive monoaminergic systems (Tanihata et al., 2000; Tanihata et al., 2003). In

this study, the pigeon was chosen firstly, due to the ease of availability, breeding and

handling and secondly, based on the previous studies that pigeon has an easily quantifiable

vomiting response (Navarra et al., 1992; Preziosi et al., 1992; Tanihata et al., 2000).

1.6.2. Suncus murinus:

Suncus murinus (S. murinus, House musk shrew, family, Soricidae) locally known as

Chuchunder, has been used in emesis research since 1980s (Matsuki et al., 1988), to study

the mechanisms of chemotherapy induced vomiting. S. murinus is an acceptable animal

model for the study of vomiting induced by chemotherapy (Sam et al., 2003). The vomiting

response can be observed with emetic challenge by motion (Ueno et al., 1988), X-irradiation

(Ito et al., 2003), copper sulphate, nicotine and cancer chemotherapeutic agents (Rudd et al.,

1999; Sam et al., 2003). We therefore selected S. murinus as second animal model for

studying the anti-emetic potential of selected plant extracts.

1.7. Aims and objectives of the study:

1. To screen various extracts of Cannabis sativa, Bacopa monniera and Zingier officinale

for their intrinsic anti-emetic activity against cisplatin induced Retching plus Vomiting

(R + V) in animal model(s).


29

2. To find out the effects of various combinations of Cannabis sativa, Bacopa monniera

and Zingiber officinale on the spectrum of anti-emetic activity against cisplatin induced

Retching plus Vomiting (R + V) in the vomit model(s).

3. To examine the involvement of gastrointestinal motility/gastric emptying in cisplatin

induced vomiting in pigeon model.

4. To evaluate the impact of Cannabis sativa, Bacopa monniera, Zingiber officinale

extracts and their combinations on neurotransmitters implicated in cisplatin induced

Retching plus Vomiting (R + V) in specific brain areas and intestine in pigeon model.

5. To observe the effect of BM extracts alone and in combination with Cannabis sativa

active constituent (herbal/synthetic) on C-fos protein expression in specific brain areas

involved in the act of vomiting in Suncus murinus.

Chapter 2 Methods

30

Chapter 2

Methods

Chapter 2 Methods

31

2.1. Animal husbandry:

2.1.1. Pigeon:

Pigeons of either sex or breed were kept in the breeding facility of the Department of

Pharmacy, University of Peshawar (Fig 2.1). The pigeon houses/holes were constructed of

wood with openings to allow the pigeon’s liberty for the purpose of exercise, food and water

acquisition and their re-entry to the house without special help from the keeper. At the same

time the houses were constructed in a way to protect the pigeons from harsh weather and

provide them nesting places to raise their squabs while, the wire mesh enclosure of that

specific locality keep the pigeons safe from predators. The habitat contained upto 40 houses,

habitat and each house was having dimensions of 12 × 8 and 1.5 × 1 feet respectively

(Figure 2.1) and is having proper heat control, ventilation and dryness. Keeping in view the

purpose of breeding, the pigeons were kept on specific diet composed of Millet + Wheat

(locally available food).

For experimental purpose, pigeons of either sex or breed weighing between 250 – 350g were

used. They were housed in groups of eight at 22 - 26 ˚C under a 12 hr light/dark cycle and

had free access to food and water before and during experimentation (Figure 2.2).

Chapter 2 Methods

32

Breeding facility for pigeon at Department of Pharmacy, University of Peshawar:

Figure 2.1 Breeding facility for pigeons at Department of Pharmacy, University of

Peshawar; composed of 40 houses having dimensions of 1 × 1.5 feet each and a wire mesh

covered area of 22 × 16 feet with availability of food and water.

8 f

eet

12 feet

1.5 feet

1 f

eet

22 feet

16 feet

Chapter 2 Methods

33

2.1.2. Suncus murinus:

Suncus murinus (S. murinus) were bred at the “Animal care and laboratory services” The

Chinese University of Hong Kong (CUHK), under controlled conditions and were

transported through online request to animal holding room of the School of BioMedical

Sciences (SBS), in special cages having dimensions of 1.5 × 1 feet.

For experimental purpose, male S. murinus (40 – 60 grams) obtained from the breeding

facility of the Chinese University of Hong Kong were housed in a temperature controlled

room (24 ± 2oC) with artificial lighting (0600 – 1800), humidity level being maintained at

50 ± 5 %. Pelleted cat chow (Feline Diet 5003, PMIR Feeds, St. Louis, U.S.A) and water

were available ad libitum.

2.2. Video recording setup:

2.2.1. Recording setup for pigeon experiments:

All the behavioral experiments on pigeon model were conducted in “Bioassay laboratories”

at Department of Pharmacy, University of Peshawar. The video recording setup consisted of

an IPIR camera connected with PC through additional lane port with saltec powerlink

uninterrupted power supply. The camera was fixed in a way to monitor and record animal’s

behavior (n = 8) in confining cages made of stainless steel having dimensions of (1 × 1 × 1)

feet and painted white. The specially designed confining cages were arranged in a stand of

two shelves each containing four cages. Stand made of wood having dimensions (4 × 3 × 1)

feet and painted white for colour uniformity and properly labeled showing the number of

animals (Figure 2.3).

Chapter 2 Methods

34

Specially designed confining cages for video recording of pigeon experiments:

Figure 2.2 Specially designed confining cages (1 × 1 × 1 feet) made of stainless steel

arranged in wood stand (4 × 3 × 1 feet) in two shelves for video recording of pigeon

experiments.

*

*

4 feet

3 f

eet

1 feet

1 f

eet

1 feet

1 feet

Chapter 2 Methods

35

Video recording setup for pigeon experiments:

Figure 2.3 Recording setup for pigeon experiments at “Bioassay Laboratories”, Department

of Pharmacy, University of Peshawar; consisting of Cats eye camera connected with PC

through lane port.

2.2.2. Recording setup for Suncus murinus experiments:

All experiments on S. murinus were conducted in a quiet room having light intensity of 15 ±

2 Lux. Animals were transferred to clear perplex observation chambers (21 × 14 × 13 cm3)

before experimentation and 12 hr acclimatization time was given to each group (n = 5 - 6).

Food and water were available before and during experiment. The animal behavior was

Chapter 2 Methods

36

recorded using closed circuit cameras (Panasonic WV-PC240, China) mounted above each

chamber which were connected to a hard disk recorder (Everfocus, EDSR900, Socio, Ind.

Ltd, Taiwan) coupled with a desktop computer (Dell OPTIPLEX GX 270) (Figure 2.4).

Image of each animal and the center of gravity were tracked by the camera. Analogue video

signals were digitalized by the computer. Etho Vision Color-Pro software (Version 3.1,

Noldus Information Technology, Netherland) was used for the automated tracking and

analysis of animal movement and activity. From the video recordings, the position and

center of gravity of each animal was sampled every 0.2 second to calculate the total distance

moved and average velocity of the animal with a cut up filter of 2 cm during data analysis.

Video recording setup for Suncus murinus experiments:

Figure 2.4 Recording setup for S. murinus experiments at Brain Gut laboratory, School of

BioMedical Sciences, the Chinese University of Hong Kong; composed of Panasonic close

circuit cameras mounted on each chamber and connected to hard disc recorder.

Chapter 2 Methods

37

2.3. Quantification of vomiting:

2.3.1. Quantification of vomiting in Pigeon:

On the day of experiment, the pigeons were transferred to individual cages specially

designed for video observation (Figure 2.2) and cisplatin (7 mg/kg) was administered

intravenously via the brachial wing vein (Tanihata et al., 2000). The dose of cisplatin was

selected on the basis of our studies which induced vomiting in all the animals tested (chapter

3). The behavior of the pigeon was recorded with a video recording setup upto 24 hr. Food

and water were available during the observation period and each animal was used once. The

vomiting response (Figure 2.5) with or without oral expulsion was considered as one

vomiting episode (Preziosi et al., 1992); One vomiting episode comprised of 2 to 80 jerks

(vomiting behaviors). Latency to first emesis, emetic episodes and jerks were recorded. The

parameter to split two emetic episodes was the complete relaxation of the animal.

Chapter 2 Methods

38

Pigeon showing the act of vomiting:

Figure 2.5 Pigeon showing the act of vomiting, an acceptable animal model (adult weight

250 – 350 grams) for screening of chemical entities for their anti-emetic potential. Photo

taken at Bioassay laboratory, Department of Pharmacy, University of Peshawar.

2.3.2. Quantification of vomiting in Suncus murinus:

Vomiting episodes were characterized by rhythmic abdominal contractions that were either

associated with the expulsion of vomitus (vomiting) or not associated with the expulsion of

any material (retching). The number of Retching plus Vomiting (R + V) episodes and the

latency to first vomit were recorded. Episodes of R + V were considered separate when there

was a delay of 2 s or when animal changed its position in the observation cage.

Chapter 2 Methods

39

Suncus murinus showing the act of vomiting:

Figure 2.6 A Photograph of a vomiting S. murinus (house musk schrew), a small mammal

having weight of 50 - 80 grams that is an acceptable model for the study of nausea and

vomiting and for the development of new anti-emetic drugs. Photo taken at School of

BioMedical Sciences, The Chinese University of Hong Kong.

2.4. Measurement of locomotor activity in Suncus murinus:

Total distance moved and average velocity of the animal during the observation period was

calculated with the help of Etho vision color-pro software (Version 3.1, Noldus Information

Technology, Netherland).

Chapter 2 Methods

40

2.5. Plant collection and extraction:

2.5.1. Cannabis sativa:

The plant was collected at a farm, from Malakand Division (Khyber Pukhtoonkhwa,

Pakistan) at its bloom season. The plant was authenticated by Prof. Dr. Muhammad Ibrar,

Department of Botany, University of Peshawar and a specimen was deposited at the

herbarium with voucher No 8717. Leaves and flowering tops were separated, shade dried,

coarsely grinded and then extracted with different solvents based on increasing order of

polarity as shown in the extraction scheme (Doorenbos et al., 1971).

Chapter 2 Methods

41

Extraction scheme for Cannabis sativa:

Cannabis sativa (coarsely grinded plant material)

Figure 2.7 Extraction scheme for Cannabis sativa to get hexane, n-butanol and methanolic

fractions.

2.5.2. Bacopa monniera:

The plant was collected in November from Rumalee stream near Quaid-e-Azam University,

Islamabad Pakistan. The plant was authenticated by Prof. Dr. Muhammad Ibrar, Department

of Botany, University of Peshawar and a specimen was deposited at the herbarium with

voucher No 7421. The Ariel parts were separated, shade dried and coarsely powdered.

Marc

Extract

Evaporated

CS-HexFr (yield 101 grams) n-butanol X2

Extract

Marc Evaporated

CS-ButFr (yield 15 grams)

Methanol X2

Extract

Evaporated

CS-MetFr (yield 18 grams)

Marc

01 kg, n-hexane X2

Chapter 2 Methods

42

Soxhlet apparatus was used for extraction purpose. Furthermore, fractionation was done to

get the n-butanol fraction which is reported to be bacoside rich fraction (Kahol et al., 2004).

The extraction scheme to get the methanol and n-butanol fraction is given hereby.

Extraction scheme for Bacopa monniera:

Step 1:

Bacopa monniera (coarsely grinded plant material)

Extract

Marc Evaporated

Residue (yield 103 grams)

Acetone

Extract

Evaporated

Residue (yield 61.12 grams)

Marc

Extract

Methanol (soxhlet apparatus) X3

Marc Filtered, Evaporated

Residue (yield 60 grams)

01 kg, n-hexane

Chapter 2 Methods

43

Step 2:

Methanol extract (Semi solid)

Figure 2.8 Extraction scheme for Bacopa monniera methanolic (step 1) and n-butanol

fraction (step 2).

200 grams

1L methanol, filtered

Filtrate

500mL, acetone, filtration, X5

Ppt (bacosides) Filtrate

+ H2O

Aq solution

+ n-butanol, X3

Aq Phase n-butanol Phase

Evaporated


Chapter 2 Methods

44

2.5.3. Zingiber officinale:

The plant rhizome were purchased from a local market at Mardan and was authenticated by

Prof. Dr. Muhammad Ibrar, Department of botany, University of Peshawar, a specimen was

preserved in the herbarium for future reference (voucher No 20017 - pup) . The rhizomes

were crushed and dried under shade and extracted using maceration method (Sharma et al.,

1997). The extraction scheme is given below.

Extraction scheme for Zingiber officinale:

Zingiber officinale (coarsely grinded rhizomes)

Figure 2.9 Extraction scheme for Zingiber officinale acetone fraction.

01 kg, acetone, X3

Marc Extract

Evaporated


Chapter 2 Methods

45

2.6. Chemicals and drugs:

Table 2.1

S.No Name of chemical/drug Source Use

1. Acetone (commercial grade) Haq Chemicals,

Peshawar, Pakistan

Extraction

2. Acetonitrile (HPLC grade) Fisher scientific U.K Component of HPLC mobile

phase

3. Bacosides A3, II and

Bacosaponin C

Mississippi University,

U.S.A

Bacoside standards

4. Cisplatin Korea United Pharm.

Inc Korea

Highly emetogenic anti-

neoplastic agent for induction of

vomiting in Pigeon and S.

murinus

5. Charcoal Haq Chemicals,

Peshawar, Pakistan

GIT motility studies

6. Carbachol Sigma Gmbh, Germany Cholinergic agonist

7. Dopamine (DA) Acros organics,

Belgium

Neurotransmitter standard

8. De ionized water Applied Pharmacology

lab, Department of

Pharmacy, University

of Peshawar, Pakistan

For preparation of mobile phase

& drug solutions

9. Ethylene diamine tetra acetic

acid (EDTA)

Merck (local distributer

in Pakistan)

Chelating agent

10. Homovanillic acid (HVA) Acros organics,

Belgium


11. Hydrogen per oxide (H2O2) Merck (local distributor

in Hong Kong)

Cleaning agent in histology

12. Methanol (commercial grade) Haq Chemicals,

Peshawar, Pakistan

Extraction

Chapter 2 Methods

46

13. Metoclopramide (MCP) GlaxoSmithKline,

Pakistan, Ltd

Anti-emetic (D2 blocker)

14. Methanol (HPLC grade) Fisher scientific, U.K Component of HPLC mobile

phase

15. Noradrenaline (NA) Alfa Aesar, U.K Neurotransmitter standard

16. n-butanol (commercial grade) Haq Chemicals,

Peshawar, Pakistan

Extraction

17. n-hexane (commercial grade) Haq Chemicals,

Peshawar, Pakistan

Extraction

18. N-(2-Mercaptopropionyl)

glycine

Sigma Aldrich Gmbh,

Germany

Antioxidant

19. Palonosetron (PalS) Helsinn Heathcare SA,

Switzerland

5HT3 receptor antagonist

20. Paraformaldehyde (PFA) Electron Microscopy

Science, China

Fixative

21. per mount Panreac Quimica SA,

Switzerland

Mounting medium

22. Potassium chloride (KCl) Merck (local distributor

in Hong Kong)

Component of PBS solution

23 Potassium dihydrogen

orthophosphate (KH2PO4)

Merck (local distributor

in Hong Kong)


24. Sodium chloride (NaCl) Merck (local distributor

in Hong Kong)


25. Sodium dihydrogen

orthophosphate (NaH2PO4)

Merck (local distributor

in Hong Kong)

PH Buffer

26. Triton-X 100 Sigma Gmbh, Germany Detergent

27. Vectastain Elite ABC kit Vector laboratories,

Burlingame, U.S.A

C-fos staining kit

28. WIN 55, 212-2 Sigma Gmbh, Germany Cannabinoid receptor type 1

(CB1) agonist

29. 0.45µ filter Sartorious, Germany Filtration

Chapter 2 Methods

47

30. 1-octane sulphonic acid Fisher scientific, U.K Ion pairing agent

31. 3, 4-dihydroxy phenyl acetic

acid (DOPAC)

Acros organics,

Belgium


32. 5-hydroxytryptamine (5HT) Acros organics,

Belgium


33. 5-hydroxy indolacetic acid

(5HIAA)

Acros organics,

Belgium


2.7. Instruments and apparatus:

Table 2.2

S.NO Item Source

1. Analytical balance AX 200 Shimadzu, Japan

2. Boeco Rotary Evaporator 400 SD Boeco, Germany

3. Column Teknokroma; Tracer extrasil ODS1 (4.6 mm x 150

mm, 3 μm)

(Barcelona), Spain

4. Centrifuge Centurion scientific, Ltd,

U.K

5. Cover slips Thermo Scientific, China

6. Column, Peurospher Star RP.C18e, HibarR RT 250-4.6(5

μm)

Merck, Germany

7. Desktop computer, Dell OPTIPLEX GX 270 U.S.A

8. ECD detector, Coulchem III, model 5300, along with dual

analytical cells, model 5011

ESA, U.S.A

9. Freezing microtome (Cryostat) Shandon

10. hard disk recorder, Everfocus, EDSR900 Taiwan

Chapter 2 Methods

48

11. High Performance Liquid Chromatography (HPLC) system,

including

CBM (communication boss module)-20A

Double Pumps-LC-20AT

Injection port, 7725i (Rheodyne)

Shimadzu, Japan

12. Homogenizer A. Daigger & company,

Inc. U.S.A

13. Lux meter Extech Instrument,

Hong Kong

14. Micropipette (10 – 200 μL) Treff Lab, France

15. pH meter, 3505 Jenway, U.K

16. Panasonic WV-PC240 for S. murinus experiments China

17. Refrigerator (- 80o C) IlShin, DF 8517, Korea

18. Refrigerator PEL Co. Pakistan

19. Soxhlet Apparatus Locally made (Peshawar

Pakistan)

20. Shaker (Heidolph Promax 2020) Lab Plant, U.K

21. Super frost microscope slides Thermo Scientific

22. UV detector, SPD-20A Shimadzu, Japan

23 Vacuum Pump Roeker 300, Taiwan

24. Vacuum filtration assembly Boeco, Germany

25. Video recording camera, Cats eye IPIR for Pigeon

experiments

Korea

26. Vortex mixer, Gyromixer Pakland scientific,

Pakistan

27. Ziess Axioskop-2 Plus microscope equipped with Zeiss

Axioskop-2 camera

Carl Zeiss Inc.

Thornwood, U.S.A

28. 24-wel plates Nunc, Denmark

Chapter 2 Methods

49

2.8. Drug formulation:

Cisplatin was dissolved in normal saline by heating upto 60oC and then cooled upto

40 - 45oC before administration. Cannabis Sativa fractions were dissolved in absolute

ethanol, mixed with emulsifier and made the volume with distilled water in such a way that

the final mixture consists of ethanol : emulsifier : distilled water in a ratio of 5 : 5 : 90

(Feigenbaum et al., 1989). The reference drug palonosetron, methanol (BM-MetFr) and n-

butanol (BM-ButFr) fractions of Bacopa monniera (BM) and acetone fraction of Zingiber

officinale (ZO) were dissolved in normal saline by gentle agitation and sonication was

carried out to dissolve the extract/drug and to obtain uniform solutions.

2.9. Drugs administration:

Cotton wool and methylated spirit were used to sterilize the skin of pigeon prior to all drug

administrations. Intravenous and intramuscular routes were used in the pigeon except for

charcoal which was administered orally for the assessment of gastrointestinal motility, while

subcutaneous and intraperitoneal routes were used in S. murinus.

2.9.1. Intravenous administration:

In pigeons, cisplatin was administered though brachial wing vein by following the method

of Tanihata et al (Tanihata et al., 2000) using 1 mL non-pyrogenic syringe with sharp

painless needles of 27G × 1/2". The pigeon was firmly griped in the hand in a way to keep

the pigeon relax. The wing vein was made prominent using swab, soaked in methylated

spirit before injection and after injection the swab was placed on the injection site for some

time, to avoid un-necessary bleeding from the vein.

Chapter 2 Methods

50

2.9.2. Intramuscular administration:

All the drugs (extracts/standards) in emetic and gastrointestinal motility studies in pigeon

were administered intramuscularly using chest muscle except for cisplatin and charcoal

which were administered intravenously and orally, respectively. 1 - 2 mL non-pyrogenic

syringes with sharp painless needles of 23G × 1" were used.

2.9.3. Intraperitoneal administration:

In S. murinus, cisplatin solution was administered intraperitoneally. During intraperitoneal

administration cisplatin was directly administered in to the peritoneal space surrounding the

abdominal organs, avoiding direct injection into the organs. The animals (S. murinus) were

held firmly from the loose skin behind the neck and holding the animal tail firmly in the

little finger exposing the ventral side of the animal. The needle of the syringe was gently

inserted in the lower right quadrant of the abdomen. The plunger was then pressed to

discharge the contents into the animal peritoneal cavity. 1 mL non-pyrogenic syringes with

sharp painless needles of 26G × 1" were used.

The procedure needs proper training for handling S. murinus, as the animals in colony are

often engaged in alarm vocalization, wrestling, tail biting and body biting. Furthermore, the

animals are having sharp teeth that they use for biting objects coming in contact with their

body and especially during handling for experimental purpose. In this regard, lack of

experience may lead to unexpected animal distress or injury to the concern person and for

safety purpose; thicker protective gloves (made of leather) are used in handling.

Chapter 2 Methods

51

2.9.4. Subcutaneous administration:

The drugs (extracts/standards) were administered through subcutaneous route in S. murinus.

In this procedure, animals were held firmly on the table in such a way that the loose skin

behind the neck was lifted up with fingers and the drugs administered into the subcutaneous

area through syringe. The plunger was gently pressed to discharge the drugs/contents in the

specified subcutaneous area. 1 mL non-pyrogenic syringes with sharp painless needles of

26G × 1" were used.

2.10. Measurement of gastrointestinal motility:

The pigeons of either sex or breed (n = 6 - 8) weighing 250 – 350 grams were used in GIT

motility study. Animals were starved from food for 18 hours prior to experiment, but were

allowed free access to water. After 80 minutes of CS-HexFr or normal saline (SAL)

administration, 2 mL of a 10% charcoal slurry in 5% gum acacia was administered to each

pigeon orally (Singh et al., 1996). For antagonism, MCP (30 mg/kg) or carbachol (0.1

mg/kg) was administered intramuscularly 5 minutes before drug administration. Pigeons

were killed 20 minutes after being administered with charcoal meal, abdomen was opened,

and small intestine was dissected out, and was placed on a clean surface. The distance

travelled by the charcoal meal from the pylorus was measured. The entire length of the small

intestine was also measured. The percentage distance travelled by the charcoal plug along

the small intestine (from the pylorus to the ceacum) was then estimated for saline, CS-HexFr

and combination of CS-HexFr with MCP or carbachol groups. Percent GIT motility was

calculated with the help of following formula:

Chapter 2 Methods

52

% GIT motility = (Distance travelled by charcoal through small intestine/total length of

small intestine) x 100

2.11. Standardization of Bacopa monniera extracts for bacoside “A” major

components:

Bacopa monniera methanolic fraction (BM-MetFr) and n-butanol fraction (BM-ButFr) were

screened for bacoside “A” major components (bacoside A3, bacoside II and bacosaponin C)

by High Performance Liquid Chromatography (HPLC) with UV using the method of Rauf et

al with little modification (Rauf et al., 2011).

2.11.1. High Performance Liquid Chromatography (HPLC) system for bacoside

quantification:

The HPLC system was composed of LC - 20AT double pump (Shimadzu, Japan) and SPD -

20A UV Visible detector, a Rheodyne injector with 20 μL loop connected with a

communication bus module (model 20 A). The HPLC system had inbuilt Shimadzu software

“LC Solution” Version 1.2 for data analysis.

2.11.2. Preparation of standards:

Standard solutions of all three bacosides (bacoside A3, bacoside II and bacosaponin C) were

prepared by dissolving 2 mg/ mL of HPLC standards of Bacopaside II, Bacoside A3, and

Bacopasaponin C in HPLC grade methanol. Working standard solutions were prepared by

dilution with HPLC grade methanol in seven different strengths ranging from 1 μg to 500

μg/mL.

Chapter 2 Methods

53

2.11.3. Preparation of samples:

Briefly, 50 mg of methanolic extract of BM (BM-MetFr) was dissolved in 10 mL of HPLC

grade methanol and was centrifuged for ten minutes at 3000 rpm. Then the centrifuged

solution was filtered through 0.45 u filter, and was injected into HPLC system for analysis.

In case of butanolic fraction (BM-ButFr) the sample was diluted before injection into the

HPLC system to avoid column overloading. 10 mg of BM-ButFr was taken and dissolved in

10 mL of HPLC grade methanol, centrifuged and filtered through 0.45 µ filter. From this

filtered solution 300 µL was taken and made up the final volume upto 5 mL with HPLC

grade methanol, mixed well using vortex and injected into HPLC system for analysis.

2.11.4. Chromatographic conditions:

The method of Rauf et al (Rauf et al., 2011) was used with little modifications for the

quantification of bacosides. Column used was purospher C18 column (250 x 4.6 mm, 5 μ)

and the mobile phase consisted of phosphoric acid 0.2 % and acetonitrile (62:38 v/v), pH

adjusted to 3.0 with 3 M NaOH. The HPLC system was run at 0.6 mL/ min flow rate using

wavelength of 205 nm. All the peaks were secured in 33 minutes run time. The peaks were

first confirmed by spiking the samples with standard bacosides. The above method was

revalidated for linearity, specificity, accuracy and recovery.

Chapter 2 Methods

54

2.12. High Performance Liquid Chromatography (HPLC) method for

neurotransmitter analysis:

2.12.1. Sample collection & handling:

Animals were killed by decapicitaion and whole brain was excised onto an ice freezing plate

and specified brain areas i.e. area postrema and brain stem and intestinal sample were taken

and stored at - 80O

C refrigeration facility. Since neurotransmitters are highly prone to

degradation because of exposure to light, oxygen and temperature, all brain samples were

collected on ice cold slabs and immediately stored in labeled eppendorf tubes at – 80O C to

avoid neurotransmitters degradation. Samples were handled in such a way to ensure

minimum loss of neurotransmitters due to exposure to light, heat and oxygen.

