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
xiv
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
xv
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
Top Related