ANTIMICROBIAL INVESTIGATION OF CRUDE

EXTRACTS AND PURIFIED COMPOUNDS OF

GARCINIA GRIFFITHII T. ANDERS AND

ARTOCARPUS ALTILIS (PARKINSON) FOSBERG,

MALAYSIAN TROPICAL PLANTS

BY

TARA K. JALAL

A thesis submitted in fulfilment of the requirement for

the degree of Master of Science Biotechnology Sciences

Kulliyyah of Science

International Islamic University

Malaysia

APRIL 2013

ii

ABSTRACT

The present study focuses on the bioassay guided isolation of antimicrobial

compounds from Garcinia griffithii and Artocarpus altilis. The hexane, ethyl acetate,

dichloromethane and methanol extracts were obtained from the dried (leaves of G.

griffithii and twigs of A. altilis) by using Soxhlet extraction. The plants crude extracts

were evaluated for in vitro antimicrobial activity against six pathogenic

microorganisms (two Gram-positive strains: Staphylococcus aureus (ATCC-25923)

and Bacillus cereus (ATCC-11778), two Gram-negative strains Escherichia coli

(ATCC-35218) and Pseudomonas aeruginosa (ATCC-27853), and two fungal strains:

Candida albicans (ATCC-10231) and Cryptococcus neoformans (ATCC-90112))

using the disc diffusion method at concentration of 2 mg/disc. The antimicrobial

activity of G. griffithii methanol extract showed the highest zone of inhibition of

18±2.4mm, while hexane and dichloromethane extracts of A. altilis was 14.6±0.2mm.

Whereas minimum inhibition concetration (MIC) and minimum bactericidal/minimum

fungicidal concetration (MBC/MFC) for isolated compounds and the extracts were

ranged from 62.5-500 µg/ml and 250-2000 µg/ml, respectively. Bioassay guided

isolation of the methanol extract of G. griffithii afforded betulinic acid and from

hexane and dichloromethane (DCM) extracts of A. altilis afforded cycloartenyl acetate

and stigmasterol respectively. Their structures were identified by 1H-NMR,

13C-

NMR, IR and the spectra obtained were compared with previous data. This study

forms a strong basis for declaring G. griffithii and A. altilis as antimicrobial potential

sources to treat pathogenic microorganisms.

iii

ملخّص البحث

و كارسينيا كرفتي من للميكروبات المضادةالحيويه المركبات عزل على الدراسة هذه تركزمن والميثانول الميثان اسيتات، إيثيل الهكسين،مستخلصات تم استخراج.التيليساورتوكاربس

باستخدام التيليس من اورتوكاربس المجففة والأغصان المجففةكارسينيا كرفتي وراقا ستة ضد المختبر في الميكروبات مضادات لنشاط النباتات مستخلصات قيمت. سوكسهليت

واثنين من سلالات :غرام إيجابية سلالات اثنين من) للأمراض المسببة الدقيقة الكائنات من-Bacillus cereus (ATCC و Staphylococcus aureus (ATCC-25923)سلبية غرام

11778), Escherichia coli (ATCC-35218) و Pseudomonas aeruginosa

(ATCC-27853), واثنين من سلالة الفطريات: Candida albicans (ATCC-10231) و Cryptococcus neoformans ( (ATCC-90114.2±81 مم تثبيط منطقة أعلى

، الميثانوللمستخلص وكان نشاط الميكروبات. القرص/ملغ4 القرص نشر أسلوب باستخدام الأدنى الحد تثبيط التركيزو (MIC) .اورتوكاربوس التيليس من مستخلص والميثان الهكسين بينماالأدنى فطريات / جراثيم الأدنى (MBC/MFC) )الحد تراوحتوحين 2.4±82.1 مم

مل للمركبات المعزوله/ميكروغرام 522-14.5مل للمستخلصات و /ميكروغرام 452-4222كارسينيا الميثانول من اوراق نبات يتولينيك من مستخلص الب حمض عزل تم .التوالي على

