Date post: | 11-Apr-2015 |
Category: |
Documents |
Upload: | saleheen1978 |
View: | 39 times |
Download: | 1 times |
ANTISPASMODIC, ANTIDIARRHEAL and LD50
DETERMINATION of Syzygium guineense in
ANIMAL MODELS
By
BIZUAYEHU NIGATU
A thesis submitted to School of Graduate studies in partial fulfillment of the
degree of Master of Science in Pharmacology, Department of
Pharmacology, Faculty of Medicine, AAU.
DECEMBER 2004
ANTISPASMODIC, ANTIDIARRHEAL and LD50
DETERMINATION of Syzygium guineense in
ANIMAL MODELS
By
BIZUAYEHU NIGATU
A thesis submitted to School of Graduate studies in partial fulfillment of the
degree of Master of Science in Pharmacology, Department of
Pharmacology, Faculty of Medicine, AAU.
Under the Supervision of Eyasu Makonnen, PhD., Professor of
Pharmacology, Department of Pharmacology, Faculty of Medicine, AAU;
Asfaw Debella, PhD., Researcher, Department of Drug Research, EHNRI
and Yalemtsehay Mekonnen, PhD., Associate Professor, Director of
Institute of Pathobiology, AAU.
December 2004
i
DEDICATION
To my Son, Nahom and
beloved Wife, Gelila Sahlu
ii
ACKNOWLEDGEMENTS I praise the name of Almighty God who gave me power and patience in every endeavor of my
life.
I would like to express my genuine thank to my advisors Professor Eyasu Makonnen,
Dr. Asfaw Debella and Dr.Yalemtsehay Mekonnen for their advice and encouragement
while I was working on my thesis research.
My heartfelt thanks go to my wife W/o Gelila Sahlu and my mother W/o Berhane Gebre
Tsadik, whose moral support was an immense help throughout my work.
I am very much grateful to Dr. Abebe Aberra, who helped me during the pilot study.
I want to thank the laboratory technician, Ato Yohannes Negash, who was often around me
during my experimental works and helped me a lot on the operation of the Polygraph
machine.
I would like to deeply thank staff members of Pharmacology Department, AAU and EHNRI
(Drug Research Department) for their technical assistance rendered to me and also for their
cooperativeness to use the facilities.
I would like also to thank the laboratory technician of Core Lab, W/t Betlehem Tefera, for her
cooperation.
My indebtedness is also extended to the animal attendants of Biology Department and
Pharmacology Department, Ato Molla Wale and W/t Hiwot Berhe, respectively, for their
constant care of the experimental animals and supports.
iii
I wish to thank the school of Graduate Studies of AAU for providing me the fund for the
experimental work. I further wish to give my gratitude to the Department of Pharmacology of
AAU for providing me with laboratory equipment, chemicals, and other supports.
Eventually, I would like to thank all my academic colleagues for their supportive opinions and
suggestions.
Finally, I would deeply thank Gondar University, for sponsoring me.
iv
TABLE OF CONTENTS
DEDICATION………….…………………………………………………………………….i ACKNOWLEDGEMENTS………………………………………………………………….ii
LIST of FIGURES…………………………………………………………………………….iv LIST of TABLES……………………………………………………………………………vi
ABBREVIATIONS………..……………………………………………………………… vii ABSTRACT………………………………………………………………………………… ix 1 Introduction……………………………………………………………………………….1
1.1 General………………………………………………………………………………1 1.2 Ethnomedical information of traditional medicine and scientific investigation…….3
1.2.1 Extraction, biological activity and chemical constituents’ evaluation……….4 1.3 Traditional medicine in Ethiopia……………………………………………………7 1.4 Herbal Remedies for Gastrointestinal Motility Disorders and Diarrhea…………….4
1.5 The Genus Syzygium……………………………………………………………….117 1.5.1 Syzygium guineense…………………………………………………………128
2 Objective of the study………………………………………………………………….151 2.1 General objective……………………...………………………………………..21
2.2 Specific obhectives…………………………………………………………………21
3 Materials and Methods………………………………………………………………… 16 3.1. Materials…………………………………………………………………………. 16
3.2 Methods……………………………………………………………………………16 3.2.1 Collection of plant materials…..…………………………………………….. 22 3.2.2 Preparation of extracts…...……………………………………………………23
3.2.2.1 Aqueous extracts...........................................................................................17 3.2.2.2 Hydro-alcoholic extracts...............................................................................17 3.2.2.3 Fractionation of the aqueous extract.............................................................18
3.2.2.4 Experimental animals acclimatization………………...………………...24 3.2.2.5 Pharmacological screening……………………………………………....24
3.2.2.5.1 In vitro testing on Guinea pig Ileum.........................................................19 3.2.2.5.2 In vivo Small intestine transit determination: Charcoal meal Test ........21 3.2.2.5.3 Antidiarrhoeal activity/Castor oil-induced Diarrhea/ ..............................21 3.2.2.5.4 LD50 determination...................................................................................22 3.1.2.4.5 Pilot study on effective dose determination..............................................22
3.2.2.6 Phytochemical Screening…………………………………………………..28 3.2.2.6.1 Identification by chemical means .............................................................23 3.2.2.6.2 Identification of secondary metabolites by Thin-layer chromatography....23
3.2.2.7 Statistical analysis………………………………………………………...29 4 Results……………………………………………………………………………………24
4.1 Phytochemical screening of Syzygium guineense ……………………………….24 4.2 In vitro effects on Guinea-pig Ileum (GPI) ……………………………………….1
4.3 Effect of the extracts on small intestinal transit…………………………………… 14 4.4 Effect of extracts on castor-oil induced diarrhea in mice………………………….. 17
4.5 LD50 determination in mice…………………………………………………………215 5 Discussion……………………………………………………………………………… 24 6 Conclusion……………………………………………………………………………… 31 References…………………………………………………………………………………. 33
v
LIST of FIGURES
Page
Figure 1. Flow chart of sequence for the study of plants used in TM……………………. 6
Figure 2. The plant Syzygium guineense with its green lanceolate, opposite leaves containing red and black fruits (The picture taken from Arebaminche Bush)…………….. 19 Figure 3. The plant Syzygium guineense with ellipsoid drupe, purplish fruit (The picture taken from Abebe and Debela, et al (2003)……………………………………….………. 19
Figure 4. Effect of increasing concentrations of twigs aqueous extracts on the cumulative dose–response sigmoid curves of acetylcholine…………………………………………… 36 Figure 5. Effect of increasing concentrations of twigs 80% methanolic extracts on the cumulative dose–response sigmoid curves of acetylcholine………………………………. 37
Figure 6. Effect of increasing concentrations of stem bark aqueous extracts on the cumulative dose–response sigmoid curves of acetylcholine ……………………………….38 Figure 7. Effect of increasing concentrations of stem bark 80% methanolic extracts on the cumulative dose–response sigmoid curves of acetylcholine………………………………. 39
Figure 8. Effect of increasing concentrations of fruit aqueous extracts on the cumulative dose–response sigmoid curves of acetylcholine…………………………………………… 40 Figure 9. Effect of increasing concentrations of fruit 80% methanolic extracts on the cumulative dose–response sigmoid curves of acetylcholine………………………………. 41 Figure 10. Effect of increasing concentrations of twigs aqueous extracts on the cumulative dose–response sigmoid curves of histamine…………………………………………………42 Figure 11. Effect of increasing concentrations of twigs 80% methanolic extracts on the cumulative dose–response sigmoid curves of histamine……………………………………43 Figure 12. Effect of increasing concentrations of stem bark aqueous extracts on the cumulative dose–response sigmoid curves of histamine……………………………………44 Figure 13. Effect of increasing concentrations of stem bark 80% methanolic extracts on the cumulative dose–response sigmoid curves of histamine…………………………………… 45
Figure 14. Effect of increasing concentrations of fruit aqueous extracts on the cumulative dose–response sigmoid curves of histamine…………………………………………………46 Figure 15. Effect of increasing concentrations of fruit 80% methanolic extracts on the cumulative dose–response sigmoid curves of histamine……………………………………47 Figure 16. Antidiarrheal effect of aqueous extracts of Syzygium guineense in Castor oil-induced mice……………………………………………………………………………….. 53 Figure 17. Antidiarrheal effect of 80% methanolic extracts of Syzygium guineense in Castor oil-induced mice…………………………………………………………………………….54 Figure 18. Antidiarrheal effect of twig aqueous fractionates of Syzygium guineense in Castor oil-induced mice…………………………………………………………………………….54 Figure 19. Probit transformed responses of twig aqueous extracts ……………………….. 55 Figure 20. Probit transformed responses of twigs hydroalcoholic extracts……………….. 56 Figure 21. Probit transformed responses of stem bark aqueous extracts………………….. 56 Figure 22. Probit transformed responses of stem bark hydroalcolic extracts………………57
vi
LIST of TABLES
Page
Table 1. Results of phytochemical identification by using chemical means……………….31
Table 2. Results of TLC analysis of crude extracts and fractionates of Syzygium guineense
..33 Table 3 Inhibition of gastro-intestinal motility by Syzygium guineense crude extracts……49 Table 4. Inhibition of gastro-intestinal motility by Syzygium guineense twigs aqueous fractionates…………………………………………………………………………………. 50 Table 5. Effect of crude aqueous extracts and fractionates in onset of diarrhea……………52
Table 6. Effect of crude hydroalcoholic extracts in onset of diarrhea………………………53
vii
ABBREVIATIONS
5-HT – 5- Hydroxy Tryptamine
AAU – Addis Ababa University
Ach - Acetylcholine
AD - After Death
ANOVA - One-way analysis of variance
BF- n-Butanol fraction
cAMP - Cyclic Adenosine Monophosphate
Con – Control
Conc. - Concentration
EHNRI – Ethiopian Health and Nutrition Research Institute
Faq – Fruit aqueous extracts
Fm – Fruit 80% methanolic extracts
g/kg- gram per kilogram
GPI – Guinea-pig ileum
H- Histamine receptor
His – Histamine
IBS - Irritable Bowel Syndrome
IR- Infra Red
Laq – Leaf tips aqueous extracts
LD50 – Lethal Dose 50
Lm – Twigs 80% methanolic extracts
LPRD - Loperamide
mg/ml - milligram per milliliter
min- minute
viii
mm/min - millimeter per minute
mg/kg – milligram per kilogram
M – Muscarinic
MS- Mass Spectrophotometer
NMR- Nuclear Magnetic Resonance
Rf – Retention Factor
SBaq – Stem bark aqueous extracts
SBm – Stem bark 80% methanolic extracts
SEM - Standard Error of Mean
SPSS – Statistical Package for the Social Sciences
SR- Solid residue
µg/ml - microgram per milliliter
TLC – Thin Layer Chromatography
TM – Traditional Medicine
UV- Ultra-violet
WR- Water residue
WHO – World Health Organization
ix
ABSTRACT
Aqueous and hydroalcoholic extracts of dried twigs, stem barks and fruits of Syzygium
guineense were tested on contraction of isolated guinea pig ileum (GPI) in vitro; intestinal
transit and castor oil-induced diarrhea test in mice in vivo. Different concentrations of each
extract of the plant were used in the presence of agonist controls:- ACh and histamine (in
GPI) as contraction stimulators in vitro, atropine and dexchlorpheniramine were used in GPI
and atropine and loperamide in the intestinal transit and antidiarrheal test, respectively, as
positive control. The leaf aqueous (Laq) and hydroalcoholic (Lm) extracts, exhibited
significant dose-dependent reductions in ACh and histamine-induced commulative
contractions (P< 0.001). The spontaneous agonist-induced contractions of GPI were greatly
reduced by Laq and Lm at maximal dosages suggesting the spasmolytic property of the crude
extracts. All the extracts except Laq and Lm (with dose of 100 and 200µg/ml) were less
potent than atropine (6.66x10-9 M) and dexchlorpheniramine (1.3x10-9 M) in the experiment
of GPI. Most doses of the extracts (i.e Laq, Lm, SBaq) showed antitransit activity in the
intestine of mice and Laq (200mg/g) showed comparable effects with that of atropine. All the
six extracts (i.e. Laq, Lm, SBaq, SBm, Faq and Fm) showed significant antidiarrheal activity
against castor oil-induced diarrhea. The LD50 of Laq, Lm, Sbaq, SBm, and Fm were 14.10,
2.91, 5.12, 8.77 and >10.0g/kg respectively. The present study suggested that the plants
studied possess spasmolytic, antitransit and antidiarrheal properties due to the presence of
flavonoid, tannin or cumarins shown to be present in some extracts. The results also support
the traditional folk use of the twigs and stem bark parts of the plants for stomach pains,
intestinal cramps and diarrhea. Further study should be pursued in order to find the exact
mechanism of action and to characterize major secondary metabolites responsible for the
activity observed.
1
1 Introduction
1.1 General
In the struggle for survival, man had to identify the plants that are not only deleterious to
health but also those which would serve for his well being, i.e., as a source of food as well as
drugs that would mitigate pain or symptoms of ill health (Abebe and Ayehu, 1993).
Man knows the application of plants for medicinal purpose since time immemorial. Medicinal
plants represent element of unequaled reservoir of new substances with potentially useful
properties. The dependence on plants as source of medicine is still relied in many parts of the
world, as it is indeed in Africa. Ethiopia, with its diverse topography including great mountain
ranges, has rich endemic elements in its flora; approximately, 7,000 higher plants species are
known to occur. Traditional herbal remedies not only represent part of struggle of the people
to meet essential drug needs but they are also an integral component of their culture beliefs
and attitudes (Abebe and Hagos, 1991).
Fossil records date human use of plants as medicines at least to the Middle Paleolithic age
some 60,000 years ago (Solecki et al., 1975). From that point the development of traditional
medical (TM) systems incorporating plants as a means of therapy can be traced back to only
as far as recorded documents of their likeness. However, the value of these systems is much
more than a significant anthropologic or archeological fact. According to World Health
Organization (WHO), almost 65% of the world’s populations have incorporated medicinal
plants into their primary modality of health care (Farnsworth et al., 1985).
2
WHO estimates that 80 % of the population living in developing countries depends on TM for
their primary health care (PHC) needs (WHO; Harare, 2001). The goals of using plants as
sources of therapeutic agents include:- (1) isolation of bioactive compounds for direct use as
drugs, e.g., digoxin, digitoxin, morphine, reserpine, taxol, vinblastine, vincristine etc., (2)
production of bioactive compounds of novel or known structures as lead compounds for
semisynthesis to produce patentable entities of higher activity and/or lower toxicity, e.g.,
metformin, nabilone, oxycodone, taxotere, teniposide, verapamil, and amiodarone, which are
based, respectively, on galegine,9-tetrahydro-cannabinol, morphine, taxol, podophyllotoxin,
khellin, and khellin; (3) use of agents as pharmacologic tools, e.g., lysergic acid diethylamide,
mescaline, yohimbine; and (4) use of the whole plant or part of it as a herbal remedy, e.g.,
cranberry, paperminit oil, echinacea, feverfew, garlic, ginkgo biloba, St. John’s wort and saw
palmetto ( Fabricant and Farnsworth, 2001).
