Memoire Online - Evaluation of the hypoglycemic, hypolipidemic and anti alpha amylase effects of extracts of the twigs and fruits of ficus ovata vahl (moraceae)

To my late parents, Mama FOUONDO MOMO Antoinette and Dr FOUONDO COCKNEY. Thanks for always standing by me. It is because of your conviction that I got where I am today, I will never forget you.

To my lovely husband DOUNTSOP KENNE Bruno and kids KENNE FOUONDO Rolland Martin, KENNE MOMO James Steve and KENNE KENNE Israel Nathan

ACKNOWLEDGEMENT

A research document like this could not have been realized without the assistance of certain people to whom I testify my sincere gratitude.

I wish to acknowledge the assistance of my supervisor Prof OBEN ENYONG Julius, who inspite of his numerous responsibilities, corrected and guided me throughout this project. Accept my sincere gratitude.

I wish to extend my immense appreciation and thanks to my director Dr FOKUNANG Charles, who designed my research topic and closely followed up this work to its completion. His critical review has contributed to the output of my write up.

Many thanks to Pr ETOA François Xavier, Head of Department of Biochemistry and to all the lecturers of the Department of Biochemistry, University of Yaounde I, who provided suitable lectures necessary for my training.

My profound gratitude and appreciation also goes to Dr NGONDI Judith Laure for her technical and moral support for the success of this work.

Great appreciation also goes to Dr NGAMENI, Mr. NONO BORGIA, Mr. NANA Frederick and all the elders and colleagues of the Laboratories of Natural Product-Phytochemistry and Toxicology (FMBS) who assisted in the harvest, extraction and phytochemical screening of Ficus ovata vahl.

My profound gratitude also goes to Miss NYANGONO Christine for all her devotion, who welcome me in the field of research and took me through the laboratory set up.

Many thanks to Pr FOKOU Elie , Pr. MINKA Samuel, Mrs MBONG Angie and to all the members of the Laboratory of Food Science and Metabolism especially my close colleagues: BEYEGUE Eric, ESSOLA Nadine, ESSOUMAN Florine, NGUIMKENG SIGNING Boris, MAPTOUOM Laure, MECHUM Pamela, NGO NLEND Marguerite, NGUELE Raymond, NKOUGNI Jacob, for their advice and encouragement.

To my friends of the Masters II promotion for their criticisms and social interaction.

I wish to extend my love and gratitude to my brothers and sisters respectively; NDE Robertson, TEITA Daniel, TCHOUPOU Marie-Sollange, POUFONG Daniel, LONSTIE Alex, MAFFO Lumiere and my aunt TOUKEM Bertine for their multiple support and tons of prayer for my success.

I wish to appreciate my family-in-law especially my mother-in-law Mrs. KENNE SONNA Julienne, brothers and sisters in-law, especially, Mr. MANE Olivier and miss KENNE Laure for their support and encouragement.

To my family friends Mr. DUCOS Rogers, Mr. KAMDEM Olivier, Mr. YMELE Allen, Mr. YAMO Gil, Mr. NDONGBOU Daniel, Mrs. PEKAKOUMCHE Mariama, LAMYA Glory, FOMEKONG Caroline, ASONGANI Adeline, Joel KAMDEM, NCHE Eleanor for their advice and friendship.

For those that are not cited here expressis verbis kindly accept my sincere gratitude.

TABLE OF CONTENT

ABREVIATION

LIST OF FIGURES

LIST OF TABLES

ABSTRACT

The phytochemical components of many medicinal plants have most often been linked to the modulation of biomarkers associated to diabetes type 2. This study was aimed at evaluating the anti á-amylase, antihyperglycemic, hypoglycemic and antihyperlipidemic activities of twigs and fruits extracts of Ficus ovata.

Hydroethanolic and ethanolic extracts of twigs and fruits of F. ovata were prepared and used for the phytochemical analysis and antioxidant potential screening in vitro. The most active extracts were selected for the evaluation of in vitro antiamylase activity, acute toxicity study as well as their effects on fasting (hypoglycemic test) and postprandial (antihyperglycemic test) blood glucose levels. In addition, the preventive effects of the extracts against some biomarkers of diabetes (body weight, fasting blood glucose, lipid profile, endothelia dysfunction, hepatic and renal toxicities) were evaluated at dose of 300mg/kg of body weight in rats fed on high fructose-high cholesterol diet for 14 days.

Phytochemical screening revealed the presence of alkaloids, tannins, saponins, glycosides, phenols and flavanoids in all extracts except phlobatannin that was absent in the fruit extracts. Fruits extracts had the highest polyphenol content (EF= 718.142 #177; 12.910 mg CatEq vs HF= 486.876 #177; 8.606 mg CatEq; P< 0.05) and the best DPPH antiradical scavenging effect (IC₅₀; EF= 2.7mg/ml, HF=0.70mg/ml) compared to twigs (P< 0.05). Hydroethanolic twigs and fruits (FOHT and FOHF) extracts selected were the most active for each plant part. FOHF had significantly high (p=0.05) antiamylase effects as compared to FOHT and both FOHT and FOHF were weakly toxic since no dead was recorded at LD50>5000mg/Kg of BW. In addition, both extracts had hypoglycemic and antihyperglycemic effects (percentage decrease 8.908% vs 5.747% and 21.566% vs 8.208% respectively) with FOHT being more active than FOHF(p=0.05). Finally, the preventive study results show that, co-administration of extracts especially FOHF was observed to significantly reduce fasting blood glucose (p<0.05), Triglyceride (p<0.05), Total & LDL-Cholesterol (p<0.05), creatinine and total protein levels, and significantly increase HDL-Cholesterol (p<0.05) and Nitric oxide (plasma; P< 0.05) levels. Thus the extracts enable us to maintain or ameliorate these changes to nearly normal levels and reveal its preventive effects.

These results suggest that FOHF and FOHT could be of interest in the prevention of hyperlipidemia and hyperglycemia associated with type 2 diabetes .

Key words: antihyperglycemia, antihyperlipidemia, diabetes, Ficus ovata, hypoglycemia.

RESUME

Les composés bioactifs de plusieurs plantes médicinales sont de plus en plus considérés comme des facteurs de choix dans la stratégie de prévention du diabète de type 2. Le but de cette étude était donc d'évaluer les activités antihyperglycémiante, hypoglycémiante et antihyperlipidémiante des extraits de fruits et de tiges de Ficus ovata.

Les extraits éthanoliques et hydroethanoliques des fruits et tiges de F. ovata ont été préparés et les screenings phytochimiques et antioxydant in vitro évalués. A l'issue des tests précédents, les extraits les plus actifs ont été sélectionnés pour la suite des expériences à savoir ; l'activité antiamylasique in vitro, la toxicité aiguë, les tests aigus d'évaluation des activités hypoglycémiantes et antihyperglycémiantes. Enfin, l'effet préventif des extraits sélectionnés sur quelques biomarqueurs du diabète de type 2 (le poids corporel, la glycémie a jeun, les marqueurs du profil lipidique, de la dysfonction endothéliale, toxicité hépatique et toxicité rénale) a été évalué à la dose 300mg/kg de poids corporel chez des rats nourris à base d'une alimentation enrichie en fructose et graisses pendant 14 jours.

Le criblage phytochimique a révélé la présence des alcaloïdes, tannins, saponines, glycosides, phénols et flavonoïdes à l'excepté phlobatannin qui était absent dans les fruits. La teneur en polyphénols (FOEF=718.142 #177; 12.910 mg EqCat vs FOHF= 486.876 #177; 8.606 mg EqCat; P< 0.05) et l'effect inhibitrice du radical DPPH des extraits de fruits (IC₅₀; FOEF= 2.7mg/ml, FOHF=0.70mg/ml) était significativement plus élevée comparativement aux extraits de tiges. Les extraits hydroéthanoliques des fruits et des tiges (FOHF et FOHT) étaient les plus actives pour chaque partie du plant. FOHF avait une activité antiamylasique significativement élevés (p=0,05) par rapport à l'extrait FOHT et ces deux extraits étaient faiblement toxiques car aucun décès n'a été enregistré à la dose administrée (LD50= 5000mg/kg PC). En outre, bien que ces deux extraits aient présenté un effet hypoglycémiant et antihyperglycémiant (pourcentage de baisse 8.908% vs 5.747% et 21.566% vs 8.208% respectivement), l'extrait FOHT était plus efficace (p=0.05). Par ailleurs, les résultats de l'étude préventive ont montré que l'administration concomitante des traitements, particulièrement FOHF a entrainé une diminution significative (P< 0,05) de la glycémie, le taux de triglycerides, cholesterol total, cholestérol LDL, créatinine et protéine total (P< 0,05), et augmentais le taux de cholestérol HDL (P<0,05) et Oxyde Nitrique (plasma; P< 0.05) donc révèle les effets préventives.

Ces résultats suggèrent que les extraits hydroéthanoliques de tiges et fruits de F. ovata pourraient prévenir l'hyperglycémie et l'hyperlipidémie associées au diabète de type 2.

Mot clés: antihyperglycémiant, antihyperlipidemiant, diabète, Ficus ovata, hypoglycémiant.

GENERAL INTRODUCTION

INTRODUCTION

Diabetes mellitus is one of the most common non communicable diseases, and its epidemic proportion has placed it at the forefront of public health challenges currently facing the world (Craig et al., 2009). The increasing prevalence of diabetes mellitus, the emergence of diabetes complications as a cause of early morbidity and mortality, and the enormous and mounting burden on health care systems make diabetes a priority health concern (Craig et al., 2009).

The world prevalence of diabetes among adults (aged 20-79 years) was estimated to rise from 6.4%, affecting 285 million adults, in 2010, to 7.7%, affecting 439 million adults by 2030. Between 2010 and 2030, there was to be a 69% increase in numbers of adults with diabetes in developing countries and 20% increase in developed countries (Shaw et al., 2010). In Cameroon, recent estimations situate the prevalence rate at 4.3%, with an increased prevision of 4.7 % by the year 2025 (Shaw et al., 2010)

This epidemic has been attributed to high fat and high sugar intakes in modern diets, correlating with the increased use of fructose as a sweetener including lack of physical activity and sedentary life style (Jatin et al., 2011). Diabetes can be managed by exercise and diet which in case of failure, pharmaceutical drugs such as insulin, insulin secretagogues, insulin sensitizers and á-glucosidase can be use. These drugs are either too expensive or have undesirable sides effects or contraindications. The search for more effective and safer hypoglycemic agents therefore has continued to be an area of research of interest (Krishna et al., 2004). Alternative strategies to the current modern pharmacotherapy of diabetes mellitus are urgently needed, because of the inability of existing modern therapies to control all the pathological aspects of the disorder, as well as the enormous cost and poor availability of the modern therapies for many rural populations in developing countries (Krishna et al., 2004). The World Health Organization (WHO) has recommended and encouraged the use of alternative therapy especially in countries where access to the conventional treatment of diabetes is not adequate (Claudia et al., 2006).

Plants used in traditional medicine to treat diabetes mellitus represent a valuable alternative for the control of this disease (Paul et al., 2006). The use of medicinal plant is quasi-general throughout the continent; however some of the plants reputed in the indigenous system of medicine are not scientifically established for their activities (Kuete et al., 2009). In this context, a number of medicinal plants and herbs have been studied and validated for their hypoglycaemic potential using experimental animal models of diabetes as well clinical studies involving diabetic patients.The plants used include the members of the Moraceae family and within this family, the genus Ficus is well documented for its biological activities such as hypoglycemia and antihyperglycemia (Vivek et al., 2010), antidiabetes (Mohana et al., 2010), antioxidant and antimicrobial (Al-Fatimi et al., 2007 ; Changwei et al ., 2008), anticancer ( Al-Fatimi et al., 2007), antidiarhoeal, antiplasmodial, anti-pyretic, antiulcer, gastroprotective (Rao et al., 2008), etc. Ficus ovata, another plant of the Ficus specie found in the savanna woodland, forest edges, river side forest and secondary forest, up to an altitude of 2100 m is distributed in the subtropical Africa. Ficus species is known as elephant tree and Punjab in English (Tchinda, 2010). Ficus ovata is use widely for street ornament in Dakar (Kuete et al., 2009). Traditionally the decoction of the stem bark and leaves of this plant is used for the treatment of infectious diseases, gastrointestinal infections, diarrhea, anti-poison and as lactation stimulant (Kuete et al., 2009).

PROBLEMATIC AND HYPOTHESIS

Formulation of problem

Even though there is no specific cure for diabetes mellitus, there exist ways of reducing the blood sugar level and to prevent long-term complications such as stroke and cardio vascular diseases (CVD). Numerous curative effects of F. ovata have been discovered, but no scientific investigation was focused on the antidiabetic activity. This work is therefore orientated towards the research of the ability of F. ovata to inhibit reaction favoring the digestion, absorption of glucose and fat and its presence in blood leading to the hypoglycemic and hypolipidemic in laboratory rats.