ومن مستخلص الهكسين والدايكلوروميثان تم عزل خلات السايكلو اورتينيل و كرفتيوتم تحدديد هياكلها الستيكما ستيرول على التوالي من اغصان النبات اورتوكاربوس التيليس

والاشعه تحت الحمراء ين الكاربون المغناطيسيباستخدام الرنين الهيدروجين المغناطيسي و الرننبات ان والاطياف بالمقارنه مع بيانات سابقة وتشكل هذه الدراسه اساسا قويا لاعلان

صدر محتمل لعلاج ات ومبكمضاد للميكرو رفتي و نبات اورتوكاربوس التيليس كارسينيا ك ة.الدقيقالتي تسببها الكائنات الامراض

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion it conforms

to acceptable standards of scholarly presentation and is fully adequate, in scope and

quality, as a dissertation for the degree of Master of Science (Biotechnology

Sciences).

________________________

Muhammad Nor Omar

Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and is fully adequate, in scope and quality as a

dissertation for the degree of Master of Science (Biotechnology Sciences).

________________________

Muhammad Khan Ayob

External Examiner

________________________

Qamar Uddin Ahmed

Internal Examiner

This thesis was submitted to the Department of Biotechnology Science and is accepted

as a fulfilment of the requirement for the degree of Master of Science (Biotechnology

Sciences).

________________________

Zarina Zainuddin

Head, Department of

Biotechnology Sciences

This thesis was submitted to the Kulliyyah of Sciences and is accepted as a fulfilment

of the requirement for the degree of Master of Science (Biotechnology Sciences).

________________________

Kamaruzzaman Yunus

Dean, Kulliyyah of Science

v

DECLARATION

I hereby declare that this dissertation is the results of my own investigations, except

where otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Tara K. Jalal

Signature……………………… Date……………………..

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright @ 2013 by Tara K. Jalal. All rights reserved.

ANTIMICROBIAL INVESTIGATION OF CRUDE EXTRACTS

AND PURIFIED COMPOUNDS OF GARCINIA GRIFFITHII T.

ANDERS AND ARTOCARPUS ALTILIS (PARKINSON) FOSBERG,

MALAYSIAN TROPICAL PLANTS

No part of this unpublished research may be reproduced, stored in a retrieval system,

or transmitted, in any form or by any means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder except

as provided below:

1. Any material contained in or derived from this unpublished research may only

be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or

electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and

supply copies of this unpublished research if requested by other universities

and research libraries.

Affirmed by Tara K. Jalal

.............................................. ...............................

Signature Date

vii

I dedicate my master thesis to ALLAH (s.w.t.) the almighty, to my beloved mother,

late Mrs. Roanak Baker Sami, my father (Kamal Jalal), my husband (Ahmed Shakir)

and my loving daughters (Mariam, Zainab and Zahra).

viii

ACKNOWLEDGMENTS

Alhamdulillah, all praises are due to Allah (s.w.t.) for His mercies, blessings and

guidance in completing this successfully.

First and foremost, I would like to express my sincere gratitude to my

supervisor, Assoc. Professor Dr. Muhammad Nor Omar for his unrelentless supports

and constructive criticism throughout the period of my research. During this time, he

showed me patience and enthusiasm, infact he is a great source of knowledge and

inspiration.

In the course of this research I have been blessed with many helpful persons,

who always render one form of assistance or the other to me any time the need arose

they also shared with me useful suggestions; they include brother Ahmad Muzammil,

Dr. Naik (a post doc.) and so may Allah reward you all aboundantly.

I also wish to thank everyone at Kulliyyah of Science, especially, the Dean of

Kulliyah of Science for great working atmosphere and being so helpful at any time.

Last and not least, once more, I want to thank my loving family including my

dear husband Ahmed and my wonderful children Mariam, Zainab and Zahra. I thank

you for being so patient with me when I was busy with the project.

