EFFECTS OF BETA-CARYOPHYLLENE FROM PIPER GUINEENSE ON OXIDATIVE STRESS MARKERS IN MUSCLE TISSUES OF WISTAR RATS TREATED WITH OXALIPLATIN

EFFECTS OF BETA-CARYOPHYLLENE FROM PIPER GUINEENSE ON OXIDATIVE STRESS MARKERS IN MUSCLE TISSUES OF WISTAR RATS TREATED WITH OXALIPLATIN BY ANYANKA PROVIDENCE IKECHUKWU MATRIC NO: 2019/120228/REGULAR A PROJECT SUBMITTED TO THE DEPARTMENT OF HUMAN PHYSIOLOGY FACULTY OF BASIC MEDICAL SCIENCES ABIA STATE UNIVERSITY, UTURU AUGUST, 2024 EFFECTS OF BETA-CARYOPHYLLENE FROM PIPER GUINEENSE ON OXIDATIVE STRESS MARKERS IN MUSCLE TISSUES OF WISTAR RATS TREATED WITH OXALIPLATIN BY ANYANKA PROVIDENCE IKECHUKWU MATRIC NO: 2019/120228/REGULAR A PROJECT SUBMITTED TO THE DEPARTMENT OF HUMAN PHYSIOLOGY FACULTY OF BASIC MEDICAL SCIENCES ABIA STATE UNIVERSITY, UTURU IN PARTIAL FULFILLMENT FOR THE REQUIREMENT OF THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE IN HUMAN PHYSIOLOGY SUPERVISED BY: ONWUKA KELECHI COLLINS AUGUST, 2024 DECLARATION I, ANYANKA PROVIDENCE IKECHUKWU, an undergraduate student of the Department of Human Physiology, with matriculation number 2019/120228/REGULAR, do hereby declare, on my honor that this project work has not been previously presented, either wholly or in part for the award of any other degree, certificate or publication in any university, other higher institution or elsewhere. Sign .............................. Date ...................... ANYANKA PROVIDENCE IKECHUKWU (MAT NO: 2019/120228/REGULAR) CERTIFICATION We certify that this research titled Effect of Beta-Caryophyllene on Oxidative Stress Markers in Muscle Tissue of Wistar Rats Treated with Oxaliplatin by ANYANKA PROVIDENCE IKECHUKWU with matriculation number 2019/120228/REGULAR was approved by the Department of Human Physiology, Faculty of Basic Medical Sciences, Abia State University. However, we affirm that the student ANYANKA PROVIDENCE IKECHUKWU with the matriculation number 2019/120228/REGULAR bears full responsibility for the contents of this work. Sign....................................... Date ........................... Onwuka Kelechi Collins (Supervisor) Sign........................................ Date ........................... Dr. Chris Akunneh-Wariso (Head of department) Sign........................................ Date ........................... External Examiner DEDICATION This work is dedicated to God Almighty. ACKNOWLEDGEMENTS I would like to extend my heartfelt gratitude to my supervisor, Mr. Onwuka Kelechi Collins, whose guidance, expertise, and encouragement were invaluable throughout the course of this research. His commitment to excellence and patient mentorship have profoundly influenced this work. I am also deeply thankful to Dr. Chris Akunneh-Wariso, the HOD of Human Physiology, for his unwavering support and guidance throughout this journey. His leadership and insights have been crucial to the successful completion of this project. My heartfelt thanks go to my colleagues and friends for their assistance and encouragement during the course of this study. Lastly, I am deeply indebted to my family for their unconditional love, constant support, prayers and encouragement, without which this work would have not been possible. This accomplishment is as much theirs as it is mine. TABLE OF CONTENTS Cover page i Title page ii Declaration iii Certification iv Dedication v Acknowledgement vi Table of content vii List of tables ix List of abbreviations x Abstract xi CHAPTER ONE: INTRODUCTION 1.1 Background of the study 1 1.2 Statement of problem 2 1.3 Aim of the study 2 1.4 Specific objectives 2 1.5 Significance of study 3 CHAPTER TWO: LITERATURE REVIEW 2.1 Oxaliplatin 4 2.2 Mechanism of oxaliplatin therapy 5 2.2.1 Neuronal damage 5 2.2.2 Mitochondrial dysfunction 5 2.2.3 Oxidative stress 6 2.2.4 Altered ion channel activity 6 2.3 Oxidative stress and its effects in muscle tissues 6 2.4 Impact of oxidative stress in muscle 7 2.5 Consequences of oxidative stress in muscles 8 2.6 Defense mechanism against oxidative stress 8 2.7 Potential therapeutic intervention for oxidative stress: Beta-caryophyllene 9 2.7.1 Sources of Beta-caryophyllene 10 2.7.2 Black pepper (Piper Guineense) 10 2.7.3 Chemical properties of Beta-caryophyllene 11 2.7.4 Mechanism of action 11 2.7.5 Rationale for investigating Beta-caryophyllene 12 CHAPTER THREE: MATERIALS AND METHODS 3.1 Ethical clearance 13 3.2 Location of study 13 3.3 Purchase of animals 13 3.4 Purchase of duloxetine 13 3.5 Purchase of oxaliplatin 13 3.6 Purchase and processing of Piper Guineense fruit 13 3.6.1 Preparation of Piper Guineense