Study interest

· To recognize and valorize resources that our African environment has offered to us;

· To arouse a true complementary collaboration between biochemist, microbiologist, phytochemist, pharmacists and traditional pharmacopoeia, so that it can be beneficial to the society.

Hypothesis

From the previous study of the antidiabetic activities of other plants such as Tournefortia hartwegiana, compound isolation enables the identification of some metabolites with their anti-diabetic activities. Other studies have identified some of these metabolites in our plant of interest such that F. ovata extract, being our dependent variable and antidiabetic activity, our independent variable, our hypothesis is that Ficus ovata has hypoglycemic and hypolipidemic effects on rats.

OBJECTIVES

General objective

This study is aimed at evaluating the antihyperglycemic, hypoglycemic antihyperlipidemic and in vitro anti á- amylase effects of the twigs and fruits extracts of F. ovata

Specific objectives

· To conduct Phytochemical and antioxidant potential screenings of ethanolic and hydroethanolic extracts of F. ovata in order to select the most active extracts.

· To evaluate the antiamylase, hypoglycemic and antihyperglycemic activities of the most active extracts

· To assess the preventive effects of the selected extracts on some biomarkers of diabetes (hyperglycemia, hyperlipidemia and endothelial dysfunction) on rats fed on high fructose-high cholesterol diet.

CHAPTER I. LITERATURE REVIEW

I.1.Generalities on diabetes mellitus

I.1.1. Glucose metabolism

I.1.1.1. Digestion and absorption of carbohydrates

Dietary polysaccharides are hydrolysed in the gastrointestinal tract by the enzyme alpha amylase to produce oligosaccharides and disaccharides. The resulting disaccharides are further hydrolysed by alpha glucosidase enzymes to produce glucose and other monosaccharides as shown below

Glucose and other monosaccharides (fructose and galactose) resulting from digestion of carbohydrates are absorbed through the small intestine into the hepatic portal vein. This results in elevation of the postprandial blood glucose level (Hannan et al., 2007).

I.1.1.2. Role of the pancreas in glucose metabolism

The pancreas plays a primary role in the metabolism of glucose by secreting the hormones, insulin and glucagon (Figure 1.). Elevated postprandial blood glucose level stimulates pancreatic beta cells to secrete insulin which then facilitates the entry of glucose into the muscle and adipose tissues, thereby clearing excess glucose from the circulation. Insulin also stimulates the processes of glycolysis (catabolism of glucose) and glycogenesis (synthesis of glycogen from glucose) and inhibits both hepatic gluconeogenesis and glycogenolysis thereby reducing the hepatic glucose output (kimber et al., 2006). The actions of insulin are opposed by glucagon, a hormone produced by the pancreatic alpha cells when the blood glucose level tends to be low. Glucagon inhibits glycogenesis and stimulates both gluconeogenesis and glycogenolysis which releases blood glucose into the blood circulation thereby raising the blood glucose level (kimber et al., 2006).

Figure 1: The role of the pancreas in glucose homeostasis (Cheng and Fantus, 2005)

I.1.1.3. Metabolic actions of insulin

Metabolic actions of insulin result from its interaction with the insulin receptor (IR) found in all insulin responsive target cells (liver, muscle and adipose tissue). Insulin binds to the alpha-subunit of IR and activates the intrinsic tyrosine kinase activity of the beta-subunit of the receptor. Activated IR results in the subsequent phosphorylation of intracellular substrates including insulin receptor substrates, phosphatidylinositol (PI) 3-kinase, and protein kinase B (PKB). Normal insulin action leads to increased glycogen synthesis, glucose transport, and lipogenesis, and decreased gluconeogenesis, glycogenolysis, and lipolysis (Cheng and Fantus, 2005).

I.1.2. Prevalence of diabetes mellitus

Chronic non transmissible diseases are diseases that have evolved for many years and they require long term management. These diseases include diabetes, cardiovascular diseases, high blood pressure and cancer. They are the direct consequences of our daily behavioral activities such as lack of physical activities, obesity, malnutrition, cigarette smoking and alcoholism (Craig et al., 2009). Diabetes mellitus an example of such disease whose prevalence among adults (aged 20-79 years) was estimated to rise from 6.4%, affecting 285 million adults, in 2010, to 7.7%, affecting 439 million adults by 2030. Between 2010 and 2030, there was to be a 69% increase in numbers of adults with diabetes in developing countries and a 20% increase in developed countries (Shaw et al., 2010). In Cameroon, recent estimations situated the prevalence rate at 4,3%, with an increased prevision of 4,7% by the year 2025 (Shaw et al., 2010). This epidemic has been attributed to high fat/ high sugar intake in modern diet including sedentary lifestyle and lack of physical activity (Jatin et al., 2011).

I.1.3. Definition, classification of diabetes mellitus and other categories of glucose regulation

Diabetes mellitus is a group of metabolic diseases of multiple etiology characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (Craig et al., 2009).The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (Craig et al., 2009).

Assigning a type of diabetes to an individual often depends on the circumstances present at the time of diagnosis, and many diabetic individuals do not easily fit into a single class. Thus, for the clinician and patient, it is less important to label the particular type of diabetes than it is to understand the pathogenesis of the hyperglycemia and to treat it effectively (ADA, 2009, Rasilainen et al., 2004).

Stages (WHO, 1999) Types	Normoglycemia	Hyperglycemia
	Normal glucose tolerance	Impaired Glucose regulation IGT and/or IFG	Diabetes mellitus
			Not insulin requiring	Insulin requiring for control	Insulin requiring for survival
Type 1 · Autoimmune · Idiopathy Type 2 · Predominantly insulin resistance · Predominantly insulin secretary defects Other specific types
Gestational diabetes

I.1.4. Signs and symptoms

The classical symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger) weight loss, and blurred vision. Impairment of growth and susceptibility to certain infections may also accompany chronic hyperglycemia (Cooke et al., 2008). Symptoms may develop rapidly (weeks or months) in type 1 diabetes while in type 2 diabetes they usually develop much more slowly and may be subtle or absent. Type 1 should always be suspected in cases of rapid vision change, whereas with type 2 changes are generally more gradual, but should still be suspected (Cooke et al., 2008).

I.1.5. Diagnostic criteria for diabetes mellitus

Three ways to diagnose diabetes are possible, and each, in the absence of unequivocal hyperglycemia, must be confirmed, on a subsequent day, by any one of the three methods.

1. Symptoms of diabetes plus casual plasma glucose concentration =11.1 mmol/l (200 mg/dl).Casual are defined as any time of day without regard to time since last meal.

The test should be performed as described by WHO, 1999, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water or 1.75 g/kg of body weight to a maximum of 75 g (Rasilainen et al., 2004).

Corresponding values (mmol/L) are =10.0 for venous whole blood =11.1 for capillary whole blood and =6.3 for both venous and capillary whole blood.

I.1.6. Etiological of disorders of glycemia

Etiological types designate defects, disorders or processes that often result in diabetes

I.1.6.1. Type 1 diabetes

Type 1 indicates the processes of ß-cell destruction that may ultimately lead to diabetes mellitus in which insulin is required for survival to prevent the development of ketoacidosis, coma and death. Type 1 is usually characterized by the presence of anti-glutamic acid decarboxylase (anti-GAD) antibodies, islet cell or insulin antibodies which identify the autoimmune processes that lead to ß-cell destruction. Consequently, the pancreas secretes little or no insulin (Thunander et al., 2008). Most cases are primarily due to T-cell mediated pancreatic islet â-cell destruction, which occurs at a variable rate, and becomes clinically symptomatic when approximately 90% of pancreatic beta cells are destroyed (Craig et al., 2009). When the clinical presentation is typical of type 1 diabetes but antibodies are absent, then the diabetes is classified as Type 1B (idiopathic). Most idiopathic cases are of African or Asian ancestry; however other forms of diabetes should also be considered (Dunger et al., 2004).

I.1.6.2. Type 2 diabetes mellitus

This form of diabetes, which accounts for 90-95% of those with diabetes, previously referred to as non-insulin-dependent diabetes, type II diabetes, or adult-onset diabetes, encompasses individuals who have insulin resistance and usually have relative (rather than absolute) insulin deficiency (Harris et al., 2003). Patients with type 2 diabetes generally are older, although there is an alarming increase in the incidence of type 2 diabetes in children and adolescents." Patients with type 2 diabetes have insulin resistance syndrome (e.g., central obesity, hypertension, hyperlipidemia) for many years (Harris et al., 2003). There are three major pathophysiological abnormalities in patients with type 2 diabetes, that include early loss of first-phase insulin production associated with defective beta cell secretion, peripheral resistance to insulin action primarily in muscle tissue and the liver, and excessive hepatic glucose production as disease progresses. Normally, first-phase insulin secretion exerts an inhibitory effect on hepatic glucose production and output. When a patient has beta cell defects, first-phase insulin secretion is impaired and eventually lost, which results in fasting hyperglycemia (Hadi et al., 2007).The body's attempt to moderate blood glucose levels results in enhanced second-phase insulin secretion, and hyperinsulinism occurs. Beta cells may secrete high levels of insulin to normalize blood glucose levels and successfully maintain normoglycemia for many years. Gradually, however, the beta cells may begin to falter, and insulin secretion decreases. As hepatic glucose production increases, both fasting and postprandial glucose levels become elevated (Hadi et al., 2007).

Insulin resistance implies that the body's cells (insulin receptors) are less sensitive to the action of insulin. Insulin resistance, defined as the decreased ability of insulin to promote glucose uptake in skeletal muscle and adipose tissue and to suppress hepatic glucose output, may be present for many years before the development of any abnormality in plasma glucose levels (Haffner, 2003). Consequently, blood glucose levels rise, even though the beta cells produce more insulin. In patients with insulin resistance, however, hyperinsulinemia does not suppress gluconeogenesis, and chronic hyperglycemia develops. Insulin sensitivity can decline by at least 70% before fasting plasma glucose concentrations become abnormal, and it may take up to 20 years to reach that point (Haffner, 2003). Experts are not certain yet about the mechanism underlying insulin resistance, but they know that obesity, particularly central obesity, increases insulin resistance. They further speculate that defects in intracellular signalling prevent glucose from entering cells (Mukhyaprana et al., 2004).

- Radethnicity (e.g., African American, Hispanic, Native American, Asian American, Pacific Islander) Age 45 years

- High-density lipoprotein cholesterol level I < 35 mg per dL (0.9 mmol per L) or a triglyceride level = 250 mg per dL (2.83 mmol per L)

- History of gestational diabetes mellitus or delivery of babies above 4.032 g (Mukhyaprana et al., 2004).

I.1.6.3. Other specific types

Other specific types are currently less common causes of diabetes mellitus, but are conditions in which the underlying defect or disease process can be identified in a relative specific manner (WHO, 1999). They include:

- Diseases of the exocrine pancreas, such as cancer of the pancreas, cystic fibrosis and fibrocalculous pancreatopathy (a form of diabetes that was formerly classified as one type of malnutrition-related diabetes mellitus) and many others.

I.1.7. Complications of diabetes mellitus

Uncontrolled hyperglycemia in both type 1 and type 2 diabetes lead to the development of both acute and long term complications (Weiss and Sumpio, 2006).^1(*) Acute complications of diabetes mellitus include ketoacidocis (type 1) or nonketotic hyperosmolar coma (type 2). Long term complications include cardiovascular diseases, hypertension, chronic renal failure, retinal damage, nerve damage, erectile dysfunction and macrovascular damage which may cause poor healing of wounds particularly of the feet and can lead to gangrene which may require amputation (WHO, 1999). Chronically elevated blood glucose levels lead to increase production of mitochondrial reactive oxygen species (ROS), which activate a number of metabolic pathways whose end products contribute to the development of long term complication of diabetes (Weiss et al., 2006).

I.1.8. Diabetes mellitus in special groups and circumstances

I.1.8.1. Metabolic syndrome and type 2 diabetes mellitus

Often a person with abnormal glucose tolerance (IGT or diabetes) will be found to have at least one or more of the other cardiovascular disease risk factors such as hypertension, central (upper body) obesity, and dyslipidemia. This clustering has been labelled diversely as the metabolic syndrome, syndrome X, or the insulin resistance syndrome (WHO, 2006). Alone, each component of the cluster conveys increased cardiovascular disease risk, but as a combination they become much more powerful. This means that the management of persons with hyperglycemia and other features of the metabolic syndrome should focus not only on blood glucose control but also include strategies to reduce the impact of other cardiovascular disease risk factors (WHO, 2006). The metabolic syndrome with normal glucose tolerance identifies the subject as a member of a group at very high risk of future diabetes. Thus, vigorous early management of the syndrome may have a significant impact on the prevention of both diabetes and cardiovascular disease, especially as it is well documented that the features of the metabolic syndrome can be present for up to 10 years before glycemia disorder is detected (Miller et al., 2010).