JAZAKUMULLAHU KHAIRAH.

ix

TABLE OF CONTENTS

Abstract in English .............................................................................................. ii

Abstract in Arabic ................................................................................................ iii

Approval Page ..................................................................................................... iv

Declaration Page .................................................................................................. v

Dedication ........................................................................................................... vi

Copyright Page .................................................................................................... vii

Acknowledgements ............................................................................................. viii

List of Tables ....................................................................................................... xii

List of Figures ..................................................................................................... xiii

List of Abbreviations ........................................................................................... xiv

CHAPTER ONE: INTRODUCTION ............................................................... 1

1.1 Back ground and justification ............................................................. 1

1.2 Antimicrobial agents from plants ........................................................ 2

1.3 Significance of study .......................................................................... 3

1.4 General objective ................................................................................ 5

1.5 Specific objectives .............................................................................. 5

1.6 Research hypotheses ........................................................................... 6

CHAPTER TWO: LITERATURE REVIEW .................................................. 7

2.1 Medicinal plants ................................................................................. 7

2.2 Traditional medicine ........................................................................... 8

2.3 Drug discovery from medicinal plants ................................................ 9

2.4 Phytochemicals in plants .................................................................... 12

2.4.1 Phenolic compounds ................................................................. 12

2.4.2 Flavones, Flavonoids and Flavonols .......................................... 13

2.4.3 Tannin ...................................................................................... 13

2.4.4 Coumarin .................................................................................. 14

2.4.5 Alkoloids .................................................................................. 14

2.4.6 Terpenes ................................................................................... 14

2.4.6.1 Triterpenes ...................................................................... 15

2.5 Garcinia ............................................................................................. 16

2.6 Botany and uses of Garcinia ............................................................... 16

2.6.1 Botany ...................................................................................... 16

2.6.2 Uses of Garcinia ....................................................................... 17

2.6.3 Chemical constituents of Garcinia griffithii .............................. 18

2.7 Artocarpus (Moraceae) ....................................................................... 19

2.7.1 Artocarpus altilis (Parkinson) (Fosberg) ................................... 20

2.8 Botany and uses of Artocarpus altilis ................................................. 20

2.8.1 Botany ...................................................................................... 20

2.8.2 Uses of Artocarpus altilis.......................................................... 21

2.8.3 Chemical constituents of A. altilis .................................................... 21

2.9 Antimicrobial constituents of plants .................................................. 23

x

2.10 Microorganisms used in the antimicrobial tests ................................. 30

2.10.1 Bacillus cereus ........................................................................ 30

2.10.2 Escherichia coli ...................................................................... 30

2.10.3 Staphylococcus aureus ............................................................ 30

2.10.4 Pseudomonas aeruginosa ........................................................ 31

2.10.5 Candida albicans .................................................................... 31

2.10.6 Cryptococcus neoformans ....................................................... 32

2.11 Extraction ......................................................................................... 32

2.12 Antimicrobial activity ....................................................................... 33

2.13 Analytical methods and structure elucidation .................................... 35

2.13.1 Column chromatography ......................................................... 36

2.13.2 Thin layer chromatography ..................................................... 37

2.14 Bioassay ........................................................................................... 37

2.14.1 Bioassay guided fractionation-disc diffusion assay and broth

assay.................................................................................................. 38

