University Of Nigeria Nsukka - OBETA, JUDITH NNEDIMKPA … PROJECT FOR... · 2015-08-29 · 2 studies on the effect of aqueous extract of millettia aboensis leaves on lomotil –

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OBETA, JUDITH NNEDIMKPA

(PG/M.Sc/12/61880)

Ogbonna Nkiru

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

FACULTY OF BIOLOGICAL SCIENCES

DEPARTMENT OF BIOCHEMISTRY

STUDIES ON THE EFFECT OF AQUEOUS

EXTRACT OF MILLETTIA ABOENSIS LEAVES

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STUDIES ON THE EFFECT OF AQUEOUS EXTRACT OF MILLETTIA

ABOENSIS LEAVES ON LOMOTIL – INDUCED CONSTIPATION IN

WISTAR ALBINO RATS

BY

OBETA, JUDITH NNEDIMKPA

(PG/M.Sc/12/61880)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT ON THE

REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE

(M.Sc.) DEGREE IN PHARMACOLOGICAL BIOCHEMISTRY

FACULTY OF BIOLOGICAL SCIENCES

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISORS: PROF. OFC NWODO AND DR. PARKER E. JOSHUA

OCTOBER, 2014

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CERTIFICATION

Obeta, Judith Nnedimkpa, a post graduate student of the Department of Biochemistry,

University of Nigeria Nsukka, with Registration Number PG/M.Sc./12/61880 has

satisfactorily completed the requirements for the award of the Degree of Master of Science

(M. Sc.) in Pharmacological Biochemistry. The work embodied in this thesis is original and

has not been submitted in part or full for any other diploma or degree of this or any other

University to the best of our knowledge.

…………………………… …………………………….

PROF. OFC NWODO DR. PARKER E. JOSHUA

(Supervisor) (Supervisor)

………………………….. …………………………….

PROF. OFC NWODO EXTERNAL EXAMINER

(Head of Department)

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DEDICATION This work is solely dedicated to God Almighty for His knowledge, direction, grace,

protection and love during the course of this programme and also to my beloved parents,

siblings and husband for their encouragements and support.

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ACKNOWLEDGEMENTS

May the name of God the Almighty be glorified for seeing me through this programme. He is

indeed a faithful Lord. I would like to express my sincere gratitude to my supervisors, Prof.

O.F.C. Nwodo and Dr. Parker E. Joshua for their fatherly motivations, directions, corrections

and constructive criticism that contributed in the successful completion of this research work

despite their tight schedules. I am indeed very indebted to you for your support.

To all my great lecturers of the Department of Biochemistry, I appreciate the

encouragement, contributions, love and goodwill given to me during this period. I specially

thank the current head, Prof. O.F.C. Nwodo, Prof. L. U.S. Ezeanyika, Prof. I. N. E. Onwurah,

Prof. F.C. Chilaka, Prof. O. U Njoku, Prof. E. A. Alumanah, Prof. P N Uzoegwu, Dr. Parker

E. Joshua, Dr. V. N. Ogugua, Dr. B. C. Nwanguma, Dr. (Mrs.) C. A. Anosike, Dr. O. C.

Enechi, Prof. H. A. Onwubiko, Dr. S.O. O. Eze, Dr. C. S. Ubani, Mr. P. A. C. Egbuna, Dr.

(Mrs.). C. Ezekwe, Mr. O. E. Ikwuagwu, Mrs. U. O Njoku, Mrs. M. N. Awachie, Dr. V. E. O.

Ozougwu, Mr. C. Agu, Mr. C. C. Okonkwo and a host of others, for their immense support in

my quest for knowledge.

To my friends and colleagues who contributed in numerous ways to the successful

completion of this work; Tochi, Somadina, Paul, Obiora, Mary, Robert, Oge, Emenike,

Kachi, Geraldine, Chidimma, Ebere, and others. May the good Lord bless you all. To my

brethren in Catholic Association of Postgraduate Students (CAPS), I say thanks for your love

and prayers. Finally, my deep gratitude goes to my lovely parents Mr. & Mrs. M. C. Obeta,

husband and siblings for their support, encouragement and prayers.

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ABSTRACT

Millettia aboensis leaf is a rich source of reducing sugar, tannins, glycosides and flavonoids and

has been reported to have medicinal property as well as some physiological potentials. The leaf

also has been used by traditional herbalists for general healing of diseases including ulcer and

laxatives. This study evaluated the effect of aqueous extract of M. aboensis leaves on lomotil-

induced constipation in Wistar albino rats. The qualitative phytochemical constituents of the

extract showed the relative presence of reducing sugars, tannins and flavonoids in relatively high

concentrations; alkaloids, steroids and glycosides in moderately high concentrations; soluble

carbohydrates, saponins and hydrogen cyanides were in low concentrations while terpernoid was

not detected. The median lethal dose effect (LD50) of the extract recorded no death at dose of

5000 mg/kg b.w. Assay of Aspartate Amino transferase and Alanine Amino transferase activities

in serum of treated rats (groups 2 and 3) given 100 and 1000 mg/kg b.w. of the extract showed

significant increase (p<0.05) compared to the control group 1 (normal saline). The ALP activity

in serum of the mice in groups 2 and 3 administered 100 and 1000 mg/kg b.w. of the extract

exhibited neither significant increase nor decrease (p>0.05) compared to the control group 1

mice. Triacylglycerol and High density lipoprotein concentrations in serum of the mice treated

with 100 and 1000 mg/kg b.w. of the extract showed non-significant increase (p>0.05) compared

to the control group while the LDL and total cholesterol concentrations of the groups 2 and 3

given 100 and 1000 mg/kg b.w. of the extract showed non-significant decrease (p>0.05)

compared to the control group. The potassium ion concentration showed a non-significant

increase (p>0.05) in the groups 2 and 3 mice administered 100 and 1000 mg/kg b.w. of the

extract compared to the control group while the serum level of sodium ion showed a non-

significant increase (p>0.05) in group 2 mice administered 100 mg/kg b.w. of the extract and a

significant increase (p<0.05) in group 3 mice that received 1000 mg/kg b.w. of the extract

compared to the control mice. There was neither a significant decrease nor increase (p>0.05) in

the serum level of glucose of the mice in groups 2 and 3 administered 100 and 1000 mg/kg b.w.

of the extract compared to the control. The result of the aqueous extract of M. aboensis on the

mean value of the faecal droppings on lomotil-induced constipation in rats showed neither a

significant decrease nor increase (p>0.05) in groups 2 (standard drug of lomotil), 3 (100 mg/kg

b.w. of extract), 5 (100 mg/kg of extract + 5 mg/ml of lomotil, 7 (5 mg/ml of lomotil + 200 mg/kg

b.w. of extract) and 8 (5mg/ml of lomotil + 200 mg/kg of extract) compared to the negative

control (normal saline) while group 4 mice (200 mg/kg b.w. of extract) showed a non-significant

increase (p>0.05) and group 6 (200 mg/kg b.w. of extract + 5 mg/ml of lomotil) showed a

significant increase (p<0.05) compared to the positive control group 2 (standard drug of 5 mg/ml

of lomotil). The study of the effects of the aqueous extract of M. aboensis on transport of glucose

across everted rat intestine showed significant increase (p<0.05) in the glucose influx into the

everted intestinal sac (serosal compartment) in a dose-dependent manner in all the treated groups

contained in groups 3, 4 and 5 everted in 100, 200 and 400 µg/ml of the extract compared to the

control group 1 while in group 2 (standard drug metformin), there was a significant decrease

(p<0.05) compared to the control. Sodium transport across the everted rat intestine showed a

significant increase (p<0.05) in the influx of sodium ions into the serosal compartment of groups

2, 3 and 4 everted in 100, 200 and 400 µg/ml of the extract compared to the control group while

potassium transport across everted rat intestine showed significant increase (p<0.05) in the efflux

of potassium ions into the mucosal compartment of all the treated groups 2, 3 and 4 everted in

100, 200 and 400 µg/ml of the extract compared to the control group 1. The significant increase

in the frequency of faecal droppings on the extract treated groups may prove that the extract

contains some bioactive compounds that have the properties of laxative effects when

administered and therefore support the claim that this plant is used in folk medicine for the

treatment of constipation.

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TABLE OF CONTENTS

Title Page - - - - - - - - - - - i

Certification - - - - - - - - - - ii

Dedication - - - - - - - - - - iii

Acknowledgement - - - - - - - - - iv

Abstract - - - - - - - - - - v

Table of Contents - - - - - - - - - vi

List of Figures - - - - - - - - - - xii

List of Tables - - - - - - - - - - xiv

List of Abbreviations - - - - - - - - - xv

CHAPTER ONE: INTRODUCTION

1.1. Plant description - - - - - - - - 2

1.1.2. Scientific classification of Millettia aboensis - - - - - 3

1.2. Phytochemistry - - - - - - - - 4

1.2.1. Phytochemical components in plants - - - - - 4

1.2.1.1. Flavonoids - - - - - - - - - 5

1.2.1.2. Tannins - - - - - - - - - 5

1.2.1.3. Hydrogen cyanides - - - - - - - - 6

1.2.1.4. Alkaloids - - - - - - - - - 6

1.2.1.5. Steroids - - - - - - - - - 7

1.2.1.6. Saponins - - - - - - - - - 7

1.2.1.7 .Glycosides - - - - - - - - - 8

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1.2.1.8 .Reducing sugars - - - - - - - - 8

1.3. Constipation - - - - - - - - - 9

1.3.1. Definition of constipation - - - - - - - 9

1.3.2. Constipation in children - - - - - - - 9

1.3.3. Types of constipation - - - - - - - - 10

1.3.3.1. Occasional Constipation - - - - - - - 10

1.3.3.2 Chronic constipation - - - - - - - - 10

1.3.4. Causes of constipation - - - - - - - 10

1.3.4.1. Diet cause - - - - - - - - - 10

1.3.4.2. Medication cause - - - - - - - - 11

1.3.4.3 .Metabolic and muscular cause - - - - - - 11

1.3.4.4. Psychological cause - - - - - - - - 11

1.3.5. Diagnosis of constipation - - - - - - - 11

1.3.5.1. Rome II criteria for constipation - - - - - - 12

1.3.6. Prevention of constipation - - - - - - - 12

1.3.7. Treatment of constipation - - - - - - - 12

1.3.7.1. Dietary fibre - - - - - - - - - 12

1.3.7.2. Lubricant laxatives - - - - - - - - 13

1.3.7.3. Emollient laxatives (Stool softeners) - - - - - 13

1.3.7.4. Hyperosmolar Laxatives - - - - - - - 13

1.4. Biochemical parameters - - - - - - - 14

1.4.1. Body electrolytes - - - - - - - - 14

1.4.2. Depletion and absorption of sodium - - - - - - 14

1.4.3. Depletion and absorption of potassium - - - - - 15

1.5. Na+/K

+ - ATPase - - - - - - - - 15

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1.5.1. Sodium – potassium pumps - - - - - - - 15

1.5.2. Functions of Na+/K

+ - ATPase - - - - - - 15

1.5.2.1 Resting potential - - - - - - - - 16

1.5.2.2. Transport - - - - - - - - - 16

1.5.2.3. Controlling cell volume - - - - - - - 16

1.5.3. Mechanisms of Na+ - K

+ - ATPase pump - - - - - 16

1.6. Blood glucose - - - - - - - - - 17

1.6.1. Regulation of blood glucose - - - - - - - 17

1.6.2. Abnormality in blood glucose level - - - - - - 17

1.6.2.1. High blood sugar (Hyperglycemia) - - - - - - 17

1.6.2.2. Low blood sugar (Hypoglycemia) - - - - - - 17

1.7. Liver - - - - - - - - - - 18

1.7.1. Functions of the liver - - - - - - - 18

1.8. Liver markers - - - - - - - - - 18

1.8.1. Alanine transaminase - - - - - - - - 19

1.8.2. Alkaline phosphatase - - - - - - - 19

1.8.3. Aspartate transaminase - - - - - - - 19

1.9. Lipid profile - - - - - - - - - 20

1.9.1. High density lipoprotein - - - - - - - 20

1.9.2. Triacylglycerol - - - - - - - - 20

1.9.3. Low density lipoprotein - - - - - - - 20

1.9.4. Cholesterol - - - - - - - - - 21

1.10. Objectives of the research - - - - - - - 22

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CHAPTER TWO: MATERIALS AND METHODS

2.1. Materials - - - - - - - - - 23

2.1.1. Plant materials - - - - - - - - 23

2.1.2. Animals - - - - - - - - - 23

2.1.3. Equipment - - - - - - - - - 23

2.1.4. Chemical reagents - - - - - - - - 24

2.2. Methods - - - - - - - - - 24

2.2.1. Experimental design - - - - - - - - 24

2.2.2. Effect of the aqueous extract of Millettia aboensis on glucose, sodium and potassium

transport across everted rat intestine - - - - - - - 24

2.2.3. Reagents - - - - - - - - - - 25

2.2.4. Glucose saline - - - - - - - - - 25

2.2.5. Extraction of plant material - - - - - - - 26

2.2.6. Acute toxicity - - - - - - - - - 27

2.2.7. Qualitative phytochemical analysis - - - - - - 27

2.2.7.1. Test for glycosides - - - - - - - - 27

2.2.7.2. Test for steroids - - - - - - - - 27

2.2.7.3. Test for tannins - - - - - - - - 28

2.2.7.4. Test for carbohydrates - - - - - - - 28

2.2.7.5. Test for saponins - - - - - - - - 28

2.2.7.6. Test for alkaloids - - - - - - - - 28

2.2.7.7. Test for flavonoids - - - - - - - - 29

2.2.7.8. Test for terponoids - - - - - - - - 29

2.2.7.9. Test for reducing sugars - - - - - - - 29

2.2.8. Quantitative phytochemical analysis - - - - - - 29

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2.2.8.1. Determination of glycosides - - - - - - - 29

2.2.8.2. Determination of steroids - - - - - - - 30

2.2.8.3. Determination of tannins - - - - - - - 30

2.2.8.4. Determination of soluble carbohydrates - - - - - 30

2.2.8.5. Determination of saponins - - - - - - - 30

2.2.8.6. Determination of alkaloids - - - - - - - 30

2.2.8.7. Determination of flavonoids - - - - - - - 31

2.2.8.8. Determination of terponoids - - - - - - - 31

2.2.8.9. Determination of hydrogen cyanides - - - - - 31

2.2.8.10. Determination of reducing sugar - - - - - - 31

2.2.9. Biochemical parameters - - - - - - - 31

2.2.9.1 .Determination of total cholesterol - - - - - - 31

2.2.9.2 .Determination of low density lipoprotein concentration - - - 33

2.2.9.3. Determination of triacylglycerol - - - - - - 34

2.2.9.4. Determination of high density lipoprotein - - - - - 35

2.2.9.5. Assay of alanine aminotransferase activity - - - - - 36

2.2.9.6. Assay of alkaline phosphatase activity - - - - - 37

2.2.9.7. Assay of aspartate aminotransferase activity - - - - 38

2.2.9.8. Blood glucose concentration - - - - - - 39

2.2.9.9. Serum concentration of sodium ion - - - - - - 39

2.2.9.10. Serum Concentration of Potassium ion - - - - - 40

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CHAPTER THREE: RESULTS

3.1. Extraction yield - - - - - - - - 42

3.2. Acute toxicity test - - - - - - - - 42

3.3. Qualitative and quantitative phytochemical composition of

aqueous extract of M. aboensis - - - - - - 42

3.4. Toxicological Effect of M. aboensis extract on AST activity in mice - 44

3.5. Toxicological Effect of M. aboensis extract on ALT activity in mice - 46

3.6. Toxicological Effect of M. aboensis extract on ALP activity in mice - 48

3.7. Toxicological Effect of M. aboensis extract on total cholesterol concentration in mice-

- - - - - - - - - - - 50

3.8. Effect of M. aboensis extract on a low density lipoprotein concentration in mice 52

3.9. Toxicological Effect of M. aboensis extract on HDL concentration in mice - 54

3.10. Toxicological Effect of M. aboensis extract on TAG concentration in mice - 56

3.11. Toxicological Effect of M. aboensis extract potassium concentration in mice - 58

3.12. Toxicological Effect of M. aboensis extract on sodium ion concentration in mice 60

3.13. Toxicological Effect of M. aboensis extract on glucose concentration in mice - 62

3.14. Effect of M. aboensis extract on glucose transport across everted rat intestine- 66

3.15. Effect of M. aboensis extract on sodium transport across everted rat intestine 68

3.16. Effect of M. aboensis extract on Potassium transport across everted rat intestine 70

CHAPTER FOUR: DISCUSSION

4.1. Conclusion - - - - - - - - - 77

4.2. Suggestions - - - - - - - - - - 77

Reference - - - - - - - - - - 78

Appendices - - - - - - - - - - 89

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LIST OF FIGURES

Fig 1: Structural view of the plant Millettia aboensis - - - - 3

Fig 2: Toxicological Effect of M. aboensis on aspartate aminotransferase activity in mice

