HYPOTENSIVE ACTIVITY AND TOXICOLOGICAL PROFILE OF STEM BARK EXTRACT OF SYZYGIUM GUINEENSE (Willd, D.C. Myrtaceae) IN LABORATORY ANIMALS

ABSTRACT

Hypertension of unknown cause is called essential or primary hypertension while hypertension of a known cause is called secondary hypertension. Hypertension is called a silent killer because it is usually asymptomatic. It is a global problem affecting 1 in 3 adults in the United States. It is preventable and treatable disease. Orthodox drugs for its management are expensive and have associated side effects. Alternative source with good efficacy and minimum toxicity from natural products are explored. Hypotensive activity and toxicological profile of methanolic stem bark extract of Syzygium guineense (Willd, D.C. Myrtaceae) were evaluated. It is a medicinal plant used in folk medicine to treat hypertension, diarrhoea and infection. Phytochemical analysis of the stem bark extract was carried out using standard procedures. Bioassy-guided fractionation and finger printing of the crude extract were carried out. Five fractions(SGF1-SGF5) were isolated. The oral and intraperitoneal acute toxicity studies were carried out in mice using Lorke‟s method with modification. Subchronic oral toxicity studies of the methanolic stem bark extract in male and female Wistar rats for 90 days. The assessment of the toxic effect was evaluated at 42 and 90 days respectively, on clinical observations, feed and water intake, body weight, heamatology, blood chemistry, urinalysis, gross necropsy, organ weight and histopathology. The in vivo assessment of hypotensive activity of extract and fractions were carried in anaesthesized normotensive cat. The in vitro investigations to assess the possible mechanisms of hypotensive action of the extract were carried out on isolated tissue experiments. These included: guinea pig atria, rabbit perfused heart, rabbit ileum and rat uterus. The oral median lethal dose of the methanolic stem bark extract of Syzygium guineense was greater than 5000mg/Kg body weight while the intraperitoneal median lethal dose was 2154.1mg/Kg. There was significant (p<0.05) increase in feed intake in extract- treated male and female rats when compared to the corresponding control. There was also significant increase in water intake in male and female rats treated with Syzygium guineense in a dose-dependent manner in 90 days. The extract produced significant (p<0.05) increase in body weight of male and female rats. The relative organ body weight ratio values of extract-treated rats were not significantly different from those in the control groups. The haematological parameters and the biomarkers of liver function test and lipid profile were all normal in male and female rats when compared to the control. There was also, no significant change in the serum levels of electrolytes, urea and creatinine in male and female rats. All the values were within normal range. The histopathology results revealed no significant changes in all the organs. Phytochemical tests revealed the presence of carbohydrates, alkaloids, saponins, tannins, phytosterols, anthranol glycosides, cardiac glycosides, phenols, terpenoids, and flavonoids. The extract and fraction F3 significantly (p<0.0001) reduced both systolic and diastolic blood pressure of anaesthetized normotensive cat. The extract significantly (P<0.05) reduced adrenaline induced rise in systolic and diastolic pressure in anaesthetized cat. Atropine competitively reversed the hypotensive effect of the extract towards basal rhythm obtained with normal saline. The extract produced significant (p<0.05) concentration-dependent decrease in height of contraction of guinea pig atria. The extract also produced a concentration-dependent decrease in contraction of perfused rabbit heart. Adrenaline alone increased the height of contraction of the heart muscles. In the presence of the methanolic stem bark extract of Syzygium guineense the height of the amplitude produced was markedly lower than that due to adrenaline alone. The extract abolished all the spontaneous contractions of the rat uterus. These results showed that methanolic extract Syzigium guineense is safe and possesses significant blood pressure lowering effect in anaesthetized normotensive cat thus justifying its folkloric use in the treatment of hypertension.

