PHYTOCHEMICAL AND ANTICONVULSANT STUDIES OF METHANOL LEAF EXTRACT OF HYMENOCARDIA ACIDA, TUL (EUPHORBIACEAE)
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ABSTRACT
The research was on the phytochemical and anticonvulsant studies of methanol leafextract of Hymenocardia acida, Tul, (Euphorbiaceae) used inNorthern Nigeria for the treatment of headache, rheumatic pain, sickle cell crisis, malaria, epilepsy and cancer. The preliminary phytochemical screening of crude methanol extract (CME) using standard methods revealed the presence of terpenoids, tannins, saponins, alkaloids and flavonoids.Phytochemical evaluations were carried out using silica gel column chromatography, preparative thin layer chromatography and gel filtration using sephadex LH-20. The CME was partitioned successively with n-hexane, chloroform, ethyl acetate and n-butanol to yield different fractions. Extensive phytochemical investigation of n-Hexane soluble fraction of the leaf using silica gel column chromatography, gel filtration and preparative thin layer chromatography led to the isolation of Lupeol. The structure of the isolated compound was elucidated with the help of 1HMR and 13C NMR analysis. The oral median lethal dose (LD50)in mice was found to be greater than 5000mg/kg, suggesting the crude extract is practically non-toxic. Anticonvulsant activity was studied using maximum electroshock test (MEST) in chicks and pentylenetetrazole (PTZ) induced seizure model in mice. The CME of Hymenocardia acida at doses of 150mg/kg, 300mg/kg and 600mg/kg did not exhibit significant activity against MES convulsion because none of the chicks was protected against the seizure but there was 90% protection with the standard drugPhenytoin at a dose of 20mg/kg while the extract produced a dose independent activity in the PTZ induced seizure in mice which was significant at (p<0.05) which was seen as percentage protection against seizure as 50, 33.33, 16.67% at doses of 150mg/kg, 300mg/kg and 600mg/kg respectively i.e the lower the dose of the extract the higher the protection. The standard control, Sodium Valproate 200mg/kg protected the mice 100% . The finding of the study suggests that the CME of Hymenocardia acida possesses significant anticonvulsant activity which might be due to the phytochemical constituents. This provides some scientific rationale for the ethnomedicinal claim of the use of the plant in the management of epilepsy.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Natural Product
Natural Productscan be defined as organic compounds and other chemicals synthesized by plants through metabolic processes aided by sunlight, involving CO2, H2O vapour and chlorophyll. Generally, natural products are characterized by specific functions they perform in plants and animals. Classical natural product chemistry methodologies enabled the discovery of a vast array of bioactive secondary metabolites from various source materials including terrestrial plants, terrestrial micro-organisms, marine organisms, and terrestrial vertebrates and invertebrates (Tyler et al., 1988). Natural products have been used since ancient times and in folklore for the treatment and prevention of many diseases and illnesses. They are the most successful source of potential drugleads (Haefner, 2003; Butler, 2004; Cragg andNewman,2005; Mishra andTiwari, 2011;Rey-ladino et al., 2011).
Therefore, natural product continue to provide unique structural diversity in comparison to standard combinatorial chemistry, and presents opportunities for discovering mainly novel low molecular weight lead compounds. Since less than 10% of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery with the challenge being how to access this natural chemical diversity (Cragg andNewman,2005).
The earliest records of natural products were depicted on clay tablets in cuneiform from Mesopotamia (2600 B.C.) which documented oils from Cupressus sempervirens (Cypress) and Commiphora species (myrrh) which are still used today to treat coughs, colds and inflammation (Cragg andNewman,2005). The Ebers Papyrus (2900 B.C.) is an Egyptian pharmaceutical record, which documents over 700 plant-based drugs ranging from gargles,
pills, infusions to ointments (Cragg andNewman,2005). The Chinese Materia Medica written sometimes around 1100 B.C. “Wu Shi Er Bing Fang” contains 52 prescriptions, Shennong Herbal (~100 B.C.) contain 365 drugs and the Tang Herbal (659 A.D) gave the records of 850 drugswhich are documented records of the uses of natural products (Cragg andNewman,2005). The Greek physician, Dioscorides, recorded the collection, storage and the uses of medicinal herbs, whilst the Greek philosopher and natural scientist, Theophrastus (~300 B.C.) dealt with medicinal herbs (Cragg andNewman,2005).