I.1.8.2. Hyperlipidemia and type 2 diabetes mellitus

Hyperlipidemia refers to elevated levels of lipids and cholesterol in the blood, and is also identified as dyslipidemia, to describe the manifestations of different disorders of lipoprotein metabolism. Lipids in blood are either free or bound to other molecules. They are either provided by diet or are endogenously synthesized. Lipids, such as cholesterol and triglycerides, are insoluble in plasma. Circulating lipid is carried in lipoproteins that transport lipid to various tissues for energy use, lipid deposition, steroid hormone production and bile acid formation. The lipoprotein consists of esteri?ed and unesteri?ed cholesterol, triglycerides, phospholipids, and proteins. Abnormalities in lipoprotein metabolism are a major predisposing factor to atherosclerosis, increasing risk for CHD (Cromwell et al., 2006).

Five major lipoproteins exist, each with a di?erent function: chylomicrons carry triglycerides from the intestine to the liver, skeletal muscles and to adipose tissues; very low density lipoproteins (VLDLs) carry (newly synthesized) triglycerides from the liver to adipose tissue; intermediate density lipoproteins (IDLs) are intermediate between VLDL and low density lipoprotein (LDL). LDL is the main carrier of circulating cholesterol within the body. It is used by extra hepatic cells for cell membrane and steroid hormone synthesis. Once the LDL is taken up by LDL receptors, free cholesterol is released and accumulates within the cells. High density lipoprotein (HDLs) collects cholesterol from body tissues and brings it back to the liver. The protein components of the lipoprotein are known as apolipoproteins or apoproteins. The different apolipoproteins serve as cofactors for enzymes, and ligands for receptors. Defects in apolipoprotein metabolism lead to abnormalities in lipid handling (Lucy et al, 2005).

Three main pathways are responsible for the generation and transport of lipids within the body: exogenous, endogenous, and reverse cholesterol transport (Nicole et al., 2008). Although elevated low density lipoprotein cholesterol (LDLc) is thought to be the best indicator of atherosclerosis risk, dyslipidemia can also describe elevated total cholesterol (TC) or triglycerides (TG), or low levels of high density lipoprotein cholesterol (HDLc) (Cromwell et al., 2006). The most common lipid pattern in type 2 diabetes consists of hypertriglyceridemia, low high-density lipoprotein cholesterol (HDLc) and normal plasma concentrations of low-density lipoprotein cholesterol (LDLc). However, in the presence of even mild hyper-TG, LDLc particles are typically small and dense and may be more susceptible to oxidation (Grundy, 2006). Chronic hyperglycaemia promotes the glycation of LDLc and both these processes are believed to increase the atherogenicity of LDLc.

Under normal circumstances, insulin activates the enzyme lipoprotein lipase and hydrolyses triglycerides. However, in insulin deficient subjects, it fails to activate the enzyme and causes hypertriglyceridemia. In insulin deficient diabetics, the plasma free fatty acid concentration is elevated as a result of increased free fatty acid outflow from fat depots, where the balance of the free fatty acid esterification-triglyceride lipolysis circle is displaced in favour of lipolysis (Jie et al., 2007). Excess plasma NEFA can inhibit insulin-stimulated glucose utilization in muscle and promote hepatic production of glucose. Whereas, reduction of plasma NEFA concentration improves glucose utilization, enhances the suppression of hepatic glucose production by insulin (Jie et al., 2007).

I.1.8.3. Oxidative stress, endothelia dysfunction and diabetes

Oxidative stress is caused by a relative overload of oxidants, i.e., reactive oxygen species especially from glycation or lipoxidation processes and decreased enzymatic and non enzymatic antioxidant defence system. Evidence has accumulated suggesting that diabetic patients are under oxidative stress and that complications of diabetes seem to be partially mediated by oxidative stress (Hadi et al., 2007). Several mechanisms seem to be involved in the development of an oxidative stress in the presence of elevated glucose concentrations, namely glucose autoxidation, protein glycation, AGE formation and the polyol pathway (Hadi et al., 2007). Thus, it has been shown that oxygenated free radicals are able to alter vascular function (endothelia dysfunction), especially by inhibiting synthesis and action of nitric oxide (NO
·). Endothelia dysfunction comprises a number of functional alterations such as impaired vasodilatation, inflammation activation and increase plasma level of endothelia products all of which are usually associated to cardiovascular disease. A key feature of endothelial dysfunction is the inability of arteries and arterioles to dilate appropriately in response to stimuli. This limits the delivery of nutrients and hormones to the distal tissues (Wineke et al., 2009). Insulin resistance may be associated with intracellular production of free radicals which in turn could be responsible for deterioration of insulin action thus leading to a vicious cycle (Ghufran et al., 2011).

Figure 6 : Hyperglycemia induced endothelial dysfunction (Hadi et al., 2007)

I.1.9. Prevention and Management of type 2 diabetes mellitus

I.1.9.1. Strategies for treatment and control of diabetes

There exist a primary, secondary and tertiary prevention of diabetes mellitus. Primary prevention of type 2 diabetes is possible and includes Lifestyle changes aimed at weight control and increased physical activity. The benefits of reducing body weight and increasing physical activity are not confined to type 2 diabetes, they also play a role in reducing heart disease and high blood pressure.

Secondary and tertiary preventions are keys to reducing the risk of costly diabetic complications, as well as their associated disabilities (Craig et al., 2009). The primary purpose of secondary prevention activities such as screening is to identify individuals without symptoms who already have the disease, who are at high risk of developing complications related to the primary disease, and where intervention could have a beneficial effect.

Tertiary prevention of diabetes includes every action taken to prevent or delay the consequences of diabetic complications, such as blindness, foot amputation and adverse pregnancy outcomes. Strategies for tertiary prevention involve prevention of the development of complications by strict metabolic control, education and effective treatment. They also involve screening for early stages of complications, when intervention and treatment are generally more effective (Craig et al., 2009). Diabetes mellitus type 2 should not be managed based on symptoms alone.

The goal of treatment of diabetes mellitus is to control blood glucose and ultimately prevent long-term complications. Provided hyperglycemia is mild in type 2 diabetes, patients may be given at least a one month trial of diet, exercise and weight management in order to control hyperglycemia. If this regimen does not lead to adequate blood glucose control, the physician will need to prescribe oral anti-hyperglycaemic agents and/or insulin (Reaven et al., 2009). It is now well established that multiple metabolic abnormalities associated with insulin resistance and increased cardiovascular risk, such as dyslipidemia, obesity and hypertension, are already present at diagnosis. Results of many intervention studies have demonstrated marked benefit from antihypertensive, lipid-lowering and anti-platelet therapy (Reaven et al., 2009).

Exercise is extremely important in the management of diabetes because of its effect on blood glucose and free fatty acids. Exercise burns calories and helps to control weight, eases stress and tension, and maintains a feeling of well-being. In addition, regular exercise improves the body's response to insulin and may make oral anti-diabetic drugs and insulin more effective. It also promotes circulation, and lowers cholesterol and triglyceride levels, thus reducing the risk of cardiovascular disease (Eldor et al., 2009).

I.1.9.2. Mechanism of action of conventional oral hypoglycemic drugs

Oral hypoglycemia agents exert their glucose lowering effects via a variety of mechanisms (Figure 6). These mechanisms of action include: reduction of hepatic glucose production, (metformin, a biguanide), enhancement of insulin secretion by pancreatic beta cells, (insulin secretagoges) improvement of insulin sensitivity (metformin) and inhibition of intestinal glucose digestion and absorption (alpha glucosidase inhibitors) (Baby et al., 2011). The use of these drugs is however, limited by the fact that they have adverse side effects, such as potential hypoglycemia (e.g. sulfonylurea), weight gain, (meglitinides, sulfonylurea and thiazolidinesdiones), gastro-intestinal discomforts (alpha glucosidase inhibitors, and alpha amylase inhibitors) and lactoacidosis (TZDs and metformin) (Cheng and Fantus, 2005). In addition to their potential side effects, many of the oral anti-diabetic agents have higher secondary failure rates.

Figure 7 : Action site of western medicine in diabetes treatment (Baby et al., 2011)

Oral agents may counteract insulin resistance, improve ß-cell glucose sensing and insulin secretion, or control the rate of intestinal glucose absorption. Combinations of oral agents, in particular sulfonylureas plus metformin or thiazolidinediones plus metformin, have improved the care of diabetic patients, and may be used when monotherapy is ineffective (Craig et al., 2009).

I.1.9.2 Medicinal plants and herbs for diabetes

As it's the case with other diseases, medicinal plants have been used since ancient times to treat and manage diabetes mellitus in traditional medical systems of many cultures throughout the world (Jung et al., 2006). Currently, medicinal plants continue to play an important role in the management of diabetes mellitus, especially in developing countries, where many people do not have access to conventional antidiabetic therapies (Acharya and Shrivastava, 2008). In developed countries the use of antidiabetic herbal remedies is reported to have been declining since the introduction of insulin and synthetic oral hypoglycemic agents during the early part of the twentieth century. However, in recent years, there has been a resurgence of interest in medicinal plants with hypoglycemic potential in these countries (Paul et al., 2006). This renewed interest in herbal antidiabetic remedies in developed countries is believed to be motivated by several factors, including, the side effects, high secondary failure rates and the cost of conventional synthetic antidiabetic remedies (Paul et al., 2006).

I.1.9.3. Bioactive ingredients (principles) of antidiabetic medicinal plants

Ivorra et al (1989) studied the structure of 78 different compounds isolated from plants with attributed hypoglycemic activity. They classified these compounds according to the following chemical groups:

Similarly, Bailey and Day (1989) listed 29 compounds that contained 14 polysaccharides, 5 alkaloids, 4 glycosides and 6 other compounds. Grover, (2002) reviewed 45 medicinal plants of India with confirmed antidiabetic potential. Of the 17 hypoglycemic principles isolated and identified in this review 5 compounds are amino acids and related compounds, 5 compounds are glycosides, and 3 compounds are phenolic (flavonoids) compounds. The remaining compounds are alkaloids (2 compouds), terpenoids (1 compound) and polysaccharides (1 compound). Bnouham et al., (2006) also reviewed 178 medicinal plants with potential antidiabetic activity.

I.1.9.4. Mechanism of action of antidiabetic medicinal plants and their components

There are several possible mechanisms through which these herbs can act to control the blood glucose level (Tanira, 1994). The mechanisms of action can be related, generally, to the ability of the plant in question (or its active principle) to lower plasma glucose level by interfering with one or more of the processes involved in glucose homeostasis. The reported mechanisms whereby herbal antidiabetic remedies reduce blood glucose levels are more or less similar to those of the synthetic oral hypoglycemic drugs and are summarized as follows (Tanira, 1994; Bastaki, 2005; Bnouham et al ., 2006):

iii) improvement of insulin sensitivity (enhanced glucose uptake by fat and muscle cells)

iv) alteration of the activity of some enzymes that are involved in glucose metabolism

I.1.9.5. Investigation of some mechanism of action of antidiabetic plant extracts

Below is a brief description of some procedures used to investigate the in vivo effects of plant materials on insulin secretion, digestion and absorption of glucose, activation of the insulin receptor and the activity of some carbohydrate metabolizing enzymes.

In most published studies, investigation of the effect of medicinal plant extract on insulin secretion in vivo has involved the use of streptozotocin or alloxan induced animal models of diabetes (Eidi et al., 2006). Both alloxan and streptozotocin causes destruction of pancreatic beta cells resulting in reduced insulin secretion (Fröde and Medeiros, 2008). In streptozotocin and alloxan induced animal models of diabetes, insulin is markedly depleted but not absent (Fröde and Medeiros, 2008). For this reasons these animal models have been widely used to study the effect of antidiabetic remedies on insulin secretion in vivo.

In order to investigate the effect of an antidiabetic plant extract on intestinal digestion and/or absorption of carbohydrates, study animals are usually divided into experimental and control groups. Experimental animals are given a plant extract under investigation while control animals are given a vehicle. An hour later, both groups of animals are given a fixed amount of glucose, sucrose or starch. Thereafter, blood glucose levels are measured at 0.5, 1, 1.5, 2 and 3 hrs after administration of the carbohydrate. Areas below the oral glucose tolerance curves of experimental groups are then calculated and compared with those of control groups (Hannan et al., 2007). Alternatively, a glucose tolerance test can be determined in the same group of animals before and after oral administration of the plant extract (Karato et al., 2006). A comparison of the glucose tolerance curve before and after oral administration of the plant extract will indicate whether or not the plant extract contribute to the delay in carbohydrate digestion and subsequent lowering of the blood glucose level glucose.

