UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES SCHOOL OF MEDICINE AND DENTISTRY ANALGESIC EFFECTS OF ANNONA MURICATA LEAF EXTRACT IN PACLITAXEL AND STREPTOZOTOCIN-INDUCED DIABETIC NEUROPATHY IN MURINE MODELS BY KOOMSON, FREDERICK ALEXANDER (10600286) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL PHARMACOLOGY DEGREE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY AUGUST 2022 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh ii ABSTRACT Background: Annona muricata have demonstrated antinociceptive and anxiolytic effects in animal models through its leaf extracts. This study evaluates the aqueous leaf extract of the plant for possible analgesic properties in hyperalgesia and allodynia associated with paclitaxel-induced neuropathy in mice and streptozotocin (STZ)- induced diabetic neuropathy in rats. Methods:10kg of the coarse crushed leaves was soaked in 3 liters of distilled water to make a decoction, cooled, filtered and freeze dried for use. Sub-acute toxicity test was carried out for 14- days after which blood samples were taken and examined for haematological analysis. Phytochemistry of the extract was conducted and analgesic property was accessed using hot plate test. Irwin test was also conducted to observe alterations in behavior and physiological activity, neurotoxicity and mortality. Diabetic- induced neuropathy in Sprague-Dawley rats was accomplished by injecting 55mg/kg body weight of STZ followed by 120mg/kg body weight of nicotinamide to achieve type 2 diabetes mellitus. Paclitaxel-induced neuropathy was also achieved by injecting ICR mice with 2mg/kg body weight body weight of paclitaxel continuously for 5 days. Parameters which include cold allodynia mechanical hyperalgesia and thermal hyperalgesia were measured before the administration of paclitaxel and on day 1 – 5 and after the administration of paclitaxel. In STZ-induced diabetic neuropathy experiment parameters were measured before the administration of STZ and after the administration of STZ on day 2, 4, 6, 8, 10, 12 and 14. These animals were then treated with Annona muricata extract (AME) (30, 100 and 300 mg/kg body weight), pregabalin (10, 30 and 100 mg/kg body weight) and distilled water as a vehicle daily for 5 days and 14 days continuously in paclitaxel- and diabetic-induced peripheral neuropathy respectively. Pain thresholds were measured on day 1, 2, 3 and 5 in paclitaxel-induced neuropathy experiment and that of STZ-induced - diabetic neuropathic experiment, it was measured from day University of Ghana http://ugspace.ug.edu.gh iii 1-7. Results: Annona muricata Extract (AME) showed no toxicity as no death were observed during the 14-day study period in sub-acute toxicity studies. Preliminary phytochemical screening of the extract indicated the presence of secondary metabolites which includes alkaloids, saponins, flavonoids, tannins, glycosides, triterpenoids and sterols. The extract showed analgesic property during the hot plate test. CNS safety pharmacology using Irwin test indicated no mortality when experimental animals were observed for 24 hours after various treatment doses were employed. Observable physiological/ pharmacological effects were noted which include straub tail, defecation, sniffing among others. Relative organ weight of the experimental animals also indicated no obvious abnormally when compared to the control during Irwin’s test. AME and pregabalin produced analgesic properties which was exhibited in paclitaxel and STZ-induced - neuropathy as increased paw withdrawal latencies to mechanical, cold-water stimuli and thermal hyperalgesic tests. Conclusions: The findings from this study suggest that aqueous extract of Annona muricata is sub acutely safe with observable CNS physiological effect and no observable CNS toxicity. Again, the extract possesses an analgesic property as seen in both paclitaxel- and STZ-induced diabetic neuropathy in animal models which may contribute to its traditional use in managing neuropathic pain. University of Ghana http://ugspace.ug.edu.gh iv DEDICATION This work is dedicated to the Almighty God and the Department of Pharmacology and Toxicology, University of Ghana. ACKNOWLEDGEMENTS My sincere gratitude goes to the Almighty for the strength and energy through this program and for making it a success. I am enormously grateful to Dr. Patrick Amoateng and Prof. Major George Asare for their excellent supervision, patience, guidance and dedication towards my training in scientific research. I also want to thank the Department of Pharmacology and Toxicology, School of Pharmacy, University of Ghana, for the training, exposure and experience they offered me during my postgraduate studies. I am especially grateful to the staff of Noguchi Memorial Institute of Medical Research (NMIMR). I am thankful to my parents, siblings, friends and colleagues for their support and encouragements. University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS DECLARATION .............................................................................................................................. i ABSTRACT ..................................................................................................................................... ii DEDICATION ................................................................................................................................ iv ACKNOWLEDGEMENTS ............................................................................................................. iv TABLE OF CONTENTS ................................................................................................................ v LISTS OF FIGURES ..................................................................................................................... ix LISTS OF TABLES ........................................................................................................................ xii LIST OF ABBREVIATIONS ...................................................................................................... xiii CHAPTER ONE .............................................................................................................................. 1 INTRODUCTION ........................................................................................................................... 1 1.1 Background ............................................................................................................................ 1 1.2 Problem Statement and Justification ................................................................................... 2 1.3 Hypothesis .............................................................................................................................. 4 1.4 Aim ......................................................................................................................................... 4 1.5 Specific Objectives ............................................................................................................ 5 CHAPTER TWO ............................................................................................................................ 6 LITERATURE REVIEW ............................................................................................................... 6 2.1 Background ............................................................................................................................ 6 2.2 OVERVIEW OF PAIN ......................................................................................................... 7 2.2.1 Neurophysiology of pain ................................................................................................ 8 2.2.2 Peripheral pain processes .............................................................................................. 9 2.2.3 Central processing of pain ........................................................................................... 10 2.2.4 Inhibiting pain mediation ............................................................................................ 11 University of Ghana http://ugspace.ug.edu.gh vi 2.2.5 Classification of pain .................................................................................................... 12 2.2.6 Nociceptive pain ............................................................................................................ 13 2.2.7 Neuropathic pain .......................................................................................................... 13 2.3 Paclitaxel-induced Peripheral Neuropathy ....................................................................... 15 2.3.1 Paclitaxel and peripheral neuropathy ........................................................................ 15 2.3.2 Pathogenesis of paclitaxel-induced peripheral neuropathy ...................................... 16 2.3.3 Paclitaxel and microtubule interference ..................................................................... 17 2.3.4 Paclitaxel and mitochondrial dysfunction .................................................................. 18 2.3.5 Paclitaxel and axon degeneration ................................................................................ 18 2.3.6 Paclitaxel and calcium homeostasis ............................................................................ 19 2.3.7 Paclitaxel and peripheral nerve excitability ............................................................... 19 2.3.8 Paclitaxel, immune processes and neuroinflammation ............................................. 19 2.3.9 Genetics and paclitaxel-induced peripheral neuropathy .......................................... 20 2.4 Diabetic-Induced Peripheral Neuropathy ......................................................................... 20 2.4.1 Diabetes and diabetic neuropathy ............................................................................... 20 2.4.2 Free radicals and diabetic neuropathy ....................................................................... 23 2.4.3 Anatomy of diabetic neuropathic pain ....................................................................... 24 2.4.4 Pathophysiology of diabetic neuropathy .................................................................... 25 2.4.5 Mechanisms leading to the development of DNP ....................................................... 26 2.4.6 Clinical features ............................................................................................................ 28 2.5 Pharmacotherapy of Diabetes and Paclitaxel-induced Neuropathic Pain ...................... 34 2.5.1 Antidepressants ............................................................................................................ 34 2.5.2 Tramadol ....................................................................................................................... 34 2.5.4 Opioids .......................................................................................................................... 35 University of Ghana http://ugspace.ug.edu.gh vii 2.5.5 Antiepileptic drugs ....................................................................................................... 35 2.6 Use of Plants as Analgesic Agents in the Management of Pain ....................................... 36 2.6.1 Annona muricata ........................................................................................................... 36 2.7 Ethnobotanical uses of Annona muricata .......................................................................... 40 2.7.1 Pharmacological studies of Annona muricata ............................................................ 41 CHAPTER 3 .................................................................................................................................. 48 MATERIALS AND METHODS .................................................................................................. 