EVALUATION OF EFFECTS OF HEXANE AND METHANOL EXTRACTS OF Trigonella Faenum-Graecum L. ON SOME CARDIOVASCULAR PARAMETERS IN CATS AND RABBITS

Abstract:

Trigonella Foenum-gaecum L. contains a broad spectrum of therapeutic properties. It is used as galactagogue and antidiabetic among others in traditional medicine practice. Effects of methanol and hexane extracts of the seeds of Trigonella Foenum-gaecum L. on blood pressure was evaluated in normotensive male adult cats. Intravenously administered doses: (0.1 mg/ml, 1mg/ml, 10 mg/ml and 100 mg/ml) of the extracts to anaesthetized normotensive cats produced decrease in mean diastolic, and systolic and mean arterial pressures in a graded dose response manner but the decrease was significant as on the dose of 100mg/ml on diastolic blood pressure 14.50 ± 0.50 mmHg (hexane extract) and 13.50 ± 2.50 mmHg (methanol extract) while dose of Ach 10μg/ml produced a fall in mean diastolic, systolic, and means arterial pressure of 13.5 ± 2.50 mmHg, 22 ± 4.0 mmHg, and 16.3 ± 2.5 mmHg respectively. Atropine (25mg/ml) at all tolerable doses did not block the hypotensive effect of both extracts but rather potentiated the effect of the extracts. The methanol and hexane extracts of the seeds of Trigonella Foenum-gaecum L. decreases both the force and rate of myocardial contraction in a concentration dependent manner. The results obtained on Isolated Rabbit Prefused Heart Experiment shows that Methanol and Hexane extracts’ blood pressure and myocardial relaxation effects may be by the combined effects of myocardial depression, restorative function of the plant high vitamin c content or/ and muscarinic receptor mediated vascular smooth muscle relaxation. Results of the experiment indicated that the plant Trigonella Foenum-gaecum L. has blood lowering properties properties, and therefore, lend pharmaco-physiological credence to folkloric, ethnomedical use of the plant in management and/or control of high blood pressure in some rural communities. Keywords: Fenugreek, Trigonella Foenum-gaecum, blood pressure, myocardic contractility.

           CHAPTER ONE

1.0 Introduction

Cardiovascular diseases account for 17 million deaths annually worldwide and are known to be number one group of ‘killer disease’ (Ananya, 2014). Persistent high blood pressure is one of the leading causes of disability, mortality, and morbidity along the population through strokes and heart attacks It is the most common chronic illness among the world faces (Carretero and Oparil, 2001; Chobanian et al., 2003).

 

Cardiovascular disease is a class of diseases that involve the heart or blood vessels (arteries, capillaries, and veins) (Malton et al., 1993; WHO, 2014). Cardiovascular disease refers to any disease that affects the cardiovascular system, principally cardiac disease, vascular diseases of the brain and kidney, and peripheral arterial disease (Kelly and Valentin, 2010). The causes of cardiovascular disease are diverse but atherosclerosis and/or hypertension are the most common. Additionally, with aging come a number of physiological and morphological changes that alter cardiovascular function and lead to subsequently increased risk of cardiovascular disease, even in healthy asymptomatic individuals (Dantas et al., 2012).

 

Cardiovascular deaths and disease have increased at a fast rate in low- and middle-income countries (Mendis et al., 2011). Although cardiovascular disease usually affects older adults, the antecedents of cardiovascular disease, notably atherosclerosis, begin in early life, making primary prevention efforts necessary from childhood.

 

Free radicals produced at the end of metabolic processes: poly unsaturated fatty acids occur as a major part of low density lipoproteins in blood and oxidation of these LDL play a vital role in pathologies including atherosclerosis, coronary artery disease, myocardium infraction, heart failure, renal insufficiency, and stroke and dissecting aneurysm of aorta (Beevers  et al. 2001), with high level of oxidized lipids, blood vessels are damaged due to the reactions  and can lead to generation of foam cells  and plaques damaging endothelial cells and hardening of the affected vessels with High blood pressure as the most common end result (Esterbuer et al., 1991).