The number of higher plant species (angiosperms and gymnosperms) on Earth is estimated at
250,000 (Ayensu et al., 1978.) with a lower level at 215,000 (Cronquist, 1988) and an upper
level as high as 500,000 (Tippo and Stern, 1972). Of these, only about 6% have been screened
for biological activity and 15% evaluated phytochemically (Verpoorte, 2000).
Chemical diversity of secondary plant metabolites that results from plants evolution may be
equal or superior to that found in synthetic combinatorial chemical libraries (Farnsworth,
2001). It was estimated that in 1991 in the United States, for every 10,000 pure compounds
(most likely synthetic origin) that are biologically evaluated (primarily in vitro), 20 would be
tested in animal models, and 10 of these would be clinically evaluated, and only one would
reach U.S. Food and Drug Administration approval for marketing. The time required for this
process was estimated to be 10 years at a cost of 231 million U.S dollars (Vagelos, 1991).
3
1.2 Ethnomedical information of traditional medicine and scientific
investigation
In 1985 there was a proposed approach, based on ethnomedical information, to
experimentally prove plants as a possible source of drugs. The approach was designed
primarily for implementation by developing countries, where lack of hard currency often
prevents sophisticated types of research from being conducted. The possibility of drug
development in the form of stable, standardized crude extracts and eventual development of
the active principles from these plants was envisioned (Figure 1) (Farnsworth et al., 1985).
The most reliable type of information arises from in-depth studies carried out on the
ethnomedical use of plants of a particular ethnic group living in a given area through frequent
communication, preferably in their own language. It should be noted, however, that an
extensive knowledge of TM may exist in only in a few members of the community and a
focus on this group would yield greater results.
Before such knowledge can be investigated scientifically, the information provided will often
need clarification and standardization. Data on the part of the plant used, time of collection,
method of preparation (formulation) and methods of application are also necessary since they
all affect the nature and amount of any biologically active compounds (Samuelsson, 1987;
Trease et al., 2002).
A typical example of success reported in drug discovery based on ethnopharmacological
approach is the discovery of artemisinin. Artemisia annua is a plant which was recorded
during 281-340 AD for treating malaria (WHO 2001, China). In 1976, artemisinin compounds
4
were identified and their mechanism of action elucidated. Artemisinin acts against malarial
parasites in a very different way from quinine and most of the synthetic quinoline
antimalarials. Several large trial studies have shown the efficacy of artemisinin but the more
soluble analogue artemether and artesunates are now widely used and are recommended by
WHO as antimalarias in chloroquine resistant areas (WHO, Geneva; 2001).
1.2.1 Evaluation of extraction, biological activity and chemical constituents’
The extract used for testing should approximate as closely as possible to that obtained from
the traditional way of preparation. In many cases, this will be simple extraction with hot
water. But a variety of other solvents as well as various additives may be used in the treatment
of materials before use. In most instances however, it is likely that fairly polar compounds
will be extracted, although the solubility of less polar substances may be increased
considerably due to solubilizing compounds. After preparation of the extracts a particular
assay, or series of in vitro bioassay techniques are designed on the basis of the physiology of
the origin, biochemistry or molecular biology of the disease (Samuelsson, 1987; Trease et al.,
2002). Chemical examination should be linked with tests for biological activity.
Chemotaxonomy is one approach that facilitates the proportion of plants to be screened, thus
saving time and money. This is so because, specific secondary metabolites, such as
flavonoids, are often restricted in distribution, being found only in groups of related plants.
E.g. isoflavnoids are common in species of the Fabaceae, but are found in few other plant
families. Of the over 5500 types of alkaloids known, many are confined to a single genus or
subfamily. Only a single alkaloid has been found in the many species of Bombacaceae tested
so far, but the Solanaceae, Rubiacea and Ranunculaceae are the source of hundreds of distinct
5
forms (Martin, 1995). The presence of different secondary metabolites in a plant can be
screened by the use of appropriate chromogenic reagents after separation (Trease et al., 2002).
1
Ethnopharmacological information or Name of the plants of frequently used in catalogue
Review and evaluate literature
Decide on need to test
Select test
Establish priorities for testing
Collect plants
Carry out tests for safety and toxicity
protocols for safety and toxicity
Develop criteria for safety and toxicity tests
Determine safety from published
Collect plants
Determine type of biological activity
Prepare, stabilize, & standardize extracts
Isolate & identify active
principles
Carry out human studies
Develop methods
of industrial production
1
1.3 Traditional medicine in Ethiopia
The introduction of medicine to Ethiopia dates back to the 16th century during the regime of
Emperor Libne Dingel (1508-1540) restricted to introducing drugs. The first government that
ran modern health care was established in 1906 with the opening of Menelik II Hospital in
Addis Ababa. Since then the government has taken the formal responsibility of delivering
health care to the population and health institutions were established in the different regions
of the country. However, the growth and development of modern health care in Ethiopia as a
whole has been very slow and to date, its coverage is less than 50% of the population. The
vast majority of the rural population, therefore, still depends on TM and its practitioners
(Shiferaw, 1996).
The beginning of Ethiopian TM could not be established with certainty due to lack of
adequate written sources. The early report on the Ethiopian TM practices was the one
provided by Francisco Alvares in the early 16th century in which he mentioned that,
Ethiopians knew about the use of bleeding and cupping and about the use of various herbs as
purgatives (Desta, et al., 1996). However, Pankrust noted that, it would seem reasonable to
assume that the country's medical lore was then already well established. He also added that,
despite the probable long established nature of Ethiopian traditional remedies, the earliest
known texts are the Geez "Matshafa Faws" of mid-seventeenth century and "Matshafa
Madhanit" of the early 18th century. These medical texts contain several references to plants,
animal products and minerals as well as magic and superstition (Pankhrust, 1976).
Hagenia abyssinica Gmel (Kosso in Amharic) initially taken from Ethiopia, was introduced
into the international world of medicine as an age-old tested medicament. Richard Pankhurst
2
wrote at length how the crude extract of this plant began to be utilized in Europe. He wrote
that "the first foreign medical man to interest himself in Kosso" was a French physician
called Dr. A Brayer around 1816. Brayers' first acquaintance with Kosso was from a contact
he had with an old Armenian merchant called Karabet in Constantinople (now Istanbul) who
told him that the "... Ethiopians cured themselves with the aid of the flowers of a plant which
... was known by the word which also signified the taenia itself " (Pankhrust, 1975).
Jesuit travelers of the early seventeenth century provided substantially more details on
traditional Ethiopian medical practice. The Portuguese missionary Manoel de Almeida
reported the existence in the country of “many purgative herbs”, as well as other plants
known to heal wounds. His compatriot, and fellow missionary, Manoel Barradas specified
several Ethiopian medicinal plants by name, among them the ‘enkoy’ (Ximenia americana),
the ‘decuma’ (Syzygium guineense), and the ‘waginos’ (Brucea antidysenterica). Subsequent
observers also recorded the use of these, and other plants (Pankhrust, 1975).
The root barks of “Waginos” (in Geez) and “Abalo” (in Amharic) were used by people living
in northern Ethiopia for treating dysentery for many centuries. A British traveler and amateur
physician called James Bruce who stayed in Ethiopia from 1769 - 1771 was attacked by
dysentery when he was about to leave Ethiopia. He tried to cure himself with the help of the
medicines he had brought along from Europe but was not successful. Knowing that he would
not be able to make it to Europe traveling through the hot landmass of Sudan and Egypt, the
chief of Ganhar of Shanqilla informed him to take a well-established local drug known as
“Waginos”. The root barks of this plant were cleaned, dried in the sun, and ground into
powder. James Bruce was then made to take two spoonfuls of the powder with camel's milk.
After the sixth or seventh day Bruce regained his health and was able to continue his journey
3
to England. On his way back, he took some of the powder and fruits of “Waginos”; the
powder, he used whenever his companions and himself fell sick and the fruits were delivered
to a botanist at the British Museum called Daniel Solander, who, noting that it represented a
taxon not known in Europe planted it in several British gardens. The plant was later named
Brucea antidysenterica J.K Miller in honor of James Bruce and with the specific epithet
indicating the medicinal property of the plant (Tadesse, 1986).
Ethiopia is the home of many nationalities and remarkably diverse flora, including numerous
endemic species that are utilized in the different traditional medical practices of which the
two systems are important to be considered in studying the effects of the herbal medicine
(Abebe, 1986).
4
1.4 Herbal Remedies for Gastrointestinal Motility Disorders and Diarrhea
According to Christen, (1990) currently available antispasmodics often called spasmolytics
can be classified into three major subclasses:
1. Antimuscarinics (e.g., cimetropium, prifinium)
2. Smooth muscle relaxants (i.e., drugs that directly inhibit smooth muscle contractility,
e.g., by increasing cyclic AMP levels or interfering with the intracellular calcium
pool: tiropramide, papaverine-like agents (Singh et al., 2003), and
3. Ca+2-channel blockers (especially L-type Ca+2-channel blockers such as nifedipine or
pinaverium and peppermint oil (Christen, 1990).
The plant kingdom is rich in chemical constituents’ of antispasmodics that relieve colicky
pain. Infact, most remedies used in conventional medicine include at least one antispasmodic
of plant origin. They form a very important part of the treatment of gastrointestinal motility
disorders such as dyspepsia (indigestion), spasms of intestine such as colic; peptic and
duodenal ulceration; nausea and vomiting; constipation and irritable bowel syndrome (IBS)
(Williamson et al., 1996; Sadraei et al., 2003b). The antispasmodics are considered useful for
relieving or calming colicky pains resulting from spasms of the gut muscles and diarrhea due
to hypermotility of the gastrointestinal tract (Gilani et al., 1994a), and other features of IBS.
Among the wide range of plant-derived drugs that have relaxant activities on various smooth
muscles, papaverine ( Papaver somniferum) is the one, which is used in the treatment of colic
(Mustafa et al., 1995). It is a non-selective smooth muscle relaxant and is used as a control
drug for antispasmodic effects. Muscarinic antagonists like atropine (Atropa belladonna)
5
inhibit the contractions of gastrointestinal tract induced by acetylcholine (Ach). This partial
inhibition of gastrointestinal motility by atropine drugs has led to their widespread use as
antispasmodics in the treatment of disorders associated with intestinal hypermotility (Ghosh et
al., 1993; Broadley and Kelly, 2001). The other important mediator in contraction of
gastrointestinal tract is histamine via H1 receptors activation, which was dominantly found in
gut (Zavecz and Yellin, 1982). Drugs such as metoclopromide also affect the serotonin
receptors to exert a prokinetic or an antispasmodic effect. The development of serotonin 5-
HT3 receptor antagonists offers enormous therapeutic potential as antiemetic, antidiarrhoeal
agents, in the control of abdominal pain and discomfort and rectification of gastrointestinal
motility (Gwee and Read, 1994). Blocking 5-HT3 receptors leads to reduced smooth muscle
contractility (i.e. an antispasmodic effect), which is of clinical significance in chronic diarrhea
(De Ponti and Tonini, 2001; Singh et al., 2003). 5-HT7 inhibitors are also involved in the
inhibitory effect of serotonin in guinea-pig ileum and the ligands acting on the receptor may
prove to be useful antispasmodic agents to treat gastrointestinal motility disorders such as IBS
(Tuladhar et al., 2003).
There are also a number of plant extracts tested for activity against some of the conditions that
mainly involve testing the plant extracts for antispasmodic activity and antidiarrheal
properties. Plant-derived antispasmodics include some tropane alkaloids (atropine, hyoscine
or scopolamine, hyoscycamine), opium alkaloids (papaverine, codeine, morphine), flavonoids
(luteolin, cirsimartin, quercetin, rutin, apigenin, kaempferol, genkwanin) and essential oils
(peppermint, caraway, dill, garlic, chamomile, anise) (Sanchez de Rojas et al., 1994;
Williamson et al., 1996). Nowadays, a lot of scientific articles can be cited that report the
antispasmodic, muscle relaxant and antidiarrheal effects of plant extracts, or their active
chemical constituents.
6
The presence of smooth muscle relaxant agents isolated from species of Malaysian medicinal
plants (Mustafa et al., 1995), Mexican medicinal plants (Estrada et al., 1999; Rodriguez-
Lopez et al., 2003) and United Arab Emirates Medicinal plants (Tanira et al., 1996) were
evidenced by their inhibitory effect of the cholinergic, histaminergic, nitriergic and ion-
induced smooth muscle contractions of guinea pig and rat duodenum, and rabbit jejunum.
Aqueous extracts of many plants are widely used in therapy in complementary medicines of
antispasmodics (Dire et al., 2003).
Current therapy for some gastrointestinal disorders is directed towards inhibition of smooth
muscle contractions. It is well known that aqueous herbal medicines are traditionally used for
their spasmolytic and antidiarrheal activity in various countries (Hajhashemi et al., 2000;
Sadraei et al., 2003b). In an in vitro experiment, aqueous extracts of Prunus spinosa
L.branches were tested to have diminished response to ACh and histamine as spasmogens in
mouse duodenum and guinea pig ileum (Rodriguez et al., 1986). A room temperature aqueous
extract of the roots of Taverniera abyssinica (“Dengetegna”) antagonized ACh and histamine
induced contractile responses of the guinea pig ileum and relaxed the smooth muscle of rabbit
duodenum, which is suggestive of its ethnomedical use in stomachache treatment (Noamesi et
al., 1990). The aqueous extract of Linum usitatissimum (“Telba”) seed was observed to show
significant spasmolytic acivitiy and protective effects against experimental ulcerogenesis in
guinea pig ileum and mouse stomach (Makonnen, E. 1996). The aqueous extract of Evodia
rutaecarpa fruit was used to examine its effects on castor oil-induced diarrhea and to compare
with its anti-transit effect in mice. The results indicated that the extracts had both anti-transit
effect and antidiarrheal effects (Li-Li Yu and Liao et al., 2000). The antihistaminic and
anticholinergic activities of aqueous extract of barberry fruits (Berberis vulgaris) were
7
investigated on isolated guinea-pig ileum and found to be anticholinergic and antihistaminic
(Shamsa and Khosrokhavar, et al., 1999). The constipating and spasmolytic effects of khat
leaves extract (Catha edulis Forsk) were investigated and it was found that the khat extract
antagonizes the spasmogenic effects of both histamine and carbachol on isolated guinea pig
ileum and whole mice in a concentration dependent manner (Makonnen, 2000).
