CHEMICAL TRANSFORMATION AND PHYTOCHEMICAL ...

CHEMICAL TRANSFORMATION AND PHYTOCHEMICAL STUDIES OF

BIOACTIVE CONSTITUENTS FROM EXTRACT OF CALLISTEMON

CITRINUS (CURTIS) SKEELS

by

LARAYETAN ROTIMI ABISOYE

A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY (CHEMISTRY)

DEPARTMENT OF CHEMISTRY

FACULTY OF SCIENCE AND AGRICULTURE

UNIVERSITY OF FORT HARE

ALICE 5700

SOUTH AFRICA

Supervisor: Professor Omobola O. Okoh

Co-supervisor: Professor Alexander Sadimenko

February, 2018

Declaration

I, Larayetan Rotimi Abisoye declare that this thesis entitled “Chemical

Transformation and Phytochemical Studies of Bioactive Constituents from

Extract of Callistemon citrinus (Curtis) Skeels” was carried out by me and

submitted to the University of Fort Hare for the degree of Doctor of

Philosophy in Chemistry in the Faculty of Science and Agriculture, School of

Science, and the work enclosed therein is my original work with exclusion to

the citations and that this work has not been submitted at any other

academic institution in partial or total for the award of any degree.

Name: Larayetan Rotimi Abisoye

Signature ….……………..…………………...

Date: February, 2018

ii

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Declaration on Plagiarism

I, Larayetan Rotimi Abisoye, student number: 201516683 hereby declare

that I am fully aware of the University of Fort Hare‟s policy on plagiarism

and I have taken every precaution to comply with the regulations.

Signature …………………………………

Date …………………………………

Declaration of Research Ethical Clearance

I, Larayetan Rotimi Abisoye, student number 201516683 hereby declare

that I am fully aware of the University of Fort Hare‟s policyon research ethics

and I have taken every precaution to comply with the regulations. I confirm

that my research constitutes an exemption to rule G17.6.10.5 and an ethical

certificate with reference number is not required.

Signature……………………………………………… Date………………………………..

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Certification

This thesis entitled “Chemical Transformation and Phytochemical Studies

of Bioactive Constituents from Extract of Callistemon citrinus (Curtis)

Skeels” meets the regulations governing the award of degree of Doctor of

Philosophy of the University of Fort Hare and is approved for its contribution

to scientific knowledge and literary presentation.

--------------------------------------------- ---------------------------

Prof. Omobola O. Okoh Date

Major supervisor

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Acknowledgements

First and foremost my gratitude goes to Jehovah-El-Shaddai, whose

wisdom and assistance is greatly used in the successful completion of this

thesis and inspiration throughout my years of study in University of Fort

Hare and also for sparing my life even up to this present moment, may His

name be praised in my life.

My sincere and deep gratitude goes to my supervisors Professor

Omobola. O. Okoh, Professor Anthony Okoh, and Professor Alexander. P.

Sadimenko for their help and support in terms of the thorough supervision

given to me on this thesis, their immense contribution, guidance,

understanding and patience throughout the course of this program, you

were more than a supervisors to me; a father, mother and a mentor, thanks

for the unflinching support given to me despite my flares, you are all too

wonderful to be forgotten. May the Almighty God reward you all with great

success.

In addition, I have received strong support and encouragement from my

parent late Pastor David Adeyemi Larayetan and Mrs Comfort Larayetan, I

depend so much on you morally and spiritually and you never let me down,

you are too precious, you are more than a parent to me. May you eat from

the fruit of your labour in life. Amen

Worth mentioning are my colleagues in Chemistry Department Dr.

Abdulrazaq Yahaya, Dr. Taofeek Salaudeen, Dr Olufemi Ademoyegun, Mr.

Lam Olaniyan, Dr. Mike Ojemaye, Dr. Adeniji Goke, Mr. Ezekiel Agoro, Miss

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Dorcas Mutukwa and Mrs. Remi Gbede, University of Fort Hare South Africa

for the stimulation and encouragement, may the good Lord be with you all.

I would like to acknowledge the financial support from Tertiary Education

Trust Fund Nigeria (Tetfund), Kogi State University (KSU) Anyibga Nigeria, Prof

Aothony Okoh of AEMREG UFH and Govan Mbeki Research and Development

Centre, University of Fort Hare, Alice, I am indeed very grateful to these

bodies without their financial support; the journey could have been a bit

rough.

I am extremely grateful to my „Angel‟, my darling and beautiful wife, Mrs

Larayetan Omolayo Elizabeth (nee Olori) for her unfailing love, standing in

the gap during my absence, understanding, motivation and words of

encouragement from her, you are just my perfect and missing ribs, and may

the good Lord that we serve be with you. This acknowledgement would not

be complete if I fail to mention my lovely children, Rehoboth Larayetan,

Israel Larayetan and Hephzibah Larayetan for their understanding and

patience throughout my absence during the course of this program. You will

surely get to your promised land in Jesus name.

I also want to thank all my siblings: Iretiolu Ajao (nee Larayetan), Joy

Oladejo (nee Larayetan), Mercy Adeyemi (nee Larayetan), Abayo Larayetan,

Gospel Larayetan, Dunsin Akinsoto (nee Larayetan) and the baby of the

house Sunday Larayetan for their prayers, advice, concern and words of

encouragement, may the God of our father continue to bind us together in

love.

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I am greatly indebted to the Christ Apostolic Church Campus Fellowship,

KSU branch, Kogi State, Nigeria for their prayers and unflinching love shown

to my family during the course of my absence, my God will surely reward

you.

Finally, I give praise to my maker who has brought me thus far and

making this great dream a reality. I adore the trinity for being my teacher,

my comforter and for the precious gift of life, for keeping my family intact

during my absence; I ascribe all glory to Him.

God is not a man, that he should lie; neither the son of man, that he

should repent: hath he said, and shall he not do it? Or hath he spoken, and

shall he not make it good? Numbers 23:19

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Dedication

This thesis is dedicated to the Almighty God, the Alpha and the Omega, the

first and the last, the beginning and the end and also to my parent Late

Pastor and Mrs. Larayetan for giving me the best legacy in the world -

Education!

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Contributions to Knowledge

1. Paper Published (Peer Reviewed)

Rotimi A. Larayetan, Omobola O. Okoh, Alexender Sadimenko and

Anthony I. Okoh (2017) Terpene constituents of the aerial parts, phenolic

content, antibacterial potential, free radical scavenging and antioxidant

activity of Callistemon citrinus (Curtis) Skeels (Myrtaceae) from Eastern

Cape Province of South Africa. BMC 17(292): 1-9.( DOI 10.1186/s12906-

017-1804-2).

2. Paper Submitted for Consideration

Rotimi A. Larayetan, Omobola O. Okoh and Alexender Sadimenko Effect of

Seasonal Variation on the Secondary Metabolites and Antioxidant Activity of

Callistemon citrinus (Curtis) Skeels (Myrtaceae) grown in Eastern Cape of

South Africa (Journal of Essential oil Bearing Plant).

Rotimi A. Larayetan, Omobola O. Okoh and Alexender Sadimenko

Chemical components, antitrypanosomal, antiplasmodial and antibacterial

potencies of the seed, leaf and flower volatile oils of Callistemon citrinus

(Journal of Essential oil Research).

Larayetan Rotimi, Mike O. Ojemaye, Omobola O. Okoh and Anthony

I.Okoh.Synthesis, characterization, antimalarial, antitrypanocidal and

antimicrobial properties of gold nanoparticle (Journal of Food Science)

3. Papers Presented at Conferences

9th-12th July, 2017: Attendance and oral presentation of paper entitled:

Rotimi A. Larayetan, Omobola O. Okoh, Alexender Sadimenko and

Anthony I. Okoh (2017). Terpene Constituents of the Aerial Parts, Phenolic

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Content, Antibacterial Potential, Free Radical Scavenging and Antioxidant

Activity of Callistemon citrinus (Curtis) Skeels (Myrtaceae) from Eastern

Cape Province of South Africa. 20th Indigenous Plant Use Forum, held at the

Batter Boys Village, Montana, Pretoria, South Africa.

12th-16th September, 2017: Attendance and oral presentation of paper

entitled: Rotimi A. Larayetan, Omobola O. Okoh, Alexender Sadimenko

and Anthony I. Okoh (2017). Effect of Seasonal Variation on the Secondary

Metabolites and Antioxidant Activity of Callistemon citrinus (Curtis)

(Myrtaceae) grown in Eastern Cape of South Africa. 3rd African

Biotechnology and Biomedical Conference (AIBBC–2017), held at Nairobi

Kenya.

4. Award

Best oral presentation 3rd African Biotechnology and Biomedical

Conference (AIBBC–2017), Nairobi Kenya.

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List of Abbreviations and Notations

ABTS 2, 2‟-azino-bis (3-ethylbenzothiazoline-6-sulfonic

acid

AEMREG: Applied and Environmental Microbiology Research

Group

AgNPs silver nanoparticles

AuNPs gold nanoparticles

BHA n-butylated hydroxyl anisole

BHT n-butylated hydroxyl toluene

CC column chromatography

C.citrinus Callistemon citrinus

DPPH 2, 2-diphenyl-1-picrylhydrazyl

EDS energy dispersive X-ray

EOs essential oils

FTIR Fourier-transform infrared spectroscopy

DMSO dimethyl sulfoxide

GC-MS gas chromatography-mass spectrometry

GC gas chromatography

GHz Gigahertz

GMRDC Govan Mbeki Research and Development Centre

GPC gel permeation chromatography

HAT human African trypanosomiasis

HOAc acetic acid

HPAS heteropolyacid

xii

HPLC high performance liquid chromatography

HPMo phosphomolybdic acid

HPW Phosphotungistic Acid

I nuclear magnetic spin

IC50 inhibitory concentration at 50 %

MBC minimum bactericidal concentration

MIC minimum bactericidal concentration

MS mass spectrometry

OH hydroxyl group

OPC overall phenolic content

P.falciparium: Plasmodium falciparium

PPS potassium persulfate

RBC red blood cells

ROS reactive oxygen species

SAMRC South African Medical Council

SEACREG Synthetic, Environmental and Applied Chemistry

Research Group

SEM scanning electron microscopy

T.b Trypanosoma brucei

TEM transmission electron microscopy

TLC thin layer chromatography

UV ultraviolet

UFH University of Fort Hare

WHO World Health Organization

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XRD X-ray diffraction

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Table of Contents

Title Page ............................................................................................ ii

Declaration .......................................................................................... ii

Declaration on Plagiarism ..................................................................... iii

Certification ........................................................................................ iv

Acknowledgements .............................................................................. v

Dedication ........................................................................................ viii

Contributions to Knowledge .................................................................. ix

List of Abbreviations and Notations ........................................................ xi

General Abstract ............................................................................. xxxii

Chapter 1 ........................................................................................... 1

Introduction ........................................................................................ 1

1.1 Background ................................................................................ 1

1.2 Introduction on Malaria and Trypanosomiasis .................................. 4

1.2.1 Intermediary Agents of Plasmodium ......................................... 6

1.2.2 Life cycle of Plasmodium falciparium ......................................... 6

1.2.3 Intermediary Agents of Trypanosomiasis ................................... 7

1.2.4 Life Cycle of Tsetse Fly ........................................................... 8

1.3 Motivation/Rational for the Study .................................................. 8

1.4 Hypothesis ................................................................................. 9

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1.5 Problem Statement ...................................................................... 9

1.6 Aims and Objectives of Research .................................................. 10

Chapter 2 .......................................................................................... 12

Review of Literature ............................................................................ 12

2.1 Drug Discovery .......................................................................... 12

2.2 Natural products as a foundation of novel drugs ............................. 13

2.3 Demands for Aromatic/Therapeutic Plants as Source of ................... 15

Medicine ......................................................................................... 15

2.4 Definition of the Volatile Oil ......................................................... 16

2.5 Groups of Volatile Oil Compounds ................................................. 18

2.5.1 Hydrocarbons ....................................................................... 18

2.5.2 Esters .................................................................................. 18

2.5.3 Oxides ................................................................................. 19

2.5.5 Alcohols ............................................................................... 19

2.5.6 Phenols ................................................................................ 20

2.5.7 Aldehydes ............................................................................ 20

2.5.8 Ketones ............................................................................... 20

2.6 Secondary Metabolites of Aromatic Plants with Unique .................... 21

Reference to Terpenoids ................................................................... 21

2.6.1 Monoterpenes ....................................................................... 21

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2.6.2 Diterpenes ........................................................................... 22

2.6.3 Triterpenes ........................................................................... 22

2.7 Factors Affecting Composition of Volatile Oils ................................. 23

2.7.1 Harvest Time ........................................................................ 24

2.7.2 Method of extraction .............................................................. 24

2.7.3 Temperature ........................................................................ 25

2.8 Methods of Administration of Volatile Oils ...................................... 25

2.9 Isolation of Bioactive Compounds ................................................. 26

2.10 Overview of Analytical Methods Used in Natural Product ................ 26

Research ......................................................................................... 26

2.10.1 Pre-extraction Preparation of Samples ................................... 26

2.10.2 Extraction ........................................................................... 27

2.11 Extraction Techniques for Crude Extract ...................................... 28

2.11.1 Maceration ......................................................................... 29

2.11.2 Cold Pressing ...................................................................... 29

2.11.3 Steam Distillation ................................................................ 29

2.11.4 Microwave Assisted Extraction .............................................. 30

2.11.5 Solvent Extraction ............................................................... 30

2.11.6 Hydrodistillation .................................................................. 31

2.12 Different Chromatography and Spectroscopy Techniques ............... 32

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2.12.1 High Performance Liquid Chromatography (HPLC) ................... 32

2.12.2 Thin Layer Chromatography (TLC) ......................................... 33

2.12.3 Preparative Thin Layer Chromatography (PTLC) ...................... 33

2.12.4 Gel Permeation Chromatography (GPC) .................................. 34

2.12.5 Column Chromatography (CC) .............................................. 35

2.12.6 Fourier Transform Infrared Spectroscopy (FTIR) ...................... 36

2.12.7 Mass Spectrometry (MS) ...................................................... 37

2.12.8 Nuclear Magnetic Resonance (NMR) Spectroscopy ................... 37

2.13 Plant Products and Their Transformation ...................................... 38

2.13.1 Pinenes .............................................................................. 39

2.13.2 Eucalyptol .......................................................................... 43

2.13.3 Limonene ........................................................................... 45

2.13.4 α-Terpineol ......................................................................... 48

2.14 Chemical Transformations in Plants ............................................. 49

2.15 Antioxidant Activity ................................................................... 51

2.15.1 Mechanism of Action of Antioxidants ...................................... 53

2.15.2 Generation of Free Radicals in the Body ................................. 53

2.15.3 Natural versus Synthetic Antioxidants .................................... 54

2.15.4 Plants as Sources of Antioxidants .......................................... 54

2.15.5 Examples of Natural Antioxidants .......................................... 56

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2.16 New Antioxidants ...................................................................... 62

2.16.1 Polyphenols ........................................................................ 63

2.16.2 Flavonoids .......................................................................... 64

2.16.3 Diphenyl Picryl Hydrazyl (DPPH) Free Radical .......................... 65

2.17 Plant Empire as Foundation of Leading Drugs ............................... 68

2.17.1 Introduction ........................................................................ 68

2.17.2 Plant under Study (The Genus Callistemon) ............................ 69

2.17.3 Callistemon citrinus ............................................................. 70

Chapter 3 .......................................................................................... 85

Experimental ..................................................................................... 85

3.1 Plant Collection ........................................................................ 85

3.2 Microbial Strains....................................................................... 85

3.3 Reagents Used ......................................................................... 85

3.4 Separation of Volatile Oils.......................................................... 86

3.5 Gas Chromatography - Mass Spectrometry .................................. 86

3.6 GC-MS Determination of Bioactive Compounds ............................ 86

3.7 Detection of Components .......................................................... 87

3.8 Preparation of Plant Extracts ...................................................... 87

3.9 Extraction ................................................................................ 88

3.10 Synthesis of Silver Nanoparticles ............................................. 92

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3.11 Synthesis of Gold Nanoparticles ............................................... 93

3.12 Characterization .................................................................... 93

3.13 In vitro Antioxidant Action ...................................................... 94

3.14 In vitro Antibacterial Action ..................................................... 95

3.15 Qualitative Phytochemical Screening ........................................ 96

3.16 Preparation of McFarland Turbidity Standard ............................. 96

3.17 Quantification of Total Phenolic Content .................................... 97

3.18 Quantification of Total Tannin Content ...................................... 98

3.19 Quantification of Total Flavonoids Content................................. 98

3.20 Quantification of Total Flavonols Content .................................. 99

3.21 Phosphomolybdate Assay ........................................................ 99

3.22 Pilot Phytochemical Tests ...................................................... 100

3.23 Time Kill Assay .................................................................... 101

3.24 Plasmodium falciparum Culture and Maintenance ..................... 102

3.25 Antiplasmodial Activity ......................................................... 102

3.26 Antitrypanosomal Activity ..................................................... 103

3.27 Single Concentration Screening ............................................. 104

3.28 Dose Response .................................................................... 105

3.29 Cytotoxicity Assay ................................................................ 105

3.30 Statistical Analysis ............................................................... 106

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Chapter 4 ........................................................................................ 107

Results and Discussion ...................................................................... 107

4.1 Composition, Antibacterial and Antioxidant Activity of Essential Oils of

Callistemon citrinus from Eastern Cape Province of South Africa .......... 107

4.1.1 Abstract .......................................................................... 107

4.1.2 Background ..................................................................... 107

4.1.3 Constituents of the Volatile Oil .............................................. 108

4.1.4 Overall Phenolic Content ...................................................... 113

4.1.5 Antibacterial Activities of the Volatile Oil Obtained from Leaves and

Flowers ...................................................................................... 114

4.1.6 In vitro Antioxidant Action .................................................... 116

4.1.7 Conclusion ......................................................................... 119

4.2 Effect of Seasonal Variation on the Secondary Metabolites and

Antioxidant Activity of Callistemon citrinus ........................................ 120

4.2.1 Abstract .......................................................................... 120

4.2.2 Background ..................................................................... 120

4.2.3 Chemical Composition of the Volatile Oil from Callistemon citrinus

(January-December, 2016) ........................................................... 123

4.2.4 Effect of Seasonal Variation on Percentage Yield of the Volatile Oil

................................................................................................. 130

4.2.5 Effect of Seasonal Variation on Volatile Oil Content .................. 130

xxi

4.2.6 Effect of Flowering Phase on Percentage Yield and Volatile Oil

Content ...................................................................................... 133

4.2.7 Effect of Seasonal Variation on the Antioxidant Activity of

Callistemon citrinus ..................................................................... 133

4.2.8 Conclusion ....................................................................... 135

4.3 Determination of Bioactive Compounds, Phytochemical Constituents,

Antioxidant Capacity and In Vitro Antimicrobial Potential of Crude Extracts

Isolated from Callistemon citrinus .................................................... 136

4.3.1 Abstract .......................................................................... 136

4.3.2 Background ........................................................................ 137

4.3.3 Constituents of the Extracts.................................................. 138

4.3.4 Phytochemical Screening ...................................................... 142

4.3.5 Overall Tannin Content ........................................................ 143

4.3.6 Overall Antioxidant Capacity ................................................. 143

4.3.7 Overall Phenolic Content ...................................................... 144

4.3.8 Overall Flavonoid Content ................................................. 144

4.3.9 Overall Flavonol Content ...................................................... 145

4.3.10 Antioxidant Activities of the Crude Extracts ........................... 145

4.3.11 Antibacterial Activity of the Extracts .................................... 147

4.3.12 Minimum Inhibitory Concentration ....................................... 151

4.3.13 Time Kill ........................................................................... 152

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4.3.14 Conclusion ........................................................................ 154

4.4 Chemical Components, Antitrypanosomal, Antiplasmodial and

Antibacterial Potencies of the Seed, Leaf and Flower Volatile Oils of

Callistemon citrinus ........................................................................ 155

4.4.1 Abstract .......................................................................... 155

4.4.2 Background ........................................................................ 155

4.4.3 Components of the Volatile Oils ............................................ 156

4.4.4 Antiplasmodial Activity ......................................................... 158

4.4.5 Antitrypanosomal Activity ..................................................... 159

4.4.6 Cytotoxicity Activity ............................................................. 161

4.4.7 Antibacterial Potency of Seed Volatile Oil ................................ 162

4.4.8 Conclusion ......................................................................... 164

4.5 Phytochemical Investigation, Isolation and Characterization of

Bioactive Compounds Responsible for Antiplasmodial and Antitrypanosomal

Action from Crude Extracts of Callistemon citrinus ............................. 165

4.5.1 Abstract ............................................................................. 165

4.5.2 Background ........................................................................ 165

4.5.3 Percentage Yield ................................................................. 167

4.5.4 Isolation of Fractions ........................................................... 169

4.5.5 Antitrypanosomal Action ...................................................... 169

4.5.6 Antimalarial Activity ............................................................ 172

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4.5.7 Cytotoxicity Activity ............................................................. 174

4.5.8 Phytochemical Analysis ........................................................ 178

4.5.9 Description of Active Principle ............................................... 180

4.5.10 FT IR Spectrum of Hexane Crude Extract of Callistemon citrinus

................................................................................................. 181

4.5.11 Conclusion ........................................................................ 182

4.6 Silver Nanoparticles Mediated by Callistemon citrinus extracts:

Antimalarial, Antitrypanosomal, and Antibacterial Efficacy ................... 182

4.6.1 Abstract ............................................................................. 182

4.6.2 Background ........................................................................ 183

4.6.3 Synthesis and Characterization ............................................. 187

4.6.4 Antitrypanosomal Activity ................................................... 193

4.6.4 Antiplasmodial Action ........................................................ 195

4.6.6 Cytotoxicity Activity ............................................................. 197

4.6.7 Antibacterial Activity .......................................................... 198

4.6.8 Determination of the Minimum Inhibitory Concentration and

Minimum Bactericidal Concentration .............................................. 199

4.6.9 Conclusion ......................................................................... 203

4.7 Synthesis, Characterization, Antimalarial, Antitrypanocidal and

Antimicrobial Properties of Gold Nanoparticles ................................... 203

4.7.1 Abstract ............................................................................. 203

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4.7.2 Background ........................................................................ 204

4.7.4 Antitrypanosomal and Cytotoxicity Activities ......................... 209

4.7.5 Antiplasmodial Properties ................................................... 211

4.7.6 In vitro Antibacterial Activity .............................................. 211

4.7.7 Conclusion ......................................................................... 213

Chapter 5 ........................................................................................ 214

General Discussion, Conclusions and Recommendation .......................... 214

5.1 General Discussion and Main Findings ......................................... 214

5.2 Future Trends, Prospects and Recommendations .......................... 222

References ...................................................................................... 223

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List of Table

TABLE 4.1: FRACTIONAL COMPOSITION OF CONSTITUENTS OF THE LEAVES, FLOWERS AND

STEMS OILS OF CALLISTEMON CITRINUS. ................................................ 108

TABLE 4.2: MAJOR COMPONENTS OF VOLATILE OIL OF CALLISTEMON CITRINUS FROM

VARIOUS PARTS OF THE WORLD ........................................................... 112

TABLE 4.3: INHIBITION ZONE (MM) SHOWING ANTIBACTERIAL ACTIVITIES OF THE

VOLATILE OILS AND CIPROFLOXACIN ..................................................... 115

TABLE 4.4: IC50 PROFILE OF THE LEAVES AND FLOWERS‟ OIL OF CALLISTEMON CITRINUS

(MG /ML) ..................................................................................... 118

TABLE 4.5: FRACTIONAL COMPOSITION OF THE CONSTITUENTS OF THE LEAVES OF

CALLISTEMON CITRINUS (JANUARY-DECEMBER). ....................................... 124

TABLE 4.6: EFFECT OF SEASON ON ANTIOXIDANT CAPACITY OF CALLISTEMON CITRINUS

VOLATILE OIL IC50 (MG ML-1). .......................................................... 135

TABLE 4.7: COMPONENTS OF METHANOLIC EXTRACT OF CALLISTEMON CITRINUS. ...... 139

TABLE 4.8: COMPONENTS OF ETHYL ACETATE EXTRACTS OF CALLISTEMON CITRINUS. . 140

TABLE 4.9: QUALITATIVE PHYTOCHEMICAL SCREENING OF ETHYL ACETATE EXTRACT OF

CALLISTEMON CITRINUS. ................................................................... 142

TABLE 4.10: QUANTITATIVE PHYTOCHEMICAL CONSTITUENTS OF ETHYL ACETATE AND

METHANOL EXTRACTS OF CALLISTEMON CITRINUS. ..................................... 143

TABLE 4.11: ANTIRADICAL ABILITY OF EXTRACTS FROM CALLISTEMON CITRINUS. ....... 146

TABLE 4.12: REGION OF INHIBITION (MM) SHOWING ANTIBACTERIAL ACTIVITIES AGAINST

BACTERIAL TEST ORGANISMS. ............................................................. 148

TABLE 4.13: MINIMUM INHIBITION CONCENTRATION OF CALLISTEMON CITRINUS EXTRACTS

AGAINST MICROBIAL STRAINS. ............................................................ 151

xxvi

TABLE 4.14: TIME OF KILL FOR THE STANDARD DRUGS CIPROFLOXACIN (0.1 MG ML-1)

AGAINST BACTERIAL ORGANISMS. ........................................................ 153

TABLE 4.15: TIME OF KILL FOR ETHYL ACETATE EXTRACT (0.1 MG ML-1) AGAINST

BACTERIAL ORGANISM. ...................................................................... 153

TABLE 4.16: CHEMICAL COMPOSITION OF THE VOLATILE SEED OIL OF CALLISTEMON

CITRINUS. .................................................................................... 156

TABLE 4.17: INHIBITION ZONE (MM) FOR SEED VOLATILE OIL OF CALLISTEMON CITRINUS

AND CIPROFLOXACIN (STANDARD DRUG). ............................................... 163

TABLE 4.18: PERCENTAGE YIELD OF VARIOUS CRUDE EXTRACTS. ......................... 168

TABLE 4.19: IC50 FOR A-E. ................................................................... 170

TABLE 4.20: IC50 FOR F1-E2. ............................................................... 171

TABLE 4.21: IC50 FOR A-E. ................................................................... 173

TABLE 4.22: IC50 FOR F1-S2. ............................................................... 173

TABLE 4.23: PERCENTAGE VIABILITY FOR F1-E2. ........................................... 174

TABLE 4.24: PERCENTAGE VIABILITY FOR FRACTIONS A-E. ................................ 175

TABLE 4.25: IC50 FOR FRACTIONS A-E. ..................................................... 176

TABLE 4.26: PERCENTAGE VIABILITY FOR FRACTIONS F1-E2. ............................. 177

TABLE 4.27: IC50 FOR FRACTIONS F1- S2.................................................. 177

TABLE 4.28: ANTIMALARIAL, ANTITRYPANOSOMAL CHARACTERISTICS AND CYTOTOXICITY

OF THE CRUDE EXTRACTS AND FRACTIONS OF CALLISTEMON CITRINUS. ............. 178

TABLE 4.29: PHYTOCHEMICAL ANALYSIS OF VARIOUS EXTRACTS OF CALLISTEMON

CITRINUS. .................................................................................... 179

xxvii

TABLE 4.30: INHIBITION ZONE (MM) SHOWING ANTIBACTERIAL ACTIVITIES OF THE

NANOPARTICLES DERIVED FROM CALLISTEMON CITRINUS WITH THE STANDARD DRUG

CIPROFLOXACIN AGAINST BACTERIAL TEST ORGANISMS. .............................. 200

TABLE 4.31: MINIMUM INHIBITORY CONCENTRATION (MIC) VALUES (MG/ML) FOR

NANOPARTICLES AND STANDARD DRUG. ................................................. 202

TABLE 4.32: MINIMUM BACTERICIDAL CONCENTRATION (MBC) VALUES (MG/ML) FOR

NANOPARTICLES AND STANDARD DRUG. ................................................. 202

TABLE 4.33: ZONE OF INHIBITION OF THE SYNTHESIZED AUNPS FROM CALLISTEMON

CITRINUS AND THE STANDARD DRUG. .................................................... 212

xxviii

List of Figures

FIGURE 3.1 : COLUMN CHROMATOGRAPHY APPARATUS USED IN THIS EXPERIMENT ....... 91

FIGURE 4.1: ANTIBACTERIAL ACTIVITY OF LEAF OIL OF CALLISTEMON CITRINUS. ....... 116

FIGURE 4.2: ANTIBACTERIAL ACTIVITY OF FLOWER OIL OF CALLISTEMON CITRINUS. ... 116

FIGURE 4.3: ABTS SCAVENGING ACTION. ................................................... 118

FIGURE 4.4: DPPH SCAVENGING ACTION. ................................................... 118

FIGURE 4.5: KEY COMPONENTS IDENTIFIED IN THE VOLATILE OIL OF CALLISTEMON

CITRINUS LEAVES. ........................................................................... 129

FIGURE 4.6: BIOSYNTHETIC PATHWAY OF SOME OF THE COMPONENTS OF CALLISTEMON

CITRINUS VOLATILE OIL. ................................................................... 132

FIGURE 4.7: ANTIRADICAL EFFECT OF THE LEAF OILS ISOLATED FROM CALLISTEMON

CITRINUS. .................................................................................... 135

FIGURE 4.8: ANTIRADICAL EFFECTS OF ETHYL ACETATE AND METHANOL EXTRACTS AND

STANDARD DRUG (VITAMIN C) ON DPPH RADICALS. ................................. 147

FIGURE 4.9: ANTIRADICAL EFFECTS OF ETHYL ACETATE AND METHANOL EXTRACTS AND

STANDARD DRUG (VITAMIN C) ON ABTS RADICALS. .................................. 148

FIGURE 4.10: ANTIBACTERIAL ACTIVITY OF ETHYL ACETATE LEAF EXTRACT. ............ 149

FIGURE 4.11: ANTIBACTERIAL ACTIVITY OF METHANOL LEAF EXTRACT. .................. 150

FIGURE 4.12: PLDH MALARIA ASSAY (SINGLE CONCENTRATION). ....................... 159

FIGURE 4.13: DOSE-RESPONSE CURVE FOR TRYPANOSOME ASSAY 1 ..................... 160

FIGURE 4.14: SINGLE ASSAY CONCENTRATION FOR CYTOTOXICITY. ..................... 161

FIGURE 4.15: DOSE RESPONSE FOR TRYPANOSOMAL ASSAY FOR A-E. ................... 171

FIGURE 4.16: DOSE RESPONSE FOR TRYPANOSOMAL ASSAY FOR F1-E2. ................ 171

FIGURE 4.17: DOSE RESPONSE PLDH ASSAY FOR A-E. .................................... 172

xxix

FIGURE 4.18: DOSE RESPONSE PLDH ASSAY FOR F1-S2. ................................ 173

FIGURE 4.19: PLDH ASSAY SINGLE CONCENTRATION FOR F1-E2. ....................... 174

FIGURE 4.20: CYTOTOXICITY ASSAY: SINGLE CONCENTRATION SCREEN. ................ 175

FIGURE 4.21: CYTOTOXICITY ASSAY: IC50 FOR CRUDE A-E. ............................. 176

FIGURE 4.22: CYTOTOXICITY ASSAY FOR FRACTION F1-E2: SINGLE CONCENTRATION

SCREEN. ...................................................................................... 176

FIGURE 4.23: CYTOTOXICITY ASSAY: IC50 FOR FRACTIONS F1-E2. .................... 177

FIGURE 4.24 : 1H NMR SPECTRAL EXAMINATION OF COLUMN FRACTION 17 (HEXANE

FRACTION OF C. CITRINUS LEAVES). ..................................................... 180

FIGURE 4.25: 13C SPECTRAL EXAMINATION OF COLUMN FRACTION 17 (HEXANE

FRACTION OF LEAVES OF C. CITRINUS). ................................................. 181

FIGURE 4.26: CRYSTAL IMAGE OF THE SUSPECTED PURE COMPOUND. ................... 181

FIGURE 4.27: FT IR SPECTRUM OF THE HEXANE CRUDE LEAF EXTRACT. ................. 182

FIGURE 4.28: X-RAY DIFFRACTOGRAM OF (A) CALLISTEMON CITRINUS EXTRACT AND

AGNPS OBTAINED FROM (B) LEAVES, (C) FLOWERS, AND (D) SEEDS. ............. 188

FIGURE 4.29: FT IR SPECTRUM OF (A) CALLISTEMON CITRINUS EXTRACT AND AGNPS

OBTAINED FROM (B) LEAVES, (C) FLOWERS, AND (D) SEEDS. ...................... 190

FIGURE 4.30 : TEM MICROGRAPHS OF CALLISTEMON CITRINUS MEDIATED AGNPS

OBTAINED (A) LEAVES, (B) FLOWERS, AND (C) SEEDS. .............................. 191

FIGURE 4.31: SEM IMAGES OF CALLISTEMON CITRINUS MEDIATED AGNPS OBTAINED (A)

LEAVES, (B) FLOWERS, (C) SEEDS, AND (D) EDS OF AGNPS. ..................... 192

FIGURE 4.32: ABSORPTION SPECTRA OF AGNPS OBTAINED WITH DIFFERENT PLANT

PARTS. ........................................................................................ 193

FIGURE 4.33: SINGLE CONCENTRATION OF TRYPANOSOME ASSAY. ....................... 194

xxx

FIGURE 4.34: DOSE-RESPONSE CURVE FOR TRYPANOSOME ASSAY. ...................... 194

FIGURE 4.35: DOSE-RESPONSE CURVE FOR PLDH ASSAY.* .............................. 196

FIGURE 4.36: SINGLE ASSAY CONCENTRATION FOR CYTOTOXICITY. ...................... 198

FIGURE 4.37: FTIR SPECTRA OF PLANT EXTRACT AND GOLD NANOPARTICLES. ......... 207

FIGURE 4.38: ABSORPTION SPECTRA OF GOLD NANOPARTICLES. ......................... 208

FIGURE 4.39: IMAGES OF AUNPS UNDER (A) SEM, (B) EDS, AND (C) TEM. ......... 209

FIGURE 4.40: DOSE RESPONSE CURVE FOR TRYPANOSOME ASSAY.1 ..................... 211

xxxi

Keywords

ABTS; AgNPs; Antimalarial; Antimicrobial; Antioxidant; Antiplasmodial

action; Antitrypanosomal activity; AuNPS; Bioactive compounds;

Callistemon citrinus; Cell cytotoxicity; Crude extracts; Cytotoxicity activity;

DPPH; FTIR; GC-MS; Hela; Hydro distillation; IC50; OPC; Phytochemicals;

Seasonal variation; SEM; TEM; Volatile oil; XRD.

xxxii

General Abstract

Callistemon citrinus belongs to the family Myrtaceae and exhibits

therapeutic activities. The aerial parts of this plant are used to treat different

ailments, among them are parasitic infections. The leaves, flowers and

stems of Callistemon citrinus were subjected to hydrodistillation. The oils

collected were studied by GC-MS analysis for the essential constituents. The

overall phenolic content of the leaves oil, radical scavenging, antibacterial

action and antioxidant activities of the essential oils of Callistemon citrinus

were determined using standard methods, with free radical DPPH or ABTS as

reference antioxidants. Chemical transformation of the components was

examined for a whole year. A relationship between the chemical change in

the volatile oil constituents, antioxidant capacity, percentage yield of the oil

of Callistemon citrinus and fluctuation in season has been established. Active

phytochemicals present in both ethyl acetate and methanolic extracts of

Callistemon citrinus were determined spectrophotometrically. The

antimicrobial properties, time of kill, and antioxidant activity of the extracts

were explored. The bioactive components were characterized by high level

of fatty acids. Squalene, a triterpenoid synthesized in human liver was

obtained in the two extracts at varying amounts. The ethyl acetate extract

demonstrated strong activity against P. aeruginosa ACC (28.7 ± 1.2 mm),

Listeria ACC (26.0 ± 2.0 mm) and Escherichia coli ATCC 35150 (24.0 ± 3.5

mm). Qualitative phytochemical screening revealed the presence of

alkaloids, glycosides, saponins, steroids and triterpenoids, fats and oils,

flavonoids, phenols and tannins in them. In the quantitative phytochemical

xxxiii

determination (total tannin, total flavonoids and flavonols, total phenolic and

total antioxidant capacity) were carried out. The minimum time needed to

kill the tested bacterial strains totally ranged from 15 to 24 hours.

The aqueous extracts used for biosynthesis of nanoparticles were

obtained from the fresh aerial parts of the plant. The biosynthesized gold

and silver nanoparticles (AuNPs and AgNPs) of the aqueous extracts of the

seed, flower and leaf of the plant, which are active as reducing and capping

agents, were characterized using UV-VIS spectrophotometry, XRD, SEM,

EDS, TEM, and FT IR. The XRD analysis revealed that the AgNPs were

crystalline and the TEM showed that the shapes were spherical with an

average size of 29 nm. For AuNPs, an average particle size of about 37 nm

was confirmed by the TEM while the morphology and composition of the

AuNPs were ascertained by SEM and EDS micrographs; uneven spherical

shaped nanoparticles were established by the SEM. Both SEM and EDS

demonstrated triangular shaped materials made up of silver and oxygen

only. Absorption spectra confirmed by UV-VIS signify the dispersed nature of

the synthesized nanoparticles with absorption band observed at 280 nm for

the leaf AgNPs. FT IR had absorption bands at about 1700 cm-1 establishing

the C=O stretching due to the amide bond while the FT IR for the AuNPs

showed an absorption peak at 230 cm-1 confirming the presence of gold

nanoparticles.

The phytochemical investigation, isolation and characterization of the

bioactive compounds of various organic crude extracts like hexane,

dichloromethane, methanol and ethyl acetate were as well carried out, and

xxxiv

the compounds responsible for their medicinal actions were determined. The

results from different experiments revealed that the leaves and flowers of

Callistemon citrinus possessed phenolic compounds and cyclic ethers with a

variety of pharmacological action. The ethyl acetate and methanol crude

extracts were found to possess broad spectrum of antimicrobial activities

and pharmaceutically essential bioactive components with striking

antioxidant capacities that may be used in the synthesis of novel drugs for

the management of different ailments.

The AuNPs and AgNPs synthesized from the seed, flower and leaf extracts

of Callistemon citrinus where found to have prominent antimalarial,

antiplasmodial, and antibacterial activities. The biosynthesized nanoparticles

inhibit all the bacterial strains used and they were not cytotoxic to Hela

cells, confirming their prospect for use as an excellent source for naturally

occurring drugs against malaria, cell cytotoxicity, trypanosomes, and

microbial infection. Similarly the crude organic extracts and the fractions

derived from them exhibited high antimalarial and antitrypanosomal

activities, but they were toxic to Hela cells. This is an indication that they

will not be safe for use as targeted drugs for mammalian organism.

1

Chapter 1

Introduction

1.1 Background

A wealth of knowledge on the use of plant materials employed in

promoting health has increased over the years and this information is

readily accessible for present researchers who are engaged in drug

discovery. The most primitive evidence of human use of plants as remedy

for healing dated back to the Neanderthal age, where plants were used as

medicine in the form of crude drugs such as tinctures, powders, teas,

poultice and other herbal formulation (Ramawat and Merrillon, 2008;

Winslow and Kroll, 1998).

In South America, Greece, Egypt, China, and Rome plants were

broadly used as a basis of both food and medicine (Grabley et al., 1999,

Newman and Cragg, 2009, Atanasov et al., 2015). Current drugs and

conventional medicines are principally based on natural products making

nature the keystone of drug development with roughly 70% of the entire

drugs in the market linked with natural products. Detection of novel

natural product based drugs encompasses isolation of new plant

metabolites obtained from diverse sources such as marine organisms,

plants and bacteria. Nature is seen as a dynamic biochemical factory

where both primary and secondary metabolites are generated

biosynthetically (Ogita et al., 2015).

Study conducted on plants with medicinal properties along with the

recognition of the chemical constituents accountable for their various

2

activities have validated the primordial traditional healing knowledge and

have confirmed the enduring curative potential of several plant medicines

(Babu et al., 2009).

Herbs derived from plants have been found to interact positively with

human body thereby producing beneficial effects in terms of health

promotion. Captivatingly, it has been observed that vertebrates other

than man eat some types of plants as self-medication under diseased

conditions (Fowler et al., 2007; Krief et al., 2010; Raman and Kandula,

2008). This observation of eating behavior and unanticipated manners in

wild animals has led to the detection of plants with healing potentials.

The study of self-medication observed in animals may lead to a new

approach in drug discovery for man. For example, new antimalarial

compounds were isolated from Trichilia rubescens leaves due to a

behavioral study on chimpanzees from a natural population in Uganda

(Pan et al., 2015).

Roughly 80% of potentially active drugs like immunosuppressive,

anticancer, antimicrobial, and cardiovascular drugs are derived from plant

origin (Devasagayam et al., 2004; Gordaliza, 2009; Liu, 2003). In the

same vein 80% of drug components are also directly obtained from

natural products (Maridass and De Britto, 2008), while 50% of

pharmaceuticals are imitative of compounds which were first isolated as

active ingredient from plants, animals, insects, and organisms (Krief et

al., 2010).

3

Ethnobotanical studies conducted throughout Africa substantiate that

indigenous plants are the chief constituents of traditional African

medicines (Cunningham, 2001) About 60% of the population from South

Africa utilize therapeutic plants for their health care needs and roughly

3,000 species are employed by approximately 200,000 indigenous

traditional healers (Van Wyk, et al., 1997).