 

Antioxidants like B-carotene, vitamin C and Vitamin E play important role in restorative and protection of the vessels (Neuzil et al., 1997). An elevated arterial pressure is an important public health issue in developed countries. Although it is common, asymptomatic and readily detectable but it can often lead to lethal complication, if left untreated.

 

Evidence suggests a number of risk factors for heart disease: age, gender, high blood pressure, high serum cholesterol levels, tobacco smoking, excessive alcohol consumption, sugar consumption (Howard and Wylie-Rosett, 2002; Finks et al., 2012), family history, obesity, lack of physical activity, psychosocial factors, diabetes mellitus, air pollution (Kelly and Valentin, 2010) While the individual contribution of each risk factor varies between different communities or ethnic groups the consistency of the overall contribution of these risk factors to epidemiological studies is remarkably strong (Yusuf et al., 2004).

Some of these risk factors, such as age, gender or family history, are immutable; however, many important cardiovascular risk factors are modifiable by lifestyle change, drug treatment or social change.

 

Because of high incidence and morbidity, various drugs and regimes have been advocated for the control of persistent high blood pressure. Many new drugs have been introduced which may demonstrate better efficacy but possess side effects. Recently attention has been focused towards herbal and mineral preparations which are traditionally used as potential therapeutic agents in the prevention and management of cardiovascular diseases (Pierdomenico et al., 2009).

 

Arterial Blood Pressure is determined by two parameters, systolic and diastolic, which depend on whether the heart muscle is contracting (systole) or relaxing between beats (diastole) and equate to a maximum and minimum pressure each in the heart during cardiac cycle, respectively. Normal blood pressure at rest is within the range of 100-140 mmHg systolic (top reading) and 60-90 mmHg diastolic (bottom reading). High blood pressure is said to be present if it is persistently at or above 140/90 mmHg ( Chobanian et al., 2003).

 

Persistent high blood pressure is one of the risk factors for strokes, heart attacks, heart failure, and arterial aneurysm, and is a leading cause of chronic kidney failure (Pierdomenico et al., 2009). Moderate elevation of arterial BP leads to shortened life expectancy. Both dietary and lifestyle changes as well as medicines can improve BP control and decrease the risk of associated health complications.

 

Figure 1.1: Some Factors Leading to High Blood Pressure (Blausen, 2014)

 

Evidence for genetic influence on blood pressure is reported from various studies (Corvol et al., 1999).  There is greater similarity in blood pressure within families than between families, which indicates a form of inheritance (Feinleib et al., 1977). And it was proved that this finding wasn’t due to shared environmental factors (Biron et al., 1976). Single gene mutations are proved to cause Mendelian forms of high and low blood pressure. Almost 10 genes have been identified to cause these forms of hypertension (Lifton et al., 2001; Wilson et al., 2001).These mutations affect blood pressure by altering renal salt handling. Recently and with the aid of newly developed genetic analysis techniques researchers found statistically significant linkage of blood pressure to several chromosomal regions, including regions linked to familial combined hyperlipidaemia (Hsueh et al., 2000; Hunt et al., 2002).

Overall, however, identifiable single-gene causes of hypertension are uncommon, consistent with a multifactorial cause of essential hypertension (Niu et al., 1999; Luft, 2000).

 

The best studied monogenic cause of hypertension is the Liddle syndrome, a rare but clinically important disorder in which constitutive activation of the epithelial sodium channel predisposes to severe, treatment-resistant hypertension (Shimkets et al., 1994). Epithelial sodium channel activation resulting in inappropriate sodium retention at the renal collecting duct level. Patients with the Liddle syndrome typically present with volumedependent, low renin, and low aldosterone, and hypertension. Screenings of general hypertensive populations indicate that the Liddle syndrome is rare and does not contribute substantially to the development of hypertension in the general population (Melander et al., 1998).

