i

EVALUATION OF ANTIDIABETIC PROPERTIES OF FRACTIONS

OF GONGRONEMA LATIFOLIUM (FAMILY ASCLEPIADACEAE)

METHANOLIC EXTRACT ON DIABETIC AND NON-DIABETIC

RATS

BY

UZODINMA, SAMUEL UCHENNA

PG./M.PHARM/06/41681

BEING A PROJECT REPORT SUBMITTED TO THE

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY,

FACULTY OF PHARMACEUTICAL SCIENCE UNIVERSITY OF

NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF A MASTER OF

PHARMACY DEGREE IN PHARMACOLOGY OF THE

UNIVERSITY OF NIGERIA, NSUKKA

PROFESSOR PETER A. AKAH

(SUPERVISOR)

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

APRIL, 2012

ii

CERTIFICATION

This project report titled “evaluation of antidiabetic properties of fractions of

Gongronema latifolium (family asclepiadaceae) methanolic extract on

diabetic and non-diabetic rats” is hereby certified as meeting the

requirements for the award of Master of Pharmacy degree in the Department

of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences,

University of Nigeria, Nsukka.

____________________ _____________________

PROF. PETER A. AKAH PROF. C. O. OKOLI

Supervisor Head of Department

iii

DEDICATION

This work is dedicated to the glory of God the father, the son

and the Holy Spirit through whom all things are possible and to

my family for their love and support

iv

ACKNOWLEDGEMENT

I wish to express my profound gratitude to my supervisor, Prof. P. A.

Akah for his patience and understanding, and for supervising this project

work. I am grateful to my entire lecturers such as Prof. C. O. Okoli, Dr.

(Mrs.) Adaobi Ezike etc who have contributed immensely to my progress by

the knowledge imparted to me.

My thanks and appreciation also goes to my wife Mrs. Nkiru

Uzodinma and my family for their love, prayers, support and understanding

during this programme. I must not forget to thank God for my colleagues.

Mr. Christain Okolo, Pharm John Ibeabuchi, Pharm .O. Chukwu, Pharm.

S.U. Nduka, Dr. E.C. Ugwu, Dr. Mama, Pharm. T. Maduka, Pharm

Ogbonna, Pharm Mbaoji, and Mr. Ik Odo for their cooperation and support.

My thanks also goes to Mr. Sabastine Igboeme, Pharm. C. Ubaka, Pharm

(Rev) Emeka Ezea, Mr.A. Ozioko and Mr. Ugwuozor of Botany herbarium,

UNN, for their technical assistance in the course of carrying the project

work. My deep gratitude goes to My friends – Pharm (Dr.) Mathew Okonta,

Prof. C. Esimone and Dr. .I. Uzochukwu for their encouragement during this

project.

Above all, I will never forget to thank God Almighty for His endless, love,

protection, Mercies and graces which He continuously showered on me,

v

ABSTRACT

Antidiabetic activities of G. latifolium were investigated in rat model.

A whole methanolic extract of the leaves of G. latifolium was prepared by

soxhlet extraction and was separated into fractions to yield the methanol,

chloroform and n-hexane soluble fractions (MF, CF and HF) respectively.

An aqueous extract of the plant was prepared by cold maceration. The

extract and its fractions where screened for phytochemical constituents

according to standard procedures. The acute toxicity (LD50) of the methanol

extract was determined in mice. The aqueous extract and methanol extract

including the fractions were tested for antidiabetic effect in rats. Diabetes

was induced by intraperitonial injection of alloxan monohydrate for a period

of 7 days. The blood glucose levels were analyzed as indices of diabetes.

After 7 days, alloxan monohydrate increased the blood glucose level

(P<0.05) of rats indicating hyperglycaemia. Treatment of the rats with

extract (1000 mg/kg and 2000 mg/kg) of G. latifolium decreased

significantly (P<0.05) the blood glucose level within 32 hours of treatment.

The standard antidiabetic, glybenclamide also showed similar effect. The

antidiabetic potency of the extract and fractions were in the order of

MF>ME>AE>HF>CF. The phytochemical screening revealed the presence

of proteins, saponins, alkaloids, terpenoids and steroids and absence of

tannins, reducing sugar and acidic compounds in the crude extract. The MF

was rich in proteins, saponins, carbohydrates and glycosides. The CF

contains resins and alkaloids while HF was very rich in resins, terpenoids

and steriods. The extract and fractions did not lower the blood glucose level

of non-diabetic rats. This study concludes that the leaves of G. latifolium can

be used as traditional treatment for diabetes mellitus as claimed by local

users.

vi

TABLE OF CONTENTS

TITLE PAGE……………………………………………………………..i

DEDICATION……………………………………………………………ii

ACKNOWLEDGEMENT………………………………………………iii

ABSTRACT………………………………………………………………iv

TABLE OF CONTENTS…………………………………………………v

CHAPTER ONE: INTRODUCTION……..…………………………….1

1.1 Definition and etiology of diabetes mellitus………………………...1

1.1.2 Epidemiology of diabetes mellitus…………………………………..2

1.2 Classification of diabetes mellitus…………………………………..3

1.2.1 Type 1 diabetes mellitus……………………………………………..4

1.2.2 Type 2 diabetes mellitus……………………………………………..5

1.2.3 Gestational diabetes mellitus………………………………………...6

1.2.4 Other types…………………………………………………………...6

1.2.5 Sign and symptoms…………………………………………………..7

1.3 Genetics of diabetes mellitus………………………………………...9

1.4 Diagnosis of diabetes mellitus……………………………………...11

1.4.1 Diagnostic tests……………………………………………………..11

1.4.2 Screening test……………………………………………………….12

1.4.3 Monitoring test……………………………………………………...13

1.5 Prevention, treatment and management …………………………...13

1.6 Prognosis…………………………………………………………….24

1.6.1 Acute complications………………………………………………...24

1.6.2 Chronic complications……………………………………………...27

1.7 Phytotherapy and diabetes mellitus management………………….27

vii

1.7.1 Pharmacognostic profile of Gongronema Latifolium taxonomy……34

1.7.2 Medicinal and non medicinal use………………………………….36

1.7.3 Geographical distribution…………………………………………...37

1.7.4 Chemical constituents of G. Latifolium…………………………… 37

1.8 Aim of research ……………………………………………………38

CHAPTER TWO: MATERIALS AND METHODS…………………...39

2.1 Drugs, chemicals and reagents……………………………………..39

2.2 Collection of plant material…………………………………………39

2.3 Extraction and fractionation………………………………………...40

2.4 Phytochemical analysis …………………………………………….40

2.5 Experimental animals……………………………………………….40

2.6 Acute toxicity test …………………………………………………41

2.6.1 Induction of diabetes mellitus………………………………………41

2.6.2 Effect of aqueous extract of G. latifolium on mean fasting blood Sugar

of normal and alloxanized Rats……………….…………….…….42

2.6.3 Effect of methanol extract of G. latifolium on the mean fasting blood

glucose on normal and alloxanized rats……………………………42

2.6.4 Effect of the fractions on mean fasting blood glucose on alloxanized

rats………………………………………………………………….43

2.7 Statistical Analysis………………………………………………….43

CHAPTER THREE: RESULTS…………………………………………44

3.1 Results of extraction and fractionation…………………………….44

3.2 Result of phytochemical analysis…………………………………..44

3.3 Result of acute toxicity test……………………………………..….45

3.4 Result of blood glucose in glycaemic rats administered with aqueous

extract of G. latifolium…………………………………….………45

viii

3.5 Result of blood glucose of glycaemic and norglycaemic rats given

methanol extracts…………………………………………………..45

3.6 The results of blood glucose in glycaemic treated with fractions of G.

latifolium……………………………………………………………………47

CHAPTER FOUR: DISCUSSION AND CONCLUSION……………..50

4.1 Discussion…………………………………………………………...50

4.2 Conclusion…………………………………………………………..52

REFERENCES……………………………………………………………53

1

CHAPTER ONE: INTRODUCTION

1.1: Definition and Etiology of Diabetes Mellitus

Diabetes mellitus often referred to simply as diabetes is a syndrome of

disorder in metabolism, usually due to a combination of hereditary and

environmental causes, resulting in abnormal high blood sugar level

(hyperglycaemia) (Tierney et al, 2002). Paulsen (1998) saw diabetes mellitus

as a syndrome of disturbed intermediary metabolism caused by inadequate

insulin secretion or impaired insulin action, or both.

Blood glucose levels are controlled by a complex interaction of multiple

chemicals and hormones in the body including the hormone insulin made in

the beta cells of the pancreas. Diabetes mellitus consists of a group of

syndromes characterized by hyperglycaemia, altered metabolism of lipids,

carbohydrates, and proteins; and an increased risk of complications from

vascular disease. Criteria for the diagnosis of diabetes mellitus have been

proposed by several medical organizations (WHO, 1999). The American

Diabetes Association criteria include symptoms of diabetes mellitus as

polyuria, polydipsia, and unexplained weight loss, a random plasma glucose

concentration of greater than 200 mg/dl (11.1mM), a fasting plasma glucose

concentration of greater than 126 mg/dl (7mM), or a plasma glucose

concentration of greater than 200 mg/dl (11mM) 2 hours after the ingestion

2

of a oral glucose load (Expert Committee on the Diagnoses and Treatment of

Diabetes Mellitus, 2003)

1.1.2 Epidemiology of Diabetes Mellitus

In recent years, developed and developing nations have witnessed

an explosive increase in the prevalence of diabetes mellitus predominately

related to life style changes and the resulting surge in obesity (King et al,

1998). The metabolic consequences of prolonged hyperglycemia and

dyslipidemia, including accelerated atherosclerosis, chronic kidney disease

and blindness pose an enormous burden on patients with DM and on the

public health system (Goodman and Gilman, 2006)

In 2000, according to the World Health Organization, at least 171

million people worldwide suffer from diabetes, or 2.8% of the population.

(Roglic et al, 2004). Its incidence is increasing rapidly, and it is estimated

that by the year 2030, this number will almost double. (Roglic et al, 2004).

Diabetes mellitus occurs throughout the world but is more common

(especially type 2) in the more developed countries (Rother, 2007). The

greatest increase on prevalence is, however, expected to occur in Asia and

Africa, where most patients will likely be found by 2030 (Roglic et al,

2004). The increase in incidence of diabetes in developing countries follows

the trend of urbanization and lifestyle changes, perhaps most importantly a

“western-style” diet.

3

For past 20 years, diabetes rates in North America have been

increasing substantially. In 2008 there were about 24 million people with

diabetes in the United State alone, of which 5.7 million people remain

undiagnosed. Over one million people are estimated to have pre-diabetes.