It has been documented that naturally occurring metabolites exhibit

superior biochemical specificity and chemical variety compared to

synthetic drugs (Koehn and Carter, 2005). Several of these metabolites

as well as their products of their transformation have exceptionally useful

therapeutic properties. Primary metabolites play a fundamental role in the

metabolism and replication of cells and they are significant in the survival

and metabolic process of an organism whereas secondary metabolites are

definite for some limited variety of species.

Primary metabolites consist of protein, carbohydrate, nucleic acid,

lipids, steroids and fatty acids, which occur in the course of biosynthetic

pathways and play a crucial role in plant metabolism and reproduction of

cells, the secondary metabolites comprises of phenols, flavonoids,

saponins, alkaloids and terpenoids that serve in protection, predation and

communications to an organism. (Zivanovic, 2012). A number of

metabolites have been isolated from plants and many of them have

influential physiological effects in humans and are used as medicines.

Secondary metabolites have been documented to constitute the active

component of curative plants (Kinghorn et al., 2003).

4

Volatile oil also referred to as concentrate, essential oil, etheric oil or

aetheroleum is a complex blend of essential constituents produced by

living organisms. They are so called because of their ability to evaporate

when subjected to heat, which makes them different from fixed oils which

do not evaporate and cannot be distilled. Essential oils are odoriferous in

nature and commonly found in the plant kingdom, in unique plant parts

like the secretory hair, glands, ducts and resin ducts (Ahmadi et al.,

2002; Bezic et al., 2009; Ciccarelli et al.).

1.2 Introduction on Malaria and Trypanosomiasis

Mosquito is the chief vector among the several vector-borne diseases

affecting both animal and man. There are two main mosquito borne

diseases: the viral diseases transmitted principally by the Aedes aegypti,

which are responsible for yellow and dengue fever and also the parasitic

disease malaria which is an exceptionally precarious one parasitic caused

by Plasmodium falciparium parasite and transmitted by the Anopheles

species.

Malaria simply means „bad air‟ and is coined from two Italian words

„mal‟ and „aria‟ (Kolli and Sunderarajan, 2013). Humans are affected by

Plasmodium through the bite of an anopheles mosquito (Ongecha, et al.,

2006). It has been documented that about 350-500 million people are

infected by malaria annually leading to about 1 million deaths of African

children alone. An estimated amount of about 220 million people are also

affected with dengue fever claiming approximately 12,500-25,000 people

worldwide (Gupta et al., 1996; Hammami et al., 2011).Yellow fever leads

5

to about 200,000 ailment killing about 30,000 worldwide as estimated by

WHO. Majority of this death is recorded in areas not vaccinated

predominantly in Africa (Meyer et al., 1982).

Over hundred countries of the World including Africa, South and middle

America, hot and mild hot zones, Southeast Asia and Oceania to mention

a few, were pervasive of malaria in the 20th century. Among the three

most precarious ailment found among humans, malaria is ranked along

with AIDs and tuberculosis (Howitt et al., 2012).

Mosquitoes need water for the completion of their life cycle and larval

stage. Consequently, the best way to manage or bring to an end diseases

spread by mosquitoes is to disrupt or completely destroy their life cycle at

the larva stage (Mohammed et al., 2009).

Different plant extracts have been used as medication to combat

malaria; among them are quinine, chloroquine and artemisinin. Quinine

was the first successful antimalaria medicine extracted from plant

(Cinchona tree). In 1972 another effective antimalarial drug artemisinin

was obtained from Artemisia annua, a Chinese plant (White et al., 2008).

These drugs along with the others derived from them were used to

combat malaria ailment. Disappointingly, in 2009 artemisinin resistance

was first documented on the Thai-Cambodia border and this again

gingered the need for other antimalarial drugs (Noedl et al., 2008).

The year 2015 happened to be an astonishing year in the management

of malaria due to the three significant occurrences that took place.

Youyou Tu won the noble prize for his breakthrough in the invention of

6

artemisinin, the drastic decline in malaria infection particularly in the Sub-

Saharan Africa and RTS,S first vaccine against P. falciparium malaria,

although the RTS,S vaccine was not effective against P. vivae and only

partly protect against P. falciparium malaria (Benelli and Mehlhorn, 2016).

Mosquito treated net along with artemisinin derivatives brought about

rapid decrease in death initiated by malaria in 2010 as recorded by WHO.

1.2.1 Intermediary Agents of Plasmodium

There are five genus of Plasmodium causing infections in man, they

are Plasmodium falciparium, Plasmodium malaria, Plasmodium vivex,

Plasmodium ovale and Plasmodium knowlesi (Shapiro et al., 2013; WHO,

2014). The most dangerous and deadly species among the Plasmodium

genus is the P. falciparium particularly when the infection is found in

pregnant women and young children with less defensive immune system.

Although, P. knowlesi is predominantly found in monkeys, it has the

ability of affecting man and this has been a serious problem in Southeast

Asia (WHO, 2015).

1.2.2 Life cycle of Plasmodium falciparium

The parasite causing malaria depends on two hosts. The infested

female anopheles mosquito during the cause of blood meal, while feeding

on human blood deposits sporozoites into the blood stream of man

through its salivary glands. The deposited sporozoites travel to the liver

from the blood stream where they metamorphose and proliferate through

the asexual reproduction route into schizonts (Tarun et al., 2006).

7

The schizonts breaks up into merozoites (for other species of

Plasmodium like P. vivax and P. ovale, the sporozoites stay dormant in

the liver in a „hypnozoites‟ form for several months or years before they

attack the red blood cells (RBC).

The merozoites through the erythrocyctic cycle permeate the red blood

cells and undergo asexual enlargement inside the RBC. The erythrocyctic

cycle starts through a minute ring form and blossom into a sizeable

amoeba referred to as the „trophozoites‟ and later develops into schizonts

which ends up breaking into merozoites. The cycle is repeated causing

fever each time the parasites breaks free and invades the blood cells.

Again when mosquitoes pierce an infected person, they devour the

gametocytes and develop into mature sex cells referred to as gametes.

The developed female gametes form the „ookinetes‟ that penetrate

through the mosquito midgut wall to produce oocysts on the outer

surface. In the oocyst interior, several hundreds of sporozoites are

established; the rupturing of the oocyst releases the sporozoites which

swim to the mosquito salivary glands. The cycle of human infection starts

again when the mosquito pierces another person.

1.2.3 Intermediary Agents of Trypanosomiasis

There are two sub-types of Trypanosoma brucei which are structurally

identical and are the source of different disease mode in humans (Urbina,

1994). The first is T. b. rhodesiense that is responsible for East African

sleeping sickness and T. b. gambiense accountable for West African

sleeping sickness, the third type of the Trypanosoma brucei referred to as

8

T. b. brucei rarely affects man, they are majorly found in tropical African

countries and causes nagana in cattle, horses, dogs and pigs.

1.2.4 Life Cycle of Tsetse Fly

Tsetse flies are the major vector (Glossina species) that transmits

„salivarian trypanosomes (Peacock et al., 2011). They require two hosts

to survive and replicate. Their life cycle begins when they feed on the

blood of mammalian host. They introduce „metacyclic trypomastigotes‟

into the skin tissues from their salivary glands, this find its way into the

bloodstream and from there enters into the various body fluids like the

lymphatic, blood or spinal cord fluid where they are changed into

bloodstream trypomastigotes and replicate by binary fission. The second

stage commence when a new tsetse fly ingests the infected blood of

humans, the bloodstream trypomastigotes from the first human bitten by

tsetse fly above changes into procyclic trypomastigotes in the midgut of

the tsetse fly, where they replicate and transform into metacyclic

trypomastigotes and divide into several others by binary fission.

The immune system cannot eradicate trypanosomal brucei owing to

the fact that it contains a glycoprotein (VSG) covering that renders the

cell membrane very thick and rigid to recognize. In addition to this, it

recurrently modifies its configuration ahead of the immune system

reaction and possible attack.

1.3 Motivation/Rational for the Study

Callistemon citrinus can provide an alternative source for antimicrobial,

antiplasmodial and antitrypanosomal drugs and can be a source of 1, 8-

9

cineole. However, it can only be developed, if its bioactive ingredients are

known and standardized. Therefore there was need to investigate C.

citrinus in order to understand better its chemical composition, properties,

safety and efficacy. Another important motivation is the incomplete

information relating to the chemical transformation in the constituents of

the essential oil.

1.4 Hypothesis

According to the existing data, Callistemon citrinus should contain

antimicrobial, antiplasmodial, antitrypanosomal, and analgesic compounds

that can be isolated and their action tested in-vitro.

1.5 Problem Statement

Callistemon citrinus is used traditionally to treat gastrointestinal

distress, pain and infectious diseases from bacteria, fungi, virus and

parasites. The methanolic and ethanolic leaf extracts of Callistemon

citrinus possesses antibacterial and cardio protective activities in rat. The

ethanolic extract of the stem was also reported to show free radical

scavenging activity and elastase inhibition. The bark extract has

cytotoxicity against A549 cells line. The leaf oil of this plant is also known

to possess antimicrobial, anti-inflammatory, fungi toxicity, antinociceptive

activities. However, the compounds responsible for the physiological and

medicinal actions of this traditional remedy are not known. This has not

only hindered the standardization and development of this herb, but also

made its recognition; acceptance and utilization remain locally restricted.

There is also paucity of information on the antiplasmodial,

10

antitrypanosomal and bioactive properties of EOs of the most indigenous

plants such as Callistemon citrinus used in folk medicine for management

of diseases in Africa as well as the effects of seasons on them.

1.6 Aims and Objectives of Research

The aim of this work was to isolate volatile oils, determine the

chemical transformation, characterize the constituent compounds in the

leaves, flowers and seed of Callistemon citrinus, which are responsible for

its medicinal properties and to also synthesize plant metallic nanoparticles

from the plant.

The specific objectives are as follows:

1. To collect Callistemon citrinus leaves, seeds and flowers during spring,

summer, winter and autumn season.

2. To extract the essential oil of the leaves, flowers, seeds and stem of

Callistemon citrinus using hydro distillation method and analyze the

components using GC-MS.

3. To test the antimicrobial activity of the volatile oil against some gram

positive and gram negative bacteria: Aeromonas hydrophila (ACC),

Escherichia coli (ATCC 35150), Salmonella typhi (ACC), Listeria

monocytogenes (ACC), Vibro alginolyticus (DSM 2171), Staphylococcal

enteritis (ACC), Pseudomonas aeruginosa (ACC) and Staphylococcus

aureus (ACC).

4. To evaluate the radical scavenging and antioxidant capacity of the

volatile oil of the leaves and flowers of this plant against two different

radicals (DPPH and ABTS) by spectrophotometric method.

11

5. To determine the chemical transformation taking place in the

constituents of the volatile oil of the leaves of this plant.

6. To isolate triterpenoids from the aerial part of the plants.

7. To synthesize silver and gold nanoparticles from the aerial part of the

plant, characterized the nanoparticles chemically, and test for their

antiplasmodial, antitrypanosomal and antibacterial activities.

12

Chapter 2

Review of Literature

2.1 Drug Discovery

Plants are indispensable and play a vital role for the continued

existence of life on planet earth because they are responsible for giving

oxygen to different organisms and man. They also provide some non-

nutritional components better called secondary metabolites, which do not

take part in cell metabolism and are formed from different precursors like

amino acids or through various enzymatic reactions (Harborne, 1993).

Present day drugs and traditional medicines have their foundation in

natural products. Nature is recognized as the basis for drug development,

with 70% of the entire drugs found in the market connected with natural

products, whereas 30% of novel drugs are purely synthetic (Newman,

2008).

New natural product-based drugs consist of new metabolites derived

from different natural sources which include plants, aquatic organisms

and bacteria, in addition to the structurally stimulated development of

earlier recognized natural compounds. Pharmaceutical industry had

engaged itself in the development of improved new drugs via synergistic

chemistry and screening on molecular targets, but it has been established

that natural products are still found to be distinctive and irreplaceable (Ji

et al., 2009; Hong, 2011). Compounds from natural products possess

superior drug like properties when compared with those formulated

through combinatorial chemistry. Furthermore, metabolites from natural

13

materials like plants, aquatic organisms and many others exhibit better

biochemical specificity coupled with chemical diversity when compared to

synthetic drugs found in the market (Koehn and Carter, 2005).

2.2 Natural products as a foundation of novel drugs

Natural products have been used as a source of traditional medicine

since time immemorial. For instance, volatile oil from aromatic plants was

used by earliest Egyptians in embalming, for the inhibition of bacterial

growth and impediment of decay. In Greece, China, Rome and South

America, plants were engaged as a source of food and medicine (Cragg

and Newman, 2009; Grabley et al., 1999), yet less than 10% of plant

species are used as source of food by both humans and animals while

greater amount are utilized for therapeutic purposes (Cowan, 1999).

Majority of the anticancer drugs these days have their root in natural

origin (Cragg et al., 2006). Not less than 67-70% of the world population

usually make use of plant derived orthodox medicines intended for their

primary health care as recorded by WHO (Cragg et al 2009). People of

rural areas rely solely on traditional medicine and this is ascribed to

cultural and economic reasons. They are so poor that they cannot afford

the high cost of modern drugs nor are able to access western health care

facilities, thereby are left with only the traditional form of health care

which remains affordable (Matu and Van Staden, 2003.)

Herbal therapy or the use of plants or extracts obtained from plant for

medicinal purpose was dated back to history and has been widely

accepted in drug therapy in the last few decades. Natural products are

14

considered to be very vital owing to the fact that they are the chief source

of novel drugs (Kingston, 2011).

Organic compounds obtained from natural products are basically from

aquatic organisms, plants, vertebrates and invertebrates (Mehta et al.,

2010). Nature is seen as a biochemical plant for the production of both

primary and secondary metabolites since molecules derived from them

possess several biological functions. Primary metabolites are usually

found in many organisms or cells and they play a great role in the

metabolic activities, growth, development and reproduction of cells. Their

physiological function is vital for the continued existence and metabolic

processes of that organism. Examples include proteins, nucleic acids, fatty

acids, lipids carbohydrates and steroids, which are generated through an

essential biosynthetic pathway whereas the secondary metabolites are

organic derived compounds found in small amounts and are not directly

concerned with the normal growth, reproduction, or development of that

organism. Their absence does not lead to the immediate death of the

organism but results in a long term injury. They are also very

instrumental to the plant defense against herbivore, attract some animals

to the plant for pollination or seed dispersal, signals compounds and as

well restrain or arouse biological processes in other organisms. The

secondary metabolites are associated with the organism‟s dealings with

its environment and this generates novel chemical compounds that

encourage their survival (Hanson, 2003; Mishra and Tiwari, 2011).

15

Secondary metabolites exhibit vast array of structural diversity and

include about 2500 of monoterpenes, 5000 of sesquiterpenes, 2500 of

diterpenes, 5000 of triterpenes, 700 of non-protein amino acids, 12000 of

alkaloids, 150 of alkyl amides, 100 of amines, 60 of cyanogenic

glycosides, 4000 of flavonoids, 100 of glucosinolates, and others (Wink,

2003). It is possible that a particular group of secondary metabolites

leads within a known taxon. They are usually concerted in a given region

of a plant like the fruits, roots, flowers, bark or the glandular hair, but

when found in different organs of the same plant they normally exhibits

diverse chemical profile (Araujo et al., 2003).

2.3 Demands for Aromatic/Therapeutic Plants as Source of

Medicine

The desirability of therapeutic and aromatic plants is constantly

growing owing to rising consumers demand and importance of these

plants for culinary, healing, and other anthropogenic usage. Since

consumers are becoming more and more educated about issues of food,

health, and nutrition, they are also being exposed to the benefits and

potential relevance of therapeutic and aromatic plants and their

metabolites. These aforementioned plants generate a large variety of

secondary metabolites; which includes essential oils. Essential oils could

replace or complement synthetic compounds of the chemical industry,

without inducing the same secondary effects (Dhifi et al., 2016)

Aromatic plants had been used since primeval times for their

preservatives and therapeutic properties and to impart fragrance and

16

flavor to food and beverages, or to restore to health both body and mind

for thousands of years (Baris et al.,2006; Margaris et al., 1982;

Tisserand, 1978; Wei and Shibamoto, 2010). Documentation findings in

Mesopotamia, China, India, Persia and ancient Egypt confirm their uses

for many treatments in diverse forms. The therapeutic properties of

aromatic plants are partly ascribed to their volatile oils. The functions of

this secondary metabolite (volatile oil) in plants are as follows: for

chemical signals enabling the plant to manage or control its environment

(ecological role); to attract pollinating insects and repel predators,

inhibition of seed germination, or communication between plants by

emitting chemical substances as indicator of the presence of herbivores.

All the aerial parts of aromatic plant such as the flowers, seeds, leaves,

bark, rhizome, fruits may contain essential oil.

2.4 Definition of the Volatile Oil

Volatile oils are hydrophobic, soluble in alcohol, non-polar or weakly

polar solvents, waxes and oils, which are only faintly soluble in water.

They are mostly colorless or pale-yellow, with the exception of the blue

volatile oil of chamomile (Matricaria chamomilla), and are usually liquid of

lower density than water (sassafras, vetiver, cinnamon and clove

essential oils being exceptions) (Martin et al., 2010; Gupta et al., 2010).

Owing to their molecular structures (presence of olefenic double bonds

and functional groups like, hydroxyl, aldehyde, ester), volatile oils are

readily oxidizable by light, heat and air (Skold et al., 2006; Skold et al.,

2008).

17

The overall oil content present in plants is extremely small and hardly

ever go beyond 1% except in some particular cases, for example in

Callistemon citrinus, Syzygium aromaticum, Myristica fragrans can reach

up to 10% or more.

Essential oils are complex mixtures of volatile constituents produced by

living organisms; they are colorless and liquid at room temperature and

are made up of about two hundred chemical compounds which contain

carbon, hydrogen and oxygen as their building blocks. They can be

classified into two groups: terpenoids and non-terpenoids. The two

sources are both made up of hydrocarbons or their oxygenated

derivatives and may sometimes contain sulfur or nitrogen. They may be

present in the form of hydrocarbons (e.g. pinene, limonene, myrcene),

alcohols (e.g. linalol, santalol), acids (e.g. benzoic acid, geranic acid),

aldehydes (e.g. citral), cyclic aldehydes (e.g. cuminal), ketones (e.g.

camphor), lactones (e.g. bergaptene), phenols (e.g. eugenol), phenolic

ethers (e.g. anethole), oxides (e.g. 1, 8 cineole) and esters (e.g. geranyl

acetate) (Deans et al., 1990).

Fluctuation in the chemical composition of volatile oils have been

documented with relation to soil type, harvest time, extraction method,

seasonal variations, plant organs, degree of maturity of the plant,

geographical origins and genetic. The factors that determine volatile oil

yield and the type of constituents in it are many; which are difficult to

separate from each other because they influence each other and are

18

interconnected (Anwar et al., 2009; Hussain et al., 2008; Marotti et al.,

1994).

2.5 Groups of Volatile Oil Compounds

2.5.1 Hydrocarbons

The bulk of volatile oils are classified under this category, which are

made up of hydrogen and carbon only. They are further classified into

terpenes (monoterpenes C10, sesquiterpenes C15, and diterpenes C20).

They can exist in the alicyclic (monocyclic, bicyclic or tricyclic), acyclic or

aromatic form. Examples in this group are α- and β-myrcene, α- and β-

pinene, α-sabinene, α-phellandrene, thujane, fenchane, farnesene,

cadinene, azulene and p-cymene. These chemical components are linked

to various therapeutic activities like stimulant, antiviral, antitumor,

decongestant, antibacterial, hepatoprotective (Baser and Buchbauer,

2015; Bowles, 2003; Burt, 2004; Deans et al., 1990; Edris, 2007; Griffin

et al., 1999; Ozbek, 2003; Pengelly, 2004; Svoboda et al., 1999).

2.5.2 Esters

Esters possess aroma imparted to the oils and are common in most

volatile oils; examples include linalyl acetate, geranyl acetate, bornyl

acetate, and eugenol acetate. Their therapeutic activities include

antifungal, spasmolytic, sedative, anesthetic, and anti-inflammatory

action (De Sousa et al., 2011; Ghelardini et al., 2001; Peana et al., 2002;

Pengelly, 2004; Sugawara et al., 1998).

19

2.5.3 Oxides

Oxides are also known as cyclic ethers are by far the strongest

odorants and the most commonly found in essential oils are eucalyptol,

bisabolene oxide, linalool oxide, ascaridole, and sclareol oxide. Their

medicinal values are anti-inflammatory, expectorant and stimulant of the

nervous system (DeSousa, 2011; Ghelardini et al., 2001; Pengelly,

2004).

2.5.4 Lactones

They are commonly found in pressed oils and are of moderately high

molecular weights. Citroptene, nepetalactone, bergaptene, costuslactone,

dihydronepetalactone, alantrolactone, epinepetalactone, aesculatine, and

psoralen are common examples. They exhibit medicinal values like

antimicrobial, antiviral, antipyretic, sedative, hypotensive, and analgesic

properties (DeSousa, 2011; Gomes et al., 2009; Miceli et al., 2005;

Pengelly, 2004).

2.5.5 Alcohols

In addition to sweet pleasant aroma, alcohols are by far the most

useful therapeutically with no record of contraindication, their therapeutic

usefulness include antiseptic, antimicrobial, tonifying, balancing,

spasmolytic, anesthetic, and anti-inflammatory activities. Examples in this

category are citronellol, linalool, menthol, borneol, santalol, nerol and

geraniol (De Sousa, 2011; Ghelardini et al., 1999; Peana et al., 2002;

Pengelly, 2004; Sugawara et al., 1998).

20

2.5.6 Phenols

Phenols are comparable in properties to alcohols but are more

pronounced. In addition to their irritation especially to the skin and

mucous membrane, they are by far the most reactive and are potentially

toxic. They have antimicrobial, spasmolytic, anesthetic, irritant, immune

stimulating effect. The most common are eugenol, carvacrol, thymol, and

chavicol (De Sousa, 2011; Ghelardini et al., 1999; Pengelly, 2004)

2.5.7 Aldehydes

They are common in volatile oil constituents, but are very unstable and

can easily oxidize. They are found in most culinary herbs like cumin and

cinnamon and possess a characteristic syrupy fragrance. Medicinally, they

are antiviral, hypotensive, antimicrobial, tonic, calming, antipyretic,

sedative, and spasmolytic in nature. They are known as vasodilators.

Myrtenal, neral, geranial, benzaldehyde, citral, and cumin aldehyde are

some of the few examples in this category (Dorman & Deans, 2000;

Pengelly, 2004).

2.5.8 Ketones

Ketones are not frequently found in volatile oils; they are

comparatively stable molecules and are not mostly vital as fragrances or

flavor substances. They may be neurotoxic and abortifacient, e.g.

camphor and thujone (Gali-Muhtassib et al., 2000) but exhibit some

beneficial effects. Some of their therapeutic properties are mucolytic, cell

regenerating, sedative, antiviral, analgesic, and digestive. As a result of

their stability, they are not easily metabolized by liver. Frequent examples

21

found in volatile oils include carvone, menthone, pulegone, fenchone,

camphor, thujone and verbenone.

2.6 Secondary Metabolites of Aromatic Plants with Unique

Reference to Terpenoids

Among the secondary metabolites, terpenoids class represents a large

and broadly distributed group of the natural compounds having their

skeleton obtained from the C5 isoprene units. The number of C5 isoprene

unit is used as a basis for classification for the terpenes; monoterpenoids

(C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids /

steroids / saponins (C30/C27), and tetraterpenoids (C40). The total

number of terpenoids is above 22,000 at present (Hanson, 2003; Wink,

1999a; Wink, 1999b; Wink, 2008b).

2.6.1 Monoterpenes

Monoterpenes are composed of two isoprene units and are broadly

distributed in nature particularly in volatile oils. They are useful

components in the flavor and perfumery industries. They are not only

present in plant matter but also in marine organisms. The sesquiterpenes

are made up of three isoprene units.

The mono- and sesquiterpenes are volatile in nature and can be

extracted as essential oils; they are principally abundant in plant families

like the Myrtaceae, Asteraceae, Apiaceae, Rutaceae, Lamiceae,

Cupressaceae, Pinaceae, Lauraceae, Santalaceae, Piperaceae, Hypericeae

and Zingiberaceae (Wink, 2004).

22

The essential oils in plants are mainly lipophilic and are found in oil

cells, cavities, secreting ducts, glandular hair, trichomes, resin channels,

or other dead cells (Wink, 1997; Wink, 2004).

The mono-, sesqui-, and diterpenes constituents of volatile oils are

poisonous to both microbes and herbivores and may serve as defense

compounds in resistance to such organisms (Tholl et al., 2004).They also

attracts pollinating insects (Wink, 2004).

Biosynthesis of some monoterpenes is outlined (Degendardt, 2009).

2.6.2 Diterpenes

Diterpenes usually contain 20 carbon atoms in their basic skeleton and

are made up of four isoprene units obtained from geranyl pyrophosphate

that are mainly found in resins like abietic and pimaric acid, but are

scarcely encountered in genuine volatile oils obtained by hydro distillation

owing to their low volatility.

More energy is required for the diterpenes to change to their vapor

form. As a result of this extended distillation times are needed for their

recovery. Some diterpenes like phytol are found in volatile oil constituents

(Dewick, 2009; Hanson, 2003).

2.6.3 Triterpenes

The triterpenes belong to a group of compounds made up of three

terpenes units with a molecular formula of C30H48. They are believed to be

composed of six isoprene units and Squalene is the most significant

triterpene. They are broadly classified according to the number of rings

23

present in them but the five membered rings (pentacyclic) tend to

dominate.

Both triterpenes and steroids may occur as aglycons but more

frequently occur as saponins, while free triterpenes and steroids exist as

lipophilic molecules, saponins are soluble in water and amphiphilic

molecule capable of causing a leakage to the biomembrane.

Consequently, they exhibit antimicrobial and anti-herbivore action (Wink,

2004).

Volatile oils have been employed as a therapeutic agent in the

treatment of diseases throughout the world for centuries (Rios and Recio,

2005) and their components have been documented (Ashour et al., 2009;

Raman et al., 1995; Svoboda et al., 1999). It has been established that

plant secondary metabolites are useful in medical procedures and relevant

in the food, cosmetic and pharmaceutical industries (Dorman and Deans,

2000).

2.7 Factors Affecting Composition of Volatile Oils

Environmental factors likes mode of extraction, relative humidity,

irradiance, photoperiod, location, soil composition, climate and plant

cultivation methods have immense impact on the volatile oil quality and

composition (Panizzi et al., 1993). Various factors mentioned above have

been used to determine why accurate specification of the constituents of

volatile oils is not acceptable.

24

2.7.1 Harvest Time

One of the prominent factors influencing the quality of volatile oils is

harvest time (Marino et al., 2001). The developmental stage of the plant

(ontogeny) largely affects the quantity of volatile oil composition. Early or

late harvest of crops is linked to the low yield of volatile oil content, as

over matured and immature crop leads to poor yield of herbs and oil

content. It has been reported that to get a high yield of volatile oils, it is

preferable to harvest the plant after flowering so as to acquire high

quantity of the oil (Panizzi et al., 1993). In addition to harvest time, the

numbers of harvest carried out per year also influence both the yield and

composition of oil (Ibrahim et al., 2014).

2.7.2 Method of extraction

Method of extraction also plays a vital influence on the variability and

composition of the components of essential oil and explains the reason

behind the differences observed in products derived from steam

distillation and that originally present in the secretory organs of the plant

(Harborne, 1998). Distillation parameters like the parts of the plant

distilled, method of distillation, time of the day and stage of growth, when

plant was harvested and the length of distillation all influence both the

components and quality as well as its medicinal effect (Clark and Menary,

1984). It was reported that about one and half hour is needed for the

distillation of lavender oil but if shorter than that, roughly 18-20 % of the

oil chemical constituents would be missing (Harborne, 1998).

25

2.7.3 Temperature

During distillation process, low temperature should be encouraged

because of thermolabile aromatic components of an essential oil, which

are easily destroyed or altered by high temperatures (Duriyaprapan et al.,

1986). High temperatures are not encouraged as they seem to cause

harshness in the oil composition. The pH, electropositive and

electronegative balances of the volatile oils is seriously affected by the

effect of temperature. For lavender and cypress, temperature should not

exceed 118.3 °C during distillation process since it had been established

that high temperature causes a decrease in the oil yield (Okoh, 2010).

2.8 Methods of Administration of Volatile Oils

Various methods have been employed in the administration of volatile

oil such as aromatic baths compresses, absorption through the skin,

inhalation and ingestion. The method adopted solely depends on the need

or condition for application (Harborne, 1998). Lots of French practitioners

have established that ingesting the oils internally is highly effective as it is

absorbed and transported to the rest of the body. However, care should

be taken due to the potential toxicity of some oils (Schiller and Schiller,

1996). Volatile oil is also capable of entering the body via inhalation due

to their volatility, when inhaled it is carried to the respiratory tracts and

lungs and afterwards distributed to the blood stream (Moss et al., 2003).

Peppermint oil, administered orally has shown to be very effective for

indigestion (Schuler, 1990). At times all the three methods of application

26

(topical, inhalation and ingestion) are interchangeable and may produce

similar benefits.

2.9 Isolation of Bioactive Compounds

Subjecting a large number of extracts to different tests to ascertain

whether they exhibit a biological effect is generally one of the first steps

in detecting bioactive compounds in plants. Thus, all the extracts obtained

are subjected to biological testing to determine if they possess any

activity. Those having positive biological activity are processed further

until a bioactive component is obtained in an uncontaminated form (Nepf

and Ghisalberti, 2008).

2.10 Overview of Analytical Methods Used in Natural Product

Research

Both quantitative and qualitative studies carried out on bioactive

compounds derived from plant materials predominantly depend on the

selection of appropriate methods. A number of commonly used techniques

in the isolation of natural product are discussed below.

2.10.1 Pre-extraction Preparation of Samples

The first stride in the study of medicinal plants is the preliminary

preparation of the plant samples so as to preserve the secondary

metabolites in the plant before extraction is done. Extraction can be

carried out from the fresh or dried plant samples of the leaves, flowers,

stems, bark, roots and fruits. Before extraction is carried out, preliminary

preparation of plant samples like drying and grinding are very important

because they have the capability to influence the preservation of the

27

secondary plant metabolites in the final extract (Okoh et al., 2008). Both

dried and fresh samples could be used in the study of medicinal plants but

most frequently dried samples are preferred due to the time researchers

need for the experimental design. It has been documented that at least

three hours is needed to maintain freshness of plant samples between the

space of harvesting and the time for experimental work (Sulaiman et al.,

2011). Grinding of the plant samples reduces the particle size resulting in

a larger surface contact between the sample and extraction solvents.

Powdered samples of about 0.5 mm are more homogenized with smaller

particles thereby ensuring a better surface interaction with the solvent

used for extraction.

2.10.2 Extraction

Extraction is simply the separation of the medicinal parts of the plants

by carefully adopting the right solvents through standard procedures

(Handa et al., 2008). Extraction plays a significant and crucial role on the

final outcome of the study. Extraction methods are sometimes referred to

as sample preparation techniques. It is true that the development of

modern chromatographic and spectrometric techniques makes bioactive

compound analysis easier than before but the success still depends on the

extraction methods, input parameters and the exact nature of plant parts.

The most common factors affecting extraction processes are the matrix

properties of the plant part, the solvents used, temperature, and

extraction time. It is only possible to conduct further separation,

identification, and characterization of bioactive compounds if the

28

extraction process has been appropriately done. Bioactive compounds

from plant materials can be extracted by various classical extraction

techniques. Most of these techniques are based on the extracting power

of different solvents used and the application of heat and/or mixing. The

commonly used standard methods are: Soxhlet extraction, maceration

and hydrodistillation to obtain a crude extract which is then concentrated

using a rotary evaporator (Azmir et al., 2013).

2.11 Extraction Techniques for Crude Extract

Extraction method is a process whereby homogenized plant tissues are

soaked in a desired solvent to remove the phytochemicals present in the

plant matrix. Different solvent may be used based on the type of phyto-

compounds that are targeted for extraction (Pandey and Tripathi, 2014).

Polar solvents usually remove polar compounds and this is equally true

for non-polar solvents and non-polar compounds. There are different

polarities of solvents just like the phyto compounds. The three main

polarity strength of solvent are: polar, medium and non-polar. Examples

of polar solvents are water, ethanol and methanol whereas medium-polar

solvents are dichloromethane, acetone and ethyl acetate and non-polar

solvents are petroleum ether, hexane, chloroform and toluene (Pandey

and Tripathi, 2014). It is also possible to mix different solvents for the

extraction of a particular sample at different ratio based on the targeted

compound.

29

2.11.1 Maceration

Maceration is the process of preparing an extract by drenching parts of

the plants in either an organic solvent, water, or vegetative oil for a

specific period of time like 3 h or more, the plant part in the solvent may

be agitated or allowed to stand at ambient temperature until the solvent

has fully penetrated and softened the cell structures thereby breaking the

cell wall of the plant to discharge the soluble phyto compound. The

soluble constituents of the plant are now dissolved in the solvent used.

The extract can now be removed from the plant matter by decantation

(Trendafilova et al., 2010).

2.11.2 Cold Pressing

Cold pressing is used to extract volatile oils from lemon, grapefruits,

orange and bergamot. The rinds are usually separated from the fruits and

grounded or chopped and then hard pressed to take out the liquid extract.

The watery mixture of the essential oil and the liquid derived from this

process is separated. It is interesting to note that the shelf life of such oil

obtained through this method is moderately short (Yilmaz, 2017).

2.11.3 Steam Distillation

Steam distillation is a process of extracting active substances from

both medicinal and aromatic plants. A steam distillation apparatus has a

steam generation part which furnishes the needed heat to the mixture of

plant matter and solvent in the flask from beneath. The steam helps to

dissolve the phyto-compounds present in the plant parts and find its way

into the condenser where it is condensed into a liquid mixture and

30

collected in a separator (Stichlmair and Fair, 1998). It can be used to

extract water insoluble compounds. A rotary evaporator may be employed

to separate the solvent from the desired extract.

2.11.4 Microwave Assisted Extraction

Microwave assisted extraction makes use of microwave energy to aid

separation of analyte from plant matrix into a solvent used for extraction

(Trusheva et al., 2007). Microwave radiation is generated from

electromagnetic field with a frequency range of about 0.3 to 300 GHz

(Camel, 2001), the energy from the microwave is channeled to the plant

material via molecular interactions with the electromagnetic field. The

electromagnetic energy is converted into thermal energy (Thostenson and

Chou, 1999). This method helps to lessen extraction time and the volume

of the solvent including the quantity of plant materials used when put side

by side with the conventional method like maceration and Soxhlet

extraction, in addition it brings about an improvement in the amount of

medicinal components obtained from the plant matrix and it is easily

reproducible. The right conditions for extraction must be adhered to avoid

thermal degradation (Kaufmann and Christen, 2002).

2.11.5 Solvent Extraction

Solvent extraction is a method used in extracting oils from oil bearing

plants through solvents like hexane, benzene or ethers. The plant may be

first grounded and then thoroughly mixed with the solvent. Selection of

solvents to be used depends on factors such as the characteristics of the

components being extracted, cost, and environmental issues. The

31

resulting mixture obtained from this process is filtered and concentrated

in vacuum or by evaporation. This method is not too suitable for the

extraction of volatile oils because small amount of the oil can be left

behind during extraction. The pure volatile oils obtained from this method

are depressurized to avoid missing important components (Yilmaz, 2017).

2.11.6 Hydrodistillation

Hydrodistillation is a conventional means of extraction whereby the

plant materials are soaked in water and heat is directly supplied in a

Clevenger type distillation apparatus. It is used in isolating volatile and

non-volatile polar constituents from aromatic or odoriferous plants

(Grosso et al., 2007). As heat is supplied to the round-bottomed flask

containing the mixture of the plant matter and water, the plant cells get

ruptured thereby releasing the oils (Mohammad et al., 2016). The volatile

oil produced is carried by steam in the vapor phase to the condenser,

upon condensation, the liquid mixture moves into the separators where

the water and volatile oil are separated by density differences

(Mohammad et al., 2016). The temperature adopted for this method must

be strictly monitored so as to prevent thermal degradation of some

compounds; also prolong heating of the plant material in the flask can

lead to hydrolysis of esters, decomposition of other components or

polymerization of aldehyde. This method is inexpensive because no

organic solvent is required, but it is energy consuming.

32

2.12 Different Chromatography and Spectroscopy Techniques

2.12.1 High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography is a versatile form of column

chromatography widely used for isolation of natural products (Cannell,

1998). Rather than allowing the solvent to trickle down through the

column by gravity, it is pushed through under pressure of about 400

atmospheres making the solvent to travel more rapidly. The particle size

for the column packing material is of smaller size with larger surface area

and this allows more interaction between the components in the crude

extract and the stationary phase thereby resulting in a better separation

of the components mixture (Harvey, 2000).

HPLC instrument is segmented in design and it is made up of an auto

sampler, a solvent delivery pump, an analytical column, a guard column,

a detector, and a printer. Separation is achieved in HPLC on the basis of

different migration rates of the different components in the extract while

the extent of separation is based on the choice of both the stationary and

mobile phases (Harvey, 2000). Preparative HPLC is gaining popularity

among researchers for isolation and purification of crude extract, even

though it is similar to the analytical HPLC, but rather than applying a little

quantity of the sample to maximize resolution, a large amount of the

sample is used in preparative HPLC to get the most out of purification

products and minimize quantity of the solvent used (Mbayeng et al,

2008).

33

2.12.2 Thin Layer Chromatography (TLC)

Thin layer chromatography is a quick, easy and inexpensive procedure

that enables researchers to know how many components are in the crude

extract. It is mostly used to examine the fractions derived from column

chromatography so as to ascertain if the said fractions contained more

than one component or if the fractions can be pulled together without

disturbing their purity (Kenkel, 2003). Separation on TLC occurs on the

basis of relative affinity of the various constituents towards both the

stationary and mobile phases (Harvey, 2000). The components

interacting with the mobile phase are carried by capillary action and thus

travel over the surface of the stationary phase. During the cause of the

movement, components with a higher affinity to the stationary phase

move slower than those with a lower affinity, which move faster on the

stationary phase, resulting in separation of the various components in the

crude mixture. Immediately separation is achieved, the individual

constituents are visualize as a spot on the TLC plate either by spraying of

iodine vapor, spraying solutions, or viewing under UV light.

2.12.3 Preparative Thin Layer Chromatography (PTLC)

Preparative thin layer chromatography is a method employed to

separate and isolate larger quantity of extract than what is obtainable in a

normal analytical TLC (Rabel and Sherma, 2017). The amount of extract

that can be applied on PTLC could be up to 10 mg or even greater than

one gram. Basically, both PTLC and analytical TLC use the same principle,

the only difference between them is that PTLC has a thicker stationary

34

phase and can accommodate more amounts of extract than the analytical

TLC. The extract to be isolated or separated are loaded on the PTLC plate

as streaks rather than the conventional spots of TLC. A suitable solvent

system is used and separation is achieved on the basis of the relative

affinity of the different compounds in the extract to both the stationary

and mobile phase just like the normal TLC. The compound of interest may

be obtained by scraping the sorbent layer from the PTLC plate by a

spatula and the compound can be cleaned through filtration,

centrifugation or crystallization. In some cases, the purity of the targeted

compound may be adequate for the purpose of identification and

structural determination using NMR, GC-MS, LC-MS, and/or FT-IR.

2.12.4 Gel Permeation Chromatography (GPC)

Gel permeation chromatography (size exclusion chromatography)

depends on the capability of molecules to travel through a column of gel

that contains pores of evidently defined size. Separation on GPC is on the

basis of sizes of the component mixture; those molecules having larger

size are restricted from entering the pores thereby making their

movement faster through the column and are eluted first. Slightly smaller

molecules are allowed to enter some pores but take a longer time to elute

from the column while small molecules enters the pores but are delayed

more than the slightly smaller molecules before elution takes place. The

advantage of this method is that large molecules speedily elute, it‟s

simple and isocratic, although the column is costly and sensitive to

contamination. The most frequently used gel for natural product isolation

35

is sephadex LH-20 used to separate bioactive compound or chlorophyll

from compounds of interest (Guiochon, 2001).

2.12.5 Column Chromatography (CC)

Column chromatography is made up of column particulate material like

alumina or silica, through which solvent has passed through at

atmospheric, medium or lower pressure. Column chromatography is used

as a purification technique in order to isolate bioactive compounds from

the crude extract (Kenkel, 2003). It is made up of a stationary phase (a

solid adsorbent) introduced into a vertical glass column and the mobile

phase (a liquid) usually added from the top and flows down through the

column by gravity or external pressure (Kenkel, 2003). The active

biological components found as minor entities in the crude extract are

purified through column chromatography. This is done by applying the

crude at the top of the column while the eluent is made to pass through

the column by gravity or through the use of air pressure. When balance is

established between the solute adsorbed on the adsorbent and the eluent

flowing down the column, separation is now brought about and this is

made possible due to the difference in interactions of the components of

the mixture with both stationary and mobile phases. The components or

eluates are collected in the form of a solvent drips from beneath the

column (Harvey, 2000). The frequently used adsorbents for column

chromatography are silica gel (SiO2) and alumina (Al2O3) available in

different mesh sizes. Influence of the solvent polarity used in the column

is of great significance as this affects the relative rates, by which the

36

components move through the column (Harvey, 2000). When the solvent

is too polar, its movement becomes too fast making separation of the

components in the crude difficult to achieve and if the solvent is not polar

enough, no component is eluted from the column. Therefore appropriate

selection of the solvent type is vital for the successful utilization of column

chromatography as separation techniques. It is common to first use a

non-polar solvent to elute the less polar components and once this is

achieved a more polar solvent is applied to the column to elute the more

polar compounds from the column (Kenkel, 2003).