2.12.2. Preparation of stock solutions:

Stock solutions of all neurotransmitters were prepared by dissolving known quantities of the

neurotransmitters (5 mg/ 10 mL) in 0.2 % Perchloric Acid (PCA) and immediately stored at

– 80O C in labeled glass containers properly covered to protect from light. For calibration

purposes, stock solutions were diluted using known volumes of cold 0.2 % Perchloric Acid

(PCA) to prepare 1.0 μg/ mL solution. These 1.0 μg/ mL solutions were diluted such to give

seven different calibrations between ranges of 100 Pico grams to 400 nano grams. The

different dilutions of standard were used for preparation of calibration curves and further for

spiking of the biological samples during analysis to verify the peaks of respective analyte.

Chapter 2 Methods

55

2.12.3. Sample preparation:

For analysis, the stored brain and intestinal samples were taken, weighed and homogenized

in ice cold 0.2 % PCA at 5000 rpm with a Teflon-glass homogenizer (Wise stir HS 30E).

The samples were then centrifuged at 12000 rpm/ minute (4oC) (Centurion, UK) for thirty

minutes and filtered through 0.45 μ filter. The filtrate was injected directly into the HPLC

system or stored in labeled eppendorf tubes at - 80o C refrigeration facility.

2.12.4. Chromatography:

The High Performance Liquid Chromatography (HPLC) (Shimadzu, Japan), consisted of

Communication Bus Module (CBM, Model 20 A), dual pumps (Model LC - 20AT), an

analytical column Teknokroma; Tracer extrasil ODS1 (4.6 mm x 150 mm, 3 μm), a

Rheodyne injector with 20 μL loop attached to an electrochemical detector (ECD; ESA,

Coulochem III, model 5300) set with an analytical cell (model 5011 A). The

chromatographic data was analyzed using Shimadzu software LC Solution Version 1.2.

Electrodes 1 and 2 of the analytical cell were set at + 200 and − 200 mV respectively, with a

sensitivity of 2 μA, while the guard cell (model 5020) potential was set at 500 mV. The

mobile phase consisted of 94 mM sodium dihydrogen orthophosphate, 40 mM citric acid,

2.3 mM sodium 1-octane sulphonic acid, 50 μM EDTA, and 10 % acetonitrile, pH adjusted

to 3. The above method was revalidated for linearity, specificity, accuracy and recovery.

Chapter 2 Methods

56

2.13. C-fos immunohistochemistry:

2.13.1. Immunohistochemical procedure:

At the end of experiment, the S. murinus were anesthetized using phenobarbitone at the dose

of 40 mg/kg i.p. and intracardially perfused with 100 mL (approx) ice-cold saline followed

by Paraformaldehyde (PFA, 4 %) in Phosphate Buffer Saline (PBS). The brains were then

removed carefully and kept for overnight incubation in 4 % PFA at 4o C. Brain tissues were

embedded in Optimal Cutting Temperature (OCT) compound with the help of liquid

nitrogen and then sectioned at 40 µm in the coronal plane using freezing microtome

(Shandon) and collected in 10 mM PBS. The brain sections were rinsed thrice, each 5 min

with PBS and were incubated in 0.3 % H2O2 in PBS for 60 min at room temperature under

orbital shaking to quench endogenous peroxidase activity. The sections were rewashed with

10 mM PBS three times, each 5 min and blocked with 1.5 % normal goat serum (Vectastain

Elite ABC kit, Vector laboratories, Burlingame, U.S.A) containing 0.3 % triton-X 100 in

PBS for one hour at room temperature under orbital shaking. Without washing, the sections

were incubated in rabbit C-fos antibody (1:10,000, Ab5, Oncogene Research Products,

Cambridge, U.S.A) for 48 hr at 4oC. Subsequently, the sections were rinsed with PBS (10

mM) thrice, each 5 min and transferred to secondary goat rabbit antibody (1:200, Vector

laboratories) for 1 hr at room temperature with gentle agitation. C-fos expression was

visualized using peroxidase substrate (Vector VIP kit, Vector laboratories, Burlingame,

U.S.A, Figure 2.10) (Chan et al., 2013).

Chapter 2 Methods

57

Vectastain Elite ABC kit:

Figure 2.10 Vectastain Elite ABC kit, used in C-fos immunohistochemical procedure.

2.13.2. Quantification of c-fos immunoreactivity:

Ziess Axioskop-2 Plus microscope (Carl Zeiss Inc. Thornwood, U.S.A) equipped with Zeiss

Axioskop-2 camera, was used for the quantification of C-fos immunoreactivity (C-fos-IR).

C-fos-IR was counted in the areas of brain stem including nucleus tractus solitarius (NTS),

Area postrema (AP), and dorsal motor nucleus of vagus nerve (DMVN) and in the forebrain

area hypothalamus. C-fos positive cells were manually counted at X20 magnification (De

Jonghe and Horn, 2009).

2.13.3. Image acquisition and processing:

Zeiss Axioskop-2 plus microscope (Carl Zeiss Inc. Thornwood, U.S.A) equipped with Zeiss

Axiocam-2 camera was used to get histological images under bright field. All images were

processed using Adobe Photoshop to enhance its brightness and contrast, but not otherwise

manipulated.

Chapter 2 Methods

58

2.14. Ethical approval:

Pigeons of either sex in weight range of 250 - 350 grams, bred in the animal house,

Department of Pharmacy, University of Peshawar were used in the experiments. All

procedures were approved by the ethical committee (5/pharm), Department of Pharmacy,

University of Peshawar. Furthermore, all the experiments on S. murinus were conducted

under the license (11-237) in DH/HA & P/8/2/1 Pt. 18 provided by the ministry of health

Hong Kong, SAR, China. In both the cases, Animals were kept at approved standards,

temperature 22 ± 2 °C, with 12 hr light/dark cycle, with free access to food and water.

Chapter 3 Emetic potential of cisplatin in pigeon and S. murinus

59

Chapter 3

Studies on the emetic potential of cisplatin in

pigeon and Suncus murinus


60

3.1 Introduction:

Nausea (an unpleasant sensation often associated with less intense stimulation of vomiting

center) and vomiting (an urge to expel the gastrointestinal contents) are encountered either

together or separately as symptoms of various diseases and treatments. One of the most

prominent example of treatment induced nausea and vomiting is that induced by anti-

neoplastic agents (cisplatin, cyclophosphamide). Lack of efficacious anti-emetics in clinical

settings against both nausea and vomiting necessitate further research in this area; though,

there is continuing interest to develop new anti-emetic agents to combat nausea and

vomiting and to understand the mechanisms involved.

The progress in development and understanding the physiology and pharmacology of new

chemical entities having anti-emetic potential have come from a series of experiments

conducted in animals that demonstrate the predictive value of animal models. Chemotherapy

induces a biphasic vomiting response in humans, where the acute phase is well controlled by

5HT3 receptor antagonists while delayed phase is still a challenge in clinics (Gralla et al.,

1999). Cisplatin induced vomiting (response with expulsion of gastrointestinal contents) and

retching (response without expulsion of gastrointestinal contents) models have been

developed in dogs (Topal et al., 2005; Yamakuni et al., 2002), ferrets (Higgins et al., 2012),

piglets (Grelot and Esteve, 2009), pigeons (Tanihata et al., 2003; Tanihata et al., 2004), S.

murinus (house musk schrew) (Sam et al., 2003) and least schrew (Cryptotis parva)

(Darmani, 2001). Each model is having its own drawbacks, but they have provided

important data to prove the effectiveness of different legends against both the phases of

cisplatin induced vomiting.


61

Cisplatin is one of the Highly Emetogenic Chemotherapy (HEC) agents belonging to

platinum analogues and induces a biphasic vomiting response (early and delayed response)

in animals and human (Ullah et al., 2012). Though the pathogenesis for the acute vomiting

response (especially the role of neurotransmitter serotonin) of cisplatin induced vomiting

(Topal et al., 2005) is well understood, but the mechanisms responsible for triggering the

delayed phase and the nausea are the matters of debate and need to be explored further.

Keeping in view the discrepancies present in vomiting models, we selected pigeon (avian,

non-mammal) and S. murinus (insectivore, mammals) to study the impact of various plant

extracts alone and in combination in these animals.

3.1.1. Cisplatin induced Retching plus Vomiting (R + V) in pigeon:

Cisplatin induced vomiting in clinics, has also been established in pigeon model and this

was first coined by Navarra et.al (Navarra et al., 1992). Pigeon, a member of the avian class,

is found to be sensitive to various emetogenics including reserpine (Coronas et al., 1975),

cardiac glycosides (Hanzlik and Wood, 1929), 5HT receptor agonists (Wolff and Leander,

1995), and the oncolytic agents including cisplatin (Ullah et al., 2012) and

cyclophosphamide (Wolff and Leander, 1997). Cisplatin induces emesis either administered

by intravenous or intra cerebro-ventricular route in pigeon and numerous studies indicate the

involvement of reserpine sensitive-monoaminergic neurotransmitters in the mediation of

vomiting (Tanihata et al., 2003). Furthermore, NK1 receptor antagonists (Tanihata et al.,

2003), prokinetics (Ullah et al., 2012), CB1 receptor agonists (Feigenbaum et al., 1989;

Ferrari et al., 1999), corticosteroids (Tanihata et al., 2004), and 5HT1A receptor agonists


62

(Wolff and Leander, 1995) have been screened for their potential to attenuate cisplatin

induced vomiting in the pigeon as vomit model.

3.1.2. Cisplatin induced Retching plus Vomiting (R + V) in Suncus murinus:

Suncus murinus (house musk schrew), a species of insectivore also has been used to study

the mechanisms of cisplatin induced vomiting and currently is an acceptable model for the

study of cisplatin induced Retching plus Vomiting (R + V) for prolong time periods; upto 72

hours (Sam et al., 2003). The studies in S. murinus has provided evidences for the

involvement of free radicals and subsequent release of 5HT from enterochromaffin (EC)

cells in the etiology of cisplatin mediated vomiting (Matsuki et al., 1993; Mutoh et al.,

1992). The S. murinus is found to be practically useful model to study the involvement of

oxidative stress (Torii et al., 1993), serotonin (Nakayama et al., 2005), substance P (Rudd et

al., 1999), arginine vasopressin (AVP) (Ikegaya and Matsuki, 2002) and cannabinoids

(Bolognini et al., 2012) in emetic circuits and ultimately, for the development of new anti-

emetic drugs.

3.2 Aims and objectives of the study:

The objective of this study was to select a dose of cisplatin that reliably induces vomiting

response in the pigeon and Suncus murinus (house musk schrew) with no mortality upto the

desired period of time.


63

3.3 Materials and methods:

3.3.1. Animals:

3.3.1.1. Pigeon:

The Pigeons bred at the animal house facility, Department of Pharmacy, University of

Peshawar were used in the study.

For more details See Chapter 2: Methods, section 2.1.1.

3.3.1.2. Suncus murinus (House musk shrew):

Adult male S. murinus were provided by the animal care and laboratory services, the

Chinese University of Hong Kong (CUHK), Hong Kong.

For further details See Chapter 2: Methods, section 2.1.2.

3.3.2. Video recording setup:

Two separate video recording setups were used each for pigeon and S. murinus at “Bioassay

laboratories”, Department of Pharmacy, University of Peshawar and “School of biomedical

sciences”, Faculty of Medicine, The Chinese University of Hong Kong, respectively.

For more details See Chapter 2: Methods, section 2.2.

3.3.3. Drug formulation and administration:

For details See Chapter 2: Methods, section 2.8 & 2.9.


64

3.3.4. Quantification of vomiting:

Different animal models have different vomiting response to emetogenic stimuli. Pigeon

shows a very prominent and clear cut vomiting response induced by various emetogenic

substances, which is easily quantifiable. Cisplatin the highly emetogenic chemotherapeutic

agent (HEC) also induces a reliable vomiting, the intensity of which increases with

increasing dose of cisplatin. The criterion for the quantification of vomiting in the pigeon is

described in Chapter 2: Methods, section 2.3.1.

3.4 Results:

3.4.1. Induction of vomiting by intravenous administration of cisplatin in pigeon:

Cisplatin reliably induced vomiting at doses as low as 5 mg/kg, where 60 % response was

observed in pigeons. Nonetheless, 7 mg/kg, cisplatin induced a 100 % response in pigeons

tested which comprised approximately 43 mean episodes following a mean latency of ~ 67

min (Table 5.2, Figure 3.1A). The increase in dose of cisplatin from 7 mg/kg onward

resulted only in the increase in the number of Retching plus Vomiting (R + V) episodes

without affecting the latency time. Furthermore, the high doses of cisplatin were more

distressing to the pigeons and were having negative impact on survival upto the observation

period. Regardless of the dose, all responding animals appeared to vomit within the first two

hours after cisplatin administration, with the most intense period occurring around the first

hour (Figure 3.1B). None of the animals died during 24 hr of observation period by the

selected dose of cisplatin (7 mg/kg).


65

Figure 3.1. (A) Dose-response relationship of cisplatin (2 - 10 mg/kg) to induce vomiting

in pigeons and (B) profile of cisplatin (7 mg/kg i.v.) induced vomiting during a 24 hr

observation period. Data represents mean ± s.e.m. of 17 determinations.


66

3.4.2. Induction of vomiting by intraperitoneal administration of cisplatin in Suncus

murinus:

Cisplatin at the dose of 30 mg/kg i.p. induced a reliable vomiting in all the animals tested

(Table 8.1) without lethality, with peak vomiting response at 1 - 2 hr and 47 hr, respectively

(Fig. 3.2). The response comprised of 9.8 ± 1.5 R + V episodes in t 0 - 24 hr while 3.8 ± 1.1

R + V during the t 24 - 48 hr period with a latency of 59 ± 3.3 min (Table 8.1). The dose 30

mg/kg of cisplatin was selected from the preliminary studies conducted in this lab (Sam et

al., 2003). Saline (0.9 % w/v) injected at 10 mL/kg i.p. did not induce emesis in these

animals.

Figure 3.2. The profile of cisplatin induced Retching plus Vomiting (R + V) in Suncus

murinus during a 48 hr observation period. Cisplatin was administered intraperitoneally.

Results represent the mean ± s.e.m. of the total numbers of R + V occurring during 0 – 48 hr

(n = 5).


67

3.5 Discussion:

In our studies we successfully used pigeon and Suncus murinus (house mush schrew)

models to induce a reliable vomiting upto the observation period without any lethality. The

pigeon specie “Columba livia” has been used for elaboration of brain areas, but in our

studies we used either breed or sex of pigeons keeping in view the sensitivity differences

which are in parallel with the humans (Tanihata and Uchiyama, 2003). Some studies have

reported the induction of vomiting by 4 mg/kg of cisplatin dose in pigeon intravenously

(Tanihata et al., 2000), but in our studies we got the vomiting response in only 60 % of

animals tested at the dose of 5 mg/kg (figure 3.1A), this difference with respect to previous

studies may be attributed to species differences, environmental factors and diet. In this study

however, 7 mg/kg dose of cisplatin induced robust Retching plus Vomiting (R + V) in all the

animals (100 %) tested upto 24 hr of observation period without any lethality. Previous

studies report no mechanistically distinct acute or delayed phase of chemotherapy induced

vomiting in the pigeons, even though some studies followed upto 72 hours of observation

(Tanihata et al., 2003; Tanihata et al., 2004). In our studies we observed the animals for 24

hours to comply with the ethical use of animals.

Suncus murinus is frequently used in emesis research since long. S. murinus is indigenous to

Asian countries and belongs to insectivora, having adult weight ~ 60 grams. S. murinus has

been in extensive use since 1990s after its first use by matsuki (Matsuki et al., 1988) at

University of Tokyo, Japan. Uptill now serotonergic mechanisms of Chemotherapy Induced

Vomiting (CIV) are well elaborated in above cited model, as 5HT3 receptor antagonists are

proving itself to be effective in preventing acute phase of vomiting successfully. In S.

murinus we got the first peak of vomiting response at 1 - 2 hr, after which the response


68

gradually decreased and then the second peak appeared at ~ 47 hr. The 30 mg/kg dose of

cisplatin which was selected on the basis of preliminary studies conducted in this lab (Sam

et al., 2003). The dose of 30 mg/kg in our studies induced a reliable vomiting response upto

the observation period without any lethality. In our studies we observed the behavior of the

animal upto 48 hr to know the possibility for its effectiveness against delayed phase of

vomiting in vomit models of dogs and ferrets.

In summary, based on this study 7 mg/kg cisplatin administered i.v. was selected for

induction of vomiting in pigeon, while 30 mg/kg was used in case of S. murinus.

Chapter 4 Quantification of bacoside “A” major components

69

Chapter 4

Standardization of Bacopa monniera extracts

for bacoside “A” major components


70

4.1. Introduction:

The biological effects of Bacopa monniera (BM, family Scrophulariaceae) have been

authenticated by the traditional as well as scientific literature. The important effects of the

isolated bacosides are well known in inflammation (Channa et al., 2006), pain (Rauf et al.,

2012; Subhan et al., 2010), anxiety (Bhattacharya and Ghosal, 1998; Calabrese et al., 2008;

Sheikh et al., 2007), cognitive deficits (Raghav et al., 2006) and in management of

convulsive disorders (Mathew et al., 2010) and has been in use in India since 3000 years

where locally known as “Brahmi” (Mathew et al., 2010; Russo and Borrelli, 2005). In

Pakistan BM is known by the name “Jal neem booti” (Qureshi and Raza Bhatti, 2008;

Subhan et al., 2010). BM plant extracts and isolated bacosides (bacoside A3, bacoside II &

bacosaponin C) have been screened for their various neuropharmocological activities in

several laboratories and the reports are available for their nootropic action (Russo and

Borrelli, 2005). In addition BM is famous for its memory vitalizing property and is highly

valued for debilitating conditions in CNS.

BM is currently being marketed in western countries as a memory enhancing agent

(Bacomind®) and it has been proved that the herb contains many active constituents,

however, the major bioactive components are the steroidal saponins, especially bacoside

“A”, which is a mixture of three components (bacoside A3, bacoside II and bacosaponin C)

(Deepak et al., 2005). Furthermore, the standardized extract of BM is also available for

clinical use in India approved by the central drug research institute (Russo and Borrelli,

2005).


71

4.2. Aims and objectives of the study:

Keeping in view the neuropharmocological profile of Bacopa monniera (BM), the increasing

interest in this herbal drug and also based on our previous findings from this laboratory, this

study was designed to examine the contents of Bacoside “A” major components in the

methanolic extract (BM-MetFr) and n-butanol extract (BM-ButFr) of indigenously found BM,

by using High Performance Liquid Chromatography coupled with UV detector

(HPLC - UV) and the method already developed and revalidated by our laboratory (Rauf et al.,

2011).

4.3. Materials and methods:

4.3.1. Chemicals and reagents:

All the chemicals and bacoside standards used for the quantification of bacoside “A” major

components were of HPLC grade and were handled carefully.

For further details see Chapter 2, Methods, section 2.6.

4.3.2. High Performance Liquid Chromatography (HPLC) system:

The High Performance Liquid Chromatography coupled with UV- detector (HPLC - UV)

was used for the quantification of bacoside “A” major components (bacoside A3, bacoside II

& bacosaponin C) in both the BM methanolic fraction (BM-MetFr) and n-butanolic fraction

(BM-ButFr).

Further details of the HPLC system are given in sections 2.11, Chapter 2, Methods.


72

4.3.3. Sample handling:

See Chapter 2: Methods, section 2.11.3.

4.3.4. Preparation of stock solutions:

For details see Chapter 2, Methods, section 2.11.2.



quantification of bacosides in both methanolic and n-butanol fractions.

For further details see Chapter 2, Methods, section 2.11.4.

4.4. Results:

4.4.1. Standardization of Bacopa monniera methanolic fraction (BM-MetFr):

In this study, the HPLC - UV analysis of BM methanol fraction provided finger prints for the

presence of bacoside “A” major components including bacoside A3, bacoside II and

bacosaponin “C”. Our results indicated the presence of these bacosides in concentrations of

24 ± 1.1 µg/mg, 4.76 ± 0.03 µg/mg, 1.23 ± 0.01 µg/mg (n = 3) for bacoside A3, bacoside II

and bacosaponin C, respectively. Likewise, the total concentration of bacoside “A” major

components in BM-MetFr was 29.99 ± 2.1 µg/mg.


73

4.4.2. Standardization of Bacopa monniera n-butanol fraction (BM-ButFr):

The HPLC analysis of BM n-butanol fraction revealed that it is bacoside rich fraction

containing bacoside “A” major components bacoside A3, bacoside II and bacosaponin C in

concentrations of 57.91 ± 3.2 µg/mg, 40.60 ± 0.9 µg/mg, and 17.23 ± 1.7 µg/mg (n = 3),

respectively. Similarly, the total concentration of bacoside “A” three major components was

115.74 ± 3.9 µg/mg of n-butanol fraction or 38.37 ± 0.7 µg/gm of dry powder. These values

closely relate with the values reported by Khalid Rauf (Rauf et al., 2011).

Figure 4.1A: HPLC chromatogram showing peaks of standard Bacosides:

HPLC chromatogram of standard bacosides; showing peaks of bacoside “A” major

components bacoside A3 (1), bacoside II (2) and bacosaponin C (3).

1

2 3

Time (minutes)

Abso

rban

ce (

205nm

)


74

Figure 4.1B: HPLC chromatogram showing peaks of bacosides in sample:

HPLC chromatogram of sample (BM n-butanol fraction) showing peaks of bacoside “A”

major components bacoside A3 (1), bacoside II (2) and bacosaponin C (3).

4.5. Discussion:


quantification of bacosides in the BM methanolic and n-butanol fractions. All the bacosides

were eluted in the order; bacoside A3 (22.5 minutes), bacoside II (24.5 minutes) &

bacosaponin C (31.0 minutes), while the complete run time was 33 minutes. Each fraction

was diluted before injection to prevent column over loading. Our results are indicative of the

high concentrations of bacoside “A” major components in the BM-ButFr (115.74 µg/ mg of

extract) as compared to BM-MetFr (29.99 µg/ mg of extract) proving BM-ButFr as the

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

-1000

-750

-500

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

1

2

3

Time (minutes)

Abso

rban

ce (

205nm

)


75

bacoside rich fraction. Our results are in coincidence with the findings of Rauf et al (Rauf et

al., 2011) who also reported the bacosides in the BM methanolic and n-butanol fraction and

concluded the n-butanol fraction to be the bacosides rich fraction.

Chapter 5 Anti-emetic effect of C. sativa, B. monniera & Z. officinale

76

Chapter 5

Effect of Cannabis sativa, Bacopa monniera or

Zingiber officinale (ginger) extracts and their

combinations on cisplatin induced Retching

plus Vomiting (R + V) in pigeons


77

5.1. Introduction:

Cancer is the second leading cause of deaths in the United States and since 1990s 22 %

increase in the incidence of cancer has been reported with the four frequent cancers being

breast, colorectal, lungs and stomach (Parkin, 2001). The chemotherapy for the treatment of

carcinomas in clinics have deleterious side effects of nausea and vomiting especially

considering the use of Highly Emetogenic Chemotherapy (HEC) agents like cisplatin.

Chemotherapy induces biphasic vomiting in humans; acute and delayed, where the acute

phase is sensitive to 5HT3 receptor antagonists but the delayed phase is not well controlled

though recently NK1 receptor antagonists have shown promising results (Grelot and Esteve,

2009; Higgins et al., 2012). 5HT3 receptor antagonists in combination with NK1 receptor

antagonists and dexamethasone are proving to be useful for the management of

Chemotherapy Induced Vomiting (CIV) in clinical setups, but the goal of complete control

is not achieved yet (Markman, 2002; Pfister et al., 2004) thus necessitating the search for

new cost effective chemical entities or combinations having broad spectrum anti-emetic

activity.

Drug discovery from medicinal plants is an important and considerable area, due to which

the isolation of early drugs like artemether, cocaine, quinine and galantamine has been

carried out successfully and are still in use (Butler, 2004; Newman et al., 2000).

Furthermore, they are providing templates for the synthesis of new molecules as well. Uptill

now, the isolation and characterization of pharmacologically active compounds from herbal

origin is the substantial focus point and more recently, the standardization techniques have

been applied to get insight into the active moieties responsible for therapeutic actions.

Keeping in view the multifactorial CIV and to search for the cost effective remedy from


78

herbal origin, we selected Cannabis sativa (CS), Bacopa monniera (BM) and Zingiber

officinale (ZO) to screen them alone and in combination against cisplatin induced Retching

plus Vomiting (R + V) in the pigeon. Studies have shown that cannabis extract, Δ9-

tetrahydocannabinal (Δ9-THC) and synthetic analogues (e.g. Nabilone, Sativex

®, Marinol

®)

have been in use for the management of chemotherapy induced vomiting (Stark, 1982; Ware

et al., 2008). Furthermore, in comparison to metoclopramide and prochlorperazine cannabis

preparations have proved to be superior (Russo, 2001). Since two decades, the research on

the therapeutic potential of cannabis is reached to its peak because of the discovery of

cannabinoid receptors (Abalo et al., 2011; Van Sickle et al., 2003) and endocannabinoid

system (Mackie and Stella, 2006; Pacher et al., 2006).

The plant Bacopa monniera (BM) is a renowned medicinal plant in ayurvedic system of

medicine belonging to family “Scrophulariaceae”. The plant has been screened for its safety

and tolerability profile and the preparations (e.g. Bacomind®) are available in the market for

the management of memory impairments (Limpeanchob et al., 2008) and cognitive disorders

(Calabrese et al., 2008). In addition, BM preparations have also clinical utility for anxiety

and epilepsy (Mathew et al., 2010). The bacosides (bacoside A3, bacoside II & bacosaponin

C) are the active moieties responsible for the pharmacological profile; the same has been

quantified previously (Phrompittayarat et al., 2007) and by our laboratory (Rauf et al.,

2011b). BM has been proved to be having anti-oxidant (Bhattacharya et al., 2000) and anti-

dopaminergic (Rauf et al., 2011b) activity, which steered to formulate the hypothesis for its

anti-emetic activity against cisplatin induced vomiting in pigeon vomit model. Moreover,

the oxidative stress induced by cisplatin (Gupta and Sharma, 1996; Santos et al., 2007) and

the subsequent release of serotonin (Minami and Endo, 2003), substance P (Saito et al.,


79

2003) and dopamine (Darmani et al., 2003a) caused by cytotoxic agents are the triggering

mechanisms in the mediation of the vomiting act induced by cisplatin.