- - - - - - - - - - - 45

Fig 3: Toxicological Effect of Aqueous extract of M. aboensis on Alanine aminotransferase

activity - - - - - - - - - - - 47

Fig 4: Toxicological Effect of aqueous extract of M. aboensis on alkaline phosphatase in

mice - - - - - - - - - - - 49

Fig 5: Toxicological Effect of Aqueous extract of M. aboensis on total cholesterol

concentration in mice - - - - - - - - 51

Fig 6: Effect of Aqueous extract of M. aboensis on low density lipoprotein concentration in

mice - - - - - - - - - - - 53

Fig 7: Toxicological Effect of Aqueous extract of M. aboensis on high density lipoprotein

concentration in mice - - - - - - - - - - 55

Fig 8: Toxicological Effect of Aqueous extract of M. aboensis on triacylglycerol

concentration in mice - - - - - - - - - 57

Fig 9: Toxicological Effect of Aqueous extract of M. aboensis on Potassium ion

concentration in mice - - - - - - - - - 59

Fig 10: Toxicological Effect of Aqueous extract of M. aboensis on Sodium ion concentration

in mice - - - - - - - - - - - - 61

Fig 11: Toxicological Effect of Aqueous extract of M. aboensis on Glucose concentration in

mice - - - - - - - - - - - 63

Fig 12: Effect of Aqueous extract of M. aboensis on Glucose transport

across everted rat intestine - - - - - - - 67

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Fig 13: Effect of Aqueous extract of M. aboensis on Sodium transport


Fig 14: Effect of Aqueous extract of M. aboensis on Potassium transport


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LIST OF TABLES

Table 1: Qualitative phytochemical composition of aqueous extract of M. aboensis - 42

Table 2: Quantitative phytochemical composition of aqueous extract of M. aboensis - 43

Table 3: Effects of different doses of the aqueous extract of Millettia aboensis on the

frequency of defecation of lomotil-induced constipation in rats - - - 64

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LIST OF ABBREVIATIONS

ADP- Adenosine diphosphate

AEMA- Aqueous extract of Millettia aboensis

ALP- Alkaline phosphatase

ALT- Alanine aminotransferase

AST- Aspartate aminotransferase

ATP- Adenosine triphosphate

BM- Bowel movement

BDCP- Bioresources Development Conservation Programme

DNA- Deoxyribonucleic acid

ECF- Extracellular fluid

EDTA- Ethylenediaminetetraacetic acid

HCL- Hydrogen chloride

HCN- Hydrogen cyanide

HCO2—

Hydrogen carbonates

HDL – High density lipoprotein

HMG-COA – 5-hydroxy-3-methylglutaryl-coenzyme A

HPO42-

- Hydrogen tetraoxophosphate

H2SO4 – Hydrogen tetraoxosulphate

KHB – Krebs Henseleit bicarbonate

GK –Glucokinase

GPO –Glycerol phosphate oxidase

GPT –Glutamate pyruvate transaminase

LDL –Low density lipoprotein

LFT –Liver function test

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POD -Peroxidase

PVS –Polyvinyl sulphate

TAG -Triacylglyceride

VLDL –Very low density lipoprotein

CHAPTER ONE

INTRODUCITON

Traditional medicine according to (Treben, 1998) is defined as the knowledge, skills

and practices of holistic health care, recognized and accepted for its role in the maintenance

of health and the treatment of disease conditions. The application of herbs to improve man’s

health must have come from early man in the most non-scientific way. Since that period, the

application of herbs has been known and accepted by all individuals and nations (Theiss and

Peter, 2000).

Herbal medicine also called botanical medicine or phytomedicine refers to using a

plant’s seeds, berries, roots, leaves, bark or flowers for medicinal purposes. Herbalism has a

long tradition of use outside of conventional medicine. It is becoming more mainstream as

improvements in analysis and quality control along with advances in clinical research

showing the value of herbal medicine in the treating and preventing disease (Izzo et al., 2009)

Medicinal plants are plants in which one or more of their plant contain substances that

can be used for the therapeutic purposes or which are precursors for the synthesis of useful

drugs (WHO, 2005). However, some contain excipients in addition to the active ingredients.

Medicinal plants as a group comprise approximately 8000 species and account for about 50%

of higher flowering plant species (Treben, 1998). Some examples of medicinal plants include

Napolliena imperalis, Digoxin lancta, Chenopodium ambrosoides, Morinda lucida, Zingiber

officinale etc. Medicinal plants are used commonly in modern medicine and pharmacology.

One of the early attempts to store information on medicinal plants have long been on going in

Nigeria in an attempt to set appropriate pharmacopoeia standards and obtain the quantity of

plants in our natural environment (Katzung et al., 1995).

Constipation (also known as dyschezia) refers to bowel movements that are infrequent

or hard to pass. It could also be referred to as infrequent passage (less than three per week),

difficulty in expulsion or unusually hard stool, feeling of incomplete evacuation and need for

manual evacuation of stool (Chatoor and Emmanuel, 2009). Constipation is a common cause

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of painful defecation. Severe constipation includes obstipation (failure to pass stools or gas)

and fecal impaction, which can progress to bowel obstruction and become life-threatening

(Walia et al., 2009).

The genus Millettia appears in the African pharmacopeia since centuries. It has a wide

range of biological activities such as antitumoral, anti-inflammatory, antiviral, bactericidal,

insecticidal and pest-destroying (Burkill, 1995). Thus, the multiplicity of these activities,

beginning to be confirmed by pharmacological studies in laboratory, confers on this genus a

great interest in traditional medicine as well as in the research of new biologically active

compounds. (Okafor and Ham, 1999).

1.1 Millettia aboensis belongs to the family of fabacaea. It is popularly known as

“Otoroekpo or Uturuekpa” among the indigenous people of the Nsukka senatorial district of

Enugu state of Nigeria. They are perennial evergreen non-climbing trees of 30 – 40 feet high

and up to 2 feet in girth but usually 12m high with reddish-brown pubescence on the petioles,

branches, inflorescence and fruits (Burkill, 1995). They are found commonly in low land rain

forest. The flowers are purple in erect woody racemes up to 18in long. It has conspicuously

rusty-hairy leaves and handsome purple flowers in erect terminal racemes at branch (Burkill,

1995). Millettia aboensis have been used by traditional medicinal practitioners to manage

constipation, respiratory difficulties, colds and headaches (Neuwinger, 2000). The ethanol

extract of the root is also used in the study of anti-inflammatory, antioxidant and

antimicrobial activity and also macerated root in alcohol is used to treat hernias and jaundice

(Lock, 1989).

Studies have also shown that the leaf, stem and roots mixed with other plant materials

(herbs) are used to cure veneral diseases such as gonorrhea, syphilis and so on. (Neuwinger,

2000).

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Fig 1: Structural view of the plant Millettia aboensis. (Otoroekpo).

1.1.2 Scientific classification of Millettia aboensis

Millettia aboensis is a member of the family fabaceae. This dicotyledonous plant is classified

scientifically as;

Domain Eukaryota

Kingdom Plantae

Subkingdom Viridaeplantae

Phylum Tracheophyta

Subphylum Euphyllophytina

Class Spermatopsida

Subclass Rosidae

Superorder Rosanae

Order Fabales

Family Leguminosae

Subfamily Giseke

Tribe Milletieae

Genus Millettia

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Specific epithet aboensis

Botanical Name Millettia aboensis

Lock, (1989).

1.2. Phytochemistry

Phytochemicals are natural bioactive substances that are found in plants. These

natural substances have shown to work with nutrients and dietary fibers to protect animals

and man against diseases. These plant products which are derived from plant parts such as

fruits, leaves, roots, stem bark, seeds and so on have been part of phytomedicine, thereby

showing that any part of plant may contain these important active substances (Njoku and

Aku, 2007). Phytochemicals are also non-nutritive chemicals that have protective or disease

preventive property. (Harborne, 1998). Phytochemicals comprise of a number of substances

such as carotenoids (carotene, lutein, and lycopene), alkaloids (caffeine, theobromine,

theophylline etc), phenolics (flavorioids, anthocyanins, catechins) and sterols (campesterol,

sitosterol, and stigmasterol) (Akinmoladun et al., 2007).

1.2.1. Phytochemical components in plants

Phytochemical compounds of plants have shown to be formed during the plant normal

metabolic processes. These substances are often referred to as secondary metabolites such as

Alkaloids, flavonoid, Saponins, Tannins, Glycosides, Steroids, Hydrogen Cyanides,

Terpenoids, Reducing sugars, soluble carbohydrates and so on (Harborne, 1998; Okwu,

2004). The qualitative and quantitative studies of aqueous extract of Millettia aboensis leaves

showed to contain the above chemical compounds (Harborne, 1998).

Phytochemicals are naturally occurring and are believed to be effective in fighting or

preventing disease due to their antioxidant effect (Halliwell and Gutteridge, 1992). Thus, the

medicinal values of these plants depend in their component phytochemicals that produce the

definite physiological actions on the human body. The most essential of these phytochemicals

are flavonoids tannins, phenolic and alkaloids compounds (Hill, 1952)

Some of these naturally occurring phytochemicals are anticarcinogenic and while

some have other useful properties, some prevent oxidation by free radicals and therefore

known as chemopreventers. Among these chemopreventers are some plant polyphenols,

vitamins and pigments such as flavorioids, carotenoids and chlorophylls (Farombi, 2000).

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1.2.1.1. Flavonoids Flavorioids are polyphenolic compounds that are common in nature and are

categorized according to chemical structure into flavones, flavonols, flavonones, catechins,

chalcones, isoflavones and anthocyanidins. They are plant Secondary metabolites occurring

at relatively high amounts in several kinds of fruits, grains and vegetables harvested for

human consumption (Manach et al., 2004). Flavonoids are widely distributed throughout

plants and give the flowers and fruits of many plants their vibrant colours. They also play

important roles in protecting the plants from microbes and insect attacks. At the cellular level,

flavonoids have been found to exert a variety of biological effects (Middleton et al., 2000),

likely mediated by specific interaction with molecular targets. Flavonoids can be nutritionally

helpful by triggering enzymes that reduce the risk of certain cancers, heart diseases and age-

related degenerative diseases. Some studies also shows flavonoids may help to prevent tooth

decay and reduce the occurrence of common ailments such as the flu. Hence, the capacity of

flavonoids to act as antioxidants depends upon their molecular structure. The position of

hydroxyl groups and other features in the chemical structure of flavonoids are important for

their antioxidant and free radical activities. For instance, Quercetin is the most abundant

dietary flavonoid and is a potent antioxidant because of the presence of all the right structural

features for free radical scavenging activity.

1.2.1.2. Tannins

Tannins are an astringent bitter plant polyphenolic compound that binds to precipitate

proteins and various other organic compounds including amino acids and alkaloids, (Petridis,

2010). The word Tannin is widely applied to any large polyphenolic compound containing

sufficient hydroxyls and other suitable groups (such as carboxyl) to form strong complexes

with proteins and other macromolecules. These compounds are widely distributed in many

plants where they play a role in protection from predation perhaps also as pesticides and in

plant growth regulation (Katie et al., 2006). Tannins are also polymerized phenols with

defensive properties. In tanning, collagen proteins are bound together with phenolic groups to

increase the hide’s resistance to water, heat and microbes (Heldt and Heldt, 2005). Thus,

property of astringency from tannins is what causes the dry and unripe fruit or red wine

(McGee, 2004). Tannins are incompatible with heavy metals, iron, lime water, alkalis,

metallic salts, zinc sulfate, strong oxidizing agents and gelatin since they form complexes and

precipitate in aqueous solution (Bisanda et al., 2003). Many human physiological activities,

22

such as stimulation of phagocytic cells, host-mediated tumour activity and a wide range of

anti-infective actions, have been assigned to tannins (Haslam 1996)

1.2.1.3. Hydrogen cyanides

Hydrogen cyanide is an inorganic compound with chemical formal HCN. It is a

colorless extremely poisonous liquid hydrogen cyanide is a linear molecule, with a triple

bond between carbon and nitrogen. It has a bitter, faint and almond like color with some

people are unable to detect owing to a genetic character. The volatile compound has been

used as inhalation rodenticide and human poison. Cyanide ions interfere with iron-containing

respiratory enzymes (Mathews, 2004; Morocco, 2005).

The most important toxic effect of hydrogen cyanide is inhibition of the metal

containing enzymes such as cytochromoxidase. This enzyme system is responsible for the

energy providing processes in the cell where oxygen is utilized that is respiration. Thus, when

cell respiration ceases, it is no longer possible to keep normal cell functions, which may

result to cell death (Baud et al., 2002).

1.2.1.4. Alkaloids

Alkaloids are a group of natural organic bases found in plants, characterized by their

specific physiological action and toxicity used by many plants as a defense against herbivores

like insects. They are one of the most diverse groups of secondary metabolites found in living

organisms and have an array of structural types, biosynthetic pathways, and pharmacological

activities (Robert, 1998). Alkaloids may be rated to various organic bases, the most one being

quinolone, isoquinoline pyrrole, pyridine and other more complicated derivatives. Most

alkaloids are crystalline solids, others volatile liquids and some are gums. They contain

nitrogen as part of a ring, and have general properties of amines. They also include some

related compounds with neutral and even weakly acidic properties (Raj, 2004). Alkaloids

generally exert pharmacological activity particularly in mammals. Presently, many of most

commonly used drugs are alkaloids from natural sources and new alkaloid drugs are alkaloid

drugs are alkaloid drugs are still being developed for clinical use (Robert, 1998). Most

alkaloids with biological activity in humans affect the nervous system, particularly the action

of neural transmitters, e.g. adrenaline, dopamine, serotonin, acetylcholine and so on (Richard,

1999)

23

1.2.1.5. Steroids

A steroid is a type of organic compound that contains a characteristic arrangement of

four cycloalkane rings that are joined to each other. Examples of steroids are the dietary fat

cholesterol, the sex hormones estradiol and testosterone and the anti-inflammatory drug

dexamethasone. (Zollner et al., 2006). The core of steroids is composed of twenty carbon

ions bond atoms bonded together that take the form of four fused rings, three cyclohexane

rings. The steroids vary by the functional groups attached to this four-ring core and by the

oxidation state of the rings. Sterols are special forms of steroids, with a hydroxyl group at

position 3 and a skeleton derived from cholestane (Rossie, 2006). Sterols were used to be

considered to be animal substances that are similar to sex hormones but in recent years, an

increasing number of such compounds have detected in plant tissues. Sterols have important

functions in all eukaryotes. For instance, free sterols are integral components of the

membrane lipid bilayer where they play an important role in the regulation of membrane

fluidity and permeability (Corey et al., 1993). Thus, distinct of steroids are found in plants,

animals and fungi. Therefore, all steroids are made in cells either from the sterols lanosterol

(animals and fungi) or from cycloaterol. (Plants).

1.2.1.6. Saponins

Saponins are natural glycosides of steroid or triterpene which showed many different

biological and pharmacological activities. Saponins a characterized by bitter, foaming

properties, haemolytic effect on red blood cells and cholesterol binding properties (Okwu,

2005). Saponins can also activate the mammalian immune system, which have led to

significant interest in their potential as vaccine adjuvants. Thus, sapins are glycosides of

triterpenoid or steroidal aglycones with a varying number of sugar side chains. Saponins are

especially enriched in plant epidermal cells forming a protective surfactant that forms a soapy

froth when mixed with water (Wegner et al., 2002). Extracts of plants rich on saponins have

been shown to have cholesterol lowering and anticancer properties but are also known to

reduce the digestibility in ruminants and to be generally toxic to cold blooded animals and

insects (Kerwin, 2004).

24

1.2.1.7. Glycosides

Glycosides are molecules in which a sugar is bound to another functional group

through a glycosidic bond. They are also a variety of natural occurring substances in which a

carbohydrate portion, consisting of one or more sugars or an ironic acid that is a sugar acid is

combined with a hydroxyl compound. The hydroxyl compound, usually a non-sugar entity

(aglycon), such as a derivative of phenol or an alcohol, may also be another carbohydrate as

in cellulose, starch or glycogen which consist of many glucose unites thus, many glycosides

occur in plants often as fruit pigment, for instance, anthocyanins, various medicines,

condiments and dyes from plants occurs as glycosides; of great value are the heart stimulating

glycosides of digitalis and strophan, members of a group known as cardiac glycosides

(Arewang et al., 2007).

Glycosides derived from glucuronic acid (the uronic acid of glucose) and steroids are

constituents of normal animal urine. Compounds (nucleosides) derived from the partial

breakdown of nucleic acids are also glycosides. The most important synthetic enzymes in

nature are glycosyltransferases (Polakova et al., 2004).

1.2.1.8. Reducing sugars

A reducing sugar is any sugar that either has an aldehyde group or is capable of

forming one in solution through isomerism. The reducing property of a reducing sugar is due

to the presence of an aldehyde functional group. However, the cyclic hemiacetral forms of

aldoses can open to reveal an aldehyde and certain ketoses can undergo tautomerization to

become aldoses. (Campbell and Farrell, 2012).

25

1.3. Constipation

1.3.1. Definition of constipation

Constipation is referred to as infrequent bowel movements (Typically three times or

fewer per week). It could be referred to as the difficulty during defecation (Straining during

more than 25% of bowel movement or a subjective sensation of had stools) or sensation of

incomplete bowel evacuation (Chatoor et al., 2009). Constipation is a common digestive

complaint and often a chronic functional gastrointestinal disorder. The estimation revealed

that the occurrence is 2% to 20% of the population (Sonnenberg and Koch, 1989). It is also a

common clinical problem comprising a constellation of symptoms that include excessive

straining, hard stools, and feeling of incomplete evacuation or infrequent defecation (Higgins

and Johanson, 2004). Constipation is not only discomforting but also a cause of abdominal

distension, vomiting, restlessness, gut obstruction and perforation and may be associated with

aspiration or fatal pulmonary embolism (Mostafa et al., 2003). Constipation may be caused

by a number of conditions such as metabolic problems, fiber deficiency, anorectal problems,

and drugs. But the three possible causes of constipation are congenital, primary and

secondary while the most common cause is primary and is not life-threatening (Leung, 2007).

Constipation can be treated by water and fibre intake either as dietary source or as

supplements (Hsieh, 2005).

