Effect of Bacopa Monnieri on Morphine Dependence and Tolerance to Analgesia in Animal Models

EFFECT OF BACOPA MONNIERI ON MORPHINE DEPENDENCE AND TOLERANCE TO ANALGESIA IN ANIMAL MODELS

PhD Thesis

Khalid Rauf

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR

SESSION 2012

EFFECT OF BACOPA MONNIERI ON MORPHINE DEPENDENCE AND TOLERANCE TO ANALGESIA IN ANIMAL MODELS

Khalid Rauf

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

Doctor of Philosophy in Pharmacy

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR

SESSION 2012

DEDICATION

To my parents for their, love, guidance, support and prayers that

have always been an unstinting source of inspiration for me

ACKNOWLEDGEMENTS

With profound gratitude, I heartily acknowledge that the successful completion of this thesis is one the many blessings of Allah (subhanaho wa Taalaa) that He continuously showers upon me since the time I did not had the capacity to realize and acknowledge it. And it’s really a hard undertaking to look for words so eloquent to express my gratitude for my Creator. I sincerely acknowledge and appreciate the academic guidance, support, and the continuous intellectual patronage of my supervisor

Professor Dr. Fazal Subhan. Without his profound skills and insight of the subject this work would not have been possible. I heartily acknowledge his continuous commitment to help me achieve this goal otherwise the accomplishment of this work would have been a distant point in my life.

I offer my profound gratitude to the members of the graduate study committee (GSC) and advanced studies & research board (ASRB) for their cooperation and guidance during my efforts to complete the research work. I heartily acknowledge and sincerely thank all my course tutors at the Department of Pharmacy, University of Peshawar for their help, support and care they extended to me during their course works.

I am extremely obliged to Prof. Dr. Ikhlas.A.Khan, the National Center for Natural

Products Research, Mississippi, USA for the gift of HPLC standards of Bacosides. I am also thankful to the whole teaching staff, Lab. & administrative staff of department of Pharmacy, University of Peshawar for their continuous help and support. I am deeply grateful to all my research colleagues, seniors and juniors for their immeasurable emotional support, kindness and for adding some really unforgettable moments to my life and making my stay comfortable in the laboratory and campus. I am really thankful to Ikhlaque Ahmed Pharm.D for his help in acquisition of morphine and for his continuous help and logistic support in acquisition and procurement of rats and mice from National Institute of health Islamabad.

Not least but the last to write, this work is backed and emotionally financed by many prayers and good wishes of my parents whom love and support kept me going. My each and every family member has extensive contribution in enabling me completing this task. The supportive role of Ministry of Health, Pakistan and Ministry of

Narcotics control, Pakistan, in acquisition of Morphine sulphate is gratefully acknowledged. I sincerely thank M/S Punjab Drug House labs, for the gift of

Morphine sulphate through proper channel. Many thanks to the Higher Education commission of Pakistan for granting me indigenous PhD scholarship to complete my studies.

Khalid Rauf

Summary

Bacopa monnieri (BM) is a renowned ayurvedic herb found in shady marshy places and fresh water streams across Europe, Asia, including India and most parts of

Pakistan. BM has century’s old clinical utility for various neuropsychiatric illnesses like, insomnia, loss of memory, anxiety, epilepsy and depression and a special repute as nootropic and memory enhancer. BM has a documented safety profile in multiple clinical trials, including old age individuals. BM neuropharmacological profile is attributed to saponins called Bacoside A, found in its methanolic and n-butanol extract.

The series of studies outlined in this thesis include quantification of Bacoside A major components i.e. Bacoside A3, Bacopaside II and Bacopasaponin C, using a revalidated High Performance Liquid Chromatography (HPLC) with UV detection method, in methanol and n-butanol extract of the of locally available BM.

Toxicological studies were performed in mice to calculate the LD50 of methanolic extract (Mt-ext BM) and n-butanol extract (n-Bt-ext BM) of the locally available BM.

Effects of acute and sub chronic (one week) administration of BM both extracts on dopamine and serotonin turnover were also investigated in mice. For the study of neurotransmitters modulations in various brain areas a precise, specific and linear, reverse phase HPLC method was developed and validated using dual electrode electrochemical detection, for simultaneous determination of Dopamine (DA)

Dihydroxyphenylacetic acid (DOPAC), Homovanillic acid (HVA), 5- hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5HIAA) and noradrenaline

(NA). The method was highly reproducible with high sensitivity, selectivity, precision and accuracy. BM both extracts were found to have antinociceptive effects, with n-Bt-ext BM having capacity to enhance morphine analgesia. Both extracts Mt-ext BM and n-Bt- ext BM were found to be effective in amelioration of acquisition of morphine tolerance in mice. BM n-butanol fraction highest dose had analgesia comparable to morphine and no tolerance to antinociceptive effect of BM was observed during one week treatment in mice. Both extracts were found to depress locomotor activity in saline treated animals without altering DA and 5-HT turn over in mice striatum, while both extracts were found to inhibit morphine induced hyperlocomotion and enhanced dopaminergic and serotonergic transmission in mice striatum.

Effects of both extracts were studied on behavioral signs of naloxone precipitated morphine withdrawal in rats and we found that both acute and chronic administration of both extracts significantly decreased somatic signs of naloxone precipitated morphine withdrawal as compared to saline treated groups. Rats brain areas involved in morphine reward, dependence, and withdrawal were screened for DA and 5-HT turn over. Effects of both extract on noradrenaline concentration in frontal cortex and hippocampus were also investigated in rats undergoing naloxone precipitated morphine withdrawal. In addition to lowering somatic signs of morphine withdrawal, a significant increase in DA and 5-HT turn over in striatum and nucleus accumbens with significant decrease in noradrenaline contents in frontal cortex induced by BM, was observed during naloxone precipitated morphine withdrawal. The behavioral and neurotransmitters data implied a fair relationship between withdrawal somatic signs and neurotransmitters changes induced by BM treatments in naloxone precipitated morphine withdrawal.

The behavioral and neurotransmitters findings of this work concluded, significant effects of BM on acquisition and expression of morphine tolerance, enhancement of morphine analgesia, inhibition of morphine induced enhanced dopaminergic and serotonergic activity with concomitant depression of locomotor activity. These findings highlights a newer potential role of BM in the clinical management of morphine tolerance, opiates prescriptions abuse and treatment of chronic malignant and non malignant pains by BM alone or in combination with other opiates. The significant finding in suppression of behavioral signs of naloxone precipitated morphine withdrawal with concomitant enhanced dopamine and serotonin turnover picture shows BM potential role in clinical management of opioid detoxification, dependence, withdrawal and relapse.

LIST OF ABBREVIATIONS

5HIAA 5 Hydroxy indolacetic acid

5-HT 5-Hydroxytryptamine

BM Bacopa monnieri ca Calcium

CNS Central Nervous system

CVS cardiovascular system

DBH dopamine beta hydroxylase

DOPAC 3, 4-Dihydroxyphenylacetic acid

ED50 Median effective dose

Ext Extract

GABA Gamma amino butyric acid

GIT Gastrointestinal tract

HPLC High Performance Liquid Chromatography

HVA Homovanillic Acid i.e Id Est (that is)

Kg Kilogram

L-NAME L-NG-Nitroarginine methyl ester

LD50 Median lethal dose

LOD limit of detection

LOQ limit of quantification

MP Morphine

Mt-ext Methanolic extract n Numbers of animals in a group

NA Noradrenaline

NAc Nucleus Accumbens

NMDA N-methyl-D-aspartate NLX Naloxone

NO Nitric oxide

PCA Perchloric acid

ONOO− peroxynitrite

PKC Protein Kinase C

POMC Proopio-melanocortin

SAL Saline

SEM Standard error of mean

VTA Ventral tegemental area

α2 Alpha-2

δ Delta

ε Epsilon

κ Kappa

σ Sigma

Table of Contents

Chapter 1.Introduction

1.1. Introduction 2 1.1.1.Opioids 4 1.1.2.Endogenous peptides 5 1.1.3.Opioids receptors 6

1.2. Morphine tolerance 7

1.3. Morphine dependence 9 1.3.1.Dopamine (DA) 11 1.3.2.Serotonin 12 1.3.3. Noradrenaline (NA) 13

1.4. Morphine withdrawal 14

1.5. Animal models for drug tolerance and dependence 16 1.5.1.Animal as models of disease 16 1.5.2.Animals models for morphine tolerance and dependence 18 1.5.2.1.Mice 19 1.5.2.2.Rats 20

1.6. Current therapeutic options for the treatment of morphine dependence 21 1.6.1.Detoxification 21 1.6.2.Abrupt detoxification 22 1.6.2.1.Tapering technique 23

1.7. Bacopa monnieri 24 1.7.1.Traditional uses of Bacopa monnieri (BM) 29 1.7.2.Memory enhancer 29 1.7.3.Tranquilizing, antidepressant and sedative effects 30 1.7.4.Antiepileptic effects 31 1.7.5.Anti inflammatory and antinociceptive effects 32 1.7.6.Effects on GIT 32 1.7.7.Antioxidant and adaptogenic effect 32

1.8. Aims and objectives of the study 33 Chapter 2. Methodology

2.1.Chemicals and Reagents 35

2.2.Instruments & Apparatus 36

2.3. Plant collection, extraction and fractionation 36 2.3.1.Preparation of methanolic extract 37 2.3.2.Preparation of n-butanol fraction 37

2.4. Standardization of selected plant extracts for Bacopasides 38 2.4.1.High performance liquid chromatography (HPLC) system 39 2.4.1.1.Preparation of standards 39 2.4.1.2.Sample preparation 39 2.4.1.3.Chromatographic conditions 40 2.4.1.4.Method validation 40

2.5.Dose preparation of Bacopa monnieri extract 40

2.6.Drug administration 40 2.6.1.Drug administration 41 2.6.2.Intraperitoneal administration 41

2.7. Method development and validation of neurotransmitters analysis by HPLC 42 2.7.1.Sample handling 42 2.7.2.Preparation of stock solution 42 2.7.3.Sample preparation 43 2.7.4.Chromatography 43 2.7.5.Method validation 44

2.8. Acute toxicity test 45

2.9. Effect of acute and sub chronic treatment with Bacopa Monnieri on neurotransmitters 45 2.9.1.Acute treatment group 45 2.9.1.Sub chronic treatment group 45 2.9.2.Sample preparation for neurotransmitters analysis 46

2.10. Determination of antinociceptive activity (Hot Plate method) 46 2.10.1.Induction and evaluation of morphine tolerance 46 2.10.2.Assessment of effect of Bacopa monnieri on development of morphine tolerance 47 2.10.3.Assessment of effect of Bacopa monnieri on expression of morphine tolerance 47 2.10.4.Antinociceptive effect of Bacopa monnieri and its combination with morphine 47 2.10.5.Evaluation of tolerance to the antinociceptive effect of Bacopa monnieri 48

2.11. Measurement of locomotor activity 48 2.11.1.Sample preparation for neurotransmitters analysis 48

2.12. Morphine dependence protocol 49 2.12.1.Dose preparation of morphine 49 2.12.2.Treatment groups 49 2.12.3.Evaluation of behavioral signs of naloxone precipitated withdrawal 50 2.12.3.1. Weight loss 50 2.12.3.2. Jumping behavior 50 2.12.3.3. Wet dog shakes 51 2.12.3.4. Abdominal writhes 51 2.12.3.5. Diarrhea 51 2.12.3.6. Squeal on touch 51 2.12.3.7. Raring 52 2.12.3.8. Salivation 52 2.12.4. Sample preparation for neurotransmitters analysis 52

2.13. Ethical approval 52

Chapter 3. HPLC method revalidation and chromatographic analysis of Bacopasides

3.1. Introduction 54

3.2. Materials and methods 54

3.3. Results 54 3.3.1. Chromatography 54

3.4. Method validation 55 3.4.1. Sample preparation 55 3.4.2.Selectivity 55 3.4.3.Recovery 58 3.4.4.Sensitivity 59 3.4.5.Precision 59 3.4.6.Linearity 61 3.4.7.Stability 63 3.5. Discussion 63

Chapter 4. HPLC method development and validation for simultaneous determination of dopamine, serotonin and their metabolites with noradrenaline in rat brain tissues

4.1. Introduction. Error! Bookmark not defined.

4.2. Materials and methods. 65

4.3. Results 65 4.3.1.Chromatography 65 4.3.2.Selection of analytical cell potential 67

4.4. Method validation 68 4.4.1.Selectivity 68 4.4.2.Recovery. 68 4.4.3.Sensitivity 69 4.4.4.Linearity 69 4.4.5.Precision 73

4.5. Stability Studies 75

4.6. Application of the method 77

4.7. Discussion 77 4.8. Conclusion 79

Chapter 5. Effect of Bacopa monnieri on morphine tolerance

5.1. Introduction 81

5.2.Materials and methods 82

5.3. Results 83

5.3.1.Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C 83 5.3.2. Induction and evaluation of morphine tolerance 84 5.3.2.1. Antinociceptive effect of morphine sulphate in hot plate test on day one 84 5.3.2.2. Antinociceptive effect of morphine sulphate in hot plate test on day six 85 5.3.3. Effect of n-Bt-ext of Bacopa Monnieri on development of morphine tolerance 86 5.3.4. Effect of n-Bt-ext of Bacopa monnieri on expression of morphine tolerance 87 5.3.5.Antinociceptive effect of n-Bt-ext of Bacopa monnieri in hot plate test 88 5.3.6.Antinociceptive effect of n-Bt-ext of Bacopa monnieri in combination with morphine 89 5.3.7. Development of tolerance to the antinociceptive effect of n-Bt-ext Bacopa monnieri 90 5.3.8.Effect of Meth ext BM on development of morphine tolerance 91 5.3.9. Effect of acute administration of Meth ext BM on acquisition of morphine tolerance 92 5.3.10.Antinociceptive effect of Mt-ext BM in hot plate test 93 5.3.11.Antinociceptive Effect of Meth ext BM in combination with morphine 94 5.3.12.Development of tolerance to the antinociceptive effect of Meth ext BM 95 5.4. Discussion 96

Chapter 6. Effect of Bacopa monnieri on morphine induced locomotor hyperactivity and neurotransmitters

6.1. Introduction 100

6.2. Materials and methods 101

6.3. Results 101 6.3.1.Chromatographic analysis of methanolic extract of Bacopa monnieri (Mt-ext BM) for Bacopasides 101 6.3.2.Effect of Mt-ext BM alone and in combination with morphine on locomotor activity 101 6.3.3.Effect of Mt-ext BM on striatal DA and its metabolites DOPAC and HVA in mice 102 6.3.4.Effect of Mt-ext BM on 5-HT and its metabolite 5HIAA in the striatum 103 6.3.5.Chromatographic analysis of n-butanol extract of Bacopa monnieri (n-Bt-ext BM) for Bacopasides 104 6.3.6.Effect of n-Bt-ext BM alone and in combination with morphine on locomotor activity 105 6.3.7.Effect of n-Bt-ext BM on DA and its metabolites DOPAC and HVA in mouse striatum 105 6.3.8.Effect of n-Bt-ext BM on 5-HT and its metabolite 5HIAA in the striatum 106 6.5. Discussion 108

6.5. Conclusion 111

Chapter 7. Effect of acute and sub chronic administration of Bacopa monnieri on neurotransmitters

7.1 Introduction 113

7.2. Materials and methods 114

7.3. Results 114 7.3.1.Quantification of Bacoside A major components in Mt-ext BM and n-Bt-ext BM 114 7.3.2.Effect of acute treatment of Mt-ext BM on whole brain neurotransmitters 114 7.3.3.Effect of sub chronic treatment of Mt-ext BM on whole brain neurotransmitters 115 7.3.4.Effect of n-Bt-ext acute treatment on whole brain neurotransmitters in mice

116 7.3.5.Effect of n-Bt-ext sub chronic treatment on whole brain neurotransmitters in mice 117 7.4 Discussion 118

Chapter 8. Effect of Bacopa monnieri on naloxone precipitated morphine withdrawal and neurotransmitters

8.1. Introduction 123

8.2. Materials and methods 124

8.3. Results 124

8.3.1. Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C in Mt-ext BM and n‐Bt-ext BM 124 8.3.2. Effect of Mt-ext BM on body weight loss in naloxone precipitated morphine withdrawal 125 8.3.3.Effect of Mt-ext BM on naloxone precipitated morphine withdrawal jumping behavior 127 8.3.4. Effect of Mt-ext BM on naloxone precipitated morphine withdrawal induced writhes 129 8.3.5.Effect of Mt-ext BM on Squeal on touch behavior in naloxone precipitated morphine withdrawal 131 8.3.6.Effect of Mt-ext BM treatment on naloxone precipitated morphine withdrawal salivation 133 8.3.7.Effect of Mt-ext BM treatment on raring behavior in naloxone precipitated morphine withdrawal 135 8.3.8.Effect of Mt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal 137 8.3.9.Effect of Mt-ext BM treatment on incidence of teeth chattering in naloxone precipitated morphine withdrawal 139 8.3.10.Effect of Mt-ext BM treatment on incidence of wet dog shake behavior in naloxone precipitated morphine withdrawal 141 8.3.11. Effect of n-Bt-ext BM treatment on body weight loss in naloxone precipitated morphine withdrawal 143 8.3.12. Effect of n-Bt-ext BM treatment on incidence of jumping behavior in naloxone precipitated morphine withdrawal 145 8.3.13. Effect of n-Bt-ext BM treatment on incidence of writhes in naloxone precipitated morphine withdrawal 147 8.3.14. Effect of n-Bt-ext BM treatment on incidence of Squeal on touch behavior in naloxone precipitated morphine withdrawal 149 8.3.15. Effect of n-Bt-ext BM treatment on incidence of teeth chattering in naloxone precipitated morphine withdrawal 151 8.3.16. Effect of n‐Bt-ext BM treatment on incidence of wet dog shakes in naloxone precipitated morphine withdrawal 153 8.3.17. Effect of n-Bt-ext BM treatment on incidence of salivation in naloxone precipitated morphine withdrawal 155 8.3.18. Effect of n-Bt-ext BM treatment on incidence of raring in naloxone precipitated withdrawal 157 8.3.19. Effect of n-Bt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal 159 8.3.20. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally) on frontal cortex levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 161 8.3.21.Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 162 8.3.22 Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 164 8.3.23.Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 166 8.3.24. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine morphine withdrawal 168 8.3.25. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on striatum tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 169 8.3.26. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on Hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 170 8.3.27. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 172 8.4. Discussion 173

Chapter 9. Acute toxicological studies on Bacopa monnieri

9.1. Introduction 179

9.2. Materials and methods 179

9.3. Results 179 9.3.1. Acute toxicological effect ofMt-ext BM 179 9.3.2.Acute toxicological effect of n-Bt-ext BM 180 9.4. Discussion 181

Chapter 10. General discussion

10.1.General Discussion 183

10.2.Future Work 188

References 191

Appendices 216

LIST OF TABLES

S.No Topic Page No 2.1 Instruments & Apparatus 36

Recovery of Bacoside A3, Bacopaside II, Bacopasaponin C 3.1 59 from spiked methanolic extract

Precision and intra-day and inter-day data of Bacoside A3, 3.2 60 Bacopaside II, Bacopasaponin C Recovery of NA, DOPAC, DA, 5HIAA, HVA, 5-HT from 4.1 69 spiked samples Effect of inter days and intraday on NA, DOPAC, DA, 4.2 74 5HIAA, HVA, 5-HT precision in spiked samples Stability of NA,DA,DOPAC,5HIAA,HVA,5-HT in PCA with, 4.3 76 and without antioxidant Effect of normal saline and Mt-ext BM (10, 20 or 30 mg/kg) 6.1 on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 104 5HIAA in mice Effect of normal saline, morphine (MP, 10 mg/kg) and morphine (MP, 10 mg/kg) + Mt-ext BM (10, 20 and 30 mg/kg) 6.2 104 on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA Effect of normal saline and n-Bt-ext BM (5, 10 or 15 mg/kg) 6.3 on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 107 5HIAA in mice Effect of normal saline, morphine (MP, 10 mg/kg) and morphine (MP, 10 mg/kg) + n-Bt-ext BM (5, 10 or 15 mg/kg) 6.4 107 on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 5HIAA. Effect of acute administration of Mt-ext BM on DA, 5-HT and 7.1 115 their metabolites in mice whole brain Effect of sub chronic (one week) administration of Mt-ext BM, 7.2 116 On DA, 5-HT and their metabolites in mice whole brain Effect of acute administration of n-Bt-ext BM on DA and 5- 7.3 117 HT turn over Effect of chronic administration of n-Bt-ext BM on DA and 5- 7.4 118 HT turn over Effect of normal saline and Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) on frontal cortex tissue levels of NA, DA, 8.1 161 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal 8.2 Effect of normal saline and Mt-ext BM chronic treatment (10, 162 20 or 30 mg/kg orally) on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM acute treatment (10, 20 and 30 mg/kg orally) on striatal tissue levels of NA, DA, 8.3 163 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM Chronic treatment (10, 20 or 30 mg/kg orally) on Striatal tissue levels of NA, DA, 8.4 164 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) on Hippcampal tissue levels of NA, DA, 8.5 165 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM Chronic treatment (10, 20 or 30 mg/kg orally) on Hippocampus tissue levels of NA, 8.6 165 DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) on Nucleus Accumbens tissue levels of 8.7 166 NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and Mt-ext BM Chronic treatment (10, 20 or 30 mg/kg orally) on Nucleus Accumbens tissue levels of 8.8 167 NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on Frontal Cortex tissue levels of NA, 8.9 168 DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM Chronic treatment (10, 20 or 30 mg/kg orally) on Frontal Cortex tissue levels of 8.10 169 NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on Striatal tissue levels of NA, DA, 8.11 170 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal Effect of normal saline and n-Bt-ext BM Chronic treatment (5, 8.12 10 or 15 mg/kg orally) on Striatal tissue levels of NA, DA, 170 DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on Hippocampus tissue levels of NA, 8.13 171 DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM Chronic treatment (5, 10 or 15 mg/kg orally) on Hippocampus tissue levels of NA, 8.14 171 DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on Nucleus Accumbens tissue levels of 8.15 172 NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal Effect of normal saline and n-Bt-ext BM Chronic treatment (5, 10 or 15 mg/kg orally) on Nucleus Accumbens tissue levels of 8.16 173 NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated opioid withdrawal

TABLE OF FIGURES

S.No TOPIC Page No 1.1 Bacopa monnieri image 26

1.2 Bacoside A3 27 1.3 Bacopasaponin C 28 1.4 Bacopaside II 28

Chromatogram showing Bacoside A3, Bacopaside II, 3.1 56 Bacopasaponin C standards 1.75µg/mL

Chromatograms showing Bacoside A3, Bacopaside II, 3.2 57 Bacopasaponin C in n-Butanol fraction (1) and standards(2).

Chromatogram showing Bacoside A3 (1), Bacopaside II (2), 3.3 58 Bacopasaponin C (3) in methanolic extract

3.4 Calibration curve Bacoside A3, in a range of 500 ng-2.ug 61 3.5 Calibration curve Bacopaside II, in a range of 500 ng-2 ug 62 3.6 Calibration curves Bacopasaponin C, range 500 ng-2 ug 62 Chromatogram showing NA, DOPAC, DA, 5HIAA, HVA and 5- 4.1 HT as peak A, B, C, D, E. respectively in 1 ng / mL standards 66 sample. Chromatogram showing, NA, DOPAC, DA, 5HIAA, HVA and 5- 4.2 HT denoted as, peak A, B, C, D,E and F, respectively in saline 67 treated mice striatum Volatammogram showing all six peaks. NA, DOPAC, DA, 4.3 68 5HIAA, HVA , 5-HT 4.4 Calibration curve of DOPAC, in a range of 1 ng to 300 ng. 70 4.5 Calibration curve of 5HIAA, in a range of 1 ng to 300 ng. 70 4.6 Calibration curve of HVA, in a range of 1 ng to 300 ng. 71 4.7 Calibration curve of 5-HT, in a range of 1 ng to 300 ng 71 4.8 Calibration curve of DA, in a range of 1 ng to 300 ng. 72 4.9 Calibration curve of NA, in a range of 1 ng to 300 ng 72 Day 1 analgesia of morphine group and saline group in mice. 5.1 84 Animals Antinociceptive effect of morphine sulphate in hot plate test on day 5.2 85 six Effect of n Bt-ext of Bacopa Monnieri on development of 5.3 86 morphine tolerance Effect of n Bt-ext of Bacopa monnieri on expression of morphine 5.4 87 tolerance 5.5 . .Antinociceptive effect of n Bt-ext of Bacopa monnieri in hot 88 plate test Antinociceptive effect of n Bt-ext of Bacopa monnieri in 5.6 89 combination with morphine Development of tolerance to the antinociceptive effect of n Bt-ext 5.7 90 Bacopa monnieri Effect of meth ext BM on development of morphine tolerance 5.8 91

Effect of acute administration of Meth ext BM on acquisition of 5.9 92 morphine tolerance 5.10 Antinociceptive effect of Meth-ext BM in hot plate test 93 Antinociceptive Effect of Meth-ext BM in combination with 5.11 94 morphine Development of tolerance to the antinociceptive effect of meth ext 5.12 95 BM Effect of Mt-ext BM (10, 20 and 30 mg/kg orally) on locomotor 6.1 activity in saline and morphine (10 mg/kg intraperitoneally) in 102 mice Effect of n Bt-ext BM (5, 10 and 15 mg/kg i.p) on saline (SAL) 6.2 105 and morphine (MP, 10 mg/kg) induced locomotor activity in mice Effect of Mt-ext BM on body weight loss in naloxone precipitated 8.1 125 morphine withdrawal Effect of Mt-ext BM on body weight loss in naloxone precipitated 8.2 126 morphine withdrawal Effect of Mt-ext BM on naloxone precipitated morphine 8.3 127 withdrawal jumping behavior. Effect of Mt-ext BM on naloxone precipitated morphine 8.4 128 withdrawal jumping behavior. Effect of Mt-ext BM on naloxone precipitated morphine 8.5 129 withdrawal induced abdominal constrictions behavior Effect of Mt-ext BM on naloxone precipitated morphine 8.6 130 withdrawal induced abdominal constrictions behavior Effect of Mt-ext BM on Squeal on touch behavior in naloxone 8.7 131 precipitated morphine withdrawal. Effect of Mt-ext BM on Squeal on touch behavior in naloxone 8.8 132 precipitated morphine withdrawal Effect of Mt-ext BM treatment on naloxone precipitated morphine 8.9 133 withdrawal salivation Effect of Mt-ext BM treatment on naloxone precipitated morphine 8.10 134 withdrawal salivation 8.11 Effect of Mt-ext BM treatment on raring behavior in naloxone 135 precipitated morphine withdrawal Effect of Mt-ext BM treatment on raring behavior in naloxone 8.12 136 precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of diarrhoea in 8.13 137 naloxone precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of diarrhoea in 8.14 138 naloxone precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of teeth chattering in 8.15 139 naloxone precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of teeth chattering in 8.16 140 naloxone precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of wet dog shake 8.17 141 behavior in naloxone precipitated morphine withdrawal Effect of Mt-ext BM treatment on incidence of wet dog shake 8.18 142 behavior in naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on body weight loss in naloxone 8.19 143 precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on body weight loss in naloxone 8.20 144 precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of jumping behavior 8.21 145 in naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of jumping behavior 8.22 146 in naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of abdominal 8.23 constrictions behavior in naloxone precipitated morphine 147 withdrawal Effect of n-Bt-ext BM treatment on incidence of abdominal 8.24 constrictions behavior in naloxone precipitated morphine 148 withdrawal Effect of n-Bt-ext BM treatment on incidence of squeal on touch 8.25 149 behavior in naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of squeal on touch 8.26 150 behavior in naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of teeth chattering in 8.27 151 naloxone precipitated withdrawal Effect of n-Bt-ext BM treatment on incidence of Teeth chattering8. 8.28 152 in naloxone precipitated withdrawal Effect of n-Bt-ext BM treatment on incidence of wet dog shakes in 8.29 153 naloxone precipitated morphine withdrawal 8.30 Effect of n-Bt-ext BM treatment on incidence of wet dog shakes in 154 naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of salivation in 8.31 155 naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of salivation in 8.32 156 naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of raring in naloxone 8.33 157 precipitated withdrawal Effect of n-Bt-ext BM treatment on incidence of raring in naloxone 8.34 158 precipitated withdrawal Effect of n-Bt-ext BM treatment on incidence of diarrhoea in 8.35 159 naloxone precipitated morphine withdrawal Effect of n-Bt-ext BM treatment on incidence of diarrhoea in 8.36 160 naloxone precipitated morphine withdrawal median lethal dose of methanolic extract (155 mg/ kg) of bacopa 9.1 180 monnieri in mice. Median Lethal dose of n-Butanol extract of Bacopa monnieri in 9.2 180 mice (81.1 mg/Kg).

Chapter 1 Introduction

Chapter 1

Introduction

Chapter 1 Introduction

1.1. Introduction

The history of early humans depicts the presence of diseases, which were mainly attributed to the anger of the many gods they worshiped, and believed in their existence, anger, pleasure and rewards (Sigerist, 1970). Since the dawn of history man had known the plant that heals all sorrows and placates crying children and has been reported in annals of history that dates back to 5000 B.C depicting its cultivation in Mesopotamia to extract its juice called Jill which means joy

(Brownstein, 1993). Opium poppy medicinal properties were known to humans in

Egypt and Persia around 1550 B.C. It was the Greeks that coined the word “opion” a diminutive of “opos” which meant vegetable juice (Brownstein, 1993). Opium is the oldest of the psychoactive substances known to humanity; it was used medicinally as cough suppressant, sedative, tranquilizer and analgesic during renaissance in Europe.

Opium is actually dried exudates obtained from unripe capsules of poppy plant

“Papaver Somniferum”. Morphine being a drug derived from opium juice is considered to be the most potent and effective of all analgesics used clinically for the management of chronic pains like cancer (Brownstein, 1993; Hamilton and Baskett,

2000). As morphine has strong rewarding properties like causing relaxation and induction of euphoria it has an extensive abuse liability. Opium is known to human society as addictive drug since the dawn of human history, and till now opiates dependence in its more huge and complex form is serious challenge to the social and economic fabric of society (Wall et al., 2000). Pakistan being third world country has a huge population of opioid addicts and their use and number increasing as lack of financial resources precludes a holistic and comprehensive solution of this problem that continuously translates itself in the form of multitude of socioeconomic and

Chapter 1 Introduction health problems like, mortality, morbidity, HIV, unemployment, co morbidity and interpersonal breakdowns, crimes and legal issues (Ahmad et al., 2001; Gillis and

Mubbashar, 1995; Gossop, 1989; Strathdee et al., 2003). That’s why opioid addiction has been termed as chronic relapsing disease and presents a substantial social and health challenge to the world in general and Pakistan in special (Gillis and

Mubbashar, 1995; Gossop, 1989).

As opiates are extensively used in clinical practices for management of both chronic and acute pains, its prescription trend is on a continuous rise for the last one decade

(McCabe et al., 2009; McCabe et al., 2008) and clinical settings are facing a newer challenge of prescription opiates abuse. There are clinical evidences that 3-16% patients taking long term opiates as therapy have genetic vulnerability of drug dependence (Savage, 1996). It has been observed that chronic opiates therapy induces dependence in about 24% patients treated for chronic backache, apart from old age people who chronically uses clinically prescribed opiates and have social or mental problems they help support their predisposition to dependence (Manchikanti and Singh, 2008; Martell et al., 2007). Non medical prescription drug abuse is on the rise, especially in countries where state has least/ineffective control over drugs of abuse (Johnston et al., 2008; Veilleux et al., 2010). According to Dr Johnston and

Passik, findings (Johnston et al., 2008; Passik, 2009) opiates are the second to marijuana as illicit drug in United States. Moreover according to another report

(Brands et al., 2004; Mendelson et al., 2008), patients using opiates as part of therapy are also at higher risk of addiction (Katz et al., 2008; Veilleux et al., 2010).

Additionally poly substance abuse is more common problem among opiates (Van den Brink and Haasen, 2006a) as 92 % of all opioid addicts have poly substance abuse like nicotine or others. In another survey it has been reported that 40 to 80 % 3

Chapter 1 Introduction of all opiates addicts have a co morbid other psychiatric illnesses, like anxiety, depression and other affective disorder are the most extensively reported illnesses

(Passik, 2009; Strain, 2002).

Opioid dependence also called a chronic relapsing disorder as there is 60 percent relapse among heroin addicts seeking treatment (Benich III, 2010; Johnston et al.,

2008; Mark et al., 2001; Van den Brink and Haasen, 2006a). The issue is even worse in developed world also as the opioid dependence annually costs half a trillion to

U.S. economy as direct burden of dependence in the form of treatments, health costs, unemployment, family break ups, criminal activities, and justice (Birnbaum et al.,

2006; Mark et al., 2001; Ruetsch, 2010).

1.1.1. Opioids

Opioids is a term employed to denote all natural peptides found indigenously in body

, acquired from Papaver Somniferum or synthetic analogues having morphine like activity. Opiates are drugs obtained from opium like morphine, codeine, their semi synthetic congeners like oxycodone etc and all synthetic molecules having morphine like effects. Opioids receptors theory was propounded by Goldstein and his co- workers (Goldstein et al., 1971) which further translated in detail by three independently working research groups (Pert and Snyder, Simon and co-workers,

Terenius and co-workers) that propounded distinct biding sites for opioids in both human and animals brain (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973).

The discovery of Opioids receptors further sharpened the quest of scientific community for endogenous legends for these receptors, and in 1975, Dr John Hughes

(Hughes, 1975) reported two opioid peptides Leu-enkaphalin and Met-enkaphalins with the inhibitory effect on acetylcholine release in guinea pig ileum. In the

Chapter 1 Introduction upcoming era, researchers made substantial progress in search of endogenous legends and beta endorphin, dynorphins, enkaphalins, and their diverse chemical and genetical structures and roles were defined (Bodnar, 2009). These peptides have different affinity for opioid receptors and produce diverse actions based on physiologic and anatomical positioning of the tissues where the receptors are located.