It has been establish that some antidiabetic remedies, for example, metformin exert its blood glucose effects by inhibiting endogenous glucose production by the liver through the process of gluconeogenesis and glycogenolysis (Bastaki, 2005). For this reason, as part of efforts to find out the possible mode of action of antidiabetic remedies, several researchers have investigated the effect of plant extracts on the activities of gluconeogenic enzymes: glucose 6-phosphatase, fructose 1,6-bisphosphatase; the glycogenolytic enzyme; glycogen phosphorylase and hepatic glucokinase. In order to investigate the effect of medicinal plant extract on key enzymes involved in glucose homeostasis in vivo, the study design used are similar to the one describe above for the study of the effect of plant extract on stimulation of insulin except that at the end of the feeding period blood and selected tissues are also collected for the measurement of the activity of selected enzymes in plasma or tissue homogenates in vitro.

I.1.9.6 Extraction of plant material

The choice of the extraction solvent depends mainly on the polarity and hence the solubility of the bioactive compound(s) of interest. Although water is generally used as an extractant in many traditional protocols, organic solvents of varying polarities are often used (either alone or in different combinations) in modern methods of extraction to exploit the various solubilities of plant constituents. The polarity and chemical profiles of most of the common extraction solvents have been determined (Eloff et al., 1998) and are summarized in Table II.

Thus, if the polarity or the solubility of the compound(s) of interest is known, information such as the one in the table below can be used to select a suitable extractant solvent or a mixture of two or more solvents of different polarity. Alternatively, a solvent such as acetone, which has the capacity to extract both polar and non-polar substances, and has been recommended by Eloff (1998) for the extraction of most polar and non-polar compound.

If the polarity of the compounds of interest is not known, the powdered plant material can be extracted simultaneously with a mixture of different proportions of two or more solvents of different polarity. Alternatively, the powdered plant material can be extracted sequentially with solvent of different polarity in what is known as a sequential extraction procedure (Bruneton, 1999).

The choice of the extraction procedure depends on the nature of the source material and the compound to be isolated. Solvent extraction procedures applied to plant natural products include but not limited to maceration, percolation, soxhlet extraction, steam distillation and sequential solvent extraction (Jones and Kinghorn, 2005).

Table II : Polarity and chemical profiles of most of the common extraction solvents

I.1.10. Experimental diabetes

They are used for many decades. The can either be spontaneous or provoked. This constitutes laboratory rodents and mammals. Small ruminants are objects for many medical researches but are not used in diabetology because their herbivoral metabolism is very different from that of omnivores. Spontaneous model are rare in animals and the type of diabetes is not always the same as the one found on man. Certain species of animals were created for medical use.

Induced methods are obtained by administration of the toxic agent on the endocrine pancreas or by pancreatectomy.

I.1.10.1. Chemical induction model

Streptozotocin (STZ) is an antibiotic, anti-tumoral of synthesis use in anticancerous chemotherapy in man. In animals, streptozotocin selectively destroys the pancreatic insulin-secreting â-cells, leaving less active cells. STZ diabetic mice are one of the animal models of human insulin-dependent diabetes mellitus characterized by high fasting blood glucose levels and drastic reduction in plasma insulin concentration (Jie et al., 2007).

Alloxan is one of the usual substances used for the induction of diabetes mellitus apart from streptozotocin. Alloxan has a destructive effect on the beta cells of the pancreas (Vivek et al., 2010). Alloxan causes a massive reduction in insulin release by the destruction of â-cells of the islets of langerhans, thereby inducing hyperglycemia. Insulin deficiency leads to various metabolic alterations in the animals via increased blood glucose, increased cholesterol, increased levels of alkaline phosphate and transaminases (Vivek et al., 2010).

I.1.10.2. Diet induction model

Experimental cardiovascular risk factors of diabetes complications are usually induced by the consumption of high fat diet, high sucrose diet, and high cholesterol diet depending on the pathological state that is to be induced. For instance, it has been shown that rats fed on high fructose diet mimic the progression of type 2 diabetes seen in humans including glucose intolerance, increased oxidative stress, hypertension, and reduced myocardial and vascular compliance (Jatin et al., 2011).

I.1.11. Metabolism of fructose, glucose and type 2 diabetes mellitus

Dietary fructose undergoes rapid metabolism by the liver since most cells lack glut-5 transporter which transport fructose into cells. In contrast, hepatic glucose metabolism is limited by the capacity to store glucose as glycogen and, more importantly, by the inhibition of glycolysis and further glucose uptake resulting from the effects of citrate and ATP to inhibit phosphofructokinase. Because fructose uptake by the liver is not inhibited at the level of phosphofructokinase, fructose consumption results in larger increases of circulating lactate than does consumption of a comparable amount of glucose (Angela et al., 2007). Low-dose fructose has also been found to restore the ability of hyperglycaemia to regulate hepatic glucose production. In addition, fructose ingestion results in smaller postprandial glycemic excursions compared to glucose and glucose-containing carbohydrates (starches) that are rapidly absorbed as glucose. However, increased blood fructose concentrations could also contribute to glycation and diabetic complications (Angela et al., 2007).

In contrast to low doses of fructose, when much larger amounts of fructose are consumed (e.g., in sucrose- and HFCS- sweetened beverages), fructose continues to enter the glycolytic pathway distal to phosphofructokinase and hepatic triacylglycerol production is facilitated. Thus, unlike glucose metabolism, in which the uptake of glucose is negatively regulated at the level of phosphofructokinase, high concentrations of fructose, can serve as a relatively unregulated source of acetyl-CoA (George et al., 2007). Thus, fructose is more lipogenic than glucose, an effect that might be exacerbated in subjects with existing hyperlipidemia or insulin resistance or type 2 diabetes (Heather et al., 2005). In contrast to glucose, dietary fructose does not stimulate insulin or leptin (which are both important regulators of energy intake and body adiposity). Therefore, the decrease in insulin responses to meals and leptin production associated with chronic consumption of diets high in fructose may have deleterious long-term effects on the regulation of energy intake and body adiposity (Jatin et al., 2011). Another recent report has proposed a hypothesis relating fructose intake to the long-known relation between uric acid and heart disease. The ADP formed from ATP after phosphorylation of fructose on the 1-position can be further metabolized to uric acid. The metabolism of fructose in the liver drives the production of uric acid, which utilizes nitric oxide, a key modulator of vascular function (George et al., 2007).

Figure 8 : Hepatic fructose metabolism: highly lipogenic pathway (Heather et al., 2005)

Stimulated triglyceride synthesis is likely to lead to hepatic accumulation of triglyceride, which has been shown to reduce hepatic insulin sensitivity, hepatic insulin resistance, glucose intolerance as well as increased formation of VLDL particles due to higher substrate availability, increased apoB stability, and higher MTP, the critical factor in VLDL assembly (Kimber et al., 2008).

I.2. Botanical review of experimental plant: Ficus ovata

I.2.1. Botanical Aspect of Moraceae

The family of Moraceae belongs to plant kingdom, branch of phanerogames, sub-branch of angiosperm, class of dicotyledonous, sub-class of monochlamides and order of urticals. It takes its name from the genus Murier or Morus in Latin and in Greek it is called Moreas. Moraceae are constituted of trees, sub-trees or herbs which can be dioic or monoic with or without a latex (Mensbruge, 1966). The leaves are disposed in a spiral form; their nervation is palmated, pinnated or radial. The young ones of Moraceae plants are characterised by their first leaves which is simple opposite or sub-opposite (Human et al., 1985). Their unisexual flowers are dioic or monoic and it is fixed on the plant directly. The fruits are dehiscent; the grains which are with or without endosperms have equal or unequal cotyledons. This family of Moraceae counts about 50 genus and 900 to 1000 species. In Cameroon, about 13 genus and 99 species, are represented and amongst the most spread genus we have; Morus, Artocapus, Ficus and Dorstenia (Chang et al., 1998).

I.2.2. Botanical Aspect of the genus Ficus

Ficus or fig tree is the name of some shrubs or trees of the family Moraceae producing a milky juice and best known for their fleshy and edible fruits. Leaf shape is very variable. The shape may be whole or lobed and the edges rough or smooth. In some tropical species, leaf shape changes during growth of the tree. The flowers are minute and unisexual (male or female), they cluster on flat or hollow receptacle. Male flowers have one or two stamens, rarely more. In female flowers, the stamens are numerous and pedicellate. In fact, the shape of fruit that develops from inflorescences is varied. The seeds, embedded in the fruit are very numerous. The ovary of Ficus has a lateral style. The branches are covered with a fluffy greenish grey bark. The Ficus is found mainly in tropical forests, but they also exist in temperate regions, especially around Mediterranean (Tchinda et al., 2010). The genus Ficus includes 850 species of which about 60 are present in Cameroon (Sabatie, 1985).

I.2.3. Botanical Aspect of Ficus ovata

Ficus ovata, another plant of the Ficus specie found in the savanna woodland, forest edges, river side forest and secondary forest, up to an altitude of 2100 m is distributed in the subtropical Africa. Ficus species is known as elephant tree and Punjab in English (Hanelt et al., 2001), Ficus ovata is use widely for street ornament in Dakar in Senegal (Kuete et al., 2009). In Africa, Ficus ovata is found in Senegal, southern Ethiopia, Kenya, North of Angola, Zambia, Malawi, Mozambique, and Cameroon.

Figure 8 : Ficus ovata (Tchinda et al., 2010)

· Life;embryophyta(plant);angiospermae

(flowering plant);eudicotyledons

· Order;Rosales

· Family.Moraceae

· Genus;Ficus

· Subgenus;Urogstigma

· Section;Galoglychia

· Subsection;Caulocarpae

· Specie; ovata

· Botanique name; Ficus ovata

Common name; punjab

The sites of Ficus ovata in Cameroon (Aubreville, 1964) include Dschang, Bafang , Limbe, Bayangam, Nkongsamba, Meiganga, Maroua, Bipindi, Yaounde.

I.2.4. Uses of some Ficus in traditional pharmacopoeia to treat diabetes

Table III: Uses of some Ficus in traditional pharmacopoeia to treat diabetes

Plants	Parts used	Indigenous use (Kiran et al., 2011)
Ficus bengalensis L	Aerial roots, bark	1. The stem bark is extracted in hot water and extract is given orally to the patient. 2. By eating fruits to reduce blood glucose. 3. Regular chewing of fresh root tips can reduce blood glucose level.
Ficus racemosa Roxb	Bark, Fruit	1. Decoction of ripe fruits use in diabetes. 2. Decoction of stem bark reduces blood glucose.
Ficus lacor Ham	Fruit	Powder of dried ripe fruits is used to treat diabetes
Ficus religiosa L.	Bark	The bark boiled in hot water and the extract given orally to the diabetic person
Ficus microcarpa L.f.	Fruit, leaves	Fresh leaves and fruits taken in equal quantity, grind them, and taken orally is best remedy to treat diabetes
Ficus virens Dryand	Leaves	Leaves are used to treat diabetes
Ficus carica L.	Leaves	The decoction of leaves used to cure diabetes.
Ficus hispida L.f.	Bark	Infusion of bark used as remedy to treat diabetes

The multiple and diverse uses of Ficus species in traditional pharmacopoeias were definitely the starting point of several scientific studies carried out until today.

I.2.5. Uses of Ficus ovata in traditional pharmacopoeia

The decoction of leaves of Ficus ovata Vahl is used to treat infectious diseases and facilitate childbirth. The decoction of the bark stems is used in the treatment of gastrointestinal infections, diarrhea and as antipoison. In Benin, the leaves of Ficus ovata are used against external hemorrhoids, sprains and jaundice (yellowing) (Kuete et al., 2009) and its leaves are used in Ivory Coast against the psychoneuroses. For this, we must drink a glass of the decoction of the leaves, morning, noon and night, wash your body with this and make a decoction enema decoction of roots (Assi et al., 1990). Fruits are used to stimulate milk production in cows and stem back use as food for mastication (Hanelt et al., 2001).

I.2.6. Previous work on biological activities of some Ficus

We have put together some previous phytochemical and biological work on the genus Ficus, which has lead to the isolation of a number of secondary metabolites belonging to several classes of compounds and responsible in one way or the other for their biological activities.