Relatively the less polar solvents like methanol and ethanol are very appropriate in extracting
the spasmolytic agents of plants. The ethanol extract of Capparis cartilaginea inhibited the
submaximal contractions of ileum induced by ACh, histamine or serotonin (Gilani and Aftab,
1994). Gilani et al. (1994a) also showed that pure compounds from leaf ethanol extracts of
Moringa oleifera were found to have inhibitory effect on isolated ileum in a concentration
dependent manner. Dichloromethane extracts of Inula crithmoides L. (Barrachina et al.,
1995a) and, later on, methanol and dichloromethane extracts of Teucrium species (Barrachina
et al., 1995b) were known to produce a significant inhibition in the maximal contractile
effects of ACh, histamine and serotonin in the guinea pig ileum and rat duodenum. The crude
methanol extracts of Erythrina sigmoidea stembark were found to have potent anticholinergic
effects by decreasing the tone and spontaneous activity of isolated rat ileum induced by
carbachol and acetylcholine (Nkeh et al., 1993). They were also found to inhibit histamine-
induced contraction of the same tissue showing their potency of antihistamine effect (Nkeh et
al., 1996). The leaf ethanol extract of Moringa stenopetala was shown to have a potential
antispasmodic effect on guinea pig ileum (Mekonnen, 1999).
Some of the chemical constituents of plants having antispasmodic and antidiarrheal effects are
summarized as follows. Flavonoids are natural products, which exhibit various
8
pharmacological effects. Quercetin, one of the flavonoids isolated from aerial parts of Conzya
flaginoides, caused a concentration dependent inhibition of spontaneous contractions of rat
ileum (Mata et al., 1997), and showed antidiarrheal activity against castor oil-induced
diarrhea in mice. It also exerted inhibitory effects on guinea pig ileum contractile response
(Galvez et al., 1996). Rutin, another flavonoid in Artemisia scoparia, was found to cause a
concentration dependent inhibition of spontaneous movements of rabbit jejunum (Gilani et
al., 1994b). Flavone cirsimartin, which is isolated from Artemisia judaica, Artemisia
capillaris, Artemisia xerophytica and Artemisia scoparia is responsible for the spasmolytic
activity of isolated guinea pig ileum and thus support their use in folk medicine for certain
gastrointestinal disorders such as ulcer and acute diarrhoea (Abdalla and Abu Zarga, 1987).
Four flavonols with spasmolytic activity were isolated from the aerial parts of Artemisia
abrotanum, which are the active principles for smooth muscle relaxing activity of the plant
(Bergendorff and Sterner, 1995; Harborne and Williams, 2000). The flavone luteolin, isolated
from Colchicum richii, caused a concentration dependent relaxation of the tone of ileum
(Abdalla et al., 1994). There are also alkaloids reported in plants for their spasmolytic effect.
For example bisnordihydrotoxiferine and villosimine are the indole alkaloids isolated from the
roots of Strychnos divaricans, which antagonized ACh and histamine responses in the guinea
pig ileum (da Silva et al., 1993). Another chemical constituent, 7-methoxy coumarin, has
been reported to be a smooth muscle relaxant responsible for the spasmolytic activity of
Lavandula stoechas L. extract (Gilani et al., 2000). Tannins and mucilaginous substances in
the fruits of Aegle marmelos a component of Ayurvedic medicine are reported to show
antidiarrheal activity against castor oil diarrhea in mice (Pallavi and Subhash, 2003).
9
Results from investigations with animal tissues also suggested that the essential oils;
peppermint oil and caraway oil (as categories of essential oils) were found to relax
gastrointestinal smooth muscle by reducing Ca2+ influx (Gwee and Read, 1994; Micklefield et
al., 2000). The essential oil of Achillea ageratum L. was found to be an effective spasmolytic
agent capable of inhibiting ACh and BaCl2 induced contraction of isolated rat duodenum (de
la Puerta and Herrera, 1995). Satureja hortensis L. essential oil was investigated to have
antispasmodic effect on rat-isolated ileum in vitro. It was found to inhibit the maximum
response due to ACh, relax ileum contraction due to depolarization by KCl, and inhibit castor
oil-induced diarrhoea (Hajhashemi et al., 2000). Essential oil of Ocimum gratissimum was
investigated and found to reversibly relax the basal tone of isolated guinea pig ileum and
reverse the tonic contractions induced by KCl and ACh in concentration dependent manner
(Madeira et al., 2002). The leaves extract has also showed antidiarrheal effect against castor
oil induced diarrhea in mice (Veronica N. et al., 1999). The essential oils of Artemisia
thuscula Cav. flowers (Perfumi et al., 1995) and Artemisia alba (Perfumi et al., 1999) were
investigated and shown to have dose-dependent and essentially non-competitive spasmolytic
effects in guinea pig ileum. The essential oil of Pycnocycia spinosa was also reported to have
antispasmodic and antidiarrheal activity (Sadraei and Naddafi et al., 2003).
Most medicinal plants, which were reported to be anthelmintics, were also found to be
antispasmodics. Anthelmintics, like smooth muscle relaxants, act by depressing the smooth
muscle or by inhibiting the metabolic processes. For instance, the aqueous extract of Bersama
abyssinica antagonized the spasmogenic effect of histamine on guinea pig ileum in a non-
reversible manner. The mechanisms of taenicidal action of Hagenia abyssinica ('Kosso') was
suggested to be due to its inhibitory effect on contraction of isolated guinea pig ileum
(Arragie et al., 1983). Kosotoxin, a constituent of this plant was tested for spasmolytic
10
properties on guinea pig ileum and rabbit jejunum in vitro and was found to inhibit their
spontaneous contractions in a concentration dependent manner (Bogale, et al., 1996).
11
1.5 The Genus Syzygium
Since the plant belonging to the genus Syzygium is the main topic of investigation for this
thesis, a concise account of this, genus is described as follows.
Hypoglycemic effects were seen in Syzygium cumini (Eugenia jambolama), it is used in folk
medicine for diabetes in India (Prince et al., 1998). The same effect was seen in Syzygium
jambos prepared from infusion of the leaves in clinical studies (Teixeira et al., 1990).
The members of this genus are well known for their antimicrobial effects when tested in vitro.
Among the members Syzygium aromaticum (Clove) (Dorman et al., 2000) and Syzygium
guineense (Tsakala et al., 1996) both had potent antibacterial against diarrhea causing
bacteria. The methanolic leaf extracts of Suzygium aromaticum has also shown antibacterial
activity against Gram-negative anaerobes (Cai et al., 1996). Betulinic, dihydrobetulinic,
platanic and oleanolic acids from Syzygium claviflorum leaves exhibited extremely potent
anti-HIV activity (Fujioka et al., 1994; Kashiwada et al., 1998). Herpes inhibition was
reported by eugeniin isolated from Geum japonicum and Syzygium aromaticum effect in mice
(Kurokawa et al., 1998; Namba et al., 1998). Syzygium aromaticum has also antifungal in
vitro and anticytomegalovirus effect in mice (Yukawa et al., 1996; Montes-Belmont et al.,
1998; Shirak et al., 1998). Out of 50 Puerto Rican plants tested against Mycobacterium
tuberculosis in vitro, Syzygium jambos was reported to be active (Frame et al., 1998).
Other effects reported for the members of this genus include; anti-inflammatory (Lee et al.,
1995), larvicidal in mosquito vector (Pushpalata et al., 1995), weight reduction (Palit et al.,
1999) and diuretic effect in rats (Silva- Netto, 1998) by the leaves of Syzygium jambolanum.
12
Inhibition of the aggregation and alteration of arachidonic acid metabolism in human blood
platelets by acetyl eugenol from oil of cloves (Syzygium aromaticum) (Srivastava et al., 1991)
and inhibition of the anaphylaxis effects by flower bud aqueous extract of the same plant in
rats, possibly by the lowered serum histamine level were also reported (Kim et al., 1998).
Antifertility and spermatogenesis activities of the oleanolic acid isolated from flowers of
Syzygium cumuni were also reported. This acid also stopped spermatogenesis in male rats
without changes in body weight or reproductive organ weights (Rajasekaran et al., 1988).
Castor oil induced diarrhea was reduced by bark extracts of Syzygium cumini (Mukherjee et
al., 1998).
1.5.1 Syzygium guineense
Syzygium guineense is one of the plants that belong to the family Myrtaceae that has a
synonym Sysygium owariense commonly known as water berry in English (Agwu and Okeke,
1996), “Dokma” in amharic, “Ocha” in Gamo and “Karava” in Gurage. Syzygium guineense
(water berry tree), a dense, leafy forest tree around 30 meters high; bark flaky, grayish-white;
leaves broadly lanceolate, opposite, entire to the branch; fruit ellipsoid drupe, purplish in
color (Bekele, 1993; Abebe and Debela et al. 2003). The plant is found in the altitude range
2,300 - 2,700 m. It has edible fruit, with higher nutritional value (Figure 2 and Figure 3) (Saka
and Msonthi, 1994; Tadesse and Hedberg, 1995; Nievergelt et al. 1998).
13
Figure 2. The plant Syzygium guineense with its green lanceolate, opposite leaves
containing red and black fruits (The picture was taken in Arebaminche Bush).
Figure 3. The plant Syzygium guineense with ellipsoid drupe, purplish fruit (The picture was
taken from Abebe and Debela, et al (2003).
Traditionally the decoction of the bark of the plant was used in treatment of diarrhea (Abebe,
and Debela et al, 2003) and also the twigs and aerial roots were used for different stomach
ailments (such as stomachache) (Kassu, 2002). Syzygium guineense was reported to have
potent antibacterial effect against diarrhea causing bacteria (Tsakala et al., 1996). The
aqueous extract showed an antibacterial activity against some bacterial strains: Salmonella E.,
14
Shigella D., Shigella F., E. Coli., Enterobacter A. It did not show any activity against
Citrobacter F., Proteus M., Klebsiella P. The in vitro results showed very strong effect on
diarrhea causing pathogenic bacteria (Tsakala, et al., 1996). Evaluation of the 7% powdered
leaf of this plant also showed complete inhibition of S. aureus, S. flexineri and S. boydii test
strains within 8 to 12 hrs. It also had a marked retarding effect on the growth of B. cereus, S.
tyhpimurium and E. coli (Ashebir and Ashenafi, 1999).
The ethanolic extracts of stem bark of Syzygium guineense showed molluscicidal activity
(Oketch-Rabah, and Dossaji, 1998). Methanol extract of S. guineense bark (collected in
Tanzania) inhibited intrinsic contractions in isolated ileum tissue of rabbit. The inhibition, at
bath concentrations of 0.5-2.0 mg/ml, was dose-related but non-linear. It also produced
sustained hypotension in anaesthetized rats. A dose of 5µg of this plant lowered systolic,
diastolic and mean blood pressure by 16%, 22% and 17%, respectively. Maximum effect was
obtained at a dose of 40µg; the systolic, diastolic and mean blood pressures are lowered by
23%, 36% and 28%, respectively. The greater fall in blood pressure was in diastolic rather
than in systolic blood pressure ( Malele, et al ., 1997 ).
Since large portion of the herbal remedies in the health care system of Ethiopia is not yet
explored, efforts to evaluate scientifically herbal remedies of traditionally used plants are of
paramount importance for their possible application in the future (Gedif and Hahn, 2002). In
view of these facts, the aim of the present study is to test the possible antidiarrheal and
antispasmodic effects. The study on antispasmodic effect might help to deduce the possible
mechanism of action related to the secondary metabolites in both in vivo and in vitro studies.
15
2 Objective of the study
2.1 General objective
To test for the antispasmodic and antidiarrheal effects of the aqueous and 80% methanol
extracts of Syzygium guineense and carry out acute toxicity test on the extracts to justify the
possible safe use traditionally.
2.2 Specific objectives
� To test for antispasmodic effect of the aqueous and 80% methanolic extract of the tip
of the leaf (twigs), stem barks, fruits on isolated ileum of guinea pig in vitro.
� To test for antispasmodic effect of the aqueous and 80% methanolic extract of the tip
of the leaf (twigs), stem barks, ripe and unripe fruit in vivo in mice.
� To evaluate the acute toxicity of the aqueous and 80% methanolic extracts and there
by determine LD50 in mice.
� To screen the antidiarrheal effect of the above extract in mice in vivo.
16
3 Materials and Methods
3.1 Materials
Chemical and solvents
Methanol (TechnoPharmchem, Bahadargarh, India), Distilled water, NaCl, KCl, MgCl2,
NaHCO3, NaHPO4, Glucose, CaCl2, Castor oil, Gum acacia (Riedel-De Haen AG, Sec Lze,
Hannover, Germany), Charcol(The British Drug House, Ltd., London).
Experimental animals
Male and female albino mice and male Guinea pigs were bought from EHNRI, Addis Ababa
and used in present study.
Standard drugs
Atropine (Sigma-Aldrich Chemie GMbH, Germany), Hisatmine (Sigma-Aldrich Chemie
GMbH, Germany), Acetylcholine Chloride (Sigma-Aldrich Chemie GMbH, Germany),
dexchlorpheniramine (LOBA CHMIE Pvt. Ltd., India) and Loperamide (Sigma-Aldrich
Chemie GMbH, Germany).
3.2 Methods
3.2.1 Collection of plant materials
The leaves tips (twigs), stem barks and unripe and ripe fruits of Syzygium guineense plant
samples were collected during January12-14, 2004. The leaf was collected from Wondogenet
and Arbaminch, and the stem bark and the fruit used for the study were collected from
17
Arbaminch. The collected plant identified by a taxonomist (Herbarium No. SG-2112) and
voucher specimen representing each part were deposited in the National Herbarium,
Department of Biology, AAU and Department of Research, EHNRI.
3.2.2 Preparation of extracts
3.2.2.1 Aqueous extracts
Aqueous extracts of twigs, stem bark and fruits of the plant were prepared by maceration of
40 gm of the powdered plant material, respectively in 100ml of distilled water. The aqueous
extracts were intermittently agitated for 24 hours at room temperature. After 24 hrs, each
sample was filtered out using a gauze and lyophilized to give amorphous powdered material
yield were twigs (Laq., 9.7%), stem bark (SBaq., 4.2%) and fruit (Faq., 7.3%) (Debella,
2002). The extracts were kept in desiccators at room temperature until the in vitro and in vivo
experiments were done.
3.2.2.2 Hydro-alcoholic extracts
The powdered twigs, stem bark and fruit of Syzygium guineense weighing 100 grams each
were macerated by 80% (V/V) methanol separately. The maceration was carried out for 48hrs
with intermittent agitation. The extracts were then filtered with the aid of filter paper
(Whatman No 3). The filtrates were concentrated using Rota-vapor (BÜCHI Rota-vapor R-
205, Switzerland) and further dried using water bath. The percentage yield was calculated to
give twigs (Lm., 12.5%), stem bark (SBm., 11.6%) and fruit (Fm., 11.2%) respectively, these
were kept in a refrigerator until the experiments were done (Debella, 2002).