CHAPTER

1.0 INTRODUCTION

1.1 Preamble

Hypertension (HTN) or high blood pressure is a chronic medical condition in which the blood pressure (BP) in the arteries is elevated and sustained. It is the second leading cause of premature death; kills 1 in 6 adults in the United States; and 1 in 5 Americans is completely unaware of the risk (Olives et al., 2013). Significant differences across the United States were revealed in the first ever analysis of awareness, treatment and control of hypertension conducted by the Institute for Health Metrics and Evaluation (IHME) at the University of Washington. Also, in the united states, estimate trends in prevalence, awareness, treatment and control were derived from data on National Health and Nutrition Examination Survey (NHANES) and Behavioral Risk Factor Surveillance System (BRFSS). The estimate of median prevalence of total hypertension in 2009 was; 37.6% (range: 26.5 to 54.4%) in men

and 40.1% (range 28.5 to 57.9%) in women. The median country-level control was 57.7% (range 43.4 to 65.9%) in men and 57.1% (range 43.0 to 65.46%) in women. Although control of hypertension is on the rise, prevalence of total hypertension continues to increase in the US (Olives et al., 2013).

Hypertension is a multifactorial disease involving a complex interplay of genetic and environmental factors (Lerman et al., 2005); it is one of the most common global Health ailments affecting more than 10% of the world‟s population (Chen et al., 2007). It is the leading preventable cause of cardiovascular diseases such as stroke, myocardial infarction, left ventricular hypertrophy, nephropathy, retinopathy and dementia. Every 20mmHg of systolic or 10mmHg diastolic increase in blood pressure above 115mmHg/75mmHg in middle and old age groups, results in doubling of mortality from ischemic heart disease and stroke (Lewington et al., 2002).

Over 50 million Americans have, as indicated by the data from the National Health and Nutrition Examination survey (NHANES), hypertension that warrant treatment (Burt et al., 1995; Hajjar and Kotchen, 2003). The global prevalence estimates for hypertension is about 1 billon individuals; and approximately 7.1 million deaths per year may be attributable to it (W H O, 2002). Prevalence increases with advancing age; more than half of people 60-69 years of age and approximately three quarter of those 70 years and older are affected (Burt et al., 1995). The life time risk of hypertension is approximately 90 percent for men and women who were non-hypertensive at 55 or 65 years and survived to the age of 80-85 years (Vasan et al., 2002).

The reduction in cardiovascular, and renal morbidity and mortality is the ultimate goal of antihypertensive therapy. A number of drugs are available for reducing blood pressure. Two or more antihypertensive agents from different classes of drugs are required in most hypertensive individuals before a control can be attained (Cushman et al., 2002; Hansson et al., 1998; Black et al., 2003; Dahlof et al., 2002; Materson et al., 1993).

Advances have been made in the efficacy and tolerability of orthodox antihypertensive drugs. Nevertheless, target blood pressure (i.e. <140/90mmHg) is not achieved in 25% of individuals treated with them. (Evans et al., 2005); and apart from being expensive, each agent is associated with relative drawbacks, often within the context of various patient characteristics such as age, co-morbidities and risks status (Germino, 2009).

Plants have been used for prevention and treatment of various diseases since ancient times. The use of different plants in the form of botanical drugs, dietary suppliments, and medicinal foods is increasing (Raskin et al., 2002). Human use of plants as medicines is dated from fossil records at least to the middle Paleolithic age some 60,000 years ago (Solecki and Shenidar, 1975).

Almost 65% of the world’s population have, according to the World Health Organisation, incorporated medicinal plants into their primary modality of health care (Farnsworth et al., 1985). The World Health Organisation (WHO) also estimates that 80% of the population living in developing countries depend on traditional medicine for their primary health care needs (WHO, 2001).

Natural products can be considered as one of the potential sources for more effective and better tolerated antihypertensive agents. The present study is attempts to evaluate the antihypertensive effect of methanolic stem bark extract of Syzygium guineense to support its folkloric use in the treatment of hypertension.

1.2 Statement of Research Problem

Hypertension is a global cardiovascular disease. It affects as many as 73 million Americans including children (Rosamond et al., 2008). It increases the risks of serious health conditions, including strokes, heart attacks, heart and kidney failure. Symptoms of hypertension are often undetectable, many people live with untreated high blood pressure and hence the disease is called silent killer disease.