2.15 Structure elucidation and spectroscopy ............................................. 39

CHAPTER THREE: MATERIALS AND METHODS ................................... 41

3.1 Experiment design .............................................................................. 41

3.2 Materials ............................................................................................ 41

3.2.1 Reagents and chemicals ............................................................ 41

3.2.2 Apparatus and chemicals for antibacterial testing ...................... 41

3.3 Methods ............................................................................................. 42

3.3.1 Plant Materials .......................................................................... 42

3.3.2 Extraction ................................................................................. 42

3.3.2.1 Preparing of G. gfiffithii and A. altilis exracts .................. 42

3.4 Source of microorganisms .................................................................. 43

3.4.1 Storage and maintenace of bacteria and fungal stock cultures .......... 43

3.5 Antimicrobial activity of the crude extracts of G. griffithii and

A. altilis .................................................................................................... 43

3.5.1 Samples preparation .................................................................. 44

3.5.2 Bacterial inoculum preparation ................................................. 44

3.5.3 Fungal inoculum preparation..................................................... 45

3.5.4 Disc diffusion method ............................................................... 45

3.5.5 Determination of (MIC) and (MBC/MFC)…………………….. 46

3.6 Bioassay guided isolation of compounds from G. griffithii and

A. altilis .................................................................................................... 47

3.6.1 Chromatographic purification of G. griffithii methanolic extract47

3.6.2 Chromatographic purification of A. altilis extracts .................... 48

3.7 Antimicrobial activities for isolated compounds FAC, DEC and HFC 49

3.7.1 Disc diffussion method ............................................................. 49

3.7.2 Determination of (MIC) of isolated compounds ........................ 49

3.7.3 (MBC/MFC) of isolated compounds ......................................... 50

3.8 Structure identification ....................................................................... 50

3.8.1 Nuclear magnetic resonance spectroscopy (NMR) ................ 50

3.8.2 NMR sample preparation .......................................................... 50

3.9 Statistical analysis ............................................................................ 51

xi

CHAPTER FOUR: RESULTS.......................................................................... 52

4.1 Yield of crude extracts of G. griffithii ................................................. 52

4.1.1 Yield of crude extracts of A. altilis ............................................ 52

4.2 Antimicrobial activities of the crude extracts of G. griffithii and

A. altilis ................................................................................................... 53

4.2.1 Antimicrobial activities of methanol extract and fractions of

G. griffithii ........................................................................................ 63

4.2.2 Antimicrobial activity compounds FAC, DEC and HFC ........... 64

4.2.3 MIC compounds FAC, DEC and HFC ...................................... 65

4.3 Melting points of compound FAC, DEC and HFC .............................. 67

4.4 IR analysis of compound FAC, DEC and HFC ................................... 67

4.5 Structure elucidation of compounds FAC, DEC and HFC ................... 68

4.5.1 Compound FAC ........................................................................ 68

4.5.2 Compound DEC ....................................................................... 70

4.5.3 Compound HFC ........................................................................ 72

CHAPTER FIVE: DISCUSSION ..................................................................... 74

5.1 Percentage yield of plant extracts ........................................................ 74

5.2 Antimicrobial activities ...................................................................... 74

5.2.1. Antimicrobial activities of the crude extracts............................ 74

5.3 Antimicrobial activities of compounds FAC, DEC and HFC ............... 79

5.4 Identification of antimicrobial compounds from G. griffithii and

A. altilis .................................................................................................... 82

5.4.1 Compound FAC ........................................................................ 82

5.4.2 Compound DEC ....................................................................... 84

5.4.3 Compound HFC ........................................................................ 86

CHAPTER SIX CONCLUSION AND RECOMMANDATION ..................... 88

6.1Conclusion .......................................................................................... 88

6.2 Suggestions for future and future work ............................................... 89

BIBLIOGRAPHY .............................................................................................. 91

PUBLICATIONS ............................................................................................... 105

APPENDICES ................................................................................................... 106 1H-and

13C-NMR spectra of compound FAC ............................................ 106

COSY correlations H-H and HMBC of compound FAC .......................... 108 1H-and

13C-NMR spectra of compound DEC ............................................ 110

COSY correlations H-H and HMBC of compound DEC .......................... 112 1H-and

13C-NMR spectra of compound HFC ............................................ 114

COSY correlations H-H and HMBC of compound HFC .......................... 116

xii

LIST OF TABLES

Table No. Page No.