(CDC, 2000). About 5%-10% of diabetes cases in North America are types 1

with the rest being type 2. The American Diabetes Association point out the

2003 assessment of the National Center of Chronic disease prevention and

Health promotion that 1 in 3 Americans born after 2000 will develop

diabetes in their lifetime (Narayan et al, 2003)

The vast majority of diabetic patients have type 2 diabetes mellitus. In

the United State, about 90% of all diabetic patients have type 2 diabetes

mellitus. Both type 1 and type 2 DM are increasing in frequency. The reason

for the increase of type 1 DM is not known. The genetic basis of type 2 DM

cannot change in such a short time thus other contributory factors including

increasing age, obesity, sedentary lifestyle, and low birth weight, must

account for this dramatic increase.

1.2: Classification of Diabetes Mellitus

1.2.1: Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is characterized by loss of the insulin-producing

beta cells of the islets of Langerhans in the pancreas leading to insulin

deficiency. This type of diabetes can be further classified as immune-

4

mediated or idiopathic. The majority of type 1 diabetes is of the immune-

mediated nature, where beta cell loss is a T-cell mediated autoimmune attack

(Rother, 2007). There is no known preventive measure against type 1

diabetes, which causes approximately 10% of diabetes mellitus cases in

North America and Europe. Most affected people are otherwise healthy and

of a healthy weight when onset occurs. Sensitivity and responsiveness to

insulin are usually normal, especially in the early stages. Type 1 diabetes can

affect children or adults but was traditionally termed "juvenile diabetes"

because it represents a majority of the diabetes cases in children. (Expert

Committee on the Diagnosis and Treatment of Diabetes Mellitus, 2003)

1.2.2: Type 2 Diabetes Mellitus

Type 2 diabetes mellitus is characterized differently and is due to

insulin resistance or reduced insulin resistance or reduced insulin sensitivity,

combined with relatively reduced insulin secretion which in some case

becomes absolute. The defective responsiveness of body tissues to insulin

almost certainly involves the insulin receptor in cell membranes. However,

the specific defects are not known. Diabetes mellitus due to known specific

defect are classified separately.

In the early stage of type 2 diabetes, the predominant abnormality is

reduced insulin sensitivity, characterized by elevated levels of insulin in the

blood. At this stage hyperglycaemia can be reversed by a variety of

5

measures and medications that improve insulin sensitivity or reduced

glucose production by the liver. As the disease progresses, the impairment of

insulin secretion worsens, and therapeutic replacement of insulin often

becomes necessary.

There are numerous theories as to the exact cause and mechanism in

type 2 diabetes (Riserus, 2009). Central obesity (fat concentrated around the

waist in relation to abdominal organ, but not subcutaneous fat) is known to

predispose individuals to insulin resistance. Abdominal fat is especially

active hormonally, secreting a group of hormone called adipokines that may

possibly impair glucose tolerance. Obesity is found in approximately 55% of

patients diagnosed with type 2 diabetes.

Other factors include aging (about 20% of elderly patients in North

America have diabetes) and family history (type 2 is much more common in

those with close relatives who have had it). In the last decade, type 2

diabetes has increasingly begun to affect children and adolescents, likely in

connection with the increased prevalence of childhood obesity seen in recent

decades in some places (Rosenbloon and Silvestein, 2003). Environmental

exposures may contribute to recent increases in the rate of type 2 diabetes. A

positive correlation has been found between the concentration in the urine of

bisphenol A, a constituent of polycarbonate plastic, and the incidence of type

2 diabetes.

6

1.2.3: Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) resembles type 2 diabetes in

several respects, involving a combination of relatively inadequate insulin

secretion and responsiveness. It occurs in about 2%-5% of all pregnancies

and may improve or disappear after delivery. Gestational diabetes is fully

treatable but requires careful medical supervision throughout the pregnancy.

About 20%-50% of affected women develop type 2 diabetes later in life

(Lawrence et al, 2005).

Even though it may be transient, untreated gestational diabetes can

damage the health of the fetus or mother. Risks to the baby include

macrosomia (high birth weight), congenital cardiac and central nervous

system anomalies, and skeletal muscle malformations. A 2008 study

completed in the United State found that the more American women are

entering pregnancy with pre-existing diabetes (Lyssenko et al, 2008). In fact

the rate of diabetes in expectant mothers has more than double in the past 6

years (Lawrence et al, 2005). This is particularly problematic as diabetes

raises the risk of complication during pregnancy as well as increasing the

potential that the children of diabetic mothers will also become diabetic in

future.

7

1.2.4: Other types.

Most cases of diabetes mellitus fall into the two broad etiologic

categories of type 1 or type 2 diabetes however, many types of diabetes

mellitus have known specific cause, and thus fall into separate categories as

diabetes due to a specific cause. As more research is done into diabetes,

many patients who were previously diagnosed as type 1 or type 2 diabetes

will be reclassified as diabetes due to their known specific cause

Some case of diabetes are caused by the body’s tissue receptors not

responding to insulin (even when insulin levels are normal, which is what

separates it from type 2 diabetes); this form is very uncommon. Genetic

mutations (autosomal or mitochondrial) can lead to defects in beta cell

function. Abnormal insulin action may also have been genetically

determined in some cases. Diseases associated with excessive secretion of

insulin-antagonistic hormones (for example, chronic pancreatitis and cystic

fibrosis) can cause diabetes (which is typically resolved once the hormone

excess is removed). Many drugs impair insulin secretion and some toxins

damage pancreatic beta cells. The diagnostic entity, malnutrition-related

diabetes mellitus (MRDM) was profound by the World Health Organization

when the current taxonomy was introduced in 1999. (WHO 1999).

8

Different forms of Diabetes mellitus

1: Type 1 diabetes mellitus also called insulin dependent diabetes mellitus

example autoimmune type 1 diabetes mellitus (tpye 1A) and

non- autoimmune or idiopathic type I diabetes mellitus (type 1B)

2: Type 2 diabetes mellitus also called non-insulin dependent diabetes

mellitus example are

Specific- denatured gene mutations

Maturity-onset diabetes of youth (MODY)

MODY1 Therapeutic nuclear factor 4α (HNF4A) gene mutations

MODY 2 glucokinase (GCK) gene mutations

MODY 3 -6

MODY X unidentified gene mutation

Insulin gene mutations

Insulin receptor gene mutations

3: Diabetes secondary to pancreatic disease examples

Chronic pancreatic

Tropical diabetes (chronic pancreatitis) associated with nutritional and /or

toxic factors.

4: Diabetes secondary to other endocrinopathics

Cushing’s disease

Glucocorticoid administration

9

Acromegaly

5: Diabetes secondary to immune suppression

6: Diabetes associated with genetic syndromes e.g prader-willi syndrome

7: Diabetes associated with drug therapy

1.2.5: Sign and Symptoms

The classical triad of diabetes symptoms include polyuria, polydipsia,

and polyphagia, which are, respectively, frequent urination, increased thirst

and consequent increased fluid intake, and increase appetite symptoms may

develop quite rapidly (weeks or months) in type I diabetes particularly in

children. However, in type 2 diabetes symptoms usually develop much more

slowly and may be subtle or completely absent. Type I diabetes may also

cause a rapid and significant weight loss (despite normal or even increased

eating) and irreducible fatigue. All of these symptoms except weight loss

can also manifest in type 2 diabetes in patients whose diabetes is poorly

controlled (Santaguida et al, 2008).

When the glucose concentration in the blood is raised beyond its renal

threshold, reabsorption of glucose in the proximal renal tubuli is incomplete,

and part of the glucose remains in the urine (glycosuria). This increases the

osmotic pressure of the urine and inhibits reabsorption of water by the

kidney, resulting in increased urine production (polyuria) and increased

thirst (Tarnow et al, 2008).

10

Prolonged high blood glucose leads to changes in the shape of the

lenses of the eyes, resulting in vision changes; sustained sensible glucose

control usually returns the lens to its original shape. Blurred vision is a

common complaint leading to a diabetes diagnosis. Type 1 DM should

always be suspected in cases of rapid vision change (Theodore et al, 2008).

Patients (usually with type 1 diabetes) may also initially present with

diabetes ketoacidosis (DKA), an extreme state of metabolic dysregulation

characterized by the smell of acetone on the patient’s breath; a rapid, deep

breathing known as Kussmaul breathing; polyuria, nausea, vomiting and

abdominal pain, and any of many altered states of consciousness or arousal

(such as hostility and mania or, equally, confused and lethargy). In severe

DKA, coma may follow, progressing to death. Diabetic ketocidosis is a

medical emergency and required immediate hospitalization. A rarer but

equally severe possibility is hyperosmolar nonketotic state which in more

common in type 2 diabetes and is mainly the result of dehydration due to

loss of body water. Often, the patient has been drinking extreme amounts of

sugar-containing drinks, leading to a vicious circle in regard to the water loss

(Genuth, 2006; Sniderman et al, 2007).

11

1.3. Genetics of diabetes mellitus

Over 20 regions in the human genome are associated with Type 1

diabetes, but makes little contribution to overall susceptibility toType 1

diabetes (Davies et al, 1994; Concannon et al, 1998). The strongest linkage

with Type 1 diabetes is shown by the human leucocyte antigen (HLA) gene

cluster in the major histocompatibility complex (MHC) located on

chromosome 6p21 (Ghosh and Schork, 1996). HLA antigens are cell-surface

glycoproteins that play a crucial role in presenting auto antigen peptide

fragments to T lymphocytes and thus initiate an auto immune response

(Nerup et al, 1977). They comprised of two classes, class I and class II,

which are encoded by different genes within the HLA region and thus differ

fundamentally in structure. Class I molecules comprise the HLA A, B, C

while class II molecules comprise HLA DP, DQ and DR and are coded by

their respective genes (Nerup et al, 1974). The HLA class II molecules are

central to the human immune response because they present peptide antigens

to T-helper (CD 4 positive) cells. There are two types of class II genes: those

encoding - polypeptides and those encoding -polypeptides which

together form the functional class II -heterodimer. This results in a

variety of genes (Yamagata et al, 1996).

Type 2 diabetes shows a clear familial aggregation but it does not

segregate in a classical Mendelian fashion. It is polygenic, with different

12

combinations of gene defects. Genetic and environmental factors may affect

insulin biosynthesis, insulin secretion and insulin action. The complex

interactions between genes and the environment complicate the task of

identifying any single genetic susceptibility factor for Type 2 diabetes

(Walley et al, 2006). The maintenance of normal glucose homeostasis

depends on a precisely balanced and dynamic interaction between tissue

sensitivity to insulin (especially in muscle and liver) and insulin secretion.

The molecular circuitry that maintains glucose homeostasis depends on the

result of several combined gene defects, or from the simultaneous action of

several susceptible alleles, or else from combinations of frequent variants at

several loci that may have deleterious effects when predisposing

environmental factors are present.

It is generally accepted that insulin resistance (IR) precedes the failure of

insulin secretion and exacerbates this by imposing an increased secretory

burden on the -cells (Ferrannini, 1998). However, subtle abnormalities in

-cell function have been demonstrated early in the course of Type 2

diabetes mellitus (Vionnet et al, 1992), and even in first degree relatives of

individuals with Type 2 diabetes mellitus - suggesting a possible basis for an

inherited component for -cell failure (Kalsilamdrof and Tentouris, 2003).