Enkaphalins have high propensity for delta receptors, while dynorphins and β- endorphins predominantly have high affinity for kappa and mu receptors respectively.

Later on Zadina et al, (Zadina et al., 1997) reported two more peptides, endomorphin-

1 and endomorphine-2 having high propensity for mu receptors (Bodnar, 2009).

1.1.2. Endogenous peptides

The discovery of endogenous opiate receptors led to the discovery of endogenous opiate transmitters that were called Endorphins (Endogenous morphine), enkaphalins and dynorphins. Opioids peptides are produced by three genetically distinct large precursors’ proopiomelanocortin (POMC), proenkaphalin, and prodynorphin. The opioid peptides mainly comprise β-endorphin, enkaphalins (Met-enkaphalin and

Leu-enkaphalin) and Dynorphins (dynorphins A, dynorphins B, alpha neo endorphin). All opioid peptides after production in nucleus are transported to nerve ending via microtubule transport. After cleavage by intrinsic proteases system the peptides are released from nerve ending where they occupy the specified receptors, stimulating second messenger system. Endogenous Opioids peptides are extensively distributed and found in different brain areas like brain stem, limbic system, hypothalamus and spinal cord. Enkaphalins are found in spinal cord, cortex, hippocampus amygdala, and Locus coeruleus. Dynorphins is found in hippocampus, striatum, and spinal cord. Endogenous opioids peptides are also found peripherally

Chapter 1 Introduction like in immune cells, gastrointestinal tract, adrenal medulla and pancreas. These peptides apart from imparting analgesia, also have many other important roles like mediation of stress, behavior, memory, seizures, mood, drug tolerance and dependence (Olson et al., 1997).

1.1.3. Opioids receptors

There are five types of distinct opioid receptors i.e., mu (µ), kappa (κ), delta (δ), sigma (σ) and epsilon (є), which are all G-protein coupled receptors. After coupling with receptor the endogenous peptides apart from producing analgesia, also produce euphoria, respiratory depression and mood elevation. Mu receptors are mainly associated with ensuring analgesia, reward, motor control and opioid withdrawal syndrome, inhibition of gastrointestinal tract motility, respiratory and physical depression. Mu receptors are found in close proximity to reward pathways, analgesia and dopaminergic circuitry (Arvidsson et al., 1995). There are two types of mu receptors, µ1 and µ2. The µ1 receptors are mainly concerned with mediation of analgesia at spinal and supra spinal level and have a high degree of selectivity for morphine. While µ2 are mainly associated with respiratory depression. Morphine acts as exogenous legend while enkaphalin and endorphin are endogenous legends for these receptors. Delta (δ) receptors are of two types, δ1 and δ2 mainly responsible for mediation of analgesia both spinally and supra spinally, hallucination and psychostimulant effects. Morphine is an exogenous while enkaphalins are endogenous legend for these receptors. Kappa receptors (κ) are mainly involved with dysphoria, analgesia (spinal) sedation, respiratory depression and miosis. There are three subtypes of Kappa receptors (κ), i.e. κ1, κ2, and κ3 receptors. Spinal analgesia is mainly mediated by κ1; supra spinal analgesia by κ2 and role of κ3 receptors is not

Chapter 1 Introduction clear. Delta (δ) receptors and Kappa receptors (κ) are found in some part of brain and spinal cord (Arvidsson et al., 1995). Sigma receptors are mainly involved in the mediation of opiates side effects like dysphoria, miosis etc. The Epsilon (є) receptors are involved with mediating analgesia but their contribution is less pronounced as compared to other receptors. The structures of all major receptors has been elucidated and cloned and there is 70 percent homology between chemical structures of all these receptors (Chen et al., 1996; Kieffer et al., 1992; Thompson et al., 1993). All receptors share common effector system and produced their pharmacological effects by inhibiting inwardly potassium conductance and also inhibiting voltage gated calcium conductance (Grudt and Williams, 1993).

The endogenous opioid system is the substrate for all opiates and all effects of natural or synthetic opiates are mediated via this system. In healthy humans also this system plays an essential role in regulation of nociception, emotional behavior, learning and reward mediation.

1.2. Morphine tolerance

One of the most common and distressing side effect associated with prolong clinical use of morphine is tolerance to its analgesic effect. The same tolerance to euphoric and analgesic effects is experienced by opiate addicts also. Upon development of tolerance continuously larger doses are required to produce the same analgesic effect produced with smaller dose at the start of the therapy. Another rather bizarre aspect of morphine tolerance is development of hyperalgesia associated with development of tolerance (Laulin et al., 1999; Mao et al., 1994). Both humans and animals data reflects that there are two distinct phases of opiates tolerance expression, first phase

Chapter 1 Introduction may occur with first dose and dissipates swiftly as therapy is discontinued, while the second phase takes weeks in development and expression. This development of tolerance predisposes the opiates using patient or abuser to extensive side effects like sedation, respiratory depression, constipation, risk of addiction, and poor quality of life (Foley, 1995). Tolerance mechanism is rather complex and not a monolithic phenomenon. It has a strong association with neuroadaptation, receptor desensitization through adaptive changes and altered genes expression at both central and peripheral levels (Christie, 2008; Williams et al., 2001). Although there are multiple other agents like β Arrestin and Protein Kinase C (PKC), that may lead to acceleration of morphine induced µ receptor desensitization (Bailey et al., 2004).

Activation of PKC by NMDA receptors or G protein coupled receptors is imperative for development of morphine tolerance, and conversely reversal of morphine tolerance by PKC inhibitors (Bailey and Connor, 2005; Hong et al., 2010; Wang et al., 1997).

Current research highlights the prominent role of nitro oxidative stress in pain etiologies and acquisition and expression of opiates tolerance due to prolong exposure to opiates for clinical or non clinical indications. After chronic morphine exposure, there is Peroxynitrite (ONOO-) over production and its control by drugs that disrupts this cycle has promising results in inhibiting morphine tolerance (Watkins et al.,

2007). Chronic morphine administration activates glial cells that produce more pro inflammatory cytokines (Chao et al., 1994). All those compounds including bioflavonoid and saponins that have strong scavenging reactivity towards

Peroxynitrite (ONOO-) are reported to inhibit morphine tolerance. These bioflavonoid either scavenge or disrupt its production cycle, and so helps restore opioid analgesia

Chapter 1 Introduction and reverse tolerance (Anjaneyulu and Chopra, 2003; Dai et al., 2007; Naidu et al.,

2003; Pavlovic and Santaniello, 2007).

1.3. Morphine dependence

Chronic use of morphine for both clinical or non clinical indications leads to drug tolerance, dependence and addiction (Nestler, 1996). There is close relation between opiate dependence and subsequent behavioral changes in maternity, siblings, society as a whole. Physical dependence is displayed when opiates are discontinued or some antagonists like Naloxone is administered (Nestler, 1996). Electrophysiological and molecular studies have revealed that chronic opioids administration leads to biochemical adaptive changes in locus coeruleus (LC) and mesolimbic dopamine systems with key role played by G-protein cyclic AMP, and coupled receptors

(Nestler, 1996, 2004; Nestler and Aghajanian, 1997; Nestler et al., 1994; Nestler et al., 1993). Among opiates the molecules that have higher propensity for µ receptors have higher abuse potential than less preferring one (Devine and Wise, 1994).

Mesolimbic dopamine system is primarily attributed as reward pathway to have a critical role in opioid dependence (Koob and Bloom, 1988; Nestler, 2004).

Activation of dopaminergic neurons in mesolimbic system by opiates induce positive enforcements (Koob and Bloom, 1988; Nestler, 2004). Stimulation of opioid receptors in VTA leads to extensive release of dopamine in nucleus accumbens

(NAc) with a prominent behavioral display of excitement and euphoria. Additionally morphine and other opiates occupy µ receptors found on the gamma amino butyric acid (GABA) neurons throughout the brain structures, thus lowering GABA- inhibition in Ventral tegemental area (VTA) leading to upsurge of DA in nucleus accumbens. While this dopaminergic activity drops dramatically during opioids

Chapter 1 Introduction withdrawal, during dependence there are biochemical modulation of indigenous anti- opioid system which is drastically altered by chronic opiates exposure (Rothman,

1992). There is a critical interplay between N-methyl-D-aspartate receptors (NMDA) and nitric oxide and NMDA receptors not only modulate nitric oxide but its blockade also inhibits opioid withdrawal (Bredt and Snyder, 1992; Toda et al., 2009).

Molecular studies imply that chronic opiates exposure induced biochemical changes in locus coeruleus and mesolimbic system (Nestler, 2004; Nestler et al., 1994;

Nestler et al., 1993). The main target mediator of neurochemical changes are G- proteins cyclic AMP and this protein phosphorylation induces drug dependence, tolerance and other manifestations of opioid withdrawal (Guitart and Nestler, 1993).

Mesolimbic reward pathways plays major role in mediation of opiates rewards, dependence and withdrawal behavior through direct and indirect modulation of dopamine (Everitt and Robbins, 2000; Koob, 1992; Nestler, 2004; Robbins, 2000;

Shaw et al., 1984). Additionally VTA and NAc are the key players in the mediation of rewarding properties of opiates. Accordingly opioid antagonists, injected directly into either VTA or nucleus accumbens lower drug seeking behavior in opioid dependent animals (Young et al., 2011).

Opiates have also been found to negatively modulate and impair biochemical parameters in human blood (Kouros et al., 2010). Chronic use of opiates also impair natural oxidative stress homeostasis in human body leading to DNA damage, mast cells destabilization, focal glumerulosclerosis, hepatotoxicity with reduced glutathione peroxidase, glutathione, catalase and superoxide dismutase levels

(Kriegstein et al., 1999; Patel et al., 2003; Pereska et al., 2007; Poeggeler et al., 1993;

Xu et al., 2006).

Chapter 1 Introduction

The interplay of various neurotransmitters and neuropeptides in induction of opiates dependence is highly complex and intricate phenomena. The brief overview of role of major neurotransmitters i.e. DA, 5HT, and NA in opioid dependence is as follows.

1.3.1. Dopamine (DA)

Dopamine is said to be prime indicator of reward process and all drugs of abuse including opiates, raise intra synaptic dopamine levels in nucleus accumbens and striatum. Current research has further clarified a breadth of dopamine role in signaling the behavioral features including, aversive, rewarding, unprecedented, and unpredictable events (Hyman and Malenka, 2001; Robinson and Berridge, 2000,

2008). Acute and chronic administration of opiates induces an upsurge of dopamine and its metabolites in mice striatum with an associated increase in locomotor activity

(Babbini and Davis, 1972; Fadda et al., 2005; Gauchy et al., 1973; Kuschinsky and

Hornykiewicz, 1974; Rethy et al., 1971). Dopamine transmits its effects primarily through a series of pre and post synaptic G protein coupled receptors. Up to seven dopamine receptor have been confirmed so for but major five are reported to influence drug reward and withdrawal. D1 and D5 primarily stimulate adenylate cyclase activity while D2, D3 and D4 inhibit adenylate cyclase activity. D1 and D5 are located post synaptically while D2, D3 and D4 are located pre synaptically. The role of D1, D2 and D3 is primarily associated with reward and withdrawal (Harris and

Aston-Jones, 1994). The D2 receptors have a well pronounced role in opiates dependence withdrawal and relapse. Researchers have reported that D2 receptor agonists provoke a relapse in opiate dependent individuals (Harris and Aston-Jones,

1994). It has also been reported through Positron Emission Tomography that chronic opiates exposure have propensity to lower D2 receptor density in ventral striatum

Chapter 1 Introduction

(Wang et al., 1997). This DA turn over quantification is one analytical parameter for assessing direct or indirect effect of centrally active drugs on dopaminergic pathways in key brain areas (Frank and Srinivasa, 2011).

1.3.2. Serotonin

Although serotonin is primarily involved in behavioral inhibition, appetite, locomotion, mood, anxiety like behaviors, sensory reactivity, emotional stabilization, sexual behavior, pain perception and cognition. Serotonin has not been reported to be directly involved in motivation and reward pathways, but affects motivation and reward phenomena through modulation of dopamine. Conversely some researchers have reported Serotonin to have a key modulation role in opioid analgesia, dependence, withdrawal and tolerance (Capasso, 2008, 2009). Serotonin has been found to have close interplay with dopamine and cerebral infusion of serotonin in ventral tegemental area raises dopamine concentration in nucleus accumbens via 5-

HT2 receptors and drugs altering serotonin may modify opiates analgesia, dependence, tolerance and withdrawal phenomenon. Additionally both acute and chronic administration of opiates leads to an upsurge of serotonin, 5-

Hydroxyindoleacetic acid (5HIAA), and its turnover in mice brain and serotonin role in induction and withdrawal through serotonin altering drugs is well established

(Carboni et al., 1988; Eric J, 1996; Guan and McBride, 1989; Hui et al., 1996; Neal and Sparber, 1986; Pinelli et al., 1997; Samanin et al., 1980). Although some evidences support the direct role of serotonin and noradrenalin in mediation, establishment and expression of opioid dependence and withdrawal (Auclair et al.,

2004; Carboni et al., 1988; Gray, 2002; Tassin, 2008).

Chapter 1 Introduction

Around fourteen subtypes of serotonin receptors have been reported with a diverse range of associated effects (Terry Jr et al., 2008). As 5HT2A receptor antagonist has been reported to inhibit morphine induced locomotor effects. Recent studies in isolated tissues have validated the findings of significant impact of serotonin on opioid dependence and withdrawal (Capasso, 2009).

1.3.3. Noradrenaline (NA)

Noradrenaline (NA) an important neurotransmitter produced mainly in locus coeruleus from DA by the action of an enzyme dopamine beta hydroxylase (DBH) in the brain stem having a wider role and implication in opioid dependence and withdrawal symptoms. Drugs affecting noradrenalin receptors are among the mainstay of current therapeutic regimens used for management of opioid withdrawal

(Greenwell Tn Fau - Walker et al.; Streel et al., 2006). It also works as hormone in blood produced by adrenal medulla. It’s a legend for G protein coupled adrenoceptors that are either α adrenoceptors or β adrenoceptors. There are two main subtypes of these α receptors, i.e. α1 and α2, each having three sub types. The current research has highlighted a broader role of NA in mediation of opioid dependence. In

this respect mice lacking α1B have been reported to be free from morphine addictive properties and do not self administer opiates in drug discrimination paradigm as normal mice do (Goodman, 2008).

Clonidine, an α2A agonist has been reported to inhibit morphine induced conditioned place preference (CPP) (Sahraei et al., 2004). Moreover clonidine has been reported to inhibit morphine withdrawal upon infusion into locus coeruleus area of morphine dependent mice (Taylor et al., 1988). Mice deficient in DBH have been reported to be free from morphine induced CPP. Additionally α2A agonists have been reported to 13

Chapter 1 Introduction attenuate stress induced reinstatement of drug seeking behavior in opioid dependent animals (Belujon and Grace, 2011; Highfield et al., 2001). Venlafaxine, a dual NE/5-

HT reuptake inhibitor has been reported to inhibit morphine induced CPP in animal models (Goodman, 2008). Animals express the euphoric effects of morphine through locomotor hyperactivity, and morphine has been reported to mediate this effect via noradrenaline (Lanteri et al., 2007). Drugs intended to treat opioid dependence are screened for interaction with noradrenaline also (Goodman, 2008).

1.4. Morphine withdrawal

Mesolimbic system is the center of emotions, seat of major reward pathways and opiates extend their abuse liable potential and withdrawal effects through this system

(Koob et al., 1989; Stinus et al., 1990). One major functional component of this reward pathway is nucleus accumbens that mediates both reward (positive reinforcement) and aversive behavior during withdrawal, and is also known as a critical target for mediating aversive stimulus properties of opioid withdrawal (Koob et al., 1989; Stinus et al., 1990). It has been reported that dopamine transmission drops considerably during morphine withdrawal in nucleus accumbens (NAc)

(Acquas et al., 1991; Crippens and Robinson, 1994; Pothos et al., 1991). This decline in dopamine concentration has been reported in various microdialysis studies in animals undergoing withdrawal (Pothos et al., 1991). This decline in dopamine is exhibited as aversive behavior by the animals undergoing withdrawal (Crippens and

Robinson, 1994; Koob et al., 1989; Stinus et al., 1990). It has been reported that drugs used in opioid withdrawal like Clonidine prevents the drastic decline of dopamine in NAc thus ameliorating aversive behavior and associated distress both in animals and humans (Pothos et al., 1991). Receptors level studies have confirmed a

Chapter 1 Introduction broader role for DA receptors in NAc during opioid withdrawal (Druhan et al., 2000;

Elwan and Soliman, 1995; Georges et al., 1999).

Earlier it was reported that activation of D2 receptor in chronic opiates user prevents somatic signs of opioid withdrawal syndrome, while D2 receptor antagonists precipitates opioid withdrawal with a prominent signs like wet dog shakes, teeth chattering etc in rats. (Harris and Aston-Jones, 1994). This reflects the involvement of D2 receptors located in the shell region of the NAc in teeth chattering and wet dog shake behavior exhibited by animals undergoing opioid withdrawal (Harris and

Aston-Jones, 1994). Additionally like NAc dopamine levels also drops considerably in striatum of opiate dependents animals. Striatum being a key area of basal ganglia has key role in controlling motor activity and upsurge of opiates induced dopamine and serotonin is translated as hyper ambulatory response behavior in rodents, a typical display of euphoric state by rodents. Moreover all voluntary movements are also regulated from this center. Striatum also mediates some key stereotype behavioral signs of opioid withdrawal like, lethargic gate, wet dog shakes, and withdrawal jumps. Furthermore D1 receptors stimulation leads to enhanced grooming behavior in animals (Sibley, 1999). Several reports have highlighted a broader role of dopamine transmission and modulation of both D1 and D2 receptors densities during opiates dependence, and withdrawal (Bhargava and Gulati, 1990; Elwan and

Soliman, 1995; Medina and Reiner, 1995).

Chapter 1 Introduction

1.5. Animal models for drug tolerance and dependence

Various animals are used as models to assess the effects of abuse liable drugs, drugs producing antinociceptive effects, safety, therapeutic window and pharmacodynamic studies of newer prospective molecules.

1.5.1. Animal as models of disease

As ethically humans cannot be used as first hand experimental model for studying various diseases, and their treatment probabilities through new legend synthetic or herbal molecules, animals are used as models to study pathology, prognosis, physiology and responses of newer molecules as treatment options (Mitruka et al.,

1976). Since the advent of eighteen century animals models were inducted to assess newer potential legend molecules for prevention and treatment of various diseases.

Furthermore attempts were made to induce pathologies with similarity to human diseases to better understand molecular level prognosis and treatment options

(Nomura, 1997). In nineteen century this animal usage underwent a real boom and newer models were screened and approved to study diverse pathological disorders

(Mitruka et al., 1976).

An animal models is selected based on its capability that a spontaneous or induce behavior, normative biology, or pathology can be studied and have both behavioral, neuro histochemical resemblance of the pathology, signs and progression of the disease comparable to humans. The induced pathology or behavior in animals must have similarity factor in common with human diseases. Whether homologous or isomorphic the model is considered suitable based on the best model is characterized

Chapter 1 Introduction as affordable, easy to breed, and similarity with human subjects (Mitruka et al., 1976;

Nomura, 1997).

Dr Claude Bernard and Dr Robert Koch a Physiologist made tremendous contribution by publishing extensively on animal models for study of organs physiology and pathologies and are respected and acknowledged for their contribution across the globe in scientific communities (Emmett-Oglesby et al.,

1990; Gardner, 2000).

The emergence of genetically modified models brought an unprecedented revolution in the pursuit of exploring and understanding of human pathologies and organ physiologies and molecular level mechanisms of actions of drugs (Mitruka et al.,

1976; Nomura, 1997).

Nowadays all potential legend molecules as prospective candidates needs passing preclinical tests in various approved preclinical animal models with approved standards both qualitatively and quantitatively (McBride and Li, 1998).

Animal models offer many advantages over other biochemical approaches like isolated tissues, cell lines, or enzymes, organites or receptors that animals offer a homologous intact system offering systemic integrity for drugs, pathologies or physiological studies (McBride and Li, 1998; Medina and Reiner, 1995; Mitruka et al., 1976; Nomura, 1997).

One primary criterion for an animal to be accepted as animal model of disease is that it should be isomorphic or homologous. An animal model is declared as Isomorphic when it exhibits holistically or in part the clinical picture (behavioral, neurochemical, anatomo pathological or electrophysiological) observed in human disease, even

Chapter 1 Introduction though the cause of pathophsiology, behavior or biochemical change may be different in animals than humans (McBride and Li, 1998; Medina and Reiner, 1995;

Mitruka et al., 1976; Nomura, 1997).

While in homologous system both cause and induced behavioral, neurochemical, biochemical and anatomopathological changes are same in both humans and in animal models. The homologous models are more preferred and authentic for pre clinical assessment of new legend molecules or therapies (Emmett-Oglesby et al.,

1990; Gardner, 2000).

Nowadays many national and 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 for publication experimental data, 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).

1.5.2. Animals models for morphine tolerance and dependence

As animals are declared as indispensable means of analyzing drug dependence, prognosis, etiology and treatment alternatives through various therapeutic approaches. In study of opiates dependence various animal models are used based on requirement of study, homology and isomorphism of the model and affordability of the animals. Animals are extensively used in studying substrates of drug dependence, behavioral and neurochemical modulation and site of action of the proposed treatment molecules (Emmett-Oglesby et al., 1990; Markou et al., 1993).

Chapter 1 Introduction

Although we select an animal model as a homologous or isomorphic based on degree of similarity in induction and behavioral dependence paradigms, despite the fact that human psychology is highly complex and delicate and animals dependence never holistically portray human dependence and tolerance picture.

But to date scientific data reveals that rodents are best models to study various paradigms of morphine dependence. Many models have been used and tried for various paradigms and aspects of analyzing morphine dependence and tolerance but rodents specially mice and rats offer many advantages over higher animals like monkey in studying and conducting research in morphine dependence (Gardner,

2000).

1.5.2.1 Mice

Mice are extensively used animal models for morphine, analgesia, tolerance, dependence, withdrawal, receptor density studies like up or down regulation. Since the development of genetically modified mice as animal models is the choice of researchers in drug dependence studies. Mice are good models for studying drug induced stereotype drug discrimination behaviors e.g. Studies, like conditioned place preference, conditioned place aversion, drug self administration, drug discrimination and subsequent histochemcial analysis of central reward pathways like frontal cortex, striatum and nucleus accumbens etc. Since the advent of microdialysis studies, they are still one of the fore most choices of researchers based on its affordability, ease to breed nature and availability. The mice are considered as best model to study conditioned place preference and withdrawal behavior. Additionally morphine induces same neurotransmitters modulation in mice reward related discreet brain areas as found in humans (Frenois et al., 2002). Additionally mice withdrawal

Chapter 1 Introduction behaviors, tolerance to analgesia, drug seeking behavior have strong and distinct behavioral similarities with human subjects. Since the development of highly sensitive electrochemical analytical techniques for the measurements of neurotransmitters, scientists are expanding the use of mice as morphine dependence model (Gardner, 2000).

1.5.2.2. Rats

Rats were the first among the rodents established as morphine addiction models. As they are larger in size than mice, their brains comparably bigger than mice and their drug seeking behavior, withdrawal, and neurochemical picture was more prominent they were among the most widely used model as compared to mice (Espejo et al.,

1995).

Rats have a well established thoroughly studied drug discrimination, and withdrawal behavior and are extensively used by laboratories for studying morphine induced conditioned place preference, avoidance, drug self administration, drug seeking behavior and withdrawal.

Rats are one of the most widely used model to study drug self administration. Rats provide an affordable and reliable model to study loss of control over drug use and to study compulsive drug taking behavior (Espejo et al., 1995). Rats are also extensively used to study opioid receptors, down or up regulation through western blotting and other techniques. Rats being affordable easy to breed and availability of genetically modified models are best choice for molecular level studies in morphine dependence (Emmett-Oglesby et al., 1990; Gardner, 2000).

Chapter 1 Introduction

1.6. Current therapeutic options for the treatment of morphine

dependence

As the management of drug dependence is a highly complex and multifaceted overarching medical challenge, which is ever expanding in both its size and socioeconomic impact, afflicting all segments of society. Medical community although primarily focuses on pharmacological interventions for the management of opioid dependence, but its genetic and psychosocial element can neither be ignored nor undermined. There are group of clinicians that advocates only psychosocial solution as fundamental approach to the management of this issue.

There are multiple treatment approaches with variable treatment outcomes, strengths and weaknesses, depending on nature of pharmacotherapy, psychosocial factors and length of the treatments. Treatment strategies of lactating opiates or with other psychopathological problems further complicates the treatment and lowers therapeutic success. Generally the following approaches are adopted with variable treatment outcomes.

1.6.1. Detoxification

The objective of detoxification is to remove the opiate in controlled, professional and a humane and properly tailored way to ensure maximum treatment retention and minimum discomfort (Amato et al., 2003, 2004). As opiates overdose is the major cause of death among opiate dependent individuals, only detoxification can help avoid this fatal outcome of dependence. Detoxification is associated with strong psychosomatic signs and symptoms which include diarrhea, chills, sweating, anxiety, and irritability, in varying magnitude. These signs of withdrawal are so distressful for

Chapter 1 Introduction the addicts and are one major cause of relapse and therapeutic failure in opioid dependence treatment. The detoxification is not fatal, but its psychosomatic symptoms are really discouraging and frightful for the addicts. (Gowing et al., 2008).

Although the detoxification is deemed as sole therapy in opioid dependence management but clinically its value is less as management of the stress and depression that follows this phase of detoxification is the parameter that that determines the success of the therapy (van den Brink and Haasen, 2006b).

Detoxification is the primary and initial step towards dependence treatment, and detoxification can be achieved as abrupt detoxification, or tapering technique.

1.6.2. Abrupt detoxification

As name indicates, this approach uses immediate cessation of opiates with some antagonist like naloxone and concomitant use of alpha2 adrenergic agonist to minimize intensity of withdrawal syndrome precipitated by opioid antagonist. This approach has the advantage that although it’s more distressing to patient but has been found to be shorter in getting detoxification. Although addicts experience side effects of alpha2 adrenergic agonists, like sedation, hypotension, etc. Some clinicians also consider addition of anesthesia to help relieve addicts experience less pain of withdrawal, although major challenge of respiratory and cardiac arrest discourages this approach (Gowing et al., 2006a, b; Strain and Stitzer, 2005).

The use of naloxone in various studies is not significantly higher than placebo, although strategies of addition of buprenorphine enhances therapy success rates and facilitates detoxification phase more bearable for the patient. Due to higher degree of emotional distress, anxiety, current literature stress for the management and control of negative emotional behaviors during withdrawal phase. 22

Chapter 1 Introduction

1.6.2.1. Tapering technique

In this approach, the dose of opioid is gradually tapered as per a systemic protocol and methadone is added as replacement and over a specified time space till the complete replacement is achieved. This approach has more patient satisfaction although treatment with methadone is costly even in advanced countries (Amato et al., 2003).

During tapering process methadone is the drug of choice and most extensively used replacement therapy. Although it’s full opioid agonist, still counted and trusted as first line therapy. Methadone has been rated as first line therapy with higher and longer retention time (Elkader and Sproule, 2005; Zweben Je Fau - Payte and Payte,

1990).

Despite extensive utility methadone has abuse potential and entails risk of dependence, overdose induced side effects like respiratory depression, etc (Elkader and Sproule, 2005; Krabbe et al., 2003).

Additional psychosocial support from community, friends, and family, is needed to achieve high degree of compliance, therapy completion, low chances of relapse and higher success rate of treatment outcomes. A partial agonist buprenorphine is another option because it’s rather safe from respiratory depression (Gowing et al., 2006a). It has also been found to be superior to placebo and naltrexone in randomized clinical trials (Gowing et al., 2006a). Although its treatment cost is higher than methadone, but still offer a viable option in patients where higher doses of methadone may not be administered.

Chapter 1 Introduction

Levo-α-acetylmethadol (LAAM) is another full opioid agonist that has greater capacity to suppress heroin seeking behavior than methadone, but incidence of side effects and tolerability issues narrows its use spectrum among clinicians treating opioid addicts. Another other draw-back associated with LAAM, is “torsade de pointes,” a fatal ventricular disorder mainly attributed as LAAM side effect.

However in a recent randomized clinical trial LAAM has been found more efficacious as compared to methadone (Gonzalez et al., 2002; Michels et al., 2007).

Additionally in China Chinese herbal medicine are extensively used as monotherapy

(One herb) or combination (Polytherapy) for the management of opioid withdrawal.

A Meta analysis comparing Chinese herbal therapies with conventional therapies like

α2 adrenergic agonists and full opioid agonist like methadone and LAAM (Michels et al., 2007). The findings of the data reflect that herbal therapies are equally effective, more tolerable, with higher efficacy in relieving psychosomatic signs of withdrawal with comparable affordability, least dropping from treatment because of withdrawal distress (Guo et al., 1995; Hao and Zhao, 2000; Kang et al., 2008; Shi, 2006; Shi et al., 2008) .

As opioid dependence issue is multifaceted so in its management therapeutic and psychosocial aspects needs complete and holistic management.

1.7. Bacopa monnieri

Bacopa monnieri (BM), locally known as Jal Neem bootee and Brahmi in Pakistan 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). This plant belongs to Scrophulariaceae family, has been

Chapter 1 Introduction reported to have 220 genera and 3000 species. Bacopa Monnieri has a century’s old clinical utility in Ayurvedic system of medicine for various mental diseases, like anxiety, epilepsy, insomnia, diuretic and cardiac and nervous tonic. Bacopa monnieri major ayurvedic indication is for the management of poor cognition, and lack of concentration, as nootropic and gastric aid. Bacopa Monnieri has a folkloric utility to improve memory, respiration and learning. Bacopa Monnieri has many active moieties including alkaloids, sterols and saponins. Alkaloids mainly extracted include Brahmine, nicotine and Herpestine. The major bioactive component of

Bacopa Monnieri, 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 components, i.e. Bacoside II, Bacopaside A3, Bacopasaponin C and an isomer of

Bacopasaponin C (Deepak et al., 2005). Additionally currently new saponins

Bacopaside 1, II, III, VI and V have been isolated.

Chapter 1 Introduction

Figure 1.1. Bacopa monnieri Islamabad

Binomial Name: Bacopa monnieri Local Name : Jalneem Booti (Qureshi and Raza Bhatti, 2008) Order: Lamiales Family: Scrophulariaceae Genus: Bacopa Species: Bacopa monnieri Class: Magnoliopsida Division: Magnoliophyta Kingdom: Plantae Part used: Aerial parts and whole plant Synonym(S): Herpestis monniera, Bramia monnieri, Gladiola monnieri, Bramia monniera, Lysimachia monnieri, Septas repens Other Species: Bacopa caroliniana, Bacopa aquatica , Bacopa myriophylloides , Bacopa laxiflora, Bacopa madagascariensis, Bacopa repens,

Chapter 1 Introduction

Bacopa diffuses, Bacopa crenata, Bacopa laxiflora, Bacopa australis

Figure 1.2. Bacoside A3

Chapter 1 Introduction

Figure 1.3. Bacopasaponin C

Figure 1.4. Bacopaside II

Chapter 1 Introduction

1.7.1. Traditional uses of Bacopa monnieri (BM)

Bacopa Monnieri a creeping herb found across sub continent in shady wet places and fresh water streams. BM has century’s old clinical image as medhyarasyana (drug to improve intellect) and utility in ayurvedic system of medicine for various nervous system disorders, like epilepsy, depression, insomnia, poor cognition, hysteria, obsessive compulsive disorder and panic attacks (Russo and Borrelli, 2005). In ayurvedic it has always been used for anxiety, poor concentration, diuretic, memory booster and energizer for heart and brain. Apart from being a nerve tonic, BM has also been used as antipyretic, digestive aid and to treat insanity and asthma. BM has also been used as astringent and cardiotonic (Gohil and Patel, 2010). The roots of

BM are also used as diuretic and kidney tonic in folkloric therapy in subcontinent

(Gohil and Patel, 2010). During the last few decades BM has been extensively screened preclinically for various pharmacological activities (Russo and Borrelli,

2005).

1.7.2. Memory enhancer

BM has extensively been studied clinically in various age group individuals as herbal therapy for poor cognition, to boost memory and cognition defects. In a double blind placebo controlled trial in seventy six healthy volunteers BM was found to significantly delay forgetfulness of newly acquired information and improve cognition (Nathan et al., 2001). In another double blind 12 weeks trial single dose of

300 mg BM was found to significantly improve memory consolidation, verbal memory improvement and speed of information processing (Roodenrys et al., 2002).

BM has been screened in children with attention deficit hyperactivity disorder, and has been found to be significantly effective and tolerable (Negi et al., 2000). BM has

Chapter 1 Introduction also been used and found safe, effective and well tolerated in elderly anxious and depressed patients (Calabrese et al., 2008).

BM primary image in ayurvedic system has been as nootropic drug, which protects against loss of memory and improves both memory and cognition. BM has been found to improve stress; drug induced memory impairments and improves memory, information consolidation (Bhattacharya et al., 1999; Holcomb et al., 2006; Uabundit et al., 2010). BM has been reported to delay onset and progression of Alzheimer disease in experimental models most probably through its strong antioxidant effect

(Dhanasekaran et al., 2007; Prabhakar et al., 2008). There are strong evidences that

BM modulates cholinergic function significantly (Saraf et al., 2010) and might have a plausible role in improving cognition. BM has been reported to have protectant effect against drug induced, neurotoxin induced and immobilization stress induced memory impairments (Singh et al., 2010). BM has been found to improve memory by helping information retrieval, logical memory, and paired association learning in normal doses and has been found to be safe and tolerable in old age people also

(Jyoti et al., 2007; Saraf et al., 2011).

