UNIVERSITI PUTRA MALAYSIA

ANTIMICROBIAL AND WOUND HEALING ACTIVITIES OF THREE

CASSIA SPECIES

ELYSHA NUR ISMAIL

FPSK(M) 2004 7

ANTIMICROBIAL AND WOUND HEALING ACTIVITIES OF THREECASSIA SPECIES

By

ELYSHA NUR ISMAIL

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysiain Fulfilment of the Requirement for the Degree of Master of Science

November 2003

DEDICATION

"Dedicated especially to Dr Nazrul Hakim Abdullah (supervisor), my parents

Ismail Ibrahim and Siti Mahfuzah, Mr. Reezal Ishak whose sacrifice and

support has enabled me to complete this study successfully".

ii

Abstract of thesis presented to the Senate ofUniversiti Putra Malaysia in fulfilmentof the requirement for the degree of Master of Science

ANTIMICROBIAL AND WOUND HEALING ACTIVITIES OF THREECASSIA SPECIES

By

ELYSHA NUR ISMAIL

November 2003

Chairman: Associate Professor Dr. Muhammad Nazrul Hakim Abdullah, Phd

Faculty : Medicine and Health Sciences

Cassia alata, Cassia fistula and Cassia auriculata are largely used in traditional

medicine for centuries, to improve health and well being of human civilizations in

rural areas of developing countries worldwide. The objective of this study is to

investigate the antimicrobial and wound healing activities of all three Cassia species.

The first two experiments were conducted to investigate the antibacterial and

antifungal activities of three Cassia species using disc diffusion methods. Ethanol

and aqueous extract of all three Cassia species were tested in vitro against the

bacteria Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Bacillus

subtilis, and the fungi, Candida albicans, Candida tropicalis, Microsporum canis

and Aspergillus fumigatus. All three Cassia species were effective against

Staphylococcus aureus and Bacillus subtilis in a dose dependent manner, and were

not effective against Escherichia coli and Salmonella enteritidis. The results were

compared with commercial antibiotics chloramphenicol (30 mg/ml), ampicilin (10

mg/ml), penicillin G (10 mg/ml), erythromycin (15 mg/ml), tetracycline (30 mg/ml)

iii

'\

and enrofloxacin (5 mg/ml). All three plants were only effective against the Gram­

positive bacteria. The ethanol leaf extract of the plants at concentration 80 mg/ml can

be compared to the commercial antibiotic, penicillin against Bacillus subtilis. Only

Cassia alata and Cassia auriculata has exhibited antifungal activity. Cassia fistula

has no effect against all tested fungi. The ethanol and aqueous bark extracts from

Cassia alata was only effective against Candida albicans, whereas the ethanol leaf

and bark extract from Cassia auriculata was only effective against Microsporum

canis. When comparing the two plants against each other, we found that the plant

extracts was selective and has a very narrow spectrum against the tested fungi. The

ethanol leaves extracts from all Cassia species were chosen for the third test, which

was the wound healing activity in mice. Topical application over an incised wound

showed progressive infiltration of inflammatory cells, increased blood vessel

formation, and enhanced proliferation of cells because of treatment with Cassia

fistula extract and acriflavine. Wound contraction in specimens from the groups

treated, respectively, with Cassia fistula, acriflavine and control, showed significant

structural improvement when compared to Cassia alata and Cassia auriculata, in

which there were no signs of healing at the end of the experiment. It can be

concluded that, ethanol extracts of the leaf and bark of Cassia alata, Cassia fistula

and Cassia auriculata showed antibacterial and antifungal activity which may be

attributed to the presence of chemical constituent such as flavonoids, chrysophanol

antharaquinones, and chrysarobin. This study also showed the promising wound

healing activity of Cassia fistula in mice and warrants detailed experimental and

clinical studies. It also provides a rationale for the use of Cassia fistula in

preparations of traditional medicine to promote wound healing.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