A prospective study in Pima Indians showed that the progression from

normal to IGT and finally to Type 2 diabetes was accompanied by a

13

progressive decline in -cell secretory capacity (Weyer et al, 1998). The

mechanisms underlying -cell failure in Type 2 diabetes however remain

elusive. Type 2 diabetes being an extremely heterogenous disorder,

phenotypically, and pathogenetically, is polygenic in nature (Hanson et al,

1997). This means that multiple genes (polymorphism), each insufficient in

themselves, must be present in order to cause diabetes. Such genes might

affect -cell apoptosis, regeneration, glucose sensing, glucose metabolism,

ion channels, energy transduction, and other islet proteins necessary for

synthesis, packaging, movement and release of secretory granules (Barret,

2008). Many rare forms of defective glucose metabolism have been shown

to be caused by gene defects involving the -cell and the insulin receptor

(Defronzo and Prato, 1996). Of these the most common and important form

is the maturity onset diabetes of the young (MODY).

1.4: Diagnosis of Diabetes Mellitus

Diabetes mellitus is characterized by recurrent or persistent hyperglycemia,

and is diagnosed by demonstrating any one of the following: (WHO, 1999)

Fasting plasma glucose level ≥ 7.0 mmol/L (126 mg/dL).

Plasma glucose ≥ 11.1 mmol/L (200 mg/dL) two hours after a 75 g

oral glucose load as in a glucose tolerance test.

14

Symptoms of hyperglycemia and casual plasma glucose

≥ 11.1 mmol/L (200 mg/dL).

Glycated hemoglobin (Hb A1C) ≥ 6.5%.

A positive result, in the absence of unequivocal hyperglycemia, should be

confirmed by a repeat of any of the above-listed methods on a different day.

It is preferable to measure a fasting glucose level because of the ease of

measurement and the considerable time commitment of formal glucose

tolerance testing, which takes two hours to complete and offers no

prognostic advantage over the fasting test (Seydah et al, 2001). According

to the current definition, two fasting glucose measurements above

126 mg/dL (7.0 mmol/L) is considered diagnostic for diabetes mellitus.

People with fasting glucose levels from 100 to 125 mg/dL (5.6 to

6.9 mmol/L) are considered to have impaired fasting glucose. Patients with

plasma glucose at or above 140 mg/dL (7.8 mmol/L), but not over

200 mg/dL (11.1 mmol/L), two hours after a 75 g oral glucose load are

considered to have impaired glucose tolerance. Of these two pre-diabetic

states, the latter in particular is a major risk factor for progression to full-

blown diabetes mellitus as well as cardiovascular disease. (Mathew, 1998).

15

1.4.1: Diagnostic Tests.

Some of the commonly employed tests in the diagnosis of diabetes

mellitus include oral glucose tolerance test (OGTT) and in some cases,

fasting blood glucose (FBS).

A. Oral glucose tolerance test (OGTT).

This is the most accepted and widely applied test for the diagnosis of

diabetes mellitus. In this test, the patients must fast for 14 hours and should

discontinue glucose-altering medication at least 3days prior to test. The

patients must not smoke cigarette or drink alcohol or coffee just before and

during the test, and patient must not be carbohydrate depleted 3 days prior to

the test (George, 1992). After fasting for about 14 hours, the patient is

given an oral glucose load of 75-100 g and blood sample is withdrawn every

30 minutes for the next 2 hours since in 2 hours the blood glucose level

(<200 mg%) in a non-diabetic patient is expected to have normalized. But in

diabetic, the plasma glucose level is higher than 200 mg%. (Aguwa and

Omole, 2004), and returns to the baseline more slowly than it does in normal

or non-diabetics (Ganong, 1999).

B. Fasting blood glucose (FBG)

Blood sample is collected and analyzed after the patient has fasted

over night, after a period of unimpaired carbohydrate intake. The normal

16

range of fasting blood glucose is 70-110 mg% when collected from the

venous blood (Aguwa and Omole, 2004).

1.4.2: Screening Test

Person that should be screened include those with strong family

history of diabetes mellitus, persons severely obese, mothers with babies

above 3.7 kg body weight at birth and patients scheduled for surgical

operations. (George, 1992).

A. Postprandial blood glucose test

This involves withdrawing of blood sample form the patient 2 hours

after feeding on heavy carbohydrate meal or being fed on 100 g glucose

load. In a non-diabetic patient, the blood glucose level returns to normal (70-

110 mg%) after 2 hours while in diabetics, hyperglycaemia becomes

apparent after 2 hours (Aguwa and Omole, 2004).

B. Urine test

Testing of early morning urine with clinistix or clinitest strips to

determine the presence of glucose in urine can be done with as little as

0.25% glucose in the urine. Clinitix (glucose oxidase) is glucose specific and

the best quantitative estimation method for urine glucose level while the

clinitest (copper reduction method) is non-specific for glucose only. The

17

urine test method is gradually being relegated for the more concise and

glucose specific automated electronic devices.

C. Random blood glucose test (RBG)

Blood sample is withdrawn from the patient and analyzed at any time

of the day irrespective of meal that was taken when the blood glucose level

is above 250 mg%. The patient is further tested with a method for diagnosis.

1.4.3: Monitoring Test

This method is mainly employed to monitor the therapeutic outcomes

in management of diabetes mellitus and to enable the health personnel to

choose the right drug (s) especially in ambulatory patients.

a. Glycosylated haemoglobin measurement.

Glucose has been found to bind to proteins irreversibly and non-

enzymatically thus causes chemical alteration in the proteins. The non-

enzymatic glycosylation of the proteins occur by direct reaction between the

aldehyde groups of the reducing sugars and primary amino groups in

proteins to form Schiff bases that is rearranged to form stable protein

ketoamine derivatives. This contributes to diabetes complications because it

is an oxidative process (Odukoya and Ogbeche, 2002). In normal individual,

the glycosylated haemoglobin (HBAIC) is between 3-6% while in a diabetic

patient, the level may be as high as 18-20%. It is used to monitor therapy

compliance in diabetics considering their blood glucose control.

18

1.5: Prevention, Treatment and Management

A: prevention: type 1 diabetes risk is known to depend upon a genetic

predisposition based on HLA type, an unknown environmental trigger

(suspected to be infection, although none has proven definitive in all cases),

and an uncontrolled autoimmune response that attacks the insulin producing

beta cell. Some research has suggested that breast feeding decreased the risk

factor in later life. Various other nutritional risk factors are being studied,

but no firm evidence has been found (Virtanen and Knip, 2003). Giving

children 2000iu of vitamin D during their first year of life is associated with

reduced risk of type I diabetes, though the causal relationship is obscure.

(Hypponen et al, 2001)

Children with antibodies to beta cell protein (ie at early stage of an

immune reaction to them) but no overt diabetes, and treated with vitamin B3

(niacin), had less than half the diabetes onset incidence in a 7 year time span

as did the general population, and an even lower incidence relative to those

with antibodies as above, but when received no vitamin B3 (Eliott et al,

1996)

Type 2 diabetes risk can be reduced in many cases by making changes

in diet and increasing physical activity (Lindstrom et al, 2006; Knowler et al,

2002). The American Diabetes Association (ADA) recommends maintaining

a healthy weight, getting at least 21/2 hours of exercise per week, having a

19

modest fat intake, and eating sufficient fibre. The ADA does not recommend

alcohol consumption as a preventive, but it is interesting to note that

moderate alcohol intake may reduce the risk, a similarly confused

connection between low does alcohol consumption and heart disease is

termed the French paradox

There is inadequate evidence that eating foods of low glycogenic

index is clinically helpful despite recommendations and suggested diet

emphasizing this approach. (Bantle et al, 2006). There are numerous studies

which suggest connections between some aspects of type 2 diabetes with

ingestion of certain foods or with some drugs. Some studies have shown

delayed progression to diabetes in predisposed patients through prophylactic

use of metformin (Knowler et al, 2002), rosiglitazone (Gerstein et al, 2006),

or valsartan (Kjeldsen et al, 2006). In patents on hydroxyl chloroquine for

rheumatoid arthritis, incidence of diabetes was reduced by 77% though

caused mechanism is unclear (Wasko et al, 2007). Breastfeeding may also be

associated with the prevention of type 2 of the disease in mothers (Stuebe et

al, 2005). Clear evidence for these and any of many other connections

between foods and supplements and diabetes is sparse to date; none, despite

secondary claims for or against is sufficiently well established to justify as a

standard clinical approach.

b. Treatment and Management.

20

Diabetes mellitus is currently a chronic disease without a cure, and

medical emphasis must necessarily be on managing/avoiding possible short-

term as well as long-term diabetes-related problems. There is an

exceptionally important role for patient education, dietetic support sensible

exercise, self monitoring of blood glucose, with the goal of keeping both

short-term blood glucose levels, and long term levels as well, within

acceptable bounds. Careful control is needed to reduce the risk of long term

complications. This is theoretically achievable with combinations of diet,

exercise and weight loss (type 2) various oral diabetic drugs (type 2 only),

and insulin use (type 1 and for type 2 not responding to oral medication,

mostly those with extended duration diabetes). In addition, given the

associated higher risks of cardiovascular disease, lifestyles modifications

should be undertaken to control blood pressure (Alder et al, 2000) and

cholesterol by exercising more, smoking less or ideally no at all, consuming

an appropriate diet, wearing diabetic socks, wearing diabetic shoes, and if

necessary, taking any of the several drugs to reduce blood pressure. Many

type I treatment include combination use of regular or NPH insulin, and/or

synthetic insulin analogs eg Humalog, Hovolog or Apidra in combinations

such as Lantus/Levemir and Humalog, Novolog or Apidra. Another types

treatment option is the use of the insulin pump eg Deltec Cozmo, Animas,

Medtronic minimed, insulet Omnipod, or ACCU-CHEK. A blood lancet is

21

used to pierce the skin in order to draw blood to test it for sugar levels

(Nathan et al, 2005).

In countries using a general practitioner system, such as the United

Kingdom, care may take place mainly outside hospitals, with hospital-based

specialist care used only in case of complications, difficult blood sugar

control, or research project (DCCTRG,1995). In other circumstance, general

practitioners and specialists, share care of a patient in a team approach.

Optometrists, podiatrists/chiropodists, dietitians, physiotherapists, nursing

specialist, nurse practitioners, or certified diabetes, educators, may jointly

provide multidisciplinary expertise (Walker, 2007).

ORAL HYPOGLYCEMIC AGENTS

History: In contrast to the systematic studies that led to the isolation of

insulin, the sulfonylurea: were discovered accidentally. Jaben and colleagues

noted that some sulfonamides caused hypoglycemia in experimental

animals. Soon thereafter, I butyl -3-sulfonylurea (carbutamide) became the

first clinically useful sulfonylurea for the treatment of diabetes. Although

later withdrawn because of adverse effects on the bone marrow, this

compound led to the development of the entire class of sulfonylareas. The

sulfonylureas are divided into two groups or generations of agent. The first

group include tolbutamide, acetohexamide, tolazamide, and chlorpropamide.

