ANALGESIC, ANTI-INFLAMMATORY AND OTHER PHARMACOLOGICAL ACTIVITIES OF METHANOL LEAF EXTRACT OF OLAX SUBSCORPIOIDEA OLIV (OLACACEAE) IN LABORATORY ANIMALS

ABSTRACT

Synthetic analgesic and anti-inflammatory drugs have major side effects such as constipation, nausea and vomiting, sedation and mental clouding, etc.,which have significantly limited their use.There is therefore, an intensification of search for newer analgesic and anti-inflammatory agents from the huge array of medicinal plant resources with better efficacy and fewer side effect profiles. Medicinal plants such as Olax subscorpioidea have been used traditionally for the management of pains, inflammatory diseases, yellow fever, cancer and rheumatism. The study aims at establishing the analgesic and anti-inflammatory potentials of methanol leaf extract of Olax subscorpioidea; and elucidating its possible mechanism of actions. The methanol extract and its fractions were subjected to phytochemical screening; oral and intra-peritoneal median lethal dose (LD50) determination; evaluation of analgesic activities using acetic acid-induced writhing, formalin induced pain and hot plate tests in mice; and evaluation of anti-inflammatory activity using carrageenan-induced hind paw oedema model in rats. The doses (oral) used for these studies were 250, 500 and 1,000 mg/kg for the methanol extract, residual aqueous and butanol fractions; while doses of 150, 300 and 600 mg/kg were used for the hexane fraction. The residual aqueous and butanol fractions (1,000 mg/kg, orally) were subjected to sub-acute inflammation studies using cotton-pellet induced granuloma in rats; also the concentrations of inflammatory cytokines in the tissue exudates of rats following carrageenan induced paw oedema was investigated. The roles of opioidergic, (α1, α2 and β)-adrenergic, serotonergic, ATP-sensitive potassium channels and nitric oxide-l-arginine pathways in the analgesic activities of the butanol fraction (1,000 mg/kg, oral) were further investigated. Results of the preliminary phytochemical screening of the methanol extract and the fractions indicated the presence of various phytochemicals such as carbohydrates, cardiac glycosides, tannins, flavonoids, alkaloids, saponins, steroid and triterpenes. The oral LD50 of the methanol extract, residual aqueous and butanol fractions was estimated to be greater than 5,000 mg/kg in both rats and mice; that of hexane fraction was estimated to be 2,200 and 3,800 mg/kg in mice and rats respectively. The intraperitoneal LD50 in mice was estimated to be 3,800 mg/kg for the methanol extract; 2,200 mg/kg for the residual aqueous fraction and 1,300 mg/kg for butanol and hexane fractions; it was estimated in rats to be 3,800 mg/kg for the methanol extract, residual aqueous and butanol fractions; and 2,200 mg/kg in the hexane fraction. The acetic acid induced writhes and the formalin induced pain licking effect were significantly (p<0.05, p<0.01 and p<0.001) reduced by the methanol extract and the fractions in a dose-dependent manner. The thermal pain latency was also significantly (p<0.05, p<0.01 and p<0.001) increased by the methanol extract and its fractions (except hexane fraction). The paw oedema was also significantly (p<0.05, p<0.01 and p<0.001) reduced by the methanol extract and the fractions across the time. The residual aqueous and butanol fractions (1,000 mg/kg) significantly (p<0.01 and p<0.001) reduced granuloma formation in the cotton pellet-induced granuloma studies in rats. The residual aqueous and butanol fractions significantly (p<0.05 and p<0.01) decreased the concentrations of vascular endothelial growth factor (VEGF); the butanol fraction significantly decreased ((p<0.05) concentrations of epidermal growth factor (EGF) and interleukin-1α (Il-1α). The residual aqueous and butanol fractions also significantly (p<0.05 and p<0.01) increased the concentration of Il-1β, IL-5 and interferon-γ (IFN-γ) while the residual aqueous fraction significantly(p<0.05) increased the concentration of IL-6 in the rats‘ paw tissue exudates. The pretreatment of mice with l-arginine and metergoline significantly (p<0.01 and p<0.05, respectively) decreased the analgesic effect of the butanol fraction; while pretreatment with naloxone, prazosin, yohimbine, propranolol and glibenclamide, each, had no significant effect on its analgesic activity. The results of the studies revealed that Olax subscorpioidea possesses marked analgesic and anti-inflammatory activities; the anti-inflammatory activity is mediated via the inhibition of pro-inflammatory cytokines such as IL-1α, IL-1β, VEGF and EGF and/or via the stimulation of the synthesis of anti-inflammatory cytokines such as IL-5, IL-6 and IFN-γ. These results also suggest the possible involvement of serotonergic and nitric oxide pathways in the analgesic effect of Olax subscorpioidea. CHAPTER ONE