In 2005, a report by the National Health Examination Surveys (NHANES), in the United State indicated that the population aged 20 years or older, an estimated 41.9 million men and 27.5 million women have prehypertension, 12.8 million men and 12.2 million women have stage I hypertension and 4.1 million men and 6.9 million women have stage II hypertension (Qureshi et al., 2005). Approximately 20% of the world adults are estimated to have hypertension when hypertension is defined as blood pressure [BP] in excess of 140/90 mmHg. The prevalence increases in patients over 60 years. It is also estimated to cause approximately 7.1 million death annually (WHO, 2002). Prescription (synthetic) drugs normalize the blood pressure but do not treat it. They are expensive and have associated side effects which could be life ridden (Barton, 2008).

Medicinal plants have, since time immemorial, been used in virtually all cultures as a source of medicine. In most developing countries, the use of traditional medicine and medicinal plants as normative basis for the maintenance of good health has been observed (UNESCO, 1996). Dietary change is important in lowering blood pressure. The Dietary Approaches to Stop Hypertension (DASH) includes fruits and vegetables, low fat dairy food, beans and nuts as well as sodium restriction (Wong, 2008).

Natural remedies have been used in traditional medicine for thousands of years to support the health of the heart and circulatory system. Herbal medicines that contribute to the commercial drug preparations manufactured today include, ephedrine from Ephedra sunica, Salicin (source of aspirin) from Salix alba, digitoxin from Digitalis purpurea and reserpine from Rauwolfia serpentina (Nick et al., 1998). A number of published clinical studies demonstrated the ability of range of herbs to support cardiovascular system and to maintain blood pressure within normal range (Leuchtgens, 1993). Further studies demonstrated the benefits of certain herbs in maintaining cardiac health and blood flow (Hempel et al., 2005).

Different parts of Syzygium guineense have various folkloric uses. Decoction of the bark is used as antidiarrhoeal and antihypertensive (Orwa et al., 2009). The seed of the plant is used to treat Diabetes Mellitus (Adebayo, 2009). It is used in combination with other medicinal plants to treat Herpes zoster (Kisangau et al, 2007; Kazlila and Hedmbi, 2010). It is used to treat malaria, has antibacterial effect (Tsalaka, 1997) and molluscicidal activity (Oketch-Rabah and Dossaji 1998).

1.3 Justification

Hypertension is gradually becoming a global killer disease. The incidence is increasing in the developed countries, in spite of the availability of effective antihypertensive drugs. Nearly one in three Americans has, according to federal health officials; high blood pressure and more than half of those cases are “out of control” (Rosamond et al., 2008; Olives et al., 2013). In developing countries especially Africa, access to effective, inexpensive and good quality medicines is vital especially at primary care level. In Nigeria prevalence of hypertension ranges from 8% – 46.6% depending on the study target population, type of measurement and cut-off value used for defining hypertension. Prevalence is similar in men and women (7.9% – 50.2%) vs 3.5% – 68.8% respectively) and in the urban (8.1% – 42.0%) and rural setting (13.5% – 46.4%). Awareness, treatment and control are generally low with attendant high burden of hypertension related complications (Ogah, et al., 2012).

In developing countries, conventional treatment of hypertension is expensive, inaccessible to most rural populace and is associated with severe adverse or toxic effects. Poorly treated or untreated cases of hypertension especially primary (essential) type predisposes to serious end organ damage such as heart failure, kidney damage, hypertensive encephalopathy and hypertensive retinopathy. In Sub-Saharan Africa, more than 80% of the population relies on traditional medicines and healers as the primary source of health care (WHO, 2002). This is mainly due to easy accessibility and affordability of consulting with traditional healers. The use of plant remedies has steadily increased worldwide in recent years, as well as the search for new phytochemicals that potentially could be developed as useful drugs for the treatment of hypertension and other chronic diseases. Problems of access to and high cost of conventional antihypertensive therapy, particularly by the rural dwellers in low income settings like Nigeria necessitated this research into the hypotensive activity and toxicological evaluation of methanolic extract of Syzygium guineense stem bark in experimental animals.

1.4 Theoretical Frame Work

Phytotherapy is a modern botanical medicine that applies scientific research and professional standards to the practice of herbal medicine. The methods used to evaluate herbal medicines are similar to those used by orthodox medicines. A herb may however, contain many active ingredients and may have several actions to support the body‟s health, unlike the single chemical in a drug. The initial evaluation of phytotherapeutic agents is acute toxicity studies which may reveal clinical manifestations elicited by the drug on investigation and provide viable information for determining median lethal dose (LD50) based on method described by Lorke (1983) with slight modifications. The results could also be used to identify the different dose levels for chronic or sub-chronic toxicity studies.