2.1 Antimicrobial Compounds from Plants 27

4.1 Yields of G. griffithii extracts with hexane, 52

ethyl acetate and methanol

4.2 Yields of A. altilis extracts with hexane, 53

dichloromethane and methanol

4.3 Screening of antimicrobial activity of G. griffithii extracts 56

4.4 Screening of antimicrobial activity of A. altilis extracts 57

4.5 MIC of G. griffithii extracts on bacterial and fungal strains 58

4.6 MBC/MFC of G. griffithii extracts on bacterial and fungal strains 59

4.7 MIC of A. altilis extracts on the bacterial and fungal strains 60

4.8 MBC/MFC of A. altilis extracts on bacterial and fungal strains 61

4.9 Antimicrobial activity of the methanol extract 62

4.10 Antimicrobial activity of F1 from methanol extract 63

4.11 Antimicrobial activities of isolated compounds 64

4.12 MIC of isolated compounds 66

4.13 MBC of isolated compounds 66

4.14 Melting points of compound FAC, DEC and HFC 67

4.15 IR data of compounds FAC, DEC and HFC 67

4.16 1H- and

13C-NMR chemical shift of compound FAC (Betulinic Acid) 69

4.17 1H- and

13C-NMR chemical shift of compound DEC (Stigmasterol) 71

4.18 1H- and

13C-NMR chemical shift of compound HFC 73

(Cycloartenyl acetate)

xiii

LIST OF FIGURES

Figure No. Page No.

2.1 Terpenoid compounds from Artocarpus 16

2.2 Leaf of Garcinia griffithii 19

2.3 Artocarpus altilis tree 28

4.1 Plates showing Zones of Inhibitions formed by the crude 55

extracts of G. griffithii on S. aureus, B. cereus and E. coli

4.4 Bacterial and Fungal zones of inhibition of G. griffithii extracts 61

4.3 Bacterial and Fungal zones of inhibition of A. altilis extracts 62

4.2 Graph shows the activity of isolated compounds against S. aureus 65

5.1 Structure of compound FAC (Betulinic acid) 84

5.2 Structure of compound DEC (Stigmasterol) 85

5.3 Structure of compound HFC (Cycloartenyl acetate) 87

xiv

LIST OF ABBREVIATION

UV Ultra-violet

TLC Thin Layer Chromatography

IR Infrared Spectroscopy

KBr Potassium bromide

NMR Nuclear Magnetic Resonance

VLC Vacuum Liquid Chromatography

MIC Minimum Inhibitory Concentration

MBC Minimum Bacteria Concentration

MFC Minimum Fungicidal Concentration

2D-NMR Two Dimensional Techniques NMR

1H Proton NMR

13C Carbon-13 NMR

ATCC American Type Culture Collection

DNA Deoxyribonucleic acid

SD Standard Deviation

H2SO4 Sulfuric acid

NA Nutrient agar

PDA Potato Dextrose Agar

NB Nutrient Broth

PDB Potato Dextrose Broth

MeOH Methanol

EtOAc Ethyl Acetate

xv

DCM Dichloromethane

CHCl3 Chloroform

CDCl3 Deuterated Chloroform

CC Column Chromatography

Perp TLC Preparative Thin Layer Chromatography

HIV Human immunodeficiency virus

AIDS Acquired Immune Deficiency Syndrome

SAR Structure Activity Relationship

NCI National Cancer Institute

ADME Absorption, Distribution, Metabolism, and Excretion

μl/well microliter per well

CFU/ml Colony Forming Unit per millilitre

mg/disc milligram per disc

SPSS Statistical Predictive Analytics Software

IIUM International Islamic University of Malaysia

1

CHAPTER ONE

INTRODUCTION

1.1 BACK GROUND AND JUSTIFICATION

The term natural products is closely related to herbs and medicinal plants, which can

be used as dietary supplements, traditional or alternative medicines (Holt and

Chandra, 2005). For years it has been used to treat a variety of human diseases across

the globe. Although the history of medicine/drugs could be traced back to early period

of human civilization, modern medicines have gradually developed through scientific

discoveries. Over 11,000 herbal remedies have been discovered in China for the past

5000 years, while around 8000 medicines have been formulated in India ayurvedic

remedies system. Later, during the civilization of Roman, Greece, European, Arabian

and Egypt, the use of traditional medicine became popular (Willcox et al., 2004).

