ANTI-DIABETIC EFFECT OF METHANOLIC EXTRACT OF Parkia biglobosa LEAF IN ALLOXAN INDUCED DIABETIC RAT

ANTI-DIABETIC EFFECT OF METHANOLIC EXTRACT OF Parkia biglobosa LEAF IN ALLOXAN INDUCED DIABETIC RAT


ABSTRACT

Parkia biglobosa is used traditionally in Nigeria to treat a range of ailments including diarrhoea, pneumonia, ulcers, burns, coughs, jaundice and diabetes. In present study, anti-hyperglycaemic effect of Parkia biglobosa crude methanolic leaf extract were investigated against Alloxan induced diabetic rats. A total of twenty five (25) rats were randomly allocated to 5 groups of 5 rats each. Groups 4-5 were treated with methanolic crude extract of Parkia biglobosa leaf (100mg and 300mg). Group 3 served as standard control (glibenclimide 2.5mg/kg b.wt) and group 2 served as diabetic control, while Group 1 served as the normal control. All treatments were administered orally for 28 days. Fasting blood glucose level and body weight were monitored during the study period. Results reveal that diabetic untreated rats didn’t have any significant decrease (p<0.05) in body weight and significant increase (p<0.05) in the blood glucose level. However, treatment with P. biglobosa leaf  significantly decreased (p<0.05) blood glucose level when compared with the untreated control. The 300mg of methanolic extract had the best activity with significant reduction (p<0.05) in the blood glucose level when compared to the normal control. Conclusively, Parkia biglobosa leaf possess significant anti-hyperglycaemic properties against Alloxan induced diabetic rat with the 300mg of methanolic being the most active, thus can serve as a potent anti-hyperglycaemic and is therefore recommended for further isolation of the active components.

 




 

 CHAPTER   ONE

INTRODUCTION AND LITERATURE REVIEW

1.1  Introduction

Diabetes mellitus (DM) is characterized by chronic hyperglycaemia with disturbances of carbohydrates, fat, and protein metabolism resulting from defect from insulin secretion, insulin action, or both. The prevalence of diabetes is rapidly rising all over the globe at an alarming rate. There is an increase in the prevalence of type 1 diabetes also, but the main cause of diabetic epidemic is type 2 diabetes mellitus. The international Diabetes federation(IDF) estimates the total number of diabetic subjects to be around 40.9 million in India and this is further set to rise to 60.9 million by the year 2025 (Sicree et al., 2006). The majority of cases of diabetes fall on two broad etiopathogenetic categories now called type 1 and type 2 diabetes mellitus. The effects of diabetes mellitus includes long–term damage, dysfunction and failure of various organs. Diabetes mellitus may present with characteristic symptoms such as thirst, polyuria, blurring of vision, and weight loss (Ganie, 2005). In its most severe forms, ketoacidosis or a non ketotic hyperosmolar state may develop and lead to stupor, coma and, in absence of effective treatment, death. Often symptoms are not severe, or may be absent, and consequently hyperglycemia sufficient to cause pathological and functional changes may be present for a long time before the diagnosis is made. The long–term effects of diabetes mellitus include progressive development of the specific complications of retinopathy with potential blindness, nephropathy that may lead to renal failure, and/or neuropathy with risk of foot ulcers, amputation, Charcot joints, and features of autonomic dysfunction, including sexual dysfunction (McCance et al., 1997). Animal studies show islet damage following prolonged exposure to hyperglycemia, which can be prevented by treatment, and several studies show an improved insulin response after the correction of hyperglycemia (Nattrass and Bailey, 1999). 

Nature has presented to humanity the gift of vast therapeutic and wide varieties of medicinal plant. There are over 250,000 species of flowering plant on earth, 155,000 of which are found in tropics (Cordell et al., 2008) .Plants are the first and true medicine that have been found useful in many ways. However it is widely believed that these valuable medicinal resources in plants are largely untapped because of inadequate scientific technical and commercial infrastructures in developing countries (Olayiwola, 1993).

Parkia biglobosa belongs to the plant family Mimosaceae of the order Leguminisae.  Parkia biglobosa popularly known as the African locust bean tree is known in Yoruba as Igba, or Irugba, in Hausa as Dorowa and in lbo as Origili. The fermented seeds of P. Biglobosa are used in all parts of Nigeria and indeed the West Coast of Africa for seasoning traditional soups. Similarly, both trees form a crown so are often grown as shade trees, The roots, barks, leaves, stems, flowers, fruits and seeds of P. biglobosa are all used medicinally to treat a range of ailments including diarrhoea, ulcers, pneumonia, burns, coughs, jaundice, diabetes e.t.c. (Sacande and Clethero, 2007). The pulp contains higher cellulose and sucrose but less ascorbic acid than the cotyledons. The pulp also contains simple sugars except maltose (Alabi et al., 2005). The seeds of P. biglobosa on fermentation are used in cooking stew and soup. The sweet yellow pulp contains 60% sugar when ripe and the seeds contain 30% protein as well as vitamins and minerals (Sacande and Clethero, 2007). The fruit pods are used to produce an insecticide powder for treating crops. Parkia species have found use traditionally as foods, medicinal agents and are of high commercial value. The pulverized bark of P. bicolor is employed in wound healing. P. biglobosa is known to provide an ingredient that is used in treating leprosy, and for treating hypertension. In Gambia, the leaves and roots are used in preparing a lotion for sore eyes. A decoction of the bark of P. biglobosa is also used as a bath for fever, as a hot mouthwash to steam and relieve toothache. The pulped bark is used along with lemon for wound and ulcers (Irvine, 2009). Parkia plants have been identified as source of tannins, saponins, gums, fuel and wood Seeds of various species of Parkia have also been investigated for their protein and amino acid contents (Fetuga et. al., 1999) 


1.2 Statement of Problem

Diabetes mellitus is known to be an epidemic disease that has plagued humanity. It has no permanent cure and is only managed to improve life expectancy. The therapeutic cost and side effect accompanying the use of standard anti-diabetic drugs have constituted a major challenge in the management of diabetes; and the bid to improve the life expectancy of people living with it on a global scale has been left vague.