48 3.1 Reagents/Drugs/Apparatus ................................................................................................. 48 3.2 Experimental Animals and Housing Conditions .............................................................. 48 3.3 Time of Experimentation .................................................................................................... 48 3.4 Plant Collection and Extraction ......................................................................................... 49 3.5 Toxicity Studies of Annona muricata Extract ................................................................... 49 3.5.1 Sub-Acute Toxicity ....................................................................................................... 49 3.6 Parameters Investigated ..................................................................................................... 49 3.6.1 Clinical observations and body weights ...................................................................... 49 3.7 Clinical Pathology ............................................................................................................... 50 3.7.1 Haematology ................................................................................................................. 50 3.7.2 Macroscopic and microscopic examinations .............................................................. 50 3.7.3 Histological examination of isolated organs ............................................................... 51 3.8 Phytochemical Screening of Annona muricata Extract ..................................................... 51 3.9 Primary Observation and Safety Pharmacology Assessment Using Irwin Test ............ 51 3.10 Establishing the Analgesic Activity of AME Using Hot Plate Test ............................... 52 3.11 Investigating the Effect of AME on Paclitaxel-induced Neuropathic Pain .................. 54 3.11.1 Inducing peripheral neuropathy using paclitaxel .................................................... 54 University of Ghana http://ugspace.ug.edu.gh viii 3.11.2 Treatment with AME, pregabalin and saline on paclitaxel - Induced neuropathic ICR mice ................................................................................................................................ 55 3.12 Investigating the Effect of AME on Diabetic-Induced Peripheral Neuropathy. .......... 56 3.12.1 Inducing diabetic neuropathy using STZ ................................................................. 56 3.12.2 Extract/drug treatment of streptozotocin-induced neuropathic pain .................... 57 3.13 Statistical Analysis ............................................................................................................. 57 RESULTS ...................................................................................................................................... 58 4.1 Toxicity ................................................................................................................................. 58 4.1.1 Sub-acute toxicity ......................................................................................................... 58 4.1.1.3 Clinical pathology ...................................................................................................... 61 4.1.1.3.1 Macroscopic Examinations .................................................................................... 61 4.1.1.3.2 Microscopic Examinations ..................................................................................... 62 4.2 Phytochemical Screening of AME ..................................................................................... 66 4.3 Irwin Test ............................................................................................................................. 66 4.4 Assessing the Analgesic Effect Annona muricata using Hot Plate Test ........................... 67 4.5.1.1 inducing neuropathy using paclitaxel (cold allodynia test) .................................... 69 4.5.1.2 Inducing neuropathy using paclitaxel (hot plate test) ............................................ 70 4.5.1.3 Inducing neuropathy using paclitaxel (mechanical hyperalgesia test) .................. 71 4.5.1.4 Effects of AME on cold allodynia in PIPNE ............................................................ 72 4.5.1.5 Effects of AME on hot plate test in PIPNE ............................................................. 74 4.5.1.6 Effects of AME on mechanical hyperalgesia in PIPNE .......................................... 76 4.5.2 Diabetic-Induced Peripheral Neuropathy ...................................................................... 78 4.5.2.1 STZ-induced diabetic neuropathy (cold allodynia Test) ........................................ 78 4.5.2.3 STZ-induced diabetic neuropathy (Mechanical hyperalgesia) .............................. 80 University of Ghana http://ugspace.ug.edu.gh ix 4.5.2.5 Effects of AME on Thermal hyperalgesia (DIPN) .................................................. 83 4.5.2.6 Effects of AME on mechanical hyperalgesia (DIPN) .............................................. 85 CHAPTER FIVE ........................................................................................................................... 88 DISCUSSION, CONCLUSION AND RECOMMENDATIONS .............................................. 88 5.1 Discussion ............................................................................................................................. 88 5.2 CONCLUSION .................................................................................................................... 93 5.3 RECOMMENDATIONS .................................................................................................... 93 REFERENCES .......................................................................................................................... 94 LISTS OF FIGURES Figure 2.1: Illustration of the processes of pain initiating from the periphery to the brain .............. 9 Figure 2.2: An illustration of the types of pain ............................................................................... 13 Figure 2.3: A diagrammatic representation of mechanism of action of taxanes causing peripheral neuropathy. ............................................................................................................................... 18 Figure 2.4: An illustration of the features of diabetic peripheral neuropathy ................................. 22 Figure 2.5: Stocking Glove Configuration of DPN . ....................................................................... 24 Figure 2.6: Mechanisms of diabetic neuropathy ............................................................................. 34 University of Ghana http://ugspace.ug.edu.gh x Figure 2.7: Diagram showing leaves and fruits of Annona muricata .............................................. 38 Figure 4.1: Photomicrographs of the livers isolated from of Sprague-Dawley rats after AME treatment at various doses.. .. ................................................................................................. 62 Figure 4.2: Photomicrographs of the kidneys harvested from Sprague-Dawley rats after treatment with various doses of AME. .. ................................................................................................ 64 Figure 4.3: Effect exhibited by AME (30 -1000 mg/kg body weight, p.o), morphine (0.3-10mg/kg body weight) and the Vehicle (veh) on the evaluation of analgesia using %MPE (A) and AUC (B) in the hot plate test. ............................................................................................................ 67 Figure 4.4: A comparison of tail withdrawal as cold allodynia) on day 1 and day 5 post paclitaxel- induced neuropathy.... .............................................................................................................. 69 Figure 4.5: A comparison of paw withdrawal (thermal hyperalgesia) on day 1 and day 5 after paclitaxel-induced neuropathy……………………………………………………………..70 Figure 4.6: A comparison of paw withdrawals as a measure of the onset of mechanical hyperalgesia on day 1 and day 5 after paclitaxel administration………………………...71 University of Ghana http://ugspace.ug.edu.gh xi Figure 4.7: The effect of AME (30, 100, 300 mg/kg body weight, p.o) and PGB (10, 30, 100 mg/kg body weight, p.o) on cold allodynia in paclitaxel-induced neuropathic rats…….72 Figure 4.8: The effect of AME (30, 100, 300 mg/kg body weight, p.o) and PGB (10, 30, 100 mg/kg body weight, p.o) on thermal hyperalgesia in paclitaxel neuropathic rats……... 74 Figure 4.9: The effect of AME (30 – 300 mg/kg body weight, p.o) and PGB (10 – 100 mg/kg body weight, p.o) on mechanical hyperalgesia in paclitaxel-induced neuropathic mice.. ................ 76 Figure 4.10: A comparison of tail withdrawal (as a measure of cold allodynia) on day 1 and day 14 after STZ-induced diabetes... ................................................................................................... 77 Figure 4.11: A comparison of paw withdrawals (as a measure of the onset of thermal hyperalgesia) on day 1 and day 7 after STZ administration.. ......................................................................... 78 Figure 4.12: A comparison of paw withdrawals (as a measure of the onset of mechanical hyperalgesia) on day 1 and day 7 after STZ administration. ................................................... 79 Figure 4.13: The effect of AME (30, 100, 300 mg/kg body weight, p.o) and PGB (10, 30, 100 mg/kg body weight, p.o) on cold allodynia in diabetic neuropathic rats………………...81 Figure 4.14: The effect of AME (30,100, 300, mg/kg body weight, p.o) and PGB (10, 30, 100 mg/kg body weight, p.o) on thermal hyperalgesia in diabetic neuropathic rats………...83 University of Ghana http://ugspace.ug.edu.gh xii Figure 4.15:. The effect of AME (30 – 300 mg/kg body weight, p.o) and PGB (10 – 100 mg/kg body weight, p.o) on mechanical hyperalgesia in diabetic neuropathic rats…………….85 LISTS OF TABLES Table 3: Animal groupings and mode of drug administration ......................................................... 52 Table 4.1: The effects of AME on relative weights of major organs (g) isolated from rats in a 14- day sub-acute toxicity study. .................................................................................................... 57 Table 4.2: Haematological analysis of AME (100, 300 and 1000 mg/kg body weight) after a 14- day observation period ............................................................................................................. 58 Table 4.3: The effects of AME (100, 300 and 1000 mg/kg body weight) on the change in body weights t ................................................................................................................................... 61 Table 4.4: Physiological and pharmacological effect of AME in Irwin .......................................... 65 Table 4.5: The effects of AME (10, 30, 100, 300, 1000 and 3000mg/kg body weight) on relative weights of major organs (g) from the mice in a preliminary pharmacological study using Irwin test .................................................................................................................................. 66 University of Ghana http://ugspace.ug.edu.gh xiii Table 4.6: ED50 of AME and MOR in the Hot plate experiment confirming the extract’s analgesic effect ........................................................................................................................................ 68 Table 4.7: ED50 of AME and PRG in paclitaxel-induced neuropathy experiment ......................... 77 Table 4.8: ED50 of AME and PRG in STZ-induced diabetic neuropathic experiment .................. 