Many of modern medicines, like Aspirin are derived directly or indirectly from

medicinal plants. Plants are directly used as medicines by a majority of cultures

around the world especially in Chinese and Indian medicines. Today, all traditional

medicines have their roots in folk medicines and household remedies. World Health

Organization (WHO) has listed 20,000 medicinal plants used in different parts of the

world. Other estimates indicate the number to range between 35,000 and 70,000

worldwide (Bhattarai and Karki, 2004). In terms of medicinal usage, it does not mean

that synthetic drugs are not used in western medicine but rather some of them are

derived from plants (Gurib, 2006). The global market for traditional medicines was

estimated at US$ 83 billion annually in 2008, with an exponential rate of increament

(WHO, 2011). From 1983 to 1994, ca. 39% of the new approved drugs were of natural

2

origin, including natural and synthetic products based on the natural product models

(Cragg et al., 2003). Today, there are 121 pure chemicals extracted from about 130

species of higher plants that have been used in the modern pharmaceutical drugs

foundaction throughout the world. Out of these, 89 are plant derived drugs, currently

used in modern medicine, and originally discovered through the study of traditional

cures and folk knowledge of native people (Choudhary and Atta-ur-Rahman, 2002;

Bhattarai and Karki, 2004). On the other hand, numerous studies have reported that

medicinal plants produce a large number of secondary metabolites with antimicrobial

effects on pathogens (Greathead, 2003).

Conservative estimates suggest that there are more than 250,000 species of

higher plants exist on this planet, and only a very small percentage of plants have been

exhaustively studied for their potential value as sources of drugs (Faccini, 2001).

Obviously natural products will continue to be extremely important as sources of

medicinal agents. In addition to the natural products which have found direct

medicinal application as drug entities, many others can serve as chemical models or

templates for the design, synthesis, and semi synthesis of novel substances for treating

human diseases. Although there are some new approaches to drug discovery, such as

combinatorial chemistry and computer-based molecular modelling design, none of

them can replace the important role of natural products in drug discovery and

development (Pezzuto, 1997).

1.2 ANTIMICROBIAL AGENTS FROM PLANTS

An antimicrobial agent is a chemical or biological agent that either destroys or inhibits

the growth of microorganisms (Iwu et al., 1999). Plants have been used for centuries

to treat infection of microorganisms. Since prehistoric time, spices and herbs have

3

been used not only for flavouring foods but also for antiseptic or medicinal purposes.

Their preservative effects suggest the presence of antioxidative and antimicrobial

activity (Nakatani, 2000). In a study done by Nascimento et al. (2000) in which a large

number of plant extracts were investigated for their antimicrobial properties against

various bacteria and fungi. The interest in plants as a therapeutic agent is increasing

because of the public awareness of the problem related to over prescription as well as

to the incorrect usage of antibiotics; many people now practice autonomy in terms of

medical care and botanical therapeutics (Cowan, 1999). Current trend of research is

concentrating on preparing plant extracts to produce medicinal drugs based on known

scientific criteria. Besides, these extracts overcome the harmful effects encountered

with the use of antibiotics and the resistance of microbes to the antibiotics.

This resistance encourages researchers to practice alternatives medicine

obtained from plant sources with low cost to overcome microbial infections. The

mechanisms by which plant extracts control bacterial activity take many possible

routes (Al-Zubaydi et al., 2009).

1.3 SIGNIFICANCE OF STUDY

Plants from Guttiferae (Clusiaceae) family are widely distributed of chiefly tropical

trees and shrubs and vines that produce oils and resins and some usable timber, could

be found mostly in tropics of Asia and America. Garcinia is the biggest genus in the

family Guttiferae with about 400 species widely distributed in tropical Asia, Australia,

tropical and southern Africa and Polynesia (Merza et al., 2004). Garcinia species are

known to be a rich source of oxygenated and prenylated phenol derivatives including

xanthones, flavonoids, benzophenones, lactones and phenolic acids (Ji et al., 2007).