During the Dark and Middle Ages the monasteries in England, Ireland, France and Germany preserved this Western knowledge whilst the Arabs preserved the Greco-Roman knowledge and expanded the uses of their own resources, together with Chinese and Indian herbs unfamiliar to the Greco-Roman world (Cragg andNewman,2005).
It was the Arabs who were the first to privately own pharmacies in the 8th century with Avicenna, a Persian pharmacist, physician, philosopher and poet, contributing much to the sciences of pharmacy and medicine through works such as the CanonMedicinae (Cragg andNewman,2005).
1.2 Traditional Medicine
Traditional medicine refers to health practices, knowledge and beliefs incorporating plants, animals and mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being (WHO, 2005; NNMDA, 2008).The world Health organization estimated that 80 percent of people worldwide still rely on plant-based traditional medicines for some aspect of their primary health care (Farnsworth and Soejarto, 1985).
Medicinal plants are plants containing substances which can be used for medication or as precursor of drug synthesis (Sofowora, 1982). Medicinal plants can be referred to as: ‘all higher plants that have been alleged to have medicinal properties, i.e. effects that relate to health, or which have been proven to be useful as drugs by western standards, or which contain constituents that are used as drugs’ (Farnsworth and Soejarto, 1991). The term ‘medicinal’ as applied to a plant indicates that it contains a substance or substances which modulate beneficially the physiology of sick mammals, and that it has been used by man for that purpose (Fellows, 1991). Medicinal plants have been a source of medicine to human health since ancient time, whereas about 60-75% of world populations require plant for carrying health (Farnsworth, 1994; Joy et al., 1998; Harvey, 2000). Plants and microbes are the main source of natural products (Hayashi et al., 1997; Armaka et al., 1999; Lin et al.,1999a ;Lin et al.,1999b: Basso et al., 2005), and consistently become main source of the newest drugs (Harvey 2000). Many methods of investigation or drug development from natural sources are based on the bioassay-guided isolation of natural products on traditional uses of local plants (Ataur Rahman and Choudhary 1999). Ayurveda is the most ancient health caresystem and is practiced widely in India,Srilanka and other countries (Chopra and Doiphode, 2002). Atharvveda (around 1200 BC), Charak Samhita and Sushrut Samhita (100 – 500 BC) are the main classics that give detailed descriptions of over 700 herbs (Dash et al., 2001). In the western world, documentation of use of natural substances for medicinal purposes can be found as far back as 78 A.D., when Dioscorides wrote “De Materia Medica”, describing thousands of medicinal plants (Tyler et al., 1988).
This treatise included descriptions of many medicinal plants that remain important in modern medicine, not because they continue to be used as crude drug preparations, but because they serve as the source of important pure chemicals that have become mainstays of modern therapy.
The knowledge associated with traditional medicine (complementary or alternative herbal products) has promoted further investigations of medicinal plants as potential medicines and has led to the isolation of many natural products that have become well known pharmaceuticals. Many modern pharmaceuticals have been modeled on or derived from chemicals found in plants. An example is the heart medication digoxin (I) derived from foxglove (Digtialis purpurea), quinine (II), an antimalarial agent isolated from the bark of Cinchona succirubra tree. Paclitaxel (III) (Taxol®), a drug used for breast cancer was isolated from the bark of Taxus brevifolia (Pacific Yew) (Cragg, 1998). Taxol® is present in limited quantities from natural sources, its synthesis (though challenging and expensive) has been achieved (Cragg, 1998). It is widely known in ethnomedicine that various parts of a plant can possess different healing properties, for instance, the bark of Rauwolfiamombasiana is used for the treatment of malaria. The root of the same plant is used for the treatment of fever and anxiety states.The root, stem and leaves of another species of this plant, R. vomitoria, are used for fever (Iwu, 1993). Morphine (IV) used for pain relief was derived from Papaver somniferum;a potent antimalarial drug named Artemisinin (V) was isolated from Artemisia annua as a remedy against the multidrug resistant strains of Plasmodium.