Plants	Research goal	Extract	Activity
Ficus ovata	Test of antimicrobian activity of a crude extracts, fractions and compounds	Methanol bark and trunk extract	The crude extract and certain compounds inhibited the activity of steptococus faecalis, candida albicans, microsporum audouini, staphylococus aureus *(Kuate et al.,* 2009).**
Ficus Glomerata	-Hypoglycemic activity in alloxan induced diabetic -Antihyperglycemic activity in streptozotocin induced diabetic rats	Ethanolic leaves bark and aqueous extract	It has significant antihyperglycemic effect in experimental model of diabetes *(Vivek et al.,* 2010). antihyperglycemic activity in experimental animals (Faiyaz et al., 2008)**.
Ficus hispida Linn.	Hypoglycemic activity in normal and diabetic rats and probable mechanism	Ethanolic bark extract	Hypoglycemic activity. Increased glycogenesis and enhanced peripheral uptake of glucose are the probable mechanisms *(Ghosh et al.,* 2004).**
Ficus exasperata	Glycemic effect in fructose induce glucose intolerance in Sprague-Dawley rats	aqueous leaves extract	The extract ameliorated glucose intolerance induced by fructose feeding in rats *(Idowu et al.,* 2010).**
Ficus racemosa Linn.	hypoglycemic and in vitro antioxidant activity	ethanolic Fruits extract	It was suggested that it has both hypoglycaemic and antioxidant potential *(Abu et al., 2011).*
Ficus Carica	Hypoglycemic Effect In normal and Streptozotocin Induced Diabetic Rat	Water Leaves extract	Oral consumption of aromatic water leaves of Ficus carica decreased blood glucose level in normal and diabetic rats *(Rashidi et al.,* 2011)**.
Ficus krishnae L.	Anti-Diabetic and Antihyperlipidemic Activity in alloxan Induced Diabetic Rats	leaves	Ficus krishnae have an anti-diabetic effect in alloxan induced diabetic rats and their effect was equivalent to that of reference drug glibenclamide *(Mohana et al., 2010)*.

Herbal extracts contain different phytochemicals with biological activity that can be of valuable therapeutic index. Much of the protective effect of fruits and vegetables has been attributed by phytochemicals, which are the non-nutrient plant compounds. Different phytochemicals have been found to possess a wide range of activities, which may help in protection against chronic diseases. For example, glycosides, saponins, flavonoids, tannins and alkaloids have hypoglycemic activities; anti- inflammatory. Reports show that saponins possess hypocholesterolemic and antidiabetic properties. (Poongothai et al., 2011)

Flavanoids are reported to regenerate damaged pancreatic beta cells and glycosides stimulate the secretion of insulin in beta cells of pancreas. Glycoside of leucopelargonidin isolated from the bark of F. bengalensis demonstrated significant hypoglycemic, hypolipidemic and serum insulin raising effects. Phenolic compounds including quercetin and luteolin are effective in diabetic treatment where they present capacity to scavenge superoxide radical. Reports suggest that Quercetin and tannins treatment has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic beta cell integrity. Also Plant polyphenols have been known to exert anti-diabetic action and promote insulin action (Abu et al., 2011). Findings indicated that quercetin improved insulin signalling and sensitivity and thereby promoted the cellular actions of insulin in an acquired model of insulin resistance.

In previous pharmacological investigations, Ivorra et al. (1989) reported that â-sitosterol induced the uptake of insulin from â-cells and produced an anti-hyperglycemic effect. On the other hand, stigmasterol, lupeol, ursolic and oleanolic acids showed to have hypoglycemic activity. Oleanolic acid and semi-synthetic derivatives were described as â-glucosidase inhibitors. Finally, triterpenoids induced an anti-diabetic effect by different pathways, and their combination could provoke a synergic effect.

CHAPTER II. MATERIALS AND METHODS

II.1. Collection and identification of plant materials

The fruits and twigs of Ficus ovata were collected from Mount Kala, Centre region of Cameroon. The plant was identified at the Cameroon National Herbarium, Yaounde, where a voucher specimen was conserved under the reference number 26996SRF/Cam. The collected plant parts were separated from undesirable materials. They were dried under the shade separately. The plant parts were ground into tiny debris with the help of a suitable grinder, kept in a cool and dry place until analysis commenced.

We weight 125g of each plant part and macerated separately in 1L of ethanol 95% or 1L of ethanol: water solution in the ratio 1:1 for 48hours. The whole mixture was successively filtered through a piece of clean, white cotton material. The filtrates (ethanolic and hydroethanolic extracts of the twigs and fruits) obtained were evaporated to dryness in a drying room. We obtained four extracts as shown below (figure 9).

Figure 9 : protocol for the extraction by maceration in ethanol and hydroethanol (1:1)

II.2. In vitro study

II.2.1. Phytochemical screening of the extracts

In view of determining the different secondary metabolites responsible for the biological activity of the plant, extracts underwent phytochemical screening as follows;

1- Test for Phenol was done by dissolving 250 mg of each extracts in 4 ml of distilled water and the content heated for 15 minutes. After cooling and filtration 2 drops of freshly prepared ferric cyanate solution (1 ml FeCl₃ 1% and 1ml KFe (CN)₆) was added to 1 ml of each filtrate. The presence of a greenish-blue coloration indicated the presence of polyphenols (Harbone, 1976).

2- Test for Alkaloids (Mayer test) was done by heating 100 mg of the each extracts in 2 ml of H₂SO₄ 2% for 2 minutes after which the content was filtered. Few drops of the Mayer reagent (1.358g HgCl₂+500 ml H₂O and 0.8g KI +200 ml H₂O) were added in 1ml of the filtrate and the presence of a white precipitate or turbid solution was an indicator of the presence of alkaloids (Odebeyi and Sofowora, 1978).

3- Test for Saponines was done by adding 250 mg of the extract in 5 ml of distilled water. After vigorous homogenisation the mixture was heated to boil, the appearence of foam that persisted 20 minutes after cooling was an indicative of the presence of saponines (Wall et al., 1954).

4- Test for Tannins was done by adding 100 mg of each extracts in 2 ml of distilled water followed by heating in a water bath and then filtering. Few drops of 3% ferric chloride were added in 1 ml of filtrate and the observation of a blue-black or greenish-dark coloration indicated the presence of tannins (Trease and Evans, 1989).

5- Test for phlobatannins was done by dissolving 100 mg of each extract in 2 ml of distilled water. After filtration, to 0.5 ml of each filtrate was added 1 ml of hydrochloric acid 1% and the deposit of a red precipitate was an indicator of the presence of phlobatannins (Trease and Evans, 1989).

6- Test for glycosides was done by dissolving 100mg of each extract in 5 ml of HCl then neutralised by 5 ml of 5% caustic soda (soude). Drops of Fehling's solution [A (40 g of CuSO₄ 5 H₂O per litre) + B (160 g of tartrate double of sodium and potassium + 130 g of NaOH per litre)] were added one after another and the appearance of a red precipitate was an indicative of the presence of glycosides (Trease and Evans, 1978).

7- Test for flavonoids was done by dissolving 250 mg of each extract in 5 ml of sodium hydroxide 1N.The observation of a yellow solution is a preliminary evidence of the presence of flavonoids and the decolourisation of the yellow colour observe on the addition of a few drops of concentrated HCl confirms its presence (Trease and Evans, 1978).

The mother solution (5 mg/ml) was prepared by dissolving 100 mg of the pure extract with 20 ml of water. After vigorous homogenising, the following daughter solutions were prepared. It is from this working solution that the test below was carried on.

Tubes	1	2	3	4	5	6	7	8	9
Concentration (mg/ml)	5	2.5	1.5	0.75	0.5	0.25	0.05	0.025	0.012
Volume of extract (ml)	5	2.5	1.5	0.75	0.5	0.25	0.05	0.025	0.012
Volume of solvent (ml)	0	2.5	3.5	4.25	4.5	4.75	4.95	4.975	0.980
Final volume (ml)	5	5	5	5	5	5	5	5	5

II.2.2. Determination of the antioxidant potential of the plant extracts

II.2.2.1. Determination of polyphenol content using Folin ciocalteu method

Principle: This method is based on the reduction of a phosphomolibdic-tungstinic chromogene by an antioxidant and a change of colour with the absorbance measured at 750nm using a spectrophotometer. This reagent constitute of a mixture of tungstinic and phosphomolybdic acids. In alkali medium (sodium carbonate), it developed a blue colouration of which the absorbance is measured at 750nm. Ethanol (0.3 ml) in the place of extract is used as the blank. The antioxidant activity is expressed as the number of equivalents of catechin (Singleton and Rossi, 1965).

A stock solution Folin reagent of (10 ml) of concentration 2 N was introduced in a conical flask of 100 ml and the volume adjusted with water to 100ml so as to obtain a solution of 0.2 N.

Catechin (2 mg) was dissolved in 10 ml of methanol to obtain a solution of concentration 1mM from which other solutions with diverse concentration were prepared.

Procedure: The polyphenolic concentration of the extracts was determined using folin-ciocalteu reagent (sigma chemical Co St Louis, MO) diluted 10 times before use. To determine the total polyphenol concentration, 10 ul of the hydrolyse extract was added in 1ml of Folin solution diluted 10 times , after 30 minutes of incubation the absorbance was measured at 750 nm using the spectrophotometer. Catechine was used as a standard.

II.2.2.2. DPPH (1, 1-diphenyl-2-picrihydrazyl) antiradical activity

Principle: DPPH is relatively stable nitrogen centered free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH antioxidant assay is based on the ability of 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for a visible deep purple colour. When DPPH accepts an electron donated by an antioxidant, the DPPH is decolorized, and can be quantitatively measured due to the changes in absorbance (Katalinie et al., 2003).

Procedure: 20 ul of none hydrolysed aqueous extract was introduced in 2 ml of methanolic solution of DPPH (0.3 mM). After 30 minutes of incubation in the dark, the absorbance was measured with a spectrophotometer at 517nm. A control was also made (DPPH with water only). The percentage of inhibition of the DPPH radical by the specimen was calculated using the formular of Yen and Duh, (1994) as follows:

Where Ac is absorbance at time = 0 min and Ae is the absorbance after 30 minutes of incubation.

II.2.3.The anti-á-amylase effect of Ficus ovata extracts

Principle: The enzymatic activity was measured by the colorimetric technique base on the disappearance of the substrate in the reaction medium. This involved the inhibitory effect of extract on pancreatic alpha-amylase ability to hydrolyse starch (starches exists in the form of granules, composed of millions of molecules of amylopectin and a higher number of molecules of amylose) into products principally maltose that is colourless as seen in the reaction below. The remaining substrate was thus quantified by adding iodine solution (iodine and potassium iodide) in the reaction medium and the presence of a blue colour is an indicator of the quantity of substrate remaining (Komaki et al., 2003).

Figure 10 : Mechanism of pancreatic alpha-amylase activity (Tormo et al, 2004)

Irish (sigma) starch (5g) was introduced in a beaker containing 100 ml of distilled water after which it was heated for 30 minutes on a hot plate. The volume was completed to 500ml with distilled water

CaCl₂0.(56 g) of and 3.04 g of Tris was introduced in a beaker containing 490 ml of distilled water, after complete solubilisation, the pH was adjusted to 6.9 with dilute HCl.

One gram (1g) of potassium iodide and 100 mg of iodine are put in a beaker containing about 490ml of distilled water. After complete dissolution the solution was acidified to a pH of 1 with non title dilute HCl and the volume was adjusted to 500 ml with distilled water.

This was prepared to a concentration of 30ug/ml from the pure pancreatic alpha-amylase of pig type V1-A (sigma).

For each daughter solution there was an essay and a blank (no substrate). A standard was also prepared where enzyme and substrate were absent. 20 ul of á-amylase solution (30ug/ml) and the corresponding volumes of Tris-HCl (0.05M, pH 6.9) and extract was introduced in each tube. The mixture was pre-incubated at 37°C for 15 minutes and a fixed volume of starch (1%) was then added in essay tubes followed by incubation at 37°C for 15 minutes. The reaction was stopped by adding 2 ml of acidified iodine solution and potassium iodide (pH 1). The intensity of colour of each tube was determined against the blank through a spectrophotometer at an absorbance of 580nm. Methodology can be presented in a table as shown below.

Tubes	standard	E0	BE0	E1	BE1	E2	BE2	E3	BE3...	E9	BE9
Enzyme (ul)	0	20	20	20	20	20	20	20	20	20	20
Extract (ul)	0	0	0	20	20	30	30	30	30	30	30
Buffer (ul)	1400	1380	1480	1350	1450	1350	1450	1350	1450	1350	1450
PREINCUBATION AT 37°C FOR 15 MINUTES
Starch (ul)	100	100	0	100	0	100	0	100	0	100	0
INCUBATION AT 37°C FOR 15 MINUTES
Iodine+KI(ml)	2	2	2	2	2	2	2	2	2	2	2
Final vol (ml)	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5	3.5

The optical density OD for the final solutions was measured using a spectrophotometer. The intensity of the colour of each tube is determined against the blank.

OD of the initial substrate (standard) - OD of the remaining substrate (essay and blank)

This enables the elimination of non specific reaction between the enzyme and the extract.

Where A is the decrease of absorbance in the absence of extracts and B is the decrease of absorbance in the presence of extracts.

II.3. In vivo study

II.3.1. Experimental animals

Three month old male Wistar Albino rats weighing 175-225g were obtained from the animal house of the laboratory of Biochemistry, Department of Biochemistry, University of Yaoundé I, Cameroon. All animals were kept in an environmentally controlled room with a 12h light/12h dark cycle .The animals had free access to water and standard rat diet. These rats were used for the BGT, OGTT and the acute preventive treatment. Small male Wistar rats of age 2 months with average weight 100-120 g was use for acute toxicity study.