18
3.2.2.3 Fractionation of the Leaf aqueous extract
Aqueous extract (20.3g) was dissolved in water (700 ml) and extracted with n-butanol (1.4
Lts) saturated with water. The yields for aqueous (WR), n-butanol (BF) fractions and Solid
residue (SR) after solvent evaporation were (52%, w/w) and (28%, w/w), (12%, w/w)
respectively.
3.2.2.4 Experimental Animals Acclimatization
Male and female animals of Swiss albino mice (25-30 grams) and Guinea pigs (300-500
grams) of specific age were purchased from EHNRI, Addis Ababa. The mice were
acclimatized to the environment of the animal house in the Department of Pharmacology,
AAU, Faculty of Medicine and the guinea pigs were acclimatized to the environment of the
animal house in the Department of Biology, Faculty of Science, AAU.
They were acclimatized under uniform conditions of 12/12hrs light and dark cycle and housed
at a temperature of 24 + 2.00C. They were fed a standard pellet diet and tap water ad libitum
(Makonnen, 1998).
Pharmacological screening
19
3.2.2.5.1 In vitro testing on Guinea pig Ileum
Six fasted (24 hrs) guinea pigs weighing (250-400g) were killed by a gentle blow at the back
and allowed to bleed. The abdominal cavity of the animal was opened by midline incision
every time a tissue was required, and the ileum 2-2.5 cm in length was removed immediately
and trimmed from surrounding tissues. The contents of the intestine were washed with a
Physiological Salt Solution (PSS) called Tyrode solution. The isolated tissue preparations
were used according to the technique of Perry (1982) and Williamson et al. (1996).
Segments of ileum were tied with silk threads at both ends (ileum tied in opposite directions)
and suspended in a thermoregulated 25 ml organ bath, maintained at 370 C, containing a
Tyrode solution of the following composition (g/l): NaCl, 8 gram; KCl, 0.2 gram; MgCl2, 0.1
gram; NaHCO3, 1 gram; NaHPO4, 0.05 gram; Glucose, 1 gram; CaCl2, 0.2 gram. One end of
the ileum was attached to a tissue holder at the base of the organ bath and the other end to the
isometric recording device. The tissues were constantly bubbled with air mixture of 95% O2
and 5% CO2. A suitable weight or resting tension of 1 gram was applied to the individual
tissue (de la Puerta and Herrera, 1995 and Galvez et al. 1996).
The suspended ileum was allowed to equilibrate for 30-45 minutes before adding
acetylcholine or histamine or the particular plant extract or the standard drugs. After the initial
equilibration period, histamine or Acetylcholine (10-9 to 10-4 M) was added to the organ bath
and the control cumulative concentration-response curve for each one (histamine or
acetylcholine) was constructed. Each time the added concentration of the acetylcholine or
histamine was left in contact with the tissues for 30 seconds before adding the next
concentration. Then the tissue was washed two times with Tyrode solution at the interval of
20
10 minutes. It was left to resume its normal contraction. After a stabilized regular contraction,
extracts of Syzygium guineense (Laq., Lm, SBaq., SBm, Faq., and Fm) at doses of 50, 100,
and 200µg/ml were added; dexchlorpheniramine or atropine was then added to the organ bath
5 minutes before the corresponding concentration curve was recorded. After rhythmic
contraction of the tissue resumed, the control ACh or histamine was again added in order to
establish the reversible contraction capacity of the tissue and also to test the subsequent
concentration of the plant extract. The same procedure was repeated whenever different plant
extracts at different final organ bath concentrations were tested.
The plant extracts were prepared in physiological Tyrode salt solutions while the stock
solutions of all drugs (ACh, histamine, Atropine and dexchlorpheniramine) were made in
distilled water and then serially diluted with PSS. The final dilutions of the drugs were made
fresh on the day of the experiment. The calculated concentrations of each plant extracts and
standard drugs were final organ bath concentration.
Isometric contractions were recorded with a Grass FT-03 strain gauge transducer coupled to a
Grass 79 Polygraph which is equipped with preamplifier, main amplifier, oscillograph and
time and event marker (Mekonnen, 1999). The chart speed was 5mm/minute.
In addition, the antihistaminic effects of the extracts were compared with
dexchlorpheniramine (1.3x 10-9 M) and the anticholinergic effects of the extracts were
compared with atropine (6.6x10-9 M) (Shamsa and Khosrokhavar et al., 1999).
21
3.2.2.5.2 In vivo Small intestine transit determination: Charcoal meal
Test
The effect of the extract on small intestinal transit was studied in overnight fasted mice (25-
30g) of either sex, which were divided into five groups containing six mice in each group.
These groups were control (vehicle); 50,100 and 200 mg/kg of the extracts; 10mg/kg atropine
sulphate in oral route. Five minutes after treatment, mice were given 0.5 ml charcoal meal
(5% charcoal in 5% gum acacia) by oral route. All animals were scarified after 30 min;
movements of charcoal from pylorus to caecum were measured and the results were expressed
as a percentage distance travelled compared with the negative control (i.e mice in vehicle
group) (Williamson et al., 1996).
3.2.2.5.3 Antidiarrhoeal activity/Castor oil-induced Diarrhea/
Mice of either sex weighing 25-28 grams were divided into control (vehicle) and test groups.
Each animal was placed in individual cage, the floor of which was lined with soft tissue and
replaced every hour. Diarrhea was induced by administering 0.3 ml of castor oil orally to the
mice (Rouf et al., 2003). The control group received distilled water for aqueous extract group
or 3% Tween 80 for hydroalcoholic extract group. The positive control groups received
Loperamide 2 mg/kg; the other groups received each of the extracts at 50,100 and 200 mg/kg
body weight orally 40 min before the administration of castor oil. Onset of diarrhea and the
number of diarrheal episodes were recorded for each animal, for a total of 4 hrs. The results
were recorded by taking the vehicle groups as 100% and calculated as percentage inhibition
for the extracts and positive control groups.
22
3.2.2.5.4 LD50 determination
Batches of 60 mice (24-27g) of both sexes and similar age were divided randomly into six
groups, each of ten mice five females and five males, with one group serving as control. The
extracts were given orally after 3 hrs of fasting. Five groups of animals received 1000, 2000,
4000, 6000 and 10,000 mg/kg of the extracts and some extracts even higher doses (>10,000)
in a volume of 1ml, while the control groups received distilled water for the aqueous extract
group and 3% Tween 80 in the case of hydroalcoholic extracts group at the same volume. The
general signs of symptoms of toxicity and mortality were recorded for 24 hrs (Williamson et
al., 1996).
3.2.2.5.5 Pilot study on effective dose determination
The pilot study was performed with 25, 50, 100, 200 and 500mg/kg of Syzygium guineense
twigs, fruits and stems bark 80% methanolic and aqueous extracts in both antidiarrheal and
intestinal transit test. The dose 50mg/kg showed the minimum effect and there was no much
difference in the anti-diarrhea activity of 500mg/kg compared with 200mg/kg. All the extracts
in castor oil-induced diarrhea at the dose of 25mg/kg did not inhibit diarrhea significantly
when compared with castor oil alone. The same was observed for intestinal transit test. From
these preliminary works the doses 50, 100 and 200mg/kg were selected for all subsequent
studies.
The same procedure was followed for the in vitro test in which the minimum and maximum
effective doses were found to be 50µg/ml and 300µg/ml respectively. From this finding the
dose 50, 100 and 200µg/ml were selected for all subsequent studies.
23
3.2.2.6 Phytochemical Screening
3.2.2.6.1 Chemical method of screening
The powdered plant materials of twigs and stem barks; the crude aqueous and hydroalcoholic
extracts of twigs, stem barks and fruits of Syzygium guineense were tested according to a
compiled screening method of phytochemical identification (Sofowora, 1982; Deballa, 2002).
3.2.2.6.2 Screening by Thin-layer chromatography (TLC)
The crude extracts and fractionates of Laq of Syzygium guineense were tested for presence of
secondary metabolites according to a compiled screening methods of chromatographic
analysis (Deballa, 2002).
3.2.2.7 Statistical analysis
Ileum contractions of the GPI (in vitro test) were measured as maximum changes in tension
from pre-drug baseline within the contact time of 30 seconds intervals just before addition of
the next concentration of the standard spasmogen (either Histamine or Acetylcholine). The
results were expressed as percentage maximum contraction by comparing with specific
concentrations of spasmogen.
Contractions were expressed as percentage of the maximal contraction obtained from the
corresponding control curve, each point represents the Mean + standard error of the mean
(S.E.M.) of six experiments of each dose of the extract of in vitro tests of guinea pig ileum.
The level of significance was calculated by one-way analysis of variance (ANOVA) followed
24
Schiffe post-hoc test for dose response comparisons of the various plant extracts (SPSS 11.0).
The histamine and Acetylcholine dose response curves, with and without the agonists, were
plotted using Prism, version 3.0. Schiffe post-hoc test was used for analysis of in vivo data.
The Lethal Dose 50 (LD50) was calculated by the probit analysis (Prism 3.0). Differences
were considered statistically significant when p-value was less than 0.05.
4 Results
The phytochemical screening showed the presence of tannins, phytosteroids, flavonoids and
saponins in some of the crude extracts and fractionates. The nature of spasmolytic effect of
the plant extracts was studied in isolated guinea pig ileum preparations. Antidiarrhea and
intestinal transit test were performed in mice for in vivo study, which showed the following
results.
4.1 Phytochemical screening of Syzygium guineense
The powdered materials, aqueous and hydroalcoholic twigs, stem bark and fruit crude extracts
and fractionates of twigs subjected to phytochemical screening chemical methods (Table 1)
showed the presence of polyphenol, saponins, flavonoid and steroidal compound; however,
the powdered materials, extracts and fractionates indicated negative results for the presence of
alkaloids, cyanogenic glycosides and phenolic glycosides (data are not shown).
25
1
Results Secondary metabolites
Reagents
Powdered leaf Powdered Stem bark
Laq Lm SBaq SBm Faq Fm
Polyphenol
1% FeCl2 & 1% K3Fe (CN)6
+ve +ve +ve +ve +ve +ve +ve +ve
Saponins
Honey comb froth formation
-ve -ve -ve +ve -ve +ve -ve -ve
Phytosteroid
+ve +ve +ve +ve +ve +ve +ve +ve
Free Anthraquinones
+ve +ve +ve -ve +ve +ve -ve -ve
Flavonoid
+ve -ve +ve +ve -ve -ve -ve -ve
Tannin
K3Fe(CN)6 /conc. NH3
+ve +ve +ve +ve +ve +ve -ve -ve
Cardiac glycosides
Keller
+ve +ve -ve -ve +ve -ve -ve -ve
Salkowski
+ve +ve +ve -ve +ve -ve -ve -ve
Liberman
+ve -ve -ve -ve +ve -ve -ve -ve
Note : (+ve ) indicates the presence and ( -ve ) indicates absence of particular metabolites
2
1
TLC test with aqueous and hydroalcoholic extracts of twig, stem bark and fruit; and twigs
aqueous fractionates showed the presence of steroidal and phenolic compounds; flavonoids,
cumarins and tannins that were detected by spraying the TLC pates (Silica gel 60 F254) with
reagents sensitive to this class of compounds (Table 2). Best separation of the components of
the crude extracts and fractionates were achieved using a mobile phase of
Chloroform/Ethylacetate/Methanol/ Formic acid (4:3:2:1) and Chloroform/Methanol/Water
(80:20:0.5) respectively (Table 2). The mobile phases which resulted in poor separation were
Chloroform/Methanol/water (60:40:0.5), n-butanol/acetic acid/ water (6:4:1) and
Chloroform/Ethylacetate/Methanol/Formic acid (2:2:4:2).
2
1
Table 2. Results of TLC analysis Secondary metabolites of crude extracts and fractionates of Syzygium guineense
Plant material used
Mobile phases Spraying reagents/ UV light Secondary metabolites
Number components and Rf
UV λ254 Five dark spaots Rf =0.0,0.11, 0.23, 0.33 &0.93 Vanillin/H2SO4 Steroidal compounds Three, Rf =0.0, 0.11, 0.55 &0.43
Fast Blue B/ 0.1N NaOH Flavonoids Four, Rf =0.0, 0.26,0.33 &0.55 5 % KOH in MeOH Cumarins Three, Rf =0.0, 0.24 & 0.27
BF
Chloroform/Methanol/
water (80:20:0.5)
FeCl3 + Potassium ferocyanide
Phenolic compounds Five, Rf =0.0, 0.19, 0.26, 0.33 & 0.59
UV λ 254 Two dark spots, Rf =0.0 & 0.14 Vanillin/H2SO4 Steroidal compounds Three, Rf =0.0, 0.5 & 0.14 Fast Blue B/ 0.1N NaOH Flavonoids Two, Rf =0.0, 0.06
WR Chloroform/Methanol/ water (80:20:0.5)
FeCl3 + Potassium ferocyanide Phenolic compounds Two, Rf =0.0, 0.26 UV λ254 Three dark spots, Rf =0.06,
0.15 &0.63 Vanillin/H2SO4 Steroidal compounds 0ne, Rf =0.0
Fast Blue B/ 0.1N NaOH Flavonoids Three, Rf =0.0, 0.06 & 0.15 5 % KOH in MeOH Cumarins Two, Rf =0.0 & 0.15
Laq
Chloroform/Ethylacetate/ Methanol/ Formic acid (4:3:2:1) FeCl3 + Potassium ferocyanide
Phenolic compounds Four, Rf =0.0, 0.06, 0.15 &
0.63
2
Table 2 Continued…
Plant material used
Mobile phases Spraying reagents/ UV light Secondary metabolites Number components and Rf
UV λ254 Five dark spots Rf =0.14,0.42, 0.48, 0.65 &0.87
Vanillin/H2SO4 Steroidal compounds Six, Rf =0.0, 0.08, 0.42, 0.48, 0.65 & 0.79
Fast Blue B/ 0.1N NaOH Flavonoids Seven, Rf =0.06, 0.15,0.46, 0.59,0.66, 0.73 &0.81
5 % KOH in MeOH Cumarins Six, Rf =0.0, 0.06, 0.18, 0.49, 0.7 & 0.83
Lm Chloroform/Ethylacetate/ Methanol/ Formic acid (4:3:2:1)
FeCl3 + Potassium ferocyanide
Phenolic compounds Seven, Rf = 0.0, 0.06, 0.15,0.46, 0.59,0.66 & 0.73
UV λ 254 One fluoresced spot, Rf =0.06 Vanillin/H2SO4 Steroidal compounds Four, Rf =0.0, 0.08, 0.28 &
0.73 Fast Blue B/ 0.1N NaOH Flavonoids One, Rf = 0.06 5 % KOH in MeOH Cumarins Two, Rf =0.0, 0.06
SBaq Chloroform/Ethylacetate/ Methanol/ Formic acid (4:3:2:1)
FeCl3 + Potassium ferocyanide
Phenolic compounds Three, Rf = 0.0, 0.25 & 0.73
UV λ254 one fluoresced spot and one dark spot, Rf =0.06 & 0.21
Vanillin/H2SO4 Steroidal compounds Four, Rf =0.0, 0.08, 0.28 &0.73 Fast Blue B/ 0.1N NaOH Flavonoids Five, Rf =0.0, 0.06,0.21,0.43 &
0.79 5 % KOH in MeOH Cumarins Five, Rf =0.0 , 0.06, 0.14,0.35&
0.76
SBm
Chloroform/Ethylacetate/ Methanol/ Formic acid (4:3:2:1)
FeCl3 + Potassium ferocyanide
Phenolic compounds Four, Rf =0.0, 0.08, 0.24 & 0.69
3
Note:- For both aqueous and hydralcoholic extracts of fruits only the base line showed the presence of steroidal compounds.