1.3.2. Constipation in children

Constipation in children usually occurs at three distinct points in time, after starting

formula or processed foods (while an infant), during toilet training in toddlerhood, and soon

after starting school (as in kinder garden) (Cohn, 2010).

Studies have shown that after birth, most infants pass 4-5 soft liquid bowel

movements (BM) a day. Thus, Breast – fed infants usually tend to have more bowel

movement compare to formula – fed infants. Some breast - fed infants have a bowel

movement after each feed, whereas others have only one bowel movement every 2 -3 days.

Hence, infants who are breast –fed rarely develop constipation (Cohn, 2010).

26

1.3.3. Types of constipation

There are two main kinds of constipation;

Occational constipation and

Chronic constipation

1.3.3.1. Occational constipation

As the name of the constipation suggests, it is a type of constipation that does not

happen every day. However, while uncomfortable, it is a short – term condition that may only

temporarily interrupt usual – routine. Thus, this type of constipation can often be relieved

through changes to diet, exercise regimen or through the use of over-the counter medications

(Chang, 2006)

1.3.3.2. Chronic constipation

Chronic constipation on the other hand, almost becomes a new routine of its own.

Moreover, most people who have chronic constipation still experiences the “typical”

symptoms – straining, hard or lumpy stool, feeling like not being empty after having a bowel

movement, but they happen on an ongoing basis (symptoms last more than 3 months).

(Chang, 2006).

1.3.4. Causes of constipation

Primary or functional constipation is ongoing symptoms for greater than six months

not due to any underlying cause such as medication side effects or and underlying medical

conditions. Thus, it is not associated with abdominal pain thus distinguishing it from irritable

bowel syndrome. It is the most common cause of constipation. (Longstreth et al., 2006).

1.3.4.1. Diet cause

Constipation can be caused or exacerbated by a low fibre diet, low liquid intake or

dieting (Bharucha, 2007).

27

1.3.4.2. Medication cause

Many medications have constipation as a side effect. Some include (but are not

limited to); opioids (e.g. common pain killers), diuretics, antidepressants, antihistamines,

antispasmodics, anticonvulsants and aluminum antacids (Lee et al., 2010).

1.3.4.3. Metabolic and muscular cause

Constipation has a number of structural (mechanical, morphological, anatomical)

causes including; spinal cord lesions, Parkinsons, colon cancer, anal fissures, proctitis and

pelvic floor dysfunction.

Constipation also has functional (neurological) causes including; animas, descending,

perineum syndrome, and Hirschsprung’s diseases. Infants, Hirschspruny’s disease is the most

common medical disorder associated with constipation. Anismus occurs in a small minority

of person with chronic constipation or obstructed defecation (Schouten et al., 1997).

1.3.4.4. Psychological cause

Voluntary withholding of the stool is a common cause of constipation. The choice to

withhold can be due to factors such as fear or pain, fear of public restrooms, or laziness.

However, when a child holds in the stool a combination of encouragement, fluids, fiber and

laxatives may be useful to overcome the problem (Longstreth et al., 2006).

1.3.5. Diagnosis of constipation

The diagnosis of constipation is essentially made from the patient’s description of the

symptoms. Thus, bowel movements that are difficult to pass, very firm, or made up of small

hard pellets (like those excreted by rabbits) qualify as constipation, even if they occur every

day, thus, other symptoms related to constipation can include bloating, distension, abdominal

pain, headaches, a feeling of fatigue and nervous exhaustion, or a sense of incomplete

emptying (Arce et al., 2002).

Moreover, inquiring about dietary habits will often reveal a low intake of dietary

fibre, inadequate amounts of fluids, poor ambulation or immobility, or medication that are

associated with constipation.

Studies also showed that during physical examination, scybala (manually palpable

lumps of stool) may be detected on palpation of the abdomen. Rectal examination gives an

impression of the anal sphincter tone and whether the lower rectum contains any faeces or

not. Rectal examination also gives information on the consistency of the stool, presence of

28

hemorrhoids, admixture of blood and whether any tumors, polyps or abnormalities are

present. Physical examination may be done manually be the physician or by using a

colonoscope, X-rays of the abdomen, generally only performed if bowel obstruction

suspected may reveal extensive impacted faecal matter in the colon and confirm or rule other

causes of similar symptoms (Barish et al., 2010).

1.3.5.1. Rome II criteria for constipation

The Rome II criteria for constipation require at least two of the following symptoms

for 12 weeks or more over the period of a year

Straining with more than one – fourth of defecations

Hard stool with more than one-fourth of defecations

Feelings of incomplete evacuation with more than one-fourth of defecations.

Sensation of anorectal obstruction with more than one-fourth of defecations

Manual maneuvers to facilitate more than one-fourth of defecations

Fewer than three bowel movements per week

Insufficient criteria for irritable bowel syndrome (Sonnenberg, 1989).

1.3.6. Prevention of constipation

Constipation is usually easier to prevent than to treat. Following the relief of

constipation, maintenance with adequate fluid intake and high fibre diet is recommended.

Children benefit from scheduled toilet breaks, once early in the morning and 30 minutes after

meals (Camilleri and Deiteren, 2010).

1.3.7. Treatment of constipation

There are many treatments for constipation and the best approach lies on a clear

understanding of the underlying cause.

1.3.7.1. Dietary fiber

The best way of adding fiber to the diet is by increasing the quantity of fruits and

vegetables that are eaten. This means a minimum of five servings of fruits or vegetables

every day

However, the amount of fruits and vegetable that are necessary may be inconveniently

large or may not provide adequate relief from constipation. In this case, fiber supplements

can be useful (Bharucha, 2007).

29

Thus, fiber is defined as a material made by plants that is not digested by the human

gastrointestinal tract. Fiber is one of the mainstays in the treatment of constipation. Many

types of fiber within the intestine bind to water and keep the water within the intestine. The

fiber adds bulk (volume) to the stool and the water softens the stool (Bharucha, 2007). There

are different sources of fiber and the type of the fiber varies from source to source. Types of

fiber can be categorized in several ways, for instance by their source. The most common

source of fiber includes: Fruits and vegetable, Wheat or oat bran.Psyllium seed (For example,

Metamucil, Konsyl). Synthetic methyl cellulose (for example Citrucel) and polycarbophil (for

example, Equilactin, Konsyl fiber).

However, the fiber should be started at a low dose and increased every one to two

weeks until either the desired effect on the stool is achieved or troublesome flatulence

interferes. Hence when increasing amounts of fiber are used, it is recommended that greater

amounts of water be consumed (for instance, a full glass with each dose). In theory, the water

prevents “hardening” of the fiber and blockage (obstruction) of the intestine (Hsieh, 2005).

1.3.7.2. Lubricant laxatives

Lubricant laxatives contain mineral oil as either the plain oil or an emulsion

(combination with water) of the oil. The oil stays within the intestine, coats the particles of

stool and presumably prevents the removal of water from the stool. This retension of water in

the stool results in softer stool (Selby, 2010).

1.3.7.3. Emollient laxatives (Stool softeners)

Emollient laxatives are generally known as stool softeners. They contain a compound

called docusate (for example, Colace). Docusate is a welting agent that improves the ability

of water within the colon to penetrate and mix with stool. This increased water within the

stool softens the stool. Although, studies have not shown docusate to be consistently effective

in relieving constipation (Emmanuel et al., 2009).

1.3.7.4. Hyperosmolar laxatives

Hyperosmolar laxatives are undigestible, unabsorbable compounds that remain within

the colon and retain the water that already is in colon. The result is softening of the stool. The

most commonly hyperosmolar laxatives are lactulose (for example kristalose), sorbitol and

polyethylene glycol (for example Miralax) and are available by prescription only. (Emmanuel

et al., 2009).

30

1.4. Biochemical parameters

1.4.1. Body electrolyte

Chemically, electrolytes are substances that become ions in solution and acquire the

capacity to conduct electricity. Thus, they are present in the human body and the balance of

electrolyte in the body is essential for normal function of the cells and organs. Electrolyte

solutions can also result from the dissolution of some biological (e.g. DNA, Polypeptides)

and synthetic polymers (e.g. Polystyrene sulfonate), termed polyelectrolytes which contain

charged functional groups.

Physiologically, the primary ions of electrolytes are sodium(Na+), potassium(K

+),

calcium(Ca2+

), magnesium(Mg2+

), chloride(Cl-), hydrogen phosphate(HPO4

2-), and hydrogen

carbonate(HCO2-

) (Kamil et al., 2011).All known higher life forms require a subtle and

complex electrolyte balance between the intracellular and extracellular environment.

Electrolyte activity between the extracellular fluid and intracellular fluid helps in activation

of muscles and neurons (Syzdek and Jaroslaw, 2000). Moreover, the maintenance of precise

osmotic gradients of electrolytes is important. Thus, such gradients affect and regulate the

hydration of the body as well as blood pH, and are critical for nerve and muscle function. In

humans, electrolyte homeostasis is regulated by hormones such as antidiuretic hormone,

aldosterone and parathyroid hormone. (Syzdek and Jaroslaw, 2010).The common electrolytes

that are measured with blood testing include; sodium, potassium, chloride and bicarbonate.

1.4.2. Depletion and absorption of sodium

Sodium is the most abundant extracellular cation and with its associated anions,

accounts for the most of the osmotic activity of the extra cellular fluid (ECF), it is important

in determining water distribution across cell membranes. Osmotic activity depends on

concentration, and therefore on the relative amounts of sodium and water in the extracellular

fluid compartment, rather than the absolute quantity of either. An imbalance between the two

causes either hyponatraemia or hypernatraemia and therefore brings about changes in

osmolality. The daily water and sodium intakes are very variable, but in an adult amount to

about 1.5 to 2 litres and 60 to 150mMol respectively (Philip, 1994). Sodium regulates the

total amount of water in the body and the transmission of sodium into and out of individual

cells also plays a role in critical body functions. Many processes in the body, especially in the

brain nervous system, and muscles, require electrical signals for communication. The

movement of sodium is critical in generation of these electrical signals. Too much or too little

31

sodium therefore can cause cells to malfunction, and extremes in the blood sodium levels (too

much or too little) can be fatal.

1.4.3. Depletion and absorption of potassium

Potassium (K+) is the most abundant intracellular cation. Only about 2% of the total

body K+ is extracellular. Since most intracellular K

+ is contained within muscle cells, total

body K+ is roughly proportional to lean body mass. An average 70kg adult has about

3500mEq of K+ (Harrison et al., 2005). The normal potassium intake is about 60 to 100mMol

a day (Philip, 1994).

1.5. Na+

/K+

-ATPase

Na+

/K+

-ATPase (sodium-potassium adenosine triphosphatase, also known as Na+

/ K

+

pump, sodium-potassium pump, or sodium pump) is an antiporter enzyme (an electrogenic

transmembrane ATPase) located in plasma membrane of all animal cells. The Na+

/K+

-

ATPase enzyme pumps sodium out of cells, while pumping potassium into cells.

1.5.1. Sodium-potassium pumps

Active transport is responsible for cells containing relatively high concentrations of

potassium ions but low concentrations of sodium ions (Tian et al., 2006). The mechanism

responsible for this is the sodium-potassium pump, which moves these two ions in opposite

directions across the plasma membrane. It is also showed that the concentrations of sodium

and potassium ions in the two sides of the membrane are interdependent, suggesting that the

same carrier transports both ions (Forrest et al., 2012). Thus, it is known that the carrier is an

ATPase and that it pumps three sodium ions out of the cell for every two potassium ions

pumped in (Forrest et al., 2012).

1.5.2. Functions of Na+

/K+

-ATPase

The Na+

/K+

-ATPase helps to maintain resting potential, avail transport, and regulate

cellular volume. It also functions as signal transducer/integrator to regulate MAPK pathway,

ROS as well as intracellular calcium. However, in most animal cells, it is responsible for

about 1/5 of the cell’s energy expenditure (Howarth et al., 2012). For neurons, the Na+

/K+

-

ATPase can be responsible for up to 2/3 of the cell’s energy expenditure (Howarth et al.,

2012).

32

1.5.2.1. Resting potential

In order to maintain the cell membrane potential, cells keep a low concentration of

sodium ions and high levels of potassium ions within the cell (intracellular). The sodium-

potassium pump moves 3 sodium ions out and 2 potassium ions in, thus in total removing one

positive charge carrier from the intracellular space. Moreover, the action of the sodium-

potassium pump is not the only mechanism responsible for the generation of the resting

membrane potential. Also the selective permeability of the cell’s plasma membrane for the

different ions plays important role (Lee et al., 2001).

1.5.2.2. Transport

Export of sodium from the cell provides the driving force for several secondary active

transporters, membrane transport proteins, which import glucose, amino acids and other

nutrients into the cell by use of the sodium gradient. Na+

-K+

pump also provides a Na+

gradient that is used by certain carrier processes. For instance, in the gut, sodium is

transported out of the reabsorbing cell on the blood (interstitial fluid) side via Na+

-K+

pump,

whereas, on the reabsorbing (luminal) side, the Na+

-glucose symporter uses the created Na+

gradient as a source of energy to import both Na+

and glucose which shows to be far more

efficient than simple diffusion (Lee et al., 2001).

1.5.2.3. Controlling cell volume

Failure of the Na+

-K+

pumps can result to swelling of the cell. A cell’s osmolarity is

the sum of the concentrations of the various ion species and many proteins and other organic

compounds inside the cell. When this is higher than the osmolarity outside of the cell, water

flows into the cell through osmosis. This can result to the cell swell up and lysis. Therefore,

the Na+

-K+

pump helps to maintain the right concentrations of ions (Li et al., 2009).

1.5.3. Mechanism of Na+

-K+

ATPase pump

The pump while binding ATP, binds 3 intracellular Na+

ions. The ATP is hydrolysed

leading to phosphorylation of the pump at a highly conserved aspartate residue and

subsequent release of ADP. A conformational change in the pump exposes the Na+

ions to

the outside. The phosphorylated form of the pump now has a low affinity for Na+

ions, and

thus they are released. The pump binds 2 extracellular K+

ions. This brings about the

dephosphorylation of the pump and reverting it to its previous conformational state

transporting the K+

ions into the cell (Skou, 1957)

33

1.6. Blood glucose

Blood sugar concentration or blood glucose level is the amount of glucose (sugar) that

is present in the blood of a human or animal (Boily et al., 2006). The body naturally tightly

regulates blood glucose levels as a part of metabolic homeostasis. Glucose is the primary

source of energy for the body’s cells, and blood lipids (in the forms of fats and oils) are

primarily a compact energy store (Eiler, 2004). Glucose is transported from the intestine or

liver to body cells through the bloodstream and is made available for cell absorption through

the hormone insulin, produced by the body primarily in the pancreas.

In the measurement of blood glucose, normally the values ranges may vary slightly

among different laboratories. Thus, many factors affect a person’s blood sugar level. A

body’s homeostatic mechanism, when operating normally, restores the blood sugar level to a

narrow array of about 4.4 to 6.1mmol/L (79.2 to 110mg/dL) as measured by a fasting blood

glucose test American Diabetes Association, (2006).

1.6.1. Regulation of blood glucose

The body’s homeostatic mechanism keeps blood glucose interacting within a narrow

range. It is composed of several interacting systems of which hormone regulation is the most

important. Thus, there are two types of mutually antagonistic metabolic hormones affecting

blood glucose levels; catabolic hormones (such as glucagon, cortisol and catecholamines)

which increase blood glucose and one anabolic hormone (insulin) which decrease blood

glucose (Comell et al., 1988).

1.6.2. Abnormality in blood glucose level

1.6.2.1. High blood sugar (Hyperglyceamia)

The high concentration of blood sugar in the blood above normal range is referred to

as hyperglyceamia. If blood sugar levels remain too high, the body suppresses appetite over

the short term. As a result of long term hyperglyceamia it causes many of the long-term

health problems such as heart disease, eye, kidney, and nerve damage (Daly et al., 1998).

1.6.2.2. Low blood sugar (Hypoglyceamia)

A situation whereby the blood sugar levels drop too low, a potentially fatal condition

called hypoglyceamia results. Thus, the following are the likely symptoms; lethargy, shaking,

twitching, weakness in arm and leg muscles, pale complexion, sweating, paranoid or

aggressive mentality and loss of consciousness (Daly et al., 1998).

34

1.7. Liver

The liver is a vital organ present in vertebrates and some other animals. It has a wide

range of functions including detoxification, protein synthesis, and production of biochemical

necessary for digestion (Cotran et al., 2005). Anatomically, it is a reddish- brown organ with

four lobes of unequal size and shapes. A human liver normally weighs 1.44-1.66kg (3.2-

3.7lb) and located in the right upper quadrant of the abdominal cavity, resting just below the

diaphragm (Kmiec, 2001).

1.7.1. Functions of the liver

The liver plays a major role in metabolism and has a number of functions in the body,

including glycogen storage, decomposition of red blood cells, plasma protein synthesis,

hormone production, and detoxification. It lies below the diaphragm in the abdominal-pelvic

region of the abdomen. It produces bile, an alkaline compound which aids in digestion

through the emulsification of lipids. The liver's highly specialized tissues regulate a wide

variety of high-volume biochemical reactions, including the synthesis and breakdown of

small and complex molecules, many of which are necessary for normal vital functions

(Maton et al., 1993). Due to the unique and consideration of the reserve capacity of liver,

even a moderate cell injury is not reflected by measurable change in its metabolic function.

Thus, some of its functions are so sensitive that abnormalities start appearing depending on

the nature and degree of initial result.