Zingiber Officinale commonly known as ginger (family, Zingiberaceae) is known

worldwide for its use as spice and flavoring agent (Tyler, 1988), while in Chinese and

Unani’s Tibb system is indicated for the treatment of anorexia, constipation and vomiting

(Tyler, 1993). The active component gingerol is responsible for its therapeutic effects, which

is mixture of many components including 6-gingerol and galanolactone (Abdel-Aziz et al.,

2006; Tyler, 1988). Animal studies showing the anti-emetic activity of ginger when induced

by cisplatin in dogs (Sharma et al., 1997) and cyclophosphamide in Suncus murinus

(Yamahara et al., 1989) were encouraging to enroll this plant extract in the present study.

5.2. Aims and objectives:

The aim of the present study was to screen various extracts of Cannabis sativa (CS), Bacopa

monniera (BM) and Zingier officinale (ZO) for their intrinsic anti-emetic activity against

cisplatin induced Retching plus Vomiting (R + V) in pigeon and to find out the effects of

various combinations of CS, BM and ZO on the spectrum of their anti-emetic activity.


5.3.1. Animals:

Mixed breed pigeons of both sex bred at the animal house facility at Department of

Pharmacy, University of Peshawar were used in the studies.

For details see Chapter 2: Methods, section 2.1.1.


80

5.3.2. Plants extraction:

The maceration method was used for the extraction of Cannabis sativa and Zingiber

officinale (ginger) while Kahol method (Kahol et al., 2004) was used for the extraction of

Bacopa monniera.

For detail extraction procedures, see Chapter 2: Methods, sections 2.5.1, 2.5.2 & 2.5.3.

5.3.3. Drugs and chemicals:

See Chapter 2: Methods, section 2.6.

5.3.4. Drug formulation:


5.3.5. Drug administration:

Intramuscular and intravenous routes were used for drug administration. In all the cases

cisplatin was administered intravenously while the test samples were administered

intramuscularly.

For detail, See Chapter 2: Methods, section 2.9.

5.3.6. Video recording setup & quantification of vomiting and retching:

Video recording setup for recording the behavior of animals upto the desired period of time,

at “Bioassay laboratories”, Department of Pharmacy, University of Peshawar was used.

Based on our previous study (chapter 3) cisplatin at the dose of 7 mg/kg was used for the


81

induction of vomiting (figure 3.1B) and the increase in cisplatin dose only resulted in the

increase in Retching plus Vomiting (R + V) intensity.

For more details about the recoding setup and quantification of R + V, see Chapter 2:

Methods, section 2.2 & 2.3.

5.3.7. Data analysis:

The differences between means were evaluated using “one way analysis of variance”

(ANOVA) followed by Dunnett or Tukey multiple comparison tests. P < 0.05 was

considered as statistically significant. The animals which showed complete suppression of

R + V were not included in statistical analysis for latency. Data represent the mean ± s.e.m.

unless otherwise indicated.

5.4. Results:

5.4.1. Anti-emetic effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol

fraction (CS-ButFr) or methanol fraction (CS-MetFr):

Cisplatin at the dose of 7 mg/kg induced reliable R + V in all the animals tested. In these

experiments, cisplatin induced R + V following a latency of ~ 69 minutes that comprised ~

44 episodes. CS hexane fraction (CS-HexFr 5, 10 and 15 mg) attenuated cisplatin induced

R + V in non-dose dependant manner (Figure 5.1), showing significant reduction with 10

mg/kg once (OD) and twice (BD) doses upto 19 ± 3.9 (55.45 % protection) and 13.7 ± 3.2

(68.86 % protection), respectively (P < 0.01; Table. 5.1) during 24 hr of observation period

(Figure 5.2 A). The n-butanol fraction (CS-ButFr 5 & 10 mg) and methanol fraction (CS-

MetFr 10 & 15 mg) however, failed to suppress the R + V any significantly (Figures 5.2 B &


82

C). The CS-HexFr was found to be effective as it suppressed R + V upto 16 hr of observation

period (Figure 5.2A), while standard metoclopramide provided protection upto 8 hr.

None of the treatment induced vomiting when administered alone.

Percent protection provided by Cannabis sativa hexane fraction:

Figure 5.1. Percent protection observed by either once daily dose of Cannabis sativa hexane

fraction (OD; 5, 10 and 15 mg/kg) or twice daily (BD; 10 mg/kg) 80 minutes before

cisplatin challenge. The values represent mean ± s.e.m of 5 - 8 determinations.


83

Table 5.1: Effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction

(CS-ButFr) or methanol fraction (CS-MetFr) on cisplatin-induced Retching plus

Vomiting in pigeons:

Drug treatment Dose and route Pigeons

n/ vomited

R + V Episodes

Mean ± sem

Latency (min)

Mean ± sem

Jerks

Mean ± sem

Wt loss (%)

Mean ± sem

Saline + Cisplatin 02ml/kg i.m. +

07mg/kg i.v. 8/8 44 ± 3.1 69 ± 3.7 595 ± 70 15.5 ± 1.1

MCP + Cisplatin 30mg/kg i.m. +

07mg/kg i.v. 8/8 24 ± 1.3** 204 ± 61.3* 351 ± 21 12.3 ± 1.4

CS-HexFr +

Cisplatin

5mg/kg i.m. +

07mg/kg i.v. 8/8 35 ± 6.6 195 ± 67 435 ± 92 9.4 ± 1.5

10mg/kg i.m. +

07mg/kg i.v. 5/5 19 ± 3.9** 289 ± 126 328 ± 94 9.5 ± 2.7

15mg/kg i.m. +

07mg/kg i.v. 8/8 29 ± 3.1 234 ± 49 444 ± 62 10 ± 1.7

10mg/kg i.m. BD

+ 07mg/kg i.v. 8/8 13.7 ± 3.2** 271 ± 72* 238 ± 77* 9.2 ± 1.2*

CS-ButFr +

Cisplatin

5mg/kg i.m. +

07mg/kg i.v. 6/6 38 ± 5.4 105 ± 10.7 614 ± 107 10.7 ± 2.7

10mg/kg i.m. +

07mg/kg i.v. 6/6 42 ± 6.7 91 ± 12.6 771 ± 168 13.7 ± 1.5

CS-MetFr +

Cisplatin

10mg/kg i.m. +

07mg/kg i.v. 7/7 31 ± 4.7 164 ± 76 460 ± 104 11.1 ± 1.9

15mg/kg i.m. +

07mg/kg i.v. 7/7 31 ± 4.2 116 ± 27 339 ± 69 7.7 ± 2.7*

Effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction (CS-ButFr),

methanolic fraction (CS-MetFr) or standard metoclopramide (MCP) on cisplatin induced

Retching plus Vomiting (R + V) and jerking during a 24 hr observation period. The latency

to first vomit, number of R + V episodes, jerks and % weight loss is shown for the 24 hr


84

observation period. Values significantly different compared to cisplatin control are indicated

as *p < 0.05, **p < 0.01 (ANOVA followed by Tukey post hoc analysis).

Effect of Cannabis sativa hexane fraction, n-butanol fraction or methanol fraction on

cisplatin-induced retching plus vomiting in pigeons:


85

Figure 5.2. The effect of (A) Cannabis sativa hexane fraction (CS-HexFr; 5, 10 & 15

mg/kg), (B) n-butanol fraction (CS-ButFr; 5 & 10 mg/kg) and (C) methanol fraction (CS-

MetFr; 10 &15 mg/kg), on cisplatin-induced R + V during a 24 hr observation period;

standard metoclopramide (MCP, 30 mg/kg) is also shown. Each bar represents the mean ±

s.e.m of R + V episodes occurring during 4 hr periods (n = 5 - 8). Values significantly

different compared to cisplatin control are indicated as *p < 0.05, 2*p < 0.01

3*p < 0.001

(ANOVA followed by Tukey post hoc test).

5.4.2. Effect of Cannabis sativa hexane fraction (CS-HexFr), n-butanol fraction (CS-

ButFr) or methanol fraction (CS-MetFr) on cisplatin induced jerking and weight loss:

In cisplatin control group, animals lost ~ 15 % of their starting body weight. The body

weight loss in standard MCP (30 mg/kg) treated group was 12.3 ± 1.4 %, while CS-HexFr

(10 mg/kg BD) and CS-MetFr (15 mg/kg) reduced body weight loss upto 9.2 & 7.7 % (P <

0.05, Table 5.1). All other treatments failed to effect body weight loss any significantly. The


86

jerking behavior (vomiting behavior; one vomiting episode may contain 2 – 80 jerks)

observed in cisplatin control and standard MCP group were 595 ± 70 & 351 ± 21,

respectively, while no treatment decreased the jerking behavior upto the observation period

(24 hr) except CS-HexFr (10 mg/kg BD) where the jerking episodes were reduced (595 ± 70

→ 238 ± 77 (P < 0.05, Table 5.1).

5.4.3. Anti-emetic effect of standard anti-oxidant N-(2- mercaptoprpionyl) glycine

(MPG), Bacopa monniera methanol fraction (BM-MetFr) & n-butanol fraction (BM-

ButFr):

From the preliminary studies (chapter 3), cisplatin was selected at the dose of 7 mg/kg to

evaluate the anti-emetic potential of BM fractions. In these experiments, cisplatin induced R

+ V following a latency of ~ 67 min and comprised a total of 43 episodes. Bacopa monniera

methanolic fraction (BM-MetFr) at 10, 20 and 40 mg/kg, dose dependently reduced

cisplatin-induced R + V (Figure 5.3), with the highest dose delaying the onset of vomiting

by approximately 194 min and the total number of R + V episodes up to 13 ± 2.9 (66.3 %

protection) (P < 0.05; Table. 5.2) during the 24 hr period; the anti-emetic action appeared to

last for up to 16 hr (Figure. 5.4 B). Similarly, Bacopa monniera n-butanol fraction (BM-

ButFr) 5 - 20 mg/kg reduced cisplatin-induced R + V up to 12 ± 2.2 (71.6 % protection) and

delayed the onset by approximately 67 min. Moreover, the anti-emetic action was evident

for up to 24 hr in animals treated with 5 and 10 mg/kg (P < 0.001; Figure. 5.4 C). BM-ButFr

(10 mg) proved to be superior as it suppressed the response at least in one animal

completely. MCP at 30 mg/kg delayed the onset of vomiting by 130 min (P > 0.05) and

reduced R + V during the 24 hr observation period by 48.9 % (P < 0.001). Furthermore,


87

unlike BM-MetFr and BM-ButFr, standard metoclopramide (MCP; 30 mg/kg) was only

found to be significantly effective to reduce R + V during the first 8 hr period (Figure. 5.4

A). Moreover, standard anti-oxidant N-(2- mercaptoprpionyl) glycine (MPG) at the dose of

10 mg/kg attenuated cisplatin induced R + V up to 11 ± 5.6 (76.5 % protection) (P < 0.001,

Table 5.2) and delayed the onset of R + V by 347 mins (P > 0.05), but the R + V suppression

was observed upto 12 hr of observation period (Figure. 5.4 A).

Dose response relationship of Bacopa monniera:

Figure 5.3. Dose response relationship of Bacopa monniera methanolic fraction (BM-

MetFr) & n-butanol fraction (BM-ButFr) expressed as % protection against cisplatin

induced Retching plus Vomiting (R + V) in pigeons. Data represents the mean ± s.e.m of

7 - 8 determinations.


88

Table 5.2: Effect of Bacopa monniera methanol fraction or n-butanol fraction on

cisplatin-induced retching plus vomiting in pigeons:

Drug Treatment Dose & route Pigeons

n/ vomited

R + V

Mean ± sem

Latency (min)

Mean ± sem

Jerks

Mean ± sem

Wt loss (%)

Mean ± sem


07mg/kg i.v 6/6 47 ± 5.8 74 ± 6.3 647 ± 162 15.3 ± 1.4


07mg/kg i.v 8/8 24 ± 1.3** 204 ± 61.3 351 ± 21 12.3 ± 1.4

MPG + Cisplatin 10mg/kg i.m. +

07mg/kg i.v 8/8 11 ± 5.6*** 421 ± 163 225 ± 109* 4.7 ± 1.9**

Saline + Cisplatin 02ml/kg i.m.

+ 7mg/kg iv

6/6 41 ± 5.2 70 ± 6.9 614 ± 115 12 ± 2

BM-MetFr +

Cisplatin

10mg/kg i.m. +

7mg/kg iv 7/7 27 ± 5.6 270 ± 136 610 ± 161 9.1 ± 1.7

20mg/kg i.m. +

7mg/kg iv 7/7 18 ± 4.4* 156 ± 42 405 ± 167 10.2 ± 1.6

40mg/kg i.m. +

7mg/kg iv 8/8 13 ± 2.9* 264 ± 132 268 ± 108 8.9 ± 1.7


7mg/kg iv 5/5 43 ± 5.8 59 ± 5.3 509 ± 67 16.8 ± 2

BM-ButFr +

Cisplatin

5mg/kg i.m. +

7mg/kg iv 8/8 15 ± 3.2*** 152 ± 35 309 ± 83 8.3 ± 1.6*

10mg/kg i.m. +

7mg/kg iv 7/6 13 ± 3.8*** 142 ± 46 326 ± 137 5.2 ± 1***

20mg/kg i.m. +

7mg/kg iv 8/8 12 ± 2.2*** 126 ± 14.9 185 ± 38 5.6 ± 1.6***

Effect of Bacopa monniera methanolic fraction (BM-MetFr; 10, 20 & 40 mg), n-butanol

fraction (BM-ButFr; 5, 10 & 20 mg), standard metoclopramide (MCP; 30 mg) and

antioxidant N-(2- mercaptoprpionyl) glycine (MPG; 10 mg) on cisplatin induced R + V and


89

jerking during a 24 hr observation period. The latency to first vomit, number of R + V

episodes, jerks and % weight loss is shown for the 24 hr observation period. Values

significantly different compared to cisplatin control are indicated as *p < 0.05, **p < 0.01

***p < 0.001 (ANOVA followed by Tukey post hoc test).


90

Effect of standard metoclopramide, antioxidant N-(2- mercaptoprpionyl) glycine,

Bacopa monniera methanolic fraction or n-butanol fraction on cisplatin-induced

retching plus vomiting in pigeons:


91

Figure 5.4. The effect of (A) standard metoclopramide (MCP; 30 mg/kg) and N-(2-

mercaptoprpionyl) glycine (MPG; 10 mg/kg) (B) Bacopa monniera methanolic fraction

(BM-MetFr; 10, 20 & 40 mg/kg) and (C) n-butanol fraction (BM-ButFr; 5, 10 & 20 mg/kg)

against cisplatin-induced R + V during a 24 hr observation period; each bar represents the

mean ± s.e.m of R + V episodes occurring during 4 hr periods (n = 7 - 8). Values

significantly different compared to cisplatin control are indicated as *p < 0.05, 2*p < 0.01

3*p < 0.001 (ANOVA followed by Tukey post hoc test).

5.4.4. Effect of standard metoclopramide (MCP), anti-oxidant N-(2-

mercaptoprpionyl) glycine (MCP), Bacopa monniera methanol fraction (BM-MetFr) or

n-butanol fraction (BM-ButFr) on cisplatin induced jerking and weight loss:

Control cisplatin treated animals lost ~ 14.7 % of their starting body weight while, animals

treated with BM-ButFr at 5, 10 & 20 mg/kg and MPG 10 mg/kg lost less than 9 % of their

starting body weight. These differences compared to cisplatin control were found to be


92

statistically significant (P < 0.05 - 0.001, Table 5.2). In the control cisplatin treated animals

there were ~ 590 jerking episodes during the 24 hr observation period. No fraction of BM at

any dose reduced significantly jerking episodes except MPG (P < 0.05, Table. 5.2).

5.4.5. Anti-emetic effect of Zingiber officinale acetone fraction (ZO-ActFr):

Zingiber officinale acetone fraction (ZO-ActFr) was screened for its anti-emetic activity

against cisplatin induced R + V in pigeon. The dose of 50 mg/kg provided maximum

protection against the R + V episodes which was ~ 58.13 % (18 ± 4.2 episodes) (P < 0.05)

as compared to cisplatin control. The attenuation with the 25 & 100 mg doses observed was

44.18 % (24 ± 4.1 episodes) and 27.9 % (31± 5.6 episodes) respectively, but the

suppression was found to be statistically non-significant (P > 0.05, Table 5.3). The standard

MCP reduced the R + V episodes ~ 48.83 % (22 ± 4.3 episodes) (P < 0.05) as compared to

cisplatin control. Furthermore, only the standard MCP significantly increased (P < 0.01) the

latency time as compared to cisplatin control.

In this study neither the standard MCP nor the treatments failed to provide complete

vomiting suppression, as all the animals tested showed the vomiting response. ZO-ActFr 25

& 50 mg provided protection upto 16 hr and 12 hr, respectively, while standard MCP was

also found effective upto 12 hr of observation period (Figure 5.5).


93

Table 5.3: Effect of Zingiber officinale acetone fraction (ZO-ActFr) on cisplatin-

induced retching plus vomiting in pigeons:

Drug Treatment Dose and route Pigeons

n/ vomited

R + V

Mean ± sem

Latency (min)

Mean ± sem

Jerks

Mean ± sem

Wt loss (%)

Mean ± sem

Saline +

Cisplatin

02ml/kg i.m. +

07mg/kg i.v. 6/6 43 ± 4.3 68 ± 3.7 407 ± 64 16.6 ± 1.8


07mg/kg i.v. 8/8 22 ± 4.3* 230 ± 84** 447 ± 103 11.5 ± 1.5

ZO-ActFr +

Cisplatin

25mg/kg i.m. +

07mg/kg i.v. 7/7 24 ± 4.1 124 ± 21 246 ± 92 8.1 ± 1.0*

50mg/kg i.m. +

07mg/kg i.v. 7/7 18 ± 4.2* 77 ± 15 376 ± 97 11.3 ± 2.3

100mg/kg i.m.

+ 07mg/kg i.v. 8/8 31 ± 5.6 85 ± 15 569 ± 125 9.1 ± 1.6

Effect of Zingiber officinale acetone fraction (ZO-ActFr, 25, 50 & 100 mg) and standard

metoclopramide (MCP, 30 mg/kg) on cisplatin induced Retching plus Vomiting (R + V) and

jerking during a 24 hr observation period. The latency to first vomit, number of R + V

episodes, jerks and % weight loss is shown for the 24 hr observation period. Values

significantly different compared to cisplatin control are indicated as *p < 0.05, **p < 0.01

(ANOVA followed by Tukey post hoc test).


94

Effect of Zingiber officinale acetone fraction on cisplatin induced retching plus

vomiting in pigeons:

Figure 5.5. The effect of Zingiber officinale acetone fraction (ZO-ActFr; 25, 50 & 100

mg/kg) and standard metoclopramide (MCP; 30 mg/kg) on cisplatin induced Retching plus

Vomiting (R + V) during a 24 hr observation period. Each bar represents the mean ± s.e.m

of R + V episodes occurring during 4 hr periods (n = 6 - 8). Values significantly different

compared to cisplatin control are indicated as *p < 0.05, 2*p < 0.01

3*p < 0.001 (ANOVA

followed by Tukey post hoc test).

5.4.6. Effect of Zingiber officinale acetone fraction (ZO-ActFr) & standard

metoclopramide (MCP) on cisplatin-induced jerks and weight loss:

Zingiber officinale acetone fraction (ZO-ActFr) did not reduced jerking episodes at any dose

treated and similarly the standard MCP also failed to reduce the jerking episodes as

compared to cisplatin control. In cisplatin control group animals lost upto 16 % of their


95

starting body weight, while in treated groups only ZO-ActFr at the dose of 25 mg attenuated

the weight loss significantly (P < 0.05, Table 5.3), while with other dose treatments the

reduction observed was statistically non-significant (P > 0.05, Table 5.3).

5.4.7. Anti-emetic effect of CS-HexFr 10 mg + BM-MetFr 10 mg (combination 1), BM-

ButFr 5 mg + ZO-ActFr 25 mg (combination 2), ZO-ActFr 25 mg + CS-HexFr 10 mg

(combination 3) or CS-HexFr 10 mg + BM-ButFr 5 mg (combination 4):

In an attempt to get enhanced suppression, we tested various combinations of Cannabis

Sativa (CS), Bacopa monniera (BM) and Zingiber officinale (ZO) extracts against cisplatin

induced R + V in the pigeon model. These combinations were 1) CS-HexFr (10 mg) + BM-

MetFr (10 mg), 2) BM-ButFr (5 mg) + ZO-ActFr (25 mg), 3) ZO-ActFr (25 mg) + CS-

HexFr (10 mg), 4) CS-HexFr (10 mg) + BM-ButFr (5 mg).

In the cisplatin control group the mean R + V response was 44 ± 1.9 with a latency of 66 ±

8.4 minutes and all the animals tested showed R + V for the entire observation period (Table

5.4). Standard metoclopramide (MCP) reduced the vomiting behavior upto 47.72 % and

increased the latency to first vomit upto 182 minutes, but in both the cases the differences

were found to be statistically non-significant (P > 0.05) as compared to cisplatin control.

The maximum protection ~ 88.63 % (05 ± 0.1 episodes) (P < 0.001, Table 5.4) was observed

with combination 4 (CS-HexFr 10 mg + BM-ButFr 5 mg) against cisplatin induced vomiting

and was found to be synergistic when calculated (see calculations) using Limpel equation

(Limpel et al., 1962) and increased the latency time ~ 303 minutes (P < 0.01), while

combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg) & 3 (ZO-ActFr 25 mg + CS-HexFr 10

mg) provided ~ 72.72 % (12 ± 0.4 episodes) (P < 0.01) and 56.81 % (19 ± 0.2 episodes)


96

protection against cisplatin induced R + V (P < 0.05, Table 4.4) and was also proved to be

antagonistic, respectively as compared to cisplatin control. Furthermore, the combination 1

(CS-HexFr 10 mg + BM-MetFr 10 mg) was also proved to be antagonistic. All the

combinations failed to increase the latency significantly as compared to cisplatin control.

The combination 4 (CS-HexFr 10 mg + BM-ButFr 5 mg) proved to be synergistic as it

provided enhanced inhibition as compared to expected inhibition when calculated (see

calculations) and also provided complete suppression of vomiting in at least one animal out

of six while the mean vomiting response was lowered as compared to other combinations

but the difference was found to be non-significant (Table 5.4, Figure 5.7).

Calculation for synergism:

Several mathematical methods are in use for testing the additivity/synergism of drug

combinations. This section presents a method which facilitates calculating "expected"

responses of drug combinations. The "expected" response for drug combinations can be

calculated as follows (Colby, 1967)

A Percent inhibition by drug A

B Percent inhibition by drug B

E The expected percent inhibition by combination A + B can be calculated using

equation

E = A + B – AB/100


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When the observed response is greater than expected, the combination is synergistic; when

less than expected, it is antagonistic. If the observed and expected responses are equal, the

combination is additive.

The calculations to conclude the synergism, antagonism and addition for the combinations

used in this study are

Combination 1 (CS-HexFr 10 mg + BM-MetFr 10 mg):

A Percent inhibition by CS-HexFr (10 mg) 56.81 %

B Percent inhibition by BM-MetFr (10 mg) 34.14 %

E Expected percent inhibition by Combination

E = A + B – AB/100

= 56.81 + 34.14 – 56.81 × 34.14/100

= 71.56 %

Observed percent inhibition 31.81 %

Result No synergism

Combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg):

A Percent inhibition by BM-ButFr (05 mg) 65.11 %

B Percent inhibition by ZO-ActFr (25 mg) 44.18 %


E = A + B – AB/100

= 65.11 + 44.18 – 65.11 × 44.18/100

= 80.53 %


Result No synergism


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Combination 3 (ZO-ActFr 25mg + CS-HexFr 10mg):

A Percent inhibition by ZO-ActFr (25 mg) 44.18 %

B Percent inhibition by CS-HexFr (10 mg) 56.81 %


E = A + B – AB/100

= 44.18 + 56.81 – 44.18 × 56.81/100

= 75.90 %


Result No synergism

Combination 4 (CS-HexFr 10 mg + BM-ButFr 5mg):

A Percent inhibition by CS-HexFr (10 mg) 56.81 %

B Percent inhibition by BM-ButFr (05 mg) 65.11 %


E = A + B – AB/100

= 56.81 + 65.11 – 56.81 × 65.11/100

= 84.94 %


Result Synergistic


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Table 5.4: Effect of various combinations of CS Hexane fraction, BM methanolic

and bacoside rich n-butanol fraction and ZO acetone fraction on cisplatin induced

R + V in pigeons:

Drug Treatment Dose & route Pigeons

n/ vomited

R + V

Mean ± sem

Latency (min)

Mean ± sem

Jerks

Mean ± sem

Wt loss (%)

Mean ± sem


7mg/kg i.v. 6/6 44 ± 1.9 66 ± 8.4 542 ± 84 15.5 ± 1.8

MCP + Cisplatin 30mg/kg i.m.

+ 7mg/kg i.v. 7/7 23 ± 0.3 248 ± 95 411 ± 112 10.8 ± 1.6

(CS-HexFr + BM-

MetFr) + Cisplatin

(10+10mg/kg

i.m.) +

7mg/kg i.v.

7/7 30 ± 1.1 131 ± 16 672 ± 124 5.1 ± 2.5**

(BM-ButFr + ZO-

ActFr) + Cisplatin

(5+25mg/kg

i.m.) +

7mg/kg i.v.

6/6 12 ± 0.4** 69 ± 21 598 ± 194 9.6 ± 2.4

(ZO-ActFr + CS-

HexFr) + Cisplatin

(25+10mg/kg

i.m.) +

7mg/kg i.v.