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Use of herbs as alternate medicine in developed countries has expanded sharply in the latter half of the twentieth century. Monographs on selected herbs are available from a number of sources, including the German Commission E (Blumenthal et al., 1998), European Scientific Cooperative on Phytotherapy (ESCOP, 1999) and the World Health Organization (WHO, 1999) The WHO monographs, for example, describes the herbs (including synonyms and vernacular names) and the herb part commonly used, its geographical distribution, tests used to identify and characterize the herb, the active principles (when known), dosage forms and dosing, medicinal uses, pharmacology, contra-indications and adverse reactions, which is found in every society irrespective of its level of development and sophistication (Odugbemi, 2006).
1.2.1 Medicinal plants and epilepsy
Plants have a long history in the management of epilepsy. A typical example of which is Valariana officinalis used as a herbal treatment of epilepsy in Europe and America (Murray, 1998). A number of African medicinal plants have been reported to possess bioactive constituents capable of exerting anticonvulsant action hence making them relevant in the management of seizure disorders. These plants include; Glycyrrhyza glabra (Ambawade et al., 2002), Dalbergia saxatilis (Yemitan and Adeyemi, 2006), Cassia occidentalis, Heliotropium indicum and Xylopia aethiopica (Mann et al., 2003).
1.3 Epilepsy
Epilepsy, also known as the ‘falling sickness’, not only has a much older history than any of the other individual nervous or mental disorders, but it has also occupied people’s minds to a much larger extent than the majority of ailments to which the genus Homo sapiens is susceptible to (Kinnier-Wilson and Reynolds, 1990).
Epilepsy is thought to be a common and diverse set of chronic neurological disorders characterized by seizures, as such there are various definitions of epilepsy but most definitions require that the seizure be recurrent and unprovoked (Chang and Lowenstein, 2003), while others require only a single seizure combined with brain alterations which increases the chances of future seizures (Fisher et al., 2005). The term epilepsy has also bee defined as a disorder of brain function characterized by periodic and unpredictable occurrences of seizures.Brain dysfunctions, whether primary or secondary to malfunction of other systems, are a major concern of human society, and a field in which pharmacological intervention plays a key role (Rang et al., 2003). Epilepsy is estimated to affect about 50 million people worldwide and is the second most common neurological disorder after stroke (Harvey and Champe, 2006). Close to 80 % cases of epilepsy are found in developing countries (WHO, 2012).
1.3.1 Etiology
The causes of epilepsy are summarized in three general etiological groups:
The first one is the threshold, which determines the susceptibility of individual brains to generate seizures in response to epileptogenic perturbations. This will determine what is called “primary” or “idiopathic”epilepsy, when it is not the result of some other brain abnormality. They are usually benign and often remit spontaneously or after uninterrupted pharmacological treatment with Antiepileptic drugs(AEDs). The duration between onset and remission can vary from 2 to 12 years (ILAE, 1994; Engel and Pedley, 1997). The second group is related to a specific epileptogenic abnormality, which could be an acquired lesion of the brain, congenital malformations of the brain or genetic disorders other than epilepsy. This “secondary” or “symptomatic” epilepsy is very common in developing countries, where it is responsible for the difference in terms of prevalence and prognosis. Risk factors are dominated by poor perinatal care, head trauma, and intracranial infections, including parasitic infestations (such as neurocysticercosis, neuromalaria), and these are far more common than in industrialized countries. Their control requires, in addition to AEDs, specific care of the aetiology which can either be medical and/or neurosurgical (ILAE, 1994; Engel and Pedley, 1997). The third group represented by epileptic disorders that are probably symptomatic, but the causes have not been identified with existing diagnostic means, and are therefore called CRYPTOGENIC (which means hidden cause) with a high suspicion of a genetic (but no identifiable) factor (ILAE, 1994; Engel and Pedley, 1997).