1.0 INTRODUCTION

Pain is defined by the International Association for the Study of Pain (IASP) as an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage (Loeser and Treede, 2008). The British Pain Society (BPS) has defined pain as an emotion experienced in the brain; it is not like touch, taste, sight, smell or hearing. It can be perceived as a warning of potential damage, but can also be present when no actual harm is being done to the body (Moffat and Rae, 2010). It is the most common symptom of injuries and diseases (Haddad, 2007). It is a multidimensional sensory experience that is intrinsically unpleasant and associated with hurting and soreness. It may vary in intensity (mild, moderate or severe), quality (sharp, burning or dull), duration (transient, intermittent or persistent) and referral (superficial or deep, localized or diffuse). Although it is essentially a sensation, it has strong cognitive and emotional components; it is linked to or described in terms of suffering. It is also associated with avoidance motor reflexes and alterations in autonomic output. All of these traits are inextricably linked in the experience of pain (Woolf, 2004). Pain triggers various responses in the spinal cord and the brain, including reflexes, conscious perception, cognitive learning and memory processes, emotional reactions such as depression, and drug addiction (Gu et al., 2005).

Inflammation is considered as the primary physiologic defense mechanism that helps the body to protect itself against infection, burns, toxic chemicals, allergens or other noxious stimuli. An uncontrolled and persistent inflammation may act as an etiologic factor for many chronic illnesses (Kumar et al., 2004). Inflammation is the immune system‘s response to infection and injury and has been implicated in almost all chronic diseases, such as cancer, cardiovascular diseases, arthritis, inflammatory bowel syndrome, atherosclerosis and autoimmune diseases (Luo et al., 2010; Ricciotti and FitzGerald 2011; Viljoen et al., 2012). It is an intrinsically beneficial event that leads to removal of offending factors and restoration of tissue structure and physiological function (Ricciotti and FitzGerald, 2011). A disturbance that is successfully cleared results in a return to basal homeostatic set points. When conditions that induce inflammation are persistent or resolution mechanisms fail, a state of chronic inflammation ensues that can lead to loss of normal physiological functions (Hotamisligil and Erbay, 2008). The inflammatory response is characterized by redness, heat, swelling, loss of function and pain (Gautam and Jachak, 2009). Redness and heat result from an increase in blood flow, swelling is associated with increased vascular permeability and pain is the consequence of activation and sensitization of primary afferent nerve fibres (Calixto et al., 2003).

1.1 Statement of Research Problem

Pain is the most common symptom of injuries and diseases (Haddad, 2007). Virtually all known disease conditions are accompanied by pain (Donkor et al., 2013). Pain imposes significant financial burden due to its long-term treatment (Bhangoo and Swanson, 2012). It is one of the most common conditions limiting efficiency and diminishing quality of life (Caraceni et al., 2002; Mert et al., 2013). It is the main reason for visiting the emergency department of a hospital in more than 50% of cases (Cordell et al., 2002) and it is present in 30% of family practice visits (Hasselström et al., 2002).

Several epidemiological studies from different countries have reported widely varying prevalence rates for chronic pain, ranging from 12-80% of the population (Abu-Saad, 2010). It becomes more common as people approach death. Pain affects more people than diabetes, heart disease and cancer combined (Institute of Medicine Report, 2011). It is a leading cause of disability and it is a major contributor to health care costs (National Center for Health Statistics, 2006). It is estimated that 20% of adults suffer from pain and another 10% are diagnosed with chronic pain every year. Pain affects all populations, regardless of age, sex, income, race/ethnicity or geography; it is not distributed equally across the globe. Pain can lead to depression, inability to work, disrupted social relationships and suicidal thoughts (Goldberg and McGee, 2011).