for extraction 13 3.6.2 Extraction of essential oil from Piper Guineense 14 3.6.3 Phytochemical screening presence of terpene in essential oil 14 3.6.4 Extraction of trans-caryophyllene from essential oil of Piper Guineense fruit 14 3.7 Acute toxicity study of Beta-caryophyllene 15 3.8 Administration of Beta-caryophyllene 15 3.9 Oxaliplatin and pharmacological treatments 15 3.10 Experimental design 16 3.11 Collection of specimen for determination of biomarkers 16 3.12. Studies on oxidative stress 16 3.12.1 Estimation of superoxide dismutase (SOD) activity 16 3.12.2 Malondialdehyde (MDA) determination 16 3.12.3 Estimation of glutathione peroxidase (GPX) 17 3.12.4 Estimation of catalase (CAT) 17 3.13 Statistical analysis and data analysis 18 CHAPTER FOUR: RESULTS 4.1 Acute toxicity evaluation 19 4.2 Effect of beta caryophyllene administration on muscle tissue oxidative stress markers in oxaliplatin treated Wistar rats 20 CHAPTER FIVE: DISCUSSION, CONCLUSION, AND RECOMMENDATIONS 5.1 Discussion 21 5.2 Conclusion 22 5.3 Recommendations 22 5.4 Contribution to Knowledge 22 REFERENCES 23 LIST OF TABLES Table 3.1: Animal grouping and Treatment 16 Table 4.1a: Stage 1 Acute toxicity (LD50) evaluation of Beta Caryophyllene in rats 19 Table 4.1b: Stage 2 Acute toxicity (LD50) evaluation of Beta Caryophyllene in rats 19 Table 4.2: Effect of Beta Caryophyllene administration on muscle tissue oxidative stress markers 20 LIST OF ABBREVIATIONS ANOVA - Analysis of Variance ASCO - American Society of Clinical Oncology BCP - Beta-Caryophyllene CAT - Catalase CIPN - Chemotherapy-Induced Peripheral Neuropathy DRG – Dorsal Root Ganglion GPx - Glutathione Peroxidase H2O2 - Hydrogen Peroxide HCL - Hydrochloride LD50 - Lethal Dose for 50% of the population MDA - Malondialdehyde OXA - Oxaliplatin ROS - Reactive Oxygen Species SEM - Standard Error of Mean SOD - Superoxide Dismutase SPSS - Statistical Package for Social Sciences U/L - Units per Liter U/ml - Units per Milliliter ABSTRACT This study showed the impact of Beta-caryophyllene on oxidative stress biomarkers (SOD, CAT, MDA, GPx) in muscle tissue of Wistar rats undergoing oxaliplatin therapy. After a period of 14 days of acclimatization, 35 Wistar rats were allocated to five groups (n=7). Group 1 (10mg of normal saline), group 2 (4mg/kg oxaliplatin only), group 3 (1mg/kg duloxetine and 4mg/kg oxaliplatin), group 4 (25mg/kg BCP and 4mg/kg oxaliplatin), group 5 (50mg/kg BCP and 4mg/kg oxaliplatin). The rats were subjected to oxaliplatin intraperitoneally followed by oral administration of BCP for 21 days. After treatment period, muscle tissue homogenates were collected and oxidative stress markers analyzed using standard biochemical assays. The impact of BCP on oxidative stress markers was scrutinized. The data revealed that BCP, in a dose- dependent manner, augmented the levels of SOD, CAT, MDA, and GPx in contrast to the oxaliplatin treated group. The study posits that beta-caryophyllene was efficacious in mitigating oxidative stress induced by oxaliplatin in gastrocnemius muscle of Wistar rats. Keywords: Beta-Caryophyllene, Oxaliplatin, Oxidative Stress 2 CHAPTER ONE INTRODUCTION 1.1 Background of study Peripheral neuropathy refers to the many conditions that involve damage to the peripheral nervous system, which is a vast communication network that sends signals between the central nervous system (The brain and Spinal cord) and all other parts of the body, resulting in symptoms such as numbness, tingling and pain in the extremities (Smith, 2020). It is a common adverse effect caused by chemotherapy, diabetes, infections, traumatic injuries, exposure to toxins, and genetic disorders. Peripheral neuropathy in cancer patients is most often due to neurotoxic chemotherapeutic agents leading to chemotherapy-induced peripheral neuropathy (CIPN). CIPN is a side effect frequently observed in therapies with platinum compounds, taxanes and vinca alkaloids, which leads to symptoms such as numbness, tingling, pain, and weakness in the hands and feet, significantly affecting patients’ quality of life and potentially limiting the dosage of chemotherapy that can be administered (Park, Lee & Kim, 2013; Burgess et al., 2021). Oxaliplatin (OXA), a platinum-based chemotherapy agent widely used in the treatment of colorectal, pancreatic, and gastric cancers, despite its efficacy, is particularly notable for its ability to cause acute neuropathy in up to 90% of cancer patients. (Gebremedhn et al., 2018). The pathophysiology underlying chemotherapy-induced peripheral neuropathy are multifactorial