ORJI, EJIKE CELESTINE (PG/M.Sc/08/48274) … Ejike.pdfThis suggested that the mechanism of action might not be related to the inhibition of cell wall synthesis. The ethanol fraction

$Page 1: ORJI, EJIKE CELESTINE (PG/M.Sc/08/48274) … Ejike.pdfThis suggested that the mechanism of action might not be related to the inhibition of cell wall synthesis. The ethanol fraction$
PARTIAL ISOLATION OF AN ANTIBIOTIC AGAINST

STREPTOCOCCUS PYOGENES

A THESIS SUBMITTED TO THE DEPARTMENT OF

BIOLOGICAL

Webmaster

ORJI, EJIKE CELESTINE

(PG/M.Sc/08/48274)


STREPTOCOCCUS PYOGENES AND SOME BACTERIA

��

A THESIS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY

BIOLOGICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

Webmaster

Digitally Signed by Webmaster’s Name DN : CN = Webmaster’s name O= University of Nigeria, NsukkaOU = Innovation Centre

JUNE, 2011


AND SOME BACTERIA

BIOCHEMISTRY, FACULTY OF

DN : CN = Webmaster’s name O= University of Nigeria, Nsukka

ii

TITLE

PARTIAL ISOLATION OF AN ANTIBIOTIC AGAINST STREPTOCOCCUS PYOGENES

AND SOME BACTERIA FROM ZAPOTECA PORTORICENSIS.

A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (M.Sc) IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY, UNIVERSITY OF NIGERIA,

NSUKKA

BY

ORJI, EJIKE CELESTINE

(PG/M.Sc/08/48274)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: DR. H. A. ONWUBIKO

iii

JUNE, 2011

CERTIFICATION

ORJI, EJIKE CELESTINE, a postgraduate student of the Department of Biochemistry with the Reg.

No. PG/M.Sc/08/48274 has satisfactorily completed the requirement of research work, for the degree

of Master of Science (M.Sc) in Biochemistry and Molecular Biology. The work embodied in this

project (dissertation) is original and has not been submitted in part or full for any other diploma or

degree of this or any other university.

DR. H. A. ONWUBIKO PROF. L.U. S. EZEANYIKA

(Supervisor) (Head of Department)

EXTERNAL EXAMINER

iv

DEDICATION

To:

God Almighty, the source and sink of life, the repertoire and fountain of knowledge, and to all

who will find in this work a source of knowledge.

v

ACKNOWLEDGEMENT

I owe unalloyed gratitude and appreciation to my dynamic and ebullient supervisor, Dr H. A.

Onwubiko for his goodwill, assistance and contribution to this work. I am very grateful to the Head

of Department, Prof. L. U. S. Ezeanyika and the entire staff of the Biochemistry Department. I also

wish to thank Dr (Mrs) Onwubiko for her input and support throughout the period of this work.

I am most indebted to my parents: Chief Gabriel Okonkwo Orji and Mrs Theresa Orji, whose

love for academics have been a pillar to my academic furtherance. Their immense contributions

(financially and otherwise) to my academic pursuit, by the grace of God, have given rise to the

successful completion of this programme. May the Almighty God reward them immensely.

Finally, my warm regards go to my coursemates and all postgraduate students of the

Department. Their outstanding spirit of co-operation and teamwork is worthy of note.

vi

ABSTRACT

This research is aimed at isolating a new, potent antibiotic against Streptococcus pyogenes and other

bacteria from the root of the plant, Zapoteca portoricensis. It has been proven that the crude

methanol extract of Zapoteca portoricensis is very active on Staphylococcus aureus, Candida

albicans and E. coli, but no attempt has been made towards testing its activity on Streptococcus

pyogenes; furthermore isolation and characterization of the active compound from the plant root is

still to be demonstrated. Crude ethanolic extract of the plant root was active on both gram-negative

and gram-positive bacteria. Serial solvent extraction of the crude extract was done using chloroform,

ethyl acetate, acetone, and ethanol, but only the ethanol fraction of the crude extract was active on

both gram-negative and gram-positive bacteria. This suggested that the mechanism of action might

not be related to the inhibition of cell wall synthesis. The ethanol fraction was active on

Streptococcus pyogenes, Streptococcus pneumonia, E. coli, Salmonella typhi, Klebsiella pneumonia

and Bacillus subtilis. The MICs of the crude extract are 25mg/ml, 25mg/ml, 6.25mg/ml, 50mg/ml,

25mg/ml 6.25mg/ml for Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia,

E. coli, Klebsiella pneumonia and Salmonella typhii respectively. The UV-spectroscopy, Infrared

spectroscopy and NMR-spectroscopy of ethanol fraction were carried out. Both IR- spectroscopy and

the NMR-spectroscopy were used to determine the functional group present in the ethanol fraction.

The following groups were found to be present under the application IR-spectroscopy and NMR-

spectroscopy result: aromatic, sulfuryl, halogen, carboxyl and amide groups. Some double and triple

bonded carbons were also evident.

vii

TABLE OF CONTENTS

PAGE

Title Page ... .. .. .. .. .. .. .. .. .. i

Certification ... .. .. .. .. .. .. .. .. .. ii

Dedication ... .. .. .. .. .. .. .. .. .. iii

Acknowledgements . .. .. .. .. .. .. .. .. iv

Abstract ... .. .. .. .. .. .. .. .. .. v

Table of Contents ... .. .. .. .. .. .. .. .. vi

List of Figures ... .. .. .. .. .. .. .. .. .. xi

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

List of Abbreviations ... .. .. .. .. .. .. .. .. xiii

CHAPTER ONE: INTRODUCTION

1.0 Introduction ... …. ... ... ... ... ... ... 1

1.1 Antibiotics … … … … … … … … … 2

1.1.1.0 Mechanisms of actions of clinically used antimicrobial drugs... …. ... …. 3

1.1.1.1 Selective toxicity … … … … … ….. …. ….. …. 3

1.1.2 Bacterial cell wall … … … … … ….. …. …. 5

1.1.2.1 Antibiotics with beta-lactam ring …. … … …. ….. …. .. 7

1.3.2.0 Reactions and side-effects of antibiotics with beta-lactam ring …. … 8

1.3.2.1 Penicillins … … … … … … … ….. ….. 8

1.4.3 Cephalosporin … … … … … … … …. … 11

1.4.4 Carbapenems … … … …. …. …. … … … 15

1.4.4.1 Imipenem …. … … … … … … …. … 16

1.4.4.2 Meropenem … … … …. ….. …. …. …. … 16

1.4.4.3 Aztreonam … …. …. …. …. … …. … … 17

1.4.5.0 Sulphanilamides and trimethoprim-sulfamethoxazole (co-trimoxazole) …. 17

1.4.6 Quinolones …. …. …. …. …. …. …. …. … 19

1.4.7 Aminoglycosides …. …. …. ….. ….. …. ..... . 27

1.4.8 Tetracyclines ….. …. ….. ….. …… …… …… ….. …. 28

1.4.9 Chloramphenicol …. …… …… …… …… ….. … … 30

viii

1.4.10 Macrolides ….. ….. ….. …. …… …… …… …… …. 32

1.4.11 Clindamycin …. …. …. …. … …. ….. ….. …. 33

1.4.12 Vancomycin …. …. …. …. …. …. …. …. … 35

1.4.13 Polymyxin-B ….. …. …. ….. ….. ….. ….. ….. …. 36

1.5 Plant products as antimicrobial agents … …. …. …. … 36

1.5.1 Major groups of antimicrobial compounds from plants …. … …. 37

1.3.1 Phenolics and polyphenols …. …. ….. ….. ….. ….. … 43

1.3.2 Quinones ….. ….. ….. …. …. …. …. …. …. 43

1.3.3 Alkaloids ….. ….. …. …. …. …. ….. …. … 44

1.5.0 The plant ….. ….. …. …. …. …. …. …. … 45

1.5.1 Zapoteca portoricensis …. …. …. …. …. …. …. 45

1.6.0 Streptococcus pyogenes …. …. …. …. …. ….. …. 47

1.6.2 Classification of streptococci …… ….. …. …. …. …. …. 48

1.6.3 Post streptococcal sequelae ….. ….. ….. …. …. …. …. 55

1.6.4 Host defences …. …. …. …. ….. ….. …. …. 56

1.6.5 Adherence (colonization) surface macromolecules …. … … … 58

1.6.6 Production of toxins and other systemic effects ….. …. …. .. 58

1.6.7 Suppurative conditions ….. ….. …. ….. ….. … …. 58

1.6.8 Non-suppurative sequelae …… ….. ….. ….. ….. ….. …. 58

1.6.9 Treatment and prevention ….. ….. ….. …… …… …… ….. 59

1.7.0 Mechanisms of antibiotic resistance in bacteria …. …. … … 59

1.8 Research objectives … … … … … …. ... …. 62

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials … … … … … … … … 64

2.1.1 Plant materials … … … … … … 64

2.1.3 Instruments/Equipment … … … … … … 64

2.2 Methods …… …….. … … … … … 65

2.2.1 Extraction procedure … ... ... ... ... ... 65

2.2.2 Determination of extract yield … … … … … 65

2.2.3 Fractionation of the crude extract …. … … … …. 66

2.2.4.0 Preliminary phytochemical analysis … … … … 66

2.2.4.1 Preparation of Reagents for Phytochemical and Macronutrient Analyses 66

ix

2.4.1 Test for alkaloids … … … … … … 67

2.4.2 Test for glycosides … … … … … … 68

2.4.4 Test for flavonoids … … … … … … 68

2.4.5 Test for resins … … … … … … 69

2.4.6 Test for tannins … … … … … … … 69

2.4.7 Test for saponins … … … … … … … 69

2.4.8 Test for terpenoids and steroids … … … … … 70

2.4.9 Test for acidic compounds … … … … … 70

2.4.10 Test for proteins … … … … … … ... 70

2.4.10.2 Test for carbohydrates … … … … … 70

2.4.10.3 Test for reducing sugar … … … … … … 70

2.4.10.4 Test for fats and oils … … … … … … 71

2.5.0 Test microorganisms … … … … … … 71

2.5.1 Preparation of culture media … … ….. …… ….. …. 71

2.5.2 Determination of antimicrobial activity … … … … 71

2.5.3 Agar well diffusion method: Agar-well diffusion … … … 72

2.6 Determination of the functional groups present in the ethanol fraction

using IR and NMR spectrostroscopy ....... ...... ..... .... ..... 72

CHAPTER THREE: RESULTS 73

3.1 Extraction and fractionation ….. …. …. … … 73

3.2 The classes of phytochemical compounds and macronutrients … 73

3.2 Results of the antimicrobial activities of the crude ethanol extract … 75

3.3 Results of the Minimum Inhibitory Concentration (MIC) … … 76

3.4 Antimicrobial activities of the fractions … … … … 77

CHAPTER FOUR: DISCUSSION

4.1 Discussion … … … … … … … … 85

4.2 Conclusion …. …. … … … … .. … 88

REFERENCES … … … … … … … … 89

x

LIST OF FIGURES

PAGE

Fig. 1 Effects of bacteriostatic versus bactericidal antibiotics on logarithmically

growing bacterial culture … … … … ….. 3

Fig. 2 A proposed common mechanism of killing by bactericidal antibiotics… 5

Fig. 3 Comparison of the structure of cell walls … … … … 6

Fig. 4 Structure of bacterial cell wall …. …. … …. … … … 7

Fig. 5 Structure of beta-lactam of penicillin G …. ….. … … 7

Fig. 6 The structure of penicillins and their enzymatic products … …. 10

Fig. 7 The structure of cephalosporins. The different R groups allow for versatility

and improved effectiveness … … …. …. … … 11

Fig. 8 Schematic structures of first generation of cephalosporins … …. 13

Fig.9 Schematic structures of second generation of cephalosporins … 14

Fig. 10 Schematic structures of third generation of cephalosporins …. …. 15

Fig. 11 A schematic structure of metropenem a monobactams … … 17

Fig. 12 Folic Acid Analogues … … …. … … … … 18

Fig. 13 Scheme for the mechanism of action of sulphanilamide and trimethoprim 19

Fig. 14 The structure of chloramphenicol …. …. …. …. …. 31

Fig. 15 The structure of vancomycin … … … … … … 35

Fig. 16 Scheme that shows the mechanism of action of vancomycin …. 35

Fig. 17 Schematic structure of Bacitracin …. ….. ….. … …. 36

Fig. 18 Structures of common antimicrobial plant chemicals …. …. … 44

Fig 19 Reaction for the conversion of tyrosine to quinine … …. …. 45

Fig. 20 Zapoteca portoricensis … … … … … … 48

Fig. 21 Streptococcus pyogenes … … … … … … 48

Fig. 22 Cell surface structure of Streptococcus pyogenes and secreted products

involved in virulence …… …… ….. ….. ….. …. ….. 52

Fig. 23 Pathogenesis of Streptococcus pyogenes infections …. …. …. 56

Fig. 24 Phagocytosis of Streptococcus pyogenes by a macrophage …. … 57

Fig. 25 Mechanisms of antibiotic resistance in bacteria … … … 60

Fig. 26 Mechanisms of horizontal gene transfer (HGT) in bacteria …. 63

xi

Fig. 27 Minimum Inhibition Diameter of the crude ethanol extract on the

microorganisms used … …. … …. … … …. 75

Fig. 28 Inhibition Zone Diameter of chloroform fraction on the microorganisms used. . …. …. …. …. …. …. …. …. … 76

Fig. 29 Inhibition Zone Diameter of ethyl acetate fraction on the microorganisms

Used …… …… …… …… …… ….. ….. …… ….. 77

Fig. 30 Inhibition Zone Diameter of acetone fraction on the microorganisms used . 77

Fig. 31 Inhibition Zone Diameter of ethanol fraction on the microorganisms used 78

Fig. 32 Inhibition Zone Diameter of streptomycin (10µg/ml) fraction on the

microorganisms used. …. …. …. …. …. …. … 78

Fig. 33 Growth inhibition by ethanol fraction,..... ....... …. ….. …. 79

Fig.3. 12 NMR spectra for h1 at 199.9671 MHz……….. … … … 81

fig.3. 13 Infrared Spectra the ethanol fraction .……. ….. …. …. 82

Fig.3. 13 13C NMR spectra of ethanol fraction of the plant roots extract …. 83

Fig.3.14 13C NMR spectra of ethanol fraction of the plant roots extract … 84

xii

LIST OF TABLES

Table 1.1 Plants containing antimicrobial activity … … … ……. . 38

Table 1.2 Mechanisms of antibiotic resistance in bacteria … …. … 60

Table 3.1 Phytochemical screening of secondary metabolites present in the crude

extract of the plant roots. …… ….. …. …. …. … 74

Table 3.2 Antimicrobial activities of different concentrations of the crude ethanol

extract …… … …. …. … … .. …. 73

Table 3.3 Minimum Inhibitory Concentration (MIC) of the crude ethanol extract… 76

Table 3.4 Inhibitory Zone Diameter (IZD) for the fractions …. …. … 77

xiii

LIST OF ABBREVIATIONS

-OH Hydroxyl group

AIDS Acquired Immune Deficiency Syndrome

HIV Human Immunodeficiency Virus

DNA Deoxyribonucleic acid

NAM N-acetylmuramic acid.

NAG N-acetylglucosamine

MRSA Methicillin resistant S. aureus

CSF Cerebrospinal fluid

PABA para-aminobenzoic acid

OH. Hydroxyl radical

G6PD Glucose 6-phosphate dehydrogenase

SIADH Syndrome of inappropriate anti-diuretic hormone

GIT Gastro intestinal tract

LTA Lipoteichoic acids

SPE Streptococcal pyrogenic exotoxins

AMI Antibody mediated immunity

CMI Cell-mediated immunity

HGT Horizontal gene transfer

DMSO Dimethyl sulphur oxide

NMR Nuclear magnetic resonance

UV Ultra-violet spectrophotometer

IR Infrared spectrophotometer

xiv

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.0 INTRODUCTION

There has been alarming reports of multiple drug resistance in medically important strains of bacteria

and fungi (Ozumba 2003, Aibinu at al., 2004). The persistent increase in antibiotic resistant strains of

organisms has led to the development of more potent synthetic antibiotics. These new antibiotics are

scarce, costly and so not affordable particularly in developing countries. This therefore, makes

compliance to these antibiotics difficult. There is the need for continuous search for new, effective

and affordable antimicrobial agents. Local medicinal plants provide a source of new possible

antibiotics that may be potent for some of these bacteria.

Among the streptococci/enterococci, resistance to wide variety of antibiotics has emerged, with some

strains resistant to all available antibiotics. There is no clinically proven treatment effective against

streptococci/enterococci, multiple resistances to lactams, aminoglycosides and vancomycin. The

emergence of these organisms poses a stunning management dilemma. Further research into the

mechanism of resistance and new class(es) of antibiotic is very essential and is the focus of this

work.

Antibiotics are different organic compounds that are formed and secreted by plants and various

species of microorganisms, which are toxic to other microorganisms. Most plants naturally

synthesize these carbon compounds, basically for physiological functions or for use as chemical

weapons against disease-causing organisms, insects and predators (Fatope, 1995). It is estimated that

there are 250,000 to 500,000 species of plant on earth (Borris, 1996). Relatively small percentages (1

to 10%) of these are used as food by humans and animal species. It is possible that even more are

used for medicinal purposes (Moerman, 1996). Plants have an almost limitless ability to synthesize

aromatic substances, most of which are phenols or their oxygen-substituted derivatives (Geissan,

1963). Most are secondary metabolites of plants, of which at least 12,000 have been isolated, a

number estimated to be less than 10% of the total (Schulte, 1978). The investigation of these plant

bioactive secondary metabolites is a viable area of research, which aims at discovering new clinical

use and commercial importance of plant products. Most of these plants have been used by traditional

herbalists to cure many diseases, but these herbalists use these plant materials crude, or at most a

crude extract of methanol, ethanol and water. These plants in addition to these useful compounds

contain poisons, which are dangerous to health. Therefore, it is important to conduct the

xv

phytochemical screening of these plants and isolate their active principles. It is probable that a large

number of plants with biological activities remain untested.

One of these very important plants is Zapoteca portoricensis. This plant belongs to the family of

Fabaceae/mimosidea, and genus Zapoteca. Literature search indicates that this plant has been used

traditionally to treat diseases such as tonsillitis (sore throat), fever, convulsion, breast engorgement,

stomach disorder, purgative and amenorrhoea. Some phytochemical constituents of this plant have

been reported to have medicinal or antimicrobial activity. However, there is no report of the

antimicrobial activity of the plant in relation to Streptococcus pyogenes (the organism that causes

tonsillitis). This organism has been reported to have resistance to penicillin, which is the common

antibiotic used against it. Therefore, this study aims at isolating, identifying and the possibly produce

a new antibiotics from Zapoteca portoricensis against Streptococcus pyogenes and other resistance

bacteria. This would, help to manage antibiotic resistance among Streptococci/enterococci.

1.1 ANTBIOTICS

The word antibiotic came from the Greek word anti, meaning 'against,' and bios, meaning 'life' (a

bacterium is a life form). Antibiotics are known as antibacterial agents, agents or drugs used to treat

infections caused by bacteria (Annon, 2009). Modern medicine is dependent on chemotherapeutic

agents-chemical agents that are used to treat disease. Chemotherapeutic agents destroy pathogenic

microorganisms or inhibit their growth at concentrations low enough to avoid undesirable damage to

the host. Most of these agents are antibiotics (Prescott et al., 2002). Antibiotics are microbial

products or their derivatives that can kill susceptible microorganisms or inhibit their growth.

