UKWUEZE, STANLEY EJIKE (PG/PhD/05/40421) … stanley Ejike.pdfLEAVES UKWUEZE, STANLEY EJIKE (PG/PhD/05/40421) 2 ... reported in literature for this plant, guava, and the trivial names

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Digitally Signed by: Content manager’s Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Nwamarah Uche

FACULTY OF PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACEUTICAL

CHEMISTRY

PHYTOCHEMICAL AND BIOACTIVITY-GUIDED

EVALUATION OF THE ANTIBACTERIAL

CONSTITUENTS OF PSIDIUM GUAJAVA (LINN.) AND

LORANTHUS MICRANTHUS (LINN.) LEAVES

UKWUEZE, STANLEY EJIKE

(PG/PhD/05/40421)

2

PHYTOCHEMICAL AND BIOACTIVITY-GUIDED EVALUATION OF

THE ANTIBACTERIAL CONSTITUENTS OF PSIDIUM GUAJAVA

(LINN.) AND LORANTHUS MICRANTHUS (LINN.) LEAVES

UKWUEZE, STANLEY EJIKE

(PG/PhD/05/40421)

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY,

FACULTY OF PHARMACEUTICAL SCIENCES,

UNIVERSITY OF NIGERIA, NSUKKA.

SUPERVISOR: PROF. (MRS.) P. O. OSADEBE

MARCH, 2014.

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PHYTOCHEMICAL AND BIOACTIVITY-GUIDED EVALUATION OF

THE ANTIBACTERIAL CONSTITUENTS OF PSIDIUM GUAJAVA

(LINN.) AND LORANTHUS MICRANTHUS (LINN.) LEAVES

UKWUEZE, STANLEY EJIKE

(PG/PhD/05/40421)

A THESIS SUBMITTED TO

THE DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL

CHEMISTRY, FACULTY OF PHARMACEUTICAL SCIENCES,

UNIVERSITY OF NIGERIA, NSUKKA.

IN FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

DOCTOR OF PHILOSOPHY (PhD) DEGREE IN PHARMACEUTICAL

& MEDICINAL CHEMISTRY.

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CERTIFICATION

Ukwueze, Stanley Ejike, a postgraduate student with registration number:

PG/PhD/05/40421 has satisfactorily completed the requirement for the award

of the degree of Doctor of Philosophy (PhD) of the Department of

Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences,

University of Nigeria, Nsukka.

The research embodied in this thesis is original and has not been submitted in

part or in full for the award of any other diploma or degree of this or any other

University.

_____________________________ ______________________

Prof. (Mrs.) P. O. Osadebe Prof. C. J. Mbah

(Supervisor) (Head of Department)

_____________________________

(External Examiner)

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DEDICATION

To my beloved wife (Odimnobi), Nchedo, and my lovely roots: Osinachi,

Munachiso, Oluoma and Uchechi who have remained the priceless jewels and

adorable ornaments of my life.

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ACKNOWLEDGEMENT

To God be the Glory, great thing He has done! He has shown me His

faithfulness once again by making it possible for me to attain the zenith of this

tortuous and seemingly endless race. I will forever consecrate my life to Him!

My immeasurable gratitude goes to my supervisor, Prof. Mrs. P. O. Osadebe,

who God has used to guide and illuminate my academic path since my

undergraduate years. She and her inspirational husband, Rev. Prof. N. N.

Osadebe, have been more of motivators and mentors not just to me, but also to

my entire family in diverse realms. I will always remain indebted.

My appreciation also goes to my ever loving parents, Ichie and Mrs. S. K. C.

Ukwueze, who God has kept alive to witness their life-long dream of having

one of their children attain this academic summit. May God continue to

preserve their life for us!

My special acknowledgement goes to my brother, friend, colleague and

schoolmate, Dr. Okoye, F. B. C. who has always been of great support in times

of need. His role in carrying out the spectral analysis in Germany and the

eventual structure elucidations cannot be quantified. My appreciation also goes

to Dr. Peters at the Institute of Organic Chemistry, Heinrich-Heine-University

for the NMR measurements. I also acknowledge the contributions of the

Institute of Pharmaceutical Biology and Biotechnology, University of Heirich-

Heine-Düsseldorf for the HPLC/ESI-MS recordings.

I would like at this juncture to appreciate the professional and elderly roles

played by some of my senior colleagues who kept prodding me on even when I

appeared distracted. Worthy of mention here include Prof. C. O. Onyeji (my

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VC), Prof. Cyril Usifo (my amiable Prof), Assoc. Prof. Eseyin (my Oga), Prof.

Orisakwe, Prof. S. I. Ofoefule, Prof. Vincent Okore and Prof. O. K. Udeala (my

able Dean). The list is quite endless!

It will be so unfair if I shall end this eulogy without the appreciation of all the

laboratory staff that played one role or the other towards the accomplishment of

this study. Names like Chief Hassan and Mrs. Caleb come tops here. Also, my

colleagues in the Department of Pharmaceutical and Medicinal Chemistry,

University of Port Harcourt deserve special recognition.

Finally, my greatest felicitation goes to my beloved wife, Nchedo, and my

vibrant children: Osinachi, Munachiso, Oluomachukwu and Uchechi. I will

always remember the angelic voice of my sweet one (Odimnaheart) constantly

reminding me of her joy in seeing this work completed. Thank God it finally

came to a glorious end! My siblings equally deserve special recognition: Sis.

Vicky, Rev. Leo, Eby, Ify, Iyke, Chika and Eyinwa.

To anyone I might have forgotten to mention, please accept my apology and lets

all join hands to say three ‘Gbosas’ to one another for a job well done.

Merci! Danke!!

Ukwueze Stanley Ejike. February, 2014.

ABSTRACT

Antibiotics have remained the mainstay of drug therapy of infectious diseases

worldwide. Their use is, however, limited by their numerous adverse effects and rapid

development of microbial resistance. Identification of natural products from plants that

may serve as valuable sources of antimicrobial agents for medicinal or agricultural uses

seems to be a viable alternative to the conventional antibiotics. To achieve this goal,

biological assays should be carried out in order to identify promising plant extracts,

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guide the separation and isolation, and to evaluate "lead" compounds. Psidium guajava

Linn. and Loranthus micranthus Linn. have been employed traditionally in Nigeria and

other parts of the globe for the treatment of various human ailments such as wounds,

gastrointestinal tract disorders and other forms of infective and non-infective disorders.

The main objective of this study was to identify and isolate the antibacterial compounds

from the leaves of Psidium guajava and Loranthus micranthus L. The specific objectives

were to: (i) carry out phytochemical evaluation and isolation of antibacterial

constituents of the plants using standard methods, (ii) elucidate the structures of the

isolated secondary metabolites, and (iii) carry out antibacterial assay of the isolated

compounds.

Fresh leaves of Loranthus micranthus (Linn.) parasitic on the stem of Persea americana

were collected at Nsukka while those of Psidium guajava were collected from the bio-

resource area of the University of Port Harcourt in June 2010. The leaves were then

cleaned, air-dried for 14 days and milled to coarse powder. The powdered materials

(800 g each) were defatted with n-hexane (5 L) and extracted in a soxhlet extractor with

90.0 % methanol. The methanol extract was further fractionated to yield the

chloroform, ethyl acetate, acetone and methanol soluble fractions. Each of the fractions

was screened for antibacterial activity using Agar-well diffusion method. Phytochemical

tests were carried out using standard procedures. The fractions that had the best

antibacterial activity were subjected to column chromatographic separation and

monitored by analytical thin layer chromatography (TLC). The ethyl acetate fraction

(PsG-EF) from P. guajava that gave satisfactory bioassay result was subjected to further

Sephadex-LH 20 chromatographic fractionation and purification to afford ten fractions

(PsG-EF1 to PsG-EF10) which were pooled. Fractions PsG-EF4, PsG-EF5 and PsG-EF7

that had good antibacterial activity were subjected to semi-preparative reverse phase

high pressure liquid chromatography (HPLC) purification to isolate the phenolic

compounds; I-V. The structures of these compounds were elucidated by analytical and

spectral techniques which included: ultra violet (UV), proton nuclear magnetic

resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), distortionless

enhancement by polarization transfer (DEPT), proton-proton correlation spectroscopy

(1H-1HCOSY), heteronuclear multiple quantum correlation (HMQC), heteronuclear

multiple bond correlation (HMBC) and electron spray ionization-mass spectroscopy

(ESI-MS) analyses. The isolated compounds were screened against standard strains of

Staphylococus aureus (ATCC 25923) and Escherichia coli (ATCC 35219) using broth

dilution assay method, and the MIC values determined and compared with ceftriaxone.

All data obtained were analyzed by GraphPad Prism® 5 using differences in mean by

two-way ANOVA and further subjected to Bonferroni post-tests to compare replicate

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means. The results were presented as mean ± SEM. Differences between means were

considered significant at P<0.05.

The results showed that the ethyl acetate fraction (PsG-EF) from P. guajava yielded one

known compound (IV) and four novel phenolic compounds (I, II, III & V). The isolated

compounds were elucidated as : 2,4-dihydroxy-6-O-βD-glucopyranosyl benzophenone

(I); 2,4-dihydroxy-3-methyl-6-O-βD-glucopyranosylbenzophenone (II); 2,4-dihydro xy-

3-methyl-6-O-βD-glucopyranosylbenzophenone (4→5", 6'→1") benzene-2",3",4",5"-

tetraol (III); quercertin-3-O-αL-arabinofuranoside (IV) and 2,4-dihydroxy-6-O-βD-

glucopyranosylbenzophenone (4→5", 6'→1") benzene-2",3",4",5"-tetraol (V).

Compounds I, II, III, and V are new natural products which have not been previously

reported in literature for this plant, guava, and the trivial names Guajaphenone A, B, C

and D were proposed, while Compound IV has been previously reported as Guaijaverin.

The various fractions of Psidium guajava L. exhibited significant (p < 0.05) antibacterial

activities while for Loranthus micranthus L., its various fractions showed significantly

lower values (p > 0.001) when compared with the control (ceftriaxone) suggestive of a

generally weak or negligible antibacterial action. All the isolated compounds from P.

guajava were also found to have moderate antibacterial activities against E. coli and S.

aureus in comparison with ceftriaxone whlie I and IV showed lower MICs than those of

the other isolates against the test organisms.

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Table of Contents

Page

TITLE PAGE i

CERTIFICATION iii

DEDICATION iv

ACKNOWLEDGEMENT v

ABSTRACT vii

TABLE OF CONTENT ix

LIST OF FIGURES xiv

LIST OF TABLES xvii

CHAPTER ONE: GENERAL INTRODUCTION 1

1.0 PREAMBLE 1

1.1 INFECTIOUS DISEASES AND CONVENTIONAL ANTIBIOTIC THERAPY 2

1.2 LIMITATIONS OF CONVENTIONAL ANTIBACTERIAL

AGENTS 6

1.2.1 Bacterial Resistance 6

1.2.2 Adverse Reactions by Agents 7

1.3 HIGHER PLANTS AS ANTIMICROBIAL AGENTS 8

1.3.1 Medicinal Plants with Antibacterial Activity 9

1.3.2 Plant Secondary Metabolites Associated with Antibacterial Effects 10

1.3.2.1 Flavones, Flavonoids and Flavonols 11

1.3.2.2 Alkaloids 12

1.3.2.3 Terpenoids and Essential Oils 13

1.3.2.4 Tannins 15

1.3.2.5 Miscellaneous Plant Constituents 17

1.4 LITERATURE REVIEWS OF PLANTS USED 21

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1.4.1 Loranthus micranthus Linn 21

1.4.1.1 Taxonomy of L. Micranthus 21

1.4.1.2 Description of the Family, Genus and Species of L. micranthus 23

1.4.1.3 Ethnomedicinal Uses and Pharmacological Studies on

L. micranthus 24

1.4.2 Psidium guajava Linn 25

1.4.2.1 Taxonomy of Psidium guajava 25

1.4.2.2 Morphology of Psidium guajava 26

1.4.2.3 Ethnomedicinal Uses and Pharmacological Studies of

Psidium guajava 28

1.4.2.4 The Phytochemistry of Psidium guajava 32

1.5 BIOASSAY-GUIDED CHARACTERIZATION OF ANTI-BACTERIAL CONSTITUENTS FROM HIGHER PLANTS 37

1.5.1 Principles of Bioassay 38

1.5.2 Screening Methods for Antibacterial Agents from Higher Plants 38

1.5.2.1 Test Organisms and Culture Media 38

1.5.2.2 Antibacterial Testing 40

1.6 STRUCTURE ELUCIDATION OF BIOACTIVE PLANT

METABOLITES 42

1.6.1 Preliminary Analysis 42

1.6.2 Application of Modern Analytical Techniques 43

1.7 STATEMENT OF PROBLEM 44

1.8 JUSTIFICATION OF THE STUDY 44

1.9 AIMS AND SCOPE OF THE WORK 45

CHAPTER TWO: MATERIALS AND METHODS 46

2.1 MATERIALS 46

2.1.1 Plant Materials 46

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2.1.2 Microorganisms Used 46

2.1.3 Solvents and Reagents 47

2.1.4 Materials and General Instruments 48

2.2 METHODS 50

2.2.1 Extraction and Fractionation of Plant Materials 50

2.2.2 Isolation and Purification of the Active Constituents from PsG-EF 54

2.2.3 HPLC Analysis of Active Constituents from PsG-EF 55

2.2.4 Electron Spray Ionization Mass Spectrometry (HPLC/ESI-MS)

of Isolates 55

2.2.5 Nuclear Magnetic Resonance (NMR) Spectroscopy of Isolates 56

2.2.6 Preliminary Screening of Extracts and Fractions

for Antimicrobial Activity 56

2.2.7 Phytochemical Screening of Plant Extracts and Fractions 57

2.2.8 Antibacterial Screening of Isolates 57

2.2.9 Statistical Analyses 58

CHAPTER THREE: RESULTS 59

3.1 Extraction and Solvent Fractionation 59

3.2 Preliminary Phytochemical and Antibacterial Screening Results 61

3.3 Isolation of Bioactive Constituents 73

3.4 Structure Elucidation of Isolated Compounds 74

3.4.1 PsG-EF4A (Compound I) 74

3.4.2 PsG-EF4B (Compound II) 76

3.4.3 PsG-EF7A (Compound III) 78

3.4.4 PsG-EF7D (Compound IV) 80

3.4.5 PsG-EF7E (Compound V) 81

3.5 Antibacterial Profile of Bioactive Constituents 83

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CHAPTER FOUR: DISCUSSION AND CONCLUSION 84

4.1 Discussion 84

4.2 Conclusion 98

REFERENCES 100

APPENDICES 113

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LIST OF FIGURES

page

Fig. 1.0: Flowers and Leaves of Loranthus micranthus 22

Fig. 2.0: Loranthus micranthus parasitic on a host tree 23

Fig. 3.0 Psidium guajava Tree 27

Fig. 4.0 Leaves and Flowers of Psidium guajava 27

Fig. 5.0 Fruit of Psidium guajava 28

Fig. 6: Schematic Diagram of the Extraction/Fractionation 52

Procedure for L. micranthus Leaves.

Fig. 7: Schematic Diagram of the Extraction/Fractionation 53

Procedure for P. guajava Leaves.