1.8. Liver markers

Liver function tests (LFTs or LFs) are groups of clinical biochemistry laboratory

blood assays designed to give information about the state of a patient’s liver (McClatchy,

2002). The liver related enzymes or liver biomarkers, aspartate aminotransferase (AST),

alkaline phosphatase (ALP), gamma glutamyl peptidyl transferase (ALT) are indirect

measures of liver homeostasis.Most liver diseases cause only mild symptoms initially, but it

is vital that these diseases can be detected early. Hepatic (liver) involvement in some diseases

can be of vital importance. Some tests are related with functionality (e.g., albumin), some

with cellular integrity (e.g., transaminase) and some with conditions linked to the biliary tract

(gamma-glutamyl transferase and alkaline phosphatase) (Nyblom et al., 2006).

35

1.8.1. Alanine transaminase

Alanine transferase is an enzyme that transfers an amino group from the amino acid

alanine to a ketoacid acceptor (oxaloacetate). The enzyme was formerly called serum

glutamic pyruvic transaminase (SGPT) after the products formed by this reaction. Although

ALT is present in other tissues besides liver, its concentration in liver is far greater than any

other tissue, and blood levels in nonhepatic conditions rarely produce levels of a magnitude

seen in liver disease. The enzyme is very sensitive to necrotic or inflammatory liver injury.

Consequently, if ALT or direct bilirubin is increased, then some form of liver disease us

likely. If both are normal, then liver disease is unlikely (Henry, 2001).

1.8.2. Alkaline phosphatase

Alkaline phosphatase is increased in obstructive liver diseases, but it is not specific

for the liver. Increases of a similar magnitude (three to four-fold normal) are commonly seen

in bone diseases, late pregnancy, leukemia, and some other malignancies. The enzyme

gamma-glutamyl transferase (GGT) is used to help differentiate the source of an elevated

ALP. GGT is greatly increased in obstructive jaundice, alcoholic liver disease, and hepatic

cancer. When the increase in GGT is two or more times greater than the increase in ALP, the

source of the ALP is considered to be from the liver. When the increase in GGT is five or

more times the increase in ALP, these points to a diagnosis of alcoholic hepatitis. GGT, but

not AST and ALT, is elevated in the first stages of liver inflammation due to alcohol

consumption, and GGT is useful as a marker for excessive drinking. GGT has been shown

to rise after acute persistent alcohol ingestion and then fall when alcohol is avoided (Wallach,

2000).

1.8.3. Aspartate transaminase

Aspartate aminotransferase, formerly called serum glutamic oxaloacetic transaminase

(SGOT), is not as specific for liver disease as is ALT, which is increased in myocardial

infarction pancreatitis, muscle wasting diseases, and many other conditions. However,

differentiation of acute and chronic forms of hepatocellular injury is aided by examining the

ratio of ALT to AST, called DeRitis ratio. In acute hepatitis, Reye’s syndrome, and infectious

mononucleosis, the ALT predominates. However, in alcoholic liver disease, chronic hepatitis

and cirrhosis, AST predominates (Lee, 2009).

36

1.9. Lipid profile

Lipid profile or lipid panel is the collective term given to the estimation of total

cholesterol, low-density lipoprotein cholesterol, triglycerides and high-density lipoprotein.

An extended lipid profile may include very low-density lipoprotein. This is used to identify

hyperlipidemia (various disturbances of cholesterol and triglyceride levels), many forms of

which are recognized risk factors for cardiovascular disease and sometimes pancreatitis.

1.9.1. High density lipoprotein

High-density lipoprotein (HDL) is one of the five major groups of lipoproteins, which

in order of sizes, largest to smallest, are chylomicrons, LDL, HDL and VLDL, which enable

lipids like cholesterol and triglycerides to be transported within the water-based bloodstream.

In healthy individuals, about thirty percent of blood cholesterol is carried by HDL (Mard-

Soltani et al., 2012).

Blood tests typically report HDL-C level that is the amount of cholesterol contained

in HDL particles. It is often contrasted with LDL cholesterol or LDL-C, HDL particles are

able to remove cholesterol from within artery atheroma and transport it back to the liver for

excretion or re-utilization, which is the main reason why the cholesterol carried within HDL

particles (HDL-C) is sometimes called “good cholesterol. Individual with higher levels of

HDL-C seem to have fewer problems with cardiovascular diseases (Lewington et al., 2007),

while those with low HDL-C cholesterol levels (less than 40mg/dL or about 1mmol/L) have

increased rates for heart disease (Lewington et al., 2007).

1.9.2. Triacylglycerol

Triglycerides are the main constituents of vegetable oil (typically more unsaturated)

and animal fats (typically more saturated). In humans, triglycerides are a mechanism for

storing unused calories, and their high concentration in blood correlates with the consumption

of starchy and other high carbohydrate food. Triglycerides are a major component of human

skin oils. Triglycerides are formed by combining glycerol with three molecules of fatty acid.

1.9.3. Low density lipoprotein

Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein, which

enable transport of multiple different fat molecules, including cholesterol, within the water

around cells and within the water-based bloodstream. Studies have shown that higher levels

of type-B LDL particles (as opposed to type-A LDL particles) promote health problems and

37

cardiovascular disease, they are often informally called the bad cholesterol particles, (as

opposed to HDL particles, which are frequently referred to as good cholesterol) (John et al.,

2007).Because LDL particles can also transport cholesterol into the artery wall, which are

retained there by arterial proteoglycans and thus attract macrophages that engulf the LDL

particles and start the formation of plaques, increased levels are associated with

atherosclerosis. Over time vulnerable plaques rupture, activate blood clothing and produce

arterial stenosis, which can result to heart attack, stroke and peripheral vascular disease

symptoms and major debilitating events, LDL particles are formed as VLDL lipoproteins lose

triglyceride through the action of lipoprotein lipase (LPL) and they become smaller and

denser (i.e. fewer fat molecules with same protein transport shell), containing a higher

proportion of cholesterol esters (Segrest et al., 2001).

1.9.4. Cholesterol

Cholesterol is important for all animal life, each cell synthesizes it from simpler

molecules, a complex 37-step process which starts with the intracellular protein enzyme

HMG-CoA reductase. However, normal and especially high levels of fats (including

cholesterol) within the blood circulation, depending on how it is transported within

lipoproteins, are strongly associated with progression of atherosclerosis. However, most

ingested cholesterol is esterified, and esterified cholesterol is poorly absorbed. The body also

compensates for any absorption of additional cholesterol by reducing cholesterol synthesis.

For these reasons, cholesterol intake in food has little, if any effect on total body cholesterol

content or concentrations of cholesterol in the blood (Lecerf and Lorgeril, 2011).

Cholesterol is recycled. Thus, the liver excretes it in a non-esterified form (via bile) into the

digestive tract. Typically about 50% of the excreted cholesterol is reabsorbed by the small

bowel back into the bloodstream. Some plants make cholesterol in very small amounts. Plants

manufacture phytosterols (substances chemically similar to cholesterol produced within

plants), which can compete with cholesterol for reabsorption in the intestinal tract, thus

potentially reducing cholesterol reabsorption. However, phytosterols are foreign to animal

cells and, if absorbed, accelerate the progression of atherosclerosis. When intestinal lining

cells absorb phytosterols, in place of cholesterol, they usually excrete the phytosterol

molecules back into the GI tract, an important protective mechanism (John et al., 2007).

38

1.10. Aim and objectives of the research

This study is aimed at evaluating the effect of aqueous extract of Millettia aboensis leaves on

Lomotil-induced constipation using Wistar albino rats.

The following objectives are designed to achieve the aim of this research:

� To determine both qualitatively and quantitatively the phytochemical composition of

the aqueous extract of Millettia aboensis leaves.

� To determine the effect of the aqueous extract of Millettia aboensis on the liver

marker enzymes.

� To determine the effect of the aqueous extract of Millettia aboensis on the serum level

of some body electrolytes

� To determine the lethal dose (acute/ sub-acute toxicity) of the aqueous extract of

Millettia aboensis.

� To determine the effect of the aqueous extract of Millettia aboensis on the serum

levels of lipid profile (HDL, LDL, Cholesterol and Triacylglyceride).

� To determine the effect of the aqueous extract of Millettia aboensis on the blood

glucose concentration.

� To determine the effect of the aqueous extract of Millettia aboensis on Lomotil

induced constipation in rat

� To determine the effect of the aqueous extract of Millettia aboensis on the transport of

glucose, sodium and potassium ions across everted rat intestine.

39

CHAPTER TWO

MATERIALS AND METHODS

2.1. Materials

2.1.1. Plant material

The leaves of Millettia aboensis were used for this research. The leaves were

collected from Imilike Enu in Udenu Local Government Area and were identified by

Mr. Alfred .E. Ozioko of Bioresources Development and Conservation Programme

(BDCP) Research Centre, Nsukka.

2.1.2. Animals

Thirty six (36) adult albino rats were used for the haematological studies and

Eighteen (18) adult albino mice were used for the acute/sub-acute toxicity (LD50)

study. All the animals used were obtained from the Animal house of Faculty of

Biological Sciences, University of Nigeria Nsukka. The rats were acclimatized for

seven days in the Department of Home Science animal house under adequate

ventilation at room temperature before the experiment. They were fed with rat pellets

(Grand cereals and oil mills Ltd, Jos Nigeria) and water.

2.1.3. Equipment

The equipment used for this experiment were obtained from Faculty of Biological

Sciences, University of Nigeria and other laboratory and scientific shops in Nsukka.

They include:

Equipment Manufacturer

Centrifuge Vickas Ltd, England

pH meter Ecoscan

Refrigerator Thermocool, England

Spectrophotometer (E312 model Jenway, UK

Sunction pump Gallenkamp, London

Water bath Gallenkamp, London

Weighing balance Vickas Ltd, England

40

2.1.4. Chemical reagents

All chemicals and reagents used for this research were of analytical grade. Standard

commercial test kits were also used for this study.

2.2. Methods

2.2.1. Experimental design

A total of thirty six Wistar albino rats were used for this study. They were

acclimatized for seven (7) days with free access to water and feed. After acclimatization, they

were divided into four main phases of the experiment. After which, in each phase the rats

were grouped and the extract was administered via oral route with the aid of incubation tube.

The groups and doses administered are summarized below;

Group 1: Rats administered 5 ml/kg of normal saline (negative control)

Group 2: Rats administered 5 mg/kg of standard drug Lomotil (positive control)

Group 3: Rats administered 100 mg/kg extract of Millettia aboensis in normal saline

Group 4: Rats administered 200 mg/kg extract of Millettia aboensis in normal saline

Group 5: Rats administered 100 mg/kg extract of Millettia aboensis before 5 mg/kg of

Lomotil

Group 6: Rats administered 200 mg/kg extract of Millettia aboensis before 5 mg/kg of

Lomotil

Group 7: Rats administered 5mg/kg of Lomotil before 100 mg/kg of extract of Millettia

aboensis

Group 8: Rats administered 5 mg/kg of Lomotil before 200 mg/kg of extract of Millettia

aboensis

2.2.2: Effect of the aqueous extract of Millettia aboensis on glucose, sodium and

potassium transport across everted rat intestine.

In this phase, the rats were divided into three groups of 2 rats each and fasted for 18

hours before they were sacrificed. The tissue used was the small intestine of the rats and

involved three stages according to the method of Wilson and Wiseman (1954)

Stage 1: Preparation of tissue

Stage 2: Preparation of everted sacs

Stage 3: Filling of the everted sacs

41

2.2.3. Reagents

Phosphate –saline: a solution of Krebs Henseleit bicarbonate (KHB) modified by Botting and

Salzmann (1974).

2.2.4. Glucose-saline

0.9% Nacl containing 0.3% glucose

Stage I. Preparation of tissue

The white Wister albino tats were killed by a blow on the head and the throat was

severed. Then the abdomen was opened by a midline incision. The small intestine was

removed by stripping the mesentery from the intestine. Thereafter, the entire small intestine

was washed with glucose saline at a room temperature to remove debris, blood and so on.

Stage II. Preparation of everted sacs

A narrow glass rod (about 1.5mm diameter) with a double thickening at the end was

inserted into one end of the intestine. A ligature was tied over the thickened part of the rod

and everted the intestine by pushing the rod gently through the entire length of the intestine.

A ligature was formed on the both ends of the everted gut with thread. The everted gut was

placed into a dish containing glucose saline for glucose and potassium transports while in the

case of sodium, it was placed into a containing Krebs-solution at room temperature

respectively. Length of intestine (3.4cm) was tied off with thread then an open end cut from

the main length.

Stage III. Filling of everted sacs

In this case, the second ligature was placed loosely round the opened of the sac. Then

a blunt needle attached to a 1ml syringe was introduced into the open end of the sac and a

loose ligature tightened over the needle. 0.6ml of the test fluid (Krebs solution) was injected

into the sac in the case of potassium transport, Glucose saline in transport of sodium and

normal saline for transport of glucose and the ligature was tied tightly while the needle was

withdrawn. The followings are the groupings for the everted rat intestine study;

Glucose transport:

Group 1: Column A containing glucose saline and B normal saline (negative control)

Group 2: Column A containing glucose saline, 250 mg/ml of metformin and B normal saline

42

Group 3: Column A containing glucose saline, 100 µg/ml extract of Millettia aboensis and B

normal saline

Group 4: Column A containing glucose saline, 200 µg/mg extract of Millettia aboensis and B

normal saline


normal saline

Sodium transport:

Group 1: Column A containing glucose saline and B Krebs solution (negative control)


Krebs solution


Krebs solution


Krebs solution

Potassium transport:

Group 1: Column A containing Krebs solution and B glucose saline (negative control)

Group 2: Column A containing Krebs solution, 100 µg/ml extract of Millettia aboensis and B

glucose saline


glucose saline


glucose saline

2.2.5. Extraction of plant material

A quantity 1350.3g of the fresh leaves of Millettia aboensis was boiled in 7 litres of

distilled water for 1hour. The solution was filtered with Whatman No1 filter paper and the

filterate was concentrated ton a semi-solid residue in an oven at 60⁰C. The percentage yield

of the extract was determined by weighing the fresh leaves of the Millettia aboensis before

extraction and the concentrated extract obtained after evaporating to dryness and was

calculated using the following formula:

43

2.2.6. Acute toxicity

The LD50 determination of the extract was done using the modified method of Lorke

(1983). The evaluation was done in two phases, the first phase; three groups of three mice

each were treated with 10, 100 and 1000 mg/kg b.w. orally of the extract, respectively.

Clinical signs, symptoms of toxic effect and mortality were observed within 24hrs. In phase

two, four groups of three mice, in group one as control and two mice each in the rest of the

groups were treated with 1600, 2900 and 5000 mg/kg b.w. orally of the extract, respectively.

Clinical signs, symptoms of toxic effect and mortality were observed within 24hrs. The

geometric mean of the consecutive doses for which 0 and 100% survival mice were recorded

in both phases. After this, the groups treated with 100, 1000 mg/kg b.w. and control were

further treated for sub-acute toxicity study.

2.2.7. Qualitative phytochemical analysis

2.2.7.1. Test for glycosides

A quantity 2.0g of aqueous extract of Millettia aboensis was mixed with 30ml of distilled

water and 15ml of dilute sulphuric acid, respectively and heated in a water bath for 5 min.

The mixture was filtered and the filterate was used for the following test; To 5ml each of

filterate, 0.3ml of Fehling’s solutions A and B was added until it turned alkaline (tested with

litmus paper) and heated on a water bath for 2 minutes. A brick-red precipitate shows the

presence of glycosides.

2.2.7.2. Test for steroids

Ethanol (9ml) was added to 1g of aqueous extract of Millettia aboensis, refluxed for a

few minutes and filtered. The filterate was concentrated to 2.5ml on a boiling water bath and

5ml of hot water was added. The mixture was allowed to stand for an hour. The filterate was

extracted with 2.5ml of chloroform using a separating funnel. 0.5ml aqueous extract in a test

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

reddish-brown interfered indicates the presence of steroids.

44

2.2.7.3. Test for tannins

A known weight, (2g) of aqueous extract of Millettia aboensis was boiled with 5ml of

45% of ethanol for 5 min. Then the mixture was allowed to cool and filtered and the filterate

was treated with the following solutions. Bromine water: 1ml of filterate was added to 0.5ml

water and a pale brown precipitate was observed. Ferric chloride solution: 1ml of filterate

was diluted with distilled water and then 2 drops of ferric chloride solution was added. A

transient greenish to black colour shows the presence of tannins. Lead sub acetate solution: to

1ml of the filterate, 3 drops of lead sub acetate solution was added. A gelatinous precipitate

indicates the presence of tannins.

2.2.7.4. Test for carbohydrate

A quantity, 0.1g of the sample was shaken vigorously with water and filtered. To the

aqueous filterate was added few drops of Molisch reagent followed by vigorous shaking

again. Then, 1ml of concentrated sulphuric acid was carefully added down the side of the test

tube to form a layer below the aqueous solution. A brown ring at the interface indicates the

presence of carbohydrate.

2.2.7.5. Test for saponins

A quantity, 0.1g of aqueous extract of Milliettia aboensis was boiled with 5ml of distilled

water for 5 min. The mixture was filtered while still hot. The filterate was used for the

following tests: Frothing test: 1ml of the filterate was diluted with 4ml of distilled water.

The mixture was shaken vigorously and then observed on standing for a stable froth.

Emulsion test: 1ml of the filterate was added to two drops of olive oil. The mixture was

shaken and observed for the formation of emulsion.

2.2.7.6. Test for alkaloids

Aqueous extract of Millettia aboensis (0.2g) was boiled with 5ml of 2% Hcl on a

steam bath. The mixture was filtered and 1ml of the filterate was treated with the

following reagents: Dragendorff’s reagents: An orange precipitate shows the presence of

alkaloids. Mayer’s reagents: A creamy-white precipitate indicates the presence of

alkaloids Wagner’s reagents: A reddish-brown precipitate indicates the presence of

alkaloids. Picric acid (1%): A yellow precipitate shows the presence of alkaloids.