1.3   Justification of the study

Based on the side effects and other limitations accompanying the use of conventional diabetic drugs; and the need to exploit our immediate environment for new and efficient drugs to manage diabetes which is a worldwide epidemic, the need to investigate the hypoglycaemic  effect of this plant (Parkia biglobosa) became relevant.

1.4 Aim and Objectives

Aim

To investigate the anti-diabetic effect of methanolic extract of Parkia biglobosa in the management of diabetic in Alloxan induced diabetics rats.

The objectives are:

To perform crude extraction using methanol.

To determine the phytochemical constituent of the plant 

To compare the body weight of the diabetic rats and the normal rats.

To determine the in vivo anti-hyperglycemic study of the methanolic extract of Parkia biglobosa leaf. 

1.5 LITERATURE REVIEW

1.5.1 Diabetes Mellitus

The term diabetes mellitus describes a metabolic disorder of multiple causes characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long–term damage, dysfunction and failure of various organs. Diabetes mellitus may present with characteristic symptoms such as thirst, polyuria, blurring of vision, and weight loss (Alberti, 1996). In its most severe forms, ketoacidosis or a non ketotic hyperosmolar state may develop and lead to stupor, coma and, in absence of effective treatment, death. Often symptoms are not severe, or may be absent, and consequently hyperglycemia sufficient to cause pathological and functional changes may be present for a long time before the diagnosis is made. The long–term effects of diabetes mellitus include progressive development of the specific complications of retinopathy with potential blindness, nephropathy that may lead to renal failure, and/or neuropathy with risk of foot ulcers, amputation, Charcot joints, and features of autonomic dysfunction, including sexual dysfunction (McCance et al., 1997). People with diabetes are at increased risk of cardiovascular, peripheral vascular and cerebrovascular disease several pathogenetic processes are involved in the development of diabetes. These include processes which destroy the beta cells of the pancreas with consequent insulin deficiency, and others that result in resistance to insulin action. The abnormalities of carbohydrate, fat and protein metabolism are due to deficient action of insulin on target tissues resulting from insensitivity or lack of insulin (Molbak et al., 1994).

There are different types of diabetes mellitus and they are categorically divided into two types. 

1.5.1.1 Type 1 diabetes mellitus: 

Type 1 diabetes mellitus, results from insulin deficiency following destruction of the insulin-producing pancreatic beta cells. It most commonly presents in childhood but one-fourth of cases are diagnosed in adults. The incidence of type 1 diabetes varies depending upon various factors like age, family history, environmental factors etc. Incidence rates in children <14years ranging from 0.1/100,000 per year in China to 37/100,000 per year in Finland (Karvonen et al., 2000). The incidence of childhood type 1 disease is rising worldwide, with reported annual increases of 2 to 5 percent in Europe, the Middle East, and Australia (Mohammad, 2012). Type 1A diabetes mellitus results from autoimmune destruction of the insulin-producing beta cells in the islets of Langerhans. In genetically susceptible subject, this process is probably triggered by one or more environmental agents. Type 1B diabetes mellitus refers to non-autoimmune. Type 1 diabetes can be present in several different ways (Haller et al., 2005).Classic new onset, diabetic keto acidosis or as asymptomatic incidentally discovered diabetes. Classic new onset presents as hyperglycemia without acidosis. Symptoms are caused by hyperglycemia and include polyuria, polydipsia, and weightloss despite increased appetite initially. Children with type 1 diabetes often present with diabetic ketoacidosis (hyperglycemia and keto acidosis). The International Society for Pediatric and Adolescent Diabetes (ISPAD) in2007 defined the following biochemical criteria for the diagnosis of DKA (Dunger et al.,2004).Hyperglycemia, blood glucose of >200 mg/dL (11 mmol/L) a metabolic acidosis, defined as a venous pH <7.3 and/or plasma bicarbonate <15 meq/L(15 mmol/L). These abnormalities are accompanied by hyper ketosis (concentration of total ketone bodies >5 mmol/L) and hyper osmolality. Some children will be diagnosed with type 1 diabetes before the onset of clinical symptoms.

1.5.1.2 Type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is the most common form of diabetes. It is characterized by disorders of insulin action and insulin secretion, either of which may be the predominant feature. The risk of developing T2DM increases with age, obesity, and physical inactivity. T2DM shows strong familial aggregation, so that persons with a parent or sibling with the disease are at increased risk, other individuals with obesity, hypertension, or dyslipidemia and women with a history of gestational diabetes are also at increased risk of developing T2 DM. (Ganie, 2005). T2 DM is now considered to be a facet of Syndrome X (Reaven’s syndrome) comprising of hyper insulinemia, dyslipidemia, hypertension and hyperglycemia.T2DM frequently goes undiagnosed for many years because the hyperglycemia develops gradually and in the earlier stages is not severe enough to produce the classic symptoms of diabetes; however, such patients are at increased risk of developing macrovascular and microvascular complications (Owen, 1998). The classic symptoms of polyuria, thirst, recurrent blurred vision, paresthesias, and fatigue are manifestations of hyperglycemia and osmotic diuresis and are present late in the course of disease. Diabetes should be suspected in women with chronic Candida vulvovaginitis as well as in those who have delivered large infants (4.1 kg) or have had polyhydramnios, pre-eclampsia, or unexplained fetal losses (Owen, 1998). 