Antibiotics can as well be defined as molecules that kill, or stop the growth of microorganisms,

including both bacteria and fungi. Antibiotics that kill bacteria are called "bactericidal" while

antibiotics that stop the growth of bacteria are called "bacteriostatic", as shown in Figure 1.1. These

substances can be semi-synthetic, wherein a molecular version produced by the microbe is

subsequently modified by chemists to achieve desired properties. Furthermore, some antimicrobial

compounds, originally discovered as products of microorganisms, can be synthesized entirely by

chemical means (Todar, 2009).

The modern era of antimicrobial chemotherapy began in 1929, with Fleming’s discovery of the

powerful bactericidal substance, penicillin, and Domagk’s discovery in 1935 of a synthetic chemical

(sulphanilamides) with broad antimicrobial activity. In the early 1940s, spurred partially by the need

for antibacterial agents in World War II, penicillin was isolated, purified and injected into

experimental animals, where it was found not only to cure infections but also to possess incredibly

low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy, and an

intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in

the treatment of infectious diseases. The rapid isolation of streptomycin, chloramphenicol and

tetracycline antibiotics was clinically useful (Todar, 2008).

1.1.1.0 MECHANISMS OF ACTION OF CLINICALLY USED ANTIMICROBIAL DRUGS

1.1.1.1 SELECTIVE TOXICITY

An ideal antimicrobial agent exhibits selective toxicity. This term implies that a drug is harmful to a

pathogen without being harmful to the host. Often, selective toxicity is relative rather than absolute;

this implies that a drug in a concentration tolerated by the host may damage an infecting

microorganism (Brook’s et al., 2002).

Selective toxicity may be a function of a specific receptor required for drug attachment, or may

depend on the inhibition of biochemical events essential to the organism but not the host. These

mechanisms of action can be placed under six headings:

xvii

1. Agents that inhibit synthesis of bacterial cell wall; these include the penicillins and

cephalosporins, which may be structurally similar or dissimilar agents such as cycloserine,

vancomycin, bacitracin, and the azole antifungal agents (e.g. clotrimazole, fluconazole).

2. Agents that act directly on the cell membrane of the microorganism, affecting permeability

and leading to leakage of intracellular compounds; these include the detergents such as

polymyxin and the polyene antifungal agents nystatin and amphotericin B, which bind to cell-

wall sterols.

3. Agents that affect the function of 30S or 50S ribosomal subunits to cause reversible inhibition

of protein synthesis; these bacteriostatic drugs include chloramphenicol; the tetracyclines,

erythromycin and pristinamycins.

4. Agents that bind to the 30S ribosomal subunit and alter protein synthesis, which eventually

leads to cell death; these include the aminoglycosides.

5. Agents that affect bacterial nucleic acid metabolism, such as the quinolones, which inhibit

topoisomerases.

6. The antimetabolites, including trimethoprim and the sulphonamides.

1.1.2. BACTERIAL CELL WALL

Bacteria possess a rigid outer layer, the cell wall. It maintains the shape of the microorganism and

‘corsets’ the bacterial cell, which has high internal osmotic pressure. The internal pressure is three to

five times greater in Gram-positive than in Gram-negative bacteria, as shown in Figure 1.3. Injury to

cell wall (e.g. by lysozyme) or inhibition of its formation may lead to lysis of the cell. In a hypertonic

environment (e.g., 20% sucrose), damaged cell wall formation leads to formation of spherical

bacteria ‘protoplasts’ from gram-positive organisms or ‘spheroplasts’ from gram-negative organisms;

these forms are limited by the fragile cytoplasmic membrane. If such protoplasts or spheroplasts are

placed in an environment of ordinary tonicity, they take up fluid rapidly, swell and explode.

1.3.2.0ANTIMICROBIAL ACTION THROUGH INHIBITION OF CELL WALL

SYNTHESIS

The cell wall contains a chemically distinct complex polymer ‘mucopeptide’ (‘peptidoglycan’)

consisting of polysaccharides and highly cross-linked polypeptides. The polysaccharides regularly

contain the amino sugars N-acetylglucosamine and acetylmuramic acid. The latter is found only in

bacteria. To the amino sugars are attached polypeptide chains. The final rigidity of the cell wall is

imparted by cross-linking of peptide chains (e.g., through pentaglycine bonds) as in transpeptidation

reactions carried out by several enzymes. The peptidoglycan layer is much thicker in the wall of

gram-positive than of gram-negative bacteria.

Figure 2: A proposed common mechanism of killing by bacteria antibiotics. Antibiotics

with diverse targets (ribosome for aminoglycosides, DNA gyrase for quinolone and

penicillin-binding proteins for Beta-lactam) trigger NADH depletion and superoxide (O2-)

formation by hyperactivation of the electron transport chain. Free-radical damage of iron-

sulphur clusters releases ferrous ion, inducing the generation of highly destructive hydroxyl

radicals (OH-) and cell death. (Collins, J. 2007).

xix

The walls of bacteria are made of a complex polymeric material called peptidoglycan, as shown in

the diagram above. It contains both amino acids and amino sugars. The amino sugars are of two

kinds: N-acetylglucosamine (NAG) its close relative and N-acetylmuramic acid (NAM).

1.1.2.1 ANTIBIOTICS WITH BETA-LACTAM RING

The beta-lactams get their name from the characteristic ring structure. The arrow in figure 1.5 shows

the bond that is broken by the beta-lactamases, the enzymes that are synthesized by many penicillin-

resistant bacteria. There are antibiotics with the beta-lactam, ring namely, penicllins, cephalosporins

and carbapenems. Under penicillins there are penicillin G, (a natural product) produced by the

fungus Penicillium chrysogenum, ampicillin, (a semi-synthetic compound), amoxicillin, (a semi-

synthetic compound). Cephalosporins have over two dozen types in current use. Most are semi-

synthetic compounds derived from the secretion of the mould Cephalosporium. Some examples of

cephalosporins are: cephalexin (e.g., keflex®), cefaclor (e.g., ceclor®), cefixime (e.g., suprax®).

Under carbapenems we have: meropenem (merrem), ertapenem (invanz).

The beta-lactams all work by interfering with the synthesis of the bacterial cell wall — a structure

that is not found in eukaryotes. The beta-lactam antibiotics bind to and inhibit enzymes needed for

the synthesis of the peptidoglycan wall. While they have little effect on resting bacteria, they are

lethal to dividing bacteria as defective walls cannot protect the organism from bursting in hypotonic

surroundings.

1.3.2.0 REACTIONS AND SIDE-EFFECTS OF ANTIBIOTICS WITH BETA-LACTAM

RING

1.3.2.1 PENICILLINS

In 1928 Alexander Fleming observed that a culture plate on which Staphylococci were being grown

had become contaminated with a mould of the genus Penicillium, and that bacterial growth in the

vicinity of the mould had been inhibited. He isolated the mould in pure culture and demonstrated that

it produced an antibacterial substance, which he called penicillin. This substance was extracted by

Florey and Chain in 1940 from Penicillium notatum and they showed that it had powerful

Figure 5: Structure of beta-lactam of Penicillin G

Figure 4 The structure of bacterial cell wall

xxi

chemotherapeutic properties in infected mice and that it was non-toxic. Its remarkable antibacterial

effects in man were clearly demonstrated in 1941 in a policeman who had Staphylococcal and

Streptococcal septicemia with multiple abscesses. Ten years later virtually unlimited quantities of

penicillin G were available for clinical use. After the isolation of the nucleus, 6-aminopenicillanic

acid, numerous semi-synthetic penicillins were developed that are stable at acid pH, resistant to �-

lactamase, and active against both Gram-positive and Gram negative bacteria. The penicillins are

classified as �-lactam drugs because of their unique four-member �-lactam ring. Structural integrity

of the 6-aminopenicillanic acid nucleus is essential for the antibacterial activity.

Penicillins are hypersensitive and can cause anaphylaxis. They are the most common cause of drug

allergy. All types, dosages and forms of administration of penicillins can potentially cause allergic

reactions. Cross-reactions among the penicillins and between them and the cephalosporins are not

uncommon. Local reactions to intramuscular injection may be seen. Oral administration may cause

nausea and diarrhoea. Toxic effects are minimal.

Penicillin G and V are effective against Gram-positive cocci (Streptococcus, Neisseria meningitides,

but not Enterococcus), Trepenoma pallidum (syphilis), Borrelia burgdorferi (Lyme disease), some

anaerobes (Clostridium, Corynebacterium diphtheriae, Actinomyces, Bacillus anthracis, Listeria),

but they are not effective against Bacteroides fragilis. They are susceptible to penicillinase and so not

effective against Staphylococcus aureus, since it synthezises the enzyme. It is given orally (penicillin

V is less vulnerable to gastric acid) or parenterally. Adding procaine or benzathine prolongs the

effect of penicillin. Penicillin is best absorbed in an empty stomach. It is used to treat infections by

the above named bacteria. It is also used as prophylaxis to prevent recurrent rheumatic fever, and in

contact of patients with syphilis, gonorrhoea and streptococcal infections. It is used as prophylaxis

against infective endocarditis for susceptible people before dental or surgical procedures. Interactions

with probenecid causes increased concentration in Jarisch-Herxheimer reaction seen in the majority

of patients with secondary syphilis on receiving the first dose of penicillin. Flu-like symptoms –

chills, fever, muscle aches, headache, joint pain – with enhanced colour of the lesions of syphilis, last

for up to 48 hours and do not recur with later doses. The may be due to antigens released by the

breakdown of spirochetes, this may not be a reason to stop penicillin treatment; aspirin will help to

control the symptoms.

Clinically, penicillin G has the following limitations: it is unstable at acidic pH, it is susceptible to

destruction by �-lactamase (penicillinase) and it is relatively inactive against Gram-negative bacilli.

The diagram below shows that the site of action of the enzyme is the �-lactam ring of penicillin.

Some penicillin are resistant to penicillinase; these are methicillin, nafcillin, oxacillin, cloxacillin,

and dicloxacillin. They are effective against Gram-positive cocci (less so than penicillin V or G),

including Staphylococcus aureus. Methicillin resistant S. aureus (MRSA) is treated by vancomycin.

It is given orally (best absorption is on an empty stomach) and parenterally. Methicillin may cause

interstitial nephritis.

Ampicillins, which are a type of penicillin, have the following examples, amoxicillin and

bacampicillin. They are effective also against some Gram-negative organisms – Hemophilus (E. coli),

Proteus, also Salmonella and Listeria. They are susceptible to �-lactamase – and so not effective for

Staphylococcus infections. They are given orally (best absorption is on an empty stomach) and

parenterally. Ampicillin causes rashes in patients with infectious mononucleosis. Allopurinol will

also increase chances of rashes with ampicillin. Ampicillin and clavulanic acid inactivate �-lactamase

and so prevent breakdown of the antibiotic. Both compounds are effective against Staphylococcus

organisms.

Anti-Pseudomonas penicillins are also effective against Pseudomonas, Enterobacter and Proteus, but

they are susceptible to �-lactamase. The commonest examples are carbenicillin and ticarcillin.

Carbenicillin increases risk of heart failure, due to its ability to cause hypernatraemia and

hypokalaemia. It also promotes the aggregation of platelets. Ticarcillin has fewer side effects and so

is preferred; it is given orally and parenterally. A preparation of ticarcillin and clavulanic acid (for

parenteral use) is more effective against Bacteroides, S. aureus and Gram-negative bacilli. The

Figure 6: The structure of penicillins and its enzymatic products (Suzer, 2008)

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broad-spectrum penicillins are effective against Klebsiella and some other Gram-negative organisms.

The examples are mezlocillin and piperacillin. They are susceptible to beta-lactamase and are given

only parenterally.

1.4.3.0 CEPHALOSPORIN

Cephalosporins are the most frequently prescribed class of antibiotics. They are structurally and

pharmacologically related to the penicillins. Like the penicillins, cephalosporins have a beta-lactam

ring structure that interferes with synthesis of the bacterial cell wall and so are bactericidal (which

means that they kill bacteria). Cephalosporin compounds were first isolated from cultures of

Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist Giuseppe Brotzu.

The first agent cephalothin (cefalotin) was launched by Eli Lilly in 1964. Cephalosporins are derived

from cephalosporin C which is an acid-stable molecule with antibacterial activity and is produced

from Cephalosporium acremonium.

1.4.3.1 Mode of action

Cephalosporins are bactericidal agents and have the same mode of action as other beta-lactam

antibiotics (such as penicillins). All bacterial cells have a cell wall that protects them. Cephalosporins

disrupt the synthesis of the peptidoglycan layer of the bacterial cell walls, and cause the walls to

break down and eventually the bacteria to die.

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Figure 7: The structure of cephalosporins. The different R groups allow for versatility and improved

effectiveness.

1.4.3.2 Side Effects

Cephalosporins generally cause few side effects. Common side effects involve mainly the digestive

system and they include mild stomach cramps or upset, nausea, vomiting, and diarrhoea. These side

effects are usually mild and go away over time. Cephalosporins can sometimes cause overgrowth of

fungus normally present in the body. This overgrowth can cause mild side effects such as a sore

tongue, sores inside the mouth, or vaginal yeast infections.

More serious but infrequent reactions that can sometimes occur with cephalosporins include black

tarry stools, chest pain, fever, painful or difficult urination, allergic reactions and serious colitis.

Serious colitis is a rare side effect that includes severe watery diarrhoea (sometimes containing blood

or mucus), severe stomach cramps, fever, and weakness or faintness. Because the cephalosporins are

structurally similar to the penicillins, some patients allergic to penicillins may be allergic to a

cephalosporin antibiotic. The incidence of cross-sensitivity is approximately 5–10% (Bayarski,

2010).

1.4.3.3 Indications

Cephalosporins are indicated for the treatment of bacterial infections caused by susceptible

organisms. First generation cephalosporins are predominantly active against Gram-positive bacteria,

and successive generations have increased activity against Gram-negative bacteria (often with

reduced activity against Gram-positive organisms).

Bacteria are classified in several ways. One of the way is by their colour after a particular chemical

stain (Gram stain) is applied. Some bacteria stain blue and are called Gram-positive, others stain pink

and are called Gram-negative. Gram-negative bacteria have a unique outer membrane that prevents

many drugs from penetrating them, making Gram-negative bacteria generally more resistant to

antibiotics than are Gram-positive bacteria. Gram-negative bacteria are able to become resistant to

antibiotics, Gram-positive bacteria are usually slow to develop such resistance.

Cephalosporins are used to treat a wide variety of bacterial infections, such as respiratory tract

infections (pneumonia, strep throat, tonsillitis, bronchitis), skin infections and urinary tract infections.

They are sometimes given with other antibiotics. Cephalosporins are also commonly used for

surgical prophylaxis - prevention of bacterial infection before, during, and after surgery.

1.4.3.4.0 Classification of Cephalosporins

Cephalosporins are grouped into "generations" based on their spectrum of antimicrobial activity. The

first cephalosporins were designated first generation while later, more extended spectrum

cephalosporins were classified as second generation cephalosporins. Each newer generation of

cephalosporins has significantly greater Gram-negative antimicrobial properties than the preceding

generation, in most cases with decreased activity against Gram-positive organisms. Fourth generation

cephalosporins, however, have true broad spectrum activity. The newer agents have much longer

half-lives resulting in the decrease of dosing frequency.

1.4.3.4.1 First generation

First generation cephalosporins are moderate spectrum agents. They are effective alternatives for

treating staphylococcal and streptococcal infections and therefore are alternatives for skin and soft-

tissue infections, as well as for streptococcal pharyngitis. The first generation cephalosporins are:

Cefadroxil, Cephalexin, Cephaloridine, Cephalothin, Cephapirin, Cefazolin and Cephradine.

Cefazolin is the most commonly used first generation cephalosporin. The other first generation

cephalosporins have similar efficacy to Cephalexin, but must be dosed more often, and are therefore

not as commonly prescribed.

First generation cephalosporins R1 R2

Figure 8: Schematic structures of first generation of cephalosporins (Suzer, 2008)

1.4.3.4.1 Second generation

The second generation cephalosporins have a greater gram-negative spectrum while retaining some

activity against gram-positive bacteria. They are also more resistant to beta-lactamase. They are

useful agents for treating upper and lower respiratory tract infections, sinusitis and otitis media.

These agents are also active against E. coli, Klebsiella and Proteus, which makes them potential

alternatives for treating urinary tract infections caused by these organisms. Cefoxitin is a second

generation cephalosporin with anaerobic activity, and although seldom used as a therapeutic agent, it

may be useful for prophylaxis in gastrointestinal surgery. The second generation cephalosporins are:

Cefaclor, Cefoxitin, Cefprozil and Cefuroxime.

1.4.3.4.2 Third generation

Third generation cephalosporins have a broad spectrum of activity and further increased activity

against gram-negative organisms. Some members of this group (particularly those available in an oral

formulation) have decreased activity against gram-positive organisms. The parenteral third

generation cephalosporins (ceftriaxone and cefotaxime) have excellent activity against most strains of

Streptococcus pneumoniae, including the vast majority of those with intermediate and high level

resistance to penicillin. These agents also have activity against N. gonorrhoeae. Ceftazidime has

Figure 9: Schematic structures of second generation of cephalosporins (Suzer, 2008).

useful antipseudomonal activity. The third generation cephalosporins are: Cefdinir, Cefixime,

Cefpodoxime, Ceftibuten, Ceftriaxone and Cefotaxime.

1.4.3.4.3 Fourth generation

Fourth generation cephalosporins are extended spectrum agents with similar activity against Gram-

positive organisms as first generation cephalosporins. They also have a greater resistance to beta-

lactamases than the third generation cephalosporins. Many can cross blood brain barrier and are

effective in meningitis.

Cefepime is more active against Gram-negative with somewhat enhanced activity against

Pseudomonas but slightly lesser activity against pneumococci. Cefpirome is more active against

pneumococci and has somewhat lesser activity against pseudomonas. These drugs also have activity

against nosocomial pathogens such as Enterobacter and Acinetobacter and their use should therefore

be restricted to the setting of nosocomial sepsis. The fourth generation cephalosporins are: cefepime,

cefluprenam, cefozopran, cefpirome and cefquinome.

1.4.4 CARBAPENEMS

Figure 10: Schematic structures of third generation of cephalosporins (Suzer, 2008).

xxviii

The carbapenem class of antibiotics was discovered in the late 1970s following research by two

separate groups. At Beecham Pharmaceuticals, Brown et. al., (1976) found that the olivanic acids,

produced by Streptomyces olivaceus, could behave as inhibitors of bacterial �-lactamases. At the

same time, research carried out by Merck showed that the potent antibiotic, thienamycin isolated

from Streptomyces cattleya also contained the carbapenem ring structure (Albers-Schonberg et. al.,

1978). Not only was thienamycin shown to be broad spectrum in its activity, but like the olivanic

acids, it is also stable in the presence of �-lactamases. In order to increase chemical stability at high

concentrations and thereby enable its clinical use, various derivatives of the amino group of

thienamycin have investigated. The N-formimidoyl derivative, or imipenem retains the broad

spectrum of activity of the natural product, although it is usually given in combination with an

inhibitor of renal dihydropeptidase, cilastatin to prevent its metabolism in the kidneys (Kropp et. al.,

1980; Kahan et. al., 1983). The chemistry of a large number of modified carbapenems produced by

total chemical synthesis, such as panipenem, biapenem and meropenem, and the antibacterial

activities of meropenem in particular, has been reviewed recently (Neu, 1994; Coulton and Hunt,

1996). Carbapenems and monobactams were developed to deal with �-lactamase producing Gram-

negative organisms, which were resistant to broad spectrum and extended spectrum penicillins.