7/7 19 ± 0.2* 85 ± 12 415 ± 108 7.3 ± 1.9*

(CS-HexFr + BM-

ButFr ) + Cisplatin

(10+5mg/kg

i.m.) +

7mg/kg i.v.

6/5 05 ± 0.1*** 369 ± 123** 99 ± 47 10.6 ± 1.7

Effect of various combinations of CS Hexane fraction (CS-HexFr), BM methanolic fraction

(BM-MetFr) & bacoside rich n-butanol fraction (BM-ButFr) and ZO acetone fraction (ZO-

ActFr) on cisplatin induced vomiting and jerking during a 24 hr observation period.

Standard metoclopramide (MCP; 30 mg/kg) is also shown. Values significantly different

compared to cisplatin control are indicated as *p < 0.05, **p < 0.01 ***p < 0.001 (ANOVA

followed by Tukey post hoc test). Combination 1 (CS-HexFr 10 mg + BM-MetFr 10 mg),

Combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg), Combination 3 (ZO-ActFr 25mg +

CS-HexFr 10mg) & Combination 4 (CS-HexFr 10 mg + BM-ButFr 5mg).


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Effect of various combinations of Cannabis sativa Hexane fraction, Bacopa monniera

methanolic fraction & bacoside rich n-butanol fraction and Zingiber officinale acetone

fraction on cisplatin-induced Retching plus Vomiting (R + V) in pigeons:

Figure 5.6. Effect of various combinations of CS Hexane fraction (CS-HexFr), Bacopa

monniera methanolic fraction (BM-MetFr) & bacoside rich n-butanol fraction (BM-ButFr)

and Zingiber officinale acetone fraction (ZO-ActFr) on cisplatin induced Retching plus

Vomiting (R + V) during a 24 hr observation period. Standard metoclopramide (MCP; 30

mg/kg) is also shown. Each bar represents the mean ± s.e.m of vomiting episodes occurring

during 4 hr periods (n = 6 - 7). Values significantly different compared to cisplatin control

are indicated as *p < 0.05, 2*p < 0.01

3*p < 0.001 (ANOVA followed by Tukey post hoc

test). Combination 1 (CS-HexFr 10 mg + BM-MetFr 10 mg), Combination 2 (BM-ButFr 5

mg + ZO-ActFr 25 mg), Combination 3 (ZO-ActFr 25 mg + CS-HexFr 10 mg) and

Combination 4 (CS-HexFr 10 mg + BM-ButFr 5 mg).


101

Vomiting suppression sketch of various combinations of Cannabis sativa Hexane

fraction (CS-HexFr), Bacopa monniera methanolic fraction (BM-MetFr) and bacoside

rich n-butanol fraction (BM-ButFr) and Zingiber officinale acetone fraction (ZO-

ActFr) on cisplatin induced Retching plus Vomiting (R + V) in pigeons:


102


103

Figure 5.7. Vomiting suppression sketch of combinations on cisplatin induced vomiting

during 24 hr of observation period. A. Cisplatin control, B. Combination 1 (CS-HexFr 10

mg + BM-MetFr 10 mg), C. Combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg), D.

Combination 3 (ZO-ActFr 25 mg + CS-HexFr 10 mg), E. Combination 4 (CS-HexFr 10 mg

+ BM-ButFr 5 mg) (n = 6 – 7).

5.4.8. Effect of CS-HexFr 10 mg + BM-MetFr 10 mg (combination 1), BM-ButFr 5 mg

+ ZO-ActFr 25 mg (combination 2), ZO-ActFr 25 mg + CS-HexFr 10 mg (combination

3), CS-HexFr 10 mg + BM-ButFr 5 mg (combination 4) on cisplatin-induced jerks and

weight loss:

In the cisplatin control group, animals lost upto 15 % of their starting body weight, while in

the treatment groups combination 1 (CS-HexFr 10 mg + BM-MetFr 10 mg) & 3 (ZO-ActFr

25 mg + CS-HexFr 10 mg) lost less than 8 % of their body weight which was found

statistically significant as compared to cisplatin control (P < 0.05 - 0.01, Table 5.4), while


104

combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg) & combination 4 (CS-HexFr 10 mg +

BM-ButFr 5 mg) and standard metoclopramide (MCP) failed to reduce the weight loss any

significantly. In the cisplatin control group the jerking episodes observed were 542 ± 84,

where no treatment significantly reduced the jerking episodes, while good suppression was

observed with combination 4 (CS-HexFr 10 mg + BM-ButFr 5 mg) which reduced the

jerking episodes upto 99 jerks but the difference was found to be statistically non-significant

as compared to cisplatin control.

5.5. Discussion:

In this study the extracts of Cannabis sativa (CS), Bacopa monniera (BM) and Zingiber

officinale (ZO) were screened against cisplatin induced Retching plus Vomiting (R + V) in

pigeons. All of the extracts provided protection, where the protection provided by BM was

an interesting new finding. Cannabis sativa preparations have been used against vomiting as

reported by Mechoulam et al (Mechoulam and Feigenbaum, 1987). In the present study, we

screened three different fractions (n-hexane, n-butanol & Methanol) of CS against cisplatin

induced R + V in pigeon vomit model, where the n-hexane fraction (CS-HexFr) was found

to be effective to attenuate cisplatin induced R + V. CS-HexFr at the dose of 10 mg/kg

single and twice daily dosing provided upto 55.45 % (19 ± 3.9 episodes) & 68.86 % (13.7 ±

3.2) protection, respectively (Table 5.1). The n-hexane extract contains cannabis major

active constituent Delta-9-tetrahydrocannabinol (Δ9- THC) which has been in use for the

treatment of various diseases including 1) anti-emetic for the management of cancer

Chemotherapy Induced Vomiting (CIV) in clinics and 2) the enhancement of appetite. Δ9-

THC is also found to have anti-inflammatory, apasmolytic, analgesic and anti-glaucoma


105

activity (Carlini, 2004). Furthermore, Sallan and his co-workers proved that the active

component of CS (Δ9- THC) have anti-emetic property (Sallan et al., 1975), which resides in

its ability to stimulate presynaptic cannabinoid CB1 receptors (Darmani, 2001) and

subsequent inhibition of monoamine neurotransmitters (serotonin, norepinephrine,

dopamine) and acetylcholine release (Darmani et al., 2003b). In our study, we were not able

to standardize CS hexane fraction for its active constituent Δ9- THC because of its

categorization in controlled substances which led to the failure to acquire HPLC standard.

Bacopa monniera (BM) is a perennial herb (family - Scrophulariaceae), which is found

around the world including Pakistan (Qureshi and Raza Bhatti, 2008). BM extracts have

been screened for its active components bacosides and our results are indicative of the high

concentration of bacosides in the n-butanol fraction (Chapter 4, section 4.4.2) and the same

has been reported by Rauf et al, as well from this laboratory (Rauf et al., 2011b). BM is

found to be a well tolerated phytomedicine in various clinical trials (Calabrese et al., 2008)

and currently is available in different herbal formulations alone or in combination with other

plant extracts for the management of cognitive disorders and is having safety and tolerability

profile. BM is reported to have strong antioxidant activity (Bhattacharya et al., 2000) and is

having inhibitory effects on hyperactivity mediated by dopamine receptors (Sumathi et al.,

2007). In this study, bacosides at the dose of ~ 700 µg/kg was found to be effective to

attenuate the R + V response upto the observation period (24 hr) in pigeons which support

its usefulness as anti-emetic/adjunct for the management of chemotherapy induced vomiting

in clinics. The above results reveal that BM may be better than metoclopramide and its

promising results as anti-emetic in our studies might be due to its antioxidant (Bhattacharya

et al., 2000) and antidopaminergic activity as reported in rodents (Rauf et al., 2011b) and in


106

the present study in pigeons (chapter 7). The doses of BM used in this study are based on

previous study on BM extract conducted in this laboratory (Rauf et al., 2011a).

Metoclopramide (MCP), which is a clinically relevant anti-emetic with dopamine and 5-HT3

receptor antagonist properties (Al-Zubaidy and Mohammad, 2005) was used as a positive

control. The dose of MCP that we selected is higher than required to antagonize cisplatin

induced emesis in other species (Zhang et al., 2006), and was based on a previous study in

the pigeon showing activity against reserpine-induced emesis (Coronas et al., 1975). The

metoclopramide was selected as standard because of the intrinsic emetic activity of 5HT3

receptor antagonists in pigeon (unpublish data).

Zingiber officinale (ZO; family Zingiberaceae) commonly known as ginger; the plant

rhizome and is distributed and cultivated in Pakistan, India, Malaysia, China, Taiwan and

Bangladesh. Ginger has been in use for medicinal purposes since long and is an important

plant in Chinese and Indian pharmacopoeias. In our study the gingerols rich acetone extract

(ZO-ActFr) of the ginger (Sharma et al., 1997) was tested against cisplatin induced R + V in

pigeon vomit model. The standardization of ginger extract is reporting the quantities of

gingerols in concentration ~ 60 mg/g of extract (Rai et al., 2006). Ginger have been screened

in postoperative nausea and vomiting in clinics and is found to be superior to placebo and

equally effective as metoclopramide (Ernst and Pittler, 2000). In this study, the dose of 50

mg was found to be highly effective in attenuating cisplatin induced R + V, nonetheless

longer protection i.e. upto 16 hr was observed with 25 mg dose. There are several lines of

evidences explaining the anti-emetic effect of ginger; in animal models ginger is shown to

enhance gastrointestinal transport i.e. having gastroprokinetic properties. Furthermore,

ginger is having anti-hydroxytryptamine activity in the isolated ileal segments (Abdel-Aziz


107

et al., 2006) as galanolactone, one of the component of ginger, has been proved to be a

competitive antagonist at ileal 5HT3 receptors. Thus anti-emetic effects could be brought

about by it antagonism at 5HT3 receptors in the gastrointestinal tract (Abdel-Aziz et al.,

2006; Yamahara et al., 1989). Furthermore, ginger has also been found to be having

inhibitory action on substance P and the expression of NK1 receptors (Qiu-hai et al., 2010).

The common use as spice, flavoring agent and food stuff is suggesting that ginger could be

free of serious side effects. The British herbal compendium reports no adverse effects of

ginger (Bradley, 1992). Our results of current study are indicative of promising anti-emetic

activity of ginger acetone extract against cisplatin induced vomiting in the vomit model of

pigeon.

Chemotherapy induced vomiting in clinics is regarded a multifactorial phenomenon and a

single anti-emetic fails to rectify the vomiting. Therefore, combination regimen including

5HT3 receptor antagonist (e.g. ondansetron), NK1 receptor antagonist (e.g. aprepitant) and

dexamethasone is recommended for the effective management of this distressing side effect.

Further in this study, the plant extracts in various combinations were tested that provided

evidences for the synergistic action of CS-HexFr (10 mg) with BM-ButFr (5 mg)

(combination 4) i.e. ~ 88 % Protection (05 ± 0.1 episodes; P < 0.001) (Figure 5.7D, Table

5.4) whereas the protection observed alone was ~ 55.45 % and 68.08 % protection,

respectively. Combination 2 (BM-ButFr 5 mg + ZO-ActFr 25 mg) was also found to be

effective though less significant (P < 0.01) to combination 4 (CS-HexFr 10 mg + BM-ButFr

5 mg).


108

In summary, the extracts of CS, ZO and BM (current study) have promising anti-emetic

effect against cisplatin induced vomiting in pigeon vomit model. Moreover, the combination

of CS-HexFr (10 mg) with BM-ButFr (5 mg) provided highly significant anti-emetic effect.

The availability of cannabis preparations (Marinol®

, Sativex & Nabilone), BM preparations

(Bacomind®) and ginger a well accepted traditional medicine and cost effectiveness are

justifying/defending the use of these extracts in the management of chemotherapy induced

vomiting.

Chapter 6 Role of gastrointestinal motility in cisplatin induced vomiting

109

Chapter 6

Effect of Cannabis sativa on gastrointestinal

motility and consequent influence on cisplatin

induced Retching plus Vomiting (R + V) in

pigeons


110

6.1. Introduction:

Cannabis sativa (CS) preparations have been used against vomiting (Mechoulam and

Feigenbaum, 1987) and the anti-emetic effect of the active component of Cannabis sativa

Delta-9-tetrahydrocannabinol (Δ9 THC) and related compounds have been confirmed

clinically (Tramer et al., 2001). The active component of Cannabis sativa “Δ9 THC” is

reported to act on cannabinoid CB1 receptors (Darmani, 2001a). The CB1 receptors are

located presynaptically and the stimulation of which results in the inhibition of

neurotransmitters (serotonin, norepinephrine, dopamine) and acetylcholine release (Darmani

et al., 2003). This inhibition of ongoing contractile transmitter (acetylcholine) release in the

enteric nervous system leads to depression of gastrointestinal (GIT) motility and motor

activity in the stomach (Pertwee, 2001a). The suppressive effect of Δ9 THC on GIT is

reported to be reversed by CB1 receptor antagonists (rimonibant, SR 141716A etc)

indicating the involvement of CB1 receptors in the mediation of delayed gastric emptying

and decrease in GIT motility (Hornby and Prouty, 2004). The same GIT suppression by Δ9

THC has also been confirmed in human by delay in gastric emptying of radiolabeled solid

food (McCallum et al., 1999).

The highly emetogenic chemotherapeutic agent cisplatin dose dependently inhibits gastric

emptying in rats and mice; per se (Sharma, 1998), while the distention of the stomach

caused by cytotoxic agents like cisplatin has been shown parallel to nausea and vomiting

(Roos et al., 1981) accompanying GIT symptoms such as abdominal discomfort in patients

(Andrews et al., 1990). In fact, several lines of evidences are fully supporting the dogma that

the prokinetics are potentiating the anti-emetic effects of other chemical entities devoid of

such property like ferulic acid which enhances intestinal motility (Badary et al., 2006) and is


111

proved to be effective in attenuating cisplatin induced vomiting and gastrointestinal

discomfort caused by these cytotoxic agents. In addition prokinetic property of

metoclopramide by agonism of 5HT4 receptors is positively contributing to its anti-emetic

action as well which is mediated via 5HT3 and D2 receptors centrally (Frisch et al., 1995).

6.2. Aims and Objectives:

Keeping in view the involvement of gastrointestinal motility/gastric emptying, the present

study was designed to find out the role of gastrointestinal motility suppression caused by

Cannabis sativa hexane fraction (CS-HexFr) in vivo by charcoal propulsion method and

further to investigate the impact of its antagonism by prokinetic/cholinergic agonist on the

anti-emetic spectrum of the Cannabis sativa hexane fraction against cisplatin induced

Retching plus Vomiting (R + V) in pigeon.


6.3.1. Animals:

Mixed breed pigeons of both sex bred at the animal house facility of Department of

Pharmacy, University of Peshawar were used in the studies.

For details see Chapter 2: Methods, section 2.1.1.

6.3.2. Materials and drugs:



112

6.3.3. Extraction of Cannabis sativa:

For detail extraction procedure see Chapter 2: Methods, section 2.5.1.


Intramuscular and intravenous routes were used for drug administration, while charcoal was

administered orally. In all the cases cisplatin was administered intravenously while the

treatments were administered intramuscularly.

For details, See Chapter 2: Methods, section 2.9.

6.3.5. Video recording setup & quantification of vomiting:

Video recording setup for recording the behavior of the pigeons upto the desired period of

time, at Bioassay laboratories, Department of Pharmacy, University of Peshawar was used.

Cisplatin the highly emetogenic chemotherapeutic agent induced a reliable vomiting

response at the dose of 7 mg/kg (chapter 3) and the increase in cisplatin dose resulted only in

the increase in R + V.

For more details about video recording setup and the criterion for the quantification of

vomiting in pigeon see Chapter 2: Methods, section 2.2.1 & 2.3.1.

6.3.6. Measurement of gastrointestinal motility:

Charcoal propulsion method was used for the measurement of gastrointestinal motility to

estimate suppression caused by Cannabis sativa hexane fraction (CS-HexFr; 10 mg) and its

antagonism by metoclopramide (MCP; 10 & 30 mg) and carbachol (0.1 mg).

Further details are in Chapter 2: Methods, section 2.10.


113

6.4. Results:

6.4.1. Gastrointestinal suppression caused by Cannabis sativa hexane fraction (CS-

HexFr) and its antagonism by metoclopramide and carbachol:

The normal gastrointestinal motility (GIT) assessed by charcoal propulsion method was

39. 35 ± 4.6 %. CS-HexFr at the dose of 10 mg/kg caused suppression of GIT motility upto

26.62 ± 1.02 % as compared to saline. In antagonism studies, MCP (10 & 30 mg/kg) and

carbachol (0.1 mg/kg) antagonized the suppression caused by CS-HexFr (10 mg/kg),

significantly (P < 0.001, Figure 6.1).

Gastrointestinal suppression caused by Cannabis sativa hexane fraction:

Figure 6.1. Percent suppression in gastrointestinal (GIT) motility caused by Cannabis sativa

hexane fraction (CS-HexFr; 10 mg) and its antagonism by metoclopramide (MCP; 10 & 30

mg) and carbachol (0.1 mg). Values significantly different compared to cisplatin control are

indicated as 3*p < 0.001 (ANOVA followed by Tukey post hoc test, n = 6 - 8).


114

6.4.2. Impact of Cannabis sativa hexane fraction (CS-HexFr) in combination with

metoclopramide (MCP) and carbachol on cisplatin induced Retching plus Vomiting

(R + V):

Cannabis sativa hexane fraction (CS-HexFr; 10 mg/kg) in combination with

metoclopramide (MCP; 30 mg/kg) and carbachol (0.1 mg/kg) showed enhanced anti-emetic

activity against cisplatin induced R + V after second dose at 12th

h, while no

synergism/potentiation was seen at the first dosing at t = 0 (Figure 6.2C & D). Carbachol

(0.1 mg/kg) did not induce vomiting by itself when tested alone (unpublish data). The 4 hr

sketch of vomiting episodes indicates more pronounced suppression in R + V after the

second dose at 12 hr (Figure 6.3).


115

Table 6.1: Effect of Cannabis sativa hexane fraction (CS-HexFr) and its

combinations on cisplatin induced Retching plus Vomiting (R + V) in pigeons:

Drug treatment Dose and route Pigeons

n/ vomited

R + V

Mean ± sem

Latency (min)

Mean ± sem

Jerks

Mean ± sem

Wt loss (%)

Mean ± sem

Saline + Cisplatin 02 ml/kg i.m. +

7 mg/kg i.v. 8/8 44 ± 3.1 69 ± 3.7 595 ± 70 15.5 ± 1.1

MCP + Cisplatin 30 mg/kg i.m. +

7 mg/kg i.v. 8/8 21 ± 1.3** 217 ± 61.3* 331 ± 21 12.1 ± 1.1

CS-HexFr + Cisplatin 10 mg/kg i.m. +

7 mg/kg i.v. 8/8 13.7 ± 3.2** 271 ± 72* 238 ± 77* 9.2 ± 1.2*

(CS-HexFr + MCP) +

Cisplatin

(10 mg + 30 mg)

i.m. +

7 mg/kg i.v.

8/8 14 ± 2.1** 164 ± 39 212 ± 41 13.6 ± 2.6

(CS-HexFr + carbachol)

+ Cisplatin

(10 mg + 0.1mg)

i.m. +

7 mg/kg i.v.

6/6 19 ± 2.9** 132 ± 28.3 339 ± 59 8.13 ± 2.2

Effect of Cannabis sativa hexane fraction (CS-HexFr) and its combinations administered

twice daily on cisplatin-induced Retching plus Vomiting (R + V) and jerking during a 24 hr

observation period, standard metoclopramide (MCP) is also shown. The latency to first

vomit, number of vomiting episodes and jerks and % weight loss is shown for the 24 hr

observation period. Values significantly different compared to cisplatin control are indicated

as *p < 0.05, **p < 0.01 (ANOVA followed by Tukey post hoc analysis).


116

Effect of Cannabis sativa hexane fraction (CS-HexFr) 10 mg and its combination with

MCP and carbachol on cisplatin-induced Retching plus Vomiting (R + V):


117

Figure 6.2. The effect of (A) Cisplatin control (B) CS-HexFr 10 mg (C) CS-HexFr 10 mg +

MCP 30 mg/kg (combination 1) (D) CS-HexFr 10 mg + Carbachol 0.1 mg/kg (combination

2), administered twice daily on cisplatin-induced vomiting during a 24 hr observation

period. Each bar represents the mean ± s.e.m of Retching plus Vomiting (R + V) episodes

occurring during 1 hr period (n = 6 - 8). The arrow indicates dosing time.


118

Effect of Cannabis sativa hexane fraction and its combinations, on cisplatin-induced

Retching plus Vomiting (R + V) in pigeons:

Figure 6.3: The effect of CS-HexFr (10 mg) and its combinations, on cisplatin-induced

Retching plus Vomiting (R + V) during a 24 hr observation period; MCP at 30 mg/kg is also

shown. Each bar represents the mean ± s.e.m of vomiting episodes occurring during 4 hr

periods (n = 5 - 8). Values significantly different compared to cisplatin control are indicated

as *p < 0.05, 2*p < 0.01

3*p < 0.001 (ANOVA followed by Tukey post hoc test). Arrow

indicates dosing time. Combination 1 (CS-HexFr 10 mg + MCP 30 mg), Combination 2

(HexFr 10 mg + Carbachol 0.1 mg).

6.5. Discussion:

Cisplatin, one of the chemotherapeutic agents has been in use for the management of various

carcinomas like ovarian, testicular, head and neck carcinomas (Muggia, 2009) having one of

the severe limiting side effect of nausea and vomiting (Topal et al., 2005). The vomiting

caused by chemotherapeutic drugs is multifactorial. Among these factors the delay in gastric


119

emptying and decrease in gastrointestinal motility are playing its role inpart. Cisplatin dose

dependently causes suppression of gastric emptying (Sharma and Gupta, 1998). The anti-

emetics in clinical use like metoclopramide and serotonin receptor antagonists have been

shown to alter the gastric motility produced by cisplatin in rats.

Delta-9-tetrahydrocannabinol (Δ9 THC) and other synthetic cannabinoids have been

screened for their anti-emetic activity against cisplatin induced vomiting in various animal

models. The cannabinoids whether from natural sources or synthetic are reported to act

through the activation of presynaptically located CB1 receptors, which leads to the inhibition

of various transmitters release in the gastrointestinal tract (Darmani, 2001b).

In our studies we used pigeon as a vomiting model for assessment of the emetic potential of

cisplatin as this specie has been used in emesis research for many years (Gupta and Dhawan,

1960; Preziosi et al., 1992). We screened various crude fractions of Cannabis sativa for its

potential to suppress cisplatin induced Retching plus Vomiting (R + V) in this specie where

CS-HexFr at the dose of 10 mg/kg showed upto 68.86 % protection against cisplatin induced

vomiting (P < 0.01, Figure 5.1).

Cannabinoids have been reported to cause the suppression of gastrointestinal motility (Abalo

et al., 2011), as it causes the inhibition of ongoing contractile transmitter release (Pertwee,

2001). It is hypothesized that this suppression may antagonize the anti-emetic activity as

cisplatin is causing delay in gastric emptying, per se (Sharma and Gupta, 1998). In the

present study, CS-HexFr 10 mg/kg suppressed the gastrointestinal motility upto 26.62 % as

compared to saline. Similarly we observed the reversal of inhibition by MCP and carbachol.

MCP 10 and 30 mg/kg and carbachol at 0.1 mg/kg produced significant reversal (P < 0.001,


120

Figure 6.1); similar efficacy of MCP and carbachol in combination with CS-HexFr 10

mg/kg was also observed in our studies against cisplatin induced R + V in pigeon, where the

enhanced attenuation was observed after the peak of acute phase (Figure 6.2).

In conclusion, CS-HexFr at the dose of 10 mg/kg provided maximum protection against

cisplatin induced vomiting in pigeon but caused the suppression of GIT motility. MCP (30

mg/kg) and carbachol (0.1 mg/kg) antagonized the gastrointestinal suppression caused by

CS-HexFr (10 mg/kg) and enhanced its anti-emetic profile. These observations may indicate

the involvement of suppression of cholinergic mechanism responsible for delay in gastric

emptying and suppression in GIT motility by CS-HexFr.

Chapter 7 Effect of plants extracts on neurotransmitters

121

Chapter 7

Effect of Cannabis sativa, Bacopa monniera or

Zingiber officinale (ginger) extracts on

neurotransmitters implicated in vomiting

circuits in pigeons


122

7.1. Introduction:

Cytotoxic agents like cisplatin and cyclophosphamide are having the side effects of nausea

and vomiting most feared by patients undergoing chemotherapy (Hesketh and Grunberg,

2003). The D2 receptor blocker “metoclopramide” was found to be effective against

Chemotherapy Induced Vomiting (CIV) at higher doses, where the anti-emetic effect is

reported to be mediated through antagonism of 5-hydroxy tryptamine type 3 (5HT3)

receptors (Coronas et al., 1975; Miner and Sanger, 2012). These findings of 5HT3 mediated

anti-emetic effect of metoclopramide led to the discovery of 5HT3 receptor antagonists

(ondansetron, granisetron, tropisetron and palonosetron). The 5HT3 receptor blockers have

proved to be effective in the control of acute phase (~ 24 hr) only (Nakayama et al., 2005),

while their relative resistance at the delayed phase (24 hr +) has led to the incomplete

control and this have surely jeopardized the acceptance of such compounds in the

management of CIV. Currently, the NK1 receptor antagonists have proved to be effective in

the control of CIV, especially considering the delayed phase of vomiting (Gardner et al.,

2012). In addition, dexamethasone has also shown promising results in combination with

other anti-emetics (Tanihata et al., 2004). The failure of single anti-emetic agent for the

control of CIV is steering the etiology to be multifactorial, and there are evidences for the

involvement of many neurotransmitter systems including serotonergic (Higgins et al., 2012;

Percie du Sert et al., 2011), dopaminergic (Darmani and Crim, 2005; Osinski et al., 2005)

and neurokininergic (Grelot and Esteve, 2009; Tanihata et al., 2003) systems acting in

emetic circuitry at different time pointes.