22

A second, more potent ones include glyburide (glibenclamide), glipizide,

gliclazide, and glimepride.

In 1997, repaglimide, the first number of a new class of oral insulin

secretagogues called meglitinide was approved for clinical use. This agent

has gained acceptance as a fast-acting premeal therapy to limit postprandial

hyperglycemia. Another example of the class is Nateglamide (Kalbag et al,

2001).

The goat’s plant (Galega officinalis), used to treat diabetes in Europe

in medieval times was found in the early twentieth century to contain

guanidine. Guanidine has hypoglycemic properties but was too toxic for

clinical use. During the 1920s, biguanides were investigated for use in

diabetes, but they were overshadowed by the discovery of insulin (Defronzo

and Goodman, 2001). Shortly after the introduction of the sulfenylureas the

first biguanides became available for clinical use which includes phenformin

and Metformin. Phenformin was withdrawn from the market because of an

increased frequency of lactic acidosis associated with its use. Metformin has

been used extensively in Europe without significant adverse effects and was

approved for use in the United State in 1995.

Thiazolidinedions were introduced in 1997 as the second major class

of insulin sensitizers.” These agents bind to peroxisome proliferator-

activated receptors (principally PPARY), resulting in increased glucose

23

uptake in muscle and reduced endogenous glucose production. The first of

this agent troglitazone was withdrawn from use in the United States in 2000

because of an association with hepatic toxicity. Two other agents of this

class, rosiglitazene and pioglitazone, have not been associated with

widespread liver toxicity and are used worldwide.

Mechanism of Action

Sulfonylureas cause hypoglycemia by stimulating insulin release

from pancreatic B-cells. It binds to the SUR1subunits and blocks the ATP-

sensitive K+ channel (Aguilar-Bryan et al, 1995). The drugs thus resemble

physiological secretagogues (e.g glucose, leucine), which also lower the

conductance of this channel. Reduced K+ conductance causes membrane

depolarization and influx of Ca2+ through voltage-sensitive Ca2+ channels.

Sulfonylureas also may further increase insulin levels by reducing

hepatic clearance of the hormone. Sulfonylureas also stimulate release of

somatostation, and they may suppress the secretion of glucagons slightly

(Philipson and Steiner, 1995). Although extrapanreatic effects of

sulfonylureas can be demonstrated, they are of main clinical significant in

the treatment type 2 DM Patients

Repaglimide Meglitimides: Like sulfonylurea, repaglimide stimulates insulin

release by closing ATP- dependent potassium channels in pancreatic cells.

24

The drug is absorbed rapidly from the gastrointestinal tract, and peak blood

levels are obtained within 1 hour. The half –life of the drug is about 1hour.

These features of the drug allow for multiple preprandial uses as compared

with the classical once-or twice-daily dosing of sulfonylureas.

Biguanides (metformm): Metfomin is antihyperglycasemic, not

hypoglycaemic (Bailey 1992). It does not cause insulin release from the

pancreas and generally does not cause hypoglycemia, even in large doses.

Metformin has no significant effects in the secretion of glucagons, cortisol,

growth hormone, or somatostatin. Metformin reduces glucose levels

primarily by decreasing hepatic glucose production and by increasing insulin

action in muscle and fat. The mechanism by which metformin reduces

hepatic glucose production is controversial, but most data indicate an effect

on reducing gluconeogenesis (Stumvoll et al 1995). Metformin also may

decrease plasma glucose by reducing the absorption of glucose from the

intestine, but this action has not been shown to have clinical relevance.

Thazolidinediones: They are selective agents for nuclear peroxisone

proliferation-activated receptor-y (PPARY). These drugs bind to PPARY,

which activates insulin-responsive genes that regulate carbohydrate and lipid

metabolism. It requires insulin to be present for their action

thiazolidinediones exert their principal effects by increasing insulin

sensitivity in peripheral tissue but also may lower glucose production by the

25

liver. Thiazolidinediones increase glucose transport into muscle and adipose

tissue by enhancing the synthesis and translocation of specific forms of the

glucose transporter. Although muscle is a major insulin-sensitive tissue,

PPARY is vertically absent in skeletal muscle. The first-generation

sulfonylureas vary considerably in their half-lives and extent of metabolism.

The half life of acetohexamide is short but the drug is reduced to an active

compound whose half-life is similar to those of tolbutanide and tolazamide

(4-7hrs).It may be necessary to take these drugs in divided daily dose.

Chlorpropamide has a long half-life (24 to 48 hrs). The second-generation

agents are approximate 100 times more potent than those in the first group.

Although their half-lives are short (3 to 5 hours), their hypoglycemic effects

are evident for 12 to 24 hrs, and they often can be administer once daily. The

reason for the discrepancies between their half-lives and duration of action is

not clear.

All the sulfonylurea is metabolize by the liver, and the metabolites are

excreted in the Urine. Metabolism of chlopropamide is incomplete, and

about 20% of the drug is excreted unchanged. Thus sulfonylureas should be

administered with caution to patients with either renal or hepatic

insufficiency.

Therapeutic uses

26

Sulfonylureas are used do control hyperglycemia in type 2 DM

patients who cannot achieve appropriate control with changes in diet alone.

In all patients, continued dietary restrictions are essential to maximize the

efficacy of the sulfonylureas. Contraindication to the use of these drugs

include type 1 DM, pregnancy, lactation, and for the older preparations,

significant hepatic or renal insufficiency.

Thiazolidinediones

Three thiazolidinediones have been used in clinical practice

(tioglitazone, rosiglitazone, and pioglitazone); however, troglitazone was

withdrawn from use because it was associated with severe hepatic toxicity.

Rosiglitazone and pioglitazone can lower haemoglobin Aic levels by 1% to

1.5% in patients with type 2 DM. The thiazolidone tend to increase high-

density lipoprotein (HDL) cholesterol but have variable effects on

triglyceredinediones and low-density lipoprotem (LDL) cholesterol. Hence

thiazolidinediones have been reported to cause anemia, weight gain, edema

and plasma volume expansion (Ruderman and Prentki, 2004).

Glucagon-like peptides (incretins)

Over some decades ago, Melutyre et al, reported that oral as compared

with intraveneus delivery of glucose produced a greater release of insulin.

Subsequent work identified two hormone-glucose-dependent insulinotropic

polypeptide gastric inhibitory polypeptide and glucagon-like peptide- 1

27

(GLP -1)- that are released from the upper and lower bowel that augment

glucose dependent insulin secretion (Mayo et al, 2003).

These hormones are termed incretins. The two incretins deferentially

stimulate insulin secreting. GLP has little effect on increasing insulin

secretion in type 2 DM, whereas GLP-1 significantly augments glucose

dependent insulin secretion. Consequently, GLP-1 has become an attractive

target for therapeutic development in type 2 DM. GLP-1 also reduces

glucagon secretion, slow gastric emptying, and decrease appetite. Incretins

are inactivated by dipeptidyl peptidase IV enzyme (DPP-IV) within (1-2

minutes) of its release in. Consequently, considerable work has been

performed to produce GLP-1 reception agonist that maintain the physiologic

effects of the native incretin but are resistant to actions of DPP-IV i.e

incretin mimetics. The GLP receptor is expressed in the pancreatic islet, as

well as, the gut, adipose tissue, heart, pituitary, adrenal cortex and the brain

(Usdai et al 1993).

Incretin mimetics. (exenatide). Incretins are hormones produced from the

gastrointestinal tract that act to enhance the usual release of insulin after the

oral ingestion of carbohydrates (Nauk et al 1986; Drucker 2003). They also

slow the gastric absorption of nutrients and act to promote a feeling of

satisfy that can lead to weight loss in overweight individuals. These agents

work to lower glucose levels without causing hypoglycemia, but with

28

gradual weight loss due to decrease in caloric intake. Exenatide augment the

hypoglycemic effects of sulphonylureas when co-administered, but on its

own, will not course hypoglycemia, and do not when used in combination

with metformin. Liraglutide is a human long –acting form of glucagon-like

peptide-1 (GLP-1) that is similar in action to extinitide

ALPHA- GLUCOSIDASE INHIBITORS

Alpha-glucosidase inhibitors act by inhibiting the enzyme alpha

glucosidase found in the brush border cells that line the small intestine,

which cleaves more complex carbohydrates like starch, dextrin, and

disaccharides into sugar (Davis et al, 1991). Because they inhibit the

breakdowns and subsequent absorption of carbohydrates from the gut

following meals, these drugs impact on postprandial hyperglycemia more

reasonably and modestly on fasting plasma glucose level. α-glucosidase

inhibitors do not stimulate insulin release and therefore do not result in

hypoglycemia. They can significantly improve hemoglobin A1c levels in

severely hyperglycemia type 2diabetes mellitus patients.

The serious side effect observed with these agents is gastrointestinal

effects such as abdominal discomfort, bloating, flatulence and diarrhoea.

Examples include Acarbose (PRECOSE) and migletol (glyset). Acarbose

treatment has been linked to elevation in serum transaminase level and the

29

use of this agent is contraindicated in patients with liver cirrhosis (Chiasson

et al, 2002).

Mechanism of action of anti-diabetic drugs

The present treatment of diabetes is focused on controlling and lowering

blood glucose to a normal level. The mechanisms of both western medicines

and the Traditional medicines to lower blood glucose are

(1) To stimulate B. cell of pancreatic islet to release insulin

(2) To resist the hormones which rise blood glucose

(3) To increase the number or rise the appetency and sensitivity of insulin

receptor site to insulin,

(4) To decrease the leading-out of glycogen

(5) To enhance the use of glucose in the tissue and organ

(6) To clear away free radicals, resist lipid peroxidation and convert the

metabolic disorder of lipid and protein,

(7) To improve microcirculation in the body.

Based on the above-mentioned mechanisms, the drugs clinically used

to treat diabetes can be mainly divided into insulin, insulin secretagogues,

insulin sensitivity improvement factor, insulin like growth factor, aldose

reductase inhibitors, α- glucosidase inhibitors, protein glycation inhibitors,

almost all of which are chemical and biochemical drugs (Zhao, 1999). The

effect of these drugs is only aimed to lower the level of blood glucose.

30

Moreover, in most cases, side effect such as hypoglycemia, lactic acid

intoxication and gastrointestinal upset appear after patients took these

medicines. The drugs commonly used in clinic to treat or control diabetes

are the following.