86 LIST OF ABBREVIATIONS ABTS 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid AME Annona muricata extract AST Aspartate aminotransferase ATP Adenosine triphosphate AUC Area Under Curve CIPN Chemotherapy induced peripheral neuropathy CNS Central nervous system DIPN Diabetic-induced peripheral neuropathy DN Diabetic neuropathy DPN Diabetic Peripheral Neuropathy DSP Diabetic sensorimotor polyneuropathy DNA Deoxyribonucleic acid DRG Dorsal root ganglion ED Effective dose ED50 Effective dose for 50% University of Ghana http://ugspace.ug.edu.gh xiv FeCl3 Ferric (III) Chloride ORAC Oxygen Radical Absorbance Capacity HCT Hematocrit HDL High-density lipoprotein HGB Hemoglobin IASP International Association for the Study of Pain ICR Imprint control region IL Interleukin Ip. Intraperitoneal IV Intravenous K3Fe (CN)6 Potassium ferricyanide L-NAME NG-nitro-L-arginine methyl ester LD Lethal dose LDL Low density lipoprotein LYM Lymphocytes MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volume MPE Maximal possible effect MPV Mean platelet volume MOR Morphine NO Nitric oxide PIPNE Paclitaxel-induced peripheral neuropathy experiment University of Ghana http://ugspace.ug.edu.gh xv PGB Pregabalin ROS Reactive oxygen species SNRI Serotonin-Norepinephrine reuptake inhibitors STZ Streptozotocin T1DM Type 1 Diabetes Mellitus T2DM Type 2 Diabetes Mellitus USDA-ARS United States Department of Agriculture-Agricultural Research Service WHO World Health Organisation University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 Background The development of peripheral neuropathy in patients with long term paclitaxel usage and diabetes mellitus have different symptoms, mechanism of neurologic involvement, conduit, risk covariates, pathologic modification and underlying mechanisms (Tesfaye et al., 2010). Neuropathic pain is as a result of a damage in the peripheral nerves or central nervous system (Daousi et al., 2004). Neuropathic pain causes may include diabetes mellitus (Pop-Busui et al., 2017), shingles (Woolf. et al., 1999), multiple sclerosis (Mori et al., 2010), spinal cord injuries (Wilmshurst et al., 2019) stroke (Ocean & Vahdat, 2004) HIV infection (Lefaucheur et al., 2017), cancer and cancer treating drugs (Rang, 2003). Peripheral neuropathy and neuropathic pain are used interchangeably. It is estimated that 25% of patients suffering from diabetes might experience neuropathy (Boulton et al., 2004). According to Van Hecke et al. (2013) with reference to paclitaxel-induced peripheral neuropathy, 6.9% to 10% of the world’s population suffers from it. The clinical signs are distal, symmetrical and are usually with nocturnal exacerbations such as deep aching electric-like shock and a burning sensation with hyperalgesia and often allodynia upon examination (Boulton et al., 2004). Major treatment options normally entail treating the underlying cause and managing the pain symptomatically. For instance, lowering hyperglycemia or halting the use of paclitaxel usage could be the first line treatment approach when it comes to diabetic-induced peripheral neuropathy (DIPN) and paclitaxel-induced peripheral neuropathy (PIPN) respectively (Finnerup et al., 2015). Relief from pain is often between 30% and 50% even in patients taking higher doses of pain relief medications (Attal, 2019). Uncontrolled diabetes caused by type-1 diabetes or type-2 diabetes is primarily one of the major University of Ghana http://ugspace.ug.edu.gh 2 contributions to diabetic complications in various organs and systems including the somatosensory nervous system (Backonja, 2004). Type-1 diabetes mellitus arises as a result of total inability of the pancreatic beta cells to secrete insulin (Casellini & Vinik, 2007) whiles type-2 diabetes is caused by the resistance of beta cells to insulin (Huang et al., 2016). It can also be caused by loss of pancreatic beta cells as a result of infections mostly of viral source or toxic damage producing insulin insufficiency. Hyperglycemia-induced oxidative and nitrosative stress serves as a major link between diabetes and diabetic complications (Casellini & Vinik, 2007), this release free radicals from autoxidation and glycosylation of glucose and proteins respectively (Zimmermann, 2001). Paclitaxel, an anticancer agent possessing tubulin-stabilizing effect, is usually used in diseases such as ovarian cancer, breast cancer, non-small cell lung carcinoma and stomach cancer. However, the use of paclitaxel is often limited by incidence of severe adverse reactions which includes peripheral neuropathy characterized by frequently occurring sensory neuropathies like dysesthesias, numbness, pain and thermo hyperesthesia in the feet and hands and usually mild motor neuropathies including muscle weakness and reduction of motor skill for instance buttoning a shirt (Ocean & Vahdat, 2004). 1.2 Problem Statement and Justification Aside acute kidney shock and depression of the bone marrow, one of the major reasons for not complying with anti-cancer therapy or changing the dose regimen can be largely attributed to the neurotoxic side-effects of some common chemotherapeutic agent. A typical side effect associated with these agents includes neuropathic pain. Diabetes mellitus affects approximately 132 million people as of 2010 with 1.9% of the population having one of its developing complications as peripheral neuropathy (Van Acker et al., 2009). Peripheral neuropathy as caused by paclitaxel usage University of Ghana http://ugspace.ug.edu.gh 3 or long-standing diabetes mellitus complication can significantly interfere with function of peripheral nerves and can compromise the quality of life of individuals who suffers from it. Usually, the symptoms show a predominant sensory or sensory-motor neuropathy which sometimes occur together with the dysfunction of the autonomic nervous system. There is currently no cure for this type of nerve damage. Management is tailored symptomatically and is normally treated with the use of antidepressants, opioid analgesics, anticonvulsants among others (Attal et al., 2006). Pharmacovigilant study of these drugs used clinically have been indicative of some undesirable adverse effects and sometimes unsafe when employed long term for pain management. Some of these undesirable effects includes; drowsiness, constipation, dependence, dizziness, dry mouth, headaches, heart burn, and palpitations, insomnia, cardiac myopathies among others. (Attal et al., 2006). They are also very expensive for the average individual to afford especially in Ghana (Amoateng et al., 2017). Considering the above setback in the treatment of neuropathy, there is a need to find agent(s) with high safety profile, cheaper and of better therapeutic value compared to the existing drugs on the market for clinical use. In identifying new agent/s to curb the existing burden, medicinal plants usage has empirically been identified to be a promising area in disease management where conventional medicine seems to be struggling, especially with respect to pain management. A review by Fatemah Forouzanfar et al in April 2018 have suggested common medicinal plants employed in the management of neuropathic pain. These include Acorus calamus, Artemisia dracunculus, Butea monosperma, among others. Annona muricata which is one of the readily available plant in the tropics has undergone quite a number of studies as far as pain and the nervous system is concern, its folkloric use and animal studies has been channeled in the line of anti-depressant, anticonvulsant, analgesic, anti-diabetic as well as anti tumour activities (Alali et al., 1999). Its anti-inflammatory University of Ghana http://ugspace.ug.edu.gh 4 effect is same as the action of indomethacin, a non-steroidal anti-inflammatory drug (De Sousa et al., 2010; Poma et al., 2011). The antinociception of the hydro-ethanolic extract has also been demonstrated using different chemical and thermal nociceptive models (Hamid et al., 2012). Anxiolytic and anti-stress effects have also been found (Oviedo et al., 2009). Traditionally it has also been used as a sedative (Hasrat et al., 1997). In Brazil Martinique, Mexico and Nicaragua the leave stock is also used as an analgesic (Coria-Tellez et al., 2018). The above research findings indicate clearly that A. muricata have a nervous system effect and possesses an analgesic property making it a suitable candidate for other forms of neurological screening especially that of peripheral neuropathy. Several agents from plant origin with potential therapeutic properties in the treatment of neuropathic pain have been identified unsuitable in the line of safety pharmacology, (Sengupta et al., 2012). In this regard, the plant of concern will go through sub-acute toxicological studies and subsequent Irwin’s test to ascertain its neurological effect. To a very large extend this research will focus on safety Annona muricata aqueous leaf extract and it potential in the management of paclitaxel and diabetic-induced peripheral neuropathy in animal models. 1.3 Hypothesis H1: The Aqueous leaf extract of Annona muricata possesses an effect on the nervous system and will be useful in the management of peripheral neuropathy caused by paclitaxel and diabetes mellitus using animal models. 1.4 Aim Evaluate the analgesic effect of aqueous extract of Annona muricata on paclitaxel and diabetic- induced peripheral neuropathy using animal models. University of Ghana http://ugspace.ug.edu.gh 5 1.5 Specific Objectives 1.To conduct sub-acute toxicity studies on A. muricata. 2. To perform preliminary phytochemical screening on the extract of A. muricata 3. To investigate the general CNS safety pharmacology using Irwin test. 4. To demonstrate the effect of the aqueous extract of A. muricata on paclitaxel-induced neuropathy. 5. To investigate the effect of aqueous leaf extract of A. muricata on diabetic-induced peripheral neuropathy. University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO LITERATURE REVIEW 2.1 Background Sensation of pain resulting from a lesion or disease of the somatosensory nervous system have been attributed to several causes including a variety of systemic, metabolic and toxic agents. Some of the commonest treatable causes are diabetes mellitus, hypothyroidism, drugs and nutritional deficiencies (Baron et al., 2010). Accurate diagnosis involves careful clinical assessment, judicious laboratory testing and electrodiagnostic studies or nerve biopsy. Systematic investigation begins with localization of the lesion to the peripheral nerves, identification of fundamental etiology and the exclusion of potential causes which are treatable (Azhary et al., 2010). Analysis on cerebrospinal fluids and lumbar puncture have been useful in diagnosing Guillain-Barre syndrome and chronic inflammatory demyelinating neuropathy (Sainaghi et al., 2010). Electrodiagnostic analysis such as nerve conduction studies and electromyography contribute to the differentiation of axonal and demyelinating or mixed neuropathy. Treatment targets fundamental disease processes, nutritional deficiencies and provide symptomatic pharmacological intervention (Chichkova & Katzin, 2010). Diabetes which is characterized by persistence high blood glucose affects the peripheral nervous system. Its neuropathic complication is largely involved in almost all of impairments occurring in the peripheral nerves. Diabetic sensorimotor polyneuropathy (DSP) and diabetic neuropathy (DN) are used synonymously. Patients with DSP typically have numbness, tingling, pain and/or weakness that begin in the feet and spread proximally in a length-dependent fashion (stocking and glove distribution) (Bouhassira et al., 2013). It has been estimated that diabetic peripheral neuropathy (DPN) occurs between 10% and 20% of patients suffering from diabetes whiles about 40% to 60% experience neuropathy (Callaghan, 2012). University of Ghana http://ugspace.ug.edu.gh 7 However, these statistics may be underrated because about 12% of diabetic patients suffering from DPN do not inform their health care providers about this condition. Clinical symptoms observed during examination are allodynia and hyperalgesia (Brown & Asbury, 1984). Some neuropathic pains are differentiated based on electric-stabbing sensations with insensitivities or without insensitivities. Chemotherapeutic agents can also induce toxic action on peripheral nerves. The gravity can span from loss of sensory activity and lenient paresthesia to neuropathic pain, extreme ataxia and frailty resulting in conspicuous disability (Zilliox, 2017). Autonomic nerve fiber activity with orthostatic hypotension, impotence and incontinence can worsen the quality of life of suffers (Jost, 2003). Several cytotoxic agents are outlined as neurotoxic however only a few like the paclitaxel has its peripheral neuropathy as a dose-limiting side-effect (Cata et al., 2006). Paclitaxel, an antineoplastic drug obtained from the bark of the western yew tree Taxus brevifolia, is active against various tumors such as carcinoma of the ovary, breast, lung and the head and the neck. Paclitaxel exhibits antitumor action by fostering microtubule convergence (Park et al., 2014) making neuropathy one of its untoward effects. Peripheral sensory neuropathy is habitually reported neurotoxic effect of paclitaxel which restricts treatment with high and cumulative doses when administered singly or together with other neurotoxic antineoplastic drugs such as cisplatin. 