DUAL EFFECTS OF MUSA PARADISIACA

PHYTOCHEMICAL PROPERTIES AND DUAL EFFECTS OF MUSA PARADISIACA (AND IN COMBINATION WITH CATECHOLAMINES) ON PEPTIC ULCERATION IN ALBINO WISTAR RATS

BY

HERBERT EZE BARNABAS, MB.Sc (Hons), Hse, iissn DEPARTMENT OF HUMAN PHYSIOLOGY FACULTY OF BASIC MEDICAL SCIENCE MADONNA UNIVERSITY

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DECLARATION I hereby declare that this thesis on “Phytochemical properties and dual effects of methanolic extract of Musa Paradisiaca (and in combination with catecholamines) on peptic ulcer in wistar albino rats” was written by me and is a record of my research work. References made to published literatures were duly acknowledged.

© Herbert Eze Barnabas OCTOBER, 2009

herbertbarnabas@yahoo.com The above declaration is hereby confirmed: Olorunfemi Joyce Oluwadare, B.sc, M.sc, Ph.D Head of Department for Department of Human/Medical Physiology Madonna University, Port Harcourt, Nigeria.

Professor Arthur Nwafor, B.Sc, M.Sc., Ph.D Research Consultant/Visiting professor University of Porth Harcourt, Porth Harcourt City, Nigeria.

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DEDICATION This thesis is dedicated to the memory of my late father, who taught me that the best kind of knowledge to have is that which is learned for its own sake. It is also dedicated to my mother, who taught me that even the largest task can be accomplished if it is done one step at a time.

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ACKNOWLEDGEMENTS There are many people who helped to make this book possible and to guide me to its completion.

First I would like to express my gratitude to my supervisor, Olorunfemi Joyce Oluwadare, whose expertise, understanding, and patience, added considerably to my first undergraduate project experience. I appreciate his vast knowledge and skills in many areas (e.g gastrointestinal physiology, reproductive physiology, biostatistics, instrumentation) and his assistance, which have on occasion made me “GREEN� with envy. I would like to thank also members of staff of the department of medical physiology, Madonna University particularly, Mr. Ezeokafor Emmanuel, Mr. Raymond, and Mr. Chibueze for the assistance they provided at all levels of the research project, particularly in the areas of laboratory practice, animal handling and drug extraction. They provided me with direction, technical support and were more like friends to me than lecturers.

Very special thanks goes out to Very Rev. Fr. Professor Emmanuel M P Edeh, CSSP, OFR, without whose motivation and encouragement I would not have considered a degree in Human physiology.

Father Edeh is the one professor/teacher who truly made a difference in my life. It was under his tutelage that I developed a focus and became interested in vision and human factors. I doubt that I will ever be able to convey my appreciation fully, but I owe him my eternal gratitude. I would also like to thank my project group members and my course mates, Onyeka Gibson Okoloh, Mmachukwu Oforka, Adebari Adebola, Samson Solomon Kaakilan and Emmanuel Okafor, for our physiological debates, exchange of knowledge, skills, and venting of frustration during my undergraduate program, which helped enrich the experience.

My indefatigable thanks also go out to Chidinma Lilian Onuebunwa my best friend, without whose love, encouragement and typing assistance, I would not have finished this thesis. Her steadfast support of this project was greatly needed and deeply appreciated. Chinwe Jessica Obi also helped me more than she would ever know; my gratitude goes to her also in a very special way.

I would also like to thank my family for the support they provided me through my entire life and in particular I must acknowledge my sweet mother, Mrs. Basilia Herbert Egbeyi and my elder brothers, James Herbert and Christian Herbert (krizway), if I received any form of financial assistance in the course of this research work, it was solely from them, my express gratitude goes to them in a special way.

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In conclusion, I recognize this research would not have been less strenuous without the prayerful assistance of the members of the confraternity of the Miraculous Infant Jesus of Prague, Madonna University chaplaincy, Elele Campus, and choristers of Madonna Central Choir, Elele campus, your prayers provided me with a blessed assurance at times of critical need and this aided the research process in innumerable ways.

Herbert Eze Barnabas October, 2009.

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ABSTRACT This study is designed to investigate the potential prevention and healing effects of the methanolic extract of Musa paradisiaca (and in combination with catecholamines) on indomethacin-induced peptic ulcer. Unripe plantain pulp was processed to extract its constituents using methanol. The extract was subjected to quantitative and qualitative phytochemical analysis and was found to contain pharmacological and bioactive compounds such as glycosides, alkaloids, tannins, carbohydrate, protein and saponins. The mean lethal dose of the extract was also determined.

Forty wistar rats (150 – 250g) of both sexes were used for the experiments; 20 rats for prophylaxis and 20 rats for healing assessment.

The pylorus ligation technique was used for cytoprotective action of the extract. The experiment comprised of 4 groups of 5 rats in each group. Group I (Ulcerated control) received distilled water, Group II - IV received 0.5 ml of the plant extract orally for 14 days. Animals were fasted for 48 hours after the end of the second week. 50mg/kg of adrenalin (Epinephrine) was administered to members of group II and 50mg/kg of dopamine was administered to members of group III. One hour later the animals were sacrificed, the stomachs were removed by laparastomy. The gastric lesions in the glandular region were assessed and measured to determine the ulcer index. Pylorus ligation in the group II, III and IV showed significant (p < 0.05) reduction in the ulcer index compared to group I. The ulcer index of Group I was 14.8±3.5 compared to Group II (8.2±1.4), Group III (4.8±1.7), and Group IV (3.0±1.1). The extract also showed 67.57% ulcer protection index.

The rats used for healing assessment were divided into five groups of four rats in each group. 50 mg/kg of indomethacin was administered orally to the rats after a 48 hours fast to induce ulcer.

Group I (ulcerated control) rats were sacrificed one hour after the administration of indomethacin (NSAID) drug and the gastric lesions in the glandular region of the stomach assessed. Group II (ulcerated untreated control) was administered with normal saline (0.9%), Group III (positive control) was administered with 200 mg/kg of Cimetidine, Group IV and V was given the extract for 14 days with feed and water ad libitum.

After the 14 days of drug treatment, the stomach glandular region was assessed for healing gastric lesions. The ulcer index of the control was 29.5 ± 0.39 and 21 ± 0.85 in the ulcerated untreated control. The percentage ulcer inhibition of Cimetidine was 89.83% with an ulcer index of 3.0 ± 0.46.

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The extract at 100 mg/kg produced a significant (p < 0.05) dose dependent healing effect (81.35% inhibition) at a mean ulcer index of 5.5 ± 0. 93 and a significant (p < 0.05) mean ulcer index from 29.5 ± 0.39 and 21 ± 0.85 to 2.0 ± 1.15 in group V (93.22%) at 200 mg/kg.

The results suggested that the methanolic extract of Musa paradisiaca possess both ulcer prevention and healing effect. This effect was however decreased when the extract is administered with catecholamines.

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TABLE OF CONTENT Title page…………………………………………………………………………………………………………………………………………….I Declaration……………………………………………………………………………………………………………………………………II Certification……………………………………………………………......................................................................III Dedication………………………………………………………………...................................................................IV Acknowledgment………………………………………………………………………………………………………………………....V Abstract……………………………………………………………………...................................................................VII Table of Content…………………………………………………………………………………………………………………………..IX List of Tables .…..…………………………………………………………………………………………………………………………XII CHAPTER ONE: INTRODUCTION 1.1 Background of the Study…………………………………………………………………………………………………………1 1.2 Scope of the study………………………………………………………………………………………………………………….3 1.3 Aims and Objectives……………………………………………………………………………………………………………….3 1.4 Significance of the study…………………………………………………………………………………………………………4 CHAPTER TWO: LITERATURE REVIEW 2.1 Musa paradisiaca..………………………………………………………………………………………………………………5 2.1.1 Description……………………………………………………………...............................................................6 2.1.2 Origin and Distribution…………………………………………………………………………………………………………7 2.1.3 Taxonomy…………………………………………………………………………………………………………………………….8 2.1.4 Active ingredients………………………………………………………………………………………………………………..8 2.1.4.1 Importance of some of the Bioactive constituents……………………………………………………………9 2.1.5 Properties and General uses……………………………………………………………………………………………..10 2.1.5.1 Nutritional value and uses……………………………………………………………………………………………..13 2.1.5.2 Medical and Therapeutic Values…………………………………………………………………………………….15 2.1.6 Toxicities………………………………………………………………………………………………………………………………….15 2.1.7 Interactions…………………………………………………………………………………………………………………………16

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2.1.8 Unripe plantain and Gastric ulceration……………………………………………………………………………….16 2.2 Catecholamine……………………………………………………………………………………………………………………….17 2.2.1 Synthesis…………………………………………………………………………………………………………………………….17 2.2.2 Function……………………………………………………………………………………………………………………….......18 2.2.3 Effect of catecholamines……………………………………………………………………………………………………18 2.2.4 Function in plants………………………………………………………………………………………………………………19 2.2.5 Structure……………………………………………………………………………………………………………………………19 2.2.6 Degradation………………………………….…………………………………………………………………………………….19 2.2.7 Effect of Catecholamines on gastro intestinal lesions………………………………………………………..19 2.3 Peptic ulcer…………………………………………………………………………………………………………………………..21 2.3.1 Concept of Peptic Ulcer……………………………………………………………………………………………………….23 2.3.2 Classification……………………………………………………………………………………………………………………...22 2.3.2.1 Types of peptic ulcer……………………………………………………………………………………………………….22 2.3.3 Etiology of Peptic ulcer……………………………………………………………………………………………………….22 2.3.4 Complications of peptic ulcer……………………………………………………………………………………………..25 2.3.5 Signs and Symptoms……………………………………………………………………………………………………………25 2.3.6 Diagnosis…………………………………………………………………………………………………………………………….26 2.3.7 Management/ Treatment of ulcer……………………………………………………………………….…………….27 2.3.8 Epidemiology………………………………………………………………………………………………………………………30 2.3.9 History………………………………………………………………………………………………………………………………..30 CHAPTER THREE 3.1 Materials……………………………………………………………………………………………………………………………….32 3.2 Preparation of plant material…………………………………………………………………………………………………33 3.3 Phytochemical screening…………………………………………………………………………………………..…………..33 3.3.1 Preparation of Reagents for Phytochemical analysis……………………………………………………………33 3.3.2 Qualitative Phytochemical analisis of the Extract………………………………………………………………..34

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3.3.3 Quantitative Phytochemical analisis of the Extract……………………………………………………………40 3.4 Acute Toxicity Test…………………………………………………………………………………………………………………42 3.5 Animal monitoring and feeding……………………………………………………………………………………………43 3.6 Experimental procedures………………………………………..…………………………………………………………….43 3.6.1 Assessment for healing effect of Musa paradisiaca…………………………………………………………..44 3.7 Sample collections and assessment………………………………………………………………………..……………44 3.8 Statistical analysis………………………………………………………………………………………………………………….45 CHAPTER FOUR 4.1 Results…………………………………………………………………………………………………………………………………46 CHAPTER FIVE DISCUSSION AND CONCLUSION 5.1 Discussion………………………………………………………………………………………………………………………………56 5.1.1 Phytochemical Screening……………………………………………………………………………………………………54 5.1.2 Acute Toxicity……….……………………………………………………………………………………………………………54 5.1.3 Ulcer induction using NSAID.……………………………………………………………………………………………..54 5.1.4 Prophylactic effect of Musa paradisiaca (and in combination with catecholamines on peptic ulceration……………………………………………………………………………………………………………………………………55 5.1.5 Gastric ulcer healing effect of Musa paradisiaca……………………………………………………………..….57 5.2 Conclusion………………………………………………………………………………………………………………………….…58 5.3 Recommendation……………………………………………………………………………………………………………….…59 References……………………………….………………………………………………………………………………………………...60

LIST OF FIGURES AND TABLES

FIGURE 1.1. Photograph of a bunch of plantain…………………………………………….7

FIGURE1.2

Photograph

of

a

peptic

ulcer

taken

during

an

endoscopy………………………………….………………………………………………23

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upper

TABLE I: Qualitative phytochemical analysis of unripe plantain (musa paradisiaca)………………….………………….……………………………………………………49

TABLE 2: Quantitative Phytochemical analysis of unripe plantain (musa paradisiaca)…………………...………………………………………………………………………51

TABLE 3: Dual effect of musa paradisiaca on peptic ulcer and in combination with catecholamine……………………………………………………………………………...52

TABLE 4: Healing effect and % inhibition of unripe plantain extract………………………………………………………………………………………53

CHARTS…………………………………………………………………………………...54

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CHAPTER ONE INTRODUCTION 1.1 BACKGROUND OF STUDY Gastro duodenal ulceration is a common disease in both developed and developing countries (Pavo et al., 2000). Many factors have been implicated in the development of gastric ulcers. Among these factors is smoking, caffeine, alcohol, stress, Helicobacter pylori and non steroidal anti inflammatory drugs (NSAIDs) such as aspirin, Ibuprofen and naproxen sodium (Barr et al., 1983). Most of these factors either weakens the stomachâ&#x20AC;&#x;s protective mucous and make it more susceptible to the damaging effect of acid and pepsin or stimulate the secretion of gastric acid (Dâ&#x20AC;&#x2122;souza and Dhume, 1991). The hypersecretion of gastric acid has been discovered to be a strong factor in the development of both acute and chronic gastric mucosal lesions (Lam, 1984). This is shown in the fact that suppression of gastric acid by surgical and pharmacological means provides effective and rapid healing of ulcers (Rabon and Michael, 1990; Walsh and Peterson, 1995; Bastaki et al; 2000).

For centuries, extracts from leaves, seeds, fruits, barks and roots of plants have been used in the preparation of syrups and infusions in traditional medicine. These preparations have been used to treat cases ranging from the common cold to malaria, liver cirrhosis, hypertension, etc (Okwuonu et al., 2007). Today, natural product chemists use a combination of laboratory techniques and information from folklore and herbal medicine to make compounds with effects similar to those plant- derived compounds in the search for new and more effective drugs. Over 50% of all modern chemical drugs are of natural plant origin, and is essential in drug development programs of the pharmaceutical industry (Burton et al., 1983). The initial identification of more than 20,000 species of tropical forests origin by the W.H.O, in 1978 has contributed immensely to the knowledge of different uses rooted in traditional medicine, which plays a major part in maintaining the health and welfare of both rural and urban dwellers in developing countries (Oluyemi et al., 2007).

Generally, drug plants are unique in containing compounds that are end-products of long biosynthetic pathways and are usually not needed in such plants metabolic processes. These compounds are called secondary metabolites: alkaloids, glycosides, essential oils and other organic constituents (Davis and Heywood, 1963) which are usually produced in different parts of the plants like the root, leaves, fruits and seeds and then transported to other parts of the plants for storage (Kochlar, 1981). The search for novel, non toxic antiulcer preparations from medicinal plant is

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currently in vogue in order to obtain alternative sources of medicine for the management of gastric hyper secretion and gastro duodenal ulcers (Tan et al., 2005). In developing nations, this turn of events has been prompted in part by high cost of modern antiulcer medication as well as the multiple side effects that may result from their prolonged use (Tan et al., 2005).

Musa paradisiaca is a monocotyledonous perennial and important crop in the tropical and subtropical world regions (Strosse et al., 2006). It is a tropical tree-like herb, with large leaves of which the overlapping bases form the so-called false trunk. Fully grown, the stem reaches a height of 10 â&#x20AC;&#x201C; 30 feet. The flower bud is very large and shaped like a heart, and as it grows, it slowly unfolds and shows about one hundred small blossoms, which are long and narrow and grow together in clusters or groups (Lewis et al., 1998). The average weight of a bunch is about 25 Ibs. Each plantain plant bears fruit only once. The propagation is through shoots from rhizomes, since most of the species are sterile (Okwuonu et al., 2007). The unripe fruit of plantain, rich in starch, is cooked as food or dried and ground into flour. On ripening of the fruit, the starch turns into sugar (Sanya et al., 1964). Although the banana family is of more interest for its nutrient than its medical properties, it has some value in traditional medicine (Aremu and Udoessien, 1990). The red protecting leaves of the bud are used against heavy menstrual bleeding (menorrhagia). Other applications are against: diarrhea, dysentery, migraine, hypertension, asthma and jaundice (Suriname, 2002). Plantain is the perfect supplement for active people. It provides large amounts of vitamins most commonly lost during strenuous exercise (Okwuonu et al, 2007).