Neurotransmitters are very important to be considered in understanding the vomiting

circuitry especially the involvement of dopamine (DA), 5-hydroxytryptamine (5HT) and


123

neuropeptide substance P. In the past decades, major advances have been made in the

understanding of the neuro-pharmacology of the emetic pathways. In general, the

identification of 5HT3 receptor blockers flourished the research in the investigations of

vomiting mechanisms and consequently the reappraisal of the involvement of brain areas

(area postrema, nucleus tractus solitarius & dorsal motor nucleus of vagus nerve). The

neurotransmitter “Serotonin” (5HT) is the primary culprit in the initiation of vomiting

response especially considering CIV (Grunberg and Koeller, 2003). Upto 95 % of 5HT is

present in the enterochromaffin (EC) cells in the gastrointestinal mucosa along with

substance P (Diemunsch and Grelot, 2000; Minami and Endo, 2003), which is released by

the noxious stimulus caused by Moderate Emetogenic Chemotherapy (MEC) and Highly

Emetogenic Chemotherapy (HEC) agents like cyclophosphamide and cisplatin, respectively

(Percie du Sert et al., 2011; Wolff and Leander, 1997). The released 5HT then activates

5HT3 receptors on vagal afferents which stimulate the brain centers to initiate the vomiting

response (Hesketh and Van Belle, 2003). Furthermore, in human and animal studies, there

are evidences for the increased level of 5-Hydroxy Indole Acetic Acid (5HIAA, urine)

(Cubeddu et al., 1995; Veyrat-Follet et al., 1997), 5HT in the intestinal mucosa (ileal

segment), Tryptophan Hydroxylase (TPH, ileum), Aromatic L-amino Decarboxylase

(AADC, ileum) (Endo et al., 1993) and in the brain stem (Minami, 1995) following cisplatin

treatment, while a decrease in Monoamine Oxidase (MAO, ileum) has also been reported

(Endo et al., 1993). This enhancement in 5HT biosynthesis and reduction in degradation

ultimately lead to the upsurge of serotonin which is imperative in the mediation of vomiting

act.


124

Dopamine (DA) is also among the several neurotransmitters, which theater its role in

keeping the emotional balance, regulation of cognition, food intake, reward and sexual

behavior (Baptista et al., 2002). Moreover, DA plays integral role in the genesis of vomiting

as well, through selective activation of D2 receptors, localized in the limbic system,

hypothalamus, amygdala and in the brain stem emetic circuitry (Le Moine and Bloch, 2004).

Dopaminergic agonists like apomorphine has been reported to be emetic in a variety of

species including dogs (Foss et al., 1998), ferrets (Osinski et al., 2003; Osinski et al., 2005)

and human (Schofferman, 1976). The emetic action of apomorphine and loperamide has

been suggested to be mediated in the chemoreceptor trigger zone/area postrema through

stimulation of dopamine receptors, as ablation of this area abolished the vomiting response

(Miller and Leslie, 1994; Yoshikawa et al., 1996). In continuation, the delayed phase of

cisplatin induced vomiting does not depend on vagal afferents but is mediated via the area

postrema (Percie du Sert et al., 2009). Moreover, area postrema is also known to be the site

for vomiting induction by apomorphine and loperamide (Foss et al., 1998).

In CIV, the early vomiting majorly involves the monoaminergic neurotransmitters especially

serotonergic system, while the late phase is associated with monoaminergic system

excluding the serotonergic system (Tanihata et al., 2000). Furthermore, substantial

evidences are there for the overlapping of serotonergic, dopaminergic and neurokininergic

mechanisms for the entire time course of cisplatin induced vomiting (Darmani and Crim,

2005; Higgins et al., 2012; Saito et al., 2003).


125


Considering the relevance of DA and 5HT in cisplatin induced vomiting, this study was

designed to evaluate the participation of these monoamine neurotransmitters and their

metabolites in cisplatin induced vomiting, and to examine the impact of Cannabis sativa

(CS), Bacopa monniera (BM), Zingiber officinal (ZO) extracts alone and in combination on

neurotransmitters implicated in the act of vomiting in specific brain areas and intestine in

pigeons.


7.3.1. Chemicals and reagents:

All the chemicals used in analysis including neurotransmitter standards, were of analytical

grade.

Further details are in Chapter 2, Methods, section 2.6.

7.3.2. High Performance Liquid Chromatography (HPLC) system:

The High Performance Liquid Chromatography (HPLC, Shimadzu, Japan) coupled with

electrochemical detector (ECD) was used for the quantification of neurotransmitters and

their metabolites in specific brain areas and intestinal samples.

Details about the HPLC system are given in section 2.12, Chapter 2, Methods.

7.3.3. Sample preparation, handling and preparation of stock solutions:

See Chapter 2: Methods, sections 2.12.1, 2.12.2, 2.12.3.


126


The chromatographic method already developed by our lab (Rauf et al., 2011) was used for

the quantification of neurotransmitters and their metabolites in specific brain areas involved

in the act of vomiting and intestine of pigeons.

Details about the chromatography are given in Chapter 2 Methods, section 2.12.4.

7.4. Results:

7.4.1. High Performance Liquid Chromatography (HPLC), method reproducibility:

The HPLC method used for the quantification of neurotransmitters and their metabolites,

already developed by this lab (Rauf et al., 2011) was highly reproducible. All of the

neurotransmitters and their metabolites were separated within 13 minutes, in the following

order; nor-adrenaline (NA; 4.4 minutes), dihydroxy phenyl acetic acid (DOPAC; 5.6 minutes),

dopamine (DA; 7.2 minutes), 5-hydroxy indole acetic acid (5HIAA; 9.3 minutes), homovanillic

acid (HVA; 10 minutes) & 5-hydroxy tryptamine (5-HT; 11.2 minutes) (Figure 7.1A & B).


127

Figure 7.1A: HPLC chromatogram showing peaks of standard neurotransmitters and

their metabolites:

Chromatogram showing the peaks of nor-adrenaline (NA), dihydroxy phenyl acetic acid

(DOPAC), dopamine (DA), 5-hydroxy indole acetic acid (5HIAA), homovanillic acid (HVA)

and 5-hydroxy tryptamine (5-HT) denoted by A, B, C, D, & E, respectively of standard (100 ng /

mL).

0.0 2.5 5.0 7.5 10.0 min

0

2500

5000

7500

10000

12500

15000

17500

20000A

B

C

D

E

F

Time (minutes)

Curr

ent

(Arb

itra

ry u

nit

s)


128

Figure 7.1B: HPLC chromatogram showing peaks of neurotransmitters and their

metabolites in sample:

Chromatogram showing the peaks of nor-adrenaline (NA), dihydroxy phenyl acetic acid

(DOPAC), dopamine (DA), 5-hydroxy indole acetic acid (5HIAA), homovanillic acid (HVA)

and 5-hydroxy tryptamine (5-HT) denoted by A, B, C, D, & E, respectively in sample (Area

postrema).

0.0 2.5 5.0 7.5 10.0 min

0

2500

5000

7500

10000

12500

15000

17500A

B

C D

E

F

Time (minutes)

Curr

ent

(Arb

itra

ry u

nit

s)


129

7.4.2. Effect of metoclopramide (MCP), BM methanolic fraction (BM-MetFr),

butanolic fraction (BM-ButFr), CS Hexane fraction (CS-HexFr), ZO acetone fraction

(ZO-ActFr) or combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) on Basal level

of neurotransmitters and their metabolites (ng/mg tissue wet weight) at specific brain

areas (AP & BS) and intestine of pigeon:

7.4.2.1. Effect of standard MCP on basal neurotransmitters and their metabolites in

the brain areas and intestine:

The standard MCP treatment reduced the concentration of 5HIAA in the areas of AP (Table

7.1A) and BS (Table 7.1B) with significance of (P < 0.05) and (P < 0.001), respectively as

compared to basal level. In addition, the decrease in the concentration of HVA was also

observed in the AP, which was found to be statistically significant (P < 0.05) with respect to

basal HVA concentration (Table 7.1A).

7.4.2.2. Effect of BM methanolic fraction (BM-MetFr) or butanolic fraction (BM-

ButFr) on basal neurotransmitters and their metabolites in the brain areas and

intestine:

As shown in table (7.1A, B & C), BM-MetFr at doses 10, 20 & 40 mg/kg & BM-ButFr at

doses 5, 10 & 20 mg/kg treatments failed to alter the basal level of neurotransmitters (NA,

DA & 5HT) and their metabolites (DOPAC, HVA & 5HIAA) any significantly, in the brain

areas (AP & BS) and intestine. However, BM-MetFr at doses 10, 20 & 40 mg/kg and BM-

ButFr at dose of 10 mg/kg decreased the level of 5HIAA significantly (P < 0.01) at the level

of BS (Table 7.1B). Moreover, NA showed upsurge with 20 mg/kg dose of BM-ButFr (P <

0.001) in the intestine (Table 7.1C).


130

7.4.2.3. Effect of CS Hexane fraction (CS-HexFr) on basal neurotransmitters and their

metabolites in the brain areas and intestine:

As shown in table (7.1A, B & C), treatment with CS-HexFr (10 mg/kg) had no significant

effects on NA, DA and its metabolites DOPAC and HVA, 5HT and its metabolite 5HIAA in

the brain areas (AP & BS) and intestine. Though, the concentration of DA at the level of AP

and intestine was increased significantly (P < 0.001) as compared to basal level.

7.4.2.4. Effect of ZO acetone fraction (ZO-ActFr) on basal neurotransmitters and their

metabolites in the brain areas and intestine:

Zingiber officinale acetone fraction (ZO-ActFr) at the dose of 50 mg/kg did not altered the

basal neurotransmitter level except a decrease in the concentration of 5HIAA in the brain

area of BS, where the difference was found to be statistically significant (P < 0.05) as

compared to basal level (Table 7.1B).

7.4.2.5. Effect of combination (CS-HexFr 10 mg + BM-ButFr 5 mg) on basal

neurotransmitters and their metabolites in the brain areas and intestine:

Cannabis sativa hexane fraction (CS-HexFr; 10 mg) in combination with Bacopa monniera

n-butanol fraction (BM-ButFr; 5 mg) decreased the 5HIAA level only in the brain area of

BS, which was found to be statistically significant (P < 0.05) as compared to basal level

(Table 7.1B).


131

Table 7.1A: Effect of metoclopramide (MCP), BM methanol fraction (BM-MetFr), n-

butanol fraction (BM-ButFr), CS hexane fraction (CS-HexFr), ZO acetone fraction

(ZO-ActFr) or combination (CS-HexFr 10 mg + BM-ButFr 5 mg) on basal level of

neurotransmitters and their metabolites at the brain level of AP in pigeons:

Treatment NA DOPAC DA 5HIAA HVA 5HT

Saline 0.610 ± 0.014 0.382 ± 0.111 0.590 ± 0.146 0.158 ± 0.036 0.913 ± 0.095 0.062±0.034

MCP 30mg 0.023 ± 0.005 0.017 ± 0.006 0.025 ± 0.012 0.005 ± 0.001* 0.121±0.063* 0.023±0.001

BM-MetFr 10mg 0.058 ± 0.017 0.047 ± 0.010 0.070 ± 0.007 0.017 ± 0.002 0.346 ± 0.047 0.014±0.002

BM-MetFr 20mg 0.106 ± 0.039 0.168 ± 0.074 0.186 ± 0.066 0.050 ± 0.025 0.238 ± 0.117 0.054±0.034

BM-MetFr 40mg 0.336 ± 0.174 0.092 ± 0.025 0.216 ± 0.089 0.032 ± 0.011 0.476 ± 0.151 0.035±0.014

BM-ButFr 05mg 0.261 ± 0.031 0.043 ± 0.010 0.044 ± 0.105 0.207 ± 0.041 0.426 ± 0.072 0.146±0.050

BM-ButFr 10mg 0.044 ± 0.023 0.115 ± 0.034 0.277 ± 0.054 0.047 ± 0.006 0.356 ± 0.098 0.020±0.004

BM-ButFr 20mg 0.909 ± 0.165 0.313 ± 0.087 0.802 ± 0.210 0.066 ± 0.028 0.854 ± 0.440 0.105±0.057

CS-HexFr 10mg 0.579 ± 0.500 0.094 ± 0.026 1.888±0.547*** 0.260 ± 0.087 1.335 ± 0.323 0.126±0.106

ZO-ActFr 50mg 0.372 ± 0.036 0.122 ± 0.047 0.191 ± 0.037 0.039 ± 0.006 0.874 ± 0.204 0.045±0.008

(CS-HexFr 10mg+

BM-ButFr 5mg)

1.491 ± 1.382 0.408 ± 0.276 0.225 ± 0.088 0.100 ± 0.044 1.096 ± 0.507 0.147±0.095

Effect of BM-MetFr (10, 20 & 40 mg/kg), BM-ButFr (5, 10 & 20 mg/kg), CS-HexFr (10

mg/kg), ZO-ActFr (50 mg/kg) or combination of CS-HexFr (10 mg) with BM-ButFr (5 mg)

administered 30 minutes before saline administration, on the basal level of neurotransmitters

and their metabolites (ng/mg tissue wet weight) at the brain level of area postrema (AP) in

pigeons at t = 3 hr (n = 6 - 8). Standard MCP is also shown. Values significantly different

compared to basal level are indicated as *p < 0.05, ***p < 0.001 (ANOVA followed by

Tukey post hoc analysis).


132

Table 7.1B: Effect of metoclopramide (MCP), BM methanol fraction (BM-MetFr), n-



neurotransmitters and their metabolites at the level of BS in pigeons:


Saline 0.094 ± 0.022 0.060±0.020 0.175±0.078 0.060 ± 0.021 0.060 ± 0.016 0.010 ± 0.003

MCP 30mg 0.119 ± 0.033 0.027±0.006 0.044±0.012 0.007± 0.001*** 0.066 ± 0.031 0.019 ± 0.002

BM-MetFr 10mg 0.040 ± 0.020 0.017±0.006 0.063±0.027 0.011 ± 0.003** 0.051 ± 0.025 0.037 ± 0.019

BM-MetFr 20mg 0.147 ± 0.091 0.052±0.036 0.058±0.040 0.005 ± 0.001** 0.037 ± 0.021 0.019 ± 0.005

BM-MetFr 40mg 0.020 ± 0.009 0.035±0.002 0.022±0.018 0.003 ±0.001*** 0.022 ± 0.015 0.010 ± 0.002

BM-ButFr 05mg 0.108 ± 0.010 0.015±0.001 0.698±0.407 0.067 ± 0.014 0.021 ± 0.003 0.167±0.014***

BM-ButFr 10mg 0.054 ± 0.016 0.021±0.006 0.046±0.011 0.011 ± 0.001** 0.032 ± 0.022 0.011 ± 0.002

BM-ButFr 20mg 0.156 ± 0.097 0.084±0.050 0.178±0.110 0.031 ± 0.001 0.243±0.077*** 0.041 ± 0.005

CS-HexFr 10mg 0.012 ± 0.003 0.098±0.002 0.342±0.039 0.038 ± 0.003 0.015 ± 0.000 0.031 ± 0.000

ZO-ActFr 50mg 0.056 ± 0.055 0.128±0.127 0.082±0.054 0.011 ± 0.001* 0.088 ± 0.021 0.020 ± 0.001

(CS-HexFr 10mg

+BM-ButFr 5mg)

0.160 ± 0.115 0.031±0.000 0.428±0.157 0.012 ± 0.003* 0.104 ± 0.042 0.020 ± 0.006




and their metabolites (ng/mg tissue wet weight) at the brain level of BS in pigeons at t = 3 hr

(n = 6 - 8). Standard MCP is also shown. Values significantly different compared to basal

level are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post

hoc analysis).


133

Table 7.1C: Effect of metoclopramide (MCP), BM methanol fraction (BM-MetFr), n-



neurotransmitters and their metabolites at the level of intestine in pigeons:


Saline 0.194 ± 0.059 0.067±0.020 0.090 ± 0.064 0.076 ± 0.058 0.056±0.025 0.049 ± 0.016

MCP 30mg 0.138 ± 0.039 0.054±0.025 0.059 ± 0.018 0.097 ± 0.022 0.198±0.102 0.062 ± 0.013

BM-MetFr 10mg 0.117 ± 0.047 0.106±0.047 0.089 ± 0.045 0.061 ± 0.016 0.069±0.032 0.236 ± 0.103

BM-MetFr 20mg 0.114 ± 0.040 0.110±0.053 0.329 ± 0.125 0.077 ± 0.014 0.032±0.012 0.158 ± 0.022

BM-MetFr 40mg 0.015 ± 0.006 0.026±0.003 0.011 ± 0.005 0.028 ± 0.004 0.013±0.009 0.044 ± 0.007

BM-ButFr 05mg 0.204 ± 0.033 0.005±0.001 0.123 ± 0.052 0.268±0.068** 0.077±0.025 0.848±0.187***

BM-ButFr 10mg 0.290 ± 0.083 0.144±0.109 0.289 ± 0.196 0.121 ± 0.028 0.247±0.107 0.106 ± 0.026

BM-ButFr 20mg 1.328±0.271*** 0.090±0.044 0.244 ± 0.162 0.051 ± 0.014 0.142±0.052 0.145 ± 0.036

CS-HexFr 10mg 0.021 ± 0.001 0.063±0.029 1.291 0.273*** 0.219 ± 0.045 0.102±0.042 0.011 ± 0.002

ZO-ActFr 50mg 0.334 ± 0.131 0.037±0.028 0.356 ± 0.113 0.050 ± 0.008 0.231±0.110 0.103 ± 0.015

(CS-HexFr 10mg

+ BM-ButFr 5mg)

0.248 ± 0.040 0.123±0.045 0.056 ± 0.001 0.029 ± 0.010 0.119±0.115 0.063 ± 0.021




and their metabolites (ng/mg tissue wet weight) at the level of intestine in pigeons at t = 3 hr

(n = 6 - 8). Standard MCP is also shown. Values significantly different compared to basal

level are indicated as **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey post hoc

analysis).


134

7.4.3. Effect of metoclopramide (MCP), CS Hexane fraction (CS-HexFr), BM

methanolic fraction (BM-MetFr), butanolic fraction (BM-ButFr), ZO acetone fraction

(ZO-ActFr) or combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) on level of

neurotransmitters and their metabolites (ng/mg of tissue wet weight) at specific brain

areas and intestine of pigeon at acute time point (3rd

hour):

7.4.3.1. Effect of standard MCP on neurotransmitters and their metabolites in the

brain areas and intestine at 3rd

hour of cisplatin treatment:

Cisplatin treatment significantly increased (P < 0.001) the concentration of 5-hydroxy

tryptamine (5HT) in the brain stem (BS; Table 7.2B) and intestine (Table 7.2C) as compared

to basal level, while a non-significant increase was observed in the area postrema (AP; Table

7.2A). The treatment with standard MCP at the dose of 30 mg/kg failed to change the

concentration of NA, DOPAC , DA, 5HIAA and HVA in all the brain areas (AP & BS) and

intestine, but reduced the concentration of 5HT in the BS and intestine significantly (P <

0.001) as compared to cisplatin control (Table 7.2B & C). In addition to its inhibitory effects

on 5HT, MCP also decreased 5HIAA concentration in both the brain areas (AP & BS) and

intestine significantly (P < 0.01-0.001, Table 7.3A, B & C).

7.4.3.2. Effect of CS-HexFr on neurotransmitters and their metabolites in the brain

areas and intestine at 3rd


Cisplatin treatment caused significant increase of 5HT (P < 0.001) and 5HIAA (P < 0.001)

in the brain areas (AP & BS) and intestine, without affecting the concentrations of NA,

DOPAC & DA (Table 7.3A, B & C) as compared to basal level, except in the AP where the

increase in the 5HT concentration was found to be statistically non-significant (P > 0.05,


135

Table 7.3A). CS-HexFr (10 mg/kg) significantly reduced the 5HIAA (P < 0.001) and 5HT

(P < 0.001) concentrations in the brain areas (AP & BS) and intestine while no effects were

seen on the levels of NA and DOPAC. On the contrary, CS-HexFr (10 mg) treatment caused

increase in the concentration of DA in AP, BS and intestine that was significant (P < 0.001)

as compared to cisplatin control (Table 7.3A, B & C).

7.4.3.3. Effect of BM-MetFr or BM-ButFr on neurotransmitters and their metabolites

in the brain areas and intestine at 3rd


BM-MetFr (10, 20 & 40 mg/kg) and BM-ButFr (5, 10 & 20 mg/kg) treatments reduced the

concentration of 5HT in the brain area of BS (P < 0.001, Table 7.2B) and intestine (P <

0.001, Table 7.2C) as compared to cisplatin control, without any significant effects on NA,

DOPAC, HVA and 5HIAA. Furthermore, no significant alteration was observed in the brain

area of AP.


136

Table 7.2A: Effect of standard metoclopramide (MCP), BM methanolic fraction

(BM-MetFr) or n-butanolic fraction (BM-ButFr) on neurotransmitters and their

metabolites at the brain level of AP at 3rd



Saline 0.571 ± 0.072 0.389±0.108 0.543 ± 0.130 0.290 ± 0.059 1.374±0.485 0.011 ± 0.001

Cisplatin 1.411 ± 1.160 0.299±0.132 0.073 ± 0.023 0.211 ± 0.079 3.859±3.373 0.316 ± 0.093

MCP 30mg 0.116 ± 0.078 0.106±0.040 0.223 ± 0.103 0.024 ± 0.005 0.069±0.045 0.023 ± 0.005

BM-MetFr 10mg 0.537 ± 0.144 0.129±0.032 0.245 ± 0.066 0.035 ± 0.010 1.301±0.515 0.020 ± 0.009

BM-MetFr 20mg 0.682 ± 0.297 0.358±0.222 0.844 ± 0.585 0.109 ± 0.061 0.447±0.215 0.042 ± 0.009

BM-MetFr 40mg 0.250 ± 0.082 0.206±0.009 0.699 ± 0.143 0.035 ± 0.007 0.393±0.092 0.027 ± 0.006

BM-ButFr 05mg 0.960 ± 0.146 0.492±0.088 0.558 ± 0.128 0.238 ± 0.039 1.603±0.341 1.990 ± 1.646

BM-ButFr 10mg 0.445 ± 0.098 0.110±0.031 0.197 ± 0.058 0.038 ± 0.017 0.319±0.217 0.025 ± 0.011

BM-ButFr 20mg 0.109 ± 0.032 0.058±0.012 0.122 ± 0.020 0.025 ± 0.006 1.317±0.414 0.020 ± 0.003

Effect of BM-MetFr (10, 20 & 40 mg/kg), BM-ButFr (5, 10 & 20 mg/kg) or standard MCP

(30 mg/kg) administered 30 mins before cisplatin challenge, on the level of

neurotransmitters and their metabolites (ng/mg tissue wet weight) at the brain level of Area

Postrema (AP) of pigeons at t = 3 hr of cisplatin administration (n = 6 - 8).


137

Table 7.2B: Effect of standard metoclopramide (MCP), BM methanolic fraction


metabolites at the brain level of BS at 3rd



Saline 0.071 ± 0.004 0.073±0.012 0.069 ± 0.023 0.017 ± 0.015 0.022±0.010 0.031 ± 0.002

Cisplatin 0.080 ± 0.019 0.130±0.098 0.032 ± 0.001 0.049 ± 0.013 0.016±0.008 0.138±0.018###

MCP 30mg 0.044 ± 0.016 0.015±0.005 0.018 ± 0.011 0.041 ± 0.001 0.042±0.002 0.021±0.001***

BM-MetFr 10mg 0.152 ± 0.050 0.053±0.021 0.011 ± 0.025 0.016 ± 0.004 0.102±0.028 0.012±0.004***

BM-MetFr 20mg 0.145 ± 0.071 0.057±0.021 0.093 ± 0.030 0.025 ± 0.005 0.220±0.062 0.007±0.001***

BM-MetFr 40mg 0.132 ± 0.007 0.097±0.006 0.797 ± 0.086 0.025 ± 0.003 0.222±0.128 0.018±0.001***

BM-ButFr 05mg 0.037 ± 0.010 0.013±0.001 0.010 ± 0.001 0.021 ± 0.004 0.024±0.001 0.090 ± 0.020*

BM-ButFr 10mg 0.068 ± 0.011 0.139±0.126 0.052 ± 0.040 0.055 ± 0.001 0.215±0.077 0.017±0.002***

BM-ButFr 20mg 0.037 ± 0.004 0.083±0.014 0.076 ± 0.016 0.018 ± 0.001 0.289±0.023 0.006±0.001***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of Brain Stem

(BS) of pigeons at t = 3 hr of cisplatin administration (n = 6 - 8). Values significantly

different compared to cisplatin control are indicated as *p < 0.05, ***p < 0.001, while

Values significantly different as compared to basal level are indicated as ###p < 0.001

(ANOVA followed by Tukey post hoc analysis).


138

Table 7.2C: Effect of standard metoclopramide (MCP), BM methanolic fraction


metabolites at the level of intestine at 3rd



Saline 0.374 ± 0.184 0.105±0.040 0.137 ± 0.054 0.410 ± 0.269 0.054±0.022 0.044 ± 0.016

Cisplatin 0.208 ± 0.032 0.015±0.002 0.002 ± 0.000 0.285 ± 0.020 0.035±0.003 0.821±0.137###

MCP 30mg 0.119 ± 0.044 0.060±0.059 0.164 ± 0.127 0.033 ± 0.005 0.086±0.030 0.045±0.006***

BM-MetFr 10mg 0.152 ± 0.050 0.053±0.021 0.116 ± 0.025 0.016 ± 0.004 0.102±0.028 0.012±0.004***

BM-MetFr 20mg 0.229 ± 0.116 0.048±0.031 0.066 ± 0.044 0.028 ± 0.004 0.348±0.185 0.009±0.001***

BM-MetFr 40mg 0.235 ± 0.066 0.101±0.036 0.530 ± 0.235 0.082 ± 0.010 0.290±0.112 0.048±0.021***

BM-ButFr 05mg 0.051 ± 0.024 0.016±0.007 0.040 ± 0.023 0.208 ± 0.026 0.040±0.026 0.420±0.069***

BM-ButFr 10mg 0.087 ± 0.026 0.024±0.013 0.041 ± 0.026 0.021 ± 0.008 0.498±0.353 0.005±0.002***

BM-ButFr 20mg 0.238 ± 0.036 0.105±0.066 0.112 ± 0.035 0.023 ± 0.004 0.784±0.291 0.027±0.008***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of intestine in

pigeons at t = 3 hr of cisplatin administration (n = 6 - 8). Values significantly different

compared to cisplatin control are indicated as ***p < 0.001, while Values significantly

different as compared to basal level are indicated as ###p < 0.001 (ANOVA followed by

Tukey post hoc analysis).