Experimental food: The animals where fed on standard laboratory diet made up of corn flour (60%), wheat (10%), fish (12%), soya beans (15%), palm oil (2%), vitamin complex (0.5%), and minerals (1.5%).

II.3.2. Evaluation of the acute toxicity effect of hydroethanolic extracts

This was aimed at evaluating the toxic effect of a single dose of a product administrated once. The protocol use was that of test limit proposed by OECD (2004). It recommended the administration of a single dose (=5000 mg/kg of the body weight) of a substance to experimental animals (rats) after which an intensive observation of the physiological changes was done for 48 hours. The animals were observed for 2 weeks and their physiological parameters registered. After two weeks those that survived were sacrificed and their blood collected for appropriate analysis (markers of toxicity). The rats were treated as follows; 3 groups of 5 rats were constituted as seen below (table VII)

II.3.4. Effect of extracts on glycemia

Glycemia was evaluated using a glucose meter (SD-check) and blood glucose test strips. The principle includes quantifying the blood glucose based on the enzymatic conversion of glucose into gluconic acid in the presence of glucose oxydase (300unites/100strip) and potassium ferrocyanide (mediator 9.0mg/100strip). The strip is made up of electrodes that measure the level of glycemia. Glucose in blood mixed with reagent on the test strip creates a small electric current. The intensity of the current created depends on the quantity of glucose in blood. The quantity of gluconic acid formed, measured by electric impedance is directly proportional to the quantity of glucose contained in the test zone (Ellen et al., 2003).

Procedure: A drop of fresh blood placed on the microplate will migrate by capillarity and filled the test zone. Few seconds after, the quantity of glucose appears on the screen of the apparatus in mg/dl.

II.3.4.1. Evaluation of hypoglycemic activity in hyperglycemic rats

For this, 3 groups of 5 hyperglycemic rats where constituted and treated as follows:

All the rats being at fasted state throughout the experiment, the fasting blood glucose test of the various rats was done before administration of our extracts (0h). The extracts were administrated orally by intra-gastric route using an oesophageal probe followed by successive glucose test measurement determined at instants of 2h, 5h, by incision of the tail and placing a drop of blood on the glucose strips and the glucose meter reading registered.

II.3.4.2. Evaluation of the antihyperglycemic activity in normoglycemic rats

Test of antihyperglycemia provoked by oral route in normal rats (oral glucose tolerance test): To evaluate the capacity of the organism to manage glucose, 3 groups of 5 rats were constituted and treated as shown in table IX below:

All the rats being at fasted state throughout the experiment, the fasting blood glucose test of the various rats was done before administration of our extracts (0h). The extracts were administrated orally by intra-gastric route using an oesophageal probe, 30mins after, 2g/kg body weight of glucose (test 1 and 2) was administration followed by successive glucose test measurement determined at instants of 0.5h, 1h, 2h. Glucose measurement was done by incision of the tail, a drop of blood placed on the glucose strip and the glucose meter reading registered.

II.3.5. Evaluation of the modulation effects of hydroethanolic fruits and twigs on some biomarkers of diabetes type 2 on rat subjected to high fructose-high cholesterol diet

Table X: Slightly modify food composition as proposed by Dhandapani (2007)

Composition	Control (%)	Atherogenic diet + fructose
Proteins (powder milk)	22	20
Carbohydrates (peel corn flour and wheat flour (3:2)	55	50
Fructose	0	5
Lipids (hydrogenated fats and margarine)	15	17
Mineral salts (bone flour and NaCl (3:1)	4	4
Vitamins	1	1
Fibres (Cellulose)	3	3

Male Wistar rats (175-225g) were divided into four groups of rats. Food and water was provided ad libitum. The body weights of rats were measured after 12 hours fasting period before experimentation begins. The control group received just distilled water while the three other groups received fructose and cholesterol by the intra-gastric route. Test groups received FOHT and FOHF. The positive control received no treatment. Fructose was administrated one hour before the administration of the various extracts. The weights were taken again on day 7 and day 14 (Dhandapani et al., 2007, Idowu et al., 2010).

At the end of the experimental period (14 days), animals were kept 12 hours without food and water and their blood glucose taken. Fasting rats were sacrificed under light anaesthesia with ether vapour. Blood collected in EDTA tubes from the jugular artery was centrifuged for 10min at 3400 rpm and the supernatant constituting the plasma was collected into dry eppendorf tubes and stored at -20°C for some biochemical analysis. Tissue homogenate was done by collecting the heart free from fat, and blood by rinsing in a solution of 0.9% (w/v) NaCl. After anatomical, macroscopical, and comparative observation, 1g of the organ was grinded in a mortar and homogenized in 10 ml of 0.9% (w/v) NaCl. The resulting homogenate was put in a centrifuge tube, allowed to sediment for about 1 hour before centrifuging at 3400 rpm for 10 minutes. The resulting supernatant which constitutes the homogenate was kept in eppendorf tubes and conserve -20°C for later use in biochemical assay.

II.5. Biochemical experimentation

II.5.1. Determination of plasmatic lipid profile

II.5.1.1. Quantitative determination of cholesterol (chronolab kits).

Principle The cholesterol present in the sample generates a coloured complex as shown.

The intensity of the colour formed is proportional to the cholesterol level (Naito, 1984).

Procedure: Total cholesterol was determined by first preparing the working reagent (mixture of 2 reagents). The blank (1mL WR), standard (1 mL WR and 10 uL standard) and the samples (1 mL WR and 10 uL sample) were prepared. The solution was mixed and incubated for 10 min at room temperature. The spectrometer was adjusted to zero with distilled water. The absorbance (A) of samples and standard were read against the blank at 500nm. The colour was stable for at least 60 minutes.

II.5.1.2- Quantitative determination of Triglycerides (chronolab kits).

Principle Sample triglycerides incubated with lipoprotein lipase (LPL) liberate glycerol and free fatty acids. Glycerol is converted to glycerol- 3-phosphate (G3P) and adenosine -5-diphosphate (ADP) by glycerol kinase and ATP. Glycerol-3-phosphate (G3P) is then converted by glycerol phosphate dehydrogenase (GPO) to dehydroxyacetone phosphate (DAP) and hydrogen peroxide (H₂O₂). In the last reaction, hydrogen peroxide (H₂O₂) reacts with 4-aminophenazone (4-AP) and p-chlorophenol in the presence of peroxidise (POD) to give a red colour dye: The intensity of the colour formed is proportional to the Triglycerides concentration in the sample (Fossati et al., 1982).

Procedure: Triglycerides were determined by first preparing the working reagent (mixture of 2 reagents). The blank (1mL WR), standard (1 mL WR and 10 uL standard) and the samples (1mL WR and 10 uL sample) were prepared. The solutions were mixed and incubated for 10 min at room temperature. The spectrometer was adjusted to zero with distilled water. The absorbance (A) of the samples and standard were read against the blank at 500nm. The colour was stable for at least 60 minutes.

II.5.1.3- Quantitative determination of HDL Cholesterol (chronolab kits)

Principle: The very low density (VLDL) and low density (LDL) lipoproteins from serum or plasma are precipitated by phosphotungstate in the presence of magnesium ions. After removed by centrifugation the clear supernatant containing high density lipoproteins (HDL) is used for the determination of HDL cholesterol (Young, 2001).

Procedure: Precipitation was done by pipeting 100 uL of the reagent and 1mL of sample into the centrifuge tube. The solution was well mixed and allowed to stand for 10 minutes at room temperature followed by centrifuge at 4000 rpm for 10 minutes. The supernatant was collected and tested for HDL cholesterol (HDLc).

II.5.1.4- Quantitative determination of VLDL and LDL Cholesterol

II.5.2. Determination of Markers of hepatic and renal toxicity

II.5.2.1. Determination of total protein (Biuret method)

Principle: Copper ions react in alkali solution, with protein peptide bonds to give a purple coloured Biuret complex. The amount of complex formed is directly proportional to the amount of protein in the specimen (Henry et al., 1974).

Procedure: Total protein (TP) content was measured by preparing reagent blank (20 ul distilled water and 1000 ul Biuret reagent), standard (1000 uL Biuret reagent and 20 uL calibrator) and the sample (1000uL Biuret reagent and 20 uL sample). After mixing and incubating for 30 min at 20-25°C the absorbance (A) of the samples and standard were read against the blank at 550nm.

II.5.2.2. Determination of creatinine level (chronolab kits)

Principle: The assay is based on the reaction of creatinine with sodium picrate as described by Jaffe. Creatinine reacts with alkali picrate forming a red complex. The time interval chosen for measurement avoids interference from other serum constituents (Murray et al., 1984a).

The intensity of the colour formed is proportional to the creatinine level in the sample.

Procedure: The blank (1 ml WR), standard (1 ml WR and 100 uL standard) and sample (1 ml WR and 100 ul sample) were pipette into cuvettes. After mixing, the stop watch was started and the absorbance (A ₁) read after 30 seconds and 90 seconds (A ₂) at 500nm.

II.5.2.3. Determination of transaminase activities (aspartate aminotransferase ASAT and alanine aminotransferase ALAT). (chronolab kits)

Principle: Aspartate aminitransferase (ASAT) or Glutamate oxaloacetate (GOT) catalyses the reversible transfer of an amino group from aspartate to á-ketoglutarate forming glutamate and oxaloacetate. The oxaloacetate produced is reduced to malate by malate dehydrogenase (MDH) and NADH (Murray et al., 1984b).

Alanine aminotransferase (ALAT) or Glutamate pyruvate transaminase (GPT) catalyses the reversible transfer of an amino group from alanine to á-ketoglutarate forming glutamate and pyruvate. The pyruvate produced is reduced to lactate by lactate dehydrogenase (LDH) and NADH.

The rate of decrease in concentration of NADH, measured photometrically is proportional to the catalytic concentration of ASAT and ALAT respectively.

Procedure: The same procedure was use both for ASAT and ALAT where after pipetting 1mL of the WR and 100 uL of sample into a cuvette it was mixed and incubates for 1 minute. The spectrometer was adjusted to zero with distilled water. The initial absorbance (A) of the sample was read, stopwatch started and the absorbance at 1 minute intervals read thereafter for 3 minutes at 340nm. The difference between absorbance and the average absorbance differences per minutes (?A/min) were calculated.

Units: one international unit (IU) is the amount of enzyme that transforms 1umol of substrate per minute, in standard conditions. The activity is expressed in units per litre of sample (U/L).

II.5.3. Determination of Nitric Oxide level

Principle: This is based on the reaction of diazotization described by Griess in 1879. It describes the chemical reaction between sulphanilamide and naphtylethylenediamine dihydrochloride (NED) under acidic conditions (phosphoric acid). Sulphanilamide and NED compete for nitrite in the Griess reaction. This reagent detects NO₂^- in a variety of biological and experimental liquid samples such as plasma, serum, urine and tissue culture. This system detects the nitrite formed which is one of the primary stable and non volatile compounds from degradation of nitric oxide in biological mediums (Manish et al., 2006).

A solution of sulphanilamide and dichloro N-1-naphtylethylene diamine was prepared and allowed for 15-30 minutes at room temperature.

Procedure: In 100 uL of sample, 100 uL of sulphanilamide solution was added and pre-incubated for 5-10 minutes at room temperature while protected from light. After pre-incubation, 100 uL of dichloro N-1-naphtylethylene diamine was added and incubated for 5-10 minutes at room temperature and protected from light.

The presence of a purple/mangenta colour is an indicator of the presence of nitrite formed.

II.6. Statistical analysis

SPSS program: version 10.0 for Windows was used and the results presented as mean #177; SEM. Differences between means were done by a one-way analysis of variance (ANOVA) followed by post hoc test using Tamhane and LSD. Values of p< 0.05 were taken to imply statistical significance.

CHAPTER III. RESULTS AND DISCUSSION

III.1.Results

III.1.1. Yield of extraction and phytochemical screening

Two solvent system were used for the extraction of the fruits and twigs of F. ovata which where ethanolic and hydroethanolic solvent systems. The above results show that the hydroethanolic solvent gave higher yield than the ethanolic solvent for extraction of the fruits and twigs.

	Alkaloids	Saponins	Flavonoids	Tannins	Phlobatannins	Glycosides	Phenols
FOEF	+	+	+	+	-	+	+
FOET	-	+	+	+	+	+	+
FOHF	+	+	+	+	-	+	+
FOHT	+	+	+	+	+	+	+

FOEF= Ficus ovata ethanolic fruits; FOET= Ficus ovata ethanolic twigs; FOHF= Ficus ovata hydroethanolic fruits; FOHT= Ficus ovata hydroethanolic twigs

Generally, extracts of fruits and twigs of Ficus ovata contain groups of bioactive compounds such as alkaloids, glycosides, saponins, and polyphenols such as flavonoids, tannins and phenols. Phlobatannins were absent in the fruit extracts of Ficus ovata and alkaloids where absent in ethanolic fruits.