Musa paradisiaca is most well known as a supplier of potassium. Potassium is very important to muscle function and is the nutrient most often associated with relieving muscle cramps (Sanya et al., 1964). The potassium in Musa paradisiaca may also reduce the risk of hypertension and stroke, and because plantain is also high in dietary fiber, the herb may also reduce the risk of certain types of malignant diseases, especially in the colon (Suriname, 2002). Phytochemical analysis of the leaves reveal that it is rich in saponins, tannins, glycosides, alkaloids, flavonoids, resins and other organic constituents (Obadoni et al., 2002; Adeniyi and Odufowa, 2000; Iwo, 1993).

Ulcers result due to loss or destruction of epidermis and/or subcutaneous tissue (Das, 1993). The common sites for ulcerations are the stomach, duodenum, intestinal ulcers in typhoid fever, intestinal tuberculosis, bacillary and amoebic dysentery, ulcers of the legs due to varicose veins etc. (Das,

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1993). Despite the finding that a bacterial infection is the cause of ulcers in 80% of cases, bacterial infection does not appear to explain all ulcers and researchers continue to look at stress as a possible cause, or at least a complication in the development of ulcers (Levenstein, 1998). The discovery that Helicobacter pylori are a cause of peptic ulcer has tempted many to conclude that psychological factors are unimportant.

Recent studies with plantain (Musa paradisiaca) have indicated its ulcer-protective and healing activities through its predominant effect on various mucosal defensive factors (Sanya et al, 1964).

The extract showed significant antiulcer effect and antioxidant activity in gastric mucosal homogenates, where it reversed the increase in ulcer index, lipid peroxidation and super oxide dismutase values induced by stress (Brzozowski et al., 2002). Also because the use of herbs and herbal therapies in Africa and other part of the world including the U.S is escalating, it is essential to be aware of clinical and adverse effects, doses and potential drug interactions. Most practicing physicians have little knowledge of herbal treatments or adverse effects (Wharton et al, 1986). A consumer poll in 1998, however, indicated that nearly one-third of respondents use botanical remedies (Brevoort, 1998). Moreover, of those who take prescription medications, nearly one in five uses herbs, high-dose dietary supplements or both (Eisenberg et al, 1998), suggesting an estimated 15 million adults are at risk for adverse interactions involving prescription medications and herbs or vitamin supplements.

1.2 SCOPE OF THE STUDY This study is limited to the assessment of the glandular region of the stomach of wistar rats which were induced ulcer by indomethacin and pylorus ligation.

1.3 AIM AND OBJECTIVES In Africa, there has been many sponsored ethnobotanical survey which revealed the presence of many plants purported by traditional practitioners to be efficient in the management of complications arising from peptic ulcer (Tan et al., 2005). This study is under taken to determine if the medicinal properties of unripe plantain extract alone and in combination with catecholamine can prevent or completely eradicate ulcer induced by indomethacin in rats.

1.4 SIGNIFICANCE OF STUDY

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This work is designed towards providing a good means of promoting, healing and prevention of ulcer reoccurrence through the use of herbal plant extract such as unripe plantain (Musa paradisiaca).

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CHAPTER TWO LITERATURE REVIEW 2.1 MUSA PARADISIACA Banana plants are monocotyledonous perennial and important crops in the tropical and subtropical world regions (Strosse et al., 2006). They include dessert banana, plantain and cooking bananas.

Traded plantain (Musa paradisiaca AAB) and other cooking bananas (Musa Serpientum ABB) are almost entirely derived from the AA.BB hybridization of Musa acuminate (AA) and Musa balbisiana (BB) (Stover and Simmonds 1987; Robinson 1996). Plantain and cooking bananas (Musa Cavendish AAA) in exterior appearance, although often larger; the main differences in the former being that their flesh is starchy rather than sweet, they are used unripe and require cooking (Emaga et al., 2007). Dessert bananas are consumed usually as ripe fruits; whereas ripe and unripe plantain fruits are usually consumed boiled or fried (Surga et al., 1998).

Banana plantations constitute extensive crops in tropical and Caribbean countries, where they are used as basic food. In Venezuela, the commercial plantain cultivar used is the Harton variety. It is difficult to establish the production volumes, as the areas where it is grown are dispersed aver the country, either in artesanian familiar small areas (conucos) or in large areas where it is produced for export. The production of plantain in Venezuela during the last ten years, excluding dessert banana fruits (cambur), has been estimated at 5850000 ton (FAO, 2004; Agrevo, 2008; MAT, 2008). New high yield cultivars allow banana plants to be grown more extensively, resulting in a higher economic value, as they respond to plant improvement methods, fertilization and pest and disease control (Gwanfogbe et al., 1988). From the nutritional point of view, these fruits are among the green vegetables with the richest iron and other nutrients contents (Aremu and Udoessien, 1990).

However, they are highly perishable and subjected to fast deterioration as their moisture content and high metabolic activity persist after harvest (Demirel and Turhan, 2003).

Plantain, Plantago major, was considered to be one of the nine sacred herbs by the ancient Saxon people, and has been celebrated in Anglo-Saxon poetry as the “mother of herbs” (Culpeper et al, 1990). There are more than 200 species of plantain and nearly as many recorded uses for this herb (Culpeper et al, 1990). Plantain is native to northern and central Asia and Europe. Early colonists brought plantain to North America as one of their favoured healing remedies (Duke et al, 1997).

Native Americans called this persistent herb “White man‟s foot” as it is often found growing along

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well-trodden foot paths (Culpeper et al, 1990). The Latin generic name means “Sole of the foot” (Culpeper et al, 1990). The indigenous Americans adopted many of the traditional European uses for this beneficial herb. They also used the plant to draw out the poison of rattlesnake bite, to soothe rheumatic pain, as a poultice to treat battle wounds, and as an eye wash (Elias et al, 1996).

Plantain is a member of the plantaginaceae family. Some of the familiar species, naturalized throughout North America, are: Plantago major, commonly Known as common plantain, dooryard plantain, broad leave plantain, greater plantain, round-leafed plantain, way bread, devils shoe string, bird seed, snake weed, and white man‟s foot, Plantago media l., known as hoary plantain, and Plantago lanceolata l., also known as English plantain, lance-leaf plantain, buckhorn, chimneysweeps, headsman, ribgrass, ribwort, ripple grass, hen plant, snake plantain, fire weed, and soldier herb (Pelletier et al. 2002). Two species of plantain, valued medically primarily for the seed, are Plantago psyllium l. and Plantago indica, also known as flea seed (Culpepper et al. 1990). The dried, ripe seed of these species, generally called psyllium, is high in mucilage and is widely used as a bulk-forming laxative (Duke et al., 1997).

2.1.1

DESCRIPTION

Plantains are large plants that grow in the tropical part of South America, Asia, and Africa, where the climate is warm and damp the year round. It grows ten to forty feet in height and has enormous broad green leaves that are sometime ten feet long (Lewis et al., 1998). The flower bud is very large and shaped like a heart, and as it grows, it slowly unfold and shows about one hundred small blossoms, which are long and narrow and grow together in clusters or groups (Lewis et al., 1998).

From the centre of the crown spring the flowers. Only flowers develop into a plantain fruit that vary in length from about 4 – 12 inches (Okwuonu et al., 2007). The average weight of a bunch is about 25 1bs. Each plantain bears fruit only once. The propagation is through shoot from the rhizomes, since most of the seeds are sterile (Okwuonu et al., 2007). Some of the clusters of Musa paradisiaca grow into a great bunch of fruits, and each bunch is called a „hand‟, because it resembles a hand with the separate plantain as fingers (Okwuonu et al., 2007). Musa paradisiaca is a tropical tree-like herb, with large leaves of which the overlapping bases form the so-called false trunk. Fully grown, the stem reaches a height of 10 – 30 feet (Aremu and Udoessien, 1990). Musa paradisiaca is also packed with natural energy and phytonutrients.

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Plantain bears regular flower, usually in elongated spike. The calyx and corolla are chaffy. The four stamens

have

long,

threadlike

filament

(Aremu

and

Udoessien,

1990).

The trunk of the plantain tree is not a stem at all but may overlapping leaf bases. These are often fleshly, containing large amount of starch and useful substances (Moser et al., 2008). The leaves consist of a broad blade with parallel vein running perpendicular to a midrib. The midrid extends into apetiole or stalk, and a sheating base (Aremu and Udoessien, 1990). The bases of the leaves overlap tightly, forming a rigid pseudostem. The fruits are picked when they are unripe and starch rich, but when they are starch turns into simple sugar (Sucrose, glucose, and fructose) (Ngo, 2008).

Musa paradisiaca produces green or greenish-yellow seedless fruits, so fruits develop parthenorcarpically (in the absence of seed development). The name â&#x20AC;&#x153;plantainâ&#x20AC;? refers to Musa paradisiaca l., which requires cooking before it is eaten. An intoxicating drink can be prepared from the fruit. The leaves are cut into strips and woven into mat and bags (Levenstein, 1998).

2.1.2

ORIGIN AND DISTRIBUTION

Plantain is native to Southeast Asia India and cultivated in tropical regions (Whelan et al., 2008).

The plantain family, with two genera and about 40 species, occur in distribution habitants in the old world tropics (Aremu and Udoessien, 1990). It has unisexual, often but it is now an important crop throughout the moist tropics, but as a local staple food and as an export crop. Plantain have sterile flower, and the fruits develop unfertilized, so plantain contain no seeds (Okwuonu et al., 2007).

Production of new plant is by vegetative means, and the bases of the old plants.

The plant is widely distributed throughout the tropical region. It is native to India and Burma throughout the Malay Archipelago to New Guinea, America, Australia, Samona, and tropical Africa.

However, the cultivation is limited to Florida, the Canary Island, Egypt, Southern Japan, and South Brazil (Dvorkin et al., 2008).

2.1.3

TAXONOMY

According to Chesman, who in 1948 pioneered the Modern classification of bananas most edible bananas and plantain belong to the Eumusa section of the genus Musa (family Musaceae) and derive from the species Musa acuminate and Musa balbisiana, which correspond roughly to two species originally described by Linnaeus in the general botanical work systema Naturae (1758) to which he

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gave the names Musa sapientum and Musa paradisiaca, the first referring to a plant producing hornshaped fruit and similar to the modern “French plantain”, and the second popular dessert banana of the tropics the “silk fig” (Simmonds, 1996).

A commercial different group evolved from the Australimusa section of the musa genus, the so called fei bananas, common in the pacific and composed of a group of cultivars characterized by the red cap of the plant and, chiefly the fact that its fruit is produced in erect bunches rather than the hanging bunches typical of all Eumusa types (Simmonds, 1996).

2.1.4 ACTIVE INGREDIENTS These include tannins, eugenol, and tryamine (Ngo, 2008). Tannins are naturally occurring phenolic compounds which precipitate protein. In general, tannins are high molecular weight (mr>500) and have many phenolic groups (Hagerman et al., 1997). They are abundant in young and leaves and in many unripe fruit. As the fruit ripen the tannins disappear and are converted into glucose and other substances (Lewis et al., 1998).

Tannin is a bitter substance, and this is why very strong tea fruits of myrobalans taste bitter. It is an aseptic that is free from the attack of parasitic fungi and insect (Reed, 1995). High content in plantain and unripe fruit has antibiotic activity (Ngo, 2008). Serotonin, levarterenol, and Dopamine are available in the ripe fruits and peel (Morton, 2005). A 200g portion of unripe plantain is a rich source of vitamin B6, K and Ca good source of folate and copper; a source selenium; provide 12g of dietary fiber; supplies 240kcal(100kj) (Adams, 1992). Serotonin, levarterenol and Dopamine are available in the ripe fruit and peel. Other chemical constituents are alkaloids, steroidal lactones and iron (Morton, 1987). Together, these constituents are thought to give plantain mild antiinflammatory, antimicrobial, anti-hemorrhagic and expectorant action (Scott et al., 1970).

2.1.4.1

IMPORTANCE OF SOME OF THE BIOACTIVE CONSTITUENTS ALKALOIDS: These are complex nitrogenous substances and occur in combination with some organic acid, mostly in the seeds and roots of some plants. They have an intensely bitter taste and many are extremely poisonous. A few of them are liquids. There are over 200 known alkaloids found in plants. They often produce dramatic physiological effect in humans and other animals. A few examples are quinine and cinchonine in the root and stem of cinchoma, strychnine; conine and tubocurarine are infamous toxins, whereas morphine,

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codeine, atropine and vincristine are important therapeutic drugs. They are component of arrow poisons; used as muscle relaxant during surgery, cough suppressant and other uses (Uno et al, 2001).

FLAVONOIDS: these are 15-carbon compounds which occur naturally and are widely distributed in the plant kingdom appearing in flower, fruits, stem, leaves, roots and plant derived beverages such as tea and wine (Mckenzie et al, 1985). These are ubiquitous in occurrence in nearly all plants; the ease with which they are isolated and identified even from small amounts of plant materials as well make this chemical the most used for medicinal purposes. Flavonoids protect plants against external pathogens, ultra-violet light or heat. The most important class of flavonoids include: anthocyanides flavones, flavanones, flavan-301(also known as catehin) (Bate-Smith and Swain, 1962).

Flavonoids possess anti-inflammatory properties and act as modulators of the immune system in a number of biological systems. This stems from the fact that they are powerful antioxidants protecting biosystems against damaging effects of free radicals. Most flavonoids belong to a group of chemicals, called polyhenols, and their antioxidant properties are dependent on this polyphenolic chemical structure (Bohan and Kocipai, 1994).

PHENOLS: These are characterized by the presence of the hydroxyl group (-OH) and itâ&#x20AC;&#x;s derived from hydrocarbons (aromatic hydrocarbons) by the presence of the hydroxyl group (Davis and Heywood, 1963). Common examples of phenols include the following: resorcinol, hydroquinone 0 â&#x20AC;&#x201C; cresol. Most of the phenols are noted for their germicidal properties and cresol is used widely as a wood preservative (Obute, 2005).

Plants produce many thousands of compounds which contain one or more phenolic residues, these compounds can be divided into major groups, according to the number of carbon atom in their skeleton (Davis and Heywood, 1963).

TANNINS: Tannins are naturally occurring phenolic compounds which precipitates protein.

In general, tannins are high molecular weight (Mr > 500) and have many phenolic groups (Hagerman et al, 1997). They are abundant in young and old leaves and in many unripe fruits. As the fruits ripen the tannins disappear and are converted into glucose and other substances.

~ 20 ~

RESINS: These are mostly found in the stems of conifers and occur in abundance in special canals or ducts; known as resin-ducts. They are yellowish solid, insoluble in water but soluble in alcohol, turpentine and spirit. They occur associated with small quantity of turpentine which is removed by distillation, and residue is pure resin (which rapidly heals wounds). Resins also help plants to defend themselves (Uno et al., 2001).

GLYCOSIDES: Secondary metabolites often occur in plants in combination with one or more systems. Such combination molecules are called glycosides. For example, digitoxose (used to produce digitalis for heart patients) is found in purple foxglore (Digitalis purpurea) and apiose is unique to parsey (petroselinum) and its relatives (Uno et al., 2001). Cyanogenic glycosides are sugar-containing compounds that release cyanide gas when broken down.

These compounds occur in many families, but they are especially common in the pea and rose families (Uno et al., 2001).