 

Autonomic nervous system plays a central role in maintaining cardiovascular homeostasis via pressure, volume, and chemoreceptor signals. It does this by modifying peripheral vasculature and the function of the kidneys, which affect cardiac output, vascular resistance, and fluid retention. Problems with this system, such as excess activity of the sympathetic nervous system, increase blood pressure and contribute to hypertension (Esler, 2000; Takahashi, 2008). In addition, increased activity of the sympathetic accompanied by reduced activity of the parasympathetic has been associated with many metabolic and hemodynamic abnormalities that result in increased cardiovascular morbidity and mortality (Esler, 2000; Brook and Julius 2000). Arterial baroreceptors are reset to a higher pressure in hypertensive patients, and this peripheral resetting reverts to normal when arterial pressure is normalized (Xue et al., 2007; Lifton et al., 2001). Furthermore, there is central resetting of the aortic baroreflex in hypertensive patients, resulting in suppression of sympathetic inhibition after activation of aortic baroreceptor nerves. Additional small-molecule mediators that suppress baroreceptor activity and contribute to exaggerated sympathetic drive in hypertension include reactive oxygen species and endothelin (Chapleau et al., 1992), Some studies shown that hypertensive patients manifest greater vasoconstrictor responses to infused norepinephrine than normotensive controls (Ziegler et al., 1991).And that hypertensive patients do not show the normal response to increased circulating norepinephrine levels which generally induces down regulation of noradrenergic receptor, and its believed that this abnormal response is genetically inherited (Calhoun et al., 1993).

 

Exposure to stress increases sympathetic outflow, and repeated stress-induced vasoconstriction may result in vascular hypertrophy, leading to progressive increases in peripheral resistance and blood pressure (Carretero and Oparil, 2001).This could partly explain the greater incidence of hypertension in lower socioeconomic groups, since they must endure greater levels of stress associated with daily living. Persons with a family history of hypertension manifest augmented vasoconstrictor and sympathetic responses to laboratory stressors, such as cold pressor testing and mental stress that may predispose them to hypertension. This is particularly true of young African Americans. Exaggerated stress responses may contribute to the increased incidence of hypertension in this group (Fujino et al., 2004).

 

Another system maintaining the extracellular fluid volume, peripheral resistance and that if disturbed may lead to hypertension, is the renin-angiotensin-aldosterone system (Brenner and Rector, 2004; McConnaughey et al., 1999; Segura and Ruilope, 2007). The activity of local renin–angiotensin systems and alternative pathways of angiotensin II formation may make an important contribution to remodeling of resistance vessels and the development of target organ damage (i.e. left ventricular hypertrophy, congestive heart failure, atherosclerosis, stroke, end-stage renal disease, myocardial infarction, and arterial aneurysm) in hypertensive persons (Hasegawa and  Komuro, 2009; Saitoh, 2009).

 

The endothelium of blood vessels produces an extensive range of substances that influence blood flow and, in turn, is affected by changes in the blood and the pressure of blood flow. For example, local nitric oxide and endothelin, which are secreted by the endothelium, are the major regulators of vascular tone and blood pressure (O’Brien et al., 2007).

 

Evidence suggests that oxidant stress alters many functions of the endothelium, including modulation of vasomotor tone. Inactivation of nitric oxide (NO) by superoxide and other reactive oxygen species (ROS) seems to occur in conditions such as hypertension (Nakazono et al., 1991; Laursen et al., 1997). Normally nitric oxide is an important regulator and mediator of numerous processes in the nervous, immune and cardiovascular systems, including smooth muscle relaxation thus resulting in vasodilation of the artery and increasing blood flow, suppressor of migration and proliferation of vascular smooth-muscle cells. It has been suggested that angiotensin II enhances formation of the oxidant superoxide at concentrations that affect blood pressure minimally (Cai and Harrison 2000).

Endothelin is a potent vasoactive peptide produced by endothelial cells that has both vasoconstrictor and vasodilator properties. Circulating endothelin levels are increased in some hypertensive patients, particularly African Americans and persons with hypertension (Ergul  et al., 1996; Campia et al., 2004).

