ALUATION of ANTI-ULCER PROPERTIES of METHANOL EXTRACT of Terminalia Superba Engl

ALUATION OF ANTI-ULCER PROPERTIES of METHANOL EXTRACT of Terminalia superba Engl. & Diels (Combretaceae) STEM BARKEV

A Dissertation Submitted to the Department of Pharmacognosy and Environmental Medicine, Faculty of Pharmaceutical Sciences, University of Nigeria in partial fulfilment of the requirements for the Award of Master of Pharmacy Degree.

CHUKUMA MICHAEL ONYEGBULAM

REG.NUMBER PG/M.PHARM./12/63223.

SUPERVISOR: PROFESSOR C.O.EZUGWU

SEPTEMBER 2014 ii

CERTIFICATION

THIS PROJECT REPORT TITLED EVALUATION OF PHYTOCHEMICAL AND

ANTI-ULCER PROPERTIES OF Terminalia superba, ENGL. & DIELS (Combretaceae),

IS AN ORIGINAL RESEARCH WORK DONE BY

CHUKUMA MICHAEL ONYEGBULAM

REG.NUMBER PG/M. PHARM./12/63223.

IT IS HEREBY CERTIFIED AS MEETING THE REQUIREMENTS FOR THE AWARD

OF MASTER OF PHARMACY (M.PHARM) DEGREE OF THE DEPARTMENT OF

PHARMACOGNOSY AND ENVIRONMENTAL MEDICINE, FACULTY OF

PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA.

------

Prof. C. O. Ezugwu

Supervisor/HOD

Dept. of Pharmacognosy and Environmental Medicine iii

DEDICATION

This research is dedicated to the glory of the ONLY LIVING GOD WHO MAKE ALL

THINGS BEAUTIFUL IN HIS OWN TIME

ACKNOWLEDGEMENTS

Above all I thank almighty GOD for His protection and provision throughout this course and research work. I thank my family, especially wife Mrs. Harriet Ihuoma Chukuma, for their love, prayers, support and sacrifice towards the success of this project.

I cannot thank the following enough for their intellectual input, direction and support-

My supervisor Prof. C. O. Ezugwu sir, your patience, availability and readiness to discuss this project have been exceptional, Prof. S. I. Ofoefule, Prof. P.A. Akah, Dr. Mrs. U.E Odo, and Dr. Michele, Mr. Atogba James Mr. Austin N. Okorie, all the staff of the Department of

Pharmacognosy and Environmental Medicines. My appreciation of your sacrifice and selfless commitment to this project cannot be sufficiently expressed in words. Without your encouragement and support this project would have been mere wishful thinking.

My friends Barr. Jackson Agbai Abba, Mr. Oko Ogbuja, Mr. Rowland Ojukwu, Mr.

Nwokocha Obewu, Mr. John Eleanya Onuoha, Miss Deborah Nwanne Ibe, your contributions to the success of this project merit special mention.

GOD BLESS YOU ALL.

TABLE OF CONTENTS

Title Page ------i

Certification ------ii

Dedication ------iii

Acknowledgements ------iv

Table of Contents ------v

List of Tables------vii

List of Figures ------viii

Abstract ------ix

Chapter One ------1

1.1 Introduction ------2 1.2 The Plant Terminalia superba ------4

1.2.1 Uses ------9

1.3 Some previous researches on T. superba Engl. & Diels ------13

1.4 Ulcers ------17

1.4.1 Pathophysiology of stomach ulcers ------17

1.4.2 Treatment of ulcers ------18

1.5 In vivo models used for evaluation of potential anti-gastro

duodenal ulcer agents ------30

1.6 Acute toxicity testing ------33

Chapter Two: Materials and Methods

2.1 Reagents and Equipments ------34

2.2 Experimental procedure ------35

2.2.1 Collection, Identification and Preparation------35 vi

2.2.2 Microscopy ------35

2.2 3 Extraction ------35

2.2.4 Preliminary phytochemical screening of powdered bark ------36

2.2.5 Acute toxicity test of crude extract ------39

2.2.6 Fractionation ------40

2.2.7 Evaluation of antiulcer activity ------41

Chapter Three: Results

3.1 Macroscopy ------46

3.2 Microscopy ------48

3.3 Phytochemical screening------54

3.4 Acute toxicity ------58

3.5 Antiulcer activity ------59

Chapter Four: Discussion and Conclusion

4.1 Discussion ------64

4.2 Conclusion ------66

References ------67

Appendix ------80

vii

LIST OF TABLES

Table 1: Some previous researches on T. superba ------13

Table 2: Review of some anti-ulcer researches involving other plants ------21

Table 3: Some plant derived tannins with antiulcer property ------26

Table 4: Some plant derived flavonoids with antiulcer property ------27

Table 5: Some plant derived saponins with antiulcer property ------28

Table 6: Some active constituents isolated from plants ------29

Table 7: Preliminary Phytochemical of Screening of Powdered

T. superba Stem Bark ------55

Table 8: Phytochemical screening of T superba stem bark

crude methanol extract ------56

Table 9: Phytochemical screening of T superba stem Bark methanol Fraction ---- 57

Table 10: Phytochemical screening of T superba Stem Bark ethyl acetate fraction 58

Table 11: Acute toxicity result ------59

Table 12: Antiulcer activity of crude methanol extract of Terminalia superba ----- 61

Table 13: Effect of Methanol Fraction on Aspirin-Induced Ulcer ------62

Table 14: Comparison of effect of fractions of T. superba stem bark ------63

Table 15: Administration of crude methanol extract ------80

Table 16: Administration of doses of different fractions ------81

Table 17: Administration of methanol fraction ------82

Table 18: Phytochemical screening of T superba

stem bark ethyl acetate fraction ------83

viii

LIST OF FIGURES

Fig 1: Photograph of T. superba ------6

Fig 2: Internal surface of T. superba stem bark ------46

Fig 3: Outside surface of T. superba stem bark ------46

Fig 4: Internal surface of T. superba leaf ------47

Fig 5: External surface of T. superba leaf ------47

Fig 6: Branch showing characteristic turfs ------47

Fig 7: Large coloured cork cells------48

Fig 8: Fork-shaped fibres ------48

Fig 9: Diamond-shaped Ca oxalate ------48

Fig 10: Rosette calcium oxalate crystals ------49

Fig 11: Large rosette calcium oxalate crystal ------49

Fig 12 Showing sclerieds ------49

Fig 13: Showing fragment of phloem cells ------50

Fig 14: Showing fibres with narrow lumen ------50

Fig 15: Showing fragment of phloem parenchyma cell ------51

Fig 16: Showing cork cells showing underlying group of fibres ------51

Fig 17: Showing large phloem parenchyma cells with calcium

oxalate crystals ------52

Fig 18: Showing fragment of phloem cells ------52

Fig 19: Showing phloem parenchyma with biserate medullary ray ------53

ABSTRACT

Terminalia superba Engl. & Diels (Combretaceae), is a member of the genus Terminalia that comprises around 100 species distributed in tropical regions of the world. In Africa it is found along the coast of west and central Africa. It has different uses in traditional medicine such as antimalarial, anti-diabetic, anti-fungal, and anti-hypertensive in the areas where it is found. Most of these uses are yet to be scientifically investigated. The powdered stem bark of Terminalia superba was extracted by maceration using methanol. The crude extract was chromatographically fractionated using n-hexane, ethyl acetate, and methanol. Phytochemical analysis was conducted on the crude methanol extract, n-hexane, methanol, and ethyl acetate fractions using standard procedures. The LD50 of the crude methanol extract was determined using Lorke’s method. The phytochemical analysis showed the presence of alkaloids, saponins, glycosides, flavonoids, tannins, terpenoids, resins and reducing sugars. The crude extract, the methanol and ethyl acetate fractions were investigated for anti-ulcer activity using the ethanol, stress, and aspirin induction models. The parameters evaluated were ulcer index and percentage protective index. The data was statistically analysed. There was significant difference (p < 0.05) between the group treated with the crude extract and the control group. The microscopy showed the presence features characteristic of a bark. The anti-ulcer screening showed that the methanol extract of Terminalia superba possesses antiulcer property and its use in traditional medicine for treatment of stomach ulcer is justified. 1

CHAPTER ONE

1.1: Introduction

The fact that nature has bestowed us with abundant provision and resources for healing through herbs is not in doubt and cannot be over emphasized. Man has used plants as sources of food and medicine since creation. Many applications of plants as medicines are not scientifically evaluated but are based on reported success in healing/cure over time. The use of plants and plant parts for medicinal purposes can be described with different names, such as Traditional Medicine, Complimentary Alternative Medicine (CAM) but the contents are the same, natural substances.

Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses…in some Asian and African countries, 80% of the population depend on traditional medicine for primary healthcare (WHO , 2008). Almost 65% of the world’s population has incorporated the value of plants as a methodology of medicinal agents into their primary modality of health care

(Lanfranco, 1992).

Whether critics look at traditional medicine (complimentary alternative medicine) as folklore, trick, or manipulation and exploitation of the ignorant, the fact remains that there have been, and there are still herbs with undeniable therapeutic efficacy around us, e.g. Digitalis purpurea L. foxglove-source of the cardiac glycoside digitoxin, Papaver somniferum L.

(opium poppy)-source of the drug morphine and codeine, Cinchona succirubra –source of the anti-malarial drug quinine , Artemisia annua, source of artemisinin, now one drug used as part of combination therapy (ACT) for treatment Plasmodium falciparum infection, Panax gingseng, Gingko biloba, Atropa belladonna-source of atropine, Erythroxylum coca–source 2 of cocaine, Ephedra species–source of ephedrine, Pilocarpus jaborandi (Holmes) – source of pilocarpine Physostigma venenosum –source of physostigmine, Pacific yew tree, Taxus brevifolia source of paclitaxel (Taxol® )

What may be lacking is information on these herbs. In today’s world in which the trend is like “back to the roots” the need for evaluation of herbal materials to verify and authenticate claims of pharmacological properties and therapeutic values (claims) is a necessity. Only

0.4% of the total number of MEDLINE-listed articles for the period 1966 – 1996, refer to research concerning natural drugs and herbs (World Health Organization, 2005). Recent screening with plants has revealed many compounds like flavonoids, alkaloids, saponins, terpenoids, monoterpenoids (linalool), glycoproteins, polysaccharides, tannins, essential fatty acids, phenolic compounds and vitamins having pronounced antioxidant, antineoplastic, antiulcer, anti-inflammatory and immune stimulating potential (Dashputre and Naikwade,

2011).

Natural products serve in various capacities as drugs and starting materials for drugs. Review of all approved agents during the time frame of more than 25 years from 01/1981 to 06/2006 for all diseases worldwide and from 1950 (earliest so far identified) to 06/2006 for all approved antitumor drugs worldwide reveals the utility of natural products as sources of novel structures, but not necessarily the final drug entity (Newman and Cragg, 2007). Out of

255 drugs which are considered as basic and essential by the World Health Organization

(WHO), 11% are obtained from plants and a number of synthetic drugs are also obtained from natural precursors (World Health Organization, 2005). The evaluation and assessment of phytochemical properties of a plant has standard and established methods which may vary slightly but have the same basic chemical principles. Phytochemical evaluation involves mainly the tests for secondary metabolites-alkaloids, glycosides, steroids, flavonoids, tannins, saponins, proteins, carbohydrates, fats and oils. 3

Phytochemical evaluation will be incomplete or ineffective if the various constituents present in an extraction liquor are not separated from one another. Among the separation techniques available and/or practiced, chromatography is the most common, easiest, and cheapest.

For further characterization advanced techniques like HPLC, Two-Dimensional

Chromatography, Gel Electrophoresis Chromatography, GC/MS, LC/IR, LC/Nmr, 2D Nmr,

Tandem MS etc. are employed. With developments in analytical chemistry and its principles- extraction techniques, separation techniques, purification techniques, isolation and characterization techniques, more plants have been deeply evaluated for pharmacological and therapeutic properties. Some have been re-evaluated for properties that have not been investigated before, with some positive and justifiable results.

A woman was interview on the Network Service of Radio Nigeria, 7’0 clock news, on 4th

February 2013 (World Cancer Day 2013). She said that she was diagnosed of cancer and went to traditional health practitioners. Her case worsened and complicated and by the time she sought conventional healthcare the cancer had spread. Whatever she was given may be active against another ailment, or may have no pharmacological activity at all, or may even be carcinogenic. This is not to say that there are no natural anticancer drugs. This is one case in support for the call for investigation and standardization of herbal products in the country.

There is the urgent need for scientific investigation of the ingredients of our traditional medicine system to determine their pharmacologically active constituents, hence, therapeutic applications, their efficacy or lack of it, as well as their safety. The aim of this research is to investigate the phytochemical properties and anti-ulcer activity of the methanol extract of the stem bark of Terminalia superb Engl. & Diels (Family Combretaceae). The result of this research will strengthen or discourage the use of Terminalia superba in the treatment of stomach ulcers and add to available knowledge data base on our plants. 4

Available literature was searched for current and relevant information on the plant Terminalia superb Engl. & Diels, recent researches on it, researches on antiulcer activity of other plants, techniques and principles of methods to be involved in the research (for example methods of ulcer induction, methods of extraction of medicinal actives from plants, etc.). This search in as much as it tried to be extensive and desired to be exhaustive, does claim to have assessed all materials available.