Extracts and pure isolates of Garcinia species exhibited various biological activities

4

such as antimicrobial (Naldoni et al., 2009), anticancer (Han et al., 2008), anti-

inflammatory (Obolskiy et al., 2009), From previouse studies reported that Garcinia

spp. possessed antimicrobial acivities sucha as Garcinia cola exhibit antibacterial

against Streptococcus pyogenes, Staphylococcus aureus, Plesiomonas shigelloides and

Salmonella typhimurium (Seanego and Ndip, 2012), extract of leaves, and bark

Garcinia mangostana L. showing strong antibacterial against Listeria monocytogenes

and Staphylococcus aureus (Palakawong et al., 2010) and fruit flesh of asam kandis

Garcinia diocia possessed antimicrobial activities against Staphylococcus aureus

Bacillus subtilis, Bacillus cereus, Escherichia coli and Candida albicans (Puji et al.,

2012).

One of the two samples that have been used for this study is Garcinia griffithi,

this plant has been used traditionally by local communities to treat various diseases

including gout and also previously reported that Garcinia griffithii (stem bark)

possessed antioxidant activity (Susanti et al., 2011). But no work regarding the

antimicrobial activity has been reported on Garcinia griffithi leaves. Thus, the present

study aims to investigate antimicrobial properties of leaf of Garcinia griffithii.

Second sample that has been used in this study is Artocarpus altilis

(Breadfruit) from the mulberry family (Moraceae) is widely distributed over the

tropical regions of the Southeast Asia including Malaysia, Indonesia, Thailand and the

Pacific Islands, (Eusoso and Bamiro, 1995). Artocarpus altilis is used traditionaly in

Malaysia for instance the toasted flowers are rubbed on the gums around aching teeth

to ease pain. Latex is massaged into the skin to treat broken bones and sprains and is

bandaged on the spine to relieve sciatica. It is commonly used to treat skin ailments

and fungal diseases such as thrush. The latter is also treated with crushed leaves.

5

Diluted latex is taken internally to treat diarrhoea, stomach-ache and dysentery (Orwa

et al., 2009).

Studies of Artocarpus were performed on anti-inflammatory activity (Fang et

al., 2008; Wel et al., 2005), antibacterial activity (Khan et al., 2003), antioxidant

properties (Ko et al., 1998) and antidiabetic activity (Fernando et al., 1991). The

chemical constituents of Artocarpus species have earlier been reviewed and one of the

principal compounds reported of this plant are phenolic type compounds (Hakim et

al., 2006).

Studies of Artocarpus altilis showed that Artocarpus altilis (fruits) exhibited

antioxidant activity (Firdose et al., 2011), while the root part of Artocarpus altilis

showed antitubercular and antimalarial activity (Surat et al., 2007). But no work

regarding the antimicrobial activity has been reported on Artocarpus altilis twigs.

Thus, the present study aims to investigate antimicrobial properties of twigs of

Artocarpus altilis.

1.4 GENERAL OBJECTIVE

The aim of this study is to evaluate the antimicrobial activity of G. griffithii T. Anders

(leaves) and A. altilis (twigs) on various bacteria and fungi associated with some

human pathogens in-vitro.

1.5 SPECIFIC OBJECTIVES

1. To prepare G. griffthii’s leaves and A. altilis’s twigs extracts by using

diffirent solvents (hexane, ethyl acetate/dichloromethane and methanol).

6

2. To determine antimicrobial activities of the crude extracts on selected

pathogenic bacterial and fungal strains comprising, two Gram-positive

strains- Staphylococcus aureus (ATCC-25923) and Bacillus cereus

(ATCC-11778); two Gram-negative strains- Escherichia coli (ATCC-

35218) and Pseudomonas aeruginosa (ATCC-27853), and two fungal

strains: Candida albicans (ATCC-10231) and Cryptococcus neoformans

(ATCC-90112) notable for causing some severe human diseases.

3. To fractionate the most potent G. griffithii and A. altilis crude extract with

the intention of isolating the antimicrobial compounds.

4. To determine the chemical structures of biologically active compound(s)

isolated from G. griffithii and A. altilis.

1.6 RESEARCH HYPOTHESES

G. griffithii and A.altilis contain antimicrobial compound(s) against selected bacteria

and fungi that might be exploited in the treatment of diseases caused by pathogenic

microorganisms.