Fig. 8: Graph of Mean IZD (mm) ± SEM of the extracts/fractions of

L. micranthus leaves against bacteria 68

Fig. 9a: Structure of Compound I 74

Fig. 9b: Numbering of Carbon Skeleton of Compound I 75

Fig. 10a: Structure of Compound II 76

Fig. 10b: Numbering of Carbon Skeleton of Compound II 77

Fig. 11a: Structure of Compound III 78

Fig. 11b: Numbering of Carbon Skeleton of Compound III 79

Fig. 12: Structure of Compound IV 80

Fig. 13: Structure of Compound V 81

Appendix 1: ESI-MS /UV Spectra of Compound I 113

Appendix 2: H-NMR Spectrum (500MHz; MeOD) of Compound I 114

Appendix 3: H-NMR Spectrum (600MHz; MeOD) of Compound I 115

Appendix 4: H-NMR Spectrum (600MHz; DMSO-d6) of Compound I 116

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Appendix 5: 2-D COSY of Compound I 117

Appendix 6: C-13 NMR Spectrum of Compound I 118

Appendix 7: HMQC Spectrum of Compound I 119

Appendix 8: HMBC Spectrum of Compound I 120

Appendix 9: ESI-MS /UV Spectra Compound II 121

Appendix 10: H-NMR Spectrum (500MHz; MeOD) of Compound II 122

Appendix 11: H-NMR Spectrum (600MHz; DMSO) of Compound II 123

Appendix 12: 2-D COSY of Compound II 124

Appendix 13: C-13 NMR Spectrum of Compound II 125

Appendix 14: ESI-MS /UV Spectra Compound III 126

Appendix 15: H-NMR Spectrum (500MHz; MeOD) of Compound III 127

Appendix 16: H-NMR Spectrum (600MHz; DMSO) of Compound III 128

Appendix 17: 2-D COSY of Compound III 129

Appendix 18: C-13 NMR Spectrum of Compound III 130

Appendix 19: HMQC Spectrum of Compound III 131

Appendix 20: HMBC Spectrum of Compound III 132

Appendix 21: UV Spectrum of Compound IV 133

Appendix 22: ESI-MS Spectra of Compound IV 134

Appendix 23: H-NMR Spectrum (500MHz; MeOD) of Compound IV 135

Appendix 24: 2-D COSY of Compound IV 136

Appendix 25: H-NMR Spectrum (500MHz; MeOD) of Compound V 137

Appendix 26: 2-D COSY of Compound V 138

Appendix 27: 2-D COSY [aromatic region] of Compound V 139

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LIST OF TABLES Page

Table 1: Some plant species with potential antimicrobial activities... 9

Table 2: Yield from extracts/fractions of the leaves of L. micranthus. 59

Table 3: Yield from extracts/fractions of the leaves of P. guajava …. 60

Table 4: Results of phytochemical tests on the leaf extract of

Loranthus micranthus parasitic on different host trees.... 61

Table 5: Results of the anti-microbial screening of extracts of

mistletoe from six different host plants... 62

Table 6: Results of phytochemical tests on the leaf extract of

Loranthus micranthus harvested at different seasons... 63

Table 7: Results of the anti-microbial screening of leaf extracts of

Loranthus harvested at different seasons... 64

Table 8: Result of MICs of leaf extracts of African mistletoe harvested

from P. americana against some fungi... 65

Table 9: Results of phytochemical tests on the solvent fractions of L.

micranthus leaves harvested from P.americana... 66

Table 10: Result of mean IZD (mm) ± SEM for L. micranthus

extracts/fraction… 67

Table 11: Results of phytochemical tests on the leaf extract of

P. guajava harvested at different seasons.... 69

Table 12: Results of the anti-microbial screening of leaf extracts of

P. guajava harvested at different seasons... 70

Table 13: Results of phytochemical tests on the solvent fractions

of Psidium guajava leaves... 71

Table 14: Table of Mean IZD (mm) +/- SEM for P. guajava

extracts/fractions... 72

Table 15: 1H and

13C-NMR data of Compound I... 75

Table 16: 1H and

13C-NMR data of Compound II... 77

Table 17: 1H and

13C-NMR data of Compound III... 79

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Table 18: 1H and

13C-NMR data of Compound V... 82

Table 19: Antibacterial profile of the isolated compounds against

Staphylococcus aureus and E. coli... 83

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CHAPTER ONE

GENERAL INTRODUCTION

1.0 PREAMBLE

Over the years, medicines and medicinal agents derived from plants have made

large contributions to human health and well-being. This is because they are

either used directly as phytomedicines for the treatment of various ailments or

they may become the base and the natural blueprint for the development of new

drugs (Cseke et al, 2006).

Herbal medicine also called phytotherapy or phytomedicine has been around

since the beginning of recorded history. It has also been described as the

therapeutic use of medicinal plants referred to as herbs (Thea et al, 2008).

Herbal medicine has become an integral part of standard health care, based on a

combination of time honored traditional usage and ongoing scientific research.

Surging interest in medicinal herbs has increased scientific scrutiny of their

therapeutic potential and safety. Some of the medicinal plants are believed to

enhance the natural resistance of the body to infections (Atal et al, 1986).

According to the World Health Organisation (WHO), herbal medicines could

also be referred to as phytopharmaceuticals sold as over the counter products in

modern dosage forms such as tablets, capsules, syrups or liquids for oral use or

dietary supplements containing herbal products, also called nutraceuticals

available in modern dosage forms, or even referred to as medicines consisting of

other crude, semi processed or processed medicines, which have a vital place in

primary health care and developing countries like Nigeria.

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Traditional medicines are finished drug products intended for self-medication or

application that contain, as the active principles, herbal ingredients that have

received relatively little attention in world scientific literature, but for which

traditional or folkloric use is well documented in herbal references. It may

contain chemically defined or herbal based materials in addition to the active

principles (Canada, 1989).

The medicinal properties of plant have been investigated in the light of recent

scientific development throughout the world due to their potent pharmaceutical

activities and low toxicity. Today many countries still rely on the medical

values of herbs and use of medicinal plants for their therapeutic practices (Thea

et al, 2008). In this same vein, Nigeria which is having a vast heritage of

knowledge and expertise in herbal medicines is not an exception.

Finally, it has been variously established that the identification of these natural

products from plants that may serve as valuable sources of bioactive agents for

medicinal and agricultural uses largely depends on bioactivity-directed isolation

(Cseke et al, 2006).

1.1 INFECTIOUS DISEASES AND CONVENTIONAL ANTIBIOTIC

THERAPY

Infectious diseases, also known as contagious diseases or transmissible diseases,

and include communicable diseases, comprise clinically evident illness (i.e.,

characteristic medical signs and/or symptoms of disease) resulting from the

infection, presence and growth of pathogenic biological agents in an individual

host organism. In certain cases, infectious diseases may be asymptomatic for

much or their entire course. Infectious pathogens include some viruses, bacteria,

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fungi, protozoa, multicellular parasites, and aberrant proteins known as prions.

These pathogens are the cause of disease epidemics, in the sense that without

the pathogen, no infectious epidemic occurs.

Transmission of pathogen can occur in various ways including physical contact,

contaminated food, body fluids, objects, airborne inhalation, or through vector

organisms (Ryan and Ray, 2004). Infectious diseases that are especially

infective are sometimes called contagious and can be easily transmitted by

contact with an ill person or their secretions. Infectious diseases with more

specialized routes of infection, such as vector transmission or sexual

transmission, are usually regarded as contagious but do not require medical

quarantine of victims.

The term infectivity describes the ability of an organism to enter, survive and

multiply in the host, while the infectiousness of a disease indicates the

comparative ease with which the disease is transmitted to other hosts; and as

such, an infection is not synonymous with an infectious disease, as some

infections do not cause illness in a host (Ryan and Ray, 2004).

Among the almost infinite varieties of microorganisms, relatively few cause

disease in otherwise healthy individuals. Infectious disease results from the

interplay between those few pathogens and the defenses of the hosts they infect.

The appearance and severity of disease resulting from any pathogen depends

upon the ability of that pathogen to damage the host as well as the ability of the

host to resist the pathogen. Clinicians therefore classify infectious

microorganisms or microbes according to the status of host defenses - either as

primary pathogens or as opportunistic pathogens:

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Primary pathogens cause disease as a result of their presence or activity within

the normal, healthy host, and their intrinsic virulence (the severity of the disease

they cause) is, in part, a necessary consequence of their need to reproduce and

spread. Many of the most common primary pathogens of humans only infect

humans; however many serious diseases are caused by organisms acquired from

the environment or which infect non-human hosts.

Organisms which cause an infectious disease in a host with depressed resistance

are classified as opportunistic pathogens. Opportunistic disease may be caused

by microbes that are ordinarily in contact with the host, such as pathogenic

bacteria or fungi in the gastrointestinal or the upper respiratory tract, and they

may also result from (otherwise innocuous) microbes acquired from other hosts

(as in Clostridium difficile colitis) or from the environment as a result of

traumatic introduction (as in surgical wound infections or compound fractures).

An opportunistic disease requires impairment of host defenses, which may

occur as a result of genetic defects (such as chronic granulomatous disease),

exposure to antimicrobial drugs or immunosuppressive chemicals (as might

occur following poisoning or cancer chemotherapy), exposure to ionizing

radiation, or as a result of an infectious disease with immunosuppressive

activity (such as with measles, malaria or HIV disease). Primary pathogens may

also cause more severe disease in a host with depressed resistance than would

normally occur in an immunosufficient host.

Many human diseases are caused by pathogenic organisms resulting sometimes

in high mortality figures. Among these pathogens, bacteria account for a

reasonable percentage of causative organisms implicated in human infectious

22

diseases. Over the years, infections have been managed by the conventional

antibiotics. Antibiotics are microbial metabolites or synthetic analogues inspired

by them that, in small doses, inhibit the growth and survival of microorganisms

without serious toxicity to the host. They therefore exhibit selective toxicity. In

many cases, the clinical utility of natural antibiotics has been through medicinal

chemistry manipulations of the original structure leading to broader

antimicrobial spectrum, greater potency, lesser toxicity, more convenient

administration, and additional pharmacokinetic advantages. Through customary

usage, the many synthetic substances that are unrelated to natural products but

still inhibit or kill microorganisms are referred to as antimicrobial agents instead

(Martin, 1998).

Antibiotics are used to treat infections caused by organisms that are sensitive to

them, usually bacteria or fungi. They may alter the normal microbial content of

the body (e.g. in the intestine, lungs, bladder) by destroying one or more groups

of harmless or beneficial organisms, which may result in infections (such as

thrush in women) due to overgrowth of resistant organisms. These side-effects

are most likely to occur with broad-spectrum antibiotics (those active against a

wide variety of organisms). Resistance may also develop in the microorganisms

being treated; for example, through incorrect dosage or over-prescription.

Antibiotics should, therefore, not be used to treat minor infections, which will

clear up unaided. Some antibiotics may, in addition, cause allergic reactions

(Hendricks and Nemeth, 2010).

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1.2 Limitations of Conventional Antibacterial Agents

1.2.1 Bacterial Resistance

Resistance is the failure of microorganisms to be killed or inhibited by

antimicrobial treatment. Resistance can either be intrinsic (exist before exposure

to drugs) or acquired (develop subsequent to exposure to a drug). Resistance of

bacteria to the toxic effects of antimicrobial agents and to antibiotics develops

fairly easily both in the laboratory and in the clinic and is an ever-increasing

public health hazard.

In clinical practice, resistance more commonly takes place by Resistance (R)

factor mechanisms. In more lurid examples, enzymes are elaborated that attack

the antibiotic and inactivate it. Mutations leading to resistance occur by many

mechanisms. They can result from point mutations, insertions, deletions,

inversions, duplications and transpositions of segments of genes or by

acquisition of foreign DNA from plasmids, bacteriophages, and transposable

genetic elements.

These mechanisms can convert an antibiotic-sensitive cell to an antibiotic -

resistant cell. This can take place many times in a bacterium's already short

generation time.

Bacterial resistance generally is mediated through one of three mechanisms:

• Failure of the drug to penetrate into or stay in the cell

• Destruction of the drug by defensive enzymes, or

• Alterations in the cellular targets of the enzymes.

24

All these call for conservative but aggressive application of appropriate

antimicrobial chemotherapy. In many cases, however, a resistant microorganism

can still be controlled by achievable, though higher, doses than are required to

control sensitive populations. These higher doses must be cautiously employed

as they may predispose the patient to antibiotic adverse reactions that can be

life-threatening.

1.2.2 Adverse Reactions by Agents

Many patients placed on conventional antibiotics have reported various cases of

adverse drug reactions ranging from mild to severe/life-threatening reactions

including arrhythmias, hepatotoxicity, acute renal failure, and antiretroviral

therapy-induced lactic acidosis (Granowitz et al, 2008). Adverse reactions

associated with drug use include allergies, toxicities, and side effects. An

allergy is a hypersensitivity reaction to a drug. Many allergies are IgE-mediated

and occur soon after drug administration. Examples of IgE-mediated type 1

hypersensitivity reactions include early-onset urticaria, anaphylaxis,

bronchospasm, and angioedema. Non-IgE-mediated reactions include hemolytic

anemia, thrombocytopenia, acute interstitial nephritis, serum sickness,

vasculitis, erythema multiforme, Stevens-Johnson syndrome, and toxic

epidermal necrolysis. Toxicity, which is generally due to either excessive

dosing or impaired drug metabolism, is a consequence of administering a drug

in quantities exceeding those capable of being physiologically ‘‘managed’’ by the

host. Examples of toxicity caused by excessive dosing include penicillin-related

neurotoxicity (e.g. twitching, seizures) and the toxicities caused by

aminoglycosides. Side effects include adverse reactions that are neither

25

immunologically mediated nor related to toxic levels of the drug. An example is

the dyspepsia caused by erythromycin.

Various forms of frequently encountered toxicities/adverse reactions include

anaphylaxis, cardiotoxicity, nephrotoxicity, adverse heamatological and

dermatological reactions, neurotoxicity, hepatotoxicity, muscoskeletal tocxicity,

electrolyte and glucose abnormalities, fever, antibiotic-associated

diarrhea/colitis, etc (Granowitz et al, 2008).

1.3 HIGHER PLANTS AS ANTIMICROBIAL AGENTS

The emergence of pathogenic microbes with increased resistance to established

antibiotics provides a major incentive for the discovery of new antimicrobial

agents. Antimicrobial screening of plant extracts and phytochemicals then

represents a starting point for antimicrobial drug discovery.

Main-stream medicine is increasingly receptive to the use of antimicrobial and

other drugs derived from plants, as traditional antibiotics (products of

microorganisms or their synthesized derivative) become ineffective and as new,

particularly viral, diseases remain intractable to this type of drug. Another

driving factor for the renewed interest in plant antimicrobials in the past 20

years has been the rapid rate of (plant) species extinction (Lewis and Elvin-

Lewis, 1995). There is a feeling among natural-products chemists and

microbiologists alike that the multitude of potentially useful phytochemical

structures which could be synthesized chemically is at risk of being lost

irretrievably. There is a scientific discipline known as ethnobotany (or

ethnopharmacology), whose goal is to utilize the impressive array of knowledge

assembled by indigenous peoples about the plant and animal products they have

26

used to maintain health (Rojas et al, 1992). Lastly, the ascendancy of the human

immunodeficiency virus (HIV) has spurred intensive investigation into the plant

derivatives which may be effective, especially for use in underdeveloped

nations with little access to expensive Western medicines.

1.3.1 Medicinal Plants with Antibacterial Activity

Many tropical and non-tropical plants have been evaluated for their

antimicrobial activity. For want of space, representatives of diverse plant

families with documented antibacterial activity and the organisms tested are

tabulated below.

Table 1: Some plant species with potential antimicrobial activities

Common/Botanical name Family Bacterial Susceptibility

Celosia argentea L. Amaranthaceae K. pneumoniae

Tylophora indica (Burm.f.) Merr. Asclepiadaceae K. Pneumoniae

Vernonia anthelmintica (L.) Willd. Asteraceae K. Pneumoniae

Balanites aegyptiaca (L.) Del. Balanitaceae K. Pneumoniae, S. typhimurium

Spathodea campanulata Beauv Bignonaceae K. Pneumoniae

Cassia fistula L. Caesalpiniaceae K. Pneumoniae, P. mirabilis

Beta vulgaris L. Chenopodiaceae K. Pneumoniae

Spinacia oleracea L. Chenopodiaceae K. Pneumoniae, P. mirabilis

Commelina benghalensis L. Commelinaceae K. Pneumoniae

Rourea santaloides (Vahl.) Connaraceae E. aerogenes; K. Pneumoniae; P.

mirabilis

Cressa cretica L. Convolvulaceae K. Pneumoniae

Lepidium sativum L. Cruciferae S. typhimurium

27

Momordica charantia L. Cucurbitaceae E. aerogenes; K. Pneumoniae; P.

mirabilis

Cyperus scarious R.Br. Cyperaceae E. aerogenes; K. Pneumoniae; P.

mirabilis

Ricinus communis L. Euphorbiaceae K. Pneumoniae, P. mirabilis

Arachis hypogaea L. Fabaceae E. aerogenes; K. Pneumoniae;

Vigna radiata L. Fabaceae K. Pneumoniae; P. mirabilis

Fumaria indica (Haussk.) Pugsley. Fumariaceae K. Pneumoniae; P. mirabilis

Ocimum kilimanjaricum L. Labiatae

E. aerogenes; E. coli; K.

Pneumoniae; P. mirabilis; P.

vulgaris.

Artocarpus hetrophyllus Lam. Moraceae E. aerogenes; P. mirabilis

Ficus elastica Roxb. Moraceae K. Pneumoniae; P. mirabilis

Piper longum L. Piperaceae E. aerogenes; K. Pneumoniae; P.

mirabilis; S. typhimurium .

Gardenia resinifera Roth. Rubiaceae K. Pneumoniae; P. mirabilis

Mesua ferra Linn. Guttiferae E. aerogenes; K. Pneumoniae; P.

mirabilis; P. vulgaris.

Alchornea cordifolia Euphorbiaceae K. Pneumoniae; E. coli; B. subtilis;

S. aureus

Chromolaena odorata Asteraceae Propionibacterium canes;

Mycobacterium spps.

(Parekh and Chanda, 2007; Okoye and Ebi, 2007)

1.3.2 Plant Secondary Metabolites Associated with Antimicrobial Effect

Plants have been shown to possess an amazing potential to synthesize aromatic

substances, most of which are phenols or their oxygenated-substituted

derivatives. These substances have been reported to consist of mostly secondary

metabolites, of which at least 12,000 have been isolated, a number estimated to

still be less than 10% of the total (Schultes, 1978).

28

Plant secondary metabolites, in many cases, serve as plant defense mechanisms

against predation by microorganisms, insects, and herbivores. Some, such as

terpenoids give plants their odours; others (e.g. quinones and tannins) are

responsible for plant pigmentation. Many of these compounds are also

responsible for plant flavour. It is therefore not surprising that useful

antimicrobial phytochemicals have been derived from these plant secondary

metabolites.

