CHUKWUMA, UCHENNA VALENTINE … Uchenna...extracts of Euphorbia hirta to rats in group 3 to 6 and...

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Digitally Signed by: Content manager’s Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Nwamarah Uche

Faculty of Biological scicnces

Department of Biochemistry

EFFECTS OF ETHANOL EXTRACTS OF EUPHORBIA HIRTA

HERB ON SOME OXIDATIVE AND BIOCHEMICAL

PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

CHUKWUMA, UCHENNA VALENTINE

(PG/M.Sc/08/48973)

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TITLE

EFFECTS OF ETHANOL EXTRACTS OF EUPHORBIA HIRTA HERB

ON SOME OXIDATIVE AND BIOCHEMICAL PARAMETERS IN

ALLOXAN-INDUCED DIABETIC RATS

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR AWARD OF THE DEGREE OF MASTER OF

SCIENCE (M.Sc) IN MEDICAL BIOCHEMISTRY, UNIVERSITY OF

NIGERIA,

NSUKKA

BY

CHUKWUMA, UCHENNA VALENTINE

(PG/M.Sc/08/48973)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: DR. V. N. OGUGUA

APRIL, 2013

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CERTIFICATION

CHUKWUMA, Uchenna Valentine, a postgraduate student with Registration Number

PG/M.Sc/08/48973 in the Department of Biochemistry has satisfactorily completed the

requirements for the research work for the degree of Master of Science (M.Sc) in Medical

Biochemistry. The work embodied in this report is original and has not been submitted in

part or full for any other diploma or degree of this or any other university.

DR V.N. OGUGUA PROF L.U.S. EZEANYIKA

(Supervisor) (Head of Department)

EXTERNAL EXAMINER

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DEDICATION

My sincere dedication goes specially to God Almighty.

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ACKNOWLEDGEMENTS

I am very grateful to God for the gift of life and good health throughout the course of this

work. I am also grateful to my parents who provided the funds for the work. Great thanks go to my

supervisor Dr. V.N. Ogugua for his guidance at every stage of the work. Finally, I wish to thank Dr.

Parker Elijah Joshua, (Medical/Pharmacological Biochemistry Unit, University of Nigeria, Nsukka), Dr.

Olive Obienu (Consultant Gastroenterologist, U.N.T.H, Ituku/Ozalla), Dr. Francis Ukekwe (Consultant

Histopathologist, U.N.T.H, Ituku/Ozalla), and Dr. M.C. Ugonabor (Consultant Chemical Pathologist,

U.N.T.H, Ituku/Ozalla) for their wonderful contributions to the success of the work.

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ABSTRACT

Diabetes mellitus produces a lot of highly reactive oxygen species which have been attributed

to the aetiology and pathophysiology of the disease. In view of the adverse effects associated

with synthetic drugs and natural medicine being considered to be safer, cheaper and more

effective, traditional antidiabetic plants can be explored. The results of the experiment showed

that there were significant increases (P<0.05) in the concentrations of total cholesterol, low

density lipoprotein (LDL) and triacylglycerol (TAG) in group 2 rats (diabetic untreated)

compared with normal control rats (group 1). Administration of 300 mg/kg b.w. of ethanol

extracts of Euphorbia hirta to rats in group 3 to 6 and 0.01mg/kg b.w of voglibose to rats in

group 7 showed significant reduction (p<0.05) in total cholesterol, LDL and TAG

concentrations. On the other hand, there was significant decrease (p<0.05) in high density

density (HDL) concentrations in the group 2 (diabetic untreated) compared with group 1

(normal rats). However, administration of 300 mg/kg b.w of ethanol extracts of E. hirta to rats

in group 3 to 6 and 0.01 mg/kg b.w to rats in group 7 showed significant increase (p<0.05) in

HDL concentration. There was no significant increase (p>0.05) in sodium and bicarbonate ion

concentrations but significant increase (p<0.05) in potassium and chloride ion concentrations

in diabetic untreated rats (group 2) compared with rats in normal control group. There was

significant increase (p<0.05) in serum urea and creatinine concentrations in diabetics untreated

rats (group 2) compared with normal rats (group 1). Administration of 300 mg/kg b.w. of

ethanol extract of E. hirta to groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to group 7

resulted in significant decrease (p<0.05) in serum urea and creatinine concentrations. There

was significant decrease (p<0.05) in serum catalase and superoxide dismutase activities and

vitamin C concentration with significant increase (p<0.05) in serum malondialdehyde

concentration in group 2 (diabetics untreated rats) compared with normal rats (group 1).

However, addition of 300 mg/kg b.w. of ethanol extract of E. hirta to Groups 3 to 6 and 0.01

mg/kg b.w. of voglibose to group 7 resulted in significant increase (p<0.05) in serum catalase

and superoxide dismutase activities and vitamin C concentration, with significant decrease

(p<0.05) in MDA concentration compared with the diabetic untreated rats (group 2). There was

significant increase (p<0.05) in blood glucose concentration in rats of group 2 to 7 before

administration of ethanol extracts of E. hirta and voglibose compared with normal rats (group

1). When 300 mg/kg b.w. of ethanol extract of E. hirta was administered to groups 3 to 6 and

0.01 mg/kg b.w. of voglibose to group 7, there was significant decrease (p<0.05) in blood

glucose concentration compared with diabetic untreated (group 2). The administration of 300

mg/kg b.w. of ethanol extract of E. hirta and 0.01 mg/kg b.w. of voglibose showed significant

increase (p<0.05) in the body weights of the rats in groups 4 to 7 compared with that of normal

control. No significant increase (p>0.05) in the body weights of rats in group 2 and 3 compared

with normal rats (group 1). When 300 mg/kg b.w. of ethanol extract of E. hirta and 0.01

mg/kg b.w. of voglibose were administered to rats in groups 3 to 7, there was significant

increase (p<0.05) in the body weights of the rats compared with diabetic untreated rats (group

2).

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TABLE OF CONTENTS

PAGE

Title Page .. .. .. .. .. .. .. .. .. .. i

Certification .. .. .. .. .. .. .. .. .. .. ii

Dedication .. .. .. .. .. .. .. .. .. .. iii

Acknowledgements .. .. .. .. .. .. .. .. .. iv

Abstract .. .. .. .. .. .. .. .. .. .. v

Table of Contents .. .. .. .. .. .. .. .. .. vi

List of Figures .. .. .. .. .. .. .. .. .. .. xi

List of Tables .. .. .. .. .. .. .. .. .. .. xii

List of Plates .. .. .. .. .. .. .. .. .. xiii

List of Abbreviations .. .. .. .. .. .. .. .. .. xiv

CHAPTER ONE: INTRODUCTION

1.1 History of diabetes mellitus ... ... ... ... ... ... ... 1

1.2 Types of diabetes mellitus ... ... ... ... ... ... ... 3

1.2.1 Type 1 diabetes mellitus (insulin dependent diabetes mellitus) ... ... 3

1.2.1.1 Immune mediated type 1 diabetes mellitus ... ... ... ... ... 3

1.2.1.2 Idiopathic type 1 diabetes mellitus ... ... ... ... ... ... 3

1.2.2 Type 2 dibetes mellitus (Non-insulin dependent diabetes mellitus) ... ... 3

1.2.3 Gestational diabetes mellitus ... ... ... ... ... ... ... 3

1.2.4 Miscellanous types of diabetes mellitus ... ... ... ... ... 3

1.2.4.1 Pre-diabetes (Impaired glucose tolerance) ... ... ... ... ... 3

1.2.4.2 Genetic type ... ... ... ... ... ... ... ... ... 4

1.2.4.3 Secondary diabetes ... … … ... ... ... ... ... 4

1.3 Clinical features of diabetes mellitus ... ... ... ... ... 4

1.4 Diagnosis of diabetes mellitus … ... ... ... ... ... 4

1.5 Complications of diabetes mellitus … ... ... ... ... ... 5

1.5.1 Acute complications diabetes mellitus ... ... ... ... ... 5

1.5.2 Chronic complications of diabetes mellitus ... ... ... ... ... 5

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1.5.2.1 Diabetes Macroangiopathy ... … ... ... ... ... ... 5

1.5.2.2 Diabetic microangiopathy … ... ... ... ... ... ... 6

1.5.2.3 Infections … … … … ... ... ... ... ... 6

1.6 Euphorbia hirta herb … … … ... ... ... ... ... 6

1.6.1 Medicinal application of Euphorbia hirta ... ... ... ... ... 7

1.7 Alpha glucosidase inhibitors … ... ... ... ... ... 7

1.7.1 Voglibose in the management of type 2 diabetes … … … … 7

1.8 Oxidative stress in diabetes … … … … … … … 7

1.8.1 Catalase … … … ... ... ... ... ... ... 8

1.8.2 Glutathione perosidase and glutathione reductase … ... ... ... 8

1.8.3 Lipid peroxidation … … … … … … … … 8

1.8.4 Superoxide dismutase … … … … … … … 9

1.8.5 Vitamins … … … … ... ... ... ... ... 9

1.9 Justification of the research … … … … … … … 10

1.10 Aim and objectives of the study ... ... ... ... ... … 10

1.10.1 Aim of the study … … … … … … … … 10

1.10.2 Specific objectives of the study … ... ... ... ... ... 10

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials … … … … … … … … … 11

2.1.1 Plant materials (Euphorbia hirta herbs) … … … … … 11

2.1.2 Animals … … … … … … … … … 11

2.1.3 Chemicals/Reagents/Samples … … … … … … 11

2.1.4 Instruments/Equipment … … … … … … … 11

2.2 Methods … … … … … … … … … 12

2.2.1 Experimental design … … … … … … … … 12

2.2.2 Composition of diet fed experimental rats … … … … … 12

2.2.3 Preparation of normal saline … … … … … … … 13

2.2.4 Extraction of Euphorbia hirta herbs … … … … … … 13

2.2.5 Induction of diabetes … … … … … … … … 13

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2.2.6 Acute toxicity studies (LD50) … … … … … … … 13

2.2.7 Phytochemical analysis … … … … … … … 14

2.2.7.1 Test for carbohydrate (Molisch’s test) … … … … … 14

2.2.7.2 Test for alkaloids (General tests) … … … … … … 15

2.2.7.3 Test for glycosides (Fehling’s test) … … … … … 16

2.2.7.4 Test for saponins (Kokate, 2001) … … … … … 16

2.2.7.5 Test for tannins (Ferric chloride method) … … … … … 17

2.2.7.6 Test for flavonoids (Ammonium test method) … … … … 17

2.2.7.7 Test for resins (Precipitation test) … … … … … 17

2.2.7.8 Test for proteins (Millon’s test) … … … … … … 17

2.2.7.9 Test for oils … … … … … … … … 17

2.2.7.10 Test for steroids and terpenoids … … … … … … 18

2.2.8 Proximate analysis … … … … … … … 18

2.2.8.1 Crude protein … … … … … … … … 18

2.2.8.2 Crude fat … … … … … … … … … 20

2.2.8.3 Moisture … … … … … … … … 20

2.2.8.4 Ash/mineral matter … … … … … … … 21

2.2.8.5 Crude fibre … … … … … … … … 21

2.2.8.6 Carbohydrate or nitrogen free extract (NFE) … … … … 22

2.2.9 Determination of total cholesterol concentration … … … … 23

2.2.10 Low density lipoprotein-cholesterol concentration … … … 24

2.2.11 High density lipoproteins (HDL)–cholesterol concentration … … 25

2.2.12 Determination of triacylglycerol concentration … … … … 26

2.2.13 Assay of superoxide dismutase activity … … … … 27

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2.2.14 Assay of catalase activity … … … … … … … 29

2.2.15 Determination of malondialdehyde concentration … … … 30

2.2.16 Determination of ascorbic acid concentration … … … … 31

2.2.17 Determination of sodium ion concentration … … … … 32

2.2.18 Determination of potassium ion concentration … … … … 33

2.2.19 Determination of chloride ion concentration … … … … 33

2.2.20 Determination of bicarbonate ion concentration … … … 34

2.2.21 Determination of serum urea concentration … … … … 34

2.2.22 Determination of serum creatinine concentration … … … … 36

2.2.23 Determination of fasting and random glucose concentration … … 37

2.2.24 Body weight … … … … … … … … 38

2.2.25 Histopathological examination … … … … … … 38

2.3 Statistical analysis … … … … … … … … 40

CHAPTER THREE: RESULTS

3.1 Phytochemical analysis … … … … … … … 41

3.2 Percentage proximate composition … … … … … … 42

3.3 Acute toxicity studies … … … … … … … 43

3.4 Effects of ethanol extracts of Euphorbia hirta on serum total cholesterol

concentration of normal and diabetic rats … … … … … 44

3.5 Effects of ethanol extracts of Euphorbia hirta on serum high density

lipoprotein concentration of normal and diabetic rats … … … 46

3.6 Effects of ethanol extracts of Euphorbia hirta on serum low density

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3.7 Effects of ethanol extracts of Euphorbia hirta on serum triacylglycerol


3.8 Effects of ethanol extracts of Euphorbia hirta on serum sodium ion


3.9 Effects of ethanol extracts of Euphorbia hirta on serum potassium ion


3.10 Effects of ethanol extracts of Euphorbia hirta on serum chloride ion


3.11 Effects of ethanol extracts of Euphorbia hirta on serum bicarbonate ion


3.12 Effects of ethanol extracts of Euphorbia hirta on serum urea


3.13 Effects of ethanol extracts of Euphorbia hirta on serum creatinine


3.14 Effects of ethanol extracts of Euphorbia hirta on serum catalase

activity of normal and diabetic rats … … … … … … 64

3.15 Effects of ethanol extracts of Euphorbia hirta on serum superoxide

dismutase activity of normal and diabetic rats … … … … 66

3.16 Effects of ethanol extracts of Euphorbia hirta on malondialdehyde


3.17 Effects of ethanol extracts of Euphorbia hirta on vitamin C


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3.18 Effects of ethanol extracts of Euphorbia hirta on body glucose concentration

of normal and diabetic rats before and after the experiment … … … 72

3.19 Effects of ethanol extracts of Euphorbia hirta on body weights of normal

and diabetic rats before and after the experiment … … … … 74

3.20 Histopathological examination … … … … … … 76

CHAPTER FOUR: DISCUSSION

4.1 Discussion … … … … … … … … … 84

4.2 Conclusion … … … … … … … … … 89

4.3 Recommendations … … … … … … … 89

REFERENCES … … … … … … … … … 90

APPENDICES … … … … … … … … … 97

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LIST OF FIGURES

PAGE

Fig. 1 Euphorbia hirta plant … … … … … … … … 6

Fig. 2 Effects of ethanol extracts of Euphorbia hirta on serum total cholesterol


Fig. 3 Effects of ethanol extracts of Euphorbia hirta on serum high density


Fig. 4 Effects of ethanol extracts of Euphorbia hirta on serum low density


Fig. 5 Effects of ethanol extracts of Euphorbia hirta on serum triacylglycerol


Fig. 6 Effects of ethanol extracts of Euphorbia hirta on serum sodium ion


Fig. 7 Effects of ethanol extracts of Euphorbia hirta on serum potassium ion


Fig. 8 Effects of ethanol extracts of Euphorbia hirta on serum chloride ion


Fig. 9 Effects of ethanol extracts of Euphorbia hirta on serum bicarbonate ion


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Fig. 10 Effects of ethanol extracts of Euphorbia hirta on serum urea