2.1.4 PROPERTIES AND GENERAL USES The parts of Musa paradisiaca commonly used are the roots, leaves, fruit and stem (Dvorkin et al., 2008). The root is anthelmentic, depurative and tonic, and is useful in venereal diseases, helminthiasis, scabies, leprosy, skin disease and debility. The tender leaves are useful in scabies, inflammations, opthalmopathy blister and burns (Davids et al., 1998). The fresh leaves, crushed and applied to wounds, sore, insect bites, bee and wrap stings, eczema, and sunburn are healing to tissue because of high allantoin content (Culpeper et al., 1990). Plantain is an ancient remedy used widely for relieving cough, urinary infections, and digestive problems (Duke et al., 1997). The infusion has been used as a blood purifying tonic, a mild expectorant, and a domestic (Duke et al., 1997). The juice from crushed leaves may also stem the flow of blood from cuts, and soothe the itch of poison ivy or the sting of nettle (urtica dioca) (Pelletier et al., 2002). The root of the herb has been used to relieve tooth ache (Duke et al., 1997). The juice may relieve ear ache (Alebiowu et al., 2002). A decoction of plantain has been used in douche preparations to relieve leucorrhea, and inflammation of the intestines (Elias et al., 1996).

Plantain is used throughout the world. It is an effective treatment for chronic colitis, acute gastritis, enteritis, and enterocolitis according to the Russian Ministry of Healthy. The German commission E, an advisory panel on herbal medicines for that country, lists plantain as a safe and effective herb with demulcent, astringent and antibacterial properties (Tyler et al., 1994). A poultice (salve

~ 21 ~

prepared from the leaf) or an infusion used as a skin wash, have been shown to reduce pain, itching, and bleeding from hemorrhiods (Elias et al., 1996). Studies in Italy and Russia have confirmed plantain`s usefulness as a weight-loss remedy (Tyler et al., 1994). In Chinese medicine plantain is considered a remedy for male impotence (Culpeper et al., 1990). The species P. major and P. lanceolata contain mucilage, the iridiod glycosides, cubin and catapol, flavonoids, tannins, and silica (Pelletier et al., 2002).

The fruits of Musa paradisiaca are astringent, emollient, cooling, anthelmintic, aphrodisiac, antiscorbutic, demulcent and tonic. They are useful in vitiated conditions of pitta, dipsia, helminthiasis, scabies, pruritus, strangury and general debility (Dvorkin et al., 2008). The ashes obtained by burning the plant are antiscorbutic, anthelmintic and stomachic, colic and verminosis (Dvorkin et al., 2008). The flower is good for dysentery, diabetes, ascites, and dropsy. The inflorescence axis (stem) is very specific for renal and vesical calculi (Dvorkin et al. 2008).

The root is acrid, anthelmintic, tonic, increases appetite; useful in â&#x20AC;&#x153;kaphaâ&#x20AC;? and biliousness, pain in the ear, menstrual disorder, disease of the blood, diabetes insipidus, acid dyspepsia, and leprosy (Ngo, 2008). The juice of the root is also anthelmintic. The juice of the stem is cooling, astringent to the bowels, antidysenteric; useful in thirst, strangury, urinary discharges, leprosy, disease of the ear, the blood, the uterus and vagina (Dvorkin et al., 2008). Young plantain leaves are used as a cool dressing for blisters, burns, and to retain the moisture of water dressing. They may also be used in opthalmia and other eye diseases (Ngo, 2008).

The ashes produced by burning the dried leaves,

the stem, or the entire plants are antiscorbutic; they are used in acidity, heartburn, colic and intestinal worm (Culpeper et al., 1998). The juice of the flower mixed with curd is used in dysentery and menorrhagia (David et al., 1998). The gum obtained from unripe plantain mixed with rice water is used in diarrhea; the sap of the fresh stem is largely used in nervous affections via hysteria, epilepsy, etc (Dvorkin et al, 2008).

The leaves are used for cough and bronchitis. The root can arrest hemoptysis and possess strongly astringent and anthelmintic properties (Whelan, 2008). Plantain juice is used as an antidote for snake bite (Ngo, 2008). Other uses are asthma, burns, diabetes, dysentery, excessive menstrual flow, fever, gangrene, gout, headache, hemorrhage, inflammation, insomnia, intestinal parasites, sore, syphilis, tuberculosis, ulcers, and warts (Morton, 2005).

In Surinameâ&#x20AC;&#x;s traditional medicine, the red

~ 22 ~

protecting leaves of the bud was used against heavy menstrual bleeding (Menorrhagia). Other therapeutic uses were against diarrhea, dysentery, migraine, hypertension, asthma and jaundice (David et al., 1998). Other uses are asthma, burns, diabetes, dysentery, excessive menstrual flow, fever, gangrene, gout, headache, hemorrhage, inflammation, insomnia, intestinal parasites, sores, syphilis, tuberculosis, ulcers and warts (Morton, 2005). In Surinameâ&#x20AC;&#x;s traditional medicine, the red protecting leaves of the bud was used against heavy menstrual bleeding (Menorrhagia). Other therapeutic uses were against diarrhea, dysentery, migraine, hypertension, asthma and jaundice (David et al, 1998).

Studies in rats demonstrate effectiveness for stone lysis (Ngo, 2008). In animal studies, the extract of Musa paradisiaca green fruits reduced hyperglycemia in normal and diabetic mice (Adewunmi, 2003), and protected the gastric mucosa from aspirin-induced erosion stimulating gastric and colonic mucosa (Lewis et al, 1993), and nonspecific relaxing and inhibiting effect on aortic and portal smooth muscles. There was evidence in vivo antimicrobial activity of Musa paradisiaca L. root extract (Goel, 1989). The root extracts show in vitro anti microbial activity. Plantain seed, particularly those of the species P. psyllium and P. ovate soaked in water and ingested are widely used as a gentle and safe bulk laxative, and anti-diarrhea (Tyler et al, 1994). Plantago seeds from these two species are listed in the United States Pharmacopoeia XXVII as an official laxative herb.

Psyllium is found in numerous commercial laxative preparations (Tyler et al, 1994). Psyllium seed has also been proven beneficial in reducing high levels of blood cholesterol (PDR for Herbal Medicines, 1998). Psyllium seeds contain high mucilage content in addition to other phytochemicals including monoterpene, alkaloids, glycosides, sugars, triterpenes, fixed oil, fatty acids, and tannins (Culpeper et al, 1990).

The entire plant may be used with an alum mordant to dye wool a bronze-gold colour (Alebiowu et al, 2002). A newer use of plantain starch is in the manufacture of pharmaceuticals; like corn starch, plantain starch can be used as an inert ingredient to mix with drugs in order to form tablets containing consistent measured doses of drugs (Alebiowu et al, 2002). The potassium in plantain may help reduce the risk of hypertension and stroke, and because plantain is also high in dietary fiber, the herb may also reduce the risk of certain types of malignant diseases, especially in the colon (Best et al., 1999). Plantain is also packed with natural energy and phytonutrients.

2.1.5.1

NUTRITIONAL VALUE AND USES

~ 23 ~

Plantain fruit is composed mainly of water (around 75 percent for plantain and 65 percent for banana) and carbohydrates from 22 percent for banana and 32 percent for plantain. It contains several vitamins, including A, B, and C, and is very low in protein and fat but rich in minerals, particularly potassium (around 400mg/100g). It is cholesterol free, high in fiber, and low in sodium.

Chemical composition varies not only among cultivars but also according to climatic and other conditions (Kagy, 1999). Ripe fruit is usually consumed fresh simply peeled and eaten as a snack or dessert, in salad mixed with other fruits and with breakfast cereals, but I also lends itself to more elaborate dishes ranging from ice cream to pie fillings (Morton, 1987).

Plantain, can be eaten ripe or unripe, but many countries have developed commercial processes to provide a wide variety of products from both fruits puree, flour, jam, jelly, chips, crisps, flakes, dried, catsup, relishes or spreads, preserves, vinegar and even wine. Banana flour, both from green and ripe fruit, has a great industrial potential and, enriched with sugar, powdered milk, minerals and vitamins, and artificial flavouring, is much used in the manufacture of dairy products, such as cakes, banana-flavoured drinks, baby food and diverse sauce (Robinson, 1996). In Uganda, the country with the highest per capita consumption of plantain and banana in the world in 1996: 243kg while people in most European countries only average between 7 and 15kg â&#x20AC;&#x201C; an important part of the diet comes from unripe plantain that are first peeled, then steamed wrapped in their own leaves, and finally pounded to a starchy paste called matoke that constitutes the main dish. Both Uganda and Tanzania produce and consume large quantities of beer brewed from local highlands plantains. A plantain and soybean mixture, SOYAMUSA, combining carbohydrate and proteins has been recently developed in Nigeria to be used as a weaning food for toddlers. All told, plantains represent more than 25 percent of the food energy requirement of Africa (Frison and Sharrock, 1999).

Tostones is a very popular dish in the Caribbean Island: slices of green plantain are double fried (flattening slices with a wooden press between frying), producing a tasty side dish used in lieu of the ubiquitous French-fried potato. Mfong is a typical Puerto Rican dish made from fried green plantain, pork and garlic. Finely ground and roasted dried green plantain has been utilized as a coffee substitute in some countries (Morton, 1987). Although the fruit is the main economic product, many parts of the plantain can be used as food, fodder, or for industrial purposes. Throughout the tropics male buds, young flowers, and even the pseudo stem of some cultivars are eaten cooked as vegetable. Flowers and ashes from burned green leaves and pseudo stems are used in curies in Southeast Asia. The possibility of using the raquis to prepare flour for human consumption and

~ 24 ~

making marmalade from plantain peel is being studied in Columbia. Leaves are used for wrapping other food during steaming or other cooking, such as in preparing the Venezuelan hallaca and many pit steamed or pit roasted meat and vegetables typical and among the pacific Islanders. Banana leaves are also used in environmentally friendly â&#x20AC;&#x153;disposable platesâ&#x20AC;? in southeast India, where in fact several cultivars (mainly AAB or ABB plantain types) are grown exclusively for leaf production (Singh, 1996).

2.1.5.2

MEDICINAL AND THERAPEUTIC VALUES

The early digestibility and nutritional content make ripe plantain an excellent food particularly suitable for young children and elderly people. In the green stage (and after liquefying) it is used in Brazil to treat dehydration in infants, as the tannins in the fruit tend to protect the lining of the intestinal tract against further loss of liquids. In general, the banana is appropriate for consumption when a low fat, low sodium, and/or cholesterol-free diet is required, making it particularly recommendable for people with cardiovascular and kidney problems, arthritis gout or gastrointestinal ulcers (Robinson, 1996).

As the fruit is easy to carry and peel, it is of great value to athletes as a quick and healthy method of replenishing energy because of its high energy value, 75-115kcal/100mg of pulp (the lower range for banana and the higher for plantain). Plantain contains complex carbohydrates capable of replacing glycogen and important vitamins, particularly B6 and C, and minerals (potassium, calcium, magnesium, and iron). Ripe fruit has been used to treat asthma and bronchitis, and as mentioned, in the control of diarrhea, boiled and mashed ripe fruit, especially when mixed with other appropriate plants, is also good remedy against constipation (Robinson, 1996). The juice extracted from the male bud is thought to be good for stomach problem. The peel of ripe plantain has antiseptic properties and can be used to prepare a poultice for wounds or even applied directly to a wound in an emergency. The plantain pseudo stem is also cooked in India as a dish called khich khach, to be taken monthly to prevent constipation. Fresh leaves have reportedly been used medically for a whole range of disorders from headaches to urinary tract infections. At one time stem juice was considered a remedy for gonorrhea (Morton, 1987).

~ 25 ~

2.1.6

TOXICITIES

No toxicities and contraindications are reported in humans yet. Musa paradisiaca is a non toxic plant (ASPCA Poison Control Centre, 2004). In an animal study, Musa paradisiaca l. pseudo stalk extract was used in rats to evaluate toxicities but the product showed no toxic effect when rats took a dose of 2g/kg orally (Guevara et al, 2003).

In one animal study however, Musa paradisiaca, containing many polyvalent cations, reduced the absorption of quinine antibiotic (Nwafor et al., 2003).

Also Psyllium seed and plantain have been reported to cause allergic reactions in sensitive persons (Pelletier et al, 2002) and a self-limiting diarrhea has been reported as an adverse effect (Goel et al, 2002).

2.1.7 INTERACTIONS Plantain has been reported to decrease the absorption of digoxine (Elias et al, 1996). Its vitamin K content may interfere with blood-thinning medications (anticoagulants) (Duke et al, 1997). Plantain should not be taken together with prescription diuretics as it increases the risk of potassium loss from the bloodstream (hypokalemia) (Culpeper et al, 1990).

2.1.8 UNRIPE PLANTAIN AND GASTRIC ULCERATION Sanya et al, have reported the anti-ulcerogenic activity of dried powder of banana pulp (DRBP) against ulcers induced by histamine in guinea pigs and, phenylbutazone, restraint stress and prednisolone in rats (Sanya et al, 1991). Other workers like Elliott and Heward (1976) and Best, et al (1984) have also confirmed the anti-ulcerogenic activity of plantain against histamine induced gastric ulcers in mice and aspirin-induced gastric ulcers in rats respectively.

The effect of the biological variables like size, season and soil on the anti-ulcerogenic activity of dried plantain banana pulp has been evaluated and it was reported that the activity was primarily observed with unripe, mature, green plantain banana (Goel et al, 2002). Recently the methanolic extract of banana was reported to have anti-oxidant effect but not anti-Helicobacter pylori in vitro (Goel et al, 2001). Dried unripe plantain pulp extract was reported to have no activity on offensive acid pepsin secretion and the effect was mostly ascribed to increase in gastric mucous secretion quantified in terms of total carbohydrate : protein ratio (Tc : P ratio) (Goel et al, 1985).

~ 26 ~

Further studies with unripe plantain extract on the changes induced by ulcerogenic agents like aspirin (ASP), phenybutazone, indomethacin and prednisolone in the dissolved mucosubstances of gastric juice showed that it not only increased the Tc : P ratio induced by ulcerogenic drugs (Goel et al, 1985). While, there was no change in the individual carbohydrates there is a significant decrease in protein content in Tc: P ratio (Goel et al, 2002). Decrease in protein content signifies decreased leakage from gastric mucosal barrier. Increase in glycoprotein content of the mucosa and cell shedding in the gastric juice were also reported as further evidence for strengthening of mucosal resistance (Goel et al, 1986). Apart from mucosal resistance unripe plantain extract was also reported to increase cell proliferation as observed from increase in DNA and [3H] – thymidine uptake by the mucosal cells and increase in mucosal thickness. This property was also reported to be involved in healing of ulcers (Mukhopadhyay et al, 1987).

2.2

CATECHOLAMINE

Catecholamines are sympathomimetic (Dorland’s Medical Dictionary) “fight-or-flight” hormones that are released by the adrenal glands in response to stress (University of California Health Library). They are part of the sympathetic nervous system. They are called catecholamines because they contain a catechol group, and are derived from the amino acid tyrosine (Purves et al, 2008).

The most abundant catecholamines are epinephrine (adrenaline), norepinephrine (noradrenaline) and Dopamine, all of which are produced from phenylalanine and tyrosine. Catecholamines are watersoluble and are 50% bound to plasma proteins, so they circulate in the bloodstream. Tyrosine is created from phenylalanine by hydroxylation, by the enzyme phynylalanine hydroxylase (Tyrosine is also ingested directly from dietary protein). It is then sent to catecholamine-secreting neurons. Here, many kinds of reactions convert it to Dopamine, to norepinephrine, and eventually to epinephrine (Ann, 1987).

2.2.1 SYNTHESIS Catecholamines are produced mainly by the chromaffin cells of the adrenal medulla and the postganglionic fibers of the sympathetic nervous system (Andrew Holman, 2005). Dopamine, which acts as a neurotransmitter in the central nervous system, is largely produced in neuronal cell bodies in two areas of the brainstem: the substantia nigra and the ventral tegmental area (Ann, 1987).