 

Persistent high blood pressure puts a strain on the heart, leading to hypertensive heart disease and coronary artery disease if untreated. It is also a major risk factor for stroke, aneurysms of the arteries (e.g. aortic aneurysm), and peripheral arterial disease and is a cause of chronic kidney disease. Even moderate elevation of arterial blood pressure is associated with a shortened life expectancy. Dietary and lifestyle changes can improve blood pressure control and decrease the risk of associated health complications, although drug treatment is often necessary in people for whom lifestyle changes are not enough or not effective. A proportion of people with high blood pressure report headaches (particularly at the back of the head and in the morning), as well as light headedness, vertigo, tinnitus (buzzing or hissing in the ears), altered vision or fainting episodes (Diao et al., 2012). These symptoms, however, might be related to associated anxiety rather than the high blood pressure itself (Arguedas et al., 2009).

 

On physical examination, hypertension may be suspected on the basis of the presence of hypertensive retinopathy detected by examination of the optic fundus found in the back of the eye using ophthalmoscopy. Classically, the severity of the hypertensive retinopathy changes is graded from grade I–IV, although the milder types may be difficult to distinguish from each other (Fisher and Williams, 2005). Ophthalmoscopy findings may also give some indication as to how long a person has been hypertensive (Diao et al., 2012).

 

Figure 1.2: Complications of persistent high blood pressure (Haggstrom, 2014)                          

1.1     Trigonella Foenum-graecum L.

Trigonella foenum-graecum L. (also known as Fenugreek in English, methi in India, Erun in Yoruba and Kimba in Hausa) is an annual plant in the family Fabaceae. The plant has small round leaves, is cultivated worldwide as a semi-arid crop, and is a common ingredient in dishes from the Indian Subcontinent. Major fenugreek-producing countries are India,

Iran, Nepal, Bangladesh, Pakistan, Argentina, Egypt, France, Spain, Turkey, Morocco and China (Parthasarathy et al., 2008). The largest producer of fenugreek in the world is India, fenugreek grows an average height of two feet. The leaves consist of three small obovate to oblong leaflets; leaves and seeds mature in long pods, are used to prepare extracts or powders for medicinal use (Deepika et al., 2014). Fenugreek has three culinary uses: as a herb (dried or fresh leaves), as a spice (seeds), and as a vegetable (fresh leaves, sprouts, and microgreens). Sotolon is the chemical responsible for fenugreek’s distinctive sweet smell. Fenugreek seeds are used both whole and in powdered form and are often roasted to reduce their bitterness and enhance their flavor (Sharma et al., 1990).

 

Figure 1.3: Fenugreek- leaves and seeds (Anitha, 2013)

 

Fenugreek has been used for its medicinal properties for thousands of years. The earliest recorded use of fenugreek dates back to Egypt in 1500 B.C., according to the National

Center for Complementary and Alternative Medicine (NCAM). The Food and Drug Administration has not approved the use of fenugreek for medicinal purposes, but some doctors and pharmacists recommend it for certain medical conditions. Fenugreek is often used as a food flavoring  (Jill, 2014); They have a strong aroma and a bitter taste. But when used in small quantities they impart flavour to food (Anitha, 2013).

 

The chemical constituents of fenugreek include: Alkaloides such as trimethylamine, neurin, trigonelline, choline, gentianine, carpaine and betain. Amino acids including isoleucine, 4Hydroxyisoleucine, histidine, leucine, lysine, L-tryptophan, argenine. Saponins such as graecunins, fenugrin B, fenugreekine (Colin, 2013; Deepika et al., 2014), trigofoenosides A-G. Steroidal sapinogens : yamogenin, diosgenin, smilagenin, sarsasapogenin, tigogenin, neotigogenin,gitogenin, neogitogenin, yuccagenin, saponaretin. Flavonoids:

quercetin,rutin,vetixin isovetixin. Fibers: gum, neutral detergent. Fibres. Other  constituent include  coumarin, lipids, vitamins, minerals. 28% mucilage; 22 % proteins; 5 % of a stronger-swelling, bitter fixed oil (Oncina et al., 2000).