1
4.2 In vitro effects on Guinea-pig Ileum (GPI)
Some of the tested extracts showed a considerable inhibition of cumulative ACh and His-
induced contraction towards higher concentrations, while still others exhibited only slight
inhibition as compared to the control. All the extracts (except Faq and Fm) significantly
inhibited the ACh and His-induced contraction as compared with the control. Laq and Lm
extracts exhibited significant inhibition (P < 0.001) (Figures 4, 5, 11 and 12). SBaq and Sbm
extracts exhibited moderate inhibitions with lower concentration of standard spasmogen (P <
0.001) and higher concentration of standard spasmogen (P<0.05) (Figure 6, 7, 13 and 14).
While Fm showed slight inhibition (P < 0.05) (Figure 9 and 16), selected doses of Faq
exceptionally did show a slight spasmolytic activity (P < 0.05) at lower concentration of
standard spasmogen (Figure 8 and 15). However, a dose of 50µg/ml both for Faq and Fm
extracts showed spasmogenic activity at higher concentration of standard spasmogen.
The dose-dependent effects of both hydroalcoholic and aqueous extracts of twigs of leaf and
stem bark showed for all the three doses in both Ach and His-induced. But no significant
dose-dependent effects were shown in case of fruit extracts. The aqueous extract, particularly
that of leaves was found to show the most inhibitory activity on the isolated GPI.
The doses of 100 and 200µg/ml of aqueous and hydroalcoholic twigs extracts showed
significant spasmolytic activity than atropine (6.66x10-9 M). The other remaining extracts did
not show significant spasmolytic activity compared to atropine (figure 4 and 5). The doses of
100 and 200µgm/ml of the aqueous and hydroalcoholic twigs extracts showed significant
spasmolytic activity at lower concentration of His compared to dexchlorpheniramine (1.3 x
10-9 M) (Figure 10 and 11).
2
-10 -9 -8 -7 -6 -5 -4 -3
0
20
40
60
80
100
Control
50100
200
Atropine
Log C (M) of Ach
% M
ax.
Co
ntr
acti
on
Figure 4. Effect of increasing concentrations of Twigs aqueous extracts on the cumulative
dose–response sigmoid curves of acetylcholine in guinea-pig ileum. The dose of
extracts expressed in µg/ml and the concentration of atropine is 6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All doses of the twig aqueous extracts showed significant spasmolytic action (P< 0.001) at all
concentrations of Ach compared with control. The dose of 100 and 200µg/ml showed
significant spasmolytic effect as compared to atropine P<0.05.
3
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100Control
50100200
Atropine
Log C ( M ) of Acetylcholine
% m
ax.
Co
ntr
acti
on
Figure 5. Effect of increasing concentrations of Twig 80% methanolic extracts on the
cumulative dose–response sigmoid curves of acetylcholine in guinea-pig ileum.
The dose of extracts expressed in µg/ml and the concentration of atropine is
6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
Almost all doses of the twig 80% methanolic extracts showed significant spasmolytic action
(P< 0.05) at all concentrations of Ach compared to Control. The doses of 100 and 200µg/ml
showed significant spasmolytic effect as compared to atropine P<0.05.
4
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100Control50
100200
Atropine
Log C ( M ) of Acetylcholine
% M
ax.
Co
ntr
acti
on
Figure 6. Effect of increasing concentrations of Stem bark aqueous extracts on the
cumulative dose–response sigmoid curves of acetylcholine in guinea-pig ileum.
The dose of extracts expressed in µg/ml and the concentration of atropine is
6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
Almost all the doses of the stem bark aqueous extracts showed significant spasmolytic activity
at all concentrations of Ach compared to control (P<0.05) and all the three doses of the
extracts did not show significant spasmolytics activity as compared to atropine.
5
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 Control50100200Atropine
Log C ( M ) of Acetylcholine
% M
ax.
Co
ntr
acti
on
Figure 7. Effect of increasing concentrations of Stem bark 80% methanolic extracts on the
cumulative dose–response sigmoid curves of acetylcholine in guinea-pig ileum.
The dose of extracts expressed in µg/ml and the concentration of atropine is
6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the stem bark 80% methanolic extracts showed spasmolytic effect at all
concentrations of Ach compared to control (P< 0.05) and all the three doses of the extracts did
not show significant spasmolytics activity as compared to atropine.
6
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
Control50100
200Atropine
Log C ( M ) of Acetylcholine
% M
ax.
Co
ntr
acti
on
Figure 8. Effect of increasing concentrations of Fruit aqueous extracts on the cumulative
dose–response sigmoid curves of acetylcholine in guinea-pig ileum. The dose of
extracts expressed in µg/ml and the concentration of atropine is 6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the fruit aqueous extracts at concentrations of Ach (10-9 and 10-8 M)
showed significant spasmolytic activity (P<0.05), the doses of 50 and 100µgm/ml at Ach
concentration of 10-7 and 10 -6 M did not show significant spasmolytics activity (P>0.05) and
all the three doses of the extracts did not show significant effect at 10-5 M Ach concentration
compared to the control (P>0.05). The 50µgm/ml extracts showed spasmogenic activity at
higher concentration of acetylcholine instead of spasmolytic activity compared to control; all
the three doses of the extracts did not show significant spasmolytics activity compared to
atropine.
7
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
Control
50
100
200Atropine
Log C ( M ) of Acetylcholine
% M
ax.
Co
ntr
acti
on
Figure 9. Effect of increasing concentrations of Fruit 80% methanolic extracts on the
cumulative dose–response sigmoid curves of acetylcholine in guinea-pig ileum.
The dose of extracts expressed in µg/ml and the concentration of atropine is
6.66x10-9 M.
Control Ach Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by ACh prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the fruit 80% methanolic extracts at concentrations of Ach (from 10-9 to
10-7 M) showed significant spasmolytic action (P< 0.05) and also a dose of 50µgm/ml at Ach
concentration of 10-6and all the three doses of the extracts at 10-5 M Ach concentration did not
show significant spasmolytics activity compared to control (P>0.05). The 50µgm/ml extracts
showed spasmogenic activity at 10-5 M concentration of acetylcholine compared to control.
Also, all the three doses of the extracts did not show significant spasmolytic activity as
compared to atropine.
8
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 Control50
100
200
dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 10. Effect of increasing concentrations of Twig aqueous extracts on the cumulative
dose–response sigmoid curves of histamine in guinea-pig ileum. The dose of
extracts expressed in µg/ml and the concentration of dexchlorpheniramine is
1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by His prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the twig aqueous extract at concentrations of His (from 10-9 to 10-6 M)
and the dose of 100 and 200µgm/ml at His concentration of 10-5 and 10-4M showed significant
spasmolytic action compared to control (P< 0.05). A dose of 50µgm/ml at His concentration
of 10-5 M was not significant compared to control (P>0.05). All the three doses of the extracts
showed significant spasmolytic activity at lower concentration of His compared to
dexchlorpheniramine (P<0.05).
9
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 Control
50100
200dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 11. Effect of increasing concentrations of Twig 80% methanolic extracts on the
cumulative dose–response sigmoid curves of histamine in guinea-pig ileum. The
dose of extracts expressed in µg/ml and the concentration of
dexchlorpheniramine is 1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by His prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the twig 80% methanolic extract at concentrations of His (from 10-9 to
10-6 M) showed significant spasmolytic activity (P<0.05). The dose of 100 and 200µgm/ml at
His concentration of 10-5 and 10-4M also showed significant spasmolytic activity (P< 0.05).
The 50 µgm/ml extracts showed spasmogenic effect at 10-5 M concentration of His instead of
spasmolytic activity as compared to control. The doses of 100 and 200µgm/ml of the extracts
showed significant spasmolytic activity at lower concentration of His compared to
dexchlorpheniramine (P<0.05).
10
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
Control50
100200
dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 12. Effect of increasing concentrations of Stem bark aqueous extracts on the
cumulative dose–response sigmoid curves of histamine in guinea-pig ileum. The
dose of extracts expressed in µg/ml and the concentration of dexchlorpheniramine
is 1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by His prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the stem bark aqueous extracts at concentrations of His (from 10-9 to 10-
7 M) and the dose of 100 and 200µgm/ml at His concentration of 10-5 showed significant
spasmolytic action (P< 0.05) compared to control. The 50µgm/ml extracts showed
spasmogenic effect at 10-6 and 10-5 M concentration of histamine instead of spasmolytics
activity compared to control. All the three doses did not show significant spasmolytic effect at
10-5 M His concentration compared to control. Also, all the three doses of the extracts did not
show significant spasmolytic activity as compared to dexchlorpheniramine.
11
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
Control
50100200
dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 13. Effect of increasing concentrations of Stem bark 80% methanolic extracts on the
cumulative dose–response sigmoid curves of histamine in guinea-pig ileum. The
dose of extracts expressed in µg/ml and the concentration of dexchlorpheniramine
is 1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by His prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the stem bark 80% methanolic extracts at concentrations of His (from
10-9 to 10-5 M) only showed significant spasmolytic action compared to control (P< 0.05).
Also, all the three doses of the extracts did not show significant spasmolytic activity as
compared to dexchlorpheniramine.
12
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
Control50
100
200
dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 14. Effect of increasing concentrations of Fruit aqueous extracts on the cumulative
dose–response sigmoid curves of histamine in guinea-pig ileum. The dose of
extracts expressed in µg/ml and the concentration of dexchlorpheniramine is
1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by His prior to the addition of the
plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
A dose of 200µgm/ml at lower concentration of His showed significant spasmolytic action
(P< 0.05), but at higher concentration of histamine showed spasmogenic activity compared to
control. The doses of 50 and 100µgm/ml at all concentration of His showed spasmogenic
activity compared to control. Also, all the three doses of the extract did not show significant
spasmolytic activity as compared to dexchlorpheniramine.
13
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
120
Control50
100
200
dexchlorpheniramine
Log C ( M ) of Histamine
% M
ax.
Co
ntr
acti
on
Figure 15. Effect of increasing concentrations of Fruit 80% methanolic extracts on the
cumulative dose–response sigmoid curves of histamine in guinea-pig ileum. The
dose of extracts expressed in µg/ml and the concentration of
dexchlorpheniramine is 1.3x10-9 M.
Control Histamine Contact time = 5 minutes
Responses were expressed as % of the initial contractions induced by spasmogen His prior to the
addition of the plant extracts. Data were given in Mean + SEM of six guinea-pig preparations.
All the three doses of the fruit 80% methanolic extract at concentrations of His (10-9 and 10-8
M) and also the dose of 100 and 200µgm/ml at His concentration of 10-7 showed significant
spasmolytic action compared to control (P<0.05). A dose of 50µgm/ml at His concentration of
10-7 did not show significance compared to control. All the three doses of the extracts showed
spasmogenic instead of spasmolytic activity at His concentration above 10 -7 M compared to
control. Also, all the three doses of the extract did not show significant spasmolytic activity as
compared to dexchlorpheniramine.
14
4.3 Effect of the extracts on small intestinal transit
All the three doses of Laq and SBaq extracts of Syzygium guineense in dose-dependent
manner inhibited intestinal transit significantly (P<0.001). The Lm extracts showed similar
effect at doses of 50 and 200mg/kg. The dose of 200mg/kg of Laq and Lm showed similar
inhibition as that of atropine (10mg/kg). The dose of 50 for SBm and Fm, 100 and 200mg/kg
of Faq did not show significant inhibition of intestinal transit (Table 3). In case of aqueous
fractionates of leaf; water residue and butanolic fractionates showed moderate inhibition of
intestinal transit (P<0.05) dose dependently, which is not comparable to crude twig aqueous
extracts. The solid residue (i.e. the insoluble residue left during solvent partition of the
aqueous partitioning with the n-butanol and water) did not show significant inhibition of
intestinal transit (Table 4).
15
Table 3. Inhibition of gastro-intestinal motility by Syzygium guineense crude extracts (n=6)
Movement of charcoal meal as percentage of full intestinal length (%)
Items
Dose (in mg/kg)
80% Methanolic extracts Aqueous extracts
Control
Leaf
Stem bark
Fruit
Atropine
-
50
100
200
50
100
200
50
100
200
10
83.23 + 1.44
67.66 + 1.66**
55.21 + 1.575**
67.98 + 2.39**
72.94 + 4.04NS
58.51 + 2.03**
56.94 + 1.38**
74.79 + 2.463NS
72.14 + 1.877*
58.72 + 1.962**
23.87 + 1.518**
76.15 + 1.149
49.95 + 1.587**
35.66 + .805**
29.74 + 1.325**
59.29 + 2.343**
32.53 + 1.540**
30.20 + 2.055**
66.91 + 2.492*
77.50 + 1.663NS
71.80 + 1.790NS
23.87 + 1.518**
*P< 0.05**P< 0.001 NS= not significant
16
Table 4. Inhibition of gastro-intestinal motility by Syzygium guineense twigs aqueous
fractionates (n=6)
Items
Dose
(in mg/kg)
Movement of charcoal meal as
percentage of full intestinal length
Control
Water residue (WR)
n-Butanol fraction(BF)
Solid residue(SR)
Atropine
-
50
100
200
50
100
200
50
100
200
10
83.23 + 1.44
63.77+ 2.11**
55.383 + 1.687**
48.133 + 0.95**
59.748 + 2.34**
51.733 + 1.91**
41.916 + 1.157**
88.931+ 1.31NS
81.466 + 1.08NS
73.083 + 1.37*
23.87 + 1.518**
*P< 0.05**P< 0.001 NS= not significant
17
4.4 Effect of extracts on castor-oil induced diarrhea in mice
In vivo study, all the mice in the control groups had diarrhea within 30 minutes after
administration of castor oil. However, mice pretreated with both aqueous and hydroalcoholic
crude extracts of Syzygium guineense had a delay in the onset of diarrhea and some of them
had no wet defecation at all (Figure 16 and 17; Table 5 and 6).