Fig. 11 Effects of ethanol extracts of Euphorbia hirta on serum creatinine


Fig. 12 Effects of ethanol extracts of Euphorbia hirta on serum catalase

activity of normal and diabetic rats … … … … … … 65

Fig. 13 Effects of ethanol extracts of Euphorbia hirta on serum superoxide

dismutase activity of normal and diabetic rats … … … … 67

Fig. 14 Effects of ethanol extracts of Euphorbia hirta on serum malondialdehyde


Fig. 15 Effects of ethanol extracts of Euphorbia hirta on vitamin C


Fig. 16 Effects of ethanol extracts of Euphorbia hirta on body glucose concentration

of normal and diabetic rats before and after the experiment … … … 73

Fig. 17 Effects of ethanol extracts of Euphorbia hirta on body weights of normal

and diabetic rats before and after the experiment … … … … 75

LIST OF TABLES

Table 1: World health organisation diabetes criteria … … … … 5

Group 1 = Normal Control Group 2 = Diabetic Untreated

Group 3 = 300mg/kg b.w. of flower Euphorbia hirta Group 4 = 300mg/kg b.w. of leaf Euphorbia hirta

Group 5 = 300mg/kg b.w. of stem Euphorbia hirta Group 6 = 300mg/kg b.w. of flower+leaf+stem extract

Group 7 = Standard drug (0.01mg/kg b.w. of Voglibose)






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Table 2: Procedure for malondialdehyde concentration determination … … 31

Table 3: Qualitative phytochemical analysis of ethanol extracts of Euphorbia hirta 41

Table 4: Percentage proximate composition of ethanol extracts of Euphorbia hirta 42

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LIST OF PLATES

Page

Plate 1: Photomicrograph of pancreas of normal control rats (group 1)

showing an unremarkable islet of langerhans surrounded by

normal acini of exocrine pancreas tissue (EP) … … … … 77

Plate 2: Photomicrograph of pancreas of group 2 rats (diabetic untreated)

showing moderate to severe lymphocytic infiltration of islet of langerhans … 77

Plate 3: Photomicrograph of pancreas of group 3 diabetic rats treated

with 300 mg/kg b.w. of the flower extract showing mild to moderate

lymphocytic infiltration of islet of langerhans … … … … 78


with 300 mg/kg b.w. of the leaf extract showing mild lymphocytic

infiltration of islet of langerhans … … … … … … 80


with 300 mg/kg b.w. of the stem extract showing mild lymphocytic

infiltration (LI) of islet of langerhans … … … … … … 81


with 300 mg/kg b.w. of the flower, leaf and stem extract showing scanty

lymphocytic infiltration (LI) of islet of langerhans … … … … 82

Plate 7: Photomicrograph of pancreas of group 7 rats (0.01 mg/kg b.w. of

standard drug, voglibrose showing scanty lymphocytic infiltration

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of an enlarged islet of langerhans … … … … … 83

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LIST OF ABBREVIATIONS

CAT Catalase

CHOL Cholesterol

Cl– Chloride Ion

HDL High density lipoprotein

K+ Potassium ion

LDL Low density Lipoprotein

MDA Malondialdehyde

Na+ Sodium ion

NFE Nitrogen free extract

SOD Superoxide dismutase

TAG Triacylglycerol

VLDL Very low density lipoprotein

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CHAPTER ONE

INTRODUCTION

Euphorbia hirta herb is traditionally used to treat asthma, respiratory tract infections

and cough (Ogbulie et al., 2007) but has been recently reported to have antidiabetic effect

which may be related to its antioxidant capacity and its alpha glucosidase inhibitory properties

(Widharna et al., 2010). Some established alpha glucosidase inhibitors within the intestinal

brush border, attenuates post-prandial blood glucose peaks (Balfour and Tavish, 1993).

Diabetes mellitus produces a lot of highly reactive oxygen species which have been attributed

to the aetiology and pathophysiology of the disease. Antioxidant enzymes such as catalase,

glutathione peroxidase and superoxide dismutase help to neutralize harmful free radicals

(Nelson and Cox, 2005). In view of the adverse effects associated with synthetic drugs and

natural medicine being considered to be safer, cheaper and more effective, traditional

antidiabetic plants can be explored (Kamboj, 2000).

1.1 HISTORY OF DIABETES MELLITUS

The term diabetes was coined by Aretaeus of Cappodocia susbruta (6th

Century B.C)

identified diabetes and classified it as med humelia and identified it with obesity sendentary

life; hence, advising exercise to cure (Dwired et al., 2007). Medieral Persia Aricenna (980-

1037) provided a detailed account on diabetes in the canon of medicine, describing the

abnormal appetite and the collapse of sexual function. He also recognised a primary and

secondary diabetes, and described diabetes gangrene. He treated diabetes using a mixture of

lupine trigonella (Fenugreek) and zedoary seed which produced a reduction in excretion of

sugar. He described diabetes insupidus very precisely for the first time but Johann Peter Frank

(1745-1821) differenciated between diabetes mellitus and diabetes insupidus (Nabipour, 2003).

Diabetes was first recorded in English in the form diabete, in a medical text written

around 1425. In 1675, Thomas Willis added two words mellitus from the Latin origin meaning

“honey”, a reference to the sweet taste of the urine. Matthew Dabson confirmed that the sweet

taste was because of an excess of a kind of sugar in the urine and of people with diabetes

(Dobson, 1776). Aretaeus did attempt to treat it but could not give a good prognosis; He

commented on life (with diabetes) is short and disgusting (Medvei, 1993).

The discovery of a role for the pancreas in diabetes was described by Joseph Von

Mering and Oskar Minkowski, who in 1889 found that dogs whose pancreas was removed,

developed all the signs and symptoms of diabetes and died shortly after wards (Von Mering

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and Minkowski, 1890). In 1910, Sir Edwin Albert sharpey-Schafer suggested that people with

diabetes were deficient in a single chemical that is normally produced by the pancreas. He

proposed calling this substance insulin, from the Latin insula meaning island, but the endocrine

role of insulin was not clarified until 1921 when Sir Fredrick Grant Banting and Charles

Herbert Best repeated the work of Von Mering and Minkowski and demonstrated they could

reverse induced diabetes in dogs by giving them extracts from the pancreatic islets of

Langerhans of healthy dogs (Bating et al., 1991).

In 1869, Paul Langerhans, a medical student of Berlin, was studying the structure of the

pancreas under microscope when he identified some previously unnoticed tissue climps

scattered throughout the bulk of the pancreas and were known as islets of Langerhans. In 1901,

another major step was taken by Eugene Opie when he clearly established the link between the

islet of Langerhans and diabetes. The distinction between what is now known as type 1

diabetes and type 2 diabetes was first clearly made by Sir Harold Perciral (Harry) Hims Worth

and in January, 1936 (Himsworth, 1936).

Bating and Best purified the hormone insulin from Biovin pancreas at the University of

Toronto (Bating et al., 1991) and in plants such as Safflower. The first synthetic insulin was

produced simultaneously in the labs of Panaroitis Katsovannis at the University of Pittsburgh

and Helmut, Zahn at RWTH, Aachen University in the early 1960. The first genetically

engineered synthetic human insulin was produced in a Laboratory in 1977 by Herbert Boyer

using E. coli, and in 1980, a U.S. biotech company Genentech, founded by Boyer and Eli Lily

developed human insulin under the brand name Humulin. The insulin was isolated from

genetically altered bacterial which produced large quantities of insulin.

Other land mark discoveries include:

• Identification of the first of the sulfonylureas in 1942 by Marcel Janbon and co-workers

(Janbon et al., 1942) and it induced hypoglycaemia in animals (Patlak, 2002).

• Determination of the amino acid sequence of insulin by Sir Fredick Sanger

• The radioimmuno assay for insulin as discovered by Rosaly Jallow and Solomon

Berson, gaining Jallow a Nobel Prize in Physiology or Medicine in 1977.

• Dr. Gerald Reaven identified the constellation of symptoms now called metabolic

syndrome in 1988.

1.2 Types of Diabetes Mellitus

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There are four main types of diabetes mellitus: Type 1 diabetes, Type 2 diabetes,

gestational diabetes and miscellanous types of diabetes.

1.2.1 Type 1 diabetes mellitus (insulin dependent diabetes mellitus)

Type 1 diabetes is characterised by loss of the insulin-producing beta cells of the islets

of the langerhans in the pancreas leading to insulin deficiency apparently mediated by white

cell production of active oxygen species (Oberley, 1988). This type of diabetes can be further

classified as:

1.2.1.1 Immune-mediated type 1 diabetes mellitus

This type of diabetes mellitus accounts for majority of type 1 diabetes where the beta

cell loss is a T-cell mediated autoimmune attack (Rother, 2007).

1.2.1.2 Idiopathic type 1 diabetes mellitus

In this type of diabetes mellitus, no aetiology has been clearly implicated.

1.2.2 Type 2 diabetes mellitus (Non-insulin dependent diabetes mellitus)

This type of diabetes mellitus result from insulin resistance, a condition in which cells

fail to use insulin properly, sometimes combined with an absolute insulin deficiency. There is a

strong inheritable genetic connection in type 2 daibetes.

1.2.3 Gestational diabetes mellitus

This type of diabetes mellitus occurs when pregnant women, who have never had

diabetes before have high blood glucose level during pregnancy. Even though it may be

transient, untreatable gestational diabetes can damage the health of the foetus or mother. Risks

to the baby includes: Macrosomia (high birth weight), congenital cardiac anomalies, central

nervous system anomalies and skeletal muscle malformation. Infact, the rate of diabetes in

expectant mothers has more than doubled in the past 6 years (Lawrence et al., 2008).

1.2.4 Miscellaneous types of diabetes mellitus

1.2.4.1 Pre-diabetes (Impaired glucose tolerance)

This is a condition that occurs when a person’s blood glucose levels are higher than

normal, but not high enough for a diagnosis of type 2 diabetes. Many people destined to

develop type 2 diabetes spend many years in a state of pre-diabetes and have risks of

22

cardiovascular complications (ADA, 2002) which have been termed “America’s largest health

care epidemics (Jellinger, 2009).

1.2.4.2 Genetic Type

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

function. Abnormal insulin action may also have been genetically determined in some cases

(Tominaga, 1999).

1.2.4.3 Secondary diabetes

Any disease that causes extensive damage to the pancreas may lead to diabetes. Such

examples include chronic pancreatitis, cystic fibrosis, acromegaly, haemochromatosis,

Cushing’s syndrome (Iwasaki et al., 2008), thyrotoxicosis, aging (Jack et al., 2004), high fat

diet (Lovejoy, 2002) and less active life (Hu, 2003). Obesity has been found to contribute to

approximately 55% of cases of type 2 diabetes (CDC, 2004) and decreasing consumption of

saturated fats and transfatty acids, while replacing them with unsaturated fats, may decrease

risks (Riserus et al., 2009).

1.3 Clinical features of diabetes mellitus

The classical symptoms of diabetes mellitus include: Polyuria (frequency urination),

polydipsia (increased thirst), polyphagia (frequent hunger), fatigue and weight loss (Cooke,

2008). Other important features include: collapse of sexual function (Nabipour, 2003), blurring

of vision and skin rashes collectively known as diabetes dermadromes and develop in 30 to

70% of diabetic patients (Izaki, 2000).

1.4 Diagnosis of Diabetes Mellitus

Diabetes is characterized by recurrent or persistent hyperglycemia and is diagnosed by

demonstrating any one of the following: (WHO, 1999).

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

• Plasma glucose ≥ 11.1mmol/L (200 mg/dL) 2 hours after a 75g oral glucose load as in a

glucose tolerance test.

• Symptoms of hyperglycemia and casual plasma glucose ≥ 11.1mmol/L (200 mg/dL).

• Glycerated haemoglobin (HbAIC ≥ 6.5%).

Also, 2006 WHO diabetes criteria

Table 1: World Health Organisation diabetes criteria

23

Condition 2 hours glucose mmol/L

(mg/dL)

Fasting glucose mmol/L

(mg/dL)

Normal < 7.8 (< 140) < 6.1 (<110)

Impaired Fasting glycaemia < 7.8 (<140) ≥ 6.1 (≥ 110) and < 7.0 (<126)

Impaired glucose tolerance ≥ 7.8 (≥ 140) < 7.0 (< 126)

Diabetes mellitus ≥ 11.1 (≥ 200) ≥ 7.0 (≥ 126)

For pre-diabetes, fasting plasma glucose level from 6.1 mmol/L (110 mg/dL) to 6.9

mmol/L (125 mg/dL) (DCDM, 2005). It is preferred to measure a fasting glucose level because

of the ease of measurement and the considerable time commitment of formal glucose tolerance

testing which takes 2 hours to complete and offers no prognostic advantage over the fasting test

(Saydah et al., 2001). Glycosylated haemoglobin (HbAIC) has emerged as an accepted marker

of glycemic control and clinical efficacy in studies of diabetes (Horie, 2009).

1.5 Complications of Diabetes Mellitus

1.5.1 Acute complications diabetes mellitus

Diabetes without proper treatment can cause many complications such as

hypoglycaemia, diabetes ketoacidosis or non ketotic hyperosmolar coma:

1.5.2 Chronic complications of diabetes mellitus

1.5.2.1 Diabetes Macroangiopathy

Diabetes macroangiopathy affects small muscular arteries, especially arteries of the

lower leg and foot. A toe may be gangrenous in the presence of normal female and popliteal

pulses due to the fact that relatively small vessels are narrowed by atheroma. This result in

atherosclerotic cardiovascular disease like coronary arthery disease, cerebrovascular disease

and peripheral vascular disease.

1.5.2.2 Diabetic Microangiopathy

24

Diabetes microangiopathy affects large arteries and affects diabetes of all types,

appears to be related to the duration of the diseas and is properly aggravated by poor diabetic

control. It is responsible for diabetic retinopathy, neuropathy and nephropathy (MacSween and

Whaley, 1992), coronary heart disease and hypertension (Adler et al., 2000).