2.12.6 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR is used by both organic and inorganic chemists to determine the

chemical functional groups (bonds) or compounds found in an unknown

mixture of crude extract. It is also a popular tool for structural elucidation

and compound identification (Eberhardt et al., 2007; Hazra et al., 2007).

The spectrum of an unknown compound can be identified by matching

their spectrum with a reference spectrum in the library.

Preparation of liquid samples is done by sandwiching a drop of the

sample between two plates of high purity sodium chloride which forms a

thin film between the plates while solid samples may be crushed with

potassium bromide (KBr) to remove large scattering effects from large

crystals, the powdered mixture is then compacted by a mechanical die

press to form a translucent thin pellet through which the beam of the

spectrometer can pass or the solid sample dissolved in a solvent like

methylene chloride and the resulting solution introduced onto a single salt

37

plate where the solvent is evaporated leaving behind a thin film of the

original material on the plate (Hazra et al., 2007).

2.12.7 Mass Spectrometry (MS)

Mass spectrometry is an analytical method that helps to produce

charged particles (ions) from the analyte. The substance to be analyzed is

bombarded with an electron beam having sufficient energy to fragment

the molecule into the positive fragments (cations or radical cations) thus

generated are accelerated into a vacuum through a magnetic field and

sorted on the basis of mass to charge ratio (m/z ratio) (Bruice, 2000).

The fact about how these positively charged particles are separated and

detected varies according to the specific design of the mass analyzer

segment of the instrument. The output of the mass spectrometer shows a

plot of relative intensity versus the mass to charge ratio (m/z). The most

prominent peak in the spectrum is referred to as the base or parent peak

and other peaks are reported relative to its intensity. MS is used to

determine both molar mass and molecular formula. Apart from

determination of molecular compound it is also used to identify what

isotopes of an element may be present in a sample (Kenkel, 2003).

2.12.8 Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear magnetic resonance spectroscopy is a technique which deals

with the reorientation of magnetic nuclei in a magnetic field and this is

brought about by absorption of energy after the nucleus of an atom is

excited from its lowest energy spin state to the next higher one (Carey,

2000). Atomic nuclei possess spin angular moment, which is dependent

38

upon the nuclear spin quantum number I and may have values of zero,

half integers or whole integers. Many nuclei have I = 0 and possess no

angular momentum. These include all nuclei with both an even atomic

and even mass numbers such as 12C and 16O. Since NMR spectroscopy is

dependent upon the magnetic properties of the nucleus, the study cannot

be carried out on these nuclei. In organic chemistry, the most commonly

encountered nuclei with I = ±1/2 are 1H, 13C, 19F, 29Si, and 31P and can in

theory be observed by NMR. The two elements that are mainly common in

organic molecules (carbon and hydrogen) have isotopes 1H and 13C

capable of giving NMR spectra that are wealthy in structural information.

Proton nuclear magnetic resonance (1H NMR) spectrum informs us

regarding the environments of the different hydrogen atoms in a molecule

while carbon-13 nuclear magnetic resonance (13C NMR) spectrum does

the same for the carbon atoms (Carey, 2000). Collectively, 1H and 13C

NMR are used in determining the molecular structure of a compound. It is

regularly used in combination with the other spectrometric methods like

MS and FTIR.

2.13 Plant Products and Their Transformation

A foundation for plant studies is based on its taxonomy, determination

of the chemical components of the plant and elucidation of the structure

of its constituents. Often, secondary metabolites obtained from plants

represent novel classes of chemical compounds. Also, several plant

metabolites and a number of products of their transformation have very

useful properties. Those secondary plant metabolites whose chemically

39

transformed components have been found useful economically are the

essential oils (Shults et al., 2007). Essential oils are generally isolated

from plants by means of steam distillation or hydrodistillation, and

recently, microwave-assisted extraction. Whichever method employed,

there is always some chemical transformation of components in the final

products. In the further few sections general synthesis and properties of

the key components of volatile oil of Callistemon citrinus are considered.

2.13.1 Pinenes

Both α- and β-pinenes are bicyclic terpenes. They are prepared by

distillation of turpentine oils. α-Pinene and β-pinene being in isomerization

equilibrium can be transformed also by isomerization route to camphene

and limonene respectively (Corma et al., 2007; Flores-Holguin et al.,

2008). The last is able to isomerize to terpinolene and α-terpinene as

shown in equation 1 (Okoh, 2010). Numerous routes are identified to lead

to various monocyclic and tricyclic terpenes. Nevertheless, camphene and

limonene remain the main products, and selectivity of their formation

depends on the nature of the acidic heterogeneous catalyst. The pathway

leading to camphene and other related products is the ring-expansion

route ultimately leading to such a valuable substance as camphor. The β-

pinene pathway leads to monocyclic terpenes. The route is

heterogeneously catalyzed by different acidic oxide catalysts including

mesoporous molecular sieves with different Si/Al ratios (Wang et al.,

2010). Acidic β-zeolites give preference to camphene (Yilmaz et al.,

2005); particularly with Brønsted acidic surface sites (Gunduz et al.,

40

2005). Usually weak surface acids favor camphene, while strong acids are

selective with respect to the monocyclic terpenes. Tin (IV), titanium (IV),

and zirconium (IV) polyphosphate catalysts, in contrast, support the

limonene pathway (Costa et al., 1996).

(1)

Epoxidation of α-pinene via t-butyl hydroperoxide or cumyl hydro-

peroxide is also a heterogeneously catalyzed reaction leading to a mixture

of products, α-pinene oxide, campholenic aldehyde, 1, 2-pinanediol, and

verbenone, as shown in the chain of transformations of equation 2 (Chiker

et al., 2003).

(2)

41

Atmospheric photooxidation and ozonolysis of α-pinene give

diaterpenylic acid acetate and terpenylic acid as shown in equation 3

(Claeys et al., 2009). The hydroxyl radical addition to the double bond of

α- or β-pinene instigates their degradation (Atkinson and Arey, 1998).

The same function belongs to β-hydroxyalkoxy radicals (Dibble, 2001).

Consequently α-pinene with hydroxyl radicals yields formaldehyde,

acetaldehyde, acetone, campholene aldehyde and pinon aldehyde, and β-

pinene – formaldehyde, nopinone, acetaldehyde, acetone, trans-3-

hydroxypinone, perilaldehyde, and myrtanal.

(3)

Hydration of α-pinene may go via two routes depending on the way of

isomerization, either to the hydrated product of borneol, or hydrated

products of α-, β-, and γ-terpineol as well as 1, 8-terpine (equation 4)

(Castanheiro et al., 2005).

42

(4)

Dehydrogenation of α-pinene accompanied by isomerization in the

presence of the platinum or palladium catalysts supported on the surface

of silica, alumina, or zeolites (Roberge et al., 2001). The first step is

isomerization of α-pinene to terpinolene, then dehydrogenation to α-and

γ-terpinenes takes place, and finally p-cymene and other products are

afforded (equation 5) (Okoh, 2010). Hydrogenation of α-pinene gives

pinane, an important substance in the flavor and fragrance industry

(Surburg and Paten, 2016). Pyrolysis of α-pinene results in ocimene and

alloocimene while that of β-pinene gives myrcene. β-Pinene adds

formaldehyde to yield nopole (Okoh, 2010).

(5)

43

2.13.2 Eucalyptol

Eucalyptol (1, 3, 3-trymethyl-2-oxabicyclo [2.2.2] octane, 1, 8-

Cineole, 1, 8-epoxy-p-menthane) is a monoterpene cyclic ether. Its

molecule has Cs symmetry, and its dipole moment is in the mirror plane

of the oxygen-containing ring. The electron density evolves over the

oxygen ring (Aparicio et al., 2007). 1,8-Cineole may be prepared from α-

terpineol in the presence of heteropolyacid supported on silica

H3PW12O40/SiO2 along with 1, 4-cineole (equation 6) (Lana et al., 2006).

In natural conditions, 1, 8-cineole can be produced from limonene

through the steps of α-terpineol and 1, 8-terpine (equation 7). Steps are

slow and reversible but the final yield of 1, 8-cineole can be substantial

(Farina et al., 2005)

(6)

(7)

Biooxidation leads to 2-hydroxo- and 2-oxo-1, 8-cineole, potential

synthons for organic chemistry (equation 8) (Rodriguez et al., 2006;

Garcia et al., 2009). Chemically, the oxidation process can be extended to

cineolic acid and its mono-methyl ester as well as anhydride (Rae and

Redwood, 1974). Cineolic acid can be transformed into cyclic N-

44

phenylimides by a sequence of reactions (equation 9) involving carboxylic

group protection and cyclization (Silvestre et al., 1997).

(8)

(9)

Another chain of transformations starts with chemical oxidation of 1,8-

cineole using hydrogen peroxide in the presence of manganese(III)

porphyrin (Cavaleiro et al., 1996) or chromyl acetate (De Boggiato et al.,

1987) to yield 3-keto-1,8-cineole. Treatment of the product by potassium

hydroxide in ethanol yields seudenone (equation 10) (Silvestre et al.,

2000).

45

(10)

In atmospheric conditions, oxidation of 1,8-cineole proceeds even

further to diaterebic acid acetate and diaterpenylic acid acetate (Linuma

et al., 2008).

(11)

2.13.3 Limonene

Limonene (1-methyl-4-prop-1-en-2-ylcyclohexene) is a cyclic

monoterpene having the molecular formula C10H16, with a molecular

weight of 136.237 g mol-1. It is a naturally occurring hydrocarbon found

in the rinds of citrus fruit like grape fruit, lemon, lime and oranges. It is

biosynthetically obtained from geranyl pyrophosphate through cyclization

of the neryl carbocation or its equivalent, followed by a loss of proton

from the cation to form the alkene (equation 12) (Mann et al., 1994)

46

(12)

Limonene hydration and acetylation brought about by phosphotungstic

(H3PW12O40⋅nH2O) and phosphomolybdic acid (H3PMo12O40.nH2O) catalysts

supported on silica titanium dioxide was reported (Avila et al., 2008). The

phosphotungstic acid produces larger quantity of hydration and

acetylation products than the phosphomolybdic acid catalyst.

Transformation reaction of limonene starts with exocyclic carbocation

production which may be transformed into limonene isomers or either

react with water to produce alcohols. The alcohol produced may react

with acetic acid to form two corresponding acetates (equation 13) (Avila

et al., 2008).

47

(13)

In the same vein epoxidation of limonene via two titanium silicate

zeolite type catalysts, microporous (TS-1) and mesoporous (Ti-SBA-15)

was also documented (equation 14) (Pena et al., 2012).

(14)

An electrophilic attack occurs on the double bond (position 1-2) in

limonene molecules giving rise to 1, 2-epoxylimonene and on further

hydration of this product, 1, 2-epoxylimonene diol was produced (Duetz

et al., 2003; Pena et al., 2012). In another process, carveol was formed

48

as a result of cyclic allylic hydrogen abstraction (allylic oxidation-

hydroxylation at position 6) of limonene molecule (Duetz et al., 2003;

Pena et al., 2012; Rothenberg et al., 1998). Likewise if the allylic

hydrogen abstraction (allylic oxidation-hydroxylation is carried out at

position 7) perillyl alcohol os formed (Duetz et al., 2003; Pena et al.,

2012). Carvone is also observed as one of the products in another

process (Ramos-Fernandez et al., 2014).

2.13.4 α-Terpineol

Brønsted and Lewis acids, ion exchange resins and zeolites have been

used as acid catalysts for the chemical transformation of terpenoids.

These are accessible and cheap catalysts that usually lead to new

pathway of terpenoids transformation. There is little attention drawn to

the use of clay as catalyst for terpenoids transformation, although some

publications have documented their use for catalyzing different organic

reactions (Dasgupta and Torok, 2008). Heteropolyacids have attracted

interest for the synthesis of fine chemicals (Kozhevnikov, 2002).

Heterogeneous catalysis brought about by H3PW12O40 (heteropolyacid)

gave a high catalytic activity in different terpenoids reactions like

acetoxylation, hydration and isomerization. Isomerization of α-terpineol

derived by heteropolyacid in both homogenous and heterogeneous

system gave a good yield of 1, 8 and 1, 4-cineole (equation 6) (Kumar

and Agarwal, 2014).

49

2.14 Chemical Transformations in Plants

Chemical transformation is the modification of known plant metabolites

in order to enhance the original activity of the chemical compound or

obtain derivatives that exhibit other activities. Often secondary

metabolites isolated from plants represent novel classes of chemical

compounds and these plant metabolites and products of their

transformations possess very useful properties. Monoterpenes such as

cis-α-ocimene, α-myrcene, and 4-trans-6-trans-alloocimene, which are

chemically transformed products of essential oils, have pleasant odors;

hence they are synthesized on a large scale for use in perfumery industry

(Terry et al., 2005). At elevated temperatures, β-myrcene is converted to

β-pinene (equation 15) (Liu and Hammond, 1964; Dauben et al., 1969).

(15)

Kinetic studies have shown that linalool, geraniol, and nerol,

interconvert to acids via an allylic rearrangement to yield linalool and α-

terpineol, respectively (Mitzner et al., 1976). On oxidation, citronellol

gives citronellal, while citronellal cyclizes to give isopulegol in good yield

with sulphuric acid (equation 16) (Sully, 1964). On partial hydrogenation

of geraniol, the monounsaturated alcohol citronellol was obtained

(equation 17). Condensation of citronellal with acetone gives, via aldol-

type pathway, dihydropseudoionone, which readily cyclizes to

50

dihydroionone, a substance with a fresh smell of flowers (equation 18)

(Monteiro et al., 2004).

(16)

(17)

(18)

Plants emit a wide range of volatile organic compounds whose common

precursor is isoprene (Fehsenfeld et al., 1992). Isoprene emission is

generally related to photosynthesis because both processes are light and

carbon dioxide dependent (Sharkey et al., 1991). Monoterpenes are

released from specialized organs such as resin ducts, oil glands and

glandular trichomes (Tingey et al., 1991). In many cases, monoterpenes

are emissions found in field studies (Loreto et al., 1996).Temperature is

the main factor affecting the emission of monoterpenes (Tingey et al.,

1980; Tingey et al., 1991). Terpenes have the general formula (C5H8)n

and are biosynthesized from isoprene units in the form of isopentyl

pyrophosphate. The parent terpenes and their oxidation products such as

epoxides, alcohols, aldehydes, and ketones constitute one of the largest

51

classes of organic compounds found in biological systems. Monoterpenes

(n=2) are more volatile than their sesquiterpenoid (n=3) homologues.

The volatile diterpenes such as squalene (n=4) and larger terpenoids

have important biological activities, e.g., some are hormones or

precursors to hormones. Many mono- and sesquiterpenoid compounds

found naturally in plant essential oils are sought after fragrances and

flavorings due to their distinctive pleasant odors and taste notes

(Charlwood and Charlwood, 1991). Verbenol, verbenone, and myrtenol

are derived from the oxidation of α-pinene (Bell et al., 2003; Colocousi et

al., 1996). Myrtenol arises from oxidation of C10 group. Monoterpenes are

biogenic volatile organic compounds that play an important role in

atmospheric chemistry (Hoffmann et al., 1997; Lee et al., 2006; Pinto et

al., 2007; Yu et al., 1999).

2.15 Antioxidant Activity

Although oxygen is very crucial to the cells of human body, however,

when in contact with free radicals it results to oxidative damage of the

cell. Consequently, there is need for shielding of these cells by antioxidant

from the effect of free radicals. Roughly about four thousand antioxidant

compounds are found in foods, vitamin E, vitamin C (ascorbic acid), beta-

carotene, selenium, and carotenoids were the largely studied antioxidant

compounds in the past (Abdalla and Roozen, 1999). Oxidation is a

chemical reaction leading to the loss of electrons, this reaction usually

generates free radicals in body leading to various chain reactions, and

these chain reactions thus generated are the source of injury or death to

52

body cells. Antioxidants are capable of inhibiting these chain reactions by

getting rids of the free radical intermediates thus hindering other

oxidation reactions. They are substances that help halt the oxidation of

other substances; they also help in mopping up free radicals and prevent

them from causing damages to the cells (Hyldgaard et al., 2012; Paul,

2011). They inhibit lipid peroxidation and several others free radical-

mediated processes, thereby shielding the body from a lot of diseases

linked to the reactions of free radicals. This is made possible by being

oxidized themselves; therefore antioxidants are themselves reducing

agents (Sies, 1997). Free radicals generate molecular alterations that are

connected to different human diseases like cancer, arteriosclerosis,

Alzheimer‟s and Parkinson‟s disease, diabetes, asthma, arthritis, immune

deficiency diseases, and ageing (Lobo et al., 2010; Mahmood et al.,2010;

Mimica-Dukic et al.,2010; Sachdev et al.,2008). Unevenness between

oxidizing agents and natural antioxidants or inhibition of antioxidant

enzymes leads to oxidative stress and activate pathogenic progression of

some cruel diseases.

Animals and plants retain a complex system of diverse kinds of

antioxidants which include vitamins A, C, E, glutathione as well as

enzymes like peroxidase, superoxide dismutase and catalase.

Antioxidants are employed industrially as food preservatives, in cosmetics

and to stop the degradation of rubber and petrol (Dabelstein et al., 2007).

53

2.15.1 Mechanism of Action of Antioxidants

There are two mechanisms of action proposed for antioxidants (Rice-

Evans and Diplock, 1993). The first is the chain-breaking mechanism

through which the primary antioxidant gives an electron to the free

radical present in the systems while the second mechanism deals with the

abstraction of reactive nitrogen species initiators (secondary antioxidants)

by quenching chain-initiating catalyst. It has been discovered that

antioxidants may bring to bear their effects on any biological systems by

different mechanisms like metal ion chelation, gene expression regulation,

electron donation and co-antioxidant (Krinsky, 1992).

2.15.2 Generation of Free Radicals in the Body

Free radicals and other ROS are generated either through normal

interior important metabolic processes in human body or from exterior

sources like exposure to X-ray, ozone, through cigarette smoking or

industrial chemicals, and through air pollutant in the environment (Bagchi

and Puri, 1998). Free radical formation takes place constantly in the cells

as a result of both enzymatic and non-enzymatic reactions. Enzymatic

reaction that produces free radicals is that involved in the respiratory

chain, in phagocytosis, in prostaglandin synthesis, and in the cytochrome

P-450 system, while the generation of free radicals through non-

enzymatic reactions includes those investigated by ionization reactions or

by the addition of oxygen to organic compounds (Liu et al., 1999).

54

2.15.3 Natural versus Synthetic Antioxidants

Synthetic antioxidants like butylated hydroxytoluene, butylated

hydroxyanisole, propyl gallate and tert-butyl hydroquinone and natural

antioxidants such as α-tocopherol (in vegetables, broccoli and mangoes)

are commonly used in the preservation of food. However the use of

synthetic antioxidants is progressively getting restricted due to their

toxicity and damaging effect on health (Tawaha et al., 2007). There is an

increasing investigation of antioxidant compounds of novel origin that can

be used as food supplement as well as preservatives in the food industry,

since they can be added to foods containing fats and oils to stop them

from becoming rancid and discolored. It has been reported that the intake

of antioxidants can boost thyroid hormone action, normalize copper, zinc

and blood sugar level, reinstate proper cell oxygen level, restore libido,

regulate blood clotting, avert breast fibrocysts, avoid breast and

endometrial cancer, boost the possibility of embryo survival and sustain

secretory endometrium (Maurin et al., 2003).

2.15.4 Plants as Sources of Antioxidants

Dating back to 1950s, research has been conducted on the antioxidant

potentials of some herbs and spices, since then several studies have

emerged on a vegetable source in other to extract a novel antioxidant

compound from it. This has led to the characterization of different

antioxidants from the leaves of plants which are as effective as the

synthetic butylated hydroxyl anisole (BHA) and butylated hydroxyl

toluene (BHT) (Kuti and Konuru, 2014; Vekiari et al., 1993). Different

55

plant parts may contain natural antioxidants. Fruits, spices, nuts, seeds,

leaves, bark, roots and vegetables are all potential sources of natural

antioxidants. Antioxidants have also been found in the following oil seeds:

flaxseed, sunflower, soybean, cottonseed and canola. The bulk of natural

antioxidants found in plants are phenolic compounds and the most

significant groups are the flavonoids, tocopherols and phenolic acids

which are widespread to all plant sources (Naczk and Shahidi, 2006).

Since natural antioxidants are found in food eaten by man since time

immemorial, they are presumed to be safe, useful in cancer prevention

and helping to stop oxidation reactions in the body.

Phenolic compounds found in most plants possess both primary and

secondary antioxidant actions. They act as chain breaking antioxidants

through the process of contributing hydrogen atoms to lipid radicals

(radical scavenging) or by averting initiation reactions through chelating

metals (Luzia et al., 1998; Rice-Evans et al., 1997; Sugihara et al.,

1999). Phenolic compounds acting as both radical scavenging and

chelating agents are made possible as a result of their structural features.

Phenolic extracts from different plant sources have been revealed to

possess antioxidant actions. These include fruits, sunflower seeds

vegetable and medicinal plant, tomatoes, garlic and ginger (Abushita et

al., 1997; Aruoma et al., 1997; Velioglu et al., 1998).

Plants may be a chief source of natural dietary antioxidants. Plants

containing compounds like tannins, flavonoids and other phenolic

components have been documented to prevent cells against oxidative

56

damages brought about by free radicals (Kahkonen et al., 1999).

Phenolic compounds contain conjugated ring structures and hydroxyl

groups playing a key role in antioxidative activity through scavenging

superoxide anion, singlet oxygen and lipid peroxyl radicals and regulate

free radicals either by hydrogenation or forming complex with oxidizing

agents (Liu et al., 2008). Dietary antioxidant compounds like vitamins,

minerals and pigments found in vegetables and fruits are capable of

thwarting and restraining possible threats from oxidative damage when

ingested regularly (Dasgupta and Bratati, 2007; Edziri et al., 2012; Gupta

and Prakash 2009; Shyamala et al., 2005). Certain severe ailments can

be minimized through the normal intake of vegetables rich in dietary

antioxidants (Hunter and Fletcher, 2002).

2.15.5 Examples of Natural Antioxidants

2.15.5.1 Beta Carotene and Carotenoids

Plants contain a group of pigments called the carotenoids. The most

common of the carotenoids are β-carotene 1, lycophene 2, lutein 3, and

zeaxanthin 4 (Abdalla and Roozen, 1999), which are distinguished by

their orange, yellow or red color. Carotenoids are fat soluble nutrients

that inhibit free radical damage, we have about 600 known carotenoids,

although β-carotene is converted to vitamin A 5 in the body but they are

not regarded as vitamins. This useful antioxidant cannot be synthesized

by the body of animals or humans but can be derived from the diet we

ingest; it is believed that they work best with other phytochemicals and

vitamins (Clement, 1998).

57

Carotenoids operate as antioxidant in the body shielding against

cellular damage, effect of ageing and even some chronic diseases. β-

Carotene assists to lessen the risk of cancer, is anti-inflammatory in

nature and helps to strengthen the immune system. Zeaxanthin and

lutein on the other hand are found in the retina and lens of the human

eyes, they help improve eye health and shield the retina from dangerous

radiations. Lycophene help reduce prostate cancer. Carotenoids are

commonly present in deeply colored vegetables and fruits (Larson, 1988).

β-Carotene is principally found in fruits and vegetables likes mangoes,

carrots, sweet potatoes and in some leafy vegetables such as spinach and

collard green (Brown and Rice-Evans, 1998). Lycophene is mainly

common in tomatoes, guava, papaya, water lemon and red pepper while

lutein and zeaxanthin are present in dark green leafy vegetables like

collard green, broccoli, pumpkin and Brussel sprouts.

58

2.15.5.2 Ascorbic Acid (Vitamin C)

Vitamin C 6 also known as ascorbic acid is a water soluble vitamin and

a powerful antioxidant. It is passed out through urine when the

concentration is high through the blood. It is naturally found in some

foods, but added to others or could be available as a dietary supplement.

People unlike most animals are incapable of synthesizing this important

dietary component endogenously (Li and Schellhorn, 2007). This dietary

component is needed for the biosynthesis of collagen, L-carnite and some

neurotransmitters in addition to its function in protein metabolism (Carr

and Frei, 1999; Li and Schellhorn, 2007). Collagen an indispensable

constituent of connective tissue plays a fundamental role in wound

healing. Vitamin C is similarly a powerful scavenger of air pollutants and

an essential physiological antioxidant; it has been reported to avert free

radical-related diseases like cancer, as well as annulling disease

conditions (Frei, 1999). It works with vitamin E to obstruct the damaging

reactions that are generated by free radicals (Diplock, 1995) and assists

in the absorption of minerals such as iron (Essawi and Srour, 2000).

Other diseases that vitamin C guards against are heart disease, obesity,

59

and weight loss, osteoarthritis, high blood pressure and diabetes.

Vegetables and fruits like oranges, grape fruits, mangoes, the dark green

vegetables, red cabbage, and chili peppers are the greatest sources of

vitamin C. Deficiency of vitamin C results to scurvy, a disease brought

about by breakdown in collagen; others include easy bruising, slow wound

healing, dry splitting hair, nose bleeds, dry red spots on the skin and

bleeding gum.

2.15.5.3 Vitamin E

Vitamin E 7, a fat-soluble vitamin is a collective name for a set of fat-

soluble compounds with unique antioxidant activities (Traber, 2006); it is

naturally found in some foods and obtainable as a dietary supplement.

Vitamin E exists in eight chemical forms (α, β, γ, δ-tocopherols and α-, β-

, γ-, δ-tocotrienols) with different levels of biological activity (Traber,

2006). α-Tocopherol is the only form that is known to meet human

requirements. Free radicals generated in the body are capable of

damaging cells and may contribute to the development of cardiovascular

disease and cancer (Verhagen et al., 2006). The unshared electrons of

free radicals are greatly energetic and react speedily with oxygen to form

reactive oxygen species (ROS).

60

The body produces ROS endogenously when converting food to energy,

and antioxidants are capable of protecting the cells from the damaging

effects of ROS. Other sources of free radicals that could be detrimental to

the body are cigarette smoke, air pollution, and ultraviolet radiation from

the sun. Vitamin E a fat-soluble antioxidant is capable of inhibiting the

production of ROS formed when fat undergoes oxidation, so vitamin E has

antioxidant property (Yepez et al., 2002). Antioxidants protect cells from

the damaging effects of free radicals, which are molecules that contain an

unshared electron. Free radicals‟ damage cells and might contribute to the

development of cardiovascular disease and cancer (Verhagen et al.,

2006). In addition to this, it also acts as cell stabilizer by preventing the

oxidation of low-density lipoprotein cholesterol (LDL-Cholesterol). This is

known to reduce risk of atherosclerosis and heart attack (Thabrew et al.,

1998). Most of the research on vitamin E as an antioxidant is associated

to free radical induced heart diseases. This condition was found to be

reversed with vitamin E ingestion. Several foods provide vitamin E. Nuts,

seeds, and vegetable oils are among the greatest sources of alpha-

tocopherol a component of vitamin E, and considerable amounts are also

available in green leafy vegetables and fortified cereal.

61

2.15.5.4 Selenium

Selenium is a mineral needed in very minute amounts to make

important enzymes that are critical for good health. It is present in some

foods, including plant foods cultivated in selenium-rich soil, and some

meats and seafood. Selenium is nutritionally important for humans and a

constituent of scores of seleno proteins that plays a crucial role in

reproduction, thyroid hormone metabolism, DNA synthesis, and protection

from oxidative damage and infection (Rayman, 2012). It has also been

reported to possess antioxidant activity that helps the body protect

against cellular damage from free radicals in addition to being a cofactor

of an essential antioxidant enzyme in the body called glutathione

peroxidise (Nakatani, 2000). Record shows that taking food rich in

selenium has positive antiviral effects, necessary for successful male and

female fertility and reproduction and also lowers the risk of cancer,

autoimmune and thyroid disease (Rayman, 2012). Deficiency in selenium

can affect thyroid function resulting to ailments such as keshan disease

(enlarge heart and poor heart function) (Chen, 2012; Rayman, 2008),

Kashin-Beck disease (osteoarthropathy) (Coates et al. 2010; Jirong et al.,

2012; Rayman, 2008) and myxedematous endemic cretinism (results in

mental retardation) (Coates et al., 2010; Sunde, 2006). Selenium is

contained in vegetables such as onions, cucumber, mushroom, garlic,

whole grains, wheat germ and animal products like egg, milk, chicken,

sea foods, etc.

62

2.15.5.5 Uric Acid

Uric acid accounts for approximately half the antioxidant capability of

plasma. In fact, uric acid may possibly have substituted for ascorbate in

human development. However, like ascorbate, uric acid can also mediate

the production of active oxygen species (Jaeschke et al., 2002).

2.16 New Antioxidants

Natural antioxidants derived from herbs and spices have been found to

be effective against oxidative stress and consequently play a significant

role in the chemoprevention of ailments resulting from lipid peroxidation

(Nakatani et al., 2000). Flavonoids found in medicinal plants are

responsible for the therapeutic properties of such plants in addition to

both organic and inorganic compounds like phenolic acids; coumarins and

antioxidant trace nutrients like Cu, Zn, or Mn (Czinner et al., 2001).

Flavonoids and other polyphenols obtained from plant materials have

potentially valuable effects on human health. These compounds are

referred to as secondary plant metabolites, a description signifying that

majority of these substances are considered as non-essential and hence

are secondary in function, but have been found to be a significant part of

human diet and are regarded to be active components of some medicinal

plants. Flavonoids are powerful inhibiting agents against superoxide anion

(O2-), hydroxyl (OH-), peroxyl (ROO-), alkoxyl (RO-) radicals. Those

having multiple OH- substitution possess very strong antioxidant activities

against peroxyl radicals (Alanko et al., 1999). It is now believed that

there are several hundreds of other antioxidants that occur naturally in

63

foods and beverages. Most of them are not regarded as nutrients, but

phytochemicals (meaning plant chemicals). Phytochemicals are now being

discovered as powerful disease fighters. Flavonoids, polyphenols and

anthocyanins are some of the names found in relation to the new

antioxidants (Bravo, 1998).

2.16.1 Polyphenols

Polyphenols belong to a group of phytochemicals obtained in high

concentrations in wine, grapes and a wide array of other plants which are

linked to heart disease and cancer prevention (Danyi et al., 2009).

Generally, phenolic compounds or polyphenols possess a similar basic

structural chemistry including an aromatic or phenolic ring structure. It is

also imperative to note that at least 8,000 phenolic compounds are

accountable for the brightly colored pigments of various fruits and

vegetables. In addition to this, they protect plants from diseases and

ultraviolet light and help avoid damage to seeds pending when they

germinate (Gordon, 1996; Haslam, 1996).

Polyphenols are capable of forming complexes with reactive metals

such as iron, zinc, and copper, thus, reducing their absorption. This

seems to be a negative side effect (reducing nutrient absorption), but

excess levels of such elements (metal cations) in the body can catalyze

the production of free radicals and contribute to oxidative damage of cell

membranes and cellular DNA (Lebeau et al., 2000). In addition to

chelating effect on metal cations, polyphenols also act as potent free

64

radical scavengers inside the body, where they are capable of causing

cellular damage.

Natural polyphenols can range from simple molecules such as phenolic

acid to huge extremely polymerized compounds, such as tannins.

Conjugated forms of polyphenols are the most common, where various

sugar molecules, organic acids, and lipids (fats) are joined with the

phenolic structure. Differences in this conjugated chemical structure

accounts for different chemical taxonomy and disparity in the method of

action and health properties of various compounds. It has been asserted

that polyphenols assist in cancer prevention, defense from heart disease,

hypertension, antibiotic/antiviral action, anti-inflammation activity, and

reduced risk of cardiovascular diseases and cancer (Bouchet et al., 1998).

2.16.2 Flavonoids

Flavonoids are either water soluble polyphenolic compounds found in

plants (fruits and vegetables) or hydroxylated phenolic compounds that

are produced by plants in reaction to microbial infection (Dixon et al.,

2005). Flavonoids are the most significant plant pigments that are

responsible for flower coloration, producing yellow, red, or blue

pigmentation in petals intended to attract pollinator animals. Flavonoids

generally consist 15-carbon skeleton 8, made up of two phenyl rings (A

and B) linked together by oxygen containing heterocyclic ring (C).

65

The functional hydroxyl group found in flavonoid compounds is

responsible for their antioxidant activity through scavenging of free

radicals achieved by hydrogen donation mechanism as shown by the

equation below (equation 19) or by chelating metal ions (Kumar et al.,

2013; Kumar and Pandey, 2013). Flavonoids refer to the most frequent

and broadly distributed group of phenolic compounds in plants, occurring

nearly in all plant parts, especially in the photosynthesizing plant cells.

They are essential parts of human and animal diet. Flavonoids are

phytochemicals and for this reason cannot be produced by humans and

animals (Koes et al., 2005). Consequently, flavonoids found in animals

are of plant origin rather than being biosynthesized in situ. The most

abundant flavonoids in food are the flavonols.

(19)

2.16.3 Diphenyl Picryl Hydrazyl (DPPH) Free Radical

The body of a man is composed of billions of molecules bound together

by electromagnetic forces. The chemical bonds between these molecules

are produced by paired electrons, but free radicals are molecules that

66

have lost an electron and they are very unstable and highly reactive. They

can either donate an electron to or receive an electron from other

molecules, hence acting as an oxidants or reductants (Davies, 2000). A

single free radical is capable of damaging a million or more molecules via

a chain reaction known as radical propagation; this leads to oxidation or

what is referred to as oxidative stress. It is not all free radicals generated

by the body that are harmful, some are beneficial, in reality free radicals

produced by the immune system aid in the destruction of bacteria and

viruses, whereas others are concerned with the production of vital

hormones and they trigger enzymes that are required for life itself (Ratty

et al., 1988). Free radicals become a problem when produced in excess

by human body leading to cell and tissue damage; their overabundance

production creates more radicals in the body (Cotelle et al., 1996). 2, 2-

Diphenyl-1-picrylhydrazyl abbreviated as DPPH is a dark colored

crystalline power and a cell permeable stable free radical at ambient

temperature. It is a well-recognized radical and a trap (scavenger) for

other radicals. DPPH has one major application: in the evaluation of

chemical reactions involving radicals, mainly used for common antioxidant

assay for detecting free radical scavenging activity (Brand-Williams et al.,

1995).

67

DPPH has a strong absorption band at around 520 nm (Brand-Williams

et al., 1995), and is characterized by a deep violet color in solution. When

mixed with a substance that can donate hydrogen atom (antioxidant), it

reacts with it and is reduced by a loss of this violet color to colorless or

pale yellow. Depicting the DPPH radical by Z and the antioxidant donor

molecule by AH, the primary reaction is given below (equation 20):

Z + AH → ZH + A (20)

ZH is the reduced form and A is the new radical generated in the first

step. This new free radical undergoes additional reactions, determining

the overall stoichiometry. That is the number of DPPH molecules reduced

(decolorized by the molecule of the reductants). The reaction above is the

type of reaction taking place in an oxidizing system like auto-oxidation of

a lipid or other unsaturated substances: the DPPH molecule Z acts as a

free radical formed in the system whose activity is to be suppressed by

the substance AH (Basnet et al., 1997).

If, on the other hand, the molecule has two neighboring sites for

hydrogen abstraction that are internally connected, as is the case with

ascorbic acid (vitamin C), then there might be a further hydrogen

abstraction reaction after the first one, as depicted in the equation below.

This results to a 2:1 stoichiometry (equation 21). That is two molecules of

DPPH being reduced by one molecule of ascorbic acid.

68

(21)

One parameter that has been initiated for interpretation of the results

from the DPPH method is the effective concentration or IC50. This is

defined as the concentration of the extract required to scavenge 50% of

DPPH activity (Thabrew et al., 2005). The IC50 parameter has the

drawback in that the higher the antioxidant activity, the lower the IC50

(Mensor et al., 2001).

2.17 Plant Empire as Foundation of Leading Drugs

2.17.1 Introduction

Plants used for traditional remedy contain broad range of substances

that can be used to treat chronic as well as contagious diseases. The plant

empire is a virtual goldmine of prospective drug targets and other useful

molecules waiting to be discovered. About 10-15% of the 750,000 known

species of higher plants have been assessed for biologically active

compounds (Duraipandiyan et al., 2006). Natural products formed by

plants, fungi, bacteria, insects and animals have been isolated as

biologically active pharmacophores. Important considerations have been

given to natural products particularly from plant in the pharmaceutical

industry for their broad structural variety as well as their wide range of

69

pharmacological activities. Natural products obtained from therapeutic

plants, either as standardized or unadulterated compounds, provide

numerous chances for new drug leads and new chemical entities due to

the unmatched availability of chemical diversity (Butler, 2004; Newman,

2008)

2.17.2 Plant under Study (The Genus Callistemon)

Callistemon obtained its name from a Scottish botanist by name Robert

Brown (1773–1858), who contributed significantly to the study of Botany

through the utilization of microscope. Callistemon genus belongs to the

Myrtaceae family and is made up of about thirty-four species. The word

Callistemon is from two Greek words (Kallistos meaning most beautiful

and stema meaning a stamen) (Khanna et al., 1990). This evergreen

shrub possesses lanceolate and aromatic leaves and is widespread in

Australia, although some of them have been introduced to other region

like USA and Africa. The plant is known as bottle brush due to its

traditional brush that looks like a conventional bottle brush (Gilman,

1999, Nel et al., 2004).

Naturally, Callistemon is commonly found either along watercourse or

the edge of a swamp. This plant enjoys moist conditions and grows very

well in gardens when watered regularly, although some of their species

are drought resistant. It can be planted either by cutting or from its seeds

and the flowering time of the plant is usually in spring or early summer

(October-December). Other conditions may make this plant to bring out

flowers at other times of the year. They are grown as decorative plants in

70

gardens, houses, streets or offices because of their red attractively

colored flowers. The leaf of this plant releases a refreshing fragrance

when crushed with hands (Spencer et al., 1991, Wrigley and Fagg, 1993)

Callistemon genus has about 140 genera with approximately 3800

species and had been employed as an anticough and antibronchitis in

traditional rural settings. Its antiphylococcal, nematocidal, larvicidal,

pupicidal, antithrombotic and antioxidant activities of the class

Callistemon species and also the antimicrobial properties of their volatile

oils had been widely documented (Larayetan et al., 2017).

2.17.3 Callistemon citrinus

Callistemon citrinus, which is generally recognized as Crimson bottle

brush, is an evergreen tree or flowering shrub, from the Myrtaceae family.

It can reach a height of up to 6-15 m and 1.3-1.5 m in girth with prickly

pointed mid-green leaves. Possession of beautiful red flowers with red

dark anthers is one of the characteristic features of this plant (Larayetan

et al., 2017; Wrigley and Fagg, 2007).

The various parts of this plant have been employed for the treatment

of common diarrhea, dysentery and rheumatism, in addition to this; it is

used as an anticough, water accent, antibronchitis and as an insecticide in

tradition medicine (Mohmoud et al., 2002).

The brilliant red flower spikes of this plant are very rich in nectar and

this tends to draw insects and birds to the plant (Spencer et al., 1991,

Wrigley and Fagg, 1993). C. citrinus is also rich in polyphenols (Salem et

al., 2017). Flower heads differ in color based on the species. Majority of

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the flowers are red, while some are green, orange, yellow or white, the

flower head possess woody capsules that appear like bead bracelets on

the bark. Callistemon citrinus has a lanceolate and aromatic leaves, they

are commonly planted as an ornamental plant in South Africa. They are

referred to as crimson bottle-brush (Larayetan et al., 2017).

2.17.3.1 Taxonomy of Callistemon citrinus

Callistemon citrinus belongs to the kingdom Plantae, subkingdom:

Tracheobionta, super division: Spermatophyte, grouped under

Magnoliophyta division, classified under Dicotyledons class, has a subclass

of Rosidae, ranked under the order of Myrtales. The genus Callistemon

belongs to the Myrtaceae family and the species are Callistemon citrinus

Curtis, Metrosideros citrina Curtis, Callistemon lanceolatus (Sm) Sweet

(Kumar et al., 2015).

2.17.3.2 Examples of Some Callistemon Species

Generally, some species of Callistemon comprise of Callistemon

acuminatus (tapering-leaved bottlebrush), Callistemon brachyandrus

(Lindl) (prickly bottlebrush), Callistemon citrinus (Curtis) Skeels

(crimson bottlebrush), Callistemon chisholmii, Callistemon coccineus,

Callistemon comboynensis (cliff bootlebrush), Callistemon flavovirens

(Green Bottlebrush), Callistemon formosus, Callistemon forresterae,

Callistemon genofluvialis, Callistemon glaucus, Callistemon kenmorrisonii

(betka bottlebrush), Callistemon linearis, Callistemon montanus

(mountain bottlebrush), Callistemon nervosus, Callistemon nyallingensis,

Callistemon pachyphyllus (wallum bottlebrush), Callistemon pallidus

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(lemon bottlebrush), Callistemon pauciflorus (desert bottlebrush),

Callistemon pearsonii, Callistemon pinifolius (pine-leaves bottlebrush),

Callistemon pityoides (alpine bottlebrush), Callistemon polandii,

Callistemon pungens, Callistemon recurvus, Callistemon rigidus (stiff

bottlebrush), Callistemon rugulosus (scarlet bottlebrush), Callistemon

salignus (willow or white bottlebrush), Callistemon shiressii, Callistemon

sieberi (river bottlebrush), Callistemon subulatus, Callistemon teretifolius

(needle or flinders ranges bottlebrush), Callistemon wimmerensis

(wimmera bottlebrush) Callistemon pallidus, Callistemon viminalis

(weeping bottlebrush). (www.treenames.net).

