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Study Level: BTech, BSc, BEng, BA, HND, ND or NCE

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The leaf of Mitracarpusvillosus (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 ethylacetateleaf extract of Mitracarpusvillosus 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 ethylacetateleaf extract of Mitracarpusvillosus (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 ethylacetateleaf extract of Mitracarpusvillosus on mouse behaviour was examined using various in-vivo models whichinclude 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 ethylacetateleaf extract of Mitracarpusvillosustested 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. Ethylacetateleaf extract of Mitracarpusvillosus 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 ethylacetateleaf extract of Mitracarpusvillosusat 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 ethylacetateleaf extract of Mitracarpusvillosus are probably mediated via the benzodiazepine-GABA-ergic (BDZ-GABA) pathways. The results from this study provide scientific evidence that the ethylacetate extract of Mitracarpusvillosus 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.



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 facilitiesimplies that hitherto unknown casesbecome 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). Literature review of scientific reports for current epidemiological data on neurological and psychiatric disorders in Nigeria is inadequate, but inferences on the prevalence of neurological conditions can be made from studies conducted in different regions of the country. Although hospital medical statistics do not reflect the true prevalence of a particular disease in developing countries where people tend to seek help from traditional medicine practitioners before doing so at the hospitals (Osemene et al., 2011), thedatathat can be derived from the hospitals however will provide some information on the rate of prevalence on neurological and neuropsychiatric conditions.Abiodun and Ogunremi(1990) and Mohammed et al.,(2014) reported that about one third of in-patients with physical illnesses at general hospitals had comorbid psychiatric conditions and in many of these cases, the psychiatric disorders were unrecognised by attending physicians.Additionally, certain types of neurological disorders could have been under-represented because of inadequate facilities for detailed neurological diagnosis (Wammanda et al., 2007). Thus, there is need to address neurological conditions including psychiatric and pain disorders with a view to finding effective treatment for the management of these diseases.