with key mechanisms including mitochondrial dysfunction and oxidative stress believed to play a central role in its pathogenesis. (Broyl et al., 2011). Chemotherapeutic agents, including Oxaliplatin, induce oxidative stress in peripheral nerves by generating reactive oxygen species (ROS), leading to mitochondrial dysfunction, DNA damage, and ultimately neuronal injury and death. (Canta et al., 2015). Oxaliplatin-induced peripheral neuropathy (OIPN) is particularly challenging to manage due to its complex pathophysiology and lack of effective treatments (Hershman et al., 2014). According to the American Society of Clinical Oncology (ASCO) Guidelines for Chemotherapy-Induced Peripheral Neuropathy, there are no effective drugs for the prevention of peripheral neuropathy and the only drug recommended for use in treatment is duloxetine (Loprinzi et al., 2020). Therefore, there is still a strong need for novel therapeutic approaches that can prevent or mitigate the onset and severity of CIPN. Beta-caryophyllene (BCP), a member of sesquiterpene lactone found in cannabis and non- cannabis plants, also in large amounts in the essential oils of curry, cloves, cinnamon, black pepper, cannabis, and guava (Basha et al., 2015) has emerged as a potential candidate for the management of OIPN due to its several pharmacological effects such as antidiabetic, hepatoprotective, neuroprotective, anti-inflammatory, and antioxidant properties in various experimental models (Basha et al., 2020). 1.2 Statement of problem Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating side effect experienced by cancer patients undergoing treatment with oxaliplatin. This condition often leads to sensory and motor dysfunction, neuropathic pain, and a diminished quality of life for affected individuals. While beta-caryophyllene has shown promise as a protective agent due to its antioxidant properties, its efficacy specifically in preventing or attenuating CIPN induced by oxaliplatin is not well understood. Therefore, the primary problem addressed by this study is to evaluate the potential of beta-caryophyllene as a protective agent against oxidative stress in the context of oxaliplatin-induced peripheral neuropathy. 1.3 Aim of the study The aim of the study is to investigate the Effects of Beta-caryophyllene on Oxidative stress from the gastrocnemius muscle in Wistar rats treated with oxaliplatin. 1.4 Specific objectives The objectives of the study are: i. To determine the mean superoxide dismutase level in the gastrocnemius muscle across the experimental groups. ii. To determine and compare the mean malondialdehyde level in peripheral nerve tissues across the experimental groups. iii. To determine and compare the mean glutathione peroxidase level in gastrocnemius tissue homogenates of the rats exposed to OXA treated with beta-caryophyllene to control groups. iv. To determine and compare the mean catalase level in gastrocnemius tissue homogenates of the rats exposed to OXA treated with beta-caryophyllene to control groups. 1.5 Significance of study This study seeks to provide valuable insights into the development of therapeutic strategies for managing Chemotherapy-induced Peripheral Neuropathy and improving the quality of life for cancer patients undergoing treatments. CHAPTER TWO LITERATURE REVIEW 2.1 Oxaliplatin Oxaliplatin (Trans-1-diaminocyclohexane oxalate platinum II) is a third-generation platinum derivative patented in 1976, it is indicated for the treatment of digestive tract tumors (advanced colorectal, esophageal, stomach, liver, and pancreatic cancers). It was first synthesized in Japan and later developed in Europe as a less toxic and more effective alternative to other platinum derivative in treatments against colorectal cancer. (Wiseman et al., 1999; Nora et al., 2010). Despite its efficacy against colorectal cancer, oxaliplatin is one of the most prominent neurotoxic chemotherapy agents that interferes with tumor cell proliferation by forming DNA-platinum adducts, which leads to the destruction of cancer cells and also healthy ones (Pachman et al., 2015). Therapy with oxaliplatin causes major side effects such as; myelotoxicity, gastrointestinal reactions, and peripheral neurotoxicity. (Aubin et al., 2013). These side effects may result in the termination of treatment plans and a reduction in the compliance of colorectal cancer patients during the treatment. Oxaliplatin-induced peripheral neuropathy is a common side effect associated with oxaliplatin therapy occurring in over 85-96% patients. (Han et al., 2016). OIPN produces