2.17.3.3 Uses of Callistemon Species

Callistemon viminalis is used as a diuretic to ease problems of the

urinary tract. It is also used by women as douche to cleanse the

genitourinary tract from excessive menstruation or mucosal discharge as

leucorrhea and as well as for urinary incontinence and bed-wetting in

children (Abd, 2012). In Jamaica, the decoction is used as hot tea

treatment of gastroenteritis, diarrhea, and skin infections (Salem et al.,

2013), whereas it is used in Kenya for treating toothache (Kamau et al.,

2016)

Callistemon citrinus is extensively cultivated as an ornamental in hot to

warm temperate region (Whistler, 2000). The leaves of this plant are

used as a tea alternative and contain a pleasantly revitalizing flavor

(Quadri et al., 2013). The flowers serve as a source of tan dye, which

does not require a mordant, but when used with a mordant the color

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turns green. A cinnamon dye is acquired from the leaves, other member

of this genus can also be used (Grae, 1994).

2.17.3.4 Traditional Uses

The aerial parts of this plant have been used in frequent remedies for

management of diarrhea, dysentery and rheumatism. The plant is used by

traditional healers in India to treat respiratory ailment such as cough,

bronchitis and also employed as insecticides, while its essential oil is used

as an antimicrobial herbal drug (Chopra., et al., 1956; Netala et al.,

2015; Shaha and Salunkhe, 2014). About 500 mL of the fresh leaves

decoction of Callistemon citrinus is drunk by the adult people of Uganda

to treat cough (Namukobe et al., 2011). It is commonly used as

traditional Chinese medicine pills for treating hemorrhoids in China (Ji,

2009). Callistemon is used in farm land as weed control and as bio

marker for environmental management (Burchet et al., 2002, Wheeler,

2005). It is also used for tool handles and as fuel (Manandhar, 2002).

2.17.3.5 Medicinal Uses

Volatile oil from Callistemon citrinus has been confirmed to possess

antinociceptive and anti-inflammatory activities in an experiment

conducted on rats, in which it reduced paw volume in paw edema; it also

demonstrated a higher activity more than the synthetic antibiotic

clotrimazole and miconazole (Brophy et al., 1998; Oyedeji et al., 2009;

Stanaland et al, 1996; Sudhakar et al., 2004). Chromogenic bioassay

carried out on the methanolic extract of this plant revealed anti-thrombin

activity (Chistokhodova et al., 2002). The ethyl acetate extract from this

74

plant may be helpful in management of type-1 and type-2 diabetes linked

with abnormalities in lipid profiles C. citrinus has anti-hyperglycemic

property in addition to its ability to enhance body weight, liver profile,

renal profile with total lipid levels in alloxan-diabetic rats (Kumar et al.,

2011). This plant has been documented to possess anti-thrombin, anti-

candida, anti-inflammatory, fungi toxicity and antinociceptive actions

(Chistokhodova et al., 2002; Dutta et al., 2007; Netala et al., 2015;

Shaha and Salunkhe, 2014). It is also used to combat gastro-intestinal

disorder, pains and contagious diseases brought about by virus, fungi,

bacteria and similar pathogens (Goyal et al., 2012). Moreover, it is a

valuable natural herbicide because it contains a phytotoxin called

leptospermone belonging to β-triketones (Vogler et al., 1998).

Some of the components of the volatile oils obtained from the aerial

parts of this plant have therapeutic actions. Eucalyptol which has been

reported globally as the major component of the different parts of this

plant has anti-viral, anti-tussive, bronchodilator and mucolytic actions

(Harris et al., 2007; Sokovic et al., 2010). α-Pinene is also found to be

one of the major components of the oil and it possesses some therapeutic

potentials like anti-bacterial, anti-cancer, anti-inflammatory, antioxidant,

and antinociceptive properties (Bae et al., 2012; Dorman et al., 2000;

Him et al., 2008; Wang et al., 2008; Wang et al., 2012)

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2.17.3.6 Pharmacological activities

2.17.3.6.1 Antifungal action

Volatile oils obtained from two species of Callistemon and Eucalyptus

from Cameroun (Callistemon rigidus, C. citrinus, Eucalyptus camaldulensis

and Eucalyptus saligna) were screened for their antifungal activity and

found to be very potent against Aspergillus flavus (Dongmo et al., 2010).

On the other hand, the methanolic extract of Callistemon citrinus leaf

from Australia was unable to inhibit Aspergillus niger and Candida

albicans (Cock, 2012).

2.17.3.6.2 Acute Toxicity, Brine Shrimp Cytotoxicity

Cock (2012) determined the toxicity of C. citrinus using the Artemia

franciscana nauplii bioassay. No LC50 values were recorded for the C.

citrinus leaf or flower extracts in the experiment because there was no

considerable increase in mortality higher than the seawater controls,

which indicated that the extract was non-toxic. The crude methanol

extract of the plant was reported harmless at a concentration of 250

mg/mL or less. The brine shrimp cytotoxicity assay meant that the plant

species could be a source of cytotoxic agents (Ali et al., 2011).

2.17.3.6.3 Antibacterial Activity

The antibacterial properties of the leaf and flower oils of C. citrinus

(Larayetan et al., 2017) against various pathogenic bacteria including

Aeromonas hydrophila (ACC), Escherichia coli (ATCC 35150), Salmonella

typhi (ACC), Listeria monocytogenes (ACC), Vibro alginolyticus (DSM

2171), Staphylococcal enteritis (ACC) and Staphylococcus aureus (ACC)

76

by agar well diffusion method were documented. The performance of the

oils in terms of inhibitory action was found potent on the entire set of

tested bacteria indicating that the plant had very broad spectrum of

action against both gram-positive and gram-negative bacteria. Highest

inhibitory action for Vibro alginolyticus DSM 2171 (67.0 ± 2.0 and 60.0 ±

5.0 mm) and Aeromonas hydrophila ACC (58.0 ± 0.3 and 52.0 ± 1.0

mm) as well as the gram-positive bacteria such as Staphylococcal

enteritis ACC (62.0 ± 0.5 and 55.0 ± 2.0 mm) were reported. Both the

leaves and flowers oils demonstrated maximum inhibitory action on gram-

negative bacteria.

In another study methanolic and ethanolic extracts from C. citrinus

were screened against nine pathogenic bacteria which were Streptococcus

pyogenes, Bacillus cereus, Bacillus anthracis, Salmonella typhi, Kelebsiella

pneumoniae, Streptococcus epidermidis, Escherichia coli, Pseudomonas

aeruginosa and Listeria monocytogenes by disc diffusion method and the

results obtained showed a promising antimicrobial activity against the

listed pathogenic bacteria especially on S. typhi, B. cereus, S.

epidermidis, and B. Anthracis. The extracts were more potent on gram-

positive than gram-negative bacteria (Seyydnejad et al., 2010).

2.17.3.6.4 Antioxidant activity

The inhibition of the DPPH radical through in vitro antioxidant activity

of both the leaf and flower oils of C. citrinus (Larayetan et al., 2017)

revealed that they were concentration dependent. The DPPH radical was

reduced by 50% having an IC50 of 1.49 and 1.13 mg/mL respectively as

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against the standard β-carotene and ascorbic acid with IC50 of 1.28 and

3.57 mg/mL The ability of the DPPH radical scavenging of the flowers oil

on the basis of percentage inhibition and IC50 was higher than those of

the leaves oil and the standard antioxidant drugs used. In the same vein,

the methanolic extract of this plant demonstrated strong elastase

inhibition and DPPH radical scavenging activities (Kim et al., 2009).

2.17.3.6.5 Total Phenolic Content

The total phenolic content of the leave oil of this plant from South

Africa was determined using Folin-Ciocateau method and was found to be

899 μg/mg gallic acid equivalent higher than the same species from India

(Larayetan et al., 2017). The ethanolic extract from the leaves of C.

citrinus shows 95.8 ± 1.2 mg/g (Abdelhady et al., 2011).

2.17.3.6.6 Description of the Plant

Callistemon citrinus is considered a synonym of Melaleuca citrina by

the Royal Botanic Garden Kew (Govaerts, 2013). It was formerly called

Callistemon lanceolatus and is the most cultivated among the

bottlebrushes. It usually grows into a small tree-proportion when

conditions are favorable and can reach a height of about 10-15 feet tall

with fine texture. The branches are numerous, long, slender and have a

hard fibrous bark when young. The bark is usually grey in color with

interlacing ridges (Mabhisa et al., 2016). The flowers of this plant are

mostly bright red, but some may be green, orange, yellow or white

depending on the species. The flower head generates a profusion of

tripled celled seed capsules about the stem which stays on the plant with

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the seeds closed; the seeds are only open if there is a fire outbreak or

any external factor, which may cause the death of the plant. Callistemon

citrinus has the ability of flowering twice a year if well-watered, but its

main flowering season starts in early November. Flowering is also possible

in autumn or winter if conditions for the plant are favorable and the red

brilliant appearance of the flower reaches its peak in late summer. The

leaves arrangement is alternate, sharp pointed, simple with lanceolate or

linear contour and the leaf venation is pinnate. It is an evergreen plant

with leaf blade length of about 2-4 inches; the veins of the leaves are

clearly visible on both sides (Mabhisa et al., 2016). The fruits are brown

in color with a round shape having a length of about 0.5 inches. The

capsules of the fruit are opened only when the part of the plant bearing

these capsules are dead (Brophy et al., 2013, PlantNET, 2014). One of

the cultivation parameters for this plant is full sunlight, the plant has soil

tolerance and can grow in either clay, loamy, acidic or well-drained soil,

and it is also drought resistant (Sutar et al., 2014).

2.17.3.6.7 Volatile Oil Extraction

Plant parts are subjected to hydrodistillation method for 3-4 hours

using Clevenger-type apparatus, in order to extract the volatile oils from

the different plant parts (leaves, stem, bark, fruits and flowers). The oils

derived from these plant parts are dried over anhydrous Na2SO4, and kept

in the vial at 4°C. Analyses of the components of the volatile oils are done

via GC-MS (Mabhisa et al., 2016).

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2.17.3.6.8 Chemical Constituents

The volatile oil of the leaves, flowers and stem of C. citrinus have been

comprehensively studied. In Eastern Province of South Africa the leaf

essential oil component was documented to consist of eucalyptol

(48.98%), α-pinene (20.02%), α-terpineol (8.10%), isopinocarveol

(5.75%) and pinocarvone (2.81%), while the flower oil contains

components like α-eudesmol (12.93%), caryophyllene (11.89%), bornyl

acetate (10.02%), eucalyptol (8.11%), bicyclogermacrene (5.71%) and

γ-eudesmol (5.21%), and the seed oil components were eucalyptol

(56.00) and α-pinene (31.03) (Larayetan et al., 2017). The oil obtained

from the flowers part of this plant shows no similarity in its constituents

when compared with others in the literature. The flowers volatile oil from

Iran have α-pinene (25.70%) and eucalyptol (18.10%) as the key

components (Minar et al., 2014), whereas flower oil from Himalaya have

eucalyptol (36.60%) and α-pinene (29.70%) as its leading components

(Kumar et al., 2015). Leaf volatile oils from Reunion Island and lower

region of Himalaya have eucalyptol (68.0 and 66.30%) as the major

component with other components like α-Pinene (12.80 and 18.70 %)

and α-terpineol (10.6%) ranking behind it (Chane-Ming et al., 1998,

Srivastava et al., 2003). There is a particular trend of eucalyptol being

the major components of this plant in almost all oils obtained from

various part of the world.

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2.17.3.7 Phytochemical Studies

From the phytochemical point of view, this plant is found to be rich in

several secondary metabolites with potentials for pharmaceuticals,

industrial and food processing industry. Some examples of secondary

metabolites with pharmacological relevance are the terpenoids,

triterpenoids, flavonoids, saponins and steroids (Goyal et al., 2012).

Flowers include flavonoids (pelargonidin-3,5-diglucoside 10 (R = H),

cyanidin-3,5-diglucoside 10 (R= OH) and kaempferol 11);

monoterpenoids (β-pinene 12 and 1,8-cineole 13); tannins (pyrogallol 14

and catechol 15); and triterpenoids (betulinic acid 16, α-amyrin 17,

oleanolic acid 18 and β-sitosterol 19). Fruits consist of monoterpenoids

(1,8-cineole 13 and α-terpineol 20) and triterpenoids (α-amyrin 17,

betulinic acid 16, oleanolic acid 18, and β-sitosterol 19. Leaves feature

flavonoids (quercetin 21, 3‟, 4‟, 7-trihydroxyflavone 22);

monoterpenoids (1,8-cineole 13, α-pinene 23 and limonene 24); and

triterpenoids (23–hydroxyursolic acid 25, ursolic acid 26, and uvaol

27). Constituents of seeds are tannins (gallic acid 28 and ellagic acid

29).

81

82

Ahmed F. et al. (2016) from Bangladesh also reported the isolation of

three triterpenoids and one steroidal glycoside from the methanolic crude

extract and carbon tetrachloride fraction of the leaves of C. citrinus; they

are erythrodiol 30, betulinic acid 16, β-sitosterol-3-O-β-D-glucoside 31

and taraxerol 32.

83

Cuong et al. (2016) from Vietnam documented also the isolation of one

new flavonoid callistine A 33 (R = H), six recognized flavonoids, 6,7-

dimethyl-5,7-dihydroxy-4'-methoxyflavone 33 (R = Me), astragalin 34,

quercetin 21, catechin 35, eucalyptin 36 (R = Me), and 8-

demethyleucalyptin 36 (R = H), along with five triterpenoids, 3-β-

acetylmorolic acid 37, 3β-hydroxy-urs-11-en-13(28)-olide 37, betulinic

acid 38, diospyrolide 39 and ursolic acid 40.

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85

Chapter 3

Experimental

3.1 Plant Collection

Fresh leaves with flowers were collected from their natural habitation

in the vicinity of the University of Fort Hare in Alice, Eastern Cape

Province of South Africa from January to December of 2016. The plant

was identified by Dr. Maeyikeso of the Department of Botany and voucher

sample (Larayetan 1) was kept in the Giffen herbarium, University of Fort

Hare, South Africa for record purposes. The volatile oils were isolated

through hydrodistillation for about 3 h using Clevenger apparatus

according to the suggested method (British Pharmacopoeia, 1998).

3.2 Microbial Strains

Pure isolates of Aeromonas hydrophila (ACC), Escherichia coli (0157:

H7: ATCC 35150), Salmonella typhi (ACC), Listeria Ivanovii ATCC 19119,

Mycobacterium smegmatis ATCC 19420), Listeria monocytogenes (ACC),

Vibro alginolyticus (DSM 2171), Staphylococcal enteritis (ACC) and

Staphylococcus aureus (ACC) were obtained from the Department of

Biochemistry and Microbiology. All the cultures were maintained on

nutrient agar for further use.

3.3 Reagents Used

2, 2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium

salt, 2, 2-diphenyl-1-picrylhydrazyl, silver nitrate, sodium acetate, barium

chloride, aluminium trichloride, gold (III) chloride, ammonium molybdate

and potassium persulfate were purchased from Sigma-Aldrich (St Louis,

86

USA) for this study. All other analytical grade reagents used were

sourced from Merck (Germany).

3.4 Separation of Volatile Oils

500 g of the leaves, 250 g of the flowers and 150 g of the stems of this

plant were sequentially subjected to hydro- distillation process for three

hours in Clevenger apparatus in accord with the requirements (European

Pharmacopoeia Commission, 2004). The volatile oil was consecutively

extracted using 7 L, 4 L and 2L of water. The oils collected from the

various plant parts were immediately subjected to GC-MS analyses.

3.5 Gas Chromatography - Mass Spectrometry

GC-MS analyses were run on a Hewlett-Packed HP 5973 mass

spectrometer connected with an HP-6890 gas chromatograph. Operating

conditions for this analysis were as follows: original column temperature

70oC, highest temperature 240oC, equilibration time 3min, ramp 4

oC/min, concluding temperature 240oC. Inlet mode: split less, initial

temperature 220oC, pressure 8.27 psi, flush out flow 30 mL/minute, flush

out time 0.20 min, gas helium, capillary column 30 m x 0.25 mm, coat

thickness 0.25 µm, original flow 0.7 mL/min, linear velocity 32 cm/s. MS:

EI method at 70 eV.

3.6 GC-MS Determination of Bioactive Compounds

Presence of bioactive components in the two extracts was revealed by

GC-MS examination carried out on a multi-dimensional gas

chromatography coupled with gas chromatography-mass

87

spectrophotometer (Shimadzu Japan, 2010). This machine possesses

polar doubled capillary and non-polar column (25.0 m×0.25 μm internal

diameter, 0.25 μm film thickness). Helium of high purity was used as

carrier gas at a flow rate of 0.99 mL/min. Starting and final temperatures

were 60oC and 280oC at a heated rate of 3oC/min which was constant

isothermally for 6 min, solvent cut time was set at 3 min, E.I mode was

70 eV while linear velocity for the column was set at 36.8 cm/s.

3.7 Detection of Components

Chemical components of these oils were identified on the basis of their

individual retention times with a reference to homologues series of n-

alkanes in the robust NIST Library 2014. The mass spectra fragmentation

of the compounds was compared to the available data (McLafferty, 1989;

Adams, 1997; Joulain and Konig, 1998).

3.8 Preparation of Plant Extracts

Fresh leaves of the plant were air-dried for 21 days at room

temperature. The dried leaves were crushed by mechanical grinder to

powder and used to prepare two different extracts as highlighted below.

Methanol extract. 250 g of the powdered dried leaves was soaked in

800 mL of methanol for 72 h, the mixture was shaken on an orbital

shaker (Model 420 Series, Thermo Fisher Scientific) at 250 rpm, filtered

with Whatman No.1 filtered paper and concentrated using rotary vacuum

evaporator. The extract was then preserved in tightly stoppered bottle in

the refrigerator at 4oC until needed for analysis.

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Ethyl acetate extract. 250 g of the powdered dried leaves was

equally soaked in 800 mL of ethyl acetate for 72 h. The same procedure

as stated above was followed to obtain dry ethyl acetate extract, which

was stored in an air-tight bottle and kept in the refrigerator until further

analysis.

Extract for nanoparticles of silver and gold. The fresh plant part

(leaves, flowers and seeds) of Callistemon citrinus were obtained from the

University of Fort Hare vicinity and were air dried for about 27 days at

ambient temperature, then grinded with a mechanical grinder (Polymix,

PX-MFC 90D). About 50 g of the crude powdered samples were soaked in

400 mL distilled water and agitated on an orbital shaker for 24 hours. The

extracts were filtered with Whatman No 1 filter paper and the filtrates

were then lyophilized into dry power, preserved in tightly stoppered

centrifuge tubes, refrigerated at 4°C until needed for the nanoparticles

synthesis.

3.9 Extraction

The leaves and seeds obtained from the plant were air dried at

ambient temperature for 21 days; it was blended into powder by using a

Polymix (PX-MFC-90D). The powered plant (leaves, seeds and flowers,

900 g each) was consecutively extracted with n-hexane, dichloromethane,

ethyl acetate and methanol for five days. Each extraction was improved

by carrying out the maceration twice; the solvent extracts obtained from

the above process were filtered and evaporated to dryness via a rotary

evaporator under reduced pressure. The marc of the powered materials

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was re-soaked in the solvent recovered and the extraction was carried out

2-3 times, extracts obtained from each step were stored in a brown vial

until needed for analysis. Thus hexane, dichloromethane, ethyl acetate,

methanol leaf extracts and DCM seed extract were obtained and labelled

as HE, DCM, EA, ME and DCM seed extracts, respectively. The common

extraction procedure used for Callistemon citrinus plant materials (leaves,

seed and flowers) in this work is highlighted below in scheme1.

Scheme 3.1: General Extraction Protocol Adopted for Isolation of Callistemon citrinus Plant Material.

Ethyl acetate (EA), dichloromethane (DCM), hexane (HE), methanol

(ME), deuterated chloroform (CDCl3), dimethyl sulfoxide (DMSO) were the

solvents employed for the extraction of the crude extracts and isolation of

different fractions. Hexane and ethyl acetate were used to run the thin

90

layer chromatography plate, while DMSO and CDCl3 were used for the

bioassay and NMR analysis, respectively.

Column chromatography (Figure 3.1) was carried out on the hexane

leaf and DCM seed extracts showing considerable antiplasmodial and

antitrypanosomal activities as recorded from the bioassay conducted on

the five crude extracts. These extracts were separated into their various

fractions, while silica gel of mesh size 60 (0.040-0.63 mm) obtained from

Macherey- Nagel was employed as the stationary phase. About 75 g of

silica gel was mixed with n-hexane-ethyl acetate (80-20%) solution to

obtain a homogenous slurry, the resulting mixture was stirred well to

eliminate air bubbles, and then introduced into the glass column, a little

washed sand was added to the top of the column to give a smooth flat

surface that will hinder agitation of the already packed silica gel in the

column when the extract is introduced into it. The column was eluted with

100% hexane and subsequently the polarity was increased by adding

10% ethyl acetate. 20 mL of each of the fractions were collected in a 30

mL glass vials and were subjected to rotary evaporator at 40oC, so as to

concentrate the fractions to dryness.

91

Figure 3.1 1: Column Chromatography Apparatus Used in this Experiment.

TLC plate (silica gel 60, F254 aluminium from Macherey- Nagel,

Germany) was used to run the TLC. About two to three spots of the crude

extracts were cautiously applied onto a thin layer chromatographic plate

(coated with silica) and allowed to dry. Hexane and ethyl acetate (8:2,

7:3, 6:4, etc.) were used in the TLC chamber (except otherwise stated).

After about 4 min, the plate was dipped in a suitable solvent that enables

the compounds in the spot to travel upwards by capillary attraction. The

plate was then removed from the solvent and allowed to dry. The

positions of different chromatograms were observed by fluorescence

under UV-light (both short and long 254 nm and 365 nm) followed by

dipping in a molybdate stain, a Ryobi heating gun (HG 2000) was used to

supply heat unto the TLC plate for color development. The extracts

showing plain resolved bands were subjected to bioassay for detection of

antiplasmodial and antitrypanosomal active spots in the various crude

extracts.

92

1H, 13C and all 1D NMR spectroscopy were recorded by means of a 500

MHz Bruker spectrometer at the University of Johannesburg, Chemistry

Department, Auckland Park. All the spectra were recorded at room

temperature using deuteriochloroform (CDCl3). 25 mg of the suspected

pure compounds were dissolved in 1 mL of a deuterated (CDCl3) solvent,

it was then filtered through a Pasteur pipette equipped with a cotton wool

plug so as to discharge into a 5 mm NMR tubes which were labelled

visibly with a concentric label. The rationale behind the filtration was to

get rid of any undissolved particulates or dust from the solution, which

can affect the resolution and the line shapes of the NMR spectra. All

spectra were referenced according to the central line of deuterated

chloroform at δH 7.24 for 1H NMR spectra and δC 77.20 for 13C NMR

spectra. All the spectra were analyzed and the results obtained from it

were compared with published information in literature, so as to elucidate

the structures of the isolated compounds.

The IR spectrum of the crude sample was derived and documented

using a Perkin-Elmer Spectrum. FT IR spectrophotometer Samples were

adjusted against an air background and samples were directly positioned

on the window and then analyzed.

3.10 Synthesis of Silver Nanoparticles

Exactly 12.5 mL each of the extracts (21.2 mg/mL) was added to 90

mL of 1 mM solution of silver nitrate salt (AgNO3). The mixtures in conical

flasks were kept under continuous stirring condition (10 rpm) for about 5

h at room temperature with the reaction vessel covered with aluminium

93

foil to avoid auto-reduction of the AgNO3 due to photosensitivity. The

initial color when the flower, leaf and seed extracts were added to the

aqueous AgNO3 solution were ox-blood, pale cream and cream

respectively. The appearance of darkish-brown, reddish-brown and deep-

brown coloration confirmed the formation of various nanoparticles. The

resultant mixtures were centrifuged at 15000 rpm for 15 min at room

temperature; the pellet obtained was washed twice with distilled water

and air dried.

3.11 Synthesis of Gold Nanoparticles

The production of gold nanoparticles was achieved by adding 12.5 mL

of the plant extract to 90mL of gold (III) chloride solution (0.001 M), the

mixture was incubated for 6 h with continuous stirring. After the

expiration of 6 h, the mixture was centrifuged at 15000 rpm for 15 min at

room temperature; the pellet obtained was washed twice with distilled

water and air dried.

3.12 Characterization

The synthesized materials were characterized with a number of

techniques to ascertain the composition, structure and morphology of the

materials. Bruker D8 advanced X-ray diffractometer was used to

determine the crystallinity and size of the materials. Perkin-Elmer

Universal ATR 100 Fourier transform infra-red spectrophotometer (FT-IR)

was employed to observe the vibrations of the samples, while scanning

electron microscope and electron diffraction spectrophotometer images

were obtained using JOEL JSM-6390 LVSEM. SEM and EDS were used to

94

ascertain the morphology and composition of the materials, whereas

transmission electron microscope images were recorded using JOEL 1210

transmission electron microscope at 100 kV accelerating voltage. This is

required to have information on the shape and size of the synthesized

materials. UV-Visible spectra were obtained with Perkin-Elmer Universal

absorption spectrophotometer.

3.13 In vitro Antioxidant Action

DPPH assay: The radical scavenging and antioxidant activities of the

oils from both the leaves and flowers of the plant were evaluated against

the free radical DPPH. Five different concentrations (0.025-0.40 mg mL-1)

of the oils and commercial antioxidants (β-carotene and vitamin C) were

incubated with a DMSO solution of DPPH for about 30 min at ambient

temperature in the dark. The mixture was shaken thoroughly with a

vortex machine and the absorbance was taken at 517 nm. The volatile oil

ability to scavenge DPPH free radical was calculated using the equation

below.

% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑣𝑜

𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙× 100

Where Acontrol is the absorbance of DPPH+DMSO; and Avo is the absorbance

of DPPH with volatile oil or the commercial antioxidant. The dose response

curve was plotted and the IC50 value of the commercial antioxidant and

volatile oil was calculated (Okoh et al., 2011).

ABTS assay. The modified method of Witayapen (Nantitanon et al.,

2007) was used to evaluate the ABTS activity of the volatile oil extracts.

95

The working solution was obtained by oxidation of ABTS stock solution (7

mM) with 2.4 mM of potassium persulfate in equivalent amounts and the

mixture was permitted to react for 12 h at 25oC. A portion (1 mL) of the

resultant solution was further diluted using 60 mL of methanol to obtain

an absorbance of 0.706 ± 0.001 at 734 nm after 7 min using a UV-

spectrophotometer. Summarily, five different concentrations (0.025, 0.05,

0.1, 0.2 and 0.4 mg mL-1) of each of the volatile oils were mixed with

methanolic solution of ABTS for 7 minutes at 25oC in the dark. The

absorbance was then measured spectrophotometrically at 734 nm and the

% inhibition of ABTS radical by the volatile oils and commercial

antioxidants (β-carotene and vitamin C) was calculated using the equation

described for DPPH assay above.

3.14 In vitro Antibacterial Action

Antibacterial activity of the volatile oil was tested by means of the agar

well diffusion method (Collins et al., 2004). The microbial cultures used in

this study were inoculated in nutrient broth (Oxoid) and incubated for 24

h at 37 ± 0.1oC. Sufficient amount of Muller Hilton Agar (Oxoid) was

poured into sterile petri dishes and permitted to solidify under aseptic

situation. Using a sterilized cork borer, five 6 mm diameter wells were

evenly distributed in freshly prepared and solidified Mueller Hilton agar

(Oxoid) in petri dishes. The bacterial culture was adjusted to 0.5 Mc

Farland turbidity standard and the test microbes (0.1 mL) were inoculated

with a germ-free swab on the exterior of the appropriate solid medium in

each of the petri dishes. Varying concentrations of the volatile oil made

96

from the stock ranging from 0.04 to 0.025 mg mL-1 were prepared and

introduced into each of the wells and labelled appropriately. The

inoculated petri dishes were incubated at 37oC for 24 h. All the petri

dishes were then examined for zones of growth inhibition surrounding the

individual wells and the average diameter of these zones was measured in

millimetres. All tests were performed under hygienic conditions.

3.15 Qualitative Phytochemical Screening

The ethyl acetate extract of the plant was subjected to examination for

the detection of phytochemical compounds by employing the published

procedures (Evans, 2006; Harborne, 2007). The qualitative detection of

various phytochemicals was carried out by using Mayer's and Wagner‟s

reagents (alkaloids). Other tests carried out include the modified Born-

Trager and Keller-Killiani tests for glycosides, foam test for saponins,

Salkowski and Liebermann-Burchard tests for steroids and triterpenoids,

stain test for fats and oils, ferric chloride test for phenols and tannins and

lead acetate test for flavonoids.

3.16 Preparation of McFarland Turbidity Standard

McFarland turbidity standard used in this study is in accord with the

published procedure (Cheesbrough, 2005). 1% v/v solution of sulfuric

acid was prepared by adding 1 mL of concentrated sulfuric acid to 99 mL

of water, shaken carefully, while the solution of barium chloride (1% w/v)

was prepared by dissolving 0.5 g of BaCl2⋅2H2O in 50 mL of distilled water.

McFarland turbid solution was therefore obtained by adding a portion (0.6

97

mL) of the 1% barium chloride solution prepared to 99.4 mL of the 1%

sulfuric acid solution. The solution was transferred to a screw-cup bottle

and preserved until needed for analysis.

3.17 Quantification of Total Phenolic Content

The overall phenolic content of the leaves‟ volatile oil was determined

by the procedure of Folin-Ciocateau. Exactly 1 mL of Folin-Ciocateau

reagent was mixed with 1 mL aliquot of the volatile oil and 46 mL of

distilled water. After 3 minutes 3 mL of (2% w/v) of Na2CO3 solution was

added to the mixture, shaken and incubated for 2 h in the dark.

Absorbance of the resulting mixture was taken on a UV-visible

spectrophotometer at 760 nm against the blank and the overall phenolic

content was presented as gallic acid equivalent in µg mg-1 (Govindappa

and Poojashri, 2011).

The phenolic content of the extracts was examined via a

spectrophotometric method (Kim et al., 2003). About 1 mg/mL of each

extract in 1 mL of solvent was added to 1 mL of Folin-Ciocalteau reagent

in different test-tubes and the mixture was left for about 4 minutes, then

10 mL of 7% Na2CO3 solution and 13 mL of deionized distilled water were

added to the above mixture. The tubes were vortex-mixed for about 25

seconds and kept in the dark at 25oC for colour development, after which

the absorbance was read at 750 nm. The analyses were carried out in

triplicate and the results were expressed as mg of gallic acid

equivalent/100 g of gallic acid using a prepared calibration curve with

linear equation as presented below.

98

y = 0.009x + 0.012 (R2= 0.999)

Here x is the concentration and y is the Gallic acid equivalent.

3.18 Quantification of Total Tannin Content

Tannin determination was evaluated according to the method

described (Van Buren and Robinson, 1999) with minor alteration (Kaur

and Arora, 2009) using tannic acid as a standard. 250 mg of the extract

was added to 50 mL of distilled water in a conical flask. The mixture was

agitated for 1 h using a mechanical shaker, filtered into a 50-mL

volumetric flask and made up to the final volume by addition of distilled

water. An aliquot (1 mL) of the filtrate was mixed with 4 mL of distilled

water and treated with 2 mL (10-fold dilution) of 0.1 M FeCl3 in 0.1 M HCl

and 0.008 M potassium ferrocyanide. The resultant solution was mixed

thoroughly and allowed to stay for 10 min; the absorbance was measured

at 605 nm against the blank. The quantification was carried out based on

the 7-point standard calibration curve of tannic acid (20, 40, 60, 80, 100,

140, 200 mg/L) in distilled water. The tannin content was expressed as

tannic acid equivalent in mg per 100 g of dry material.

3.19 Quantification of Total Flavonoids Content

The method (Ordonez et al., 2006) was employed to determine the total

flavonoids content. About 0.5 mL of 2% AlCl3 in ethanol solution was

mixed with 0.5 mL of the extracts and kept at room temperature for 1 h.

Absorbance was measured at 420 nm and the flavonoids content was

expressed as mg rutin equivalent/100g of rutin using the equation below.

y= 0.023x + 0.022 (R2= 0.982)

99

Here x is the concentration and y is the Rutin equivalent.

3.20 Quantification of Total Flavonols Content

Evaluation of the total flavonols content in the leave extracts was done

in accord with the method (Kumaran and Karunakarah, 2007), in which

2.0 mL of the sample, 3.0 mL of sodium acetate (50 g/L) and 2.0 mL of

aluminium trichloride prepared in ethanol were mixed together. The

absorbance of the mixture was measured at 440 nm after 2.5 h at 20oC.

Total flavonols content was then calculated as mg quercetin equivalent/

100 g of quercetin from the calibration curve using the equation:

y = 0.003 x - 0.003 (R2 = 0.998)

Here x is the concentration and y is the quercetin equivalent.

3.21 Phosphomolybdate Assay

The total antioxidant ability of C. citrinus extracts was examined by

phosphomolybdate method with ascorbic acid as a standard

(Umamaheswari and Chatterrjee, 2008). A portion (0.1 mL) of the

sample extracts was mixed with 1 mL of a reagent solution containing 0.6

M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium

molybdate. Different tubes containing the mixture were covered with an

aluminium foil and incubated in a water bath at 95°C for 90 min. The

test-tubes were brought out of the water bath and allowed to cool to

ambient temperature; the absorbance of the mixture was then measured

at 765 nm against the blank, using ascorbic acid as the standard.

100

3.22 Pilot Phytochemical Tests

Preliminary screenings of the phytochemical compounds present in the

five crude extracts of Callistemon citrinus and likely group of

antiplasmodial and antitrypanosomal active components were conducted

by using standard methods (Kamal, 2014; Madhukar, 2013).

Tannins (Braymer‟s test) 2 mL extract + 2 mL H2O + 2-3 drops FeCl3

(5%). Green precipitate indicates the presence of tannin.

Flavonoids 1 mL extract + 1 mL Pb (OAc)4 (10%). Yellow coloration

indicates the presence of flavonoids

Terpenoids 2 mL extract + 2 mLl (CH3CO)2O + 2-3 drops conc. H2SO4.

Deep red coloration indicates the presence of terpenoids

Saponins (foam test) (a) 5 mL extract + 5 mL H2O + heat. Froth

appears. (b) 5 mL extract + olive oil (few drops). Emulsion forms.

Steroids (Salkowski test) 2 mL extract + 2 mL CHCl3 + 2 mL H2SO4

(conc.). Reddish-brown ring at the junction indicates the presence of

steroids.

Glycosides (Liebermann‟s test) 2 mL extract + 2 mL CHCl3 + 2 mL

CH3COOH. Yellow coloration indicates the presence of glycosides.

Alkaloids (Hager‟s Test) 2 mL extract + few drops of Hager‟s reagent.

Yellow precipitate indicates the presence of alkaloids.

101

3.23 Time Kill Assay

Determination of the rate of kill was conducted according to the known

method (Okoli and Iroegbu, 2005). This was aimed at determining the

minimum time required to completely kill the tested microbial strains

used in this study under laboratory conditions (in vitro analysis). The

tested bacterial strains were adjusted to McFarland 0.5 turbidity standard

and 0.1 mg mL-1 of the ethyl acetate crude extract was prepared as well

by dissolving 800 mg in 10 mL of distilled water. Each of the tested

bacterial strains was inoculated into separate micro tubes for 0, 3, 6, 9,

12, 15, 18 and 24 h, respectively in a rotary shaker at 120 rpm. After

each of the incubation periods (h), the microbial suspension inoculated in

the presence of the ethyl acetate crude extract was then inoculated onto

Mueller Hinton agar in petri dishes and incubated for 24 h at 37oC. The

petri dishes were labelled against the period of incubation in the micro-

tubes and the tested isolates. The experiment was conducted under strict

aseptic conditions in other to avoid contaminations, as this will

compromise the results of the time kill assay. A control experiment was

set up without inoculating the microbial strain suspension in ethyl acetate

extract prior to inoculation on Mueller-Hinton agar. The time kill assay of

each of the isolates was determined from the petri dishes showing no

growth after inoculation from the micro test tubes suspensions containing

ethyl acetate crude extract (0.1mg mL-1). The petri dishes that showed no

growth were recorded as the minimum time (time kill for that particular

102

organism) required to completely eliminate the bacterial strain by the

antimicrobial agent (ethyl acetate crude extract).

3.24 Plasmodium falciparum Culture and Maintenance

The malaria parasites (Plasmodium falciparum strain 3D7) were

preserved in RPMI 1640 medium containing 2 mM L-glutamine and 25 mM

hepes (Lonza). The medium was further supplemented with 5% albumax

II, 20 mM glucose, 0.65 mM hypoxanthine, 60 μg/mL gentamycin and 2-

4% hematocrit human red blood cells. The parasites were cultured at

37oC under an atmosphere of 5% CO2, 5% O2, 90% N2 in sealed in T25 or

T75 culture flasks. Parasitaemia (the concentration of parasites in the

culture) was measured by light microscopy of giemsa-stained thin blood

smears.

3.25 Antiplasmodial Activity

Parasite viability was measured using parasite lactate dehydrogenase

(pLDH) activity according to the described method (Makler et al., 1993).

Chloroquine (Sigma Aldrich) or artemisinin (Sigma Aldrich) was used as

positive controls. Plasmodium falciparum strain 3D7 from Malaria

parasites were maintained in RPMI 1640 medium containing 2 mM L-

glutamine and 25 mM hepes (Lonza). The medium was further

supplemented with 5% albumax II, 20 mM glucose, 0.65 mM

hypoxanthine, 60 μg/mL gentamycin and 2-4% hematocrit human red

blood cells. The condition for the parasites culture was 37oC under an

atmosphere of 5% CO2, 5% O2, 90% N2 in sealed T25 or T75 culture

flasks.

103

Screening of the samples against malaria parasites, which were added

to the parasite cultures in 96-well plate and incubated for 48 h in a 37oC

CO2 incubator, was carried out at a concentration of 50 μg/mL. After the

expiration of 48 h, the plate was removed from the incubator. 20 μL of

the culture was removed from each well and added to 125 μL of a mixture

of Malstat and NBT/PES solutions in a fresh 96-well plate. These solutions

were used to determine the activity of the parasite lactate dehydrogenase

(pLDH) enzyme in the cultures. A purple product was formed when pLDH

was present, and this product could be quantified in a 96-well plate

reader at an absorbance of 620 nm (Abs620). The Abs620 reading in each

well was thus a sign of the pLDH activity and number of parasites in that

well.

3.26 Antitrypanosomal Activity

The main cause of African sleeping sickness in human (human African

trypanosomiasis and nagana (animal African trypanosomiasis) in cattle is

Trypanosoma brucei (T. b.) parasites. The subspecies accountable for

Nagana (T. b. brucei) rarely affects humans and is commonly used for

drug screening. To evaluate anti-trypanocidal activity, the synthesized

nanoparticles were added to in vitro cultures of T. b. brucei in 96-well

plate at a fixed concentration of 50 μg/mL. The mixture was incubated for

about 48 h and the number of parasites that can survive the drug

exposure was determined by adding a resazurin-based reagent, this

reagent was reduced to resorufin by living cells. Resorufin is a fluorophore

(Excitation560/Emission590) and can thus be quantified in a multi-well

104

fluorescence plate reader. The results obtained were expressed as %

parasite viability and this was achieved by comparing resorufin

fluorescence in compound-treated wells relation to untreated controls.

The test was carried out in duplicate wells, and standard deviation (SD)

was also calculated. By and large, extracts that decreased parasite

viability to < 10-20% were considered for additional testing (e.g. dose-

response and cytotoxicity assays). Pentamidine (an existing drug

treatment for trypanosomiasis) was used as a positive control drug

standard.

3.27 Single Concentration Screening

The single concentration approach was adopted where the compound

of interest was added to the parasites and incubated for 48 h. This was a

fast way to verify the anti-malarial activity (if any) of the compound of

interest at a particular concentration and mainly suitable when large

numbers of samples were needed to be screened. As a rule of thumb, if a

compound does not reduce parasite numbers by >80% at 10 μM (for pure

compounds) or 50 μg/mL (for natural extracts), it is improbable to have a

promising anti-malarial activity. For each test sample concentration, the

cell viability or percentage parasitaemia was calculated. The test was

carried out in triplicate wells and standard deviation (SD) was derived.

The % parasitaemia of the extracts were compared with any of these

standard drugs: chloroquine (an anti-malarial drug) or emetine (which

induced cell apoptosis) or pentamidine (an existing drug used in the

treatment of trypanosomiasis) depending on the type of assay conducted.

105

3.28 Dose Response

This assay was carried out to determine the IC50 concentrations of the

compounds (50% inhibitory concentration, or the concentration of the

compound needed to kill 50% of the parasites in a culture). As a general

rule, a compound with an IC50 < 1 μM can be considered a promising

anti-malarial, while an IC50 < 0.1 μM is very good. A standard drug like

chloroquine or artemisinin with IC50 values of approx. 0.02 μM is used for

comparison. For natural extracts an IC50 values ≤ 20 μg/mL are

promising and < 1 μg/mL very good. For each sample, percentage

viability was plotted against logarithm of extract concentration and the

IC50 (50% inhibitory concentration) resolved from the resulting dose-

response curve by non-linear regression using Prism 5 for Windows,

Version 5.02 (Graph Pad Software, Inc.) program. Chloroquine,

pentamidine, or emetine was used as positive standards drugs based on

the type of a test carried out. Chloroquine, pentamidine and emetine gave

IC50 values in the range of 0.00001–100 μM. The samples were tested in

concentration range of 250 to 0.11 μg/mL (3-fold-dilutions) for

antitrypanosomal/antiplasmodial and from 125 to 0.057156 μg/mL (also

in a three-fold dilution series) for cytotoxic assays.