1.5.2.3 Infections

There is an increased susceptibility of bacterial and fungal infections. Boils, carbuncles

and urinary tract infections sometimes complicated by pylonephritis and renal papillary

necrosis are of frequent occurrence and may precipitate diabetic coma.

1.6 Euphorbia hirta Herb

Euphorbia hirta herb, commonly called Asthma weed is a very common annual herb. It

has a hairy plane that grows up to 2 inches in height; it has numerous small flowers clustered

together with opposite oblong leaves. The young yellow fruit is a small hairy capsule with 3

reddish-brown seeds. The plant flowers and fruits all year long. Fig. 1 shows the picture of the

plant with its leaves, stem and flower.

Fig. 1: Euphorbia hirta plant

1.6.1 Medicinal Application of Euphorbia hirta

25

Euphorbia hirta herb is traditionally used to treat asthma, bronchitis, worm infestation,

conjunctivitis and dysentery (Ogbulie et al., 2007) but has been recently reported to have

antidiabetic effect which may be related to its antioxidant capacity and its alpha glucosidase

inhibitory properties (Widharna et al., 2010).

1.7 Alpha Glucosidase Inhibitors

Alpha glucosidase inhibitors act by delaying the absorption of complex carbohydrates

and disaccharides to absorbable monosaccharide from the small intestine. They lower post

prandial blood glucose and insulin levels by reversibly inhibiting glucosidases in intestinal

brush (Balfour and Tavish, 1993). This process leads to a reduction of glucose absorption and

subsequent reduction in postprandial hyperglycemia (Van de Larr, 2005).

1.7.1 Voglibose in the Management of Type 2 Diabetes

Voglibose is an alpha glucosidase inhibitor and is an ideal agent for the management of

type 2 diabetes due to its direct hypoglycaemic effect through decreased absorption and

hypolipidemic effect via improved insulin sensitity (Shinozaki, 1996).

Voglibose reduces cartid in time media thickness with decrease in HbAiC hence

reducing the incidence of chronic vascular complications in diabetic patients (Yibchok-anun,

2009). A study conducted by Satoh and co-workers on 30 type 2 diabetic patients suggested

that voglibose reduces oxidative stress generated and soluble intercellular adhesion molecule in

obese type 2 diabetic patients (Satoh, 2006).

1.8 Oxidative Stress in Diabetes

Increasing evidence in both experimental and clinical studies suggest that increased

oxidative stress is a widely accepted participant in the development and progression of diabetes

and its complications (Baynes, 1991). Diabetes is usually accompanied by increased

production of free radicals (Chang et al., 1993), or impaired antioxidant defenses (Mc Lennan

et al., 1991).

Common antioxidants includes the vitamin A, C and E and the enzymes superoxide,

dismutase, catalase, glutathione peroxidase and glutathione reductase (Maritin et al., 2003).

Other antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10 (Brownlee, 2001).

Several bioflavonoids, antioxidants minerals (copper, zinc, manganese and selenium) and the

cofactors (folic acid, vitamin B1, B2, B6, B12). They work in synergy with each other and

against different types of radicals. Vitamin E suppresses the propagation of lipid peroxidation

26

(Hensley et al., 2000). Vitamin C, with vitamin E inhibits hydroperoxide formation. Metal

complexing agents, such as penicillamine bind transitional metals involve in some reactions in

lipid peroxidation and inhibit Fenton and Haber-weiss-type reactions (Laight et al., 2000);

vitamins A and E scavenge free radicals (Chow, 1991).

The involvement of oxidative stress in the pathology of diabetes from its associated

cardiovascular dysfunctions, nephropathy, retinopathy (leading to blindness) and embryopathy

or congenital malfunctions, suggests that potential management of diabetes could benefit from

use of dietary biofactors in medicinal and food plants (Okezie et al., 2007). The effects

of antioxidants on oxidative stress are measured through some observable biomarkers which

include:

1.8.1 Catalase

Catalase located in peroxisomes, decomposes hydrogen peroxide to water and oxygen

(Winterbourn, 1993). Catalase activity is consistently found to be elevated in the heart (Sanders

et al., 2001), Aorta (Kocak et al., 2000), as well as brain (Aragno et al., 1999) of diabetic rats.

1.8.2 Glutathione Peroxidase and Glutathione Reductase

Glutathione peroxidase and reductase are two enzymes that are found in the cytoplasm,

mitochindria and nucleus. Glutathione peroxidase metabolizes hydrogen peroxide to water by

using reduced glutathione as a hydrogen donor (Sies, 1993). Glutathione disulphide is recycled

back to glutathione reductase, using the co-factor NADPH generated by glucose-6-phosphate

dehydrogenase (Santini et al., 1997). Glutathione peroxidase activity is seen to be elevated in

liver (Rauscher et al., 2000), kidney (Rauscher et al., 2000), aorta (Kocak et al., 2000), blood

(Mohan and Das, 1998) and red blood cells (Sailaja and Suresh, 2000) whereas decreased

activity was seen in heart (Kaul et al., 1996) and retina (Obrosova et al., 2000).

1.8.3 Lipid peroxidation

Hydroperoxides have toxic effects on cells both directly and through degradation to

highly toxic hydroxyl radicals. They may also react with transitional metals like iron or copper

to form stable aldehydes such as malondialdehydes that will damage cell membranes. Peroxyl

radicals can remove hydrogen from lipids, producing hydroperoxides that further propagate the

free-radical pathway (Halliwell and Guttetidge, 1990).

Induction of diabetes in rats with streptozotocin (STZ) or alloxan uniformily result in

an increase in thiobarbituric acid reactive substances (TBARS) an indirect evidence of

intensified free-radical production. Increase in TBARS associated with diabetes is presented by

27

treatment with nicotinamide (Melo et al., 2000), aspirin (Caballero et al., 2000), sodium

nitroprusside (Mohan and Das, 1998), captoprin, enalapril (Kedziora-Kornatowska et al.,

2000), if this treatment is given before or immediately after the diabetogen (Mekinova et al.,

1995).

1.8.4 Superoxide Dismutase

Isoforms of SOD are variously located within the cells. CuZn-SOD is formed in both

the cytoplasm and the nucleus. Mn-SOD is confined in the mitochodria but can be released into

extracellular space (Reiter et al., 2000). SOD converts superoxide anion radical produced in

the body to hydrogen peroxide, thereby reducing the likelihood of superoxide anion interacting

with nitric oxide to form reactive peroxynitrite. Alternations of SOD activity in diabetic

animals are normalised by captopril (Kedziora-Kornatowska et al., 1998), lipoic acid

(Obrosova et al., 2000), all of which were administered prior to or concomitant with the

diabetogen. Treatment with vitamin C, vitamin E and β-carotene for eight weeks elevates

hepatic SOD activity in diabetic rats, which is normal without treatment (Mekinova et al.,

1995).

In the heart, which is an important target in diabetes and prone to diabetic

cardiomyopathy leading to chronic heart failure, SOD and glutathione peroxidase expression as

well as activities are decreased (Maritim et al., 2003), whereas catalase is increased in

experimental models of diabetes (Hayden and Tyagi, 2003).

1.8.5 Vitamins

Vitamins A, C and E are diet-derived and detoxify free radicals directly. They also

interact in recycling processes to generate reduced form of the vitamins. Α-tocopherol is

reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid which is

generated is recycled to glutathione. Vitamin E, a component of the total peroxyl radical-

trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet

oxygen and protect membranes from lipid peroxidation (Weber et al., 1997).

Treatment with vitamin C and E was shown to decrease urinary albumin excretion,

glomerular basement membrane thickness and kidney weight in STZ-induced diabetic rats

(Kedziora-Kornatowska et al., 2003).

1.9 Justification of the Research

Good herbal remedy for the treatment of diabetes mellitus is a welcomed development.

Most of the severe complications of diabetes are due to the hyperglycaemic effects and the

28

effects of free radicals produced as a result of the pathogenesis of diabetes mellitus. Hence, a

good herbal drug with antioxidant and alpha glucosidase inhibition actions could prevent

complications of diabetes.

1.10 AIM AND OBJECTIVES OF THE STUDY

1.10.1 Aim of the Study

This research is carried out to assess the effects of ethanol extracts of the leaves,

flowers and stems of Euphorbia hirta on the blood glucose levels, body weight, oxidative and

biochemical parameters in alloxan-induced diabetic rats.

1.10.2 Specific Objectives of the Study

This research work is therefore set out to achieve the following objectives:

1. To determine the effect of 300 mg/kg b.w. of the flower, leaf and stem ethanol extracts

of E. hirta on serum electrolyte concentrations, lipid and renal profiles in comparison

with 0.01 mg/kg b.w. of the standard drug, voglibose.

2. To determine the effect of 300 mg/kg of the flower, leaf and stem ethanol extracts of E.

hirta on the variation of the blood glucose and body weight rats compared with 0.01

mg/kg b.w of the standard drug, voglibose.

3. To determine the effect of the 300 mg/kg b.w. of the ethanol extracts on some oxidative

parameters of the rats compared with the 0.01 mg/kg b.w. of standard drug, voglibose.

29

CHAPTER TWO

MATERIALS AND METHODS

2.2 Materials

2.2.1 Plant materials (Euphorbia hirta Herbs)

Large quantities of the flowers, leaves and stems of Euphorbia hirta herb were

collected from the University of Nigeria, Nsukka environ and were identified by Mr. Ozioko of

the herbarium Botany Dept., U.N.N. The different parts of the Euphorbia hirta herb were air-

dried separately at room temperature, then into powdery form using electrical grinding

machine.

2.2.2 Animals

Adult male Wistar albino rats of between 10 and 16 weeks with weights of 160–200g were

obtained from the Animal House of the Faculty of Biological Sciences, University of Nigeria, Nsukka.

The animals were acclimatised for about 7 days under standard environmental conditions, with a 12

hour light/dark cycle maintained on a regular feed (vital feed) and water ad libitum.

2.2.3 Chemicals/reagents/samples

All chemicals used in this study were of the analytical grade and products of May and

Baker, England; BDH, England and Merck, Darmstadt, Germany. Reagents used for all the

assays were commercial kits and products of Randox, USA; QCA, Spain; Teco (TC), USA;

Biosystem Reagents and Instruments, Spain.

All other chemicals were obtained from the usual suppliers.

2.2.4 Instruments/Equipment

Rotary evaporation (Model Modulyo 4K, Edward, England), Water Bath (Gallenkamp,

England), Chemical Balance (Gallenkamp, England), Conical Flasks (Pyrex, England), Hotbox

(Gallenkamp, England), Centrifuge (3,500 rpm, PIC, England), Digital Photo Calorimeter (EI 312 Model,

Japan), Adjustable Micropipette (Perfect, U.S.A.), Refrigerator (Kelvinator, Germany), pH Meter (Pye,

Unicam 293, England), , multi-well microtiter plate reader (Tecan, Austria).

30

2.2 Methods

2.2.1 Experimental design

Forty (40) albino Wistar rats were acclimatized at the same conditions of temperature

and pressure, and the same animal feeds were used for all the rats. The rats were divided into

seven (7) groups as shown below:

Group 1 Normal Rats (Control)

Group 2 Diabetic Rats (Not treated)

Group 3 Diabetic Rats + 300 mg/kg b.w. of ethanol flower extract.

Group 4 Diabetic Rats + 300 mg/kg b.w. of ethanol leaf extract.

Group 5 Diabetic Rats + 300 mg/kg b.w. of ethanol stem extract.

Group 6 Diabetic Rats + 300 mg/kg b.w. of ethanol (flower + leaf + stem) extract.

Group 7 Diabetic Rats + 0.01 mg/kg b.w. of voglibose.

2.2.2 Composition of diet fed experimental rats

The proximate composition of the normal rat diet given to the animals in the control group

was as follows:

Crude protein - 14.5g %

Crude fat - 4.8g %

Crude fibre - 7.2g %

Crude ash - 8.0g %

Phosphorus - 0.62g %

Lysine - 0.6g %

Methionine - 0.29g %

Methionine + Cystine - 0.52g %

Calcium - 0.8g %

Vitamin E - 15mg/100g

Vitamin C - 50mg/100g

31

Manganese - 30mg/100g

Zinc - 30mg/100g

Sodium - 0.15g %

2.2.3 Preparation of normal saline

This was prepared by dissolving 0.89g of sodium chloride (NaCl) in 50ml of water and the

volume made up to 100ml of distilled water.

2.2.5 Extraction of Euphorbia hirta Herbs

Large quantities of the flowers, leaves and stems of Euphorbia hirta herb were

collected from the University of Nigeria, Nsukka environ and were identified by Mr. Ozioko of

the herbarium Botany Dept., U.N.N. The different parts of the Euphorbia hirta herb were air-

dried separately at room temperature, then into powdery form using electrical grinding

machine. The ground samples extracted with 95% ethanol solution, using cold maceration

techniques. The samples were filtered using Whatman filter paper. The filtrates concentrated to

solid matter using rotary evaporators, which then become the stock sample of the flowers

leaves, and stem ethanol extracts which were used for the analysis. These extracts were stored

in the refrigerator compartment.

2.2.5 Induction of Diabetes

Diabetes was induced in the rats by intraperitoneal injection of Alloxan in a dose of about

160mg/kg body weight in Normal Saline (Chougale et al., 2007) or 65mg/kg body weight of

intravenous injection of alloxan in Normal Saline (Gruppuso et al., 1990). The blood glucose

concentrations and body weights of the rats were measured on days 0, 5, 10, 15, 20, 25, 30 while the

rats were treated with the extracts and the standard drug (Voglibose) from day 15 and sacrificed on

the day 30. Their sera were used for oxidative and biochemical analyses.

2.2.6 Acute Toxicity Studies (LD50)

32

Acute toxicity studies (LD50) was measured using method of Lorke (1989). The animals were

divided into two groups, A and B, with each group subdivided into four groups made up of three

animals each.

Experimental Protocol for Acute Toxicity Studies

Phase I:

Group 1 : Mice were administered with 10mg/kg of body weight of the ethanol flowers,

leaves and stem extracts of Euphorbia hirta herb.

Group 2 : Mice were administered with 100mg/kg of body weight of the

ethanol flower, leaf and stem extracts of Euphorbia hirta herb..

Group 3 : Mice were administered with 1000 mg/kg of body weight of the ethanol

flower, leaf and stem extracts of Euphorbia hirta herb.

Group 4 : Mice were administered with 1000 mg/kg of body weight of distilled water.

Phase II







Group 4 : Mice were administered with 5000 mg/kg of body weight of distilled water.