1.3.2.1 Flavones, Flavonoids and Flavonols

Flavones are phenolic structures containing one carbonyl group (as opposed to

the two carbonyls in quinones). Addition of a 3-hydroxy group yields a flavonol

while flavonoids are also hydroxylated phenolic substances but occur as a C6-

C3 unit linked to an aromatic ring. These compounds have been known to be

synthesized by plants in response to microbial infection and have equally been

found in vitro to be effective antimicrobial substances against a wide array of

microorganisms (Dixon et al, 1983; Cowan, 1999). Their activity is probably

due to their ability to complex with extracellular and soluble proteins and to

complex with bacterial cell walls, often leading to the inactivation of the

proteins, loss of function and cell lysis (Stern et al, 1996). More lipophilic

flavonoids may also disrupt microbial membrane (Tsuchiya et al, 1996).

Example of flavonoid compounds with known antimicrobial activity includes

the catechins. These are the most reduced form of the C-3 unit in flavonoid

compounds that have been extensively researched due to their occurrence in

oolong green teas. Teas have been reported to exert antimicrobial activity and

also contain a mixture of catechin compounds which inhibited in vitro activity

29

of Vibrio cholerae 01, Streptococcus mutans, Shigella, and other bacteria and

microorganisms (Toda et al, 1989; Batista et al, 1994; Borris, 1996; Sakanaka

et al, 1989; Sakanaka et al, 1992; Vijaya et al, 1995).

Examples of compounds in these groups with antimicrobial activity include

flavone (I), catechin (II), chrysin (III) and quercetin (IV).

I II

III IV

1.3.2.2 Alkaloids

These are basically heterocyclic nitrogenous compounds. Many of these

compounds found in higher plants have shown promising antibacterial activity.

For instance, diterpenoid alkaloids, commonly isolated from the plants of the

Ranunculaceae, or buttercup family, are commonly found to have antimicrobial

30

properties (Omulokoli et al, 1997). Some of the highly aromatic planar

quaternary alkaloids such as berberine (V) have their mechanism of action

attributable to their ability to intercalate with DNA (Phillipson and O'Neill,

1987). Other alkaloids with antimicrobial actions are Harmane (VI), Piperine

(VII), etc.

V VI

VII

1.3.2.3 Terpenoids and Essential Oils

Essential oils are secondary metabolites that are highly enriched in compounds

based on an isoprene structure. The observed fragrance in most plants is

contained in their essential oil fraction. They consist mainly of compounds

belonging to the chemical group called terpenes. When the compounds contain

31

additional elements, usually oxygen, they are called terpenoids. Terpenoids,

even though synthesized from acetate units, differ from fatty acids by their

extensive branching and cyclization.

Many plant terpenoids have been found to be active against bacteria, fungi,

viruses and protozoa (Amaral et al, 1998; Habtemariam et al, 1993; Himejima

et al, 1992; Mendoza et al, 1997; Kubo et al, 1993; Hasegawa et al, 1994;

Ghoshal et al, 1996; Rao et al, 1993; Sun et al, 1996; Tassou et al, 1995; 2000).

Although the mechanism of their antibacterial action is not yet fully understood,

terpenes are speculated to act by disrupting cell membranes due to their

lipophilic nature. In fact, it has been reported as far back as 1977 that of all the

essential oil derivatives being examined, 30% were inhibitory to bacteria while

60% inhibited fungi (Chaurasia and Vyas, 1977). For instance, capsaicin

[(VIII); a terpenoid constituent found in Chiles peppers] in addition to its wide

range of biological activities in humans, has been shown to clearly inhibit

various bacteria to differing extents; and although possibly detrimental to the

human gastric mucosa, it is bactericidal to Helicobacter pylori (Cichewicz and

Thorpe, 1996; Jones et al, 1997). Also, the ethanol-soluble fraction of purple

prairie clover yields a terpenoid called petalostemumol, which showed excellent

activity against Bacillus subtilis and Staphylococcus aureus but lesser activity

against Gram-negative bacteria as well as Candida albicans (Hufford et al,

1993). In the same vein, two diterpenoid compounds isolated from the roots of

Plectranthus hereroensis were found to have good activity against S. aureus, V.

cholerae, P. aeruginosa, and Candida spp (Batista et al, 1994); while another

diterpene, trichorabdal A, isolated from a Japanese herb by Kadota et al (1997)

32

was found to directly inhibit H. Pylori. Terpeoids like menthol (IX) and

artemisin (X) have also shown antimicrobial activity.

VIII

IX X

1.3.2.4 Tannins

The term 'tannin' is used to describe a group of polymeric phenolic substances

capable of tanning leather or precipitating gelatin from solution. The property is

known as astringency. Tannins which are found in almost every plant part are

divided into hydrolyzable and condensed tannins. Hydrolyzable tannins are

based on gallic acid, usually as multiple esters with D-glucose; while the more

numerous condensed tannins (often called proanthocyanidins) are derived from

flavonoid monomers (Cowan, 1999). Generally, tannins may be formed by

33

condensation of flavan derivatives which have been transported to woody

tissues of plant, or alternatively, by polymerization of quinone units (Geissman,

1963).

Tannins have been shown to act at the molecular levels by complexing with

microbial proteins through so-called non-specific forces such as hydrogen

bonding and hydrophobic effects, as well as covalent bond formation (Haslam,

1996; Stern et al, 1996). Thus, their mode of antimicrobial action may be

related to their ability to inactivate microbial adhesins, enzymes, cell envelope

transport proteins, etc. They may also complex with polysaccharide (Ya et al,

1988). Tannins have also shown to act via direct inactivation of microorganisms

(eg. low tannin concentrations modify the morphology of germ tubes of

Crinipellis perniciosa (Brownlee et al, 1990).

According to various documented studies reviewed by Scalbert (1991), tannins

were found to be toxic to filamentous fungi, yeasts and bacteria. Condensed

tannins have been described to bind cell walls of ruminal bacteria, preventing

growth and protease activity (Jones et al, 1994). Though still speculative,

tannins are considered to be wholly or partially responsible for the antibiotic

activity of various aqueous and solvent extracts of many tropical and temperate

plants scattered across the globe (Taylor et al, 1996). Pentagalloyl glucose (XI;

hydrolysable tannin) and procyanidine (XII; condensed tannin) have shown

remarkable antimicrobial activities (Cowan, 1999).

34

XI XII

1.3.2.5 Miscellaneous Plant Constituents

Other major groups of antimicrobial compounds from plants include the simple

phenols and phenolic acids, quinones, coumarins, lectins and polypeptides; and

mixtures of all these groups. There abound many documented plant derivatives

belonging to these chemical groups that have proven antimicrobial activity.

The common herbs terragon and thyme both contain caffeic acid (XIII; a

phenylpropane-derived phenolic compound), which is effective against viruses,

bacteria and fungi (Wild, 1994; Brantner et al, 1996). Catechol (XIV) and

pyrogallol both are hydroxylated phenols, shown to be toxic to microorganisms;

while eugenol (XV; a phenolic compound possessing a C3 side chain at lower

level of oxidation but also classified as an essential oil) is considered

bacteriostatic against both fungi and bacteria (Duke, 1985). Gallic acid (XVI)

has also proven to be toxic to some microorganisms.

35

XIII XIV

XV XVI

Quinones are aromatic rings with two ketone substitutions which are ubiquitous

in nature and are characteristically highly reactive. In addition to providing a

source of stable free radicals, they are known to complex irreversibly with

nucleophilic amino acids in proteins, often leading to inactivation of the protein

and loss of action (Stern et al, 1996). For these reasons, the potential range of

quinone antimicrobial effects is great. Kazmi et al (1994) described an

anthraquinone from Cassia italica, a Pakistani tree, which was bacteriostatic for

Bacillus anthracis, Corynebacterium pseudodiphthericum and Pseudomonas

aeruginosa but bactericidal for Pseudomonas pseudomalliae. Also, Hypericin

(XVII), an anthraquinone from St. John’s wort (Hypericum perforatum), has

received much attention in the scientific journals lately as an antidepressant.

This compound has, however, been reported by Duke (1985) to possess general

36

antimicrobial properties. Cowan (1999) reported that rhein (XVIII) which is an

anthraquinone compound has broad antimicrobial effects.

XVII XVIII

Coumarins (XIX) are phenolic substances made up of fused benzene and α-

pyrone rings. They are responsible for the characteristic odour of hay. As a

group, coumarins have been found to stimulate macrophages which could have

an indirect negative effect on infections (Casley-Smith, 1997). Hydroxycinamic

acids, related to coumarins, seem to be inhibitory to Gram-positive bacteria

(Fernandez et al, 1996). Also, phytoalexins, which are hydroxylated derivatives

of coumarins, are produced in carrots in response to fungal infection and can be

presumed to have antifungal activity (Hoult and Paya, 1996). General

antimicrobial activity was equally documented in coumarin compounds found

in woodruff (Galium odoratum) extracts (Thompson, 1978). Although data

about specific antibiotic properties of coumarins are scarce, many reports give

reasons to believe that some utility may reside in these phytochemicals (Cowan,

1999; Hamburger and Hostettmann, 1991).

37

XIX

Peptides which are inhibitory to microorganisms were first reported by Balls et

al (1942). They are often positively charged and contain disulfide bonds. Their

mechanism of action may be the formation of ion channels in the microbial

membrane, or by competitive inhibition of adhesion of microbial proteins to

host polysaccharide receptors (Zhang and Lewis, 1997; Sharon and Ofek,

1986). Inhibition of bacteria and fungi by these macromolecules (e.g. peptides

from the herbaceous Amaranthus) has been documented (De Bolle et al, 1996).

Also, thionins, which are peptides commonly found in barley and wheat,

consisting of 47 amino acid residues are toxic to yeasts and both Gram-negative

and Gram-positive bacteria (Fernandes de Caleya et al, 1972). Fabatin, a

recently identified 47-residue peptide from fava beans, appears to be structurally

related to γ-thionins from grains and inhibits E. coli, P. aeruginosa and

Enterococcus hirae but not Candida or Saccharomyces (Zhang and Lewis,

1997).

38

The antimicrobial activity of several extracts from plants has been linked to

compounds belonging to more than one chemical group. For instance, the

chewing sticks which are widely used in many African countries as an oral

hygiene aid come from different species of plants, and within one stick, the

chemically active component may be heterogeneous (Akpata and Akinrimisi,

1977). Crude extracts of one species used for this purpose, Serindeia werneckei,

inhibited the periodontal pathogens Porphyromonas gingivalis and Bacteroides

melaninogenicus in vitro (Rotimi et al, 1988). Also, the active component of

one of the Nigerian chewing sticks (Fagara zanthoxyloides) was found to

consist of various alkaloids (Odebiyi and Sofowora, 1979). Pawpaw (Carica

papaya) yields a milky sap, often called latex, which is a complex mixture of

chemicals (Cowan, 1999). Chief among them is papain, a well-known

proteolytic enzyme. It also contains carpaine (an alkaloid) and terpenoids

(Thomson, 1978). All these compounds in papaya have been shown to

contribute to the antimicrobial properties of its latex which was found to be

bacteriostatic to B. subtilis, Enterobacter cloacae, E. coli, Salmonella typhi,

Staphylococcus aureus and Proteus vulgaris (Osato et al, 1993).

1.4 LITERATURE REVIEWS OF PLANTS USED

1.4.1 Loranthus micranthus Linn

1.4.1.1 Taxonomy of L. Micranthus

The botanical profile of Loranthus micranthus is as summarized below:

Kingdom: Plantae

Phylum: Angiosperm

39

Sub-Phylum: Dicotyledons

Order: Santalales

Family: Loranthaceae

Sub-Family: Lorantheae

Genus: Loranthus

Species: micranthus

The mistletoe plant is an evergreen obligate parasite with over 700 species

which depends on its hosts for minerals and water only, as it can

photosynthesize its carbohydrate by means of its green leaves (Gill, 1973;

Griggs, 1991).

The most common species include: European mistletoe (Viscum album L.);

American mistletoe (Phoradendron flavescens); Australian/Argentine mistletoe

(Ligaria cuneifolia R et. T); African mistletoe, e.t.c.

Figure 1.0: Flowers and Leaves of Loranthus micranthus

40

Figure 2.0: Loranthus micranthus parasitic on a host tree

1.4.1.2 Description of the Family, Genus and Species of L. micranthus

The Loranthaceae family consists of parasites with green leaves found in both

tropical and temperate regions. They are mostly small semi-parasitic shrubs

attached to their hosts by suckers or haustoria (usually regarded as modified

adventurous roots). The family is fairly large with over 36 genera and 130

species, most of which are quite omnivorous in their choice of hosts, but a few

are restricted to one or two. Few members of Loranthaceae family root in the

earth (e.g. the Western Australia Christmas tree -Nuytsia floribunda, which

grows into a small tree of up to 10 metres high). For most others that root on

hosts, there is commonly an outgrowth, often of considerable size and

complicated in shape, where the parasite root joins the host. The roots of the

parasites often branch within the tissue of the host (as in Viscum).

41

The genus, Loranthus, consists of several species scattered in many parts of

Africa. It belongs to the sub-family, Lorantheae, which is characterized by the

presence of stem without secretory canals and has extraxylary phloem. Their

flowers have below the petals an outgrowth from the axis in form of small ring

or fringe called calyculus. After some weeks of the seeds germinating on

branches of its host, the Loranthus plant produces proper flowers which are

generally bright red and conspicuous, although one species produces yellow

flowers with red tips. The flowers are soon followed by small, fleshy,

drupaceous fruits which are much sought after by birds. The red Loranthus is a

common sight in many parts of West Africa, particularly in cocoa and cola

plantations, where whole branches are often covered with this medicinal herb.

The African mistletoe species, micranthus, is found mainly in the Southeastern

part of Nigeria. It grows on a large number of hosts including kola nut (Kola

acuminata), avocado (Persea americana), dogoyaro/neem (Azadirachta indica),

oil bean (Pentaclethra macrophylla), ogbono (Irvigia gabonensis),

lemons/citrus, etc. It produces sympodial, often dichasical, stem and the leaves

are usually evergreen and leathery. The cymose inflorescences are in spikes,

with the flowers on the internodes as well as on the nodes. Thus, they have

clusters of narrowly tubular flowers that are bright-red which appear as clusters

of coloured ‘matches’.

1.4.1.3 Ethnomedicinal Uses and Pharmacological Studies on L. micranthus

Several ethnomedicinal usages have been attributed to Loranthus micranthus.

These include: blood pressure control (antihypertensive activity), anti-diabetic

42

activity, anticancer, antimicrobial and in many other metabolic diseases which

qualified mistletoe as an “all-purpose herb” (Kafaru, 1993; Obatomi et al, 1994;

Oliver-Bever, 1986; Dalziel, 1955). Different research works have been carried

out on the several species of the plant to demonstrate and support the existence

of many of the ethno medicinal claims (Obatomi et al, 1996; Osadebe and

Ukwueze, 2004; Osadebe and Akabogu, 2006; Osadebe et al, 2004, 2010, 2012;

Ukwueze and Osadebe, 2012; Agbo et al, 2013; Omeje et al, 2012). Previous

works on Loranthus micranthus have equally shown that some of its medicinal

activities vary with the particular host tree from which it is harvested (Osadebe

and Ukwueze, 2004; Osadebe et al, 2004). Other factors that have been shown

to affect the phytochemical composition and pharmacological activities of

mistletoe plant include species, harvesting season, etc. (Obatomi et al, 1994;

Wagner et al, 1996; Osadebe et al, 2008).

1.4.2 Psidium Guajava Linn

1.4.2.1 Taxonomy of P. guajava

The botanical profile of Psidium guajava is as summarized below:

Kingdom: Plantae

Phylum: Angiosperms

Sub-Phylum: Eudicots

(unranked): Rosids

Order: Myrtales

Family: Myrtaceae

43

Subfamily: Myrtoideae

Tribe: Myrteae

Genus: Psidium

Species: guajava

Binomial name: Psidium guajava L.

Psidium guajava L, a fruit-bearing tree commonly known as guava, of the

family Myrtaceae, is a native of tropical America. The French call it goyave or

goyavier; the Dutch, guyaba or goeajaaba; the Surinamese, guave or goejaba;

and the Portuguese, goiaba or goaibeira. Hawaiians call it guava or kuawa. In

Guam, it is abas. In Malaya, it is generally known either as guava or jambu batu

(Morton, 1987).

1.4.2.2 Morphology of P. guajava

Cultivated varieties grow about 10 m in height and produce fruits within 4

years. Wild trees grow up to 20 m high and are well branched. The guava tree

can be easily identified by its distinctive thin, smooth, copper-colored bark that

flakes off, showing a greenish layer beneath. The trees might have spread

widely throughout the tropics because they thrive in a variety of soils, propagate

easily and bear fruits quickly. The fruits are enjoyed by humans, birds and

monkeys, which disperse guava seeds and cause spontaneous dumps of guava

saplings to grow throughout the rainforest (Wealth of India, 2003).