1.2.        Traditional and Herbal Medicines

medicine is defined as the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness and herbal medicines include herbs, herbal materials, herbal preparations and finished herbal products that contain as active ingredients parts of plants or other plant materials or combinations (WHO, 2005). The use of plants as sources of medicines is as old as the history of man. Man has used plants as food and medicines and acquired the knowledge of medicinal plants with healing properties by probably learning through trial and error, spiritual inspiration, observing primates and other animals repeatedly consume certain plant species and then passed on this knowledge from one generation to the next through myths, traditions, writings and symbols used to categorize those plants that can treat diseases (Halberstein, 2005; Mamedov, 2012). The evolution and preservation of traditional plant based systems of treating illnesses within local communities gave birth to medical systems such as Ayurverda, Unani, Siddha, Amchi, aromatherapy medical systems;these systems are unique to each local area because they are deeply embedded into the cultures and traditional belief systems of the people (Nagori et al., 2011). In present day, traditional medicine is progressively gaining popularity in both developed and developing countries. An increasing number of the population in many industrialized countries now regularly use some form of traditional medical system (United States – 42%, Australia – 48%, France – 49%, Canada – 70%); and factors such as variety, flexibility, accessibility, broad acceptance, relative low cost, relative low side effects, rising economic importance, limitations of conventional orthodox treatment for chronic ailments, the desire for holistic or natural treatments, embedding of cultural belief systems, increased advertising and media coverage of traditional medicine practices are some of the elements advancing the course of traditional medicine (Coss et al., 1998, Hussain and Malik, 2013). In countries such asChina, Korea, and Vietnam, insurance fully covers traditional treatment services (Hussain and Malik, 2013), and increasing number of insurers in theUnited States of America and managed care organizations also offer alternative medicine programs (Shirwaikar et al., 2013). In Africa, traditional medicine plays a key role in the health of the people and this system of alternative medicine has been described as one of the effective measures that can be utilized to attain total healthcare coverage of the world population as it is more accessible to the rural and urban poor communities and also gaining wide acceptability in the industrialized worlds (Shirwaikaret al, 2013). The scepticism that has trailed this practice due to the secrecy surrounding the methods used by the traditional medicine healers is beginning to wane largely due to improved education of the operators of these systems; this education has made the custodians of traditional medicine practices to employ series of strategies like trainings to ensure the integration of their practices into national health care systems and they are responsive to subjecting their recipes to scientific validation (Awodele et al., 2011). Besides, progress in the fields of pharmaceutical sciences such as pharmacology and pharmacognosy which provide scientific basis for the methods employed by traditional medicine practitioners (Bologa et al., 2013; Dhami, 2013) have led researchers and other practitioners of orthodox medicine to appreciate that herbal medicine may possibly play a vital role in patient care and could be effective in treating chronic illnesses and should be recognised by governments (Awodele et al., 2012). Nigeria is a multicultural society with its indigenous traditional medical systems which were developed from the use of available resources to meet the health care requirement of the people. In Nigeria, both the traditional and orthodox medical systems enjoy large patronage and a study on the comparative assessment of herbal and orthodox medicines in Nigeria revealed that about 41% of the respondents took herbal medicines as first drug of choice (Osememe et al., 2011), while about 95% use it concurrently with western medicine either as dietary supplements, health food/ drinks or drugs; thus there is a need to formulate a national health policy to integrate traditional medicine practices with the orthodox medical practice that hinges on a collaboration, rather than competition (Adefolaju, 2014). Regulation of herbal medicines was introduced in Nigeria in 1993 in Decree No. 15, and revised in 1999 (WHO, 2005). The Federal Government of Nigeria had approved a policy frame work that would regulate the practice of traditional medicine in Nigeria and also endorsed a bill for the establishment of Traditional Medical Council of Nigeria (TMPA, 2004). The use of traditional medicine alongside or even in place of conventional medicine has been advocated for treatment/management of long term illnesses where conventional drugs have resulted in intolerable side effects or in cases which have not been effectively managed with orthodox medicine based therapy (Siddiqui et al., 2014). Herbal medicines are regarded as natural rather than synthetic therefore are considered to be safer with fewer side effects and have often been promoted as food supplements; therefore they are seen as part of a healthy life style.However, ingredients once used for symptomatic management in traditional healing are now used in developed countries as part of health promotion or disease prevention strategies; thus, acute treatments have been replaced by chronic exposure thus the potential of developing toxicity effects as a result of exposure to very high doses with an example of herbal products used for weight loss (Allison et al., 2001). Indiscriminate applications of these products have been associated with deleterious effects (Ekor, 2013). Moreover, several herbal drugs produced and sold currently in the developing countries do not meet the required quality standard and therefore have no consistency in quality in batch to batch products and do not have well defined and characterized composition (Shirwaikaret al, 2013).

1.3.        Statement of Research Problem

According to WHO, about 70 – 90 % of the world’s population relies on plants for their primary health care of which 35,000 to 70,000 species have been used as medicaments, a figure corresponding to 14 – 28% of the 250,000 plant species estimated to occur around the world and thus far only about 17% have been investigated for medical potential.The chemical and biological diversity of plants represent a potentially limitless renewable source for use in the development of new pharmaceuticals (Mamedov, 2012) and serve as a reservoir for identification of novel lead compounds for drug development (Melva, 2013) because in today’s global market, several drugs were derived from tropical plants (De Padua et al., 1999). Mental and substance use disorders were the fifth leading cause of DALYs in the year 2010 (Whiteford et al., 2015). In Nigeria, studies in a Lagos primary health care setting reporteda 4 – 15% prevalence of generalized anxiety disorder (Lasebikan et al., 2012). Co-morbidityof ailments is prevalent in neurological disorders (Hesdorffer, 2016) and neuropsychiatric conditions are incurable illnesses with the conditions of many patients deteriorating over time thereforethe patients present with several debilitating symptoms and functional limitations. Many agents used in the management of neurological conditions are expensive, limited in availability, have incapacitating side effects and adverse drug reactions and could be highly subjected to dependence (Von Moltke and Green blatt, 2003, Lakhan and Vieira, 2010).