1.2 The Plant Terminalia superb Engl. & Diels

Taxonomy: kingdom: Plantae Division: Mangnoliophyta Class: Mangnoliopsida Order: Myrtales Family: Combretaceae Genius : Terminalia Species : T. superba Current name: Terminalia superba Authority: Engl. & Diels Common names (English) : Black korina, limba, white afara (French) : Frakè, limba (German) : Limba (Spanish) : Akom (Swahili) : Mwalambe (Trade name) : Korina, limba (Yoruba) : Afa, afara : (www.worldagroforestrycentre.org, 2013) Synonym(s) Terminalia altissima A. Chev

(http://www.plantnames.unimelb.edu.au/new/Sorting/Terminalia.html, 2013

Origin and geographic distribution

Terminalia superba is a tree of about 30-50 m high. It is a member of the genus Terminalia that comprises around 100 species distributed in tropical regions of the worldwide (Victor et al, 2010). In Africa it is found in West and Central Africa, from Guinea Bissau east to DR

Congo and south to Cabinda (Angola) (Kimpouni, 2009) In Nigeria it is Indigenous to Cross

River State (Burkill., 1985) 6

Figure 1: T. superba Tree ( Magnification = 1.0)

MAIN LOCAL NAMES

Countries Local Names

Benin AZINII

Cameroon AKOM

Central African Rep. N’GANGA

Congo LIMBA

Côte d’Ivoire FRAKE

Dem. Rep. of Congo LIMBA

Equatorial Guinea AKOM

Ghana OFRAM

Nigeria AFARA

Nigeria WHITE AFARA

Sierra Leone KOJAGEI

France FRAKE

France LIMBO

France NOYER DU MAYOMBE

USA KORINA

(www.ecochoice.co.uk/pdf, 2014)

Nigerian Vernacular Names

EDO ẹ̀ghọẹ̀n-nófūá, nófūó: white; referring to the flaking bark

EFIK àfia étò = white tree

IGALA uji-oko (H-Hansen)

IGBO èdò (auctt.) èdò óchạ́ = white edo (Amufu)ojiloko (Nkalagu) ojiroko

(Owerri) èdò óchạ́ = white edo (Egbema) apaụpaụ tịín (Tiemo)

ISEKIRI egonni

NUPE eji

URHOBO unwon ron

YORUBA afaa , afara (www.ecocrop.fao.org/ecocrop)

Botanical Description

Terminalia superba is a large tree, up to 50 m tall and 5 m in girth, bole cylindrical, long and straight with large, flat buttresses, 6 m above the soil surface; crown open, generally flattened, consisting of a few whorled branches, leaves simple, alternate, in tufts at the ends of the branches. Bark fairly smooth, greying, flaking off in small patches; slash yellow, bark surface smooth and grey in young trees, but shallowly grooved and with elongated, brownish grey scales, inner bark soft-fibrous, pale yellow (Kimpouni, 2009 ). Root system frequently fairly shallow, and as the tree ages the taproot disappears. Buttresses, from which descending roots arise at some distance from the trunk, then support the tree. Leaves simple, alternate, in tufts at the ends of the branches; deciduous, leaving pronounced scars on twigs when shed.

Petiole 3-7 cm long, flattened above, with a pair of sub-opposite glands below the blade; lamina glabrous, obovate , 6-12 x 2.5-7 cm, with a short acuminate apex. Nerves 6-8 pairs; secondary reticulation inconspicuous. Inflorescence a 7-18-cm, laxly flowered spike, 9 peduncle densely pubescent; flowers sessile, small, s greenish-white; calyx tube saucer shaped, with 5 short triangular lobes. Petals absent. Stamens usually twice the number of calyx lobes (usually 10), in 2 whorls, glabrous; filaments a little longer than calyx; intra- staminal disc annular, flattened, 0.3 mm thick; densely woolly pubescent. Fruit a small, transversely winged, sessile, golden-brown smooth nut, 1.5-2.5 x 4-7 cm (including the wings). Nut without the wing about 1.5 x 2 cm when mature, usually containing 1 seed. The generic name comes from the Latin ‘terminalis’ (ending), and refers to the habit of the leaves being crowded at the ends of the shoots (Burkill, 1985). Some of the above botanical descriptions are shown in Fig.3 to Fig. 4 below.

Ecology

Terminalia superba is most common in moist semi-deciduous forest, but can also be found in evergreen forest. It occurs up to 1000 m altitude. It is most common in disturbed forest. It is found in regions with an annual rainfall of (1000–) 1400–3000 (–3500) mm and a dry season up to 4 months, and mean annual temperatures of 23–27°C. Terminalia superb prefers well- drained, fertile, alluvial soils with pH of about 6.0, but it tolerates a wide range of soil types, from sandy to clayey-loamy and lateritic. It does not tolerate prolonged water logging, but withstands occasional flooding (Richter and Dallwitz, 2000).

1. 2. 1 USES

GENERAL USES

The wood, usually traded as ‘limba’, ‘afara’, ‘ofram’ or ‘fraké’, is valued for interior joinery, door posts and panels, mouldings, furniture, office-fittings, crates, matches, and particularly for veneer and plywood. It is suitable for light construction, light flooring, ship building, interior trim, vehicle bodies, sporting goods, toys, novelties, musical instruments, food containers, vats, turnery, hardboard, particle board and pulpwood. It is used locally for 10 temporary house construction, planks, roof shingles, canoes, paddles, coffins, boxes and domestic utensils. It is suitable for paper making, although the paper is of moderate quality.

The wood is also used as firewood and for charcoal production. A yellow dye is present in the bark; it is used traditionally to dye fibres for matting and basketry. The bark is also used for dyeing textiles blackish. In Côte d’Ivoire Terminalia superba is occasionally used as a shade tree in cocoa and coffee plantations, and in DR Congo it is used as shade tree in coffee, cocoa and banana plantations (Kimpouni, 2009).

ETHNO-MEDICINAL USES

Bark decoctions and macerations are used in traditional medicine to treat wounds, sores, haemorrhoids, diarrhoea, dysentery, malaria, vomiting, gingivitis, bronchitis, aphthae, swellings and ovarian troubles, and as an expectorant and anodyne. The leaves serve as diuretic and roots as laxative (Richter and Dallwitz, 2009). Terminalia superba is generally used in traditional medicine to treat bacterial, fungal and viral infections. The bark of this plant is used to eradicate intestinal worms and treat gastrointestinal disorders such as enteritis, abdominal pain, diarrhoea, fever, headache, conjunctivitis. In the Southwest of Côte d’Ivoire the bark of T. superba, called "tree of malaria", (Orewa et al 2009). In Cameroon it is locally used in the treatment of various ailments, including diabetes mellitus, gastroenteritis, female infertility and abdominal pains (Adjanohoun et al., 1996).

The uses of the different parts of the plant can be summarized as follows:

Bark

Medicines: anti-emetics; diarrhoea, dysentery; dropsy, swellings, oedema, gout; generally healing; oral treatments; pain-killers; pregnancy, anti-aborifacients, pulmonary troubles

Phytochemistry: alkaloids

Products: dyes, stains, inks, tattoos and mordants

Root bark

Phytochemistry: tannins, astringents

Leaf

Medicines: abortifacients, ecbolics

Root

Medicines: laxatives, etc.

Phytochemistry: resins

Wood

Products: fuel and lighting; household, domestic and personal items; pulp and paper

(www.plants.jstor.org, 2013).

PHYTOCHEMISTRY

The phytochemical screening revealed the presence of polyterpens, polyphenols, flavonoids, tannins catechic, alkaloids and saponins (Kouakou et al., 2013).

Previous works revealed presence of compounds that have been characterised. Two compounds isolated following bio-assay guided fractionation namely 3,4'-di-O-methylellagic acid 3'-O-β-D-xylopyranoside and 4'-O-galloy-3,3'-di-O-methylellagic acid 4-O-β-D- xylopyranoside (Kuete et al., 2010). Methanol extract of the stem bark of Terminalia superba led to the isolation of four new triterpene glucosides ( which were characterized as 2α,3β- 12 dihydroxyolean-12-en-28-oic acid 28-O-β-D-lucopyranoside , 2α,3β, 21β-trihydroxyolean-

12-en-28-oic acid 28-O-β-D-glucopyranoside , 2α,3β, 29-trihydroxyolean-12-en-28-oic acid

28-O-β-D-glucopyranoside and 2α,3β,23,27-tetrahydroxyolean-12-en-28-oic acid 28-O-β-D- glucopyranoside (Turibio et al., 2009)

Table 1: Some previous researches on T. superba

Title Aim Result Author

1 Acute toxicity and This study was These results Kouakou et al., 2013

anti-ulcerogenic aimed to evaluate suggested that the

activity of an the acute toxicity preventive anti-ulcer

aqueous extract from and gastric anti- activity of AETs

the stem bark of ulcer activity of an may be due to a

Terminalia superba aqueous extract of cytoprotective effect.

Engl. and Diels Terminalia superba

(Combretaceae)

2 Phytochemical In this research, The data in the present Bamisaye et al.,2013

constituents and aqueous extract of T. study indicate that the

antidiarrheal effects superba leaves was aqueous extract of T.

of the aqueous extract investigated for the superba leaves

of Terminalia superba treatment of possessed antidiarrheal

leaves on diarrhoea in wistar properties

wistar rats rats.

3 Antifungal activity of To locate the true Aqueous extracts and Ahon et al., 2011

the aqueous and potential anti- hydro-alcoholic extract

hydro-alcoholic microbial in general, of T. superba have a

extracts of T. superba but especially anti- dose-dependent

Engl. on the in vitro fungal extracts of T. fungicidal activity

growth of clinical superba on the in- against clinical fungal

isolates of pathogenic vitro growth of C. isolates used

fungi albicans,

A. fumigatus, C. 14

neoformans and

T. mentagrophytes

4 The aqueous extract of To Investigate the The aqueous extract of Tom et al., 2011

Terminalia superba hypotensive and the the stem bark of T.

(Combretaceae) antihypertensive superba

prevents glucose- effects of the exhibits hypotensive

induced hypertension aqueous extract of and anti-hypertensive

in rats. the stem bark of properties

Terminalia superba.

5 Protective role of Investigation of the The results suggest Ngueguim et al., 2011

Terminalia superba protective role of that, ethyl acetate

Ethyl Aetate against Terminalia superba extract of T. superba

Oxidative Stress Type ethyl acetate extract lower blood glucose

2 Diabetes against and hyperlipidemia,

streptozotocin- prevent oxidative

nicotinamide induced stress and reduce

type 2 diabetes. blood pressure in

diabetic conditions.

5 Antimycobacterial, To evaluate the Provide promising Kuete et al., 2010

antibacterial and methanol extract baseline information

antifungal activities of from the stem bark for the potential use of

Terminalia superba of Terminalia the crude extract from

(Combretaceae) superba (TSB), T. superba, in the

fractions (TSB1–7) treatment of

for antimicrobial tuberculosis, bacterial

activity and fungal infections 15

7 Anti-diabetic activity The present study Terminalia superba Padmashree et al., 2010

of methanol/methylene was undertaken to leaf extract possess

chloride extract of investigate the anti- antidiabetic

Terminalia superba hyperglycemia effect properties

leaves on of the

streptozotocin-induced methanol/methylene

diabetes in rats chloride extract of

Terminalia superba

leaf

8 in vivo assessment of To investigate the This extract Momo and Oben 2009

hypoglycaemic and possible actions of demonstrates

antioxidant activities aqueous extract of significant

of aqueous extract of the roots of hypoglycaemic effect

Terminalia superba in Terminalia superba thus reduces the

alloxan-diabetic rats on glucose antioxidant parameters

homeostasis and on in alloxan-induced

MDA, SOD and diabetes rats

catalase homolysate

of diabetes rats

9 Antioxidant properties The evaluations of For all the plants Momo et al., 2009

and α-amylase the antioxidant tested, at least one Inhibition of potential and α- extract inhibited the

Terminalia superba, amylase inhibitory activity of α-amylase.

Albiziz sp., Cola activity of these The most effective was

odorata and Harunga extracts were also the hydroethanolic

madagascarensis used carried out extract of T. superba.

in the management of 16

diabetes

10 α-Glucosidase To identify All the isolated Wansi et al., 2007 inhibitory constituents α-glucosidase compounds were

from stem bark of inhibitory evaluated for their Terminalia superba constituents from glycosidase inhibition (Combretaceae) stem bark of activities. Gallic acid Terminalia superba and methyl gallate showed significant α-glucosidase inhibition activity.