1.3.2 Pathophysiology
Despite extensive researches that led recent breakthroughs in the understanding of the mechanisms involved in the pathophysiology of epilepsy, the specific causes of several types of epilepsy are still unknown (Engelborgs et al., 2000). According to Ditcher and Brodie (1996), the hypersynchronous discharges during a seizure may begin in a very discrete region of cortex and then spread to neighbouring regions. Seizures initiation is characterized by two concurrent events: high frequency bursts of action potentials and hypersynchronization of a neuronal population.
1.3.2 Mechanisms of Ictogenesis and Epileptogenesis
Ictogenesis is a transient and direct event that induces seizures due to excessive discharges from groups of neurons. Such discharges are initiated by the sequential opening of the voltage-dependent Na+ channels due to membrane depolarization resulting from K+ and/or Ca2+ channel-mediated events or via neurotransmitters and/or activation of ionic glutamate receptors. On the other hand, epileptogenesis involves long-lasting and prolonged histological/biochemical alterations of neuron network and reorganization of neuronal matrices, with the process ranging from months to years (Sasa, 2006). According to Sasa (2006), drugs currently used in the management of epilepsy (antiepileptic drugs) are classified as drugs against ictogenesis (anti-seizure) which is different from the concept of epileptogenesis because they are unable to stop the progression of epilepsy.
1.3.4 Classification of Epilepsy
Epilepsies have been classified as described below (Tripathi, 2008).
1.3.4.1Generalized seizures
Generalized tonic-clonic seizures (GTCS), major epilepsy, grand mal: This is the commonest and lasts for 1-2 minutes. The usual sequence is aura-cry-unconsciousness-tonic spasm of all body muscles-clonic jerking followed by prolonged sleep and depression of all CNS functions.
Absence seizures (minor epilepsy, petit mal): prevalent in children, lasts about 60 seconds. Momentary loss of consciousness, patient apparently freezes and stares in one direction, no muscular or little bilateral jerking.
Atonic seizures (Akanitic epilepsy): Unconsciousness with relaxation of all muscles due to excessive inhibitory discharges. Patient may fall.
Myoclonic seizures: Shock-like momentary contraction of muscles of a limb or the whole body.
Infantile spasms (Hypsarrhythmia): seen in infants, probably not a form of epilepsy.
Intermittent muscle spasm and progressive mental deterioration.
1.3.4.2Partial seizures
Simple partial seizures (SPS, cortical focal epilepsy)
Complex partial seizures (CPS, temporal lobe epilepsy, psychomotor).
Simple partial or complex partial seizures secondarily generalized
1.3.5 Status Epilepticus
Status epilepticus (SE) is a life threatening emergency characterized by a prolonged continuous state of convulsions. It is defined as a continuous seizure activity or multiple seizures without regaining consciousness for more than 30 min (Delgado-Escueta et al., 1983). If untreated can lead to brain damage and death. It can either be generalized convulsive SE or non-convulsive SE (Delorenzo et al., 1992). The pathophysiology of SE is not clearly understood but excess excitatory (glutamate) neurotransmission and loss of normal inhibitory (GABA) neurotransmission are thought to be the most likely mechanisms. The first-line therapies of choice are intravenous benzodiazepines (e.g. diazepam and lorazepam), which potentiate the inhibitory responses mediated by GABA-A receptors (Brophy et al., 2012).