7

CHAPTER TWO

LITERATURE REVIEW

2.1 MEDICINAL PLANTS

Medicinal plants are potential resources of new drugs. It is estimated that there are

more than 250,000 flower plant species. Studying medicinal plants helps to

understand plant toxicity and protect humans and animals from natural poisons.

Cultivation and preservation of medicinal plants through metabolic engineering means

optimizing of genetic and regulatory processes within cells to increase the cells

production of a certain substance of plants to protect their biological diversity (Yang

et al., 1998). The medicinal effects of plants are due to metabolites especially

secondary compounds produced by plant species. Plant metabolites can be divided

into: primary metabolites and secondary metabolites. Primary metabolites are

intracellular compounds (nucleic acids) that play important roles in the metabolism

and/or reproduction of plant cells (Hanson, 2003). Secondary metabolites are organic

compounds that are not directly involved in the normal growth, development or

reproduction of an organism (Neog et al., 2011).

Many valuable drugs have been isolated from medicinal plants through

scientific studies which include; asprin, atropine, morphine, reserpine and quinine.

Due to the general awareness of the widespread toxicity and harmful effects

associated with the long use of synthetic drugs, extracts from natural sources are

preferred rather than the synthetic. Unfeigned effort is actually needed to explore

more plants and eventually isolate, purify and characterize the structure of their

bioactive compounds, using different modern analytical techniques. These active

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compounds could belong to any class of the organic compounds such as flavonoids,

terpenoids, steroids, alkaloids and fatty acids. Natural products provide good models

for the development of drugs. Moreover, validation of the traditional prescription by

applying modern scientific knowledge to obtain the leads for the new drugs

development gains momentum as the numbers of pure compounds with better

therapeutic activities are discovered. It is also believed that the drugs developed from

natural therapeutic agent would be relatively safer with fewer side effects. Keeping

the active compounds as the model molecules, chemical modifications are affected as

part of structure activity relationship (SAR) studies. These modifications can be

achieved by different methods such as derivatization, alteration of functional groups

with the help of computer drug aided design softwares (molecular modeling). Through

certain modifications; one can enhance the activity and lower the toxicity in the

modified molecule (analogue) as compared to the initial lead compound. A

considerable number of new therapeutically active drugs that are presently being used

have been developed through these scientific innovations (Balunas and Kinghorn,

2005).

2.2 TRADITIONAL MEDICINE

Plants which are consumed in the form of crude remedies such as tinctures, teas,

poultices, powders, and other herbal formulations have been utilized as medicines for

thousands of years. The specific plants to be used and the methods of application for

particular ailments are passed down from generation to generation. Eventually

information regarding medicinal plants was recorded in herbal pharmacopoeias

(Balunas and Kinghorn, 2005).

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Modern allopathic medicine has its roots in ancient medicine, and it is likely

that many important new remedies will be discovered and commercialized in the

future, as it has been till now, by following the leads provided by traditional

knowledge and experiences (Joanne et al., 2007). While European traditions are

particularly well known and have had a strong influence on modern western

pharmacognosy, almost all societies have well established herbal traditions, some of

which have hardly been studied at all. The study of these traditions will not only

provide an insight into how the field has developed but it is also a fascinating example

of our ability to develop a diversity of cultural practices (Balunas and Kinghorn,

2005).

2.3 DRUG DISCOVERY FROM MEDICINAL PLANTS

Numerous methods have been used to produce drugs from plants and other natural

sources, synthetic chemistry, combinatorial chemistry and molecular modelling

(Geysen et al., 2003; Lombardino and Lowe, 2004). The specific plants to be used and

the methods of application for particular ailments were passed down orally from the

ancestors. Eventually information regarding medicinal plants was recorded in herbal

pharmacopoeias (Balunas and Kinghorn, 2005). Drug discovery from medicinal plants

has envolved to include numerous fields of inquiry and various methods of analysis.

The process typically begins with a botanist, ethnobotanist, ethnopharmacologist, or

plant ecologist who collects and identifies the plant(s) of interest. Collection may

involve species with known biological activity for which active compound/s have not

been isolated (i.e traditionally used herbal remedies) or may involve taxa collected

randomly for a large screening program.