1.7.3. Tranquilizing, antidepressant and sedative effects

Bacopa Monnieri has been found to have a profound sedative and tranquilizing effect as outlined by Chowdhuri and co-workers in animals (Chowdhuri et al., 2002). The tranquilizing effect in preclinical models has also been reported by Achliya,et al

(Achliya et al., 2004). The hydroethanolic and aqueous extracts both have been reported to exert sedative effect in animals models (Malhotra and Das, 1959). Bacopa monnieri anxiolytic effects dates back to its centuries old clinical utility as anxiolytic

(Russo and Borrelli, 2005). In various preclinical studies, role and efficacy of BM as 30

Chapter 1 Introduction anxiolytic has been reported (Sairam et al., 2002; Zhou et al., 2007b). In another study

BM anxiolytic effect was compared with Lorazepam and was found to be equally effective with an added benefit of tolerability and improving memory and cognition

(Bhattacharya et al., 1999). BM antidepressant effect was evaluated in comparison to imipramine and was found to be equally effective at dose of 20-40 mg/kg per day

(Gohil and Patel, 2010; Shen et al., 2009; Zhou et al., 2007a). BM has been reported to have a strong adaptogenic effect in both acute and unpredictable stress in animal models also. In this respect BM has been reported to correct acute and chronic unpredictable stress induced modulation of biogenic amines in rats and normalize stress induced cortisol upsurge (Sheikh et al., 2007).

1.7.4. Antiepileptic effects

BM has a renowned folkloric image and an extended clinical usage as antiepileptic since time immemorial (Shanmugasundaram et al., 1991). BM has been reported to offer protection against seizures at higher doses in animal models . In another study

BM higher doses were found to have anticonvulsant effect when administered chronically while acute dosing did not exhibit anticonvulsant effect in animal models

(Ganguly and Malhotra, 1969). BM protectant role against epilepsy has been reported in association with a specified gene expression in hippocampus, in animals studies

(Paulose et al., 2008). BM has also been found effective in preventing pilocarpine induced epilepsy in animal models (Khan et al., 2008). Moreover the protectant role of BM in epilepsy in animals has been confirmed in recent findings by Dr Mathew and co-workers (Mathew et al., 2010). Currently mechanism elucidation of BM role in epilepsy is under investigation and BM has been reported to modulate epileptic

Chapter 1 Introduction behavior through its down regulation of 5-HT2C receptors in rat brain (Krishnakumar et al., 2009).

1.7.5. Anti inflammatory and antinociceptive effects

BM has been reported to have naloxone reversible antinociceptive effect and the effects are opioidergic in nature as its antinociception was found to be reversed by naloxone (Subhan et al., 2010a). Apart from its central effects BM also induces peripheral analgesia, by inhibition of pro inflammatory mediators, and so prevents inflammation (Pawar et al., 2007; Viji and Helen, 2008; Viji et al., 2011; Viji et al.,

2010; Vohora et al., 1997).

1.7.6. Effects on GIT

BM has been reported to lower gastrointestinal tract (GIT) motility (Subhan et al.,

2010b) and this motility blockage was reversed by picrotoxin reflecting the presence of GABA neurotransmitters in the mediation of anti GIT motility effects of BM

(Subhan et al., 2010b). BM has been reported to have relaxant effect on GIT via calcium channel inhibition, although BM failed to modulate intracellular calcium

(Channa et al., 2003; Dar and Channa, 1997, 1999). BM has also protectant and curative effects against gastric ulcer, having multi facet effects in ulcer therapy (Goel et al., 2003; Gohil and Patel, 2010; Sairam et al., 2001).

1.7.7. Antioxidant and adaptogenic effect

BM has been reported to have a strong antioxidant effect, and animals’ data reflects the protective effect of BM against strong oxidative stress homeostasis modulators, like morphine, nicotine, aluminum, and anticancer drugs. Literature search reveals that BM mediates antioxidant effect via some key enzymes (Gohil and Patel, 2010).

Chapter 1 Introduction

1.8. Aims and objectives of the study

Keeping in view the pharmacological profile of BM, and its folkloric use this behavioral and neurochemical study was designed,

1. To quantify Bacoside A major components, i.e. Bacoside A3, Bacopaside ll and Bacopasaponin C in methanolic and n-butanol fraction of BM using high performance liquid chromatography with UV.

2. To develop methodology for simultaneous determination of neurotransmitters i.e. noradrenaline, dopamine, serotonin, 5-hydroxyindolacetic acid, homovanillic acid and dihydroxyphenylacetic acid using High Performance Liquid

Chromatography with electrochemical detector.

3. To evaluate the effect of acute and sub chronic administration of Bacopa monnieri on dopamine and serotonin turn over in mice whole brain.

4. To test methanolic and n-butanol fraction of Bacopa monnieri for its impact on morphine analgesia and expression and acquisition of morphine tolerance.

5. To assess the effect of Bacopa monnieri on morphine induced locomotor hyperactivity and its impact on dopamine and serotonin turn over in mice striatum.

6. To evaluate and examine the effect of Bacopa Monnieri on behavioral signs of opioid withdrawal and detoxifying potential, in animal models of morphine withdrawal syndrome.

7. To explore the behavioral and neurochemical basis of Bacopa monnieri in naloxone precipitated morphine withdrawal in animal models by measuring neurotransmitters implicated in the development of dependence and withdrawal.

Chapter 2 Methodology

Chapter 2

Methodology

Chapter 2 Methodology

2.1. Chemicals and Reagents

Neurotransmitters including Dopamine (DA), 3, 4-Dihydroxyphenylacetic acid

(DOPAC), Homovanillic Acid (HVA), 5-hydroxytryptamine, (5-HT) and 5-

Hydroxyindolacetic acid (5HIAA) were acquired from Acros Organics local distributor while Noradrenaline (NA) was supplied by local distributor for Alfa Aesar.

While HPLC grade Acetonitrile (Fischer Scientific) methanol (Fischer Scientific),

Perchloric acid, 1-Octane Sulfonic Acid sodium salt (Fischer Scientific),

Ethylenediaaminetetraacetic acid (Fischer Scientific), Citric Acid (Fischer Scientific), sodium Dihydrogen orthophosphate mono sodium Di Hydrate (Fischer Scientific),

Ascorbic Acid (Sigma Aldrich) and naloxone hydrochloride (Sigma Aldrich) were supplied by Sigma Local distributor. Deionized water was double distilled (JK-SSD-

20 Stainless Steel Distiller, china) and filtered through 0.45 µ filter (Sartolon

Polyamind Sartorius Germany). Commercial grade solvents, like methanol, n-butanol, n-hexane and acetone for extraction were purchased from Haq Chemicals Limited

Peshawar. Bacoside A3, Bacopaside II and Bacopasaponin C were gifted by Prof Dr

Ikhlas.A.Khan, School of Pharmacy, Mississippi University USA. Morphine Sulphate

(Germany) was generously gifted through proper channel by M/S Punjab Drug House laboratories Lahore.

Chapter 2 Methodology

2.2. Instruments & Apparatus

Table 2.1.

S.N0 Description Source 1 Analytical Balance AX 200 Shimadzu, Japan

2 Vacuum Filtration Assembly Boeco, Germany 3 Vacuum Pump Roeker 300, Taiwan Pakland scientific, 4 Vortex mixer, Gyromixer Pakistan High performance liquid chromatography (HPLC) system, including 5 CBM (communication boss module)-20A Double Pumps-LC-20AT Injection port, 7725i (Rheodyne, USA) Shimadzu Japan ECD detector, Coulchem 111, model 5300, (ESA USA) along with dual analytical cells, model 5011 (ESA USA) and UV detector, SPD-20A (Shimadzu, Japan) 6 Micropipette (10-200µL) Treff Lab, France JENWAY, United 7 pH meter, 3505 Kingdom Pakland scientific, 8 Vortex mixer, Gyromixer Pakistan Column, Peurospher Star RP.C18e, HibarR RT 250- Merck, Germany 9 4.6(5µM) 10 Column MD_150; (3mm x 150 mm, 3µm) ESA,U.K Shim Pack C column ( 250 x 4.6 mm, 5 µm particle 11 18 Japan size) University of Peshawar. 12 Soxhlet Apparatus Pakistan 13 Refrigerator (-80 oC ) IlShin, DF 8517, Korea

14 Refrigerator PEL Co. Pakistan

15 Boeco Rotary Evaporator 400 SD BOECO Germany

Havard apparatus 16 Hot Plate Apparatus Germany

2.3. Plant collection, extraction and fractionation

Bacopa monnieri was collected from Rumli stream near Quaid-e-Azam University

(Islamabad) in April 2007. The plant was authenticated by Prof Dr Muhammad Ibrar

Chapter 2 Methodology and a voucher no.2147 was acquired upon deposition of plant sample in herbarium of department of Botany, University of Peshawar, Pakistan. Aerial parts of the plant were separated, washed; shades dried and then coarsely grinded

2.3.1. Preparation of methanolic extract

The shade dried, coarsely grinded plant material weighing 8.00 kg was taken and was first treated with n-hexane to defat it (yield 824 grams). Then this plant material was further treated with commercial grade acetone to remove chlorophyll type pigments

(yield 489 grams). Then this plant material was extracted with methanol in Soxhlet apparatus for twenty four hours. On next day, the extract was filtered through muslin cloth and then through filter paper and filtrate was collected. This procedure of methanolic extraction was repeated three times. Three extracts were mixed together and were concentrated at 50oC, under reduced pressure using rotary evaporator. The concentrated extract was then dried on water bath maintained at 50oC to a semisolid mass. The semisolid mass was transferred to a wide mouth bottle, sealed and stored in the refrigerator.

The percent yield of methanolic extract was thus calculated by following formula:

Weight of semisolid extract = W1 = 512 g

Weight of coarsely grinded powder of aerial part of Bacopa monnieri taken for extraction = W2 = 8000 g

Percentage yield = W1/W2 x 100 = 512/8000 x 100

Percentage yield = 6.5 %

2.3.2. Preparation of n-butanol fraction

The 100 g of semisolid methanolic extract (representing 1.65 kg of dried aerial parts) of Bacopa monnieri was dissolved in 800 ml methanol and was then filtered. The

Chapter 2 Methodology filtrate was then transferred to separating funnel and 500 ml acetone was added successively three times to the separating funnel, and was rigorously shaken, to help precipitate Bacosides. This precipitate was then left for drying and the fried precipitete was dissolved in distilled water and transferred to a separating funnel. To this separating funnel 700 ml of n-butanol was transferred separating funnel was again rigorously shaken to help transfer Bacoside from aqueous to organic phase. This process was repeated thrice. The n-butanol fraction phase was collected and dried under reduced pressure to convert it into semisolid mass.

Weight of semisolid extract of n-butanol fraction= 500 mg

Percentage yield methanolic extract = Weight of n-butanol fraction /Weight of methanolic extract x 100 = 0.5/100x100

Percentage yield from methanolic extract =0.5%

Percentage yield from dried aerial parts of BM = Weight of n-butanol fraction

/Weight of dried aerial powder x 100 = 0.5/1650x100

Percentage yield from dried aerial parts of BM = 0.0303 %

2.4. Standardization of selected plant extracts for Bacopasides

The contents of Bacopaside ll, Bacoside A3, and Bacosaponin C were quantified in both methanolic and n-butanol fraction using High performance Liquid

Chromatography (HPLC) with UV using Phrompitayyarat method with some modifications in the choice of column and mobile phase composition

(Phrompittayarat et al., 2007) .

Chapter 2 Methodology

2.4.1. High performance liquid chromatography (HPLC) system

The HPLC system consisted 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 software

Schimadzu software LC Solution Version 1.2 for data analysis.

2.4.1.1. Preparation of standards

Standard solutions of all three Bacosides were prepared by dissolving 2 mg/ mL of

HPLC standards of Bacopaside ll, 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 nano grams per mL.

2.4.1.2. Sample preparation

Briefly, 50 mg of methanolic extract of BM was dissolved in 10 mL methanol (HPLC

Grade) and was then centrifuged for ten minutes at 3000 rpm. Then this solution was filtered through 0.45 u filter, and was injected directly into HPLC system.

As n-butanol fraction is rich in Bacosides, its much diluted sample was injected in

HPLC system. In case of n-butanol fraction 10 mg of the extract was dissolved in 10 ml HPLC grade methanol, and was centrifuged and filtered as described in methanolic extract sample preparation. From this filtered solution, 20 µL was taken and diluted with HPLC grade methanol to make up the final volume up to 10 mL. After mixing for five minutes at vortex mixer, this solution was injected into HPLC system for analysis

Chapter 2 Methodology

2.4.1.3. Chromatographic conditions

The acceptable separation was achieved by using Shim Pack C18 column (250 x 4.6 mm, 5 µm particle size). The mobile phase consisted of phosphoric acid 0.2% and acetonitrile (60:40 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 22 minutes run time. The peaks were first confirmed by spiking the samples with standards Bacosides.

2.4.1.4. Method validation

The above method was revalidated for linearity, specificity, accuracy and recovery.

2.5. Dose preparation of Bacopa monnieri extract

Preparations containing 80 mg n-butanol fraction extract dissolved in 10 ml normal saline were used in antinociceptive, locomotor, tolerance and opioid withdrawal experiments. The extracts were thoroughly sonicated and were stored in labeled containers in refrigerator till final analysis.

180 mg of methanolic extract was dissolved in 10 mL normal saline and sonication was carried out to help dissolve the extract and obtain uniform solution. This solution was used in locomotor, tolerance and opioid withdrawal experiments. Extracts were administered orally in all experiments except toxicity experiments where extracts were administered intraperitoneally.

2.6. Drug administration

Drugs were administered to animals through various routes based on procedural requirements of the experiments.

Chapter 2 Methodology

2.6.1. Drug administration

Bacopa monnieri extracts were administered orally to all animals during antinociceptive, tolerance, locomotor and opioid withdrawal experiments. The extracts dissolved in normal saline were administered by oral gavage method. The drugs were administered through feeding tubes No 4 (Mice) and No 6 (Rat), connected to 5cc syringe. Feeding tubes # 4 and # 6 was used for mice and rats respectively. Animals were held firmly by the loose skin of their neck and back with left hand animal tail firmly held between little and ring finger. The animals were held vertically; in such a way that tube was inserted from the side of the mouth, and gently passed on through the esophagus into the stomach. The plunger of the syringe was gently pressed to discharge the contents into the stomach. The oral administration in mice specifically needs high degree of dexterity and confidence.

2.6.2. Intraperitoneal administration

Morphine and normal saline were administered by intraperitoneal administration during experiments to both mice and rats. During toxicity studies Bacopa monnieri extract was administered by this route. During intraperitoneal administration drugs were directly administered in to the peritoneal space surrounding the abdominal organs, avoiding direct injection to the organs. The animals were held firmly from the loose skin behind the neck and holding the animal tail firmly secured in the little finger exposing the ventral side of the animal. The needle of the syringe was directly and gently inserted in the lower right quadrant of the abdomen. The plunger was gently pressed to discharge the contents in the animal peritoneal cavity. The procedure needs proper training and dexterity in mice handling especially as lack of experience may lead to unexpected animal distress or injury to the concerned person.

Chapter 2 Methodology

2.6.3. Subcutaneous administration

During some experimental procedures drugs were administered to into the subcutaneous tissues. During this procedure animal were held firmly on the table in such a way that the loose skin behind the neck is lift up with fingers and the drugs administered into the subcutaneous area through syringe. The plunger is gently pressed to discharge the drugs in the specified subcutaneous area.

2.7. Method development and validation of neurotransmitters analysis by HPLC

2.7.1. Sample handling

Animals were killed by decapicitaion and whole brain excised onto an ice chilled plate and specified brain areas like, striatum, hippocampus, nucleus accumbens or frontal cortex were separated and stored at -80 oC 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 eppendorfs tubes at - 80 oC 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.

2.7.2. Preparation of stock solution

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 - 80 oC in labeled glass containers properly covered to avoid exposure to light. For calibration purposes, known volumes of cold 0.2 % Perchloric

Acid (PCA) was added to stock solutions to prepare 1.0 µg mL -1 solution. These

Chapter 2 Methodology

1.0 µg mL -1 solutions were such diluted to give seven different calibrations between ranges of 200 Pico grams to 500 nano grams. The so prepared seven dilutions were used for preparation of calibration curves and intermittent spiking during analysis.

2.7.3. Sample preparation

For analysis, animal whole brain was excised and areas of interest separated, 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 g

/minute (4oC) (Centurion UK) for twenty minutes and filtered through a 0.45 micron filter. The samples obtained were injected directly into the HPLC system or stored in labeled eppendorfs tubes at -80 oC refrigeration facility.

2.7.4. Chromatography

The High Performance Liquid Chromatography (HPLC) (Shimadzu, Japan), consisted of Communication Bus Module (model 20 A), two independently working pumps (model LC-20AT), an analytical column MD_150; (3mm x 150 mm, 3µm), a

Rheodyne injector with 20 µL loop attached to an electrochemical detector (ESA

Choulchem III model 5300) equipped with an analytical cell (model 5011 A). The chromatographic data was analyzed using Schimadzu 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 3).

Chapter 2 Methodology

2.7.5. Method validation

The chromatographic method was validated for linearity, specificity, precision

(Intermediate precision and repeatability), robustness and reproducibility. To confirm method specificity and linearity all six neurotransmitters i.e. NA, DA, DOPAC, HVA,

5HT and 5HIAA, were dissolved in 0.2 % PCA and their peak separation and retention times were confirmed. After this 20 mg striatal tissue was extracted with

PCA and was assessed for contents of all six compounds. The same striatal tissue aliquot 100 μL was spiked with 100 μL of standard solution of all six compounds and peaks separation and retention time were noted.

Linearity of the method was determined by spiking blank (striatum 20 mg tissue) with three known concentration of all six compounds and analyzed in triplicates. From the chromatograms of the spiked analyses slope (m) intercept and correlation co efficient and standard error were determined.

To quantify percent recovery, blank samples were spiked with three known standard mixture concentration of analytes and extracted and analyzed in triplicate. Response ratios of indigenous analytes were subtracted from spiked responses and was divided by corresponding mixtures of analytes and multiplied by 100 to calculate percent recovery.

Method intermediate precision was confirmed by injecting three different spiked concentrations, at three different timings, i.e. 08:00, 15:00 and 22:00 hours for three days and response were expressed as mean and percent Residual Standard Deviation

(%RSD). Method sensitivity was assessed by quantifying Limit Of Detection (LOD) and Limit of Quantification (LOQ) based on signal to noise ratio (S/N). LOD was thrice the value of response to noise while LOQ reflected a response ten times higher than noise.

Chapter 2 Methodology

Individual and spiked samples stability was evaluated at 25 oC, -10 oC, and -54 oC with or without added antioxidant. Robustness of the method was evaluated by the deliberate changes in ambient temperature, modulation of aqueous and organic components of mobile phase and changes in mobile phase flow rate.

2.8. Acute toxicity test

Acute toxicity of methanolic and n-butanol extract was tested in mice. Methanolic extract was administered intraperitoneally to Balb C mice (n=8) of either sex weighing 23-28 grams in doses of 30, 40, 60, 80,100, 120, and 140 mg/kg. Likewise, n-butanol extract was administered to Balb C mice, weight ranges (23-28g) intraperitoneally in doses, of 20, 40, 60, 80, 100, and 120 mg/kg body weight .The no of animal death in twenty four hours post injection was noted.

2.9. Effect of acute and sub chronic treatment with Bacopa Monnieri on neurotransmitters

2.9.1. Acute treatment group

In these experiments mice (23-27g) groups were given, single dose of 10, 20, and 30 mg /kg of methanolic extract or 5, 10, and 15 mg/ kg of n-butanol extract orally.

Control group received normal saline. Animals were killed by cervical dislocation one hour after oral drug administration and whole brain was excised and stored at -80 0C till analysis of neurotransmitters.

2.9.1. Sub chronic treatment group

In these experiments, mice groups received single daily dose of 10, 20, and 30 mg/kg methanolic extract or 5, 10, and 15 mg/ kg of n-butanol fraction orally for seven days.

On day seven one hour after the dose administration all animals were killed by

Chapter 2 Methodology cervical dislocation, whole brain excised and stored at -80c for analysis. Control group received normal saline for seven days.

2.9.2. Sample preparation for neurotransmitters analysis

The whole brain was excised weighed, and processed for neurotransmitters analysis as described on Page 43.

2.10. Determination of antinociceptive activity (Hot Plate method)

Antinociceptive response of various doses of methanolic and n-butanol fraction was assessed by hot plate method using Harvard hot plate (Harvard apparatus U.K.) at 54

± 0.10C. Animals were acclimatized to the laboratory conditions for at least two hours before the start of experiments. All animals were screened for pre test latency before drug administration. Only those animals having a pre test latency of less than 15 seconds were included in the experiment. A cut off time of 30 seconds was set to avoid any injury to the animal. After 30 minutes of pre-testing, animals were administered with standard drug morphine sulphate, plant extract or saline and were tested for latency on hot plate maintained at 54 ± 0.10C at 30 and 60 minutes after drug administration (Eddy and Leimbach, 1953). Percent Analgesia was calculated with the help of following formula:

Percent Analgesia = (Test Latency-Control latency) / (Cut-off time- control latency) x100

2.10.1. Induction and evaluation of morphine tolerance

Tolerance to morphine analgesia was produced in animals using a five day schedule of 20 mg/kg morphine sulphate twice daily injection. Antinociceptive response of all groups was assessed at 30 minutes and 60 minutes, on day one and day six by hot

Chapter 2 Methodology plate method using 10 mg/kg morphine sulphate intraperitoneally. Control group received normal saline intraperitoneally.

2.10.2. Assessment of effect of Bacopa monnieri on development of morphine tolerance To assess the effect of Bacopa monnieri on induction of morphine tolerance, animals

(n=8) received single dose of n Bt-ext BM (5, 10, or 15 mg/kg orally), Mt-ext BM

(10, 20, and 30 mg/kg orally) or saline daily along with morphine sulphate 20 mg/kg twice daily injection of for five days. Antinociceptive response to 10 mg/kg of morphine sulphate was tested on hot plate as described earlier on day one and day six.

Control group received normal saline along morphine tolerance induction schedule.

2.10.3. Assessment of effect of Bacopa monnieri on expression of morphine tolerance

Animals (n=8) received 20 mg/kg twice daily morphine sulphate for five days and on day six separate groups received a single dose of saline or n Bt-ext BM (5, 10, or 15 mg/kg orally), Mt-ext BM (10, 20, or 30 mg/kg orally) 60 minutes before 10mg/kg morphine dose. Control group received normal saline 30 minutes before morphine dose 10 mg/kg on day six. Antinociceptive response was tested at 30 minutes and 60 minutes interval.

2.10.4. Antinociceptive effect of Bacopa monnieri and its combination with morphine

Antinociceptive effect of n-Bt-ext BM (5, 10, or 15 mg/kg orally ), Mt-ext BM (10,

20, and 30 mg/kg orally) or saline alone or in combination with 10 mg/kg morphine was also tested on hot plate at 30 minutes and 60 minutes time post injection as described earlier.

Chapter 2 Methodology

2.10.5. Evaluation of tolerance to the antinociceptive effect of Bacopa monnieri

To assess the antinociceptive effect after repeated administration of Bacopa monnieri extracts, animals were administered twice daily, 15 mg/kg n-Bt-ext BM or 30 mg/kg

Mt-ext BM orally (maximum tolerable dose) for maximum period of time, i.e. seven consecutive days. Antinociceptive effect of the 15 mg/kg n Bt-ext BM and 30 mg/kg

Mt-ext BM was assessed on hot plate on day one and day eight at 60 minutes and 90 minutes interval. Control group received 15 mg/kg n Bt-ext BM or 30 mg/kg Mt-ext

BM on day one and day eight, and normal saline twice daily for seven days.

2.11. Measurement of locomotor activity

Locomotor activity was measured in a box measuring 50cm × 40cm × 44cm (length × width × height) with a smooth floor divided by lines into four equal rectangular zones.

Mice were acclimatized under red light (40 watt) to the laboratory conditions one hour before the start of experiments. Animals were administered saline, morphine (10 mg/kg) intraperitoneally, or plant extract orally. Locomotor activity evaluated as line- crossings was performed thirty minutes after intraperitoneal drug administration and sixty minutes after oral drug administration. Selected groups received doses of saline or plant extract sixty minutes before morphine (10mg/kg) dosing. Thirty minutes after intraperitoneal administration of morphine, or saline, the mice were placed in the activity box and group mean line-crossing counts were subsequently noted between 1 and 30 minutes.

2.11.1. Sample preparation for neurotransmitters analysis

The whole brain was excised and specified brain areas were separated, weighed, and processed for neurotransmitters analysis as described on Page 43.

Chapter 2 Methodology

2.12. Morphine dependence protocol

Morphine dependence was induced in rats, (200-250 grams) of either sex, using a well established eight days protocol of administering progressively higher doses of morphine sulphate (Subhan et al., 2009). Briefly rats received twice daily injection of morphine starting from 10 mg/kg twice daily on day one, 20 mg/kg twice daily on day two, 30 mg/kg twice daily on day three, 40 mg/kg twice daily on day four, 50 mg/kg twice daily on day five, 65 mg/kg twice daily on day six, day seven and day eight. On day nine all groups received last injection of 65mk/kg morphine sulphate. All groups

(n=6) received morphine as per dependence schedule except control group that received normal saline twice daily for eight days similar to morphine dependence schedule.

2.12.1. Dose preparation of morphine

Morphine sulphate 200 mg was dissolved in 10 ml normal saline and was administered intraperitoneally during dependence induction schedule.

2.12.2. Treatment groups

Saline control group received normal saline twice daily for eight days and on day ninth, received 0.2 mg/kg of naloxone hydrochloride, thirty minutes after normal saline injection. Morphine group received morphine sulphate dependence scheduled treatment for eight days and received naloxone hydrochloride 0.2 mg/kg subcutaneously on day nine thirty minutes after morphine sulphate dose 65 mg/kg. In acute groups (n=6) three separate groups received morphine sulphate as per dependence protocol, on day nine received Bacopa monnieri extract (methanolic/ n- butanol) in three doses per oral route sixty minutes before last morphine sulphate

Chapter 2 Methodology dose. In chronic treatment protocol separate groups received scheduled morphine sulphate dependence protocol.

All three chronic groups identically received daily a single dose of Bacopa monnieri

(methanolic/ n-butanol) per oral route daily sixty minutes before first dose of morphine sulphate. Naloxone hydrochloride was administered subcutaneously after thirty minutes of last morphine/saline dose on day ninth.

2.12.3. Evaluation of behavioral signs of naloxone precipitated withdrawal

After completion of morphine dependence induction protocol as outlined above, naloxone precipitated withdrawal was induced in animals by subcutaneous administration of naloxone 0.2 mg/kg and behavioral signs of opioid withdrawal were noted for next thirty minutes.

2.12.3.1. Weight loss

Animals were weighted before naloxone administration and were weighed thirty minutes after naloxone administration, and weight change noted as percent weight loss during opioid withdrawal.

2.12.3.2. Jumping behavior

The animals display a stereotype behavior of jumping during opioid withdrawal.

Numbers of jumps were noted during thirty minutes time after naloxone administration.

Chapter 2 Methodology

2.12.3.3. Wet dog shakes

Wet dog shakes is another sign displayed by animals during opioid withdrawal behavior. The number of wet dog shakes behavior is noted for thirty minutes after naloxone administration

2.12.3.4. Abdominal writhes

Animals display severe abdominal constriction called writhing behavior, in which the animal constricts its abdomen with simultaneous stretching of the hind limbs, during opioid withdrawal, which is a prominent sign of opioid withdrawal behavior

(Stevenson et al., 2006). Numbers of writhes were counted in each group for thirty minutes after naloxone dose.

2.12.3.5. Diarrhea

Diarrhea is another prominent sign of opioid withdrawal. No of times stool passed is noted in thirty minutes after naloxone administration. Diarrhea is ranked as mild, moderate, or severe. No of stools passed in 30 minutes if less than or equal to six is termed as mild, no of stools passed less than ten times in thirty minutes is termed as moderate and no of stools passed more than ten times per thirty minutes is termed as severe.

2.12.3.6. Squeal on touch

One of the most distinguishing characteristic of opioid withdrawal is squeal, (cry) of the animal upon touching which reflects animal’s dysphoria, hyperalgesia and distress. The animal is gently held and its squeal behavior was noted every five minutes till thirty minutes after naloxone injection. Again the squeal was subjectively rated as mild moderate or severe depending on animal behavior. And for statistical 51

Chapter 2 Methodology analysis of this behavior, squeal is subjectively quantified as mild, moderate, and severe, denoted as 5, 10, and 15 respectively.

2.12.3.7. Raring

Raring is another prominent behavior during opioid withdrawal. Raring behavior was noted as time spent while standing on back foot and leaning against the box walls.

The time spent was noted in minutes.

2.12.3.8. Salivation

Salivation is another important sign of morphine withdrawal syndrome. The salivation is observed and rated subjectively as mild moderate or severe during thirty minutes post naloxone injection

2.12.4. Sample preparation for neurotransmitters analysis

The whole brain was excised and specified brain areas were separated, weighed, and processed for neurotransmitters analysis as described on Page 43.

2.13. Ethical approval

Mice (Balb C) weighing 23-28 grams and Sprague Dawley rats (150-250 grams) of either sex, bred in the animal house facility, Department of Pharmacy, University of

Peshawar were used in the experiments. All procedures were approved by the ethical committee, Department of Pharmacy, University of Peshawar. Animals were kept at approved standards, of temperature 22±2 °C, with 12 h light/12 h dark cycle, with free access to food and water.

Chapter 2 Methodology

2.1. Chemicals and Reagents 35

2.2. Instruments & Apparatus 36

2.3. Plant collection, extraction and fractionation 36 2.3.1. Preparation of methanolic extract 37 2.3.2. Preparation of n-butanol fraction 37

2.4. Standardization of selected plant extracts for Bacopasides 38 2.4.1. High performance liquid chromatography (HPLC) system 39 2.4.1.1. Preparation of standards 39 2.4.1.2. Sample preparation 39 2.4.1.3. Chromatographic conditions 40 2.4.1.4. Method validation 40

2.5. Dose preparation of Bacopa monnieri extract 40

2.6. Drug administration 40 2.6.1. Drug administration 41 2.6.2. Intraperitoneal administration 41

2.7. Method development and validation of neurotransmitters analysis by HPLC 42 2.7.1. Sample handling 42 2.7.2. Preparation of stock solution 42 2.7.3. Sample preparation 43 2.7.4. Chromatography 43 2.7.5. Method validation 44

2.8. Acute toxicity test 45

2.9. Effect of acute and sub chronic treatment with Bacopa Monnieri on neurotransmitters 45 2.9.1. Acute treatment group 45 2.9.1. Sub chronic treatment group 45 2.9.2. Sample preparation for neurotransmitters analysis 46

2.10. Determination of antinociceptive activity (Hot Plate method) 46 2.10.1. Induction and evaluation of morphine tolerance 46 2.10.2. Assessment of effect of Bacopa monnieri on development of morphine tolerance 47 2.10.3. Assessment of effect of Bacopa monnieri on expression of morphine tolerance 47 2.10.4. Antinociceptive effect of Bacopa monnieri and its combination with morphine 47 2.10.5. Evaluation of tolerance to the antinociceptive effect of Bacopa monnieri 48

2.11. Measurement of locomotor activity 48 2.11.1. Sample preparation for neurotransmitters analysis 48

2.12. Morphine dependence protocol 49 2.12.1. Dose preparation of morphine 49 2.12.2. Treatment groups 49 53

Chapter 2 Methodology

2.12.3. Evaluation of behavioral signs of naloxone precipitated withdrawal 50 2.12.3.1. Weight loss 50 2.12.3.2. Jumping behavior 50 2.12.3.3. Wet dog shakes 51 2.12.3.4. Abdominal writhes 51 2.12.3.5. Diarrhea 51 2.12.3.6. Squeal on touch 51 2.12.3.7. Raring 52 2.12.3.8. Salivation 52 2.12.4. Sample preparation for neurotransmitters analysis 52

2.13. Ethical approval 52

Chapter 3 Chromatographic analysis of Bacopasides

Chapter 3 HPLC method revalidation and chromatographic analysis of

Bacopasides

53 Chapter 3 Chromatographic analysis of Bacopasides

3.1. Introduction

Bacopa monnieri a renowned ayurvedic plant has been extensively used for the treatment

of various neuropsychiatric disorders (Russo and Borrelli, 2005). The major bioactive

molecule that carries the neuropharmacological activities is Bacoside A, which is a

mixture of Bacoside A3, Bacopaside II, Bacopasaponin C and an isomer of

Bacopasaponin C (Deepak and Amit, 2004). Several methods have been reported for

simultaneous determination of Bacoside A using HPLC, but very few analytical methods

are reproducible (Deepak et al., 2005; Ganzera et al., 2004). We tried to quantify the

Bacoside A3, Bacopaside II and Bacopasaponin C using The Phrompittayarat et al

method (Phrompittayarat et al., 2007). However using the prescribed chromatographic

conditions outlined by Phrompittayarat et al the Bacoside A3 and Bacoside II could not be

properly separated. So the objective of this work was to revalidate Phrompittayarat et al

method for linearity, specificity, sensitivity, precision, recovery and stability of the

compounds under our chromatographic conditions.

3.2. Materials and methods

Materials and methods details are available on page 39-40, Methodology chapter 2.

3.3. Results

3.3.1. Chromatography

The acceptable separation was achieved by using Peurospher C18 column ( 250 x 4.6 mm,

5 µm particle size). The mobile phase consisted of phosphoric acid 0.2% and acetonitrile

(60:40 v/v), pH adjusted to 3.0 with 3 M NaOH. The HPLC system was run at 0.6 mL

54 Chapter 3 Chromatographic analysis of Bacopasides

/min flow rate using wavelength of 205 nm. All the peaks were secured in 22 minutes run

time. The peaks were first confirmed by spiking with standards Bacosides.