11 Antimicrobial Antibacterial The isolation and Tabopda et al., 2009

Pentacyclic bioassay-guided characterization of

Triterpenoids from fractionation of the four new triterpene

Terminalia superba methanol extract of glucosides

the stem bark of

Terminalia superba

12 Analgesic Activities of To evaluate the n-BuOH fraction of T. Dongmo et al., 2006

the Stem Bark Extract analgesic activities of superba stem bark of Terminalia superba the extract could be beneficial in

Engl and Diels obtained from this the management of

(Combretacea) plant by in-vivo pain

screening methods

Other members of the Terminalia species have also been shown to possess anti-ulcer roperties. T. chebula showed reduction in lesion index, total affected area and percentage of lesion in comparison with control groups in the aspirin, ethanol and cold restraint stress- induced ulcer models. The T. chebula extract increased mucus production in aspirin and ethanol-induced ulcer models and showed anti-secretory activity in pylorus ligated model leading to a reduction in the gastric juice volume, free acidity, total acidity, and significantly increased gastric pH (Sharma et al., 2011. Raju. et al 2009). In pyloric ligation induced ulcer model, oral administration of ethanolic extract of T. catappa in two different doses showed significant reduction in ulcer index, gastric volume, free acidity, total acidity and PH as compared to the control group. (Bharath et al., 2014), Terminalia pallida Brandis has also been demonstrated to possess anti-ulcer activity (Gupta et al., 2005).

1 .4 ULCERS

1.4.1 PATHOPHYSIOLOGY OF STOMACH ULCERS

Pathophysiology of ulcer is due to an imbalance between aggressive factors (acid, pepsin, H. pylori and NSAID's) and local mucosal defensive factors (mucus bicarbonate, blood flow and prostaglandins). (Walker and Whittlesea, 2012). The underlying pathophysiology associated with H. pylori infection involves the production of cytotoxin associated gene A (cag A) proteins and vacuolating cytotoxins such as vac. A which activate the inflammatory cascade

(Maury et al., 2012). Alcohol causes secretion of gastric juice and decrease mucosal resistance due to which protein content of gastric juice is significantly increased by ethanol

(Maity et al., 2003). Ethanol readily penetrates the gastric mucosa due to its ability to solubilize the protective mucous and expose the mucosa to the proteolytic and hydrolytic actions of hydrochloric acid and pepsin, causing damage to the membrane. Moreover, alcohol stimulates acid secretion and reduces blood flow leading to micro vascular injuries, through disruption of the vascular endothelium and facilitating vascular permeability; it also increases 18 activity of xanthine oxidase (Sener et al., 2004). NSAIDs inhibits the PG synthesis of gastric mucosa, PG gives cytoprotection. Enhancement of leukotriene synthesis, exhibits damage effect. Aspirin also inhibit gastric peroxidase and may increase mucosal H2O2 and hydroxyl ions level to cause oxidative mucosal damage (Datta et al., 2002). Stress can arise from prolonged anxiety, tension, and emotion, severe physical discomfort, haemorrhage and surgical shock, burns and trauma, thereby resulting in severe gastric ulceration. Recently research has shown that resistant cold stress causes severe haemorrhage ulcer through derangement of the mucosal antioxidant enzyme such as super oxide, dismutase and peroxides. This is the stress condition arising mainly from physiology discomfort and the mechanism of ulceration caused in this case should be different from ulcer caused due to other factors. The stress generate highly reactive OH- radicals that causes oxidative damage of the gastric mucosa (Udaya et al., 1999).

Recently oxidative free radicals have been implicated in mediating NSAID, H. Pylori, ethanol, and cold restraint stress induced gastric injury (Huilgol and Jamadar, 2013). Stress has also been found to decrease the quality and amount of mucus adhering to the gastric mucosa. It has been suggested that, in conditions of emotional tension, there is not only a greater destruction of mucus and decreased synthesis of its components, but also a quality change that affects the translation, acylation, and glycosylation of the ribosomal peptides

(Peters and Richardson, 1983).

1.4.2 TREATMENT OF ULCERS

Treatment of endoscopically proven uncomplicated peptic ulcer disease has changed dramatically in recent years. Curing of H. pylori infection and discontinuation of NSAIDS are key elements for the successful management of peptic ulcer disease (Maury et al., 2012)

Antiulcer agents can be grouped into the following pharmacological classes;

Histamin H2-Receptor Antagonists e.g. cimtetidine, ranitidine, famotidine, nizatidine. 19

Proton Pump Inhibitors e g. Omeprazole, Lansoprazole, Rabeprazole, Pentoprazole

Cytopretective Agents e.g. Sucralfate, Bismuth chelate

Prostaglandine analogoues e.g. Misoprostol

Antacids e.g. Magnesium trisilicate, Aluminium hydroxide gel

Antibiotics e.g. Amoxycillin, Clarithromycin, Metronidaziole

Muscarinic receptor Blockers e.g. Pirezepine, and Telenzepine

These drugs are broadly classified into two, those that decrease or counter acid pepsin secretion and those that afford cytoprotection by virtue of their effects on mucosal defensive factors. These drugs act by different mechanisms. Most of the commonly used drugs such as

H2-blockers (ranitidine, famotidine etc.), M1-blockers (pirenzepine, telenzepine etc), proton pump inhibitors (omeprazole, lansaprazole etc), decrease secretion of acid while, drugs like sucralfate and carbenoxolone promote mucosal defence. It is now assumed that these drugs ultimately balance the aggressive factors (acid, pepsin, H. pylori, bile salts) and defensive factors (mucin secretion, cellular mucus, bicarbonate secretion, mucosal blood flow and cell turnover). (Goel and Bhattacharya, 2002). The standard first-line therapy is a one week

"triple therapy" consisting of proton pump inhibitors such as omeprazole and the antibiotics clarithromycin and amoxicillin. In Helicobacter Pylori Infection a typical regime is lansoprazole 30 mg + amoxicillin 1 g + clarithromycin 500 mg PO q12hr for 10-14 days.

Dual therapy (clarithromycin-resistant): lansoprazole 30 mg + amoxicillin 1 g PO q8hr for 14 days. Penicillin allergy: lansoprazole 30 mg + clarithromycin 500 mg + metronidazole 500 mg q12hr for 10-14 days (emedicine.medscape.com, 2014).

REVIEW OF PLANT-DERIVED ANTI-ULCER AGENTS

Recent screening of plants has revealed many compounds like flavonoids, alkaloids, saponins, terpenoids, monoterpenoids (linalool), glycoproteins, polysaccharides, tannins, essential fatty acids, phenolic compounds with antiulcer properties (Neetesh et al., 2010).

Natural Remedies

Fresh cabbage juice is an excellent ulcer treatment. It produces an amino acid that increases blood flow to the lining of the stomach. Honey has been used for hundreds of years as a topical preparation to promote the healing of wounds. When ingested, it heals and strengthens the stomach lining and kills harmful bacteria. Unripe plantains promote strong stomach linings by producing a mucoid substance that coats the stomach lining, giving it protection against acids. Bananas offer protection in the same manner. Eating a diet that is fibre-rich is an added ulcer protection. ((African traditional herbal clinic, 2013) Fruits, vegetables, legumes and whole grains produce substance, which help to protect the stomach lining

(Borrelli and Izzo, 2000). Among herbal drugs, liquorice, aloe gel and capsicum (chilli) have been used extensively and their clinical efficacy documented. Also, ethno-medical systems employ several plant extracts for the treatment of peptic ulcer. (African traditional herbal clinic, 2013)

Botanical compounds with anti-ulcer activity include flavonoids (i.e. quercetin, naringin, silymarin, anthocyanosides, sophoradin derivatives), saponins (i.e. from Panax japonicus and

Kochia scoparia), tannins (i.e. from Linderae umbellatae), gums and mucilages (i.e. gum guar and myrrh). (Borrelli and Izzo, 2000).

Table 2: Review of some recent anti-ulcer researches involving other plants

Plant Plant Extract Ulcer Model Reference part Cayratia trifolia L. Leaves petroleum Pylorus ligation and Gupta et al.,2012

ethanol (MECT) (Vitaceae) ether and

hydro-

alcohol

(30:70)

Emblica officinalis Fruit Ethanol Pylorus ligation, Al-Rehaily et al., 2002

Gaertn., syn: indomethacin,

Phyllanthus emblica hypothermic restraint

(Euphorbiaceae), stress and necrotizing

agents (80% ethanol,

0.2 M NaOH and 25%

NaCl).

Nigella sativa Linn Seed Alcoholic Pylor ligation and Rajkapoor et al.,2002

Family: Ranunculaceae aspirin

Abutilon indicum L. Leaves ethanolic Pylorus ligatIion and Dashputre et al.,2011

(Family: Malvaceae), extract ethanol

Capsicum frutescenes Fruit ethanolic aspirin Dass , et al.,2008

Mimosa pudica , leaves Methanolic, Aspirin, Alcohol and Vinothapooshan and K

(Fabaceae), chloroform pyloric ligation model Sundar 2011

and diethel

ether extracts 22

Aegle marmelos Fruit Methanolic Indomethacin induced Ganesh et al, 2011

seed and aqueous ulceration, stressed

induced ulceration and

pylorus ligation

Picrasma quassioides Whole Aqueous Aspirin-pylorus Hwisa et al., 2013

(D. Don) Bennett plant Extract ligation, HCl-ethanol, family Simaroubaceae water immersion-stress

Garcinia kola seeds, Methanolic Ethanol, Ige et al, 2012

Family: Guttifera.

Entandrophragma utile Bark Aqueous Aspirin, John et al 2012

immobilization, cold-

restraint, histamine-

induced, pylorus

ligation, necrotizing

substances

Cayratia trifolia methanolic pyloric ligated and Jyoti , et al 2012

ethanol

Tinospora cordifolia whole Alcoholic Pyloric ligation, Bairy et al.2001

plant extract, ibuprofen and cold

restraint

Falcaria vulgaris leaves Ethanolic Ethanol Khazaei et al, 2006

Family :Umbelliferae and extract

stems

Barleria prionitis Linn Leaves Methanol Ethanol and Manjusha et al,2013

Family Acanthaceae) Indomethacin

Morinda citrifolia Linn Fruit Ethyl acetate Ethanol,A spirin and Muralidharan and 23

Rubiaceae, Pyloric ligation, Srikanth, 2009

Cysteamine HCl,

Carpolobia lutea leaf Ethanol Indomethacin, Ethanol, Nwidu and Nwafor,

(polygalaceae) G. Don Reserpine in 0.5% 2009

Acetic acid, Stress,

Serotonin and

Diethylthiocarbamate

Cassia singueana Leaves Methanol Indomethacin Ode, 2011

Hibiscus cannabinus Leaves methanolic Pylorus ligation and Silambujanaki et al

Family: Malvaceae, Indomethacin 2010

Falcaria vulgaris leaves hydro ethanol (50%) Khazaei, and Salehi

Family: Umbelliferae and alcoholic 2006.

stems

Aloe vera Leaf gel Etanolic Indomethacin and Subramanian et al.,

extract Ethanol 2007

Bauhinia racemosa fruit aqueous Paracetamol Borikar et al 2009

powder extract

"Parsley" Petroselinum Aerial Ethanolic Pyloric ligation, Al-Howiriny et al, crispum, parts hypothermic- restraint- 2003

stress, 24

Aspilia africana C.D. Leaf Aqueous Ehanol, indomethacin Ubaka et al., 2010

Adams, (Compositae) and aspirin

Croton zambesicus leaf ethanolic indomethacin, ethanol Okokon et al, 2011.

Muell Arg. and histamine

(Euphorbiaceace) (syn

C. amabilis Muell.

Arg. C. gratissimus

Burch)

Boswellia serrata bark Ppetroleum aspirin Zeeyauddin , 2011

(Family Bursera-ceae) ether

(250mg/kg)

and aqueous

extracts

Moringa oleifera leaves acetone Ethanol, Cold Devaraj et al.., 2007

Lam (Moringaceae) and extract restraint stress,

fruits and indomethacin,

methanol Pylorus ligation,

extract

Momordica Fruits olive oil indomethacin DENG‹Z and charantia L.- extract Nesrin ,2005

(cucurbitaceae)

Barleria prionitis L. Leaves methanolic ethanol and Manjusha et al,

Family Acanthaceae indomethacin 2013

Kigelia africana, Leaves Ethanolic aspirin Orole et al., 2013.