two distinct manifestations of peripheral neuropathy: an acute cold-enhanced form and chronic distal sensory neuropathy. (Avan et al., 2015). The acute form is one affecting 80-90% of oxaliplatin treated patients (Ewertz et al., 2015). It occurs within hours of infusion and lasts for the following 7 days, it is characterized by cold- sensitive paresthesia/dysesthesia, with motor symptoms associated with prolonged muscular contractions, tetanic spasm, and fasciculations. (Grolleau et al., 2001). The incidence of chronic OPIN is said to occur in approximately 10% of patients receiving cumulative doses of oxaliplatin ranging from 780 to 850 mg/m2 (Grothey, 2003). It is characterized by sensory loss, sensory ataxia, and changes in proprioception. Common symptoms include: numbness, tingling, and mild or burning pain in hands and feet (Grisold et al., 2016). This chronic form of OIPN persists throughout treatment and increases in intensity with cumulative dose. Symptoms are resolved within 6-12 months of cessation of therapy, but in a small group of patients, symptoms persist for more than one year. (Saif & Reardon, 2005). 2.2 Mechanisms of Oxaliplatin Therapy Earlier studies tell the mechanisms by which oxaliplatin exerts its anti-tumor effects are largely due to its ability to damage DNA and generate excess reactive oxygen species (ROS) that lead to apoptosis in cancer cells (Conklin, 2004). While the mechanisms are crucial for the drug’s anti- cancer activity, they can also have unintended consequences on healthy cells, including those in the peripheral nervous system, where peripheral neuropathy is initiated by the accumulation of platinum adducts in the Dorsal Root Ganglion (DRG) and trigeminal ganglion (TG) neurons, where it interacts with the DNA of organelles such as the nucleus and mitochondria of neuron cells, affecting DNA replication, block cell cycle, inhibiting DNA repair, and inducing neuronal apoptosis. (Rabik & Dolan, 2007; Jaggi & Singh, 2012). Based on preclinical studies, many different hypotheses have been suggested to explain oxaliplatin’s pathogenesis. The main common mechanisms of pathogenesis include mitochondrial dysfunction, oxidative stress, neuronal damage, altered ion channel activity, and apoptosis induction (Areti et al., 2014). I. Neuronal damage Once oxaliplatin accumulates in the dorsal root ganglion (DRG) neurons, it interacts with the nuclear DNA to form DNA-platinum adducts (Ta et al., 2006). DRG neurons require a high level of active transcription to maintain their normal structure and function. However, accumulation of oxaliplatin induces nucleolar DNA damage which leads to global transcriptional arrest of neuronal cells, which may activate apoptosis pathways, leading to neuronal atrophy, thereby disrupting normal nerve function. (Pasetto et al., 2006; Yan et al., 2015). II. Mitochondrial Dysfunction After entering neuronal cells, oxaliplatin combines with mitochondrial DNA (mDNA) to form platinum-mDNA adducts which impairs the physiological replication and transcription of mDNA, leading to the synthesis of abnormal proteins and impairment of the respiratory chain in mitochondria (Canta et al., 2015). The impairment of the mitochondria physiological function leads to decreased cellular metabolism, an increased production of reactive oxygen species (ROS) and oxidative stress which contributes to reduced energy production and muscle fatigue (Di Cesare Mannelli et al., 2012). III. Oxidative stress Oxidative stress is a key pathogenic mechanism involved in Oxaliplatin-induced peripheral neuropathy which causes the excessive production of reactive oxygen species (ROS), leading to an imbalance between oxidation and antioxidant systems. (Areti et al., 2014). The impairment of the mitochondrial function and structure caused by oxaliplatin leads to an increase in the production of free radicals and bioenergy depletion, antioxidant depletion, biomolecular damage, demyelination, neuroinflammation, mitophagy impairment, and alterations of cellular protein, lipid, and DNA that ultimately lead to apoptosis (Wei et al., 2021). IV. Altered ion channel activity The development of peripheral neuropathy may be partially induced by the altered action of sodium channels (NaV), potassium channels (KV), and transient receptor potential (TRP) channels. The voltage-gated sodium channels are necessary to facilitate the initiation and propagation of action potentials in neurons and other excitable cells (Descoeur et al., 2011). Oxaliplatin alters the function of voltage-gated sodium channels, leading to hyperexcitability and spontaneous firing of sensory neurons. This hyperexcitability contributes to the development of neuropathic pain and sensory disturbances as seen in oxaliplatin-induced neuropathy (Descoeur et al., 2011). 