(i)Insulin: There are many kinds of preparations

Sulfanylureas: Tolbutanide (ovurase), glibenclanide (glyburide,

Daonil), glidazide (Diamicron), glibenses (minidiab),

glimepiride e.t.c

(ii)Biguanide: Phenformin (Diabenede), Dimethylbiguanide (Diaformin)

Metformin Hydrochloride (glucophage)

(iii)-glucosidase inhibitor (-GD1): glucobay (Acarbose), glyset, miglet

(iv)Aldose reductase inhibitor: Tolrestat, Alredase, kinedak, imprestat e.t.c

(v)Thazolidienedions: Troglitazone, Rosigitazone, Pioglitazine e.t.c

(vi)Carbamoylmethyl benzoic acid: repaglinide

(vii)Insulin-like growth factor: igf-1

(viii)Other: Dichloroacetic acid

1.6: Prognosis

Patients education, understanding, and participation is vital since the

complications of diabetes are far less common and less severe in people who

have well-controlled blood sugar levels. (Nathan et al, 2005). Wider health

problems accelerate the deleterious effects of diabetes. These include

31

smoking, elevated cholesterol levels, obesity, high blood pressure and lack

of regular exercise. According to a study, women with high blood pressure

have a threefold risk of developing diabetes.

1.6.1 Acute complication

Diabetic ketoacidosis (DKA) is an acute and dangerous complication that

is always a medical emergency. Low insulin levels cause the liver to turn to

fat for fuel (ie, Ketosis); ketone bodies are Intermediate subtracts in that

metabolic sequence. This is normal when periodic, but can become a serious

problem if sustained. Elevated levels of ketone bodies in the blood decrease

the blood’s PH, leading to DKA (TopNew, 2009). Abdominal pain is

common and may be severe. The level of consciousness is typically normal

until late in the process, when lethargy may progress to coma. Ketoacidosis

can easily become severe enough to cause hypotension shock, and death.

Urine analysis will reveal significant levels of ketone bodies (which have

exceeded their renal threshold blood level to appear in the urine, often before

other overt symptoms). Prompt, proper treatment usually results in full

recovery, through death can results from inadequate or delayed treatment, or

from complications (e.g., brain edema). DKA is always a medical

emergency and requires medical attention. Ketoacidosis is much more

common in type 1 diabetes than type 2. (Rich, 2006).

32

Hyperosmolar nonketotic state (HNS) is an acute complication sharing

many symptoms with DKA, but an entirely different origin and different

treatment. A person with very high (usually considered to be above

300mg/d1 (16 mmo1/L)) blood glucose levels, water is osmotically drawn

out of cells into the blood and the kidneys eventually begin to dump off

water and an increase in blood osmolarity. If fluid is not replaced (by mouth

or intravenously), the osmotic effect of high glucose level combined with the

loss of water, will eventually lead to dehydration. The body’s cells become

progressively dehydrated as water is taken from them and excreted.

Electrolyte imbalances are also common and are always dangerous. As with

DKA, urgent medical treatment is necessary, commonly beginning with

fluid volume replacement. Lethargy may ultimately progress to a coma,

though this is more common in type 2 diabetes than type 1. (Centofari 1995)

A. Hypoglycaemia

Hypoglycemia, or abnormally low blood glucose, is an acute

complication of several diabetes treatments. It is rare otherwise, either in

diabetic or non-diabetic patients. The patient may become agitated, sweaty,

weak, and have many symptoms of sympathetic activation of the autonomic

nervous system resulting in feelings akin to dead and immobilized panic.

Consciousness can be altered or even lost in extreme cases, leading to coma,

seizures, or even brain damage and death may or can result. In patients with

33

diabetes this may be caused by several factors, such as too much or

incorrectly timed insulin, too much or incorrectly timed exercise (exercise

decrease insulin requirements) or not enough food (specifically glucose

containing carbohydrates). The variety of interactions makes identification

difficult in many instances (Foss et al, 2001).

It is more accurate to note that iatrogenic hypoglycemia is typically the

result of the interplay of absolute (or relative) insulin, excess and

compromised glucose counter-regulation in type 1 and advanced type 2

diabetes. Decrease in insulin, increase in glucagon, and absent of the latter,

increase in epinephrine are the primary glucose counter regulatory factors

that normally prevent or (more or less rapidly) correct hypoglycemia. In

insulin-deficient diabetes (exogenous) insulin levels do not decrease as

glucose levels fall, and the combination of deficient glucagon and

epinephrine responses causes defective glucose counter regulation (Chang

and Halter, 2003).

Furthermore, reduced sympathoadrenal responses can cause

hypoglycemia unawareness. The concept of hypoglycemia-associated

autonomic failure (HAAF) in diabetes posits that recent incidents of

hypoglycemia cause both defective glucose counter regulation and

hypoglycemia unawareness. By shifting glycemic thresholds for the

sympathoadrenal (including epinephrine) and the resulting neurogenic

34

responses to lower plasma glucose concentrations, antecedent hypoglycemia

tends to a vicious cycle of recurrent hypoglycemia and further impairment of

glucose counter regulation. In many cases (but not all), short-term avoidance

of hypoglycemia reverses hypoglycemia unawareness in affected patients,

although this is easier in theory than in clinical experience (Taubes, 2008;

Liew et al, 2009)

Respiratory infections: The immune response is impaired in

individuals with diabetes mellitus. Cellular studies have shown that

hyperglycemia both reduces the function of immune cells and inflammation.

The vascular effect of diabetes also tend to alter lung function, all of which

leads to an increase in susceptibility to respiratory infections such as

pneumonia and influenza among individuals with diabetes several studies

also show diabetes associated with a worse disease course and slower

recovery from respiratory infections (Ahmed et al, 2008).

1.6.2. Chronic Complications

Chronic elevation of blood sugar level leads to damage of blood

vessels. The endothelial cells lining the blood vessels take in more glucose

than normal, since they don’t depend on insulin. They then form more

surface glycoproteins than normal and cause the basement membrane to

35

grow thicker and weaker. In diabetes, the resulting problems are grouped

under microvascular disease and macrovascular disease.

However, some research challenges the theory of

hyperglycemia as the cause of diabetes complications. About 40% diabetics

who carefully control their blood sugar nevertheless develop neuropathy.

(Centofani, 1995), and that some of those with good blood sugar control still

develop nephropathy, (Rich, 2006), requires explanation. It has been

discovered that the serum of diabetics with neuropathy is tonic to nerves

even if its blood sugar content is normal (Pittinger et al, 1993). Recent

research suggests that in type 1 diabetics, the continuing autoimmune

immune disease which initially destroyed the beta cells of the pancreas may

also cause retinopathy (Kastelan et al 2007), neuropathy (Granberg et al,

2005), and nephropathy (Ichinose et al, 2007).

The damage to small blood vessels leads to a microagiopthy which

can cause one or more of the following phototherapy and diabetes mellitus

managed.

1.7: Phytotherapy and diabetes mellitus management

Since ancient times, traditional medicines all over the world have

advocated the use of plants to treat diabetes. Plants and many of their

derivatives are being used as natural remedies and folk medicines for the

treatment of diabetes globally (Akah et al, 2002). More than 1200 plant

36

derived compounds have been tested for their ability to lower blood sugar

levels. Many have been found to contain chemical constituents that have

hypoglycemic activity in animal model (Li et al, 2004).

Plant derivatives with purported hypoglycemic properties have been

used in folk medicine and traditional healing systems around the world [eg.

Native American Indian, Jewish (Yaniv et al 1987), Chinese (Covington,

2001), East Indian and Mexican] Many modern Pharmacenter used in

conventional medicine today also have national plant origins. Among them,

salega officinalis (goat’s rue or French Lilac), which was a common

traditional remedy for diabetes (Pandey et al, 1995; Oubre et al, 1997).

Similarly, the use of vitamin and mineral supplements for primary or

secondary disease prevention is of increasing interest (O’Connell, 2001).

However, there is relatively little known regarding efficacy and safety

of herb, vitamin, or other dietary supplements for diabetes. Two prior

reviews by Ernst et al examined plants with hypoglycemic activity in

humans, including 22 clinical trials (5 randomized controlled trials [RCTS]).

of the single herbs the higher-quality RCTS (with Jadad serves of 3 or

greater) are available for Coccinia indica, Ginseng species, Bauhinia

forficate, and myrcia uniflora are RCT for Allium sativum is also of adequate

quality but was conducted in nondiabetic individuals. Other herbs, Allium

cepa, Ocimum sarctum, Ficus carica, Silibum maricanum, Opuntia

37

streptacartha, and Trigenella foren, have been studied in poorer-quality

RCTS. Gymnema Sylvestve and Momordica charantia have been studied in

only nonrandomized controlled trials.

Coccinia indica

Coccmia indica (ivy gourd) is a creeping plant that grows wildly in

many parts of the India subcontinent, and is used to treat “sugar urine”

(madhumeha) in Ayurveda, a traditional East Indian healing system. The

mechanism of action of Coccinia indica is not well understood, but the herb

appears to have insulin-mimetic properties (Kamble et al, 1996). The one

RCT of this herb (n=32), conducted in India, reported significant changes in

glycaemic control following 6 weeks use of powder from locally obtained

crushed dried leaves in poorly controlled or otherwise untreated patients

with type 2 diabetes (Azad Khan et al, 1979). Another three-arm controlled

clinical trial (n=70) compared 12 weeks’ use of dried herb pellets made fresh

leaves with no treatment and oral hypoglycemic agents (chlorpropamide) in

patients with Type 2 diabetes (Kamble et al, 1996). The magnitude of

change seen with the herb was similar to that with a conventional drug.

Ginseng species

Several different plant species are often referred to as ginseng.

These include Chinese or Korean ginseng (panax ginseng), Siberian ginseng

(Eleutherocolcus senticosus), American ginseng (P. quiquefolius), and

38

Japanese guising (P. japonicus). Panax sepecies (from the root panacea) are

often touted further “cure-all” adaptogenic properties, immune stimulant

effects, and their ability to increase stamina, concentration, longevity, and

overall wellbeing (Ernst, 1997). Principal components are believed to be the

triterpenoid saponin glycosides (ginsenoside or panaxosides). Hypoglycemic

effects have been shown in streptozotoccin rat models (Shapiro and Gong,

2002). Reported mechanisms of action include decreased rate of

carbohydrate absorption into the portal hepatic circulation, increased glucose

transport and uptake mediated by intricacies, increased glycogen storage,

and modulation insulin secretion (Shapiro and Gong, 2002).

Allium Species: Satirum and cepa

Allium sativum (garlic), a member of the lily family is the most

commonly used worldwide for flavorful cooking. Much of the clinical

literature on garlic has focused on its potential antioxidant activity and

microcirculatory effects (eg, Allicin and ajoene for use in hypertension and

hyperlipidemia). Few studies have examined its effects on insulin and

glucose handling, although some attention has been given to allyl propyl

disulfide, a volatile oil, and S-allyl-cysteine sulfoxide, a sulfur containing

amino acid (Shane-McWhorter, 2001). Experiments in animal models with

alloxan-induced diabetes have shown moderate reductions in blood glucose;

no effect is seen in pancreatectomized animals (Shella and Augusti, 2001).