139

7.4.3.4. Effect of ZO-ActFr on neurotransmitters and their metabolites in the brain

areas and intestine at 3rd


Zingiber officinale acetone fraction (ZO-ActFr) treatment at the dose of 50 mg/kg reduced

the level of 5HIAA (P < 0.001) and 5HT (P < 0.05 - 0.001) in the brain (AP & BS; Table

7.3A & B) and intestine (Table 7.3C) as compared to cisplatin control. No significant

alteration was seen in NA, DOPAC, DA and HVA in the brain (AP & BS) and intestine,

except DA and its metabolite DOPAC which increased significantly (P < 0.001) in the

intestine (Table 7.3C).


neurotransmitters and their metabolites in the brain areas and intestine at 3rd

hour of

cisplatin treatment:

CS-HexFr (10 mg) administered in combination with BM-ButFr (5 mg) decreased the

concentration of 5HT (P < 0.05 – 0.001) and its metabolite 5HIAA (P < 0.001) in both, brain

(AP & BS) and intestine (Table 7.3A, B & C). However, no significant effect was observed

on the level of NA, DOPAC, DA and HVA in the brain (AP & BS) and intestine, except

DOPAC which was increased (P < 0.05) at the level of intestine (Table 7.3C).


140

Table 7.3A: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-

HexFr), ZO acetone fraction (ZO-ActFr) or combination of CS-HexFr (10 mg) with

BM-ButFr (5 mg) on neurotransmitters and their metabolites at the brain area of AP

at 3rd


Treatment NA Dopac DA 5HIAA HVA 5HT

Saline 0.605 ± 0.298 0.217 ±0.100 0.618 ± 0.218 0.087 ± 0.039 0.805±0.166 0.113 ± 0.060

Cisplatin 1.879 ± 1.622 0.312 ±0.183 0.080 ± 0.030 0.316 ± 0.101# 0.555±0.188 0.282 ± 0.120

MCP 30mg 0.116 ± 0.078 0.142 ±0.050 0.310 ± 0.137 0.026 ± 0.006** 0.040±0.021 0.030 ± 0.005*

CS-HexFr 10mg 0.265 ± 0.034 0.638 ±0.133 2.142±0.387*** 0.045±0.012*** 1.140±0.162 0.030 ± 0.010**

ZO-ActFr 50mg 0.605 ± 0.221 0.080 ±0.027 1.147 ± 0.615 0.013±0.001*** 0.171±0.133 0.006 ± 0.002*

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.166 ± 0.139 0.192 ±0.088 0.339 ± 0.144 0.046 ± 0.019** 0.443±0.181 0.048 ± 0.022*

Effect of CS-HexFr (10 mg/kg), ZO-ActFr (50 mg/kg) or combination of CS-HexFr (10 mg)

with BM-ButFr (5 mg) administered 30 mins before cisplatin challenge, on the level of


Postrema (AP) of pigeons at t = 3 hr of cisplatin administration (n = 6 - 8). Standard MCP is

also shown. Values significantly different compared to cisplatin control are indicated as *p <

0.05, **p < 0.01 ***p < 0.001, while Values significantly different compared to basal level

are indicated as #p < 0.05 (ANOVA followed by Tukey post hoc analysis).


141

Table 7.3B: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-


BM-ButFr (5 mg) on neurotransmitters and their metabolites at the level of BS at 3rd



Saline 0.119±0.043 0.031 ±0.030 0.253 ± 0.152 0.013 ± 0.001 0.090±0.016 0.018 ± 0.000

Cisplatin 0.094±0.024 0.173 ±0.136 0.030 ± 0.001 0.036±0.004### 0.028±0.003 0.131±0.020###

MCP 30mg 0.041±0.021 0.039 ±0.003 0.013 ± 0.002 0.021±0.001*** 0.023±0.001 0.008±0.000***

CS-HexFr 10mg 0.026±0.001 0.013 ±0.001 0.436±0.020*** 0.009±0.001*** 0.013±0.004 0.010±0.003***

ZO-ActFr 50mg 0.633±0.050 0.015 ±0.002 0.133 ± 0.077 0.018±0.002*** 0.147±0.054 0.037±0.004***

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.089±0.007 0.011 ±0.001 0.119 ± 0.069 0.003±0.002*** 0.018±0.003 0.006±0.002***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of BS in

pigeons at t = 3 hr of cisplatin administration (n = 6 - 8). Standard MCP is also shown.

Values significantly different compared to cisplatin control are indicated as ***p < 0.001,

while Values significantly different compared to basal level are indicated as ###p < 0.001



142

Table 7.3C: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-


BM-ButFr (5 mg) on neurotransmitters and their metabolites at the level of intestine at

3rd



Saline 0.337 ± 0.045 0.087 ± 0.035 0.133 ± 0.031 0.032 ± 0.010 0.089±0.046 0.048 ± 0.051

Cisplatin 0.222 ± 0.044 0.015 ± 0.003 0.022 ± 0.005 0.295±0.024### 0.038±0.004 0.665±0.125###

MCP 30mg 0.109±0.040* 0.029 ± 0.001 0.246 ± 0.183 0.031±0.006*** 0.067±0.030 0.041±0.005***

CS-HexFr 10mg NA 0.067 ± 0.039 0.920±0.130*** 0.003±0.001*** 0.030±0.012 0.001±0.000***

ZO-ActFr 50mg 0.328 ± 0.036 0.088±0.109*** 1.005±0.055*** 0.030±0.013*** 0.124±0.066 0.049±0.021***

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.266 ± 0.104 0.047 ± 0.275* 0.399 ± 0.232 0.003±0.001*** 0.004±0.002 0.007±0.006***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of intestine in


Values significantly different compared to cisplatin control are indicated as *p < 0.05, ***p

< 0.001, while Values significantly different compared to basal level are indicated as ###p <

0.001 (ANOVA followed by Tukey post hoc analysis).


143

7.4.4. Effect of standard metoclopramide (MCP), CS Hexane fraction (CS-HexFr),

BM methanolic fraction (BM-MetFr), n-butanolic fraction (BM-ButFr), ZO acetone

fraction (ZO-ActFr) or combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) on

level of neurotransmitters and their metabolites at specific brain areas and intestine of

pigeon at delayed time point (18th

hour):

7.4.4.1. Effect of standard MCP on neurotransmitters and their metabolites in the

brain areas and intestine at 18th


Cisplatin increased the level of DA highly significantly (P < 0.001) in the AP (Table 7.4A),

while a non-significant trend towards increase was observed in the areas of BS and intestine

(Table 7.4B & C). 5HT concentrations were also raised in the brain area of BS (P < 0.05)

and intestine (P < 0.001), without effecting the levels of NA, DOPAC, 5HIAA, HVA in the

areas of BS and intestine and 5HT in the AP (Table 7.4A, B & C). Treatment with standard

metoclopramide (MCP; 30 mg/kg) significantly decreased the upsurge of DA at the brain

area of AP (P < 0.001; Table 7.4A) and BS (P < 0.01; Table 7.4B). Furthermore a decrease

in the concentration of 5HIAA and 5HT was also observed in the brain area of AP (P < 0.05

– 0.01; Table 7.4A, 7.5A) and intestine (P < 0.001; Table 7.4C) as compared to cisplatin

control.


144

7.4.4.2. Effect of CS-HexFr on neurotransmitters and their metabolites in the brain

areas and intestine at 18th


Cannabis sativa hexane fraction (CS-HexFr) at the dose of 10 mg/kg decreased significantly

(P < 0.001) the upsurge in the concentration of DA in the brain area of AP (P < 0.001; Table

7.5A) while decrease in 5HT was observed in the brain area of BS (P < 0.001; Table 7.5B)

and intestine (P < 0.001; Table 7.5C).

7.4.4.3. Effect of BM-MetFr or BM-ButFr on neurotransmitters and their metabolites

in the brain areas and intestine at 18th


Both the fractions of BM i.e. methanolic (BM-MetFr; 10, 20 & 40 mg) and butanolic (BM-

ButFr; 5, 10 & 20 mg) were found effective in reducing the DA concentration highly

significantly (P < 0.001) with respect to cisplatin control at the level of AP (Table 7.4A).

Similar effect however, was also seen in the area of BS but with variable statistical

significance (P < 0.05- 0.001; Table 7.4B). However, BM-MetFr 20 and 40 mg/kg failed to

attenuate the DA concentration any significantly at the brain level of BS (Table 7.4B).

Moreover, at the level of intestine, significant reduction (P < 0.001) was observed in the

level of 5HT with the BM methanolic and butanolic fractions with all the doses tested,

except BM-MetFr 40 mg and BM-ButFr 5 mg (Table 7.4C). Furthermore, BM-MetFr 40 mg

and BM-ButFr 20 mg significantly (P < 0.05) decreased the level of 5HIAA in the brain area

of AP (Table 7.4A).


145

Table 7.4A: Effect of standard metoclopramide (MCP), BM methanolic fraction

(BM-MetFr) or n-butanol fraction (BM-ButFr) on neurotransmitters and their

metabolites in the brain area of AP at 18th



Saline 0.599 ± 0.084 0.458±0.160 0.535 ± 0.168 0.347 ± 0.084 1.763±0.743 0.012 ± 0.001

Cisplatin 0.247 ± 0.059 0.022±0.008 13.43±4.528### 0.164 ± 0.042 0.395±0.104 0.147 ± 0.044

MCP 30mg 0.161 ± 0.070 0.052±0.023 0.048±0.024*** 0.021 ± 0.002* 0.276±0.157 0.010 ± 0.003

BM-MetFr 10mg 0.493 ± 0.166 0.150±0.019 0.365±0.061*** 0.059 ± 0.004 1.164±0.272 0.057 ± 0.014

BM-MetFr 20mg 0.467 ± 0.208 0.193±0.058 0.350±0.063*** 0.053 ± 0.010 0.576±0.202 0.046 ± 0.009

BM-MetFr 40mg 0.903 ± 0.170 0.075±0.018 0.155±0.033*** 0.030 ± 0.004* 0.198±0.045 0.025 ± 0.007

BM-ButFr 05mg 0.808 ± 0.104 0.765±0.152 0.746±0.254*** 0.223 ± 0.045 1.177±0.248 0.750 ± 0.243

BM-ButFr 10mg 0.233 ± 0.055 0.039±0.031 0.062±0.044*** 0.074 ± 0.032 1.056±0.403 0.037 ± 0.015

BM-ButFr 20mg 0.215 ± 0.036 0.068±0.023 0.146±0.026*** 0.024 ± 0.006* 0.901±0.255 0.018 ± 0.003




Postrema (AP) of pigeons at t = 18 hr of cisplatin administration (n = 6 - 8). Values

significantly different compared to cisplatin control are indicated as *p < 0.05, ***p <

0.001, while Values significantly different compared to basal level are indicated as ###p <

0.001 (ANOVA followed by Tukey post hoc analysis).


146

Table 7.4B: Effect of standard metoclopramide (MCP), BM methanolic fraction


metabolites at the level of BS at 18th



Saline 0.069 ± 0.005 0.086±0.017 0.094 ± 0.033 0.018 ± 0.021 0.025±0.017 0.011 ± 0.006

Cisplatin 0.068 ± 0.003 0.021±0.001 0.258 ± 0.057 0.036 ± 0.003 0.019±0.003 0.121 ± 0.008#

MCP 30mg 0.101 ± 0.094 0.001±0.001 0.013 ± 0.013** 0.014 ± 0.004 0.082±0.052 0.014 ± 0.002

BM-MetFr 10mg 0.074 ± 0.066 0.054±0.050 0.021 ± 0.019** 0.035 ± 0.006 0.371±0.161 0.030 ± 0.005

BM-MetFr 20mg 0.241 ± 0.146 0.073±0.033 0.135 ± 0.126 0.024 ± 0.008 0.168±0.051 0.033 ± 0.008

BM-MetFr 40mg 0.384 ± 0.139 0.064±0.015 0.109 ± 0.040 0.015 ± 0.006 0.243±0.100 0.110 ± 0.096

BM-ButFr 05mg 0.072 ± 0.002 0.030±0.002 0.007±0.002*** 0.034 ± 0.002 0.010±0.001 0.141 ± 0.005

BM-ButFr 10mg 0.054 ± 0.024 0.025±0.025 0.021 ± 0.016** 0.018 ± 0.003 0.390±0.101 0.007 ± 0.001*

BM-ButFr 20mg 0.128 ± 0.033 0.147±0.030 0.080 ± 0.035* 0.025 ± 0.003 0.216±0.090 0.025 ± 0.003


(30 mg/kg) administered 30 mins before cisplatin challenge, on the level of neurotransmitters

and their metabolites (ng/mg tissue wet weight) at the brain level of BS of pigeons at t = 18 hr

of cisplatin administration (n = 6 - 8). Values significantly different compared to cisplatin

control are indicated as *p < 0.05, **p < 0.01 ***p < 0.001, while Values significantly

different compared to basal level are indicated as #p < 0.05 (ANOVA followed by Tukey post

hoc analysis).


147

Table 7.4C: Effect of standard metoclopramide (MCP), BM methanolic fraction


metabolites at the level of intestine at 18th



Saline 0.288 ± 0.237 0.060±0.015 0.058 ± 0.021 0.656 ± 0.403 0.059±0.031 0.045 ± 0.026

Cisplatin 0.228 ± 0.027 0.005±0.001 0.397 ± 0.173 0.390 ± 0.044 0.053±0.006 0.588±0.163###

MCP 30mg 0.177 ± 0.078 0.011±0.003 0.020 ± 0.010 0.022 ± 0.005 0.433±0.384 0.030±0.006***

BM-MetFr 10mg 0.405 ± 0.129 0.281±0.130 0.064 ± 0.330 0.039 ± 0.019 1.112±0.685 0.058±0.024***

BM-MetFr 20mg 0.336 ± 0.144 0.309±0.146 0.122 ± 0.051 0.114 ± 0.023 0.095±0.031 0.188 ± 0.035**

BM-MetFr 40mg 0.545 ± 0.254 0.220±0.104 0.159 ± 0.067 0.113 ± 0.034 0.111±0.043 0.317 ± 0.072

BM-ButFr 05mg 0.152 ± 0.027 0.075±0.034 0.069 ± 0.029 0.227 ± 0.028 0.221±0.131 0.641 ± 0.067

BM-ButFr 10mg 0.103 ± 0.039 0.031±0.031 0.090 ± 0.067 0.066 ± 0.028 1.737±0.360 0.017±0.004***

BM-ButFr 20mg 0.294 ± 0.068 0.235±0.073 0.333 ± 0.161 0.126 ± 0.010 0.594±0.301 0.140±0.024***


(30 mg/kg) administered 30 mins before cisplatin challenge, on the level of neurotransmitters

and their metabolites (ng/mg tissue wet weight) at the level of intestine in pigeons at t = 18 hr

of cisplatin administration (n = 6 - 8). Values significantly different compared to cisplatin

control are indicated as **p < 0.01 ***p < 0.001, while Values significantly different

compared to basal level are indicated as ###p < 0.001 (ANOVA followed by Tukey post hoc

analysis).


148

7.4.4.4. Effect of ZO-ActFr on neurotransmitters and their metabolites in the brain

areas and intestine at 18th


Treatment with Zingiber officinale acetone fraction (ZO-ActFr) at the dose of 50 mg/kg

reduced the contents of DA in the brain area of AP (P < 0.001; Table 7.5A) and 5HT in the

areas of BS and intestine ((P < 0.001; Table 7.5B & C) as compared to cisplatin control. No

significant alteration was seen in the contents of 5HIAA, DA, DOPAC and NA at the level

of BS and intestine.


neurotransmitters and their metabolites in the brain areas and intestine at 18th

hour of

cisplatin treatment:

The combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) was found effective in

decreasing the upsurge of DA in the brain area of AP (Table 7.5A) and 5HT at the level of

BS (Table 7.5B) and intestine (Table 7.5C) highly significantly (P < 0.001) as compared to

cisplatin control. However, no significant alteration was seen on neurotransmitters and their

metabolites, except NA in the brain area of BS (Table 7.5B) which was significantly

increased (P < 0.001).


149

Table 7.5A: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-


BM-ButFr (5 mg) on neurotransmitters and their metabolites in the brain area of AP

at 18th



Saline 0.494 ± 0.063 0.337±0.138 0.491 ± 0.169 0.219 ± 0.030 0.854±0.121 0.010 ± 0.001

Cisplatin 0.268 ± 0.073 0.026±0.010 7.366±1.500### 0.188 ± 0.053 0.556±0.114 0.181 ± 0.052##

MCP 30mg 0.182 ± 0.092 0.062±0.030 0.098±0.029*** 0.019 ± 0.002** 0.322±0.178 0.008 ± 0.003**

CS-HexFr 10mg 0.392 ± 0.052 0.254±0.154 0.818±0.232*** 0.104 ± 0.016 0.294±0.144 0.105 ± 0.011

ZO-ActFr 50mg 1.304 ± 1.014 0.229±0.081 0.846±0.407*** 0.082 ± 0.044 1.513±0.817 0.127 ± 0.071

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.471 ± 0.174 0.166±0.066 0.504±0.362*** 0.072 ± 0.012 1.388±0.370 0.107 ± 0.029




Postrema (AP) of pigeons at t = 18 hr of cisplatin administration (n = 6 - 8). Standard MCP

is also shown. Values significantly different compared to cisplatin control are indicated as

**p < 0.01 ***p < 0.001, while Values significantly different compared to basal level are

indicated as ##p < 0.01 ###p < 0.001 (ANOVA followed by Tukey post hoc analysis).


150

Table 7.5B: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-


BM-ButFr (5 mg) on neurotransmitters and their metabolites at the level of BS at 18th



Saline 0.072 ± 0.005 0.074±0.015 0.070 ±0.030 0.118 ± 0.021 0.027±0.014 0.001 ± 0.000

Cisplatin 0.067 ± 0.004 0.001±0.000 0.175 ±0.026 0.034 ± 0.003 0.009±0.001 0.121±0.010###

MCP 30mg 0.008 ± 0.003 0.002±0.001 0.019 ±0.030 0.011 ± 0.002 0.082±0.052 0.014±0.002***

CS-HexFr 10mg 0.138 ± 0.021 0.033±0.014 0.116 ±0.045 0.022 ± 0.005 0.210±0.122 0.024±0.006***

ZO-ActFr 50mg 0.014 ± 0.008 0.001±0.000 0.080 ±0.068 0.001 ± 0.000 0.009±0.005 0.002±0.000***

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.277±0.094*** 0.007±0.002 0.074 ±0.074 0.014 ± 0.002 0.022±0.020 0.022±0.004***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of BS in






151

Table 7.5C: Effect of standard metoclopramide (MCP), CS hexane fraction (CS-


BM-ButFr (5 mg) on neurotransmitters and their metabolites at the level of intestine at

18th



Saline 0.289 ±0.181 0.119±0.053 0.162 ± 0.071 0.001 ± 0.000 0.033±0.020 0.053 ± 0.025

Cisplatin 0.223 ±0.036 0.005±0.001 0.151 ± 0.042 0.329 ± 0.054 0.060±0.007 0.463±0.098###

MCP 30mg 0.177 ±0.078 0.011±0.001 0.020 ± 0.020 0.022 ± 0.005 0.433±0.384 0.030±0.006***

CS-HexFr 10mg 0.392 ±0.052 0.254±0.154 0.818 ± 0.232 0.104 ± 0.016 0.294±0.144 0.105±0.011***

ZO-ActFr 50mg 0.172 ±0.064 0.001±0.000 0.485 ± 0.218 0.009 ± 0.006 0.050±0.030 0.017±0.011***

(CS-HexFr 10mg +

BM-ButFr 5mg)

0.464 ±0.060 0.001±0.001 1.308 ± 0.240 0.020 ± 0.011 0.113±0.112 0.047±0.027***



neurotransmitters and their metabolites (ng/mg tissue wet weight) at the level of intestine of






152

7.5. Discussion:

In this study, the BM fractions both methanolic (BM-MetFr) and n-butanolic (BM-ButFr),

Cannabis sativa hexane fraction (CS-HexFr), Zingiber officinale acetone fraction (ZO-

ActFr) and the combination (CS-HexFr 10 mg + BM-ButFr 5 mg) have shown their impact

on neurotransmitters and their metabolites especially dopamine and serotonin (5HT) in the

specific brain areas involved in the act of vomiting and in the intestine in pigeon model.

These neurotransmitters have been shown to be the important mediators of nausea and

vomiting induced by emetogenic chemotherapeutic agents like cisplatin (Darmani et al.,

2009). All the chemotherapeutic agents especially the Highly Emetogenic Chemotherapy

(HEC) agents such as lomustine, cyclophosphamide and cisplatin cause release of various

neurotransmitter mediators for induction of vomiting. Neurotransmitter of prime importance

is 5HT for the acute phase of vomiting whose peak occurs at 3rd

hour of cisplatin

administration in animal models and human (Gralla et al., 1999; Grelot and Esteve, 2009;

Percie du Sert et al., 2011; Sam et al., 2001).

BM treatments decreased the concentration of 5HT at the level of BS (Table 7.2B) and

intestine (Table 7.2C) at 3rd

hour of cisplatin treatment, while a trend towards decrease in the

concentration of 5HIAA was also observed where the difference was found to be statistically

non-significant as compared to cisplatin control. BM has been proved to be having

antioxidant potential (Ghosh et al., 2007; Jyoti and Sharma, 2006) and this is one of the

probable mechanism by which BM-MetFr and BM-ButFr, might have reduced the

concentration of 5HT comparable to MCP in the brain stem (BS) and intestine at 3rd

hour of

treatment (Table 7.2B & C). The decrease of 5HT concentration might be resulting by

protection of enterochromaffin (EC) cells from oxidative damage at the level of intestine is


153

important to be considered as 95 % of 5HT is present in the EC cells of the gastrointestinal

mucosa (Veyrat-Follet et al., 1997). The significant decrease of 5HT by BM-MetFr (10, 20

& 40 mg) and BM-ButFr (5, 10 & 20 mg) at the level of BS is convincing in that this area is

considered part of the final common pathway (NTS) for the initiation of vomiting act

(Minami, 1995). Furthermore, there are evidences for the comparatively high density of

5HT3 receptors in the NTS and DMV (Himmi et al., 1998; Kwiatkowska et al., 2004) that

signify the effect of BM treatments in the area of BS in relation to its anti-emetic effect.

In this study, with BM treatments have no significant effect on neurotransmitters (NA, DA

& 5HT) and their metabolites (DOPAC, HVA & 5HIAA) in the brain area of AP at peak

acute time (3rd

hour) of cisplatin treatment. The AP has been shown to be involved in the

delayed phase of vomiting. Accordingly, ablation of this area resulted in suppression of

delayed vomiting in animal model (Percie du Sert et al., 2009). Moreover, the

neurotransmitter “serotonin” is reported to be the primary and important culprit in the

mediation of acute vomiting response acting via brain area of NTS (Nakayama et al., 2005;

Rudd and Naylor, 1994). Furthermore, in the vomiting circuitry of the brain, AP is

considered the seat for induction of vomiting by morphine and apomorphine mediating their

effects through dopamine receptors present in the area postrema (King, 1990; Miller and

Leslie, 1994; Yoshikawa et al., 1996).

In the same fashion, the reduction in 5HT and 5HIAA by CS-HexFr (10 mg), ZO-ActFr (50

mg) and combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) in the brain areas (AP &

BS) and intestine are supportive for their anti-emetic activity at the acute vomiting response

(3rd

hour). Similar effect was observed by standard MCP, except CS-HexFr (10 mg) that

caused increase in the concentration of dopamine in the brain areas and intestine (Table


154

7.3A, B & C). Furthermore, the same was observed with ZO-ActFr (50 mg) and

combination (CS-HexFr 10 mg + BM-ButFr 5 mg) at intestine.

A number of studies are suggesting the involvement of CB1 receptor activation for the

antiemetic action of Cannabis sativa (Δ9-THC) (Darmani, 2001) against various emetogenic

agents, which are co-localized with 5HT3 receptors in the NTS and GIT (Hermann et al.,

2002), that inhibit the release of monoamines especially 5HT in the least schrew model

(Darmani and Johnson, 2004) and Pigeon model (present study). Our present results

indicating the decrease in concentration of 5HT and 5HIAA by CS-HexFr (10 mg) are

supportive for its anti-emetic effect by suppressing behavioral signs of cisplatin induced

Retching plus Vomiting (R + V) in pigeon model (chapter 5).

The active components present in ZO acetone fraction (ZO-ActFr) collectively known as

gingerols especially 6-gingerol and galanolactone have previously been demonstrated for

their effects on 5HT3 (Sharma et al., 1997) and NK1 receptors (Qiu-hai et al., 2010). Our

present results demonstrating the decrease in concentration of 5HT and 5HIAA by ZO-

ActFr (50 mg) are supportive and in parallel for their anti-emetic profile in pigeon model

(chapter 5).