1.4.        Justification of the Study

Neuropsychiatric disorders produce a range of symptoms and operational restrictions 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 those contending with these maladies (Ojagbemi et al., 2013). These disease conditions are lifelong necessitating prolonged treatment which may cause non-compliance with drug administration, thereby adversely affecting health care outcomes and resulting in treatment failure (Jimmy and Jose 2011). One of the important areas in which herbal medicines enjoy a high patronage worldwide is in the treatment of neurological and psychiatric disorders (Etkin, 1988, Magaji et al, 2008). Medicinal plants are used to modify moods, feelings, and behaviour in tribal ceremonies;many ethnic cultures also frequently maintain within their collections of herbal medicines substances valued as analgesics, tonics and stimulants, and these constitute potentially valuable but unexploited sources of psychotropic drugs (Cragg and Newman, 2013, Danjuma et al., 2014). Traditional medical practitioners make claims of successful treatments of diseases using a variety of herbal medicines often with insufficient supporting evidence. A proper scientific evaluation of these herbal medicines with pertinent emphasis on established pharmacological and toxicological paradigms is imperative in order to determine their efficacy and safety. Mitracarpus villosus is a plant of varied medicinal applications. The use of the plant for the treatment of headaches in traditional medicine has been reported in literature and co-morbidity of headaches and psychiatric conditions have been reported (Shoib et al., 2014). Literature search revealed that a triterpenoid compound, ursolic acid had been isolated from this plant (Ekpendu et al., 1993). This compound has been reported to have sedative, antitumour, anti-diabetic, anti-inflammatory, vasorelaxant and antioxidant properties (Wozniak et al., 2015). Further search showed inadequate scientific documentation in literature of the effect of Mitracarpus villosus on the central nervous system (CNS). This finding motivated the interest to investigate the anxiolytic properties and safety of this plant as a step towards the identification and isolation of the biologically active components and to complement earlier studies on this important medicinal plant that is already in common use in order to document the pharmacological profile of the plant.

1.5         Theoretical Framework


The pharmacological activities exhibited by plants have been attributed to their various phyto-constituents, therefore, extraction, separation and phytochemical tests are conducted to identify the pharmacologically active principles present in plants (Sheikh et al., 2013).

1.5.2      Toxicity studies

The objective of these studies is to determine the toxicity profile of a plant substance. This can be determined using acute, sub-acute and chronic toxicity studies. The acute toxicity studies aims at elucidating the dose that causes major adverse effects and an estimation of the minimum dose causing lethality. This is expressed as LD50 – dose that produces lethality to 50% of the test subjects (Sathya et al, 2012). The results allow a substance to be ranked and classified according to their intrinsic toxicity following the Globally Harmonised System for the classification of chemicals which cause acute toxicity (OECD, 2001); whereas the main objective of repeated toxicity studies is to characterize the toxicological profile of the test compound after repeated administration with a duration of exposure of up to 28 days. The information that can be obtained from these studies include identification of target organs of toxicity, dose dependence, exposure-response relationship and the potential reversibility of toxic effects (CPMP, 2000). This information can be used to estimate an initial safe starting dose and dose range for human trials and to identify parameters for clinical monitoring for potential adverse effects (FDA, 2010), and the possible health hazards likely to arise from repeated exposure over a relatively limited period of time (OECD, 2008)

1.5.3      General behavioural studies Irwin primary observation tests – This method described by Irwin (1962, 1968) can be employed to detect potential adverse effects of drugs on the central nervous system (CNS). When applied at an early stage of drug development, is particularly suitable to screen and select compounds against unwanted CNS effects, understand the mechanisms underlying these effects and possibly, discover novel therapeutic effects. By using an appropriate dose range for each test molecule, it is possible to obtain information on its pharmacological profile, on the intensity and the duration of its effects, and on the specificity of these effects (De Ron et al., 2008; Moser, 2011). Assessment of general behavioural and locomotor activity of rodents can be achieved using the open field test (OFT) and mouse staircase. The open field examines an animal’s response to a new environment and consequently its locomotion activity as the animal will tend to explore the new environment. Conversely, a novel wide open space may induce fear and anxiety in the animal resulting in changes in locomotion, exploration, rearing and grooming behaviours (Kharade et al., 2011; Kishore et al., 2012). The parameters monitored in the OFT have been used to determine sedative or stimulant effects of a pharmacological agent (Steru et al., 1987; Martinez-Vazquez et al., 2012). The hole-board experiment measures exploratory behaviour in animals. A decrease in number of head dips reveals sedative behaviour (File and Pellow, 1985) and is thus a measure of CNS depressant activity, while an increase in the number is an indication of CNS excitement (Ezekiel et al., 2010).