39

Allium cepum (onion) also contains allyl propyl disulphide and has similar

purported hypoglycemic properties. Reported mechanisms of allium species

include increased secretion or slowed degradation of insulin, increased

glutathione peroxidase activity, and improved liver glycogen storage (Shane-

McWhorter, 2001; Bailey and Day 1989). The lanceted data provide

conflicting evidence for allium species in glycemic control.

Opuntia streptacantha

Opuntia streptacantha (nopal) or the prickly pear cactus can be found

in arid regions throughout the western hemisphere, and is commonly used

for glucose control by those of Mexican descent. It has a high-soluble fiber

and pectin content, which may affect intestinal glucose uptake, partially

accounting for its hypoglycemic actions (Shapiro and Gong, 2002). Animal

models have reported decrease in postprandial glucose and HbAic with

synergistic effects with insulin. Studies in pancreatectomized animals report

that hypoglycemic activity is not dependent on the presence of insular (Frati

et al, 1991). The limited data suggests a possible hypoglycemic effect of

nopal; however, longer-term clinical trials are needed.

Pterolarpus marsupium (Indian kino, pitasara, Venga)

The tree is the source of the Kino of the European pharmacopeia.

The gum-resin looks like dried blood (Dragon’s blood) much used in Indian

medicine. This herb has a long lnstory of use in India as a treatment for

40

diabetes. The flavanoid, (-) - epicatechin, extracted from the bark of this

plant has been shown to prevent alloxan-induced beta cell damage in rats.

Both epicatechin and a crude alcohol extract of Pterocarpus marsupium

have actually been shown to regenerate functional pancreatic beta cells. No

other drug or natural agent has been shown to generate this activity.

Momordica charantia (Bitter Melon).

Bitter melon, also known as balsam pear, is a tropical vegetable

widely cultivated in Asia, Africa and South American and has been used

extensively in folk medicine as a remedy for diabetes. The blood sugar

lowering action of the fresh juice or extract of the unripe front has been

clearly established in both experimental and clinical studies.

Better melon is composed of several compounds with confirmed

anti-diabetic properties. Charantia, extracted by alcohol, is a hypoglycemic

agent composed of mixed steroids that is more potent than the drug

tolbutamide which is often used in the treatment of diabetes. Momordia also

contains an insular like polypeptide, polypeptide-P, which lowers blood

sugar levels when injected subcutaneously into type 1 diabetic patients. The

oval administration of 50-60ml of the juice has shown good results in

clinical trials.

Some plants with anti diabetic properties are shown in Table 1 .

41

Table1. Plants with antidiabetic effect

Plant species Part of plant used Reference

Allium cepa Bulb Augustin et al, 1974

Allium sativum Bulb Shane-mcwhorter,

2001

Aloe vera Aloe geland sap from

leaves

Yongchaiyudha at al

1996. Ajabnor, 1990

Anarcardium

occidentale

Bark and leaves Esimone et al, 2001

Akah et al, 2002

Anemarrhena Rhizomes Li et al, 2004

Astragalus

membranaceus

Roots Li et al, 2004

Atractylodes japonica Rhizomes Li et al, 2004

Azadirachta indica Fruits, leaves and bark Akah et al, 2002

Bauhinia forficate Leaves Barbosa-filho et al,

2005

Bouvardia terniflora Stem bark Barbosa-filho, et al,

2005

Brickellia

veroricaefolia

Entire plant Barbosa-filho, et al,

2005

Bridelia ferruginea

benth, linn

Roots, bark and leaves Iwu, 1980

Cecropia obtusifolia Leaves Barbosa-filho, et al,

2005

Coccinia grandis Tuberous roots Jan and Sharma, 1967

Coccuina indica Whole plan Grover et al, 2002

Coptis chinesis Rhizomes Li et al, 2004

Cornus officinalis Pulps Li et al, 2004

Croton cajucara Not stated Farias et al, 1997

Cuminum nigrum Seeds Gilani el al, 2000

Dioscorea dumertorum Tubers Iwu, 1992. Undie and

Akubue, 1986

Eugenia Jambolana Fruits Sanjay, 2002

Gymnema sylvestre Leaves Shanmugasundaram

et al, 1990.

Lagerstroemin

speciosa

Leaves Okada, 2003

Momordica Charanta Fruits Sanjay, 2002

Jayasoonya, 2000

42

Murraya koenigiil Seeds and stems Barbosa –filho et al,

2005

Musa sapientum Flowers and peels Jani et al, 1967 Akah

et al, 2002

Ocimum gratissimum Leaves Akah et al, 2002

Opuntia streptacantha Fruits and stems Frati et al, 1990

Panax genus Root Dey et al, 2002.

Shibata, 2001

Phyllatus embira Leaves Barbosa –filho, et al

2005

Plantago psyllium Pods Frati-munari et al

1985.

Polygonatum odoratum Rhizomes Li et al, 2004

Polygonatum sibricum Rhizomes Li et al, 2004

Psiduim guajaval Leaves Li et al, 2004

Pterocarpus

marsupium

Leaves Sanjay, 2002

Pueraria lobata Roots Li et al, 2004

Rehonannia glutinosa Roots Li et al, 2004

Salacia reticulate Root and stem Yoshikawa, et al

1997

Stevia rebaudiana Stem, leaves Barbosa-filho, et al,

2005

Tacoma sans Leaves and bark Akah et al, 2002

Trchosanthes kirilowii Roots Li et al, 2004

Trigonella foenum

graecum

Seeds Broca, 1999 Sharma

et al, 1990

Vernonia amygdalina Leaves Akah et al, 1992;

Gyang, et al, 2004

1.7.1: Pharmacognostic profile of Gongronema latifolium taxonomy

Domain: Eukaryota

Kingdom: Plantae

Subkingdom: Virideaplantae

Phylum: Magnoliophyta

43

Subphylum: Spermatophytina

Class: Magnoliopsida

Subclass: Lamiidae

Superorder: Gentiananae

Order: Gentianales

Family: Asclepiadaceae

Genus: Genus

Specific: Latifolium

Botanical Name: Gongronema latifolium benth

Common Names

Igbo- utazi

Yoruba – arok

Kissi (Sierra leone)-ndondopole

Mende (Sierra leone )-buli-yeyako

Senegal-gasub

Ghana –Aborode- aborode.

Members of the genus Gongronema

G. angolense, G. obscurum, G. Filipes, G. taylorii, G. gazense, G. wullichii,

G. hemsleyama, G. welwitschii, G. latifolium, G. multibracteolatum, G.

napalense, G. nepalense

44

1.7.2: Medicinal and non medicinal uses.

Gongronema latifolium has been used for many generation for medicinal

and non medicinal purposes. They are used in Sierra leone as chew-sticks,

and cutup and boiled with lime juice or infused in water over three days, the

liquor is taken as a purge for colic and stomach-pains, and symptoms

connected with worm-infection. The infusion is taken as a cleansing purge

by Mohammedans during Ramadan. It is given to new born babies in the

Joru area of Sierra leone to make them grow rapidly.

In Ghana, the leaves are rubbed on the joints of small children to help

them walk and in southern Nigeria, the leaves serve as a vegetable. The bark

contains a quantity of latex and though it has been viewed with potential

interest for its rubber, it has apparently never been exploited. The bark of G.

latifoliun merits examination as an official dyspeptic. In Ghana, the boiled

fruits are put into soup as a laxative (Burkill, 1985)

The leaves of G. latifolium are primarily used as spice and vegetable

in traditional folk medicine. (Ugochukwu and Babady, 2002; Ugochukwu et

al, 2003)

Reports by various authors showed that G. latifolium contains

essential oils, saponins and pregnones among others (Morebise et al, 2002).

Ugochukwu and Babady (2003) reported that aqueous and ethanolic extract

45

of G. latifolum had hypoglycaemic effect. Morebise et al (2002) showed that

it has anti-inflammatory properties.

Anti-lipid peroxidative activity of G. latifolium in streptozotocin-

induced diabetes was reported by Nwanjo et al (2006). Antibacterial activity

has also been reported for G. latifolium (Eleyimni, 2007). Hepatoprotective

and hypolipidemic effects have also been reported. (Ugochukwu and

Babady, 2003; Ugochukwu et al, 2003; Nwanjo, 2005, Nwanjo and

Alumanah, 2005).

Geopraphical distribution.

G. latifolium is a climber from a tuberous base, of deciduous and

secondary forests from Guinea-Bissau to West Cameroons, and Nigeria and

widely dispersed elsewhere in tropical Africa. The stems are soft and pliable.

1.7.5: Chemical constituents of Gongronema latifolium

G. latifolium is a good source of protein. Its protein content (27.2%

dry matter) (Eleyinmi, 2007) is quite high and compares favourable with

percent dry matter values reported for chickpea (24.0%), cowpea (24.7%),

lentil (26.1%), green pea (24.9%), fluted pumpkin leaves (22.4%),

Tamarindus indica (24.3%), mucuna flagellipes (24.9%), Hibiscus

esculentus (23%) and Parkia biglobosa (20.9%). (Glew et al, 1997;

Akwaowo et al, 2000; Ajayi et al, 2006; Igbal et al, 2006). Consumption of

100g dry matter of G. latifolium may be capable of providing 27 g of protein

46

which satisfies recommended daily allowance of protein for children. Thus,

G. latifolium leaves appear to represent a potentially rich source of some, but

not all, of the essential amino acids that are essential for humans.

The fat crude fat content of G. latifolium (6.07%) (Eleyinmi, 2007)

compare favourable with percent dry matter values reported for leafy

vegetables like Brachystegia eurywma (5.87%) and Tamarindus indica

(7.70%) (Ajayi et al, 2006). A child consuming 100g of G. latifolium would

be ingesting approximately 6.07g of fatty acid which translates into 54-56

kcal of energy or about 3%-3.5% of their daily total energy requirement.

Apart from providing energy, the lipid fraction of G. latifolium

contains modest but useful amounts of the essential fatty acid, linoleic acid.

(31.1%) (Eleyinmi, 2007).

1.8: Aims of research.

Most of the investigations done so far on G. latifolium focused mainly

on the hypolipidaemia, hepatoprotective, antibacterial and

antihyperglycaemia effect on the crude extract.The hypoglycaemic effect of

the crude extract have been elucidated by some researchers.

This present study aimed to evaluate the hypoglycaemic and

antihyperglycaemic activities of G. latifolium in normal and alloxan induced

diabetes in rats and possibly identifying the active fraction of the extract

responsible for the hypoglycaemic effect.

47

CHAPTER TWO

MATERIALS AND METHOD

2.1 Drugs, chemicals and reagents.

The following chemicals and drugs were used in the course of this

research.

Alloxan monohydrate (Sigma USA)

Glibendamide (NGC, Nigeria)

Methanol (Sigma USA)

n-Hexane (Sigma USA)

Chloroform (Sigma USA)

Crystalline copper (ii) Sulphate

Rochelle salt

Potassium bismuth

Iodide solution (Dragendrof reagent).