III.1.2. Result of the antioxidant potential of our plant extracts

III.1.2.1. Polyphenol content of our extracts

Table XIV above shows that all extracts had polyphenols with FOEF having a higher content (718.142 mg catechin Eq). We also note a significant difference (P<0.05) in polyphenols content for all the extracts.

III.1.2.2. DPPH (1, 1-Diphenyl-2-Picrilhydrazyl) antiradical activity of extracts

Figure 11 below represent the inhibition percentages obtain after an evaluation of antiradical activity of the ethanolic and hydroethanolic fruits and twigs extracts respectively.

Figure 11 above shows that for both plant parts, the hydroethanolic solvent was the best system with a high antiradical activity compared to ethanolic solvent system (p<0.05) and the scavenging activity increases as concentration increases. Moreover the inhibition profile of the hydroethanolic fruits was the best with an IC 50 of 0.701 mg/ml as compared to the others and this IC 50 was significantly different (p<0.05) from all the other extracts.

III.1.3. The effect of extracts on starch digestion in vitro.

Figure 12 below represents the variation of the inhibition of activity of á-amylase (digestive enzyme) activity in the presence of different concentrations of extracts.

Figure 12 : Effect of extracts on the inhibition of pancreatic á-amylase activity

From figure 12 above, we observed that the percentage inhibition of the alpha amylase activity increased with increase in concentration for both extracts with that of hydroethanolic fruits being significantly higher compared to that of hydroethanolic twigs. Also, when the concept of IC50 values was used, hydroethanolic fruit extract showed the best result with an IC50 of 0.4727 mg/ml as compared to that of the twigs having an IC50 of 0.7473mg/ml. á-Amylase inhibitory activity was measured at various concentrations and the inhibition was observed at all doses (Fig. 12). When comparing the total water soluble phenolic concentration of hydroethanolic extracts with the á-amylase inhibitory activity, a correlation was observed.

III.1.4. Acute toxicity study of the hydroethanolic fruits and twigs extracts

III.1.4.1. Effect of extracts on the behaviour of experimental animals

The behavioural reactions of rats under acute toxicity are represented in table XV.

Observations	Negative control	FOHT	FOHF
General mobility	Normal	Normal 30min after	Normal 4hrs after
sleep	Normal	Normal	Normal
feeding	Normal	Normal 1hr after	Normal 6hrs after
faeces	Normal	Normal	Normal
Sensibility to sound	Jump immediately	Jump immediately	Jump immediately
death	No dead	No dead	No dead

From the above result we observe that these extracts at 5000mg/Kg of BW taken at a single dose do not lead to death. The other parameters remain comparable to the negative control.

III.1.4.2. Effect of extracts on the variation of body weight

We also evaluate the effects of the dose 5000mg/Kg BW of our extract on the variation of body weight and the results are presented as follows.

From the above figure (figure 13), we observe that, the weight gain of the test groups was reduced as compared to that of the negative control and the difference was significant (p<0.05).

III.1.4.3. Effect of extracts on transaminases activities and creatinine

Table XVI below represent the result of markers of toxicity after acute toxicity.

Table XVI: Effect of extracts on markers of toxicity (ASAT, ALAT and Creatinine)

Groups	ASAT (U/L)	ALAT (U/L)	Creatinine (mg/dl)
Control	8.632 #177; 0.052	91.956 #177; 0.262	31.913 #177; 0.770
FOHT (5000mg/Kg BW)	5.652 #177; 0.354^*	85.651 #177; 1.097^*	24.880 #177; 0.919^*
FOHF (5000mg/Kg BW)	6.867 #177; 0.420^*	80.704 #177; 0.196^*	37.248 #177; 2.688^*

*: significant difference between the control and the test groups at the threshold of 0. 05;

From the above results we notice a significant decrease in the concentration of ALT and AST in the test groups and equally a decrease in the concentration of creatinine for the extract FOHT. We also observe an increase in the concentration of creatinine for the FOHF extract.

III.1.5. The effect of extracts on glycemia

III.1.5.1 Hypoglycemic effect of extracts on hyperglycemic rats

The evaluation of hypoglycemic effect of FOHT and FOHF on hyperglycemic rats revealed the following results.

	Glycemia (mg/dl)
Time	0 Hour	2 Hours	5 Hours
Control	121.200 #177;1.162	102.800 #177; 2.991	79.000 #177; 6.380
FOHT	116.600 #177; 1.275	107.200 #177; 4.615	89.400 #177; 0.266
FOHF	115.600 #177; 5.568	106.800 #177; 1.554	105.600 #177; 5.140^*

*: significant difference between the control and the test groups at the threshold of 0. 05;

There was a decrease in glycemia for all groups after the administration of extracts from time 0 hour to the 5^th hour. This decrease was not significantly different from the 0 to 2^nd hour between the control and the test groups. From the 2^nd to the 5^th hour, the decrease was still not significant between the control and the FOHT group but there was a significant increase between the control and the FOHF group. The percentage decrease for the extract FOHT was significantly higher (8.908%) than that of the extract FOHF (5.747%).

III.1.5.2. Antihyperglycemia effects of extracts on normal rats

The evaluation of the effect of FOHT and FOHF extracts in the regulation of glucose intolerance in rats revealed the following results (table XVIII).

	Glycemia (mg/dl)
	0 min	30 min	60 min	120 min
Negative control	88.000#177;3.705	105.600 #177; 2.970	100.900 #177; 2.770	93.300#177;1.164
Positive control	82.400#177;3.173^*a	175.400#177;3.809^*a	111.300#177;2.958^*a	91.100#177;2.642^*a
FOHT	99.300 #177; 1.685^b	124.700 #177; 2.905^b	141.800 #177; 5.495^b	98.300#177;1.824^a
FOHF	89.300 #177; 1.271^b	171.700 #177; 7.256^b	182.200 #177; 10.914^b	143.000#177;3.977^b

a and b : significant difference between the control and the groups FOHT and FOHF at the threshold of 0,05;

The variation of glycemia during the glucose tolerance test was significantly higher in the positive control as compared to the negative control. Glucose level increased significantly 30 minutes after administration in the positive control as compared to the treated groups but this glucose level decreased significantly from 30 minutes up to 120 minutes. The percentage of decrease was higher with the FOHT extract (21.566%) as compared to the FOHF extract (8.208%).

III.1.6. Modulatory effects of extracts on some biomarkers of type 2 diabetes

After 14 days of experimentation, the results of the biochemical parameters and the body weight evaluated between the control groups revealed the importance of the implication of diet (atherogenic diet, high fructose and high cholesterol) in the pathology of our animals.

III.1.6.1. Effect of extracts on the body weight of experimental rats

Concerning body weight parameters, it was notice that the negative control had a significant increase of about 31.448 g (16.24%) from the first to the fourteenth day of experimentation as compared to 18.146 g (9.27%) observed in rats of the positive control group. The FOHT and FOHF extracts showed a significant decrease in the variation of body weight as compared to the positive control (XIX).

	Weight T0 (day 1)	Weight T2 (day 7)	Weight T3 (day 14)
Negative control	193.614 #177; 5.780	214.284 #177; 2.206	225.062 #177; 2.924
Positive control	195.672 #177; 7.495	207.506 #177; 7.551^*a	213.818 #177; 7.291^*a
Treated groups
FOHT(300mg/Kg)	195.942 #177; 10.866	177.912 #177; 9.405^b	182.546 #177; 12.375^b
FOHF(300mg/Kg)	194.214 #177; 10.329	186.784 #177; 12.715^b	191.372 #177; 12.973^b

a and b : significant difference between the positive control and the groups FOHT and FOHF at p< 0,05.

III.1.6.2.Effect of extracts on fasting blood glucose after experimentation

The fasting blood glucose at the end of experimentation was not significantly different (p<0.05) between the negative and the positive control group. The fasting blood glucose was significantly suppressed by the hydroethanolic fruits extract of F. Ovata as compared to the positive control but the FOHT extract shows no significant (figure 14).

Figure 14 : Effect of extracts on fasting blood glucose after experimentation

a and b : significant difference between the control and the groups FOHT and FOHF at the threshold of 0,05;

III.1.6.3. Effect of extracts on markers of lipid profil after experimentation

A significant increase (p<0.05) in plasmatic concentration of total cholesterol (TC),triglycerides (TG), LDL-cholesterol and the atherogenic index TC/ HDL-cholesterol and LDL-cholesterol/ HDL- cholesterol in the positive control was observed as compared to the negative control (table XX).

Table XX: Effect of extracts on the markers of lipid profile after experimentation

Groupes	Total cholesterol (mg/dl)	Triglycerides (mg/dl)	HDL-cholesterol (mg/dl)	LDL-cholesterol (mg/dl)	VLDL- cholesterol (mg/dl)
Negative control	56.732#177;7.943	19.500#177;4.184	34.928#177;1.069	17.903#177;6.817	3.900#177;0.836
Positive control	86.169#177;2,053*^a	37.000#177;4,728*^a	28.026#177;2,857*^a	50.743#177;4,789*^a	7.400#177;0.945*^a
Treated groups
FOHT(300mg/Kg)	69.751#177;4.380^b	30.791#177;7.783^a	26.823#177;6.507^a	36.769#177;4.938^a	6.153#177;1.556^a
FOHF(300mg/Kg)	65.830#177;5.256^b	11.333#177;0.825^b	45.542#177;6.329^b	18.020#177;5.922^b	2.266#177;0.165^b

*: significant difference between the positive control and the negative control. Chol: Cholesterol

a and b : significant difference between the control and the test groups at the threshold of 0,05;

The consumption of this atherogenic diet and high fructose-high cholesterol, lead to a significant increase (p<0.05) in the concentration of total cholesterol which is contrary to the normal food. Also, we also observed a significant (p<0.05) decrease of this parameter in the test groups as compared to the negative control (table XX).

Equally, we observed a significant increase (p<0.05) in the concentration of triglyceride, LDL-cholesterol and VLDL-cholesterol in the positive control as compared to the negative control. Only the extract FOHF significantly reduced (p<0.05) the concentration of triglyceride, LDL- and VLDL- cholesterol.

Furthermore, we observed a significant increase (p<0.05) in the plasmatic concentration of HDL- cholesterol in the test group FOHF as compared to the positive control.

The atherogenic index CT/HDL- chol and LDL-chol/HDL-chol reduced significantly (p<0, 05) for the FOHF extract as compared to the positive control. There was no significant difference between the test group that receives the FOHT extract and the positive control.

III.1.6.4. Effect of extracts on the activity of transaminases (ASAT, ALAT), creatinine and total protein levels

A significant increase (p<0.05) in, the creatinine and protein levels were observed in the positive control as compared to the negative control. This translates abnormality in the functions of the kidney. The activity of transaminase ASAT/ALAT was not significantly different between negative and the positive control.

Table XXI: Effect of extracts on the activity of transaminases (ASAT, ALAT), creatinine and total protein levels

Groups	ASAT (U/L)	ALAT (U/L)	Creatinine (mg/dl)	Proteins (g/l)
Negative control	87.532#177;0,977	33.406 #177;0.977	1.202#177;0,442	68.476#177;3.041
Positive control	91.529#177;3,316^a	37.674#177;2,048^a	2.877#177;0,468^*a	83.258#177;3.419^*a
Treated groups
FOHT(300mg/Kg)	80.025#177;2.419^b	32.941#177;2.907^a	0.647#177;0.576^b	72.181#177;4.754^b
FOHF(300mg/Kg)	90.598#177;2.882^a	31.777#177;0.553^a	1.017#177;0.379^c	79.040#177;1.837^a

a, b and c : significant difference at the threshold 0,05 between positive control and the test groups.

There was a significant decrease (p<0, 05) in the concentration of creatinine in the test groups as compare to the positive control. The concentration of ALAT and ASAT of the test groups was not significantly different (p<0, 05) from that of the positive control.

In addition, the concentration of protein was significantly (p<0,05) reduced in the test group FOHT as compared to the positive control but there was no significant difference between the test group FOHF and the positive control.

The above results enable us to suggest that our atherogenic diet and high fructose-high cholesterol intake has a positive effect on the induction of hyperlipidemia and CVD.

III.1.6.7. Effect of extracts on nitric oxide level

From the figure below, we observed that nitric oxide level was significantly (p<0, 05) reduced in the positive control as compared to the negative control in the plasma but not the heart. This implies that the diet has produced a positive effect in the endothelia dysfunction.