1.3.6 Anticonvulsant Studies
1.3.6.1 Animal Models for Anticonvulsant Studies
The use of animal seizure models is essential in the discovery and development of new drugs for the treatment of epileptic seizures. These models can be either in vivo or in vitro. Discovery of a new therapeutic agent begins with the hypothesis that there is a relationship between the experimental seizure model and the initiation and propagation of the seizure and that the experimental seizure approximates the pathophysiology underlying the human condition. Phenytoin, carbamazepine and valproate were identified by relatively simple screening procedures that do not provide insight into a drug’s mechanisms of action. Two concepts were assumed: either seizure spread or seizure threshold was affected. When animal models were developed that used electrical stimulation or chemoconvulsants, they were systematically validated using the then-known clinically effective compounds. The pharmacologic activity of the potential anticonvulsants was then profiled to predict their utility in the various types of human epilepsy. The therapeutic activity as well as the toxicity of these new agents was then demonstrated in various animal models and species (Harvey, 2000). The anticonvulsive pharmacology of novel test substances can be characterized using variations of two basic test methods of animals: blockade of electroshock-induced convulsive seizures and the blockade of chemically-induced convulsive seizures. Variations of the two basic methods have led to the classification of the experimental models into acute and chronic seizure models (Mody and Schwartzkroin, 1997).
1.3.6.2 Acute seizure models
Acute seizure approaches to the understanding of seizure-like activity which has been extremely important in the history of epileptic investigation, because they represent the first best method to explore basic mechanisms. In addition, some of these models have become the standards against which antiepileptic drugs are evaluated for efficacy (Mody and Schwartzkroin, 1997).For this study, one electrical (MEST) and one chemical method of acute seizure induction was employed i.e the PTZ-induced seizures. The maximal electroshock test (MEST), developed by Toman and collaborators in 1946 and modified by Swinyard and Kupferberg (1985) and Browning (1992), is probably the best validated pre-clinical test that predicts drugs effective against generalized seizures of the tonic-clonic (grand mal) type (Loscher and Schmidt 1988; White, 2003; Mares and Kubova, 2006). It allows evaluation of the ability of an agent to prevent seizure spread through neural tissue of the CNS (Swinyard and Kupferberg, 1985). The MES test is simple and can be conducted easily with a minimal investment in equipment and technical expertise and is well standardized (Mares and Kubova, 2006). Several standard and newly developed drugs are effective in MES test, hence making it possible to quantify their anticonvulsant potency after both single and combined application (White, 2003; Borowich, 2007).Agents that act on sodium channels e.g. carbamazepine, phenytoin, oxcarbazepine and lamotrigine are known to suppress hind limb tonic extension induced by maximal electroshock (Rho and Sankar, 1999). Pentylenetetrazole (PTZ) is a chemoconvulsant agent that induces seizures in experimental animals by the non-competitive inhibition of Gamma Amino Butyric Acid (GABA) receptors and is a widely accepted experimental model for absence seizure (Loscher et al., 1991). PTZ, a tetrazole derivative is the prototype agent in the class of systemic convulsants that when administered parenterally has consistent convulsant actions in mice, rats, cats and primates (DeDeyn et al., 1992). PTZ was introduced as a screening test for anticonvulsant in part because the antiabsence seizure drug ethosuximide, which is effective against PTZ induced seizures, fails to alter MES thresholds. In contrast, some drugs effective against MES induced seizures such as phenytoin and carbamazepine are ineffective against PTZ induced seizures (DeDeyn et al., 1992).
1.4 Antiepileptic Drugs (AEDs)
The term antiepileptic is used synonymously with anticonvulsantto describe drugs that are used to treat epilepsy (which does not necessarily cause convulsions) as well as non-epileptic convulsive disorders (Rang et al., 2003). Management of epilepsy usually requires the use of antiepileptic drugs, however, in cases of refractory epilepsy, non-pharmacological methods can be employed and they include surgery, ketogenic diet and implantation of medical devices e.g. Vagus nerve stimulation (VNS).
1.4.1 Mechanisms of Action of AEDs
AEDs protect against seizures through interactions with a variety of cellular targets. The actions on these targets are often categorized into three major classes: (Ranget al.,2007).