 

The phytochemistry of Fenugreek reveals: a. stem: Fenugreek contains a number of steroidal sapogenins. The diosgenin were found in the oily embryo. Two furastanol glycosides, F-ring opened precursors of diosgenin have been reported, as also hederagin glycosides. The alkaloid trigonelline, trigocoumarin, trimethyl coumarin and nicotinic acid are present in stem (Shailendra and Garima 2014). Mucilage is a prominent constituent of the seeds. About 28 % mucilage; 5 % of a stronger-smelling, bitter fixed oil, 22 % proteins; a volatile oil; two alkaloids, Trigonelline and Choline, and a yellow colouring substanceare present in stem (Helambe and Dand, 2003).  b. leaf: The leaves contain 7 saponins, known as graecunins. These compounds are glycosides of diosgenin. Leaves contain moisture

86.1%, protein 4.4%, fat 0.9%, minerals 1.5%, fiber 1.1%, and carbohydrates 6%. The mineral and vitamins contents are calcium, iron, phosphorous, carotene, thiamine, riboflavin, niacin and vitamin C (Mehrafarin et al., 2011).  c. seed: Fenugreek Seeds are aromatic, bitter, carminative, galactogouge, antibacterial and may be eaten raw or cooked. Bulk of the seed is dietary fiber (50%) and protein (30%) both of which have no taste or flavor. The chemical components of fenugreek seeds include a large carbohydrate fraction (mucilaginous fiber, galactomannan); 20-30% proteins high in tryptophan and lysine; pyridine-type alkaloids; flavonoids; free amino acids (4-hydroxyisoleucine, arginine, lysine, histidine); saponins; glycosides; vitamins, minerals, (28%) mucilage, (22 %) proteins, 5 % of a stronger-smelling, bitter fixed oil. volatile oils. Bitterness is mainly due to the oil, steroidal saponins and alkaloids. Historically used as a culinary and medicinal herb, recent research studies have shown its effectiveness in reducing blood glucose levels, promoting lean body mass, lowering cholesterol, and treating gastrointestinal disorders. Studies has shown the role of Fenugreek seeds in adjustment of some enzymes, including those related to glucose and lipid metabolism. Preliminary research with type-1 diabetics suggest that fenugreek may aid insulin secretion and may reduce total cholesterol and LDL cholesterol levels (Vats et al., 2002 and Zia et al., 2001).

 

Seeds of Fenugreek contain 0.1% to 0.9% diosgenin and are extracted on a commercial basis. Plant tissue cultures from seeds grown under optimal conditions have been found to produce as much as 2% diosgenin with smaller amounts of gitongenin and trigogenin. The seeds also contain the saponin fenugrin B. Several coumarin compounds have been identified in fenugreek seeds as well as a number of alkaloids (eg, trigonelline, gentianine, carpaine) (Ragni et al, 2016). A large proportion of the trigonelline is degraded to nicotinic acid and related pyridines during roasting. These degradation products are, in part, responsible for the flavor of the seed. The seeds also yield as much as 8% of a fixed, foulsmelling oil. Three minor steroidal sapogenins also have been found in the seeds: smilagenin, sarsapogenin, and yuccagenin  (Oncina et al., 2000).

 

Fenugreek seeds contain moisture 13.7%, protein 26.2%, fat5.8%, minerals (calcium, carotene, thiamine, niacin and riboflavin) 3.0%, fiber 7.2% and carbonhydrates 44.1% per 100g. Fenugreek leaves contain moisture 86.1%, protein 4.4%, fat 0.9%, minerals(calcium, carotene, thiamine, niacin and riboflavin phosphorous vitaminC) 1.5%, fiber 1.1%, and carbonhydrates 6.0 per 100g (Mullaicharam et al., 2013). It also contains lecithin, choline, minerals, B. Complex, iron, Phosphates, PABA (Para-Amino Benzoic Acid) and vitamins A, D and a rich source of vitamin K (Anwesha, 2011).