~ 27 ~

Dopamine is the first catecholamine to be synthesized from the biosynthetic pathway.

Norepinephrine and epinephrine, in turn, are derived from further modifications of Dopamine. It is important to note that the enzyme Dopamine hydroxylase requires copper as a cofactor and DOPA decarboxylase requires PLP. Catecholamine synthesis is inhibited by alpha â&#x20AC;&#x201C; methyltyrosine, by inhibiting tyrosine â&#x20AC;&#x201C; 3 monooxygenase (Purves et al., 2008).

2.2.2 FUNCTION Two catecholamines; norepinephrine and Dopamine, act as neuromodulator in the central nervous system and as hormones in the blood circulation. The catecholamine norepinephrine is a neuromodulator of the peripheral sympathetic nervous system but is also present in the blood (mostly through "spillover" from the synapses of the sympathetic system) (Schultz, 2002).

High catecholamine levels in blood are associated with stress, which can be induced from psychological reactions or environmental stressors such as elevated sound levels, intense light, or low blood sugar levels (Ann, 1987). Extremely high levels of catecholamine (also known as catecholamine toxicity) can occur in central nervous system trauma due to stimulation and/or damage of nuclei in the brainstem, in particular those nuclei affecting the sympathetic nervous system. In emergency medicine, this occurrence is widely known as catecholamine dump (Schultz, 2002). Extremely high levels of catecholamine can also be caused by neuroendocrine tumours in the adrenal medulla, a treatable condition known as pheocromocytoma (Wood, 2008).

High levels of catecholamines can also be caused by monoamine oxidase A deficiency. This is the gene responsible for degradation of these neurotransmitters and thus increases the circulation of them considerably. It occurs in the absence of pheochromocytoma, neuroendocrine tumors, and carcinoid syndrome, but it looks similar to carinoid syndrome such as facial flushing, aggression, and ADHD (Schultz, 2002).

2.2.3 EFFECT OF CATECHOLAMINES Catecholamines cause general physiological changes that prepare the body for physical activity (fight-or-flight response). Some typical effects are increase in heart rate, blood pressure, blood glucose levels, and a general reaction of the sympathetic nervous system. Some drugs like tolcapone (a central COMT-inhibitor) raise the levels of all the catecholamines.

~ 28 ~

2.2.4 FUNCTION IN PLANTS Catecholamines have been found in 44 plant families, but no essential metabolic function has been established for them. They are precursors of benzo[c]phenanthridine alkaloid, which are the active principal ingredients of many medicinal plant extracts. Catecholamine has been implicated to have a possible protective role against insect predators, injuries, and nitrogen detoxification. They have been shown to promote plant tissue growth, somatic embryogenesis from in vitro cultures, and flowering. Catecholamine inhibits indole-3-acetic acid oxidation and enhances ethylene biosynthesis. They have also been shown to enhance synergistically various effects of gibberellins (Kuklin et al, 1995).

2.2.5 STRUCTURE Catecholamines have the distinct structure of a benzene ring with two hydroxyl groups, an intermediate of ethyl chain, and a terminal amine group.

2.2.6 DEGRADATION Catecholamines have a half-life of approximately a few minutes when circulating in the blood.

Monoamine oxidase (MAO) is the main enzyme responsible for degradation of catecholamine.

Amphetamines and MAOIs bind to MAO in order to inhibit its action of breaking down catecholamine. This is primarily the reason why the effects of amphetamines have a longer lifespan than those of cocaine and other substances. Amphetamines not only cause a release of Dopamine, epinephrine, and norepinephrine into the blood stream but also suppress re-absorption.

2.2.7

EFFECT OF CATECHOLAMINES ON GASTRO INTESTINAL LESIONS

Previous biochemical and pharmacological studies have suggested the involvement of Dopamine in the pathogenesis of experimental duodenal and gastric ulcers in the rat (Takeuchi et al, 1990).

However, Szabo and colleagues demonstrated that Dopamine itself and its agonists markedly reduced the severity of cysteamine - induced duodenal lesions. Hernadez et al, (1990) showed a significant protection by Dopamine of the gastric mucosa against stress induced ulceration. The protective action of Dopamine in such ulcer models may be associated with inhibition of motility alterations of these tissues during lesion formation, but not with the amelioration of secretory disorders caused by cysteamine or stress (GutiĂŠrrez-Cabano, Candido A. 1995).

~ 29 ~

Acidified ethanol (60% ethanol in 150 mM HCL, per os) induced elongated bands of hemorrhagic lesions along the long axis of the stomach within 1hr in rats. Pretreatment with Dopamine hydrochloride (DA : 1 – 10 mg/kg, subcutaneously) dose- dependently reduced the severity of these lesion (Takeuchi et al, 1987). In parallel study, DA had no effect on acid secretion but inhibited gastric motor activity in a dose-related manner (Takeuchi et al, 1987). Similarly to DA, both norepinephrine (NEI : 1mg/kg, subcutaneously) showed inhibition of the motor activity and gastroprotection against acidified ethanol, but these effects are significantly attenuated by yohimbine, an inhibitor of α₂-adrenoceptors (5 mg/kg, subcutaneously), but not by prazosin, haloperidol, or indomethacin (Takeuchi et al, 1987). A highly significant relationship was found between the inhibitory effects of DA, NE, and EPI on the motor activity and the mucosal lesions. In addition administration of gentian violet (0.5% w/v, per os) stained the mucosa deep blue as elongated wide bands in the corpus region, and such localized staining was significantly prevented

by DA, suggesting a flattening of the mucosal folding in the presence of DA (Takeuchi et al, 1987).

These results suggest that DA (and other catecholamines) protects the rat gastric mucosa against injury caused by acidified ethanol, probably through inhibition of gastric motor activity mediated with stimulation of α₂ - adrenoceptors.

Of interest also is the finding that incubation of rat stomach, fundus and other tisssues with catecholamines in vitro increased the production of prostaglandin E₂ (Pace-Asciak, 1972; Collier, McDonald-Gibson and Seed, 1976) which has the property of protecting gastric mucosa against injury (Robert et al., 1979).

~ 30 ~

2.3 2.3.1

PEPTIC ULCER CONCEPT OF PEPTIC ULCER

A peptic ulcer, also known as PUD disease is an abrasion of the mucosa caused principally by the digestive action of the gastric juice (Peek and Blaster, 1997). As much as 80% of ulcers are associated with Helicobacter Pylori, a spiral shaped bacterium that lives in the acidic environment of the stomach, however only 20% of those cases go to a doctor. Ulcers can also be caused or worsen by drugs such as Aspirins and other NSAIDs (Non steroidal anti â&#x20AC;&#x201C; inflammatory drugs). Contrary to general believe, more peptic ulcers arise in the duodenum (first part of the small intestine, just after the stomach) than in the stomach (G.I Consult, 2007). Acid peptic disorders are very common in the United States, with 4 million individuals (new cases and recurrences) affected per year. Life time prevalence of PUD in the United States is approximately 12% in men and 10% in women. Also, an estimated 15, 000 deaths occur per year as a result of complicated peptic ulcer disease (PUD).

Financial impact of these common disorders has been substantial with an estimated burden of health care costs of more 15 billion dollars per year in the United States (Walsh and Peterson, 1995).

An ulcer is an excoriated area of the mucosa caused principally by the digestive action of gastric juice (Barr et al, 1983). An ulcer in the lining of the stomach or duodenum where hydrochloric acid pepsin are present is referred to as peptic ulcer (Barr et al, 1993) when the ulceration is in the stomach it is called gastric ulcer and when it is present in the duodenum, it is referred to as duodenal ulcer. Gastric and duodenal ulcers are together called peptic ulcers. And although ulcers can occur in the esophagus (reflux esophagitis), gastric and duodenal ulcers are more common. Peptic ulcer can be acute or chronic. Acute peptic ulcer is characterized by multiple and superficial lesion. It may or may not be asymptomatic and often heals without scaring (Ohara et al., 1995). Chronic type is characterized by single and deep lesion which heals with scar formation (Ohara et al, 1995). It is persistent and symptomatic.

Duodenal ulcer is characterized by pain which follows a consistent pattern. It occurs at a particular time of the day or during episode of emotional stress but can be relieved by food. The pain of gastric ulcer does not follow a consistent pattern and is aggravated by food intake (Hollander et al, 1994).

~ 31 ~

2.3.2

CLASSIFICATION OF PEPTIC ULCER

A peptic ulcer may arise at various locations - Stomach (called gastric ulcer) - Duodenum (called duodenal ulcer) - Esophagus (called esophageal ulcer) - Meckelâ&#x20AC;&#x;s Diverticulum (called Meckelâ&#x20AC;&#x;s diverticulum ulcer)

Types of peptic ulcers

2.3.2.1 -

Type I: Ulcer along the lesser curve of the stomach Type II: Two ulcers present; one gastric, one duodenal Type III: Pre pyloric ulcer Type IV: Proximal gastro-esophageal ulcer (G.I Consult, 2007).

2.3.3

ETIOLOGY OF PEPTIC ULCER

In the Industrial world the prevalence of Helicobacter Pylori infections in the general population rises steadily with age and in the UK, approximately 50% of those over the age of 50 years are infected (Feldman et al., 2002). Around 90% of ulcer patients and 70% of gastric ulcer patients are infected with Helicobacter Pylori; the remaining 30% of gastric ulcer are due to non-steroidal antiinflammatory drugs [NSAIDs] (Feldman et al., 2002). The usual cause of peptic ulceration is an imbalance between the rate of secretion of gastric juice and the degree of protection afforded by the gastro duodenal mucosal barrier and the neutralization of the gastric acid by duodenal juices (Peck and Blaser, 1997).

Other specific causes of peptic ulcer include: -

Helicobacter Pylori

-

Non- steroidal anti- inflammatory drugs [NSAIDs]

-

Smoking

-

Alcohol

-

Foods; such as cassava containing meals, spices, coffee and caffeine containing beverages.

-

Increase secretion of acid-peptic juices.

-

Stress.

~ 32 ~

1.

HELICOBACTER PYLORI

According to the University of Maryland medical journal (2008), 80% of gastric ulcers and 90% of duodenal ulcers developed as a result of infection with helicobacter pylori which colonizes the antral mucosa. Bacterial infection by helicobacter pylori breaks down the gastro-duodenal mucosal barrier both by its physical ability to burrow through the barrier and by releasing bacterial digestive enzymes that liquefy the barrier (Bowen, 2003). As a result, the strong acidic digestive juices of the stomach secretions can then penetrate into the underlying epithelium and literally digest the epithelial cells. In several cases they even digest the deeper tissues and this leads to peptic ulceration (Peek and Blaser, 1997; Bowen, 2003).

2.

NON-STAROIDAL ANTI-INFLAMMATORY DRUGS [ NSAIDS]

Another major cause is the use of non-steroidal anti-inflammatory drugs [NSAIDs] such as aspirin, Ibuprofen, and Naproxen sodium. The stomach and duodenum with a layer of mucosa wall protects itself from the gastric acid secreted by the parietal cells. Gastric acid is one of the main secretions of the stomach, together with several enzymes and intrinsic factors (Dâ&#x20AC;&#x2122; Souza and Dhume, 1991) chemically, it is an acid solution consisting mainly of hydrochloric acid (Hcl) and small quantities of potassium chloride (Kcl) and sodium chloride (Nacl) (David, 1951). Mucous secretion is stimulated by certain prostaglandins.

NSAIDs block the function of cyclooxygenase [COX -1], which is essential for the production of these prostaglandins (Cullen et al., 1997). NSAIDs such as Aspirin are widely used to combat pain and treat arthritis, but have a strong propensity for breaking down the mucosal barrier (Hojgaard et al, 1996).

3.

SMOKING

Smoking leads to atherosclerosis and vascular spasms, causing vascular insufficiency and promoting the development of ulcers through ischemia (Peek and Blaser, 1997). Also, studies have shown that smoking increases the leaving process of already existing smokes and contributes to ulcer reoccurrence. This is due to increase nervous stimulation of stomach secretory glands (Barr et al, 1983).

~ 33 ~

4.

ALCOHOL Although no proven link has been found between alcohol consumption and peptic ulcers, ulcers are more common in people who have liver cirrhosis, a disease that occur as a result of heavy alcohol consumption (Pavo et al, 2000; Barr et al, 1983).

5.

INCREASED SECRETION OF ACID- PEPTIC JUICES

In most people, who have peptic ulcer in the initial portion of the duodenum, the rate of gastric acid secretion is greater than normal, sometimes as much as twice normal. Although

part of this

increased secretion may be stimulated by bacterial infection, experiments in animals plus evidence of excess neutral stimulation of gastric acid secretion in human beings with peptic ulcer suggest that excess secretions of gastric juices for any reason [for instance, even in psychic disturbances] is often a primary cause of peptic ulceration (Peek and Blaser, 1997).

6.

STRESS

Although emotional stress is no longer thought to be a cause of ulcers, people often report that emotional stress increases ulcer pain (Barr et al, 1983). Physical stress however may increase the risk of developing ulcers especially in the stomach. For example, people with injuries such as burns and people undergoing major surgery often require rigorous treatment to prevent ulcers and ulcer complications.

The acute diffuse lesion caused by stress is pathologically and clinically distinct from chronic peptic ulcer disease, stress such as sepsis, shock severe trauma, surgery and excessive burns [CURLING ULCER] tend to cause peptic lesion. Cushingâ&#x20AC;&#x;s ulcer associated with brain injury such as surgery, traumatic and cerebrovascular accidents, ischemia, mucus deficiency and breaking of barriers to hydrogen have been involved in the pathogenesis of stress lesion (IVY, 1981; Morton, 1987).

2.3.4

SIGNS AND SYMPTOMS

Although some types of gastric lesion like acute peptic ulcers may not always cause symptoms (Brody et al, 1998), the most common ulcer symptoms include gnawing or burning pain in the abdomen between the breast bone and the navel, and often occur between meals in the early hours of the morning (Ohara et al, 1995). It may last for four minutes to a few hours.

~ 34 ~

Less common ulcer symptoms include, belching, nausea, vomiting, poor appetite, loss of weight, feeling tired and general body weakness. Duodenal ulcers are exacerbated by it. However, the symptoms of gastric and duodenal ulcers may resemble other digestive disturbances, therefore, consultation with a physician is necessary when the above symptoms are noticed (Brody et al, 1998).

Other signs and symptoms of ulcer may include; bloating and abdominal fullness, water brash (rush of saliva after an episode of regurgitation to dilute the acid in esophagus), hematesis (Vomiting of blood; this can occur due to bleeding directly from a gastric ulcer, or from damage to the esophagus from severe/continuing vomiting), melena (tarry, foul-smelling faeces due to bioxidized iron from hemoglobin), and although very rare, an ulcer can lead to a gastric or duodenal perforation. This is extremely painful and requires immediate surgery.

2.3.5

COMPLICATIONS OF PEPTIC ULCER

If quick treatment is not administered, an individual with ulcers may experience serious complications which may include: I.

HEMORRHAGE: acid gastric juice may cause the development of many tiny ulcers, or gastric erosions, leading to multiple anemias (Ros,s 1970). When a major artery is eroded, a serious and possibly life threatening hemorrhage may occur causing; shock, hematemesis, and melena (Ross, 1970).

II.

PERFORATION: When an ulcer erodes through the full thickness of the wall, of the stomach and duodenum, their contents enter the peritoneal cavity, causing acute peritonitis. Infected inflammatory materials may collect under the diaphragm, forming a subphrenic abscess and the infection may spread through the diaphragm to the pleural cavity (Ross, 1970).

III.

PYLORI STENOSIS: chronic ulceration could lead to pylori and duodenal stenosis. This causes blockage of food passage into the duodenum. The obstruction leads to dilatation of the stomach and this may proceed until the victus fills the abdomen (Ross, 1970).

IV.

PENETRATION INTO OTHER ORGANS: penetration occurs when the ulcer has extended into the adjacent organs rather than into free peritoneal space. Penetration occurs particularly into the head of the pancreas. This occurs frequently with duodenal failure (Charles and Bruce 1969).

