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 justifie
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
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 principlesextraction
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
5
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)
7
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)
8
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 óc̣ há = white edo (Amufu)ojiloko (Nkalagu) ojiroko
(Owerri) èdò óc̣ há = 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; intrastaminal
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 welldrained,
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).
11
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-Dxylopyranoside
(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-Dglucopyranoside
(Turibio et al., 2009)
13
Title Aim Result Author
1 Acute toxicity and
anti-ulcerogenic
activity of an
aqueous extract from
the stem bark of
Terminalia superba
Engl. and Diels
(Combretaceae)
This study was
aimed to evaluate
the acute toxicity
and gastric antiulcer
activity of an
aqueous extract of
Terminalia superba
These results
suggested that the
preventive anti-ulcer
activity of AETs
may be due to a
cytoprotective effect.
Kouakou et al., 2013
2 Phytochemical
constituents and
antidiarrheal effects
of the aqueous extract
of Terminalia superba
leaves on
wistar rats
In this research,
aqueous extract of T.
superba leaves was
investigated for the
treatment of
diarrhoea in wistar
rats.
The data in the present
study indicate that the
aqueous extract of T.
superba leaves
possessed antidiarrheal
properties
Bamisaye et al.,2013
3 Antifungal activity of
the aqueous and
hydro-alcoholic
extracts of T. superba
Engl. on the in vitro
growth of clinical
isolates of pathogenic
fungi
To locate the true
potential antimicrobial
in general,
but especially antifungal
extracts of T.
superba on the invitro
growth of C.
albicans,
A. fumigatus, C.
Aqueous extracts and
hydro-alcoholic extract
of T. superba have a
dose-dependent
fungicidal activity
against clinical fungal
isolates used
Ahon et al., 2011
Table 1: Some previous researches on T. superba
14
neoformans and
T. mentagrophytes
4 The aqueous extract of
Terminalia superba
(Combretaceae)
prevents glucoseinduced
hypertension
in rats.
To Investigate the
hypotensive and the
antihypertensive
effects of the
aqueous extract of
the stem bark of
Terminalia superba.
The aqueous extract of
the stem bark of T.
superba
exhibits hypotensive
and anti-hypertensive
properties
Tom et al., 2011
5 Protective role of
Terminalia superba
Ethyl Aetate against
Oxidative Stress Type
2 Diabetes
Investigation of the
protective role of
Terminalia superba
ethyl acetate extract
against
streptozotocinnicotinamide
induced
type 2 diabetes.
The results suggest
that, ethyl acetate
extract of T. superba
lower blood glucose
and hyperlipidemia,
prevent oxidative
stress and reduce
blood pressure in
diabetic conditions.
Ngueguim et al., 2011
5 Antimycobacterial,
antibacterial and
antifungal activities of
Terminalia superba
(Combretaceae)
To evaluate the
methanol extract
from the stem bark
of Terminalia
superba (TSB),
fractions (TSB1–7)
for antimicrobial
activity
Provide promising
baseline information
for the potential use of
the crude extract from
T. superba, in the
treatment of
tuberculosis, bacterial
and fungal infections
Kuete et al., 2010
15
7 Anti-diabetic activity
of methanol/methylene
chloride extract of
Terminalia superba
leaves on
streptozotocin-induced
diabetes in rats
The present study
was undertaken to
investigate the antihyperglycemia
effect
of the
methanol/methylene
chloride extract of
Terminalia superba
leaf
Terminalia superba
leaf extract possess
antidiabetic
properties
Padmashree et al., 2010
8 in vivo assessment of
hypoglycaemic and
antioxidant activities
of aqueous extract of
Terminalia superba in
alloxan-diabetic rats
To investigate the
possible actions of
aqueous extract of
the roots of
Terminalia superba
on glucose
homeostasis and on
MDA, SOD and
catalase homolysate
of diabetes rats
This extract
demonstrates
significant
hypoglycaemic effect
thus reduces the
antioxidant parameters
in alloxan-induced
diabetes rats
Momo and Oben 2009
9 Antioxidant properties
and α-amylase
Inhibition of
Terminalia superba,
Albiziz sp., Cola
odorata and Harunga
madagascarensis used
in the management of
The evaluations of
the antioxidant
potential and α-
amylase inhibitory
activity of these
extracts were also
carried out
For all the plants
tested, at least one
extract inhibited the
activity of α-amylase.
The most effective was
the hydroethanolic
extract of T. superba.
Momo et al., 2009
16
diabetes
10 α-Glucosidase
inhibitory constituents
from stem bark of
Terminalia superba
(Combretaceae)
To identify
α-glucosidase
inhibitory
constituents from
stem bark of
Terminalia superba
All the isolated
compounds were
evaluated for their
glycosidase inhibition
activities. Gallic acid
and methyl gallate
showed significant
α-glucosidase
inhibition activity.