 

1.2     Statement of the Research Problem

By 2030, more than 23million people may die annually of CVD worldwide. The major causes of CVD are tobacco use, physiocal inactivity, an unhealthy diet and harmful use of alcohol (WHO, 2014). Of WHO’s six regions, Africa has the highest prevelence of CVD particularly High blood pressure of extimated 46% of adults aged ≥25 The problem caused by high blood pressure worsen on those who are unaware of the risk or cannot afford checkups (WHO, 2011).

Although oxidation reaction is crucial to life, it produces free radicals that damage cells and body function in general. The systems of complex and multiple types of antioxidants, such as glutathione, vitamin C, vitamin A, vitamin E as well as enzymes like catalase, peroxidases and superoxide dismutase, are widely used in dietary supplements and have been investigated for prevention of diseases like cancer, coronary heart diseases and neurodegenerative diseases (Jha et al., 1995).

In the last three decades, a lot of concerted efforts have been channeled into researching into local plants with hypotensive or antihypertensive therapeutic values. The hypotensive or antihypertensive effects of some of these medicinal plants have been validated and others disproved. Attempts by the low-income group, particularly the rural dwellers in the developing countries, to control high blood pressure and its attendant complications in the face of the scarce socioeconomic resources, have led more people opting for herbal remedy. However, more scientific research needs to be done to verify the effectiveness and elucidate the safety profile of such herbal remedies (Farnworth et al., 2001).                                        

 1.3     Justification of the Study

People in low and middle-income countries who suffer from CVDs and other no communicable diseases have less access to effective and equitable health care service which responds to their needs. As a result, many people in low and middle-income countries are detected late in the course of the disease and die younger from CVDs and other non communicable diseases, often in their most productive years (WHO, 2014).

Attempts by the low-income group, particularly the rural dwellers in the developing countries, to control high blood pressure and its attendant complications in the face of the scarce socioeconomic resources, have led more people opting for herbal remedy (WHO, 2011).

Fenugreek is known to contain many medicinal properties. The seed extract displayed a positive effect as positive cardiotonic, hypoglycaemic, diuretic, antiphlogistic and hypotensive agents (Duke and Ayensu, 1985). Moreover, a more recent research suggests that Trigonellafoenum-graecum L. has antioxidant, antihypertensive, anticancer, antithromolic effect (Doshi et al., 2012). There different ways of preventing and treating

CVDs, beside drug therapy and life style changing, current study’s interest focus on plant based natural drug treatment exploiting the said properties of Fenugreek.

Also, the chemical constituents of fenugreek include: B-carotene, vitamin C, Vitamin E and Vitamin A; all of which are potent antioxidants and are expected to preserve endothelial function via free radical scavenging activity among others (Muhammad et al., 2013).

Therefore with the dearth of information suggests that Trigonella foenum-graecum L. would have not only Blood Pressure lowering effect but also possesses preventive and restorative effect on cardiovascular system.

 

1.4     Aim and Objectives

1.4.1 Aim

This research work was undertaken to determine effects of Hexane and Methanol extracts of Trigonella foenum graecum L. on blood pressure and cardiac contractility.

 

1.4.2 Objectives

1. To determine acute toxicity level of Hexane and Methanol extracts of Trigonella foenum graecum L.
2. To determine effects of Hexane and Methanol extracts of Trigonella foenum graecum on cat blood pressure.

• To determine effects of Hexane and Methanol extracts of Trigonella foenum graecum L. on cardiac contractility of rabbit.

                                               

1.5     Research Hypothesis

Hexane and Methanol extracts of Trigonella foenum graecum L. have no effect on cardiovascular system.

 

EVALUATION OF EFFECTS OF HEXANE AND METHANOL EXTRACTS OF Trigonella Faenum-Graecum L. ON SOME CARDIOVASCULAR PARAMETERS IN CATS AND RABBITS

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