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

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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 By 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.
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