3.4. Method validation

3.4.1. Sample preparation

Samples were prepared by dissolving both standards, and extracts (methanolic and n-

Butanol) in HPLC grade methanol. The dissolved extracts were centrifuged at 3000 rpm

for ten minutes. The solution was filtered through 0.45 u filter to avoid column choking.

Additionally as n-Butanol fraction is rich in bacosides so 20 µL of n-Butanol fraction was

first diluted with 10 mL methanol (HPLC grade) to avoid column overloading and

subsequent deformed peaks.

3.4.2. Selectivity

Under the prescribed chromatographic conditions all three peaks were separated that

appeared in the following order, i.e. Bacoside A3 (18 min), Bacopaside II (19 min), and

Bacopasaponin C(22 min) as shown in Fig 3.1.

55 Chapter 3 Chromatographic analysis of Bacopasides

Figure 3.1. Chromatogram showing Bacoside A3, Bacopaside II, Bacopasaponin C standards 1.75µg/mL.

56 Chapter 3 Chromatographic analysis of Bacopasides

Figure 3.2. Chromatograms showing Bacoside A3, (A) Bacopaside II (B), Bacopasaponin C (C) in n-Butanol fraction of BM (1) and standards (2).

57 Chapter 3 Chromatographic analysis of Bacopasides

Figure 3.3. Chromatogram showing Bacoside A3 (A), Bacopaside II (B), Bacopasaponin C (C) in methanolic extract.

3.4.3. Recovery

The recovery of Bacoside A3, Bacopaside II, and Bacopasaponin C were quantified by adding three concentrations (1 µg, 2 µg, 3 µg) in 5 mg/ml methanolic extract, and the process was repeated in quintuplicate (5). The recovery of all compounds was between 98 to 101 percent (Table 3.1).

58 Chapter 3 Chromatographic analysis of Bacopasides

Table 3.1

Recovery of Bacoside A3, Bacopaside II, Bacopasaponin C from spiked methanolic extract (mean Recovered Conc±S.D) Conc Bacoside A3 Bacopasie II (µg/mL) Bacopasaponin C (µg/mL) µg/ml (µg/mL) 1 µg/ml 0.99±0.04 0.99±0.02 1.03±0.01

2 µg/ml 2.0±0.02 1.99±0.04 2.1±0.03

3 µg/ml 2.99±0.03 3.1±0.09 3.0±0.03

Recovery of Bacoside A3, Bacopaside II, Bacopasaponin C from spiked methanolic extract. Data shows mean conentration ±S.D (n=5).

3.4.4. Sensitivity

The method was validated for sensitivity also. Standard solutions (1 µg/mL) of all three

Bacosides i.e. Bacoside A3, Bacopaside II, Bacopasaponin C were serially diluted and

injected into HPLC system and signal to noise ratio equal to three was calculated. The

Limit of detection (LOD) for all three Bacosides was found as 350 ng and Limit of quantification (LOQ) was found as 1.1 µg/mL.

3.4.5. Precision

The precision both inter day and intraday was found in acceptable range as outlined in table 3.2.

59 Chapter 3 Chromatographic analysis of Bacopasides

Table 3.2

Precision and intra-day and inter-day data of Bacoside A3, Bacopaside II, Bacopasaponin C

Bacosides† Spiked Mean observed Inter Day Accuracy Mean observed Intra Accuracy Conc(µg/ml) Conc (n=5) % Conc Day(n=5) % %RSD %RSD Bacoside A3 1 1.10 1.06 110.00 1.00 1.40 100.00 2 2.10 0.90 105.00 2.20 4.50 110.00 3 3.10 1.20 103.33 3.00 2.10 100.00 Bacopaside II 1 0.98 1.40 98.00 0.96 3.40 96.00 2 1.99 0.70 99.50 2.00 5.10 100.00 3 3.10 0.80 103.33 2.97 2.10 99.00 Bacopasaponin C 1 1.10 1.20 110.00 1.10 4.50 110.00 2 2.00 0.400 100.00 2.00 2.00 100.00 3 3.10 1.8 103.33 3.10 1.10 103.33 Table showing inter day and intraday precision of Bacoside A3, Bacopaside II, Bacopasaponin C.†=(n= 6)

60 Chapter 3 Chromatographic analysis of Bacopasides

Table showing inter day and intraday precision of Bacoside A3, Bacopaside II, Bacopasaponin C.†=(n=5)

3.4.6. Linearity

All three Bacosides standards were dissolved in methanol ( HPLC Grade ) and calibration curve was drawn by injection of sample in the concentration range of 0.5 µg -2.0 µg to the HPLC system. Each strength of individual Bacoside was injected in triplicate. Calibration curve showed good linearity, with r2 ≤ 0.99.

Bacoside A3 200000

150000

y=83323x+120 100000 R2=0.998 Area

50000

0 0.0 0.5 1.0 1.5 2.0 2.5 Conc (ng/mL)

Figure 3.4. Calibration curve of Bacoside A3, in a range of 0.5 µg -2.0 µg

61 Chapter 3 Chromatographic analysis of Bacopasides

Bacopaside II 300000

200000

Area y=140711x+3530 100000 R2=0.997

0 0.0 0.5 1.0 1.5 2.0 2.5 Concentration (ng/mL)

Figure 3.5. Calibration curve of Bacopaside II, in a range of 0.5 µg -2.0 µg.

µg.

Bacopasaponin C 400000

300000

y=162728x-3321 200000 2 R =0.999 Area

100000

0 0.0 0.5 1.0 1.5 2.0 2.5 Conc (ng/mL)

Figure 3.6. Calibration curves Bacopasaponin C, range 0.5 µg -2.0 µg. .

62 Chapter 3 Chromatographic analysis of Bacopasides

3.4.7. Stability

All three Bacosides were found to be stable in methanol (HPLC grade) for one week at 25 oC, while standard solutions remained stable for one month both at -10 oC and 15 oC.

3.5. Discussion

As we were unable to get proper separation with mobile phase composition as outlined by

Phrompittayarat et al, (Phrompittayarat et al., 2007) The methods was modified by replacing column and increasing organic phase concentration from 35 % to 40 % and was revalidated for linearity, selectivity, sensitivity, specificity, stability and recovery. In the reported

Phrompittayarat et al, (Phrompittayarat et al., 2007) the bacosides A3 and Bacopsaide II could

not be separated, However by changing mobile phase composition and so mobile phase flow rate

the peaks were separated effectively. The modified method was used to quantify Bacoside A3,

Bacopaside II and Bacopasaponin C in both methanolic and in n-Butanol fraction successfully.

The method run time was 23 minutes and the compound eluted in the following order! Bacoside

A3, Bacopaside II, Bacopasaponin C. The n-Butanol fraction was very rich in Bacosides so 20

µL was diluted in 10 mL methanol and was then injected into HPLC system. Additionally

injection of n-Butanol fraction directly into HPLC system gave deformed peaks and strongly overloaded the column. The peaks were well resolved in C18, 5 µ, 250 mm length column as

compared to C18, 5 µ, and 150 mm column. The increase in organic portion of mobile phase by

2% decreased sensitivity, run time and selectivity, and likewise decrease in organic phase

concentration by 2% decreased selectivity and increased retention time. Lowering in pH lowered retention time but decreased selectivity, and increase in mobile phase pH increased both

retention time and subsequently run time.

3.1. Introduction 54

63 Chapter 3 Chromatographic analysis of Bacopasides

3.2. Materials and methods 54

3.3. Results 54 3.3.1. Chromatography 54

3.4. Method validation 55 3.4.1. Sample preparation 55 3.4.2. Selectivity 55 3.4.3. Recovery 58 3.4.4. Sensitivity 59 3.4.5. Precision 59 3.4.6. Linearity 61 3.4.7. Stability 63

3.5. Discussion 63

TABLE 3.1 59 TABLE 3.2 60

FIGURE 3.1. CHROMATOGRAM SHOWING BACOSIDE A3, BACOPASIDE II, BACOPASAPONIN C STANDARDS 1.75µG/ML. 56 FIGURE 3.2. CHROMATOGRAMS SHOWING BACOSIDE A3, (A) BACOPASIDE II (B), BACOPASAPONIN C (C) IN N- BUTANOL FRACTION OF BM (1) AND STANDARDS (2). 57

FIGURE 3.3. CHROMATOGRAM SHOWING BACOSIDE A3 (A), BACOPASIDE II (B), BACOPASAPONIN C (C) IN METHANOLIC EXTRACT. 58

FIGURE 3.4. CALIBRATION CURVE OF BACOSIDE A3, IN A RANGE OF 0.5 µG -2.0 µG 61 FIGURE 3.5. CALIBRATION CURVE OF BACOPASIDE II, IN A RANGE OF 0.5 µG -2.0 µG. 62 FIGURE 3.6. CALIBRATION CURVES BACOPASAPONIN C, RANGE 0.5 µG -2.0 µG. 62

64 Chapter 4 HPLC method development for neurotransmitters assay

Chapter 4

HPLC method development and validation for simultaneous determination of dopamine, serotonin and their metabolites with noradrenaline in rat brain tissues

64 Chapter 4 HPLC method development for neurotransmitters assay

4.1. Introduction.

In neuropsychiatric research, the simultaneous determination of catecholamines, indoleamines and their metabolites are inevitable to understand disease states and treatment strategies (Kobayashi, 2001; Lanteri et al., 2007). Biogenic amines are being measured by HPLC for the last four decades, using various detectors, analytical cells and mobile phases. Very few reproducible methods are available using HPLC with electrochemical detection (ECD) that analyses, DA, DOPAC, HVA, 5-HT,

5HIAA and NA simultaneousely using electrochemical system. Most of the available methods have rather long run times like more than 25 minutes at flow rate of 1 ml/ minute. Moreover available reported methods have either single electrode or use very advance dual electrode analytical cell system.

The aim of this study was to develop a rapid, sensitive, precise and specific method for simultaneous determination of DA, DOPAC, HVA, 5-HT, 5HIAA and NA in rat brain tissues in comparatiely short time.

4.2. Materials and methods.

The materials and methods are described in detail in Methodology chapter Page 42- 44.

4.3. Results

4.3.1. Chromatography Complete separation of all neurotransmitters with good resolution was achieved on an analytical column MD_150; (3mm x 150 mm, 3µm) using mobile phase, consisting

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 3). All neurotransmitters eluted within 8 minutes, in the following order, NA (2.5 minutes),

65 Chapter 4 HPLC method development for neurotransmitters assay

DOPAC (3.3) DA (4.1 minutes) 5HIAA (4.2 minutes) HVA (6.2) and 5-HT (7.5) minutes) Fig 4.1.

Figure 4.1. Chromatogram showing NA, DOPAC, DA, 5HIAA, HVA and 5-HT as peak A, B, C, D, E. respectively in 1 ng / mL standards sample.

66 Chapter 4 HPLC method development for neurotransmitters assay

Figure.4.2. Chromatogram showing, NA, DOPAC, DA, 5HIAA, HVA and 5-HT denoted as, peak A, B, C, D, E and F, respectively in saline treated mice striatum.

4.3.2. Selection of analytical cell potential Each and every neurotransmitter (NA, DOPAC, DA, HVA, 5-HT, 5HIAA) were injected separately and tested on various voltages of both channels starting from 50 mvolts, than 100 mvolts and with a gradual increase of 100 mv up to 1000 mv were screened and response recorded. An optimum potential setting for both the electrode was selected at +200 and −200 mV respectively, with a sensitivity of 2 µA. A representative Voltammogram is shown in Fig 4.3.

67 Chapter 4 HPLC method development for neurotransmitters assay

Figure. 4.3. Voltammogram of NA, DOPAC, DA, 5HIAA, HVA, 5-HT peaks shown in chromatograms. Chromatograms are denoted as A,B,C,D,E,F,G,H, with applied potential of 50(A), 100(B), 200(C), 300(D), 400(E), 500(F), 600 (G), 700 mV(H).

4.4. Method validation

4.4.1. Selectivity As shown in Fig, 4.1, 4.2, and 4.3, all peaks were separated and high selectivity was observed under prrescribed chromatographic conditions. Peaks were separated both in standard mixture and striatal tissue analysis, that appeared in the folowing order,i.e.

NA, DA, DOPAC, 5HIAA, HVA and 5-HT.

4.4.2. Recovery. The recovery of all six neurotransmitters (NA, DA, DOPAC, 5HIAA, HVA, 5-HT) were measured by spiking 10 mg striatal tissue with three concentration (1ng, 50 ng,

100 ng) of standard neurotransmitters (NA, DA, DOPAC, 5HIAA, HVA, 5-HT), and

68 Chapter 4 HPLC method development for neurotransmitters assay

the process repeated in quintuplicate (n=5) as shown in table 4.1. All compound

recovery was good and above 95% as shown in table 4.1.

Table 4.1

Recovery (ng/mL) of NA, DA, DOPAC, 5HIAA, HVA, 5-HT from spiked striatal tissues (mean Recovered Conc±S.D) Concentration NA DA DOPAC 5HIAA HVA 5-HT s 1 ng/ml 0.93±0.0 0.99±0.0 0.97±0.0 0.96±0.0 0.95±0.0 1.03±0.0 2 4 4 5 5 1 50 ng/mL 49.3±0.0 50±0.04 48±0.04 48.4±0.0 47.3±0.0 49.4±0.0 2 2 3 2 100ng/mL 99±0.03 101±0.03 98±0.09 99±.03 96±0.03 101±0.03 Recovery of NA, DOPAC, DA, 5HIAA, HVA, 5-HT from spiked samples. Data shows mean conentration ±S.D (n=5).

4.4.3. Sensitivity Sensitivity of the method was assessed by calculating signal to noise ratio at 3 for all

compunds termed as Limit of Detection (LOD) by simple dilution of the working

standards of all neurotransmitters NA, DOPAC, DA, 5HIAA, HVA, 5-HT,

respectively. Additionally Limit of Quantification was assessed as LOD X 10/3.

The LOD for NA, DOPAC, DA, 5HIAA, and 5-HT was found to be 11 pg/mL while

LOD for 5HIAA was found 19 pg/mL. The LOQ for NA, DOPAC, DA, 5HIAA, 5-

HT was found 40 pg/ mL, while LOQ for HVA was 65 pg/mL.

4.4.4. Linearity Each neurotransmitter (NA, DOPAC, DA, 5HIAA, HVA, 5-HT) was dissolved in 0.2

% PCA, and analyzed in range of 1-300 ng concentration range as shown in the

figures (4.4 to 4.9) with linear correlation value, r2=0.99 was obtained for all

individual neurotransmitters, reflecting good linearity .

69 Chapter 4 HPLC method development for neurotransmitters assay

DOPAC 40000000

30000000 102599x+90680 R2=0.999 20000000 Area

10000000

0 0 100 200 300 400 Conc (ng/mL)

Figure 4.4. Calibration curve of DOPAC, in a range of 1 ng to 300 ng. Standard was dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99. .

5HIAA 4000000

3000000

74108X-2404 2000000 2

Area R =0.999

1000000

0 0 100 200 300 400 Conc (ng/mL)

Figure 4.5. Calibration curve of 5HIAA, in a range of 1 ng to 300 ng. Standard was dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99.

70 Chapter 4 HPLC method development for neurotransmitters assay

HVA 800000.0

600000.0 2508X+452 400000.0 R2=0.999 Area

200000.0

0.0 0 100 200 300 400 Conc (ng/mL)

Figure 4.6. Calibration curve of HVA, in a range of 1 ng to 300 ng. Standard was dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99.

5-HT 20000000

15000000

10000000 y=52666x+49496 Area R2= 0.999

5000000

0 0 100 200 300 400 Conc (ng/mL)

Figure 4.7. Calibration curve of 5-HT, in a range of 1 ng to 300 ng. Standard were dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99.

71 Chapter 4 HPLC method development for neurotransmitters assay

1.01007

8000000.0 28525X+8480.9 6000000.0 R2=0.999

Area 4000000.0

2000000.0

0.0 0 100 200 300 400 Conc (ng/mL)

Figure 4.8. Calibration curve of DA, in a range of 1 ng to 300 ng. Standard was dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99.

NA 20000000

15000000 y=60664x+28813 R2=0.999 10000000 Area

5000000

0 0 100 200 300 400 Conc (ng/mL)

Figure 4.9. Calibration curve of NA, in a range of 1 ng to 300 ng. Standard was dissolved in 0.2% PCA and linear regression was obtained. r2 = 0.99.

72 Chapter 4 HPLC method development for neurotransmitters assay

4.4.5. Precision

Precision (Inter days ) and Intra day and Accuracy of the method (Table 4.2) conforms to the standards of precision as outlined by Épshtein (Épshtein, 2004).

73 Chapter 4 HPLC method development for neurotransmitters assay

Table 4.2 Effect of inter days and intraday on NA, DOPAC, DA, 5HIAA, HVA, 5-HT precision in spiked samples. (n=5)

Precision and intra-day and inter-day data of NA, DOPAC, DA, 5HIAA, HVA, 5-HT Inter Day Intra Spiked Mean observed Accuracy Mean observed Accuracy NEUROTRANSMITTERS (n=5) Day(n=5) Concentration(ng/ml) Concentration % Concentration % %RSD %RSD 10 10.1 1.06 101 10.2 1.4 102 NA 50 50.3 0.9 100.6 50.2 4.5 100.4 100 101 1.2 101 103 2.1 103 10 9.8 1.4 98 9.6 3.4 96 DOPAC 50 49.7 0.7 99.4 47.2 5.1 94.4 100 99.8 0.8 99.8 95.1 2.1 95.1 10 10.2 1.2 102 10.1 4.5 101 DA 50 50.3 0.4 100.6 50 2 100 100 100.1 1.8 100.1 100.2 3.1 100.2 10 9.8 1.2 98 10.2 6.1 102 5HIAA 50 50.3 0.9 100.6 50.1 3.3 100.2 100 100.1 0.8 100.1 99.8 1.3 99.8 10 9.6 1.4 96 9.5 2.1 95 HVA 50 49.7 1.2 99.4 48 2.3 96 100 99.8 0.9 99.8 97.1 4 97.1 10 10.3 0.9 103 10.3 5.1 103 5-HT 50 50.2 1.1 100.4 50.3 4.6 100.6 100 100.1 0.6 100.1 100 5.2 100

74 Chapter 4 HPLC method development for neurotransmitters assay

4.5. Stability Studies

All neurotransmitters (NA, DOPAC, DA, 5HIAA, HVA, 5-HT) were stable for 30 days at -54 oC and percent loss calculated was less than 3 %. All neurotransmitters were found to be stable at

-10 oC and remained within their limits (5%) for two weeks. At 25 oC all neurotransmitters were degraded above 20% in sixty minutes with DOPAC, and HVA, more rapidly and extensively degraded than the rest of neurotransmitters. Addition of 0.5 mM ascorbic acid improved neurotransmitters stability especially at room temperature, but addition of ascorbic acid made detection and quantification of NA peak impossible as addition of ascorbic acid increases the size of solvent front manifold making NA peak invisible (McKay et al., 1984).

75 Chapter 4 HPLC method development for neurotransmitters assay

Table 4.3 Stability of NA, DA, DOPAC, 5HIAA, HVA, 5-HT in PCA with, and without antioxidant o o o 25 C a -10 C b -54 C c Without Antioxidant Without Antioxidant Without Antioxidant Neurotransmitters.† Conc.(ng/mL) antioxidant Added* antioxidant Added* antioxidant Added* 1 77 97 99 99 100 99 NA 10 79 10 101 100 101 101 100 68 99 98 101 99 101 1 57 95 98 99 99 100 DOPAC 10 78 100 100 99 97 99 100 69 98 99 101 95 101 1 78 101 100 101 101 100 DA 10 80 99 101 99 101 99 100 79 95 100 100 100 100 1 79 94 98 96 101 99 5HIAA 10 80 94 99 102 102 101 100 76 98 100 101 99 103 1 55 91 98 101 98 99 HVA 10 73 93 97 99 99 101 100 69 93 96 94 94 97 1 69 100 99 101 101 99 5-HT 10 77 99 100 100 99 102 100 79 96 100 101 99 101

Stability studies of NA, DA, DOPAC, 5HIAA, HVA, 5-HT in PCA at room temperature (25 oC), -10 oC, and -54 oC with, and without antioxidant. a = 01 hours, b = one week, c =one month. Antioxidant added* 0.5 mM Ascorbic acid. †= (n=6)

76 Chapter 4 HPLC method development for neurotransmitters assay

The method was found to be highly reproducible in brain tissues analysis of rats used in

morphine withdrawal experiments. Moreover, slight intentional variation of mobile composition

(organic: aqueous) ratio changed both sensitivity, and selectivity of the method. The method was tried with C18 columns of 5 µ particle sizes. The mobile phase gave complete separation of all

neurotransmitters and their metabolites (NA, DA, DOPAC, 5HIAA, HVA, 5-HT ) in same order,

in C18 columns of 5 µ particle sizes with 150 mm length (ShimPack) and 250 mm length

(Peurospher) as evidenced in MD-150 column chromatograms. The overall run time with

ShimPack was found 17 minutes at 2.0 mL/minute flow rate while 20 minutes at 2ml/minutes

flow rate . The method gave complete separation with same elution order in C18 columns of 5 µ

particle size columns.

4.6. Application of the method

Currently these neurotransmitters NA, DOPAC, DA, 5HIAA, HVA, 5-HT are screened in both

preclinical research individually and or their turn over, to understand neurobiology of disease or impact of treatment. The method developed in this study was successfully employed to assess the

effects of Bacopa monnieri (BM) on NA, DOPAC, DA, 5HIAA, HVA, 5-HT in mice striatum.

Additionally this method was successfully used to assess the effects of acute and chronic

treatment of BM on naloxone induced morphine withdrawal and subsequent serotonin and

dopamine turn over in Sprague Dawley rats’ discrete brain areas like frontal cortex, striatum,

Hippocampus and nucleus accumbens.

4.7. Discussion

The developed method is highly reproducible and economical as HPLC-UV grade solvents

instead of ECD grade have been used during the assay which is far more affordable than ECD

77 Chapter 4 HPLC method development for neurotransmitters assay

grade. The method offers reproducibility options for third world countries research labs where

HPLC with ECD grade chemicals are either unavailable or may not be afforded in Labs having

stringent chemicals budget. The method has good sensitivity and can be successfully employed

to assess the dopaminergic or serotonergic modulations in mice/rat discreet brain areas.

Moreover the method has high sensitivity for NA also as evident from the calibration curves of

all neurotransmitters that show acceptable linearity, and acceptable range of precision (inter

days, intraday variation) so no internal standard was used. One limitation associated with the

method that brain samples extracted and preserved with 0.5 mM Ascorbic acid gives very high

solvent front thus overlapping NA peak (McKay et al., 1984). Accordingly caution should be exercised while choosing antioxidant for neurotransmitter stability, and use of other antioxidant

than ascorbic acid is recommended for satisfactory quantification of NA. Moreover a better

response for 5-HT and 5HIAA was achieved at +200 and -300 mV but this leads to NA

overlapping by solvent front and causing serious problem in NA analysis.

Extraction was performed with PCA to deproteinate the brain sample and standards were also

dissolved in 0.2% PCA, though PCA has been reported to support 5HIAA degradation in long

term storage (Boix et al., 1997). Although no antioxidant was used for sample storage, and

stability was purely conferred through, management of temperature (-10 oC and -54oC) protection from light and acidic medium. Samples showed high stability at -10 oC and -54oC, while all

neurotransmitters degraded above 15 % at room temperature. Addition of Acetonitrile by two

percent further lowered overall run time but lowered both sensitivity and selectivity of DOPAC, and DA.

Increasing of sodium 1-octane sulphonic acid by 10 percent had no effect on run time of all other

neurotransmitters but increased serotonin retention time by 4 minutes. Moreover slight intentional

variation of mobile composition (organic: aqueous) ratio changed both sensitivity, and selectivity

78 Chapter 4 HPLC method development for neurotransmitters assay

of the method. The method has been tried with C18 columns of 5 µ particle sizes. The mobile

phase gave complete separation of all compounds (NA, DA, DOPAC, 5HIAA, HVA, 5-HT ) in same order, in C18 columns of 5 µ particle sizes with 150 mm length (ShimPack) and 250 mm

length (Peurospher) as evidenced in MD-150 column chromatograms. The overall run time with

ShimPack was found 17 minutes at 2.0 mL/minute flow rate while 23 minutes at 2ml/minutes

flow rate in 250 mm length (Peurospher) column . The method gave complete separation with

same elution order in C18 columns of 5 µ particle size columns.

The overall run time of the method is shorter than the available methods, like 25 min (Baig et al., 1991),

20 min (Brautigam et al., 1998) 25 min (Zhang et al., 2003) and 32 min (Yoshitake et al., 2003) and 11

minutes (Hubbard et al., 2010).

4.8. Conclusion

The method has been validated for linearity, specificity, selectivity, recovery, economy and

precision, and conforms to international standards. Additionally it’s the shortest of the available

methods (8 minutes) reported till now.

79 Chapter 4 HPLC method development for neurotransmitters assay

4.1. Introduction. 65

4.2. Materials and methods. 65

4.3. Results 65 4.3.1. Chromatography 65 4.3.2. Selection of analytical cell potential 67

4.4. Method validation 68 4.4.1. Selectivity 68 4.4.2. Recovery. 68 4.4.3. Sensitivity 69 4.4.4. Linearity 69 4.4.5. Precision 73

4.5. Stability Studies 75

4.6. Application of the method 77

4.7. Discussion 77

4.8. Conclusion 79

TABLE 4.1 69 TABLE 4.2 EFFECT OF INTER DAYS AND INTRADAY ON NA, DOPAC, DA, 5HIAA, HVA, 5-HT PRECISION IN SPIKED SAMPLES. (N=5) 74 TABLE 4.3 76

FIGURE 4.1. CHROMATOGRAM SHOWING NA, DOPAC, DA, 5HIAA, HVA AND 5-HT AS PEAK A, B, C, D, E. RESPECTIVELY IN 1 NG / ML STANDARDS SAMPLE. 66 FIGURE.4.2. CHROMATOGRAM SHOWING, NA, DOPAC, DA, 5HIAA, HVA AND 5-HT DENOTED AS, PEAK A, B, C, D, E AND F, RESPECTIVELY IN SALINE TREATED MICE STRIATUM. 67 FIGURE. 4.3. VOLTAMMOGRAM OF NA, DOPAC, DA, 5HIAA, HVA, 5-HT PEAKS SHOWN IN CHROMATOGRAMS. CHROMATOGRAMS ARE DENOTED AS A,B,C,D,E,F,G,H, WITH APPLIED POTENTIAL OF 50(A), 100(B), 200(C), 300(D), 400(E), 500(F), 600 (G), 700 MV(H). 68 FIGURE 4.4. CALIBRATION CURVE OF DOPAC, IN A RANGE OF 1 NG TO 300 NG. STANDARD WAS DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 70 FIGURE 4.5. CALIBRATION CURVE OF 5HIAA, IN A RANGE OF 1 NG TO 300 NG. STANDARD WAS DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 70 FIGURE 4.6. CALIBRATION CURVE OF HVA, IN A RANGE OF 1 NG TO 300 NG. STANDARD WAS DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 71 FIGURE 4.7. CALIBRATION CURVE OF 5-HT, IN A RANGE OF 1 NG TO 300 NG. STANDARD WERE DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 71

80 Chapter 4 HPLC method development for neurotransmitters assay

FIGURE 4.8. CALIBRATION CURVE OF DA, IN A RANGE OF 1 NG TO 300 NG. STANDARD WAS DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 72 FIGURE 4.9. CALIBRATION CURVE OF NA, IN A RANGE OF 1 NG TO 300 NG. STANDARD WAS DISSOLVED IN 0.2% PCA AND LINEAR REGRESSION WAS OBTAINED. R2 = 0.99. 72

81 Chapter 5 Effect of Bacopa monnieri on morphine tolerance

Chapter 5 Effect of Bacopa monnieri on morphine

tolerance

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.1. Introduction

Clinical management of chronic malignant and non malignant pains is still a major challenge to the medical communities across the globe, with an economic impact of around 100 billion annually (Renfrey et al., 2003). Opioids are still the drugs of choice for the management of chronic refractory malignant and non malignant pains.

One major limitation that narrows the therapeutic value of opioids for the management of chronic pains is development of tolerance that needs escalating doses to produce same analgesic effects consequently causing cumbersome side effects, including poor quality of life, sedation, constipation, and respiratory depression.

There is growing concern among clinicians regarding clinical use of opioids, their tolerance, subsequent addiction and abuse among patients taking opioids. As there are meager chances of replacement of opioid in near future by some new magical panacea for chronic pains, the medical communities are confronting with challenges of opioid tolerance, addiction and non medical use (Walwyn et al., 2010).

As plants provide a prolific source of chemically diverse bioactive compounds, there is renewed interest in phytomedicines with central effects for the management of opioid tolerance and other drug of abuse management (Abenavoli et al., 2009;

Almeida et al., 2001; Muscoli et al., 2010) and many plants have been preliminarily screened for this purpose and a lot of work is underway, with some encouraging and significant results (Ernst, 2006; Liu et al., 2009). Bacopa monnieri (L.) Wettst.

(Brahmi) is perennial herb from Scrophulariaceae family, found in marshy places across Asia and Europe including Pakistan (Qureshi and Raza Bhatti, 2008). Bacopa monnieri having reputed nootropic effects has been extensively used for various diseases in ayurvedic system of medicine since time immemorial (Russo and Borrelli,

2005). The plant has many active moieties but its major bioactive putative component

Chapter 5 Effect of Bacopa monnieri on morphine tolerance is Bacoside A mainly responsible for its neuropharmacological effects (Deepak et al.,

2005). Bacoside A is a mixture of major components, Bacoside A3, Bacopaside II and

Bacopasaponin C and isomer of Bacopasaponin C (Deepak et al., 2005). The plant has been reported to have analgesic (Subhan et al., 2010; Vohora et al., 1997) anxiolytic

(Calabrese et al., 2008), antidepressant (Abbas et al., 2011; Sairam et al., 2002), anti- inflammatory (Channa et al., 2006) hepatoprotective (Sumathi and Nongbri, 2008) curative effect in ulcer (Sairam et al., 2001) and memory enhancing properties

(Uabundit et al., 2010). The plant has been found to be safe and well tolerated in humans as herbal products are commercially available for enhancing memory in old age patients (Pravina et al., 2007). Bacopa monnieri has been reported to have a protective effect against morphine toxicity at various organs level, including, liver, kidneys and brain (Sumathi and Niranjali Devaraj, 2009).

The aim of this study was, firstly to quantify Bacoside A concentration in methanolic and n-butanol fraction of locally available Bacopa monnieri, secondly to investigate whether tolerance develops to the antinociceptive effects of Bacopa monnieri

(methanolic and n-butanol fraction) with its repeated administration, and thirdly to investigate the effects of Bacopa monnieri (methanolic and n-butanol fraction) on acquisition and expression of morphine tolerance in mice.

5.2. Materials and methods

The details of materials and methods are available in Chapter 2, “Methodology” page 46-48.

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3. Results

5.3.1.Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C The HPLC analysis revealed that Mt-ext BM contained Bacopasides were 1.3 µg

(Bacopasaponin C), 1.4 µg (Bacoside A3), and 1.3µg (Bacopaside ll), in each gram of

Mt-ext BM. The assay of n-Bt-ext BM revealed that this fraction wass Bacoside A rich as it contains Bacoside A3 (3.49 ug/mg), Bacopasaponin C (2.4 µg/mg) and

Bacopaside ll as (3.6 µg/mg), likewise the total quantity of Bacoside A three major components was 9.5 µg /mg of n-Butanol fraction equivalent 15.67ug/gram of dry powder.

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.2. Induction and evaluation of morphine tolerance

5.3.2.1. Antinociceptive effect of morphine sulphate in hot plate test on day one Antinociceptive response of a single dose 10 mg/kg morphine sulphate was almost similar in both pre treatment groups (n=8) at both 30 minutes and 60 minutes time on day one, as depicted in Fig 5.1.

100 Morphine group 80 SAL group

20 Percent Nociception 0 30 60

Time (minutes)

Figure 5.1. Day 1 analgesia of morphine group and saline group in mice. Animals received morphine sulphate (10 mg/Kg i.p.) or saline on day one and were tested for antinociceptive response at 30 minutes and 60 minutes after morphine administration. Each point represents mean±±SEM of percent protection (n= 8).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.2.2. Antinociceptive effect of morphine sulphate in hot plate test on day six As shown in the figure 5.2, animals that received 20 mg/kg twice daily schedule elicited a decreased antinociceptive response both at 30 minutes and 60 minutes time, on day, six, exhibiting development of tolerance to the antinociceptive effect of morphine Fig 5.2. Two way ANOVA clearly indicated that there was significant decrease in an antinociceptive response (***p<0.001).

100 SAL group 80 Morphine group

60 *** *** 40

20 Percent Nociception 0 30 60 Time (minutes)

Figure 5.2. Effect of 10 mg/kg morphine sulphate (i.p.) on day six in morphine treated group and saline group in mice. Animals received morphine sulphate (10 mg/Kg i.p.) or saline on day six and were tested for antinociceptive response at 30 minutes and 60 minutes of morphine administration. Each point represents mean±SEM of percent protection (n=8). (***p<0.001, values significantly different as compared to saline (Two way ANOVA).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.3. Effect of n‐Bt‐ext of Bacopa Monnieri on development of morphine tolerance As shown in the figure 5.3, n-Bt-ext BM acute treatment significantly (***p<0.001) inhibited development of tolerance to the antinociceptive effect of morphine in all three (5, 10, and 15 mg/kg) treated groups as compared to saline groups on day six

(ANOVA followed by Bonferroni post tests).