45

2.2.7.7. Test for flavonoids

A quantity, 0.2g of aqueous extract of Millettia aboensis was heated with 10ml of

ethyl acetate in boiling water for 3 min. The mixture was filtered and the filterate was

used for the following tests: Aluminum chloride test: 4ml of the filterate was shaken with

1ml of 1% aluminum chloride solution and observed for light yellow colouration that

shows the presence of flavonoids. Ammonium test: 4ml of the filterate was shaken with

1ml of dilute ammonium solution to obtain two layers. The layers were allowed to

separate. A yellow precipitate observed in the ammonium layer indicates the presence of

flavonoids.

2.2.7.8 .Test for terponoids

The presence of terpenoids was determined by dissolving a little quantity of the

sample in an ethanol. Then, 1ml of acetic anhydride was added followed by addition of

concentrated of H2SO4. A change in colour from pink to violet indicates the presence of

terpenoids.

2.2.7.9. Test for reducing sugars

The presence of reducing sugar was determined by adding 5ml of a mixture of equal

parts of Fehling’s solutions A and B to 5ml each of aqueous extract of Millettia aboensis.

These were heated in a water bath for 5 min. A brick red precipitate shows the presence of

reducing sugars.

2.2.8. Quantitative phytochemical analysis

2.2.8.1. Determination of glycosides

Glycosides were determined according to the method described by Harborne (1973). A

known weight, 1g of the sample M. aboensis was macerated with 20ml of distilled water and

2.5ml of 15% lead acetate was added to the filterate, it was shaken vigorously with 2.5ml of

chloroform. The lower layer was collected and evaporated to dryness. The residue was

dissolved with 3ml of glacial acetic acid, 0.1ml of 5% ferric chloride which followed by

0.25ml of concentrated H2SO4. It was shaken and put in the dark for 2hr. The absorbance of

the solution was read at 530nm using Spectrophotometer.

46

2.2.8.2. Determination of steroids

Steroid contents were determined according to the method of Harborne (1984). A

quantity, 5g of M. aboensis was macerated in 20ml of ethanol. It was then filtered and 2ml of

chromate solution was added to 2ml of the filterate and was allowed to stand for 30min and

its absorbance was read at 550nm using Spectrophotometer.

2.2.8.3. Determination of tannins

Tannin contents were determined using the method of Pearson (1976). A quantity, 1g

of the sample Millettia aboensis was macerated in 50ml 0f methanol. 0.3ml of 0.1N ferric

chloride in 0.1N Hcl was added to 5ml of the filterate followed by 0.3ml of 0.008M

potassium ferric cyanide. The absorbance was read at 720nm within 10min using

Spectrophotometer.

2.2.8.4. Determination of soluble carbohydrate

Soluble carbohydrate was determined using the method of Harborne (1984). A known

weight, 5g of the sample M. aboensis was macerated in 50ml of distilled water. It was filtered

and 2ml of saturated aqueous solution of picric acid was added to 1ml of the filterate. The

absorbance of the solution was read at 580nm using Spectrophotometer.

2.2.8.5. Determination of saponins

Saponins were determined according to the method described by Harborne (1973). A

quantity, 5g of M. aboensis was macerated with 10ml of petroleum either twice in beaker.

The filterate was combined and evaporated to dryness. The residue was dissolved in 6ml of

ethanol. From the dissolved residue, 2ml was collected and put in a test tube, and then 2ml of

chromate solution was added to it. It was allowed to stand for 30min and the absorbance was

read at 550nm against an ethanol blank using Spectrophotometer.

2.2.8.6. Determination of alkaloids

Alkaloids were determined according to the method described by Pearson (1976). This

was done by maceratingn5g of the sample Millettia aboensis with a mixture of 20ml of

ethanol and 20% H2SO4 (1:1 v/v). 5ml of 60% H2SO4 was added to 1ml of filterate. After 5

min, 5ml of 0.5% formaldehyde in 60% H2SO4 was added to the filterate, mixed and allowed

to stand for 3hours. The absorbance was read at 565nm using Spectrophotometer.

47

2.2.8.7. Determination of flavonoids

Flavonoids of the extract were determined according to the method of Harborne

(1973). A quantity, 1g of Millettia aboensis was macerated with 20ml of ethylacetate for 5

minutes, 5ml of dilute ammonium was added to 5ml of filterate. This was shaken for 5

minutes and the upper layer was collected. The absorbance was read at 490nm using

Spectrophotometer.

2.2.8.8. Determination of terpenoids

Terpenoid content of the extract was determined according to the method described by

Harborne (1984). A quantity, 1g of the sample M. aboensis was macerated with 50mls of

ethanol. 2.5ml of aqueous phosphomolybdic acid solution and 2.5ml of concentrated H2SO4

were gradually added to 2.5ml; of the filterate and allowed to stand for 30 min and 5mls of

ethanol was added to it. The absorbance was read at700nm using Spectrophotometer.

2.2.8.9. Determination of hydrogen cyanides

Hydrogen cyanide of the sample was determined according to the method of Harborne

(1998). A known weight, 1g of the sample Millettia aboensis was macerated with 50ml of

distilled water. Then 4mls of alkaline picrate solution was added to 1ml of the filterate and

boiled for 5 min. It was allowed to cool at room temperature after which the absorbance was

read at 530nm using Spectrophotometer.

2.2.8.10. Determination of reducing sugar

Reducing sugar of the extract was determined according to the method described by

Harborne (1984).1g of the sample M. aboensis was macerated with 20ml of distilled water

and 1ml of alkaline copper reagent was added to the filterate and boiled for 5min, allowed to

cool and 1ml of phosphomolybdic acid reagent was added to the filterate followed by 7ml of

distilled water and the absorbance was read at 420nm using Spectrophotometer.

2.2.9. Biochemical parameters

2.2.9.1. Determination of total cholesterol

The cholesterol concentration was determined by the method of Allian et al. (1974) as

outlined in the Randox commercial kits.

48

Clinical significance

The measurement of cholesterol is used in the diagnosis and treatment of lipid

lipoprotein metabolism disorders. Lipid play an important role in the body; they serve as

hormones or hormones precursors, aid in digestion, provide energy, storage and metabolic

fuels, acts as structural and functional component in bio membranes and form insulation to

allow nerve conduction and prevent heat loss.

Principle

The cholesterol is determined after enzymatic hydrolysis and reaction in which the

indicator quinonemine is formed from hydrogen peroxide and 4-amino antipyrime in the

presence of phenol and peroxidase.

Cholesterol ester + H2O Cholesterol esterase cholesterol + fatty acids

Cholesterol + O2 Cholesterol oxidase cholesterol-3-one + H2O2

2H2O2 + phenol + 4-aminoantipyrine Peroxidase Quinoneimine + 4H2O

Reagents

Contents Initial concentration of solution

4-Aminoantipyrine 0.30mmol/l

Phenol 6mmol/l

Peroxidase ≥0.5U/ml

Cholesterol esterase ≥0.15U/l

Cholesterol oxidase ≥0.1U/l

Pipes Buffer 80mmol/l; pH6.8

Procedure

Three test tubes in the test tube rack were labeled reagent blank (So) standard (SI) and

sample (S2). 10µl of distilled water was pipetted into the test tube labeled (So), 10µl of the

standard reagent was pipetted into the test tube labeled (S1) while 10µl of serum sample was

added to the test tube labeled (S2). The 100µl of working reagent was added into each of the

labeled test tube (S0, S1 and S2). The reaction medium was mixed properly and incubated for

10minutes at 37⁰C. The absorbance of the sample was read against reagent blank at

wavelength 546 within 60min using Spectrophotometer.

49

Calculation

Concentration of cholesterol in sample = ∆Asample × concentration of standard

Normal values of cholesterol in human serum/plasma

Value Interpretation

<5.17mmol/l (200mg/dl) desirable blood cholesterol

5.17-6.18mmol/l (200-239mg/dl) border line-high blood cholesterol

≥6.20mmol/l (240mg/dl) high blood cholesterol

2.2.9.2. Determination of low density lipoprotein (LDL) concentration

The low density lipoprotein concentration was determined by the method of Assmann et al.

(1984) as outlined in the QCA commercial Enzyme kit.

Principle

Low density lipoprotein-cholesterol (LDL-Cholesterol) was 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

Reagent

Contents Concentration of solutions

Precipitation Reagent:

Polyvinyl sulphate 0.7g/l

EDTA Na2 5.0Mm

Polyethyleneglycol monomethyl ether 170g/l

Stabilizer

The steps used were as follows:

(1) Precipitation reaction

The precipitation solution (3 drops or 1ml) 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 min at room

temperature (20-25⁰C). Then, the mixture was centrifuged at 2, 000 rpm for 15 min

and the cholesterol concentration in the supernatant was determined.

50

(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)

2.2.9.3. Determination of triacylglycerol

The triacylglycerol concentration was determined by the method of Allian et al

(1974) as outlined in the Randox commercial kits.


The measurement of triacylglycerol is used in the diagnosis and treatment of

diseases involving lipid metabolism and various endocrine disorders e.g. nephrosis, liver

obstruction and diabetes mellitus.

Principle

The triacylglycerol assay is determined based on the enzymatic hydrolysis by the

action of lipases. Quinoneimine an indicator is formed from hydrogen-peroxide, 4-

aminophenazone and 4-chlorophenol under the catalytic influence of peroxidase.

Triglycerides + H2O Lipases glycerol + fatty acids

Glycerol + ATP GK glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 GPO dihyroxyacetone phosphate + H2O2

H2O2 + 4-aminoatipyrine + chlorophenol POD Quinoneimine + H2O

Reagents

Content Concentration in the test

Pipes buffer 40mmol/l

4-chloropphenol 5.5mmol/l

Magnesium-ions 17.5mmol/l

Enzyme reagent

51

4-aminophenazone 0.5mmol/l

ATP 1.0mmol/l

Lipases ≥150U/ml

Glycerol-kinase ≥0.4U/ml

Glycerol-3-phosphate oxidase ≥1.5U/ml

Peroxidase ≥0.5U/ml

Procedure

One vial of enzyme reagent was reconstituted with 15ml of buffer to form th reagent

mixture. Three test tubes were labeled as reagent blank (S0), standard (S1) and sample (S2).

10µl of serum sample was pipetted into sample (S2), 10µl of CAL standard was added to test

tube and 100µl of the reagent mixture was added to each of the test tube and then mixed

thoroughly. Incubate for 10 minutes at temperature 37⁰C in a water bath for 5 min. The

absorbance of the test sample and the standard was read against reagent blank at 500nm

within 60 min using Spectrophotometer.

Calculation

Triglyceride concentrations were calculated using a factor,

11.95mmol/l × ∆ Absorbance.

2.2.9.4. Determination of high density lipoprotein

The high density lipoprotein (HDL) concentration was determined by the method of

Allian et al (1974) as outlined in the Randox commercial kits.

.


High density lipoproteins (HDL) are composed of a number of heterogenic particles,

including cholesterol and vary with respect to size and content of lipid and apolipoprotein.

HDL functions to remove cholesterol from the peripheral cells to the liver where the

cholesterol is converted to bile acids and excreted into the intestine.

Principle

Low density lipoprotein (LDL and VLDL) and chylomicron fractions are lipoprotein

precipitated quantitatively by the addition of phosphotungstic acid in the presence of

52

magnesium ions. The cholesterol concentration in the high density lipoprotein (HDL) fraction

which remains in the supernatant is determined.

Cholesterol ester + H2O cholesterol esterase cholesterol + fatty acids

Cholesterol + 1/2O2 Cholesterol oxidase cholestene-3-one + H2O2

2H2O2 + phenol + 4-aminoantipyrine DCFS peroxidase quinoneimine

Reagent

Contents Initial Concentration of Solution

Phosphotungstic Acid 0.55mmol/l

Magnesium chloride 25mmol/l

Procedure

The procedure involved two steps

1 Precipitation step

Three centrifuge tubes were labeled (S1 and S2 ). 100µl of distilled water was pipetted into

the test tubes labeled (SO), 200µl of the standard reagent was pipetted into the test tube

labeled (S1) and 200µl of serum sample was also pipetted in a test tube labeled (S2). A drop

of precipitate solution reagent was added, 0.55mmol/l of phosphotungstic acid 1M

magnesium chloride was added to each of the centrifuge tubes.

2 Colorimetric step

The content of the various tubes were thoroughly mixed and incubated for 10 min at

room temperature (20-25⁰C), then centrifuged for 2 min at 12, 000rpm. The absorbance was

measured at 500nm against the reagent blank within 60 min.

2.2.9.5. Assay of Alanine Aminotransferase Activity

Alanine aminotransferase (ALT) activities were assayed by the method of Reitman

and Frankel, (1957) as described in the Randox commercial kit.

Principle

Alanine aminotransferase is measured by monitoring the concentration of pyruvate

hydrazine formed with 2, 4-dinitrophenylhydrazine.

53

α- oxoglutarate + L-alanine GPT L-glutamate + pyruvate

Reagent

Content Initial concentration of solution

Buffer

Phosphate buffer 100mmol/l, pH 7.4

L- alanine 200mmol/l

α –oxoglutarate 2.0mmol/l

2, 4-dinitrophenylhydrazine 2.0mmol/l

Procedure

Two test tubes were labeled as reagent blank (BR) and test tube (ST) placed in a test

tube rack. To 0.1ml of the serum pipetted into the test tube labeled (ST), 0.5ml of buffer

solution reagent was added to each of the test tube and then 0.1ml of distilled water was

added to the test tube labeled (BR). It was thoroughly mixed and incubated for 30 min at

37⁰C. 0.5ml of 2, 4-dinitrophenylhydrazine reagent solution was added to each of the test tube

and again mixed thoroughly and allowed to stand for 20 min at room temperature. After

which 5ml of sodium hydroxide was added to each of the test tubes, mixed and the

absorbance of the sample was read at 546nm against the reagent blank after 5 min.

2.2.9.6. Assay of Alkaline Phosphatase Activity

Alkaline phosphatase (ALP) activities were assayed by the method of Babson et al,

(1966) as outlined in the QCA commercial kit.

Principle

The principle of alkaline phosphatase is based on the reaction involving serum alkaline

phosphatase and a colourless substrate of phenolphthalein, giving rise to phosphoric acid and

phenolphthalein which at alkaline pH values, turns pink that can be spectrophotometrically

determined.

Reagents

Contents Concentration in the reagent solution

2-Amino-2-methyl-1-propanol pH 11 7.9M

54

Phenolphthalein monophosphate 63mM

Na2HPO4 80mM

Method

Two test tubes labeled as sample (SA) and standard (ST). To 1ml of distilled water pipetted

into each of the test tubes, a drop of substrate reagent was added, mixed and incubated for 5

min at 37⁰C. 0.1ml of standard reagent was added to the test tube labeled (SA), mixed and

incubated for 20 min at a temperature of 37⁰C. After which 5ml of colour developer was

added to each of the test tubes. The absorbance was read at 550nm using Spectrophotometer.

Calculations

SA Abs. × 30 = U/L of Alk. Phosphatase

ST Abs.

Normal values for alkaline phosphatase in the human blood;

Adults: 9-35U/L

Children: 40-90U/L

2.2.9.7. Assay of Aspartate Aminotransferase Activity

Aspartate aminotransferase (AST) activities were carried out by the method of

Reitman and Frankel (1957) as outlined in the Randox commercial kit.

Principle

Aspartate aminotransferase is measured by monitoring the concentration of

oxaloacetate hydrozone formed with 2, 4-dinitrophenylhyrdazine.

Reagent

Contents Initial concentration of solution

Buffer

Phosphate buffer 100mmol/l, pH 7.4

L-aspartate 100mmol/l

α- oxoglutarate 2mmol/l

2, 4-dinitrophenylhydrazine 2mmol

55

Method

The blank and sample test tubes were set up in duplicate. 0.1ml of serum was pipetted

into the sample tubes. 0.5ml of reagent 1 was pipetted into both sample and blank tubes. The

mixtures were thoroughly mixed and incubated for exactly 30 min at 37⁰C and pH 7.4. 0.5ml

of reagent 2 containing 2, 4-dinitrophenylhydrazine was added into all the test tubes followed

by 0.1ml of sample into the blank tubes. The tubes were mixed thoroughly and incubated for

exactly 20 min at 25⁰C. 5.0ml of sodium hydroxide solution was then added to each tube and

mixed. The absorbance was read against the blank after 5 min at 546nm using

Spectrophotometer.

2.2.9.8. Blood glucose concentration

Determination of blood glucose was carried using Accu-chek advantage glucometer.

Principle

Glucose in the blood sample is oxidized in the reactive zone of the strip to gluconolactone.

During this reaction, oxidized hexacyanoferrate (3) is reduced to hexacyanoferrate (2). A

voltage is applied between the two palladium electrodes contained in the strip. This leads to

the hexacyanoferrate (2) being reoxidized to hexacyanoferrate (3) with release of electrons.

The small electric current that is generated is measured by the meter and converted into a

glucose concentration reading.

2.2.9.9. Serum concentration of Sodium Ion

Serum concentration of sodium ion was carried out by the method of Terri and Sesin,

(1958) as described in the TECO DIADNOSTIC kit.

Principle

The present method is based on modification of the method described by Maruna and

Trinda, in which the sodium is precipitated as the triple salt, sodium magnesium uranyl

acetate, with the excess uranium then being reacted with ferrocyanide, producing a

chromophore whose absorbance varies inversely as the concentration of sodium in the test

sample.