44

Figure 4.0: Leaves and Flowers of Psidium guajava

Figure 3.0: Psidium guajava Tree

45

Figure 5.0: Fruit of Psidium guajava

1.4.2.3 Ethnomedicinal Uses and Pharmacological Studies of P. guajava

Psidium guajava is a medicinal plant used in tropical and subtropical countries

to treat many health disorders. In the indigenous system of medicine, different

parts of the plant are used for the treatment of various human ailments such as

wounds, ulcers, bowels and cholera (Begum et al., 2002a). Investigations have

indicated that its bark, fruit and leaves possess antibacterial, hypoglycaemic,

anti-inflammatory, analgesic, antipyretic, spasmolytic and CNS depressant

activities ( Begum et al., 2002b). It has indeed been variously reported that

Psidium guajava leaf extract has a wide spectrum of biological activities such as

anticough, antibacterial, haemostasis (Jaiarj et al., 1999; 2000), antidiarrhoeal

and narcotic properties (Lozoya et al.,1990), and antioxidant properties (Qian

and Nihorimbere, 2004). According to Lutterodt and Maleque (1998) and

Meckes et al., 1996, the leaf extract is used to treat diarrhoea, abdominal pain,

convulsions, epilepsy, cholera, insomnia and has hypnotic effect.

46

The long history of guava use has led modern-day researchers to intensify their

study on guava extracts. Its traditional use against diarrhea, gastroenteritis and

other digestive complaints has been validated in numerous clinical studies. In a

study including 17 Thai medicinal plants on anti-proliferative effects on human

mouth epidermal carcinoma and murine leukemia cells using MIT assay, guava

leaf showed anti-proliferative activity, which was 4.37 times more than

vincristine (Manosroi et al. , 2006).

Bark and leaf extracts were shown to have in vitro toxic action against

numerous bacteria. Gallocatechin isolated from the methanol extract of guava

leaf showed antimutagenic activity against E. coli (Matsuo et al., 1994). Water

and chloroform extracts of guava were effective in activating the mutagenicity

of Salmonella typhimurium (Grover and Bala, 1993). The antimicrobial

activities of P. guajava and leaf extracts, determined by disk diffusion method

(zone of inhibition), were compared to tea tree oil (TTO), doxycycline and

clindamycin antibiotics. It was shown that P. guajava leaf extracts might be

beneficial in treating acne especially those that have anti-inflammatory activities

(Qadan et al., 2005). The active flavonoid compound-quercetin-3-O-alpha-l-

arabinopyranoside (guaijaverin) - extracted from guava leaves has high potential

antiplaque activity by inhibiting the growth of Streptococcus mutans (Limsong

et al., 2004). Guava leaf extract also inhibited the growth of Streptococcus

aureus in a study carried out by disc diffusion method (Abdelrahim et al.,

2002). In several other studies, guava showed significant antibacterial activity

against common diarrhea-causing bacteria such as Staphylococcus, Shigella,

Salmonella, Bacillus, E. coli, Clostridium and Pseudomonas. Indeed, the

aqueous, alcohol and chloroform extracts have been found to be effective

against Aeromonas hydrophila, Shigella spp. and Vibrio spp., Staphylococcus

aureus, Sarcinta lutea and Microbacterium phlei (Jaiarj et al., 1999). In a more

recent study, the aqueous and ethanol:water extracts of P. guajava leaves, roots

and stem bark were found to be active against the Gram-positive bacteria

47

Staphylococcus aureus and Bacillus subtilis, but virtually inactive against the

Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa

(Sanches et al.,2005).

A double-blind clinical study of the effects of a Phytodrug (QG-5) developed

from guava leaf showed a decrease in duration of abdominal pain, which was

attributed to antispasmodic effect of quercetin present in leaf extract (Xavier et

al., 2002). Guava leaf extracts and fruit juices have also been clinically studied

for infantile diarrhea. In a clinical study with 62 infants with infantile rotaviral

enteritis, the recovery rate was 3 days (87.1%) in those treated with guava, and

diarrhea ceased in a shorter period than controls. It was concluded in the study

that guava has 'good curative effect on infantile rotaviral enteritis' (Wei et al.,

2000). Lectin chemicals in guava were shown to bind to E. coli (a common

diarrhea-causing organism), preventing its adhesion to the intestinal wall and

thus preventing infection and resulting diarrhea (Rodriguez et al., 2001). Guava

leaf extract has also shown to have tranquilizing effect on intestinal smooth

muscle, inhibit chemical processes found in diarrhea and aid in the re-

absorption of water in intestines. In another research, an alcoholic leaf extract

was reported to have a morphine-like effect, by inhibiting the gastrointestinal

release of chemicals in acute diarrheal disease. This morphine-like effect was

thought to be related to a chemical, quercetin. The effective use of guava in

diarrhea, dysentery and gastroenteritis can also be related to guava's

documented antibacterial properties (Tona et al., 2000). In a study carried out

with leaf extract of the plant, inhibition of gastrointestinal release of

acetylcholine by quercetin present in extract was suggested as a possible mode

of action in the treatment of acute diarrheal disease (Lutterodt, 1992).

48

Guava fruit and leaf showed antioxidant and free radical scavenging capacity

(Hui-Yin and Gow-Chin, 2007). A study of aqueous extract of P. guajava in

acute experimental liver injury induced by carbon tetrachloride, paracetamol

and thioacetamide, showed its hepatoprotective activity. The effects observed

were compared with a known hepatoprotective agent, silymarin. Histological

examination of the liver tissues supported hepatoprotection (Roy et al., 2006).

During various episodes of screening of medicinal plants, extract from P.

guajava leaves was found to exhibit significantly inhibitory effect on the protein

tyrosine phosphatase1B (PTP1B). Significant blood glucose lowering effects of

the extract were observed after intraperitoneal injection of the extract at a dose

of 10mg/kg in both 1-and 3-month-old Lepr(db)/Lepr(db) mice (Oh et al. ,

2005). In a study undertaken to investigate the hypoglycemic and hypotensive

effects of P. guajava leaf aqueous extract in rats, it showed hypoglycemic

activity. The hypoglycemic effect of plant extract was examined in normal and

diabetic rats, using streptozotocin (STZ)-induced diabetes mellitus model

(Ojewole, 2005). Also, i.p. treatment with 1g/kg guava juice produced a marked

hypoglycemic action in normal and alloxan-treated diabetic mice (Cheng and

Yang, 1983). In two randomized human studies, the consumption of guava fruit

for 12 weeks was shown to reduce blood pressure by an average 8%, decrease

total cholesterol level by 9%, decrease triglycerides by almost 8% and increase

HDL cholesterol by 8%; while a randomized, single-blind, controlled trial

conducted to examine the effects of guava fruit intake on blood pressure and

blood lipids in patients with essential hypertension showed the possibility that

an increased consumption of guava fruit can cause a substantial reduction in

49

blood pressure and blood lipids without decreasing HDL-cholesterol level

(Singh et al., 1992, 1993).

Leaf extract of guava had shown ionotropic effect on guinea pig atrium

(Conde-Garcia et al., 2003). Some studies reported that the leaf extract and its

derivative identified as quercetin has effect on the intracellular calcium levels in

gastrointestinal smooth muscle (Lozoya et al., 1990), in cardiac muscle cell

(Apisariyakul et al., 1999) and in neuromuscular junction (Chaichana and

Apisariyakul, 1996). In other animal studies, guava leaf extracts have shown

central nervous system (CNS) depressant activity (Shaheen, 2000). Guava leaf

extract showed anticough activity by reducing the frequency of cough induced

by capsaicin aerosol (Jaiarj et al., 1999).

1.4.2.4 The Phytochemistry of Psidium guajava

Guava has been found to be rich in tannins, phenols, triterpenes, flavonoids,

essential oils, saponins, carotenoids, lectins, vitamins, fibre and fatty acids.

According to Olajide et al (1999), the leaves of P. guajava contain an essential

oil rich in cineol, tannins, triterpenes and flavonoids. Various reports of

phytochemical screening of Psidium guajava leaf showed tannins in aqueous

extract; and anthocyans, alkaloids, flavonoids, tannins and steroids/terpenoids in

ethanolic extract.

More than twenty identified compounds from Psidium guajava leaf have been

reported (Seshadri and Vasishta, 1965; Osman et al., 1974; Lutterodt and

Maleque, 1988). The major components are: β-selinene (XX), β-caryophyllene

(XXI), caryophyllene oxide (XXII), squalene (XXIII), selin-11-en-4α-ol

(XXIV), guaijavarin (XXV), isoquercetin (XXVI), hyperin (XXVII), quercitrin

(XXVIII) and quercetin-3-O-gentobioside; morin-3-O-α-L-lyxopyranoside

(XXIX), morin-3-O-α-L-arabopyranoside (XXX); β-sitosterol (XXXI), uvaol

50

(XXXII), oleanolic acid (XXXIII), ursolic acid (XXXIV) and one new

pentacyclic triterpenoid: guajanoic acid (Lozoya et al., 1994; Meckes et al.,

1996; Arima and Danno, 2002; Begum et al., 2004).

XX XXI

XXII XXIII

XXIV XXV

51

XXVI XXVII

XXVIII XXIX

XXX XXXI

XXXII XXXIII

52

XXXIV

Guava fruit is higher in vitamin C than citrus fruits (80 mg of vitamin C in 100g

of fruit) and contains appreciable amounts of Vitamin A as well and is also a

good source of pectin (Sunttornusk, 2002).

The bark of guava tree contains considerable amounts of tannins (11-27%), and

hence is used for tanning and dyeing purposes. Leucocyanidin (XXXV), luectic

acid, ellagic acid (XXXVI) and amritoside (XXXVII) have been isolated from

the stem bark.

Other compounds that have been isolated from guava plant include avicularin

(XXXVIII; 3-L-4-4-arabinofuranoside), α-pinene (XXXIX), β-pinene (XL),

limonene (XLI), terpenyl acetate (XLII), isopropyl alcohol (XLIII),

longicyclene (XLIV), β-bisabolene, β-copanene, farnesene, humulene,

cardinene, curcumene, mallic acids, ursolic, crategolic, guayavolic acids,

cineol, etc (Shruthi et al., 2013).

53

XXXV XXXVI

XXXVII XXXVIII

XXXIX XL XLI

XLII XLIII XLIV

54

1.5 BIOASSAYBIOASSAYBIOASSAYBIOASSAY----GUIDED CHARACTERIZATION OF ANTIGUIDED CHARACTERIZATION OF ANTIGUIDED CHARACTERIZATION OF ANTIGUIDED CHARACTERIZATION OF ANTIBACTERIALBACTERIALBACTERIALBACTERIAL

CONSTITUENTS FROM HIGHER PLANTSCONSTITUENTS FROM HIGHER PLANTSCONSTITUENTS FROM HIGHER PLANTSCONSTITUENTS FROM HIGHER PLANTS

The driving force behind much phytochemical research is the discovery of new

biological active compounds for medicinal or agricultural uses. Biological

assays then must be carried out in order to identify promising plant extracts, to

guide the separation and isolation, and to evaluate lead compounds.

Identification of natural products from plants that may serve as valuable sources

of bioactive agents for medicinal and agricultural uses largely depends on

bioactivity-directed isolation (Cseke et al, 2006).

The choices of bioassays depend a great deal on the amounts of materials to be

tested and the time and effort necessary to carry out the assays. Obviously, an in

vivo assay using the organism afflicted (humans or animals) would provide the

most meaningful results. However, exploratory screening using whole animals

is impractical (or unethical), and various in vitro screening methods have been

developed to provide guided separation and identification of lead compounds.

The latter have the advantage in that they can be automated with robotics and

miniaturized, leading to rapid throughput screening of large numbers of

samples. In addition, the in vitro bioassays may provide activity information

that is precluded by poor bioavailability using a whole-animal in vivo assay. For

instance, natural products that inhibit the growth of tumor cells or bacteria in an

in vitro assay may identify promising molecular structures that would benefit

from semi-synthetic modifications (Cseke et al, 2006).

55

1.5.1 Principles of Bioassay

Bioassay (or biological assay) is the estimation of the activity or potency of a

drug or other substance (e.g. plant extract) by comparing its effects on a test

organism with that of a standard preparation. It is a type of scientific experiment

conducted to measure the effects of a substance on a living organism and is

essential in the development of new drugs and other scientific monitoring.

Bioassays may be qualitative or quantitative.

1.5.2 Screening Methods for Antibacterial Agents from Higher Plants

The discovery of promising plant extracts and the subsequent activity-guided

isolation of constituents put specific requirements on the bioassays to be used

for that purpose. They have to be simple, rapid, reproducible and inexpensive in

order to be compatible with the large number of assays to be performed.

Antimicrobial activity of plants can be detected by observing the growth

response of various microorganisms to those plant tissues or extracts which are

placed in contact with them. Many methods for detecting such activity are

available, but since they are not equally sensitive or even based upon the same

principle, the results obtained will also be profoundly influenced not only by the

method selected, but also by the microorganisms used to carry out the test

(Vanden-Berghe and Vlietinck, 1991). In general, biological assays or

evaluation can be carried out much more efficiently on water-soluble, pure

crystalline substances than on mixtures like plant extracts.

1.5.2.1 Test Organisms and Culture Media

The purpose of any antimicrobial investigation will obviously determine to a

great extent the choice of test organisms to be used. For an investigation of a

56

general character, the test organisms selected should be as diverse as possible

and preferably representative of all important groups of pathogenic bacteria

according to their physical and chemical composition and resistance pattern.

Most screening studies on plant extracts, however, have been carried out on one

or two bacteria, including strains of Staphylococcus aureus and Escherichia

coli, although such findings may not adequately predict an interesting broad-

spectrum activity or a selective but pronounced activity against some of the

problem-pathogenic bacteria in chemotherapy such as resistant S. aureus, P.

aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Neisseria gonorrhoeae,

Candida albicans and others.

Most bacteria (and yeasts) can be cultivated on standard Mueller-Hinton agar or

diagnostic sensitivity test agar (DST) and American type culture collection

(ATCC) or similar standard microorganisms are available. Only few bacteria

(e.g. Neisseria gonorrhoeae and Campylobacter fetus) require special growth

factors which should be included in the standard medium.

In general, standard microorganisms should be preferably used as test bacteria

during screening for new antimicrobially-active plant components for ease of

reproducibility of results by other researchers. If the interest, however, is in

finding new products which are selectively active against problem

microorganisms causing certain diseases, e.g. resistant P. aeruginosa, it is

clearly appropriate to employ the corresponding isolated pathogenic

microorganisms (Rwangabo et al., 1988).

57

1.5.2.2 Antibacterial Testing

The currently available antimicrobial screening methods fall into three broad

groups, including diffusion, dilution and bioautographic methods (Rios et al,

1988). These testing methods will only give an idea of the presence or absence

of substances with antimicrobial activity in the plant extracts, as the potency of

the active ingredients can only be determined on pure compounds using

standardized methodologies. The results obtained using any of the methods,

however, are influenced by such factors such as extraction method, inoculum

volume, culture medium composition, pH and incubation temperature.

In the diffusion technique, a reservoir (e.g. filter paper disc, porcelain/stainless

steel cylinder or hole punched in the media) containing the plant extract to be

tested is brought into contact with an inoculated medium (e.g. agar) and, after

incubation, the diameter of the clear zone around the reservoir (inhibition zone

diameter) is measured. In order to lower the detection limit using this method,

the inoculated system is kept at low a temperature during several hours before

incubation, which favors diffusion over microbial growth and thus increases the

inhibition diameter. In most studies, the inhibition zones obtained are compared

with those obtained for antibiotics so as to establish the sensitivity of the test

organism to the extract. Advantages of the diffusion methods are the small size

of the sample used in the screening and the possibility of testing up to five or six

compounds per plate against a single microorganism.

For the dilution methods, samples being tested are mixed with a suitable

medium, which has previously been inoculated with the test organism. After

incubation, growth of the microorganism may be determined by direct visual or

58

turbidimetric comparison of the test culture with a control culture which did not

receive an addition of the sample being tested, or by plating out both test and

control cultures (Kavanagh, 1963). Usually a series of dilutions of the original

sample in the culture medium is made and then inoculated with the test

organism. After inoculation, the endpoint of the test (MIC-value) is taken as the

highest dilution which will just prevent perceptible growth of the test organism

(Vanden-Berghe and Vlietinck, 1991). In comparison, several different test

microorganisms may be tested simultaneously on the same dilution as against

diffusion methods in which several substances or dilutions of one substance

may be tested simultaneously against one test microorganism. The agar dilution

method is thus very quick, time saving and also very useful to guide the

isolation of antimicrobially active components from plant extracts (Bakana et

al, 1987; Rwangabo et al, 1988).

Bioautographic methods are employed to localize antibacterial activity on a

chromatoGram. The procedures are based on the agar diffusion technique,

whereby the antimicrobial agent is transferred from the thin layer or paper

chromatoGram to an inoculated agar plate through a diffusion process. Zones of

inhibition are then visualized by appropriate vital stains. Although very suitable

for testing highly active antibiotics (MIC-values < 10ug/ml), bioautographic

methods might not be very promising for testing plant extracts, which often

contain much less potent antimicrobial agents than the currently available

antibiotics (Vanden-Berghe and Vlietinck, 1991).