1.4.1 In-Vitro Studies

These are experiments that are conducted using component of an organism (tissue) that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Tissue organ bath are used to maintain the integrity of the tissue for several hours, in a temperature controlled environment while physiological measurements are performed. Experiments involve the addition of drugs to the organ-bath or direct/field stimulation of the tissue. Reaction of the tissue is by contraction or relaxation. Isolated tissue preparations are used to study the effects of drugs on specific type of receptors. They are also used for bioassay of drugs, characterisation of specific receptors or its subtypes to determine concentration response curve of an agonist, to study antagonism of drug in a new drug discovery. Other advantages of isolated tissue over intact animals are that several preparations can be tested on tissue obtained from single animal; relatively small amount of test materials is required and the drug effect is tested directly and its action as the drug is free from the factors of absorption, metabolism and excretion (Narkhede et al., 2012). Tissues can be sourced from various species (e.g. rabbit, guinea pig, rat, chick or toad). Some of the common tissues that are studied with organ bath system include: heart tissues, aortic rings, ileum, phrenic diaphragm and gastrenemius muscles (www.adinstruments.com).

1.4.2 In-Vivo Research

In vivo research is experimentation using a whole living organism. It is better suited for observing the overall effect of drugs and chemical agents on living organisms. Experiments are conducted in animal models to support clinical drug candidate selection. Clinical trial is also a form of in vivo research. Pharmacodynamic and pharmacokinetic properties of drugs in normal pathophysiological animal models are studied. Toxicology in vivo research includes acute, sub-chronic and chronic studies which are usually designed to meet international guidelines (OECD, FDA etc.).

1.4.3 Sub-chronic toxicity Study

In drug discovery research, the primary objective of toxicology studies is to evaluate the safety of potential drug candidates. The ultimate goal is to translate the animal responses into the under of the risk for human subject.

Subchronic toxicity studies are 12-26 weeks duration. The animals are dosed daily by the same route (oral) that the chemical substance would normally be administered to humans. They are then observed for any physical changes as well as changes in body weight or food consumption. At the end of the dosing regimen, the animals are euthanisized and their tissues evaluated for evidence of toxicity.

1.4.4 High-Performance liquid Chromatography

This is a chromatographic technique used to separate mixture of compounds with the purpose of identifying, quantifying and purifying the individual components of the mixture. Chemical separation can be accomplished by utilising the fact that certain compounds have different migration rates given a particular column and mobile phase. Compounds can be separated; the extent or degree of separation is determined by the choice of stationary phase and mobile phase. One method of separation would involve a column packing that is polar so that a solution of solute of differing polarity would have the solutes held on the column packing for differing periods of time, depending on the polarity of the solute. Thus less polar solutes would be eluted first and very polar solutes would be eluted last.

1.5 Objective of the study

The main objective is to determine the hypotensive activity and safety profile of methanolic stem bark extract of Syzygium guineense. Specific objectives include:

i. To evaluate the acute toxicity of the extract to detect behavioural signs of toxicity and determine the median lethal dose (LD50) following oral and intraperitoneal administration in rats.
ii. To carry out fractionation of the crude extract using Column Chromatography.

iii. To carry out phytochemical screening of the crude extract.

iv. To determine the effect of the extract and fractions on anaesthetized normotensive cats‟ blood pressure.

v. To determine the effect of the extract and fractions on the adrenergic and cholinergic receptors on isolated guinea pig atria.

vi. To determine the effect of the extract and fractions on isolated pefussed rabbit heart.

vii. To determine the effect of the extract and fractions on the adrenergic and cholinergic receptors on isolated rabbit ileum.

viii. To determine the effect of the extract and fractions on isolated non-pregnant rat uterus.

ix. To carry out sub-chronic (90 days) toxicity studies of the crude extract to determine its long term effects in rats.

1.6 Research Hypothesis

Syzygium guineense stem bark is safe and contains pharmacologically active chemical constituent(s) with hypotensive activity.