Nauclea latifolia and extracts 25

Staudtia stipitata

Ginger (Zingiber Ginger Aqueous aspirin Wang et al. 2011 officinale) powder

Etandrophagma utile fresh aqueous Ethanol or John et al 2012

bark histamine

Aegle marmelos fruits aqueous Aspirin Das and Roy 2012

(AM), family:

Rutaceae

Researches have been done on plants with antiulcer effect, some such researches are reported

Table 3-4. Some even extending to identification of chemical groups responsible for activity, table 6 below,

Table 3: Some Plants containing tannins with anti-ulcer activity

Botanical name Part Ulcer model Reference

plant

Calliandra Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal portoticensis 1988

Entandrophragma Bark Ethanol John and utile Onabanjo,1990

Linderae umbellatae Stem Stress Ezaki et al.,1985

Mallotus japonicas Bark (Clinical study) Saijo et al., 1989

Rhigiocarya Leaves Indomethacin, reserpine, Aguwa, 1985, racemifera serotonin

Veronica officinalis Aerial Indomethacin, reserpine Scarlat et al 1985

parts

Table 4: Some plants containing flavonoids with anti-ulcer activity

FLAVONOID ULCER MODEL REFERENCE

Anthocyanosides Pylorus-ligated, Reserpine, Magistretti et al 1998 Phenylbutazone Catechin Stress Lorenz et al., 1975 Genistin Phenylbutazone, Serotinine, Rainova et al., 1988 Pylorus-ligated, Stress, Reserpin Hypolaetin-8-glucoside Stress, Ethanol, Alcaraza and Hoult, 1985 Acetylsalicylic acid Kaempferol Ethanol Izzo et al., 1994 Leucocyanidin Aspirin Lewis et al., 1999 Luteolin-7-glycoside Pylorus-ligated, Stress, Rainova et al., 1988 Reserpine, Phenylbutazone, Serotinin 5-Methoxyflavone Indomethacin Blank et al., 1997 Myricetin3-0-D- Stress, Pylorous-ligated, Reyes et al., 1996 Galactoside Ethanol Naringin Ethanol, Stress, Pylorous Martin et al 1993 ligation Quercertin Stress, Ethanol, Reserpin Izzo et a.,l 1994 Rutin Stress Izzo et al., 1994 Silymarin Ethanol Alarcon 1992 Ternatin Ethanol, Indomethacin, Stress Rao et al., 1997 Vexibinol HCL, Ethanol Yamahara et al 1990

Table 5: Some Plants Containing Saponins with Anti-Ulcer Activity

Botanical Name Part Plant Ulcer Model Reference

Calendula officinalis Rhizome Caffeine-arsenic, butadiene, Iatsyno et al., 1978 pylorus-ligated Calliandra Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal 1988 portoticensis Kochia scoparia Fruit Ethanol, indomethacin Mastuda et al., 1998 Panax binnatifidus Rhizome Psychological stress Nguyen et al., 1996; Panax japonicus Rhizome Stress, HCL Yamahara et al., 1987 Pyrenacantha Leaves Indomethacin, serotonin, Aguwa and Okunji 1986 stauditii stress Rhigiocarya Leaves Indomethacin, reserpine, Aguwa, 1985 racemifera serotonin Spartium junceum Flowers Ethanol Yesilada and Takaishi, 1999 Veronica officinalis Aerial Indomethacin, reserpine Scarlat et a l, 1985 parts

Table 6: Some active constituents isolated from plant (Goel and Sairamanti, 2002)

Plants and plant part Active Constituents Models

Tectona grandis Linn Lapachol IS-ASP-induced GU in rats and (Trunk bark and wood HIST- induced DU in rats and GP chips repectively Rhamnus procumbens Kaempferol PL-, ethanol, IS- and CRS- (Whole plant) induced GU in rats and HIST- induced GU and DU in GP. Rhamnus triquerta Wall Emodin RS-, PL- and IS- induced GU in (Whole plant) rats Datura fastuosa (Leaves) Withafstuosin E CRS-, PL- and ASP-induced GU in rats Flueggea Bergenin/norbergenin PL- and ASP- induced GU in rats microcarpa and CRS- induced GU in rats and (Leaves and GP. roots) Azadirachta indica Nimbidin ASP-, prednisolone-, indomethacin-, serotonin stress- and acetic acid induced GU in rats. HIST- induced DU in GP. CYS- induced DU in rats Ocimum basilum Fixed oil ASP-, indomethacin-,ethanol, HIST-,reserpine-, Serotonin-, PL- and stress-induced GU in rats Bacopa monniera (Whole Standardized extract of CRS-, ethanol, ASP- and PL- plant) bacoside A (35%) induced GU in rats ASP-aspirin; ce-chloroform; CRS-cold restraint stress; CYS-cysteamine; DU-duodenal ulcer; GP- guinea pig; GU-gastric ulcer; HIST-histamine; IS-immobilization stress; PL-pylorus ligation; RS- restraint stress;

1.5 In Vivo Models Used for Evaluation of Potential Anti-gastro duodenal

Ulcer Activity

Animal models represent an attempt to imitate the pathologies associated with human disease states in a preclinical setting. In using animal models, it is therefore important to create a test system that allows the basic mechanism of pathology to be systemically manipulated so as to obtain a better understanding of its biological basis. An important issue in this regard is to construct validity-the degree to which the model corresponds to the clinical state in humans.

So, in general, experimentally induced gastric and duodenal ulcers should resemble the appearance, complications, development, and mode of healing to humans.

The rat stomach shows an obvious division into two parts: the upper non-secretory portion rumen and the lower glandular secretory portion which is analogous to the body of the stomach in man both anatomically and functionally. The rat being omnivorous resembles man nutritionally (Lahiri and Plit, 2012). Peptic ulcers can be induced by physiological, pharmacological or surgical manipulations in several animal species. However, most experiments in peptic ulcer studies are carried out in rodents. For preventive models, it is advisable to compare the potential drug or test material with cyto-protectant reference drugs such as misoprostol and sucralfate that are known to prevent peptic ulcers. The case of healing, or curative studies, the use of histamine receptor antagonists such as cimetidine or ranitidine, and proton-pump inhibitors such as omeprazole, is recommended as reference drugs (Adinortey et al., 2013).

Several models are used experimentally for testing or investigating anti-peptic ulcer activity of chemical compounds and they include the following:

Ø water-immersion-stress or cold-water-restraint or cold-restraint stress

Ø NSAIDs- (indomethacin, aspirin, and ibuprofen) induced gastric ulcers

Ø ethanol-induced gastric ulcers

Ø acetic acid-induced gastric ulcers

Ø histamine-induced gastric ulcers

Ø reserpine-induced gastric ulcers

Ø serotonin-induced gastric ulcers

Ø pylorus-ligated-induced peptic ulcers

Ø diethyldithiocarbamate- (DDC)-induced peptic ulcers

Ø methylene blue-induced ulcers

Ø ischemia-reperfusion- (I-R-) induced gastric ulcers

Ø cysteamine-induced duodenal ulcers

Ø indomethacin-histamine-induced duodenal ulcers

Ø ferrous iron-ascorbic acid-induced gastric ulcers

Ø acetic acid-H. pylori-induced ulcers

Ø HCl/ethanol- induced ulcer

Ø H. pylori-induced ulcers

Procedure for Aspirin model

Albino rats of either sex weighing between 150-200 gm. are divided into five groups of six animals in each group. The animals are fasted for 24 hours. The test drug in varying concentrations based on the design of the experiment is administered orally in 2% gum acacia solution thirty minutes prior to aspirin at dose of 200 mg/kg. 4 hours later the rats are sacrificed by using anaesthetic ether and their stomachs dissected for the determination of gastric lesions (Datta et al., 2002).

Procedure for Ethanol-induced ulcer model.

Albino rats of either sex weighing between (150-200 g) are divided into six groups of animals in each group. The animals are fasted for 24 hours with free access water. Animals are given test drugs or standard drug. 1 hour later 1ml/200 gm of 99.80% alcohol is administered orally to each animal. Animal are sacrificed 1 hour after, alcohol administration, stomach is isolated and cut open along the greater curvature and pinned on a soft board. The length of each gastric lesion is measure in mm. The percentage inhibition is expressed as sum of the length of the control-mean lesion index of test / mean lesion index of control × 100 (Datta et al.,

2002).

Procedure for Cold-Restraint-Stress-induced ulcer model

Albino Wistar rats of either sex weighing between (150-200 gm.) are divided into five groups of six animals in group. Cold-resistance-stress (CRS) ulcer was induced ulcer to 18 hours fasted rats, cold resistance stress is given by strapping the rats on a wooden plank and keeping them for 2 hours at 4oC -6oC. The animals are then sacrificed by cervical dislocation and ulcers are scored on the dissected stomachs (Datta et al., 2002) 33

1.6 ACUTE TOXICITY TESTING

There are different methods used in determination of LD50 which include Arithmetical method of Karber , Lorke method, Arithmetical method of Reed and Muench, Graphical method of Miller and Tainter, Graphical method of Litchfield and Wilcoxon, Acute Toxic

Class Method (OECD/OCDE 423, 2001) Fixed Dose Procedure (OECD/OCDE 420 , 2001) and Up-and-Down Procedure (OECD/OCDE 425, 1998).

LORKE’S METHODS of ACUTE TOXICITY TESTING.

The study was conducted in two phases using a total of sixteen male rats. In the first phase, nine rats were divided into 3 groups of 3 rats each. Groups 1, 2 and 3 animals were given

10 mg/kg, 100 mg/kg and 1000 mg/kg body weight of the extract, respectively, to possibly establish the range of doses producing any toxic effect. Each rat was given a single dose after at least 5 days of adaptation. In addition, a fourth group of three rats was set up as control group and animals in the group were not given the extract. In the second phase, further specific doses (1600 mg/kg, 2900 mg/kg and 5000 mg/kg body weight of the extract were administered to three rats (one rat per dose) to further determine the correct LD50 value

(Lorke, 1983).

CHAPTER TWO

MATERIALS AND METHODS

2.1 Reagents and Equipments

Aspirin and Ranitidine obtained from Professor S I. Ofoefule of the Department of

Pharmaceutical Technology and Industrial Pharmacy of the Faculty of Pharmaceutical

Sciences, University of Nigeria, Nsukka

Methanol Absolute

Diethylether from Sigma-Aldrich Laboratory, GmBH ,

Chloroform from Scharlau S.A. Spain

Naphtol in ethanol (Molisch reagent)

Concentrated Sulphuric acid

Fehling’s Reagents I and II

Ethanol 50% and 96%

Dragendoff’s reagent

Dilute ammonia solution

20% Potassium hydroxide solution

Ferric chloride solution

Lead sub-acetate solution

Ethyl acetate

Million’s reagent

Acetone AR

Ethanol Absolute n-Hexane 35

Distilled water

Silica Gel for Column Chromatography 70/230

Photomicroscope

Whatman filter paper

Vital feed animal finisher by Grand cereals Limited, Jos Plateau, State Nigeria

Electronic scale

All AR reagents were bought from Joechem Venture CO (NIG), 45 Enugu Road. Nsukka,

Enugu State, Nigeria

2.2 EXPERIMENTAL PROCEDURES

2.2.1 Collection, Identification and Preparation

The stem bark of Terminalia superba was collected in Nsukka, Enugu State in Eastern

Nigeria in July 2013. The plant material was identified by Mr. A.O. Ozioko, a taxonomist who retired from the Department of Botany of the Faculty of Biological Sciences, now working with Interceed, Nsukka. A specimen has been deposited with the herbarium of the

Department of Pharmacognosy and Environmental Medicine of the Faculty of

Pharmaceutical Sciences, UNN,

Preparation

It was air-dried in the shade and reduced to a powdery form by pounding with mortar and pestle.

2.2 .2 Microscopy

The dried and powdered stem bark of Terminalia superba were examined microscopically using photomicroscope. 36

2.2.3 Extraction

The bark was cut into small pieces and dried under shade. A portion of the dried powdered material (1100 g) was macerated in five litres of methanol by cold extraction for 3 days in large amber bottles with intermittent shaking. The extract was filtered after 24 hours and a fresh solvent was added and the procedure repeated for another 24 hours.

2.2.4 Preliminary phytochemical screening of powdered stem bark

The crude drug was used to perform the preliminary phytochemical tests. The method was according Trease and Evans (2009) and Sengar et al 2009. Portions of the sample were used to test for alkaloids, saponins, tannins, glycosides, flavonoids, resins, proteins, oils, steroids, carbohydrates and reducing sugars.

1 Test for Carbohydrates

A 0.1 g portion of the powdered material was boiled with 2ml of water and filtered. S

Few drops of naphthol in ethanol (Molisch’s reagent) was added to the filtrate.

Concentrated sulphuric was then gently poured down the side of the test tube to form

a lower layer. A purple interfacial ring indicating the presence of carbohydrate was

observed.

2 Test for Reducing Sugars

A 0.1 g portion of the powder was shaken vigorously with 5ml of distilled water and

filtered. The filtrated was used in the following test.

Fehling’s Test: To 1 ml portion of the filtrate was added equal volumes of Fehling’s

solution I and II, and boiled on water bath for a few minutes. A brick red precipitate

indicating the presence of reducing sugars was observed 37

3 Test for Alkaloids

(i) A 20 ml of 5% sulphuric acid in 50% ethanol was added to 2g portion of the

powdered material and heated on a boiling water bath for 10 minutes cooled, and

filtered. 2ml of the filtrate was treated with Dragendorff’s reagent. Red precipitates

were observed, indicating the presence of alkaloids

(ii) The remaining filtrate was placed in 100ml separating funnel and made alkaline with

dilute ammonia solution. The aqueous alkaline solution was separated and extracted

with two 5ml portions of dilute sulphuric acid.