Anxiety studies – The mouse staircase evaluates anxiety and sedation. Increase in the number of rearing denotes anxiety while a reduction in the number of steps climbed is a suggestion of sedative activity (Gahlot et al., 2011). Natural aversion of rodents from brightly lit places will be evaluated in the light/dark transition model. (Akindele et al., 2012). This test is sensitive to the detection of anxiolytic/anxiogenic agents. Anxiolytic drugs increase the number of transitions between the light/dark compartments and/or increase the time spent in the light area. (Crawley et al., 1981). The Elevated plus maze (EPM) is sensitive to the anxiolytic/ anxiogenic effects of drugs. The percentage of time spent on the open arms is the parameter used to measure anxiety. These are increased by anxiolytics and decreased by anxiogenic agents (Pellow and File, 1986; File, 1991). Motor co-ordination (Rota rod) test  This method provides a quantitative measure of motor co-ordination and offers some estimation of CNS related side effects (Mortari et al., 2007). The rotarod test provides an index of skeletal muscle relaxation (Woodeet al., 2011). Diazepam induced sleep – A large number of substances are able to either stimulate the CNS and cause excitation or cause a decrease in activity thereby producing drowsiness, thus indicating sedation and CNS depression. Standard drugs such as diazepam act selectively on GABAA receptor, which mediates fast inhibitory synaptic transmission throughout the central nervous system (Ngo Bum et al., 2012). The sedative property of a plant can be confirmed by its ability to potentiate the duration of diazepam induced sleep (Ngoupaye et al., 2013).

1.5.4      Pain Studies

Orofacial formalin test  This method assesses visceral pain response and is considered an acceptable representation of clinical pain in comparison to methods which elicit thermal pain e.g. hot plate (Besra et al., 1996, Morrow et al., 1998). The orofacial region is one of the most densely innervated areas of the body by the trigeminal nerve which focuses some of the most acute pain. It is the site of frequent chronic and referred pains such as migraine (Munoz et al., 2010). Increased pain in response to noxious stimulation following peripheral tissue injury depends on an increase in the sensitivity of primary afferent nociceptors at the site of injury i.e peripheral sensitization and on an increase in the excitability of neurons in the CNS i.e central sensitization (Coderrre and Melzack, 1992). The acetic acid induced abdominal writhing assesses peripheral anti-nociceptive activity (Wani et al., 2012). Carageenan induced hyperalgesia which exhibits a biphasic response is a method that assess inflammatory pain without any injury to the inflamed tissue (Gill et al., 2013).

1.5.5      Anti-Convulsant studies

An evaluation of the potential pro and anti convulsant potential of a test compound is often included in the CNS safety pharmacology of drugs. These tests can identify the convulsant/anticonvulsant effects of novel compounds. The pentylenetetrazole test is a primary test that identifies compounds that prevent and/ raise seizures threshold (White, 1999).

1.6. Aim/Objectiv

The aim of the sudy is to investigate the anxiolytic effects of the leaf extracts of Mitracarpus villosus (MV) and its active fractions(s) in rodents.

1.6.1.     Specific Objectives

The specific objectives of this study are to:

  • the constituents of the leaf extract of Mitracarpus villosus and its fraction using phytochemical analysis.
  • Carry out preliminary/pilot tests to determine the effect of the hexane, methanol and ethylacetate leaf extracts of Mitracarpus villosus on diazepam induced sleep, hole-board and staircase test in mice.
  • Evaluate the safety profile of the most active fraction of Mitracarpus villosus leaf by means of acute toxicity (LD50) and sub-chronic toxicity testing of the extract
  • Examine the anxiolytic effects of the ethylacetate extract of Mitracarpus villosus and its active fraction using Hole-board, Open Field Test, Staircase test, Light/dark Box and Elevated PlusMaze.
  • Elucidate the possible mechanism(s) underlying the anxiolytic actions of the ethylacetate leaf extract of Mitracarpus villosusif any by interaction with antagonists.
  • Test the effects of theethylacetate leaf extract of Mitracarpus villosus on pain, inflammation and pyrexia.

1.7 Research Hypothesis

The  plant  Mitracarpus  villosus contains  pharmacologically active  principles  with anxiolytic effects and it is non-toxic.


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