Carbapenems are derived from Streptomyces species and one example is the semisynthetic imipenem

which acts in the same way as the other �-lactams.

1.4.4.1 IMIPENEM

Imipenem has a very broad spectrum of antimicrobial activity being active against many aerobic and

anaerobic Gram-positive and Gram-negative organisms including Listeria, Pseudomonas, and most

Enterobacteriaceae. It is used when other antibiotics fail to eradicate the bacteria in question.

Meticillin resistant Staphylococci are less susceptible to imipenem. Imipenem is partly broken in the

kidney by a dehydropeptidase in the proximal tubule, and is therefore given intravenously in

combination with cilastatin, a specific inhibitor of this enzyme to prevent breakdown in the renal

tubules. Pamipenem another type of carbapemem is being investigated.

1.4.4.2 MEROPENEM

Meropenem is a similar antibiotic to imipenem, but it is not degraded by dehydropeptidase, thus no

cilastatin is needed. Excessive levels of it in kidney failure can cause seizures with imipenem but not

with meropenem.

1.4.4.3 AZTREONAM

The main monobactam is aztreonam, which is resistant to most �-lactamases. It has an unusual

spectrum, being active only against Gram-negative aerobic rods including Pseudomonas, N.

menengitidis and H. influenza. Aztreonam is also a wide spectrum �-lactam antibiotic. It is not

usually among first line antibiotics. New injectable monobactams under investigation are

carumonam and tigemonam.

1.4.5.0 SULPHANILAMIDES AND TRIMETHOPRIM-SULFAMETHOXAZOLE (CO-

TRIMOXAZOLE)

Sulphanilamides are pharmaceuticals used extensively as antibacterial compounds, which have, in

general, very high chemotherapeutic coefficients, so that over dosage can cause digestive or renal

accidents. They are bacteriostatic against a wide range of Gram-positive and Gram-negative, but

many organisms are resistant to them. Both bacteria and their human hosts require folic acid for

nucleic acid synthesis (it is converted into purines and thymidine) as well as protein synthesis

(precursor of the amino acids methionine and glycine).

However, bacteria synthesize their folic acid starting with para-aminobenzoic acid (PABA), while

human hosts must ingest folic acid already formed; that is, for the human host, it is a vitamin.

Sulphanilamides, and the other sulfa drugs, are analogues of PABA; they compete with PABA and,

when chosen, block the synthesis of folic acid. Mammals ignore PABA and its analogues and thus

can tolerate sulfa drugs.

Figure 11: A schematic structure of metropenem a monobactams

These synthetic molecules block the final step in the conversion of PABA to folic acid so they, too,

block nucleotide and protein synthesis in bacteria but not in mammals.

Trimethoprim is one of several antibacterials in current use. These folic acid analogues are often used

in combination with a sulfa drug. They act by preventing synthesis of folic acid. Trimethoprim and

sulfamethoxazole (a sulphonamide) together are synergistic in inhibiting folic acid synthesis and

bacteria show less resistance to them. They have good oral absorption. It is also important for

patients to be well hydrated and with its dose reduced (or avoided) in patient with renal insufficiency.

Sulphonamides are effective against Nocardia and Toxoplasma. They can replace penicillin for

prophylaxis in penicillin-sensitive patients.

1.4.5.1 Side effects

Sulfanilamides can cause lack of appetite, nausea and vomiting. They can also cause kernicterus in

neonate. If given to neonate or pregnant women, headache, depression, hallucinations can occur.

Figure 12: Folic Acid Analogues

1.4.5.2 Toxic effects

Sulphinalamides can cause hypersensitivity reactions in the body. They can also cause

agranulocytosis and aplastic anaemia. Other side effects include; nausea, fever, joint pain and

rashes.

1.4.5.3 Contraindication

Sulphanilamide may cause acute haemolysis in patients with glucose-6-phosphate dehydrogenase

deficiency. Interactions of sulphanilamide with other drugs can increase effects of oral

anticoagulants, sulfuryl group, urea and hydration in the body. Examples of Sulphanilamides and

their uses are as follows: sulfisoxazole (used topically in the eye, with erythromycin for otitis media

and for urinary tract infections) and sulfasalazine is used in inflammatory bowel disease.

Sulfacetamide is used topically in the eye. Silver sulfadiazine is used on burns or decubitus (pressure)

ulcers to prevent secondary bacterial or fungal infection. Trimethoprim-sulfamethoxazole is used in

�

�

Figure 1.12 Scheme for the mechanism of action of sulfanilamides and trimethoprim.

13

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paediatric and in urinary tract infections. It is also effective against Pneumocystis carinii in AIDS

patients. It is used for prophylaxis in neutropenic patients.

1.4.6 QUINOLONES

The first quinolone, nalidixic acid (NegGram), was introduced in 1962. Since then, structural

modifications have resulted in second-, third-, and fourth-generation fluoroquinolones, which have

improved coverage of gram-positive organisms (Oliphant and Green 2002). Other examples of

quinolones are ciprofloxacin, ofloxacin and norfloxacin. Quinolones rapidly inhibit DNA synthesis

by promoting cleavage of bacterial DNA in the DNA-enzyme complexes of DNA gyrase and type IV

topoisomerase, resulting in rapid bacterial death. As a general rule, gram-negative bacterial activity

correlates with inhibition of DNA gyrase, and gram-positive bacterial activity corresponds with

inhibition of DNA type IV topoisomerase (Oliphant and Green 2002). They are well absorbed orally;

they can also be given parenterally. They are effective against E. coli, Shigella, Salmonella,

Enterobacter, Campylobacter and Neisseria. Ciprofloxacin is more effective against Pseudomonas,

Enterococcus and Pneumococcus. Quinolones also effective against intracellular bacteria such as

Chlamydia, Mycoplasma, Mycobacterium, Legionella and Brucella.

1.4.6.1 Pharmacokinetics of quinolones

Like aminoglycosides, the quinolones exhibit concentration-dependent bacterial killing. Bactericidal

activity becomes more pronounced as the serum drug concentration increases to approximately 30

times the minimum inhibitory concentration (MIC) (Hooper et al., 2000; Turnidge, 1999). Higher

drug concentrations paradoxically inhibit RNA and protein synthesis, thereby reducing bactericidal

activity (Hooper et al., 2000). Quinolones have a postantibiotic effect of about one to two hours.

When used in combination with agents from other antibiotic classes, such as beta-lactams and

aminoglycosides, the quinolones are not predictably synergistic (Hackbarth et al., 1986). Although

the effects of most combinations are indifferent, ciprofloxacin (Cipro) and rifampin (Rifadin) appear

to be antagonistic (Hackbarth et. al., 1986).

Quinolones are well absorbed following oral administration, with moderate to excellent

bioavailability (Hooper et. al., 2000; Turnidge, 1999). Serum drug levels achieved after oral

administration are comparable to those with intravenous dosing, which allows an early transition

from intravenous to oral therapy and a potential reduction of treatment costs (Walker, 1999).

xxxiii

Food does not impair the absorption of most quinolones. However, quinolones chelate with cations

such as aluminum, magnesium, calcium, iron, and zinc. This interaction significantly reduces

absorption and bioavailability, resulting in lower serum drug concentrations and less target-tissue

penetration (Hooper et. al., 2000; Turnidge, 1999).

Elimination half-lives for the quinolones vary from 1.5 to 16 hours. Therefore, most of these drugs

are administered every 12 to 24 hours. The quinolones are eliminated by renal and nonrenal routes.

To avoid toxicity, dosages often need to be adjusted in patients with renal or hepatic impairment

(Hooper et. al., 2000; Turnidge, 1999). The majority of quinolones are excreted renally; however,

sparfloxacin (Zagam), moxifloxacin (Avelox), and trovafloxacin (Trovan) are excreted hepatically.

Quinolones are widely distributed throughout the body. Tissue penetration is higher than the

concentration achieved in plasma, stool, bile, prostatic tissue, and lung tissue. Intracellular

concentration is exceptional in neutrophils and macrophages. Quinolones also penetrate well in urine

and kidneys when renal clearance is the route of drug elimination. Penetration into prostatic fluid,

saliva, bone, and cerebrospinal fluid does not exceed serum drug levels. Because cerebrospinal fluid

levels of quinolones are predictably poor, these agents are inadequate for first-line treatment of

meningitis (Hooper et. al., 2000; Turnidge, 1999; Alghasham and Nahata 1999).

1.4.6.2 Antimicrobial activity of quinolones

The quinolones can be classified into four generations based on antimicrobial activity (Owens and

Ambrose, 2000). First-generation agents, which are used less often today, have moderate gram-

negative activity and minimal systemic distribution. Second-generation quinolones have expanded

gram-negative activity and atypical pathogen coverage, but limited gram-positive activity. These

agents are most active against aerobic gram-negative bacilli. Ciprofloxacin remains the quinolone

most active against Pseudomonas aeruginosa (Hooper, 2000). Third-generation quinolones retain

expanded gram-negative and atypical intracellular activity but have improved gram-positive

coverage. Finally, fourth-generation agents improve gram-positive coverage, maintain gram-negative

coverage, and gain anaerobic coverage (Ambrose et. al., 1997).

Marginal susceptibility and acquired resistance limit the usefulness of second-generation quinolones

in the treatment of staphylococcal, streptococcal, and enterococcal infections (Hooper, 2000). The

presently available fluoroquinolones with in vitro activity against Streptococcus

pneumoniae (including current penicillin-resistant strains) are levofloxacin (Levaquin), sparfloxacin,

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gatifloxacin (Tequin), moxifloxacin, and trovafloxacin. Levofloxacin and sparfloxacin exhibit

inferior in vitro streptococcal activity compared with gatifloxacin, moxifloxacin, and trovafloxacin.

Gatifloxacin is two to four times more active than levofloxacin against S. pneumoniae in vitro, and

moxifloxacin is four to eight times more active. Compared with ciprofloxacin and levofloxacin, the

fluoroquinolones gatifloxacin, moxifloxacin, and trovafloxacin have greater in vitro activity

against S. aureus and some Enterococcus strains (Eliopoulos, 1999).

Although gatifloxacin and moxifloxacin have in vitro anaerobic activity, only trovafloxacin is labeled

for the treatment of anaerobic infections. Clinafloxacin, an investigational fluoroquinolone, has the

most potent in vitro anaerobic activity (Applebaum, 1999).

Ciprofloxacin, ofloxacin (Floxin), and the newer fluoroquinolones have exceptional intracellular

concentrations. Moxifloxacin, gatifloxacin, levofloxacin, and the investigational drug gemifloxacin

have exceptional activity against Legionella, Chlamydia, Mycoplasma, and Ureaplasma species

(Hooper, 2000). Intracellular respiratory pathogens such as Chlamydia pneumoniae, Mycoplasma

pneumoniae, and Legionella pneumophila are predictably susceptible to fluoroquinolones. These

antibiotics are regarded as second-line antituberculous agents and should be reserved for the

treatment of resistant tuberculosis.

1.4.6.3 Therapeutic uses of quinolones

1.4.6.3.1 Genitourinary infections

Because of their extensive gram-negative coverage, quinolone antibiotics were initially used to treat

urinary tract infections. The higher genitourinary drug concentrations that occur with renally cleared

quinolones promote their effectiveness in the treatment of genitourinary infections. Given in three- to

10-day courses, most quinolones are as effective as trimethoprim-sulfamethoxazole (Bactrim, Septra)

in treating uncomplicated urinary tract infections caused by susceptible Escherichia coli (Wolfson

and Hooper 1989; Hooper, 1991).

Complicated urinary tract infections include those in patients with stones or obstructive uropathies

and in patients with catheter-related infections. These infections are often associated with

nosocomial, antibiotic-resistant gram-negative pathogens and gram-positive bacteria, and with

Candida species. Because ciprofloxacin, ofloxacin, lomefloxacin (Maxaquin), enoxacin (Penetrex),

levofloxacin, and gatifloxacin have higher renal clearance and greater renal concentration, they are

optimal choices for the treatment of complicated urinary tract infections (Hooper, 2000).

xxxv

Ciprofloxacin has been shown to be more effective than trimethoprim-sulfamethoxazole and

aminoglycosides in seven- to 10-day courses for the treatment of complicated urinary tract infections.

However, few patients maintain sterile urine six weeks after any antibiotic therapy (Hooper,

2000). Bacterial resistance and Candida superinfection often limit treatment in complicated urinary

tract infections, with an estimated failure rate of at least 2 percent (Wolfson and Hooper 1989; Fang

et al., 1991). Failure rates as high as 20 percent may be encountered with infections caused by

pathogens such as P. aeruginosa (Hopper, 2000).

A seven- to 10-day course of orally administered norfloxacin (Noroxin) or ofloxacin has been

successful in the treatment of uncomplicated pyelonephritis, with a bacteriologic cure rate equal to

that for trimethoprim-sulfamethoxazole. In the treatment of acute uncomplicated pyelonephritis in

non-pregnant women, similar efficacy has been shown for levofloxacin, in a dosage of 250 mg per

day for seven to 10 days, and ciprofloxacin, in a dosage of 500 mg twice daily for 10 days. However,

relapses were more common with levofloxacin (Hopper, 2000). Gatifloxacin, in a dosage of 400 mg

per day, has compared favorably with ciprofloxacin, in a dosage of 500 mg twice daily, in the

treatment of complicated urinary tract infections and pyelonephritis, with cure rates of 93 percent and

91 percent, respectively.

Fluoroquinolones, especially levofloxacin and ciprofloxacin, are valuable in the treatment of

complicated urinary tract infections and pyelonephritis. Yet bacterial resistance, relapse of infections,

and recurrent infections remain critical issues. Complex genitourinary tract infections continue to be

a niche for this antibiotic class.

1.4.6.3.2 Prostatitis

Quinolones are effective in the treatment of prostatitis because of their excellent penetration into

prostatic tissue. When taken for four to six weeks, norfloxacin, ciprofloxacin, levofloxacin, and

ofloxacin have eradication rates of 67 to 91 percent (Hopper, 2000; Sabbaj, 1986). Treatment failures

have been associated with shorter treatment courses (e.g., two weeks) and less susceptible bacteria,

specifically P. aeruginosa and Enterococcus species ( Schaeffer and Darras, 1999 ).

Levofloxacin is an excellent first-line agent in the treatment of prostatitis. Ciprofloxacin should be

reserved for use in patients with resistant gram-negative, pseudomonal, and enterococcal prostatitis,

because of its superior activity against P. aeruginosa and enterococci.

1.4.6.3.3 Respiratory diseases

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Acute bacterial sinusitis may be the complication of an initial viral illness. The primary bacterial

isolates are S. aureus, S. pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The U.S.

Food and Drug Administration (FDA) has labeled gatifloxacin, moxifloxacin, sparfloxacin, and

levofloxacin for use in the treatment of acute bacterial sinusitis. Clinical trials comparing

fluoroquinolones with amoxicillin-clavulanate potassium (Augmentin), cefuroxime axetil (Ceftin),

and clarithromycin (Biaxin) have demonstrated the efficacy of the quinolone antibiotics (Wolfson

and Hooper, 1989). However, we believe that quinolones should not be used as first-line agents in the

treatment of acute bacterial sinusitis because of the potential for development of bacterial resistance.

Acute bronchitis may follow a viral illness, but antimicrobial therapy generally is not warranted

unless the patient has underlying pulmonary disease. Fluoroquinolone therapy for acute bacterial

bronchitis has been effective against H. influenzae and M. catarrhalis, the primary pathogens

(Hopper, 2000). The use of ciprofloxacin for S. pneumoniae and P. aeruginosa bronchitis has

resulted in clinical treatment failures and the development of bacterial resistance. Generally,

levofloxacin, sparfloxacin, ofloxacin, gatifloxacin, and moxifloxacin have compared favorably with

cefuroxime, cefaclor (Ceclor), amoxicillin-clavulanate potassium, and amoxicillin (Hopper, 2000).

Community-acquired pneumonia is the sixth leading cause of death in the United States. Even with

optimal therapy, this illness is associated with mortality rates of approximately 14 percent in

hospitalized patients and less than 1 percent in patients not requiring hospitalization. S. pneumoniae,

H. influenzae, and M. pneumoniae are the pathogens most commonly identified in community-

acquired pneumonia; less commonly isolated pathogens include viruses, S. aureus, C. pneumoniae,

M. catarrhalis, Klebsiella pneumoniae, and L. pneumophila (Bartlett at. al., 2000; Heffelfinger,

2000). The pathogens most often responsible for death in patients with community-acquired

pneumonia are S. pneumoniae, S. aureus, and L. pneumophila.

Antibiotic choices for outpatient and in-patient treatment of pneumonia were stratified in a recent

consensus statement from the Infectious Diseases Society of America (IDSA) (Bartlett et. al., 2000)

and in guidelines formulated by the Centers for Disease Control and Prevention (CDC) (Heffelfinger,

2000). Preference was not given to a specific antibiotic class. Listed antibiotic choices for outpatient

treatment included macrolides, doxycycline (Vibramycin), and fluoroquinolones. Antibiotic choices

for hospitalized patients included fluoroquinolones or extended-spectrum penicillins (piperacillin

[Pipracil], piperacillin azobactam [Zosyn], or ampicillin sulbactam [Unasyn]), carbapenems

(meropenem [Merrem] and imipenem cilastatin [Primaxin]) and cephalosporins, plus adjunctive

xxxvii

macrolides, aminoglycosides, clindamycin (Cleocin), or metronidazole (Flagyl) (Bartlett at. al.,

2000; Heffelfinger, 2000).

For treatment of community-acquired pneumonia in patients hospitalized in a general ward, IDSA

recommends a macrolide with an extended-spectrum cephalosporin. Beta-lactam/beta lactamase

inhibitor combined with a macrolide, or a fluoroquinolone alone could as well be used. For the

treatment of patients hospitalized in an intensive care unit, the IDSA guidelines recommend a

macrolide or a fluoroquinolone (levofloxacin, gatifloxacin, or moxifloxacin) plus an extended-

spectrum cephalosporin (cefotaxime or ceftriaxone) or a beta-lactam/beta-lactamase inhibitor.

For the treatment of atypical pneumonias, macrolides are likely to be equivalent to fluoroquinolones

and are currently more cost-effective. Quinolones provide exceptional coverage against atypical

pathogens when infection with these organisms is suspected in patients with community-acquired

pneumonia. However, ofloxacin has been associated with treatment failures, and ciprofloxacin has

displayed reduced activity against Chlamydia species (Hopper, 2000).

Compared with other quinolones, moxifloxacin and gatifloxacin have been shown to have superior in

vitro activity against pneumococci. Although this activity may make moxifloxacin or gatifloxacin an

attractive choice for pneumococcal infections, these agents should probably be reserved for treatment

of infections with atypical pathogens or for life-threatening pneumonias (Jones and Pfaller, 2000;

Appelbaum, 2000).

Of the fluoroquinolones, ciprofloxacin and trovafloxacin have been studied most extensively in the

treatment of nosocomial pneumonia. Ciprofloxacin has been found to be comparable in efficacy to

imipenem cilastatin in mechanically ventilated patients, especially those infected with pathogens

from the Enterobacteriaceae family. It has also been associated with poorer responses and higher

clinical failure rates in patients with nosocomial pneumonia caused by S. aureus or P. aeruginosa.

The efficacy of the newer quinolones (moxifloxacin and gatifloxacin) in the treatment of nosocomial

pneumonia is currently being assessed in clinical trials. At present, quinolones are best used in

combination antimicrobial therapy for nosocomial pneumonia. Fluoroquinolone mono-therapy may

worsen the increasing problem of antibiotic resistance in the nosocomial setting.