2.3 Oxidative Stress and its effects in Muscle tissue Oxidative stress is a phenomenon caused by an imbalance between production and accumulation of reactive oxygen species (ROS) in cells and tissues and the ability of a biological system to detoxify these reactive products (Sies, 2015). Reactive oxygen species (ROS) are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them (Konig et al., 2001). These free radicals, or reactive oxygen species are highly reactive molecules with one or more unpaired electron(s) formed when oxygen interacts with specific molecules. They severe bodily functions, including cellular signaling and fighting off pathogens. Superoxide radicals (O2 -), hydrogen peroxide (H2O2), hydroxyl radicals (OH), and singlet oxygen (1O2) are commonly defined reactive oxygen species (Sato et al., 2013). They’re generally considered harmful by-products of oxygen metabolism having important physiological functions in signal transduction and immunological response. When ROS production increases, they start showing harmful effects on important cellular structures like proteins, lipids, and nucleic acids (Wu et al., 2013). Mitochondrial dysfunction induced by oxaliplatin results in the production of ROS, exacerbating oxidative stress and neuronal damage. ROS production occurs through various mechanisms, including impairment of electron transport chain complexes and disruption of mitochondrial DNA integrity (Cavaletti et al., 2007). Oxidative stress reflects the imbalance due to an excess of ROS or oxidants, or downregulation of antioxidant systems, or both (Sies, 2015). The implications of ROS in cancer and cancer treatment are complex because ROS participate as signaling molecules in physiological processes of proliferation and survival. ROS can activate pro-tumorigenic signaling, enhance cell survival and proliferation, and result in DNA damage and genetic instability. However, ROS can also promote anti-tumorigenic signaling, initiating Oxidative stress-induced tumor cell death (Moloney & Cotter, 2018). Depending on the degree of oxidative stress, it results in the modification of intracellular signal pathways or in functional damage mediated by macromolecular oxidation (Reuter et al., 2010). Chronic oxidative stress alters the function of voltage-gated sodium channels in peripheral nerves, affecting neuronal excitability and action potential propagation (Descoeur et al., 2011). ROS-mediated modifications to ion channels contribute to hyperexcitability and hypersensitivity of peripheral nerves, triggering an inflammatory response, characterized by the release of pro- inflammatory cytokines and chemokines, further exacerbating neuronal damage (Di Cesare Mannelli et al., 2012). The interplay between oxidative stress and inflammation therefore creates a vicious cycle, amplifying neurotoxicity and perpetuating peripheral neuropathy symptoms. 2.4 Impact of Oxidative stress in Muscle I. Lipid peroxidation: Oxidative stress induces lipid peroxidation, damaging cell membranes. The peroxidation of lipids compromises membrane integrity, leading to increased cellular permeability and muscle cell damage. This process results in muscle fiber degeneration and impaired muscle function (Schneider et al., 2011). II. Protein oxidation: Oxidative modifications to muscle proteins disrupt their structure and function, impairing muscle contraction and metabolic processes. Protein oxidation affects key muscle enzymes and structural proteins, contributing to muscle weakness and fatigue (Chiarugi & Moskowitz, 2002). III. DNA Damage: Reactive oxygen species (ROS) can induce mutations and strand breaks in DNA, triggering cell cycle arrest and apoptosis. In muscle tissue, this DNA damage hampers muscle cell regeneration and repair, leading to muscle atrophy and decreased muscle mass over time (Bai et al., 2019). 