56

Reagent composition

Filtrate Reagent: Uranyl Acetate 2.1mM and Magnesium Acetate 20mM in ethyl alcohol.

Acid Reagent: A diluted acetic acid. Sodium Color Reagent: Potassium Ferrocyanide,

non-reactive stabilizers, and fillers. Sodium Standard: Sodium Chloride

solution.150mEq/L of sodium

Procedure

Filtrate Preparation

The test tubes were labeled: blank, standard, control, sample etc. 1.0 ml of Filtrate Reagent

was added to all tubes. 50µl of sample was added to all tubes and distilled water to the blank.

All tubes were shacked vigorously and mix continuously for 3 min. The tubes were

centrifuged at high speed for 10 min and the supernatant fluid was tested as described below;

Colour Development

The test tubes were labeled corresponding to the above Filtrate tubes. 1.0ml Acid Reagent

was added to all tubes. 50µl of Supernatant was added to the respective tubes and mixed.

Followed by 50µl of Color Reagent to all tubes and mixed. The colorimeter was zeroed with

distilled water at 550nm

1. The absorbance of all the tubes was read and recorded.

CALCULATIONS

Abs. of Blank – Abs. of S × Conc. of STD (mEq/L) = Conc. of S 5(mEq/L)

Abs. of Blank – Abs. of STD

2.2.9.10. Serum concentration of Potassium Ion

Serum concentration of potassium ion was carried out by the method of Terri and

Sesin, (1958) as described in the TECO DIADNOSTIC kit.

Principle

The amount of potassium is determined by using solution tetraphenylboron in a

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

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

57

Reagent composition

Potassium reagent: Sodium Tetraphenylboron 2.1mM, preservatives and thickening agents.

Potassium Standard: Equivalent to 4mEq/L.

Procedure

The test tubes were labeled: standard, control, test samples, etc. A blank is necessary. 1.0ml

of potassium Reagent was added to all tubes. 0.01ml (10µl) of samples was added to the

respective tubes. It was mixed and allowed to stand at room temperature for 3 min. After 3

min, the wavelength of spectrophotometer was set to 500 nm, spectrophotometer was zeroed

with reagent blank. The absorbance of all tubes was read and recorded.

58

CHAPTER 3

RESULTS

3.1. Extraction Yield The percentage yield of aqueous extract of Millettia aboensis was 5.13%.

3.2. Acute toxicity test In the median lethal dose (LD50) test for the plant extract, no mortality or any

observable behavioural change of the animals used in this study was recorded up to

5000mg/kg body weight of the extract.

3.3. Qualitative and Quantitative Phytochemical Composition of aqueous extract of M.

aboensis The qualitative and quantitative phytochemical composition of aqueous extract of

M. aboensis showed relatively high concentration of bioactive compounds such as reducing

sugar, tannins and flavonoids (Table 1 and 2). The alkaloids, steroids and glycosides were

moderately present in concentration, while the soluble carbohydrates, saponins and hydrogen

cyanides were slightly present in low concentration and terpenoids were not detected.

Table 1: Qualitative phytochemical composition of aqueous extract of M. aboensis.

Phytochemical Bioavailability

Reducing sugar +++

Tannins +++

Flavonoids +++

Alkaloids ++

Steroids ++

Glycosides ++

Soluble carbohydrates +

Saponins +

Hydrogen cyanides +

Terpenoids -

Bioavailability Key - = Not detected

+ = present in low concentration

++ = present in moderately high concentration

+++ = present in very high concentration

59

Table 2: Quantitative phytochemical composition of aqueous extract of M. aboensis.

Phytochemical Bioavailability

Reducing sugar 38.35±0.45

Tannins 18.02±0.35

Flavonoids 5.35 ± 0.28

Alkaloids 4.69 ± 0.36

Steroids 3.31 ± 0.31

Glycosides 4.87 ± 0.32

Soluble carbohydrates 2.87 ± 0.31

Saponins 1.81 ± 0.55

Hydrogen cyanides 1.77 ± 0.25

60

3.4. Toxicological Effect of M. aboensis Extract on Aspartate Amino transferase Activity

in Mice.

Fig. 2 shows significant increase (p<0.05) in the AST activities of groups 2 and 3 mice fed

100 and 1000 mg/kg b.w of the extract respectively compared with the AST activities of mice

in group 1. However, non-significant difference (p>0.05) was observed in the AST activities

of the test mice in groups 1 and 3.

61

Fig. 2: Toxicological effect of aqueous extract of Millettia aboensis leaves on aspartate

aminotransferase activity in mice

Group1= Normal Control

Group2= 100mg/kg b.w. of aqueous extract of Millettia aboensis leaves


62

3.5. Toxicological Effect of M. aboensis Extract on Alanine Amino transferase Activity

in Mice

Alanine aminotransferase (ALT) activities increased significantly (p<0.05) in the treated

mice under groups 2 and 3 administered 100 and 1000 mg/kg b.w. of aqueous extract of

Millettia aboensis leaves respectively compared with that of normal control mice as shown in

Fig.3. In the same vein, significant increase (p<0.05) was noticed in the AST activities of

group 2 mice compared with that of group 3.

63

Fig 3: Toxicological effect of aqueous extract of Millettia aboensis leaves on alanine

aminotransferase activity in mice




64

3.6. Toxicological Effect of M. aboensis Extract on Alkaline phosphatase Activity in

Mice

.Non-significant increase (p>0.05) was recorded, as shown in Fig. 4, in the activities of

alkaline phosphatase (ALP) of mice (group) fed 100 mg/kg b.w. of the extract compared with

the ALP activities of the control mice in group 1. Conversely, non-significant decrease

(p>0.05) was observed in the ALP activities of group 3 mice administered 1000 mg/kg b.w.

of the extract compared with that of the normal control mice.

65

Fig. 4: Showing the toxicological effect of AEMA on Alkaline phosphatase activity in

mice




AEMA= Aqueous Extract of Millettia aboensis

66

3.7. Toxicological Effect of M. aboensis extract on total cholesterol concentration in mice

Non-significant decrease (p>0.05) was observed as shown in Fig 5, in the serum level of total

Cholesterol concentrations of the mice in groups 2 and 3 administered 100mg/kg and

1000mg/kg b.w of the extract compared with the total Cholesterol concentrations of the

normal control mice in group 1.

67




68

3.8. Effect of M. aboensis extract on low density lipoprotein concentration in mice

From Fig 6, Non-significant decrease (p>0.05) was recorded in the low density lipoprotein

concentrations of mice in groups 2 and 3 fed with 100mg/kg and 1000mg/kg b.w. of the

extract compared with the LDL concentrations of the control mice in group 1.

69




70

3.9. Toxicological Effect of M. aboensis extract on high density lipoprotein concentration

in mice

Fig 7, showed non-significant decrease (p>0.05) in the high density lipoprotein

concentrations of mice (group 2) fed 100mg/kg b.w. of the aqueous extract of Millettia

aboensis compared with the HDL concentrations of the control mice in group 1. In the same

vein, non-significant increase (p>0.05) was observed in the HDL concentrations of group 3

mice fed 1000mg/kg b.w. of the extract compared with that of the normal control mice.

71




72

3.10. Toxicological Effect of M. aboensis extract on triacylglycerol concentration in mice

Non-significant decrease (p>0.05) was recorded as shown in Fig 8, in the concentrations of

triacylglycerol of mice (group 2) administered 100mg/kg b.w of the extract compared with

the TAG concentrations of the control mice in group 1. On the other hand, non-significant

increase (p>0.05) was observed in the TAG concentrations of group 3 mice administered

1000mg/kg b.w. of the extract compared with that of the control mice in group 1.

73




74

3.11. Toxicological Effect of M. aboensis extract on potassium ion concentration in mice

From Fig 9, non-significant increase (p>0.05) was recorded in the serum level of potassium

ion concentrations in groups 2 and 3 mice fed 100mg/kg and 1000mg/kg b.w. of the aqueous

extract compared with that of the mice in the normal control (group 1).

75



Group3= 1000mg/kg b.w. of aqueous extract of Millettia aboensis leave

76

3.12. Toxicological Effect of M. aboensis extract on sodium ion concentration in mice

Fig. 10 shows dose-dependent significant increase (p<0.05) in the concentrations of serum

sodium ion (Na+) of mice across the groups compared to the control group 1.

77




78

3.13. Toxicological Effect of M. aboensis extract on glucose concentration in mice

Non-significant decrease and increase (p>0.05) were observed in the blood glucose

concentrations of groups 2 and 3 mice administered 100mg/kg and 1000mg/kg b.w. of the

extract respectively compared with the blood glucose concentration of the normal control

mice in group 1.

79

0

20

40

60

80

100

120

Group 1 Group 2 Group 3

Me

an

Glu

co

se

Co

nc (

mm

ol/L

)

Treatment Group

Fig. 11: Toxicological effect of aqueous extract of Millettia aboensis leaves on serum

glucose concentration in mice



Group3= 1000mg/kg b.w. of aqueous extract of Millettia aboensis leave

80

Table 3: Effects of different doses of the aqueous extract of Millettia aboensis on the

frequency of defecation of lomotil- induced constipation in rats.

Mean value

Percentage

of defecation

Groups Number of Stools after enhancement

(%)

1st hour 2

nd hour 3

rd hour 4

th hour

1 0.25±0.50 0.00±0.00 0.00±0.00 0.00±0.00 0.0625 3.45

2 0.25±0.50 0.00±0.00 0.00±0.00 0.00±0.00 0.0625 3.45

3 0.75±0.50 0.00±0.00 0.00±1.00 0.00±0.00 0.3125 17.25

4 0.25±0.50 0.25±0.50 1.50±1.00 0.25±0.50 0.5625 31.03

5 0.25±0.50 0.50±1.00 0.00±0.00 0.00±0.00 0.1875 10.34

6 0.00±0.00 1.75±1.50 0.00±0.00 1.00±1.41 0.4375 24.14

7 0.00±0.00 0.25±0.50 0.00±0.00 0.00±0.00 0.0625 3.45

8 0.00±0.00 0.25±0.50 0.25±0.50 0.00±0.00 0.125 6.91

Group 1= 5ml/kg Normal saline (Negative control)

Group 2= 5mg/ml of lomotil (Positive control)

Group 3= 100mg/kg of extract

Group 4= 200mg/kg of extract

Group 5= 100mg/kg of extract+ 5mg/ml of lomotil

Group 6= 200mg/kg of extract + 5mg/ml of lomotil

Group 7 = 5mg/ml of lomotil + 100mg/kg of extract

Group 8 = 5mg/ml of lomotil + 200mg/kg of extract

81

Table 3. The above table shows a dose –dependent increase (P<0.05) on the percentage

enhancement of the faecal droppings across the rats treated groups (4, 6, 3, 5, 8 and 7) fed

200mg/kg and 100mg/kg b.w. of the extract respectively compared with the normal negative

and positive control groups (1 and 2). In other words, the extract was able to ameliorate

constipation on the lomotil-induced rats by increasing the number of faecal droppings.

82

3.14. Effect of M. aboensis extract on glucose transport across everted rat intestine.

A significant increase (p<0.05) was recorded as shown in Fig. 12, on the influx of glucose

transport of rats treated groups (3,4 and 5) administered 100µg/ml, 200µg/ml and 400µg/ml

of the aqueous extract compared with the glucose transport of the control rats in group 1. On

the other hand, a significant decrease (p<0.05) was observed on the glucose transport of

group 2 rats fed 250mg/ml of metformin compared with that of the positive normal control

rats group 1.

83

Fig 12. Effect of aqueous extract of Millettia aboensis on the transport of glucose

concentration of rats’ intestine


Group2= 250mg/ml of Metformin

Group3= 100µg/ml of aqueous extract of Millettia aboensis leave

Group4= 200µg/ml of aqueous extract of Millettia aboensis

Group5= 400µg/ml of aqueous extract of Millettia aboensis

84

3.15. Effect of M. aboensis extract on sodium transport across everted rat intestine.

A significant increase (p<0.05) was recorded as shown in Fig. 13, on the influx of sodium


of the aqueous extract compared with the sodium transport of the control rats in group 1.

85

Fig 13. Effect of aqueous extract of Millettia aboensis on the transport of sodium



Group2= 100µg/ml of aqueous extract of Millettia aboensis leaves



86

3.16. Effect of M. aboensis extract on potassium transport across everted rat intestine.

A significant increase (p<0.05) was observed as shown in Fig. 14, on the efflux of potassium


of the aqueous extract compared with the potassium transport of the control rats in group 1.

87

Fig 14. Effect of aqueous extract of Millettia aboensis on the transport of potassium






88

CHAPTER FOUR

DISCUSSION

In the recent years, there has been an increasing cognizance of the usefulness of

medicinal plants in the treatment and management of disease conditions. The plant kingdom

is paragon house of potential drugs. Drugs from plants are readily available, safe, less

expensive and efficient. Some plants have been selected for medicinal uses and therefore

constitute the most obvious choice in the present search for therapeutically effective agents.

New drugs synthesized from plant include: antimicrobial drugs, anti-inflammatory and

antihepatotoxic drugs (Arunkumar and Muthuselvam, 2007), anticancer drugs, (Philipson and

Wright, 1996). The use of herbal preparation by trado-medicinal practitioners that are not

scientifically proven for the treatment of diseases is very common in some rural areas. Hence,

it is therefore very important to use experimental screening method in order to find out the

true information about the safety and efficacy of these products and to establish the active

component of these herbal medicaments. In this research, studies were carried out to

investigate the use of aqueous extract of Millettia aboensis leaves for the treatment of

constipation using animals. Results showed no death on an oral LD50 of 5000 mg/kg body

weight which indicates that the extract is relatively safe at this level of dose administration.

Qualitative and quantitative phytochemical constituents of the plant leaves were

investigated. The results of the preliminary phytochemical analyses revealed the presence of

soluble carbohydrates, reducing sugars, tannins, saponins, flavonoids, terpenoids, alkaloids,

hydrogen cyanides, steroids and glycosides. The qualitative phytochemical analysis showed

relatively high concentration of tannins, flavonoids and reducing sugar. Moderate presence of

alkaloids, steroids, glycosides in the extract. Soluble carbohydrate, saponins and hydrogen

cyanides were detected at low concentration, terpenoids were not detected. Flavonoids

(5.35±0.28mg/100g) have been shown to exhibit their actions through effects on membrane

permeability and by inhibition of membrane bound enzymes such as the ATPase and

phospholipase A2 (Li et al., 2003) and this property may explain the mechanisms of

antioxidative action of M. aboensis. Flavonoids serve as health promoting compound as a

result of its active radicals scavenging potential (Hausteen, 1983). Flavonoids are

hydroxylated phenolic substances known to be synthesized by plants in response to microbial

infection and they have been found to have antimicrobial activity against a wide array of

microorganisms. Their activity is probably due to their ability to complex with extracellular

and soluble proteins and to complex with bacterial cell wall (Marjorie, 1996). They are also

89

effective antioxidants and show strong anticancer activities (Salah et al., 1995). Their

antioxidant activity helps in the removal of oxidant free radicals (Cai et al., 2004) which may

be the reason for protection against cardiovascular and degenerative diseases. Tannins are

important astringent bitter plant polyphenolic compound that binds to and precipitate proteins

and various other organic compounds such as amino acids and alkaloids. Tannins play a role

in protection from predation, perhaps also as pesticides and in plant growth regulation

(Kadam et al., 1990). Tannins are known to be useful in the treatment of inflamed or

ulcerated tissues and they have remarkable activity in cancer prevention (Ruch et al., 1989).

M. aboensis showed high concentration of tannins with a mean value of 18.02±0.35mg/100g.

The results also revealed the leaf extract to contain high concentration of reducing sugars

with a value of 38.35±0.45mg/100g. Reducing sugar has an aldehyde group or is capable of

forming one in solution through isomerism. The aldehyde group allows the sugar to act as a

reducing agent. The aldehyde can be oxidized through a redox reaction in which another

compound is reduced during oxidation of aldoses. Reducing sugar helps to reduce certain

oxidizing agents (Campbell and Farrell, 2012). The results indicated that the leaves possess

some biologically active compounds which could serve as potential sources of drugs and their

phytochemicals may exert some biological activities when ingested by animals.

Millettia aboensis aqueous extract had a moderate concentration of alkaloids with a

value of 4.69±0.36mg/100g which has been associated with medicinal uses for periods.

Alkaloids are known for their analgesic, antibacterial and antispasmodic properties

(Harborne, 1974). They often have pharmacological effects and are used as medications,

recreational drugs, anticancer compounds, antihypertension agents, and antiasthmatic

therapeutics, etc. They act on a diversity of metabolic systems in humans and invoke a bitter

taste (Rhoades, 1979) which may be responsible for the protection against predator and

parasite. However, they inhibit certain mammalian enzyme activities such as

phosphodiesterase; thus prolonging the action of cyclic AMP (cAMP) (Okaka et al., 1992).

The aqueous extract was shown to contain glycosides with a mean of 4.87±0.32mg/100g.

Glycosides play numerous important roles in living organisms. Many plants store chemicals

in the form of inactive glycosides which can be activated by enzyme hydrolysis causing the

sugar part to be broken off, making the chemical available for use (Brito-Arias, 2007) and

glycosides-containing plants can be used as medication. Glycosides are known to lower the

blood pressure and heart performance (Nyarko and Addy, 1990). The presence of glycosides

in this plant could make it useful in the treatment of constipation as one of the classes of

glycosides anthraquinone glycosides have been reported to have a laxative property (Brito-

90

Arias, 2007). The extract analysis confirmed the presence of saponins (1.81±0.55mg/100g).