KEGIATAN ANTIMICROB DAN PENYEMBUHAN LUKA TIGA SPESIESCASSIA

Oleh

ELYSHA NUR ISMAIL

November 2003

Pengurusi: Professor. Madya Dr. Muhammad Nazrul Hakim Abdullah, PhD

Fakulti : Perubatan dan Sains Kesihatan

Cassia alata, Cassia fistula, dan Cassia auriculata sudah diguna di luar bandar di

seluruh dunia, selama beberapa abad dalam pengubatan tradisional untuk

meningkatkan kesihatan dan kesejahteraan manusia. Objektif kajian ini ialah

menyelidik kegiatan antiimikrob dan penyembuhan luka tiga species Cassia. Dua

ujikaji dijalankan untuk menyelidik kegiatan antibakteria dan antifungus ketiga-tiga

Cassia ini dengan mengguna kaedah resapan cakera. Ekstrak etanol dan akues untuk

ketiga-tiga spesies Cassia ini diuji secara in vitro terhadap Escherichia coli,

Salmonella enteritidis, Staphylococcus aureus dan Bacillus subtilis, dan terhadap

fungus Candida albicans, Candida tropicalis, Microsporum canis dan Aspergillus

fumigatus. Ketiga-tiga Cassia ini berkesan terhadap Staphylococcus aureus dan

Bacillus subtillis, dan secara bersandarkan dos, dan tidak berkesan terhadap

Escherichia coli dan Salmonella enteritidis. Penemuan daripada ujian ini

dibandingkan dengan antibiotik komersial kloramfenikol (30 mg/ml), ampisilin (10

v

mg/ml), penisilin G (l0 mg/ml), eritromisin (15 mg/ml), tetrasiklin (30 mg/ml), dan

enroflosaksin (5 mg/ml). Ekstrak etanol daun daripada ketiga-tiga tumbuhan pada

kepekatan 80 mg/ml boleh dibandingkan kepada antibiotik komersial, penisilin

terhadap Bacillus subtilis. Hanya Cassia alata dan Cassia auriculata menunjukkan

aktiviti antikulat. Cassia fistula tidak menunjukkan kesan terhadap kesemua kulat

yang diuji. Ekstrak etanol dan akues batang daripada Cassia alata hanya berkesan

terhadap Candida albican, manakala ekstrak etanol daun dan batang daripada Cassia

auriculata hanya berkesan terhadap Microsporum canis. Apabila dibandingkan

kedua-dua tumbuhan itu sesama sendiri, kami dapati bahawa extrak tumbuhan

memilih dan mempunyai spectrum yang kecil terhadap kulat yang diuji. Ekstrak

etanol daun daripada ketiga-tiga Cassia dipilih untuk ujian ketiga, iaitu kegiatan

penyembuhan luka pada mencit. Penggunaan secara topikal kepada luka hirisan

menunjukkan penyusupan sel keradangan secara progresif, dan peningkatan

pemproliferatan sel yang diperlaku dengan ekstrak Cassia fistula dan akriflavin.

Pengecutan luka dalam spesimen daripada kumpulan terperlaku dengan Cassia

fistula, akriflavin, dan kawalan, menunjukkan pembaikkan struktur secara tererti

apabila dibandingkan dengan Cassia alata dan Cassia auriculata, di mana tiada

petanda yang menunjukkan bahawa berlakunya penyembuhan pada ahkir ujikaji

tersebut. Apa yang boleh disimpulkan ialah, ekstrak etanol untuk daun dan batang

Cassia alata, Cassia fistula dan Cassia auriculata menunjukkan kegiatan

antibakteria dan antifungus spectrum luas tererti, disabitkan kepada wujudnya unsur

kimia seperti flavonoid, krisofanol, antrakuinon dan krisorabin yang mungkin

bertanggungjawab kepada kegiatan antimikrob tersebut. Kajian ini mengesahkan

bahawa kegiatan penyembuhan luka Cassia fistula berpotensi dalam mencit dan

adalah wajar ujikaji dan kajian klinikal yang lebih mendalam dijalankan. Kajian ini

vi

juga menunjukkan yang penggunaan Cassia fistula dalam persediaan ubatan

tradisional untuk merangsang penyembuhan luka adalah rasional.

VII

ACKNOWLEDGEMENTS

In the name ofAllah, Most gracious, Most merciful

All gratifications are referred to Allah

The preparation of this thesis has been aided by significant contribution from many

individuals, which are gratefully acknowledged.