1.4.6.3.4 Sexually transmitted diseases

Based on 1998 guidelines from the CDC, ceftriaxone is the agent of choice for treatment of

uncomplicated Neisseria gonorrhoeae urethritis and cervicitis. A single dose of ciprofloxacin or

ofloxacin should be considered as alternative treatment in patients with penicillin allergy. Recently,

xxxviii

gatifloxacin was reported to be as effective as ofloxacin against N. gonorrhoeae. A seven-day course

of ofloxacin or sparfloxacin has been found to be as effective as doxycycline in the treatment of C.

trachomatis infections. Finally, ciprofloxacin has been reported to be as effective as trimethoprim-

sulfamethoxazole for treating chancroid caused by Haemophilus ducreyi. Pelvic inflammatory

disease is a polymicrobial infection. Quinolone treatment options include ofloxacin plus

metronidazole, ofloxacin plus cefoxitin (Mefoxin), and ciprofloxacin plus clindamycin.

Fluoroquinolone monotherapy is incomplete (Hopper, 2000).

1.4.6.3.5 Gastroenteritis

Prophylactic antimicrobial therapy is not recommended for the prevention of diarrhea in travelers.

Norfloxacin or ciprofloxacin has been found to be comparable to trimethoprim-sulfamethoxazole in

the treatment of traveler's diarrhea caused by Shigella species, enterotoxigenic E. coli,

or Campylobacter jejuni. Ciprofloxacin and ofloxacin are the agents of choice for treatment of enteric

typhoid fever. Norfloxacin has been found to be superior to both trimethoprim-sulfamethoxazole and

doxycycline in the treatment of Vibrio cholerae infection (Hopper, 2000).

1.4.6.3.6 Skin and soft tissue infections

Because of limited data, the role of fluoroquinolones in the treatment of skin and soft tissue

infections remains uncertain. Most fluoroquinolones have limited gram-positive activity; thus, they

should not be considered first-line agents for skin and soft tissue infections. Diabetic foot infections,

which are polymicrobial, can be treated with quinolones in combination with other antibiotics

(Hopper, 2000).


Quinolones can cause nausea, headache, dizziness and abdominal discomfort. They can also increase

risk of convulsions in patient with epilepsy.

1.4.6.5 Interactions

Theophylline or non-steroidal anti-inflammatory drugs with a quinolone can cause hallucinations,

delirium and even seizures. Ciprofloxacin increases concentration of theophylline.

1.4.7 AMINOGLYCOSIDES.

Aminoglycosides are among the most commonly used broad-spectrum antibiotics in the anti-infective

armamentarium. The vast majority of aminoglycosides are bactericidal. They have predictable

pharmacokinetics, and they often act in synergy with other antibiotics- properties that make them

xxxix

valuable as anti-infectives. Furthermore, despite the potential for renal toxicity, ototoxicity, and

bacterial resistance, several members of this family of antibiotics have enjoyed clinical use for

several decades (Lakshmi et al., 2000).

Aminoglycosides are multifunctional hydrophilic sugars that possess several amino and hydroxy

functionalities. The amine moiety is mostly protonated in biological media; hence, these

antibiotics can be considered polycationic species for the purpose of understanding their biological

interactions. Since they are polycationic, they show a binding affinity for nucleic acids. Specifically,

aminoglycosides possess high affinities for certain portions of RNAs, especially the prokaryotic

rRNA (Recht et al., 1999). In addition, aminoglycosides bind to the hammer-head ribozyme (Stage

et. al., 1995; Tor et. al., 1998), tRNAPhe (Kirk et al., 1999), the Rev response element (RRE)

transcriptional activation region in human immunodeficiency virus (HIV) (Cho and Rando 1999;

Hendrix, 1997), the ribozyme from hepatitis delta virus (Rogers et al., 1996), and group I self-

splicing introns (Cech, 1990; von Ashen, 1991; von Ashen et al., 1991). Aminoglycosides are not

absorbed orally but given parenterally or topically.

Streptomycin was the first aminoglycoside introduced in 1944, followed by neomycin (1949),

kanamycin (1957), gentamicin (1963), tobramycin (1968), amikacin (1972), and netilmicin in 19751.

Tobramycin, gentamicin, amikacin and netilmicin are used in Gram-negative bacteraemic patients,

while streptomycin is used as a mycobactericidal agent. Examples of aminoglycosides are

gentamicin, netilmicin, amikacin, streptomycin, tobramycin and neomycin. They are effective against

aerobic Gram-negative organisms; amikacin has the widest spectrum (Sandhu et al., 2007).

1.4.7.1 Uses

Gentamicin, tobramycin, amikacin and netilmicin are all used in pyelonephritis, hospital-acquired

pneumonia, meningitis (by Gram-negative bacteria, given intrathecally), peritonitis and sepsis.

Tobramycin is also used topically in the eye. Streptomycin was the prototype; due to resistance, it is

now used mostly in tuberculosis, tularaemia and plague. Neomycin is used only topically or orally (to

clean the intestines before abdominal surgery) because of its severe toxicities.


Ototoxicity of aminoglycosides can act on both auditory and a vestibular portion of cranial nerve

VIII; damage is usually irreversible and main effect is hearing loss. Nephrotoxicity of these

antibiotics are usually mild and reversible; the main problem is that decreased clearance increases

xl

risk of ototoxicity. They can as well cause neuromuscular blockade with apnoea. Streptomycin may

also cause damage to the optic nerve and peripheral neuritis.


Furosemide and ethacrynic acid are synergistic for hearing loss with aminoglycosides.

1.4.8 TETRACYCLINES

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of

microorganisms including gram-positive and gram-negative bacteria such as chlamydiae,

mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been

used extensively in the prophlylaxis and therapy of human and animal infections and also at

subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium,

Shigella dysenteriae, was isolated in 1953 ( Chopra and Marilyn, 2001). Tetracyclines have

a broad-spectrum antibiotics activities, but their general usefulness has been reduced with the

onset of bacterial resistance. Despite this, they remain the drug of choice for some specific

indications. They are bacteriostatic against Gram-positive and Gram-negative organisms,

including Rickettsia, Chlamydia, Mycoplasma pneumonaea, Brucella, Hemophilusducreyi,

Legionella, Helicobacter pylori, Trepenoma pallidum, Borrelia bungdorferi and Urea plasma.

Examples are tetracycline, doxycycline, minocycline and demeclocycline.

1.4.8 1 Uses

Tetracyclines may be used in the treatment of infections of the respiratory tract, sinuses, middle ear,

urinary tract, intestines, and also gonorrhoea, especially in patients allergic to �-

lactams and macrolides. However, their use for these indications is less popular than they once were

due to widespread resistance development in the causative organisms. Tetracycline derivatives are

currently being investigated for the treatment of certain inflammatory disorders.


Tetracyclines should be used with caution in those with liver impairment and may worsen renal

failure (except doxycycline and minocycline). Antacids and milk reduce the absoption of

tetracyclines. Drugs in the tetracycline class become toxic over time, so expired prescriptions of these

drugs should be discarded after the expiration date has passed. It damages intestinal flora and may

xli

cause fungal overgrowth, cause nausea, vomiting, diarrhoea epigastric pain and abdominal

discomfort (better if given with food); esophagitis and ulcers have been observed. Severe

thrombophlebitis may occur with intravenous use. Demeclocycline and doxycycline may cause

photosensitivity.


Tetracycline has the following toxic effect; Liver damage – especially in pregnant women, Renal

damage (this may worsen existing disease), Demeclocycline may cause nephrogenic diabetes

insipidus (and so is used to treat the Syndrome of inappropriate Anti-diuretic Hormone (SIADH)),

Pigmentation of the teeth may occur in small children if

tetracyclines are given to them or to their mothers during pregnancy. Out-dated tetracycline can cause

a form of Fanconi’s syndrome.


When ingested, it is usually recommended that tetracyclines should be taken with a full glass of water

two hours after eating, and one hour before eating. This is partly due to the fact that tetracycline

binds easily with magnesium, aluminium, iron, and calcium, which reduces its ability to be

completely absorbed by the body. Dairy products or preparations containing iron are not

recommended directly after taking the drug. When tetracylines are given with dairy foods, antacids,

iron compounds, sulcrafate or bismuth compounds, the metal will chelate and decrease the absorption

of the antibiotic.

1.4.8.5 Contraindications

Tetracycline use should be avoided in pregnant or lactating women, and in children with developing

teeth because they may result in permanent staining (dark yellow-gray teeth with a darker horizontal

band that goes across the top and bottom rows of teeth).

1.4.9 CHLORAMPHENICOL

Chloramphenicol is a broad-spectrum antibiotic. It is active against a wide variety of organisms. It

interferes with the production of proteins that the bacteria need to multiply. This inhibits the ability of

the bacteria to grow and therefore stops the spread of the infection. Chloramphenicol is effective

against a wide variety of microorganisms, but due to serious side-effects (e.g., damage to the bone

marrow) in humans, it is usually reserved for the treatment of serious and life-threatening infections

(e.g., typhoid fever). It is also used in eye drops or ointment to treat bacterial conjunctivitis.

Chloramphenicol is a bacteriostatic drug. It targets bacterial ribosomes, which are structures in the

bacteria necessary for the manufacture of new proteins. The ribosomes are the site where amino acids

are assembled to form proteins. By preventing the amino acids from interacting with the ribosome,

chloramphenicol halts protein synthesis.

Neisseria and Pneumococcus.

1.4.9.1 Uses of chloramphenicol

Chloramphenicol is a potent antibiotic that is effective against a wide array of bacteria (

2004). It is equally effective against gram

against anaerobic bacteria, which are bacteria that do not need oxygen to live. Chloramphenicol,

however, can cause some serious side effects and is generally only used for severe infections that

have proven resistant to other antibiotics. Because some bacteria ar

bacteria may need to be cultured and their susceptibility to the drug tested before chloramphenicol is

used.

1.4.9.2 Administration and interactions

Chloramphenicol can be taken orally or injected into the body. Oral chl

form of chloramphenicol palmitate. In the gastrointestinal tract, this compound is broken down and

the free chloramphenicol is absorbed. Chloramphenicol is injected in the form of chloramphenicol

succinate, which is then broken down to yield chloramphenicol in the blood. Chloramphenicol is

processed by the liver to form inactive breakdown products and is excreted via the urine.

Chloramphenicol increases half-lives of dicumarol, phenytoin, chlorpropamide an

half-life is decreased by rifampin or phenobarbital.

Figure 14: The structure of chloramphenicol



proteins. By preventing the amino acids from interacting with the ribosome,

chloramphenicol halts protein synthesis. Chloramphenicol may be bactericidal against Hemophilus,

Chloramphenicol is a potent antibiotic that is effective against a wide array of bacteria (

2004). It is equally effective against gram-negative and gram- positive bacteria and also has activity

anaerobic bacteria, which are bacteria that do not need oxygen to live. Chloramphenicol,


have proven resistant to other antibiotics. Because some bacteria are resistant to chloramphenicol, the


1.4.9.2 Administration and interactions

Chloramphenicol can be taken orally or injected into the body. Oral chloramphenicol is taken in the



down to yield chloramphenicol in the blood. Chloramphenicol is


lives of dicumarol, phenytoin, chlorpropamide an

life is decreased by rifampin or phenobarbital.

: The structure of chloramphenicol



proteins. By preventing the amino acids from interacting with the ribosome,

Chloramphenicol may be bactericidal against Hemophilus,

Chloramphenicol is a potent antibiotic that is effective against a wide array of bacteria (Robert et al.,

positive bacteria and also has activity

anaerobic bacteria, which are bacteria that do not need oxygen to live. Chloramphenicol,


e resistant to chloramphenicol, the


oramphenicol is taken in the



down to yield chloramphenicol in the blood. Chloramphenicol is


lives of dicumarol, phenytoin, chlorpropamide and tolbutamide. Its

xliii

1.4.9.3 Side effects of chloramphenicol

The most severe side effect of chloramphenicol, is suppression of the bone marrow (Robert et al.,

2004). Because the bone marrow produces red and white blood cells as well as platelets, patients can

develop anemia, clotting disorders and a weakened immune system. Most often this bone marrow

suppression is temporary and will go away once therapy is stopped. However, in approximately one

out of every 125,000 cases, irreversible idiosyncratic aplastic anemia will develop, which will result

in permanent bone marrow depression. This condition can develop even after the initial course of

therapy has been stopped.

1.4.10 MACROLIDES

The most commonly used macrolide antibiotics consist of a macrocyclic lactone ring containing 14,

15 or 16 atoms with sugars linked via glycosidic bonds (Elks and Ganellin, 1991). The clinically

useful macrolide antibiotics can be conveniently classified into three groups based on the number of

atoms in the lactone nucleus. Erythromycins A, B, C, D, E and F, oleandomycin, roxithromycin,

dirithromycin, clarithromycin and flurithromycin are 14-membered macrolides whereas azithromycin

is a 15-membered compound. 16-Membered macrolides include josamycin, rosaramicin,

rokitamycin, kitasamycin, mirosamicin and spiramycin, the latter two compounds being used almost

exclusively in veterinary medicine (Reynolds, 1993). They are usually bacteriostatic and effective

against Gram-positive organisms, Chlamydia, Mycoplasma and Legionella; azithromycin and

clarithromycin are also effective against Hemophilus and Mycobacterium-avium-intracellular. They

are given orally in capsules or with enteric coating as gastric acid breaks the drug down they are also

given parenterally or give on an empty stomach.

Except for rosaramicin and mirosamycin, which are isolated from Micromonospora species, and the

semisynthetic derivatives of erythromycin A (roxithromycin, dirithromycin, clarithromycin,

flurithromycin and azithromycin), macrolides are produced from various Streptomyces organisms.

Consequently, the macrolide antibiotics obtained from macrolide-producing organisms commonly

consist of mixtures of homologous components.

All these macrolide antibiotics display similar antibacterial properties and are active against Gram-

positive and some Gram-negative bacteria and are particularly useful in the treatment of

Mycoplasmas, Haemophilus influenzae, Chlamydia species and Rickettsia. In particular, macrolide

antibiotics constitute an important alternative for patients exhibiting penicillin sensitivity and allergy.

xliv

1.4.10.1 Uses of microlides

Macrolides are used for treatment of pertussis. It is also used for treatment of prophylaxis in

penicillin-sensitive patients, particularly in pregnant women.

1.4.10.2 Side effects of microlides

Microlides can cause Gastro Intestinal Tract (GIT) effects which might result to abdominal pain,

nausea, vomiting and hypersensitivity. Cholestatic hepatitis could occur after stopping the

treatment.


Erythromycin increases effects of carbamazepine, steroids, cyclosporine, digoxin, theophylline,

valproic acid, warfarin and more via cytochrome P450.

1.4.11 CLINDAMYCIN

Clindamycin is a semisynthetic antibiotic derived from lincomycin by 7s - chloro substitution of the 7

r - hydroxyl group of the lincomycin. Clindamycin is therefore an antibiotic of the lincosamides

class and possesses similar properties to its sister compound lincomycin. The blueprint for any

protein structure comes from the cell’s DNA. The relevant area of DNA (which is double stranded)

opens and is transcribed to form a strand of messenger RNA. The messenger RNA travels from the

cells nucleus outward to where a group of cell organelles called ribosomes can become attached. The

ribosomes grab the strand of messenger RNA and link the appropriate amino acids (bound to the

transfer RNA) into the desired protein (Brooks, 2007).

Clindamycin and benzoyl peroxide have activity against Propionibacterium acnesin in-vitro. This

organism binding to the 50S subunit of the bacterial ribosome. It is given orally, topically and

parenterally.

1.4.11.1 Uses of clindamycin

Clindamycin is used for the treatment of infections caused by Propionibacterium acnesin. They are

also has been associated with acne vulgaris. Benzoyl peroxide releases free-radical oxygen which

oxidizes bacterial proteins in the sebaceous follicles decreasing the number of anaerobic bacteria and

decreasing free fatty acids. Clindamycin reversibly binds to 50S ribosomal subunits preventing

peptide bond formation. Thereby inhibiting bacterial protein synthesis. It could be bacteriostatic or

bactericidal depending on drug concentration, infection site, and organism. Its bacteriostatic effect is

by interfering with bacterial protein synthesis, in a similar way as erythromycin and chloramphenicol,

by used for the treatment of oral and tropical acne and for oral and tropical treatment of bacterial

vaginosis.

1.4.11.2 Side effects of clindamycin

Clindamycin is the major cause of pseudomembranous colitis. It also causes skin rash and when

given intravenously, thrombophlebitis may also occur.

1.4.12 VANCOMYCIN

Vancomycin is a glycopeptide antibiotic that inhibits the final steps of peptidoglycan biosynthesis by

binding to the D-Ala-D-Ala dipeptide termini of peptidoglycan precursors so that they cannot be

further processed (Anderson et al., 1965). Vancomycin is produced by Streptococcus orientalis. It is

only active against Gram-positive bacteria except Flavobacterium. It is also effective against

Staphylococcus, Streptococcus, Enterococcus, Corynebacterium and Clostridium. Vancomycin

inhibits cell wall synthesis by binding firmly to the D-Ala-D-Ala terminus of nascent peptidoglycan

penta-peptide. This inhibits the transglycosylase, preventing further elongation of peptydoglycan and

cross-linking. Vancomycins are given intravenously and orally for the treatment of pseudo-

membranous colitis.

�

1.4.12.1 Uses of vancomycin

Vancomycin is used for the treatment of pseudo-membranous colitis. It is also used for the treatment

of infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA).

Figure 16: The structure of Vancomycin

1.4.12.2 Side effects of vancomycin

One of the known side effects of vancomycin is hypersensitivity.

1.4.12.3 Toxic effects of vancomycin

At high concentration vancomycin can cause ototoxicity which may be reversible. It can also cause

flushing, tachycardia and hypotension which occurs with too rapid intravenous infusion. Kidney

damage may occur if given with nephrotoxic drugs or to patient with renal disorder.

1.4.12 BACITRACIN

Bacitracin is topically used only due to severe nephrotoxicity if given parenterally. It is effective

against Gram-positive aerobes. It inhibits cell wall formation by interfering with dephosphorylation

in cycling of the lipid carrier that transfers peptidoglycan subunits to the growing cell wall.

Bacitracin is markedly nephrotoxic if administered systemically. It is poorly absorbed in topical use.

Thus topical application results in local antibacterial activity without significant systemic toxicity.

Bacitracin is used to treat eyes and skin diseases.

1.4.13 POLYMYXIN-B

Just like bacitracin polymyxin B is used topically only due to severe nephrotoxicity if given

parenterally. It is effective against Gram-negative bacteria, especially Pseudomonas.

1.4.13 Uses of polymycin-B

Polymyxin B is used for the treatment of ears, eyes and skin diseases.

�

Figure 1.13 Schematic structure of bacitracin Figure 17: Schematic structure of bacitracin

xlviii

1.5 PLANT PRODUCTS AS ANTIMICROBIAL AGENTS

Since the advent of antibiotics in 1950s, the use of plant derivatives as antimicrobials has been

virtually non-existent. Currently, one-quarter to one-half of all pharmaceuticals dispensed in the

United States have higher-plant origins. Very few of these are intended for use as antimicrobials,

since bacterial and fungal are sources relied on for these activities (Cowan, 1999).