Saponins are glycosides with a distinctive foaming characteristic which are found in many

plants. They are known to produce inhibitory effects on inflammation (Just et al., 1998).

Some of the characteristics of saponins include formation of foams in aqueous solutions,

haemolytic activity, bitterness and cholesterol-binding properties (Sodipo et al., 2000; Okwu,

2004 a), they are major ingredients in traditional Chinese medicine and thus are responsible

for most of the observed biological effects (Liu and Henkel, 2002). The results obtained in

this study, showed that aqueous extract of Millettia aboensis little or no effect toxicological

effect on the studied indices and thus suggest that the identified phytochemical compounds

may be the bioactive constituents. This plant is proving to be an increasingly valuable

reservoir of bioactive compounds of substantial medicinal importance.

The management of activities of marker or diagnostic enzymes in tissues plays a

significant and well-known role in diagnosis, disease investigation and in the assessment of

drug or plant extract for safety/toxicity risk. The enzymes considered in this research are

useful marker enzymes of liver cytolysis and damage to the plasma membrane of the liver

cells.

The liver marker enzymes assayed after 7 days of treatment showed a significant

increase (p<0.05) in the AST and ALT activities of groups 2 and 3 rats compared with that of

the normal control rats as shown in Figs 1 and 2, respectively. The significant increase of 100

and 1000 mg/kg body weights of the aqueous extract of Millettia aboensis could be due to de

novo synthesis of the enzyme molecules or an adaptation by the liver to the assault from the

plant extract leading to the activities higher than the normal. Studies by (Conigrave et al.,

1995; Edwards et al., 1997) have shown that persistent increase of serum ALT, ALP and

AST activities are reliable markers for hepatotoxicity. In other words, the results of ALT and

AST activities showed that the aqueous extract of Millettia aboensis leaves could have a

negative effect on the hepatocytes when there is consistent administration of the extract.

However, alkaline phosphatase is considered a maker enzyme for the plasma membrane and

endoplasmic reticulum. The non-significant differences in the serum activity of ALP in

groups 2 and 3 showed that the extract did not have a negative effect on the hepatocytes.

Electrolytes are substances that become ions in solution and acquire the capacity to

conduct electricity. They are present in the human body and their balance in the body is very

essential for normal functions of the cells and organs. In this study, the concentration of

serum levels of sodium and potassium were analysed in mice on administration of the

aqueous extract of Millettia aboensis after seven days of treatment. The results of the sodium

91

showed a non-significant increase in the group administered 100mg/kg body weight of the

mice and significant increase in the group administered 1000mg/kg as compared to the

control group. This implies that the increase in sodium concentration is dependent on the

dosage increament and also that the extract could be used for anti-constipation. However, the

results for potassium showed a non-significant increase in groups administered with 100 and

1000mg/kg of body weight of the mice compared to the control groups. In other words, this

implies that the extract has no negative effect on the body electrolyte levels of the animals.

Assessment of plasma lipid profile is required for the state of wellbeing of every

individual as cardiovascular parameters can be used to determine how toxic a compound can

be to the blood parameters. Some phytochemicals may have deleterious effects on the blood

cells and to this end, it was necessary to determine the effect of the aqueous leaves extracts of

Millettia aboensis on haematological parameters (lipid profile). It was found that there was a

dose-dependent change in lipid profile parameters. From the study, it was observed that

1000mg/kg body weight of extract showed non-significant decrease on the low density

lipoprotein cholesterol concentration while a dose of 1000mg/kg body weight of extract

showed non-significant increase on the high density lipoprotein concentration. However, a

dose of 100mg/kg body weight of the extract showed a non-significant increase on the

triacylglycerol while a dose of 1000mg/kg body weight of extract showed a non-significant

decrease on the cholesterol concentration. Therefore, this dose sensitive alteration of serum

lipid of the extract could be justified that the extract was not toxic to the lipid profile but

rather could be used in relieving some cardiovascular associated diseases. This was in

accordance with the research work done by Nwankwo and Omodamiro, 2013. It has been

established that hypercholesterolemia is a risk factor for cardiovascular diseases such as

atherosclerosis and myocardial infarction which are causes of morbidity and mortality. The

presence of flavonoids has been shown to have the ability to scavenge free radicals which

help to prevent cardiovascular diseases by interfering with the oxidation of LDL which is one

of the chief engineers of artherosclerosis. According to Nwankwo and Omodamiro, (2013),

the leaf extract of V. africana showed an increase in the concentration of TAG which implies

that the plant had the ability to increase the rate of lipid breakdown; lipolysis leading to the

accumulation of TAG’s. However, extract of Millettia abeonsis leaves was in contrast to this

finding.

Moreover, the study of the effect of the aqueous extract on M. aboensis leaves on

lomotil induced-constipation on rats showed an increase on the frequency of stooling on the

lomotil+AEMA (aqueous extract of Millettia aboensis) when compared to the untreated,

92

thereby showing the effectiveness of the extract on treatment of constipation. This result was

in accordance with Kim et a., 2013 who worked on the laxative effect of L .platyphylla on

loperamide-induced constipation of SD rats. However, the AEMA caused a reduction of key

factors level on the muscarinic acetylcholine receptors (MAchRs) signaling pathway in

Lomotil+AEMA treated groups relative to the Lomotil+vehicle treated group. Also, AEMA

was able to increase GIT motility and stooling frequency without induction of diarrhoea as a

side effect. However, the presence of secondary metabolites present could be responsible for

the biological activities observed and the increase of the intestinal motility could be useful in

the treatment of constipation. Also, the presence of tannins in the plant M. aboensis which

have astringent property may decrease purgative action of anthraquinone glycosides thereby

rendering the laxative effect mild.

The sodium-potassium pump is a form of active transport in that it uses ATP to

“pump” 3 sodium ions (3Na+) out of the cell (against the flow of diffusion) and 2 potassium

ions (2K+) into the cell (also against the flow of diffusion). The sodium-potassium pump is

important in the movement of ions across cell membranes of muscle cells (to help muscle

contraction) and also for creating charge imbalances across the cell membranes of nerve cells

(generating electrical impulses).

In this work, the research was carried out to ascertain the effect of aqueous extract of

Millettia aboensis on transport of glucose, sodium and potassium ions across everted rat

intestines. The study showed an increase in the influx of glucose and sodium. In other words,

there was a transport of glucose and sodium into the serosal compartment. The mechanism of

transport of molecules across membrane showed glucose and sodium to have a symport

mechanism of transport. Hence, this may presume that some phytochemical components of

the leave extracts of Millettia abeonsis may have triggered some glucose carriers (protein

symbol GLUT) example GLUT2 a membrane carrier protein located in the basolateral

membrane of small intestine and also the Na+-ATPase molecule to bring about the transport

of these molecules across the intestinal membrane. However, there was a significant increase

in the efflux of potassium that is transport of potassium into the mucosal compartment. Thus,

this may also be as a result of bioactive compounds in the extract that may have likely

triggered the mechanism of Na+-K

+ pump in the antiport mechanism of movement of these

ions across the intestinal membrane.

93

4.2. Conclusion

Aqueous extract of Millettia aboensis leaves have been claimed by the folk medicine in

treatment of constipation. This research was carried out to ascertain this claim. Following the

effects exhibited by the extract in the enhancement of the transport of glucose, sodium and

potassium ions across the intestinal membrane of the rats and as well significant increase in

the frequency of faecal droppings on the extract treated groups in lomotil-induced

constipation in rats, this may prove that the extract contains some bioactive compounds that

have the properties of laxative effects when administered and therefore support the claim that

this plant is used in folk medicine for the treatment of constipation.

4.3. Suggestions for further studies

The effects of aqueous extract of the leaves of Millettia aboensis on lomotil-induced

constipation was investigated in this study and it is therefore suggested that further studies be

done on

� Elucidating the nature of the active constituents in the plant (roots, stems, etc.)

and assay them to determine the mechanism of actions.

� A comparative study of both aqueous and methanol extract of M.aboensis

could also be investigated. This will help to know the solvent that may have a

significant effect on gastrointestinal disorder (constipation).

� The use of purified forms of the aqueous extract of the Millettia aboensis on

lomotil-induced constipation.

� Identifying the particular phytochemical constituents responsible for the

effects of aqueous extract of Millettia aboensis leaves on lomotil-induced

constipation

94

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APPENDICES

Oneway {Toxicological Studies}

Appendix I: Descriptives

N Mean

Std.

Deviation

Std.

Error

AST Group 1=Normal

Control

3 132.6667 17.92577 10.34945

100 mg/kg b.w. of

Extract

3 170.0000 6.00000 3.46410

1000 mg/kg b.w. of

Extract

3 163.3333 5.77350 3.33333

Total 9 155.3333 19.87461 6.62487

ALT Group 1=Normal

Control

3 76.6667 12.85820 7.42369

100 mg/kg b.w. of

Extract

3 183.3333 9.86577 5.69600

1000 mg/kg b.w. of

Extract

3 119.3333 6.11010 3.52767

Total 9 126.4444 47.29459 15.76486

ALP Group 1=Normal

Control

3 34.3333 3.21455 1.85592

100 mg/kg b.w. of

Extract

3 36.3333 1.52753 .88192

1000 mg/kg b.w. of

Extract

3 30.0000 2.00000 1.15470

Total 9 33.5556 3.46811 1.15604

Cholesterol Group 1=Normal

Control

3 3.8667 .56862 .32830

100 mg/kg b.w. of

Extract

3 3.0333 .20817 .12019

1000 mg/kg b.w. of

Extract

3 3.0667 .66583 .38442

Total 9 3.3222 .60782 .20261

HDL Group 1=Normal

Control

3 .4000 .10000 .05774

100 mg/kg b.w. of

Extract

3 .3000 .00000 .00000

1000 mg/kg b.w. of 3 .4000 .00000 .00000

106

Extract

Total 9 .3667 .07071 .02357

LDL Group 1=Normal

Control

3 2.7333 .40415 .23333

100 mg/kg b.w. of

Extract

3 2.0333 .20817 .12019

1000 mg/kg b.w. of

Extract

3 1.8667 .64291 .37118

Total 9 2.2111 .56001 .18667

TAG Group 1=Normal

Control

3 1.4333 .20817 .12019

100 mg/kg b.w. of

Extract

3 1.3000 .20000 .11547

1000 mg/kg b.w. of

Extract

3 1.5667 .20817 .12019

Total 9 1.4333 .21213 .07071

Sodium Group 1=Normal

Control

3 190.0000 27.83882 16.07275

100 mg/kg b.w. of

Extract

3 230.0000 20.00000 11.54701

1000 mg/kg b.w. of

Extract

3 300.0000 70.00000 40.41452

Total 9 240.0000 61.99798 20.66599

Potassium Group 1=Normal

Control

3 12.5333 2.27450 1.31318

100 mg/kg b.w. of

Extract

3 14.3333 .11547 .06667

1000 mg/kg b.w. of

Extract

3 15.0667 2.72274 1.57198

Total 9 13.9778 2.10344 .70115

Glucose Group 1=Normal

Control

3 104.6667 13.05118 7.53510

100 mg/kg b.w. of

Extract

3 92.0000 13.11488 7.57188

1000 mg/kg b.w. of

Extract

3 109.3333 3.51188 2.02759

Total 9 102.0000 12.20656 4.06885

107

Descriptives

95% Confidence Interval for

Mean

Minimu

m

Maximu

m

Lower

Bound Upper Bound

AST Group 1=Normal

Control

88.1366 177.1968 112.00 144.00

100 mg/kg b.w. of

Extract

155.0952 184.9048 164.00 176.00

1000 mg/kg b.w. of

Extract

148.9912 177.6755 160.00 170.00

Total 140.0564 170.6103 112.00 176.00

ALT Group 1=Normal

Control

44.7251 108.6082 62.00 86.00

100 mg/kg b.w. of

Extract

158.8254 207.8413 172.00 190.00

1000 mg/kg b.w. of

Extract

104.1550 134.5117 114.00 126.00

Total 90.0906 162.7983 62.00 190.00

ALP Group 1=Normal

Control

26.3479 42.3187 32.00 38.00

100 mg/kg b.w. of

Extract

32.5388 40.1279 35.00 38.00

1000 mg/kg b.w. of

Extract

25.0317 34.9683 28.00 32.00

Total 30.8897 36.2214 28.00 38.00

Cholesterol Group 1=Normal

Control

2.4541 5.2792 3.40 4.50

100 mg/kg b.w. of

Extract

2.5162 3.5504 2.80 3.20

1000 mg/kg b.w. of

Extract

1.4126 4.7207 2.50 3.80

Total 2.8550 3.7894 2.50 4.50

HDL Group 1=Normal

Control

.1516 .6484 .30 .50

100 mg/kg b.w. of

Extract

.3000 .3000 .30 .30

1000 mg/kg b.w. of

Extract

.4000 .4000 .40 .40

Total .3123 .4210 .30 .50

LDL Group 1=Normal 1.7294 3.7373 2.50 3.20

108

Control

100 mg/kg b.w. of

Extract

1.5162 2.5504 1.80 2.20

1000 mg/kg b.w. of

Extract

.2696 3.4637 1.40 2.60

Total 1.7806 2.6416 1.40 3.20

TAG Group 1=Normal

Control

.9162 1.9504 1.20 1.60

100 mg/kg b.w. of

Extract

.8032 1.7968 1.10 1.50

1000 mg/kg b.w. of

Extract

1.0496 2.0838 1.40 1.80

Total 1.2703 1.5964 1.10 1.80


Control

120.8445 259.1555 165.00 220.00

100 mg/kg b.w. of

Extract

180.3172 279.6828 210.00 250.00

1000 mg/kg b.w. of

Extract

126.1104 473.8896 230.00 370.00

Total 192.3441 287.6559 165.00 370.00


Control

6.8832 18.1835 10.00 14.40

100 mg/kg b.w. of

Extract

14.0465 14.6202 14.20 14.40

1000 mg/kg b.w. of

Extract

8.3030 21.8303 12.00 17.20

Total 12.3609 15.5946 10.00 17.20

Glucose Group 1=Normal

Control

72.2457 137.0876 90.00 115.00

100 mg/kg b.w. of

Extract

59.4208 124.5792 80.00 106.00

1000 mg/kg b.w. of

Extract

100.6093 118.0573 106.00 113.00

Total 92.6172 111.3828 80.00 115.00

109

ANOVA

Sum of

Squares df

Mean

Square F Sig.

AST Between

Groups

2378.667 2 1189.333 9.133 .015

Within Groups 781.333 6 130.222

Total 3160.000 8

ALT Between

Groups

17294.222 2 8647.111 86.471 .000

Within Groups 600.000 6 100.000

Total 17894.222 8

ALP Between

Groups

62.889 2 31.444 5.660 .042

Within Groups 33.333 6 5.556

Total 96.222 8

Cholesterol Between

Groups

1.336 2 .668 2.473 .165

Within Groups 1.620 6 .270

Total 2.956 8

HDL Between

Groups

.020 2 .010 3.000 .125

Within Groups .020 6 .003

Total .040 8

LDL Between

Groups

1.269 2 .634 3.070 .121

Within Groups 1.240 6 .207

Total 2.509 8

TAG Between

Groups

.107 2 .053 1.263 .348


Total .360 8

Sodium Between

Groups

18600.000 2 9300.000 4.593 .062

Within Groups 12150.000 6 2025.000

Total 30750.000 8

Potassium Between

Groups

10.196 2 5.098 1.214 .361

Within Groups 25.200 6 4.200

Total 35.396 8

Glucose Between

Groups

482.667 2 241.333 2.041 .211

110

Within Groups 709.333 6 118.222

Total 1192.000 8

111

Post Hoc Tests

Appendix II:

Multiple Comparisons

Dependent

Variable

(I) Group (J) Group

Mean

Differen

ce (I-J)

Std.

Error Sig.

95% Confidence

Interval

Lower

Bound

Upper

Bound

AST LS

D

Group

1=Normal

Control

100 mg/kg b.w.

of Extract

-

37.3333

3*

9.317

45

.007 -

60.1323

-

14.5344

1000 mg/kg

b.w. of Extract

-

30.6666

7*

9.317

45

.017 -

53.4656

-7.8677

100 mg/kg b.w.

of Extract

Group

1=Normal

Control

37.3333

3*

9.317

45

.007 14.5344 60.1323

1000 mg/kg

b.w. of Extract

6.66667 9.317

45

.501 -

16.1323

29.4656

1000 mg/kg

b.w. of Extract

Group

1=Normal

Control

30.6666

7*

9.317

45

.017 7.8677 53.4656

100 mg/kg b.w.

of Extract

-6.66667 9.317

45

.501 -

29.4656

16.1323

ALT LS

D

Group

1=Normal

Control

100 mg/kg b.w.

of Extract

-

106.666

67*

8.164

97

.000 -

126.645

6

-

86.6877

1000 mg/kg

b.w. of Extract

-

42.6666

7*

8.164

97

.002 -

62.6456

-

22.6877

100 mg/kg b.w.

of Extract

Group

1=Normal

Control

106.666

67*

8.164

97

.000 86.6877 126.645

6

1000 mg/kg

b.w. of Extract

64.0000

0*

8.164

97

.000 44.0210 83.9790

1000 mg/kg

b.w. of Extract

Group

1=Normal

Control

42.6666

7*

8.164

97

.002 22.6877 62.6456

100 mg/kg b.w.

of Extract

-

64.0000

8.164

97

.000 -

83.9790

-

44.0210

112

0*

ALP LS

D

Group

1=Normal

Control

100 mg/kg b.w.

of Extract

-2.00000 1.924

50

.339 -6.7091 2.7091

1000 mg/kg

b.w. of Extract

4.33333 1.924

50

.065 -.3758 9.0424

100 mg/kg b.w.

of Extract

Group

1=Normal

Control

2.00000 1.924

50

.339 -2.7091 6.7091

1000 mg/kg

b.w. of Extract

6.33333* 1.924

50

.017 1.6242 11.0424

1000 mg/kg

b.w. of Extract

Group

1=Normal

Control

-4.33333 1.924

50

.065 -9.0424 .3758

100 mg/kg b.w.

of Extract

-

6.33333*

1.924

50

.017 -

11.0424

-1.6242

Cholest

erol

LS

D

Group

1=Normal

Control

100 mg/kg b.w.

of Extract

.83333 .4242

6

.097 -.2048 1.8715

1000 mg/kg

b.w. of Extract

.80000 .4242

6

.108 -.2381 1.8381

100 mg/kg b.w.

of Extract

Group

1=Normal

Control

-.83333 .4242

6

.097 -1.8715 .2048

1000 mg/kg

b.w. of Extract

-.03333 .4242

6

.940 -1.0715 1.0048

1000 mg/kg

b.w. of Extract

Group

1=Normal

Control

-.80000 .4242

6

.108 -1.8381 .2381

100 mg/kg b.w.

of Extract

.03333 .4242

6

.940 -1.0048 1.0715

HDL LS

D

Group

1=Normal

Control

100 mg/kg b.w.

of Extract

.10000 .0471

4

.078 -.0153 .2153

1000 mg/kg

b.w. of Extract

.00000 .0471

4

1.000 -.1153 .1153

113

Oneway {In}

Appendix III: Descriptive

N Mean

Std.