100 *** Morphine + SAL *** 80 *** *** Morphine + 5mg n-Bt-ext BM *** Morphine + 10mg n-Bt-ext BM 60 *** Morphine + 15mg n-Bt-ext BM

20 Percent Analgesia

0 30 60

Time (minutes)

Figure 5.3. Effect of chronic treatment of n-Bt-ext BM 5, 10, and 15 mg/kg orally on development of morphine tolerance as compared to morphine group. Animals received 5, 10 and 15 mg/kg n-Butanol single oral dose daily along with morphine tolerance induction schedule of 20 mg/kg morphine sulphate twice daily (i.p.) and were tested for antinociceptive response at 30 minutes and 60 minutes of administration in hot plate test. Each point represents mean±SEM of percent protection (n=8). (***p<0.001, Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.4. Effect of n‐Bt‐ext of Bacopa monnieri on expression of morphine tolerance

As depicted in the figure 5.4, single injection of n-Bt-ext BM (5, 10, and 15 mg/kg) significantly (***p<0.001) attenuated expression of morphine tolerance as compared to saline treated groups (ANOVA followed by Bonferroni post tests).

Morphine + SAL Morphine + 5mg n-Bt-ext BM 100 Morphine +10mg n-Bt-ext BM *** Morphine + 15mg n-Bt-ext BM 80 *** *** * *** 60 * 40

20 Percent nociception 0 30 60 time (minutes)

Figure 5.4. Effect of Acute treatment of 5, 10, and 15 mg/kg n-Bt-ext orally on expression of morphine tolerance as compared to saline group. Animals (post morphine induced tolerance) received single oral dose of n-Bt-ext BM 5, 10 and 15 mg/kg on day six, 60 minutes prior to 10 mg/kg morphine (i.p.) challenge dose and were tested for antinociceptive test. In saline group 10 mg/kg morphine (i.p.) challenge dose was administered 30 minutes after saline (i.p) treatment. Each point represents mean±SEM of percent protection (n=8). ANOVA followed by Bonferroni post tests revealed significant difference between treatment groups and control group (***p<0.001, *p<0.05).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.5. Antinociceptive effect of n‐Bt‐ext of Bacopa monnieri in hot plate test As shown in the figure 5.5, n-Bt-ext BM exhibited a significant antinociceptive response in hot plate test. Two way ANOVA followed by Bonferroni post tests revealed significant antinociceptive activity as compared to saline group(***p<0.001) at all doses tested.

100 SAL 80 5mg n-Bt-Ext BM 10mg n-Bt-Ext BM 60 15mg n-Bt-Ext BM *** *** ** *** 40 ** ***

20 Percent nociception 0 60 90 Time(minutes)

Figure 5.5. Antinociceptive effect of n-Bt-ext BM in mice. Animals were administered, n-Bt-ext, (5, 10 and 15 mg/kg orally) and were screened on hot plate for percent protection at 60 and 90 minutes. Each point represents mean±SEM of percent protection (n=8). (**p<0.01 and ***p<0.001, values significantly different as compared to saline (Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.6. Antinociceptive effect of n‐Bt‐ext of Bacopa monnieri in combination with morphine As depicted in the figure 5.6, n-Bt-ext (5, 10 and 15mg/kg orally) significantly augmented morphine antinociceptive activity. Two way ANOVA followed by

Bonferroni post tests revealed significantly augmented antinociceptive activity

(*p<0.05, ***p<0.001) at all doses tested (5, 10 and 15mg/kg).

100 Morphine + SAL 80 *** Morphine + 5mg n-Bt-ext BM *** * *** Morphine + 10mg n-Bt-ext BM *** 60 * Morphine + 15mg n-Bt-ext BM

20 Percent nociception 0 30 60 Time (minutes)

Figure 5.6. Antinociceptive effect of n-Bt-ext BM in combination with morphine sulphate (10 mg/kg i.p) in mice. Animals received 5, 10 and 15mg/kg of n-Bt-ext BM orally 60 minutes before morphine administration and were tested for antinociceptive effect at 30 and 60 minutes. Each point represents mean±SEM of percent protection (n=8). (*p<0.05 and ***p<0.001, values significantly different as compared to control (Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.7. Development of tolerance to the antinociceptive effect of n‐Bt‐ext Bacopa monnieri As depicted in the figure 5.7, n-Bt-ext BM exhibited no tolerance to antinociceptive response in hot plate test both on day one and day seven. Student t test revealed that tolerance did not develop to maximum tolerable dose of n-Bt-ext (15 mg/kg orally ) twice daily in seven days and the comparison of n-Bt-ext BM treatment group on day seven and control group antinociceptive activity was found to be statistically non significant

100 15mg/kg n-Bt-Ext BM 80 SAL

0 60 Time (minutes) 90

Figure 5.7. Antinociceptive effect of n-Bt-ext on day seven. Animals were administered n-Bt-ext for seven days (15 mg/kg orally ) twice daily and were screened for antinociceptive effect on hot plate at 60 minutes and 90 minutes. Each point represents mean±SEM of percent protection (n= 8)..

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.8. Effect of Meth ext BM on development of morphine tolerance As shown in fig 5.8, chronic administration of Meth ext BM (10, 20, or 30 mg/kg orally) significantly inhibited development of tolerance to the antinociceptive effect of morphine as compared to saline group. Two way ANOVA followed by Bonferroni post tests, revealed the effect of treatment as significant *p<0.05.

100 Morphine + SAL 80 Morphine + 10 mg Mt-ext BM * * ** **** Morphine + 20 mg Mt-ext BM 60 Morphine + 30 mg Mt-ext BM

20 Percent nociception 0 30 60

Time (minutes)

Figure 5.8. Effect of chronic administration of Meth ext BM 10, 20, and 30 mg/kg on development of morphine tolerance as compared to morphine group. Animals received 10, 20, and 30 mg/kg Mt-ext BM orally single daily dose along with morphine tolerance induction schedule of 20 mg/kg morphine sulphate twice daily, and were tested for antinociceptive response at 30 minutes and 60 minutes after administration morphine (10 mg/kg i.p.) in hot plate test. Each point represents mean±SEM of percent protection (n=8). (*p<0.05, (**p<0.01 ***P<0.001, Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.9. Effect of acute administration of Meth ext BM on acquisition of morphine tolerance

As shown in Fig 5.9, acute treatment of 10 and 20 mg/kg single oral dose of Meth ext

BM failed to inhibit expression of morphine tolerance. However, 30 mg/kg dose significantly suppressed (p<0.05 expression of morphine tolerance as revealed by

Two way ANOVA followed by Bonferroni post tests.

70 Morphine + SAL * 65 Morphine + 10 mg Mt-ext BM Morphine + 20 mg Mt-ext BM 60 Morphine + 30 mg meth extract * 55

50 Percent nociception 45 30 60 Time (Minutes)

Figure 5.9. Effect of Acute treatment of Mt-ext BM 10, 20, and 30 mg/kg Meth ext BM on expression of morphine tolerance as compared to morphine group. Animals (post morphine induced tolerance) received single oral dose of Mt-ext BM 10, 20, and 30 mg/kg on day six, 60 minutes prior to 10 mg/kg morphine (i.p.) challenge dose and were tested for antinociceptive test. Each point represents mean±SEM of percent protection (n=8). ANOVA followed by Bonferroni post tests revealed significant difference between treatment groups and control group (***p<0.001).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.10. Antinociceptive effect of Mt‐ext BM in hot plate test

As depicted in Fig 5.10, all three doses of Meth ext BM exhibited significant antinociceptive response in hot plate test as compared to saline group. Results were statistically highly significant when assayed by Two way ANOVA followed by

Bonferroni post tests.

60 SAL *** *** *** *** 10 MG Mt-ext BM *** *** 40 20 mg Mt-ext BM 30 mg Mt-ext BM

20 Percent nociception 0 60 90

Time (Minutes)

Figure 5.10. Antinociceptive effect of Meth ext, (10, 20, and 30 mg/kg) in mice. Animals were administered Meth ext, (10, 20, and 30 mg/kg orally) and were screened after 60 minutes on hot plate for percent protection at 60 and 90 minutes. Each point represents mean±SEM of percent protection (n=8). (*p<0.05 and ***P<0.001, values significantly different as compared to saline (Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.11. Antinociceptive Effect of Meth ext BM in combination with morphine

As shown in Fig 5.11, Meth ext 10 or 20 mg/kg oral dose failed to enhance significantly morphine antinociceptive effect, however 30 mg/kg oral dose significantly enhanced morphine antinociceptive effect (p<0.05). The results were statistically significant when assayed by two way ANOVA followed by Bonferroni post tests.

90 * Morphine + SAL Morphine + 10 mg Mt-ext BM Morphine + 20 mg Mt-ext BM 80 * Morphine + 30 mg Mt-ext BM

70 Percent nociception 60 30 60 time (minutes)

Figure 5.11. Antinociceptive effect of Mt-ext BM in combination with morphine sulphate (10 mg/kg i.p.) in mice. Animals received 10, 20 or 30 mg/kg of Mt-ext BM orally 60 minutes before morphine administration and were tested for antinociceptive effect at 30 minutes and 60 minutes. Each point represents mean±SEM of percent protection (n=8). *p<0.05 values significantly different as compared to control (Two way ANOVA followed by Bonferroni post tests).

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.3.12. Development of tolerance to the antinociceptive effect of Meth ext BM As shown in Fig 5.12, upon one week treatment with max oral dose Mt-ext BM 30 mg/kg twice daily, no tolerance was observed as compared to saline group. The difference in treatment group and saline group was insignificant when analyzed by

Student t test.

100 30 mg Mt-ext BM 80 SAL

20 Percent nociception 0 30 60 Time (minutes)

Figure 5.12. Antinociceptive effect of Mt-ext BM on day six. Animals were administered Mt-ext BM for seven days (30 mg/kg) twice daily and were screened for antinociceptive effect on hot plate at 60 minutes and 0 minutes. Each point represents mean±SEM of percent protection (n= 8). Student t test revealed that tolerance did not develop to maximum tolerable dose Mt-ext BM in seven days and the comparison of Mt-ext BM on day seven and control group antinociceptive effect was found to be statistically non significant.

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.4. Discussion

In this study HPLC analysis of locally available BM n-Bt-ext indicates the presence of around 15.67ug of Bacoside A (Bacopaside A3, Bacopaside II and Bacopaside

C)/gram of dry powder. Whereas 1.3 µg (Bacopasaponin C), 1.4 µg (Bacoside A3), and 1.3µg (Bacopaside ll), in each gram of Mt-ext BM.

Administering Bacoside A in doses above 50 µg /kg has been found to equally effective in acute pain models in mice, this might have important future clinical implications in management of both chronic malignant and non malignant pains. The above results reveal that Bacopa monnieri has an antinociceptive effect comparable to morphine in acute pain models, with an added benefit that it enhances morphine antinociceptive effect also. This effect highlights a newer facet of research on the role of BM in clinical cases where opioids are to be used. Animals were administered a maximum tolerable dose of 15 mg/kg n-Bt-ext BM and 30 mg/kg Meth ext BM, for seven days, to assess development of tolerance to its antinociceptive effect and concluded that no tolerance developed to the antinociceptive effect of BM . In mice administration of more than 15 mg/kg of n-Bt-ext BM and 30 mg/kg Meth ext BM led to death of animals within three to four days. As BM has got both antinociceptive and anti-inflammatory effects, it’s high time to evaluate BM role in management of pain models either as substitute or adjuvant to opioids with an objective to minimize opioids dose, tolerance and cumbersome side effects. Furthermore BM has been found to be effective in neuropathic pain models (Sahoo et al., 2010) .

Bacopa monnieri has got calcium channel blocking effect (Channa et al., 2003) also, this might be one contributing factor, as calcium channels blockers have capability to diminish morphine tolerance (Smith et al., 1999). Additionally sustained Ca2+ channel blockade results in modulation in the adenylyl cyclase effectors system

Chapter 5 Effect of Bacopa monnieri on morphine tolerance activated by μ-opioid receptor activation, leading to the suppression of opioid tolerance and restoration of super-sensitivity (Hurlé et al., 2000). Nitric oxide modulates the actions of morphine and related analgesics and tolerance to morphine analgesia is likely prevented by nitric oxide synthase inhibition (Zafar et al., 2002) and tolerance to morphine analgesia is potentiated by nitric oxide (Dambisya and Lee,

1996; Toda et al., 2009). Nitric oxide and peroxynitrite (ONOO−) are reported as the key players that current literature suggests have a major role in both acquisition and expression of morphine tolerance (Bryant et al., 2009). Peroxynitrite has been the center of all current research in the pursuit of newer molecules from both natural and synthetic origin, for the management of chronic pains, neuropathic pains, and morphine tolerance (Salvemini, 2009; Salvemini and Neumann, 2009).

Bacopa monnieri has been reported that it protects against NO induced oxidative stress and rotenone induced oxidative stress in dose dependent manner (Bhattacharya et al., 2000; Kapoor et al., 2009; Russo and Borrelli, 2005; Shinomol, 2011). Bacopa monnieri has been found to be an effective and well tolerated phytomedicine in various clinical trials, including old age patients also (Calabrese et al., 2008; Deb et al., 2008). Currently Bacopa monnieri is available as standardized herbal product alone or in combination with other plant extracts for various cognitive disorders and preliminary clinical data of safety and tolerability in various age groups has promising results (Pravina et al., 2007; Qureshi et al., 2010). Additionally, Bacopa monnieri not only suppresses acquisition and development of morphine tolerance but also protects liver, kidneys and brain from toxic effects of chronic morphine use (Nebelkopf, 1987;

Sumathi and Niranjali Devaraj, 2009; Sumathy et al., 2001). Addition of Bacopa monnieri as an adjuvant therapy for the management of opioid tolerance has many

Chapter 5 Effect of Bacopa monnieri on morphine tolerance added benefits like it is effective both as single dose or repeated dose and has the capacity to enhance morphine analgesia.

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

5.1. Introduction 81

5.2. Materials and methods 82

5.3. Results 83

5.3.1.Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C 83 5.3.2. Induction and evaluation of morphine tolerance 84 5.3.2.1. Antinociceptive effect of morphine sulphate in hot plate test on day one 84 5.3.2.2. Antinociceptive effect of morphine sulphate in hot plate test on day six 85 5.3.3. Effect of n-Bt-ext of Bacopa Monnieri on development of morphine tolerance 86 5.3.4. Effect of n-Bt-ext of Bacopa monnieri on expression of morphine tolerance 87 5.3.5. Antinociceptive effect of n-Bt-ext of Bacopa monnieri in hot plate test 88 5.3.6. Antinociceptive effect of n-Bt-ext of Bacopa monnieri in combination with morphine 89 5.3.7. Development of tolerance to the antinociceptive effect of n-Bt-ext Bacopa monnieri 90 5.3.8. Effect of Meth ext BM on development of morphine tolerance 91 5.3.9. Effect of acute administration of Meth ext BM on acquisition of morphine tolerance 92 5.3.10. Antinociceptive effect of Mt-ext BM in hot plate test 93 5.3.11. Antinociceptive Effect of Meth ext BM in combination with morphine 94 5.3.12. Development of tolerance to the antinociceptive effect of Meth ext BM 95

5.4. Discussion 96

FIGURE 5.1. DAY 1 ANALGESIA OF MORPHINE GROUP AND SALINE GROUP IN MICE. ANIMALS RECEIVED MORPHINE SULPHATE (10 MG/KG I.P.) OR SALINE ON DAY ONE AND WERE TESTED FOR ANTINOCICEPTIVE RESPONSE AT 30 MINUTES AND 60 MINUTES AFTER MORPHINE ADMINISTRATION. EACH POINT REPRESENTS MEAN±±SEM OF PERCENT PROTECTION (N= 8). 84 FIGURE 5.2. EFFECT OF 10 MG/KG MORPHINE SULPHATE (I.P.) ON DAY SIX IN MORPHINE TREATED GROUP AND SALINE GROUP IN MICE. ANIMALS RECEIVED MORPHINE SULPHATE (10 MG/KG I.P.) OR SALINE ON DAY SIX AND WERE TESTED FOR ANTINOCICEPTIVE RESPONSE AT 30 MINUTES AND 60 MINUTES OF MORPHINE ADMINISTRATION. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (***P<0.001, VALUES SIGNIFICANTLY DIFFERENT AS COMPARED TO SALINE (TWO WAY ANOVA). 85 FIGURE 5.3. EFFECT OF CHRONIC TREATMENT OF N-BT-EXT BM 5, 10, AND 15 MG/KG ORALLY ON DEVELOPMENT OF MORPHINE TOLERANCE AS COMPARED TO MORPHINE GROUP. ANIMALS RECEIVED 5, 10 AND 15 MG/KG N-BUTANOL SINGLE ORAL DOSE DAILY ALONG WITH MORPHINE TOLERANCE INDUCTION SCHEDULE OF 20 MG/KG MORPHINE SULPHATE TWICE DAILY (I.P.) AND WERE TESTED FOR ANTINOCICEPTIVE RESPONSE AT 30 MINUTES AND 60 MINUTES OF ADMINISTRATION IN HOT PLATE TEST. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (***P<0.001, TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 86 FIGURE 5.4. EFFECT OF ACUTE TREATMENT OF 5, 10, AND 15 MG/KG N-BT-EXT ORALLY ON EXPRESSION OF MORPHINE TOLERANCE AS COMPARED TO SALINE GROUP. ANIMALS (POST MORPHINE INDUCED TOLERANCE) RECEIVED SINGLE ORAL DOSE OF N-BT-EXT BM 5, 10 AND 15 MG/KG ON DAY SIX, 60 MINUTES PRIOR TO 10 MG/KG MORPHINE (I.P.) CHALLENGE DOSE AND WERE TESTED FOR ANTINOCICEPTIVE TEST. IN SALINE GROUP 10 MG/KG MORPHINE (I.P.) CHALLENGE DOSE WAS ADMINISTERED 30 MINUTES AFTER SALINE (I.P) TREATMENT. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). ANOVA FOLLOWED BY

Chapter 5 Effect of Bacopa monnieri on morphine tolerance

BONFERRONI POST TESTS REVEALED SIGNIFICANT DIFFERENCE BETWEEN TREATMENT GROUPS AND CONTROL GROUP (***P<0.001, *P<0.05). 87 FIGURE 5.5. ANTINOCICEPTIVE EFFECT OF N-BT-EXT BM IN MICE. ANIMALS WERE ADMINISTERED, N- BT-EXT, (5, 10 AND 15 MG/KG ORALLY) AND WERE SCREENED ON HOT PLATE FOR PERCENT PROTECTION AT 60 AND 90 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (**P<0.01 AND ***P<0.001, VALUES SIGNIFICANTLY DIFFERENT AS COMPARED TO SALINE (TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 88 FIGURE 5.6. ANTINOCICEPTIVE EFFECT OF N‐BT-EXT BM IN COMBINATION WITH MORPHINE SULPHATE (10 MG/KG I.P) IN MICE. ANIMALS RECEIVED 5, 10 AND 15MG/KG OF N-BT-EXT BM ORALLY 60 MINUTES BEFORE MORPHINE ADMINISTRATION AND WERE TESTED FOR ANTINOCICEPTIVE EFFECT AT 30 AND 60 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (*P<0.05 AND ***P<0.001, VALUES SIGNIFICANTLY DIFFERENT AS COMPARED TO CONTROL (TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 89 FIGURE 5.7. ANTINOCICEPTIVE EFFECT OF N-BT-EXT ON DAY SEVEN. ANIMALS WERE ADMINISTERED N-BT-EXT FOR SEVEN DAYS (15 MG/KG ORALLY ) TWICE DAILY AND WERE SCREENED FOR ANTINOCICEPTIVE EFFECT ON HOT PLATE AT 60 MINUTES AND 90 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N= 8).. 90 FIGURE 5.8. EFFECT OF CHRONIC ADMINISTRATION OF METH EXT BM 10, 20, AND 30 MG/KG ON DEVELOPMENT OF MORPHINE TOLERANCE AS COMPARED TO MORPHINE GROUP. ANIMALS RECEIVED 10, 20, AND 30 MG/KG MT-EXT BM ORALLY SINGLE DAILY DOSE ALONG WITH MORPHINE TOLERANCE INDUCTION SCHEDULE OF 20 MG/KG MORPHINE SULPHATE TWICE DAILY, AND WERE TESTED FOR ANTINOCICEPTIVE RESPONSE AT 30 MINUTES AND 60 MINUTES AFTER ADMINISTRATION MORPHINE (10 MG/KG I.P.) IN HOT PLATE TEST. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (*P<0.05, (**P<0.01 ***P<0.001, TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 91 FIGURE 5.9. EFFECT OF ACUTE TREATMENT OF MT-EXT BM 10, 20, AND 30 MG/KG METH EXT BM ON EXPRESSION OF MORPHINE TOLERANCE AS COMPARED TO MORPHINE GROUP. ANIMALS (POST MORPHINE INDUCED TOLERANCE) RECEIVED SINGLE ORAL DOSE OF MT-EXT BM 10, 20, AND 30 MG/KG ON DAY SIX, 60 MINUTES PRIOR TO 10 MG/KG MORPHINE (I.P.) CHALLENGE DOSE AND WERE TESTED FOR ANTINOCICEPTIVE TEST. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). ANOVA FOLLOWED BY BONFERRONI POST TESTS REVEALED SIGNIFICANT DIFFERENCE BETWEEN TREATMENT GROUPS AND CONTROL GROUP (***P<0.001). 92 FIGURE 5.10. ANTINOCICEPTIVE EFFECT OF METH EXT, (10, 20, AND 30 MG/KG) IN MICE. ANIMALS WERE ADMINISTERED METH EXT, (10, 20, AND 30 MG/KG ORALLY) AND WERE SCREENED AFTER 60 MINUTES ON HOT PLATE FOR PERCENT PROTECTION AT 60 AND 90 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). (*P<0.05 AND ***P<0.001, VALUES SIGNIFICANTLY DIFFERENT AS COMPARED TO SALINE (TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 93 FIGURE 5.11. ANTINOCICEPTIVE EFFECT OF MT-EXT BM IN COMBINATION WITH MORPHINE SULPHATE (10 MG/KG I.P.) IN MICE. ANIMALS RECEIVED 10, 20 OR 30 MG/KG OF MT-EXT BM ORALLY 60 MINUTES BEFORE MORPHINE ADMINISTRATION AND WERE TESTED FOR ANTINOCICEPTIVE EFFECT AT 30 MINUTES AND 60 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N=8). *P<0.05 VALUES SIGNIFICANTLY DIFFERENT AS COMPARED TO CONTROL (TWO WAY ANOVA FOLLOWED BY BONFERRONI POST TESTS). 94 FIGURE 5.12. ANTINOCICEPTIVE EFFECT OF MT-EXT BM ON DAY SIX. ANIMALS WERE ADMINISTERED MT-EXT BM FOR SEVEN DAYS (30 MG/KG) TWICE DAILY AND WERE SCREENED FOR ANTINOCICEPTIVE EFFECT ON HOT PLATE AT 60 MINUTES AND 0 MINUTES. EACH POINT REPRESENTS MEAN±SEM OF PERCENT PROTECTION (N= 8). STUDENT T TEST REVEALED THAT TOLERANCE DID NOT DEVELOP TO

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Chapter 5 Effect of Bacopa monnieri on morphine tolerance

MAXIMUM TOLERABLE DOSE MT-EXT BM IN SEVEN DAYS AND THE COMPARISON OF MT-EXT BM ON DAY SEVEN AND CONTROL GROUP ANTINOCICEPTIVE EFFECT WAS FOUND TO BE STATISTICALLY NON SIGNIFICANT. 95

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Chapter 6 Bacopa monnieri and locomotor activity

Chapter 6 Effect of Bacopa monnieri on morphine induced locomotor hyperactivity and

neurotransmitters

Chapter 6 Bacopa monnieri and locomotor activity

6.1. Introduction

The mesolimbic system is the seat of emotion and dopaminergic modulation induced by morphine and other psychoactive drugs are expressed by an increased ambulatory response (Babbini and Davis, 1972). Acute morphine administration increases the levels of, DOPAC, HVA, and 5-HIAA as well as DA and 5-HT reflecting an elevated turnover of these neurotransmitters in mouse striatum (Babbini and Davis, 1972;

Fadda et al., 2005; Gauchy et al., 1973; Kuschinsky and Hornykiewicz, 1974; Rethy et al., 1971). Repeated morphine administration leads to reverse tolerance and an increased morphine-induced locomotor response in animal models. Measurement of this hyperactivity is one parameter for assessing psychotoxic potential of abuse liable drugs (Robinson and Becker, 1986; Sanchis-Segura and Spanagel, 2006). Chronic morphine dosing also leads to post synaptic DA receptor super sensitivity (Puri and

Lal, 1973) and episodic withdrawal promotes psychomotor sensitization (Rothwell et al., 2010). Moreover, intermittent morphine exposure induced upregulation of dopamine receptor regulated G protein in the forebrain has a key role in morphine sensitization (Narita et al., 2003). Hence, there is a close relationship between drug induced sensitization, subsequent addiction, drug seeking behavior and relapse

(Robinson and Becker, 1986).

Bacopa monnieri has also 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

(Sumathi, 2007). Apart from enhancing morphine analgesia (Rauf et al., 2011), BM has also been reported to protect major organs like liver, kidneys , and brain from chronic morphine toxicities (Sumathi and Devaraj, 2009). As BM is a renowned nootropic, has a clinical and folkloric standing for memory enhancement, scientists

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Chapter 6 Bacopa monnieri and locomotor activity are exploring newer role for nootropic and cognitive enhancers for the management of opioid dependence (Dhonnchadha and Kantak, 2011; Myers and Carlezon Jr, 2010).

The aim of this study was to investigate the effects of the major active BM component

Bacoside A, found in methanolic extract (Mt-ext BM) and n-butanol fraction (n-Bt- ext BM), on morphine induced locomotor hyperactivity in relation to striatal dopamine (DA), 5-hydroxytryotamine (5-HT) and their metabolites dihydroxyphenyl acetic acid (DOPAC), Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-

HIAA) which are implicated in locomotor behavior.

6.2. Materials and methods

The details of each step, procedures, and materials are given in Chapter methodology, Page 48.

6.3. Results

6.3.1. Chromatographic analysis of methanolic extract of Bacopa monnieri (Mt‐ext BM) for Bacopasides

In this study the HPLC analysis revealed that Mt-ext BM contained Bacoside A, major components i.e. Bacoside A3, Bacopasaponin c and Bacopaside II. Our results further indicated that the quantity of these Bacopasides were 1.6 µg (Bacopasaponin

C), 5 µg (Bacoside A3), and 1.8 µg (Bacopaside ll), in each gram of Mt-ext BM.

6.3.2. Effect of Mt‐ext BM alone and in combination with morphine on locomotor activity

Methanolic extract of Bacopa monnieri significantly (*p<0.05) reduced locomotor activity in saline treated animals. The results indicated (Fig.6.1) that oral administration of all three doses i.e., 10, 20 or 30 mg/kg Mt-ext BM significantly reduced locomotor activity in saline and morphine treated mice. However the 30 mg

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Chapter 6 Bacopa monnieri and locomotor activity

/kg Mt-ext BM inhibition of ambulation was far more significant (***p<0.001) than

10 and 20 mg/kg Mt-ext BM in both saline and morphine treated animals (Fig.6.1).

60 Saline

Mt-ext BM 10 mg/ kg

Mt-ext BM 20 mg/ kg 40 * ** ** Mt-ext BM 30 mg/ kg

MP 10 mg/kg + Mt-ext BM 10 mg/ kg

20 MP 10 mg/kg + Mt-ext BM 20 mg/ kg

* * ** MP 10 mg/kg + Mt-ext BM 30 mg/ kg MP 10 mg/kg + Saline No of lines crossed in 30 minutes 30 in crossed lines of No 0

Figure 6.1. Effect of Mt-ext BM (10, 20 and 30 mg/kg orally) on locomotor activity in saline and morphine (10 mg/kg i.p.) in mice (n=6). Values are expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05, **p<0.01

6.3.3. Effect of Mt‐ext BM on striatal DA and its metabolites DOPAC and HVA in mice

Oral treatment of mice with Mt-ext BM (10, 20 and 30 mg/kg orally) in all three doses (10, 20, 30 mg/kg) failed to change DA, DOPAC and HVA, in mice striatum as compared to saline treated group as shown in table 6.1. However while in morphine

(10 mg/kg) treated groups Mt-ext BM all three doses significantly lowered DA

,DOPAC and HVA as compared to morphine treated animals as shown in table

6.2.The dose dependent impact of Mt-ext BM was clearly evident from changes in dopamine level as compared to morphine treated groups. The inhibition of morphine induced upsurge of DOPAC and HVA was highly significant also but the effect did not clearly display dose dependency (Table 6.2).

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Chapter 6 Bacopa monnieri and locomotor activity

6.3.4. Effect of Mt‐ext BM on 5‐HT and its metabolite 5HIAA in the striatum

As shown in table 6.1, Mt-ext BM all three doses (10, 20 or 30 mg/kg orally) did not alter 5-HT and 5HIAA concentration in mice striatum as compared to saline treated group. In morphine treated group all three doses of Mt-ext BM significantly lowered

5HIAA concentration although the inhibition picture did not clearly portray a dose dependent response as shown in table 2. A down ward trend in 5-HT contents was seen in morphine treated groups but this inhibition was statistically insignificant

(Table 6.2).

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Chapter 6 Bacopa monnieri and locomotor activity

Table 6.1. Effect of normal saline and Mt-ext BM (10, 20 or 30 mg/kg) on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 5HIAA in mice

Treatment DA DOPAC HVA 5-HT 5HIAA Saline 4225±235 637±42 22±7 463±46 133±23 Mt-ext BM 10 4496±229 368±66 17±2 281±49 120±14 mg/kg Mt-ext BM 20 3913±321 667±14 19±2 451±14 143±35 mg/kg Mt-ext BM 30 3592±392 847±44 18±4 431±74 169±29 mg/kg † Concentration levels are expressed as Mean ± S.E.M ng/gram of wet tissue

Table 6.2. Effect of normal saline, morphine (MP, 10 mg/kg) or morphine (MP, 10 mg/kg) + Mt-ext BM (10, 20 and 30 mg/kg) on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 5HIAA.

Striatal tissue concentrations†

Treatment DA DOPAC HVA 5-HT 5-HIAA Saline 4225±135 637±42 22±7 463±46 133±23 MPa 6180±142 1435±62 564±43 401±69 287±15 MPa + Mt-ext 10 mg/kg 4496±235* 561±15** 97±41*** 347±51 166±04** MPa Mt-ext 20 mg/kg 3913±121** 323±23*** 120±25*** 253±26 119±13*** MPa + Mt-ext 30 mg/kg 3592 ±192*** 189±29*** 71±18*** 263±34 97±10*** † Concentration levels are expressed as Mean ± S.E.M ng/gram of wet tissue, *p< 0.05, **p<0.01, ***p < 0.001. Values are significant as compared to morphine treated group. MPa = morphine 10 mg/kg intraperitoneally.

6.3.5. Chromatographic analysis of n-butanol extract of Bacopa monnieri (n-Bt-ext BM) for Bacopasides

In this study chromatographic analysis of n-Bt-ext BM revealed that this fraction was

Bacoside A rich, as it contained Bacopasaponin C (3.2 µg/mg) Bacoside A3 (4.14 ug/mg), and Bacopaside II (4.4 µg/mg). Thus the total quantity of these three

Bacoside A major components was 11.74 µg/mg in n-Bt-ext BM.

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Chapter 6 Bacopa monnieri and locomotor activity

6.3.6. Effect of n-Bt-ext BM alone and in combination with morphine on locomotor activity

As depicted in Fig 6.3, n-Bt-ext BM significantly reduced (***p<0.001) locomotor activity in saline treated mice at doses of 5, 10, and 15 mg/kg. Additionally, Bt-ext

BM in doses of 5, 10, and 15 mg/kg also reduced morphine (10 mg/kg) induced locomotor activity in a highly significant fashion (***p<0.001)

Figure 6.2. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg i.p) on saline (SAL) and morphine (MP, 10 mg/kg) induced locomotor activity in mice (n = 6). Values are expressed as Mean ± S.E.M. (***p<0.001; ANOVA, followed by Tukey’s post hoc test).

6.3.7. Effect of n-Bt-ext BM on DA and its metabolites DOPAC and HVA in mouse striatum

As shown in table 6.4, acute morphine administration (10mg/kg) caused a significant increase in the striatal tissue concentration of DA, HVA, and DOPAC. Pretreatment with n-Bt-ext BM (5, 10, and 15 mg/kg) significantly lowered (p<0.001) the morphine induced HVA upsurge as compared to the morphine treated group. Additionally,

105

Chapter 6 Bacopa monnieri and locomotor activity treatment with n-Bt-ext BM (15 mg/kg) also significantly inhibited the morphine induced rise in DA and DOPAC level in the striatum (P<0.05). Moreover, a trend towards a decrease in DA concentration was also noted in the 10 and 5 mg/kg n-Bt- ext BM treatment groups though the effect never achieved statistical significance.

Interestingly, n-Bt-ext BM failed to induce any significant decrease in DA or its metabolites in the saline control group as shown in table 7.6.3.

6.3.8. Effect of n-Bt-ext BM on 5-HT and its metabolite 5HIAA in the striatum As shown in table 6.4, Morphine treatment (10 mg/kg) raised the level of both

5-HT and its metabolite, 5HIAA, but only the increase in 5HIAA achieved statistical significance (p<0.001), for all three doses of n-Bt-ext BM within the groups. Furthermore, n-Bt-ext BM treatment appeared to inhibit the serotonin upsurge induced by morphine but it was not statistically significant. Additionally, none of the n-Bt-ext BM doses modified serotonin or 5HIAA, striatal concentrations in saline treated mice, as shown in table 6.3

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Chapter 6 Bacopa monnieri and locomotor activity

Table 6.3. Effect of normal saline and n-Bt-ext BM (5, 10 or 15 mg/kg) on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 5HIAA in mice

Striatal tissue concentrations† Treatments DA DOPAC HVA 5-HT 5HIAA Saline 2658 ± 157 166.7 ± 48 323.5 ± 36 505 ± 36 241± 29 n-Bt-ext BM (5mg/kg) 2629 ± 321 283 ± 49 274.5 ± 30 508 ± 34 235. ± 20 n-Bt-ext BM (10 mg/kg) 2602 ± 648 317 ± 53 381.9 ± 52 499± 52 245 ± 26 n-Bt-ext BM (15 mg/kg) 2443 ± 659 388 ± 45 292 ± 30 438 ± 15 261 ± 25

† Concentration levels are expressed as Mean ± S.E.M ng/gram of wet tissue

Table 6.4. Effect of normal saline, morphine (MP, 10 mg/kg) or morphine (10 mg/kg) + n-Bt-ext BM (5, 10 or 15 mg/kg) on striatal tissue levels of DA, DOPAC, HVA, 5-HT and 5HIAA.