Wansi et al., 2007
11 Antimicrobial
Pentacyclic
Triterpenoids from
Terminalia superba
Antibacterial
bioassay-guided
fractionation of the
methanol extract of
the stem bark of
Terminalia superba
The isolation and
characterization of
four new triterpene
glucosides
Tabopda et al., 2009
12 Analgesic Activities of
the Stem Bark Extract
of Terminalia superba
Engl and Diels
(Combretacea)
To evaluate the
analgesic activities of
the extract
obtained from this
plant by in-vivo
screening methods
n-BuOH fraction of T.
superba stem bark
could be beneficial in
the management of
pain
Dongmo et al., 2006
17
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 stressinduced
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).
20
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).
21
Table 2: Review of some recent anti-ulcer researches involving other plants
Plant Plant
part
Extract Ulcer Model Reference
Cayratia trifolia L.
(MECT) (Vitaceae)
Leaves petroleum
ether and
hydroalcohol
(30:70)
Pylorus ligation and
ethanol
Gupta et al.,2012
Emblica officinalis
Gaertn., syn:
Phyllanthus emblica
(Euphorbiaceae),
Fruit Ethanol Pylorus ligation,
indomethacin,
hypothermic restraint
stress and necrotizing
agents (80% ethanol,
0.2 M NaOH and 25%
NaCl).
Al-Rehaily et al., 2002
Nigella sativa Linn
Family: Ranunculaceae
Seed Alcoholic Pylor ligation and
aspirin
Rajkapoor et al.,2002
Abutilon indicum L.
(Family: Malvaceae),
Leaves ethanolic
extract
Pylorus ligatIion and
ethanol
Dashputre et al.,2011
Capsicum frutescenes Fruit ethanolic aspirin Dass , et al.,2008
Mimosa pudica ,
(Fabaceae),
leaves Methanolic,
chloroform
and diethel
ether extracts
Aspirin, Alcohol and
pyloric ligation model
Vinothapooshan and K
Sundar 2011
22
Aegle marmelos Fruit
seed
Methanolic
and aqueous
Indomethacin induced
ulceration, stressed
induced ulceration and
pylorus ligation
Ganesh et al, 2011
Picrasma quassioides
(D. Don) Bennett
family Simaroubaceae
Whole
plant
Aqueous
Extract
Aspirin-pylorus
ligation, HCl-ethanol,
water immersion-stress
Hwisa et al., 2013
Garcinia kola
Family: Guttifera.
seeds, Methanolic Ethanol, Ige et al, 2012
Entandrophragma utile
Bark Aqueous Aspirin,
immobilization, coldrestraint,
histamineinduced,
pylorus
ligation, necrotizing
substances
John et al 2012
Cayratia trifolia methanolic pyloric ligated and
ethanol
Jyoti , et al 2012
Tinospora cordifolia whole
plant
Alcoholic
extract,
Pyloric ligation,
ibuprofen and cold
restraint
Bairy et al.2001
Falcaria vulgaris
Family :Umbelliferae
leaves
and
stems
Ethanolic
extract
Ethanol Khazaei et al, 2006
Barleria prionitis Linn
Family Acanthaceae)
Leaves Methanol Ethanol and
Indomethacin
Manjusha et al,2013
Morinda citrifolia Linn Fruit Ethyl acetate Ethanol,A spirin and Muralidharan and
23
Rubiaceae, Pyloric ligation,
Cysteamine HCl,
Srikanth, 2009
Carpolobia lutea
(polygalaceae) G. Don
leaf Ethanol Indomethacin, Ethanol,
Reserpine in 0.5%
Acetic acid, Stress,
Serotonin and
Diethylthiocarbamate
Nwidu and Nwafor,
2009
Cassia singueana
Leaves Methanol Indomethacin Ode, 2011
Hibiscus cannabinus
Family: Malvaceae,
Leaves methanolic Pylorus ligation and
Indomethacin
Silambujanaki et al
2010
Falcaria vulgaris
Family: Umbelliferae
leaves
and
stems
hydro
alcoholic
ethanol (50%) Khazaei, and Salehi
2006.
Aloe vera Leaf gel Etanolic
extract
Indomethacin and
Ethanol
Subramanian et al.,
2007
Bauhinia racemosa fruit
powder
aqueous
extract
Paracetamol Borikar et al 2009
“Parsley” Petroselinum
crispum,
Aerial
parts
Ethanolic Pyloric ligation,
hypothermic- restraintstress,
Al-Howiriny et al,
2003
24
Aspilia africana C.D.
Adams, (Compositae)
Leaf Aqueous Ehanol, indomethacin
and aspirin
Ubaka et al., 2010
Croton zambesicus
Muell Arg.
(Euphorbiaceace) (syn
C. amabilis Muell.
Arg. C. gratissimus
Burch)
leaf ethanolic indomethacin, ethanol
and histamine
Okokon et al, 2011.