The mice were monitored closely for 24 hours for signs of toxicity and lethality

2.2.7 Phytochemical analysis

Preliminary Phytochemical analysis was carried out based on the procedures outlined by

Harborne (1973), and modified by Trease and Evans (1996). The following phytochemical tests were

carried out:

33

2.2.7.1 Test for carbohydrate (Molisch’s test)

To 2ml of the flowers, leaves and stems of Euphorbia hirta extract filtrate, 2 drops of alcoholic

solution of α-napthol were added, the mixture was thoroughly shaken and 1ml of concentrated

tetraoxosulphate (vi) acid was added slowly along the sides of the test tubes and allowed to stand. A

violet ring indicates the presence of carbohydrates.

2.2.7.2 Test for alkaloids (General tests)

A known weight, 50 mg of solvent free flowers, leaves and stems of Euphorbia hirta extract

was stored with 0.5ml of dilute hydrochloric acid and filtered. The filtrate was tested carefully with

various alkaloidal reagents as follows:

(a) Mayer’s test (potassium mercuric iodide solution): To 0.5ml of the flowers, leaves and

stems of Euphorbia hirta filtrate, a drop of Meyer’s reagent was added by the side of the

test tubes. A white or cream precipitate indicates the test as positive.

Meyer’s reagents:- Mercuric chloride (1.358g) was dissolved in 60 ml of water and

potassium iodide (5.0g) is dissolved in 10ml of water. The two solutions were mixed and made

up to 100ml with water.

(b) Dragendorff’s test (bismuth potassium iodide solution): To a few of the filtrate, 1ml of

Dragendorff’s reagent was added. A prominent yellow precipitate indicates the test as

positive.

Dragendorff’s reagents:-

Stock solution:- Bismuth carbonate (5.2g) and sodium iodide (4g) were boiled for 5

minutes with 50ml glacial acetic acid. After 12 hours, the precipitate, sodium acetate crystals

are filtered off using a sintered glass funnel. Clear, red-brown filtrate, 40ml is mixed with 160

ml of ethyl acetate and 1ml of water and stored in amber-coloured bottles.

Working solution:- A volume, 10ml, of the stock solution was mixed with 20 ml of acetic

acid and made up to 100ml with water.

34

(c) Wagner’s test (iodine in potassium iodide solution): To 0.5ml of the filtrate, 2 drops of

Wagner’s reagent were added by the side of the test tubes. A reddish-brown precipitate

indicates the test as positive.

Wagner’s reagent:- Iodine (1.27 g) and potassium iodide (2 g) was dissolved in 5 ml of

water and made up to 100ml with dissolved water

(d) Hagner’s test (A saturated solution of picric acid in cold water): To 0.5 ml of the filtrate, 1

ml of Hager’s reagent (saturated aqueous solution of picric acid) was added. A prominent

yellow and characterised crystalline precipitate indicates the test as positive.

The remaining filtrate was placed in 100 ml separating funnel and made alkaline with dilute

ammonia solution. The aqueous alkaline solution was separated and extracted with two 5 ml portions

of dilute sulphuric acid. The sample was tested with a few drops of Mayer’s, Wagner’s and

Dragendorff’s reagent.

2.2.7.3 Test for glycosides (Fehling’s test)

A quantity, 50mg, of the concentrated flowers, leaves and stems of Euphorbia hirta herb

extract was hydrolysed with concentrated hydrochloric acid for 2 hours on a water bath, filtered and

the hydrolysate was subjected to the following tests:

A. Borntrager’s Test (Evans and Trease, 1989):

To 2ml of filtered hydrolysate, 3ml of chloroform was added and shaken, chloroform

layer was separated and 10% ammonia solution was added to it. Pink colour indicates the

presence of glycosides.

B. Legal’s Test: A known weight, 50 mg, of the extract was dissolved in pyridine, sodium

nitroprusside solution was added and made alkaline using 10% sodium hydroxide. Presence of

glycosides is indicated by pink colour.

2.2.7.4 Test for saponins (Kokate, 2001)

35

A known weight, 50mg, of the concentrated flowers, leaves and stems of Euphorbia hirta

extract was diluted with distilled water and made up to 20 ml. The suspension is shaken in a

graduated cylinder for 15 minutes. A 2 cm layer of foam indicates the presence of saponins.

2.2.7.5 Test for tannins (Ferric chloride method)

Concentrated flowers, leaves and stems of Euphorbia hirta herb extract (1 g) was boiled with

50 ml of water, filtered and used for the ferric chloride test proper. To 3 ml of the filtrate, a few drops

of ferric chloride were added. Presence of cream gelatinous precipitate and a light green coloration

which changes shortly to black indicate a positive test.

2.2.7.6 Test for flavonoids (Ammonium test method)

Ethylacetate (10 ml) was added to 0.2g of the concentrated flowers, leaves and stems of

Euphorbia hirta extract sample and heated on a water bath for 3 minutes. The mixture was cooled,

filtered and the filtrate used for the ammonium test proper:

A volume, 4 ml, of the filtrate was shaken with 1 ml of dilute ammonia solution. The layers

were allowed to separate. A yellow solution with sodium hydroxide which turns colourless with dilute

hydrochloric acid confirms the presence of flavonoids.

2.2.7.7 Test for resins (Precipitation test)

The concentrated flowers, leaves and stems of Euphorbia hirta (0.2 g) was extracted with 15ml

of 95% ethanol. The alcoholic extract was then poured into 20 ml of distilled water in a beaker.

Precipitate formed at the bottom of the test tube indicates positive test.

2.2.7.8 Test for proteins (Millon’s test)

To 2ml of the filtrate, 2 drops of Millon’s reagent were added. A white precipitate

indicates the presence of proteins.

Millon’s Reagent: Mercury (1g) is dissolved in 9ml of fuming nitric acid. When the

reaction was completed, equal volume of distilled water was added.

36

2.2.7.9 Test for oils

The concentrated flowers, leaves and stems of Euphorbia hirta (0.1 g) material was pressed

between a filter paper and the paper was observed for translucency.

2.2.7.10 Test for steroids and terpenoids

Ethanol (9 ml) was added to 1g of the flowers, leaves and stems of Euphorbia hirta sample and

refluxed for a few minutes and filtered. The filtrate was concentrated down to 2.5 ml on a boiling

water bath and 5ml of hot water was added to it and mixed thoroughly. The mixture was allowed to

stand for 1 hour after which the waxy matter filtered off. The filtrate was shaken with 2.5 ml of

chloroform in a separating funnel. The lower layer was drawn out. To 0.5 ml of the chloroform extract

in a test tube was carefully added 1 ml of concentrated sulphuric acid to form a lower layer.

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and

heated with 3 ml of concentrated sulphuric acid for 10 minutes on a water bath. A reddish brown

interface indicates positive result for steroids while a grey coloration indicates a positive test for

terpenoids.

2.2.8 Proximate analysis

Percentage concentrations of protein, carbohydrate, crude fibre, moisture and ash in the

flowers, leaves and stems of Euphorbia hirta extracts were determined using the AOAC method

(1990).

2.2.8.1 Crude protein

Principle

The crude protein content was determined using the micro Kjeldahl method. The method is

generally used to determine nitrogen (N) in substances which contain N as ammonium salts, nitrates

or organic N compounds. Since it measures the total amount of N in a compound only a rough

indication of the total protein content can be obtained and was termed crude protein. The quantity of

N measured was then multiplied by 6.25 to obtain the protein content of the compound. The

multiplication factor can vary with some materials (AOAC, 1990).

37

The N of protein and other compounds were converted into ammonium sulphate by acid

digestion with boiling H2SO4.The acid digest was cooked, diluted with water and made strongly basic

with NaOH. Ammonium was released and distilled into a 4% boric acid solution. The amount of

ammonium borate formed was determined with standard H2SO4 or HCl.

The indicator used, bromocresol green, gave a pink colour end point at a hydrogen ion

concentration corresponding to a solution of NH4Cl. Boric acid is so weak that it has no appreciable

influence on the pH concentration. The reactions are represented as:

NH3 + HB03 NH4 + BO3-

H+ + B02 HB02 (AOAC, 1990)

The method involved three major steps:

(a) Digestion of the sample

(b) Distillation of the ammonia into a trapping solution.

(c) Quantification of the ammonia by titration.

(a) Digestion: A small quantity of samples of the flowers, leaves and stems of Euphorbia hirta

herb extracts (0.1 g) was weighed in a Kjeldhal flask containing 2.0g sodium sulphate/copper

sulphate as catalyst. Concentrated H2SO4 (20 ml) was introduced into the flask and the

contents gently heated. The heating was increased until the contents of the flask were

completely digested giving a clear solution.

(b) Distillation: The content of the flask was washed with 220 ml distilled water into a distillation

flask and cooled under ice blocks. To the flask, 100 ml of 4% boric acid first added; later, 3

drops of screened methyl red were also added.

(c) Back titration: After cooling, 40% NaOH (50 ml) was added and the distillate was titrated

against 0.5 N Na2SO4 solution.

The percentage nitrogen was calculated using the relationship

% Nitrogen = 100××××

SampleofWeight

MWNDfNT

Where

38

T = Titre volume

N = Normality of acid

Df = Dilution factor

MWN = Molecular weight of nitrogen

% Protein = % Nitrogen × 6.25

Where 6.25=conversion factor of nitrogen to protein

2.2.8.2 Crude fat

Principle

The sample was continuously extracted with ether. After extraction, the ether extract was

evaporated to dryness and the residue designated the ether extract. This is referred to as the fat

portion of the sample. Even though, the ether extract also contains organic acids, oils, pigments,

alcohols and fat-soluble vitamins and it is referred to as crude fat. Many of the complex lipids, such as

phospholipids are not completely extracted in this procedure (Ensimger and Olentine, 1978).

Method

A washed, dried and cooled quick-fit flask was weighed. Flowers, leaves and stems of

Euphorbia hirta extracts were weighed into the extraction thimble and placed in the quick-fit soxhlet

apparatus. The solvent flask containing 250 ml of diethyl ether was connected to a condenser. The set-

up was heated for 16hrs for complete extraction. The extract was evaporated at 70oC to remove any

remaining solvent present. The apparatus was reweighed and percentage fat calculated as follows:

% Crude Fat = 100×SampleofWeight

OilofWeight

2.2.8.3 Moisture

39

Method

Two grammes (2g) of freshly collected samples of flowers, leaves and stems of Euphorbia hirta

were weighed and dried in the oven at 110oC to a constant weight. The dishes and samples were

cooled and reweighed and percentage moisture content calculated using. The relationship is as

follows:

% Moisture = 1001

32 ×−

W

WW

Where

W1 = Weight of sample

W2 = Initial weight of sample and dish

W3 = Final weight of dry sample

2.2.8.4 Ash/Mineral matter

Principle

Ash is defined as the mineral matter of a feed or material since it includes for the most part

the inorganic or mineral components of the feed or material (Ensiminger and Olentine, 1978; Cullison,

1982). The sample was heated at 600oC to burn off all organic materials. The inorganic material which

did not volatilize at this temperature was designated ash.

Method

Into previously weighed porcelain dishes were put 2g samples of flowers, leaves and stems of

Euphorbia hirta extracts and reweighed. The crucible and samples were placed in a muffle furnace at

600oC for 3 hrs. The ashes and crucible were cooled in a desiccator and reweighed. The percentage

ash content was calculated using:

% Ash = 12

13

WW

WW

−

−

Where

W1 = Weight of crucible

40

W2 = Weight of crucible and sample

W3 = Weight of crucible and ash

2.2.8.5 Crude fibre

Principle

This fraction was designed to include those materials in food which were of low digestibility

namely cellulose, certain hemicelluloses and some of the lignin, if present. Some of the lignin,

however, may be included in the nitrogen free extract. A moisture – free, ether extract is digested first

with weak acid solution (1.25% H2SO4) and then with a weak base solution (1.25% NaOH). The organic

residue left after digestion is collected. The loss of weight on ignition was called crude fibre.

Method

Samples of the flowers, leaves and stems of Euphorbia hirta extracts (2 g) each was weighed

into 500 ml beakers containing pre-heated dilute H2S04 about (40 ml). The content was boiled for 30

mins and filtered. The residue was washed three times with hot water, then 150 ml of pre-heated KOH

and drops of antifoam agent (loctanol) were added to the sample in the beaker and heated to boiling.

The mixture was boiled slowly for more 30 mins, filtered and washed three times with hot water.

Acetone was then used in washing it three times in cold extraction unit and the content dried at 130oC

for an hour.

After ashing the content at 500oC, the ash was weighed and the percentage fibre calculated as

follows:

% Crude fibre = 100×SampleofWeight

FibreofWeight

2.2.8.6 Carbohydrate or Nitrogen Free Extract (NFE)

Principle

41

Proximate analysis of carbohydrate is also known as Nitrogen Free Extract (NFE)

determination. It includes mostly sugars and starches and also some of the more soluble

hemicelluloses and some of the more soluble lignin (Cullison, 1982). Since this fraction was designed

to include the more soluble carbohydrates, it is sometimes referred to as the carbohydrate portion of

the material being analysed.

Method

NFE was calculated by subtracting the sum of the other fractions from 100 as follows:

100 – (% moisture + % crude protein + % crude fat + crude fibre + % ash) = % NFE.

2.2.9 Determination of Total Cholesterol Concentration

Total cholesterol concentration was determined using the method of Allain et al. (1976) as

contained in QCA commercial kits.

Principle

The total cholesterol determination using QCA commercial enzyme kit is based on the

assay principle that total cholesterol is determined after enzymatic hydrolysis and oxidation.

The indicator, coloured quinonic derivative is formed from hydrogen peroxide and 4-

aminoantipyrine in the presence of p-hydroxybenzoic acid and peroxidase.

[ ]

acidsFatty lCholestero OH esters-lCholesteroEsterase Chol.

2 + →+

22

oxidase Chol.

22 OH neCholesteno O OH lCholestero + →++

04H derivated quinonic Coloured acid zoicHydroxyben-p yrineAminoantip-4 OH 2

Peroxidase

22 + →++

Procedure

Blank (BL), sample (SA) and standard (ST) were the three sets of labelled test tubes. A

quantity, 0.01 ml, of the serum sample was pipetted into the sample (SA) test tube. Also, 0.01

ml of the standard was introduced into the standard (ST) test tube with a corresponding

42

addition of 1 ml of working reagent into each of the test tubes. The solutions in the different

sets of test tubes were well mixed and allowed to stand for 5 minutes at 37oC (or 10 minutes at

room temperature). The absorbance was read at the wavelength of Hg 546 nm.

Calculations

The total cholesterol concentration in the sample was calculated using the following general

formula:

lcholestero totalof mg/dl 200 x O.D.ST

O.D.SA=

Where SA is Sample

ST is Standard

OD is Optical density

200 is a constant

SI Units = (mg/100 ml) × 0.0259 = mmol/L

2.2.10 Low Density Lipoprotein-Cholesterol Concentration

Low density lipoprotein – cholesterol concentration was determined using the method

of Assman et al. (1984) using QCA commercial kit.