The suppression of diarrhea for most crude extracts was dose dependent. All the doses of fruit
aqueous extracts; 200mg/kg of stem bark aqueous, twig and fruit hydroalcoholic extracts
showed 100% suppression of diarrhea (P<0.001).
The suppression of diarrhea with all fractionates of leaf aqueous extracts were not significant
compared to vehicle, but the solid residue showed moderate suppression of diarrhea (Figure
18 and Table 5). The onset of diarrhea for all fractionates is similar to that of vehicle group.
18
Table 5. Effect of crude aqueous extracts and fractionates on onset of diarrhea.(n=6)
Treatment Dose (mg/kg) Onset of diarrhea (in min)
Castor oil + distilled water(control) - 29.5 + 4.2
Castor oil + Leaf aqueous ext.
50
100
200
45.4 + 7.2
58.1 + 3.3
112 + 2.1 Castor oil +Stem bark aqueous ext.
50
100
200
54 + 1.7
123 + 3.8
ND
Castor oil + Fruit aqueous ext.
50
100
200
ND
ND
ND
Castor oil + water residue (WR)
50
100
200
45 + 6.48
58.3 + 3.37
45 + 5.22
Castor oil + Butanolic fraction (BF)
50
100
200
42.8 + 8.67
52.9 + 2.11
65.33 + 4.27
Castor oil + solid residue (SR)
50
100
200
44.67 + 5.33
88 + 1.99
112 + 4.23
Loperamide 10 ND
19
Table 6. Effect of crude hydroalcolic extracts in onset of diarrhea (n=6)
Treatment group Dose (in mg/kg) Onset of diarrhea (in min)
Castor oil + 3 % Tween 80(control) - 34.5 + 7.1
Castor oil + Leaf meth.(80%)
50
100
200
102.43 + 3.65
118.44 + 2.44
ND
Castor oil + Stem bark meth.(80%)
50
100
200
41.78 + 7.89
58.67 + 6.67
100 + 3.33
Castor oil + Fruit meth.(80%)
50
100
200
137 + 4.13
146 + 2.11
ND
Loperamide 10 ND
ND= Not Determined
0
10
20
30
40
50
60
70
80
90
100
50 100 200 50 100 200 50 100 200 10
Laq Sbaq Faq LPRD
Dose in mg/kg
% i
nh
ibit
ion
of
Dia
rrh
ea
% inhibition
Figure 16. Antidiarrheal effect of aqueous extracts of Syzygium guineense in Castor oil-
induced mice
All of them were significant (P<0.05)
20
0
20
40
60
80
100
50
10
0
20
0
50
10
0
20
0
50
10
0
20
0
10
Lm Sbm Fm LPRD
Dose in mg/kg
% i
nh
ibit
ion
of
dia
rrh
ea
% inhibition
NS
Figure 17. Antidiarrheal effect of 80% methanolic extracts of Syzygium guineense in Castor
oil-induced mice.
All the others were significant at P<0.05. NS= not significant
0
10
20
30
40
50
60
70
80
90
100
50 100 200 50 100 200 50 100 200 10
WR BF SR LPRD
Dose in mg/kg
% i
nh
ibit
ion
of
dia
rrh
ea
% inhibition
*
*
*
* *
*
Figure 18. Antidiarrheal effect of twig aqueous fractionates of Syzygium guineense in Castor
oil-induced mice.
* P <0.05
21
4.5 LD50 determination in mice
During 24 hours of observation most mice showed anorexia, hypoactivity and piloerection
before death, but the remaining ones remained healthy in 24 hrs.
The LD50 of fruit hydroalcolic extract was > 10.0g/kg as no mouse died at dose of 10.0g/kg.
For the Laq, Lm, SBaq, SBm extracts the LD50 were determined using probit analysis and
gave 14.10, 2.91, 5.12 and 8.77g/kg, respectively (Figure 19-22). But, the LD50 for Faq is not
determined because of lack of enough amounts of extracts.
2.5 3.0 3.5 4.0 4.50.0
2.5
5.0
7.5
Log dose
Pro
bit
scale
Log LD50
Figure 19. Probit transformed responses of twig aqueous extracts.
Slope 3.934 ± 0.4759
Y-intercept -11.19 ± 1.905
r² 0.9716
P value 0.0143
LD50= 14.10g/kg
Y= -11.19+3.9 x
22
2.0 2.5 3.0 3.5 4.03
4
5
6
Log dose
Pro
bit
Scale
Log LD50
Figure 20. Probit transformed responses of twigs hydroalcoholic extracts.
Slope 1.746 ± 0.06437
Y-intercept -1.051 ± 0.2210
r² 0.9973
P value 0.0014
LD50= 2.91g/kg
Y= -1.1 + 1.7 x
3 .0 0 3 .2 5 3 .5 0 3 .7 5 4 .0 0 4 .2 5 4 .5 03
4
5
6
7
L o g L D 5 0
L o g D o s e
Pro
bit
Scale
Figure 21. Probit transformed responses of Stem bark aq. Extract
Slope 2.129 ± 0.3511 Y-intercept -3.411 ± 1.292 r² 0.9484 P value 0.0261 LD50= 5.12g/kg
Y= -3.4 + 2.1 x
23
2 .5 3 .0 3 .5 4 .0 4 .5 5 .03
4
5
6
7
Log LD 50
Log D oseP
rob
it S
cale
Figure 22. Probit transformed responses of stem bark hydroalcoholic extracts
Slope 1.285 ± 0.1586
Y-intercept 0.2392 ± 0.5636
r² 0.9563
P value 0.0039
LD50 = 8.77g/kg
Y= 0.24 + 1.29 x
24
5 Discussion
On Guinea pig ileum (GPI) in vitro experiments, twig aqueous extracts of Syzygium guineense
showed more inhibition of ACh and histamine induced contractions of the tissues than any of
the other extracts. The inhibitory activities of all extracts except Faq and Fm were also
significant in a dose dependent manner from 50 - 200 µg/ml. These results show the
spasmolytic properties of the extracts. Similar results were reported for the plant by different
investigators. As reported by Malele, et al., (1997) the methanolic extract of stem barks of
Syzygium guineense caused a concentration dependent reduction in the contraction of isolated
ileum tissue of rabbit.
In the present study, the aqueous extract of Syzygium guineense showed more spasmolytic
activity in a dose dependent manner than the hydroalcoholic extract which is contrary to most
findings (Gilani and Aftab, 1994; Gilani et al., 1994a and Gilani et al. 1999), this may be due
to the presence of chemical components responsible for spasmolytic activity in more polar
solvents like water.
The difference in the parts of the plant used might also account for differential spasmolytic
effects. Caceres et al. (1992) reported that the seed parts of Moringa oleifera showed
significant antispasmodic activity on the intestinal spasm of rat duodenum while the other
parts (leaves, root, stalk and flowers) didn't. Both the aqueous and hydroalcoholic twig and
stem bark extracts of Syzygium guineense had spasmolytic effect on GPI, while, its fruit
extract showed spasmogenic effect at lower dose and no effect at higher doses in both Ach
and Histamine induced contraction. This finding is consistent with the traditional uses in
Ethiopia for gastrointestinal disorders (Debela and Dawit et al., 2003; Kassu, 2002) of this
25
plant parts. Since the antispasmodic activity of the plant is present mainly in the twigs,
collecting leaves does not pose a great danger to the existence of an individual plant when
compared with the collection of underground part, stem bark or whole part. Studies have
shown that removal of up to 50% of tree leaves does not significantly affect the growth of the
species studied (Poffenberger et al., 1992) and also does not pose any threat on the survival of
the plants (Abebe and Ayehu, 1993).
Both the aqueous and hydroalcoholic extracts of the twig and stem bark on GPI seemed to
have spasmolytic effects at lower doses. Sadraei et al. (2003) showed that hydroalcoholic
extract of P. spinosa has a concentration dependent spasmolytic activity in GPI. But fruit
extracts seemed to have spasmogenic effect at higher concentration of Ach and Hisatmine.
The inhibitory effects of both aqueous and hydroalcoholic leaf and fruit extracts were long
lasting and completely reversible by intermittent washing of the spontaneous contractions GPI
preparation for 30-60 min. Nevertheless, for stem bark aqueous and hydroalcoholic extract the
inhibitory effect was partial to complete abolishment even after many washing with
increasing concentration. The reversible effect of the leaf and fruit extracts could be explained
in features of the nature of reverse antagonism. By the same token, the irreversible effect of
stem bark extracts could explain the nature of irreversible antagonism. According to
Mekonnen (1999) spontaneous and rhythmic contractions of both mouse duodenum and GPI
were abolished by leaf ethanol extract of Moringa stenopetala. The results of this study
indicate a similar rightward shift in the dose– response curves of acetylcholine in the presence
of leaf aqueous extract. The maximal effects of histamine and acetylcholine that were
depressed in the presence of Laq, Lm and SBaq and SBm extracts; Laq, Lm and SBm were
achieved by increasing the concentrations of histamine and acetylcholine respectively.
26
In the in vitro study with total crude extracts, the spasmolytic action may be associated with
the combined effect of several chemical constituents in a synergistic way. For instance, the in
vitro antispasmodic effect of the hydroalcoholic extract of Pycnocyla spinosa on ileum
contractions was suggested to be associated with components in the alkaloid fraction to a
greater extent, flavonoid-rich fraction to a lesser extent, and saponin-rich fraction to the least
extent (Sadraei et al., 2003a). The antispasmodic effect of polyherbal preparation, SJ-200 was
also ascribed to such combination of active chemical constituents (Venkantaranganna et al.,
2002). The same explanation may be given to presently studied twig and stem bark of
Syzygium guineense. But this explanation may not be given for the fruit extracts. Since, in the
phytochemical study, both the aqueous and hydroalcoholic fruit extracts were found to
contain little secondary metabolites. Commonly in fruits, primary metabolites known to be
found. This might be related to the nature of the fruit being spasmogen at selected doses.
In the in vitro experiment, extract Laq and Lm at concentration of 100 and 200 µg/ml were
found to have comparable antispasmodic effect as that of atropine and dexchlorpheniramine.
All the extracts are shown to have more anticholinergic than antihistaminic effects. This may
be due to the higher affinity of the active component(s) to muscarinic than to histaminic
receptors. In the present study, Syzygium guineense twig and stem bark extracts were found to
reduce the spontaneous contraction of the isolated guinea-pig ileum. It has been established
that the spontaneous contractions of the intestinal smooth muscle are regulated by cycles of
depolarisation and repolarisation. Action potentials are generated at the peak of depolarization
and constitute a fast influx of calcium ions through the voltage-activated calcium channels
(Walsh and Singer, 1980; Brading, 1981). Therefore, the extract may contain compounds,
which interfere with the calcium channels activity. The extract also reduced contractions of
GPI induced by Acetylcholine and histamine. Similar inhibitory effects of Ferula sinaica root
27
extract on rat and guinea pig uterine smooth muscle contractions were reported by Aqel et al.
(1991). However, the exact mechanism of action of Syzygium guineense extract is not yet
known. Acetylcholine and histamine cause depolarization and tonic contractions of intestinal
smooth muscles. It is generally accepted that an increase in concentration of cytoplasmic-free
calcium ions is indispensable for smooth muscle contraction. The activation of muscarinic
receptors of longitudinal smooth muscle of guinea-pig small intestine produces an increased
frequency of action potential discharge and depolarisation, which results in a contraction
(Reddy et al., 1995). The acetylcholine-evoked contraction is generally regarded as mediated
via M3 subtype of muscarinic receptor although the muscle has a preponderance of M2
subtype muscarinic binding sites. Histamine-induced contraction occurs via H1 receptor
activation (Zavecz and Yellin, 1982) and this leads to increased influx of calcium through L-
type voltage-operated channels (Gilani et al., 1994). In short, calcium ions gain access to the
cytoplasm through voltage-activated or receptor-operated calcium channels (Triggle, 1985).
In the antidiarrheal study, the hydroalcoholic and aqueous crude extracts of Syzygium
guineense given orally, exhibited significant inhibitory activity against castor oil-induced
diarrhea at all doses. The results were dose-dependent and comparable with that of standard
drug loperamide. But the fractions of the twig aqueous extracts did not show significant
antidiarrheal effect and all the three fractionates did not show significant antitransit in the
small intestine of mice. The antidiarrheal effect of the crude extracts, therefore, might be
attributed to the combined effect of all fractions. The observation that the solid residue
fraction showed more antidiarrheal effect than the other two fractions may be due to Kaolin
like effect of the solid residue fraction.
28
Castor oil causes diarrhea through its active metabolite ricinolic acid (Ammon et al., 1974;
Watson and Gordon, 1962), which stimulates the peristaltic activity of small intestine leading
to changes in electrolyte permeability of intestinal mucosa. Its action is also associated with
stimulation of release of endogenous prostaglandins (Galvez et al., 1993). Laq , Lm, and SBaq
had significantly reduced intestinal motility as observed by the decrease in intestinal transit
motility of charcoal meal. The reduction in the intestinal motility by Laq , Lm, and SBaq may
be responsible for the antidiarrhoea activity. Other factors may have played role in the anti-
diarrhea effect of Fm. However, the more antidiarrheal activity of fruit extracts compared
with that of twig and stem bark extracts might not be explained at the moment because other
experimental models of diarrhea such as MgSO4, Prostaglandin–induced antidiarrheal test
should be exhausted. Probably Laq, Lm, and SBaq increased the reabsorption of NaCl and
water by decreasing intestinal motility as observed by the decrease in intestinal transit by
charcoal meal and by their anticholinergic and antihistaminic effects. This study may support
the claimed traditional use of Syzygium guineense as an antidiarrhoea agent (Abebe and
Debela, et al. 2003). It also coincided with the result of other plants in the same species.
According to Mukherjee et al. (1998) castor oil induced diarrhea was reduced by the bark
extracts of Syzygium cumini.
In some cases, it has been found that antidiarrhoeal activity is associated with the
antimicrobial activity of leaf and bark extracts of Syzygium guineense which was reported to
have potent antibacterial effect against diarrhea caused by bacteria (Tsakala et al., 1996;
Ashebir and Ashenafi, 1999) which might enhance the antidiarrheal use of this plant.
29
The median lethal dose was also determined. The results of LD50 were Laq=14103mg/kg,
Lm=2914mg/kg, Sbaq=5122mg/kg, Sbm=8770mg/kg and Fm>10,000mg/kg. Some adverse
effects, such as hypoactivity, were observed immediately after the administration, while
anorexia was more pronounced at the higher doses and persisted until death. The difference of
LD50 values obtained might be due to difference in the presence of compounds, which were
toxic in different parts of the plants. No significant difference in LD50 values was found for
males and females. This finding suggests that the twig aqueous and fruit hydroalcoholic
extracts are relatively safe when given orally.