Assoc. Prof. Dr. Muhd. Nazrul Hakim Abdullah, my sincere appreciation for

his support and guidance, constantly providing valuable advice and ideas for this

project.

Assoc. Prof. Dr. Abdul Rahim Mutalib for providing facilities at the

Bacteriology Laboratory.

Dr. Wan Nordin Wan Mahmud for providing facilities at the Histology

Laboratory.

Pn. Indu Bala and Encik Razali from Malaysian Agricultural and Research

Development Institute (MARDI) for the identification of the plants.

I am delighted to acknowledge all the staff of Histology Laboratory; Puan

Sapiah, Physiology Laboratory; En. Kufli, the staff of Bacteriology Laboratory: En

Hajaraih and Encik Jefri.

I wish to express my sincere gratitude to my friends, Azmahani Abdullah,

Nor Shahida Abdul Rahman, and Soleha Mohd Hassan and most of all to Reezal

Ishak for his everlasting support and guidance throughout this course.

Last but not least to my beloved parents, Ismail Ibrahim and Siti Mahfuzah

bte. Sheikh Abdul Kadir for their supports.

viii

I certify that an Examination Committee met on 21 5t November, 2003 to conduct thefinal examination of Elysha Nur Ismail on her Master of Science thesis entitled"Antimicrobial and Wound Healing Activities of Three Cassia Species" in accordancewith Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti PertanianMalaysia (Higher Degree) Regulations 1981. The Committee recommends that thecandidate be awarded the relevant degree. Members of the Examination Committee areas follows:

Johnson Stanslas, Ph.D.LecturerFaculty of Medicine and Health ScienceUniversiti Putra Malaysia(Chairman)

Muhammad Nazrul Hakim Abdullah, Ph.D.Associate ProfessorFaculty of Medicine and Health ScineceUniversiti Putra Malaysia(Member)

Dato' Abdul Salam Abdullah, Ph.D.ProfessorFaculty of Veterinary MedicineUniversiti Putra Malaysia(Member)

Abdul Manan Mat Jais, Ph.D.ProfessorSekolah Sains IntegratifKolej Universiti Teknologi dan Pengurusan Malaysia (KUTPM)Blok A, Jalan Equestrian B/52 off Jalan Persiaran SukanSeksyen 1340 I00 Shah Alam, Selangor

Abdul Rahim MutaIib, Ph.D.Associate ProfessorFaculty of Veterinary MedicineUniversiti Putra Malaysia(Member)

Wan Nordin Wan Mahmud, Ph.D.Faculty of Veterinary MedicineUnivesiti Putra Malaysia(Member)

Date:

ix

o5 MAR 2004

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted asfulfilment of the requirement for the degree of Master of Science. The members ofthe Supervisory Committee are as follows:

Muhd Nazrul Hakim Abdullah, PhDAssociate ProfessorDepartment of Biomedical SciencesFaculty of Medicine and Health SciencesUniversiti Putra Malaysia(Chairman)

Dato' Abdul Salam Abdullah, PhDProfessorFaculty of Veterinary MedicineUniversiti Putra Malaysia(Member)

Abdul Manan Mat Jais, PhDAssociate ProfessorDepartment of Biomedical SciencesFaculty of Medicine and Health SciencesUniversiti Putra Malaysia(Member)

Abdul Rahim Mutalib, PhDAssociate ProfessorDepartment of Pathology and MicrobiologyFaculty of Veterinary MedicineUniversiti Putra Malaysia(Member)

Wan Nordin Wan Mahmud, PhDLecturerFaculty of Veterinary MedicineUniversiti Putra Malaysia(Member)

AINI IDERIS, Ph.DProfessor/ DeanSchool of Graduate StudiesUniversiti Putra Malaysia

Date: 0 5 HAY 2004

x

DECLARATION

I hereby declare that the thesis is based on my original work except for quotationsand citations which have been duly acknowledged. I also declare that it has not beenpreviously or concurrently submitted for any other degree at UPM or otherinstitutions.