2.5 Consequences of Oxidative stress in Muscles I. Muscle Fatigue and Weakness: Muscle fatigue is a common side effect of oxaliplatin therapy, resulting from impaired mitochondrial function and oxidative damage to muscle proteins. Persistent oxidative stress reduces ATP production, essential for muscle contractions, leading to muscle weakness (Argyriou et al., 2013). II. Muscle Atrophy: Chronic oxidative stress activates proteolytic pathways, such as the ubiquitin-proteasome system, which degrade muscle proteins. Simultaneously, oxidative stress inhibits muscle protein synthesis, contributing to muscle atrophy and reduced muscle mass (Bonifati et al., 2010). III. Inflammation and Pain: Oxidative stress activates inflammatory pathways leading to the release of pro-inflammatory cytokines. This inflammation exacerbates muscle damage and contributes to muscle pain (myalgia), further reducing the quality of life in patients undergoing oxaliplatin therapy (Areti et al., 2014). Oxidative stress emerges as a primary effector in the mechanism of oxaliplatin therapy, contributing to mitochondrial dysfunction, neuronal injury, altered ion channel function, and inflammation. Targeting oxidative stress pathways may offer promising therapeutic strategies for preventing or mitigating and improving side effects of oxaliplatin treatment. 2.6 Defense Mechanisms against Oxidative Stress Under normal physiological conditions, the production of ROS is balanced by the activity of antioxidant enzymes and other molecules that help to reduce oxidation. Antioxidants are a class of compounds able to counteract oxidative stress and mitigate its effects on individuals’ health, they play a crucial role in safeguarding the body against damage resulting from oxidative stress induced by free radicals (Osoy et al., 2008; Halliwell & Gutteridge, 2015). Excessive ROS is promptly removed from the cell to prevent their potentially harmful effects. Numerous antioxidant enzymes which catalyze reactions that convert ROS into less harmful molecules, thereby protecting cells from oxidative damage include; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR), as well as non-enzymatic endogenous antioxidants which directly scavenge ROS and reactive nitrogen species (RNS), providing immediate protection against oxidative damage like ascorbic acid, tocopherols, reduced glutathione (GSH), and uric acid, participate in mechanisms responsible for eliminating or regulating the levels of ROS within the cell (Halliwell & Gutteridge, 2015). I. Superoxide Dismutase (SOD): SOD is a critical antioxidant enzyme that protects cells from oxidative stress by catalyzing the dismutation of superoxide radicals into oxygen and hydrogen peroxide. Superoxide radicals are highly reactive molecules produced as byproducts of various cellular processes. The enzyme helps mitigate potential damage caused by these radicals, thereby playing a key role in cellular defense and longevity (Carillon et al., 2013). II. Catalase (CAT): Catalase is another crucial enzyme that decomposes hydrogen peroxide into water and oxygen. Hydrogen peroxide, if accumulated, can lead to cellular damage and oxidative stress. Catalase works alongside other antioxidants to maintain cellular health and prevent oxidative damage (Glorieux & Calderon, 2017). III. Glutathione Peroxidase (GPx): GPx is an important enzyme found in the cytoplasm and mitochondria of mammalian cells, that reduces hydrogen peroxide and organic peroxides to water and corresponding alcohols, using glutathione, a powerful antioxidant as a substrate. GPx helps to neutralize harmful peroxides and maintain cellular redox balance, thus protecting cells from oxidative stress and damage (Handy & Loscalzo, 2022). IV. Malondialdehyde (MDA): MDA is a byproduct of lipid peroxidation, which occurs when free radicals attack lipids in cellular membranes. MDA is commonly used as a biomarker for oxidative stress and cellular damage, as increased levels of MDA can indicate significant oxidative damage to cell membranes (Del et al., 2005). 2.7 Potential Therapeutic Intervention for Oxidative Stress: Beta-Caryophyllene In response to the growing recognition of oxidative stress as a key contributor to neuropathic damage, antioxidant therapy has gotten considerable interest as a potential therapeutic strategy for alleviating neuropathic symptoms. Among the natural compounds explored, beta- caryophyllene emerged as a promising candidate for neuropathy management (Basha et al., 2020). Beta-caryophyllene (BCP), is a non-psychoactive sesquiterpene lactone present in cannabis and non-cannabis plants such as oregano, cinnamon, cloves, and black pepper, with significant antioxidant, anti-inflammatory, neuroprotective and anti-proliferative effects, making it a promising candidate for mitigating OIPN (Sharma et al., 2016). BCP is a cannabis-derived compound that interacts with the endocannabinoid system which plays an important role in regulating central physiological processes that underlie learning and memory, anxiety, depression, addiction, appetite and feeding, pain, neuronal excitability and protection in humans (Gertsch et al., 2010). BCP has been reported to selectively bind to the cannabinoid receptor type 2 (CB2) which in peripheral tissues in the body, is a therapeutic target for treatment of inflammation, pain, atherosclerosis, osteoporosis and have been implicated in the regulation of oxidative stress (Andre et al., 2016). 