Principle

Low density lipoprotein–cholesterol (LDL–cholesterol) can be determined as the

difference between total cholesterol and cholesterol content of the supernatant after

precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the presence of

polyethyleneglycol monomethyl ether.

LDL-cholesterol = Total cholesterol – cholesterol in the supernatant

Reagents

Content Initial Concentration of Solutions

1. Precipitation Reagent:-

43

Polyvinyl sulphate 0.7 g/L

EDTA Na2 5.0 mM

Polyethyleneglycol monomethyl ether 170 g/L

Stabilizers

Procedure

(1) Precipitation reaction

The precipitation solution (3 drops or 0.1 ml) was carefully measured into test tubes labeled

accordingly. The serum sample (0.2 ml) was added to the labeled test tubes. The contents were

thoroughly mixed and left to stand for 15 minutes at room temperature (20–25oC). Then, the mixture

was centrifuged at 2,000 × g for 15 minutes and the cholesterol concentration in the supernatant was

determined.

(2) Cholesterol determination

The concentration of the serum total cholesterol was determined according to the QCA

CHOD–PAP method.

Calculations

The LDL–cholesterol concentration in the sample was calculated using the following general

formula:

LDL–cholesterol (mg/dl) = Total cholesterol (mg/dl) – 1.5 × supernatant cholesterol (mg/dl).

2.2.11 High Density Lipoproteins (HDL)–Cholesterol Concentration

High density lipoprotein–cholesterol concentration was determined using the method of

Albers (1978) as contained in QCA commercial kit.

Principle

44

Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) are lipoproteins

precipitated from the serum by the action of a polysaccharide in the presence of divalent cations.

Then, high density lipoprotein–cholesterol (HDL–Cholesterol) present in the supernatant, is

determined.

acidFatty lCholestero OH esters-lCholesteroesterase chol.

2 + →+

22

oxidase chol.

22 OH neCholesteno OH O2

1 lCholestero + →++

04H neQuinoneimi DCFS yrineAminoantip-4 OH2 2

eperoxidase

22 + →++

Procedure

The procedure took two steps:

(A) Precipitation step

The serum sample (0.3 ml) was pipetted into labeled centrifuge tubes. Also, a drop of the

precipitant solution or reagent (10g/L of dextran sulphate, 1M of magnesium acetate and stabilizers)

was added to each of the centrifuge tubes.

(B) Colorimetric step

Then contents in the various tubes were thoroughly mixed and allowed to stand for 15

minutes at room temperature (20–25oC); then centrifuged at 2,000 × g for 15 minutes (or 10,000 × g

for 2 minutes). The concentration of cholesterol in the supernatant was determined.

Calculations

The HDL cholesterol concentration in the sample was calculated using the following general

formula:

45

lCholestero - HDL mg/dl 52.5 x A

A

standard

sample=

Or

lCholestero - HDL mmol/dl 1.36 x A

A

standard

sample=

Where

52.5 and 1.36 are constants.

2.2.12 Determination of Triacylglycerol Concentration

Triacylglycerol concentration was determined using method of Albers et al. (1978) as

contained in Randox commercial kit.

Principle

The triacylglycerol concentration was determined after enzymatic hydrolysis with lipases. The

indicator is a quinoneimine formed from hydrogen –peroxide, 4-aminophenazone and 4-

chlorophenol under the catalytic influence of peroxide.

Triglycerides + H2O Lipases

Glycerol + fatty acids

Glycerol + ATP GK

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 GPO

Dihydroxyacetone + phosphate + H2O2

2H2O2 + 4-aminophenazone + 4-chlorophenol POD

Quinoneimine + HCl + 4H2O.

A known volume of 100 µl of the reagent was pipetted into the reagent blank tube, standard tube and

the sample tubes. The standard (10 µl) was then added to the standard tube while 10 µl of the sample

was pipetted into the sample tube. The mixtures in the three tubes were mixed and incubated at 20 –

250C for 10 minutes. Then, the absorbance of the sample and the standard were measured against the

reagent blank within 60 minutes at 546 nm.

Triacylglycerol concentration (mmol/l) =

Standard

Sample

A

A

∆

∆ × 2.29

ASample = Absorbance of sample

46

AStandard= Absorbance of standard

2.2.13 Assay of Superoxide Dismutase Activity

Superoxide dismutase (SOD) activity was assayed using the method as described by

Fridorich (1989) as contained in Randox commercial kit.

Principle

The role of superoxide dismutase (SOD) is to accelerate the dismutation of the toxic

superoxide radical (O2·), produced during oxidative energy processes, to hydrogen peroxide and

molecular oxygen. This method employs xanthine and xanthine oxidase (XOD) to generate superoxide

radicals which react with 2–(4–iodophenyl)–3–(4–nitrophenyltetrazolium chloride (I.N.T.) to form a

red formazan dye. The superoxide dismutase activity is then measured by the degree of inhibition of

this reaction. One unit of SOD is that which causes a 50% inhibition of the rate of reduction of INT

under the conditions of the assay.

Xanthine Uric acid + O2·

I.N.T. Formazan dye

OR

O2· + O2

·+ 2H

+ O2 + H2O2

Reagent Composition

Contents Initial Concentration of Solutions

R1a Mixed Substrate

XOD

O2·

SOD

47

Xanthine

I.N.T.

0.05 mmol/l

0.025 mmol/l

R1b Buffer

CAPS

EDTA

40 mmol/l; pH 10.2

0.94 mmol/l

R2 Xanthine Oxidase 80 U/l

CAL Standard

Procedure

Wavelength 505 nm

Cuvette 1 cm path length

Temperature 37oC

Measurement Against air

Three sets of test tubes were set up and labeled as sample diluent (SD), standards (S2 –

S6) and diluted sample (DS). A volume of 0.05 ml of the diluted whole blood sample was pipetted into

the test tube labelled DS. This was accompanied by the introduction of 0.05 ml of the standards into

the test tube labeled S2 – S6 (standards). The Ransod sample diluent (0.05 ml) was also added to the

test tube labeled SD. A corresponding volume of 1.7 ml each of the mixed substrate (R1) was added to

the labelled test tubes respectively. The reaction mixture was mixed thoroughly.

After thorough mixing of the reaction medium, a volume of 0.25 ml of xanthine oxidase

(R2) was also added to labeled test tubes. Again, the content was thoroughly mixed and the initial

absorbance (A1) read after 30 seconds and the timer started simultaneously. The final absorbance (A2)

was read after 3 minutes.

Calculation

48

3

12 AA − = ΔA/min of standard or sample

Sample diluent rate (S1 rate) = Rate of uninhibited reaction = 100%.

All standard rate and diluted sample rates were converted into percentages of the sample

diluent rate, and subtracted from 100% to give a percentage inhibition.

100 – )(

)100(

min/1

min/

dS

Std

A

A

∆

×∆ = % Inhibition

100 – )(

)100(

min/1

min/

dS

sample

A

A

∆

×∆ = % Inhibition

Percentage inhibition plot for each standard was plotted against Log10 (Standard

concentration in SOD units/ml). The percentage inhibition of sample was used to obtain units of SOD

from the standard curve.

SOD units/ml of whole blood = SOD units/ml from standard curve × dilution factor.

Converting to SOD units/g Haemoglobin

mlnHaemoglobig

mlUnitsSOD

/

/ = SOD units/g Haemoglobin.

2.2.14 Assay of Catalase Activity

Catalase activity was assayed using the method of Aebi (1983).

Principle

49

The ultraviolet absorption of hydrogen peroxide can be easily measured at 240 nm. On the

decomposition of hydrogen peroxide with catalase, the absorption decreases with time and from this

decrease catalase activity can be measured.

Reagents

Phosphate buffer, pH 7.0

3.522 g KH2PO4 and 7.268 g Na2HPO4.2H20 in 1000 ml of water

Hydrogen peroxide solution

0.085 ml of 30% hydrogen peroxide in 25 ml phosphate buffer.

Procedure

Red blood cell lysate was prepared by adding 1.2 ml of distilled water to 0.2 ml of RBC. Then

500 fold dilution of RBC lysate by phosphate buffer was made before the determination of catalase

activity. Immediately following the addition of 1 ml phosphate buffer (blank) or hydrogen peroxide

solution into 2 ml RBC diluted lysate, the change of absorbance of RBC against blank at 240 nm was

recorded every 15 seconds for 1 minute on a UV spectrophotometer. The activity of catalase was

calculated using the following equation:

Catalase catalytic concentration (U/L) = 00693.0

)/23.0 21 ALogA×

Where A1 = A240 at t = 0

A2 = A240 at t = 15 seconds

0.23 and 0.00693 = Constants

2.2.15 Determination of Malondialdehyde Concentration

Lipid peroxides assay was done by determining the concentration of malondialdehyde

(MDA) formed using the method of Varshney and Kale (1990).

50

Principle

Malondialdehyde (MDA) reacts with thiobarbituric acid to form a red or pink coloured

complex which, in acid solution, absorbs maximally at 532nm.

MDA + 2TBA MDA:TBA adduct + H2O

Reagent Preparation

i. 1.0% Thiobarbituric acid (TBA): A quantity, 1.0 g, thiobarbituric acid was dissolved in 83 ml of

distilled water on warming. After complete dissolution the volume was made up to 100 ml with

distilled water.

ii. 25% Trichloroacetic acid (TCA): A quantity, 12.5 g, of trichloroacetic acid was dissolved in

distilled water and made up to 50 ml in a volumetric flask with distilled water.

iii. Normal saline solution (NaCl): A quantity, 0.9 g, of NaCl was dissolved in 10 ml of distilled

water and made up to 100 ml with distilled water.

Procedure

To 0.1 ml of plasma in test tube was added 0.45 ml of normal saline and mixed thoroughly

before adding 0.5 ml of 25% trichloroacetic acid (TCA) and 0.5 ml of 1% thiobarbituric acid. The same

volume of tricholoracetic acid, and saline was added to the blank. 0.1 ml of distilled water was also

added to the blank instead of plasma. Then, the mixture was heated in a water bath at 95 0C for 40

minutes. Turbidity was removed by centrifugation. The mixture was allowed to cool before reading

the absorbance of the clear supernatant against reagent blank at 532 nm. Thiobarbituric acid reacting

substances were quantified as lipid peroxidation product by referring to a standard curve (Appendix 1)

of MDA concenteation (i.e. equivalent generated by acid hydrolysis of 1,1,3,3-tetraethoxypropane

(TEP) prepared by serial dilution of a stock solution).

Table 2: Procedure for malondialdehyde concentration determination

Pipette into cuvette___________________________________________________

Blank Test

Plasma --- 0.10 ml

51

Distilled water 0.10 ml ---

Normal saline 0.45 ml 0.45 ml

25% TCA 0.50 ml 0.50 ml

1% TBA 0.50 ml 0.50 ml___

2.2.16 Determination of Ascorbic Acid Concentration

Ascorbic acid (vitamin C) concentration was determined using the method of Tarvay

(1970).

Procedure

To a quantity, 10 g, of each sample was added 80 ml of ethanol and 20 ml of distilled water,

this was covered and shaken for 2 hours. After 2 hours, it was filtered and the filtrate measured. 5ml

of the filtrate was dispensed into a conical flask. 50ml of distilled water, 2.5 ml of 1 M H2SO4 and 1 ml

of 10% starch indicator were added and. The mixture was titrated with 0.05 M iodine solution till blue-

black colour appeared.

Calculation

Vitamin C (ascorbic acid) content was calculated according to the formula.

Vitamin C = )(

10000886.0..

gUsedSampleofWeightCuvetteofVolume

ExtractedofVolumeVT

×

×××

Where T.V. = Titre value

1 ml of 0.05 M iodine solution consumes 0.00886 g of vitamin C

2.2.17 Determination of Sodium Ion Concentration

52

Sodium ion concentration was determined using the method of Tietz (1983) as

contained in Teco diagnostic kit. In this present method, sodium is precipitated as the triple

salt, sodium magnesium uranyl acetate, with the excess uranium then being reacted with

ferrocyanate, producing a chromophore whose absorbances varies inversely as the

concentration of sodium in the test specimen.

Different test tubes were labelled as standard (S), control (C), test (T) and blank (B). The

filtrate reagent (1.0 ml) was pipetted to all test tubes. The sample (50 μl) was added to all test tubes

and distilled water was then added to the blank. All the test tubes were shaken vigorously and mixed

continuously for 3 minutes. All the test tubes were centrifuged at high speed of 1,500 G for 10

minutes.

A volume of 1.0 ml of acid reagent was pipetted to all test tubes at the colour development

stage. The supernatant (50 μl) was added to the respective tubes and mixed thoroughly. The colour

reagent (50 μl) was also added to all the test tubes and mixed thoroughly. The spectrophotometer was

zeroed with distilled water; after which, the absorbance readings were taken at a wavelength of 550

nm.

2.2.18 Determination of Potassium Ion Concentration

Potassium ion concentration was determined using the method of Tietz (1983) as contained in

Teco diagnostic kit. The amount of potassium is determined by using sodium tetraphenylborone in a

specifically prepared mixture to produce a colloidal suspension. The turbidity of which is proportional

to potassium concentration in the range of 2 – 7 mEq/L.

The test tubes were well labelled as standard, control, test, etc. Also, a blank was provided. A

volume of 1.0ml of potassium reagent was pipetted into all tubes. Also, 0.01ml (10μl) of samples was

added to the respective test tubes and mixed thoroughly. The test tube mixtures were allowed to stay

under room temperature for 3 minutes. After 3 minutes, the absorbance of the solutions in the

different test tubes was read at a wavelength of 500nm with the reagent blank

2.2.19 Determination of Chloride Ion Concentration

Chloride ion concentration was determined using the method of Tietz (1983) as

contained in Teco diagnostic kit.

Principle:

Hg(SCN)2 + 2Cl– HgCl2 + 2SCN

–

53

3SCN– + Fe

3+ 4 Fe(SCN)3 Red Complex

Chloride ions form a soluble, non-ionized compound, with mercuric ions and will displace

thiocyanate ions from non-ionized mercuric thiocyanate. The released thiocyanate ions react with

ferric ions to form a colour complex that absorbs light at 480 nm. The intensity of the colour produced

is directly proportional to the chloride concentration.

Reagents:

1. Chloride Reagent (Active Ingredients):

Mercuric Nitrate 0.058 mM

Mercuric Thiocyanate 1.75 Mm

Mercuric Chloride 0.74 mM

Ferric Nitrate 22.3mM

Non-reactive ingredients and stabilizers in dilute acid and methanol.