Striatal tissue concentrations† Treatments DA DOPAC HVA 5-HT 5-HIAA

Saline 2658 ± 157 167 ± 48 324 ± 36 505 ± 36 241 ± 29

MPa 4782 ± 218 1577 ± 168 7614 ± 232 760 ± 70 6387 ± 642 MPa + n-Bt-ext 5 3863 ± 255 518 ± 40 784 ± 89** 685 ± 12 185 ± 38*** mg/kg MPa + n-Bt-ext 10 3616 ± 237 613 ± 66 772 ± 46** 490 ± 40 234 ± 34*** mg/kg MPa + n-Bt-ext 15 2277± 31* 440 ± 76* 868 ± 31** 398 ± 43 164 ± 17*** mg/kg † Concentration levels were expressed as mean ± S.E.M ng/gram of wet tissue, *p< 0.05, **p< 0.01, ***p< 0.001. Values are significant as compared to morphine treated group. MPa = morphine 10 mg/kg intraperitoneally

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Chapter 6 Bacopa monnieri and locomotor activity

6.5. Discussion

Almost all psychostimulants and opiates, with any propensity for drug abuse, induce hyperactivity which is regarded as an indication of euphoria in rodents which is attributable mainly to their effects on dopaminergic transmission in the striatum and mesocorticolimbic system (Koob, 1992). Thus, morphine and other psychostimulants have been found to increase extracellular dopamine, in the striatum and nucleus accumbens. BM has been reported to inhibit, morphine hyperactivity, tolerance, reverse tolerance, dopamine receptor supersensitivity, and apomorphine induced climbing behavior in rats (Sumathi, 2007). Single and repeated administration of abuse liable drugs like morphine and cocaine also induce DNA damage within major body organs including brain, liver, and blood (Alvarenga et al., 2010; Ammon-Treiber and Höllt, 2005). In the current study, BM both extracts (n-Bt-ext BM and Mt-ext

BM) decreased locomotor activity in both saline-treated as well as morphine-treated animals at all three doses tested (n-Bt-ext BM 5, 10 and 15 mg/kg, Mt-ext BM 10, 20,

30 mg/kg ) as shown in fig. 6.2 & 6.3. This latter outcome closely corroborates that of

Sumathi et al., (Sumathi, 2007) who observed an inhibitory response of BM alcoholic extract on morphine induced hyperactivity.

Our results also showed that Mt-ext BM all three doses significantly lowered morphine induced increase in DA, DOPAC, HVA and 5HIAA. Furthermore n-Bt-ext

BM has a significant antidopamine effect, since it significantly reduced the morphine- induced upsurge of DA as well as DOPAC at the highest dose employed (15 mg /kg) and also HVA at all doses (table 6.2). The ability of n-Bt-ext BM to suppress locomotor activity by itself with no inherent effect on striatal DA or its metabolites, may implicate a DA-release independent mechanism in this response. However this does not exclude the possibility of an action at postsynaptic DA receptors since an

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Chapter 6 Bacopa monnieri and locomotor activity alcoholic extract of BM will inhibit apomorphine-induced dopaminergic behavior and cage climbing (Sumathi, 2007). The reduction of morphine hyperlocomotor activity observed in the current study, might conceivably stem either from a functional interaction between BM (n-Bt-ext BM, Mt-ext BM) locomotor suppression and morphine hyperactivity, a direct postsynaptic negative dopaminergic action, or via the reduction in morphine induced DA, HVA and DOPAC upsurge observed in Table 6.2

& 6.4.

The antidopamine effect might arise from the fact that Bacoside A reduces nitric oxide (NO) mediated oxidative stress both at the cytoplasmic and mitochondrial level

(Shinomol and Muralidhara, 2010); (Hosamani and Muralidhara, 2009; Russo et al.,

2003). There is linked evidence of a glutamatergic-dopaminergic interplay (Koob,

1992) which is an essential element of sensitization to opioids and psychostimulants

(Pierce and Kalivas, 1997). In this context, glutamate regulates the release of DA in the striatum (Avshalumov et al., 2003) and it has been demonstrated that nitric oxide

(NO) plays a role in this process (Dominguez et al., 2004; Hanbauer et al., 1992).

Furthermore, L-arginine, from which NO is generated in vivo, augments morphine induced hyperactivity, whereas L-NG-Nitroarginine methyl ester (L-NAME), a specific nitric oxide synthase (nos) inhibitor, diminishes this opioid-evoked activity in mice (Calignano et al., 1993; Pogun et al., 1994). L-arginine has also been found to increase dopamine concentrations in the nucleus acumbens and striatum, and correspondingly, this effect is inhibited by L-NAME (Pierce and Kalivas, 1997)

There is evidence that the serotonergic system is implicated in opioid hyperlocomotion (Christophe et al., 2008; Filip et al., 2010). The current data shows that even though both Mt-ext BM and n-Bt-ext BM lowered striatal tissue levels of 5-

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Chapter 6 Bacopa monnieri and locomotor activity

HIAA in morphine treated mice, which is suggestive of a decrease in serotonin turnover; it did not significantly modify the morphine-induced serotonin upsurge.

However, our results do accord with those in rat cerebellum whereby 5-HT concentrations remain unmodified following BM extract treatment although they are reduced in epileptic animals and subsequently restored to control levels by BM extract

(Krishnakumar et al., 2009).

Bacoside A possesses calcium channel inhibitory activity (Dar and Channa, 1999) which has been found to potentiate opioid analgesia and delay tolerance onset without increasing respiratory depression (Michaluk et al., 1998). Calcium channel blockers themselves inhibit morphine hyperlocomotion (Zhang et al., 2003) in addition to being antidopaminergic/antiserotonergic and they also inhibit the opioid induced norepinephrine upsurge in the hypothalamus (Pucilowski, 1992). Whether the calcium channel blocking effect of Bacoside A is linked to its influence on neurotransmitters such as norepinephrine remains to be established and this warrants further study

In summary, n-Bt-ext BM and Mt-ext BM which contains Bacoside A, not only inhibited locomotor activity per se in mice, but also reduced morphine induced hyperlocomotion. Moreover, analysis of mouse striatal neurotransmitter and metabolite concentrations suggested that n-Bt-ext BM and Mt-ext BM also reversed morphine stimulated dopamine and serotonin turnover. It is a possibility that the antidopaminergic effect of Bacoside A may have promising implications for morphine sensitization and dependence especially since the Bacosides have a favorable safety and tolerability profile (Calabrese et al., 2008; Das et al., 2002; Goel et al., 2003;

Joshua Allan et al., 2007; Raghav et al., 2006).

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Chapter 6 Bacopa monnieri and locomotor activity

6.5. Conclusion

The findings of the locomotor activity reflect that Bacopa monnieri both extracts, i.e. methanolic and n-butanol fraction significantly inhibits locomotion in both saline and morphine treated animals. Additionally the degree of inhibition is significantly higher in n-butanol fraction than in methanolic extract group. Furthermore neurotransmitters results indicate that Bacopa monnieri both extracts in all three doses did not significantly alter, DA, DOPAC, HVA, 5-HT and 5HIAA in saline treated animals.

While both extracts in all three doses inhibits morphine induced upsurge of DA,

DOPAC, HVA and 5HIAA significantly.

TABLE 6.1. EFFECT OF NORMAL SALINE AND MT-EXT BM (10, 20 OR 30 MG/KG) ON STRIATAL TISSUE LEVELS OF DA, DOPAC, HVA, 5-HT AND 5HIAA IN MICE ..... 104 TABLE 6.2. EFFECT OF NORMAL SALINE, MORPHINE (MP, 10 MG/KG) OR MORPHINE (MP, 10 MG/KG) + MT-EXT BM (10, 20 AND 30 MG/KG) ON STRIATAL TISSUE LEVELS OF DA, DOPAC, HVA, 5-HT AND 5HIAA...... 104 TABLE 6.3. EFFECT OF NORMAL SALINE AND N-BT-EXT BM (5, 10 OR 15 MG/KG) ON STRIATAL TISSUE LEVELS OF DA, DOPAC, HVA, 5-HT AND 5HIAA IN MICE ..... 107 TABLE 6.4. EFFECT OF NORMAL SALINE, MORPHINE (MP, 10 MG/KG) OR MORPHINE (10 MG/KG) + N-BT-EXT BM (5, 10 OR 15 MG/KG) ON STRIATAL TISSUE LEVELS OF DA, DOPAC, HVA, 5-HT AND 5HIAA...... 107

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Chapter 6 Bacopa monnieri and locomotor activity

6.1. Introduction 100

6.2. Materials and methods 101

6.3. Results 101 6.3.1. Chromatographic analysis of methanolic extract of Bacopa monnieri (Mt-ext BM) for Bacopasides 101 6.3.2. Effect of Mt-ext BM alone and in combination with morphine on locomotor activity 101 6.3.3. Effect of Mt-ext BM on striatal DA and its metabolites DOPAC and HVA in mice 102 6.3.4. Effect of Mt-ext BM on 5-HT and its metabolite 5HIAA in the striatum 103 6.3.5. Chromatographic analysis of n-butanol extract of Bacopa monnieri (n-Bt-ext BM) for Bacopasides 104 6.3.6. Effect of n-Bt-ext BM alone and in combination with morphine on locomotor activity 105 6.3.7. Effect of n-Bt-ext BM on DA and its metabolites DOPAC and HVA in mouse striatum 105 6.3.8. Effect of n-Bt-ext BM on 5-HT and its metabolite 5HIAA in the striatum 106

6.5. Discussion 108

6.5. Conclusion 111

FIGURE 6.1. EFFECT OF MT-EXT BM (10, 20 AND 30 MG/KG ORALLY) ON LOCOMOTOR ACTIVITY IN SALINE AND MORPHINE (10 MG/KG I.P.) IN MICE (N=6). VALUES ARE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P<0.05, **P<0.01 102 FIGURE 6.2. EFFECT OF N-BT-EXT BM (5, 10 AND 15 MG/KG I.P) ON SALINE (SAL) AND MORPHINE (MP, 10 MG/KG) INDUCED LOCOMOTOR ACTIVITY IN MICE (N = 6). VALUES ARE EXPRESSED AS MEAN ± S.E.M. (***P<0.001; ANOVA, FOLLOWED BY TUKEY’S POST HOC TEST). 105

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

Chapter 7 Effect of acute and sub chronic administration of Bacopa monnieri on neurotransmitters

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

7.1 Introduction

Normal Behavior is an outcome of a discreet and sensitive balance of neurotransmitters in specified brain areas (Stricker and Zigmond, 2010). The maintenance of this very delicate balance is imperative as neurotransmitters modulation control developmental, behavioral, emotional and hormonal states directly or indirectly (Berridge, 2004; Stricker and Zigmond, 2010). The Dopamine (DA) being major neurotransmitter is responsible for emotional balance, and regulation of human cognition (Colzato et al., 2010). DA mainly controls food intake, endocrine functions, emotional states, reward and sexual behavior (Baptista et al., 2002).

Likewise 5-Hydroxytryptamine (5-HT) also plays an integral and pivotal role in controlling emotions, food intake, anxiety behaviors and endocrine functions

(Bjorvatn et al., 2002). The disturbance of this delicate balance between DA and 5-HT leads to many pathological conditions translating itself in the form of multiple neuropsychiatric disorders (Esposito, 2006; Wood and Wren, 2008). Serotonin per se is an important neurotransmitter and any disturbance of 5-HT may cause series of neurologic and psychiatric illnesses, like hallucination, anxiety, depression and migraine (Hou et al., 2006). Phytochemcials that either disrupt balance between DA and 5-HT or Modulate DA or Serotonin metabolism also leads to behavioral and endocrine disturbances and subsequent pathologies (Farias et al., 2010; Ganong,

1980; Veldhuis et al., 1997; Verma et al., 2007).

Keeping in view the neuropharmacological profile of BM and our previous findings, this study was designed to examine the contents of Bacopaside A in the methanolic extract (Mt-ext) and n-butanol extract (n-Bt-ext) of indigenously found BM and also to assess the effect of acute and sub chronic (one week) administration of the

113

Chapter 7 Effect of Bacopa monnieri on neurotransmitters methanolic extract and n-butanol extract on dopamine and serotonin turn over in mice whole brain.

7.2. Materials and methods

The details of material and methods are available on page 45-46 in chapter Methodology.

7.3. Results

7.3.1 Quantification of Bacoside A major components in Mt‐ext BM and n‐Bt‐ext BM In this study the HPLC analysis revealed that Mt-ext BM contained Bacoside A, major components and the quantity of these Bacopasides were 1.3 µg (Bacopasaponin

C), 1.4 µg (Bacoside A3), and 1.3µg (Bacopaside ll), in each gram of Mt-ext BM. Chromatographic analysis of n-Bt-ext BM revealed that this fraction was Bacoside A rich, as it contained Bacopasaponin C (3.2 mg/g) Bacoside A3 (4.14 mg/g), and Bacopaside II (4.4 mg/g). Thus the total quantity of these three Bacoside A major components was 11.74 mg/g in n-Bt-ext BM.

7.3.2 Effect of acute treatment of Mt‐ext BM on whole brain neurotransmitters As shown in the table 7.1, acute treatment of Mt-ext BM had no significant effect on

DA, 5-HT and their metabolites HVA, DOPAC, and 5HIAA in mice whole brain.

Moreover, there was also no change in the ratios of HVA/DA, DOPAC/DA,

HVA+DOPAC/DA and 5HIAA/5-HT as compared to saline treatment group (table

7.1).

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

Table 7.1. Effect of acute treatment of Mt-ext BM on DA, 5-HT their metabolites and turnover in mice whole brain Effect of saline and Mt-ext BM (acute) on neurotransmitters Neurotransmitters† Saline 10 mg/kg 20 mg/kg 30 mg/kg DA 700±73 731±123 971±135 1014±175 DOPAC 39 ±05 27±6 46±18 31±08 HVA 77±12 51±25 82±16 112±11 5-HT 216±28 181±22 300±59 282±45 5HIAA 75±03 65±11 78±22 57±10 Effect of saline and Mt-ext BM (acute) on neurotransmitters turnover neurotransmitters† Saline 10 mg/kg 20 mg/kg 30 mg/kg HVA/DA 0.11±0.01 0.05±0.02 0.19±0.08 0.12±0.06 DOPAC/DA 0.07±0.02 0.03±0.00 0.06±0.03 0.03±0.00 HVA+DOPAC/DA 79.53±13 50.78±24 182.2±44.0 112.6±60 5HIAA/5-HT 0.47±0.17 0.36±0.04 0.34±0.11 0.24±0.07 †Neurotransmitters concentration (ng/ 100 mg of wet tissue) are expressed as mean ± S.E.M.

7.3.3 Effect of sub chronic treatment of Mt‐ext BM on whole brain neurotransmitters Sub chronic (7 days) treatment of Mt-ext BM also failed to alter DA, 5-HT and their metabolites DOPAC, HVA and 5-HIAA in mice whole brain (table 7.2). Furthermore, there was also no significant effect in the ratio of HVA/DA, DOPAC/DA,

HVA+DOPAC/DA and 5HIAA/5-HT as compared to saline treatment group (table

7.2).

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

Table 7.2. Effect of sub chronic (one week) treatment of Mt-ext BM, On DA, 5- HT their metabolites and turnover in mice whole brain

Effect of saline and Mt-ext BM (sub chronic) on neurotransmitters

Neurotransmitters† Saline 10 mg/kg 20 mg/kg 30 mg/kg DA 570±98 900±170 567±50 830±98 DOPAC 39±05 35±04 58±08 29±4 HVA 62±12 91±17 58±15 80±14 5-HT 183±19 220±40 226±37 183±19 5HIAA 112±29 96±13 58±05 46±06 Effect of saline and Mt-ext BM (sub chronic) on neurotransmitters turnover Neurotransmitters Saline 10 mg/kg 20 mg/kg 30 mg/kg turnover† HVA/DA 0.12±0.02 0.13±0.03 0.08±0.03 0.14±0.02 DOPAC/DA 0.10±0.07 0.05±0.01 0.07±0.02 0.05±0.00 HVA+DOPAC/DA 72.±08 91±17 57±14.80 80±14 5HIAA/5-HT 0.34±0.08 0.48±0.05 0.46±0.05 0.29±0.05 †Neurotransmitters concentration (ng/ 100 mg of wet tissue) are expressed as mean ± S.E.M.

7.3.4. Effect of n-Bt-ext acute treatment on whole brain neurotransmitters in mice As shown in table 7.3, acute treatment of n-Bt-ext BM 10 and 15 mg/kg significantly lowered DA and 5HIAA contents in whole brain, while 5 mg/kg acute treatment failed to modulate DA, 5-HT, or their metabolites. Moreover acute administration of n-Bt-ext BM 10 and 15 mg/kg significantly raised DOPAC/DA and

HVA+DOPAC/DA ratio level in mice whole brain, while 5 mg/kg acute treatment failed to affect DA or serotonin turn over (Table 7.3). Acute treatment with n-Bt-ext

BM all three doses failed to significantly alter serotonin turn over in mice whole brain

(7.3).

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

Table 7.3. Effect of acute treatment of n-Bt-ext BM on DA, 5-HT and their metabolites in mice whole brain Effect of saline and n-Bt-ext BM (acute) on neurotransmitters

Neurotransmitters† Saline 5 mg/kg 10 mg/kg 15 mg/kg

DA 255±11 266±09 167±15* 191±11*

DOPAC 19 ±02 27±02 15±01 30±02 HVA 54±04 22±02 64±03 84±13 5-HT 110±14 126±10 75±07 94±04 5HIAA 38±02 41±05 23±03** 25±05* Effect of saline and n-Bt-ext BM (acute) on neurotransmitters Neurotransmitters† Saline 5 mg/kg 10 mg/kg 15 mg/kg HVA/DA 0.098±0.00 0.010±0.00 0.128±0.01 0.159±0.01 DOPAC/DA 0.20±0.01 0.06±0.00 0.40±0.04* 0.46±0.08** HVA+DOPAC/DA 0.30±0.01 0.16±0.01 0.53±0.04* 0.61±0.09** 5HIAA/5-HT 0.31±0.02 0.23±0.01 0.25±0.05 0. 54±0.14

†Neurotransmitters concentration (ng/ 100 mg of wet tissue) are expressed as mean ± S.E.M, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01

7.3.5. Effect of n‐Bt‐ext sub chronic treatment on whole brain neurotransmitters in mice As shown in table 7.4, sub chronic treatment with n-Bt-ext BM, 5 and 10 mg/kg did alter DA concentration in mice whole brain while 15 mg/kg significantly raised the

DA concentration as shown in table 7.6. Moreover all three doses significantly raised

HVA and lowered 5HIAA, in mice whole brain. Sub chronic treatment with n-Bt-ext all three doses failed to alter 5-HT or DOPAC concentration whole brain, as shown in table 7.4. More over sub chronic treatment with n-Bt-ext, failed to alter 5HIAA/5-HT,

HVA/DA turnover but significantly raised DOPAC/DA and HVA+DOPAC/DA turnover with 10 and 15 mg/kg doses (Table 7.4).

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

Table 7.4. Effect of sub chronic (one week) treatment of n-Bt-ext BM, On DA, 5- HT and their metabolites in mice whole brain Effect of saline and n-Bt-ext BM (sub chronic) on neurotransmitters

Neurotransmitters† Saline 5 mg/kg 10 mg/kg 15 mg/kg DA 267±9 297±14 229±3 353±15*** DOPAC 26±3 27±4 24±8 33±4 HVA 62±12 104±2* 96±15* 232±14*** 5-HT 123±19 150±8 110±12 143±16 5HIAA 45±2 18±1*** 19±2*** 16±1*** Effect of saline and n-Bt-ext BM (sub chronic) on neurotransmitters

Neurotransmitters† Saline 5 mg/kg 10 mg/kg 15 mg/kg HVA/DA 0.091±0.00 0.090±0.00 0.103±0.00 0.95±0.00 DOPAC/DA 0.21±0.01 0.35±0.01 0.42±0.06* 0.66±0.05*** HVA+DOPAC/DA 0.29±0.04 0.44±.02 0.53±0.06* 0.75±0.05*** 5HIAA/5-HT 0.34±0.01 0.45±0.14 0.10±0.00 0.05±0.03

†Neurotransmitters concentration (ng/ 100 mg of wet tissue) are expressed as mean ± S.E.M, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, ***p<0.005

7.4 Discussion

In this study, HPLC analysis of available sample BM plant showed the presence of all the major components of Bacoside A, i.e. Bacopasaponin C, Bacoside A3, and

Bacopaside ll calculated as 1.3 µg, 1.4 µg, and 1.3µg respectively in each gram of Mt- ext BM, while quantity of these three Bacoside A major components was 11.74 mg/g in n-Bt-ext BM.

Assessment of DA and 5-HT turnover can be judged from the rates of accumulation of their metabolites such as DOPAC, HVA and 5-HIAA. In this respect it has been reported that the metabolites and ratios of metabolites to neurotransmitters are more

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters sensitive measure as compared to steady state levels of neurotransmitters

(Baldessarini et al., 1992). As all those agents that increase DA and its metabolites concentration in mesolimbic system have abuse potential like opiates, cocaine, and the compounds that lowers DA concentration induce cognitive, behavioral and motor coordination defects (Berridge and Robinson, 1998; Esposito, 2006). In this study we found that acute and sub chronic administration of Mt-ext BM does not significantly change DA, DOPAC, HVA and ratios of DOPAC/DA, HVA/DA, in mice whole brain. It reflects the safety and subsequent tolerability of BM in preclinical models.

Apparently based on these findings it can be concluded that BM is free from such untoward and toxic effects associated with changes in these neurotransmitters.

Since compounds having psychoactive potential modulate the balance of brain 5-HT and DA in mice whole brain at both acute and chronic administration (Ahtee and

Attila, 1987; Babbini and Davis, 1972; Fadda et al., 2005; Kuschinsky and

Hornykiewicz, 1974; Rethy et al., 1971; Sulzer, 2011; Tejada et al., 2011). In this study the balance between DA, and its metabolites and serotonin and its metabolites portray an image that BM maintains monoamines homeostasis and does not induce

DA and 5-HT imbalance which contributes to various neurologic, behavioral and hormonal anomalies in both acute and sub chronic use.

Additionally both acute and sub chronic administration of Mt-ext BM does not significantly alter 5H-T, 5HIAA, or ratio of 5HIAA/5-HT in mice which further strengthen nootropic action of BM as drugs increasing serotonin metabolism are associated with development and augmentation of retrograde amnesia and cognition problems (Semba et al., 2005). Moreover drugs lowering 5-HT synthesis induce emotional, behavioral and neurologic abnormalities (Hou et al., 2006). As treatment with of Mt-ext BM does not alter dopaminergic and serotonergic systems in mice thus

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters further validating the safety and tolerability of BM usage in ayurvedic system of medicine for various neuropsychiatric disorders.

Furthermore, n-Bt-ext BM acute administration (10, 15 mg/kg) lowered DA and

5HIAA concentration significantly, while significantly increased DOPAC/DA and

HVA+DOPAC/DA ratio in mice whole brain (table 7.3&7.4). The change in

DOPAC/DA and HVA+DOPAC/DA ratio seen in this study does not imply change in striatum as earlier we have reported no effect of acute treatment of n-Bt-ext BM on striatal DA turn over (Chapter 7).

Conversely sub chronic treatment with n-Bt-ext BM increased DA at highest dose (15 mg/kg orally). Similarly chronic treatment with n-Bt-ext BM HVA was significantly raised by all three doses except 5HIAA that decreased significantly. Interestingly

DOPAC/DA and HVA+DOPAC/DA turn over in mice whole brain was significantly increased by 10 and 15 mg/kg doses while 5HIAA/5-HT turnover was not altered significantly. The apparent raised DA turn over, induced by n-Bt-ext BM might be because of very high concentration of Bacoside A, i.e. 11.74 mg/gram which is around 1000 times higher as compared to its concentration in methanolic extract. The n-Bt-ext BM effect in amelioration of morphine tolerance, and augmentation of morphine analgesia was significantly higher than methanolic extract, might be an attribute of high concentration of Bacoside A. As evident from our toxicological studies (chapter 9) the calculated LD50 of n-Bt-ext BM was 81.1 mg/kg and 15 mg/kg for one week might be a sub chronic toxic dose and the altered DA turnover might be due to toxicity induced by higher doses. The brain specified parts DA and 5-HT turnover with behavioral assessments might exhibit a clearer outlook of n-Bt-ext BM impact on DA and 5-HT turn over preferably through microdialysis. Mostly methanolic extracts are used in ayurvedic system of medicine for various central

120

Chapter 7 Effect of Bacopa monnieri on neurotransmitters nervous system diseases (Russo and Borrelli, 2005). In another report BM Meth-ext has been reported to have an adaptogenic effect (Rai et al., 2003), and restores NA,

DA and 5-HT modulations induced by acute unpredictable stress and chronic stress in rats striatum (Sheikh et al., 2007). There are clinical trials that have reported the safety and tolerability of BM in human thus signifying the safety and lack of central untoward effects that could be attributed to mild effect of Mt-ext BM on neurotransmitters like DA or 5-HT or their metabolism (Calabrese et al., 2008;

Nathan et al., 2001; Raghav et al., 2010; Stough et al., 2008; Stough et al., 2001).

In summary, acute and sub chronic treatment of methanolic extract of Bacopa monnieri containing Bacopaside A components i.e. Bacopasaponin C, Bacoside A3, and Bacopaside ll have no significant effect on the levels of DA, 5-HT and their metabolites in mice whole brain. It can be implied that Bacopa monnieri may not have DA and 5-HT modulation in healthy preclinical models, although further studies are warranted to assess DA and serotonin turn over in discrete brain areas through microdialysis.

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Chapter 7 Effect of Bacopa monnieri on neurotransmitters

7.1 Introduction 113

7.2. Materials and methods 114

7.3. Results 114 7.3.1 Quantification of Bacoside A major components in Mt-ext BM and n-Bt-ext BM 114 7.3.2 Effect of acute treatment of Mt-ext BM on whole brain neurotransmitters 114 7.3.3 Effect of sub chronic treatment of Mt-ext BM on whole brain neurotransmitters 115 7.3.4. Effect of n-Bt-ext acute treatment on whole brain neurotransmitters in mice 116 7.3.5. Effect of n-Bt-ext sub chronic treatment on whole brain neurotransmitters in mice 117

7.4 Discussion 118

TABLE 7.1. EFFECT OF ACUTE TREATMENT OF MT-EXT BM ON DA, 5-HT THEIR METABOLITES AND TURNOVER IN MICE WHOLE BRAIN ...... 115 TABLE 7.2. EFFECT OF SUB CHRONIC (ONE WEEK) TREATMENT OF MT-EXT BM, ON DA, 5-HT THEIR METABOLITES AND TURNOVER IN MICE WHOLE BRAIN ...... 116 TABLE 7.3. EFFECT OF ACUTE TREATMENT OF N-BT-EXT BM ON DA, 5-HT AND THEIR METABOLITES IN MICE WHOLE BRAIN ...... 117 TABLE 7.4. EFFECT OF SUB CHRONIC (ONE WEEK) TREATMENT OF N-BT-EXT BM, ON DA, 5-HT AND THEIR METABOLITES IN MICE WHOLE BRAIN ...... 118

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Chapter 8

Effect of Bacopa monnieri on naloxone precipitated morphine withdrawal and

neurotransmitters

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.1. Introduction

Morphine dependence is a chronic relapsing disease caused by its chronic illicit or medical use, which is associated with tolerance, dependence and withdrawal syndrome upon cessation (Redmond Jr and Krystal, 1984). Mechanistically morphine dependence is a complex interplay of neuroadaptation produced by chronic morphine usage, comprising of serotonergic, dopaminergic, cholinergic, GABAergic and peptidergic transmissions, involving both mesolimbic and mesocortical brain areas

(Goeldner et al., 2011). Opiates dependence is on alarming increase across the globe including Pakistan, posing a huge socio economic and health challenge to the world in general and Pakistan in special (Emmanuel et al., 2004; Emmanuel et al., 2003; Mufti et al., 2004). Opioid dependence treatment although costly, needs holistic management (Redmond Jr and Krystal, 1984) as half of all heroin users have HIV infection also (Krantz and Mehler, 2004). Previously withdrawal was assumed as the major proponent behind drug seeking behavior but current research highlights other more complex neuroadaptation induced by chronic use of opiates (Gray, 2002; Shalev et al., 2001). Although methadone remains at mainstay in opioid withdrawal therapy but in many parts of the world this therapy is either not available or not affordable

(Jittiwutikarn et al., 2004; Justo et al., 2006). Additionally the methadone has another major limitation that it causes dependence in individuals using it as substitute, thus further limiting its degree of acceptance (Justo et al., 2006; Lenné et al., 2001).

Moreover Torsades de Pointes (TdP) a type of polymorphic ventricular tachycardia is one common problem associated with clinical use of methadone and as the use of methadone increases a parallel rise in methadone associated TdP events reporting is also increasing (Justo et al., 2006). Due to these reasons search for an alternative, safe and affordable therapy is the primary need of the day. Recently Chinese herbal

123

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal medicines have been found to be safer, cost effective and easily available alternate to conventional therapies for opioid detoxification in multiple clinical trials (Liu et al.,

2009). Many researchers have reported a newer role for the cognitive enhancers in the management of opioid withdrawal (Dhonnchadha and Kantak, 2011; Myers and

Carlezon Jr, 2010).

The objective of this work was firstly to assess Bacopa monnieri a renowned ayurvedic nootropic plant extracts for the contents of Bacoside A3, Bacopasaponin C and Bacopaside II, secondly to assess its effect on behavioral signs of opioid withdrawal, and thirdly to examine changes in the levels of neurotransmitters ( DA,

HVA, DOPAC, 5-HT, 5HIAA, and NA) in the brain areas of rats implicated in naloxone precipitated morphine withdrawal syndrome.

8.2. Materials and methods

Details of material and methods are available in Chapter 2 Methodology, page 49-52.

8.3. Results

8.3.1. Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C in Mt-ext BM and n-Bt-ext BM In this study the HPLC analysis of Mt-ext BM revealed the presence of Bacoside A, major components i.e. Bacoside A3, Bacopasaponin c and Bacopaside II. Our results further indicated that the quantity of these Bacopasides were 1.6 µg (Bacopasaponin

C), 5.4 µg (Bacoside A3), and 1.7 µg (Bacopaside ll), in each gram of Mt-ext BM.

While n-Bt-ext BM was found to have Bacoside A3 (3.56 ug/mg), Bacopasaponin C

(2.60 µg/mg) and Bacopaside ll as (3.30 µg/mg), likewise the total quantity of

Bacoside A three major components was 9.5 µg /mg of n-Butanol fraction equivalent

15.67ug/gram of dry powder.

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.2. Effect of Mt-ext BM on body weight loss in naloxone precipitated morphine withdrawal As shown in figure 8.1 & 8.2, both acute and chronic treatment of Mt-ext BM all three doses (10, 20 or 30 mg/kg orally) significantly reduced body weight loss in rats undergoing naloxone precipitated morphine withdrawal. ANOVA followed by

Tukey’s Post hoc analysis revealed reduction in body weight loss statistically significant P<0.01 as compared to saline treated group. Additionally chronic treatment of Mt-ext BM (10, 20 or 30 mg/kg orally) also significantly reduced body weight loss during naloxone precipitated morphine withdrawal (Fig 8.2).

15 MP.+SA L(Acute)+NLX

MP+Mt-ext BM 10 mg (Acute) +NLX MP+ Mt-ext BM 20 mg(Acute) +NLX

MP+ Mt-ext BM 30 mg(Acute) +NLX 10 * * ** **

5 Weight Loss (grams)

Figure 8.1. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on body weight loss in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 or 30 mg/kg orally, one hour before naloxone challenge. Treatment groups were significantly different from morphine naloxone treatment group **p<0.01.Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. **p<0.01

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL (chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10 * * *

5 Weight (grams) Loss

Figure 8.2. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on body weight loss in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Treatment groups were significantly different from morphine naloxone treatment group *p< 0.05.Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.3. Effect of Mt-ext BM on naloxone precipitated morphine withdrawal jumping behavior As depicted in Fig 8.3 and 8.4, both acute and chronic treatment of Mt-ext BM all three doses tested (10, 20 or 30 mg/kg orally) significantly lowered number of jumps in naloxone precipitated morphine withdrawal, in rats (n=6) as compared to saline treated group.

40 MP.+SAL (Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM 20 mg(Acute)+NLX 30 MP+ Mt-ext BM 30 mg(Acute)+NLX

20 * ** * No of Jumps of No

Figure 8.3. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Jumping behavior in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 or 30 mg/kg orally, one hour before naloxone challenge. Treatment groups were significantly different from morphine naloxone treatment group .Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01,

127

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

40 MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX 30 MP+ Mt-ext BM 30 mg ( Chronic)+NLX

20 * ** Number of jumps of Number 10

Figure 8.4. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on jumping behavior in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Treatment groups were significantly different from morphine naloxone treatment group .Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis.*p< 0.05

128

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.4. Effect of Mt-ext BM on naloxone precipitated morphine withdrawal induced writhes

As shown in Fig 8.5, Acute treatment with Mt-ext BM all three doses tested (10, 20 or 30 mg/kg orally) failed to show any significant effects on abdominal constrictions induced by naloxone precipitated morphine withdrawal in rats, However chronic treatment with only 30 mg / kg dose exhibited significant decrease (*p< 0.05) in abdominal constrictions in naloxone precipitated morphine withdrawal (n=6), as compared to saline (Fig 8.6).