3.29 Cytotoxicity Assay

The overt cytotoxicity of the synthesized nanoparticles was estimated

against Hela (human cervix adenocarcinoma) (Keusch et al., 1972). Stock

solutions of the nanoparticles (20 mg/mL) were prepared in DMSO and

later diluted with culture medium to 50 μg/mL and were incubated in 96-

106

well plate containing Hela (human cervix adenocarcinoma) cells for about

48 h. The amounts of cells that were able to outlive exposure to the drugs

were also established via resazurin-based reagent and reading resorufin

fluorescence in a multi-well plate reader. The obtained results were

articulated as % viability (the resorufin fluorescence in compound-treated

wells compared to untreated controls). This test was carried out in

duplicate wells and standard deviation was generated for the targeted

compounds. The results for the cytotoxicity assay were also expressed as

% cell viability (obtained from fluorescence reading in treated wells

versus untreated control wells). Emetine (which induces cell apoptosis)

was employed as a positive standard drug.

3.30 Statistical Analysis

Statistical analyses were carried out using Microsoft Excel 2007.

Alternatively, analysis of data was done using original software in the

statistical computing system. This software put into consideration the

adjustment of the regression coefficient square, R2 (Ojemaye et al.,

2017a).

107

Chapter 4

Results and Discussion

4.1 Composition, Antibacterial and Antioxidant Activity of

Essential Oils of Callistemon citrinus from Eastern Cape Province

of South Africa

4.1.1 Abstract

The essential constituents of the volatile oils obtained from the aerial

parts of the plant as well as their antioxidant activity, free radical

scavenging, phenolic content, and the antibacterial potential of the oils

are examined.

4.1.2 Background

Volatile oils are complex and of natural origin having a strong odor,

they are usually formed from odoriferous medicinal plants, apart from

volatile oil, aromatic plants possess phenolic compounds with numerous

pharmacological activities. The study of the constituents of therapeutic

plants is of immense significance needed for the production of novel drugs

(Yadav and Agarwala, 2011). Eucalyptol (a cyclic monoterpenoid ether)

has been found to be the dominant constituent of this plant with various

degrees of pharmacological effects and hence it is used as a marker for

medicinal essential oil classification (Sadlon and Lamson, 2010).

However, there is dearth of information on the chemical profile of the

volatile oils of Callistemon citrinus obtained from the flowers and stems,

and thus the present study was undertaken with the aim of investigating

the essential constituents of its volatile oil as well as its antioxidant, free

108

radical scavenging, phenolic content and the antibacterial potentials of

both the leaves and flowers of Callistemon citrinus, respectively from

Eastern Cape Province of South Africa.

4.1.3 Constituents of the Volatile Oil

Hydro distillation of the leaves, flowers and stems of Callistemon

citrinus produced a clear light, pale yellow and light oil with percentage

yields of 0.70% (leaves), 0.80% (flowers) and 0.50% (stems) v/w of the

wet samples. The identified components, retention times and percentage

compositions of the chemical compounds are given in Table 4.1. From the

table it is obvious that qualitative and quantitative constituents of the oils

differ from each other.

Table 4.1: Fractional Composition of Constituents of the Leaves, Flowers and Stems oils of Callistemon citrinus.

Retention time Constituents Oil composition

Leaves Flowers Seeds

3.43 3.89 3.97 3.98 4.12 4.25 4.29 4.34 4.52 4.69 4.73 4.77 4.97 5.21 5.23 5.44 5.67 5.73 5.76 5.85 5.88 5.94

Isopentyl acetate β-Thujene α-Pinene

1R-α-Pinene Camphene

Vinyl amyl carbinol β-Phellandrene

β-Pinene 3,4-dimethylheptane

o-Cymene Limonene Eucalyptol

γ-Terpinene 3-Carene β-linalool α-Fenchol

Pinocarveol (+)-2-Bornanone Camphenilanol

Pinocarvone Borneol

Terpinen-4-ol

0.17 -

20.02 -

0.63 - -

1.10 0.81

- -

48.98 0.21 0.17 0.57 0.93 5.75

- 0.27 2.81

- 0.79

- 0.65

- 4.57 3.81 0.14 0.37 2.26

- 1.22

- 8.11 1.76 0.05

- - -

4.35 - -

1.75 0.77

- -

31.03 - - - -

1.18 - -

3.97 56.00

- -

1.08 -

0.51 - - - -

0.77

109

6.03 6.09 6.22 6.23 6.30

6.42 6.75 7.11

7.23 7.42 7.77 7.89 7.93 7.98 8.03 8.08 8.16 8.25 8.36 8.52 8.65 8.76 8.80 8.82 8.86 8.87 8.91 8.96

9.02 9.07 9.11 9.22 9.28

9.38

9.52

9.82

9.93

10.69

α-Terpineol Myrtenol

cis-Carveol (+)-Camphene

cis-p-metha-1(7), 8-diene-2-ol Geraniol

(-)-Bornyl acetate trans -2-Acetoxyl-1, 8-

cineole 4-Carene Copaene

Caryophyllene Aromadendrene

α-Gurjinene Humulene

Alloaromadendrene α-Elemene

α-Farnesene Bicyclogermacrene

(+)-δ-Cadinene Elemol

(+)-Valencene Spathulenol (-)-Globulol (+)-Ledene γ-Gurjinene

(+)-Viridiflorol Rosifoliol

3,4-Dimethoxy-benzoic acid methyl ester

β-Eudesmol γ-Eudesmol

Hinesol α-Eudesmol

cis-1(7),8-p-mentha-diene-2-ol 5-Amino-1-

phenylpyrazole p-Cymene-3-propionic

acid, α-methyl ester 3-Carene, 4-isopropenyl

cis-Calemenene Geranyl-α-Terpinene

8.01 0.29 0.70

- 0.53

0.40

- 0.18

0.76

- - - - - - - - - - - -

0.19 0.39

- 0.20

- -

1.72

0.26 - - - - - - - - -

0.22 - -

0.26 - -

10.02 - -

0.34 11.89 3.80 0.70 1.05 0.89 0.51 0.42 5.71 2.10 0.58 0.37 3.16

- 2.33

- 0.49 0.50

- -

5.21 2.03

12.93 1.05

1.15

0.35

0.34

0.42 0.28

3.69 - - - - - - - - - - - - - - - - - - - -

1.10 0.65

- - - - - - - - - - - - - - -

Total 96.84 98.92 99.98

Hydrocarbons Monoterpene 22.89 15.24 36.18

Oxygenated Monoterpenes 70.01 26.27 62.05

Sesquiterpene Hydrocarbons 0.20 30.53 -

110

Oxygenated Sesquiterpenes 0.84 24.90 1.75

Others 2.88 1.98 -

Twenty-six components for the leaf oil amounting to 96.84%, forty-

one components for the flowers oil representing 98.92% and ten

components for the stems oil amounting to 99.98% were identified in the

three oil samples. The leaves oil was composed of mainly oxygenated

monoterpenes (70.01%) followed by monoterpene hydrocarbons

(22.89%), sesquiterpene hydrocarbon (0.20%), and oxygenated

sesquiterpenes (0.84%). The dominant constituents in the leaf oil

samples were eucalyptol (48.98%), α-pinene (20.02%), α-terpineol

(8.01%) and pinocarveol (5.75%). Other notable components found were

pinocarvone (2.81%) and β-pinene (1.10%). Minor constituents include

α-fenchol (0.93%), terpinen-4-ol (0.79%), camphene (0.63%) and β-

linalool (0.57%). Table 4.1

The flower oil was found rich in sesquiterpene hydrocarbons (30.53%),

followed by oxygenated monoterpenes (26.27%), oxygenated

sesquiterpenes (24.90%) and monoterpene hydrocarbons (15.24%). The

main constituents characterizing the floral oil were α-eudesmol (12.93%),

caryophyllene (11.89%), (-)-bornyl acetate (10.02%), eucalyptol

(8.11%), bicyclogermacrene (5.71%), γ-eudesmol (5.21%) and 1R-α-

Pinene (4.57%). Table 4.1

The stem oil comprises oxygenated monoterpenes (62.05%) and

monoterpene hydrocarbons (36.18%). The key components dominating

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the stem oil were eucalyptol (56.00%), α-pinene (31.03%), limonene

(3.97%) and α-terpineol (3.69%). Table 4.1

The flower volatile oil of our sample has α-Eudesmol as the major

component. It does show high voltage-gate calcium channel blocker

activity, which is a foremost problem in anti-migraine treatment (Asakura

et al., 2000a) and is available to attenuate post-ischemic brain injury in

rats (Asakura et al., 2000b).

Volatile oil of the leaves of Callistemon citrinus in this study showed

similar profile compared to those reported earlier in literature as shown in

Table 4.2 (Oyedeji et al., 2009; Kumar et al., 2015; Zandi-Sohani et al.,

2012; Minar et al., 2014; Chane-Ming et al., 1998; Srivastava et al.,

2003; Riaz and Chaudhary et al., 1990). Eucalyptol was found to be the

main constituent though in varying amounts except for Himalaya where

the dominant component of the leaves oil was α-pinene (32.30%) but the

flowers oil from this region was still rich in eucalyptol. This could be

attributed to environmental factors like variable ecological and climatic

conditions in the regions, as well as the nature of the plant and the

processing method (Ogundajo et al., 2013).

112

Table 4.2: Major Components of Volatile Oil of Callistemon citrinus from

Various Parts of the World

Origin Major Components Reference

South Africa f

α-Eudesmol (12.93%), caryophyllene (11.89%), bornyl-acetate (10.02%),

eucalyptol (8.11%), bicyclogermacrene (5.71%) and α-eudesmol (5.21%)

Larayetan et al., 2017

South Africa l

Eucalyptol (48.98%), α-pinene (20.02%), α-terpineol (8.10%), isopinocarveol (5.75%) and pinocarvone (2.81%)

Larayetan et al., 2017

South Africa s

Eucalyptol (56.00) and α-pinene (31.03). Larayetan et al., 2017

Himalaya f

Eucalyptol (36.6%), α-pinene (29.7%) Kumar et al.,2015

Himalaya l

α-Pinene (32.30%), limonene (13.1%), α-terpineol (14.6%)

Kumar et al.,2015

Iran l Eucalyptol (34.20%), α-pinene (29.0%), α-terpineol (16.70%), α-phellandrene (9.0%)

Zandi et al., 2012

Iran l Eucalyptol (67.60%), α-pinene (9.40%), β-

pinene (4.70%) Minar et al., 2014

Iran f α-Pinene (25.70%), eucalyptol (18.10%), β-

pinene (7.30%), linalool (5.30%). Minar et al., 2014

Iran s Eucalyptol (41.30%), α-pinene (19.10%), α-terpineol (4.10%)

Minar et al., 2014

Reunion Island l Eucalyptol (68.0%), α-pinene (12.80%), α-

terpineol (10.6%) Chane-Ming et al., 1998

Lower Region of Himalaya l

Eucalyptol (66.30%), α-pinene (18.70%) Srivastava et al., 2003

South Africa l

Eucalyptol (61.20%), α-pinene (13.40%), β-pinene (4.70%)

Oyedeji et al., 2009

Pakistan Eucalyptol a, α-terpineol a Riaz and Chaudhary, 1998 l leaves, f flowers, s stems, a quantitative data not available

α-Eudesmol (12.93%), caryophyllene (11.89%) and bornyl acetate

(10.02%) were the dominant components found in the volatile oil of the

flowers of this plant under investigation. Eucalyptol was also notably

present in the flower oil but the content was low (8.11%) compared to

the stems and leaf oils which were (56.00%) and (48.98%) respectively.

113

The oil collected from the flowers shows no homogeneity in constituents

in comparison with others in the literature (Table 4.2). While the flowers

essential oil from Iran showed α-pinene (25.70%) and eucalyptol

(18.10%) as the principal components (Minar et al., 2014), another from

Himalaya recorded eucalyptol (36.60%) and α-pinene (29.70%) as its

foremost components (Kumar et al., 2015). The present study reveals

that α- eudesmol (12.93%), caryophyllene (11.89%) and bornyl acetate

(10.02%) are the major constituents in the flower oil. This might be

related to the effects of several factors like relative humidity, irradiance,

photoperiod, method of extraction, plant cultivation techniques, soil

structure and climate which could greatly influence the composition and

quality of essential oil (Panizzi et al., 1993).

4.1.4 Overall Phenolic Content

The overall phenolic content in the leaves volatile oil was found to be

899.00 µg mg-1 gallic acid equivalents, which was higher than the same

species from India that recorded 261 mg/g (Kumar et al., 2015). This

shows the presence of phenolic compounds such as α-terpineol and

pinocarveol in the leaves‟ oil. Phenolic compounds in plants are known to

increase its antioxidant activity (Tawata et al., 1996). They have a major

responsibility in scavenging free radicals that cause oxidative stress due

to their antioxidant capacity against peroxyl radicals. This helps them

scavenge electrophiles and active oxygen species, limit auto-oxidation by

chelating metal ions and increase the ability to adjust some enzymes

action (Mediani et al., 2013).

114

4.1.5 Antibacterial Activities of the Volatile Oil Obtained from

Leaves and Flowers

The present work examined the in vitro antibacterial activities of the

volatile oils from leaves and flowers of Callistemon citrinus on the four

gram-negative and three gram-positive bacteria as shown in Table 4.3).

The activities of the oils in terms of inhibitory zones were effective on all

the tested bacteria showing that the plant under study has very wide

spectrum of action against both gram-positive and gram-negative

bacteria (Figure 4.1 and 4.2). The inhibitory effect of oils both from the

leaves and flowers was highest against gram-negative bacteria like Vibro

alginolyticus DSM 2171 (67.0±2.0 and 60.0±5.0 mm) and Aeromonas

hydrophila ACC (58.0±0.3 and 52.0±1.0 mm), as well as the gram-

positive bacteria such as Staphylococcal enteritis ACC (62.0±0.5 and

55.0±2.0 mm) at a concentration of 0.4 mg/mL, while the lowest effect

was recorded for Escherichia.coli ATCC 35150 (27.0±3.0 and 20.0±4.0

mm). Oil from both leaves and flowers showed highest inhibitory effect on

gram-negative bacteria which was contrary to some reports published in

literature (Cock, 2012). The antibacterial properties of these volatile oils

were found comparable to those of the volatile oil from leaves of the

Western part of South Africa which gave inhibition zone ranging between

13.3 and 26.3 mm for S. aureus (ATCC 3983), a gram-positive bacteria,

also E.coli (ATCC 4983) and P. aeruginosa (ATCC 7700) which are gram

negative bacteria (Oyedeji et al., 2009). The antibacterial effect observed

in this plant may be linked to some bioactive compounds such as

115

alkaloids, tannins, terpenoids, ether and phenolic compounds like

flavonoids, which are considered to be bacteriostatic and fungistatic

(Okwu et al., 2004; Okwu and Morah, 2007). This effect correlates with

its folkloric uses and shows that it is an efficient antimicrobial plant that

can be employed in alternative medicine for the treatment of bacterial

infection (Doughari et al., 2009).

Table 4.3: Inhibition Zone (mm) Showing Antibacterial Activities of the

Volatile Oils and Ciprofloxacin

Microorganism

Positive control Zone of inhibition of volatile oils and standard drug

Ciprofloxacin (mg /mL) Leaves’ volatile oil (mg/mL) Flowers’ volatile oil(mg/mL)

0.4 0.1 0.025 0.4 0.1 0.025 0.4 0.1 0.025

Gram-negative bacteria

Aeromonas hydrophila

40.0 ± 5.0 32.0 ± 1.0 28.± 0.2 62.0 ±0.5 45.0 ± 0.1 25.0 ± 0.4 55.0 ± 2.0 38.0 ± 0.9 22.0 ± 0.9

Escherichia coli 38.0 ± 4.0 32.0 ± 4.0 24.0 ± 0.9 27.0 ± 3.0 18.0 ± 3.0 13.0 ± 3.0 20.0 ± 4.0 12.0 ± 4.0 10.0 ± 3.0

Vibro alginolyticus

33.0 ± 2.0 29.0 ± 2.0 23.0 ± 0.4 67.0 ± 2.0 48.0 ± 5.0 28.0 ± 4.0 60.0 ± 5.0 42.0 ± 0.8 23.0 ± 1.0

Salmonella typhi

40.0 ± 1.0 34.0 ± 0.6 28.0 ± 3.0 53.0 ± 0.5 40.0± l.0 20.0 ± 2.0 48.0 ± 0.5 35.0 ± 2.0 14.0 ± 0.5

Gram-positive bacteria

Staphylococcal enteritis

40.0 ± 5.0 32.0 ± 1.0 28.0 ± 0.2 62.0 ± 0.5 45.0 ± 0.1 25.0 ± 0.4 55.0 ± 2.0 38.0 ± 0.9 22.0 ± 0.9

Staphylococcus aureus

29.0 ± 2.0 24.0 ± 0.0 17.0 ± 2.0 54.0 ± 4.0 26.0 ± 1.0 18.0 ± 2.0 53.0 ± 3.0 24.0 ± 5.0 16.0 ± 2.0

Listeria monocytogenes

44.0 ± 6.0 38.0 ± 0.2 32.0 ± 1.0 54.0 ± 2.0 42.0 ± 2.0 21.0 ± 1.0 50.0 ± 2.0 39.0 ± 3.0 18.0 ± 3.0

Zone of inhibition (millimeter), ACC: Aemreg Culture Collection, ATCC:

American Type Collection Center, values are mean ± SD, n = 3

116

Figure 4:1: Antibacterial Activity of Leaves‟ Oil of Callistemon citrinus.

Figure 4.2: Antibacterial Activity of Flower Oil of Callistemon citrinus.

4.1.6 In vitro Antioxidant Action

The in vitro antioxidant activities of the volatile oils were evaluated

using DPPH radical scavenging test. The violet color production of DPPH

dissolved in DMSO is due to its unpaired nitrogen electrons. The DPPH

radical is in the process reduced to DPPH-H, turning from violet to yellow

in the presence of antioxidant compound (Edziri et al., 2012)

117

The inhibition of the DPPH radical by the volatile oil of the leaves and

flowers was concentration-dependent. The inhibition percentage of the

volatile oils at different concentrations (0.025, 0.05, 0.1, 0.2, 0.4 mg

mL-1) ranged between 38.3% and 76.2% for the leaves oil and from

40.7% to 80.6% for the flowers oil. The percentage of inhibition for both

the ascorbic acid and β-carotene varied as 18.1%–54.04% and 32.4% –

77.45% respectively (Figure 4.3 and 4.4). The leaves and the flowers oils

were capable of reducing the DPPH radical by 50% with IC50 of 1.49 and

1.13 mg mL-1 compared to β-carotene and ascorbic acid which have an

IC50 of 1.28 and 3.57 mg mL-1 (Table 4.4). The capacity of the DPPH

radical scavenging of the flowers oil in terms of percentage inhibition and

IC50 was higher than those of the leaves oil and the two synthetic

antioxidant drugs (Figure 4.4). The inhibition of DPPH free radical by both

the leaves and flowers oils were higher than that reported for the volatile

oil of same species in Iran (Minar et al., 2014). For the ABTS assay, the

essential oils collected from the leaves and flowers of the plant under

study showed free radical scavenging activities which were dose-

dependent, having a maximum activity of 79.47% at 0.4 mg mL-1 for the

leaves‟ oil and 95.61% for the flowers‟ oils (Figure 4.4). The oils from

both plant parts showed a 50% reduction of 0.14 and 0.03 mg mL-1

respectively which implies that the flowers‟ oil possesses higher

antioxidant capacity than the leaves oil and other typical antioxidants

(vitamin C & BHT) with IC50 of 0.13 and 0.19 respectively (Table 4.4).

The high scavenging activity of the leaves oil over BHT (standard

118

antioxidant) could be due to the high content of eucalyptol in the leaves‟

oil (Mimica-Dukic et al., 2003).

Figure 4.3: ABTS Scavenging Action.

Table 4.4: IC50 Profile of the Leaves and Flowers‟ Oil of Callistemon

citrinus (mg /mL)

S/N Activity

Callistemon citrinus Standard antioxidant (positive control)

Leaves’ oil (IC50)

Flowers’ oil (IC50)

Vitamin C (IC50)

β-Carotene (IC50)

BHT (IC50)

1 DPPH 1.49 1.13 3.57 1.28 -

2 ABTS 0.14 0.03 0.13 - 0.19

Figure 4.4: DPPH Scavenging Action.

The antioxidant activities of volatile oils are reportedly not only due to

phenolic content of the oil but also some other constituents like

119

monoterpene alcohols, ketones, aldehydes, hydrocarbons and ethers,

which are known to contribute to the free radical scavenging activity of

some volatile oil (Edris, 2007). Volatile oils of Thymus caespititus, Thyme

camphorates, and Thyme mastichina, which contain high contents of

linalool and eucalyptol, have high antioxidant activities almost equal to

those of α-tocopherol (Migue et al., 2004). Similarly, the M. aquatic high

scavenging activity was due to the presence of eucalyptol in the volatile

oil (Mimica-Dukic et al., 2003). The essential oil of the plant in the

present study also showed high level of eucalyptol (monoterpenoid ether)

which might be responsible for its antioxidant activity.

4.1.7 Conclusion

This study represents the first analyses of the volatile constituents of

the essential oils from Callistemon citrinus leaves, flowers and stems to

the best of our knowledge in the Eastern Province of South Africa. Aside

from the traditional uses of the extract of the plant, its volatile oil

possesses high-quality antioxidant potential and may possibly compete

well with synthetic antioxidant drugs in the market. Observation drawn

from this experiment shows clearly that the leaves and flowers of

Callistemon citrinus possess substantial quantity of the phenolic

compounds and cyclic ethers with several pharmacological patterns. The

present investigation showed that the studied plant is a good traditional

herb of potential value for the cure of various ailments.

120

4.2 Effect of Seasonal Variation on the Secondary Metabolites

and Antioxidant Activity of Callistemon citrinus

4.2.1 Abstract

A relationship between the chemical disparity observed in the volatile

oil constituents, antioxidant capacity, percentage yield of the oil of

Callistemon citrinus and the fluctuation in season taking place yearly has

been established.

4.2.2 Background

Majority of consumers all over the world still depend on medicinal

plants as a way out of the health problems militating against them

(Verma and Singh, 2008). Callistemon citrinus (Curtis) Skeels has been

naturalized in South Africa and it is found grown in almost every province

of the country. The plant possesses abundant of polyphenols in its

essential oil and organic extracts. Volatile oils are made up many

lipophilic and extremely volatile constituents like terpenoids (mono-, bi-,

tricyclic mono-, and sesquiterpenoids), small chain hydrocarbons and

phenylpropanoids obtained from vast varieties of chemical groups of

compounds recognized to be predisposed to alteration and degradation. If

the defensive compartmentation in the plant matrix is deprived, volatile

components would be made prone to chemical transformation, oxidative

damage and polymerization brought about by heat, light, air, or the

developmental stage of the plant which could lead to disparity in the

chemical constituents of the oil (Miguel et al., 2004). Also due to

structural organization common in the same chemical clusters,

121

components of volatile oils have been recognized to simply change into

each other by cyclization, isomerization, oxidation, dehydration, and

dehydrogenation brought about by chemical or enzymatic reactions.

Constituents of volatile oil undergo chemical transformation during the

course of growth or during oil distillation, thus probing the legitimacy of

the genuineness of the oil. Aromatic and medicinal plants exhibit changes

in their active components at different seasons of the year (autumn,

spring, summer and winter), which could be as a result of the fluctuation

of different environmental variables like temperatures and rainfall (Ahmad

et al., 2011; Szakiel et al., 2011).

Research conducted on Pelargonium graveolens leaves gave the best

yield and maximum geraniol content of (29.87%) in winter (Mittal et al.,

2013). Similarly, spathulenol and caryophyllene oxide which are the

major components of Eugenia uniflora leaves were found higher in the oil

obtained from the leaves in dry seasons (April- September) as a result of

the biotic pressure that is capable of altering the plant volatiles (Da Silva

et al., 2012). The volatile oil of Melissa officinalis exhibited disparity in

percentage yield monthly possibly because of the influence of the sun and

shade in the microenvironment where the plants grow (Saeb and

Gholamrezaee, 2012). Higher percentages of (2.5 and 1.95 %) were

obtained in the extraction of the volatile oils from Eucalyptu.

camadulensis and Eucalyptus cinera respectively during summer season.

This may be linked with the physical and chemical strain encountered by

plants during the period, in which secretion to various defense

122

components of the secondary metabolite particularly the

terpenes/terpenoids compounds occurs (Soni et al., 2015). Mentha

canadensis yielded the maximum menthol content (5.3%) in February

and lowest in May (3.5%) owing to the plant ontogeny, environmental

regulation and seasonal fluctuation which affects the genetic expression of

oil production (Del Carmen et al., 2013; Kooke and Keurentjes, 2012),

likewise thymol which is the major component of Origanum syriacum was

the highest in summer (46.70%) while p-cymene was the highest in early

spring (62.18%) (Toncer et al., 2010).This development was attributed to

the long photoperiods which could be responsible for the increase in

volatile oil of the foliage and phenolic monoterpenes in the oil (Fischer et

al., 2011; Regnault-Roger et al., 2012).

To circumvent variations in oil components with respect to fluctuation

in seasons, all collected leaves were obtained from fully established plants

with no visual damage. Although, a number of researchers have

documented disparity in the chemical constituents of volatile oils with

respect to their origin, environmental circumstances, and the

developmental phase of the collected plant materials, there is a dearth of

information on the seasonal fluctuations of the chemical constituents,

antioxidant capacity and percentage yield of oil of Callistemon citrinus.

Our aim was to ascertain if there were a relationship between the

chemical disparity of the volatile oil constituents, antioxidant capacity,

percentage yield of the oil of Callistemon citrinus and the fluctuation in

season taking place yearly.

123

4.2.3 Chemical Composition of the Volatile Oil from Callistemon

citrinus (January-December, 2016)

Hydrodistillation of the fresh leaves of Callistemon citrinus gave a pale

yellow volatile oil with a strong fragrance; about ninety-seven

components were identified in the twelve treatments analyzed each

month for a period of one year. The results show that the content of the

essential oil varied throughout the months and seasons of the year (Table

4.5). The key components were pinocarvone (1.25-6.17%), pinocarveol

(0.10-9.56%), α-terpineol (24-9.94%), α-pinene (7.45-22.75%),

limonene (24.08), and eucalyptol (14.69-72.35%) (Figure 4.5), the

compositional profile of the leaves of Callistemon citrinus (January-

December) treatments under investigation revealed marked qualitative

and quantitative differences. The oils of the twelve treatments were rich

in monoterpene hydrocarbon (8.45-63.74%), oxygenated monoterpenes

(13.37-84.86%), sesquiterpene hydrocarbons (0.03-7.95%), oxygenated

sesquiterpenes (0.43-8.77%), diterpenes (0.08-0.31%), esters (0.35-

5.73%), and alcohols (0.03-0.20%). The highest monoterpene

hydrocarbon content (63.74%) was recorded in August (winter period)

while the highest oxygenated monoterpene (84.86%) was obtained in

September (spring). Ester content of the oil had the highest percentage of

(5.73%) in October (spring). Diterpenes are scarcely encountered in

genuine volatile oils obtained through distillation due to their low volatility

but phytol (C20H40O), an acyclic diterpene alcohol was seen in trace

amount (0.08-0.31%) for the first time in the volatile oil of Callistemon

124

citrinus grown in South Africa. Triterpenoids and higher terpenoids like

sterols or carotenoids are only present in the non-volatile extracts such as

plant resins or gums (Crozier et al., 2006).

Table 4.5: Fractional Composition of the Constituents of the Leaves of

Callistemon citrinus (January-December).

Month of Harvest

RT

Jan Feb Mar April May June

Treatment 1 2 3 4 5 6

% Yield 0.27 0.31 0.23 0.25 0.22 0.18

Compounds Percentage Composition of Various Components

3-Hexen-1-ol 3.301 - - 0.03 0.11 - 0.10

Isoamyl acetate 3.436 1.72 0.17 0.71 1.46 0.91 0.66

Methyl 4-methyl, methyl valerate

3.551 - - 0.04 0.06 0.05 0.03

Butyl isobutanoate 3.724 - - - - - -

β-Thujene 3.908 - - 0.36 0.06 0.04 -

α-Pinene 3.986 14.85 20.02 22.75 22.32 13.60 12.39

Camphene 4.177 - 0.63 0.18 0.30 0.63 0.39

Sabinene 4.316 - - 0.11 - - -

β-Pinene 4.382 0.60 1.10 5.87 1.28 2.33 0.96

Heptane-3,4-dimethyl 4.523 - 0.81 - - - -

α-Phellandrene 4.541 - - 0.61 - - -

Terpinolene 4.645 - - 0.34 - - -

4-Hexen-1-ol, acetate 4.674 - - - 0.09 - -

o-Cymene 4.698 0.74 - - - - -

Limonene 4.789 - - - - - -

Eucalyptol 4.795 63.72 48.98 37.17 51.92 27.25 26.43

α-Terpinene - - - - - - -

γ-Terpinene 4.647 - 0.21 0.61 0.23 0.43 0.17

α-Terpinolene - - - - - - 0.18

3-Carene 5.217 - 0.17 - - 1.49 -

4-Carene 5.265 - 0.76 2.61 - - -

Linalool 5.307 0.51 0.57 2.07 0.59 0.83 0.43

α-Pinene oxide 5.386 - - - 0.04 - -

Cis-p-metha-1(7), 8-diene-2-ol

5.446 0.30 0.53 - 0.29 1.20 0.76

α-Fenchol 5.487 0.58 0.93 0.13 0.11 1.25 0.81

Campholenal 5.565 - - - 0.11 0.50 0.26

Cis-P-2,8-Menthadien-1-ol

5.502 - - 0.44 0.03 0.11 0.09

Trans-p-2,8-Menthadien-1-ol

5.523 - - - - 0.18 -

125

α-Fenchene 5.639 - - 0.04 - - -

Pinocarveol 5.679 3.98 5.75 0.10 2.76 6.75 5.29

Pinocarvone 5.755 1.83 2.81 - 1.25 3.76 2.74

Camphenilanol 5.765 - 0.27 - - - -

Terpinen-4-ol 5.976 0.63 0.79 1.31 0.58 1.33 0.72

Myrtenol 6.095 - 0.29 - 0.20 0.76 0.48

α-Terpineol 6.104 6.18 8.01 8.47 5.24 9.94 6.78

Cis-Carveol 6.227 0.44 0.70 - 0.41 1.58 0.95

α-Bergamontene 6.383 - - 1.29 - - -

Nerol 6.421 0.27 - - - - -

D-Carvone 6.429 - - - - - -

Geraniol 6.479 - 0.40 1.60 0.38 0.90 0.46

Citral 6.588 - - - - - -

Longifolene 6.574 - - 0.05 - - -

Carvacrol 6.635 - - - - - 0.04

β-Guaiene 6.683 - - 0.08 - - -

β-Bisabolene 6.753 - - 0.05 - - -

Bornyl acetate 6.761 - - - - 0.26 0.04

3,4-Xylenol 6.766 - - - - - -

Thymol 6.782 - - - - 0.08 -

Methyl nerolate 6.926 - - - 0.10 0.35 -

Methyl geranate 6.932 - - 0.27 - - 0.12

α-Sinensal 7.018 - - - - - -

Adamantan-2-ol 7.022 - - - - 3.53 -

2-Acetoxy-1, 8-cineole 7.123 - 0.18 0.28 0.30 0.64 0.32

α-Farnesene 7.196 - - 0.07 - - -

Neryl acetate 7.197 - - - - - -

Eugenol 7.220 - - - - - -

Geranyl acetate 7.320 - - 0.47 0.07 0.08 0.03

α-Himachalene 7.373 - - 0.03 - - -

Methyl cinnamate 7.418 - - 0.18 0.04 0.11 0.04

Pivarose 7.458 - - - - 0.08 -

β-Phenylethyl acetate 7.478 - - - - - -

α-Santalol 7.640 - - - - 0.10 -

Di-epi-α-cedrene 7.688 - - - - - -

α-Gurjunene 7.689 - - 0.06 - - -

Caryophyllene 7.765 - - 0.54 - - -

Aromandendrene 7.893 - - 0.2 - 0.11 -

Humulene 7.988 - - 0.13 - - -

Alloaromandendrene 8.040 - - 0.11 - 0.31 0.03

α-Elemene 8.077 - - - - - -

Trifluoroacetyl-lavandulol

8.169 - - - - - -

γ-Gurjenene 8.176 - - 0.77 - - -

1,4-dimethyl tetralin 8.178 - - - - - -

Bicyclogermacrene 8.247 - - 0.70 - - -

Germacrene 8.252 - - - - - -

Durohydroquinone 8.322 - - 0.14 0.05 0.07 0.02

126

δ-Cardinene 8.365 - - 0.13 - - -

Epizonarene 8.399 - - - - - -

Elemol 8.503 - - - - 0.07 -

Epiglobulol 8.658 - 0.20 - 0.06 0.24 0.33

Valencene 8.704 - - - - - -

Palustrol 8.721 - - - - 0.24 -

Aromandendr-1-ene 8.723 - - 0.17 - - -

Spathulenol 8.780 - 0.19 0.93 0.14 1.07 -

Globulol 8.831 - 0.39 0.71 0.30 1.56 0.67

Viridiflorol 8.882 - - 0.08 0.14 0.71 -

Rosifoliol 8.902 - - - - 0.90 -

Ledol 8.936 - - - - - -

1-acetyl-2-amino-3-cyano-7-isopropyl-4-

methylazulene 8.996 - - - - 4.70 2.42

β-Caryophylladienol 9.103 - - - - 0.40 -

Viridiflorene 9.247 0.21 - - - - -

Trans-Farnesol 9.440 - - - - - -

Cedran-diol (8S,14) 9.475 - - - - 0.02 -

Eudesma-4,11-dien-2-ol 9.549 - - - - - -

Benzyl benzoate 9.969 - - - - 0.04 -

Farnesol acetate 10.05 - - - - - -

(Z,Z)-α-Farnesene 10.24 - - - - - -

Phytol 11.40 - - - - - -

(%) Totals 96.56 94.86 92.99 91.02 91.49 65.14

Hydrocarbons Monoterpene

16.19 22.89 33.48 24.19 18.52 14.09

Oxygenated Monoterpenes

78.44 70.03 51.29 63.91 59.95 46.24

Sesquiterpene Hydrocarbons

0.21 - 4.38 - 0.82 0.03

Oxygenated Sesquiterpenes

- 0.78 - 0.64 4.89 1.00

Diterpene - - - - - -

Hydrocarbon 0.81 - - - -

Esters 1.72 0.35 1.95 2.03 2.48 1.24

Alcohol - - 0.03 0.2 - 0.10

Aldehyde - - - - -

Others - - 1.85 0.05 4.83 2.44

127

Month of Harvest

RT

July Aug Sept Oct Nov Dec

Treatment 7 8 9 10 11 12

% Yield 0.20 0.19 0.24 0.25 0.25 0.30

Compounds Percentage Composition of Various Components

3-Hexen-1-ol 3.301 0.12 0.03 - 0.06 0.02 0.20

Isoamyl acetate 3.436 0.01 0.05 0.51 1.01 0.58 2.38

Methyl 4-methyl, methyl valerate

3.551 - - - 0.06 0.03 0.12

Butyl isobutanoate 3.724 - - - - - 0.02

β-Thujene 3.908 0.46 3.29 - - - 0.03

α-Pinene 3.986 9.44 10.83 7.45 9.88 14.85 10.77

Camphene 4.177 - 0.16 0.20 0.30 0.48 0.69

Sabinene 4.316 - 0.47 - 0.16 - -

β-Pinene 4.382 2.75 8.43 0.63 5.83 1.10 1.26

Heptane-3,4-dimethyl 4.523 - - - - - -

α-Phellandrene 4.541 3.47 10.64 - - 0.03 -

Terpinolene 4.645 - - - - - -

4-Hexen-1-ol, acetate 4.674 - - - - - -

o-Cymene 4.698 - - - - - -

Limonene 4.789 - 24.08 - - - -

Eucalyptol 4.795 38.77 - 72.35 14.69 40.89 39.99

α-Terpinene - - - - - - -

γ-Terpinene 4.647 0.77 2.78 0.17 1.54 - 0.15

α-Terpinolene - - - - - - -

3-Carene 5.217 - - - - - 1.22

4-Carene 5.265 0.12 3.06 - 2.84 0.93 -

Linalool 5.307 1.48 - 0.28 2.88 0.51 1.09

α-Pinene oxide 5.386 - - - 0.09 - 0.21

Cis-p-metha-1(7), 8-diene-2-ol

5.446 0.56 0.06 - 0.05 0.10 1.36

α-Fenchol 5.487 1.02 - 0.48 0.34 1.08 1.87

Campholenal 5.565 0.10 0.03 0.20 0.13 0.38 0.56

Cis-P-2,8-Menthadien-1-ol

5.502 0.17 - - - 0.10 0.18

Trans-p-2,8-Menthadien-1-ol

5.523 - - - 0.20 - -

α-Fenchene 5.639 - - - - - -

Pinocarveol 5.679 2.35 0.16 3.86 - 8.07 9.56

Pinocarvone 5.755 2.20 - 1.40 - 4.09 6.17

Camphenilanol 5.765 0.21 0.11 - - - -

Terpinen-4-ol 5.976 4.23 3.17 - 2.45 0.76 0.86

Myrtenol 6.095 0.62 - 0.13 - 0.62 0.87

α-Terpineol 6.104 7.38 7.39 5.48 8.89 8.26 8.75

Cis-Carveol 6.227 0.79 - 0.68 - 1.12 1.55

α-Bergamontene 6.383 - - - - - -

Nerol 6.421 - 0.23 - - - -

D-Carvone 6.429 - - - - - 0.92

128

Geraniol 6.479 0.99 2.20 - 3.12 0.55 -

Citral 6.588 - 0.02 - 0.11 - -

Longifolene 6.574 - - - - - -

Carvacrol 6.635 0.03 - - - - -

β-Guaiene 6.683 - - - - - -

β-Bisabolene 6.753 - - - - - -

Bornyl acetate 6.761 - - - 0.14 0.07 0.08

3,4-Xylenol 6.766 - - - - 0.05 -

Thymol 6.782 0.33 - - - - -

Methyl nerolate 6.926 - - - - - -

Methyl geranate 6.932 - - - 1.24 - -

α-Sinensal 7.018 - - - - - 3.01

Adamantan-2-ol 7.022 - - - - - -

2-Acetoxy-1,8-cineole 7.123 0.29 0.26 0.23 0.90 0.35 0.47

α-Farnesene 7.196 - - - - 2.54 -

Neryl acetate 7.197 - 0.22 - 0.25 - -

Eugenol 7.220 0.47 - - - - -

Geranyl acetate 7.320 0.35 1.85 - 1.41 0.07 0.14

α-Himachalene 7.373 - - - - - -

Methyl cinnamate 7.418 - - - 0.56 0.06 0.08

Pivarose 7.458 - - - - 0.06 -

β-Phenylethyl acetate 7.478 - - - 0.13 - -

α-Santalol 7.640 - - - 0.05 - -

Di-epi-α-cedrene 7.688 - - - 0.16 - -

α-Gurjunene 7.689 - - - - - -

Caryophyllene 7.765 0.04 0.41 - 1.61 - -

Aromandendrene 7.893 0.04 - - 0.65 - -

Humulene 7.988 - - - 0.44 0.11 -

Alloaromandendrene 8.040 0.09 - - 1.72 - -

α-Elemene 8.077 - - - 0.20 - -

Trifluoroacetyl-lavandulol

8.169 - - - 0.24 - -

γ-Gurjenene 8.176 - 0.08 - - - -

1,4-dimethyl tetralin 8.178 - - - - 0.06 -

Bicyclogermacrene 8.247 - - - - - -

Germacrene 8.252 - - - 2.30 - -

Durohydroquinone 8.322 0.11 - - - 0.03 0.05

δ-Cardinene 8.365 - - - 0.46 - 0.15

Epizonarene 8.399 - - - 0.14 - -

Elemol 8.503 - - - - - -

Epiglobulol 8.658 0.15 0.35 - 0.38 0.12 0.19

Valencene 8.704 - 0.54 - - - -

Palustrol 8.721 - - - 0.65 0.11 0.12

Aromandendr-1-ene 8.723 0.16 - - - - -

Spathulenol 8.780 0.22 1.21 - 1.98 0.57 0.45

Globulol 8.831 0.82 1.81 0.43 2.18 0.72 0.71

Viridiflorol 8.882 0.46 1.20 - 1.62 0.38 0.36

Rosifoliol 8.902 - 1.59 - - 0.43 -

129

Ledol 8.936 0.11 2.15 - - - -

1-acetyl-2-amino-3-cyano-7-isopropyl-4-

methylazulene 8.996

- - - 7.48 - -

β-Caryophylladienol 9.103 - - - - - -

Viridiflorene 9.247 - 0.33 - 0.25 - -

Trans-Farnesol 9.440 0.04 0.34 - 0.39 - -

Cedran-diol (8S,14) 9.475 - - - - - -

Eudesma-4,11-dien-2-ol 9.549 - 0.12 - - - -

Benzyl benzoate 9.969 - 0.02 - - 0.01 0.04

Farnesol acetate 10.05 - 0.01 - 0.03 - -

(Z,Z)-α-Farnesene 10.24 - - - 0.02 - -

Phytol 11.40 - - - 0.31 - 0.08

(%) Totals 81.72 89.68 94.48 82.52 90.29 96.71

Hydrocarbons Monoterpene

17.01 63.74 8.45 20.55 17.39 14.12

Oxygenated Monoterpenes

61.23 13.37 84.86 32.95 66.53 73.94

Sesquiterpene Hydrocarbons

0.33 1.36 - 7.95 2.65 0.15

Oxygenated Sesquiterpenes

1.80 8.77 0.43 7.25 2.33 1.83

Diterpene - - - 0.31 - 0.08

Hydrocarbon - - - 0.06 -

Esters 0.65 2.41 0.74 5.73 1.23 3.29

Alcohol 0.12 0.03 - 0.06 0.07 0.20

Aldehyde - - - - 3.01

Others 0.58 - - 7.72 0.03 0.09

Figure 4.5: Key Components Identified in the Volatile Oil of Callistemon

citrinus leaves.