The extract was tested with a few drops of Mayer’s, Wagner’s, and Dragendorff’s

reagents. Red precipitates were observed with Dragendorff’s reagent, milky

precipitates were formed with Mayer’s reagent, while reddish brown precipitates were

formed with Wagner’s reagent indicating the presence of alkaloids

4 Test for Glycosides

A 5 ml of dilute sulphuric acid was added to 0.1g of the powdered material in a test

tube and boiled for 15 minutes on a water bath, cooled, and neutralized with 20%

Potassium hydroxide. 10 ml of a mixture of equal parts of Fehling’s solutions I and II

was added and boiled for 5 minutes. A dense brick red ppt. was formed indicating the

presence of glycosides.

5 Test for Saponins

A 20 ml of distilled water was added to 0.25g portion of the powdered material in 100

ml beaker boiled gently for 2 minutes. The mixture was filtered and allowed to cool.

5 ml of the filtrate was diluted with 20 ml of distilled water and shaken vigorously for

15 minutes. A stable foam was formed which indicates the presence of saponins. 38

6 Test for Tannins

A 1.0 g portion of the powdered material was boiled with 50 ml of distilled water,

filtered and subjected to the following tests:

(I) Ferric Chloride Test

Few drops of Ferric were added to 3 ml of the filtrate. Greenish black precipitates

indicate the presence of tannins was formed.

(II) Lead Acetate Test

A few drops of lead sub-acetate was added to 3 ml of the filtrate. The presence

of tannins was indicated by the formation of cream precipitates.

7 Test for Flavonoids

A 10 ml of ethyl acetate was added to 0.2 g of the powdered material and heated on a

water bath for 3 minutes. The mixture was cooled, filtered and the filtrate used for the

following tests:

Ammonium Test:

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

were allowed to separate. The ammonium layer became yellow indicating the

presence of flavonoids.

1% Aluminium Chloride Solution Test

A 4 ml portion of the filtrate was shaken with 1 ml of 1% Aluminium chloride

solution. The layers were allowed to separate. The Aluminium chloride layer became

yellow due to the presence of flavonoids. 39

8 Test for Resins

A 0.2 g portion of the powdered material was extracted with 15 ml of 96% ethanol.

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

Precipitates were formed due to the presence of resins.

9 Test for Proteins

To a little portion of the filtrate from 8 above was added 2 drops of Million’ reagent.

White precipitates were formed due to the presence of proteins.

10 Test for Oils

A 0.1 g portion of the powdered material was pressed between a filter paper. No

translucency or staining was observed implying the absence of oils.

11 Test for Steroids

A 9 ml measure of ethanol was added to1.0 g of the powdered material and refluxed

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

water bath and 5 ml of hot water was added. The mixture was allowed to stand for 1

hour and the waxy matter filtered off. The filtrate was extracted with 2.5 ml of

chloroform using a separating funnel.

Test for Terpenoids

To 0.5 ml of the chloroform extract in a test tube was carefully added 1 ml of

concentrated sulphuric acid to form a lower layer. A reddish brown interface was

observed indicating the presence of steroids. 40

12 Test for Terpenoids

A 0.5 ml of the chloroform extract from 11 above was evaporated to dryness in a

water bath and heated with 3 ml of concentrated Sulphuric acid for 10 minutes on a

water bath. A grey colour was observed, indicating the presence of terpenoids.

2.2.5 Acute Toxicity

Albino mice of both sexes, weighing between 97 – 136 g and albino mice weighing between

18.5 – 24.5 g purchased from University of Nigeria Nsukka, animal house, were used in this study. The animals were housed in cross ventilated room in cages at 22 ± 2.5°C with 12 h dark/12 h light cycles and were feed with standard growers mash feeds. Animals were acclimatized for one week and fasted overnight, with free access to water prior to experiments.

Acute toxicity (LD50) Test

The acute toxicity test of the methanol extract of Terminalia superba stem bark was estimated in mice .The tests involved two phases. The first phase is determination of the toxic range. The mice were placed in three groups of three animals per group and were given Methanol extract of stem bark of Terminalia superba (10 mg/kg, 100 mg/kg and

1000 mg/kg; p.o.). The treated mice were observed for 24 h for number of deaths. The death pattern in the first phase determines the doses to be used for the second phase.

Since there was no deaths recorded in the first phase, a fresh batch of three mice received

1600 mg/kg, 2900 mg/kg, and 5000 mg/kg of the extract. The treated animals were observed for lethality or signs of acute intoxication for 24 hours (Lorke, 1983).

The LD50 calculated as the geometric mean of the highest non-lethal dose (1600mg) and the lowest lethal dose (2900mg). 41

2.2.6 Fractionation

The crude methanol extract of T. superba was fractionated using wet packing column chromatographic techniques. Silica gel for column chromatography 70/230 was made into a free flowing slurry using n-hexane and quickly poured into a glass column measuring 150 cm in length and 5 cm internal diameter already containing some n-hexane. The tap was opened and the liquid eluted to a height of about 2 cm above the silica gel in the column. 60 g of the crude extract was dissolved in a little volume of n-Hexane mixed with three times its weight of silica and the solvent was allowed to evaporate leaving a dry powder. The powder was packed on top of the silica inside the column and covered with chromatographic sand. The column was eluted with the three solvents, starting with n-hexane, followed by ethyl acetate, and then methanol. n-Hexane was run through the column with tap open and the eluent collected in a flask. This was done until the liquid coming out of the column became colourless. The mobile phase n-hexane was replaced by ethyl acetate and the procedure repeated. Finally Methanol was used and the fraction collected as. The different liquid fractions were concentrated and dried using rotary evaporator to get the fractions as dry powders.

2.2.7 Evaluation of antiulcer activity

A Evaluation of antiulcer activity of crude methanol extract

Aspirin- induced gastric ulcer:

Ratus ratus rats of either sex weighing between 97 and 156 g were divided into five groups of six animals in group. The animals were well fed and water given freely for three days, then fasted for 24 hours. They were housed in steel cages at room temperature 30 + 2oC under

12/12 hours light dark cycle and were well fed with pelleted standard feed and water given freely. 42

Procedure

The procedure adopted was that of Datta et al (2002) modified by the addition of ranitidine

50mg/kg according to the method of Muralidharan et al (2009). The crude methanol extract or methanol fraction, or ethyl acetate fraction of T. superba stem back was used to make a

100 mg/ml stock solution. The solution was administered in volumes equivalent to 100 mg/kg, 200 mg/kg, and 400 mg/kg, to three different groups of animals consisting of five rats per group. Ranitidine at a dose of 50mg/kg used as standard and was given to a forth group.

A fifth group of six rats received only water. Both the test substance and the standard

(ranitidine) were administered orally 30 minute prior to administration of aspirin at dose of

200 mg/kg. Four hours later the rats are sacrificed by using anaesthetic ether and their stomachs dissected and examined for the determination of gastric lesions.

Macroscopic evaluation of stomach

The stomachs were opened along the greater curvature, rinsed with normal saline to remove gastric contents and blood clots and examined by a 10x magnifier lens to assess the formation of ulcers. The numbers of ulcers were counted and scored.

Scoring of ulcer will be made as follows:

Normal stomach ------(0)

Pinhole ------(1.0)

Spot ulcer ------(1.5)

Haemorrhagic streak ------(2.0)

Small erosion ------(2.5)

Large erosions ------(3.0)

Perforation ------(3.5) 43

Mean ulcer score for each animal was used to express ulcer index. The Protection Index (PI) percentage of ulcer protection was determined as follows:

Uc −Ut Protection Index (PI) = × 100 Uc

Where, ut = ulcer index of treated group. Uc = ulcer index of control group.

Cold-restraint- stress-induced ulcer model

Ratus ratus rats of either sex weighing between 68 and 95 g were divided into five groups of six animals in group. The animals were well fed and water given freely for three days, then fasted for 24 hours.

Procedure

The procedure adopted was that of Gupta et al (1985). The crude extract of T. superba stem bark, the methanol fraction and ethyl acetate fractions, were used to make 100 mg/ml stock solutions. The solutions was administered in volumes equivalent to 100 mg/kg, 200mg/kg, and 400 mg/kg to three different groups of animals consisting of six rats per group.

Ranitidine at a dose of 50mg/kg used as standard and was given to a forth group. A fifth group of six rats received only water. Cold-restraint-tress ulcer induced by strapping the rats on a wooden plank and keeping them for 2 hours at 4oC.One hour later, the animals were sacrificed by cervical dislocation and ulcers were examined on the dissected stomachs

(Goodman and Gilman, 1996).

Macroscopic evaluation of stomach

Scoring of ulcer will be made as follows:

Normal stomach ------(0)

Pinhole ------(1.0)

Spot ulcer ------(1.5)

Haemorrhagic streak ------(2.0)

Small erosion ------(2.5)

Large erosions ------(3.0)

Perforation ------(3.5)

Mean ulcer score for each animal was used to express ulcer index. The percentage of ulcer protection was determined as follows:

Uc −Ut Protection Index (PI) = × 100 Uc

Where, ut = ulcer index of treated group. Uc = ulcer index of control group.

Ethanol-induced ulcer model.

Ratus ratus rats of either sex weighing between 60 and 132 g were divided into five groups of six animals in group. The animals were well fed and water given freely for three days, then fasted for 24 hours.

Procedure

The procedure adopted was that of Datta et al (2002). The dried residue from the methanol fraction and ethyl acetate fractions, and the crude extract of T. superba bark were used to make 100 mg/ml stock solutions. The solutions was administered in volumes equivalent to

100 mg/kg, 200mg/kg, and 400 mg/kg to three different groups of animals consisting of six rats per group. Ranitidine at a dose of 50mg/kg used as standard and was given to a forth group. A fifth group of six rats received only water. One hour later 1ml/200gm of 99.80% alcohol is administered p.o to each animal. 45

The animals were sacrificed 1 hour after alcohol administration by using anaesthetic ether and their stomachs dissected and examined for the determination of gastric lesions and scoring.

Macroscopic evaluation of stomach

Scoring of ulcer will be made as follows:

Normal stomach ------(0)

Pinhole ------(1.0)

Spot ulcer ------(1.5)

Haemorrhagic streak ------(2.0)

Small erosion ------(2.5)

Large erosions ------(3.0)

Perforation ------(3.5)

Mean ulcer score for each animal was used to express ulcer index. The percentage of ulcer protection was determined as follows:

Uc −Ut Protection Index (PI) = × 100 Uc

Where, ut = ulcer index of treated group. Uc = ulcer index of control group.

Statistical analysis

The experimental data were tested for statistical significance by means of analysis of variance with one way ANOVA followed by Turkey contrast analysis and Dunnett multiple 46 comparison tests (p<0.05) using SPSS 20. Results were expressed as Mean ± SEM. A probability level of less than 5% was considered significant (P≤0.05).

CHAPTER THREE

RESULTS

3.1 MACROSCOPY

Fig 2: Internal surface of T. superba stem bark Fig 3: External surface of T. superba stem bark

Magnification = 1.0

Fig 4: Internal surface of T.superba leaf Fig 5: External surface of T.superba leaf

Magnification = 1.5

Fig 6: Branch showing characteristic turfs

3.2 MICROSCOPY

The microscopy revealed the presence of coloured cork cells, phloem parenchyma cells, fibres in bundles with some of them fork-shaped and some with narrow lumen, Calcium oxalate crystals in different shapes-rosette and diamond shaped. These results are shown in

Figures 7 to 19

Fig 7: Large coloured cork cells Magnificatio = 1.0

Fork-shaped fibres Diamond-shaped calciun oxalate

Fig 8: Fork-shaped fibres Fig 9: Diamond-shaped Ca Oxalate

Magnification = 1.2 Magnification = 1,2 50

Rosette-shaped Calciun oxalate crystal

Mag = 1.7

Fig 10: Rosette Calcium oxalate crystals Fig 11: Large Rosette Calcium oxalate

crystal

Magnification = 1.2

sclerieds

Fig 12: Showing sclerieds (Mag. = 1.2)

Fig 13: Showing Bundle of fibres with adjoining cork cells (

Mag = 1.0)

Narrow lumen

Fig 14: Showing Fibres with narrow lumen (Magnification = 1.2) 52

Fig 15: Showing Fragment of phloem parenchyma cell (Magnification = 1.2)

Fig 16: Showing Cork cells showing underlying group of fibres (Magnification = 1.6)

Calciun oxalate crystals

Fig 17: Showing large phloem parenchyma cells with Calcium oxalate crystals

(Magnification = 2)

Fig 18: Showing fragment of phloem cells (Magnification = 1.2)

Bi-serated medullary ray

Fig 19: Showing phloem parenchyma with bi-serate medullary ray

3.3 PHYTOCHEMICAL RESULTS

The phytochemical screening revealed the presence of polyterpens, polyphenols, flavonoids, tannins, glycosides, carbohydrates, reducing sugars, resins, steroids, proteins, alkaloids and saponins.