- Enhancement of GABA action g. Phenobarbital (VI), Diazepam (VII).
- Inhibition of sodium channel function g. Valproate (VIII), Phenytoin (IX).
- c) Inhibition of calcium channel function g. Ethosuximide (X), Gabapentine (XI).
| O | H | O | HO | |
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N
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(IX)
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1.5 Statement of Research Problem
Epilepsy is one of the oldest conditions known to mankind and still the most common neurological condition affecting individuals of all ages. At any given time, it is estimated that 50 million individuals worldwide are diagnose of epilepsy (WHO, 2001). Epilepsy is a major neurological disorder that accounts for 0.5 % of global burden of disease with close to 80 % of the cases worldwide found in developing countries (WHO, 2012). World Health Organisation estimates that the proportion of the general population with active epilepsy (i.e. continuing seizures or the need for treatment) at a given time is 4 to 10 per 1000 people. However, some studies in developing countries suggest that the proportion is 6 to 10 per 1000 (WHO, 2012). In developed countries, annual new cases are 40 to 70 per 100,000 people in the general population. In developing countries, this figure is often close to twice as high due to high risk of experiencing conditions that can lead to permanent brain damage (WHO, 2012). The major causes of epilepsy include meningitis, tumours and traumas especially due to automobile traffic accidents. Statistics has shown that Nigeria and some East African countries have the highest automobile accident cases in the world with attendant increase in post-traumatic epilepsy (Ogunrin, 2006). As with many other neurological disorders, epilepsy is usually managed not cured with AEDs. These pharmacological agents inhibit seizures and thus are also referred to as antiseizure drugs. Whether these drugs prevent the development of epilepsy (epileptogenesis) is uncertain (Cascino, 1994). Approximately, 20 to 30 % of patients are refractory to therapies using currently available AEDs (Sasa, 2006) and 88 % of such patients suffer severe side effects like renal failure, due to long term management of the condition with such drugs (Baker et al., 1997). According to Meldrum (1997), plant extracts can be an important source of natural and safer drugs for the treatment of epilepsy. Medicinal plant extracts, fractions and pure compounds have been used traditionally for the treatment of epilepsy and have demonstrated anticonvulsant properties that need to be further investigated (Raza et al., 2001; Kumar et al.,2012). The study was therefore designed to investigate the activity of the plant extract and to contribute to drug development.
1.6 Justification for the Study
AEDs are the mainstay in management of epilepsy and may have great impact on the quality of life of epileptic patients. Despite the continued development and release of new antiepileptic drugs, many patients have seizures that do not respond to drug therapy or have related side effects that preclude continuous use (Perucca et al., 2007). Even in patients in whom pharmacotherapy is efficacious, current AEDs do not affect the progression of epilepsy (Loscher and Schmidt, 2006). These factors and more therefore, call for development and search for new AEDs especially from medicinal plant sources which may have fewer side effects and greater efficacy. There are many medicinal plants employed locally in the management of epilepsy but with limited scientific evidences for their safety and effectiveness (WHO, 2008). This necessitates the need to scientifically evaluate the anticonvulsant profile of Hymenocardia acida in order to validate its folkloric use around Shika, Kaduna state of Nigeria.
1.7 Aim and Objectives of the Study
1.7.1 Aim: To isolate and characterize some ofthe bioactive compound(s) of the plant and to validate scientifically, the ethnomedicinal claim for the use of the plant in the management of epilepsy.
1.7.2 Specific Objectives
- To identify the phytochemical constituents present in Hymenocardia acida
- To isolate and characterize some of the bioactive compound(s).
- To determine the median lethal dose (LD50) of the crude methanol extract.
- To determine the anticonvulsantactivity of the crude methanol extract.
1.8 Statement of Research Hypothesis
The methanol leaf extract of Hymenocardia acida contains bioactive constituent(s) with anticonvulsant activity.
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