XI

Date: l-5 t1AY ZOO4

TABLE OF CONTENTS

PageDEDICATION iiABSTRACT 1lI

ABSTRAK vACKNOWLEDGEMENTS Vlll

APPROVALS ixDECLARATION xiLIST OF TABLES xvLIST OF FIGURES xviLIST OF ABBREVIATIONS/NOTAIONS/GLOSSARY OF TERMS XVlll

CHAPTER

I

II

INTRODUCTIONObjective of Study

LITERATURE REVIEWThe Plants

Cassia alataPlant DescriptionHabitatUsesPreviously Isolated Active CompoundsFolk MedicineContemporary Medicine

Cassia fistulaPlant DescriptionHabitatUsesPreviously Isolated ConstituentFolk MedicineContemporary Medicine

Cassia auriculataPlant DescriptionHabitatUsesPreviously Isolated Active CompoundsFolk MedicineContemporary Medicine

xii

I5

66111112121213141818181919202124242525252526

Bacteria 28Gram-positive Bacteria 28

Staphylococcus aureus 28Bacillus subtilis 30

Gram-negative Bacteria 31Escherichia coli 31Salmonella enteriditis 32

Fungi 33The Moulds 34

Aspergillus fumigatus 34Microsporum canis 35

The Yeast 36Candida albicans and Candida tropicalis 36

III IN VITRO ANTIBACTERIAL ACTIVITY OF CASSIA 39SPP

Introduction 39Materials and Methods 40

Preparation of Plant Extracts 40Agar Preparation 41Microorganism and Medium 42Antibacterial Sensitivity Test 42Statistical Analysis 42

Results 43Cassia alata 43Cassia fistula 45Cassia auriculata 47

Discussion 61

IV IN VITRO ANTIFUNGAL ACTIVITY OF CASSIA SPP 66Introduction 66Materials and Methods 67

Preparation of Plant Extracts 67Agar Preparation 67Microorganism and Medium 67Antifungal Sensitivity Test 68Statistical Analysis 69

Results 69Discussion 78

V IN VIVO WOUND HEALING ACTIVITY OF THREE 82CAESALPINIACEAE

Introduction 82Materials and Methods 83

Preparation of Plant Extracts 83Chemicals 83Experimental Animals 83Skin Samples Taken at Day 0, 3, 6 and 9 85Statistical Analysis 86