Literature about the delayed phase of cisplatin induced vomiting is indicative for presence of

overlapping mechanisms involving the role of substance P and dopamine (Darmani et al.,

2003; Diemunsch and Grelot, 2000). BM has been shown to inhibit hyperactivity, dopamine

receptor supersensitivity induced by morphine and climbing behavior by apomorphine in

rats (Sumathi et al., 2007) and the same has also been reported by our laboratory (Rauf et al.,

2011). In the present studies, cisplatin treatment in pigeon resulted in the increase in the


155

concentration of dopamine at 18th

hour of treatment in the brain areas (AP & BS; Table 7.4A

& B) and 5HT in the intestine only (Table 7.4C). BM-MetFr and BM-ButFr treatments

decreased the dopamine upsurge at the level of AP and BS (Table 7.4A & B). In AP at 18th

hr the BM-MetFr (10 mg) significantly reduced (P < 0.001) the dopamine upsurge caused by

cisplatin and also resulted in the suppression of V + R episodes but the suppression was

found to be statistically non-significant (Table 5.2). These results provide evidences for the

effectiveness of BM extracts as anti-emetic for prolong protection against the vomiting

caused by cisplatin as the large body of evidences suggests dopamine in the etiology of

vomiting. The antidopaminergic effect of BM extracts observed in this study is in line with

our previous reported studies from this laboratory (Rauf et al., 2012; Rauf et al., 2011).

Furthermore, decrease in the concentration of 5HIAA and 5HT by BM treatments in the

brain area of AP and intestine at 18th

hour of cisplatin treatment, respectively might be

additional mechanism involved in the anti-emetic properties of BM extracts.

CS-HexFr, ZO-ActFr or combination of CS-HexFr with BM-ButFr suppressed the dopamine

concentration in the brain area of AP (Table 7.5A) while no significant dopaminergic

suppression was seen in the BS (Table 7.5B) and intestine (Table 7.5C) at 18th

hour of

cisplatin treatment. However, BM was found to be effective in suppressing the dopamine

upsurge at the level of BS (Table 7.4B) as well. Furthermore, 5HT concentration was

decreased in the brain area of BS and intestine thus showing the dominant anti-serotonergic

effect of CS-HexFr, ZO-ActFr & combination of CS-HexFr with BM-ButFr treatments in

the brain area of BS and in the intestine (Table 7.5B & C). The combined anti-serotonergic

and anti-dopaminergic effect of combination observed in this study may explain its prolong

protection against cisplatin induced vomiting in pigeon model (Table 5.4).


156

In this study, treatments with BM, CS, ZO extracts and combination (CS-HexFr 10 mg +

BM-ButFr 5 mg) failed to alter basal neurotransmitters level and their metabolites

significantly, except the rise in dopamine concentration by CS-HexFr (10 mg) at the level of

AP and intestine. Furthermore, the decrease in the concentration of 5HIAA by MCP (30

mg), BM-MetFr (10, 20 & 40 mg), BM-ButFr (10 mg), ZO-ActFr (50 mg) and combination

(CS-HexFr 10 mg + BM-ButFr 5 mg) was observed at the brain area of BS. The lack of any

significant effect by treatment with CS, BM and ZO extracts on basal neurotransmitters and

their metabolites may explain their safety and tolerability. Furthermore, synthetic analogue

of Δ9-THC-dronabinol (Marinol

®) and Nabilone are already in use for the control of cancer

CIV. BM is also available in various formulations (e.g. Bacomind®) for the treatment of

neuropathic pains and as memory enhancer, while ginger is used as spice/flavoring agent

and has long been used for the management of gastrointestinal disorders.

In conclusion, extracts of CS, BM and ZO are having anti-serotonergic and anti-

dopaminergic effects in a blended manner at the two different time points. At the acute time

point, dominantly the anti-serotonergic effects were observed by all the treatments including

CS-HexFr, BM-MetFr, BM-ButFr, ZO-ActFr and the combination (CS-HexFr 10 mg + BM-

ButFr 5 mg), while at the delayed time point anti-dopaminergic effects were seen by all the

BM treatments. Moreover, anti-serotonergic effects were observed with CS-HexFr, ZO-

ActFr and the combination (CS-HexFr 10 mg + BM-ButFr 5 mg).

Chapter 8 Attenuation of vomiting & C-fos-IR by BM extracts

157

Chapter 8

Attenuation of cisplatin induced Retching plus

Vomiting (R + V) and C-fos immunoreactivity

(C-fos-IR) by bacosides containing Bacopa

monniera fractions in Suncus murinus


158

8.1. Introduction:

Nausea and vomiting are some of the complications observed in patients undergoing cancer

chemotherapy. Since 1980’s 5HT3 receptor antagonists (e.g. ondansetron, granisetron and

palonosetron) in combination with glucocorticoids (e.g. dexamethasone) and NK1 receptor

blockers like aprepitant and natupitant are in practice clinically for the prophylaxis and

treatment of nausea and vomiting (Gora-Harper et al., 1999; Hesketh and Grunberg, 2003).

Suncus murinus (S. murinus; house musk schrew), a species of insectivore is an acceptable

model to study the mechanism of CIV and has been proved to be highly useful for providing

evidences for the involvement of free radicals and subsequent release of 5-HT from

enterochromaffin cells in the etiology of vomiting (Matsuki et al., 1993; Mutoh et al., 1992).

Moreover, the expression of C-fos immunoreactivity (C-fos-IR) is considered a marker for

neuronal excitation and can be labeled by immunohistochemical procedures for examining

the neural circuit involved in the act of vomiting. The basal level of C-fos though low but

can be rapidly induced by different stimuli. Cisplatin induces acute C-fos in vomiting

species in the hind brain areas including area postrema (AP), nucleus tractus solitaious

(NTS) and dorsal motor nucleus of vagus nerve (DMV) and in the forebrain area

“hypothalamus” (Ariumi et al., 2000; De Jonghe and Horn, 2009; Miller and Ruggiero,

1994a). The studies in rodents are also providing evidences for the expression of C-fos by

cisplatin in the hind brain areas (Endo and Minami, 2004).

Recently, Bacopa monniera has been reported to possess inhibitory effects on morphine-

induced pharmacological activities such as hyperactivity, tolerance, reverse tolerance,

dopamine receptor sensitivity, and apomorphine induced climbing behavior in rats (Rauf et


159

al., 2011a; Sumathi, 2007) and is protective against aluminum-induced oxidative stress,

which is mechanistically similar to the oxidative stress induced by cisplatin (Jyoti et al.,

2007; Jyoti and Sharma, 2006; Kharbangar et al., 2000; Santos et al., 2007).


Keeping in view the pharmacological profile of BM and our previous findings on the

suppression of cisplatin induced vomiting in pigeon model by standardized extracts of BM,

this study was designed to investigate the effects of the BM methanol fraction (BM-MetFr)

& bacoside rich n-butanol fraction (BM-ButFr) and the combination of Δ9 THC synthetic

analogue (WIN,55-212-2) with BM-ButFr against cisplatin induced Retching plus Vomiting

(R + V) in S. murinus and also to observe its effects on C-fos-IR in specific brain areas in

the S. murinus.


8.3.1. Animals:

Male S. murinus provided by the animal care and laboratory services of the Chinese

University of Hong Kong (CUHK) were used in the study.

Further details regarding animal husbandry are given in Chapter 2, Methods, section 2.1.2.

8.3.2. Materials and drugs:



160

8.3.3. Extraction of Bacopa monniera:

Kahol method (Kahol et al., 2004) was used with some modifications for the extraction of

BM. Two major fractions methanol and the bacoside rich n-butanol fraction were obtained.

For detailed extraction procedure of BM, see Chapter 2: Methods, section 2.5.2.

8.3.4. Drug formulation:



Intraperitoneal and subcutaneous routes were used for drug administration. In all the cases

cisplatin was administered intraperitoneally while the treatments were administered

subcutaneously.

For further details, See Chapter 2: Methods, section 2.9.

8.3.6. Experimental setup for behavioral studies:

Video recording setup for recording the behavior of the animals for the desired period of

time, at the Brain-Gut Laboratory, “School of BioMedical sciences”, Faculty of Medicine,

The Chinese University of Hong Kong (CUHK), Hong Kong, was used.

For more details See Chapter 2: Methods, section 2.2.2.

8.3.7. Measurement of Retching plus Vomiting (R + V) and locomotor activity:

Animal models vary in vomiting response to different emetogenic stimuli. The response in

S. murinus remains for very short period of time, which needs expertise for recognition and


161

counting. Cisplatin induces a reliable vomiting at the dose of 30 mg/kg (Sam et al., 2003).

Furthermore, locomotor activity was measured through automated tracking system by

Ethovision software.

The details about the quantification of V + R and measurement of locomotor activity are

described in Chapter 2: Methods, section 2.3.2 & 2.4.

8.3.8. C-fos immunohistochemistry:

The detail immunohistochemical procedure, quantification of C-fos immunoreactivity (C-

fos-IR), Image acquisition and processing is given in Chapter 2, Methods, section 2.13.

8.3.9. Data analysis:

The V + R episodes, latency, weight loss and locomotor activity data were analyzed by “one

way analysis of variance” (ANOVA), in case of significance followed by Tukey’s multiple

comparison test or Dunnett’s multiple comparison test (GraphPad Prism version 5.0, Inc.

Version, California, USA). The animals which showed complete suppression of V + R were

not included in statistical analysis for latency. Values are expressed as the mean ± s.e.m.

unless otherwise stated. In all the cases, the differences were considered significant when P

< 0.05.


162

8.4. Results:

8.4.1. Effect of palonosetron, Bacopa monniera methanol fraction (BM-MetFr), n-

butanol fraction (BMButFr) or combination of (WIN 55,212-2 + BMButFr) on cisplatin

induced Retching plus Vomiting (R + V):

The effect of BM-MetFr (10, 20 & 40 mg) and BM-ButFr (5, 10 & 15 mg) were tested

against cisplatin challenge and compared with standard anti-emetic palonosetron. BM

treatments dose dependently suppressed cisplatin induced Retching plus Vomiting (R + V)

as compared to cisplatin control (Figure 8.1). Palonosetron (PalS; 0.5 mg/kg) and BM-MetFr

(40 mg/kg) reduced the R + V episodes upto 02 ± 0.7 (79.5 % protection; P < 0.001) and 4.2

± 1.6 (57.1 % protection; P < 0.05), respectively at t 0-24 hr (Table 8.1A), while BM-ButFr

provided protection upto 75.0 % (2.2 ± 0.6 episodes; P < 0.05) as compared to cisplatin

control (Table 8.1B). Interestingly, BM-ButFr at the dose of 5 & 10 mg/kg provided

complete (P < 0.05) protection at t 24-48 hr (Table 8.1B), while BM-MetFr and BM-ButFr

(20 mg/kg) provided upto 63.1% (1.4 ± 1.1 episodes; P > 0.05) and 92.3 % (0.2 ± 0.2

episodes; P > 0.05) protection, respectively. Only palonosetron significantly increased (P <

0.05 - 0.001) the latency time while other BM fractions failed to do so. BM-ButFr proved to

be superior to BM-MetFr as its (5, 10 mg) & (20 mg) dose completely attenuated the

vomiting in 40 % and 20 % of animals respectively, while palonosetron provided complete

protection in 20 % animals. No significant differences were observed for body weight loss

and locomotion among cisplatin control and treated groups (Table 8.1A & B). The

combination of BM-ButFr (5 mg) with WIN 55,212-2 (10 mg) proved to be antagonistic

when calculated (see calculations) using Lampel equation (Limpel et al., 1962).


163

Calculation for synergism:

Here calculations are presented to know about the expected response and then decided for its

synergistic activity as we have done for other combination studies in Chapter 5.

Potential combination (WIN 55,212-2 + BMButFr):

A Percent inhibition by WIN 55,212-2 (10 mg) 50.40 %.

B Percent inhibition by BM-ButFr (05 mg) 52.84 %


E = A + B – AB/100

= 50.40 + 52.84 – 50.40 × 52.84/100

= 76.61 %


Result Non-synergistic


164

Dose response relationship of Bacopa monniera extracts:

Figure 8.1. Dose response relationship of Bacopa monniera methanol fraction (BM-MetFr)

and n-butanol fraction (BM-ButFr) to protect Suncus murinus from cisplatin induced

Retching plus Vomiting (R + V) during 48 hr of observation period. Data represents the

mean ± s.e.m of 5 - 6 determinations.


165

Table 8.1A: Effect of Bacopa monniera methanol fraction (BM-MetFr) on cisplatin induced Retching plus Vomiting (R +

V) in Suncus murinus.

Drug Treatment Dose and route animals

n/ vomited

V + R

Latency (min)

mean ± s.e.m

Locomotion (cm)

mean ± s.e.m

Wt loss (%)

mean ± s.e.m

t 0-48 t 0-24 t 24-48

Saline + Cisplatin 10 ml/kg i.p.

+30 mg/kg i.p.

5/5 13.8 ± 1.8 9.8 ± 1.5 3.8 ± 1.1 59 ± 3.3 52454 ± 13842 7.1 ± 2.4

PalS + Cisplatin 0.5 mg/kg s.c.

+30 mg/kg i.p.

5/4 4.0 ±1.6** 2.0 ±0.7** 2.4 ± 1.2 575 ± 116* 28615 ± 11165 10.4 ± 3.2

BM-MetFr + Cisplatin

10 mg/kg s.c.

+ 30 mg/kg i.p.

5/5 7.0 ± 0.9 5.2 ± 0.9 1.8 ± 0.7 155 ± 96 39426 ± 15609 8.6 ± 3.4

20 mg/kg s.c.

+ 30 mg/kg i.p.

5/5 7.2 ± 2.7 5.6 ± 1.4 1.6 ± 1.3 86 ± 13.1 38366 ± 12831 12.0 ± 3.0

40 mg/kg s.c.

+ 30 mg/kg i.p.

5/5 5.6 ± 1.3* 4.2 ± 1.6* 1.4 ± 1.1 254 ± 177 37742 ± 11773 9.2 ± 2.2

Effect of standard palonosetron (PalS) & Bacopa monniera methanol fraction (BM-MetFr) on cisplatin induced Retching plus

Vomiting (R + V) during 48 hr observation period. The latency to first vomit, % weight loss and locomotive activity is shown for

the t 0 - 48 hr while number of V + R is shown for t 0 - 48, 0 - 24, 24 - 48 hr observation period. Values significantly different

compared to cisplatin control are indicated as *p < 0.05, **p < 0.01 (ANOVA followed by Tukey post hoc analysis).


166

Table 8.1B: Effect of Bacopa monniera n-butanol fraction (BM-ButFr) and combination of WIN 55, 212-2 (10 mg) with

BM-ButFr (5 mg) on cisplatin induced Retching plus Vomiting (R + V) in Suncus murinus.

Drug Treatment Dose and route animals

n/ vomited

V + R

Latency (min)

mean ± s.e.m

Locomotion (cm)

mean ± s.e.m

Wt loss (%)

mean ± s.e.m

t 0-48 t 0-24 t 24-48

Saline + Cisplatin 10 ml/kg i.p.

30 mg/kg i.p. 6/6 12.3 ± 2.3 9.6 ± 0.5 2.6 ± 0.9 54 ± 7.0 50539 ± 31869 9.4 ± 1.6

PalS + Cisplatin 0.5 mg/kg s.c.

30 mg/kg i.p. 6/4 2.6 ± 1.0* 1.1 ± 0.6 1.5 ± 0.7 630 ± 209*** 35787 ± 12752 12.2 ± 0.9

WIN 55,212-2 + Cisplatin 10 mg/kg s.c.

30 mg/kg i.p. 6/6 6.1 ± 1.3 2.9 ± 1.1 3.1 ± 0.7 78 ± 12 44215 ± 29190 11.1 ± 4.2

BM-ButFr +Cisplatin

5 mg/kg s.c.

30 mg/kg i.p. 5/3 5.8 ± 3.7 5.8 ± 3.7 0.0 ± 0.0* 54.6 ± 9.6 44426 ± 14247 9.4 ± 1.3

10 mg/kg s.c.

30 mg/kg i.p. 5/3 3.6 ± 1.8 3.8 ± 1.9 0.0 ± 0.0* 58.6 ± 3.6 62140 ± 27311 9.8 ± 1.7

20 mg/kg s.c.

30 mg/kg i.p. 5/4 2.6 ± 0.8* 2.2 ± 0.6* 0.2 ± 0.2 57 ± 4.7 60479 ± 32673 8.9 ± 1.8

(WIN 55,212-2 +

BMButFr) + Cisplatin

10 + 5 mg/kg s.c.

30 mg/kg i.p. 5/5 4.0 ± 1.9 3.6 ± 0.9 0.4 ± 0.1 55.8 ± 8.9 59439 ± 25323 9.1 ± 2.1

Effect of standard palonosetron (PalS), n-butanol fraction (BM-ButFr) and combination of WIN 55, 212-2 (10 mg) with BM-

ButFr (5 mg) on cisplatin induced Retching plus Vomiting (R + V) during 48 hr observation period. The latency to first vomit,


167

% weight loss and locomotive activity is shown for the t 0 - 48 hr while number of V + R is

shown for t 0 - 48, 0 - 24, 24 - 48 hr observation period. Values significantly different

compared to cisplatin control are indicated as *p < 0.05, ***p < 0.001 (ANOVA followed

by Tukey post hoc analysis).

8.4.2. Induction of C-fos by cisplatin:

Cisplatin induced long term C-fos immunoreactivity (C-fos-IR; ~ 48 hr) in the hindbrain

areas including area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor

nucleus of vagus nerve (DMV) and in the forebrain area hypothalamus (HP) including

dorsomedial (DMH) and ventromedial (VMH) nucleus of hypothalamus (Figure 8.2). C-fos-

IR was increased significantly in AP, DMV and HP (P < 0.05-0.001) except NTS where the

activity was found to be statistically non-significant. Moreover, no activity was observed in

cingulum and cingulate cortex.


168

Representative photomicrographs showing C-fos-IR:

Treatment AP NTS + DMV HP

Saline

+

Saline

Saline

+

Cisplatin

PalS(0.5mg)

+

Cisplatin

AP

NTS

DMV

HP

DMH

VMH


169

BM-MetFr

(10 mg)

+

Cisplatin

BM-MetFr

(20 mg)

+

Cisplatin

BM-MetFr

(40 mg)

+

Cisplatin

BM-ButFr

(5 mg)

+

Cisplatin


170

BM-ButFr

(10 mg)

+

Cisplatin

BM-ButFr

(20 mg)

+

Cisplatin

Figure 8.2. Representative photomicrographs showing C-fos immunoreactivity (C-fos-IR;

blue black reaction product in the nuclei of the cell, shown in circles) in the hind brain

including area postrema (AP), nucleus tractus solitarius (NTS), and dorsal motor nucleus of

vagus nerve (DMV) and in the forebrain area of hypothalamus (HP) including dorsomedial

(DMH) and ventromedial (VMH) nuclei. Magnifications 10X.

8.4.3. Effect of palonosetron, Bacopa monniera methanol fraction (BM-MetFr) & n-

butanol fraction (BMButFr) on cisplatin induced C-fos expression:

All the BM fractions proved to be effective in reducing C-fos count and the results were

comparable with the standard palonosetron. Palonosetron and BM-MetFr at the dose of 10 &

20 mg significantly reduced (P < 0.01) the C-fos count in AP (figure 8.3A), while only 10

mg dose of BM-MetFr was effective in DMV (P < 0.05; figure 8.3C). All the doses of BM-


171

MetFr significantly reduced the C-fos count at the level of hypothalamus (P < 0.01 - 0.001)

and the same effect was observed with palonosetron (figure 8.3D), while the reduction in

NTS was found to be non-significant (figure 8.3B). BM-ButFr (5, 10 & 20 mg) significantly

reduced (P < 0.01 - 0.001) C-fos count at the level of AP and hypothalamus, while

significant reduction (P < 0.05) was observed at the level of NTS (5, 10 mg of BM-ButFr)

and DMV (BM-ButFr 5 mg) (Figure 8.3A, B , C & D).

A. Effect of palonosetron or Bacopa monniera methanolic fraction (BM-MetFr) or

n-butanolic fraction (BM-ButFr) on cisplatin induced C-fos count in the area postrema

(AP):


172

B. Effect of palonosetron or Bacopa monniera methanolic fraction (BM-MetFr) or

n-butanolic fraction (BM-ButFr) on cisplatin induced C-fos count in the brain area of

nucleus tractus solitaious (NTS):

C. Effect of palonosetron or Bacopa monniera methanolic fraction (BM-MetFr) or

n-butanolic fraction (BM-ButFr) on cisplatin induced C-fos count in dorsomedial

nucleus of vagus nerve (DMV):


173

D. Effect of palonosetron or Bacopa monniera methanolic fraction (BM-MetFr) or

n-butanolic fraction (BM-ButFr) on cisplatin induced C-fos count in the hypothalamus

of the forebrain area (HP):

Figure 8.3. Effect of palonosetron (PalS; 0.5 mg/kg), Bacopa monniera methanol fraction

(BM-MetFr; 10, 20 & 40 mg/kg) or n-butanol fraction (BM-ButFr; 5, 10 & 20 mg/kg) on

cisplatin induced C-fos-IR, quantified at 48 hr in hindbrain areas including (A) area

postrema (AP), (B) nucleus tractus solitarius (NTS), (C) dorsal motor nucleus of vagus

nerve (DMV) and (D) hypothalamus (HP) of the forebrain area. Values significantly

different compared to cisplatin control are indicated as *p < 0.05, **p < 0.01 ***p < 0.001,

while Values significantly different as compared to basal level are indicated as #p < 0.05,

###p < 0.001 (ANOVA followed by Tukey post hoc analysis).

8.5. Discussion:

The present study is the first of its kind to investigate the anti-emetic potential of BM extracts

against cisplatin induced vomiting in the S. murinus model. We selected S. murinus as vomit

model due to its reliability and widespread use in the studies employing cisplatin as vomiting


174

inducing agent (Matsuki et al., 1988). We used cisplatin at the dose of 30 mg/kg to induce

Retching + Vomiting (R + V) in the S. murinus as this dose has been tested and considered to

be suitable to induce R + V upto prolong time period (~ 72 hrs), where no lethality was

observed upto the observation period (Sam et al., 2003). Moreover, in the S. murinus model

the incidence rate of cisplatin induced R + V is found to be analogous with that of human

(Hesketh, 1996; Kris and Tyson, 1985).

This study, regarding the screening of Bacopa monniera (BM) extracts alone and in

combination with WIN 55, 212-2 (10 mg; Δ9-tetrahydrocannabinal synthetic analogue) in S.

murinus against cisplatin induced vomiting, is the extension of our previous work in pigeon

model, where Bacopa monniera methanolic fraction (BM-MetFr) and n-butanol fraction

(BM-ButFr) dose dependently suppressed the behavioral signs of cisplatin induced R + V

(chapter 5).

BM has been in use since ancient times for the management of many disorders, where

bacosides have been proved to be the major components responsible for its pharmacological

activities and our results about HPLC - UV analysis of n-butanol fraction indicated the high

concentration of bacoside “A” major components including bacoside A3, bacoside II and

bacosaponin C in the n-butanol fraction (chapter 4). The potent anti-emetic activity of BM-

ButFr by suppressing the behavioral signs of cisplatin induced vomiting more significantly as

compared to BM-MetFr in S. murinus may be attributed to its high concentration of

bacosides as BM active component bacosides have been reported to be having potent anti-

oxidant activity (Ghosh et al., 2007), neuroprotective effects (Jyoti et al., 2007) and

anticancer effects (D'Souza et al., 2002). BM has been found effective to protect against

aluminium induced oxidative stress which is having close similarity with the oxidative stress


175

induced by cisplatin (Jyoti and Sharma, 2006). BM also restores the normal antioxidant

defense mechanisms of the body (Shinomol, 2010). In our study, BM treatments attenuated

cisplatin induced R + V dose dependently (Figure 8.1). BM-ButFr proved to be superior to

BM-MetFr as the former in doses of 5 & 10 mg was found to be more effective in

suppressing the vomiting for prolong time periods (P < 0.05, Table 8.1) and provided upto 75

% protection against the first intense cluster of R + V. BM-MetFr provided ~ 57 % protection

at t 0-24, while no significant suppression was observed at t 24-48 (P > 0.05, Table 8.1A).

The BM antioxidant property may contribute to its anti-emetic activity as cisplatin causes the

generation of free radicals and ultimately oxidative stress, which is the primary cause for the

release of 5HT from enterochromaffin (EC) cells in the gut mucosa and finally the triggering

of vomiting response mediated through vagus nerve (Mehendale. Sangeeta R Aung et al.,

2004; Torii et al., 2012). Furthermore, anti-oxidants/free radical scavengers have been proved

to be effective in the suppression of CIV evidenced by others (Gupta and Sharma, 1996; Torii

et al., 1993) and our findings in pigeon model as well, where 2-mercaptopropionyl glycine

(MPG; potent anti-oxidant) abolished the vomiting response induced by cisplatin (chapter 5).

Moreover, BM has also been proved to be having anti-dopaminergic effects as it suppressed

the morphine induced pharmacological activities like hyperactivity, dopamine receptor

supersentivity and apomorphine induced climbing behavior in rodents (Sumathi et al., 2007),

the same has also been reported by our laboratory (Rauf et al., 2011b). In our previous study

in pigeon model, BM treatments showed anti-dopaminergic/anti-serotonergic effects in the

brain areas involved in the act of vomiting and intestine (chapter 7), which are the important

culprits in the mediation of cisplatin induced vomiting (Darmani et al., 1999; Minami and

Endo, 2003; Osinski et al., 2005).


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The 5HT3 receptor antagonist palonosetron was selected as a standard along with the BM

plant extracts for comparison as palonosetron is proved to be an effective anti-emetic agent

against CIV in dogs (Macciocchi et al., 2005), ferrets (Percie du Sert et al., 2011), S. murinus

(De Jonghe and Horn, 2009) as well as humans (Grunberg and Koeller, 2003) and is having

long half life (~ 40 h).Furthermore, 5HT3 receptor antagonists like palonosetron have no

intrinsic emetic activity in Suncus murinus as was observed in pigeon model (unpublish

data).

Studies have indicated the expression of C-fos immunoreactivity (C-fos-IR) as a sensitive

marker of neuronal excitation which is currently in use in mapping neuronal pathways. Brain

C-fos-IR is found to be increased after cisplatin treatment for at least 48 h in the caudal hind

brain areas in rat (a non vomiting specie). Furthermore C-fos-IR has also been reported in

vomiting species like cat (Miller and Ruggiero, 1994b), ferret (Billig et al., 2001) and least

schrew (Cryptotis parva) (Ray et al., 2009). Antineoplastic agent cisplatin causes both, acute

and long term increases in C-fos-IR at hind brain areas including area postrema (AP), nucleus

tractus solitarius (NTS), dorsal motor nucleus of vagus nerve (DMV) and in the forebrain

areas including dorsomedial (DMH) and ventromedial (VMH) nucleus of hypothalamus (HP)

(De Jonghe and Horn, 2009).