130

4.2.4 Effect of Seasonal Variation on Percentage Yield of the

Volatile Oil

The oil yields of the twelve treatments generally ranged from 0.18-

0.31% with the highest percentage recorded in summer period (0.31%)

and the lowest in winter (0.18%). The lower yield of the volatile oil in

winter may be linked to the presence of moisture content in the leaves as

a result of high relative humidity, which is known to reduce the volatile oil

production as a result of surplus water (Carneiro et al., 2010). The results

in this study (Table 4.5) showed seasonal fluctuation in the yields and this

corroborates the report of other researchers on volatile oil yields and

chemical composition which are majorly influenced by factors such as like

light, rainfall, liming, time of harvest, soil, altitude and developmental

phase of plant (Blank et al., 2005; Gobbo-Neto et al., 2007; Lakusic et

al., 2011; Lima et al., 2003; Verma et al., 2011)

4.2.5 Effect of Seasonal Variation on Volatile Oil Content

Effect of seasonal fluctuation was also established in the volatile oil

contents of this study as it was revealed that maximum oil content was

recorded in December–February (94.86-96.63%) summer period in South

Africa (Table 4.5), which gave credence to a previous study where it was

reported that extraction of the volatile oil of Thymus serpyllum L

produced highest percentage of oil content in summer season (Verma et

al., 2011). The lowest oil content from this study was recorded in June

(58.72%) (Table 4.5) when winter began in South Africa, this might be

related to excess of water, which has the tendency to reduce volatile oil

131

production (Carneiro et al., 2010). Seasonal effects on volatile oil

production usually make summer stands out as the season with the

maximum volatile oil content this could be linked to the positive influence

of higher temperature in this season coupled with precipitation which is

capable of affecting the vegetative growth of the plants (Botrel et al.,

2010; Santos et al., 2012). Eucalyptol, a cyclic monoterpenoid ether, with

various degrees of pharmacological effects which also serves as a marker

for medicinal essential oil classification (Sadlon and Lamson, 2010) is the

dominant component of the volatile oils. It compared favorably well with

the volatile oil component from previous study of the same plant from

South Africa (Larayetan et al., 2017), it reached its highest value

(72.35%) in Spring (September) and the lowest value (48.98%) in

summer (February) (Table 4.5) substantiating the findings documented

by (Soni et al., 2015).

Sudden disappearance of eucalyptol in the month of August and the

appearance of limonene (24.08%), which could not be detected in any

other month, may be due to the reversal of eucalyptol to α-terpineol and

loss of water (dehydration) from α-terpineol leading back to α-terpinyl

cation and further loss of a proton from this cation giving rise to limonene

(Figure 4.6) (Croteau et al., 1994; Degenhardt et al., 2009;

Rajaonarivony et al., 1992). Formation of α-phellandrene as a result of

the disappearance of eucalyptol in the month of August may also be due

to the α-terpinyl cation following the 1, 7-hydride shift pathway leading to

132

the formation of phellandryl cation and further loss of a proton from one

of the carbon of the phenyl ring to give α-phellandrene (Figure 4.6).

Figure 4.6: Biosynthetic Pathway of Some of the Components of

Callistemon citrinus Volatile Oil.

The next key component in the volatile oil of this plant was α-pinene,

which gave a range of 7.45-22.75%; it has maximum value in the month

of March (autumn) 22.75% and minimum value in the month of

September (spring) 7.45% (Figure 4.6). α-Terpineol an unsaturated

133

volatile monocyclic alcohol is the third chief component with utmost value

in the month of May (9.94%) and least value in the month of April

(5.24%). Its anti-hypernociceptive behavior and anti-inflammatory

activity has been documented (Perez-Sanchez et al., 2012). It can be

concluded from Table 4.5 that seasons affect the volatile oil components

of this plant.

4.2.6 Effect of Flowering Phase on Percentage Yield and Volatile

Oil Content

Another significant aspect to be considered is the flowering phase of

Callistemon citrinus. According to the field surveillance of this

investigation, the flowering of this plant occurs during spring and summer

period and remains in autumn. Both volatile oil content and percentage

yield suggest that they might be linked to the flowering period because

the highest percentage yield of 0.31% in February, 0.30% in December,

0.25% in November and 0.25% in April, coupled with the highest oil

content of 96.63% in December, 96.56% in January, 92.99% in March

and 90.29% in November, fell between the summer, spring and autumn

period of South African season Table 4.5. Comparable behavior during the

flowering phase has been recorded for Lamiaceae species (Botrel et al.,

2010; Lakusic et al., 2011; Perez-Sanchez et al., 2012).

4.2.7 Effect of Seasonal Variation on the Antioxidant Activity of

Callistemon citrinus

Antioxidant capacity of the volatile oil of Callistemon citrinus leaves

also demonstrated significant influence of seasonal variation on its

134

activity. The most significant activity was recorded in the month of

September (spring) with an IC50 of 0.50 mg mL-1, followed by January

(summer) IC50 0.88 mg mL-1, April (autumn) IC50 1.35 mg mL-1. The

least activity was observed in June (winter) collection with IC50 of 1.45

mg mL-1 (Table 4.6 and Figure 4.7). It has been documented that

antioxidant activity of volatile oils is not only due to phenolic content of

the oil but constituents like monoterpene alcohols, ketones, aldehydes,

hydrocarbons, and ethers, which also add to the free radical scavenging

activity of some volatile oil (Edris, 2007). The volatile oils of Thymus

caespititus, Thyme camphorates, and Thyme mastichina which have high

contents of linalool and eucalyptol were shown to have high antioxidant

activity, which were almost equal to that of α-tocopherol (Miguel et al.,

2004). Similarly, the high scavenging activity of M. aquatic was also

linked to eucalyptol in the volatile oil (Mimica-Dukic et al., 2003). The

volatile oil of the plant in this study also yielded high content of eucalyptol

(cyclic monoterpenoid ether) in the month of September (72.35%),

January (63.72%) and April (51.92%), which were spring, summer and

autumn seasons, respectively. This might contribute to its high

antioxidant activity in addition to phenolic compound like terpenoids and

pinocarveol. Similar behavior have been reported for the antioxidant

capacity of Bellis perennis flowers which exhibited highest antioxidant

capacity for samples collected from spring to autumn. The discrepancy in

the antioxidant action of this plant was reportedly due to the fluctuation in

environmental factors such as day and night temperature, rainfalls,

135

drought and the duration / intensity of sunshine (Siatka and Kasparova,

2010). The effect of seasonal fluctuation on the antioxidant activity in the

present study revealed that the least activity recorded in the winter was

in variance with that reported for Ocimum basilicum which exhibited it

highest antioxidant capacity in winter season with an IC50 value of 4.8 µg

mL-1 (Hussain et al., 2008).

Table 4.6: Effect of Season on Antioxidant Capacity of Callistemon

citrinus Volatile Oil IC50 (mg mL-1).

Oil Summer leaf oil Winter Leaf oil Spring Leaf oil Autumn Leaf oil

DPPH. 0.88 ± 0.05 1.45 ± 0.00 0.50 ± 0.04 1.35 ± 0.04

Figure 4.7: Antiradical Effect of the Leaf Oils Isolated from Callistemon

citrinus.

4.2.8 Conclusion

Season brings about chemical disparity in oil yield, antioxidant capacity

and oil content of Callistemon citrinus from South Africa. The dominance

of eucalyptol, in the different treatments (January-December) (except for

136

the month of August) of the leaf makes it an excellent marker for

Callistemon citrinus species. Taking into account that summer, spring and

autumn gave the highest yields of volatile oil, antioxidant capacity as well

as maximum content of eucalyptol, it can be said that these seasons are

most suitable to get a better quality of the oil. The leaves of this plant

possess volatile oil which differs in quantity and quality as a result of

seasonal fluctuation. It is important for researchers to know the season

with the highest quality of volatile oil as this tends to give the best

biological activity.

4.3 Determination of Bioactive Compounds, Phytochemical

Constituents, Antioxidant Capacity and In Vitro Antimicrobial

Potential of Crude Extracts Isolated from Callistemon citrinus

4.3.1 Abstract

Active phytochemicals present in both ethyl acetate and methanolic

extracts of Callistemon citrinus were examined. The antimicrobial,

bioactive determination using GC-MS, time of kill and antioxidant

activities were explored. The bioactive components were characterized by

high amount of fatty acids (52.88 and 62.48%). Squalene, a triterpenoid

synthesized in human liver was obtained in both extracts at a varying

amount. The ethyl acetate extract demonstrated strong activity against

Paeudomonas aeruginosa ACC (28.7 ± 1.2 mm), Listeria ACC (26.0 ± 2.0

mm) and Escherichia coli ATCC 35150 (24.0 ± 3.5 mm). Qualitative

phytochemical screening revealed alkaloids, glycosides, saponins, steroids

and triterpenoids, fats and oils, flavonoids, phenols and tannins.

137

Quantitative phytochemical determination (total tannin, total flavonoids

and flavonols, total phenolic and total antioxidant capacity) was

performed using spectrophotometry. The minimum time needed to totally

kill the tested bacterial strains ranges from 15 to 24 hours.

4.3.2 Background

Plants are used by humans to ease and treat several diseases,

nowadays in several countries of the world, traditional medicines are used

as substitute to conventional medicine (Ramawat and Merrillon, 2008;

Winslow and Kroll, 1998). Countless medicinal plants possessing

antioxidant activity now draws the attention of many researchers to

investigate their role in the fight against numerous diseases like

Alzheimer's disease, cancer, atherosclerosis, cerebral cardiovascular

events, diabetes and hypertension to mention just a few (Liu, 2013;

Devasagayam et al.,2004).

Plants serve as reservoir for potentially valuable chemical compounds

which can be used to produce drugs resulting into leading molecules used

for current design and synthesis (Arun et al., 2011; Varier, 1995). Plant

extracts together with the phytochemicals possess antimicrobial

properties which are of great importance for therapeutic treatment

(Nagesh and Santhamma, 2009). Most pharmacological activities of

medicinal plants are traced to their secondary metabolites which are

smaller in molecules when compared to the constituents of primary

metabolites like proteins, carbohydrates, and lipids. Secondary

metabolites like alkaloids, terpenoids, tannins, saponins, flavonoids, and

138

cardiac glycosides from both medicinal and aromatic plants can be used

for the synthesis of various antimicrobial and antifungal drugs which are

relatively less harmful to man (Kalimuthu et al., 2010). Plants with

medicinal basis are usually utilized by traditional practitioners in most

rural areas of developing countries of the world (Gupta et al., 2005;

Sandhu and Heinrich, 2005).

Phytochemical analysis carried out on the leaves of Callistemon citrinus

revealed the presence of alkaloids, terpenoids, steroids and flavonoids

(Shinde et al., 2012). There is however, a dearth of information with

regards to the comparative assessment of the antioxidant and

antibacterial properties, time of kill, and particularly the nature of the

bioactive components of the extracts of Callistemon citrinus, which

necessitated the present study, in addition to the antioxidant and

antibacterial capabilities of the plant.

4.3.3 Constituents of the Extracts

Exactly twenty and twenty-two bioactive compounds were found in the

methanol and ethyl acetate extracts of the plant of study by GC-MS

investigation, respectively. The retention indexes, molecular formula,

molar mass, peak area (%) and nature of the compounds are presented

in Tables 4.7 and 4.8. Although, the level of fatty acids found in the two

extracts was high (52.88 and 62.48%), it is apparent that the main

compounds characterizing both extracts are qualitatively and

quantitatively different. Other components of the methanol and ethyl

acetate extracts include esters (21.48 and 8.06%), oxygenated

139

monoterpenoids (8.00 and 4.48%), triterpenes (2.98 and 2.52%),

respectively. Oleic acid, a fatty acid was identified as one of the major

components from the GC-MS results of the two extracts (6.42 and

28.23%). It is a monosaturated omega-9 fatty acid that has a lot of

health benefits and is carefully employed as one of the constituents in the

preparation of cosmetics (Liebert, 1987). It is also capable of preventing

ulcerative colitis (De Silver et al., 2014), protects cell from free radical

damage (Haug et al., 2007), reduce blood stress (Ruiz-Gutierrez et al.,

1996) and augment fat burning (Lim et al., 2013). Palmitic acid, a

saturated lengthy chain fatty acid with sixteen carbon atoms was also

found abundantly in both extracts of the Callistemon citrinus (32.87 and

13.57%). It is one of the major and mostly spread natural saturated acids

present in plants like palm oil, palm kernel oil, odoriferous plants, Moringa

oleifera seed oil, in animals and animal-derived products like cheese,

milk, meat and microorganisms (Lim et al., 2013). It is also used in the

production of cosmetics (Fassett and Irish, 1963).

Table 4.7: Components of Methanolic Extract of Callistemon citrinus.

Name of

Compound RI

Peak Area (%)

Molecular Formula

Molar Mass (g mol-1)

Compound Nature

4-Carene 919 0.22 C10H16 136 Monoterpene

Eucalyptol 1059 2.22 C10H18O 154 Oxygenated

monoterpenoid

2-Nonenal 1112 0.27 C9H16O 140 Unsaturated

aldehyde

Limonene diepoxide

1128 0.31 C10H16O2 168 Oxygenated

monoterpenoid

Pinocarveol 1131 0.46 C10H16O 152 Oxygenated

monoterpenoid

α-Terpineol 1143 1.22 C10H18O 154 Oxygenated

monoterpenoid

4-Methyl-isopulegone 1252 0.27 C10H18O 166 Oxygenated

140

monoterpenoid

trans-2-Decenol 1266 0.92 C10H20O 156 Unsaturated

alcohol

Aromadendrene 1386 0.46 C15H24 204 Sesquiterpene

(8E,10Z)-1,8-Pentadecatriene

1518 27.00 C15H26 248 Hydrocarbon

β-Eudesmol 1593 0.55 C15H26O 222 Oxygenated

sesquiterpenoid

Methyl 14-methyl-pentadecanoate

1814 2.04 C17H34O2 270 Ester

Palmitic acid 1968 32.87 C16H32O2 256 Fatty acid

9-Octadecenal 2007 0.91 C18H34O 266 Unsaturated

aldehyde

Stearic acid, methyl ester

2077 1.07 C19H38O2 298 Ester

trans-Vaccenic acid, methyl ester

2085 3.46 C19H36O2 296 Ester

Methyl linoleate 2093 1.49 C19H34O2 294 Ester

Stearic acid 2167 13.59 C18H36O2 284 Fatty Acid

Oleic acid 2175 6.42 C18H34O2 282 Fatty acid

Squalene 2914 2.98 C30H50 410 Triterpene

Table 4.8: Components of Ethyl Acetate Extracts of Callistemon citrinus.

Name of Compound RI Peak

Area (%) Molecular

formula Molar mass

(g mol-1) Compound

Nature

4-Carene 919 0.35 C10H16 136 Monoterpene

2,6-Dimethyl-3,7-octadien-2-ol,

1041 0.31 C10H18O 154 Oxygenated

monoterpenoid

Eucalyptol 1059 4.55 C10H18O 154 Oxygenated

monoterpenoid

2-Nonenal 1112 1.53 C9H16O 140 Unsaturated

aldehyde

trans-Pinocarveol 1131 0.69 C10H16O 152 Oxygenated

monoterpenoid

α-Terpineol 1143 1.56 C10H18O 154 Oxygenated

monoterpenoid

Myrtanal 1126 0.51 C10H16O 152 Oxygenated

monoterpenoid

4-methyl-isopulegone 1252 0.38 C10H18O 166 Oxygenated

monoterpenoid

Aromadendrene 1386 0.46 C15H24 204 Sesquiterpene

Patchulane 1393 0.29 C15H26 206 Sesquiterpene

Viridiflorol 1530 1.17 C15H26O 222 Sesquiterpene

Methyl-2-hydroxytetradecanoate

1842 7.18 C15H30O3 258 Hydroxyl ester

Methyl hexadecanoate 1878 3.76 C17H34O2 270 Ester

Palmitic acid 1968 13.57 C16H32O2 256 Fatty acid

141

9-Octadecenal 2007 1.70 C18H34O 266 Unsaturated

aldehyde

Stearic acid, methyl ester 2077 2.03 C19H38O2 298 Ester

trans-Vaccenic acid, methyl ester

2085 5.67 C19H36O2 296 Ester

Methyl linoleate 2093 2.84 C19H34O2 294 Ester

Stearic acid 2167 10.43 C18H36O2 284 Fatty acid

Oleic acid 2175 28.23 C18H34O2 282 Fatty acid

Sterolic acid 2184 10.25 C18H32O2 280 Fatty acid

Squalene 2914 2.52 C30H50 410 Triterpene

Stearic acid, a saturated fatty acid having 18-carbon chain was present

in a substantial amount in both methanolic and ethyl acetate extracts of

the plant (13.59 and 10.43%). It has an IUPAC name of octadecanoic

acid, and is mostly used in the production of detergent, soaps and

cosmetics such as shampoos and sharing cream products. Soap is not

made directly from stearic acid but indirectly by saponification of

triglycerides containing the stearic acid esters. Surfactants, cosmetics and

personal hygiene products are in fact prospects of stearic acid (Gunstone,

2004).

Squalene is a triterpene and also possesses antioxidant and chemo-

preventive activity against colon carcinogenesis (Amarowicz, 2009; Rao et

al., 1998). It was found in a relatively lower amount in both extracts of

Callistemon citrinus (2.98 and 2.52%). Various components of both

extracts of the plant of study have been associated with different

therapeutic activities. Eucalyptol which appeared in a significant amount

in the two leaf extracts of the plant has notable bronchodilator effects,

antiviral activity, antitussive effect, mucolytic, mucociliary effects and

anti-inflammatory activity. These sets of bioactive compounds possess

142

synergistic effects, which may be accountable for the therapeutic benefits

of Callistemon citrinus as employed by traditionalists.

4.3.4 Phytochemical Screening

The phytochemical study of the ethyl acetate plant extract revealed

the presence of different bioactive components such as alkaloids,

glycosides, saponins, steroids and triterpenoids, fats and oils, flavonoids,

phenols and tannins (Table 4.9). Bioactive compounds stored in the plant

possess biological antibacterial activity that has no record of unpleasant

effect on human being (Doughari et al., 2009). These compounds have

been reported to bestow resistance in opposition to microbial pathogens

and this establishes the demonstration of the antibacterial activity by the

leaf extract in the present study (Anibijuwon and Udeze, 2009).

Secondary metabolites like terpenoids have been reported to have anti-

inflammatory, antimalaria, antibacterial, antiviral activities and inhibition

of cholesterol synthesis (Mahato and Sen, 1997). Previous phytochemical

studies on the leaf of Callistemon citrinus revealed the presence of

alkaloids, flavonoids, terpenoids and steroids (Shinde et al., 2012).

Table 4.9: Qualitative Phytochemical Screening of Ethyl Acetate Extract of

Callistemon citrinus.

Phytochemical Constituents Test Results

Saponins Foam test +

Glycosides Modified Borntrager and Keller-Killiani test

+

Alkaloid Mayer and Wagner test +

Steroids and Triterpenoids Salkowski test +

Phenols and Tannins Ferric Chloride test +

Flavonoids Lead acetate +

Fats and Oils Stain test +

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4.3.5 Overall Tannin Content

Tannins, polyphenolic compounds are present in different plant parts

(Waterman and Mole, 1994). Tannin displays antioxidant, antimicrobial

and anti-inflammatory properties (Okwu and Okwu, 2004). Eating of food

rich in tannin will offer a lot of curative and beneficial effects to man.

Overall tannin content was higher in ethyl acetate extract than methanol

extract as shown in Table 4.10.

4.3.6 Overall Antioxidant Capacity

The overall antioxidant capacity is a quantitative way to determine the

degree of reduction of Mo (VI) to Mo (V). The OAC of both ethyl acetate

and methanol extracts in this study are 1568.73 ± 61.03 and 3031.53 ±

133.07 mg AA/100g, respectively (Table 4.10). This implies that both

extracts will have to contain as much of antioxidant compounds as

equivalents of ascorbic acid to efficiently reduce the oxidant in the

reaction matrix. Antioxidant ability of ascorbic acid was employed as a

reference standard with which plant extracts with potential antioxidant

were compared (Aderogba et al., 2005).

Table 4.10: Quantitative Phytochemical Constituents of Ethyl Acetate and

Methanol Extracts of Callistemon citrinus.

Extracts Overall Tannin

Content (mg TAE/100g)

Overall Phenolic Content

(mg GAE/100g)

Overall Flavonoid Content

(mg RE/100g)

Overall Flavonol Content

(mg RE/100g)

Overall Antioxidant

Capacity (mg AA/100g)

Ethyl acetate 12000 ± 65.34 10964.11 ± 40.22 368.12 ± 14.48 438.38 ± 11.73 1568.73 ± 61.03

Methanol 8000 ± 28.67 22511.23 ± 105.12 688.37 ± 39.92 512.90 ± 11.00 3031.53 ± 133.07

144

4.3.7 Overall Phenolic Content

Phenolic substances are ubiquitous secondary metabolites in plants.

They possess antioxidant activity (Okudu et al., 1994; Tawata et al.,

1996; Tepe et al., 2006). The result obtained in this study shows that the

leaf extracts of Callistemon citrinus contained phenolic compounds and

the content was higher in the methanolic than in the ethyl acetate

extract. This might be due to the influence of extraction solvent on the

overall content of the phenolic compounds. The overall phenolic content in

both ethyl acetate and methanolic leaves extract of this present study

were found to be 10,964.11 ± 40.22 and 22,511.23 ± 105.12 mg

GAE/100g, respectively (Table 4.10). The results were higher than those

reported for the same species from India that recorded 261 mg/g (Kumar

et al., 2015), possibly because of the influence of environmental factors

on the phenolic content.

4.3.8 Overall Flavonoid Content

Flavonoids, a secondary metabolite that refer to a class of naturally

occurring polyphenols are found in plants. They are utilized for the

manufacture of pigments that attract insects for pollination in plants.

They cannot be synthesized by animals and man because they are

phytochemicals (Koes et al., 2005). They are usually accountable for

taste, color, impediment of fat oxidation and prevention of enzymes and

vitamins degradation in food (Yao et al., 2004). In addition, they also

exhibit significant anti-inflammatory, anti-allergic and anti-cancer

145

activities (Crozier and Ashiharu, 2006). The most abundant flavonoids in

food are the flavonols.

The overall flavonoids contents determined in both ethyl acetate and

methanol leaf extracts were 368.12 ± 14.48 and 688.37 ± 39.92 mg

RE/100g, making polar methanol extract of this present study higher than

the apolar ethyl acetate as shown in Table 4.10.

4.3.9 Overall Flavonol Content

Flavonols are light-yellow and weakly soluble substances found in

leaves, fruits, berries and flowers of 80% higher plants. The evaluation of

total flavonols content in the extracts of the plant of study was expressed

as rutin equivalent. The higher flavonols content as presented in Table

4.10 was observed in the methanol extract (512.90 ± 11.00 mg

RE/100g), and the lower content was seen in the ethyl acetate extract

(438.38 ± 11.73 mg RE/100g).

4.3.10 Antioxidant Activities of the Crude Extracts

The DPPH• antioxidant assay is based on the principle that any

substance capable of donating an atom of hydrogen or an electron is an

antioxidant or antiradical species and its potency is demonstrated as

DPPH•. Color is transformed from purple to yellow in the test sample due

to formation of neutral DPPH-H molecule upon the uptake of an hydrogen

atom from an antioxidant specie (Guerrini et al., 2009).

It has been documented that it is preferable to use up to two methods

when carrying out test on antioxidant activity (Schlesier, 2002). The

evaluation of the antioxidant activity of the ethyl acetate and methanol

146

crude extracts was carried out in vitro via two radical models (DPPH and

ABTS) and the antioxidant capacity of the two extracts were measured

based on their efficient IC50 concentration which correspond to the

extracts concentration capable of reducing the initial DPPH• absorbance by

50%.

The IC50 of the ethyl acetate extract (2.41 ± 0.25) was lower than

that of the methanol extract (1.33 ± 0.24) in the DPPH assay, but the two

extracts showed a better activity than the standard drug (vitamin C) with

IC50 of 2.43 ± 0.49 (Table 4.11). In the case of the ABTS assay, ethyl

acetate extract with IC50 of 0.80 ± 0.36 was also found to scavenge the

radicals less than the methanol extract having IC50 of 0.52 ± 0.53. Like

the DPPH assay, the two extracts under ABTS also exhibited a good

activity than the standard drug (vitamin C) with IC50 (4.60 ± 0.24). A

better and more efficient result was recorded for the ABTS assay as

shown in the IC50 results for the two experiments (Table 4.11).

Percentage inhibitions of these radicals by the extracts and reference

standard (vitamin C) were concentration dependent (0.025 to 0.4mg mL-

1) articulated in percentage inhibition versus concentration as illustrated

in Figures 4.8 and 4.9.

Table 4.11: Antiradical ability of extracts from Callistemon citrinus.

Activity Callistemon citrinus Reference compound

Ethyl acetate extract Methanol extract Vitamin C

ABTS 0.80 ± 0.36 0.52 ± 0.52 4.60 ± 0.24

DPPH 2.41 ± 0.25 1.33 ± 0.24 2.43 ± 0.49

147

Figure 4.8: Antiradical Effects of Ethyl Acetate and Methanol Extracts and

Standard Drug (Vitamin C) on DPPH Radicals.

4.3.11 Antibacterial Activity of the Extracts

The two extracts of Callistemon citrinus showed varying degree of

antibacterial activities against the test organisms as shown in Table 4.12.

Ethyl acetate extracts had the highest region of inhibition for the different

bacterial strains as determined by the diameter of the zone of inhibition.

It gave the highest zone of inhibition with P. aeruginosa ACC (28.7± 1.2

mm), Listeria ACC (26.0 ± 2.0 mm) and Escherichia coli ATCC 35150

(24.0 ± 3.5 mm) and the lowest inhibitory activity for Vibro alginolyticus

DCM 2171 (19.0 ± 1.0 mm) and Salmonella typhi ACC (18.0 ± 2.0 mm)

at a concentration of 0.4 mg/mL (Fig 4.10 and 4.11). A similar activity

was also seen for methanolic leaf extract as it gave highest inhibitory

activities for both P. aeruginosa ACC (22.7 ± 1.2 mm) and Aeromonas

hydrophilia ACC (19.7 ± 1.5 mm) and lowest inhibitory action for

Escherichia coli ATCC 35150 (13.7 ± 1.5 mm) at a concentration of 0.4

mg/mL (Figure 4.8 and 4.9).

148

Figure 4.9: Antiradical effects of Ethyl Acetate and Methanol Extracts and

Standard Drug (Vitamin C) on ABTS Radicals.

Table 4.12: Region of Inhibition (mm) Showing Antibacterial Activities

against Bacterial Test Organisms.

Microorganism Concentration

Positive control Zone of inhibition of extracts and standard drug

Ciprofloxacin (mg mL-1) Ethyl acetate extract (mg mL-1) Methanol extract (mg m-L1)

0.4 0.1 0.025 0.4 0.1 0.025 0.4 0.1 0.025

Gram-negative Bacteria

Aeromonas hydrophila ACC

34.0 ±0.6

26.0 ±2.0

20.0 ± 0.5 22.7 ±2.3 15.0 ± 1.0 11.3 ± 1.2 19.7 ±1.5 15.0 ± 1.0 10.0 ±

0.3

Escherichia coli ATCC 35150

38.0 ±4.0

32.0 ±4.0

24.0 ± 0.9 24.0 ± 3.5 17.0 ± 1.0 11.3 ± 1.2 13.7 ± 1.5 11.2 ± 0.8 7.3 ± 1.2

Vibro alginolyticus DSM 2171

33.0 ±2.0

29.0 ±2.0

23.0 ± 0.4 19.0 ± 1.0 14.0 ± 1.0 10.3 ± 1.5 17.0 ± 1.0 13.0 ± 1.0 8.7 ± 1.2

Salmonella typhi ACC

40.0 ±1.0

34.0 ±0.6

28.0 ± 3.0 18.0 ± 2.0 15.3 ± 3.1 8.7 ± 1.2 14.3 ± 0.6 10.3 ± 0.6 8.0 ± 0.6

Pseudomonas aeruginosa ACC

38.0 ±2.0

36.0 ±1.0

33.0 ± 3.0 28.7 ± 1.2 17.3 ± 2.1 14.7 ± 1.2 22.7 ± 1.2 17.2 ± 1.9 14.0 ±

1.0

Gram positive Bacteria

Staphylococcal enteritis ACC

40.0 ±5.0

32.0 ±1.0

28.0 ± 0.2 20.3 ± 0.6 14.7 ± 1.2 11.0 ± 1.7 21.0 ± 1.0 15.7 ± 1.5 12.3 ±

0.6

Staphylococcus aureus ACC

29.0 ±2.0

24.0 ±0.0

17.0 ± 2.0 21.3 ± 1.6 16.7 ± 2.3 12.7 ± 1.2 19.3± 1.2 12.7 ± 1.2 9.5 ± 0.9

Listeria monocytogenes

ACC

44.0± 6.0

38.0± 0.2

32.0 ± 1.0 26.0 ± 2.0 17.3 ± 1.2 12.3 ± 1.5 16.7 ± 1.2 12.0 ± 2.0 9.3 ± 1.2

Zone of inhibition (millimeter), ACC Aemreg culture collection, ATCC

American type collection center, values are mean ± SD, n = 3

149

Figure 4.10: Antibacterial Activity of Ethyl Acetate Leaf Extract.

Although both extracts showed highest inhibitory effect on gram-

negative bacteria, which was contrary to some reports published in

literature (Cock, 2012), but they still exhibited satisfactory inhibition for

the gram-positive bacteria. This might be due to the single cell-wall layers

of gram-positive bacteria that could be easily penetrated by the extracts

than the gram-negative bacteria, which possess an extra

lipopolysaccharide and protein cell-wall that provide permeability blockade

to the antibacterial agents (Kaur and Arora, 2009; Adwan and Abu-

Hassan, 1998).

150

Figure 4.11: Antibacterial Activity of Methanol Leaf Extract.

The two extracts from this study demonstrated the strongest inhibitory

properties against P. aeruginosa and Escherichia coli as stated above. P.

aeruginosa, a recognized pathogen, is linked to chronic obstructive

pulmonary disease associated with intense inflammation (Croxen and

Finlay, 2012; WHO, 2012) and Escherichia coli an Enterobacteriaceae

family is responsible for diseases like gastro-intestinal and urinary tract

infections (Croxen and Finlay, 2012; Kaper et al., 2004; WHO, 2012).The

extracts also showed a good inhibitory effect for a gram positive

bacterium (S. aureus), which is responsible for lower respiratory tract

infection, ventilator-assisted pneumonia and osteomyelitis (Shito, 2006).

Findings from this study indicate that the leaves of C. citrinus contain

antibacterial components that justify traditional and medicinal usage of

the plant to guard against infections caused by both gram-positive and

gram-negative bacteria.

151

4.3.12 Minimum Inhibitory Concentration

The MIC of the extracts was carried out to determine the lowest

concentration that showed no visible growth when compared with the

control containing no extract as the antimicrobial agent. The MIC is also

helpful in ascertaining the level of resistance of a particular bacterial

strain and thus serves as a pointer to the use of certain antimicrobial

agents. The ethyl acetate leaf extract MIC values of 0.025 ± 0.00, 0.025

± 0.00, 0.025 ± 0.00, 0.025 ± 0.010, 0.100 ± 0.000, 0.025 ± 0.010,

0.100 ± 0.000, 0.025 ± 0.000 reveal that it is more active against the

different bacterial strains Aeromonas hydrophila (ACC), Escherichia coli

(ATCC 35150), Vibro alginolyticus (DSM 2171), Salmonella typhi (ACC),

Pseudomonas aeruginosa (ACC), Staphylococcal enteritis (ACC),

Staphylococcus aureus (ACC) and Listeria monocytogenes (ACC), than

the methanol leaf extract with MIC values of 0.100 ± 0.010, 0.100 ±

0.010, 0.100 ± 0.010, 0.025 ± 0.010, 0.100 ± 0.010, 0.100 ± 0.010,

0.025 ± 0.010, 0.100 ± 0.010 mg mL-1 as shown in Table 4.13. The two

extracts recorded the same MIC values of 0.025 ± 0.000 and 0.100 ±

0.000 mg mL-1 for Salmonella typhi ACC and Pseudomonas aeruginosa

ACC, showing that they were inhibited at the same concentration.

Table 4.13: Minimum Inhibition Concentration of Callistemon citrinus

Extracts against Microbial Strains.

Bacteria Ethylacetate leaf

extract Methanol leaf extract

Ciprofloxacin Positive control

Aeromonas hydrophila ACC

0.025 ± 0.00 0.100 ± 0.01 0.100 ± 0.02

Escherichia coli ATCC 35150

0.025 ± 0.00 0.100 ± 0.00 0.100 ± 0.00

152

Vibro alginolyticus DSM 2171

0.025 ± 0.00 0.100 ± 0.00 0.100 ± 0.01

Salmonella typhi ACC 0.025 ± 0.01 0.025 ± 0.00 0.100 ± 0.01

Pseudomonas aeruginosa ACC

0.100 ± 0.00 0.100 ± 0.00 0.100 ± 0.02

Staphylococcal enteritis ACC

0.025 ± 0.01 0.100 ± 0.00 0.100 ± 0.00

Staphylococcus aureus ACC

0.100 ± 0.00 0.025 ± 0.01 0.100 ± 0.01

Listeria monocytogenes ACC

0.025 ± 0.00 0.100 ± 0.01 0.100 ± 0.01

4.3.13 Time Kill

The time kill assay was aimed at determining the minimum time

required to completely kill the tested microbial isolates used in this study

under laboratory conditions (in vitro) (Table 4.14 and 4.15). The time of

kill was conducted against the tested isolates in this study using

ciprofloxacin at a concentration of 0.1 mg mL-1 (from the average MIC

value obtained for most of the tested isolates) as the positive control. The

findings further confirmed that the positive control drug (ciprofloxacin) at

concentration of 0.1 mg mL-1 was effective in eliminating the isolates used

in this study at a minimum incubation period of 15 h (Table 4.14). The

time of kill was also conducted against the tested isolates using ethyl

acetate leaf extract at a concentration of 0.1 mg mL-1 (from the average

MIC value obtained for most of the tested isolates). The minimum

incubation period required to completely kill the tested isolates using the

extract ranged from 15 to 24 h except for Vibro alginolyticus (DCM 2171),

S. typhi ACC, S. aureus ACC and S. enteritis ACC that were completely

killed in 18 h (Table 4.15). The result revealed that despite the fact that

ethyl acetate crude extract at a concentration of 0.1 mg mL-1 was able to

153

kill the isolates, the incubation period varied among the microorganisms,

although the control (ciprofloxacin) required less incubation period than

the crude extract.

Table 4.14: Time of Kill for the Standard Drugs Ciprofloxacin (0.1 mg mL-

1) against Bacterial Organisms.

Bacteria strains Period of incubation (h)

0 3 6 9 12 15 18 21 24

Escherichia coli ATCC 35150

+ + + + + - - - -

Listeria monocytogenes

ACC + + + + + - - - -

Aeromonas hydrophila ACC

+ + + + + - - - -

Pseudomonas aeruginosa ACC

+ + + + + - - - -

Salmonella typhi ACC

+ + + + + + - - -

Staphylococcus aureus ACC

+ + + + + - - - -

Staphylococcal enteritis ACC

+ + + + + - - - -

Vibro alginolyticus (DCM .32171

+ + + + + + - - -

Table 4.15: Time of Kill for ethyl acetate extract (0.1 mg mL-1) against

bacterial organism.

Bacteria strains Period of incubation (h)

0 3 6 9 12 15 18 21 24

Escherichia coli ATCC 35150

+ + + + + - - - -

Listeria monocytogenes

ACC + + + + + - - - -

Aeromonas hydrophila ACC

+ + + + + - - - -

Pseudomonas aeruginosa ACC

+ + + + + - - - -

Salmonella typhi ACC

+ + + + + + + - -

154

Staphylococcus aureus ACC

+ + + + + - + - -

Staphylococcal enteritis ACC

+ + + + + - + - -

Vibro alginolyticus (DCM .32171

+ + + + + + + - -

4.3.14 Conclusion

Phytochemical tests conducted on the ethyl acetate extract showed the

presence of typical bioactive compounds such as alkaloids, saponins,

tannins terpenoids / triterpenoids and phenolic compounds like flavonoids,

which are considered to be bacteriostatic and fungistatic. The inhibition

property of the extracts resides primarily in the bioactive compounds in

plants. Some of the bioactive constituents (e.g. alkaloids, tannins,

terpenoids like eucalyptol, α-terpineol and different fatty acids) may be

responsible for the therapeutic, antiseptic, anti-inflammatory,

antinociceptive and anticough properties of this plant.

The inhibitory role observed on the various microorganisms by the

crude extract of C. citrinus is an indication that it contains a broad

spectrum of antimicrobial constituents which if properly standardized can

be used against some array of pathogen. This study has also provided a

rational for the use of the plant not only in the traditional medicine but

also as a backup of scientific information to justify its different folkloric

uses in the traditional rural settings. Further studies are ongoing on this

plant to isolate, categorize, characterize and elucidate the major

components and structures of its bioactive compounds.

155

4.4 Chemical Components, Antitrypanosomal, Antiplasmodial

and Antibacterial Potencies of the Seed, Leaf and Flower Volatile

Oils of Callistemon citrinus

4.4.1 Abstract

The antitrypanosomal, antiplasmodial and cell cytotoxicity of the

flower, leaf and seed oils of Callistemon citrinus were evaluated and found

to exhibit antitrypanosomal activities with IC50 ranging from 16.67 to

92.23 µg/mL. Three oil samples were found not to be cytotoxic to Hela

(human cervix adenocarcinoma) cells /HEK 293 (human embryonic

kidney) cells. They were also unable to bring about a significant decrease

in pLDH at a concentration of 50 μg/mL to less than 20%.

4.4.2 Background

Pentamidine, suramin, melarsoprol, mel B and arsobal are some of the

modern drugs produced some years ago to combat trypanosomiasis.

However, several setbacks like inadequate supply, resistance by

trypanosomal parasites, expensive cost and some toxic effect have been

reported (Nwodo et al., 2015; Welburn et al., 2009). As a result of these

shortcomings some medicinal plants of African origin that can combat

trypanosomiasis have been documented (Atawodi et al., 2009). This has

led to an intensive search by researchers into a novel leading drug

particularly from Africa plant for trypanosomiasis disease or a compound

that will have antitrypanosomal action (Hoet et al. 2004).

The aim of this work is to assess the antitrypanosomal, antiplasmodial

and cell cytotoxicity of the flower, leaf and seed volatile oils of

156

Callistemon citrinus with a view of assessing if it could be used as an

alternative medicine to the commonly available synthetic drugs in use for

the purpose.

4.4.3 Components of the Volatile Oils

The oil obtained by hydrodistillation from the fresh seed of Callistemon

citrinus gave a pale-yellow color with a minty smell and the yield was

0.95% v/w of the wet sample. Forty-one components were identified in

the seed volatile oil amounting to 95.78% of the entire oil content. The

key components of the seed volatile oil were eucalyptol (37.56%), α-

pinene (13.20%), α-terpineol (8.11%), and terpinen-4-ol (5.99%) as

shown in Table 4.16. There were no major differences in the dominant

components of the volatile oils from the leaves, flowers and stems

(Larayetan et al, 2017). Some notable components like linalool (2.23%),

α-pinene (4.36%) and thymol (0.75%) were also present in the seed oil

(Table 4.16).

Table 4.16: Chemical Composition of the Volatile Seed Oil of Callistemon

citrinus.