~ 35 ~

V.

OBSTRUCTION: obstruction, the ulcer blocks the path of food trying to leave the stomach. Minor degrees of obstructions are present in about 20 â&#x20AC;&#x201C; 25% of patients with duodenal ulcers (Russel and Norman, 1993).

VI.

DEVELOPMENT OF A MALIGNANT TUMOR: this may complicate gastric ulceration.

2.3.6

DIAGNOSIS

An esophagogastroduodenoscopy [EGD], a form of endoscopy also known as gastroscopy, is carried out on patients in whom a peptic ulcer is suspected. And because treatment protocols may be different for different types of ulcers (Walsh and Perterson, 1995), it is advisable to adequately diagnose before commencing treatment. For example, NSAIDs induced ulcer treatment is quite different from the treatment for a peptic ulcer caused by the bacterium Helicobacter pylori (Walsh and Peterson 1995; Hollander, 1994).

By direct visual identification, the location and severity of the ulcer can be described. Moreover, if no ulcer is present, EGD can often provide an alternative diagnosis. There are a number of options available for diagnosing ulcer and testing for H. pylori bacterium. These include: a) Upper gastrointestinal series in an x-ray of the esophagus, stomach and duodenum to locate an ulcer which is made visible on the X-ray image by swallowing barium. b) Urea breathe test. This is non invasive and does not require EGD c) Direct culture from an EGD biopsy specimen. d) Endoscopy. Here, a small flexible instrument with a camera at the end is inserted through the mouth into the oesophagus, stomach and duodenum to view the entire upper GIT (Ohara et al, 1995). e) Direct detection of urease activity in a biopsy specimen by rapid urease test. f) Measurement of antibody levels in blood. This too does not require EGD. g) Stool antigen test. h) Histological examination and staining of an EGD biopsy.

~ 36 ~

i) Blood and stomach tissue test can be performed to detect the presence of H. pylori. However, some of these tests may give false negative result in people who recently took omeprazole or bismuth (Walsh and Peterson, 1995).

The possibility of other causes of ulcer, notably malignancy [gastric cancer] needs to be kept in mind. This is especially true in ulcers of the grater [large] curvature of the stomach; most are also a consequence of chronic H. pylori infection. If a peptic ulcer perforates, air will leak from inside the gastrointestinal tract [which always contains some air] to the peritoneal cavity [which normally never contains air]. This leads to free gas within the peritoneal cavity. If the patient stands erect, as when having a chest X-ray, the gas will float to a position underneath the diaphragm. Therefore gas in the peritoneal cavity, shown on an erect chest X-ray or supine lateral abdominal X-ray is an omen of perforated peptic ulcer disease (Marshall, 1983).

2.3.7

MANAGEMENT / TREATMENT OF ULCER

The management of a patient with peptic ulcer disease involves a detailed discussion with the patient to confirm that his/her story is in agreement with most of the already discussed causes and clinical presentations. Specific treatment will be determined by a physician based on:  Age, overall health, and medical history of the patient.  Patients‟ tolerance for specific medication procedures or therapies.  Expectations of the cause of the disease.  Extent of the disease.  Patient‟s opinion or preferences.

Treatment of peptic ulcer involves general measures and specific measures (Edwards et al., 1995; Flashe and Akinkugbe, 2000) a.

GENERAL MEASURES: the patients are advised to quit smoking. Aspirin and other NSAIDs

are discouraged because they cause gastrointestinal distress. Diet modification and regulation has been demonstrated to promote relief from pain and promote healing. Strong coffee or tea should be avoided because they stimulate nocturnal gastric secretion. Also heavy consumption of alcohol is discouraged as well as food such as high pepper diet, coffee and caffeine-containing drinks.

~ 37 ~

b.

SPECIFIC MEASURES:

This involves eradicating H. pylori [bacterium] organisms using

drugs such as colloidal bismuth subcitrate; one tablet (400mg) 8-hourly for two weeks; these drugs are used in combination.

When H. pylori infection is present, the most effective treatments are combinations of 2 antibiotics (e.g. clarithromycin, amoxicillin, tetracycline, and metronidazole) and 1 proton pump inhibitor (PPI), sometimes together with a bismuth compound. In complicated, treatment-resistant cases, 3 antibiotics (e.g. amoxicillin + clarithromycin + metronidazole) may be used together with a PPI and sometimes with bismuth compound. An effective first time therapy for uncomplicated causes would be amoxicillin + metronidazole + rabeprazole (a PPI). In the absence of H. pylori, long term higher dose PPI are often used.

Another combination of drugs that could be used are as follows; Amoxycillin (500mg) 6-hourly and omeprazole 20mg 12-hourly for four weeks. Treatment of H. pylori usually leads to clearing of infection, relief of symptoms and eventual healing of ulcers. Recurrence of infection can occur and retreatment may be required if necessary with other antibiotics. Since the widespread use of PPI, in the 1990s, surgical procedures like â&#x20AC;&#x153;highly selective vagotomyâ&#x20AC;? for uncomplicated peptic ulcers became obsolete. Patients who are taking non steroidal anti-inflammatory drugs (NSAIDs) may be prescribed a prostaglandin analogue (misoprostol) in order to help prevent peptic ulcer, which may be a side effect of the NSAIDs. Other drugs that can be used to relieve symptoms include; I.

ANTACIDS: these are drugs which on ingestion neutralize the acids of the gastric content and thus lower the acidity of the content. By changing the acidity of the gastric content to pH 4.0 and above, antacids prevents irritation of the ulcer and relieves pain (Kazung, 2001). Antacids includes; -

Aluminium hydroxide gel

-

Magnesium salts

-

Magaldrate

-

Sodium bicarbonate

-

Calcium hydroxide and calcium carbonate

-

Bismuth subcitrate

~ 38 ~

II.

HISTAMINES {H2} â&#x20AC;&#x201C;RECEPTOR ANTAGONISTS: the stimulation of gastric acid secretion induced by histamines is due to activation of histamines H2- receptor blocking drugs. These drugs inhibit gastric acid secretion (Kazung, 2001). They include: Cimetidine, Ranitidine, Famotidine, Nizatidine, Roxatidine (Kazung, 2001).

III.

ANTIMUSCARINIC DRUGS [Antispamodics]: Atropine-like drugs blocks the action of acetylcholine on the muscarinic receptors and by this mechanism reduce the tone and motility of the gut. They may reduce the volume of gastric acid secretion but not the concentration (Kazung, 2001). Atropine is rarely used because of its side effects.

Dicyclomine, Ozyphencycline and Propantheline have been found useful in peptic ulcer, but are more often used as adjunct in gastrointestinal disorders characterized by smooth muscle spasm (Edwards et al., 1995)

IV.

MUCOSAL PROTECTIVE AGENTS: these compounds known as cytoprotective compounds have several actions that enhance mucosal protective mechanisms, thereby preventing mucosal injury, reducing inflammation and healing existing ulcers (Brunton, 1996). These agents include sucralfate, colloidal bismuth etc.

V.

PROTEIN PUMP INHIBITORS [PPIs]: These groups of drugs irreversibly inhibit the gastric parietal cell proton pump, H+ |K+ ATPase, the final pathway for hydrogen ion secretion. A single daily dose can effectively inhibit 100% of gastric acid secretion (Brunton, 1996; Ewart, 1990). The proton pump inhibitors currently available are Omeprazole, Lansprazole, Ransprazole, and Pantoprazole (Kazung, 2001).

Other

antiulcer

agents

includes:

metoclopramide,

Domperidone,

Cisapride,

Carbenoxolone sodium, tricyclic antidepressants, Somatostatin etc (Kazung, 2001).

2.3.8

EPIDEMIOLOGY

In Western countries the prevalence of H. pylori infections roughly matches ages (20% at age 20, 30% at age 30, 80% at age 80 etc). Prevalence is higher in third world countries. Transmission is by food, contaminated ground water and through human saliva [such as from kissing or sharing food utensils] (Huwez et al., 2008).

2.3.9

HISTORY

~ 39 ~

John Lykoudis, a general practitioner in Greece, treated patients for peptic ulcer disease with antibiotics, beginning in 1958, long before it was commonly recognized that bacteria were a dominant cause for the disease (Marshall, 2002).

Helicobacter pylori were rediscovered in 1982 by two Australian scientists, J. Robin Warren and Barry J. Marshall, as a causative factor for ulcers (Marshall, 1983). In their original paper, they contended that most stomach ulcers and gastritis were caused by colonization with this bacterium, not by stress or spicy food as had been assumed before (Marshall and Warren, 1984). The H. Pylori hypothesis was poorly received so in an act of self-experimentation, Marshall drank a Petri dish containing a culture of organisms extracted from a patient and soon developed gastritis. His symptoms disappeared after two weeks, but he took antibiotics to kill the remaining bacteria at the urging of his wife, since halitosis is one of the symptoms of infection (Van Der Weyden et al., 2005).

In 1997, the centers for disease control and prevention, with other government agencies, academic institutions and industry, launched a national education campaign to inform health care providers and consumers about the links between Helicobacter Pylori and ulcers. This campaign reinforced the news that ulcers are curable infection and that health can be greatly improved and money saved by disseminating information about H. Pylori (Marshall 1983).

In 2005, the karolinska institute in Stockholm awarded the nobel price in physiology/medicine to Dr. Marshall and his long time collaborate Dr. Warren for their discovery of the bacterium, Helicobacter pylori and its role in gastritis and peptic ulcer disease. Professor Marshall continues research about H. pylolri and runs a molecular biology laboratory at U.W.A in Perth, Western Australia. It was previously a widely accepted misunderstanding that the use of chewing gum resulted to peptic ulcers. The medical profession believed that this was because the action of mastication on gum caused the over stimulation of production of hydrochloric acid, which is believed to cause erosion of the stomach lining in the absence of food thus causing the development of gastric ulcers (Toohey, 1974).

On the other hand, in recent past, some believed that natural tree resin extract, mastic gum, actively eliminates the H. pylori bacteria (Humez et al., 1998). However, multiple subsequent studies have found no effect of using mastic gum on reducing H. pylori levels (Loughlin et al., 2003; Bebb et al., 2003).

~ 40 ~

CHAPTER THREE 3.1

MATERIALS The materials used for this research include the following: 

Unripe plantain



Formaldehyde



Distilled water



0.9% Normal saline



Cotton wool and thread



Beakers and test tubes



Syringes and needles



Dissecting set and board



Tap water



Weighing balance



10ml pipette and measuring cylinders



Mortar and pestle



Wooden and plastic cages



Sodium bicarbonate



Thiopental



Epinephrine(Adrenaline)



Dopamine



Cimetidine



Methanol



Air-tight bottles



Rotary Evaporator



Manual grinding machine

~ 41 ~

3.2

PREPARATION OF PLANT MATERIAL

Musa paradisiaca was purchased from the local market in Elele, Rivers State. The fruits were peeled and the pulps were sliced and dried under the sun for ten days. The dried plantain pulps were grinded into a coarse form using a corona Japan made grinding machine, and were pounded into a powder form using a mortar and a pestle. The pounded material weighing 500g was stored in airtight bottles for extraction. 150g of the powdered materials was packed into a soxhlet apparatus and extracted by maceration with 350ml of methanol for 48 hours. The methanol was evaporated using a rotary evaporator. The filtrate was further concentrated using an electric incubator. The yield obtained was 30.25g. Consequently the residue from the extract was dissolved in saline and used in the study.

3.3

PHYTOCHEMICAL SCREENING

3.3.1

Preparation of Reagents for Phytochemical Analysis

i.

5% Ferric Chloride Solution A quantity (2.5g) of ferric chloride was dissolved in 50ml of distilled water.

ii.

Ammonium Solution A quantity (375ml) of the stock concentrated ammonium solution was dissolved in 62.5ml of distilled water and made up to 1000ml.

iii.

45% Ethanol A quantity (45ml) of absolute ethanol was mixed with 55ml of distilled water.

iv.

Aluminium chloride solution A quantity (0.5g) of aluminium chloride was dissolved in 100ml of distilled water.

v.

Dilute sulphuric acid A quantity (10.4ml) of concentrated sulphuric acid was mixed with 5ml of distilled water and made up to 100ml.

~ 42 ~

vi.

Lead sub acetate solution A quantity (45ml) of 15% lead acetate solution (i.e. 7.5g of lead in 50ml of distilled water) was dissolved in 20ml of absolute ethanol and 35ml of distilled water.

vii.

Wagner’s reagent A quantity (2g) of iodine crystals and 3g of potassium iodide, were dissolved in 100ml of distilled water.

viii.

Mayer’s reagent A quantity (1.35g) of mercuric chloride was dissolved in 60ml of distilled water. Also, 5g of potassium iodide were dissolved in 20ml of distilled water. The solutions were mixed and the volume made up to 100ml.

ix.

Dragendorff’s reagent A quantity (0.85g) of bismuth carbonate was dissolved in 100ml of glacial acetic acid and 40ml of distilled water, to give solution A. another solution called solution B was prepared by dissolving 8.0g of potassium iodide in 20ml of distilled water. Both solutions were then mixed to give a stock solution-Dragendorff‟s reagent.

x.

Molisch reagent A quantity (0.1g) of a-naphthol was dissolved in 100ml of ethanol.

xi.

2% Hydrochloric acid A quantity (2ml) of concentrated hydrochloric acid was dissolved in distilled water and made up to 100ml.

3.3.2 Qualitative Phytochemical Analysis of the Extract The phytochemical analysis was carried out by testing for the presence or absence of plant constituents as glycosides, proteins, alkaloids, saponins, flavonoids, carbohydrates, reducing sugars, and tannins (Aliyu, 1998).

~ 43 ~

a) Tests for Flavonoids 10ml of ethylacetate were added to 0.2g of the powdered plant material and heated on a water bath for 3 min. The mixture was cooled, filtered and the filtrate used for the following tests.

Ammonium Test 4ml of the filtrate were shaken with 1ml of dilute Ammonia solution. The layers were allowed to separate and the yellow colour in the ammoniacal layer indicates the presence of flavonoids. % Aluminium Chloride Solution Test 4ml portion of the filtrate was shook with 1ml of 1% aluminum chloride solution. The layers were allowed to separate. A yellow colour in the aluminum chloride layer indicates the presence of flavonoids. b) Test for Proteins Millonâ&#x20AC;&#x2122;s Test To a little portion of the filtrate in a test tube, two drops of millions regent was added. A white precipitate indicates the presence of proteins.

Xanthoproteic Reaction Test 5ml of the filtrate was heated with few drops of concentrated nitric acid; a yellow colour which changes to orange on addition of an alkali indicates the presence of protein.

Biuret Test A crystal of copper sulphate was added to 2ml of the filtrate, and then 2 drops of potassium hydroxide was added. A purple or pink colour shows the presence of proteins.

Picric Acid Test To a little portion of the filtrate was added a few drops of picric acid. A yellow precipitate indicates the presence of proteins. c) Test for Alkaloids

~ 44 ~

1g of the plant material was boiled for 2 minutes with 5 ml of 2% hydrochloric acid in a steam bath and the material filtered. A volume (1ml portion) of the filtrate was treated with 2 drops of the following reagents and observed for precipitate.

Mayer‟s reagent (potassium mercuric iodide solution) Dragendorff‟s reagent (bismuth potassium iodide solution) Wagner‟s reagent (iodine in potassium iodide solution) 1% picric acid solution The remaining was placed in 100ml separatory funnel and made alkaline with dilute ammonia solution. The aqueous alkaline solution was allowed for ten minutes and added 25 ml chloroform, shaken and allowed for 4 minutes. The chloroform layer was separated and extracted with 5ml portions of dilute sulphuric acid. The extract was tested with few drops of Mayer‟s, Wagner‟s and Dragendorff‟s reagent. Alkaloids gives a milky precipitate with one drop of Mayer‟s reagent, reddish-brown precipitate with one drop of Wagner‟s reagent, yellow precipitate with one drop of picric acid reagent or brick red precipitate with one drop of Dragendorff‟s reagent. d) Test for Saponins 1g of the powder was boiled with 5ml of distilled water for 5 minutes and the mixture was filtered hot and allowed to cool. The filtrate was then used for the following tests: Frothing Test 1ml of the filtrate was diluted with 4ml of distilled water, shaken vigorously and observed on standing. A stable froth (foam) indicates the presence of saponins.