20 MP.+SAL(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM20mg(Acute)+NLX 15 MP+ Mt-ext BM 30 mg(Acute)+NLX

Number of writhes of Number 5

Figure 8.5. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on writhes in animals undergoing naloxone precipitated withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge.

129

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

20 MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX

15 MP+ Mt-ext BM 30 mg ( Chronic)+NLX

10 * Number of writhes 5

Figure 8.6. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Abdominal constrictions in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 or 30 mg/kg orally along with morphine dependence schedule.Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

130

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.5. Effect of Mt-ext BM on Squeal on touch behavior in naloxone precipitated morphine withdrawal Acute treatment with Mt-ext BM (10, 20 mg/kg orally) did not significantly reduce squeal on touch behavior in rats undergoing naloxone precipitated morphine withdrawal, except the highest dose 30 mg/kg, as shown in Fig 8.7. Nevertheless chronic treatment with Mt-ext BM ameliorated squeal on touch behavior significantly at the doses 20 and 30 mg/kg orally, while 10 mg /kg chronic dosing failed to affect squeal on touch behavior induced by naloxone precipitated morphine withdrawal in rat (n=6) Fig 8.8.

15 MP.+SAL (Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX 10 MP+ Mt-ext BM20mg(Acute)+NLX MP+ Mt-ext BM 30 mg(Acute)+NLX

Squeal onSqueal touch 5

Figure 8.7. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Squeal in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge. Values were expressed as mean±SEM.

131

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX * MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10 * Squeal on Squeal touch

Figure 8.8. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on squeal on touch in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. In Treatment groups 20 or 30 mg/kg significantly lowered squeal on touch as compared morphine naloxone treatment group. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. *p< 0.05

132

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.6. Effect of Mt-ext BM treatment on naloxone precipitated morphine withdrawal salivation Both acute and chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) failed to show any significant impact on salivation significantly in naloxone precipitated morphine withdrawal animals as compared to saline treated group as shown in Fig 8.9 and Fig 8.10.

15 MP.+SAL(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM 20 mg(Acute)+NLX MP+ Mt-ext BM 30 mg(Acute)+NLX 10 Salivation 5

Figure 8.9. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on salivation in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge.

133

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SA L(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10 Salivation 5

Figure 8.10. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on salivation in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule.

134

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.7. Effect of Mt-ext BM treatment on raring behavior in naloxone precipitated morphine withdrawal Acute treatment with all three doses of Mt-ext BM (10, 20 or 30 mg/kg orally) slightly enhanced raring behavior but could not achieve statistical significance Fig

8.6, similarly chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) failed to affect significantly raring behavior in animals undergoing morphine withdrawal, although a lowering trend was present but suppression of raring is statistically insignificant Fig 8.13.

20 MP.+SA L(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM20mg(Acute)+NLX 15 MP+ Mt-ext BM 30 mg(Acute)+NLX

10 Rearing

Figure 8.11. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on raring in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge. Treatment groups are not significantly different from morphine naloxone treatment group. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test.

135

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10 Rearing

Figure 8.12. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on raring behavior in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 or 30 mg/kg orally along with morphine dependence schedule. Values were expressed as mean±SEM.

136

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.8. Effect of Mt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal Both acute and chronic treatment at all three doses of Mt-ext BM (10, 20 or 30 mg/kg orally) were ineffective in reducing incidence of diarrhoea significantly in naloxone precipitated morphine withdrawal animals (n=6) as compared to saline treated group

(Fig 8.13 & 8.14).

20 MP.+SAL(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM20mg(Acute)+NLX 15 MP+ Mt-ext BM 30 mg(Acute) +NLX

10 Diarrhoea score 5

Figure 8.13. Effect of aute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on diarrhoea in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge. Treatment groups are not significantly different from morphine naloxone treatment group. Values were expressed as mean±SEM. .

137

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX 10 MP+ Mt-ext BM 30 mg ( Chronic)+NLX

5 Diarrhoea score

Figure 8.14. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on diarrhoea in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Values were expressed as mean±SEM.

138

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.9. Effect of Mt-ext BM treatment on incidence of teeth chattering in naloxone precipitated morphine withdrawal Acute and chronic treatment of Mt-ext BM (10, 20 or 30 mg/kg orally) failed to affect significantly teeth chattering induced by naloxone precipitated morphine withdrawal in rats (8.15), except 30 mg / kg chronic dose significantly lowered Teeth chattering in naloxone precipitated morphine withdrawal (n=6), as compared to saline

(Fig 8.16).

15 MP.+SAL(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM 20 mg(Acute)+NLX MP+ Mt-ext BM 30 mg(Acute)+NLX 10

5 Teeth chattering

Figure 8.15. Effect of Acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Teeth Chattering in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically Mt-ext BM 10, 20 and 30 mg/kg orally, one hour before naloxone challenge. Treatment groups are not significantly different from treatment morphine naloxone group. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test.

139

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 M P.+SA L(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10

5 Teeth chattering

Figure 8.16. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Teeth Chattering in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Only 30 mg/kg dose significantly lowered teeth chattering as compared to morphine naloxone group *p<0.05.Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test.

140

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.10. Effect of Mt-ext BM treatment on incidence of wet dog shake behavior in naloxone precipitated morphine withdrawal Acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) had no significant effect on wet dog shake behavior in animals undergoing naloxone precipitated morphine withdrawal Fig 8.17, similarly chronic treatment of 10 mg/kg Mt-ext BM failed to affect significantly wet dog shake behavior in animals, However larger doses 20 and

30 mg/kg significantly lowered wet dog shake behavior in animals in naloxone precipitated morphine withdrawal Fig 8.18.

MP.+SAL(Acute)+NLX MP+Mt-ext BM 10 mg(Acute)+NLX MP+ Mt-ext BM 20 mg(Acute)+NLX 10 MP+ Mt-ext BM 30 mg(Acute)+NLX

5 Wet dog shakes

Figure 8.17. Effect of acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on wet dog shake in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Values were expressed as mean±SEM..

141

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL(Chronic)+NLX MP+Mt-ext BM 10 mg (Chronic)+NLX MP+ Mt-ext BM 20 mg ( Chronic)+NLX MP+ Mt-ext BM 30 mg ( Chronic)+NLX 10

* *

5 Wet dog shakes

Figure 8.18. Effect of chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) on Wet dog shake behavior in animals undergoing naloxone precipitated morphine withdrawal (n=6). All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received Mt-ext BM 10, 20 and 30 mg/kg orally along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

142

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.11. Effect of n-Bt-ext BM treatment on body weight loss in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (10 and 15 mg/kg orally) significantly reduced body weight loss (p<0.05, ANOVA, followed by Tukey’s Post hoc analysis) during naloxone precipitated morphine withdrawal in animals except 5 mg/kg n-Bt-ext BM failed to affect body weight loss during withdrawal any significantly Fig 8.19.

Moreover ANOVA with Tukey’s post hoc analysis revealed that chronic treatment with n-Bt-ext BM reduced body weight loss highly significantly at doses 5, 10mg/kg

(p<0.01) and p<0.001 at 15 mg/kg, during naloxone precipitated morphine withdrawal Fig 8.20.

15 MP.+SAL(Acute)+NLX MP+ nBt-ext BM 5 mg(Acute) +NLX MP+ nBt-ext BM 10 mg(Acute) +NLX

MP+ nBt-ext BM 15 mg(Acute) +NLX 10 * *

5 Weight loss (grams) Weight loss

Figure 8.19. Effect of acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on body weight loss during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 and 15 mg/kg orally) one hour before naloxone challenge. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

143

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

10 MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX ** ** MP+ nBt-ext BM 10 mg (Chronic) +NLX *** MP+ nBt-ext BM 15 mg (Chronic) +NLX

Weight Loss Weight 5

Figure 8.20. Effect of chronic treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on body weight loss during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight . Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. **p< 0.01, ***p< 0.001

144

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.12. Effect of n-Bt-ext BM treatment on incidence of jumping behavior in naloxone precipitated morphine withdrawal Both acute and chronic treatment of n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly inhibited jumping behavior in naloxone precipitated morphine withdrawal in animals as shown in Fig 8.21 and 8.22. ANOVA followed by Tukey’s

Post hoc analysis revealed significant reduction after acute treatment 5, 10 mg/kg

(*p< 0.05), 15 mg/kg (**p< 0.01) as well as chronic treatment at doses 5, 10 and 15 mg/kg (**p< 0.01).

40 MP.+SA L(A cute)+NLX MP+ nBt-ext BM 5 mg(Acute) +NLX MP+ nBt-ext BM 10 mg(Acute) +NLX 30 MP+ nBt-ext BM 15 mg (Acute)+NLX

20 * * ** Number of jumps of Number 10

Figure 8.21. Effect of acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on Jumping behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) one hour before naloxone challenge. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p< 0.01

145

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

30 MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX MP+ nBt-ext BM 10 mg (Chronic) +NLX 20 MP+ nBt-ext BM 15 mg (Chronic) +NLX

** ** Number of Jumps of Number 10 **

Figure 8.22. Effect of acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on jumping behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. **p< 0.01.

146

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.13. Effect of n-Bt-ext BM treatment on incidence of writhes in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) failed to show any significant effect on abdominal constrictions induced by naloxone precipitated morphine withdrawal as shown in Fig 8.23. Nevertheless, chronic treatment with n-

Bt-ext BM at all three doses significantly lowered (*p< 0.05), withdrawal induced abdominal constrictions in rats undergoing naloxone induced morphine withdrawal as shown in Fig 8.24.

20 M P.+SA L(A cute)+NLX MP+ nBt-ext BM 5 mg(Acute) +NLX MP+ nBt-ext BM 10 mg(Acute) +NLX 15 MP+ nBt-ext BM 15 mg(Acute) +NLX

Number of writhesNumber of 5

Figure 8.23. Effect of acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on writhing behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Values were expressed as mean±SEM.

147

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX 10 * * * MP+ nBt-ext BM 10 mg (Chronic) +NLX MP+ nBt-ext BM 15 mg (Chronic) +NLX

Numberwrithes of 5

Figure 8.24. Effect of chronic treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on writhing behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

148

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.14. Effect of n-Bt-ext BM treatment on incidence of Squeal on touch behavior in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) did not significantly lower squeal on touch on touch behavior induced by naloxone precipitated morphine withdrawal as shown in Fig 8.25. However chronic treatment with n-Bt-ext BM all three doses significantly (*p< 0.05), decreased squeal on touch behavior in rats as shown in Fig 8.26.

M P.+SA L(A cute)+NLX 15 MP+ nBt-ext BM 5 mg(Acute) +NLX MP+ nBt-ext BM 10 mg(Acute) +NLX MP+ nBt-ext BM 15 mg(Acute) +NLX

10 Squeal on touchSqueal 5

Figure 8.25. Effect of acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on squeal on touch behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. All three doses failed to significantly lower squeal on touch behavior in animals as compared to morphine naloxone group. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test.

149

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX 10 MP+ nBt-ext BM 10 mg (Chronic) +NLX MP+ nBt-ext BM 15 mg (Chronic) +NLX *** *** *** Squeal on touch Squeal 5

Figure 8.26 Effect of chronic treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on squeal on touch behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. ***p< 0.001

150

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.15. Effect of n-Bt-ext BM treatment on incidence of teeth chattering in naloxone precipitated withdrawal Acute treatment with n-Bt-ext BM (5 and 10 mg/kg orally) had no significant effect on teeth chattering induced by naloxone precipitated morphine withdrawal except 15 mg/kg dose as shown in Fig 8.27. However chronic treatment of n-Bt-ext BM all three doses significantly decreased withdrawal induced teeth chattering behavior in rats undergoing naloxone induced morphine withdrawal as shown in Fig 8.28.

15 MP.+SAL (Acute)+NLX MP+ nBt-ext BM 5 mg (Acute)+NLX MP+ nBt-ext BM 10 mg(Acute) +NLX MP+ nBt-ext BM 15 mg(Acute) +NLX 10

5 Teeth chattering

Figure 8.27. Effect of acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on teeth chattering behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge). Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. *p< 0.05

151

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

15 MP.+SAL (Acute)+NLX MP+ nBt-ext BM 5 mg (Acute)+NLX MP+ nBt-ext BM 10 mg(Acute) +NLX MP+ nBt-ext BM 15 mg(Acute) +NLX 10

5 Teeth chattering

Figure 8.28. Effect of chronic treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on teeth chattering behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. **p< 0.01, ***p< 0.001),

152

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.16. Effect of n-Bt-ext BM treatment on incidence of wet dog shakes in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5 and 10 mg/kg orally) did not significantly lower wet dog shakes induced by naloxone precipitated morphine withdrawal, but 15 mg /kg dose significantly lowered ( *p< 0.05) wet dog shakes in animals, as shown in Fig

8.29. Additionally chronic treatment of n-Bt-ext BM all three doses (5, 10 or 15 mg/kg orally) also significantly lowered withdrawal induced wet dog shakes behavior in rats undergoing naloxone induced morphine withdrawal as shown in Fig 8.30.

10 MP.+SAL (Acute)+NLX MP+ nBt-ext BM 5 mg(Acute) +NLX * MP+ nBt-ext BM 10 mg(Acute) +NLX MP+ nBt-ext BM 15 mg (Acute)+NLX 5 Wet dog shakes

Figure 8.29. Effect of acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on wet dog shakes behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05

153

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

10 MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX * MP+ nBt-ext BM 10 mg (Chronic) +NLX ** MP+ nBt-ext BM 15 mg (Chronic) +NLX 5

Wet dog shakes ***

Figure 8.30. Effect of chronic treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on wet dog shakes behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p< 0.01, ***p< 0.005

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.17. Effect of n-Bt-ext BM treatment on incidence of salivation in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) did not significantly decrease salivation induced by naloxone precipitated morphine withdrawal as shown in Fig 8.31. However chronic treatment with n-Bt-ext BM all three doses significantly

(*p< 0.05) lowered salivation in rats undergoing naloxone induced morphine withdrawal as shown in Fig 8.32.

M P.+SA L(A cute)+NLX MP+ nBt-ext BM 5 mg(Acute) +NLX MP+ nBt-ext BM 10 mg (Acute)+NLX 10 MP+ nBt-ext BM 15 mg(Acute) +NLX Salivation 5

Figure 8.31. Effect of acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on salivation behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Values were expressed as mean±SEM.

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

6 MP.+SAL(Chronic)+NLX * ** MP+ nBt-ext BM 5 mg (Chronic) +NLX 4 MP+ nBt-ext BM 10 mg (Chronic) +NLX MP+ nBt-ext BM 15 mg (Chronic) +NLX Salivation

Figure 8.32. Effect of chronic treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on salivation behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Newman- Keuls Multiple Comparison Test. *p< 0.05

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.18. Effect of n-Bt-ext BM treatment on incidence of Raring in naloxone precipitated withdrawal

Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) did not significantly lower raring induced by naloxone precipitated morphine withdrawal as shown in Fig

8.33, but chronic treatment with all three doses lowered raring behavior highly significantly (***p< 0.001) in rats undergoing naloxone induced morphine withdrawal (Fig 8.34).

20 MP.+SAL+NLX MP+ nBt-ext BM 5 mg +NLX MP+ nBt-ext BM 10 mg +NLX 15 MP+ nBt-ext BM 15 mg +NLX

10 Rearing

Figure 8.33. Effect of acute treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on raring behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Values were expressed as mean±SEM.

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

MP.+SAL(Chronic)+NLX MP+ nBt-ext BM 5 mg (Chronic) +NLX *** *** *** MP+ nBt-ext BM 10 mg (Chronic) +NLX Rearing MP+ nBt-ext BM 15 mg (Chronic) +NLX 5

Figure 8.34. Effect of chronic treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on raring behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. ***p< 0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.19. Effect of n-Bt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) did not significantly lower diarrhoea induced by naloxone precipitated morphine withdrawal as shown in

Fig 8.35. Nevertheless chronic treatment with n-Bt-ext BM at the highest dose tested i.e. 15 mg mg/kg dose significantly decreased diarrhoea in rats undergoing naloxone induced morphine withdrawal (Fig 8.36).

MP.+SAL(Acute)+NLX 10 MP+ nBt-ext BM 10 mg(Acute) +NLX MP+ nBt-ext BM 10 mg (Acute)+NLX MP+ nBt-ext BM 15 mg (Acute)+NLX Diarrhoea score 5

Figure 8.35. Effect of acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) on diarrhoea behavior during naloxone precipitated morphine withdrawal. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received identically n-Bt-ext BM (5, 10 or 15 mg/kg orally) orally, one hour before naloxone challenge. Values were expressed as mean±SEM.

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MP.+SAL (chronic)+NLX 10 * * * MP+ nBt-ext BM 5 mg (chronic)+NLX MP+ nBt-ext BM 10 mg(chronic) +NLX MP+ nBt-ext BM 15 mg(Chronic) +NLX

5 Diarrhoea score

Figure 8.36. Effect of chronic treatment with n-Bt-ext BM (5, 10 and 15 mg/kg orally) on incidence of diarrhoea during naloxone precipitated morphine withdrawal.. All groups were made dependent on morphine (twice daily), as per eight days schedule starting from 8 mg/kg/day and gradually increasing to 65 mg/kg/day on day eight. Saline group received saline one hour before naloxone treatment while rest groups received n-Bt-ext BM (5, 10 or 15 mg/kg orally) along with morphine dependence schedule. Values were expressed as mean±SEM, applying ANOVA followed by Newman-Keuls Multiple Comparison Test. *p< 0.05

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8.3.20. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally) on frontal cortex levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal As shown in table 8.1 and 8.2, both Acute and chronic treatment of Mt-ext BM (10,

20 or 30 mg/kg orally) highly significantly lowered (***p< 0.001) NA concentration in frontal cortex of animals undergoing naloxone precipitated morphine withdrawal.

Additionally 10 mg/kg acute treatment significantly (*p< 0.05) decreased DA also, while 20 mg/kg and 30 mg/kg identically increases HVA and 5HIAA levels.

Moreover chronic treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) significantly decreased 5-HT and 5HIAA levels.

Table 8.1. Effect of normal saline and Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Frontal cortex tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT

Saline 358±21 39±05 205±32 75±17 17±03 145±28

Mt-ext 10mg/kg) 145±19*** 13±10 138±60* 56±08 42±06* 127±16

Mt-ext (20 g/kg) 110±30*** 41±14 146±14 43±09 64±08*** 121±90

Mt-ext 30mg/kg) 159±16*** 44±08 223±48 190±19*** 21±5 111±11 † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue. Values are significantly different as compares to saline treated group, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, ***p<0.001

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Table 8.2. Effect of normal saline and Mt-ext BM chronic treatment (10, 20 or 30 mg/kg orally) on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Frontal cortex tissue concentrations† DOPA Treatments NA DA 5HIAA HVA 5-HT C Saline 372±17 32±12 275±25 98±15 18±03 178±13 Mt-ext 10mg/kg) 129±12*** 54±19 215±10 54±16* 150±13 93±10*** Mt-ext (20 g/kg) 124±60*** 33±15 299±24 29±10*** 134±19 62±19*** Mt-ext 30mg/kg) 159±16*** 44±08 231±48 75±10 37±15 112±11** † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, ***p<0.001

8.3.21. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal As depicted in table 8.3 and 8.4, both acute and chronic treatment with Mt-ext BM treatment (10, 20 and 30 mg/kg orally) significantly increased NA concentration in striatal tissues, additionally acute treatment with 10 mg/kg dose also significantly raised DA and DOPAC concentration. Moreover acute treatment with 20 mg/kg dose of Mt-ext BM also significantly raised HVA and 5HIAA concentration and chronic treatment with Mt-ext BM (10, 20 and 30 mg/kg orally) significantly raised DOPAC and HVA concentration in striatal tissues as compared to saline treated group.

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Table 8.3. Effect of Mt-ext BM acute treatment (10, 20 and 30 mg/kg orally) and saline on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Striatal tissue concentrations†

Treatments NA DOPAC DA 5HIAA HVA 5-HT

Saline 42±13 148±17 1815±62 50 ±13 33 ±15 85±10

Mt-ext 10mg/kg) 99 ±33 355±38*** 526±38* 73±20 41±17 57±10

Mt-ext (20 g/kg) 99±24* 111±12 1789±42 170±45* 110±56*** 85±20

Mt-ext 30mg/kg) 129±29** 273±76** 1602±66 76±17 34±13 95±19

† Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying anova followed by tukey’s post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

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Table 8.4. Effect of Mt-ext BM chronic treatment (10, 20 or 30 mg/kg orally) and saline and on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal Striatal tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 42±21 148 ±17 1781±122 50 ±13 33±15 85±10 Mt-ext 10mg/kg) 273±30*** 72±25* 1845±113 31±16 123±37* 97±13 Mt-ext (20 g/kg) 126±13* 264±68** 1736±101 32±08 118±27* 93±14 Mt-ext 30mg/kg) 146±16** 273±76** 1436±151 76±17 34±13 95±19 † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

8.3.22 Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal Acute treatment with Mt-ext BM (30 mg/kg orally) significantly increased NA,

5HIAA, HVA, and 5-HT while, (20 mg/kg orally) significantly raised DOPAC and

5HIAA in hippocampus. Acute treatment with Mt-ext BM 10 mg/kg dose also significantly raised DOPAC in hippocampus of the animals undergoing naloxone precipitated morphine withdrawal (Table 8.5). chronic treatment with Mt-ext BM at all three doses tested significantly increased NA concentration, while a upsurge trend in DA was visible but only 10 mg/kg dose produced statistically significant increase in DA in hippocampal tissues (Table 8.6).

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Table 8.5. Effect of Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) and saline on hippocampal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Hippocampus tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 36±14 24±06 143±34 27±02 37±03 46±11 Mt-ext 10mg/kg) 43±16 190±16*** 137±36 35±02 53±07 33±06 Mt-ext (20 g/kg) 42±19 89±04*** 120±24 161±23*** 53±16 56±17 Mt-ext 30mg/kg) 61±6* 41±11 91±25 49±06** 77±20** 69±12* † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001 Table 8.6. Effect of normal saline and Mt-ext BM chronic treatment (10, 20 or 30 mg/kg orally) on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Hippocampus tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT

Saline 40±10 24±03 150±25 20±05 39±16 68±11

Mt-ext 10mg/kg) 165±10*** 37±09 223±18* 36±08 24±15 50±13

Mt-ext (20 g/kg) 116±14*** 34±11 201±25 45±06 21±4± 66±13

Mt-ext 30mg/kg) 124±20*** 23±16 189±20 43±18 57±15 69±10 † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.23. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal Acute treatment with Mt-ext BM (10, 20 or 30 mg/kg orally) significantly raised NA,

DA, DOPAC, HVA and 5HIAA in nucleus accumbens (Table 8.7). Chronic treatment also significantly raises NA, DOPAC, DA and HVA in nucleus accumbens. Chronic treatment also induced an upsurge trend in 5HIAA, but only 20 mg/kg dose induced upsurge attained statistical significance (Table 8.8).

Table 8.7. Effect of Mt-ext BM acute treatment (10, 20 or 30 mg/kg orally) and saline on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Nucleus accumbens tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 50±11 14±03 102±08 41±18 22±12 71±14 Mt-ext 126±12*** 44±06*** 242±20*** 101±6** 76±15*** 115±17 10mg/kg) Mt-ext (20 119±10** 29±04* 153±19* 92±17* 53±19* 91±10 g/kg) Mt-ext 154±18*** 49±07*** 209±22*** 90±14* 34±13* 112±19 30mg/kg) † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05,**p<0.01, ***p<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Table 8.8. Effect of saline and Mt-ext BM chronic treatment (10, 20 or 30 mg/kg orally) on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and

5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Nucleus accumbens tissue concentrations†

Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 55±14 18±12 102±09 41±17 22±2 71±14 Mt-ext 10mg/kg 105±15*** 44±09* 111±10 61±8 55±15*** 62±19 Mt-ext (20 g/kg 111±17*** 41±10* 159±15** 81±10* 46±14** 154±14*** Mt-ext 0mg/kg 101±18*** 61±11*** 189±12*** 71±12 73±17*** 82±12 † Concentration levels were expressed as mean ± S.E.M ng/500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05, **p<0.01, ***p<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.24. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly decreased

NA, 5-HT (10 and 20 mg/kg) and increased, DA DOPAC and HVA in rats frontal

cortex. An upward trend was noted in 5HIAA but only 15 mg/kg dose effect on

5HIAA attained statistical significance (Table 8.9). Chronic treatment of n-Bt-ext BM

(5, 10 or 15 mg/kg orally) significantly decreased NA and significantly increased

5HIAA, DOPAC, and HVA levels in fontal cortex. A strong upward trend in DA was

visible but could not gain statistical significance (Table 8.10).

Table 8.9. Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Frontal cortex tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 368±21 39±11 205±32 75±16 17±3 145±09 n-Bt-ext mg/kg 192±18*** 195±21*** 714±78*** 99±18 265±14*** 48±16*** n-Bt-ext 10 g/kg 195±12*** 104±19** 545±12** 99±13 113±19*** 45±08*** n-Bt-ext 15 g/kg 197±11*** 152±12*** 448±22* 175±15*** 215±44*** 211±11 † Concentration levels were expressed as mean± S.E.M ng/500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *P<0.05, **P<0.01, ***P<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Table 8.10. Effect of normal saline and n-Bt-ext BM chronic treatment (5, 10 or 15 mg/kg orally) on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Frontal cortex tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT

Saline 372±17*** 32±02 275±25 65±16 18±03 178±13

n-Bt-ext 5 mg/kg 223±11*** 62±09* 392±38 110±30* 137±15*** 168±24

n-Bt-ext 10 mg/kg 231±21*** 82±15*** 370±25 119±23** 151±11*** 128±12

n-Bt-ext 15 mg/kg 215±5*** 62±14** 373±25 113±16** 100±08*** 147±14 † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05, **p<0.01, ***p<0.005

8.3.25. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on striatum tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly increased

NA, 5HIAA, and HVA in striatum. An upward trend was visible in DOPAC also but only 10 and 15 mg/kg dose induced changes in DOPAC were found to be statistically significant (Table 8.11). Chronic treatment of n-Bt-ext BM statistically raised NA,

HVA, and 5HIAA in striatum, while statistically significant descending trend was observed in DOPAC and DA. Only 15 mg dose induced serotonin upsurge which was statistically significant (Table 8.12).

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Table 8.11. Effect of normal saline and n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Striatal tissue concentrations† (Acute) Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 42±05 148 ±17 1798±48 50 ±03 33±5 85±10

n-Bt-ext 5mg/kg 165±12*** 199±12 1489±41 165±15*** 190±16*** 89±16

n-Bt-ext 10 mg/kg 258±27*** 209±12* 1701±48 136±30*** 151±17*** 83±18

n-Bt-ext 15 mg/kg 257±23*** 391±19*** 1629±51 172±15*** 153±28*** 216±24*** † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05, ***p<0.001 Table 8.12. Effect of normal saline and n-Bt-ext BM chronic treatment (5, 10 or 15 mg/kg orally) on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Striatal tissue concentrations† (Chronic)

Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 42.±05 148±17 1781±12 50.±03 33.±05 85.±10 n-Bt-ext mg/kg 323±26*** 20±05*** 310±34*** 145±3*** 120±14*** 174±13*** n-Bt-ext 10 mg/kg 153±22** 56±07*** 344±21*** 138±8*** 110±17*** 153±16*** n-Bt-ext 15 mg/kg 160±17** 16±03*** 153±19*** 90±12** 119±15*** 110±10* † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

8.3.26. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on Hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly increase

NA, DOPAC, HVA, and 5 HIAA (Table 13). An upward trend is visible in DA, but

only 5 mg/kg dose induced DA change which is statistically significant (Table 8.13).

Chronic treatment of Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally)

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal significantly raised NA and 5HIAA in hippocampus (Table 8.14). An upward trend is seen in DA and 5-HT but only 5 and 10 mg /kg attains statistical significance (Table

8.14).

Table 8.13. Effect of n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) and normal saline on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Hippocampus tissue concentrations† Acute Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 36±14 24±06 143±19 27±02 37±30 46±12 n-Bt-ext 5mg/kg 215±24*** 173±14*** 399±49*** 63±08** 246±12*** 53±07 n-Bt-ext 10 235±30*** 127±12*** 242±46 59±12** 133±13*** 72±`15 mg/kg n-Bt-ext 15 110±10* 89±10** 205±23 55±13* 145±17*** 57±10 mg/kg † Concentration levels were expressed as mean± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p<0.05, **p<0.01, ***p<0.001 Table 8.14. Effect of n-Bt-ext BM chronic treatment (5, 10 or 15 mg/kg orally) and saline on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Hippocampus tissue concentrations† Chronic Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 40±09 24±13 150±15 20±05 39±16 68±14 n-Bt-ext 581±16*** 62±18*** 571±71*** 136±10*** 45±10 113±13 5mg/kg n-Bt-ext10 208±11* 35±30 163±16 67±19** 115±04*** 169±19*** mg/kg n-Bt-ext 15 255±14** 21±04 158±12 51±06* 98±06*** 137±11 mg/kg † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

8.3.27. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly increased

NA, DOPAC,DA and 5HIAA in nucleus accumbens, while a statistically significant increase was observed in 5-HT and 5HIAA only at the highest dose 15 mg/kg (Table

8.15). Chronic treatment also induced a significant increase in NA, DA, 5HIAA, and

HVA. Moreover upward trend was seen in 5-HT that achieved statistical significance at 5 and 10 mg/kg doses (Table 8.16).

Table 8.15. Effect of n-Bt-ext BM acute treatment (5, 10 or 15 mg/kg orally) and saline on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Nucleus accumbens tissue concentrations† Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 50±05 14±03 102±08 41±09 22±03 71±14 n-Bt- 255±16*** 140±22*** 405±34*** 79±11 261±18*** 75±10 ext5mg/kg n-Bt-ext 10 163±07*** 104±14** 373±27*** 78±10 151±21*** 69±13 g/kg n-Bt-ext 15 227±29*** 116±21** 518±37*** 187±16*** 122±16*** 249±12*** g/kg † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. **p<0.01, ***p<0.001

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

Table 8.16. Effect of normal saline and n-Bt-ext BM chronic treatment (5, 10 or 15 mg/kg orally) on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in rats undergoing naloxone precipitated morphine withdrawal

Nucleus accumbens tissue concentrations†

Treatments NA DOPAC DA 5HIAA HVA 5-HT Saline 50±05 14±03 102±08 41±08 22±02 71±14 n-Bt-ext mg/kg 389±34*** 22±05 277±26** 121±06*** 164±29*** 157±15** n-Bt-ext 10 135±07* 21±05 294±22** 116±11*** 83±08* 153±06** g/kg n-Bt-ext 15 128±15* 16±03 228±08* 89±03*** 119±05*** 95±10 g/kg † Concentration levels were expressed as mean ± S.E.M ng/ 500 mg of wet tissue, applying ANOVA followed by Tukey’s Post hoc analysis. *p< 0.05, **p<0.01, ***p<0.001

8.4. Discussion

Our results indicate that acute and chronic treatment of methanolic extract at all three doses tested (10, 20 or 30 mg/kg orally) significantly inhibited behavioral parameters of naloxone precipitated morphine withdrawal in rats. All three doses significantly lowered major parameters of opioid withdrawal like weight loss, jumping behavior, squeal on touch, and wet dog shake behavior through chronic treatment although acute dosing of Mt-ext BM also significantly inhibits body weight loss and jumping behavior in animals undergoing withdrawal (Fig 8.2-36). Acute and chronic treatment of Mt-ext BM failed to affect naloxone precipitated morphine withdrawal induced teeth chattering (Fig 8.15-8.16) diarrhoea (Fig 8.13-8.14) and salivation (Fig 8.9-8.10) and abdominal constriction (Fig 8.5-8.6, except 30 mg/kg chronic dosing).

Additionally one prominent effect that is note worthy is significant lowering of squeal on touch behavior by Mt-ext BM 30 mg/ kg acute dosing as shown in Fig 8.7. This might be because of its antinociceptive effect, although BM has already been reported

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal to have significantly lowered diabetic neuropathic pains and hyperalgesia. Our findings suggest its significant effect in lowering withdrawal induced hyperalgesia and discomfort.

Further more Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly inhibited major parameters of opioid withdrawal in all three doses, i.e. body weight loss ( Fig 8.19-8.20) and jumps (Fig 8.21-8.22) which is a promising sign although squeal on touch (Fig 8.25) and wet dog shakes (Fig 8.29) were also inhibited significantly by the 15 mg /kg acute dose of n-Bt-ext BM.

Like Mt-ext BM acute effects, n-Bt-ext BM (5, 10 or 15 mg/kg orally) failed to lower significantly the diarrhoea (Fig 8.35), abdominal constrictions (Fig 8.23), raring (Fig

8.33), salivation (Fig 8.31) except teeth chattering (Fig 8.27).The major signs of opioid withdrawal are the result of Locus coeruleus (LC) hyperactivity, with major involvement of noradrenergic pathways hyperactivity (Gerra et al., 2001). Many till now reports highlight that serotonergic pathways indirectly or Alpha adrenoreceptor agonists directly inhibits noradrenergic hyperactivity elicited by naloxone precipitated morphine withdrawal in LC and hypothalamus (Devoto et al., 2002; Gerra et al.,

2001; Raith and Hochhaus, 2004). There is a close mechanistic correlation between acute stress and opioid withdrawal, varying only in placement of stimuli (Erb, 2010), and BM has been has been reported to have an adaptogenic effect also (Sheikh et al.,

2007) , so the contribution of the adaptogenic effect of BM cannot be ruled out in its ameliorating effect in opioid withdrawal (Sheikh et al., 2007). BM is also a renowned nootropic agent, and recently researchers have highlighted a newer potential role of nootropic in opioid withdrawal and some new interesting findings have a promising future outlook (Dhonnchadha and Kantak, 2011; Myers and Carlezon Jr, 2010) .