2. Chloride Calibrator:

Sodium Chloride 100mEq/L

Procedure:

The different test tubes were labeled “blank”, “calibrator”, “patient”, etc. A quantity of 1.5ml

of chloride reagent was pipetted to each test tube. Also, a known volume, 0.01ml of calibrator or

sample was pipetted to the respective test tube and mixed thoroughly which was followed by

incubated for at least 5 minutes. The spectrophotometer was set at 480 nm and zero with reagent

blank. The absorbance readings were read at 480 nm and recorded all readings of all test tubes.

Calculation:

Conc. of Chloride (mEq/L) = Calibrator ofion ConcentratCalibrator of Absorbance

Unknownof Absorbance×

54

2.2.20 Determination of Bicarbonate Ion Concentration

Bicarbonate ion concentration was determined using the method of Tietz (1983) as

contained in Teco diagnostic kit.

2.2.21 Determination of Serum Urea Concentration

Serum urea concentration was determined using the method of Tietz (1994) as

contained in Randox diagnostic kit.

Principle:

This method is based on the principle that urea in the serum is hydrolysed to ammonia in the

presence of urease. The ammonia is then measured photometrically by Berthelot’s reaction.

Urea + H2O 2NH3 + CO2

NH3 + Hupochlorite + Phenol Indophenol (Blue Compound)

Reagents Composition

Reagents

Contents Concentration of Solutions

1. EDTA

Sodium nitroprusside

Urease

116 mmol/L

6 mmol/L

1 g/L

2. Phenol (Diluted) 120 mmol/L

3. Sodium hydrochlorite (diluted)

Sodium hydroxide

27 mmol/L

0.14 N

CAL Standard Specific instruction

Urease

55

Procedure

Three sets of test tubes labelled Blank (B), Standard (ST) and Sample (SA) were set up. A

volume of 10 μl each of the serum sample was pipetted into the test tubes labelled SA, ST and B

respectively. Reagent 1 (10 μl) which is made up of EDTA, sodium nitroprusside and urease was

pipetted into all the three sets of test tubes labelled B, ST and SA respectively. The contents of the

three test tubes were thoroughly mixed and incubated at 37oC for 10 min. After which, 2.50 ml each of

Reagents 2 (diluted phenol) and 3 (diluted sodium hypochloride and sodium hydroxide) were added to

the three sets of test tubes. The contents were also thoroughly mixed and incubated at 37oC for 15

min. The absorbance of the sample (Asample) and standard (Astandard) against the blank was read at a

wavelength of 546 nm against the blank.

Urea Conc. = ×Standard

sample

A

A Standard Conc

Where:

A sample = Absorbance of the Sample

A standard = Absorbance of the Standard

2.2.22 Determination of Serum Creatinine Concentration

Serum creatinine concentration was determined using the method of Tietz (1994) as

contained in Randox diagnostic kit. The modified Jaffe method (Blass et al., 1974) for the in vitro

determination of creatinine in serum using the Quimica Clinica Applicada (QCA) creatinine test kit was

employed in the determination of creatinine concentration.

Principle

Creatinine in alkaline solution reacts with picrate to form a coloured complex.

Sample

56

Serum

Reagents

Reagent A = Alkaline solution containing NaOH and Na2CO3

Reagent B = Picric acid solution

Reagent C= Standard, an aqueous solution equivalent to 2mg/dl of creatinine.

Methodology

A working reagent composed of equal volumes of Reagent A and B (Alkaline solution and

picric acid solution) was prepared. For each determination, 0.5ml of Reagent A mixed with 0.5 ml of

Reagent B gave 1ml of working reagent for each serum sample, 0.1ml of sample was added to 1.0ml

of working reagent in a clean test tube. It was mixed properly and transferred to a cuvette, a stop

watch was started and absorbance was read at the 20th

and 80th

seconds against a working reagent

blank at 546 nm. Two standards were prepared and run by adding 0.1ml of the standard (Reagent C)

to 1 ml of working reagent in a test tube. It was mixed properly and transferred to a cuvette, a stop

watch was started and absorbance read at the 20th

and 80th

seconds against a working reagent blank

at 546nm for the samples. The mean of the standards was used as the standard.

The following formula was used to calculate the serum creatinine concentration of each

sample.

Serum Creatinine Conc. (mg/dl) = 1

2

Standard of Sec)20th -(80th Absorbance of Change

Sample of Sec)20th -(80th Absorbance of Change×

Where 2 =standard aqueous solution of creatinine

2.2.23 Determination of Fasting and Random Glucose Concentration

Fasting and random blood sugar using Accu-chek Active glucometer by Roche

Diagnostic according to the method of Marks and Dawson (1965).

Principle

57

Glucose Gluconic acid + H2O2

H2O2 H2O + O

O + Acceptor Coloured Complex + H2O

The method is based on the reaction of glucose and oxygen in the presence of glucose oxidase

to yield gluconic acid and hydrogen peroxide. Hydrogen peroxide subsequently oxidizes the dyes in a

reaction mediated by peroxidase producing a blue coloured form of the dyes. The intensity of the blue

colour is proportional to the glucose concentration in the sample and is measured and read by the

ONE TOUCH meter.

The One-Touch glucometer was essentially a reflectance meter. The amount of light reflected

in reagent area of the dextrostix measured in a readout meter scale was a measure of the

concentration of glucose in the blood. Snips were made on the tail of the animal to release blood on

the sensitive spot on the glucometer.

Reagents

ONE TOUCH Glucometer (Lifescan Inc. Johnson – Johnson Company, USA) and test strips were

used. The composition of the test strips is:

• Glucose oxidase (14/U)

• Peroxidase (11/U)

• 3-methyl-2-benzothiazolinonehydrazone hydrochloride (0.06mg)

• 3-dimethylaminobenzoic acid (0.12mg).

Procedure

i) Insert the code key into the glucometer code key opening.

Glucose oxidase

Peroxidase

O-toluidine

58

ii) Insert a test strip to make sure that the code on the glucometer matches the code on the

test strip.

iii) Insert a fresh new strip with the orange pad facing up until it goes no further into the

glucometer opening for test strips.

iv) Wait until the image of a flashing blood appears on the glucometer screen; that signifies

that the glucometer is ready. Then put a drop of blood collected with a capillary tube on

the centre of the square of the orange pad.

v) An hour glass symbol appears on the glucometer screen followed after 5 seconds by the

test result.

vi) Copy the test result as the blood glucose level in g/dl.

2.2.24 Body Weight

According to duration of study, both control and the test animals were weighed before

and after the experiment. Therefore, the difference in body weight was recorded and compared.

2.2.25 Histopathological Examination The histological examination of the tissues of the pancreas of Wistar male albino rats

was done using the method of Drury et al. (1967) as described below:

A. Fixation and Washing

Formalin (10%) was used as the fixative and for the purpose of preservation. A thin section of

the tissue (about 1 to 2 cm in diameter) was trimmed with a sharp razor blade. The small pieces of the

tissue were placed in the 10% formalin, the container was shaken gently several times to make sure

that the fluid had reached all surfaces and that pieces were not sticking to the bottom. This was then

incubated at 250C for 24 hours, to allow proper fixing. The fixed tissue pieces were washed with

running water for 24 hours to free them from excess fixatives.

B. Dehydration

Water was removed from the tissue before embedding the tissue in paraffin. The dehydration

was achieved by immersing the thin sections of the tissue in automatic tissue processor containing 12

jars.The first three (3) jars contained 70, 90 and 95% absolute alcohol respectively. This was done to

59

remove the water content in the tissues. The absolute alcohol reduced the shrinking that occurred in

the tissue. The time for each step was 30 minutes. A second change of absolute alcohol was included

to ensure complete removal of water. This was achieved in the second three (3) jars of the automatic

tissue processor.

C. Clearing

Solutions of xylene were used for clearing the tissue sections. This step was achieved in the

third three (3) jars of the automatic tissue processor. Because the alcohol (ethanol) used for

dehydration would not dissolve or mix with molten paraffin, the tissue was immersed in xylene

solution which was miscible with both alcohol and paraffin before infiltration could take place.

Clearing was done to remove opacity from dehydrated tissue. A period of 15 minutes was

allowed to elapse before the tissue was removed from the solution for infiltration with paraffin.

D. Infiltration with Paraffin

Paraffin wax at 50 to 52°C was used to infiltrate the tissue. The tissue was transferred directly

from the clearer to a bath containing melted paraffin. After 30 - 60 minutes incubation in the first

bath, the tissue was then removed to a fresh dish of paraffin contained in the fourth three jars of the

automatic tissue processor for a similar length of time.

E. Embedding (Blocking) with Paraffin

As soon as the tissue was thoroughly infiltrated with paraffin, it (paraffin) was allowed to

solidify around and within the tissue.

F. Paraffin Sectioning

The embedded blocks were trimmed into squares and fixed in the microtome knives for

sectioning after which the sections were floated on a water bath.

60

G. Mounting

Glass slides were thoroughly cleaned and a thin smear of albumen fixative was made on the

slides. The albumenized slide was used to collect the required section from the rest of the ribbon in

the water. The section on the glass slide was kept moist before staining.

H. Staining with Haematoxylin

The slides were passed through a series of jars containing alcohols of decreasing strength and

various staining solutions.

I. Microscopic Observation of Slide

The slides prepared were mounted on photomicroscope, one after the other and viewed at

different magnification power of the microscope. Photograph of each of the slides was taken.

2.3 Statistical Analysis

The results were expressed as mean±SD and test of statistical significance was carried out

using one–way analysis of variance (ANOVA). The means was separated using Duncan multiple Test.

The statistical packaged used was the statistical package for social sciences (SPSS), version 17.

61

CHAPTER THREE

RESULTS

3.1 Phytochemical Analysis

Phytochemical analysis of the crude ethanol extracts of Euphorbia hirta shows the

presence of flavonoids, steroids, alkaloids, tannins, saponin, carbohydrate, reducing sugar and

proteins. This is shown in Table 3.

Table 3: Qualitative phytochemical analysis of ethanol extracts of Euphorbia hirta

Stem extract Leaf extract Flower extract

Carbohydrate + ++ ++

Reducing sugars ++ + Nd

Proteins + ++ ++

Saponins ++ ++ ++

Glycoside Nd + Nd

Flavonoid + ++ +

Tannin + ++ Nd

Alkaloid + + +

Fats/oil Nd + +

Acidic compounds Nd Nd Nd

+ = Low in abundance

++ = Moderate in abundance

+++ = High in abundance

nd = Not detected

3.2 Percentage Proximate Composition

62

The percentage proximate composition of the three components of ethanol extract of

Euphorbia hirta shows that carbohydrate has the greatest percentage in all three extracts,

followed by protein, and fibre is the least. This is shown in Table 4.

Table 4: Percentage proximate composition of ethanol extracts of Euphorbia hirta

Stem extract % Leaf extract % Flower extract %

Moisture 6.0 6.3 5.0

Ash 1.5 1.8 0.9

Fibre 1.0 0.6 0.5

Fat 2.0 6.0 10.0

Protein 7.88 16.636 13.572

Carbohydrate 81.62 68.664 70.028

3.3 Acute Toxicity Studies

This study showed no mortality up to a dose of 2000 mg/kg body weight. Hence, the

extracts are considered safe for long term administration.

63

3.4 Effects of Ethanol Extracts of Euphorbia hirta on Serum Total Cholesterol

Concentration of Normal and Diabetic Rats

In Fig. 2, significant (P<0.05) increase was observed in the total cholesterol

concentration of rats in the diabetic untreated group (group 2) compared with normal control

(group 1). However, non-significant decrease (P>0.05) was observed in the total cholesterol

concentration of diabetic rats (group 7) treated with the standard drug (voglibose) compared

64

with the diabetic untreated rats. Total cholesterol concentrations of all test and control animals

decreased significantly (P<0.05) compared with the total cholesterol concentration of animals

in the diabetic untreated group. Similarly, significant (P<0.05) elevation of total cholesterol

concentration of was observed in the diabetic rats of all the test groups except group 6 rats

administered 300mg/kg b.w. of Euphorbia hirta leaf extract compared with the total

cholesterol concentration of rats in the normal control group (Fig. 2 and Appendix II).

65

Fig. 2: Effect of ethanol extracts of Euphorbia hirta Leaves, stem

and flowers on total cholesterol concentration in alloxan-induced

diabetic rats

0

50

100

150

200

250

300

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

Group

Me

an

To

tal

Ch

ol.

Co

nc

(m

g/d

l)

3.5 Effects of Ethanol Extracts of Euphorbia hirta on Serum High Density Lipoprotein






66

There was significant (P<0.05) reduction in the high density lipoprotein (HDL)

concentration of rats in all the control and test groups except group 4 rats administered

300mg/kg b.w. of E. hirta leaf extract compared with the high density lipoprotein (HDL)

concentration of rats in group 1 (normal control) as shown in Fig. 3. Conversely, the HDL

concentration of rats in all the control and test groups increased significantly (P<0.05)

compared with the HDL concentration of rats of diabetic untreated rats (group 2). Fig. 3 also

shows non-significant (P>0.05) variations in the high density lipoprotein (HDL) concentration

of rats in all the test groups compared with the HDL concentration of group 7 rats administered

the standard drug after diabetes induction. Also, there were no significant (P>0.05) alterations

in the HDL concentrations of rats across the different test group (groups 3, 4, 5 and 6) as

shown in Fig. 3.

67

Fig. 3: Effect of ethanol extracts of Euphorbia hirta Leaves, stem

and flowers on high density lipoprotein concentration in alloxan-

induced diabetic rats

0

20

40

60

80

100

120


Group

Me

an

HD

L C

on

c (

mg

/dl)

3.6 Effects of Ethanol Extracts of Euphorbia hirta on Serum Low Density Lipoprotein


Fig. 4 shows significant (p<0.05) increase in the low density lipoprotein (LDL)

concentration of rats in the all control and test groups compared with the low density

lipoprotein concentration of normal control rats (group 1). However, there was significant





68

(p<0.05) decrease in the LDL concentration of rats in all the control and test groups compared

with the LDL concentration of rats in the diabetic untreated group (group 2). It was also shown

in Fig. 4 that non-significant (p>0.05) variations were observed in the LDL concentration of

diabetic rats in all the test groups administered varying doses of the extract compared with

standard control (group 1, voglibose). There was significant (p<0.05) decrease in the LDL

concentration of rats (group 6) administered 300 mg/kg b.w. of flower, leaf and stem extracts

after the induction of diabetes compared with the LDL concentration of rats of group 5

administered 300 mg/kg b.w. of E. hirta stem extract after the induction of diabetes.