The results in the in vitro GPI experiments, intestinal transit and antidiarrheal test revealed
that crude extracts of Syzygium guineense must have active chemical constituents with
possible spasmolytic, antitransit and antidiarrheal effects. According to Abdalla and Abu
Zarga (1987); Gilani et al., (1994b); Abdalla et al., (1994), flavonoid constituents of different
plants showed spasmolytic activity in different tissues preparations in vitro. Quercetin, one of
the flavonoids isolated from the aerial parts of Conyza flaginoides exerted inhibitory effects
on GPI contractile response, and also caused a concentration dependent inhibition of the
spontaneous contractions of rat ileum (Galvez et al., 1996; Mata et al., 1997). The distinct
relaxant effect of Satureja obovota varieties on the isolated rat duodenum was suggested to be
due to the presence of the polar compounds, flavonoids (Sanchez de Rojas et al., 1994).
According to Harborne and Williams (2000) and Karamenderes and Apaydin (2003) the
antispasmodic effect of total extract of Achillea nobilis L. on rat duodenum, was reported to
be due to the inhibitory effects of some flavonoids present in the plant. These flavonoids are
responsible for other useful medicinal properties in the folk medicine. Lozoya et al. (2002)
reported about the relationship of the spasmolytic, antimotility and antidiarrhoeic activity of
Psidium guajava folia extract with quercetin flavonoids present in the plant. The significant
antidiarrhoeal activity of the methanolic fraction of unripe fruits of Psidium guajava extract
30
was also connected to the flavonoids that inhibit ACh release in GI tract (Lutterodt, 1989
cited in Ghosh et al., 1993). Gilani et al.,(2000) reported about the relationship between the
presence of cumarins with spasmolytic activity too. In the current phytochemical study;
flavonoid and cumarins were found in twigs and stem barks of Syzygium guineense. These
might be responsible for the observed spasmolytic and antidiarrheal activity of the extracts.
The presence of tannins and mucilaginous substances in the fruits of Aegle marmelos was
reported to show antidiarrheal activity against castor oil diarrhea in mice (Pallavi and
Subhash, 2003). Laq, Lm, SBaq and SBm of Syzygium guineense were found to have tannins,
which might be responsible for the observed antidiarrheal activity.
The Geiger’s criteria for the acceptance of a drug as an antidiarrheal include: (1) inhibition of
the production of wet or unformed faeces in animals; (2) inhibition of the production of
watery stool or fluid evacuation in animal and (3) inhibition of gastrointestinal propulsive
action. (Akah, 1988). The twig aqueous extracts, therefore, meets the Geiger’s criteria as
observed from its results.
31
6 Conclusion
In this study the possible spasmolytic effects of Laq, Lm, SBaq and Sbm extracts of Syzygium
guineense were demonstrated. Laq extracts have shown higher spasmolytic activities while
Lm, SBaq and Sbm have shown low antispasmodic effect. Phytochemical screenings using
chemical test and thin-layer chromatographic analyses indicated that different classes of
compounds including flavonoids, cumarins, saponins, tannins occurred in both hydralcoholic
and aqueous extracts of Syzygium guineense. The spasmolytic and antidiarrhea activities
shown by twigs and stem bark extracts might be related to the presence of active chemical
components such as flavonoids, cumarins or tannins as demonstrated by fractionating the leaf
aqueous extracts. However, further investigations should be carried out in order to isolate and
confirm the identities of compounds by spectroscopic methods such as UV, IR, MS and
NMR.
The spasmolytic effects of plants can be affected by different factors like solvent employed,
concentration of extracts used and even parts of the plant tested. Further studies should be
conducted by combining each fraction in order to clarify synergistic effects of each fraction
that are responsible for the observed activities. To trace the exact chemical constituents that
result in the observed activities, it should have to be isolated through pharmacological guided
fractionation. This is very useful to identify the real spasmolytic agent in the traditional folk
use of the plant that is responsible for the remedy of many gastrointestinal ailments.
All crude extracts of Syzygium guineense and solid residue of the twigs aqueous fractionate
decrease the frequency and onset of diarrhea. This finding strongly supports the use of the
plant as antidiarrheal in traditional medicine. The other finding related to the acute toxicity of
32
different extracts showed that the twig aqueous and fruit hydroalcoholic extracts were
relatively safe. Further studies are required to confirm the underlying mechanisms of action.
33
References
Abdalla, S., Abu Zarga, M., and Sabri, S. (1994). Effects of the flavone luteolin, isolated from
Colchicum richii, on guinea pig isolated smooth muscle and heart and on blood flow.
Phytother.Res. 8: 265-270.
Abdalla, S. and Abu Zarga, H. (1987). Effects of cirsimartin, a flavone isolated from
Artemisia judaica, on isolated guinea pig ileum. Planta Med. 53 (4): 322-324.
Abebe, D. (1986), Traditional medicine in Ethiopia: The attempts being made for its effective
and better utilization, SINET: Ethiop. J. Sci. 9 (supp. l): 61-69.
Abebe, D. and Ayehu, A. (1993). Medicinal Plants and Enigmatic Health Practices of
Northern Ethiopia. BSPE. Addis Ababa. pp. 1-4.
Abebe, D. and Hagos, E. (1993). Plant Genetic Resources of Ethiopia. pp 1-12, Chapman and
Hall Ltd, UK.
Abebe, D., Debella, D., and Urga, K. (2003). Medicinal Plants of Ethiopia, p 63, Camerapix
Publishers Interanational, Nairobi, Kenya.
Agwu, C. O. C. and Okeke,G. I. (1996). Pollen analytical and thin-layer chromatographic
study of honey from three savanna zones of northern Nigeria. Nigerian Journal of
Botany 9-10: 25-36.
Akah, P.A., Aguwa, C.N., Agu, R.U. (1999). Studies on the antidiarrhoeal properties of
Pentaclethra macrophylla leaf extracts. Phytother. Res. 13: 292–295.
34
Ammon, H.V., Thomas, P.J., Phillips, S. (1974). Effects of oleic and recinoleic acids net
jejunal water and electrolyte movement. Journal of Clinical Investigation.53: 374-
/379.
Aqel, M.B., Al-khalil, S., Afifi, F., 1991. Effects of a Ferula sinaica root extract on the
uterine smooth muscle of rat and guinea pig. J Ethnopharmacol. 31: 291–297.
Arragie M., Metzner, J. and Bekemier, H. (1983). Antispasmoidc effect of Hagenia
abyssinica. Planta Med. 47: 240-241.
Ashebir,M., Ashenafi,M. (1999). Evaluation of the Antibacterial Activity of Crude
Preparations of Foeniculum vulgare, Ruta chalepensis And Syzygium guineense on
Some Food-Borne Pathogens. Eth Phar J. 17(1): pp. 37-43.
Ayensu ES., DeFilipps RA. (1978). Endangered and Threatened Plants of the United States.
Washington, DC: Smithsonian Institution. pp. 48-52.
Barrachina, M.D., Bello, R., Martinez-Cuesta, M.A., and Esplugues, J. (1995b).
Antinflamatory activity and effects on isolated smooth muscle of extracts from
different Teucrium species. Phytother.Res. 9: 368-371.
35
Bekele, T. (1993). Vegetation ecology of remnant Afromontane forests on the Central Plateau
of Shewa, Ethiopia. Acta Phytogeographica Suecica.79: 1-64. Dep. Ecol. Botany,
Uppsala Univ., Villavagen 14, S-752 36 Uppsala, Sweden; Stromgaard, P. (1985).
Biomass, growth, and burning of woodland in a shifting cultivation area of South
Central Africa. Forest Ecology And Management.12(3-4): 163-178.
Bergendorff, O. and Sterner, O. (1995). Spasmolytic flavonols from Artemisia abrotanum.
Planta Med. 61 (4): 370-371.
Bogale, M., Dagne, E., Izzo, A.A., Capasso, F., and Mascolo, N. (1996). Spasmolytic activity
of kosotoxin in the guinea pig ileum and rabbit jejunum in vitro. Phytother.Res. 10
(supplement 1): S112-S113.
Brading, A.R. (1981).How do drugs initiate contraction in smooth muscle? Trends in
Pharmacological Sciences. 2: 262–265.
Broadley, K.J. and Kelly, D.R. (2001). Muscarinic Receptor Agonists and Antagonists.
Molecules 6: 142-193.
Cai L, Wu CD. (1996). Compounds from Syzygium aromaticum possessing growth inhibitory
activity against oral pathogens. J Nat Prod. 59:987-90.
Christen M. O. (1990) .Action of pinaverium bromide, a calcium-antagonist, on
gastrointestinal motility disorders. Gen. Pharmacol. 21: 821–825.
36
Cronquist A. (1988). An Integrated System of Classification of Flowering Plants. New
York:Columbia University Press, 1981,7.The Evolution and Classification of
FloweringPlants. Bronx, NY:New York Botanical Garden. pp. 100-7.
da Silva, B.A., de Araujo, A.P. Mukherjee, R. and Chiappeta, A.A. (1993).
Bisnordihydrotoxiferine and villosimine from Strychnos divaricans root:
spasmolytic properties of Bisnordihydrotoxiferine. Phytother.Res. 7: 419-424.
Fabricant, D.S. and. Farnsworth, N.R. (2001).The Value of Plants Used in Traditional
Medicine for Drug Discovery. Environ Health Perspect. 109: (suppl 1):69–75
de la Puerta, R. and Herrera, M.D. (1995). Spasmolytic action of essential oil of Achillea
ageratum L. in rats. Phytother. Res. 9: 150-152.
De Ponti, F. and Tonini, M. (2001). Irritable Bowel Syndrome: New agents for targeting
serotonin receptor subtypes. Drugs.61(3): 317-332
Debella A. (2002). Manual for Phytochemical screening of Medicinal Plants. Dept. of Drug
Research, EHNRI, Addis Ababa, Ethiopia. pp.1-72.
Desta, Y., Debella, A. and Assefa, G. (1996).Traditional Medicine: Global and National
perspectives. In: Proceedings of the workshop on Development and utilization of
Herbal Remedies in Ethiopia, Dawit Abebe (Ed), Ethiopian Health and Nutrition
Research Institute, Addis Ababa, pp. 1-19.
37
Dire, G.F., Lima, E.A.C., Gomes, M.L., Moreno, S., Faria M.V.C., Jales, R.L., Catanho,
M.T.J.A. and Filho, M.B. (2003). Evaluation of Biological effects of a Natural
extract of Chayotte (Sechium edule): A molecular and cellular analysis. Pakistan
Journal of Nutrition. 2(4): 249- 253.
Dorman, HJ., Deans, SG. (2000). Antimicrobial agents from plants: antibacterial activity of
plant volatile oils. J Appl Microbiol. 88:308-16.
Edwards, S., Tadesse, M., Hedberg (1995). Flora of Ethiopia and Eritrea ,Canellaceae to
Euphorbiaceae; Editor(s): I Source: (2 Part 2): pp. 106-71; Addis Ababa: The
National Herbarium.
Estrada, S., Rojas, A., Mathison, Y., Israel, A., and Mata, R. (1999). Nitric oxide / cGMP
mediates the spasmolytic action of 3, 4’- dihydroxyl -5, 5’- demethoxy bibenzyl
from Scaphyglottis livida. Planta Med. 65: 109-114.
Farnsworth, NR., Akerele, O., Bingel, AS., Soejarto, DD., Guo, Z. (1985). Medicinal plants
in therapy. Bull W H O. 63:965–981.
Frame, AD., Rios-Olivares, E., De Jesus, L., Ortiz, D., Pagan, J., Mendez, S. (1998). Plants
from Puerto Rico with anti-Mycobacterium tuberculosis properties. P R Health Sci
J. 17: 243-52.
38
Fujioka, T., Kashiwada, Y., Kilkuskie, RE., Cosentino, LM., Ballas, LM., Jiang, JB., Janzen,
WP., Chen, IS., Lee, KH. (1994). Anti-AIDS agents, 11. Betulinic acid and
platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV
activity of structurally related triterpenoids. J Nat Prod. 57(2):243-7.
Galvez, J., Zarzuelo, A., Crespo, M.E., Lorente, M.D., Ocete, M.A., Jimenez, J.( 1993).
Antidiarrhoeic activity of Euphorbia hirta extract and isolation of an active
flavanoid constituent. Planta Med. 5: 333-336.
Galvez, J., Duarte, J., de Medina, S., Jimenez, J. and Zarzuelo, A. (1996). Inhibitory effects of
Quercetin on the Guinea pig ileum contractions. Phytother.Res. 10: 66-69.
Gedif, T., Hahn, H.J. (2003), The use of medicinal plants in self- care in rural central
Ethiopia. J. Ethnopharmaco. 87: 155-161.
Ghosh, T.K., Sen, T., Das, A., Dutta, A.S., and Nag Chaudhuri, A.K. (1993). Antidiarrhoeal
activity of the methanolic fraction of the extract of unripe fruits of Psidium
guajava Linn. Phytother.Res.7: 431-433.
Gilani, A.H. and Aftab, K. (1994). Hypotensive and Spamolytic activities of Ethanolic extract
of Capparis cartilaginea. Phytother.Res. 8: 145-148.
Gilani, A.H., Aftab, K., Suria, A., Siddiqui, S., Salem, R., Siddiqui, B.S., and Faizi, S.
(1994a). Pharmacological studies on Hypotensive and Spasmolytic activities of
Pure compounds from Moringa oleifera. Phytother.Res.8: 87-91.
39
Gilani, A.H., Aziz, N., Ahmad, M., Alam, M.T., Rizwani, G.H. (1999). Spasmogenic and
Spasmolytic constituents in Sida pakistanica. Pharmaceutical Biology. 37(1): 173-
180.
Gilani, A.H., Aziz, N., Khan, M.A., Shaheen, F. Jabeen, Q., Siddiqui, B.S., and Herzig, J.W.
(2000). Ethnopharmacological evaluation of the anticonvulsant, sedative and
antispasmodic acitivities of Lavandula Stoechas L. J. Ethnopharamcol. 71: 161-
167.
Gilani, A.H., Janbez, K.H., Lateef, A. and Zaman, M. (1994b). Ca2+ Channel Blocking
activity of Artemisia scoparia extract. Phytother.Res. 18:161-165.
Gwee, K.-A. and Read, N.W. (1994). Rolling review: disorders of gastrointestinal motility -
therapeutic potentials and limitations. Ailment Pharmacol.Ther. 8: 105-118.
Hajhashemi, V., Sadraei, H., Ghannadi, A.R., and Mohseni, M. (2000). Antispasmodic and
Antidiarrhoeal effect of Satureja hortensis L. essential oil. J. Ethnopharmacol. 71:
187-192.
Harborne, J.B. and Williams, C.A. (2000). Advances in flavonoid research since 1992.
Phytochemistry. 55: 481-504.
40
Karamenderes, C. and Apaydin, S. (2003). Antispasmodic effect of Achillea nobilis L.
subp.sipylea (O.Schwarz) Bassler on the rat isolated duodenum. J. Ethnopharmacol.