TABLE 7: Preliminary phytochemical of screening of powdered T. superba stem bark

Test Reagent Observation Inference Test for Alkaloids Mayer’s reagent Milky precipitate and Dragendorff’s Brick red precipitate + reagent Test for Glycosides Fehling’s solution I Dense brick red and II precipitate + Test for Tannins Ferric chloride Greenish black Lead sub-acetate precipitate with ferric chloride , and cream + precipitate with lead sub acetate Test for Saponins Distilled water Frothing and Stable foam + Test for Flavonoids Ethyl acetate, A yellow colour in ammonium both the ammonical solution, and 1% and aluminium layers + Aluminium chloride solution Test for Carbohydrates Molisch reagent, Purple interface + and conc. H2SO4 Test for Steroids Ethanol, Reddish brown chloroform, conc. interface observed + H2SO4 Test for Terpenoids Ethanol, Grey colour observed + chloroform, conc.

H2SO4 Test for Resins 96% ethanol and Solution became water cloudy yellow because of formation of + precipitate Test for Proteins Million’s reagent White precipitate + Test for Mucilage Reducing sugars Fehling’s reagent Brick red precipitate + + = Present; - = Absent

TABLE 8: Phytochemical Analysis of T superba stem bark crude methanol extract

Test Reagent Observation Inference

Test for Alkaloids Mayer’s reagent Brick red precipitate. and Dragendorff’s + reagent Test for Glycosides Fehling’s solution Brick red precipitate. I and II + Test for Tannins Ferric chloride Green precipitate

Lead sub-acetate + Test for Saponins Distilled water Stable foam + Test for Flavonoids Ethyl acetate, Yellow lower ammonium Ammonium layer solution, and 1% + Aluminium chloride solution Test for Carbohydrates Molisch reagent, Dark colouration and conc. H2SO4 + Test for Steroids Ethanol, Reddish brown chloroform, conc. interface observed + H2SO4 Test for terpenoids Ethanol, Grey colour observed chloroform, conc. + H2SO4 Test for Resins 96% ethanol and Cloudy because of water precipitate + Test for Proteins Million’s Reagent Yellow precipitate + Reducing sugars Fehling’s reagent Brick red precipitate + + = Present; - = Absent

TABLE 9: phytochemical screening of T superba stem bark methanol fraction

Test Reagent Observation Inference Test for Alkaloids Mayer’s reagent Brick red precipitate and Dragendorff’s + reagent

Test for Glycosides Fehling’s solution Brick red precipitate I and II + Test for Tannins Ferric chloride Green precipitate Lead sub-acetate +

Test for Saponins Distilled water Stable foam + Test for Flavonoids Ethyl acetate, Yellow lower ammonium Ammonium layer solution, and 1% + Aluminium chloride solution

Test for Carbohydrates Molisch reagent, Dark colouration and conc. H2SO4 + Test for Steroids Ethanol, Reddish brown chloroform, conc. interface observed + H2SO4 Test for Terpenoids Ethanol, Grey colour observed chloroform, conc. + H2SO4 Test for Resins 96% ethanol and Cloudy because of water precipitate. +

Test for Proteins Million’s Reagent Yellow precipitate. + Reducing sugars Fehling’s reagent Brick red precipitate + + = Present; - = Absent

Table 10: Phytochemical screening of T superba stem bark ethyl acetate fraction

Test Reagent Observation Inference Test for Alkaloids Mayer’s reagent and Brick red precipitate Dragendorff’s reagent +

Test for Glycosides Fehling’s solution I and II Brick red precipitate + Test for Tannins Ferric chloride No Green precipitate Lead sub-acetate -

Test for Saponins Distilled water Stable foam + Test for Flavonoids Ethyl acetate, ammonium Yellow lower solution, and 1% Ammonium layer Aluminium chloride + solution

Test for Molisch reagent, and conc. Dark colouration Carbohydrates Sulphuric acid +

Test for Steroids Ethanol, chloroform, conc. Reddish brown H2SO4 interface observed +

Test for Terpenoids Ethanol, chloroform, conc. Grey colour observed H2SO4 +

Test for Resins 96% ethanol and water Clear solution - Test for Proteins Million’s Reagent Yellow precipitate + Reducing sugars Fehling’s reagent Brick red precipitate. + + = Present; - = Absent

3 .4 ACUTE TOXICITY TEST

Table 11: Acute toxicity result

PHASE 1

DOSE 10 mg/kg 100 mg/kg 1000 mg/kg NO. OF ANIMALS 3 3 3

NO. OF DEATHS 0 0 0

% DEATH 0 0 0

PHASE 2

DOSE 1600 mg/kg 2900 mg/kg 5000 mg/kg NO. OF ANIMALS 1 1 1

NO. OF DEATHS 0 1 1

% DEATH 0 100 100

LD50 Calculation;

This data shows that Terminalia superba stem bark is toxic at doses above 2154mg.

3.5 EVALUATION OF ANTIULCER / GASTROPROTECTIVE

ACTIVITY

The results as presented in Table 9 below showed the pre-treatment with and without the methanol extract of Terminalia superba bark on aspirin-induced gastric ulcerations. The mean ulcer score was significantly lower in groups pre-treated with the extract at

100 mg/kg (P < 0.023), 200 mg/kg (P < 0.005) and 400 mg/kg (P < 0.001) dose levels.

The stress and ethanol models also showed similar pattern of activity as shown below

The groups pre-treated with the various doses of the bark extract showed a progressive decline in ulcer indices relative to experimental control. The preventive index (percentage protection) conferred by the extract using the different doses was found to increase in a dose- dependent manner. The result for the mean ulcer index was expressed as Mean ± S.E.M

Table 12: Antiulcer activity of crude methanol extract of Terminalia superba

TREATMENT ASPIRIN-INDUCED ULCER Dose (mg/kg) TOTAL ULCER MEAN ULCER % PROTECTIVE SCORE SCORE (UI) INDEX 100 27 4.5 ± 1.4* 55

200 20 3.3 ± 0.7* 67

400 14 2.3 ± 0.3* 77

Ranitidine 50 4 0.67 ± 0.33* 93 (Standard) Water 5 ml/kg 60 10.1 ± 2.5 - (Control)

ETHANOL-INDUCED ULCER 100 100 20 ± 1.9* 47.8

200 63.5 13.2 ± 3.0* 66.8

400 47.5 9.5 ± 1.6* 75.2

Ranitidine 50 24 4.8 ± 0.96* 87.5 (Standard) Water 5l/kg 191.5 38.3 ± 1.81 - (Control)

STRESS-INDUCED ULCER 100 142 28.4 ± 5.13 12.88

200 76.5 15.3 ±6.56* 53.07

400 25 5.0 ± 1.61* 84.66

Ranitidine 50 30.5 6.1 ± 1.72* 81.29 (Standard) Water 5 ml/kg 163 32 ± 4.88 - (Control)

Results are expressed as Mean ± SEM, n= 6 ,*p<0.05

The stomach of members of the group that received 400mg of the extract per kg body weight

in the aspirin model was swollen and contained yellowish mucous substance. This group also

experienced the highest protection against aspirin induced ulceration, they had the highest

protective index, one of the parameters investigated. This can be interpreted as an indication

of the mechanism of action of the active chemical in the bark, possibly gastro protective

through mucous secretion. This was also shown by the methanol fraction.

Table 13: Effect of Methanol Fraction on Aspirin-Induced Ulcer Treatment Dose TOTAL MEAN ULCER % PROTECTIVE INDEX (mg/kg) ULCER SCORE (UI) SCORE 100 52.5 10.3 ± 2.06 41.47

200 48.2 8.5 ± 2.33* 51.70

400 45 5.9 ± 2.1* 66.47

Ranitidine(Standard) 50 11.5 2.4 ± 0.5* 86.93

Distilled water (5ml /kg) ( Control) 88 17.6 ± 3.17 -

Results are expressed as Mean ± SEM, n= 6 (for each group),*p<0.05

When the crude extract, the methanol fraction, the ethyl acetate fraction were compared using ranitidine as the standard the crude extract showed more activity than both the methanol fraction and the ethyl acetate fractions.

Compared antiulcer activity of different fractions

Table 14: Comparison of effect of fractions of T. superba stem bark

Crude Ethyl +ve Control -ve Control Fraction Methanol extract in Acetate (Ranitidine) (Water) water

Total Score 71 52 38 15.1 105.5

Mean Score 11.8 8.8 ± 2.3 6.3 ± 1.3 2.5± 0.37 19.08 ± 3.14 (UI) ±1.5

%Protective 32.7 50.7 63.98 85.68 - Index

Results are expressed as Mean ± SEM, n = 6;*p<0.05

STATISTICAL ANALYSIS

The statistical analysis showed that there is significant difference p < 0.05 between the animals treated with crude methanol extract, or the fractions, and those not treated. This is an indication of antiulcer effect of the test substance. A further analysis showed that the methanol fraction has higher activity than the ethyl acetate fraction.

CHAPTER FOUR

4. 1 DISCUSSION

Stomach ulcer is a major disease that is wide spread in the world and is not restricted to any particular sex or age group. The predisposing factors are all around us, be it stress, alcohol

NSAIDs, H .pylori infection, etc. Much effort has been made to find treatments for stomach ulcer. This has yield results that have helped many people who have the disease. The diversity of factors implicated in the aetiology of stomach ulcers coupled with the different mechanisms of action of the currently available drugs indicate that all is not yet known about stomach ulcer treatment. The etiology of peptic ulcer is unidentified in most of the cases, yet it is generally accepted that it results from an imbalance between aggressive factors and the maintenance of mucosal integrity through the endogenous defence mechanisms. (Piper and

Stiel, 1986). To recuperate the balance, different therapeutic agents are used to obstruct the gastric acid secretion or to boost the mucosal defence mechanisms by increasing mucosal production, stabilizing the surface epithelial cells or interfering with the prostaglandin synthesis (Kumar et al 2014). An increase in the acid secretion, a decrease in the gastric mucosal protection and an induction of oxidative stress in gastric mucosa are the important factors that are implicated in the pathogenesis of peptic ulcers. (Bairy et al., 2001)

The gastro-protective potential of the extract and fractions may in part be due to their chemical constituents which includes tannins, saponins and flavonoids, and their cytoprotective mechanism on gastric mucosa. The crude methanol extract and the methanol fraction of T. superba was very consistent and effective in all experimental ulcer models studied and may be useful in the treatment of peptic ulcer in man. It is possible that one of the mechanisms of anti-ulcerogenic effects of the extracts may be their ability to mobilize prostaglandins in gastric mucosa. Suppression of alcohol-induced ulceration indicated that the extract suppress lipoxygenase path-way, this may in part be one of its mechanisms of 65 action. (Nwidu and Nwafor, 2009). In the present study, Terminalia superba stem bark methanol extract, ethyl acetate and methanol fractions reduced the production of gastric ulcers, even in the presence of aspirin, ethanol, or stress.

Compounds found in plants which have shown anti-ulcer properties include saponins, tannins, and flavonoids (Borrelli and Izzo, 200) and phytochemical screening of the

Terminala superba stem bark showed the presence of these compounds. Therefore, it is possible that the ulcer-protective activity of Terminalia superb stem bark is, at least in part, due to their presence. Various members of the Terminala species e.g. T. catappa, (Bharath et al., 2014), Terminalia pallida Brandis (Gupta et al., 2005), T. chebula (Sharma et al., 2011.

Raju. et al 2009) Terminalia belerica (Choudhary, 2012), Terminalia arjuna (Paarakh, 2010) have been proved to possess anti-ulcer properties. Therefore, the discovery by this research that it has anti-ulcer property is not surprising.

In this research the choice of method of extraction took into consideration the fact that thermo- labile substances may be present in the sample and the solvent, methanol was chosen to ensure that as much of the present compounds as possible were extracted. Considering the various structural elements that make up the stem bark a yield of 15% w/w is not low. And the presence of coloured cork cells and the presence of bi-serated medullary rays, and both diamond- and rosette-shaped calcium oxalate crystals may help as quality control and identification features for Terminalia superba bark powder. The pharmacological properties of T. Superb stem bark coupled with other activities reported by researches listed in table 1 show that the plant Terminalia superb is a rich source of compounds with pharmacological activity including anti-ulcerogenic activity as revealed by this research. Considering the possession of this activity by other members of the combretaecea family and Terminalia species this result will be considered to be in line with other findings concerning the family.

4. 2 CONCLUSION:

The present study was undertaken to see if the methanol extract of T. superba stem bark could show antiulcer effect in three different models of peptic ulcers. The extract showed comparable antiulcer effects to the standard drug (ranitidine) in aspirin, ethanol, and stress induced ulcers.