Retention Time (Minutes) Chemical Constituents Oil composition (%) Seed

3.441` Isoamyl acetate 0.18

3.729 Isobytyric acid 0.37

3.907 β-Thujene 1.06

3.992 α-Pinene 13.20

4.356 β-Pinene 4.36

4.555 α-Phellandrene 5.15

4.785 Eucalyptol 37.56

5.000 γ-Terpinene 2.26

5.229 (+)-4-Carene 0.93

5.260 Linalool 2.23

5.455 Fenchol 0.43

5.495 trans-2-Menthenol 0.13

157

5.539 α-Campholenal 0.17

5.631 cis-2-Menthenol 0.11`

5.673 Pinocarveol 1.36

5.883 (+)-Borneol 1.17

5.959 Terpinen-4-ol 5.99

6.054 α-Terpineol 8.11

6.139 Sabinyl acetate 0.57

6.231 cis-Carveol 0.18

6.248 cis-Geraniol 0.18

6.304 cis-p-metha-1(7), 8-diene-2-ol 0.08

6.428 Geraniol 2.03

6.476 Carvoisomethone 0.27

6.634 Thymol 0.75

6.736 Bornyl acetate 0.04

7.109 Exo-2-hydroxycineole 0.19

7.215 Eugenol 0.49

7.306 Geranyl acetate 0.39

8.307 Durohydroquinone 0.23

8.645 Epiglobulol 0.20

8.693 Spathulenol 2.07

8.807 Globulol 1.36

8.856 Viridiflorol 0.65

8.897 Rosifoliol 0.17

8.959 α-Selinene 0.50

9.206 Alloaromandendrene 0.13

9.291 Isoaromandendrene epoxide 0.15

9.427 Farnesol 0.10

9.547 Eudesma-4,11-dien-2-ol 0.08

9.766 Benzyl benzoate 0.2

Total composition (%) 95.78

Oil yield (%) 0.95

Initial phytochemical evaluation of the bark of Callistemon citrinus

shows the presence of some secondary metabolite like steroids,

terpenoids and flavonoids which might be responsible for majority of its

pharmacological activity (Netala et al., 2015). Alkaloids are known to

possess antimalarial activity and quinine which belongs to an alkaloid

class (secondary metabolite) is the most essential and oldest antimalarial

drug (Mazid et al., 2011). Records show that linalool and thymol which

are components of terpenoids found in volatile oils exhibit antiplasmodial

158

activity in vitro (Babili et al., 2011; Goulart et al., 2011). β-Pinene,

linalool and 1,8-cineole obtained from the essential oil of Cymbopogon

species from Benin have been reported to have antitrypanosomal

activities with IC50 of 47.37, 39.32 and 83.15 μg/mL (Kpoviessi et al.,

2014a). This is also in agreement with our work as these components

stated above with other secondary metabolites in Callistemon citrinus

may be responsible in their synergistic activity to bring about the

antiplasmodial and antitrypanosomal action in the leaf, flower and seed

volatile oils.

4.4.4 Antiplasmodial Activity

The antiplasmodial activity of the volatile oils derived from the aerial

part (flowers, leaves and seeds) of Callistemon citrinus was not revealed

against the malaria parasite at 50 μg/mL, and their percentage viability

values were 78.01 ± 8.05%, 74.36 ± 4.30% and 93.09 ± 2.02%,

respectively. Only those samples which are able to bring about a

significant decrease in pLDH at a concentration of 50 μg/mL to less than

20% are considered active against the malaria parasite (Figure 4.12). The

standard drug chloroquine used as positive control and for basis of

comparison has an IC50 of 0.012 μM. Different volatile oils extracted from

several aromatic plants have shown antiplasmodial activity and this have

been documented in literature. (Babili et al., 2011; Milhau et al., 1997;

Tchoumbougnang et al., 2005; Valentin et al., 1995).

159

Figure 4.12: pLDH Malaria Assay (Single Concentration).

4.4.5 Antitrypanosomal Activity

Three volatile oils obtained by hydrodistillation from the flower, leaf

and seed of Callistemon citrinus affected the viability of T. b. brucei at a

fixed concentration of 25 μg/mL with percentage of viable parasites

approximated to be 0.51, 0.35 and 1.92% thereby displaying

antitrypanosomal property (Figure. 4.13). The antitrypanosomal activity

of the flower oil was the highest with IC50 of 16.67 μg/mL, while the

activity of leaf oil was the next displaying a moderate activity with an

IC50 of 26.00 μg/mL. The seed oil had the least activity with IC50 of

92.23 μg/mL. Bero et al. (2011) reported that samples with IC50 value of

≤ 20 µg/mL are considered as good or very potent whereas those

between 20-60 μg/mL are known to be moderate, and others with IC50>

100 μg/mL are seen to be non-active The pentamidine used as positive

control exhibited IC50 of 0.0066 μM. The essential oil components

postulated to be responsible for the antitrypanosomal activity of volatile

oils (β-pinene, linalool and 1, 8-cineole) were present in the three volatile

160

oils (Kpoviessi et al., 2014a; Larayetan et al., 2017). Available

information on the antitrypanosomal activity of the Callistemon genus is

very scarce.

Figure 4.13: Dose-Response Curve for Trypanosome Assay 1

1 X8 - Flower volatile oil, X9 - Leaf volatile oil and X10 - Seed volatile oil

Some components of volatile oils such as eucalyptol, cyclobutane, 6-

octane and citral obtained through GC-MS from the essential oils of

Eucalyptus citriodora, Eucalyptus camaldulensis, Cymbopogon citrates

and Citrus sinensis were suggested to be responsible for the in-

vitroantitrypanosomal brucei brucei and antitrypanosomal evansi action of

the various oils (Habila et al., 2010). The antitrypanosomal and

antiplasmodial effect of Ocimum grastissimum have been documented;

the volatile oil in the full flowering stage exhibits a moderate activity with

IC50 of 76.32 μg/mL against Trypanosome brucei (Kpoviessi et al.,

2014b).

161

4.4.6 Cytotoxicity Activity

Three essential oils were screened against Hela (human cervix

adenocarcinoma) cells/HEK 293 (human embryonic kidney) cells at a

concentration of 50 μg/mL prepared out of the stock solutions of the

volatile oils. All the three volatile oil samples (flower, leaf and seed)

tested were not cytotoxic at that concentration (Figure 4.14). None of

them did cause any significant cytotoxic effects at concentration of 50

μg/mL (reduced the viability of HeLa cells to below 50%) because their

percentage cell viability were greater than 70%.

Figure 4.14: Single Assay Concentration for Cytotoxicity.

Their non-cytotoxicity might be a hint of their safety as targeted drugs

for mammalian organisms, although there is need for an extensive

additional examination on Callistemon citrinus particularly in the area of

bioassay-guided fractionation on the aerial parts of the plant to see which

will yield an antitrypanosomal lead drug. Oral intake of volatile oils

demands some level of caution due to the potential toxicity of some

essential oils (Abdelouaheb and Amadou, 2012). So there is a need for a

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thorough cytotoxicity test to determine the appropriate concentration at

which any volatile oil could be recommended for oral use. When taken

orally, the volatile oil may possibly be transported to all other parts of the

body via the bloodstream, once in the body they link up with the

physiological functions in three different ways. The first mode of their

action is through the biochemical means where they interrelate with the

blood stream and intermingle with enzymes and hormone in the body.

The second mode of action is through physiological means; here they

operate on definite physiological function like the case of fennel volatile oil

that contains estrogen component which may be of help for female with

lactation or menstrual problem. Their third means of approach is via

psychological pathway whereby the volatile oil is inhaled and this triggers

a positive effect on the olfactory area of the brain (limbic system) by

experiencing an action stimulated by the inhaled volatile oil, this then

makes the mental and emotional performance of the individual to change

due to the message sent by the neuro-transmitter (Buchbauer, 1993;

Johnson, 2011; Shibamoto et al, 2010).

4.4.7 Antibacterial Potency of Seed Volatile Oil

The potency of the seed volatile oil and the antibacterial sensitivity

were quantified by determining the zone of inhibition as shown in Table

4.17. Extract from plants exhibit higher potency against gram-negative

than gram-positive bacteria in terms of zone of inhibition (Abdelhady and

Aly, 2012; Haque et al., 2012). The seed essential oil of this plant

displays a very good activity against gram-negative Vibro alginolyticus

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DSM 2171, Salmonella typhi ACC and Escherichia coli 0157:H7:ATCC

35150 with zone of inhibitions of 62.0 ± 4.0 mm, 57.0 ± 0.5 mm and

55.0 ± 1.0 mm, respectively with the highest activity recorded for Vibro

alginolyticus DSM 2171 as shown in the Table 4.17. These results were

consistent with the report about the oils from the leaf and aerial part of

Callistemon citrinus from Ethiopia (Aweke and Yeshanew, 2016). The

lowest activity was documented for Mycobacterium smegmatis ATCC

19420 at an inhibition zone of 38.0 ± 1.0 mm. The seed oil was found to

be more effective in terms of its ability to inhibit bacteria growth than the

standard drug ciprofloxacin. This might be as a result of various

components of the seed oil including eucalyptol, α-pinene, terpinen-4-ol,

α-terpineol, and α-phellandrene. These components have been confirmed

to possess antimicrobial potency (Chane-Ming et al., 1998; Riaz and

Chaudhary, 1990). This effect shows a relationship with the folkloric

utilization of this plant and confirms that it is an effective antimicrobial

plant that can be engaged as a substitute medicine for the management

of bacterial infection (Doughari et al., 2009)

Table 4.17: Inhibition Zone (mm) for Seed Volatile Oil of Callistemon

citrinus and Ciprofloxacin (Standard Drug).

Microorganism Concentration

Positive control Ciprofloxacin (mg mL-1)

Seed volatile oil (mg mL-1)

0.4 0.1 0.025 0.4 0.1 0.025

Escherichia coli 0157:H7:ATCC

35150 38.0 ± 4.0 32.0 ± 4.0 24.0 ± 0.9 55.0 ± 1.0 40.0 ± 1.0 20.0 ± 0.8

Vibro alginolyticus DSM 2171

33.0 ± 2.0 29.0 ± 2.0 23.0 ± 0.4 62.0 ± 4.0 45.0 ± 0.8 22.0 ± 0.5

Salmonella typhi 40.0 ± 1.0 34.0 ± 0.6 28.0 ± 3.0 57.0 ± 0.5 39.0 ±2.0 17.0 ± 0.5

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ACC

Staphylococcal enteritis ACC

40.0 ± 3.0 32.0 ± 1.0 28.0 ± 0.2 52.0 ± 2.0 40.0 ±0.9 25.0 ± 0.8

Staphylococcus aureus ACC

29.0 ± 1.0 24.0 ± 0.0 10.0 ± 3.0 56.0 ± 3.0 30.0 ± 5.0 19.0 ± 2.0

Listeria Ivanovii ATCC 19119

44.0 ± 4.0 38.0 ± 0.5 30.0 ± 1.0 48.0 ± 2.0 36.0 ± 3.0 20.0 ± 3.0

Mycobacterium smegmatis ATCC

19420 36.0 ± 0.6 25.0 ± 2.0 18.0 ±1.0 38.0 ± 1.0 21.0 ± 2.0 14.0 ± 0.8

4.4.8 Conclusion

The efficacy of the volatile oils of the leaves, flowers and seeds from

Callistemon citrinus was evaluated against trypanosomal and plasmodial

parasites. To the best of our knowledge, this is the first time the

antitrypanosomal and cell cytotoxicity of the essential oils of the seeds,

leaves and flowers of Callistemon citrinus would be reported. The three

oils were found not active against Plasmodium falciparum strain 3D7 in

vitro but exhibited good to moderate activity against trypanosomal brucei.

It is also noteworthy to say that the three oils were not cytotoxic to Hela

(human cervix adenocarcinoma) cells/HEK 293 (human embryonic

kidney) cells. The seed oil exhibited a very good antibacterial activity

against the various bacterial strains used which confirms its traditional

usage as an antimicrobial plant and proves that it can be used as an

alternative herbal drug to combat bacterial infections due to its non-toxic

nature.

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4.5 Phytochemical Investigation, Isolation and Characterization of

Bioactive Compounds Responsible for Antiplasmodial and

Antitrypanosomal Action from Crude Extracts of Callistemon

citrinus

4.5.1 Abstract

Different crude portion of Callistemon citrinus were extracted and

subjected to antimalarial, antitrypanosomal and cell cytotoxicity assay.

The methanol crude extract had the highest % yield (8.95%), followed by

the ethyl acetate extract (4.36%), and the least was DCM extract

(0.81%). Crude A and E (hexane leaf and DCM seed) extracts have the

highest antimalarial and antitrypanosomal activities with IC50 of (2.30

and 3.22) for antimalarial and (1.19 and 0.39) for antitrypanosomal

action. All the crude extracts were cytotoxic. Attempts to isolate

phytochemical compounds responsible for these activities were

unsuccessful.

4.5.2 Background

Plants serve as an imperative source of different pharmacologically

bioactive components with medicinal potential (Harvey, 2008).

Investigation of bioactive compounds from curative plants gives support

to the development of phytotherapeutic means of fighting different

ailments (Fennel et al., 2004). The World Health Organisation (WHO)

wholly recognizes the importance of herbal drugs for human health care

and has printed various policies guiding principle and criteria for these

botanical drugs (Hosseinzadeh et al., 2015).

166

The main cause of malaria infection is plasmodium parasite; they

contaminate the red blood cells (RBC) and bring about symptoms like

pain, chills, fever and sometime swelling. There are a lot of modern drugs

used to combat malaria but the strong resistance posed by malaria

parasites to conventional antimalaria drugs has spurred the interest of

several local societies living in rural areas and researchers to seek an

alternative in plants from their vicinities. Chloroquine which was very

cheap and effective is now considered ineffectual as a result of

multiplication of some resistant strains (Haddad et al., 2017). This reason

mentioned above is posing a serious challenge to the eradication of

malaria because the disease always resurfaces where it had been initially

stamped out and emerges in formally unaffected vicinities. The resistance

of P.falciparium to well recognised antimalarial drugs is now a common

thing in nearly every area of its prevalence (Maregesi et al., 2010; WHO,

2006). In the case of trypanosomiasis, the known drugs like homidium,

suramin, pentamidine and several others have also shown resistance to

the parasite causing this disease (Nwodo et al., 2015; Welburn et al.,

2009).

Due to the increase occurrence of malaria and trypanosomiasis in sub

Saharan Africa, there was a need to seek for an alternative to the current

drugs available in the market which are used to combat these diseases.

Such an alternative plant that could be used as traditional medicine must

be simple, cost-effective, possess higher efficiency, and be able to

overcome the effect of resistance posed by parasites to modern synthetic

167

drugs. The plant of interest must be able to combat these parasites by

inhibiting or totally destroying their growth without necessarily affecting

or killing human cells. We have about 121 genera with approximately

3000-5000 species comprising of trees and shrubs belonging to the

Myrtaceae family. Callistemon citrinus falls within this group.

Records have shown that plant can be used as an herbal remedy

against diarrhea; rheumatism, dysentery, cough and bronchitis, its cardio

protective and relaxant properties coupled with its herbicidal and

antimicrobial action have been documented. (Sutar et al., 2014; Netala et

al., 2015;Oyedeji et al., 2009; Ali et al., 2011; Goyal et al., 2012).

Present work seeks to evaluate the crude and active fractions of this

plant as an alternative herbal medicine to curb plasmodial and

trypanosomal parasites and examines their in vitro cytotoxicity against

Hela cells.

The aim of this work was to isolate the phytochemical compounds in

the crude extracts of Callistemon citrinus and to verify the antimalarial,

antitrypanosomal and cell cytotoxicity of both the crude extracts and the

bioactive compounds isolated from the various crudes. There is scanty

information on the antitrypanosomal, antimalaria and cytotoxicity

activities using Hela (human cervical carcinoma) cells of Callistemon

citrinus.

4.5.3 Percentage Yield

900 g of the powered leaves were used for the extraction, the various

percentage yield are given in Table 4.18. Both the leaves and seeds of the

168

plant were used to isolate the bioactive compounds via column

chromatography and thin layer chromatography.

Table 4.18: Percentage Yield of Various Crude Extracts.

Amount of Material used

Hexane Crude (% Yield)

DCM Crude (% Yield)

EA Crude (% Yield)

Me Crude (% Yield)

Leaves (900)g 2.71 0.81 4.36 8.95

Seed(500)g - 0.97 - -

Various plants from the Myrtaceae family have been used to seek an

alternative to modern antimalaria drugs. It has been recorded that

Eucalyptus robusta and a compound (robustadial B) isolated from this

plant exhibit good antimalaria activity (Xu et al., 1984). The stem bark

from Psidium guajava (Myrtaceae) has been shown to be very active

against chloroquine sensitive P. falciparum D 10 strain with an IC50 of

10-20 μg/mL (Nundkumar and Ojewole, 2002;). The flora buds from

Eugenia caryophyllis (Clove pepper) from the Myrtaceae family is also

used to treat malaria (Gbadamosi et al., 2011). The presence of alkaloid

secondary metabolite is responsible for the antimalaria activity of most

medicinal plants (Ajaiyeoba et al., 2006). Since ancient times alkaloids

have been the most significant among secondary metabolites used in the

production of drugs. A good example of this is quinine derived from the

Cinchona succirubra from the family of Rubiaceae. For over three hundred

years now, quinine has been employed for the treatment of malaria. The

same is recorded for the flavonoids found to inhibit the malaria parasite

growth via L-glutamine (Elford, 1986; Kaur et al., 2009).

169

4.5.4 Isolation of Fractions

About 46.75 mg DCM seed and 60 mg of the hexane leaf extracts of

Callistemon citrinus were prepared by dissolving in about 5 mL

dichloromethane-ethyl acetate (5:1) and hexane-ethyl acetate mixture

(8:2). They were loaded on the sand-silica gel bed in the column using a

Pasteur pipette. The extract was eluted with different mixtures of hexane-

ethyl acetate and dichloromethane-ethyl acetate solution as the mobile

phase (90:10-10:90) about 53 fractions of hexane leaf and 48 fractions of

DCM seed of 20 mL each in a test-tube were collected from the column.

Fractions with similar TLC behavior were pooled together based on the

result of the TLC guide, about four sub-fractions were obtained for the

DCM seed and were labelled F1-F4 (F1: 1, F2: 8-13, F3: 14-21, F4: 22-

29) and also four sub- fractions for the hexane leaf E1-E2 (E1: 15, E2:

19-32) and S1-S2 (S1: 19-31, S2: 32-51). All the fractions obtained

above were mixtures as shown by the TLC plate and visualized under UV

lamp. Some of the fractions were re-columned and eluted with different

solvent mixtures, on the TLC plate and visualized under UV lamp. They

appear to be a single spot but it was discovered after the NMR analysis

that they were still mixtures.

4.5.5 Antitrypanosomal Action

The antitrypanosomal action of the crude extracts and fractions were

tested via Trypanosomal brucei assay (Table 4.19 and Figure 4.15; Table

4.20 and Figure 4.16). The % viability of the crude extracts and the

various fractions at 50 μg/mL were able to reduce the trypanosomal

170

parasites to less than 20%, the IC50 of both extracts and fractions were

less than 20 μ/mL (Table 4.19) and ranges from 1.19 to 0.41. Crude

extracts A and E have the highest antitrypanosomal activities with IC50 of

1.19 and 0.39 and were selected them for isolation in column

chromatography. The IC50 values less or equal to 20 μg/mL are

considered very potent against the targeted parasite (Bero et al., 2013).

Table 4.19: IC50 for A-E.

Compound IC50 (μg/mL for samples, μM for

pentamidine)

A 1.190

B 3.023

C 0.4046

D 0.4140

E 0.3911

Pentamidine 0.003911

The crude extracts and fractions obtained from it exhibited very good

antitrypanosomal activities, but the result obtained from this work needed

to be checked in line with cytotoxicity to be sure that the crudes and

fractions are not toxic on Hela cell lines or any other cells. By so doing,

we were able to establish that their cytotoxicity is not due to general

toxicity on both the parasites and human.

171

Figure 4.15: Dose Response for Trypanosomal Assay for A-E.

Figure 4.16: Dose Response for trypanosomal assay for F1-E2.

Table 4.20: IC50 for F1-E2.

Compound IC50 (μg/mL for samples, μM for

pentamidine)

F1 0.6902

F2 0.4472

F3 2.662

F4 9.068

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S1 12.32

S2 3.537

E1 15.75

E2 41.04

Pentamidine 0.006274

4.5.6 Antimalarial Activity

The antiplasmodial activity against malaria parasite of Plasmodium

falciparium 3D7 strains were examined by using both the crude extracts

and fractions derived from the crude (Figures 4.17-4.19; Tables 4.21-

4.23). They were both able to decrease the viability of Plasmodium

falciparium to below 20%. Their IC50 were ≤ 10 μg/mL. Cos et al. (2006)

opined that IC50 below 10 μg/mL shows a very promising antimalarial

action. The IC50 for the crude extracts A-E ranges from 2.30 to 4.98 with

crude A and E (2.30 and 3.22) having the highest antimalarial activities

and were selected for isolation.

Figure 4.17: Dose Response pLDH Assay for A-E.

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Table 4.21: IC50 for A-E.

Compound IC50

μM for Chloroquine, μg/mL for sample

A 2.305

B 3.611

C 4.359

D 4.988

E 3.224

Chloroquine 0.01182

Table 4.22: IC50 for F1-S2.

Compound IC50

μg/mL for samples, μM for Chloroquine

F1 8.507

F2 2.878

F3 8.145

S1 0.897

S2 1.209

Chloroquine 0.01127

Figure 4.18: Dose Response pLDH Assay for F1-S2.

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Figure 4.19: pLDH Assay Single Concentration for F1-E2.

Table 4.23: Percentage Viability for F1-E2.

Compound Viability % SD

F1 12.529490 8.307570

F2 8.628767 6.193087

F3 12.888330 3.138833

F4 82.622860 13.795830

S1 12.788650 7.076471

S2 17.493440 2.246051

E1 88.603520 4.003422

E2 109.256700 1.353269

4.5.7 Cytotoxicity Activity

The cytotoxicity the crude extracts (A-E) and the fractions derived

from it (F1-E2) were assessed against Hela (human cervix

adenocarcinoma) cells at a concentration of 50 μg/mL, (Figures 4.20-

4.23; Tables 4.24-4.27). It was discovered that all the crude and some of

the fractions were cytotoxic except for fractions F4, S1, E1 and E2. They

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caused significant cytotoxic effect because they were able to reduce the

viability of Hela cells to below 50% as seen in their cell viabilities which

were less than 70%. Table 4.28 generalizes antimalarial and

antitrypanosomal properties, as well as cytotoxicity of the crude extracts

and fractions of Callistemon citrinus.

Figure 4.20: Cytotoxicity Assay: Single Concentration Screen.

A-D: Hex, EA, DCM and ME leaf extracts, E: DCM seed extract

Table 4.24: Percentage Viability for Crude A-E.

Compound at 50 μg/mL Viability % SD

A 27.812150 0.9301538

B 26.739700 6.201189

C 10.933230 0.5868806

D 7.778297 2.968739

E 19.358250 0.08377581

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Figure 4.21: Cytotoxicity Assay: IC50 for Crude A-E.

Table 4.25: IC50 for Crude A-E.

Compound (μM for Emetine, μg/mL for Samples)

IC50

Emetine 0.01864

A 92.08

B 57.4

C 37.39

D 41.07

E 32.45

Figure 4.22: Cytotoxicity Assay for fraction F1-E2: Single Concentration

Screen.

F1-F4: fractions from DCM seed, S1-S2, E1-E2: Fractions from Hex leaf

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Table 4.26: Percentage Viability for Fractions F1-E2.

Compound at 50 μg/ml Viability % SD

F1 -3.099163 1.946565

F2 12.855970 1.236943

F3 11.858250 2.195205

F4 105.390600 1.802407

S1 100.147900 3.293803

S2 10.848490 2.445191

E1 95.171180 4.765754

E2 82.250690 3.921810

Figure 4.23: Cytotoxicity Assay: IC50 for Fractions F1-E2.

Table 4.27: IC50 for Fractions F1- S2.

Compound (uM for Emetine,

ug/ml for Samples) IC50

F1 44.63

F2 93.04

F3 82.53

S2 64.81

Emetine 0.02897

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Table 4.28: Antimalarial, Antitrypanosomal Characteristics and

Cytotoxicity of the Crude Extracts and Fractions of Callistemon citrinus.

Samples Anti-

malarial cell viability

Anti-trypanosomal cell viability

Cyto-toxicity cell viability

(%)

Anti-plasmodial activity

IC50 value

Anti-trypanoso

mal activity

IC50 value

Cyto-toxicity activity

IC50 value

S.I on 3D7. (S.I on T. b brucei)

Crude A 1.85 ± 1.22 -4.34 ±0.91 27.81 ± 0.93 2.30 1.19 92.1 40.04 (30.49)

Crude B 8.43 ± 7.98 -1.11 ± 0.64 26.74 ± 6.20 3.61 3.02 57.4 15.90

(48.23)

Crude C 4.75 ± 1.72 4.54 ± 3.52 10.93 ± 0.59 4.56 0.40 37.4 8.20 (93.50)

Crude D 6.36 ± 3.35 2.27 ± 1.15 7.78 ± 2.97 4.98 0.41 41.1 12.76 (105.38)

Crude E 7.69 ± 3.14 10.22 ± 1.44 19.35 ±0.08 3.22 0.39 32.5 6.52

(79.26)

Fractions

F1 12.53 ± 8.30 -1.54 ± 0.09 -3.09 ± 1.95 8.51 0.69 44.63 5.24 (64.68)

F2 8.63 ± 6.19 -1.46 ± 0.01 12.86± 1.24 2.88 0.45 93.04 32.30 (206.75)

F3 12.89 ± 3.14 -1.05 ± 0.42 11.86 ± 2.19 8.15 2.66 82.53 10.12 (31.02)

F4 82.62 ±13.79 -1.84 ± 0.00 105.39 ± 1.80 - 9.07 - -

S1 12.79 ± 7.08 -1.52 ± 0.02 100.15 ± 3.29 0.89 12.32 - -

S2 17.49 ± 2.25 -0.45 ± 0.16 10.85 ± 2.45 1.21 3.54 64.81 53.56 (18.30)

E1 88.60 ± 4.00 -1.66 ± 0.02 95.17 ± 4.77 - 15.75 - -

E2 109.26 ±1.35 -1.79 ± 0.04 82.25 ± 3.92 - 41.04 - -

4.5.8 Phytochemical Analysis

The phytochemical tests carried out on the five different extracts of

Callistemon citrinus shown in Table 4.29 reveals the presence of a variety

of bioactive secondary metabolites in the leaves of this plant and may be

accountable for their medicinal attributes (Benavente-Garcia et al., 1997).

Among these phytochemicals, glycosides, steroids were absent in all the

five extracts of this plant, alkaloids were present in all the plant extracts,

saponins and phenols were absent in hexane, dichloromethane (leaf and

seed) and ethyl acetate extracts but present in the methanol extracts.

Terpenoids were present in varying amount in all five extracts, flavonoids

were also present in all extracts except for dichloromethane seed extract,

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and tannin was present in all extracts except for ethyl acetate. It has

been documented that secondary metabolites contribute considerably to

the biological activities of therapeutic plants such as hypoglycemic,

antidiabetic, antioxidant, antimicrobial, anti-inflammatory,

anticarcinogenic, antimalarial, anticholinergic, antileprosy activities etc.

(Dasgupta et al., 2013; Dose et al., 2009) . Consequently, the preliminary

result indicates that activity may be due to alkaloids or terpenoids.

Table 4.29: Phytochemical Analysis of Various Extracts of Callistemon

citrinus.

Phytochemicals Hexane leaf Dichloromethane

leaf Ethyl acetate

leaf Methanol

leaf DCM seed

Alkaloids + + + + +

Steroids - - - - -

Phenols - - - + -

Terpenoids ++ ++ + + +

Flavonoids + + + + -

Saponins - - - + -

Tannins + + - + +

Glycosides - - - - -

+ Present; ++ present in more quantity; - absent

Tannins possess amazing stringent properties which include anti-

diarrheal, anti-hemostatic and anti-hemorrhoid activity and may be

responsible for its broad spectrum anti-microbial properties against

bacteria, fungi and viruses. They are also recognized to accelerate the

healing of wounds and swollen mucous membranes (Kumari et al., 2012;

Negi et al., 2011; Salah et al., 1995). Flavonoids are also present in all

the extracts of these medicinal plants. They function as potent water-

soluble antioxidants and free radical scavengers, which thwart oxidative

cell damage and also possess strong anticancer activity (Kumari et al.,

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2012; Negi et al., 2011; Salah et al., 1995). In addition to these, they

also aid in managing diabetes induced oxidative stress. Terpenoids are

basically lipids and are recognized for their aromatic and fragrance

qualities, their functions also includes growth regulating, color, odor and

antimicrobial activity, they are known to contribute positively in the

prevention of several diseases including cancer (Satyanarayana et al.,

2008; Setchell and Cassidy, 1999; Shah et al., 2009).

4.5.9 Description of Active Principle

The bioactive fractions were greenish and yellowish crystalline

powdered. The characterization and elucidation showed it was a mixture

and not a pure compound (Figure 4.24-4.26).

Figure 4.24: 1H NMR Spectral Examination of Column Fraction 17

(Hexane Fraction of C. Citrinus Leaves).

181

Figure 4.25: 13C Spectral Examination of Column Fraction 17 (Hexane

Fraction of Leaves of C. Citrinus).

Figure 4.26: Crystal Image of the Suspected Pure Compound.

4.5.10 FT IR Spectrum of Hexane Crude Extract of Callistemon

citrinus

The FT IR spectrum of the hexane crude leaf extract of Callistemon

citrinus shown in Figure 4.27 below exhibited characteristic absorption

band at about 1700 cm-1 which can be attributed to C=O stretching due

to amide bond in the plant, it also showed absorption band at about 3400

182

cm-1 representing O-H stretching of alcohol and phenol, while the peak

at about 893 cm-1 signifies =C-H bending of alkenes.

Figure 4.27: FT IR Spectrum of the Hexane Crude Leaf Extract.

4.5.11 Conclusion

All the crude extracts and the fractions derived from them exhibited

high antimalaria and antitrypanosomal activities, but were toxic to Hela

cells. This is an indication that they are not safe to be used as targeted

drugs for mammalian organisms. Column chromatography attempt on the

fractions that were not cytotoxic produced no pure compounds.

4.6 Silver Nanoparticles Mediated by Callistemon citrinus

extracts: Antimalarial, Antitrypanosomal, and Antibacterial

Efficacy

4.6.1 Abstract

The three biosynthesized AgNPs acquired from the reduction of AgNO3

by the aqueous crude extracts of Callistemon citrinus were characterized

by means of SEM, TEM, EDS, XRD, UV-visible and FTIR. The XRD revealed

183

that the AgNPs were crystalline in nature and the TEM showed that the

shapes were spherical with an average size of 29 nm. The SEM and EDS

demonstrated triangular shaped materials and that the AgNPs were made

up of silver and oxygen only, absorption spectra confirm by UV-VIS

signifies the dispersed nature of the synthesized nanoparticles with

absorption band observed at 280 nm for the leaf. FTIR had absorption

bands at about 1700 cm-1 in all spectra‟s establishing the C=O stretching

owing to amide bond, another remarkable peak at 3400 cm-1 was seen in

the crude extract which was ascribed to the O-H stretching from water as

a result of the aqueous nature of the plant extracts used. It is interesting

to know that this peak was not seen in the AgNPs demonstrating the

development of calcined AgNPs, in addition to this, peak at 420 cm-1 was

observed for all the three nanoparticles synthesized and this shows the

successful synthesis of the AgNPs. The antimicrobial activities of the of

the AgNPs was also confirm via both gram-positive and gram-negative

bacteria strains with a very significant inhibitory action, MIC values of

7.8125 mg/mL were documented for all the silver nanoparticles. Potent

antiplasmodial activities with IC50 ranging from 2.99-5.34 μg/mL were

also recorded and a poor IC50 of 107.30 μg/mL for antitrypanosomal

activity of the leaf AgNP was also documented.

4.6.2 Background

The significant role of nanotechnology in present day research is

gaining popularity among different researchers; this has led to the growth

of research in different areas such as drug design, environmental

184

remediation, mechanics, cosmetics, medicine, etc. Nanoparticles are

inorganic/organic materials with size between 1-100 nm; their small size

in relation to their large surface to volume ratio makes them to be

exceptionally significant. Several applications of nanoparticles in sensor

technology, pharmaceuticals industry, processed food, optoelectronics,

molecular biology, drug delivery and biomedical system, biomimetric

materials, production of polymeric membranes for filtrations, gas

separation and waste treatment due to the large surface energy,

extensive Plasmon excitation and specific electron structures induced by

the efficient transition linking the molecular and metallic states (Choi and

Wang, 2011; Kim et al., 2014; Ng et al., 2013; Phoon and Jasimah,

2010; Stark et al., 2015; Wilczewska et al., 2012).

Nanoparticles are synthesized through various methods like solvent

dispersion, ionic gelation, supercritical fluid extraction, solid state

reactions, chemical reactions, co-precipitation and polymerization

techniques. These procedures involve the use of chemicals that are costly,

non-biodegradable and environmentally unfriendly. For this reason

researchers have been busy seeking for alternative methods of synthesis

using non-toxic and environmentally friendly biological methods and

materials (green synthesis). Biogenic mode of using different plant

extracts and microorganisms through the route of green synthesis is

useful due to its reduced environmental impact coupled with the

generation of large quantity of nanoparticles that are not contaminated

but are cost-effective, simple energy conserving with well-defined size,

185

morphology and compatible in the area of food and medical application

(Hutchison, 2008; Tagad et al., 2013; Yamini et al., 2011).

Nanoparticles produced from green synthetic route are also found to

have antioxidant properties (Naveena and Prakash, 2013), curative

potential (Zhang et al., 2008) and antimicrobial activity (Morones et al.,

2005; Muthuswamy et al., 2010; Nabikhan et al., 2010; Ruparelia et al.,

2008; Savithramma et al., 2011; Sondi and Salopek-Sondi, 2004).

Different plants parts like seeds, flower, stem, fruits, skin or even their

extracts are now used for making nanoparticles production through green

synthesis.

The function of these extracts or plant parts is to reduce and stabilize

the nanoparticles (Kumar and Yadav, 2009). This is brought about as a

result of the diverse plant metabolites like amino acids, alkaloids, tannins,

saponins, flavonoids, enzymes, vitamins and terpenoids embedded in the

plant which are already established to possess therapeutic activity

(Kulkarni and Muddapur, 2014). Plant extracts have the ability to reduce

metal ions, and this has brought about extensive concentration to green

synthesis over the years (Iravani et al., 2011; Kumar and Yadav, 2009;

Li, 2010; Marshall et al., 2007; Park et al., 2011)

Several application of synthesized nanoparticles obtained from

different techniques have been found to have in vitro diagnostic relevance

(Chen et al., 2012; Doria et al., 2012; Youns et al., 2011). It has been

observed that silver nanoparticles exhibit antimicrobial properties against

animal and human pathogens (Kandasamy et al., 2012, Lara et al., 2011;

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Singhal et al., 2011).The efficacy of nanoparticles derived from silver

metal against microbial and cancer ailment have been reported (Ghosh et

al., 2012a, 2012b). Nanoparticles are recognized to obstruct protein and

DNA replication (Chaloupka et al., 2010).

Numerous applications of metal nanoparticles in agriculture and crop

production, antimicrobial food packaging, in wastewater effluent

treatment etc have all been documented (Duran et al., 2007; Espitia et

al., 2012; Khot et al., 2012; Nair et al., 2010; Ojemaye et al., 2018).

Silver nanoparticles have been established to boost larvicidal activity

against filariasis and malaria vector (Rajakumar and Rahuman, 2011;

Santhoshkumar et al., 2011). The antiplasmodial, anticancer and

antifungal activities of silver nanoparticles have been documented

(Ponarulselvam et al., 2012; Ravindra et al., 2010; Subramanian, 2012;

Vivek et al., 2011). The antimicrobial ability of silver nanoparticles relies

on the size and environmental states like pH and ionic strength. The

mechanism of antimicrobial potency of silver nanoparticles is through the

slow discharge of toxic silver ions initiated by oxidation inside and outside

the cell, which tend to affect the membrane permeability of the microbial

cells (Liu et al., 2011).

From a variety of metals exhibiting antimicrobial properties, silver has

the most efficient antibacterial activity and has been found to be the least

toxic to animal cells; this is why it is enormously used for therapeutic

treatment. During World War 1 it was used to treat wounded soldiers in

order to stall microbial growth (Ankanna et al., 2010), the therapeutic

187

effectiveness have been well acknowledged for over 200 decades (Prabhu

and Poulose, 2012). Silver is employed in a nitrate form to bring about

antimicrobial action but when added to plant extracts through green

synthetic route, it amplifies the antimicrobial potency of the nanoparticles

formed due to larger surface area brought about by the smaller size. A

considerable disparity in the chemical component of plant extracts from

the same species collected from different locations or environment may

bring about diverse result in the application of silver nanoparticles.

In this study, we report on the synthesis, characterization and

antimicrobial, antiplasmodial and antitrypanosomal properties of silver

nanoparticles brought about by the reduction of AgNO3 using plant parts

(leaves, flowers and seeds) of Callistemon citrinus. To the best of our

knowledge, no study has reported the antimicrobial, antiplasmodial and

antitrypanosomal properties of the green synthesized AgNPs of

Callistemon citrinus using its different plant parts.

4.6.3 Synthesis and Characterization

The synthesized AgNPs obtained from the reduction of AgNO3 by the

plant parts extracts of Callistemon citrinus were characterized by a

number of techniques prior to testing them for their antimicrobial,

antiplasmodial and antitrypanosomal properties.

X-ray diffraction spectra of silver nanoparticles obtained from the

reduction of AgNO3 by aqueous extract of (B) leaf (C) flower and (D) seed

parts of Callistemon citrinus is presented in Figure 4.28. It can be

observed that all the synthesized materials are crystalline in nature upon

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the reduction of AgNO3. Peaks observed at 2 values of 28o and 34o for

Figures 4.28 (B), (C) and (D) indicate the successful synthesis of AgNPs.

These peaks are not observed in the diffraction pattern of Figure 4.28 (A).

Ojemaye et al, 2017a reported that diffraction peaks at (111), (200),

(220) and (311) are characteristic peaks of metal nanoparticles and these

peaks are also observed in the diffractogram in Figure 30 confirming the

successful synthesis of AgNPs. The crystallite size of all synthesized

materials with (111) diffraction peak using Scherer formula showed that

all synthesized materials are in the size range of 25-32 nm.

Figure 4.28: X-ray Diffractogram of (A) Callistemon citrinus Extract and

AgNPs Obtained from (B) Leaves, (C) Flowers, and (D) Seeds.

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Figure 4.29 shows the FTIR spectra of (A) plant extract, (B) leaf-

AgNPs, (C) flower-AgNPs, and (D) seed-AgNPs. Adsorption bands

observed at 1700 cm-1 in all spectra are characteristic of the C=O

stretching due to amide bond in the plant. A broad peak observed at 3400

cm-1 in the spectra of plant extract (Figure 4.29A) is attributed to O-H

stretching from water since the plant extract is an aqueous one. This

band is obviously missing from the spectra of Figures 4.29 B, C, and D

indicating the formation of calcined AgNPs. Also, stretching frequency

observed at 420 cm-1 in Figures 4.29 B, C and D which is attributed to Ag-

O band further confirms the successful synthesis of silver nanoparticles,

this band is however not observed in the infrared spectra of the plant

extract (Figure 4.29A). Result similar to this was reported recently

(Ojemaye et al., 2017b, Devaraj et al., 2013). The FTIR further confirms

that some specific phytochemicals were accountable for the synthesis and

stabilization of AgNPs as shown from the different peaks explained above.

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Figure 4.29: FT IR Spectrum of (A) Callistemon citrinus Extract and AgNPs

Obtained from (B) Leaves, (C) Flowers, and (D) Seeds.

Transmission electron microscope (TEM) was used to determine the

shape, size and morphology of the synthesized materials (Figure 4.30).

From the micrographs, it can be observed that spherical shaped materials

were synthesized with an average size of 29 nm. The average size

obtained from the measurement using TEM complements the result

obtained from XRD therefore confirming that nanosized materials have

been successfully synthesized. Worth of noting is that the plant part of

Callistemon citrinus employed for the reduction of AgNO3 did not influence

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the shape and size of the synthesized materials. The same observation

was reported (Jyoti et al., 2016).

Figure 4.30: TEM Micrographs of Callistemon citrinus Mediated AgNPs

Obtained (A) Leaves, (B) Flowers, and (C) Seeds.

Scanning electron microscope (SEM) and electron diffraction

spectrophotometer (EDS) were used to assess the morphology and

composition of the materials obtained from the reduction of AgNO3 by

plant parts of Callistemon citrinus (Figure 4.31). From the images, it can

be seen that roughly triangular spherical shaped materials were

synthesized; this morphology is characteristic of AgNPs (Mittal et al.,

2016). Interestingly, EDS results show that the materials are composed

only of silver and oxygen (from water molecule that might be left in the

A B

C

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material after drying) with silver showing to possess about 89% of the

overall composition of elements in the different materials.

Figure 4.31: SEM images of Callistemon citrinus Mediated AgNPs Obtained

(A) Leaves, (B) Flowers, (C) Seeds, and (D) EDS of AgNPs.

Absorption spectra of the silver nanoparticles were measured using

UV-Visible spectrophotometer between 200–400 nm (Figure 4.32). Broad

peaks were observed around 235 nm for seed and flower mediated AgNPs

indicating the dispersed nature of the materials synthesized using these

plant parts. Absorption band observed at 280 nm for the leaf

nanoparticles indicates the slow reduction rate of AgNO3 using this plant

part. Similar observation for the absorption maxima of AgNPs are

obtained from other plants (Hyllested et al., 2015; Philip and Unni, 2011).

A B

C D

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Figure 4.32: Absorption Spectra of AgNPs Obtained with Different Plant

Parts.

4.6.4 Antitrypanosomal Activity

The antitrypanosomal activity of the three synthesized nanoparticles

from Callistemon citrinus leaves, flowers and seeds were examined by

Trypanosoma brucei assay, it was found that their % viability at 50 μg/mL

were correspondingly (19.740 ± 4.09%, 35.043 ± 0.76% and 76.520 ±

6.90%). Only leaf nanoparticles brought about a significant decrease to

20% in trypanosome parasites at a concentration of 50 μg/mL; the other

two synthesized nanoparticles (flower and seed) could not do so and were

therefore considered inactive (Figure 4.33).

