BIZUAYEHU NIGATU

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.

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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

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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)


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)


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)


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.




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


% M

ax.

Co

ntr

acti

on

Figure 6. Effect of increasing concentrations of Stem bark aqueous extracts on the



6.66x10-9 M.




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


% M

ax.

Co

ntr

acti

on

Figure 7. Effect of increasing concentrations of Stem bark 80% methanolic extracts on the



6.66x10-9 M.




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


% 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.




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


% M

ax.

Co

ntr

acti

on

Figure 9. Effect of increasing concentrations of Fruit 80% methanolic extracts on the



6.66x10-9 M.




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


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


% 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.




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


% M

ax.

Co

ntr

acti

on

Figure 12. Effect of increasing concentrations of Stem bark aqueous extracts on the


dose of extracts expressed in µg/ml and the concentration of dexchlorpheniramine

is 1.3x10-9 M.




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


% M

ax.

Co

ntr

acti

on

Figure 13. Effect of increasing concentrations of Stem bark 80% methanolic extracts on the


dose of extracts expressed in µg/ml and the concentration of dexchlorpheniramine

is 1.3x10-9 M.




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


% 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.




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


% M

ax.

Co

ntr

acti

on

Figure 15. Effect of increasing concentrations of Fruit 80% methanolic extracts on the


dose of extracts expressed in µg/ml and the concentration of

dexchlorpheniramine is 1.3x10-9 M.


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

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