Terminalia. superba stem bark methanol extract has antiulcer/gastro-protective properties.

It is therefore a good candidate for screening for new anti-ulcer drugs

Further work should be carried out to isolate, purify and possibly characterize the active principle responsible for the observed effect. Also additional work should be embarked upon with a view to elucidate the possible mechanism of action of the extract.

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APPENDIX

APPENDIX 1 ADMINISTRATION OF DOSES

Table: 15 Administration of crude methanol extract

ANIMAL ASA EXTRACT Animal Animal Wt. (mg) Vol. of Wt. Vol. of Mark wt. (g) Stock (mg) Stock 100mg/ml 100mg/ml Sol.(ml) Sol.(ml) 100 mg/kg Head 97.86 19.58 .2 9.78 .10 Tail 114.37 22.88 .3 11.4 .11 Trunk 120.98 24.20 .24 12.1 .12 Right. Side 116.70 23.29 .24 11.7 .12 Left Side 23.34 .23 11.7 .12 Unmarked 100.04 20.00 .2 10.04 .10 200 mg/kg Head 120.06 24.01 .24 24.01 .24 Tail 135.82 27.16 .27 27.16 .27 Trunk 159.5 31.9 .32 31.9 .32 Right Side 116.5 23.3 .23 23.3 .23 Left Side 104.03 20.81 .21 20.81 .21 Unmarked 108.7 21.75 .22 21.75 .22 Ranitidine 50 mg/kg Head 113.21 22.64 .23 45.20 .45 Tail 104.55 20.91 .21 41.02 .41 Trunk 104.85 20.97 .21 41.94 .42 Right Side 101.81 20.36 .20 40.72 .41 Left Side 105.92 21.18 .21 42.37 .42 Unmarked 121.43 24.29 .25 40.57 .41 400 mg/kg Head 127.14 25.43 .25 6.35 .64 Tail 118.32 23.67 .24 5.9 .59 Trunk 101.64 20.33 .20 5.08 .51 Right Side 107.46 21.49 .21 5.37 .51 Left Side 123.08 24.60 .25 6.15 .62 Unmarked 136.68 27.34 .27 6.83 .68 Water 5 ml/kg Head 133.09 26.62 .67 Tail 104.24 20.85 .52 Trunk 113.86 22.77 .67 Right Side 114.18 22.84 .57 Left Side 122.94 24.59 .61 81

Unmarked 115.03 23.01 .77

COMPARISON OF FRACTIONS

Table 16 Administration of doses of different fractions

ANIMAL ASA EXTRACT Animal Animal Wt. (mg) Vol. of Wt. Vol. of Mark wt. (g) Stock (mg) Stock Sol. Sol. (ml) (ml) Methanol fraction 200 mg/kg Head 188.84 37.77 .38 37.77 .38 Tail 196.13 39.23 .39 39.23 .39 Trunk 200.17 40.03 .40 40.03 .40 Right. 160.67 32.11 .32 32.11 .32 Side Left. Side 176.40 35.28 .35 35.28 .35 Unmarked 202.49 40.50 .41 40.50 .40 Ethyl acetate fraction 200 mg/kg Tail 118.29 23.66 .24 23.66 .24 Trunk 88.18 17.64 .18 17.64 .18 Right Side 98.02 19.60 .20 19.60 .20 Left Side 92.63 18.53 .19 18.53 .19 Unmarked 113.40 26.68 .27 26.68 .27 Ranitidine 50 mg/kg Head 196.75 39.35 .39 9.84 .39 Tail 203.96 40.79 .41 10.2 .41 Trunk 160.97 32.19 .32 8.05 .32 Right Side 260.87 52.17 .52 13.04 .52 Left Side 209.35 41.87 42 10.47 .42 Unmarked 199.79 39.96 .40 9.99 .4 Crude methanol extract 200 mg/kg Head 168.02 33.60 .34 33.60 .34 Tail 184.06 36.81 .37 36.81 .37 Trunk 174.40 34.88 .35 34.88 .35 Right Side 155.38 31.08 .31 31.08 .31 Left Side 272.82 54.56 .55 54.56 .55 Unmarked 237.24 47.45 .48 .48 .48 Water 5 ml/kg Head 139.42 27.88 .28 .7 Tail 115.03 23.00 .23 .6 Trunk 156.87 31.37 .31 .8 82

Right Side 166.80 33.36 .33 .8 Left Side 143.24 28.65 .29 .7 Unmarked 165.88 33.18 .33 .8

Table 17: Administration of methanol fraction

ANIMAL ASA EXTRACT Animal Animal Wt. (mg) Vol. of Wt. Vol. of Mark wt. (g) Stock (mg) Stock Sol. Sol.(Ml) (ml) 100 mg/kg Head 98.14 19.63 .196 9.8 .32 Tail 81.73 16.35 .164 8.17 .27 Trunk 92..49 18.50 .185 9.25 .31 Right Side 84.04 16.81 .168 8.4 .28 Left. Side 99.95 16.81 .2 10.0 .33 200 mg/kg Head 90.08 18.02 .18 18.02 .6 Tail 79.63 15.93 .16 15.93 .53 Trunk 92.81 18.56 .19 18.44 62 Right. 97.52 17.51 .18 17.50 .06 Side Left Side 85.24 17.05 .17 17.01 .57 Ranitidine 50 mg/kg Head 71.28 14.26 .14 28.51 2.85 Tail 96.07 19.33 19 38.67 3.87 Trunk 88.15 17.63 .18 35.26 3.53 Right. 80.52 16.10 .16 32.23 3.22 Side Left Side 62.49 12.50 .13 25.0 2.50 400 mg/kg Head 109.52 21.88 .21 5.47 .36 Tail 72.09 14.42 .14 3.6 .24 Trunk 84.18 16.84 .17 4.2 .28 Right. 84.06 16.81 .17 4.2 .28 Side Left. Side 86.55 17.31 .17 4.3 .29 Water 5 ml/kg Head 93.30 18.66 .19 .47 Tail 92.48 18.5 .19 .46 83

Trunk 90.56 18.11 .18 .45 Right Side 94.06 18.81 .19 .47 Left Side 89.35 17.87 .18 .46

APPENDIX 2 PHYTOCHEMICAL ANALYSIS

Table 18: Phytochemical screening of T. superba

Test Sample

Powdered Crude Methanol Ethyl acetate bark extract Fraction Fraction

1 Alkaloids + + + +

2 Glycosides + + + +

3 Tannins + + + -

4 Saponins + + + +

5 Flavonoides + + + +

6 Carbohydrates + + + +

7 Steroids + + + +

8 Terpenoids + + + +

9 Resins + + + - 84

10 Proteins + + + +

11 Mucilages + + + +

12 Reducing sugars + + + +

(+) Indicates Presence; (-) Indicates Absence

APPENDIX 3

Table 11: EVALUATION OF ANTI-ULCER PROPERTIES

EFFECT OF T superba STEM BARK METHANOL FRACTION ON ASPIRIN- INDUCED ULCER

Ulcer scores

Dose 100 200 400 Ranitidine Water Head 5.5 7.5 13.5 2 29 Tail 11.5 5.5 3 3.5 19 Trunk 5.5 7.5 8 3.5 13.5 Right. Hind 15.5 17.5 2 1.5 16 Left .Hind 13.5 4.5 3 1 10.5 Total Score 51.5 42.5 29.5 11.5 88 Mean Score (UI) 10.3±2.05 8.5±2.41 5.9±2.17 2.3±0.51 17.6±3.17 %PI 41.48 51.71 66.48 86.93

EFFECT OF T superba STEM BARK CRUDEMETANOL EXTRACT ON ASPIRIN-INDUCED ULCER

DOSE Ulcer scores

100 200 400 Ranitidine Water 85

Head 11 3 2 0 6.5 Tail 4.5 6 2.5 0 15 Trunk 2.5 2.5 3 1 3.5 Right. Hind 1 2 2.5 0 8 Left .Hind 3 2 1 1 7.5 Unmarked 5 4.5 3 2 19.5 Total Score 27 20 14 4 60 Mean Score (UI) 4.5±3.3 3.3±1.6 2.3±.8 .7±0.8 10.1±6.0 %PI 55 67 77 93

EFFECT OF CRUDE T superba STEM BARK METHAOL EXTRACT ON

STRESS-INDUCED ULCER

100 200 400 Ranitidine Water Head 47.5 8.5 4 4.5 38 Tail 27.5 10 4 5.5 16 Trunk 27.5 41.5 0 7 35 Left Side 22 9 9 12 29 Right Side 17.5 7.5 8 1.5 45 Total Ulcer Score 142 76.5 25 30.5 163 Mean score (UI) 28.4±5.13 15.3±6.56 5.0±1.61 6.1±1.72 32.6±4.88 % PI 12.88344 53.06748 84.66258 81.28834

EFFECT OF T superba STEM BARK CRUDE METHANOL EXTRACT ON

ETHANOL-INDUCED ULCER

Ulcer scores

Head 25.5 11 5 4 45 Tail 19 10 8 3 37 Trunk 16 25 9.5 8.5 37.5 Left Side 16.5 8 10.5 4 38 86

Right Side 23 9.5 14.5 4.5 34 Total Ulcer Score 100 63.5 47.5 24 191.5 Mean score (UI) 20±1.85 13.2±3.02 9.5±1.56 4.8±0.96 38.3±1.81 % PI 47.78068 66.84073 75.19582 87.46736 0

COMPARISON OF FRACTIONS

Ulcer scores Ethyl acetate Methanol Ranitidine Crude Water Head 17.5 6.5 1.5 9 29 Tail 14.0 2.5 4 6.5 28 Trunk 12 18.5 2.1 4.5 13.5 Right. Hind 11.5 4 2.3 1.5 3 L.eft Hind 9.5 9 3.2 11 12 Unmarked 6.5 11.5 2 5.5 10 Total Score 71 52 15.1 38 105.5 Mean Score (UI) 11.83 8.67 2.52 6.33 1756 %PI 32.70 50.71 85.69 63.98

APPENDIX 4

STATISTICAL ANALYSIS

ANOVA and POST HOC RESULT OF CRUDE METHANOL EXTRACT

Descriptives Score

N Mean Std. Deviation Std. Error 95% Confidence Interval for Minimu Maximum Mean m

Lower Bound Upper Bound

1 6 4.500 3.4928 1.4259 .834 8.166 1.0 11.0 2 6 3.333 1.6021 .6540 1.652 5.015 2.0 6.0 3 6 2.333 .7528 .3073 1.543 3.123 1.0 3.0 4 6 .667 .8165 .3333 -.190 1.524 .0 2.0 5 6 10.000 6.0000 2.4495 3.703 16.297 3.5 19.5 Total 30 4.167 4.4048 .8042 2.522 5.811 .0 19.5

ANOVA Score

Sum of Squares df Mean Square F Sig.

Between Groups 302.667 4 75.667 7.276 .000 88

Within Groups 260.000 25 10.400 Total 562.667 29

Post Hoc Tests

Multiple Comparisons Dependent Variable: Score

(I) Group (J) Group Mean Difference (I- Std. Error Sig. 95% Confidence Interval

J) Lower Bound Upper Bound

2 1.1667 1.8619 .969 -4.301 6.635

3 2.1667 1.8619 .771 -3.301 7.635 1 4 3.8333 1.8619 .269 -1.635 9.301

5 -5.5000* 1.8619 .048 -10.968 -.032

1 -1.1667 1.8619 .969 -6.635 4.301

3 1.0000 1.8619 .983 -4.468 6.468 2 Tukey 4 2.6667 1.8619 .614 -2.801 8.135 HSD 5 -6.6667* 1.8619 .011 -12.135 -1.199

1 -2.1667 1.8619 .771 -7.635 3.301

2 -1.0000 1.8619 .983 -6.468 4.468 3 4 1.6667 1.8619 .896 -3.801 7.135

5 -7.6667* 1.8619 .003 -13.135 -2.199

1 -3.8333 1.8619 .269 -9.301 1.635 4 2 -2.6667 1.8619 .614 -8.135 2.801 89

3 -1.6667 1.8619 .896 -7.135 3.801

5 -9.3333* 1.8619 .000 -14.801 -3.865

1 5.5000* 1.8619 .048 .032 10.968

2 6.6667* 1.8619 .011 1.199 12.135 5 3 7.6667* 1.8619 .003 2.199 13.135

4 9.3333* 1.8619 .000 3.865 14.801 1 5 -5.5000* 1.8619 .023 -10.354 -.646 Dunnet 2 5 -6.6667* 1.8619 .005 -11.520 -1.813 t t (2- 3 5 -7.6667* 1.8619 .001 -12.520 -2.813 sided)b 4 5 -9.3333* 1.8619 .000 -14.187 -4.480

*. The mean difference is significant at the 0.05 level. b. Dunnett t-tests treat one group as a control, and compare all other groups against it.

Homogeneous Subsets

Score

Group N Subset for alpha = 0.05

1 2

4 6 .667

3 6 2.333

2 6 3.333 Tukey HSDa 1 6 4.500

5 6 10.000

Sig. .269 1.000

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 6.000.

