ABSTRACT
The total alkaloid, tannins contents and antiulcer activity of the extracts from four selected medicinal plants were investigated. The total alkaloids content (TAC) was evaluated according to the chloride colometric method in which atropine was used as standard. While the total tannins content (TTC) was also determined using Folin Ciocalteau assay in which gallic acid was used as a standard. The antiulcer activity of the extracts was investigated using the ethanol induced model in wistar albino rats. Parameters such as gastric volume, pH and ulcer index were used as indicators for the antiulcerogenic activity of the extracts. The animals were orally treated with distilled water (Normal control group), ranitidine 5 mg/kg (standard control group), 0.5 mg/kg of ethanol (Negative control group) and 100,200 and 400 mg/kg of the extracts, 1 h before oral administration of absolute ethanol to induce gastric mucosal injury. The result showed that E. deightonii extract has the richest source of alkaloids and tannins (0.850 ± 0.001 mg AE/g and 0.133 ± 0.001 mg GAE/g respectively, while A. hispidum and P. staudtii has the least TAC and TTC (0.800 ± 0.001 mg AE/g) and (0.124 ± 0.001 mg GAE/g) respectively. The extract was considered safe with the LD50 greater than 5000 mg/kg for E. deightonii, 2154, 3808 and 2154 mg/kg for A. hispidum, P. staudtii and P. lunatus respectively. The extracts at dose levels of 100, 200 and 400 exhibited significant decrease *(P˂ 0.05) in the gastric volume, while the pH of the gastric juice was significant increase *(P˂ 0.05) in the ethanol induced model. The extracts showed minimum inhibition of gastric acid ranging from 16- 90%. The results showed that the methanol extracts of the selected plants possessed antiulcer as well as cytoprotective ability which could be attributed to the presence of the secondary metabolites.
TABLE OF CONTENTS
Title page i
Approval page ii
Certification iii
Dedication iv
Acknowledgement v
Table of Contents vi
List of Tables x
List of Figure xi
List of Abbreviations xii
Abstract xiii
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Background of Study 2
1.2 Statement of the Problem 5
1.3 Objectives of the Study 5
1.4 Justification of the study 6
CHAPTER TWO
2.0 LITERATURE REVIEW 7
2.1 Acanthospermum hispidum DC 7
2.2 Pachypodanthium staudtii Engl & Diels 7
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2.3 Phaseolus lunatus 8
2.4 Euphorbia deightonii Croizot 8
2.5 Taxonomical classification of the Plants 9
2.6 Photochemistry 11
2.6.1 Alkaloids 12
2.6.1.1 Classifications of Alkaloids 13
2.6.1.2 Properties of Alkaloids 17
2.6.1.3 Identification of Alkaloids 18
2.6.1.4 Plant Alkaloids 20
2.6.2 Tannins 26
2.6.2.1 Classifications of Tannins 27
2.6.2.2 Properties of Tannins 28
2.6.2.3 Identification of Tannins 28
2.6.2.4 Plant Tannins 29
2.6.2.5 Pharmaceutical uses of Tannins 30
2.7 Ulcer 30
2.7.1 Peptic ulcer 31
2.7.3 Therapy for peptic ulcer 32
CHAPTER THREE
3.0 EXPERIMENTAL 40
3.1 General 40
3.2 Plant collection and Identification 41
3.3 Preparation of plant materials and extracts 41
3.4 Preparation of reagents 42
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3.4.1 Folin Ciocalteau reagent 42
3.4.2 2 N of Sodium Hydroxide solution 42
3.4.3 Bromo cresol green solution 42
3.4.4 2 M of Sodium phosphate solution 42
3.4.5 0.2 M of Citric acid solution 43
3.4.6 Phosphate Buffer solution 43
3.4.7 0.9% of Normal saline 43
3.5 Qualitative phytochemical analysis of the extracts 43
3.5.1 Test for alkaloids 43
3.5.2 Test for tannins 44
3.6 Quantitative phytochemical analysis of the extracts 44
3.6.1 Determination of alkaloid contents 44
3.6.2 Determination of tannin contents 44
3.7 Acute toxicity of the extracts 45
3.8 Antiulcer assay 46
3.8.1 Macroscopical evaluation of stomach 46
3.8.2 Gastric volume and pH measurement 47
3.9 Statistical Analysis 47
CHAPTER FOUR
4.0 RESULTS 48
4.1 Qualitative phytochemical analysis of the extracts 48
4.1.1 Atropine and Gallic acid standards for the calibration curve 49
4.1.2 Total alkaloid and Total tannin content 51
4.2 Acute toxicity (LD50) tests on the plant extract 54
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4.3 Antiulcer activity of the plant extracts 60
4.4 Histology of the stomach of the experimental animals 63
4.5 Discussion 69
CHAPTER FIVE
5.0 CONCLUSION 71
5.1 Recommendation 71
REFERENCES 72
CHAPTER ONE
1.0 INTRODUCTION
Plants are gifts from God and have been utilized by human beings for basic preventive and curative healthcare since time immemorial1. In recent years, following the demonstration of the presence of active principles against many diseases and infections in a variety of plants extracts, there have been a great surge of public interest in the use of herbs and plants. This phenomenon has been viewed by some scientists as modern Herbal-renaissance2. Though, pharmaceutical industries are moving towards synthetics and biotechnology research in search of novel medicine in the 21st century, consumers are expressing a greater awareness of the risk of using synthetic products3. Therefore, attention is now focused on the various medicinal plants, their potentials, safety and their levels of toxicities if any 4.