69

Fig. 4: Effect of ethanol extracts of Euphorbia hirta leaves, stem and

flowers on low density lipoprotein concentration in alloxan-induced

diabetic rats

0

20

40

60

80

100

120

140

160

180


Group

Me

an

LD

L C

on

c (

mg

/dl)

3.7 Effects of Ethanol Extracts of Euphorbia hirta on Serum Triacylglycerol






70

Triacylglycerol (TAG) concentration increased significantly (p<0.05) in the diabetic

untreated rats (group 2) compared with the TAG concentration of the normal control rats

(group 1) as shown in Fig. 5. Fig. 5 also shows that the TAG concentration of rats in all the test

groups and group 7 rats (standard drug) decreased significantly (p<0.05) compared with the

TAG concentration of the diabetic untreated rats. Non-significant (p>0.05) change was

observed in the TAG concentration of rats in all the test groups compared with the TAG

concentration of group 1 rats (normal control). Similarly, non-significant (p>0.05) difference

was observed in the TAG concentration of rats across the different test groups as shown in Fig.

5.

71


flowers on triacylglycerol concentration in alloxan-induced diabetic

rats

0

50

100

150

200

250


Group

Me

an

TA

G C

on

c (

mg

/dl)

3.8 Effects of Ethanol Extracts of Euphorbia hirta on Serum Sodium Ion






72

As shown in Fig. 6, significant (P<0.05) reduction was observed in the sodium ion

(Na+) concentration of the rats in all the test groups (groups 3, 4, 5 and 6) administered varying

doses of the extracts compared with the sodium concentrations of rats in the normal control

group (group 1) and group 7 rats treated with the standard drug. Fig. 6 also shows relative

increase in the sodium ion concentration of untreated diabetic rats (group 2) compared with the

sodium ion concentration of rats in group 1; however, such increase was not significant

(P>0.05). There was non-significant (P>0.05) variation in the sodium ion concentrations of rats

across the different test group.

73


flowers on sodium ion concentration in alloxan-induced diabetic

rats

0

20

40

60

80

100

120

140

160


Group

Me

an

Na

+ C

on

c (

mE

q/L

)

3.9 Effects of Ethanol Extracts of Euphorbia hirta on Serum Potassium Ion






74

Potassium ion (K+) concentrations increased significantly (P<0.05) in all the test rats

administered varying doses of Euphorbia hirta extracts compared with the K+ concentration of

rats in the normal control group (group 1) as shown in Fig. 7. Also, significant (P<0.05) was

observed in the K+ concentration of rats in groups 2 (diabetic untreated) and 7 (standard drug)

compared with the K+ concentration of rats in group 1. On the other hand, there was neither

significant (P>0.05) increase nor decrease in the K+ concentration of diabetic untreated rats

(group 2) compared with the K+ concentration of rats treated with the standard drug after

induction of diabetes (Fig. 7).

75


flowers on potassium ion concentration in alloxan-induced diabetic

rats

0

1

2

3

4

5

6

7

8


Group

Me

an

K+ C

on

c (

mE

q/L

)

3.10 Effects of Ethanol Extracts of Euphorbia hirta on Serum Chloride Ion






76

Fig. 8 shows significant (P<0.05) elevation in the chloride ion (Cl–) concentration of

group 2 rats (diabetic untreated) compared with the chloride ion (Cl–) concentration of group 1

rats (normal control). The chloride ion (Cl–) concentration of all the test group rats except

group 6 (300mg/kg b.w. of flower+leaf+stem extracts) decreased significantly (P<0.05)

compared with the chloride ion (Cl–) concentration of rats in all the control groups as observed

in Fig. 8.

77


flowers on chloride ion concentration in alloxan-induced diabetic

rats

0

20

40

60

80

100

120

140


Group

Mean

Cl- C

on

c (

mE

q/L

)

3.11 Effects of Ethanol Extracts of Euphorbia hirta on Serum Bicarbonate Ion






78

It was observed in Fig. 9 that there was non-significant difference (P>0.05) in

bicarbonate ion concentration of the rats in the test groups administered varying doses of the

different extracts compared with the bicarbonate ion concentrations of the rats in all the control

group. Similarly, there were non-significant (P>0.05) changes in the bicarbonate ion

concentration of diabetic untreated rats (group 2) and group 7 rats treated with the standard

drug after the induction of diabetes compared with normal control rats (group 1). Fig. 9 shows

significantly (P<0.05) elevation in bicarbonate ion concentration of groups 3 and 6 rats

administered 300mg/kg b.w. of flower extract of Euphorbia hirta and 300mg/kg b.w. of flower

+ leaf + stem extract of Euphorbia hirta respectively compared with bicarbonate ion

concentration of group 4 rats administered 300mg/kg b.w. of leaf extract of Euphorbia hirta.

79


flowers on bicarbonate ion concentration in alloxan-induced

diabetic rats

0

5

10

15

20

25

30

35


Group

Me

an

HC

O3- C

on

c (

mm

ol/

L)

3.12 Effects of Ethanol Extracts of Euphorbia hirta on Serum Urea Concentration of

Normal and Diabetic Rats





80

Fig. 10 shows dose-dependent significant (P<0.05) decrease in the serum urea

concentration of rats in the different test groups compared with the serum urea concentration of

diabetic untreated rats (group 2). There was significant (P<0.05) increase in the serum urea

concentration of groups 3 and 4 rats administered 300mg/kg b.w. of flower extract of

Euphorbia hirta and 300 mg/kg b.w. of leaf extract of Euphorbia hirta respectively compared

with the serum urea concentration of normal rats in group 1. It was observed, as shown in Fig.

10, that the serum urea concentration of rats in all the test groups and the two control groups

(normal and standard drug) decreased significantly (P<0.05) compared with the serum urea

concentration of positive control rats (diabetic untreated).

81

Fig. 10: Effect of ethanol extracts of Euphorbia hirta leaves, stem

and flowers on urea concentration in alloxan-induced diabetic rats

0

10

20

30

40

50

60

70

80

90


Group

Me

an

Ure

a C

on

c (

mg

/dl)

3.13 Effects of Ethanol Extracts of Euphorbia hirta on Serum Creatinine Concentration

of Normal and Diabetic Rats

There was a dose-dependent significant (P<0.05) decrease in the serum creatinine

concentration of test rats in groups 3, 4 and 5 administered varying doses of the extract





82

compared with the serum urea concentration of the group 2 rats (diabetic untreated) as shown

in Fig. 11. In the same vein, significant (P<0.05) decrease was also observed in the serum

creatinine concentration of rats in the other control and test groups compared with the diabetic

untreated rats. Creatinine concentration of groups 5 (300 mg/kg of stem Euphorbia hirta) and 6

(300 mg/kg of flower+leaf+stem extracts of Euphorbia hirta) rats had significant (P<0.05)

decrease compared with the serum creatinine concentration of normal control rats.

83


and flowers on creatinine concentration in alloxan-induced diabetic

rats

0

0.5

1

1.5

2

2.5

3


Group

Me

an

Cre

ati

nin

e C

on

c (

mg

/dl)

3.14 Effects of Ethanol Extracts of Euphorbia hirta on Serum Catalase Activity of






84

Fig. 12 shows dose-dependent significant (P<0.05) increase in the serum catalase

activities of test rats in all the test groups and group 7 diabetic rats treated with the standard

drug compared with the serum urea concentration of the group 2 rats (diabetic untreated).

There was significant (P<0.05) decrease in the serum catalase activities of diabetic untreated

rats (group 2) compared with the serum catalase activities of normal control rats (group 1) as

shown in Fig. 12. Conversely, significant (P<0.05) increase was observed in the serum catalase

activities of the diabetic rats treated with the standard drug compared with the serum catalase

activities of normal control rats (group 1) and diabetic untreated rats. However, there was no

significant (P>0.05) difference in the serum catalase activities of rats in all the test groups

compared with the serum catalase activities of normal control group as shown in Fig. 12.

85


and flowers on catalase activity in alloxan-induced diabetic rats

0

0.5

1

1.5

2

2.5

3

3.5

4


Group

Mea

n C

ata

las

e A

cti

vit

y (µ

/mg

)

3.15 Effects of Ethanol Extracts of Euphorbia hirta on Serum Superoxide Dismutase

Activity of Normal and Diabetic Rats





86

There was significant (P<0.05) reduction, as shown in Fig. 13, in the serum superoxide

dismutase (SOD) activities of group 2 diabetic untreated rats compared with the SOD activities

of the normal rats (group 1) and rats (group 7) administered standard drug. Non-significant

(P>0.05) increase was observed in the SOD activities of rats (group 7) administered standard

drug compared with the SOD activities of rats in the normal control group. Superoxide

dismutase (SOD) activities of test rats in groups 3 and 4 administered 300 mg/kg b.w. of

flower and leaf extracts of Euphorbia hirta respectively increased significantly (P<0.05)

compared with the SOD activities of rats in all the control groups (groups 1, 2 and 7).

However, the SOD activities of test rats in groups 5 and 6 administered 300 mg/kg b.w. of

Euphorbia hirta stem extract and combined extract (flower + leaf + stem) respectively showed

non-significant (P>0.05) reduction in the SOD activities of rats in group 1 and 7 as shown in

Fig. 13.

87


and flowers on superoxide dismutase activity in alloxan-induced

diabetic rats

0

1

2

3

4

5

6


Group

Me

an

SO

D A

cti

vit

y (µ

/mg

)

3.16 Effects of Ethanol Extracts of Euphorbia hirta on Serum Malondialdehyde


Observation carried out in Fig. 14 showed increased concentration of malondialdehyde

(MDA) in diabetic untreated rats (group 2) compared with the MDA concentration of rats in





88

groups 1 (normal control) 7 (standard drug). There was no significant (P>0.05) decrease in the

MDA concentration of rats in all the test groups except group 3 rats administered 300 mg/kg

b.w. of the flower extract compared with the MDA concentration of the normal control rats. On

the other hand, the MDA concentration of rats in all the test groups decreased significantly

(P<0.05) compared with the MDA concentration of diabetic untreated rats found in group 2.

89


and flowers on malondialdehyde concentration in alloxan-induced

diabetic rats

0

1

2

3

4

5

6


Group

Me

an

MD

A C

on

c (

mm

ol/

L)

3.17 Effects of Ethanol Extracts of Euphorbia hirta on Vitamin C Concentration of






90

Results shown in Fig. 15 revealed that vitamin C concentration in group 2 rats (diabetic

untreated) decreased significantly (P<0.05) compared with the vitamin C concentration of rats

in group 1 (normal control) and group 7 (standard drug). Conversely, rats in all the test groups

had significant (P<0.05) reduction in their vitamin C concentration compared with the vitamin

C concentration of diabetic untreated rats. However, there was no significant variation in the

concentration of vitamin C of diabetic rats (group 7) treated with the standard drug compared

with the vitamin C concentration of normal control rats as shown in Fig. 15.

91


and flowers on vitamin C concentration in alloxan-induced diabetic

rats

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8


Group

Mea

n V

it C

Co

nc

(m

g/d

l)

3.18 Effects of Ethanol Extracts of Euphorbia hirta on Body Glucose Concentration of

Normal and Diabetic Rats Before and After the Experiment





92

Fig. 16 shows significant (P<0.05) rise in the blood glucose concentration of rats in all

the test groups and other control groups compared with the blood glucose concentration of

normal control rats (group 1) before the experiment. However, there was neither significant

(P>0.05) increased nor decrease in the blood glucose concentration of rats in all the groups

compared with the blood glucose concentration of diabetic untreated rats (group 2) before the

experiment.

Significant (P<0.05) decrease was observed in the blood glucose concentration of rats

in all the groups compared with the blood glucose concentration of rats in the diabetic

untreated rats after the experiment as shown in Fig. 15. There was non-significant (P>0.05)

variation in the blood glucose concentration of rats in all the test groups and group 7 rats

(standard drug) compared with the blood glucose concentration of normal control rats.

Similarly, non-significant (P>0.05) change was observed in the blood glucose concentration of

across the test groups after the experiment as shown in Fig. 15.

93

0

50

100

150

200

250

300

350


Group

Me

an

Glu

co

se

Co

nc

(m

g/d

l)

Glucose Before

Glucose After

Fig. 16: Effect of ethanol extracts of Euphorbia hirta leaves, stem and flowers on serum

glucose level of alloxan-induced diabetic rats

3.19 Effects of Ethanol Extracts of Euphorbia hirta on Body Weights of Normal and

Diabetic Rats Before and After the Experiment

As shown in Fig. 17, the body weights of rats in the diabetic untreated group (group 2)

decreased compared with the body weights of rats in group 1 (normal control ); however, such





94

decrease was not significant (P>0.05) before the experiment. The body weights of rats in all the

test groups had significant (P<0.05) increase, except group 3 rats administered 300 mg/kg of

flower extract of Euphorbia hirta, compared with the body weights of rats in group 2 before

the experiment. Results (Fig. 17) after the experiment showed that the body weights of rats in

all the test and control groups increased significantly (P<0.05) compared with the body weights

of diabetic untreated rats. There was observable decrease in the body weights of rats in all the

test groups, except group 3, compared with diabetic rats (group 7) treated with standard drug

after the experiment. The body weights of rats in the test groups before the experiment

exhibited dose-dependent increase before the experiment as shown in Fig. 17.

95

0

50

100

150

200

250

300


Group

Me

an

Bo

dy

We

igh

t (g

)

Weight Before

Weight After

Fig. 17: Effect of ethanol extracts of Euphorbia hirta leaves, stem and flowers on body weight

of alloxan-induced diabetic rats

3.20 Histopathological Examination





96

Plate 1 shows unremarkable islet of langerhans of normal rats in group 1. Moderate to

severe lymphocytic infiltration of islet of langerhans was observed in group 2 alloxan-induced

diabetic untreated rats compared with the tissues of normal control rats in group 1 as shown in

Plate 2. Group 3 diabetic rats showed mild to moderate lymphocytic infiltration of islet of

langerhans after treatment with 300mg/kg of ethanol flower extract (Plate 3). Plates 4 and 5

showed mild lymphocytic infiltration of islet of langerhans in Groups 4 and 5 diabetic rats

administered 300mg/kg of ethanol leaf extract and 300 mg/kg of ethanol stem extract

respectively. Scanty lymphocytic infiltration of islet of langerhans was observed in diabetic

rats (Group 6) treated with combined therapy of 300 mg/kg of ethanol flower, leaf and stem

extract as shown in Plate 6. Group 7 diabetic rats (standard control) treated with 0.01mg/kg

b.w. of voglibose showed scanty lymphocytic infiltration of an enlarged islet of langerhans

(Plate 7).

97

Plate 1: Photomicrograph of pancreas of normal control rats (group 1) showing an

unremarkable islet of langerhans (IL) surrounded by normal acini of exocrine pancreas (EP) (H

& E stain x 400).

98

Plate 2: Photomicrograph of pancreas of group 2 rats (diabetic untreated) showing moderate to

severe lymphocytic infiltration (LI) of islet of langerhans (IL (H & E stain x 400).