Clinical biochemists and microbiologists have two reasons to be interested in antimicrobials from

plant extracts. First, it is very likely that these phytochemicals will find their way into the arsenal of

antimicrobial drugs prescribed by physicians; several of them are already being tested in humans. It

is reported that on the average, two or three antibiotics derived from microorganisms are launched

each year. After a down-turn in that pace in recent decades, the pace is again quickening as scientists

realize that the effective life of any antibiotic is limited. Worldwide spending on finding new anti-

infective agents is expected to increase. New sources, especially plant sources, are being investigated.

Second, the public is becoming increasingly aware of problems with the over-prescription and misuse

of traditional antibiotics. In addition, many people are interested in having more autonomy over their

medical care. A multitude of plant compounds are in natural food stores. The use of plant extracts has

enjoyed increasingly great popularity since the late 1990s.

1.5.1 MAJOR GROUPS OF ANTIMICROBIAL COMPOUNDS FROM PLANTS

Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenol or

their oxygen-substituted derivatives. Most are secondary metabolites, of which at least 12,000 have

been isolated. In many cases, these substances serve as plant defence mechanisms against predation

by microorganisms, insects, and herbivores. Some, such as terpenoids, give plants their odours;

others (Quinone and tannins) are responsible for the same herbs and spices used by humans to season

food and yield useful medicinal compounds (Cowan, 1999). Useful antimicrobial phytochemicals can

be divided into several categories, described below and summarized in table 1.

1.3.1 PHENOLICS AND POLYPHENOLS

1.3.1.1 SIMPLE PHENOLS AND PHENOLIC ACIDS

Some of the simplest bioactive phytochemicals consist of a single substituted phenolic ring. Cinnanic

and caffeic acids are common representatives of a wide group of phenylpropane-derived compounds

which have the highest oxidation state (figure 18).The common herbs tarragon and thyme both

contain caffeic, which is effective against viruses (Wild, 1994), bacteria (Burdick, 1971,

Thomson,1978) and fungi (Duke, 1985). Cowan (1999) observed that catechol and pyrogallol which

are hydroxylated phenols, are toxic to microorganisms. Catechol has two –OH groups, and pyrogallol

has three. The sites and number of hydroxyl group on the phenol group are related to their relative

xlix

toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity.

In addition, he found that more highly oxidized phenols are more inhibitory. The mechanism thought

to be responsible for phenol toxicity to microorganisms include enzyme inhibition by the oxidized

compounds, possibly through reaction with sulfhydryl group or through more nonspecific interaction

with the protein. Phenolic compounds possessing no oxygen are classified as essential oils and often

cited as antimicrobial as well. Eugenol is a well characterized representative found in clove oil

(figure18). Eugenol is considered bacteriostatic against both fungi and bacteria.

Common name

Scientific name

Compound

Class

Activity

Relative toxicity

Alfalfa Allspice Aloe Apple Ashwagandha Aveloz Bael tree Balsam pear

Medicagosativa Pimenta dioica Aloe barbadensis, Aloe vera Malussylvestris Withaniasomniferum Euphorbia tirucalli Aeglemarmelos

? Eugenol Latex Phloretin Withafarin A ? Essential oil ?

Essential oil Complex mixture Flavonoid derivative Lactone - Terpenoids Alkaloid

Gram-positive organisms General Corynebacterium, Salmonella, Streptococcus, S. aureus General Bacteria, fungi - S. aureus Fungi

2.3 2.5 2.7 3.0 0.0 0.0 - 1.0

Table I: Plants containing antimicrobial activity

l

Barberry Basil Bay Betel pepper Black pepper Blueberry Brazilian pepper tree Buchu Burdock Buttercup Caraway Cascara sagrada Cashew Castor bean Ceylon Chamomile Cinnamon

Momordica charantia Berberis vulgaris Ocimumbasilicum Laurusnobilis Piper betel Piper nigrum Vaccinium spp. Schinustere binthifolius Barosmasetulina Arctiumlappa Ranunculus bulbosus Carumcarvi Rhamnuspur shiana Anacardiumpulsatilla Ricinuscommunis Cinnamo mumverum Matricaria chamomilla Larreatri dentata

Berberine Essential oils Essential oils Catechols, eugenol Piperine Fructose Terebinthone Essential oil Protoanemonin Tannins Salicylic acids ? Essential oils, others Anthemic acid Nordihydroguai-aretic acid Capsaicin Eugenol

Terpenoids Terpenoids Essential oils Alkaloid Monosaccharide Terpenoids Terpenoid Polyacetylene, tannins, terpenoids Lactone Coumarins Polyphenols Anthraquinone Polyphenols Glutamate Terpenoids, tannins Phenolic acid Coumarins

General Bacteria, protozoa Salmonella, bacteria Bacteria, fungi General Fungi, Lactobacillus, Micrococcus, E. coli, E. faecalis E. coli General General Bacteria, fungi, viruses General Bacteria, fungi, viruses Viruses, bacteria, fungi P. acnes Bacteria, fungi General M. tuberculosis, S. typhimurium

2.0 2.5 0.7 1.0 1.0 1.0 2.0 2.3 2.0 1.0 2.0 - 0.0 2.0 2.3

Table I: continues

li

Chapparal Chili peppers, paprika Clove Coca Cockle Coltsfoot Coriander, cilantro Cranberry Dandelion Echinacea Eucalyptus Fava bean Gamboge Garlic Ginseng Glory lily Goldenseal Gotu kola Grapefruit peel

Capsicum annuum Syzygium aromatic Erythroxylum coca Agrostem magithago Tussilagofarfara Coriandrum sativum Vaccinium spp. Taraxacu mofficinale Anethum gravens Echinaceae angustifolia Viciafaba Garciniahanbuv Allium sativum Panaxnoto ginseng Gloriosa superba Hydrastis canadensis --- ?

Cocaine ? ? ? Fructose Other ? Essential oil ? Tannin Fabatin Allicin, ajoene Colchicine ? ? ? Berberine Asiatocoside Catechin

Lignan Terpenoid Terpenoid Alkaloid

?

? ? Monosaccharide Terpenoid Polyphenol Terpenoid Thionin Sulfated terpenoids Saponins Alkaloid Alkaloids Alkaliods Terpenoid Terpenoid

S.aureus, helminths Viruses Skin bacteria Bacteria General Gram-negative and –positivecocci General General Bacteria, fungi Bacteria C. albicans, S. cerevisiae Bacteria General Bacteria, viruses Bacteria General E. coli, Sporothrix schenckii, Staphylococcus, Tricho- phyton

2.0 2.0 1.7 0.5 1.0 2.0 2.7 3.0 1.5 - 0.5 2.7 0.0 2.0 1.7 2.0 0.5 2.7 1.7

Table I: continues

lii

Green tea Harmel, rue Hemp Henna Hops Horseradish Hyssop (Japanese) herb Lantana — Lavender-cotton Legume (West Africa) Lemon balm Lemon verbena _ Licorice Lucky nut, yellow Mace, nutmeg

Centella asiatica Citrus paradisa Camellia sinensis Peganum harmala Cannabis sativa Lawsonia inermis Armora ciarusticana Hyssopu sofficinalis Rabdosiatrichocarpa Lantana camara Lawsonia Santolinachamaecyparissus Millettia thonningii Melissa officinalis Aloysiatriphylla Glycyrrhizaglabra Thevetiaperuviana Myristic

�-Resercyclic acid Gallic acid Lupulone, humulone — — Trichorabdal ? Lawsone Alpinumisoflavone Tannins Essential oil Glabrol ? ? ? ? Helanins Tannins

Flavonoid Organic acid Phenolic Phenolic acids (Hemi) terpenoids Terpenoids Terpenoids Terpene Quinone Flavone Polyphenols Terpenoid ? Phenolic alcohol

Bacteria, Giardia duodenale, Fungi General Shigella Vibrio S. mutans Viruses Bacteria, fungi Bacteria and viruses S. aureus Viruses Helicobacter pylori General M. tuberculosis Gram-positive bacteria, Candida Schistosoma Viruses Ascaris E. coli, M. tuberculosis,

2.0 1.0 1.0 1.5 2.3 1.0 1.0 — 1.0 1.5 2.0 0.0 1.5 2.7

liii

Marigold Mesquite Mountain tobacco Oak Olive oil Papaya Potato Prostrate knotweed Purple prairie Rauvolfia, chandra Turmeric Valerian Willow

afragrans Calendula officinalis Prosopis juliflora Arnica montana Oleaeuropaea Carica papaya Solanumtuberosum Polygonum aviculare Petalostemum Cinchona sp. Rauvolfia serpentina Curcuma longa Valeriana officinalis Salix alba

Quercetin (available commercially) Hexanal Latex ? ? Quinine Reserpine Curcumin Essential oil Salicin ?

Lactone Polyphenols Flavonoid Aldehyde Mix of terpenoids, organic acids, alkaloids Flavonol Alkaloid Alkaloid Terpenoids Terpenoid Phenolic glucoside Coumarin

S. aureus S. aureus, M. tuberculosis Plasmodium General General Bacteria General General General General General Bacteria, fungi Bacterial Bacteria, fungi

1.5 2.0 2.0 1.0 3.0 2.0 2.0 2.0 2.0 1.0 2.7 3.0 2.5

1.3.2 QUINONES

Quinones have aromatic rings with two ketone substitutions (figure 1.18). They are ubiquitous in

nature and characteristically reactive. These compounds, being coloured, are responsible for the

browning reaction in cuts or injured fruits and vegetables and are an intermediate in the melanin

synthesis pathway in human skin (Schmidt, 1988). The switch between diphenol (hydroquinone) and

diketone (quinone) occurs easily through oxidation and reduction reactions. The individual redox

potential of the particular quinone-hydroquinone pair is very important in many biological systems.

Ubiquinone (coenzyme Q) plays an important role in mammalian electron transport system. Vitamin

K is a complex naphthoquinone.

Its antihemorrhagic activity may be related to its ease of oxidation in body tissues. Hydroxylated

amino acids may be made into Quinone in the presence of suitable enzymes, such as a

polyphenoloxidase (Vamos-Vigyazo,1981). The reaction for the conversion of tyrosine to Quinone is

shown in (figure 1.18).

In addition to providing stable free radicals, Quinone is known to complex irreversibly with

nucleophilic amino acid in protein, often leading to inactivation of the protein (Stern et. al.,1996.)

and its loss of function. For that reason, the potential ranges of Quinone antimicrobial effects are

great. Probable targets in the microbial cells are surface-exposed adhesions, cell wall polypeptides,

and membrane-bound enzymes. Quinones may also render substrate unavailable to the

microorganism. As with all plant-derived antimicrobial, the possible toxic effects of Quinones must

be thoroughly examined.

Kazmi et. al. (1994) described an anthraquinone from Cassiaitalica, a Pakistani tree, which was

bacteriostatic for Bacillus anthracis, Corynebacterium pseudodiphenicum, and Pseudomonas

aeruginosa and bactericidal for Pseudomonas pseudomalliae. Hyperricim, an anthraquinone

(Hypericum perforatum), has received much attention in the popular press as an antidepressant, and

Duke reported in 1985 that it has general antimicrobial properties.

Figure 19: Reaction for the conversion of tyrosine to quinine (Cowan, 1999).

lvi

1.3.3 ALKALOIDS

Most heterocyclic nitrogen compounds are called alkaloids and the first medically useful example of

an alkaloid was morphine, isolated in 1805 from the opium poppy Papaver somniferum. The name

morphine came from the Greek Morpheus (Fessenden et al., 1982), which means god of dreams.

Codeine and heroin are both derivatives of morphine. Diterpenoid alkaloids, commonly isolated from

the plants of the Ranunculaceae, or buttercup family (Jones et al., 1986), are found to have

antimicrobial properties (Omulokoli et al., 1997). Solamargine, a glycoalkaloid from the berries of

Solanum khasianum, and other alkaloids may be useful against HIV infection (McMahon et al., 1995,

Sethi, 1979) as well as intestinal infections associated with AIDS (McDevitt et al., 1996). While

alkaloids have been found to have microbiocidal effects (including against Giardia and Entamoeba

species), its antidiarrheal effect is probably due to their effects on transit time in the small intestine.

Berberine is also an important representative of the alkaloid group. It is potentially effective against

trypanosomes (Freiburghaus et al., 1996) and plasmodia (Omulokoli et al., 1997). The mechanism of

action of highly aromatic planar quaternary alkaloids such as berberine and harmane (Hopp et al.,

1976) is attributed to their ability to intercalate with DNA (Phillipson and Neill. 1987).

1.5.0 THE PLANT

1.5.1 Zapoteca portoricensis

Zapoteca portoricensis is a perennial shrub with a climbing stem which is woody at its base. This

plant belongs to the family of Fabeceae/mimosidea, and genus Zapoteca. Z. portoricensis is

commonly called dead awakener (English). However the Yorubas call it Ule, while the Igbos in

Nsukka area, where the plant is majorly used by traditional doctors, call it Azonta.

Other names of the plant are as follows;

• Acacia portoricensis

• Anneslia portoricensis

• Calliandra portoricensis

• Feuilleea portoricensis

• Mimosa portoricensis

1.4.2 Scientific classification of Zapoteca portoricensis

Kingdom Plantae – Plants

Subkingdom Tracheobionta – Vascular plants

Superdivision Spermatophyta – Seed plants

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Division Magnoliophyta – Flowering plants

Class Magnoliopsida – Dicotyledons

Subclass Rosidae

Order Fabales

Family Fabaceae – Pea family

Genus Zapoteca – white stickpea

Species Zapotecaportoricensis – Schott's stickpea

Variety Zapotecaformosa – Schott's stickpea

1.5.3 Geographical range of Zapoteca portoricensis

Zapoteca portoricensis is widely distributed in tropical rain forest. It is found in tropical Africa:

Nigeria, Ghana, Togo, and the Caribbean Islands especially British Virgin Island, Dominican

Republic, Grenada, Haiti, Jamaica and Puerto Rico.

1.5.4 Ethnopharmacological Uses of Zapoteca portoricensis

The plant has been used traditionally to treat diseases like tonsillitis (sore throat), fever, convulsion,

breast engorgement, stomach disorder, purgative, and amenorrhoea.

1.5.5 Previous Chemical and Pharmacological Investigations on Zapoteca portoricensis

Aqueous and alcoholic extracts of the leaf have been reported to be used in the treatment of gastro

intestinal disorders, spasmodic and in the treatment of tonsillitis (Nwakile, 2004). Reported

phytochemical analysis of the roots indicated the presence of a number of secondary metabolites such

as saponins, resins, glycosides, flavonoids, alkaloids, terpenoids and steroids. Recently, the

antimicrobial and anti-inflammatory activities of Zapoteca portoricensis have been authenticated in

experimental models. These preliminary results, coupled with the folkloric use of the plant in the

treatment of tonsillitis have suggested the formulation of the root extract of Zapoteca portoricensis

into lozenges for the possible treatment of mouth and throat infections (Esimone, 2009).

1.6.0 STREPTOCOCCUS PYOGENES

Streptococcus pyogenes (Group A streptococcus) is a Gram-positive, nonmotile, nonspore forming

coccus that occurs in chains or in pairs of cells. Individual cells are round-to-ovoid cocci, 0.6-1.0

micrometre in diameter (Figure 1.21). Streptococci divide in one plane and thus occur in pairs or

(especially in liquid media or clinical material) in chains of varying lengths. The metabolism of S.

pyogenes is fermentative. The organism is a catalase-negative, aerotolerant anaerobe (facultative

anaerobe), and requires enriched medium containing blood in order to grow. Group A streptococci

typically have a capsule composed of hyaluronic acid and exhibit beta (clear) haemolysis on blood

agar.

Streptococcus pyogenes is one of the most frequent pathogens of humans. It is estimated that between

5-15% of normal individuals harbour the bacterium, usually in the respiratory tract, without signs of

disease. As normal flora, S. pyogenes can infect when defences are compromised or when the

Figure 20: Zapoteca portoricensis

Figure 21: Streptococcus pyogenes; (Left). Gram stain of Streptococcus pyogenes in

a clinical specimen; (Right). Colonies of Streptococcus pyogenes on blood agar

exhibiting beta (clear) haemolysis (Todar, 2009).

lix

organisms are able to penetrate the constitutive defences. When the bacteria are introduced or

transmitted to vulnerable tissues, a variety of types of suppurative infections can occur (Todar, 2009).

In the last century, infections by S. pyogenes claimed many lives especially since the organism was

the most important cause of puerperal fever (sepsis after childbirth). Scarlet fever was formerly a

severe complication of streptococcal infection, but now, because of antibiotic therapy, it is little more

than streptococcal pharyngitis accompanied by rash. Similarly, erysipelas (a form of cellulitis

accompanied by fever and systemic toxicity) is less common today. However, there has been a recent

increase in variety, severity and sequelae of Streptococcus pyogenes infections, and a resurgence of

severe invasive infections, prompting descriptions of "flesh eating bacteria" in the news media. A

complete explanation for the decline and resurgence is not known. Today, the pathogen is of major

concern because of the occasional cases of rapidly progressive disease and because of the small risk

of serious sequelae in untreated infections. These diseases remain a major worldwide health concern,

and effort is being directed toward clarifying the risk and mechanisms of these sequelae and

identifying rheumatogenic and nephritogenic strains of streptococci (Todar, 2009).

Acute Streptococcus pyogenes infections may be present as pharyngitis (strep throat), scarlet fever

(rash), impetigo (infection of the superficial layers of the skin) or cellulitis (infection of the deep

layers of the skin). Invasive, toxigenic infections can result in necrotizing fasciitis, myositis and

streptococcal toxic shock syndrome. Patients may also develop immune-mediated post-streptococcal

sequelae, such as acute rheumatic fever and acute glomerulonephritis, following acute infections

caused by Streptococcus pyogenes.

Streptococcus pyogenes produces a wide array of virulence factors and a very large number of

diseases. Virulence factors of Group A streptococci include:

(1) M protein, fibronectin-binding protein (Protein F) and lipoteichoic acid for adherence,

(2) hyaluronic acid capsule as an immunological disguise and to inhibit phagocytosis;

(3) invasins such as streptokinase, streptodornase (DNase B), hyaluronidase, and streptolysins,

(4) Exotoxins, such as pyrogenic (erythrogenic) toxin which causes the rash of scarlet fever and

systemic toxic shock syndrome.

1.6.2 CLASSIFICATION OF STREPTOCOCCI

1.6.2.1 Haemolysis on blood agar

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The type of haemolytic reaction displayed on blood agar has long been used to classify the

streptococci. Beta -haemolysis is associated with complete lysis of red cells surrounding the colony,

whereas alpha-haemolysis is a partial or "green" haemolysis associated with reduction of red cell

haemoglobin. Nonhemolytic colonies have been termed gamma-haemolytic. Group A streptococci

are nearly always beta-haemolytic; related Group B can manifest alpha, beta or gamma haemolysis.

Most strains of S. pneumonia are alpha-haemolytic but can cause ß-haemolysis during anaerobic

incubation. Most of the oral streptococci and enterococci are non haemolytic. The property of

haemolysis is not very reliable for the absolute identification of streptococci, but it is widely used in

rapid screens for identification of S. pyogenes and S. pneumonia.

1.6.2.2 Antigenic types

The cell surface structure of Group A streptococci is among the most studied of any bacteria (Figure

1.22). The cell wall is composed of repeating units of N-acetylglucosamine and N-acetylmuramic

acid, the standard peptidoglycan. Historically, the definitive identification of streptococci has rested

on the serologic reactivity of "cell wall" polysaccharide antigens as originally described by Rebecca

Lancefield. Eighteen group-specific antigens (Lancefield groups) were established. The Group A

polysaccharide is a polymer of N-acetylglucosamine and rhamnose. Some group antigens are shared

by more than one species. This polysaccharide is also called the C substance or group carbohydrate

antigen.