2.7.1 Sources of Beta-Caryophyllene The approximate quantity of caryophyllene in the essential oil of each source is given in square brackets ([ ]): Cannabis (Cannabis sativa) [3.8–37.5% of cannabis flower essential oil] Black caraway (Carum nigrum) [7.8%] Cloves (Syzygium aromaticum) [1.7–19.5% of clove bud essential oil] Hops (Humulus lupulus) [5.1–14.5%] Basil (Ocimum spp.) [5.3–10.5% O. gratissimum; 4.0–19.8% O. micranthum] Oregano (Origanum vulgare) [4.9–15.7%] Black pepper (Piper nigrum) [7.29%] Lavender (Lavandula angustifolia) [4.62–7.55% of lavender oil] Rosemary (Rosmarinus officinalis) [0.1–8.3%] True cinnamon (Cinnamomum verum) [6.9–11.1%] Malabathrum (Cinnamomum tamala) [25.3%] Ylang-ylang (Cananga odorata) [3.1–10.7%] Copaiba oil (Copaifera) (National Center for Biotechnology Information, 2020). 2.7.2 Black Pepper (Piper Guineense) Piper guineense, is also called West African black pepper, Ashanti pepper, Guinea cubeb, and Benin pepper. It is native to the tropical rain forest of Africa, where it thrives mainly in the wild. It is, however, partly cultivated in Southern Nigeria, where the leaves and seeds are used to flavor soup. (Ameh et al., 2011) Piper plants (Piperaceae) are most commonly used as a food in almost all African regions. Two species, Piper capensis and Piper guineense (West African black pepper), are the most famous species in Africa (Noumi et al, 2011). They grow in many home gardens and crop plantations and have numerous culinary uses, including flavoring of soups, meat, fish, eggs, salads, and sauces. Another widely and frequently used Piper species in Africa is Piper nigrum, commonly known as black pepper, white pepper, green pepper, peppercorn, or Madagascar pepper (Chaveerach et al, 2006). A large number of Piper species have been reported to possess anti-inflammatory and antinociceptive activities. Black pepper essential oil has been reported to exhibit significant antinociceptive properties in the acetic acid–induced writhing test, and it also reduced acute inflammation in the carrageenan-, dextran- and formalin-induced chronic models of inflammation (Jeena et al., 2014). The phytochemical studies of the plant revealed the presence of proteins, carbohydrates, alkaloids, steroids, glycosides, saponins, flavonoids, tannins and phenolic compounds. It also contains vitamins, minerals and fat. Various studies have been done on the plant to determine its pharmacological and therapeutic properties such as antibacterial, antioxidant, anti-inflammatory, hepatoprotective, fertility, aphrodisiac, anticonvulsant and larvicidal properties (Morufu et al., 2016). 2.7.3 Chemical Properties of Beta-Caryophyllene (BCP) BCP has a molecular formula of C15H24, a molecular weight of 204.35 g/mol and a boiling point of approximately 160-180oC and density of around 0.930g/cm3. It is a bicyclic compound comprising a cyclobutane ring fused to a cyclohexane ring. BCP is a colorless to pale yellow liquid with characteristic spicy, peppery aroma (Matsubara & Hayashi, 2004). It is a lipophilic molecule which makes it readily available to cross cell membranes and interact with intracellular targets (Gertsch et al., 2008). 2.7.4 Mechanism of action BCP exerts its effects primarily through the activation of the cannabinoid receptor type 2 (CB2). These CB2 receptors are a type of receptor found predominantly outside the central nervous system (CNS), on immune cells, peripheral nerve terminals, and other tissues throughout the body (Mackie, 2008). They are part of the endocannabinoid system, involved in various physiological processes including pain modulation, immune response, and inflammation. Unlike other cannabinoids, BCP selectively binds to CB2 receptors without producing psychoactive effects, which are typically associated with cannabinoid receptor type 1 (CB1). This binding reduces inflammation and oxidative stress, which are key contributors to neuropathic pain and neuronal damage (Gretsch et al., 2008). 