1.5.2 Insulin secretion in Type 2 diabetes

Insulin secretion is the end result of a complex cascade of actions within the beta-cell. Nutrient metabolism by the beta-cell increases ATP concentrations, which results in closure of the ATP-sensitive potassium channels. The decrease in potassium ion leads to depolarization of the cell membrane, allowing the voltage-dependent of calcium ions. This in turn leads to the extrusion of insulin. There is no evidence to suggest that this series of steps required for insulin secretion differs qualitatively between normal subjects and Type 2 diabetes patients. Quantitatively, however, there is a major difference in the relation of stimulus to resultant secretion. The interpretation of basal insulin levels and insulin secretion in response to stimuli such as oral or intravenous glucose in Type 2 diabetes patients is problematical. Some findings of elevated insulin levels in patients with Type 2 diabetes are not consistent with studies using highly specific insulin assays (Williams et al., 1991).Much of the apparent insulin measured by the non-specific antibodies used in radio-immunoassays can be accounted for by pro-insulin or split pro-insulin products, as shown when specific immune radio-metric assays are employed.  If intravenous glucose is used as a stimulus to insulin secretion, the situation is somehow clearer. The normal response to intravenous glucose is biphasic (Nattrass and Bailey, 1999). In Type 2 diabetes, first-phase insulin secretion is lost, but the second phase may be normal or even exaggerated. An inadequate first-phase insulin release inevitably results in hyperglycemia and thus a greater stimulus to second-phase secretion. At a later stage of the disease, the second-phase response also becomes impaired. Impaired insulin secretion could arise from either beta-cell dysfunction or reduced beta-cell mass. In animals, more than 85% of the islet tissue must be destroyed to give insulinopaenic diabetes, and it is likely therefore that both lesions co-exist. Animal studies show islet damage following prolonged exposure to hyperglycemia, which can be prevented by treatment, and several studies show an improved insulin response after the correction of hyperglycemia (Nattrass and Bailey, 1999).

1.5.3 Insulin action in Type 2 diabetes

The first step in any action of insulin is its binding to a specific receptor (Cheatham and kahn, 1991).There follows instigation and amplification of the signal via second-messenger pathways that control the various membranal, cytoplasmic and genomic actions of insulin Crucial for signal transmission is the activation of insulin receptor tyrosine kinase activity and the auto phosphorylation of tyrosine residues in the receptor. Several intracellular proteins have been implicated as insulin receptor substrates, the phosphorylation of which directs the insulin signal into different intracellular pathway (Cheatham and kahn, 1991). 

1.5.3 Management of Diabetes Mellitus

Diabetes mellitus is condition associated with number of complications including coronary heart disease, retinopathy, neuropathy etc. It is now clear that tight control of blood glucose significantly reduces the risk of complications of diabetes. Therefore, multidisciplinary approach, involving dieticians, endocrinologists /diabetologists, cardiologists, nephrologists, ophthalmologists, chiropodists etc. is needed for management of diabetes mellitus. Insulin is the only therapy available for patients with type 1 diabetes. Insulin replacement in patients with type 1 diabetes has been less than optimal because it is not possible to completely reproduce the normal physio-logic pattern of insulin secretion into the portal vein. The problem of achieving optimal insulin delivery remains unsolved with the present state of technology. The management of type 2 diabetes mellitus involves the use of therapeutic drugs such as the alpha glucosidase inhibitors and example of alpha glucosidase inhibitors are the acarbose, miglitol, and voglibose. Alpha glucosidase inhibitors are particularly useful for managing mild hyperglycemia(high blood glucose) in newly diagnosed people with type 2 diabetes patient as well as for managing after meal hyperglycemia(Rewers and Gottlieb, 2009).

1.5.4 Mode of Action of Alloxan

Alloxan has two distinct pathological effect: it selectively inhibits glucose-induced insulin secretion through specific inhibition of glucokinase. Streptozotocin is an antimicrobial agent and has also been used as a chemotherapeutic alkylating agent (White, 1963; Schein et al., 1967; Schein at al., 1974). In 1963, Rakieten et al. reported that Alloxan  is diabetogenic. Again, this insulinopenia syndrome, called ‘Alloxan diabetes’ (Schein et al., 1967), is caused by the specific inhibition of the pancreatic beta cells and Alloxan has been the agent of choice for the induction of diabetes mellitus in animals ever since (Arison et al., 1967; Lenzen, 1996). Streptozotocin inhibits insulin secretion and causes a state of insulin-dependent diabetes mellitus. Both effects can be attributed to its specific chemical properties, namely its alkylating potency. As with alloxan, its beta cell specificity is mainly the result of selective cellular uptake and accumulation.

1.5.5 Mechanism of Diabetes

Several mechanisms surround the hyperglycemic state present in diabetic patients. It could be as a result of insulin resistance, or damage to the pancreatic β-cells (as a result of drugs or other agents that cause necrosis to the islets of Langerhans), or absence/insufficient production of the hormone insulin (which is the hormone responsible for converting excess blood glucose into glycogen, which is then stored in the tissues). 

1.5.6 Conventional Antihyperglycemic Drugs

Incretin Mimetics : Incretin effect is the difference in insulin secretory response from an oral glucose load in comparison to glucose administered intravenously. The incretin effect is responsible for 50–70% of total insulin secretion after oral glucose intake (Nauck and Meier, 2016). The two naturally occurring incretin hormones that play important roles in the maintenance of glycemic control: glucose-dependent insulinotropic polypeptide (GIP, or incretin) and glucagon-like peptide (GLP-1); these peptides have a short half-life, as these are rapidly hydrolyzed by DPP-4 inhibitors within 1½ min. In patients with T2DM, the incretin effect is reduced or absent. In particular, the insulinotropic action of GIP is lost in patients with T2DM. Incretins decrease gastric emptying and causes weight loss. Because of impact on weight loss, these medications may find increasing use in diabetes.