1.6.2.3 Pathogenesis

Streptococcus pyogenes owes its major success as a pathogen to its ability to colonize and rapidly

multiply and spread in its host while evading phagocytosis and confusing the immune system. Acute

diseases associated with Streptococcus pyogenes occur chiefly in the respiratory tract, bloodstream,

or the skin. Streptococcal disease is most often a respiratory infection (pharyngitis or tonsillitis) or a

skin infection (pyoderma). Some strains of streptococci show a predilection for the respiratory tract;

others, for the skin. Generally, streptococcal isolated from the pharynx and respiratory tract do not

cause skin infections. Figure 1.23 describes the pathogenesis of S. pyogenes infections.

S. pyogenes is the leading cause of uncomplicated bacterial pharyngitis and tonsillitis commonly

referred to a strep throat. Other respiratory infections include sinusitis, otitis, and pneumonia.

Infections of the skin can be superficial (impetigo) or deep (cellulitis). Invasive streptococci cause

joint or bone infections, destructive wound infections (necrotizing fasciitis) and myositis, meningitis

and endocarditis. Two post streptococcal sequelae, rheumatic fever and glomerulonephritis, may

follow streptococcal disease, and occur in 1-3% of untreated infections. These conditions and their

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pathology are not attributable to dissemination of bacteria, but to aberrant immunological reactions to

Group A streptococcal antigens. Scarlet fever and streptococcal toxic shock syndrome are systemic

responses to circulating bacterial toxins.

The cell surface of Streptococcus pyogenes accounts for many of the bacterium's determinants of

virulence, especially those concerned with colonization and evasion of phagocytosis and the host

immune responses. The surface of Streptococcus pyogenes is incredibly complex and chemically-

diverse. Antigenic components include capsular polysaccharide (C-substance), cell wall

peptidoglycan and lipoteichoic acid (LTA), and a variety of surface proteins, including M protein,

fimbrial proteins, fibronectin-binding proteins, (e.g. Protein F) and cell-bound streptokinase.

The cytoplasmic membrane of S. pyogenes contains some antigens similar to those of human cardiac,

skeletal, and smooth muscle, heart valve fibroblasts, and neuronal tissues, resulting in molecular

mimicry and a tolerant or suppressed immune response by the host. The cell envelope of a Group A

streptococcus is illustrated in Figure 1.22.

In Group A streptococci, the R and T proteins are used as epidemiologic markers and have no known

role in virulence. The group carbohydrate antigen (composed of N-acetylglucosamine and rhamnose)

has been thought to have no role in virulence, but emerging strains with increased invasive capacity

produce a very mucoid colony, suggesting a role of the capsule in virulence.

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Figure 22: Cell surface structure of Streptococcus pyogenes and secreted products involved in

virulence (Todar, 2009).

The M proteins are clearly virulence factors associated with both colonization and resistance to

phagocytosis. More than 50 types of S. pyogenes M proteins have been identified on the basis of

antigenic specificity, and it is the M protein that is the major cause of antigenic shift and antigenic

drift in the Group A streptococci. The M protein (found in fimbriae) also binds fibrinogen from

serum and blocks the binding of complement to the underlying peptidoglycan. This allows survival of

the organism by inhibiting phagocytosis.

The streptococcal M protein, as well as peptidoglycan, N-acetylglucosamine, and group-specific

carbohydrate, contain antigenic epitopes that mimic those of mammalian muscle and connective

tissue. As mentioned above, the cell surface of recently emerging strains of streptococci is distinctly

mucoid (indicating that they are highly encapsulated). These strains are also rich in surface M

protein. The M proteins of certain M-types are considered rheumatogenic since they contain

antigenic epitopes related to heart muscle, and they therefore may lead to autoimmune rheumatic

carditis (rheumatic fever) following an acute infection.

1.6.2.4 The Hyaluronic Acid Capsule

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The capsule of S. pyogenes is non-antigenic since it is composed of hyaluronic acid, which is

chemically similar to that of host connective tissue. This allows the bacterium to hide its own

antigens and to go unrecognized as antigenic by its host. The Hyaluronic acid capsule also prevents

opsonized phagocytosis by neutrophils or macrophages.

1.6.2.5 Adhesins

Colonization of tissues by S. pyogenes is thought to result from a failure in the constitutive defences

(normal flora and other nonspecific defence mechanisms) which allows establishment of the

bacterium at a portal of entry (often the upper respiratory tract or the skin) where the organism

multiplies and causes an inflammatory purulent lesion.

It is now realized that S. pyogenes (like many other bacterial pathogens) produces multiple adhesins

with varied specificities. There is evidence that Streptococcus pyogenes utilizes lipoteichoic acids

(LTA), M protein, and multiple fibronectin-binding proteins in its repertoire of adhesins. LTA is

anchored to proteins on the bacterial surface, including the M protein. Both the M proteins and

lipoteichoic acid are supported externally to the cell wall on fimbriae and appear to mediate bacterial

adherence to host epithelial cells (Todar, 2009). The fibronectin-binding protein, Protein F, has also

been shown to mediate streptococcal adherence to the amino terminus of fibronectin on mucosal

surfaces.

Identification of Streptococcus pyogenes adhesins has long been a subject of conflict and debate.

Most of the debate was between proponents of the LTA model and those of the M protein model. In

1972, Gibbons and his colleagues proposed that attachment of streptococci to the oral mucosa of

mice is dependent on M protein. However, Ofek and Beachey (1978) argued that lipoteichoic acid

(LTA), rather than M protein, was responsible for streptococcal adherence to buccal epithelial cells.

In 1996, Hasty and Courtney proposed a two-step model of attachment that involved both M protein

and teichoic acids. They suggested that LTA loosely tethers streptococci to epithelial cells, and then

M protein and/or other fibronectin (Fn)-binding proteins secure a firmer, irreversible association. The

first streptococcal fibronectin-binding protein (Sfb) was demonstrated in 1992. Shortly thereafter,

protein F was discovered. Most recently (1998), the M1 and M3 proteins were shown to bind

fibronectin (Todar, 2009).

1.6.2.6 Extracellular products: invasins and exotoxins

Colonization of the upper respiratory tract and acute pharyngitis may spread to other portions of the

upper or lower respiratory tracts resulting in infections of the middle ear (otitis media), sinuses

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(sinusitis), or lungs (pneumonia). In addition, meningitis can occur by direct extension of infection

from the middle ear or sinuses to the meninges or by way of bloodstream invasion from the

pulmonary focus. Bacteraemia can also result in infection of bones (osteomyelitis) or joints

(arthritis). During these aspects of acute disease the streptococci bring into play a variety of secretory

proteins that mediate their invasion.

For the most part, streptococcal invasins and protein toxins interact with mammalian blood and tissue

components in ways that kill host cells and provoke a damaging inflammatory response. The soluble

extracellular growth products and toxins of Streptococcus pyogenes (see Figure 1.22, above), have

been studied intensely. Streptolysin S is an oxygen-stable leukocidin; Streptolysin O is an oxygen-

labile leukocidin. NADase is also leukotoxic. Hyaluronidase (the original "spreading factor") can

digest host connective tissue hyaluronic acid, as well as the organism's own capsule. Streptokinases

participate in fibrin lysis. Streptodornases A-D possesses deoxyribonuclease activity; Streptodornases

B and D possess ribonuclease activity as well. Protease activity similar to that in Staphylococcus

aureus has been shown in strains causing soft tissue necrosis or toxic shock syndrome. This large

repertoire of products is important in the pathogenesis of S. pyogenes infections. Even so, antibodies

to these products are relatively insignificant in protection of the host.

Streptococcal invasins lyse eukaryotic cells, including red blood cells and phagocytes; they lyse other

host macromolecules, including enzymes and informational molecules; they allow the bacteria to

spread among tissues by dissolving host fibrin and intercellular ground substances.

1.6.2.7 Pyrogenic Exotoxins

Three streptococcal pyrogenic exotoxins (SPE), formerly known as Erythrogenic toxin, are

recognized: types A, B, C. These toxins act as superantigens by a mechanism similar to those

described for staphylococci. As antigens, they do not require processing by antigen presenting cells.

Rather, they stimulate T cells by binding class II MHC molecules directly and non-specifically. With

superantigens about 20% of T cells may be stimulated (vs. 1/10,000 T cells stimulated by

conventional antigens) resulting in massive detrimental cytokine release. SPE A and SPE C are

encoded by lysogenic phages; the gene for SPE B is located on the bacterial chromosome.

The erythrogenic toxin is so-named for its association with scarlet fever which occurs when the toxin

is disseminated in the blood. Re-emergence in the late 1980's of exotoxin-producing strains of S.

pyogenes has been associated with a toxic shock-like syndrome similar in pathogenesis and

manifestation to staphylococcal toxic shock syndrome, and with other forms of invasive disease

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associated with severe tissue destruction. The latter condition is termed necrotizing fasciitis.

Outbreaks of sepsis, toxic shock and necrotizing fasciitis have been reported at increasing frequency.

The increase in invasive streptococcal disease was associated with emergence of a highly virulent

serotype M1 which is disseminated world-wide. The M1 strain produces the erythrogenic toxin (Spe

A), thought to be responsible for toxic shock, and the enzyme cysteine protease which is involved in

tissue destruction. Because clusters of toxic shock were also associated with other serotypes,

particularly M3 strains, it is believed that unidentified host factors may also have played an important

role in the resurgence of these dangerous infections.

1.6.3 POST STREPTOCOCCAL SEQUELAE

Infection with Streptococcus pyogenes can give rise to serious nonsuppurative sequelae: acute

rheumatic fever and acute glomerulonephritis. These pathological events begin 1-3 weeks after an

acute streptococcal illness, a latent period consistent with an immune-mediated aetiology. Whether

all S. pyogenes strains are rheumatogenic is controversial; however, clearly not all strains are

nephritogenic (Todar, 2009).

Acute rheumatic fever is a sequel only of pharyngeal infections, but acute glomerulonephritis can

follow infections of the pharynx or the skin. Although there is no adequate explanation for the precise

pathogenesis of acute rheumatic fever, an abnormal or enhanced immune response seems essential. In

addition, persistence of the organism on pharyngeal tissues (i.e., the tonsils) is associated with an

increased likelihood of rheumatic fever. Also, acute rheumatic fever can result in permanent damage

to the heart valves. Less than 1% of sporadic streptococcal pharyngitis infections result in acute

rheumatic fever; however, recurrences are common, and life-long antibiotic prophylaxis is

recommended following a single case.

The occurrence of cross-reactive antigens in S. pyogenes and heart tissues possibly explains the

autoimmune responses that develop following some infections. The antibody mediated immune

(AMI) response (i.e., level of serum antibody) is higher in patients with rheumatic fever than in

patients with uncomplicated pharyngitis. In addition, cell-mediated immunity (CMI) seems to play a

role in the pathology of acute rheumatic fever.

Acute glomerulonephritis results from deposition of antigen-antibody-complement complexes on the

basement membrane of kidney glomeruli. The antigen may be streptococcal in origin or it may be a

host tissue species with antigenic determinants similar to those of streptococcal antigen (cross-

reactive epitopes for endocardium, sarcolemma, and vascular smooth muscle). The incidence of acute

glomerulonephritis in the United States is variable, perhaps due to cycling of nephritogenic strains,

but it appears to be decreasing. Recurrences are uncommon, and prophylaxis following an initial

attack is unnecessary.

1.6.4 HOST DEFENCES

S. pyogenes is usually an exogenous secondary invader, following viral disease or disturbances in the

normal bacterial flora. In the normal human the skin is an effective barrier against invasive

FIGURE 23: Pathogenesis of Streptococcus pyogenes infections. Adapted

from Baron's Medical Microbiology Chapter 13, Streptococcus by Maria

Jevitz Patterson.

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streptococci, and nonspecific defence mechanisms prevent the bacteria from penetrating beyond the

superficial epithelium of the upper respiratory tract. These mechanisms include mucociliary

movement, coughing, sneezing and epiglottal reflexes.

The host phagocytic system is a second line of defence against streptococcal invasion. Organisms can

be opsonized by activation of the classical or alternate complement pathway and by anti-

streptococcal antibodies in the serum. S. pyogenes is rapidly killed following phagocytosis enhanced

by specific antibody. The bacteria do not produce catalase or significant amounts of superoxide

dismutase to inactivate the oxygen metabolites (hydrogen peroxide, superoxide) produced by the

oxygen-dependent mechanisms of the phagocyte. Therefore, they are quickly killed after engulfment

by phagocytes. The streptococcal defence must be to stay out of phagocytes.

In immune individuals, IgG antibodies reactive with M protein promote phagocytosis which results

in killing of the organism. This is the major mechanism by which AMI is able to terminate Group A

streptococcal infections. M protein vaccines are a major candidate for use against rheumatic fever,

but certain M protein type’s cross-react antigenically with the heart and themselves may be

responsible for rheumatic carditis. This risk of autoimmunity has prevented the use of Group A

streptococcal vaccines. However, since the cross-reactive epitopes of the M-protein are now known,

it appears that limited anti-streptococcal vaccines are on the horizon.

FIGURE 24: Phagocytosis of Streptococcus pyogenes by a macrophage (Todar 2009).

The hyaluronic acid capsule allows the organism to evade opsonization. The capsule is also an

antigenic disguise that hides bacterial antigens and is non antigenic to the host. Actually, the

hyaluronic acid outer surface of S. pyogenes is weakly antigenic, but it does not result in stimulation

of protective immunity. The only protective immunity that results from infection by Group A

streptococcus comes from the development of type-specific antibody to the M protein of the fimbriae,

which protrude from the cell wall through the capsular structure. This antibody, which follows

lxviii

respiratory and skin infections, is persistent. Presumably, protective levels of specific IgA are

produced in the respiratory secretions while protective levels of IgG are formed in the serum.

Sometimes, intervention of an infection with effective antibiotic treatment precludes the development

of this persistent antibody. This accounts, in part, for recurring infections in an individual by the

same streptococcal strain. Antibody to the erythrogenic toxin involved in scarlet fever is also long

lasting.

1.6.5 ADHERENCE (COLONIZATION) SURFACE MACROMOLECULES

The following are the adherence surface macromolecules of S. pyogenes to the host; M protein,

Lipoteichoic acid (LTA), Protein F and Sfb (fibronectin-binding proteins)

The defence against host immune responses are antigenic disguise and tolerance provided by

hyaluronic acid capsule.

1.6.6 PRODUCTION OF TOXINS AND OTHER SYSTEMIC EFFECTS

Toxic shock: Exotoxin is superantigen that binds directly to MHC II (without being processed) and

binds abnormally to the T cell receptor of many (up to 20% of) T cells. Exaggerated production of

cytokines causes the signs of shock: fever, rash, low blood pressure, aberrant interaction between

toxin, macrophage, and T cells. Induction of circulating, cross-reactive antibodies: some of the

antibodies produced during infection by certain strains of streptococci cross-react with certain host

tissues. These antibodies can indirectly damage host tissues, even after the organisms have been

cleared, and cause autoimmune complications.

1.6.7 SUPPURATIVE CONDITIONS

Active infections associated with S. pyogenes pus occur in the throat, skin, and systemically.

A. Throat

Streptococcal pharyngitis is acquired by inhaling aerosols emitted by infected individuals. The

symptoms reflect the inflammatory events at the site of infection. A few (1-3%) people develop

rheumatic fever weeks after the infection has cleared.

B. Skin

Impetigo involves the infection of epidermal layers of skin. Pre-pubertal children are the most

susceptible. Cellulitis occurs when the infection spreads subcutaneous tissues. Erysipelas is the

infection of the dermis. About 5% of patients will develop more disseminated disease. Necrotizing

fasciitis involves infection of the fascia and may proceed rapidly to underlying muscle.

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C. Systemic

Scarlet fever is caused by production of erythrogenic toxin by a few strains of the organism. Toxic

shock is caused by a few strains that produce a toxic shock-like toxin.

1.6.8 NON-SUPPURATIVE SEQUELAE

Some of the antibodies produced during the above infections cross-react with certain host tissues.

These can indirectly damage host tissues, even after the organisms have been cleared, and cause non

suppurative complications. Rheumatic fever: M protein cross reacts with sarcolemma. Antibodies

cross-react with heart tissue, fixes complement, and cause damage. Glomerulonephritis: Antigen-

antibody complexes may be deposited in kidney, fix complement, and damage glomeruli.

1.6.9 TREATMENT AND PREVENTION

Penicillin is still uniformly effective in treatment of Group A streptococcal disease. It is important to

identify and treat Group A streptococcal infections in order to prevent sequelae. No effective vaccine

has been produced, but specific M-protein vaccines are being tested.

1.7.0 MECHANISMS OF ANTIBIOTIC RESISTANCE IN BACTERIA

Several mechanisms have evolved in bacteria which confer them with antibiotic resistance. These

mechanisms can chemically modify the antibiotic, render it inactive through physical removal from

the cell, or modify target site so that it is not recognized by the antibiotic (Todar, 2008).

The most common mode is enzymatic inactivation of the antibiotic. An existing cellular enzyme is

modified to react with the antibiotic in such a way that it no longer affects the microorganism. An

alternative strategy utilized by many bacteria is the alteration of the antibiotic target site. These and

other mechanisms are shown in the figure and accompanying table below.

Figure 25: Mechanisms of antibiotic resistance in bacteria

Table 1.2 Mechanisms of antibiotic resistance in bacteria

Antibiotic Method of resistance

Chloramphenicol reduced uptake into cell

Tetracycline active efflux from the cell

�-lactam, Erythromycin,

Lincomycin eliminates or reduces binding of antibiotic to cell target

�-lactams,

Aminoglycosides,

Chloramphenicol

enzymatic cleavage or modification to inactivate antibiotic

molecule

Sulphonamides,

Trimethoprim metabolic bypass of inhibited reaction

Sulphonamides,

Trimethoprim overproduction of antibiotic target (titration)

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1.7.1 THE ACQUISITION AND SPREAD OF ANTIBIOTIC RESISTANCE IN

BACTERIA

The development of resistance is inevitable following the introduction of a new antibiotic. Initial

rates of resistance to new drugs are normally on the order of 1%. However, modern uses of

antibiotics have caused a huge increase in the number of resistant bacteria. In fact, within 8-12 years

after wide-spread use, strains resistant to multiple drugs become widespread. Multiple drug resistant

strains of some bacteria have reached the proportion that virtually no antibiotics are available for

treatment.

Antibiotic resistance in bacteria may be an inherent trait of the organism (e.g. a particular type of cell

wall structure) that renders it naturally resistant, or it may be acquired by means of mutation in its

own DNA or acquisition of resistance-conferring DNA from another source.

A. Inherent (natural) resistance: Bacteria may be inherently resistant to an antibiotic. For example,

an organism lacks a transport system for an antibiotic; or an organism lacks the target of the

antibiotic molecule; or, as in the case of Gram-negative bacteria, the cell wall is covered with an

outer membrane that establishes a permeability barrier against the antibiotic.

B. Acquired resistance: Several mechanisms are developed by bacteria in order to acquire resistance

to antibiotics. This require, either the modification of existing genetic material or the acquisition of

new genetic material from another source.

C. Vertical gene transfer: The spontaneous mutation frequency for antibiotic resistance is on the

order of about of about 10-8- 10-9. This means that one in every 108- 109 bacteria in an infection will

develop resistance through the process of mutation. In E. coli, it has been estimated that streptomycin

resistance is acquired at a rate of approximately 10-9 when exposed to high concentrations of

streptomycin. Although mutation is a very rare event, the very fast growth rate of bacteria and the

absolute number of cells attained means that it doesn't take long before resistance is developed in a

population.