Boswellia serrata
(Family Bursera-ceae)
bark Ppetroleum
ether
(250mg/kg)
and aqueous
extracts
aspirin Zeeyauddin , 2011
Moringa oleifera
Lam (Moringaceae)
leaves
and
fruits
acetone
extract
and
methanol
extract
Ethanol, Cold
restraint stress,
indomethacin,
Pylorus ligation,
Devaraj et al.., 2007
Momordica
charantia L.-
(cucurbitaceae)
Fruits olive oil
extract
indomethacin DENG‹Z and
Nesrin ,2005
Barleria prionitis L.
Family Acanthaceae
Leaves methanolic ethanol and
indomethacin
Manjusha et al,
2013
Kigelia africana,
Nauclea latifolia and
Leaves Ethanolic
extracts
aspirin Orole et al., 2013.
25
Staudtia stipitata
Ginger (Zingiber
officinale)
Ginger
powder
Aqueous aspirin Wang et al. 2011
Etandrophagma utile fresh
bark
aqueous Ethanol or
histamine
John et al 2012
Aegle marmelos
(AM), family:
Rutaceae
fruits aqueous Aspirin Das and Roy 2012
26
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
plant
Ulcer model Reference
Calliandra
portoticensis
Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal
1988
Entandrophragma
utile
Bark Ethanol John and
Onabanjo,1990
Linderae umbellatae Stem Stress Ezaki et al.,1985
Mallotus japonicas Bark (Clinical study) Saijo et al., 1989
Rhigiocarya
racemifera
Leaves Indomethacin, reserpine,
serotonin
Aguwa, 1985,
Veronica officinalis Aerial
parts
Indomethacin, reserpine Scarlat et al 1985
27
Table 4: Some plants containing flavonoids with anti-ulcer activity
FLAVONOID ULCER MODEL REFERENCE
Anthocyanosides Pylorus-ligated, Reserpine,
Phenylbutazone
Magistretti et al 1998
Catechin Stress Lorenz et al., 1975
Genistin Phenylbutazone, Serotinine,
Pylorus-ligated,
Stress, Reserpin
Rainova et al., 1988
Hypolaetin-8-glucoside Stress, Ethanol,
Acetylsalicylic acid
Alcaraza and Hoult, 1985
Kaempferol Ethanol Izzo et al., 1994
Leucocyanidin Aspirin Lewis et al., 1999
Luteolin-7-glycoside Pylorus-ligated, Stress,
Reserpine, Phenylbutazone,
Serotinin
Rainova et al., 1988
5-Methoxyflavone Indomethacin Blank et al., 1997
Myricetin3-0-DGalactoside
Stress, Pylorous-ligated,
Ethanol
Reyes et al., 1996
Naringin Ethanol, Stress, Pylorous
ligation
Martin et al 1993
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
28
Table 5: Some Plants Containing Saponins with Anti-Ulcer Activity
Botanical Name Part Plant Ulcer Model Reference
Calendula officinalis Rhizome Caffeine-arsenic, butadiene,
pylorus-ligated
Iatsyno et al., 1978
Calliandra
portoticensis
Leaves Stress, pylorus ligated, E. coli Aguwa and Lawal 1988
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
stauditii
Leaves Indomethacin, serotonin,
stress
Aguwa and Okunji 1986
Rhigiocarya
racemifera
Leaves Indomethacin, reserpine,
serotonin
Aguwa, 1985
Spartium junceum Flowers Ethanol Yesilada and Takaishi,
1999
Veronica officinalis Aerial
parts
Indomethacin, reserpine Scarlat et a l, 1985
29
Table 6: Some active constituents isolated from plant (Goel and Sairamanti, 2002)
Plants and plant part Active Constituents Models
Tectona grandis Linn
(Trunk bark and wood
chips
Lapachol IS-ASP-induced GU in rats and
HIST- induced DU in rats and GP
repectively
Rhamnus procumbens
(Whole plant)
Kaempferol PL-, ethanol, IS- and CRSinduced
GU in rats and HISTinduced
GU and DU in GP.
Rhamnus triquerta Wall
(Whole plant)
Emodin RS-, PL- and IS- induced GU in
rats
Datura fastuosa (Leaves) Withafstuosin E CRS-, PL- and ASP-induced GU
in rats
Flueggea
microcarpa
(Leaves and
roots)
Bergenin/norbergenin PL- and ASP- induced GU in rats
and CRS- induced GU in rats and
GP.
Azadirachta indica Nimbidin ASP-, prednisolone-,
indomethacin-, serotonin stressand
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-, PLand
stress-induced
GU in rats
Bacopa monniera (Whole
plant)
Standardized extract of
bacoside A (35%)
CRS-, ethanol, ASP- and PLinduced
GU in rats
ASP-aspirin; ce-chloroform; CRS-cold restraint stress; CYS-cysteamine; DU-duodenal ulcer; GPguinea
pig; GU-gastric ulcer; HIST-histamine; IS-immobilization stress; PL-pylorus ligation; RSrestraint
stress;
30
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).
31
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
32
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).
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