Biguanide: Galega officinalis, a herbaceous plant, was found to contain guanidine, galegine, and biguanide, which decreased blood glucose levels (Viollet et al., 2012). Metformin is a biguanide that is the main first-line oral drug of choice in the management of T2DM across all age groups. Metformin activates adenosine monophosphate-activated protein kinase in the liver, causing hepatic uptake of glucose and inhibiting gluconeogenesis through complex effects on the mitochondrial enzymes (Viollet et al., 2012). Metformin is highly tolerated and has only mild side effects, low risk of hypoglycemia and low chances of weight gain. Metformin is shown to delay the progression  of T2DM, reduce the risk of complications, and reduce mortality rates in patients by decreasing hepatic glucose synthesis (gluconeogenesis) and sensitizing peripheral tissues to insulin (Viollet et al., 2012). Furthermore, it improves insulin sensitivity by activating insulin receptor expression and enhancing tyrosine kinase activity. Recent evidence also suggests that metformin lowers plasma lipid levels through a peroxisome proliferator-activated receptor (PPAR)-α pathway, which prevents CVDs (Viollet et al., 2012). Reduction of food intake possibly occurs by glucagon-like peptide-1 (GLP-1)-mediated incretin-like actions. Metformin may thus induce modest weight loss in overweight and obese individuals at risk for diabetes.

Metformin has an excellent safety profile, though may cause gastrointestinal disturbances including diarrhea, nausea, and dyspepsia in almost 30% of subjects after initiation. Introduction of metformin at low doses often improve tolerance. Extended release preparations seldom cause any gastrointestinal issues. Very rarely, metformin may cause lactic acidosis, mainly in subjects with severe renal insufficiency. Another potential problem arising from the use of metformin is the reduction in the drug’s efficiency as diabetes progresses. Metformin is highly efficient when there is enough insulin production; however, when diabetes reaches the state of failure of β-cells and resulting in a type 1 phenotype, metformin loses its efficacy.

GLP-1 Receptor Agonists:The currently GLP-1 receptor agonists available are exenatide and liraglutide. These drugs exhibit increased resistance to enzymatic degradation by DPP4. In young patients with recent diagnosis of T2DM, central obesity, and abnormal metabolic profile, one should consider treatment with GLP-1 analogs that would have a beneficial effect on weight loss and improve the metabolic dysfunction. GLP-1 analogs are contraindicated in renal failure.

Exenatide: Exenatide, an exendin-4 mimetic with 53% sequence homology to native GLP-1, is currently approved for T2DM treatment as a single drug in the US and in combination with metformin ± sulfonylurea. Because of its half-life of 2.4 h, exenatide is advised for twice-daily dosing. Treatment with 10 µg exenatide, as an add-on to metformin, resulted in significant weight loss (−2.8 kg) in comparison to patients previously treated with metformin alone. Exenatide is generally well tolerated, with mild-to-moderate gastrointestinal effects being the most common adverse effect.

Sulfonylureas: Sulfonylureas lower blood glucose level by increasing insulin secretion in the pancreas by blocking the KATP channels. They also limit gluconeogenesis in the liver. Sulfonylureas decrease breakdown of lipids to fatty acids and reduce clearance of insulin in the liver (Proks et al., 2002). Sulfonylureas are currently prescribed as second-line or add-on treatment options for management of T2DM. They are divided into two groups: first-generation agents, which includes chlorpropamide, tolazamide, and tolbutamide, and second-generation agents, which includes glipizide, glimepiride, and glyburide. The first-generation sulfonylureas are known to have longer half-lives, higher risk of hypoglycemia, and slower onset of action, as compared to second-generation sulfonylureas. Currently, in clinical practice, second-generation sulfonylureas are prescribed and more preferred over first-generation agents because they are proven to be more potent (given to patients at lower doses with less frequency), with the safest profile being that of glimepiride.

Hypoglycemia is the major side effect of all sulfonylureas, while minor side effects such as headache, dizziness, nausea, hypersensitivity reactions, and weight gain are also common. Sulfonylureas are contraindicated in patients with hepatic and renal diseases and are also contraindicated in pregnant patients due to the possible prolonged antihyperglycemic effect to infants. Drugs that can prolong the effect of sulfonylureas such as aspirin, allopurinol, sulfonamides, and fibrates must be used with caution to avoid hypoglycemia. Moreover, other oral antidiabetic medications or insulin can be used in combination with sulfonylurea and can substantially increase the risk of hypoglycemia.

Pramlintide: an amylin analog, is an agent that delays gastric emptying, blunts pancreatic secretion of glucagon, and enhances satiety. It is a Food and Drug Administration (FDA)-approved therapy for use in adults with T1DM. Pramlintide induces weight loss and lowers insulin dose. Concurrent reduction of prandial insulin dosing is required to reduce the risk of severe hypoglycemia. Other medications that may lower blood sugar include bromocriptine, alpha-glucosidase inhibitors like voglibose and acarbose, and bile acid sequestrants like colesevelam. It may be noted that metformin sequesters bile acids in intestinal lumen and thus has a lipid-lowering effect; also the same mechanism may contribute to gas production and gastrointestinal disturbances.

1.5.7 Medicinal plants

Medicinal plants are plants used as natural medicines. medicinal plants can be described as any plant in which one or more of its organ contains substances that can be used for the therapeutic purpose or which are precursors for the synthesis of useful drugs(UNESCO, 1996).Examples includes food, spices, perfumery plants, microscopic plants like fungi, actinomycetes used for isolating drugs especially antibiotics, fibre plants like cotton, flax, and jute used for preparation of surgical dressings .Plants are the first and only true medicines and have been found useful in several ways. They are used as teas or in other extracted forms for their chemical constituents, Also they are use as agent in drug synthesis. Nigerian flora has already made and will continue to make a great contribution to the health care of Nigerians (Abolaji et al., 2007).Infact, the indigenous medicinal plants forms an important component of the natural wealth of Nigeria.

Medicinal plant for example contain bitter substances that stimulates digestion, anti-inflammatory compounds that reduces swellings and pain, phenolic compounds that can act as antioxidants and venotonics, antibacterial and antifungal tannins that act as natural antibiotics, diuretic substances that enhance the elimination of waste products and toxins and alkaloids that enhance mood and give a sense of well-being (Gurib-Fakim, 2006).