Once the resistance genes have developed, they are transferred directly to all the bacteria's progeny

during DNA replication. This is known as vertical gene transfer or vertical evolution. The process is

strictly a matter of Darwinian evolution driven by principles of natural selection: a spontaneous

mutation in the bacterial chromosome imparts resistance to a member of the bacterial population. In

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the selective environment of the antibiotic, the wild type (non mutants) is killed and the resistant

mutant is allowed to grow and flourish.

D. Horizontal gene transfer: Another mechanism beyond spontaneous mutation is responsible for

the acquisition of antibiotic resistance. Lateral or horizontal gene transfer (HGT) is a process

whereby genetic material contained in small packets of DNA can be transferred between individual

bacteria of the same species or even between different species.

There are at least three possible mechanisms of HGT, equivalent to the three processes of genetic

exchange in bacteria. These are transduction, transformation or conjugation.

Conjugation occurs when there is direct cell-cell contact between two bacteria (which need not be

closely related) and transfer of small pieces of DNA called plasmids takes place. This is thought to be

the main mechanism of HGT.

Transformation is a process where parts of DNA are taken up by the bacteria from the external

environment. This DNA is normally present in the external environment due to the death and lysis of

another bacterium.

Transduction occurs when bacteria-specific viruses (bacteriophages) transfer DNA between two

closely related bacteria.

lxxiii

Figure 26: Mechanisms of horizontal gene transfer (HGT) in bacteria (Todar, 2009).

The combined effects of fast growth rates to large densities of cells, genetic processes of mutation

and selection, and the ability to exchange genes, account for the extraordinary rates of adaptation and

evolution that can be observed in the bacteria. For these reasons bacterial adaptation (resistance) to

the antibiotic environment seems to take place very rapidly in evolutionary time. Bacteria evolve

fast!

1.8 RESEARCH OBJECTIVES.

Zapoteca portoricensis has been used by traditional herbalist to treat tonsillitis, an ailment caused

mainly by Streptococcus pyogenes. The organism also has been resistant to penicillin which is the

common antibiotic used against it. In search for a potent antibiotic this plant was considered a

candidate, since it has been used to treat tonsillitis. The objectives of the research include to:

1. Determine the phytochemical constituents of the plant root.

2. Isolate of an antibiotics against Streptococcus pyogenes.

3. Characterize the active compounds and the functional groups present.

4. Determine of the possible mechanism of action of the compound on both gram-negative and

gram-positive bacteria.

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Plant Materials

2.1.1.1 Field collection of plant material: Plant roots were collected from areas surrounding Nru

village, Nsukka. The plant was identified by Mr Alfred Ozioko of the Department of Botany,

University of Nigeria, Nsukka. The materials were cleaned off adhering soil and dust in the field by

shaking and were dried at room temperature for one week.

2.1.2 Instruments/Equipment

Test tubes

Scientific 210 VGP, Buck

Beakers Pyrex

Bijou bottle

Conical Flasks Pyrex

Filter paper Wattman, England

lxxiv

Glass funnel Kimax

Hot plate Gallenkamp

Measuring Cylinder Pyrex England

Micro Pipette Perfect (p) U.S.A

Incubator Fisher

Oven, Model 301 Fisher

pH Meter Pyeunicam 293, England

Electric balance

Swap stick

Pipette Pyrex

Petri dishes Pyrex

Refrigerator Haier Thermocool

Spatula Pyrex

Spectrophotometer

Autoclave

Rotary evaporator

Spectronic 20-Model,

Bausch & Comb,

Gallenkamp.

Infrared. Spectrometer Gallenkamp, England

Syringe (1 ml, 2 ml) DANA JET, Nigeria

Nuclear Magnetic Rasonance Spectrometer Gallenkamp, England

2.2 METHODS

2.2.1 Extraction Procedure

The roots were washed and air dried at room temperature for two weeks. The dried roots were

pulverized using a mechanical grinder and the weight of the root powder was 550g. The powder was

soaked (macerated) in 3.5 litres of absolute ethanol. The mixture was kept for three days under room

temperature. Filtration was done using Wattman No 1 filter paper. The resulting extract was

concentrated under fan at room temperature to avoid denaturation of the active ingredients to obtain a

semi-solid mass. The extract was stored in a refrigerator at temperature below 0oC until it was needed

for analysis.

2.2.2 Determination of Extract Yield

The percentage yield of the extracts was determined by weighing the pulverized dry leaves

before extraction and the concentrated extracts obtained after extraction and then calculated using the

formula:

Percentage yield =

2.2.3 Fractionation of the crude extract

The crude ethanol extract was fractionated using serial solvent fractionation method (Jamil et al.,

2009). The dried crude extract weighed 6.5g. This dried extract was mixed with silica gel (60GF for

column), in the ratio of 1:2 (w:w). The solvents: chloroform, ethyl acetate, acetone and ethanol

respectively, were used to wash the mixture. For a particular solvent the washing was done until a

colourless filtrate was obtained, then the solvent was changed to another one in the order above, after

air drying the mixture. The fractions were concentrated by allowing the solvents to evaporate under

room temperature. The chart below shows a brief outline of the fractionation procedure.

2.4 Preliminary Phytochemical Analysis

Weight (g) of extract

Weight (g) of pulverized roots 100%

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2.2.4.0 Preliminary Phytochemical Analysis

The crude ethanol extract of the roots were subjected to phytochemical analysis according to the

method outlined by Harborne (1998). The phytochemical analysis was done to detect the presence of

secondary metabolites, such as alkaloids, tannins, saponins, resins, flavonoids, steroids, glycosides

and terpenoids.

2.2.4.1 Preparation of Reagents for Phytochemical and Macronutrient Analyses

� Ammonium Solution: A quantity, 187.5 ml, of the stock concentrated ammonium solution was

dissolved in 31.25 ml of distilled water and made up to 500 ml.

� 45% Absolute Ethanol: A quantity, 45 ml of absolute ethanol was mixed with 55 ml of distilled

water.

� 0.5% Aluminum Chloride Solution: A quantity, 0.5 g of aluminum chloride was dissolved in

some distilled water and made up to 100 ml.

� Dilute Sulphuric Acid: Concentrated sulphuric acid, 10.9 ml, was mixed with 5 ml of distilled

water and made up to 100 ml.

� Dragendorff’s Reagent: Bismuth carbonate, 0.85 g, was dissolved in 100 ml of glacial acetic

acid and 40 ml of distilled water to give solution A. Another solution called solution B was

prepared by dissolving 8.0 g of potassium iodide in 20 ml of distilled water. Both solutions were

then mixed to give a stock solution.

� 5% Ferric Chloride Solution: Ferric chloride, 5 g was dissolved in a small amount of distilled

water and made up to 100 ml with distilled water

� 2% Hydrochloric Acid: A volume of 2 ml of concentrated hydrochloric acid was dissolved in a

small amount of distilled water and made up to 100 ml with distilled water

� 1% Hydrated Copper Sulphate: Anhydrous copper sulphate, 1 g, was dissolved in a small

amount of distilled water and made up to 100 ml with distilled water

� Lead Sub-acetate Solution: B volume of 45 ml, of 15% lead acetate solution (i.e. 7.5 g of lead

acetate in 50 ml of distilled water) was dissolved in 20 ml of absolute ethanol and 35 ml of

distilled water.

� Mayer’s Reagent: Mercuric chloride, 1.35 g, was dissolved in 50 ml of distilled water. Also, 5g

of potassium iodide was dissolved in 20 ml of distilled water. The solutions were mixed and the

volume made up to 100 ml.

lxxvii

� Molisch Reagent: One gram of -naphthol was dissolved in 100 ml of ethanol.

� 40% Sodium Hydroxide: A volume of 40 ml of concentrated sodium hydroxide was diluted

with 60 ml of distilled water.

� Wagner’s Reagent: A quantity, 2 g of iodine crystals and 3 g of potassium iodide were

dissolved in a small amount of distilled water and made up to 100 ml with distilled water

� 2.4.1 Test for Alkaloids

A quantity of 0.2 g of the sample was added to 5 ml of 2% hydrochloric acid and heated on a boiling

water bath for 3 minutes, it was allowed to cool and then filtered. A portion of the filtrate (1ml) was

treated with 2 drops of the following reagents.

i. Dragendorff’s reagent: a red precipitate indicates the presence of alkaloids.

ii. Mayer’s reagent: a creamy white coloured precipitate indicates the presence of alkaloids.

iii. Wagner’s reagent: a reddish-brown precipitate indicates the presence of alkaloids.

iv. Picric acid (1%): a yellowish precipitate indicates the presence of alkaloids.

2.4.2 Test for Glycosides

i. Fehling’s test: A quantity, 2 g of sample was mixed with 30 ml of water and heated on a water

bath for 5 minutes and filtered. About 5 ml of a mixture of equal parts of Fehling’s solutions A and B

was added to 5 ml of the filtrate until it turned alkaline (tested with litmus paper) and then boiled on a

water bath for 5 minutes. A brick-red precipitate indicates the presence of glycosides.

ii. Hydrolytic test: A volume of 5 ml of dilute hydrochloric acid was added to 0.1 g of the sample

in a test tube and boiled for 15 minutes on a water bath, then cooled and neutralized with 20%

potassium hydroxide solution. A volume, 10 ml of a mixture of equal parts of Fehling’s solutions A

and B was added and boiled for 5 minutes. A more dense brick-red precipitate indicates the presence

of glycosides.

2.4.4 Test for Flavonoids

Ethyl acetate, 10 ml, was added to 0.2 g of the sample and heated on a water bath for 3 minutes. The

mixture was allowed to cool, filtered and the filtrate used for the following tests.

i. Ammonium test: A volume of 4 ml of the filtrate was shaken with 1 ml of dilute ammonia

solution. The layers were allowed to separate. A yellow colour in the ammoniacal layer indicated the

presence of flavonoids.

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lxxviii

ii. Aluminium chloride test: A portion of the filtrate, 4 ml, was shaken with 1 ml of 1% aluminium

chloride solution. The layers were allowed to separate. A yellow colour in the aluminium chloride

layer indicated the presence of flavonoids.

2.4.5 Test for Resins

i. Precipitate test: A quantity of 0.2 g of the sample was extracted with 15ml of 96% ethanol. The

alcoholic extract was then poured into 20 ml of distilled water in a beaker. A precipitate occurring

indicated the presence of resins.

ii. Colour test: A quantity, 0.2 g of the sample was extracted with chloroform and the extract

concentrate to dryness. The residue was redissolved in 3 ml of acetone and 3 ml of concentrated

hydrochloric acid was added. The mixture was heated in a water bath for 30 minutes. A pink colour

which changes to magenta red indicated the presence of resins.

2.4.6 Test for Tannins

A quantity, 2 g of the sample was boiled with 5 ml of 45% ethanol for 5 minutes. The mixture was

cooled and then filtered; the filtrate was then treated with the following solutions.

i. Lead sub-acetate solution: To 1 ml of the filtrate 3 drops of lead sub acetate solution was added.

A red gelatinous precipitate indicated the presence of tannins.

ii. Bromine water: To 1 ml of the filtrate, 0.5 ml of bromine water was added and then observed for

a pale brown precipitate.

iii Ferric chloride solution: A volume of 1 ml of the filtrate was diluted with distilled water and

then 2 drops or ferric chloride were added. A transient greenish to black colour indicated the presence

of tannins.

2.4.7 Test for Saponins

A quantity of 0.1 g of sample was boiled with 5 ml of distilled water on a water bath for 5mitues. The

mixture was filtered hot and allowed to cool. The filtrate was used for the following tests.

i. Emulsion test: To 1 ml of the filtrate, 2 drops of olive oil was added and the mixture shaken

vigorously. The formation of emulsion indicates the presence of Saponins.

ii. Frothing test: A volume of 1 ml of the filtrate was diluted with 4 ml of distilled water. This

mixture was shaken vigorously and then observed on standing for a stable froth.

2.4.8 Test for Terpenoids and Steroids

A volume, 9 ml of absolute ethanol was added to l g of sample and refluxed for 5 minutes and

filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath and then 5 ml of hot water

was added. The mixture was allowed to stand for 1 hour and the waxy matter was filtered off. The

filtrate was extracted with 2.5 ml of chloroform using a separating funnel. To 0.5ml of the

lxxix

chloroform extract in a test tube was carefully added 1 ml of concentrated sulphuric acid to form a

lower layer. A reddish brown interface shows the presence of steroids.

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and heated with

3 ml of concentrated sulphuric acid for 10 minutes on a water bath. A grey colour indicates the

presence terpenoids.

2.4.9 Test for Acidic Compounds

A quantity, 0.l g was placed in a clean dry test tube and sufficient water added. This was warmed in a

hot water bath and then cooled. A piece of water-wetted (moist) litmus paper was dipped into the

filtrate and the colour change on the litmus paper observed.

2.4.10 Macronutrient Analyses

The tests below were carried out to determine the presence of macronutrients in the fresh leaves of

the plant samples.

2.4.10.1 Test for Proteins

A volume, 5 ml of distilled water was added to 0.1 g of the samples. This was left to stand for 3 hours

and then filtered. The filtrate was used for the following tests.

i Millons Test: To 2 ml portion of the filtrate was added 0.1 ml Millon’s reagent. It was shaken

and kept for observation. A yellow precipitate indicates the presence of proteins.

ii Biurette Test: Another 2 ml portion of the filtrate was put in a test tube and 5 drops of 1%

hydrated copper sulphate was added. A volume of 2 ml of 40% sodium hydroxide was also added

and the test tube shaken vigorously to mix the contents. A purple colouration shows the presence of

proteins (presence of two or more peptide bonds).

2.4.10.2 Test for Carbohydrate

A quantity, 0.1 g of the sample was shaken vigorously with water and then filtered. To the aqueous

filtrate was added few drops of Molisch reagent, followed by vigorous shaking again. Then, 1 ml of

concentrated sulphuric acid was carefully added through the side of the test tube to form a layer

below the aqueous solution. A brown ring at the interface indicates the presence of carbohydrate.

2.4.10.3 Test for Reducing Sugar

A quantity, 0.1 g of sample was shaken vigorously with 5ml of distilled water and filtered. Equal

volumes of Fehling’s solutions A and B were added to 1 ml portion of the filtrate. The mixture was

shaken vigorously. A brick red precipitate indicates the presence of reducing sugars.

2.2.10.4 Test for Fats and Oil

lxxx

A quantity of 0.1 g of sample was pressed between filter paper and the paper observed. A control was

also prepared by placing 2 drops of olive oil on filter paper. Translucency of the filter paper indicates

the presence of fats and oil.

2.5 Test microorganisms

The clinical isolates were obtained from the Faculty of Veterinary Medicine, University of Nigeria,

Nsukka, Enugu State, Nigeria. Test microorganisms consisted of Streptococcus pyogenes,

Streptococcus pneumonia, Bacillus subtilis, Salmonella typhi, Staphylococcus aureus, E. coli,

Klebsiella pneumonia.

2.5.1 Preparation of culture media

The materials for the experiment were sterilised in an autoclave. They were allowed to cool before

being used. Nutrient agar, pH 7.4 (a product of Oxoid laboratories, England) was used in the study.

A quantity, 5.6g of dried nutrient agar was weighed. This was dissolved in 200ml of water. The

suspension was well mixed by stirring in the water. The solution was heated to dissolve and to purify

media. The media were sterilised by autoclaving at 50oC. The plates were thereafter inoculated with

test microorganisms and incubated at 37oC for 24 hours.

2.5.2 Determination of antimicrobial activity

The antimicrobial activities of the crude extract were determined using NCCLS method (WHO,

2000). For determination of antibacterial activity, bacterial cultures were adjusted to 0.5 McFarland

turbidity standards and inoculated to 15cm diameter nutrient agar (Oxoid) plates. The crude ethanol

extract of the plant’s roots was dissolved in dimethyl sulfoxide (DMSO); (500mg of the extract was

constituted with 5ml of 100% DMSO to prepare stock solution). The concentration of stock was

100mg/ml. Different concentrations (50mg/mL, 25mg/mL, 12.5mg/mL, 6.25mg/mL), of the plant

root extract were prepared using serial dilution in DMSO. The control had DMSO alone without any

extract. The method used was agar well diffusion method (Arwa et al., 2008). Susceptibility testing

was carried out by measuring the inhibitory zone diameters on the media. The inhibitory zone

diameters were rounded of to the nearest whole numbers (mm) for analysis. The sensitivity of extract

was compared to that a standard antibiotic, streptomycin. The antimicrobial activities of the different

fractions, each at a concentration 100mg/ml, were carried out, using the method described above.

2.5.3 Agar well diffusion method

The assay was conducted as described by Perez et al. (1990). Briefly, microorganisms from growth

on nutrient agar incubated at 37°C for 18 h were suspended in saline solution, 0.85% NaCl and

lxxxi

adjusted to a turbidity of 0.5 Mac Farland standards (108 cfu/ml). The suspension was used to

inoculate 15cm diameter Petri plates with a sterile non toxic cotton swab on a wooden applicator. Six

millimetre diameter wells were punched in the agar and filled with fractions. The dissolution of the

extract was aided by 1% (v/v) DMSO which did not affect microorganisms’ growth, according to our

control experiments. Commercial antibiotic, streptomycin was used as positive reference standard to

determine the sensitivity of the strains. The extract was introduced into the wells. Plates were

incubated in at 37°C for 24 hr. Antibacterial activities were evaluated by measuring inhibition zone

diameters. The experiments were conducted twice. All tests were performed in duplicate and the

antibacterial activity was expressed as the mean diameter of inhibition zones (mm) produced by the

plant root extracts.

2.6 Use of Ultraviolet, Infra-Red and NMR spectroscopy to determine the functional groups in

the ethanol fraction

Ultraviolet/visible spectroscopy was carried out on the fraction to determine the functional group

present in the fraction. The extract was dissolved in ethanol and ethanol was used as a control.

Readings were taken, starting from 200nm to 350nm, at the intervals of 50nm. Infrared spectra were

obtained between the frequencies: 399.19 to 3999.64, the sensitivity is 50.00. The infrared

spectroscopy was done at the Central Research Laboratory, University of Uyo, Akwa-Ibom, Nigeria.

Proton NMR spectra were obtained in DMSO, at frequency of 199.9671071MHz at ambient

temperature. 13C-Specrta was obtained in DMSO, at frequency of 50.2817571MHz, at ambient

temperature. Two-dimensional NMR experiment was performed in the same solvent as were 13C

experiment.

CHAPTER THREE

RESULTS

3.1 EXTRACTION AND FRACTIONATION

lxxxii

The percent extract yield of the plant roots was 1.56%. After fractionation the following fractions

were obtained: chloroform fraction, acetone fraction, ethyl acetate fraction and ethanol fraction.

3.2 Result of phytochemical analysis and macronutrients in the crude extract of the plant

roots

Phytochemical screening of the plant roots showed that the plant contains alkaloids, glycosides,

reducing sugar, carbohydrates, steroids, terpenoids, saponins, proteins, and fats and oil. The roots

lacked flavonoids, tannins and acidic compounds. The chemical classes of compounds in the plant

roots as indicated by the phytochemical analysis are listed in Table 3.

Table 3: Phytochemicals present in the crude extract of the plant’s roots.