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Our neurotransmitters data indicate that all three doses of Mt-ext BM, in both acute and chronic treatment significantly lowered nor adrenaline in frontal cortex of the animals undergoing withdrawal (Table 8.11-8.12). Acute treatment with Mt-ext BM led to further lowering of DA and chronic Mt-ext BM led to lowering of 5-HT contents in frontal cortex. Mt-ext BM significantly raised contents of NA in striatum in both acute and chronic treatments, while significantly lowering of DA was observed in striatum of animals given Mt-ext BM in acute dosing regimen. In both acute and chronic dosing of Mt-ext BM, HVA contents were significantly increased reflecting DA turn over Table (8.5-8.6).

Hippocampus data reflect a paradox image as chronic treatment of Mt-ext BM led to the significant upsurge of NA contents, while acute treatment failed to modulate NA.

Previously Selective Serotonin Reuptake Inhibitors (SSRI) have been reported to lower somatic signs of opioid withdrawal, without altering NA contents in hippocampus (Gray, 2002). Additionally DA turnover was increased as significant upsurge in DOPAC was observed in hippocampus with acute treatment with Mt-ext

BM. Both acute and chronic treatment of Mt-ext BM significantly raised DA,

DOPAC and NA in nucleus accumbens (table 8.7 & 8.8) which validates the findings that paucity of DA during withdrawal leads to formation of many somatic signs of opioid withdrawal including dysphoria irritability, teeth chattering, wet dog shakes and depression (Pothos et al., 1991) . Chronic treatment of Mt-ext BM also raised 5-

HT level in nucleus accumbens (NAc) table 8.4 , thus depicting a reversal of morphine effects on NAc serotonergic system and thus alleviating somatic signs of opioid withdrawal (Spampinato et al., 1985) .

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Likewise both acute and chronic treatment of n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly lowered NA contents in frontal cortex of animals undergoing opioid withdrawal, with an added feature of raising significantly DA, DOPAC, HVA and

5HIAA (only 30 mg/kg dose) in acute treatment. While in chronic treatment NA was significantly lowered while DOPAC, HVA and 5HIAA were raised significantly with an upsurge trend for DA also but this upsurge was statistically insignificant (Table

8.6). This lowering of NA has great significance in mediation of major somatic signs of opioid withdrawal, and all drugs that lower NA in frontal cortex, lowers intensity of opioid withdrawal syndrome (Delfs et al., 2000; Jasmin et al., 2006; Rossetio et al.,

1993). Lowering of dopamine turnover in frontal cortex has close relationship with severity of some somatic signs of opioid withdrawal (Lyvers and Yakimoff, 2003), and n-Bt-ext BM induced upsurge of DA turn over with simultaneous decrease in NA might have contributed to the lowering of somatic signs of opioid withdrawal (Fig

8.19-34). Acute treatment with n-Bt-ext BM significantly raised NA, HVA, and

5HIAA in striatal tissues in all three doses and raised DOPAC with 10 mg/kg and 15 mg/kg dose Table 8.7. While chronic treatment significantly raised NA, HVA, 5-HT and 5HIAA striatum in all three doses, while significantly lower DA, and DOPAC concentration as shown in Table 8.8.

In hippocampus acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly increased DA, DOPAC, HVA, 5HIAA and NA, while 5-HT remained unaffected. While chronic treatment of n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly raised NA and 5 HIAA in all three doses and DA, DOPAC were significantly raised by 5 mg/kg chronic dosing only, while HVA was raised by 10 and

15 mg/kg chronic dosing. Although hippocampus DA turnover picture is somewhat elusive and is not clear in chronic treatment with n-Bt-ext BM (5, 10 and 15 mg/kg

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal orally), but somatic signs data have shown significant lowering of opioid withdrawal signs.

Acute treatment with n-Bt-ext BM (5, 10 or 15 mg/kg orally) significantly raised NA,

DOPAC, DA, HVA in NAc in all three doses, while only highest dose 15 mg/ kg significantly raised 5-HT and 5HIAA. The significant increase in DA, and serotonin in nucleus accumbens by acute treatment validate the behavioral findings in which Acute treatment with n-Bt-ext

BM (5, 10 or 15 mg/kg orally) significantly lowered opioid withdrawal signs in naloxone precipitated morphine withdrawal in animals. While in chronic treatment also n-Bt-ext BM (5,

10 or 15 mg/kg orally) significantly raised HVA, and 5HIAA, while 5-HT was also raised significantly by 5 mg/kg dose and 10 mg/kg dose table 8.15-8.16. Our findings reflect that

BM successfully ameliorates somatic signs of opioid withdrawal syndrome, accompanied by significant upsurge of DA, DOPAC, and 5HIAA in both nucleus accumbens and striatum.

The current findings further validate the preliminary findings, of future potential role of nootropics in the management of opioid withdrawal and dependence. The significant reduction of major signs of opioid withdrawal by BM might be due to, its adenosinergic (Sahoo et al., 2010), calcium channel blocking (Dar and Channa, 1999) and antioxidant effect (Bhattacharya et al., 2000) of BM as adenosinergic, nitric oxide synthase inhibitors and calcium channel blockers have been reported to lower physical signs of opioid withdrawal. Furthermore nitric oxide synthase inhibitory effect (Russo et al., 2003; Zarrindast et al., 2003) or GABAergic effect (Subhan et al.,

2010) of BM might have individually or collectively contributed to the inhibitory effects of BM in naloxone precipitated morphine withdrawal. The findings of the current in vivo work further validate the in vitro findings of Sumathi et al (Sumathi et al., 2002) which reported the significant effects of BM in isolated tissues of naloxone precipitated morphine withdrawal.

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8.1. Introduction 123

8.2. Materials and methods 124

8.3. Results 124

8.3.1. Chromatographic analysis of Bacoside A3, Bacopaside II, and Bacopasaponin C in Mt-ext BM and n‐Bt-ext BM 124 8.3.2. Effect of Mt-ext BM on body weight loss in naloxone precipitated morphine withdrawal 125 8.3.3. Effect of Mt-ext BM on naloxone precipitated morphine withdrawal jumping behavior 127 8.3.4. Effect of Mt-ext BM on naloxone precipitated morphine withdrawal induced writhes 129 8.3.5. Effect of Mt-ext BM on Squeal on touch behavior in naloxone precipitated morphine withdrawal 131 8.3.6. Effect of Mt-ext BM treatment on naloxone precipitated morphine withdrawal salivation 133 8.3.7. Effect of Mt-ext BM treatment on raring behavior in naloxone precipitated morphine withdrawal 135 8.3.8. Effect of Mt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal 137 8.3.9. Effect of Mt-ext BM treatment on incidence of teeth chattering in naloxone precipitated morphine withdrawal 139 8.3.10. Effect of Mt-ext BM treatment on incidence of wet dog shake behavior in naloxone precipitated morphine withdrawal 141 8.3.11. Effect of n-Bt-ext BM treatment on body weight loss in naloxone precipitated morphine withdrawal 143 8.3.12. Effect of n-Bt-ext BM treatment on incidence of jumping behavior in naloxone precipitated morphine withdrawal 145 8.3.13. Effect of n-Bt-ext BM treatment on incidence of writhes in naloxone precipitated morphine withdrawal 147 8.3.14. Effect of n-Bt-ext BM treatment on incidence of Squeal on touch behavior in naloxone precipitated morphine withdrawal 149 8.3.15. Effect of n-Bt-ext BM treatment on incidence of teeth chattering in naloxone precipitated withdrawal 151 8.3.16. Effect of n‐Bt-ext BM treatment on incidence of wet dog shakes in naloxone precipitated morphine withdrawal 153 8.3.17. Effect of n-Bt-ext BM treatment on incidence of salivation in naloxone precipitated morphine withdrawal 155 8.3.18. Effect of n-Bt-ext BM treatment on incidence of Raring in naloxone precipitated withdrawal 157 8.3.19. Effect of n-Bt-ext BM treatment on incidence of diarrhoea in naloxone precipitated morphine withdrawal 159 8.3.20. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally) on frontal cortex levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 161 8.3.21. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on striatal tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 162 8.3.22 Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal 164 8.3.23. Effect of Mt-ext BM treatment (10, 20 or 30 mg/kg orally on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 166

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8.3.24. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on frontal cortex tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal 168 8.3.25. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on striatum tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal 169 8.3.26. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on Hippocampus tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated morphine withdrawal 170 8.3.27. Effect of n-Bt-ext BM (5, 10 and 15 mg/kg orally) treatment on nucleus accumbens tissue levels of NA, DA, DOPAC, HVA, 5-HT and 5HIAA in naloxone precipitated withdrawal 172

8.4. Discussion 173

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TABLE 8.1. EFFECT OF NORMAL SALINE AND MT-EXT BM ACUTE TREATMENT (10, 20 OR 30 MG/KG ORALLY) ON FRONTAL CORTEX TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 161 TABLE 8.2. EFFECT OF NORMAL SALINE AND MT-EXT BM CHRONIC TREATMENT (10, 20 OR 30 MG/KG ORALLY) ON FRONTAL CORTEX TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 162 TABLE 8.3. EFFECT OF MT-EXT BM ACUTE TREATMENT (10, 20 AND 30 MG/KG ORALLY) AND SALINE ON STRIATAL TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 163 TABLE 8.4. EFFECT OF MT-EXT BM CHRONIC TREATMENT (10, 20 OR 30 MG/KG ORALLY) AND SALINE AND ON STRIATAL TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 164 TABLE 8.5. EFFECT OF MT-EXT BM ACUTE TREATMENT (10, 20 OR 30 MG/KG ORALLY) AND SALINE ON HIPPOCAMPAL TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 165 TABLE 8.6. EFFECT OF NORMAL SALINE AND MT-EXT BM CHRONIC TREATMENT (10, 20 OR 30 MG/KG ORALLY) ON HIPPOCAMPUS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 165 TABLE 8.7. EFFECT OF MT-EXT BM ACUTE TREATMENT (10, 20 OR 30 MG/KG ORALLY) AND SALINE ON NUCLEUS ACCUMBENS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 166 TABLE 8.8. EFFECT OF SALINE AND MT-EXT BM CHRONIC TREATMENT (10, 20 OR 30 MG/KG ORALLY) ON NUCLEUS ACCUMBENS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 167 TABLE 8.9. EFFECT OF NORMAL SALINE AND N-BT-EXT BM ACUTE TREATMENT (5, 10 OR 15 MG/KG ORALLY) ON FRONTAL CORTEX TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 168 TABLE 8.10. EFFECT OF NORMAL SALINE AND N-BT-EXT BM CHRONIC TREATMENT (5, 10 OR 15 MG/KG ORALLY) ON FRONTAL CORTEX TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 169 TABLE 8.11. EFFECT OF NORMAL SALINE AND N-BT-EXT BM ACUTE TREATMENT (5, 10 OR 15 MG/KG ORALLY) ON STRIATAL TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 170 TABLE 8.12. EFFECT OF NORMAL SALINE AND N-BT-EXT BM CHRONIC TREATMENT (5, 10 OR 15 MG/KG ORALLY) ON STRIATAL TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 170 TABLE 8.13. EFFECT OF N-BT-EXT BM ACUTE TREATMENT (5, 10 OR 15 MG/KG ORALLY) AND NORMAL SALINE ON HIPPOCAMPUS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 171 TABLE 8.14. EFFECT OF N-BT-EXT BM CHRONIC TREATMENT (5, 10 OR 15 MG/KG ORALLY) AND SALINE ON HIPPOCAMPUS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 171 TABLE 8.15. EFFECT OF N-BT-EXT BM ACUTE TREATMENT (5, 10 OR 15 MG/KG ORALLY) AND SALINE ON NUCLEUS ACCUMBENS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 172 180

Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

TABLE 8.16. EFFECT OF NORMAL SALINE AND N-BT-EXT BM CHRONIC TREATMENT (5, 10 OR 15 MG/KG ORALLY) ON NUCLEUS ACCUMBENS TISSUE LEVELS OF NA, DA, DOPAC, HVA, 5-HT AND 5HIAA IN RATS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL 173

FIGURE 8.1. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON BODY WEIGHT LOSS IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 OR 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. TREATMENT GROUPS WERE SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP **P<0.01.VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. **P<0.01 125 FIGURE 8.2. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON BODY WEIGHT LOSS IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. TREATMENT GROUPS WERE SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP *P< 0.05.VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05 126 FIGURE 8.3. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON JUMPING BEHAVIOR IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 OR 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. TREATMENT GROUPS WERE SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP .VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05, **P<0.01, 127 FIGURE 8.4. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON JUMPING BEHAVIOR IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. TREATMENT GROUPS WERE SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP .VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS.*P< 0.05 128 FIGURE 8.5. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON WRITHES IN ANIMALS UNDERGOING NALOXONE PRECIPITATED WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE

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(TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. 129 FIGURE 8.6. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON ABDOMINAL CONSTRICTIONS IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 OR 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE.VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05 130 FIGURE 8.7. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON SQUEAL IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM. 131 FIGURE 8.8. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON SQUEAL ON TOUCH IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. IN TREATMENT GROUPS 20 OR 30 MG/KG SIGNIFICANTLY LOWERED SQUEAL ON TOUCH AS COMPARED MORPHINE NALOXONE TREATMENT GROUP. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. *P< 0.05 132 FIGURE 8.9. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON SALIVATION IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. 133 FIGURE 8.10. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON SALIVATION IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. 134 FIGURE 8.11. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON RARING IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT

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WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. TREATMENT GROUPS ARE NOT SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. 135 FIGURE 8.12. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON RARING BEHAVIOR IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 OR 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM. 136 FIGURE 8.13. EFFECT OF AUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON DIARRHOEA IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. TREATMENT GROUPS ARE NOT SIGNIFICANTLY DIFFERENT FROM MORPHINE NALOXONE TREATMENT GROUP. VALUES WERE EXPRESSED AS MEAN±SEM. 137 FIGURE 8.14. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON DIARRHOEA IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM. 138 FIGURE 8.15. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON TEETH CHATTERING IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY MT-EXT BM 10, 20 AND 30 MG/KG ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. TREATMENT GROUPS ARE NOT SIGNIFICANTLY DIFFERENT FROM TREATMENT MORPHINE NALOXONE GROUP. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. 139 FIGURE 8.16. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON TEETH CHATTERING IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. ONLY 30 MG/KG DOSE SIGNIFICANTLY LOWERED TEETH CHATTERING AS COMPARED TO MORPHINE NALOXONE GROUP *P<0.05.VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. 140

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FIGURE 8.17. EFFECT OF ACUTE TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON WET DOG SHAKE IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM.. 141 FIGURE 8.18. EFFECT OF CHRONIC TREATMENT WITH MT-EXT BM (10, 20 OR 30 MG/KG ORALLY) ON WET DOG SHAKE BEHAVIOR IN ANIMALS UNDERGOING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL (N=6). ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED MT-EXT BM 10, 20 AND 30 MG/KG ORALLY ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05 142 FIGURE 8.19. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON BODY WEIGHT LOSS DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05 143 FIGURE 8.20. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON BODY WEIGHT LOSS DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT . SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. **P< 0.01, ***P< 0.001 144 FIGURE 8.21. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON JUMPING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05, **P< 0.01 145 FIGURE 8.22. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON JUMPING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. **P< 0.01. 146

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

FIGURE 8.23. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON WRITHING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM. 147 FIGURE 8.24. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON WRITHING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05 148 FIGURE 8.25. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON SQUEAL ON TOUCH BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. ALL THREE DOSES FAILED TO SIGNIFICANTLY LOWER SQUEAL ON TOUCH BEHAVIOR IN ANIMALS AS COMPARED TO MORPHINE NALOXONE GROUP. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. 149 FIGURE 8.26 EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON SQUEAL ON TOUCH BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. ***P< 0.001 150 FIGURE 8.27. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON TEETH CHATTERING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE). VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. *P< 0.05 151 FIGURE 8.28. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON TEETH CHATTERING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. SALINE GROUP RECEIVED SALINE ONE

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Chapter 8 Effect of Bacopa monnieri on morphine withdrawal

HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. **P< 0.01, ***P< 0.001), 152 FIGURE 8.29. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON WET DOG SHAKES BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. 153 FIGURE 8.30. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON WET DOG SHAKES BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY TUKEY’S POST HOC ANALYSIS. *P< 0.05, **P< 0.01, ***P< 0.005 154 FIGURE 8.31. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON SALIVATION BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM. 155 FIGURE 8.32. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON SALIVATION BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. *P< 0.05 156 FIGURE 8.33. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON RARING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM. 157 FIGURE 8.34. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON RARING BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE

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DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. ***P< 0.001 158 FIGURE 8.35. EFFECT OF ACUTE TREATMENT WITH N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ON DIARRHOEA BEHAVIOR DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED IDENTICALLY N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ORALLY, ONE HOUR BEFORE NALOXONE CHALLENGE. VALUES WERE EXPRESSED AS MEAN±SEM. 159 FIGURE 8.36. EFFECT OF CHRONIC TREATMENT WITH N-BT-EXT BM (5, 10 AND 15 MG/KG ORALLY) ON INCIDENCE OF DIARRHOEA DURING NALOXONE PRECIPITATED MORPHINE WITHDRAWAL.. ALL GROUPS WERE MADE DEPENDENT ON MORPHINE (TWICE DAILY), AS PER EIGHT DAYS SCHEDULE STARTING FROM 8 MG/KG/DAY AND GRADUALLY INCREASING TO 65 MG/KG/DAY ON DAY EIGHT. SALINE GROUP RECEIVED SALINE ONE HOUR BEFORE NALOXONE TREATMENT WHILE REST GROUPS RECEIVED N-BT-EXT BM (5, 10 OR 15 MG/KG ORALLY) ALONG WITH MORPHINE DEPENDENCE SCHEDULE. VALUES WERE EXPRESSED AS MEAN±SEM, APPLYING ANOVA FOLLOWED BY NEWMAN-KEULS MULTIPLE COMPARISON TEST. *P< 0.05 160

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Chapter 9

Acute toxicological studies on Bacopa

monnieri

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Chapter 9 Acute toxicological studies on Bacopa monnieri

9.1. Introduction

As a part of product development process all potential molecules are first screened pre clinically for toxicity and its per kg LD50 and ED50 are calculated and are used in same per kg body weight in other preclinical models with slight variation of route of administration or dosage forms as per protocols requirements (Fox, 2007). As mice are cheap, easy to breed and scientifically thoroughly explored for individual tissues and organs physiology. Moreover mice are the best choice and most extensively used acute toxicity models, evaluation of acute, chronic and individual organ or tissue toxicity (Fox, 2007). Acute LD50 calculated in mice is used and accepted internationally. Mice are the best option for toxicokinteic studies as well. Acute toxicity, chronic toxicity or therapeutic window calculated in mice can be used for dose calculation in per kg for other rodents or animal models (Fox, 2007). Although

Bacopa monnieri (BM) has been extensively used in ayurvedic clinical practice and has been thoroughly documented, preclinically and clinically safe herb (Russo and

Borrelli, 2005). The objective of this work was to calculate LD50 of methanolic extract and n-butanol extract of locally available BM.

9.2. Materials and methods

The details of material and methods are described on page 45, Chapter 2 Methodology.

9.3. Results 9.3.1. Acute toxicological effect of methanolic extract of Bacopa Monnieri (Mt-ext BM

Separate animals (n=8) were administered single intraperitoneal dose of saline, 20, 40,

60, 80,100,120,140,160 mg/kg Mt-ext BM, and number of deaths with in twenty four hours of extract administration was noted and LD50 was calculated, as 150 mg/kg

(Fig 9.1)

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Chapter 9 Acute toxicological studies on Bacopa monnieri

250

200

150

100 155 mg/kg 50

Cuumulative PercentCuumulative Death 0 0 500 1000 1500 Dose (mg/Kg)

Figure 9.1. Median Lethal dose of methanolic extract (155 mg/ Kg) of Bacopa monnieri in mice.

9.3.2. Acute toxicological effect of n-Butanol extract of Bacopa Monnieri

Separate Animals (n=8) were administered single intraperitoneal dose of saline, 20,

40, 60, 80,100,120,140,160mg/kg n-Bt-Ext BM, and number of deaths with in twenty four hours of extract administration was noted and LD50 of n-Butanol extract was calculated, as 80 mg/kg

450 400 350 300 250 200 150 100 81.1.m g/kg 50

Cumulative PercentCumulative Death 0 0 40 80 120 160 Dose (mg/Kg)

Figure 9.2. Median Lethal dose of n-Bt-ext BM of Bacopa monnieri in mice (81.1 mg/Kg).

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Chapter 9 Acute toxicological studies on Bacopa monnieri

9.4. Discussion

The acute toxicity experiments were performed to know about the toxicity profile of locally available BM. Additionally the extraction method was a modified form of

Kahol et al (Kahol et al., 2004) so the objective of also to see the impact of changed extraction method on acute toxicity in animal models. Balb C mice were used which are universally accepted, thoroughly documented and extensively used. BM has a documented safety profile both in animals and humans and extensive clinical data have proven safety of BM in human volunteers and old age individuals (Calabrese et al., 2008; Morgan and Stevens, 2010). BM safety has further been validated by findings that sub chronic use of BM does not alter DA and 5-HT turn over in mice brain (For details see Chapter 7). Although BM has been earlier reported and having

LD50 232 mg/kg for methanolic extract and 227 mg /kg for n-Bt-Ext BM , our finding corroborate with the previous findings also. In folkloric therapy too BM is considered safe, well tolerated, and free from untoward side effects. Currently herbal capsules are available for nootropic indication both in east and west also (Abascal and Yarnell,

2011; Pravina et al., 2007).

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Chapter 9 Acute toxicological studies on Bacopa monnieri

9.1. Introduction 179

9.2. Materials and methods 179

9.3. Results 179 9.3.1. Acute toxicological effect of methanolic extract of Bacopa Monnieri (Mt-ext BM 179 9.3.2. Acute toxicological effect of n-Butanol extract of Bacopa Monnieri 180

9.4. Discussion 181

182

Chapter 10 General Discussion

Chapter 10

General discussion

181

Chapter 10 General Discussion

10.1. General Discussion

Opioid dependence is a universal health problem, which afflicts both developed and underdeveloped societies across the globe (Jaffe and O'Keeffe, 2003). This multi facet problem is globally on the rise and in spite of immense pool of health resources, advanced countries like U.S has failed to contain the ever increasing burden of opioid addicts in its society (Mark et al., 2001). Since the clinical use of opiates for the management of chronic malignant and non malignant pain is universally accepted and trusted therapy, its use is another major area that transforms and produces opioid dependent individuals (Chabal et al., 1997). One major limiting factor regarding clinical use of opiates is the development of tolerance to its analgesic effect, thus necessitating higher doses to produce same analgesic response (chapter 1).

Poly substance abuse (concurrent use of more than one psychoactive substance) is another bizarre aspect of opioid dependence, concomitant use of cannabis, alcohol, stimulant and sedatives is an integral feature of opioid dependence phenomena. This polysubstance abuse for nicotine as added substance is common in 92% opioid addicts. This polysubstance abuse further adds to the length, cost, success and complexity of clinical management of dependence.

The mainstay therapy i.e. methadone, due to its cost, safety profile, and subsequent clinical outcomes, has much lower safety profile, narrow therapeutic window with inherent addiction potential and subsequent cardiac problems (Justo et al., 2006;

Lenné et al., 2001; Wolstein et al., 2009). Additionally humans studies have found profound hyperalgesia in patients on methadone (Doverty et al., 2001), and this management of hyperalgesia adds to the cost, duration and success of the therapy

(Angst and Clark, 2006; Hay et al., 2010; Hay et al., 2009). Additionally relapse

182

Chapter 10 General Discussion among patients treated with methadone ranges between to 60 to 75 percent, which is really alarming.

The search for newer molecules is underway worldwide to treat morphine tolerance and dependence. Since the findings of newer role of nootropics in drug dependence,

Bacopa monnieri candidacy has further been strengthened (Dhonnchadha and Kantak,

2011; Myers and Carlezon Jr, 2010; Russo and Borrelli, 2005). Our findings of effect of BM on expression and acquisition of morphine tolerance in animal models is of great significance in planning a clinical trials in chronic pain patients, as BM has been clinically examined and found safe in various clinical trials including old age people signifying good safety and tolerability profile (Calabrese et al., 2008). As BM has been found to exert its antinociceptive effect via activation of adenosine 1A receptors

(Sahoo et al., 2010), and adenosinergic drugs have been reported to enhance morphine analgesia in addition to lowering morphine tolerance (Ahlijanian and Takemori,

1985), which is in agreement with our findings (Chapter 5). BM has been reported to have an antinociceptive effect, partially reversible by naloxone and the analgesia produced by n-butanol fraction has a potency equivalent to morphine in hot plate experiments. The impact of BM on expression of morphine tolerance as reported in

Chapter 5, might have immense clinical implications because BM apart from having adenosinergic effects, have nitric oxide synthase inhibitory and calcium channel blocking effect also. Additionally adenosinergic drugs are termed as analgesics of the future, and our preclinical work indicates that tolerance does not develop to continuous use of BM that reflects significance of the BM, in clinical management of pain as single or combination therapy in comparison to existing therapies (Chapter 5).

The three main constituents of Bacoside A (Bacoside A3,Bacopaside II and

Bacosaponin C) in the locally available specie of BM, was quantified, (Chapter 4),

183

Chapter 10 General Discussion and our findings imply that extraction protocol have a significant effect on contents of

BM from the same source collected at the same time (Subhan et al., 2010). In this extraction performed through Kahol et al., method (Kahol et al., 2004) and analgesia was quantified at 10 mg/Kg body weight in methanolic extract was comparable to another method 40 mg/kg bodyweight. Additionally n-Butanol extract of BM obtained through Kahol et al., method produced analgesia at 5 mg/kg body weight while plant collected from same source and same time of year but extracted with an other technique produced analgesia at 80 mg/kg body weight (Subhan et al., 2010).

BM is a renowned ayurvedic herb with centuries old clinical usage for the management of neuropsychiatric disorder. Still its usage in rural communities for its ethno pharmacological image is very high and it’s demand as published by the Indian agriculture board is 6621 tons annually (Sharma et al., 2010). Due to its extensive clinical utility for the management of neuropsychiatric illnesses, BM has been declared as threatened species and alternate sources and state sponsored growing has been recommended to avoid extinction or emergency shortfall in its availability

(Tiwari et al., 2001). Many other plants, like Passiflora incarnata has been screened for their potential role in lowering opioid withdrawal syndrome and in a double blind study randomized control trial have been found to be safe, effective and well tolerated therapy (Akhondzadeh et al., 2001). Moreover Nigella sativa has also been screened clinically in opioid withdrawal and has been documented safe and effective therapy in lowering significantly somatic signs of opioid withdrawal (Sangi et al., 2008). These plants, Nigella sativa and passiflora, none have intrinsic strong antinociceptive effect as BM which is an added benefit with BM apart from lowering somatic signs of naloxone precipitated withdrawal syndrome.

184

Chapter 10 General Discussion

The study of Drug sensitization and its mechanistic elucidation is the primary center of current research; sensitization is gate way to induction of drug seeking behavior with neural plasticity, dependence and relapse after long phase of abstinence from opiates (Listos et al., 2011; Robinson and Berridge, 1993). Till now morphine sensitization and subsequent DA upsurge in mesolimbic system and role of DA receptors, nitric oxide and intracellular calcium have been extensively explored. Our behavioral finding depicts that BM has significant effect in lowering morphine induced locomotor hyperactivity. Our neurotransmitters data reflects the antidopaminergic/serotonergic affect with plausible role of adenosine 1A receptors, nitric oxide, and intracellular calcium and GABAergic effects of BM. The critical analysis of antidopaminergic/serotonergic effect of BM (Chapter 6), with subsequent lowering in DA and serotonin turnover has an apparent impression that BM antidopaminergic effect may be attained with BM doses that does not induce antinociceptive effect, though this impression needs experimental exploration in animal’s models. A finding of the antidopaminergic/serotonergic effect of BM is of high significance and may contribute to the treatment of opioid dependence management.

In this study we also found that methanolic extract and n-Butanol extract of BM, the n-Butanol extract has a stronger analgesia, and subsequent more potent effect in lowering morphine tolerance than methanolic extract (Chapter 6). However effects of both extracts on morphine induced locomotor hyperactivity and morphine withdrawal syndrome were highly effective, but not significantly different. The successful amelioration of opioid withdrawal somatic signs with associated DA and 5-HT up surge in both striatum and nucleus Accumbens can partly be attributed to its direct effect on adenosine 1A receptors (Sahoo et al., 2010), calcium channel blocking (Dar

185

Chapter 10 General Discussion and Channa, 1999) and nitric oxide synthase inhibitory effect (Govindarajan et al.,

2005; Saraf et al., 2009) . Recent findings imply that Adenosine receptors have a substantial role in mediation of both addictive and aversive behaviors expression as examined in preclinical models through a close interplay with opioid receptors.

Adenosine agonists and opioid both have been reported to inhibit Gamma Amino

Butyric acid (GABA) and glutamate in presynaptic terminals in Nucleus Accumbens

(Chieng and Williams, 1998; Manzoni et al., 1998; Yuan et al., 1992). The production of quasi opioid withdrawal behavior by adenosine antagonist in saline treated animals further strengthens the plea that adenosinergic effect of BM has contributed significantly in ameliorating naloxone precipitated withdrawal behavior in animals

(Collier et al., 1974). Additionally both methanolic extract and n-Butanol extract of

BM have successfully lowered major somatic signs of opioid withdrawal, with subsequent lowering of Noradrenalin in frontal cortex (Chapter 9), with an added significant increase in DA and serotonin turn over in striatum and nucleus accumbens implying low propensity for opioid withdrawal induced depression (Goeldner et al.,

2011) although this effects needs evaluation using specified depression evaluation paradigms. As BM has been reported to have antidepressant effect comparable to standard antidepressants like Selective Serotonin Reuptake Inhibitors (SSRI) it can be implicated that BM will lower opioid withdrawal depression also, although this idea needs thorough screening in animal models of opioid withdrawal induced depression.

As animals experience acute stress during opioid withdrawal (Krabbe et al., 2003) and this stress is one strong proponent of relapse (Van Bockstaele et al., 2010), BM has been reported to have an adaptogenic effect during acute and chronic unpredictable stress with moderating effect on stress induced cortisol and neurotransmitters changes

(Sheikh et al., 2007). This role of BM have favorable theoretical implications and

186

Chapter 10 General Discussion needs evaluation/ validation in preclinical models. Although BM has been reported to be highly effective in diabetes induced hyperalgesia, mainly through its direct effect on adenosine 1A receptors (Sahoo et al., 2010), our findings of lowering of squeal on touch (Chapter 9) are encouraging but not in accordance to the findings of Sahoo et al., (Sahoo et al., 2010). This altered and a comparatively weaker response of lowering naloxone precipitated withdrawal induced hyperalgesia by BM might be reversal act of naloxone, as naloxone has been reported to reverse BM analgesia

(Subhan et al., 2010) and in naloxone precipitated opioid withdrawal this role of naloxone cannot be ruled out. Our findings in analgesia, tolerance, locomotor effects, and withdrawal are in accordance to the reported neuropharmacological profile of

BM, role as a nootropic and its role in drug cue extinction need evaluation in specified models preclinically.

Our findings of acute and sub chronic administration of BM on DA and 5-HT turn over further validate the clinical and preclinical findings advocating safety and tolerability of BM.

10.2. Future Work

The behavioral and neurochemical work can be extended to multifaceted molecular work comprising many areas like

1. This work can be receptor specific in terms of type of receptor for specific

neurons and its impact on adenosine 1A auto receptors and heteroreceptors,

with specific correlation to 5-HT, Noradrenalin and dopamine in key brain

areas like frontal cortex, Locus coeruleus, brain amygdala and striatum. This

work should be followed by microdialysis work in acute, and chronic use, and

in animals undergoing withdrawal.

187

Chapter 10 General Discussion

2. The other optional work can be oxidative stress centered focusing mainly on

NOO- cycle and its correlation to plant in normal and morphine dependent

animals. As during morphine tolerance oxidative stress in spinal cord is raised.

This study shall mainly focus on studying neurochemical and oxidative

parameters involved in drug tolerance like, NMDA, GABA, and oxidative

stress parameters status in dependent animals’ spinal cords.

3. As Bacopa is known to have lowering effect on Gastrointestinal tract (GIT)

motility, Work needs to be done on impact of Bacoside on git

neurotransmitters and GIT motility. Impact of Bacopasides on 5-HT receptors

regulation, and overall turnover of 5-HT in opioid addict animal gut.

4. As Bacopa monnieri is used as herbal therapy, its long term impact on blood

and platelets neurotransmitters needs to be evaluated in both animals and

humans with and without opioid addiction.

5. As Bacopa has been reported to be having a strong calcium channels blocking

effect comparable to verapamil, its potential neuropharmacological role in

management of bipolar disorders needs screening at molecular level.

6. Work on opioid receptor down regulation during opioid addiction and

subsequent impact of Bacopa monnieri needs screening.

7. Impact of Bacopa monnieri on indigenous neuropepetides and its impact on P

glycoprotein in normal and opioid addict animals is also a grey area that needs

exploration.

188

Chapter 10 General Discussion

8. Impact of Bacopa monnieri on NMDA receptors and GABA also needs

exploration to help understand its role in morphine tolerance and antiepileptic

folkloric utility.

9. Preclinical work needs to be initiated to add Bacopa with methadone and

compare its effects with simple methadone and then tapering methadone and

subsequently stopping methadone, and comparing it with just methadone alone

therapy, and evaluating its potential role in avoiding methadone dependence.

10. Role of Bacopa monnieri needs to be screened in ameliorating or decreasing

the extra pyramidal side effects of antipsychotics.

11. Effect of Bacopa needs screening for its role in preventing diabetic

neuropathies and in preventing cisplatin nephrotoxicity.

189

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