Emulsion Test 2ml of Olive oil was added to 1ml of the filterate, shaken vigorously and observed. The formation of emulsion indicates the presence of saponins.

Haemolysis Test 1ml of the filtrate was added to 1 ml of a solution of the blood in normal saline. The occurrence of homolysis (shown by the sedimentation of the cells) indicates the presence of saponins.

~ 45 ~

e) Test for Glycosides Fehlingâ&#x20AC;&#x;s Test: To 5ml of a mixture of equal parts (50:50) of Fehlingâ&#x20AC;&#x;s solution I and II, was added 5ml of the extract and then heated on a water bath for 5 minutes, a brick red precipitate indicates the presence of a reducing sugar.0.1g of the plant material was boiled with 3ml of dilute sulphuric acid for 35 minutes.

Hydrolysis Test: 5ml dilute sulphuric acid were added to 0.1g of the powder in a test tube and boiled for 15 minutes in a water bath, then cooled and neutralized with 20% potassium hydroxide solution. 10 ml of a mixture of equal parts of Fehlingâ&#x20AC;&#x;s solutions I and II were added and boiled for 5 minutes. A more dense brick red precipitate indicates the presence of glycosides.

Cardiac glycosides 0.1g of ground sample was warmed at 45o C in 5ml of chloroform in a water bath for fifteen minutes and later decanted. The decanted solution was evaporated to dryness on a water bath and the residue dissolved in 3ml of glacial acetic acid containing a drop of ferric chloride solution. The solution was carefully poured into the second tube containing 3ml of concentrated sulphuric acid. A reddish-brown ring at the interface indicates presence of cardiac glycosides.

Cyanogenic Glycosides 10g of the ground sample was put in a conical flask and about 15ml of distilled water was added to cover the sample. A piece of sodium picrate paper was suspended in the flask placed in a boiling water bath for an hour. Change in the colour of the sodium picrate paper from yellow to orange shows the presence of cyanogenic glycosides.

O- and C-glycosides 0.1g of the ground sample was heated in 5ml of water in a boiling water bath for fifteen minutes, cooled and filtered. The filtrate was treated with 5ml of 25% HCL and the solution heated again for fifteen minutes and then cooled in 10ml of diethylether in a separating funnel. Two layers were formed namely acid layer and organic layer. Both layers were then examined for the presence of a purple red colour.

~ 46 ~

The ether extract occupying the lower layer (2ml) was mixed with 5ml of 3.5 % dilute ammonia solution and shaken. To an aliquot of the acid phase (2ml) was added to 0.5g of ferric chloride crystals, heated for thirty minutes in a water bath and then cooled. The solution was extracted with 10ml of chloroform and washed with 5ml of distilled water. 3ml of 3.5% dilute ammonia solution was then added. A dark-red precipitate indicates the presence of O-and C-glycosides while a colourless solution indicates absence of O-and Cglycosides. f) Test for Terpenoids 9ml of ethanol were added to 1g of the powder extract and refluxed for a few minutes and then filtered. The filtrate was concentrated to 2.5ml on a boiling water bath and 5ml of hot water added. The mixture was allowed to stand for 1hr and the waxy matter filtered off. The filtrate was extracted with 2.5ml of chloroform using separatory funnel.

To 0.5ml of the chloroform extract in a test tube was added 1ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface shows the presence of steroids.

Another 0.5ml of the chloroform extract was evaporated to dryness on a water bath and heated with 3ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the presence of terpenoids. g) Test for Reducing Sugars 0.1g of the extract was shaken vigorously with 5ml of distilled water and filtered. The filtrate was used for the following test: Fehling’s Test To 1ml portion of the filtrate were added equal volumes of the Fehling solutions I and II and boiled on a water bath for a few minutes. A brick red precipitate indicates the presence of reducing sugar.

Benedict’s Test To 1ml portion of the filtrate, 2ml of Benedict‟s reagent were added. The mixture was shaken, heated on a water bath for 5 minutes. A rusty brown precipitate indicates the presence of reducing sugars.

~ 47 ~

h) Test for Starch Iodine Test 0.1g of the extract was mixed with a drop of iodine solution. A blue-black colour indicates the presence of starch. i) Test for Resins Precipitation Test 0.2g of the powered material was extracted with 15ml of 96% ethanol. The alcoholic extract was then poured into 20ml of distilled water in a beaker. The formation of precipitate indicates the presence of resins.

Colour Test 0.2g of the powdered material was extracted with chloroform and the extract concentrated to dryness. The residue was re-dissolved in 3ml Acetone and 3ml of concentrated HCl added.

This mixture was heated in a water bath for 30 minutes. A pink colour which changes to magenta, red indicates the presence of resins. j) Test for the presence of Tannins 2g of the ground plant material was boiled in 5ml of 45% ethanol for five minutes; the mixture was cooled and then filtered. The resulting filtrate was used for the following tests.

Bromine water 0.5ml of bromine water was added to 1ml of the filtrate and then observed. A pale brown precipitate indicates the presence of tannins.

Lead sub-acetate Three drops of lead sub-acetate solution were added to 1ml of the filtrate. A gelatinous precipitate indicates the presence of tannins.

Ferric chloride 1ml of the filtrate was diluted with 5ml of distilled water and two drops of ferric chloride solution were added. A transient colour midway between green and black indicates the presence of tannins.

~ 48 ~

3.3.3

Quantitative Phytochemical analysis of the Extract

Determination of total phenolic contents Total phenolics were determined using Folin-Ciocalteu reagent as described by Velioglu et al.

(1998), with slight modifications. Folin-Ciocalteu reagents (FCR) consist of a yellow acidic solution containing complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids. Dissociation of a phenolic proton in a basic medium leads to a phenolate anion, which reduces FCR forming a blue coloured molybdenum oxide. The colour intensity is directly proportional to the phenolic contents (Huang et al., 2005). Briefly, 100μl of the extract dissolved in methanol (1mg/ml) was mixed with 750μl of Folin-Ciocalteu reagent (diluted 10-fold in dH2O) and allowed to stand at 22 oC for 5 min; 750μl of Na2CO3 (60 g/l) solution was then added to the mixture. After 90 min the absorbance was measured at 725nm. Results were expressed as gallic acid equivalents.

Determination of tannin contents Tannin content in each sample was determined using insoluble polyvinyl-polypirrolidone (PVPP), which binds tannins as described by Makkar et al. (1993). Briefly, 1 ml of extract dissolved in methanol (1mg/ml), in which the total phenolics were determined, was mixed with 100mg PVPP, vortexed, left for 15 min at 4 oC and then centrifuged for 10 min at 3000 rpm using a Fischer Scientific centrifuge. In the clear supernatant the non-tannin phenolics were determined the same way as the total phenolics (Velioglu et al., 1998). Tannin content was calculated as a difference between total and non-tannin phenolic content.

Determination of flavonoids and flavonols The flavonoids content was determined according to the method described by Kumaran and Karunakaran (2006) with slight modifications. This method is based on the formation of a flavonoid-aluminum complex, which absorbs maximally at 415nm. Briefly, 100μl of plant extracts in methanol (10mg/ml) was mixed with 100μl of 20% aluminium trichloride in methanol and a drop of acetic acid, and then diluted with methanol to 5ml. The absorption at 415nm was read after 40 min. Blank samples were prepared from 100μl of plant extracts and a drop of acetic acid, and then diluted to 5ml with methanol. The absorption of standard rutin solution (0.5mg/ml) in methanol was

~ 49 ~

measured under the same conditions. The amount of flavonoids in plant extracts in rutin equivalents (RE) was calculated by the following formula:

Where A is the absorption of plant extract solution, Ao is the absorption of standard rutin solution, m is the weight of plant extract, mg and mo is the weight of rutin in the solution, mg. The flavonoid content is expressed in mg rutin equivalents/mg plant extract. The content of flavonols was also determined as described by Kumaran and Karunakaran (2006) with slight modifications. Briefly, 1ml of each methanolic plant extracts (10 mg/ml) was mixed with 1 ml aluminium trichloride (20 mg/ ml) and 3 ml sodium acetate (50 mg/ ml). The absorbance at 440 nm was read after 2.5 h. The absorption of standard rutin solution (0.5mg/ml) in methanol was also measured under the same conditions. The amount of flavonols in plant extracts in rutin equivalents (RE) was calculated by the same formula for flavonoids.

3.4

ACUTE TOXICITY TEST The test was carried out according to the method of Dietrich Lorke (1981). 12 mice weighing

between 23 and 30g of both sexes and obtained from the animal house of the department of Medical physiology, university of Nigeria, Enugu campus, Enugu State were used for the study. Animal care and treatment were conducted in conformity with the institutional guidelines that are in compliance with international laws and policy. The experiment involves two stages.

STAGE I: This involves nine mice. The animals were divided into three groups of three rats each.

GROUP A: The mice in this group received 10 mg/kg body weight of the extract.

GROUP B: This group of mice received 100 mg/kg body weight of the extract.

GROUP C: Mice in this group received 1000 mg/kg body weight of the extract.

The animals were closely monitored for a period of twenty four hours and the number of deaths noted in each group.

~ 50 ~

STAGE TWO: This stage was carried out based on the results of the first stage and it involves three mice. The animals were grouped into three groups of one animal in each group.

GROUP A: This received 1500mg/kg of the extract.

GROUP B: This received 3000mg/kg of the extract.

GROUP C: This received 5000mg/kg of the extract.

The animals were monitored for another twenty four hours and the number of deaths were also noted.

3.5

ANIMAL MONITORING AND FEEDING

The animals used for this research were albino wistar rats. Forty of them were obtained from the animal house, department of physiology, university of Nigeria, Enugu campus, Enugu State. Upon arrival at Madonna University, Elele campus, they were weighed.

These rats of weight between 80 and 150g were randomly assign into four groups and kept in wooden cages with slated floor and fine iron netting, which were cleaned regularly. The animals were allowed three weeks of acclimatization to the animal house (25Ë&#x161;C to 30Ë&#x161;C; 14 hours light; 10 hours dark cycle).

During this period, they were fed with normal rat chow (Guinea feeds, with composition; protein, 14.5%; fat, 4.8; fiber, 7.2%; calcium, 0.8%; phosphorus, 0.62%; sodium, 0.15% and metabolizable energy, 2,300kcal/kg and water ad libitum.

3.6

EXPERIMENTAL PROCEDURES

The gastroprotective activity of the plant extract was assessed against indomethacin induced gastric lesions in the conscious rat.

On commencement of the experiment, the rats were fed as follows: Group 1: This served as the control cage. Members of this group were fed with normal rat chow and water ad libitum for the two weeks of the experiment.

~ 51 ~

Group 2: This group of rats was fed with the normal rat chow and 50 mg/kg of the extract for the two weeks of the research. Feeding stops 48 hours prior to the end of the second week. 50 mg/kg body weight of a 100% solution of adrenaline was injected intraperitonially into the rats 30 minutes before ulcer induction. Ulcer induction was by administration of indomethacin using oral route.

Group 3: The rats in this group were fed as in group 2 except that Dopamine (50 mg/kg body weight of a 100% solution) was injected into the rats in place of epinephrine.

Group 4: The same as in groups 2 and 3 except that there was no administration of catecholamine into the rats before ulcer induction.

3.6.1

ASSESSMENT FOR HEALING EFFECT OF MUSA PARADISIACA

Twenty Wistar albino rats were used for the experiment. The animals were fasted for 48 hours but were given water ad libitum prior to the experiment and then, the animals were given 50mg/kg of indomethacin orally. The animals were divided into 5 groups with 4 rats in each group.

Group I – which is the negative or ulcerated control were sacrificed after one hour with an overdose of thiopental. The stomachs were removed and the gastric lesions produced were measured.

Group II – received 1.0ml/kg normal saline (0.9% NaHCOȝ) orally for 14 days as the vehicle or ulcerated untreated control.

Group III – treated with 200mg/kg cimetidine orally for 14 days as the positive control.

Group IV– treated with the plant extract at a dose of 100mg/kg orally for 14 days.

Group V – treated with the plant extract at a dose of 200mg/kg orally for 14 days.

3.7

SAMPLE COLLECTION AND ASSESSMENT

After the fourteenth day, the animals were sacrificed with overdose of thiopental anesthesia. The stomach were removed, cut open along the greater curvature and the extent of cytoprotection and gastric ulcer healing were assessed (after fixing in 10% formalin) (Alphine and Word, 1969). The gastric lesions were measured in millimeter and scored as described by Kulkarni, 1996. The ulcer index for each rat was taken as the mean ulcer score. The surface area of each lesion was also determined as well as the percentage ulcerated surface.

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The percentage inhibition was calculated using 100 â&#x20AC;&#x201C; (A/B x 100/1) Where, A = Group treatment mean value (ulcer index) B = Control mean value (ulcer index)

3.8

STATISTICAL ANALYSIS

The results obtained from this study were analyzed using the Statistical Package for Social Sciences (SPSS) version 17.0 for Windows. Analysis of variance (ANOVA) was used to compare means, and values were considered significant at P<0.05. Post Hoc multiple comparisons for differences between groups within groups were established using least significant difference (LSD), Tukey, Scheffe and Duncan. Results are presented as mean Âą S.E.M.

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CHAPTER FOUR RESULTS

The table below shows the outcome of the phytochemical analysis and the experiments.

TABLE I: QUALITATIVE PHYTOCHEMICAL ANALYSIS OF UNRIPE PLANTAIN (MUSA PARADISIACA) CONSTITUENTS

DISTINGUISHING FACTOR

Glycosides

Brick red precipitate

++

Acidity

Red in litmus paper

-

Alkaloids

Cream precipitate

+++

RELATIVE PRESENCE

Redish brown precipitate Orange red precipitate Flavonoids

Yellow colour

+

Tannins

Light Brown precipitate

++

Cyanogenic glycosides

Orange colour

-

Resins

Precipitate and magnet red

-

O &C glycosides

Dark red precipitate

++

Terpenoids

Grey colour

-

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Carbohydrate

Blue black colour

+++

Protein

White precipitate

+

Saponins

Hemolysis

NB:

+++

+

= Present in low amount

++

= Present in moderate amount

+++

=

Present in high amount

-

=

Absent

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TABLE 2: QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF UNRIPE PLANTAIN (MUSA PARADISIACA) CONSTITUENTS

AMOUNT



Total phenol

0.13



Tannin

0.12



Non- Tannin

0.01



Flavonoid

0.01



Flavonol

NB:

0.09

* mg gallic and equivalent/mg dry weight extract 

mg rutin equivalent/mg dry weight extract

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TABLE 3: TOXICITY TEST RESULTS Stage

Group

№ of Mice

Dose (mg/kg)

№ of Deaths

I

A

3

10

mg/kg

0

B

3

100 mg/kg

0

C

3

1000 mg/kg

0

A

1

1500 mg/kg

0

B

1

3000 mg/kg

0

C

1

5000 mg/kg

0

II

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TABLE 4: DUAL EFFECT OF MUSA PARADISIACA ON PEPTIC ULCER AND IN COMBINATION WITH CATECHOLAMINE Group

N

Treatment Group

Nature treatment

I

5

Ulcerated control

_

_

II

5

Extract+ Epinephrine

Prophylaxis

50 mg/kg 8.2± 1.4*

44.60%

III

5

Extract + Dopamine Prophylaxis

50 mg/kg 4.8 ± 1.7*

67.57%

IV

5

Extract alone

50 mg/kg 3.0 ± 1.1*

79.73%

Prophylaxis

of Dose (mg/kg)

Ulcer index % Inhibition 14.8±3.5*

0%

*Statistical significant relative to control if p < 0.05 N = number of rats The oral administration of 50mg/kg of indomethacin produced macroscopically visible hemorrhagic lesion in the glandular portion of the stomach. The ulcer index of the control was 14.8 ± 3.5. The percentage ulcer inhibition of Musa paradisiaca in combination with epinephrine and Dopamine was 44.60% and 67.57% respectively at a mean index of 8.2 ± 1.4 and 4.8 ± 1.7. While the extract alone produced a significant (p < 0.05) cytoprotective effect (79.73% inhibition) at a mean ulcer index of 3.0 ± 1.1. There is also a significant (p < 0.05) decrease in mean ulcer index from 14.8 ± 3.5 to 1.2 ± 0.49 in group V (91.89%).