1.5.8 Parkia biglobosa

Parkia biglobosa also known as dawadawa (Hausa), African locust beans (English), Igba/Iyere (Yoruba), Nere  have been known to be a native of Africa and is an important multipurpose tree of West African Savannah land and one of the most common species of the parkland agro-forestry system (Sacande and Clethero, 2007).The roots, barks, leaves, stems, flowers, fruits and seeds of P. biglobosa are all used medicinally to treat a range of ailments including diarrhoea, ulcers, pneumonia, burns, coughs, jaundice, diabetes e.t.c. (Sacande and Clethero, 2007). The pulp contains higher cellulose and sucrose but less ascorbic acid than the cotyledons. The pulp also contains simple sugars except maltose. In Gambia, the leaves and roots are used in preparing a lotion for sore eyes.


Figure 1: Parkia biglobosa tree Source: (Sacande and Clethero, 2007).

1.5.8.1 Taxonomic Description of Parkiabiglobosa

Kingdom:   Plantae   

Subkingdom: Viridiplantae

Super kingdom: Embryophyta

Division: Tracheophyta

Class: Magnoliopsida

Order: Fabales

Family:  Fabaceae

Genus: Parkia

Species: Parkia speciosa, parkia timoriana, parkia filicoidea, parkia bicolor 

1.5.8.2 Traditional uses

The traditional uses for P. biglobosa can be defined as non timber forest products (NTFP), which include wood energy (fuel wood and charcoal) and all other tangible products other than timber (Builders et al., 2014). Non-timber forest products derived from P. biglobosa are food, medicine, animal fodder, soil amendments, charcoal, and firewood. The most significant product from P. biglobosa is food. The food products collected from P. biglobosa are especially important due to the seasonality of fruit maturation and food availability. The seeds are used in preparation of dawadawa, a protein and fat rich food. The yellow starchy pulp that surrounds the seed is an important food supplement rich in Vitamin C and carbohydrates. The dried powder is often mixed with water to produce a drink called dozim.

1.5.8.3 Phytochemistry

The stem bark is reported to contain flavonoids, tannins, terpenes, saponins, sterols, phenols and reducing sugars (Builders et al., 2012).elemental analysis showed the presence of magnesium, calcium, iron, zinc, potassium, sodium and copper (Builders et al., 2012). Leaf of African locust bean tree contains flavonoids, tannins, saponins, cardiac glycosides, alkaloids and reducing sugars (Builders et al., 2011) Long chain ester of trans-ferulic acid, a mixture of long-chain cis-ferulates and different kinds of catechins (catechins and ferulates) were identified in the stem bark of P. biglobosa (Tringali et al., 2000). Phytochemical results indicated that the root bark of the plant contained a lot of glycosides and tannins, appreciable amounts of saponins and traces of alkaloids. (Udobi et al., 2009). Also saponins, carbohydrates, tannins and flavonoids were detected in the root of P. biglobosa (Udobi et al., 2009).The chemical composition of the African plant Parkia biglobosa (Fabaceae) roots and barks by Liquid Chromatography–Electrospray Ionization and Direct Injection Tandem Mass Spectrometry analysis was also investigated (Tala et al., 2013).Mass spectral data indicated that B-type oligomers are present, namely procyanidins and prodelphinidins, with their gallate and glucuronide derivatives, some of them in different isomeric forms. The yellowish fruit pulp is very rich in carbohydrate (60%), 10-20% of which is sucrose, 291mg Vitamin C. The seeds contain 35% proteins, 29% lipids, 16% carbohydrates, calcium and have good organo leptic properties. A non-toxic oil of variable composition is also present. Some sources indicate arachidic acid as the most abundant fatty acid, accompanied by behenic, stearic, palmitic and linoleic acids. (Elemo et al., 2011).The proximate analysis of the nutritive contents of P. biglobosa seeds indicated the presence of high amount of lipid, crude protein, pure protein, carbohydrates, total soluble sugar and starch (Alabi et al., 2005).

1.5.8.4 Antiplasmodial and antipyretic activity

The antiplasmodial activities of the methanol extract and methanol fraction of the stem bark of African locust bean tree were evaluated against malaria model Plasmodium berghei berghei and clinical isolates of Plasmodium falciparum. The crude extract and methanol fraction exhibited dose dependent reduction of parasitaemia at the different doses administered. Methanol fraction showed higher reduction of parasitaemia (Builder et al., 2012).Antipyretic properties of the crude extract and methanol fractions were studied; reduction in yeast induced hyperpyrexia was produced by the extract and the fractions. The methanol fraction exhibited a significant reduction in yeast induced elevated temperature (Builder et al., 2012).The antiplasmodial activity of the leaves of P. biglobosa was evaluated in vivo and in vitro against Plasmodium berghei berghei and clinical isolates of Plasmodium falciparum respectively. There was a dose dependent inhibition of parasitaemia in the in vivo antiplasmodial tests. The in vitro screening demonstrated a weak and concentration dependent activity of the extract against P. falciparum (Builders et al., 2012).

1.5.8.5 Analgesic and anti-inflammatory activity

Then N-hexane extract from the bark of P. biglobosa had some analgesic and anti-inflammatory effects (Kouadio et al., 2000).Intra peritoneal administration of the methanol extracts of P. biglobosa stalk significantly antagonized the formation of croton pellet granuloma in a dose-dependent manner. The extract also showed a dose-dependent inhibition of the croton oil ear inflammation in test animals, there was also appreciable inhibition of carrageenin-induced rat pawoedema compared with controls. The extracts of P. biglobosa further inhibited the arachidonic acid induced paw oedema in a dose-dependent manner comparable to the dual blocker, phenidone .Thus, suggesting that the observed anti-inflammatory activities may be produced by the inhibition of the lipo-oxygenase pathways, the cyclo oxygenase pathways or both which are involved in metabolism of arachidonic acid (Nwaehujor et al., 2011)

1.5.8.6 Antisnake venom

A water-methanol extract of P. biglobosa stem bark had been shown to possess antisnake venom activity. This extract also reduced the loss of responses to acetylcholine (Ach), carbachol and potassium chloride (KCl),which are normally blocked by N. nigricollis venom, and significantly reduced the contractures of the preparation induced by venom (Asuzu et al., 2003).