According to the World Health Organization (WHO), a medicinal plant is any plant which, in one or more of its organs contain substances that can be used for therapeutic purposes or which are precursors for the syntheses of useful drugs5. Furthermore, medicinal plants can be seen as herbal preparations produced by subjecting plant materials to extraction, fractionation, purification, concentration or other physical or biological processes and which may be produced for immediate consumption or as a basis for the herbal products6. About 80% of the world‟s population uses medicinal plants in the treatment of diseases and in African countries, the rate is even much higher7.
In Nigeria, due to the high rate of poverty amongst the populace, various synthetic drugs which have been produced for the treatment of ulcer are beyond the reach of the average citizen. Hence there is a heavy dependence on plants and herbal products for the
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treatment of diseases especially ulcer. It is in this light that critical attention should be given to some indigenous medicinal plants such as Ancanthospermum hispidum DC, Pachypodanthium staudtii Engl & Diels, Phaseolus lunatus and Euphorbia deightonii Croizot in our locality with the hope of providing a fairly lasting solution to the health care problems.
1.1 BACKGROUND OF THE STUDY
Acanthospermum hispidum (DC), (Family Asteraceae) is a medicinal plant commonly called “bristly starburr” and locally known as “ewe onitan meta” or “kaashinyaawo” in Western and Northern Nigeria respectively8. The leaves are used locally for the treatment of jaundice, malaria, vomiting, cephalgias, head-ache, abdominal pain, convulsion, stomach-ache, constipation, eruptive fever, snake bite, epilepsy, blennorrhoea, hepato-binary disorder, microbial infection and viral infection9. The plant has been reported to possess sesquiterpene lactones such as acanthospermal B, acanthospermal B epoxide, hispidunolide A and B10-14.
Furthermore, Acanthospermum hispidum contains phytochemicals such as carbohydrates, alkaloids, glycosides, flavoniods, tannins, terpenoids and saponins that may be useful adjuvant for antibiotic, antiviral, antitrypsomal, antiplasmodial, antimicrobial, antitumor and anthelmentic formulations15,16. It is also used for the treatment of skin aliments, cough and bronchitis9; though the antiulcer activity has not been reported.
Pachypodanthium staudtii Engl & Diels, (Family Annonaceae) is a medicinal plant predominantly found in southern Nigeria. It is commonly called “Pepper tree” and locally called “Ntoko neto” in Eastern Nigeria. The stem bark and roots bark are used locally in
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the treatment of gastro-intestinal pains, headache, chest pains, tumors, toothache, bronchitis and oedema17 .The leaves, stem and bark of this plant contain the following phytochemicals: anthocyanines, anthraquinones, flavoniods, phenols, sterols, triterpenes, saponins18, tannins and alkaloids
The plant also possesses antibacterial activity; It was reported to be active against the following bacteria Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Providencia stuartii and Pseudomonas aeruginosa17,18.The plant has been proven to be biologically active and its bioactive components are as follows; pachypodol, 2,4,5-trimethoxystyrene, pachypophyllin, pachypostaudins A and B18, pachypodanthine19, pachysonto20, staudin21, sabinene, β-elemene, E-β-caryophyllene, β-selinene, β-bisabolene, δ-cadinene, 2,4,5-trimethoxy-1-vinylbenzene22. Despite the enormous importance of Pachypodanthium staudtii there has been no record on its antiulcer activity.