2.2 OVERVIEW OF PAIN Pain can be described as unpleasant sensation which is often a response to external or internal stimulus having the potential to cause tissue damage. Pain has its origin from the Latin word ‘poena’ which means punishment and it depicts the damaging consequences that can be imposed on the body. Pain is defined by the World Health Organization (WHO) and International Association for the Study of Pain (IASP) as any unpleasant experience either sensory or emotional connected to actual or potential tissue damage or described in terms of such damage” (Taxonomy, 2014). Even though University of Ghana http://ugspace.ug.edu.gh 8 pain can be unpleasant, overbearing or present as a symptom to numerous medical conditions, it is an essential, adaptive warning sign that provides protection. Alerting the patient by the stimulation of immune function promotes healing and prevents further damages to the tissue (Tripathi et al., 2016). It is usually an uninterrupted reaction to an outward activity often associated with harm to tissues caused by infection, injury, inflammation, cancer among others. Sometimes it is with no known cause (e.g., trigeminal neuralgia). Clinically, pain has been added to the four different vital sign assessment which are temperature, pulse, blood pressure and respiratory rate (Fitzgibbon et al., 2010). Unfortunately, there isn’t any objective test to measure pain making it sometimes difficult for clinicians to assess it. (McCaffery, 1990). Pain evaluation is a vital phase when offering clinical intervention (Lacroix et al., 2017). There are several suggestions and instructions which determine what appropriate pain assessment protocols must include. Unfortunately, some of these protocols seem impractical. It is essential then that health care professionals select the adequate characteristics of pain assessment in relation to the presented clinical situation. 2.2.1 Neurophysiology of pain Pain is beyond the nociceptive neuronal transmission from injury site to where it is generated and perceived which is the brain. Pain involves numerous physiological processes including the somatosensory and limbic systems. It is also subjective because every person experience pain in different ways (Muthuraman et al., 2008). Numerous painful conditions are linked with an impairment of normal physiological pain pathway. This pathway is composed of the peripheral and central processes. The peripheral involves activities influencing nerve terminals whiles central pathway influences transmission in the synapse along the dorsal horn to the brain (Rang, 2003). University of Ghana http://ugspace.ug.edu.gh 9 Figure 2.1: Illustration of the processes of pain initiating from the periphery to the brain (Reddi et al. 2013). Briefly in figure 2.1, primary afferent neurons transmit noxious stimulus via the spinothalamic tract to the sensory cortex of the brain for interpretation and adequate response. 2.2.2 Peripheral pain processes 2.2.2.1 Pain transmission in the dorsal horn of the spinal cord The dorsal horn of the spinal cord contains Aδ and C fibers synapse with secondary afferent neurons. Histologically, the dorsal horn has ten layers known as Rexeed laminae. The synapse fibers Aδ and C sends information to Rexeed lamina I, II and other laminae via nociceptive-specific neurons. (Reddi et al., 2013). Primary afferent terminals secrete and release different excitatory neurotransmitters, calcitonin gene-related peptide (CGRP) and somatostatin. Diverse reactions involving inter-neurons, afferent neurons as well as descending modulatory pathways occur inside University of Ghana http://ugspace.ug.edu.gh 10 dorsal horn (Reddi et al., 2013). These reactions influence activities in the dorsal horn including processes of the secondary afferent neurons. Laminae II of the dorsal horn has been found to be the primary source of inhibition or facilitation of the transmission of pain. 2.2.2.2 Pain transmission in the ascending tracts in the spinal cord Second-order brain cells travels through contra-laterally in the spinothalamic, spinoreticular and spinomesencephalic tracts and transmits information to the supraspinal centers; hypothalamus, thalamus, periaqueductal grey, locus coeruleus and cerebral cortex. Here nociceptive signals are localized and generated together with sympathetic, thermoregulatory and arousal responses. They then synapse with third-order neurons in the somatosensory cortex (Meriaux et al., 2018; Reddi et al., 2013). 2.2.3 Central processing of pain Pain processing in the central nervous system is mediated by nucleus raphe magnus and periaqueductal grey matter. In addition, nociceptive inhibition of neurons in the dorsal horns which block nociception-transmitting neurons also contributes to the central processing of pain. Transmission of pain via the spinal cord is promoted by the lateral spinothalamic tract pathway for nociceptive information to reach the brain. The lateral spinothalamic tract pathway is divided into two different pathways. One of which is the neospinothalamic tract for "fast spontaneous pain". This pathway controls fast pain which travels through type Aδ fibers to discontinue on the dorsal horn of the spinal cord where they form a synapse with the dendrites of the neospinothalamic tract (Millan, 1999). The axons of these neurons pass through the spine to the brain and cross the midline via the anterior white commissure, through the contralateral anterolateral columns and then stops on the ventrobasal complex of the thalamus which synapses with the dendrites of the somatosensory cortex. University of Ghana http://ugspace.ug.edu.gh 11 The second pathway is the paleospinothalamic tract. This pathway slowly advances pain through type C fibers. It also transmits the sensation of pain to the dorsal horn through laminae II and III. Type C fiber together with laminae II and III are called the substantia gelatinosa. Electrical impulses from nerve fibers passes through lamina V in the dorsal horn. Lamina V synapses with brain cells that connect with fibers from the fast pathway. The fast pathway transmits the impulse to the opposite side through anterior white commissure. This then travels up anterolateral pathway. These brain cells or neurons are linked to one-tenth of fibers in brain regions including the thalamus and medulla (Kivell & Prisinzano, 2010; Millan, 1999). 2.2.4 Inhibiting pain mediation Processes work to restrict pain mediation in the spinal cord and through descending blocking from higher centers. 2.2.4.1 Gate control theory of pain The gate control theory of pain was described by Melzack and Wall in 1965. It illustrates a mechanism showing that controlling pain at the spinal cord level is possible (Reddi et al., 2013). The theory also explains the reason banging our head feels better when we rub it against an obstacle and states that Aβ fibers inhibitory interneurons activation in the dorsal horn blocks the transmission of pain signals through the C fibers (Reddi et al., 2013) 2.2.4.2 Descending inhibition Parts or regions of the brain that has an impact on descending inhibitory are the periaqueductal grey and rostral ventromedial medulla (RVM). These areas have numerous endogenous opioids as well as opioid receptors demonstrating why opioids are considered as analgesics. Some pathways descend University of Ghana http://ugspace.ug.edu.gh 12 through the dorsal horn which contains adrenergic, serotoninergic and opioid receptors inhibiting pain medication. Since the pathways involves noradrenaline and serotonin it can be concludes that they are monoaminergic (Reddi et al., 2013). 2.2.5 Classification of pain Pain is classified into nociceptive and neuropathic. Other types of pain are classified according to their duration, which are the acute and chronic pain. In addition, some other types of pain also exist outside these classification e.g. pain from fibromyalgia (Rang, 2003) Figure 2.2: An illustration of the types of pain (www.Painscience.com) Figure 2.2 indicates three main pain classification which includes nociceptive pain, neuropathic pain and pain arising from other noxious stimuli. They all exhibit different classical signs but some of their clinical characteristics appears to be the same description in all pain types. University of Ghana http://ugspace.ug.edu.gh 13 2.2.6 Nociceptive pain Nociceptive pain refers to pain caused by activities in the neuronal pathways which are secondary to tissue damage or stimuli that are harmful to tissues. It is activated by factors such as inflammation or diseases. Examples of nociceptive pain are arthritic or surgical pain or lower bac pain (Rang, 2003). Nociceptors are involved in the identification of dangerous stimuli and conveying them into electrical impulses they are transmitted to the brain. Nociceptors are free nerve endings sensory receptors of core sensory afferent neurons possessing a single cell body and found inside dorsal root ganglion. There are three main groups of afferent neurons which are as follows; Group A, further classified into α, β, γ, δ, group B, and group C. Noxious or harmful stimuli are responded to by the primary sensory afferent fibers such as tiny diametric, unmyelinated C-polymodal fibers and thinly myelinated, small diametric (Aδ) fibers. These primary sensory afferent fibers are connected to nociceptors and are triggered by stimuli including pressure, chemicals and heat (Koltzenburg, 2000). Damaged tissues also release inflammatory mediators such as cytokines, bradykinin and H+ which stimulates or sensitizes nociceptors by decreasing the threshold for their activation (Reddi et al., 2013). The expression of pain is two-fold. It’s either first pain or second pain. First pain is conducted through Aδ nociceptors. Consequently, its fast, sharp, localized and short-lived. the second pain is conducted and transmitted by the C-fiber, polymodal nociceptors. It is slow, diffuse, persistent, burning and not short-lived like first pain but long-lived and can even last after termination of the stimulus (Ma & Zhang, 2010). 