59

1.6 STRUCTURE ELUCIDATION OF BIOACTIVE PLANT

METABOLITES

Chemical compounds, usually derived from plants and other natural sources,

have been used by humans for thousands of years to alleviate pain, diarrhea,

infection, and various other maladies. Until recently, these ''remedies" were

primarily crude preparations of plant material of unknown constitution. The

revolution in the synthetic organic chemistry during the nineteenth century

produced a concerted effort towards identification of the structures of the active

constituents of these naturally derived medicinals and synthesis of what were

hoped to be more efficacious agents.

By determining the molecular structures of the active components of these

complex mixtures, it is hoped that a better understanding of how these

components work can be elucidated (Knittel and Zavod, 2008).

1.6.1 Preliminary Analysis

Bioassay-directed fractionation is the process of isolating pure active

constituents from some type of biomass (eg. plants, microbes, marine

invertebrates, etc.) using a decision tree that is dictated solely by bioactivity

(Kinghorn, 2008). A variety of chromatographic separation techniques are

available for these purposes, including those based on adsorption on sorbents,

such as silica gel, alumina, Sephadex, and more specialized solid phases, and

methods involving partition chromatography inclusive of counter-current

chromatography. Recent improvements have been made in column technology,

automation of high-performance liquid chromatography (HPLC; a technique

60

often used for final compound purification) and compatibility with HTS

methodology (Butler, 2004).

1.6.2 Application of Modern Analytical Techniques

Routine structure elucidation is performed using combinations of spectroscopic

procedures, with particular emphasis on one and two-dimensional 1H- and

13C-

nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS).

2D NMR spectra, generally, provide more information about a molecule than

1D NMR spectra and are especially useful in determining the structure of a

molecule, particular for molecules that are too complicated to work with using

1D NMR. Thus, for the structural elucidation of a compound, a simple 1D NMR

(H1 and C

13) may not be sufficient. Advanced techniques like correlation

spectroscopic methods, when analyzed properly, provide the perfect way to

determine the structure of a given formula or an entirely unknown compound.

The combination of some or all of these 1D and multi-dimensional NMR

spectra are all very useful in identification and characterization of the structure,

and orientation of bonds in a molecule (Becker, 2000).

Considerable progress has been made in the development of cryogenic and

microcoil NMR probe technology for the determination of structures in sub-

milligram amounts of natural products (Koehn and Carter, 2005). In addition,

the automated processing of spectroscopic data for the structure elucidation of

natural products is a practical proposition (Steinbeck, 2004).

Another significant advance is the use of "hyphenated" analytical techniques for

rapid determination of the structure of natural products without the need for a

separate isolation step, such as liquid chromatograph-nuclear magnetic

61

resonance (LC-NMR) and LC-NMR-MS (Koehn and Carter, 2005; Butler,

2004).

1.7 STATEMENT OF PROBLEM

Antibiotics have remained the mainstay of clinical therapy of infectious diseases

worldwide. Many microorganisms, however, are becoming increasingly

resistant to most of these agents (Jawetz et al., 1989). Another serious limitation

of these chemotherapeutic agents is the numerous adverse effects associated

with their administration (Snavely and Hodges, 1984). These limitations have,

thus, made it imperative that extensive efforts must be made to uncover new

antimicrobial agents from alternative sources whose structures and modes of

action may very likely differ from those of microbial sources (antibiotics).

Compounds extracted from higher plants have the potential of fulfilling this

purpose and that has led to the increasing screening studies on several plant

extracts in search of new ‘leads’. Unfortunately, most of these works are

preliminary investigations of the pharmacological actions of the crude plant

extracts, rather than the isolation, identification or the characterization of the

active constituents of these extracts.

1.8 JUSTIFICATION OF THE STUDY

Several works have been carried out to verify and confirm the traditional

antibacterial uses of Psidium guajava and Loranthus micranthus growing in the

tropical rain forest region of Nigeria, but researches on the isolation,

62

characterization and detailed chemical investigations of their pharmacologically

active components have remained inexhaustive.

There is, therefore, the need for comprehensive chemical and bio-molecular

studies on these active constituents, and hence the justifications for this present

research.

1.9 AIMS AND SCOPE OF THE WORK

Aims of the Study

• To carry out a detailed bioactivity-guided phytochemical evaluation of

the extracts/solvent fractions of the leaves of Psidium guajava and

Loranthus micranthus.

• To identify and isolate the antibacterial compounds from the leaves of

the plants.

• To characterize and elucidate the structure of the isolates.

• To compare the antibacterial potentials of these isolates with standard

antibiotics with a view to developing derivatives of the ‘lead’ compounds

and optimize same for activity with respect to potency and selectivity,

especially against bacteria causing opportunistic AIDS infections [ e.g.

resistant strains of Pseudomonas aeruginosa, and Staphylococcus

aureus; including the multi-drug resistant (MDR) strains of methicillin-

resistant Staphylococcus aureus (MRSA), and Mycobacterium avium

complex (MAC)].

63

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Plant Materials

The leaves of Loranthus micranthus L. were collected in mid-June at Nsukka,

South-Eastern Nigeria, from the stem of Persea americana and authenticated by

Mr. A. Ozioko, a taxonomist with the Bio-resources Development and

Conservation Project (BDCP), Nsukka, Nigeria. Voucher specimen was

deposited in the herbarium of the Faculty of Pharmaceutical Sciences,

University of Nigeria, Nsukka. The leaves of Psidium guajava L. were collected

at the same period from the bio-resource area of the University of Port Harcourt

in South-South region of Nigeria and authenticated by the Plant Science

Biology (PSB) Department of the University. The voucher specimen was

deposited in the herbarium of the Faculty of Pharmaceutical Sciences,

University of Port Harcourt.

The leaves were air-dried at room temperature to a constant weight, pulverized

and passed through a 1mm sieve. The powdered materials were stored in air-

tight containers and kept in a refrigerator.

2.1.2 Microorganisms Used

The studies were performed with standard cultures of Staphylococus aureus

(ATCC 25923), Pseudomonas aeruginosa (ATCC 27833) and Escherichia coli

(ATCC 35219) obtained from the Nigerian Institute of Medical Research

(NIMR),Yaba, Lagos, Nigeria. The clinical isolates of other microorganisms

64

were used. All the microorganisms were grown in nutrient broth (Biotec,

Suffolk, UK) at 37oC and maintained on nutrient agar (Biotec, Suffolk, UK)

slants at 4oC. The standardized cultures of the organisms were used throughout

the experiment.

2.1.3 Solvents and Reagents

N-Hexane (Sigma-Aldrich, South Africa), Chloroform (Sigma-Aldrich, South

Africa), Ethyl acetate (BDH, England), Methanol (Sigma-Aldrich, South

Africa), Acetone (BDH, England), DMSO (Sigma-Aldrich, USA), Acetic acid

(Hopkins and Williams, England). Cutter mill (Manesty, England), Electronic

Balance (Sartorius, Germany) and Weighing balance (Ohans, USA). Other

reagents used were freshly prepared in the laboratory according to official

specifications.

Solvents Used for Isolation and Characterization of Active Ingredients

Dichloromethane (DCM), ethyl acetate, hexane and methanol. These solvents

were purchased from the Institute of Chemistry, University of Duesseldorf,

Germany. They were distilled before use and special grades were used for

spectroscopic measurements.

Solvents for HPLC

Methanol (LiChroSolv HPLC; Merck) and nano-pure water (distilled and heavy

metals free water obtained by passing distilled water through nano- and

ionexchange filter cells; Barnstead, France).

Solvents for NMR

Deuterated methanol (Uvasol, Merck) and DMSO-d6 were used for NMR

measurements.

65

2.1.4 Materials and General Instruments

Chromatography

Pre-coated TLC plates (Silica Gel 60 F254, layer thickness 0.2mm) Merck

Silica Gel 60, 40−63 μm mesh size Merck

Sephadex LH 20, 25−100 μm mesh size Merck

General instruments

Analytical balances MC-1 Sartorious

Half-micro and analytical balance MC-1 Sartorious

Glass ware Schott Duran

Drying Oven ET6130 Heraeus

Ultra sonicator RK 510H Bandelin

UV-Lamp (254 and 366 nm) Camag

Rotary evaporator Büchi Rotavapor R-200

Vacuum pump CVC 2000 Vacuubrand

Centrifuge Pico Heraeus

Nitrogen generator UHPN 3001 Nitrox

Air generator ZA 20 WGA

Fraction collector Retriever II ISCO

Lyvac GT2 (Freeze dryer) Steris

Vacuum pump Trivag D10E (Freeze dryer) Leybol

Syringe Hamilton 1701 RSN

Magnetic stirrer Variomag

HP Behrotest PH 10-Set Multipoint

Semi-preparative HPLC

66

Pump: L-7100 Merck/Hitachi

Detector: UV-L7400 (Photodiode array detector) Merck/Hitachi

Printer: Chromato-Intergartor D-2000 Merck/Hitachi

Column: Eurospher 100-C18, [10 μm; 300 mm × 8 mm] Knauer

Pre-column: Eurospher 100-C18, [10 μm; 30 mm × 8 mm] Knauer

Analytical HPLC

Pump: P 580A LPG Dionex

Autosampler: ASI-100T (injection volume = 20 μL) Dionex

Detector: UVD 340S (Photodiode array detector) Dionex

Column oven: STH 585 Dionex

Column: Eurospher 100-C18, [5 μm; 125 mm × 4 mm] Knauer

Pre-column: Vertex column, Eurospher 100-5 C18 [5-4 mm] Knauer

Software: Chromeleon (V. 6.30)

HPLC-MS

Analytical HPLC: Agilent 1100 series (Photodiode array detector) Agilent

MS: Finigan LCQ-DECA Thermoquest

Ionizer: ESI and APCI Thermoquest

Vacuum pump: Edwards 30 BOC

Column: Eurospher 100-C18, [5 μm; 227 mm × 2 mm] Knauer

Pre-column: Vertex column, Eurospher 100-5 C18 [5−4 mm] Knauer

NMR

DRX-500 Bruker

67

2.2 METHODS

2.2.1 Extraction and Fractionation of Plant of Materials

A portion of the powdered material (800 g) from L. micranthus plant was

defatted with n-hexane (4.5 L; to yield HFM) and the dried marc (540 g)

extracted in a soxhlet extractor with absolute methanol (3.5L). The dried

methanol soluble extract (MFM; 60.0 g) was reconstituted in 30 mL of

methanol, made up to 250 mL with distilled water, shaken for about 30 minutes

and subjected to successive liquid-liquid extraction with chloroform (3 X 750

ml), ethyl acetate (3 X 750 ml) and acetone (3 X 750 ml) to yield the

chloroform (CFM; 11.4 g), ethyl acetate (EFM; 21.3 g) and acetone (AFM; 17.4

g) soluble fractions respectively. An aqueous portion (8.7 g) was left behind

after the fractionation process. The fractions were concentrated by evaporating

over water-baths set at 40oC. The concentrated/dried fractions were stored in a

refrigerator for further analysis.

The pulverized air-dried leaves (600 g) from Psidium guajava were defatted

with n-hexane (2.5L) and the dried marc (450 g) extracted with 5 L of 90 %

methanol for 4 days at room temperature (250C) and the extract concentrated in

vacuo with rotary evaporator. The dried methanol extract (35 g, 7.7 % w/w) was

reconstituted in 20 mL of methanol, made up to 200 mL with distilled water,

shaken for about 30 minutes and subjected to successive liquid-liquid extraction

with chloroform (3 x 750 ml), ethyl acetate (3 x 750 ml) and acetone (3 x 750

ml) to yield PsG-CF (6.0 g; 1.3% w/w), PsG-EF (10.7g; 2.4% w/w) and PsG-

68

AF (8.6 g; 1.9 % w/w) fractions respectively. An aqueous portion (6.8 g) was

left behind after the fractionation process.

Another portion (300 mg of the crude powder from L. micranthus) was

macerated in 90% methanol (3.5 L) for 48 hours and the filtrate concentrated in

vacuo to yield the crude methanol extract, CMFM.

Similarly, 100 mg of the crude powder from P. guajava was macerated in 90 %

methanol (1.5 L) for 48 hours and the filtrate concentrated in vacuo to yield the

crude methanol extract, PsG-CMF.

The schematic representaions for the extraction and fractionation procedures for

the plants are presented in Figures 6 & 7.

69

Dried Marc; 540g

Methanol; 5.5L

Hexane Fraction

(HFM); 44.2g

Chloroform; 3 X 750 ml

Methanol Fraction

(MFM); 60.0g

N-Hexane; 4.5L

Dried Marc

Acetone Fraction

(AFM); 17.4g

Ethyl acetate Fraction

(EFM); 21.3g

Chloroform Fraction

(CFM); 11.4g

Acetone; 3 X 750 ml

Ethyl acetate; 3 X 750 ml

Methanol;

3.5L

Crude Methanol

Fraction (CMFM);

31.4g 800g

PLANT

MATERIAL 300g

Fig. 6: Schematic Diagram of the Extraction/Fractionation Procedure for L. micranthus Leaves.

Aqueous Residue

(8.7g)

70

Plant Material (100g)

Dried Marc; 450g

Methanol; 5L

Hexane Fraction

(PsG-HF); 32.6g

Chloroform; 3 X 750 ml

Methanol Fraction

(PsG-MF); 35g

N-Hexane; 2.5L

Dried Marc

Acetone Fraction

(PsG-AF); 8.9g

Ethyl acetate Fraction

(PsG-EF); 10.9g

Chloroform Fraction

(PsG-CF); 6.2g

Acetone; 3 X 750 ml

Ethyl acetate; 3 X 750 ml

Methanol;

1.5L

Crude Methanol

Fraction (PsG-

CMF); 14.4g 600g

Fig. 7: Schematic Diagram of the Extraction/Fractionation Procedure for P. guajava Leaves.

Aqueous Residue

(6.8g)

71

2.2.2 Isolation and Purification of the Active Constituents from PsG-EF

Thin layer and column chromatography

TLC was performed on pre-coated TLC plates with Silica gel 60 F254 (layer

thickness 0.2 mm, E. Merck, Darmstadt, Germany) with either CH2Cl2: MeOH

(9:1) for semi-polar compounds or n-Hexane: EtOAc (8:2) for non-polar

compounds as mobile phase. The compounds were detected by their UV

absorbtion at 254 and 366 nm or by spraying the TLC plates with anisaldehyde

reagent followed by heating at 1100C.

Column chromatography was carried out using Sephadex® LH-20. The ethyl

acetate fraction (PsG-EF) was separated on a sephadex LH-20 column (3 X 60

cm) eluted with Dichloromethane: MeOH (1:1) to afford 10 pooled fractions

PsG-EF1 to PsG-EF 10. Briefly, 200 g of Sephadex LH-20 was sonicated with

300 mL of DCM: MeOH (1:1) for about 30 min and the gel gradually

transferred into column (3 X 60 cm) with the tab open. Solvent was allowed to

drain until the gel was firmly formed. The sample to be separated was dispersed

in 2 mL of DCM: MeOH (1:1) sonicated for 10 min and then centrifuged. The

supernatant was transferred into the column and allowed to permeate the gel,

when fresh DCM: MeOH (1:1) was added and the whole set up connected to

Fraction collector (Retriever II ISCO, Germany) and adjusted to the flow rate of

0.2 mL/min.

72

2.2.3 HPLC Analysis of Active Constituents from PsG-EF

Analytical high pressure liquid chromatography (HPLC)

The fractions (PsG-EF1 to PsG-EF 10) were subjected to analytical HPLC. The

solvent gradient used started with 10:90 (MeOH: nanopure water (adjusted to

pH 2 with phosphoric acid) increasing to 100 % MeOH in 45 minutes. The

compounds were detected by an UV-VIS Diode Array detector.

Semi-preparative high pressure liquid chromatography (HPLC)

Antibacterial bioactive fractions PsG-EF4 and PsG-EF7 were subjected to semi-

preparative HPLC purification to isolate the compounds PsG-EF4A, PsG-EF4B,

PsG-EF7A, PsG-EF7D and PsG-EF7E. Briefly, the separation column (125 x

21.4 mm, ID) was prefilled with Eurospher C-18 (Knauer, Berlin, Germany) or

Dynmax (250 x 21.4 mm, L.ID). The mobile phase used comprised a linear

gradient of nanopure water and methanol. 50 µL of approximately 40 mg/mL

solution of the substance was injected for each time. The flow rate was

stabilized at 5 mL/min, and the paper speed of the recorder was 5 mm/min. The

eluted peaks were collected respectively by manual work based on the records

of a UV-vis detector.