1.5.8.7 Anti-diarrhoeal activity

Research showed that P. biglobosa had anti-diarrhoeal properties in mice. Anti-diarrhoeal activities of the aqueous stem bark extract of P. biglobosa and its fractions designated PF1-PF4 investigated in mice indicated that the extract and its column chromatographic fraction F3 significantly (p < 0.05) and dose-dependently reduced frequency of stooling in castor-oil induced diarrhoea, castor-oil-induced intestinal fluid accumulation and intestinal transit (Tijani et al., 2009).

1.5.8.8 Anti-bacterial activity

The anti-bacterial activity of the stem bark and leaves of P.biglobosa has been conducted on four strains of Staphylococcus aureus isolated from patients in the National Hospital Yalgado Ouagadougou, Burkina Faso (Millogo-Kone et al., 2006). lso Antibacterial evaluation of the methanol extract and aqueous fractions of the leaf, stem bark and root of the African locust bean tree, Parkia biglobosa was carried out using the agar- well diffusion method. The extracts and their fractions were tested against two gram positive organisms – Staphylococcus aureus and Bacillus subtilis and two gram negative organisms–Escherichia coli and Pseudomonas aeruginosa. Results obtained confirmed a broad spectrum of activity as all the organisms used were inhibited by the extracts and their aqueous fractions (Udobi et al., 2010).

1.5.8.9 Anti-microbial activity

Ethanol extract of the P. biglobosa exhibited antimicrobial activities against the multi-drug resistant isolates (Adebayo et al., 2008).The antimicrobial screening of the leaves P. biglobosa was done using standard strains of microorganisms. The extracts exhibited a concentration dependent antibacterial, inhibiting the growth of the gram – positive bacteria used in the study (Ajaiyeoba, 2002).Antimicrobial activities of the aqueous stem bark extract of P. biglobosa and its fractions designated PF1-PF4 were investigated in against selected diarrhoea-causing organisms, the crude extract as well as fractions F3 and F4 strongly inhibited growth of selected microorganisms (Tijjani et al., 2009).

1.5.9 Antioxidant activity

Antioxidant activities of the leaves and stem barks were determined by the means of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay (Millogo-kone et al., 2009) The result of the antioxidant activities of the methanol extract and methanol fraction indicated that the sensitivity of the antioxidant activity of the methanol fraction is higher than that of crude un-fractionated methanol extract(Builders et al., 2012). Study on the antioxidant properties of the stem bark of P .biglobosa showed that the radical- scanvenging potential of P. biglobosa was dose-dependent; this activity was higher than that of ards (rutin, ascorbic acid, butylated hydro-anisole (BHA) and alpha tocopherol) (Ajaiyeoba et al., 2002).The stalk of P. biglobosa also showed in vitro anti-oxidant activities using the DPPH.

1.5.10 Anti-diabetic activity

The hypoglycaemic effect of fermented seeds of Parkia biglobosa, a natural nutritional condiment that features frequently in some African diets as a spice, was investigated in alloxan- induced diabetic rats(Odetola et al., 2006).

1.5.11 Hypertension

An alcohol extract of crude seeds of P. biglobosa showed anti-hypertensive activity and contractile effect on smooth muscles of the intestine, and increased the tonus and mobility of the uterus. Ichthyo toxic and molluscicidal activities have been recorded for the seeds due to the presence of saponins (Sina et al., 2012).

1.5.12 Hyperlipidaemia

The hypolipidemic effect and the improvement in serum lipid profile of triton-induced hyperlipidemic rats by Parkia biglobosa saponins were investigated. The result indicated that P. biglobosa-mediated therapeutic effects may be associated with its hypolipidemic components (Komolafe et al., 2013).The cardio protective effect of P. biglobosa stem bark used on iso proterenol (ISO) induced myocardial infarction in rats was evaluated. P. biglobosa ameliorated positively biochemical alterations, prevented oxidative stress and histological and morphological changes induced by iso proterenol (Adi et al., 2013).

1.6 Toxicity

The acute and sub-acute toxicity profile of the water and alcohol extracts of the stem bark of P. Biglobosa was investigated. The result of this study showed that the lethal dose (LD50)was greater than5000mg/kg per oral (p.o) for both extracts and the toxicity characteristics of the methanol and water extracts of the stem bark P. biglobosa in short time treatment with the extracts  (Builders et al., 2012).The result of acute toxicity study of the stem, leaf and root of P. biglobosa indicated that LD50 fell within the range of 500 – 5000 mg/kg body weight confirming them to be only slightly toxic and hence not potentially dangerous  (Abalaka et al., 2010).The toxicity of aqueous and ethanol extracts of Parkia biglobosa pods on Clarias gariepinus was investigated. It was concluded that aqueous and ethanol extracts of P. biglobosa pods are toxic to C. geriepinus juveniles with the ethanol extract being more toxic, which shows that apart from the bark of P. biglobosa, the pods has piscicidal property and can be put into use in the control and management of fish ponds to eradicate predators by farmers (Abalaka et al., 2010)




 

CHAPTER TWO

2.0 MATERIAL AND METHODS

2.1 Materials

2.1.1 Plant Collection

The freshly harvested leaf of Parkia biglobosa was collected from Aliero local Government Area of Kebbi State, Nigeria.

2.1.2 Reagent and Chemicals

All the reagent and chemicals used were of analytical grade and were products of Sigma Chemical Co., USA. The reagents were freshly prepared, and distilled water was used throughout the experiment.