Results 87Discussion 97

xiii

VI GENERAL DISCUSSION AND CONCLUSIONSummary

BIBLIOGRAPHYAPPENDICESBIODATA OF THE AUTHOR

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101104106114138

LIST OF TABLES

Table Page

Chemical constituent of three Cassia plants 10

2 The folk use of Cassia alata 14

3 The folk use of Cassiajistula 21

4 Toxin and enzymes produced by Staphylococcus 29aureus

5 The inhibitory zone value (mean ± S.O) of Cassia alata 45against tested bacteria.

6 The inhibitory zone value (mean ± S.O) of Cassia 47fistula against tested bacteria

7 The inhibitory zone value (mean ± S.O) of Cassia 49auriculata against tested bacteria

8 The inhibitory zone value of standard antibiotics 61

9 The inhibitory zone value of Cassia alata against tested 71fungi.

10 The inhibitory zone value of Cassia auriculata against 72tested fungi.

11 The inhibitory zone value of standard antifungal drugs 79

12 Wound healing division of mice in groups often 87

13 Attributed score of inflammation and cicatrisation. 88

14 Score of inflammation 89

15 Score of cicatrisation 89

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LIST OF FIGURES

Figure Page

Medicinal plant used in this study, Cassia alata. 7

2 Medicinal plant used in this study, Cassiajistula. 8

3 Medicinal plant used in this study, Cassia auriculata 9

4 Inhibition zone of ethanol extracts of Cassia alata against 50Staphylococcus aureus.

5 Inhibition zone of ethanol extracts of Cassia alata against 51Bacillus subtilis.

6 Inhibition zone of ethanol extracts of Cassiajistula against 52Staphylococcus aureus.

7 Inhibition zone of ethanol extracts of Cassiajistula against 53Bacillus subtilis.

8 Inhibition zone of ethanol extracts of Cassia auriculata against 54Staphylococcus aureus.

9 Inhibition zone of ethanol extracts of Cassia auriculata against 55Bacillus subtilis.

10 The mean of ethanol leaf extracts of Cassia alata in different 56concentration.

11 The mean of ethanol leaf extracts of Cassiajistula in different 57concentration.

12 The mean of ethanol bark extracts of Cassia jistula in different 58concentration.

13 The mean of ethanol leaf extracts of Cassia auriculata in 59different concentration.

14 The mean of ethanol bark extracts of Cassia auriculata in 60different concentration.

15 The inhibitory zone values of ethanol and aqueous extract of 73Cassia alata against Candida albicans.

XVI

LIST OF FIGURES

16 The inhibitory zone values of ethanol and aqueous extraction of Cassia 74auriculata against Microsporum canis.

17 The mean ofethanol bark extracts of Cassia alata in different 75concentration.

18 The means of aqueous bark extracts of Cassia alata in different 76concentration.

19 The mean of ethanol leaf extracts of Cassia auriculata in different 77concentration.

20 The mean ofethanol bark extracts of Cassia auriculata in different 78concentration.

21 Effects of inflammation at day 3 91

22 Effects of inflammation at day 6 92

23 Effects of inflammation at day 9 93

24 Effects of cicatrisation at day 3 94

25 Effects of cicatrisation at day 6 95

26 Effects of cicatrisation at day 9 96

xvii

LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS

AIDS

ANOVA

ALP

APACHE

CNS

DNA

IBS

MARDI

MIC

P

SD

UPM

Acquired Immunodeficiency Syndrome

Analysis of variance

Alkaline phosphatase

Acute Physiology and Chronic Health Evaluation

Central nervous system

Deoxyribonucleic acid

Institute of Bioscience

Malaysian Agricultural and Research Development Institute

Minimum inhibitory concentration

Significant value

Standard division

Universiti Putra Malaysia

xviii

CHAPTER I

INTRODUCTION

Medicinal plants are widely used by all sections of people either directly as

folk remedies in different indigenous system of therapy or indirectly in the

pharmaceutical preparation of modern medicines. Recent years have witnessed an

upsurge in worldwide used of medicinal plants, plant parts and/or active ingredients

interest among scientific institutions, biological research societies and in health care

providers (Fransworth et aI., 1985).

In developing countries, particularly in the rural areas and among the urban

poor, herbal medicine is, in most cases, the only form of health care. It is woven with

magical-religious elements and sick persons would immediately turn to "someone

who knows" and only as a last resort will consult a regular physician.

Many medical practitioners with training in pharmacology and

pharmacognosy are well aware of the number of modern therapeutic agents that have

been derived from tropical forest species. In fact, over 120 pharmaceutical products

currently in use are plant-derived; some 75% of these discovered by examining the

use ofthese plants in traditional medicine (Fransworth et aI., 1985).

Herbs have been used for centuries to improve health and well being of

civilization (Lindley, 1981). Initially, the term "herb" only applied to non-woody

plants, but today, "herb" refers to any part of a plant used for medicinal or flavouring

purposes. An "herb" may be a fruit, a bark, a flower, a leaf or a root as well as non­

woody plant (Usher, 1974).

It is estimated that fifty-six percent of low-income world's population use

herbal medicine and supplementation for their primary health care (Planta et aI.,

2000).

Respiratory infections, diarrhea, fungal/bacterial infections, diabetes and

malaria are among the common health problems occurring in the rural communities

in tropical developing countries. Numerous tropical medicinal plants are used

traditionally and have been shown in vivo to possess biological activities against

these diseases (Pinn, 2000).

Antimicrobial agents are the synthetic and natural compounds/drugs to

suppress growth of microorganism. A bactericidal agent kills bacteria, whereas a

bacteriostatic agent inhibits their growth but does not kill them (Levinson and

Jawetz, 1998).

Bacteria are minute, unicellular, plant-like, microscopic organisms, which

differ from true plants in that they lack chlorophyll. They reproduce by binary

fission. They are widely distributed in soil, air, water, and milk, on the surface of

2

fruits and vegetables, and in various parts of the body such as the alimentary canal

and skin (Davies, 1994).

