ANXIOLYTIC PROPERTIES OF MITRACARPUS VILLOSUS (SW) DC. LEAF EXTRACTS IN MICE AND RATS

ABSTRACT

The leaf of Mitracarpus Villosus (Sw.) DC. (Rubiaceae) is used in West African folk medicine for the management of a plethora of stress-related diseases including headaches and toothaches. In this study, preliminary phytochemical analysis of the ethylacetate leaf extract of Mitracarpus Villosus and its fractions were conducted. Vacuum liquid chromatography (VLC), column chromatography (CC) and thin layer chromatography (TLC) using solvents of varying polarities were employed to separate the constituents of the plant extract. The intraperitoneal and oral lethal doses (LD50) of the extracts were determined by Lorke’s method in mice and rats. The sub-chronic toxicity studies were carried out on male and female Wistar albino rats by daily administration of ethylacetate leaf extract of Mitracarpus Villosus (312 – 1250 mg/kg) for 28 days during which body weight, feed and water consumption were monitored. After 28 days, effects of the extract on biochemical and haematological parameters were evaluated and the histological changes of the vital organs were also examined. The effect of the ethylacetate leaf extract of Mitracarpus Villosus on mouse behaviour was examined using various in-vivo models which include the hole-board (exploratory behaviour), open field test (OFT), staircase test (SCT), light/dark box (LDB), elevated plus maze (EPM), diazepam-induced sleep and rota rod tests. The anti-nociceptive effects of the ethylacetate extract were tested on acetic acid-induced abdominal writhing, oro-facial formalin-induced pain as well as carageenaan–induced hyperalgesia in mice and rats. The effects of the extract on inflammation and body temperature were determined using formalin induced paw oedema and Baker‘s yeast induced pyrexia respectively in mice. The ethylacetate leaf extract of Mitracarpus Villosus tested positive for alkaloids, steroids, terpenes, flavonoids, cardiac glycosides, resins and tannins while the biologically active fraction tested positive for terpenes and steroids. The intraperitoneal LD50 of the extract was calculated to be 1264.91 mg/kg and the oral LD50 was estimated to be greater than 5000 mg/kg in mice and rats, respectively. Ethylacetate leaf extract of Mitracarpus Villosus at doses of 312, 625 and 1250 mg/kg did not cause changes of food and water intake or body weight, but significant (p<0.001) increase of the weight of the liver and levels of the mean corpuscular volume (MCV) were recorded at 1250 mg/kg. Similarly, significant (p<0.05) changes in the levels of the hepatic enzyme alkaline phosphatase (ALP) and the renal index creatinine were recorded. There was no significant difference in levels of electrolytes (Na, K, Cl, HCO3) and urea. Histological evaluation of the organs presented with distortion of the structures of kidney and liver tissues at high doses of up to 1250 mg/kg. Graded doses of the extract (6.25 – 25 mg/kg) exhibited significant (p<0.001) anxiolytic effects by causing increase in locomotion and central square crossings in the open field test. Staircase test produced increase in locomotion and rearing. The percentage of time spent in the light compartment was prolonged both in the LDB and EPM tests when compared to control. At higher doses, the extract (100 – 400 mg/kg) significantly (p<0.01) and dose-dependently prolonged the duration of diazepam-induced sleep (p<0.05), decreased the number of both peripheral and central squares crossed in the OFT, decreased (p<0.001) number of head-dips in the hole-board test and reduced steps climbing in SCT (p<0.05) in mice. The most active fraction E2, exhibited a similar pattern of behavioural actions comparable to the crude extract. The ethylacetate leaf extract of Mitracarpus Villosus at the doses tested had no effect on motor co-ordination as observed in the rota-rod assay in mice. The extract at 100 – 400 mg/kg significantly (p< 0.05) and dose-dependently inhibited acetic acid-induced writhing, decreased the time of face rubbing induced by formalin in mice and increased the paw withdrawal threshold of carageenan induced hyperalgesia in rats. Paw thickness induced by formalin was also significantly (p<0.001) reduced. Hyperthermia induced by baker‘s yeast was significantly (p< 0.05) reversed by the extract. The actions exhibited by the ethylacetate leaf extract of Mitracarpus Villosus are probably mediated via the benzodiazepine-GABA-ergic (BDZ-GABA) pathways. The results from this study provide scientific evidence that the ethylacetate extract of Mitracarpus Villosus leaf may contain psychoactive principles that are sedative in nature with potential anxiolytic effects attributable to the presence of terpenoidal and steroidal compounds. The sedative and anxiolytic effects may be mediated through the benzodiazepine site of the GABAA receptor channel complex as the effect of the extract on diazepam induced sleep was reversed by flumazenil. The extract exhibited anti-nociceptive effects against neurogenic and inflammatory mediated pain with anti-inflammatory and hypothermic effects. These findings support the further appraisal of the biologically active principles of the plant as analgesic, anxiolytic and anti-inflammatory agents.