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TABLE 5: HEALING EFFECT AND % INHIBITION OF UNRIPE PLANTAIN EXTRACT Treatment

N

Ulcerated control

4

Ulcerated untreated control

4

Cimetidine

4

Extract Extract

Dose

Ulcer Index ± S.E.M

% Ulcer Healing

-

29.5 ± 1.04*

0

0.5 ml

21.0 ± 1.96*

28.81

100 mg/kg

3.0 ± 0.41*

89.83

4

100 mg/kg

5.5 ± 1.26*

81.35

4

200 mg/kg

2.0 ± 0.81*

93.22

Normal saline

*Statistically significant relative to control if p < 0.05 N = number of rats The oral administration of 50mg/kg of indomethacin produced macroscopically visible hemorrhagic lesion in the glandular portion of the stomach. The ulcer index of the control was 29.5 ± 1.04 and 21 ± 1.96 in the ulcerated untreated control. The percentage ulcer inhibition of Cimetidine was 89.83% with an ulcer index of 3.0 ± 0.41. The extract at 100 mg/kg produced a significant (p < 0.05) dose dependent healing effect (81.35% inhibition) at a mean ulcer index of 5.5 ± 1.26 and a significant (p < 0.05) mean ulcer index from 29.5 ± 1.04 and 21 ± 1.96 to 2.0 ± 0.81 in group V (93.22%) at 200mg/kg.

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CHARTS 18 16 Mean Ulcer Index

14 12 10 8 6 4 2 0 Ulcerated Control

Extract + Epinephrine

Extract+ Dopamine

Extract Alone

Treatment Groups

% Inhibition

Fig: Dual effects of Musa paradisiaca and in combination with catecholamines on indomethacin induced peptic ulcer. Result shown are mean ulcer index.

90 80 70 60 50 40 30 20 10 0 Ulcerated Control

Extract + Epinephrine

Extract + Dopamin

Extract alone

Treatment Groups

Fig: Percentage inhibition of the methanolic extract of unripe plantain and in combination with catecholamines on indomethacin induced peptic ulceration in wistar albino rats.

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35

Ulcer index

30 25 20 15 10 5 0

Ulcerated Control Ulcerated ulcerated Control

Cimetidine

Extract (100 mg)

Extract (200 mg)

Treatment Groups

Fig. 1: Healing effect of the methanolic extract of Musa paradisiaca on indomethacin induced ulceration. Results shown are the mean ulcer index.

120

% Inhibition

100 80 60 40 20 0 Ulcerated Control

Ulcerated untreated control

Cimetidine

Extract 100 mg/kg Extract 200 mg/kg

Treatment Groups

Fig. 2: Percentage inhibition of the methanolic extract of Musa paradisiaca on indomethacin induced ulceration in wistar albino rats.

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CHAPTER FIVE DISCUSSION AND CONCLUSION 5.1

DISCUSSION

5.1.1 PHYTOCHEMICAL SCREENING From the phytochemical screening, Musa paradisiaca tested positive for tannins, flavonoids, glycosides, alkaloids, carbohydrate, protein, and saponins. It however tested negative for acidity, cyanogenic glycosides, resins and terpenoids.

According to Ngo (2008), high tannin content in plantain has antibiotic activity. Tannin in the fruit also help, as explained by Robinson (1996) to protect the lining of the intestinal tract against loss of liquids and so is potent in the treatment of dehydration in infants.

Flavonoids have been reported to possess both anti ulcer and anti-inflammatory activities (Kontureck et al., 1986; Goel et al., 1988). Also according to Lewis et al (1999) a flavonoid, leucocyanidin isolated from unripe plantain banana pulp has an anti ulcer activity. Bate-Smith and Swain (1962), also showed that flavonoids help to protect plants against external pathogens, ultra-violet light or heat and that Flavonoids present in plantains possess anti-inflammatory properties and act as modulators of the immune system. This stems from the fact that they are powerful antioxidants protecting biosystems against damaging effects of free radicals as was observed by Bohan and Kocipai, (1994). Human studies as explained by the work of Nishino et al (1984) suggested that the flavonoids present in plantain may help reduce the risk of cardiovascular disease and stroke.

Ajali (2004) described glycosides as compounds containing a carbohydrate and noncarbohydrate residue in the same molecule. Ghosal and coworkers in 1984 reported the anti-ulcerogenic activity of sterylacylglycosides 1 â&#x20AC;&#x201C; IV isolated from Musa paradisiaca Linn against ulcers in rats and humans. According to literature plantain serves as a rich source of energy because of its high energy value, 75 â&#x20AC;&#x201C; 115kcal/100mg of pulp and also contains complex carbohydrates capable of replacing glycogen and important vitamins. This is also supported by the work of Robinson (1996).

Literature has equally shown the biological activities of alkaloids to include hypoglycermia, hypolipidermia and hypotension among other biological activities as shown by Oladele et al (1995) and Sudheesh et al., (2005). Ajali (2004) also reported that the alkaloid present in plantain is a very effective pain killer and can also be used as a cough suppressant and as an antidiarrheal drug.

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On the basis of recent experimental investigations, saponins are claimned to have hypocholesteromic, immunostimulatory and anticarcinogenic properties (Ajali, 2004). Saponins nonetheless have harmful effects. Certain pasture weeds contain substantial quantities of dangerous saponins and result in life threatening toxicities for certain animal species. Symptoms of toxicity of saponins range from listlessness, anorexia, weight loss, gastroenteritis to coma and death (Cheeke, 1998). It is interesting, however, that human beings do not suffer severe poisoning from saponins (Ajali, 2004). A new application of saponins in animal husbandry has been explored. It involves the use of extracts containing large amount of saponins in treating protozoan disease. The principle underlying this is the fact that saponins form strong insoluble complexes with cholesterols in the cell membrane of protozoans found in the gut, thus destroying the protozoan causing disease as described by Peter (1998).

Plantainâ&#x20AC;&#x;s low fat content also makes it recommendable for people with cardiovascular and kidney problems, arthritis gout or gastrointestinal ulcers particularly when a low fat, low sodium, and/or cholesterol-free diet is required as explained by Robinson (1996). Plantain can also be a good source of protein.

5.1.2 ACUTE TOXICITY TEST Test for toxicity of the extract showed that at a dose of 5000mg/kg, it had no lethal effect on the experimental animals thereby supporting the reports of ASPCA Poison Control Centre (2004) and Guevara et al., (2003) that Musa paradisiaca is a non toxic plant and has neither toxic effect nor contraindications in humans.

5.1.3 ULCER INDUCTION USING NSAID Aspirin is the most widely used member of a class of drugs known as non-steroidal antiinflammatory drugs (NSAIDS). Other members of this class are indomethacin and ibuprofen. These drugs produce their effects because they specifically inhibit cyclo-oxygenase enzyme that is needed for prostaglandin synthesis (Stuart Ira Fox, 2002). Through this action, the drugs inhibit inflammation but produce some unwanted side effects including gastric bleeding (Stuart Ira Fox, 2002) 5.1.4 PROPHYLACTIC EFFECT OF MUSA PARADISIACA (AND IN COMBINATION WITH CATECHOLAMINES) ON PEPTIC ULCERATION

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The results obtained from the study of the anti ulcer effect of the methanolic extract of Musa paradisiaca and in combination with epinephrine and Dopamine offered a significant (p < 0.05) ulcer inhibition in the glandular region of wistar albino rats. The ulcer index decreased from, 14.8 ± 3.5 (ulcerated control) to 8.2 ± 1.4 and 4.8 ± 1.7 and gastric lesions inhibition of 44.60% and 67.57% in groups II and III respectively (Table 4). Administration of the extract alone showed significant (p < 0.05) ulcer inhibition with a decrease in the ulcer index from 14.8 ± 3.5 (ulcerated control) to 3.0 ± 1.1 and gastric lesions inhibition of 79.73%. The result confirms the report of Best, et al (1984), that unripe plantain extract possess an anti-ulcerognic and cytoprotective effect on aspirin-induced gastric lesions.

And as Miller (1982) and Jain et al, (2002) had observed, it is possible that this cytoprotective action of unripe plantain is mediated by the action of endogenous prostaglandin which promotes mucus secretion and plays an important role in maintaining mucosal integrity against the actions of various damaging agents. It can be deduced also from the work of Best et al (1984) that the cytoprotective effect of the extract result from the enhancement of the mucosa barrier through the increased production of prostaglandins thereby antagonizing the actions of NSAIDS. The decrease in percentage inhibition effect of the extract in combination with the catecholamine, epinephrine and Dopamine in groups two and three respectively compared to percentage inhibition of the extract alone in group four could be as a result of the initial activation of the sympathoadrenal system as was demonstrated by Hans Selye a Canadian physiologist in 1936. From Selye‟s hypothesis it has been revealed that the sympathoadrenal system becomes activated, with increased secretion of epinephrine and nor-epinephrine in response to stressors that challenge an organism to respond physically. According to Bugajski et al (2001), catecholamines are known to increase during physiological and psychological stress.

From available literature it has been revealed that under stressful conditions, there is increased ACTH (adrenocorticotrophic Hormones) release from the anterior pituitary and thus there is increased secretion of glucocorticoids from the adrenal cortex (Stuart Ira Fox, 2002).

Glucocorticoids (a class of corticosteroids including cortisol) in turn exert permissive effects on the actions of catecholamines. And when these permissive effects are not produced because of abnormal low glucocorticoids, catecholamines will not be effective as they are normally (Stuart Ira Fox, 2002). However, on the other hand, increased secretion of glucocorticoids from the adrenal cortex stimulates the production of bleeding peptic ulcers as was described by Hans Selye, (1936).

~ 64 ~

From this theory it is possible that having subjected the rats to a 48 hours fast, the effect of the hunger stress on the pituitary adrenal axis coupled with the exogenous administration of catecholamine in groups II and III (chapter four) exacerbated the indomethacin induced ulceration giving rise to complications that affected, invariably, the effects of the extract in combination with the catecholamine (Epinephrine and Dopamine). This collaborates with Takeuchi et al, (1990) who shows that Dopamine and other catecholamines are involved in the pathogenesis of experimental duodenal and gastric ulcers in rats. But it disagrees with Szabo and colleagues (1990), who demonstrated that dopamine and its agonists markedly reduced the severity of duodenal lesions and protects the gastric mucosa against injury through inhibition of gastric motor activity mediated wit stimulation of α₂ - adrenoceptors. The work also disagrees with Pace – Asciak (1972), Collier et al (1976) who reported the finding that incubation of rat stomach, fundus and other tisues with catecholamines in vitro increased the production of prostaglandin E₂ which according to Robert et al., (1979) has the property of protecting gastric mucosa against injury.

However more research

needs to be carried out in this regard to determine the direct mechanism of action of these hormones in combination with the extract.

5.1.5 GATRIC ULCER HEALING EFFECT OF MUSA PARADISIACA The oral administration of 50mg/kg of indomethacin produced macroscopically visible hemorrhagic lesion in the glandular portion of the stomach. The ulcer index of the control was 29.5 ± 1.04 and 21 ± 1.96 in the ulcerated untreated control. The percentage ulcer inhibition of Cimetidine was 89.83% with an ulcer index of 3.0 ± 0.41. The extract at 100mg/kg produced a significant (p < 0.05) dose dependent healing effect (81.35% inhibition) at a mean ulcer index of 5.5 ± 1.26 and a significant (p < 0.05) mean ulcer index from 29.5 ± 1.04 and 21 ± 1.96 to 2.0 ± 0.81 in group V (93.22%) at 200mg/kg. This result indicated that the extract healed the ulcers and that it must have done so by stimulating the growth of the gastric mucosa as described by Goel et al (1985). The results obtained showed a dose-response relationship in that the aspirin controls gave an ulcer index value of 29.5 ± 1.04 (control) and 21 ± 1.96 (ulcerated untreated control) compared with 5.5 ± 1.26 at a dose of 100mg/kg (group four) and 2.0 ± 0.81 at a dose of 200mg/kg (group five). At 100mg/kg, the extract showed an 81.35 % inhibition compared to 89.93% inhibition of Cimetidine but at 200mg/kg the extract showed a 93.22 % inhibition compared to Cimetidine. It is possible that endogenous substances such as cyclic AMP and prostaglandins may be involved in the mechanism of the healing action of plantain as described by Best et al., 1984.

~ 65 ~

Ulcer healing is a complex process that involves combination of wound retraction and reepithelialization (Szabo and Hollander, 1989). It also involves other factors, such as growth factors and angiogenesis. Sitoindoside IV was also reported to significantly mobilize and activate peritoneal macrophages with increase in DNA and [3H]-thymidine uptake in different organs indicating possible role of macrophages aiding wound healing, which may account for plantainâ&#x20AC;&#x;s anti ulcer activity (Chattopadhyay et al, 1987). Goel et al, 1985 studied the effect of dried unripe plantain pulp extract and reported that is has no activity on offensive acid pepsin secretion. They ascribed the effect mostly to increase in gastric mucous secretion quantified in terms of total carbohydrate: protein ratio (Tc: P ratio).

Further studies with unripe plantain extract on the changes induced by ulcerogenic agents like aspirin (ASP), phenybutazone, indomethacin and prednisolone in the dissolved mucosubstances of gastric juice showed that it not only increased the Tc : P ratio induced by ulcerogenic drugs (Goel et al, 1985). While, there was no change in the individual carbohydrates there is a significant decrease in protein content in Tc: P ratio (Goel et al, 2002). Decrease in protein content signifies decreased leakage from gastric mucosal barrier. Increase in glycoprotein content of the mucosa and cell shedding in the gastric juice were also reported as further evidence for strengthening of mucosal resistance (Goel et al, 1986). Apart from mucosal resistance unripe plantain extract was also reported to increase cell proliferation as observed from increase in DNA and [3H] â&#x20AC;&#x201C; thymidine uptake by the mucosal cells and increase in mucosal thickness. This property was also reported to be involved in healing of ulcers (Mukhopadhyay et al, 1987).

Finally this work conforms also with the theory that the ulcer healing effect of Musa paradisiaca against aspirin induced gastric ulcers in rats was due to its ability to stimulate the growth of gastric mucosa since the constituents were thermobile, water soluble and insoluble in organic solvents as was reported by Best et al., (1984).

5.2 CONCLUSION The results of the studies with the methanolic extract of Musa paradisiaca and in combination with catecholamines together with its phytochemical properties makes it a potent herbal drug for the treatment of peptic ulcer disease and prompts that chemistry of plantain pulp be studied more

~ 66 ~

extensively to find out the active principle(s), which can be promising ulcer healing drug(s). Also, further studies should be made to see if this effect is true for all ulcer models including those caused

by Helicobacter pylori. Till then one should not hesitate to use its dried powder in the treatment of peptic ulcer disease as the powder seem to be safe and potent. Ulcer patients should take care to see that they are not exposed to catecholamine prescription before commencing (or during) treatment with Musa paradisiaca as the extract does not seem to show a favourable drug interaction with dopamine and epinephrine.

5.3 RECOMMENDATION It is apparent that experimental evaluation of herbal drugs for treatment of gastric ulcer is rather impressive, but very few have reached clinical trials and still few have been marketed. This shows that the benefits of research are not reaching the people to whom medical research is directed and hence the time, man power and resources are not efficiently utilized.