Gram-positive bacteria are prokaryotic cell whose cell wall consists chiefly of

peptidoglycan and lack of the outer membrane of gram-negative cells. Gram-positive

bacteria is, bacteria that take the initial stain of the Gram stain and are not

decolourised, so that they appear purple (Madigan et ai., 1997). Two common

bacteria, which cause serious diseases in human, were chosen for this study,

Staphylococcus aureus and Bacillus subtilis.

Gram-negative bacteria are prokaryotic cell whose cell wall contains

relatively little peptidoglycan but has an outer membrane composed of

lipopolysaccharide, lipoprotein, and other complex macromolecules. The lack of

peptidoglycan, makes the bacteria loses the initial stain of the Gram stain, is

decolourised, and takes the colour of the final stain; red (Madigan et ai., 1997). Two

common Gram negative bacteria, which cause serious diseases in human, were

chosen for this study, Escherichia coli and Salmonella enteritidis.

Fungi are nonphototrohic eukaryotic microorganisms that contain rigid cell

walls. Fungi can be differentiated from prokaryotes by the fact that fungal cells are

usually much larger and contain a nucleus, vacuoles, and mitochondria, which are

typical of eukaryotic cells. There are three groups of fungi with major practical

importance, the moulds, yeast and mushrooms. In this study, only the moulds and

yeast are used (Madigan et ai., 1997).

3

The habitats of fungi are quite diverse. Some are aquatic, living primarily in

fresh water, and a few marine fungi are known. Most fungi have terrestrial habitats,

in soil or on dead plant matter, and these types often play crucial roles in the

mineralization of organic carbon in nature. A large number of fungi are parasites of

terrestrial plants, a few fungi are parasitic on animals, including humans, although in

general fungi are less significant as animal pathogens that are bacteria and viruses

(Madigan et aI., 1997).

Moulds are filamentous fungi. They grow as long filament (hyphae) and

become mat (mycelium). They are widespread in nature and are commonly seen on

stale bread, cheese, or fruit. Aspergillus fumigatus and Microsporum canis are used

in this study. Both moulds are from clinical isolates.

Yeast are unicellular (single cells) fungus and grows asexual budding, most

of them are classified with the Ascomycetes. Yeast usually flourishes in habitats

where sugars are present, such as fruits, flowers, and the bark of trees. A number of

yeast species live symbiotically with animals, especially insects, and a few species

are pathogenic for animals and humans. The yeast used in this study is Candida

albieans and Candida tropicalis. Both Candida species are from clinical isolates.

Wound healing is a complex sequence of events is initiated by the stimulus of

injury to tissues. A positive stimulus may result from the release of some factors by

the wounding of tissues (Allison, 1992). This sequence of physiologic events occurs

by a process of connective tissue repair. These events involve the migration,

proliferation, adhesion and differentiation ofthe epithelial cells (Raghow, 1994).

4

Objective of the study

I) To evaluate the effects of ethanol and aqueous of leaves and barks extracts

from Cassia alata, Cassia jistula and Cassia auriculata against several

bacteria in vitro.

2) To evaluate the effects of ethanol and aqueous of leaves and barks extracts

from Cassia alata, Cassiajistula and Cassia auriculata against fungi in vitro.

3) To determine the ethanol leaves extracts of Cassia alata, Cassia jistula and

Cassia auriculata in promoting wound healing in mice.

5

CHAPTER II

LITERATURE REVIEW

The plants

Cassia is an herbaceous plant belonging to Caesalpiniaceae tribe of Leguminose

family. Cassia alata, Cassia fistula and Cassia auriculata are largely used in

traditional medicine for centuries, to improve health and well being of civilizations in

rural areas of developing countries worldwide. All three plants continued to be used

as major source of medicine in primary health care. Their usage varies around the

world and concentrated on treatment of skin diseases (ringworm) externally (Usher,

1974).

The extracts of Cassia alata, Cassia fistula and Cassia auriculata have

similar composition, with a slight variation in the amount of their active components

and each is purported to have different therapeutic properties. However, little has

been done to compare the effectiveness of the three species (Percival, 2000).

Although many of the active components of Cassia have been identified

(Table 1), their mechanism of action is not completely known nor is the

bioavailability, relative potency or the synergistic effects of the active compounds

known (Giron, 1991).

6