CHAPTER ONE

1.0 INTRODUCTION

The rising burden of neurological disorders in developing countries has been ascribed to increasing life expectancy and urbanization of population; the availability of better diagnostic facilities implies that hitherto unknown cases become diagnosed and

recorded (Wasay and Ali, 2010). Furthermore, the complexity and challenges of everyday life in modern society in addition to the rising spate of insecurity along with natural disasters has led to various degrees of anxiety, insomnia and depression (WHO, 2012). Psychosocial and other neurological disorders have been found to be associated with pains, headaches, stress and related debilitating psychiatric disorders among people in both developed and developing countries (Sinanovic, 2012).

Neurological conditions produce a range of symptoms and functional limitations that greatly limit the ability of individuals to perform normal daily activities thereby posing serious challenges to the individuals and the family members that support them. In addition, neurological conditions pose an economic burden to society from both direct (health care expenditures) and indirect costs i.e. monetary value due to loss of productive years as a result of premature death or loss of activity because of long term disability (CIHI, 2007, Ojagbemi et al., 2013).

1.1. Epidemiology of Neurological Diseases

The burden of neurological, mental and substance use disorders in the world has been estimated to account for 10.4% of global Disability Adjusted Life Years (DALYs) and 28.5% of global years living with disability (YLDs) (Whiteford, 2015). The World Health Organisation (WHO, 2006) reported that neurological conditions contribute to an estimated 92 million DALYs in 2005 and it is projected to increase to 103 million in 2030.

Neurological conditions contributed to the global burden of disease in the African region, an estimated 2.9 % in 2005. Some reports have suggested that neurological diseases account for more than 20% of the world’s disease burden with a greater majority of people affected living in Africa (Tegueu et al, 2013). The life expectancy of persons with psychiatric disorders tend to be shorter compared with non-sufferers due to associated factors that include health risky behaviours like obesity, smoking, substance use (Markkula et al., 2012)with anxiety and depression being the most common psychiatric disorders presenting in behavioural neurology (Sinanovic, 2012).

The International Neuropsychiatry Association (INA) defined Neuropsychiatry as a field of medicine with interest in the complex relationship between human behaviour and brain function and attempts to understand behavioural disorders on the basis of an interaction of neurobiological and psychosocial factors. Neuropsychiatric conditions include dementias, cognitive impairment, sleep and movement disorders, psychiatric disorders such as anxiety, depression (Sachdev, 2005; Beletsky and Mirsattari, 2012); while psychiatric/mental disorders are chronic, malfunctioning conditions that present primarily as abnormalities of thought, feeling or behaviour, producing either distress or impairment of function(WHO, 2006).

Neurological and neuropsychiatric conditions are closely related with a common background in neuroscience. The advancement of the field of behavioural neurology has made it possible to study morphological links to personality traits and neuro-psychiatric symptoms and connect this to genetic, biochemical and neuro-receptor characteristics of these conditions (Aarli, 2005).