Deviation

Std.

Error


Control

2 .0720 .00141 .00100

Group 2=0.005g of

Extract

2 .0705 .00071 .00050

Group 3=0.01g of

Extract

2 .1050 .00141 .00100

Group 4=0.02g of

Extract

2 .1230 .00141 .00100

Total 8 .0926 .02387 .00844


Control

2 .0460 .00283 .00200

Group 2=0.005g of

Extract

2 .0485 .00212 .00150

Group 3=0.01g of

Extract

2 .0555 .00071 .00050

Group 4=0.02g of

Extract

2 .0615 .00071 .00050

Total 8 .0529 .00664 .00235

Descriptive


Mean

Minimu

m

Maximu

m

Lower

Bound Upper Bound


Control

.0593 .0847 .07 .07

Group 2=0.005g of

Extract

.0641 .0769 .07 .07

Group 3=0.01g of

Extract

.0923 .1177 .10 .11

Group 4=0.02g of

Extract

.1103 .1357 .12 .12

Total .0727 .1126 .07 .12

Potassium Group 1=Normal .0206 .0714 .04 .05

114

Control

Group 2=0.005g of

Extract

.0294 .0676 .05 .05

Group 3=0.01g of

Extract

.0491 .0619 .06 .06

Group 4=0.02g of

Extract

.0551 .0679 .06 .06

Total .0473 .0584 .04 .06

ANOVA

Sum of

Squares df

Mean

Square F Sig.

Sodium Between

Groups

.004 3 .001 816.692 .000


Total .004 7

Potassium Between

Groups

.000 3 .000 29.173 .004


Total .000 7

115

Post Hoc Tests

Appendix IV: Multiple Comparisons

Dependent Variable (I) Group (J) Group Mean

Difference

(I-J)

Sodium LSD Group 1=Normal

Control

Group 2=0.005g of

Extract

.00150

Group 3=0.01g of

Extract

-.03300*

Group 4=0.02g of

Extract

-.05100*

Group 2=0.005g of

Extract

Group 1=Normal

Control

-.00150

Group 3=0.01g of

Extract

-.03450*

Group 4=0.02g of

Extract

-.05250*

Group 3=0.01g of

Extract

Group 1=Normal

Control

.03300*

Group 2=0.005g of

Extract

.03450*

Group 4=0.02g of

Extract

-.01800*

Group 4=0.02g of

Extract

Group 1=Normal

Control

.05100*

Group 2=0.005g of

Extract

.05250*

Group 3=0.01g of

Extract

.01800*

Potassium LSD Group 1=Normal

Control

Group 2=0.005g of

Extract

-.00250

Group 3=0.01g of

Extract

-.00950*

Group 4=0.02g of

Extract

-.01550*

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00250

Group 3=0.01g of

Extract

-.00700*

Group 4=0.02g of -.01300*

116

Extract

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00950*

Group 2=0.005g of

Extract

.00700*

Group 4=0.02g of

Extract

-.00600*

Group 4=0.02g of

Extract

Group 1=Normal

Control

.01550*

Group 2=0.005g of

Extract

.01300*

Group 3=0.01g of

Extract

.00600*


Dependent Variable (I) Group (J) Group Std.

Error


Control

Group 2=0.005g of

Extract

.00127

Group 3=0.01g of

Extract

.00127

Group 4=0.02g of

Extract

.00127

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00127

Group 3=0.01g of

Extract

.00127

Group 4=0.02g of

Extract

.00127

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00127

Group 2=0.005g of

Extract

.00127

Group 4=0.02g of

Extract

.00127

Group 4=0.02g of

Extract

Group 1=Normal

Control

.00127

Group 2=0.005g of

Extract

.00127

Group 3=0.01g of

Extract

.00127

117


Control

Group 2=0.005g of

Extract

.00184

Group 3=0.01g of

Extract

.00184

Group 4=0.02g of

Extract

.00184

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00184

Group 3=0.01g of

Extract

.00184

Group 4=0.02g of

Extract

.00184

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00184

Group 2=0.005g of

Extract

.00184

Group 4=0.02g of

Extract

.00184

Group 4=0.02g of

Extract

Group 1=Normal

Control

.00184

Group 2=0.005g of

Extract

.00184

Group 3=0.01g of

Extract

.00184

118


Dependent Variable (I) Group (J) Group Sig.


Control

Group 2=0.005g of

Extract

.305

Group 3=0.01g of

Extract

.000

Group 4=0.02g of

Extract

.000

Group 2=0.005g of

Extract

Group 1=Normal

Control

.305

Group 3=0.01g of

Extract

.000

Group 4=0.02g of

Extract

.000

Group 3=0.01g of

Extract

Group 1=Normal

Control

.000

Group 2=0.005g of

Extract

.000

Group 4=0.02g of

Extract

.000

Group 4=0.02g of

Extract

Group 1=Normal

Control

.000

Group 2=0.005g of

Extract

.000

Group 3=0.01g of

Extract

.000


Control

Group 2=0.005g of

Extract

.245

Group 3=0.01g of

Extract

.007

Group 4=0.02g of

Extract

.001

Group 2=0.005g of

Extract

Group 1=Normal

Control

.245

Group 3=0.01g of

Extract

.019

Group 4=0.02g of

Extract

.002

Group 3=0.01g of

Extract

Group 1=Normal

Control

.007

119

Group 2=0.005g of

Extract

.019

Group 4=0.02g of

Extract

.031

Group 4=0.02g of

Extract

Group 1=Normal

Control

.001

Group 2=0.005g of

Extract

.002

Group 3=0.01g of

Extract

.031


Dependent Variable (I) Group (J) Group 95%

Confidence

Interval

Lower

Bound


Control

Group 2=0.005g of

Extract

-.0020

Group 3=0.01g of

Extract

-.0365

Group 4=0.02g of

Extract

-.0545

Group 2=0.005g of

Extract

Group 1=Normal

Control

-.0050

Group 3=0.01g of

Extract

-.0380

Group 4=0.02g of

Extract

-.0560

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0295

Group 2=0.005g of

Extract

.0310

Group 4=0.02g of

Extract

-.0215

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0475

Group 2=0.005g of

Extract

.0490

Group 3=0.01g of

Extract

.0145

120


Control

Group 2=0.005g of

Extract

-.0076

Group 3=0.01g of

Extract

-.0146

Group 4=0.02g of

Extract

-.0206

Group 2=0.005g of

Extract

Group 1=Normal

Control

-.0026

Group 3=0.01g of

Extract

-.0121

Group 4=0.02g of

Extract

-.0181

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0044

Group 2=0.005g of

Extract

.0019

Group 4=0.02g of

Extract

-.0111

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0104

Group 2=0.005g of

Extract

.0079

Group 3=0.01g of

Extract

.0009



Confidence

Interval

Upper Bound


Control

Group 2=0.005g of

Extract

.0050

Group 3=0.01g of

Extract

-.0295

Group 4=0.02g of

Extract

-.0475

Group 2=0.005g of

Extract

Group 1=Normal

Control

.0020

Group 3=0.01g of

Extract

-.0310

Group 4=0.02g of -.0490

121

Extract

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0365

Group 2=0.005g of

Extract

.0380

Group 4=0.02g of

Extract

-.0145

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0545

Group 2=0.005g of

Extract

.0560

Group 3=0.01g of

Extract

.0215


Control

Group 2=0.005g of

Extract

.0026

Group 3=0.01g of

Extract

-.0044

Group 4=0.02g of

Extract

-.0104

Group 2=0.005g of

Extract

Group 1=Normal

Control

.0076

Group 3=0.01g of

Extract

-.0019

Group 4=0.02g of

Extract

-.0079

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0146

Group 2=0.005g of

Extract

.0121

Group 4=0.02g of

Extract

-.0009

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0206

Group 2=0.005g of

Extract

.0181

Group 3=0.01g of

Extract

.0111

*. The mean difference is significant at the 0.05 level.

122

Oneway {Out}

Appendix V: Descriptives

N Mean

Std.

Deviation

Std.

Error


Control

2 .1355 .00354 .00250

Group 2=0.005g of

Extract

2 .1435 .00212 .00150

Group 3=0.01g of

Extract

2 .1450 .00424 .00300

Group 4=0.02g of

Extract

2 .1540 .00566 .00400

Total 8 .1445 .00767 .00271


Control

2 .0105 .00071 .00050

Group 2=0.005g of

Extract

2 .0240 .00141 .00100

Group 3=0.01g of

Extract

2 .0270 .00000 .00000

Group 4=0.02g of

Extract

2 .0300 .00141 .00100

Total 8 .0229 .00801 .00283

123

Descriptives


Mean

Minimu

m

Maximu

m

Lower

Bound Upper Bound


Control

.1037 .1673 .13 .14

Group 2=0.005g of

Extract

.1244 .1626 .14 .14

Group 3=0.01g of

Extract

.1069 .1831 .14 .15

Group 4=0.02g of

Extract

.1032 .2048 .15 .16

Total .1381 .1509 .13 .16


Control

.0041 .0169 .01 .01

Group 2=0.005g of

Extract

.0113 .0367 .02 .03

Group 3=0.01g of

Extract

.0270 .0270 .03 .03

Group 4=0.02g of

Extract

.0173 .0427 .03 .03

Total .0162 .0296 .01 .03

ANOVA

Sum of

Squares df

Mean

Square F Sig.

Sodium Between

Groups

.000 3 .000 6.866 .047


Total .000 7

Potassium Between

Groups

.000 3 .000 131.667 .000


Total .000 7

124

Post Hoc Tests

Appendix VI: Multiple Comparisons

Dependent Variable (I) Group (J) Group Mean

Difference

(I-J)


Control

Group 2=0.005g of

Extract

-.00800

Group 3=0.01g of

Extract

-.00950

Group 4=0.02g of

Extract

-.01850*

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00800

Group 3=0.01g of

Extract

-.00150

Group 4=0.02g of

Extract

-.01050

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00950

Group 2=0.005g of

Extract

.00150

Group 4=0.02g of

Extract

-.00900

Group 4=0.02g of

Extract

Group 1=Normal

Control

.01850*

Group 2=0.005g of

Extract

.01050

Group 3=0.01g of

Extract

.00900


Control

Group 2=0.005g of

Extract

-.01350*

Group 3=0.01g of

Extract

-.01650*

Group 4=0.02g of

Extract

-.01950*

Group 2=0.005g of

Extract

Group 1=Normal

Control

.01350*

Group 3=0.01g of

Extract

-.00300*

Group 4=0.02g of -.00600*

125

Extract

Group 3=0.01g of

Extract

Group 1=Normal

Control

.01650*

Group 2=0.005g of

Extract

.00300*

Group 4=0.02g of

Extract

-.00300*

Group 4=0.02g of

Extract

Group 1=Normal

Control

.01950*

Group 2=0.005g of

Extract

.00600*

Group 3=0.01g of

Extract

.00300*


Dependent Variable (I) Group (J) Group Std.

Error


Control

Group 2=0.005g of

Extract

.00409

Group 3=0.01g of

Extract

.00409

Group 4=0.02g of

Extract

.00409

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00409

Group 3=0.01g of

Extract

.00409

Group 4=0.02g of

Extract

.00409

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00409

Group 2=0.005g of

Extract

.00409

Group 4=0.02g of

Extract

.00409

Group 4=0.02g of

Extract

Group 1=Normal

Control

.00409

Group 2=0.005g of

Extract

.00409

Group 3=0.01g of

Extract

.00409

126


Control

Group 2=0.005g of

Extract

.00106

Group 3=0.01g of

Extract

.00106

Group 4=0.02g of

Extract

.00106

Group 2=0.005g of

Extract

Group 1=Normal

Control

.00106

Group 3=0.01g of

Extract

.00106

Group 4=0.02g of

Extract

.00106

Group 3=0.01g of

Extract

Group 1=Normal

Control

.00106

Group 2=0.005g of

Extract

.00106

Group 4=0.02g of

Extract

.00106

Group 4=0.02g of

Extract

Group 1=Normal

Control

.00106

Group 2=0.005g of

Extract

.00106

Group 3=0.01g of

Extract

.00106

127


Dependent Variable (I) Group (J) Group Sig.


Control

Group 2=0.005g of

Extract

.122

Group 3=0.01g of

Extract

.081

Group 4=0.02g of

Extract

.011

Group 2=0.005g of

Extract

Group 1=Normal

Control

.122

Group 3=0.01g of

Extract

.733

Group 4=0.02g of

Extract

.062

Group 3=0.01g of

Extract

Group 1=Normal

Control

.081

Group 2=0.005g of

Extract

.733

Group 4=0.02g of

Extract

.093

Group 4=0.02g of

Extract

Group 1=Normal

Control

.011

Group 2=0.005g of

Extract

.062

Group 3=0.01g of

Extract

.093


Control

Group 2=0.005g of

Extract

.000

Group 3=0.01g of

Extract

.000

Group 4=0.02g of

Extract

.000

Group 2=0.005g of

Extract

Group 1=Normal

Control

.000

Group 3=0.01g of

Extract

.047

Group 4=0.02g of

Extract

.005

Group 3=0.01g of

Extract

Group 1=Normal

Control

.000

128

Group 2=0.005g of

Extract

.047

Group 4=0.02g of

Extract

.047

Group 4=0.02g of

Extract

Group 1=Normal

Control

.000

Group 2=0.005g of

Extract

.005

Group 3=0.01g of

Extract

.047



Confidence

Interval

Lower

Bound


Control

Group 2=0.005g of

Extract

-.0194

Group 3=0.01g of

Extract

-.0209

Group 4=0.02g of

Extract

-.0299

Group 2=0.005g of

Extract

Group 1=Normal

Control

-.0034

Group 3=0.01g of

Extract

-.0129

Group 4=0.02g of

Extract

-.0219

Group 3=0.01g of

Extract

Group 1=Normal

Control

-.0019

Group 2=0.005g of

Extract

-.0099

Group 4=0.02g of

Extract

-.0204

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0071

Group 2=0.005g of

Extract

-.0009

Group 3=0.01g of

Extract

-.0024

129


Control

Group 2=0.005g of

Extract

-.0164

Group 3=0.01g of

Extract

-.0194

Group 4=0.02g of

Extract

-.0224

Group 2=0.005g of

Extract

Group 1=Normal

Control

.0106

Group 3=0.01g of

Extract

-.0059

Group 4=0.02g of

Extract

-.0089

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0136

Group 2=0.005g of

Extract

.0001

Group 4=0.02g of

Extract

-.0059

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0166

Group 2=0.005g of

Extract

.0031

Group 3=0.01g of

Extract

.0001

130



Confidence

Interval

Upper Bound


Control

Group 2=0.005g of

Extract

.0034

Group 3=0.01g of

Extract

.0019

Group 4=0.02g of

Extract

-.0071

Group 2=0.005g of

Extract

Group 1=Normal

Control

.0194

Group 3=0.01g of

Extract

.0099

Group 4=0.02g of

Extract

.0009

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0209

Group 2=0.005g of

Extract

.0129

Group 4=0.02g of

Extract

.0024

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0299

Group 2=0.005g of

Extract

.0219

Group 3=0.01g of

Extract

.0204


Control

Group 2=0.005g of

Extract

-.0106

Group 3=0.01g of

Extract

-.0136

Group 4=0.02g of

Extract

-.0166

Group 2=0.005g of

Extract

Group 1=Normal

Control

.0164

Group 3=0.01g of

Extract

-.0001

Group 4=0.02g of

Extract

-.0031

131

Group 3=0.01g of

Extract

Group 1=Normal

Control

.0194

Group 2=0.005g of

Extract

.0059

Group 4=0.02g of

Extract

-.0001

Group 4=0.02g of

Extract

Group 1=Normal

Control

.0224

Group 2=0.005g of

Extract

.0089

Group 3=0.01g of

Extract

.0059

*. The mean difference is significant at the 0.05 level.

Documents

University Of Nigeria Nsukka - OBETA, JUDITH NNEDIMKPA … PROJECT FOR... · 2015-08-29 · 2 studies on the effect of aqueous extract of millettia aboensis leaves on lomotil –