2.7.5 Rationale for Investigating Beta-Caryophyllene (BCP) Preclinical studies investigating the therapeutic potential of BCP in neuropathic pain models have yielded promising results as it has been shown to attenuate neuropathic pain behaviors, reduce neuro-inflammation, and improve nerve function in animal models of cisplatin-induced neuropathy (Horváth et al., 2012). Due to its lipophilicity, BCP is said to easily penetrate cell membranes while still presenting good oral bioavailability (Fidyt et al., 2016) and because of its selectivity to CB2 receptor, BCP lacks psychoactive effects and still has anti-inflammatory and analgesic effects in different experimental models of inflammation and pain (Bento et al., 2011). In a preclinical experiment conducted by Horváth et al. (2012), it was found that BCP attenuated cisplatin-induced nephrotoxicity through the activation of cannabinoid 2 receptors, leading to reduced oxidative stress. This suggests that similar protective mechanisms may be involved in BCP‘s effects on neuropathy induced by other chemotherapeutic agents like oxaliplatin. In a preclinical experiment on tumor-bearing mice, Agnes et al. (2023), showed that BCP has pharmacological potential to treat OIPN with mechanisms involving CB2 receptors, as well as regulating inflammatory and redox homeostasis in the spinal cord. BCP also prevented mechanical and cold allodynia when administered at the onset of oxaliplatin therapy (Agnes et al., 2023). This study showed BCP as an attractive candidate against OIPN. In conclusion, while preclinical evidence supports BCP’s potential as a neuropathic pain modulator and a novel adjunctive therapy for OIPN, further research is warranted to elucidate its clinical utility which will be essential for improving neuropathic symptom management and enhancing patient outcomes in oncology. CHAPTER THREE MATERIALS AND METHODS 3.1 Ethical Clearance Ethical clearance was obtained from the Faculty of Basic Medical Sciences, College of Medicine and Health Sciences, Abia State University, Uturu. 3.2 Location of Study Department of Human Physiology, Faculty of Basic Medical Sciences Abia State University, Uturu. 3.3 Purchase of Animals Fifty-three (53) Wistar rats (200 ± 10g) were purchased and used for this main experimental and acute toxicity study respectively. They were acclimatized for two weeks before commencement of the experiment. The rats for the experimental work were grouped into five groups of 7 rats. 3.4 Purchase of Duloxetine Duloxetine was purchased from a pharmaceutical store in Enugu. It was prepared according to the modified method of Meng et al. (2019). Duloxetine HCL was prepared in 0.9% sterile saline and administered orally at a dose of 25 mg/kg as the standard drug of this experiment. 3.5 Purchase of Oxaliplatin Oxaliplatin was ordered from a pharmaceutical store in Enugu. For storage purposes for 30 days, a stock solution of 1mg/ml in 5% dextrose solution was prepared for administration by injection as against unstable saline solutions (Mehta et al., 2015). 3.6 Purchase and Processing of Piper Guineense Fruit Piper guineense fruit was procured from the local market and was identified and authenticated by a plant scientist in the Department of Botany, Abia State University, Uturu, Nigeria. A sample of it was kept in the herbarium for reference purposes. 3.6.1 Preparation of Piper Guineense for Extraction The fruit was prepared according to the method of Liu et al. (2018). They were dried to a moisture content of 2000 mg/kg. Details on observed behaviors of the animals during the acute toxicity test are presented in table 4.1a and 4.1b Table 4.1a: Stage 1 Acute toxicity (LD50) evaluation of Beta Caryophyllene in rats Group Dose (mg/kg) No. of Deaths Percentage of mortality Observations 1 25 0/3 0.00 No mortality observed, instead animals remained active and physically stable. 2 100 0/3 0.00 No mortality observed, instead animals remained active and physically stable 3 500 0/3 0.00 No mortality observed, instead animals remained active and physically stable Table 4.1b: Stage 2 Acute toxicity (LD50) evaluation of Beta Caryophyllene in rats Group Dose (mg/kg) No. of Deaths Percentage of mortality Observations 1 560 0/3 0.00 No mortality observed, instead animals remained active and physically stable. 2 1000 0/3 0.00 No mortality observed, instead animals remained active and physically stable. 3 2000 0/3 0.00 No mortality observed. Animals were initially calm but regained physical activity within one hour of administration. LD50>2000 mg/kg 4.2 Effect of Beta Caryophyllene administration on muscle tissue oxidative stress markers in oxaliplatin treated wistar rats The result obtained for the level of oxidative stress markers in Table 4.2, showed significant (p