2.2.4 Electron Spray Ionization Mass Spectrometry (HPLC/ESI-MS) of

Isolates

HPLC/ESI-MS was carried out using a ThermoFinningan LCQ-Deca mass

spectrometer connected to an UV detector. The samples were each dissolved in

MeOH and injected to the HPLC/ESI-MS set up. A solution of the sample was

then sprayed at atmospheric pressure through a 2-5 kV potential. HPLC was run

73

on a Eurospher C-18 (6 x 2 mm, L. ID) reversed phase column. The mobile

phase was H2O containing 0.1% Formic acid (A), to which MeOH (B) or ACN

(C) was added by a linear gradient: initial, 0% of B; 45 min, 80% of B; 55 min,

100% of B. The flow rate was at 400 μL/min and the absorbance detected at 254

nm. ESI (electrospray ionization) was performed at a capillary temperature of

200 0C and drift voltage of 20eV. Since the molecular ion peak is the most

abundant ion in ESI spectra, it is also possible to perform MS/MS experiments.

Measurements were done at Institute of Pharmaceutical Biology and

Biotechnology, University of Heirich-Heine-Düsseldorf.

2.2.5 Nuclear magnetic resonance spectroscopy (NMR) of Isolates

The 1H NMR and

13C NMR spectra were recorded at 300

0K on DRX 500 or

NMR spectrometers. All 1D and 2D spectra were obtained using the standard

Brüker software. The sample was dissolved in a deuterated methanol (CD3OD),

or hexadeuterated dimethylsulfoxide (DMSO-d6) the choice of which is

dependent on the solubility of the sample. TMS was used as internal standard

reference signal. The observed chemical shifts (δ) were recorded in ppm and the

coupling constants (J) were recorded in Hz.

2.2.6 Preliminary Screening of Extracts and Fractions for Antimicrobial

Activity

Antibacterial Activity Testing

Each extract or fraction was screened for antibacterial activity using the

microorganisms provided. The inhibition zone diameters (IZDs) of the extracts

74

and reference antibiotics (Ceftriaxone - MAY & BAKER PLC, Nigeria) were

determined by agar-well diffusion method (Lovian, 1980). The standardized

broth culture (0.1ml) of the test microorganism was introduced into a sterile

petri-dish and 20ml of molten agar added. The content was mixed thoroughly

and allowed to solidify. Four 2-fold serial dilutions of each extract (including

the fractions/ sub-fractions) and reference antibiotics were obtained from their

stock solutions (20mg/ml in DMSO). Four quadrants were marked on each

petri-dish and a cup (6mm) was bored on each quadrant using a sterile cork-

borer. Two drops of each dilution were placed in each cup using Pasteur

pipettes, allowed to diffuse for about an hour and then incubated at 37oC. This

procedure was repeated for each of the microorganisms. The IZDs were

recorded after 24 hours of incubation.

2.2.7 Phytochemical Screening of Plant Extracts and Fractions

Phytochemical Tests

Phytochemical tests were carried out to detect the presence of steroids,

alkaloids, tannins, glycosides, reducing sugars, flavonoids and saponins. These

were carried out according to the procedures outlined by Harbourne (1998).

2.2.8 Antibacterial Screening of Isolates

Standard cultures of S. aureus and E. coli in Mueller-Hinton broth that had been

incubated for 24 hours were diluted 1000-fold with the same broth. Aliquots of

the dilution were mixed with 0.1 ml of the solutions of the isolated compounds

that have been dissolved or suspended in 10% aqueous dimethyl sulfoxide

(DMSO) in sterilized culture tubes. The mixtures were incubated for 24 hours at

37oC and the growth of each test bacterium determined by turbidity. The

75

minimum inhibitory concentration (MIC) of each compound against a particular

organism was expressed as the least concentration of that compound which did

not show turbidity. Ceftriaxone was used as the standard antibacterial agent.

2.2.9 Statistical Analyses

Statistical Analysis

All the data obtained were analyzed by GraphPad Prism® (Model 5) using two-

way ANOVA and subjected to Bonferroni post-tests to compare replicate

means. The statistical results were presented as mean ± SEM. Differences

between means were considered significant at P<0.05.

76

CHAPTER THREE

RESULTS

3.1 Extraction and Solvent Fractionation Result

The yield from the extraction and fractionation carried out on the leaves of

Loranthus micranthus using various solvents is presented in Table 2 below.

Table 2: Yield from Extracts/Fractions of the leaves of L. micranthus.

Extract/Fraction Weight of

Powder/Marc

(g)

Yield (g) % Yield

HFM 800.0 44.2 5.5

MFM 540.0* 61.0 11.3

CFM 540.0* 11.4 2.1

EFM 540.0* 21.3 3.9

AFM 540.0* 17.4 3.2

CMFM 300.0 31.4 10.5

Key: HFM = Hexane extract, MFM = Defatted Methanol extract, EFM= Ethyl acetate soluble

fraction, AFM = Acetone soluble fraction, CFM = Chloroform soluble fraction, CMFM

= Crude Methanolic Extract; * = Portion of marc obtained after defatting with

hexane.

77

The yield from the extraction and fractionation carried out on the leaves of

Psidium guajava using various solvents is presented in Table 3 below.

Table 3: Yield from Extracts/Fractions of the leaves of P. guajava.

Extract/Fraction Weight of

Powder/Marc

(g)

Yield (g) % Yield

PsG-HF 600.0 32.6 5.3

PsG-MF 450.0* 35.7 7.9

PSG-CF 450.0* 6.2 1.3

PsG-EF 450.0* 10.9 2.4

PsG-AF 450.0* 8.9 2.0

PsG-CMF 100.0 14.8 14.8

Key: PsG-HF = Hexane extract, PsG-MF = Methanol soluble fraction, PsG-EF=Ethyl

acetate soluble fraction, PsG-AF = Acetone soluble fraction, PsG-CF = Chloroform

soluble fraction, PsG-CMF = Crude Methanolic Extract; * = Portion of marc obtained after defatting with hexane.

78

3.2 Preliminary Phytochemical and Antibacterial Screening Results

The results of the preliminary investigations carried out on the leaf

extracts/fractions of Loranthus micranthus and Psidium guajava are presented

below.

The result of the comparative study on the phytochemical and anti-microbial

properties of leaves of Loranthus micanthus harvested from different host trees

is shown in Tables 4 & 5.

Table 4: Results of Phytochemical Tests on the Leaf extract of Loranthus

micranthus parasitic on different host trees.

HOST TREE Steriods Alkaloids Glycosides Reducing sugars Flavonoids Saponins Tannins

K. acuminata + ++++ + + ++ + ++

P. americana + ++++ ++ + + + ++

B. nitida - + + + + + +

P.macrophylla - + + + + + +

I. gabonensis - ++ + + + + ++

A. indica + + ‽ + + - +

- = Absent; + = Present in small quantity; ++ = moderately present; +++ =

Present in large quantity; ‽ = not detected.

79

Table 5: Results of the anti-microbial screening of extracts of mistletoe

from six different host plants.

Each value represents the mean ± SEM;*: P <.05, **: P < 0.01, ***: P < 0.001 significantly lower

when compared with control 1; blank spaces indicate no observable inhibition; IG = I. gabonensis;

PM = P. macrophylla; KA = K. acuminata; AI = A. indica; PA = P. americana; BN = B. nitida; AMX

= amoxicillin; KTZ = ketoconazole

Host tree Minimum Inhibitory Concentrations (MIC, mg/ml) ± SEM

S. aureus B. subtilis P. aeruginosa S. typhi A. niger C. albicans

IG 4.45±0.20* 4.19±0.30 6.08 ± 2.34 5.55± 0.55 - -

PM 5.76 ± 1.27 4.13 ± 0.59 7.97±1.81 - - -

KA 4.01 ± 0.17* 3.99 ± 0.30** 5.16± 2.03 6.48±.0.68 2.56± 0.14 7.98±1.86

AI 6.08 ± 0.23 4.53 ± 0.19 7.41±1.21 - - -

PA 3.76±0.25* 3.53±0.84** 4.53 ± 2.34*** 4.94± 0.2 4.63±1.0 6.05±0.32

BN 7.28±0.50 7.70±1.38 10.32±0.32 - - -

AMX 4.95±0.64 4.19±0.04 7.05±0.74 2.43± 0.49 - -

KTZ - - - - 0.44±0.07 2.26± 0.05

80

The result of the study on the seasonal variations of the phytochemical and anti-

microbial constituents of L. miranthus is presented in Tables 6 & 7.

Table 6: Results of Phytochemical Tests on the Leaf extract of Loranthus

micranthus harvested at different seasons

MONTHS Tannins Flavonoids Alkaloids Reducing sugars

Terpenoids Saponins Glycosides

Jan + + - + + + +

April ++ ++ ++ + + + +

July ++ +++ ++ + + + +

Nov + ++ - + + + +

Key: - = Absent; + = Present in small quantity; ++ = Moderately present; +++ = present

in large quantity.

81

Table 7: Results of the anti-microbial screening of leaf extracts of

Loranthus harvested at different seasons.

Months Inhibition Zone Diameter (IZD, mm) ± SEM

S. aureus B. subtilis P. aeruginosa S. kapemba E. coli

Jan. 14.7±0.0.67 13.3±0.33*** 16.0±0.58 10.7±0.67*** 18.3±0.88

April 16.7±1.67 17.0±0.58 17.3±0.67 20.7±0.67 16.0±0.58*

July 16.7±0.67 18.0±0.00 16.0± 0.58 19.7±.0.33 19.0± 0.58

Nov. 14.7±0.33 16.3±0.33* 16.0±1.0 18.7±0.33* 14.7±0.33**

Each value represents the mean ± SEM,*: P < 0.05, **: P < 0.01, ***: P < 0.001

significantly lower when compared with values obtained at the other months.

82

The result of the preliminary study to verify the folkloric utilization of the

leaves of the African mistletoe (L. micranthus) as antifungal agent is presented

in Table 8.

Table 8: Result of MIC of L. micranthus against some fungi

Each value represents the mean ± SEM; n = 3.

Extract MIC (mg/ml)

Candida Aspergillus

K. acuminata 7.73 ± 0.59 4.98 ± 2.02

P. americana 5.85 ± 0.26 4.07 ± 1.17

I. gabonensis 9.73 ± 2.31 8.14 ± 1.96

Ketoconazole (control) 1.09 ± 0.13 0.22 ± 0.11

83

The results of the phytochemical and antimicrobial investigations on the

fractions from Loranthus micranthus are presented in Tables 9 & 10 below.

Table 9: Results of Phytochemical Tests on the solvent fractions of

Loranthus micranthus leaves harvested from Persea Americana

FRACTIONS Tannins Flavonoids Alkaloids Reducing

sugars

Terpenoids Saponins Glycosides

CMFM + ++ ++ + + + +

HFM + + - - + - -

MFM + + + + + + +

EFM + + - + - - +

AFM + ++ + + + + +

CFM + + - - + - -

Key: - = Absent; + = Present in small quantity; ++ = Moderately present; +++ = Present in

large quantity; CMFM = Crude Methanolic Extract, HFM = Hexane extract, MFM

= Defatted Methanol extract, EFM= Ethyl acetate soluble fraction, AFM = Acetone

soluble fraction, CFM = Chloroform soluble fraction.

84

Table 10: Result of Mean IZD (mm) +/- SEM for L. micranthus

Extracts/Fraction

FRACTIONS

{20mg/ml} S. aureus B. subtilis P. aeruginosa E. coli

CMFM 16.00 ± 0.58*** 18.00 ± 0.58 18.00 ± 0.58*** 10.00 ± 1.16***

HFM 1.98 ± 0.88*** 3.82 ± 1.20*** 2.10 ± 0.33*** 0.00 ± 0.00***

MFM 9.67 ± 1.20*** 13.00 ± 2.31*** 10.00 ± 2.08*** 5.67 ± 1.20***

EFM 4.67 ± 1.20*** 6.67 ± 1.20*** 6.67 ± 0.33*** 6.33 ± 0.88***

AFM 7.33 ± 1.20*** 9.67 ± 1.20*** 7.67 ± 0.88*** 5.00 ± 1.15***

CFM 2.67 ± 0.33*** 5.67 ± 0.67*** 3.00 ± 0.58*** 1.33 ± 0.33***

Control (10mg/ml)

26.00 ± 0.58 20.00 ± 0.58 33.67 ± 2.03 27.00 ± 1.15

CMFM = Crude Methanolic Extract, HFM = Hexane extract, MFM = Defatted Methanol

extract, EFM= Ethyl acetate soluble fraction, AFM = Acetone soluble fraction,

CFM = Chloroform soluble fraction, Control = Ceftriaxone; * P<0.05; ** P<0.01 and

*** P<0.001 significantly lower when compared with Control

85

Fig. 8.0: Graph of Mean IZD (mm) ± SEM of the extracts/fractions of L.

micranthus leaves against bacteria.

86

The results of the phytochemical and antimicrobial investigations on the leaves

of Psidium guajava harvested at differents seasons prevalent in Nigeria are

presented in Tables 11 & 12 below.

Table 11: Results of Phytochemical Tests on the Leaf extract of Psidium

guajava harvested at different seasons

MONTHS Tannins Flavonoids Alkaloids Reducing sugars

Terpenoids Saponins Glycosides

Jan ++ - + + + - +

April ++ + - ++ + + +

July ++ ++ - ++ + + +

Nov ++ + + ++ + - +

- = Absent; + = Present in small quantity; ++ = Moderately present; +++ = Present in

large quantity

87

Table 12: Results of the anti-microbial screening of leaf extracts of Psidium

guajava harvested at different seasons

Months Inhibition Zone Diameter (IZD, mm) ± SEM

S. aureus B. subtilis P. aeruginosa S. kapemba E. coli

Jan. 31.0 ± 1.40 19.3 ± 0.47* 16.0 ± 1.40*** 26.0 ± 1.41 20.33 ± 0.40

April 31.0 ± 1.0 20.3 ± 0.44 20.0 ± 0.00 29.5 ± 0.45 22.3 ± 0.45

July 25.7 ± 0.24* 24.0 ± 1.41 18.7 ± 0.40* 15.3 ± 0.04*** 18.7 ± 0.59*

Nov. 19.3 ± 0.45*** 20.3 ± 0.04 24.7 ± 0.45 18.7 ± 0.04** 21.7 ± 0.47

Each value represents the mean ± SEM,*: P < 0.05, **: P < 0.01, ***: P < 0.001 significantly

lower when compared with values obtained at the other months

88

The results of the phytochemical and antimicrobial investigations on the

fractions from Psidium guajava leaf are presented in Tables 13 & 14 below.

Table 13: Results of Phytochemical Tests on the solvent fractions of

Psidium guajava leaves

FRACTIONS

Tannins Flavonoids Alkaloids Reducing sugars

Terpenoids Saponins Glycosides

PSG-CMF ++ ++ + ++ + + ++

PSG-MF ++ ++ + + + + ++

PSG-EF ++ ++ - + - + ++

PSG-AF ++ + - + - + +

PSG-CF + - - + + + +

Key: PSG-CMF = Crude Methanolic Extract, PSG-MF=Methanol soluble fraction, PSG-

EF=Ethyl acetate soluble fraction, PSG-AF = Acetone soluble fraction, PSG-CF =

Chloroform soluble fraction;- = Absent; + = Present in small quantity; ++ = Moderately

present; +++ = Present in large quantity.

89

Table 14: Table of Mean IZD (mm) +/- SEM for P. guajava

Extracts/Fractions

Fractions

(10mg/ml)

S. aureus B. subtilis P. aeruginosa E. coli

PSG-CMF 23.67 +/- 0.88 17.67 +/- 0.88 14.33 +/- 0.88*** 6.67 +/- 0.67***

PSG-MF 16.67 +/- 0.72*** 14.67 +/- 0.54*** 12.33 +/- 0.62*** 10.00 +/- 0.94***

PSG-EF 25.00 +/- 1.25 19.00 +/- 0.82 15.00 +/- 1.25*** 12.00 +/- 0.94***

PSG-AF 21.00 +/- 0.94** 17.33 +/- 0.27 14.33 +/- 0.72*** 9.67 +/- 0.54***

PSG-CF 9.67 +/- 0.54*** 7.00 +/- 0.94*** 5.67 +/- 0.72*** 1.67 +/- 0.27***

Control (10mg/ml)

26.00 +/- 0.58 20.00 +/- 0.58 33.67 +/- 2.03 27.00 +/- 1.15

Key: PSG-CMF = Crude Methanolic Extract, PSG-MF=Methanol soluble fraction, PSG-

EF=Ethyl acetate soluble fraction, PSG-AF = Acetone soluble fraction, PSG-CF =

Chloroform soluble fraction; Control = Ceftriaxone; * P<0.05; ** P<0.01 & ***

P<0.001 significantly lower when compared with Control.

90

3.3 Isolation of Bioactive Constituents

The ethyl acetate fraction (PsG-EF) from P. guajava that gave the best

antibacterial bioassay result was subjected to further Sephadex-LH 20

chromatographic fractionation and purifications to afford ten pooled fractions

(PsG-EF1 to PsG-EF10). Fractions PsG-EF4, PsG-EF5 and PsG-EF7 that had

good antibacterial potential were subjected to semi-preparative reverse phase

HPLC purification to isolate the phenolic compounds I - V.

The structures of these compounds were elucidated using a combination of

analytical and spectral techniques which included: UV, 1H-NMR,

13C-NMR,

DEPT, 1H

1HCOSY, HMQC, HMBC and ESI-MS analyses (see Appendices for

spectra details).