ANOVA For CRUDE METHANOL EXTRACT ETHANOL MODEL

Descriptives 90

SCORE

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

1 5 20.000 4.1382 1.8507 14.862 25.138 16.0 25.5 2 5 13.200 6.7602 3.0232 4.806 21.594 8.0 25.0 3 5 9.500 3.4821 1.5572 5.176 13.824 5.0 14.5 4 5 4.800 2.1389 .9566 2.144 7.456 3.0 8.5 5 5 38.300 4.0559 1.8138 33.264 43.336 34.0 45.0 Total 25 17.160 12.5737 2.5147 11.970 22.350 3.0 45.0

ANOVA SCORE

Sum of Squares df Mean Square F Sig.

Between Groups 3410.460 4 852.615 44.419 .000 Within Groups 383.900 20 19.195 Total 3794.360 24

Post Hoc Tests

Multiple Comparisons Dependent Variable: SCORE

(I) GROUP (J) Mean Difference (I- Std. Error Sig. 95% Confidence Interval

GROUP J) Lower Bound Upper

Bound

2 6.8000 2.7709 .142 -1.492 15.092

3 10.5000* 2.7709 .009 2.208 18.792 1 4 15.2000* 2.7709 .000 6.908 23.492 Tukey HSD 5 -18.3000* 2.7709 .000 -26.592 -10.008

1 -6.8000 2.7709 .142 -15.092 1.492 2 3 3.7000 2.7709 .673 -4.592 11.992 91

4 8.4000* 2.7709 .046 .108 16.692

5 -25.1000* 2.7709 .000 -33.392 -16.808

1 -10.5000* 2.7709 .009 -18.792 -2.208

2 -3.7000 2.7709 .673 -11.992 4.592 3 4 4.7000 2.7709 .458 -3.592 12.992

5 -28.8000* 2.7709 .000 -37.092 -20.508

1 -15.2000* 2.7709 .000 -23.492 -6.908

2 -8.4000* 2.7709 .046 -16.692 -.108 4 3 -4.7000 2.7709 .458 -12.992 3.592

5 -33.5000* 2.7709 .000 -41.792 -25.208

1 18.3000* 2.7709 .000 10.008 26.592

2 25.1000* 2.7709 .000 16.808 33.392 5 3 28.8000* 2.7709 .000 20.508 37.092

4 33.5000* 2.7709 .000 25.208 41.792 1 5 -18.3000* 2.7709 .000 -25.646 -10.954

2 5 -25.1000* 2.7709 .000 -32.446 -17.754 Dunnett t (2-sided)b 3 5 -28.8000* 2.7709 .000 -36.146 -21.454

4 5 -33.5000* 2.7709 .000 -40.846 -26.154

*. The mean difference is significant at the 0.05 level. b. Dunnett t-tests treat one group as a control, and compare all other groups against it.

Homogeneous Subsets

SCORE

GROUP N Subset for alpha = 0.05

1 2 3 4

4 5 4.800

Tukey HSDa 3 5 9.500 9.500

2 5 13.200 13.200 92

1 5 20.000

5 5 38.300

Sig. .458 .673 .142 1.000

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 5.000.

ANOVA OF CRUDE METHANOL EXTRACT ON COLD-RESTRAINT-STRESS

MODEL

Descriptives 93

ULCER SCORE

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

1 5 28.400 11.4695 5.1293 14.159 42.641 17.5 47.5 2 5 15.300 14.6740 6.5624 -2.920 33.520 7.5 41.5 3 5 5.000 3.6056 1.6125 .523 9.477 .0 9.0 4 5 6.100 3.8633 1.7277 1.303 10.897 1.5 12.0 5 5 32.600 10.9225 4.8847 19.038 46.162 16.0 45.0 Total 25 17.480 14.6710 2.9342 11.424 23.536 .0 47.5

ANOVA ULCER SCORE

Sum of Squares df Mean Square F Sig.

Between Groups 3189.340 4 797.335 8.069 .000 Within Groups 1976.400 20 98.820 Total 5165.740 24

Post Hoc Tests

Multiple Comparisons Dependent Variable: ULCER SCORE

(I) (J) Mean Std. Sig. 95% Confidence

ANIMAL ANIMA Difference Error Interval

GROUP L (I-J) Lower Upper

GROUP Bound Bound

2 13.1000 6.2871 .265 -5.713 31.913

3 23.4000* 6.2871 .010 4.587 42.213 1 4 22.3000* 6.2871 .015 3.487 41.113

5 -4.2000 6.2871 .961 -23.013 14.613

1 -13.1000 6.2871 .265 -31.913 5.713

3 10.3000 6.2871 .492 -8.513 29.113 2 4 9.2000 6.2871 .596 -9.613 28.013

5 -17.3000 6.2871 .081 -36.113 1.513

1 -23.4000* 6.2871 .010 -42.213 -4.587

2 -10.3000 6.2871 .492 -29.113 8.513 Tukey HSD 3 4 -1.1000 6.2871 1.000 -19.913 17.713

5 -27.6000* 6.2871 .002 -46.413 -8.787

1 -22.3000* 6.2871 .015 -41.113 -3.487

2 -9.2000 6.2871 .596 -28.013 9.613 4 3 1.1000 6.2871 1.000 -17.713 19.913

5 -26.5000* 6.2871 .003 -45.313 -7.687

1 4.2000 6.2871 .961 -14.613 23.013

2 17.3000 6.2871 .081 -1.513 36.113 5 3 27.6000* 6.2871 .002 8.787 46.413

4 26.5000* 6.2871 .003 7.687 45.313 1 5 -4.2000 6.2871 .905 -20.867 12.467

2 5 -17.3000* 6.2871 .041 -33.967 -.633 Dunnett t (2-sided)b 3 5 -27.6000* 6.2871 .001 -44.267 -10.933

4 5 -26.5000* 6.2871 .002 -43.167 -9.833

*. The mean difference is significant at the 0.05 level. b. Dunnett t-tests treat one group as a control, and compare all other groups against it.

Homogeneous Subsets

ULCER SCORE

ANIMAL GROUP N Subset for alpha = 0.05

1 2

3 5 5.000

4 5 6.100

2 5 15.300 15.300 Tukey HSDa 1 5 28.400

5 5 32.600

Sig. .492 .081

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 5.000.

ANOVA of EFFECT OF METHANOL FRACTIION ON ASPIN-INDUCTION MODEL

Descriptives SCORE

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

1 5 10.3000 4.60435 2.05913 4.5829 16.0171 5.50 15.50 2 5 8.3000 5.40370 2.41661 1.5904 15.0096 3.50 17.50 3 5 5.9000 4.85283 2.17025 -.1256 11.9256 2.00 13.50 4 5 2.3000 1.15109 .51478 .8707 3.7293 1.00 3.50 5 5 17.6000 7.10106 3.17569 8.7829 26.4171 10.50 29.00 Total 25 8.8800 6.93914 1.38783 6.0157 11.7443 1.00 29.00

ANOVA SCORE

Sum of Squares df Mean Square F Sig.

Between Groups 652.840 4 163.210 6.492 .002 Within Groups 502.800 20 25.140 Total 1155.640 24

Post Hoc Tests

Multiple Comparisons Dependent Variable: SCORE

(I) GROUP (J) GROUP Mean Difference (I- Std. Error Sig. 95% Confidence Interval

J) Lower Bound Upper Bound

2 2.00000 3.17112 .968 -7.4892 11.4892

3 4.40000 3.17112 .642 -5.0892 13.8892 1 4 8.00000 3.17112 .125 -1.4892 17.4892

5 -7.30000 3.17112 .185 -16.7892 2.1892

1 -2.00000 3.17112 .968 -11.4892 7.4892

3 2.40000 3.17112 .940 -7.0892 11.8892 2 4 6.00000 3.17112 .353 -3.4892 15.4892

5 -9.30000 3.17112 .056 -18.7892 .1892

1 -4.40000 3.17112 .642 -13.8892 5.0892

2 -2.40000 3.17112 .940 -11.8892 7.0892 Tukey HSD 3 4 3.60000 3.17112 .786 -5.8892 13.0892

5 -11.70000* 3.17112 .011 -21.1892 -2.2108

1 -8.00000 3.17112 .125 -17.4892 1.4892

2 -6.00000 3.17112 .353 -15.4892 3.4892 4 3 -3.60000 3.17112 .786 -13.0892 5.8892

5 -15.30000* 3.17112 .001 -24.7892 -5.8108

1 7.30000 3.17112 .185 -2.1892 16.7892

2 9.30000 3.17112 .056 -.1892 18.7892 5 3 11.70000* 3.17112 .011 2.2108 21.1892

4 15.30000* 3.17112 .001 5.8108 24.7892 Dunnett t (2- 1 5 -7.30000 3.17112 .100 -15.7068 1.1068 98

b sided) 2 5 -9.30000* 3.17112 .028 -17.7068 -.8932

3 5 -11.70000* 3.17112 .005 -20.1068 -3.2932

4 5 -15.30000* 3.17112 .000 -23.7068 -6.8932

*. The mean difference is significant at the 0.05 level. b. Dunnett t-tests treat one group as a control, and compare all other groups against it.

Homogeneous Subsets

SCORE

GROUP N Subset for alpha = 0.05

1 2

4 5 2.3000

3 5 5.9000

2 5 8.3000 8.3000 Tukey HSDa 1 5 10.3000 10.3000

5 5 17.6000

Sig. .125 .056

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 5.000.

ANOVA FOR COMPARISON of FRACTIONS ON ASPIRN-INDUCTION ODEL

Descriptives SCORE

N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

1 6 11.8333 3.76386 1.53659 7.8834 15.7833 6.50 17.50 2 6 8.6667 5.81951 2.37580 2.5595 14.7739 2.50 18.50 3 6 2.5167 .91524 .37365 1.5562 3.4772 1.50 4.00 4 6 6.3333 3.35659 1.37032 2.8108 9.8559 1.50 11.00 5 5 18.5000 9.21954 4.12311 7.0524 29.9476 10.00 29.00 Total 29 9.2621 7.15554 1.32875 6.5402 11.9839 1.50 29.00

ANOVA SCORE

Sum of Squares df Mean Square F Sig.

Between Groups 792.960 4 198.240 7.426 .000 Within Groups 640.688 24 26.695 Total 1433.648 28 100

Post Hoc Tests

Multiple Comparisons Dependent Variable: SCORE

(I) (J) GROUP Mean Std. Error Sig. 95% Confidence Interval

GROUP Difference Lower Upper

(I-J) Bound Bound

2 3.16667 2.98303 -5.6214 11.9547

3 9.31667* 2.98303 .034 .5286 18.1047 1 4 5.50000 2.98303 .373 -3.2881 14.2881

Tukey 5 -6.66667 3.12862 .240 -15.8837 2.5504 HSD 1 -3.16667 2.98303 .824 -11.9547 5.6214

3 6.15000 2.98303 .269 -2.6381 14.9381 2 4 2.33333 2.98303 .933 -6.4547 11.1214

5 -9.83333* 3.12862 .033 -19.0504 -.6163 101

1 -9.31667* 2.98303 .034 -18.1047 -.5286

2 -6.15000 2.98303 .269 -14.9381 2.6381 3 4 -3.81667 2.98303 .706 -12.6047 4.9714

5 -15.98333* 3.12862 .000 -25.2004 -6.7663

1 -5.50000 2.98303 .373 -14.2881 3.2881

2 -2.33333 2.98303 .933 -11.1214 6.4547 4 3 3.81667 2.98303 .706 -4.9714 12.6047

5 -12.16667* 3.12862 .006 -21.3837 -2.9496

1 6.66667 3.12862 .240 -2.5504 15.8837

2 9.83333* 3.12862 .033 .6163 19.0504 5 3 15.98333* 3.12862 .000 6.7663 25.2004

4 12.16667* 3.12862 .006 2.9496 21.3837 1 5 -6.66667 3.12862 .129 -14.8008 1.4674

* Dunnett t 2 5 -9.83333 3.12862 .015 -17.9674 -1.6992 b (2-sided) 3 5 -15.98333* 3.12862 .000 -24.1174 -7.8492

4 5 -12.16667* 3.12862 .002 -20.3008 -4.0326

*. The mean difference is significant at the 0.05 level. b. Dunnett t-tests treat one group as a control, and compare all other groups against it.

Homogeneous Subsets

SCORE

GROUP N Subset for alpha = 0.05

1 2 3

3 6 2.5167

4 6 6.3333 6.3333

2 6 8.6667 8.6667 Tukey HSDa,b 1 6 11.8333 11.8333

5 5 18.5000

Sig. .286 .392 .217

Means for groups in homogeneous subsets are displayed. 102

a. Uses Harmonic Mean Sample Size = 5.769. b. The group sizes are unequal. The harmonic mean of the group sizes is used. Type I error levels are not guaranteed.