STUDIES ON THE EFFECT OF AQUEOUS EXTRACT OF MILLETTIA ABOENSIS LEAVES ON LOMOTIL – INDUCED CONSTIPATION IN WISTAR ALBINO RATS

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.

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
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
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

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
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

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

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
Fig 13: Effect of Aqueous extract of M. aboensis on Sodium transport
across everted rat intestine – – – – – – – 69
Fig 14: Effect of Aqueous extract of M. aboensis on Potassium transport
across everted rat intestine – – – – – – – 71

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

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
POD -Peroxidase
PVS –Polyvinyl sulphate
TAG -Triacylglyceride
VLDL –Very low density lipoprotein

CHAPTER ONE
INTRODUCTION
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 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).