Phaseolus lunatus, (Family Fabaceae) is a new world legume that has been domesticated in areas corresponding to present day Peru and Mexico and currently cultivated in many tropical regions of the world including Nigeria23. It is commonly called “lima beans” and locally called “Awuje” or “Eree” in Southern and Eastern Nigeria respectively. Phaseolus lunatus like many other legumes is a good source of dietary fiber, and virtually a fat-free source of high-quality protein. Lima beans contain both soluble fiber, which helps regulate blood sugar levels and lowers cholesterol, and insoluble fiber, which aids in the prevention of constipation, digestive disorders, irritable bowel syndrome and diverticulitis24. Lima bean is a nitrogen-fixing legume that sheds its leaves copiously and is thus valuable for restoring soil fertility. It also contains phytochemicals like tannins, saponins, oxalate, phytic acid25 and alkaloids. Phaseolus lunatus, like any other
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leguminous plant is rich in protein, thus it serves as a major source of food for man and livestock26.
Phaseolus lunatus features two inducible indirect defenses to protect itself against herbivores. Besides the emission of plant volatiles, extra floral nectar is secreted to attract carnivorous anthropods to herbivore that damage plants. The activation of both putative defenses efficiently protects lima beans from leaf damage27.
Despite the great potentials of this crop, it is highly underutilized in the country and it has not received much attention in terms of crop improvement and so local cultivars are still being grown by the farmers. Lima bean has not received the benefit of intensive research programme as devoted to cowpea and soybean28.There is therefore, urgent need to extensively research on the medicinal properties of lima seeds with particular interest on its antiulcer properties.
Euphorbia deightonii Croizot, (Family Euphorbiaceae) is a medicinal plant commonly called “Africa never dies” (because of its ability to survive in any weather condition) and locally called “oro agogo” in Northern Nigeria. The importance of Euphorbia deightonii Croizot is enormous. It is used for horticultural purposes in making hedges, markers and ornamental flowers29. In medicine, it is also used in the treatment of leprosy, skin mucosae, infertility in women and used as an Aphrodisiac30. The plant also possesses copious white latex which is said to be poisonous. Its poison is sometimes extracted and smeared on edges of arrows, darts, spears or sword as poison. Euphorbia deightonii Croizot contains phytochemicals like resin, exudate-gum, steroids30, tannins and alkaloids. Over the years, little efforts have been put in place in order to harness the medicinal principles of Euphorbia deightonii Croizot. It is in this light that this study was
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carried out in order to investigate the effect of Euphorbia deightonii Croizot leaf extract on the experimental model of acute ulcer injury on adult Wistar albino rats.
1.2 STATEMENT OF THE PROBLEM.
Over the years, medicinal plants have grown to be of great importance to the health of humans globally. In Nigeria, medicinal plants play an important role in the health care delivery. About 70-80% of its population depend on them for the treatment of various ailments particularly ulcer31. There is need therefore, to authenticate the potency of some selected medicinal plants in order to meet the growing health challenges. These plants are: Acanthospermum hispidum, Pachypodanthium staudtii, Phaseolus lunatus and Euphorbia deightonii. A number of pharmacological investigations have been reported on various parts of the selected medicinal plants listed above. However there is need to evaluate the gastroprotective activity of the extracts of the selected medicinal plants on experimental adult wistar albino rats.
1.3 OBJECTIVES THE OF STUDY.
This study was intended to achieve the following specific objectives;
I. Extraction of Acanthospermum hispidum leaf, Pachypodanthium staudtii stem bark, Phaseolus lunatus seed and Euphorbia deightonii leaf with methanol.
II. Qualitative determination of alkaloids and tannins of the crude extracts obtained from these medicinal plants.
III. Quantitative determination of alkaloids and tannins of the crude extracts obtained from these medicinal plants.
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IV. Determination of the lethal dose (LD50) of these medicinal plants extracts on Wistar albino mice.
V. Determination of the antiulcer activity of these medicinal plants extracts using the Ethanol-Induced model on adult Wistar albino rats.
1.4 JUSTIFICATION OF THE STUDY
The medicinal value of plants have assumed an important dimension in the past few decades owing mainly to the discovery that extracts from plants contain not only minerals and primary metabolites but also a diverse array of secondary metabolites with antioxidant potentials32.
In developing countries and particularly in Nigeria a large segment of the population still rely on traditional medicine to treat serious diseases including infections, cancers, ulcer and different types of inflammations. Due to the high dependence on medicinal plants as an important source of primary health care delivery, it is imperative that sufficient scientific research should be done on medicinal plants found within our localities.
In the last few years, efforts have been taken to identify new antiulcer drugs from natural sources like plants33. The need for the scientific validity of the antiulcer property of Acanthospermum hispidum leaf, Pachypodanthium staudtii stem bark, Phaseolus lunatus seed and Euphorbia deightonii leaf in ethanol induced ulcer on adult wistar albino rats will justify this research.
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