In the current study, cisplatin (30 mg/kg i.p.) induced C-fos-IR in the hind brain area

including AP, NTS and DMV and in the forebrain area hypothalamus of the S. murinus

(Figure 8.2 & 8.3) as well as vomiting response thus these brain areas are implicated in the

mediation of vomiting act. Furthermore, the C-fos-IR was significantly suppressed by 5HT3

receptor antagonist palonosetron (Figure 8.2 & 8.3) showing the involvement of 5HT3

receptors in the mediation of vomiting by cisplatin which is in agreement with the previously


177

reported studies (De Jonghe and Horn, 2009). The long term suppression (~ 48 hr) of

cisplatin induced C-fos-IR by BM treatments is in parallel with its anti-emetic effect against

cisplatin induced R + V and is comparable with the standard palonosetron, indicates the

involvement of at least in part of the C-fos expression in the activation of emetic pathways.

Currently, BM is available in a variety of formulations and has been reported to be safe in all

respect. Apart from its anti-emetic activity against cisplatin induced R + V in S. murinus, BM

is having other added benefits as it is also having antinociceptive (Vohora et al., 1997),

antidepressant (Sheikh et al., 2007), antidopaminergic (Rauf et al., 2011b) and anticancer

effects (Charmandari et al., 2005).

In summary, the present study is indicative of the anti-emetic effect of BM extracts

containing bacosides against cisplatin induced vomiting for prolong time periods in S.

murinus. Attenuation of C-fos-IR observed in this study is in parallel with its suppression of

behavioral signs of vomiting in S. murinus. Furthermore, BM is having good safety and

tolerability profile and may be a potential anti-emetic alone or in combination for the

prophylaxis and treatment of CIV in human, especially the late cluster of vomiting which is

still a challenge in clinical setups (Yamakuni et al., 2006), warrants its further investigations

in the more established vomit models of ferret and dog.

Chapter 9 General discussion

178

Chapter 9

General discussion


179

9.1. General discussion:

Nausea and vomiting caused by cancer chemotherapeutic agents especially the Highly

Emetogenic Chemotherapy (HEC) like cisplatin are having the distressing undesirable

effects observed in patients undergoing chemotherapy. The brutality of these unpleasant side

effects often lead to non-compliance and even refusal of curative treatment (Hesketh and

Van Belle, 2003). The cytotoxic agent cisplatin is indicated for the management of ovarian

(Muggia, 2009), testicular, and head & neck carcinomas (Lajer and Daugaard, 1999). In

clinical setups, cisplatin is administered along with anti-emetics to compensate the stressful

adverse effect of nausea and vomiting prophylactically before starting the chemotherapy and

onward intermittently during the course of chemotherapy.

Anti-emetics are of very importance to be considered for the control of Chemotherapy

Induced Vomiting (CIV) and numerous anti-emetic agents are in practice for the

management of this dilemma. The introduction of 5-hydroxy tryptamine receptor type 3

antagonists (5HT3; ondansetron, granisetron, palonosetron etc) revolutionized the control of

CIV especially the acute phase response (~ 24 hr) while failed to show any shielding effects

against delayed phase response (24 hr +) (Hesketh and Van Belle, 2003; Topal et al., 2005).

Currently, the invention of NK1 receptor blockers (aprepitant, natupitant, vofopitant etc)

have shown broad spectrum anti-emetic activity and better control against the cisplatin

induced delayed phase of vomiting in animal models and in clinics (Gardner et al., 2012;

Grelot and Esteve, 2009). Furthermore, corticosteroids (e.g. dexamethasone) in combination

with existing anti-emetics have also been proved to enhance the protection against CIV

(Gralla et al., 1999; Smith et al., 1991). According to the American Society of Clinical

Oncology (ASCO) guidelines, 5HT3 receptor antagonists in combination with NK1 receptor


180

blockers and dexamethasone have been recommended to be used to get better control of CIV

in clinics (Gralla et al., 1999; Pfister et al., 2004). Despite, to the compliance of ASCO

guidelines the anti-emetic combination regimens are still failing to provide complete

remission of CIV and a considerable proportion of patients still vomit after cancer

chemotherapy (Ballatori et al., 2007; Glaus et al., 2004).

The struggle to look for the new chemical entities and combinations to be cost effective and

possessing broad spectrum anti-emetic activity, convinced us to look for anti-emetic

potential of some selected plants extracts indigenous to Pakistan and their combinations

against CIV in vomit models of pigeon and Suncus murinus.

Cisplatin which belongs to the highly emetogenic class of cancer chemotherapeutic agents is

in use for the screening of anti-emetic potential of current anti-emetic agents, their

combinations and particularly for novel chemical entities in animal models. Cisplatin (4 – 10

mg/kg) has been used by several investigators to induce vomiting in pigeons (Feigenbaum et

al., 1989; Wolff and Leander, 1995). However, Tanihata et al (Tanihata et al., 2000) used

the lower dose of 4 mg/kg and longer observation periods (~ 72 hr). In fact, our colony of

pigeons had shown a reliable vomiting response at 7 mg/kg upto 24 hr of observation period

(Ullah et al., 2012), while the vomiting response was observed with doses as low as 5 mg/kg

where only 60 % of the animals showed the vomiting response (chapter 3). The difference in

dose of cisplatin used in our study to induce reliable vomiting with respect to previous

studies may be attributed to species differences, environmental factors and diet. In the S.

murinus we followed the studies of Sam et al (Sam et al., 2003) and the dose of 30 mg/kg

was used.


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Cannabis sativa (CS) and Zingiber officinale (ZO) are well known for their anti-emetic

property while Bacopa monniera (BM) in this study provided new avenue for its anti-emetic

activity against cisplatin induced vomiting in the vomit models of pigeon and S. murinus. CS

hexane fraction (CS-HexFr; 10 mg) showed protection against CIV in pigeon model while

the n-butanol and methanol fractions were found to be ineffective. The anti-emetic activity

of CS in this study is in accordance with the previous findings (Russo, 2001), where the

active component of cannabis, Δ9-tetrahydrocannabinol (Δ

9-THC) has already been proved

for attenuation of cisplatin induced vomiting in animal models (Darmani and Crim, 2005;

Wang et al., 2009) and progress has been made up to the extent that CS preparations

(Marinol®, Sativex) are now available in the market with registered indications for the

control of CIV and anorexia. Furthermore, the active component of cannabis (Δ9-THC) has

also been proved to be having anti-inflammatory (Costa et al., 2004; Klein, 2005), analgesic

(Blake et al., 2006; Karst et al., 2003) and anti-oxidant activity (Hampson et al., 1998;

Marsicano et al., 2002). Uptill now two cannabinoid receptors; CB1 & CB2 have been

identified , where CB1 receptor agonism has been shown to be responsible for the mediation

of anti-emetic effect of cannabinoids (Phyto & synthetic), distributed in high density in the

central nervous system (Tramer et al., 2001). The presynaptically located CB1 receptors

agonism leads to the inhibition of neurotransmitters release and subsequent suppression of

vomiting (Darmani, 2001).

As evident from the literature, neurochemical mediators of various types are responsible in

vomiting circuits for CIV, especially serotonin (5HT), dopamine, substance P and

prostaglandins are postulated to contribute in its genesis. The emetogenic anti-cancer agent

cisplatin induces biphasic vomiting both in human (Hesketh and Van Belle, 2003) and in


182

other vomiting species (Darmani et al., 2009; De Jonghe and Horn, 2009; Navari, 2013;

Qiu-hai et al., 2010). Neural analysis of cisplatin control group in pigeon model indicated

the upsurge of serotonin in the brain areas of BS and intestine at acute time point (03 hr)

suggesting the neurotransmitter serotonin (5HT) as the triggering mediator for acute phase

response in pigeons (chapter 7). The increase in 5HT concentration in our study is in line

with the previous findings in animal models where 5HT3 receptor antagonists (ondansetron,

granisetron, palonosetron etc) are found to be effective against acute phase of CIV

(Grunberg and Koeller, 2003). Furthermore, at delayed time point (18 hr) in pigeon the

increase in the concentration of dopamine at the level of AP and serotonin in the brain area

of BS and in the intestine is indicating the differential involvement of neurotransmitters at

this time point; as CIV is thought to be a multifactorial phenomenon and both the phases of

vomiting are mechanistically different (Darmani et al., 2009), that’s why the prophylactic

and intermittent administration of single antiemetic agent fails to provide complete control

against cisplatin induced vomiting in clinics.

The correlation of neurotransmitter data in our study for suppressive effect of Cannabis

sativa hexane fraction (CS-HexFr) on behavioral signs of cisplatin induced vomiting in

pigeon model is supportive for its anti-emetic effect. Although, the anti-emetic potential of

CS is known (Darmani and Crim, 2005; Wang et al., 2009), but the present study is

providing neural evidences for the mediation of its anti-emetic action in specific brain areas

involved in the act of vomiting and intestine in pigeon model. In this study, CS-HexFr (10

mg) was found to decrease the concentration of 5HT and 5HIAA in all the brain areas (AP +

BS) and intestine at acute time (03 hr; chapter 7), probably through agonism of cannabinoid

CB1 receptors, which are co-localized with 5HT3 receptors (Darmani and Johnson, 2004)


183

and inhibit the release of 5HT from enterochromaffin (EC) cells in the gut mucosa.

Furthermore, at delayed time point (18 hr) CS-HexFr (10 mg) decreased the concentration of

dopamine in AP, while the decrease in the concentration of 5HT was observed at the level of

BS and in the intestine (chapter 7).

Cannabinoids are having undesirable peripheral effect of reduction in gut motility and

secretions both in physiological and patho-physiological conditions as cannabinoids have

been shown to inhibit the release of ongoing contractile transmitter release in the gut (Abalo

et al., 2011; Pertwee, 2001). The suppression of gastrointestinal (GIT) motility is speculated

to be weakening the anti-emetic profile of CS. In the present study, treatment of CS-HexFr

(10 mg) resulted in the suppression of gastrointestinal (GIT) motility. Additionally, cisplatin

also leads to the gastric stasis and distention dose dependently, by release of serotonin and

vagal afferents stimulation, inhibition of calcium calmodulin complex and nitric oxide

synthase activation, which collectively leads to stomach distention/inhibition of gastric

emptying and subsequent vomiting (Jarve and Aggarwal, 1997; Sharma and Gupta, 1998).

The antagonism of the suppression caused by CS-HexFr by prokinetic agents in our study

resulted in the enhancement in anti-emetic profile of CS-HexFr (10 mg) against cisplatin

induced R + V in pigeons at delayed time point (18 hr) as the delayed gastric emptying and

decreased GIT motility is one of the suggested mechanism involved in part in the etiology of

delayed sickness (Kris et al., 1994).

Further in this study, BM methanol fraction (BM-MetFr) and n-butanol fraction (BM-ButFr)

showed promising suppression of vomiting induced by cisplatin in pigeon model and the

same was also observed in the S. murinus, but the n-butanol fraction was found to be more

potent than methanolic fraction in both the vomit models. The more pronounced effects of


184

BM n-butanol fraction on the suppression of behavioral signs of CIV in pigeon (chapter 5)

and S. murinus (chapter 8) as compared to methanol fraction might be due to the presence of

high concentration of bacosides; as n-butanol fraction in this study has been analyzed to be

rich in bacosides major components including bacoside A3, bacoside II & bacosaponin C

(chapter 4). BM is having plethora of therapeutic benefits and is found to be effective in the

management of pain (Singh et al., 2013; Subhan et al., 2010), epilepsy (Mathew et al.,

2010), depression (Sairam et al., 2002) cognitive disorders (Jyoti and Sharma, 2006; Raghav

et al., 2006) and inflammation (Channa et al., 2006). BM is also well known for anti-oxidant

potential as it modules endogenous cytoplasmic and mitochondrial oxidative markers

(Shinomol, 2010) and prevents from aluminium induced oxidative stress, which is

mechanistically similar to oxidative stress induced by cisplatin (Jyoti et al., 2007). The

oxidative stress induced by cisplatin is one of the mechanisms which trigger the release of

serotonin form enterochromaffin cells in the intestinal mucosa (Gupta and Sharma, 1996).

BM is also proved to be effective in reducing morphine induced hyperactivity and dopamine

receptor supersensitivity reported by others (Sumathi et al., 2007) and our laboratory as well

(Rauf et al., 2011). The anti-oxidant (Bhattacharya et al., 2000)(chapter 5) and anti-

dopaminergic (Rauf et al., 2011) (chapter 7) properties of BM are hereby justified to be

responsible for the suppression of cisplatin induced vomiting in vomit models of pigeon and

S. murinus. The neurotransmitter data in pigeon model regarding BM-MetFr (10, 20 & 40

mg) and BM-ButFr (5, 10 & 20 mg) treatments showed anti-serotonergic effect by

decreasing the concentration of serotonin in the brain area of BS and in the intestine,

probably due to its anti-oxidant potential (Ghosh et al., 2007; Shinomol, 2010) protecting

EC cells from the oxidative stress induced by cisplatin, while no effect in the brain area of


185

AP was observed, where the brain area of AP is well known to be the site of vomiting

induction by dopaminergic agonists like apomorphine (Ariumi et al., 2000; Borison et al.,

1984; Yoshikawa et al., 1996) and blood born substances. The absence of any significant

increase in the serotonin concentration in the brain area of AP by cisplatin at acute time (03

hr) might be due to the comparatively lower density of 5HT3 receptors with respect to

nucleus tractus solitaious (NTS). Furthermore, the brain area of AP is proved to be involved

in the etiology of delayed response; as ablation of AP resulted in the suppression of delayed

vomiting (Percie du Sert et al., 2009). In this study, BM treatments resulted in the decrease

of dopamine upsurge in the brain area of AP caused by cisplatin at delayed time point (18

hr; chapter 7), authenticating the involvement of dopaminergic component in the brain area

of AP in the mediation of delayed response in pigeons. Furthermore, the anti-dopaminergic

effects were also seen in the brain area of BS by few doses of BM treatments but with

variable statistical significance. The anti-dopaminergic effect shown by BM methanol

fraction (BM-MetFr) and bacoside rich n-butanol fraction (BM-ButFr) is in accordance with

the study of Sumathi et al (Sumathi et al., 2007) and also coincide with the results from our

laboratory (Rauf et al., 2011).

The expression of C-fos immunoreactivity (C-fos-IR) which is a sensitive marker of

neuronal excitation, implicate the involvement of the brain areas including area postrema

(AP), nucleus tractus solitaious (NTS) and dorsal motor nucleus of vagus nerve (DMV) and

hypothalamus of the forebrain area in the long term sickness induced by chemotherapy agent

cisplatin. In the S. murinus model (a specie with the vomiting response), cisplatin (30

mg/kg) induced R + V behavior upto 48 hr of observation period and our results indicated

the expression of early gene C-fos immunoreactivity (C-fos-IR) in the brain areas involved


186

in the act of vomiting including AP, NTS and DMV, the same is also reported by Ito et al

(Ito et al., 2003) in the S. murinus and in rats (a non-vomiting specie) by Horn et al (Horn et

al., 2007). Furthermore, cisplatin also resulted in the expression of C-fos-IR at 48 hr after

injection in the forebrain area of hypothalamus (HP) including dorsomedial (DMH) and

ventromedial nucleus of hypothalamus (VMH). Treatment of BM-MetFr (10, 20 & 40 mg),

BM-ButFr (5, 10 & 20 mg) and combination of WIN 55, 212-2 (10 mg) with BM-ButFr (5

mg) significantly inhibited the cisplatin induced C-fos-IR in the brain areas of AP, NTS and

DMV and in the forebrain areas including DMH & VMH (chapter 8), providing additional

evidence for the effectiveness of BM treatments and combination for prolong time periods in

S. murinus model. This study is the first to show long term (48 hr) inhibition of cisplatin

induced R + V behavior and C-fos-IR in the brain areas by BM extracts containing bacosides

in the S. murinus model. C-fos-IR is known to play a role in cisplatin induced vomiting and

the inhibition of this response by BM extracts, is suggesting the involvement of C-fos

expression at least in part in the activation of vomiting pathways.

ZO acetone fraction (ZO-ActFr; rich in gingerols) (Sharma et al., 1997) have also been

found to be effective to attenuate cisplatin induced R + V in pigeons and its use in cooking

as spice and flavoring agent is indicative to be having safe, and tolerable profile. The ginger

has been advocated for the management of nausea and vomiting, where the constituents

gingerols, shagoals and galanolactone illustrate the antagonistic action at 5HT3 receptors

(Abdel-Aziz et al., 2006) and prokinetic effects (Ghayur and Gilani, 2005; Sharma and

Gupta, 1998), which collectively mediate the anti-emetic activity of ginger and is found to

be equally effective as metoclopramide (Ernst and Pittler, 2000). The pungent constituents

collectively known as gingerols have also been reported to be having inhibitory effects on


187

the upsurge of substance P both centrally and peripherally and NK1 receptors expression in

the vomit model of mink (Qiu-hai et al., 2010). In our study, ZO-ActFr at the dose of 50 mg

provided maximum protection against cisplatin induced R + V in pigeons (chapter 5), where

the protection observed was ~ 58.13 %. Furthermore, the impact of ZO-ActFr on the major

neurotransmitter mediators including dopamine and serotonin is encouraging and is in

support for its anti-emetic effect where ZO-ActFr (50 mg) decreased the concentration of

5HT and 5HIAA in the brain areas (AP & BS) and intestine at acute time point (3 hr).

Moreover, at delayed time point (18 hr) treatment of ZO-ActFr (50 mg) resulted in the

decreased concentration of dopamine in the brain area of AP, while 5HT decrease was

observed at the level of BS and in the intestine (chapter 7).

Keeping in view the mechanistic complexity of vomiting and the differential participation of

neurotransmitters through the time course of cisplatin induced vomiting in human (Hesketh

and Van Belle, 2003), our studies regarding the combinations of Plants extracts showed

synergism and the enhancement of the anti-emetic spectrum. The combinations of CS, BM

and ZO extracts provided variable protection against cisplatin induced vomiting in vomit

models of pigeon and S. murinus and the combination of CS-HexFr (10 mg) with BM-ButFr

(5 mg) was found to be synergistic in pigeon model. The previous discussion about the

neurotransmitter data is justifying the synergic effect of CS-HexFr (10 mg) with BM-ButFr

(5 mg), where CS-HexFr & BM-ButFr are having anti-serotonergic (03 hr & 18 hr) and anti-

dopaminergic (18 hr) effects predominantly, respectively in pigeons subsequently resulting

in good suppression of R + V behavior induced by cisplatin (chapter 5).

In summary, there is a host of currently available, new investigational entities and

combinations having cost effectiveness and broad spectrum anti-emetic profile to be


188

evaluated for prolong inhibition of vomiting response and suppression of the neural system

involved in CIV. We found that Cannabis sativa (CS), Bacopa monniera (BM) and Zingiber

officinale (ZO; ginger) might be the promising anti-emetic herbal remedy, where CS

preparations (e.g. Marinol®) are available in the market with registered indications for CIV

and anorexia. Moreover, the common use of ginger in cooking as spice and flavoring agent

advocates being free of serious side effects. The anti-emetic activity of BM extracts

containing bacosides against cisplatin induced vomiting in the vomit models of pigeon and

S. murinus in this study is reported for the first time and provided a new avenue to be

explored further in more established vomit models; as BM preparations (e.g. Bacomind®) are

available for use as memory enhancer in old age patients. The effect of CS, BM and ZO

treatments on basal neurotransmitter level in pigeon model further reflects the safe & sound

and acceptable sketch of these extracts, where no significant alteration was seen in any of

the neurotransmitters and their metabolites in the specific brain area and intestine except few

alterations (chapter 7). The promising anti-emetic activity of CS, BM and ZO extracts alone

and the synergistic combination of CS-HexFr (10 mg) with BM-ButFr (5 mg) in the vomit

models of pigeon by suppressing the behavioral signs of R + V, reducing the upsurge of

serotonin and dopamine induced by cisplatin necessitate to be further explored in the vomit

models of ferret and dog and subsequently in human; as the extracts are having safe and

tolerable profile.

Chapter 9 Future work

189

9.2. Future work:

The current study comprising of a series of experiments in the vomit models of pigeon

(avian) and Suncus murinus (S. murinus; mammal) for screening the anti-emetic potential of

some selected plant extracts and their combinations to look for a promising herbal remedy

for the management of Chemotherapy Induced Vomiting (CIV) in clinics, which is still a

challenge especially the delayed phase of vomiting. The behavioral, gastrointestinal,

neurochemical and C-fos immunohistochemistry (C-fos-IR) work in this study not only

provides sufficient evidences towards the use of this herbal remedy but has also opened new

areas of interest to be investigated. Some of the avenues which needs to be further explored

include

The findings related to the anti-emetic potential of BM extracts in vomit model of

pigeon and S. murinus in this study are reported for the first time and needs to be

further explored in more established vomit models of ferret and dog and afterward in

human. In addition, there are avenues to explore mechanisms for the mediation of its

prolong protection against cisplatin induced Retching plus Vomiting (R + V).

In present study, the bacoside rich n-butanol fraction of BM (BM-ButFr) potently

inhibited behavioral signs of cisplatin induced R + V as compared to methanolic

fraction (BM-MetFr) in the pigeon and S. murinus models. These findings strengthen

the involvement of bacosides in the mediation of anti-emetic effect by BM extracts

against cisplatin induced R + V in pigeon & S. murinus and establish the need to

further study pure bacosides in cisplatin induced vomiting in vomit models.


190

The combination of Cannabis sativa hexane fraction (CS-HexFr; 10 mg) with

Bacopa monniera n-butanol fraction (BM-ButFr; 5 mg) provided synergistic anti-

emetic effect in the pigeon against cisplatin induced R + V making it potential

combination to be evaluated further for its suppression on the behavioral signs of

vomiting in dogs and ferrets.

BM treatments and combination of Cannabis sativa hexane fraction (CS-HexFr; 10

mg) with Bacopa monniera n-butanol fraction (BM-ButFr; 5 mg) or WIN 55, 212-2

(Δ9-tetrahydocannabinal synthetic analogue; 10 mg) needs to be screened in rats (a

non-vomiting specie) for their probable effects on nausea behavior using conditioned

taste aversion and pica paradigms.

As gastrointestinal motility and gastric emptying are the important aspects to be

considered in chemotherapy induced vomiting reported by others and in this study as

well. Keeping in view the involvement of gastrointestinal motility and gastric

emptying there are avenues to study the suppression caused by cisplatin and anti-

emetic treatments. The rectification of which will result in enhanced anti-emetic

profile.

Microdialysis is the more sensitive, direct and accurate technique to be implied for

the measurement of neurotransmitters and their metabolites in specific brain areas in

animal models. The technique is of high significance to know more clearly about the

impact of CS, BM, ZO extracts and combination of Cannabis sativa hexane fraction

(CS-HexFr; 10 mg) with Bacopa monniera n-butanol fraction (BM-ButFr; 5 mg) or

WIN 55, 212-2 (Δ9-tetrahydocannabinal synthetic analogue; 10 mg) on the

neurotransmitters involved in the act of vomiting in the vomit models.


191

Along with the neurotransmitters, neuropeptides are also of great importance

especially neuropeptide “substance P” which is known to be the important culprit in

the mediation of chemotherapy induced delayed vomiting response. The study has

been conducted for Zingiber officinale where gingerol (ginger active constituent) has

shown inhibitory effect on “substance P” and the expression of NK1 receptors in

mink vomit model. In this regard, the combination of CS-HexFr (10 mg) with BM-

ButFr (5 mg) or WIN 55, 212-2 (10 mg), and especially the BM extracts needs to be

screened for their probable effect on the levels of neuropeptide “substance P” and

NK1 receptor expression in the specific brain areas of S. murinus.

BM has been proved to be having anti-dopaminergic effect reported by others and

our laboratory as well. Furthermore, the current study is also indicative for anti-

dopaminergic effect by BM treatments as it decreased the cisplatin induced dopamine

upsurge in the area postrema (AP) located in the brain stem, where the AP is known

to be involved in the delayed sickness, supporting the effectiveness of BM treatments

for prolong time period against cisplatin induced vomiting. In continuation, the BM

treatments need further investigations on its effects on dopamine receptor expression

in specific brain areas involved in the act of vomiting in S. murinus.

Keeping in view the anti-dopaminergic effect of BM extracts evidenced by previous

studies, the reports from this laboratory and furthermore, by the current study in

pigeon model is making the bacoside rich fraction of BM (BM-ButFr) the potential

candidate to be screened against emetogenics like morphine and apomorphine. The

emetogenic agents’ morphine and apomorphine induces vomiting through activation

of dopaminergic receptors centrally at the brain level of area postrema.


192

C-fos immunoreactivity (C-fos-IR) is one of the markers for neuronal excitation and

is used for mapping neural pathways involved in the etiology of cisplatin induced

nausea and vomiting. Our results in S. murinus are indicative of C-fos-IR by cisplatin

in the specific areas of brain involved in the act of vomiting and the subsequent

attenuation by treatments is showing the way further to look for the effects of the

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Appendices

216

Appendices

Appendices

217

PUBLICATION RELATED TO THE WORK IN THIS THESIS:

PUBLISHED:

Ullah I, Subhan F, Rauf K, Badshah A, Ali G. Role of gastrointestinal motility/gastric

emptying in cisplatin-induced vomiting in pigeon. African journal of pharmacy and

pharmacology (2012) 6:2592-2599

MANUSCRIPTS IN PREPARATION:

Ullah I; Subhan F; Rudd J A; Rauf K; Alam J. Methanol and n-butanol fraction of Bacopa

monniera attenuate cisplatin induced vomiting in the pigeon.

Ullah I; Subhan F; Rudd J A. Attenuation of cisplatin-induced retching plus vomiting and

C-fos immunoreactivity by bacosides containing bacopa monniera fractions in

suncus murinus

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