Potassium iodide (Wagners reagent)

Potassium mercuric iodide (Meyers reagent)

Prcric acid (Hagers reagent)

Silica Gel (60 x 200 mesh size)

One Touch Glucometer (lifescan USA)

2.2: Collection of plant material

Fresh leaves of the floral part of G. latifolium was collected by Mr A.

Ozioko of Bioresource Development and Conservation Programme (BDCP)

48

laboratory, Nsukka. It was subsequently identify by Mr Ugwuozor of Botany

Department, University of Nigeria Nsukka.

The fresh leaves where dried under shade and was milled into coarse

powder using John Wiley laboratory miller (model 4).

2.3: Experimental animals.

Males and females albino rats (110-200)g were used for the study.

The rats were purchased form the animal house of the department of

Pharmacology and Toxicology UNN. They had continuous access to food

and water during the period of experimentation.

2.4: Extraction and fractionation

One kilogram of the powdered dried leaves of G. latifolium was

extracted using soxhlet extraction as described by Trease and Evans (1999).

The powdered leaves was packed in the thimble and plugged with cotton

wool and was macerated at ambient temperature with 2.0 litres of methanol

for 3 days

After the third day, the extract was almost colourless and was

concentrated to solid form using RE: 52 Rotatory evaporator.

The solid methanolic extract was fractionated using the following

solvent in the order of increasing polarity viz chloroform, n-hexane and

methanol,

49

Aqueous extract was also prepared with 1kg of a dried leaves sample

using 100mls of distilled water

2.5: Phytochemical analysis

The whole methanolic extract and the individual solvent fractions

were subjected to phytochemical investigation, using the method described

by Harbone (1989). The tests carried out were to confirm the pressure or

absence of alkaloids, saponins flavonoids, tannins, glycosides, resins,

triterpenes, steroids, carbohydrates, fats and oil, reducing sugars and acidic

compounds.

2.6: Acute toxicity test

The acute toxicity (LD50) of the extract was determined in order to

define the range of the lethal dose and the safe range for the extract. The

methanol extract was administered in normal saline. The test was carried out

in two stages as described by Lorke (1983) using a total of 12 mice of

weight 15-32 g.

In the first stage, the animals were divided into 3 groups of 3 mice

each, and the extract was administered at three dose level (10, 100 and 1000

mg/kg) body weight. The animals were then monitored for 24 hours. Based

on the results obtained from this first stage, the remaining animals were then

grouped into 3 groups of 1 animal each for second stage of the test.

50

In the second state, three dose ranges were also used 1600, 2900 and

5000 mg/kg body weight. Each dose was administered to one specific group

only and the animals were examined again for another 24 hours. The number

of death (s) was noted for each group.

2.6.1: Induction of diabetes mellitus

Alloxan monohydrate was used to induce diabetes in rats. Alloxan

was first weighed and then solubilized with 0.2ml normal saline just prior to

injection. Diabetes was induced by injecting a dose of 120mg/kg body

weight intraperitonially. (Kannur et al, 2006). The alloxanized rats were kept

for 7 days for hyperglycaemia to develop with free access to food and water.

The rats were fasted on the 8th day for 12 hours and their blood glucose

levels were determine using one Touch Glucometer (Lifescan, USA). Rats

with glucose levels above 120 mg/kg were recruited for the study.

2.6.2: Effect of Aqueous extract of G. latifolium on mean fasting blood

glucose of normal and alloxanized rats.

The normoglycamic and glycaemic rats were fasted for 24 hours but had

access to water ad libitum thought the experiment. Five groups of 5 rats per

group were used and was treated as follows

Normoglycaemic glycaemic

Group 1 200mg/kg 200mg/kg.

Group 2 400mg/kg 400mg/kg.

51

Group 3 800mg/kg 800mg/kg.

Group 4 2mg/kg 2mg/kg

(glybenclamide) (glybenclamide)

Group 5 2mglkg (normal saline) 2mglkg (normal saline)

Blood samples were withdrawn from the animal tail vein at fixed time

intervals of 2, 4, 8, 16, 32 hours interval after the administration of the

respective drugs and the blood glucose levels determined.

2.6.3: Effect of methanol extract of G. latifolium on the mean fasting

blood glucose on normal and alloxanized rats.

The normoglycaemic and glycaemic rats were also fasted for 24 hours

but had access to water ad libitum throughout the experiment. Five groups of

5 rats per group were used and were treated as described above.

Blood samples were collected from the animal tail vein at fixed time

intervals of 2,4,8,16,32 hours after the administration of the respective drugs

and the blood glucose levels determine as well.

2.6.4: Effect of the fractions on mean fasting blood glucose on

alloxanized rats.

The glycaemic rats were fasted for 24 hours but had access to water

ad libitum throughout the experiment and was treated as follows

Group one received 400mg/kg of methanol fraction

Group two received 400mg/kg of n-hexane fraction

52

Group three received 400mg/kg of chloroform fraction

Group four received 2mg/kg of glybenclamide

Group five received 2ml/kg of normal saline

Blood samples were collected form the animal tail vein at fixed

intervals of 2, 4, 8, 16 and 32 hours after the administration of the respective

drugs and the blood glucose levels determined.

2.7: Statistical analysis

Results are given as mean blood glucose levels SEM (standard error

of mean). One way ANOVA with post hoc Dunnet’s multiple comparison

tests. P values of 0.05 and less were taken to imply statistical significance

between the means. Analysis was done using statistical programme for

social sciences (SPSS) version 7.

53

CHAPTER THREE

RESULTS

3.1 Extraction and fractionation

The green coloured methanol extract of G. latifolium gave an overall

yield of 385.22 g from the 1kg-dried leaves used for the extraction. The

solvent graded fraction of the 300g of crude extract yielded as follows:

n-Hexane soluble fraction = 43g (14.3%)

Chloroform soluble fraction = 94g (31.3%)

Methanol soluble fraction = 122g (40.6%)

3.2: Phytochemical analysis.

Phytochemical test was carried out on the whole methanolic extract as

well as the individual solvent fractions. The results are shown in Table 1.

Phytochemical screening of all the extract and fractions of G. latifolium

showed the presence of various chemical constitutions mostly saponins,

proteins, carbohydrates, resins, alkaloids, flavanoids. Terpenoids saponins,

alkaloids, resins and steroids are conspicuously present in large amount in

the crude extract. The methanol fraction possesses proteins, saponins and

glycosides in large amount and contains terpenoids, steroids and alkaloids in

moderate amount. The chloroform fraction contains resins, flavonoids and

alkaloids in moderate quantity but is devoid of other phytoconstituents. The

54

n- hexane fraction possesses terpenoid, steroids and resins in large quantity

also but is devoid of other constituents. (Table 1)

3.3 Acute toxicity test

Death was recorded in 1000 mg/kg dose therefore a geometric mean

of the dose where death occurred and the dose preceding the recorded death

was calculated and the LD50 was found to be 0. 9 g/kg.

3.4 Effect of the aqueous extract on hyperglycaemic and non-

glycaemic rats

The effect of aqueous extract on the blood sugar level of glycaemic

rats is presented in Table 2. There was a gradual decrease in the blood sugar

level from 0 hour to 32 hour. The decrease seems to be drastic in the

800mg/kg dose which is comparable with the standard antiglycaemic,

glybenclamide. There was no significant (P<0.05) decrease in the group

treated with normal saline (Table 2). There was no significant decrease in

the blood sugar level of normaglycaemic rats treated with G. latifolium

aqueous extract (Table 3)

3.5 Effect of methanol extract on diabetic and non-diabetic rats

Administration of alloxan monohydrate led to elevation of blood

glucose level. The anti-hyperglycaemic effect of the methanolic extract of

the leaves of G.latifolium (200 mg/kg, 400 mg/kg) and glibenclamide (2

mg/kg) on blood glucose levels of diabetic and non-diabetic rate were shown

55

in Tables 4 and 5. The antidiabetic effect of the test drug and glibenclamide

exhibited effect in dose dependent manner. Administration of methanol

extract (200gm/kg) recorded a slight increase in the blood glucose level in 2

hour time but later decreased in the 16th hour to 32

nd hour. The 800mg/kg of

methanol extract showed a significant (P < 0.05) decrease in the blood sugar

level and is comparable with the standard antiglycaemic. (Table 4)

In normal rats, the reduction in the blood glucose levels was found to

be insignificant (P < 0.01) as compared to the normal control.

The percentage reduction of hyperglycaemic was significant (P <

0.05) on 8th, 16

th and 32

nd hour after treatment with 800mg/kg of G-

latifolium. The percentage was also significant (P < 0.05) on 4th, 8

th, 16

th and

32nd

hour of treatment with glibenclamide. (Fig 2 and 3).

3.6 Effect of the fraction on hyperglycaemic rats

The anti-hyperglyaemic effect of methanol fraction (MF) chloroform

fraction (CF) n-hexane fraction (HF) and glibenclamide is represented in

Table 6. The MF recorded a marked decrease in the blood glucose level of

glycaemic rats on 2nd

hour till 32nd

hour (P < 0.05). The n-hexane fraction

and choloroform fraction showed an insignificant (P > 0.05) on 2nd

hour to

16th hour for treatment but was significant (P < 0.05) on the 32

nd hour. The

anti-hyperglycaemic effect observed in methanol fraction is comparable with

56

the effect of the standard antidiabetic, glibenclamide (Table 6).The treatment

with glibenclamide showed a significant (P < 0.05) decrease in blood sugar

level of glycaemic rats on 2nd

hour to 3nd

hour.

57

Table 1: Results of Phytochemical Analysis

(-) => Not Present

(+) => Present in small concentration

(++) => Present in moderately high concentration

(+++) => Present in high concentration

(++++) => Abundantly Present.