99

Plate 3: Photomicrograph of pancreas of group 3 diabetic rats treated with 300 mg/kg b.w. of

the flower extract showing mild to moderate lymphocytic infiltration (LI) of islet of langerhans

(H & E stain x 400).

100


the leaf extract showing mild lymphocytic infiltration (LI) of islet of langerhans (H & E stain x

400).

101


the stem extract showing mild lymphocytic infiltration (LI) of islet of langerhans (H & E stain

x 400).

102


flower, leaf and stem extracts showing scanty lymphocytic infiltration (LI) of islet of

langerhans (H & E stain x 400).

103

Plate 7: Photomicrograph of pancreas of group 7 rats (0.01 mg/kg b.w. of standard drug,

voglibrose, showing scanty lymphocytic infiltration (LI) of an enlarged islet of langerhans (H

& E stain x 400).

104

CHAPTER FOUR

DISCUSSION

The results of the experiment showed that there was significant increase (p<0.05) in the total

cholesterol concentration in group 2 rats (diabetic untreated) compared with normal control rats

(group 1). This increase in total cholesterol concentration can be attributed to diabetes-induced

hypercholesterolaemia (Mayne, 1991). Administration of 300 mg/kg b.w. of ethanol extracts of E. hirta

to rats in Groups 3 to 6, and 0.01 mg/kg b.w. of voglibose in rats in group 7 showed significant

reduction (p<0.05) in the total cholesterol concentration compared with rats in group 2 (diabetic

untreated). The decrease in total cholesterol is more pronounced in group which is a combination of

the three extracts. The significant decrease may be due to increased breakdown of the cholesterol in

the liver, and decreased absorption of cholesterol via the chylomicrons due to inhibition of α-

glucosidase enzymes. Conversely, there was significant decrease (p<0.05) in high density lipoprotein

(HDL)-cholesterol concentration in the Group 2 (Diabetic untreated) compared with Group 1 (normal

rats). This decrease may be due to the inhibition of HDL-C production in diabetes. However,

administration of ethanol extracts of E. hirta in Groups 3 to 6 and voglibose in Group 7 showed

significant increase in HDL-C concentration. This significant increase may be due to increase

cholesterol transport pathway (Nelson and Cox, 2008).

There was also significant increase (p<0.05) in low density lipoprotein (LDL-C)-cholesterol

concentration in Group 2 (diabetic untreated) compared with Group 1 (normal control rats). This can

be attributed to increase in concentration LDL-C resulting from diabetes induction. However, there

was corresponding significant decrease (p<0.05) in LDL-C with the administration of 300 mg/kg b.w. of

ethanol extracts of E. hirta to rats in Groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to rats in Group 7

compared with diabetic untreated rats (Group 2). This decrease is more in Group 6 rats which were

administered a mixture of the three ethanol extracts. This significant decrease in LDL-C may be due to

the increase in the reverse cholesterol transport pathway, and decreased cholesterol concentration

from the intestine in the chylomicrons due to α-glucosidase inhibition. Similarly, there was significant

increase (p<0.05) in triacylglycerol concentration in Group 2 (Diabetic untreated) compared with the

normal untreated rats (Group 1). This may be due to diabetes-induced hypertriglyceridaemia.

However, administration of 300 mg/kg b.w. of ethanol extracts of E. hirta to rats in Groups 3

to 6 and 0.01 mg/kg b.w. of voglibose to rats in Group 7 caused significant reduction (p<0.05) in the

triacylglycerol concentration. This decrease in LDL-C concentration is more in Group 6 which is a

105

combination of the three ethanol extracts. This significant decrease in LDL-C may be attributed to the

reduced absorption of the triacylglycerol rich chylomicrons from the intestinal brush borders due to

inhibition of the α-glucosidase in the brush borders. Voglibose has also been postulated to decrease

triacylglycerol (Satoh et al., 2006).

It can be noticed that there was non-significant change (p>0.05) in the sodium ion

concentration in diabetic untreated rats (Group 2) compared with rats in normal control group. This

may be due to the fact that diabetes may not have a direct effect on serum sodium ion concentration.

However, administration of 300 mg/kg b.w. of ethanol extracts of E. hirta to rats in Groups 3 to 6 and

0.01 mg/kg b.w. of voglibose to rats Group 7 resulted in significant reduction (p<0.05) in serum

sodium ion concentration. The relative hyponatraemia may be due to the diuretic property of E. hirta

ethanol extracts which is related to the angiotensin converting enzyme inhibition (Edwards et al.,

1995).

Furthermore, there was significant increase (p<0.05) in serum potassium ion concentration in

diabetic untreated rats (Group 2) compared with the potassium ion concentration of normal rats

(Group 1). This may be due to diabetes induced hyperkalaemia due to poorly controlled diabetes with

possibilities of diabetic ketoacidosis resulting (Mayne, 1991). However, there were no significant

change (p<0.05) in serum potassium concentration on administration of 300 mg/kg b.w. of ethanol

extracts of E. hirta to rats in Group 3 to 6 and 0.01 mg/kg b.w. of voglibose to rats in Group 7. The loss

of significance in potassium ion concentration shows that neither ethanol extracts of E. hirta nor

voglibose has effect on the serum potassium ion concentration. Moreover, there was a significant

increase (p<0.05) in serum chloride ion concentration in diabetic untreated animals compared with

the animals in the normal control group. This may be attributed to diabetes-induced hyperchloraemic

acidosis where sodium is reabsorbed with chloride ion in the tubules, in exchange with hydrogen ion

or potassium ion (Mayne, 1991). When ethanol extracts of E. hirta were added to Groups 3 to 6, there

was significant reduction in serum chloride ion concentration compared with rats in diabetic

untreated group. This may be attributed to the diuretic effect of E. hirta extract where sodium and

chloride ions are exchanged for potassium and hydrogen ions respectively. However, the

administration of 0.01 mg/kg b.w. of voglibose to rats in Group 7 showed non-significant change

(p>0.05) in the serum chloride ion concentration compared with the chloride ion concentration of

Group 2 rats (diabetic untreated).

Furthermore, there was no significant change (p>0.05) in the serum HCO3– concentration in

diabetic untreated rats (Group 2) compared with HCO3– concentration of normal rats (Group 1). Also,

there was no significant change (p>0.05) in HCO3– concentration when 300 mg/kg b.w. of the ethanol

106

extracts were administered to rats in Groups 3 to 6 and 0.01 mg/kg b.w. of voglibose to rats in Group

7 compared with HCO3– concentration of diabetic untreated rats (Group 2). Conversely, there was

significant increase (p<0.05) in the serum urea concentration in diabetic untreated rats (Group 2)

compared with urea concentration of normal untreated rats (Group 1). This may be attributed to the

acute phase of diabetic nephropathy with reduction in the glomerular filtration rate (Mayne, 1991).

However, there were significant decrease (p<0.05) in the serum urea concentration when ethanol

extracts of E. hirta were added to Groups 3 to 6 and voglibose to Group 7 compared with diabetic

untreated rats (Group 2) which is an indication of the resolution of the acute phase of diabetic-

induced nephropathy.

Moreover, there was significant increase (p<0.05) in the serum creatinine concentration in

diabetic untreated rats (Group 2) compared with Group 1 (normal control rats). This may be due to

diabetes-induced nephropathy with reduced GFR. However, administration of ethanol extracts of E.

hirta to Groups 3 to 6 and voglibose to Group 7 caused significant reduction in the serum creatinine

concentration. This is a clear indication of the resolution of the acute phase of diabetes induced

nephropathy.

There was significant decrease (p<0.05) in the serum catalase activity in Group 2 (diabetic

untreated rats) compared with Group 1 normal control rats. This may be attributed to the increasing

production of free radicals due to the diabetic state (Oberley, 1988). However, there were significant

increases (p<0.05) in the serum catalase activity when ethanol extracts of E. hirta were added to

Groups 3 to 6 and voglibose to Group 7 compared with diabetic untreated rats. This can be caused by

the scavenging action of the ethanol extracts of E. hirta. Voglibose caused highest increase in serum

catalase activity as it has been known for its ability to reduce oxidative stress (Satoh et al., 2006).

Significant decrease (p<0.05) was observed in the serum superoxide dismutase activity of

group 2 diabetic untreated compared with normal control rats (Group 1). This may be attributed to

the increased production of free radicals due to the diabetic state. On administration of 300 mg/kg

b.w. of ethanol extracts of E. hirta to rats in Groups 3, 4, 5 and 6, and 0.01 mg/kg b.w. of voglibose to

Group 7, there was significant increase (p<0.05) in the serum superoxide dismutase (SOD) activity

compared with diabetic untreated rats (Group 2). This can be caused by the antioxidant scavenging

action of the ethanol extracts of E. hirta.

Nevertheless, there was significant increase (p<0.05) in the malondialdehyde (MDA)

concentration in diabetic untreated rats (Group 2) compared with the MDA concentration of Group 1

animals (normal rats). This may be due to increased lipid peroxidation (Gallou et al., 1993) which was

107

induced by diabetes resulting in increased reactive oxygen species (Baynes, 1991). When 300 mg/kg

b.w. of ethanol extracts of E. hirta was administered to rats in Groups 3, 4, 5 and 6, and 0.01 mg/kg

b.w. of voglibose to Group 7, there was significant decrease (p<0.05) in MDA concentration compared

with diabetic untreated rats (Group 2). This could be attributed to the antioxidant scavenging activities

of ethanol extracts of E. hirta resulting in reduced lipid peroxidation.

Finally, there was significant reduction in vitamin C concentration in Group 2 (diabetic

untreated) compared with the normal control animals. This is due to diabetes-induced free radical

production. However, when 300 mg/kg. b.w. of ethanol extracts of E. hirta were administered to rats

in Groups 3, 4, 5 and 6, and 0.01 mg/kg b.w. voglibose to Group 7 rats, there were significant increases

(p<0.05) in vitamin C concentration compared with the normal control rats (Group 2). This may be

caused by the antioxidant scavenging action of ethanol extracts of E. hirta.

It can be observed that there was significant increase (p<0.05) in blood glucose concentration

in rats of Groups 2 to 7 before the administration of 300 mg/kg b.w. of ethanol extract of E. hirta and

0.01 mg/kg b.w. of voglibose compared with Group 1 (normal rats). This increase in blood glucose

could be attributed to the induction of diabetes with alloxan in the respective groups.

However, when 300 mg/kg b.w. of ethanol extracts of E. hirta were administered to rats in

Groups 3, 4, 5 and 6, and 0.01 mg/kg b.w. of voglibose to rats in Group 7, there were significant

decreases (p<0.05) in the blood glucose concentration after the administration of the extracts to the

rats compared with Group 2 rats (diabetic untreated). This has shown that ethanol extracts of E. hirta

and voglibose exhibit hypoglycaemic activities (Kim et al., 2004).

It can be observed that before the administration of 300 mg/kg b.w. of ethanol extracts of E.

hirta and 0.01 mg/kg b.w. of voglibose, there was significant increase (p<0.05) in the body weights of

rats in Groups 4, 5, 6 and 7 with non-significant increase (p>0.05) in the body weights of rats in Group

3 compared with the body weights of normal control rats. There was no significant change (p<0.05) in

the body weights of diabetic untreated rats (Group 2) compared with the body weights of rats in

Group 1 (normal control). However, after the administration of 300 mg/kg b.w. of ethanol extracts of

E. hirta to rats in Groups 3, 4, 5 and 6, and 0.01 mg/kg b.w. voglibose to Group 7, there was significant

increase (p<0.05) in body weights of all rats in Groups 1, 3, 4, 5, 6 and 7 compared with diabetic

untreated rats (Group 2). The increase in Group 1 can be attributed to normal growth to normal feed

high in carbohydrate and fats while increase in Group 3 to 7 was probably due to increases

glycogenesis and lipogenesis with diabetes treatment.

108

The induction of diabetes by alloxan in the diabetes rats (as shown in Rats 2 of Plate 2) caused

destruction of the islet of Langerhans which histologically resulted in moderate to severe infilteration

of the islet of Langerhans by lymphocytes (shown by the multiple black spots in the islets on Plate 2

when compared with Plate 1).

However, addition of different ethanol extracts of Euphorbia hirta resulted in resolution of the

damage on the islets of Langerhans which is shown histologically by reduction in lyphocytic

infilteration (black spots) of the islets of Langerhans. This results in mild to moderate lymphocytic

infiltration when flower extract was added (plate 3), mild lymphocytic infiltration when leaf and stem

extracts were added (Plates 4 & 5) and scanty lymphocytic infiltration when equal mixture of flower,

leaf and stem extracts and voglibose (standard drug) were added (Plates 6 & 7).

These resolutions tend to restore the insulin secretory capacity of the islets of Langerhans, reverses

the diabetic condition with reversal of biochemical parameters in rats of Groups 3 to 7 towards

normal ranges when compared with diabetic untreated rats in Group 2.

4.2 CONCLUSION

From the results of the tests, we can deduce that the 300 mg/kg. b.w. of ethanol extracts of

Euphorbia hirta has lipid lowering activity by reducing serum total cholesterol, triacylglycerol and low

density lipoprotein-cholesterol while increasing high density lipoprotein-cholesterol concentrations by

increasing the reverse cholesterol transport pathway. Dose of 300 mg/kg b.w. of ethanol extracts of E.

hirta also improves renal function by reducing serum urea and creatinine concentrations; hence,

reducing incidences of acute diabetic nephropathy. Ethanol extracts (300 mg/kg b.w.) of E. hirta

exhibit antioxidant scavenging action by increasing catalase and superoxide dismutase activities, and

vitamin C concentration; thereby reducing malondialdehyde concentration, hence reducing lipid

peroxidation. Finally, 300 mg/kg b.w. of ethanol extracts of E. hirta has hypoglycaemic action which

are identical to the hypoglycaemic action of voglibose via α-glucosidase inhibition. The decrease in

serum Na+ and Cl

– concentrations has shown that ethanol extracts of E. hirta exhibits diuretic

peoperties which is suspected to be due to the inhibition of the angiotensin converting enzyme.

4.3 RECOMMENDATIONS

Further experiments have to be done to fractionate and elucidate the different compounds of

the ethanol extracts of the flower, leaf and stem of Euphorbia hirta so as to identify the actual

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component(s) of the herb with the diuretic, hypoglycaemia, lipid lowering, antioxidative activities with

reduced urea and creatinine profile. Comparison with other antidiabetic drugs like glibenclamide,

metformin and soluble insulin should be undertaken to access the extent of hypoglycaemic actions of

ethanol extracts of E. hirta and the extent of reduction of diabetic complications.

CHUKWUMA, UCHENNA VALENTINE … Uchenna...extracts of Euphorbia hirta to rats in group 3 to 6 and...

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