2.1.4 Experimental animal

Healthy albino rats of average weight (120-150)g were purchased from Sokoto, Sokoto State. The rats were kept in clean plastic cages and maintained under standard laboratory conditions in the biochemistry laboratory, Kebbi State University of Science and Technology Aliero. They are allowed to acclimatize to laboratory conditions for two weeks and an access to rat pellets and water to feed ad libitum.

2.1.5 Equipment used for research work

Glucometer, glucometer strips (active accu check), weighing balance, oven, conical flask, beaker, water bath, retort stand, sample bottles, and other general equipment was used.

2.2 Methods

2.2.1 Sample preparation and extraction

The fresh leaf of Parkia biglobosa were rinsed in clean water and cut into pieces, and air dried in the laboratory for two weeks. The dried leaves of Parkia biglobosa were pounded to coarse powder using a mortar and pestle. Extraction of plant materials was performed by weighing 400g of powdered plant and extracted using 1500ml of methanol. The resulting extracts were concentrated in oven and the solvent were also evaporated. The concentrated extracts were stored in airtight vials.

2.2.2 Qualitative Phytochemical Screening

The Parkia biglobosa leaf extracts were analyzed for glycosides, alkaloids, saponins, tannins, flavonoids, steroids, phenols and resins using standard procedures.

2.2.2.1 Test for Glycosides: 2ml of acetic acid was added to 2ml of the extract. The mixture was cooled in cold water bath. 2ml of concentrated H2S04 was then added, colour development from blue to bluish green indicates the presence of glycosides (Valsala and Karpagaganapathy, 2002).

2.2.2.2 Test for Alkaloids: 1.27g of iodine and 2g of potassium iodide dissolved in distilled water (Wagner’s reagent), this was added to the plant extract. Appearance of reddish brown colour confirmed the presence of alkaloids (Harbone, 1998).

2.2.2.3 Test for saponins: To 2ml of the extract, 2ml of distilled water was added and agitated in a test tube for 5minutes. The formation of foams indicates the presence of saponin (Valsala and Karpagaganapathy, 2002).


2.2.2.4 Test for tannins: 5 drops of 0.1% ferric chloride was added to 2ml of extract, a brownish green or blue black coloration indicates the presence of tannins (Valsala and Karpagaganapathy, 2002)

2.2.2.5 Test for Flavonoid: 2ml of 10% Sodium hydroxide was added to 2ml of the extract in a test tube. An intense yellow colour was formed which turned colorless upon addition of 2ml of dilute hydrochloric acid indicating the presence of flavonoid (Valsala and Karpagaganapathy, 2002).

Test 2.2.2.6 for Steroids: To 1ml of the extract, 0.5ml of acetic anhydride and 0.5ml chloroform were added and concentrated sulphuric acid later added. Formation of a brownish green ring at the contact of the two liquids indicates the presence of steroids (Harbone, 1998).

2.2.2.7 Test for Phenols: 2ml of the extract was mixed was mixed with ferric chloride solution. Green or dirty green precipitates indicate the presence of phenolic compounds (Elmahmood and Doughari, 2008).

2.2.2.8 Test for Terpenoids: 5ml of the extract was added to 2ml chloroform.3ml of conc.H2SO4 was carefully added to form a layer. Reddish brown coloration of the interface indicates the presence of terpenoids.

2.2.3 Animal studies 

2.2.3.1 Induction of the rats

The rats were first weighed and labelled for easy recognition. 0.25g of Alloxan (150mg/kg) was dissolved as 5ml in distilled water. The   albino rats were then induced with diabetes through intraperitoneal after a 12hour fasting at a dose of 45mg/kg body weight. It involved treatment with the extracts for 72hours after induction of diabetes in the rats. 30 rats (of which 25 came up with diabetes mellitus) were divided into 5 groups of five (5) each.

 2.2.3.2 Treatments with the methanolic extract of parkia biglobosa leaf

Group 1 (Control): Rats in this group received no medication, distilled water only.

Group 2 (Negative Control): Rats in this group received alloxan at a dose of 45mg  

Group 3: Rats in this group received glibenclamide at a dose of 2.5mg body weight

Group 4: Rats in this group received 100mg of methanolic extract of Parkia biglobosa leaf

Group 5: Rats in this group received 300mg of methanolic extract of  Parkia biglobosa leaf

The treatment lasted for 28 days, with their weights and fasting blood sugar (FBS) being determined weekly. On the 2 days, the animals were sacrificed under chloroform anesthesia.

2.2.3.4 Blood collection

The animal was scarificed after 29 days the blood of animal was collected and centrifuge. The serum was used for Biochemical analysis

 

2.2.3.5 Method for analysing biochemical parameters

ASP(µ/L)

Reitman and Frankel,1957


ALT(µ/L)

Reitman and Frankel,1957


ALP(µ/L)

Lowry et al., 1946


TOTAL PROTEIN (g/dl)

Gomail et al.,1949


TOTAL BILIRUBIN(mg/dl)

Jendrassik and Grof, 1936



2.2.3.6 Statistical Analysis

The results were reported as mean ± standard error of mean (SEM). The values were analysed using Statistical Package for Social Sciences (SPSS) windows program version 20. Statistical significance of difference between means were carried out using one-way analysis of variance (ANOVA). p<0.05 was considered to be significant. 