PHYTOCHEMICAL

COMPOUNDS

RESULTS

Alkaloids ++++

Glycosides +++

Reducing sugar ++

Carbohydrate +++

Flavonoids -

Fat and oil +

Steroids ++++

Terpenoids ++++

Protein +

Tannins -

Saponins +

Resins +++ Key; - : Absent, +: Present, ++ : Low concentration, +++ : Moderate Concentration, ++++: High concentration. 3.2 The antimicrobial activities of the crude ethanol extract.

Table 3.3 indicates the inhibition zone diameter (IZD) of the crude ethanol extract on the

microorganisms used. The crude ethanol extract of the plant roots was found to possess antimicrobial

activities on both Gram-positive and Gram-negative bacteria. The crude extract was active on S.

Pyogenes, Streptococcus pneumonia, Klebsiella pneumonia, Staphylococcus aureus, Salmonella

typhi and E. coli. The crude extract was not active on Bacillus subtilis. There were significant

differences (p<0.05) between the standard antibiotics and crude extract.

Table 4: Antimicrobial activity of different concentrations of the crude ethanol extract.

MICROORGANISMS

INHIBITORY ZONE DAIMETER (IZD ) OF DIFFERENT CONCENTRATUIONS IN mm

100mg/ml

50mg/ml

25mg/ml

12mg/ml

6.25mg/ml

DMSO

STREPTOMYCIN

(at 10.0µg/ml)

Bacillus subtilis - - - - - - 15.50

Staphylococcus aureus 9.0 8.0 0.6 - - - 15.0

Streptococcus pyogenes 11.0 9.0 6.0 - - - 18.50

Streptococcus pneumonia 11.0 10.0 10.0 8.5 8.0 - 5.5

Escherichia coli 16.5 11.0 - - - - 18.0

Klebsiela pneumonia 10.1 7.4 6.0 - - - 12.0

Salmonella typhi 16.5 12.0 10.0 10.0 10.0 - 10.0

3.3 The Minimum Inhibitory Concentrations (MIC) of the crude ethanol

MICs of the crude extract are 25mg/ml, 100mg/ml, 6.25mg/ml, 50mg/ml, 25mg/ml 6.25mg/ml for

Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia, E. coli, Klebsiella

pneumonia and Salmonella typhi respectively (Table 5).

Table 5: Minimum inhibitory concentrations (MIC) of the crude ethanol extract

MICROORGANISMS MIC (mg/ml)

Bacillus subtilis -

Staphylococcus aureus 25mg/ml

Streptococcus pyogenes 25mg/ml

Streptococcus pneumonia 6.25mg/ml

Escherichia coli 50mg/ml

Klebsiella pneumonia 25mg/ml

Salmonella typhi 6.25mg/ml

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MINIMUM INHIBITION CONCENTRATION

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lxxxiv

3.4 Antimicrobial activities of the fractions

The chloroform fraction was active on Staphylococcus aureus and on Streptococcus pneumonia, but

it was not active on other microorganisms. The ethyl acetate fraction was active only on

Streptococcus pneumonia while acetone fraction did show any significant activity on the

microorganisms used. The ethanol fraction showed significant activity on all the bacteria. Only

ethanol fraction of the crude extract was active on both Gram-negative and Gram-positive bacteria

(Table 6).

TABLE 6. INHIBITORY ZONE DIAMETER OF THE FRACTIONS (100mg/ml) MICROORGANISMS

INHIBITION ZONE DIAMETER OF DEFFERENT FRACTION (mm)

CHLOROFORM FRACTION

ETHYL ACETATE FRACTION

ACETONE FRACTION

ETHANOL FRACTION

STREPTOMYCIN

(at 10.0µg/ml)

Bacillus subtilis - - - 21.0 15.50

Staphylococcus aureus 13.0 - - 18.0 15.0

Streptococcus pyogenes - - - 25.0 18.50

Streptococcus pneumonia 13.0 - - 13.0 5.5

Escherichia coli - - - 11.0 18.0

Klebsiella pneumonia - 11.0 - 20.5 12.0

Salmonella typhi - - - 22.0 10.0

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Figure 28: Inhibition Zone Diameter of chloroform fraction on the microorganisms used.

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Figure 29: Inhibition Zone Diameter of ethyl acetate fraction on the microorganisms used.

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Figure 31: Inhibition Zone Diameter of ethanol fraction on the microorganisms used.

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Fig.33: (A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show that there were no growth inhibition by acetone, ethyl acetate and chloroform fractions on the organism, pyogenes

A

B

C D

Fig.33: (A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show thatwere no growth inhibition by acetone, ethyl acetate and chloroform fractions on the organism, Salmonella typhi

A

B

C

D

Fig.34: (A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show thatthere were no growth inhibition by acetone, ethyl acetate and chloroform fractions on the organism, Klebsiella pneumonia

A

B

C

D

A

B

C

D

(A) Shows growth inhibition by ethanol fraction , (B), (C), were no growth inhibition by acetone, ethyl

acetate and chloroform fractions on the organism, Streptococcus

(A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show that therewere no growth inhibition by acetone, ethyl acetate and chloroform fractions on the

(A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show thatwere no growth inhibition by acetone, ethyl acetate and chloroform fractions on

there

(A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show that were no growth inhibition by acetone, ethyl acetate and chloroform fractions on

3.5 ULTRAVIOLET, INFRA-RED

When scanned in ultraviolet spectrophotometer, the extract absorbed maximally at

199.9671071 MHz was observed and the highest peaks were between 2

peaks between 180-56 ppm. It was noticed that the following group

aromatic group, sulfuryl group, halogen group, carboxyl group and some double and triple bonded

carbons (Fig. 7-10).

Fig.36: (A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show thatwere no growth inhibition by acetone, ethyl acetate and chloroform fractions on the organism, Streptococcus species

A

B

C

D

ED AND NUCLEAR MEGNETIC RESONANCE

When scanned in ultraviolet spectrophotometer, the extract absorbed maximally at

199.9671071 MHz was observed and the highest peaks were between 2-6 ppm. The

56 ppm. It was noticed that the following groups are present in the fraction;


(A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show thatwere no growth inhibition by acetone, ethyl acetate and chloroform fractions on the

ESONANCE SPECTRA

250nm. The H1 at

6 ppm. The 13CNMR showed

are present in the fraction;


(A) Shows growth inhibition by ethanol fraction , (B), (C), (D), show that there were no growth inhibition by acetone, ethyl acetate and chloroform fractions on the

Table 7

Peak results for Ethanol fraction of the roots

Frequency: 399.19-3999.64, threshold: 26.186, sensitivity: 50

Peak result table:

Peak 1

Fig.38: INFRARED SPECTRA

xc

Position 3391.32

Height 14.413

TABLE 8.

THE SUSPECTED FUNCTIONAL GROUPS FROM INFRARED SPECTRA

Absorption

maximum cm-1

Function groups Structures

3400 Hydroxyl group O-H

3000-2800 Aldehyde, CH stretch C-H, C=O, CH2 , CH3 , C=O

H

1800-1600 Carbonyl groups C=O

2200-2000 Double, cyanide, C=C, C=N

1200-1000 Primary amine -NH2

1500-1400 Benzene ring C=C of benzene ring

PPM FREQUENCY FUNCTIONAL GROUP STRUCTURE

Fig. 40: 13C NMR SPECTRA OF ETHANOL FRACTION OF THE PLANT ROOTS EXTRACT. Pulse Sequence: apt, Solvent: cdc13, Ambient temperature, Mercury-200BB “oauife”, 1st pulse 180.0 degrees, 2nd pulse 92.9 degrees, Acq. Tme 1.498 sec, Width 12500.0 Hz, 3000 repetitions, OBSERVE 13C, 50.2817571MHz. DECOUPLE H1, 199.9671274 MHz, Power 39 dB on during acquisition. WALTZ-16 modulated, DATA PROCESSING, Line broadening 1.0Hz, Total time 1hr, 25min, 2 sec.

Fig. 39: 13C NMR SPECTRA OF ETHANOL FRACTION OF THE PLANT ROOTS EXTRACT. Pulse Sequence: s2pul, Solvent: DMSO, Ambient temperature, Mercury-200BB “oauife”, pulse 92.9 degrees, Acq. Tme 1. 498 sec, Width 12500.0 Hz, 2000 repetitions, OBSERVE C13, 50.2817571MHz. DECOUPLE H1, 199.960773MHz, Power 39 dB, continuosly on WALTZ -16 modulated DATA PROCESSING, Line broadening 1.0 Hz, FT size 65536, Total time 56min, 5 sec.

xcii

TABLE 9. THE SUSPECTED FUNCTIONAL GROUP FROM NMR SPECTRA

CHAPTER FOUR

DISCUSSION AND CONLUSION

4.1 DISCUSSION

Streptococcus pyogenes is one of the most common and ubiquitous human pathogens. It is

responsible for the majority of cases of sore throat in paediatric patients. Also, it is the causative

agent of some severe life threatening infections (sepsis, necrotizing fasciitis, toxic shocks syndrome)

and non-suppurative sequelae such as rheumatic fever and acute glomerulonephritis

(Descheermaeker et al., 2000). The choice of antibiotic for the treatment of streptococcal pharyngitis

40-50 38.883,40.3,40.717,39.048 DMSO (SOLVENT)

60-80 60.284,61.126,-83.281 SULFULRYL, CARBOXYL,

HALIDES, AMIDE, AMINE,

CH-N-, CH2-O-,

C-S-, CH-S-, C-O-,

C-Hal, - NH2

80-100 104.599 ALKENES C= C

160-180 AROMATIC GROUP Benzene ring

xciii

is penicillin, due to its proven efficacy, safety, and low cost. For patients allergic to penicillin,

erythromycin or other macrolides are the drugs of choice (Yamanaka, 2004). Lately, however, an

increase in macrolide resistance has been observed in different countries.

Over the last 20 years, Streptococcus pnuemonniae resistant to penicillin and other antibiotics such as

microlides have become a global therapeutic problem. Microlide resistnce in S. pnuemonniae is

generally due to either target site modification by erm methylases or active efflux (Farrell et al.,

2006). These modifications have resulted in two major phenotypes, designated as MLSB and M. In

the MLSB phenotype methylation of 23 S rRNA cuases resistance to microlide, lincosamide and

streotogramin B antibiotics. MLSB phenotype can be constitutive or inducible. The M phenotype, on

the other hand, is due to the presence of mef genes and causes resistance to microlides only. Target

modification by ribosomal gene mutation is a relatively rare mechanism (Gulay et al., 2008).

This increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of

strains with reduced susceptibility to antibiotics raises the spectre of untreatable bacterial infections

and adds urgency to the search for new infection-fighting strategies (Sieradski et al. 1999).

According to a study performed by the WHO based on publications on pharmacopoeias and

medicinal plants in 91 countries, the number of medicinal plants is nearly 20,000. These plants if

properly purified could provide a number of medicinal phytochemical compounds. The

characteristics of the plants that inhibit the growth/activities of microorganisms and are important for

human health have been researched in laboratories since 1926. Traditional medical treatments in

daily life are now being used with empiric methods. Zapoteca portoricensis is one these numerous

plant used by traditional herbalist to treat sore throat. Over the years the efficacy of the roots of this

plant in treating this ailment has been proven. Since the ethanol fraction of the crude extract of the

root of the plant, Zapoteca portoricensis was active on Streptococcus pyogenes, isolation of the

active compound could lead to the discovery of antibiotic against S. pyogenes. This plant has been

used by traditional herbalists in Nsukka of Enugu State to treat tonsillitis. The root part of this has

been very effective in this regard. This is why the plant root was considered as a potential source of

an antibiotic agent against Streptococcus pyogenes, the organism implicated in tonsillitis. The root of

this plant, having been used by traditional medical practitioners to cure sore throat, could be a

possible source of antibiotic against S. pyogenes and related bacteria.

The ethanol fraction of the plant root extract was active on Streptococcus pyogenes, Streptococcus

pneumonia, Salmonella typhi, Staphylococcus aureus, E. coli and Klebsiella pneumonia, as shown in

xciv

Table 6. Since, the ethanol fraction was active on both gram-negative and gram-positive bacteria; it is

an indication that the active substance has a broad spectrum activity. It also suggested an alternative

mechanism to that of penicillin, which inhibits the synthesis of cell wall in gram-positive bacteria.

Eugenol is an essential oil known to be bacteriostatic and fungistatic (Beuchat, 1989; Cowan, 1999).

Catechol, on the other hand, is a hydroxylated phenol which is toxic to microorganisms (Cowan,

1999). It is thought that enzyme inhibition by the phenolic compounds is the mechanism for

microorganism inhibition observed in these substances. This is possibly done by the oxidized

compounds through reaction with sulfhydryl groups or through nonspecific interactions with the

proteins (Cowan, 1999). The active compound in the root extract of Z. portoricensis might be an

antimetabolite just like sulfonilamides and trimathoprines which interfere with the synthesis of

tetrahydrofolate by inhibiting enzymes dihydropterate synthetase and dihydrofolate reductase

respectively. Tetrahydofolate is used in synthesis of nucleic acids. The inhibition of the synthesis of

nucleic acids leads to cell death.

Phytochemical constituents such as tannins, saponins, flavonoids, alkaloids and several other

aromatic compounds are secondary metabolites of plants that serve as defense mechanisms against

predation by many microorganisms, insects and other herbivores (Afolayan and Meyer, 1997,

Lutterodt et al., 1999, Marjorie, 1999 and Bonjar et al., 2004). Alkaloids, saponins, glycosides,

tannins and steroids were present in the extract and fractions of the root of the plant, Z. portorecesis.

Antibacterial properties of several plant extracts have been attributed to secondary metabolites such

as some of these (Cowan, 1999; Okoli and Iroegbu, 2005). The presence of secondary metabolites in

plants, produce some biological activity in man and animals and it is responsible for their use as

herbs (Sofowora, 1986). Thus, the spectra of activities displayed by these extracts on microorganisms

used can be explained by the presence of any or combination of alkaloids, tannins, glycosides,

saponins and steroids. It signifies the potential of Zapoteca portoricensis as a source of therapeutic

agents which may provide leads in the on going search for antimicrobial agent from plants.

According to Ebana et al (1991) and Cushnie and Lamb (2005) both alkaloids and glycosides exhibit

antimicrobial activities. Tannins are important in herbal medicine in treating wounds and to arrests

bleeding (Nguyi, 1988). The results obtained showed that ethanolic extracts of Z. portoricensis

exhibited inhibitory activities against the tested bacteria (Staphylococcus aureus, Streptococcus

pyogenes, Streptococcus pneumonia, E. coli, Klebsiella pneumonia and Salmonella typhi) with

different degrees as demonstrated by measuring the diameters of inhibition zones and these results

are in conformity with the results obtained by traditional herbalists who have used the roots of this

plant to treat the bacterial disease, streptococcal pharyngitis otherwise called sore throat.

xcv

The demonstration of antimicrobial activity against both Gram-positive and Gram-negative bacteria

may be indicative of the presence of broad spectrum antibiotic compounds (Cichewicz and Thorpe,

1996). Catechol and pyrogallol are hydroxylated phenols, shown to be toxic to microorganisms;

catechol has two –OH groups while pyrogallol has three. The site(s) and number of hydroxyl groups

on the phenol group are thought to be related to their relative toxicity to microorganisms, with that,

increased hydroxylation results in increased toxicity. In addition, some authors have found that more

highly oxidized compounds show more inhibition to the growth of the microorganisms. The

mechanism thought to be responsible for phenolic toxicity to microorganisms include enzyme

inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or through

more nonspecific interaction with the proteins (Cowan, 1999). The IR-Spectra of the ethanol fraction

showed a broad band at 3391.32cm-1, which indicates the presence of –OH group. Also, the band at

1400cm-1, indicates the presence of aromatic group. This possibly is responsible for the

antimicrobial activity of the ethanol fraction. There are aldehyde, amide and primary amine shown in

the infrared spectra. In addition, the Nuclear Magnetic Resonance spectra showed peaks between 60-

80ppm, 80-100ppm and 100-110ppm which indicate the presence of aromatic group, sulfuryl group,

aldehydes, halogen groups, amides, and carboxyl. These functional groups are also the same with that

of the IR-spectra, which showed bands at 3000cm-1 for aldehydes, at around 1600cm-1 for amides.

Most likely the antimicrobial activity of the active substance or factor is by enzyme inhibition or

through negative interaction with the DNA metabolism or protein synthesis. This is because of its

activity on both gram-negative and gram-positive bacteria (Table 6).

Medicinal plants have been the subject of human curiosity and need (Omino and Kokwaro, 1991;

Khalil, 2007). This is because of the ability of plants to produce secondary metabolites. These

secondary metabolites are important bioactive substances that can produce definite physiological

action on human body; some of them have antimicrobial activities. The antimicrobial activity of the

crude extract could be due to the presence of these secondary metabolites as was evident by the

phytochemical screening. The phytochemical screening showed the presence of alkaloids, steroids,

terpenoids, carbohydrate, and resins. Others such as, proteins, saponins, fats and oil were present but

in a very low concentration. Flavonoids and tannins were absent in the crude extract. Antimicrobial

activities exhibited by the plant could be due to the presence of alkaloids, glycosides, carbohydrate,

steroids present in the plant root extract (Fennel et al., 2004). Among compounds of pharmacological

interest occurring in plants, are alkaloids. Alkaloids have good antimicrobial activity against both

gram-negative and gram-positive bacteria (Karou et al., 2005). The presence of alkaloids in high

concentration could have likely contributed to the antimicrobial activity of the ethanol extract of the

xcvi

plant root. There is no inhibitory zone observed in the Bacillus subtilis plate, with the crude extract,

but the ethanol fraction produced inhibitory zone on all the test microorganisms. This might be due

the presence of several other compounds in the crude extract that suppressed the activity in B. subtilis

(Table 4). The minimum inhibitory concentration of the crude extract is lowest in Streptococcus

pneumonia and Salmonella typhi, this shows highest activity. This is followed by Streptococcus

pyogenes, Staphylococcus aureus, Klebsiella pneumonia, and lastly E. coli (Table 5). The ethanol

fraction was active on all the test microorganisms; this indicates a higher activity than the crude

extract (Table 6). It is possible that carbohydrate in the crude extract facilitated growth of the

microorganisms and hence antagonizing the antimicrobial activity of the active compounds in the

extract. There are no reports of antimicrobial activities of Zapoteca portoricensis in relation to

Streptococcus pyogenes. Also, no attempt has been made to isolate and characterize the active

compound responsible for the activity of the extract of the plant roots; hence there should be more

research in these areas, to introduce a new potent antibiotic against drug resistant Streptococcus

pyogenes.

4.2 CONCLUSION

This study substantiated the antimicrobial activity of Zapoteca portoricensis against Streptococcus

pyogenes and other related bacteria. Among ethyl acetate, chloroform, acetone and ethanol fractions

of the extract, the ethanol fraction was found to exert the most potent antibacterial activity. Some

functional groups detected in the extract that might contribute to this activity include; aldehyde,

amide, hydroxyl, sulfuryl and sulfhydryl groups. It is not clear the cause of antimicrobial activity of

the extract but any or combination of alkaloids, glycosides, steroids, terpenoids, resins and

carbohydrate could have been responsible. The results suggest a possible discovery of a new

antibiotic that could overcome the resistance posed by S. pyogenes. Further purification, isolation and

characterization could introduce another antibiotic that is potent against Streptococcus pyogenes as

well as other drug resistant bacteria.

xcvii

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ORJI, EJIKE CELESTINE (PG/M.Sc/08/48274) … Ejike.pdfThis suggested that the mechanism of action might not be related to the inhibition of cell wall synthesis. The ethanol fraction