84: 175-179.
Kashiwada, Y., Wang, HK., Nagao, T., Kitanaka, S., Yasuda, I., Fujioka, T., Yamagishi, T.,
Cosentino, LM., Kozuka, M., Okabe, H., Ikeshiro, Y., Hu, CQ., Yeh, E., Lee,
KH.(1998). Anti-AIDS agents. 30. Anti-HIV activity of oleanolic acid, pomolic
acid, and structurally related triterpenoids. J Nat Prod. 61:1090-5.
Kassu, A. (2002), Ethnobotanical survey and the medicinal plants of some areas in South and
Central Ethiopia. Focus GCMS newsletter. 2(4): 50-63.
Kim, HM., Lee, EH., Hong, SH., Song, HJ., Shin, MK., Kim, SH., Shin, TY. (1998). Effect of
Syzygium aromaticum extract on immediate hypersensitivity in rats. J
Ethnopharmacol. 60:125-31.
Kurokawa, M., Hozumi, T., Basnet, P., Nakano, M., Kadota, S., Namba, T., Kawana, T.,
Shiraki, K.(1998). Purification and characterization of eugeniin as an anti-
herpesvirus compound from Geum japonicum and Syzygium aromaticum. J
Pharmacol Exp Ther. 284:728-35.
Lee, GI., Ha, JY., Min, KR., Nakagawa, H., Tsurufuji, S., Chang, IM., Kim, Y. (1995).
Inhibitory effects of Oriental herbal medicines on IL-8 induction in
lipopolysaccharide-activated rat macrophages.Planta Med. 61:26-30.
41
Li-Li, Y., Jyh-Fei, L., Chen, CF. (2000). Anti-diarrheal effect of water extract of Evodiae
fructus in mice. J Ethnopharmacol.73:39-45.
Lozoya, X., Reyes-Morales, H., Chavez-Soto, M.A., Martinez-Garcia, M.C., Soto- Gonzalez,
Y. and Doubova, S.V. (2002). Intestinal antispasmodic effect of a phytodrug of
Psidium guajava folia in the treatment of acute diarrhoeic disease. J Ethnopharmacol.
83:19-24.
Madeira, S.V.F., Matos, F.J.A., Leal-Cardoso, J.H. and Criddle, D.N. (2002). Relaxant effects
of the essential oil of Ocimum gratissimum on isolated ileum of guinea pig. J
Ethnopharmacol. 81: 1-4.
Malele, R. S., Moshi, M. J., Mwangi, J. W., Achola, K. J., Munenge, R. W. (1997).
African Journal of Health Sciences, 4: 43-45.
Mata, R., Rojas, A., Acevedo, L., Estrada, S., Calzada, F., Rojas, I, Bye, R. and Linares, R.
(1997). Smooth muscle relaxing flavonoids and terpenoids from Conyza
flaginoides. Planta Med. 63: 31-35.
Makonnen, E. (1996). Is Linum usitatissimum seed a Potential Medicine in the Therapy of
Peptic ulcer? Ethiop.J.Health Dev.10 (2): 79-82.
Makonnen, E. (2000). Constipating and Spasmolytic effects of Khat (Catha edulis Forsk) in
experimental animals. Phytomedicine. 7(4): 309-312.
42
Mekonnen, Y. (1999). Effects of Ethanol Extract of Moringa stenopetala leaves on Guinea
pig and Mouse smooth muscle. Phytother.Res. 13: 1-3.
Micklefield, G.H., Greving I., and May, B. (2000). Effects of Peppermint oil and Caraway oil
on gastrointestinal motility. Phytother.Res. 14: 20-23.
Montes-Belmont R, Carvajal M. (1998). Control of Aspergillus flavus in maize with plant
essential oils and their components. J Food Prot. 61:616-9.
Mukherjee, PK., Saha, K., Murugesan, T., Mandal, SC., Pal, M., Saha, BP. (1998). Screening
of anti-diarrhoeal profile of some plant extracts of a specific region of West
Bengal, India. J Ethnopharmacol. 60(1): 85-9.
Mustafa, M.R., Mohamad, R., Din, L. and Wahid, S. (1995). Smooth Muscle Relaxant
activities of compounds from Malaysian Medicinal plants on rat aorta and guinea
pig ileum. Phytother.Res. 9: 555-558.
Namba, T., Kurokawa, M., Kadota, S., Shiraki, K. (1998). Development of antiviral
therapeutic agents from traditional medicines. Yakugaku Zasshi. 118:383-400.
Nievergelt, B., Good, T. & Güttinger, R. (1998). A survey of the Flora and Fauna of the
Simen Mountains National Park, Ethiopia. Special Issue of Walia. Journal of the
Ethiopian Wildlife and Natural History Society. Addis Ababa, Ethiopia.
43
Nkeh, B., Kamanyi, A., Bopelet, M., Ayafor, J.F. and Mbafor, J.T. (1993). Anticholinergic
effects of the methanol stembark extract of Erythrina sigmoidea on isolated rat
ileal preparations. Phytother.Res. 7: 120-123.
Nkeh, B., Kamanyi, A., Bopelet, M., Ayafor, J.F. and Mbafor, J.T. (1996). Inhibition of
histamine-induced contraction of rat ileum by promethazine and methanol
stembark extract of Erythrina sigmoidea (Hua). Phytother.Res. 10: 444-446.
Noamesi, B.K., Bogale M., and Dagne E.(1990). Intestinal Smooth muscle Spasmolytic
actions of the aqueous extract of the roots of Taverniera abyssinica. J
Ethnopharmacol. 30(1): 107-113.
Oketch-Rabah, H.A.; Dossaji, S.F. (1998 ). Molluscicidal activity of some Kenyan medicinal
plants. South African Journal of Science. 94: 299-301.
Palit, P., Furman, BL., Gray, AI. (1999). Novel weight-reducing activity of Galega officinalis
in mice. J Pharm Pharmacol. 51:1313-9.
Pallavi, B. and Subhash, B. (2003). Gastrointestinal effects of Mebarid®, an ayurvedic
formulation, in experimental animals. J Ethnopharmacol. 86: 173-176.
Pankhrust, R. (1975). Historical Anecdote: Dr Brater and Europes' Discovery of Kosso,
Ethio. Med. J. 13 (1): 29-34.
44
Pankhrust, R. (1976). Historical reflection of traditional Ethiopian pharmacopoeia,
Ethiop.Pharm J. 2:29-32.
Perfumi, M., Paralleli, F., and Cingonali, M.L. (1995). Spasmolytic activity of essential oil of
Artemisia thuscula Cav. from the Canary Islands. J.Essent.Oil. Res. 7(4): 387-392.
Perry, W.L.M. (1982). Pharmacological Experiments on Isolated Preparations. Churchill
Livingstone Press. London and New York. pp. 55-86, 94,95.
Poffenberger, M., McGean, B., Khare, A., Campbell, J. (1992). Field Method Manual,
Volume II. Community Forest Economy and Use Pattern: Participatory Rural
Appraisal (PRA) Methods in South Gujarat,India. Society for Promotion of
Wastelands Development, New Delhi.
Prince, PS., Menon, VP., Pari, L. (1998). Hypoglycaemic activity of Syzigium cumini seeds:
effect on lipid peroxidation in alloxan diabetic rats. J Ethnopharmacol. 61(1): 1-7.
Pushpalatha E, Muthukrishnan J. (1995 ). Larvicidal activity of a few plant extracts against
Culex quinquefasciatus and Anopheles stephensi. Indian J Malariol. 32(1):14-23.
Rajasekaran, M., Bapna, JS., Lakshmanan, S., Ramachandran Nair, AG., Veliath, AJ.,
Panchanadam, M. (1998). Antifertility effect in male rats of oleanolic acid, a
triterpene from Eugenia jambolana flowers. J Ethnopharmacol. 24:115-21.
45
Rodriguez, R., Lasheras, B. and Cenarruzabeitia, E. (1986). Pharmacological activity of
Prunus spinosa on isolated tissue preparations. Planta Med. 52(4): 256-259.
Rodriguez-Lopez, V., Salazar, L. and Estrada, S (2003). Spasmolytic activity of several
extracts obtained from some Mexican medicinal plants. Fitoterapia. 74: 725-728.
Sadraei, H., Naddafi, A., Ashgari, G. (2003). Relaxant effect of Essential oil and Hydroalcolic
extracts of Pycnocycia spinosa. Phytother. Res. 17: 645-649.
Sadraei, H., Ghannadi, A., and Malekshashi, K. (2003b). Relaxant effects of essential oil of
Melissa officinalis and citral on rat ileum contractions. Fitoterapia. 74: 445-452.
Saka, J. D. K. and Msonthi, J. D. (1994). Nutritional value of edible fruits of indigenous wild
trees in Malawi. Forest Ecology and Management 64(2-3): 245-248.
Samuelsson, G. (1987). Plants used in traditional medicine as sources of drugs. Bull. Chem.
Soc. Ethiop. 1(1): 47-54 & 57.
Sanchez de Rojas, V.R., Ortega, T. and Villar, A. (1994). Activity of the Extracts of Two
Satureja obovata varieties on isolated smooth muscle preparations. Phytother.Res.
8: 212-217.
Shamsa, F., Ahmadiani, A., Khosrokhavar, R. (1999). Antihistaminic and anticholinergic
activity of barberry fruit (Berberis bulgaris) in the guinea-pig ileum. J
Ethnopharmacol. 64: 161-166.
46
Shiferaw, M. (1996), The role of health professionals in the development of traditional
medicine in Ethiopia. In: Proceedings of the workshop on Development and
utilization of Herbal Remedies in Ethiopia, Dawit Abebe (Ed), Ethiopian Health
and Nutrition Research Institute, Addis Ababa, pp. 15-18.
Shiraki, K., Yukawa, T., Kurokawa, M., Kageyama, S. (1998). Cytomegalovirus infection and
its possible treatment with herbal medicines. Nippon Rinsho. 56:156-60.
Silva-Netto, CR., Lopes, RA., Pozetti, GL. (1986). Effects of extract of dried leaves of
Jambolao (Syzygium Jambolanum) on renal excretion of water, sodium and
potassium in rats. Preliminary results. Rev Fac Odontol Ribeiro Preto. 23:213-5.
Singh, R.K., Pandey, H.P., and Singh, R.H. (2003). Irritable Bowel Syndrome: Challenge
ahead. Current Science. 84(2): 1525-1533.
Sofowora, A. (1982). Medicinal plants and Traditional Medicine in Africa, pp 142-146, John
Willey and Sons Press, U.S.A.
Solecki, R., Shanidar, IV. (1975). A Neanderthal flower burial in northern Iraq. Science.
190:880–881.
Srivastava, KC., Malhotra N. (1991). Acetyl eugenol, a component of oil of cloves (Syzygium
aromaticum L.) inhibits aggregation and alters arachidonic acid metabolism in
human blood platelets. Prostaglandins Leukot Essent Fatty Acids. 42: 73-81.
47
Tadesse, M. (1986). Some Medicinal plants of Central Shoa and South-Western Ethiopia,
SINET: Ethiop.J.Sci. 9 (Suppl): 143-167.
Tanira, M.O., Ali, B.H., Bashir, A.K., Wasfi, I.A., and Chandranath, I. (1996). Evaluation of
the relaxant activity of some United Arab Emirates Plants on intestinal smooth
muscle. J Pharm. Pharmacol. 48(5): 545-550.
Teixeira, CC., Pinto, LP., Kessler, FH., Knijnik, L., Pinto, CP., Gastaldo, GJ., Fuchs, FD.
(1997). The effect of Syzygium cumini (L.) skeels on post-prandial blood glucose
levels in non-diabetic rats and rats with streptozotocin-induced diabetes mellitus. J
Ethnopharmacol. 56(3):209-13.
Tippo O, Stern WL. (1977). Humanistic Botany. New York:W.W. Norton,.,9. Schultes RE.
The future of plants as sources of new biodynamic compounds. In: Plants in the
Development of Modern Medicine(Swain T, ed). Cambridge, MA:Harvard
University Press, 1972;103–124.
Trease, G.E and Evans, W.C. (2002), Pharmacognosy, ELBS, Oxford. pp. 67-69.
Triggle, D.J., 1985. Calcium ions and respiratory smooth muscle function.Br. J Clin.
Pharmac. 20: 213s–219s.
Tsakala, TM., Penge, O., John, K. (1996). Screening of in vitro antibacterial activity from
Syzygium Guineense (Willd) hydrosoluble dry extract. Ann Pharm Fr.54:276-9.
48
Tuladhar, B.R., Ge, L., and Naylor, R.J. (2003). 5-HT7 receptors mediate the inhibitory effect
of 5-HT on peristalisis in the isoalted guinea pig ileum. Br.J. Pharmac.138 (7):
1210-1214.
Vagelos PR. (1991). Are prescription drug prices high? Science. 252:1080–1084.
Venkataranganna, M.V., Anturlikar, S.D., Gopumadhavan, S., Prakash, N.S., Girish, M.R.,
Murthy, S.and Mitra S.K. (2002). Antispasmodic activity of SJ (Himcospaz): an
herbal preparation. Pharmaceutical Biology. 40(6): 416-421.
Verpoorte R. (2000). Pharmacognosy in the new millennium: leadfinding and biotechnology.
J Pharm Pharmacol 52:253–262.
Walsh Jr., J.V., Singer, J.J. (1980). Calcium action potentials in single freshly isolated smooth
muscle cells. Am. J Phys. 239: C162–C174.
Watson, W.C., Gordon, R. (1962). Studies on the digestion absorption and metabolism of
castor oil. Biochemistry and Pharmacology. 11: 229-236.
Waynforth, H.B. (1980). Experimental and Surgical Technique in the Rat. Academic Press,
London.
WHO. (2001). Report on Antimalarial drug development, World Health Organization
Regional office for the Western Pacific, Shanghai, China.
49
WHO. (2001). The use of antimalarial drugs, Report of WHO Informal consultation, World
Health Organization, Geneva.
WHO. (2001). Promoting the role of traditional medicine in health systems: A strategy for
the African Region, WHO Regional Office for Africa, Harare.
Williamson, E.M., Okpako, D.T., and Evans, F.J. (1996). Selection, Preparation and
Pharmacological Evaluation of Plant Materials.Vol.1.pp.1-8, 25-45,169-189,191-
216.
Yukawa, TA., Kurokawa, M., Sato, H., Yoshida, Y., Kageyama, S., Hasegawa, T., Namba,
T., Imakita, M., Hozumi, T., Shiraki, K. (1996). Prophylactic treatment of
cytomegalovirus infection with traditional herbs. Antiviral Res. 32:63-70.
Zavecz, J.H., Yellin, T.O. (1982). Histamine receptors in the myenteric plexus-longitudinal
muscle of the response to electrical stimulation. J Pharmacol. Exp. and Ther. 223:
177–182.