The IC50 of the leaf nanoparticle obtained from the dose response

curve was poorly active against trypanosomes at 107.30 μg/mL (Figure

4.34). Bero et al. (2011) recorded that IC50 value of ≤ 20 μg/mL are regarded

as good or very potent while IC50 of between 20-60 μg/mL are considered as

194

moderate but IC50 > 100 μg/mL is termed not active. The

antitrypanosomal action of methanolic extract of Solanum schimperianum

from the Kingdom of Saudi Arabia gave an IC50 of 0.61 μg/mL and

another plant from the same region C. tuberculata showed an IC50 of

0.5 μg/mL (Abdel-Sattar et al., 2009). The presence of some secondary

metabolites such as alkaloids, terpenoids, flavonoids, saponins, tannins,

steroids and many others in plants may be responsible for their

antitrypanosomal activities (Atawodi and Alafiatayo, 2007; Atawodi and

Ogunbusola, 2009; Stephen, 2009).

Figure 4.33: Single Concentration of Trypanosome Assay.

Figure 4.34: Dose-Response Curve for Trypanosome Assay.

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4.6.4 Antiplasmodial Action

Three silver nanoparticles prepared from the leaf, flower, and seed

extracts of Callistemon citrinus were subjected to in vitro screening of

antiplasmodial activity against the malaria parasites (Plasmodium

falciparum strain 3D7), The synthesized leaf nanoparticles obtained from

Callistemon citrinus at a concentration of 50 μg/mL strongly decreased

the viability of Plasmodium falciparum, while that of the synthesized

flower also strongly decreased Plasmodium falciparum at the same

concentration to (0.423 ± 1.125). In addition, the synthesized seed

nanoparticle also showed strong % viability against same Plasmodium

falciparum strain. The synthesized nanoparticles were tested in duplicate

wells, and standard deviations (SD) were derived. Their percentage

viabilities were then plotted against logarithm of synthesized

nanoparticles concentration 250 to 0.11 µg/mL, 3-fold-dilutions and the

IC50 (50% inhibitory concentration) were obtained from the resulting

dose-response curve by non-linear regression (Figure 4.35). For

comparative purposes, chloroquine (an anti-malarial drug) was used as a

comparative drug standard and IC50 values yielded were in the range

0.01-0.05 μM. The IC50 for the leaf, flower, and seed silver nanoparticles

all showed strong antiplasmodial activity of (3.14, 2.99 and 5.34 μg/mL,

respectively). For crude extracts, IC50 values should definitely be below

100 μg/mL (Cos et al., 2006), although most promising antimalaria

extracts display IC50 values under 10 μg/mL (Krettli, 2009; Soh and

Benoit-Vical, 2007). The three nanoparticles exhibited an IC50 less than

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10 µg/mL showing they are all promising candidates for antiplasmodial

lead drug. Some medicinal plants of South Africa origin like Mimusops

caffra, Hypoxis colchicifolia and M.obtusifolia have been used by

traditional healers in the Zulu communities to treat malaria, but among

these three medicinal plants, it was established that Hypoxis colchicifolia

did not exhibit any antiplasmodial action as asserted by the Zulu

traditional healers (Grubben and Denton, 2004). Several medicinal plants

from Mali and Sao-Tome like Feretia apotanthera, Securidaca

longepedunculata, Guiera senegalensi and Morinda citrofolia have been

recommended by traditional healers in these regions to combat against

malaria parasite strains (Ancolio et al., 2002). Perusal of literature on the

antiplasmodial activity of both crude and silver nanoparticles obtained

from Callistemon citrinus revealed no report or documentation has been

done up to date.

Figure 4.35: Dose-Response Curve for pLDH Assay.*

* X4, X5 and X6 represent Ag nanoparticles of leaf, flower and seed

extracts, respectively

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4.6.6 Cytotoxicity Activity

The leaf, flower and seed nanoparticles with the crude samples they

were derived from were evaluated against Hela (human cervix

adenocarcinoma) cells and HEK 293 (human embryonic kidney) cells at a

concentration of 50 μg/mL (Figure 4.36). It was revealed that the

nanoparticles and the crude samples did not show any sign of cytotoxicity

since the samples did not cause any significant cytotoxic effects at a

concentration of 50 μg/mL (they did not reduce the viability of HeLa cells

to below 50%) because their percentage cell viability were greater than

70%.This might be a hint of their safety as targeted drugs for mammalian

organisms. The synthesized nanoparticles indicate a strong antiplasmodial

and very poor antitrypanosomal activities devoid of toxicity on HeLa cells

which strongly indicates that the effects on parasite cultures was not a

result of a general cytotoxic effect of the synthesized nanoparticles.

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Figure 4.36: Single Assay Concentration for Cytotoxicity.

4.6.7 Antibacterial Activity

Agar well diffusion method as described by Collin et al. (2004) was

used to test for the zone of inhibition of the nanoparticles material.

Microorganisms were cultured and inoculated in Nutrient Broth (oxoid),

this was incubated for a day (24 h) at a temperature of 37°C. McFarland

0.5 turbidity standard was used to standardized the inoculum before

inoculation to give a confluent growth. About 38 g of Mueller Hilton Agar

(Oxoid) was dissolved in 1 L of distilled water and the resultant mixture

was autoclave for about 30 min at 15 Ibs and 121°C, it was allowed to

cool for about one hour and about 15-20 mL were dispensed into

sterilized petri dishes and permitted to solidify in the various petri dishes.

In all the petri dishes containing Muller Hinton Agar, 6 mm diameter wells

were made via an uncontaminated cork borer. The bacterial culture was

adjusted to 0.5 McFarland turbidity standard and the test microbes (0.1

mL) were inoculated with a clean swab on the outer surface of the solid

medium in the petri dishes. Into each of the wells was fed 62.5-15.625

mg/mL of the nanoparticles extracts obtained from the stock solution and

labelled accordingly. The inoculated petri dishes were inverted and

incubated at 37°C for 24 h. After all-night incubation, the diameter of

each zone was measured and recorded. All tests were performed under

hygienic conditions.

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4.6.8 Determination of the Minimum Inhibitory Concentration

and Minimum Bactericidal Concentration

The microdilution procedure was adopted in order to evaluate the

minimum inhibitory concentration of different samples. To achieve this

250 μL of Mueller-Hinton broth was dispensed into each of Eppendorf

tubes. Concentrations ranging from 62.50 to 15.625 mg/mL of the

samples were prepared from the stock solution of 125 mg/mL in DMSO by

two fold serial dilution. Aliquot of 250 μL from the highest concentration

of the sample was added into the first tubes containing MHB to bring the

final volume to 500 μL. From this same tube 250 μL of the mixture was

removed and added to the second tube, the same thing was done for the

third tube through a twofold serial dilution and the contents were carefully

vortexed. Exactly 20 μL from the inoculums‟ suspension of each bacterial

strain (0.5 McFarland, ∼1 × 108 colony forming units (CFU) mL−1) was

afterwards added and vortexed to allow sufficient mixing of the extract

and broth. The positive and the negative control that were used are

ciprofloxacin and DMSO, respectively. The test was carried out in

duplicate and incubated at 37oC for 24 h. The MIC values of the extracts

were defined as the lowest concentration that showed no visible growth

when compared with the control containing only MHB while the MBC was

ascertained by the pour-plate method of all tube content without visible

growth in the MIC method above onto fresh Mueller-Hinton agar plates

and the culture was then incubated for 24 hours at 37°C. The lowest

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concentration of extracts that did not show any colony growth on the solid

medium after incubation period of 24 h was regarded as the MBC.

The synthesized nanoparticles from the aerial part of Callistemon

citrinus demonstrated significant inhibitory activities (Table 4.30) against

both gram-positive and gram-negative bacteria strains (Escherichia coli

0157:H7:ATCC 35150, Vibro alginolyticus DSM 2171, Salmonella typhi

ACC, Staphylococcal enteritis ACC, Staphylococcus aureus ACC, Listeria

Ivanovii ATCC 19119 and Mycobacterium smegmatis ATCC 19420.

Table 4.30: Inhibition Zone (mm) Showing Antibacterial Activities of the

Nanoparticles Derived from Callistemon citrinus with the Standard Drug

Ciprofloxacin against Bacterial Test Organisms.

Microorganism concentration

Positive control Ciprofloxacin (mg/mL)

Nano Leaf (mg/mL)

62.5 31.25 15.625 62.5 31.25 15.625

Gram-Negative Bacteria Strains

Escherichia coli 0157:H7:ATCC

35150 35.0 ± 4.0 30.0 ± 4.0 18.0 ± 0.9 18.0 ±3.0 12.0 ± 3.0 7.0 ± 3.0

Vibro alginolyticus DSM 2171

33.0 ± 2.0 25.0 ± 2.0 13.0 ± 0.4 13.0 ± 2.0 11.0 ± 5.0 9.0 ± 4.0

Salmonella typhi ACC

35.0 ± 1.0 32.0 ± 0.6 18.0 ± 3.0 13.0 ± 0.5 10.0 ± l.0 8.0 ± 2.0

Gram-Positive Bacteria Strains

Staphylococcal enteritis ACC

40.0 ± 5.0 32.0 ± 1.0 17.0 ± 0.2 20.0 ± 0.5 16.0 ± 0.1 8.0 ± 0.4

Staphylococcus aureus ACC

20.0 ± 2.0 15.0 ± 0.0 11.0 ± 2.0 10.0 ± 4.0 8.0 ± 1.0 7.0 ± 2.0

Listeria Ivanovii ATCC 19119

35.0 ± 6.0 30.0 ± 0.2 22.0 ± 1.0 18.0 ± 2.0 12.0 ± 2.0 10.0 ± 1.0

Mycobacterium smegmatis ATCC

19420 35.0 ± 6.0 32.0 ± 0.0 24.0 ± 1.0 13.0 ± 0.4 `10.0 ± 0.5 8.0 ± 1.0

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Microorganism concentration

Nano Seed (mg/ mL) Nano Flower(mg/mL)

62.5 31.25 15.625 62.5 31.25 15.625

Gram-Negative Bacteria Strains

Escherichia coli 0157:H7:ATCC

35150 18.0 ± 3.0 15.0 ± 3.0 8.0 ± 3.0 25.0 ± 3.0 18.0 ± 3.0 10.0 ± 3.0

Vibro alginolyticus DSM 2171

15.0 ± 2.0 12.0 ± 5.0 6.0 ± 4.0 15.0 ± 2.0 10.0 ± 5.0 8.0 ± 4.0

Salmonella typhi ACC

20.0 ± 0.5 18.0 ± 1.0 10.0 ± 2.0 15.0 ± 0.5 10.0±1.0 8.0 ± 2.0

Gram-Positive Bacteria Strains

Staphylococcal enteritis ACC

25.0 ± 0.5 23.0 ± 0.1 18.0 ± 0.4 22.0 ± 0.5 18.0 ± 0.1 15.0 ± 0.4

Staphylococcus aureus ACC

15.0 ± 4.0 15.0 ± 1.0 13.0 ± 2.0 10.0 ± 4.0 8.0 ± 1.0 8.0 ± 2.0

Listeria Ivanovii ATCC 19119

18.0 ± 2.0 15.0 ± 2.0 13.0 ± 1.0 25.0 ± 2.0 20.0 ±2.0 18.0 ± 1.0

Mycobacterium smegmatis ATCC

19420 15.0 ± 3.0 10.0 ± 0.1 8.0 ± 1.0 15..0 ± 0.5 15.0 ± 0.4 12.0 ± 0.5

The nanoparticles synthesized from the flower exhibited the highest

inhibitory activities (25.0 ± 3.0 mg/mL and 25.0 ± 2.0 mg/mL) against

Escherichia coli 0157:H7: ATCC 35150 a gram-negative bacteria and

Listeria Ivanovii ATCC 19119, a gram-positive bacteria strain at a

concentration of 62.5 mg/mL among the nanoparticles. Seed and flower

nanoparticles displayed the same inhibitory activity of 15.0 ± 2.0 mg/mL

against Vibro alginolyticus DSM 2171 at the highest concentration, while

seed AgNPs demonstrated good activities of 20.0 ± 0.5 mg/mL and 18.0

± 0.5 mg/mL against gram negative Salmonella typhi ACC at 62.5 mg/mL

and 31.25 mg/mL, respectively. The lowest activities of 10.0 ± 4.0

mg/mL were recorded for leaf and flower nanoparticle against gram-

negative Staphylococcus aureus ACC at 62.5 mg/mL. Same MIC values of

7.8125 mg/mL were recorded for all the bacterial strains (Table 4.31).

Similarly 31.25 mg/mL of leaf AgNPs was able to kill (bactericidal)

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Escherichia coli 0157:H7: ATCC 35150 but twice the amount was needed

by flower AgNPs to completely exterminate same Escherichia coli

0157:H7: ATCC. On the other hand the leaf AgNPs were bactericidal at a

concentration of 31.25 mg/mL against Staphylococcus aureus ACC and

the flower AgNPs were bacteriostatic against the same bacterial strain at

the same concentration (Table 4.32).

Table 4.31: Minimum Inhibitory Concentration (MIC) Values (mg/mL) for

Nanoparticles and Standard Drug.

Bacteria Nano leaf Nano seed Nano flower Ciprofloxacin DMSO

Escherichia coli 0157:H7:ATCC

35150 7.8125 7.8125 7.8125 15.625 0.5 mL VG *

Vibro alginolyticus DSM 2171

7.8125 7.8125 7.8125 15.625 0.5 mL VG

Salmonella typhi ACC

7.8125 7.8125 7.8125 7.8125 0.5 mL VG

Staphylococcal enteritis ACC

7.8125 7.8125 7.8125 15.625 0.5 mL VG

Staphylococcus aureus ACC

7.8125 15.625 7.8125 7.8125 0.5 mL VG

Listeria Ivanovii ATCC 19119

7.8125 7.8125 7.8125 15.625 0.5 mL VG

Mycobacterium smegmatis ATCC

19420 7.8125 7.8125 7.8125

15.625

1.5 mL

VG

* VG – visible growth

Table 4.32: Minimum Bactericidal Concentration (MBC) Values (mg/mL)

for Nanoparticles and Standard Drug.

Bacteria Nanoparticle

(Leaf) Nanoparticle

(Seed) Nanoparticle

(Flower) Ciprofloxacin

Positive control

DMSO Negative control

Escherichia coli 0157:H7:ATCC

35150

Bactericidal at 31.25 NG

Bactericidal at 31.25 NG *

Bactericidal at 62.5 NG

Bactericidal at

≤ 15.625 NG 0.5 mL VG *

Vibro alginolyticus DSM 2171

Bactericidal at 31.25 NG

Bactericidal at 31.25 NG

Bactericidal at 31.25 NG

Bactericidal at

≤15.625 NG 0.5 mL VG

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Salmonella typhi ACC

Bactericidal at 31.25 NG

Bactericidal at 31.25 NG

Bactericidal at 15.625 NG

Bactericidal at

≤15.625 NG 0.5 mL VG

Staphylococcal enteritis ACC

Bactericidal at 62.5 NG

Bactericidal at 31.25 NG

Bactericidal at 15.625 NG

Bactericidal at

≤15.625 NG 0.5 mL VG

Staphylococcus aureus ACC

Bactericidal at 31.25 NG

Bactericidal at 31.25 NG

Bacteriostatic at 31.25 VG

Bactericidal at

≤15.625 NG 0.5 mL VG

Listeria Ivanovii ATCC 19119

Bactericidal at 62.5 NG

Bacteriostatic at 15.75 VG

Bactericidal at 61.5 NG

Bactericidal at

≤15.625 NG 0.5 mL VG

Mycobacterium smegmatis ATCC 19420

Bactericidal at 62.5 NG

Bactericidal at 62.5 NG

Bacteriostatic at 31.25 VG

Bactericidal at

≤15.625 NG 0.5 mL VG

* NG= No growth ** VG= Visible growth

4.6.9 Conclusion

The synthesized silver nanoparticles from the leaves, flowers and

seeds of Callistemon citrinus exhibited exceptional antiplasmodial and

antibacterial activities devoid of any cytotoxic effect. It confirms the use

of this plant by traditional healers, so that the AgNPs could be used as an

excellent alternative to synthetic antiplasmodial and antimicrobial agent

to combat malaria and infectious diseases from different bacterial strains.

4.7 Synthesis, Characterization, Antimalarial, Antitrypanocidal and

Antimicrobial Properties of Gold Nanoparticles

4.7.1 Abstract

Biosynthesis of gold nanoparticles can be achieved by using

Callistemon citrinus seed extract as both reducing and capping agent.

Characterization of the AuNPs, in vitro antiplasmodial, antitrypanosomal

and the antibacterial action of both the biosynthesized gold nanoparticles

and the crude extracts of the plant were performed. The biosynthesized

AuNPs was verified by a color change immediately after the seed extract

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was added to the gold (III) chloride solution. Characterization of the

AuNPs was done by UV-Vis, X-ray diffraction (XRD), scanning electron

microscopy (SEM), energy dispersive X-ray (EDS), transmission electron

microscopy (TEM) and Fourier transform infrared (FTIR). The FT IR

showed an absorption peak at 230 cm-1 indicating absorption band for

gold nanoparticles, the morphology and composition of the AuNPs was

ascertained by SEM and EDS micrographs; uneven spherical shaped

nanoparticles were established by the SEM analysis and an average

particle size of about 37 nm as confirmed by the TEM analysis. The crude

extracts exhibited antitrypanosomal activities with an IC50 of 11.06,

33.66 and 37.1 μg/mL for the seed, flower and leaf. A poor

antitrypanosomal action was observed for the AuNPs, both the crude and

AuNPs were inactive against plasmodial parasite, but the antibacterial

activities of the nanoparticle were potent against gram positive and gram

negative bacterial strains.

4.7.2 Background

During the past decade metal nanoparticles have triggered serious

interest among researchers as a result of their well-defined physical,

chemical and biological properties. Natural products like biodegradable

polymers (chitosan), bacteria, fungi and extracts of different plant genus

are now adopted as stabilizing and reducing agents to serve as an

alternative in the inorganic synthesis of nanoparticles (Ahmed S. et al.,

2016a; Ahmed S. et al., 2016b; Krishnaswamy et al., 2014). The reason

for this is that the green route synthesis of nanoparticles is eco-friendly,

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simple, economical and comparatively reproducible (Kulkarni and

Muddapur, 2014; Mittal et al., 2014).

Biogenesis of gold nanoparticles via plant extracts is becoming more

accepted due to their compelling antibacterial activity, low-risk for clinical

research and the ease of gold salt reduction. The technique is

straightforward, it is a one-step approach, and it is profitable for a large

scale production. Gold nanoparticles possess electrical and harmonious

optical characteristic. They display surface plasmon resonance, which is

employed in drug conveyance, trace detector and diagnostic probes. Gold

nanoparticles through biogenic route possess a superb biocompatibility

and non-toxicity. Shapes of gold nanoparticles include nanoprisms,

nanotriangles, nanoplates, nanowires, nanocages, nanospheres,

nanostars, nanobelts, etc. Shape and size have a great impact on their

properties, particularly optical properties (Cao, 2004; Thakkar et al.,

2010). More attractive optical characteristic are exhibited by triangular

shaped nanoparticles than the spherically shaped (Ganeshkumar et al.,

2012).

Gold nanoparticles have transformed the field of medicine owing to

extensive application in direct drug delivery, imaging, analysis and

curative purposes brought about by the enormously small size, solidity,

tunable optical, non-cytotoxic, appreciative uppermost layer, physical and

chemical properties (Huang and El-Sayed, 2010; Thakkar et al., 2010).

The synthesis of gold, nickel oxide and mercuric oxide nanoparticles using

Callistemon viminalis have been reported (Kumar et al., 2011; Sone et

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al., 2016). The use of silver oxide and silver nitrate to prepare

nanoparticles mediated by Callistemon citrinus have also been

documented (Ravichandran et al., 2016; Paosen et al., 2017). The

secondary metabolites embedded in plant such as terpenoids, tannins,

flavonoids, proteins, amino acids, enzymes may be responsible for the

bioreduction of Au+ ions of HAuCl4⋅4H2O into metallic Au0 nanoparticles

using extracts from plant (Thakkar et al., 2010).

Callistemon citrinus is known as a rich source of compounds with

bioactive components since it produces different secondary metabolites

with important biological activities. Formation of strong antioxidant agents

and free radicals are major expressions observed from the growth of this

plant in different environment, this is made possible as a result of harsh

growth conditions and combination of high oxygen concentration and light

(Rajasulochana et al., 2012). This study reports on the synthesis and

characterization of gold nanoparticles by the reduction of aqueous HAuCl4

using the seed part of Callistemon citrinus extract and their anti-

plasmodium, anti-trypanocidal and anti-microbial activities were

assessed. Interestingly, no study to the best of our knowledge as

reported the bio-synthesis of gold nanoparticles using Callistemon citrinus

for their application for anti-plasmodium, anti-trypanocidal and anti-

microbial properties.

FT IR was employed for the determination of the functional groups on

plant extract and AuNPs (Figure 4.37).

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Figure 4.37: FTIR Spectra of Plant Extract and Gold Nanoparticles.

The FTIR spectra of AuNPs showed vibration frequencies at about 1680

cm-1 and 3400 cm-1 attributed to carbonyl stretch and N-H stretch

vibrations respectively arising from amide bonds from protein. These

stretching frequencies indicate that amino acid residues and protein

peptides can effectively bind to the surface of metals thereby functioning

as a coating agent on the surface of the nanoparticles (preventing

agglomeration) and serving as a reducing agent. The reducing ability

ensured that a peak was observed at about 500 nm attributed to Au-O

band. This Au-O band is however not observed in the spectra of the plant

extract (Figure 4.35). Similar observation was reported (Abdel-Raouf et

al., 2017).

Successful synthesis of gold nanoparticles was confirmed by the

change in color from Au (I) to Au (0) followed by scanning with UV-Visible

spectrophotometer. The UV-Visible spectrophotometer measurement

208

(Figure 4.38) showed an absorption band at 230 nm for gold

nanoparticles formed by plant extract reduction. This band has been

demonstrated to be the absorption band for gold nanoparticles with sizes

ranging from 1 to 50 nm (Henglein 1993).

Figure 4.38: Absorption Spectra of Gold Nanoparticles.

In other to determine the morphology and composition of the

synthesized material, SEM and EDS micrographs of the bio-synthesized

gold nanoparticles were recorded (Figure 4.39A and 4.39B). Irregular

spherically shaped nanoparticles were seen to have been formed from the

SEM micrograph (Figure 4.39A). Although, some agglomerations of the

particles were observed which could be as a result of the uneven

distribution of the plant extract in solution, the particles formed were still

well dispersed. EDS micrograph (Figure 4.39B) showed that pure AuNPs

were obtained after the bio-reduction of gold chloride salt. This result

agrees with the observations (Thirumurugan et al., 2012).

209

Figure 4.39: Images of AuNPs under (A) SEM, (B) EDS, and (C) TEM.

TEM image of AuNPs were observed to ascertain the morphology and size

of the synthesized material (Figure 4.39C). It can be observed that a

spherically shaped material was mainly formed. Other than these shapes,

small amount of triangular and rectangular nano-shaped material was

also formed. Size determination indicates that an average particle size of

around 37 nm was obtained for this bio-synthesized material.

4.7.4 Antitrypanosomal and Cytotoxicity Activities

The gold nanoparticles synthesized from the seed extract of

Callistemon citrinus were investigated with Trypanosoma brucei assay, it

was revealed that the % viability at 50 μg/mL was (103.19 ± 0.56%) and

this value was not able to bring a considerable reduction at 50 μg/mL of

A B

C

210

trypanosome parasites and are considered inactive, but the crude seed

from which it was synthesized from was able to reduce trypanosome

parasites to (0.54 ± 0.01%) which were considered very active. Similar

results were recorded for the crude flowers and leaves of the plant: 22.06

± 0.375% and 4.79 ± 0.82%, respectively. The three crudes were further

subjected to dose response test to ascertain the concentration of the

compound needed to kill 50% of the parasites in a culture (Figure 4.40).

It was discovered from the dose response curve that the IC50 of the

crude seeds, flowers and leaves of this plant were 11.06 μg/mL, 33.66

μg/mL and 37.1 μg/mL, respectively. An existing literature document

(Bero et al., 2013) stated that IC50 value of ≤ 20 μg/mL are considered

as good or very potent while IC50 of between 20-60 μg/mL are

considered as fair and The IC50> 100 μg/mL is termed not active. Based

on this observation the crude seed showed a very good activity while the

crude flowers and leaves showed moderate activity.

The AuNPs (seeds) were tested against Hela (human cervix

adenocarcinoma) cells/ HEK 293 (human embryonic kidney) cells at an

exact concentration of 50 μg/mL. It was found that the synthesized gold

(seeds) nanoparticles were not cytotoxic since they were not able to

reduce the viability of Hela cells to below 50% (89.66 ± 1.55%). One can

reliably establish from the above result that the killing of the parasite

cultures was not as a result of general cytotoxicity of the seed AuNPs.

211

Figure 4.40: Dose Response Curve for Trypanosome Assay.1

1 X1, X2 and X3 are crude seeds, flowers and leaves respectively

4.7.5 Antiplasmodial Properties

The antiplasmodial activity of the gold seed nanoparticle was also

examined, the % viability at 91.58 ± 8.04% were discovered to be

inactive as they were not able to bring reduction of pLDH to less than

20%.

4.7.6 In vitro Antibacterial Activity

The aqueous extract of the seed of Callistemon citrinus plant brought

about the reduction of gold ions. This may be due to the presence of high

amount of eucalyptol with α-terpineol and terpinen-4-ol present in the

seed. The in-vitro antibacterial potency of the synthesized gold seed

nanoparticles were evaluated against three gram-negative and four gram-

positive bacterial strains (Escherichia coli 0157:H7:ATCC 35150, Vibro

alginolyticus DSM 2171, Salmonella typhi ACC, Staphylococcal enteritis

ACC, Staphylococcus aureus ACC, Listeria Ivanovii ATCC 19119 and

212

Mycobacterium smegmatis ATCC 19420). It was discovered that the

AuNPs has a broad spectrum of activity against the bacteria strains.

The recorded inhibitory effect was highest for Staphylococcal enteritis

ACC and Staphylococcus aureus ACC (20.0 ± 0.5 and 20.0 ± 0.4) at a

concentration of 62.5 mg/mL and was found to compete favorably well

with the standard drug used as positive control while the lowest inhibitory

action of 13.0 ± 2.0 and 13.0 ± 0.0 was documented for Vibro

alginolyticus DSM 2171 and Mycobacterium smegmatis ATCC 19420

(Table 4.33). The smaller size of the AuNPs coupled with the large surface

area could be responsible for the enhanced membrane permeability and

subsequent cell damage (Kasthuri et al., 2009).

Table 4.33: Zone of Inhibition of the Synthesized AuNPs from Callistemon

citrinus and the Standard Drug.

Microorganism Concentration

Positive control Ciprofloxacin (mg mL-1)

Au Seed nano (mg mL-1)

62.5 31.25 15.625 62.5 31.25 15.625

Gram-negative bacteria strains

Escherichia coli 0157:H7:ATCC

35150 35.0 ± 4.0 30.0 ± 4.0 18.0 ± 0.9 15.0 ± 3.0 12.0 ± 3.0 7.0 ± 3.0

Vibro alginolyticus DSM 2171

33.0 ± 2.0 25.0 ± 2.0 13.0 ± 0.4 13.0 ± 2.0 12.0 ± 5.0 9.0 ± 4.0

Salmonella typhi ACC

35.0 ± 1.0 32.0 ± 0.6 18.0 ± 3.0 15.0 ± 0.5 9.0 ± 1.0 7.0 ± 2.0

Gram-positive bacteria strains

Staphylococcal enteritis ACC

40.0 ± 5.0 32.0 ± 1.0 17.0 ± 0.2 20.0 ± 0.5 13.0 ± 0.1 9.0 ± 0.4

Staphylococcus aureus ACC

20.0 ± 2.0 15.0 ± 0.0 11.0 ± 2.0 20.0 ± 4.0 10.0 ± 1.0 8.0 ± 2.0

Listeria Ivanovii ATCC 19119

35.0 ± 6.0 30.0 ± 0.2 22.0 ± 1.0 15.0 ± 2.0 12.0 ± 2.0 10.0 ± 1.0

Mycobacterium smegmatis ATCC

19420 35.0 ± 6.0 32.0 ± 0.0 24.0 ± 1.0 13.0 ± 0.0 11.0 ± 1.0 9.0 ± 0.3

213

4.7.7 Conclusion

AuNPs was successfully synthesized from the seed extract of

Callistemon citrinus. It was tested against plasmodial, trypanosomal

parasites, as well as some bacterial strains. TEM analysis indicated an

average size of about 37 nm and the SEM analysis showed that most of

the nanoparticles synthesized were of irregular spherical shape. Although

AuNPs were not active against plasmodial and trypanosomal parasites,

they were able to inhibit all the bacterial strains used. This confirms the

usage of the plant seeds as an excellent source for naturally occurring

cytotoxic and antimicrobial drugs.

214

Chapter 5

General Discussion, Conclusions and Recommendation

5.1 General Discussion and Main Findings

The aim of this work was to know the bioactive compounds responsible

for the different therapeutic activities of Callistemon citrinus from the

family Myrtaceae. The hypothesis tested was that a number of active

therapeutic compounds, which would have antimicrobial, antioxidant,

antimalarial and antitrypanosomal properties can be isolated and tested

in-vitro confirming the ethnobotanical/ethnomedicinal uses of the plant as

claimed by traditional healers in rural settings. The rationale of this work

was not only to isolate the bioactive compounds of high therapeutic

potentials but also to perform biosynthesis of both gold and silver

nanoparticles using the biogenic route and test for their toxicity,

antimicrobial, antimalarial and antitrypanosomal potentials, which are

scarcely investigated, as far as Callistemon citrinus is concerned in this

part of the world. Although, therapeutic plants are generally regarded to

be safe, nonetheless, there is a need for the assessment of the crude

extract from this plant on a normal cell line, so as to allow a proper

determination of their toxicity.

In this study a practicable scientific validation and justification on the

ethnomedicinal potentials and uses of Callistemon citrinus were

established. The plant has been used across Africa, China, India and other

countries to treat diarrhea, dysentery and rheumatism. It is also used by

traditional healers in India to treat gastro-intestinal disorder, respiratory

215

ailment such as cough, bronchitis, pain, hemorrhoids and infectious

diseases caused by bacteria, fungi, virus and other pathogens.

Callistemon citrinus has also been found useful as an insecticide and its

essential oil as an antimicrobial herbal.

Hydrodistillation using Clevenger apparatus allowed was employed

toextract the volatile oil from the aerial part of this plant while GC-MS was

used to characterize the chemical components. In vitro antioxidant

potential of the plant was determined by DPPH and ABTS method,

antibacterial potential was obtained through agar well diffusion method.

UV-Vis, X-ray diffraction (XRD), scanning electron microscopy (SEM),

energy dispersive X-ray (EDS), transmission electron microscopy (TEM)

and Fourier transform infrared (FT IR) were used to characterize the gold

and silver nanoparticles.

The crude extracts from the aerial parts of the plant were extracted

using organic solvents of different polarities. Organic and aqueous crude

extracts as well as the volatile oils obtained from the aerial parts of the

plants exhibited very wide spectrum of action against gram-positive

bacteria like Listeria monocytogenes (ACC), Staphylococcal enteritis

(ACC) and Staphylococcus aureus(ACC) and gram negative-bacteria Vibro

alginolyticus (DSM 2171), Aeromonas hydrophila (ACC), Escherichia coli

(ATCC 35150) and Salmonella typhi (ACC), which are basically linked with

gastro-intestinal disorder, respiratory, urinary tract infections, pain, cough

and dysentery.

216

The volatile oil showed remarkable inhibitory zones that were effective

on all the tested bacteria listed above, indicating that the plant under

study possesses wide ranging spectrum of action against the two

categories of bacteria. The inhibitory effects of oils obtained from the

leaves and flowers were highest against gram-negative bacteria like Vibro

alginolyticus DSM 2171 (67 ± 2.0 and 60 ± 5.0 mm) and Aeromonas

hydrophila ACC (58 ± 0.3 and 52 ± 1.0 mm), as well as the gram-positive

bacteria such as Staphylococcal enteritis ACC (62 ± 0.5 and 55 ± 2.0

mm) at a concentration of 0.4 mg/mL, while the lowest effect was

recorded for E.coli ATCC 35150 (27 ± 3.0 & 20 ± 4.0 mm).

The antibacterial effect observed in this plant may be linked to some

bioactive compounds such as alkaloids, tannins, terpenoids, ether and

phenolic compounds like flavonoids, which are considered to be

bacteriostatic and fungistatic. This effect correlates with its folkloric uses

and shows that it is an efficient antimicrobial plant that can be employed

as alternative medicine for the treatment of bacterial infection.

The DPPH inhibition percentage of the volatile oils at different

concentrations (0.025, 0.05, 0.1, 0.2, 0.4 mg mL-1) ranged between

38.3% and 76.2% for the leaves oil and from 40.7% to 80.6% for the

flowers oil. The percentage of inhibition for both the ascorbic acid and β-

carotene varied as 18.1%–54.04% and 32.4% –77.45% respectively. The

oils from the leaves and flowers were capable of reducing the DPPH

radical by 50% with IC50 of 1.49 and 1.13 mg mL-1 compared to β-

carotene and ascorbic acid which have an IC50 of 1.28 and 3.57 mg mL-1.

217

The capacity of the DPPH radical scavenging of the flower oils in terms of

percentage inhibition and IC50 was higher than those of the leaf oils.

The leaves and flowers of the plant demonstrated free radical

scavenging activities which were dose dependent under ABTS assay,

having a maximum activity of 79.47% at 0.4 mg mL-1 for the leaves oil

and 95.61% for the flower oils. The oils from both plant parts showed a

50% reduction with IC50 of 0.14 and 0.03 mgmL−1 respectively, which

implies that the flower oils possess higher antioxidant capacity than the

leaf oils and other typical antioxidants (vitamin C and butylated hydroxyl

toluene) with IC50 of 0.13 and 0.19, respectively. The high scavenging

activity of the leaf oils over BHT (standard antioxidant) could be due to

the high content of eucalyptol in the leaf oil.

In another experiment the chemical transformation taking place in the

volatile oil was confirmed through GC-MS analysis for a period of one

year. The hydro-distillation of the fresh leaves of Callistemon citrinus gave

a pale-yellow volatile oil with a strong scenting fragrance. About ninety-

seven components were identified in the twelve treatments analyzed in

one year. The key components were pinocarvone (1.25-6.17%),

pinocarveol (0.10-9.56%), α-Terpineol (5.24-9.94%), α-Pinene (7.45-

22.75%), limonene (24.08) and eucalyptol (14.69-72.35%). The

compositional profile of the leaves of Callistemon citrinus varied between

January and December. Treatments under investigation revealed

markedly qualitatively and quantitatively differences. The obtained results

218

show that seasonal variations affected the chemical compositions, oil yield

and also the antioxidant activities of the volatile oil of Callistemon citrinus

leaves. Therefore, it is important to consider such effects for industrial

and therapeutic purposes.

Active phytochemicals compounds present in both ethyl acetate and

methanolic extracts of Callistemon citrinus were examined. The

antimicrobial, bioactive determination using GC-MS, time of kill and

antioxidant activities of the plant were also explored in the study. The

bioactive components from both extracts were determined using GC-MS.

Both ethyl acetate and methanol extracts were characterized by high

amount of fatty acids (52.88 & 62.48%). Other noticeable components in

the methanol and ethyl acetate extracts include esters (21.48 and

8.06%), oxygenated monoterpenoids (8.00 and 4.48%), triterpenes (2.98

and 2.52%), respectively. Oleic acid, a fatty acid was identified as one of

the major components from the GC-MS results of the two extracts (6.42

and 28.23%), others in varying quantities were palmitic (32.87 &

13.57%) and stearic acids (13.59 &10.43%)

The antibacterial results of the ethyl acetate extract demonstrated

strong activity against P. aeruginosa ACC (28.7± 1.2 mm), Listeria ACC

(26.0 ± 2.0 mm) and Escherichia coli ATCC 35150 (24.0 ± 3.5 mm), just

as similar action was recorded for methanol extract against P. aeruginosa

ACC (22.7± 1.2 mm) and Aeromonas hydrophilia ACC (19.7± 1.5 mm).

Furthermore, the biosynthesis of AuNPs and AgNPs using aqueous

extracts obtained from the aerial parts of the plants was successful,

219

followed by the characterization of the two nanoparticles and the in vitro

evaluation of antitrypanosomal, antimalarial, antibacterial activities as

well as cell cytotoxicity of the green synthesized nanoparticles.

Characterization of the AuNPs and AgNPs was achieved via several

analysis like UV-Visible spectrophotometry, X-ray diffraction (XRD),

scanning electron microscopy (SEM), energy dispersive X-ray (EDS),

transmission electron microscopy (TEM) and Fourier transform infra-red

spectroscopy (FTIR). The biosynthesized nanoparticles displayed very

good antitrypanosomal, antibacterial and antimalarial action and were

devoid of being cytotoxic to Hela (human cervix adenocarcinoma) cells.

Several biological investigations of Callistemon citrinus have been

reported in the literature, but the antimalarial, antitrypanosomal and cell

cytotoxicity using Hela (human cervix adenocarcinoma) cells of the

biosynthesized AgNPs and AuNPs from the aerial parts of Callistemon

citrinus are to the best of our knowledge reported for the first time. The

XRD showed that the AgNPs were crystalline in nature while the TEM

showed that the shapes were spherical with an average size of 29 nm.

The SEM and EDS demonstrated triangular shaped materials and that the

AgNPs were made up of silver and oxygen only. Absorption spectra

confirmed by UV-vis signify the dispersed nature of the synthesized

nanoparticles with absorption band observed at 280 nm for the leaf. FTIR

had absorption bands at about 1700 cm-1 in all spectra‟s establishing the

C=O stretching owing to amide bond, another remarkable peak at 3400

cm-1 was seen in the crude extract which was ascribed to the O-H

220

stretching from water as a result of the aqueous nature of the plant

extracts used. It is interesting to know that this peak was not seen in the

AgNPs demonstrating the development of calcined AgNPs. In addition to

this, peak at 420 cm-1 was observed for all the three nanoparticles

synthesized and this shows the successfully synthesis of the AgNPs.

The antimicrobial actions of the of the AgNPs was also confirm through

both gram-positive and gram-negative bacteria strains like Escherichia

coli 0157:H7:ATCC 35150, Vibro alginolyticus DSM 2171, Salmonella

typhi ACC, Staphylococcal enteritis ACC, Staphylococcus aureus ACC,

Listeria Ivanovii ATCC 19119 and Mycobacterium smegmatis ATCC

19420. Significant inhibitory action from the flower AgNPs of this plant

was recorded, exhibiting highest inhibitory activities (25.0 ± 3.0 mg/mL

and 25.0 ± 2.0 mg/mL) against Escherichia coli 0157:H7: ATCC 35150 a

gram-negative bacteria and Listeria Ivanovii ATCC 19119, a gram-positive

bacteria strain among the nanoparticles. Seed and flower AgNPs displayed

the same inhibitory activity of 15.0 ± 2.0 mg/mL against Vibro

alginolyticus DSM 2171 at the highest concentration. Minimum inhibitory

concentration values of 7.8125 mg/mL were documented for all the

AgNPs. Potent antiplasmodial activities with IC50 ranging from 2.99 to

5.34 μg/mL were also recorded and a poor IC50 of 107.30 μg/mL for

antitrypanosomal activity of the leaf AgNPs as well.

The biosynthesized AuNPs were verified by a color change immediately

after the seed extract was added to the gold (III) chloride solution. The

FT IR showed an absorption peak at 230 cm-1 indicating absorption band

221

for gold nanoparticles. The morphology and composition of the AuNPs was

ascertained by SEM and EDS micrographs; uneven spherical shaped

nanoparticles were established by the SEM analysis and an average

particle size of about 37 nm was confirmed by the TEM analysis. The

crude extracts exhibited antitrypanosomal activities with an IC50 of

11.06, 33.66 and 37.1 μg/mL for the seed, flower and leaf. A poor

antitrypanosomal action was observed for the AuNPs. The crude seed

extract and AuNPs were inactive against plasmodial parasite; also the

antibacterial activities of the nanoparticle were potent against gram-

positive and gram-negative bacterial strains.

The crude organic extracts and the fractions obtained from them

exhibited high antimalarial and antitrypanosomal activities. The IC50 for

antimalarial activity of the crude extracts A-E ranges from 2.30 to 4.98

with crude A and E extracts (2.30 and 3.22) having the highest

antimalarial activities were selected for isolation. Similarly, crude extracts

A and E have the highest antitrypanosomal activities with IC50 of 1.19

and 0.39 and these crudes were selected for isolation in column

chromatography. Unfortunately, all the crude extracts were toxic to Hela

(human cervix adenocarcinoma) cells. This indicates that they are not

safe to be used as targeted drugs for mammalian organisms. Column

chromatography attempt on the fractions that were not cytotoxic

produced no pure compounds.

222

5.2 Future Trends, Prospects and Recommendations

In view of the findings in this study, the following recommendations

are suggested and future prospects are highlighted.

(1) The crude organic extract should be partitioned by water; the sub-

partitioning can be done using organic solvents of different

polarities.

(2) Bio-guided assay and isolation should be carried out on the stem,

bark and root of Callistemon citrinus as the organic leaf extract

contains a lot of chlorophyll which causes a drag on TLC plate and

makes isolation difficult to achieve.

(3) Isolation should also be attempted on the aqueous crude extract

because all the organic crude extracts apart from those from the

volatile oils and some fractions from the crude were cytotoxic to

Hela (human cervix adenocarcinoma)/HEK 293 (human embryonic

kidney) cells but the aqueous crude extracts from the aerial parts of

the plant were not toxic and were therefore used for the

biosynthesis of AuNPs and AgNPs.

(4) The development of polymeric nanocarriers that can be used for

drug delivery should be embarked upon in the future since the

aqueous crude extract of the plant was not toxic.

223

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