CHAPTER THREE

 3.0 RESULTS 

3.1 PRELIMINARY PHYTOCHEMICAL SCREANING

The result of phytochemical analysis of methanolic of  parkia biglobosa leaf extract revealed the presence of some secondary metaboliteas show in the table below 













Table 2.1:  Phytochemicals Constituents of Parkia biglobosa Methanolic leaf Extract

Phytochemical

Observation


  Glycoside

                  + + +


Alkaloid

                    +


Flavonoid

                  + + +


Terpenoid

                  + + 


Steroids

                  + +


Phenols

                    +


Anthraquinones

                     _


Tannin

                   + + +


Saponin

                   + + 


Key: (+): present, (-): Not detected, (+ +): moderately present, (+ + +): abundantly




 

3.2 Anti-Diabetics Activity of Methanolic Extract of Parkia biglobosa Leaf on fasting blood glucose in Alloxan-Induced Diabetic Rats 

The results revealed that diabetic untreated rats had progressive decrease in body weight and significantly increase in the blood sugar levels. However treatments with with methanolic extract of Parkia biglobosa significantly decrease the blood glucose and improves the body weight of the diabetic rat compared to the untreated control. 300mg of methanolic extract of Parkia biglobosa has the best hypoglycaemic activity with significant decrease (p<0.05) in the blood sugar level.


 



Table 3.2: Glucose Levels of Rats Administered with methanolic extract of Parkia biglobosa leaf

GROUPS

DAY 7

DAY 14

DAY 21

DAY 28


Control

95.16 ± 2.63

94.01 ± 1.92

93.97 ± 0.88

92.92 ± 0.89


100mg/kg of Alloxan

243.51 ± 7.39*

243.90 ± 6.32*

209.86 ± 9.54**

215.18 ± 8.88**


2.5mg/kg of Glibenclamide

229.18 ± 15.22*

167.06 ± 3.54*

99.89 ± 4.22

92.07 ± 3.32


100mg of P.B

237.000 ± 7.76*

185.00 ± 4.00*

119.97 ± 0.48*

115.13 ± 1.10*


300mg of P.B

217.52 ± 7.85*

185.80 ± 3.04*

112.43 ± 2.00*

103.34 ± 2.09


results are presented as mean ± SEM. * shows values that are significantly different from the control. Data was analyzed using ANOVA by GraphPad Instat (Version 16) and p<0.05 is considered significant

 


Figure 3.1: Effect of Parkia biglobosa Leaf Fractions on Fasting blood Glucose Level  in Alloxan- Induced Diabetic Rats


 




 Table 3.3: Body Weights of Rats Used

GROUPS

BEFOR INDUCTION

AFTER 14      DAYS

AFTER 28 DAYS


Control

135.64 ± 5.62

136.98 ± 4.28

144.30 ± 2.87


100mg/kg of Alloxan

142.12 ± 5.29

135.98 ± 4.30

134.42 ± 3.37


2.5mg/kg  Glibenclamide

153.48 ± 2.29

154.10 ± 1.68

161.54 ± 1.98


100mg of P.B

156.06 ± 5.53

      159.68 ± 3.26

164.20 ± 3.45


300mg of P.B

166.20 ± 5.33

161.50 ± 5.34

172.80 ± 3.83



Results are presented as mean ± SEM. Data was analyzed using ANOVA by GraphPad Instat (Version 16).





Figure 3.2: Body Weights of Rats over 28 days

                  


 

Table 3.4: effect of methanolic leaf extract of parkia biglobosa on some biochemical  parameters 

PARAMETERS

CONTROL

100mg

300mg


ASP (U/L)

114.30 ± 0.02

118.01± 1.01

116.11 ± 1.20


ALT (U/L)

65.92 ± 0.81

69.48± 73

62.73 ± 0.40


ALP (UL)

260-41± 3.12

265.66 ± 0.2

259.49± 0.41


 TOTAL PROTEIN (g/dl)

6.55± 0.11

      6.18 ± 1.18

4.49 ± 0.67


TOTAL BILIRUBIN

1.04 ± 0.22

0.70 ± 0.31

0.51 ± 0.22












CHAPTER FOUR

4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

4.1    Discussion

It is widely accepted that the use of plants-derived principles will offer access to effective medical care for the treatment and management of diseases through self medication. However, the major overriding measures as suggested by world health organization in the selection of herbal medicines for health care need is safety (WHO, 2010). In addition to medicinal properties, plants extracts should be safe for consumption without having any adverse effects on the body tissues or organs especially the liver which is the main organ responsible for metabolism of the plant when injested. Results obtained from this studies which  shows a significantly decrease in the blood glucose level of the rats when treated with methanolic exract of Parkia biglobosa leaf  also correspond to the work carried out  by Odetola et al., 2006. The hypoglycaemic effect of fermented seeds of Parkia biglobosa, a natural nutritional condiment that features frequently in some African diets as a spice, was investigated in alloxan- induced diabetic rats (Odetola et al., 2006).According to the work carried out by Builders et al., the results of the antioxidant activities of the methanol extract indicated that the sensitivity of the antioxidant activity of the methanol fraction is higher than that of Aqueous extract (Builders et al., 2012). Study carried out  on the anti-hyperglycemic effects of parkia biglobosa seed was used to treat alloxan- induced diabetic rats and there was a decrease in the blood glucose level which also correspond to this research work. (Ajaiyeoba et al., 2002).

The result obtained from these study shows that blood glucose level and body weight monitored in the Alloxan induced diabetic rat are useful markers for assessment of the hypoglycaemic activity of the extract. The untreated diabetic rats had a decrease in body weight and high increase in blood glucose level. However, treatment  with parkia biglobosa extract significantly decrease the blood sugar level and improves body weight of the diabetic rat when compared with untreated control. The 300mg of  methanolic extract had the best activities with significant reduction of blood glucose level. Although the standard possessed the highest antihyperglycemic activity and have the highest activity to reduce the level of blood glucose compere to parkia biglobosa extract

4.2 Conclusion

Conclusively, A methanolic extract of parkia biglobosa leaf possess significant anti-hyperglycemic properties against Alloxan induced diabetic rats with the 300mg of  methanolic extract been the most potent, thus can serve as a lead for the development of new drug  for diabetes.

4.3 Recommendation

Based on the the results obtained from this study, it is recommended that further study be conducted with the view of determining the effect of this extract upon chronic administration. The Parkia biglobosa leaf could also be used in dishes to manage diabetes, since it is known to have anti hyperglycemic effect.


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