A summary of the spectral data and the elucidated structures of the isolates are

presented in pages 74- 82 while the detailed interpretation of analytical data

leading to the predicted structures is presented in chapter 4 under “Discussion”.

91

3.4 Structure Elucidation of Isolated Compounds

3.4.1 PsG-EF4A (Compound I: Yellow semi-solid; 3.0mg)

1H-NMR (500 MHz, MeOD) δ = 7.70 (m, 2H), 7.52 (t, J=7.4, 1H), 7.41 (t, J=7.7, 2H), 6.21

(d, J=2.1, 1H), 6.07 (d, J=2.0, 1H), 4.81 (d, J=7.7, 1H), 3.85 (m, 1H), 3.66 (dd, J=5.7, 12.1,

1H), 3.22 (m, 1H), 2.83 (m, 1H).

1H-NMR (600 MHz, MeOD) δ = 7.70 (m, 2H), 7.52 (m, 1H), 7.42 (t, J=7.8, 2H), 6.22 (d,

J=2.1, 1H), 6.07 (d, J=2.1, 1H), 4.81 (d, J=7.8, 3H), 3.85 (dd, J=2.2, 12.1, 1H), 3.66 (dd,

J=5.6, 12.1, 1H), 3.22 (dd, J=6.6, 11.1, 1H), 2.84 (dd, J=7.8, 9.1, 1H).

EI.MS m/z: 392.8 [M+1], 391.3 [M-1], 231.1 [M+1 - 162]

UV λmax (MeOH) nm (Ɛ): 254; 296.

O O

HO OH

O

OH

OHHO

OH

[Molecular Formula: C19H20O9; Formula Mass = 392.4]

Fig. 9a: Structure of Compound I

92

Table 15: 1H and

13C-NMR data of compound I

Position δδδδH δδδδC HMBC

1 - 107.7

2 - 161.0

3 6.07 d (2.1) 96.6 1, 2, 4, 5

4 - 162.0

5 6.22 d (2.1) 94.2 1, 3, 4, 6

6 - 156.9

7 - 194.8

8 - 138.7

9/13 7.7 d 128.9 7, 11, 10, 12

10/12 7.42 t (7.8) 128.2 8, 9, 13

11 7.52 t 132.4 9, 13

1' 4.81 d (7.8) 100.6 6

2' 2.83 dd 73.1 1', 3'

3' 3.32 # 76.5 2', 4'

4' 3.22 t 69.4 6'

5' 3.34 # 77.1 4'

6' 3.66 dd Ha

3.86 dd Hb

60.6 5'

4'

NMR was measured at 500 MHz (1H) and 150 MHz (13C) (CD3OD). # = overlapped with the broad solvent peak.

Fig. 9b: Numbering of Carbon Skeleton for Compound I [XXVII]

93

3.4.2 PsG-EF4B (Compound II: Yellow semi-solid; 3.5mg)

1H NMR (500 MHz, MeOD) δ = 7.57 (m, 2H), 7.48 (t, J=7.4, 2H), 7.38 (dd, J=7.7, 15.3,

2H), 6.20 (s, 1H), 4.72 (d, J=7.7, 1H), 3.87 (dd, J=2.1, 12.0, 1H), 3.65 (dd, J=5.9, 12.0, 1H),

3.25 (d, J=9.1, 1H), 3.14 (t, J=9.4, 1H), 2.51 (dt, J=7.1, 14.3, 1H), 2.02 (s, 3H).

1H NMR (600 MHz, MeOD) δ = 7.58 (dd, J=3.4, 5.0, 2H), 7.49 (m, 1H), 7.39 (m, 3H), 6.20

(s, 1H), 4.73 (d, J=7.7, 1H), 3.87 (dd, J=2.2, 12.0, 1H), 3.66 (dd, J=6.0, 12.0, 1H), 3.28 (ddd,

J=5.8, 10.9, 19.6, 2H), 3.16 (m, 2H), 2.52 (dt, J=8.2, 16.3, 1H), 2.02 (d, J=5.7, 3H).

EI.MS m/z: 406.8 [M+1], 405.3 [M-1], 245.2 [M+1 - 162]

UV λmax (MeOH) nm (Ɛ): 250; 302.

O O

HO OH

O

OH

OHHO

OH

[Molecular Formula: C20H22O9; Formula Mass- 406.38328]

Fig. 10a: Structure of Compound II

94

Table 16: 1H and

13C-NMR data of compound II

Position δH δC HMBC

1 - 107.5

2 - 158.0

3 - 113.6

4 - 160.1

5 6.20 d (2.1) 94.2 1, 3, 4, 6

6 - 155.3

7 - 197.1

8 - 140.4

9/13 7.58 d (3.4; 5.0) 128.7 7, 11, 10, 12

10/12 7.39 m 127.6 8, 9, 13

11 7.49 m 131.5 9, 13

1' 4.73 d (7.7) 100.1 6

2' 2.52 dt 72.9 1', 3'

3' 3.28 # 73.4 2', 4'

4' 3.16 m 69.9 6'

5' 3.30 dd 77.0 4'

6'

3-CH3

3.66 dd Ha

3.87 dd Hb

2.02 s (5.7)

61.4

7.9

5'

4'

2, 3, 4 NMR was measured at 500 MHz (

1H) and 150 MHz (

13C) (CD3OD).

# = overlapped with the broad solvent peak.

Fig. 10b: Numbering of Carbon Skeleton for Compound II

95

3.4.3 PsG-EF7A (Compound III: Brownish-yellow semi-solid; 2.5mg)

1H NMR (500 MHz, MeOD) δ = 7.50 (m, 2H), 7.41 (d, J=7.4, 1H), 7.27 (t, J=7.7, 2H), 7.10

(s, 1H), 6.15 (s, 1H), 4.76 (d, J=7.7, 1H), 4.55 (d, J=11.9, 1H), 4.34 (m, 1H), 3.50 (s, 2H),

2.50 (m, 1H), 2.02 (s, 3H).

1H NMR (600 MHz, MeOD) δ = 7.50 (d, J=7.3, 2H), 7.41 (q, J=7.4, 1H), 7.27 (t, J=7.7,

2H), 7.10 (s, 1H), 6.15 (s, 1H), 4.77 (d, J=7.7, 2H), 4.55 (dd, J=1.9, 11.9, 2H), 4.35 (dd,

J=4.6, 12.0, 1H), 3.51 (m, 1H), 3.32 (dd, 1H), 3. 28 (dd, 1H), 2.51 (dd, J=9.1, 17.8, 1H), 2.02

(s, 3H).

EI.MS m/z: 545.0 [M+1], 543.3 [M-1]

UV λmax (MeOH) nm (Ɛ): 262; 362

[Molecular Formula: C26H24O13; Formula Mass- 544.46]

Fig. 11a: Structure of Compound III

96

Table 17: 1H and

13C-NMR data of compound III

Position δH δC HMBC

1 - 105.0

2 - 157.3

3 - 115.5

4 - 161.3

5 6.15 s 93.6 1, 3, 4, 6

6 - 156.4

7 - 177.7

8 - 138.5

9/13 7.50 d (7.3) 128.4 7, 11, 10, 12

10/12 7.27 t (7.7) 127.6 8, 9, 13

11 7.41 q (7.4) 134.2 9, 13

1' 4.77 d (7.7) 101.8 6

2' 2.51 dd 72.8 1', 3'

3' 3.28 dd 76.2 2', 4'

4' 3.32 dd 70.3 6'

5' 3.51 m 73.7 4'

6'

1''

2''

3''

4''

5''

6''

3-CH3

4.35 Ha (dd)

4.55 Hb (dd)

-

-

-

-

-

7.10 s

2.02 d (5.7)

69.0

148.4

145.6

120.7

145.2

165.8

108.6

8.0

5'

4'

4, 1'', 2'', 4'', 5''

2, 3, 4

NMR was measured at 600 MHz (1H) and 150 MHz (

13C) (CD3OD).

Fig. 11b: Numbering of Carbon Skeleton for Compound III

97

3.4.4 PsG-EF7D (Compound IV: Brownish-Yellow, needle-like solid; 4.5mg).

1H NMR (500 MHz, MeOD) δ = 7.52 (1H,s), 7.50 (1H,d,J=8.3), 6.9

(1H,d,J=8.4), 6.39 (1H,s), 6.20 (1H,s), 5.47 (1H,s), 4.85 (1H, d, J=7.3), 4.33

(1H, d, J=2.8), 3.90 (1H, d, J=3.1),3.87 (1H, m), 3.81 (1H, m), 3.50 (2H, t,

J=3.6), 3.35 (1H, m).

EI.MS m/z: 435.0 [M+1], 433.1 [M-1], 303.1 [(M+1) - 132]

UV λmax (MeOH) nm (Ɛ): 257 (26, 595), 354 (23, 595).; m.p. 252-2630C.

PsG-EF7D was elucidated as the previously reported compound: Quercertin-

3-O-αL- arabinofuranoside with the chemical name: 2-(3,4-Dihydroxy

phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl-α-L-arabinofuranoside. [Trivial name: guaijaverin].

Fig. 12: Structure of Compound IV

98

3.4.5 PsG-EF7E (Compound V: Yellow semi-solid; 2.1mg)

1H NMR (500 MHz, MeOD) δ = 7.61 (d, J=8.4, 2H), 7.44 (s, 1H), 7.31 (t,

J=7.8, 2H), 7.09 (s, 1H), 6.16 (s, 1H), 6.05 (s, 1H), 4.50 (s, 1H), 4.33 (s, 1H),

3.58 (s, 1H), 2.77 (s, 1H).

UV λλλλmax (MeOH) nm (ƐƐƐƐ): 265; 294.

O O

OH

O

OH

OHOH

O

O O

OH

OH

OH

[Molecular Formula: C25H22O13; Formula Mass- 530.43]

Fig. 13: Structure of Compound V

99

Table 18: 1H and

13C-NMR data of compound V

Position δH δC HMBC

1 - 105.0

2 - 157.3

3 6.16 s 115.5

4 - 161.3

5 6.05 s 93.6 1, 3, 4, 6

6 - 156.4

7 - 177.7

8 - 138.5

9/13 7.61 d (8.4) 128.4 7, 11, 10, 12

10/12 7.31 t (7.8) 127.6 8, 9, 13

11 7.44 s 134.2 9, 13

1' 4.70 s 101.8 6

2' 2.77 s 72.8 1', 3'

3' 3.18 s 76.2 2', 4'

4' 3.42 s 70.3 6'

5' 3.58 s 73.7 4'

6'

1''

2''

3''

4''

5''

6''

4.33 dd Ha

4.50 dd Hb

-

-

-

-

-

7.09 s

69.0

148.4

145.6

120.7

145.2

165.8

108.6

5'

4'

4, 1'', 2'', 4'', 5''

NMR was measured at 500 MHz (1H) and 150 MHz (13C) (CD3OD).

100

3.5 Antibacterial Profile of Bioactive Constituents

Table 19: Antibacterial profile of the isolated compounds against

Staphylococcus aureus and E. coli.

Compound

MIC (µg/ml)

S. aureus E. coli

I 250 650

II 400 900

III 850 1950

IV 250 750

V 650 1200

Ceftriaxone 20 25

101

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 Discussion

The results of the preliminary investigations carried out on the leaf

extracts/fractions of Loranthus micranthus and Psidium gajava gave some basic

but revealing data that proved very vital for further assay on the plant materials.

First, the comparative study of the phytochemical and anti-microbial properties

of leaves of Loranthus micanthus harvested from different host trees showed

significant variations in the parameters reviewed (Tables 4 & 5). The extracts

from K. acuminata and P. americana exhibited appreciable activities against

some of the bacteria, but, showed only mild activity against the fungi. The

extract from P. americana showed significant activity (P < 0.05) against some

Gram-negative bacteria (P. aeruginosa) while maintaining moderate activity

against the Gram-positive bacteria. Also, alkaloids, terpenoids and tannins were

found to be relatively preponderant in the extracts from P. americana and K.

acuminata as against that from other hosts. Thus, the study indicated that L.

micranthus leaves to be used for the treatment of non-specific infections should

be preferentially sourced from either P. americana or K. acuminata.

In the same vein, the study on the seasonal variations of the phytochemical and

anti-microbial constituents of the plants revealed some interesting parameters.

The investigation on leaves of L. micranthus (parasitic on P. americana)

harvested at different seasons of the year showed variations across the seasons

(Tables 6 & 7). Some phytoconstituents like alkaloids, terpenoids and tannins

102

were found to show significant variations across the seasons. These constituents

were found to be more abumdant in the extracts from the leaves harvested in

April and July. Higher antimicrobial activities were also observed within these

seasons. In effect, better antimicrobial activity was exhibited by the extracts of

the leaves harvested in April and July. This might then suggest that Loranthus

leaves intended for use in the treatment of non-specific infections may be

preferentially harvested either at the onset (April) or the peak (July) of the rainy

season. On the other hand, the seasonal variation study data (Tables 11& 12) on

the phytochemical and antimicrobial activities of Psidium guajava were non-

significant, even though the extracts harvested in April (onset of rainy season)

showed slightly better bioactivities than the other seasons. This may be as a

result of the relatively similar amount of the major phytoconstituents like

terpenoids, tannins, flavonoids, etc. found in the extracts across the seasons.

The results (Table 8) of the study for determination of the folkloric utilization of

the leaves of the African mistletoe (L. micranthus) as an all-purpose

antimicrobial agent showed that it had negligible antifungal activity and thus its

paste should not be recommended for the treatment of wounds or any other

topical infections where pathogenic fungal organisms might be implicated.

Tables 9 & 10 on the investigation of the crude extract and various fractions of

L. mircranthus for antibacterial activity showed that although the plant had

some level of activity against the test organisms, when compared to the control,

all the extracts/fractions showed statistically lower values (up to P<0.001)

suggestive of a generally weak antibacterial action. Among the

extracts/fractions, there was a conspicuous and progressive loss of antibacterial

103

activity with the initial methanol extracts (CMFM and MFM) having the highest

activities while the other solvent fractions showed diverse but significantly

lower values (Figure 8.0). For the fractions, that of acetone (AFM) exhibited the

highest level of antibacterial activity. The activity trend is therefore CMFM >

MFM > AFM > EFM > CFM > HFM. An attempt at further fractionation of the

acetone soluble fraction (AFM) from Loranthus yielded sub-fractions with no

observable zones of inhibition. This suggested total loss of antibacterial activity

at this stage, and consequently, the bioactivity-guided isolation process could

not proceed any further. The phytochemical screening results followed the same

trend as above with CMFM and MFM showing the presence of most of the

constituents screened (Table 9). CFM and HFM only showed traces of

terpenoids and phenolic compounds. AFM with a slightly higher activity than

the other fractions tested positive for such constituents like tannins, alkaloids,

terpenoids and saponins, while the presence of tannins and flavonoids in EFM

might have contributed to its improved antibacterial activity over CFM and

HFM.

The prevailing data for L. micranthus assay showed therefore that the observed

antibacterial activity in mistletoe might have arisen as a result of interactions

among the various plant constituents identified rather than that of any one in

isolation. As previously reported by different researchers, the antimicrobial

activity of several extracts from plants has been linked to compounds belonging

to more than one chemical group. For instance, the chewing sticks which are

widely used in many African countries as an oral hygiene aid come from

different species of plants, and within one stick, the chemically active

104

component may be heterogeneous (Akpata and Akinrimisi, 1977). Also, the

active component of one of the Nigerian chewing sticks (Fagara

zanthoxyloides) was found to consist of various alkaloids (Odebiyi and

Sofowora, 1979). Pawpaw (Carica papaya) yields a milky sap, often called

latex, which is a complex mixture of chemicals (Cowan, 1999). All the

compounds in papaya have been shown to contribute to the antimicrobial

properties of its latex which was found to be bacteriostatic to B. subtilis,

Enterobacter cloacae, E. coli, Salmonella typhi, Staphylococcus aureus and

Proteus vulgaris (Osato et al, 1993). It follows therefore, using bioassay as a

guide, that the utilization, in isolation, of any of the constituents present in

Loranthus for the management of microbial infections might not only lead to

treatment failure but could equally result to the development of resistance

among pathogenic organisms being treated. We therefore recommended the use

of the crude, aqueous or alcoholic extracts of mistletoe leaves in the

management of non-complicated community acquired bacterial infections

(Ukwueze et al, 2013; Ukwueze and Osadebe, 2013).

Conversely, the results of the antimicrobial screening of the solvent fractions

and extracts from P. guajava yielded very positive significant data (Table 14).

Compared with control, all the test materials (except the chloroform-soluble

fraction, PsG-CF) showed antibacterial activities comparable to that of

ceftriaxone at similar concentrations. Most of the fractions from P. guajava

showed similar or slightly higher antibacterial activity than the crude methanol

extract (PsG-CMF) with the ethylacetate-soluble fraction (PsG-EF) exhibiting

the highest antibacterial activity against the test organisms and also showing a

statistically comparable activity with the control. Phytochemical analysis of the

P. guajava extracts/fractions showed that flavonoids and phenolic compounds

105

might be mainly responsible for the antimicrobial activities as the highly active

fractions had a preponderance of these phytoconstituents (Table 13). These

findings were in consonance with literature reports on the plant (Limsong et al.,

2004; Olajide et al 1999; Xavier et al., 2002; Arima and Danno, 2002; Begum

et al., 2004).