2.2.7 Neuropathic pain Neuropathic pain may be induced by lesions or diseases to the nerves of the somatosensory neurons. The injury to these nerves can be attributed to factors such as an infection, trauma, diseases like University of Ghana http://ugspace.ug.edu.gh 14 diabetes mellitus, surgery or chemotherapy. Neuropathic pain is more likely to be spontaneous and is experienced as a burning or ‘like an electric shock’. It is experienced in response to a stimulus which might not necessarily cause pain (allodynia),or may be experienced as an exaggerated response to a painful stimulus (hyperalgesia) (Colloca et al., 2017). Pain generated by peripheral nerve damage is interrelated with neuropathic pain and as such they are used synonymously even though it may include central pain linked to injuries to the CNS. Neuropathic pain changes the patient’s quality of life by interrupting the mental wellness. Due to the chronic nature of neuropathic pain it presents a setback in clinical setting other factors that may contribute to this setback includes the gravity and reduced efficacy of some classical analgesics (Backonja, 2004). With reference to the manifestations of this type of pain, mechanical allodynia consists of marked disruption in sensation. In addition, cold allodynia may be prominent particularly in sympathetically stimulating episodes (Bennett & Xie, 1988). 2.2.7.1 Etiology of neuropathic pain Neuropathic pain has been classified into two. The first classification is based on the etiology of injury to the nervous system whiles the second is based on its anatomical distribution. This classification is helpful in differential diagnosis and disease-modifying treatment. However, it does not have a framework for clinical management of the pain. The connections between its etiology, mechanisms, and symptoms is very intricate (Devor et al., 1994). The manifestation of pain in various disease states may occur through common mechanisms. These mechanisms are the consequence of a particular disease process. Even though, few patients are affected by neuropathic pain there are no predictors for its development. Some fundamental causes of neuropathic pain are excessive intake of alcohol, surgery on the spinal cord, chemotherapy (Boland et al., 2010), diabetes University of Ghana http://ugspace.ug.edu.gh 15 mellitus, facial nerve problems, HIV/AIDS infection and syphilis (Sharif-Alhoseini et al., 2012). 2.2.7.2 Symptoms of neuropathic pain Most people suffering from neuropathic pain shows continuous or paroxysmal pain without a stimulus. This type of pain may be striking, piercing or burning and is determined by the type of activity the sympathetic nervous system may be involved in. Spontaneous activity in the fibers of nociceptor C causes a burning sensation and sensitizes neurons of the dorsal horn. Spontaneous activity in large, myelinated A fibers is often linked with stimulus-independent paresthesia and the sensitization of the central nervous system to pain and dysesthesias. Pain which is induced by a stimulus is characterized by peripheral nerve impairment attributed to hyperalgesia and allodynia. Allodynia in isolation does not implicate a specific process it is therefore essential to view as a subset of hyperalgesia so that the clinical manifestations can be diagnosed based on the mechanism involved. Stimulus-induced hyperalgesia has been divided into mechanical, thermal or chemical. Mechanical hyperalgesia can occur as dynamic, static or punctate hyperalgesia. 2.3 Paclitaxel-induced Peripheral Neuropathy 2.3.1 Paclitaxel and peripheral neuropathy Taxanes, an antineoplastic drug works on microtubules by interrupting sensory dominant nerves by acting on sensory fibers with small diameters. Manifestation usually involves microtubule depolymerization and repolymerization which causes damage. Such damages include dysesthesias, paresthesias, changes in proprioception, numbness and loss of dexterity in the toes and fingers. sometimes other localizations including effect on motor and autonomic activity may occur. Clinical manifestations usually begin just few days after the first dose and its dose dependent. The effect ameliorates when treatment ceases. For some people symptoms can last for up to 3 years after completing therapy however for some it continues the entire life. Examples include paclitaxel, University of Ghana http://ugspace.ug.edu.gh 16 docetaxel and cabazitaxel. These drugs are approved for treating cancer such as cancer of the breast, ovary and prostate. The occurrence of taxanes can causes chemotherapy-induced peripheral neuropathy (CIPN) which may rise between the range of 11% and 87% with Paclitaxel recording the greatest rate. Paclitaxel fostered neuropathy is marked by various sensory alterations for instance the occurrence of mechanical allodynia where light pressure or touch usually seen as harmless elicits pain. The exact pathobiology of CIPN is not fully clear, however, current studies points to “terminal arbor degeneration” (Bobylev et al., 2015), oxidative stress (Han & Smith, 2013), mitochondrial impairment and mitotoxicity (Ocean & Vahdat, 2004). 2.3.2 Pathogenesis of paclitaxel-induced peripheral neuropathy Paclitaxel causes microtubule interference, which inhibits axonal transport and results in Wallerian degeneration. This affects the action of ion channels such as sodium and potassium causing excitability of peripheral neurons. Mitochondrial injury caused by Paclitaxel increases the levels of reactive oxygen species which causes impairment in calcium homeostasis of neurons and damages to enzymes, proteins and lipids. This causes apoptotic alteration and demyelination of peripheral nerves. Paclitaxel also activates microglia and astrocytes which in turn attracts and activates immune cells. It also causes the secretion and elevation of pro-inflammatory cytokines which can lead to nociceptor sensitization and ultimately to the development of neuroinflammation. University of Ghana http://ugspace.ug.edu.gh 17 Figure 2.3: A diagrammatic representation of mechanism of action of taxanes causing peripheral neuropathy (Zajączkowska et al., 2019). Figure 3 illustrates the MOA of taxanes causing neuropathic pain, briefly taxanes causes peripheral neuropathy via two major pathways which includes microtubule disruption and activation of microglia and astrocytes which eventually results in neuroinflammation and altered excitability of peripheral neurons resulting in peripheral neuropathy. 2.3.3 Paclitaxel and microtubule interference Microtubule interference is the primary mode of activity of taxanes and it accounts for their antineoplastic effect, it is also linked with the evolution of peripheral neuropathy (Gornstein, 2017). Its assembling and packaging of the microtubules results in changes in cell shape and cell strength University of Ghana http://ugspace.ug.edu.gh 18 which inhibits transport of synaptic vesicles in the axons (Scott et al., 2011). 2.3.4 Paclitaxel and mitochondrial dysfunction Mitochondria damage in neurons and other cells causes oxidative stress and produces reactive oxygen species (ROS) including hydroxyl radicals and superoxide. Axonal transport of important cellular components and mRNA deficits (Bobylev et al., 2015) to distal neuronal regions because of microtubule interruption may have notable influence on this mechanism. Elevated levels of ROS are found in the spinal cord and sensory neurons. These elevated levels can activate apoptotic pathways and can disrupt cell structure and demyelination. These activities inhibit signal transmission and immune response activation leading to increased secretion of cytokines (pro-inflammatory cytokines). This mechanism amplifies itself in that the above mechanisms can initiate further mitochondrial injury. (Bulua, 2011;Areti, 2014). In recent studies, paclitaxel is known to cause swelling of the mitochondria as well as vacuolation and loss of mitochondrial structure (Gilardini et al., 2012). 2.3.5 Paclitaxel and axon degeneration Numerous studies have stated that the administration of paclitaxel causes damage to peripheral nerves, decreases neuronal fibers and leads to. The disruption of microtubule and the resulting impairment in axonal transport of some cellular components can lead to the degeneration of distal nerve segments (Wallerian degeneration) and the restructuring of axonal membrane (Bober, 2015). Boyette et al. demonstrated that there is a reduced number of intra-epidermal fibers in paclitaxel- induced CIPN murine models (Boyette-Davis, 2012). Ferrari also showed that there is a restricted corneal innervation in these murine models. Signaling of cytokines and chemokines may also be implicated in the degeneration of axons as Zhang et al. have revealed that a decrease in the levels of chemokine MCP1/CCL-2 decreases nerve degeneration and CIPN behaviors in a murine model University of Ghana http://ugspace.ug.edu.gh 19 (Ferrari, 2013). 2.3.6 Paclitaxel and calcium homeostasis An impairment in Ca2+ hemostasis is involved in PIPN pathogenesis. An impairment in intracellular Ca2+ has been noticed in models illustrating paclitaxel neuropathy. Endoplasmic reticulum and mitochondria are rich in intracellularly Ca2+. Paclitaxel administration can lead to the release of Ca from the mitochondria through the activation of mitochondrial permeability transition pore (mPTP). This can cause a rapid depolarization in the mitochondria (Kidd, 2002). The is a high probability that in the ER paclitaxel can cause the release of Ca2+ through the regulation of 1,4,5-trisphosphate receptor (IP3R) (Boehmerle, 2006). This process leads to an increased expression of CaV3.2 channels in rats. The repression of these process leads to an overturn of hyperalgesia (Peltier & Russell, 2002). 2.3.7 Paclitaxel and peripheral nerve excitability Modification in the expression and activity of NaV, TPR and KV ion channels is an alternate process that can account for the development of PIPN. A reduction in the expression of K+ ion channel was found in the DRG of paclitaxel evoked CIPN model. This caused a spontaneous function of nociceptors. Activation of TRPV1 and TRPA1 cation channels plays a significant role in pain signaling and are identified in DRG neurons (Materazzi, 2012). TRPA1 antagonists are known to relieve paclitaxel-induced inflammation, cold allodynia and hyperalgesia. Paclitaxel also increases the levels of NaV1.7 contributing to the development of CIPN (Hara, 2013) and hence restricting this channel reduces hyperalgesia in rats. 2.3.8 Paclitaxel, immune processes and neuroinflammation Paclitaxel increases pro-inflammatory cytokines (TNF alpha and IL-1 beta) and reduces anti- University of Ghana http://ugspace.ug.edu.gh 20 inflammatory cytokines (IL-4 and IL-10) ( Doyle, 2012; Areti, 2014) . This mechanism attract and activate immune cells consequently leading to neuroinflammation (Krukowski, 2016). Krukowski (2016) demonstrated that IL-10 decreases paclitaxel-induced CIPN. Paclitaxel also activates microglial and astrocyte as well as increasing macrophage number of DRG in neuronal and non- neuronal cells (Gornstein, 2017). 2.3.9 Genetics and paclitaxel-induced peripheral neuropathy It has been found in recent studies that low frequency variants of EPHA6, EPHA5 and EPHA ephrin gene receptors and its associated severity in taxanes induced neuropathy (Apellániz-Ruiz et al., 2015; Leandro-García et al., 2012). Genes playing an essential role in paclitaxel-induced neuropathy includes glycogen synthase kinase-3β gene (GSK3β) (Park et al., 2014), Charcot-Marie-Tooth disease gene ARHGEF10 (Boora et al., 2016) and VAC14 which codes for parts of a trimolecular complex regulating the levels of phosphatidylinositol 3,5-bisphosphate (Komatsu et al., 2015). 