Unresolved inflammatory processes may be involved in the pathogenesis and progression of many inflammatory diseases, including asthma, atherosclerosis, cancer, rheumatoid arthritis, multiple sclerosis, heart disease, gouty arthritis, rhinitis and ischemia–reperfusion injury (Iwalewa et al., 2007; Medzhitov, 2008; Medzhitov, 2010; Alessandriet al., 2013). The costs of unrelieved pain and inflammatory diseases can result in longer hospital stays, increased rates of re-hospitalization, increased outpatient visits, and decreased ability to function fully leading to lost income. As such, patient’s unrelieved chronic pain and inflammatory problems often result in an inability to work and maintain sound health (Strigo et al., 2000).

Some of the analgesic and anti-inflammatory drugs available are often expensive, inaccessible and cause undesired and serious adverse effects (Babu et al., 2009; Donkor et al., 2013). For example, most of the opiate analgesics used clinically activate µ opioid receptors and the various central nervous system (CNS) side effects resulting from the use of opioids have been attributed to the µ opioid receptors. This development has directed research in favour of the more selective, safe and efficacious δ opioid receptors (Amrani, 2011). NSAIDs exert their analgesic effect by inhibiting the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to leukotrienes and prostaglandins (PG), which are known to sensitize nociceptors near the location of the pain. Under physiological conditions, PG mediate many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity and fertility. Dysregulated PG synthesis or degradation has been associated with a wide range of pathological conditions (Ricciotti and FitzGerald, 2011). Therefore, adverse effects of NSAIDs mediated through the gastrointestinal and renal systems are as a result of the inhibition of COX.

1.2 Justification of the Study

Pain, when untreated can negatively affect all aspects of daily life, including physical activities, school attendance, sleep patterns, family interactions and social relationships and can lead to distress, anxiety, depression, insomnia, fatigue or mood changes, such as irritability and negative coping behaviour (WHO, 2012). The problems associated with drugs used in pain management are of serious concern, which necessitate the need for development of new drugs and variety of treatment option from bioactive constituents obtained from plants used in traditional medicine (Stark et al, 2013).

Plants represent still a large untapped source of structurally novel compounds that might serve as lead for the development of novel drugs (Ahmad et al., 1992). Despite the immense technological advancement in modern medicine, many people in developing countries still rely on traditional medicine for their daily health care needs (Louwet al., 2007; Premanathan et al., 2000).

Herbal medicine is thus gaining popularity, but lack of knowledge of the mechanism action and side effects of these preparations may undermine their utilization (Boullata and Nace, 2000). Understanding the cellular and molecular mechanisms of analgesic and anti-inflammatory actions of herbs will permit the discovery of promising targets for the development of new drugs to treat chronic pain and inflammatory diseases (Calixto et al., 2003).

A good number of plant products with anti-inflammatory and analgesic activity have been documented, but very few of these compounds have reached clinical use due to scant scientific evidence that could explain their mode of action (Bellik et al., 2013). The anti-inflammatory and analgesic activities of O. subscorpioidea have not been established scientifically. Similarly, the mechanism of analgesic and anti-inflammatory actions of O. subscorpioidea has not been studied before; thus, there is need for the studies.

1.3 Aim and Objectives

1.3.1 Aim

The aim of the study is to establish the analgesic and anti-inflammatory potentials of methanol leaf extract of Olax subscorpioidea and elucidate its possible mechanisms of analgesic and anti-inflammatory actions.

1.3.2 Specific objectives

1. To identify the class of phytoconstituents present in the methanol leaf extract and fractions of O. subscorpioidea.

2. To determine the acute toxicity of the methanol leaf extract and fractions of O. subscorpioidea.

3. To assess the analgesic effect of the methanol leaf extract and fractions of O. subscorpioidea.

4. To assess the anti-inflammatory effect of the methanol leaf extract and fractions of O. subscorpioidea.

5. To determine the possible mechanisms of analgesic and anti-inflammatory activities of O. subscorpioidea.

1.4 Research Hypothesis

The methanol leaf extract of Olax subscorpioidea possess analgesic activity which is mediated through the involvement of nitric oxide-l-arginine, serotonergic, adrenergic and opioidergic pathways; and possess anti-inflammatory activity mediated through the inhibition of pro-inflammatory cytokines and/or stimulation of anti-inflammatory cytokines.