The ethyl acetate fraction (PsG-EF) from P. guajava that gave a satisfactory

bioassay result was subjected to further Sephadex-LH 20 chromatographic

fractionation and purifications to afford ten pooled fractions (PsG-EF1 to PsG-

EF10). Fractions PsG-EF4, PsG-EF5 and PsG-EF7 that had good antibacterial

yield were subjected to semi-preparative reverse phase HPLC purification to

isolate the phenolic compounds I - V. The structures of these compounds were

elucidated using a combination of analytical and spectral techniques which

included: UV, 1H-NMR,

13C-NMR, DEPT,

1H

1HCOSY, HMQC, HMBC and

ESI-MS analyses.

The phytochemical and spectral analysis of the isolated compounds indicated

that they were mainly phenolic glycosides having either hexose or pentose

sugars as the glycone portion. Ultraviolet (UV) spectral analysis of most

reported phenolic glycosides show that the nucleus of chromone (benzo-γ-

pyrone) constitutes the parent chromophore in flavonoids and other related

phenolic compounds like xanthones and anthocyanins. These chromones which

occur naturally in the hydroxylated or glucosylated forms have in general two or

three UV bands near 250, 300 and 350 nm. They represent the principal bands

but could be modified by substitutions as could be seen in the UV spectral of

these isolates. Also, the presence of prominent mass peaks of fragments in the

EI-MS spectra of the compounds indicative of loss of either pentose (-132 m/z)

or pyranose (-162 m/z) molecules confirm that they were glycosides. Other

spectral data of the compounds equally supported this assertion (figures 9-13

and the appendices).

106

Compound I was isolated as yellowish viscous liquid with UV maxima at λmax

254.0 and 296.0 nm. The molecular formula of I was deduced as C19H20O9

based on the ESI-MS molecular ion peak at 392.8 [M+H]+. The

1H-NMR

spectrum of compound I showed aromatic signals of an AA’BB’X system at δH

7.7 (m, 2H), δH 7.42 (t, J=7.8 Hz) and δH 7.52 ((t, J=7.4 Hz) respectively. Each

of the AA’BB’ signals were found to be integrating for two protons assigned to

H-9/H-13 and H-10/H-12 respectively while the X signal at δH 7.52 integrated

for one proton assigned to H-11 (Table 15 & figure 9b). These five protons are

assigned to ring A of the aglycone nucleus as shown in the structure below

(XLIII).

XLV

Another set of aromatic signals at δH 6.22 (d, J=2.1) and δH 6.07 (d, J=2.1)

integrating for one proton each were assigned to H-5 and H-3 respectively

attached to ring B of the phenolic nuclei (fig. 9b). The 2D-COSY spectrum and

the coupling constant (J = 2.1) of these protons indicated that they were meta-

coupled, while the HMBC showed no correlation between these protons and the

carbonyl carbon (δC 194.8; C-7). The various spectral correlations including the

fragmentation pattern of the ESI-MS spectrum correctly predicted the structure

of ring B of the aglycone backbone as shown (XLVI).

107

XLVI

The correlations equally showed that unlike in many traditional flavonoid nuclei

where we have fused structures, rings A & B were actually linked by a carbonyl

group. A careful correlation of the HMBC spectrum as presented in Table 15

informed our proposal of a benzophenone skeleton as shown (XLVII).

XLVII

The 1H-NMR of compound I further showed signals from oxygenated C-Hs (δH

2.84-4.81). This strongly suggested the presence of a sugar moiety [Hobley et

al., 1996]. The signal at δH 4.81 is typical of proton attached to the anomeric

carbon (δC 100.6) assigned to H-1'. The signals at δH 3.85 (dd, J=2.2, 12.1),

3.66 (dd, J=5.6, 12.1), 3.34 (dd), 3.32 (dd), 3.22 (dd, J=6.6, 11.1), 2.84 (dd,

108

J=7.8, 9.1) were assigned to H-6'b, H-6'a, H-5', H-3', H-4' and H-2' respectively.

The spectral correlations and the molecular ion fragment peak at 231.1 m/z

[M+1 (- 162)] indicated the presence of a pyranose sugar, which could be either

glucose or galactose [Qian et al., 2008; Al-Fadhli et al., 2006]. The HMBC

spectrum of the compound that showed no correlation between H-4'of the sugar

moiety and signal at δH 6.07 (H-5) of ring B confirmed this sugar to be d-

glucose [Li et al., 2013]. Galactose with its proton at C-4' projecting outwards

would have produced the reverse correlation. This is evident from the structures

of these sugars (XLVIII).

XLVIII

The attachment of the d-glucopyranosyl moiety (sugar unit) at the C-6 position

of the benzophenone was based on the HMBC correlations of H-5 signal to C-1,

C-3, C-4 and C-6 and that of H-1' to only C-6. Also, in the 1H-NMR spectrum,

the anomeric proton signal of glucose was observed as a doublet at δH 4.81 with

diaxial coupling constant J1’,2’ = 7.7 Hz, which confirmed a β configuration and

the pyranose form of sugar unit [Markham and Geiger, 1994; Olszewska and

Wolbis, 2002]. Compound I was thus elucidated as a benzophenone glycoside

109

with the name 2,4-dihydroxy-6-O-β-D-glucopyranosylbenzophenone for which

the trivial name guajaphenone A is proposed. To the best of our knowledge,

this compound is being isolated for the first time from Psidium guajava plant.

Our search also revealed that compound 1 is closely related to another

benzophenone glycoside, 2,6-dihydroxy-4-O-β-d-glucopyranosylbenzophenone,

which was isolated for the first time from the leaves of Psidium guajava. (Fu et

al., 2010).

Compound II was isolated as brownish-yellow viscous liquid with UV maxima

at λmax 250.0 and 302.0 nm and a molecular formula of C20H22O9 which was

deduced based on the ESI-MS molecular ion peak at 406.8 [M+H]+. This puts

the difference between the mass of I and II at exactly +14. This value is

equivalent to the mass of a methylene group (-CH2) and could imply the

replacement of a proton in compound I with a methyl (-CH3) group. A

comparison of the 1H and

13C NMR data of I and II showed that their spectra

have similar chemically equivalent signals in almost all the regions with just

few variations (Table 16). The 1H-NMR spectrum of compound II showed only

one signal at δH 6.20 (1H, s) assigned to H-5 of ring B as against two signals at

δH 6.22 (d, J=2.1, 1H) and 6.07 (d, J=2.1, 1H) assigned respectively to H-5 and

H-3 in compound I (figure 10b). This confirms the replacement of a proton in

compound II at the ring B region. The 1H-NMR spectrum of II also showed an

additional signal at the SP3 hybridized C-H region (δH 2.02: d, J=5.7) integrating

for three protons indicating the presence of a methyl group. 2-D COSY, DEPT,

110

HMQC and HMBC correlations confirmed the assignment of groups in ring B

of the compound as illustrated in this structure (XLIX):

XLIX

A strong HMBC correlation of the methyl proton signals to C-3 supported a

methyl group substitution at this position. Further 1H-

1H COSY, DEPT, HMQC

and HMBC correlations confirmed II as a methyl substituted analogue of I and

was elucidated as 2,4-dihydroxy-3-methyl-6-O-β-D-glucopyranosylbenzophen-

one, for which we have proposed the trivial name guajaphenone B. This

compound is being reported for the first time in literature.

Compound III spectra are a little bit different from the previous ones, even

though there exists reasonable degree of symmetry among the spectra. It was

isolated as brownish viscous liquid with UV maxima at λmax 262.0 and 362.0 nm

and a molecular formula of C26H24O13 which was deduced based on the ESI-MS

molecular ion peak at 545.0 [M+H+]. The

1H-NMR spectrum of this compound

showed signals similar to that of Compound II in most of the analytical regions.

The proton signals at δH 7.50, 7.41 and 7.27 were basically identical as those

111

found at the same region in the previous compounds which corresponded to the

proton arrangement already discussed for ring A of the glycosides. Compared to

compound II, however, there was an aromatic singlet proton signal at δH 7.10

which neither 2D-COSY nor other heteronuclear correlations were able to

assign it as part of rings A or B of that compound. This suggested the presence

of another aromatic nucleus (ring C) in compound III. Its 13

C-NMR spectrum

confirmed this ring with signals between δC 115-125 ppm which were clearly

absent in the spectra of Compounds I & II (Appendices 6, 13 & 18). HMBC

spectrum of the compound showed that this ring C proton is remotely linked to

the signals at δC 108.6. Thus, HMBC and COSY correctly predicted the

structure of ring C of Compound III as in (L) below:

L

The EI-MS spectrum also showed a prominent fragment peak of about 153 m/z

which was closely linked to ring C fragment (Appendix 14). A combination of

all the spectral correlations thus elucidated Compound III as 2,4-dihydroxy-3-

112

methyl-6-O-βD-glucopyranosylbenzophenone (4→5", 6'→1") benzene-

2",3",4",5"-tetraol.

Compound IV spectra presented quite a different correlation from the

compounds so far discussed. The positive ionization from the EI-MS data was

435 [M+1] while the negative ionization gave 433 [M-1] implying that the

molar mass was 434. The major fragment of 435 was 303 [(M+1) -132]. This

signified a loss of 132 m/z corresponding to the loss of a pentose sugar. The

mass of the aglycone was thus 302 m/z. This with the UV absorption spectrum

confirmed the compound to be a glycoside. The 1H-NMR spectrum suggested

that there were two aromatic spin systems [one ABX (δ 7.52, 7.50 and 6.90: two

are otho-coupled while two are meta-coupled) and one AX (δ 6.39 and 6.20: the

two are meta-coupled)]. The 2D-COSY therefore predicted the structures (LI) of

the AX (ring A) and ABX (ring C) aromatic systems to be:

LI

However, the EI-MS data and other spectral information indicated the presence

of another conjugated system (ring B) without proton signals. This presented the

possibility of a fused system, typical of most flavonoids as indicated below

(LII):

113

LII

The spin system from δH 5.45, 4.33, 3.90, 3.87 and 3.50 belonged to the sugar (a

pentose) protons. From the spectral correlations and literature reports, this could

either be rhamnose or arabinose. The absence of a C-methyl signal in the 1H-

NMR spectrum rules out the presence of rahmnose whose occurrence could

equally have caused an upfield shift of H-2' and H-6' signals of the flavonoid

nucleus (Markham and Geiger, 1994). Comprehensive analysis of the NMR

data of IV showed that the compound is the previously reported quercertin-3-O-

α-L-arabinofuranoside with the trivial name, guaijaverin. The structure of the

compound was confirmed by comparison of its 1D and 2D NMR data with

those reported in the literature (Olszewska and Wolbis, 2002; Sanches et al.,

2005; Begum et al., 2002a).

Compound V was isolated as golden-brown viscous liquid with UV maxima at

λmax 265.0 and 294.0 nm and a molecular formula of C25H22O13 which was

deduced based on the ESI-MS molecular ion peak at 531.4 [M+H+]. As

observed with compound II, the difference between the calculated positive

ionization peak for III and V is -14, a value that is equivalent to the mass of a

114

methylene group (-CH2) and which could imply the loss of a methyl (-CH3)

group for a proton in this instance. The 1H-NMR spectrum of the compound

showed signal for this additional proton at δH 6.05 (1H, s) assigned to H-3 of

ring B as against the single signal at the same aromatic region in compound III

(Table 18). Other analytical data were very similar to that of Compound III in

most of the regions. Compound V was thus elucidated as the demethylated

analogue of Compound III with the name 2,4-dihydroxy-6-O-βD-

glucopyranosylbenzophenone (4→5", 6'→1") benzene-2",3",4",5"-tetraol.

The antibacterial profiling of the isolated compounds showed that they all had

activities against S. aureus and E. coli but at different degrees. Compounds I &

IV showed activities closest to that of ceftriaxone against both organisms at

similar concentrations (Table 19). It appeared, however, that substitution (in this

case methylation) generally decreased antibacterial action in each case as can

be seen between I versus II and V versus III with the demethylated forms

showing lower MIC values against the bacteria. It was equally observed that the

compounds all had better antibacterial action against Staphylococcus aureus

than against Escherichia coli.

115

4.2 Conclusion

Several medicinal plants have been used in traditional practice across Africa to

combat infectious disorders prevalent in that region. Currently, many of these

herbal remedies are available in Nigerian and other West African markets with

bogus claims of efficacy, hence the need for scientific validation of these claims

and establishment of the principle components and mechanisms behind their

activities.

Several works have been carried out in the past to verify the folkloric use of the

African mistletoe in the management of microbial infections. Previous works by

our research team on the crude plant powder and some of its fractions have

established low levels of antibacterial activity by L. micranthus, but with

negligible anti-fungal property. The optimal harvesting seasons as well as the

host trees of choice for mistletoe as an antimicrobial agent were equally well

documented. Further bioactivity-guided analysis on the antibacterial potential of

mistletoe, however, showed that the observed antibacterial activity in mistletoe

might have arisen as a result of interactions among the various plant

constituents identified rather than that of any one in isolation. We therefore

recommended the use of the crude, aqueous or alcoholic extracts of mistletoe

leaves in the management of non-complicated community acquired bacterial

infections.

The ethyl acetate fraction, as well as the Sephadex LH-20 fractions from P.

guajava exhibited significant (P < 0.05) antibacterial activity. The isolated

compounds (I-V) from the active fractions of P. guajava displayed moderate

antibacterial activities. The chemical names of the isolated compounds were

116

elucidated as : 2,4-dihydroxy-6-O-βD-glucopyranosylbenzophenone (I); 2,4-

dihydroxy-3-methyl-6-O-βD-glucopyranosylbenzophenone (II); 2,4-dihydroxy-

3-methyl-6-O-βD-glucopyranosylbenzophenone (4→5", 6'→1") benzene-

2",3",4",5"-tetraol (III); quercertin-3-O-α-L-arabinofuranoside (IV) and 2,4-

dihydroxy-6-O-β-D-glucopyranosylbenzophenone (4→5", 6'→1") benzene-

2",3",4",5"-tetraol (V). Compounds I, II, III and V are new natural products

which have not been previously reported in literature, and the trivial names

guajaphenone A, B, C and D were proposed, while Compound IV has been

previously reported. Compound I was found to be closely related to a new

benzophenone glycoside, 2,6-dihydroxy-4-O-β-d-glucopyranosylbenzophenone,

isolated for the first time from the leaves of Psidium guajava (Fu et al., 2010).

All the isolated compounds from P. guajava were also found to have moderate

antibacterial activities against E. coli and S. aureus in comparison to

ceftriaxone, with I and IV showing lower MICs than that of the other isolates

against the organisms.

The results of this study are therefore a major contribution to the field of

medicinal chemistry of drugs used in infectious disease states, considering the

observed limitations of the contemporary antibiotic agents. It is expected that

these isolated compounds could be optimized and developed into useful

therapeutic agents in management of infectious disease states and that further

works should be carried out in order to maximize the full potentials of this

study.

117

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APPENDICES

Appendix 1: ESI-MS /UV Spectra of Compound I

131

Appendix 2: H-NMR Spectrum (500MHz; MeOD) of Compound I

132

Appendix 3: H-NMR Spectrum (600MHz; MeOD) of Compound I

133

Appendix 4: H-NMR Spectrum (600MHz; DMSO-d6) of Compound I

134

Appendix 5: 2-D COSY of Compound I

135

Appendix 6: C-13 NMR Spectrum of Compound I

136

Appendix 7: HMQC Spectrum of Compound I

137

Appendix 8: HMBC Spectrum of Compound I

138

Appendix 9: ESI-MS /UV Spectra Compound II

139

Appendix 10: H-NMR Spectrum (500MHz;MeOD) of Compound II

140

Appendix 11: H-NMR Spectrum (600MHz; DMSO) of Compound II

141

Appendix 12: 2-D COSY of Compound II

142

Appendix 13: C-13 NMR Spectrum of Compound II

143

Appendix 14: ESI-MS /UV Spectra Compound III

144

Appendix 15: H-NMR Spectrum (500MHz; MeOD) of Compound III

145

Appendix 16: H-NMR Spectrum (600MHz; DMSO) of Compound III

146

Appendix 17: 2-D COSY of Compound III

147

Appendix 18: C-13 NMR Spectrum of Compound III

148

Appendix 19: HMQC Spectrum of Compound III

149

Appendix 20: HMBC Spectrum of Compound III

150

Appendix 21: UV Spectrum of Compound IV

151

Appendix 22: ESI-MS Spectra of Compound IV

152

Appendix 23: H-NMR Spectrum (500MHz; MeOD) of Compound IV

153

Appendix 24: 2-D COSY of Compound IV

154

Appendix 25: H-NMR Spectrum (500MHz; MeOD) of Compound V

155

Appendix 26: 2-D COSY of Compound V

156

Appendix 27: 2-D COSY [aromatic region] of Compound V