Figure 15 : Effect of extracts on nitric oxide level in the plasma and heart

a and b : significant difference at the threshold 0,05 between positive control and the test groups

We also observed that at the level of the plasma, the positive control was significant lower (p<0, 05) than in the test groups whereas at the level of the heart the concentration of nitric oxide was not significantly different between the positive control and the test groups

III.2.Discussion

The present study deals with two dimensions of the antidiabetogenic effects of the plant extracts of F. ovata. In one dimension, the hypoglycemic effect was measured. In the other dimension hypolipidemic potential of this plant extracts was studied as there is a close correlation between hyperglycemia and hyperlipidemia.

Preliminary, all four extracts screened for phytochemical, revealed the presence of groups of bioactive compounds such as alcaloids, glycosides, saponins, and polyphenolic compounds such as flavonoids, tannins and phenols. Phlobatannins were absent in the fruit extracts of F. ovata. These results correlate with that of Poongothai (2011) whose investigation on the preliminary phytochemical screening of Ficus racemosa linn bark reveal the presence of the above compound and stated that they possess a variety of biological activity including hypoglycaemia. Previous studies done on methanolic bark of F. ovata extract reveal the presence of certain triterpenoids such as â sitosterol, lupeol, and oleanolic acid (Kuate et al., 2009) which are known to possess some antidiabetic activities..

Generally the polyphenolic content test and the DPPH antiradical activity test showed that hydroethanolic extracts of fruits and twigs had the best solvent system as compared to the ethanolic extracts for each plant part. When comparing the total water soluble phenolic concentration with the DPPH radical scavenging antioxidant activity of the fruits and twigs extracts (table XIII and figure11), no positive correlation was observed. This goes to support the hypothesis of Brand Williams et al. (1995) that the DPPH kinetic is proportional to the amount of OH group present on the phenolic compound (Claudia et al., 2008). Thus, the hydroethanolic extracts may be rich in phenolic coumpounds that have many OH groups leading to it high DPPH scavenging activity. These compounds act as hydrogen donors to free radicals by stopping lipid peroxidation at the stage of initiation (Claudia et al., 2008).

Concerning acute toxicity, the extracts administsrated at a unique dose of 5000mg/Kg lead to no deaths. Following the classification of OECD (2001), which states that substances administstared at a dose =5000mg/Kg of BW and that does not lead to a lethal effect, can be presented as weakly toxic. This suggests that our extracts could be considered as being weakly toxic.

In this study, we observed the inhibition of alpha amylase activity by the hydroethanolic extracts with the fruits showing a high inhibition profile as compared to the twigs. Also, concerning the hypoglycemic test (BGT) in hyperglycemic rats, we did not observe a significant reduction of glycemia between the control and the test groups throughout the 5 hours of experiment. The percentage decrease on the blood glucose level of the hydroethanolic twigs (8.908%) was higher than that of hydroethanolic fruits (5.747%). The antihyperglycemia test done on normal rats showed that our extracts have high lowering effect on glycemia 30 minutes after glucose loads as compared to the positive control. From the 60^th minutes to the 120^th minutes, the glucose level was higher in the test groups than the positive control. This may be due to the slow metabolism of glycosides present in our plant that increases the blood glucose level. The percentage decrease on the blood glucose level of the hydroethanolic twigs (21.566%) was higher than that of hydroethanolic fruits (8.208%). This percentage decrease in blood glucose in the hypoglycemic test and the improved glucose tolerance may be due to various phytochemicals found to possess a wide range of activities, which may help in protection against chronic diseases. For example, glycosides, saponins, flavonoids, tannins and alkaloids have hypoglycemic activities; anti- inflammatory activities. The terpenoids have also been shown to decrease blood sugar level in animal studies (Poongothai, 2011). This made us think that the hydroethanolic extract may act by stimulating the secretion of insulin in beta cells of pancreas, increasing insulin sensitivity in addition to inhibition of the alpha amylase activity and many other enzymes involved in the transformation of dietary carbohydrate or glycogen to glucose. This correlate with the study done by Tormo et al. (2004) where after the administration of the polyphenolic extracts of fruits which had presented a high inhibition of the alpha amylase activity in rats, showed a lowering effect on the blood glucose of treated rats. This also correlates with the work by Ortiz-Andrade et al. (2006) on Glucosidase inhibitory activity of the methanolic extract from Tournefortia hartwegiana where Pharmacological investigations, reported that â-sitosterol induced the uptake of insulin from â-cells and produced an anti-hyperglycemic effect. On the other hand, stigmasterol, lupeol, ursolic and oleanolic acids showed to have hypoglycemic activity. Oleanolic acid and semi-synthetic derivatives were described as â -glucosidase inhibitors.

It is now well established that fructose feeding causes insulin resistance in experimental animals. For this study the fasting blood glucose at the end of experimentation was not significantly different between the control groups and FOHT extract but that of the FOHF extract was significantly lower than that of the positive control group. This could be explained by the fact that the time of experimentation was not long enough to result to insulin resistance or glucose intolerance as the glycemia after experimentation for the positive control was less than 110mg/dl. This result is contrary to that of Idowu et al. (2010) whose work on the glycemic effect of Ficus exasperata in fructose induced glucose intolerance and found that the extract ameliorated glucose intolerance. Inspite of the absent of glucose intolerance, FOHF extract showed a blood glucose lowering activity in vivo and this could be due to the fact that the extracts may stimulate insulin secretion by the pancreas or/and enhance insulin sensitivity in various organs especially the muscle and the live in a manner similar to sulfonylureas.

The consumption of high fat diet by rats associated with cholesterol and fructose throughout the sub acute experiment resulted in a group of metabolic disorders which was felt at the level of some plasma biochemical parameters in the absence of treatment. During the experimental period, an increase in body weight variation was observed in the negative control compared to positive control. This is contrary to the result obtained by Raneva and Shimasaki (2005) who obtained the increase of body weight in mice using high-fat diet during their study on the effects of green tea catechins on lipid peroxidation on the organs of mice. In the treated groups, the extracts FOHT and FOHF showed a significantly low weight variation. Phytochemical Screening of the extracts revealed significant (p <0.05) presence of polyphenols which could be involved in various mechanisms leading to reduced energy reserves and thus reducing the variation of BW. They may stimulate hepatic lipid metabolism and low accumulation of fatty acids in the liver and visceral organs as shown by Murase et al. (2002).

The results of lipid profile showed that the atherogenic diet, high fructose-high cholesterols significantly increased (p<0.05) plasma concentrations of total cholesterol, triglycerides and LDL cholesterol in the positive control compared to negative control (Table XIX). These results are comparable to those of Czerwinski et al. (2004) who show increased consumption of dietary cholesterol resulted in a high cholesterol, high triglyceride levels, high plasma lipid peroxides and atherogenic index chol-LDL/chol-HDL. These high levels of LDL cholesterol in the positive control could be attributed to their lack of recognition by their receptors on the cell membrane. If LDL is not recognized by it receptors could cause oxidation and endothelial dysfunction promoting leukocyte and platelet adhesion and release of growth factors necessary for atherogenesis (Lavoie, 2003). The treatment with the extract FOHF resulted in a significant decrease (p <0.05) in plasma TC, triglycerides, VLDL and LDL-cholesterol. But this decrease was not significant with the extract FOHT. HDL-cholesterol concentration increased significantly with the administration of the extract FOHF (Table XX). This improvement in lipid profile can be explained by the presence of saponins in the extracts FOHF as shown by Dhandapani (2007), working on the hypolipidemic activity of extracts of Eclipta prostrata leaves in male albino rats of Wistar strain. Reports show that saponins possess hypocholesterolemic and antidiabetic properties. Increased atherogenicity indices TC / HDL-chol and chol-LDL/chol-HDL was observed in rats of untreated group receiving the atherogenic diet (positive control), which corroborates Dhandapani (2007) which showed that consumption of high fat diet increased the atherogenic index. The administration of the extract FOHF resulted in a significant decrease (p <0.05) of these index with increase in the protection against the development of atherosclerosis. Hyperlipidemia is attributable to excess mobilization of fat from the adipose tissue due to the under utilization of glucose (Mohana et al., 2010). Regarding the mechanism of action saponins found in F ovata may enhance the activity of enzymes involved in bile acid synthesis and its excretion by precipitating cholesterol from micelles and interfere with enterohepatic circulation of bile acids making it unavailable for intestinal absorption (Santosh et al., 2009). Moreover, a significant decline in plasma LDL-cholesterol in treated groups could be correlated with saponin content of F. ovata, saponins may enhance the hepatic LDL-receptor levels, increase hepatic uptake of LDL-cholesterol and aid its catabolism to bile acids. Also saponins may lower TG by inhibiting pancreatic lipase activity (Santosh et al., 2009). Furthermore, the decline in VLDL cholesterol levels in treated groups could be directly correlated to a decline in TG levels of these groups, as it is well established that VLDL particles are the main transporters of TG in plasma (Santosh et al., 2009). Thus, a simultaneous decline in both TG and VLDL-cholesterol in treated groups indicates the possible effect of saponins (Mohana et al., 2010).

The concentrations of total protein, creatinine and activity of the transaminases ASAT and ALAT, reflect the degree of renal and hepatic damage generated upon exposure to risk factors of cardiovascular disease (Wasan et al., 2001) and can lead to various complications. In this study, there was no significant difference (p <0.05) in the markers of hepatic toxicity (ASAT and ALAT activities) as compared to the positive control after the sub acute experiment. There was a significant decrease (p=0.05) in the markers of renal toxicity (total protein and creatinine levels) as compared to the positive control. These could be attributed to polyphenols in the extracts of F. ovata that may act in the regeneration of reduced glutathione as a proton donor to counteract the action of free radicals.

The low concentration of nitric oxide in the positive control as compared to the test groups at the level of the plasma could be due to increased concentration of reactive oxygen species which lead to endothelia dysfunction especially by inhibiting the synthesis and action of nitric oxide. This correlates with the work done by Hadi et al. (2007). The concentration of Nitric oxide was high in the extract FOHT than in the extract FOHF.

CONCLUSION

In conclusion, the results from this study whose objective was to evaluate the antihyperglycemic, hypoglycemic, antihyperlipidemic and in vitro anti á-amylase properties of twigs and fruits extracts of Ficus ovata showed that, firstly hydroethanolic fruits and twigs were the most active extracts due to their high antioxidant and antiamylase activities. Moreover, the acute toxicity study showed that FOHT and FOHF were weakly toxic since no death was recorded at the experimental dosage of 5000mg/kg BW. In addition, acute hypoglycemic and antihyperglycemic studies revealed that hydroethanolic twigs extract was most active compared to the fruits extracts. Finally the sub acute preventive study showed that the hydroethanolic fruit extract was most active in ameliorating the rise in blood lipids and glucose levels induced by the high fructose high cholesterol diet.

These results suggest that hydroethanolic extracts of Ficus ovata twigs and fruits could be of interest in the prevention of hyperlipidemia and hyperglycemia associated to type 2 diabetes

PESPECTIVES

· determination of precise active substance(s), site(s) and mechanism(s) of its pharmacological effect;

· Evaluation of antidiabetic activity on streptozotocin induced diabetic models;

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APPENDIX

APPENDIX 1: Composition of polyvitamin and amino acids in hydrosoluble powder

APPENDIX 2: Reagents for the determination of total cholesterol

Working reagent (WR): The content of the reagent 2 was dissolved with the corresponding volume of reagent 1 followed by capping and mixing gently to dissolve contents. Stability of the WR: 4 months at +2 to +8°C or 40 days at room temperature.

APPENDIX 3: Reagents for the determination of Triglycerides

Working reagent (WR): The content of the reagent 2 was dissolved with the corresponding volume of reagent 1 followed by capping and mixing gently to dissolve contents. Stability of the WR: 6 weeks at +2 to +8°C or 1 week at room temperature.

APPENDIX 4: Reagents for the determination of HDL cholesterol

APPENDIX 5: Reagents for the determination of Total protein

Buiret reagent: add 100mls of distilled water to one vial of Biuret concentration. Stable for 12 month when sealed and stored at +15-25°C. Protected from light

Blank reagent: dilute the content of blank reagent bottle with 200mls of distilled water, rinsing the bottle thoroughly. Stable for 12 months when sealed and stored at +15-25°C. Standard is ready for use.

APPENDIX 6: Reagents for the determination of creatinine

Working reagent (WR): Equal volume of the reagent 2 was mixed with the corresponding volume of reagent 1 followed by capping and mixing. Stability of the WR: 10 days at +15 to +25°C.

APPENDIX 7: Reagents for the determination of AST and ALT respectively

Working reagent (WR): The content of Reagent 2 was dissolved with the corresponding volume of Reagent 1 (buffer substrate) followed by capping and mixing to dissolve contents. Stability: 21 days at +2 to +8°C or 3 days at room temperature (+15 to +25°C).

Evaluation of the hypoglycemic, hypolipidemic and anti alpha amylase effects of extracts of the twigs and fruits of ficus ovata vahl (moraceae)

DEDICATION