Hence, pharmacologists need to take more active interest in evaluation of herbal drugs for potential anti-ulcer activity and standardization of such herbal drugs to be clinically effective and globally competitive.

REFERENCES Adams KL (1992) Food dehydration options. Value added Technical Note. www.attra.org/ attra.pub/PDF/dehydrate.pdf (08/28/2007).

Agrevo (2008) Servicios. Cultivos: Plátano y Cambur. Breve reseña del cultivo en el país. www.reshet.net/agrevo/02b05 cont. html Andrew Holman (2005),Dopamine from Parkinson‟s Disease to fibromyagia, Fibromyalgia Frontiers (Volume 13, Number). National Fibromyalgia Partnership, inc.

Aremu CY, Udoessien EI (1990) Chemical estimation of some inorganic elements in selected tropical fruits and vegetables. Food Chem. 37: 229-240.

ASPCA Poison Control Centre, (2004) Toxic and Non-Toxic Plant. Animal Hot Spot website.

Avalable at http://www.people.ku.edu/~peace03/toxicplants.htm. Accessed May 4, 2005.

Bebb, J. R., Bailey-Flitter, N., Ala Aldeen, D., and Atherton J. C. (2003). Mastic Sum has no Effect on Helicobacter pylori Load in vivo: J. Antimicrob. Chemother; 52(3):522-3.

Brevoort P, (1998). The booming U.S. botanical market: a new overview. Herbal Gram.

~ 67 ~

1998;44:33–46 Champion, J. M.(1967) "Botanique et Génétique des Bananiers." Tome I. Notes et Documents sur les Bananiers et leur Culture. I.F.A.C. Setco.

Chattopadhyay, S., Chaudhuri, S., and Ghosal, S. (1987). Activation of peritoneal macrophages by sitoindoside-IV, an anti-ulcerogenic acylsteryl glycoside from Musa Paradisiaca: Planta Medica;53:16-8.

D‟souza and Dhumes V. G.(1991). Garlic Cytoprotection. Indian journal of physiology and pharmacology. 35, 889.

Davies, G. (1994) "Banana and Plantains in the East African Highlands." In Bananas and Plantains, edited by S. Gowen, 493–508. London: Chapman and Hall.

Elliott GB, Heward GJF (1976). The influence of a banana supplement diet on gastric ulcers in mice.

Pharmacological Res Comm ;8:167-71.

Emaga E. T., Herinavalona A.R., Wathelet, B., Tchago, T. J., and Paquot, M. (2007). Effect of the Stage of Maturation and Varieties on the Chemical Composition of Banana and Plantain Peels: Food Chem; 103(9): 590 – 600.

FAO (2004) Anuario Estadístico FAO. www.faostat.fao.org. (02/2008).

Frazer, J. G. citant A. C. Kruijt (1913). The Belief in Immortality, 1. excerpted in Infomusa 8 (1999): 30.

(1)

Frison, E. A., and S. L. Sharrock (1998). "Introduction: The Economic, Social and Nutritional Importance of Banana in the World." In Bananas and Food Security, edited by C. Picq, E. Fouré, and E. A. Frison, 21–35. International Symposium, Douala, Cameroon, 10–14 November, 1998.

G.I Consult, (2007). Perforated Peptic Ulcer. Retrieved on 2007 – 08- 26.

Ghosal S, Saini KS (1984). Sitoindosides I and II, two new antiulcergenic sterylacyl from Musa paradisiaca.

glucosides

Goel RK (1983). Effect of vegetable banana on gastric secretion and ulceration: an experimental and clinical study. Ph.D thesis submitted to Banaras Hindu University.

Goel RK, Bhattacharya SK, (2002). Gastro duodenal mucosal defence and mucosal defence and mucosal protective agents. Indian J. EXP Biol; 29; 701 – 714 pub med.

Goel RK, Chakrabarthy A, Sanyal AK (1985). The effect of biological variables on the antiulcerogenic effect of vegetable plantain banana. Planta Med 1985;2:85-8.

~ 68 ~

Goel RK, Govinda D, Sanyal K, (1985). In vivo antimicrobial activity of Musa paradisiaca L root extracts.

Fitoterapia 1989;60(2):157-8.

Goel RK, Govinda Das D, Sanyal AK (1985). Effect of vegetable banana power on changes induced by ulcerogenic agents on dissolved mucosubstances in gastric juice. Indian J Gastroenterol 1985;4:249-51.

Goel RK, Gupta S, Shankar R, Sanyal AK (1986). Antiulcerogenic effect of banana powder (Musa sapientum var. paradisiaca) and its effect on mucosal resistance. J Ethnopharmacol;18:33-44.

Goel RK, Maiti RN. Gastric ulcer protective effect of tamrabhasma (1992), an Indian Ayurvedic preparation of copper and plantain banana. Naples, Italy: Proceedings: First International Symposium on natural drugs and the digestive tract; p. 73-6.

Goel RK, Tavares IA, Benntt A (1989). Stimulation of gastric and colonic mucosal eicosanoid synthesis of plantain banana. J Pharm Pharmacol ;41:747-50.

Gopinath, C. Y. (1995) "How to Cook a Musa Pseudostem." Infomusa 4 (2) (1995): 31.

Guevara O, Rodriguez T, Perez C, (2003) Oral acute toxicity assay of a phytomedicine elaborated with an extract of Musa paradisiaca pseudo-stem. Acta Farmaceutica Bonaerense;22(1):57-9.

Gwanfogbe PN, Cherry JP, Simmons JG, James C (1988) Functionality and nutritive value of composite plantain (Musa paradisiaca) fruit and glandless cottonseed flours. Tropic. Sci. 28: 51-66.

Hernandez, Daniel E. (1990) The Role of Brain Peptides in the Pathogenesis of Experimental Stress Gastric Ulcers. Annals of the New York Academy of Sciences 597(1 neurobiology) Huang D, Ou, B. and Prior, R.L. (2005). The chemistry behind antioxidant capacity assays. J Agric Food Chem., 53: 1841-1856.

Huwez Fu, Thrill Well D, Cockayne, Ala „Alden DA, 2008. Mastic gum kills Helicobacter pylori (Letter to the editor, not a peer review scientific article) N Engl. J. Med. 339 (26); 1994. PMID 987617. Retrieved on 2008-09-06.

Kagy, V. (1998). "Importance Alimentaire, Socio-économique et Culturelle de la Banane dans la Société Kanak de Nouvelle-Calédonie." In Bananas and Food Security, edited by C. Picq, E. Fouré, and E. A. Frison, 437–445. International Symposium, Douala, Cameroon, 10–14 November, 1998.

Katzung, 2001, Basic and Clinical Pharmacology, 9th edition.

Kuklin A. I. and B. V. Conger, "Catecholamines in plants," Journal of Plant Growth Regulation, Springer New York ISSN 0721-7595 (Print), ISSN 1435-8107 (Online), Issue Volume 14, Number 2 / June, 1995 DOI 10.1007/BF00203119, pp. 91-97.

~ 69 ~

Kumaran, A. and Karunakaran, J. (2006). In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT-Food Sci Technol., 40: 344–352.

Levenstein S, (1998). Stress and peptic ulcer; life beyond Helicobacter BMJ 316(7130); 538-41.

PMID 9501720.

Lewis D, Field W, Shaw G. (1998) A natural flavonoid present in unripe plantain banana pulp (Musa sapientum L. var. paradisiaca L) protects the gastric mucosa from aspirin-induced erosion.

Journal of Ethnopharmacology 1999;65(3):283-8.

Lounglin MF, Ala „Aldeen DA, Jenks PJ, (2003). Monotherapy with Mastic Dog not eradicate Helicobacter pylori infection from mice. J. Antimicrob. Chemother. 51 (2);; 367-71. PMID 12562704.

Makkar, H. P. S., Bluemmel, M., Borowy, N. K., and Becker, K. (1993). Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. Journal of the Science of Food and Agriculture, 61: 161–165.

Marshal, B., and Robin, J. (2005). Peptic Ulcer Disease. American College of Gastroenterogy: 301313.

Marshall B. J., Warren J. R.(1984); Unidentified curved Bacilli in the stomach ulcer patients with gastritis and peptic ulceration. Lancet 1(8390): 1311-5. Doi: 10.1016/s0140-6736(84)91816-6.

PMID 6145023.

Marshall, 1983 Marshall B. J. ed.(2002); „Helicobacter pioneers; firsthand accounts from scientist who discovered Helicobacter‟. 1892-1982. ISBN 0-86793-035-7.

MAT (2008) Dirección de Estadísticas. Oficina de Planificación y Desarrollo. Ministerio de Agriculturay Tierras. Caracas, Venezuela. (Unpubl. data).

Miller, T.A. (1982). Protective effect of prostaglandin against mucosal damage; current knowledge and proposed mechanism. AM. J. Physiol. 245 (Gastrointestinal liver physiology, 8); G601 – G629.

Moreira, R. S (1999). Banana, Teoria e Prática de Cultivo. Fundação Cargill. Sao Paulo, Brazil.

CD-ROM. Morton, J. (2005). Atlas of Medical Plants of Middle America. Bahamas to Yucatan: Musaceae.

Illinois. Charles Thomas: 101, 170.

Morton, J. (2005). Atlas of Medical Plants of Middle America. Bahamas to Yucatan: Musaceae.

Illinois. Charles Thomas: 101, 170.

Morton, J. F. (1987). Fruits of Warm Climates. Winterville, N.C.: Creative Resources Systems (distributors); Miami, Fla.

~ 70 ~

Morton, J. J., and Malone M. H. (1987). Evaluation of vulnerary activity by open wound procedure in rats. Arch Int. Pharmacodyn Ther: 196; 117.

Moser, S., Thomas, M., Marc-Oliver, E., Stephen, J., Nicholas, J. T., and Benmhard, K. (2008). Bleu Luminescence of Ripening Banana: Angewante Chemic International Edition; 47(46): 89548957.

Mukhopadhyay K, Bhattacharya D, Chakrabarti A, Goel R K, Sanyal A K (1987). Effect of banana powder (Musa sapientum var. paradisiaca) on gastric mucosal shedding. J Ethnopharmacol;21:11-9.

Ngo Phuong, Lana Dvorkin, Julia Whelan (2008), Musa paradisiaca, Boston Healing Landscape Project, a centre for the study of cultural, religious and medical pluralism.

Nwafor S, Esimone C, Amadi C, et al.(2003) In vivo interaction between ciprofloxacin hydrochloride and the pulp of unripe platain (Musa paradisiaca L). Eur Journal of Drug Metabolism&Pharmarcol.2003;28(4):253-258.

Purves, Dale, George J. Augustine, David Fitzpatrick, William C. Hall, Anthony-Samuel LaMantia, James O. McNamara, and Leonard E. White (2005). Neuroscience. 4th ed. Sinauer Associates. pp. 137–8. ISBN 978-0-87893-697-7.

Robinson JC (1996) Bananas and Plantains. Crop Production Science in Horticulture Series Nº 5.

CABI. Wallingford, UK. pp. 1-33.

Sanyal AK, Banerjee CR, Das PK (1965). Studies on peptic ulceration. Part II- Role of banana in restraint and prednisolone-induced ulcer in albino rats. Arch Int Pharmacodyn ;155:244-8.

Sanyal AK, Banerjee CR, Das PK (1965). Studies on peptic ulceration. Part II- Role of banana restraint and prednisolone-induced ulcer in albino rats. Arch Int Pharmacodyn 155:244-8.

in

Sanyal AK, Das PK, Sinha S, Sinha YK (1961). Banana and gastric secretion. J Pharm Pharmacol ;13:318-9.

Sanyal AK, Gupta KK, Chawdhury NK (1964). Studies of peptic ulceration Part I: Role of banana in phenylbutazone induced ulcers. Arch Int Pharmacodyn;149:393-400.

Sanyal AK, Gupta KK, Chawdhury NK (1964). Studies of peptic ulceration Part I: Role of banana in phenylbutazone induced ulcers. Arch Int Pharmacodyn 149:393-400.

Sanyal AK, Gupta KK, Chowdhury NK (1963). Banana and gastric secretion. J Pharm Pharmacol ;15:283-4.

Schultz W (2002). "Getting formal with dopamine and reward". Neuron 36 (2): 241–263. doi:10.1016/S0896-6273(02)00967-4. PMID 12383780.

Scott KJ, Blake, JR, Stracha n, G Tugwell, BL and McGlasson WB (1971) Transport of Bananas at Ambient Temperatures using Polyethylene Bags. Tropical cha Agriculture (Trinidad ): 48; 163– 165.

~ 71 ~

Simmonds, N. W. (1966). Bananas. 2d ed. London: Longmans.

Singh RP, Heldman DR (2001) Introduction to Food Engineering. 3rd ed. Academic Press. San Diego, CA, USA. pp. 565, 567.

Singh, H. P. (1996)"Growing Banana for Leaf." Infomusa 5 (1): 27–28.

Stover R H, Simmonds NW (1987) Bananas. 3rd ed. Wiley. New York, USA. pp. 97-103 Strosse, H. Schoofs, H., Panis, B., Andre, E., Reyniers, K., and Swennen, R. (2006). Development of Embroyogenic Cell Suspension from Short Meristematic Tissue in Banana and Plantains: Plant Sci; 170(6): 104-112.

Stuart Ira Fox (2002); Text Book of Human Physiology, McGraw Hill Company.

Surga J, Bolívar A, Trujillo LV (1998) Caractérisation de la production et de la commercialization des Musa au Venezuela In Picq C, Fouré E, Frison E (Eds.) Bananas and Food Security. Proc.

Int. Symp. Bananas and Food Security. INIBAP. Douala, Cameroun. pp. 68-85.

Takeuchi, Koji (1990) Different effects of cytoprotective drugs on ethanol- and aspirin-induced gastric mucosal injury in pylorus-ligated rats. Digestive Diseases and Sciences 35(2)

Tan Pr., Nyase B., Dimo T., Mezui C. et al(2005); Gastric cytoprotective anti-ulcer effects of the leaf methanolic extract of ocimum suave(lamiaceae) in rats.

Tewari PV, Kumar N, Sharma RD, Kumar A (1996). Treatment of Amlapitta (Khila-Sthana) In Kasyapa Samhita, Tewari P V, editors. Varanasi: Chaukhambha Visva Bharati; p. 630-5.

Thompson, A. K., and O. J. Burden (1995); "Harvesting and Fruit Care." In Bananas and Plantains, edited by S. Gowen, 403–433. London: Chapman and Hall.

Toohey, (1974) Medicine for Nurses.

University of California Health Library Valmayor, R. V., S. H. Jamaluddin, B. Silayoi, S. Kusumo, L. D. Dahn, O. C. Pascua, and R. R. C. Espino (2000). Banana Cultivar Names and Synonyms in Southeast Asia. Los Baños, Laguna, Philippines: INIBAP. Van der Weyden, M. B., Armsrtong, R. M., and Gregory, A.T. (2005). Ulcer Diagnosis and Treatment CDC Bacterial Mycotic Disease: Med. J. Aust; 183(11-12): 612-4.

Velioglu,Y. S., Mazza,G., Gao, L. and Oomah, B. D. (1998).Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural & Food Chemistry; 46: 4113–4117.

~ 72 ~

Washirat, W., Hanucharuenkul, S. and Suriyawonigpaisal P. (2003). Stress, but not Helicobacter pylori is Associated with Peptic Ulcer Disease in a Thai Population: J. Med. Assoc Thai; 86(7):672-85.

White, Lynton Dove (1994). Canoe Plants of Ancient Hawaii: Mai'A. Wood PB, Patterson JC 2nd, Sunderland JJ, Tainter KH, Glabus MF, Lilien DL (2008); Reduced presynaptic dopamine activity in fibromyalgia syndrome demonstrated with positron emission tomography: a pilot study.J Pain. Jan;8(1):51-8.

~ 73 ~