2.4 Diabetic-Induced Peripheral Neuropathy 2.4.1 Diabetes and diabetic neuropathy The effect of diabetes mellitus on the peripheral nervous system manifests diversely. Diabetic neuropathy is largely involved in damages to the peripheral nerves. Symptoms of diabetic neuropathy include tingling sensation, numbness, pain with/without weakness. These symptoms generally start from the feet and travels to the fingers (Albers & Pop-Busui, 2014). These symptoms correspond with sensory manifestations which are more conspicuous than motor involvement. Majority of patients experience a condition of insensibility and excruciating sensitivity simultaneously. However, this condition varies within patients (Callaghan et al., 2012). Diabetic-induced neuropathic pain and numbness results in imbalance making patients fall. This University of Ghana http://ugspace.ug.edu.gh 21 makes it a major factor that increases the number of falls in patients suffering from diabetic neuropathy (D'Silva et al., 2016). It has been discovered that patients suffering from diabetic neuropathy have increased susceptibility to falls than patients suffering from diabetes without neuropathy (Peltier et al., 2014). In severe cases, patients are more likely to acquire foot ulcers and lower extremity amputations in disease state (Dabkana et al., 2018). Diabetes is the leading cause of lower extremity amputations, which is more highly probable to occur in patients also suffering from neuropathic pain (Al-Rubeaan et al., 2015). Many patients with diabetic foot ulcers undergo lower extremity amputations globally (Ferreira-Chamorro et al., 2018). Diabetes affects the health standard of patient and becomes worse in patients also suffering from neuropathic pain (Alleman, 2015). DN incapacitates patients and because there are no therapeutic agents to treat it makes patient more devastated (Goldman & Appell, 1999). In the diabetic population, about 20% of them suffer from diabetic neuropathy whiles 40% to 60% have reported neuropathy (Vincent et al., 2011). These figures may be under-recorded since 12% of patients with DNP informs their healthcare provider about their situation. The symptoms of DNP are similar to other forms of neuropathic pain but are distinguished by electric-stabbing sensations with or without insensibilities (Brown & Asbury, 1984). Diabetic neuropathy is based on the length of axon and begins in toes travelling upwards to the calf and finally to the fingertips (Edwards et al., 2008). University of Ghana http://ugspace.ug.edu.gh 22 Figure2.4: An illustration of the features of diabetic peripheral neuropathy Figure 2.4 illustrates the clinical manifestation of diabetes peripheral neuropathy which is characterized by allodynia and hyperalgesia and can be illicited using Von Frey test, hindpaw withdrawal test and tail flick test in animal modules. (Azhary, H. et al 2010) University of Ghana http://ugspace.ug.edu.gh 23 Figure 2.5: Stocking Glove Configuration of DPN. (easd.org) Figure 2.5 illustrates the pattern with which diabetes peripheral neuropathy affects certain specific body areas. It is realized that the pattern of neuropathy manifestations is with respect to the extremities(limbs). 2.4.2 Free radicals and diabetic neuropathy Reactive oxygen species which includes hydroxyl radicals, peroxide, superoxide and stress triggered by single oxygen are actively involved in the development of diabetic neuropathy. Oxidative stress induced by long-term hyperglycemia is a direct linkage that presents a unified mechanism involving tissue damage (Negi, 2011). Biological markers of oxidative stress undergo significant alteration in DN. Also, there is a nerve dysfunction which may be caused by the over expression of superoxide and peroxynitrite in sciatic nerves (Pacher et al., 2005). The over expression of superoxide decreases vascular activity which can be a hindrance to nutrient supply to the sciatic nerve. A reduction in glutathione and antioxidant enzymes has also been found to contribute to diabetic neuropathy (Yagihashi et al., 2011). DNA fragmentation has been identified in peripheral nerve sections of University of Ghana http://ugspace.ug.edu.gh 24 animals induced with diabetic neuropathy (Sullivan et al., 2007). In addition, natural cell death of the dorsal root ganglion (DRG) and vagus ganglion was recorded in streptozotocin-induced diabetic animals (Guo et al., 2004). However, similar report was not recorded in peripheral neurons of models showing elevated ROS from chronic hyperglycemia (Zherebitskaya et al., 2009). It has been stated by Zherebitskaya and his team that elevated levels of blood glucose decreases the activity of antioxidant enzymes. This created distortions in DRG which was regulated by ROS but there was no recorded natural cell death. The occurrence of natural cell death in peripheral nerves and diabetes has not been fully understood. However, a correlation between chronic hyperglycemia and elevated ROS has been established. Therefore, oxidative biological markers can be used as indicators for accurate diagnoses and progression of neuropathy in diabetes (Yagihashi et al., 2011). 2.4.3 Anatomy of diabetic neuropathic pain The sensation of pain indicates the presence of actual or potential tissue injury caused by a stimulus. Sensory afferent nerves transmit pain through myelinated fibres from the skin and other parts of the body. Large, myelinated fibres such as A-alpha and A-beta are involved in limb proprioception and the transmission of sensations from limb proprioception respectively. Also, large, myelinated A- delta fibres and small C unmyelinated fibres transmit nociceptive sensations. Pain that is superficial, producing stinging or pricking sensation is transmitted by A-delta fibres whiles pain that is deep- seated, burning and itching travels through slow, unmyelinated C fibres. This type of pain is often followed by hyperalgesia and allodynia. Damage to tissues causes the secretion and release of inflammatory chemicals such as prostaglandins, cytokines, bradykinins, and histamines at the site of inflammation. This leads to the depolarization of nociceptors causing an action potential (Willis & Westlund, 1997). Consequently, action potential transmits nociceptive sensation via dorsal root University of Ghana http://ugspace.ug.edu.gh 25 ganglion (DRG) to the spinal cord through its dorsal horn. The release of glutamate and substance P causes transmission of nociceptive sensations through spino-thalamic tract, thalamus and the cortex. Pain is perceived and intercepted in the cortex (Willis & Westlund, 1997). Neuropathic pain is a consequence of lesion or disease attacking the somatosensory system not necessarily by a stimulus. It can be classified as peripheral neuropathic pain or central pain. Peripheral neuropathic pain is marked by the activation of pathways of pain in the peripheral nerves and posterior roots. Central pain is marked by the activation of pain pathway in the spinal cord and brain (Treede et al., 2008). Conversely, nociceptive pain is a reaction to damages to tissues such as skin and muscles caused by a stimulus. It usually stops when the injury is healed. Based on the nature of origin pain and its etiology, the symptoms of pain can be focal, multifocal or generalized (Aslam et al., 2014). 2.4.4 Pathophysiology of diabetic neuropathy The development of Diabetic Peripheral Neuropathy (DPN) remains unclear. However, recent studies show DPN is as a result of an impact exerted by physiologic and metabolic dysfunction in somatosensory nerves. The effect of diabetes impacts these nerves and the microvasculature supplying the nerves (vasa nervorum) (Van Dam et al., 2013). Chronic hyperglycemia, characterized by an increase in plasma glucose levels (Britland et al., 1992) plays a vital role in nerve injury. It initiates aberrant biochemical mechanisms such as dyslipidemia (Vincent et al., 2013;Hinder et al., 2013), secretion of advanced glycated end products (Jack & Wright, 2012), protein kinase C (Geraldes & King, 2010), inflammation (Pučić et al., 2011) dysfunction of insulin signaling (Kim & Feldman, 2012), production of ROS by the mitochondria (Sena & Chandel, 2012), hyperactivity of the polyol pathway (Oates, 2002) and ER stress (Lupachyk et al., 2013). These abnormalities interfere cellular homeostasis and promote the development of diabetic neuropathy eventually progresses. Vascular disorders are known to cause neuropathy in diabetic animal models (Tesfaye University of Ghana http://ugspace.ug.edu.gh 26 et al., 2010). As DPN progress, neuronal impairment is linked to the development of endoneurial microangiopathy (Malik et al., 1994), vascular irregularities promote reduced oxygen tension and hypoxia resulting in neuronal ischemia. Recent findings shows that DPN in both type-1 and type-2 diabetic patients may be inherently separate disorders (O'Brien et al., 2014a). Another clinical study evaluated the effectiveness of regulating glucose on the prevalence of DPN. It has been found that regulating glucose lessened the onset and progression of DPN in type-1 diabetes mellitus (T1DM). This has not been recorded in people suffering from type-2 diabetes mellitus (T2DM). This shows that DPN in T1DM and T2DM are different from each other (Callaghan et al., 2012). 2.4.5 Mechanisms leading to the development of DNP 2.4.5.1 Polyol Pathway Aldose reductase is an enzyme that reduces toxic aldehydes into inactive alcohols and its the main enzyme in polyol pathway. When a cell becomes extremely hyperglycemic, glucose is converted to sorbitol by aldose reductase. Sorbitol is then oxidized to fructose. This process involves a cofactor of aldose reductase, nicotinamide adenine dinucleotide phosphate (NADPH). NADPH produces glutathione which reduces intracellular oxidative stress (Brownlee, 2005). In a study conducted for a period of five years, aldose reductase inhibitor administered to diabetic dogs resulted in an impairment of nerve conduction velocity as its observed in diabetic patients. It was reported that the induced diabetic impairment in the velocity of nerve conduction was ameliorated after treatment (Engerman et al., 1994). University of Ghana http://ugspace.ug.edu.gh 27 2.4.5.2 Advanced glycated end products (AGE) Damages to cells by the precursors of AGE involves three different mechanisms. Firstly, endothelial cell which is its main target changes intracellular proteins for instance proteins taking part in gene transcription. Secondly, the AGE diffuses from cells and changes the molecules of extracellular matrix altering signaling of the cells and matrix which causes cellular impairment. This process is identified by the cross linkage of collagen, consequent tendon and ligament pathology. Lastly, products of AGE disperse from cells and changes proteins that are circulating in the blood including albumin. These proteins trigger AGE’s resulting in the secretion of inflammatory cytokines as well as growth factors creating a vascular dysfunction (Winocour et al., 1988). 2.4.5.3 Protein Kinase C Pathway Protein Kinase C is triggered by an increase in the synthesis of diacylglycerol by hyperglycemia. This actuates protein kinase C cofactors. PKC produce an effect on the expression of genes such as causing a reduction in endothelial nitric oxide (NO). It also increases vasoconstrictor endothelin-1 and modifies cellular metabolism and axonal flow of Schwann cells (Greene et al., 1990). 2.4.5.4 Hexosamine Pathway Hexosamine pathway is the final pathway hastened by high blood glucose which is metabolized by glycolysis. Metabolites like fructose 6-phosphate from glycolysis is transported into signaling pathway and converted into Serine and threonine by GFAT enzyme. Serine and threonine are then bound by uridine phosphate (UDP) N-acetyl glucosamine causing alterations to gene expression and subsequent nerve irregularities (Nawroth et al., 2018). University of Ghana http://ugspace.ug.edu.gh 28 2.4.6 Clinical features 2.4.6.1 Microvascular ischemic changes Pathological alteration of diabetic nerves include the thickening of capillary basement membrane, endothelial cell hyperplasia, neuronal ischemia and even neuronal death (Pallas & Larson, 1996). 