Crude

Extract

Methanol

Fraction

Chloroform

Fraction

n-

hexane

Fraction

1. Saponins + + + + + + - -

2. Proteins + + + + + + + - -

3. Tannins - - - -

4. Carbohydrates + + + + + + - -

5. Reducing Sugars - - - -

6. Resins + + + + + + + + + + +

7. Flavonoids + + + + + -

8. Alkaloids + + + + + + + + -

9. Glycosides + + + + + + - -

10. Terperoids + + + + + - + + + +

11. Steroids + + + + + - + + + +

12. Fats and oils + - - + +

13. Acidic compounds - - - -

58

Table 2: Effect of the aqueous extract on hyperglycaemic rats

Blood Glucose level (mg/dl)

Groups 0hr 2hr 4hr 8hr 16hr 32hr

1

200mg\kg

196.66±30.24 187.33±53.26 160.33±42.66 157.00±40.26 155.67±38.73 156.33±40.15

2

400mg\kg

247.33±44.42 206.67±10.00 225.00±10.00 217.00±7.81 212.00±5.56 175.00±29.51*

3

800mg\kg

284.00±55.97 221.33±22.47 211.67±15.27 193.00±25.35 182.33±19.04* 152.33±40.52*

4

Glybenclamide

283.67±39.07 254.67±19.50* 212.33±10.26 163.67±9.88* 148.67±30.50* 138.00±21.63*

5

Normal saline

216.33±25.50 191.33±21.50 190.00±15.00 200.67±25.01 198.66±30.00 201.00±24.06

Each value represents Mean±SEM

* represent statistically significance when compared to control

One way ANOVA followed by Dunnets T-test

59

Table 3: Effect of the aqueous extract on non-glycaemic rats

Blood Glucose level (mg/dl) Groups 0hr 2hr 4hr 8hr 16hr 32hr

1

200mg\kg

57.67±11.24C 47.07±19.55

C 40.00±9.16

C 38.00±7.94

C 37.67±6.66

C 36.33±4.73

C

2

400mg\kg

54.00±1.00 C

44.33±10.26C 40.00±3.61

C 37.67±4.93

C 37.00±6.08

C 36.00±6.00

C

3

800mg\kg

64.67±10.79C 49.67±3.5

C 52.00±4.00

C 49.33±3.51

C 48.33±5.51

C 47.67±4.05

C

4

Glybenclamide

42.33±6.02 C

34.67±5.68 C

38.00±5.2C 34.00±2.00

C 34.33±2.89

C 34.67±3.21

C

5

Normal saline

10 ml/kg

35.00±8.54 33.00±9.64 35.33±7.0 32.33±8.0 31.66±7.51 33.66±4.04

Each value represents Mean±SEM

* represent statistically significance when compared to control

One way ANOVA followed by Dunnets T-test

c represent

statistically insignificance when compared to control

60

Table 4: Effect of methanol extract on hyperglycaemic rats

Blood Glucose level (mg/dl)

Groups 0hr 2hr 4hr 8hr 16hr 32hr

1

200mg\kg

178.00±39.50

186.66±22.50

183.06±23.28

184.33±22.14

181.67±23.18

179.33±21.13*

2

400mg\kg

210.33±38.73

202.67±29.14

199.67±27.50

197.00±24.56

193.00±22.11*

195.00±22.72*

3

800mg\kg

219.33±36.90

182.00±41.79

155.33±27.53*

146.33±22.50*

145.66±20.59*

143.67±22.14*

4

Glybenclamide

2mg/kg

202.00±19.04

180.00±53.56

153.33±41.05*

137.33±27.54*

131.33±28.18*

126.0±30.26*

5

Normal saline

198.20±33.20

189.13±32.22

180.67±35.55

174.47±35.55

172.75±37.39

172.40±40.87

Each value represents Mean±SEM

* represent statistically significance when compared to control

One way ANOVA followed by Dunnets T-test

61

Table 5: Effect of methanol extract on hypoglycaemic rats

Blood Glucose level (mg/dl)

Groups 0hr 2hr 4hr 8hr 16hr 32hr

1

200mg\kg

91.67±12.66

81.00±13.75C

78.33±12.86C

76.33±16.44C

77.67±11.59C

78.33±11.02

C

2

400mg\kg

87.33±30.03

81.33±19.63C

81.33±19.63C

75.00±28.93C

72.67±27.43C

72.00±27.33

C

3

800mg\kg

98.00±18.08

82.00±27.22C

73.33±12.42C

66.67±16.65C

64.67±16.29C

63.33±14.22

C

4

Glybenclamide

2mg/kg

94.67±14.50

58.33±12.01C

56.67±10.21C

55.00±6.93C

43.67±5.85C

49.67±8.96*

5

10 ml/kg

Normal saline

73.33±4.5

70.67±2.08

70.00±7.21

71.67±4.61

70.67±5.68

71.33±5.85

Each value represents Mean±SEM

* represent statistically significance when compared to control

One way ANOVA followed by Dunnets T-test

62

Table 6: Effect of the fractions on hyperglycaemic rats

Blood Glucose levels (mg/dl)

Group 0hr 2hr 4hr 8hr 16hr 32hr

1

400mg\kg

Methanol

154.00±44.23

155.33±48.34*

158.33±45.65*

157.67±45.39*

164.67±53.35*

142.00±12.72*

2

400mg\kg

n-Hexane

149.66±14.01

136.00±4.00 C

128.33±8.02 C

126.67±8.5 C

127.66±9.07C

132.33±9.6*

3

400mg\kg

Chloroform

221.00±19.28

220.66±24.00C

218.33±13.31C

202.00±17.08C

193.33±24.13C

187.00±85.00*

4

Glybenclamide

2mg/kg

186.00±32.05

177.00±27.83*

151.33±23.58*

135.67±17.21*

133.67±14.19*

105.00±64.11*

5

10ml/kg

Normal saline

178.67±24.01

177.66±26.31

191.67±35.92

210.66±18.50

212.50±4.95

220.50±5.85

Each value represents Mean±SEM

* represent statistically significance when compared to control

One way ANOVA followed by Dunnets T-test c represent

statistically insignificance when compared to control

63

F ig 1: B lood S ug ar L evel of Aqueous E x trac t T reated Hyperg lyc aemic R ats

-10

0

10

20

30

40

50

60

0hr 2hrs 4hrs 8hrs 16hrs 32hrs

T ime (Hours)

Pe

rce

nta

ge

Re

du

cti

on

(%

)

200mg /kg

400mg /kg

800mg /kg

G lybenc la mide

Norma l sa line

F ig 2: B lood S ug ar L evel of Normog lyc aemic R ats T reated with

Methanol E x trac t

-10

0

10

20

30

40

50

60

0hr 2hrs 4hrs 8hrs 16hrs 32hrs

T ime (Hours)

Pe

rce

nta

ge

Re

du

cti

on

(%

)

200mg/kg

400mg/kg

800mg/kg

G lybenc lamide

Normal s aline

64

F ig 3: B lood S ug ar L evel of G lyc aemic R ats T reated with Methanol E x trac t

0

5

10

15

20

25

30

35

40

45

0hr 2hrs 4hrs 8hrs 16hrs 32hrs

T ime (Hours)

Pe

rce

nta

ge

Re

du

cti

on

(%

)

200mg/kg

400mg/kg

800mg/kg

G lybenc lamide

Normal s aline

F ig 4: B lood S ug ar L evel of G lyc aemic R ats T reated with

F rac tions of G -latifolium

-40

-30

-20

-10

0

10

20

30

40

0hr 2hrs 4hrs 8hrs 16hrs 32hrs

T ime (Hours)

Pe

rce

nta

ge

Re

du

cti

on

(%

)

400mg/kg MF

400mg/kg C F

400mg/kg HF

G lybenc lamide

Normal s aline

65

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1: Discussion

This study investigated the antidiabetic activity of Gongronema

latifolium leaves extracts and fractions. Plants are well known in traditional

herbal medicine for their hypoglycaemic activities and available literature

indicate that there are more than 800 plant species showing hypoglycaemic

activity (Rajapogal and Sasikala, 2008). There have been increasing demand

for use of plant products with antidiabetic activity due to low cost, easy

availability and lesser side effects (Sharma et al, 2010). Therefore plants

materials are continuously scrutinized and explored for their effect as

antidiabetic agents.

Hyperglycaemia (diabetes) is a major degenerative disease in the

world today (Ogbonnia et al, 2008), affecting at least 15 million people and

having complications which include hypertension, atherosclerosis and

microcirculatory disorders. (Edem, 2009).

In this investigation, alloxan was used to induce diabetes in rats.

Alloxan (2,4,5,6, tetraoxypyrimidine; 2,4,5,6- pyrimidinefetrone) is an

oxygenated pyrimidine derivative (Lenzen 2008) and was originally isolated

66

in 1818 by Brugnatelli and got its name in 1838 by Friedrich Wohler and

Sustus von Liebig. Alloxan is a toxic glucose analogue, which selectively

destroy insulin-producing cells in the pancreas when administered to rodents

and many other animal species. Alloxan is one of the usual substances used

for the induction of diabetes mellitus apart from streptozotocin. It has a

destructive effect on the beta cells of the pancreas.

Pancreas is the primary organ in sensing the organisms dietary and

energetic states via glucose concentration in the blood and in response to

elevated blood glucose, insulin is secreted (Edem, 2009). Insulin deficiency

leads to various metabolic alterations in the animals viz increased blood

glucose, increased cholesterol, increased levels of alkaline phosphate and

transaminases (Shanmugasundaram et al 1990). In this study, results showed

that 72 hours after alloxan 12 mg/kg between administrations, serum glucose

increased.

The result of the present study indicate that G. latifolium extract and

fractions was found to reduce the glucose levels in rats made diabetic with

alloxan. Alloxan has been shown to induce free radical production and cause

tissue injury. The pancreas is especially susceptible to the action of alloxan

induced free radical damage. The methanolic extract and aqueous extract

demonstrated a significant (P<0.05) decrease in blood glucose level at varied

doses. The decrease in glucose level was progressive giving the highest

67

effect after 16 hours of G. latifolium therapy. A dose of 800 mg/kg of

methanol extract gave the highest effects after the 32 hours. The aqueous

extract reduced the blood glucose level but showed no significant effect.

Administration of the same doses of G. latifolium extracts in normal rats did

not alter (nor cause significant decrease or increase) the glucose level. This

means that ingestion of G. latifolium leaf as vegetable have no harmful

effects.

Similar observations were made with the fractions. The methanol,

chloroform and n-hexane fractions reverse the alloxan induced diabetes in

rats but at different rates. Methanol fraction was able to return the blood

sugar level to normal ranges while the chloroform and n-hexane fractions

also reduced the blood glucose level but could not return it to its normal

ranges.

The effects of the methanol extract, methanol fraction and

glibenclamide in the diabetic rats when compared with the control (negative

control) were similar after 16 hours of treatments. Phytochemical screening

of the extract and fractions of G. latifolium showed the presence of various

chemical constituents mostly saponins, proteins, carbohydrates, resins,

alkaloids and flavanoids. Terpenoids and steroids were conspicuously

present in large amount in the crude extract. The literature reports reveal that

68

flavonoids and terpenoids present in the plant extract is known to possess

antidiabetic activity. (Sharma et al, 2010; Sikarwar and Patil, 2010).

Although the exact mechanism of action of the extract is unknown, the

effect exhibited suggests a possible stimulation of insulin release from the

residual B-cells and glucagons inhibition. In addition, the extract might have

insulin-like effect acting by improving the glucose uptake and metabolism or

by inhibiting gluconeogensis thereby exerting the hypoglyeamic effect.

Further studies however need to be carried out to identify the exact

mechanism of action of G-latifolium and the compounds responsible for the

hypoglycaemic effect.

4.2: Conclusion.

The speedy and progressive decrease in the blood glucose levels of

rats, formally increased by alloxan monohydrate, by the treatment with the

leavesof G. latifolium may be due to the presences of phytochemicals found

in abundance in the plant.

This present study therefore support the claim of the local use of G.

latifolium leaves in the treatment of Diabetes mellitus.

69

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