2.4.6.2 Advanced glycosylation end products Chronic intracellular hyperglycemia releases glycating agents known as advanced glycosylation end products. Advanced glycosylation end products can form inside and around peripheral nerves. They can disrupt axonal transport, resulting in decreased velocity of nerve conduction. Advanced glycosylation end products can also decrease NADPH through the activation of NADPH oxidase. This process can produce hydrogen peroxide and oxidative stress (Zochodne, 1999) 2.4.6.3 Inflammatory microvasculopathy Numerous studies have suggested different forms of diabetic neuropathies such as asymmetrical neuropathies, mononeuritis multiplex and diabetic amyotrophy can be induced by inflammatory vasculopathy. Diabetic nerves emerge having high responsiveness to immune factors (cellular and humoral), which involves removal of immunoglobulin, actuation of lymphocytes and complement trigger (Greene et al., 1990). 2.4.6.4 Growth factor and insulin deficiency To keep the structure of the nerves, its activities and restoration following an injury, neurotrophic factors are needed. Decrease in the number of nerve growth factor and insulin-like growth factors 1 have been connected to serious forms of diabetic neuropathy in murine models. Insulin have University of Ghana http://ugspace.ug.edu.gh 29 demonstrated neurotrophic activities and its insufficiency can account for the evolution of neuropathy (Brown & Asbury, 1984). 2.4.6.5 Neuronal membrane ion channel activity Aberrant calcium channel action is implicated in cellular damage and death observed in different diseases. Elevated functions of voltage-gated calcium channels are illustrated in diabetic neuropathy that results in tissue damage. Sodium channel defect plays a vital function in initiating painful neuropathy, a frequent observation in diabetic sufferers. 2.4.6.6 Essential fatty acids Essential fatty acid from linolenic acid, prostaglandins and thromboxane have been found to be unbalanced in diabetic patients. This causes several cellular abnormality like anomaly in membrane fluid, cell membrane modifications of red blood and decreased levels of prostaglandin E2 (Edwards et al., 2008). 2.4.6.7 Distal predominant sensory polyneuropathy Distal predominant sensory polyneuropathy is the commonest type of diabetic neuropathy that occurs because of “dying-back” axopathy. a length-dependent procedure. Paresthesias and numbness begins stealthily and continues steadily, starting from the feet and proceeds with time. Loss of touch usually precedes loss of perception and vibration. Serious forms of neuropathy and uncoordinated movements can occur. Sensory deficits can result in complexities like unhealing sores and neuropathic arthropathy. Motor nerves are often impacted late, resulting in muscle weakness and reduction in tissues. Notable motor frailty signals a coincidental neuropathy from a different source such as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (Peltier et al., 2014). University of Ghana http://ugspace.ug.edu.gh 30 2.4.6.8 Autonomic neuropathy Autonomic neuropathy is rarely reported in diabetes but occurs. Some of its clinical symptoms are distal anhidrosis, orthostatic hypotension, and erectile weakness in men. Comorbidity with cardiac disease may possibly account for death in diabetic suffers because of silent myocardial ischemia and arrhythmia. Gastrointestinal defects such as serious constipation, or diarrhea may be observed in some cases. 2.4.6.9 Compressive mononeuropathy compressive injury has a higher incidence in diabetic nerves. Carpal tunnel syndrome occurs in one- third of patients. Likewise, ulnar nerve disorders occur across the elbow whiles nerve damage occur across the fibular head. Results from diabetic patients with carpal tunnel syndrome can be successful after surgery with clear focal modification of demyelination in the median nerve across the carpal tunnel (Callaghan et al., 2012). 2.4.6.10 Noncompressive focal and multifocal neuropathies Diabetic amyotrophy, truncal neuropathies, cranial neuropathies, mononeuropathies, and mononeuropathy multiplex are examples of clinical spectrum of multifocal neuropathies in diabetes. These may be as a result of nerve ischemia and occlusion of the vasa nervorum. Diabetes is connected to accelerated atherosclerosis, which may be implicated in evolution of neuropathy. Current studies have indicated that mononeuropathy multiplex and diabetic amyotrophy are as a result of inflammatory vasculopathy which acts on vasa nervorum, as such, immune therapy has been recommended. Similar pathogenetic mechanisms may be the cause for the development of other noncompressive focal neuropathies. Unfortunately, pathological examinations in this regard University of Ghana http://ugspace.ug.edu.gh 31 are not sufficient to draw any relevant conclusions (Tesfaye et al., 2010). 2.4.6.11 Diabetic amyotrophy Diabetic amyotrophy is uncommon but disabling neuropathy that shows in type-2 diabetics. The manifestations are usually sub-acute with pain and asymmetric frailty and atrophy of proximal lower limb muscles. Muscles and limbs such as distal lower extremity muscles and infrequently upper limb muscles respectively may be affected. Whiles the resulting pain is severe, sensory impairments are comparatively less severe. Unintended reduction in weight at the beginning is a frequent occurrence. Symptoms can be present for about 6 months after which there may be a steady improvement over 2-3 years. Some experiments indicate that immunosuppressant therapy can be helpful, especially in early treatment. For instance, pulse corticosteroid shows fast pain relief and rapid motor recuperation. The treatment is with less adverse events and does not induce hyperglycemia (Al- Rubeaan et al., 2015). It was found in a study that 7 out of 8 people who took a 2-hour glucose tolerance test had an increased glucose level in their serum in the deficient glucose tolerance range. They also had normal levels of fasting blood glucose and HbA1c. This shows that sporadic hyperglycemia and glucose tolerance deficiency may be implicated in cases where diabetes is absent. 2.4.6.12 Mononeuritis multiplex and multifocal neuropathy Mononeuropathy multiplex is a condition of more than two peripheral nerve trunks causing multifocal sensory motor impairment. Sensory motor impairment often occurs in collagen vascular disorders. it can also occur as a symptom of nonsystemic vasculitis in peripheral nerves. Mononeuritis multiplex unlike diabetic amyotrophy shows muscle extremity activity, stepwise progression and sensory-motor multifocal irregularities. Diabetic amyotrophy however, proceeds quickly involving predominant proximal and striking motor. Inflammatory vasculopathy with University of Ghana http://ugspace.ug.edu.gh 32 multifocal axon loss are fundamental in the pathogenesis of mononeuritis multiplex in diabetes. In one study 3 out of 4 patients on corticosteroids and chlorambucil demonstrated dramatic and faster amelioration. 2.4.6.13 Cranial neuropathies Cranial neuropathy is frequently seen as oculomotor nerve palsy in diabetes. It usually produces pupil-sparing third nerve palsy. Some cranial nerves have been implicated. These neuropathies occurring suddenly have fine course with steady sudden recovery. 2.4.6.14 Other non-compressive focal neuropathies Truncal neuropathy shows sudden initiation of pain in the truncal nerve. Truncal neuropathy may occur independently or cofound with diabetic amyotrophy or mononeuritis multiplex. Unintended reduction in weight can take place. Ulnar, peroneal, femoral, and sciatic mononeuropathies may occur acutely or sub acutely. Pathophysiological of these conditions are not completely understood but may be connected to axon loss, possibly because of loss of oxygen to nerves. Treatment of these focal neuropathies includes pain control and regulation of hyperglycemia; the results are usually good (Woolf. et al., 1999). 2.4.6.15 CIDP in diabetes CIDP is often seen in patients exhibiting sub-acute and severe motor neuropathy with proximal frailty not like the “typical” diabetic neuropathy which usually is sensory and length dependent. Diabetes in CIDP and vice versa are very common in the general population. The frequent occurrence of nerve conduction irregularities together with axon loss and demyelination have made the diagnoses of CIDP in diabetics difficult. This difference is essential, as patient’s response to University of Ghana http://ugspace.ug.edu.gh 33 immune treatments can be comparable to patients with CIDP but not diabetic (Callaghan et al., 2012) Figure 2.6: Mechanisms of diabetic neuropathy (https://ars.els-cdn.com/content/image/1- s2.0-S1474442212700650-gr3.jpg) Figure 2.6 illustrates the MOA of diabetic neuropathy. Briefly hyperglycaemia can cause neuropathy via osmotic stress resulting in ROS hence causing cell damage including nerve cells. University of Ghana http://ugspace.ug.edu.gh 34 2.5 Pharmacotherapy of Diabetes and Paclitaxel-induced Neuropathic Pain 2.5.1 Antidepressants Antidepressant also produce analgesic effects because of their influence on modulatory inhibitory controls. Several antidepressants are used to treat NP. Such antidepressants include tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOI). Duloxetine through numerous studies have been recommended to treat pain from diabetic neuropathy. Its use is not only limited to diabetic neuropathy but also to other forms of NP including paclitaxel-induced neuropathy (Attal & Bouhassira, 2015). Some common adverse effects attributed mostly to TCA are somnolence, constipation, dry mouth and dizziness. In addition, tertiary amine TCA produce sedation, postural hypotension and anticholinergic effects. Duloxetine on the other hand is more associated with nausea (Attal et al., 2016). 2.5.2 Tramadol Tramadol which is of the opioid group possess analgesic property. They exert their influence by inhibiting serotonin and norepinephrine reuptake like tapentadol. These drugs are often misused and abused with a high probability of dependency compared to other opiods. Tramadol has been efficiently used to treat peripheral NP. However, it is contraindicated in geriatrics because it may cause confusion and in patients already on antidepressants because they may cause serotonin syndrome (Vinik et al., 2014). 2.5.3 Cannabinoids It has been demonstrated that treatment with Oro-mucosal cannabinoids (2.7 mg delta-9- tetrahydrocannabinol and 2.5 mg cannabidiol) is effective against pain associated with multiple sclerosis and also against refractory peripheral NP with allodynia ( Rog et al., 2007; Attal et al., University of Ghana http://ugspace.ug.edu.gh 35 2016). However, negative results have been observed in some studies (Attal et al., 2016). Side effects such as dizziness, fatigue and nausea are known to occur. Cannabis in some situations can increase the severity of psychiatric conditions therefore, it is contraindicated in patients suffering from psychiatric disorders (Zilliox, 2017). 2.5.4 Opioids Opioids such as oxycodone and morphine are averagely effective against peripheral Neuropathic pain (Eisenberg et al., 2005). Side effect from these opioids include constipation, nausea and somnolence. After several years of administering high doses of opioids there is a high probability of its abuse. It may also cause cognitive deficits, modification of endocrine and immunologic functions (Provenzano & Viscusi, 2014; Pujol et al., 2018). Some other factors that call for attention to increased prescription of opioid are overdose mortality, morbidity and misuse (Zilliox, 2017). Daily dose of morphine equivalence must be carefully evaluated especially when patients require higher daily doses. 2.5.5 Antiepileptic drugs It has been found in animal studies that the analgesic effect of pregabalin and gabapentin are linked with decreased central sensitization and nociceptive transmission through alpha-2- delta subunit of calcium channels (Luo, 2002; Wijayasinghe et al., 2016). They are highly effective against peripheral or central NP even though negative results have been detected in some recent studies (Simpson et al., 2010). Extended release of gabapentin formulations (enacarbil) produced similar results as gabapentin used in clinical trials, which was administered twice daily. Similar activity in comparison with TCA has been stated with frequent side effects such as somnolence and weight gain (Attal et al., 2016). Anti-epileptics like pregabalin and gabapentin have not been consistent with its treatment against Neuropathic pain. However, that has not been the case with carbamazepine University of Ghana http://ugspace.ug.edu.gh 36 in trigeminal neuralgia (Attal et al., 2016). 2.6 Use of Plants as Analgesic Agents in the Management of Pain Plants are good sources of bioactive substances which are useful in treatment and management of different diseases. These bioactive compounds accounts for the reason why 90% of drugs are from plant sources (Vittalrao et al., 2011). Medications from plant sources are noted to be relatively safer (Sengupta et al., 2012). Plants contain several compounds which have been found to possess analgesic properties and with lower toxic profiles and higher therapeutic effects (Sengupta et al., 2012). Examples of these natural compounds are tannins, alkaloids, flavonoids and saponins, (Jain et al., 2011; Ranjan et al., 2010; Zulfiker et al., 2010). Plants like Manilkara zapota, Scoparia dulcis L, Ficus racemose, Allium stracheyi, Murraya paniculate have analgesic effect and are traditionally used to treat pain (Borikar et al., 2009; Jain et al., 2011). 2.6.1 Annona muricata Annona muricata also called soursop, ‘Graviola’ in Portuguese and ‘Guana ́bana’ in Latin America. In Ghana, Akans call it “Aluguintuguin”. Taxonomically it is classified; Kingdom: Plantae, Division: Angiosperms Magnoliophyta: Class: Magnolids, Order: Magnoliales, Family: Annonaceae, Genus: Annona, Species: Muricata. 2.6.1.1 Botanical description of A. muricata Annona muricata is always green and has a height of about 5–8 m, 15-83 cm in diameter with low branches, features an open, roundish canopy with large, glossy, dark green leaves (Coria-Tellez et al., 2018). It has large fruits, heart-shaped and green in color. The diameter of the fruits varies between 15 and 20 cm. It flowers and fruits all year round but has some definite seasons during which it blossoms (Pinto et al., 2005). The plant commonly located in West Africa, Central and University of Ghana http://ugspace.ug.edu.gh 37 South America and also in Southeast Asia. The plant grows in regions with altitudes less than 1200 m above sea level, temperatures between 25 and 28°C, relative humidity between 60 and 80% and annual rainfall above 1500 mm. Its fruit is edible and dark green in color, the average weight is 4 kg in some countries (Pinto et al., 2005) but in México (Arenas- Ocampo et al., 2003), Venezuela (Ojeda et al., 2007) and Nicaragua (Coria-Tellez et al., 2018). The seeds in each fruit are about 55- 170 when fresh and become light brown when dry (Awan et al., 1980). It has white and creamy flesh with pleasant fragrance and flavor (Pinto et al., 2005). Figure 2.7: Diagram showing leaves and fruits of Annona muricata 2.6.1.2 Geographical distribution The exact origin A. muricata is unknown (Vijayameena et al., 2013) but may be native to Central America and Northern South America (Hanelt et al., 2001). It is cultivated in the warm lowlands of the Caribbean, East Africa, West Africa, temperate and tropical Asia, North America and South- University of Ghana http://ugspace.ug.edu.gh 38 Central Pacific Islands (USDA ARS-2014). It been stated as exotic to the Caribbean and West Indies including Puerto Rico (Acevedo-Rodríguez. P. & Strong, 2012). It has also been listed as native to Puerto Rico by United States Department of Agriculture-Agricultural Research Service (USDA ARS-2014). 2.6.1.3 Phytochemical constituents Studies have shown that A. muricata is made of 212 bioactive compounds. The notable ones are acetogenins, alkaloids, and phenols. Most of the phytochemicals have been isolated from organic extracts but lately the direction has been on aqueous extracts (Orwa et al., 2009) also mentioned the presence of carbohydrates and essential oils. 2.6.1.3.1 Acetogenins Studies have shown that there are about 120 acetogenins found in the ethanolic, methanolic or other organic extracts of different parts of the plant (Beg et al., 2011). Pulp and fruit peel acetogenins can be identified by their aliphatic chains of about thirty-five to thirty-eight carbon atoms with γ-lactone α ring attached (Alali et al., 1999). A large portion of acetogenins in A. muricata has tetrahydrofuran ring with two THF rings. Acetogenins are found predominantly in leaves and fruits ( Liaw & Wiener, 2002; Höllerhage et al., 2009), some are in seeds (Wu et al., 1995). Acetogenins are the main bioactive compounds found in Annonaceae family (Alali et al., 1999). It has been demonstrated through research that acetogenins are more cytotoxic comparted to alkaloids and rotenone (synthetic cytotoxic compound). Acetogenins and alkaloids are much researched on because of their therapeutic and neurotoxic effects (Mohanty et al., 2008). 2.6.1.3.2 Alkaloids University of Ghana http://ugspace.ug.edu.gh 39 Alkaloids such as reticuline and coreximine are found in A. muricata (Nawwar et al., 2012). The leaves of Annona muricata has the highest alkaloid content (Matsushige, 2012). Alkaloids have been identified its stems, roots (Nawwar et al., 2012) and fruits (Hasrat et al., 1997). They are usually in the form of isoquinoline, aporphine and protoberberine (Mohanty et al., 2008). In vitro analysis exhibit that the alkaloids have high affinity for 5-HT1A receptors and are involved in the synthesis of dopamine (Hasrat et al., 1997). This suggests that they may be responsible for the plant’s antidepressant-like effect (Hasrat et al., 1997). It has also been proven to have cytotoxic impact (Matsushige, 2012). Some alkaloids may have neurotoxic activity which may cause them to evoke neuronal death via apoptosis (Mohanty et al., 2008). 2.6.1.3.3 Phenolic compound A. muricata has about thirty-seven phenolic compounds with the essential ones being, quercetin (Nawwar et al., 2012), gallic acid, flavonoids and lipophilic compounds (Correa et al., 2012). Asare and his team in 2014 suggested that there are differences in the quantity of total phenols extracted when using organic or aqueous extracts. This is vital reason been that habitual uses of medicinal plants are in aqueous infusion coupled with the fact that most phenols are known to be water soluble. Phenolic compounds are believed to be responsible for plant free radical scavenging activity (Asare et al., 2015). 2.6.1.3.4 Other compounds The leaves, seeds and pulp of A. muricata contains vitamins and carotenoids (Correa et al., 2012; Vijayameena et al., 2013). Amide N-p-coumaroyl tyramine and cyclopeptides seen in seeds possesses anti-inflammatory and anti-cancer activities ( Wu et al., 1995; Wélé et al., 2004). The pulp of A. muricata has 37 volatile compounds. Almost all of these volatile compounds are aromatic and University of Ghana http://ugspace.ug.edu.gh 40 aliphatic esters (Cheong et al., 2011). Some essential oils that are predominantly sesquiterpenes derivates from the leaves produce cytotoxic effect against human breast carcinoma cell line (MCF- 7) (Jaramillo et al., 2000; Kossouoh et al., 2007). 2.7 Ethnobotanical uses of Annona muricata The plant has been screened and found to possess numerous medicinal properties (Badrie & Schauss, 2009; Gbaguidi et al., 2017). Traditionally, the bark, root, seed or leaf has different applications. In Indonesia (Boyom et al., 2011a), the Caribbean islands (Boulogne et al., 2011) and South Pacific countries, its leaves treat skin ailments (Cano & Volpato, 2004). In countries like Mauritius, New Guinea and Ecuador the leaves are applied topically (Sreekeesoon & Mahomoodally, 2014). In Brazil Martinique, Mexico and Nicaragua the leave stock is used as an analgesic (Coria-Tellez et al., 2018), while in several countries like Benin, the Caribbean, Cuba (Joyeux et al., 1995) and México (Waizel-Bucay & Waizel-Haiat, 2009) they are useful in curing colds, flu and asthma. In Malaysia leaves of A. muricata are efficient in healing infections of cutaneous external and internal parasites. In Ghana, the plants is decocted into a mixture and used to bath for therapeutic purposes (Asare et al., 2015). The fruit is not only used as food but used to treat diarrhea, heart and liver pathologies as well. In South America, it is used in the management of Intestinal parasites (Badrie & Schauss, 2009). Recently, the leaves of A. muricata is being used in the treatment of hypertension (Badrie & Schauss, 2009), diabetes and cancer (Alonso-Castro et al., 2011). The unripe fruit, seeds, leaves and roots of the plant are being used as pesticides, insecticides and insect repellents (Isman & Akhtar, 2007). The plant has also been recommended for the control lepidopteran larvae, aphids and thrips (Falistocco & Ferradini, 2020). University of Ghana http://ugspace.ug.edu.gh 41 2.7.1 Pharmacological studies of Annona muricata 2.7.1.1 Cytotoxic effect The anticancer property have been mentioned by Asare and others and it is believed to influence the cytotoxic property of A. muricata (Asare et al., 2015). Some extracts have shown toxic action to cancer cell lines (Betancur-Galvis et al., 1999). It has been found through research that a concentration of 1.6 µg/ml and 50µg/ml hydroethanolic leaf extract of the plant increases the viability of non-cancerous cells. However, 100µg/ml of the hydroethanolic leaf extract left their viability unchanged. In tumor cells, the healing period of the plant increased whiles in rodents the healing time of induced wound is reduced (Paarakh et al., 2009). Hydroethanolic extract of the plant influence results obtained; organic solvents such as pentanolic and ethanolic, were the most effective plant extracts against cancer cells grown in vitro. The effect is higher in pentanolic and ethanolic extracts than that of the aqueous extract (Ménan et al., 2006). Extracts with LC50 less than10 µg/ml are highly cytotoxic but extract from plants with LC50 values ≤20 µg/ml can be used to treat cancer (Falistocco & Ferradini, 2020). Ethyl acetate leaf extract of the plant possess an inhibitory property against U-937 cell line. According to Osorio and his colleagues A. muricata extracts exhibits good cytotoxic action however, there are more plants with potent cytotoxic activity, for instance, Thevetia ahouai has LC50 <1µg/ml (Calderón et al., 2007). Hexane leaf extract from the plant have high levels of flavonoids and much efficient in inhibiting cell proliferation relative to methanol or chloroform extracts. Moghadamtousi et al., 2015 has stated that the mechanism of action of plant extract shows interference of mitochondrial membrane to suppress apoptosis and cells in G0/G1 phase. University of Ghana http://ugspace.ug.edu.gh 42 2.7.1.2 Anti-protozoal activity The plant exhibits antiprotozoal activity against different protozoans such as genera Plasmodium (Boyom et al., 2011a), Leishmania , Biomphalaria (Luna et al., 2006) Trypanosoma, and Entamoeba (Shivas et al., 2015). The antiplasmodial action is especially important because of the need to find antimalarial agents in tropical regions. Methanolic extract showed activity against the parasites in vitro even though it was not as potent as chloroquine or artemisinin (Boyom et al., 2011b). The most therapeutic effect are its seed extracts as the alkaloids (Fofana et al., 2013), acetogenin, anonaine, and gallic acid screened from the plant has antiplasmodial action (Yamthe et al., 2015). Phenolic compounds inhibit enzymes involved in fatty acid bio